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https://openalex.org/W4286907039	https://zenodo.org/records/5559436/files/PJEST-014-GICCL-21(1).pdf	English	null	FUZZY BASED APPROACH FOR THE PARAMETRIC ESTIMATION OF THE MEMS BASED THERMO PNEUMATIC MICRO PUMP	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	3,258	ABSTRACT Over the last few decades, technological advancements and the tendency toward miniaturization have resulted in the development of a new subject known as micro-electro- mechanical systems (MEMS). MEMS have many applications but our focus in this research was on MEMS-based thermopneumatic micro pump. It consists of a chamber, loaded with vapors or solution, a diaphragm, and a temperature control system. It has many advantages like low power consumption while maintaining high performance and accurate dose control. MATLAB fuzzy logic controller (FLC) is used for the parametric estimation of the MEMS-based thermopneumatic micropump. The effect of voltage and frequency applied on the micropump were studied on the back pressure and flow rate. The increase in voltages and frequency were decreased the back pressure and fluid flow rate. Similarly, with a decrease in voltages and frequency were increased the back pressure and fluid flow rate. The difference between the MAMDANI calculated value and the simulated value from the rule viewer is 1.61% for both the output of back pressure and flow rate. Corresponding Author: Ghulam Muhiudin: gmuhiudin786@gmail.com Original Research Article Keywords: MEMS, Thermopneumatic micropump, Fuzzy logic controller Pakistan Journal Emerging Sciences and Technologies (PJEST) by Govt. Islamia College Civil Lines Lahore, Pakistan is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License. PAKISTAN JOURNAL OF EMERGING SCIENCE AND TECHNOLOGIES (PJEST) October, 2021, 1(1) ISSN 2788-8630 (On-Line) FUZZY BASED APPROACH FOR THE PARAMETRIC ESTIMATION OF THE MEMS BASED THERMO PNEUMATIC MICRO PUMP Syed Asad Ali Shah1, Maira Syed2, Ghulam Muhiudin1, Firdous Ahmad1, Maham Akhlaq1 1 Department of Physics (Electronics), Government College University, Lahore 2 Centre of Excellence in Solid State Physics, University of the Punjab, Lahore October, 2021, 1(1) October, 2021, 1(1) Correspondence: gmuhiudin786@gmail.com ARTICLE INFORMATION Citation: Received: 15thSeptember 2021 Revised and Accepted: 03-October-2021 Published On-Line 10-October-2021 Corresponding Author: Ghulam Muhiudin: gmuhiudin786@gmail.com Original Research Article ARTICLE INFORMATION Citation: Received: 15thSeptember 2021 Revised and Accepted: 03-October-2021 Published On-Line 10-October-2021 *Corresponding Author: Ghulam Muhiudin: gmuhiudin786@gmail.com Original Research Article Introduction: Many micropumps have already been created utilizing various techniques for use in MEMS. The thermopneumatic technique is one of the various techniques, significant in the categorization of mechanical micropump [1]. Looking for the benefits of the thermopneumatic actuator it consists of compact size and cheap expense as well as low energy efficiency while maintaining high performance and accurate dose control [2]. In addition, it can easily Page 108 of 10 Page 108 of 10 manufacture by the lithography method. This kind of actuator has a chamber, loaded by vapors or solution, diaphragm and the temperature control system [3]. This system includes the heater and air conditioning module this will alter the pressure within the air chamber as a result solution will be pumped back and forth between pump chamber using input and exit valves [4]. In response, the diaphragm moves with a moderate voltage level. When temperature rises with the heater, deforming the diaphragm and pressing the pump chamber in the process. In cooling process, the lowering temperature will lower the pressure exerted on the diaphragm. Therefore, the diaphragm returns to its normal position [5]. This change in pressure in the micropump serve as the metrics for analyzing the performance. The quantity of solution pump is primarily determined by displacement of diaphragm, that in turn determined by the torque produced by the actuators [6]. The change in pressure is described by the following equation (1). ∆𝑃= 𝐸(𝛽∆𝑇− ∆𝑉 𝑉) (1) (1) Where ∆𝑃 represents the change pressure, 𝛽 represents thermal expansion, ∆𝑇 represents the temperature change, and ∆𝑉 𝑉 represents a percentage of volume change [7]. The resonance frequency is governed by the stiffness characteristics of material which in turn influenced by geometrical characteristics such as thickness and area of the micropump [8]. The diagrammatic representation of the thermopneumatic micropump is shown below. Fig. 1. Diagrammatic representation of thermopneumatic micropump [9] Fig. 1. Diagrammatic representation of thermopneumatic micropump [9] The pump chamber, on the other hand, causes heat to build up in the chamber. When heat production is not an issue, thermopneumatic pump is a good choice for the application Furthermore, the low thermal response is an inherent characteristic of this pump [10]. Thermopneumatic micropumps has a lengthy thermal time constant, which has restricted its application. As a result, Woias et al in 2005 described the maximum frequency of thermopneumatic pump to be around 50 Hz [11]. Introduction: Van De Pol et al. (1990) was the first researcher reported the thermopneumatic micropump in which fluid velocity was measured at 34 L/min and temperatures reached almost 30 degrees Celsius at 6 V [9]. The efficiency of the system decreases above 100Hz as thermopneumatic micropump may not be capable to cool enough the fluid [12]. The researchers of 2015 have demonstrated the thermopneumatic micropump without using the mechanical parts although this pump is classified under the mechanical pumps. Parthasarathy et al. demonstrated that such pump operates based on Charles Law, which specifies that volume is direct proportion to the temperature, the pressure remain Page 109 of 10 constant [13]. Chin and Tan et al. uses the thermopneumatic actuation method for their applications as Biomedical and microfluidic devices respectively. Abi-Samra et al. represented the TPP for the application as centrifugal microfluidic disc platforms [14]. The LC resonator driven TPP was constructed by Chee et al. simulated findings showed that the heat chamber reached 46.7°C in very little time of 40s following activating the micro heater [15-16]. Fuzzy Logic Controller (FLC) is used for simulation because it provides a very valuable flexibility for reasoning[17]. Fuzzy logic is used to handle fractional or partial truths, where the truth value may be completely true or false or may be in between of both. Relative importance of precision is the core of the fuzzy logic. It is a technique to impose human-like thinking into a controlled experiment[18][19]. In this work, a fuzzy logic controller was used to perform parametric estimate for the development of a MEMS-based thermopneumatic micropump. When an electric voltage is provided to the heater, the temperature of the air inside the cavity rises, which causes the diaphragm to deflect. The pressure is created by the diaphragm deflection, and this pressure is responsible for the fluid flow through the valve. The increase in voltages and frequency will decrease the back pressure and fluid flow and decrease in voltages and frequency will increase the back pressure and fluid flow. Fuzzy system design: To obtain exact output variable data based on the change in given inputs, input variables have been defined in the fuzzy logic controller. Voltages, frequency, back pressure, and fluid flow are the two input and output variables on the fuzzy inference editor, as illustrated in Fig.2. It's crucial because fuzzy logic has a wide range of values between 0 and 1, which makes it almost optimal. Fig.2. Fuzzy Inference system for the thermopneumatic micropump The input and output variable graphs were created using the MATLAB membership function. A graph has been generated for each of the two unique entities. Figures 3-6 show the graphs for the input voltage and frequency, as well as the output back pressure and flow rate. The range for the input parameters voltage and frequency were 1-30V and 1-20Hz, respectively. The range for the output parameters back pressure and flow rate were 1-6kPa and 1-40ul/min, respectively. Fi 2 F I f t f th th ti i Fig.2. Fuzzy Inference system for the thermopneumatic micropump Fig.2. Fuzzy Inference system for the thermopneumatic micropump The input and output variable graphs were created using the MATLAB membership function. A graph has been generated for each of the two unique entities. Figures 3-6 show the graphs for the input voltage and frequency, as well as the output back pressure and flow rate. The range for the input parameters voltage and frequency were 1-30V and 1-20Hz, respectively. The range for the output parameters back pressure and flow rate were 1-6kPa and 1-40ul/min, respectively. Page 110 of 10 Fig. 3. Membership function graph showing voltage 1(1-30V) Fig. 4. Membership function graph showing frequency(1-20Hz) Fig. 3. Membership function graph showing voltage 1(1-30V) Fig. 4. Membership function graph showing frequency(1-20Hz) Fig. 5. Membership function graph showing Back pressure (1-6KPa) Fig. 3. Membership function graph showing voltage 1(1-30V) Fig 4 Membership function graph showing frequency(1 20Hz) Fig. 4. Membership function graph showing frequency(1-20Hz) Fig. 5. Membership function graph showing Back pressure (1-6KPa) Fig. 5. Membership function graph showing Back pressure (1-6KPa) Page 111 of 10 Fig. 6. Membership function graph showing Flow Rate (1-40ul/min) Fig. 6. Membership function graph showing Flow Rate (1-40ul/min) Fig. 6. Membership function graph showing Flow Rate (1-40ul/min) g p g p g There are a total of 9 (32 = 9) rules for designing the fuzzy logic controller because there are two input variables and three membership functions. Fuzzy system design: The input linguistic variables are these nine membership functions, and each set has a corresponding output linguistic variable. The Hagen–Poiseuille equation is used to define the output linguistic variable. These rules are used to generate the MATLAB FLC simulation results. The defined rules are shown in Table I. There are a total of 9 (32 = 9) rules for designing the fuzzy logic controller because there are two input variables and three membership functions. The input linguistic variables are these nine membership functions, and each set has a corresponding output linguistic variable. The Hagen–Poiseuille equation is used to define the output linguistic variable. These rules are used to generate the MATLAB FLC simulation results. The defined rules are shown in Table I. Table I: Rules selected for MATLAB fuzzy logic simulations Voltage(V) Frequency (Hz) Back Pressure (KPa) Flow Rate(ul/min) Low Low High High Low Medium High High Low High Medium Medium Medium Low Medium Medium Medium Medium Medium Medium Medium High Low Low High Low Medium Medium High Medium Low Low High High Low Low Table I: Rules selected for MATLAB fuzzy logic simulations Page 112 of 10 3D graphs are investigated using the rules. We have a three-dimensional graph as shown in Fig. 7 and 8 in which voltage and frequency are the two inputs and back pressure and flow rate is the output respectively. Page 113 of 10 Fig. 7. 3D graph between the input voltage and frequency applied to the micropump with respect to the output back pressure. Fig. 8. 3D graph between the input voltage and frequency applied to the micropump with respect to the output flow rate. Fig. 7. 3D graph between the input voltage and frequency applied to the micropump with respect to the output back pressure. Fig. 7. 3D graph between the input voltage and frequency applied to the micropump with respect to the output back pressure. ph between the input voltage and frequency applied to the micropump with respect ack pressure. Fig. 8. 3D graph between the input voltage and frequency applied to the micropump with respect to the output flow rate. Fig. 8. 3D graph between the input voltage and frequency applied to the micropump with respect h fl Fig. 8. 3D graph between the input voltage and frequency applied to the micropump with respect to the output flow rate. Fuzzy system design: Page 113 of 10 Page 113 of 10 Page 113 of 10 Fig. 7 and 8 show that the back pressure will be high as the applied input voltage and frequency will low, and back pressure will be low as the applied input voltage and frequency will high. For output 2 Table II: Comparison between simulated and calculated value and their percentage error Output Simulated Value Calculated Value Error Back Pressure 3.48 3.537 1.61% Voltage 20.4 20.734 1.61% Table II shows the comparison between the simulated and calculated value and their percentage error. The Crisp value of the output 1 from rule viewer is 3.48 and calculated value of Mamdani model is 3.537. The error between the simulated value and the calculated value is 1.61%. The Crisp value of the output 2 from rule viewer is 20.4 and calculated value of Mamdani model is 20.734. The error between the simulated value and the calculated value is 1.61%. Results and Discussion The MATLAB Rule viewer displays the rules defined in the rule editor. The corresponding output crisp value of flow rate and back pressure for any two crisp values of the input variables voltage and frequency can be viewed in the rule viewer as illustrated in Fig. 9. Fig. 9. Rule viewer to show up two outputs for the corresponding two inputs For the calculations of the results, the input crisp values are selected, and their corresponding output crisp values are noted. The selected input crisp values for voltage are 14.5V and frequency 12.8 Hz and the corresponding output crisp values for back pressure are 3.48 KPa and flow rate 20.4 ul/min as shown in Fig. 9. These values of membership functions are calculated as: Fig. 9. Rule viewer to show up two outputs for the corresponding two inputs Fig. 9. Rule viewer to show up two outputs for the corresponding two inputs For the calculations of the results, the input crisp values are selected, and their corresponding output crisp values are noted. The selected input crisp values for voltage are 14.5V and frequency 12.8 Hz and the corresponding output crisp values for back pressure are 3.48 KPa and flow rate 20.4 ul/min as shown in Fig. 9. These values of membership functions are calculated as: For the calculations of the results, the input crisp values are selected, and their corresponding output crisp values are noted. The selected input crisp values for voltage are 14.5V and frequency 12.8 Hz and the corresponding output crisp values for back pressure are 3.48 KPa and flow rate 20.4 ul/min as shown in Fig. 9. These values of membership functions are calculated as: A1= 30-14.5/30=0.5166 V A2= 1-A1= 0.4833 V A3= 20-12.8/20=0.36 Hz A4= 1-A3= 0.64 Hz A4= 1-A3= 0.64 Hz For the corresponding membership function values A1, A2, A3 and A4, the following 4 rules out of total nine rules are selected. The Mamdani formula is used to calculate the minimum membership function value (Gi) and the Singleton value (Vi). The total of all minimal membership function values and singleton values is calculated, and by using these values the crisp value of output is derived as shown below: Page 114 of 10 For output 1 For output 2 Conflicts of Interest: The authors declare no conflict of interest. Acknowledgement: The authors would like to thank the Nano-Electronics lab, Department of Physics (Electronics), GC university Lahore for assistance with the collection of data. Acknowledgement: The authors would like to thank the Nano-Electronics lab, Department of Physics (Electronics), GC university Lahore for assistance with the collection of data. Conclusion MEMS-based micropumps have gained a significant importance due to its wide applications in biomedical sector as well as in industrial sector. In biomedical sector it is used for Page 115 of 10 Page 115 of 10 drug delivery within implantable systems. In industrial sector it is used during the manufacturing processes for the delivery of small amount of glue. In this work MATLAB fuzzy logic controller is used for the parametric estimation of the MEMS-based thermopneumatic micro pump. The effect of voltage and frequency applied on micro pump were studied on the back pressure and flow rate. The increase in voltages and frequency were decreased the back pressure and fluid flow rate. Similarly with a decrease in voltages and frequency were increased the back pressure and fluid flow rate. The difference between the MAMDANI calculated value and the simulated value from the rule viewer are 1.61% for both the output of back pressure and flow rate. Author’s Contribution: G.M., Conceived the idea; S.A.A.S., G.M., designed the simulated work in Fuzzy Logic Controller, and M.A., F.A., did the acquisition of data; F.A., G.M., M.A., M.S., executed simulated work, F.A., did the data analysis; G.M., M.S., wrote the basic draft, S.A.A.S., did the language and grammatical edits or Critical revision. Funding: The publication of this article was funded by no one. Conflicts of Interest: The authors declare no conflict of interest. REFERENCES [1] O. C. Jeong and S. S. Yang, “Fabrication of a thermopneumatic micropump with aluminum flap valves,” J. Korean Phys. Soc., vol. 37, no. 6, pp. 873–877, 2000, doi: 10.3938/jkps.37.873. [2] H. K. Bardaweel and S. K. Bardaweel, “Dynamic simulation of thermopneumatic micropumps for biomedical applications,” Microsyst. Technol., vol. 19, no. 12, pp. 2017–2024, 2013, doi: 10.1007/s00542-012-1734-3. [3] Y. J. Yang and H. H. Liao, “Development and characterization of thermopneumatic peristaltic micropumps,” J. Micromechanics Microengineering, vol. 19, no. 2, 2009, doi: 10.1088/0960- 1317/19/2/025003. [4] C. Joshitha, B. S. Sreeja, and S. Radha, “A review on micropumps for drug delivery system,” Proc. 2017 Int. Conf. Wirel. Commun. Signal Process. Networking, WiSPNET 2017, vol. 2018- Janua, pp. 186–190, 2018, doi: 10.1109/WiSPNET.2017.8299745. [5] P. K. Das and A. B. M. T. Hasan, “Mechanical micropumps and their applications: A review,” AIP Conf. Proc., vol. 1851, 2017, doi: 10.1063/1.4984739. [6] S. Mohith, P. N. Karanth, and S. M. Kulkarni, “Experimental investigation on performance of disposable micropump with retrofit piezo stack actuator for biomedical application,” Microsyst. Technol., vol. 25, no. 12, pp. 4741–4752, 2019, doi: 10.1007/s00542-019-04414-2. [7] M. W. Ashraf, S. Tayyaba, and N. Afzulpurkar, “Micro Electromechanical Systems (MEMS) based microfluidic devices for biomedical applications,” Int. J. Mol. Sci., vol. 12, no. 6, pp. 3648– 3704, 2011, doi: 10.3390/ijms12063648. [8] S. Mohith, P. N. Karanth, and S. M. Kulkarni, “Recent trends in mechanical micropumps and their applications: A review,” Mechatronics, vol. 60, no. February, pp. 34–55, 2019, doi: 10.1016/j.mechatronics.2019.04.009. [9] R. Barua, S. Datta, A. R. Chowdhury, and P. Datta, “Advances in MEMS and Micro-Scale Technologies for Application in Controlled Drug-Dosing Systems,” no. May, pp. 165–179, 2018, doi: 10.4018/978-1-5225-4969-7.ch007. 10] C. Huo, C. Bai, and P. Zhang, “Micropumps for Microfluidic Devices and BioM C. Bai, and P. Zhang, “Micropumps for Microfluidic Devices and BioMEMS,” J. Phys Page 116 of 10 Page 116 of 10 Conf. Ser., vol. 1626, no. 1, 2020, doi: 10.1088/1742-6596/1626/1/012040. [11] P. 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https://openalex.org/W2950485079	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0203238&type=printable	English	null	Impact of fruit-tree shade intensity on the growth, yield, and quality of intercropped wheat	PloS one	2,019	cc-by	8,869	Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This work was funded by the National Natural Science Foundation of China (Grant Nos. 31560587, 31660370, 31760364), Special Fund for Agro-scientific Research in the Public Interest (201503116) and the Modern Agricultural Industry Technology System (CARS-03-49). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Impact of fruit-tree shade intensity on the growth, yield, and quality of intercropped wheat Xu QiaoID1,2, Lihan Sai2, Xingwu Chen2, Lihua Xue2, Junjie LeiID2* 1 Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing, China, 2 Institute of Grain Groups, Xinjiang Academy of Agricultural Sciences, Urumqi, Xinjiang, China Xu QiaoID1,2, Lihan Sai2, Xingwu Chen2, Lihua Xue2, Junjie LeiID2* Xu QiaoID1,2, Lihan Sai2, Xingwu Chen2, Lihua Xue2, Junjie LeiID2* 1 Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing, China, 2 Institute of Grain Groups, Xinjiang Academy of Agricultural Sciences, Urumqi, Xinjiang, China 1 Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing, China, 2 Institute of Grain Groups, Xinjiang Academy of Agricultural Sciences, Urumqi, Xinjiang, China * leijunjie@sohu.com a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 RESEARCH ARTICLE Abstract Agroforestry is a common traditional practice in China-especially in the southern Xinjiang of Northwest China. However, the productivity of many agroforestry systems has been lower than expected in recent years, highlighting the need for an actionably deep mechanistic understanding of the competition between crops and trees. Here, three different fruit tree/ wheat (jujube/wheat, apricot /wheat, and walnut /wheat) intercropping agroforestry systems were chosen to investigate influence of different fruit tree shade intensity on the growth, yield and quality of intercropping wheat. Compared to the monoculture wheat system, the mean daily shade intensity of the jujube-, apricot-, and walnut-based intercropping systems were, respectively, 23.2%, 57.5%, and 80.7% shade. The photosynthetic rate of wheat in the jujube-, apricot-, and walnut-based intercropping systems decreased by, respectively, 11.3%, 31.9%, and 36.2% compared to monoculture wheat, and the mean number of fertile florets per spike decreased by 26.4%, 37.4%, and 49.5%. Moreover, the apricot- and walnut- based intercropping systems deleteriously affected grain yield (constituent components spike number, grains per spike, and thousand grain weight) and decreased the total N, P, and K content of intercropping wheat. Tree shading intensity strongly enhanced the grain pro- tein content, wet gluten content, dough development time, and dough stability time of wheat, but significantly decreased the softening degree. Strong negative linear correlations were observed between tree shade intensity and the number of fertile florets, grain yield related traits (including spike number, grains per spike, and thousand grain weight), nutrient content (N, P and K), and softening degree of wheat. In contrast, Daily shade intensity was positively linearly correlated with grain protein content, wet gluten content, dough development time, and dough stability time. We conclude that jujube-based intercropping systems can be practi- cal in the region, as they do not decrease the yield and quality of intercropping wheat. Editor: Guangyuan He, Huazhong University of Science and Technology, CHINA Editor: Guangyuan He, Huazhong University of Science and Technology, CHINA Received: August 14, 2018 Accepted: January 29, 2019 Published: April 2, 2019 Received: August 14, 2018 Accepted: January 29, 2019 Published: April 2, 2019 Copyright: © 2019 Qiao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OPEN ACCESS Citation: Qiao X, Sai L, Chen X, Xue L, Lei J (2019) Impact of fruit-tree shade intensity on the growth, yield, and quality of intercropped wheat. PLoS ONE 14(4): e0203238. https://doi.org/10.1371/journal. pone.0203238 Editor: Guangyuan He, Huazhong University of Science and Technology, CHINA Fruit tree shade decrease intercropping wheat yield and quality having significant potential to provide a range of environmental services, including reductions in nutrient leaching, improvements in soil erosion and water loss [3], enhancement of soil nutrient status and nutrient cycling [4], sequestration of carbon [5], increases in soil organic carbon, increases in soil microbial community diversity and abundance [6], and increases in the effects of the activity of beneficial soil organisms [7]. Additionally, agroforestry systems can provide windbreaks, thereby reducing wind speed [1,8]. Tree-based intercropping systems also promote larger earthworm populations compared to monoculture crops [9]. Zizyphus jujuba–Triticum aestivum agroforestry systems are frequently used to improve land-use effi- ciency and increase economic returns in southern Xinjiang Province [10]. Competing interests: The authors have declared that no competing interests exist. Friday and Fownes [11] reported that competition for light is the main cause of reductions in maize yields in hedgerow/maize intercropping systems in the USA. Kittur et al. [12] reported that low understory photosynthetically active radiation (PAR) was the dominant factor contrib- uting to reductions in the growth of turmeric in denser bamboo stands compared to widely spaced bamboo in India. Similar results were reported in Paulownia systems on the North China Plain and Loess Plateau [13–15]. Jose et al. [16] observed that maize yields were reduced by 35% and 33% when intercropped with black walnut and red oak, respectively, compared to monoculture treatments. Smethurst et al. [8] also found that competition for light was the main factor causing lower crop yields compared to a monoculture configuration in a temperate agro- forestry system, and that C4 crops (e.g., maize) were more vulnerable to shading compared to C3 crops (e.g., wheat). Wang et al. [17] reported that the yields of both jujube and wheat were lower in 3-, 5-, and 7-year-old jujube tree–wheat intercropping systems, and that the wheat yield decreased as the distance from the jujube trees decreased. Thus, it is important to investi- gate the mechanisms of aboveground competitive interactions in agroforestry systems. By 2012, the total area of fruit trees in southern Xinjiang Uygur Autonomous Region, North- west China, had reached more than 1 million hectares [10]. Fruit tree-based intercropping sys- tems are widely favored by the local population, and more than 80% of fruit trees have been planted as intercropping systems. However, as the fruit trees have grown, the productivity of the intercropping crops in many of the agroforestry systems has been lower than expected in recent years [10,15,17]. The widespread planting of fruit trees has consequences for food security, and has challenged the ability of the region to feed the local population. Therefore, it is important to highlight the need for a systematic understanding of belowground and aboveground interactions under different agroforestry systems to guide practices that can achieve high yields and efficiency. Although many of the competitive pathways in alley cropping systems have been identified, not all have been adequately quantified. In this study, we compared three different varieties of fruit-tree (jujube, apricot, and walnut) intercropping with wheat to examine the aboveground interactions and likely response mechanisms. Fruit trees and wheat were selected for study because of their importance as the main economic and food crops in southern Xinjiang Uygur Autonomous Region, Northwest China. The objectives of the study were to determine (1) whether the fruit trees had a significant effect on the growth and yield of the companion crop (wheat) via shading; (2) whether the yield of the intercropped plants could be increased in this agroforestry system, and what possible solutions are available to minimize aboveground inter- species competition; (3) whether this planting mode is suitable, and which fruit tree-based intercropping system offers the best option in the region; and (4) the effects of this agroforestry system on the quality of the intercropped wheat. Introduction Agroforestry is a land-use system in which woody perennials are grown in association with agricultural crops or pastures, in which there are both ecological and economic interactions between trees and the other components [1,2]. Agroforestry systems are increasingly viewed as 1 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 Competing interests: The authors have declared that no competing interests exist. Experimental design All fields were fertilized with farmyard manure (15,000 kg hm-2, N: P2O5: K2O = 0.37%: 0.41%: 0.46%), urea (275 kg N hm-2), triple superphosphate (150 kg P2O5 hm-2), and potassium sulphate (150 kg K2O hm-2); all were applied homogeneously throughout the fields before sowing wheat (40% of the N was applied initially, with the remain- ing 60% of the N fertilizer applied at the wheat stem elongation stage). In 2011, monoculture wheat and the wheat for the 3 intercropping systems were sown on 8 October, 2010 and harvested on 11 June, 2011. In 2012, the wheat was sown on 3 October, 2011 and harvested on 9 June, 2012. All fields were fertilized with farmyard manure (15,000 kg hm-2, N: P2O5: K2O = 0.37%: 0.41%: 0.46%), urea (275 kg N hm-2), triple superphosphate (150 kg P2O5 hm-2), and potassium sulphate (150 kg K2O hm-2); all were applied homogeneously throughout the fields before sowing wheat (40% of the N was applied initially, with the remain- ing 60% of the N fertilizer applied at the wheat stem elongation stage). Site description Field experiments were conducted in 2011 and 2012 at the fourth village of Zepu County (38˚ 05´N, 77˚10´E), Kashi Prefecture, Xinjiang Uygur Autonomous Region, China. Altitude is 1,318 m above sea level. Annual mean temperature is 11.6˚C (1961–2008). Cumulative temper- ature above 0˚C is 4,183˚C. The mean frost-free period is 212 days. Annual precipitation is 54.8 mm, potential evaporation is 2,079 mm. This region has a typical arid climate, and the soil type is arenosol. Some chemical properties of the soil are presented in S1 Table. Materials and methods Ethics Statement: (1) there was no specific permissions were required for these locations; (2) the field studies did not involve endangered or protected species. 2 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 Fruit tree shade decrease intercropping wheat yield and quality Experimental design The field study comprising 4 planting patterns: monoculture wheat (Triticum aestivum L. Xin- dong-20), wheat intercropped with 9-year-old jujube trees (Zizyphus jujuba Mill. Junzao), wheat with 10-year-old apricot trees (Prunus armeniaca L. Saimaiti), and wheat with 10-year- old walnut trees (Juglans regia L. Wen-185). The row spacing was 0.13 m in wheat. The fruit trees were planted in north-south orientation. Basic information for the different types of fruit trees is showed in Table 1. The jujube-, apricot-, and walnut-based intercropping wheat strips were, respectively, 3.30, 5.10, and 6.00 m wide, and the distance between the jujube, apricot, and walnut tree to the nearest wheat row was 0.85, 0.95, and 1.00 m; the jujube, apricot, and walnut trees occupied 34.0%, 27.1%, and 25.0% of the gross field site areas (Fig 1). The total area for each of the four systems (monoculture wheat, jujube-wheat, etc) was 0.4 hm2. The sown density of monoculture wheat and the 3 intercropping systems were each 4.25×106 plants per hm2. The field study comprising 4 planting patterns: monoculture wheat (Triticum aestivum L. Xin- dong-20), wheat intercropped with 9-year-old jujube trees (Zizyphus jujuba Mill. Junzao), wheat with 10-year-old apricot trees (Prunus armeniaca L. Saimaiti), and wheat with 10-year- old walnut trees (Juglans regia L. Wen-185). The row spacing was 0.13 m in wheat. The fruit trees were planted in north-south orientation. Basic information for the different types of fruit trees is showed in Table 1. The jujube-, apricot-, and walnut-based intercropping wheat strips were, respectively, 3.30, 5.10, and 6.00 m wide, and the distance between the jujube, apricot, and walnut tree to the nearest wheat row was 0.85, 0.95, and 1.00 m; the jujube, apricot, and walnut trees occupied 34.0%, 27.1%, and 25.0% of the gross field site areas (Fig 1). The total area for each of the four systems (monoculture wheat, jujube-wheat, etc) was 0.4 hm2. The sown density of monoculture wheat and the 3 intercropping systems were each 4.25×106 plants per hm2. In 2011, monoculture wheat and the wheat for the 3 intercropping systems were sown on 8 October, 2010 and harvested on 11 June, 2011. In 2012, the wheat was sown on 3 October, 2011 and harvested on 9 June, 2012. Harvest and analysis Schematic illustration of planting patterns in monoculture wheat and 3 different fruit tree-wheat based intercropping systems. This figure represents a single site with each of the four planting patterns. There were 5 samples from different tree rows forming a single replication. https://doi.org/10.1371/journal.pone.0203238.g001 determined using the micro-Kjeldahl method, P concentrations by the molybdo-vanado-phos- phate colorimetrical method and K concentrations by flame photometry [18]. In 2011, 15 plants for each replication were selected to calculate shoot biomass at overwintering stage, reviving stage, jointing stage, booting stage, anthesis stage, filling stage, and maturity stage, respectively, and the shoot samples were heated at 105˚C for 30 min and then oven-dried (72 h, 75˚C) [15]. Fertile florets In both years, 21 main flowering spikes from each replicate, were harvested destructively to investigate fertile florets in the flowering period (50% anthesis). The 21 main flowering spikes were from three regions (in the middle region of the tree rows, underneath the tree of east can- opy and west canopy). Harvest and analysis Wheat was harvested by hand when mature. There were 5 samples from different tree rows forming a single replication. In 2011 and 2012, The monoculture wheat, jujube-, apricot-, and walnut-based intercropping wheat were harvested, respectively, 6.5 m2 (5.0 m length × 1.3 m width), 9.9 m2 (3.0 m × 3.3 m), 10.2 m2 (2.0 m×5.1 m), and 15 m2 (2.5 m × 6.0 m), and samples were immediately dried to a constant weight on a sunning ground to thresh seeds (in order to calculate wheat yield). In order to make wheat samples much more representative, 2 m length intercropping wheat samples from three regions (in the middle region of the tree rows, under- neath the tree of east canopy and west canopy) were harvested respectively to estimate the total spike number and grains per spike, and then all samples were threshed for seeds to estimate thousand grain weight and harvest index. The stalks (except grains) and grain samples were digested in a mixture of concentrated H2SO4 and H2O2. Nitrogen concentrations were Table 1. The basic information of the 3 fruit trees. Fruiter Spacing (m) Age (yr) DBH (cm) Trunk (m) Height (m) Crown width (m) Jujube 1.5×5.0 9 9.4 0.4 2.5 2.4–2.3 Apricot 2.0×7.0 10 17.7 0.7 5.6 5.2–5.8 Walnut 5.0×8.0 10 20.2 1.3 6.6 6.3–6.7 https://doi.org/10.1371/journal.pone.0203238.t001 Table 1. The basic information of the 3 fruit trees. Fruiter Spacing (m) Age (yr) DBH (cm) Trunk (m) Height (m) Crown width (m) Jujube 1.5×5.0 9 9.4 0.4 2.5 2.4–2.3 Apricot 2.0×7.0 10 17.7 0.7 5.6 5.2–5.8 Walnut 5.0×8.0 10 20.2 1.3 6.6 6.3–6.7 https://doi.org/10.1371/journal.pone.0203238.t001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 3 / 17 Table 1. The basic information of the 3 fruit trees. PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 3 / 17 Fruit tree shade decrease intercropping wheat yield and quality Fig 1. Schematic illustration of planting patterns in monoculture wheat and 3 different fruit tree-wheat based intercropping systems. This figure represents a single site with each of the four planting patterns. There were 5 samples from different tree rows forming a single replication. https://doi org/10 1371/journal pone 0203238 g001 Fig 1. Schematic illustration of planting patterns in monoculture wheat and 3 different fruit tree-wheat based intercropp site with each of the four planting patterns. There were 5 samples from different tree rows forming a single replication. Fig 1. PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 Grain quality analyses Grain protein content was measured in whole grains using a near-infrared reflectance analyzer (FOSS -1241, Near Infra -Red Reflectance, Sweden) calibrated respectively to combustion analysis using a LECO FP528 according to official AACC methods (Approved Methods 46– 30.1, AACC International, 2013) [19]. Aliquots of grain portions (50 g) were taken from each plot and tempered to a moisture basis of 152 g H2O kg-1 for 18–20 h before milling (Approved Methods 26–95.01 AACC International 2013). Tempered samples were milled in a Quadrumat Junior Mill (CW Branbender Instruments Inc., South Hackensack, NJ, USA). A standard shaker (Strand Shaker Co., Minneapolis, MN, USA) at 225 rpm for 90 s with the USA standard testing sieve No. 70 with the opening size of 212 μm was used to separate flour from bran, and the flour was weighed (Approved Methods 26–21.02, AACC International 2013) [19]. The extraction was carried out adopting the procedure as described in AACC (Approved Methods 38–12.02, AACC International 2013) [19]. Dough was prepared using 2% sodium chloride solution at the rate of 60% of the weight of flour. Prepared dough was kept immersed in water for 40 min. The dough was washed under stream of running water until all the starch was washed out and the wash water was clear. The viscoelastic mass obtained was wet gluten. The wet gluten content was calculated by the formula given below: Wet gluten contentðdry basis; %Þ ¼ wet gluten weight = flour weight 100% Eqn ð1Þ A 10 g flour sample (adjusted to 140 g H2O kg-1 moisture) was run in a Mixograph (National Manufacturing, Lincoln, NE, USA). Mixograph mixing time was fixed to 8 min and data were analyzed using Mixsmart software (National Manufacturing). Dough development time (Midline peak time was recorded as the time in minutes required for optimum develop- ment of dough), dough stability time (time during dough consistency is at 500 BU) and dough softening degree were measured (Approved Methods 54–40.02, AACC International, 2013) [19]. PAR measurement In 2011 and 2012, light penetration was measured at three regions, in the middle region of the tree rows, under the tree of east canopy and west canopy above wheat using a SunScan Canopy Analysis System (Delta-T Devices, Cambridge, UK). The 64 light sensors of the SunScan mea- sured individual levels of PAR, which were transmitted to a PDA and expressed as μmolm−2s−1. SunScan readings were taken when the sky was clear to avoid the interference of clouds at the filling stage. One measurement was performed every two hours from 09:00 to 19:00. 4 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 Fruit tree shade decrease intercropping wheat yield and quality Photosynthetic parameters In 2011 and 2012, the net photosynthetic rate (Pn) of the flag leaves was determined with a LI- 6400XT Portable Photosynthesis System (LI-COR, Inc., USA), and the readings were taken when the sky was clear to avoid the interference of clouds at the filling stage. The measure- ments were conducted under traditional open system [15] and under controlled conditions with a CO2 concentration of 380 μmol m-2 s-1. The PAR was set at 1200 μmol m-2 s-1, which was provided by a 6400-2B LED light source. The Pn was measured at three regions, in the middle region of the tree rows, under the tree of east canopy and west canopy. One measure- ment was performed every two hours from 09:00 to 19:00. An average value was calculated from three flag leaves from each replicate. Results Results Statistical analysis Mean daily shade intensityð%Þ ¼ ðPARmono PARintÞ= PARmono 100% Eqn ð2Þ Eqn ð2Þ Mean daily shade intensityð%Þ ¼ ðPARmono PARintÞ= PARmono 100% Eqn ð2Þ PARmono is the mean daily PAR of monoculture wheat system; PARint is the mean daily PAR of fruit tree based intercropping system. PARmono is the mean daily PAR of monoculture wheat system; PARint is the mean daily PAR of fruit tree based intercropping system. Experimental data were collected from 2011 and 2012. One way analysis of variance was per- formed on all datasets using SPSS 16.0 for Windows (SPSS Inc., Chicago, IL). Significant differ- ences between pairs of mean values were determined with Duncan’s multiple range test at the 5% level. Standard error between the replications was also calculated. Simple regression analysis PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 5 / 17 Fruit tree shade decrease intercropping wheat yield and quality was used to examine the relationships among the data of fertile florets, grain yield (including spike number, grains per spike, and thousand grain weight), nutrient uptake (N, P, and K con- tent) and grain quality (including protein content, wet gluten content, dough development time, dough stability time, and softening degree) of wheat with understory mean daily shade intensity. Light interception and photosynthetic rate Diurnal variation of the understory photosynthetically active radiation (PAR) and photosyn- thetic rate (Pn) in the three intercropping systems and the monoculture wheat system varied with time, and with single peak curves during midday (13:00–15:00) (Fig 2). Owing to Fig 2. The daily change of photosynthetically active radiation (PAR) and photosynthetic rate (Pn) of wheat in monoculture configurations and 3 different fruit tree-wheat intercropping systems at the filling stage in 2011 (a, c) and 2012 (b, d). The PAR and Pn data are the mean values of the three regions, in the middle region of the tree rows, under the tree of east canopy and west canopy, respectively. Mono, monoculture wheat system; Jiw, jujube-wheat intercropping system; Aiw, apricot- wheat intercropping system; Wiw, walnut-wheat intercropping system. https://doi.org/10.1371/journal.pone.0203238.g002 Fig 2. The daily change of photosynthetically active radiation (PAR) and photosynthetic rate (Pn) of wheat in monoculture configurations and 3 different fruit tree-wheat intercropping systems at the filling stage in 2011 (a, c) and 2012 (b, d). The PAR and Pn data are the mean values of the three regions, in the middle region of the tree rows, under the tree of east canopy and west canopy, respectively. Mono, monoculture wheat system; Jiw, jujube-wheat intercropping system; Aiw, apricot- wheat intercropping system; Wiw, walnut-wheat intercropping system. https://doi.org/10.1371/journal.pone.0203238.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 6 / 17 Fruit tree shade decrease intercropping wheat yield and quality reflectance, absorbance, and transmittance by the canopies of the three fruit tree types, the PAR of crops in the intercropping systems were lower than that in the monoculture configura- tions. For example, the mean daily PAR in the jujube-, apricot-, and walnut-based intercrop- ping systems were, respectively, just 78.7%, 45.5%, and 20.1% of the monoculture configurations in 2011 and 75.0%, 39.5%, and 18.9% of the monoculture configurations in 2012 (Fig 2A and 2B). Further, the photosynthetic rates in the jujube-, apricot-, and walnut- based intercropping systems decreased, respectively, by an average 26.2%, 36.9%, and 50.9% compared to monoculture wheat in 2011 and by 26.6%, 37.9%, and 48.2% in 2012. Fertile florets The distribution of fertile florets along the wheat spikes is shown in Fig 3. Fruit tree shade reduced the number of fertile florets on almost all spikelets, with especially pronounced reduc- tions in the middle position (spikelets 4–12 from the base of the spike). The total number of fertile florets per wheat spike in the monoculture configuration were increased by 1.12, 1.35, and 1.42 times compared to the jujube-, apricot-, and walnut-based intercropping systems in 2011 and by 1.14, 1.61, and 1.76 times in 2012, respectively (Fig 3). Furthermore, significant correlations (P < 0.001) were observed between the number of fertile florets and the mean daily shade intensity of wheat in both 2011 and 2012 (Fig 4). Fig 3. Distribution of the fertile florets along the spike of wheat in monoculture configurations and 3 different fruit tree-wheat intercropping systems in 2011 (a) and 2012 (b). The distribution of the fertile florets data are the mean values of the three regions, in the middle region of the tree rows, under the tree of east canopy and west canopy. Mono, monoculture wheat system; Jiw, jujube-wheat intercropping system; Aiw, apricot-wheat intercropping system; Wiw, walnut-wheat intercropping system. Fig 3. Distribution of the fertile florets along the spike of wheat in monoculture configurations and 3 different fruit tree and 2012 (b). The distribution of the fertile florets data are the mean values of the three regions, in the middle region of the tr west canopy. Mono, monoculture wheat system; Jiw, jujube-wheat intercropping system; Aiw, apricot-wheat intercropping sy system Fig 3. Distribution of the fertile florets along the spike of wheat in monoculture configurations and 3 different fruit tree-wheat intercropping systems in 2011 (a) and 2012 (b). The distribution of the fertile florets data are the mean values of the three regions, in the middle region of the tree rows, under the tree of east canopy and west canopy. Mono, monoculture wheat system; Jiw, jujube-wheat intercropping system; Aiw, apricot-wheat intercropping system; Wiw, walnut-wheat intercropping system. Fig 3. Distribution of the fertile florets along the spike of wheat in monoculture configurations and 3 different fruit tree-wheat intercropping systems in 2011 (a) and 2012 (b). The distribution of the fertile florets data are the mean values of the three regions, in the middle region of the tree rows, under the tree of east canopy and west canopy. Fertile florets Mono, monoculture wheat system; Jiw, jujube-wheat intercropping system; Aiw, apricot-wheat intercropping system; Wiw, walnut-wheat intercropping system. https://doi.org/10.1371/journal.pone.0203238.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 7 / 17 Fruit tree shade decrease intercropping wheat yield and quality Fig 4. Relationship between the fertile florets and mean daily shade intensity in 2011 and 2012. htt //d i /10 1371/j l 0203238 004 Fig 4. Relationship between the fertile florets and mean daily shade intensity in 2011 and 2012. Note: Mono, monoculture wheat system; Jiw, jujube-wheat intercropping system; Aiw, apricot-wheat intercropping system; Wiw, walnut-wheat intercropping system. Across all data, values with the same letter within each column are not significantly different among the treatments (P < 0.05). https://doi.org/10.1371/journal.pone.0203238.t002 https://doi.org/10.1371/journal.pone.0203238.t002 s://doi.org/10.1371/journal.pone.0203238.t002 Wheat yield components In 2011 and 2012, spike number (expressed per unit area of the monoculture wheat or the real intercropping wheat strip area—i.e., without the distance from the fruit trees to the nearest wheat row) and grains per spike were significantly higher in the monoculture wheat and jujube-based intercropping wheat systems than in the apricot- and walnut-based intercropping systems (Table 2). In both years, the thousand grain weight, harvest index (proportion of seed dry weight relative to the total above-ground dry weight), and net yield of wheat in the mono- culture wheat system were each significantly higher than in the jujube-, apricot-, and walnut- based intercropping systems (excluding the net yield of wheat in the jujube-based intercrop- ping system in 2011). Additionally, in both 2011 and 2012, strong negative linear correlations (P < 0.001) were observed between mean daily shade intensity and spike number, grains per spike, thousand grain weight, and net yield (Fig 5). Table 2. Yield components of wheat in monoculture configurations and 3 different fruit tree-wheat intercropping systems in 2011 and 2012. Year Treatment Spike number (104/ hm2) Grains per spike thousand grain weight (g) Harvest index Net yield (kg/ hm2) Gross yield (kg/ hm2) 2011 Mono 654±134a 39.1±2.5a 42.1±1.8a 0.52±0.04a 8114±845a 8114 Jiw 692±109a 37.3±3.0a 36.7±2.3b 0.49±0.03b 8215±868a 5363 Aiw 475±62b 37.4±2.8a 34.9±2.0c 0.44±0.04c 5863±657b 4272 Wiw 446±74b 32.0±4.1b 30.0±4.1d 0.39±0.05d 3555±900c 2666 2012 Mono 637±85a 38.8±4.3a 37.3±3.0a 0.45±0.03a 7506±668a 7506 Jiw 611±87a 36.3±3.8b 31.7±1.8b 0.41±0.04b 6892±825b 4549 Aiw 410±68b 32.4±3.5c 25.8±2.6c 0.37±0.04c 4479±642c 3264 Wiw 342±79c 21.6±4.3d 21.0±2.8d 0.30±0.05d 2654±805d 1990 ture configurations and 3 different fruit tree-wheat intercropping systems in 2011 and 2012. Table 2. Yield components of wheat in monoculture configurations and 3 different fruit tree-wheat intercropping system PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 8 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 Fruit tree shade decrease intercropping wheat yield and quality Fig 5. Relationship between the spike number (a), grains per spike (b), thousand grain weight (c) and net yield (d) with mean daily shade intensity of wheat in 2011 and 2012. Fig 5. Relationship between the spike number (a), grains per spike (b), thousand grain weight (c) and net yield (d) with mean daily shade intensity of wheat in 2011 and 2012. Fig 5. Relationship between the spike number (a), grains per spike (b), thousand grain weight (c) and net yield (d) with mean 2012 Fig 5. Note: Mono, monoculture wheat system; Jiw, jujube-wheat intercropping system; Aiw, apricot-wheat intercropping system; Wiw, walnut-wheat intercropping system. Across all data, values with the same letter within each column are not significantly different among the treatments (P < 0.05). https://doi.org/10.1371/journal.pone.0203238.t003 Wheat yield components Relationship between the spike number (a), grains per spike (b), thousand grain weight (c) and net yield (d) with mean daily shade intensity of wheat in 2011 and 2012 N, P, and K content In 2011 and 2012, the total N, P, and K uptake of wheat in the monoculture system and the jujube-based intercropping systems were significantly higher than in the walnut-based inter- cropping system (Table 3). For example, the N, P, and K content of wheat in the monoculture N, P, and K content In 2011 and 2012, the total N, P, and K uptake of wheat in the monoculture system and the jujube-based intercropping systems were significantly higher than in the walnut-based inter- cropping system (Table 3). For example, the N, P, and K content of wheat in the monoculture N, P, and K content In 2011 and 2012, the total N, P, and K uptake of wheat in the monoculture system and the jujube-based intercropping systems were significantly higher than in the walnut-based inter- cropping system (Table 3). For example, the N, P, and K content of wheat in the monoculture Table 3. The N, P and K contents of wheat in monoculture configurations and 3 different fruit tree-wheat intercropping systems in 2011 and 2012. Year Treatment N content (kg/ hm2) P content (kg/ hm2) K content (kg/ hm2) 2011 Mono 191±9a 29.9±3.4a 548±34a Jiw 198±8a 29.4±2.0a 583±47a Aiw 169±8b 24.3±2.7a 519.6±23a Wiw 138±19c 18.3±3.4b 366±60b 2012 Mono 189±7a 29.4±0.3a 607±13a Jiw 195±11a 28.2±3.3a 604±73a Aiw 169±16b 20.7±1.8b 473±16b Wiw 121±5c 16.8±1.2c 377±35c ts of wheat in monoculture configurations and 3 different fruit tree-wheat intercropping systems in 2011 and 2012. Table 3. The N, P and K contents of wheat in monoculture configurations and 3 different fruit tree-wheat intercrop PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 9 / 17 Fruit tree shade decrease intercropping wheat yield and quality system were, respectively, 1.55, 1.63, and 1.50 times higher than in the walnut-based intercrop- ping system in 2011 and 1.56, 1.75 and 1.61 times higher in 2012. Additionally, in both 2011 and 2012, strong negative linear correlations (P < 0.01) were observed between mean daily shade intensity of wheat and N, P, and K content (Fig 6). https://doi.org/10.1371/journal.pone.0203238.g006 Grain quality traits In 2011 and 2012, grain protein content, wet gluten content, dough development time, and dough stability time of wheat in the walnut-based intercropping system were significantly higher than in the monoculture and jujube-based intercropping system. In contrast, the high- est values for the softening degree parameter were observed in the monoculture system (Table 4). Furthermore, the mean daily shade intensity of wheat both in 2011 and 2012 was highly positively linearly correlated (P < 0.01) with grain protein content, wet gluten content, development time, and stability time (Fig 7A–7D). The softening degree was negatively line- arly correlated (P < 0.01) with mean daily shade intensity (Fig 7E). Wheat yield components system were, respectively, 1.55, 1.63, and 1.50 times higher than in the walnut-based intercrop- ping system in 2011 and 1.56, 1.75 and 1.61 times higher in 2012. Additionally, in both 2011 and 2012, strong negative linear correlations (P < 0.01) were observed between mean daily shade intensity of wheat and N, P, and K content (Fig 6). Fig 6. Relationship between the N, P and K contents with mean daily shade intensity of wheat in 2011 and 2012. https://doi.org/10.1371/journal.pone.0203238.g006 Fig 6. Relationship between the N, P and K contents with mean daily shade intensity of wheat in 2011 and 2012. https://doi.org/10.1371/journal.pone.0203238.g006 10 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 Fruit tree shade decrease intercropping wheat yield and quality Note: Mono, monoculture wheat system; Jiw, jujube-wheat intercropping system; Aiw, apricot-wheat intercropping system; Wiw, walnut-wheat intercropping system. Pro: Grain protein content; WG: Wet gluten content; DDT: Dough development time; DST: Dough stability time; SD: Softening degree. Across all data, values with the same letter within each column are not significantly different among the treatments (P < 0.05). https://doi.org/10.1371/journal.pone.0203238.t004 PAR and photosynthetic rate Light, as a primary limiting factor in tree-based intercropping systems, influences the growth and development of intercropped crops significantly [20]. Awal et al. [21] and Rey- nolds et al. [22] showed that it was difficult for maize to obtain sufficient solar energy when grown underneath the canopy of higher jujube tree components at a distance of less than 2.5 m to the tree rows. Similar results were reported in other studies of temperate agrofor- estry systems [12,23,24]. In our study, the mean daily shade intensity in jujube-, apricot-, and walnut-based intercropping systems compared to monoculture configurations was 21.3%, 54.5%, and 80.3% shade, respectively, in 2011, and 25.0%, 60.5%, and 81.1% in 2012 (Fig 2). Gao et al. [15] observed a clear, positive linear relationship between the distance from the apple tree rows and the daily mean values of PAR and net photosynthetic rate in apple–soybean and apple–peanut intercropping systems. Additionally, Kittur et al. [12] reported that the rhizome yield and understory PAR could be predicted by a linear equa- tion in Kerala, India. In our study, in the walnut tree-based intercropping system, due to its taller trunk, larger leaves, and large canopy architecture, the intercropped wheat was markedly influenced by the shading effect of the trees. The photosynthetic rate of walnut- based intercropped wheat was 50.9% and 48.2% lower than that of monoculture wheat in 2011 and 2012, respectively. Regular pruning of the fruit-tree canopy could reduce light interception, thus improving the yield of intercropped crops. heat in monoculture configurations and 3 different fruit tree-wheat intercropping systems in 2011 and 2012. The grain quality of wheat in monoculture configurations and 3 different fruit tree-wheat intercropping systems in 201 Table 4. The grain quality of wheat in monoculture configurations and 3 different fruit tree-wheat intercropping systems in 2011 and 2012. Year Treatment Pro (%) WG (%) DDT (min) DST (min) SD (FU) 2011 Mono 12.8±0.4c 28.6±1.0b 3.10±0.17b 2.35±0.30b 75.0±4.4a Jiw 12.7±0.4c 27.5±0.3b 3.15±0.31b 3.35±0.77b 67.5±7.3ab Aiw 14.4±0.3b 31.7±1.0b 3.75±0.55ab 3.50±0.20b 65.0±10.4ab Wiw 17.2±0.2a 40.9±4.5a 4.15±0.59a 5.65±0.97a 52.5±8.7b 2012 Mono 12.7±0.2b 28.2±1.0c 3.12±0.10b 2.50±0.30c 72.3±6.8a Jiw 13.5±0.4b 29.8±2.2c 3.90±0.95ab 4.70±0.62b 63.5±4.0b Aiw 14.5±1.8b 33.6±2.6b 4.75±0.85a 6.72±0.76a 52.5±3.9bc Wiw 17.1±1.3a 39.9±0.9a 4.73±0.15a 5.58±0.29a 60.0±1.8c Note: Mono, monoculture wheat system; Jiw, jujube-wheat intercropping system; Aiw, apricot-wheat intercropping system; Wiw, walnut-wheat intercropping system. Pro: Grain protein content; WG: Wet gluten content; DDT: Dough development time; DST: Dough stability time; SD: Softening degree. PAR and photosynthetic rate Across all data, values with the same letter within each column are not significantly different among the treatments (P < 0.05). PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 11 / 17 Fruit tree shade decrease intercropping wheat yield and quality Fig 7. Relationships between grain quality with mean daily shade intensity of wheat in 2011 and 2012. Pro: Protein content; WG: Wet gluten content; DDT: Dough development time; DST: Dough stability time; SD: Softening degree. https://doi.org/10.1371/journal.pone.0203238.g007 pp g y q y Fig 7. Relationships between grain quality with mean daily shade intensity of wheat in 2011 and 2012. Pro: Protein content; WG: Wet gluten content; DDT: Dough development time; DST: Dough stability time; SD: Softening degree. Fig 7. Relationships between grain quality with mean daily shade intensity of wheat in 2011 and 2012. Pro: Protein content; WG: Wet gluten content; DDT: Dough development time; DST: Dough stability time; SD: Softening degree. https://doi.org/10.1371/journal.pone.0203238.g007 https://doi.org/10.1371/journal.pone.0203238.g007 Fertile florets The grain number per spike is determined by the number of spikelets per spike and the num- ber of florets per spikelet. The environmental factors (e.g., light) that determine the spikelet number of grains have been well studied [25]. In the present study, the fruit tree-based inter- cropping system reduced the number of fertile wheat florets on almost all spikelets, particularly in the middle portions of 4–12 spikelets of the spike (Fig 3). Therefore, the development of the PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 12 / 17 Fruit tree shade decrease intercropping wheat yield and quality floret varies considerably depending on its position on the spike [25]. Furthermore, we observed significant (P < 0.001), negative correlations between the fertile florets and mean daily shade intensity from the fruit trees (Fig 4). Willey and Holliday [26] showed that light intensity significantly influenced the initiation of spikelets and floret primordia, giving rise to fewer grains. For example, shading delayed the rate of floret initiation per spike by 11.4%, con- sequently decreasing the number of florets by 22.3% and the grain weight per spike by 19% at maturity [25]. Shoot nitrogen (N), phosphorous (P), and potassium (K) uptake Light plays an important role in dry matter accumulation and the nutrient uptake of crops. Cui et al. [29] showed that shade significantly decreased the total N, P, and K contents of sum- mer maize. In the present study, the total N, P, and K contents of wheat in the walnut-based intercropping systems were significantly lower than those in monoculture wheat (Table 3). Previous studies showed that nutrient uptake by the understory crop were closely correlated with overstory tree density, the understory plant varieties, and plant nutrient demand [15,30]. Kittur et al. [12] reported decreased uptake of N, P, and K by understory turmeric with decreasing bamboo spacing. We observed a highly significant (P < 0.05), negative linear corre- lation between the N, P, and K contents of wheat and mean daily shade intensity (Fig 6). The N, P, and K concentrations of wheat stalks and grains in the walnut-based intercropping sys- tems were significantly higher than those in monoculture wheat (S2 Table). Cui et al. [29] also reported that the N, P, and K concentrations of summer maize were enhanced by shading. The fruit-tree shade intensity increased the dry matter accumulation of the intercropped wheat, which in turn increased the ability of the wheat plants to absorb nutrients. Grain yield components Understory crop yield is determined by the intercepted available light, and the efficiency of converting the intercepted light into photosynthate [15]. Peng et al. [14] reported decreases of 38% and 29% in the yields of maize and soybean, respectively, in a tree-based agroforestry intercropping system on the Loess Plateau, China. In our study, spike number, grains per spike, thousand-grain weight, and net yield were all significantly higher in the monoculture wheat and jujube-based intercropping wheat systems than those in the apricot- and walnut- based intercropping systems in both 2011 and 2012 (Table 2). Additionally, the wheat shoot biomass in the apricot- and walnut-based intercropping systems was 21.4% and 42.5% lower, respectively, at the mature stage in 2011 (S1 Fig). Yang et al. [27] reported that the yield of intercropped wheat was decreased by 25.8%, 16.5%, and 6.70% at distances of 90, 110, and 130 cm to the jujube tree rows, respectively. Other studies of temperate agroforestry systems reported significant increases in the grain yield and yield components with increasing distance to the tree rows [14,28]. We observed a highly significant (P < 0.001), negative linear correla- tion between the wheat grain yield and its components (spike number, grains per spike, and thousand-grain weight) and mean daily shade intensity (Fig 5). Thus, our study demonstrates that the shading intensity of fruit trees in the agroforestry systems had a significant, negative effect on the intercropped grain yield and its components. Conclusion We found that tree shade intensity was generally the major limiting factor for crop productiv- ity in agroforestry systems in this region. Reflectance, absorbance, and transmittance by the tree canopy dramatically reduce the PAR for the crop canopy. Fruit-tree shading resulted in decreased photosynthate accumulation, which in turn resulted in decreased nutrient uptake in the intercropped grain plants. Fruit-tree shading had a marked, negative effect on the develop- ment of fertile florets, resulting in fewer grains per spike and reduced net photosynthesis, ulti- mately resulting in decreases in grain weight, grain yield, and grain quality. Our results show that jujube-based intercropping systems offer a suitable agroforestry system in the region since they did not decrease the yield and quality of the intercropped wheat. Highly significant, nega- tive linear correlations were observed between tree shade intensity and the number of fertile florets, grain yield related traits (including spike number, grains per spike, and thousand-grain weight), nutrient content (N, P, and K), and softening degree of wheat. In contrast, daily shade intensity was positively linearly correlated with wheat grain protein content, wet gluten con- tent, and the dough development and stability times. Future research should focus on the development of shade-tolerant crop varieties and examine how regular pruning of the tree canopy structure can improve crop productivity in such systems. It would also be useful to investigate the mechanisms underlying the aboveground and belowground interspecific inter- actions in agroforestry systems further. Grain quality Lu et al. [31] reported that the protein and wet gluten contents, and the falling number of intercropped wheat increased significantly in a Paulownia-based intercropping system com- pared to a monoculture configuration. Additionally, Wang et al. [32] reported that the wheat starch and crude fat contents were enhanced in apricot tree-based agroforestry systems, and PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 13 / 17 Fruit tree shade decrease intercropping wheat yield and quality that the quality of the wheat decreased with decreasing distance to the apricot trees. In the present study, increased tree shade intensity markedly enhanced the wheat grain protein and wet gluten contents, and dough development and stability times, whereas it significantly decreased the degree of softening (Table 4). Our results are generally consistent with those from previous studies and confirm that tree shading during grain development can have a sub- stantial effect on grain yield and quality in agroforestry systems [32]. We observed a highly sig- nificant (P < 0.01), positive linear correlation between the wheat grain protein and wet gluten contents, and dough development and stability times with the mean daily shade intensity, whereas shade was negatively correlated with the degree of softening (Fig 7). Bhatta et al. [33] reported a negative correlation between grain protein content and grain yield and grain vol- ume weight. In our study, we observed a significant, negative linear correlation between the grain protein and wet gluten contents, and dough development and stability times and the grain yield and thousand-grain weight of wheat, and a positive correlation with the degree of softening (S2 and S3 Figs). Consequently, fruit tree shading resulted in a decrease in wheat yields and seed dry weight, resulting in a decrease in the quality of wheat. Friday and Fownes [11] suggested that the shade intensity from trees could be alleviated by pruning of the tree canopy, increasing the intercepted light reaching the intercropped plants. To obtain higher grain production, appropriate management measures are needed to mini- mize competition in fruit tree–crop intercropping systems, and we recommend the following measures: (1) select more suitable crop varieties (e.g., shade-tolerant, early-maturing, and low- height varieties); (2) select more suitable fruit tree varieties (e.g., low-height varieties that have small leaves); and (3) regularly prune the fruit trees to reduce the shading intensity from the fruit trees. Supporting information N, P and K concentrations in stalks and grains of wheat in monoculture configu- rations and 3 different fruit tree-wheat intercropping systems in 2011 and 2012. Note: Mono, monoculture wheat system; Jiw, jujube-wheat intercropping system; Aiw, apricot- wheat intercropping system; Wiw, walnut-wheat intercropping system. (DOCX) S2 Table. N, P and K concentrations in stalks and grains of wheat in monoculture configu- rations and 3 different fruit tree-wheat intercropping systems in 2011 and 2012. Note: Mono, monoculture wheat system; Jiw, jujube-wheat intercropping system; Aiw, apricot- wheat intercropping system; Wiw, walnut-wheat intercropping system. (DOCX) Project administration: Xingwu Chen. Project administration: Xingwu Chen. Writing – original draft: Xu Qiao, Junjie Lei. Writing – review & editing: Xu Qiao, Junjie Lei. Acknowledgments We are grateful for the support from the Extension Centre of Agricultural Technology in Zepu County, Kashi Prefecture, Xinjiang. We also would like to thank the anonymous reviewers and the editors for their helpful comments. Supporting information S1 Fig. The shoot dry weight of wheat in monoculture configurations and 3 different fruit tree-wheat intercropping systems in 2011. The data indicates the mean values of east, middle and west regions. Mono, monoculture wheat system; Jiw, jujube-wheat intercropping system; PLOS ONE \| https://doi.org/10.1371/journal.pone.0203238 April 2, 2019 14 / 17 Fruit tree shade decrease intercropping wheat yield and quality Aiw, apricot-wheat intercropping system; Wiw, walnut-wheat intercropping system. (TIF) S2 Fig. Relationships between grain quality and grain net yield of wheat in 2011 and 2012. Note: Pro: Protein content; WG: Wet gluten content; DDT: Dough development time; DST: Dough stability time; SD: Softening degree. (TIF) Aiw, apricot-wheat intercropping system; Wiw, walnut-wheat intercropping system. (TIF) S2 Fig. Relationships between grain quality and grain net yield of wheat in 2011 and 2012. Note: Pro: Protein content; WG: Wet gluten content; DDT: Dough development time; DST: Dough stability time; SD: Softening degree. (TIF) S3 Fig. Relationships between grain quality and thousand grain weight of wheat in 2011 and 2012. Pro: Protein content; WG: Wet gluten content; DDT: Dough development time; DST: Dough stability time; SD: Softening degree. (TIF) S1 Table. Nutrient status of the experimental soil. Note: Mono, monoculture wheat system; Jiw, Jujube-wheat intercropping system; Aiw, apricot-wheat intercropping system; Wiw, wal- nut-wheat intercropping system. (DOCX) S2 Table. N, P and K concentrations in stalks and grains of wheat in monoculture configu- rations and 3 different fruit tree-wheat intercropping systems in 2011 and 2012. Note: Mono, monoculture wheat system; Jiw, jujube-wheat intercropping system; Aiw, apricot- wheat intercropping system; Wiw, walnut-wheat intercropping system. (DOCX) S2 Fig. Relationships between grain quality and grain net yield of wheat in 2011 and 2012. Note: Pro: Protein content; WG: Wet gluten content; DDT: Dough development time; DST: Dough stability time; SD: Softening degree. (TIF) S3 Fig. Relationships between grain quality and thousand grain weight of wheat in 2011 and 2012. Pro: Protein content; WG: Wet gluten content; DDT: Dough development time; DST: Dough stability time; SD: Softening degree. (TIF) S1 Table. Nutrient status of the experimental soil. Note: Mono, monoculture wheat system; Jiw, Jujube-wheat intercropping system; Aiw, apricot-wheat intercropping system; Wiw, wal- nut-wheat intercropping system. (DOCX) S1 Table. Nutrient status of the experimental soil. Note: Mono, monoculture wheat system; Jiw, Jujube-wheat intercropping system; Aiw, apricot-wheat intercropping system; Wiw, wal- nut-wheat intercropping system. (DOCX) S2 Table. Author Contributions Formal analysis: Xu Qiao, Junjie Lei. 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https://openalex.org/W2064561841	https://www.frontiersin.org/articles/10.3389/fpsyt.2013.00071/pdf	English	null	Meditative Movement for Depression and Anxiety	Frontiers in psychiatry	2,013	cc-by	16,187	Peter Payne1 and Mardi A. Crane-Godreau1,2* 1 Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon, NH, USA 2 Research and Development Service, Veteran’s Administration Medical Center, White River Junction, VT, USA This review focuses on Meditative Movement (MM) and its effects on anxiety, depression, and other affective states. MM is a term identifying forms of exercise that use movement in conjunction with meditative attention to body sensations, including proprioception, inte- roception, and kinesthesis. MM includes the traditional Chinese methods of Qigong (Chi Kung) andTaijiquan (Tai Chi), some forms ofYoga, and other Asian practices, as well asWest- ern Somatic practices; however this review focuses primarily on Qigong andTaijiquan. We clarify the differences between MM and conventional exercise, present descriptions of sev- eral of the key methodologies of MM, and suggest how research into these practices may be approached in a systematic way. We also present evidence for possible mechanisms of the effects of MM on affective states, including the roles of posture, rhythm, coherent breathing, and the involvement of speciﬁc cortical and subcortical structures. We survey research outcomes summarized in reviews published since 2007. Results suggest that MM may be at least as effective as conventional exercise or other interventions in ameliorat- ing anxiety and depression; however, study quality is generally poor and there are many confounding factors. This makes it difﬁcult to draw deﬁnitive conclusions at this time. We suggest, however, that more research is warranted, and we offer speciﬁc suggestions for ensuring high-quality and productive future studies. REVIEW ARTICLE published: 24 July 2013 doi: 10.3389/fpsyt.2013.00071 REVIEW ARTICLE published: 24 July 2013 doi: 10.3389/fpsyt.2013.00071 PSYCHIATRY REVIEW ARTICLE published: 24 July 2013 doi: 10.3389/fpsyt.2013.00071 PSYCHIATRY Edited by: Edited by: Felipe Schuch, Hospital de Clinicas de Porto Alegre, Brazil Reviewed by: William R. Marchand, University of Utah, USA Petros C. Dinas, FAME Laboratory, Greece Correspondence: Mardi A. Crane-Godreau, Microbiology and Immunology, Geisel School of Medicine at Dartmouth, 1 Medical Center Drive, HB 7936, Lebanon, 03756 NH, USA e mail: mardi crane@dartmouth edu Edited by: Felipe Schuch, Hospital de Clinicas de Porto Alegre, Brazil Reviewed by: William R. Marchand, University of Utah, USA Petros C. Dinas, FAME Laboratory, Greece Correspondence: Mardi A. Crane-Godreau, Microbiology and Immunology, Geisel School of Medicine at Dartmouth, 1 Medical Center Drive, HB 7936, Lebanon, 03756 NH, USA e-mail: mardi.crane@dartmouth.edu y Felipe Schuch, Hospital de Clinicas de Porto Alegre, Brazil Felipe Schuch, Hospital de Clinicas de Porto Alegre, Brazil Reviewed by: William R. Marchand, University of Utah, USA Petros C. Dinas, FAME Laboratory, Greece Reviewed by: William R. Marchand, University of Utah, USA Petros C. Dinas, FAME Laboratory, Greece *Correspondence: p Mardi A. Crane-Godreau, Microbiology and Immunology, Geisel School of Medicine at Dartmouth, 1 Medical Center Drive, HB 7936, Lebanon, 03756 NH, USA e-mail: mardi.crane@dartmouth.edu Keywords: Qigong, Chi Kung,Taijiquan,Tai Chi, exercise, basal ganglia, default mode network, interoception INTRODUCTION INTRODUCTION as meditation, but they are distinct practices. The objective of this review is to deﬁne MM and to clarify how it dif- fers from conventional exercise and from seated meditation, to examine the evidence for its ability to ameliorate depres- sion and anxiety, and to suggest possible mechanisms for these effects. While both exercise and meditation have been acknowledged as having health beneﬁts,the category of exercise that combines med- itative focus with movement is often ignored or misunderstood. As a class of exercise it lacks a broadly accepted name. We refer to the group of practices including the traditional Chinese prac- tices of Taijiquan (1), Qigong (2), and Hatha (postural) Yoga (3), as well as Western methods such as the Alexander Technique (4) and Feldenkrais (5). It has been proposed that this form of exer- cise be called “Meditative Movement” (MM), which is deﬁned by Larkey et al. as a practice involving movement, a meditative state of mind, attention to the breath, and deep relaxation (6). We will use the designation MM except when referring to a speciﬁc discipline. METHODS To gather preliminary information for the survey of the current state of research into the effects of MM on anxiety and depression, we performed searches in PubMed, Ovid, and Google Scholar, using the key words Qigong, Taijiquan, Tai Chi, Chi Kung, exer- cise, Yoga, mindful, movement, meditative, meditation, Somatics, Alexander Technique, and Feldenkrais, combining these terms with psychological,health,psychosocial,stress,well-being,depres- sion, and anxiety. We conﬁned our search to publications in English, and prioritized reviews later than 2006. We found 14 review papers focusing solely or substantially on MM in relation to anxiety or depression (16–29), all of them relating to meditation, Yoga, Qigong, and/or Taijiquan, and used these as our principal source of information. In some cases we included general reviews of MM as long as they included a substantial focus on affective states (18, 19, 29). We address MM because of the proliferation of publications, some scholarly, some in the popular press, that claim physical or psychological beneﬁt from one or more of these practices, includ- ing reduced anxiety and depression, a more positive affective state, greater calmness of mind, greater physical relaxation, improved general health, better balance, lower blood pressure (BP), and improved biomarkers for inﬂammation and immune function [such as C-reactive protein (CRP) and cortisol] (7–13). Conventional exercise has been shown to improve depres- sion and anxiety (14, 15), but it is not clear whether these results also apply to MM, or whether the mechanisms of MM are similar or different to those of conventional exercise. Like- wise there is a substantial body of literature on (seated) med- itation; it is likely MM shares many of the same mechanisms For the purpose of this study we excluded Yoga, as well as stud- ies focusing solely on seated meditation. Due to the many systems covered by the word“Yoga”(explained in,seeYoga below) we chose to substantially limit our use of studies of Yoga. Future studies of MM could includeYoga as long as care is taken to separate the more July 2013 \| Volume 4 \| Article 71 \| 1 www.frontiersin.org www.frontiersin.org Payne and Crane-Godreau Meditative movement Qigong, and spiritual Qigong; all share the same basics, but use different techniques relevant to their speciﬁc aims. Qigong, and spiritual Qigong; all share the same basics, but use different techniques relevant to their speciﬁc aims. TAIJIQUAN Taijiquan (T’ai Chi Ch’uan,Tai Chi Chuan,Tai Chi,tie jee chwahn) is a Chinese martial art based on the same principles as Qigong; basic or modiﬁed Taijiquan is also used as a health and medita- tive exercise. Opinions differ as to whether Taijiquan should be regarded as a form of Qigong or as a separate category (16, 30); here, we consider both Qigong and Taijiquan as forms of MM. We found no relevant reviews of Somatics, Feldenkrais, or Alexander Technique. For further information on aspects of MM not mentioned in the scientiﬁc literature, we relied on the authors’ extensive libraries of books and articles on the subject, supple- mented when necessary by searches on Amazon.com and the Library of Congress. YOGA The Sanskrit word “Yoga” means “to join,” and refers to the indi- vidual’s union with God; traditionally, Yoga is the practical side of the diverse Hindu religious systems. Different forms of Yoga use differing methods to achieve union with God. Some forms of Yoga may be forms of MM as deﬁned in Section “Characteris- tics of MM” below, such as Hatha Yoga asanas and pranayama (3) and Yantra Yoga (31); others, such as Bhakti, Jnana, and Raja Yoga (32), are specialized forms of devotional practice, philosophical inquiry, or sitting meditation, and often involve moral and dietary observances. METHODS religious or philosophical aspects from the MM components and to examine carefully the speciﬁc techniques to determine whether they meet the deﬁnition of MM. We also chose not to explore the literature on seated meditation in detail, except in speciﬁc cases where it directly illuminates MM, for the reasons explained in Section “Seated Meditation.” Some academic reviews of MM practices have, however, included seated meditation (16, 30). The traditional Chinese practices of Taijiquan and Qigong conform well to the proposed deﬁnition of MM (6), therefore our study focuses principally on them. SEATED MEDITATION Seated meditation uses a mostly static posture, and involves the exploration of various mental states. Many different form of seated meditation are practiced throughout the world. There is substan- tial overlap between the techniques of MM and some forms of seated meditation, in that they may use similar mental strategies. However the term MM implies that movement or the intention of movement is involved (6), which is not true of many forms of meditation. There is a large literature on seated meditation, espe- cially the style called “mindfulness,” and comparatively little on MM per se. y g Limitations of these methods were that we did not include reviews in languages other than English; however, because sev- eral of the reviews we included did not have this limitation, we nevertheless were able to take into account much of the work published in China and Korea. We were however unable to access directly research papers in languages other than English, which is a signiﬁcant limitation of this study. Also, we focused mainly on Qigong and Taijiquan, excluding the literature on Yoga and much of the literature on meditation for the reasons explained above. A deeper investigation of the speciﬁc nature of the tech- niques of Yoga and meditation studied in speciﬁc papers might allow a more inclusive approach in the future, and might lead to changes in the deﬁnition of MM. In addition we chose not to search for every possible kind of practice that might be a form of MM. Some initial exploration found no relevant references in the scientiﬁc literature to several names of Somatics or Asian MM systems (for example, Alexander Technique, Feldenkrais, Baguazhang), and we concluded that continuing this search would be unfruitful and would take too much time, besides necessar- ily being incomplete as the authors are not familiar with all forms of practice that might be considered MM. Nevertheless, this is an acknowledged limitation of this study. A more thorough search using more terms might well discover more information of value. SOMATICS “Qigong” and “Taijiquan” often appear in Western literature with multiple spellings. There are two systems of transliteration, the earlier Wade–Giles system, and the contemporary (and ofﬁcial) Pinyin. By way of introducing these alternative spellings, we will use the Pinyin form, with the Wade–Giles and other alternate spellings in parentheses, and a phonetic approximation in italics in our deﬁnitions below. Over the past century, a number of MM practices have developed in the West. These have been referred to by the name “Somatics” (38). This term includes a wide variety of practices that, in gen- eral, share the deﬁning characteristics of MM; in addition to the Alexander Technique (4) and Feldenkrais (5),examples of Somatic practices are: Eutony (39), Mensendieck (40), Focusing (41), Sen- sory Awareness (42), Aston Patterning (43), Rolﬁng Movement (44), Continuum (45), and Authentic Movement (46). Somatic Experiencing® (47) uses the principles of MM as a therapy for trauma. At this point there are very few scientiﬁc studies of these methods, and we found none focusing on affective states. OTHER ASIAN SYSTEMS Meditative movement includes more Asian systems than those mentioned above; a full enumeration would not be useful here as the numbers run into the hundreds, but Aikido (33), Shin Tai Do (34), Baguazhang (35), SuﬁDance (36), and Buddhist walking meditation (37) are other exemplars. DEFINING THE TERMS QIGONG “Qi” (Ch’i, chee) means “life energy” or “breath.” It is a core concept throughout Chinese culture. “Qigong” (Ch’i Kung, Chi Kung, chee goong) means “work on the life energy,” and is a broad term including methods that simultaneously “regulate the body, the breath, and the mind” (1). There are four main branches of Qigong: health-maintenance Qigong, medical Qigong, martial MOVEMENT been treated by researchers as forms of exercise comparable to ordinary aerobics, stretching, or relaxation techniques. As Cather- ine Kerr pointed out (48), the understanding of the biomedical researcher as to what is happening in MM is often at variance with that of the MM teacher. A researcher might understand the MM practices as a form of exercise for generalized stress reduction, not much different in principle from going for a walk. The MM instructor, however, thinks of it as a more sophisticated process, in which the awareness of the practitioner is placed in speciﬁc regions of the body to make speciﬁc changes. This disjunction of views can lead to problems in designing experiments that ask and answer relevant scientiﬁc questions (49). been treated by researchers as forms of exercise comparable to ordinary aerobics, stretching, or relaxation techniques. As Cather- ine Kerr pointed out (48), the understanding of the biomedical researcher as to what is happening in MM is often at variance with that of the MM teacher. A researcher might understand the MM practices as a form of exercise for generalized stress reduction, not much different in principle from going for a walk. The MM instructor, however, thinks of it as a more sophisticated process, in which the awareness of the practitioner is placed in speciﬁc regions of the body to make speciﬁc changes. This disjunction of views can lead to problems in designing experiments that ask and answer relevant scientiﬁc questions (49). Meditative movement may use either prescribed movement (where the required motion is speciﬁc and must be learned and practiced) or spontaneous free-form movement (where the prac- titioner allows their body to move spontaneously on its own). In some cases, the movement used may be extremely subtle, to the point of being invisible (55). In order to move the Qi (cause certain sensations in the body), physical movement is useful but not nec- essary. A practitioner may begin by making a large and obvious motion, then make it smaller and smaller until it is impercep- tible. In this process the interoceptive/proprioceptive sensations become progressively more intense. This increased intensity may correlate with the objective effectiveness of the practice. A com- mon saying in Qigong is, “Small movement is better than large movement; no movement is better than small movement”(56). MIND “Mind” in this context means “awareness” and not “con- ceptual thought.” In MM the mind is neither engaged in conceptual activity nor focused on a future goal, nor is it in the “default mode” of mind-wandering (50), but instead is focused on direct bodily experience. We view this spa- tial/interoceptive/proprioceptive/kinesthetic focus of awareness as the principal deﬁning characteristic of MM. The practitioner’s awareness is on the kinesthetic sensations of the whole body mov- ing through space; the ﬂow of breath and blood and other visceral sensations; the experience of balance, orientation, and posture; and the felt sense of space – quite different from physical aware- ness in conventional exercise (51). Kerr (49) has referred to this focus as “mind-in-body,” to distinguish it from the more familiar concept of “mind-body”practices. This way of using the attention is similar to that used in some forms of seated meditation (52); but MM often involves additional speciﬁc mental techniques. For example, practitioners may be instructed to “feel the air as heavy” (53), as if they were moving underwater. This may be difﬁcult at ﬁrst, but with practice one can develop a vivid tactile/kinesthetic sense of viscous, almost hydraulic air movement, accompanied by a pleasurable sense of lightness, warmth, smoothness, and power (53). This indicates progress in the exercise and may be predic- tive of positive objective effects. In MM practices, the mind is also used to “direct the movement of Qi” (54). Since this is a core aim of most MM, it is important to understand this in a way that is compatible with a scientiﬁc approach. Section “Imagery” below explores this in more detail. MOVEMENT In traditional Qigong practice, quiescent seated meditation is con- sidered to be a part of Qigong, as is quiet standing. Although the distinction between“static”and“moving”practice is acknowl- edged,it is not a major distinction;in traditional Qigong,they were usually practiced together, with each supporting the other (57). In many apparently static forms of Qigong, the mind is actively employed in imagining movement, which produces certain inter- nal sensations of motion; such a practice may alternate between overt and imperceptible “movement.” In this context, it becomes hard to deﬁne exactly what is meant by the distinction between movement and stillness. We wonder whether, logical as it seems at ﬁrst, making overt movement an essential element in MM is appropriate. One possibility would be to reﬁne this part of the deﬁnition of MM to refer to the intention of movement, or to include the movement of internal sensations in the body in the deﬁnition of “movement.” The use of the term“MM,”suggested by Larkey (6),is an impor- tant step in recognizing the special features of these practices. Larkey proposed the following essential characteristics of MM: ﬁrst,a meditative state of mind,usually involving a focus of aware- ness on the body; second, some form of prescribed (or sometimes spontaneous) movement; third,explicit attention to the breathing; and fourth, a state of deep relaxation. Larkey based his deﬁnition on his familiarity with Qigong, Taijiquan, and similar practices, in an attempt to bring attention to these forms of exercise as distinct from conventional exercise. We will expand somewhat on these in order to deﬁne more precisely the features of MM, and to make it clear how radically it differs from most other exercise. We believe that, for future research, it is important to have a full and precise understanding of MM on its own terms,so that it can be accurately evaluated. CHARACTERISTICS OF MM MIND WHY “MEDITATIVE MOVEMENT?” WHY “MEDITATIVE MOVEMENT?” In the past 5 years, the number of research studies of MM has increased considerably (47); however, these practices have often July 2013 \| Volume 4 \| Article 71 \| 2 Frontiers in Psychiatry \| Affective Disorders and Psychosomatic Research Payne and Crane-Godreau Meditative movement REVIEWS OF MM’s EFFECTS ON AFFECTIVE STATES A number of reviews have looked speciﬁcally at the effects of Taiji- quan, Qigong, Yoga, and seated meditation on improving anxiety, depression,andotheraffectivemeasures(16–29).Arelativelysmall number of studies have focused on the use of MM for improving anxiety and depression, whereas a larger number of studies have investigated the effects of seated meditation (106, 107). Here we focus principally on MM rather than seated meditation. Ospina (29) reviewed the scientiﬁc quality of studies of med- itation, deﬁned as including Yoga, Taijiquan, and Qigong as well as seated meditation (30), and, applying the rigorous CONSORT standards (108, 109), concluded that most studies were of poor quality. Further scientiﬁc research into meditation required bet- ter attention to study quality as well as a more uniﬁed theoretical perspective on meditation. She stated that no ﬁrm conclusions about the effects of meditation could be drawn from the available research (30). Biological systems are “complex systems”; this is a technical term implying spontaneous self-organization (86, 87). Complex systems organize themselves to preserve optimal, rather than min- imal or maximal, levels of any number of variables (such as temperature or chemical composition). This is known as “home- ostasis,”a term coined by Cannon (88). Cortisol levels,for instance, can be an indicator of stress, a presumptively negative condition; toolittlecortisol,however,isasbadastoomuch(89,90).Greatcare is needed in pharmacological interventions to avoid unwanted side effects due to “overshoot.” The aim of MM is to restore the body’s innate mechanisms for establishing homeostasis or dynamic equi- librium (58). Keeping this simple point in mind could change a research hypothesis from “does this intervention reduce cortisol levels?” to “does this intervention move cortisol levels toward a normal range?” A more recent review and meta-analysis by Chen at al. focus- ing on MM for anxiety (16), examined 36 adequate randomized controlled trials (RCTs). This study used the classical Chinese deﬁ- nition of Qigong as“the skill of mind-body exercises that integrate body, breath, and mind adjustments into one” (16), and thus included sitting meditation practices. In evaluating studies for inclusion, Chen advocated the use of the Boutron modiﬁcation (110) of the rigorous CONSORT standards. The CONSORT stan- dards (108,109) were designed to apply to the evaluation of design quality of pharmaceutical trials,where rigorous double-blinding is easy. In studying MM however, the same level of blinding is more problematic;theBoutronmodiﬁcationgivesguidelinesspeciﬁcally for non-pharmaceutical studies. DEEP STATE OF RELAXATION (BALANCED TONE) of studies have serious limitations, and much more high quality research is needed to be able to draw deﬁnitive conclusions (11, 19, 20, 48). In English“deep relaxation”conjures images of a ﬂoppy slackness. On the other hand,we can talk of a jaguar walking through the jun- gle as “deeply relaxed”; these are two different states. The Chinese word usually translated“relax”is“song”(soong) (84). However, this word does not mean limpness (84), but rather a state of com- pletely balanced tone, “eutonis” (39, 85), in which every muscle is doing exactly what it should. This state is experienced as light, free, open, and effortless; but at the same time stable, powerful, and well-rooted. Tension is a state of hypertonus, slackness a state of hypotonus,and the outcome of successful MM practice is a state of eutonis. There are practices that involve a hypotonic state, like Hatha Yoga’s Shavasana, or “corpse posture” (3); some practices use brief maximal hypertonus. Neither of these is typical of MM, which always aims at the balanced state described above. BREATHING Awareness and control of the breath are central in Qigong and Taijiquan (58), Hatha Yoga (59), and Somatics (8). In Chinese and in many other languages, the same word can refer to both“breath” and “life energy.”“Attending to or moving the Qi” can refer either to the physical breathing, or to certain bodily, emotional, or spa- tial sensations (58). These multi-level meanings of key terms must not be ignored in approaching MM. Altering the breathing pattern may alter the functioning of the autonomic nervous system (60). Various breathing practices are said to enable emotional release (61), to calm the mind (62), or to enhance physical power (63). A central practice in most forms of Qigong is to pass the aware- ness through the body in synchrony with the breathing rhythm (64). Depending on the desired result, the breath may be slow or fast, felt in various parts of the body, imagined as having differ- ent qualities (such as warmth or coolness), or held for various lengths of time (65). In MM, the breath is described as a bridge between unconscious and conscious functions, a way for the con- scious mind to inﬂuence the unconsciously controlled functions of the autonomic nervous system (66). The mutual inﬂuence of the breath,the autonomic nervous system,and the emotions is well recognized in the scientiﬁc literature (60, 67–76), and numerous studies have been made of the effects of speciﬁc MM breathing techniques (77–83), but as far as the present authors are aware there has been no systematic review of these studies. July 2013 \| Volume 4 \| Article 71 \| 3 www.frontiersin.org Payne and Crane-Godreau Meditative movement REVIEWS OF MM’s EFFECTS ON AFFECTIVE STATES A complex system that is poorly self-regulated may fail to main- tain a variable (such as the level of hormones or other signaling agents) within a normal range; or the level of concentration may oscillate over time, between too low and too high. Increased self- regulation will reduce the magnitude of oscillations so they no longer leave the normal range; more reﬁned regulation results in smaller oscillations (91). Investigations of body sway suggest that MM may also improve postural self-regulation (92). Twenty-ﬁve of the 36 RCTs investigated by Chen (16) found meditation (seated or moving) signiﬁcantly more effective than control interventions in improving the symptoms of anxiety. Con- trol groups included standard of care only (no intervention), attention controls (using another activity unrelated to MM), and active interventions other than MM (16). Comparing Standard MeanDifference(SMD),thereweretwostatisticallysigniﬁcantdif- ferences between different groups of studies: outcomes of poorer quality studies tended to be more positive, and studies from Asian countries also tended to more positive outcomes. There were also indications (although not statistically signiﬁcant) that movement- oriented methods were more effective than static meditation, and thatgroupdeliverywasbetterthanindividual.Moststudiesdidnot focus on clinically diagnosed anxiety disorders, but used anxiety questionnaires as one among several measures, leaving it unclear how effective these interventions would be for clinically signiﬁ- cant levels of anxiety. No adverse effects were reported in any of the studies (16). In studying MM it should be kept in mind that the desired out- come is a state of increasingly reﬁned dynamic balance, and not a state characterized by maxima or minima, tension, or slackness. The word“relaxation”should be used with caution due to its ambi- guity, and the concepts from the theory of complex systems may prove useful in describing the outcomes and processes of MM. BENEFITS OF MM Meditative movement has positive effects on a wide range of mental and physical measures,although results are far from unam- biguous. There have been a number of publications on the effects of MM practices on: depression (23), anxiety (17), cognitive abil- ity (93), inﬂammation (94), immune function (95), arthritis (96), supportive cancer care (97), cardiac and pulmonary health (78, 98, 99), balance (98, 100), aerobic capacity (101), strength (102), bone density (103), ﬁbromyalgia (104), and diabetes (105). Over- all, MM seems to have positive effects on a broad range of health conditions. An equally consistent ﬁnding is that the vast majority Jahnke selected 67 RCTs for review (out of 576 considered) (19). His criteria were that the study appeared in a peer-reviewed English-language journal between 1993 and 12/2007; that it had been cited in the academic literature; and that it was designed to test the effects of Taijiquan or Qigong (exclusive of seated July 2013 \| Volume 4 \| Article 71 \| 4 Frontiers in Psychiatry \| Affective Disorders and Psychosomatic Research Meditative movement Payne and Crane-Godreau meditation). Twenty-seven of the 67 studies focused on psycho- logical outcomes, and 6 on immune and inﬂammatory outcomes. In most cases MM demonstrated improvement in measures of anxiety and depression; these changes were generally signiﬁcant compared with inactive controls, but did not usually reach signif- icance when compared to an exercise or other active therapeutic control. In six other studies, MM reduced stress markers such as cortisol, adrenaline, and noradrenaline, and inﬂammation mark- ers such as cytokines, CRP, and immunoglobulin-G (Ig-G). These results gave a biochemical conﬁrmation that MM may reduce the secretion of stress-related and inﬂammation-related biomarkers. Jahnke noted that MM rarely produced less change than active therapeutic controls, which was signiﬁcant in view of the simplic- ity, safety, cost-effectiveness, and gentle exertion of MM practice. Jahnke’s conclusion was that “this category shows promise for examining (MM’s) potential mechanisms of action for the change of psychological states.” Jahnke made a case for considering Taiji- quan and Qigong as substantially identical: his study revealed no signiﬁcant differences in outcomes between Taijiquan and Qigong over a wide range of variables. control. Anxiety scores also decreased signiﬁcantly in seven stud- ies, two of these compared to an active control. One study looked at stress-related biomarkers (cortisol, catecholamines) and found signiﬁcant reductions compared to a wait-list control. BENEFITS OF MM Quality of life (a general measure of well-being based on a questionnaire) and self-efﬁcacy (a measure of how capable and conﬁdent the subject feels in relation to daily demands) also showed signiﬁ- cant improvement, both compared to wait-list controls and to a traditional rehabilitation group (one study); in another study, Qigong trended toward being more effective than conventional exercise but not signiﬁcantly so. A meta-analysis of three stud- ies of diabetic patients demonstrated that Qigong was effective at reducing depression and anxiety and improving quality of life. The authors comment on the poor quality of most studies and the need for more careful study design, larger samples, and better con- trols. They suggested using a convincing“sham”Qigong control to reduce “frustrebo” effects (the possible negative effect on controls from not receiving the intervention they want). The use of sham MM controls is rare in MM studies, and designing such a study presents challenges (111), however a RCT by Lee et al. (9) used an effective sham Qigong control. They found signiﬁcant reduction of anxiety (as measured by Spiel- berg’s state-trait anxiety inventory, STA1-X1), as well as cortisol and aldosterone levels, in a Qigong (“Qi-training”) group as com- pared to sham Qigong controls. The control group learned exactly the same movements,but participants were not instructed how“to gather and move the Qi” (the process of intentional manipulation of interoceptive sensation). Lee states that after the 1-h training, the intervention subjects “could gather and move Qi with con- scious effort”and that“[t]he control group learned to perform the same external motions as the Qigong training group, but with- out any conscious effort to gather or move Qi.” Measures were taken shortly before and shortly after each training session; ses- sions lasted 1 h, and were repeated three times, separated by at least 2 days. Care was taken to blind the randomization as well as the collection of outcome data. This study is one of the few that give adequate details about the intervention, including the subtle factors alluded to above, as well as providing a valid double blind in the assessment of outcomes. The results suggest a contribution to anxiety reduction by factors speciﬁc to MM (“gathering and moving the Qi”). A review by Oh (21) on Qigong and depression found 10 acceptable RCT studies between 2009 and 2011. Oh excluded Taijiquan and meditation, including only moving Qigong prac- tices. BENEFITS OF MM Only two of the studies focused speciﬁcally on depression as a primary outcome; the rest used a measure of depression as one among others. Of the 10 studies, 4 found that Qigong had signiﬁcant positive effects on depression; 2 found that Qigong had the same positive effectiveness as conventional exercise, and 1 found that Qigong was as effective as conventional rehabilita- tion treatment. This latter study by Tsang (23) involved both a pilot study and a follow-up with a larger sample size, comparing Qigong to a speciﬁc rehabilitation program targeted at depres- sion. Qigong had signiﬁcantly better results than the rehabilitation group. Oh’s evaluation was that these results were inconclusive: he cited widespread problems with poor experimental design, lack of speciﬁcity in description of the intervention, small sample size, brief length of study, lack of investigation of biomarkers, lack of studies involving clinical cases,lack of three-arm studies,and inad- equate blinding. While many studies indicate that Qigong may effectively reduce stress, the litany of experimental short-comings outlined above highlights the need for greater rigor in the design of future studies. In a 2010 review focusing on Taijiquan, Fields (18) mentioned three studies showing the positive effects of Taijiquan on the auto- nomic nervous system as measured by heart rate (HR) variability frequency analysis; in two of these studies, Taijiquan was shown to havebetterresultsthanbriskwalkingorslowmovement,whichhas interesting implications for the comparison between conventional exercise and MM. Fields also mentioned four studies in which Tai- jiquan has been shown to decrease a variety of negative emotional states, as well as two of her own studies combining Taijiquan and Yoga practice for pregnant women, in which signiﬁcant alteration in EEG measurements was shown. Like all the other reviewers, she pointed out the many methodological ﬂaws in these studies, including a wide range of dosages, target populations, diagnos- tic instruments, and the frequent use of pre-post measurements rather than controls. A limitation of her review is that she does not give details of how she selected the studies to include. A 2013 review of the effects of Qigong on anxiety, depression, and well-being by Wang et al. (28) drew from both Chinese and English-language studies, since 2000 for the former and 2003 for the latter. One hundred and ﬁfty-eight studies were identiﬁed, of which 15 were ﬁnally selected for inclusion. BENEFITS OF MM His earlier studies investigated the psychological basis for improved depression. Qigong practice increased the sense of self-efﬁcacy and mastery by enabling elders to master a physical task as well as to improve competence through increased balance and strength; in addition Qigong practice provided them with increased social support and improved interpersonal relation- ships. These psychological factors may cause decreased depression (116). Tsang offered three possible neurobiological explanations for Qigong’s anti-depressive effect. First, he suggested that Qigong may act by increasing brain levels of monoamine neurotransmit- ters,as has been claimed for selective serotonin reuptake inhibitors (SSRIs). He noted that studies have shown that conventional exer- cise has this effect (117), as well as increasing the concentration of tryptophan in the brain (tryptophan is a serotonin precursor). He pointed out that the time course of Qigong’s effect is similar to that of SSRIs: the antidepressant effect takes a couple of weeks to initiate, then fades away a few weeks after ceasing the intervention. Tsang’s second explanation involved the hypothalamic-pituitary- adrenal (HPA) axis which secretes cortisol and adrenaline in response to stress, and which has been implicated in depression. Tsang pointed out that, since the hypothalamus is under control of the limbic system, the mental calming effects of Qigong may reduce limbic activation of the HPA axis, thus reducing plasma cortisol and ACTH and possible lessening depression. Tsang cited studies showing that Qigong practice may reduce cortisol and ACTH (9). Tsang’s third explanation invoked the relation between decreased neurogenesis in the hippocampus and depression. Stress and the resultant increase in plasma cortisol may downregulate brain derived neurogenesis factor (BDNF) and reduce neurogene- sis. He noted that animal studies suggest that exercise upregulates BDNF and increases neurogenesis (118), and that conventional exercise in humans may have the same effects (119, 120). Yet another review in 2010 by Wang et al. (26) searched Eng- lish and Chinese databases through 2009 for references to Taiji- quan (not Qigong) in relation to anxiety, depression, mood, self- esteem, and psychological stress. Randomized, non-randomized, and observational studies were considered. Randomized con- trolled studies were assessed for inclusion using the Jadad criteria (112). Wang performed a meta-analysis (using a bias-corrected Hedges g-score to compensate for small sample size), and dis- cussed the non-randomized studies, separately for each of the ﬁve affective measures (anxiety, depression, mood, self-esteem, and psychological stress). BENEFITS OF MM Overall results included signiﬁcant support for the positive effect of Taijiquan on all ﬁve measures. He noted that, despite the use of exercise control groups in several studies, it was not possible to say whether Taijiquan had equal or supe- rior effects compared to conventional exercise. He also noted the limitations in conducting a meta-analysis due to heterogeneity of population and dosage. The present authors note that very few of the studies tested the effects of daily practice of Taijiquan; most studies involved practice two or three times a week,which is gener- allyregarded among Taijiquan teachers as inadequate for achieving signiﬁcant results (113). g A recent 2013 review by Wang et al. (27) looked at Qigong (not Taijiquan). An extensive search of English and Chinese databases was done, inclusive of Chinese dissertations and doctoral theses. The selection of studies for inclusion in the review used narrowly focused criteria: only RCTs were included; subjects had to have a diagnosis of anxiety or depressive illness; studies of mood, self- esteem, or stress were not included; studies had to use instruments validated for the measurement of depressive or anxiety states. The Wayne checklist (103) was used to evaluate study quality; this is a checklist developed speciﬁcally for use in studies of Qigong. It includes several items clearly required by the nature of the Qigong intervention, such as a complete description of the intervention and details of the qualiﬁcations of the instructors,as well as appro- priate blinding requirements. Following data extraction, the effect sizes were calculated using Hedges g-score, and SMD was calcu- lated for pooled effects. Out of 503 studies, 12 RCTs met inclusion criteria. Three of these used conventional exercise as a control. All studies measured depressive symptoms, whereas only four evalu- ated anxiety. Pooled effects were calculated for each type of control groupused.Comparedwithconventionalexercise,Qigongshowed a moderate positive effect on measures of depression; compared with Cognitive-Behavioral Therapy, Qigong was equal in effect; Many reviews of MM have noted its effects on biomarkers of inﬂammation such as CRP, tumor necrosis factor alpha (TNF- α), and interleukin 6 (IL-6) (19, 62, 93, 94). As Tsang pointed out, this is potentially relevant to some of the effects of MM on depression, as it appears that depression is linked to inﬂammatory processes in the brain (in particular the hippocampus), which interfere with the generation or survival of new neurons in the hippocampus (neurogenesis). BENEFITS OF MM This review excluded Yoga and seated meditation, focusing exclusively on standard Qigong exercises involving obvious movement, and applied rigor- ous standards for study selection. In all but one study,subjects were healthyorhadanon-psychiatricchronicillness;onestudyinvolved depressed patients. In seven of the studies, mood and depression scores improved signiﬁcantly (using a variety of scales such as the Hamilton Depression Severity Index, the Self-Rating Depression Scale, and the CES Depression Scale). Compared to usual care, psychosocial support, and active stretching, Qigong did better in two studies, while in two studies Qigong did as well as the active July 2013 \| Volume 4 \| Article 71 \| 5 www.frontiersin.org www.frontiersin.org Meditative movement Payne and Crane-Godreau Another 2010 review by Saeed (22) looked at conventional exercise and Yoga as well as Taijiquan, Qigong, and meditation. Saeed focused on studies of clinically diagnosed affective disor- ders. Although Yoga is not within the scope of the present study, it is interesting to note that there appear to be a number of stud- ies of the effects of Yoga on clinical anxiety, depression, and other psychiatricdiseases,incontrasttotheliteratureonQigongandTai- jiquan where there are very few such studies. Saeed’s conclusion was that there was not enough evidence to support using Qigong or Taijiquan as a treatment for clinical affective disorders. Saeed’s survey excluded most of the studies included in other reviews, which measured changes in anxiety, depression, and mood among non-clinical populations. Saeed also found little evidence for the effectiveness of conventional exercise in these populations. compared with a newspaper reading control, Qigong showed a large effect; and compared with waitlist, a moderate effect. Among the four studies investigating anxiety, only one showed Qigong superior to controls. Wang cautioned that home practice was not monitored, thus dosage is hard to ascertain. Also, the Qigong intervention was always provided in a group context whereas the control groups often did not have this social exposure. Since social engagement may have a signiﬁcant effect on depressive symptoms it is necessary in future studies to provide equal social engage- ment for all groups. Wang’s overall conclusion is that evidence for the effectiveness of Qigong in treating anxiety and depression is positive but limited by numerous factors. p y A speculative 2008 review by Tsang (25) moved forwards from his earlier studies on the effects of Qigong on depression in the elderly (114, 115) to reﬂect on the possible neurobiological basis for these effects. BENEFITS OF MM In a recent review of the connec- tion between depression and inﬂammation, Krishnadas pointed out that a third of patients with major depressive disorder (MDD), July 2013 \| Volume 4 \| Article 71 \| 6 Frontiers in Psychiatry \| Affective Disorders and Psychosomatic Research Meditative movement Payne and Crane-Godreau the MM intervention itself. Because an important part of most MM practices is the cognitive/affective state of positive belief and expectation, controlling for this variable could be seen as inap- propriate, irrelevant, or unnecessary (111). Further complicating experimental design, in MM the placebo effect is not necessarily regarded as a confounding factor; rather, the factors on which the placebo effect is based are being harnessed to produce positive out- comes (111). It is possible to tease out the cognitive, affective, and motor aspects components of the effects of MM by good exper- imental design; however, MM might achieve some of its effects through the interaction of its component factors, rather than a simple addition. without any other major illness, have elevated inﬂammatory bio- markers; in particular, CRP, TNF-α, and IL-6. Moreover, MDD is epidemiologically associated with inﬂammatory medical condi- tions(121),andpatientstreatedwithcytokinesareatincreasedrisk of developing MDD. Neuroinﬂammation is linked with a number of psychiatric and neurological disorders in addition to depression (122). Since peripheral inﬂammation and central nervous system (CNS) inﬂammation involve somewhat different conditions, it is not possible to conclude from blood and salivary markers that MM might affect CNS inﬂammation. However melatonin may protect the brain against inﬂammation and promote neurogenesis (123), and meditation produced both short-term and long-term increases in melatonin levels (80, 124, 125). The second category involves difﬁculties in experimental design speciﬁc to the study of MM (21, 49, 127). The study of MM presents issues such as difﬁculties with blinding and the selection of appropriateshamcontrolactivities.Studiespresentawiderange of interventions, often poorly speciﬁed; they use diverse doses and durations; and there is usually a mixture of possible active factors in a single intervention (such as expectation,psychosocial support, skill levels, training methodology, movement, mental focus, belief, and expectation). Much of this confusion stems from the lack of a systematic and complete taxonomy of these methods, which should be based on accurate understanding of MM. The various complex processes of gathering, storing, purifying, and circulating Qi should be understood, operationalized, and brought out of the realm of apparent superstition. POSSIBLE MECHANISMS OF THE EFFECTS OF MM ON AFFECTIVE DISORDERS There are two broad categories of mechanisms proposed for the effects of MM: ﬁrst, well-recognized mechanisms shared by ordi- nary physical exercise, such as muscular or cardio-vascular load- ing; and second, complex neurological mechanisms relating either to neuronal plasticity (128–130) or neurohormonal and neuroim- munological modulation (25, 95). We think the latter offers the most productive avenue for future research, and we make several speculations along these lines below. CONCLUSION Overall, these reviews invite the following tentative conclusions: MM consistently produced reductions in measures of anxiety and depression when compared with non-active controls in studies of anxiety or depression. When matched to an exercise or other active therapeutic intervention control, MM usually performed about the same, rarely any worse, and sometimes better than the control. MM generally produced less reduction in measures of anxiety and depression in seriously ill patients and more reduction in healthy or slightly compromised subjects. However,the forms of MM used in such studies are invariably health-maintenance Qigong,and not the much more speciﬁcally targeted medical Qigong. It would be surprising if an intervention designed for mild ill health were to be effective for severe clinical conditions. Review papers usually investigate multiple studies of pharma- ceuticals or other well-deﬁned treatments. Since the administra- tion of a speciﬁc dosage of a particular drug is easy to standardize, variation of results between different studies is assumed to reﬂect randomoruncontrolledfactorsnotrelatedtothedrugitself;there- fore a statistical averaging of many studies is assumed to give more accurate results as to the true effectiveness of the drug. However, thesituationwithMMisquitedifferent.Giventhecompletelackof standardizationortaxonomyof MM,divergentresultsinstudiesof MM may reﬂect in part the differential effectiveness of the inter- ventions used rather than uncontrolled factors. For this reason, although we agree with several of the reviewers that the results are inconclusive, we feel that the remarkably positive results obtained in some studies, even when MM was compared to an active tar- geted intervention (23), warrant continued exploration of MM along the scientiﬁcally more rigorous lines we suggest below. We note in the reviews cited above the lack of complete agree- ment as to the requirements of adequate study design. Whereas some apply the rigorous CONSORT (109) standards in evalu- ating the acceptability of studies, others suggest more moderate standards such as the Boutron modiﬁcations (110) or the Jadad criteria (112), or standards speciﬁcally designed for this particular ﬁeld such as the Wayne checklist (103). Some consensus needs to be reached on this issue to further research in this ﬁeld. SHORTCOMINGS OF STUDIES OF MM All the reviewers mentioned above (16, 18, 19, 21, 22, 25–29) come to similar conclusions about the shortcomings of most of the studies of MM to date. These short-comings fall into two broad categories. BENEFITS OF MM We do not mean to suggest that the traditional concepts of Qi should be imported into the realm of science; this cannot and should not be done. But both the sub- jective and the objective phenomena associated with descriptions of Qi can be identiﬁed in a scientiﬁc way. RHYTHM The smooth rhythmic motions of MM are usually experienced as quite pleasurable. Shin Lin (102) suggests that moderate rhythmic movement may increase parasympathetic tone, whereas intense exertion causes more sympathetic activation (102). Both in the elderly and in infants, regular rhythmic motion has been shown to be calming (133). In animal behavior, moderate rhythmic motion is very common in grooming behavior, which has been observed to have a calming effect on the animal. Cats have been shown to release elevated levels of serotonin while grooming (134); it is pos- sible something similar happens in humans when activities mimic grooming behaviors. g p y A recent study of posture (141) showed that when subjects adopted a “low-power” or a “high-power” posture for a few min- utes, there were signiﬁcant shifts in behavior as well as in testos- terone and cortisol levels. The authors stated that their results indicate the reality of “cognitive embodiment,” in which volun- tary physical posture can produce not only changes in cognitive and affective experience, but neurohormonal shifts as well. This suggests that the emphasis on posture in MM may have a scien- tiﬁc basis (142). Carney et al. used typical postures of the kind one might see in a boardroom; from the viewpoint of MM, the postures were unbalanced and poorly integrated (although they do clearly express power or lack thereof). The neurochemical and behavioral shifts that occurred through adopting a “high-power” posture involved increased testosterone and a shift toward risk- taking behavior; this is no more a shift toward balance than the increased cortisol observed in the “low-power” postures. The very precise posture used in almost all MM movements is well aligned and balanced between the extremes. We speculate that just the act of holding a balanced posture for a period of time may induce behavioral, affective, and neurochemical shifts in the direction of The speed with which one performs MM is around six times a minute (0.1 Hz); this appears to be a frequency at which the breath and heart beat have the greatest tendency to come into coherence (135); in other words, at this frequency respiratory sinus arrhyth- mia (RSA) is enhanced. During MM practice there is a feeling of “being in the groove,” of moving in a strong, slow rhythm. METABOLIC EXPENDITURE The ﬁrst category has to do with general experimental design (16, 19, 28, 126). This includes ﬁrst, disagreement about the neces- sity of certain aspects of experimental design, such as blinding of the participants; and second, the clearly necessary and often inad- equate aspects of the studies, such as small sample size, inadequate description of procedures, inadequate blinding of data collection, and inadequate controls. The former issue involves the nature of Meditative movement exercises are of mild to moderate intensity, are easily controlled, need no equipment, and little space, can be done indoors in inclement weather, and involve no sudden move- ments. The level of exertion is not much more than that of a gentle walk, and it may be hard to understand how MM can have such a range of powerful effects. MM involves smooth coordinated July 2013 \| Volume 4 \| Article 71 \| 7 www.frontiersin.org www.frontiersin.org Meditative movement Payne and Crane-Godreau good description of the experience of a regular oscillation of blood volume (136). This promotes efﬁciency in the cardio-respiratory system, and may enhance parasympathetic tone when such tone is low. In states of excess vagal tone and insufﬁcient sympathetic arousal (vaso-vagal syndrome), this rhythmic breathing could bring theANS back toward balance by elevating sympathetic activ- ity (137) and possibly reducing vagal tone. This restoration of autonomic balance is likely to have a moderating effect on affec- tive symptoms, and might reduce conscious fear and anxiety by reducing the intensity of the somatic markers (the interoceptive awareness of visceral/affective states on which subjective affective experience may be based) (138). This synchronized breathing will affect measures HRV (139); a frequency analysis of HRV will tend to show a strong narrow spike in the high frequency (HF) range, which is regarded as the most reliable indicator of healthy increase in vagal tone (72),and will show reduced power through the rest of the range. This has been referred to as “coherent breathing” (140), and it may be associated with strong positive feelings and a more balanced mental state (135). Interestingly, the one outcome for which there is the greatest evidence for the effectiveness of Qigong is reduced BP; hypertension can be signiﬁcantly associated with increased anxiety and stress. movements of all parts of the body, which gently challenge the range of motion of essential joints. METABOLIC EXPENDITURE In keeping with its philoso- phy of balance, MM avoids extremes of stretch or exertion and cautions against any feelings of strain or effort (131). Studies comparing the effects of MM on physical measures such as balance, bone density, HR, heart rate variability (HRV), BP, aer- obic capacity, and strength to a no-intervention control generally found signiﬁcant improvement in these indices (increased balance and bone density, reduced HR and BP, increased HRV, aerobic capacity, and strength) with MM, whereas comparisons to con- ventional exercise interventions often showed MM to have equally positive effects as conventional exercise. However in the case of the MM intervention, the degree of exertion appeared to be substan- tially less than that used in the conventional exercise intervention. Jahnke mentions that the relatively mild leg ﬂexion and low inten- sity movements typical of MM nevertheless produced signiﬁcant effects on bone density (19, 103). Jahnke speculated that some of the positive physical effects of MM might not be due to the same mechanisms as those of conventional exercise. More precise mea- surements of the metabolic equivalent (MET) and other objective measures of exertion will be necessary for clear conclusions. Chao has measured the MET of modiﬁed Taijiquan to be three METs, a moderately low intensity level (132). MET is a measure of the energy consumption during exercise,expressed as a ratio to a stan- dard level of energy consumption approximately equivalent to that during a resting state. Thus one MET is a resting state, three METs is three times the energy consumption of the resting state. Maxi- mal exertion would be about 23 METs. Some comparisons of MM to conventional exercise demonstrate greater effects on regulating the autonomic nervous system for MM (18), again suggesting a different mechanism from that of conventional exercise. GROUNDING AND POSTURE Many MM practices involve a regular shifting of the weight form one foot to the other, or a rhythmical activity of the whole body with the weight ﬁrmly planted on both slightly bent legs. Great attention is paid to correct postural alignment and to using the bodyasawhole.Althoughthedegreeof exertionislow,themuscles are being used in a well-coordinated and conscious way, possibly re-patterning the motor nervous system. This aspect of the prac- tice is held to enhance “grounding,” which is the subjective feeling and objective state of being more stably connected to the ground. It seems likely that this experience would help increase the sense of self-efﬁcacy, balance, and conﬁdence, all of which have been shown to be improved by MM (23, 98), and might help to stabilize mood swings and reduce depression and anxiety. Frontiers in Psychiatry \| Affective Disorders and Psychosomatic Research INTEROCEPTION Acentralaspectof MMistheattentiontointeroceptiveandpropri- oceptive sensations. MM practitioners become signiﬁcantly more sensitive to tactile (129), interoceptive (128), and kinesthetic per- ceptions, and regularly experience a variety of positive and com- plex inner sensations. Damasio’s recent theory of somatic markers (145) has clariﬁed how important interoception is to affective and cognitive function; he has shown that what are called intuitions or hunches may be due to this system. Information about the state of the viscera comes up lamina 1 of the spinal cord, via the brainstem parabrachial nucleus to the ventromedial thalamus, and thence through the posterior insular cortex (the primary interoceptive cortex) to the anterior insula where the information is integrated with higher order contextual information and becomes accessi- ble to consciousness. Damasio suggests that the integrity of this pathway is necessary for many affective, interpersonal, and cogni- tive processes, since it is the basis for a clear sense of one’s own affective and autonomic state (146). Information received through this interoceptive system is the basis on which the anterior insula (as well as the anterior cingulate gyrus and the ventromedial pre- frontal cortex) generate an attentional bias, which organizes the brain to pay attention to the outer world in particular ways. This system is implicated in the capacity for empathy and compassion, as well as addiction, anxiety, and depression. Farb (147) found that improved conscious access to the interoceptive cues associated with sadness was associated with decreased depressive symptoms. Meditation and MM have been shown to enhance interoception, even to the point of altering the neuronal connections between the posterior and anterior insula, the crucial bridge through which interoceptive sensations reach consciousness (128).We speculate that positive affective changes from MM may be due in part to this process of enhanced interoception. This process is similar to an athlete’s use of motor imagery for the rehearsal of a sport (149).Visual,interoceptive,and kinesthetic imagery can produce signiﬁcant, widespread, and lasting changes in the brain. Motor imagery is known to activate areas of the brain responsible for generating internal sensations, such as the poste- rior parietal cortex and the pre-motor and supplementary motor areas (150–152). Visualization can improve motor performance signiﬁcantly (149, 153), and increase muscle strength (154). Imag- ined movement activates many of the same areas of the brain as actual motion, although the patterns of activation are not identi- cal (155). RHYTHM When RSA is strongly established, blood circulation becomes more efﬁcient; the heart rate increases during inhalation, as the blood volume moves relatively more into the lungs, and slows during exhalation, as the blood volume moves into the periph- eral circulation. This alteration in blood volume may be part of the basis for the suggestion in MM that one “breathes into the arms and legs” – an obvious physical impossibility, but perhaps a July 2013 \| Volume 4 \| Article 71 \| 8 Frontiers in Psychiatry \| Affective Disorders and Psychosomatic Research Payne and Crane-Godreau Meditative movement interoceptive/kinesthetic experience, usually with a clear hedonic component. These subjectively experienced interoceptive shifts are believed to correspond to actual changes in the physiology of the body and nervous system (58, 131). For instance a spread of pleas- ant warmth may indicate increased capillary dilation; a soft feeling in the heart may involve reduced BP and increased HRV; a feeling of weightlessness in the limbs may indicate improved control of muscle tone via the reticular activating system in the brain stem. balance. A study by Yeh et al. (143) demonstrated a signiﬁcant decrease in leukopenia in breast cancer patients using a 3-week Standing Meditation Qigong intervention (Zhan Zhuang); this practice involved simply standing for 15 min in a precisely aligned, relaxed, and well-balanced posture. In seated meditation too, the correct posture is seen as a central part of the practice (144). INTEROCEPTION The posterior parietal cortex, where the body image is constructed (156), as well as the supplementary motor area, where motor plans are elaborated, are preferentially activated by imag- ined movement (151, 157). The effects of imagined movement extend to the autonomic system; cardio-vascular and skin resis- tance changes accompany imagined exertion (150, 158), and it is possible that other autonomic changes can be triggered as well, such as altered parasympathetic regulation of the heart (140), improved autonomic regulation of the enteric nervous system, and increased capacity for social engagement through activation of the supra-diaphragmatic portion of the parasympathetic sys- tem – Porges’ ventro-vagal system (159). The positive effects of motor imagery can be predicted from the degree of autonomic responsiveness during the imagery (160). THE BASAL GANGLIA The principle functional circuits of the brain: affective, interocep- tive, motivational, attentional, executive, associative, memory, and sensorimotor all need to coordinate with each other in relation to the challenges from the environment. One needs to be able to notice and orient to a relevant stimulus, identify its meaning, adopt an appropriate physical, affective, and cognitive prepara- tory stance (posture), then choose and execute appropriate action. Recent research into the basal ganglia (BG) suggests that they are a major center for this functional integration. All cortical regions have semi-independent parallel loop circuits going down to the striatum (the input nucleus of the BG), to the globus pallidus, substantia nigra, and sub-thalamic nucleus, and returning via the thalamus to the same area of the cortex (161). This enables the BG to selectively inhibit or stimulate particular parts of the cortex, as well as to coordinate their actions. This applies to both voluntary and involuntary or habitual actions (162). The BG have a similar set of loop circuits descending to the pontine and medullary areas, extending the process of selection and coordination to brainstem functions (163). The symptoms of Parkinson’s disease (PD; which is caused by a deﬁcit in the dopamine circuits of the BG), as well as other diseases possibly related to the BG, such as Tourette’s syn- drome and attention deﬁcit hyperactivity disorder (ADHD), have associated behavioral,cognitive,and affective symptoms (161). PD The principle functional circuits of the brain: affective, interocep- tive, motivational, attentional, executive, associative, memory, and sensorimotor all need to coordinate with each other in relation to the challenges from the environment. One needs to be able to notice and orient to a relevant stimulus, identify its meaning, adopt an appropriate physical, affective, and cognitive prepara- tory stance (posture), then choose and execute appropriate action. RESEARCH DESIGN is known for restrained movement, contracted posture, depres- sion, and cognitive limitations; ADHD presents an opposite pic- ture. The terms hypophrenia and hyperphrenia have been coined to refer to the cognitive/affective dimensions of hypo- and hyper- kinetic disorders, indicating the interaction of motor, affective, and cognitive dimensions in the BG (161). As noted in Section “Reviews of MM’s Effects on Affective States” above, consensus needs to be reached as to the appropriate stan- dards for conducting and evaluating studies of MM. Recommen- dations for the design of future research include: adequate sample size, the correct use of blinding procedures, ideally use of a double blind; the use of control groups, ideally three-arm studies using an inactive and an active attention, treatment, or sham intervention control. In reporting outcomes blinding and randomized selec- tion procedures should be fully described. The great majority of studies of the effect of MM on depression or anxiety are lim- ited to about 3 months of intervention. In many cases, this may not be enough time to even begin to achieve mastery; one could say that the real “dose” does not begin until there is a reason- able degree of skill in performing the practices. Trials should be long enough to ensure subjects achieve an adequate level of skill. Skill levels attained by subjects should be measured and taken into account in analysis of the outcomes. We suggest deﬁning certain reported subjective experiences – such as sensations of lightness, ﬂow, warmth, wholeness – as indices of skill levels. Traditional Qigong teachings describe the experiences indicative of various degrees of achievement (113). Objective measures could also be made that might correlate with skill, such as analysis of postural sway or stride variation (173). Measures based on the theory of complex systems could prove productive (111, 173–176). We speculate that the focus of MM on the intentional cultiva- tion of balanced posture, enhanced interoception, and kinesthesis, the conscious focus on smooth and balanced movement, rhyth- mic breathing and positive affect, and the explicit use of intention, all acting together, may affect the whole brain via the BG and move it toward more balanced functioning. Hyperphrenia (anx- iety, ADHD) calms down and hypophrenia (depression, apathy) is energized. Excess activation in the HPA axis diminishes, and the neurochemical environment becomes well regulated. FUTURE DIRECTIONS Astheabovefactorsareimplemented,clearandsolidresultsshould begin to accumulate as to the effects of MM practices. Should these results conﬁrm the suggestions from current research, then it will be appropriate to begin to deconstruct the role of the different aspects of MM: what proportion of the results are due to the movement, the breathing, the mental focus; how much is due to social interaction, positive expectation, cultural belief, and so on. Speciﬁc elements of MM should be used in isolation to examine the exact mechanisms of action, and in combination to determine whether the components have an additive or a synergistic effect. As the deﬁnition of MM becomes clearly established, more forms of MM should be studied in depth, including speciﬁc forms of Yoga and Western Somatics. Care should be taken to discriminate between MM and other factors such as religious belief, devotional practices, and philosophical or moral practices. As more forms of MM are studied the deﬁnition of MM should be evaluated and reﬁned to make sure that it remains valid and useful. RESEARCH DESIGN In this process posture and movement play a central and fundamental role; the process of “adopting a posture toward something” (164) is a central and unifying function of the entire nervous system. Lli- nas (165) has argued that motor function is the principle reason for the existence of the CNS. IMAGERY Qigong uses phrases such as “direct the Qi,” “gather the Qi,” “move the Qi,” which may be problematic to understand from a biomedical point of view. This capacity is a principal goal of Qigong (148) and other forms of MM (where the term “Energy” is often used instead of “Qi”). A useful way of understand- ing these terms is to think of “Qi” as a dynamic interocep- tive/proprioceptive/kinesthetic/tactile sensation of tingling,vibra- tion, warmth, pressure, or ﬂow. Thus“moving the Qi”translates as the intentional use of imagery to modify the practitioner’s inner experience. A typical MM instruction might be: “While you are performing a certain coordinated movement, imagine the sensa- tion of a ﬂow of warm liquid from your pelvis up through your spine and out your arm.”Or:“place your attention in the center of your chest and imagine a ﬂower gently opening.”After a period of practice, such procedures can result in vivid physical sensations; not an abstract mental picture, but an embodied “felt sense” of July 2013 \| Volume 4 \| Article 71 \| 9 www.frontiersin.org www.frontiersin.org Payne and Crane-Godreau Meditative movement DUAL DEFAULT MODES OF THE BRAIN Recentresearchhasidentiﬁedruminationormind-wandering(the chronic semi-conscious churning of thoughts) (166) as a signif- icant factor in anxiety and depression (167–169). The “default mode” of the brain (50, 166) involves activity of the posterior cingulate cortex and the precuneus, as well as the superior and medial temporal gyri; these are both involved with internal “self- talk,” the “autobiographical” self with all its stressful stories (170). Brewer (166) has shown in a study of experienced meditators that in meditative states these regions become less active, and the dorso-lateral and medial prefrontal cortex, the dorsal ante- rior cingulate cortex, and posterior insular cortex become more active. These are areas involved with cognitive control and atten- tion to the present, particularly to interoceptive stimuli. Long- term meditation practice brings about permanent restructuring of these pats of the brain, resulting for instance in increased gray matter in areas of the cortex involved in interoception (171, 172). We suggest that MM may be effective in the short term at changing the default mode of brain activity away from rumi- nation toward present-oriented awareness, and in the long term at changing the wiring of the brain (128). If this is the case, MM could be of immense help in the therapy of depression and anxiety. Since MM uses many variables in intervention, studies need detailed descriptions not only of physical movements, but also of the nature of instructions in terms of use of mind as well as the social context. One shortcoming for all current research is the lack of some sort of taxonomy to break down elements into recog- nizable categories; until such a system is developed and adopted, it will remain difﬁcult to compare studies and gain signiﬁcant understanding of MM and disciplines like Qigong. Frontiers in Psychiatry \| Affective Disorders and Psychosomatic Research PITFALLS IN STUDYING MM AND SUGGESTIONS FOR FUTURE RESEARCH We have emphasized above the importance of an adequate under- standing of the core principles of MM, as well as the development of a systematic taxonomy. We believe this is necessary for signiﬁ- cant progress. Research should be carried out in populations with clinical diagnoses of depression or anxiety; however when dealing with clinical illness, an MM intervention should be matched to the condition it is designed to treat. This requires the help of an appropriately qualiﬁed practitioner. A confounding factor could be that the diagnosis of the patients in the research group from an MM point of view might not be congruent with that of Western medicine. REFERENCES Evid Based Complement Alternat Med (2013) 2013:134737. doi:10. 1155/2013/134737 5. Feldenkrais M. Exploring Aware- ness Through Movement [Sound Recording]. 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Evid Based Complement Alternat Med (2013) 2013:152738. doi:10.1155/ 2013/152738 21. Oh B, Choi SM, Inamori A, Rosen- thal D, Yeung A. Effects of Qigong on depression: a systemic review. CONCLUSION Meditative movement is a system of considerable scope, sophis- tication, complexity, and potential power. At this early stage it is necessary to consider, with appropriate discrimination, a wide range of explanatory mechanisms of its effects, from July 2013 \| Volume 4 \| Article 71 \| 10 Frontiers in Psychiatry \| Affective Disorders and Psychosomatic Research Payne and Crane-Godreau Meditative movement that is necessary to consider actions via the higher centers of the brain when studying MM. ordinary physiological and psychological processes, through higher brain functions, to electrical, and electromagnetic ﬁeld phenomena. Given the lack of adverse outcomes in studies that employed various forms of MM, the low costs involved in delivering MM therapies and that the outcomes of most studies are encouraging, we believe that continued research in this area could prove to be highly beneﬁcial in the ﬁeld of anxiety and depression disorder treatment. Meditative movement uses techniques that are quite different from those of conventional exercise, and there are sound possi- ble explanations for their mechanisms of action on affective and autonomic states. Investigation into these putative mechanisms could lead to signiﬁcant breakthroughs in the development of new forms of intervention for anxiety, depression, and related conditions. We have shown that culturally unfamiliar explana- tions of the therapeutic effects of MM (such as “moving the Qi”) can be translated into scientiﬁcally meaningful terms, allowing for a signiﬁcantly more thorough and effective investigation of MM disciplines than has been possible hitherto. In particular we believe CH. Tai Chi on psychological well-being: systematic review and meta-analysis. BMC Complement Altern Med (2010) 10(1):23. doi: 10.1186/1472-6882-10-23 ACKNOWLEDGMENTS We gratefully acknowledge support received from FlightAttendant Medical Research Institute (FAMRI). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. REFERENCES Effects of Qi-training on anxiety and plasma concentra- tions of cortisol,ACTH,and aldos- terone: a randomized placebo- controlled pilot study.Stress Health (2004) 20(5):243–8. doi:10.1002/ smi.1023 32. Patanjali. How to Know God: The Yoga Sutras of Patanjali. 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Ionta S, Heydrich L, Lenggen- hager B, Mouthon M, Fornari E, Chapuis D, et al. Multisensory mechanisms in temporo-parietal cortex support self-location and ﬁrst-person perspective. Neuron (2011) 70(2):363–74. doi:10.1016/ j.neuron.2011.03.009 145. Damasio A, Carvalho GB. The nature of feelings: evolutionary and neurobiological origins. Nat Rev Neurosci (2013) 14(2):143–52. doi:10.1038/nrn3403 168. Miller GA, Crocker LD, Spiel- berg JM, Infantolino ZP, Heller W. Issues in localization of brain function: the case of lateralized frontal cortex in cognition, emo- tion, and psychopathology. Front Integr Neurosci (2013) 7:2. doi:10. 3389/fnint.2013.00002 135. Lehrer PM, Vaschillo E, Vaschillo B, Lu S-E, Eckberg DL, Edelberg R, et al. Heart rate variability biofeed- back increases baroreﬂex gain and peak expiratory ﬂow. Psychosom Med (2003) 65(5):796–805. doi: 10.1097/01.PSY.0000089200. 81962.19 146. Damasio A. 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Best practice for motor July 2013 \| Volume 4 \| Article 71 \| 14 Frontiers in Psychiatry \| Affective Disorders and Psychosomatic Research Meditative movement Payne and Crane-Godreau biology approach to studying Tai Chi, physiological complex- ity and healthy aging: design and rationale of a pragmatic randomized controlled trial. Contemp Clin Trials (2013) 34(1):21–34. doi: 10.1016/j.cct.2012.09.006 as a complex system. Emer- gence (2000) 2(2):24–57. doi: 10.1207/S15327000EM0202_03 171. Holzel BK, Carmody J, Vangel M, Congleton C, Yerramsetti SM, Gard T, et al. Mindfulness prac- tice leads to increases in regional brain gray matter density. Psychi- atry Res (2011) 191(1):36–43. doi: 10.1016/j.pscychresns.2010.08.006 Citation: Payne P and Crane-Godreau MA (2013) Meditative movement for depression and anxiety. Front. Psychiatry 4:71. doi: 10.3389/fpsyt.2013.00071 176. Theise ND. July 2013 \| Volume 4 \| Article 71 \| 15 REFERENCES From the bottom up: complexity, emergence and Bud- dhist metaphysics. Tricycle Buddh Rev (2006) 15(2):24–6. fp y This article was submitted to Frontiers in Affective Disorders and Psychosomatic Research, a specialty of Frontiers in Psy- chiatry. This article was submitted to Frontiers in Affective Disorders and Psychosomatic Research, a specialty of Frontiers in Psy- chiatry. 172. Lazar SW, Kerr CE, Wasserman RH, Gray JR, Greve DN, Treadway MT, et al. Meditation experience is associated with increased corti- cal thickness. Neuroreport (2005) 16(17):1893. doi:10.1097/01.wnr. 0000186598.66243.19 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any com- mercial or ﬁnancial relationships that could be construed as a potential con- ﬂict of interest. 174. Dörner D, Schölkopf J. Control- ling complex systems; or, exper- tise as“grandmother’s know-how.” In: Ericsson KA, Smith J, editors. Toward a General Theory of Exper- tise. Cambridge: Cambridge Uni- versity Press (1991). p. 218–39. Copyright © 2013 Payne and Crane- Godreau. This is an open-access arti- cle distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are cred- ited and subject to any copyright notices concerning any third-party graphics etc. Copyright © 2013 Payne and Crane- Godreau. This is an open-access arti- cle distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are cred- ited and subject to any copyright notices concerning any third-party graphics etc. 173. Wayne PM, Manor B, Novak V, Costa MD, Hausdorff JM, Goldberger AL, et al. A systems Received: 06 April 2013; accepted: 05 July 2013; published online: 24 July 2013. Received: 06 April 2013; accepted: 05 July 2013; published online: 24 July 2013. 175. Juarrero A. Dynamics in action: intentional behavior July 2013 \| Volume 4 \| Article 71 \| 15 www.frontiersin.org
https://openalex.org/W4213377551	https://www.qeios.com/read/IY1RTI/pdf	English	null	CCL26 wt Allele	Definitions	2,020	cc-by	93	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios CCL26 wt Allele National Cancer Institute National Cancer Institute Qeios ID: IY1RTI · https://doi.org/10.32388/IY1RTI Source National Cancer Institute. CCL26 wt Allele. NCI Thesaurus. Code C49743. Human CCL26 wild-type allele in the vicinity of 7q11.23 and is approximately 3 kb in length. This allele, which encodes C-C motif chemokine 26 protein, plays a role in the chemotactaxis of eosinophils and basophils in inflammatory processes.CCL26 may contribute to eosinophil accumulation in atopic diseases. Qeios ID: IY1RTI · https://doi.org/10.32388/IY1RTI 1/1
W2040201699.txt	https://zenodo.org/records/1722180/files/article.pdf	en		Nachtrag zum «Sechsten Bericht der Commission für die Festsetzung der Atomgewichte»	Berichte der Deutschen Chemischen Gesellschaft	1,905	public-domain	365	1194 wit in Danerversuchen ohne Diaphragma a n Eisenanoden uod Kupferkathoden bei Gegenwart von Iiupfersalz aus Nitratlosangen 36-procentige Losungen von Nitrit mit einer durchechDittlicheu Stromausbeute VOII ca. 84 pCt. herstellen konnten, wenn der Elektrolyt dauernd mit Nitrat gesattigt gehalten und das auf die Kathode fdllende Kupfer im Elekrrolyten erganzt wurde. Natiirlich arbeitet hierbei auch die Anode a u f Nitrit. d a Animoniak gegenwartig ist. So giinatig diese Resultate auch aussehen, so stellen sich doch der Erzeugung von festem Nitrit auf diesem Wege noch erbebliche Srhwierigkeiten entgegen, a u f die wir hier nicbt eingehen wollen. D r e s d e n , Miirz 1905. H. L a n d o l t : N a c h t r a g eum ))Sechsten Bericht der Commission fur die F e a t s e t z u n g der Atomgewichtea I ) . 210. (Eingegsogen am 11. MBrx 1905.) In dem sechsten Bericht sind auf S. 50 bei der Aufstellung des Abstimmungs-Ergebnissee z w e i Stimmen xfiir die alleinige Tabelle H = 1 c aufgefiihrt. Hr. H. E r d m a n n 2 ) hat nun im letzten Heft dieser BBerichteCc erklart, dass die Regietrirung seiner Stimlre in diestr Klasse unrichtig war. Hiernach bat sich also nur e i n Mitglied der Grossen Atomgewichts-Commission fiir die alleinige Ausgabe der T:ibelle H = 1 ausgesprochen. Hr. E r d m a n n war bisdahin bekanntlich einer der eifrigsten Verfecbter der Wasserstoff-Atomgewichte; daher ist der Iritbum entstanden. Das w e s e n t l i c h e Ergebniss der Abstimmungwird dadurch jedoch nicht beriihrt; denn die Majoritiit fiir die Sauerstoff-Tabelle bleibt ganz die gleiche wie friilier. D e r V o r s i t z e n d e d e r A t o m g e w i c h t s- C o m ni i 8 s i o n d e r D e u t s c h e n c h e m i s c h e n G e s e I Is c h aft. I) I' Diem Berichte 38, 13-22 [1905]. Diesr Berichte 38, 978-979 [1905].
https://openalex.org/W4323288267	https://www.sciencepubco.com/index.php/ijamr/article/download/32160/17671	English	null	Analytical solutions of reaction-diffusion-convection type equations from porous media by the Laplace-Adomian method	International journal of applied mathematical research	2,022	cc-by	4,528	Analytical solutions of reaction-diffusion-convection type equations from porous media by the Laplace-Adomian method Yanick Alain Servais WELLOT1* and Gires Dimitri NKAYA2 1U i it´ M i NGOUABI C B ill 1Universit´e Marien NGOUABI Congo Brazzaville 2Universit´e Marien NGOUABI Congo Brazzaville *Corresponding author E-mail: wellotyanick@gmail.com The equation (1) becomes: Lu+Ru+Nu = h (3) (3) Where u is a unknown function of H into H (H is a Hilbert space), L and R are linear operators; and L invertible, with L−1 as inverse. N is a nonlinear operator from a Hilbert space H into H. h is a given fonction in H. Abstract The aim of this paper is to solve analytically ﬂuid ﬂow problems in a porous medium, the Laplace-Adomian method gives algorithms that converge faster to achieve the exact solution when it exists. Keywords: Laplace-Adomian method, media porous, Reaction-diffusion-convection equation. that gives the Laplace-Adomian method[11, 12, 13, 14]. 2. About the Laplace-Adomian method The mathematical modeling of physical systems leads to functional equations (ordinary differential equations (ODE), partial differential equations (PDEs), integro-differential equations and integral equa- tions, ...). The search for exact or approximate solutions, when they exist, uses several methods. Among then, we ﬁnd the Adomian decomposition methods of homotopy perturbation method, Laplace- Adomian method , variational iterations method, ... By applying the transform L of Laplace at the equation (3), we have: L (Lu)+L (Ru)+L (Nu) = L (h) (4) (4) Case of a partial differential equation ∂2 With In this paper, we focus on nonlinear partial differential equations and the use of Laplace transforms and the Adomian decompositional method to analytically solve these equations[2, 4, 5]. 1. Introduction that gives the Laplace-Adomian method[11, 12, 13, 14]. The purpose of this work is to experiment this Laplace-Adomian method by avoiding the linearization and the discretization of the space and of time for better to solve the models of the partial differ- ential equations of the porous media problems. The mathematical models associated with the ﬂow in porous medium, with possible transport of solutes are represented by the systems of partial differential equations based on reaction- diffusion-convection. The mathematical models associated with the ﬂow in porous medium, with possible transport of solutes are represented by the systems of partial differential equations based on reaction- diffusion-convection. The study of ﬂows in porous media is central to the oil industry dur- ing the exploitation of an oil or gas deposit, in the management of water resources, pollution by chemical, agricultural or radioactive wastes, and also many environmental problems. The presentation below is shows how the Laplace-Adomian method works. It is the algorithm of the method.[1, 4, 5] C id f ti l ti The study of ﬂows in porous media is central to the oil industry dur- ing the exploitation of an oil or gas deposit, in the management of water resources, pollution by chemical, agricultural or radioactive wastes, and also many environmental problems. Consider a functional equation Consider a functional equation Au = h (1) Natural porous media are heterogeneous at several scales, which complicates experimental studies, if not impossible, while predic- tions are vital and the need for reliable numerical simulation models remains [10, 15]. Au = h (1) With A = L+R+N With A = L+R+N (2) (2) International Journal of Applied Mathematical Research Website: www.sciencepubco.com/index.php/IJAMR doi: Research paper Copyright © 2018 Author. This is an open access article distributed under the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited. 3. Numerical Applications Finally, with the conditions (5), the Laplace transform, into the equation (4) we have: 3. Numerical Applications Finally, with the conditions (5), the Laplace transform, into the equation (4) we have: Finally, with the conditions (5), the Laplace transform, into the equation (4) we have: Case of a partial differential equation ∂2 Laplace transforms do not allow us to solve nonlinear equations, because there is no Laplace transform of nonlinear terms[6, 7, 8, 9]. To circumvent the difﬁculty or to overcome this insufﬁciency of the Laplace transform, a coupling is made between the Laplace trans- forms and the Adomian decomposition method. It is this coupling Let’s set Ltt(.) = ∂ ∂t2 (.) with the initial and boundary conditions, we have the following relation: u(x,0) = α1(x) et ut(x,0) = α2(x) (5) (5) International Journal of Applied Mathematical Research 44 Let us consider the following equation [2] Let us consider the following equation [2] Let us consider the following equation [2] Let us consider the following equation [2] Let: Let: s2L (u(x,t)) = su(x,0)+ut(x,0)+L (h(x,t)) −L (Ru(x,t)−L (Nu(x,t)) (7)        ∂u(x,t) ∂t = u2(x,t)∂2u(x,t) ∂x2 +u3(x,t)+ ∂u(x,t) ∂x +u(x,t) u(x,0) = sin(x) (7)        ∂u(x,t) ∂t = u2(x,t)∂2u(x,t) ∂x2 +u3(x,t)+ ∂u(x,t) ∂x +u(x,t) u(x,0) = sin(x) (15) This gives the expression: This gives the expression: (x,t)) = 1 s u(x,0)+ 1 s2 ut(x,0)+ 1 s2 L (h(x,t)) −1 s2 L (Ru(x,t))−1 s2 L (Nu(x,t)) (8) Or: ∂u(x,t) ∂t = u2(x,t)∂2u(x,t) ∂x2 +u3(x,t)+ ∂u(x,t) ∂x +u L (u(x,t)) = 1 s u(x,0)+ 1 s2 ut(x,0)+ 1 s2 L (h(x,t)) −1 s2 L (Ru(x,t))−1 s2 L (Nu(x,t)) (8) Or: ∂u(x,t) ∂t = u2(x,t)∂2u(x,t) ∂x2 +u3(x,t)+ ∂u(x,t) ∂x +u(x,t) (16) By applying the Laplace transform in relation witch (16) we have: (h(x,t)) ,t)) (8) Or: ∂u(x,t) ∂t = u2(x,t)∂2u(x,t) ∂x2 +u3(x,t)+ ∂u(x,t) ∂x +u(x,t) (16) (16) By applying the Laplace transform in relation witch (16), we have: By applying the Laplace transform in relation witch (16), we have: Or again Or again L ∂u(x,t) ∂t = L u2(x,t)∂2u(x,t) ∂x2 +u3(x,t)+ ∂u(x,t) ∂x +u(x,t) (17) L ∂u(x,t) ∂t = L u2(x,t)∂2u(x,t) ∂x2 +u3(x,t)+ ∂u(x,t) ∂x +u(x,t) L (u(x,t)) = 1 s α2(x)+ 1 s2 α1(x)+ 1 s2 L (h(x,t)) −1 s2 L (Ru(x,t))−1 s2 L (Nu(x,t)) (9) L ( ) ∂t = L u2(x,t) ( ) ∂x2 +u3(x,t)+ ( ) ∂x +u(x,t) (17) We obtain: L (u(x,t)) = 1 s α2(x)+ 1 s2 α1(x)+ 1 s2 L (h(x,t)) −1 s2 L (Ru(x,t))−1 s2 L (Nu(x,t)) (9) ∂t We obtain: (17) (17) (9) (9) We obtain: L (u(x,t) = u(x,0) s−1 + 1 s−1L (Nu(x,t))+ 1 s−1L ∂u(x,t) ∂x (18 We look for the solution u when it exists, the equation (1) in the form of a series: L (u(x,t) = u(x,0) s−1 + 1 s−1L (Nu(x,t))+ 1 s−1L ∂u(x,t) ∂x (18) We look for the solution u when it exists, the equation (1) in the form of a series: L (u(x,t) = u(x,0) s−1 + 1 s−1L (Nu(x,t))+ 1 s−1L ∂u(x,t) ∂x (18) We look for the solution u when it exists, the equation (1) in the form of a series: L (u(x,t) = u(x,0) s−1 + 1 s−1L (Nu(x,t))+ 1 s−1L ∂u(x,t) ∂x (18) We look for the solution u when it exists, the equation (1) in the form of a series: (18) With: With: u(x,t) = ∞ ∑ n=0 un(x,t) (10) (10) (10) Nu(x,t) = u2(x,t)∂2u(x,t) ∂x2 +u3(x,t) (19) (19) The application of the inverse Laplace transform gives the following result: The application of the inverse Laplace transform gives the following result: The non-linear part is also expressed as a series of polynomials: u(x,t) = et sin(x)+L −1 1 s−1L (Nu(x,t))+ 1 s−1L ∂u(x,t) ∂x (20) N(u(x,t)) = ∞ ∑ n=0 An(x,t) (11) u(x,t) = et sin(x)+L −1 1 s−1L (Nu(x,t))+ 1 s−1L where An are Adomian polynomials deﬁned by the formula . Let us consider the following equation [2] Substituting (10) and (11) in (9) we obtain the expression: where An are Adomian polynomials deﬁned by the formula . Let us consider the following equation [2] (37) Therefore: u(x,t) = 2e2tp ex −e−4x +L −1( 1 s−2L (Nu(x,t))) (50) Let’s ﬁnd the solution of u(x,t) in the form (50) u(x,t) = u0(x,t)+u1(x,t)+u2(x,t)+u3(x,t)+u4(x,t)+... (37) Therefore: u(x,t) = 2e2tp ex −e−4x +L −1( 1 s−2L (Nu(x,t))) (50) Let’s ﬁnd the solution of u(x,t) in the form Therefore: Let’s ﬁnd the solution of u(x,t) in the form Let’s ﬁnd the solution of u(x,t) in the form Let’s ﬁnd the solution of u(x,t) in the form u(x,t) = et sin(x) 1−1 2t2 +...+(−1)n t2n 2n! + et cos(x) t −1 6t3 +...+(−1)n t2n+1 (2n+1)! (38) u(x,t) = +∞ ∑ n=0 un(x,t) (51) Knowing that: +∞ u(x,t) = +∞ ∑ n=0 un(x,t) (51) (51) (51) Nu(x,t) = +∞ ∑ n=0 An(x,t) (52) That is: Nu(x,t) = +∞ ∑ n=0 An(x,t) (52) (52) That is: That is: u(x,t) = et (sin(x)cos(t)+cos(x)sin(t)) = et sin(x+t) (39) n=0 We obtain the following algorithm: u(x,t) = et (sin(x)cos(t)+cos(x)sin(t)) = et sin(x+t) (39) n 0 We obtain the following algorithm: (39) The exact solution of the problem 1 is: The exact solution of the problem 1 is: The exact solution of the problem 1 is: u(x,t) = et sin(x+t) (40) 3.2. Example 2        u0(x,t) = 2e2t√ ex −e−4x un+1(x,t) = L −1 1 s−2L (An(x,t)) (53) tion of the problem 1 is: u(x,t) = et sin(x+t) (40) e 2        u0(x,t) = 2e2t√ ex −e−4x un+1(x,t) = L −1 1 2L (An(x,t)) p u(x,t) = et sin(x+t) (40)    u0(x,t) = 2e2t√ ex −e−4x 1 u(x,t) = et sin(x+t) (40) e 2     un+1(x,t) = L −1 1 s−2L (An(x,t)) (53) u(x,t) = e sin(x+t) (40) 3.2. Example 2    un+1(x,t) = L −1 1 s−2L (An(x,t)) (53) mple 2    un+1(x,t) = L −1 1 s−2L (A Let us consider the following equation [2] Now: By the same process, the other terms are obtained. u2(x,t) = L −1 1 s−1L (A1(x,t))+ 1 s−1L (∂u1(x,t) ∂x ) (29) Now: ∂u(x,t) ∂t = Nu(x,t)+2u(x,t) (43) With : ∂u(x,t) ∂t = Nu(x,t)+2u(x,t) (43) u2(x,t) = L −1 1 s−1L (A1(x,t))+ 1 s−1L (∂u1(x,t) ∂x ) (29) ∂u(x,t) ∂t = Nu( With : (43) t) = 2u1u0 ∂2u0 ∂x2 +u2 0 ∂2u1 ∂x2 +3u1u2 0 = 0 (30) Nu(x,t) = u(x,t)∂u(x,t) ∂x x + A1(x,t) = 2u1u0 ∂2u0 ∂x2 +u2 0 ∂2u1 ∂x2 +3u1u2 0 = 0 (30) u2(x,t) = −sin(x)L −1 1 (s−1)3 (31) Nu(x,t) = u(x,t)∂u(x,t) ∂x x + 3 u(x,t)∂u(x,t) ∂x −2u2(x,t) (44) (44) u2(x,t) = −sin(x)L −1 1 (s−1)3 Applying the Laplace transform L to the equation (42), generates the following: u2(x,t) = −1 2t2et sin(x) (32) the followi ∂ (32) L ∂u(x,t) ∂t = L (Nu(x,t))+2L (u(x,t)) (45) Or: (45) and: A2(x,t) = 2u2u0 ∂2u0 ∂x2 +u2 1 ∂2u0 ∂x2 +2u1u0 ∂2u1 ∂x2 + Or: (33) sL (u(x,t))−2L (u(x,t)) = u(x,0)+L (Nu(x,t)) (46) Or again: sL (u(x,t))−2L (u(x,t)) = u(x,0)+L (Nu(x,t)) (46) Or again: (33) (46) Or again: Or again: L (u(x,t)) = 1 s−2u(x,0)+ 1 s−2L (Nu(x,t)) (47) Applying the inverse Laplace transform L −1 gives: L −1 1 (s−1)4 (34) L (u(x,t)) = 1 s−2u(x,0)+ 1 s−2L (Nu(x,t)) (47) Applying the inverse Laplace transform L −1 gives: L (u(x,t)) = 1 s−2u(x,0)+ 1 s−2L (Nu(x,t)) (47) 1 (47) u3(x,t) = −cos(x)L −1 1 (s−1)4 (34) (34) Applying the inverse Laplace transform L −1 gives: (35) u(x,t) = L −1( 1 s−2u(x,0))+L −1( 1 s−2L (Nu(x,t))) (48) Now, u3(x,t) = −1 6t3 cos(x) (35) u(x,t) = L −1( 1 s−2u(x,0))+L −1( 1 s−2L (Nu(x,t))) (48) Now, u3(x,t) = −1 6t3 cos(x) (35) u(x,t) = L −1( 1 s−2u(x,0))+L −1( 1 s−2L (Nu(x,t))) (48) Now, ( A3(x,t) = 0 u4(x,t) = 1 4!t4 sin(x) (36) L −1 1 s−2 = e2t L −1 1 s−2 = e2t (49) ( A3(x,t) = 0 u4(x,t) = 1 4!t4 sin(x) (36) L −1 1 s−2 = e2t (49) therefore: (49) (36) therefore: therefore: Recursively, we have : Recursively, we have : +u2(x,t)+u3(x,t)+u4(x,t)+... (37) u(x,t) = 2e2tp ex −e−4x +L −1( 1 s−2L (Nu(x,t))) (50) u(x,t) = u0(x,t)+u1(x,t)+u2(x,t)+u3(x,t)+u4(x,t)+... Let us consider the following equation [2] Substituting (10) and (11) in (9) we obtain the expression: Let us look for the solution of u(x,t) in the form (21) below: Let us look for the solution of u(x,t) in the form (21) below: u(x,t) = ∞ ∑ n=0 un(x,t) (21) (21) ∞ ∑ n=0 L (un(x,t)) = 1 s α1(x)+ 1 s2 α2(x)+ 1 s2 L (h(x,t)) − ∞ ∑ n=0 1 s2 L (Run(x,t))+ 1 s2 L (An(x,t)) And: And: (12) Nu(x,t) = ∞ ∑ n=0 An(x,t) (22) Nu(x,t) = ∞ ∑ n=0 An(x,t) (22) (12) (22) We obtain the following algorithm: We deduce the following Laplace-Adomian algorithm: We deduce the following Laplace-Adomian algorithm:    u0(x,t) = et sin(x) un+1(x,t) = L −1 1 s−1L (An(x,t))+ 1 s−1L ∂un(x,t) ∂x (23)                L (u0(x,t)) = 1 s α1(x)+ 1 s2 α2(x)+ 1 s2 L (h(x,t)) L (un+1(x,t)) = − 1 s2 L (Run(x,t))+ 1 s2 L (An(x,t)) ,n ⩾0 (23) (13) with: (13) A0(x,t) = u2 0 ∂2u0 ∂x2 +u3 0 = 0 (24) A0(x,t) = u2 0 ∂2u0 ∂x2 +u3 0 = 0 (24) By substitution process, we have: substitution process, we have: x,t) = L −1 1 s−1L (A0(x,t))+ 1 s−1L ∂u0(x,t) ∂x (25) u1(x,t) = L −1 1 s−1L ∂u0(x,t) ∂x (26) nce: u1(x,t) = cos(x)L −1 1 (s−1)2 (27) By applying the inverse L −1 of the Laplace transform into the expressions of u0(x,t) and un+1(x,t) are established: By applying the inverse L −1 of the Laplace transform into the expressions of u0(x,t) and un+1(x,t) are established: u1(x,t) = L −1 1 s−1L (A0(x,t))+ 1 s−1L ∂u0(x,t) ∂x (25)                              u0(x,t) = L −1 α1(x) s +L −1 1 s2 α2(x))+ 1 s2 L [h(x,t)] un+1(x,t) = −L −1 1 s2 L [Run(x,t)]−1 s2 L [An(x,t)] ,n ⩾0 (14) Let: u1(x,t) = L −1 1 s−1L ∂u0(x,t) ∂x (26) Hence: u1(x,t) = cos(x)L −1 1 (s−1)2 (27) Thus: u1(x,t) = tet cos(x) (28)                              u0(x,t) = L −1 α1(x) s +L −1 1 s2 α2(x))+ 1 s2 L [h(x,t)] un+1(x,t) = −L −1 1 s2 L [Run(x,t)]−1 s2 L [An(x,t)] ,n ⩾0 (14)                              u0(x,t) = L −1 α1(x) s +L −1 1 s2 α2(x))+ 1 s2 L [h(x,t)] un+1(x,t) = −L −1 1 s2 L [Run(x,t)]−1 s2 L [An(x,t)] ,n ⩾0 (14) Let: u1(x,t) = L − Hence: u1(x,t) = Thus: u1(x Let: u1(x,t) = L −1 1 s−1L ∂u0(x,t) ∂x (26) (26) Hence: (14) u1(x,t) = cos(x)L −1 1 (s−1)2 (27) (27) Thus: Thus: −L −1 1 s2 L [Run(x,t)]−1 s2 L [An(x,t)] ,n ⩾0 −L −1 1 s2 L [Run(x,t)]−1 s2 L [An(x,t)] ,n ⩾0 Thus: u1(x,t) = tet cos(x) (28) u1(x,t) = tet cos(x) (28) u1(x,t) = tet cos(x) (28) International Journal of Applied Mathematical Research 45 45 Now: By the same process, the other terms are obtained. 3.2. Example 2 Let us consider the following equation [8] Let us consider the following equation [8] = u(x,t)∂u(x,t) ∂x x +3 u(x,t)∂u(x,t) ∂x + A0(x,t) = u0(x,t)∂u0(x,t) ∂x x +                  ∂u(x,t) ∂t = u(x,t)∂u(x,t) ∂x x +3 u(x,t)∂u(x,t) ∂x + 2 u(x,t)−u2(x,t) u(x,0) = 2 √ ex −e−4x (41) A0(x,t) = u0(x,t)∂u0(x,t) ∂x x + 3 u0(x,t)∂u0(x,t) ∂x −2u2 0(x,t) (54) and: (54) (41) and: A1(x,t) = u0(x,t)∂u1(x,t) ∂x +u1(x,t)∂u0(x,t) ∂x x + 3 u0(x,t)∂u1(x,t) ∂x +u1(x,t)∂u0(x,t) ∂x −4u0u1(x,t) Let’s solve this equation by the Laplace-Adomian method. Let’s ask: A1(x,t) = u0(x,t)∂u1(x,t) ∂x +u1(x,t)∂u0(x,t) ∂x x + Let’s solve this equation by the Laplace-Adomian method. Let’s ask: A1(x,t) = u0(x,t)∂u1(x,t) ∂x +u1(x,t)∂u0(x,t) ∂x x + Let’s solve this equation by the Laplace-Adomian method. 3.2. Example 2 Let’s ask: u(x,t) ∂x x +3 u(x,t)∂u(x,t) ∂x + (42) x 3 u0(x,t)∂u1(x,t) ∂x +u1(x,t)∂u0(x,t) ∂x −4u0u1(x,t) ∂u(x,t) ∂t = u(x,t)∂u(x,t) ∂x x +3 u(x,t)∂u(x,t) ∂x + 2 u(x,t)−u2(x,t) (42) x 3 u0(x,t)∂u1(x,t) ∂x +u1(x,t)∂u0(x,t) ∂x −4u0u1(x,t) (55) 2 u(x,t)−u2(x,t) (55) International Journal of Applied Mathematical Research 46 The calculations made give:                                                                A0(x,t) = 0 u1(x,t) = 0 A1(x,t) = 0 u2(x,t) = 0 A2(x,t) = 0 u3(x,t) = 0 · · · un(x,t) = 0 (56) Afterwards, L (w(x,t)) = 1 (s−β)w(x,0)+ 1 (s−β)L (Nw(x,t)) (64) The application of the inverse Laplace transform gives the following result: w(x,t) = xeβt +L −1 1 s−β L (Nw(x,t)) (65) Let’s look for the solution of u(x,t) in the form (66) below : u(x,t) = ∞ ∑ n=0 un(x,t) (66) and : Nu(x,t) = ∞ ∑ n=0 An(x,t) (67) W b i h f ll i l i h Afterwards, Afterwards, L (w(x,t)) = 1 (s−β)w(x,0)+ 1 (s−β)L (Nw(x,t)) (64)            A0(x,t) = 0 u1(x,t) = 0 L (w(x,t)) = 1 (s−β)w(x,0)+ 1 (s−β)L (Nw(x,t (64) The application of the inverse Laplace transform gives the following result: w(x,t) = xeβt +L −1 1 s−β L (Nw(x,t)) (65) (65) (56) Let’s look for the solution of u(x,t) in the form (66) below : u(x,t) = ∞ ∑ n=0 un(x,t) (66) u(x,t) = ∞ ∑ n=0 un(x,t) (66) and : and : Nu(x,t) = ∞ ∑ n=0 An(x,t) (67) Nu(x,t) = ∞ ∑ n=0 An(x,t) (67) un(x,t) = 0 We obtain the following algorithm: We obtain the following algorithm: Therefore, the exact solution of the problem is then : We obtain the following algorithm: Therefore, the exact solution of the problem is then : 7)              w0(x,t) = xeβt wn+1(x,t) = L −1( 1 s−2L (An((x,t)) (68) p u(x,t) = 2e2tp ex −e−4x (57) Example 3 consider the following equation [3]              w0(x,t) = xeβt wn+1(x,t) = L −1( 1 s−2L (An((x,t)) (68) With u(x,t) = 2e2tp ex −e−4x (57)        w0(x,t) = xeβ u(x,t) = 2e2tp ex −e−4x (68) 3.3. Example 3 Let us consider the following equation [3] Let us consider the following equation [3] With : With :          ∂w(x,t) ∂t = 2w(x,t)∂2w(x,t) ∂x2 +2 ∂w(x,t) ∂x 2 −βw(x,t) w(x,0) = x (58) A0(x,t) = w0(x,t)∂2w0(x,t) ∂x2 + ∂w0(x,t) ∂x 2 (69) and : A1(x,t) = w0(x,t)∂2w1(x,t) ∂x2 +w1(x,t)∂2w0(x,t) ∂x2 +2 ∂w1(x,t) ∂x ∂w0 ∂ (70)          ∂w(x,t) ∂t = 2w(x,t)∂2w(x,t) ∂x2 +2 ∂w(x,t) ∂x 2 −βw(x,t) w(x,0) = x      ∂w(x,t) ∂t = 2w(x,t)∂2w(x,t) ∂x2 +2 ∂w(x,t) ∂x 2 −βw(x,t) A0(x,t) = w0(x,t)∂2w0(x,t) ∂x2 + ∂w0(x,t) ∂x 2 (69) d (69) and : (58) A1(x,t) = w0(x,t)∂2w1(x,t) ∂x2 +w1(x,t)∂2w0(x,t) ∂x2 +2 ∂w1(x,t) ∂x ∂w0(x ∂x (70) (58)          ∂w(x,t) ∂t = 2w(x,t)∂2w(x,t) ∂x2 +2 ∂w(x,t) ∂x 2 −βw(x,t) w(x,0) = x (59) The calculations made give:              A0(x,t) = e2βt w1(x,t) = −1 β (eβt −e2βt)          ∂w(x,t) ∂t = 2w(x,t)∂2w(x,t) ∂x2 +2 ∂w(x,t) ∂x 2 −βw(x,t) w(x,0) = x ∂t = 2w(x,t) ∂x2 +2 ∂x −βw(x,t) w(x,0) = x (59) he application of the Laplace transform L to the equation (59), nerates the following: L ∂w(x,t) ∂t = L 2w(x,t)∂2w(x,t) ∂x2 +2 ∂w(x,t) ∂x 2 +βw(x,t) ! (60) et’s put N (w(x,t)) = 2w(x,t)∂2w(x,t) ∂x2 +2 ∂w(x,t) ∂x 2 (61) e get:                                                                    A0(x,t) = e2βt w1(x,t) = −1 β (eβt −e2βt) A1(x,t) = 0 w2(x,t) = 0 A2(x,t) = 0 w3(x,t) = 0 · · · wn(x,t) = 0, ∀n ⩾2 (71) (59) The application of the Laplace transform L to the equation (59), generates the following: L ∂w(x,t) ∂t = L 2w(x,t)∂2w(x,t) ∂x2 +2 ∂w(x,t) ∂x 2 +βw(x,t) ! 3.3. Example 3 (60)                         w2(x,t) = 0 A2(x,t) = 0 w3(x,t) = 0 ( L ∂w(x,t) ∂t = L 2w(x,t)∂2w(x,t) ∂x2 +2 ∂w(x,t) ∂x 2 +βw(x,t) ! (60) Let’s put N (w(x,t)) = 2w(x,t)∂2w(x,t) ∂x2 +2 ∂w(x,t) ∂x 2 (61) We get:                                           w2(x,t) = 0 A2(x,t) = 0 w3(x,t) = 0 · · · wn(x,t) = 0, ∀n ⩾2 (7 L ∂w(x,t) ∂t = L 2w(x,t)∂2w(x,t) ∂x2 +2 ∂w(x,t) ∂x 2 +βw(x,t) ! (60) (71) L 2w(x,t)∂2w(x,t) ∂x2 +2 ∂w(x,t) ∂x 2 +βw(x,t) ! (60) (60) Let’s put Let’s put N (w(x,t)) = 2w(x,t)∂2w(x,t) ∂x2 +2 ∂w(x,t) ∂x 2 (61) get: N (w(x,t)) = 2w(x,t)∂2w(x,t) ∂x2 +2 ∂w(x,t) ∂x 2 (61) (61) We get: We get: g Finally, the sum of the terms of the series gives: g Finally, the sum of the terms of the series gives: Finally, the sum of the terms of the series gives: Finally, the sum of the terms of the series gives: sL (w(x,t))−w(x,0) = L (Nw(x,t))+βL (w(x,t)) (62) This gives: w(x,t) = w0(x,t)+w1(x,t) = (x−1 β )eβt + 1 β e2βt (72) The exact solution of the problem is then: w(x,t) = w0(x,t)+w1(x,t) = (x−1 β )eβt + 1 β e2βt (72) (62) sL (w(x,t))−w(x,0) = L (Nw(x,t))+βL (w(x,t)) (62) This gives: w(x,t) = w0(x,t)+w1(x,t) = (x−1 β )eβt + 1 β e2βt The exact solution of the problem is then: (72) This gives: The exact solution of the problem is then: The exact solution of the problem is then: (s−β)L (w(x,t)) = w(x,0)+L (Nw(x,t)) (63) w(x,t) = x−1 β eβt + 1 β e2βt (73) w(x,t) = x−1 β eβt + 1 β e2βt (73) (s−β)L (w(x,t)) = w(x,0)+L (Nw(x,t)) (63) w(x,t) = x−1 β eβt + 1 β e2βt (73) (73) International Journal of Applied Mathematical Research 47 4. Conclusion [6] Jasem Fadaei, “Application of Laplace-Adomian Decomposition Method on Linear and Nonlinear System of PDEs”, Applied Math- ematical Sciences, Vol.5, (2011), pp.1307-1315. The application of the Laplace-Adomian method has allowed us to obtain the exact solutions of the problems studied in this article. The manipulation of Adomian polynomials and Laplace transforms have allowed us to obtain efﬁcient algorithms that converge faster to the exact solution of the problem. We can conclude that this method is well suited for solving nonlinear partial differential equations not only in the case of ﬂows in porous media, but also in many physical phenomena of high complexity. [7] M. Hussain and M. MajidKhan, “Modiﬁed Laplace Decomposi- tion Method”, Applied Mathematical Sciences, Vol.4, No.36, (2010), pp.1769-1783. [8] A. Khalouta and A. Kadem, “New analytical method for solving non- linear time-fractional reaction-diffusion-convection problems”, Revista Colombiana de Matem`aticas, Vol.54, (2020), pp.1-11. [9] Onur Kiymaz, “An Algorithm for Solving Initial Value Problems Us- ing Laplace-Adomian Decomposition Method”, Applied Mathematical Sciences, Vol.3, No.30, (2009), pp.1453-1459. [10] Vincent Le Maout, “Mod´elisation d’´ecoulements multiphasiques de ﬂu- ides visco´elastiques en milieux poreux”, Th`ese de doctorat Universit´e de Bordeaux, Bordeaux le 03 Novembre, 2020. References [11] Shahid Mahmood, Rasool Shan, Hassan Khan and Muhammad Arif, “Laplace Adomian Decomposition Method for Multi Dimensional Time Fractional Model of Navier-Stokes Equation”, Symmetry MDPI, Vol.11 No.149, (2019), pp.1-15. [1] Arshad Ali, Humaira, Laila, Kamal Shah, “Analytical Solution of General Fisher’s Equation by using Laplace Adomian decomposition method”, J. Pur. Appl. Math., Vol.2, No.3, (2018), pp.1-4. [12] A. Naghipour, J. Manaﬁan, “Application of the Laplace Adomian De- composition and implicit Methods for solving Burger’s Equation”, TWMS J. Appl. Math., Vol.6, No.1, (2015), pp.68-77. pp pp [2] S. Nuseir Ameina, “Power Series Solutions for Nonlinear Systems of Partial Differential Equations”, Journal of Mathematics Sciences: Advances and Applications, Vol.61, (2020), pp.17-35. [13] P. Pue-on, “Laplace Adomian Decomposition Method for Solving Newell-Whitehead-Segel Equation”, Applied Mathematical Sciences, Vol.7, No.132, (2013), pp.6593-6600. pp pp [3] Jonas Berx and Joseph O. Indekeu, “The BLUES function method applied to partial differential equations and analytic approximants for interface growth under shear”, Institute for Theoretical Physics, KULeuven, Belgium (Dated: August 5. 2021), (2021) [14] A. M. Wazwaz and M. S. Mehanna, “The Combined Laplace-Adomian Method for handling Singular Integral Equation of Heat Transfer”, International Journal of Nonlinear Sciences, Vol.10, No.2, (2010), pp.248-252. KULeuven, Belgium (Dated: August 5. 2021), (2021) [4] B. Beyi, J. Bonazebi-Yindoula, L. Som´e, G. Bissanga, “Application of the Laplace-Adomian Method annd the SA Method to Solving Tthe Partial Differential and Integro-differential Equations” , Applied Mathematical Sciences, Vol.6, No.104, (2012), pp.5147-5159. [15] F. Zami-Pierre, R. de Loubens, M. Quintard and Y. Davit, “Polymer Flow Through Porous Media: Numerical Prediction of the Contribution of Slip to the Apparent Viscosity”, Transport in Porous Media, Vol.119, No.3, (2010), pp.521-538. ( ) pp [5] J. Bonazebi-Yindoula, A. Massamba, G. Bissanga, “Solving of Klein- Gordon Equation by Two Methods of Numerical Analysis”, Journal of Applied Mathematics and Physics, Vol.4, (2016), pp.1916-1929.
https://openalex.org/W2969934957	https://www.biorxiv.org/content/biorxiv/early/2019/07/18/601682.full.pdf	English	null	Additive genetic variance for lifetime fitness and the capacity for adaptation in an annual plant	Evolution	2,019	cc-by	21,119	. CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint Additive genetic variance for lifetime fitness and the capacity for adaptation in an 1 annual plant 2 3 Mason W. Kulbaba1,£, Seema N. Sheth1, 2, Rachel E. Pain1, Vince M. Eckhart3, Ruth G. Shaw1 4 5 £corresponding author: mason.kulbaba@gmail.com 6 1 Department of Ecology, Evolution, and Behavior, University of Minnesota, St. Paul, MN, USA. 7 2 Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, 8 USA. 3 Department of Biology, Grinnell College, Grinnell, IA, USA 9 10 11 12 13 14 15 16 17 18 19 20 21 Running head: Capacity for adaptation in an annual plant 22 23 Key words: Chamaecrista fasciculata, adaptive capacity, aster models, genotype-by- 24 environment interactions, Fisher’s Fundamental Theorem of Natural Selection 25 Additive genetic variance for lifetime fitness and the capacity for adaptation in an 1 annual plant 2 3 Mason W. Kulbaba1,£, Seema N. Sheth1, 2, Rachel E. Pain1, Vince M. Eckhart3, Ruth G. Shaw1 4 5 £corresponding author: mason.kulbaba@gmail.com 6 1 Department of Ecology, Evolution, and Behavior, University of Minnesota, St. Paul, MN, USA. 7 2 Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, 8 USA. 3 Department of Biology, Grinnell College, Grinnell, IA, USA 9 10 11 12 13 1 1 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint Introduction 43 A population’s mean absolute fitness, W̅ , in terms of the per capita contribution of 44 offspring, corresponds to its growth rate and is thus a measure of its degree of adaptation (Fisher 45 1930, pp. 25, 37; Roughgarden 1996, Ch. 4). Fisher (1930) showed that the immediate capacity 46 for further adaptation is proportional to the magnitude of a population’s additive genetic variance 47 for absolute fitness, evaluated as an individuals’ lifetime contribution of offspring to the 48 population (Fisher 1930; Price 1970, 1972; Ewens 2004). Moreover, this Fundamental Theorem 49 of Natural Selection (FTNS, Fisher 1930) quantitatively predicts the rate of adaptation under 50 prevailing environmental conditions as the ratio of VA(W) to current mean population fitness, W̅ . 51 This ratio predicts the genetically based change, from one generation to the next, in the per capita 52 rate of population growth. Such predictions are vital to understanding the adaptive process, by 53 first determining the possibility for evolutionary change in mean fitness, and second, the 54 expected magnitude of fitness increase in the wild. Thus, using FTNS to predict future adaptation 55 under ongoing natural selection requires direct, quantitative estimates of standing additive 56 genetic variance for fitness, VA(W). Studies that take such a direct and predictive approach are 57 exceedingly rare (but see Etterson 2004a,b; Winn 2004; Sheth et al. 2018). 58 A population’s mean absolute fitness, W̅ , in terms of the per capita contribution of 44 offspring, corresponds to its growth rate and is thus a measure of its degree of adaptation (Fisher 45 1930, pp. 25, 37; Roughgarden 1996, Ch. 4). Fisher (1930) showed that the immediate capacity 46 for further adaptation is proportional to the magnitude of a population’s additive genetic variance 47 for absolute fitness, evaluated as an individuals’ lifetime contribution of offspring to the 48 population (Fisher 1930; Price 1970, 1972; Ewens 2004). Moreover, this Fundamental Theorem 49 of Natural Selection (FTNS, Fisher 1930) quantitatively predicts the rate of adaptation under 50 prevailing environmental conditions as the ratio of VA(W) to current mean population fitness, W̅ . 51 This ratio predicts the genetically based change, from one generation to the next, in the per capita 52 rate of population growth. Abstract 26 The immediate capacity for adaptation under current environmental conditions is directly 27 proportional to the additive genetic variance for fitness, VA(W). Mean absolute fitness, W̅ , is 28 predicted to change at the rate VA(W) W ̅̅̅ , according to Fisher’s Fundamental Theorem of Natural 29 Selection. Despite ample research evaluating degree of local adaptation, direct assessment of 30 VA(W) and the capacity for ongoing adaptation is exceedingly rare. We estimated VA(W) and 31 W̅ in three pedigreed populations of annual Chamaecrista fasciculata, over three years in the 32 wild. Contrasting with common expectations, we found significant VA(W) in all populations and 33 years, predicting increased mean fitness in subsequent generations (0.83 to 6.12 seeds per 34 individual). Further, we detected two cases predicting “evolutionary rescue”, where selection on 35 standing VA(W) was expected to increase fitness of declining populations (W ̅̅̅̅< 1.0) to levels 36 consistent with population sustainability and growth. Within populations, interannual differences 37 in genetic expression of fitness were striking. Significant genotype-by-year interactions reflected 38 modest correlations between breeding values across years (all r < 0.490), indicating temporally 39 variable selection at the genotypic level; that could contribute to maintaining VA(W). By directly 40 estimating VA(W) and total lifetime W̅ , our study presents an experimental approach for studies 41 of adaptive capacity in the wild. 42 The immediate capacity for adaptation under current environmental conditions is directly 27 2 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint Introduction 43 Such predictions are vital to understanding the adaptive process, by 53 first determining the possibility for evolutionary change in mean fitness, and second, the 54 expected magnitude of fitness increase in the wild. Thus, using FTNS to predict future adaptation 55 under ongoing natural selection requires direct, quantitative estimates of standing additive 56 genetic variance for fitness, VA(W). Studies that take such a direct and predictive approach are 57 exceedingly rare (but see Etterson 2004a,b; Winn 2004; Sheth et al. 2018). 58 di f i l i h ll f d i f i l i d 5 Studies of ongoing selection have generally focused on traits of particular interest. Lande 59 and Arnold’s (1983) proposal of multiple regression of fitness on traits powerfully stimulated 60 research of this kind. As they noted, this approach reflects only the selection on those traits under 61 consideration, through their associations with the measure of fitness. Few such studies report the 62 extent to which the included traits account for the variation in fitness (but see Conner 1988; 63 Janzen 1993; Conner et al. 1996a,b). Natural selection likely bears on many more traits, 64 however, and this implies that the included traits typically account for a modest proportion of the 65 however, and this implies that the included traits typically account for a modest proportion of the 65 3 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint variation in fitness (see Shaw 2019). Whereas the regression of fitness on a set of traits can yield 66 insight into evolutionary change in particular traits under selection, they are ill-suited for 67 predicting the change in mean absolute fitness, i.e. the degree of overall adaptation. 68 Retrospective studies of genetic differentiation and adaptation to changes in local 69 conditions have amply demonstrated the efficacy of selection in the past. Introduction 43 These cases are 70 illuminating even when the aspect(s) of the environment that impose(d) differential selection are 71 unknown, as is often the case, given the high dimensionality of environment. When the timing of 72 an abrupt environmental change is known, the rate that adaptation proceeded can also be 73 determined, or at least bounded. Cases of rapid adaptation, within a few to tens of generations, 74 are now well known for a variety of taxa and include adaptation that followed changes in edaphic 75 conditions (Antonovics and Bradshaw 1970; Al-Hiyaly et al. 1993), introduction of herbicides 76 (Vigueira et al. 2013; Baucom 2019), predators (Fisk et al. 2007), and changing climate (Franks 77 et al. 2007; Geerts et al. 2015). These examples of rapid adaptation to local conditions reveal 78 capacity for adaptation to environmental differences from standing genetic variation (Barrett and 79 Schluter 2008). However, such studies are limited to characterizing the current degree of 80 adaptation in response to past selection and do not evaluate the potential for ongoing adaptation. 81 Quantitative prediction of the rate of adaptation from FTNS is vital to understanding 82 ongoing adaptation in wild populations, especially in assessing whether standing levels of 83 VA(W) suffice for contemporary natural selection in declining populations (i.e. W̅ < 1) to halt 84 further decline (Bradshaw 1991). Such “evolutionary rescue” (Gomulkiewicz and Holt 1995) 85 that increases fitness to 1 (i.e. individuals demographically replacing themselves) or greater 86 variation in fitness (see Shaw 2019). Whereas the regression of fitness on a set of traits can yield 66 insight into evolutionary change in particular traits under selection, they are ill-suited for 67 predicting the change in mean absolute fitness, i.e. the degree of overall adaptation. 68 4 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint environmental variation (Carlson et al. 2014). Introduction 43 ; https://doi.org/10.1101/601682 doi: bioRxiv preprint variance, may explain low heritability of fitness components (Price and Schluter 1991). 112 Nevertheless, the empirical generalization about lower heritability of individual components of 113 fitness contributed to the expectation that additive genetic variance for lifetime fitness is likely 114 be modest. This expectation may, however, be inaccurate because it does not account for the 115 dependence of sequential fitness components through the life-span. Third, when pedigrees are 116 not experimentally derived and may be uncertain (e.g. Foerster et al. 2007), logistical challenge 117 encumber efforts to both reconstruct relatedness of individuals and determine total lifetime 118 fitness. Uncertainty in the assignment of relatedness casts doubt on the estimates of quantitative 119 genetic parameters (Thomas et al. 2000). Finally, confounding between genetic and 120 environmental influences, likely in observational studies, can also bias VA(W) estimates 121 (Pemberton 2010), and therefore inferences drawn from them. 122 Whereas direct estimates of VA(W) in the wild are rare, theory bearing on whether 123 substantial genetic variation could be maintained is ambiguous. In particular, early theory 124 addressing the role of environmental variation, particularly temporal variability, indicated that 125 restrictive conditions are required to maintain genetic variation within populations (Felsenstein 126 1976; Hedrick 1986; Gillespie and Turelli 1989). More recently, models focusing on VA(W) 127 under environmentally varying selection have demonstrated maintenance of substantial VA(W) 128 (Zhang 2012; Shaw and Shaw 2014; see also Yi and Dean 2013). 129 Direct estimation of VA(W) is not a trivial endeavor, requiring sound experimental desig 130 and statistical inference. Experimental approaches that facilitate estimates of complete lifetime 131 fitness expressions from fully pedigreed individuals, and avoid confounding environmental and 132 genetic effects are essential (Shaw 2019). Further, statistical tools are needed that can validly 133 model lifetime fitness, which conforms to no conventional statistical distribution (Shaw et al. 134 variance, may explain low heritability of fitness components (Price and Schluter 1991). 112 Nevertheless, the empirical generalization about lower heritability of individual components of 113 fitness contributed to the expectation that additive genetic variance for lifetime fitness is likely to 114 be modest. This expectation may, however, be inaccurate because it does not account for the 115 dependence of sequential fitness components through the life-span. Third, when pedigrees are 116 not experimentally derived and may be uncertain (e.g. Foerster et al. Introduction 43 2007), logistical challenges 117 encumber efforts to both reconstruct relatedness of individuals and determine total lifetime 118 fitness. Uncertainty in the assignment of relatedness casts doubt on the estimates of quantitative 119 genetic parameters (Thomas et al. 2000). Finally, confounding between genetic and 120 environmental influences, likely in observational studies, can also bias VA(W) estimates 121 (Pemberton 2010), and therefore inferences drawn from them. 122 variance, may explain low heritability of fitness components (Price and Schluter 1991). 112 Nevertheless, the empirical generalization about lower heritability of individual components of 113 fitness contributed to the expectation that additive genetic variance for lifetime fitness is likely to 114 be modest. This expectation may, however, be inaccurate because it does not account for the 115 dependence of sequential fitness components through the life-span. Third, when pedigrees are 116 not experimentally derived and may be uncertain (e.g. Foerster et al. 2007), logistical challenges 117 encumber efforts to both reconstruct relatedness of individuals and determine total lifetime 118 fitness. Uncertainty in the assignment of relatedness casts doubt on the estimates of quantitative 119 genetic parameters (Thomas et al. 2000). Finally, confounding between genetic and 120 environmental influences, likely in observational studies, can also bias VA(W) estimates 121 (Pemberton 2010), and therefore inferences drawn from them. 122 Whereas direct estimates of VA(W) in the wild are rare, theory bearing on whether 123 substantial genetic variation could be maintained is ambiguous. In particular, early theory 124 addressing the role of environmental variation, particularly temporal variability, indicated that 125 restrictive conditions are required to maintain genetic variation within populations (Felsenstein 126 1976; Hedrick 1986; Gillespie and Turelli 1989). More recently, models focusing on VA(W) 127 under environmentally varying selection have demonstrated maintenance of substantial VA(W) 128 (Zhang 2012; Shaw and Shaw 2014; see also Yi and Dean 2013). 129 Direct estimation of VA(W) is not a trivial endeavor, requiring sound experimental design 130 and statistical inference. Experimental approaches that facilitate estimates of complete lifetime 131 fitness expressions from fully pedigreed individuals, and avoid confounding environmental and 132 genetic effects are essential (Shaw 2019). Further, statistical tools are needed that can validly 133 model lifetime fitness, which conforms to no conventional statistical distribution (Shaw et al. 134 Direct estimation of VA(W) is not a trivial endeavor, requiring sound experimental design 130 and statistical inference. Introduction 43 In the current context of drastic ongoing 89 environmental change, estimates of the magnitude of a populations’ VA(W) are critical for 90 predicting the genetically-based change in W̅ in response to the natural selection impinging on 91 them. Given the elevated threat to population persistence due to rapid change in climate and 92 other aspects of the environment (Shaw and Etterson 2012; Neumann et al. 2017), it is now 93 imperative to clarify the efficacy of selection in maintaining and enhancing population mean 94 fitness in situ. 95 Despite their importance, very few direct estimates of standing VA(W) exist (but see 96 Etterson 2004a,b; Winn 2004; Sheth et al. 2018). Several factors may explain this paucity of 97 empirical research. First, population mean fitness is often thought to be at or near optimum due 98 to incessant selection, which would tend to fix or eliminate alleles having directional effects on 99 fitness. Accordingly, VA(W), and therefore the capacity for adaptation, is conjectured to be 100 reduced to low or negligible levels (e.g., Mazer 1987; Barton and Keightley 2002). However, 101 even with consistent and strong selection at individual loci, fixation requires hundreds of 102 generations (e.g., Fig. 6.3 in Hartl and Clark 1997; Messer et al. 2016). The continuing response 103 to selection in the Illinois corn selection experiment (Moose et al. 2004), even after 100 104 generations, demonstrates the persistence of variation supporting a response to artificial selection 105 on biochemical constituents of kernels. However, the resilience of variation under selection in 106 nature remains an open empirical question. Second, numerous studies have found that estimates 107 of narrow-sense heritability ( 𝑉𝐴 𝑉𝑃) for individual fitness components tend to be lower than for 108 morphological traits (Roff and Mousseau 1987; McFarlane et al. 2014). Because environmental 109 5 5 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. Introduction 43 2007, 2013), we directly estimate VA(W) and 141 W̅ to quantitatively predict the rate of adaptation. To assess the generality of our findings, we 142 conducted our experiment in three populations in three successive years. Our experimental 143 design allowed us to characterize population-specific expression of VA(W) and to predict the rate 144 of future adaptation, while also evaluating temporal variation in the expression of VA(W), and 145 therefore variation in the capacity for adaptation, in three populations over three years. 146 Introduction 43 C. fasciculata produces hermaphroditic, enantiostylous flowers that 153 are buzz-pollinated (Lee and Bazzaz 1982) by bumblebees (Bombus spp). Its experimental 154 tractability has made it the focus of previous studies of gene flow (Fenster 1991a,b), quantitative 155 genetics (Kelly 1993; Etterson 2004b), selection on traits (Etterson 2004a), local adaptation 156 2008). Aster models account for the dependent nature of sequential life-history fitness 135 expressions to estimate total mean lifetime fitness (Geyer et al. 2007), and random parental 136 effects (Geyer et al. 2013) to estimate the additive genetic effects on lifetime fitness. 137 2008). Aster models account for the dependent nature of sequential life-history fitness 135 expressions to estimate total mean lifetime fitness (Geyer et al. 2007), and random parental 136 effects (Geyer et al. 2013) to estimate the additive genetic effects on lifetime fitness. 137 2008). Aster models account for the dependent nature of sequential life-history fitness 135 expressions to estimate total mean lifetime fitness (Geyer et al. 2007), and random parental 136 effects (Geyer et al. 2013) to estimate the additive genetic effects on lifetime fitness. 137 In this study, we estimate the capacity for adaptation under contemporary natural 138 selection in wild populations of the annual legume Chamaecrista fasciculata growing in their 139 native locations. Using Fisher’s Fundamental Theorem (Fisher 1930) and aster modelling to 140 analyze records of lifetime fitness (Geyer et al. 2007, 2013), we directly estimate VA(W) and 141 W̅ to quantitatively predict the rate of adaptation. To assess the generality of our findings, we 142 conducted our experiment in three populations in three successive years. Our experimental 143 design allowed us to characterize population-specific expression of VA(W) and to predict the ra 144 of future adaptation, while also evaluating temporal variation in the expression of VA(W), and 145 therefore variation in the capacity for adaptation, in three populations over three years. 146 2008). Aster models account for the dependent nature of sequential life-history fitness 135 expressions to estimate total mean lifetime fitness (Geyer et al. 2007), and random parental 136 effects (Geyer et al. 2013) to estimate the additive genetic effects on lifetime fitness. 137 In this study, we estimate the capacity for adaptation under contemporary natural 138 selection in wild populations of the annual legume Chamaecrista fasciculata growing in their 139 native locations. Using Fisher’s Fundamental Theorem (Fisher 1930) and aster modelling to 140 analyze records of lifetime fitness (Geyer et al. Introduction 43 Experimental approaches that facilitate estimates of complete lifetime 131 fitness expressions from fully pedigreed individuals, and avoid confounding environmental and 132 genetic effects are essential (Shaw 2019). Further, statistical tools are needed that can validly 133 model lifetime fitness, which conforms to no conventional statistical distribution (Shaw et al. 134 6 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint 7 2008). Aster models account for the dependent nature of sequential life-history fitness 135 expressions to estimate total mean lifetime fitness (Geyer et al. 2007), and random parental 136 effects (Geyer et al. 2013) to estimate the additive genetic effects on lifetime fitness. 137 In this study, we estimate the capacity for adaptation under contemporary natural 138 selection in wild populations of the annual legume Chamaecrista fasciculata growing in their 139 native locations. Using Fisher’s Fundamental Theorem (Fisher 1930) and aster modelling to 140 analyze records of lifetime fitness (Geyer et al. 2007, 2013), we directly estimate VA(W) and 141 W̅ to quantitatively predict the rate of adaptation. To assess the generality of our findings, we 142 conducted our experiment in three populations in three successive years. Our experimental 143 design allowed us to characterize population-specific expression of VA(W) and to predict the rate 144 of future adaptation, while also evaluating temporal variation in the expression of VA(W), and 145 therefore variation in the capacity for adaptation, in three populations over three years. 146 Materials and Methods 147 Study system 148 We estimated VA(W) and W̅ as the basis for predicting the generational change in mean 149 fitness in three populations (Fig. 1) of the annual legume Chamaecrista fasciculata, each 150 growing in its home site. The range of C. fasciculata spans from the prairies of the Northern 151 Great Plains in Minnesota to Central Mexico and to the eastern seaboard of North America 152 (Irwin and Barneby 1982). Materials and Methods 147 Study system 148 We estimated VA(W) and W̅ as the basis for predicting the generational change in mean 149 fitness in three populations (Fig. 1) of the annual legume Chamaecrista fasciculata, each 150 growing in its home site. The range of C. fasciculata spans from the prairies of the Northern 151 Great Plains in Minnesota to Central Mexico and to the eastern seaboard of North America 152 (Irwin and Barneby 1982). C. fasciculata produces hermaphroditic, enantiostylous flowers that 153 are buzz-pollinated (Lee and Bazzaz 1982) by bumblebees (Bombus spp). Its experimental 154 tractability has made it the focus of previous studies of gene flow (Fenster 1991a,b), quantitative 155 genetics (Kelly 1993; Etterson 2004b), selection on traits (Etterson 2004a), local adaptation 156 7 7 7 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint (Galloway and Fenster 2000), adaptation to climate change (Etterson and Shaw 2001; Etterson 157 2004b), evolution of range limits (Stanton-Geddes et al. 2012b, 2013), and genotype-by- 158 environment interactions (Sheth et al. 2018). 159 (Galloway and Fenster 2000), adaptation to climate change (Etterson and Shaw 2001; Etterson 157 2004b), evolution of range limits (Stanton-Geddes et al. 2012b, 2013), and genotype-by- 158 environment interactions (Sheth et al. 2018). 159 (Galloway and Fenster 2000), adaptation to climate change (Etterson and Shaw 2001; Etterson 157 2004b), evolution of range limits (Stanton-Geddes et al. 2012b, 2013), and genotype-by- 158 environment interactions (Sheth et al. 2018). 159 Genetic sources and pedigree design 160 In the fall of 2012, 2-3 fruits from 200 maternal plants no closer together than 10 m were 161 collected from a population of C. fasciculata occupying remnant prairie at Kellogg-Weaver 162 Dunes in Minnesota (44°15'43" N, 91°55'15" W; hereafter “McCarthy Lake”). Similarly the 163 following fall, fruits from 200 maternal plants were collected from populations of C. Materials and Methods 147 183 Field planting design 184 In October-November of each of three consecutive years (2014-2016), pedigreed seeds 185 from each respective population were planted into restored tallgrass prairie near to (<1 km) each 186 site of origin. To maintain the environment for the experimental populations as realistic as 187 possible, vegetation was left in place and only mown (or burned in the case of CERA in 2016 188 and McCarthy Lake in 2014) in the fall to facilitate planting. In Fall of 2014, all rows needed for 189 all three years of the study were laid in parallel two meters apart, each row 50 m long. For each 190 Fall planting, a subset of rows within blocks was chosen at random, and planting positions were 191 marked with nails at 2 m intervals. Pedigreed seeds were planted at randomly chosen positions 192 according to the following protocol. Seeds from each full-sibling family from the greenhouse 193 crosses were combined and haphazardly distributed into envelopes, such that each envelope 194 contained five seeds. We randomly assigned envelopes to positions along rows in a randomized 195 block design (eight blocks at Grey Cloud and McCarthy Lake, and four blocks at CERA 196 positioned in the same location in all years), resulting in 15-24 replications of each half-sib 197 family per block. Blocks contained 25-26 rows (13 rows at McCarthy Lake). At each position 198 logistics, the final pedigree consisted of 42 paternal half-sibling families and 124 maternal full- 180 sibling families at Grey Cloud, 48 paternal half-sibling families and 132 maternal full-sibling 181 families at McCarthy Lake, and 21 paternal half-sibling families and 60 maternal full-sibling 182 families at CERA. 183 logistics, the final pedigree consisted of 42 paternal half-sibling families and 124 maternal full- 180 sibling families at Grey Cloud, 48 paternal half-sibling families and 132 maternal full-sibling 181 families at McCarthy Lake, and 21 paternal half-sibling families and 60 maternal full-sibling 182 families at CERA. 183 Field planting design 184 In October-November of each of three consecutive years (2014-2016), pedigreed seeds 185 from each respective population were planted into restored tallgrass prairie near to (<1 km) each 186 site of origin. Materials and Methods 147 To maintain the environment for the experimental populations as realistic as 187 possible, vegetation was left in place and only mown (or burned in the case of CERA in 2016 188 and McCarthy Lake in 2014) in the fall to facilitate planting. In Fall of 2014, all rows needed for 189 all three years of the study were laid in parallel two meters apart, each row 50 m long. For each 190 Fall planting, a subset of rows within blocks was chosen at random, and planting positions were 191 marked with nails at 2 m intervals. Pedigreed seeds were planted at randomly chosen positions 192 according to the following protocol. Seeds from each full-sibling family from the greenhouse 193 crosses were combined and haphazardly distributed into envelopes, such that each envelope 194 contained five seeds. We randomly assigned envelopes to positions along rows in a randomized 195 block design (eight blocks at Grey Cloud and McCarthy Lake, and four blocks at CERA 196 positioned in the same location in all years), resulting in 15-24 replications of each half-sib 197 family per block. Blocks contained 25-26 rows (13 rows at McCarthy Lake). At each position 198 centered at the nail, five seeds from a randomly chosen full-sib family were planted at 10 cm 199 spacing, approximately 1 cm under the soil surface. 200 Field planting design 184 In October-November of each of three consecutive years (2014-2016), pedigreed seeds 185 from each respective population were planted into restored tallgrass prairie near to (<1 km) each 186 site of origin. To maintain the environment for the experimental populations as realistic as 187 possible, vegetation was left in place and only mown (or burned in the case of CERA in 2016 188 and McCarthy Lake in 2014) in the fall to facilitate planting. In Fall of 2014, all rows needed for 189 all three years of the study were laid in parallel two meters apart, each row 50 m long. For each 190 Fall planting, a subset of rows within blocks was chosen at random, and planting positions were 191 marked with nails at 2 m intervals. Pedigreed seeds were planted at randomly chosen positions 192 according to the following protocol. Seeds from each full-sibling family from the greenhouse 193 crosses were combined and haphazardly distributed into envelopes, such that each envelope 194 contained five seeds. Materials and Methods 147 fasciculata 164 at Grey Cloud Dunes Scientific and Natural Area in Minnesota (44°46'32"N, 93°01'38"W; 165 hereafter “Grey Cloud”), and 100 fruits from plants at the Conard Environmental Research Area 166 in Iowa (41°40'44.2"N 92°51'24.9"W; hereafter “CERA”). 167 To obtain pedigreed populations of seeds from each of these populations, six seeds from 168 each maternal plant were surface sterilized using an 8.9% bleach solution followed by a 70% 169 ethanol rinse. We used 100 grit sandpaper to scarify the seeds, imbibed them in sterile water for 170 three days, and then planted them in small peat pots. A total of 167 Grey Cloud, 196 McCarthy 171 Lake, and 84 CERA individuals were used to generate pedigreed plants. One seedling per 172 individual was used to represent each distinct field-collected family and was transplanted into a 173 large tree pot with 1 teaspoon of Osmocote ® slow release fertilizer (14:14:14) after producing 174 five true leaves. Plants were grown in the University of Minnesota Plant Growth Facilities 175 greenhouse under 16:8-h photoperiod. Hand crosses were made according to a nested paternal 176 half-sibling design (North Carolina Design I; Comstock and Robinson 1948), with independent 177 sets of three dams randomly assigned to sires. Hand crosses were performed daily, and all non- 178 pollinated flowers were removed. After accounting for mortality in the greenhouse and planting 179 8 8 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint logistics, the final pedigree consisted of 42 paternal half-sibling families and 124 maternal full- 180 sibling families at Grey Cloud, 48 paternal half-sibling families and 132 maternal full-sibling 181 families at McCarthy Lake, and 21 paternal half-sibling families and 60 maternal full-sibling 182 families at CERA. Materials and Methods 147 We randomly assigned envelopes to positions along rows in a randomized 195 block design (eight blocks at Grey Cloud and McCarthy Lake, and four blocks at CERA 196 positioned in the same location in all years), resulting in 15-24 replications of each half-sib 197 family per block. Blocks contained 25-26 rows (13 rows at McCarthy Lake). At each position 198 centered at the nail, five seeds from a randomly chosen full-sib family were planted at 10 cm 199 spacing, approximately 1 cm under the soil surface. 200 9 9 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint Fitness surveys 201 We regularly recorded fitness components for each planted seed throughout the growing 202 season of the following year, including emergence, survival to flowering, and reproductive 203 output (Table 1). A plant was considered present if cotyledons had emerged, and survival to 204 flowering was scored based on the presence of open flowers or evidence of flowering (e.g., 205 wilted flowers, pedicels, or fruit). At each late-season census (i.e. after flowering), we recorded 206 the number of ripe fruit collected, number of fully elongated but immature fruits collected, 207 number of fruits collected off the ground in the immediate vicinity, number of immature fruits, 208 and pedicels from dehisced fruits remaining on the plant (see Sheth et al. 2018). Herbivory, 209 ranging from light browsing to consumption of the entire plant, or severing of the stem near the 210 soil surface, was recorded throughout the growing season. When herbivory occurred early in the 211 growing season (i.e. June-July), plants sometimes recovered and eventually reproduced. 212 Therefore, we retained these plants in censuses after herbivory to account for this late season 213 seed production. Because individuals ranged widely in number of fruits set (1-68), and fruits 214 explosively dehisce at maturity, we were unable to collect all mature fruits or count all seeds 215 each plant produced. Therefore, seed counts were obtained on a subsample of fruits produced. 216 Subsampling was accounted for in the statistical analyses (see below). 217 Statistical Analyses 218 Estimates of W̅ and VA(W) for each population in each year (Geyer et al. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint Materials and Methods 147 CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint modeled as Bernoulli variables, and the number of fruits and seeds produced, which were 223 modeled with Poisson distributions. To account for subsampling of fruits, we followed Appendix 224 S1 of Stanton-Geddes et al. (2012a), incorporating in our graphical models an additional node 225 representing the total number of fruits sampled. This leads to estimates that are scaled up to the 226 total number of seeds produced per seed planted. Using this model for expression of components 227 of fitness through the lifespan, as well as the dependence of each on components expressed 228 earlier, we obtained unconditional estimates, i.e. the mean and additive genetic variance of the 229 number of seeds produced per seed planted. 230 modeled as Bernoulli variables, and the number of fruits and seeds produced, which were 223 modeled with Poisson distributions. To account for subsampling of fruits, we followed Appendix 224 S1 of Stanton-Geddes et al. (2012a), incorporating in our graphical models an additional node 225 representing the total number of fruits sampled. This leads to estimates that are scaled up to the 226 total number of seeds produced per seed planted. Using this model for expression of components 227 of fitness through the lifespan, as well as the dependence of each on components expressed 228 earlier, we obtained unconditional estimates, i.e. the mean and additive genetic variance of the 229 number of seeds produced per seed planted. 230 For each population in each year, we conducted separate aster analyses to estimate 231 VA(W), including as predictors parental (genetic) effects and block. As in Sheth et al. (2018), 232 only parental (genetic) effects were treated as random as the basis for estimating VA(W). Models 233 including factors whose levels are considered random (i.e. for which the parameter of interest is 234 variance of the levels' effects) were introduced by Fisher (1918) with the motivation of 235 estimating genetic variance of a quantitative trait. The original statistical methodology relies 236 heavily on the convenient mathematical properties of the assumed Gaussian distribution of the 237 random effects. Geyer et al. Materials and Methods 147 2007; Shaw et 219 al. 2008) we conducted aster analyses, as implemented in the package “aster” (Geyer 2018), 220 using the R 3.5.1 environment (R Core Team 2018). The graphical model for lifetime fitness 221 We regularly recorded fitness components for each planted seed throughout the growing 202 season of the following year, including emergence, survival to flowering, and reproductive 203 output (Table 1). A plant was considered present if cotyledons had emerged, and survival to 204 flowering was scored based on the presence of open flowers or evidence of flowering (e.g., 205 wilted flowers, pedicels, or fruit). At each late-season census (i.e. after flowering), we recorded 206 the number of ripe fruit collected, number of fully elongated but immature fruits collected, 207 Therefore, we retained these plants in censuses after herbivory to account for this late season 213 seed production. Because individuals ranged widely in number of fruits set (1-68), and fruits 214 explosively dehisce at maturity, we were unable to collect all mature fruits or count all seeds 215 each plant produced. Therefore, seed counts were obtained on a subsample of fruits produced. 216 Subsampling was accounted for in the statistical analyses (see below). 217 Estimates of W̅ and VA(W) for each population in each year (Geyer et al. 2007; Shaw et 219 al. 2008) we conducted aster analyses, as implemented in the package “aster” (Geyer 2018), 220 using the R 3.5.1 environment (R Core Team 2018). The graphical model for lifetime fitness 221 (Fig. 1) included seedling emergence and survival of plants to flowering, both of which were 222 Estimates of W̅ and VA(W) for each population in each year (Geyer et al. 2007; Shaw et 219 al. 2008) we conducted aster analyses, as implemented in the package “aster” (Geyer 2018), 220 using the R 3.5.1 environment (R Core Team 2018). The graphical model for lifetime fitness 221 (Fig. 1) included seedling emergence and survival of plants to flowering, both of which were 222 10 10 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . Materials and Methods 147 ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint factor and parental (genetic) effects and the interaction between parental effects and year 246 (genotype-by-year) as random factors. We tested fixed factors with likelihood ratio tests of the 247 full model vs. models excluding individual factors. To obtain estimates of additive genetic 248 variance for lifetime fitness, we first conducted separate analyses with sire and dam as random 249 factors, finding both significant and roughly comparable in magnitude. We then constructed 250 models that explicitly equated the variance due to sires with that for dams (i.e. modeling the 251 equal nuclear transmission of each parent to their offspring) and estimated a single variance 252 component for their effects (hereafter parental effects), with sire effects being (see Sheth et al. 253 2018). From separate analyses, also including blocks as fixed effects, we estimated W̅ . 254 For each population-year combination, mean fitness was represented by the estimate for 255 mean fitness (with its standard error) that is the median value among the estimates of mean 256 fitness for all the blocks of a given site. To visualize breeding values for fitness on the 257 measurement scale (i.e. number of seeds produced per seed planted), we performed non-linear 258 transformations of breeding values on the canonical parameter scale to the mean-value parameter 259 scale using a mapping function that adds a component of a fixed effect (see Table 3 for relevant 260 blocks) to each random parental effect (Geyer and Shaw 2013; de Villemereuil et al. 2016). The 261 resulting values represent estimates of breeding values on the mean-value parameter (i.e. 262 measurement scale). Estimates of VA(W) on the measurement scale were obtained via the delta 263 method following Geyer (2019). We obtained asymmetrical 95% confidence intervals using the 264 parametric bootstrap to obtain a bootstrap-t distribution from the difference between original 265 and 100 bootstrapped VA(W) values divided by their standard error (DiCiccio and Efron 1996). Materials and Methods 147 (2013) outline the theoretical and computational problems that 238 attend relaxation of the Gaussian assumption for the random effects, especially with multiple 239 random factors, in aster models and other GLMM, which are explicitly motivated by the need to 240 model cases that do not meet that assumption (see also Bolker et al. 2009). We follow the 241 recommendation of Geyer (2015, slide 3) to designate as random only those factors necessary to 242 address the questions of scientific interest, here, the parental effects, enabling estimation of 243 genetic variance for fitness. In addition, to test whether parental genetic effects differed among 244 years, we jointly analyzed the data for cohorts grown in all three years, including year as a fixed 245 For each population in each year, we conducted separate aster analyses to estimate 231 VA(W), including as predictors parental (genetic) effects and block. As in Sheth et al. (2018), 232 only parental (genetic) effects were treated as random as the basis for estimating VA(W). Models 233 including factors whose levels are considered random (i.e. for which the parameter of interest is 234 variance of the levels' effects) were introduced by Fisher (1918) with the motivation of 235 estimating genetic variance of a quantitative trait. The original statistical methodology relies 236 heavily on the convenient mathematical properties of the assumed Gaussian distribution of the 237 random effects. Geyer et al. (2013) outline the theoretical and computational problems that 238 attend relaxation of the Gaussian assumption for the random effects, especially with multiple 239 random factors, in aster models and other GLMM, which are explicitly motivated by the need to 240 model cases that do not meet that assumption (see also Bolker et al. 2009). We follow the 241 recommendation of Geyer (2015, slide 3) to designate as random only those factors necessary to 242 address the questions of scientific interest, here, the parental effects, enabling estimation of 243 genetic variance for fitness. In addition, to test whether parental genetic effects differed among 244 years, we jointly analyzed the data for cohorts grown in all three years, including year as a fixed 245 11 11 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. Materials and Methods 147 CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint were calculated for predicted changes in fitness ( VA(W) W ̅̅̅ ) following Geyer (2019). Apart from the 269 possibility of seed dormancy, our fitness records cover the entire life history of individual plants, 270 and therefore represent estimates of total absolute fitness over individual life spans. 271 To compare the genetic effects on fitness (breeding values) between years, and the 272 potential for temporal fitness trade-offs, we obtained breeding values for each sire in each year 273 and plotted each sire’s value in one year against its breeding value in another for all three 274 combinations of years. We also calculated Pearson’s product-moment correlation coefficients 275 between inferred breeding values expressed in different years. We emphasize that these 276 correlations do not account for the uncertainty associated with inferred breeding values; we 277 present them only as a coarse description of association between breeding values expressed in 278 different years. All data and scripts for the results reported here are available at 279 https://github.com/mason-kulbaba/adaptive-capacity.git 280 281 Results 282 Expression of additive genetic variance for lifetime fitness 283 We found highly significant additive genetic variance for lifetime fitness, VA(W), in all 284 three years for all three populations, as reflected by significant parental effects in all models 285 (Table 2); block effects were also significant in all cases, indicating spatial variation within sites 286 in expression of fitness. Estimates of VA(W) varied widely among years and populations, with 287 the smallest and largest estimates of VA(W) found in consecutive years for the Grey Cloud 288 population (VA(W) = 0.631 in 2016, and 6.491 in 2017). The statistical detection of VA(W) 289 . CC-BY 4.0 International license a y p ) , g p y p p p p y were calculated for predicted changes in fitness ( VA(W) W ̅̅̅ ) following Geyer (2019). Apart from the 269 possibility of seed dormancy, our fitness records cover the entire life history of individual plants, 270 and therefore represent estimates of total absolute fitness over individual life spans. Materials and Methods 147 266 Predicted changes in mean fitness from FTNS ( VA(W) W ̅̅̅ ) were added to the estimates of mean 267 factor and parental (genetic) effects and the interaction between parental effects and year 246 (genotype-by-year) as random factors. We tested fixed factors with likelihood ratio tests of the 247 full model vs. models excluding individual factors. To obtain estimates of additive genetic 248 variance for lifetime fitness, we first conducted separate analyses with sire and dam as random 249 factors, finding both significant and roughly comparable in magnitude. We then constructed 250 models that explicitly equated the variance due to sires with that for dams (i.e. modeling the 251 equal nuclear transmission of each parent to their offspring) and estimated a single variance 252 component for their effects (hereafter parental effects), with sire effects being (see Sheth et al. 253 2018). From separate analyses, also including blocks as fixed effects, we estimated W̅ . 254 For each population-year combination, mean fitness was represented by the estimate for 255 mean fitness (with its standard error) that is the median value among the estimates of mean 256 fitness for all the blocks of a given site. To visualize breeding values for fitness on the 257 measurement scale (i.e. number of seeds produced per seed planted), we performed non-linear 258 transformations of breeding values on the canonical parameter scale to the mean-value parameter 259 scale using a mapping function that adds a component of a fixed effect (see Table 3 for relevant 260 blocks) to each random parental effect (Geyer and Shaw 2013; de Villemereuil et al. 2016). The 261 resulting values represent estimates of breeding values on the mean-value parameter (i.e. 262 12 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . Materials and Methods 147 271 To compare the genetic effects on fitness (breeding values) between years, and the 272 potential for temporal fitness trade-offs, we obtained breeding values for each sire in each year 273 and plotted each sire’s value in one year against its breeding value in another for all three 274 combinations of years. We also calculated Pearson’s product-moment correlation coefficients 275 between inferred breeding values expressed in different years. We emphasize that these 276 correlations do not account for the uncertainty associated with inferred breeding values; we 277 present them only as a coarse description of association between breeding values expressed in 278 different years. All data and scripts for the results reported here are available at 279 https://github.com/mason-kulbaba/adaptive-capacity.git 280 281 Expression of additive genetic variance for lifetime fitness 283 We found highly significant additive genetic variance for lifetime fitness, VA(W), in all 284 three years for all three populations, as reflected by significant parental effects in all models 285 (Table 2); block effects were also significant in all cases, indicating spatial variation within sites 286 in expression of fitness. Estimates of VA(W) varied widely among years and populations, with 287 the smallest and largest estimates of VA(W) found in consecutive years for the Grey Cloud 288 population (VA(W) = 0.631 in 2016, and 6.491 in 2017). The statistical detection of VA(W) 289 13 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint (Table 3) implies capacity for genetic response to natural selection based on standing VA(W). 290 Probability distributions of breeding values for each of the nine cases are displayed in Fig. 2. 291 Significant genotype-by-year interactions for each population (Table 2) indicated that the 292 expression of parental genetic effects varied among years. Year-specific breeding values within 293 each population revealed large differences between years in family-specific expressions of total 294 lifetime fitness (Fig. 3). Materials and Methods 147 Breeding values exhibited small to moderate correlations between years, 295 with two cases of modest negative correlations: 2015 vs. 2016 in McCarthy Lake and 2016 vs. 296 2017 in CERA (Fig. 3). The findings of significant genotype-by-year interactions indicate that 297 families contributing disproportionately to the next generation often differ among years. 298 (Table 3) implies capacity for genetic response to natural selection based on standing VA(W). 290 Probability distributions of breeding values for each of the nine cases are displayed in Fig. 2. 291 Significant genotype-by-year interactions for each population (Table 2) indicated that the 292 expression of parental genetic effects varied among years. Year-specific breeding values within 293 each population revealed large differences between years in family-specific expressions of total 294 lifetime fitness (Fig. 3). Breeding values exhibited small to moderate correlations between years, 295 with two cases of modest negative correlations: 2015 vs. 2016 in McCarthy Lake and 2016 vs. 296 2017 in CERA (Fig. 3). The findings of significant genotype-by-year interactions indicate that 297 families contributing disproportionately to the next generation often differ among years. 298 Mean fitness and predicted changes in mean fitness 299 Mean fitness and predicted changes in mean fitness 299 The inclusion of block effects in all fixed-effects aster models for W̅ explained significantly 300 more variation than models without (Grey Cloud and McCarthy Lake: all test df = 7, all test 301 deviance > 4.26, all P < 0.05; CERA: all test df = 3, all test deviance > 27.92, all P < 0.0001). 302 The inclusion of block effects in all fixed-effects aster models for W̅ explained significantly 300 more variation than models without (Grey Cloud and McCarthy Lake: all test df = 7, all test 301 deviance > 4.26, all P < 0.05; CERA: all test df = 3, all test deviance > 27.92, all P < 0.0001). 302 As with estimates of VA(W), estimates of mean fitness varied among populations and years 303 (Table 3; Fig. 4). Mean fitness was highest in the McCarthy Lake site in 2015 (mean and 304 standard error: 3.528 ± 1.227) and lowest in Grey Cloud in 2016 (0.640 ± 0.129). This and one 305 other instance (CERA mean and standard error: 0.725 ± 0.112) indicating declining populations 306 (i.e. W̅ < 1) were found in the same year. Materials and Methods 147 307 The estimates predicting change in mean fitness from Fisher’s Fundamental Theorem 308 ( VA(W) W ̅̅̅ ), are biologically meaningful, ranging from increases of just under 1 seed per individual 309 seed planted to about 6, varying with the magnitude of both W̅ and VA(W). The largest increase 310 in mean fitness was predicted for the progeny of Grey Cloud plants after 2017 (mean increase of 311 The estimates predicting change in mean fitness from Fisher’s Fundamental Theorem 308 ( VA(W) W ̅̅̅ ), are biologically meaningful, ranging from increases of just under 1 seed per individual 309 seed planted to about 6, varying with the magnitude of both W̅ and VA(W). The largest increase 310 in mean fitness was predicted for the progeny of Grey Cloud plants after 2017 (mean increase of 311 14 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint 6.122 seeds per plant) and the smallest for progeny of McCarthy Lake plants after 2016 (mean 312 increase of 0.834 seeds per plant). Notably, we identified two predictions of evolutionary rescue 313 for the progeny generations of 2016 in Grey Cloud and CERA. This predicted increase in mean 314 fitness is consistent with population sustainability and growth of the progeny generations (Table 315 3; Fig. 4). 316 Discussion 317 The immediate capacity for population-level adaptation is strongly dependent on the 318 presence and magnitude of additive genetic variance for lifetime fitness (Fisher 1930; Ewens 319 2004). Our study detected significant and substantial capacity for immediate adaptation through 320 current natural selection on standing levels of VA(W) in three populations in three years. Among 321 these nine cases, we detected two instances of populations declining numerically, and in those 322 cases obtained predictions of evolutionary rescue increasing mean fitness to levels consistent 323 with maintaining or increasing population size. Materials and Methods 147 Genotype-by-year interactions indicated that 324 genetic effects on fitness differed among years. These interactions reflect differences among 325 years both in the magnitude of VA(W) and in genotypic fitness rankings. Predicted change in 326 mean fitness also varied among years, suggesting that the capacity for adaptation is strongly 327 influenced by temporal environmental variation. Below, we discuss the importance of these 328 findings and consider how our results from direct study of the adaptive process in the wild 329 illuminate the potential for ongoing adaptation. We also relate our findings to the potential for 330 evolutionary rescue of declining populations. Finally, we describe how our experimental and 331 analytical approaches overcome obstacles commonly associated with evaluating adaptive 332 6.122 seeds per plant) and the smallest for progeny of McCarthy Lake plants after 2016 (mean 312 increase of 0.834 seeds per plant). Notably, we identified two predictions of evolutionary rescue 313 for the progeny generations of 2016 in Grey Cloud and CERA. This predicted increase in mean 314 fitness is consistent with population sustainability and growth of the progeny generations (Table 315 3; Fig. 4). 316 The immediate capacity for population-level adaptation is strongly dependent on the 318 presence and magnitude of additive genetic variance for lifetime fitness (Fisher 1930; Ewens 319 2004). Our study detected significant and substantial capacity for immediate adaptation through 320 current natural selection on standing levels of VA(W) in three populations in three years. Among 321 these nine cases, we detected two instances of populations declining numerically, and in those 322 cases obtained predictions of evolutionary rescue increasing mean fitness to levels consistent 323 with maintaining or increasing population size. Genotype-by-year interactions indicated that 324 genetic effects on fitness differed among years. These interactions reflect differences among 325 years both in the magnitude of VA(W) and in genotypic fitness rankings. Predicted change in 326 mean fitness also varied among years, suggesting that the capacity for adaptation is strongly 327 influenced by temporal environmental variation. Below, we discuss the importance of these 328 findings and consider how our results from direct study of the adaptive process in the wild 329 illuminate the potential for ongoing adaptation. We also relate our findings to the potential for 330 evolutionary rescue of declining populations. Materials and Methods 147 It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint Expression of additive genetic variance for lifetime fitness 334 Our detection of prevalent and non-negligible additive genetic variance for lifetime 335 fitness, characterized as the number of seeds produced per seed planted, indicates differential 336 genetic contributions to the progeny generation and, via the FTNS, clear capacity for ongoing 337 adaptation under natural selection in each year. We estimated significant VA(W) in the home site 338 of all three populations in all three years of study (Fig. 2), albeit at varying magnitudes. These 339 results contrast with those of several studies that conclude limited capacity for adaptation, 340 through estimates of heritability of fecundity (e.g. Kruuk et al. 2000), or other individual fitness 341 components (Mousseau and Roff 1987; Matos et al. 2000; McCleery et al. 2004; Teplitsky et al. 342 2009). We suggest that these studies can be reconciled with our findings in part by recognizing 343 that even when additive genetic variances of individual fitness components are modest, estimates 344 of VA(W) may be considerably larger due to the compounding of fitness components through an 345 individual’s life-history (Shaw et al. 2008; Shaw and Geyer 2010). This compounding plays out 346 over the entire life-history, regardless of the extent to which there are genetically based trade- 347 offs between individual components of fitness (e.g. Rose and Charlesworth 1981). Our analysis 348 of total lifetime fitness through aster modeling enabled us to account for the dependent nature of 349 sequential fitness components in our estimates of total fitness. VA(W) may also be obscured by 350 environmental variation (Price and Schluter 1991). 351 O fi di f l t i t ti b t t d b titi d i t 352 Our finding of prevalent interactions between genotype and year can be partitioned into 352 two aspects of environmental dependence of genetic expression (Falconer 1952). First, 353 correlations between years of family-specific breeding values for lifetime fitness were generally 354 low and, in two cases, slightly negative (Fig. 3). Thus, genotypic contributions of offspring in 355 one year are not predictive of contributions in another year. Materials and Methods 147 Finally, we describe how our experimental and 331 analytical approaches overcome obstacles commonly associated with evaluating adaptive 332 capacity in the wild, and we advocate for the broader implementation of these approaches. 333 The immediate capacity for population-level adaptation is strongly dependent on the 318 presence and magnitude of additive genetic variance for lifetime fitness (Fisher 1930; Ewens 319 2004). Our study detected significant and substantial capacity for immediate adaptation through 320 current natural selection on standing levels of VA(W) in three populations in three years. Among 321 these nine cases, we detected two instances of populations declining numerically, and in those 322 cases obtained predictions of evolutionary rescue increasing mean fitness to levels consistent 323 with maintaining or increasing population size. Genotype-by-year interactions indicated that 324 genetic effects on fitness differed among years. These interactions reflect differences among 325 years both in the magnitude of VA(W) and in genotypic fitness rankings. Predicted change in 326 mean fitness also varied among years, suggesting that the capacity for adaptation is strongly 327 influenced by temporal environmental variation. Below, we discuss the importance of these 328 findings and consider how our results from direct study of the adaptive process in the wild 329 illuminate the potential for ongoing adaptation. We also relate our findings to the potential for 330 evolutionary rescue of declining populations. Finally, we describe how our experimental and 331 analytical approaches overcome obstacles commonly associated with evaluating adaptive 332 capacity in the wild, and we advocate for the broader implementation of these approaches. 333 15 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Materials and Methods 147 Interestingly, the two strongest 356 Our finding of prevalent interactions between genotype and year can be partitioned into 352 two aspects of environmental dependence of genetic expression (Falconer 1952). First, 353 correlations between years of family-specific breeding values for lifetime fitness were generally 354 low and, in two cases, slightly negative (Fig. 3). Thus, genotypic contributions of offspring in 355 one year are not predictive of contributions in another year. Interestingly, the two strongest 356 16 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint genetic correlations between years were not for consecutive years. These modest between-year 357 genetic correlations suggest that the response to selection may not be accompanied by directiona 358 change in the frequency of the same alleles over multiple years. Second, for each population, 359 estimates of VA(W) differed strikingly among years (Table 3), though we acknowledge 360 substantial uncertainty in the estimates. Nevertheless, these differences in estimated VA(W) 361 suggest that the immediate capacity for ongoing adaptation varies among years. Almost 362 certainly, many aspects of environment, whether slight or major, differed among the years at 363 each site; to determine which of these influenced genetic disparities in fitness would require 364 experimental manipulation of particular aspects of the environment, as conducted by Torres- 365 Martinez et al. (2019). 366 Whereas we found considerable VA(W) for multiple populations, explanations for the 367 maintenance of such potential variation has long been elusive (e.g. Burt 1995). One potential 368 explanation is that trade-offs between individual fitness components maintain additive genetic 369 variation (Barton and Keightley 2002). Our previous work focusing on the Grey Cloud 370 population revealed positive correlations of breeding values between fitness components and 371 also two environments (Sheth et al. 2018). This result suggests that trade-offs (i.e. negative 372 genetic correlations) do not account for the maintenance of observed levels of VA(W). Materials and Methods 147 373 Temporal environmental variation has long been conjectured to maintain genetic 374 variation, but the theoretical conditions that predict this outcome are restrictive (Felsenstein 375 1976). Experiments manipulating environmental variation have led to conflicting conclusions on 376 the role of environmental variation in the maintenance of genetic variation (Mackay 1980; 377 Yeaman et al. 2010; Huang et al. 2015). Through estimates of total lifetime fitness, instead of 378 i di id l t it t f fit lt h d t d t i t l 379 genetic correlations between years were not for consecutive years. These modest between-year 357 genetic correlations suggest that the response to selection may not be accompanied by directiona 358 change in the frequency of the same alleles over multiple years. Second, for each population, 359 estimates of VA(W) differed strikingly among years (Table 3), though we acknowledge 360 substantial uncertainty in the estimates. Nevertheless, these differences in estimated VA(W) 361 suggest that the immediate capacity for ongoing adaptation varies among years. Almost 362 certainly, many aspects of environment, whether slight or major, differed among the years at 363 each site; to determine which of these influenced genetic disparities in fitness would require 364 experimental manipulation of particular aspects of the environment, as conducted by Torres- 365 Martinez et al. (2019). 366 genetic correlations between years were not for consecutive years. These modest between-year 357 genetic correlations suggest that the response to selection may not be accompanied by directiona 358 change in the frequency of the same alleles over multiple years. Second, for each population, 359 estimates of VA(W) differed strikingly among years (Table 3), though we acknowledge 360 substantial uncertainty in the estimates. Nevertheless, these differences in estimated VA(W) 361 suggest that the immediate capacity for ongoing adaptation varies among years. Almost 362 certainly, many aspects of environment, whether slight or major, differed among the years at 363 each site; to determine which of these influenced genetic disparities in fitness would require 364 experimental manipulation of particular aspects of the environment, as conducted by Torres- 365 Martinez et al. (2019). 366 Whereas we found considerable VA(W) for multiple populations, explanations for the 367 maintenance of such potential variation has long been elusive (e.g. Burt 1995). One potential 368 explanation is that trade-offs between individual fitness components maintain additive genetic 369 variation (Barton and Keightley 2002). Materials and Methods 147 Our previous work focusing on the Grey Cloud 370 population revealed positive correlations of breeding values between fitness components and 371 also two environments (Sheth et al. 2018). This result suggests that trade-offs (i.e. negative 372 genetic correlations) do not account for the maintenance of observed levels of VA(W). 373 17 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint variation in the expression of breeding values within populations (Fig. 2; Fig. 3). Given the short 380 timeframe of our study (i.e. single generation replicated over three years), the impact of novel 381 mutations on lifetime fitness would be negligible. Additional phenomena may also contribute to 382 the conservation of genetic variation (e.g., marginal overdominance; Levene 1953; Gillespie 383 1984), but the differential and variable genetic contribution of families to the following progeny 384 generation across years (Fig. 3) is consistent with a role for temporal environmental variation in 385 impeding depletion of VA(W) by natural selection. These annual variations may contribute to the 386 maintenance of the appreciable levels of observed VA(W) in our study. 387 Our measures of immediate capacity for adaptation can alternatively be expressed as 388 evolvability (sensu Houle 1992, see Table 3) to yield the change in absolute mean fitness as a 389 proportion of the current absolute mean fitness. This measure of predicted proportional change 390 has been advocated particularly in the context of trait evolution. In the context of predicting 391 change in mean fitness, we view the original formulation of the FTNS as more informative 392 because it is directly interpretable demographically as the predicted change in the per capita 393 contribution of offspring to the population. In contrast, there is no direct demographic 394 interpretation of the corresponding evolvabilities. 395 Mean fitness and predicted changes in mean fitness 396 variation in the expression of breeding values within populations (Fig. Materials and Methods 147 2; Fig. 3). Given the short 380 timeframe of our study (i.e. single generation replicated over three years), the impact of novel 381 mutations on lifetime fitness would be negligible. Additional phenomena may also contribute to 382 the conservation of genetic variation (e.g., marginal overdominance; Levene 1953; Gillespie 383 1984), but the differential and variable genetic contribution of families to the following progeny 384 generation across years (Fig. 3) is consistent with a role for temporal environmental variation in 385 impeding depletion of VA(W) by natural selection. These annual variations may contribute to the 386 maintenance of the appreciable levels of observed VA(W) in our study. 387 Our measures of immediate capacity for adaptation can alternatively be expressed as 388 evolvability (sensu Houle 1992, see Table 3) to yield the change in absolute mean fitness as a 389 proportion of the current absolute mean fitness. This measure of predicted proportional change 390 has been advocated particularly in the context of trait evolution. In the context of predicting 391 change in mean fitness, we view the original formulation of the FTNS as more informative 392 because it is directly interpretable demographically as the predicted change in the per capita 393 contribution of offspring to the population. In contrast, there is no direct demographic 394 interpretation of the corresponding evolvabilities. 395 Mean fitness and predicted changes in mean fitness 396 Similar to estimates of VA(W), mean fitness and the predicted rate of adaptation across 397 generations varied with population and year. Predictions based on Fisher’s Fundamental 398 Theorem ( VA(W) W ̅̅̅ ) indicated that even the more modest estimates of VA(W) (e.g., Grey Cloud and 399 CERA in 2016; Table 3) were sufficient to increase mean fitness under contemporary natural 400 selection. In the case of the largest estimate of VA(W) (Grey Cloud in 2017), contemporary 401 Similar to estimates of VA(W), mean fitness and the predicted rate of adaptation across 397 generations varied with population and year. Predictions based on Fisher’s Fundamental 398 Theorem ( VA(W) W ̅̅̅ ) indicated that even the more modest estimates of VA(W) (e.g., Grey Cloud and 399 CERA in 2016; Table 3) were sufficient to increase mean fitness under contemporary natural 400 selection. Materials and Methods 147 Therefore, along with 416 Gomulkiewicz and Shaw (2012) we advocate for further empirically-based predictions of 417 adaptation, as presented in our study, to elucidate the potential for evolutionary rescue in the 418 wild. 419 Our assessments of fitness encompass components of fitness expressed across the entire life span 420 of individuals, from each seed planted through to the seeds it produced; these fitness evaluations 421 are thus uncommonly complete. Nevertheless, some aspects of fitness are not included. For 422 example, this study did not account for fitness realized through siring of seeds (male fecundity). 423 selection was predicted to drastically increase fitness to several times that of the parental 402 generation (Table 3, Fig. 4). 403 Our study identified two cases where evolutionary rescue was predicted to increase mean 404 fitness of declining populations to levels consistent with sustainability and even population 405 growth. Mean fitness of both the Grey Cloud and CERA populations during the 2016 growing 406 season was less than 1.0, indicating declining numbers of individuals to the following year. The 407 prediction of genetic change in mean fitness, however, is that fitness in the progeny generation 408 would increase to more than double the current mean fitness. Realization of this prediction 409 would not only prevent further population decline but would increase seed production to result in 410 population growth. Whereas evolutionary rescue has been directly demonstrated in controlled 411 laboratory systems (Bell and Gonzalez 2009, 2011), examples from wild systems are lacking. 412 Beginning with the work of Gomulkiewicz and Holt (1995), mathematical modeling efforts have 413 described the likelihood of evolutionary rescue under various demographic and environmental 414 scenarios, but the promise of evolutionary rescue of declining and threatened populations 415 remains unclear (Bell 2017), as does their long-term sustainability. Therefore, along with 416 Gomulkiewicz and Shaw (2012) we advocate for further empirically-based predictions of 417 adaptation, as presented in our study, to elucidate the potential for evolutionary rescue in the 418 wild. 419 Our assessments of fitness encompass components of fitness expressed across the entire life span 420 of individuals, from each seed planted through to the seeds it produced; these fitness evaluations 421 are thus uncommonly complete. Nevertheless, some aspects of fitness are not included. For 422 example, this study did not account for fitness realized through siring of seeds (male fecundity). Materials and Methods 147 In the case of the largest estimate of VA(W) (Grey Cloud in 2017), contemporary 401 Theorem ( VA(W) W ̅̅̅ ) indicated that even the more modest estimates of VA(W) (e.g., Grey Cloud and 399 CERA in 2016; Table 3) were sufficient to increase mean fitness under contemporary natural 400 selection. In the case of the largest estimate of VA(W) (Grey Cloud in 2017), contemporary 401 18 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint selection was predicted to drastically increase fitness to several times that of the parental 402 generation (Table 3, Fig. 4). 403 Our study identified two cases where evolutionary rescue was predicted to increase mean 404 fitness of declining populations to levels consistent with sustainability and even population 405 growth. Mean fitness of both the Grey Cloud and CERA populations during the 2016 growing 406 season was less than 1.0, indicating declining numbers of individuals to the following year. The 407 prediction of genetic change in mean fitness, however, is that fitness in the progeny generation 408 would increase to more than double the current mean fitness. Realization of this prediction 409 would not only prevent further population decline but would increase seed production to result in 410 population growth. Whereas evolutionary rescue has been directly demonstrated in controlled 411 laboratory systems (Bell and Gonzalez 2009, 2011), examples from wild systems are lacking. 412 Beginning with the work of Gomulkiewicz and Holt (1995), mathematical modeling efforts have 413 described the likelihood of evolutionary rescue under various demographic and environmental 414 scenarios, but the promise of evolutionary rescue of declining and threatened populations 415 remains unclear (Bell 2017), as does their long-term sustainability. Materials and Methods 147 423 In a companion study however Kulbaba and Shaw (in review) found positive genetic 424 selection was predicted to drastically increase fitness to several times that of the parental 402 generation (Table 3, Fig. 4). 403 Our assessments of fitness encompass components of fitness expressed across the entire life span 420 of individuals, from each seed planted through to the seeds it produced; these fitness evaluations 421 are thus uncommonly complete. Nevertheless, some aspects of fitness are not included. For 422 example, this study did not account for fitness realized through siring of seeds (male fecundity). 423 In a companion study, however, Kulbaba and Shaw (in review) found positive genetic 424 Our assessments of fitness encompass components of fitness expressed across the entire life span 420 of individuals, from each seed planted through to the seeds it produced; these fitness evaluations 421 are thus uncommonly complete. Nevertheless, some aspects of fitness are not included. For 422 example, this study did not account for fitness realized through siring of seeds (male fecundity). 423 In a companion study, however, Kulbaba and Shaw (in review) found positive genetic 424 19 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint correlations between lifetime fitness measured as maternal contributions (seeds set) and lifetime 425 fitness measured through paternal contributions (seeds sired), regardless of population density 426 and genetic relatedness among individuals. We therefore expect paternal-specific fitness to scale 427 proportionately with maternal-specific fitness. A second omission from our fitness estimates is 428 the potential for a delayed contribution to fitness due to seed dormancy. Partial surveys of 429 germination in later years found low emergence from seeds planted earlier than the previous Fall, 430 representing a modest proportion of each cohort. Moreover, in increasing populations, (i.e. 431 W̅ > 1), as we found in most cases, delays in an individual’s life-cycle are expected to have a 432 discounting effect on its contributions to fitness (Roughgarden 1996, Eqs. 18.31, p. 331). selection was predicted to drastically increase fitness to several times that of the parental 402 generation (Table 3, Fig. 4). 403 On the 433 temporal scale of adaptation of annual populations between consecutive years, which is our 434 present focus, it is valid to neglect such delays. To address ongoing adaptation on longer time 435 scales, further studies that include instances of delayed germination in estimates of fitness, with 436 appropriate discounting, as in Eck et al. (2015) would be worthwhile. This is especially so, in 437 view of our finding of weak correspondence of genetic selection between consecutive years (Fig. 438 3). 439 Our study has generated precise quantitative predictions of genetic change in mean 440 fitness. However, correspondence between predicted genetic change in mean fitness across 441 generations and the change in mean fitness that is realized remains to be assessed. We are 442 currently evaluating this correspondence to address the accuracy of predictions of the rate of 443 adaptation from Fisher’s FTNS for these populations. 444 Conclusions 445 correlations between lifetime fitness measured as maternal contributions (seeds set) and lifetime 425 fitness measured through paternal contributions (seeds sired), regardless of population density 426 and genetic relatedness among individuals. We therefore expect paternal-specific fitness to scale 427 proportionately with maternal-specific fitness. A second omission from our fitness estimates is 428 the potential for a delayed contribution to fitness due to seed dormancy. Partial surveys of 429 germination in later years found low emergence from seeds planted earlier than the previous Fall, 430 representing a modest proportion of each cohort. Moreover, in increasing populations, (i.e. 431 fitness measured through paternal contributions (seeds sired), regardless of population density 426 and genetic relatedness among individuals. We therefore expect paternal-specific fitness to scale 427 proportionately with maternal-specific fitness. A second omission from our fitness estimates is 428 the potential for a delayed contribution to fitness due to seed dormancy. Partial surveys of 429 germination in later years found low emergence from seeds planted earlier than the previous Fall, 430 representing a modest proportion of each cohort. Moreover, in increasing populations, (i.e. 431 W̅ > 1), as we found in most cases, delays in an individual’s life-cycle are expected to have a 432 discounting effect on its contributions to fitness (Roughgarden 1996, Eqs. 18.31, p. 331). On the 433 temporal scale of adaptation of annual populations between consecutive years, which is our 434 present focus, it is valid to neglect such delays. selection was predicted to drastically increase fitness to several times that of the parental 402 generation (Table 3, Fig. 4). 403 To address ongoing adaptation on longer time 435 scales, further studies that include instances of delayed germination in estimates of fitness, with 436 appropriate discounting, as in Eck et al. (2015) would be worthwhile. This is especially so, in 437 view of our finding of weak correspondence of genetic selection between consecutive years (Fig. 438 3). 439 O t d h t d i tit ti di ti f ti h i 440 Conclusions 445 Whereas numerous authors invoke relentless selection to explain limited heritability of 446 total fitness, or traits closely related to fitness (Kruuk et al. 2000; Coltman et al. 2005; 447 Whereas numerous authors invoke relentless selection to explain limited heritability of 446 total fitness, or traits closely related to fitness (Kruuk et al. 2000; Coltman et al. 2005; 447 20 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint McFarlane et al. 2014; de Villemereuil et al. 2019), our results demonstrate the persistence of 448 high VA(W) despite contemporary selection. Significant interactions between genotype and year 449 reflected modest correlations between years of family-specific breeding values for lifetime 450 fitness, as well as estimates of VA(W) that varied widely across years. Along with estimates of 451 VA(W), mean absolute fitness varied among sites and years. Using Fisher’s Fundamental 452 Theorem, we predicted the rate of adaptation, and two instances of evolutionary rescue from 453 standing VA(W) under contemporary natural selection. Our results reveal the power and utility of 454 Fisher’s Fundamental Theorem to identify the capacity for adaptation, and to generate 455 quantitative predictions for fitness increase in the wild. 456 Our experimental approach in combination with aster analyses for total lifetime fitness 457 provided a direct estimate of VA(W) and fitness change (Shaw 2019). . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint selection was predicted to drastically increase fitness to several times that of the parental 402 generation (Table 3, Fig. 4). 403 Many studies concerning 458 the potential for adaptation rely on indirect estimates of fitness, and often focus on one or a few 459 individual traits or fitness components, thus omitting the role of multiple life-history fitness 460 expressions. Our approach to evaluating total lifetime fitness with aster models provides a more 461 complete picture of fitness and VA(W). We recommend our empirical and analytical strategy for 462 future studies of other organisms having different life-histories to more fully characterize the 463 capacity for ongoing adaptation. 464 21 . 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Insights from population genetics for range 635 limits of a idel distrib ted nati e plant American Jo rnal of Botan 100:744 753 636 Stanton-Geddes, J., R. G. Shaw, and P. Tiffin. 2013. Insights from population genetics for range 635 Stanton-Geddes, J., R. G. Shaw, and P. Tiffin. 2013. Insights from population genetics for range 635 limits of a widely distributed native plant. American Journal of Botany 100:744–753. 636 limits of a widely distributed native plant. American Journal of Botany 100:744–753. 636 Stanton-Geddes, J., R. G. Shaw, and P. Tiffin. 2012a. Interactions between Soil Habitat and 637 Geographic Range Location Affect Plant Fitness. PLOS ONE 7:e36015. 638 Stanton-Geddes, J., R. G. Shaw, and P. Tiffin. 2012a. Literature Cited 466 Interactions between Soil Habitat and 637 , , , Geographic Range Location Affect Plant Fitness. PLOS ONE 7:e36015. 638 Geographic Range Location Affect Plant Fitness. PLOS ONE 7:e36015. 638 Stanton-Geddes, J., P. Tiffin, and R. G. Shaw. 2012b. Role of climate and competitors in limiting 639 fitness across range edges of an annual plant. Ecology 93:1604–1613. 640 29 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint Teplitsky, C., J. A. Mills, J. W. Yarrall, and J. Merilä. 2009. Heritability of fitness compo 641 a wild bird population. Evolution 63:716–726. 642 Thomas, S. C., J. M. Pemberton, and W. G. Hill. 2000. Estimating variance components i 643 natural populations using inferred relationships. Heredity 84:427–436. 644 Torres-Martínez, L., N. McCarten, and N. C. Emery. 2019. The adaptive potential of plan 645 populations in response to extreme climate events. Ecology Letters 22:866–874. 646 Vigueira, C. C., K. M. Olsen, and A. L. Caicedo. 2013. The red queen in the corn: agricul 647 weeds as models of rapid adaptive evolution. Heredity 110:303–311. 648 Winn, A. A. 2004. Natural selection, evolvability and bias due to environmental covarian 649 the field in an annual plant: Bias in phenotypic selection gradients. Journal of 650 Evolutionary Biology 17:1073–1083. 651 Yeaman, S., Y. Chen, and M. C. Whitlock. 2010. No effect of environmental heterogenei 652 the maintenance of genetic variation in wing shape in Drosophila melanogaster: 653 heterogeneity and maintenance of genetic variation. Evolution 64:3398–3408. 654 Yi, X., and A. M. Dean. 2013. Bounded population sizes, fluctuating selection and the tem 655 mode of coexistence. Proceedings of the National Academy of Sciences 110:1694 656 16950. 657 Zhang, X.-S. 2012. Fisher’s geometrical model of fitness landscape and variance in fitnes 658 within a changing environment. Evolution 66:2350–2368. 659 660 Teplitsky, C., J. A. Mills, J. W. Yarrall, and J. Merilä. 2009. Heritability of fitness components in 641 a wild bird population. Evolution 63:716–726. 642 Thomas, S. C., J. M. Pemberton, and W. G. Hill. 2000. Estimating variance components in 643 natural populations using inferred relationships. Heredity 84:427–436. 644 Torres-Martínez, L., N. McCarten, and N. C. Emery. 2019. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint Literature Cited 466 The adaptive potential of plant 645 populations in response to extreme climate events. Ecology Letters 22:866–874. 646 Vigueira, C. C., K. M. Olsen, and A. L. Caicedo. 2013. The red queen in the corn: agricultural 647 weeds as models of rapid adaptive evolution. Heredity 110:303–311. 648 Vigueira, C. C., K. M. Olsen, and A. L. Caicedo. 2013. The red queen in the corn: agricultural 647 weeds as models of rapid adaptive evolution. Heredity 110:303–311. 648 Winn, A. A. 2004. Natural selection, evolvability and bias due to environmental covariance in 649 the field in an annual plant: Bias in phenotypic selection gradients. Journal of 650 Evolutionary Biology 17:1073–1083. 651 Yeaman, S., Y. Chen, and M. C. Whitlock. 2010. No effect of environmental heterogeneity on 652 the maintenance of genetic variation in wing shape in Drosophila melanogaster: 653 heterogeneity and maintenance of genetic variation. Evolution 64:3398–3408. 654 heterogeneity and maintenance of genetic variation. Evolution 64:3398–3408. 654 Yi, X., and A. M. Dean. 2013. Bounded population sizes, fluctuating selection and the tempo and 655 mode of coexistence. Proceedings of the National Academy of Sciences 110:16945– 656 16950. 657 Zhang, X.-S. 2012. Fisher’s geometrical model of fitness landscape and variance in fitness 658 Zhang, X. S. 2012. Fisher s geometrical model of fitness landscape and variance in fitness 658 within a changing environment. Evolution 66:2350–2368. 659 within a changing environment. Evolution 66:2350–2368. 659 30 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint 663 Table 1. Range of dates for collection of fitness expression data, for three sites of Chamaecrista 665 fasciculata, over three consecutive years. 666 Year Site 2015 2016 2017 Grey Cloud June 1 – October 1 June 10 – October 7 June 5 – October 3 McCarthy Lake May 27 – September 25 June 7 – October 11 June 8 – October 5 CERA May 10 – September 23 June 2 – September 29 June 1 – October 23 667 Table 1. Range of dates for collection of fitness expression data, for three sites of Chamaecrista 665 f i l t th ti 666 667 31 31 . Literature Cited 466 CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint Table 2 668 Table 2. Summary from aster models testing the fixed effects of block and year, the random 669 factors of combined parental effects, and the interaction between parental effects and year on 670 individual lifetime fitness. Statistical significance of predictor variables was assessed using 671 likelihood ratio tests, and random parental effects were assessed from summary output of aster 672 analyses. 673 Model Test df Test deviance P Grey Cloud – G x Year model Block 23 159.91 P < 0.0001 Year 2 47.28 P < 0.0001 Parental effects -- -- P < 0.0001 Year x Parental effects -- -- P < 0.0001 McCarthy Lake – G x Year model Block 23 159.91 P < 0.001 Year 2 47.28 P < 0.001 Parental effects -- -- P < 0.0001 Year x Parental effects -- -- P < 0.0001 CERA – G x Year model Block 11 189.80 P < 0.0001 Year 2 58.95 P < 0.0001 Parental effects -- -- P < 0.0001 Year x Parental effects -- -- P < 0.0001 674 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint Table 2 668 674 32 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint Table 3 second generation determined for each of three sites and three years of Chamaecrista fasciculata. Block used to 678 estimate mean fitness indicated in parentheses after year. 679 Site/Year (Block) Number of Seeds Planted VA(W) (SE) W̅ (SE) VA(W) W ̅̅̅ (SE) Predicted Fitness of Gen. 2 Grey Cloud Dunes 2015 (Block 6) 3658 1.858 (0.558, 3.021) 1.11 (0.17) 1.67 (0.23) 2.78 2016 (Block 6) 3660 0.830 (0.193, 1.410) 0.64 (0.13) 1.30 (0.14) 1.94 2017 (Block 7) 3660 6.491 (1.365, 9.161) 1.06 (0.34) 6.11 (2.32) 7.17 McCarthy Lake 2015 (Block 4) 3445 3.528 (1.393, 16.503) 3.02 (1.23) 1.17 (0.89) 4.19 2016 (Block 5) 3480 1.572 (0.563, 5.103) 1.88 (0.57) 0.84 (0.18) 2.72 2017 (Block 6) 3516 1.410 (0.610, 2.157) 1.08 (0.18) 1.31 (0.18) 2.39 33 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint CERA 2015 (Block 1) 1845 3.204 (0.983, 6.922) 1.80 (0.24) 1.79 (0.65) 3.58 2016 (Block 2) 1750 0.856 (0.295, 2.301) 0.73 (0.11) 1.18 (0.13) 1.91 2017 (Block 3) 1750 1.573 (0.383, 3.694) 1.24 (0.25) 1.27 (0.49) 2.51 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint 34 34 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint 3 Figure 1 (A) Graphical model used to estimate lifetime fitness. Each node represents a fitness component and 4 therefore response variable, and arrows represent conditional distributions. Table 3 Probability of germination and 5 flowering (0 or 1; Bernoulli distribution), and total number of fruits and seeds produced (Poisson distribution). 6 Additional node to account for subsampling of total fruit production (not shown) also followed a Poisson 7 distribution. (B) Map of three experimental populations. Seed photos from Anna Peschel and remaining photos 8 by MWK. 9 Figure 1 (A) Graphical model used to estimate lifetime fitness. Each node represents a fitness component and Figure 1 (A) Graphical model used to estimate lifetime fitness. Each node represents a fitness component and 684 therefore response variable, and arrows represent conditional distributions. Probability of germination and 685 flowering (0 or 1; Bernoulli distribution), and total number of fruits and seeds produced (Poisson distribution). 686 Additional node to account for subsampling of total fruit production (not shown) also followed a Poisson 687 distribution. (B) Map of three experimental populations. Seed photos from Anna Peschel and remaining photos 688 by MWK. 689 Figure 1 (A) Graphical model used to estimate lifetime fitness. Each node represents a fitness component and 684 therefore response variable, and arrows represent conditional distributions. Probability of germination and 685 flowering (0 or 1; Bernoulli distribution), and total number of fruits and seeds produced (Poisson distribution). 686 Additional node to account for subsampling of total fruit production (not shown) also followed a Poisson 687 distribution. (B) Map of three experimental populations. Seed photos from Anna Peschel and remaining photos 688 by MWK. 689 35 35 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint 690 691 Figure 2. Additive genetic variation for lifetime fitness for three populations of Chamaecrista fasciculata, in 692 three consecutive years. Lines represent probability density distributions of estimated breeding values for 693 lifetime fitness (seeds set). 694 Figure 2. Additive genetic variation for lifetime fitness for three populations of Chamaecrista fasciculata, in 692 three consecutive years. Lines represent probability density distributions of estimated breeding values for 693 lifetime fitness (seeds set). 694 Figure 2. Additive genetic variation for lifetime fitness for three populations of Chamaecrista fasciculata, in 692 three consecutive years. Table 3 Lines represent probability density distributions of estimated breeding values for 693 lifetime fitness (seeds set). 694 36 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint 695 ure 3. Paternal family-specific breeding values for lifetime fitness (seeds set) compared across three nsecutive years in three populations of Chamaecrista fasciculata. Pearson correlation coefficients do not ount for error associated with estimates of breeding values and are therefore presented as a coarse Figure 3. Paternal family-specific breeding values for lifetime fitness (seeds set) compared across three 697 Figure 3. Paternal family-specific breeding values for lifetime fitness (seeds set) compared across three 697 consecutive years in three populations of Chamaecrista fasciculata. Pearson correlation coefficients do not 698 account for error associated with estimates of breeding values and are therefore presented as a coarse 699 description of the interannual relationship between breeding values. Please note differences in scale across 700 panels. 701 37 37 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 18, 2019. ; https://doi.org/10.1101/601682 doi: bioRxiv preprint 702 Figure 4. Estimated mean fitness of parental (circles with standard errors) and progeny generations (triangles 703 with standard errors) for three populations of Chamaecrista fasciculata in three consecutive years. Horizontal 704 dashed lines represent mean fitness of 1, indicating individual replacement and population stability. 705 706 Figure 4. Table 3 Estimated mean fitness of parental (circles with standard errors) and progeny generations (triangles 703 with standard errors) for three populations of Chamaecrista fasciculata in three consecutive years. Horizontal 704 dashed lines represent mean fitness of 1, indicating individual replacement and population stability. 705 706 38 38
https://openalex.org/W2967043313	https://www.e3s-conferences.org/articles/e3sconf/pdf/2019/36/e3sconf_spbwosce2019_02111.pdf	English	null	Logistic tools for optimization of the regional distribution center	E3S web of conferences	2,019	cc-by	2,847	Logistic tools for optimization of the regional distribution center Elena Makovetskaya1,* 1Tyumen Industrial University, Volodarskogo str., 38, Tyumen, 625000, Russia Abstract. The paper proposes the use of the integrated logistic approach, application of the cross-functional logistic coordination and integration in managing processes of the distribution center that promotes both to minimization of expenses and enhancing the effective functioning of the distribution center in the supply chain. The SMED analysis of logistic processes and Value Stream Mapping make it possible to identify inefficient operations and to reduce the duration of a logistics cycle. The researches of processes conducted by the author with the use of the logistic tools allowed revealing the reserves and making recommendations on how to enhance the effectiveness of the regional distribution center. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). * Corresponding author: Makov27EG@mail.ru https://doi.org/10.1051/e3sconf /201911002111 https://doi.org/10.1051/e3sconf /201911002111 , 0 2019) E3S Web of Conferences ( 110 -2018 SPbWOSCE 2111 1 Introduction In the conditions of market economy, the use of tools of logistics by economic entities is represented relevant as it promotes the forming of competitive advantages of the company. According to D.J. Bowersox, V.I. Sergeeva, in modern business the significant changes, which are characterized by the fact that not the companies but supply chains compete among themselves, happen [1, 2]. The costs for performance of the logistic functions and operations make from 35% to 48% of all expenses of the company in the sphere of retail trade. In the structure of logistic expenses in various branches of industry of economically developed countries 20-40% are the share of inventory management, transportation costs represent 15-35%, expenses on administrative and managerial functions represent 9-14% [2]. The work of the modern distribution center is characterized by the increase in the number of logistic operations, the increasing complexity of logistic functions, multiproduct deliveries and shipments. JSC Tander (trading network "Magnit") includes 37 distribution centers, 36 motor transport enterprises, more than 18 thousand shops of different formats in 65 regions of Russia [3]. In 2017 the Tyumen Distribution Centre (TDC) was open. Its total area is more than 40 thousand sq.m. and it is one of the largest multifunctional logistics centers in the Tyumen region. TDC relates to the objects of class "A" and it is characterized by high level technical equipment and standardization [4]. The researches of logistic processes conducted in TDC showed that the storage facilities are used to their maximum limit and there are inefficient logistic processes. The question of finding the internal , 0 2019) E3S Web of Conferences ( 110 -2018 SPbWOSCE 2111 , 0 2019) E3S Web of Conferences ( 110 -2018 SPbWOSCE 2111 https://doi.org/10.1051/e3sconf /201911002111 reserves to optimize the logistic expenses at increasing intensity of streams and shipments without decreasing performance indicators of activity of Tyumen Distribution Centre is particularly acute for the company. reserves to optimize the logistic expenses at increasing intensity of streams and shipments without decreasing performance indicators of activity of Tyumen Distribution Centre is particularly acute for the company. 2 Methods The general methodological principles of a research of a logistics system and processes of the distribution center include [5].  The system approach, which is manifested in the consideration of all participants, elements of logistic processes as interconnected and interacting in a logistics system towards a common goal.  The principle of total expenses is based on accounting of total logistics costs at management of material and related information, financial, service streams throughout all logistic chain. The minimum of the overall logistic costs is considered as one of the main in optimizing of logistics processes and systems.  The principle of total expenses is based on accounting of total logistics costs at management of material and related information, financial, service streams throughout all logistic chain. The minimum of the overall logistic costs is considered as one of the main in optimizing of logistics processes and systems.  The principle of logistics coordination and integration consists in the achievement of the coordinated, integrated participation of all links of the logistics system (chain) in the management of material and related streams.  The principle of logistics coordination and integration consists in the achievement of the coordinated, integrated participation of all links of the logistics system (chain) in the management of material and related streams.  The principle of modeling and information and computer support. Various models are used in the analysis and optimization of processes and elements in logistics systems: mathematical, economic-mathematical, graphic, imitating, etc.  The principle of modeling and information and computer support. Various models are used in the analysis and optimization of processes and elements in logistics systems: mathematical, economic-mathematical, graphic, imitating, etc.  The principle of stability and adaptability. The logistics system has to work steadily at tolerances of parameters and factors of the environmental factors (for example, fluctuation of orders from shops, changes of conditions of deliveries of suppliers, etc.). The logistics system of the distribution center has to adapt to new conditions, changing intensity of logistic streams, loading of the equipment and other criteria of optimization at the considerable fluctuations of factors.  The principle of a humanization of functions and technology solutions in logistics systems causes compliance of logistic processes to ecological, social requirements, etc.  The principle of a humanization of functions and technology solutions in logistics systems causes compliance of logistic processes to ecological, social requirements, etc. 2 Methods The methodology of system approach and the above-mentioned principles were applied within the research of logistic processes of the distribution center. The methodology of system approach and the above-mentioned principles were applied within the research of logistic processes of the distribution center. Processing, the analysis of the statistical and logistic information connected with functioning of the distribution center was carried out by methods of mathematical statistics, calculation and analytical methods, forecasting methods [6, 7]. The functional logistic cycle is the basis of the integrated logistics and management of supply chains. Regardless of the complexity of logistics system in general, it is necessary to investigate a configuration of a separate logistic cycle for identification of the major interrelations and lines of control. Time intervals of the separate operations entering a functional logistic cycle can be random value and all cycle is the random value submitting to a certain law of distribution. In a general view, the problem of optimization of duration of a functional logistic cycle can be presented [8, 9]. • for the average value of time of a functional logistic cycle (Т ): min, 1    n i i T Т (1) (1)  for the standard deviation of the duration of separate operations of a logistic cycle (a2T):  for the standard deviation of the duration of separate operations of a logistic cycle (a2T): 2 2 , 0 2019) E3S Web of Conferences ( 110 -2018 SPbWOSCE 2111 https://doi.org/10.1051/e3sconf /201911002111 min, 2 1 2 2        j i j i ij n i i Т      (2) (2) where Ti .ai – the average value and the standard deviation of operation i; ƞij – correlation coefficient between operation i and operation j of the logistic cycle. The probabilistic interpretation of a logistic cycle allows to determine its duration Т0 with the set probability. where Ti .ai – the average value and the standard deviation of operation i; ƞij – correlation coefficient between operation i and operation j of the logistic cycle. The probabilistic interpretation of a logistic cycle allows to determine its duration Т0 with the set probability. p p j g y obabilistic interpretation of a logistic cycle allows to determine its duration Т0 with probability. 3 Results and Discussion The researches of the logistic operations and processes in a zone of the main warehouse with use of the tools offered above by the author were conducted for the purpose of the identification of the bottlenecks in the logistics system and the optimization of the logistic processes of the Tyumen distribution center. The duration of the logistic processes of the completion of the orders, shipments of goods to consumers in TDC directly depend on the process of acceptance of the transport returnable container. Logistic process includes the operations from the moment of the installation of the car under unloading from the returnable container, and finishing with conveyance of transport container to the zones of the completion of the orders or to the zone of repair and processing. The observations were carried out behind unloading more than 400 transport trucks, 87 thermoboxes and 112 thermal covers, only the operations on opening the gates were steadily carried out (figure 2). Fig. 2. The duration of the operations the acceptance area of the transport returnable container. . 2. The duration of the operations the acceptance area of the transport returnable container. The following problems in TDC have been revealed as a result of the made observations and the analysis of logistic operations: - the repeated check of internal contents of transport container increases the time of istic cycle; - the repeated check of internal contents of transport container increases the time logistic cycle; g y - there is no information on the expected receipt of container on DC (quantity, state); - not sorted salvage in transport returnable container is led to the long acceptance; - the congestion of the DC's zone of unloading returnable container. Results of mapping of process of the completion of the order, the loss of time are presented in table 1. Results of mapping of process of the completion of the order, the loss of time are presented in table 1. The effectiveness coefficient of process of the completion of the order varies from 0.69 to 0.83. The effectiveness coefficient of process of the completion of the order varies from 0.69 to 0.83. Table 1. The analysis of the losses of time in the course of the completion. Table 1. The analysis of the losses of time in the course of the completion. КО General duration of a task, sec. Useful time, sec. Кеf Losses of 1 sort, sec. 2 Methods , 0 Т ра х Т Т   (3) (3) where хр – the indicator of normal distribution corresponding to probability Р. The SMED analysis and Value Stream Mapping were tested by the author and are recommended for the application in the distribution center as the tools of the analysis of logistic processes [10, 11]. Value Stream Map (VSM) allows visualizing logistic process and revealing the losses of time increasing duration of logistic cycles. Stages of Value Stream Mapping are presented in figure 1. Fig. 1. Stages of Value Stream Mapping. Fig. 1. Stages of Value Stream Mapping. It is necessary to pass all stream from the beginning to the end for formation of understanding of borders of process and an essence of the studied operations prior to mapping. Measurements of time of each block of the chosen logistic process or operation are reflected on the current state VSM. It is necessary to structure the duration of operation for value added time (vat) and non-value added time (nvat). The number of participants in each block/operation are surely fixed in the monitoring process and the mapping of current state VSM. The problems and factors increasing time of the duration of operation, undermining the quality of goods or caused damage, wear of the equipment are also fixed. By results of measurements of time and their fixing in the current state VSM, the effectiveness coefficient of a stream is computed. , max    nvat vat Кef (4) (4) The mapping of "future state" VCM of the process is directed to the elimination of sources of losses and idle times. The development of the actions has to be directed to the solution of the problem revealed by results of mapping which "delays" the process. Each action assumes the presence of the responsible official for realization, term of realization and the expected result. 3 , 0 2019) E3S Web of Conferences ( 110 -2018 SPbWOSCE 2111 https://doi.org/10.1051/e3sconf /201911002111 3 Results and Discussion Losses of 2 sort, sec. All losses, sec. Share of losses in the general time, % 1 647 446 0.69 68 133 201 31.1 2 788 651 0.83 64 73 137 17.4 3 1205 991 0.82 141 73 214 17.8 4 961 790 0.82 75 96 171 17.8 5 1181 878 0.74 145 158 303 25.7 https://doi.org/10.1051/e3sconf /201911002111 , 0 2019) E3S Web of Conferences ( 110 -2018 SPbWOSCE 2111 6 651 473 0.73 82 96 178 27.3 7 866 610 0.70 92 164 256 29.6 8 1017 780 0.77 120 117 237 23.3 The statistical processing of the results of the observations of the process of loading showed that the average duration of loading of one unit of the transport returnable container is 28.62 sec. (table 2). The statistical processing of the results of the observations of the process of loading showed that the average duration of loading of one unit of the transport returnable container is 28.62 sec. (table 2). able 2. The results of the statistical processing of time observations on the area of loading of goods in the Tyumen distribution center. Table 2. The results of the statistical processing of time observations on the area of loading of goods in the Tyumen distribution center. interval Xi amount of supervisions loading transport tara (ТТ) share dispersion lower upper duration average load of one ТТ 12 26 7 30 554 18.5 0.46 215.64 26.1 40 6.95 26 820 31.5 0.40 187.76 40.1 54 6.95 5 234 46.8 0.08 36.11 54.1 70 7.95 4 252 63 0.06 26.28 Total 65 1860 28.62 1 465.79 Table 2. The results of the statistical processing of time observations on the area of loading of goods in the Tyumen distribution center. The logistic process of loading of goods in TDC is characterized by high intensity of work, a maximum load on the worker and it is necessary to look for other reserves of cost saving of time for operations and observance of standards for loading of goods in a transport returnable container. 4 Conclusion With the growth of the competition and in connection with the increasing stream of goods, in general, it is necessary for the regional distribution center: • to build the effective model of the organization of all logistic process on the basis of interfunctional coordination of TDC and the retail network. • to build the effective model of the organization of all logistic process on the basis of interfunctional coordination of TDC and the retail network. • The information on quantity and condition of transport returnable container shipped from the branch stores in TDC has to be transferred online to information system of the Tyumen distribution center. • The information on quantity and condition of transport returnable container shipped from the branch stores in TDC has to be transferred online to information system of the Tyumen distribution center. • The assembly of transport returnable container by regular working DCs or on the terms of outsourcing with hourly compensation of the collector of transport returnable container will allow to reduce the duration of the logistic process of the completion of the order at the expense of sampling in ready transport returnable container. The average value of effectiveness coefficient of the process of the completion of the order will increase from 0.76 up to 0.87. p • The implementation of RFID technology to replace the technology of shaped coding will allow reducing the duration of the logistic process on all sites of the distribution center. Thus, it is represented expedient to applicate the integrated logistic approach that was approved by the author in the Tyumen distribution center and the combined use of the SMED analysis, VSM that will allow to reveal problems in logistic processes in due time, and also to make operational management decisions on elimination of bottlenecks in logistic chain. 5 5 , 0 2019) E3S Web of Conferences ( 110 -2018 SPbWOSCE 2111 https://doi.org/10.1051/e3sconf /201911002111 References 1. V.V. Dybskaya, V.I. Sergeev, Logistics and Supply Chain Management 2(84), 3-12 (2018) 2. D.J. Bowersox, D.J. Closs, Logistical Management: The Integrated Supply Chain Process (Оlimp-Business, Moscow, 2017) 3. http://magnit-info.ru/about/ 3. http://magnit-info.ru/about/ 4. E.G. Makovetskaya, N.S. Korpusopva, A.A. Gabudina, O.A. Arhipova, A theory and practice of activity of trade organizations are in the Tyumen region, Monograph (TIU, Tyumen, 2017) 5. V.V. Dybskaya, V.I. Sergeev, Logistic (Yurait, Moscow, 2019) 6. L.A. Filimonova, N.K. Skvortsova, IOP Conference Series: Materials Science and Engineering (MSE) 262, 012196 (2017) doi:10.1088/1757-899X/262/1/012196 7. E.G. Makovetskaya, E.G. Yuzikhanova, MATEC Web of Conferences 239, 04026 (2018) doi.org/10.1051/matecconf /201823904026 8. V.S. Lukinsky, A.V. Strimovskaya, Logistics and Supply Chain Management 6 (2017) 9. V.S. Lukinsky, Models and Methods of Theory of Logistic (Peter, St.P., 2007) 10. M. Imai, Gemba Kaizen: A commonsense, low-cost approach to management (Alpina P, Moscow, 2017) 11. D. Jones, J. Womack, Lean Thinking: Banish waste and create wealth in your corporation (Alpina P, Moscow, 2018) 6
https://openalex.org/W3135271442	https://link.springer.com/content/pdf/10.1007/s13202-021-01119-z.pdf	English	null	Geo-electrical investigation of the groundwater potential of Ogidi and environs, Anambra State, South-eastern Nigeria	Journal of petroleum exploration and production technology	2,021	cc-by	6,997	Abstract The electrical resistivity method was used to determine the groundwater potential of Ogidi and its environs. Thirteen sound- ings were carried out at thirteen different locations using the ABEM terrameter (SAS 1000), and adopting the Schlumberger configuration with maximum current electrode spacing of 1000 m. The lithologic logs for the boreholes were used to delineate the geologic sections. The results revealed six to nine geo-electric layers comprising laterite, clayey sandstone, sandstone, water-saturated sandstone and shale. The water-saturated sandstone forms the aquiferous unit. This unit was found to have resistivity values ranging from 363Ωm to 9107Ωm. It is deeply seated in some areas with a depth of 33.2–103 m. It was observed that the hydraulic conductivity varies between 0.001 and 0.066 m/day, while the transmissivity values vary between 0.01 and 4.7 m2/day. Among all the VES points studied, it was observed that VES 1 has the highest transmissivity value and the highest aquifer thickness. In view of the foregoing, it is expected that water will flow more from aquifer in VES 1 since groundwater flow from an aquifer is directly proportional to transmissivity. Keywords Groundwater potential · Vertical electrical sounding · Schlumberger array · Transmissivity · Geo-electric sections · Nigeria Keywords Groundwater potential · Vertical electrical sounding · Schlumberger array · Transmissivity · Geo-electric sections · Nigeria Geo‑electrical investigation of the groundwater potential of Ogidi and environs, Anambra State, South‑eastern Nigeria C. C. Onyekwelu1 · C. N. Onwubuariri2 · T. I. Mgbeojedo3 · L. S. Al‑Naimi4 · B. I. Ijeh2 · C. C. Agoha5 Received: 24 December 2020 / Accepted: 15 February 2021 / Published online: 1 March 2021 © The Author(s) 2021 https://doi.org/10.1007/s13202-021-01119-z Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 https://doi.org/10.1007/s13202-021-01119-z Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 ORIGINAL PAPER-EXPLORATION GEOPHYSICS * T. I. Mgbeojedo toomgbeojedo@gmail.com 1 Department of Physics and Industrial Physics, Nnamdi Azikiwe University, Awka, Nigeria 2 Department of Physics, Michael Okpara University of Agriculture, Umudike, Nigeria 3 Geotechnical Department, Arab Center for Engineering Studies, Doha, Qatar 4 Department of Chemistry and Earth Sciences, Qatar University, Doha, Qatar 5 Department of Geology, Federal University of Technology, Owerri, Nigeria Introduction are increasingly dependent on quantity and quality of water supply. From the study of an area like Ogidi and its environs that has a temperate climate where nature has provided water in apparently sufficient quantities, the necessity to search for groundwater reserves seems to be less obvious but it does exist (Ugochukwu et al. 2015). This is because no popula- tion can survive without adequate supply of affordable and quality portable water. In the study area, water supply prob- lems are to be expected because of the ever increasing indus- trialization and urbanization the area is undergoing. Moreo- ver, according to Okwudike (2012), in less than 15 years, the population of Ogidi which is about three hundred thousand (300,000), would have grown to about six-hundred thou- sand (600,000). During a preliminary tour of the town, it was observed that many wells drilled at different places in the area have been abortive or have dried up because of the lack of prior systematic scientific investigation. Some of the existing shallow wells reflect high drawdown during the dry season (mainly November to February each year). The draw- down or outright failure of wells, as well as the inadequacy of water supply for improvement schemes, could result to acute water shortages with attendant health and economic implications. It therefore has become necessary to study the An adequate supply of portable water is one of the prereq- uisites for every type of development programme. For this reason, the efforts connected with the location, development and conservation of ground water supplies are of funda- mental economic importance for any country, state, local council, city, town and even community. This is particularly obvious for the arid areas of the tropical and subtropical regions where both agricultural and general development * T. I. Mgbeojedo toomgbeojedo@gmail.com 1 Department of Physics and Industrial Physics, Nnamdi Azikiwe University, Awka, Nigeria 2 Department of Physics, Michael Okpara University of Agriculture, Umudike, Nigeria 3 Geotechnical Department, Arab Center for Engineering Studies, Doha, Qatar 4 Department of Chemistry and Earth Sciences, Qatar University, Doha, Qatar 5 Department of Geology, Federal University of Technology, Owerri, Nigeria * T. I. Mgbeojedo toomgbeojedo@gmail.com Vol.:(0123456789) 1 3 Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 1054 Formation (Eocene) and Imo shale with its lateral equivalent (Paleocene). The Alluvium consists of lose sands with high percentage of quartz content. Introduction It is found along the banks at the lower ridges of the River Niger. The thickness of the deposit is tens of meters in the Onitsha area (Egboka and Okpoko 1999). The Benin Formation underlies the Allu- vium. The lithology consists of thick yellow and white sands with interbedded pebbles and alternation of clays and sandy clays (Egboka and Okpoko 1999). Formation thickness is estimated to be 200 m. groundwater resource potentials of Ogidi and its environs in order to meet the projected water demand of the communi- ties within a stipulated time frame. Quality groundwater supplies, like every other kind of buried natural resources of the earth, are becoming progres- sively more difficult to locate (Keary and Brooks 1991). This owes largely to the increasing human activity resulting to the pollution of underground water sources. Accordingly, water sources located should be tested for quality, purity and satis- fied for consumption. Underlying the Benin Formation is the Ogwashi–Asaba Formation. The Ogwashi-Asaba Formation which outcrops at Onitsha, Nnewi and Ihiala consists of alternation of seams of lignite with clays and shale. The thickness is about 300 m (Ezenwa 1998). Underlying the Ogwashi-Asaba Formation is the Ameki Formation, which consists of lignite and sand- stones. The Formation thickness is estimated to be 1460 m (Ekwe et al. 2006). The Imo shale consists of thick clayey shale, fine textured, dark grey to bluish grey with occasional admixture of clay, ironstones and thin sandstone bands (Rey- ment 1965). Its sands member is the Ebenebe sandstone. The Ebenebe sandstone consists of medium to coarse-grained sandstone that forms the Ugwuoba-Ufuma-Umunze ridge. Its thickness is about 1200 m. i Any new technique which will assist in the location of borehole sites and which will eliminate to a great extent the sinking of unproductive wells is of a great value. This will complement more traditional water finding methods of geol- ogy, such as modern geophysical investigation techniques that are able to locate water-bearing formations under the circumstances that are beyond the capabilities of the older conventional methods. In principle, each of the four main methods of geophysi- cal prospecting, i.e. the magnetic, the gravity, the seismic and the electrical methods, are capable of furnishing useful information in connection with groundwater problems when applied in areas in which the physical conditions satisfy the necessary requirements. Hydrogeology of the study area Streams and rivers draining the study area are of two types; Streams and rivers draining the study area are of two types; Location and physiography 1. Those that originate from outside the study area which includes River Niger and Mamu River The study area is located in Idemili North Local Government Area of Anambra State, South-east Nigeria. The climate is tropical with an average annual rainfall of 1478 mm. The vegetation is characterized by the humid tropical rainforest belt of Nigeria. In most parts, the forests have been cleared for agriculture, industrial sites, urban development and road construction. Streams and river courses are marked by fairly thick vegetation. The survey area lies approximately between latitudes 6°05′30"N and 6°10′48"N, and longitudes 6°49′00"E and 6°55′ 30"E, and covers an area of about 7.4 Km2. The area is bounded in the north by Ogbunike, in the south by Umuoji, in the west by Nkpor and in the east by Abatete as shown in Fig. 1. 2. Those that originate from within the study area such as Idemili River and Ezigbo River (Madu 2009). The Idemili River and its tributaries drain almost a quar- ter of the area under investigation. During the rainy season, the waters of the rivers in the area are muddy. The muddy nature of the waters is as a result of sediments that are eroded into the rivers. In the dry season, due to a decrease in pre- cipitation, the level of the rivers falls and there is a marked decrease in flow rates. The surface of the waters appears less muddy as the erosive processes occurring during this period are not as intense as in the rainy season (Egboka and Okpoko 1999). Introduction In general, however, the seismic and the electric resistivity methods are mostly used because of their high-resolution power in respect of the particular problems encountered in prospecting for water (Bayowa et al. 2014). However, because of some factors like cost- effectiveness, availability of equipment and its simplicity, the electrical resistivity method was chosen over seismic method for this study. Table 1 shows the summary of the geology of Anambra State, while Fig. 2 shows a reflection of the local geology of the study area. 1 3 Geological setting The geology of Anambra State consists of Alluvium (Holo- cene), Benin Formation (Miocene-Pleistocene), Ogwashi- Asaba Formation (Oligoocene–Miocene), the Ameki 1 3 1055 Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 Table 1 Summary of the geology of Anambra State Formation name Maximum approximate thickness (N) Characteristics Holecene alluvium 90 Loose sands with quartz Miocene-Pleistocene Benin Formation 200 Thick yellow, white sands, with interbedded pebbles, clays and sandy clay Oligoocene-Miocene Ogwashi-Asaba Formation 300 Alternation of seams of lignite, clays and shales Eocene Ameki Formation 1460 Lignite and sandstone Paleocene Imo Shale 1200 Thick clayey shale, fine textured, dark grey to bluish grey, with occasional admixture of clay, ironstones and thin sandstone bands Table 1 Summary of the geology of Anambra State 1 3 Materials and method ratios, good resolution of horizontal layers and good depth sensitivity (Ward 1990). In the Schlumberger configuration (Fig. 3), C1 and C2 are the current electrodes through which artificial currents are introduced into the ground, while P1 and P2 are the potential electrodes through which the The Schlumberger configuration was chosen over Wenner configuration for this study. This is because the Schlum- berger array of electrodes provides high signal-to-noise 1 3 1 3 1056 Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 Fig. 2 Geological map of Anambra State (after Nfor et al. 2007) 1 3 Fig. 3 Schlumberger configura- tion Fig. 3 Schlumberger configura- tion Fig. 3 Schlumberger configura- tion 1 3 1057 Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 obtained in the field using Eq. (1). The terrameter is pow- ered by a 12-V dc battery. Current electrodes separation was increased at intervals during the soundings, while the poten- tial electrodes separation was kept relatively fixed. The four electrodes used were arranged in a straight line with respect to the centre and were fixed deep into the ground to ensure good electrical contact. Furthermore, the area around the electrodes was made wet (in case of dry soil conditions), to enable better current penetration. This procedure was repeated for all the thirteen VES stations. Figure 4 shows a map of the study area showing the location of the thirteen VES points. The sounding curve for each sounding point was obtained by plotting the apparent resistivity values as ordinate against half current electrodes spacing (AB/2). potential difference induced by the currents is measured. In this configuration, the potential electrodes are kept constant while current electrodes are moved about the fixed centre of the array. For Schlumberger configuration, apparent resistiv- ity is as given in the equation below (Ogungbe et al. 2012) (1) 휌a = 휋 ⎧ ⎪ ⎨ ⎪⎩ AB 2 2 − MN 2 2 MN ⎫ ⎪ ⎬ ⎪⎭ V I (1) where AB/2 is half current electrode separation, MN/2 is half potential electrode separation. Variation in electrical conductivity of the ground was investigated using electrical resistivity soundings. The Schlumberger array of electrical resistivity adopted in this study was used to delineate the vertical variations of resis- tivity at different depths. Materials and method The materials used for data collec- tion are ABEM terrameter (SAS 1000), four stainless metal stakes used as electrodes, booster, cables for current and potential electrodes, three hammers, measuring tapes and cell phones for long distance communication during meas- urement. These materials were gotten from Rural Water Sup- ply and Sanitation Agency (RUWASSA), Ministry of Public Utilities, Awka. Geo‑electric curve types The geo-electric sections obtained from VES and lithol- ogy (borehole) logs were correlated and compared to obtain the geologic sections. Table 2 shows the curve types and the curve characteris- tics for the 13 VES points. The interpreted sounding curves within this study area have about five groups of curve types. The sounding curves at VES 1 and 8 are of KH- curve type, the sounding curves at VES 2, 4, 5, 6 and 9 are of AK-curve type, VES 10, 11, 12 and 13 are of HK-curve type while VES 3 and 7 are of HA and AA curve types, respectively. Quantitative interpretation VES data were obtained at the 13 geo-electric sounding sta- tions represented as VES 1–13. Qualitative interpretation The first step in the interpretation of the resistivity sounding data was to classify the observed apparent resistivity curves into types. This classification is primarily made on the basis of the shapes of the curves. It was done by visual inspection of the sounding curves. A sound knowledge of the geol- ogy of the area under study and practical experience were needed in achieving good results. Borehole logs obtained from Ministry of Public Utilities, Awka, were used during the interpretation for comparison and correlations. Similarly, the interpretation of electrical resistivity data with the ultimate aim of determining the thickness and the true resistivity of the individual layers in the ground was carried out by means of computer iteration using WinRE- SIST version 1.0. The curves obtained were matched using master curves and were inspected to determine the number and nature of the layers. The results of the curve matching (layer resistivity and thickness) were fed into the computer as a starting model in an iterative forward modelling tech- nique using the WinRESIST software. This is to vindicate the correlation of the field curve and the theoretical curve (Ogungbe et al. 2012). Data collection Thirteen vertical electrical sounding (VES) data were col- lected using a maximum current electrode separation of AB/2 of 500 m. Apparent resistivity was obtained automati- cally by converting the resistance (R) values of the ground 1 3 1 3 1058 Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 1 3 Interpretation of VES 8 (representing KH curve type of VES 1, 8) This sounding station contains six geo-electric layers (Fig. 5 and Table 3), with laterite being interpreted as the first layer with a resistivity of 352 Ωm. The second layer which is interpreted as sandstone has a resistivity value of 3563 Ωm and a thickness of 1.29 m. The third layer with a resistivity value of 12,600 Ωm and a thickness of 7.61 m is interpreted as dry sandstone. The next layer which is the layer of inter- est (aquifer) has a thickness of 31.8 m, a resistivity of 5161 Ωm and is interpreted as water-saturated sandstone. The fifth layer is interpreted as clayey sandstone with a resistivity of 1309 Ωm. The last layer whose thickness could not be determined has a high resistivity value of 18,347 Ωm and is interpreted as sandstone. Fig. 4 Location map showing various VES points Table 2 Analysis of curve types (after Compagnie 1963) VES points Curve type Curve characteristics No of geo-electric layers 1 KH ρ1 < ρ2 > ρ3 > ρ4 7 2 AK ρ1 < ρ2 > ρ3 > ρ4 6 3 HA ρ1 > ρ2 < ρ3 < ρ4 9 4 AK ρ1 < ρ2 < ρ3 > ρ4 7 5 AK ρ1 < ρ2 < ρ3 > ρ4 7 6 AK ρ1 < ρ2 < ρ3 > ρ4 9 7 AA ρ1 < ρ2 < ρ3 < ρ4 6 8 KH ρ1 < ρ2 > ρ3 > ρ4 6 9 AK ρ1 < ρ2 < ρ3 > ρ4 6 10 HK ρ1 > ρ2 < ρ3 > ρ4 6 11 HK ρ1 > ρ2 < ρ3 > ρ4 7 12 HK ρ1 > ρ2 < ρ3 > ρ4 6 13 HK ρ1 > ρ2 < ρ3 > ρ4 6 p gy ( ) Fig. Data collection 4 Location map showing various VES points 1 3 Table 2 Analysis of curve types (after Compagnie 1963) VES points Curve type Curve characteristics No of geo-electric layers 1 KH ρ1 < ρ2 > ρ3 > ρ4 7 2 AK ρ1 < ρ2 > ρ3 > ρ4 6 3 HA ρ1 > ρ2 < ρ3 < ρ4 9 4 AK ρ1 < ρ2 < ρ3 > ρ4 7 5 AK ρ1 < ρ2 < ρ3 > ρ4 7 6 AK ρ1 < ρ2 < ρ3 > ρ4 9 7 AA ρ1 < ρ2 < ρ3 < ρ4 6 8 KH ρ1 < ρ2 > ρ3 > ρ4 6 9 AK ρ1 < ρ2 < ρ3 > ρ4 6 10 HK ρ1 > ρ2 < ρ3 > ρ4 6 11 HK ρ1 > ρ2 < ρ3 > ρ4 7 12 HK ρ1 > ρ2 < ρ3 > ρ4 6 13 HK ρ1 > ρ2 < ρ3 > ρ4 6 Interpretation of VES 8 (representing KH curve type of VES 1, 8) Interpretation of VES 8 (representing KH curve type of VES 1, 8) The fourth layer which has a very high resistivity of 18,656 Ωm and a depth of 6.10 m is inter- preted as sandstone. The layer of interest is the fifth, having a resistivity value of 4190 Ωm and a thickness of 42.6 m, and is interpreted as water-saturated sandstone. The last layer Fig. 5 Sounding curve for VES 8 Table 3 Result of interpretation of VES 8 Layer Apparent resistivity (Ωm) Thickness (m) Depth (m) Lithology 1 352 1.80 0 Top soil (laterite) 2 3563 1.29 1.8 Sandstone 3 12,600 7.61 3.09 Dry sandstone 4 5161 31.80 10.7 Saturated sand- stone 5 1309 39.40 42.5 Clayey sandstone 6 18,347 81.9 Sandstone Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 Fig. 5 Sounding curve for VES 8 Interpretation of VES 2 (representing AK‑curve type of VES 2,4,5,6 and 9) This sounding curve (Fig. 6 and Table 4) has six geo-electric layers which comprise laterite, sandstone, clayey sandstone and water-saturated sandstone. The first layer with a resistiv- ity value of 148.8 Ω and a thickness of 0.78 m is interpreted as laterite. The second layer is interpreted as sandstone with a depth, thickness and resistivity values of 0.78 m, 5.63 m and 13,018 Ωm, respectively. The third layer which has a Table 3 Result of interpretation of VES 8 Layer Apparent resistivity (Ωm) Thickness (m) Depth (m) Lithology 1 352 1.80 0 Top soil (laterite) 2 3563 1.29 1.8 Sandstone 3 12,600 7.61 3.09 Dry sandstone 4 5161 31.80 10.7 Saturated sand- stone 5 1309 39.40 42.5 Clayey sandstone 6 18,347 81.9 Sandstone Table 3 Result of interpretation of VES 8 resistivity value of 6539 Ωm and a thickness of 13.35 m is interpreted as clayey sandstone. The next layer has a high resistivity value of 21,045 Ωm and is interpreted as sand- stone. The fifth layer which has a thickness of 31.0 m and resistivity value of 9107 Ωm is interpreted as water-satu- rated sandstone (aquifer). The last layer with resistivity of 5674 Ωm and a depth of 87.34 m is interpreted as clayey sandstone. Table 3 Result of interpretation of VES 8 Layer Apparent resistivity (Ωm) Thickness (m) Depth (m) Lithology 1 352 1.80 0 Top soil (laterite) 2 3563 1.29 1.8 Sandstone 3 12,600 7.61 3.09 Dry sandstone 4 5161 31.80 10.7 Saturated sand- stone 5 1309 39.40 42.5 Clayey sandstone 6 18,347 81.9 Sandstone Interpretation of VES 10 (representing HK curve type of VES 10, 11, 12, and 13) This sounding location has six geo-electric layers (Fig. 7 and Table 5), showing laterite as the first layer with a resistivity of 76.2 Ωm and a thickness of 0.89 m. The second layer with a resistivity of 9808 Ωm and a thickness of 3.32 m is inter- preted as sandstone. The layer after the second layer has a thickness of 1.89 m and a depth of 4.21 m and is interpreted as clayey sandstone. The fourth layer which has a very high resistivity of 18,656 Ωm and a depth of 6.10 m is inter- preted as sandstone. The layer of interest is the fifth, having a resistivity value of 4190 Ωm and a thickness of 42.6 m, and is interpreted as water-saturated sandstone. The last layer Fig. 5 Sounding curve for VES 1 3 Interpretation of VES 8 (representing KH curve type of VES 1, 8) Table 2 Analysis of curve types (after Compagnie 1963) This sounding station contains six geo-electric layers (Fig. 5 and Table 3), with laterite being interpreted as the first layer with a resistivity of 352 Ωm. The second layer which is interpreted as sandstone has a resistivity value of 3563 Ωm and a thickness of 1.29 m. The third layer with a resistivity value of 12,600 Ωm and a thickness of 7.61 m is interpreted as dry sandstone. The next layer which is the layer of inter- est (aquifer) has a thickness of 31.8 m, a resistivity of 5161 Ωm and is interpreted as water-saturated sandstone. The fifth layer is interpreted as clayey sandstone with a resistivity of 1309 Ωm. The last layer whose thickness could not be determined has a high resistivity value of 18,347 Ωm and is interpreted as sandstone. Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 1059 1059 Interpretation of VES 2 (representing AK‑curve type of VES 2,4,5,6 and 9) This sounding curve (Fig. 6 and Table 4) has six geo-electric layers which comprise laterite, sandstone, clayey sandstone and water-saturated sandstone. The first layer with a resistiv- ity value of 148.8 Ω and a thickness of 0.78 m is interpreted as laterite. The second layer is interpreted as sandstone with a depth, thickness and resistivity values of 0.78 m, 5.63 m and 13,018 Ωm, respectively. The third layer which has a resistivity value of 6539 Ωm and a thickness of 13.35 m is interpreted as clayey sandstone. The next layer has a high resistivity value of 21,045 Ωm and is interpreted as sand- stone. The fifth layer which has a thickness of 31.0 m and resistivity value of 9107 Ωm is interpreted as water-satu- rated sandstone (aquifer). The last layer with resistivity of 5674 Ωm and a depth of 87.34 m is interpreted as clayey sandstone. Interpretation of VES 10 (representing HK curve type of VES 10, 11, 12, and 13) This sounding location has six geo-electric layers (Fig. 7 and Table 5), showing laterite as the first layer with a resistivity of 76.2 Ωm and a thickness of 0.89 m. The second layer with a resistivity of 9808 Ωm and a thickness of 3.32 m is inter- preted as sandstone. The layer after the second layer has a thickness of 1.89 m and a depth of 4.21 m and is interpreted as clayey sandstone. Interpretation of VES 2 (representing AK‑curve type of VES 2,4,5,6 and 9) This sounding curve (Fig. 6 and Table 4) has six geo-electric layers which comprise laterite, sandstone, clayey sandstone and water-saturated sandstone. The first layer with a resistiv- ity value of 148.8 Ω and a thickness of 0.78 m is interpreted as laterite. The second layer is interpreted as sandstone with a depth, thickness and resistivity values of 0.78 m, 5.63 m and 13,018 Ωm, respectively. The third layer which has a 1 3 1 3 060 whose thickness could not be determined is interpreted as hale with a resistivity of 189 Ωm. Interpretation of VES 7 (representing HA curve type of VES 3, 7) Six geo-electric layers were encountered in this sounding point (Fig. 8 and Table 6). The first layer has a thickness of 1.26 m and is interpreted as laterite. The second layer with a resistivity of 3478 Ωm and a thickness of 1.88 m is interpreted as sandstone. The third layer with a thickness of 9.36 m and a resistivity of 12,767 Ωm is interpreted also as sandstone. The fourth layer which is the layer of interest (aquifer) has a resistivity value of 3438Ωm and a thickness of 22.3 m and is interpreted as water-saturated sandstone. The next layer is interpreted as clayey sandstone, having a resistivity of 1577 Ωm and a depth of 34.80 m. The last layer is interpreted as shale and has a resistivity of 1228 Ωm. g. 6 Sounding curve for VES able 4 Result of interpretation of VES 2 ayer Apparent resistivity (Ωm) Thickness (m) Depth (m) Lithology 148.8 0.78 0 Top soil (laterite) 13,018 5.63 0.78 Sandstone 6539 13.35 6.41 Clayey sandstone 21,045 15.92 19.76 Sandstone 9107 31.0 35.68 Saturated sand- stone 5674 66.68 Clayey sandstone Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 1060 Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 Fig. 6 Sounding curve for VES 2 Interpretation of VES 7 (representing HA curve type of VES 3, 7) Interpretation of VES 7 (representing HA curve type of VES 3, 7) whose thickness could not be determined is interpreted as shale with a resistivity of 189 Ωm. Table 4 Result of interpretation of VES 2 Layer Apparent resistivity (Ωm) Thickness (m) Depth (m) Lithology 1 148.8 0.78 0 Top soil (laterite) 2 13,018 5.63 0.78 Sandstone 3 6539 13.35 6.41 Clayey sandstone 4 21,045 15.92 19.76 Sandstone 5 9107 31.0 35.68 Saturated sand- stone 6 5674 66.68 Clayey sandstone Table 4 Result of interpretation of VES 2 Table 4 Result of interpretation of VES 2 1 3 whose thickness could not be determined is interpreted as shale with a resistivity of 189 Ωm. Table 4 Result of interpretation of VES 2 Layer Apparent resistivity (Ωm) Thickness (m) Depth (m) Lithology 1 148.8 0.78 0 Top soil (laterite) 2 13,018 5.63 0.78 Sandstone 3 6539 13.35 6.41 Clayey sandstone 4 21,045 15.92 19.76 Sandstone 5 9107 31.0 35.68 Saturated sand- stone 6 5674 66.68 Clayey sandstone Six geo-electric layers were encountered in this sounding point (Fig. 8 and Table 6). The first layer has a thickness of 1.26 m and is interpreted as laterite. The second layer with a resistivity of 3478 Ωm and a thickness of 1.88 m is interpreted as sandstone. The third layer with a thickness of 9.36 m and a resistivity of 12,767 Ωm is interpreted also as sandstone. The fourth layer which is the layer of interest (aquifer) has a resistivity value of 3438Ωm and a thickness of 22.3 m and is interpreted as water-saturated sandstone. The next layer is interpreted as clayey sandstone, having a resistivity of 1577 Ωm and a depth of 34.80 m. The last layer is interpreted as shale and has a resistivity of 1228 Ωm. whose thickness could not be determined is interpreted as shale with a resistivity of 189 Ωm. 1 3 Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 1061 Table 5 Result of interpretation of VES 10 Layer Apparent resistivity (Ωm) Thickness (m) Depth (m) Lithology 1 76.2 0.89 0 Top soil (laterite) 2 9808 3.32 0.89 Sandstone 3 2107 1.89 4.21 Clayey sandstone 4 18,656 36.6 6.1 Sandstone 5 4190 42.6 42.7 Saturated sand- stone 6 189 85.3 Shale Fig. 7 Sounding curve for VES Geo‑electric correlation along different profiles Table 5 Result of interpretation of VES 10 Existing borehole log near the vicinity of VES 1 and VES 10 was correlated with the geo-electric sections derived from VES 1 and 10 as shown in Fig. 9. This comparison showed similar layering between the VES-modelled sections and the existing litholog A. In order to thoroughly investigate the thickness of vari- ous aquifers, geo-electric correlation was drawn along three profiles. The profiles were labelled AA’, BB’ and CC’ respectively. 1 3 1062 Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 Fig. 8 Sounding curve for VES 7 Fig. 8 Sounding curve for VES 7 VES points along profile AA’ Table 6 Result of interpretation of VES 7 Layer Apparent resistivity (Ωm) Thickness (m) Depth (m) Lithology 1 694.2 1.26 0 Top soil (laterite) 2 3478 1.88 1.26 Sandstone 3 12,767 9.36 3.14 Sandstone 4 3438 22.3 12.5 Saturated sand- stone 5 1577 26.4 34.8 Clayey sandstone 6 1228 61.2 Shale Table 6 Result of interpretation of VES 7 AA’ is a geo-electric profile drawn in the North–South direc- tion. The VES points along this profile are VES 7, 2 and 10, covering a distance of about 7 km. The VES points have six geo-electric layers each. VES 2 is an AK type, VES 10 is a HK type and VES 7 is an AA type. VES 2, VES 10 and VES 7 have aquifer thickness of 31.0 m, 42.6 m and 22.3 m, respectively. Along this profile, VES 10 holds the best pros- pect for sustainable groundwater development (Fig. 10). 1 3 1063 Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 Fig. 9 Comparison of borehole log A with VES 1 and 11 Fig. 9 Comparison of borehole log A with VES 1 and 11 VES Points along profile BB’ The VES results also revealed that the resistivity of the water-saturated sandstone (aquifer) at Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 1064 Fig. 10 Geo-electric correlation along profile AA’ Fig. 10 Geo-electric correlation along profile AA’ Fig. 10 Geo-electric correlation along profile AA’ (2011) that carried out geophysical survey in Nnokwa and Alor using vertical electrical sounding (VES) technique to investigate groundwater potentials (depth to aquifer in Nnokwa varied from 27.0 m to 82.1 m while in Alor it varied from 34.3 m to 49.3 m, respectively). The results of the VES data have aided in delineating the geo-electric sections of the area under investigations. The results revealed a minimum of six and maximum of nine geo-electric sections underlying various VES stations. The VES results also revealed that the resistivity of the water-saturated sandstone (aquifer) at VES Points along profile BB’ CC’ is a profile drawn in the North–South direction. The VES points along this profile are VES 11, 4, 5 and 1. The VES points have seven geo-electric sections each. VES 1 is a KH type, VES 4 and 5 are AK-type while VES 11 is a HK type. The thickness of aquifer for VES 1 is 72.5 m while for VES 11, 4 and 5, the thicknesses of aquifer are 6.5 m, 16.87 m and 36.5 m, respectively. VES 1 holds the best prospect for sustainable groundwater development along this profile because of the high thickness value (Fig. 12). BB’ is a profile drawn in the Northwest–Southeast direc- tion. The VES points along this profile are VES 9, 4 and 2, and it covers a distance of about 6.5 km. VES 2 and 9 have six geo-electric sections each, while VES 4 has seven geo- electric sections. The VES curves are of AK type. VES 2 has an aquifer thickness of 31.0 m, VES 4 an aquifer thick- ness of 16.87 m while VES 9 has an aquifer thickness of 39.4 m. Along the profile as shown in Fig. 11, VES 9 is the best area to site a borehole because of the highest value of aquifer thickness. 1 3 1 3 1064 Fig. 10 Geo-electric correlation along profile AA’ Discussion The geophysical method used in the study area has greatly assisted in evaluating the various aquifers, aquifer parame- ters using vertical electrical sounding techniques. The results obtained have shown a close semblance with results obtained from borehole logs obtained from the Ministry of Public Utilities Awka. Table 7 shows the summary of the aquifer parameters at the various locations across the study area. The results obtained are related to the work of Okparaeze (2011) that carried out geophysical survey in Nnokwa and Alor using vertical electrical sounding (VES) technique to investigate groundwater potentials (depth to aquifer in Nnokwa varied from 27.0 m to 82.1 m while in Alor it varied from 34.3 m to 49.3 m, respectively). The results of the VES data have aided in delineating the geo-electric sections of the area under investigations. The results revealed a minimum of six and maximum of nine geo-electric sections underlying various VES stations. Discussion The geophysical method used in the study area has greatly assisted in evaluating the various aquifers, aquifer parame- ters using vertical electrical sounding techniques. The results obtained have shown a close semblance with results obtained from borehole logs obtained from the Ministry of Public Utilities Awka. Table 7 shows the summary of the aquifer parameters at the various locations across the study area. The results obtained are related to the work of Okparaeze 1 3 1065 Fig.11 Geo-electric correlation along profile BB’ the various stations varies from as low as 363Ωm to as high as 9107 Ωm; thickness of the aquifer varies from a value of 6.5 m at VES 11 to a value of 72.5 m at VES 1. The geologic sections revealed that the first layer for the various soundings is composed of laterite. The second to the last layer at the various soundings stations contain water-saturated sandstone (aquifer) except VES 1, 7, 8 and 11 where it is at the third to the last layer. Furthermore, the results revealed that the aquifer is shallowest around Ogidi (VES 7 and 8) with val- 2 and 13) with values of 118.66 m and 75.10 m, respec- tively. Aquifer characteristics of hydraulic conductivity and transmissivity were established with hydraulic conductivity values ranging from 0.001 to 0.066 m/day while transmis- sivity values ranged from 0.006 to 4.79 m2/day. From the calculated transmissivity values, it is seen that VES 1 has the highest transmissivity value. Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 1065 Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 Fig.11 Geo-electric correlation along profile BB’ Fig.11 Geo-electric correlation along profile BB’ Fig.11 Geo-electric correlation along profile BB’ 2 and 13) with values of 118.66 m and 75.10 m, respec- tively. Aquifer characteristics of hydraulic conductivity and transmissivity were established with hydraulic conductivity values ranging from 0.001 to 0.066 m/day while transmis- sivity values ranged from 0.006 to 4.79 m2/day. From the calculated transmissivity values, it is seen that VES 1 has the highest transmissivity value. 2 and 13) with values of 118.66 m and 75.10 m, respec- tively. Aquifer characteristics of hydraulic conductivity and transmissivity were established with hydraulic conductivity values ranging from 0.001 to 0.066 m/day while transmis- sivity values ranged from 0.006 to 4.79 m2/day. From the calculated transmissivity values, it is seen that VES 1 has the highest transmissivity value. Discussion the various stations varies from as low as 363Ωm to as high as 9107 Ωm; thickness of the aquifer varies from a value of 6.5 m at VES 11 to a value of 72.5 m at VES 1. The geologic sections revealed that the first layer for the various soundings is composed of laterite. The second to the last layer at the various soundings stations contain water-saturated sandstone (aquifer) except VES 1, 7, 8 and 11 where it is at the third to the last layer. Furthermore, the results revealed that the aquifer is shallowest around Ogidi (VES 7 and 8) with val- ues of 12.70 m and 10.70 m, and deepest around Obosi (VES 1 3 1066 Fig.12 Geo-electric correlation along profile CC’ Table 7 Aquifer parameters at the various locations across the study area VES No Location Aquifer resistivity (Ωm) Depth to water (m) Aquifer thickness (m) Hydraulic conductivity (m/ day) Transmissiv- ity (m2/day) 1 Community. Primary School Umuoji 363 30.50 72.50 0.066 4.79 2 Umueze Ogidi 9107 35.68 31.00 0.002 0.08 3 Iyienu 1 Ogidi 1108 118.66 16.34 0.021 0.35 4 Iyienu 2 Ogidi 3418 63.86 16.87 0.007 0.12 5 Building material market, Ogidi 5116 27.60 36.50 0.004 0.17 6 St Paul Church, Ogidi 6412 53.31 16.25 0.003 0.06 7 Along Umuoji Ojoto Road 3438 12.50 22.30 0.006 0.16 8 St Charles’s field, Ogidi 5161 10.70 31.80 0.005 0.15 9 Behind St Barnabas Church, Ogbunike 4826 42.50 39.40 0.005 0.20 10 Ogbunike High School 4190 42.70 42.60 0.006 0.24 11 Ikenga Ogidi 5690 26.70 6.50 0.001 0.01 12 Seminary Field, Nkpor 6744 53.20 40.80 0.001 0.06 13 Opposite Holy Trinity Catholic Church, Ogidi 6009 75.10 26.90 0.003 0.10 Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 1066 Fig.12 Geo-electric correlation along profile CC’ Fig.12 Geo-electric correlation along profile CC’ Table 7 Aquifer parameters at the various locations across the study area VES No Location Aquifer resistivity (Ωm) Depth to water (m) Aquifer thickness (m) Hydraulic conductivity (m/ day) Transmissiv- ity (m2/day) 1 Community. Conclusion basement complex terrain: case study of Ekiti State, Southwestern Nigeria. Res J Eng Appl Sci 3(1):55–60 Compagnie Generale de Geophysique (1963) Master curves for electri- cal sounding, 2nd, rev edn. European Association of Exploration Geophysicists, The Hague The research work has provided information on the depth and the thickness of the aquifer units in the study area. This information will greatly assist in the development of an effective water scheme for the area. Based on all the find- ings made and the interpretations of the VES data, VES 1 (Community Primary School Umuoji) has been chosen as the most viable location for the development of groundwater resources in the study area. The thickness (72.50 m) and resistivity (363 Ωm) of the aquifer were obtained at this location. This area seems to satisfy necessary conditions that will guarantee an appreciable quantity of water for sustain- able development. Conclusively, it should be noted that aqui- fer layers were encountered in all the sounding locations, which shows that the study area has a very good groundwa- ter potential. Egboka BCE, Okpoko EI (1999) Conjunctive uses of water resources and management in Anambra State, Nigeria. Natural Appl Sci J 9 (2):2005 Ekwe AC, Onu NN, Onuoha KM (2006) Estimation of aquifer hydrau- lic characteristics from electrical sounding data: the case of Mid- dle Imo River Basin aquifers, Southeastern Nigeria. J Spatial Hydrol 6(2):121–132 Ezenwa UO (1998) Geological investigation of parts of Ogidi. Ministry of Public Utilities, Awka, p 113 Keary P, Brooks M (1991) An introduction to geophysical exploration, 2nd edn. Blackwall Scientific Publications, Oxford, p 254p i Madu CI (2009) Geoelectric sounding for predicting aquifer propertie Ministry of Public Utilities, Awka i Madu CI (2009) Geoelectric sounding for p Ministry of Public Utilities, Awka Ministry of Public Utilities, Awka Nfor BN, Olobaniyi SB, Ogala JE (2007) Extent and distribution of groundwater resources in parts of Anambra State. Appl Sci Envi- ron Manage J 11(2):215–222 Ogungbe AS, Onori EO, Olaoye MA (2012) Application of electrical resistivity techniques in the investigation of groundwater contami- nation: a case study of Ile-Epo dumpsite, Lagos, Nigeria. Int J Geomatics Geosci 3(1):30–41 Funding The authors received no specific funding for this work. Okparaeze J (2011) Geoelectric investigations of Idemili South Local Government Area of Anambra State. Appl Sci Environm Manage J 9(6):111–119 Discussion Primary School Umuoji 363 30.50 72.50 0.066 4.79 2 Umueze Ogidi 9107 35.68 31.00 0.002 0.08 3 Iyienu 1 Ogidi 1108 118.66 16.34 0.021 0.35 4 Iyienu 2 Ogidi 3418 63.86 16.87 0.007 0.12 5 Building material market, Ogidi 5116 27.60 36.50 0.004 0.17 6 St Paul Church, Ogidi 6412 53.31 16.25 0.003 0.06 7 Along Umuoji Ojoto Road 3438 12.50 22.30 0.006 0.16 8 St Charles’s field, Ogidi 5161 10.70 31.80 0.005 0.15 9 Behind St Barnabas Church, Ogbunike 4826 42.50 39.40 0.005 0.20 10 Ogbunike High School 4190 42.70 42.60 0.006 0.24 11 Ikenga Ogidi 5690 26.70 6.50 0.001 0.01 12 Seminary Field, Nkpor 6744 53.20 40.80 0.001 0.06 13 Opposite Holy Trinity Catholic Church, Ogidi 6009 75.10 26.90 0.003 0.10 Table 7 Aquifer parameters at the various locations across the study area 1067 Journal of Petroleum Exploration and Production Technology (2021) 11:1053–1067 Conflict of interest None to declare. Conflict of interest None to declare. Conflict of interest None to declare. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Reyment RA (1965) Aspects of the geology of Nigeria: the stratigraphy of the Cretaceous and Cenezoic deposits. University Press, Ibadan Ugochukwu SC, Ezeokoli FO, Okoye PU (2015) The correlation between prevailing climatic conditions and prices of building materials—a case study of Anambra State, Nigeria. Int J Appl Sci Eng Res 4(5):856–870. https://doi.org/10.6088/ijaser.04087 Ward SH (1990) Resistivity and induced polarization methods. In: Ward SH (ed) Geotechnical and environmental geophys- ics, vol 1. Society of Exploration Geophysicists. https://doi. org/10.1190/1.9781560802785.ch6l Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Declarations Okwudike JJ (2012) Hydrological surveys in Onitsha by means of electrical soundings. Unpublished MSc thesis, Anambra State University, Uli References Bayowa GO, Olorunfemi MO, Ademilua OL (2014) A geoelectric assessment and classification of the aquifer systems in a typical 1 3
https://openalex.org/W4205495212	https://research-repository.st-andrews.ac.uk/bitstream/10023/24706/1/Garrett_2022_JECS_Albion_s_Queen_by_All_Admir_d_CC.pdf	English	null	‘Albion's Queen by All Admir'd’: Reassessing the Public Reputation of Queen Charlotte, 1761‐1818	Journal for eighteenth-century studies	2,022	cc-by	12,469	© 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Journal for Eighteenth-Century Studies Vol. •• No. •• (2022) doi: 10.1111/1754-0208.12822 ‘Albion’s Queen by All Admir’d’: Reassessing the Public Reputation of Queen Charlotte, 1761-1818 NATALEE GARRETT Abstract: This article challenges contemporary and historiographical assertions about the public reputation of Queen Charlotte. Through an examination of newspaper articles and satirical prints, it traces the evolution of Charlotte’s public reputation through events such as the Regency Crisis, the Regency and ultimately her death in 1818. Charlotte’s largely positive reputation centred on repeated representations of her domesticity and de- votion to her family. Deeper analysis of public discourse reveals that a counter image of Charlotte circulated in the public sphere from 1786 onwards, one which portrayed her as an emotionally cold mother and an avaricious, politically ambitious queen. Keywords: Queen Charlotte, public opinion, royalty, newspapers, satirical prints, reputation, print culture ‘Future historians, whatever may be their particular or political prejudices, will have am- ple scope for illustrating, and handing down to ages yet unborn, the perhaps unparalleled character of CHARLOTTE, Queen of England’.1 c a acte o C O , Quee o g a d . Written on 20 November 1818, three days after the death of Queen Charlotte, this ex- tract from the Morning Herald conﬁdently asserts that future historians will support a pos- itive view of the queen’s character. The death of George III’s consort prompted dozens of British newspapers to publish panegyrics on the queen’s role as a wife and mother, her strict sense of propriety and her charitable work. These panegyrics appear to have formed the foundation of Queen Charlotte’s posthumous reputation among historians.2 Cindy McCreery has argued that Charlotte was portrayed as both a ‘superb maternal model’ and a ‘devoted wife in both written and visual commentaries’ in her lifetime.3 Linda Colley has referred to Charlotte as a ‘totem of morality’ but also as a queen conscious of her im- age as a mother, an image she encouraged through portraiture.4 Critical analysis of Charlotte’s individual reputation has been limited thus far, but modern biographies of her eldest son, George IV, have challenged her historiographical standing as a devoted ma- ternal ﬁgure.5 Recently, Marilyn Morris has highlighted that tensions between the staid king and queen and their proﬂigate sons ‘undermined the appearance of familial harmony’.6 Scholarly analysis of Charlotte’s reputation is thus varied and often secondary to analysis of George III and George IV; this article aims to synthesise a broader concep- tualisation of Charlotte’s individual contemporary reputation throughout her reign as queen. I. 1761-1786: ‘Albion’s Queen by All Admir’d’7 Charlotte of Mecklenburg-Strelitz married George III of Great Britain in 1761 and, over a period of twenty-one years, produced ﬁfteen royal children, thirteen of whom survived to adulthood. This profusion of offspring ensured the endurance of the Hanoverian dynasty, and this domestic good fortune was celebrated in the public eye by the widespread publica- tion of prints that portrayed the royal couple with their children. In some instances, these prints were copies of royally commissioned portraits, while others such as The Royal Family of Great Britain were cheap woodcuts created by enterprising printmakers to celebrate the royal family.8 Two ofﬁcial portraits of the royal family by Johan Joseph Zoffany were turned into mezzotint prints and sold in print shops: a 1771 portrait of the king, queen and their six children and an informal portrait of Queen Charlotte with the prince of Wales and the duke of York as young children.9 These images, ofﬁcial and unofﬁcial, consistently associ- ated the queen with motherhood in the British public sphere. Similarly, newspapers re- ported in detail loyal addresses made to the queen which centred on her identity as a virtuous mother. During a visit to Portsmouth in the spring of 1778, the Mayor referred to the queen as a princess ‘whose virtues adorn her sex’ and whose example the Mayor hoped would occasion ‘much public and private happiness in these kingdoms’.10 On an of- ﬁcial visit to Winchester in October 1778, the Mayor of the city referred to the queen as a ‘bright ornament’ and expressed a hope that her royal offspring should inherit the royal virtues ‘which have so deservedly rendered your majesty the delight of all your subjects’.11 Every year, celebrations of the queen’s birthday occasioned a profusion of poetry pub- lished in the press which reiterated the queen’s moral virtue and her position as a mother. Verses written by ‘Carus’ in 1778 were addressed to ‘Albion’s queen, by all admir’d’: a mother who reared her children ‘with sweet affection’ and a ﬁgure whose charity and vir- tue transcended the political partisanship of Britain.12 In 1782, the London Chronicle pub- lished a poem with the lines: ‘Behold a numerous progeny/on their fond parent lean/Oh! © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. ‘Albion’s Queen by All Admir’d’: Reassessing the Public Reputation of Queen Charlotte, 1761-1818 Written on 20 November 1818, three days after the death of Queen Charlotte, this ex- tract from the Morning Herald conﬁdently asserts that future historians will support a pos- itive view of the queen’s character. The death of George III’s consort prompted dozens of B iti h t bli h i th ’ l if d th h An examination of contemporary media culture suggests a distinctive split in popular perceptions of the queen. On the one hand, there exists the ‘ofﬁcial’ image of Queen Charlotte: a devoted wife and mother who exempliﬁed the charitable and morally upright aspects of monarchy. This representation of the queen was most apparent in the British public sphere prior to the 1780s, and it continued to endure until the queen’s death in 1818 and beyond, to form the dominant historiographical perception of Charlotte. NATALEE GARRETT 2 However, the ‘ofﬁcial’ image of the queen was challenged by a series of highly publicised events beginning in the 1780s which saw Charlotte cast in a very different light: as a queen consort who hungered for power, a greedy and parsimonious woman and a cold maternal ﬁgure who alienated the affections of her children. This ‘unofﬁcial’ image of Queen Charlotte was disseminated in satirical prints and newspaper articles, and it is these sources which form the basis of this reassessment of her reputation. Although nei- ther medium was immune to political prejudices, their persistent and sometimes contra- dictory contribution to public discourse on the royal family makes them a valuable foundation of analysis. Moments of political upheaval, in particular the Regency Crisis of 1788-1789 and the eventual implementation of a Regency in 1811, cast doubt on Charlotte’s image as a domestic, apolitical queen, while her relationships with her many children were frequently dissected by an interested and sometimes critical public sphere. Through analysis of newspaper articles and satirical prints commenting on the queen, this article seeks to re-evaluate Charlotte’s popular reputation and argues that her public im- age was complex and multifaceted. I. 1761-1786: ‘Albion’s Queen by All Admir’d’7 What a sight at once to see/the mother and the Queen’, a verse that highlighted the queen’s dynastic success as a royal consort while praising her maternal ‘fondness’ for her children.13 In the period 1761-1786, Charlotte’s ofﬁcial public image was overwhelm- ingly domestic in nature; the queen was praised for prioritising her family and the private sphere over the political and public aspects of royalty. ‘Albion’s Queen by All Admir’d’ 3 Praise of Charlotte’s studied detachment from political interference contrasted sharply with contemporary discussions of her mother-in-law, Augusta, the dowager princess of Wales. In1765, George III suffered from an unspeciﬁed but serious illness which prompted him to propose a new Regency Bill.14 The identity of the said Regent was hotly debated in the British press, and many papers gleefully noted that the king’s mother would be ex- cluded by the provision of the bill which dictated that the Regent be ‘the Queen, or one of the descendants of the late King usually resident in Great Britain’.15 Ultimately, Augusta was added to the Regency Bill, second only to Queen Charlotte, but discussions in the media reﬂected enduring popular discourse critical of Augusta’s alleged relationship with the king’s former prime minister, the earl of Bute.16 For example, in May 1765, the Westminster Journal and London Political Miscellany emphasised that fears Bute would re- gain power through the Bill were unfounded, as the king himself had excluded the dow- ager princess of Wales from acting as a Regent.17 In the early years of her reign as queen consort, Charlotte’s apolitical image therefore contrasted strongly with that of the king’s mother; while the prospect of Augusta acting as Regent provoked debate in the press, few concerns were raised about the possibility of the young queen taking on the role. Despite this seemingly overwhelming tide of public approbation for the queen, the ﬁrst hint of a satirical register against the queen’s domestic image emerged in1786, when sev- eral caricature prints depicted the king and queen as a homely farmer and his wife. In The Farmyard, published by S. W. © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. II. 1786-1788: The Queen of Hearts Cover’d with Diamonds The celebrated image of a virtuous, retiring queen who thrived in the domestic sphere was shaken in 1786, when the royal couple were implicated in the highly publicised impeach- ment trial of Warren Hastings, former governor of Bengal. In June 1786, George III was presented with a bulse containing a large diamond; this gemstone was a diplomatic gift from Nizam Ali Khan of Deccan, but as the stone was presented to the king through Has- tings, the British press interpreted the gesture as an attempt at bribery.21 In fact, the Morning Herald stated on 6 July 1786 that the king had felt compelled to return the dia- mond to Mr Hastings because ‘the publick [sic] justly conceived that the many various de- vices artfully practiced to pass the diamond as a present from the Nizam were merely a pretext to deceive His Majesty into an acceptance of it’.22 Signiﬁcantly, it appears that none of the newspaper articles discussing this scandal made reference to the queen, yet in several satirical prints produced between 1786 and 1788, Charlotte earned a starring role in the developing scandal. A pair of prints addressing the diamond scandal emerged in 1786, each one implicating the king and queen, respectively. In The Queen of Hearts Cover’d with Diamonds (1786; Fig. 2), Charlotte is envisaged as a sultaness, wearing an enormous, bejewelled turban and a variety of ostentatious diamond jewellery.23 A large, heart-shaped jewel sits in a bulse in the upper left-hand corner of the image, inextricably linking the queen’s fabulous jewels with the Hastings scandal.24 The print’s title suggests that the queen’s former pop- ularity has been jeopardised by the revelation of her supposed avarice: she is no longer the queen of hearts, but a queen who has traded the love and respect of her subjects for diamonds. Another caricature from 1788, The Diamond Eaters, Horrid Monsters!, depicted Hastings pouring diamonds labelled ‘Indian plunder’ into the mouths of George, Charlotte and the Lord High Chancellor Edward Thurlow.25 The presence of these three ﬁgures was no doubt intended to reiterate to the public the involvement of the highest political players in Hastings’s criminal deeds. The artist behind this particular print is unknown, but it is likely that this caricature and others critical of Hastings were produced by the Foxite Whigs, whose own Edmund Burke led the charge against Hastings’s alleged misdeeds in court. I. 1761-1786: ‘Albion’s Queen by All Admir’d’7 Fores, the king and queen are shown working at a farm in Windsor: the king has just placed food in a trough for pigs, while the queen feeds chickens with grain from her apron.18 A caricature published by James Phillips, The Constant Couple (1786; Fig.1), shows the king dressed as a farmer riding a horse towards Windsor, with the queen sitting pillion behind him.19 The content of these satirical prints had roots in the 1. [Anon.], The Constant Couple,1786, etching, hand-coloured, 24.5 × 35.8 cm. Courtesy of The Lewis Walpole Library, Yale University [Colour ﬁgure can be viewed at wileyonlinelibrary.com] © 2022 The Authors Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd 1. [Anon.], The Constant Couple,1786, etching, hand-coloured, 24.5 × 35.8 cm. Courtesy of The Lewis Walpole Library, Yale University [Colour ﬁgure can be viewed at wileyonlinelibrary.com] © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. NATALEE GARRETT 4 ofﬁcial image of domestic felicity that was encouraged by the king and queen, yet in these prints, that domestic image was mocked because it clashed with notions of royal grandeur. The ‘Farmer George’ images were part of a long-running visual tradition that parodied George III for his apparent yearning to lead a simple life at Windsor, but the examples which include Charlotte emphasise that, even when mocked, the queen was still depicted as a woman who existed within a decidedly domestic role at this time.20 For the ﬁrst twenty-ﬁve years of her reign as queen consort, Charlotte’s reputation as a devoted mother and sober queen appears to have been consistently bolstered by contemporary media. © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. II. 1786-1788: The Queen of Hearts Cover’d with Diamonds Indeed, the caricature The Bow to the Throne; Alias the Begging Bow, also produced in 1788, suggested that Hastings was loaning money and granting gifts to cardinals, judges, sea captains and even the Prime Minister.26 Hastings, depicted in Indian costume, sits on a gilded chamber pot and hands large bags of gold to Thurlow, William Pitt and the numerous disembodied hands grasping before him. The king and queen bow at Hastings’s feet, their eyes ﬁxed on the piles of gold littering the ﬂoor around him. In contrast, English Slavery; or, a Picture of the Times attempted to detach the king from Hastings’s guilt and emphasised his moral uprightness.27 In the image, George III and ‘Albion’s Queen by All Admir’d’ 5 5 2. Anon, The Queen of Hearts Cover’d with Diamonds, 1786, etching, hand-coloured, 14.3 × 11.1 cm. © The Trustees of the British Museum [Colour ﬁgure can be viewed at wileyonlinelibrary.com] y 5 2. Anon, The Queen of Hearts Cover’d with Diamonds, 1786, etching, hand-coloured, 14.3 × 11.1 cm. © The Trustees of the British Museum [Colour ﬁgure can be viewed at wileyonlinelibrary.com] Thurlow sit at a table on which rests a bag of gold and the diamond delivered by Hastings; the king simply declares, ‘I would not for the world touch either’, mimicking the report published in the Morning Herald in July 1786, when the king allegedly resolved to return the diamond to Hastings. Signiﬁcantly, the depiction of the queen in this image is far less ﬂattering than that of her spouse; George expresses determination to avoid Hastings’s bribery, while the queen sits at a pianoforte, singing about her love of money. The separate depictions of the royal couple in this print are signiﬁcant, as the king is cleared of guilt while Charlotte is portrayed as avaricious and immoral. It is arguable that the queen was singled out for reprobation by caricaturists who primarily aimed to criticise the king and the political status quo, but the prints examined show that caricaturists were sufﬁciently conﬁdent to directly depict the king and his ministers grovelling at the feet of Warren Hastings. That Charlotte was repeatedly represented as a key player in the Hastings diamond scandal by caricaturists suggests a signiﬁcant dimension of her public reputation which gained currency at this time. © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. II. 1786-1788: The Queen of Hearts Cover’d with Diamonds Marcia Pointon has compellingly argued that the splendour of her coronation regalia in 1761 led to Queen Charlotte being associ- ated with precious gemstones in the minds of the British public throughout her reign.28 Other satirical prints produced between 1786 and 1788 also emphasise the extent to which discussions of royal avarice circulated in the public sphere. James Gillray’s A New 6 NATALEE GARRETT Way to Pay the National-Debt, for example, was produced in response to Parliamentary dis- cussions about alleviating debts from the Civil List.29 The print depicts George III and Charlotte exiting the Treasury with thousands of coins stowed in the king’s pockets and the queen’s apron; the royal couple were both tarred with the brush of avarice, a trait which was enhanced by coverage of the Hastings scandal. In spite of the highly critical register of caricature prints linking Charlotte to the Hastings affair, many newspapers continued to publish poems and addresses that reiter- ated the established image of Charlotte as a domesticated and moral queen. Commenting on the Queen’s ofﬁcial birthday in January 1787, The Times reported that the large num- ber of individuals present at the royal levee was proof that ‘loyalty and affection increase every year’ towards the Queen.30 Signiﬁcantly, the article was at pains to suggest that the clothing of the royal and noble attendees was more indicative of economy than brilliance, suggesting that the paper wished to downplay accusations of lavish splendour at the court.31 In its coverage of the queen’s birthday, the Gazetteer and New Daily Advertiser praised Charlotte as a model wife, queen and mother, concluding that ‘her virtues are too amiable for the exertion of power’.32 This quotation underlines the fact that Charlotte’s positive public reputation was focused on her lack of interference in politics, an attribute which would have challenged notions of her as a domestic queen. While sa- tirical prints between 1786 and 1788 portrayed Charlotte in an unﬂattering, avaricious role, the majority of the British media continued to praise the queen as a paragon of fe- male virtues. However, although the satirical prints were a minority voice, their publica- tion and dissemination nonetheless created an alternate image of the queen within the British public sphere. This alternate and largely negative image would grow exponentially during the Regency Crisis in the winter of 1788-1789. III. 1788-1799: ‘A Pattern of Domestic Virtue’ The Crown-headed Ladies of Europe at present have no little sway in the respective councils of their Courts […] while our Queen is contented with the humble yet substantial virtues of domestic life, and was never known to interfere in matters of State.33 This paragraph praising Charlotte’s contentment with an apolitical domestic life was published just before the beginning of a period of signiﬁcant political upheaval known as the Regency Crisis. In November 1788, George III began to exhibit symptoms of mad- ness, prompting Parliament to plan for a Regency. The ensuing scramble for power be- tween the Foxites and Pittites saw the prince of Wales and his mother positioned at opposite sides of a political dispute that evolved into a propaganda war which played out in newspapers and satirical prints. The matter of a Regency was debated in Parliament and in newspapers throughout the winter of 1788-1789, as the Foxites fought for the rights of their supporter, the prince of Wales, and the Pittites prevaricated in hopes of the king’s swift return to health. Parlia- ment debated the extent to which the prince of Wales would hold power under a Regency. Under the ‘restricted’ Regency put forth by Prime Minister Pitt, the prince of Wales would be granted temporary executive power, while Queen Charlotte would be given control of the privy purse and the royal household, including the person of the king himself.34 The Morning Herald, an Opposition newspaper, was outspoken about the perceived injus- tices done to the prince of Wales, and it did not hesitate to criticise the queen, highlighting ‘Albion’s Queen by All Admir’d’ 7 that she was merely a ‘consort’ and that Pitt’s proposals allowed her responsibilities and powers beyond what she was due.35 The queen’s right to act in the king’s stead was again questioned in the public eye, when members of Parliament also challenged her sudden rise to power. Opposition MP Edmund Burke expressed his unease at the queen possessing the ability to manage the king’s household and the privy purse, arguing that while he respected Her Majesty, ‘he by no means thought her the most proper person to be entrusted with the giving away of such an immense sum of money’.36 Burke’s concern about the queen’s ﬁnancial trustworthiness was perhaps a subtle reference to the carica- ture prints that had dubbed Charlotte the queen of diamonds the previous year. © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. III. 1788-1799: ‘A Pattern of Domestic Virtue’ Loaded with the Spoils of India and Britain.38 Released in December 1788, the print depicts the queen as her famed pet zebra, wearing panniers full of gold and a large diamond necklace. Pitt and two other Tory ministers urge the zebra onwards to the Tower of London to split their spoils. Most signiﬁcantly, the zebra says, ‘What are Children’s rights to Ambition - I will rule in spite of them if I can conceal things at Q [Kew]’. This print directly attacks Charlotte as an unnatural mother, one who is willing to rob her children of their rights in order to advance her own cause; it is a damning contrast to the queen’s usual image as a perfect maternal ﬁgure and raises concerns about the position of queen consort in the British political sphere. Questions about the queen’s morality and domesticity also prompted contemporary media to challenge Charlotte’s loyalty to her husband. In Weird Sisters; Ministers of Dark- ness; Minions of the Moon, James Gillray portrays politicians William Pitt, Henry Dundas and Edward Thurlow watching with interest as the moon rises.39 The moon’s two faces are George III, sleeping and in shade, and Queen Charlotte, who smiles as the light shines upon her. This image literally depicts the waning of George III’s power and the ‘waxing’ of the queen’s, a controversial concept. Whether or not Queen Charlotte relished the pros- pect of enhanced inﬂuence remains unknown, but the popular response to her responsi- bilities in the case of a Regency does suggest an uneasiness at the notion of a queen consort exercising any kind of control in British life. Where Charlotte’s avarice was mer- cilessly mocked during the Hastings scandal, in caricatures addressing the queen’s role in the Regency Bill, her greed is depicted as going beyond money: she becomes a German-born princess eager to grasp the reins of political control in Britain at the very moment her husband ails. Concerns about the queen’s role in the Regency Crisis of 1789 echo similar debates about the position of George III’s mother, Augusta, during the Regency Crisis of 1765; while Charlotte avoided criticism when she was named as a potential Regent in 1765, by 1789, many press commentaries on her political role were decidedly negative, bolstered by the prince of Wales’s supporters. III. 1788-1799: ‘A Pattern of Domestic Virtue’ One of the most signiﬁcant charges laid against Charlotte in the British media at this time was her alleged plotting with Prime Minister Pitt, who was determined to prevent the prince of Wales from assuming royal authority. One of the earliest caricatures address- ing this supposed alliance appeared on 20 December 1788, at the very beginning of de- bates on the powers of the Regent. In The Prospect before Us (1788; Fig. 3), the queen is shown exiting the Treasury and stepping on a banner decorated with the prince of Wales’s seal, a sign of maternal betrayal and the usurpation of the rights of the heir to the throne.37 The queen is led by her lady-in-waiting Mrs Schwellenberg, who holds the mace and purse of the Lord Chancellor. Pitt follows behind the queen, seemingly holding onto children’s leading strings which attach to the queen’s gown. The half-crowns ﬂoating above the queen’s and Pitt’s heads emphasise their shared plot for power, but the fact that the queen is being ‘led’ by her German lady-in-waiting and ‘guided’ by Pitt could suggest that the caricaturist was willing to acknowledge that the queen had been misled by these 3. Thomas Rowlandson, The Prospect before Us, 1788, etching, hand-coloured 23.2 × 32 cm. Courtesy of Beinecke Rare Book and Manuscript Library, Yale University [Colour ﬁgure can be viewed at wileyonlinelibrary.com] 3. Thomas Rowlandson, The Prospect before Us, 1788, etching, hand-coloured, 23.2 × 32 cm. Courtesy of Beinecke Rare Book and Manuscript Library, Yale University [Colour ﬁgure can be viewed at wileyonlinelibrary.com] 8 NATALEE GARRETT untrustworthy advisors. Indeed, in the image, the queen states, ‘I know nothing of the matter. I follow Billy’s [Pitt’s] advice’. In the background of the image, a group of politi- cians and courtiers watch this powerful triumvirate; at the forefront of the crowd stands William Hastings, wearing a large turban and shouting, ‘Now my diamonds shall befriend me! Huzza!’ The inclusion of Hastings connected the Regency Crisis with the queen’s al- leged involvement in the diamond scandal earlier in 1788: in satirical prints, Charlotte’s greed thus moved from precious stones to political power. Charlotte’s supposed hunger for political power, and her willingness to step over her children’s rights to advance her position, was also depicted in The Q.A. © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. III. 1788-1799: ‘A Pattern of Domestic Virtue’ The paper argued that the Opposition ‘began a most scurrilous attack on the Queen, not only by private conversation, but through the medium of the prints in their interest’.44 After George III’s recovery in February 1789, accusations of political meddling directed at the queen largely petered out, and Linda Colley has in fact highlighted the many loyal addresses which praised the queen’s conduct during the Crisis.45 The queen also received ﬁrm support from many newspapers: for example, in April 1789, The Times claimed that ‘no Queen ever reigned more in the hearts of the people […] she is a pattern of domestic virtue which cannot be too much admired’.46 The World declared of the queen that ‘her whole conduct has been admirable! It is now cherished as referring to something more digniﬁed than any Earthly Throne, DOMESTIC MERIT, and its Reward- It has ediﬁed to every Wife and every Mother in existence- The BEST EXAMPLE in the FIRST PLACE!’47 f d l f h h An examination of newspapers and satirical prints from the Regency Crisis emphasises the pivotal role that the queen played in this very public struggle for political power. A queen once vaunted for never straying beyond her domestic duties was suddenly thrust into the public and highly politicised world of the British press. While numerous newspa- pers continued to praise the queen’s character and her moral conduct during the Crisis, it is evident that a powerful counter image of the queen emerged in this period. This counter image challenged the concept of the queen as a devoted wife and mother by insinuating that Charlotte was eager to grasp the reins of power when the opportunity presented it- self. This negative and hyperbolic image of the queen featured in caricature prints and newspapers, many of which were likely bought and paid for by supporters of the prince of Wales. These prints and articles nonetheless played upon contemporary anxiety about the political inﬂuence of elite women, an anxiety which had featured in British caricatures produced during the Westminster Election in1783.48 Regardless of the genesis of this very public attack on the queen’s character, it is evident that Charlotte’s reputation did suffer during the Regency Crisis, and the negative impact of this crisis continued after the reso- lution of the Crisis. III. 1788-1799: ‘A Pattern of Domestic Virtue’ In satirical prints, Charlotte’s supposed betrayal of her family was made possible through an unholy alliance with Prime Minister Pitt, and in each example, the onus of inﬂuence seems to rest more with the politician than with the queen. Newspaper articles from 1789 similarly point to a belief that Pitt and the queen were scheming together: in January 1789, The World reported that the queen would form a court of her own and that the Opposition now referred to Pitt as ‘her minister’.40 In February 1789, the pseudony- mous satirical commentator known as Peter Pindar published a poem in the Morning Herald, declaring that ‘PITT and the PETTICOAT shall rule together’.41 Although the king gradually regained his health and his ability to rule in February 1789, attacks on the queen and her alleged political machinations endured. The Morning Herald critiqued the queen again in March by publishing a controversial article which suggested that Queen Charlotte, in connivance with Prime Minister William Pitt, had attempted to hide news of the king’s recovery in order to establish their own political agenda.42 Whatever the ‘Albion’s Queen by All Admir’d’ ‘Albion’s Queen by All Admir’d’ 9 truth of Charlotte’s alliance with Pitt, the Crisis undoubtedly caused strain in the queen’s relationship with her eldest son, and this was well known among the court. Smith cites a letter written by Sir Gilbert Elliot, recording that the prince of Wales was initially not in- vited to a celebration for the king’s recovery, as the queen had designated it an event for those who had ‘supported’ herself and the king.43 The queen was almost certainly a victim of a smear campaign during the Crisis, and this very accusation against the Opposition was made by The Times on 15 January 1789. © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. III. 1788-1799: ‘A Pattern of Domestic Virtue’ While individualised criticisms of Charlotte declined rapidly after the resolution of the Regency Crisis, she continued to be a visible royal ﬁgure during the political turbulence of the 1790s. The queen was frequently caricatured alongside her husband in prints which revolved around the long-running spectre of royal avarice which had formed a key component of Charlotte’s popular image during the Hastings scandal. During the 1790s, the royal family was under intense scrutiny, as reform societies in Britain and the overthrow of the French monarchy raised intense discussions about the rights of mon- archy. In 1792, James Gillray attacked the king’s Proclamation against radical and sedi- tious publications by depicting the royal couple’s avarice and parsimony in a print which also satirised the ‘vices’ of the royal princes.49 Accusations of ﬁnancial greed and scheming continued to follow the king and queen: in 1798, for example, the caricature print They Are a Coming, or Deliver Your Money depicted Pitt informing the royal couple that they must use their savings to stop riots in response to the Voluntary Subscription.50 The king hides behind huge sacks of money, while Charlotte is shown wearing extrava- gant jewels and exclaiming, ‘I can never part with my Jewels twill break my poor heart!’ NATALEE GARRETT 10 Once again, Charlotte’s reputation as the queen of diamonds resurfaced during a period of signiﬁcant economic hardship for many people in Britain, occasioned by the drawn-out conﬂict with Revolutionary France.51 Although caricatures and commentaries critical to Charlotte and the royal family over- all continued to emerge in the 1790s, the overthrow of the monarchy in France appears to have encouraged loyalist newspapers to emphasise that Charlotte remained a ﬁgure much adored by the British public.52 At Charlotte’s ofﬁcial birthday in 1793, The World claimed that public celebration of the event showed that ‘the characteristic of Britons is loyalty; particularly to one, who possesses every virtue which can add splendour even to Majesty’. © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. III. 1788-1799: ‘A Pattern of Domestic Virtue’ Further emphasising loyal affection to Charlotte, the paper contrasted the queen’s happy situation with that of Queen Marie-Antoinette of France ‘immured in a sol- itary prison’ and fearing for her life.53 The Times similarly connected the queen’s birthday with loyalist sentiment: ‘the union of parties and the general junction that animates the people of England against the tyrannical republicans of France will make the court at St James’s this day one of the most splendid that has complimented Her Majesty since her coronation’.54 Nonetheless, while the loyalist press hailed Charlotte and George III as symbols of Brit- ish values during the radicalism of the French Revolution, caricaturists instead played upon well-established criticisms of the royal couple and of the queen, especially. In 1791, for example, a violent and ironic image by James Gillray depicted Charlotte and Prime Minister Pitt being hanged from a lamp post while British ‘Jacobins’ prepared to be- head George III.55 Although the image was intended as a mocking, nightmarish vision of the aims of radicals in Britain, the inclusion of Charlotte and Pitt harks back to the cari- catures which depicted their alleged political alliance during the Regency Crisis. Another image produced by Gillray in 1792 took the alliance between queen and Prime Minister even further. Inspired by John Milton’s Paradise Lost, Gillray’s Sin, Death, and the Devil de- picts the queen in the guise of a serpentine monstress (‘Sin’), throwing herself in front of Pitt (‘Death’) as Thurlow (‘the Devil’) prepares to strike him down.56 The print’s transfor- mation of the queen into a monstrous creature and the sexual undertones of the image created a scandal at court.57 It is evident that Charlotte’s supposed political alliance with Pitt endured in satirical prints long after the conclusion of the Regency Crisis, especially as the political culture of the 1790s made discussions about a power-hungry queen more pertinent than ever. The reiteration of key criticisms about Charlotte’s alleged political manoeuvrings and avarice in caricatures of the 1790s emphasises the extent to which both the Hastings scandal and the Regency Crisis had a long-term impact on the queen’s public image. IV. 1800-1818: ‘Am I Not the Queen?’ In contrast with the heated Parliamentary debates of the Crisis of 1788-1789, the com- mencement of the Regency in 1811 did not occasion a succession of concentrated vitriolic attacks on Queen Charlotte.58 Nonetheless, public critiques of the queen continued to ap- pear in newspapers and satirical prints throughout the Regency: suggestions of coldness and cruelty towards her family abounded, calling into question Charlotte’s reputation as a devoted mother. Accusations of political meddling and avarice, which had dominated rhetoric critical of Charlotte during the period 1788-1799, continued in a steady ﬂow un- til her death in1818. In the Regency period, there also emerged a signiﬁcant anti-German discourse in caricatures of the queen, which typiﬁed Charlotte as the stereotypical and ‘Albion’s Queen by All Admir’d’ ‘Albion’s Queen by All Admir’d’ 11 politically threatening ‘foreign queen’.59 Caricatures similarly mocked Charlotte’s old age, where unattractive and haggard depictions of the queen mirrored accusations of old-fashioned ideals and tyrannical behaviour in the royal household. As George III had neither died nor abdicated the throne, Charlotte occupied an unusual position within the royal family, as both the wife of the reigning king and the mother of the Regent. In response to this shift in status, the queen decided to hold a court of her own, separate from that of the Regent. In June 1813, the Morning Post disclosed that the queen had held a ‘private court’ in order to receive the Prussian minister.60 The known establishment of a queen’s court inspired satirical prints such as John Bull in the Council Chamber by George Cruikshank, published in 1813.61 The image conjures a sec- ond, secret royal court, which is discovered by an astonished ‘John Bull’, the ﬁgure who represented the everyday Englishman in this period.62 In the centre of the print, a hag- gard and greatly caricatured Charlotte sits on a throne, bearing crown and sceptre. Sev- eral attendants surround her, offering snuff and sauerkraut, as she laments: ‘Am I not the Q---n, I will not lose one jot of my prerogative’. The queen’s foot rests on an ornate stool labelled ‘Hastings diamonds’, thus resurrecting the twenty-ﬁve-year-old spectre of the Hastings scandal for the British public. IV. 1800-1818: ‘Am I Not the Queen?’ The image shows the queen’s imaginary court peopled with distasteful characters, among them leading politicians involved with the controversial Delicate Investigation into the conduct of the princess of Wales.63 It is signif- icant that this print by Cruikshank unites two of the popular critiques levelled at the queen during the period 1786-1799: hunger for political power and avarice. To these en- during critiques is added a new ‘vice’ of the queen, who was consistently criticised for her cold treatment of her daughter-in-law Caroline. On 5 June 1814, the Examiner dedicated three whole pages to a critique of the treatment that the princess of Wales had received at the hands of the royal family and politicians. The extensive article centred on the revela- tion that the Regent wrote to Queen Charlotte insisting that the princess of Wales be barred from two ofﬁcial gatherings that he himself planned to attend. Although the paper saved its greatest condemnation for the Regent, Queen Charlotte was also criticised for agreeing with her son, a move which was interpreted as contrary to the wishes of not only George III and Parliament but also the British public.64 Where political manoeuvrings had previously placed the queen and prince of Wales in opposing corners, the Regency pe- riod saw satirical artists depict mother and son as unsavoury allies. The queen’s treatment of Caroline was also criticised in satirical prints. For example, in R---l Advice (1814; Fig. 4), the artist suggests that the queen’s desire to run off her daughter-in-law was yet another grasp for power.65 In the image, the Regent asks his mother what he should say about his wife when asked by the allied sovereigns of Europe who were visiting Britain. The queen, sitting on a raised dais and taking a pinch of snuff, replies, ‘I advise you my Son, to say as little as convenient, or d—n it; say I am your R—l Wife’. The print thus presents Charlotte in the most unsavoury terms, as willing to cast aside both her own husband and her daughter-in-law in order to maintain a posi- tion of power. Unlike prints and articles produced in 1788-1789, these later representa- tions of the queen cannot be attributed to a smear campaign from a speciﬁc political party but may instead speak to the growing reform movement in Britain. © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. IV. 1800-1818: ‘Am I Not the Queen?’ These sources do, however, make evident the long-term impact of the Regency Crisis on the queen’s im- age, as accusations of political meddling continued to cling to the queen decades after the Crisis. In fact, the examples from 1811-1818 appear more interested in mocking the queen’s pretensions and foibles than accusing her of a genuine power grab: Charlotte was no longer considered a genuine threat to the political order, but rather, she had be- come a suitable object of mockery in various portions of the media. NATALEE GARRETT 12 4. John Lewis Marks, R---l Advice, 1814, etching, hand-coloured, 25.6 × 41.8 cm. Cour- tesy of The Lewis Walpole Library, Yale University [Colour ﬁgure can be viewed at wileyonlinelibrary.com] 4. John Lewis Marks, R---l Advice, 1814, etching, hand-coloured, 25.6 × 41.8 cm. Cour- tesy of The Lewis Walpole Library, Yale University [Colour ﬁgure can be viewed at wileyonlinelibrary.com] In this period, the character of the queen’s relationships with her six daughters was also increasingly called into question in newspapers and satirical prints. One of the main areas of contention was the fact that most of the royal princesses remained unmarried well into their thirties or forties, a circumstance that prompted the Examiner to declare ‘we do not like the idea of unmarried princesses, any more than that of nuns and vestal virgins’.66 The queen discouraged her daughters from marrying, claiming that such up- heaval would be detrimental to their father’s mental health.67 Nonetheless, her reluc- tance to allow her daughters to marry deprived them of the cultural expectation that they should have their own household and family. The pitiful situation of the royal prin- cesses is alluded to in numerous satirical prints from the 1790s onwards, many of which depict the princesses glumly sitting at tea with their parents or singing in the chapel at Windsor Castle.68 Rumours of affairs with courtiers and equerries were attached to each of the royal princesses, and these rumours often resurfaced in popular discourse when the princess in question eventually succeeded in marrying.69 For example, the marriage of the forty-eight-year-old Princess Elizabeth to the Landgrave of Hesse-Homburg in 1818 prompted several satirical attacks on Queen Charlotte’s morality, on the premise that the queen was instrumental in deceiving the bridegroom of the princess’s virginity. In Old Snuffy Inquiring After Her Daughter Betty by J. © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. IV. 1800-1818: ‘Am I Not the Queen?’ Lewis Marks, the queen enters the bed- chamber of Elizabeth and her new husband, the prince of Hesse-Homburg.70 Charlotte, once again depicted taking snuff, asks her daughter if the prince has discovered that she was not a virgin before her marriage. In another print titled Found It Out; or, a German P- Humbuged, Marks imagines a scenario wherein the prince of Hesse-Homburg does dis- cover the royal deceit and attempts to leave his bride, before being stopped by a furious ‘Albion’s Queen by All Admir’d’ ‘Albion’s Queen by All Admir’d’ 13 and diminutive Charlotte who ardently denies the accusations levelled at her daughter.71 The queen’s moral superiority was also satirised by Peter Pindar in a poem published in 1815. In The Cork Rump, or Queen and Maids of Honour, Pindar imagined the queen insisting that her maids of honour begin wearing excessive padding under their gowns in hopes of prompting a fashion trend that would conveniently conceal secret pregnancies among the princesses.72 Although ﬁctional and highly satirical in tone, these sources cer- tainly challenge the notion of Charlotte as a ﬁgure of ideal motherhood and spotless morality. Charlotte’s relationship with her granddaughter and namesake, Princess Charlotte, was also a popular topic of discussion in the British press between 1811 and the princess’s untimely death in1817. After Princess Charlotte broke off an engagement with the prince of Orange in 1814, the Regent declared that she would be moved to live at Windsor with her grandmother. In satirical prints, the queen is often seen standing in for the princess of Wales as a motherly ﬁgure to Charlotte, arranging meetings between the heiress to the throne and potential suitors, most of whom were of Germanic stock. In a print published in 1816, however, the artist depicts a negative view of the relationship between the queen and her granddaughter. In The Mother’s Girl Plucking a Crow, or German Flesh & English Spirit, Princess Charlotte attacks her grandmother for mistreating her mother, Caroline.73 The queen is depicted grotesquely, and the artist takes pains to emphasise that Queen Charlotte is German, while Princess Charlotte is English: where the queen is old, outdated and foreign, her granddaughter is youthful, modern and a symbol of British fortitude and morality. IV. 1800-1818: ‘Am I Not the Queen?’ Queen Charlotte’s identity as a German princess was reiterated in many carica- tures published during the Regency, with prints such as John Bull in the Council Chamber alluding to this identity through depictions of sauerkraut and the recurrent motif of the queen indulging in ‘Strasbourg’ snuff. The persistent focus on Charlotte’s Germanness in this period is a reﬂection of the rise in British nationalism that emerged as a result of the conﬂict with Revolutionary France and the ensuing Napoleonic Wars.74 Despite hav- ing lived in Britain for most of her life, Charlotte was increasingly depicted in caricatures as a foreign queen, whose foreignness may have posed a threat to the welfare of Britain and the royal family in a politically turbulent period when fears of foreign invasion preyed on the minds of the British public.75 National grief at the loss of a popular heiress to the throne rebounded on Queen Charlotte when princess Charlotte died in childbirth in November 1817. The queen was unwell and had travelled to Bath to recuperate in the winter of 1817, leading to some ac- cusations in the press that she should have been present at her granddaughter’s lying in at Claremont. The Morning Chronicle stated, ‘there was not one Lady, or even female do- mestic, resident at Claremont whose experience could authorize her to be useful to the Princess during her pregnancy or labour. Not one of them was a mother’.76 The press was largely, however, sympathetic to the queen on the loss of her granddaughter, and many reports expressed sincere concern about the impact that the news would have on the already ill queen.77 The Courier, for example, was one of several newspapers which stressed that the queen had not been present at Claremont because it was Princess Charlotte’s wish that the house not be overwhelmed by royal attendants.78 h l d h ’ l l h h h f l l These examples, centred on the queen’s complex relationships with her female rela- tives, undoubtedly challenged the idealised image of a maternal and loving queen that had been cultivated in the public sphere of Britain since the birth of the prince of Wales in 1762. © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. V. 1818: ‘The Object of Universal Esteem’79 On 17 November 1818, Charlotte died after ﬁfty-seven years as queen consort. The queen’s death was greeted by a deluge of newspaper articles and loyal addresses. A cheap and poorly printed elegy from Nottingham highlights how press coverage of the event ex- tended to those at the lower end of the social scale in Britain.80 Conscious of the national signiﬁcance of the event, The Courier claimed that ‘only one opinion exists throughout all ranks of society, as to the unblemished and irreproachable character’ of the queen.81 The Morning Herald praised the queen’s determination to exclude ‘immoral’ women from her court as ‘a more brilliant jewel in the diadem of her moral fame than any which embellished her temporal crown’.82 The Morning Post claimed that Charlotte’s virtues were ‘the object of universal esteem’ but admitted that the queen was not perfect: Faults she might have, for she was human; but they are lost in the magnitude of her good qualities and excellencies; and as long as conjugal virtues are revered, as long as parental af- fection is cherished, and the mild exercise of the loftiest dignity duly appreciated, she will be remembered with love, regret, and admiration by every feeling heart.83 In a second article published the next day, the Morning Post addressed the long-running accusations of political interference that were attributed to Charlotte from the Regency Crisis onwards. The paper asserted that ‘from politics she kept studiously aloof’ and claimed that her actions during the Regency Crisis were motivated only by a desire to sup- port her husband.84 It is signiﬁcant that even as this publication praised the late queen as a paragon of virtue, the editors felt it necessary to address the persistent rumours of polit- ical scheming, avarice and a lack of maternal sentiment that had attached themselves to Charlotte during the latter half of her reign. Even The Times, a staunch supporter of the queen, challenged notions that Charlotte was an ideal mother and a staid queen consort. © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. IV. 1800-1818: ‘Am I Not the Queen?’ While the Regency Crisis prompted some commentators to portray the queen as a mother willing to overthrow her son in order to rule, popular discourse during the Regency instead suggests that the queen was showing decidedly unmaternal sentiments NATALEE GARRETT 14 towards her daughters, daughters-in-law and granddaughter Princess Charlotte. In addition to enduring, periodic accusations of political intrigue coupled with a growing anti-German rhetoric, these public revelations of troubles within the queen’s family likely did further damage to Charlotte’s ‘ofﬁcial’ image in the years preceding her death. V. 1818: ‘The Object of Universal Esteem’79 The paper claimed that the ‘sober dignity’ of the court vanished when George III withdrew from public life in 1811, and it suggested that the queen was in support of the change from ‘grave to gay’ at court.85 The most damning claim made in the article, however, touched on Charlotte’s identity as a mother, stating that ‘much of the praise which was long bestowed upon Her Majesty, as a model of the parental and domestic character, really belonged to her illustrious consort’.86 Accord- ing to The Times, the decision of many of the queen’s children to leave her sickbed undermined the widespread belief that she had dedicated herself to ‘the sacred obligations of a mother towards her offspring’.87 On 22 November 1818, the front page of the Exam- iner was ﬁlled with an article entitled ‘Death and Character of the Queen’. A publication long critical of the queen, the Examiner began by praising The Times for ‘what is called “breaking the ice” respecting Her Majesty’s character and reputation with the public’.88 The article did not deny that the queen ‘had the virtues of appearance and may have had virtues in reality’, but it nonetheless challenged other newspapers and addresses that attempted to paint the queen in a wholly positive light.89 For example, the article stated that ‘in the great Christian virtue of charity, or generosity in money matters […] appear- ances and popular opinion are certainly against her’, before excoriating the queen for her ‘Albion’s Queen by All Admir’d’ 15 cold and un-Christian conduct towards her daughter-in-law, the duchess of Cumberland. The article concluded by stating that ‘The pretence of some of her eulogists, that she did not interfere in politics and intrigue, is refuted not only by all probability, but by what pol- iticians themselves have shewn’, a claim that challenged the accepted view that the queen did not involve herself in political disputes.90 The queen’s death thus allowed for a greater discussion of her public reputation in the British press; while many of these discussions were positive, as may be expected on the death of an individual who had existed in the public eye for almost sixty years, the opinions expressed by publications such as The Times and the Examiner unsettle any notion that Charlotte was universally adored or admired. V. 1818: ‘The Object of Universal Esteem’79 y y The queen’s death did not put a stop to public discussions about her reputation, as nu- merous posthumous biographies appeared for sale in the years following 1818. In 1820, an ‘H.W. Fitz-George’ published a satirical biography of Charlotte, entitled Memoirs of the Late Mrs. King (Otherwise the Diamond Queen!).91 Although a satirical work, the ‘biog- raphy’ did record much of the gossip that surrounded Charlotte throughout her later life, particularly her ill treatment of her daughters-in-law and her want of charity. The text concluded that Mrs King’s death ‘did not create the sensation which the loss of a good lady […] would have done; her old acquaintances, wished for her sake, it had happened twenty years before, when the presence of her husband rendered her respected’.92 The text thus suggests that the queen’s popularity and respectability declined as a direct result of George III’s gradual withdrawal from the public and political roles of kingship. Cer- tainly, Charlotte’s decision to maintain a ‘queen’s court’ and the inﬂuence that she exerted over the inner politics of the royal family were well documented and criticised in satirical prints and newspaper articles during the Regency period. Also noting the de- terioration of Charlotte’s reputation in her later years, the allegedly impartial memoirs of the queen’s life written by Walley Chamberlain Oulton stated that Charlotte was the ‘idol of the people’ in her early years but that she ‘seemed to outlive those people by whom she was thus idolized and it must be acknowledged that her popularity considerably declined previous to her decease’.93 Despite the hopes of Charlotte’s contemporary supporters, it was perhaps unrealistic to believe that the public reputation of a royal individual could re- main perfectly positive for a period of almost sixty years. © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. VI. Conclusion This article has shown that Queen Charlotte’s contemporary image evolved signiﬁcantly between 1786 and her death. The queen’s reputation was greatly impacted by factional politics during the Regency Crisis, but it appears that popular media continued to pillory her long after she ceased to be a genuine threat to the prince of Wales or to the political status quo of Britain. Queen Charlotte did not exist only as a colourless ‘totem of morality’ to the British people, as suggested by Colley: a close examination of newspaper articles and satirical prints shows that the queen possessed a complex and multifaceted reputation in the British public sphere.94 The reliability or veracity of the sources examined cannot be measured accurately, but the value of the sources lies in their ability to show us how Queen Charlotte was represented in popular media throughout her reign: positive and negative, sober and outlandish; they all form a complex image of a woman who existed at the heart of British society for almost sixty years. Extant scholarship has overwhelm- ingly labelled Charlotte as a domestic, moral and maternal ﬁgure adored by the British public throughout her life, and there is certainly evidence to support this viewpoint in many of the sources examined here. There was also, however, an alternate image of the NATALEE GARRETT 16 queen, one that emerged from the Hastings scandal of 1786 and endured until her death. In this alternate image, Charlotte’s reputation was that of an avaricious and domineering woman who longed for political power. Queen Charlotte may have been admired by many people during her lifetime, but it certainly cannot be argued that she was admired by all. NOTES I am grateful to Sarah Easterby-Smith for her helpful comments on the ﬁrst draft of this article. Many thanks are due also to the anonymous reviewers for their insightful comments and recommendations. 1. Morning Herald (20 November 1818). 2. Scholarship on Queen Charlotte is composed of general biographies and studies of her artis- tic and scientiﬁc patronage. Olwen Hedley, Queen Charlotte (London: J. Murray, 1975) remains the most comprehensive biography of Charlotte to date. Marcia Pointon has examined the cultural im- pact of diamonds on queenship, with consideration of Queen Charlotte: Marcia Pointon, Brilliant Effects: A Cultural History of Gem Stones and Jewellery (New Haven, CT: Yale University Press, 2009). For Charlotte’s role as patron, see Clarissa Campbell Orr, ‘Queen Charlotte “Scientiﬁc Queen”’, in Clarissa Campbell Orr (ed.), Queenship in Britain, 1660-1837: Royal Patronage, Court Culture and Dynastic Politics (Manchester: Manchester University Press, 2002), p.236-66; Heidi Strobel, The Artistic Matronage of Queen Charlotte (1744-1818): How a Queen Promoted Both Art and Female Artists in English Society (Lewiston, NY: Edwin Mellen Press, 2011). 3. Cindy McCreery, The Satirical Gaze: Prints of Women in Late Eighteenth-Century England (Oxford: Clarendon, 2004), p.179,198. McCreery particularly contrasts Charlotte’s domestic repu- tation with that of ‘politicised’ women such as Georgiana, duchess of Devonshire. 3. Cindy McCreery, The Satirical Gaze: Prints of Women in Late Eighteenth-Century England (Oxford: Clarendon, 2004), p.179,198. McCreery particularly contrasts Charlotte’s domestic repu- tation with that of ‘politicised’ women such as Georgiana, duchess of Devonshire. 4. Linda Colley, Britons: Forging the Nation, 1707-1837, 3rd edn (London: Pimlico, 2003), p.268. 4. Linda Colley, Britons: Forging the Nation, 1707-1837, 3rd edn (London: Pimlico, 2003), p.268. 5. E. A. Smith emphasises Charlotte’s complex, and sometimes negative, relationship with her children. E. A. Smith, George IV (New Haven, CT: Yale University Press, 1999). 5. E. A. Smith emphasises Charlotte’s complex, and sometimes negative, relationship with her children. E. A. Smith, George IV (New Haven, CT: Yale University Press, 1999). 6. Marilyn Morris, Sex, Money and Personal Character in Eighteenth-Century British Politics (New Haven, CT: Yale University Press, 2015), p.6. 6. Marilyn Morris, Sex, Money and Personal Character in Eighteenth-Century British Politics (New Haven, CT: Yale University Press, 2015), p.6. 7. A poem published for the queen’s birthday. General Advertiser and Morning Intelligencer (19 May 1778). 7. A poem published for the queen’s birthday. General Advertiser and Morning Intelligencer (19 May 1778). 8. © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. NOTES Anon, The Royal Family of Great-Britain, circa 1783, wood engraving. British Museum (2000,0723.15). 8. Anon, The Royal Family of Great-Britain, circa 1783, wood engraving. British Museum (2000,0723.15). 9. Johan Joseph Zoffany’s 1771 portrait of the royal family was available to the public in the form of mezzotint prints. Richard Earlom, after Johan Zoffany, Their Most Sacred Majesties George the IIId and Queen Charlotte, 1771, mezzotint. British Museum (Q,2.6); Anon, after Johan Zoffany, Queen Charlotte and Her Two Eldest Children, date unknown, photogravure. British Museum (1908,0714.146). See Marcia Pointon, Hanging the Head: Portraiture and Social Formation in Eighteenth-Century England (New Haven, CT: Yale University Press, 1993), p.161-7. 9. Johan Joseph Zoffany’s 1771 portrait of the royal family was available to the public in the form of mezzotint prints. Richard Earlom, after Johan Zoffany, Their Most Sacred Majesties George the IIId and Queen Charlotte, 1771, mezzotint. British Museum (Q,2.6); Anon, after Johan Zoffany, Queen Charlotte and Her Two Eldest Children, date unknown, photogravure. British Museum (1908,0714.146). See Marcia Pointon, Hanging the Head: Portraiture and Social Formation in Eighteenth-Century England (New Haven, CT: Yale University Press, 1993), p.161-7. 11. St James’ Chronicle or the British Evening Post (3-6 October 1778). 12. General Advertiser and Morning Intelligencer (19 May 1778). 12. General Advertiser and Morning Intelligencer (19 May 1778) 13. London Chronicle (17-9 January 1782). 13. London Chronicle (17-9 January 1782). 14. For analysis of the 1765 Regency Crisis, see D. Jarrett, ‘The Regency Crisis of 1765’, English Historical Review 85:335 (1970), p.282-315. 14. For analysis of the 1765 Regency Crisis, see D. Jarrett, ‘The Regency Crisis of 1765’, English Historical Review 85:335 (1970), p.282-315. 15. Jarrett, ‘The Regency Crisis of 1765’, p.300-1. ‘Albion’s Queen by All Admir’d’ 17 16. John Bullion, ‘The Origins and Signiﬁcance of Gossip about Princess Augu 1755-1756’, Studies in Eighteenth-Century Culture 21:1 (1992), p.245-65. 17. Westminster Journal and London Political Miscellany (18 May 1765). 18. Henry Kingsbury, The Farm Yard, 1786, etching, hand-coloured. British Museum (1868,0808.5522). 18. Henry Kingsbury, The Farm Yard, 1786, etching, hand-coloured. British Museum (1868,0808.5522). 19. Anon, The Constant Couple, 1786, etching, hand-coloured. Lewis Walpole Library, Yale University (786.02.24.01.1+). M. Dorothy George singles out this print at the true beginning of the ‘Farmer George’ images. M. Dorothy George, English Political Caricature: A Study of Opinion and Propaganda, 2 vols (Oxford: Clarendon Press, 1959), vol. II.190. 19. Anon, The Constant Couple, 1786, etching, hand-coloured. NOTES Lewis Walpole Library, Yale University (786.02.24.01.1+). M. Dorothy George singles out this print at the true beginning of the ‘Farmer George’ images. M. Dorothy George, English Political Caricature: A Study of Opinion and Propaganda, 2 vols (Oxford: Clarendon Press, 1959), vol. II.190. 20. Vincent Carretta has argued that Farmer George prints produced in the 1790s were a warning that the king needed to show the impressive power of the monarchy in the face of encroaching French radicalism. Vincent Carretta, George III and the Satirists from Hogarth to Byron (Athens, GA: University of Georgia Press, 1990), p.294. 20. Vincent Carretta has argued that Farmer George prints produced in the 1790s were a warning that the king needed to show the impressive power of the monarchy in the face of encroaching French radicalism. Vincent Carretta, George III and the Satirists from Hogarth to Byron (Athens, GA: University of Georgia Press, 1990), p.294. 21. Pointon, Brilliant Effects, p.192. 22. Morning Herald (6 July 1786). 23. Anon, The Queen of Hearts Cover’d with Diamonds, 1786, etching, hand-coloured. British Museum (1868,0808.5560). 23. Anon, The Queen of Hearts Cover’d with Diamonds, 1786, etching, hand-coloured. British Museum (1868,0808.5560). 24. George, English Political Caricature, p.191. 25. Anon, The Diamond Eaters, Horrid Monsters!,1788, etching, hand-coloured. British Museum (1868,0808.5740). 25. Anon, The Diamond Eaters, Horrid Monsters!,1788, etching, hand-coloured. British Museum (1868,0808.5740). 26. James Gillray, The Bow to the Throne; Alias the Begging Bow, 1788, etching, hand-coloured. British Museum (1868,0808.5726). 26. James Gillray, The Bow to the Throne; Alias the Begging Bow, 1788, etching, hand-coloured. British Museum (1868,0808.5726). 27. Anon, English Slavery; or, a Picture of the Times, 1788, etching. British Museum (1868,0612.1244). 8 P i t B illi t Eff t 8 27. Anon, English Slavery; or, a Picture of the Times, 1788, etching. British Museum (1868,0612.1244). 28. Pointon, Brilliant Effects, p.181-4. 29. James Gillray, A New Way to Pay the National-Debt, 1786, etching, hand-coloured. British Museum (1868,0808.5519). 30. The Times (20 January 1787). 31. The Times (20 January1787). In fact, the queen’s simple costume on this occasion was not a response to the Hastings scandal, but rather a tradition observed by the royal couple. The queen would dress lavishly on the king’s birthday, but modestly on her own. Likewise, the king would dress in his ﬁnest for his wife’s birthday but wear simple costume on his own birthday. 32. Gazetteer and New Daily Advertiser (19 January 1787). 33. 41. Morning Herald (23 February 1789). 40. The World (21 January 1789). © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. NATALEE GARRETT NATALEE GARRETT 42. Morning Herald (7 March 1789). The Crown ultimately pursued a libel case against the paper for this article. 42. Morning Herald (7 March 1789). The Crown ultimately pursued a libel case against the paper for this article. 43. ‘Sir Gilbert to Lady Elliot, 9 April 1789’, in Countess of Minto (ed.), Life and Letters of Sir Gilbert Elliot, 3 vols (London: Longmans, 1874), vol. I.300. Cited in Smith, George IV, p.58. 43. ‘Sir Gilbert to Lady Elliot, 9 April 1789’, in Countess of Minto (ed.), Life and Letters of Sir Gilbert Elliot, 3 vols (London: Longmans, 1874), vol. I.300. Cited in Smith, George IV, p.58. 44. The Times (15 January 1789). 45. Colley, Britons, p.270. 46. The Times (18 April 1789). 47. The World (23 February 1789). 48. The reactions of the press to the duchess of Devonshire’s involvement in the 1783 Westminster Election are a prime example of contemporary concern about the political power of elite women. See Amanda Foreman, ‘A Politician’s Politician: Georgiana, Duchess of Devonshire and the Whig Party’, in Hannah Barker and Elaine Chalus (eds), Gender in Eighteenth-Century England: Roles, Representations and Responsibilities (London: Longman, 1997), p.179-204. 49. James Gillray, Vices Overlook’d in the New Proclamation, 1792, etching, hand-coloured. British Museum (1868,0808.6199). 49. James Gillray, Vices Overlook’d in the New Proclamation, 1792, etching, hand-coloured. British Museum (1868,0808.6199). 50. Richard Newton, They Are a Coming, or Deliver Your Money, 1798, etching, hand-coloured. British Museum (1868,0808.6687). 50. Richard Newton, They Are a Coming, or Deliver Your Money, 1798, etching, hand-coloured. British Museum (1868,0808.6687). 51. Frank O’Gorman has emphasised the economic ﬂuctuations and ‘crises of subsistence’ caused by the wars with France. Frank O’Gorman, The Long Eighteenth Century: British Political and Social History 1688-1832 (London: Arnold, 1997), p.233. 52. For analysis of radicalism and caricature in the 1790s, see Diana Donald, The Age of Carica- ture: Satirical Prints in the Reign of George III (New Haven, CT: Yale University Press, 1996), p.142-83. 52. For analysis of radicalism and caricature in the 1790s, see Diana Donald, The Age of Carica- ture: Satirical Prints in the Reign of George III (New Haven, CT: Yale University Press, 1996), p.142-83. 53. The World (19 January 1793). 54. The Times (18 January 1793). 55. James Gillray, The Hopes of the Party, prior to July 14th- ‘From Such Wicked Crown & Anchor- Dreams, Good Lord Deliver Us’, 1791, etching, hand-coloured. NOTES Public Advertiser (22 October 1788). 34. George, English Political Caricature, p.199. 35. Morning Herald (19 January 1789). 36. The Times (7 February 1789). 37. Thomas Rowlandson, The Prospect before Us, 1788, etching, hand-coloured. Beinecke Rare Book and Manuscript Library, Yale University (Auchincloss Rowlandson v.3). 37. Thomas Rowlandson, The Prospect before Us, 1788, etching, hand-coloured. Beinecke Rare Book and Manuscript Library, Yale University (Auchincloss Rowlandson v.3). 38. Thomas Rowlandson, The Q.A. Loaded with the Spoils of India and Britain, 1788, etching, hand-coloured. British Museum (1868,0808.5723). 38. Thomas Rowlandson, The Q.A. Loaded with the Spoils of India and Britain, 1788, etching, hand-coloured. British Museum (1868,0808.5723). 39. James Gillray, Weird Sisters; Ministers of Darkness; Minions of the Moon,1791, etching, hand- coloured. British Museum (1851,0901.570). Extant copies of this print held in the British Museum are dated 23 December 1791. However, the contemporary memoirs of Nathaniel Wraxall state that the print was published during the Regency Crisis (1788-1789). It is possible that the extant ver- sions from 1791 were reprints of the original. See Nathaniel Wraxall, Posthumous Memoirs of His Own Time, 3 vols (London: Richard Bentley, 1818), vol. III.309-10. 41. Morning Herald (23 February 1789). 18 NATALEE GARRETT British Museum (1868,0808.6086). 56. James Gillray, Sin, Death, and the Devil. Vide Milton, 1792, etching, hand-coloured. British Museum (1868,0808.6212). 55. James Gillray, The Hopes of the Party, prior to July 14th- ‘From Such Wicked Crown & Anchor- Dreams, Good Lord Deliver Us’, 1791, etching, hand-coloured. British Museum (1868,0808.6086). 56. James Gillray, Sin, Death, and the Devil. Vide Milton, 1792, etching, hand-coloured. British Museum (1868,0808.6212). 57. M. Dorothy George, Catalogue of Political and Personal Satires Preserved in the Department of Prints and Drawings in the British Museum (London: British Museum, 1938), vol. VI.917. See also Vic Gatrell, City of Laughter: Sex and Satire in Eighteenth-Century London (London: Atlantic, 2006), p.265-6. 57. M. Dorothy George, Catalogue of Political and Personal Satires Preserved in the Department of Prints and Drawings in the British Museum (London: British Museum, 1938), vol. VI.917. See also Vic Gatrell, City of Laughter: Sex and Satire in Eighteenth-Century London (London: Atlantic, 2006), p.265-6. 58. Olwen Hedley argues that the proposed Regency Bill of 1810 did not invite the same level of political dissension as had the proposed Bill of 1789. Hedley, Queen Charlotte, p.242. 58. Olwen Hedley argues that the proposed Regency Bill of 1810 did not invite the same level of political dissension as had the proposed Bill of 1789. Hedley, Queen Charlotte, p.242. 59. Katherine Crawford, ‘Constructing Evil Foreign Queens’, The Journal of Medieval and Early Modern Studies 37:2 (2007), p.393-418. 59. Katherine Crawford, ‘Constructing Evil Foreign Queens’, The Journal of Medieval and Early Modern Studies 37:2 (2007), p.393-418. 60. Morning Post (30 June 1813). 61. George Cruikshank, John Bull in the Council Chamber, 1813, etching, hand-coloured. British Museum (1865,1111.2065). 61. George Cruikshank, John Bull in the Council Chamber, 1813, etching, hand-coloured. British Museum (1865,1111.2065). 62. For examination of the ﬁgure of John Bull in caricature, see Reva Wolf, ‘John Bull, Liberty, and Wit: How England Became Caricature’, in Todd Porterﬁeld (ed.), The Efﬂorescence of Caricature (Farnham: Ashgate, 2011), p.49-60. 62. For examination of the ﬁgure of John Bull in caricature, see Reva Wolf, ‘John Bull, Liberty, and Wit: How England Became Caricature’, in Todd Porterﬁeld (ed.), The Efﬂorescence of Caricature (Farnham: Ashgate, 2011), p.49-60. 63. For a study on Queen Caroline and the ‘Delicate Investigation’ in popular culture, see Anna Clark, ‘Queen Caroline and the Sexual Politics of Popular Culture in London,1820’, Representations 31, Special Issue: The Margins of Identity in Nineteenth-Century England (Summer, 1990), p.47-68. NATALEE GARRETT 63. For a study on Queen Caroline and the ‘Delicate Investigation’ in popular culture, see Anna Clark, ‘Queen Caroline and the Sexual Politics of Popular Culture in London,1820’, Representations 31, Special Issue: The Margins of Identity in Nineteenth-Century England (Summer, 1990), p.47-68. 64 Examiner (5 June 1814) 31, Special Issue: The Margins of Identity in Nineteenth-Century England (Summer, 1990), p.47-68. 64. Examiner (5 June 1814). 31, Special Issue: The Margins of Identity in Nineteenth-Century England (Summer, 1990), p 64. Examiner (5 June 1814). 65. John Lewis Marks, R---l Advice, 1814, etching, hand-coloured. Lewis Walpole Library, Yale University (Folio 75W87 807 v.5). 65. John Lewis Marks, R---l Advice, 1814, etching, hand-coloured. Lewis Walpole Library, Yale University (Folio 75W87 807 v.5). ‘Albion’s Queen by All Admir’d’ 19 (9 J ) 67. Jeremy Black, George III: America’s Last King (New Haven, CT: Yale University Press, 2006), p.156. 67. Jeremy Black, George III: America’s Last King (New Haven, CT: Yale University Press, 2006), p.156. 67. Jeremy Black, George III: America’s Last King (New Haven, CT: Yale University Press, 2006), p.156. 68. See, for example, James Gillray, Anti-saccharites, or John Bull and His Family Leaving Off the Use of Sugar, 1792, etching, hand-coloured. British Museum (1935,0522.4.21). See also Richard Newton, Psalm Singing at the Chapel, 1792, etching. British Museum (1868,0808.6194). 68. See, for example, James Gillray, Anti-saccharites, or John Bull and His Family Leaving Off the Use of Sugar, 1792, etching, hand-coloured. British Museum (1935,0522.4.21). See also Richard Newton, Psalm Singing at the Chapel, 1792, etching. British Museum (1868,0808.6194). 69. A. W. Purdue, ‘George III, Daughters of (Act. 1766-1857)’, in Oxford Dictionary of National Biography (23 September 2004), https://doi.org/10.1093/ref:odnb/9780198614128.001.0001/ odnb-9780198614128-e-59209 [accessed 17 February 2021]. Biography (23 September 2004), https://doi.org/10.1093/ref:odnb/9780198614128.001.0001/ odnb-9780198614128-e-59209 [accessed 17 February 2021]. 70. J. Lewis Marks, Old Snuffy Inquiring After Her Daughter Betty, 1818, lithograph, hand- coloured. British Museum (1868,0808.8384). 70. J. Lewis Marks, Old Snuffy Inquiring After Her Daughter Betty, 1818, lithograph, hand- coloured. British Museum (1868,0808.8384). 71. J. Lewis Marks, Found It Out; or, a German P- Humbuged, 1818, lithograph, hand-coloured. British Museum (1868,0808.8383). 71. J. Lewis Marks, Found It Out; or, a German P- Humbuged, 1818, lithograph, hand-coloured. British Museum (1868,0808.8383). 72. Peter Pindar, The Cork Rump, or Queen and Maids of Honour: A Poem (London, 1815). 73. George Cruikshank, The Mother’s Girl, Plucking a Crow, or German Flesh & English Spirit, 1816, etching, hand-coloured. British Museum (1868,0808.8312). 73. © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies NATALEE GARRETT George Cruikshank, The Mother’s Girl, Plucking a Crow, or German Flesh & English Spirit, 1816, etching, hand-coloured. British Museum (1868,0808.8312). 74. See Emma Vincent Macleod, A War of Ideas: British Attitudes to the Wars against Revolution- ary France 1792-1802 (Aldershot: Ashgate, 1998); see also Colley, Britons. 74. See Emma Vincent Macleod, A War of Ideas: British Attitudes to the Wars against Revolution- ary France 1792-1802 (Aldershot: Ashgate, 1998); see also Colley, Britons. 75. Clive Emsley has highlighted the British government’s persistent fears of French invasion during the wars with Revolutionary France. See Clive Emsley, ‘Repression, “Terror” and the Rule of Law in England during the Decade of the French Revolution’, The English Historical Review 100:397 (1985), p.801-25. Political anxiety over the inﬂuence of ‘foreign’ queens in eighteenth-century Europe has been examined in the case of Queen Charlotte’s contemporary, Marie-Antoinette of France, whose Austrian identity became a key motif of her trial in 1793. See Thomas E. Kaiser, ‘Who’s Afraid of Marie-Antoinette? Diplomacy, Austrophobia and the Queen’, French History 14:3 (2000), p.241-71. 76. Morning Chronicle (11 November 1817). 77. See, for example, the Morning Post (10 November 1817) and the Lancaster Gazetteer (15 November 1817). 78. The Courier cited in the Examiner (16 November 1817). 79. Morning Post (18 November 1818) 80. Charles Sambroke Ordoyno, The Lamented Death of Her Late Most Gracious Majesty, Charlotte, Queen of Great Britain (Nottingham, 1818). British Museum, London (1999,0627.4). 81. The Courier, cited in the Examiner (22 November 1818). 82. Morning Herald (20 November 1818). 83. Morning Post (18 November 1818). 83. Morning Post (18 November 1818). 84. Morning Post (19 November 1818). 84. Morning Post (19 November 1818). 85. The Times (18 November 1818). 85. The Times (18 November 1818). 86. The Times (18 November 1818). 87. The Times (18 November 1818). 88. Examiner (22 November 1818). 89. Examiner (22 November 1818). 90. Examiner (22 November 1818). 91. H.W. Fitz-George, Memoirs of the Late Mrs. King (Otherwise the Diamond Queen!) with Interest- ing and Authentic Biographical Anecdotes; Containing Some Curious and Instructive Notices of Political and Family Transactions, Hitherto Not Generally Known (London, 1820). 91. H.W. Fitz-George, Memoirs of the Late Mrs. King (Otherwise the Diamond Queen!) with Interest- ing and Authentic Biographical Anecdotes; Containing Some Curious and Instructive Notices of Political and Family Transactions, Hitherto Not Generally Known (London, 1820). 92. Fitz-George, Memoirs of the Late Mrs. King, p.63. © 2022 The Authors. 66. Examiner (9 June 1816). NATALEE GARRETT was awarded her PhD in History from the University of St Andrews in 2021 for a thesis entitled ‘“Those Scandalous Prints”: Caricatures of the Elite in France and Britain, c.1740-1795’. Her research explores visual culture, public opinion and identity in eighteenth-century Europe. 93. Walley Chamberlain Oulton, Authentic and Impartial Memoirs of Her Late Majesty, Charlotte, Queen of Great Britain and Ireland (London: J. Robins and Co., Albion Press, 1819), p.iii-v. © 2022 The Authors. Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. NATALEE GARRETT Journal for Eighteenth-Century Studies published by John Wiley & Sons Ltd on behalf of British Society for Eighteenth-Century Studies. NATALEE GARRETT NATALEE GARRETT 20 93. Walley Chamberlain Oulton, Authentic and Impartial Memoirs of Her Late Majesty, Charlotte, Queen of Great Britain and Ireland (London: J. Robins and Co., Albion Press, 1819), p.iii-v. 94. Colley, Britons, p.268. 94. Colley, Britons, p.268.
https://openalex.org/W4285592146	https://www.researchsquare.com/article/rs-1682399/latest.pdf	English	null	Comparison of dynamic and static spacers for the treatment of infections following total knee replacement: a systematic review and meta-analysis	Journal of orthopaedic surgery and research	2,022	cc-by	7,917	Research Article Keywords: Static spacers, Dynamic spacers, Periprosthetic joint infection, Total Knee Arthroplasty, meta-analysis Posted Date: May 25th, 2022 Keywords: Static spacers, Dynamic spacers, Periprosthetic joint infection, Total Knee Arthroplasty, meta-analysis Posted Date: May 25th, 2022 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/17 Page 1/17 Background Revision surgery is the most common treatment for patients who develop infection after total knee arthroplasty. Two types of spacers are often used in revision surgery: dynamic spacers and static spacers. The comparative efficacy of these two types of spacers on knee prosthesis infections is not well established. Therefore, we carried out a systematic evaluation and meta-analysis with the aim of comparing the difference in efficacy between dynamic and static spacers. Methods We conducted the literature search in PubMed, Web of Science, Cochrane Library, and Embase databases. The articles searched were clinical study comparing the difference in efficacy between dynamic spacers and static spacers for the treatment of prosthetic infections occurring after total knee arthroplasty. Results We conducted a literature search and screening based on the principles of PICOS. Ultimately, 14 relevant clinical studies were included in our current study. We use infection control rate as the primary evaluation indicator. The KSS knee scores (KSSs), KSS functional scores, bone loss and ROM are secondary indicators of evaluation. Thirteen of these included studies reported the infection control rates, with no significant difference between dynamic and static shims (RR: 1.03; 95% Cl: 0.98,1.09; P = 0.179 > 0.05). The KSSs were reported in 10 articles (RR: 5.98; 95% CI:0.52, 11.43; P = 0.032 < 0.05). 6 articles reported the KSS functional scores (RR: 13.90; 95% CI:4.95, 22.85; P = 0.02 < 0.05). 12 articles reported the ROM (RR: 17.23. 95% CI:10.18, 24.27; P < 0.0001).Six articles reported the bone loss (RR: 2.04; 95% CI:1.11, 3.77; P = 0.022 < 0.05). Conclusion Current evidence demonstrates that dynamic spacers are comparable to static spacers in controlling prosthetic joint infection. In terms of improving the functional prognosis of the knee joint, dynamic spacers are more effective than static spacers. Background For patients with severe knee injuries, total knee arthroplasty can effectively improve the function of the knee joint, relieve knee pain, and improve the quality of life of patients. Knee prosthesis joint infection is one of the most terrible complications after the total knee arthroplasty, and it is often the main reason for the failure of the total knee arthroplasty [1, 2]. The probability of prosthesis joint infection after primary knee replacement varies from 0.4–3% [3, 4]. There are many risk factors for PJI in knee prosthesis infection. Age, duration of surgery, diabetes, urinary tract infection and rheumatoid arthritis are all important risk factors for PJI after TKA surgery [5, 6]. Currently, the number of TKA operations worldwide is increasing year by year. However, due to the trend of aging population and rising obesity rate, the incidence of PJI after TKA is also increasing year by year [7, 8]. The diagnosis and treatment of infections in artificial joint prostheses is still challenging, especially in the early stages. The latest diagnosis of knee prosthesis joint infection is largely based on the diagnostic criteria proposed by the Musculoskeletal Infection Society (MSIS), and PJI is diagnosed when one of the following three conditions is met : (1)the presence of a sinus tract that communicates with the prosthesis; (2) pathogens isolated from two or more separate tissue or fluid samples obtained from the affected prosthetic joint; and (3) four of the six criteria identified are met: 1. the elevation of erythrocyte sedimentation rate (ESR) and c-reactive protein (CRP) concentrations; 2. the elevation of white blood cells (WBC); 3. the elevation of the percentage of Mesophilic polymorphonuclear leukocytes (PMN); 4. septicemia: septicemia of the affected joint; 5. isolation of microorganisms in cultures in histological analysis at 400x magnification; 6. more than 5 neutrophils present in each of the 5 high magnification fields [9–11]. Early and definitive diagnosis is the key to treating PJI. Once the diagnosis of PJI is made, the treatment options include conservative treatment with preservation of the joint prosthesis or debridement, and one or two stage prosthetic replacement surgery. The two-stage arthroprosthetic revisions is the gold standard for the treatment of PJI and is the most common treatment in clinical practice. Exclusion criteria. In addition to the inclusion criteria described above,we have also developed exclusion criteria. Articles were excluded when one of the following conditions existed: (I) only patients using dynamic spacers or static spacers in the article; (II) there was a lack of follow-up time in the article or the follow-up time was less than 12 months; (III) the outcome indicators and statistical methods associated with the article were not uniform; (IV) literature reviews, case reports or experimental animal studies; (V) the diagnostic criteria for subjects were not referenced to the Infectious Diseases Society of America ( IDSA), Musculoskeletal Infection Society (MSIS). Data extraction and management. Inclusion Criteria. (I)Clinical randomised controlled studies, retrospective case-control studies or prospective cohort studies;(II)the outcome indicators in the article included one of infection control rate, KSSs score, KSS functional score, ROM or bone loss; (III) complete clinical report; (IV) in the clinical grouping, patients in the trial group used dynamic spacers and patients in the control group used static spacers; (V)there are specific follow-up times in the article and the follow-up times are all over 12 months; (VI) all patients included in the study met the diagnostic criteria for joint prosthesis infection as defined by the Infectious Diseases Society of America (IDSA), Musculoskeletal Infection Society (MSIS). Study design and search strategy We followed the principles of The Population-Intervention-Comparators-Outcomes-Study design (PICOS) strictly when conducting our literature search.The databases used for literature searches include PubMed, Embase, Web of Science and Cochrane library databases.The time frame for the search was from database creation to 10 March 2022 and the article type were Clinical randomised controlled studies, retrospective case-control studies or prospective cohort studies of dynamic spacers and static spacers for the treatment of knee prosthesis infections. The following keywords were used in the search process: "static spacer" and "dynamic spacer" and "Knee prosthesis infection", "static spacer" and "Articulating spacer" and "PJI ". We had no restrictions on language, region or population, and all study subjects were human. All patients included in the studies met the diagnostic criteria for joint prosthesis infection as defined in the Infectious Diseases Society of America (IDSA), Musculoskeletal Infection Society (MSIS) [23, 24]. All studies that did not meet this diagnostic criterion were excluded. The specific search process is shown in Fig. 1. Background In the tow-stage of prosthetic revision, the infected prosthesis is removed and a joint spacer with antibiotics is placed into the joint to provide continuous anti-infection treatment and then a Page 2/17 Page 2/17 new prosthesis is reinserted once the infection has been controlled. The two-stage of prosthetic revision surgery has achieved satisfactory results in current clinical practice [12–14]. In the two-stage of the revision surgery, the two types of spacers that are frequently used include dynamic spacers and static spacers [15]. For static spacer, it is easier to design and the production costs of it are cheaper. In addition, static spacers are easier to implant in the joint during surgery. However, the mobility of the patient's knee joint will reduce after static spacer implantation. Static spacers are also prone to a number of complications such as causing bone loss and soft tissue damage [16, 17]. In contrast, dynamic spacers can improve the recovery of patient's knee function and reduce the incidence of associated complications. The use of spacers can provide good flexion and extension after the implantation of a new prosthesis. However, dynamic spacers are more expensive to produce. And they are not as good as static spacers in maintaining joint stability [18, 19]. In terms of infection control, some reports suggest that dynamic spacers and static spacers are similarly effective, while others suggest that dynamic spacers are less effective than static spacers in controlling infection [20– 22]. In order to further investigate whether there are any significant differences between dynamic and static spacers for the treatment of knee prosthesis infections in terms of efficacy, complications and functional impact.We conducted a comprehensive search of the literature and collated the data of inclusion literatures, then we did a corresponding meta-analysis and obtained the final results. In order to further investigate whether there are any significant differences between dynamic and static spacers for the treatment of knee prosthesis infections in terms of efficacy, complications and functional impact.We conducted a comprehensive search of the literature and collated the data of inclusion literatures, then we did a corresponding meta-analysis and obtained the final results. Identification of included studies After a literature search in PubMed, Embase, Web of Science and Cochrane library databases, a total of 353 relevant articles were initially retrieved. We first read the titles and abstracts of the articles to eliminate duplicates or articles with inconsistent content, leaving 62 articles. The content of the remaining 62 articles was carefully read by two of our researchers, and after excluding 48 of the non-compliant articles, we were left with 14 articles that met the requirements of our study [26–39]. The specific search and selection process for our included articles is referenced in Fig. 1. Data extraction and management. After determining the final inclusion literatures, we had two researchers responsible for extracting and summarising the data from the articles, and the inclusion and exclusion of literature strictly followed the criteria described above. Articles were included and excluded by reading the title, abstract and content. If there was a disagreement in the screening and data extraction process, a third researcher was responsible for negotiating a resolution. After the inclusion of literatures, data on infection control rates, KSS scores, KSS functional scores, bone loss, ROM and other relevant indicators were extracted from the literature. We extracted and integrated the data and then performed a meta-analysis. Page 3/17 Page 3/17 In addition, after identifying the 14 pieces of literature included, we assessed and scored the methodological quality of each study independently on the Newcastle-Ottawa Scale (NOS)[25]. We evaluated the quality of articles based on the NOS scale for the following items: (I) selection of study population (group), (II) comparability, and (III) how to determine which spacers to use. With a full score of 9 stars, the study is considered to be of high quality when the following conditions exist༚(I) 3 or 4 stars in the selection domain, (II) 1 or 2 stars in the comparability domain, and (II) 2 or 3 stars in the outcome or exposure domain. In addition, after identifying the 14 pieces of literature included, we assessed and scored the methodological quality of each study independently on the Newcastle-Ottawa Scale (NOS)[25]. We evaluated the quality of articles based on the NOS scale for the following items: (I) selection of study population (group), (II) comparability, and (III) how to determine which spacers to use. With a full score of 9 stars, the study is considered to be of high quality when the following conditions exist༚(I) 3 or 4 stars in the selection domain, (II) 1 or 2 stars in the comparability domain, and (II) 2 or 3 stars in the outcome or exposure domain. Outcome measurements Our primary outcome indicator for this meta-analysis was the rate of infection control in the dynam Secondary outcome indicators included KSSs scores, KSS functional scores, bone loss and ROM Our primary outcome indicator for this meta-analysis was the rate of infection control in the dynamic spacer and static spacer groups. Secondary outcome indicators included KSSs scores, KSS functional scores, bone loss and ROM after treatment with the respective spacers. Statistical analysis and data synthesis After completing the data extraction and classification summary, we used Stata SE-64 to analyse the data and finally obtained the corresponding forest plots for each group.If P < 0.05 indicates that the results are statistically significant and there is a significant difference between the two groups. If P > 0.05 indicates that the results are not statistically significant and there is no difference between the two groups. The variables obtained included dichotomous and continuous variables, and the variables were assessed so that relative risks (RR) and 95% confidence intervals (CI) were used. In terms of article heterogeneity, we used the I2 test for analysis. When I2 < 50% indicates little heterogeneity between the literature, a fixed effects model (FE) was chosen to analyse the data. When I2 > 50% indicates significant heterogeneity between the literature, a random effects model (RE) was chosen to analyse the data. Quality assessment of included studies We finally included 14 relevant clinical studies. A total of 799 patients were included in all articles, with a total of 407 patients in the treatment group using dynamic spacers and 392 patients in the group using static spacers. The average follow-up time for patients in both treatment groups was more than 12 months. Among the included studies, 9 studies were graded as high quality (≥ 7 points) and 5 studies were graded as moderate quality (≥ 4 and < 7 points). The specific characteristics and ratings of each article were shown in Table 1. Page 4/17 Page 4/17 Page 4/17 Table 1 The basic characteristics of the included studies. Study(ref.) Type of study Number of participants Age (years) (Mean ± SD) Intervention Quality of the literature Outcomes Trial Control Trial Control Trial Control Follow-up time(Trial) (months) Follow- up time (Control) (months) David 2012 [26] Retrospective 22 14 69 68 DS SS 20 22 6 KSSs, ROM Mark 2007[27] Retrospective 48 28 67 67 DS SS 71.2 62.2 7 ICR, KSSs, KSSf Jessica 2021[278] Retrospective 6 10 64.2 64.2 DS SS 36 84 7 ICR, KSSs, ROM, BL Thomas 2000[29] Retrospective 30 25 NR NR DS SS 27 36 6 ICR, KSSs, ROM, BL Cindy 2020[30] RCT 25 24 65.7 64.9 DS SS 42 42 9 ICR, KSSs, KSSf, ROM Park 2009[31] Retrospective 16 20 60.2 66.5 DS SS 29 36 8 ICR, KSSs, KSSf, ROM, BL Hsu 2007[32] Retrospective 21 7 NR NR DS SS 58 101 6 ICR, KSSs, KSSf, ROM Edward 2022[33] Retrospective 104 72 68.6 69.4 DS SS 120 228 5 ICR, KSSs, KSSf, ROM Rim 2012[34] Retrospective 14 33 64 64 DS SS 43 63 7 ICR, ROM Chiang 2011[35] Prospective 23 22 71 72 DS SS 40 40 7 ICR, ROM Roger 2002[36] Retrospective 22 26 65.1 65.7 DS SS 46 90 6 CEICR, ROM Aaron 2012[37] Retrospective 34 81 62 61 DS SS 27 66 8 ICR, KSSs, ROM, BL Jamsen 2006[38] Retrospective 22 8 68 70 DS SS 25.0 48.9 7 ICR, KSSs, KSSf, ROM, BL Kong 2021[39] Retrospective 22 20 67.2 65.5 DS SS 18 43 8 ICR, KSSs, KSSf, ROM, BL RCT: Randomized Controlled Trial; NR: not reported; AS: Dynamic Spacer; SS: Static Spacer; ICR: Infection Control Rate; KSSs: KSS knee score;KSSf༚KSS function score༛ROM༚Range of motion༛BL༚Boon loose. I f ti t l t Table 1 Infection control rate The control rate of knee prosthesis infection was the main evaluation index in this meta-analysis. In total, 13 of the 14 included literatures had reported the specific control rates of knee prosthesis infection[27–39]. From the forest plots derived from the Stata software we can see that there is no statistical difference between dynamic spacers and static spacers in terms of prosthetic infection control rates. Such results suggest that dynamic spacers and static spacers are similar in controlling prosthetic infection (RR: 1.03; 95% Cl: 0.98,1.09; P = 0.179 > 0.05), as shown in Fig. 2. Such results suggest that dynamic spacers are no less effective in controlling prosthetic infection than static spacers. The funnel diagram is shown in Fig. 3. KSS Knee score(KSSs) Discussion With the increase of total knee arthroplasty surgery year by year, the incidence of prosthetic joint infection is also increasing. When a joint prosthesis becomes infected, a poorly controlled infection can lead not only to failure of the joint arthroplasty, but in severe cases even to amputation [40, 41]. Two stage revision surgery is the most commonly used method in clinical practice and it can offers the best results in terms of treatment. In the two stage revision surgery, the commonly used spacers include dynamic spacers and static spacers and each has its own characteristics. The structure of the dynamic spacer is match to the anatomy of the knee joint, thus it can reducing adhesions and scarring of the soft tissues surrounding the knee joint. The dynamic spacer can also improve the recovery of knee function after revision surgery and reduce the incidence of some complications. However, there are also some reports suggested that dynamic spacers are less effective in controlling prosthetic joint infection. In contrast, static spacers are less prone to dislocation during fixation and it can also provide good joint stability. In addition, the static spacer can provide a high concentration of antibiotics for better infection control while maintaining limb length. Of course, there is still controversy in clinical and related research regarding the difference between dynamic and static spacers in controlling prosthetic infection and improving the prognosis of revision surgery. In the current meta-analysis, we collected relevant clinically controlled studies and performed a meta-analysis of these. Our aim is to further investigate the differences between dynamic spacers and static spacers in terms of therapeutic effect and impact on knee function. P 6/17 A total of 14 articles were included in this meta-analysis, and we grouped one primary and four secondary outcome indicators according to the indicators in each article. Finally, we did the corresponding meta-analysis for each of the five outcome indicators. The rate of infection control in prosthetic joint infection is the most important indicator of the effectiveness of revision surgery treatment. Based on the final results we can find that the treatment effect between dynamic spacers and static spacers is the same in terms of the main outcome indicator of prosthetic infection control, with no significant difference between the two groups (p = 0.179 > 0.05). KSS Knee score(KSSs) Page 5/17 Page 5/17 Of the included articles, 10 reported on the KSS Knee Scores (KSSs) of patients after receiving spacer implantation [26–31, 33, 37–39]. Based on the results of the forest plots obtained, it is known that the dynamic spacer was more effective than the static spacer in terms of KSSs scores in the knee joint after spacer implantation, with a statistically significant difference in treatment effect between the two groups (RR: 7.34; 95% CI: 1.89, 12.79; p = 0.032༜0.05). A specific forest plot is shown in Fig. 4. Of the included articles, 10 reported on the KSS Knee Scores (KSSs) of patients after receiving spacer implantation [26–31, 33, 37–39]. Based on the results of the forest plots obtained, it is known that the dynamic spacer was more effective than the static spacer in terms of KSSs scores in the knee joint after spacer implantation, with a statistically significant difference in treatment effect between the two groups (RR: 7.34; 95% CI: 1.89, 12.79; p = 0.032༜0.05). A specific forest plot is shown in Fig. 4. KSS functional score Among the secondary outcome indicators in this meta-analysis, the KSS functional score was one of the indicators to judge the effect of the spacer implantation on the patient's later functional recovery of the knee. Of the 14 articles included, a total of 6 articles reported on the KSS functional scores of patients after receiving spacer implantation[27, 30–32, 38, 39]. According to the forest plots derived from the stata software we can know that the dynamic spacer was significantly better than the static spacer in improving the patients' recovery of knee function, with statistically significant results between the two (RR: 11.16; 95% CI: 4.18, 18.13; P = 0.02 < 0.05). The forest plot is shown in Fig. 5. ROM ROM refers to the range of motion of the patient's knee joint after receiving the spacer implantation and directly reflects the recovery of the patient's knee function. It is also one of the secondary outcome indicators in our current meta-analysis. A total of 12 of these articles reported on the ROM of the knee joint after patients received spacer implantation [27, 28–35, 37–39]. The result of the meta-analysis suggested to us that the patients with dynamic spacers had significantly better ROM than those with static spacers, with statistical significance between the two groups (RR: 18.73;95% CI:11.67, 25.79༛P༜0.0001). The specific forest plot is shown in Fig. 7. Boon loose Bone loss is one of the secondary outcome indicators in this meta-analysis, and it is an indicator of the occurrence of bone loss or deficiency in and around the joint after spacer implantation. A total of six articles reported on bone loss after patients received treatment [28, 29, 31, 37–39]. We used the stata software to analyse and produce the corresponding forest plots. The results suggest that after spacer implantation in our patients, significantly fewer patients with dynamic spacers experienced bone loss than those with static spacers. There was a statistically significant difference between the two groups (RR: 2.04; 95% CI: 1.11, 3.77; p = 0.018). The specific forest plot is shown in Fig. 6. Discussion This suggests us that dynamic spacers are no less effective than static spacers for prosthetic infection control and it can also achieve the same clinical outcomes. This result is consistent with the results of many current clinical studies and further validates the efficacy of joint spacers [42– 44]. According to relevant literature reports, the concentration of antibiotics and the duration of anti-infection therapy are important factors Page 6/17 Page 6/17 in the outcome of two-stage revision surgery for prosthetic infections. The use of adequate antibiotic concentrations and duration of anti- infection therapy not only results in more satisfactory infection control, but also better reduces the recurrence of prosthetic infections [45, 46]. In addition, revision surgery at an early stage of prosthetic infection can also provide better control of prosthetic infection, and early anti-infection treatment is one of the keys to treatment and reduces the risk of surgery [47, 48]. Therefore, not only does the correct use of spacers will influence the outcome of treatment, but timely and adequate anti-infective treatment is also crucial to the success of treatment. in the outcome of two-stage revision surgery for prosthetic infections. The use of adequate antibiotic concentrations and duration of anti- infection therapy not only results in more satisfactory infection control, but also better reduces the recurrence of prosthetic infections [45, 46]. In addition, revision surgery at an early stage of prosthetic infection can also provide better control of prosthetic infection, and early anti-infection treatment is one of the keys to treatment and reduces the risk of surgery [47, 48]. Therefore, not only does the correct use of spacers will influence the outcome of treatment, but timely and adequate anti-infective treatment is also crucial to the success of treatment. Of the four secondary outcome indicators, KSSs scores, KSS functional scores and ROM were used to evaluate the impact on knee function after patients received spacer implantion, all as an indicator of post-operative recovery and efficacy. In the results of our meta- analysis, the results for all three indicators, KSSs scores, KSS functional scores and ROM, were statistically significant (p < 0.05). This indicates a significant difference between the results of the dynamic spacer group and the static spacer group in these three indicators. In terms of improved prognosis for knee function, patients with dynamic spacers had significantly better functional scores and knee mobility than those with static spacers. Discussion These results once again verified the superiority of the dynamic spacers over the static spacers, in comparison, the dynamic spacer could better improve the patient's motion function and range of motion [49–51]. Bone loss is a common complication after spacer implantation and the patient's bone loss is a judgment indicator to evaluate the impact of the spacer on the patient's bone health. Therefore, bone loss was also one of the reference indicators in our current meta-analysis. Based on the results of the meta-analysis, it is known that the patients with dynamic spacers experienced significantly less bone loss than those with static spacers (p < 0.05). There was a significant difference in the results between the two groups. This is also in line with some current findings that patients using static spacers are more likely to experience bone loss [52]. Combining these results, we can see that the dynamic spacer can achieve similar results to the static spacer in terms of infection control of the prosthesis. The performance of dynamic spacers is better than that of static spacers in improving the prognosis of knee function and preventing bone loss. For patients requiring prosthetic revision surgery, the use of dynamic spacers may provide a better prognosis and recovery of joint function. Of course, there are some limitations to our current meta-analysis. Firstly, although we included a total of 14 relevant literatures, the total number of patients studied was only 799, which may not be a large enough sample size. Perhaps we need more clinical studies with larger samples to further confirm our results. Secondly, the articles we included were not clinical randomised controlled studies, but rather retrospective clinical studies and prospective trials. The use of blinding was also not reported for the assessment of outcomes and scores related to knee function, which lacks a certain degree of concealment. In addition, there is a lack of uniformity in the assessment and the associated results may be influenced by subjective factors. Thirdly, the possible influence of relevant factors on the treatment outcomes (e.g. gender, age, height, weight, etc.) was not adequately considered in determining patient groupings. Conclusion Based on the results of the meta-analysis we finally obtained, we can know that there was no significant difference in the rate of infection control of the prosthesis between the dynamic spacer group and the static spacer group, similar results can be obtained with both types of spacers. And there are significant differences between the dynamic spacer group and the static spacer group in terms of knee function related scores and ROM. The patients using dynamic spacers can achieve better joint mobility and function. There is also a significant difference between the two groups in terms of bone loss. Patients with dynamic spacers experienced significantly less bone loss than those with static spacers. Consent for publication Not applicable. Ethics approval and consent to participate Not applicable. Declarations Ethics approval and consent to participate Competing interests The authors declare that they have no competing interests.Funding The authors declare that they have no competing interests.Funding Funding The authors declare that they have no competing interests.Funding F di Page 7/17 This work was supported by National Natural Science Youth Science Foundation of China(Grant No: 82004390) in the form of covering the consultation fees of data statistical analysis. Hang Dong received scientific funding from National Natural Science Youth Science Foundation of China (82004390). Footnotes Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Jiasheng Tao and ZIjian Yan have contributed equally as first authors. Authors' contributions Hang Dong conceived the theme and take responsibility for the article. Jiasheng Tao and Zijian Yan conceived the manuscript and wrote the manuscript. Bin Pu interpreted the patient data. Ming Chen collected and analyzed the patient data. Xiaorong Hu searched literatures. All authors read and approved the final manuscript. References Lethbridge L N, Richardson C G, Dunbar M J, et al. Measuring Surgical Site Infection From Linked Administrative Data Following Hip and Knee Replacement. The Journal of arthroplasty. 2020; 35: 528–533. [PubMed] 8. Lenguerrand E, Whitehouse M R, Beswick A D, et al. 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https://openalex.org/W2775363837	http://espace.inrs.ca/7388/1/Pawluk%202017-Disabling%20a%20Type%20I-E%20CRISPR-Cas%20Nuclease%20with%20a%20Bacteriophage-Encoded%20Anti-CRISPR%20Protein.pdf	English	null	Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein	MBio	2,017	cc-by	9,701	Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein April Pawluk,a Megha Shah,a Marios Mejdani,a Charles Calmettes,a Trevor F. Moraes,a Alan R. Davidson,a,b Karen L. Maxwella Department of Biochemistry, University of Toronto, Toronto, Ontario, Canadaa; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canadab April Pawluk,a Megha Shah,a Marios Mejdani,a Charles Calmettes,a Trevor F. Moraes,a Alan R. Davidson,a,b Karen L. Maxwella Department of Biochemistry, University of Toronto, Toronto, Ontario, Canadaa; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canadab ABSTRACT CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and ar- chaea. They provide sequence-speciﬁc detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different fami- lies of anti-CRISPR proteins have been described, each of which inhibits a partic- ular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcrip- tional repressor. Received 26 September 2017 Accepted 1 November 2017 Published 12 December 2017 Citation Pawluk A, Shah M, Mejdani M, Calmettes C, Moraes TF, Davidson AR, Maxwell KL. 2017. Disabling a type I-E CRISPR-Cas nuclease with a bacteriophage-encoded anti- CRISPR protein. mBio 8:e01751-17. https://doi .org/10.1128/mBio.01751-17. Editor Emmanuelle Charpentier, Max Planck Institute for Infection Biology Copyright © 2017 Pawluk et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Alan R. Davidson, alan.davidson@utoronto.ca, or Karen L. Maxwell, karen.maxwell@utoronto.ca. IMPORTANCE The CRISPR-Cas immune system provides bacteria with resistance to in- vasion by potentially harmful viruses, plasmids, and other foreign mobile genetic ele- ments. This study presents the ﬁrst structural and mechanistic insight into a phage- encoded protein that inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa. The interaction of this anti-CRISPR protein with the CRISPR-associated heli- case/nuclease proteins Cas3 shuts down the CRISPR-Cas system and protects phages carrying this gene from destruction. RESEARCH ARTICLE crossm RESEARCH ARTICLE ® mbio.asm.org 1 Received 26 September 2017 Accepted 1 November 2017 Published 12 December 2017 Citation Pawluk A, Shah M, Mejdani M, Calmettes C, Moraes TF, Davidson AR, Maxwell KL. 2017. Disabling a type I-E CRISPR-Cas nuclease with a bacteriophage-encoded anti- CRISPR protein. mBio 8:e01751-17. https://doi .org/10.1128/mBio.01751-17. Editor Emmanuelle Charpentier, Max Planck Institute for Infection Biology Copyright © 2017 Pawluk et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Alan R. Davidson, alan.davidson@utoronto.ca, or Karen L. Maxwell, karen.maxwell@utoronto.ca. Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein This interaction also allows the repurposing of the endogenous type I-E CRISPR system into a programmable transcriptional repressor, pro- viding a new biotechnological tool for genetic studies of bacteria encoding this type I-E CRISPR-Cas system. KEYWORDS CRISPR-Cas, Pseudomonas aeruginosa, X-ray crystallography, anti-CRISPR, type I-E CRISPR-Cas B acteria and bacteriophages (phages) coexist in a constant battle for survival. As a result, bacteria have evolved a variety of antiphage defense systems such as restriction modiﬁcation, superinfection exclusion proteins, and abortive infection mech- anisms (1, 2). While these systems provide a type of innate immunity, bacteria also possess an adaptive immune system, known as CRISPR (clustered regularly interspaced short palindromic repeats)-Cas. CRISPR and CRISPR-associated (cas) genes comprise an adaptive, RNA-guided immune system that arms bacteria with sequence-speciﬁc pro- tection against invasion by foreign nucleic acids such as phages and plasmids. CRISPR- Cas systems are broadly distributed across bacteria and archaea. They are grouped into two broad classes encompassing six types based on their phylogeny and mechanisms of activity (3). Class 1 systems (types I, III, and IV) utilize multisubunit Cas protein complexes (e.g., Cascade) for the recognition and cleavage of targeted nucleic acids, B ® mbio.asm.org 1 November/December 2017 Volume 8 Issue 6 e01751-17 ® Pawluk et al. while class 2 systems (types II, V, and VI) use a single protein (e.g., Cas9) that provides all of the required activities (3). The opportunistic human pathogen Pseudomonas aeruginosa possesses two active class 1 CRISPR-Cas systems known as type I-E and type I-F (4, 5). These CRISPR-Cas systems function in three main stages: adaptation, expression, and interference. In the adaptation stage, bacteria integrate segments of foreign DNA into their genomic CRISPR locus, comprising tandem units of short, semipalindromic repeats interspaced with invader-derived “spacer” elements (6). The next stage, expression, involves the transcription of the CRISPR locus into a long precursor CRISPR RNA and subsequent processing to single repeat-spacer units known as mature CRISPR RNAs (crRNAs). A ribonucleoprotein complex called Cascade is then formed by a set of CRISPR-associated (Cas) proteins and crRNA (7). The ﬁnal stage, interference, occurs when Cascade surveys the cell and identiﬁes target DNA molecules by complementary base pairing with the crRNA spacer (8). This allows recruitment of the Cas3 helicase/nuclease protein that destroys the foreign DNA molecule (9). Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein y While the widespread nature of CRISPR-Cas systems is expected to provide a signiﬁcant barrier to horizontal gene transfer, studies have shown that they have minimal inhibitory effect over evolutionary timescales (10). One explanation for this could be the presence of small protein inhibitors of CRISPR-Cas systems (11). These anti-CRISPR proteins have been described in association with type I-F, type I-E, type II-C, and type II-A CRISPR-Cas systems in diverse Gram-negative and Gram-positive bacteria (4, 5, 12–15). Recently, structures of three type I-F anti-CRISPR proteins were reported, revealing that each anti-CRISPR protein family possesses a unique fold and interacts with different parts of the CRISPR-Cas machinery (16–20). AcrF1 was shown to bind to the Cas7f hexamer and allosterically inhibit DNA binding, AcrF2 bound Cas8f and acted as a double-stranded DNA mimic that competes with the target DNA for the critical DNA-binding site, and AcrF3 bound Cas3, the effector nuclease and helicase protein common to all type I systems (3). Studies of type II anti-CRISPR proteins revealed two additional unique protein folds and interactions with Cas9 proteins. AcrIIC1 was shown to bind to the HNH endonuclease domain and prevent target DNA cleavage (21), while AcrIIA4 binds the protospacer-adjacent-motif (PAM)-interacting region and blocks in- teraction with the target DNA molecule (22–24). These studies highlight the structural and mechanistic diversity by which anti-CRISPR proteins function. To date, no structure has been determined for any type I-E anti-CRISPR protein. y yp p Here, we report the 2.5-Å-resolution crystal structure of anti-CRISPR protein AcrE1, which inhibits the type I-E CRISPR-Cas system of P. aeruginosa. We have identiﬁed a functional region of this protein and show that AcrE1 binds to and inactivates the Cas3 nuclease. Exploiting the ability of AcrE1 to inactivate Cas3, we developed a system where expression of AcrE1 and a promoter-targeting crRNA can convert endogenous type I-E CRISPR-Cas activity into a programmable transcriptional repression system in P. aeruginosa. November/December 2017 Volume 8 Issue 6 e01751-17 RESULTS TABLE 1 Data collection and reﬁnement statistics for Pseudomonas phage protein AcrE1 crystal structures solved in this study aHighest-resolution shell values are shown in parentheses. R factors were calculated using the formula \|Fobs Fcalc\|/Fobs. CC1/2 was calculated based on Pearson’s correlation coefﬁcient, and the cutoff value was 0.5. of helix 1 for a potential H-bond to form between the carbonyl O of Val79 and the amide N of Leu5. Residues 82 to 90 form an antiparallel -sheet that protrudes at a 90° angle from the end of helix 3. This sheet structure appears to be stabilized by multiple interactions between residues at the base of the sheet and residues in helices 1, 2, and 3 (Fig. 1b). The ﬁnal 10 residues of AcrE1 are likely disordered, as they were not well deﬁned in the electron density map and could not be modeled into the structure. The large dimeric interface is composed primarily of 18 hydrophobic residues from each monomer that are greater than 97% buried, forming a single hydrophobic core that spans the entire length of the helices (see Fig. S1a in the supplemental material). The dimeric crystal structure of AcrE1 is consistent with the native molecular weight of this protein in solution as estimated by size exclusion chromatography (SEC) (Fig. S1b). of helix 1 for a potential H-bond to form between the carbonyl O of Val79 and the amide N of Leu5. Residues 82 to 90 form an antiparallel -sheet that protrudes at a 90° angle from the end of helix 3. This sheet structure appears to be stabilized by multiple interactions between residues at the base of the sheet and residues in helices 1, 2, and 3 (Fig. 1b). The ﬁnal 10 residues of AcrE1 are likely disordered, as they were not well deﬁned in the electron density map and could not be modeled into the structure. The large dimeric interface is composed primarily of 18 hydrophobic residues from each monomer that are greater than 97% buried, forming a single hydrophobic core that spans the entire length of the helices (see Fig. S1a in the supplemental material). The dimeric crystal structure of AcrE1 is consistent with the native molecular weight of this protein in solution as estimated by size exclusion chromatography (SEC) (Fig. S1b). November/December 2017 Volume 8 Issue 6 e01751-17 RESULTS The structure of AcrE1. We previously reported that gene 34 from Pseudomonas phage JBD5, which encodes the protein AcrE1, shows anti-CRISPR activity against the type I-E system of P. aeruginosa strain SMC4386 (4). Expression of acrE1 from a plasmid or prophage allowed infection by a CRISPR-targeted phage and efﬁcient transformation of cells with a CRISPR-targeted plasmid. To gain insight into the mechanism of activity of AcrE1, we solved its three-dimensional structure by X-ray crystallography. Using single-wavelength anomalous diffraction (Br-SAD), we were able to determine the structure of AcrE1 with a C-terminal 6His tag (PDB identiﬁer [ID] 6ARZ) to a resolution of 2.5 Å (Table 1). AcrE1 displays an elongated dimeric structure (Fig. 1a). Each monomer is predom- inantly comprised of three helices. Helix 1 (residues 5 to 17) is followed by a tight turn leading into the very long helix 2 (residues 21 to 57). A short turn at the end of helix 2 leads into helix 3 (residues 62 to 80), which terminates close enough to the beginning mbio.asm.org 2 November/December 2017 Volume 8 Issue 6 e01751-17 ® A Type I-E Anti-CRISPR Protein TABLE 1 Data collection and reﬁnement statistics for Pseudomonas phage protein AcrE1 crystal structures solved in this study Parametera CHis AcrE1 NHis AcrE1 PDB code 6ARZ 6AS4 Phasing method Br-SAD MR Data collection Space group C 1 2 1 C 1 2 1 Cell dimensions a, b, c (Å) 98.14, 63.96, 59.70 96.74, 63.53, 59.46 a, b, c (°) 90, 100.94, 90 90, 100.34, 90 Solvent content (%) 52.30 45.30 Wavelength (Å) 0.9199 0.9793 Resolution (Å) 48.18–2.5 (2.59–2.50) 47.59–2.0 (2.07–2.0) I/I 24.1 (7.2) 16.25 (1.57) Completeness (%) 100 (100) 98.57 (97.59) Redundancy 7.7 (7.6) 7.7 (7.7) Rmerge 0.06 (0.296) 0.1033 (1.596) CC1/2 0.99 (0.98) 0.999 (0.529) Reﬁnement Resolution (Å) 48.18–2.5 47.59–2.0 No. of unique reﬂections 12,710 (1,257) 23,742 (2,349) Rwork/Rfree 0.18/0.22 0.19/0.23 No. of atoms 2,358 2,415 Protein 282 279 Ligands 30 0 Water 105 150 B-factors Protein 58.90 47.30 Ligands 71.10 0 Water 52.30 45.30 RMSD Bond length (Å) 0.012 0.008 Bond angle (°) 1.290 0.93 Ramachandran Favored (%) 97.10 100 Outlier (%) 0 0 aHighest-resolution shell values are shown in parentheses. R factors were calculated using the formula \|Fobs Fcalc\|/Fobs. CC1/2 was calculated based on Pearson’s correlation coefﬁcient, and the cutoff value was 0.5. RESULTS Blue shading indicates the amino acids absent from the FIG 1 Structure of AcrE1. (a) Crystal structure of the C-terminal 6His-tagged AcrE1 solved by Br-SAD reveals a predominantly helical dimeric protein. The N and C termini are noted. (b) Interactions between residues in the AcrE1 -strands (colored red) and residues in the helices (colored blue) stabilize the C-terminal -sheet. At least 10% of the surface area of each residue shown is buried by the interaction between the helices and the C-terminal region. A salt bridge is formed between E41 and R89. (c) Structural overlay of the X-ray crystal structures of the AcrE1 proteins with an N-terminal (blue) and C-terminal (red) 6His tag reveals virtually identical structures. (d) Sequence alignment of the two homologues of the AcrE1 protein family with validated activities against the type I-E CRISPR-Cas system. Asterisks indicate identical residues shared between the two proteins. Blue shading indicates the amino acids absent from the AcrE1Δ91–100 truncation mutant. molecular replacement (MR) to a resolution of 2.0 Å (Table 1). This structure was virtually identical to the C-terminally tagged structure; the two structures could be aligned over 736 backbone atoms with a root mean square deviation (RMSD) of 0.31 Å (Fig. 1c). Thus, the unusual nature of the C-terminal sheet was not a result of the presence of the 6His afﬁnity puriﬁcation tag fused to the C terminus. We also examined the packing of the C-terminal -sheet within the crystal and observed no contacts that stabilize it in this position (Fig. S3). Identiﬁcation of functionally critical surfaces of AcrE1. To identify amino acid residues in AcrE1 that are required for its function, we combined structural and sequence analyses with site-directed mutagenesis. A homologue of AcrE1 encoded by a conjugative element in P. aeruginosa that shares 62% sequence identity (AcrE1PA21) was previously found to also block type I-E CRISPR-Cas activity in P. aeruginosa strain SMC4386 (4). We identiﬁed 20 residues that were exposed on the surface of AcrE1 (accessible surface area, 35%) that were largely conserved between the two proteins and introduced mutations at each of these positions (Fig. S2a and b). Each mutant protein was expressed from a plasmid in strain SMC4386, and its ability to support replication of a CRISPR-targeted phage was assessed. None of the AcrE1 mutants tested showed any reduction in anti-CRISPR activity as measured by this assay. November/December 2017 Volume 8 Issue 6 e01751-17 mbio.asm.org 4 RESULTS The structure of AcrE1 is not similar to other previously determined anti-CRISPR proteins, and searches using the Dali server (25) did not reveal structural similarity to any other protein. The C-terminal -sheet region is unusual because both sides of the sheet are exposed to solvent, contrasting with typical -sheets, which pack a hydro- phobic surface on one side against helices or other sheets. To ensure that this structure was not a result of the presence of the C-terminal 6His tag, we also crystalized AcrE1 with an N-terminal 6His tag (PDB ID 6AS4) and determined its structure using mbio.asm.org 3 November/December 2017 Volume 8 Issue 6 e01751-17 ® Pawluk et al. FIG 1 Structure of AcrE1. (a) Crystal structure of the C-terminal 6His-tagged AcrE1 solved by Br-SAD reveals a predominantly helical dimeric protein. The N and C termini are noted. (b) Interactions between residues in the AcrE1 -strands (colored red) and residues in the helices (colored blue) stabilize the C-terminal -sheet. At least 10% of the surface area of each residue shown is buried by the interaction between the helices and the C-terminal region. A salt bridge is formed between E41 and R89. (c) Structural overlay of the X-ray crystal structures of the AcrE1 proteins with an N-terminal (blue) and C-terminal (red) 6His tag reveals virtually identical structures. (d) Sequence alignment of the two homologues of the AcrE1 protein family with validated activities against the type I-E CRISPR-Cas system. Asterisks indicate identical residues shared between the two proteins. Blue shading indicates the amino acids absent from the AcrE1Δ91–100 truncation mutant. FIG 1 Structure of AcrE1. (a) Crystal structure of the C-terminal 6His-tagged AcrE1 solved by Br-SAD reveals a predominantly helical dimeric protein. The N and C termini are noted. (b) Interactions between residues in the AcrE1 -strands (colored red) and residues in the helices (colored blue) stabilize the C-terminal -sheet. At least 10% of the surface area of each residue shown is buried by the interaction between the helices and the C-terminal region. A salt bridge is formed between E41 and R89. (c) Structural overlay of the X-ray crystal structures of the AcrE1 proteins with an N-terminal (blue) and C-terminal (red) 6His tag reveals virtually identical structures. (d) Sequence alignment of the two homologues of the AcrE1 protein family with validated activities against the type I-E CRISPR-Cas system. Asterisks indicate identical residues shared between the two proteins. RESULTS The replication efﬁciency of a CRISPR-insensitive phage, JBD93a, remained constant on all strains (data not shown). Images are representative of three independent experiments. (b) Circular dichroism (CD) spectra of AcrE1 NHis (purple) and AcrE1Δ91–100 NHis (red) reveal very similar spectra with canonical -helical signals. The spectrum of the buffer (yellow) is provided for reference. The spectra shown here are representative of three independent experiments. pletely conserved C-terminal region encompasses the short -sheet and the 10 residues at the extreme C terminus that do not form a deﬁned secondary structure. To delineate the functional importance of this C-terminal region, we created a series of C-terminal truncation mutants and assessed the anti-CRISPR activity of each. We found that the mutant with eight C-terminal residues (AcrE1Δ93–100) deleted had wild-type levels of anti-CRISPR activity (Fig. 2a). The mutant with nine residues truncated (AcrE1Δ92–100) displayed no anti-CRISPR activity. However, this activity could be almost completely recovered through addition of arabinose, which strongly induces the plasmid-borne transcriptional promoter (Fig. 2a). This suggests either that AcrE1Δ92–100 is less stable and accumulates to lower steady-state levels in the cell or that it has reduced binding afﬁnity for its target, such that a large excess of the protein is able to restore functionality. Deletion of one additional residue (AcrE1Δ91–100) led to complete loss of activity, even in the presence of arabinose (Fig. 2a). To determine if the loss of function of AcrE1Δ91–100 was a result of lack of accumu- lation in the cell due to expression defects or proteolysis, we assessed intracellular protein levels. As can be seen in Fig. 3a, wild-type AcrE1 and AcrE1Δ91–100 could be puriﬁed from cells at comparable levels when expressed from a plasmid in P. aeruginosa SMC4386. We puriﬁed these two proteins from Escherichia coli for further analyses. Similar amounts of wild-type AcrE1 and AcrE1Δ91–100 were recovered, and circular dichroism (CD) spectroscopy revealed very similar spectra with a canonical -helical signature (Fig. 2b). While wild-type AcrE1 was soluble to a high concentration, AcrE1Δ91–100 was less soluble and formed aggregates in vitro upon storage, and so no further structural work was done with this mutant. The circular dichroism data indicated that that AcrE1Δ91–100 was folded and that there were no gross changes in the secondary structure content of this truncated protein. Thus, it is likely that residues 91 and 92 of AcrE1 are involved in protein-protein interaction(s) necessary for its anti- CRISPR activity. RESULTS These results contrast with a study on anti-CRISPR protein AcrF1, in which three different single-site substitutions on one surface of the protein caused a marked inhibition of anti-CRISPR activity (16). Further comparison of the two AcrE1 homologues revealed a striking pattern of conservation: their helical regions (residues 1 to 79) share 54% sequence identity, while their C-terminal regions (residues 80 to 100) share 100% identity (Fig. 1d). This com- mbio.asm.org 4 November/December 2017 Volume 8 Issue 6 e01751-17 ® A Type I-E Anti-CRISPR Protein FIG 2 Deletion of 10 C-terminal residues from AcrE1 results in a complete loss of activity, while a signiﬁcant proportion of soluble, folded protein is retained. (a) Tenfold serial dilutions of CRISPR-targeted phage JBD8 were spotted on bacterial lawns of P. aeruginosa strain SMC4386, and the ability to replicate in the presence of an active type I-E CRISPR system was assessed. The bacteria were expressing wild-type AcrE1, the homologue of AcrE1 encoded by a conjugative element in Pseudomonas strain PA21, or one of the deletion mutants from a plasmid as noted on the left. Bacteria with low-level expression of AcrE1 (uninduced, left panels) or high-level expression (induced, right panels) were challenged with phage. Zones of clearing indicate phage replication. The replication efﬁciency of a CRISPR-insensitive phage, JBD93a, remained constant on all strains (data not shown). Images are representative of three independent experiments. (b) Circular dichroism (CD) spectra of AcrE1 NHis (purple) and AcrE1Δ91–100 NHis (red) reveal very similar spectra with canonical -helical signals. The spectrum of the buffer (yellow) is provided for reference. The spectra shown here are representative of three independent experiments. FIG 2 Deletion of 10 C-terminal residues from AcrE1 results in a complete loss of activity, while a signiﬁcant proportion of soluble, folded protein is retained. (a) Tenfold serial dilutions of CRISPR-targeted phage JBD8 were spotted on bacterial lawns of P. aeruginosa strain SMC4386, and the ability to replicate in the presence of an active type I-E CRISPR system was assessed. The bacteria were expressing wild-type AcrE1, the homologue of AcrE1 encoded by a conjugative element in Pseudomonas strain PA21, or one of the deletion mutants from a plasmid as noted on the left. Bacteria with low-level expression of AcrE1 (uninduced, left panels) or high-level expression (induced, right panels) were challenged with phage. Zones of clearing indicate phage replication. November/December 2017 Volume 8 Issue 6 e01751-17 RESULTS AcrE1 interacts with Cas3. To identify the protein interaction partners of AcrE1, we performed copuriﬁcation experiments. P. aeruginosa strain SMC4386 was transformed with plasmids expressing AcrE1, AcrE1PA21, or AcrE1Δ91–100. After induction of protein expression from the plasmids, cells were exposed to formaldehyde to cross-link protein complexes in vivo (26). Chemical cross-linker was used in these experiments because the endogenous expression level of Cas proteins in this strain was low, and we aimed to maximize capture of AcrE1-Cas protein complexes. The 6His-tagged AcrE1 proteins November/December 2017 Volume 8 Issue 6 e01751-17 mbio.asm.org 5 ® Pawluk et al. FIG 3 AcrE1 interacts with the type I-E CRISPR-Cas protein, Cas3. (a) N-terminal 6His-tagged AcrE1, AcrE1PA21, or AcrE1Δ91–100 was expressed from a plasmid in P. aeruginosa SMC4386 cells. Following in vivo formaldehyde cross-linking, anti-CRISPR proteins were afﬁnity puriﬁed using Ni-NTA agarose beads, and the resultant elutions were analyzed by SDS-PAGE followed by silver staining (upper panel) and anti-His Western blotting (lower panel). AcrE1PA21 accumulates to higher steady-state levels in the cell than AcrE1 and AcrE1Δ91–100. (b) Low levels of AcrE1 bind intracellular Cas3 to saturation. Puriﬁed 6His-tagged Cas3 expressed from a plasmid in SMC4385 cells was run as a size standard for the Cas3 protein (far right lane). Even very low levels of AcrE1 were sufﬁcient to pull down as much Cas3 as the overexpressed versions, suggesting that all the Cas3 in the cells is saturated by anti-CRISPR protein binding. Four irrelevant lanes were removed from the gel image. Numbers at left of each panel are molecular masses in kilodaltons. FIG 3 AcrE1 interacts with the type I-E CRISPR-Cas protein, Cas3. (a) N-terminal 6His-tagged AcrE1, AcrE1PA21, or AcrE1Δ91–100 was expressed from a plasmid in P. aeruginosa SMC4386 cells. Following in vivo formaldehyde cross-linking, anti-CRISPR proteins were afﬁnity puriﬁed using Ni-NTA agarose beads, and the resultant elutions were analyzed by SDS-PAGE followed by silver staining (upper panel) and anti-His Western blotting (lower panel). AcrE1PA21 accumulates to higher steady-state levels in the cell than AcrE1 and AcrE1Δ91–100. (b) Low levels of AcrE1 bind intracellular Cas3 to saturation. Puriﬁed 6His-tagged Cas3 expressed from a plasmid in SMC4385 cells was run as a size standard for the Cas3 protein (far right lane). Even very low levels of AcrE1 were sufﬁcient to pull down as much Cas3 as the overexpressed versions, suggesting that all the Cas3 in the cells is saturated by anti-CRISPR protein binding. November/December 2017 Volume 8 Issue 6 e01751-17 mbio.asm.org 6 RESULTS Four irrelevant lanes were removed from the gel image. Numbers at left of each panel are molecular masses in kilodaltons. were puriﬁed by nickel afﬁnity chromatography, and copurifying proteins were ana- lyzed by SDS-PAGE followed by silver staining. In this way, we identiﬁed a distinct band at approximately 97 kDa that copuriﬁed with AcrE1 and AcrE1PA21 but not with the inactive C-terminal truncation mutant, AcrE1Δ91–100 (Fig. 3a). This band was excised and analyzed using mass spectrometry, leading to the identiﬁcation of 55 unique peptides from Cas3 covering 67% of the protein (Tables S1 and S2). The type I-E Cas3 protein of P. aeruginosa has a calculated molecular mass of 99 kDa, in agreement with the apparent molecular mass observed on the protein gel. Thus, we concluded that AcrE1 binds to Cas3, potentially preventing its recruitment to the DNA-bound Cascade complex. We previously showed that arabinose induction of the plasmid promoter that drives AcrE1 expression was not necessary for full anti-CRISPR activity (4). This result demon- strated that a small amount of anti-CRISPR protein is sufﬁcient to completely block the CRISPR-Cas system. In support of this, we saw no increase in the level of Cas3 copurifying with AcrE1 in the presence of arabinose compared to the uninduced control (Fig. 3b), suggesting that all the Cas3 present in cells is bound to AcrE1 in both cases. AcrE1 blocks the activity of Cas3. In the absence of Cas3, Cascade can still bind DNA strongly and speciﬁcally as guided by its crRNA. Therefore, if Cascade is targeted to a promoter in the absence of Cas3, transcription is repressed as the bound Cascade complex prevents recruitment of RNA polymerase (27–29). To determine whether AcrE1 inhibits the activity of Cas3 in vivo, we designed crRNA1 and crRNA2, which targeted protospacers in the 10 and 35 regions of the promoter of phzM, a biosynthetic gene mbio.asm.org 6 ® A Type I-E Anti-CRISPR Protein FIG 4 Cas3 inactivation converts the P. aeruginosa type I-E CRISPR-Cas system into a programmable transcriptional repression platform. (a) crRNA1 and crRNA2 target the promoter region upstream of the phzM gene. (b) Conceptual model for transcriptional repression by Cascade targeted to the phzM promoter, in the case where an anti-CRISPR protein inactivates or prevents the recruitment of Cas3. The targeting of Cascade to the promoter region upstream of phzM would prevent access by RNA polymerase (RNAP), thereby locking transcription of the gene. RESULTS The same outcome would result from a cas3 deletion or inactivating mutation. (c) A conceptual model for the normal transcription of phzM resulting from the presence of an anti-CRISPR protein that blocks Cascade from binding to target DNA. The same outcome would be expected for a CRISPR-Cas deletion strain, in which the Cascade complex is absent. (d) Photographs of P. aeruginosa SMC4386 expressing crRNA1, crRNA2, or empty-vector control. In the presence of the wild-type CRISPR-Cas system (WT), cell death occurs due to self-targeting of the chromosome by crRNA1 and crRNA2. In the absence of an active CRISPR-Cas system (ΔCRISPR-Cas), normal transcription of phzM occurs, resulting in a green pigmentation. A Cas3 mutant (H89A/D90A) leads to loss of the green pigment, indicating that the Cascade complex is repressing expression of phzM. The expression of AcrE1 from prophages JBD5 and JBD79 also leads to repression of phzM expression, as indicated by the loss of green pigment. In contrast, a prophage with no anti-CRISPR protein (JBD93a) or a different type I-E anti-CRISPR protein (JBD26; AcrE3) led to cell death and normal pyocyanin production, respectively. FIG 4 Cas3 inactivation converts the P. aeruginosa type I-E CRISPR-Cas system into a programmable transcriptional repression platform. (a) crRNA1 and crRNA2 target the promoter region upstream of the phzM gene. (b) Conceptual model for transcriptional repression by Cascade targeted to the phzM promoter, in the case where an anti-CRISPR protein inactivates or prevents the recruitment of Cas3. The targeting of Cascade to the promoter region upstream of phzM would prevent access by RNA polymerase (RNAP), thereby locking transcription of the gene. The same outcome would result from a cas3 deletion or inactivating mutation. (c) A conceptual model for the normal transcription of phzM resulting from the presence of an anti-CRISPR protein that blocks Cascade from binding to target DNA. The same outcome would be expected for a CRISPR-Cas deletion strain, in which the Cascade complex is absent. (d) Photographs of P. aeruginosa SMC4386 expressing crRNA1, crRNA2, or empty-vector control. In the presence of the wild-type CRISPR-Cas system (WT), cell death occurs due to self-targeting of the chromosome by crRNA1 and crRNA2. In the absence of an active CRISPR-Cas system (ΔCRISPR-Cas), normal transcription of phzM occurs, resulting in a green pigmentation. A Cas3 mutant (H89A/D90A) leads to loss of the green pigment, indicating that the Cascade complex is repressing expression of phzM. November/December 2017 Volume 8 Issue 6 e01751-17 DISCUSSION The evolutionary arms race between bacteria and phages provides a strong selective pressure for the emergence and maintenance of anti-CRISPR proteins. Consistent with this selective pressure, 14 diverse families of anti-CRISPR inhibitors of the P. aeruginosa type I-E or I-F CRISPR-Cas system have been described (4, 5, 13). In this work, we determined the ﬁrst structure of a type I-E anti-CRISPR protein. To date, structures have been determined for three P. aeruginosa type I-F anti-CRISPR proteins (16–20), a type II-A protein from Listeria monocytogenes (22–24), and a type II-C protein from Neisseria meningitidis (21). The structure of AcrE1 does not resemble any of these previously determined structures and thus provides the sixth unique structural family of proteins that can inactivate CRISPR-Cas systems. This wide structural diversity provides further support for the independent evolution of anti-CRISPR protein families as previously proposed (29). Type I CRISPR-Cas systems are deﬁned by the presence of the gene cas3, which encodes an ~100-kDa protein with single-stranded DNA nuclease and ATP-dependent helicase activities (3). Cas3 is not part of the Cascade complex but is recruited to degrade the target DNA after Cascade binds to it (9). We found that AcrE1 binds to Cas3 using copuriﬁcation assays (Fig. 3), and the phzM transcriptional repression assays showed that this binding blocks Cas3 activity (Fig. 4). We previously showed that anti-CRISPR AcrF3 blocks activity of the type I-F CRISPR-Cas system of P. aeruginosa through an interaction with Cas3 (29), and we expect that AcrE1 works in a similar manner. Although the sequences of Cas3 proteins from type I-E and I-F systems are highly diverse, their structures are very similar (18). Thus, it is interesting that AcrE1 and AcrF3 display very different folds. They clearly bind Cas3 through distinct mechanisms, either targeting different surfaces on the Cas3 structure or utilizing different binding modes to target the same surface. We performed an extensive mutagenesis study covering all of the conserved residues with side chains exposed on the surface of AcrE1 (see Fig. S2 in the supple- mental material). All single-residue replacements displayed wild-type anti-CRISPR ac- tivity, with only the C-terminal truncation of 10 amino acids causing complete loss of activity. These results imply that the C terminus of AcrE1 forms the crucial interactions with Cas3 that lead to its inhibition. RESULTS The expression of AcrE1 from prophages JBD5 and JBD79 also leads to repression of phzM expression, as indicated by the loss of green pigment. In contrast, a prophage with no anti-CRISPR protein (JBD93a) or a different type I-E anti-CRISPR protein (JBD26; AcrE3) led to cell death and normal pyocyanin production, respectively. essential for the production of the blue-green pigment pyocyanin (Fig. 4a). In this assay, an anti-CRISPR protein that blocks the recruitment of Cas3 to the Cascade complex will result in loss of pyocyanin production due to the targeted Cascade complex blocking access of RNA polymerase (Fig. 4b). By contrast, expression of an anti-CRISPR protein that inhibits Cascade from binding to its DNA target results in normal pyocyanin production (Fig. 4c). When strain SMC4386 was transformed with plasmids expressing crRNA1 or crRNA2, cell death resulted from CRISPR-Cas targeting of the bacterial genome. However, introduction of these plasmids into the same strain containing a JBD5 or JBD79 prophage, which both express AcrE1, did not cause cell death due to the AcrE1-mediated anti-CRISPR activity. In addition, overnight cultures of these strains were completely devoid of green pigmentation, implying that the targeted phzM promoter was being repressed (Fig. 4b and d). This same behavior was observed when the experiment was performed with a strain expressing an inactive Cas3 protein, demonstrating that expression of AcrE1 phenocopies a strain lacking Cas3 activity. Expression of the crRNAs in an SMC4386 strain with its CRISPR-Cas locus completely November/December 2017 Volume 8 Issue 6 e01751-17 mbio.asm.org 7 ® Pawluk et al. deleted resulted in green pigmentation, as no Cascade complex was present to mediate phzM repression. All strains transformed by empty vector also displayed bright green pigmentation. Introduction of the crRNA-expressing plasmids into wild-type SMC4386 bearing a JBD26 prophage, which expresses a different type I-E anti-CRISPR protein (AcrE3) (4), resulted in green pigmentation, suggesting that this anti-CRISPR protein blocks Cascade DNA binding (Fig. 4c and d). In summary, these experiments demon- strate that AcrE1 functions by blocking the activity of Cas3 without inhibiting the DNA-binding activity of Cascade. AcrE3 elicits a contrasting behavior and likely abro- gates the DNA-binding activity of Cascade. Thus, AcrE1 can convert the activity of the CRISPR-Cas interference complex into a gene regulatory function. November/December 2017 Volume 8 Issue 6 e01751-17 DISCUSSION These results mirror those that we obtained in a previous study on AcrF1 in which substitutions at only 3 out of 35 surface-exposed residues tested caused a signiﬁcant reduction in anti-CRISPR activity. In both of these studies, the anti-CRISPR proteins appear to utilize a very small functional interface as deﬁned by mutagenesis. In the case of AcrF1, however, the functional interface deﬁned by mutagenesis is considerably smaller than the interaction interface between AcrF1 and the CRISPR-Cas complex observed in the co-complex structure determined by cryo-electron microscopy (17). Thus, we cannot conclude from our mutagenesis that AcrE1 forms a small interaction interface with Cas3. In this regard, it is notable that the type I-F anti-CRISPR protein, AcrF3, forms a very large interaction interface with Cas3 (18, 19), comprising an area of ~2,500 Å2. The requisite mutagenesis studies have not yet been performed to delineate the functional interface of this complex, which may also turn out to be much smaller than indicated by the structure. The ability of AcrE1 to inhibit Cas3 activity allowed us to convert the endogenous mbio.asm.org 8 November/December 2017 Volume 8 Issue 6 e01751-17 ® A Type I-E Anti-CRISPR Protein type I-E CRISPR-Cas system into a speciﬁc repressor of the phzM promoter. This experiment proved that AcrE1 inhibits Cas3 and also demonstrated the potential for a CRISPR-based tool for transcriptional repression of target genes in bacteria. Any strain possessing an active CRISPR-Cas system that can be inhibited by either AcrE1 for type I-E or AcrF3 for type I-F (29) can be transformed with a single plasmid expressing anti-CRISPR protein and a promoter-targeting crRNA to elicit speciﬁc gene repression. Anti-CRISPR proteins from P. aeruginosa have proven effective at inhibiting the type I-F CRISPR-Cas system of Pectobacterium atrosepticum, whose Cas proteins share less than 50% pairwise sequence identity to P. aeruginosa (13), indicating that the transcriptional repression system may work in many diverse bacterial lineages. Due to the small size of the crRNA molecules for these systems, targets can easily be synthesized as oligo- nucleotides and cloned into an anti-CRISPR protein-expressing plasmid, enabling quick, cost-effective, multiplexable, and robust transcriptional repression of endogenous bacterial gene expression. Our AcrE1-mediated transcriptional repression results raise the possibility that anti- CRISPR proteins could be co-opted in some species to modulate CRISPR-Cas activity for the purpose of gene regulation. MATERIALS AND METHODS Bacterial growth and phage propagation. Pseudomonas aeruginosa was grown in LB liquid medium at 37°C with shaking or on LB agar plates at 37°C overnight, unless otherwise indicated. When necessary, media were supplemented with 50 g/ml gentamicin to maintain the pHERD30T plasmid (30). Plasmids were introduced into P. aeruginosa SMC4386 by electroporation. Induction of the plasmid promoter for overexpression of anti-CRISPR genes was achieved by supplementing medium with 4 mM arabinose. Phages were propagated by mixing with P. aeruginosa SMC4386 ΔCRISPR-cas, a susceptible host. The phage-host mixture was added to LB containing 0.7% agar and 10 mM MgSO4 and poured onto thick LB agar (1.5%) plates containing 10 mM MgSO4. After overnight incubation at 30°C, plates with near- conﬂuent lysis were soaked with SM buffer for 3 h for phage extraction, followed by centrifugation at 14,000 rpm to remove cell and agar debris. Phages were stored in SM buffer over chloroform at 4°C. Phage plaque assays. To measure type I-E CRISPR-Cas activity in P. aeruginosa SMC4386, phage plaque assays were conducted as described previously (4). P. aeruginosa SMC4386 cells containing pHERD30T plasmid expressing AcrE1 were mixed with 0.7% LB agar and overlaid onto LB agar (1.5%) plates containing 10 mM MgSO4, 50 g/ml gentamicin, and (where indicated) 4 mM arabinose to induce anti-CRISPR gene expression. Tenfold serial dilutions of a CRISPR-targeted phage (JBD8) and a CRISPR- insensitive phage (JBD93a) were spotted on the surface, and the plates were incubated overnight at 30°C. Site-directed mutagenesis. Pairs of complementary oligonucleotides containing the codon to be mutated plus ﬁve codons on either side were synthesized by Euroﬁns Genomics. Pfu DNA polymerase was used to PCR amplify DNA from template plasmid pHERD30T containing the acrE1 gene. After template digestion with DpnI (New England BioLabs [NEB]), the DNA was concentrated and puriﬁed by ethanol precipitation and used to transform E. coli DH5. Mutations were conﬁrmed by DNA sequencing, and mutant plasmids were introduced into P. aeruginosa SMC4386 for phage plaque assays. AcrE1 expression and protein puriﬁcation. The AcrE1 open reading frame was cloned by ligation into pET21d() with either an N-terminal (NHis) or C-terminal (CHis) noncleavable 6His tag. E. coli BL21(DE3) cells carrying either plasmid maintained in 100 g/ml ampicillin were grown to an optical density at 600 nm (OD600) of 0.5 to 0.6, and IPTG (isopropyl--D-thiogalactopyranoside) was added to a ﬁnal concentration of 0.8 mM. DISCUSSION It is possible that both bacteria and phages encoding Cas3-targeting anti-CRISPR proteins could beneﬁt from Cascade-mediated transcrip- tional control. For example, an AcrE1-encoding phage could encode a crRNA targeting one or more host promoters in order to silence genes involved in phage resistance or to otherwise modulate host transcription in ways that would beneﬁt the phage. It is also possible that bacteria with CRISPR-Cas systems could subvert the activity of this anti-CRISPR protein and target a phage promoter to silence essential phage genes and prevent it from entering into the viral replicative cycle. Twenty-two distinct anti-CRISPR protein families have been discovered to date that inhibit four different types of CRISPR-Cas systems (4, 5, 12–15). Although relatively few have been studied in detail, it is clear that phages utilize several different strategies to inhibit the CRISPR-Cas machinery. Further structural and biochemical characterization of anti-CRISPRs will illuminate the full spectrum of inhibition mechanisms and will provide deeper insight into the functioning of diverse CRISPR-Cas systems. November/December 2017 Volume 8 Issue 6 e01751-17 MATERIALS AND METHODS The ﬁnal structure was obtained after multiple rounds of reﬁnements and building cycles using Phenix Reﬁne and Coot software packages, yielding a ﬁnal Rwork/Rfree of 0.18/0.22. The structure of NHis AcrE1 was obtained by molecular replacement using CHis as a starting model using Phaser. The ﬁnal model was generated after several rounds of model building and reﬁnement using Coot and the Phenix Reﬁne program using TLS (translation, liberation, screw), yielding a ﬁnal Rwork/Rfree of 0.18/0.23 for native NHis AcrE1. CD spectroscopy. Circular dichroism (CD) was conducted using an 0.1-cm quartz cuvette at 25°C with a measurement range of 260 to 200 nm. The scanning speed was 50 nm/min with a bandwidth of 1 nm, response time of 2 s, and data pitch of 1.0 nm. Protein concentrations for both wild-type and mutant His-tagged AcrE1 were 16 M in buffer containing 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, and 5 mM -mercaptoethanol. Triplicate measurements were recorded and averaged for each CD run. Three biological replicates were collected. In vivo formaldehyde cross-linking and afﬁnity puriﬁcation. One-hundred-milliliter cultures of P. aeruginosa strain SMC4386 containing plasmids encoding 6His-tagged anti-CRISPR AcrE1 or empty vector were grown at 37°C in LB medium. At an OD600 of 0.5, 4 mM arabinose was used to induce plasmid expression, except for the uninduced samples in Fig. 3b, to which no arabinose was added. After a 2-h incubation, 200 l of 37% formaldehyde solution was added, and cells were incubated with shaking for an additional 30 min at 37°C. Glycine was added to a ﬁnal concentration of 100 mM to quench the cross-linking reaction, and cells were pelleted. One milliliter of Novagen BugBuster reagent was used to resuspend the cell pellet, followed by 1 h of shaking incubation at 25°C and centrifugation at 14,000 rpm for 5 min. We added 50 l Ni-NTA beads (Qiagen) to each clariﬁed lysate and puriﬁed with the same buffer system as described for AcrE1 puriﬁcation above. One hundred microliters of elution buffer was added to the washed beads and incubated at 100°C for 15 min to elute proteins and reverse heat-labile formaldehyde cross-links (26). Proteins were analyzed by SDS-PAGE followed by silver staining and by SDS-PAGE followed by anti-His-tag Western blotting, as well as by trypsin digest followed by linear trap quadrupole (LTQ) (Thermo Scientiﬁc) liquid chromatography-tandem mass spectrometry (LC-MS/MS) (SPARC Biocentre, SickKids, Toronto, Canada). Transcriptional repression assay. MATERIALS AND METHODS AcrE1 CHis crystals were observed in 0.1 M sodium cacodylate buffer, pH 6.0, 0.1 M NaBr, and 25% polyethylene glycol (PEG) 3350. The crystals were further optimized with a 1:1-ratio sitting drop at 20°C under a precipitant condition composed of 0.07 M sodium cacodylate, pH 6.1, 0.5 M NaBr, 27% PEG 3350, and 15% glycerol, yielding single crystals in space group C2. Native NHis AcrE1 crystals were observed in 0.2 M ammonium citrate dibasic, 20% (wt/vol) PEG 3350. The crystals were further optimized with a 1:1-ratio sitting drop at 20°C under a precipitant condition composed of 0.2 M ammonium citrate dibasic, pH 7.0, 25% (wt/vol) PEG 3350, and 15% glycerol, yielding single crystals in space group C2. g y p g p Data collection and structure determination. CHis and NHis AcrE1 crystallographic data were collected on crystals frozen at 105 K on 08B1-1 and 08ID-1 beamlines at Canadian Light Source (CLS), respectively. Diffraction data from a total of 360 images were collected at wavelengths of 0.9199 and 0.9795 using 1° oscillations. Data were processed with the XDS package to a resolution of 2.5 and 2.0 Å for CHis and NHis AcrE1, respectively. A complete model for CHis was solved by Br-SAD with additional anomalous signal from Se atoms using Phenix AutoSol. The ﬁnal structure was obtained after multiple rounds of reﬁnements and building cycles using Phenix Reﬁne and Coot software packages, yielding a ﬁnal Rwork/Rfree of 0.18/0.22. The structure of NHis AcrE1 was obtained by molecular replacement using CHis as a starting model using Phaser. The ﬁnal model was generated after several rounds of model building and reﬁnement using Coot and the Phenix Reﬁne program using TLS (translation, liberation, screw), yielding a ﬁnal Rwork/Rfree of 0.18/0.23 for native NHis AcrE1. Data collection and structure determination. CHis and NHis AcrE1 crystallographic data were collected on crystals frozen at 105 K on 08B1-1 and 08ID-1 beamlines at Canadian Light Source (CLS), respectively. Diffraction data from a total of 360 images were collected at wavelengths of 0.9199 and 0.9795 using 1° oscillations. Data were processed with the XDS package to a resolution of 2.5 and 2.0 Å for CHis and NHis AcrE1, respectively. A complete model for CHis was solved by Br-SAD with additional anomalous signal from Se atoms using Phenix AutoSol. MATERIALS AND METHODS After 4 h of shaking incubation at 37°C, cells were pelleted and resuspended in binding buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM -mercaptoethanol) with 5 mM imidazole added. Cells were lysed by sonication, and lysates were cleared by centrifugation for mbio.asm.org 9 ® Pawluk et al. 20 min at 17,000 rpm. Nickel-nitrilotriacetic acid (Ni-NTA) beads (Qiagen) were incubated with the clariﬁed lysate for 30 min at 4°C. Column puriﬁcation at room temperature was performed with washes in wash buffer (binding buffer plus 30 mM imidazole), and the protein was eluted in elution buffer (binding buffer plus 300 mM imidazole). Binding buffer was used for overnight dialysis at room temperature. The protein was further puriﬁed by size exclusion chromatography (SEC) using either a Superdex 200 16/60 column (for large-scale puriﬁcation) or a Superdex 75 10/30 column (for analytical SEC prior to CD spectroscopy) in binding buffer. The same puriﬁcation methods and buffers were used to purify AcrE1Δ91–100 NHis. Selenomethionine (Se-Met)-labeled CHis AcrE1 was expressed using the methionine-auxotrophic E. coli BL21(DE3) B834 strain cultured in M9 minimal medium containing 0.2% glucose and trace metals supplemented with Se-Met and was puriﬁed using the same protocol as described above. Crystallization of AcrE1. Puriﬁed AcrE1 CHis or NHis (native) was initially screened with a 1:1 (protein/precipitant) ratio against the MCSG commercial suite and JCSG commercial screen using sitting drop vapor diffusion at 10 mg/ml. AcrE1 CHis crystals were observed in 0.1 M sodium cacodylate buffer, pH 6.0, 0.1 M NaBr, and 25% polyethylene glycol (PEG) 3350. The crystals were further optimized with a 1:1-ratio sitting drop at 20°C under a precipitant condition composed of 0.07 M sodium cacodylate, pH 6.1, 0.5 M NaBr, 27% PEG 3350, and 15% glycerol, yielding single crystals in space group C2. Native NHis AcrE1 crystals were observed in 0.2 M ammonium citrate dibasic, 20% (wt/vol) PEG 3350. The crystals were further optimized with a 1:1-ratio sitting drop at 20°C under a precipitant condition composed of 0.2 M ammonium citrate dibasic, pH 7.0, 25% (wt/vol) PEG 3350, and 15% glycerol, yielding single crystals in space group C2. Crystallization of AcrE1. Puriﬁed AcrE1 CHis or NHis (native) was initially screened with a 1:1 (protein/precipitant) ratio against the MCSG commercial suite and JCSG commercial screen using sitting drop vapor diffusion at 10 mg/ml. November/December 2017 Volume 8 Issue 6 e01751-17 mbio.asm.org 10 mbio.asm.org 10 MATERIALS AND METHODS Pairs of complementary oligonucleotides were designed to encode a CRISPR repeat-spacer-repeat unit in which the repeat sequence is the P. aeruginosa SMC4386 consensus repeat (5=-GTGTTCCCCACATGCGTGGGGATGAACCG-3=) and the spacer sequence matches a 32-nucleotide sequence in the phzM promoter element ﬂanked by a consensus protospacer adjacent motif (PAM), 5=-NTT-3=. The sense-strand crRNA sequences (with repeats in lowercase and spacer in uppercase) used in this study are crRNA 1 (5=-gtgttccccacatgcgtggggatgaaccgAATAAAATTACAACTTGG CTACAACCTCCGGCgtgttccccacatgcgtggggatgaaccg-3=) and crRNA 2 (5=-gtgttccccacatgcgtggggatgaacc gCTGATGCTTCCTGCAATGCCGGAGGTTGTAGCgtgttccccacatgcgtggggatgaaccg-3=). Once the oligonucle- otides were annealed by heating to 95°C and slow cooling to 10°C, sticky ends were formed that could ligate into pHERD30T predigested with EcoRI and HindIII restriction endonucleases. Ligated plasmids were sequence conﬁrmed and then used to transform SMC4386 cells by electroporation. A dramatic reduction in transformation efﬁciency of the crRNA-containing plasmids compared to the empty vector indicated that the crRNAs were expressed and processed and successfully targeted the CRISPR-Cas system to the host genome. The indicated mutant or lysogenic strains of SMC4386 were transformed with the crRNA plasmid or empty vector. To ensure that the observed phenotypes were not due to the prophage insertion site (which, for these phages, is random), all results were conﬁrmed using a second, independently isolated lysogen (data not shown). Colonies were used to inoculate King’s A liquid mbio.asm.org 10 ® A Type I-E Anti-CRISPR Protein medium (20 g/liter peptone, 10 g/liter potassium sulfate, 1.64 g/liter anhydrous magnesium chloride, and 1% glycerol) supplemented with 50 g/ml gentamicin to maintain pHERD30T and grown at 37°C. After 10 h of growth, plasmid expression of crRNAs was induced by the addition of 4 mM arabinose, and induction was allowed to proceed for 16 h at 37°C. Construction of SMC4386 cas3 H89A/D90A mutant. The cas3 gene was cloned by ligation into pHERD30T (30), and site-directed mutagenesis was performed to introduce the double mutation of H89A and D90A, which comprise the active site residues of the HD nuclease domain of Cas3. The resultant plasmid was used to transform P. aeruginosa SMC4386, and cells were grown in LB under 50-g/ml gentamicin selection for 48 h and then passaged in antibiotic-free LB medium for 72 h to cause plasmid loss. A type I-E CRISPR locus containing spacers targeting highly conserved regions of several essential genes in P. aeruginosa, including RNA polymerase and DNA polymerase, was ligated into pHERD20T, a plasmid identical to pHERD30T but with a carbenicillin (instead of gentamicin) resistance cassette. SUPPLEMENTAL MATERIAL Supplemental material for this article may be found at https://doi.org/10.1128/mBio .01751-17. Supplemental material for this article may be found at https://doi.org/10.1128/mBio 01751-17. FIG S1, TIF ﬁle, 2.6 MB. FIG S2, TIF ﬁle, 2.6 MB. FIG S3, TIF ﬁle, 2.6 MB. TABLE S1, DOCX ﬁle, 0.1 MB. TABLE S2, DOCX ﬁle, 0.1 MB. ACKNOWLEDGMENTS We thank Diane Bona and Christine Lai for technical assistance and Carina Buttner for helpful discussions. We thank members of APS at the NE-CAT beamlines 24-ID-E and 24-ID-C and CLS beamline staff at CMCF-08ID-1 for assistance with data collection and Jonathan Krieger at the SickKids SPARC Biocentre for assistance with mass spectrom- etry. This research was funded with support from the Natural Sciences and Engineering Research Council of Canada (NSERC), as well as with instrumentation and infrastructure support provided by the Canadian Foundation for Innovation (CFI) and the Ontario Ministry of Education and Innovation. A.P. was supported by an Ontario Graduate Scholarship and a Canadian Institutes of Health Research (CIHR) Doctoral Award. T.F.M. is a Canada Research Chair in the structural biology of membrane proteins. This work was supported by CIHR operating grants to A.R.D. (MOP-130482) and K.L.M. (MOP- 136845). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. A.P. designed experiments, performed experiments, and wrote the manuscript. M.S. conducted crystallographic data collection and structure determination, performed experiments, and assisted in writing the manuscript. C.C. conducted crystallographic data collection and structure determination. M.M. performed experiments. T.F.M. and A.R.D. designed and supervised experiments. K.L.M. designed and supervised experi- ments and wrote the manuscript. MATERIALS AND METHODS This plasmid was used to select for cells in which recombination of the mutated cas3 gene had occurred and CRISPR-Cas activity was lost due to the lack of nuclease function. The plasmid should not be able to transform cells with an active CRISPR-Cas system, as they would target and cleave their own genome at several key locations that are, in combination, unescapable by mutation. Sequencing was used to conﬁrm that the correct mutation in cas3 had caused the loss of CRISPR-Cas activity. 5. Bondy-Denomy J, Pawluk A, Maxwell KL, Davidson AR. 2013. Bacterio- phage genes that inactivate the CRISPR/Cas bacterial immune system. Nature 493:429–432. https://doi.org/10.1038/nature11723. 3. 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https://openalex.org/W3136578493	https://www.apsijournal.com/index.php/psyjournal/article/download/1139/707	Ukrainian	null	THE EXPERIMENTAL STUDY ON PSYCHOLOGICAL FEATURES OF GENERAL SCHOOL PUPILS’ COMMUNICATIVE SKILLS	Psihologìčnij časopis	2,020	cc-by	5,086	ISSN 2414-0023 (Print) ISSN 2414-004X (Online) ISSN 2414-0023 (Print) ISSN 2414-004X (Online) ISSN 2414-0023 (Print) ISSN 2414-004X (Online) ISSN 2414-0023 (Print) ISSN 2414-004X (Online) PSYCHOLOGICAL JOURNAL Volume 6 Issue 12 (44) 2020 DOI (Issue): https://doi.org/10.31108/1.2020.6.12 ЕКСПЕРИМЕНТАЛЬНЕ ДОСЛІДЖЕННЯ ПСИХОЛОГІЧНИХ ОСОБЛИВОСТЕЙ КОМУНІКАТИВНИХ УМІНЬ УЧНІВ ОСНОВНОЇ ШКОЛИ Зеленська Зоя Петрівна1 1 Заступник директора Києво-Печерського ліцею № 171 «Лідер», науковий кореспондент лабораторії вікової психофізіології Інституту психології імені Г.С. Костюка Національної академії педагогічних наук України, м. Київ (Україна) ORCID ID: https://orcid.org/0000-0001-7760-9437 ORCID ID: https://orcid.org/0000-0001-7760-9437 This is an Open Access journal, all articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 Interna- tional (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license. Volume 6 Issue 12 (44) 2020 Address for correspondence, e-mail: editpsychas@gmail.com p , Copyright: © Zoya Zelenska © Zoya Zelenska p , p y @g Copyright: © Zoya Zelenska This is an Open Access journal, all articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 Interna- tional (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license. DOI (Article): https://doi.org/10.31108/1.2020.6.12.9 DOI (Issue): https://doi.org/10.31108/1.2020.6.12 Volume 6 Issue 12 (44) 2020 Дослідження проводилося в межах Всеукраїн- ського експерименту: «Психологічні засади інновацій- них технологій розвитку особистості» на базі Києво- Печерського ліцею № 171 «Лідер» (Наказ МОН № 414 від 9 квітня 2015 року) (Zelenska, 2015). Першоосновою аналізу розвитку комунікатив- них умінь може бути структура спілкування, до якої, на думку Г.М. Андрєєвої, входять три головні складові: комунікативна (обмін інформацією); інтерактивна (взаємодія); перцептивна (розуміння людини людиною) (Андрєєва, 1996). Ці складові, як правило, не існують у чистому, ізольованому вигляді. Реальне спілкування включає всі складові одночасно, які переплітаються між собою. Крім цього, спілкування буває творчим і репродуктивним, глибоким і поверховим, маніпулятив- ним і суб'єктивним (Андрєєва, 1987). Для визначення психологічних особливостей розвитку комунікативних умінь нами були використані наступні стандартизовані психодіагностичні методи- ки:методика «Діагностика рівня полікомунікативної емпатії» (за М. Юсуповим) для визначення рівня про- яву емпатичних тенденцій школяра (Iusupov, 1995); методика діагностики комунікативного контролю (за М. Шнайдером) для визначення рівня комунікативного контролю особистості школяра;методика дослідження комунікативних якостей школяра («Потреба у спілку- ванні» за О.М. Орловим) для визначення рівня розвит- ку потреби учня у спілкуванні (Orlov & other, 1974);методика «Діагностика емоційних бар’єрів у мі- жособистісному спілкуванні» (за В.В. Бойко) для ви- значення наявних емоційних бар’єрів в міжособистіс- ному спілкуванні учня) (Fetyskyn & other 2002) . Аналіз спеціальної науково-психологічної літе- ратури показує, що розвитку комунікативних умінь присвячено багато праць вітчизняних та зарубіжних вчених та психологів, однак дослідження психологіч- них особливостей розвитку комунікативних умінь уч- нів основної школи не знайшло належного відображен- ня,що й визначило вибір теми нашого дослідження. Мета дослідження полягає у висвітленні ре- зультатів експериментального дослідження психологіч- них особливостей розвитку комунікативних умінь уч- нів основної школи. Діагностика показників полікомунікативної емпатії (методика М. Юсупова) в учнів експеримента- льної групи (ліцей «Лідер») – ЕГ та контрольної гру- пи – КГ (Кловський ліцей) наведено у таблиці 1. Виклад основного матеріалу. Під час дослі- дження розвитку комунікативних умінь учнів основної школи ми керувалися загальноприйнятими методологі- чними підходами в психології: системним, діяльніс- ним, особистісно-орієнтованим та особистісно- комунікативним. Основним методом нашого дослі- дження є генетико-моделюючий метод, який має на меті вивчення саме цілісної особистості, що саморозви- вається. Принципи побудови генетико-моделюючого методу: принцип аналізу за одиницями, єдності біологі- чного і соціального, креативності, рефлексивного реля- тивізму та єдності експериментальної і генетичної лі- ній розвитку особистості (Максименко, 2000). Також ми спиралися на наступні принципи: цілісності, функ- ціональної детермінованості комунікативної діяльності та її підсистем, детермінізму, єдності свідомості і дія- льності. АНОТАЦІЯ Робота присвячена проблемі комунікативних умінь учнів основної школи. Мета дослідження полягала в експе- риментальному вивченні особливостей розвитку комунікативних умінь учнів середнього шкільного віку. У статті описуються принципи дослідження та методологічні підходи до дослідження, обґрунтовується генетико- моделюючий метод як основний метод дослідження. У дослідженні застосовувалися наступні стандартизовані психодіагностичні методики: методика «Діагностика рівня полікомунікативної емпатії» (за М. Юсуповим) для визначення рівня прояву емпатичних тенденцій школя- ра; методика діагностики комунікативного контролю (за М. Шнайдером) для визначення рівня комунікативного контролю особистості школяра; методика дослідження комунікативних якостей школяра («Потреба у спілкуван- ні» за О.М. Орловим) для визначення рівня розвитку потреби учня у спілкуванні; методика «Діагностика емоцій- них бар’єрів у міжособистісному спілкуванні» (за В.В. Бойко). У результаті дослідження виявили психологічні особливості розвитку комунікативних умінь: визначені рівні розвитку емпатійних тенденцій школярів, рівні їхнього комунікативного контролю, визначені потреби у спілку- ванні школярів (комунікативні якості) та наявні емоційні бар’єри у спілкуванні. Виявлена кореляція між названи- ми складовими досліджуваної системи. Визначені перспективи подальших досліджень. Ключові слова: емпатійні тенденції школярів, емоційні бар’єри, комунікативні уміння, комунікативні якості, комунікативний контроль, учень основної школи. Проблема розвитку комунікативних умінь ви- кликає підвищений інтерес та дискусії серед психологів та педагогів. Сьогодні актуально розглядати зміст пси- хологічного поняття "комунікативних умінь" у зв'язку з активізацією процесу навчання. Школа як один із важ- ливих інститутів соціалізації особистості у навчально- виховному процесі покликана всіляко сприяти оволо- дінню учнями комунікативними уміннями і навичками. Комунікативні уміння визначаються як один із видів спеціальних умінь, пов’язаних з уміннями сприймати, відтворювати і створювати усні і письмові висловлю- вання. Постановка проблеми дослідження. В умовах сьогодення для успішної соціалізації індивіда необхід- ною умовою є оволодіння на достатньому рівні комуні- кативними уміннями. Гарно сформовані комунікативні уміння є джерелом упевненості в успішній трудовій діяльності, сімейному житті, джерелом позитивних емоційних очікувань стосовно особистісної та соціаль- ної сфер. Комунікативні уміння як складова комуніка- тивної компетентності і в широкому контексті – загаль- ної культури особистості – є запорукою активної інтег- рації та адаптації особистості в сучасному суспільстві. © Zoya Zelenska © Zoya Zelenska 92 http://www.apsijournal.com/ Volume 6 Issue 12 (44) 2020 Volume 6 Issue 12 (44) 2020 ISSN 2414-0023 (Print) ISSN 2414-004X (Online) PSYCHOLOGICAL JOURNAL Volume 6 Issue 12 (44) 2020 © Zoya Zelenska Volume 6 Issue 12 (44) 2020 DOI (Issue): https://doi.org/10.31108/1.2020.6.12 Як видно з таблиці 1, ні в учнів експеримента- льної групи, ні в учнів контрольної групи не було вияв- лено дуже високого рівня емпатійних тенденцій. Високий рівень емпатійних тенденцій мають 6,6% учнів експериментальної групи, що вказує на те, що вони з непідробним інтересом ставляться до людей, їм подобається «читати» інших, заглядати в їхнє май- бутнє. Також вони емоційно чуйні, швидко встановлю- ють контакти та знаходять спільну мову, а оточення цінує їх за щиросердність. Вони намагаються не конф- ліктувати, знаходити компромісні рішення, адекватно реагують на критику на свою адресу, а в оцінці подій більше довіряють своїм почуттям та інтуїції, ніж аналі- тичним висновкам, віддають перевагу роботі разом з іншими людьми, ніж наодинці та постійно потребують схвалення своїх дій з боку оточення. Вибірку нашого дослідження склали 126 учнів основної школи, які навчаються у 6-му і 7-му класах. З них 106 – учні Києво-Печерського ліцею № 171 «Лідер» (експериментальна група) та 20 – учні Клов- ського ліцею (контрольна група). Середній рівень емпатійних тенденцій мають 85,0% учнів контрольної групи та 65,1% учнів експери- ментальної групи. Тобто, у взаєминах із людьми вони, DOI (Article): https://doi.org/10.31108/1.2020.6.12.9 © Zoya Zelenska © Zoya Zelenska 93 http://www.apsijournal.com/ ISSN 2414-0023 (Print) ISSN 2414-004X (Online) PSYCHOLOGICAL JOURNAL Volume 6 Issue 12 (44) 2020 Volume 6 Issue 12 (44) 2020 DOI (Issue): https://doi.org/10.31108/1.2020.6.12 Таблиця 1. Показники полікомунікативної емпатії школярів № з/п Рівень ЕГ (%) КГ (%) Емпатійні тенденції школярів 1. Дуже низький 0,9 - 2. Низький 27,4 15,0 3. Середній 65,1 85,0 4. Високий 6,6 - 5. Дуже високий - - Прояв емпатії до батьків 1. Дуже низький 4,7 - 2. Низький 6,6 5,0 3. Середній 77,4 90,0 4. Високий 8,5 5,0 5. Дуже високий 2,8 - Прояв емпатії до тварин 1. Дуженизький 7,5 - 2. Низький 27,4 20,0 3. Середній 63,2 80,0 4. Високий - - 5. Дужевисокий 1,9 - Прояв емпатії до людей похилого віку 1. Дуже низький 0,9 - 2. Низький 21,7 5,0 3. Середній 73,7 95,0 4. Високий 2,8 - 5. Дуже високий 0,9 - Прояв емпатії до дітей 1. Дуже низький 2,8 - 2. Низький 13,2 15,0 3. Середній 71,8 80,0 4. Високий 9,4 5,0 5. Дуже високий 2,8 - Прояв емпатії до художніх творів 1. Дуже низький 3,8 5,0 2. Низький 34,9 20,0 3. Середній 60,4 75,0 4. Високий 0,9 - 5. Дуже високий - - Прояв емпатії до незнайомих людей 1. Дуже низький 2,8 - 2. Низький 22,7 - 3. Середній 69,8 90,0 4. ISSN 2414-0023 (Print) ISSN 2414-004X (Online) PSYCHOLOGICAL JOURNAL DOI (Issue): https://doi.org/10.31108/1.2020.6.12 Volume 6 Issue 12 (44) 2020 в учнів контрольної групи – середній та дуже низький рівні. ми відчувають низку утруднень, у тому числі надто болісно сприймають критику на свою адресу, тому вза- ємодію важко назвати ефективною. ми відчувають низку утруднень, у тому числі надто болісно сприймають критику на свою адресу, тому вза- ємодію важко назвати ефективною. Як бачимо в таблиці, в учнів контрольної групи переважають дуже високий, високий і середній рівні прояву емпатії до незнайомих людей, а в учнів експери- Таблиця 2. Отже, як показують результати дослідження, серед учнів експериментальної групи більша кількість тих, хто має високий і дуже високий рівень розвитку емпатії, а також низький і дуже низький рівень. А серед учнів контрольної групи більше тих, хто має середній рівень розвитку емпатії. Комунікативний контроль № з/п Рівень комунікативного контролю ЕГ (%) К Г (%) 1. Низький комунікативний контроль 12,3% 15,8% 2. Середній комунікативний контроль 50,9% 52,6% 3. Високий комунікативний контроль 36,8% 31,6% Комунікативний контроль Ми бачимо, що схожа тенденція розвитку ем- патичних тенденцій спостерігається в учнів і за про- явом емпатії до батьків: в учнів експериментальної гру- пи переважають дуже високий, високий, низький та дуже низький рівні прояву емпатії до батьків, а в учнів контрольної групи – середній рівень. ментальної групи – низький та дуже низький рівні. ментальної групи – низький та дуже низький рівні. За методикою діагностики комунікативного контролю (М. Шнайдер) було визначено рівень комуні- кативного контролю в учнів експериментальної (ліцей «Лідер»)та контрольної (Кловський ліцей) груп (див. табл.2). З таблиці 1 ми бачимо, що за показниками рів- ня прояву емпатії до тварин в учнів експериментальної групи переважають дуже високий, низький та дуже ни- зький рівні прояву емпатії до тварин, а в учнів контро- льної групи – середній рівень. Як ми бачимо з табл.2, високий комунікатив- ний контроль переважає у 36,8% учнів експерименталь- ної групи на відміну від 31,6% в учнів контрольної гру- пи, а середній – 52,6% –і низький –15,8% – в учнів кон- трольної групи. Схожа тенденція була виявлена і за показника- ми прояву емпатії до людей похилого віку: в учнів екс- периментальної групи переважають дуже високий, ви- сокий, низький та дуже низький рівні прояву емпатії до людей похилого віку, а в учнів контрольної групи – середній рівень. За методикою дослідження комунікативних якостей школяра («Потреба у спілкуванні» за О.М. Ор- ловим) було визначено рівень розвитку потреби у спіл- куванні в учнів експериментальної (ліцей «Лідер») та контрольної (Кловський ліцей) груп(див. табл. 3). DOI (Issue): https://doi.org/10.31108/1.2020.6.12 Високий 4,7 5,0 5. Дуже високий - 5,0 Показники полікомунікативної емпатії школярів насамперед, оцінюють їхні вчинки, менше цікавлячись мотивами вчинків. Також учні з середнім рівнем емпа- тійних тенденцій здебільшого контролюють свої емо- ції, є уважними до співрозмовників та намагаються зро- зуміти, що стоїть за їхніми словами. Однак, якщо спів- розмовники занадто проявляють свої почуття, то учні цієї групи можуть втратити терпець. У них немає розку- тості почуттів, що заважає повноцінному сприйняттю оточення та є перешкодою у встановленні повноцінних взаємин та співпраці з іншими. паніях, для них незрозумілі емоційні прояви інших, тому вони ставляться до них здебільшого з іронією. Таким особам часто важко першими розпочати розмо- ву, особливо з незнайомими людьми, їхні взаємини з оточенням є формалізованими, побудованими, як пра- вило, за статусно-рольовим принципом. Дуже низький рівень розвитку емпатії мають 0,9% учнів експериментальної групи. Учні цієї групи схильні до надмірної центрації на власній персоні, що створює труднощі у порозумінні з оточенням. Вони є прихильниками точних формулювань і раціональних рішень, віддають перевагу такій роботі, яка передбачає мінімальні контакти з іншими людьми.Учні з низьким рівнем розвитку емпатії можуть бути високопродуктив- ними в індивідуальній роботі, однак у взаємодії з інши- Низький рівень емпатійних тенденцій є вираже- нішим у 27,4% учнів експериментальної групи на відмі- ну від 15,0% учнів контрольної групи. Такі учні схильні відчувати суттєві труднощі у встановленні контактів з оточенням, вони відчувають дискомфорт у гучних ком- © Zoya Zelenska 94 http://www.apsijournal.com/ Емоційні бар’єри у міжособистісному спілкуванні Результати за шкалою «Домінування негатив- них емоцій» методики «Діагностика емоційних бар’єрів у міжособистісному спілкуванні» (за В.В. Бойко)в учнів експериментальної (ліцей «Лідер») та контрольної груп (Кловський ліцей) вказують на те, що у 100,0 % учнів контрольної групи та у 97,2% учнів експериментальної групи повністю відсутні показники домінування нега- тивних емоцій.А 2,8% учнів експериментальної групи домінування негативних емоцій, зазвичай, не заважає спілкуватися з іншими. групи на відміну від учнів контрольної групи, повністю відсутні емоційні бар’єри у міжособистісному спілку- ванні, а 9,4% вони не заважають спілкуватися. Деякі емоційні проблеми в спілкуванні мають 32,1% учнів експериментальної групи та 5,4 % учнів контрольної групи. Емоційні бар’єри, які певною мірою ускладню- ють взаємодію з людьми було встановлено у 42,5% уч- нів експериментальної групи та у 57,8% учнів контро- льної групи. А емоційні бар’єри, що шкодять встанов- ленню контактів з оточенням було виявлено у 36,8% учнів контрольної групи та у 11,3% учнів експеримен- тальної групи. Результати за шкалою «Небажання зближува- тися з людьми» методики «Діагностика емоційних бар’- єрів у міжособистісному спілкуванні» (за В.В. Бойко)в учнів експериментальної (ліцей «Лідер») та контроль- ної груп (Кловський ліцей) свідчать про те, що у 97,2% учнів експериментальної групи та у 73,6% учнів конт- рольної групи повністю відсутні показники «небажання зближуватися з людьми». А 21,1 % учнів контрольної групи та 2,8% учнів експериментальної групи «небажання зближуватися з людьми», зазвичай, не за- важає спілкуватися з іншими. Також у 5,3% учнів кон- трольної групи було виявлено «небажання зближувати- ся з людьми»у щоденному спілкуванні. Тобто, в учнів контрольної групи емоційні ба- р’єри, які ускладнюють взаємодію з людьми та шкодять встановленню контактів з оточенням є більш представ- леними, ніж в учнів експериментальної групи. Результати за шкалою «Неадекватний прояв емоцій» методики «Діагностика емоційних бар’єрів у міжособистісному спілкуванні» (за В.В. Бойко)в учнів експериментальної (ліцей «Лідер») та контрольної груп (Кловський ліцей) свідчать про те, що у 85,8% учнів експериментальної групи та у 70,0 % учнів контрольної групи емоції, зазвичай, не заважають спілкуватися з іншими, неадекватного прояву емоцій у спілкуванні практично не виникає. А у 30,0 % учнів контрольної групи та у 14,2% учнів експериментальної групи є деякі емоційні проблеми ущоденному спілкуванні, тобто, має Позаяк отримані експериментальні дані не від- повідають нормальному розподілу, то для їхньої стати- стичної обробки ми використалинепараметрич- ний статистичний критерій U-Манна Уїтні, який зазви- чай використовується для оцінки різниці між дво- ма вибірками за рівнем ознак, виміряних якісно. Цей критерій дозволяє виявити відмінності в значенні пара- метра між малими вибірками. Volume 6 Issue 12 (44) 2020 ISSN 2414-0023 (Print) ISSN 2414-004X (Online) Результати дослідження за показниками рівня прояву емпатії до дітей свідчать про те, що в учнів екс- периментальної групи переважають дуже високий, ви- сокий та дуже низький рівні прояву емпатії до дітей, а в учнів контрольної групи – середній та низький рівні. Отже, високу (3,8%) і середню (6,6%) потребу у спілкуванні мають учні експериментальної групи на відміну від учнів контрольної групи, в яких ці рівні взагалі не представлені. Низьку потребу у спілкуванні мають 68,2% учнів контрольної групи на відміну від 28,3% учнів експериментальної групи. А дуже низьку потребу у спілкуванні, навпаки, мають 61,3% учнів екс- периментальної групи на відміну від 31,8% учнів конт- Результати дослідження за показниками рівня прояву емпатії до художніх творів вказують на те, що дуже високого рівня прояву емпатії до художніх творів в учнів нашої вибірки не було виявлено. В учнів експе- риментальної групи переважають дуже високий, висо- кий, низький рівні прояву емпатії до художніх творів, а Таблиця 3. 95 КГ (%) 31,8 68,2 - - - © Zoya Zelenska 95 Volume 6 Issue 12 (44) 2020 http://www.apsijournal.com/ Комунікативні якості (потреба у спілкуванні) № з/п Рівень потреби у спілкуванні ЕГ (%) КГ (%) 1. Дуже низька потреба у спілкуванні 61,3 31,8 2. Низька потреба у спілкуванні 28,3 68,2 3. Середня потреба у спілкуванні 6,6 - 4. Висока потреба у спілкуванні 3,8 - 5. Дуже висока потреба у спілкуванні - - © Zoya Zelenska DOI (Article): https://doi.org/10.31108/1.2020.6.12.9 Комунікативні якості (потреба у спілкуванні) 95 © Zoya Zelenska © Zoya Zelenska 95 http://www.apsijournal.com/ PSYCHOLOGICAL JOURNAL Volume 6 Issue 12 (44) 2020 ISSN 2414-0023 (Print) ISSN 2414-004X (Online) DOI (Issue): https://doi.org/10.31108/1.2020.6.12 рольної групи. місцедещо неадекватний прояв емоцій. ISSN 2414-0023 (Print) ISSN 2414-004X (Online) PSYCHOLOGICAL JOURNAL Volume 6 Issue 12 (44) 2020 місцедещо неадекватний прояв емоцій. DOI (Issue): https://doi.org/10.31108/1.2020.6.12 Результати за шкалою «Негнучкість та невираз- ність емоцій» методики «Діагностика емоційних бар’є- рів у міжособистісному спілкуванні» (за В.В. Бойко)в учнів експериментальної (ліцей «Лідер») та контроль- ної груп (Кловський ліцей) засвідчили, що у 100,0 % учнів контрольної групи та у 95,3% учнів експеримен- тальної групи повністю відсутні показники негнучкості та невиразності емоцій. А негнучкість та невиразність емоцій, зазвичай, не заважають спілкуватися з іншими 4,7% учнів експериментальної групи. За методикою «Діагностика емоційних бар’єрів у міжособистісному спілкуванні» (В.В. Бойко) визнача- лась наявність емоційних бар’єрів у міжособистісному спілкуванні в учнів експериментальної (ліцей «Лідер») та контрольної (Кловський ліцей) груп(див. табл.4). Як бачимо, у 4,7% учнів експериментальної Таблиця 4. Емоційні бар’єри у міжособистісному спілкуванні DOI (Article): https://doi.org/10.31108/1.2020.6.12.9 DOI (Issue): https://doi.org/10.31108/1.2020.6.12 Юсуповим) в учнів експериментальної та контрольної груп, результати застосунку критерію U-Манна Уїтні наступні: значущі відмінності спостерігаються лише за шкалою «прояв емпатії до незнайомих людей» – в учнів контрольної групи вони є значно вищими, ніж в учнів експериментальної групи на рівні р≤0,05. Проведений кореляційний аналіз (коефіцієнт кореляції Пірсона) виявив залежність між показниками емпатійних тенденцій та статі учнів нашої вибірки. А саме такі показники, як прояв емпатії до тварин (r= 0,27; p £ 0,05), прояв емпатії до дітей (r= 0,28; p £ 0,01), прояв емпатії до художніх творів (r= 0,10; p £ 0,05), прояв емпатії до незнайомих людей (r= 0,25; p £ 0,05), емпатійні тенденції (r=0,30; p £ 0,01). Також було вияв- лено позитивний кореляційний зв'язок між показниками «прояву емпатії до художніх творів» та показниками «комунікативного контролю»(r= 0,22; p £ 0,05). Тобто, чим вищими є показники «прояву емпатії до художніх творів», тим вищими в них є «комунікативний конт- роль» і, навпаки, чим нижчого рівня в них є «прояв ем- патії до художніх творів», тим нижчим є їхній «комунікативний контроль». Відмінності показників за методикою «Діагностика емоційних бар’єрів у міжособистісному спілкуванні» в учнів експериментальної та контрольної груп свідчать про те, що в учнів контрольної групи спо- стерігаються значно вищі показники за шкалами: «неадекватний прояв емоцій», «домінування негатив- них емоцій», «небажання зближуватися з людьми» та «загальна сума емоційних бар’єрів». Для визначення взаємозв’язків між досліджува- ними показниками в учнів експериментальної та конт- рольної груп було використано кореляційний аналіз (ранговий коефіцієнт кореляції r Пірсона). В учнів експериментальної групи (ліцей «Лідер») було визначено значущий кореляційний зв'я- зок між показниками «домінування негативних емоцій» та «прояв емпатії до незнайомих людей» (r= 0,26; p £ 0,01), що вказує на те, що чим вираженішими в учнів є негативні емоції, тим виразнішим в них є прояв емпатії до незнайомих людей і, навпаки, чим меншим є прояв негативних емоцій, тим меншим буде і прояв емпатії до незнайомих людей. В учнів експериментальної групи (ліцей «Лідер») було визначено значущий кореляційний зв'я- зок між показниками «домінування негативних емоцій» та «прояв емпатії до незнайомих людей» (r= 0,26; p £ 0,01), що вказує на те, що чим вираженішими в учнів є негативні емоції, тим виразнішим в них є прояв емпатії до незнайомих людей і, навпаки, чим меншим є прояв негативних емоцій, тим меншим буде і прояв емпатії до незнайомих людей. Також було визначено, що показники«прояву емпатії до художніх творів» пов'язані негативними ко- реляційними зв’язками із показниками «невміння керу- вати емоціями» (r= -0,67; p £ 0,01). Емоційні бар’єри у міжособистісному спілкуванні Отже, стосовно показників за методикою «Діагностика рівня полікомунікативної емпатії» (за М. © Zoya Zelenska 96 http://www.apsijournal.com/ Volume 6 Issue 12 (44) 2020 ISSN 2414-0023 (Print) ISSN 2414-004X (Online) PSYCHOLOGICAL JOURNAL Volume 6 Issue 12 (44) 2020 прояви емпатії до батьків та дітей і, навпаки, більш ви- ражена здатність до гнучкості та виразності емоцій впливає на підвищення емпатії до батьків та до дітей. Volume 6 Issue 12 (44) 2020 прояви емпатії до батьків та дітей і, навпаки, більш ви- ражена здатність до гнучкості та виразності емоцій впливає на підвищення емпатії до батьків та до дітей. DOI (Issue): https://doi.org/10.31108/1.2020.6.12 Виявлені тенденції впливу та взаємовпливу описаних показників комунікативних умінь свідчать про їхню співзалежність та взаємодоповнюваність. Роз- виток одного показника підсилює інший показник і навпаки. Andreeva, H. M. (1987). Obshchenye y optymyzatsyya sovmestnoy deyatelʹnosty [Communication and optimization of joint activi- ties]. Moscow. [in Russian]. Orlov, YU. M., Shkurkyn, V. Y., Orlova, L. P. (1974) Yzmerenye potreb- nosty v obshchenyy s pomoshchʹyu oprosnyka [Measuring the need for communication using a questionnaire]. Voprosy éksperymentalʹnoy psykholohyy y ee ystoryy: Sbornyk trudov. Moscow. [in Russian]. Виявлені у цьому дослідженні психологічні особливості комунікативних умінь та попередні дослі- дження автора статті, зокрема, дослідження впливу шкільної тривожності на рівень розвитку емпатії в уч- нів основної школи (Zelenska, 2019). можуть бути пок- ладені в основу психолого-педагогічної програми роз- витку комунікативних умінь учнів основної школи у тому числі на уроках англійської мови. Використання вправ на уроках англійської мови як психологічний супровід і засіб активізації пізнавальної діяльності уч- нів та розвитку їхніх комунікативних умінь успішно впроваджено в педагогічну практику Києво- Печерського ліцею №171 «Лідер» (Zelenska, 2015). Petrovskaya, L. A. (1989). Kompetentnostʹ v obshchenyy [Competence in communication]. Sotsyalʹno-psykholohycheskyytrenynh. Mos- cow: Yzd-vo MHU. [in Russian]. Fetyskyn, N. P., Kozlov, V. V., Manuylov, H. M. (2002). Sotsyalʹno- psykholohychechkaya dyahnostyka razvytyyalychnosty y ma- lykh hrupp [Socio-psychological diagnosis of personality development and small groups]. Moscow: Yzd-vo Ynstytuta Psykhoterapyy. [in Russian]. Yusupov, Y. M. (1995). Psykholohyya émpatyy (Teoretycheskye y pry- kladnye aspekty) [Psychology of empathy (Theoretical and applied aspects)]. S.-Peterb. Hos. Un-t. SPb. [in Russian]. Перспективи подальших досліджень поляга- ють у розробці з урахуванням виявлених психологічних особливостей психолого-педагогічної програми розви- тку комунікативних умінь учнів основної школи, що уможливить формування мовленнєво-комунікативних умінь учнів на уроках, у тому числі в процесі засвоєння іншомовної лексики. DOI (Issue): https://doi.org/10.31108/1.2020.6.12 Тобто, чим більше проявляється в учнів «емпатія до художніх творів», тим менш здатними вони є до керування емоціями і навпа- ки. А показники «негнучкості та невиразності емо- цій» пов'язані позитивними кореляційними зв’язками із показниками «небажання зближуватися з людь- ми» (r=0,264; p £ 0,01), що вказує на те, що чим гнучкі- шими та виразнішими є емоції в учнів, тим міцнішим в них є бажання зближуватися з людьми і, навпаки, чим негнучкішими та невиразнішими є емоції, тим більш вираженим є небажання зближуватися з людьми. А в учнів контрольної групи було визначено інші взаємозв’язки показників. Наприклад, було визна- чено негативні кореляційні зв’язки між показниками «невміння керувати емоціями» та «прояв емпатії до художніх творів» (r= -0,67; p £ 0,01) та «емпатійні тен- денції» (r=- 0,53; p £ 0,05), що вказує на те, що чим ви- раженішим в них є невміння керувати емоціями, тим менше здатними вони є до прояву емпатії до художніх творів та взагалі до емпатії і, навпаки, чим краще вони будуть здатні керувати власними емоціями, тим вира- женішою в них буде й емпатія до художніх творів, і емпатія загалом. А показник «негнучкість та невираз- ність емоцій» має значущі кореляційні зв’язки з показ- никами «прояв емпатії до батьків» (r= 0,54; p £ 0,05) та «прояв емпатії до дітей» (r= 0,51; p £ 0,05), що свідчить про наступне: чим менш здатними є учні до гнучкості та виразності емоцій, тим менше вираженими в них є Висновки.Отже, підсумовуючи результати проведеногоемпіричного дослідження психологічних особливостей розвитку комунікативних умінь учнів основної школи, слід зазначити, що значущих розбіж- ностей в результатах учнів експериментальної групи (Києво-Печерський ліцей № 171 «Лідер») та учнів конт- рольної групи (Кловський ліцей) не виявлено. Це озна- чає, що отримані результати ми можемо розглядати як такі, що стосуються учнів основної школи будь-якого навчального закладу середньої освіти. © Zoya Zelenska 97 Volume 6 Issue 12 (44) 2020 http://www.apsijournal.com/ Zoya Zelenska Deputy Director of Kyiv-Pechersk Lyceum № 171 “Leader”, scientific correspondent of the Laboratory of Age Psychophysiology, G.S. Kostiuk Institute of Psychology, the National Academy of Educational Sciences of Ukraine, Kyiv (Ukraine) PSYCHOLOGICAL JOURNAL PSYCHOLOGICAL JOURNAL Volume 6 Issue 12 (44) 2020 chology]. Moscow: Refl-buk. [in Russian]. Volume 6 Issue 12 (44) 2020 ABSTRACT Andreeva, H. M. (1996). Sotsyalʹnaya psykholohyya [Social psychology]. Moscow: Aspekt Press. [in Russian]. © Zoya Zelenska Зеленская Зоя Петровна were used: the method examining poly-communicative empathy (M. Yusupov) to determine pupils’ empathic tendencies; the method examining communicative control (M. Schneider) to determine pupil’s communicative con- trol; the method studying pupils’ communicative qualities (“Need for Communication” by O.M. Orlov) to determine development of pupils’ need for communication; “Examining emotional barriers in interpersonal communi- cation” (VV Boyko) to determine the existing emotional barriers in pupils’ interpersonal communication. Заместитель директора Киево-Печерского лицея № 171 «Лидер», научный корреспондент лаборатории возрастной психофизиологии Института психологии имени Г.С. Костюка Национальной академии педагогических наук Украины, г. Киев (Украина) Andreeva, H. M. (1996). Sotsyalʹnaya psykholohyya [Social psychology]. Moscow: Aspekt Press. [in Russian]. The study was conducted within the All-Ukrainian research project: “The psychological principles of innova- tive technologies for personal development” on the basis of Kyiv-Pechersk Lyceum № 171 “Leader” (Order of the Min- istry of Education and Science No 414 dated April 9, 2015). Zelensʹka, Z. P., Maksymenko, S. D., Kokun, O.M. & other (2015). Vykorystannya psykholohichnykh vprav na urokakh anhliysʹkoyi movy [The use of psychological exercises in En- glish lessons]. Psykholohichnyysuprovid yak zasib aktyvizat- siyi piznavalʹnoyi diyalʹnosti uchniv. (pp. 27-36). Kyiv: DP «Informatsiyno-analitychne ahent·stvo». [in Ukrainian]. The study object: pupils’ communicative skills. The study purpose: to study experimentally the develop- ment of communicative skills of middle-age school pupils. Zelensʹka, Z. P. (2019). Doslidzhennya vplyvu shkilʹnoyi tryvozhnosti na rivenʹ ozvytku empatiyi v uchniv osnovnoyi shkoly [Study of the influence of school anxiety on the level of empathy in pri- mary school students]. Aktualʹni problemy psykholohiyi: Zbirnyk naukovykh pratsʹ Instytutu psykholohiyi imeni H.S. Kostyuka NAPN Ukrayiny. Tom. V: Psykhofiziolohiya. Psykho- lohiya pratsi. Eksperymentalʹna psykholohiya, 19, pp. 31-43. [in Ukrainian]. Zelensʹka, Z. P. (2019). Doslidzhennya vplyvu shkilʹnoyi tryvozhnosti na rivenʹ ozvytku empatiyi v uchniv osnovnoyi shkoly [Study of the influence of school anxiety on the level of empathy in pri- mary school students]. Aktualʹni problemy psykholohiyi: Zbirnyk naukovykh pratsʹ Instytutu psykholohiyi imeni H.S. Kostyuka NAPN Ukrayiny. Tom. V: Psykhofiziolohiya. Psykho- lohiya pratsi. Eksperymentalʹna psykholohiya, 19, pp. 31-43. [in Ukrainian]. The article describes the study principles and methodological approaches and substantiates the genetic modeling method used as the main study method. To achieve the study purpose and empirical objec- tives, the following standardized psychological techniques Maksymenko, S. D. (2000). Henetycheskaya psykholohyya [Genetic psy- Maksymenko, S. D. (2000). Henetycheskaya psykholohyya [Genetic psy- Maksymenko, S. D. (2000). Henetycheskaya psykholohyya [Genetic psy- © Zoya Zelenska 98 Volume 6 Issue 12 (44) 2020 http://www.apsijournal.com/ ISSN 2414-0023 (Print) ISSN 2414-004X (Online) ISSN 2414-0023 (Print) ISSN 2414-004X (Online) PSYCHOLOGICAL JOURNAL Volume 6 Issue 12 (44) 2020 Volume 6 Issue 12 (44) 2020 PSYCHOLOGICAL JOURNAL DOI (Issue): https://doi.org/10.31108/1.2020.6.12 АННОТАЦИЯ Исследование проводилось в рамках Всеукраи- нского эксперимента: «Психологические основы инно- вационных технологий развития личности» на базе Ки- ево-Печерского лицея № 171 «Лидер» (Приказ МОН № 414 от 9 апреля 2015 года). The study results: psychological features influenc- ing development of communicative skills, in particular, pupils’ empathic tendencies, their communicative control, communicative needs (communicative qualities) and exist- ing emotional barriers to communication were identified. The correlations among the named components of the stud- ied system were revealed. Объектом исследования были коммуникатив- ные умения учащихся. Цель исследования заключалась в экспериментальном изучении особенностей развития коммуникативных умений учащихся среднего школь- ного возраста. There were no significant differences between the pupils from the experimental group (Kyiv-Pechersk Lyce- um No 171 “Leader”) and the pupils from the control group (Klovsky Lyceum). This means that the obtained results can be applied to pupils from general school of any form. В статье описаны принципы исследования и методологические подходы к исследованию, обосно- ванный генетико-моделирующий метод как основной метод исследования. The revealed influence and mutual influence of the described indicators of communicative skills testify to their interdependence and complementarities. The development of one indicator reinforces other indicators and vice versa. Для достижения цели и задач эмпирического исследования применялись следующие стандартизиро- ванные психодиагностические методики: методика «Диагностика уровня поликоммуникативной эмпа- тии» (по М. Юсуповым) для определения уровня прояв- ления эмпатических тенденций школьника; методика диагностики коммуникативного контроля (по М. Шнай- дером) для определения уровня коммуникативного кон- троля личности школьника; методика исследования коммуникативных качеств школьника ( «Потребность в общении» по А.Н. Орловым) для определения уровня развития потребности ученика в общении; методика «Диагностика эмоциональных барьеров в межличност- ном общении» (по В.В. Бойко) для определения имею- щихся эмоциональных барьеров в межличностном об- щении ученика). The psychological features of communicative skills identified in the article can become the basis for a psychological and pedagogical program that develops gen- eral school pupils’ communicative skills, in particular dur- ing English lessons. Further research should include development of a psychological and pedagogical program that takes into ac- count the identified psychological features to form of gen- eral school pupils’ communicative skills during learning foreign language vocabulary. Key words: empathic tendencies, emotional barri- ers, communicative skills, communicative qualities, com- municative control, general school pupils. Результатами исследования являются выявле- ние психологических особенностей развития коммуни- кативных умений, в частности, исследованы уровни развития эмпатических тенденций школьников, уровни их коммуникативного контроля, определены потребно- © Zoya Zelenska 99 http://www.apsijournal.com/ ISSN 2414-0023 (Print) ISSN 2414-004X (Online) PSYCHOLOGICAL JOURNAL Volume 6 Issue 12 (44) 2020 Дата отримання статті: 21.09.2020 Дата рекомендації до друку: 21.11.2020 Дата оприлюднення: 30.12.2020 How to cite (як цитувати): сти в общении школьников (коммуникативные каче- ства) и имеющиеся эмоциональные барьеры в общении. Обнаружена корреляция между названными составляю- щими исследуемой системы. Zelenska, Z. (2020). THE EXPERIMENTAL STUDY ON PSYCHOLOGICAL FEATURES OF GENERAL SCHOOL PUPILS’ COMMUNICATIVE SKILLS. PSYCHOLOGICAL JOURNAL, 6 (12), 92-100. https:// doi.org/10.31108/1.2020.6.12.9 [in Ukrainian] Zelenska, Z. (2020). THE EXPERIMENTAL STUDY ON PSYCHOLOGICAL FEATURES OF GENERAL SCHOOL PUPILS’ COMMUNICATIVE SKILLS. PSYCHOLOGICAL JOURNAL, 6 (12), 92-100. https:// doi.org/10.31108/1.2020.6.12.9 [in Ukrainian] Выявлено, что значимых различий в результа- тах учащихся экспериментальной группы (Киево- Печерский лицей № 171 «Лидер») и учащихся кон- трольной группы (Кловский лицей) не обнаружено. Это означает, что полученные результаты могут рассматри- ваться применительно учащихся основной школы лю- бого учебного заведения среднего образования. Выявленные тенденции влияния и взаимовлия- ния описанных показателей коммуникативных умений свидетельствуют об их созависимость и взаимодопол- няемость. Развитие одного показателя усиливает дру- гой показатель и наоборот. Обнаруженные психологические особенности коммуникативных умений могут быть положены в ос- нову психолого-педагогической программы развития коммуникативных умений учащихся основной школы в том числе на уроках английского языка. Перспективы дальнейших исследований заклю- чаются в разработке, с учетом выявленных психологи- ческих особенностей, психолого-педагогической про- граммы развития коммуникативных умений, что позво- лит формирования коммуникативных умений учащихся основной школы, в том числе в процессе усвоения ино- язычной лексики. Ключевые слова: эмпатичные тенденции школьников, эмоциональные барьеры, коммуникатив- ные умения, коммуникативные качества, коммуника- тивный контроль, ученик основной школы. Дата отримання статті: 21.09.2020 Дата рекомендації до друку: 21.11.2020 Дата оприлюднення: 30.12.2020 Дата отримання статті: 21.09.2020 Дата рекомендації до друку: 21.11.2020 Дата оприлюднення: 30.12.2020 100 © Zoya Zelenska 100 © Zoya Zelenska http://www.apsijournal.com/
https://openalex.org/W3084579522	https://www.revistas.unipar.br/index.php/akropolis/article/download/7892/3963	Portuguese	null	O LÚDICO ENQUANTO IMPORTANTE FERRAMENTA PARA O ENSINO DA EDUCAÇÃO FINANCEIRA NA FASE INFANTIL	Akrópolis	2,020	cc-by	3,891	O LÚDICO ENQUANTO IMPORTANTE FERRAMENTA PARA O ENSINO DA EDUCAÇÃO FINANCEIRA NA FASE INFANTIL PLAYFULNESS AS AN IMPORTANT TOOL FOR TEACHING FINANCIAL EDUCATION IN THE EARLY CHILDHOOD STAGES Diego Oliveira Sales1 SALES, D. O. O lúdico enquanto importante ferramenta para o ensino da educação financeira na fase infantil. Akrópolis Umuarama, v. 28, n. 1, p. 3-7, jan./jun. 2020. Recebido em fevereiro de 2020 Aceito em março de 2020 FUNDAMENTAÇÃO TEÓRICA É difícil adentrar a uma temática em que o assunto é criança sem associá-la a atividades divertidas como correr, se esconder, pular, ou seja, brincar, pois a atividade é natural e importante para o desenvolvimento e fortalecimento das capacidades físico, social e intelectual da criança. Como anteriormente mencionado, o brincar é necessário para o desenvolvimento do indivíduo nos anos iniciais da vida e por esta razão, a ludicidade é adotada na sua maioria por pedagogos e educadores como ferramenta de apoio e desenvolvimento da criança na idade infantil. i A educação financeira é necessária e sua ausência no dia-a-dia das famílias pode os levar a sérios problemas, por isso instituições educacionais, professores e educadores são agora, por meio da Base Nacional Comum Curricular – BNCC, estimulados a levar como disciplina transversal a educação financeira para a sala de aula, dando oportunidade às crianças a entrar em contato com o tema já nos primeiros anos de vida. Para Rau (2012), a ludicidade se define pelas ações do brincar, sendo organizadas em três eixos: jogo, brinquedo e brincadeira. Ainda segundo o autor, o ensino por meio da ludicidade levar em consideração a brincadeira como atividade natural a vida humana. A brincadeira se refere a ação de brincar, comportamento espontâneo que resulta de ações não organizadas; O jogo é compreendido como brincadeira envolvida por regras que o estruturam; Brinquedo é utilizado para dar sentido ao ato de brincar; e a ludicidade abrange de forma mais ampla os conceitos apresentados anteriormente (DALLABONA; MENDES, 2004).i Diferente de nós e de nossos pais, as crianças hoje têm a oportunidade de terem acesso a temática educação financeira já nos primeiros anos iniciais da escola, o que lhes proporciona melhores oportunidades de relação com o dinheiro, evitando assim que se tornem adultos irresponsáveis e financeiramente problemáticos. i A inserção da educação financeira nas séries iniciais traz consigo a necessidade de que professores e educadores se dediquem à criação de ferramentas estratégicas para o desenvolvimento do processo ensino – aprendizagem, direcionado para alunos que têm pouco ou nenhuma experiencia com dinheiro, por serem crianças, maioria dependentes de outros responsáveis. Partindo das afirmações dos autores acima citados, as brincadeiras, o jogo e os brinquedos são ferramentas lúdicas, naturais aos indivíduos e capazes de auxiliarem no ensino e aprendizagem das crianças. DOI: 10.25110/akropolis.v28i1.7892 É comum e errôneo que associe a brincadeira, o jogo apenas como diver passa tempo ou perca de tempo, brinca jogar vai muito além destas interpretaç Para Ferreira, Misse e Bonadio (2004 brincar deve ser levado a sério, pois é m do que uma atividade sem resultado pa criança. Enquanto a criança brinca, desenv aprendizado, pois seu cérebro está constante atividade, são transferidos a consciente e inconscientemente conhecime de mundo, por meio da atenção, imaginaçã experimento. É através das brincadeiras, jogos e brinquedos manipulados diariam que as crianças desenvolvem os seus senti INTRODUÇÃO para a imaginação, para o desenvolvimento e socialização o que permite que aprendam de maneira simples e divertida. Diante dos graves problemas financeiros, éticos, morais, sociais e de saúde nos quais indivíduos e famílias inteiras estão, infelizmente submetidas, surge cada vez mais a urgente necessidade destes indivíduos receberem o direito de acesso à educação financeira, como ferramenta que proporcione ao seu usuário uma melhor relação com o dinheiro, tornando-os conscientes e responsáveis quanto ao uso de recursos nas relações de consumo e os incentive a realização de sonhos. DOI: 10.25110/akropolis.v28i1.7892 Resumo: Ao considerar os graves problemas financeiros, éticos, morais, sociais e de saúde enfrentados diariamente por indivíduos e famílias inteiras, surge a urgente necessidade de proporcionar a estes indivíduos o direito de acesso à educação financeira, como ferramenta que lhes proporcione melhor relação com o dinheiro, tornando-os conscientes e responsáveis quanto ao uso de recursos nas relações de consumo e os incentive a realização de sonhos. O presente artigo tem por objetivo mostrar que é possível ensinar educação financeira por meio de atividades lúdicas. Palavras-chave: Educação Financeira; Lúdico; Brincar. Abstract: When considering the serious problems faced on a daily basis by individuals and entire families, such as financial, ethical, moral, social and health issues, there is an urgent need to provide these individuals the right of access to financial education, as a tool that provides them a better relationship with money, making them aware and responsible regarding the use of resources in consumer relations and encouraging them to achieve their dreams. This article aims at showing that it is possible to teach financial education through playful activities. Keywords: Financial Education; Playfulness; Playing. 1Especialista em Educação Financeira com Neurociência para Docentes – Metodologia DSOSP / Universidade do Oeste Paulista. Pós-graduando em gestão financeira e controladoria / Estácio de Sá SC. Graduando em Ciências Contábeis / Universidade Cruzeiro do Sul. Graduado em Administração / Faculdade Adventista Paranaense. admdiegosales@gmail.com Recebido em fevereiro de 2020 Aceito em março de 2020 Akrópolis, Umuarama, v. 28, n. 1, p. 3-7, jan./jun. 2020 ISSN 1982-1093 3 3 SALES, D. O. SALES, D. O. INTRODUÇÃO Diante dos graves problemas financeiros, éticos, morais, sociais e de saúde nos quais indivíduos e famílias inteiras estão, infelizmente submetidas, surge cada vez mais a urgente necessidade destes indivíduos receberem o direito de acesso à educação financeira, como ferramenta que proporcione ao seu usuário uma melhor relação com o dinheiro, tornando-os conscientes e responsáveis quanto ao uso de recursos nas relações de consumo e os incentive a realização de sonhos. A educação financeira é necessária e sua ausência no dia-a-dia das famílias pode os levar a sérios problemas, por isso instituições educacionais, professores e educadores são agora, por meio da Base Nacional Comum Curricular – BNCC, estimulados a levar como disciplina transversal a educação financeira para a sala de aula, dando oportunidade às crianças a entrar em contato com o tema já nos primeiros anos de vida. DOI: 10.25110/akropolis.v28i1.7892 Diferente de nós e de nossos pais, as crianças hoje têm a oportunidade de terem acesso a temática educação financeira já nos primeiros anos iniciais da escola, o que lhes proporciona melhores oportunidades de relação com o dinheiro, evitando assim que se tornem adultos irresponsáveis e financeiramente problemáticos. A inserção da educação financeira nas séries iniciais traz consigo a necessidade de que professores e educadores se dediquem à criação de ferramentas estratégicas para o desenvolvimento do processo ensino – aprendizagem, direcionado para alunos que têm pouco ou nenhuma experiencia com dinheiro, por serem crianças, maioria dependentes de outros responsáveis. Por se tratar de um tema voltado ao público infantil, o presente artigo pretende dar visibilidade e força às atividades lúdicas como importantes ferramentas para o ensino da educação financeira. As atividades lúdicas não só abrem caminhos para um melhor relacionamento entre professor e aluno, aluno e aluno, como também são eficazes no processo de ensino aprendizagem, pois são transferidas informações por meio de práticas e atividades comuns as crianças, jogos e brincadeiras. Por meio dessas atividades, as crianças ganham espaço para a imaginação, para o desenvolvimen socialização o que permite que aprendam maneira simples e divertida. FUNDAMENTAÇÃO TEÓRICA É difícil adentrar a uma temática que o assunto é criança sem associá- atividades divertidas como correr, se escon pular, ou seja, brincar, pois a atividad natural e importante para o desenvolvim e fortalecimento das capacidades físico, so e intelectual da criança. Como anteriorm mencionado, o brincar é necessário par desenvolvimento do indivíduo nos anos ini da vida e por esta razão, a ludicidade é ado na sua maioria por pedagogos e educad como ferramenta de apoio e desenvolvim da criança na idade infantil. Para Rau (2012), a ludicidade se de pelas ações do brincar, sendo organiza em três eixos: jogo, brinquedo e brincad Ainda segundo o autor, o ensino por meio ludicidade levar em consideração a brincad como atividade natural a vida humana brincadeira se refere a ação de brin comportamento espontâneo que resulta ações não organizadas; O jogo é compreen como brincadeira envolvida por regras o estruturam; Brinquedo é utilizado para sentido ao ato de brincar; e a ludicidade abra de forma mais ampla os conceitos apresenta anteriormente (DALLABONA; MENDES, 200 Partindo das afirmações dos aut acima citados, as brincadeiras, o jogo e brinquedos são ferramentas lúdicas, natu aos indivíduos e capazes de auxiliarem ensino e aprendizagem das crianças. FUNDAMENTAÇÃO TEÓRICA Conforme consta em Significados (2019), educação é o ato de educar, instruir, é de maneira mais ampla o meio pelo qual valores e costumes são transferidos de uma geração a outra seguinte. No processo de educar, são transferidas de um agente para outro informações que levam a construção do saber. Por esse processo de transferência de informações “educação” é que são transferidos os conteúdos sobre finanças para os educandos, a fim de estabelecerem melhor relacionamento com o dinheiro. As atividades lúdicas, desde que planejadas e orientadas, são excelentes ferramentas para o processo de ensino e aprendizagem, pois de maneira divertida, ensinam e desenvolvem na criança a socialização, o físico e o intelectual. O processo de aprendizagem de acordo com Oliveira (1995, p.57): É o processo pelo qual o indivíduo adquiri informações, habilidades, atitudes, valores etc. a partir de seu contato com a realidade, o meio ambiente, as outras pessoas. É um processo que se diferencia dos fatores inatos (a capacidade de digestão, por exemplo, que já nasce com o indivíduo) e dos processos de maturação sexual, por exemplo). Em Vygotsky, justamente por sua ênfase nos processos sócios- históricos, a ideia de aprendizado inclui a interdependência dos indivíduos envolvidos no processo. (...) o conceito em Vygotski tem um significado mais abrangente, sempre envolvendo interação social. De maneira genérica, a educação financeira consiste em informar e orientar indivíduos quanto ao melhor uso de seus recursos, no que diz respeito a consumo e investimento. Para Santos, Asoo e Carvalho (2016), a educação financeira auxilia os indivíduos a planejarem e melhor usarem seus recursos. Estando certo os conceitos acima citados, trago para este trabalho uma definição que vai muito além do que comumente conhecemos. Domingos (2019) define a educação financeira como: uma ciência humana que busca a autonomia financeira, fundamentada por uma metodologia baseada no comportamento, objetivando a construção de um modelo mental que promova a sustentabilidade, crie hábitos saudáveis e proporcione o equilíbrio entre o ser, o fazer e o ter, com escolhas conscientes para a realização de sonhos. uma ciência humana que busca a autonomia financeira, fundamentada por uma metodologia baseada no comportamento, objetivando a construção de um modelo mental que promova a sustentabilidade, crie hábitos saudáveis e proporcione o equilíbrio entre o ser, o fazer e o ter, com escolhas conscientes para a realização de sonhos. FUNDAMENTAÇÃO TEÓRICA É comum e errôneo que associemos a brincadeira, o jogo apenas como diversão, passa tempo ou perca de tempo, brincar e jogar vai muito além destas interpretações. Para Ferreira, Misse e Bonadio (2004), o brincar deve ser levado a sério, pois é mais do que uma atividade sem resultado para a criança. Enquanto a criança brinca, desenvolve aprendizado, pois seu cérebro está em constante atividade, são transferidos a ela consciente e inconscientemente conhecimentos de mundo, por meio da atenção, imaginação e experimento. É através das brincadeiras, dos jogos e brinquedos manipulados diariamente que as crianças desenvolvem os seus sentidos, Por se tratar de um tema voltado ao público infantil, o presente artigo pretende dar visibilidade e força às atividades lúdicas como importantes ferramentas para o ensino da educação financeira. As atividades lúdicas não só abrem caminhos para um melhor relacionamento entre professor e aluno, aluno e aluno, como também são eficazes no processo de ensino aprendizagem, pois são transferidas informações por meio de práticas e atividades comuns as crianças, jogos e brincadeiras. Por meio dessas atividades, as crianças ganham espaço Akrópolis, Umuarama, v. 28, n. 1, p. 3-7, jan./jun. 2020 ISSN 1982-1093 4 O lúdico enquanto importante... tato, visão, olfato, além da coordenação motora, senso de equilíbrio e outras habilidades físicas e psicológicas, (PIRES; MENDES; BONADIO, 2004). entre si para o desenvolvimento e construção do conhecimento, (ROLIN; GUERRA; TASSIGNY, 2008). Diante do exposto acima, vemos que o lúdico é uma poderosa e importante ferramenta capaz de facilitar o desenvolvimento e aprendizado no ser humano em diversas áreas da vida.O brincar, proporciona à criança espaço para a imaginação, raciocínio, pensamento, interação, criatividade, conhecimento e desenvolvimento, e pode ser utilizado para o ensino da educação financeira como meio para a construção de um indivíduo consciente e responsável financeiramente. O jogo, por sua vez possibilita ao sujeito a aprendizagem e desenvolvimento por meio das orientações, regras, interações com objetos e o meio, bem como diversidade de linguagens envolvidas em sua prática, (RAU, 2012). Segundo a mesma autora, quando você entra na ação do jogo, se elabora objetivos, cria estratégias, elabora hipóteses, vivencia papéis, tenta superar obstáculos, vivencia emoções, organiza o pensamento, percebe erros e acertos e compreende os resultados (ganhando ou perdendo). Para melhor compreendermos a temática “educação financeira” é necessário que definamos educação e como ela é indispensável para o desenvolvimento do tema. CONSIDERAÇÕES FINAIS Por meio da educação financeira, velhos hábitos e comportamentos podem ser corrigidos e novos constituídos, podem mudar sua maneira de se relacionar com o dinheiro. Uma relação saudável com o dinheiro, compreende conhecer sua origem, formas legais de obtê-lo, consciência e responsabilidade no consumo, construção de reservas de emergência e prioridade na realização de sonhos. A intenção de educar financeiramente as crianças é para que se tornem adultos responsáveis, capazes de se relacionarem com o dinheiro de maneira que saibam planejar, criar reservas e investirem em sonhos. Com simulações de compra e venda em mercado fictício, diferenciação entre o necessário e o supérfluo, construção de cofrinhos com recicláveis, contação de história se é possível ensinar. Brincadeiras, jogos e brinquedos fazem parte do cotidiano do ser humano e por meio dessas ações é possível obter muito mais que diversão, conhecimento. Por meio do lúdico, brincadeiras, jogos e brinquedos é possível auxiliar na construção do melhor relacionamento da criança com o dinheiro. Atividades como simulações, por exemplo de compra e venda de produtos em um mercadinho fictício os ensinam sobre operações básicas, valor monetário, poder de compra, contagem de dinheiro e troco. A interação nesta atividade os proporciona aprender a negociar entre os demais participantes, escolher entre um produto e outro considerando qualidade, quantidade e necessidade. FUNDAMENTAÇÃO TEÓRICA Contar as crianças como eram feitas as trocas de mercadorias entre antigos povos, como o dinheiro chegou até nós, e que o trabalho é a principal fonte de recursos, os ensinarão o valor do dinheiro e do trabalho. A existência do dinheiro no dia-a-dia dos indivíduos é o que faz ser importante estudar e compartilhar a educação financeira, a fim de que sejam os indivíduos ensinados e orientados quanto a melhor maneira de se relacionar com o dinheiro. Aprender sobre dinheiro não cabe apenas aos adultos, pelo contrário, quanto mais cedo se aprende sobre o dinheiro, melhor será sua relação com ele. O dinheiro não leva desaforo para casa, ou seja, se não soubermos se relacionar com ele de forma responsável, o mais provável é que enfrentemos os diversos problemas como endividamento, inadimplência, problemas sociais, morais e até de saúde.i FUNDAMENTAÇÃO TEÓRICA dinheiro é o mesmo que proteger ou poupar o din produzidos, darão vida as sonhos, o guardar hoje, pod Além disso, a prát história serve de grande ferr da educação financeira. como eram feitas as trocas antigos povos, como o dinh e que o trabalho é a princip os ensinarão o valor do din CONSIDERAÇÕES FINAIS Por meio da educaç hábitos e comportamentos e novos constituídos, podem de se relacionar com o d saudável com o dinheiro, c sua origem, formas legais d e responsabilidade no c de reservas de emergên realização de sonhos. Com simulações de mercado fictício, diferenciaç e o supérfluo, construção recicláveis, contação de h ensinar. Brincadeiras, jogos parte do cotidiano do ser dessas ações é possível o diversão, conhecimento. . O. mentos por meio da educação do indivíduo em algum momento assará a ter contato com o dinheiro, oeda utilizado como meio de troca em omia. Este reconhecimento também or Moreira (2002), ao afirmar que o participa de todos os momentos da ômica cotidiana e que constitui parte a da vida social. existência do dinheiro no dia-a-dia dos é o que faz ser importante estudar ilhar a educação financeira, a fim de os indivíduos ensinados e orientados A construção recicláveis pode ser u importância para a desenvolver o aspec ensinará sobre prote dinheiro é o mesmo proteger ou poupar produzidos, darão vi sonhos, o guardar hoje Além disso, a história serve de grand da educação finance como eram feitas as tr antigos povos, como o SALES, D. O. SALES, D. O. comportamentos por meio da educação financeira. A construção de cofres com materiais recicláveis pode ser uma atividade de estrema importância para a criança, pois além de desenvolver o aspecto locomotor, criativo, o ensinará sobre proteção. Proteger, guardar o dinheiro é o mesmo que poupar. Ao guardar, proteger ou poupar o dinheiro nestes cofres produzidos, darão vida as suas conquistas e sonhos, o guardar hoje, pode garantir o amanhã! Todo indivíduo em algum momento da vida, passará a ter contato com o dinheiro, papel – moeda utilizado como meio de troca em uma economia. Este reconhecimento também é feito por Moreira (2002), ao afirmar que o dinheiro participa de todos os momentos da vida econômica cotidiana e que constitui parte significativa da vida social. Além disso, a prática de contação de história serve de grande ferramenta para o ensino da educação financeira. FUNDAMENTAÇÃO TEÓRICA Conforme citado anteriormente o processo de aprendizagem ocorre por meio da interação do indivíduo com o ambiente onde está inserido. Neste caso ao brincarem com outros indivíduos, sejam crianças ou adultos, aprendem a se relacionar, ao brincarem com os jogos, aprendem que na vida também existem regras e devem ser seguidas para o melhor convívio em sociedade. As atividades lúdicas são uma ótima opção para estimular a interação entre adultos e crianças e entre as crianças A realização de sonhos passar a ser o objetivo da mudança de hábitos e Akrópolis, Umuarama, v. 28, n. 1, p. 3-7, jan./jun. 2020 ISSN 1982-1093 5 SALES, D. O. comportamentos por meio da educação financeira. Todo indivíduo em algum momento da vida, passará a ter contato com o dinheiro, papel – moeda utilizado como meio de troca em uma economia. Este reconhecimento também é feito por Moreira (2002), ao afirmar que o dinheiro participa de todos os momentos da vida econômica cotidiana e que constitui parte significativa da vida social. A existência do dinheiro no dia-a-dia dos indivíduos é o que faz ser importante estudar e compartilhar a educação financeira, a fim de que sejam os indivíduos ensinados e orientados quanto a melhor maneira de se relacionar com o dinheiro. Aprender sobre dinheiro não cabe apenas aos adultos, pelo contrário, quanto mais cedo se aprende sobre o dinheiro, melhor será sua relação com ele. O dinheiro não leva desaforo para casa, ou seja, se não soubermos se relacionar com ele de forma responsável, o mais provável é que enfrentemos os diversos problemas como endividamento, inadimplência, problemas sociais, morais e até de saúde. A intenção de educar financeiramente as crianças é para que se tornem adultos responsáveis, capazes de se relacionarem com o dinheiro de maneira que saibam planejar, criar reservas e investirem em sonhos. Por meio do lúdico, brincadeiras, jogos e brinquedos é possível auxiliar na construção do melhor relacionamento da criança com o dinheiro. Atividades como simulações, por exemplo de compra e venda de produtos em um mercadinho fictício os ensinam sobre operações básicas, valor monetário, poder de compra, contagem de dinheiro e troco. A interação nesta A construção de c recicláveis pode ser uma importância para a cria desenvolver o aspecto lo ensinará sobre proteção. O lúdico enquanto importante... 4, out./dez, 2004. en las relaciones de consumo e incentivándoles a realizar sus sueños. Este artículo pretende mostrar que es posible enseñar educación financiera a través de actividades lúdicas. MOREIRA, Alice da Silva. Dinheiro no brasil: um estudo comparativo do significado do dinheiro entre as regiões geográficas brasileiras. 2002. MOREIRA, Alice da Silva. Dinheiro no brasil: um estudo comparativo do significado do dinheiro entre as regiões geográficas brasileiras. 2002. Palabras clave: Educación Financiera; Lúdico; Jugar. OLIVEIRA, Anaelize A.; PESSOA, Cristiane A. S. Educação financeira nos anos iniciais do ensino fundamental: um olhar para a formação docente. Instrumento: R Est. Pesq. Educ; Juiz e Fora, v. 20, n. 2, jul./dez. 2018. OLIVEIRA, Marta Kohl de. Vygotsky: aprendizado e desenvolvimento um processo sócio histórico. 3. ed. São Paulo: Scipione, 1995. OLIVEIRA, Marta Kohl de. Vygotsky: aprendizado e desenvolvimento um processo sócio histórico. 3. ed. São Paulo: Scipione, 1995. PIRES, R. C.; MENDES, N. S.; BONADIO, S. G. Brincar: recreação ou aprendizagem? Akrópolis, Umuarama, v. 12, n. 4, out./dez, 2004. RAU, Maria Cristina Trois Dorneles. A ludicidade na educação: uma atitude pedagógica. Curitiba: InterSaberes, 2012. ROLIN, Amanda A. M.; GUERRA, Siena S. F.; TASSIGNY, Mônica M. Uma leitura de vygotski sobre o brincar na aprendizagem e no desenvolvimento infantil. Ver. Humanidades, Fortaleza, v. 23, n. 2, p. 176-180, jul./dez, 2008. SANTOS, Bruna Neris dos; ASOO, Rafael S.; CARVALHO, Carolina G. Educação financeira infantil: a construção da cidadãos conscientes. Iturama (MG) v. 5, n. 4, p. 116-126, jul./dez. 2016. SIGNIFICADOS. O que é educação. 2019. Disponível em: https://www.significados.com.br/ educacao/. Acesso em: 17 fev. 2020. REFERENCIAS DALLABONA, Sandra R.; MENDES, Sueli Maria S. O lúdico na educação infantil: Jogar, brincar, uma forma de educar. Revista de divulgação técnico – cientifica do icpg. v. 1 n. 4, jan-mar, 2004. Em uma atividade em grupo, orientações e exemplos do que é necessário e supérfluo, podem ser compartilhados com participantes. Após primeira explicação, as crianças podem buscar em panfletos de mercados, jornais e revistas produtos que considerem necessários e supérfluos e classificá-los em um cartaz. Desta maneira aprenderão sobre prioridades e escolhas, aprenderão que existe subjetividade nestas questões, pois o que é necessário para um pode não ser para outro e o que supérfluo para um pode não ser para outro. DOMINGOS, Reinaldo. DSOP. A metodologia: conheça a educação financeira. Disponível em: https://www.dsop.com.br/educacao-financeira- dsop/. Acesso em: 17 fev. 2020. FERREIRA, Caroline M.; MISSE, Cristina H.; BONADIO, Sueli G. Brincar na educação infantil é coisa séria. Akrópolis, Umuarama, v. 12, n. FERREIRA, Caroline M.; MISSE, Cristina H.; BONADIO, Sueli G. Brincar na educação infantil é coisa séria. Akrópolis, Umuarama, v. 12, n. Akrópolis, Umuarama, v. 28, n. 1, p. 3-7, jan./jun. 2020 ISSN 1982-1093 6 EL LÚDICO COMO HERRAMIENTA IMPORTANTE PARA LA ENSEÑANZA DE LA EDUCACIÓN FINANCIERA EN LA FASE INFANTIL Resumen: Al considerar los graves problemas financieros, éticos, morales, sociales y de salud enfrentados diariamente por individuos y familias enteras, surge la urgente necesidad de proporcionar a estas personas el derecho de acceso a la educación financiera, como una herramienta que les proporcione una mejor relación con el dinero, haciéndolos conscientes y responsables del uso de recursos Akrópolis, Umuarama, v. 28, n. 1, p. 3-7, jan./jun. 2020 ISSN 1982-1093 Akrópolis, Umuarama, v. 28, n. 1, p. 3-7, jan./jun. 2020 7 7 ISSN 1982-1093
W2739666581.txt	https://journals.plos.org/ploscompbiol/article/file?id=10.1371/journal.pcbi.1005692&type=printable	en		The Stochastic Early Reaction, Inhibition, and late Action (SERIA) model for antisaccades	PLOS computational biology/PLoS computational biology	2,017	cc-by	16,579	RESEARCH ARTICLE The Stochastic Early Reaction, Inhibition, and late Action (SERIA) model for antisaccades Eduardo A. Aponte1, Dario Schöbi1, Klaas E. Stephan1,2, Jakob Heinzle1 1 Translational Neuromodeling Unit, Institute for Biomedical Engineering, University of Zurich & Swiss Institute of Technology Zurich, Zurich, Switzerland, 2 Wellcome Trust Centre for Neuroimaging, University College London, London, United Kingdom * aponte@biomed.ee.ethz.ch (EAA); heinzle@biomed.ee.ethz.ch (JH) a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Aponte EA, Schöbi D, Stephan KE, Heinzle J (2017) The Stochastic Early Reaction, Inhibition, and late Action (SERIA) model for antisaccades. PLoS Comput Biol 13(8): e1005692. https://doi. org/10.1371/journal.pcbi.1005692 Editor: Adrian M. Haith, Johns Hopkins University, UNITED STATES Received: February 5, 2017 Accepted: July 20, 2017 Published: August 2, 2017 Copyright: © 2017 Aponte et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the René and Susanne Braginsky Foundation (KES) and the University of Zurich. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abstract The antisaccade task is a classic paradigm used to study the voluntary control of eye movements. It requires participants to suppress a reactive eye movement to a visual target and to concurrently initiate a saccade in the opposite direction. Although several models have been proposed to explain error rates and reaction times in this task, no formal model comparison has yet been performed. Here, we describe a Bayesian modeling approach to the antisaccade task that allows us to formally compare different models on the basis of their evidence. First, we provide a formal likelihood function of actions (pro- and antisaccades) and reaction times based on previously published models. Second, we introduce the Stochastic Early Reaction, Inhibition, and late Action model (SERIA), a novel model postulating two different mechanisms that interact in the antisaccade task: an early GO/NO-GO race decision process and a late GO/GO decision process. Third, we apply these models to a data set from an experiment with three mixed blocks of pro- and antisaccade trials. Bayesian model comparison demonstrates that the SERIA model explains the data better than competing models that do not incorporate a late decision process. Moreover, we show that the early decision process postulated by the SERIA model is, to a large extent, insensitive to the cue presented in a single trial. Finally, we use parameter estimates to demonstrate that changes in reaction time and error rate due to the probability of a trial type (pro- or antisaccade) are best explained by faster or slower inhibition and the probability of generating late voluntary prosaccades. Author summary One widely replicated finding in schizophrenia research is that patients tend to make more errors than healthy controls in the antisaccade task, a psychometric paradigm in which participants are required to look in the opposite direction of a visual cue. This deficit has been suggested to be an endophenotype of schizophrenia, as first order relatives of patients tend to show similar but milder deficits. Currently, most models applied to experimental findings in this task are limited to fit average reaction times and error rates. Here, we propose a novel statistical model that fits experimental data from the antisaccade task, PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 1 / 36 SERIA—A model for errors and reaction times in the antisaccade task beyond summary statistics. The model is inspired by the hypothesis that antisaccades are the result of several competing decision processes that interact nonlinearly with each other. In applying this model to a relatively large experimental data set, we show that mean reaction times and error rates do not fully reflect the complexity of the processes that are likely to underlie experimental findings. In the future, our model could help to understand the nature of the deficits observed in schizophrenia by providing a statistical tool to study their biological underpinnings. Introduction In the antisaccade task ([1]; for reviews, see [2,3]), participants are required to saccade in the contralateral direction of a visual cue. This behavior is thought to require both the inhibition of a reflexive saccadic response towards the cue and the initiation of a voluntary eye movement in the opposite direction. A failure to inhibit the reflexive response leads to an erroneous saccade towards the cue (i.e., a prosaccade), which is often followed by a corrective eye movement in the opposite direction (i.e., an antisaccade). As a probe of inhibitory capacity, the antisaccade task has been widely used to study psychiatric and neurological diseases [3]. Notably, since the initial report [4], studies have consistently found an increased number of errors in patients with schizophrenia when compared to healthy controls, independent of medication and clinical status [5–8]. Moreover, there is evidence that an increased error rate constitutes an endophenotype of schizophrenia, as antisaccade deficits are also present in non-affected, first-degree relatives of diagnosed individuals (for example [5,7]; but for negative findings see for example [9,10]). Unfortunately, the exact nature of the antisaccade deficits and their biological origin in schizophrenia remain unclear. One path to improve our understanding of these experimental findings is to develop generative models of their putative computational and/or neurophysiological causes [11]. Generative models that capture the entire distribution of responses can reveal features of the data that are not apparent when only considering summary statistics such as mean error rate (ER) and reaction time (RT) [12–15]. Additionally, this type of model can potentially relate behavioral findings in humans to their biological substrate. Here, we apply a generative modeling approach to the antisaccade task. First, we introduce a novel model of this paradigm based on previous proposals [16–20]. For this, we formalize the ideas introduced by Noorani and Carpenter [17] and extend them into what we refer to as the Stochastic Early Reaction, Inhibition and late Action (SERIA) model. Second, we apply both models to an experimental data set of three mixed blocks of pro- and antisaccades trials with different trial type probability. More specifically, we compare several models using Bayesian model comparison. Third, we use the parameter estimates from the best model to investigate the effects of our experimental manipulation. We found that there was positive evidence in favor of the SERIA model when compared to our formalization of the model proposed in [17]. Moreover, the parameters estimated through model inversion revealed the complexity of the decision processes underlying the antisaccade task that is not obvious from mean RT and ER. This paper is organized as follows. First, we formalize the model developed in [17] and introduce the SERIA model. Second, we describe our experimental setup. Third, we present our behavioral findings in terms of summary statistics (mean RT and ER), the comparison between different models, and the parameter estimates. Finally, we review our findings, discuss other recent models, potential future developments, and translational applications. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 2 / 36 SERIA—A model for errors and reaction times in the antisaccade task Materials and methods Ethics statement All participants gave written informed consent before the study. All experimental procedures were approved by the local ethics board (Kantonale Ethikkomission Zürich, KEK-ZH-Nr.2014-0246). Race models for antisaccades In this section, we derive a formal description of the models evaluated in this paper. We start with a formalized version of the model proposed by Noorani and Carpenter in [17] and proceed to extend it. Their approach resembles the model originally proposed by Camalier and colleagues [21] to explain RT and ER in the double step and search step tasks, in which participants are either asked to saccade to successively presented targets or to saccade to a target after a distractor was shown. Common to all these tasks is that subjects are required to inhibit a prepotent reaction to an initial stimulus and then to generate an action towards a secondary goal. Briefly, Camalier and colleagues [21] extended the original ‘horse-race’ model [16] by including a secondary action in countermanding tasks. In [17], Noorani and Carpenter used a similar model in combination with the LATER model [22] in the context of the antisaccade task by postulating an endogenously generated inhibitory signal. Note that this model, or variants of it, have been used in several experimental paradigms (reviewed in [20]). Here, we limit our discussion to the antisaccade task. The pro, stop, and antisaccade model (PROSA) Following [17], we assume that the RT and the type of saccade generated in a given trial are caused by the interaction of three competing processes or units. The first unit up represents a command to perform a prosaccade, the second unit us represents an inhibitory command to stop a prosaccade, and the third unit ua represents a command to perform an antisaccade. The time t required for unit ui to arrive at threshold si is given by: si ¼ ri t; ð1Þ si ¼ t; ri ð2Þ where ri represents the slope or increase rate of unit ui, si represents the height of the threshold, and t represents time. We assume that, on each trial, the increase rates are stochastic and independent of each other. The time and order in which the units reach their thresholds si determines the action and RT in a trial. If the prosaccade unit up reaches threshold before any other unit at time t, a prosaccade is elicited at t. If the antisaccade unit arrives first, an antisaccade is elicited at t. Finally, if the stop unit arrives before the prosaccade unit, an antisaccade is elicited at the time when the antisaccade unit reaches threshold. It is worth mentioning that, although this model is motivated as a race-to-threshold model, actions and RTs depend only on the arrival times of each of the units and ultimately no explicit model of increase rates or thresholds is required. Thus, for the sake of clarity, we refer to this approach as a ‘race’ model, in contrast to ‘race-tothreshold’ models that explicitly describe increase rates and thresholds. Formally (but in a slight abuse of language), the two random variables of interest, the reaction time T 2 [0,1[ and the type of action performed A 2 {pro,anti}, depend only on three further random variables: the arrival times Up, Us, Ua 2 [0,1[ of each of the units. The PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 3 / 36 SERIA—A model for errors and reaction times in the antisaccade task probability of performing a prosaccade at time t is given by the probability of the prosaccade unit arriving at time t, and the stop and antisaccade unit arriving afterwards: pðA ¼ pro; T ¼ tÞ ¼ pðUp ¼ tÞpðUa > tÞpðUs > tÞ: ð3Þ The probability of performing an antisaccade at time t is given by Z t pðA ¼ anti; T ¼ tÞ ¼ pðUa ¼ tÞpðUp > tÞpðUs > tÞ þ pðUa ¼ tÞ pðUs ¼ tÞpðUp > tÞdt: ð4Þ 0 The first term on the right side of Eq 4 corresponds to the unlikely case that the antisaccade unit arrives before the prosaccade and the stop units. The second term describes trials in which the stop unit arrives before the prosaccade unit. It can be decomposed into two terms: Z t pðUa ¼ tÞ Z t pðUs ¼ tÞpðUp > tÞdt ¼ pðUa ¼ tÞ pðUs < tÞpðUp > tÞ þ pðUs ¼ tÞpðt < Up < tÞdt ð5Þ 0 0 Z t ¼ pðUa ¼ tÞ pðUs < tÞpðUp > tÞ þ pðUs < tÞpðUp ¼ tÞdt ð6Þ 0 Rt The term pðUa ¼ tÞ 0 pðUs < tÞpðUp ¼ tÞdt describes the condition in which the prosaccade unit is inhibited by the stop unit allowing for an antisaccade. Note that if the prosaccade unit arrives later than the antisaccade unit, the arrival time of the stop unit is irrelevant. That means that we can simplify Eq 4 to Z t pðA ¼ anti; T ¼ tÞ ¼ pðUa ¼ tÞ pðUp > tÞ þ pðUs < tÞpðUp ¼ tÞdt : ð7Þ 0 Eqs 3 and 7 constitute the likelihood function of a single trial, and define the joint probability of an action and the corresponding RT. We refer to this likelihood function as the PROStop-Antisaccade (PROSA) model. It shares the central assumptions of [17], namely: (i) the time to reach threshold of each of the units is assumed to depend linearly on the rate r, (ii) it includes a stop unit whose function is to inhibit prosaccades and (iii) there is no lateral inhibition between the different units. Finally, (iv) RTs are assumed to be equal to the arrive-atthreshold times. Note that the RT distributions are different from the arrival time distributions because of the interactions between the units described above. The main difference of this model compared to [17] is that we do not exclude a priori the possibility of the antisaccade unit arriving earlier than the other units. Aside from this, both models are conceptually equivalent. The Stochastic Early Reaction, Inhibition, and Late Action Model (SERIA) The PROSA model is characterized by a strict association between units and action types. In other words, the unit up leads unequivocally to a prosaccade, whereas the unit ua always triggers an antisaccade. This implies that if the distribution of the arrival times of the units is unimodal and strictly positive, the PROSA model cannot predict voluntary slow prosaccades with a late peak, or in simple words, the PROSA model cannot account for slow, voluntary prosaccades that have been postulated in the antisaccade task [23]. Similarly, it has been argued that prosaccade RT can be described by the mixture of two distributions (for example [2,22]). To account for this, we introduce the Stochastic Early Reaction, Inhibition and Late Action (SERIA) model. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 4 / 36 SERIA—A model for errors and reaction times in the antisaccade task Fig 1. Layout of the SERIA model. The presentation of a visual cue (a green bar) triggers the race of three independent units. The inhibitory unit can stop an early response. Importantly, both early and late responses can trigger pro- and antisaccades. Note that the PROSA model is a special case of the SERIA model in which πe = 1 and πl = 0, i.e. all early responses are prosaccades, whereas all late responses are antisaccades. https://doi.org/10.1371/journal.pcbi.1005692.g001 According to this model, and in analogy to the PROSA model, an early reaction takes place at time t if the early unit ue arrives before the late and inhibitory units, ul and ui, respectively. If the inhibitory or late unit arrives before the early unit, a late response is triggered at the time the late unit reaches threshold. Crucially, both early and late responses can trigger pro- and antisaccades with a certain probability. Thus, in parallel to the race processes which determine RTs, an independent, secondary decision process is responsible for which reaction is generated. Fig 1 shows the structure of the SERIA model. To formalize the concept of early and late responses, we introduce a new unobservable random variable that represents the type of response R 2 {early,late}. The distribution of the RTs is analogous to the PROSA-model, such that, for instance, the probability of an early response at time t is given by pðR ¼ early; T ¼ tÞ ¼ pðUe ¼ tÞpðUi > tÞpðUl > tÞ ð8Þ where Ue, Ui, and Ul represent the arrival times of the early, inhibitory, and late units, respectively. The fundamental assumption of the SERIA model is that a secondary decision process, beyond the race between early, inhibitory, and late units, decides the action generated in a single trial. An initial approach to model this secondary decision process is to assume that the action type (pro- or antisaccade) is conditionally independent of the RT given the response PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 5 / 36 SERIA—A model for errors and reaction times in the antisaccade task type (early or late). Hence, the distribution of RTs is not a priori coupled to the saccade type anymore; RT distributions for both pro- and antisaccades could in principle be bimodal, consisting of both fast reactive and slow voluntary saccades. Formally, the conditional independency assumption can be written down as pðA; TjRÞ ¼ pðAjRÞpðTjRÞ; ð9Þ pðA; TjRÞpðRÞ ¼ pðAjRÞpðTjRÞpðRÞ; ð10Þ pðA; T; RÞ ¼ pðAjRÞpðT; RÞ: ð11Þ The term p(A\|R) is simply the probability of an action, given a response type. We denote it as pðA ¼ projR ¼ earlyÞ ¼ pe 2 ½0; 1; pðA ¼ antijR ¼ earlyÞ ¼ 1 ð12Þ pe ; ð13Þ pðA ¼ projR ¼ lateÞ ¼ pl 2 ½0; 1; ð14Þ pðA ¼ antijR ¼ lateÞ ¼ 1 pl : ð15Þ Since the type of response R is not observable, it is necessary to marginalize it out in Eq 11 to obtain the likelihood of the SERIA model: pðA; TÞ ¼ pðA; T; R ¼ earlyÞ þ pðA; T; R ¼ lateÞ: ð16Þ The complete likelihood of the model is given by substituting the terms in Eq 16 pðA ¼ pro; T ¼ tÞ ¼ pe pðUe ¼ tÞpðUi > tÞpðUl > tÞþ Z t pl pðUl ¼ tÞ pðUe > tÞ þ pðUe ¼ tÞpðUi < tÞdt ; ð17Þ 0 pðA ¼ anti; T ¼ tÞ ¼ ð1 pe ÞpðUe ¼ tÞpðUi > tÞpðUl > tÞþ ð1 Z t pl ÞpðUl ¼ tÞ pðUe > tÞ þ ðUe ¼ tÞpðUi < tÞdt : ð18Þ 0 It is worth noting here that the PROSA model is a special case of the SERIA model, namely, it corresponds to the assumption that πe = 1 and πl = 0. The SERIA model allows for bimodal distributions, as both early and late responses can be pro- and antisaccades. Importantly, one prediction of the model is that late prosaccades have the same distribution as late antisaccades. A late race competition model for saccade type Until now, we have assumed that the competition that leads to late pro- and antisaccades does not depend on time in the sense that late actions are conditionally independent of RT. This assumption can be weakened by postulating a secondary race between late responses; this leads us to a modified version of the SERIA model, that we refer to as the late race SERIA model (SERIAlr). The derivation proceeds similarly to the SERIA model, except that we postulate a fourth unit that generates late prosaccades instead of assuming that the late decision process is time insensitive. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 6 / 36 SERIA—A model for errors and reaction times in the antisaccade task This version of the SERIA model includes an early unit ue that, for simplicity, we assume produces only prosaccades, an inhibitory unit that stops early responses ui, a late unit that triggers antisaccades ua, and a further unit that triggers late prosaccades up. As before, if the early unit reaches threshold before any other unit, a prosaccade is generated with probability pðUe ¼ tÞpðUi > tÞpðUa > tÞpðUp > tÞ: ð19Þ If any of the late units arrive first, the respective action is generated with probability: Antisaccade : pðUa ¼ tÞpðUp > tÞpðUe > tÞpðUi > tÞ: ð20Þ Prosaccade : pðUp ¼ tÞpðUa > tÞpðUe > tÞpðUi > tÞ: ð21Þ Finally, if the inhibitory unit arrives first, either a late pro- or antisaccade is generated with probability 0 1 Zt C Antisaccades : pðUa ¼ tÞpðUp > tÞB ð22Þ @ pðUi ¼ tÞpðUe > tÞdtA; 0 0 B Prosaccades : pðUp ¼ tÞpðUa > tÞ@ Zt 1 C pðUi ¼ tÞpðUe > tÞdtA: ð23Þ 0 Implicit in the last two terms is the competition between the late units, which are assumed again to be independent of each other. Formally, this competition is expressed as the probability of, for example, the late antisaccade unit arriving before a late prosaccade p(Ua = t)p(Up > t). A schematic representation of the model is shown in Fig 2. This late race is similar to the Linear Ballistic Accumulation model proposed by [24], although in that model decisions are seen as the result of a race of ballistic accumulation processes with fixed threshold but stochastic base line and increase rate. Here we only assume that the late decision process is a GO-GO race [21]. The likelihood of an action is given by summing over all possible outcomes that lead to that action: pðA ¼ pro; T ¼ tÞ ¼ pðUe ¼ tÞpðUi > tÞpðUa > tÞpðUp > tÞþ 0 pðUp ¼ tÞpðUa > tÞpðUi > tÞpðUe > tÞ þ pðUp ¼ tÞpðUa > tÞB @ Zt 1 pðUi ¼ tÞpðUe > tÞdtC A; ð24Þ 0 pðA ¼ anti; T ¼ tÞ ¼ pðUa ¼ tÞpðUp > tÞpðUi > tÞpðUe > tÞþ 0 pðUa ¼ tÞpðUp > tÞB @ Zt 1 pðUi ¼ tÞpðUe > tÞdtC A: ð25Þ 0 We have left out some possible simplifications in Eqs 24 and 25 for the sake of clarity. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 7 / 36 SERIA—A model for errors and reaction times in the antisaccade task Fig 2. Layout of the SERIAlr model. The presentation of a visual cue (a green bar) triggers the race of four independent units. The inhibitory unit can stop an early response. The late decision process is triggered by the competition between two further units. https://doi.org/10.1371/journal.pcbi.1005692.g002 The conditional probability of a late antisaccade is given by the interaction between the late units, such that Z1 pðUa < Up Þ ¼ pðUa ¼ tÞpðUp > tÞdt ¼ 1 pðUp < Ua Þ; ð26Þ 0 is analogous to the probability of a late antisaccade 1−πl in the SERIA model. This observation shows that the main difference between the SERIA and SERIAlr model is that the former postulates that the distribution of late pro- and antisaccades are equal and conditionally independent of the action performed, whereas the latter constrains the probability of a late antisaccade to be a function of the arrival times of the late units. The expected response time of late pro- and antisaccade actions is given by 1 pðUp < Ua Þ Z1 t pðUp ¼ tÞpðUa > tÞdt; ð27Þ 0 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 8 / 36 SERIA—A model for errors and reaction times in the antisaccade task 1 pðUa < Up Þ Z1 t pðUa ¼ tÞpðUp > tÞdt: ð28Þ 0 We will refer to these terms as the mean response time of pro- and antisaccade actions, in contrast to the mean arrival times, which are the expected value of any single unit. Non-decision time The models above can be further finessed to account for non-decision times δ by transforming the RT t to tδ = t−δ. The delay δ might be caused by, for example, conductance delays from the retina to the cortex. In addition, the antisaccade or late units might include a constant delay δa, which is often referred to as the antisaccade cost [1]. Note that the model is highly sensitive to δ because any RT below it has zero probability. In order to relax this condition and to account for early outliers, we assumed that saccades could be generated before δ at a rate η 2 [0,1] such that the marginal likelihood of an outlier is pðT < dÞ ¼ pðTd < 0Þ ¼ Z: ð29Þ For simplicity, we assume that outliers are generated with uniform probability in the interval [0,δ]: pðT ¼ tÞ ¼ Z if t < d: d ð30Þ Furthermore, we assume that the probability of an early outlier being a prosaccade was approximately 100 times higher than being an antisaccade. Because of the new parameter η, the distribution of saccades with a RT larger than δ needs to be renormalized by the factor 1−η. In the case of the PROSA model, for example, this means that the joint distribution of action and RT is given by the conditional probability pðA ¼ pro; T ¼ td jtd > 0Þ ¼ pðUp ¼ td ÞpðUa > td da ÞpðUs > td Þ; pðUa < 0Þ ¼ 0; pðA ¼ anti; T ¼ td jtd > 0Þ ¼ pðUa ¼ td ð31Þ ð32Þ Z da Þ pðUp > td Þ þ td pðUp ¼ tÞpðUs < tÞdt : ð33Þ 0 A similar expression holds for the SERIA models. However, in the PROSA model a unitspecific delay is equal to an action-specific delay. By contrast, in the SERIA model both early and late responses can generate pro- and antisaccades. Thus, δa represents a delay in the late actions that affects both late pro- and antisaccades. Parametric distributions of the increase rate The models discussed in the previous sections can be defined independently of the distribution of the rate of each of the units. In order to fit experimental data, we considered four parametric distributions with positive support for the rates: gamma [13], inverse gamma, lognormal [25] and the truncated normal distribution (similarly to [22] and [24]). Table 1 and Fig 3 summarize these distributions, their parameters, and the corresponding arrival time densities. We considered five different configurations: 1) all units were assigned inverse gamma distributed rates, 2) all units were assigned gamma distributed rates, 3) the increase rate of the prosaccade PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 9 / 36 SERIA—A model for errors and reaction times in the antisaccade task Table 1. Parametric density functions of the increase rates. Name Parameters Gamma k,θ Inv. gamma k,θ Log normal T. normal Z is the normalization constant Z ¼ Rate p.d.f. y k GðkÞ yk GðkÞ 2 μ,σ r y=r r k 1 2 1 lnr m s 2 ffiffiffi e ð μ,σ 0 e p1 2psr 2 R1 e r=y k 1 1 Z exp ðr mÞ2 2s2 1 r m 2 2 s e ð Þ Þ Arrival time p.d.f. yk GðkÞ y=t e y k GðkÞ e t k 1 t=y k 1 t 2 1 lntþm s 2 ffiffiffi e ð p1 2pst 1 Zt2 e 1 m 1 t s 2 Þ 2 dr. https://doi.org/10.1371/journal.pcbi.1005692.t001 and stop units (or early and the inhibitory units) was gamma distributed but the antisaccade (late) unit’s increase rate was inverse gamma distributed, 4) all the units were assigned lognormal distributed rates or 5) all units were assigned truncated normal distributed rates. All the parametric distributions considered here can be fully characterized by two parameters which we generically refer to as k and θ. Hence, the PROSA model is characterized by the parameters for each unit kp,ka,ks,θp,θa,θs. The SERIA model can be characterized by analogous parameters ke,kl,ki,θe,θl,θi and the probabilities of early and late prosaccades πe and πl. In the case of the SERIAlr model, the probability of a late prosaccade is replaced by the parameters of a late prosaccade unit kp, θp. In addition to the unit parameters, all models included the nondecision time δ, the antisaccade (or late unit) cost δa, and the marginal rate of early outliers η. Experimental procedures In this section, we describe the experimental procedures, statistical methods, and inference scheme used to invert the models above. The data is from the placebo condition of a larger pharmacological study that will be reported elsewhere. Fig 3. Illustration of probability distributions used to model increase rates. Left: Distribution of the rates based on different probability density functions: Normal (red), gamma (blue), inverse gamma (green), and log-normal (cyan). All distributions were matched to have equal mean and variance. Center: Probit plots of the same distributions. While the gamma and lognormal distributions are very close to the straight line induced by the normal distribution, the inverse gamma distribution diverges slightly more from linearity. Right: Arrival time distribution (scaled to ms). https://doi.org/10.1371/journal.pcbi.1005692.g003 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 10 / 36 SERIA—A model for errors and reaction times in the antisaccade task Participants. Fifty-two healthy adult males naïve to the antisaccade task were invited to a screening session through the recruitment system of the Laboratory of Social and Neural Systems Research of the University of Zurich. During screening, and after being debriefed about the experiment, subjects underwent an electrocardiogram, a health survey, a visual acuity test, and a color blindness test. Subjects were excluded if any of the following criteria were met: age below 18 or above 40 years, regular smoking, alcohol consumption the day before the experiment, any possible interaction between current medication and levodopa or benserazide, pulse outside the range 55–100bpm, recreational drug intake in the past 6 months, history of serious mental or neurological illness, or if the medical doctor supervising the experiment deemed the participant not apt. All subjects gave their written informed consent to participate in the study and received monetary compensation. Procedure. Each subject was invited to two sessions. During both visits, the same experimental protocol was followed. After arrival, placebo or levodopa (Madopar DR 250, 200mg of levopa + 50 mg benserazide) was orally administered in the form of shape- and color-matched capsules. The present study is restricted to data from the session in which subjects received placebo. Participants and experimenters were not informed about the identity of the substance. Immediately afterwards subjects were introduced to the experimental setup and to the task through a written document. This was followed by a short training block (see below). The experiment started 70 minutes after substance administration. Subjects participated in three blocks of 192 randomly interleaved pro- and antisaccade trials. The percentages of prosaccade trials in the three blocks were 20%, 50%, or 80%. This yielded three prosaccade probability (PP) conditions: PP20, PP50, and PP80. Thus, in the PP20 block, subjects were presented a prosaccade cue in 38 trials, while in all other 154 trials an antisaccade cue was shown. The order of the trials was randomized in each block, but the same order was used in all subjects and sessions. The order of the conditions was counterbalanced across subjects. Stimulus and apparatus. During the experiment, subjects sat in front of a CRT monitor (Philipps 20B40, distance eye-screen: ’60cm, refresh rate: 75Hz). The screen subtended a horizontal visual angle of 38 degrees of visual angle (dva). Eye movements were recorded using a remote infrared camera (Eyelink II, SR-Research, Canada). Participants’ head was stabilized with a chin rest. Data were stored at a sampling rate of 500 Hz. During the task, two red dots (0.25dva) that constituted the saccadic targets were constantly displayed at an eccentricity of ±12dva. Displaying the saccadic target before the execution of an antisaccade has been reported to affect saccadic velocity and accuracy, but not RTs [26], and arguably decreases the need for sensorimotor transformations [27]. At the beginning of each trial, a gray fixation cross (0.6 × 0.6 dva) was displayed at the center of the screen. After a random fixation interval (500 to 1000 ms), the cross disappeared, and the cue instructing either a pro- or an antisaccade trial (see below) was shown centered on either of the red dots. As mentioned above, in each block, subjects were presented with a prosaccade cue in either 20, 50, or 80 percent of the trials. The order of the presentation of the cues was randomized. The cue was a green rectangle (3.48 × 0.8dva) displayed for 500ms in either horizontal (prosaccade) or vertical orientation (antisaccade). Once the cue was removed and after 1000ms, the next trial started. Subjects were instructed to saccade in the direction of the cue when a horizontal bar was presented (prosaccade trial) and to saccade in the opposite direction when a vertical bar was displayed (antisaccade trial, see Fig 4). See [28,29] for similar task designs. Prior to the main experiment, participants were trained on the task in a block of 50 prosaccade trials, immediately followed by 50 antisaccade trials. During the training, subjects were automatically informed after each trial whether their response was correct or not (see below), or whether they had failed to produce a saccade within 500ms after cue presentation (CP). No feedback was given during the main experimental blocks. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 11 / 36 SERIA—A model for errors and reaction times in the antisaccade task Fig 4. Task design. After a variable fixation period of 500–1000ms (top) the cue (green rectangle) appeared on the screen for 500 ms. The orientation of the cue (horizontal or vertical) indicated the required response (prosaccade or antisaccade). https://doi.org/10.1371/journal.pcbi.1005692.g004 Data preparation. Data were parsed and preprocessed using the Python programming language (2.7). Saccades were detected using the algorithm provided by the eyetracker manufacturer (SR Research), which uses a velocity and acceleration threshold of 22dva/s and 3800dva/s2 [30]. We only considered saccades with a magnitude larger than 2dva. RT was defined as the time between CP and the first saccade larger than 2dva. A prosaccade trial was considered correct if the end position of the saccade was ipsilateral to the cue and, conversely, an antisaccade trial was considered correct if the end position of the saccade was contralateral to the cue. Trials were excluded from further analysis if a) data were missing, b) a blink occurred between CP and the main saccade, c) the trial was aborted by the experimenter, d) subjects failed to fixate in the interval between fixation detection and CP, e) if a saccade was detected only later than 800ms after CP, f) if the RT was below 50ms, and in the case of an antisaccade if it was below 110ms. Corrective antisaccades were defined as saccades that a) followed a prosaccade error, b) occurred no later than 900ms after CP, and c) had less than 3dva horizontal error from the red circle contralateral to the cue. Besides the fitted non-decision time δ we assumed a fixed non-decision time of 50ms for all participants [17]. This was implemented by subtracting 50ms of all saccades before being entered into the model. In order to avoid numerical instabilities, RT were rescaled from millisecond to tenths of a second during all numerical analysis. All results are presented in ms. Modeling We aimed to answer three questions with the models considered here. First, we investigated which model (i.e. PROSA, SERIA or SERIAlr) explained the experimental data best, and whether all important qualitative features of the data were captured by this model. We did not have a strong hypothesis regarding the parametric distribution of the data and hence, comparisons of parametric distributions were only of secondary interest in our analysis. Second, we investigated whether reduced models that kept certain parameters fixed across trial types were PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 12 / 36 SERIA—A model for errors and reaction times in the antisaccade task Table 2. Model families with the respective increase-rate distributions. PROSA Model Prosaccade/ stop units Anti. unit # Param. full/const. m1 =mc1 Inv. gamma Inv. gamma 15/13 m2 =mc2 Gamma Gamma 15/13 m3 =mc3 Gamma Inv. gamma 15/13 m4 =mc4 Lognorm. Lognorm. 15/13 m5 =mc5 T. norm. T. norm. 15/13 Early/stop units Late unit Inv. gamma Inv. gamma SERIA m6 =mc6 19/13 c 7 m7 =m Gamma Gamma 19/13 m8 =mc8 Gamma Inv. gamma 19/13 m9 =mc9 Lognorm. Lognorm. 19/13 m10 =mc10 T. norm. T. norm. 19/13 SERIAlr Early/stop units Late pro./anti. units m11 =mc11 Inv. gamma Inv. gamma 19/15 m12 =mc12 Gamma Gamma 19/15 m13 =mc13 Gamma Inv. gamma 19/15 m14 =mc14 Lognorm. Lognorm. 19/15 m15 =mc15 T. norm. T. norm. 19/15 Models with parameters constrained to be equal across trial types are referred through the superscript c. https://doi.org/10.1371/journal.pcbi.1005692.t002 sufficient to model the data. Third, we investigated how the probability of a trial type in a block affected the parameters of the model. Model space. Initially, we considered 15 different models as shown in Table 2. Each model was fitted independently for each subject and condition. Since our experimental design included mixed blocks, we allowed for different parameters in pro- and antisaccade trials, i.e., different increase-rate distributions depending on the trial type (TT). Under this hypothesis, the PROSA model had 12 free parameters (6 for each TT), whereas the SERIA model required 4 further parameters (πe and πl in each TT). The late race SERIAlr model included 16 parameters for the units (8 for each TT). We did not investigate the case that early reactions could trigger antisaccades but rather fixed the probability of an early antisaccade 1−πe to 10−3. The rationale behind this was that if early reactions are a priori assumed to never trigger antisaccades, rare but possible early antisaccades might cause large biases when fitting a model. Regarding the non-decision time δ, antisaccade cost δa, and rate of outliers η, we assumed equal parameters in both TT. Consequently, the full PROSA model had 15 free parameters whereas the full SERIA and SERIAlr models had both 19 free parameters. In addition to the full models, we evaluated restricted versions of each of them by constraining some parameters to be equal across TT. In the case of the SERIA model, we hypothesized that the parameters of all units were equal, irrespective of TT (i.e., that the rate of the units was not affected by the cue presented in a trial). However, we assumed that the probability that an early or late response was a prosaccade was different in pro- and antisaccade trials. Therefore, in the case of the SERIA model, instead of 12 unit parameters (6 per TT), the restricted model had only 6 parameters for the units’ rates. The parameters πe and πl were allowed to differ in PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 13 / 36 SERIA—A model for errors and reaction times in the antisaccade task pro- and antisaccade trials. In the case of the restricted SERIAlr model, the units that underlie the late decision process were allowed to vary across TT, yielding a restricted model with 4 parameters for the early and inhibitory units, and 8 for the late decision process, half of them for each trial type. In the case of the PROSA model, similarly to [17], it is possible to assume that the parameters of the prosaccade unit remain constant across TT, and that the parameters of the stop and antisaccade units depend on TT, yielding 10 parameters for the units. Prior distributions for model parameters. To complete the definition of the models, the prior distribution of the parameters was specified. This distribution reflects beliefs that are independent of the data and provides a form of regularization when inverting a model. In order to avoid any undesired bias regarding the parametric distributions considered here, we reparametrized all but the truncated normal distribution in terms of their mean and variance. We then assumed that the log of the mean and variance of the rate of the units were equally normally distributed (see Table 3). Therefore, the parametric distributions had the same prior in terms of their first two central moments. In the case of the truncated normal distribution, instead of an analytical transformation between its first two moments and its natural parameters μ and σ2, we defined the prior distribution as a density of μ and ln σ2. To ensure that μ was positive with high probability (96%) we assumed that μ * N(0.55,0.09). The variance term was distributed as displayed in Table 3. As a further constraint, we restricted the parameter space to enforce that the first two moments of the distributions of rates and RTs existed. We relaxed this constraint for the late units of the SERIAlr in order to allow for ‘flat’ distributions with possibly infinite mean and variance. This can describe a case in which the increase rate of one of the late units is extremely low. For the non-decision time δ and the antisaccade cost δa, the prior of their log transform was a normal distribution, consistent across all models. Note that the scale of the parameters δ and δa in Table 3 is tenths of a second. The fraction of early outliers η, and early and late prosaccades πe and πl were assumed to be Beta distributed, with parameters 0.5 and 0.5. Thus, for example, the prior probability of an early outlier is given by pðZÞ / Z0:5 ð1 0:5 ZÞ : ð34Þ This parametrization constitutes the minimally informative prior distribution, as it is the Jeffrey’s prior of η, πe and πl. Table 3 displays the parameters used for the prior distributions. Bayesian inference Inference on the model parameters was performed using the Metropolis-Hastings algorithm [31]. To increase the efficiency of our sampling scheme, we iteratively modified the proposal distribution during an initial ‘burn-in’ phase as proposed by [32]. Moreover, we extended this method by drawing from a set of chains at different temperatures and swapping samples across chains. This method, called population MCMC or parallel tempering, increases the statistical Table 3. Prior probability density functions. Parameter Probability density function Expected value Variance μr N ðlnmr ; 1:08; 0:97Þ 0.55 0.5 s2r N ðlns2r ; 2:64; 0:69Þ 0.1 0.01 1.25 δ N ðlnd; 1:58; 1:79Þ 0.5 δa N ðlnda ; 0:87; 1:17Þ 0.75 1.25 πe Beta(πe;0.5,0.5) 0.5 0.145 πl Beta(πe;0.5,0.5) 0.5 0.145 η Beta(η;0.5,0.5) 0.5 0.145 https://doi.org/10.1371/journal.pcbi.1005692.t003 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 14 / 36 SERIA—A model for errors and reaction times in the antisaccade task efficiency of the Metropolis-Hasting algorithm [33] and has been used in similar contexts before [34]. We simulated 16 chains with a 5-th order temperature schedule [35]. For all but the models including a truncated normal distribution, we drew 4.1 × 104 samples per chain, from which the first 1.6 × 104 samples were discarded as part of the burn-in phase. When a truncated normal distribution was included (models m5, m10, and m15), the total number of samples was increased to 6 × 104, from which 2 × 104 were discarded. The convergence of the ~ statistic of algorithm was assessed using the Gelman-Rubin criterion [33,36] such that the R the parameters of the model was aimed to be below 1.1. When a simulation did not satisfy this criterion, it was repeated until 99.5 percent of all simulations satisfied it. Models were scored using their log marginal likelihood or log model evidence (LME). This is defined as the log probability of the data given a model after marginalizing out all its parameters. When comparing different models, the LME corresponds to the log posterior probability of a model under a uniform prior on model identity. Thus, for a single subject with data y, the posterior probability of model k, given models 1 to n is pðyjmk Þpðmk Þ pðyjmk Þ pðmk jyÞ ¼ Pn ¼ Pn : pðyjm Þpðm Þ i i i¼1 i¼1 pðyjmi Þ ð35Þ Importantly, this method takes into account not only the accuracy of the model but also its complexity, such that overparameterized models are penalized [37]. A widely used approximation to the LME is the Bayesian Information Criterion (BIC) which, although easy to compute, has limitations (for discussion, see [38]). Here, we computed the LME through thermodynamic integration [33,39]. This method provides robust estimates and can be easily computed using samples obtained through population MCMC. One important observation here is that the LME is sensitive to the prior distribution, and thus can be strongly influenced by it [40]. We addressed this issue in two ways: On one hand and as mentioned above, we defined the prior distribution of the increase rates of all models in terms of the same mean and variance. This implies that the priors were equal up to their first two moments, and hence all models were similarly calibrated. On the other hand, we complemented our quantitative analysis with qualitative posterior checks [33] as shown in the results section. Besides comparing the evidence of each model, we also performed a hierarchical or random effects analysis described in [38,41]. This method can be understood as a form of soft clustering in which each subject is assigned to a model using the LME as assignment criterion. Here, we report the expected probability of the model ri, which represents the percentage of subjects that is assigned to the cluster representing model i. This hierarchical approach is robust to population heterogeneity and outliers, and complements reporting the group-level LME. Finally, we compared families of models [42] based on the evidence of each model for each subject summed across conditions. Classical statistics In addition to a Bayesian analysis of the data, we used classical statistics to investigate the effect of our experimental manipulation on behavioral variables (mean RT and ER) and the parameters of the models. We have suggested previously [11,43,44] that generative models can be used to extract hidden features from experimental data that might not be directly captured by, for example, standard linear methods or purely data driven machine learning techniques. In this sense, classical statistical inference can be boosted by extracting interpretable data features through Bayesian techniques. Frequentist analyses of RT, ER, and parameter estimates were performed using a mixed effects generalized linear model with independent variables subject (SUBJECT), prosaccade PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 15 / 36 SERIA—A model for errors and reaction times in the antisaccade task probability (PP) with levels PP20, PP50 and PP80, and when pro- and antisaccade trials were analyzed together, trial type (TT). The factor SUBJECT was always entered as a random effect, whereas PP and TT were treated as categorical fixed effects. In the case of ER, we used the probit function as link function. Analyses were conducted with the function fitglme.m in MATLAB 9.0. The significance threshold α was set to 0.05. Implementation All likelihood functions were implemented in the C programming language using the GSL numerical package (v.1.16). Integrals without an analytical form or well-known approximations were computed through numerical integration using the Gauss-Kronrod-Patterson algorithm [45] implemented in the function gsl_integration_qng. The sampling routine was implemented in MATLAB (v. 8.1) and is available as a module of the open source software package TAPAS (www.translationalneuromodeling.org/tapas). Results Behavior Forty-seven subjects (age: 23.8 ± 2.9) completed all blocks and were included in further analyses. A total of 27072 trials were recorded, from which 569 trials (2%) were excluded (see Table 4). Both ER and RT showed a strong dependence on PP (Fig 5 and Table 5). Individual data is included in the S1 Dataset and is displayed in S1 Fig. The mean RT of correct pro- and antisaccade trials was analyzed independently with two ANOVA tests with factors SUBJECT and PP. We found that in both pro- (F2,138 = 46.9, p < 10−5) and antisaccade trials (F2,138 = 37.3, p < 10−5) the effect of PP was significant; with higher PP, prosaccade RT decreased, whereas the RT of correct antisaccades increased. On a subject-by-subject basis, we found that between the PP20 and PP80 conditions, 91% of the participants showed increased RT in correct antisaccade trials, while 81% demonstrated the opposite effect (a decrease in RT) in correct prosaccade trials. Similarly, there was a significant effect of PP on ER in both prosaccade (F2,138 = 376.1, p < 10−5) as well as in antisaccade (F2,138 = 347.0, p < 10−5) trials. This effect was present in all but one participant in antisaccade trials and in all subjects in prosaccade trials. Exemplary RT data of one subject in the PP50 condition is displayed in Fig 6. Modeling Model comparison results. Initially, we considered the models outlined in Table 2. The LME over all participants (fixed effects analysis) and the posterior probability of all models Table 4. Summary of trials per subject. Valid Blink Missing Aborted FE Late S. Early S. Total Total 26503 188 60 42 249 0 30 27072 Mean 563.9 4.0 1.3 0.9 5.3 0.0 0.6 576 Std. 9.9 5.1 2.5 1.5 5.0 0.0 1.3 - Min. 536 0 0 0 0 0 0 - Max. 576 22 15 6 19 0 8 - FE: Fixation errors. Late saccades are saccades elicited after 800ms. Early saccades are prosaccades elicited before 50ms after CP or antisaccades elicited before 110ms after CP. https://doi.org/10.1371/journal.pcbi.1005692.t004 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 16 / 36 SERIA—A model for errors and reaction times in the antisaccade task Fig 5. Error rate and mean reaction time as a function of prosaccade trial probability (PP). Left panel: Mean error rates for pro- and antisaccade trials. Right panel: Mean reaction time in ms. Error bars indicate standard errors of the mean. Only correct responses are displayed. https://doi.org/10.1371/journal.pcbi.1005692.g005 and all subjects are presented in Fig 7. Independently of the particular parametric distribution of the units, the SERIAlr models had higher evidence compared to the PROSA and SERIA models. A random effects, family-wise model comparison [42] resulted in an expected frequency of r = 87% for the SERIAlr family, r = 11% for the SERIA family, and r = 2% for the PROSA family. In addition, constraining the parameters to be equal across trial types increased the model evidence irrespective of the parametric distribution assigned to the units (Fig 7). Here, the family-wise model comparison showed that models with constrained parameters had an expected frequency of r = 98%. Over all 30 models, mc13 (SERIAlr with constrained parameters, early and inhibitory increase rates gamma distributed, and late units’ rate inverse gamma distributed) showed the highest LME with ΔLME > 200 compared to all other models. Following [40], a difference in LME larger than 3 corresponds to strong evidence. To verify that the SERIAlr family was not preferred simply because the probability of early prosaccades was fixed, we considered models in the SERIA family with the same property (not displayed). We found that although fixing this value increased the LME of the SERIA family, Table 5. Summary of mean RTs and ERs. Trial type Action Reaction times [ms] PP 20 PP 50 PP80 Pro. Pro. 330(72) 319(67) 284(59) Pro. Anti. 326(68) 329(46) 336(57) Anti. Anti 354(60) 378(57) 389(61) Anti. Pro 234(50) 231(47) 225(31) Error rates [%] Pro. 26(15) 11(8) 4(4) Anti. 23(17) 35(21) 51(20) Standard deviations are shown in brackets. https://doi.org/10.1371/journal.pcbi.1005692.t005 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 17 / 36 SERIA—A model for errors and reaction times in the antisaccade task Fig 6. Exemplary histogram of the reaction times of one subject in the PP50 condition. Prosaccade trials are displayed in the upper half plane and antisaccade trials in the lower (negative) half plane. Prosaccade actions are depicted in red color, whereas antisaccade actions are shown in blue. Errors in prosaccade trials are antisaccades that for this subject occurred after the first peak of early prosaccades. Errors in antisaccade trials (lower half plane) occurred at a similar latency as early prosaccades in prosaccade trials. The histograms have been normalized to have unit probability mass, i.e., the sum of the area of all bars is one. https://doi.org/10.1371/journal.pcbi.1005692.g006 there was still a difference of ΔLME > 90 when comparing the best model of the SERIAlr family and the best model of the SERIA family with a fix probability of early prosaccades. Fits of four subjects using the posterior samples of the best PROSA (m1), SERIA (mc8 ), and SERIAlr (mc13 ) models are depicted in Fig 8. Although model m1 was the best model in the PROSA family, it clearly did not explain the apparent bimodality of the prosaccade RT distributions. Instead, RTs were explained through wider distributions. No obvious difference could be observed between the SERIA and SERIAlr models. We further examined the model fits in Fig 9 and Fig 10 by plotting the weighted fits and cumulative density functions of the reciprocal RT in the probit scale (reciprobit plot [22]) collapsed across subjects for the best model of each family. The histograms of RTs clearly show a large number of late prosaccades whose distribution is similar to the distribution of antisaccade RTs. The most pronounced–but still small—difference between the SERIA and SERIAlr models was visible in prosaccade trials in the PP20 condition (left panel, upper half plane), in which antisaccade errors displayed lower RT than correct late prosaccades. Corrective antisaccades. The RTs of antisaccades that follow an error prosaccade were not directly modeled. However, we hypothesized that corrective antisaccades are delayed late antisaccade actions, whose distribution is given by the response time distribution of late antisaccades 1 pðUa ¼ tÞpðUp > tÞ pðUa < Up Þ ð36Þ A total of 2989 corrective antisaccades were included in the analysis. The mean (±std) end time of the erroneous prosaccades was 268(±63)ms. The mean RT of corrective antisaccades was 447(±103)ms, and the weighted mean arrival time of the late antisaccade unit was 367ms. Fig 11 displays the histogram of the end time of all prosaccade errors, the RT of all corrective PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 18 / 36 SERIA—A model for errors and reaction times in the antisaccade task Fig 7. Summary of model comparison. Top: Summed LME of all subjects for all 30 models. White bars show models with all parameters free, grey bars models with constrained parameters. LMEs are normalized by subtracting the lowest LME (m5). Model mc13 (constrained SERIAlr) exceeded all other models (ΔLME > 200). Bottom: Illustration of model probability for all subjects. The posterior model probabilities for all subjects are shown as black dots. In white shading are models with all parameters free, grey bars represent models with restricted parameters. Note that in nearly all subjects, the SERIAlr models with restricted parameters showed high model probabilities. https://doi.org/10.1371/journal.pcbi.1005692.g007 antisaccades and the time shifted (+80ms) predicted response time of late antisaccades. Since we did not have a strong hypothesis regarding the magnitude of the delay of the corrective antisaccades, we selected the time shift to be the difference between the mean corrective antisaccade RT and the mean predicted response time of late antisaccades. Visual inspection strongly suggests that the distribution of corrective antisaccade RTs is well approximated by the distribution of the late responses. The short difference between corrective antisaccades’ RT and the expected response time of the late antisaccade unit (80ms) favors the hypothesis that the plan for a corrective antisaccade is initiated before the incorrect prosaccade had finished. Effects of prosaccade probability on model parameters. The effect of PP on the parameters of the model was investigated by examining the expected value of the parameters of the best scoring model (mc13 ). Initially, we considered the question of whether the mean arrival or response time of each of the units changed as a function of PP. For arrival times, this corresponds to N 1X ðE½Ui jkij ; yij þ dij Þ N j¼1 ð37Þ where i is an index over the units, j is an index over N samples collected using MCMC, and dij is the estimated delay. In the case of the late units, we considered only the response time of correct actions. Fig 12 left displays the mean arrival and response times. These were submitted to PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 19 / 36 SERIA—A model for errors and reaction times in the antisaccade task Fig 8. Fits of best PROSA (m1), SERIA (mc8 ) and SERIAlr (mc13 ) models. Columns display the normalized histogram of the RTs of pro- (red) and antisaccades (blue) in each of the conditions. Rows correspond to individual subjects named S1 to S4 for display purpose. As in Fig 6, prosaccade PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 20 / 36 SERIA—A model for errors and reaction times in the antisaccade task trials are displayed on the upper half plane, whereas antisaccade trials are displayed in the lower half plane. The predicted RT distributions based on the samples from the posterior distribution are displayed in solid (SERIAlr), broken (SERIA), and dash-dotted (PROSA) lines. Note that data from subject 3 in the PP50 condition is the same as shown in Fig 6. Early outliers are not displayed. https://doi.org/10.1371/journal.pcbi.1005692.g008 four separate ANOVA tests, which revealed that PP had a significant effect on all four units: early unit (F2,138 = 9.2, p < 10−3), late antisaccade (F2,138 = 26.6, p < 10−3), late prosaccade (F2,138 = 19.6, p < 10−3), and inhibitory unit (F2,138 = 30.9, p < 10−3). We then explored the differences across conditions through planned post hoc tests on each condition for each of the units (Table 6). The arrival times of the early unit did not change significantly between condition PP20 and PP50, but decreased significantly in the PP80 condition as compared to the PP50 block. The response times of late antisaccades increased significantly between the PP20 and the PP50 conditions but not so between the PP50 and PP80. Late prosaccades followed the opposite pattern, showing only a significant decrease in response time between the PP50 and PP80 conditions. Finally, the inhibitory unit changed significantly across all conditions. Finally, we examined how the probability of a late antisaccade p(Ua > Up) (Fig 12, right) depended on PP and TT. The estimated parameters for both pro- and antisaccade trials were analyzed with a model with factors SUBJECT, TT, PP and the interaction between TT and PP. An ANOVA test demonstrated that both PP (F2,276 = 51.2, p < 10−3) and TT (F1,276 = 985.0, p < 10−3) had a significant effect, but there was no evidence for an interaction between the two factors (F2,276 = 1.5, p < 0.23), suggesting that PP affected the probability of a late antisaccade equally in pro- and antisaccade trials. Fig 9. Fits from the best models in each family (m1 ; mc8 ; mc13 ). Model fits and RT histograms for each condition collapsed across subjects. For more details see Figs 6 and 8. https://doi.org/10.1371/journal.pcbi.1005692.g009 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 21 / 36 SERIA—A model for errors and reaction times in the antisaccade task Fig 10. Reciprobit plot of best models. Predicted and empirical cumulative density function of the reciprocal RT in the probit scale for each condition and model collapsed across all subjects. The data shown are the same as in Fig 9, but split for trial types and illustrated as cumulative distributions. Note that the y-axis is in the probit scale and that nearly all differences between the model and the data occur at very small probability values of 5% or below. https://doi.org/10.1371/journal.pcbi.1005692.g010 Subject specific parameters Finally, we investigated how some of the parameters of the model were related to each other across subjects. Because it has been commonly reported that schizophrenia is related with higher ER, but also with increased antisaccade RT, an interesting question is whether higher late-action response times are correlated with the percentage of late errors and inhibition failures, i.e., early saccades that are not stopped. We found that the response time of late pro (F1,135 = 13.6, p < 0.001) and antisaccades (F1,135 = 7.1, p < 0.01) was negatively correlated with the probability of a late error (Fig 13), but no significant interaction between PP and response time was found (pro: F2,135 = 1.7, p = 0.19; anti: F2,135 = 0.3, p = 0.76). Hence, late responders tended to make fewer late errors, suggesting a speed/accuracy trade-off in addition to the main effect of PP. We further considered the question whether the percentage of PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 22 / 36 SERIA—A model for errors and reaction times in the antisaccade task Fig 11. Empirical and predicted RT of corrective antisaccades. Left: End time of erroneous prosaccades, RTs of corrective antisaccades, and time shifted predicted response time distribution of late antisaccades. The time shift was selected to be the difference between the empirical and predicted mean response time. Center: Quantile-quantile plot of the predicted and empirical distribution of corrective antisaccades, and a linear fit to the central 98% quantiles. There is a small deviation only at the tail of the distribution. Right: Reciprobit plot of the empirical and predicted cumulative density functions of the RT of corrective antisaccades. The scale of the horizontal axis is proportional to the reciprocal RT. The vertical axis is in the probit scale. https://doi.org/10.1371/journal.pcbi.1005692.g011 inhibition failures was correlated with the expected arrival time of the late antisaccade unit in antisaccade trials (Fig 13 right). Note that the number of inhibition failures is the same in both trial types in a constrained model, but inhibition failures are errors in antisaccade trials and correct early reactions in prosaccade trials. We found that these parameters were not Fig 12. Model parameters. Left: Mean arrival or response time and standard error of the early and inhibitory units and late pro- and antisaccades. Right: Probability of a late antisaccade p(Ua > Up) in prosaccade (red) and antisaccade (blue) trials in each condition in the probit scale. https://doi.org/10.1371/journal.pcbi.1005692.g012 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 23 / 36 SERIA—A model for errors and reaction times in the antisaccade task Table 6. Post hoc comparison of the effect of PP. Early Inhib. Late pro. Late anti. Mean [ms] t138 p Mean [ms] t138 p Mean [ms] t138 p Mean [ms] t138 p PP20–PP50 4 0.6 0.50 -30 -4.0 <0.001* 3 0.5 0.55 -24 -36 <0.001* PP20–PP80 26 3.9 <0.001* -58 -7.8 <0.001* 32 5.7 <0.001* -32 -5.4 <0.001* PP50–PP80 22 3.3 0.001* -28 -3.8 <0.001* 29 5.1 <0.001* -7 -1.5 0.13 Effect of PP on the mean arrival time of the early and inhibitory unit, and for late pro- and antisaccade units in the corresponding trial type. *, p < 0.05.of a two-tailed t-test. https://doi.org/10.1371/journal.pcbi.1005692.t006 significantly correlated (F2,135 = 1.2, p = 0.26). This was also the case when we considered the expected response time of late prosaccades in prosaccade trials (not displayed; F2,135 = 0.0, p = 0.98). Fig 14 illustrates the posterior distribution of late errors and inhibition failures of two representative subjects as estimated using MCMC. Clearly, PP induced strong differences in the percentage of inhibition failures and late errors in prosaccade trials in both subjects. The effect of PP is less pronounced in late errors in antisaccade trials. The posterior distributions also illustrate how the SERIAlr model can capture individual differences: For example, the percentage of late prosaccade errors in the PP80 condition and the percentage of inhibition failures across all conditions are clearly different in each subject. Discussion In this study, we provided a formal treatment of error rates (ER) and reaction times (RT) in the antisaccade task using probabilistic models. We applied these models to data from an experiment consisting of 3 mixed blocks with different probabilities of pro- and antisaccades trials. Model comparison showed that a novel model that allows for late pro- and antisaccades Fig 13. Correlation between late arrival times and errors. Left: Percentage of late errors against late antisaccades’ response times in antisaccade trials. Center: Percentage of late errors against late prosaccades’ response time in prosaccade trials. Left: Percentage of inhibitory failures against late antisaccades’ response time in antisaccade trials. The vertical axis is in the probit scale. https://doi.org/10.1371/journal.pcbi.1005692.g013 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 24 / 36 SERIA—A model for errors and reaction times in the antisaccade task Fig 14. Posterior distribution of late errors and inhibition failures. The posterior distribution of the percentage of late and inhibition failures of two exemplary subjects (see Figs 8 and 13). Samples from the posterior distribution were obtained using MCMC. Histograms display the distributions of the samples in probit scale (horizontal axis). For these two subjects, the posterior distribution of late prosaccade and inhibitory failures clearly discriminates between the three PP conditions. https://doi.org/10.1371/journal.pcbi.1005692.g014 explains our experimental findings better than a model in which all late responses are assumed to be antisaccades. The parameter estimates of the hidden units of the model showed that changes in the inhibitory unit and the late decision process explained most of the overt changes in behavior caused by our experimental manipulation, i.e., differences in trial type probability. Moreover, we found that all units were sensitive to the PP in a block, although late responses tended to plateau when the corresponding trial type was not highly frequent. Our main finding is that two decision processes are necessary to properly model the antisaccade task: on one hand, an early race between a prepotent response towards a target and an endogenously generated signal to cancel this action, and, on the other hand, a secondary late race between two units encoding the cue-action mapping. Although the late decision process can be closely approximated by assuming that RT and actions are independent (at least in our experimental design), Bayesian model comparison demonstrated that late decisions are more accurately described by a race between two units representing different actions. The two decision processes are the sources of early errors–fast prosaccades in antisaccade trials- and late errors–late actions incongruent with the cue presented. The late decision process displays a speed/accuracy tradeoff and is biased by the probability of a trial type in a block. Moreover, PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 25 / 36 SERIA—A model for errors and reaction times in the antisaccade task this decision process predicts the RT distribution of corrective antisaccades that follow early errors. Because the extra latency of these corrective antisaccades (80ms) is relatively short, it is unlikely that corrective antisaccades are due to a restart in the decision process. Rather these are late actions that overwrite early errors. Influence of trial type probability on reaction times and error rates Our results show that both RT and ER depend on PP. While this was a highly significant factor in our study, there are mixed findings in previous reports. ER in antisaccade trials was found to be correlated with TT probability in several studies [29,46,47]. However, this effect might depend on the exact implementation of the task [47,48]. Changes in prosaccade ER similar to our study have been reported by [29] and [48]. Studies in which the type of saccade was signaled at fixation prior to the presentation of the peripheral cue do not always show this effect [47]. The results on RTs are less consistent in the literature. Our findings of increased antiand decreased prosaccade RTs with higher PP are in line with the overall trend in [29], and with studies in which the cue was presented centrally [47]. Often, there is an additional increase in RT in the PP50 condition [29,47], which was visible in our data as a slight increase in RT in the PP50 condition on top of the linear effect of PP. Overall, RTs in our study were relatively slow compared to studies in which the TT cue was separated from the spatial cue [46,47]. However, a study with a similar design and added visual search reported even slower RTs in both pro- and antisaccades [29]. Interpretation of model comparison results Formal comparison of generative models can offer insight into the mechanisms underlying eye movement behavior [11] and might be relevant in translational neuromodeling applications, such as computational psychiatry [49–53]. Here, we have presented what is, to our knowledge, the first formal statistical comparison of models of the antisaccade task. For this, we formalized the model introduced in [17] and proceeded to develop a novel model that relaxes the one-to-one association of early and late responses with pro- and antisaccades, respectively. All models and estimation techniques presented here are openly available under the GPLv3.0 license as part of the open source package TAPAS (www. translationalneuromodeling.org/tapas). Bayesian model comparison yielded four conclusions at the family level. First, the SERIA models were clearly favored when compared to the PROSA models. Second, including a late race between actions representing late pro- and antisaccades (SERIAlr) resulted in an increase in model evidence, compared to a model not including a late race (SERIA). Third, models in which the race parameters of the early and inhibitory unit were constrained to be equal across TT had a higher LME than models in which all parameters were free. Hence, the effect of the cue in a single trial was limited to the late action, and did not affect the race between an early and inhibitory process. This constitutes an important external validation, as it means that model comparison does favor a model which respects the temporal order of the experiment: Information about TT is only available after the stimulus was presented and, thus, it is unlikely to have an impact on fast reactive responses. Fourth, early responses were nearly always prosaccades. Crucially, these four conclusions are based on family-wise comparison across all parametric distribution of the increase rate of the units. A further consequence of our findings is that two independent and qualitatively different decision processes lead to an antisaccade: the race process between early and inhibitory units, and the secondary decision process that generates late responses. A separation of decisions into a ‘where’ and a ‘when’ component has been proposed by [54], but mainly in conceptual PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 26 / 36 SERIA—A model for errors and reaction times in the antisaccade task terms. However, model comparison showed that these two components (‘where’ and ‘when’) cannot be completely dissociated and that time plays a role in late decisions. Nevertheless, the assumption that action type and arrival time of late responses were independent yielded a good fit to this particular data set, suggesting that it is, in many cases, an acceptable approximation to assume a time-independent late decision process. The most obvious difference between the SERIA and SERIAlr can be observed in prosaccade trials in the PP20 condition (left panel, upper half plane Fig 9), in which late prosaccades are slower than antisaccades. We discuss this point in more detail below. Parametric distribution of reaction times. The parametric distribution of oculomotor RTs has been discussed in great detail in the literature (e.g., [13,55]). Here, we did not aim at determining the most suitable distribution, but rather opted for a practical approach by evaluating different models with a reduced number of parametric distributions. We then based our conclusions on the model with the highest LME. Nevertheless, one can consider the connection of the models presented here with other families of parametric distributions. In particular, the linear relationship si ¼t ri ð38Þ could be seen as formally inconsistent with the observation that RT are likely to be explained by stochastic accumulation processes (see for example [56,57], but [58]). This is a weaker constraint than one would expect, because under low noise conditions, for example, a linear relationship can be a good approximation of neural activity. Even if the relationship is not linear, for any continuous function ϕ with an inverse function ϕ−1, the model can be recasted as [59]: si ¼ ðtri Þ; ð39Þ 1 ðsi Þ ¼ t: ri ð40Þ In any case, linear accumulation models have been shown to yield similar conclusions to stochastic accumulation models [58]. More generally, it can be shown that if RTs follow a generalized inverse normal distribution (GIN) of the form c l 1 k pffiffiffiffiffiffiffiffi t l 1 exp GINðt; l; k; cÞ ¼ ðkt 1 þ ctÞ ð41Þ 2 2Kl ð kcÞ where λ 0, and Kλ is a modified Bessel function of the second kind, there exists a continuous diffusion process whose first hit distribution (FHD) follows the GIN [60]. A particular case of this distribution is the Wald distribution for which l ¼ 12 ; k ¼ 0. It is the FHD of the Brownian diffusion process with drift pffiffiffi Xt ¼ sct þ sWt ð42Þ where Wt denotes a Wiener process, x0 > 0, and the absorbing boundary a is zero. More relevant here, when ψ = 0 the distribution reduces to an inverse gamma distribution, the FHD of the process pffiffiffi Xt ¼ sð2l 1Þt 1 þ sWt ð43Þ PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 27 / 36 SERIA—A model for errors and reaction times in the antisaccade task with x0 > 0 and boundary a = 0 (for a detailed mathematical treatment see [60]). Thus, if the rates of a ballistic, linear processes are assumed to be gamma distributed, the RTs follow a distribution that is formally equivalent to a first hit model with stochastic updates and fixed rates. While the model presented here can be seen as a ballistic accumulation model, this equivalence suggests that it is compatible with a diffusion process with infinitesimal mean change proportional to t−1. Other antisaccade models. In broad terms, three families of antisaccade models can be distinguished (reviewed in [61]). The first set of models is based on a race process with independent saccadic and stop units. These models build on the seminal work of [16] on the stopsignal paradigm. According to this model, a ‘GO’ signal triggers a stochastic ‘race’ process that generates a response once it reaches threshold. Critically, a stop signal triggers a second process that inhibits the first ‘GO’ response if it is the first to reach threshold. Moreover, the rates of both units are assumed to be independent. This model was further extended for the antisaccade task by [17] (but see [14,21], and the review in [20]), who included a third unit such that an antisaccade is generated when a reflexive prosaccade is inhibited by an endogenously-triggered stop process. Note that the original ‘horse-race’ model has also been modified [62] to account for different competing response actions, similarly as in the antisaccade task. The models proposed here belong to this family. A second type of model relies on lateral or mutual inhibition of competing pro- and antisaccade units. In this direction, Cutsuridis and colleagues [61,63,64] proposed that lateral inhibition is implemented by inhibitory connections in the intermediate layers of the superior colliculus. Thus, saccades are the result of accumulation processes, but these are not independent of each other. Crucially, no veto-like stop signal is required. Although no formal modelfitting has been proposed for this model, qualitative agreement with data suggests that it might capture behavioral patterns relevant in translational applications [64,65]. Since no probabilistic version of this model is available, it is not yet possible to decide on the grounds of model comparison whether mutually dependent or independent race processes best explain current behavioral findings. Finally, several models that incorporate detailed physiological mechanisms have been proposed [23,66–68]. These models cannot be easily assigned to one of the above categories, as they often employ both an inhibitory mechanism that stops or withholds the reactive responses as well as competition between actions. In addition, while more realistic models possess a more fine-grained representation of the underlying neurobiology, they rely on a large number of parameters and for this reason, it is difficult to fit them to behavioral data (for discussion, see [11]). Regarding neurobiologically realistic models, the model proposed by [23] is the most similar to the SERIA model. It posits two different mechanisms that interact in the generation of antisaccades: an action-selection module and a remapping module that controls the cue-action mapping. As a consequence, this model allows for the generation of late errors that follow a similar RT distribution as correct antisaccades. Consistent with this observation, the SERIA model can quantitatively distinguish between inhibition and late cue-action mapping errors (Fig 15, left panel). A less obvious similarity between the SERIA model and [23] is that different cues do not lead directly to different dynamics in the action module, but only in the socalled ‘remapping’ module. Furthermore, the incorporation of a late race is conceptually close to the approach proposed by [23], which includes a winner-take-all competition in what we have referred here as late responses. Similarly, our model comparison results show that different cues (i.e., trial types) do not affect the GO/NO-GO process but only the late cue-action mapping. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 28 / 36 SERIA—A model for errors and reaction times in the antisaccade task Fig 15. Error sources and the correlation of response times. Left: Error rate (black line) split into the two causes predicted by the model. Inhibition errors are early actions that always trigger prosaccades. Similarly as described by [23], late errors occur when a late response leads to a prosaccade. Right: Correlation between correct antisaccades’ and late prosaccades’ response times according to the best SERIAlc model. The best linear fit is depicted as a solid line. The mean ratio of pro- and antisaccade response times (s) is displayed on the right. Although late pro- and antisaccade response times are highly correlated, their ratio is different in each condition (interaction PP and late prosaccade response time F = 9.2, p < 0.001). https://doi.org/10.1371/journal.pcbi.1005692.g015 Parameter changes across trial types One of the most salient results presented here is that models in which the parameters of the units were constrained to be equal across trial types had a larger LME than models in which all the parameters were free, suggesting that the early and inhibitory race units were not affected by the cue presented in a single trial. While visual inspection of the predicted likelihood under the posterior parameters showed that most of the prominent characteristics of the data were explained correctly, some more subtle effects were not captured accurately by the SERIA model. This is particularly clear in the PP20 condition, in which the SERIA model displays a large bias in prosaccades trials in the PP20 condition. One possible explanation is that restricting the parameters across trial types made the model too rigid to capture this effect. Fig 16 compares the fitted RT distributions for models m8 (SERIA) and m13 (SERIAlr), in which no constraint on the parameters was imposed. Both models are qualitatively almost identical, although as shown in Fig 7, the LME favored the SERIAlr model. Thereby, the qualitative similarity between both models indicates that, in our experiment, the RT of late decisions is only weakly dependent on time. In conclusion, although removing the constraint on the parameters did improve the fit, the differences are marginal and thus did not justify the additional model complexity. As mentioned above this is consistent with the notion that the information about trial type is only available to a subject once the peripheral stimulus (green bar) has been processed, presumably tens of milliseconds after the stimulus onset. In fact, this example illustrates the protection against overfitting provided by the LME, as this is a case in which simpler models were preferred over more complex models despite of slightly less accurate fits. Arguably, the constrained SERIA model fails to fully capture the RT of late prosaccade in the PP20 and PP50 conditions because of the assumption that late prosaccades have the same arrival time as late antisaccades. As shown in Fig 15, although the response time of late pro- PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 29 / 36 SERIA—A model for errors and reaction times in the antisaccade task Fig 16. Comparison between unconstrained SERIA and SERIAlr models. Comparison between models m8 (broken lines; SERIA model) and m13 (solid lines; late race SERIAlr model.). https://doi.org/10.1371/journal.pcbi.1005692.g016 and antisaccades are strongly correlated, the average ratio of the response times changes across conditions. The effect of trial type probability It is far from obvious why TT probability affects RT and ER in the antisaccade task. One possible explanation is that increased probability leads to higher preparedness for either pro- or antisaccades. Such a theory posits an intrinsic trade-off between preparations for one of the two action types that leads to higher RTs and ERs in low probability trials. Thus, a trade-off theory predicts that the arrival times of early and late responses should be negatively correlated. Although this hypothesis can explain our behavioral findings in terms of summary statistics, our model suggests a more complicated picture. The main explanation of our results is the effect of TT probability on the inhibitory unit and the probability of a late prosaccade. A higher probability of antisaccade trials leads to faster inhibition and to a higher number of late prosaccades. This resulted in higher mean RT in prosaccade trials when PP is low. In the case of antisaccades, although the mean arrival times of the late unit increased in the PP50 condition, the increased arrival time of the inhibitory unit on the PP80 condition skewed the antisaccade distribution towards higher RTs. Nevertheless, the SERIAlr implies the anticorrelation of late pro- and antisaccades in a single trial type, as these are the results of a GO-GO race. Action inhibition The biological implementation of action inhibition in the antisaccade and other countermanding tasks has received a lot of attention and is still debated [69–73]. Our work adds evidence to the theory that the antisaccade task requires a process that inhibits prepotent responses and is independent of the initiation of a late action [20]. Recent evidence from electrophysiological recordings in the rat brain ([74] reviewed by [71]) suggests that the hypothesized race between PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 30 / 36 SERIA—A model for errors and reaction times in the antisaccade task GO and inhibitory responses might be implemented by different pathways in the basal ganglia [68]. In addition to the basal ganglia, microstimulation of the supplementary eye fields tends to facilitate inhibition of saccades in the countermanding task [75]. Corrective antisaccades Although not a primary goal of our model, we considered the question of predicting corrective antisaccades. This problem has received some attention recently [18,61,65,76], as more sophisticated models of the antisaccade task have been developed. We speculated that corrective antisaccades are generated by the same mechanism as late responses. Thus, their RT distribution should follow a similar distribution. Our results strongly suggest that this is the case (see Fig 11). Moreover, the time delay of the corrective antisaccades indicates that, on average, these actions are not the result of the late unit being restarted at the end time of the erroneous prosaccade, as this would lead to much higher RTs. Rather, the planning of a corrective antisaccade might be started much before the end of the execution of an erroneous prosaccade, in accordance with the parallel planning model of the antisaccade task [46] and the ‘GO–STOP +GO’ model in [21]. Translational applications Despite the large number of studies of clinical patients using the antisaccade task, an important question remains open: What are the causes of the errors in different neurological and psychiatric conditions? For example [77,78] argued that errors in schizophrenia might be explained, at least partially, by a failure to generate a secondary late action based on several modifications of the antisaccade task. However, it was also proposed that the increased ER in schizophrenia is due to high tonic dopamine levels in the basal ganglia, that lead to decreased inhibition of early responses [68]. More generally, different neurological and psychiatric diseases, or even patients with the same condition, might be characterized by a different source of errors. For example, there is intriguing evidence [79] that patients with different diseases such as attention deficits disorders [80], Parkinson’s disease [81], and amyotrophic lateral sclerosis [82] might be characterized by different ratios of early and late errors. An interesting experimental finding in our study related to this is the considerable amount of erroneous antisaccades in prosaccade trials. An increased number of such errors could be caused by reduced cognitive flexibility leading to impaired shifting between tasks as observed for example in obsessive compulsive disorder [83]. The ability to quantify different types of errors through computational modeling might help to further characterize these diseases. Summary Here we have presented a novel model of the antisaccade task. While the basic structure of the model follows the layout of a previous model [17], we have introduced two crucial advancements. First, we postulated that late responses could trigger both pro- and antisaccades, which are selected by an independent decision process. Second, the generative nature of our model allows for Bayesian model inversion, which enables the comparison of different models and families of models on formal grounds. To our knowledge this has not been done for any of the previous models of the antisaccade task, which is of relevance for translational applications that aim at better understanding psychiatric diseases by means of computational modeling. The application of the model to a large data set yielded several novel results. First, the early and inhibitory race processes triggered by different cues are almost identical. Moreover, different PP had very different effects on the individual units, which was not obvious from the linear analysis of the mean RT and ER. Crucially, our modeling approach allowed us to look at a PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005692 August 2, 2017 31 / 36 SERIA—A model for errors and reaction times in the antisaccade task mechanistic explanation or the effects of PP by examining the individual units. In future work we aim to disentangle the mechanisms of behavioral differences caused by neuromodulatory drugs and psychiatric illnesses using formal Bayesian inference. Supporting information S1 Dataset. Table of data. Spreadsheet including all reaction times, actions and errors that entered the analysis. More details are included in the file. (XLSM) S1 Fig. Reaction time and error rate in all conditions for each subject. Mean reaction times and error rates are displayed as solid lines. (TIFF) Acknowledgments We thank Saee Paliwal for her remarks and helpful comments on an earlier version of this manuscript. Author Contributions Conceptualization: Eduardo A. Aponte, Dario Schöbi, Klaas E. Stephan, Jakob Heinzle. Data curation: Eduardo A. Aponte. Formal analysis: Eduardo A. Aponte, Jakob Heinzle. Funding acquisition: Klaas E. Stephan. Investigation: Eduardo A. Aponte. Methodology: Eduardo A. Aponte, Dario Schöbi, Jakob Heinzle. Project administration: Jakob Heinzle. Resources: Klaas E. Stephan. Software: Eduardo A. Aponte, Dario Schöbi. Supervision: Jakob Heinzle. Visualization: Eduardo A. Aponte, Jakob Heinzle. Writing – original draft: Eduardo A. Aponte, Jakob Heinzle. Writing – review & editing: Eduardo A. Aponte, Dario Schöbi, Klaas E. Stephan, Jakob Heinzle. References 1. Hallett PE. Primary and secondary saccades to goals defined by instructions. Vision Res. 1978; 18: 1279–1296. 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https://openalex.org/W2919287833	https://hal.archives-ouvertes.fr/hal-02154636/file/S%C3%A4vilammi_2019_3G_1.pdf	English	null	The Chromosome-Level Genome Assembly of European Grayling Reveals Aspects of a Unique Genome Evolution Process Within Salmonids	G3	2,019	cc-by	11,175	The chromosome-level genome assembly of european grayling reveals aspects of a unique genome evolution process within salmonids Tiina Sävilammi, Craig R. Primmer, Srinidhi Varadharajan, René Guyomard, Yann Guiguen, Simen R. Sandve, L. Asbjørn Vøllestad, Spiros Papakostas, Sigbjørn Lien Tiina Sävilammi, Craig R. Primmer, Srinidhi Varadharajan, René Guyomard, Yann Guiguen, Simen R. Sandve, L. Asbjørn Vøllestad, Spiros Papakostas, Sigbjørn Lien To cite this version: Tiina Sävilammi, Craig R. Primmer, Srinidhi Varadharajan, René Guyomard, Yann Guiguen, et al.. The chromosome-level genome assembly of european grayling reveals aspects of a unique genome evolution process within salmonids. G3, 2019, 9 (5), pp.1283-1294. 10.1534/g3.118.200919. hal- 02154636 Distributed under a Creative Commons Attribution 4.0 International License GENOME REPORT The Chromosome-Level Genome Assembly of European Grayling Reveals Aspects of a Unique Genome Evolution Process Within Salmonids Tiina Sävilammi,* Craig R. Primmer,†,‡ Srinidhi Varadharajan,§ René Guyomard,** Yann Guiguen,†† Simen R. Sandve,‡‡ L. Asbjørn Vøllestad,§ Spiros Papakostas,,1 and Sigbjørn Lien‡‡,1 Department of Biology, University of Turku, 20014 Turku, Finland, †Organismal & Evolutionary Biology Research Program, Faculty of Biological & Environmental Sciences, and ‡Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland, §Department of Biosciences, University of Oslo, 0316 Oslo, Norway, **INRA, UMR1313 GABI Génétique Animale et Biologie Intégrative, Domaine de Vilvert, 78352, Jouy-en-Josas Cedex, France, ††INRA, UR1037 Fish Physiology and Genomics, F-35000, Rennes, France, and ‡‡Centre for Integrative Genetics (CIGENE), Department of Animal and Aquacultural Sciences, Faculty of Biosciences, Norwegian University of Life Sciences, 1430 Ås, Norway ORCID IDs: 0000-0002-2033-0758 (S.V.); 0000-0001-5464-6219 (Y.G.); 0000-0003-4989-5311 (S.R.S.); 0000-0002-9389-7982 (L.A.V.); 0000-0002-5563-0048 (S.P.) ABSTRACT Salmonids represent an intriguing taxonomical group for investigating genome evolution in vertebrates due to their relatively recent last common whole genome duplication event, which occurred between 80 and 100 million years ago. Here, we report on the chromosome-level genome assembly of European grayling (Thymallus thymallus), which represents one of the earliest diverged salmonid subfam- ilies. To achieve this, we ﬁrst generated relatively long genomic scaffolds by using a previously published draft genome assembly along with long-read sequencing data and a linkage map. We then merged those scaffolds by applying synteny evidence from the Atlantic salmon (Salmo salar) genome. Comparisons of the European grayling genome assembly to the genomes of Atlantic salmon and Northern pike (Esox lucius), the latter used as a nonduplicated outgroup, detailed aspects of the characteristic chromosome evolution process that has taken place in European grayling. While Atlantic salmon and other salmonid genomes are portrayed by the typical occurrence of numerous chromosomal fusions, European grayling chromosomes were conﬁrmed to be fusion-free and were characterized by a relatively large proportion of paracentric and pericentric inversions. We further reported on transposable elements speciﬁc to either the European grayling or Atlantic salmon genome, on the male-speciﬁc sdY gene in the European grayling chromo- some 11A, and on regions under residual tetrasomy in the homeologous European grayling chromosome pairs 9A-9B and 25A-25B. The same chromosome pairs have been observed under residual tetrasomy in Atlantic salmon and in other salmonids, suggesting that this feature has been conserved since the sub- family split. KEYWORDS chromosome evolution chromosomal structure genomic rearrangements karyotype evolution retrotransposons Whole genome duplication is known to be an important driver of evolutionary novelty and speciation (e.g., Blomme et al. HAL Id: hal-02154636 https://hal.science/hal-02154636v1 Submitted on 26 May 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License The Chromosome-Level Genome Assembly of European Grayling Reveals Aspects of a Unique Genome Evolution Process Within Salmonids As such, the Atlantic salmon and European grayling genomes represent clearly distinct genome evolutionary processes that have occurred within salmonids, which demands further investigation. The Salmonidae family, also termed salmonid ﬁsh, represents an intriguing model system to study genome evolution following whole genome duplication. Salmonid ﬁsh have in common a whole genome duplication event that has occurred relatively recently, approximately 80-100 million years ago (Allendorf and Thorgaard 1984; Berthelot et al. 2014; Macqueen and Johnston 2014). It is considered that the hypothetical ancestor of salmonids had a typical diploid teleost ge- nome with approximately 50 possibly acrocentric chromosomes, and thus the duplication event resulted in approximately 100 possibly acrocentric chromosomes with tetrasomic inheritance (Phillips and Ráb 2001). Recent evidence suggests that most of the diploid inher- itance has been restored prior to lineage diversiﬁcation, albeit some regions may still be under residual tetrasomy and thus recombining (Berthelot et al. 2014; Lien et al. 2016; Robertson et al. 2017). In this study, we report the ﬁrst chromosomal-level genome as- semblyfortheEuropeangraylinganditsin-depthcomparisonwiththe Atlantic salmon genome. The assembly builds on the recently pub- lishedscaffold-levelassemblyofEuropeangraylingthatwasassembled purely from short-read sequences (Varadharajan et al. 2018). Scaffold- level genome assemblies can provide excellent source materials for chromosomal-level assembly by employing additional data sources such as long reads, linkage mapping, and synteny with closely related species. Annotating and studying this new European grayling genome assembly further revealed novel insights into the genome evolution differences between the European grayling and Atlantic salmon. Present-day salmonids have evolved drastically different karyo- types, whichsuggeststhe occurrence ofverydifferent genomeevolution processes. Lineage diversiﬁcation has resulted in three salmonid subfamilies: Thymallinae, which includes the European grayling (Thymallus thymallus); Coregoninae, which includes round whiteﬁsh (Prosopium spp.), whiteﬁsh and cisco (Coregonus spp. and Stenodus spp.); and Salmoninae, which is the subfamily of the well-studied Atlantic salmon (Salmo salar) as well as Paciﬁc salmon and trout (Oncorhynchus spp.) (Phillips and Ráb 2001; Macqueen and Johnston 2014). These subfamilies are known to radically differ in the number of chromosomes and chromosomal arms (Phillips and Ráb 2001). The European grayling represents an extreme case as it has an excep- tionally high number of chromosomes compared to other salmonids, between 2n = 98 and 2n = 102 depending on the subspecies. The Chromosome-Level Genome Assembly of European Grayling Reveals Aspects of a Unique Genome Evolution Process Within Salmonids 2006; Van de Peer et al. 2009). Whole genome duplication is also regarded as a trigger of certain dramatic consequences in genome evolution (Lien et al. 2016). A tetraploid genome, for instance, is expected to be very unstable due to a variety of reasons including multivalent pairing during meiosis, unequal separation of sister chromosomes during mitosis, and gene dosage imbalances (Comai 2005; Edger and Pires 2009; Hufton and Panopoulou 2009). Chromosomal rearrangements, which are often associated with increased transposable element activity, are expected to be frequent during this period of genomic instability to restore a Whole genome duplication is known to be an important driver of evolutionary novelty and speciation (e.g., Blomme et al. 2006; Van de Peer et al. 2009). Whole genome duplication is also regarded as a trigger of certain dramatic consequences in genome evolution (Lien et al. 2016). A tetraploid genome, for instance, is expected to be very unstable due to a variety of reasons including multivalent pairing during meiosis, unequal separation of sister chromosomes during mitosis, and gene dosage imbalances (Comai 2005; Edger and Pires 2009; Hufton and Panopoulou 2009). Chromosomal rearrangements, which are often associated with increased transposable element activity, are expected to be frequent during this period of genomic instability to restore a disomic inheritance of chromosomes, which is also called a redi- ploidization process (Ohno 1970; Semon and Wolfe 2007; Hufton and Panopoulou 2009; Lien et al. 2016). Also possibly driven by trans- posable element activity, chromosomal fusions, ﬁssions, inversions and indels can suppress, for example, multivalent pairing, and they are also expected to lead to sequence divergence and genome evolu- tion causing genomic incompatibilities among populations thus rais- ing species barriers (Rieseberg 2001; Hoffmann and Rieseberg 2008; Makhrov 2017). To this end, sequencing and comparing the genomes of taxa with a recent common genome duplication event that have evolved radically different karyotypes holds the promise to illuminate Volume 9 \| May 2019 \| 1283 questions regarding the evolutionary consequences of various types of chromosomal rearrangements (e.g., Charlesworth 2016; Wellenreuther and Bernatchez 2018). also large and metacentric (from ssa01 to ssa07) or large and acro- centric (from ssa09 to ssa20) and are thought to have resulted from Robertsonian fusions of ancestral chromosomes, that is, a fusion of two acrocentric chromosomes at their centromeres (Phillips and Ráb 2001; Lien et al. 2016). 1Corresponding authors: Centre for Integrative Genetics (CIGENE), Department of Animal and Aquacultural Sciences, Faculty of Biosciences, Norwegian University of Life Sciences, Arboretveien 6, 1430 Ås, Norway. E-mail: sigbjorn.lien@nmbu.no; Department of Biology, University of Turku, Vesilinnantie 5, 20014 Turku, Finland. E-mail: spiros.papakostas@utu.ﬁ, spiros.papakostas@gmail.com Copyright © 2019 Savilammi et al. doi: https://doi.org/10.1534/g3.118.200919 Manuscript received December 13, 2018; accepted for publication March 4, 2019; published Early Online March 4, 2019. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Supplemental material available at Figshare: https://doi.org/10.25387/ g3.7728512. 1 Copyright © 2019 Savilammi et al. doi: https://doi.org/10.1534/g3.118.200919 Manuscript received December 13, 2018; accepted for publication March 4, 2019; published Early Online March 4, 2019. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Supplemental material available at Figshare: https://doi.org/10.25387/ g3.7728512. 1Corresponding authors: Centre for Integrative Genetics (CIGENE), Department of Animal and Aquacultural Sciences, Faculty of Biosciences, Norwegian University of Life Sciences, Arboretveien 6, 1430 Ås, Norway. E-mail: sigbjorn.lien@nmbu.no; Department of Biology, University of Turku, Vesilinnantie 5, 20014 Turku, Finland. E-mail: spiros.papakostas@utu.ﬁ, spiros.papakostas@gmail.com The Chromosome-Level Genome Assembly of European Grayling Reveals Aspects of a Unique Genome Evolution Process Within Salmonids The number of European grayling chromosomes has thus remained ap- proximately the same as the number of chromosomes from the an- cestral salmonid genome straight after the salmonid-speciﬁc whole genome duplication (Phillips and Ráb 2001). European graylings also have an exceptionally high number of chromosomal arms, up to 170, which is considered to represent a marked increase over the assumed 100 arms of the hypothetical ancestral duplicated genome of salmo- nids (Phillips and Ráb 2001; Ocalewicz et al. 2013). This is assumed to be a consequence of pericentric inversions, that is, inversions con- taining the centromere of the ancestral acrocentric chromosomes (Phillips and Ráb 2001; Ocalewicz et al. 2013). The rest of the salmo- nid species have at least a third fewer chromosomes, with Atlantic salmon at the lower end of the distribution with a karyotype of n = 27 and n = 29 chromosomes in the North American and European clade, respectively, and the number of chromosomal arms as low as 72 (Phillips and Ráb 2001). Many Atlantic salmon chromosomes are MATERIAL AND METHODS Assembling the European grayling genome at the chromosome level To identify the RAD markers, we sorted the trimmed reads to sep- arate ﬁles according to barcode, removed the barcode sequence, and veriﬁedthe restrictionsitesequenceusinganin-housePerl script named barcodesplitter5.3.pl(availableinGitHub).Thereadswerethenmapped to the hybrid assembly using the Bowtie2 tool (Langmead and Salzberg 2012). Polymorphic sites were ﬁltered using the following criteria: (a) polymorphisms in parental ﬁsh were considered valid only if they were found present in fragments between 182 and 186 base pairs long (frag- ment extending to both sides from a restriction site) and had read coverage between 9 and 300 per base in both parents; (b) polymorphic sites were retained for linkage mapping when at least one of the parents was heterozygous, the polymorphism was biallelic in the offspring, and the offspring genotype distribution followed a Mendelian segregation pattern as tested by chi-square tests at 5% signiﬁcance level following correction by false discovery rate according to the Benjamini and Hochberg approach (Benjamini and Hochberg 1995). Additionally, offspring were retained in the analysis if they had at least 1,000 markers genotyped with .8 read coverage, a criterion which resulted in removal of four offspring from the analysis. Polymorphic site ﬁltering was com- pleted using R (script “RADstats_ﬁnal.R”; available in GitHub). The ﬁltered markers were mapped to linkage groups and ordered using the Lep-MAP2 software (Rastas et al. 2016). Linkage between markers was accepted at LOD $ 9, upon which additional individual markers were added at LOD $ 7. parsimonious block order, we then inverted each block and tested whether it further reduced the map length. At the end of this step we validated the ﬁnal combination of rearrangements by testing if implementing all accepted changes indeed resulted in the minimal map length. The salmonization script “salmonize_ﬁnal.R” is available in GitHub. As the ﬁnal step, all maps were further manually curated with special attention given to regions that are known to have .90% sequence similarity in Atlantic salmon, namely, the pairs of Atlantic salmon chro- mosome arms including 2p-5q, 2q-12qa, 3q-6p, 4p- 8q, 7q-17qb, 11qa-26 and 16qb-17qa (Lien et al. 2016). European grayling linkage groups corresponding to salmon chromosome arm pairs 3q-6p, 7q-17qb and 11qa-26 mapped equally well to both of their Atlantic salmon homeo- log counterparts. Assembling the European grayling genome at the chromosome level Assembly of genomic scaffolds using long-read sequence data: Using the PacBio RS2 platform, we sequenced the same DNA sample used in the recently published European grayling genome assembly (Varadharajan et al. 2018) at approximately 19x depth. The sample belonged to a single male adult ﬁsh caught from the River Glomma at Evenstad, Norway (61.42 N 11.09 E) that was killed in October 2012. The sequencing effort resulted in a total of 40 gigabase pairs of se- quence information. PacBio reads with length .5 kilobase pairs were then processed to consensus sequences using the Canu assembler (Koren et al. 2017). The resulting reads with length .10 kilobase pairs, amounting to approximately 5x depth, were used in a hybrid assembly. The PacBio reads and the previously published Illumina- based assembly (Varadharajan et al. 2018) were merged together using the PBJelly2 suite (English et al. 2012) using the noSplitSubreads, minMatch 8, minPctIdentity 70, bestn 1 and maxScore 11 parameters. Basic statistics, such as N50, L50 and the length range of the assembled sequences were calculated for each assembly using an in-house de- veloped script (contig_statistics.pl; available in GitHub). After initial mapping of the assembled scaffolds to the Atlantic salmon genome assembly (described in more detail in the linkage mapping section) and manual curation, some of the assembled scaffolds were split in cases of potential sequencing or assembly errors. Linkage mapping: Male- and female-based linkage maps were built using markers from a single European grayling family originating from the Rhine River (Obenheim, France) that included both parents, 69 female offspring and 44 male offspring that were sequenced using a restriction site associated DNA (RAD) methodology according to previously described protocol (Amores et al. 2011). The RAD frag- ments were produced by using the SbfI restriction enzyme and were sequenced using 100 base pair single-end sequencing using the Illu- mina HiSeq 2500 platform. Quality trimming of the sequence reads was performed with ConDeTri v. 2.3 (Smeds and Kunstner 2011). The RAD data consisted of a total of 4,167,787 and 7,056,371 reads for the male and female parents, respectively, and an average of 4,041,607 reads for each offspring. Scaffolds containing at least one marker covered 54% of the total length of the hybrid assembly. 1284 \| T. Sävilammi et al. Assembling the European grayling genome at the chromosome level To identify the true homologs in these linkage groups, markers were aligned separately and ordered based on each of the Atlantic salmon homeologs using nucmer and LepMap2, and the best ordering homeolog was chosen as the linkage group identity. Linkage groups corresponding to Atlantic salmon chromosome arms 2q-12qa and 4p-8q had fused linkage maps that could not be separated. Synteny-assisted genome scaffolding: The Europeangraylingscaffolds that contained markers in the ﬁnal linkage groups and the scaffolds that had a MUMmer-alignment-based position in the Atlantic salmon genome were arranged into the ﬁnal European grayling chromosomal order based on synteny with Atlantic salmon chromosomes unless there was strong evidence of a rearrangement based on the European grayling linkage map position. The alignment with Atlantic salmon was also used to orientate the scaffolds. Scaffolds were then concat- enatedintochromosome-levelsequenceassembliesbyadding100base pair gaps between each adjacent scaffold. The linkage groups were initially constructed based on recombi- nation frequencies and thereafter improved by testing the alternative ordering of markers using comparative mapping information from Atlantic salmon, a procedure hereafter referred to as salmonization. The latter was performed by ﬁrst mapping the scaffolds from the European grayling hybrid assembly to the Atlantic salmon genome assembly (Lien et al. 2016), downloaded from the NCBI Genome database (RefSeq assembly GCF_000233375.1), using the nucmer tool in MUMmer 3.0 aligner (Kurtz et al. 2004). Prior to the alignment, Atlantic salmon chromosome sequences were repeat-masked using a salmon repeat database (ssal_repeats_v2.0) and RepeatMasker v4.0.3 (Smit et al. 2013–2015). The best matching position for each European grayling scaffold in the Atlantic salmon genome was determined by adding up the number of base pairs in each hit and the number of hits within a scaffold. Second, for each linkage group, markers mapping to the most frequently associated Atlantic salmon chromosome were included in the further salmonization procedure. At each step, the correct map was assumed to be the one with the shortest female map length calculated using Lep-MAP2 (Rastas et al. 2016). During the ﬁrst step, the markers in each linkage group were initially reordered according to their locations in the Atlantic salmon assembly, and the resulting map lengths were calculated. In the second step, we inves- tigated the salmonized European grayling linkage maps where breaks in the progression of map length increase indicated possible genomic rearrangements between the European grayling and Atlantic salmon genomes (Fig. S1). Volume 9 May 2019 \| European Grayling Genome Assembly \| 1285 Assembling the European grayling genome at the chromosome level We then considered a portion of the largest breaks by applying either of the two following criteria: (a) the absolute map length difference of the break is . 10 map units for any of the markers or (b) the break length is at least eight times the standard deviation of that of all pairwise differences in adjacent markers in that linkage group. This step led to the identiﬁcation of one to eight blocks of orderly progressed markers per linkage group. In the third step, we investigated the possibility of translocations from these blocks explaining our observations. To do this, we reconstructed each link- age group by permuting the order of the corresponding blocks and selecting the solution with the minimal length as the most parsimo- nious block order. To test the possibility of inversions in the most Genome repetitiveness and repeat element assessment Kmer repetitiveness of the previously published and current genome assemblies of European grayling, Atlantic salmon and rainbow trout (Oncorhynchus mykiss) were calculated using Jellyﬁsh software v. 1.1.11 (Marçais and Kingsford 2011) using kmer size of 31. Repetitiveness was calculated by dividing counts of non-unique kmers by total kmers in the assembly. To investigate the European grayling genome in terms of repeat elements, the European grayling repeat library, containing 1,743 de novo repeats, was employed along with the repeats from RepBase v. 20.05 (Jurka et al. 2005). Transposable element sequences were curated by ﬁrst detecting the host genes that were potentially of non-transposable element origin and then classifying the remaining transposable element sequences according to the classiﬁcation system of Wicker et al. (2007). Transposable element abundances were esti- mated for both European grayling and Atlantic salmon. To remove from the ﬁnal repeat analysis any repeats that potentially originated from host genes instead of transposable elements, the transposable element sequences were compared to two different repeat databases. These databases were the REPET-formatted RepBase v. 20.05 (Jurka et al. 2005) and the Swiss-Prot database available in UniProt (as of June 1, 2018). Comparisons were conducted by using the blastn (Altschul et al. 1990) and blastx (Gish and States 1993) algorithms with parameters set to word_size = 7, and to e-value . 1·10210. A custom script named best_multi_blast_score_parser.pl (available in GitHub) was used to select the highest scoring hits for each potential transposable element sequence. Based on the best-scoring hits, each transposable element sequence was categorized as non-transposable element derived host gene and removed if it had a best-scoring hit to a Swiss-Prot sequence. The rest of the library hits were kept for further analysis. To classify the transposable element sequences, they were com- pared to the RepBase repeats using both nucleotide sequence and protein similarity. To categorize transposable element sequences to class, order, and superfamily levels, the relevant information from RepBase was used in case a sequence had an acceptable alignment hit with this database. An alignment was accepted if it suggested high similarity between query and reference repeat, deﬁned by Wicker et al. (2007). A high similarity alignment was at least 80 base pair long with at least 80% sequence similarity between query and reference repeat sequence, occupying at least 80% of the query repeat length (which we calculated after removing unknown nucleotides from the query sequence length). Genome repetitiveness and repeat element assessment These thresholds concerned the blastn search. In case of a non-acceptable nucleotide alignment for a transposable element sequence, then this sequence was searched against the RepBase database using the blastx approach, with an alignment considered valid if the hit had e-value , 1·10210 (following Lien et al. 2016). The repeat element abundance in the European grayling and Atlantic salmon genomes was assessed for each chromosome separately using the RepeatMasker v. 4.0.7 tool (Chen 2004) by using the parameter -qq. The RepeatMasker-based locations of transposable element sequences Identiﬁcation of the European grayling sex chromosome The gene named sdY for sexually dimorphic on the Y-chromosome was searched using a tblastn (Altschul et al. 1990) homology search against the European grayling chromosome assembly. The rainbow trout sdY protein sequence (GenBank: BAM24747.1) was used as bait in this search. The RAD sequences were then remapped to the chromosome-level assembly and sex-biased loci were detected from the chromosomes. Repeat library construction and genome annotation MAKER was run with default options. Functional annotation was added to the MAKER-predicted gene modelsusingBLASTagainstUniProtdatabaseanddomaininformation was added using InterProScan (Zdobnov and Apweiler 2001). MAKER- predicted gene models were then ﬁltered based on Annotation Edit Distance (AED) and the presence of known PFAM domains to retain high conﬁdence set of genes. Predicting centromere locations using the location of repeats: Repetitive element content can reveal information about the chromo- somal landscapes (Kaminker et al. 2002; Lien et al. 2016). To estimate the repeat content, copies of the generated repeat library were sought from the European grayling chromosomes using RepeatMasker. The abundances of different element classes were quantiﬁed using local regression for element abundancy over each chromosome with the R function lowess with parameter f = 0.2, and the maximum position for each element class in each of the European grayling chromosomes was extracted. These maximal density locations in each chromosome were analyzed using principal component analysis. Although centro- meres are generally epigenetic structures that cannot be observed from the nucleotide order, some transposable elements have a tendency to accumulate in certain region of the genome (Daron et al. 2014). This has been previously observed in the Atlantic salmon genome (Lien et al. 2016) where Tc1-Mariner type elements were shown to accumu- late in the centromeric regions. The chromosomal positions with the maximal abundancy of the two element classes, the centromere-related Tc1-mariner, and the most contrasting element class RTE-X were more closely inspected using the occurrences of each of the two element classes in 100 kilobase pair bins and local regression. Hypothetical centromere loci for each chromosome were predicted using the max- imal estimates of the Tc1-Mariner-richness from the local regression curves. To predict the karyotype, the long:short arm ratio was estimated for each chromosome (following Levan, Fredga and Sandberg 1964). This was performed using the peak position of the Tc1-mariner element to calculate the length of the chromosomal fragments on both sides of the peak and dividing the longer length by the shorter one. Finally, the effect of chromosome size and karyotype on re- combination frequencies was estimated using the linear model map length chromosome size + long:short arm ratio. To validate the effect of the long:short arm ratio in the full model, the chi-square test was performed to compare the full model to a reduced model with chromosome size as the only independent variable. 1286 \| T. Sävilammi et al. Repeat library construction and genome annotation p y g A comprehensive repeat library was built by combining de novo iden- tiﬁed European grayling-speciﬁc repeats as well as repeat elements identiﬁed in the Atlantic salmon genome (available at: http://web.uvic.ca/ grasp/salmon_v1.6). We initially ran the RepeatModeler software v. 1.0.11 (available at: http://www.repeatmasker.org/RepeatModeler; last accessed June 8, 2018) with default parameters. To compile a set of LTRs, we used the LTRharvest (Ellinghaus et al. 2008) and LTRdigest (Steinbiss et al. 2009) software as described in (http://weatherby.genetic- s.utah.edu/MAKER/wiki/index.php/Repeat_Library_Construction- Advanced; last accessed: June 8, 2018) and combined the results with the sequences identiﬁed by MGEscan-LTR (Lee et al. 2016). All the identiﬁed sequences were combined and ﬁltered to remove redun- dancy. The resulting de novo set of sequences was combine-queried against the Universal Protein Resource database (UniProt proteins release 2017_08, Consortium 2017) to ﬁlter out any known proteins sequences. The remaining unclassiﬁed set of sequences was then annotated using RepeatClassiﬁer, the Dfam database and TEclass (Abrusán et al. 2009). An updated reference-based set of transcripts was constructed by ﬁrst aligning the RNAseq reads to the improved assembly using STAR v. 2.6 (Dobin et al. 2013) followed by Cufﬂinks (Trapnell et al. 2010) for the prediction of transcript sequences. This along with the de novo assembled transcriptome described in Varadharajan et al. (2018) was used as an input to the PASA pipeline (Haas et al. 2003) to build a comprehensive transcript database. Further, predictions from ab initio gene ﬁnders like SNAP (Korf 2004) and GeneMark-ES (Lomsadze et al. 2005) were also used as input into MAKER v. 2.31.9 (Cantarel et al. 2008). MAKER pipeline was run for two iterations with transcript evidence from PASA transcriptome assembly and protein coding sequences from the Atlantic salmon, GTF Volume 9 May 2019 \| European Grayling Genome Assembly \| 1285 1285 in each of the two genomes were annotated with a script named “classifyGoodTEHits.R” (available in GitHub). The elements with marked difference in their abundance between the two genomes were sought out by using a linear model log2(salmon abundancy+1) log2(grayling element abundancy+1) using R (v. 3.4.0, R Core Team 2017) and elements that had residuals larger than 1.96 standard deviations from zero were considered outliers, that is, outside the 95% conﬁdence interval limits. outputs from AUGUSTUS and GeneMark-ET resulting from BRAKER (Hoff et al. 2016),the UniProt database (UniProt proteins release 2017_08, Consortium 2017) as the protein evidence and the above described repeat library. Chromosome-level European grayling genome assembly Assembly of genomic scaffolds using long-read data: By adding the PacBio long-read sequences to the published draft assembly of Varadharajan et al. (2018) and splitting 23 contigs that were deter- mined as chimeric by initial comparison to the Atlantic salmon genome assembly, we obtained a 38% increase in N50 with a 24% decrease in L50, a 62% increase in the maximum scaffold length, and a 16% increase in the total assembly length (TABLE 1). Altogether, the total number of scaffolds decreased by 25%, while the overall GC content remained almost identical at approximately 43% (TABLE 1). Linkage mapping: RAD sequencing resulted in 7,684 informative SNP markers with a median female: male ratio of map distances at 1.75:1 (sd= 3.05). Postﬁltering, 6,076 markers were assigned to the ﬁnal linkagegroups(TABLES1).Theﬁnalfemale-basedtotalmaplengthwas 3,044 centi-Morgan (cM) (Fig. S2 and TABLE S2). The average female map length per million chromosomal base pairs was 2.0 map units (TABLE S1). Predicting centromere locations using the location of the repeats: We considered the Tc1-Mariner abundance to peak around the cen- tromeric regions (Figure 1). A LINE-class RTE-X retrotransposon was also found located the furthest from Tc1-Mariner excluding some unknown and simple repeat categories, suggesting a potential subtelomeric enrichment (Fig. S3). Using the Tc1-Mariner abundances we predicted 29 telocentric and 22 sub(metacentric) karyotypes (Figure 1). After correcting for the chromosome size in megabases, the long:short arm ratio had a negative correlation with the female map length Synteny-assisted genome chromosome building: Thesynteny-assisted chromosome building step represented the biggest improvement in the n Table 1 Assembly properties of different stages of the European grayling genome assembly process. Stage 1 represents the assembly built using only short-read DNA sequencing data (from Varadharajan et al. 2018). Stage 2 involves the outcome of the hybrid assembly process, which combined short- and long-read DNA sequencing data. Finally, stage 3 corresponds to the complete genome assembly that was produced using the linkage mapping data and synteny information with the Atlantic salmon genome. Comparison to the Northern pike genome We compared the obtained European grayling chromosomes to the chromosomes of Northern pike (Esox lucius), a species that represents the closest sister group to Salmonids prior to the salmonid-speciﬁc whole genome duplication with an available genome assembly. The genome assembly was downloaded from the NCBI Genome database (RefSeq assembly GCA_000721915.1). Conserved synteny between 1286 \| T. Sävilammi et al. European grayling and Northern pike was determined by aligning European grayling and Northern pike chromosome sequences using the nucmer tool in MUMmer 3.0 aligner (Kurtz et al. 2004) and keeping hits with identity $80.0 and matchcount $100. Homeolo- gous European grayling chromosome pairs were named according to the Northern pike chromosome naming convention (Rondeau et al. 2014, Sutherland et al. 2016). genome assembly process (TABLE 1). Together with the linkage map- ping information, we managed to reconstruct all 51 European grayling chromosomes (Figure 1). Nevertheless, of the total of 18,265 scaffolds from the hybrid stage of the assembly, a large number of relatively small-sized scaffolds were left unassigned (N = 8,938 scaffolds ranging in length from 984 to 1,162,211 base pairs of which N = 3,780 scaf- folds with . 2,000 base pairs length are available at NCBI), which corresponded to a total length of 91,704,787 unassigned base pairs (or 5.8% of the total genome assembly length). Genome repetitiveness and repeat element assessment Genome repetitiveness and repeat element assessment The repetitiveness of identical sequences in the chromosome-level European grayling assembly was estimated at 11.9%. By comparison, we estimated repetitiveness at 29.3% in the Atlantic salmon genome and 9.8% in the reported genome assembly of the rainbow trout using the same software. The RepBase and Swiss-Prot searches resulted in a best-scoring hits list including 1,090 transposable element sequences, of which 32 potential host genes were removed from further transposable element analysis. Of the remaining transposable element sequences, 287 had nucleotide-based matches and 434 had protein-based matches to RepBase after ﬁltering. After the matches were combined, they contained 586 unique transposable element sequences. These clas- siﬁed transposable element sequences covered 83% of the European grayling repeat sequences. Altogether, 47.4% of the European gray- ling genome assembly and 52.3% of the Atlantic salmon genome assembly were covered by these repeats, which could be distinguished into 24 superfamilies of transposable elements(TABLE 2). Of the repeat elements identiﬁed, 14 were only found in the Atlantic salmon genome assembly, while only three were more abundant in the European gray- ling genome assembly (TABLE S3 and Figure 3). Data availability The PacBio reads, chromosome-level sequences and unmapped scaf- folds over than 2000 base pairs have been deposited at NCBI SRA and GenomesunderBioProjectIDPRJNA464295.Scriptshavebeendeposited to GitHub under the link https://github.com/tiinasa/graylinggenome. Supplemental material available at Figshare: https://doi.org/10.25387/ g3.7728512. Volume 9 May 2019 \| European Grayling Genome Assembly \| 1287 Volume 9 May 2019 \| European Grayling Genome Assembly \| 1287 DISCUSSION bl h By assembling the European grayling genome up to the level of chro- mosomes and comparing it with that of Atlantic salmon, we provided some novel insights about the very distinct genome evolution processes that have been ongoing in European grayling (Phillips and Ráb 2001) and, in particular, we reported on the complete absence of chromo- somal fusions and the somewhat frequent occurrence of chromosomal inversions (Figure 2). The absence of a fusion event in European grayling could be parsimoniously hypothesized by observing that the European grayling homeologue chromosomes always mapped to single Northern pike chromosomes (TABLE S1). This ﬁnding sug- gests the absence of chromosomal fusions in either of these two genomes since the last whole genome duplication event. Notably, a single case of chromosomal ﬁssion was observed behind the generation Chromosome-level European grayling genome assembly Numbers in brackets represent the percent increase/ decrease over the previous stage for given statistics Statistic Stage 1 Stage 2 Stage 3 N50 283,328 390,289 (38%) 33,018,251 (8,340%) L50 1,359 1,030 (-24%) 20 (-98.5%) N90 38,415 49,679 (29%) 23,618,429 (47,442%) L90 6,620 5,397 (-18%) 40 (-99.4%) Scaffolds 24,369 18,265 (-25%) 51 (-99.7%) Length Total Average sd min max 1,468,519,221 1,575,987,192 1,485,210,005 60,261 86,285 (43%) 29,121,765 (33,651%) 145,243 207,343 9,938,557 975 984 (1%) 6,483,087 (658,750%) 2,502,076 4,048,953 (62%) 44,988,017 (1,011%) Known bases 87% 95% 95% GC% 42.7% 42.8% 42.7% Volume 9 May 2019 \| European Grayling Genome Assembly \| 1287 Figure 1 The European grayling ge- nome. The tracks indicate the following information: (A) Chromosome number according to Northern pike, used as a non-WGD outgroup, and predicted chro- mosomal type (M = metacentric; SM = submetacentric; and T = acrocentric); (B) Predicted centromere (in purple) and telomere (in yellow) locations; (C) Pro- portions of the Tc1-mariner (in purple) and RTE-X elements (in yellow), which are used to predict centromere and telo- mere positions, respectively; (D) Gene density; (E) Female linkage map units; and (F) Female- (in red) and male-biased (in blue) sex-biased loci with the sdY gene region indicated. on 11A and four on 11B. Additionally, three sex-biased loci were more randomly distributed in the genome, more speciﬁcally on chromosomes 3A and 18A and in the scaffold Contig7739 (Figure 1). (estimate -1.83, P = 0.0335, F(2,45)=26.94, adjusted R-squared = 0.5246 for the whole model). The chi-square test conﬁrmed that the long:short arm ratio was indeed a signiﬁcant variable (P = 0.0283) when predicting the map length of a chromosome. This result sug- gests that the metacentric chromosomes have a relatively higher recombination rate than comparably sized telocentric chromosomes. Comparisons with the genome of Northern pike The European grayling chromosomes could be matched to the Northern pike counterparts in a 2:1 ratio except for one ancestral- duplicated chromosome that had been subsequently split in two in European grayling (13A and 13C) (TABLE S1). European gray- ling chromosomes were named according to the corresponding pike orthologs (TABLE S1). Genome rearrangements between European grayling and Atlantic salmon: Rearrangements between European grayling and Atlantic salmon chromosomes suggested conservation of the synteny within blocks of chromosome arms in both of the species, but with frequent chromosomal inversions observed between the two genomes. In particular, we detected 119 blocks from which we could conﬁdently interpret18aspericentricand24asparacentricinversionsinEuropean grayling (Figure 2, TABLE S4, and Fig. S4). Compared to Atlantic salmon, in which many chromosomal fusions have occurred after the tetraploid salmonid ancestor, the ancestral chromosome identities were conserved in European grayling with the exception of one chro- mosomal ﬁssion that was noticed (Figure 2). Identiﬁcation of the European grayling sex chromosome The relatively frequent occurrence of chromosomal inversions in the European grayling lineage was also conﬁrmed by identifying at least 51 inversions between the European grayling and Atlantic salmon genomes, which covered as much as 45% of the total length of the European grayling genome assembly (TABLE S4). Detailing the origin of these inversions, whether speciﬁc to European grayling or to Atlantic salmon, proved to be a challeng- ing task. Comparisons of the order of available markers between the genomes of these two species and that of Northern pike are often problematic due to the loss of synteny within chromosome arms and smaller number of European grayling scaffolds reliably positioned in the Northern pike genome (Fig. S4). Nevertheless, for nine of the inversions, it was clear that six were speciﬁc to the European grayling genome and three were speciﬁc to the Atlantic salmon genome (TABLE S4). Thus, chromosomal inversions appear to have played a role in the genome evolution process in both of these species, albeit more frequently identiﬁed in the European grayling genome. A relatively large number of the recognized inversions, 18 out of the 42 resolved cases, were also found to be pericentric, that is, inversions that included the centro- mere (TABLE S4). Taken together, these ﬁndings may also explain the relatively large number of chromosomal arms observed in European grayling, as a pericentric inversion of the assumedly acrocentric an- cestral chromosome would double the number of chromosomal arms (Phillips and Ráb 2001). g y Additionally, the comparison of abundances of the transposable elements between European grayling and Atlantic salmon resulted in the recognition of 14 Atlantic salmon-speciﬁc and three European grayling-speciﬁc transposable elements (Figure 3 and TABLE S3). The Atlantic salmon-speciﬁc elements include the DNA transposons DNA4-1, DNA4-2, DNA4-2B, DNA4-2C and DNA4-8, which alto- gether covered 80 megabase pairs (3.57%) in the Atlantic salmon genome, but were found to be completely absent in European grayling (TABLE S3). The DNA transposons Mariner-16, Mariner-20, and Mariner-28 were also found to be Atlantic salmon-speciﬁc (Figure 3 and TABLE S3). These elements belong to the same Tc1-Mariner superfamily, which represents one of the most abundant categories of transposable elements in salmonids, with a major suspected role in Transposable elements may have played a key role in genome evolution processes (Auvinet et al. 2018). Identiﬁcation of the European grayling sex chromosome Comparisons of the order of available markers between the genomes of these two species and that of Northern pike are often problematic due to the loss of synteny within chromosome arms and smaller number of European grayling scaffolds reliably positioned in the Northern pike genome (Fig. S4). Nevertheless, for nine of the inversions, it was clear that six were speciﬁc to the European grayling genome and three were speciﬁc to the Atlantic salmon genome (TABLE S4). Thus, chromosomal inversions appear to have played a role in the genome evolution process in both of these species, albeit more frequently identiﬁed in the European grayling genome. A relatively large number of the recognized inversions, 18 out of the 42 resolved cases, were also found to be pericentric, that is, inversions that included the centro- mere (TABLE S4). Taken together, these ﬁndings may also explain the relatively large number of chromosomal arms observed in European grayling, as a pericentric inversion of the assumedly acrocentric an- cestral chromosome would double the number of chromosomal arms (Phillips and Ráb 2001). the comparison of transposable element classes between organisms with very different genomic rearrangements, such as between European grayling and Atlantic salmon, may be of interest. We found that retrotransposons (class I transposable elements) are more abundant compared to DNA transposons (class II transposable elements) with 1.7 times and 1.3 times higher abundance in the European grayling and Atlantic salmon genomes, respectively (TABLE 2). This is similar to what has been observed in the genomes of a wide variety of other eukaryotes, such as many plants, insects, amphibians, reptiles, and mammals, which were found to have a relatively higher proportion of retrotransposons than DNA transposons (reviewed in Canapa et al. 2015). Nevertheless, this is different from what has been found in many non-salmonid ﬁsh, which were found to have DNA transpo- sons as the most abundant class (Canapa et al. 2015). The differential accumulation of transposable elements between lineages may be play- ing a signiﬁcant role in genome evolution processes, but unraveling the complexity of underlying reasons behind such differences could not be investigated in this study. of European grayling chromosomes 13A and 13C (TABLE S1). The absence of fusions agrees with previous hypotheses based on karyo- type information (Phillips and Ráb 2001) and is conﬁrmed for the ﬁrst time at the sequence level. Volume 9 May 2019 \| European Grayling Genome Assembly \| 1289 Identiﬁcation of the European grayling sex chromosome The sdY locus was mapped to 11A [2,137,039-2,136,679]; (e-value = 1.74E-54, score = 190). After remapping the RAD reads, we found the sex-biased loci to be clearly enriched at European grayling 11A close to the sdY locus and in the 11B homeolog, with four sex-biased loci found 1288 \| T. Sävilammi et al. n Table 2 Transposable element classiﬁcation and abundances in the European grayling and Atlantic salmon genomes Transposable element European grayling Atlantic salmon Class Order Superfamily base pairs % coverage base pairs % coverage RNA- transposons (class I) LINE Jockey 133311368 9.0 228445871 10.2 RTE 10629973 0.7 12021858 0.5 L1 6987652 0.5 15019466 0.7 I 1925092 0.1 2994056 0.1 LTR Gypsy 88958008 6.0 120880877 5.4 ERV 23336303 1.6 29951691 1.3 Bel-Pao 4559403 0.3 5228163 0.2 Copia 1009467 0.1 3887925 0.2 SINE tRNA 6099534 0.4 12208774 0.5 Unknown 439123 0.0 1325090 0.1 DIRS DIRS 6352447 0.4 13786775 0.6 PLE Penelope 1392065 0.1 1728304 0.1 Unknown Unknown 102556 0.0 157518 0.0 DNA- transposons (class II) TIR Tc1-Mariner 143125085 9.6 226246051 10.1 hAT 18745228 1.3 26354108 1.2 CACTA 1063048 0.1 1575430 0.1 PIF-Harbinger 606932 0.0 788870 0.0 PiggyBac 237022 0.0 555705 0.0 Unknown Unknown 5627491 0.4 90211317 4.0 Sola 148327 0.0 373468 0.0 Ginger1 59568 0.0 66177 0.0 ISL2EU 37185 0.0 0 0.0 Crypton Crypton 1763736 0.1 2838181 0.1 Maverick Maverick 4732 0.0 147407 0.0 Unknown 247405186 16.7 374107217 16.7 Total repeat coverage 703926531 47.4 1170900299 52.3 posable element classiﬁcation and abundances in the European grayling and Atlantic salmon genomes of European grayling chromosomes 13A and 13C (TABLE S1). The absence of fusions agrees with previous hypotheses based on karyo- type information (Phillips and Ráb 2001) and is conﬁrmed for the ﬁrst time at the sequence level. The relatively frequent occurrence of chromosomal inversions in the European grayling lineage was also conﬁrmed by identifying at least 51 inversions between the European grayling and Atlantic salmon genomes, which covered as much as 45% of the total length of the European grayling genome assembly (TABLE S4). Detailing the origin of these inversions, whether speciﬁc to European grayling or to Atlantic salmon, proved to be a challeng- ing task. Identiﬁcation of the European grayling sex chromosome Additionally, these ele- ments may be important in the rediploidization process by generating sequence divergence that would separate the homeologs. In particular, Volume 9 May 2019 \| European Grayling Genome Assembly \| 1289 1289 the Atlantic salmon rediploidization process (Lien et al. 2016). An- other case of an Atlantic salmon-speciﬁc element is the Copia-12 retrotransposon (Figure 3 and TABLE S3), which belongs to the Copia superfamily of retrotransposons that was recently suggested to have a role in chromosomal diversiﬁcation and speciation in other teleost ﬁshes (Auvinet et al. 2018). Among the European grayling-speciﬁc elements, hAT-10 from the hAT DNA transposon superfamily covered 123,702 base pairs (0.01%) of the assembly and was completely absent in the Figure 2 Chromosomal rearrangements between the European grayling and Atlantic salmon genomes. Each box represents an Atlantic salmon chromosome depicted on the right side of the box. The corresponding European grayling chromosomes are shown in different colors on the left side. Horizontal lines represent corresponding positions between the two genomes, with sequence identity depicted in a bluered scale on the right side of the box. Purple and yellow circles depict predicted centromere and telomere positions in the European grayling chromosomes, respectively. The axes scales represent the log2-transformed abundance in base pairs +1. Figure 2 Chromosomal rearrangements between the European grayling and Atlantic salmon genomes. Each box represents an Atlantic salmon chromosome depicted on the right side of the box. The corresponding European grayling chromosomes are shown in different colors on the left side. Horizontal lines represent corresponding positions between the two genomes, with sequence identity depicted in a bluered scale on the right side of the box. Purple and yellow circles depict predicted centromere and telomere positions in the European grayling chromosomes, respectively. The axes scales represent the log2-transformed abundance in base pairs +1. Figure 2 Chromosomal rearrangements between the European grayling and Atlantic salmon genomes. Each box represents an Atlantic salmon chromosome depicted on the right side of the box. The corresponding European grayling chromosomes are shown in different colors on the left side. Horizontal lines represent corresponding positions between the two genomes, with sequence identity depicted in a bluered scale on the right side of the box. Purple and yellow circles depict predicted centromere and telomere positions in the European grayling chromosomes, respectively. The axes scales represent the log2-transformed abundance in base pairs +1. the Atlantic salmon rediploidization process (Lien et al. 2016). 1290 \| T. Sävilammi et al. Identiﬁcation of the European grayling sex chromosome While highly accurate in repeat identiﬁcation and suitable for our purpose to sim- ply compare the element abundances between European grayling and Atlantic salmon, conventional software such as RepeatMasker that we applied has been reported to under-estimate the abundances of trans- posable elements (de Koning et al. 2011). Future studies could beneﬁt from using more sensitive approaches such as repetitive sequence clus- tering (de Koning, et al. 2011) as they may enable improved estimation of repeat element abundances. Moreover, they may allow further in- sight into repeat community structure and key element identiﬁcation using network approaches (Wacholder et al., 2014; Levy et al. 2017) thus enabling more detailed investigations of the repeat element proliferation dynamics among salmonids. stochastic in nature, the resulting effects of chromosome evolution on mutation and recombination rates can result in directed evolution (Lynch 2007) and phenotypic change. Also, following gene duplication, lineage-speciﬁc loss of duplicated gene copies (Lynch and Conery 2000) or possibly divergent expression evolution such as observed between European grayling and Atlantic salmon (Varadharajan et al. 2018), may contribute to speciation. Evidence of distinct genome evolution processes may provide avenues for further research aimed at exploring links between life history differences in salmonids and the evolution of distinct genome architectures. Transposable element activity, with lineage-speciﬁc differences such as those observed between European grayling and Atlantic salmon, is a major driver of genome evolution (Kazazian 2004) and may have been also involved in the distinct ge- nome evolution processes observed here. Furthermore, chromosomal inversions, such as those found frequently in the European grayling genome, have been suggested to have profound effects in the adaptation and speciation processes (Wellenreuther and Bernatchez 2018). For instance, they have been reported to increase genome sequence diver- gence between marine and freshwater ecotypes of a stickleback species Pungitius pungitius (Nelson and Cresko 2018) as well as between non- migratory and migratory ecotypes of Atlantic cod (Gadus morhua) (Berg et al. 2016; Kirubakaran et al. 2016). Computer simulations supported these observations and showed that chromosomal inver- sions may accelerate speciation particularly in certain conditions, such as when adaptation involves multiple genes with small individ- ual ﬁtness effects (Feder et al. 2014). Experimentation in house mouse (Mus musculus domesticus) has additionally demonstrated the possi- bility of rapid divergence mediated by Robertsonian fusions (Garagna et al. 2014). Volume 9 May 2019 \| European Grayling Genome Assembly \| 1291 Identiﬁcation of the European grayling sex chromosome An- other case of an Atlantic salmon-speciﬁc element is the Copia-12 retrotransposon (Figure 3 and TABLE S3), which belongs to the Copia superfamily of retrotransposons that was recently suggested to have a role in chromosomal diversiﬁcation and speciation in other teleost ﬁshes (Auvinet et al. 2018). Among the European grayling-speciﬁc elements, hAT-10 from the hAT DNA transposon superfamily covered 123,702 base pairs (0.01%) of the assembly and was completely absent in the in chromosomal diversiﬁcation and speciation in other teleost ﬁshes (Auvinet et al. 2018). Among the European grayling-speciﬁc elements, hAT-10 from the hAT DNA transposon superfamily covered 123,702 base pairs (0.01%) of the assembly and was completely absent in the 1290 \| T. Sävilammi et al. Figure 3 Comparison of repeat element abundances in the European grayling and Atlantic salmon genomes. The RepBase names of the elements that were more abundant in either of the two species and outside the 95% conﬁdence limit are also given. Residuals are colored to indicate element class and order categories. Figure 3 Co in the Europ The RepBas abundant in 95% conﬁden to indicate e Figure 3 Comparison of repeat element abundances in the European grayling and Atlantic salmon genomes. The RepBase names of the elements that were more abundant in either of the two species and outside the 95% conﬁdence limit are also given. Residuals are colored to indicate element class and order categories. Figure 3 Comparison of repeat element abundances in the European grayling and Atlantic salmon genomes. The RepBase names of the elements that were more abundant in either of the two species and outside the 95% conﬁdence limit are also given. Residuals are colored to indicate element class and order categories. Atlantic salmon genome assembly (Figure 3 and TABLE S3). The hAT DNA transposons, such as the Tc1-Mariner ones, are so-called cut-and- paste elements that have transposition mechanisms with the potential to actively induce genomic rearrangements in addition to indirect ways to generate homologous recombination of element copies. The accumu- lation of a particular transposable element in one of the two species may be considered as an indication of lineage-speciﬁc transposable element activity. These ﬁndings may provide unique insights to stim- ulate further research aimed at better understanding the molecular drivers of these distinct genome evolution processes. Identiﬁcation of the European grayling sex chromosome We anticipate that further salmonid-centric research in this direction aided by help from the chromosomal-level European grayling assembly that we provide will illuminate several open ques- tions that stem from these observations. Based on current knowledge we can only speculate what may be the role of the distinct European grayling chromosome architecture in the evolution of the species. Qumsiyeh (1994) hypothesizes that high diploid chromosome number leads to increased recombination rates, which in the case of the freshwater European graylings, may be asso- ciated with low differentiation, an advantageous trait in variable fresh- water environments (Phillips and Ráb 2001) (Figure 4). In contrast, reduction of chromosome numbers in the other salmonid lineages may be associated with anadromous life history strategy (Phillips and Ráb 2001). It has been suggested that periods of relaxed purifying selection, as in bottlenecked populations, may be necessary for the deleterious effects of chromosomal rearrangements to become ﬁxed (Lynch and Conery 2003; Lynch 2007). While possibly initially Casesofresidualtetrasomyandelevatedsequencesimilaritybetween homeologous chromosomes have been reported in many salmonids, suggesting that some rediploidization in these salmonid species may be Volume 9 May 2019 \| European Grayling Genome Assembly \| 1291 1291 Figure 4 Karyotypic changes among salmonid taxa. A Bayesian chronogram tree based on mitochondrial se- quence (tree obtained and edited from Shedko et al. (2013), doi: 10.5061/dryad.r42qf) of those salmonids that have diploid chromosome number (2n) and the number of chromosome arms (NF) available in Phillips and Ráb (2001). Figure 4 Karyotypic changes among salmonid taxa. A Bayesian chronogram tree based on mitochondrial se- quence (tree obtained and edited from Shedko et al. (2013), doi: 10.5061/dryad.r42qf) of those salmonids that have diploid chromosome number (2n) and the number of chromosome arms (NF) available in Phillips and Ráb (2001). Figure 4 Karyotypic changes among salmonid taxa. A Bayesian chronogram tree based on mitochondrial se- quence (tree obtained and edited from Shedko et al. (2013), doi: 10.5061/dryad.r42qf) of those salmonids that have diploid chromosome number (2n) and the number of chromosome arms (NF) available in Phillips and Ráb (2001). The evolutionary signiﬁcance of persistent residual tetrasomy remains unknown, but the existence of residual tetrasomy in the ancestral-like European grayling genome suggests that tetrasomy would be inde- pendent of chromosomal fusions typical of other salmonids (Phillips and Ráb 2001; Lien et al. 2016) and instead be favored by some other factor. 1292 \| T. Sävilammi et al. Identiﬁcation of the European grayling sex chromosome The majority of the sex-linked loci detected were found in the homeologous European grayling chromosome pair 11. Additionally, we located the sdY gene, reported as male-speciﬁc among many other salmonids (Yano et al. 2013), in chromosome 11A; thus, we concluded that chromosome 11A is the European grayling sex chromosome. ongoing (Lien et al. 2016). Although some species-speciﬁc differences in the residually tetrasomic regions have been reported, the tetraploid state appears to be conserved among salmonid species in seven to eight homeologous chromosome pairs (as summarized in Sutherland et al. 2016). Although otherwise distinctive, the karyotype evolution of European grayling was comparable to that of most salmonids in the case of residually tetrasomic regions (in chromosomes 9A & 9B homologous to ssa02q & ssa12qa, respectively and 25A & 25B ho- mologous to ssa04p & ssa08q, respectively) also being observed in the European grayling genome assembly based on shared linkage maps. Similarly, other regions (in chromosomes 2A & 2B homologous to ssa26 & ssa11a, respectively; 11A & 11B homologous to ssa6a & ssa3b, respectively; 20A homologous to ssa5b; and 23A & 23 B homologous to ssa7b and 17b, respectively) with reoccurring residual tetrasomy reported among salmonids (Sutherland et al. 2016) had elevated se- quence similarity, which has also be used as a predictor for recent or ongoing tetrasomy (Lien et al. 2016) (TABLE S1). Residual tetrasomy appears to have persisted in both Salmoninae and Thymallinae since the two lineages split, though the pace of the remaining rediploidization has been very slow since the lineage diversiﬁcation (Lien et al. 2016). In conclusion, by utilizing the novel resource of a chromosome-level genome assembly for European grayling, we were able to make some intriguing observations about the genome evolution processes in salmonids that conﬁrmed previous hypotheses and generated new questions. 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https://openalex.org/W3123842361	https://ccsenet.org/journal/index.php/ijef/article/download/20718/13978	English	null	The Attenuation of Idiosyncratic Risk under Alternative Portfolio Weighting Strategies: Recent Evidence from the UK Equity Market	International journal of economics and finance	2,012	cc-by	8,803	Abstract In this study, we investigate the attenuation of idiosyncratic risk and corresponding benefits of diversification for equally weighted and market capitalisation weighted portfolios in the UK Equity Market over 2002 - 2012. We analyse the absolute benefits of risk reduction by testing the homogeneity of variances of portfolios of different sizes using Levene's Test. Next, we perform a cost-benefit analysis to determine the return benefit of diversification from a practical perspective. We find that the absolute benefits of diversification for an equally weighted portfolio are greater in the 'crisis' than 'pre-crisis' period, but when we analyse the results from a practical perspective the benefits fall dramatically and the results are reversed. When comparing the benefits of market capitalisation weighted and equally weighted portfolios, we note that the benefits of diversification tend to be greater for an equally weighted portfolio for small portfolios but that a crossover occurs as the size of the portfolio increases. The relative benefits of diversification under these different weighting strategies are thus highly dependent upon the state of the market and further study is needed to determine why the diversification benefits for the alternative weighting strategies decay at varying rates. Keywords: portfolio diversification, idiosyncratic risk, index funds, weighting methodology Keywords: portfolio diversification, idiosyncratic risk, index funds, weighting methodology JEL Classification: C15, G01, G11, G17 Chia Rui Ming Daryl1 & Lim Kai Jie Shawn2 1 Department of Statistics, University of Warwick, Coventry, United Kingdom 2 Department of Economics, University College London, London, United Kingdom Correspondence: Chia Rui Ming Daryl, Department of Statistics, University of Warwick, Coventry, CV4 7ES, United Kingdom. Tel: 44-792-806-5816. E-mail: r.m.d.chia@warwick.ac.uk Accepted: September 11, 2012 Online Published: September 20, 2012 URL: http://dx.doi.org/10.5539/ijef.v4n11p1 Accepted: September 11, 2012 Online Published: September 20, 2012 URL: http://dx.doi.org/10.5539/ijef.v4n11p1 Received: August 13, 2012 doi:10.5539/ijef.v4n11p1 International Journal of Economics and Finance; Vol. 4, No. 11; 2012 ISSN 1916-971X E-ISSN 1916-9728 Published by Canadian Center of Science and Education International Journal of Economics and Finance; Vol. 4, No. 11; 2012 ISSN 1916-971X E-ISSN 1916-9728 Published by Canadian Center of Science and Education The Attenuation of Idiosyncratic Risk under Alternative Portfolio Weighting Strategies: Recent Evidence from the UK Equity Market Chia Rui Ming Daryl1 & Lim Kai Jie Shawn2 1 Department of Statistics, University of Warwick, Coventry, United Kingdom 2 Department of Economics, University College London, London, United Kingdom Correspondence: Chia Rui Ming Daryl, Department of Statistics, University of Warwick, Coventry, CV4 7ES, United Kingdom. Tel: 44-792-806-5816. E-mail: r.m.d.chia@warwick.ac.uk Chia Rui Ming Daryl1 & Lim Kai Jie Shawn2 1 Department of Statistics, University of Warwick, Coventry, United Kingdom 2 Department of Economics, University College London, London, United Kingdom Correspondence: Chia Rui Ming Daryl, Department of Statistics, University of Warwick, Coventry, CV4 7ES, United Kingdom. Tel: 44-792-806-5816. E-mail: r.m.d.chia@warwick.ac.uk Chia Rui Ming Daryl1 & Lim Kai Jie Shawn2 1 Department of Statistics, University of Warwick, Coventry, United Kingdom 2 Department of Economics, University College London, London, United Kingdom Correspondence: Chia Rui Ming Daryl, Department of Statistics, University of Warwick, Coventry, CV4 7ES, United Kingdom. Tel: 44-792-806-5816. E-mail: r.m.d.chia@warwick.ac.uk 2.2 Traditional Approaches to Testing the Benefits of Diversification There are 2 general approaches that have been employed in tests on diversification using simulated portfolios. The first approach pioneered by Evans and Archer (1968) focuses on the absolute benefits of diversification. This approach attempts to find the level beyond which holding more securities in a portfolio has a negligible impact on the reduction of risk. Statistical tests such as the T-Test and F-Test are often employed in these types of studies to find the point at which increasing the size of a portfolio no longer has a statistically significant impact on the reduction of risk as well as the level at which standard deviation tends to converge, or the asymptote for the graph of portfolio size vs. standard deviation. This approach has also been adopted by a number of contemporary studies on diversification and is the methodology often referenced in finance textbooks (Newbould & Poon, 1993, 1996). Lai and Seiler (2001) use this methodology to study diversification within industry groups and Benjelloun (2010) uses this approach to study the benefits of diversification from a terminal wealth perspective. The second approach pioneered by Statman (1987, 2004) takes a more practical view of the issue and seeks to find the number of securities beyond which the benefits of diversification are lower than the holding costs from an increased number of securities. This approach converts the risk of a simulated portfolio of n securities into a comparable return figure by combining the simulated portfolio with a risk-free asset to generate portfolio combinations with the same level of risk. The cost of a collective investment scheme such as a mutual fund or exchange traded fund is often used as a proxy for the cost of full diversification and is compared to the return benefit from increasing the number of securities held to obtain the optimal number of securities that one should hold in a well-diversified portfolio. This approach has also been used to study the benefits of diversification in other asset classes and markets. Gupta and Khoon (2001) adopt this approach to study the number of securities needed to form a diversified portfolio in Malaysia while Lee (2005) studies the benefits of diversification in commercial real estate portfolios. In this study, we employ both of these approaches to present the benefits of diversification from both an absolute and practical perspective. 2.2 Traditional Approaches to Testing the Benefits of Diversification We use these approaches on equally weighted random portfolios as employed in the original methodologies and extend that methodology to test the benefits of diversification using market capitalisation weighted random portfolios. 1. Introduction The benefits of employing a portfolio approach to investing plays a central role within modern finance theory. The arguments for this approach has its roots in the seminal paper by Markowitz (1957) that laid the theoretical framework for what has now evolved to become Modern Portfolio Theory. One key implication and oft quoted result from this theory is that increasing the size of one’s portfolio helps to reduce idiosyncratic risk, and it is hence possible to achieve superior risk-adjusted performance through the use of a portfolio approach to investing. However, what is the optimal number of securities that one should hold to reap the maximum benefits of diversification? This is a key question that academics grappled with in the wake of Modern Portfolio Theory. The early literature looked at the issue from 2 different perspectives. The first approach investigates the benefits of diversification by analysing the results from the simulation of numerous random portfolios and is credited to the early work of Evans and Archer (1968). The second approach seeks to find an exact solution to quantify the benefits of diversification and is credited to the analytical approach first espoused by Elton and Gruber (1977). While the ability to mathematically quantify the benefits of diversification in an exact manner is intuitively appealing, it comes at the cost of over simplification as it employs the use of many assumptions to reduce the problem to something that is mathematically tractable but not necessarily operationally useful. Hence, we have adopted a methodology in line with the first approach as it gives us greater flexibility in the crafting of our methodology and yields results that will be of interest to both academics and practitioners. In this paper, we investigate the benefits of diversification in the UK equity market through the use of a modified methodology that incorporates elements from both of these approaches. In addition, we present a more realistic model of diversification by constructing market capitalisation weighted portfolios in addition to equally-weighted portfolios that are traditionally the focus of studies on diversification. 1 1 International Journal of Economics and Finance Vol. 4, No. 11; 2012 www.ccsenet.org/ijef 2.1 Objectives and Significance of Paper 2.1 Objectives and Significance of Paper While the study of the benefits of diversification is by no means a novel topic, we propose to contribute to existing literature by suggesting improvements on the best way to quantify it. In this paper, we employ 3 changes that we believe allows us to add to the existing literature on the topic. Firstly, we conduct the experiment on the UK Equity Market using the FTSE All-Share Index as a benchmark and present the empirical results for diversification on a previously untested market over a recent time period. Next, while many studies have adopted an ex-ante approach to their investigation, we have adopted an ex-post approach in this study. Instead of asking the question what are the likely benefits in the future, we have asked the question what were the benefits during the ‘pre-crisis’ and ‘crisis’ period. This allows us to present empirical results on how the benefits from diversification and optimal number of securities to hold to reap that benefit may have changed during a period of high volatility and market uncertainty. Finally, we consider an alternative weighting scheme that allows for results that are more realistic and representative. While the traditional approach looks at equally weighted portfolios, we include in our analysis the benefits from diversification of both an equally weighted portfolio and a market capitalisation weighted portfolio. Studying the benefits of diversification for a market capitalisation weighted portfolio is more appropriate as the de facto benchmark for full diversification with the lowest possible cost would be that offered by an Exchange-Traded Index Fund, which tends to be a market capitalisation weighted investment vehicle In addition, the market capitalisation weighted portfolio is a better resemblance of a mean-variance efficient portfolio than an equally weighted one and more reflective of an approach that might be employed in practice. Hence, if we compare the cost of full diversification using an ETF with an equally weighted portfolio or use an alternative benchmark such as a mutual fund we would overstate the cost of diversification and hence lead to a misleading conclusion not representative of the decisions that would be made in reality. 2.4 Weighting Strategies The manner in which portfolios are constructed will have a tangible impact on the risk and return profile of the portfolio. In particular, alternative weighting strategies are likely to have a material influence on the risk and return characteristics of a portfolio and hence the effectiveness of a diversification strategy. In this study we consider 2 alternative weighting strategies, equally weighted portfolios and market capitalisation weighted portfolios. An equally weighted portfolio refers to a construction technique in which an equal dollar amount is invested in each holding, regardless of any differences in the fundamental characteristics of component securities. When applied to a large universe of stocks of different sizes such as in our study of the FTSE all-share index, the return profile of such portfolios will inevitably have an implicit tilt towards stocks with a small market capitalisation and may not be reflective of a portfolio that can be achieved in reality. However, equally weighted portfolios have traditionally been the focus of diversification studies as it is computationally easier to work with equally weighted portfolios when employing analytical approaches. As we are conducting our analysis using a simulation approach instead of an analytical one, the added complexity of portfolios with unequal weights is of a smaller concern. Beyond the fundamental tilt of an equally weighted portfolio, the larger problem of such a weighting strategy is the lack of a representative low-cost benchmark for full diversification. Furthermore, the correlation between stocks with a small market capitalisation and stocks with a large market capitalisation compared to stocks with similar market capitalisation tends to be lower (Huang, Eun & Lai, 2006), thus when combined into a portfolio this will exhibit lower levels of standard deviation. Hence, using an equally weighted portfolio is likely to overstate the benefits of diversification and present conclusions that may not be operationally meaningful. In order to address some of these problems, we have considered an alternative weighting strategy, that of a market capitalisation weighted portfolio. When constructing market capitalisation weighted portfolios, the proportion of each security held is according to the size of its market capitalisation. The benefit of this approach is that our benchmark, the FTSE All-Share index is by construction a capitalisation-weighted index and hence there are viable low cost options for full diversification based on the index. 2.4 Weighting Strategies Results based on market capitalisation weighted portfolios are closer to the type of decisions made in reality and are likely to have more meaningful operational implications. 2.3 Time Period of Investigation The time period employed is likely to have a material impact on the benefits of diversification. For this paper, we split our analysis into 2 time period, a 5 year period from 1 July 2002 to 30 June 2007 that we have called the 2 International Journal of Economics and Finance Vol. 4, No. 11; 2012 www.ccsenet.org/ijef ‘pre-crisis period’ and a 5 year period from 1 July 2007 to 30 June 2012 that we have called the ‘crisis period’. By using these 2 particular periods in time, we hope to present results on the benefits of diversification during a normal period of time and during a period of crisis, as well as provide a comparison on how the optimal number of securities to hold from a diversification perspective might have changed during this period of market stress. ‘pre-crisis period’ and a 5 year period from 1 July 2007 to 30 June 2012 that we have called the ‘crisis period’. By using these 2 particular periods in time, we hope to present results on the benefits of diversification during a normal period of time and during a period of crisis, as well as provide a comparison on how the optimal number of securities to hold from a diversification perspective might have changed during this period of market stress. Where ݓ௞ : Weight of security k in the n security portfolio ݓ௞ : Weight of security k in the n security portfolio ket capitalisation of security k at the start of the time period. ݉௞ : Market capitalisation of security k at the start of the time period. We also calculate the geometric mean return for the entire period considered (ܴ௣) using the following equation: lculate the geometric mean return for the entire period considered (ܴ௣) using the following equation: ܴ௣ൌexpሺ ଵ ௅ ∑ log௘ܴ௜ ௗ ௡ ௜ୀଵ ሻ (4) (4) For L = 1265 for pre-crisis and L = 1262 for crisis, where L is the number of daily return data points. For L = 1265 for pre-crisis and L = 1262 for crisis, where L is the number of daily return data points. Finally, the portfolio standard deviation (ݏ௣) is computed: Finally, the portfolio standard deviation (ݏ௣) is computed: Finally, the portfolio standard deviation (ݏ௣) is computed: Finally, the portfolio standard deviation (ݏ௣) is computed: ݏ௣ൌ ටଵ ௅ିଵ∑ ൫log௘ܴ௣െlog௘ܴௗ ௜൯ ଶ ௅ ௜ୀଵ (5) (5) We conduct 20,000 runs for each portfolio of size n for each of the 2 time periods (10,000 runs for equally weighted portfolios and 10,000 runs for market capitalisation weighted portfolios), which yields a total of 10,000,000 simulated portfolios. To increase the robustness of our results, the simulations were run with replacement. We conduct 20,000 runs for each portfolio of size n for each of the 2 time periods (10,000 runs for equally weighted portfolios and 10,000 runs for market capitalisation weighted portfolios), which yields a total of 10,000,000 simulated portfolios. To increase the robustness of our results, the simulations were run with replacement. 3.2 Absolute Benefits of Diversification In this section, we evaluate the absolute benefits of diversification by finding the level at which further increases in the number of securities held in a portfolio does not lead to a reduction in variance that is statistically significant. The traditional approach employed for tests of the homogeneity of variance between 2 samples, in this case portfolios with a different number of securities, is the F-Test. However, the F-Test has been shown to be extremely sensitive to the normality assumption (Box, 1953; C. Markowski & E. Markowski, 1990) while financial data series often exhibit characteristics that are not consistent with a normal distribution. An alternative test, Levene's test (Levene, 1960), uses the average of the absolute deviations instead of the mean square of deviations and this adjustment makes the test criterion much less sensitive to non-normal distributions (Snedecor and Cochran, 1976). Hence, we employ Levene’s Test instead to test the equality of variances between successive portfolios. The results from this test are reported in Table 3. 3.1 Creating Random Portfolios of n Securities by Simulation The aim of this study is to investigate the empirical benefits of diversification by increasing the number of securities held in a portfolio under alternative weighting strategies. In the first step of this process, we create random portfolios of n securities by simulation. The n securities are selected at random based on a uniform distribution. For each run, we calculated the daily return of the portfolio at time i (ܴ௜ ௗ) with the following equations. For equally weighted portfolios: For each run, we calculated the daily return of the portfolio at time i (ܴ௜ ௗ) with the following equati F ll i h d f li , y p ( ௜) g q For equally weighted portfolios: For equally weighted portfolios: For equally weighted portfolios: ܴ௜ ௗൌ ଵ ௡ ∑ ܴ௜ ௞ ௡ ௞ୀଵ (1) ܴ௜ ௗൌ ଵ ௡ ∑ ܴ௜ ௞ ௡ ௞ୀଵ (1) For n = 1 to 250 For n = 1 to 250 Where n is the number of securities in the portfolio. Where n is the number of securities in the portfolio. market capitalisation weighted portfolios: For market capitalisation weighted portfolios: For market capitalisation weighted portfolios: ܴ௜ ௗൌ ∑ ݓ௞ܴ௜ ௞ ௡ ௞ୀଵ (2) ݓ௞ൌ ௠ೖ ∑ ௠ೖ ೙ ೖసభ (3) ܴ௜ ௗൌ ∑ ݓ௞ܴ௜ ௞ ௡ ௞ୀଵ (2) ܴ௜ ௗൌ ∑ ݓ௞ܴ௜ ௞ ௡ ௞ୀଵ (2) ݓ௞ൌ ௠ೖ ∑ ௠ೖ ೙ ೖసభ (3) (3) ݓ௞ൌ ௠ೖ ∑ ௠ೖ ೙ ೖసభ 3 International Journal of Economics and Finance Vol. 4, No International Journal of Economics and Finance Vol. 4, No. 11; 2012 www.ccsenet.org/ijef Where 3.3 Cost-benefit Analysis While measuring the absolute benefit of diversification is important, such an approach does not take into account the fact that holding more securities comes with an added cost as well. In reality the decision of how many securities to hold is likely to be contingent upon some form of cost-benefit analysis, where the number of securities held will be increased up until the point where the marginal benefit of diversification exceeds the marginal cost of holding more securities. In this section, we approach the issue of diversification form a more practical perspective by making that explicit cost-benefit analysis. 3.3.1 Measuring the Benefit of Full Diversification We quantify the benefit of fully diversifying each n-security portfolio to the index portfolio by converting the associated reduction in standard deviation into a return figure. This is done by levering down all n-security portfolios such that its standard deviation is equal to that of the m-security population portfolio. In effect, the process transforms all n-security portfolios into ones where a portion m/p(n) of the portfolio remains invested in n-securities, while the remaining (1 - m/p(n)) is invested at the risk free rate. The net result is that the levered down portfolios will have a standard deviation equal to that of the m-security population portfolio. With all portfolios having the same standard deviation, differences in returns can therefore be attributed to diversification effects. benefit from diversification is calculated using the following equation: The benefit from diversification is calculated using the following equation: ܦ௣ሺ௡ሻൌܴ௠െሾሺܴ௣ሺ௡ሻെܴ௙ሻ ఙ೛ሺ೙ሻ ఙ೘൅ܴ௙ሿ (10) (10) ܦ௣ሺ௡ሻൌܴ௠െሾሺܴ௣ሺ௡ሻെܴ௙ሻ ఙ೛ሺ೙ሻ ఙ೘൅ܴ௙ሿ Where Dp(n) : return benefit of diversifying an n-stock portfolio to an m = 498 stock portfolio Rp(n) : geometric mean return of the n-stock portfolio Rm : geometric mean return of the index portfolio Rf : risk free rate obtained from rolling 3 month government bills over each 5 year period p(n) : standard deviation of returns of the n-stock portfolio m : standard deviation of returns of the population of m = 498 stocks m : standard deviation of returns of the population of m = 498 stocks We first deduct the relevant risk free rate from each n-security portfolio’s geometric mean return to find its market risk premium, Rp(n) – Rf. ܻప.ഥ : Mean for the n-security portfolio simulation The significance of W is tested against F(0.05, 1, 19998) where F is a quantile of the F-test distribution. We conduct this test for portfolios of successive sizes to determine the number of securities that must be added to each portfolio of n securities such that there is a statistically significant change in variance and hence benefit to diversification. 3.2.1 Levene’s Test We conduct Levene’s Test on successive portfolios with different numbers of securities. The test is conducted with the following hypotheses: ܪ଴: The 2 samples have the same variance ܪ଴: The 2 samples have the same variance ܪଵ: The 2 samples have different variances ܪଵ: The 2 samples have different variances The test statistic, W, is defined as follows: The test statistic, W, is defined as follows: ܹൌ ேି௞ ௞ିଵ ∑ ே೙ሺ௓೙.ି ௓..ሻమ ೖ೙సభ ∑ ∑ ൫௓೙ೕି ௓೙.൯మ ಿ೔ ೕసభ ೖ೙సభ (6) ܼ௡௝ൌ หܻ௡௝െ ܻ௡. തതതห (7) ܼ௡. ൌ ଵ ே೔ ∑ ܼ௡௝ ே೔ ௝ୀଵ (8) ܼ.. ൌ ଵ ே ∑ ∑ ܼ௡௝ ே೔ ௝ୀଵ ௞ ௡ୀଵ (9) ܹൌ ேି௞ ௞ିଵ ∑ ே೙ሺ௓೙.ି ௓..ሻమ ೖ೙సభ ∑ ∑ ൫௓೙ೕି ௓೙.൯మ ಿ೔ ೕసభ ೖ೙సభ (6) (6) (8) (9) ே Where W : Test statistic k : Number of groups to which the samples belong N : Total number of runs ܰ௜ : Number of runs in the n-security portfolio simulation k : Number of groups to which the samples belong N : Total number of runs ܰ௜ : Number of runs in the n-security portfolio simulation International Journal of Economics and Finance Vol. 4, No. 11; 2012 www.ccsenet.org/ijef ܻ௡௝ : Value of the j-th run from the n-security portfolio simulation ܻ௡௝ : Value of the j-th run from the n-security portfolio simulation ܻప.ഥ : Mean for the n-security portfolio simulation All values above are annualised and Dp(n) is calculated for n ranging from 1 to 250. All values above are annualised and Dp(n) is calculated for n ranging from 1 to 250. Digressing slightly from explaining the above calculation, it is of significance that the relevant risks free rate in this study is that of the annualised 5 year internal rate of return obtained by rolling 1-year UK governments securities over each 4 year period under study. Such is consistent with minimising the price and reinvestment risk present in longer dated generic government rates, and is consistent with the interpretation of the risk free rate in modern literature (Mukherji, 2011; Damodaran, 2010). 3.3 Cost-benefit Analysis This risk premium is then multiplied by the ratio of the M-security population standard deviation to the n-security portfolio standard deviation, m/p(n) , in order to lever down the portfolios’ risk premium. The risk free rate is then added back to the market risk premium of each levered down portfolio. Finally, the difference between the m-security population portfolio return and the last calculated figure is the benefit from full diversification. 4.1 Data Daily closing price and market capitalisation data were extracted from the Bloomberg Professional service for each of the FTSE All-Share Index’s constituents. In order to best reflect the ex-post return and volatility experienced by a buy-and-hold investor over each of the two periods under study, price data was extracted for the equities that constituted the index on each of the period’s start date. The relevant market capitalisation weights used in constructing the market-cap weighted portfolio were then those on each period’s start date, reflecting the proportions of each stock an investor will include in a market-cap weighted portfolio constructed at the beginning of each period. Also in line with ensuring representativeness of a buy-and-hold portfolio investment, daily price data was adjusted for normal cash dividends (regular cash, interim, income, estimated, partnership distribution, final, interest on capital, distributed and prorated), abnormal cash dividends (special cash, liquidation, capital gains, memorial, return of capital, rights redemptions, return premium, preferred rights redemption, proceeds/rights, proceeds/shares, proceeds/warrants) and capital changes (spin-offs, stocks splits/consolidations, stock dividend/bonus, rights offerings/entitlement). The price data was then screened for equities which lacked a complete dataset over each five year period under study. These equities were removed from the dataset. Eventually, what remained were 498 equities out of 703 and 698 index constituents for the pre-crisis and crisis periods respectively. These 498 equities were then inputs into our portfolio simulations for both periods. 3.3.2 Measuring the Cost of Full Diversification Diversifying fully from an n-security portfolio to an m-security portfolio incurs a cost equal to the difference in cost between holding each of the above portfolios. Given the simple, one-time transaction buy-and-hold strategy of constructing each n-security portfolio, price spread costs are negligible when spread over the entire 5 year 5 International Journal of Economics and Finance www.ccsenet.org/ijef Vol. 4, No. 11; 2012 period. Further, transaction fees are negligible vis-à-vis returns in our study given institutional-sized portfolios. We hence assume that the cost of constructing and holding the n-security portfolio is zero. period. Further, transaction fees are negligible vis-à-vis returns in our study given institutional-sized portfolios. We hence assume that the cost of constructing and holding the n-security portfolio is zero. This leaves us with the cost of full diversification being equal to the cost of holding the m-security population portfolio. Much like existing literature (Statman, 1987), we take the total expense ratio of a representative ETF as a proxy for the cost of constructing and maintaining the said m-security population portfolio. The cost of full diversification is hence the total expense ratio of a representative ETF, and in this case, the relevant ETFs – db x-trackers FTSE All-Share and the Lyxor ETF FTSE All Share ETFs – both have an expense ratio of 0.40%. This will be the cost of full diversification in our study. Finally, we find the values of n for each period and weighting methodology that best equates the benefit of full diversification with its cost – such is the point at which the marginal benefit of diversification is outweighed by the cost. 4.2 Simulation Summary Table 1 below displays the portfolio standard deviations for both the pre-crisis and crisis period for an equally weighted and market capitalisation weighted portfolio of n equities, with n ranging from 1 to 250 and n = 498 representing the population of equities under consideration, which is a proxy for all the constituents in the FTSE All-Share Index. While we are using the model generated standard deviation figures for all n between 1 and 250, for the interest of brevity, only steps of 10 are displayed beyond n = 50. International Journal of Economics and Finance Vol. 4, No. 11; 2012 www.ccsenet.org/ijef Table 1. 4.2 Simulation Summary Annualised Daily Standard Deviation of Returns Pre-Crisis Period 1 July 2002 to 30 June 2007 Crisis Period 1 July 2007 to 30 June 2012 Pre-Crisis Period 1 July 2002 to 30 June 2007 Crisis Period 1 July 2007 to 30 June 2012 Portfolio Size (n) Equally Weighted Portfolio Market-Cap Weighted Portfolio Equally Weighted Portfolio Market-Cap Weighted Portfolio Portfolio Size (n) Equally Weighted Portfolio Market-Cap Weighted Portfolio Equally Weighted Portfolio Market-Cap Weighted Portfolio 1 30.195% 30.195% 42.673% 42.662% 37 11.845% 18.137% 20.917% 30.000% 2 22.652% 25.117% 34.038% 37.967% 38 11.818% 18.101% 20.890% 29.946% 3 19.696% 23.274% 30.291% 35.926% 39 11.793% 18.076% 20.865% 29.887% 4 17.953% 22.288% 28.129% 34.768% 40 11.771% 18.048% 20.840% 29.833% 5 16.825% 21.590% 26.734% 34.010% 41 11.746% 18.014% 20.816% 29.783% 6 16.027% 21.142% 25.741% 33.422% 42 11.725% 17.982% 20.791% 29.748% 7 15.410% 20.785% 24.993% 33.084% 43 11.705% 17.957% 20.767% 29.696% 8 14.932% 20.503% 24.410% 32.748% 44 11.684% 17.924% 20.744% 29.674% 9 14.543% 20.247% 23.968% 32.493% 45 11.667% 17.902% 20.723% 29.613% 10 14.216% 20.041% 23.592% 32.265% 46 11.649% 17.874% 20.703% 29.570% 11 13.946% 19.873% 23.280% 32.134% 47 11.631% 17.845% 20.686% 29.532% 12 13.722% 19.727% 23.021% 32.070% 48 11.616% 17.820% 20.667% 29.500% 13 13.519% 19.588% 22.795% 31.951% 49 11.601% 17.798% 20.648% 29.465% 14 13.343% 19.465% 22.607% 31.805% 50 11.585% 17.777% 20.630% 29.429% 15 13.193% 19.360% 22.432% 31.656% 60 11.460% 17.585% 20.499% 29.108% 16 13.058% 19.241% 22.271% 31.568% 70 11.368% 17.407% 20.403% 28.800% 17 12.946% 19.156% 22.128% 31.407% 80 11.298% 17.266% 20.329% 28.577% 18 12.842% 19.079% 22.010% 31.294% 90 11.243% 17.154% 20.272% 28.343% 19 12.744% 19.000% 21.897% 31.182% 100 11.201% 17.056% 20.228% 28.178% 20 12.655% 18.924% 21.800% 31.088% 110 11.168% 16.973% 20.189% 28.057% 21 12.573% 18.841% 21.707% 30.982% 120 11.139% 16.902% 20.156% 27.945% 22 12.499% 18.786% 21.624% 30.907% 130 11.113% 16.831% 20.131% 27.871% 23 12.431% 18.724% 21.549% 30.827% 140 11.092% 16.782% 20.110% 27.800% 24 12.369% 18.665% 21.479% 30.707% 150 11.073% 16.727% 20.090% 27.710% 25 12.310% 18.617% 21.417% 30.643% 160 11.058% 16.681% 20.071% 27.634% 26 12.257% 18.568% 21.360% 30.567% 170 11.044% 16.644% 20.058% 27.581% 27 12.205% 18.523% 21.307% 30.513% 180 11.032% 16.609% 20.046% 27.518% 28 12.160% 18.480% 21.255% 30.469% 190 11.021% 16.578% 20.032% 27.472% 29 12.115% 18.439% 21.207% 30.434% 200 11.011% 16.547% 20.023% 27.432% 30 12.077% 18.396% 21.160% 30.357% 210 11.001% 16.519% 20.012% 27.383% 31 12.036% 18.357% 21.118% 30.282% 220 10.992% 16.496% 20.001% 27.341% 32 11.999% 18.313% 21.081% 30.241% 230 10.985% 16.472% 19.993% 27.309% 33 11.964% 18.274% 21.044% 30.177% 240 10.978% 16.448% 19.986% 27.280% 34 11.931% 18.241% 21.006% 30.123% 250 10.972% 16.428% 19.980% 27.252% 35 11.900% 18.206% 20.976% 30.080% ⋮ ⋮ ⋮ ⋮ ⋮ 36 11.873% 18.171% 20.947% 30.038% 498 10.813% 15.904% 19.814% 26.498% Table 1. 4.2 Simulation Summary Annualised Daily Standard Deviation of Returns 7 Inteernational Journaal of Economicss and Finance Vol. 4, No. 11; 2012 www.ccsennet.org/ijef Figure 1. Annnualised Daily Standard Devviation of Returrns vs. n Figure 1. Annnualised Daily Standard Devviation of Returrns vs. n As is appa increase in both the w weighting. during the arent from both n n. Further, p weighting me . Such results last decade (H h the Figure 1 ortfolio standa ethodologies, a are consistent Hjalmarsson & and Table 1, p ard deviation w and market-ca t with present & Manchev, 20 portfolio stand was larger dur ap weighting literature whi 12; Abhyanka dard deviation ring the crisis v led to larger ich investigate ar & Ho, 2007) decreases mon vis-à-vis the p standard dev es recent equit ). notonically wi pre-crisis perio viations than e ty market beh th an d for equal avior As is appa increase in both the w weighting. during the arent from both n n. Further, p weighting me . Such results last decade (H h the Figure 1 ortfolio standa ethodologies, a are consistent Hjalmarsson & and Table 1, p ard deviation w and market-ca t with present & Manchev, 20 portfolio stand was larger dur ap weighting literature whi 12; Abhyanka dard deviation ring the crisis v led to larger ich investigate ar & Ho, 2007) decreases mon vis-à-vis the p standard dev es recent equit ). notonically wi pre-crisis perio viations than e ty market beh th an d for equal avior Moving o cost-benef returns as for all n ra on to the othe fit analysis, th stated in the ta anging from 1 t er summary st e geometric m able 2. The fig to 250. tatistic central mean return of gures represent l to the input f the portfolios t the expected ts of our abso s converged to geometric me olute diversifi o the populatio ean return of a ication benefit on geometric m portfolio of si t and mean ize n, Table 2. 4.2 Simulation Summary A Eq M These retu sections 4 Annualised Dai qually Weighted P Market-Cap Weight urn and standa 3 and 4 4 belo ily Geometric M Portfolio ted Portfolio ard deviation ow Mean Returns P 1 July data are utili Pre-Crisis Period 2002 to 30 June 2 18.488% 13.021% sed as set out 2007 1 t in the metho Crisis Perio July 2007 to 30 Ju 2.568% 2.274% odology to de od une 2012 erive the resullts in Table 2. A Eq M Annualised Dai qually Weighted P Market-Cap Weight ily Geometric M Portfolio ted Portfolio Mean Returns P 1 July Pre-Crisis Period 2002 to 30 June 2 18.488% 13.021% 2007 1 Crisis Perio July 2007 to 30 Ju 2.568% 2.274% od une 2012 Table 2. AAnnualised Daily Geometric M Mean Returns These retu sections 4. urn and standa .3 and 4.4 belo ard deviation ow. data are utilised as set outt in the methoodology to deerive the resullts in These retu sections 4. urn and standa .3 and 4.4 belo ard deviation ow. data are utilised as set outt in the methoodology to deerive the resullts in 4.3 Absoluute Benefits of f Diversificationn 4.3 Absoluute Benefits of f Diversificationn 4.3 Absoluute Benefits of f Diversificationn Table 3. T Run (Note 1) 1 2 3 4 5 6 7 8 9 10 Test Statistic fro Equally Weight [02 – 07] 197.264 105.538 51.107 29.859 31.020 16.940 15.848 11.889 9.891 7.458 om Levene’s T Test Sta ted Market Ca Test on the Ho atistic from Leven apitalisation Wei 131.110 73.192 30.620 18.349 10.462 4.991 5.667 5.772 2.658 2.339 omogenity of V ne’s Test on the H ghted [02 – 07] Variances Homogeneity of V Equally Weigh [07 – 12] 248.069 113.915 61.738 47.718 25.842 26.674 17.475 14.273 13.662 7.256 Variances (Note 2 ted Market c 2) capitalisation We [07– 12] 107.892 32.825 14.900 10.836 7.139 4.513 3.571 1.842 1.891 0.551 eighted Table 3. TTest Statistic from Levene’s TTest on the Hoomogenity of VVariances Test Staatistic from Levenne’s Test on the HHomogeneity of VVariances (Note 22) 8 8 International Journal of Economics and Finance International Journal of Economics and Finance Vol. 4, No. 11; 2012 Vol. 4, No. 4.2 Simulation Summary 4, No. 11; 2012 www.ccsenet.org/ijef capitalisation weighted portfolios demonstrated less benefit from diversification compared to the equally weighted portfolios over both time periods. The table 4 below displays the benefit of full diversification for both the pre-crisis and crisis period for an equally weighted and market capitalisation weighted portfolio of n equities, with n ranging from 1 to 250 and n = 498. The table 4 below displays the benefit of full diversification for both the pre-crisis and crisis period for an equally weighted and market capitalisation weighted portfolio of n equities, with n ranging from 1 to 250 and n = 498. Table 4. 4.2 Simulation Summary Benefit of Full Diversification (Annualised % Return) Pre-Crisis Period 1 July 2002 to 30 June 2007 Crisis Period 1 July 2007 to 30 June 2012 Pre-Crisis Period 1 July 2002 to 30 June 2007 Crisis Period 1 July 2007 to 30 June 2012 Portfolio Size (N) Equally Weighted Portfolio Market-Cap Weighted Portfolio Equally Weighted Portfolio Market-Cap Weighted Portfolio Portfolio Size (N) Equally Weighted Portfolio Market-Cap Weighted Portfolio Equally Weighted Portfolio Market-Cap Weighted Portfolio 1 9.31083% 4.21277% 0.58619% 0.30285% 37 1.30872% 1.11120% 0.05789% 0.09341% 2 7.63792% 3.27864% 0.45757% 0.24153% 38 1.27723% 1.09563% 0.05652% 0.09213% 3 6.62091% 2.83606% 0.37889% 0.20986% 39 1.24835% 1.08468% 0.05527% 0.09073% 4 5.85808% 2.56841% 0.32390% 0.19024% 40 1.22284% 1.07235% 0.05401% 0.08944% 5 5.27667% 2.36371% 0.28368% 0.17666% 41 1.19373% 1.05742% 0.05286% 0.08826% 6 4.81389% 2.22497% 0.25239% 0.16570% 42 1.16894% 1.04325% 0.05156% 0.08741% 7 4.42153% 2.11027% 0.22718% 0.15923% 43 1.14539% 1.03218% 0.05036% 0.08617% 8 4.09456% 2.01648% 0.20644% 0.15265% 44 1.12135% 1.01779% 0.04919% 0.08565% 9 3.81151% 1.92895% 0.19003% 0.14759% 45 1.10055% 1.00780% 0.04816% 0.08416% 10 3.56128% 1.85691% 0.17559% 0.14298% 46 1.07900% 0.99541% 0.04712% 0.08313% 11 3.34519% 1.79716% 0.16329% 0.14031% 47 1.05765% 0.98240% 0.04627% 0.08221% 12 3.15981% 1.74434% 0.15278% 0.13898% 48 1.03961% 0.97095% 0.04529% 0.08142% 13 2.98562% 1.69304% 0.14344% 0.13653% 49 1.02217% 0.96136% 0.04432% 0.08057% 14 2.83026% 1.64733% 0.13552% 0.13349% 50 1.00289% 0.95179% 0.04342% 0.07970% 15 2.69346% 1.60775% 0.12803% 0.13036% 60 0.84925% 0.86380% 0.03668% 0.07175% 16 2.56921% 1.56241% 0.12100% 0.12848% 70 0.73513% 0.78071% 0.03171% 0.06396% 17 2.46269% 1.52944% 0.11472% 0.12504% 80 0.64700% 0.71340% 0.02782% 0.05822% 18 2.36227% 1.49936% 0.10945% 0.12260% 90 0.57679% 0.65900% 0.02481% 0.05208% 19 2.26726% 1.46831% 0.10438% 0.12018% 100 0.52282% 0.61094% 0.02250% 0.04771% 20 2.17800% 1.43833% 0.09993% 0.11813% 110 0.47870% 0.56966% 0.02043% 0.04448% 21 2.09539% 1.40502% 0.09568% 0.11579% 120 0.44152% 0.53447% 0.01866% 0.04143% 22 2.01971% 1.38312% 0.09182% 0.11412% 130 0.40772% 0.49846% 0.01731% 0.03942% 23 1.94959% 1.35770% 0.08836% 0.11234% 140 0.37932% 0.47337% 0.01616% 0.03748% 24 1.88534% 1.33398% 0.08504% 0.10965% 150 0.35438% 0.44561% 0.01510% 0.03500% 25 1.82236% 1.31402% 0.08213% 0.10823% 160 0.33475% 0.42180% 0.01409% 0.03291% 26 1.76609% 1.29377% 0.07940% 0.10651% 170 0.31583% 0.40258% 0.01338% 0.03142% 27 1.71031% 1.27507% 0.07691% 0.10528% 180 0.29989% 0.38423% 0.01270% 0.02966% 28 1.66139% 1.25713% 0.07441% 0.10426% 190 0.28474% 0.36822% 0.01199% 0.02837% 29 1.61263% 1.24037% 0.07206% 0.10347% 200 0.27076% 0.35186% 0.01146% 0.02725% 30 1.57021% 1.22188% 0.06982% 0.10171% 210 0.25785% 0.33719% 0.01088% 0.02588% 31 1.52529% 1.20568% 0.06776% 0.09999% 220 0.24621% 0.32478% 0.01028% 0.02469% 32 1.48399% 1.18696% 0.06593% 0.09904% 230 0.23578% 0.31211% 0.00987% 0.02376% 33 1.44417% 1.17037% 0.06413% 0.09755% 240 0.22695% 0.29967% 0.00948% 0.02293% 34 1.40723% 1.15635% 0.06229% 0.09629% 250 0.21927% 0.28912% 0.00913% 0.02215% 35 1.37232% 1.14117% 0.06079% 0.09528% ⋮ ⋮ ⋮ ⋮ ⋮ 36 1.34094% 1.12609% 0.05938% 0.09430% 498 0.00000% 0.00000% 0.00000% 0.00000% Data from Table 4 is also presented in Figure 2 and Figure 3. 4.2 Simulation Summary 11; 2012 www.ccsenet.org/ijef 11 5.372 2.010 7.773 0.439 12 5.228 0.967 6.001 0.483 13 6.204 1.910 5.808 0.625 14 3.285 0.706 3.934 0.381 15 4.218 0.960 5.093 0.531 16 3.170 0.638 3.312 0.957 17 2.140 0.547 3.003 0.218 18 2.665 0.879 3.155 0.341 19 2.514 0.472 3.266 0.262 20 1.789 0.340 2.971 0.483 21 2.014 0.756 2.528 0.177 22 1.647 0.440 2.632 0.188 23 1.975 0.296 2.435 0.324 24 1.783 1.038 1.409 0.178 25 1.807 0.384 1.306 0.096 26 1.281 0.809 1.969 0.081 27 1.327 0.486 1.866 0.144 28 0.693 0.253 1.236 0.189 29 1.027 0.520 0.708 0.358 30 1.022 0.245 1.198 0.298 31 0.941 0.298 1.262 0.019 32 1.140 0.450 1.150 0.142 33 0.563 0.452 1.074 0.161 34 0.678 0.426 0.768 0.134 35 0.622 0.381 0.688 0.122 36 0.694 0.239 0.806 0.043 37 0.609 0.168 1.330 0.177 38 0.678 0.239 1.107 0.165 39 0.768 0.356 1.144 0.253 40 0.516 0.196 0.868 0.141 41 0.523 0.446 0.731 0.071 42 0.779 0.387 0.557 0.151 43 0.604 0.367 0.531 0.036 44 0.554 0.389 0.899 0.290 45 0.506 0.174 0.588 0.086 46 0.823 0.286 0.386 0.049 47 0.326 0.438 0.310 0.099 48 0.621 0.573 0.677 0.072 49 0.308 0.167 0.592 0.189 50 0.586 0.497 0.306 0.083 ⋮ ⋮ ⋮ ⋮ ⋮ 246 0.011 0.014 0.012 0.021 247 0.021 0.010 0.007 0.045 248 0.007 0.029 0.013 0.018 249 0.024 0.038 0.026 0.001 Table 3 shows the results from Levene’s test. Levene's test compares 2 samples and tests for equality of variances. The benefit of Levene's test compared to other tests on the equality of variances such as the F-test is that it is robust for samples that are not normally distributed. From a diversification perspective, this section shows whether further successive increases in portfolio size results in a statistically significant change in variance. For equally weighted portfolios, increasing the portfolio size from 33 to 34 during the 'pre-crisis' period and increasing the portfolio size from 41 to 42 during the 'crisis period' no longer leads to a change in risk that is statistically significant. For market capitalisation weighted portfolios, increasing the portfolio size from 16 to 17 during the 'pre-crisis' period and increasing the portfolio size from 10 to 11 during the 'crisis period' no longer leads to a change in risk that is statistically significant. From the results, we also see that the market 9 International Journal of Economics and Finance Vol. Data from Table 4 is also presented in Figure 2 and Figure 3. Figure 2 shows how the benefit of full diversification decreases monotonically with n, and it was markedly lower during the crisis period as is 4.2 Simulation Summary Figure 2 shows how the benefit of full Table 4. Benefit of Full Diversification (Annualised % Return) Data from Table 4 is also presented in Figure 2 and Figure 3. Figure 2 shows how the benefit of full diversification decreases monotonically with n, and it was markedly lower during the crisis period as is 10 Inteernational Journaal of Economicss and Finance Vol. 4, No. 11; 2012 www.ccsennet.org/ijef consistent horizontal with the find line that repre dings of the L esents the cost Levene’s Test. of diversificat Figure 3 is a tion, equal to 0 a rescaled vers 0.40% as set ou sion of Figure ut in the sectio e 2 that includ on 3.3.2. des a Fig Figure 3. C gure 2. Benefit Cost-Benefit A t of Full Diver Analysis of Fu rsification (An ll Diversificati nnualised % Re ion (Annualise eturn) vs. N ed % Return) vvs. N Figure 2. Benefitt of Full Diver rsification (Annnualised % Reeturn) vs. N Figure 2. Benefitt of Full Diverrsification (Annnualised % Reeturn) vs. N Figure 3. Cost-Benefit AAnalysis of Fu ll Diversificatiion (Annualiseed % Return) vvs. N Figure 3. Cost-Benefit AAnalysis of Full Diversificatiion (Annualiseed % Return) vvs. N As seen fr and n = 1 pre-crisis p equally we om Figure 2 an 172 for an eq period. On the eighted and ma nd Figure 3, th qually weighte e other hand, th arket capitalisa he marginal co ed and marke he crisis period ation weighted ost of diversific et capitalisatio d saw starkly d d portfolio resp cation exceeds on weighted p dissimilar resul pectively. s the marginal portfolio respe lts, namely n = benefit for n = ectively during = 3 and n = 1 f = 133 g the for an 5.3 Alternative Weighting Strategies As is apparent from Figure 2 and Figure 3, the benefit of diversification for a market capitalization weighted portfolio is lower than that for equally weighted portfolios for small values of n, but becomes higher for larger values of n. Based on these observations, we can only conclude that the strategies considered are not systematically different in their relation to diversification benefit. The profile of benefits of diversification for equally weighted and market-cap weighted portfolios differ. The choice of weighting strategy in practical applications at the cost-benefit equivalence point is determined by whether the crossing over of the pair of lines in each period occurs above or below the cost benchmark. From this, we know that the choice between investing in a market capitalization weighted portfolio and an equally weighted portfolio is highly sensitive to the trading regime in consideration. Explaining the factors which affect the relative position between the cost benchmark and the crossover point, however, is beyond the scope of this paper, though we postulate that the cause of the crossover could be due to diversification benefits being insignificant for a small portfolio that is heavily tilted towards a few large cap stocks, and is significant only when the portfolio is larger, more evenly weighted and increasingly granular. There is certainly scope for further econometric analysis in this topic of study. 5.2 Absolute vs. Practical Benefits 5.2 Absolute vs. Practical Benefits In our study, the Levene’s Test showed how increasing the number of securities n in a portfolio led to statistically significant relative gains in diversification benefit. Such results should be interpreted with caution, however. Any form of practical portfolio construction would not be based upon the statistical significance of adding the marginal security, as set out in the seminal Evans and Archer (1968) and Benjelloun (2010), but on a cost-benefit analysis similar to that which we have performed in this paper. To stress on the importance of utilising such a cost-benefit analysis in practical applications, contrary to the Levene’s test, the cost-benefit analysis suggested a diversification extent far short of that suggested by Levene’s Test during the crisis period. This does not come as a surprise given the highly correlated trading observed during the period, and hence the lesson learnt is that practitioners should be weary of outputs from models based on relative benefits of diversification, as opposed to ones that weigh the absolute benefits and costs of diversification. 5.1 Time PPeriod The crisis compared the Levene period showe to the pre-cris e’s Test – the r ed markedly lo sis period. The relative change ower absolute e statistical sig e in diversifica benefits via t nificance of su ation benefit w the cost-benef uch a result is was significant fit analysis due strengthened b at relatively h e to diversific by our results high values of n ation from n. Such adve with the e market str practitione erse sensitivity mpirical findin ress and left-t ers, chiefly bei y of absolute ngs of present tail events (H ing that equity diversification t literature and Hwang & Min diversification n benefits to th d the prevalent n, 2012). This n benefits and he trading and t belief that co s leads to im the asset alloc d economic re orrelations inc mportant implic cation decision egime is consi crease in perio cations for m n is highly sens istent ds of arket sitive 11 International Journal of Economics and Finance Vol. 4, No. 11; 2012 www.ccsenet.org/ijef to ex-ante predictions of trading regime. Such sensitivity should be recognised in the cost-benefit analysis of any portfolio construction process, and while beyond the scope of this study, we postulate that the said margin of error can be lessened if the investor includes international equity or other asset classes that could attenuate the rise in correlation between portfolio constituents during market stress and tail events. Also beyond this study, an interesting topic for investigation is how the benefits of diversification have evolved with time and how they might evolve in the future. Such will build on studies such as that of Christoffersen et al. (2010), which considers the evolution of international diversification benefits; there is scope, however, for studies to be performed on the national level and across asset classes. 6. Conclusion In this study, we investigate the benefits of diversification using alternative weighting strategies. We simulate random portfolios of different sizes and analyse the volatility of these simulated portfolios to determine the risk reduction benefits of holding a larger number of securities. These portfolios were constructed on an equally weighted and market capitalisation weighted basis which we contend is a better approach to obtain more realistic results. We investigate the absolute benefits of risk reduction using Levene's Test and the practical benefits by performing an explicit cost-benefit comparison. We conduct this analysis on the UK equity market over the 'pre-crisis' and 'crisis' period and find that the absolute benefits of diversification for a market capitalisation weighted portfolio are smaller than the absolute benefits of diversification for an equally-weighted portfolio. When we investigate the benefits to diversification over the 2 time periods, we find that the benefits to diversification of an equally weighted portfolio are greater in the 'crisis' than 'pre-crisis' period, but when we look at it from a practical perspective the benefits fall dramatically and the results are reversed. This lends weight to the need to consider not just the absolute benefits but also the achievable benefits of diversification. When comparing the benefits of market capitalisation weighted and equally weighted portfolios, we note that the benefits of diversification tend to be greater for an equally weighted portfolio for small portfolios but that a crossover occurs as the size of the portfolio increases. While analysing the causal factors that lead to differences in values of n between the two periods and weighting methodologies is not within the scope of this paper, there was evidence during the investigation that suggested why the model might have recommended virtually zero diversification during the crisis period. Most significantly, the geometric mean annual return of the m-security population portfolio, at 2.57%, stood just 1.73 12 International Journal of Economics and Finance Vol. 4, No. 11; 2012 www.ccsenet.org/ijef times above the annual risk free rate of 1.48%. The poor market returns sharply reduced the benefit of diversification, given the accompanying cost – the average stock gave a meager if not negative return. Further still, it was apparent from the raw data and summary statistics (which showed that returns were weaker –2.27% vs. 2.57% – and standard deviation higher –26.50% vs. 6. Conclusion 19.81% – for the market-cap weighted portfolio as opposed to the equally weighted portfolio) that large market capitalisation stocks underperformed the index while having higher standard deviations. Such would further decrease the benefit of diversifying into the index, given that these poor-performing large cap stocks will be over-weighted in the portfolio. Lastly, we hypothesise that the heightened correlation between large-cap financials during the global financial crisis, exacerbated by their sizeable presence in the index and their overweighting in a portfolio would have further reduced the marginal benefit of diversification for each n-security portfolio. Further investigation will be required to determine if this is the case. References Abhyankar, A., & Ho, K. Y. (2007). Long-horizon event studies and event firm portfolio weights: evidence from U.K. rights issues re-visited. International Review of Financial Analysis, 16(1), 61-80. http://dx.doi.org/10.1016/j.irfa.2005.02.001 Benjelloun, H. (2010). Evans and Archer – Forty Years Later. Investment Management and Financial Innovations, 7(1), 98-104. Bird, R., & Tippet, M. (1986). Naive diversification and portfolio risk-A Note. Management Science, 32(2), 244-251. http://dx.doi.org/10.1287/mnsc.32.2.244 Box, G. (1953). Non-normality and tests on variances. Biometrika, 40(3), 318-335. http://dx.doi.org/10.2307/2333350 Brown, M., & Forsythe, A. (1974). Robust tests for equality of variances. Journal of the American Statistical Association, 69, 364-367. http://dx.doi.org/10.2307/2285659 Christoffersen, P., Errunza, V., Jacobs, K., & Jin, X. (2010). Is the potential for international diversification disappearing. http://dx.doi.org/10.2139/ssrn.1573345 Damodaran, A. (2010). Equity risk premiums: determinants, estimation and implications – a post-crisis update. Financial Markets, Institutions & Instruments, 18(5), 289-370. Elton, E., & Gruber, M. (1977). Risk reduction and portfolio size: an analytical solution. Journal of Business, 50(4), 415-437. http://dx.doi.org/10.1086/295964 Evans, J., & Archer, S. (1968). Diversification and the reduction of dispersion: an empirical analysis. Journal of Finance, 23(5), 761-767. http://dx.doi.org/10.2307/2325905 Gastwirth, J., Gel, Y., & Miao, W. (2009). The impact of Levene's test of equality of variances on statistical theory and practice. Statistical Science, 24(3), 343-360. http://dx.doi.org/10.1214/09-STS301 Gupta, G. S., & Khoon, C. H. (2001). How many securities make a diversified portfolio in KLSE stocks. Asian Academy of Management Journal, 6(1), 63-79. Hjalmarsson, E., & Manchev, P. (2012). Characteristic-based mean-variance portfolio choice. Journal of Banking & Finance, 36(5), 1392-1401. Huang, W., Eun, C., & Lai, S. (2006). International diversification with large- and small-cap stocks. Journal of Financial and Quantitative Analysis, 43(2), 489-524. http://dx.doi.org/10.1016/j.ribaf.2011.03.003 Hwang, Y, & Min, H. (2012). Dynamic correlation analysis of US financial crisis and contagion: evidence from four OECD countries. Applied Financial Economics, 22(24), 2063-2074. Lai, M. S., & Seiler, M. J. (2001). Is your portfolio overweighted? Know when to say when, Journal of Wealth Management, 3(4), 19-28. http://dx.doi.org/10.3905/jwm.2001.320391 Lee, S. L. (2005). The marginal benefit of diversification in commercial real estate portfolios. Working Papers in Real Estate & Planning 04/05. Levene, H. (1960). Robust tests for equality of variances. In I. Olkin (Ed.), Contributions to Probability and Statistics, 278-292. Palo Alto, California: Stanford University Press. Lim, S., & Loh, Y. (1996). A comparison of tests of equality of variances. Computational Statistical & Data Analysis, 22(3), 287-301. http://dx.doi.org/10.1016/0167-9473(95)00054-2 Markowski, C., & Markowski, E. (1990). References Conditions for the effectiveness of a preliminary test of variance. The 13 Vol. 4, No. 11; 2012 International Journal of Economics and Finance www.ccsenet.org/ijef American Statistician, 44(4), 322-326. http://dx.doi.org/10.2307/2684360 Mukherji, S. (2011). The capital asset pricing model’s risk-free rate. 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This refers to the W statistic that tests the equality of variances between successive securities. Run 1, for example, tests the equality of variances between a portfolio of 1 security and a portfolio of 2 securities. Notes Note 1. This refers to the W statistic that tests the equality of variances between successive securities. Run 1, for example, tests the equality of variances between a portfolio of 1 security and a portfolio of 2 securities. Note 1. This refers to the W statistic that tests the equality of variances between successive securities. Run 1, for example, tests the equality of variances between a portfolio of 1 security and a portfolio of 2 securities. Note 2. Critical Value of F-Statistic for this test is 0.8323066. 14 14
https://openalex.org/W2762305838	https://indieskriflig.org.za/index.php/skriflig/article/download/2262/4768	English	null	A discussion about the version of the Bible available to Muhammad	In die skriflig/In die Skriflig	2,017	cc-by	10,589	1.Surah 2:4 reads, ‘And who believe in the Revelation Sent to thee, And sent before thy time, And (in their hearts) Have the assurance of the Hereafter’ (Ali 1997:17, [author’s italics]). According to Interpretation of the Meanings of The Noble Qur’an, (Bewley & Isa Waley 2007:4, 6), and Shaykh Safi-Ur-Rahman Al-Mubarakpuri et al. (2000:1.116) the revelation ‘sent before thy time’ refers to the Torah, the Gospel, and ‘previous Messengers’. While those ‘previous Messengers’ may include ‘Arab, non-Arab, or a person of a previous Scripture’, the main emphasis is upon ‘People of the Book’ or Jews and Christians: they have a special significance … since they believe in their Books and in all the details related to that, so when such people embrace Islam and sincerely believe in the details of the religion, then they will get two rewards. (Shaykh Safi-Ur-Rahman Al- Mubarakpuri et al. 2000:1.118) Everyone else only gets one and that in only a ‘general way’. In die Skriflig / In Luce Verbi ISSN: (Online) 2305-0853, (Print) 1018-6441 In die Skriflig / In Luce Verbi ISSN: (Online) 2305-0853, (Print) 1018-6441 Page 1 of 9 Original Research Original Research Page 1 of 9 Dates: Received: 05 Apr. 2017 Accepted: 11 July 2017 Published: 11 Oct. 2017 Received: 05 Apr. 2017 Accepted: 11 July 2017 Published: 11 Oct. 2017 How to cite this article: Stoker, H.G. & Derengowski, P., 2017, ‘A discussion about the version of the Bible available to Muhammad’, In die Skriflig 51(2), a2262. https://doi.org/10.4102/ids. v51i2.2262 How to cite this article: Stoker, H.G. & Derengowski, P., 2017, ‘A discussion about the version of the Bible available to Muhammad’, In die Skriflig 51(2), a2262. https://doi.org/10.4102/ids. v51i2.2262 A discussion about the version of the Bible available to Muhammad It is a mandate that all Muslims believe in all previous revelations given by God along with the Qur’an (Surah 2:4). Relative to discussions with Christians, Muslims are required to believe the Bible. Some Muslim apologists today contend that the Bible has been ‘corrupted’ or tainted through the infusion of faulty doctrines and the exclusion of valuable texts that support Islamic ideas by dubious scribes and malicious copyists. According to them there is no way of knowing what was in the ‘original text’ of the Bible. This article offers both a response to the Muslim apologist arguments regarding biblical integrity and trustworthiness as well as explains that what Muhammad knew as the Bible through the Syriac Peshitta is essentially the same in biblical content as what most reputable Bible versions contain today. Through the efforts of labour intensive manuscript discovery and exhaustive textual criticism, both Christians and Muslims can know with precision what the early writers of both the Old and New Testament wrote as ‘inspired’ Scripture. In order for the Muslim to be consistent in following the mandate to believe all the books previously given by God as well as the Qur’an, he must believe the Syriac Peshitta, or a Bible version that is a comparable translation, in order for the Muslim mandate to make sense. Such a concession, however, places the Muslim in an extremely difficult position that needs to be discussed between Christians and Muslims if they both wish and desire to be thought of as worshiping the one true God. Corresponding author: Paul Derengowski, paul.derengowski@yahoo. com Introduction To convince his audience that Jesus is Lord and Messiah, Peter, on the day of Pentecost (Ac 2), refers several times to Old Testament prophecies that were fulfilled in Jesus Christ. The Old Testament was the source of authority in which the Jews believed and Peter declared the arrival, redemption and resurrection of our Lord on the basis thereof. From the beginning of his speech on the Areopagus, Paul connects with the beliefs and practices of the Hellenistic times. Instead of citing the Old Testament as authority, Paul uses the creation, which the Greek philosophers are engaged in their study, to bring the people to the Creator and thus to salvation in Christ (Ac 17:22ff.). It is important to know what carries weight for those who listen to you. Because the Qur’an is authoritative to Muslims, it is important for Christians in their conversations with Muslims to know what the Qur’an has say about the Bible and to what extent a connection can be found through it. Copyright: Copyright: © 2017. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. Muslims place great emphasis upon written revelation, particularly the Bible, as a guide to lead them in not only their quest for the truth, but in their relationship with Allah. Knowing what the Bible says is imperative, if not an integral part, for anyone claiming to be a follower of Islam. Muhammad ibn Abdallah ibn Abd al-Muttalib, the founder of Islam, and subsequent Muslim writers and leaders have made it a requirement to read, study and augment their understanding of the Bible (see Ahmad ibn Naqib al-Misri 1994:811; Ali 2012:147; Surah 4:136). In fact, according to Surah 2:4, a Muslim’s walk in this life as well as his eternal welfare absolutely depends upon the Christian Bible.1 Muslims seem to have a love-hate relationship when it comes to the Bible. While Muslims must extol the value of the Bible as a recorded prerequisite to the establishment of the Qur’an, they Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. Open Access http://www.indieskriflig.org.za Page 2 of 9 Original Research Original Research Page 2 of 9 that would pertain only to the Gospels or Injil. Such confidence led the highly respected Muslim scholar, Abdullah Yusuf Ali (1997), to opine as follows: demean its trustworthiness as something that has been tampered with by unscrupulous translators and dishonest scribes. In other words, they love the Bible when it supports their Islamic worldview, but they hate it when its history and doctrine run contrary to everything that they presuppose to be true. We are thus in the true line of those who follow the one and indivisible Message of the One Allah, wherever delivered. If others narrow it or corrupt it, it is they who have left the faith and created a division or schism. But Allah sees and knows all. And he will protect His own, and His support will be infinitely more precious than the support which men can give. (p. Copyright: 56) Therefore, it is the object of this article to discern what the Muslims means by the Bible, particularly as a document they define as ‘scripture’. What version of the Bible was Muhammad referring to when he spoke of ‘the Revelation sent to thee, and sent before thy time’? Was it basically the same text that Early Church authorities discovered and now comprise the current 66 books or was it something wholly other? If it was based on the same Hebrew and Greek manuscripts that make up all reputable translations currently used by Christians today, then why do most Muslims demonstrate such animosity toward them? If the translation Muhammad referred to was something wholly other, then what is the manuscript evidence to support that translation and what version of the Bible does the modern-day Muslim believe to be absolutely credible, beyond a flaw and unequivocally supportive of his beliefs that contradict Christian doctrine? While it is wonderful, at least from the Christian perspective, that anyone would make such a reassuring confession, it begs the question of just what Muhammad, A.Y. Ali and others of the Muslim faith meant or mean by believing in all those writings relative to Judeo-Christian history. Because there is no indication in the Qur’an or the Hadith – ‘Traditions relating to the deeds and utterances of the Prophet as recounted by his companions’ (Glassé 2002:159) – that Muhammad, while he was alive, understood the Bible as anything other than what the early Christians accepted it to be – meaning the original 66 books and letters that we find today as part of its constitution. What could possibly be askew about such an understanding of Muhammad’s recognition? A brief history of the biblical canon The reason for the inquiry is simple: if the Bible is such an integral part of the Islamic faith, which is even more so the case with Christians, and yet the Christians are being led astray by trusting in an aberrant text regardless of the version in which that text or translation appears, it is then incumbent upon the Muslim to divulge that superior text and bring both groups of people into harmony whereby a consistent worship of the one God is possible. Conversely, if the Muslims are unable to specify that the entire other Bible differs greatly from the original 66 books that constitute it, which is handed down from generation to generation ‘without essential loss’, then the question arises if their criticism of the various Bible versions are necessarily warranted? Much less is his claim that the Bible is an integral part of his faith necessarily true. If that is not necessarily true, then what might be the implications for other adamant statements made by Muslims that are relative to the persons and doctrines both Muslims and Christians hold to be integral? Biblical history, and specifically the Book of Acts, informs us that the gospel message was spread everywhere by Jesus’ apostles shortly after his ascension. This came on the heels of his assurance that they would be his witnesses ‘both in Jerusalem, and in all Judea and Samaria, and even to the remotest parts of the world’ (Ac 1:8). Wherever the apostles went, they shared their message in the vernacular of the people who they encountered. Hence, the original gospel message was an oral gospel that would later be written as the apostles eventually died and subsequent Christians carried on the evangelistic tradition (Ackroyd & Evans 1970:286–287; Barclay 1991:41–43; Barr 1983:12–13; Barrera 1998:104–107; Carson, Moo & Morris 1992:20-21; Comfort 1990:3; Eusebius 1953:3.39.4; Graham 1987:120–121; Guthrie 1970:222ff.; Johnson 1986:131; McDonald 1995:139; Perkins 1980:196–201; Von Campenhausen 1972:103–104). Such writing helped to preserve the authenticity and authority of the apostolic preaching and teaching. The Bible’s composition in Islam The overall consensus is that we know with extreme confidence what the New Testament gospel and text comprised.2 This led the late biblical scholar and textual critic, Sir Frederic Kenyon (1958) to write: One other fact needs to be pointed out, which is simply that the biblical canon was established and essentially closed long before Muhammad and Islam ever graced the earth with their presences. Whether one accepts the Council of Jamnia theory, which dates the closing of the Old Testament Canon at 90 AD3 or another theory that dates its closing later in the 3rd or 4th century AD, and therefore repudiates the Jamnia theory (Anderson 1959:13; Davies 1998:43–44; Harrison 1969:277–279; Leiman 1991:125ff.; McDonald 1995:35, 49–50; Rowley 1950:170; Young 1958:160), by the time Muhammad arrived on the human scene, the Law, the Prophets and the Hagiographa [the Writings] were set.4 The same applies to the New Testament. All of the books and letters that comprise its make-up were determined no later than 300 AD, with few exceptions. By 367 AD, the Early Church Father, Athanasius, records two catalogue lists of books that became widely accepted by the church as authoritative in respect to both Old and New Testament canons. Athanasius wrote that: These are the fountains of salvation ... that they who thirst may be satisfied with the living words they contain. In these alone is proclaimed the doctrine of godliness. Let no man add to these, neither let him take ought from these. (Schaff 1996:4.552) It cannot be too strongly asserted that in substance the text of the Bible is certain. Especially is this the case with the New Testament. The number of manuscripts of the New Testament, of early translations from it, and of quotations from it in the oldest writers of the Church, is so large that it is practically certain that the true reading of every doubtful passage is preserved in some one or other of these ancient authorities. This can be said of no other ancient book in the world. Scholars are satisfied that they possess substantially the true text of the principal Greek and Roman writers whose works have come down to us, of Sophocles, of Thucydides, of Cicero, of Virgil; yet our knowledge of their writings depends on a mere handful of manuscripts, whereas the manuscripts of the New Testament are counted by hundreds, and even thousands. The Bible’s composition in Islam 55) quite in such a good position … In some passages it seems certain that the true reading has not been preserved by any ancient authority, and we are driven to conjecture in order to supply it. But such passages are an infinitesimal portion of the whole. The Christian can take the whole Bible in his hand and say without fear or hesitation that he holds in it the true Word of God, handed down without essential loss from generation to generation throughout the centuries. (p. 55) are also psychologically associated: the perception of the text as sacred leads partly to a desire to preserve the text without corruption, and partly to a desire to appropriate all its incomparable riches. Furthermore, certainty that the sacred words of the text have in fact been preserved without distortion, adds to the frankness with which the very letter of the text is drawn upon for teaching purposes. (p. 41) Attention to precision and exactitude would be transfer to the transmission of the gospel message, even though initially also done orally. It would not be until a few years after the crucifixion of Jesus that the Christian world would begin to see the gospel message put upon parchment. That is not to say that the gospel would not continue to be transmitted orally. What is meant is that, as the message spread and the church grew exponentially and, as mentioned above, the apostles began to die away, the written page was used to preserve the history, integrity and teachings of both Jesus and his Apostles. Although such manual transmission included thousands of variants among the equally thousands of manuscripts, the message remained coherent and unchanged. A careful perusal through those manuscripts, using textual critical effort, reveals that, amid all the grammatical changes, errors of sight on the part of the scribe doing the transmitting, homoioteleuton [similar ending], harmonization, conflation, attempts to correct previous manuscripts and a host of other faux pas, one will encounter that the message has remained intact. Black (1994:24) concurred with Metzger’s assessment. F.F. Bruce (2000:19–20) concluded that, ‘The variant readings about which any doubt remains among textual critics of the New Testament affect no material question of historic fact or of Christian faith and practice.’ Dan Wallace (2011:55), after putting Bart Ehrman’s hyperbolic criticism into perspective and demonstrating where he agreed with his mentor, Bruce Metzger, who asserted that none of the textual variants found in the Scriptures had an effect upon Christian faith and practice, pointed out that, ‘Suffice it to say that viable textual variants that disturb cardinal doctrines found in the NT have not yet been produced.’ Therefore, if it is true that we know with more than 99% comprehension what the New Testament consisted, even without those manuscripts in hand we could reconstruct the New Testament by consulting the Early Church Fathers, and even though the 100 000+ variants found in extant manuscripts, none of them have any bearing upon New Testament belief, then despite all the presupposed criticisms to the contrary, we know what the early Christians wrote what became today’s New Testament Bible. Indeed, so extensive are these citations [from the Church Fathers] that if all other sources for our knowledge of the text of the New Testament were destroyed, they would be sufficient alone for the reconstruction of practically the entire New Testament. (p. 86) 2.Robertson (1925:21–22) argued that, whatever variants there were in the New Testament text, it only effected ‘a thousandth part of the entire text’. Metzger (1992) wrote: Others, such as Bernhard Anderson (1957:535–536); William Barclay (1991:28–29); R.T. Beckwith (1992:57, 61); Otto Eissfeldt (1965:568); Norman Gottwald (1985:113–114); La Sor, Hubbard and Bush (1982:22); Max Margolis (1948:89); W.O.E. Oesterley (1914:173–174); Oesterley and Robinson (1955:7–8); H.E. Ryle (1899:182–184); James Sanders (1972:94–95); Morton Smith (1971:1), all would agree, either in part or in full with Buhl and Pfeiffer’s assessments. Ackroyd and Evans (1970:133–135) qualify their commitment to Jamnia by presupposing an already established canon and then wrote, ‘[I]t is difficult to doubt that both the tripartite structure of the Canon and its precise contents had been settled soon after a.d. 100, if not earlier.’ Samuel Sandmel (1978:14, n.6) called the Jamnia Council ‘a convenience, not an irrefutable conclusion’. Aage Bentzen (1972:1.31) argued that ‘The synod of Jamnia did not define the Canon, but it undertook a revision’, which was his way of saying there already was a canon in existence. The councilors merely revised it. 3.Frants Buhl (1892:24) was one of the first exponents, if not the first, who advocated this view and wrote, ‘It was not until about a.d. 90 that the whole question [about the Book of Ecclesiastes] was brought up for discussion before a Synod at Jabne (Jamnia, a city not far from the coast, south of Jaffa) … At that Synod the canonicity of the whole of sacred writings was acknowledge.’ The later professor and Harvard scholar, Robert Pfeiffer (1941) was quite straightforward on the matter when he wrote in his Introduction to the Old Testament: 4.Routledge (2008:18) wish to straddle the fence on Jamnia, so to speak, and have it both ways. The Bible’s composition in Islam What is often overlooked in the transmission process of the biblical message is the fact that the early messengers committed to memory the eventual written texts that, early on, is what we commonly call the Old Testament. Meticulous precision would be the best way to describe such an effort, as the Jew was taught from a young age that the Scriptures – the Law, the Prophets and the Writings – were of divine origin and to be valued as one would value his or her own life. Such a commitment meant that very few alterations ever crept into scribal efforts to produce copies of the biblical text. So rare were the variants that Gerhardsson (1998) observed: Muhammad, the founder of Islam, is reported to have said the following: Muhammad, the founder of Islam, is reported to have said the following: Say ye: ‘We believe in Allah, and the revelation given to us, and to Abraham, Ismail, Isaac, Jacob, and the descendents [sic] (children of Jacob) and that given to Moses and Jesus and that given to (all) Prophets from their Lord: We make no difference between one and another of them: and we bow to Allah (in Islam)’. (Surah 2:136) Similar declarations are found elsewhere in the Muslim holy book (Surah 2:285; 3:84, 119; 4:136). Muhammad, in other words, recognised the importance of the biblical witnesses in both the Old and New Testament, although, in the latter case, It is just because it is the Sacred Word, the source of endless riches, which is found in the Scriptures, that each and every syllable must be both preserved and used. These two tendencies http://www.indieskriflig.org.za http://www.indieskriflig.org.za Open Access Original Research Page 3 of 9 Page 3 of 9 quite in such a good position … In some passages it seems certain that the true reading has not been preserved by any ancient authority, and we are driven to conjecture in order to supply it. But such passages are an infinitesimal portion of the whole. The Christian can take the whole Bible in his hand and say without fear or hesitation that he holds in it the true Word of God, handed down without essential loss from generation to generation throughout the centuries. (p. 3.Frants Buhl (1892:24) was one of the first exponents, if not the first, who advocated this view and wrote, ‘It was not until about a.d. 90 that the whole question [about the Book of Ecclesiastes] was brought up for discussion before a Synod at Jabne (Jamnia, a city not far from the coast, south of Jaffa) … At that Synod the canonicity of the whole of sacred writings was acknowledge.’ The later professor and Harvard scholar, Robert Pfeiffer (1941) was quite straightforward on the matter when he wrote in his Introduction to the Old Testament: At the close of the first century of our era, following the fall of Jerusalem in 70 and the resulting disorganization of Judaism, the Council at Jamnia (ca. a.d. 90), under the leadership of Johanan ben Zakkai, fixed for all times the canon of scriptures. (p. 64) Others, such as Bernhard Anderson (1957:535–536); William Barclay (1991:28–29); R.T. Beckwith (1992:57, 61); Otto Eissfeldt (1965:568); Norman Gottwald (1985:113–114); La Sor, Hubbard and Bush (1982:22); Max Margolis (1948:89); W.O.E. Oesterley (1914:173–174); Oesterley and Robinson (1955:7–8); H.E. Ryle (1899:182–184); James Sanders (1972:94–95); Morton Smith (1971:1), all would agree, either in part or in full with Buhl and Pfeiffer’s assessments. Ackroyd and Evans (1970:133–135) qualify their commitment to Jamnia by presupposing an already established canon and then wrote, ‘[I]t is difficult to doubt that both the tripartite structure of the Canon and its precise contents had been settled soon after a.d. 100, if not earlier.’ Samuel Sandmel (1978:14, n.6) called the Jamnia Council ‘a convenience, not an irrefutable conclusion’. Aage Bentzen (1972:1.31) argued that ‘The synod of Jamnia did not define the Canon, but it undertook a revision’, which was his way of saying there already was a canon in existence. The councilors merely revised it. 4.Routledge (2008:18) wish to straddle the fence on Jamnia, so to speak, and have it both ways. Georgetown University professor and defender of Islamism, John Esposito (2002) wrote: Georgetown University professor and defender of Islamism, John Esposito (2002) wrote: The inhabitants of those two towns, particularly Yathreb, had been influenced by the beliefs of Jews and Christians. The word Allah, meaning The God, was in use among them. They considered themselves to be descendants of Abraham, and were more or less acquainted with the legends of the Children of Israel and stories of the Old Testament. The story of Adam and Satan was generally known to them. They believed in the existence of angels and imagined them to be daughters – a fallacy to which the Qor’an several times alludes. (p. 35) Like Judaism and Christianity, Islam originated in the Middle East. It was not a totally new monotheistic religion that sprang up in isolation. Belief in one God, monotheism, had been flourishing for many centuries. Knowledge of Judaism, Christianity, and Zoroastrianism had been brought to Mecca in Arabia by foreign caravan trade as well as through the travels and contacts of Meccan traders throughout the Middle East. Moreover, Christian, Zoroastrian, and Jewish tribes lived in Arabia. (pp. 6–7) Christian Sociology Professor, emeritus, Alvin Schmidt (2013) observed that: By the fifth century, Arabia and Syria were known as the meeting place of Christian heresies. And by Muhammad’s time (early seventh century) numerous Christian sects were present: Arians, Ebionites, Valentinians, Basilidians, Gnostics, Carpocratians, Nestorians, Jacobites, Nazarites, Marcionites, Monophysites, Eutychians, Sabellians, Collyridians, Mariamites, Anti- Dicomariamites, and Monothelites. (p. 80) What started with Jesus, would carry over to the establishment of Christian colonies in the Arabian Peninsula. Their influence would have a great impact upon desert dwellers: The steady spread of the Gospel during the second century is evident from the fact that congregations (ekklesiai), each with its episkopos or pastoral overseer, were found in most towns and villages of the Province of Arabia when visited by Origen on various occasions during the first half of the third century. (Trimingham 1979:51) According to UC-Berkeley History Professor, Emeritus Ira Lapidus (2002): The presence of Christianity in Arabia imposed their particular ‘Christian’ views upon the Arabs, which later influenced the thinking of Islam’s founder. Islamic apologist, Karen Armstrong (1991:57), wrote, ‘At the beginning of the seventh century, the Arabs of central Arabia were surrounded by deviant forms of Christianity …’ The divisiveness of the Christian sects led Justo Gonzalez (1975) to conclude: As noted previously, Christian missionaries spread the Gospel everywhere they went. It is a mandate given by Jesus in Matthew 28:19–20 and fulfilled, starting in Acts 1:8. Although the Bible makes it clear that the Gospel spread throughout Judea, Asia Minor, ancient Macedonia unto Rome and possibly even as far as Spain, there is paltry written evidence of its infiltration into Arabia shortly after Pentecost. It is not that the Arabs are not mentioned in the Bible (see Ac 2:11); it is that the missionary effort is not readily noticeable in the biblical text and as Trimingham (1979) observed: Thus, monophysism and Nestorianism in Syria, monophysism in Egypt, and the remnants of Donatism in Africa opened the way to Islam, which was seen by many as the arm that God had caused to rise in order to chastise the Byzantine Empire. (p. 2.105) Jesus must have been in close touch with Arabs. In his homeland of Galilee he would meet them every day. His active ministry was carried on primarily among the pagan populations of Phoenicia, Ituraea, Batanaea, and the Decapolis … His itinerant ministry, though embracing Phoenicia and Lebanon, was concentrated on Arab regions, Ituraea, and in the Decapolis, among Arab peasantry rather than in Hellenistic cities. The region of Caesarea Philippi, around the present-day Banyas and near the sources of the Jordan, which was his place of retreat, less from Jews than from Galilean revolutionaries who wished to make him their leader, was inhabited by half-settled Arab Ituraeans. (p. 41) Islamic scholar Husein Haykal (1976) projected that: Islamic scholar Husein Haykal (1976) projected that: When he [Muhammad] arrived at Busrah [while in the employ of his wife, Khadijah, on a commercial trip], he came into contact with Syrian Christianity and talked to the monks and priests, some of whom were Nestorians. (p. 61) Christian Sociology Professor, emeritus, Alvin Schmidt (2013) observed that: According to UC-Berkeley History Professor, Emeritus Ira Lapidus (2002): Islamic societies were built upon the framework of an already established and ancient Middle Eastern civilization. From the pre- Islamic Middle East, Islamic societies inherited a pattern of institutions which would shape their destiny until the modern age. These institutions included small communities based upon family, lineage, clientage, and ethnic ties, agricultural and urban societies, market economies, monotheistic religions, and bureaucratic empires. The civilization of Islam, though born in Mecca, also had its progenitors in Palestine, Babylon, and Percepolis. (p. 3) The late Iranian scholar, Ali Dashti, distinguished the Bedouin from the city-dweller by observing that those outside the more populated areas were more idolatrous than those within. The reason for this was the Jewish- Christian presence in the cities. According to Dashti (1994), and particularly with reference to Mecca and Medina, Muhammad’s places of rearing and ruling: The late Iranian scholar, Ali Dashti, distinguished the Bedouin from the city-dweller by observing that those outside the more populated areas were more idolatrous than those within. The reason for this was the Jewish- Christian presence in the cities. According to Dashti (1994), and particularly with reference to Mecca and Medina, Muhammad’s places of rearing and ruling: The Bible’s composition in Islam In the case of the Old Testament we are not Such lists have remained complete and they constitute our current biblical composition. The only exception being that the Roman Catholic Church subsequently added the Apocrypha to the lists, which were recognised by the Early Church Fathers as ‘interesting’, but were not of the same weight and calibre as those adopted as inspired scripture. What bearing would this have upon the biblical version available to Muhammad? 4.Routledge (2008:18) wish to straddle the fence on Jamnia, so to speak, and have it both ways. Open Access http://www.indieskriflig.org.za Page 4 of 9 Page 4 of 9 Original Research Original Research Dashti (1994) added: While Greek was the lingua franca of most of the biblical world, not every nation or territory, visited by a Christian missionary, necessarily spoke or wrote in Koiné Greek. Judea’s neighbour to her south, Arabia, was one such territory. Arabic, which is a Semitic language, along with its different dialects, is indigenous to the Arabian Peninsula. Therefore, in order for a Christian missionary to make inroads into the Arabian culture, it required the scaling of the language barrier. One advantage the Christians may have had is the fact that Aramaic was akin to Arabic and Hebrew and was also spoken widely throughout the Middle East. Because many, if not most, of the original Christians were Jewish and hence spoke Aramaic as well as Greek, it is not improper to deduce that when they met the Peninsula Arabs, they were already familiar with their dialect. Prior to the Christian missionary effort to evangelise the Arabs, there was a large contingent of Jews already living in the Hijaz prior to Muhammad’s arrival, and especially in southern Arabia. Although removed from the immediate environs of Israel, according to Sidney Griffith (2013:11), the Arabian Jews were in ‘continuous contact with Jews elsewhere proper, and particularly in Palestine, and that they were fully aware of current Jewish traditions, both scriptural and rabbinic’. The Arabian Jews were a multilingual culture, speaking both Aramaic and its sister languages, Arabic and Syriac. Such an arrangement would allow for not only commercial trade between the Palestinian Jews to the north and Arabic-speaking Jews to the south, it would also afford the propagation and proliferation of Judaism among the pagan Arabs. As Islamic scholar, Alfred Guillame (1956), points out: The problem with such a conclusion is that no one has ever produced an Arabic text of the Bible that Christians used ‘prior to the rise of Islam’ (Griffith 2013:49, 98). What we have, according to Griffith (2013:49), are ‘tenuous extrapolations’ that amount to ‘Wishful thinking’. This is not to say that the gospel was not being preached and taught throughout the Arabian Peninsula in the Arabic dialect. As mentioned earlier, the early propagation of the biblical gospel was by word of mouth and not through the reading of a text. Dashti (1994) added: Perhaps those very priests or some others discussed the religion of Jesus with Muhammad, which had by then divided itself into several sects and parties. Ironically, such influences, over the course of time, would have a deleterious effect upon the Arabian culture that would cause it to regress back into anti-Christian thought. Such regression would turn into aggressive hostility toward both the Jews and Christians to the extent that Christian historian, Philip Schaff Furthermore these town-dwellers had adopted several Jewish practices such as circumcision, ritual ablution, avoidance of menstruating women, and observance of a rest-day, for which they chose Friday instead of Saturday. (pp. 35–36) Further evidence of the Christian missionary influence is seen in the number of heretical sects that successfully http://www.indieskriflig.org.za Open Access Page 5 of 9 Original Research Original Research According to Kashouh (2012:318), the first defence in written form of the Christian faith in Arabic was issued circa 750 AD. Because of our knowledge of such events, the first Christian texts appeared in Arabic sometime before that date. Kashouh (2012:318) argued that evidence of this is seen in two palimpsests, ‘Sinai, Ar. 514 and Codex Sinai, Ar. N.F. Par 8 and 28’, one of which (Codex Sinai) likely contained Luke’s Gospel. Although he (Kashouh 2012:318) is not absolutely certain of the discovery, ‘the text is most likely to be a Christian text and pushes back the hypothesis of the existence of the Arabic Bible to the seventh century if not earlier’. Because of ‘contaminations’ in that text, it is not only plausible, but also ‘possible’ that ‘a number of the eighth/ ninth century manuscripts originated in the seventh century’. In fact, due the exclusive nature of the Arabic text that was produced, which ‘is incompatible with biblical texts of southern Palestine the roots of which go back to the seventh century, it is indeed plausible to propose that the Arabic Gospel text first appeared in the pre-seventh century era’ (Kashouh 2012:319, [Kashouh’s italics]). (1996:4.159), described it as ‘wild, warlike’ and ‘eclectic’ – much like the religion that Muhammad would eventually establish, contrary to later claims otherwise. (1996:4.159), described it as ‘wild, warlike’ and ‘eclectic’ – much like the religion that Muhammad would eventually establish, contrary to later claims otherwise. Dashti (1994) added: As Griffith (2013:98) further argues, it would not be until after Islam’s rise and Muhammad’s death that the importance of collecting the Qur’an’s many surahs, along with the Hadith, became an issue. Suddenly there was a need to preserve the sayings and teachings of the prophet, but that would only be done in Arabic. Development of Arabic grammars and dictionaries would not occur until the second half of the 8th century (Griffith 2013:103). Translation of the Greek, Hebrew or Aramaic text of the Bible into Arabic would follow, meaning that those texts would also not come into being until at least the 8th century, and that to compete with the Qur’an. Given the number of biblical allusions to stories and characters found in the Qur’an – many of which were distorted recollections by the heretical sects already mentioned – it should come as no surprise that, later on, the followers of Muhammad would read and rehash those distortions as they made their way into the Qur’an. It is another reason why there was no effort on the part of the ‘Christians’ to produce, except possibly in note form, a Bible in Arabic that would have possibly kept in check the distortions being spread abroad among the tribal pagans prior to Islam’s rise. At the dawn of Islam the Jews dominated the economic life of the Hijaz. They held all the best land in the oases of Taima, Fadak, and Wadi-l-Qura; at Medina they must have formed half of the population…the Jews of the Hijaz made many proselytes among the Arab tribesmen. (pp. 11–12) The Syriac Peshitta is important for at least three reasons: If it is true that the Peshitta is as well attested and preserved as is contended, then what the Syrian Christian Church knew about the gospel in the 6th and 7th centuries of Muhammad’s earthly existence is exactly what biblical Christians know about it today. It is not something wholly other that the Muslim apologist wishes everyone to believe. Aside from the verbal transmission of the New Testament, the text that most likely had the greatest impact upon Arabian culture at the time Muhammad spoke of the wonders of previous ‘revelations’, ‘books’, and ‘scriptures’ was handed down by the Syrians. They gave aid to not only the Jews and Christians in Arabia, but the influence of the Old Testament, along with five versions of the New Testament they translated into Syriac, was witnessed as far as Lebanon to the north, China to the east and, of course, Arabia to the south. Although the Old Syriac version was not well attested, one particular version, the Peshitta, was The Syriac Peshitta is important for at least three reasons: The Syriac Peshitta is important for at least three reasons: • First, aside from Jewish and Christian usage, it was the version being utilised by both the Nestorians and the Jacobites (Monophysites) as they grappled over the identity of Jesus. The conclusions they drew would be reacted to by Muhammad when he taught ‘(Far is He) from having the partners they [Jews and Christians] associate (with Him)’ (Surah 9:31). Subsequent Muslims would later take up the gauntlet and ‘fight’ those foes as projections against orthodox belief. Second, the Peshitta version not only consisted of all the books common to the Hebrew Old Testament canon (along with several apocryphal works); it also contained 22 books from the New Testament canon. It included all four Gospels, the Apostle Paul’s letters and the Book of Hebrews, with only 2 Peter, 2 and 3 John, Jude and the book of Revelation left out. Due to the faithfulness of those who translated it from Greek into Syriac, anyone familiar with the former would have a good idea of what went into the latter. The Old Testament had been translated from Hebrew and Aramaic into Greek by the time both the Jews and Christians reached the Hijaz. It was more commonly known as the Septuagint (LXX). Rival stories caused conflict over the designated terminology, Septuagint: whether the naming of the translation was due to the number of Jewish scribes employed to create it or whether it had to do with the number of elders who accompanied Moses to Mount Sinai to receive the Law from God. Regardless, the Old Testament was accessible in a language the Jews were speaking and/or writing while they were dispersed abroad. The finalisation of the Old Testament text that encompassed more than just the Torah occurred early in the 2nd century AD. Such wide acceptance, though, would extend into the Christian community, only to be eventually rejected by the Jews, because ‘some Christians had based some of their criticisms against Judaism upon faulty LXX texts’ (McDonald 1995:89). Such fidelity leads to the important third reason, namely the natural rebuttal of some modern-day Muslim apologists who argue that what can be known about the Torah and the gospel is something wholly other than what Muhammad knew about them in his day. Aland and Aland (1989:194) add the following: The Peshitta version of the New Testament is the most widely attested and most consistently transmitted of the Syriac New Testament versions. The Syriac church still preserves it and holds it in reverence in this form today. (p. 194) The pre-Islamic Arabic version of the Bible He was obviously aware of something that he found worthy of adoration. But, what was it? Middle East and North Africa in the mid–7th century. Because Muhammad was dead at the time of the Arabic Bible as well as the Qur’an’s rise, what ‘books’, particularly of the Bible, was he referring to then when he testified that he believed in it and that, by necessity, all subsequent Muslims must also believe in them (Surah 2:285; 4:136; 6:92)? Moreover, how did he believe in them? Was it something written or merely audible? He was obviously aware of something that he found worthy of adoration. But, what was it? Aland and Aland (1989:194) add the following: The pre-Islamic Arabic version of the Bible Given the influx of both Jews and Christians in Arabia long before Muhammad Islamised the Hijaz, and due to the success of both groups to garner converts, even though in the latter case, the ‘Christians’ were of several heretical sects. As noted above, it must be asked if either group translated any part of the biblical canon into the native Arabic in order to spread their messages. It is a question that has provoked scholars to both affirm and deny the reality. Without rehashing the long history centred on the question of textual transmission from Greek, Aramaic or Syriac into Arabic, two of the most recent arguments from Hikmat Kashouh and Sidney Griffith – the former scholar is a proponent of a pre-Islamic Arabic version of the Bible (or at least the gospel), with the latter scholar rejecting such a proposal – will be investigated. While Kashouh’s argument shares much with other scholars on the subject (e.g. Anton Baumstark and Ifran Shahid) as ‘plausible’ or ‘possible’, Griffith’s counter-argument, pointing out the lack of tangible evidence, is enough to defeat the ‘wishful thinking’. There was no Bible written in Arabic prior to Islam’s rise and sudden expansion throughout Arabia, the http://www.indieskriflig.org.za Open Access Open Access Page 6 of 9 Original Research Page 6 of 9 copied and distributed with great vigour and faithfulness. As Bruce Metzger (1977) observed: Syrian scribes devoted great care to the transcription of the Peshitta version. A remarkable accord exists among the manuscripts of every age, there being on the average scarcely more than one important variant per chapter. (p. 49) copied and distributed with great vigour and faithfulness. As Bruce Metzger (1977) observed: Syrian scribes devoted great care to the transcription of the Peshitta version. A remarkable accord exists among the manuscripts of every age, there being on the average scarcely more than one important variant per chapter. (p. 49) Middle East and North Africa in the mid–7th century. Because Muhammad was dead at the time of the Arabic Bible as well as the Qur’an’s rise, what ‘books’, particularly of the Bible, was he referring to then when he testified that he believed in it and that, by necessity, all subsequent Muslims must also believe in them (Surah 2:285; 4:136; 6:92)? Moreover, how did he believe in them? Was it something written or merely audible? The version of the Bible available to Muhammad But, why is the Peshitta version relevant to our thematic question? As has been already established, both the Jews and the Christians occupied land and cities in the Hijaz prior to Muhammad’s existence. The texts that those Jews and Christians used as aids to memory while they propagated their message verbally were already confirmed and essentially closed by the mid–4th century AD. Although the lingua franca of the day was Greek, not all Jews or Christians necessarily spoke or wrote in Greek, but in other languages such as Latin in the West, Syriac in the East and Coptic in North Africa as they spread abroad throughout the Middle East, Asia and eventually into the Arabian Peninsula. Literally thousands of copies of biblical manuscripts, many of which are extant today, were produced as a result of the rich diversity of the languages and cultures that were encountered by both the Jews and Christians as they shared their messages. But, why is the Peshitta version relevant to our thematic question? Prophesying the coming of Muhammad The Bible has not been changed, nor has it been ‘corrupted’, in other words, if the Peshitta is the highlighted ‘revelation’ behind Muhammad’s assertion. Many of the later Muslim apologists repeatedly assert just how corrupted or tainted any current revelation is by comparing it with the ‘original text’. Nevertheless, the Syriac Peshitta is nothing more than a copy, written in another language and handed down with ‘remarkable fidelity’ (Metzger 1992:70) to Syriac-speaking Christians in the proclamation of their messages. What can be known from the contents of both the Old and New Testament is the same information as that which was known by both the Jews and Christians for hundreds of years leading up to the development of the Peshitta. Arguments raised by Muslim spokesmen such as Ajijola (1984:78) who speak of believing in the Torah, Psalms of David and the gospel, but yet denigrate them, because they supposedly do not share the ‘original form’, is misleading if not untrue. Even though Muhammad could not read the Peshitta himself, its contents is essentially the same as that found in the LXX and the Greek text from which the Peshitta was translated. Therefore, whatever charges of ‘tampering’ and/or adulteration is without merit. Again, please note Sir Frederick Kenyon’s comments above in respect to biblical and textual integrity and credibility (see above under the heading titled ‘A brief history of the biblical canon’). A second ramification of accepting the Syriac Peshitta as the Bible version available to Muhammad, alluded to as a previous ‘Revelation’ leading up to the Qur’an, is the disavowal that Muhammad was forecasted as the successor to Jesus as a ‘prophet of God’. It is not uncommon that non- Christian religious followers, and even many who claim to be Christian, wish to exalt their religious leaders to a special status in God’s economy. Typically, this status takes the form of some kind of prophet, seer or revelator. The basis for such exaltation is usually the product of biblical manipulation through poor exegesis of the biblical text coupled with a misapplication based on faulty hermeneutical principles. When the exegesis (more properly eisogesis) and interpretation are found to be wanting, the critic is either attacked personally – the biblical text is demeaned as somehow missing a plain and precious truth – or the Bible is assumed to have been tampered with somehow. 5.Although the cited Qur’anic references specifically allude to the Jews, Islamic belief includes Christians as also engaging in alleged impropriety by accusing them of falsifying (Glassé 2002:86), corrupting (Ajijola 1984:79) or introducing defects into the New Testament and thereby making it ‘obscene’ (Ali 2012:149). Anyone, however, that has spent an appreciable amount of time reading and studying the topic of textual criticism of the Bible knows immediately that such charges and accusations are without merit, if not ‘obscene’, themselves. Prophesying the coming of Muhammad All of that must be denounced when it becomes clear that what Muhammad accepted as the Bible version of his day is the same one used by the Jews and Christians prior to their entrance into the Hijaz. Khurshid Ahmad (1999:86–87) serves as a classic example of a Muslim who believes that the Bible has something to say about Muhammad’s revelation that is exegetically untenable. In his explanation on how the Qur’an influenced human history, he wrote: As long as the Muslims choose to exalt any other non-biblical revelation or to align themselves with the ‘People of the Book’, there must be a consistency in those revelations and Muslim behaviour that honours and not demeans both the Jews and Christians. The Peshitta was ‘the Book’ those people were using at the time they made progress in Arabia – both prior to and during Muhammad’s reign. No longer can the Muslims accuse the Jews of changing (Surah 2:59) or perverting (Surah 2:75) God’s revelation, or speciously writing it with their own hands (Surah 2:79), and not further accuse God of impotence over what he has revealed.5 Moreover, no longer can the Muslims look down upon the Jews as being or becoming ‘apes’ (Surah 2:65; 7:166), ‘swine’ (Surah 5:60) or cursed (Surah 5:60; 9:30; 98:6) simply because the Muslims happen to disagree with any one of a number of beliefs or doctrines they find personally distasteful. Muslims cannot call the Jews or Christians ‘losers’ (Surah 3:85) or encourage others not to befriend them (Surah 5:51; 60:1). They certainly must end their campaign of jihad against them so as to oppress them until they are either killed or ‘feel subdued’ (Surah 9:29, 73, 123; 47:4). In other words, the Muslims must ‘believe the Revelation’, as Muhammad claimed he did that would involve any reputable version In Islam religion has been perfected. That is another way of saying that with Islam the age of new revelation has come to a close, and that the age of realization of the principles revealed religion has been inaugurated. That is why in all the earlier scriptures references are to be found to the advent of the Prophet of Islam. 6.Ali (1997:1461, n. 5438), et al, contends that Paraclete is a corruption for Periclytos or ‘praised one’, referring to ‘Ahmad’ or ‘Muhammad’. However, he offers no manuscript support for his contention and there is no variant at John 14:16, 26; 15:26, or 16:7 to justify his contention. Ramifications for acknowledging Muhammad’s available ‘Revelation’ Since the Syriac Peshitta was most likely the Bible version that Muhammad alluded to in Surahs 2:4, 136, 285; 4:136, 162; 6:92, et cetera, then there are several ramifications for acknowledging it as such. We know its content and that content has not been ‘corrupted’. Any prophecies projected about the ‘prophet’ Muhammad would be dubious at best. The requirement that Muslims must read the Bible would be http://www.indieskriflig.org.za Open Access Page 7 of 9 Original Research faulty. Finally, knowledge about the persons of Jesus and God would be absent. Each of these effects will now be investigated one-by-one to judge their validity and gravity. after the order of the Peshitta or the texts upon which it was based, if they are to be consistent in their claim of following the one true religion. Inconsistency in the Muslim’s acceptance of divine revelation Given that the Syriac Peshitta was the biblical version that Muhammad sanctioned while he was alive, it follows that this version or any one of a number of other similar versions, should be ‘required’ reading for earlier and present day Muslims. Accordingly, the Bible should form the basis for Muslim belief and doctrine, which would include that found in the book Muslims believe, came straight from heaven, namely the Qur’an. There should be no variance, because God would be the author of both. Any progressive revelation would dovetail with previous revelations with the ultimate object of all revelation being the person of Jesus Christ (Lk 24:27; Jn 5:39; 2 Tm 3:15; Heb 10:7; Rv 19:10). That is not the case for the Muslims, however, as they reject, except piecemeal, anything the Bible has to say, especially when it contradicts their own presuppositions. Therefore, the conclusion can be made that today’s Muslims are at variance with what Muhammad thought about the Bible in Surah 2:4, 136, 285, et cetera. Without God’s revelation, there can be no knowledge of him either. It is why the followers of Muhammad would record him saying that: It is not fitting for a man that Allah should speak to him except by inspiration, or from behind a veil, or by the sending of a messenger to reveal, with Allah’s permission, what Allah wills: for He is most high, most wise. (Surah 42:51) If the Muslims reject the Peshitta, as well as any other textual revelation that serves as the basis upon which various Bible translations and versions are created, the mandate that stipulates belief in the Bible is without any authority to enforce it. To state as much as M.M. Ali did, namely that the Muslims are ‘required to believe’ in all the books of God, would be basically meaningless. If it is assumed the Qur’an serves as a corrector or surrogate for the Bible,it once again implies that God is impotent in preserving his previously transmitted revelation(s). Fallen humanity is capable of doing in the reverse what God is incapable of doing initially. Man’s sinful will is more decisive than God’s holy will. Furthermore, it assumes that God is mutable. In the Qur’an’s case, it would project that God somehow garnered more power, will and control over that revelation than over previous revelations. 8.After a description of the high position which Jesus occupies as a prophet, we have a repudiation of the dogma that he was Allah, or the son of Allah, anything more than a man … In Allah’s sight Jesus was as dust just as Adam was or humanity is. (Ali 1997:142, n.398) Prophesying the coming of Muhammad Students of the Bible, for instance, know that Jesus had said: ‘I have yet many things to say unto you, but ye cannot bear them now … He will guide you unto all truth: for he shall not speak of himself; but of whatsoever he shall hear, that shall he speak’ (Jn 16:12–13). A careful examination of the reference Ahmad provides as his proof text to validate Muhammad’s prophethood, reveals that not only is Muhammad not being spoken about by Jesus, but the modus operandi Ahmad used to mislead the reader. Nobody should unjustly cite the Bible to his or her own advantage – especially, if his or her own prophet has such a high esteem for the Bible. Ahmad simply excised the passage to exclude any mention of ‘the Spirit of truth’ in John 16:13 that Jesus mentioned previously in John’s Gospel as the one whom both he and the Father would send as another Paraclete (John 14:16, 17).6 With the advent of the Peshitta in Arabia, there is no room for Muhammad to be included among the biblical prophets. Anyone practicing careful exegesis and proper http://www.indieskriflig.org.za Open Access Page 8 of 9 Page 8 of 9 Original Research the Passion Week, what we know about the person of Jesus is found in the New Testament and nowhere else. The Qur’an’s recollection is highly polemical and proffers nothing of biographical value regarding the historical Jesus. In fact, in the instance of the Qur’an, Muhammad’s ‘revelation’ seems more interested in arguing with those with whom Muhammad is contending, and that with a ‘distinctive prophetology’ in mind, than providing any kind of real historical recollection.7 What little can be derived from the Qur’an in respect to Jesus, has more to do with denigrating his person (Surah 4:157, 171; 5:75, 116; 19:92) than it does in crediting those who wrote about him in their letters according to their personal experiences (1 Jn 1:1–3; 2 Pet 1:16). In other words, in the Qur’an, Jesus ends up being nothing more than an ordinary man (Surah 3:59; 43:59),8 who only came to seek and save one faction of the human race (Surah 3:49; Ali 2012:158) and never dies for anyone (Surah 4:157; 5:110), while Muhammad is viewed as Jesus’ superior, who came to comfort all humans (Surah 21:107; 61:6 cf. Ali 1997:1461, n. 5438). Prophesying the coming of Muhammad hermeneutical skill would not only see Muhammad’s absence, but upon examining the world of Islamic history and doctrine would note that whatever prophetic status he might have, it is different from those who are recognised as biblical prophets. 7.A ‘distinctive prophetology’ is a hermeneutical principle observed by Griffith (2013), whereby the Islamist recognises certain beliefs, stories and phrases common to both Jewish and Christian understanding, but have been criticised and revised with Muhammad as the focal point of absolute truth. As he put it: … it is the Qur’an’s distinctive prophetology that ultimately controls the process of scriptural recollection, determining which biblical narratives are recalled and which are ignored, a feature of the Bible in the Qur’an that is best studied in reference to well-known instances of the phenomenon rather than merely in the abstract. (pp. 58, 62, 70–71, 76, 83, 85) Conclusion Muslims claim that they are ‘required’ to believe in previous revelations concomitant with their own revelation, the Qur’an. The founder of Islam made it clear that he believed in all previous revelations, books and scriptures, which would have meant he accepted the contents of the Syriac Peshitta or the version of the Bible used by the Jews, Inconsistency in the Muslim’s acceptance of divine revelation That, however, would contradict Qur’anic revelation that God was immutable (51:58), which would, in turn, negate that he was self-sufficient (3:2; 20:111) and unified (see Hakim 1992:58) as the one being representative of deity. The only possible way for the requirement to believe in previous revelations to mean anything is for those revelations currently to exist and that there is access to those revelations to be read. Because, according to the Muslims those revelations do not exist except in alleged corrupted or tainted forms, the mandate to believe means nothing in modern-day parlance that also nullifies the words found in the Qur’an – Allah’s most perfect book. Unfortunately, the Muslims deny the continuing existence of previous revelation in the form of the Bible. To them it has been perverted beyond recognition while confessing Muhammad’s approval of the previous revelation. Because that is the case, there is no possible way to confirm later revelation. Therefore, the Qur’an can be no more a revelation from God than any other religious document, because it lacks God’s sanction. Yet, Muhammad acknowledged the existence of previous revelations from God, specifically as it is related to the Bible. The Syriac Peshitta was that relative version every Muslim today must also acknowledge or at least another version consistent with it, lest he be at odds with his prophet and without any true knowledge of God in the world. http://www.indieskriflig.org.za http://www.indieskriflig.org.za No Jesus, nor God The best and only historical document that speaks of the life of Jesus is found in the Bible and that in an extremely abbreviated account. Aside from a short birth narrative, coupled with the last three and a half years of his life that is mainly focused on http://www.indieskriflig.org.za Open Access Original Research Page 9 of 9 Page 9 of 9 Christians and the sects prevalent in the Arabian Peninsula before and after Muhammad came to notoriety. Even though Muhammad accused the Jews and the Christians of corrupting the biblical text, manuscript evidence shows that whatever corruptions there might have been were minimal at best, meaning the astute observer knows what God revealed and what he wanted humanity to retain. Bruce, F.F., 2000, The New Testament documents: Are they reliable? Eerdmans, Grand Rapids, MI. Buhl, F., 1892, Canon and text of the Old Testament, T&T Clark, Edinburgh. Carson, D.A., Moo, D.J. & Morris, L., 1992, An introduction to the New Testament, Zondervan, Grand Rapids, MI. Comfort, P.W., 1990, Early manuscripts & modern translations, Baker, Grand Rapids, MI. Dashti, A., 1994, 23 Years: A study of the prophetic career of Mohammad, Mazda, Costa Mesa, CA. Davies, P.R., 1998, Scribes and schools: The canonization of the Hebrew Scriptures, Westminster John Knox Press, Louisville. Muslims completely reject any version of the Bible, Peshitta or otherwise. To them, the Qur’an is the ‘corrector’ and replacement of all previous revelations, even though it and they supposedly share much in common. The problem, however, still remains that the trustworthiness of the Bible, which Muslims must follow and study to understand God’s message through the ages via his prophets, is denied. Because they accept the Qur’an, which alludes to the importance of the Bible, the Bible can still be used as a source of authority in discussions with Muslims who want to know more about the prophets, and especially about the Messiah, Jesus Christ. Therefore, it is a challenge for the Christian to show the Muslims that what Muhammad adored in the Bible, is the same Bible that Christians today confidently holds in their hands as the Word of God. Eissfeldt, O., 1965, The Old Testament: An introduction, Harper and Row, New York. Esposito, J.L., 2002, What everyone needs to know about Islam, Oxford University Press, Oxford. Eusebius, 1953, The ecclesiastical history, transl. K. Lake, Harvard University Press, Cambridge, MA. References Oesterley, W.O.E., 1914, The books of the Apocrypha, Robert Scott, London. Ackroyd, P.R. & Evans, C.F. (eds.), 1970, The Cambridge history of the Bible, Cambridge University Press, Cambridge. Oesterley, W.O.E. & Robinson, T.H., 1955, An introduction to the books of the Old Testament, SPCK, London. Ahmad ibn Naqib al-Misri, 1994, Reliance of the traveller, ed. and transl. Nuh Ha Mim Keller, Amana, Beltsville, MD. Perkins, P., 1980, The Gnostic dialogue: The Early Church and the crisis of Gnosticism, Paulist Press, New York. Ahmad, K., 1999, Islam: Its meaning and message, The Islamic Foundation, Leicester. Pfeiffer, R., 1941, Introduction to the Old Testament, Harpers, New York. Ajijola, A.D., 1984, The essence of faith in Islam, Presidency of Islamic Research, Riyadh. Robertson, A.T., 1925, An introduction of the textual Criticism of the New Testament, Broadman, Nashville. Aland, K. & Aland, B., 1989, The text of the New Testament, Eerdmans, Grand Rapids, MI. Routledge, R., 2008, Old Testament theology, InterVarsity, Downers Grove. Rowley, H.H., 1950, The growth of the Old Testament, Hutchinson House, London. Ali, A.Y., 1997, The meaning of the Holy Qur’an, Amana, Beltsville, MD. Ryle, H. E., 1899, The Canon of the Old Testament, MacMillan and Co., New York. Ali, M.M., 2012, The religion of Islam, Ahmadiyya Anjuman Isha, Dublin, OH. Sanders, J.A., 1972, Torah and Canon, Fortress, Philadelphia, PA. Anderson, B.W., 1957, Understanding the Old Testament, Prentice-Hall, Englewood Cliffs, NJ. Sandmel, S., 1978, The Hebrew scriptures, Oxford University Press, New Y Schaff, P., 1996, History of the Christian Church, Hendrickson, Peabody, MA. Anderson, G., 1959, A critical introduction to the Old Testament, Gerald Duckworth & Co., London. Schmidt, A.J., 2013, The American Muhammad, Concordia, St. Louis, MO. Armstrong, K., 1991, Muhammad, Phoenix, London. Armstrong, K., 1991, Muhammad, Phoenix, London. Smith, M., 1971, Palestinian parties and politics that shaped the Old Testament, Columbia University Press, New York. Barclay, W., 1991, The making of the Bible, The Saint Andrew Press, Edinburgh. Shaykh Safi-Ur-Rahman Al-Mubarakpuri, et al., (ed.), 2000, Tafsir Ibn Kathir, 10 vol., Darussalem, Riyadh. Barr, J., 1983, Holy Scripture: Canon, authority, criticism, The Westminster Press, Philadelphia, PA. Trimingham, J.S., 1979, Christianity among the Arabs in pre-Islamic times, Longman, New York. Barrera, J.T., 1998, The Jewish Bible and the Christian Bible, Brill, Leiden. Beckwith, R.T., 1992, ‘The Canon of the Old Testament’, in P.W. Comfort (ed.), The origin of the Bible, pp. 51–64, Tyndale, Wheaton, IL. Competing interests Kenyon, F., 1958, Our Bible and the ancient manuscripts, Harper & Brothers, New York. The authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. Lapidus, I.M., 2002, A history of Islamic societies, Cambridge University Press, Cambridge, 2002. La Sor, W.S., Hubbard, D.A. & Bush, F.W., 1982, Old Testament survey, Eerdmans, Grand Rapids, MI. Leiman, S.Z., 1991, The canonization of Hebrew Scripture, The Connecticut Academy of Arts and Sciences, New Haven. Authors’ contributions Margolis, M., 1948 [1922], The Hebrew Scriptures in the making, The Jewish Publication Society of America, Philadelphia, PA. P.D. as the PhD student had done the basic research; while H.G.S. as the PhD promoter supervised it and helped P.D. in the finalisation of the article. P.D. as the PhD student had done the basic research; while H.G.S. as the PhD promoter supervised it and helped P.D. in the finalisation of the article. McDonald, L.M., 1995, The formation of the Christian Biblical Canon, Hendrickson, Peabody, MA. p the finalisation of the article. Metzger, B.M., 1977, The early versions of the New Testament, Clarendon, Oxford. https://doi.org/10.1093/acprof:oso/9780198261704.001.0001 Metzger, B.M., 1992, The text of the New Testament, 3rd edn., Oxford University Press, New York. Acknowledgements Competing interests ohnson, L., 1986, The writings of the New Testament, Fortress Press, New Y Kashouh, H., 2012, The Arabic versions of the Gospels, DeGruyter, Boston, MA. No Jesus, nor God Gerhardsson, B., 1998, Memory & manuscript: Oral tradition and written transmission in Rabbinic Judaism and early Christianity, Eerdmans, Grand Rapids, MI. Glassé, C., 2002, The new encyclopedia of Islam, AltaMira Press, Walnut Creek, CA. Gonzalez, J., 1975 [1970; 1971], A history of Christian thought, Abingdon, Nashville. Gottwald, N.K., 1985, The Hebrew Bible, Fortress, Philadelphia, PA. Graham, W.A., 1987, Beyond the written word, Cambridge University Press, New York. Griffith, S.H., 2013, The Bible in Arabic, Princeton University Press, Princeton, NJ. https://doi.org/10.1515/9781400846580 Guillame, A., 1956, Islam, Penguin, Baltimore, MD. Guthrie, D., 1970, New Testament introduction, InterVarsity Press, Downers Grove. Hakim, K.A., 1992, Islamic ideology, Institute of Islamic Culture, Lahore, India. Harrison, R.K., 1969, Introduction to the Old Testament, Eerdman, Grand Rapids. Haykal, H., 1976, The life of Muhammad, Islamic Book Trust, Jaya, Malaysia. References Von Campenhausen, H., 1972, The formation of the Christian Bible, Fortress, Philadelphia, PA. Bentzen, A., 1972, Introduction to the Old Testament, G.E.C., Copenhagen. Wallace, D.B., 2011, Revisiting the corruption of the New Testament, Kregel, Grand Rapids, MI. Bewley, A. & Isa Waley, M. (eds.), 2007, Tafsir al-Jalalayn, transl. A. Bewley, Dar Al Taqwa Ltd., London. Young, E.J., 1958, ‘The Canon of the Old Testament’, in C.F.H. Henry (ed.), Revelation and the Bible, Baker, Grand Rapids. Black, D.A., 1994, New Testament textual criticism, Baker, Grand Rapids, MI. Open Access http://www.indieskriflig.org.za
https://openalex.org/W2590184723	https://www.nature.com/articles/s41598-017-00105-y.pdf	English	null	Effects of grazing on spatiotemporal variations in community structure and ecosystem function on the grasslands of Inner Mongolia, China	Scientific reports	2,017	cc-by	7,924	Effects of grazing on spatiotemporal variations in community structure and ecosystem function on the grasslands of Inner Mongolia, China Rina Su1, Junhui Cheng2, Dima Chen3, Yongfei Bai3, Hua Jin4, Lumengqiqige Chao4, Zhijun Wang4 & Junqing Li1 Received: 17 May 2016 Accepted: 6 February 2017 Published: xx xx xxxx Received: 17 May 2016 Accepted: 6 February 2017 Published: xx xx xxxx Grasslands worldwide are suffering from overgrazing, which greatly alters plant community structure and ecosystem functioning. However, the general effects of grazing on community structure and ecosystem function at spatial and temporal scales has rarely been examined synchronously in the same grassland. Here, during 2011–2013, we investigated community structure (cover, height, and species richness) and aboveground biomass (AGB) using 250 paired field sites (grazed vs. fenced) across three vegetation types (meadow, typical, and desert steppes) on the Inner Mongolian Plateau. Grazing, vegetation type, and year all had significant effects on cover, height, species richness, and AGB, although the primary factor influencing variations in these variables was vegetation type. Spatially, grazing significantly reduced the measured variables in meadow and typical steppes, whereas no changes were observed in desert steppe. Temporally, both linear and quadratic relationships were detected between growing season precipitation and cover, height, richness, or AGB, although specific relationships varied among observation years and grazing treatments. In each vegetation type, the observed community properties were significantly correlated with each other, and the shape of the relationship was unaffected by grazing treatment. These findings indicate that vegetation type is the most important factor to be considered in grazing management for this semi-arid grassland. Overgrazing has led to extreme changes in the community structure and ecosystem functioning of many grass- lands1–3. On a global scale, researchers have estimated that 60% of grasslands suffer from grazing or overgrazing, and that these conditions are more serious in some areas with high populations of grazing animals4. For example, as the largest ecosystem type in China, grasslands cover 393 million ha, or 42% of the national territory5. During the past three decades, in the grassland ecosystems in China, unsustainable land management techniques, such as overgrazing, have caused serious desertification and salinization6. For example, in the grasslands of Inner Mongolia, which has the largest area of grassland in China, approximately half of the grassland area has suffered from grazing-induced degradation during the past three decades7. Previous studies have independently demon- strated that grazing alters the community structure and ecosystem function of grasslands both at spatial and temporal scales, but the effects of grazing vary among grassland types and observed years4, 8–11. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 17 May 2016 Accepted: 6 February 2017 Published: xx xx xxxx Scientific Reports \| 7: 40 \| DOI:10.1038/s41598-017-00105-y Effects of grazing on spatiotemporal variations in community structure and ecosystem function on the grasslands of Inner Mongolia, China Rina Su1, Junhui Cheng2, Dima Chen3, Yongfei Bai3, Hua Jin4, Lumengqiqige Chao4, Zhijun Wang4 & Junqing Li1 These results have indicated that grazing-induced variations in community structure and function are more complex than originally thought and depend on both grassland context and changes in climatic conditions among observation years. Thus, it is anticipated that simultaneously studying and comparing grazing-induced variation in community 1Key Laboratory for Silviculture and Conservation of Ministry of Education, Forest College, Beijing Forestry University, Beijing, 100083, China. 2College of Grassland and Environmental Science, Xinjiang Agricultural University, Urumqi, 830052, China. 3State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China. 4Institute of Grassland Surveying and Planning, Inner Mongolia, Hohhot, 010051, China. Correspondence and requests for materials should be addressed to J.L. (email: lijunqing8100@163.com) Scientific Reports \| 7: 40 \| DOI:10.1038/s41598-017-00105-y Scientific Reports \| 7: 40 \| DOI:10.1038/s41598-017-00105-y 1 www.nature.com/scientificreports/ structure and function at both spatial and temporal scales in a given grassland will further improve our under- standing for mitigation and adaptation strategies in response to climate change in this field.f g g p g p gi A number of studies have demonstrated that, at different spatial scales or vegetation types, grazing signif- icantly altered community structure (i.e., vegetation cover, community height, and species richness) and eco- system functioning (aboveground biomass: AGB) in grasslands12, 13. At different spatial scales, the patterns and magnitude of the changes in cover, height, species richness, and AGB in response to grazing have been shown to be mediated by vegetation type and climatic conditions14–16. Researchers have generally believed that grazing will reduce cover, height, and AGB in most vegetation types17, whereas species richness will only decrease under benign conditions (i.e., meadow steppe and typical steppe) and grazing will have no significant effects on species richness under harsh conditions (i.e., desert steppe and desert18). Recent reports have also demonstrated that these responses varied depending on different locations with different climatic conditions in grassland ecosys- tems19, 20. For example, the response of ANPP to gazing at dry sites are more vulnerable to wet sites, particularly in semi-arid grasslands20. These results indicate that the spatial response of community structure and ecosystem function to grazing are context dependent, and may be influenced by factors such as elevation and the nutrient content, moisture, and bulk density of soil. In addition to vegetation type, the responses of cover, height, species richness, and AGB at various spatial scales have also been shown to be influenced by climatic gradients. Effects of grazing on spatiotemporal variations in community structure and ecosystem function on the grasslands of Inner Mongolia, China Rina Su1, Junhui Cheng2, Dima Chen3, Yongfei Bai3, Hua Jin4, Lumengqiqige Chao4, Zhijun Wang4 & Junqing Li1 For example, with increasing precipitation, the trends in species richness and AGB in the Inner Mongolian grasslands have been reported to have changed from a linear form in fenced sites to unimodal relationships in grazed sites21. These observations indicate that climatic conditions also play an important role in regulating the responses of community structure and ecosystem function to grazing. By considering the combined findings of these studies, it is further suggested that both biotic (i.e., vegetation type) and abiotic factors (i.e., climatic gradients) should be explored when explaining the influence of grazing on community structure and ecosystem function.f p p gl g g y y In addition to the spatial scale, temporal scale adds another important dimension to understand the effects of grazing on community structure and ecosystem function. For example, independent meta-analyses showed that community cover and AGB responded negatively to grazing, but that plant species richness responded neutrally at a temporal scale10, 22, 23. In addition to the differential responses of these variables to grazing, they are also com- monly modified by interannual variability in climatic conditions, such as rainfall, aridity, and microenvironmen- tal variables10, 11, 22, 23. These results indicate that the responses of community structure and function to grazing are considerably more complex than originally thought, and that they are controlled by multiple factors, including grazing conditions, vegetation type, and climatic conditions. However, we still do not know which among these factors is the most important in terms of regulating the responses of community structure and function to graz- ing, and thus additional studies are needed. g During the past decade, China has implemented a series of national project-related strategies designed to control grassland degradation and to improve grassland conditions. One important strategy is a nationwide con- servation project known as Returning Grazing Lands to Grasslands (RGLG). RGLG aims to restore these dam- aged ecosystems to a healthy state and promote an equilibrium between ecological protection and socioeconomic development24, 25. The RGLG program has established a large number of paired sites (grazed vs. fenced plot) in national grasslands to continuously monitor the recovery of cover, species richness, community height, and AGB. These paired sites include most of China’s grassland types and cover a wide range of climatic conditions, thereby providing an ideal system in which to compare the spatial and temporal responses of community structure and ecosystem function in grasslands. Effects of grazing on spatiotemporal variations in community structure and ecosystem function on the grasslands of Inner Mongolia, China Rina Su1, Junhui Cheng2, Dima Chen3, Yongfei Bai3, Hua Jin4, Lumengqiqige Chao4, Zhijun Wang4 & Junqing Li1 y g Using a long-term dataset from 250 paired sites (grazed vs. fenced) in three grassland types (meadow steppe, typical steppe, and desert steppe) in Inner Mongolia (Fig. 1), we investigated changes in the spatial and temporal patterns of community structure and ecosystem function in response to grazing and precipitation. Specifically, we addressed the following three questions: (1) How does grazing affect community structure (species richness, height, and cover) and function (aboveground biomass: AGB) among vegetation types (spatial scale) and among years (temporal scale) in the Inner Mongolian grasslands? (2) How does grazing affect the forms of the relation- ships between community properties and precipitation in the observation years? (3) How does grazing affect the forms of the relationships among community properties in different vegetation types? Results Eff Effects of grazing, vegetation type, and year on community properties. The results of three-way ANOVA showed that community cover, height, species richness, and AGB were significantly affected by the main effects of grazing treatment, vegetation type, and year, with the exception of the effect of year on species richness (all P < 0.05, Table 1). However, variation in these variables was mainly explained by vegetation type (12.38– 38.31%) and grazing treatment (4.49–13.22%), whereas year explained only 0.28–4.17% of the variation (Table 1). Compared with the fenced plots, community cover, height, species richness, and AGB declined considerably in the grazed plots of meadow and typical steppe during the period 2011–2013, with the exception of cover and species richness in meadow steppe in 2011 and species richness in typical steppe in 2013 (Fig. 2). On average, community cover, height, species richness, and AGB in grazed plots decreased by 17 ± 1% (mean ± standard error), 37 ± 2%, 21 ± 2%, and 44 ± 2%, respectively, in meadow steppe, and by 41 ± 1%, 26 ± 2%, 18 ± 1%, and 41 ± 2%, respectively, in typical steppe, during the period from 2011 to 2013.lf p y yp pp g p In addition to the influence of the main effects, community cover, height, species richness, and AGB were also significantly affected by the interactive effects of grazing treatment × year and vegetation type × year, with the exception of interactive effects of vegetation type × year on AGB (all P < 0.05, Table 1), demonstrating that the effects of grazing treatment on community properties and function depend on vegetation type. This was further affirmed by the fact that the grazing-induced decrease in cover, height, species richness, and AGB in meadow steppe was different to that in typical steppe, as mentioned above. Scientific Reports \| 7: 40 \| DOI:10.1038/s41598-017-00105-y 2 www.nature.com/scientificreports/ Figure 1. Locations of the 250 paired (fenced and grazed pairs) study sites in Inner Mongolia. (A), Meadow steppe (B), typical steppe (C), desert steppe. Map created using ArcGIS 9.3 software. (Environmental Systems Research Institute (ESRI), Redlands, CA, USA). Figure 1. Locations of the 250 paired (fenced and grazed pairs) study sites in Inner Mongolia. (A), Meadow steppe (B), typical steppe (C), desert steppe. Map created using ArcGIS 9.3 software. (Environmental Systems Research Institute (ESRI), Redlands, CA, USA). Results Eff Sources df Cover (%) Height (cm) Species richness AGB (g m−2) P SS% P SS% P SS% P SS% Grazing (G) 1 <0.0001 6.70 <0.0001 11.30 <0.0001 3.49 <0.0001 13.22 Vegetation types (V) 2 <0.0001 38.31 <0.0001 14.38 <0.0001 12.38 <0.0001 18.34 Years 2 0.0211 0.28 <0.0001 4.17 0.0835 0.27 <0.0001 1.04 GV 2 0.0032 0.42 <0.0001 1.42 0.0195 0.44 <0.0001 1.66 GY 2 0.5589 0.04 0.357 0.09 0.7043 0.04 0.433 0.07 VY 2 0.0266 0.40 0.0213 0.53 0.0004 1.16 0.233 0.25 GVY 4 0.8661 0.05 0.8264 0.07 0.6407 0.14 0.882 0.05 Table 1. Results of three-way ANOVAs using grazing treatment, vegetation types and years as fixed factors. df, P, SS% were the abbreviations of degree of freedom, P values and percentage of sum square explained by sources, respectively. Table 1. Results of three-way ANOVAs using grazing treatment, vegetation types and years as fixed factors. df, P, SS% were the abbreviations of degree of freedom, P values and percentage of sum square explained by sources, respectively. To further explore the temporal variation in grazing-induced variation among vegetation types, we compared the response ratios [RRs, indicated by ln (CG/CF), where CF and CG are the mean values of the community prop- erties in fenced and grazing plots at each site, respectively] of cover, height, species richness, and AGB over the period 2011–2013 for each of the vegetation types (Fig. 3). Our results showed that for a given vegetation type, there was no significant variation in the RRs of cover, height, species richness, and AGB during the years 2011 to 2013, with the exception that meadow steppe had the lowest RRs of cover in 2012 and desert steppe had the Scientific Reports \| 7: 40 \| DOI:10.1038/s41598-017-00105-y 3 www.nature.com/scientificreports/ ture.com/scientificreports/ Figure 2. Variation in vegetation cover, height, richness and aboveground biomass (AGB) among three vegetation types (MS: meadow steppe; TS: typical steppe; DS: desert steppe) in 2011–2013. Figure 2. Variation in vegetation cover, height, richness and aboveground biomass (AGB) among three Figure 2. Variation in vegetation cover, height, richness and aboveground biomass (AGB) among three vegetation types (MS: meadow steppe; TS: typical steppe; DS: desert steppe) in 2011–2013. highest RRs of species richness in 2012 (Fig. 3). Moreover, the RRs of species richness and AGB of meadow steppe and typical steppe were significantly lower than those of desert steppe during the period 2011–2013 (Fig. 3). Discussion Eff t f Effects of grazing, vegetation type, and year on community properties. It has been well demon- strated that high grazing intensity could trigger a significant and destructive effect on community composition and ecosystem functioning in grasslands, such as decreases in community cover and AGB26, 27, which is con- sistent with our findings for Inner Mongolian grassland. However, some previous studies have indicated that the relationships between plant variables (e.g., biomass or productivity) and grazing intensity are hump-shaped in grassland ecosystems19, 20. For example, Dangal et al.20 provided evidence that grassland ANPP can be main- tained at a grazing intensity of 0.5 and 1.0 sheep ha−1 at wet and dry sites in in semi-arid regions in Central Asia, respectively. The absence of grazing improving plant variables in the current study may be due to the relative higher grazing intensity (ca. 2.0 sheep ha−1) in Inner Mongolian grasslands. Hence, the patterns observed in our semiarid grasslands may simply represent the right side of a hump-shaped relationship. Moreover, our results also showed that in addition to grazing, community cover, height, species richness, and AGB were also signifi- cantly influenced by vegetation type and the interactive effect of grazing × vegetation type, indicating that at a spatial scale, grazing-induced decreases in cover, height, species richness, and AGB were context dependent. We found that grazing significantly decreased cover, height, species richness, and AGB in meadow and typical steppe, but had no significant effect in desert steppe, which is consistent with the findings of some previous studies in this region, which have reported that the effects of grazing on community properties are strongly mediated by vegetation type21, 24, 28. These different response patterns could be explained by variation in community compo- sition among the vegetation types and the foraging performance of livestock. In particular, the meadow steppe and typical steppe investigated in our study are dominated by species such as C. pediformis, L. chinensis, and S. baicalensis that are palatable to most livestock, whereas desert steppe is dominated by species such as S. glareosa and S. klementzii that are unpalatable to livestock. These two factors in turn lead to grazing-induced decreases in cover, height, species richness, and AGB in meadow steppe and typical steppe that are greater than those in desert steppe. Results Eff ighest RRs of species richness in 2012 (Fig. 3). Moreover, the RRs of species richness and AGB of meadow steppe nd typical steppe were significantly lower than those of desert steppe during the period 2011–2013 (Fig. 3). Effect of grazing on the relationships between community properties and precipitation. Our results showed that the forms of the relationships between community properties and precipitation were influ- enced by grazing treatment and by the year of observation (Fig. 4). Irrespective of grazing treatment, the rela- tionships between community cover and growing season precipitation (GSP) changed from a positive linear form in 2011 and 2012 (relatively low GSP, 350 and 400 mm, respectively) to a unimodal form in 2013 (relatively high GSP, 500 mm, Fig. 4). In contrast, the relationship between species richness and GSP changed from a unimodal form in 2011 and 2012 to a positive linear form in 2013 (Fig. 4). Furthermore, community height and AGB consistently exhibited unimodal relationships with GSP in fenced plots during the period 2011–2013 (Fig. 4). However, the relationship between species richness and GSP in grazed plots shifted from a unimodal form in 2011 and 2012 to a positive linear form in 2013 (Fig. 4), whereas the AGB in grazed plots also changed from a unimodal form in 2011 to a linear form in 2012 and 2013 (Fig. 4). Effect of grazing on the relationships among community properties. In addition to the significant relationship between community properties and GSP, community properties were also found to be correlated with each other in each vegetation type. However, relative to the fenced plots, grazing did not alter the forms of the relationships between AGB and cover, height, and species richness (Fig. 5). For example, a positive linear Scientific Reports \| 7: 40 \| DOI:10.1038/s41598-017-00105-y 4 www.nature.com/scientificreports/ Figure 3. Spatial pattern of grazing-induced response in vegetation cover, height, species richness and AGB among three vegetation types (MS: meadow steppe; TS: typical steppe; DS: desert steppe) in 2011–2013. Figure 3. Spatial pattern of grazing-induced response in vegetation cover, height, species richness and AGB among three vegetation types (MS: meadow steppe; TS: typical steppe; DS: desert steppe) in 2011–2013. relationship was consistently found between AGB and cover, height, and species richness in both fenced and grazed plots (Fig. Results Eff 5), with the exception that a unimodal relationship was found between AGB and species rich- ness in fenced plots in meadow steppe, between AGB and height in typical steppe, and between AGB and cover in desert steppe (Fig. 5). Discussion Eff t f Inconsistent with our findings, however, are the results from previous studies that have shown that meadow steppe was less affected by grazing than desert steppe29, 30. These discrepancies serve to indicate the com- plexity of the responses of community properties to grazing, which are controlled by multiple factors in addition Scientific Reports \| 7: 40 \| DOI:10.1038/s41598-017-00105-y 5 www.nature.com/scientificreports/ Figure 4. Relationship between vegetation cover, height, richness, aboveground biomass (AGB) and growing season precipitation at both fenced (F) and grazed (G) sites. P-value < 0.05, P-value < 0.01, P- value < 0.0001. MS: meadow steppe; TS: typical steppe; DS: desert steppe. Figure 4. Relationship between vegetation cover, height, richness, aboveground biomass (AGB) and growing season precipitation at both fenced (F) and grazed (G) sites. P-value < 0.05, P-value < 0.01, P- value < 0.0001. MS: meadow steppe; TS: typical steppe; DS: desert steppe. Figure 4. Relationship between vegetation cover, height, richness, aboveground biomass (AGB) and growing season precipitation at both fenced (F) and grazed (G) sites. P-value < 0.05, P-value < 0.01, P- value < 0.0001. MS: meadow steppe; TS: typical steppe; DS: desert steppe. to grazing intensity, including grazing history, variation in climatic conditions during the years of observation, and soil properties31–33. p p Our results also revealed that, with the exception of species richness, community properties changed among the years of observation, which is consistent with the findings of a previous study in Mongolian grassland neigh- boring our study area34. This similarity is because species in the grasslands of the Mongolian plateau are domi- nated by perennials, which have relatively slow temporal dynamics34. Moreover, we found significant interactive effects between years and vegetation types, indicating that the response of community properties to grazing at the temporal scale was also mediated by vegetation context. This conclusion is also affirmed by the variance compo- nent results, which revealed that vegetation type, rather than grazing treatment or year, was the most important factor in regulating the variation in community properties during the period in which we studied the Inner Mongolian grassland. This finding is consistent with the previous studies19, 20, 35, which predict that the plants at dry sites are deferent to wet sites because grazing would change precipitation use efficiency and nitrogen use effi- ciency. Discussion Eff t f Our results not only expand our understanding of the importance of community responses to grazing but also have important implications for grazing management in the context of different vegetation conditions in the semiarid steppe and beyond. For example, we should decrease the grazing intensity for conservation of biological diversity in the Inner Mongolian grasslands. p p Our results also revealed that, with the exception of species richness, community properties changed among the years of observation, which is consistent with the findings of a previous study in Mongolian grassland neigh- boring our study area34. This similarity is because species in the grasslands of the Mongolian plateau are domi- nated by perennials, which have relatively slow temporal dynamics34. Moreover, we found significant interactive effects between years and vegetation types, indicating that the response of community properties to grazing at the temporal scale was also mediated by vegetation context. This conclusion is also affirmed by the variance compo- nent results, which revealed that vegetation type, rather than grazing treatment or year, was the most important factor in regulating the variation in community properties during the period in which we studied the Inner Mongolian grassland. This finding is consistent with the previous studies19, 20, 35, which predict that the plants at g ghi g p p p dry sites are deferent to wet sites because grazing would change precipitation use efficiency and nitrogen use effi- ciency. Our results not only expand our understanding of the importance of community responses to grazing but also have important implications for grazing management in the context of different vegetation conditions in the semiarid steppe and beyond. For example, we should decrease the grazing intensity for conservation of biological diversity in the Inner Mongolian grasslands. Effect of grazing on the relationships between community properties and precipitation. Relationships between community properties and climatic gradients have long been debated in ecology. The reported type of relationship has varied from unimodal36, 37 to monotonous (positive or negative linear)38–40 and 6 Scientific Reports \| 7: 40 \| DOI:10.1038/s41598-017-00105-y www.nature.com/scientificreports/ Figure 5. Relationships between aboveground biomass (AGB) and vegetation cover, height, richness at both fenced (F) and grazed (G) sites. P-value < 0.05, P-value < 0.01, P-value < 0.0001. MS: meadow steppe; TS: typical steppe; DS: desert steppe. Figure 5. Relationships between aboveground biomass (AGB) and vegetation cover, height, richness at both fenced (F) and grazed (G) sites. Discussion Eff t f P-value < 0.05, P-value < 0.01, *P-value < 0.0001. MS: meadow steppe; TS: typical steppe; DS: desert steppe. to unrelated patterns41. However, our results from the Inner Mongolian grasslands revealed the existence of both monotonous and unimodal forms of relationships between cover, height, species richness, and AGB and GSP, although the specific form of the relationship varied among years and grazing treatments. Specifically, we found that the relationship between cover and GSP shifted from positively linear in the dry years (2011 and 2012) to unimodal in the wet year (2013), irrespective of grazing treatment, whereas the relationship between species richness and GSP changed from unimodal in the dry years to linear in the wet year, indicating that the form of the relationships between cover and species richness and climatic gradient is mediated by the temporal variation in climatic conditions. We noted that this observed difference in relationship between species richness and GSP in our study is inconsistent with the findings of some previous studies in this area42–44. This discrepancy could be explained by differences in the method of precipitation data collection. Previous authors have only investigated richness in a single given year and used mean annual precipitation to explore their relationships. In our study, we surveyed species richness during three consecutive years (two relatively dry years followed by one relatively wet year) and obtained GSP data for each site in each year. This method allowed us to detect temporal variations in the relationship between species richness and GSP, because species richness changed differently in the wet year after the grasslands had experienced some dry years45. to unrelated patterns41. However, our results from the Inner Mongolian grasslands revealed the existence of both monotonous and unimodal forms of relationships between cover, height, species richness, and AGB and GSP, although the specific form of the relationship varied among years and grazing treatments. Specifically, we found that the relationship between cover and GSP shifted from positively linear in the dry years (2011 and 2012) to unimodal in the wet year (2013), irrespective of grazing treatment, whereas the relationship between species richness and GSP changed from unimodal in the dry years to linear in the wet year, indicating that the form of the relationships between cover and species richness and climatic gradient is mediated by the temporal variation in climatic conditions. Materials and Methods d d i Study area and experimental design. This study was conducted in the grasslands of Inner Mongolia, which are an important part of the Eurasian steppe region (37.4°–53.38°N, 97.2°–126.07°E, Fig. 1), covering an area of 1.18 million km2 49. The grasslands of Inner Mongolian experience a continental monsoon climate, with an uneven rainfall pattern and a hot but relatively short frost-free period. From the western to eastern regions, the mean annual precipitation (MAP) in these grasslands gradually increases from 50 to 450 mm, with 70–80% of the MAP occurring during the growing season (May–August). Moreover, the mean annual temperature gradually decreases from 8 to 0 °C. With a variation in climatic conditions, soil types correspondingly change (west to east) from desert soils to brown calcic to chernozem soils. Our study included three dominant vegetation types growing in the Inner Mongolian grasslands: meadow steppe, typical steppe, and desert steppe. The meadow steppe is located in the eastern part of the Inner Mongolian grasslands and is characterized by a relatively high species richness and productivity. Typical species include Carex pediformis C. A. Mey., Filifolium sibiricum (L.) Kitam., Leymus chinensis (Trin.) Tzvel., Sanguisorba offic- inalis L, and Stipa baicalensis Roshev. The typical steppe is located in the center of the Inner Mongolian grass- lands and is characterized by an intermediate level of species richness and productivity. Typical species include Artemisia frigida Willd. Sp. P., Cleistogenes squarrosa (Trin.) Keng., L. chinensis, Stipa grandis P. Smirn., Stipa krylovii Roshev, and Thymus mongolicus Ronn. The desert steppe is distributed in the western part of the Inner Mongolian grassland and is characterized by relatively low species richness and productivity. Typical species include Allium polyrhizum Turcz. ex Regel, Stipa caucasica Schmalh, and Stipa klemenzii Roshev21, 50, 51. Experimental design and field measurement. On the basis of the spatial distribution of vegetation types and community associations, the RGLG project established a total of 250 paired sites (grazed vs. fenced) in the Inner Mongolian grassland, including 63 paired sites for meadow steppe, 148 paired sites for typical steppe, and 39 paired sites for desert steppe (Fig. 1). Each of these paired sites included two neighboring but contrasting plots: one fenced and one grazed. Since 2003, all the fenced plots have been enclosed by wire netting to prevent livestock grazing, whereas grazed plots are open and have suffered from overgrazing for a long period. Discussion Eff t f We noted that this observed difference in relationship between species richness and GSP in our study is inconsistent with the findings of some previous studies in this area42–44. This discrepancy could be explained by differences in the method of precipitation data collection. Previous authors have only investigated richness in a single given year and used mean annual precipitation to explore their relationships. In our study, we surveyed species richness during three consecutive years (two relatively dry years followed by one relatively wet year) and obtained GSP data for each site in each year. This method allowed us to detect temporal variations in the relationship between species richness and GSP, because species richness changed differently in the wet year after the grasslands had experienced some dry years45.f g p y y We also noted that the forms of the relationships between height and AGB and GSP were affected by grazing treatment. Grazing changed these relationships from a unimodal to linear form in some observation years (i.e., for height in 2013 and for AGB in 2012 and 2013). This finding is consistent with a previous study in this region, which reported that grazing could alter the relationship between AGB and precipitation21. Although we have yet to clarify the underlying mechanisms involved in regulating the forms of these relationships, our results strongly demonstrated that in Inner Mongolian grassland, the form of the relationships between cover, height, species richness, and AGB and GSP are mediated by both grazing and the temporal variation in GSP. Effect of grazing on the relationships among community properties. In this study, we found that AGB exhibited significant relationships with cover, height, and species richness, irrespective of vegetation type. Our findings indicate that some of the previous seemingly inconsistent observations in the Inner Mongolian grassland are in fact not conflicting, but instead reflect synchronous relationships. For example, we identified a unimodal relationship between AGB and species richness in meadow steppe, whereas a linear relationship was detected in typical steppe and desert steppe. These findings differ from those of some other studies, which have reported that the relationship between AGB and species richness is invariably of a unimodal or linear form46, 47. Discussion Eff t f Our results are, however, in line with an early hypothesis, which proposed that the forms of relationships between Scientific Reports \| 7: 40 \| DOI:10.1038/s41598-017-00105-y 7 www.nature.com/scientificreports/ AGB and species richness vary among vegetation types due to variations in community composition and envi- ronmental conditions48. Moreover, we also found that, compared with the fenced plots, grazing generally did not affect the relationships between AGB and cover, height, and species richness. This could be explained by the fact that grazing decreased all investigated variables in each grassland type, and thus did not influence the intrinsic relationship among these community properties. g y In this study, we performed a large-scale experiment using 250 paired plots (grazed vs. fenced) to survey community properties (cover, height, species richness, and AGB) across three vegetation types (meadow steppe, typical steppe, and desert steppe) in Inner Mongolian grassland. Our goal was to evaluate the spatial and tempo- ral effects of grazing on these variables in this region. Our results showed that in addition to grazing, vegetation type and year also had significant effects on cover, height, species richness, and AGB, and that the primary factor influencing variation in these variables was vegetation type. Moreover, the relationships between community properties and GSP were regulated by both grazing treatment and the year of observation. In each vegetation type, the observed community properties were significantly correlated with each other, and the form of the relation- ships was unaffected by grazing treatment. Our findings indicate that vegetation type plays an important role in regulating the response of community properties to grazing in this semi-arid grassland. Materials and Methods d d i f For the years 2011 to 2013, we investigated community structure (cover, height, and species richness) and function (aboveground biomass, AGB) in these 250 paired sites during the period from August 15th to 30th when the aboveground standing biomass of most plants peaks in this region43, 51. For each paired site, we initially recorded its longitude, latitude, elevation, and community type. To minimize the spatial heterogeneity, we ran- domly selected three 1 × 1 m quadrats in each of the fenced and grazed plots at each site to investigate their com- munity cover, height, species richness, and AGB. Then, we pooled the data from the three quadrats at site level for the following statistical analyses. Specifically, community cover was assessed by a visual inspection, average community height for each quadrat was measured using a portable ruler, and species richness was determined by counting the number of species present in the quadrats. After measuring these variables, we clipped the AGB at ground level in each quadrat, oven-dried the vegetation at 65 °C for 48 h, and then weighed the dried material in the laboratory. Climatic data. Climatic data. We used growing season precipitation (GSP) to reflect climatic conditions in our study, because previous studies have affirmed that it is the primary climatic factor influencing community structure and ecosystem function in this area43, 51. To obtain the GSP for these paired sites, we initially collected monthly precipitation data for the years 2011 to 2013 from 824 evenly distributed meteorological stations in China. On the basis of these collected data, we then interpreted monthly precipitation data for our investigated site via a Geographic Information System-based multiple regression method, which used latitude, longitude, and elevation as predicators52. Finally, the GSP at each site was calculated by summing the interpreted monthly precipitation data from May to September. Statistical analyses. To address our first question regarding the influence of grazing, vegetation type, and year on community structure and ecosystem function, we used a three-way ANOVA to examine the main and interactive effects of grazing, vegetation type, and year on variations in cover, height, species richness, and AGB at the plot level, respectively. If a significant main or interactive effect was detected, we then calculated its relative importance by dividing its explained sum square by the total sum square53, and then Tukey’s multiple-range test was used to compare differences in the mean values among these factors. In addition, to assess grazing-induced Scientific Reports \| 7: 40 \| DOI:10.1038/s41598-017-00105-y 8 www.nature.com/scientificreports/ variation in community structure and ecosystem function, we also compared the differences in response ratios of community cover, height, species richness, and AGB among different vegetation types in each year of observation. The response ratio was calculated as RR = ln (CG/CF), where CF and CG are the mean values of the community properties that were observed in fenced and grazing plots at each site, respectively.f p p g g p p y To address our second question regarding the effect of grazing on the forms of the relationships between community properties and GSP for each community property (i.e., cover, height, species richness, and AGB), we initially used a general linear model to examine whether a significantly positive (linear term) or unimodal (quad- ratic term) relationship existed. If the quadratic term was not statistically significant, we then considered that a linear relationship was the best-fitted model between community properties and GSP. Climatic data. If a significant quadratic term was also detected, we then used the Akaike Information Criterion (AIC) value to select the best-fitted model (model with the lowest AIC)46. 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Acknowledgements We thank Institute of Grassland Surveying and Planning of Inner Mongolia for their assistance in fieldwork. This study was supported by Special Research Program for Public-welfare Environmental Protection Projects We thank Institute of Grassland Surveying and Planning of Inner Mongolia for their assistance in fieldwork. This study was supported by Special Research Program for Public-welfare Environmental Protection Projects (201509042), Natural Science Foundation of China (31570450), and Youth Innovation Promotion Association CAS (2015061). Author Contributions Y.B., J.L. and R.S. designed the research. R.S. and D.C. wrote the main manuscript text. R.S., J.C., and D.C. performed statistical analyses. H.J., L.C. and Z.W. gave some useful comments and suggestions to this work. All authors reviewed the manuscript. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 4. Wesche, K., Ronnenberg, K., Retzer, V. & Miehe, G. 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Grazing exclusion affects soil and plant communities, but has no impact on soi carbon storage in an upland grassland. Agriculture, Ecosystems & Environment 149, 118–123 (2012). Scientific Reports \| 7: 40 \| DOI:10.1038/s41598-017-00105-y 9 Additional Informationh Competing Interests: The authors declare no competing financial interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. 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https://openalex.org/W4295969493	https://hal.inria.fr/hal-01764202/file/462132_1_En_60_Chapter.pdf	English	null	The Design for Product Service Supportability (DfPSSu) methodology: generating sector-specific guidelines and rules to improve Product Service Systems (PSSs)	Zenodo (CERN European Organization for Nuclear Research)	2,017	cc-by	5,324	The Design for Product Service Supportability (DfPSSu) Methodology: Generating Sector-Specific Guidelines and Rules to Improve Product Service Systems (PSSs) Claudio Sassanelli, Giuditta Pezzotta, Roberto Sala, Angelos Koutopes, Sergio Terzi To cite this version: Claudio Sassanelli, Giuditta Pezzotta, Roberto Sala, Angelos Koutopes, Sergio Terzi. The Design for Product Service Supportability (DfPSSu) Methodology: Generating Sector-Specific Guidelines and Rules to Improve Product Service Systems (PSSs). 14th IFIP International Conference on Product Lifecycle Management (PLM), Jul 2017, Seville, Spain. pp.679-689, 10.1007/978-3-319-72905-3_60. hal-01764202 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01764202 https://inria.hal.science/hal-01764202v1 Submitted on 11 Apr 2018 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License The Design for Product Service Supportability (DfPSSu) methodology: generating sector-specific guidelines and rules to improve Product Service Systems (PSSs) Claudio Sassanelli1,2,, Giuditta Pezzotta2, Roberto Sala2, Angelos Koutopes3, Sergio Terzi1 1 Introduction Nowadays manufacturers are always more absorbed by Service Economy. To boost their performance the paradigm of Product-Service System (PSS) has been presented to the market. PSSs are characterized by the integration of Products and Services bun- dled into unique solutions fulfilling the user’s needs [1]. However, companies are not fully actually aided by consistent PSS design methodologies, and supporting tools, which could enable them to focus on both customer’s perspective and company’s in- ternal performance but also to integrate service and product components along their whole lifecycle [2]. Some traditional PSS methodologies (e.g. [3]–[5]) tried to contin- ue going down the river of traditional product design approaches to attempt to fill this gap. Moreover, [6] proposed some more conceptual strategies to move in that direc- tion: the idea was that starting from the physical product properties and features, ser- vice design can be properly integrated in it, without neglecting a lifecycle perspective on the entire integrated solution. In such a competitive and fast changing environ- ment, concurrent engineering approaches, such as Design for X (DfX), have been proposed in literature, being more able to cope with different simultaneous issues dealing with products, processes and systems design. Overcoming the typical issues of the traditional sequential engineering, this kind of approaches can indeed adapt the physical products in various ways according to the PSS lifecycle, also addressing de- signers’ lack of knowledge in important product and service lifecycle areas [7]. A methodology generating Design Guidelines and Rules, fostering the adoption of the Design for Product Service Supportability (DfPSSu) approach [8], aims at integrating product and services with a lifecycle view. With this objective, section 2 describes the research methodology adopted and section 3 the application case characteristics. Fi- nally, section 4 presents the validation results and section 5 introduces the future re- search developments. Claudio Sassanelli1,2,, Giuditta Pezzotta2, Roberto Sala2, Angelos Koutopes3, Sergio Terzi1 1 Department of Economics, Management and Industrial Engineering, Politecnico di Milano, Via R. Lambruschini 4/b, 20156, Milano, Italy {claudio.sassanelli, sergio.terzi}@polimi.it 2 Department of Management, Information and Production Engineer- ing, University of Bergamo, Viale Marconi, 5, Dalmine (BG), 24044, Italy {claudio.sassanelli,giuditta.pezzotta, roberto.sala}@unibg.it 3 N.BAZIGOS S.A. DESIGN AND MANUFACTURING OF MOULDS 26th km Old National Road Athens - Thiva - Mandra Attikis - P.O. 19600, Greece akoutoupes@bazigosmolds.com Abstract. Nowadays manufacturers’ need to systematically develop innovative integrated solutions is increasingly pushed by new technologies, a multiple functionalities demand and a change in the customer value perception. For these reasons, it is very complex for Product Service Systems (PSS) providers to ful- fil all the design requirements: designers must consider all the objectives the PSS wants to achieve during its whole lifecycle according to different criteria, which are often to be considered according to a trade-off balance. At present, Design for X (DfX) design methods represent the most important attempt to en- hance product development according to certain characteristics or lifecycle phases: authors believe they can also support the PSS design, redesigning or enhancing products in certain X-dimensions, in particular those ones related to “service supportability”. On this basis, a methodology generating new Design for X (DfX) guidelines has been proposed: in this paper an application case in the mold industry shows how a physical product can be improved when a ser- vice has to be added and integrated. At the same time, new industry-specific PSS design guidelines and rules are proposed. Keywords: Product Service System (PSS), PSS design, Design for X (DfX), Design for Product Service Supportability (DfPSSu), Design Guideline Keywords: Product Service System (PSS), PSS design, Design for X (DfX), Design for Product Service Supportability (DfPSSu), Design Guideline 2 The methodology for generating DfPSSu Guidelines/Rules Fig. 1 summarizes the methodology mentioned above: it has the aim of creating De- sign Guidelines and Rules to enhance the design of the product features enabling and supporting the delivery of excellent services. Guidelines provide a proper basis for considering generic, non-company-specific, lifecycle oriented information to be fol- lowed during the design phases. Rules become concrete and quantitative instructions for PSS developers to be followed during their daily specific design activities, repre- senting the characterizing knowledge belonging to the company. The methodology, and its supporting tool to manage the generated Guidelines and Rules in a consistent repository, have been developed according to different research traditions [9]. The methodology is composed of 6 phases clustered in 4 main sections. Section 1: before starting with the content guideline and rule creation procedure, preliminary activities need to be performed in order to collect the basic information to be used through the adoption of the methodology. All the Design for X approaches that could be involved during the PSS design are collected: they represent the possible Abilities (A) the PSS under design could achieve and represent the starting point for the guidelines/rules definition. The DfX Ability concept is based on the “function” concept defined by [10]: they are those principles through which the PSS functions can be explicated and explained and represents what exactly the guideline addresses. Section 2: The design process can start when a PSS concept is already available. Once defined in Phase 1 the Ability/ies (A) the product under design has to achieve, an analysis must be conducted in order to create, if not existing, new suitable content guideline/s. Thus, Design and Technical Requirements (DTR) are defined: they repre- sent the practical and technical recommendations to be followed by designers and en- gineers, through which abilities could be achieved. Therefore, the new DTR has to be linked to the Ability/ies, also specifying the importance degree of the relationships. Based on the identified links between A and DTR, guidelines able to guide the de- signer/engineer activities in the Product/Service/System development must be formal- ized in text and made available as company knowledge. Fig. 1. The PSS design Guideline/Rule methodology (adapted by[9]) Fig. 1. The PSS design Guideline/Rule methodology (adapted by[9]) Section 3: in this section design rules are created. Here, the methodology must lead the designer/engineer to focus on the specific company context. 3.1 Research Methodology The application case has been conducted with the aim of testing the suitability of the presented methodology generating DfPSSu Guidelines and Rules. The paper also evi- dences the related benefits for companies and the increased efficiency, deriving from its application, in solving issues in the detailed design phase of PSS. The application case was conducted in two steps. First of all, a video has been shared with participants to train them about the methodology. Thus, the face-to-face workshop has been orga- nized to apply the methodology in the industrial context. This interactive session was led by two academics and involved two additional academics with which the company has long term relationship, in addition to the production monitoring employee and one product designer. After realizing the actual DfX methods level of use in the company, DfPSSu approach was presented. Hence, a solution, the mold digital history of re- pairs, has been detected through a concept design brainstorming, in order to enhance the company business. The methodology has been performed and design guidelines and rules supporting the design of this new PSS were obtained. Moreover, useful feedbacks on the suitability of the approach were given. 2 The methodology for generating DfPSSu Guidelines/Rules In order to create a bridge between the functionalities of the PSS to be achieved and the related lifecycle variables that need to be managed, an extended version of the Function Transfor- mation Matrices (FTM) methodology [10] is used. A series of them, all based on the same structure, is adopted to document and gauge the relationships among various factors such as: a) PSS Features (PSSF), those characteristics of the PSS components to be considered to act on DTR expressed in the Guidelines (GL); b) PSS Lifecycle Processes (LP) represent all those activities of the PSS lifecycle (from the design to disposal phase); c) PSS Process Variables (PV) are those variables which need to be detected since they affect LP. They can belong to any process of the several phases composing the PSS lifecycle. Finally, Design Rules are systematically developed based on the links found in the previous steps: their aim is the ability-driven control of lifecycle variables in order to better manage the design activities to improve the phys- ical product of a PSS. Design Rules are indeed developed to provide the links for con- trolling the variables that directly affect the PSS Ability/ies enabled by the introduc- tion of a new service on a physical product. Section 4: In this last section, the coherence of all the elements considered during the design process is verified, supporting designers and engineers in finding the right connections between the obtained high level Guidelines and the more operative Rules. For this aim, two modified X-Matrices [11] are used. 3.2 The application case: N. BAZIGOS SA The application case has been conducted in N. BAZIGOS SA, a B2B Greek company designing and manufacturing molds. Design methodologies for the product itself are long established using PLM, CAD and other software tools and methods, supported by a strong and experienced design and engineering division. Going through the ser- vitization process, their actual intent is to: reduce their environmental impact, wastes in material, energy consumption, design and machining time, time to market, frequen- cy of failure; improve customer involvement in the design and customer satisfaction; increase competitiveness and income; access to new market sectors. Indeed, in the company, PSS offerings are in early stages of adoption. The provided services are of- fered in isolation from the product, which is the mold, without considering a com- bined PSS eco-system. However, the nature of this manufacturing sector dictated up to now that the services aspect is indirectly treated. Given this lack of service-oriented approaches in the industry, the company is thus considering new PSS projects like mold delivery time estimation as a service, maintenance history per customer, joint provider-customer proactive production planning for mold modifications or opinion mining offered to customers as a service. This would enhance the monitoring and con- trol of mold lifecycle and shorten mold downtime. The methodology was applied in N. BAZIGOS SA, starting with Section 1, where some preliminary setting activities consisted in assessing the AS-IS of its design pro- cedures. The company does not adopt a really structured approach to design mold. They follow some basic principles, e.g. optimize mold cycle time (to inject, cool and eject a part). Moreover, the design guidelines and rules, that represents the company knowhow needed to implement these approaches, are not codified and written down and reside only in designers’ background. Furthermore, customers’ requirements are almost connected to production optimization, from either a quantity or quality point of view. Therefore, designers are committed to add on the basic mold some extras and to focus on certain precise aspects of the product lifecycle in a concurrent way. Most of the time the main target for the design team is to optimize, also through a consistent choice of the steel adopted, the expected number of pieces produced with the mold, minimizing downtimes. Thus, when steel hardening can be avoided, the company costs are lower, the price for the customer is lower but it will soon present more prob- lems in maintenance. 3.2 The application case: N. BAZIGOS SA Accurate fitting is required and, due to natural wear, it needs re- pair or replacement. (a) (b) Fig. 2. “2 cavity, 1 liter Seal Lid” mold: General Section (a) and fixed side (b) 3.3 Guidelines and rules generation in N. BAZIGOS SA Having analyzed the current design approach of the company, according to the meth- odology procedure in Section 1, and detected the solution to be developed, DfPSSu flows, also carries cool- ing circuit). reason, and thus they are also replaceable. 3. Hotrunner sys- tem Provided by specialized suppliers, distributes the plastic material to multi- ple cavities. Nozzle tips (and other contact points with accurate fitting are often damaged by material flow. 4. Stripper ring The component that moves relatively to the core, in order to eject the plastic part from the mold). Accurate fitting is required and, due to natural wear, it needs re- pair or replacement. (a) (b) Fig. 2. “2 cavity, 1 liter Seal Lid” mold: General Section (a) and fixed side (b) (a) (a) Fig. 2. “2 cavity, 1 liter Seal Lid” mold: General Section (a) and fixed side (b) 3.2 The application case: N. BAZIGOS SA To manage this issue, principles belonging to Design for Modu- larity and Customizability, adding changeable cups and bases, are directly linked to Maintainability. On the contrary, using thicker plates or considering other suitable so- lutions, the mold can become more reliable. However, it requires a more complex de- sign and principles as Maintainability cannot be neglected. Therefore, through the DfPSSu Methodology, it is useful to reconsider the design of an already existing mold in order to improve its functionalities (especially from a Maintainability point of view) and understand what would change. A new solution, able to meet N.BAZIGOS SA’s needs, was identified: the digital history of repairs of the mold. Thus, a product to be redesigned, referring to a customer operating in the plastic industry, was detect- ed: a “2 cavity, 1 liter Seal Lid” mold. The description of the main components (shown in Fig. 2) and the main issues with them are shown in Table 1: Table 1. “2 cavity, 1 liter Seal Lid” Mold: component and issues description Component Description Issue 1. Core & Cavity The two halves of the mold that create the plas- tic product geometry. They usually carry the center- ing elements: these are the two parts that need to be aligned properly. 2. Cooling bush An insert, that carries the injection point (hole) from which the plastic The hole is damaged by materi- al flow. They are designed as inserts for manufacturability Table 1. “2 cavity, 1 liter Seal Lid” Mold: component and issues description Component Description Issue 1. Core & Cavity The two halves of the mold that create the plas- tic product geometry. They usually carry the center- ing elements: these are the two parts that need to be aligned properly. 2. Cooling bush An insert, that carries the injection point (hole) from which the plastic The hole is damaged by materi- al flow. They are designed as inserts for manufacturability Table 1. “2 cavity, 1 liter Seal Lid” Mold: component and issues description flows, also carries cool- ing circuit). reason, and thus they are also replaceable. 3. Hotrunner sys- tem Provided by specialized suppliers, distributes the plastic material to multi- ple cavities. Nozzle tips (and other contact points with accurate fitting are often damaged by material flow. 4. Stripper ring The component that moves relatively to the core, in order to eject the plastic part from the mold). 3.3 Guidelines and rules generation in N. BAZIGOS SA Having analyzed the current design approach of the company, according to the meth- odology procedure in Section 1, and detected the solution to be developed, DfPSSu Guidelines and Rules were developed following the steps described from Section 2 to 4. Besides the aim of guiding the creation of a product consistent with the customer’s needs, the Design Guidelines and Rules are useful to limit the reworks, since they are thought to give precise information on how to design the product. Guidelines and Rules were developed following the steps described from Section 2 to 4. Besides the aim of guiding the creation of a product consistent with the customer’s needs, the Design Guidelines and Rules are useful to limit the reworks, since they are thought to give precise information on how to design the product. Fig. 3 (from solutions in the bottom part of the figure following a clockwise direc- tion) summarizes the results obtained through the application of Section 2: it begins with Phase 1, focused on Abilities definition, the five most important abilities to de- velop the solution desired have been identified. They were related to the physical product properties needed for the PSS provision: Modularity, Maintainability, In- spectability, Easy Assembly/Disassembly operations, ID Coding & Traceability. In Phase 2, six DTRs were defined to fulfill the previous Abilities: use standard components from suppliers, design and manufacture standard components for product families, connectivity of hydraulic and automatic connection, unique BOM coding, component engraving, mounting features for moving and handling. Then, the relation- ship grade between each Ability and DTR was defined. As a result, fourteen Guide- lines (GL) were obtained since only the relationships with a weight equal or higher than “3” were considered significant and thus investigated and translated in opera- tional guideline for the designer. Fig. 3. A section of the FTM - X Matrix from Solution to Guidelines Fig. 3. A section of the FTM - X Matrix from Solution to Guidelines Later, Section 3, summarized in Fig. 4 (from guidelines in the bottom part of the fig- ure following a clockwise direction), was carried out in order to create detailed rules. In Phase 3 the PSS Feature were defined. They were aimed at improving the Abilities, defined in the previous steps, of some critical components, such as: hotrunner, guid- ing components, cooling bush, sockets, centering elements, water manifold, mounting holes, centering cone. 3.3 Guidelines and rules generation in N. BAZIGOS SA Then, the team brainstormed once again to define the relation- ship between the Guidelines and the PSSF. In Phase 4, the designers’ attention moved on the Lifecycle Process (LP) steps identification, in particular seven phases were identified (Concept & Design, Manufacturing, Assembly, Validation, Use, Mainte- nance, Disposal). The definition of the relationships between the PSSF and the LP, aided engineers in understanding the value of the PSSF in all the phases of the solu- tion lifecycle. Once again, the resulting links fostered the creation of the Design Rules to be followed by the designers. 53 specific new Rules have been created (listed in part in Fig. 4). Fig. 4. 3.3 Guidelines and rules generation in N. BAZIGOS SA A section of the FTM - X Matrix from Guidelines to Rules 1 LP7_Disposal 1 2 3 3 3 3 LP6_Maintenance 3 3 3 3 3 3 1 4 1 LP5_Use 1 1 3 1 LP4_Validation (test run) 1 -2 -3 1 3 2 2 2 LP3_Assembly 2 2 3 2 2 2 3 -4 -2 -1 1 3 3 3 LP2_Manufacturing 3 3 1 1 3 3 3 1 -4 -2 -1 -2 2 2 LP1_Concept & Design 2 2 -1 -1 2 2 -2 -1 PSSF8_standard depth mounting holes PSSF7_use of water manifold PSSF6_squared shape of centering elements PSSF5_engraving non critical surfaces PSSF4_design standard sockets PSSF3_design standard cooling bushes PSSF2_validated supplier for guiding components PSSF1_validated supplier for the hotrunner component Rule 1 - To improve modularity use standard hotrunner components from validated suppliers Rule 2 - To improve modularity use guiding components from validated suppliers Rule 3 - To improve modularity design standard sockets Rule 4 - To improve modularity use standard components description Rule 5 - To improve modularity, design standards hotrunner components Rule 6 - To improve modularity design standard guiding components Rule 7 - To improve modularity design standard bushes Rule 8 - To improve modularity design standard sockets centering elements 1 4 5 5 GL1 - Use standard component to foster modularity 5 5 4 4 3 4 5 5 4 4 GL2 - Design standard components to foster modularity 4 4 5 5 2 5 3 5 5 GL3 - Use standard components to improve maintainability 2 4 5 5 GL4 - Design standard components to improve maintainability 2 2 2 GL5 - Create a unique BOM codification to foster maintainability 4 3 3 GL6 - Engrave components to foster maintainability 4 GL7 - Consider the connectivity of hydraulic and automatic connection to foster inspectability 5 4 GL8 - Consider mounting features for moving and handling to foster inspectability 4 GL9 - Consider the connectivity of hydraulic and automatic connection to foster components assembly and disassembly 5 4 GL10 - Consider mounting features for moving and handling to foster components assembly and disassembly 4 5 5 GL11 - Use standard components to foster ID coding and traceability RULES Lifecycle Processes PSS Features GUIDELINES Start Here Fig. 4. 3.3 Guidelines and rules generation in N. BAZIGOS SA Moreover, Rules related to PSSF6 (squared shape of cen- tering element) and PSSF7 (use of water manifold) were characterized by a very negative weight at the beginning of PSS lifecycle, resulting in the need of a strong ef- fort for the company: designers should consider, with a further trade-off brainstorm- ing, if it’s worth to follow them. However, many benefits could be obtained also by their achievement. For example, in order to achieve A3, Inspectabiity, DTR3, Con- nectivity of hydraulic and automatic connection, and DTR6, Mounting features for moving and handling, were considered. In particular, the relation A3-DTR3 was ex- plicated in GL7 (“Consider the connectivity of hydraulic and automatic connection to foster inspectability”). To act on this, PSSF7, Use of water manifold, was considered: this feature requires a very important effort in the beginning of the PSS lifecycle (Concept&Design and Manufacturing and less in Assembly) but makes the validation test run easier, giving also a huge improvement in the Use and Maintenance phases. Indeed, Rule 30, “To improve Inspectability, use water manifold while designing the connectivity of hydraulic and automatic connection”, contributes to GL7’s aim. 3 4 5 5 and traceability 2 4 4 4 4 GL13 - Create a unique BOM coding to foster Id coding traceability 5 4 4 GL14 - Engrave component to foster ID coding traceability 3.3 Guidelines and rules generation in N. BAZIGOS SA A section of the FTM - X Matrix from Guidelines to Rules g g handling to foster components assembly and disassembly 5 5 GL11 - Use standard components to foster ID coding and b l Finally, in Phase 5, the team checked the coherence between all the information creat- ed along the methodology thanks to the analysis of the X-Matrices. No strong contra- dictions emerged. Only one Guideline (GL5), not being linked to any PSSF, wasn’t explicated in specific Rules. Moreover, Rules related to PSSF6 (squared shape of cen- tering element) and PSSF7 (use of water manifold) were characterized by a very negative weight at the beginning of PSS lifecycle, resulting in the need of a strong ef- fort for the company: designers should consider, with a further trade-off brainstorm- ing, if it’s worth to follow them. However, many benefits could be obtained also by their achievement. For example, in order to achieve A3, Inspectabiity, DTR3, Con- nectivity of hydraulic and automatic connection, and DTR6, Mounting features for moving and handling, were considered. In particular, the relation A3-DTR3 was ex- plicated in GL7 (“Consider the connectivity of hydraulic and automatic connection to foster inspectability”). To act on this, PSSF7, Use of water manifold, was considered: this feature requires a very important effort in the beginning of the PSS lifecycle (Concept&Design and Manufacturing and less in Assembly) but makes the validation test run easier, giving also a huge improvement in the Use and Maintenance phases. Indeed, Rule 30, “To improve Inspectability, use water manifold while designing the connectivity of hydraulic and automatic connection”, contributes to GL7’s aim. 3 4 5 5 and traceability 2 4 4 4 4 GL13 - Create a unique BOM coding to foster Id coding traceability 5 4 4 GL14 - Engrave component to foster ID coding traceability Finally, in Phase 5, the team checked the coherence between all the information creat- ed along the methodology thanks to the analysis of the X-Matrices. No strong contra- dictions emerged. Only one Guideline (GL5), not being linked to any PSSF, wasn’t explicated in specific Rules. 4 Discussion Several and different results have been obtained through this application case. The main evidence is that the proposed methodology is able to solve product engineering issues, fostering the product and service features integration in the detailed PSS de- sign [9]. In particular, following the methodology, 14 new Guidelines (Fig. 3) and 53 connected Rules (Fig. 4) were obtained and checked. Feedbacks collected during the methodology application in N. BAZIGOS SA could be considered as an additional result: the difference between “Guidelines” and “Rules” could be strengthened through the way they are written (e.g. considering the use of the passive tense for the Guidelines) and the X-Matrix visualization could be enhanced (to automatically better explain its outcomes). Their main concern with the methodology regarded the effort needed to apply it, if compared to their standard procedures. N. BAZIGOS SA is a SME: designers are free to design as they want, always keeping in mind the mold manufacturability but without the need of always designing something really innova- tive. The mold, a B2B industrial product, should only satisfy the customer’s require- ments: its innovation could be considered strategic only from the service point of view, confirming the importance of the DfPSSu concept. Indeed the methodology adoption would require designers an additional amount of time to get used to the dif- ferent concepts introduced (even if it resulted to be very easy to follow) and to struc- ture the obtained knowledge in the tool repository. In companies it is difficult to change routines and to work with a new tool: experienced designers could state they don’t need to use the proposed methodology because they already know the design rules. Finally, the methodology appeared to be pretty much useful to capture brain- storming during the design phase but at the same moment it represents a very struc- tured way to govern it, decreasing a bit the sense of relax supposed to obtain new ide- as. However, according to N. BAZIGOS SA employees, this methodology can im- prove in an important way the PSS design phase mainly if applied in big multinational companies. Big companies indeed typically are more involved in the continuous pro- cess of innovation of their solutions, follow very strict requirements and have a stronger structure of resources able to exploit this procedure in a deeper way. 4 Discussion Fur- thermore, with its adoption, the problem-solving process could be simplified and speeded up also along the space, in different industrial plants scattered in diverse places, and the time, among different designers generations, and can foster collabora- tion among companies’ divisions and networks. 5 Conclusions and further developments This paper investigates how to support companies in the integration of service fea- tures already in the product design of the PSS. In order to do it and to have an empiri- cal feedback in the industrial context, the methodology generating DfPSSu guidelines and rules proposed in [9] has been adopted in an application case. This has been thus conducted in a SME producing mold for B2B market, willing to go through servitiza- tion. Thus, among the already existing products belonging to the company portfolio, the solution to be designed and provided to the customer as a PSS has been hence de- tected: the injection mold (for plastic industry) maintenance, based on the digital his- tory of repairs. Through this application case, the physical product was enhanced and service features were integrated in it: indeed the methodology confirmed to be strong- ly engineering based, being aimed at the development of a new DfX-driven approach for PSS development and at easing the problem solving process, typical of the design phase, also balancing in a trade-off the different abilities to be satisfied. The case was conducted allowing designers/engineers to freely use the methodology. According to them, the proposed methodology would yield more benefits to a large company, where designers might be based even in different countries - but required to maintain consistency in their designs. In smaller companies, where experienced designers train junior designers, day by day - working next to each other - knowledge, although valu- able, remains tacit. Based on this, a further test could be conducted in future in a mul- tinational company in order to evaluate the design methodology effectiveness not only in SMEs but also in such a different context. Finally, new sector-specific DfPSSu Guidelines and Rules were obtained: in this sense, the provision of a tool, used as a re- pository for both the generic Guidelines and the more specific Rules, can ease design- ers’ activities in protocolling the design knowledge obtained during the design phase. This knowledge can be linked, through the use of tags, either to the design project or to PSS Abilities or to other kind of concepts. Furthermore, this knowledge, consistently filtered, can also be reused for future design projects. Based on this, a tool, already used in the application case in its prototype version, is going to be developed and pro- vided to practitioners. Acknowledgments This work was funded by the European Commission through Diversity Project, GA 636692, under the H2020 program. [11] T. L. Jackson, Hoshin Kanri for the Lean Enterprise: Developing Competitive Capabilities and Managing Profit. 2006. References [1] M. Goedkoop, C. J. G. Van Halen, H. R. M. te Riele, and P. J. M. Rommens, Product Service systems, Ecological and Economic Basics, no. March. 1999. [2] G. Pezzotta, F. Pirola, A. Rondini, R. Pinto, and M.-Z. Ouertani, “Towards a methodology to engineer industrial product-service system – Evidence from power and automation industry,” CIRP J. Manuf. Sci. Technol., 2016. [3] J. C. Aurich, C. Fuchs, and C. Wagenknecht, “Modular design of technical product-service systems,” in Innovation in Life Cycle Engineering and Sustainable Development, D. Brissaud et al. (eds.), 2006, pp. 303–320. [4] N. Maussang, P. Zwolinski, and D. Brissaud, “Product-service system design methodology: from the PSS architecture design to the products specifications,” J. Eng. Des., vol. 20, no. 4, p. 349–366., 2009. g p [5] T. Hara, T. Arai, Y. Shimomura, and T. Sakao, “Service CAD system to integrate product and human activity for total value,” CIRP J. Manuf. Sci. Technol., 2009. [6] A. R. Tan, D. Matzen, T. C. McAloone, and S. Evans, “Strategies for designing and developing services for manufacturing firms,” CIRP J. Manuf. Sci. Technol., vol. 3, no. 2, pp. 90–97, 2010. [7] E. Sundin, “Life-Cycle Perspectives of Product/Service-Systems: In Design Theory,” in Introduction to product/service-system design, 2009, pp. 31–49. [8] C. Sassanelli, G. Pezzotta, F. Pirola, S. Terzi, and M. Rossi, “Design for Product Service Supportability (DfPSS) approach: a state of the art to foster Product Service System (PSS) design,” in 8th CIRP IPSS 2016, 2016, vol. 47, pp. 192– [9] C. Sassanelli, G. Pezzotta, F. Pirola, and S. Terzi, “The Design for Product Service Supportability (DfPSSu) method: Guidelines and Rules generation to enhance Product Service Systems design,” Des. Stud., p. [REVIEW], 2017. [10] A. Mital, A. Desai, A. Subramanian, and A. Mital, Product Development. Elsevier, 2008.
https://openalex.org/W4390500313	https://link.springer.com/content/pdf/10.1007/s44195-023-00059-x.pdf	English	null	The first 30 min hidden aftershocks of the 2022 September 17, ML 6.4, Guanshan, Taiwan earthquake and its seismological implications	Terrestrial, atmospheric and oceanic sciences/Terrestrial, atmospheric, and oceanic sciences	2,024	cc-by	8,551	1 Introduction In September 2022, an earthquake sequence occurred in the Eastern Taiwan. There were four earthquakes with magnitudes greater than 6 in this sequence (Fig. 1a). Among them, the Guanshan earthquake was the first large event with a magnitude of ML 6.4 occurred on Sep- tember 17, 2022. The biggest Chihshang earthquake with Correspondence: Bor‑Shouh Huang hwbs@earth.sinica.edu.tw 1 Institute of Earth Sciences, Academia Sinica, 1‑55, Nankang, P.O. BOX, Taipei 115, Taiwan 2 Seismological Center, Central Weather Administration, Taipei, Taiwan 3 Department of Geosciences, National Taiwan University, Taipei, Taiwan © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Abstract The first 30 min of the aftershock sequence following the 2022 September 17, ML 6.4, Guanshan, Taiwan earthquake is investigated by the back-projection (BP) method. Based on the Automatic Gain Control (AGC) to process individual earthquake record and sort waveforms by epicentral distance, 80 events are identified near the Guanshan earthquake epicenter to verify its temporal variation. However, most of these events cannot clearly image by the BP method to determine its spatial locations for its small amplitude and short time separation. In total, 29 aftershocks are imaged by this study to identify its spatial locations during this time period. The relocated events are compared with the Cen‑ tral Weather Bureau (CWB) rapid report catalogue. Only 5 events have been reported by the CWB rapid report catalogue. These unreported hidden aftershocks are identified to distribute surrounding the epicenter and extended to the east. This extracted information of hidden aftershocks is crucial for assessing the potential for future large aftershocks and estimating the associated seismic hazards in a given region, and provide extra message to evaluate the source physics in general. The first 30 min hidden aftershocks of the 2022 September 17, ML 6.4, Guanshan, Taiwan earthquake and its seismological implications Bor‑Shouh Huang1 , Chin‑Shang Ku1, Chin‑Jen Lin1, Shiann‐Jong Lee1, Yen‑Ling Eileen Chen2, Juen‑Shi Jiang2 and Wei‑Fang Sun3 © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Terrestrial, Atmospheric and Oceanic Sciences Terrestrial, Atmospheric and Oceanic Sciences Huang et al. Terrestrial, Atmospheric and Oceanic Sciences (2024) 35:1 https://doi.org/10.1007/s44195-023-00059-x Open Access Open Access Key Points 1. Propose and verify earthquake location using the back-projection method. 2. Discover 29 hidden events with 30 min after the Guanshan earthquake. 3. Provide extra information to study following large aftershocks and to estimate its associated seismic hazards. Keywords Guanshan earthquake, Hidden aftershocks, Back-projection, Automatic gain control, Central Weather Bureau Huang et al. Terrestrial, Atmospheric and Oceanic Sciences (2024) 35:1 Page 2 of 14 Page 2 of 14 a magnitude of ML 6.8 occurred on September 18, 2022 following. Both events have the intense shocks, caus- ing surface ruptures and multiple disasters. Although the eastern region of Taiwan is prone to earthquakes, the location where this earthquake sequence occurred is situated in an area with relatively low seismic activ- ity (Lee et al. 2023). According to early survey reports (https://fault.gsmma.gov.tw/Reports/More/63cc5a4b20 20403d9f79d3c33a7aba0c), the earthquake processes and source rupture behavior of this earthquake sequence are unique. Following a major earthquake, usually, large number of aftershocks are always triggered in the epi- central area within a short period of time. The small earthquakes that occur immediately after the mainshock (for example of the first 30 min) and are located near or within the ruptured area of the earthquake can provide significant information to explore the earthquake source physics and rupture characteristics. In Taiwan, both early warning strong motion network and regional seismic network usually play the role in detecting the aftershocks and monitor earthquake sequence. The early warning strong motion network always reports large event in first time. However, both aftershocks directly masked by the main shock’s coda wave or aftershocks with very small magnitudes, are often difficult to be detected by both seismic networks. The events that were missed to report are referred to as "hidden events" in this study and the same as Kiser and Ishii (2013). To retrieve those hidden aftershocks, the deployment of a high-density temporary seismic network, utilizing highly sensitive seismometers and complete coverage of the earthquake source area is expected. there was no extra temporary seismic stations set up in the eastern region of Taiwan at the moment of the Guan- shan earthquake occurred, it can be expected that smaller aftershocks will not be effectively detected and reported. In the study, we will examine the continuous recordings of the Taiwan Strong Motion Instrumentation Network (TSMIP), a dense seismic network originally designed for earthquake engineering applications, to extract earth- quake data for aftershock analysis. We hope to locate all aftershocks of the first 30 min and retrieve small events that were not included in the CWB rapid report catalog (https://scweb.cwa.gov.tw/zh-tw/earthquake/data/). (See figure on next page.) Fig. 1 a Schematic geological map of the source region and four major events of this earthquake sequence with magnitude greater than 6 (modified from Lee et al. 2008). Brown circle indicates the Guanshan earthquake, blue circle indicates the Chishang earthquake and two yellow circles indicate other reported events. b The recorded events of the Chishang region by CWNSN from 2022/09/01 to 2022/09/30. The event magnitudes are scaled by the size of event symbols (circle) from magnitude 6 to 1. Event depths are scaled by the color bar listed below. Both Guanshan and Chishang events are indicated as blue star and circle, respectively By using newly developed methods to analyze the recorded high spatial-density earthquake data for earthquakes in or near the source area, we are able to locate more hid- den aftershocks lost by the CWB rapid report catalog. New messages from these small earthquakes enable us to discuss the source processes of the Guanshan earth- quake, as well as to explore characteristics of earthquakes which occurred in an aseismic zone. 1.1 Regional tectonics and seismic activities in the eastern Taiwan Taiwan is located at the collisional boundary between the Eurasian Plate and the Philippine Sea Plate. The Philippine Sea Plate is moving relative to the Eurasian Plate towards the northwest direction. The fault system in eastern Taiwan is influenced by the oblique collision and strong convergence between two blocks. The domi- nate fault of this region is named as the Longitudinal Val- ley fault, it connects the two arc-trench systems of the Ryukyu and Manila trenches. The eastern region of Tai- wan is known as a high seismic risk, and has experienced multiple disastrous earthquakes in history (Shyu et al. 2007; Lee et al. 2008; Huang et al. 2009; Mozziconacci 2013a; Wen et al. 2019). The Hualien-Taitung Valley was hit by several strong earthquakes in 1951, with the largest two earthquakes reaching a magnitude of 7.3 on the Rich- ter scale. The 1951 earthquake series have caused serious damage in the source area. Over the past 70 years, this region has several earthquake sequences occurred with damage reports (https://tec.earth.sinica.edu.tw/index. php). On 2022, the Eastern Taiwan earthquake sequence occurred in the southern part of the Hualien-Taitung Val- ley (Fig. 1a). The source area of this earthquake sequence is located at an active hidden fault zone in the western In this study, we aim to investigate the aftershocks of the Guanshan earthquake. As it is the first large event of this earthquake sequence, the identification of after- shocks is not yet affected by other large earthquakes, which greatly reduces the uncertainty of the aftershock study. The earthquake rapid report catalog published by the Central Weather Bureau (CWB) mainly relied the early warning strong motion network for location deter- mination (https://www.cwa.gov.tw/V8/C/E/index.html). The complete event catalog is updated using data from the Central Weather Bureau Seismographic Network (CWBSN), a regional network, and added more small events. The final event catalog of CWBSN will be checked manually and usually delayed several months. However, Page 3 of 14 Huang et al. Terrestrial, Atmospheric and Oceanic Sciences (2024) 35:1 d on previous page ) Fig. 1 (See legend on previous page.) Fig. 1 (See legend on previous page.) Fig. 1 (See legend on previous page.) Huang et al. Terrestrial, Atmospheric and Oceanic Sciences (2024) 35:1 Page 4 of 14 Page 4 of 14 side of the Hualien-Taitung Valley suture zone near the Central Mountain Range. 1.1 Regional tectonics and seismic activities in the eastern Taiwan This fault system is adjacent to the Longitudinal Valley fault in the coastal mountain range, and seismic activities between the two fault sys- tems are often linked (Shyu et al. 2006; Mozziconacci 2013b; Lee et al. 2023). Referring to the distribution of long-term earthquake activity in Taiwan, this earthquake sequence occurred in an area with relatively low seismic- ity region. is for seismological applications, to support earthquake research in related fields by studying path effects of wave propagation and source effects of large earthquakes, including the source size and rupture process. Since 2018, the CWB has upgraded part of TSMIP stations to continuous recording and real-time transmission of data to provide earthquake intensity reports and plan for emergency response needs following future major earth- quakes. After the upgrade, a strong motion network with continuous recording has been established. Currently, there are about 400 free-field strong-motion stations, mainly distributed in the Taiwan plain and metropolitan areas (https://gdmsn.cwa.gov.tw/network_tsmip.php). 1.2 Seismic observations On September 17th, 2022, an earthquake occurred at 21:47 local time, with a magnitude of ML 6.4. The epi- center was close to Guanshan in Taitung, at latitude 23.08°N and longitude 121.16°E. On September 18th, 2022, the earthquake occurred at 14:44 local time, with a magnitude of ML 6.8. The epicenter was close to Chishang in Taitung, at latitude 23.14°N and longitude 121.2°E. In Taiwan, the reporting of felt earthquakes and the warning of large earthquakes is carried out by the early warning strong motion network of CWB (https:// www.cwa.gov.tw/V8/E/E/index.html). Till the end of September, this earthquake monitoring network detected a total of 207 felt earthquakes during the monitoring of the 2022 Eastern Taiwan earthquake sequence. The tem- poral distribution of the earthquakes was dominated by the Guanshan (foreshock) and Chishang (mainshock) earthquakes, each showing a clear mainshock-aftershock relationship (Lee et al. 2023). After the Chishang earth- quake, aftershocks continued to occur towards the north, eventually extending to the vicinity of Ruisui in Hualien (Fig. 1b). According to the reported moment tensor solu- tions (https://tecdc.earth.sinica.edu.tw/FM/AutoBATS), the source mechanisms of both events are similar. The possible fault plane is oriented in a north-northeast to south-southwest direction, with a high angle and west- ward dip, and is dominated by left-lateral slip (Fig. 1). The maximum seismic intensity of the two earthquakes reached level 6 in various parts of Taiwan, and the depth of both earthquakes was only about 7 km, which is con- sidered extremely shallow. During the 2022 eastern Taiwan earthquake sequence, the TSMIP stations continuously recorded the events and the network was fully operational. Figure 2 displays the seismic stations surrounding the epicenter of the Guanshan earthquake and the vertical component seis- mograms for seismic stations within 100 km epicenter distances. The high spatial density of stations near the source region and the resulting high-resolution seismo- grams enable a detailed examination of the Guanshan earthquake and its aftershocks. This study focuses on enhancing aftershock signals and relocating events using an array stacking approach, which eliminates the need for phase picking of emergent first arrivals of small events. The methods of analysis proposed will be explained in the next section. 2.1 Automatic gain control: signal enhancement for low amplitude aftershocks Automatic gain control (AGC) is one of the most com- mon gain recovery methods used in seismic signal pro- cessing. In this study, the concept of AGC will be applied to process earthquake multiple event seismograms, increase the strength of aftershock signals to identify the timing of aftershocks and perform location estimation. AGC is a type of limited output that uses a combination of linear amplification and compression amplification to adjust the output of the original signal. When a weak signal is input, the seismic signal is linearly amplified to ensure the output signal strength. When the input signal reaches a certain strength, the compression of the seis- mic signal is activated, causing the output amplitude to decrease. 1.3 Data collected from TSMIPh The geological environment in Taiwan is complex and diverse. In order to capture the regional seismic charac- teristics of large earthquakes, from early 1990, the CWB has continuously installed 737 free-field strong motion sensors in densely populated plain areas and named as TSMIP. Its main mission is for engineering applica- tions, to investigate site effects caused by local geologi- cal differences in Taiwan due to earthquake waves, and to establish regulations to achieve the goal of earthquake resistance and disaster reduction. Its secondary mission In this study, we proposed the Root mean square (rms) amplitude automatic gain control (or named as rms amplitude AGC) for signal enhance. This method is one of the most commonly used types of gain control (Gadal- lah and Fisher 2005, 2009). The rms amplitude AGC gain function is based on the rms amplitude of the input sig- nal within a specified time window. The gain function is Huang et al. Terrestrial, Atmospheric and Oceanic Sciences (2024) 35:1 Page 5 of 14 Fig. 2 a The selected stations of TSMIP recorded the Guanshan earthquake with epicentral distance less than 100 km. b Vertical array waveforms plotted by epicentral distances Fig. 2 a The selected stations of TSMIP recorded the Guanshan earthquake with epicentral distance less than 100 km. b Vertical array waveforms plotted by epicentral distances calculated as follows: first, the input seismic waveform is divided into fixed time windows, and the square of the amplitude of each sample in the time window is calcu- lated. Second, the average of these values is calculated, and the square root is taken. This is defined as the rms amplitude of the time window. The ratio of the desired rms amplitude (for example, a value of 2000) to the actual rms value is specified as the value of the gain function at the center of the time window. Therefore, the scaling function g(t) at the center of the time window is given by the following equation: Fig. 3 The same seismic waveforms of Fig. 2b processed by the AGC algorism to enhance aftershocks. Each trace is scaled to the same size in this record section Fig. 3 The same seismic waveforms of Fig. 2b processed by the AGC algorism to enhance aftershocks. Each trace is scaled to the same size in this Fig. 3 The same seismic waveforms of Fig. 2b processed by the AGC algorism to enhance aftershocks. 1.3 Data collected from TSMIPh Each trace is scaled to the same size in this record section Huang et al. Terrestrial, Atmospheric and Oceanic Sciences (2024) 35:1 Page 6 of 14 (1) g(t) = desired rms 1 N N i=1x2 i , detecting hidden aftershocks within a short time window following a major earthquake. (1) where xi is the amplitude of the input signal at time point i, and N is the number of samples within the window.f 2.2 Back‑projection (BP) method: application for earthquake locationh The BP method uses a grid search approach to consider all grid points within the model as possible earthquake source locations. The seismic wave travel time differences between each grid point and all seismic stations are cal- culated using an appropriate velocity model (Ishii et al. 2005). By moving all grid points appropriately to match the seismic wave travel times recorded by the seismic sta- tions, the earthquake wave energies are shifted in time and overlaid to obtain the corresponding peak values. The grid point that produces the highest peak value is identified as the earthquake source through evaluation. The calculation steps can be represented by the following formula: To demonstrate the effectiveness of our processing method, this study used the Guanshan earthquake and its aftershock data for the following 300 s as an example, as shown in Fig. 2. The seismic records from the near-source station did not show any clear aftershock signals, and the earthquake catalog from the Central Weather Bureau did not list any located aftershocks also within this 300-s time window. However, after applying the Automatic Gain Control (AGC) method proposed in this study to enhance the aftershock signals, the obtained results are shown in Fig. 3. Comparing the two figures, it is evident that multiple aftershocks near the Guanshan earthquake source occurred within this 300-s time window after the main earthquake, which were not visible in the seismic records without the AGC processing. This example high- lights the potential application of the AGC method in (2) Stacki(t) = 1 N N k=1 Sk(t −tik), (2) Fig. 4 Comparison of event locations reported by the CWB (indicated as circle) and BP (symbol: +). The spatial variation of epicenter in the EW, NS and Vertical components are shown in right panels following the BP method. This is a selected event on eastern Taiwan from CWB rapid catalog (event time: 2023/06/15/03:19:8.5) as an example. Total of 150 TSMIP waveforms with epicentral distances less than 150 km are selected by BP to locate events. The data has been previously processed by 1 Hz low pass filterer to remove incoherence signals Fig. 4 Comparison of event locations reported by the CWB (indicated as circle) and BP (symbol: +). The spatial variation of epicenter in the EW, NS and Vertical components are shown in right panels following the BP method. 2.2 Back‑projection (BP) method: application for earthquake locationh This is a selected event on eastern Taiwan from CWB rapid catalog (event time: 2023/06/15/03:19:8.5) as an example. Total of 150 TSMIP waveforms with epicentral distances less than 150 km are selected by BP to locate events. The data has been previously processed by 1 Hz low pass filterer to remove incoherence signals Fig. 4 Comparison of event locations reported by the CWB (indicated as circle) and BP (symbol: +). The spatial variation of epicenter in the EW, NS and Vertical components are shown in right panels following the BP method. This is a selected event on eastern Taiwan from CWB rapid catalog (event time: 2023/06/15/03:19:8.5) as an example. Total of 150 TSMIP waveforms with epicentral distances less than 150 km are selected by BP to locate events. The data has been previously processed by 1 Hz low pass filterer to remove incoherence signals Huang et al. Terrestrial, Atmospheric and Oceanic Sciences (2024) 35:1 Page 7 of 14 time-shift the seismic records based on predicted travel times, and stack all seismic records for this grid point. Each grid point within the model is considered a poten- tial earthquake source and the same calculations are repeated for each point of the model. If the grid point is the true hypocenter, the arrival times will align after time shifting, resulting in a constructive stack on the seismic record. For other non-source grid points, they will not align, and the stack out of phase. This repetitive process generates a 4-D BP image that includes the time his- tory of the stack amplitude at each potential hypocenter within the search 3-D volume. Finally, the earthquake hypocenter location and origin time are represented by the time point of maximum amplitude at the correspond- ing grid point. A unique feature of the BP method is where Stacki(t) represents the time series of earthquake waveform energy summed over all seismic stations, N is the number of seismic stations, k is the index of the kth seismic station, Sk(t) is the seismic wave amplitude time series of the kth seismic station, and tik is the theoretical travel time between index grid point i of the model and the kth seismic station (Ishii et al. 2007). The theoretical travel time used for stacking can be either the travel time of P-waves or S-waves. 2.2 Back‑projection (BP) method: application for earthquake locationh 6 The counted number with time plot (The N-T chart) of the first 30 minters minutes aftershocks of the 2022 September 17, ML 6.4, Guansh earthquake aftershocks during this time period on their rapid report website. that there is no need to pick arrival times for the seismic records during the earthquake location process. In this study, AGC is used in data processing to improve the visibility of aftershocks in which attenua- tion or spherical divergence has caused amplitude decay. The rms AGC is used for amplitude equalization. It gives a measurement of the overall absolute amplitude in the window, both as positive and as negative values. To per- form the automatic detection, a fixed gate length is nec- essary. After several trying, we selected 10 s for this case. Corresponded to Fig. 5a, the processed AGC Plots of seismograms is shown in Fig. 5b. It shows that each trace has similar time moveout, it indicates those events are closer to the epicenter of Guanshan earthquake. In total, 80 events have identified in this plot as events near the ML 6.4 Guanshan event. The counted event number with time (N-T plot) have been summarized as Fig. 6. In this study, we have developed a suitable program based on above procedure. The earthquake location cal- culation is performed by directly stacking seismic records from the TSMIP network with a dense array configura- tion. A 1-D regional model of Taiwan (Roecker et al. 1987) and a lateral homogeneous raytracing method (Huang 1996) are selected for the predicted travel times used in this study. Example for a medium magnitude event in the eastern Taiwan is shown in Fig. 4 (left panel), and the corresponding resolution (stacked energy) checks on the right. 2.2 Back‑projection (BP) method: application for earthquake locationh To locate an earthquake using the BP method, we need to discretize the earth model into a three-dimensional (3-D) grid. Assuming the true location of the earthquake source is within the 3-D grid. As a sample, we calculate the predicted travel times from location of a selected grid point within the model to each seismic station. We then Fig. 5 a 30-min vertical component continuous data after the ML 6.4, Guanshan earthquake collected from the TSMIP stations with epicentral distances less than 60 km. Vertical dashed lines (red) denote original time of aftershocks reported by the CWB rapid catalog. b The same network vertical-component waveforms on upper panel processed by AGC (10 s time window) to modulate aftershock amplitudes. Small events with simila alinement of mainshock are seen in this profile. It indicates all events near the epicenter and considered as near source aftershocks Fig. 5 a 30-min vertical component continuous data after the ML 6.4, Guanshan earthquake collected from the TSMIP stations with epicentral distances less than 60 km. Vertical dashed lines (red) denote original time of aftershocks reported by the CWB rapid catalog. b The same network vertical-component waveforms on upper panel processed by AGC (10 s time window) to modulate aftershock amplitudes. Small events with similar alinement of mainshock are seen in this profile. It indicates all events near the epicenter and considered as near source aftershocks Fig. 5 a 30-min vertical component continuous data after the ML 6.4, Guanshan earthquake collected from the TSMIP stations with epicentral distances less than 60 km. Vertical dashed lines (red) denote original time of aftershocks reported by the CWB rapid catalog. b The same network vertical-component waveforms on upper panel processed by AGC (10 s time window) to modulate aftershock amplitudes. Small events with similar alinement of mainshock are seen in this profile. It indicates all events near the epicenter and considered as near source aftershocks Huang et al. Terrestrial, Atmospheric and Oceanic Sciences (2024) 35:1 Page 8 of 14 Fig. 6 The counted number with time plot (The N-T chart) of the first 30 minters minutes aftershocks of the 2022 September 17, ML 6.4, Guanshan earthquake Fig. 6 The counted number with time plot (The N-T chart) of the first 30 minters minutes aftershocks of the 2022 September 17, ML 6.4, Guanshan earthquake Fig. 2.3 Analysis and results T ff l l To effectively locate earthquakes, we selected data with a high signal-to-noise ratio from nearby stations of the Guanshan earthquake epicenter (as shown in Fig. 2) for analysis. In this study, we selected data from TSMIP seismic station within 60 km of epicentral distance. In total, 87 vertical component seismograms have been selected to use in this study. For each station, a total of 30-min time trace (including the Guanshan earthquake) was extracted and plotted on a figure based on the epi- central distance of the Guanshan earthquake, as shown in Fig. 5a. Although several low-amplitude aftershock sig- nals have showed in selected stations, however, the CWB fast report catalogue only identified and marked five In this study, the method to locate aftershock for this ML 6.4 Guanshan earthquake will be the BP method proposed in this study. It will be carried out through the energy superposition of seismograms without individual picking the arrival times of seismic waves. The total aftershock location processes will be based on 30-min seismic data and processed with AGC to enhance weak signals (Fig. 5b). We extract the common time window of seismograms containing aftershock sig- nals for further earthquake location. Taking Fig. 7 as an example, we selected the waveform data from 300 to 380 s in Fig. 5b (as shown in Fig. 7a) for the following Huang et al. Terrestrial, Atmospheric and Oceanic Sciences (2024) 35:1 Page 9 of 14 Fig. 7 a AGC enhanced waveforms of event 4 (Table 1), (b) enveloped waveforms and (c). the BP location report of this study. NST: the number of stations (vertical component waveforms) selected to relocate this event ed waveforms of event 4 (Table 1), (b) enveloped waveforms and (c). the BP location report of this study. NST Fig. 7 a AGC enhanced waveforms of event 4 (Table 1), (b) enveloped waveforms and (c). the BP location report of this study. NST: the number of stations (vertical component waveforms) selected to relocate this event Page 10 of 14 Page 10 of 14 Huang et al. Terrestrial, Atmospheric and Oceanic Sciences (2024) 35:1 Fig. 8 The located events of this study (small blue circle symbols), CWB reported events (medium gray circle symbols) and source rupture slip distribution (colored contours in unit of centimeter) of the Guanshan earthquake reported by Lee et al. (2023). 2.3 Analysis and results T ff l l 8 The located events of this study (small blue circle symbols), CWB reported events (medium gray circle symbols) and source rupture slip distribution (colored contours in unit of centimeter) of the Guanshan earthquake reported by Lee et al. (2023). The CWB reported Guanshan earthquake is plotted as the symbol of big red star Fig. 8 The located events of this study (small blue circle symbols), CWB reported events (medium gray circle symbols) and source rupture slip distribution (colored contours in unit of centimeter) of the Guanshan earthquake reported by Lee et al. (2023). The CWB reported Guanshan earthquake is plotted as the symbol of big red star earthquake location determination. To ensure the sta- bility of the superposition results, we converted the selected waveform data into its envelope forms to pro- vide time traces as shown in Fig. 7b. By performing the energy superposition computation using Eq. (2) of this study, we ultimately obtained the spatial coordinates and origin time of the located aftershock, as shown in Fig. 7c. The same procedure will be applied to all dis- tinguishable time windows of aftershocks within this 30-min period. However, most of the time windows of aftershocks are too short to complete stable image superposition. In the end, a total of 29 aftershocks have located and are all plotted in Fig. 8, with their corre- sponding source parameters listed in Table 1. The results show that major aftershocks are distributed on the north and south sides of the epicenter, with a small number of them distributed in the eastern side. The overlaid beneath Fig. 8 is the coseismic slip dis- tribution of the Guanshan earthquake inferred through joint inversion of multiple geophysical data by Lee et al. (2023). The hidden earthquake distribution determined in this study is consistent with the main slip distribu- tion of the Guanshan earthquake. According to the study by Lee et al. (2023), the main rupture of this earthquake sequence mainly occurred on a west-dipping fault and 2.3 Analysis and results T ff l l The CWB reported Guanshan earthquake is plotted as the symbol of big red star within the same time period. It indicated that the ML 6.4 Guanshan earthquake has triggered a series of aftershocks in the source region. The earthquake locat- ing process of CWB primarily involves extracting the arrival times of seismic waves to simplify the seismic data. It then goes through validated and widely used computer locating programs such as HYPO71 (Lee and Lahr 1975; Lee et al. 2003), HYPOINVERSE (Klein 1978, 2003), HYPOELLIPSE (Lahr 1989, 2003), etc., to perform data inversion and confirm the epicenter location. Although the locating system of CWB adapts to regional variations and optimizes rapid reporting operations by moderately modifying existing computer locating programs, the fundamental approach still requests picking the arrival times of seismic waves and then locating the earthquake. Above earthquake loca- tion methods relying on first arrival picking is challeng- ing for small events with low signal-to-noise ratio or multiple events occurred in a short time period. To address this issue, we used an alternative proce- dure to explore, it based on the idea of migrating the energy of an earthquake back into its source position by stacking along theoretical travel-time curves. We selected also the TSMIP data which is a dense seismic array designed for seismic hazard assessment purposes and does not used for event location. In this study, employed the BP method which is a technique com- monly used to image seismic sources (Ishii et al. 2005; Meng et al. 2011; Beskardes et al. 2018). This method utilizes the recorded seismic waveforms from multiple stations and calculates the correlation between each station and potential source locations. By scanning the entire study area, this method is able to identify hidden aftershocks that may have been missed by conventional methods. In this study, we revised this method with AGC to enhance buried signals to retrieve the early aftershocks of the 2022 September 17, Guanshan Tai- wan earthquake. The findings of the study reveal that the BP method successfully detects and locates a sig- nificant number of hidden aftershocks that were not previously identified. The identified aftershocks provide valuable insights into the fault geometries and seismic activity in the region following the main earthquake. It provides an opportunity to view the evolution this earthquake sequence.h Fig. 3 Discussion and conclusions As shown in Fig. 8, the aftershocks determined in this study (29 events) presents more detailed spatial dis- tribution than that determined by CWB (5 events) Huang et al. Terrestrial, Atmospheric and Oceanic Sciences (2024) 35:1 Page 11 of 14 Table 1 Relocated events of this study (#01 is the ML 6.4 Guanshan earthquake) a DLEQ: Events(+) commonly located by Deep Learning (DL) method (Sun et al. 3 Discussion and conclusions 2023) # Year m d h mn sec lat lon dep DLEQa 01 2022 09 17 13 41 29.0 23.03 121.18 00.00 02 2022 09 17 13 43 54.0 23.08 121.09 06.00 + 03 2022 09 17 13 44 59.0 23.05 121.21 14.00 + 04 2022 09 17 13 46 35.5 23.08 121.18 02.00 + 05 2022 09 17 13 47 23.5 23.03 121.17 12.00 + 06 2022 09 17 13 47 43.0 23.05 121.19 02.00 + 07 2022 09 17 13 50 25.0 23.10 121.15 07.00 + 08 2022 09 17 13 52 46.5 23.10 121.18 08.00 + 09 2022 09 17 13 53 39.0 23.05 121.17 03.00 + 10 2022 09 17 13 54 45.5 23.05 121.21 15.00 + 11 2022 09 17 13 55 44.5 23.14 121.18 04.00 + 12 2022 09 17 13 57 05.0 23.09 121.15 02.00 + 13 2022 09 17 13 57 20.0 23.10 121.18 08.00 + 14 2022 09 17 13 57 55.0 22.95 121.11 09.00 15 2022 09 17 13 58 23.0 23.12 121.18 02.00 + 16 2022 09 17 13 59 54.0 23.05 121.19 09.00 + 17 2022 09 17 14 00 48.5 23.10 121.18 08.00 + 18 2022 09 17 14 01 07.0 23.08 121.17 13.00 + 19 2022 09 17 14 01 18.0 23.15 121.19 13.00 + 20 2022 09 17 14 01 36.0 23.12 121.18 07.00 21 2022 09 17 14 02 37.5 23.14 121.18 07.00 + 22 2022 09 17 14 04 17.0 22.99 121.12 10.00 + 23 2022 09 17 14 04 39.0 23.05 121.12 03.00 + 24 2022 09 17 14 05 29.0 23.02 121.12 03.00 + 24 2022 09 17 14 05 47.0 23.12 121.17 07.00 + 26 2022 09 17 14 06 50.5 23.05 121.19 11.00 + 27 2022 09 17 14 06 55.5 23.15 120.98 57.00 28 2022 09 17 14 07 35.0 23.08 121.18 07.00 + 29 2022 09 17 14 07 47.0 23.08 121.16 13.00 + 30 2022 09 17 14 08 30.5 23.15 120.98 57.00 Table 1 Relocated events of this study (#01 is the ML 6.4 Guanshan earthquake) sequence. For monitoring micro-seismicity on the creep- ing Chihshang Fault system and due to the sparse distri- bution of seismic stations in eastern Taiwan, NTU and IES deployed five broadband stations (red triangles in Fig. 3 Discussion and conclusions 9), the Chihshang seismic network (CSN), in early November 2021 to increase the station coverage. From September to October 2022, SeisBlue (Huang et al. 2023; Kuo-Chen et al. 2022), a deep learning-based seismologi- cal data processing platform, detects 6924 earthquakes by using the continuous seismic data from the 9 BATS stations without the CSN data but 14,454 earthquakes by combining the CSN and BATS data. Particularly on 17 September 2022 when the strong Mw 6.5 foreshock occurred, SeisBlue detects about 100 events without the CSN data but over 800 events with it. The earthquake detection ability is greatly improved by the CSN data. The details of the two-month (September to October 2022) its slip originated from the hypocenter and propagated toward the north. The Guanshan earthquake is consid- ered as a foreshock in the earthquake sequence, under- standing its detailed aftershock activity may provide an opportunity to insight the subsequent earthquake devel- opment. By examining the spatial and temporal distri- bution of these aftershocks, it may identify statistical patterns that can be used for forecasting purposes (Zhang et al. 2021) and the size distribution, and short-term fore- casting of large aftershocks. It has important implications for evaluating seismic hazards (Gulia et al. 2018). How- ever, it is beyond the scope of the initial research of this study. To validate the performance of the BP method of this study, we compared our results with new location event catalog of this earthquake sequence by Sun et al. (2023) based on an independent dataset of this earthquake Page 12 of 14 Page 12 of 14 Huang et al. Terrestrial, Atmospheric and Oceanic Sciences (2024) 35:1 Fig. 9 The 30-min aftershock seismicity right after the 2022-09-17 Guanshan earthquake occurred. The colored circles and starts are events detected by SeisBlue (Sun et al. 2023) and this study, respectively. Red triangles and squares are broadband stations. Gray squares indicated the locations of city or county. Red traces are active faults identified by Central Geological Survey, Taiwan (Lin et al. 2021) and the names of regional faults are referred to Fig. 1a deep learning-based SeisBlue earthquake catalog of the 2022 Mw 2022 Chihshang earthquake sequence would be described in Sun et al. (2023) included in this special issue. The SeisBlue earthquake catalog contains 43 after- shocks that occurred within 30 min after the Mw 6.5 Guanshan foreshock struck (Fig. 9; Table 2). 3 Discussion and conclusions Those events are located by the program HYPOCENTER (Lienert and Havskov 1995) with a 1-D velocity model by Central Weather Bureau, Taiwan (Chen and Shin 1998). Com- parison of the located event number and distribution of epicenter are similar from both BP and the deep learning (DL) location methods. However, it shows some differ- ences in detail. The original times for commonly located events showed several second variations and epicenters from BP method showed lower spatial resolutions than DL method. Both differences can be explained as differ- ent earth models are used by BP and DL methods, and the BP method used rough grids to search its epicenter. Furthermore, two events are located on western side of source region as deep events by BP method (Table 1) and do not identified by the DL method (Fig. 9). After check- ing array observations, it is found that multiple small events are identified on selected seismograms and both hypocenters are mislocated by BP stacking approach. deep learning-based SeisBlue earthquake catalog of the 2022 Mw 2022 Chihshang earthquake sequence would be described in Sun et al. (2023) included in this special issue. The SeisBlue earthquake catalog contains 43 after- shocks that occurred within 30 min after the Mw 6.5 Guanshan foreshock struck (Fig. 9; Table 2). Those events are located by the program HYPOCENTER (Lienert and Havskov 1995) with a 1-D velocity model by Central Weather Bureau, Taiwan (Chen and Shin 1998). Com- parison of the located event number and distribution of epicenter are similar from both BP and the deep learning (DL) location methods. However, it shows some differ- ences in detail. The original times for commonly located events showed several second variations and epicenters from BP method showed lower spatial resolutions than DL method. Both differences can be explained as differ- ent earth models are used by BP and DL methods, and the BP method used rough grids to search its epicenter. Furthermore, two events are located on western side of source region as deep events by BP method (Table 1) and do not identified by the DL method (Fig. 9). After check- ing array observations, it is found that multiple small events are identified on selected seismograms and both hypocenters are mislocated by BP stacking approach. Fig. 9 The 30-min aftershock seismicity right after the 2022-09-17 Guanshan earthquake occurred. 3 Discussion and conclusions Terrestrial, Atmospheric and Oceanic Sciences (2024) 35:1 Page 13 of 14 # Year m d h mn sec lat lon dep BPEQa 22 2022 09 17 13 57 17.8 23.14071 121.15299 10.66 + 23 2022 09 17 13 58 20.2 23.14677 121.14967 10.89 + 24 2022 09 17 13 58 50.7 23.03436 121.23302 17.65 25 2022 09 17 13 59 11.1 23.18617 121.15330 04.08 26 2022 09 17 13 59 27.0 22.98787 121.06712 04.12 27 2022 09 17 13 59 51.6 23.07656 121.16349 04.09 + 28 2022 09 17 14 00 18.3 23.06651 121.17497 11.73 29 2022 09 17 14 00 45.5 23.12142 121.15077 10.87 + 30 2022 09 17 14 01 04.3 23.10794 121.14091 10.56 + 31 2022 09 17 14 01 15.0 23.14824 121.15579 11.67 + 32 2022 09 17 14 01 57.3 23.17870 121.15279 06.16 33 2022 09 17 14 02 34.8 23.14763 121.15200 11.72 + 34 2022 09 17 14 03 43.6 23.04909 121.12793 11.04 35 2022 09 17 14 04 14.7 22.99771 121.09299 09.54 + 36 2022 09 17 14 04 36.0 23.04638 121.10711 04.04 + 37 2022 09 17 14 05 25.9 23.01889 121.09670 06.54 + 38 2022 09 17 14 05 44.3 23.14195 121.15247 10.56 + 39 2022 09 17 14 06 48.4 23.04987 121.15099 07.76 + 40 2022 09 17 14 07 32.4 23.10076 121.16280 05.11 + 41 2022 09 17 14 07 44.3 23.12931 121.15334 10.68 + 42 2022 09 17 14 07 58.2 23.14762 121.15622 09.56 43 2022 09 17 14 08 47.4 23.12301 121.16119 09.90 Table 2 (continued) Declarations Competing interests The authors declare that they have no competing interests. Received: 30 August 2023 Accepted: 14 December 2023 Published: 2 January 2024 In conclusion, results of this study indicate the impor- tance of utilizing advanced imaging techniques like BP method to enhance the understanding of aftershock sequences and improve seismic hazard assessments. The results contribute to the knowledge of post-earthquake processes and can assist in refining seismic monitoring strategies for large events in the future. Received: 30 August 2023 Accepted: 14 December 2023 Published: 2 January 2024 Received: 30 August 2023 Accepted: 14 December 2023 Published: 2 January 2024 Funding This study was supported by Academia Sinica, under grant AS-TP- 110-M02, the Central Weather Bureau, under grant MOTC-CWB-112-E-02, the National Science and Technology Council, Taiwan, under grant NSTC 111-2116-M-001-011. 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Red traces are active faults identified by Central Geological Survey, Taiwan (Lin et al. 2021) and the names of regional faults are referred to Fig. 1a Fig. 9 The 30-min aftershock seismicity right after the 2022-09-17 Guanshan earthquake occurred. The colored circles and starts are events detected by SeisBlue (Sun et al. 2023) and this study, respectively. Red triangles and squares are broadband stations. Gray squares indicated the locations of city or county. Red traces are active faults identified by Central Geological Survey, Taiwan (Lin et al. 2021) and the names of regional faults are referred to Fig. 1a Table 2 Earthquakes detected by SeisBlue using 14 permanent broadband stations for the first 30 min after the Mw 6.5 Guanshan foreshock occurred # Year m d h mn sec lat lon dep BPEQa 01 2022 09 17 13 43 50.1 23.07795 121.15854 07.92 + 02 2022 09 17 13 44 56.9 23.07088 121.15987 09.66 + 03 2022 09 17 13 46 07.8 23.08831 121.15965 10.57 04 2022 09 17 13 46 33.1 23.07819 121.16427 05.92 + 05 2022 09 17 13 47 20.7 23.08888 121.17744 10.06 + 06 2022 09 17 13 47 40.4 23.07638 121.16339 09.78 + 07 2022 09 17 13 49 18.2 23.04170 121.10590 04.63 08 2022 09 17 13 49 45.4 23.08904 121.15619 10.70 09 2022 09 17 13 50 22.5 23.08776 121.14108 12.32 + 10 2022 09 17 13 50 46.1 23.07298 121.16299 08.88 11 2022 09 17 13 51 14.0 23.04522 121.10281 06.59 12 2022 09 17 13 51 24.2 23.02963 121.13734 08.38 13 2022 09 17 13 51 47.6 23.09018 121.17395 07.67 14 2022 09 17 13 52 43.0 23.03156 121.17955 04.39 + 15 2022 09 17 13 53 24.8 23.08111 121.18546 07.42 16 2022 09 17 13 53 35.3 23.10270 121.16354 10.67 + 17 2022 09 17 13 54 16.0 23.09920 121.16238 07.80 18 2022 09 17 13 54 43.4 23.07831 121.16753 09.83 + 19 2022 09 17 13 55 13.8 23.10737 121.14993 10.92 20 2022 09 17 13 55 41.6 23.12378 121.16028 10.49 + 21 2022 09 17 13 57 01.9 23.09948 121.14798 07.16 + Huang et al. 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Geol Soc Am Bull 118:1447–1462. https:// doi.org/10.1130/B25905.1 Shyu JBH, Chung LH, Chen YG, Lee JC, Sieh K (2007) Re-evaluation of the surface ruptures of the November 1951 earthquake series in eastern
https://openalex.org/W2107900933	https://www.ccsenet.org/journal/index.php/ass/article/download/37507/21044/	English	null	Corporate Social Responsibilities (CSR) as a Means of Materializing Corporate Vision: A Volvo Group Approach	Asian social science	2,014	cc-by	8,987	Corporate Social Responsibilities (CSR) as a Means of Materializing Corporate Vision: A Volvo Group Approach Received: November 26, 2013 Accepted: April 1, 2014 Online Published: May 30, 2014 doi:10.5539/ass.v10n11p258 URL: http://dx.doi.org/10.5539/ass.v10n11p258 Abstract Corporate social responsibility (CSR) is considered neither an optional extra nor a philanthropic exercise; rather it is a mandatory and strategic tool by which a company can strengthen its brand, develop good relationships with stakeholders, increase its attractiveness to present as well as potential employees, and enable access to new markets which ultimately lead to materialize its vision. In this article a thorough study has been done on the exercise of CSR activities and sustainable reporting of a world renowned Swedish business conglomerate named Volvo Group with a view to know the nature as well as ways of CSR exercises. It is found that Volvo Group is not only following ‘creating shared value’ (CSV) approach but also ‘Stakeholders model’ of CSR and every CSR activity is exercised deliberately and strategically. As a result, the overall performance of the group is increasing gradually, which ultimately leads the group towards the achievement of its vision. Indeed, CSR of Volvo is related to many aspects of business management like risk management, challenge management, environment management, supply chain management, talent management, economic growth management, efficiency management and corruption management. Consequently, the competiveness of the Group is uplifting gradually. It is expected that this study will inspire the corporations of all over the world to exercise the CSR activities in strategic way not only for the greater and better benefits of the stakeholders but also for the sustainable competitive advantage of the company with a view to materialize corporate vision. Keywords: CSR, competitive advantage, stakeholders’ involvement Asian Social Science; Vol. 10, No. 11; 2014 ISSN 1911-2017 E-ISSN 1911-2025 Published by Canadian Center of Science and Education Asian Social Science; Vol. 10, No. 11; 2014 ISSN 1911-2017 E-ISSN 1911-2025 Published by Canadian Center of Science and Education 1. Introduction The authors identified and categorized three types of social investment (altruistic, selfish and strategic) through a comparison between firms, finally the authors concluded that strategic investment generates superior results for the firms that try to maximize both profit and social performance simultaneously. Strategic investment can provide extra benefits to the company in the form of skilled and dedicated workforce, differentiation of products, enhanced reputation and extraction of a premium price. This study finally point out that through socially responsible activities, a company can add necessary value and thereby develop competitive advantage, but the company must do each and every single activity strategically and CSR activities should be designed in the line with the corporate strategies. The public should see that all actions are not only adding value to products but also improving the local business environment (Porter & Kramer, 2002). Since around the world corporations are facing an increasingly competitive environment, CSR activities should be performed in such a way so that corporations can clearly differentiate their activities to generate a competitive advantage (Hillman & Keim, 2001). According to Barney (1991) positive corporate reputation can be a source of sustainable competitive advantage, since there is a direct relationship between a corporation and its stakeholders. Corporate social responsibility (CSR) can play vital role in ensuring sustainable competitiveness advantage of business and success by developing better employee performance, cooperation among the business partners, consumers and suppliers (Lamy, 2002). Globally, CSR practices are gradually shifting from traditional (philanthropy) to strategic CSR whereas in many organizations the practices of corporate social responsibility (CSR) is limited within traditional philanthropic activities such as donations to poor people, charitable organizations and religious institutions in many developing and underdeveloped countries (Mondol, 2010). But CSR is highly required for developing competitive advantage by focusing on the employee involvement in organizational activities, customer satisfaction, continuous development in quality of product, work place safety, corporate governance, labor rights, community development, environment management and fair treatment of workers which will not only differentiate the organization from its competitors but also help in the rapid industrialization and thereby ensuring sustainable development . At present it is seen that corporations are more concerned with social responsibility because of global tendency (Halme, Roome & Dobers, 2009) as well as civil society’s awareness regarding CSR (Collier & Esteban, 2007).Companies are exercising the CSR activities for greater competitiveness and better results (Ashley, 2002). 1. Introduction Corporate social responsibility (CSR) has been placed in recent times under study by the stakeholders of firm; consequently, CSR is an increasingly central concern in business decision- making and has become a widely-applied concept (Gyomlay & Moser, 2005; Cochran, 2007). It is now considered as the engine of economic growth (Lamy, 2002). It is a form of management that is defined by the ethical relationship and transparency of the company with all the stakeholders with whom it has a relationship as well as with the establishment of corporate goals that are compatible with the sustainable development of society, preserving environmental and cultural resources for future generations, respecting diversity and promoting the reduction of social problems (Ethos Institute, 2007: 78). CSR includes adaptation of manufacturing processes and products to address social values (such as eliminating excess packaging), valuing human resources (such as personal development training and Occupational Health & Safety programs),improving environmental performance through recycling and pollution abatement (such as emission reductions), and supporting community organizations (such as by sponsoring a local sporting club) (WBCSD 2004). According to Bowen (1953) business persons are responsible for the consequences of their actions in a spare wider than that covered by their profit and loss statements. Though the academic construct of CSR was first developed in the 1950s, but came to prominence in the 1970s and 1980s in conjunction with increased public scrutiny and focus upon the image of the corporation (Clark, 2000; Golob & Bartlett, 2007; World Business Council for Sustainable Development [WBCSD], 2004). Increased scrutiny has also resulted in rapid growth of the number of instruments used to manage measure, communicate, and reward corporate social responsibility (European Commission, 2004) At the inception most of the business people regard the CSR as involving in financially sustainable activities of business that go beyond legal requirements to protect the well-being of customers, employees, communities and 258 Vol. 10, No. 11; 2014 Asian Social Science www.ccsenet.org/ass the environment, but from now on, it is recognized as a source of competitive advantage (Porter & Kramer, 2006) which contributes to the business growth. According to Husted & Allen (2000) social responsibility is to be considered as an important element while formulating strategy. They also mentioned that The Harvard Business School strategy model has always included CSR for formulating strategy. 1. Introduction Michael Porter and Mark Kramer (2006) suggested that by making social and environmental contributions strategically a company can generate tangible business benefits. So, around the globe there is a competition in practicing CSR among the corporations in strategic way because of its different benefits in the form of better employee loyalty, attracting and retaining customers etc. According to Mamun & Ahmed (2009) the business enterprises should perform CSR activities for their stability, growth, prosperity and also for the development of the country. Corporate sectors must be responsible enough for ensuring investment climate, right of customers, efficiency, practicing fair employment, quality services, improved industrial life and finally protecting community interests (Mamun & Ahmed, 2009). Through literature survey it has been found that most of the corporations of underdeveloped countries are performing CSR activities in a very tradition way not in a strategic way as a result those companies are suffering. But a company can improve both financial and social performance if CSR activities are performed deliberately and strategically. Thus this case study is an attempt to focus on the nature and ways of CSR exercises of a renowned global Business Group like Volvo so that the corporations of the world can get motivation and idea in exercising CSR activities in their respective organizations in strategic way and thereby ensuring sustainable competitive advantage with a view to materializing corporate vision. This study has been conducted on Volvo Group because it is one of the top performing groups in the world having high reputation. In writing the article, the researchers studied the annual CSR report of Volvo Group, available published literatures, research monographs, journal, and magazines in this field. 1. Introduction Indeed, in the literature of management, ethics and social responsibility have been frequently connected to corporate objectives and incorporated into business speeches. Social responsibility is now considered as a universal belief in modern business (Pearce & Doh, 2005). Husted and Salazar (2006) studied strategies of CSR in many firms with the objective of maximizing both social performance and profits. The authors identified and categorized three types of social investment (altruistic, selfish and strategic) through a comparison between firms, finally the authors concluded that strategic investment generates superior results for the firms that try to maximize both profit and social performance simultaneously. Strategic investment can provide extra benefits to the company in the form of skilled and dedicated workforce, differentiation of products, enhanced reputation and extraction of a premium price. This study finally point out that through socially responsible activities, a company can add necessary value and thereby develop competitive advantage, but the company must do each and every single activity strategically and CSR activities should be designed in the line with the corporate strategies. The public should see that all actions are not only adding value to products but also improving the local business environment (Porter & Kramer, 2002). Since around the world corporations are facing an increasingly competitive environment, CSR activities should be performed in such a way so that corporations can clearly differentiate their activities to generate a competitive advantage (Hillman & Keim, 2001). According to Barney (1991) positive corporate reputation can be a source of sustainable competitive advantage, since there is a direct relationship between a corporation and its stakeholders. the environment, but from now on, it is recognized as a source of competitive advantage (Porter & Kramer, 2006) which contributes to the business growth. According to Husted & Allen (2000) social responsibility is to be considered as an important element while formulating strategy. They also mentioned that The Harvard Business School strategy model has always included CSR for formulating strategy. Indeed, in the literature of management, ethics and social responsibility have been frequently connected to corporate objectives and incorporated into business speeches. Social responsibility is now considered as a universal belief in modern business (Pearce & Doh, 2005). Husted and Salazar (2006) studied strategies of CSR in many firms with the objective of maximizing both social performance and profits. 3.1 CSR Approach in Volvo Group 3.1 CSR Approach in Volvo Group The Volvo Group has moved to the concept of creating shared value (CSV) approach of CSR from the traditionally CSR perspectives because, social welfare and corporate success are interrelated and interdependent. In essence, shared value is about creating win-win situations for society, Volvo Group and other stakeholders. A business needs educated and healthy workforce, sustainable resources and adept administration for performing effectively in the competitive business environment. To survive in the competition, profitable and competitive businesses must be developed because to perform responsibilities toward different stakeholders business must generate sufficient revenue. For example; at present majority people of the world resides in towns. Efficient transport of goods and people are specific requirement of many Cities. With the help of skills, experience, and knowledge and cooperating with the authorities of cities it is possible to form the required communication systems and structure of future. This collaboration will help the towns as well as the people residing in the cities and the business of Volvo Group. It is considered that transport is a powerful force for socio-economic development. The Group develops pioneering products and a service based on the expectations and needs of customers and intends to provide its investors a good return. Indeed, profit is very necessary for its enduring business existence and a precondition for supporting its operations, further expansion of the business and investing in new technology. The group also strives to provide service to society by using its expertise, exclusive proficiencies and resources to build up better solutions so as to rightly deal with the challenges of sustainable transport and thereby reducing risk and making profit. This is what the Group means by creating shared value. In fact, in Volvo Group, the approach of CSR is based on its core business, vision, expectations of stakeholders, operating context as well as its culture and core values. Its CSR approach encompasses value creation dimensions and risk management. CSR activities of Volvo Group enable in making a positive contribution to society as well as in ensuring sustainable development. It is the conviction of the Group that there is no disagreement between doing a profitable business concern and making contribution towards sustainable development. The Group strives to assume financial, social and environmental responsibility for its goods, services and operations. 2. A Brief History of Volvo Group The Volvo Group is one of the world’s leading manufacturers of trucks, buses and construction equip 259 Vol. 10, No. 11; 2014 Asian Social Science www.ccsenet.org/ass systems for marine and industrial applications and aerospace components. The Group also provides complete solutions for financing and service. In 1927, the first series-produced Volvo car rolled off the production line at the Gothenburg plant and in 1928 the first truck was produced. The Volvo Group employs about 115,000 people, has 65 production facilities in 20 countries and sells its products in more than 190 markets. In 2011 annual sales of the Volvo Group amounted to about SEK 310 billion. The Group’s market share of sales are 39% in Europe, 19% in North America, 11% in South America, 24% in Asia, 7% in rest of the World(Accessed online http://www.businesswire.com//news/home/20120403005688/en). systems for marine and industrial applications and aerospace components. The Group also provides complete solutions for financing and service. In 1927, the first series-produced Volvo car rolled off the production line at the Gothenburg plant and in 1928 the first truck was produced. The Volvo Group employs about 115,000 people, has 65 production facilities in 20 countries and sells its products in more than 190 markets. In 2011 annual sales of the Volvo Group amounted to about SEK 310 billion. The Group’s market share of sales are 39% in Europe, 19% in North America, 11% in South America, 24% in Asia, 7% in rest of the World(Accessed online http://www.businesswire.com//news/home/20120403005688/en). The Volvo Group was one of the first companies to become a signatory to the United Nations’ Global Compact when it was launched in 2001. The Group is committed to the principles of the initiative, and they are integrated into its Group policy and core governance document: the Volvo Group Code of Conduct. The Volvo Group’s vision is to become the world leader in sustainable transport solutions by creating value for customers in selected segments, pioneering products and services for the transport and infrastructure industries, driving quality, safety and environmental care, and working with energy, passion and respect for the individual. 3 Fi di d A l i 3. Findings and Analysis From the study it has been found that in Volvo Group, CSR activities are deeply rooted and related to many aspects of management. The findings have been presented under different broad heading such as CSR approach of Volvo Group, CSR model, Stakeholders’ engagement process, risk management, environment management, Corruption management, development of employee commitment, management of economic growth, Management of resource efficiency, supply chain management, challenge management, Management of CSR organization, research and development and management of fair competition etc. 3.1 CSR Approach in Volvo Group It is the belief of the group that it is vital to being perceived as an attractive business partner and to developing long-term relations with employees, customers, suppliers, creditors and other stakeholders (Annual CSR Report, 2011). The backbone of CSR approach of Volvo Group is its Code of Conduct. The Code of conduct states the philosophies for running business in a proper, transparent and responsible way. Code of conduct sates about minimum height of manners and is compulsory for everyone in the organization. The CSR of Volvo Group is based on nine philosophies such as; (i) Visible and strong leadership (ii) Responsibility and accountability (iii) sustainability and CSR are essential elements of its processes, business and strategy (iv) Performance is set, measured and followed up (v) Efficiency of resources (vi) Transparency (vii) 260 Vol. 10, No. 11; 2014 Asian Social Science www.ccsenet.org/ass Entertaining expectations of all stakeholders (viii) Working proactively and center on probable risks, and (ix) Partnerships and cooperation (Annual CSR report, 2011). 3.2 CSR Model in Volvo Group In stakeholder management, dialogue is a key element and a major responsibility for top managers (Szwajkowski, 2000; Wheeler & Sillanpaa, 1998; Wiedermann-Goiran et al., 2003). Communication is recognized as a vital ingredient in the CSR development in organizations, but only communication is not enough condition for ensuring meaningful CSR. Rather content of CSR is important (Szwajkowski, 2000).To survive in the competitive business with full name and fame, the opinions and views of the stakeholders are valuable. Considering the importance, systematic dialogue with all stakeholders is conducted and the opinions are honored through the development of its business. The group discloses all pertinent information timely for stakeholders. Furthermore, involving the stakeholders is one the ways of developing trust and gaining licenses to run business in a responsible way. CSR and sustainability report represents an important platform for stakeholder dialogue. The dialogue facilitates the Group to better understand the expectations of internal as well as external stakeholders with a view devise its sustainability strategies in accordance with its business objectives to minimize risk and exploit business opportunities. Since, there is a trends and global as well as internal demand for a sound CSR strategy, the management of Volvo group has been working sincerely from 2011 to develop a new CSR strategy. The goal has been to base the strategy of CSR on its ambitions, operating context and business model. Indeed, the CSR approach of the group goes beyond risk management really adds value to the business and thus contributes to its future business success. 3.3 Stakeholders’ Engagement in Volvo Group 3.3 Stakeholders’ Engagement in Volvo Group Stakeholders’ engagement is a very essential element of CSR (Lindgreen & Swaen, 2010) and companies will fail to convince stakeholders unless and until they can reveal their policies constantly attain the expected environmental, social and ethical outcomes (Collier & Esteban, 2007). There are many methods which are used for ensuring the engagement of stakeholders in Volvo Group. The details are as follows; 3.3.1 Customers Engagement Different products including goods, services and different solutions are developed as per the expectations of customers, with a view to helping the Group to increase its overall profitability and productivity. Long-term relationships are maintained with most of their customers because customer satisfaction depends not only on the quality and performance of the products, but also on how customers are treated and how service is delivered. Generally, the Group communicates with their customers in many ways such as interaction in daily operations, dialogue via social media, customer surveys, customer satisfaction surveys, and follow up on brand perception and survey on customers for knowing their level of satisfaction. Furthermore, the customer focus group is ensured in the product development phase. The Group follows up on brand image perception and level of customer satisfaction in relevant markets and industries. Brand Image Tracking and Customer Satisfaction is measured and compared with the global established industry surveys. 3.3.2 Employees Engagement 3.3.2 Employees Engagement The Group has shifted from satisfied employee concept to engaged employee concept. Because, engaged employees bring body, soul and mind to work (Collier & Esteban, 2007). Kerstin Renard, the Head of Corporate Human Resources stated, ‘We want to enable our employees to walk the extra mile- for themselves and for the company’. This sort of ‘company motto’ continuously energies the Group to ensure employee engagement with the help of some effective techniques such as: 1) Formalized forums are available in Volvo group for employee dialogue and development. Because, open dialogue helps in ensuring committed and dedicated employees through personal development. Many channels like intranet, team meetings, in-house magazines, films and webcasts are used for communication with the employees. Every single employee can ask any question openly to CEO of the Group through the Intranet. 2) The group regularly maps its strategic competences, and annually aggregate findings on a Group level to identify the most important competences for future needs. Since enhancing and securing competences is vital not only for being attractive and competitive employer but also for running the business in a profitable way. So, the Group works in different areas like capability development, health and wellbeing, talent planning, leadership training and work environment and dynamically seeks the opinions and feedback of its employees. The Group thus invests in offering an extensive variety of training and development programs to all levels of employees to ensure personal growth and advancement. Both traditional and modern training and development activities conducted that range from individual coaching and mentoring to e- based training. Talent review is conducted 261 Vol. 10, No. 11; 2014 Asian Social Science www.ccsenet.org/ass systematically and in a transparent way with a view to ensure required talent all over the organization. The process includes every single key position of all levels and leaders for providing adequate numbers of talent and skills. All managers are highly for evaluating and building necessary competencies and skills in the company. systematically and in a transparent way with a view to ensure required talent all over the organization. The process includes every single key position of all levels and leaders for providing adequate numbers of talent and skills. All managers are highly for evaluating and building necessary competencies and skills in the company. 3.3.2 Employees Engagement 3) Employee engagement index (EEI) has been introduced in the group along with employee satisfaction index (ESI) in 2011, because the business outcomes of employee engagement is well proven and include increased customer satisfaction, operational excellence and profitable growth. Issues related with business ethics, environment and CSR etc. are incorporated and entertained properly in the survey. The survey outcomes are discussed and analyzed with high priority in working groups, engaging all key employees and managers of the organization. Action plan is prepared for addressing areas of improvement throughout the year. Attitude measurement survey is conducted among all employees at least once in a year for knowing the level of employee commitment and satisfaction. In the year 2011, the overall employee engagement was 76% which was higher than the global norm of 68%. 4) Both in informal and formal forums, close relationships are maintained with a number of labor unions. All employees enjoy benefits according to performance, experience and competence regardless of race, sex, age, faith, sexual orientation, disability, union affiliation, or ethnic origin. 5) For attracting and retaining the best people the Group offers lucrative financial as well as nonfinancial benefits. As a result, at the end of 2010 the Group had 90409 regular and 14851 temporary employees and consultants, whereas; at the end of 2011 the Group had 98162 regular and 19675 temporary employees and consultants. Equal employment opportunity is the guiding principle in Volvo Group. Individual salaries are based on the market, position and the employee’s performance, and conform to the salary practice of each country. Besides fixed remuneration and variable salary, other expected benefits are usually offered such as health plans, pension solutions, recreational facilities, company cars or car allowances, insurance and so forth. 6) Different supports are provided not only for making employees healthy and energetic but also for ensuring workplace safety and employee wellbeing. There are heath coaches who continuously provide service to adopt health behavior. Stress management trainings are offered among the groups and individuals to prevent and avoid negative stress.  Non-Governmental Organizations Companies can increase their capability to deliver socially responsible solutions only when they can manage knowledge and skilled civil society who can give sufficient directions to their CSR activities and initiatives (Cambridge Programme for Industry (CPI) 2005, Demirag 2005, Collier & Esteban, 2007).Thus, the Group has developed very good relations with NGOs. For example, around the world, currently there are about 100 different cooperation projects with NGOs.  Universities and Research Institutes 3.3.5 Decision Makers On an ongoing basis, the Volvo Group communicates with authorities, politicians and institutions. This interaction includes responses to requests for comments on proposed legislation and activities. They also exchange information on the implications of legislation, incentives, etc., in order to support and enhance their business. Most of the issues related to the regulation of its products are dealt with within industry organizations in which the Volvo Group is a member.  Non-Governmental Organizations 3.3.3 Suppliers Close and long-term relationships with all suppliers is developed and maintained. The Group values the transfer of knowledge because understanding of each other’s procedures processes and values are very essential to be competitive in the market place. Different Forums like web-based supplier portal, dedicated supplier days, training sessions and contracts for formal communication are maintained. 3.3.4 Shareholders, Other Investors and Financial Analysts Regular communication is ensured with financial analysts, shareholders and other investors. There is an investor relations department that holds regular meetings with investors and issues many publications like press releases, annual reports, interim reports and a capital market day is held at least once in a year. 3.3.5 Decision Makers  Universities and Research Institutes For creating synergies at the front position of science and technology, Cooperation with academia and universities is essential because technically sophisticated products and services are being sold by Volvo Group. To go forward the technologies needed for future product development, the Group is involved in a comprehensive series of cooperative ventures with academic institutions and research bodies. Its participation 262 Vol. 10, No. 11; 2014 Asian Social Science www.ccsenet.org/ass with universities is vital for developing relationships with potential employees, students and thus for securing access to future competency. with universities is vital for developing relationships with potential employees, students and thus for securing access to future competency.  Educationist  Educationist Continual dialogues are held on educational issues with educationists at international, local, and national levels because it is the belief of the group that without the educated generation no society can prosper and progress. Thus, as part of CSR, Volvo Group supports both secondary and upper secondary schools in many locations around the world for educating the future generation. 3.4 Risk Management 3.4 Risk Management In Volvo Group, risk management is a key element of overall strategy of CSR. As part of risk management the Group tries its level best to comprehend the social, economic and political tendencies that may impact the role and reputation of the organization for making the proper strategic decisions. The Group categorized all risks under three group such as (ii) financial risks – including share evaluations, currency fluctuations, similar risks, and credit risks and (ii) external-related risks – including competition, economic cycles and regulation (iii) operational risks – related to the suppliers, introduction of new products, third-party complaints etc. The Group conducts annual internal audit. This internal corporate audit consists of about 35 internal auditors worldwide. They perform special assignments and audits as per the demand of management in line with the yearly audit plan. They issue reports periodically to the management and Audit Committee. The objectives of audit are to ensure proper effectiveness of governance processes, control and risk management. Indeed, a corporation that is adjusted with stakeholders’ priorities, expectations and concerns can respond quicker to probable risks and opportunities. 3.5 Economic Growth Management Profitable growth is a precondition for upholding competitiveness as well as for ensuring investments in the research and development of new services and products. Profitable growth is also a precondition for investing in areas and activities that contribute more to ensure long-lasting development. Since all CSR strategies are designed to exploit the market opportunities, the net sales of Volvo Group was increased from SEK 264749 Million to SEK 310367 million for the year 2010 and 2011 respectively. As a result the dividend per share increased from SEK 2.5 to SEK 3 and return on shareholder’s equity increased from 16% to 23.1% for the year 2010 and 2011 respectively. Moreover, the interest in and demand for environmentally-enhanced products have increased. The Group is well placed to exploit the opportunities of growth relating to this development. This is done by (i) developing new goods that facilitate its customers to select additional sustainable services and products (ii) expanding the service and product offer connected to the present business (iii) accessing new markets and (iv) creating the future market for sustainable transport solutions. 3.6 Management of Resources Efficiently Utilization of all resources efficiently is strategically and economically important. The group has all the time endeavored for its products and operations for having smallest harmful impact on environment. It also utilizes its existing resources in effective and efficient way with a view to make the organization competitive. The Group reduces its costs by improving resources management like water, energy and material. 3.8.3 Issues of Shortage of Raw Material and Natural Resources 3.8.3 Issues of Shortage of Raw Material and Natural Resources Around the world middle class is growing rapidly side by side the purchasing power of middle class is also increasing as a result the demand of land and other resources are also increasing. So, efficient utilization of resources is highly required to entertain the needs and expectations of people. 3.8.2 Issues of Oil Resources, Climate Change and Alternative Fuels At present climate change is considered as one of the most difficult and complex issues. For climate change, fossil fuel is highly responsible because it emits greenhouse-gas. But, oil has been considered as a reliable source of energy for long period of time, but today, the use of oil is a very controversial issue. Furthermore, oil is not only an issue for foiling environment but also for its availability. Because the reserves of oil is declining rapidly and also there is an increasing instability in the oil-producing countries. Now is not a question whether a fossil-free society would be ensured on not rather it is a question that how a fossil-free society will be ensured. Around the world many corporation are trying to develop the sources of renewable energy sources. Nevertheless, in many regions the development of renewable fuels varies largely depending on the availability of natural resources that entails a great challenge in the form of producing vehicles adapted for different types of fuel. The move towards the large-scale use of renewable fuel is also dependent on political decisions to create the necessary infrastructure. 3.8 Challenges Management It is very critical to remain competitive in a volatile business world. Nowadays, it is very essential for a company to comprehend its operating environment than ever before because any environmental change may have impact on future transport needs. So, as part of CSR, the group first identifies and defines some of the more significant challenges and the actions being undertaken by the Volvo Group to combat these challenges. The major challenges and the approaches to cope them are described bellow; 3.8.1 Issues of Urbanization, Population Growth and Megacities The population of the world is increasing constantly. It is expected that by 2050, the total number of population will exceed nine billion. At present, more than half of the population of the world resides in towns and cities and within fifty years, it is expected that the total number of population who will live in the cities and towns would be two-thirds of world population. The urbanization process is taking place very rapidly in Asia and Africa. Moreover, the number of megaregions and megacities total number of with populations surpassing ten million is rapidly increasing. It is expected that by 2015, near about 20% of the population of the world will live in cities having populations more than two million inhabitants. This increasing tendency of population is leading to an enhanced transportation need. People and big amounts of goods are to be transported daily. Cities, especially most of the cities will have particular requirements for traffic and town planning. Moreover, noise pollution and congestion issues are to be addressed for ensuring the urban environment hygienic and healthy. 3.7 Management of Environment Since 1980 the Group has been practicing internal environmental audits system for ensuring environmental performance. Since 1990 environmental data have been collected once a year from different production sites. At the end of 2011, it has been found that the Group has 65 majority-owned production plants in 20 countries, each of which was included in the data reporting. The reporting of data is in the line with global environmental standard for manufacturing plants that was introduced by Group management in 2000. Regardless of location and size, all of its production units have to conform with its minimum requirements for environmental performance and focuses on a number of important areas including energy consumption, use of chemicals, emissions to air and water, water consumption, waste, and noise. As a result, carbon dioxide emission reduced from 279,000 tons in 2010 to 255,000 tons in 2011 and nitrogen oxides decreased from 719 tons to 474 tons. Furthermore, as part of environment management, the group offers training to driver with a view to reduce fuel consumption and improve safety. It is observed that on average, drivers can save 5 to 10% in fuel consumption after participating in a course on fuel-efficient driving. Also, Volvo Group offers fuel watch that not only helps customers to use products in the most efficient way but also reduces impact on environment. It also offers various services like care track, Commute Greener to reduce the environment pollution. 263 Vol. 10, No. 11; 2014 www.ccsenet.org/ass Asian Social Science The approach of Volvo Group to adjust with the future change like urbanization The Group has been working to improve solutions of transport for the cities of the world for to enabling people and goods to be transported without enhancing emissions and congestion. Since the transport system needs to be further improved like Bus Rapid Transit (BRT) with a view to alleviate congestion, the Group is concerned in introducing telemetric solutions for more flexible transports for public and efficient distribution of goods, which will ultimately ensure more efficient planning for better traffic security, transport routes and superior planning of maintenance work. 3.8.2 Issues of Oil Resources, Climate Change and Alternative Fuels 3.8.2 Issues of Oil Resources, Climate Change and Alternative Fuels The approach of Volvo Group to adjust with the challenge like alternative fuels To combat these challenges the group is improving the efficiency in energy, logistic systems and neutrality in carbon dioxide. To reduce the emission of carbon dioxide as well as to enhance efficiency of energy the electromobility can be one of the solutions. By moving to wheel efficient renewable fuels it is possible reduce the emissions of carbon dioxide, but for shifting to the use of large-scale renewable fuels the policy decisions of governments is required because only the favorable policy as well as intention of government can help in establishing required infrastructure. For developing more efficient transport systems the group is involving in various collaborations. The Volvo Group’s approach to combat the challenge of resource shortage The Group is working to lessen the use of material as well as other resources while producing its products and thus it takes the perspective of life-cycle into consideration in the phases of product development. At the present time about thirty percent (30%) of a truck is manufactured from recycled material. New materials based on 264 Vol. 10, No. 11; 2014 www.ccsenet.org/ass Asian Social Science nanostructured compounds or alloys could create light-weight materials without losing the mechanical strength and thus create opportunities to reduce the weight of vehicles. nanostructured compounds or alloys could create light-weight materials without losing the mechanical strength and thus create opportunities to reduce the weight of vehicles. The approach of Volvo Group The Group is working to stimulate and promote the interest in the field of science and technology by reaching out to young people and supporting educations in the field of natural sciences, engineering and mathematics. 3.9 Management of CSR Organization 3.9 Management of CSR Organization 3.9 Management of CSR Organization A new CSR organization was developed in 2011 with a view to ensure faster decision making, effective governance and clearer accountability and responsibility. The new CSR organization came into effect on January 2012. On a regular basis the Board of Directors evaluates its CSR performance and considers some very relevant issues like environmental, human rights and corruption in its annual risk analysis. In the Group Executive Team, Executive Vice President (Jan-Eric Sundgre) is mainly liable for issues related to CSR. The Core Value and Public Affairs Council handle CSR issues, which is chaired by Jan-Eric Sundgren (Executive Vice President Environmental and Public Affairs) and member of the Group’s Executive Management Team. Throughout the organization, the chairman in the council is responsible for making decisions and following up the implementation of decisions. The objective of this governing body is to establish strategies, provide directions and to follow up on performance. This council also provides supports and advances the business of Volvo Group in the areas relating to its CSR, core values, public affairs and relevant emerging issues. 3.10 Management of CSR in Supply Chain 3.10 Management of CSR in Supply Chain Supply chain plays important role in ensuring the competiveness of an organization. Considering the importance, the activities of suppliers are monitored through the quality assurance and self-assessment forms, which are part of its requirements on suppliers. There is a Corporate Audit Department in the group for monitoring the compliances as per the Code of Conduct through audits and assessments. The Volvo Group has a management control system for self-evaluation of how well critical policies such as the Code of Conduct have been implemented and enforced. An annual survey is conducted to measure the level of implementation. Most of the managers in the Volvo Group state that they have taken appropriate measures within their organization to promote values and appropriate business behavior, and have held discussions with their teams on the risk of fraud and infringements of the Code of Conduct. The Volvo Group has several group-wide processes and management systems covering areas such as quality, internal control, environmental care and occupational health and safety. There are specific key performance indexes (KPIs) to monitor and follow up on the effectiveness of processes which are reported on and followed up regularly in the Core Value and Public Affairs Council. Health and safety controls and environmental care are integrated into the Production System of Volvo Group. Production system is a toolbox containing methods to increase internal efficiency and minimize losses. Development of competence of employees to meet future technical skills requirement is also an important part of Production system. Performance is measured by collecting financial, environmental and human resource data. A third party verifies data collection process. 3.11 Management of Corruption 3.8.4 Security and Safety Issues Everywhere in the world traffic safety is becoming as one of the very important issue because transportation increases. There is an increase trend among the customers regarding awareness which leads to an increasing trend of demand for safe products. The issue is of high priority for institutions and governments worldwide. In future, the focus on safety and security would be increased highly because of crime, military conflicts, natural disasters and terrorism which will affect the safety of vehicles, drivers, and goods. Volvo Group to address the issues of security and safety The approach of Volvo Group to address the issues of security and safety Presently the Group is working jointly with academia and authorities to discover novel security features and develop awareness regarding security issues both on global and local level. 3.8.5 Issue of Skilled Employees For several years, interest has waned for careers and education in the fields of engineering, natural sciences and mathematics in industrialized countries. But, the need for capable and skilled employees with these special areas will increase as services and products become sophisticated increasingly. 3.11 Management of Corruption Also, training on the Competition Law Compliance Program has been started from 2012. All the works with anti-corruption are guided by the principles of Code of Conduct, which applies to all employees and the Board of Directors. Regarding legal compliance the Volvo Group abides by the laws and regulations of that country in which it operates. In situations where the law does not give guidance, the Group applies its own corporate values and standards. In cases of conflict between mandatory law and principles contained in this code, the law prevails. The Volvo Group has an Anti-Corruption Compliance Policy that has been adopted by the Audit Committee. In addition to the policy the Group has steering documents and a hand-book that in combination with the policy constitutes its Anti-Corruption Compliance Program. The Anti- Corruption Compliance Program consists of a number of actions aimed at preventing the Volvo Group or any of its business partners from participating in corrupt activities. The program fundamentally consists of three parts such as (i) preventing corruption (ii) detecting corruption and (iii) responding to corruption. The Audit Committee, a function of the Volvo Group Board of Directors, has issued the Anti-Corruption Compliance Policy and monitors compliance. The Chief Compliance Officer is responsible for overseeing the implementation of the program, leading and participating in training and audits and leading investigations into alleged non- compliance. The Chief Compliance Officer reports to the Audit Committee on current incidents and investigations three times a year. In addition, annual reports are submitted to the Audit Committee on activities in the Anti-Corruption Program. Regular reports are also made to Group management. Moreover, whistleblowing is highly encouraged for ensuring corporate transparency because it is an important tool in fighting corruption (Mansbach, 2007). Whistleblowing is the public disclosure by a person working within an organization of acts, omissions, practices or policies by that organization that wrong or harm a third party. The aim of the disclosure is to draw the attention of the authorities or of the public to bring the unlawful activity to an end and to check further such misconduct (James, 1980; Hunt, 1998).. As per the existing Whistleblower procedure all employees within the Volvo Group are encouraged and expected to report suspected infringements of the internal policies and law to their immediate boss. 3.12 Ensuring Fair Competition Competition Law Compliance Program was introduced by the group in 2011 in order to provide its employees an l understanding of what is accepted and what is not and thereby ultimately encouraging fair and healthy competition in the markets where the Group is present. The program includes a number of actions aiming at preventing the Volvo Group from participating in activities that are contrary to competition law. The new policy explains in details regarding the behavior in relation to customers, distributors, competitors and suppliers, both as an employer and as an employee. For developing consciousness in employees regarding the policy of the company, 10,000 white-collar employees from different department like research and development, sales and marketing and product development have participated in an e-learning course on the subject. For all employees the new policy is applicable. 3.11 Management of Corruption When employees do not sense easy reporting the matter to his or her boss or a reported incident is not taken seriously the employee can report the matter directly to the Head of Corporate Audit in accordance with the Whistleblower procedure. Reports in accordance with the procedure can be submitted anonymously, to the extent permitted by law. In accordance with its Code of Conduct, the Group does not tolerate retaliation against a person for making good-faith complaints of improper behavior. In total, 30 cases were reported in 2011 under the Whistleblower procedure. 16 of these were dismissed following investigation, as there were no grounds for the allegation. In 12 cases were found with grounds for the allegations and preceded with appropriate actions. Two of these cases are still under investigation. Two cases were dealt with as potential corruption cases in 2011. These were investigated and reported to the Audit Committee. One case is still under investigation (Annual CSR report, 2011). 3.11 Management of Corruption Corruption reduction and promotion of healthy competition in the world are the aim of the group. Therefore, it is the responsibility of group to circulate its views on how to run a business in a transparent, fair and correct way. It 265 Asian Social Science Vol. 10, No. 11; 2014 www.ccsenet.org/ass is essential that its employees in all parts of the world understand the purpose of the policies and how to interpret them in everyday business. For these reasons all the white-collar employees participate in e-learning relating to the Code of Conduct and anti-corruption every three years. On-site training for selected groups is conducted every year. Messages are conveyed to the employees regarding the advantages of doing business in the right way as well as the potential consequences of non-compliance. Personal meetings and discussions are importantly conducted for creating awareness. Thousands of employees have received on-site training through the Chief Compliance Officer or other members of the compliance network. In 2011, 9,023 employees received training on the Code of Conduct and 10,107 employees were trained in anti-corruption. Also, training on the Competition Law Compliance Program has been started from 2012. All the works with anti-corruption are guided by the principles of Code of Conduct, which applies to all employees and the Board of Directors. Regarding legal compliance the Volvo Group abides by the laws and regulations of that country in which it operates. In situations where the law does not give guidance, the Group applies its own corporate values and standards. In cases of conflict between mandatory law and principles contained in this code, the law prevails. is essential that its employees in all parts of the world understand the purpose of the policies and how to interpret them in everyday business. For these reasons all the white-collar employees participate in e-learning relating to the Code of Conduct and anti-corruption every three years. On-site training for selected groups is conducted every year. Messages are conveyed to the employees regarding the advantages of doing business in the right way as well as the potential consequences of non-compliance. Personal meetings and discussions are importantly conducted for creating awareness. Thousands of employees have received on-site training through the Chief Compliance Officer or other members of the compliance network. In 2011, 9,023 employees received training on the Code of Conduct and 10,107 employees were trained in anti-corruption. 4. Conclusion It is the increasing tendency of organizations to devise prosperous and innovative ways to develop their business while simultaneously serving people in need and sustaining the fragile planet (Heslin & Ochoa, 2008). After analyzing the CSR activities of Volvo Group it is found that the CSR activities are not only confined within philanthropy rather CSR activities are performed delicately with a view to satisfy the expectations of both internal as well as external stakeholders. Consequently, the competitiveness is growing rapidly which ultimately leads the Group to the achievement of its vision. Thus, the study argues that successful CSR initiatives are derived from cautious study of each and every single organization’s unique and exclusive competencies, culture and strategic opportunities. Unfortunately, most of the organizational leaders of the world do not pay their careful and thoughtful concentration while performing CSR activities. But exercising CSR activities in a strategic way the Volvo Group yields favorable relationships with investors, increased organizational learning, growth in market share, retention of deeply engaged employees, and support from external stakeholders. Nevertheless, imitating best practices of other organizations’ strategic CSR would be unwise because benchmarking often leads to unsatisfactory results (Heslin & Ochoa, 2008). Leaders of Organization tend to improperly appreciate the delicate variations in organizational contexts and competencies whereby a given practice facilitates one organization to fly, but leads others to flounder. It is expected that this study will inspire and guide many corporate leaders of the world to devise and implement the CSR strategies in such a way so that the organization can satisfy its both internal as well as external stakeholders and thereby can survive for long period of time. 3.13 Investments in Research and Development A firm can survive in the modern competitive market place only when it can offer goods and services as per the demands of customers, including environmentally-enhanced products. And without having skilled and competent 266 Vol. 10, No. 11; 2014 Asian Social Science www.ccsenet.org/ass workforce it is not possible to develop new goods and services. Considering these facts, its investments in R&D have remained high over the last economic cycles. For example, the group invested SEK 13 billion and SEK 13.3 billion respectively in the year 2010 and 2011 for reducing the environmental impact and to develop sustainable transport solutions. Thus, the Group is continuously working for improving product performance. workforce it is not possible to develop new goods and services. Considering these facts, its investments in R&D have remained high over the last economic cycles. For example, the group invested SEK 13 billion and SEK 13.3 billion respectively in the year 2010 and 2011 for reducing the environmental impact and to develop sustainable transport solutions. Thus, the Group is continuously working for improving product performance. References Ashley, P. A. (2002). Ética e responsabilidade social nos negócios. São Paulo: Saraiva. Barney, J. (1991). Firm resources and sustained competitive advantage. Journal of Management, http://dx.doi.org/10.1177/014920639101700108 Bowen, H. R. (1953). 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https://openalex.org/W1419442049	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0133619&type=printable	English	null	Autotaxin and Endotoxin-Induced Acute Lung Injury	PloS one	2,015	cc-by	8,720	Introduction Data Availability Statement: All relevant data are within the paper. Autotaxin and Endotoxin-Induced Acute Lung Injury Marios-Angelos Mouratis1☯, Christiana Magkrioti1☯, Nikos Oikonomou1, Aggeliki Katsifa Glenn D. Prestwich2, Eleanna Kaffe1, Vassilis Aidinis1* Marios-Angelos Mouratis1☯, Christiana Magkrioti1☯, Nikos Oikonomou1, Aggeliki Katsifa1, Glenn D. Prestwich2, Eleanna Kaffe1, Vassilis Aidinis1* 1 Division of Immunology, Biomedical Sciences Research Center “Alexander Fleming”, Athens, Greece, 2 Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah, United States of America ☯These authors contributed equally to this work. * v.aidinis@fleming.gr ☯These authors contributed equally to this work. * v.aidinis@fleming.gr OPEN ACCESS OPEN ACCESS Citation: Mouratis M-A, Magkrioti C, Oikonomou N, Katsifa A, Prestwich GD, Kaffe E, et al. (2015) Autotaxin and Endotoxin-Induced Acute Lung Injury. PLoS ONE 10(7): e0133619. doi:10.1371/journal. pone.0133619 Editor: Xiao Su, Chinese Academy of Sciences, CHINA Received: February 27, 2015 Accepted: June 29, 2015 Published: July 21, 2015 Copyright: © 2015 Mouratis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Mouratis M-A, Magkrioti C, Oikonomou N, Katsifa A, Prestwich GD, Kaffe E, et al. (2015) Autotaxin and Endotoxin-Induced Acute Lung Injury. PLoS ONE 10(7): e0133619. doi:10.1371/journal. pone.0133619 Citation: Mouratis M-A, Magkrioti C, Oikonomou N, Katsifa A, Prestwich GD, Kaffe E, et al. (2015) Autotaxin and Endotoxin-Induced Acute Lung Injury. PLoS ONE 10(7): e0133619. doi:10.1371/journal. pone.0133619 Copyright: © 2015 Mouratis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE Abstract Acute Lung Injury (ALI) is a life-threatening, diffuse heterogeneous lung injury characterized by acute onset, pulmonary edema and respiratory failure. Lipopolysaccharide (LPS) is a com- mon cause of both direct and indirect lung injury and when administered to a mouse induces a lung phenotype exhibiting some of the clinical characteristics of human ALI. Here, we report that LPS inhalation in mice results in increased bronchoalveolar lavage fluid (BALF) levels of Autotaxin (ATX, Enpp2), a lysophospholipase D largely responsible for the conversion of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA) in biological fluids and chroni- cally inflamed sites. In agreement, gradual increases were also detected in BALF LPA levels, following inflammation and pulmonary edema. However, genetic or pharmacologic targeting of ATX had minor effects in ALI severity, suggesting no major involvement of the ATX/LPA axis in acute inflammation. Moreover, systemic, chronic exposure to increased ATX/LPA lev- els was shown to predispose to and/or to promote acute inflammation and ALI unlike chronic inflammatory pathophysiological situations, further suggesting a differential involvement of the ATX/LPA axis in acute versus chronic pulmonary inflammation. Mice All mice were bred at the animal facilities of the Alexander Fleming Biomedical Sciences Research Center, under specific pathogen-free conditions. Mice were housed at 20–22°C, 55±5% humidity, and a 12-h light-dark cycle; water and food were given ad libitum. Mice were bred and maintained in their respective genetic backgrounds for more than 10 generations. All experimentation in mice for this project was approved by the Institutional Animal Ethical Committee (IAEC) of Biomedical Sciences Research Center “Alexander Fleming” (#373/375), as well as the Veterinary service and Fishery Department of the local governmental prefecture (#5508). The generation and genotyping instructions of Enpp2n/n conditional knockout mice [24], CC10-Cre [18] and LysM-Cre [25] have been described previously. ATX and Acute Lung Injury National resources. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. National resources. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Autotaxin (ATX, Enpp2) is a secreted glycoprotein, widely present in biological fluids, including broncheoalveolar lavage fluid (BALF) [5, 6]. ATX is a member of the ectonucleotide pyrophosphatase-phosphodiesterase family of ectoenzymes (E-NPP) that hydrolyze phospho- diesterase bonds of various nucleotides and derivatives [7]. However and unlike other E-NPP family members, the prevailing catalytic activity of ATX is the conversion of lysophosphatidyl- choline (LPC) to lysophosphatidic acid (LPA)[8]. LPA is a phospholipid mediator [9, 10] that evokes growth-factor-like responses in almost all cell types, including cell growth, survival, differentiation and motility [11–13]. The large variety of LPA effector functions is attributed to at least six, G-protein coupled, LPA receptors (LPARs) with overlapping specificities and wide- spread distribution including the lung [14, 15]. Competing Interests: The authors have declared that no competing interests exist. differentiation and motility [11–13]. The large variety of LPA effector functions is attributed to at least six, G-protein coupled, LPA receptors (LPARs) with overlapping specificities and wide- spread distribution including the lung [14, 15]. A major role for the ATX/LPA axis has been suggested in chronic inflammation and cancer [16], while the numerous LPA effects in pulmonary cell types in vitro have implicated the axis in lung pathophysiology [17]. More importantly, genetic and pharmacologic studies in vivo [18–20] have indicated a decisive contribution of ATX/LPA in the development of pulmonary chronic inflammation and fibrosis [21–23]. Therefore, given the established role of the ATX/ LPA axis in pulmonary chronic inflammation and fibrosis in vivo, as well as the LPA effects in pulmonary cell types in vitro, in this report we evaluated a possible role for the ATX/LPA axis in endotoxin-induced acute lung injury. Data Availability Statement: All relevant data are within the paper. Acute lung injury (ALI), or mild acute respiratory distress syndrome (ARDS) [1], is a diffuse heterogeneous lung injury characterized by arterial hypoxemia, respiratory failure and low lung compliance, as well as non-cardiogenic pulmonary edema and widespread capillary leakage leading to alveolar flooding [2]. Different experimental animal models have been evolved and used to investigate the pathophysiological mechanisms of ALI, mostly based on reproducing known risk factors for the human condition, such as sepsis, acid aspiration and mechanical ventilation [3]. Among them, LPS inhalation in (C57Bl/6) mice is a well-estab- lished experimental model of ALI (LPS/ALI), characterized by acute neutrophil accumulation in lung tissue/BALF and pulmonary edema [4]. Lipopolysaccharide (LPS), a component of gram-negative bacteria cell walls and a potent TLR4 activator, is a common cause in both direct and indirect lung injury (i.e. pneumonia and sepsis respectively)[4]. Funding: This work was supported by the Hellenic Ministry for education and religion/General Secretariat of research and technology Aristia II (INFLALIPID-3311) and Synergasia 2009 (ATX- 09SYN-11-679) grants, through the Operational Programs "Education and Lifelong Learning" and “Competitiveness and Entrepreneurship” (respectively) of the National Strategic Reference Framework (NSRF), co-funded by the European Commission (European Social Fund and European Regional Development Fund respectively) and 1 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0133619 July 21, 2015 ATX and Acute Lung Injury USA). In the same day, 233 zygotes were transferred to 11 surrogate mothers F1 CBAxC57Bl/6 to generate 54 offsprings. The transgene was detected in tail DNA with PCR analysis (primers: forward 5´-ACT GCC CAT TGC CCA AAC AC-3´ and reverse 5’-TCT GAC ACG ACT GGA ACG AG-3’). From the 54 F0 mice 4 were identified as transgenic, which gave rise to the respective lines L13, L15, L16, L39. LPS-induced Acute Lung Injury Model LPS was administered by inhalation, applying a previously described method with minor mod- ifications [26, 27]. Briefly, bacterial LPS from Pseudomonas aeruginosa (serotype 10, Sigma, St. Louis, MO, USA) was dissolved in normal saline at a concentration of 2mg/ml. 5 ml of this solution was fully administered via a custom-made nebulizer at an oxygen flow-rate of 4lt/min for 25 minutes into a chamber containing 5–7 mice. For control mice, normal saline was administered as above. All measures were taken to minimize animal suffering; however and during the protocol no anaesthetics were used as no invasive or painful techniques were per- formed. After the induction of ALI, the condition of the animals was checked every two hours during the light period. No adverse effects were observed that would necessitate the use of anal- gesics and no animals died before the experimental points. Mice were sacrificed 24 hours after the induction of ALI for all experiments apart from the time course experiment where sacrifice was done at several time points between 6 and 48 hours after the induction. Sacrifice was per- formed in a CO2 chamber with gradual filling followed by exsanguination. In the pharmaco- logic study, GWJ-A-23 (dissolved in saline, 2% DMSO) was administered intraperitoneally at a dosage of 10mg/kg before exposure to LPS. The vehicle group was administered 2% DMSO in saline. Total Protein Content Determination Total protein concentration in BALF samples was measured using the Bradford protein assay (Biorad, Hercules, CA, USA) according to the manufacturer’s instructions. OD readings of samples were converted to μg/ml using values obtained from a standard curve generated with serial dilutions of bovine serum albumin (2000–125 μg/ml). PLOS ONE \| DOI:10.1371/journal.pone.0133619 July 21, 2015 Construction of the TgCC10hATX transgenic mice Vector pcDNA3.1/ZEO a1AT-hATX-BGHpA carrying the cDNA of human ATX preceded by the a1t1 promoter and followed by the Bovine Growth Hormone polyadenylation site (BGHpA) (a generous gift of G. Mills) was digested with HindIII/NaeI to isolate hATX- BGHpA. This was then ligated to pBS-CMV which had been cleaved with HindIII/EcoRV. The resulting vector pBS-CMV-hATX-BGHpA was digested with MfeI/HindIII to remove the CMV promoter and made blunt by filling the 5’ overhangs with T4 DNA polymerase. CC10 promoter was excised with a HindIII digestion from CC10-Cre-hGH and was also made blunt. Ligation followed, thus, forming the pBS-CC10-hATX-BGHpA construct. The resulting con- struct was verified with an MfeI/BssHII digestion, amplified in bacterial cultures and purified by 2xCsCl. BssHII (PauI) digestion was employed to separate the vector backbone from the transgene encoding fragment (transgenic device). The latter was then isolated by b-agarase extraction. For the production of transgenic mice from the transgenic facility of BSRC Fleming, fertil- ized CBAxC57Bl/6 hybrid (F2) zygotes were injected with the transgenic device at a concentra- tion of 5,38 ng/μl, diluted in Embryo max solution (mr-095-f, Chemicon International, CA, 2 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0133619 July 21, 2015 Immunohistochemistry Immunostaining was performed with peroxidase labelling techniques. Tissue sections were deparaffinized and endogenous peroxidase activity was blocked by incubation in 1% peroxide. The sections were preincubated with 2% Normal Goat Serum (NGS) in PBS-Tween (PBST) for 30 minutes, followed by incubation overnight at 4°C with the primary antibody against ATX (Cayman Chemical Company, Michigan, USA). Sections were then washed in PBST and incu- bated for 30 minutes with horseradish peroxidase (HRP)–conjugated anti-rabbit IgG (1:1000 dilution in PBS-T). The sections were further washed with PBST. Finally, colour was developed by immersing the sections in a solution of 0.05% 3, 3’-diaminobenzidine (DAB; Sigma) and 0.01% hydrogen peroxide in PBS. The sections were counterstained with hematoxylin. The specificity of a-ATX antibodies has been analysed in detail previously [28]. ATX activity assay ATX activity was measured using the TOOS activity assay. ATX cleaves the LPC substrate to LPA and choline. The liberated choline is oxidised by choline oxidase to betaine and hydrogen peroxide. The latter, in the presence of HRP (horseradish peroxidase), reacts with TOOS (N- ethyl-N-(2-hydroxy-3-slfopropyl)-3-methylaniline) and 4-AAP (aminoantipyrene) to form a pink quinoeimine dye with a maximum absorbance at 555nm. Briefly, 1.25x LysoPLD buffer (0.12 M Tris-HCl pH = 9, 1.25 M NaCl, 6.25 mM CaCl2, 6.25 mM MgCl2, 6.25 μM CoCl2, 1.25 mM LPC) was incubated at 37°C for 30 minutes before adding 80μl/well in 20 μl BALF in a 96-well plate. The mix was incubated at 37°C for 4 hours. At the end of the incubation, a col- our mix (5 mM MgCl2, 50 mM Tris-HCl pH = 8, 8 U/ml HRP, 0.5 mM 4-AAP, 0.3 mM TOOS, 2 U/ml choline oxidase) was prepared and 100 μl added to each well. Readings were taken every 5 minutes for 20 minutes. For each sample, the absorbance (A) was plotted against time and dA/min was calculated for the linear part of the plot. ATX activity was calculated accord- ing to the equation: Activity(u/ml) = [dA/dT(sample)—dA/dT(blank)] Vt/(32.8Vs1/2), where Vt = total volume of reaction (mL), Vs = volume of sample (mL), 32,8 = the milimolar extinction coefficient of quinoneimine dye (cm2/μmol) and 1/2 = the mols of quinoneimine dye produced by 1 mol of H2O2. 3 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0133619 July 21, 2015 ATX and Acute Lung Injury HPLC-MS/MS measurements LPA (C14:0, C16:0, C18:0, C18:1 and C20:4) and LPC species (C14:0, C16:0, C18:0, C18:1, C20:4, C22:6 and C24:0) were measured in plasma by means of HPLC-ESI/MS/MS using an RSLCnano system (Ultimate 3000 Series, Dionex Corporation, USA) coupled with an LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Waltham, MA, USA). Lipid extraction from BALF was performed as previously described with minor modifications [29]. Briefly, BALF samples (300 μL) were mixed with 700 μL PBS prior to extraction and spiked with the internal standard mix (17:0 LPA/LPC). Neutral extraction was performed twice with 2 mL ice- cold CHCl3/CH3OH (2/1, v/v) followed by 1 mL PBS saturated ice-cold CHCl3/CH3OH (2/1, v/v). Each extraction step was followed by a 60 sec vortex and a 1 min centrifugation step at 4°C at 3,000 rpm. The lower organic phases from both extraction steps were pooled and kept for LPC measurements. The remaining aqueous phase was chilled in ice for 10 min, acidified with HCl 6N το pH 3.0 and undergone further 2-step extraction with ice-cold CHCl3/CH3OH (2/1, v/v) as above. The lower organic phases were pooled and kept for LPA measurements. The neutrally extracted organic phase and the neutralized acidified lower organic phase were evaporated to dryness. Finally, the dry residues were resuspended in 0.15 mL of isopropanol for HPLC-ESI/MS/MS analysis. Recovery of LPA and LPC species ranged between 55–85% and 80–100%, respectively. The HPLC-MS/MS was performed as previously described [29]. RNA Extraction and Real-time RT-PCR Analysis RNA was extracted from the left lung lobe using the peqGOLD TriFast Reagent and treated with DNAse (RQ1 RNAse-free DNAse, Promega, Wis, USA) prior to RT-PCR according to manufacturer’s instructions. Reverse transcription was performed for cDNA synthesis using the peqGOLD MMLV H plus reverse transcriptase. All reagents were purchased from PEQLAB Biotechnologie GMBH, Germany. Real-time PCR was performed on a BioRad CFX96 Tou- chReal-Time PCR Detection System (Bio-Rad Laboratories Ltd, CA, USA). Values were nor- malized to the expression of b-2 microglobulin (B2m). Increased BALF ATX/LPA levels upon LPS-induced ALI To examine a possible involvement of ATX in the pathogenetic mechanisms underlying ALI, we first monitored its levels upon the time course of the modelled, LPS-induced, disease devel- opment. Noteworthy, and since no animal model fully represent all the clinical characteristics of human ALI, a recent American Thoracic Society workshop suggested that the main features of experimental ALI should include at least three out of the following four features: histological evidence of tissue injury (such as the accumulation of neutrophils in the alveolar or the intersti- tial space), alteration of the alveolar capillary barrier (such as the increase in total protein con- centration of the bronchoalveolar lavage fluid; BALF), an inflammatory response (such as an increase in the absolute number of neutrophils in the BALF) and evidence of physiological dys- function [32]. Accordingly, aerosolized LPS (Pseudomonas aeruginosa) was administered by inhalation to groups of littermate mice, which were then sacrificed 6, 12, 24 and 48 hours post- administration (Fig 1). Histological analysis of isolated lungs indicated that LPS inhalation resulted in alveolar wall thickening and leukocyte infiltration into the lung interstitium and alveolar space (Fig 1A), as previously reported [26, 27]. Inflammatory cells (93% neutrophils; [26]) were evident in BALFs already at the 6hr time-point and continued to increase at 48h (Fig 1A and 1B). Pulmonary microvascular leakage and edema induced by LPS was reflected in the gradual increase of BALF total protein content (Fig 1C). Interestingly, ATX activity in BALFs, as quantified with the TOOS assay on natural LPC substrates, showed a gradual increase as time progressed (Fig 1D), following total protein levels (Fig 1C) most likely reflecting a relaxation of the endothelial barrier and thus suggesting increased recruitment from the circulation. Similar findings have been reported in earlier stud- ies, upon intratracheal administration of LPS (5mg/Kg) in Sv/129 mice [33]. ATX immunohis- tochemistry (IHC) in lung tissue sections showed high constitutive expression from the bronchial epithelium, as well as a weak diffuse staining pattern in the lung parenchyma upon LPS/ALI (Fig 1E). As the main known function of ATX is the hydrolysis of LPC to LPA, the corresponding BAL fluids were analyzed with HPLC-MS/MS to identify perturbations in lysophospholipid levels upon LPS-induced ALI. LPC, the substrate of ATX and precursor of LPA, peaked at 24 hours (Fig 1F), as previously reported for the lung surfactant of guinea pigs upon LPS-induced ALI [34]. Statistical analysis Statistical significance was assessed in pair-wise comparisons with control values using a paired Student’s t-test, or a Mann-Whitney test in cases of not normal distributions, using SigmaPlot 11.0 (Systat software Inc., IL, USA), and presented as means (± S.E). In all figures, and denote p-values <0.05 and <0.001 respectively. All experiments presented are repre- sentative of two repetitions; cumulative normalized-to-control values produce identical results and conclusions. 4 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0133619 July 21, 2015 ATX and Acute Lung Injury Results and Discussion ATX was previously suggested as a candidate gene involved in the control of pulmonary func- tions, development and remodelling [30], while the lung has been suggested to be among the tissues expressing moderately high ATX mRNA levels in healthy conditions [17]. In patho- physiological conditions, increased ATX/LPA levels have been detected in fibrotic lungs, both in human patients and animal models [18, 20]. Accordingly, genetic deletion of ATX, LPAR1 or LPAR2 [18, 20, 31], as well as pharmacologic inhibition of ATX or LPAR1[18, 19], attenu- ated the development of the bleomycin (BLM)-induced modelled disease. Therefore, a major role of ATX and LPA in pulmonary chronic inflammation and fibrosis was established, attrib- uted to LPA-induced vascular leak and fibroblast recruitment [21–23]. Moreover, a number of LPA effects in pulmonary cells in vitro are consistent with a pro-inflammatory and pro-fibrotic role of ATX/LPA, although a number of reports also suggest anti-inflammatory effects of LPA [17]. Therefore, and given the role of ATX/LPA in chronic pulmonary inflammation and the LPA effects in pulmonary cell types, we reasoned a possible role of ATX/LPA in acute inflam- mation and lung injury. ATX and Acute Lung Injury Fig 1. Increased BALF ATX/LPA levels upon LPS-induced ALI. Mice were administered aerosolized LPS and were sacrificed 6, 12, 24 and 48 hours later (n = 3–5; a representative experiment out of two is shown). A. Hematoxylin & Eosin staining of lung tissue sections following LPS exposure for the indicated times resulted in alveolar wall thickening and leukocyte infiltration into the lung interstitium and alveolar space. B. Increased BALF cellularity upon LPS/ALI. C. Pulmonary microvascular leakage and edema induced by LPS was reflected in BALF protein content. D. Increased ATX activity in LPS/ALI BALFs, as measured with the TOOS assay. E. IHC for ATX in lung tissue showing constitutive expression from the bronchial epithelium, as well as a weak diffuse staining pattern in the lung parenchyma. F-G. BALF total LPC/LPA levels respectively upon LPS/ALI, as measured with HPLC-MS/MS. doi:10 1371/journal pone 0133619 g001 BALF ATX/LPA levels upon LPS-induced ALI. Mice we Fig 1. Increased BALF ATX/LPA levels upon LPS-induced ALI. Mice were administered aerosolized LPS and were sacrificed 6, 12, 24 and 48 hours later (n = 3–5; a representative experiment out of two is shown). A. Hematoxylin & Eosin staining of lung tissue sections following LPS exposure for the indicated times resulted in alveolar wall thickening and leukocyte infiltration into the lung interstitium and alveolar space. B. Increased BALF cellularity upon LPS/ALI. C. Pulmonary microvascular leakage and edema induced by LPS was reflected in BALF protein content. D. Increased ATX activity in LPS/ALI BALFs, as measured with the TOOS assay. E. IHC for ATX in lung tissue showing constitutive expression from the bronchial epithelium, as well as a weak diffuse staining pattern in the lung parenchyma. F-G. BALF total LPC/LPA levels respectively upon LPS/ALI, as measured with HPLC-MS/MS. doi:10.1371/journal.pone.0133619.g001 Increased BALF ATX/LPA levels upon LPS-induced ALI Total BALF LPA levels were also found increased (Fig 1G), as previously reported [35], correlating with and confirming BALF ATX activity levels. 5 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0133619 July 21, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0133619 July 21, 2015 Bronchial epithelium-specific ATX expression has a minor contribution to ALI pathogenesis ATX expression is localized mainly in the bronchial epithelium, both in healthy and inflamma- tory conditions (Fig 1E)[17, 18, 36], suggesting this cell type as the main pulmonary source of ATX. To confirm both its cell-specific expression, as well as to examine its possible contribu- tion to ALI pathogenesis, ATX was conditionally deleted from the bronchial epithelium by crossing the conditional knock out mouse for ATX (Enpp2n/n)[24] with the TgCC10-Cre transgenic mouse strain that expresses the Cre recombinase under the control of the mouse CC10Kd (Scgb1a1) promoter [18]. As previously reported, CC10-Cre drives conditional ATX recombination in CC10Enpp2-/- mice exclusively in bronchial epithelial cells (with an efficiency of 70–80%), while the transgenic Cre driver mouse strain itself exhibits no apparent pulmonary phenotype even under inflammatory conditions [18]. 6 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0133619 July 21, 2015 ATX and Acute Lung Injury LPS d i i d CC10E 2 / i ll ild li d di Fig 2. Genetic deletion of ATX from the bronchial epithelium has minor effects in ALI development. Aerosolized LPS was administered in mice where ATX was genetically deleted specifically from bronchial epithelial cells (CC10Enpp2-/-), as well as wild type littermates. Mice were sacrificed 24 hours later (n = 3–8; a representative experiment out of two is shown). A-C. Histological analysis (A) and BALF measurements (B & C) indicated no significant changes in the lungs of mice lacking ATX expression in the bronchial epithelium in comparison to their wild type littermates. D. Conditional deletion of ATX from the cells of the bronchial epithelium led to decreased enzyme activity of ATX in the BALF. doi:10.1371/journal.pone.0133619.g002 Fig 2. Genetic deletion of ATX from the bronchial epithelium has minor effects in ALI development. Aerosolized LPS was administered in mice where ATX was genetically deleted specifically from bronchial epithelial cells (CC10Enpp2-/-), as well as wild type littermates. Mice were sacrificed 24 hours later (n = 3–8; a representative experiment out of two is shown). A-C. Histological analysis (A) and BALF measurements (B & C) indicated no significant changes in the lungs of mice lacking ATX expression in the bronchial epithelium in comparison to their wild type littermates. D. Conditional deletion of ATX from the cells of the bronchial epithelium led to decreased enzyme activity of ATX in the BALF Fig 2. Genetic deletion of ATX from the bronchial epithelium has minor effects in ALI development. Fig 2. ATX and Acute Lung Injury Fig 3. Generation of TgCC10Enpp2 mice. A. Schematic representation of the construct used for the generation of the transgenic mice. B. Genotyping PCR of the 4 offsprings that carried the transgene, out of the 54 that were generated after the injections of the trangene-microinjected zygotes in surrogate mothers. C. All four transgenic lines contained equal copy numbers, as identified with Real-Time PCR. D. Real-Time RT-PCR confirmed the expression of the transgene (L39 is shown). E. Total ATX activity levels in the BALFs of TgCC10Enpp2 mice (L39) were found moderately upregulated with the TOOS assay. F. In the same mice, BALF LPA was also found elevated, as measured with HPLC-MS/MS. (C-F n = 3–8). doi:10 1371/journal pone 0133619 g003 Fig 3. Generation of TgCC10Enpp2 mice. A. Schematic representation of the construct used for the generation of the transgenic mice. B. Genotyping PCR of the 4 offsprings that carried the transgene, out of the 54 that were generated after the injections of the trangene-microinjected zygotes in surrogate mothers. C. All four transgenic lines contained equal copy numbers, as identified with Real-Time PCR. D. Real-Time RT-PCR confirmed the expression of the transgene (L39 is shown). E. Total ATX activity levels in the BALFs of TgCC10Enpp2 mice (L39) were found moderately upregulated with the TOOS assay. F. In the same mice, BALF LPA was also found elevated, as measured with HPLC-MS/MS. (C-F n = 3–8). doi:10.1371/journal.pone.0133619.g003 In order to further examine a role of bronchial epithelial cell-derived ATX expression, a new transgenic mouse strain was constructed, expressing human ATX driven by the CC10 pro- moter (TgCC10hEnpp2; Fig 3A) which directs expression exclusively in bronchial epithelial cells [18]. All 4 transgenic lines obtained (L13, L15, L16, L39; Fig 3B), contained 4–5 transgene copies (Fig 3C), and expressed the transgenic (hATX) mRNA in the lung tissue (Fig 3D; L39 is shown). Total ATX activity in BALFs of the transgenic mouse line was found moderately upre- gulated (Fig 3E), resulting in similar increases in BALF LPA levels (Fig 3F). To examine whether bronchial epithelial cell-derived hATX has any effect in LPS-induced ALI, LPS was administered in homozygous TgCC10hEnpp2+/+ mice (L16), which are healthy and fertile and exhibit no overt lung phenotype at 8–10 weeks after birth (Fig 4A). The moder- ate overexpression of ATX from bronchial epithelial cells had minor effects in exacerbating dis- ease symptoms such as tissue damage (Fig 4A), neutrophilic inflammation (Fig 4B) and pulmonary edema (Fig 4C), although an opposite trend in disease severity could be observed in comparison to mice with bronchial epithelial deletion of ATX (Fig 2). Therefore, bronchial epithelial expression of ATX does not seem to have a major role in LPS-induced, acute inflammation and lung injury, as opposed to its role in BLM-induced chronic pulmonary inflammation and fibrosis [18], suggesting a differential involvement of ATX/LPA in acute vs chronic pulmonary inflammation. Bronchial epithelium-specific ATX expression has a minor contribution to ALI pathogenesis Genetic deletion of ATX from the bronchial epithelium has minor effects in ALI development. Aerosolized LPS was administered in mice where ATX was genetically deleted specifically from bronchial epithelial cells (CC10Enpp2-/-), as well as wild type littermates. Mice were sacrificed 24 hours later (n = 3–8; a representative experiment out of two is shown). A-C. Histological analysis (A) and BALF measurements (B & C) indicated no significant changes in the lungs of mice lacking ATX expression in the bronchial epithelium in comparison to their wild type littermates. D. Conditional deletion of ATX from the cells of the bronchial epithelium led to decreased enzyme activity of ATX in the BALF. doi:10.1371/journal.pone.0133619.g002 doi:10.1371/journal.pone.0133619.g002 LPS was administered to CC10Enpp2-/- mice, as well as to wild type littermates, and disease severity was assessed 24 hours later. Deletion of ATX from bronchial epithelial cells had minor effects in attenuating disease development (a clear and reproducible, but not statistically signif- icant, negative trend), such as tissue damage (Fig 2A), neutrophilic inflammation (Fig 2B) and pulmonary edema (Fig 2C), despite decreased BALF ATX levels (Fig 2D). 7 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0133619 July 21, 2015 Macrophage-specific ATX expression does not contribute to ALI pathogenesis To examine if myeloid cell derived ATX contributes to the pathogenesis of ALI, the conditional knock out mouse for ATX (Enpp2n/n)[24] was crossed with a transgenic mouse strain (LysM-Cre) expressing the Cre recombinase under the control of the mouse Lysozyme M (LysM) promoter, which achieves a recombination efficiency close to 100% in granulocytes and 83–98% in macrophages [25]. No differences were observed in BALF ATX activity between 8 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0133619 July 21, 2015 ATX and Acute Lung Injury Fig 4. Genetic overexpression of ATX from the bronchial epithelium has minor effects in ALI development. Transgenic (TgCC10hEnpp2+/+) mice overexpressing hATX in the bronchial epithelium and littermate wild type mice were administered aerosolized LPS to induce ALI and were sacrificed 24 hours later (n = 3–10; a representative experiment out of two is shown). A-C. Histological analysis (A) and BALF measurements (B & C) indicated no significant changes in the lungs of mice overexpressing ATX expression in the bronchial epithelium in comparison to their wild type littermates. D. Increased enzyme activity of ATX in the transgenic mice. doi:10.1371/journal.pone.0133619.g004 Fig 4. Genetic overexpression of ATX from the bronchial epithelium has minor effects in ALI +/+ Fig 4. Genetic overexpression of ATX from the bronchial epithelium has minor effects in ALI development. Transgenic (TgCC10hEnpp2+/+) mice overexpressing hATX in the bronchial epithelium and littermate wild type mice were administered aerosolized LPS to induce ALI and were sacrificed 24 hours later (n = 3–10; a representative experiment out of two is shown). A-C. Histological analysis (A) and BALF measurements (B & C) indicated no significant changes in the lungs of mice overexpressing ATX expression in the bronchial epithelium in comparison to their wild type littermates. D. Increased enzyme activity of ATX in the transgenic mice. doi:10.1371/journal.pone.0133619.g004 g p p development. Transgenic (TgCC10hEnpp2+/+) mice overexpressing hATX in the bronchial epithelium and littermate wild type mice were administered aerosolized LPS to induce ALI and were sacrificed 24 hours later (n = 3–10; a representative experiment out of two is shown). A-C. Histological analysis (A) and BALF measurements (B & C) indicated no significant changes in the lungs of mice overexpressing ATX expression in the bronchial epithelium in comparison to their wild type littermates. D. Increased enzyme activity of ATX in the transgenic mice. Macrophage-specific ATX expression does not contribute to ALI pathogenesis LysMEnpp2-/- mice and their wild type littermates (Fig 5D), indicating no contribution of mac- rophages to BALF ATX levels upon LPS challenge. Accordingly, no differences were observed in all disease indices upon genetic deletion of ATX from macrophages (Fig 5), excluding a role of inflammatory macrophage derived ATX in LPS-induced acute lung inflammation in the lung, especially given the relatively few macrophages (<3%) in BALF infiltrates 24 hours post 9 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0133619 July 21, 2015 ATX and Acute Lung Injury Fig 5. Deletion of ATX from myeloid cells had no effects in ALI development. Aerosolized LPS was administered in mice where ATX was deleted from the myeloid cells (LysMEnpp2-/-) and wild type littermate mice. Mice were sacrificed 24 hours later. A-C. Histological analysis (A) and BALF measurements (B & C) showed no differences in inflammation and edema between mice lacking ATX expression from myeloid cells and their wild type littermates. D. Conditional deletion of ATX from myeloid cells had no effects in BALF ATX activity. doi:10.1371/journal.pone.0133619.g005 Fig 5. Deletion of ATX from myeloid cells had no effects in ALI development. Aerosolized LPS was administered in mice where ATX was deleted from the myeloid cells (LysMEnpp2-/-) and wild type littermate mice. Mice were sacrificed 24 hours later. A-C. Histological analysis (A) and BALF measurements (B & C) showed no differences in inflammation and edema between mice lacking ATX expression from myeloid cells g 5. Deletion of ATX from myeloid cells had no effects i Fig 5. Deletion of ATX from myeloid cells had no effects in ALI development. Aerosolized LPS was / Fig 5. Deletion of ATX from myeloid cells had no effects in ALI development. Aerosolized LPS was administered in mice where ATX was deleted from the myeloid cells (LysMEnpp2-/-) and wild type littermate mice. Mice were sacrificed 24 hours later. A-C. Histological analysis (A) and BALF measurements (B & C) h d diff i i fl ti d d b t i l ki ATX i f l id ll Fig 5. Deletion of ATX from myeloid cells had no effects in ALI development. Aerosolized LPS was administered in mice where ATX was deleted from the myeloid cells (LysMEnpp2-/-) and wild type littermate mice. Mice were sacrificed 24 hours later. A-C. PLOS ONE \| DOI:10.1371/journal.pone.0133619 July 21, 2015 Macrophage-specific ATX expression does not contribute to ALI pathogenesis Histological analysis (A) and BALF measurements (B & C) showed no differences in inflammation and edema between mice lacking ATX expression from myeloid cells and their wild type littermates. D. Conditional deletion of ATX from myeloid cells had no effects in BALF ATX of ATX from myeloid cells had no effects in ALI develo g y p administered in mice where ATX was deleted from the myeloid cells (LysMEnpp2-/-) and wild type littermate mice. Mice were sacrificed 24 hours later. A-C. Histological analysis (A) and BALF measurements (B & C) showed no differences in inflammation and edema between mice lacking ATX expression from myeloid cells and their wild type littermates. D. Conditional deletion of ATX from myeloid cells had no effects in BALF ATX activity. LPS [26, 27]. On the contrary macrophage derived ATX was shown to be important for the development of BLM-induced chronic pulmonary inflammation and fibrosis [18], where mac- rophages are the most abundant (>60%) infiltrating cell type during disease development [37]. 10 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0133619 July 21, 2015 ATX and Acute Lung Injury Increased systemic ATX levels exacerbate acute pulmonary inflammation Genetic deletion of ATX from bronchial epithelial cells, although diminished, did not attenuate pulmonary BALF ATX levels suggesting that a major part of BALF ATX is derived from the circulation, through the possible relaxation of endothelial and epithelial barriers upon LPS- induced ALI [4]. Therefore, and to investigate a possible role of circulating ATX in LPS/ALI, we next examined whether systemic fluctuations of ATX could modulate ALI pathogenesis. LPS was administered to homozygous transgenic mice overexpressing ATX in the liver driven by the human α1- antitrypsin inhibitor (a1t1) promoter (Tga1t1Enpp2; up to 200% of normal plasma ATX/LPA levels) [38], as well as to the heterozygous complete knock out mouse for ATX (Enpp2+/-; 50% of normal plasma ATX/LPA levels)[24]. Chronically elevated serum ATX levels in Tga1t1Enpp2 mice increased LPS-induced tissue damage (Fig 6A), BALF neutrophilic infiltration (Fig 6B) and pulmonary edema (Fig 6C) in comparison to their wild type litter- mates. However, reduced serum ATX levels in Enpp2+/- mice had only minor effects in LPS- induced ALI (Fig 6A, 6E and 6F), suggesting that a 50% reduction of systemic normal levels of ATX is not sufficient to confer resistance to ALI. Therefore, systemic, chronic exposure to increased ATX/LPA levels seems to predispose to and/or promote acute inflammation and ALI. Noteworthy, systemic ATX levels were shown not to play a role in the development of modelled chronic inflammatory diseases, such as pulmonary fibrosis and rheumatoid arthritis where local ATX expression was shown to be the crucial event [18, 28], further suggesting a dif- ferential role of ATX/LPA in acute versus chronic inflammation. Pharmacologic inhibition of ATX does not alleviate ALI It has been previously reported that pharmacologic inhibition of ATX using GWJ-A-23, a nanomolar ATX inhibitor [39, 40], attenuated both the development of BLM-induced pulmo- nary fibrosis [18], as well as triple-allergen (DRA)-induced asthma in mice [40]. Therefore, we next investigated the therapeutic potential of ATX inhibition in ALI. GWJ-A-23 was injected intraperitoneally just before LPS administration and disease indices were examined in compar- ison with vehicle treated littermate mice. As shown in Fig 7, ATX inhibition (Fig 7D) and reduction of LPA levels (Fig 7E) did not significantly attenuate ALI severity, as reflected in all disease indices (Fig 7A–7C), in agreement with the studies in Enpp2+/- mice. Likewise, pharma- cologic antagonism of LPAR1 and 3 with ki16425 had minor effects in inflammation (<25%) and no effect in pulmonary edema [35]. Therefore, the results further confirm a differential role of ATX in acute versus chronic inflammation and suggest no therapeutic potential of tar- geting the ATX/LPA axis in ALI/ARDS. ATX and Acute Lung Injury Fig 6. Systemic overexpression of ATX exacerbates ALI. Transgenic (Tga1t1Enpp2+/+) or heterozygous complete knock-out (Enpp2+/-) mice and their corresponding wild type littermate mice were administered with aerosolized LPS to induce lung injury and were sacrificed 24 hours later (n = 2–6; a representative experiment out of two is shown). A-D. Histological analysis (A) and BALF measurements (B & C) showed increased inflammation and edema in the lungs of the transgenic mice with systemic overexpression of ATX as a result of increased BALF ATX activity (D). On the contrary, histological analysis (A) and BALF measurements (E-G) showed non-significant effects in the lungs of the heterozygous knock-out mice as a result of decreased ATX activity levels. g j y Fig 6. Systemic overexpression of ATX exacerbates ALI. Transgenic (Tga1t1Enpp2+/+) or heterozygous complete knock-out (Enpp2+/-) mice and their corresponding wild type littermate mice were administered with aerosolized LPS to induce lung injury and were sacrificed 24 hours later (n = 2–6; a representative experiment out of two is shown). A-D. Histological analysis (A) and BALF measurements (B & C) showed increased inflammation and edema in the lungs of the transgenic mice with systemic overexpression of ATX as a result of increased BALF ATX activity (D). On the contrary, histological analysis (A) and BALF measurements (E-G) showed non-significant effects in the lungs of the heterozygous knock-out mice as a result of decreased ATX activity levels. Fig 6. Systemic overexpression of ATX exacerbates ALI. Transgenic (Tga1t1Enpp2+/+) or heterozygous complete knock-out (Enpp2+/-) mice and their corresponding wild type littermate mice were administered with aerosolized LPS to induce lung injury and were sacrificed 24 hours later (n = 2–6; a representative experiment out of two is shown). A-D. Histological analysis (A) and BALF measurements (B & C) showed increased inflammation and edema in the lungs of the transgenic mice with systemic overexpression of ATX as a result of increased BALF ATX activity (D). On the contrary, histological analysis (A) and BALF measurements (E-G) showed non-significant effects in the lungs of the heterozygous knock-out mice as a result of decreased ATX activity levels. doi:10.1371/journal.pone.0133619.g006 (together with fibroblast recruitment) of the observed protection from BLM-induced chronic pulmonary inflammation and fibrosis upon LPAR1 genetic deletion [20](where no inflamma- tory changes were observed, especially in early time points) or pharmacologic inhibition [19], further supporting a differential role of ATX/LPA in acute vs chronic inflammation. Conclusions LPS administration to wt C57Bl6 mice resulted in increased ATX/LPA levels in BALFs, as pre- viously reported [33, 35]. However, genetic deletion of ATX from bronchial epithelial cells or pharmacologic ATX inhibition, had minor effects in ALI pathology, as opposed to BLM- induced chronic pulmonary inflammation and fibrosis [18], suggesting a differential involve- ment of ATX/LPA in acute and chronic inflammation. Similarly, the genetic deletion or pharmacologic antagonism of LPAR1 had no effect in vas- cular leak and edema upon LPS administration [35], the major hallmark of LPS/ALI-ARDS (and minimal, <25%, effects in inflammation, possibly due to genetic background differences of control mice). On the contrary, LPA/LPAR1-induced vascular leak was the main attribute PLOS ONE \| DOI:10.1371/journal.pone.0133619 July 21, 2015 11 / 16 Acknowledgments We would like to thank I. Forster for the LysM-Cre mice, G. Mills for the Tga1t1Ennp2 trans- genic mice, G. Jiang and J. Zhang for the re-synthesis of the GWJ-A-23 ATX inhibitor. ATX and Acute Lung Injury of endothelial cells and a terminal increase of endothelial permeability that cannot be modu- lated further. The likely differential involvement of ATX/LPA in acute inflammation could possibly be also attributed in part to macrophage specific ATX expression. Very few (<3%) macrophages infiltrate LPS challenged lungs and our results have shown that they don’t contribute to the BALF ATX load nor to disease development (Fig 5). However, reducing macrophage (the most abundant, >60%, infiltrating cell type) ATX expression in BLM-induced chronic pulmonary inflammation and fibrosis reduced both ATX BALF load and disease development [18]. Moreover, a more prominent role of ATX/LPA in chronic inflammation is consistent with their role in cancer [16], given the increasing links of chronic inflammation and carcinogenesis. Conditional deletion of ATX from bronchial epithelial cells that although had minor effects in LPS-induced ALI, attenuated the development of both pulmonary inflammation and fibrosis [18], as well urethane-induced lung cancer [42]. The issue is currently investigated in a more suitable context for such studies, namely liver pathogenesis. Finally, any biological outcome of increased ATX/LPA levels would depend on the abun- dance and activity of the different LPA receptors in different cell types participating in the dif- ferent phases of an inflammatory response, especially given the reported anti-inflammatory effects of LPA/LPAR2 on innate immune responses in the lung [43] and the suggested roles of LPA in the regulation of adaptive immune responses [44]. Therefore, the complete understand- ing of the involvement of ATX/LPA in the various forms of inflammation will require precise knowledge of the spatiotemporal regulation of ATX and LPA receptors expression, as well as the cell-specific LPA effects in the different cell types involved in inflammatory responses. Author Contributions Conceived and designed the experiments: NO VA. Performed the experiments: MM NO CM AK EK. Analyzed the data: MM NO CM AK EK. Contributed reagents/materials/analysis tools: GP. Wrote the paper: MM VA. Conceived and designed the experiments: NO VA. Performed the experiments: MM NO CM AK EK. Analyzed the data: MM NO CM AK EK. Contributed reagents/materials/analysis tools: GP. Wrote the paper: MM VA. PLOS ONE \| DOI:10.1371/journal.pone.0133619 July 21, 2015 The differ- ences in LPA/LPAR1-mediated endothelial barrier functions in acute and chronic pulmonary inflammatory animal models suggest that the reported effects of LPA in endothelial 12 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0133619 July 21, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0133619 July 21, 2015 ATX and Acute Lung Injury Fig 7. Pharmacologic inhibition of ATX has no effects in ALI development. GWJ-A-23 was injected intraperitoneally before challenging mice with aerosolized LPS. Mice were sacrificed 24 hours later (n = 3–7; a representative experiment out of two is shown). A-C. Histological analysis (A) and indicated BALF measurements (B,C) suggested minor effects in ALI development, despite decreased BALF ATX activity (D) and the corresponding BALF LPA levels (E). doi:10 1371/journal pone 0133619 g007 Fig 7. Pharmacologic inhibition of ATX has no effects in ALI development. GWJ-A-23 was injected intraperitoneally before challenging mice with aerosolized LPS. Mice were sacrificed 24 hours later (n = 3–7; a representative experiment out of two is shown). A-C. Histological analysis (A) and indicated BALF measurements (B,C) suggested minor effects in ALI development, despite decreased BALF ATX activity (D) and the corresponding BALF LPA levels (E). doi:10.1371/journal.pone.0133619.g007 permeability may need chronic exposure of target cells. Indeed, LPA effects in pulmonary endothelial permeability were found to increase with time (and of course concentration)[41]. Accordingly, chronically elevated serum ATX levels in Tga1t1Enpp2 mice increased LPS- induced acute lung injury by increasing both vascular leak and inflammation (Fig 6). 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https://openalex.org/W2171833292	https://europepmc.org/articles/pmc4333262?pdf=render	English	null	Primary Synovial Sarcoma (SS) of the digestive system: a molecular and clinicopathological study of fifteen cases	Clinical sarcoma research	2,015	cc-by	5,847	Primary Synovial Sarcoma (SS) of the digestive system: a molecular and clinicopathological study of fifteen cases Salvatore Romeo1, Sabrina Rossi1, Marthelena Acosta Marín2, Fabio Canal1, Marta Sbaraglia1, Licia Laurino1, Guido Mazzoleni3, Maria Cristina Montesco4, Laura Valori1, Marta Campo Dell’Orto1, Andrea Gianatti5, Alexander Joseph Lazar2 and Angelo Paolo Dei Tos1* © 2015 Romeo et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Recently a few cases of synovial sarcoma (SS) of the abdominal viscera have been reported, raising awareness about the potential for confusion between this entity and KIT-negative gastrointestinal stromal tumors (GIST). We report the clinicopathological, immunophenotypical and molecular features of fifteen more SS occurring in the stomach (8 cases), epigastric region (one case), small intestine (one case), large intestine (three cases), involving both the terminal ileum and the caecum (one case) and liver (one case). Methods: Immunostains for SMA, DESMIN, CD34, CD117, S100, EMA, CK AE1/3, TLE1, CD56, CD99, BCL2, DOG1 were performed. Rearrangement of SS18 gene region was screened in all cases: by conventional karyotype in one case, the remaining cases were screened either by interphase FISH or Q-PCR or both. Results: Ten patients were male and five female, with an age range of 17–61 years (median 44). Tumor size ranged from 2 to 15 cm (median 8). Mitoses per 10 HPF ranged from 4 to 27 (median 9.5). Eleven tumors were monophasic fibrous SS, one biphasic SS and three poorly differentiated SS. SMA, Desmin, CD34, CD117 and S100 were negative in al cases, whereas EMA and/or CK AE1/AE3 were positive in all cases. TLE1, BCL2 and CD56 were positive in all tested cases. DOG1 was positive in one case. SS18 gene region rearrangement was demonstrated in all cases. A fusion transcript was amplified in eight cases: either SS18-SSX2 or SS18-SSX1 respectively in four cases each. Conclusions: SS is increasingly recognized at visceral sites. Molecular analyses play a key role when dealing with usual histotypes in unusual sites. Correct diagnosis is crucial for appropriate therapy. Keywords: Synovial sarcomas, Pathology, Differential diagnosis, Digestive tract, Sarcoma a hemangiopericytoma (HPC)-like vascular pattern [1]. An epithelial component is present in the biphasic vari- ant, with solid nests and glandular or tubular structures [1]. The existence of a predominantly monophasic epithe- lial pattern has been reported, too [2]. In approximately 20% of cases, SS exhibits undifferentiated, high-grade morphology and is usually indicated as “poorly differenti- ated” SS (PDSS) [3]. Three main groups of PDSS can be identified: one exhibiting round cell morphology associ- ated with necrosis and high mitotic count; another charac- terized by the presence of larger cells, with polygonal cytoplasm which may feature rhabdoid morphology; and a third group presenting as high-grade spindle cell tumors often featuring a “herringbone” growth pattern [3,4]. RESEARCH Open Access Open Access * Correspondence: apdeitos@ulss.tv.it 1Department of Pathology, Treviso Regional Hospital, Piazza Ospedale 1, 31100 Treviso, Italy Full list of author information is available at the end of the article Romeo et al. Clinical Sarcoma Research (2015) 5:7 DOI 10.1186/s13569-015-0021-3 Romeo et al. Clinical Sarcoma Research (2015) 5:7 DOI 10.1186/s13569-015-0021-3 CLINICAL SARCOMA RESEARCH Introduction Synovial sarcoma (SS) is a mesenchymal malignant tumour that accounts for approximately 10% of all soft tissue sar- comas [1]. It usually occurs in the lower limbs of children and young adults, with the knee region being the most frequently affected area [1]. Three main histological vari- ants of SS have been recognized: the monophasic, bi- phasic and poorly differentiated subtypes [1]. Both the monophasic and biphasic variants feature a spindle cell population set in a variable collagenous background with * Correspondence: apdeitos@ulss.tv.it 1Department of Pathology, Treviso Regional Hospital, Piazza Ospedale 1, 31100 Treviso, Italy Full list of author information is available at the end of the article Page 2 of 8 Romeo et al. Clinical Sarcoma Research (2015) 5:7 Romeo et al. Clinical Sarcoma Research (2015) 5:7 SS subtypes share a common genetic alteration: a trans- location involving chromosomes X and 18. This transloca- tion results in three alternative fusion products of the SS18 gene (previously known as SYT) (on chromosome 18) with either SSX1, or SSX2 or SSX4 gene (on chromo- some X) [1]. This knowledge provides useful ancillary diagnostic tools [1] for identification of the specific trans- location by interphase FISH analysis, with probes flanking the breakpoints, and amplification of the specific chimeric transcript by RT-PCR techniques [5]. records were retrieved. Follow-up information was avail- able for 11 patients (Table 1). All samples were handled in a coded fashion, and all procedures were performed ac- cording to the ethical guidelines of the local institutions. Pathology assessment and immunohistochemistry All the cases were reviewed for diagnostic confirmation and both necrosis extent and mitoses count evaluated, grading provided according to French National Feder- ation of Cancer Centers (FNLCC) (Table 2). In a subset of cases neoadjuvant chemotherapy was applied and pre- chemo biopsies were not available (Table 2); the values for mitoses and necrosis are no longer relevant for these cases as pre-treated specimens cannot be accurately graded under the FNCLCC system. SS rarely occurs in unusual sites including: the head and neck region [6,7], mediastinum [8], larynx and hy- popharynx [9], nerves [10], blood vessels [11,12], heart [13], abdominal cavity [14], gastrointestinal tract [15-23] and liver [24,25]. In routine activity it may be difficult to distinguish SS occurring in the digestive tract from other mesenchymal neoplasms, mainly GIST (gastrointestinal stromal tumour). However, this distinction is crucial to ensure a correct therapeutic approach. Here we report the clinicopathological, immunohistochemical and mo- lecular genetic data of fifteen cases of SS occurring in the digestive system. We aim to improve knowledge on this entity and stress the importance of correct differen- tial diagnosis for appropriate therapeutic management. Immunostaining was performed for EMA, cytokeratin AE1/AE3, SMA, Desmin, CD34, CD117, S-100, CD99, CD56 and TLE1. Four-μm sections of formalin-fixed paraffin-embedded material were used according to standard laboratory procedures. Details of the antibodies used are given in Table 3. iven in centimeter; NR: not reported, Follow Up is in months; DOD: dead of disease; NED: not evidence of disease; AWD: alive with dise Legend: Size is given in centimeter; NR: not reported, Follow Up is in months; DOD: dead of disease; NED: not evidence of disease; AWD: alive with disease; Lost: lost to follow up Conventional karyotype For case 14 conventional karyotype was performed. Cell culture, harvest conditions, and karyotyping were per- formed according to standard protocols. Legend: Size is given in centimeter; NR: not reported, Follow Up is in months; DOD: dead of disease; NED: not evidence of disease; AWD: alive with disease; Lost: lost to follow up. Interphase FISH Fifteen cases of SS of the abdominal viscera were col- lected from three Italian institutions, the consultation files of one of the Authors (APDT) and Departments of Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA. Patients’ clinical Fluorescent in situ hybridisation (FISH) was performed on 5 μm paraffin-embedded tissue sections using the LSI SYT (18q11.2) Dual Color Break Apart Rearrange- ment Probe set (Vysis, Downers Grove, IL, USA). Table 1 Clinicopathologic features of 14 synovial sarcomas of the digestive system Case # Site Size Gender Age Follow up Metastases 1 Gastric body 8 F 50 Lost 2 Cardias 6 M 36 AWD@36 Liver 3 Gastric 2 M 37 Recent 4 Gastric NR M 26 AWD@185 Liver, Lungs 5 Gastric 10 M 58 DOD@6 6 Gastric 10 M 21 Lost@48 7 Gastric 6 M 36 Lost@12 8 Gastric 3.8 F 54 Recent 9 Epigastrium 13 F 57 AWD@7 10 Ileum 8 M 49 DOD@60 11 Large bowel 5.5 M 40 NED@132 12 Rectosigmoid colon 6.3 F 44 DOD@47 13 Rectosigmoid colon 6.3 F 44 DOD@47 14 Ileum/Colon 7.5 M 17 NED@108 15 Liver 15 M 61 AWD@12 Legend: Size is given in centimeter; NR: not reported, Follow Up is in months; DOD: dead of disease; NED: not evidence of disease; AWD: alive with disease; Lost: lost to follow up. Table 1 Clinicopathologic features of 14 synovial sarcomas of the digestive system Page 3 of 8 Page 3 of 8 Romeo et al. Interphase FISH Clinical Sarcoma Research (2015) 5:7 Table 2 Morphological features of 15 synovial sarcomas of the digestive system Case # Type Mitoses Necrosis Grading Involvement of Perivisceral soft tissue Peritoneum Adjacent organ 1 M 7 1 2 None None None 2 PD 11 1 2 Adventitial tissue Yes None 3 M 6 0 2 None None None 4 M P P P Adventitial tissue Yes Pancreas 5 M 12 1 2 Adventitial tissue Yes Pancreas 6 M P P P None None None 7 B 27 1 3 None None None 8 M 14 1 2 None None None 9 M 8 2 3 Stomach, duodenum and liver Yes None 10 M 5 1 2 None None None 11 M 13 1 2 Perivisceral adipose tissue None None 12 PD P P P Perirectal adipose tissue None None 13 PD P P P None None None 14 M 4 1 2 None None None 15 M P P P None None None Legend: M: monophasic, B: biphasic, PD: poorly differentiated, mitoses are per 10 HPF, P: pretreated. Necrosis is reported as: 0 for no necrosis, 1 for <50% tumor necrosis, 2 for ≥50% tumor necrosis. Table 2 Morphological features of 15 synovial sarcomas of the digestive system of 15 synovial sarcomas of the digestive system reated. Necrosis is reported as: 0 for no necrosis, 1 for <50% tumor Hybridisation was performed according to the manufac- turer’s protocol. Slides were mounted and counterstained with anti-fade DAPI (Vysis, Downers Grove, IL, USA), vi- sualized using an epifluorescent microscope (Olympus BX61) and analysed with FISH analysis software (Genetix- Cytovision 4.5.1). 300 interphase nuclei were analyzed. percentage of tumor cells, as calculated from the HE- stained slides, was at least 70%. Subsequently, RNA was extracted with TRIzol-LS Reagent (Gibco BRL), accord- ing to the manufacturer’s instructions. RNA pellets were resuspended in 10–20 μl of RNAse-free water and stored at −80°C. 5 μg of total RNA were reverse transcribed in a total volume of 20 μl using specific reverse primers for SSX and BETA2M genes, respectively. Samples were in- cubated at 42°C for 1 hour, then at 72°C for 15 minutes. PCR amplification of each sample and a 1:20 dilution were performed in duplicate using 96-well plates in 25 μl reaction mixture containing 300 nM of each primer, 200 nM of each probe (SSX1-SSX2) or 100 nM probe BETA2M and 2X TaqMan Gene Expression Master Mix Results h d All cases showed SS18 gene region rearrangement. Case 14 showed 46, XY, t(X; 18)(p11;q11) karyotype (data not shown). The remaining cases were assessed ei- ther by interphase FISH (Figure 1E) or by RT-PCR or both. Eight cases were analyzed by RT-PCR: either SS18- SSX2 fusion transcript or SS18-SSX1 fusion transcript was identified in 4 cases each (Figure 1F, Table 5). In the identified fourteen cases of digestive system SS a male prevalence was found: male/female ratio was 3:1. Age at clinical presentation ranged from 17 to 61 years (median 44 years) (Table 1). Size ranged from 2 cm to 15 cm (median 8 cm) (Table 1). Eight cases were occur- ring in the stomach, one case in the epigastric region, one case in the ileum, three cases in the large intestine, one case was involving both the terminal ileum and the caecum and one case in the liver (Table 1). Follow up was available for 11 patients: range from 6 to 185 months (47 median) (Table 1). Based on clinical, morphological, immunophenotypical and molecular data a diagnosis of primary SS of the di- gestive tract was formulated: monophasic synovial sar- coma in eleven cases, biphasic SS in one case and poorly differentiated SS in three cases. Five of the eight tumors arising in the stomach tract were confined to the wall (without serosal involvement); one extended into the perivisceral soft tissue, periton- eum and omentum (Figure 1A) and two involved the pancreas (Table 2). The tumor located in the epigastric region was adherent to the stomach and duodenal wall and focally attached to the liver (Table 2). Three tumors were involving the large bowel with extension in the perivisceral adipose tissue in two of them. The case af- fecting the ileum was limited to the ileum wall, similarly also the case involving both ileum and caecum was lim- ited to the visceral wall (Table 2). The one in the liver was confined to the parenchyma without ulceration of the Glisson’s capsule (Table 2). Q-PCR Ten to fifteen 15 μm-thick sections from paraffin- embedded tissue were de-paraffinized twice using xy- lene, washed twice with absolute ethanol followed by TNE1X, resuspended in 250 μl of ATL buffer (Qiagen) with the addition of proteinase K (Qiagen), and incu- bated for 72 hours at 55°C under moderate shaking. The Table 3 Details of the antibodies used in this study Antibody Clone Producer Diluition Antigen retrieval EMA E29 Dako, Glostrup, Denmark Prediluted Flex (Dako) CKAE1/AE3 Polyclonal Dako, Glostrup, Denmark Prediluted Flex (Dako) SMA 1A4 Dako, Glostrup, Denmark Prediluted Flex (Dako) DESM D33 Dako, Glostrup, Denmark Prediluted Flex (Dako) CD34 QBend-10 Dako, Glostrup, Denmark prediluited Flex (Dako) CD117 Polyclonal Dako, Glostrup, Denmark 1/700 Flex (Dako) S-100 Polyclonal Dako, Glostrup, Denmark Prediluted Flex (Dako) TLE1 c-9121 Santa Cruz Biochemicals, Santa Cruz, CA, USA 1:100 Flex (Dako) BCL2 124 Dako, Glostrup, Denmark Prediluted Flex (Dako) CD56 123C3 Dako, Glostrup, Denmark Prediluted Flex (Dako) CD99 MIC2 Dako, Glostrup, Denmark Prediluted Flex (Dako) DOG1 K9 Novocastra, NewCastle, UK 1:100 Flex (Dako) Table 3 Details of the antibodies used in this study Romeo et al. Clinical Sarcoma Research (2015) 5:7 Romeo et al. Clinical Sarcoma Research (2015) 5:7 Page 4 of 8 Romeo et al. Clinical Sarcoma Research (2015) 5:7 Mitotic count ranged from 4 to 27 mitoses/10 HPF (median 9.5) (Table 2). Immunoreactivity for EMA was found in all tested cases ranging from focal to strong and diffuse (Figure 1D) (Table 5). Focal positiv- ity for immunostains for cytokeratin AE1-AE3 was found in 8 cases (Table 5). All tested cases were posi- tive for TLE1, BCL2 and CD99 (Table 5). No expression of CD117, SMA, DESMIN, CD34 and S-100 protein was found. DOG1 was focally expressed in one case (case 9, Table 5). (Applied Biosystems, CA), on Applied Biosystems Step- OnePlus Real-Time PCR Systems (Applied Byosystem). (For primer and probe sequences, see Table 4). Thermal cycling conditions were 2 minutes at 50°C, 10 minutes at 95°C, then 50 cycles for three PCR steps consisting of 30 seconds at 95°C and 1 minute at 60°C. Eight cases were studied by Q-PCR, for all of them a positive control prod- uct was amplified. Discussion SS is characterized by a complex, relatively distinctive immunophenotype, which includes co-expression of mesenchymal (vimentin) and epithelial markers (cyto- keratins and EMA). Since morphological features of epi- thelial differentiation may be very subtle, immunostains are a valuable diagnostic aid. Cytokeratins tend to decor- ate most biphasic synovial sarcomas, but when dealing with the monophasic subtype, the percentage of immu- nopositivity falls to 60%-70%. Interestingly, cytokeratin immunoreactivity has been demonstrated only in 50% of PDSS [3] and high molecular weight cytokeratins proved to be more sensitive than low molecular weight cytokera- tins. The most sensitive marker of epithelial differentiation is EMA, which stains most cases of PDSS, including those that fail to express cytokeratins [3]. Between 30% and 60% of SS express S-100 protein leading to potential confusion when dealing with the differential diagnosis between monophasic spindle-cell SS and MPNST [26,27]. To avoid diagnostic pitfalls caused by the use of single antibody, it is therefore strongly recommended to perform a panel of immunohistochemical markers. Microscopically, eleven of the tumours consisted of monotonous spindle cell proliferation, with scant intercel- lular eosinophilic collagenous stroma (Figure 1B) (Table 2). A focal HPC-like vascular network was observed. Three cases showed poorly differentiated features (Figure 1C) and one showed biphasic features with both spindle cells components and tubules formation (Table 2). Table 4 Sequences of the primers and probes used in this study Table 4 Sequences of the primers and probes used in this study PRIMER/PROBE name Sequence SS18 (forward) AGAGGCCTTATGGATATGACCAGAT SSXC (reverse) CRTTTTGTGGGCCAGATGC BETA2M+ (forward) TGACTTTGTCACAGCCCAAGATA BETA2M- (reverse) AATCCAAATGCGGCATCTTC SSX1 probe TCCCTTCGAATCATTTTCGTCCTCTGCT SSX2 probe TCTGGCACTTCCTCCGAATCATTTCCTT BETA2M probe TGATGCTGCTTACATGTCTCGATCCCA The diagnosis of biphasic synovial sarcoma is usually straightforward, even for cases occurring in the digestive system. Regarding monophasic SS in the digestive sys- tem, the main differential diagnosis is with GIST: any mesenchymal lesion arising in the GI tract would natur- ally be suggestive of a diagnosis of GIST and CD117 negativity “per se” does not rule out such a possibility Page 5 of 8 Romeo et al. Clinical Sarcoma Research (2015) 5:7 Figure 1 Digestive tract SS features: A- Neoplastic cells were involving gastric wall with focal extension to nearby perivisceral soft tissue and serosal ulceration (case 2, 12.5 X original magnification, HE staining). B- Most of the cases were composed of monomorphic spindle cells (case 9, 400 X original magnification, HE staining). Discussion C- Poorly differentiated SS featuring monomorphic round cells with elevated mitotic rate were encountered in 3 cases (case 4, 400 X original magnification, HE staining). D- Focal positivity for DOG1 staining was found in one case(Case 9, 400 X original magnification). E- Interphase FISH showed split a part of the two signal in most of the nuclei (case 9, original 1000X magnification). F- A fusion transcript SS18-SSX1 was amplified by Q-PCR (plot showing the amplification curves of SS18-SSX1, SS18-SSX2 and beta-microglobulin). Figure 1 Digestive tract SS features: A- Neoplastic cells were involving gastric wall with focal extension to nearby perivisceral soft tissue and serosal ulceration (case 2, 12.5 X original magnification, HE staining). B- Most of the cases were composed of monomorphic spindle cells (case 9, 400 X original magnification, HE staining). C- Poorly differentiated SS featuring monomorphic round cells with elevated mitotic rate were encountered in 3 cases (case 4, 400 X original magnification, HE staining). D- Focal positivity for DOG1 staining was found in one case(Case 9, 400 X original magnification). E- Interphase FISH showed split a part of the two signal in most of the nuclei (case 9, original 1000X magnification). F- A fusion transcript SS18-SSX1 was amplified by Q-PCR (plot showing the amplification curves of SS18-SSX1, SS18-SSX2 and beta-microglobulin). Page 6 of 8 Page 6 of 8 Romeo et al. Discussion Clinical Sarcoma Research (2015) 5:7 Table 5 Results of the performed immunostains and fusion type in 15 synovial sarcomas of the digestive system Case # EMA CKAE1/AE3 SMA DESMIN CD34 CD117 S-100 BCL2 CD99 CD56 DOG1 TLE1 Fusion type 1 + - - - - - - + + + - + SS18-SSX1 2 + - - - - - - + + + - + N/A 3 + + - - - - - + + + - + N/A 4 + - NP NP NP - - NP NP NP NP NP N/A 5 + - NP - - - - NP NP NP NP NP SS18-SSX1 6 + - NP NP NP - - NP NP NP NP NP N/A 7 NP + NP NP - - NP NP NP NP NP NP SS18-SSX2 8 + + - - - - - + + + - + SS18-SSX1 9 + + - - - - - + + + + + N/A 10 + + - - - - - + + + - + SS18-SSX2 11 + - - - - - - + + + - + SS18-SSX2 12 + + NP NP NP - - + + NP NP NP SS18-SSX2 13 + + NP NP NP - - + + NP NP NP SS18-SSX1 14 + - NP - - - - + + NP NP NP N/A 15 + + - - - - - + + + - + N/A [28]. GISTs are usually composed of short spindled and/ or epithelioid cells with perinuclear vacuolization and nuclear palisading. Recognition of the histological fea- tures, and the combination of CD117 with DOG1 stain- ing is sufficient in the majority of cases to confirm the diagnosis of GIST [29]. However caution should be used in interpreting the results of immunohistochemistry since synovial sarcoma of the digestive system may show focal positivity for DOG 1 [30], as also exemplified in our case series. Remarkably sporadic GIST cases have been reported to be positive for cytokeratin [31,32], however EMA positivity is exceptional in GIST [23]. Leiomyosarcomas and malignant spindle cell melanomas are considered in the differential diagnosis with mono- phasic SS of the digestive system. However they are characterized by a higher pleomorphism and stronger immunostaining for smooth muscle markers and mela- nocytic markers, respectively, can usually confirm the diagnoses. References 1. Suurmeijer AJH, de Bruijn D, Geurts van Kessel A, Miettinen MM. Synovial Sarcoma. Lyon: IARC Press; 2013. 1. 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PD SS may resemble other small round cell tumors in- cluding Ewing Sarcoma/PNET, neuroblastoma, rhabdo- myosarcoma and lymphoma [1]. Remarkably CD99 antigen is found also in SS [35] and epithelial markers may be absent in PDSS and focally present in Ewing Sarcoma/PNET [1]. In this setting, demonstration of re- arrangement of SSX18 or EWSR1 is crucial for differenti- ating respectively PDSS from Ewing Sarcoma/PNET [1]. Also it should be remembered that CD99 positivity in syn- ovial sarcomas does not feature the typical crisp mem- brane staining most often observed in Ewing’s sarcoma. Cytogenetically, all SS variants are characterized by the reciprocal translocation t(X;18)(p11.2;q11.2), which leads, at molecular level, to the fusion between the synovial sar- coma translocation gene on chromosome 18 (SS18) and one of the synovial sarcoma X breakpoint (SSX) genes on chromosome X: SSX1, SSX2 and rarely with SSX4 [36-38]. Interestingly, the SS18-SSX1 translocation seems to be associated with the biphasic type [39]. The SS18 gene is unrelated to any other known gene but contains a glutamine-proline-glycine-rich region, suggestive of a transcriptional activation domain. The SSX1, SSX2 and SSX4 genes are also unrelated to other known genes and encode proteins that show a remarkable homology. Sarcomatoid carcinoma may also be considered in the differential diagnosis, however it often exhibits conspicu- ous pleomorphism, stronger expression of epithelial markers, and area of conventional carcinoma are often as- sociated with the sarcomatoid component. Gastrointestinal clear cell sarcoma may be very diffi- cult to distinguish form digestive system SS [33,34], also because, as previously mentioned, SS may be positive for S100 staining [26,27]. However gastrointestinal clear cell sarcomas are usually negative for epithelial markers and show rearrangement of the EWSR1 gene [33,34]. Re- markably clear cells sarcomas occurring in the gastro- intestinal tract differ from clear cell sarcomas of the soft tissue. In fact they display scattered osteoclast-type giant Despite initial attempt to correlate fusion type with a sig- nificantly longer disease-free survival [39,40] morphological grading is still the most important prognostic indicator [41]. Furthermore, tumor size (>5 cm), presence of neural infiltration and vascular invasion, p53 overexpression, and Page 7 of 8 Page 7 of 8 Romeo et al. Discussion Clinical Sarcoma Research (2015) 5:7 high Ki67 proliferation index identify subsets of SS pa- tients with increased risk of tumor relapse [42-44]. high Ki67 proliferation index identify subsets of SS pa- tients with increased risk of tumor relapse [42-44]. 14. Fetsch JF, Meis JM. Synovial sarcoma of the abdominal wall. Cancer. 1993;72:469–77. 15. Amr SS, Shihabi NK, Al HH. Synovial sarcoma of the esophagus. Am J Otolaryngol. 1984;5:266–9. 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References Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W2013308985	https://europepmc.org/articles/pmc3913455?pdf=render	English	null	Atherosclerosis Burden in Patients with Acute Chest Pain: Obesity Paradox	ISRN Obesity	2,014	cc-by	5,368	1. Introduction defining obesity, it is not surprising that evidences of ability of obesity to be a risk factor and a poor prognosticator are inconsistent among studies. There have been multiple studies describing “obesity paradox,” a protective effect of obesity, with various clinical surrogates and outcomes in different populations of CAD including asymptomatic population [5, 7], myocardial infarction [8–10], and patients treated with revascularization [11, 12]. However, in patients with acute chest pain undergoing coronary artery calcification (CAC) evaluation, there have been sparse studies.h Obesity has been believed to be one of the major risk factors and adverse prognosticators, associated with increased mor- tality risk, for atherosclerotic diseases, especially coronary artery disease (CAD) [1, 2]. Extensive studies have shown that obesity is an independent predictor for CAD and car- diovascular death in multiple populations including a large- scale epidemiological study and a systemic review [3, 4]. Obesity is not only associated with prevalence and death in CAD itself but also related to its major risk factors including hypertension, diabetes mellitus, and dyslipidemia [5]. Body mass index (BMI) which is the index most commonly used in majority of the studies to define obesity has also been shown to be positively associated with increased risk of CAD even in the normal weight range [6]. Pathophysiology of obesity and cardiovascular diseases is complicated and it involves several pathways particularly cardiovascular hemodynamics, systemic inflammation, and leptin metabolism [5]. Given its complexity of interaction and arguable robustness of BMI in p This study seeks to primarily examine relationship between obesity and significant CAD in patients with acute chest pain of unknown cardiac significance who were admit- ted in an observation unit to further support or reject an idea of obesity paradox in this particular population. 1 Department of Medicine, The Methodist Hospital, Houston, TX 77030, USA 2 Methodist DeBakey Heart & Vascular Center, The Methodist Hospital, Houston, TX 77030, USA Correspondence should be addressed to Su Min Chang; smchang@houstonmethodist.org Received 15 September 2013; Accepted 21 October 2013; Published 12 January 2014 Academic Editors: K.-C. Huang and J. Saleh Copyright © 2014 Kongkiat Chaikriangkrai et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Obesity paradox has been described in various populations of coronary artery disease, mainly asymptomatic subjects. However, relationship between obesity and coronary artery calcification detected by cardiac CT in symptomatic patients has rarely been demonstrated. This study seeks to investigate whether the paradoxical relationship between obesity and coronary artery calcification exists in patients with acute chest pain. A final cohort of 1030 chest pain patients presenting at our emergency department who underwent coronary evaluation by multidetector cardiac CT were examined. With absent-to-mild coronary calcification (CAC score < 100) as a referent, multivariable analysis showed that presence of obesity (OR 0.564; 95% CI 0.395, 0.806; 𝑃0.002), body mass index (OR 0.945; 95% CI 0.920, 0.971; 𝑃< 0.001), body weight (OR 0.987; 95% CI 0.979, 0.995; 𝑃0.001), and body surface area (OR 0.582; 95% CI 0.369, 0.920; 𝑃0.020) were inversely associated with moderate-to-severe coronary calcification (CAC score ≥100). This study extends the concept of obesity paradox to symptomatic patients undergoing coronary artery calcium score assessment. However, biological explanation(s) of this paradox remains unanswered. Hindawi Publishing Corporation ISRN Obesity Volume 2014, Article ID 634717, 7 pages http://dx.doi.org/10.1155/2014/634717 Hindawi Publishing Corporation ISRN Obesity Volume 2014, Article ID 634717, 7 pages http://dx.doi.org/10.1155/2014/634717 Kongkiat Chaikriangkrai,1 Mahwash Kassi,1 Sayf Khaleel bala,2 Faisal Nabi,2 and Su Min Chang2 1 Department of Medicine, The Methodist Hospital, Houston, TX 77030, USA 2 Methodist DeBakey Heart & Vascular Center, The Methodist Hospital, Houston, TX 77030, USA 3. Results 3.1. Study Population Characteristics. A total of 5066 patients presented to our ED with acute chest pain between September 2005 and February 2008. The final cohort consisted of 1030 patients. Studied clinical characteristics are shown in Table 1. 2.3. CAC Scoring. CAC score was calculated with recon- structed axial images of 2.5 mm thickness as previously described by Agatston et al. [15]. A 16-slice multidetector CT scanner (Philips Precedence, Philips Healthcare, Eindhoven, The Netherlands) was used. Images were acquired during a single breath hold, using prospective ECG gating with imaging triggered at 75% of the R-R interval. Patients were categorized into 2 groups based on their CAC extent: absent- to-mild CAC (CAC score < 100) or moderate-to-severe CAC (CAC score ≥100). The median CAC score in our study population was 0 with 25th and 75th percentile of 0 and 35.75, respectively. Most (624 of 1030; 60.6%) had absent CAC, followed by 21.7% (223 of 1030) with mild CAC (0 < CAC score < 100) and 17.8% (183 of 1030) with moderate-to-severe CAC (CAC score ≥100). Patients with moderate-to-severe CAC had expected results for most clinical characteristics. They were older (𝑃< 0.001) and tended to be male (𝑃0.015). They had significantly more history of hypertension (𝑃< 0.001), history of dyslipidemia (𝑃< 0.001), history of stroke (𝑃< 0.001), and history of peripheral arterial disease (PAD) (𝑃< 0.001). Other risk factors for CAD were comparable between 2 groups which are history of diabetes (𝑃0.359), history of cigarette smoking (𝑃0.099), and family history of CAD (𝑃0.405). Compared to patients with absent-to- mild coronary calcification, patients with moderate-to-severe coronary calcification had higher median Framingham CAD 10-year risk score (𝑃< 0.001), higher median TIMI risk score (𝑃< 0.001), higher proportion of high TIMI risk score (𝑃< 0.001), and higher proportion of moderately high and high risk in ATP III criteria (𝑃< 0.001). There were also significantly higher prevalence of uses of cardiovascular medications including aspirin (𝑃 < 0.001), angiotensin converting enzyme inhibitors (ACEIs)/angiotensin receptor blockers (ARBs) (𝑃< 0.001), beta blockers (𝑃< 0.001), calcium channel blockers (𝑃< 0.001), and statins (𝑃< 0.001) but not diuretics (𝑃0.101) in moderate-to-severe CAC group compared to absent-to-mild CAC group. 2.4. Data Gathering and Processing. 2. Materials and Methods Indices for body mor- phology used in this study were height in meter, weight in kilogram, body surface area (BSA) in meter2 calculated by DuBois and DuBois formula [14], and BMI. BMI of each subject was calculated by the following formula: weight in kilogram ÷ (height in meter)2. Definition of obesity was set at BMI at least 30 kg/m2. 2. Materials and Methods 2.1. Study Population. This study is a prospective obser- vational cohort study conducted from September 2005 to 2 2 ISRN Obesity coronary syndrome, and frequency with percentage for cate- gorical variables including binary cardiovascular risk factors and uses of cardiovascular medications. Student’s 𝑡-test was used to identify differences in mean. Mann-Whitney 𝑈test was used to examine differences in medians. 𝜒2 analysis was used to identify significant heterogeneity in the frequencies. Binary logistic regression was performed to examine rela- tionship between covariables and presence of moderate-to- severe CAC. Odd ratio and 95% confidence interval (CI) were used to describe the relationship. Body morphology indices that had statistical significant difference between CAC groups were included as testing variables. All baseline characteristics that are significant univariable predictors at the level of 𝑃< 0.1 were entered into multivariable models for each testing variable to identify ability to be an independent predictor. Framingham CAD risk score was entered into multivariable model to represent age, gender, smoking history, dyslipi- demia, and hypertension. Other baseline characteristics that were included are history of stroke and history of PAD. All statistical analyses were performed with IBM SPSS/PASW Statistics 20 (SPSS Inc., Chicago, IL). 𝑃value < 0.05 was considered statistically significant. February 2008. Subjects were patients older than 18 years old who were admitted under observational status for further evaluation of acute chest pain suggestive of myocardial ischemia within the previous 24 hours. The evaluation was performed with single-photon emission computed tomog- raphy (SPECT) and CAC scoring by multidetector cardiac CT. Exclusion criteria were subjects with noncardiac chest pain based on clinical assessment, elevated troponin on initial blood samples, new or presumably new ST-segment eleva- tion or depression (≥1 mm) on baseline electrocardiogram, hemodynamic or clinical instability defined by systolic blood pressure <90 mmHg or clinical significant atrial/ventricular arrhythmia, history of coronary artery disease based on pre- vious coronary angiography or prior coronary revasculariza- tion, and subjects with known or suspected pregnancy. More detailed materials and methods were described elsewhere [13]. 2.2. Body Morphology and Obesity. Indices for body mor- phology used in this study were height in meter, weight in kilogram, body surface area (BSA) in meter2 calculated by DuBois and DuBois formula [14], and BMI. BMI of each subject was calculated by the following formula: weight in kilogram ÷ (height in meter)2. Definition of obesity was set at BMI at least 30 kg/m2. 2.2. Body Morphology and Obesity. 3. Results During observation period, all clinical information was collected including demo- graphic information, cardiovascular history (i.e., cardiovas- cular symptoms, history of hypertension, history of diabetes mellitus, history of dyslipidemia, smoking history, history of peripheral arterial disease, history of carotid artery disease, history of abdominal aortic aneurysm, family history of coronary artery disease, and current cardiovascular medica- tion profiles), and blood samples for lipid profiles, cardiac biomarkers, and renal function tests. Clinical information was used to calculate Framingham coronary heart disease 10-year risk score [16], TIMI risk score for acute coronary syndrome [17], and ATP III risk score for coronary artery disease [18]. 2.5. Statistical Analysis. Descriptive statistics for studied variables are presented as mean with standard deviation (SD) for normally distributed variables, including age, height, weight, BSA, and BMI, median with range for nonnormally distributed variables, including Framingham coronary heart disease 10-year risk score and TIMI risk score for acute 3 ISRN Obesity Table 1: Baseline characteristics. Clinical characteristics All patients (𝑁= 1030) Absent-to-mild CAC (𝑁= 847) Moderate-to-severe CAC (𝑁= 183) 𝑃value Mean age, year (SD) 54.0 (13.5) 51.2 (11.8) 66.8 (13.2) <0.001 Male, no. (%) 413 (40.1) 522 (61.6) 95 (51.9) 0.015 Risk factors for CAD, no. (%) History of hypertension 590 (57.3) 454 (53.6) 136 (74.3) <0.001 History of diabetes 152 (14.8) 121 (14.3) 31 (16.9) 0.359 History of dyslipidemia 352 (34.2) 261 (30.8) 91 (49.7) <0.001 History of cigarette smoking 192 (18.6) 150 (17.7) 42 (23.0) 0.099 Family history of CAD 52 (5.0) 45 (5.3) 7 (3.8) 0.405 History of stroke 35 (3.4) 19 (2.2) 16 (8.7) <0.001 History of PAD 12 (1.2) 5 (0.6) 7 (3.8) <0.001 Framingham risk score, median (range) 4 (1–30) 2 (1–30) 16 (1–30) <0.001 TIMI risk score, median (range) 1 (1–4) 1 (1–4) 2 (1–4) <0.001 1, no. (%) 655 (63.6) 600 (70.8) 55 (30.1) <0.001 2, no. (%) 258 (25.0) 185 (21.8) 73 (39.9) 3, no. (%) 106 (10.3) 58 (6.8) 48 (26.2) 4, no. (%) 11 (1.1) 4 (0.5) 7 (3.8) ATP III groups, no. (%) Low 366 (35.5) 353 (41.7) 13 (7.1) <0.001 Moderate 305 (29.6) 258 (30.5) 47 (25.7) Moderately high 156 (15.1) 97 (11.5) 59 (32.2) High 203 (19.7) 139 (16.4) 64 (35.0) Baseline cardiovascular medication use, no. 3. Results (%) Aspirin 190 (18.4) 132 (15.6) 58 (31.7) <0.001 ACEIs/ARBs 325 (31.6) 249 (29.4) 76 (41.5) 0.001 Beta blockers 164 (15.9) 118 (13.9) 46 (25.1) <0.001 Diuretics 182 (17.7) 142 (16.8) 40 (21.9) 0.101 Calcium channel blockers 139 (13.5) 97 (11.5) 42 (23.0) <0.001 Statins 240 (23.3) 167 (19.7) 73 (39.9) <0.001 obesity: OR 0.651, 95% CI 0.469, 0.905, 𝑃0.011). Multivari- able analysis was then evaluated by entering Framingham CAD risk score, history of stroke, and history of PAD into the model to explore confounding effect on each testing univariable predictor. All the testing univariable predictors did not only remain independent predictors for moderate- to-severe CAC compared to absent-to-mild CAC but also showed stronger relationship with greater magnitude in OR and 95% CI (body weight: OR 0.98, 95% CI 0.979,0.995, 𝑃 0.001; BSA: OR 0.582, 95% CI 0.369,0.920, 𝑃0.020; BMI: OR 0.945, 95% CI 0.920,0.971, 𝑃< 0.001; presence of obesity: OR 0.564, 95% CI 0.395,0.806, 𝑃0.002). 3.2. Body Morphology Indices and Obesity. Overall, median BMI was 29.2 kg/m2 with 25th and 75th percentile of 25.5 kg/m2 and 34.2 kg/m2, respectively. There were 223 (21.7%) normal weight patients, 336 (32.6%) overweight patients, and 471 (45.7%) obese patients. Other body mor- phology indices, differences in median BMI, and differences in mean height, mean weight, and mean BSA are shown in Table 2. Patients with moderate-to-severe CAC had sig- nificantly lower median body weight (𝑃0.005), BSA (𝑃 0.037), and BMI (𝑃0.001) than those with absent-to-mild CAC. Consistently with BMI, percentage of obesity in the moderate-to-severe CAC group was significantly lower than that in absent-to-mild CAC group (𝑃0.010). 3.2. Body Morphology Indices and Obesity. Overall, median BMI was 29.2 kg/m2 with 25th and 75th percentile of 25.5 kg/m2 and 34.2 kg/m2, respectively. There were 223 (21.7%) normal weight patients, 336 (32.6%) overweight patients, and 471 (45.7%) obese patients. Other body mor- phology indices, differences in median BMI, and differences in mean height, mean weight, and mean BSA are shown in Table 2. Patients with moderate-to-severe CAC had sig- nificantly lower median body weight (𝑃0.005), BSA (𝑃 0.037), and BMI (𝑃0.001) than those with absent-to-mild CAC. Consistently with BMI, percentage of obesity in the moderate-to-severe CAC group was significantly lower than that in absent-to-mild CAC group (𝑃0.010). Binary logistic regression analysis was used to examine these associations. Results are shown in Table 3. 3. Results Body weight, BSA, BMI values, and presence of obesity are inverse uni- variable predictors for moderate-to-severe CAC compared to absent-to-mild CAC (body weight: OR 0.989, 95% CI 0.982, 0.997, 𝑃0.005; BSA: OR 0.636, 95% CI 0.415, 0.974, 𝑃0.038; BMI: OR 0.956, 95% CI 0.933, 0.981, 𝑃0.001; presence of 4. Discussion Our final cohorts of 1030 patients under observation status for acute chest pain evaluation confirmed the concept of obesity paradox in this population. Other body morphology indices including body weight and BSA also showed inverse rela- tionship with moderate-to-severe CAC as well consistently ISRN Obesity 4 Table 2: Body morphology indices and obesity categorized by extent of CAC. Table 2: Body morphology indices and obesity categorized by extent of CAC. Body indices All patients (𝑁= 1030) Absent-to-mild CAC (𝑁= 847) Moderate-to-severe CAC (𝑁= 183) 𝑃value BMI, median (range) 29.2 (14.8–68.6) 29.4 (17.4–68.6) 27.5 (14.8–57.8) 0.001 Obesity, no. (%) 471 (45.7) 403 (47.6) 68 (37.2) 0.010 Body weight (kg) (SD) 86.4 (23.2) 87.4 (23.5) 82.1 (21.5) 0.005 BSA (m2) (SD) 2.73 (0.39) 2.74 (0.39) 2.67 (0.38) 0.037 Height (meters) (SD) 1.68 (0.11) 1.68 (0.11) 1.68 (0.11) 0.616 Table 3: Association between body morphology indices and moderate-to-severe CAC. Body indices Univariable analysis 𝑃value Multivariable analysis∗ 𝑃value BMI 0.956 (0.933, 0.981) 0.001 0.945 (0.920, 0.971) <0.001 Obesity 0.651 (0.469, 0.905) 0.011 0.564 (0.395, 0.806) 0.002 Body weight 0.989 (0.982, 0.997) 0.005 0.987 (0.979, 0.995) 0.001 BSA 0.636 (0.415, 0.974) 0.038 0.582 (0.369, 0.920) 0.020 ∗Adjusted for Framingham CAD risk score, and history of stroke, and history of PAD. Table 3: Association between body morphology indices and moderate-to-severe CAC. before by Lee et al. that BMI has a U-shaped relationship with CAC [29]. In their report, it was shown that, in young asymptomatic subjects without CAD, initial BMI on enroll- ment was positively associated with CAC at final follow-up in a linear fashion but BMI at follow-up on years 5, 10, and 15 became progressively U shape. BMI at the final follow-up at year 20 was inversely associated with CAC at final follow-up. Nevertheless, our findings in the present study are consistent with multiple prior coronary angiography-based studies [5, 7, 9–11, 30] which described a paradoxical relationship between CAC and body morphology, mainly BMI as well. with a recent coronary angiography-based study. Despite the fact that patients with moderate-to-severe CAC were older and had more history of hypertension, dyslipidemia, stroke, and PAD, body morphology indices including BMI were lower in this group. With adjusted logistic regression analysis, presence of obesity, BMI, body weight, and BSA remained independent predictors for moderate-to-severe CAC. In our study, we did not find significant differences in any body composition indices between patients with and without short-term cardiac events. 4. Discussion We chose to use Framingham CAD risk score for an adjustment as a surrogate for age, gender, history of hyper- tension, and history of dyslipidemia because, with a well- validated risk score as the Framingham CAD risk score, we aimed to minimize confounding effect and interaction between each other in these factors. We also added history of stroke and history of PAD to the adjusted analysis as they are not included in the Framingham risk score and the differ- ences in these baseline characteristics were also significant. We specifically chose moderate-to-severe CAC defined by CAC score of at least 100 as our cutoff for significant CAD as natural history of CAC is over time progressing as suggested by a recent study [19], so robustness of mild CAC in older age group in identifying clinical significant CAD might be arguable. CAC score of at least 100 has also been shown to be associated with significantly higher risk of increased cardiac events including CAD death, nonfatal myocardial infarction, and unstable angina pectoris requiring coronary revascularization [20, 21]. Our study extends the concept of obesity paradox with significant CT-derived CAC to symptomatic patients. The findings in this study support the fact that there is obesity paradox in various CAD populations including stable CAD [7], patients referred for exercise stress testing [11, 31], CAD with acute coronary syndrome requiring coronary angiog- raphy [8–10], and CAD requiring revascularization [11, 12]. Whether this concept is an unanswered phenomenon or just an artifact from heterogeneous composition of BMI is still controversial to date. Most studies that described obesity paradox used BMI as a surrogate for obesity. Given that BMI comprises body fat and fat-free mass, “obesity” defined by BMI can theoretically be actually muscle mass which could well explain why subjects with higher BMI in studies had better clinical outcomes or had lower CAC compared to sub- jects with lower BMI. In evidence, there have been multiple studies focused on cardiorespiratory fitness as it is one of the indices that possibly differentiate higher BMI secondary to body fat from higher BMI secondary to fat-free mass. It has been shown that, in subjects with higher cardiorespiratory fitness, there is no obesity paradox as opposed to subjects with lower cardiorespiratory fitness who demonstrated the paradox [32–34]. References [1] H. B. Hubert, M. Feinleib, P. M. McNamara, and W. P. Castelli, “Obesity as an independent risk factor for cardiovascular disease: a 26-year follow-up of participants in the Framingham Heart Study,” Circulation, vol. 67, no. 5, pp. 968–977, 1983. [2] G. Whitlock, S. Lewington, P. Sherliker et al., “Body-mass index and cause-specific mortality in 900 000 adults: collaborative analyses of 57 prospective studies,” The Lancet, vol. 373, no. 9669, pp. 1083–1096, 2009. [3] P. W. Wilson, R. B. D’Agostino, L. Sullivan, H. Parise, and W. B. Kannel, “Overweight and obesity as determinants of cardiovascular risk: the Framingham experience,” Archives of Internal Medicine, vol. 162, no. 16, pp. 1867–1872, 2002. [4] R. P. Bogers, W. J. E. Bemelmans, R. T. Hoogenveen et al., “Association of overweight with increased risk of coronary heart disease partly independent of blood pressure and cholesterol levels: a meta-analysis of 21 cohort studies including more than 300 000 persons,” Archives of Internal Medicine, vol. 167, no. 16, pp. 1720–1728, 2007. Summary The present study showed that, in patients with acute chest pain admitted for observation, obesity was an inverse inde- pendent predictor for coronary artery calcification detected by cardiac CT. The authors declare that there is no conflict of interests. The authors declare that there is no conflict of interests. What Is New? (i) Our study is a large-scaled study on symptomatic patients that demonstrated paradoxical association between cardiac CT-derived coronary artery calcifi- cation and obesity as well as other body morphology indices. i In contrast to BMI, BSA has not been studied extensively for associations with CAC or cardiac events. BSA is also a product of weight and height as BMI, although it is a product of multiplication as opposed to division in BMI which might not reflect obesity being as robust as BMI. The results of our study support a recent study by Roy et al. [40] which examined a relationship between BSA and CAC in patients who underwent cardiac CT for coronary artery evaluation. However, the relationship between BSA and CAC score was inverse in our study but positive in their study. They also demonstrated that BSA, but not BMI, is an independent predictor for presence of CAC.h (ii) Most of the previous cardiac CT-based studies were conducted on asymptomatic subjects and showed positive association between obesity and coronary artery calcification. ISRN Obesity The basis of this theory begins with the hypothesis that bigger body size necessitates bigger bony skeletal support. Bigger body size has been shown to be positively associated with higher bone mineralization [35]. In linking all these findings and the theory together, Broussard and Magnus proposed that the obesity paradox might be related to the vascular calcification paradox. This paradox seems to possibly involve Klotho gene which has been shown in mice and human to regulate bone mineralization and vascular calcification [36– 38]. The possibility of this theory has also been supported in patients who use bisphosphonate which demonstrated increased bone mass but decreased vascular calcification [39]. and significant CT-derived CAC. These findings extend the concept of obesity paradox to this population. However, bio- logical explanation(s) of this paradox remains unanswered. Further studies are needed to examine the mechanism of this concept. Novelty and Significance What Is Relevant? Obesity paradox in cardiovascular diseases including hyper- tension has been repeatedly described. Our study extends this concept to a different population with a different imaging modality. The limitation of this study is the single-center cross- sectional nature of examining association between body morphology and CAC. For all body morphology indices including BMI, we used a single measurement for each patient at the time of presentation to correlate with CAC. All the lack of association between body morphology indices and coronary calcium score in our study cannot reflect changes in these indices in temporal fashion. Also we did not use other body morphology measurements such as waist circumference, hip circumference, or waist-to-hip ratio which might show different results. Conflict of Interests gf Perspectives: in contrast to several previous CT-based studies examining the relationship between BMI and CAC which show positive association in asymptomatic subjects, our study is one of the first large-scale studies which demon- strated that the association is reversed in patients with acute chest pain. However, this reverse relationship between CAC and BMI was described before in symptomatic patients, though with CAC detected by angiography. This raises the possibility of obesity paradox with CAC detected by cardiac CT in this population as well as questions regarding robust- ness between the two imaging modalities in detecting CAC. Also there could possibly be differences in disease stages or biological mechanisms between asymptomatic subjects and symptomatic patients that could potentially explain this discrepancy. Further investigations are needed to confirm this paradoxical association and to find its biological expla- nation(s). 4. Discussion Other proposed biological mechanisms to explain inverse relationship between BMI and CAC in significant CAD involve another puzzle called “vascular calcification paradox” proposed by Kovacic et al. [30] which describes the phenomenon that increased bone mineral- ization is inversely associated with vascular calcification. In contrast to several prior CT-based studies which demonstrated positive relationship between BMI and CAC score [22–28], our study found that increased BMI was associated with lower CAC. However, most of the previous studies were conducted on asymptomatic subjects without significant CAD who were at low risk of developing CAD. 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https://openalex.org/W4322757531	https://www.biorxiv.org/content/biorxiv/early/2022/12/21/2022.12.21.521379.full.pdf	English	null	O-GlcNAc glycosylation orchestrates fate decision and niche function of bone marrow stromal progenitors	eLife	2,023	cc-by	20,714	. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint Mansky9, Hai-Bin 5 Ruan1,10,* 6 7 1Department of Integrative Biology and Physiology, University of Minnesota Medical School, 8 Minneapolis, MN 55455, USA 9 2Laboratory of Gastrointestinal Microbiology, Jiangsu Key Laboratory of Gastrointestinal Nutrition and 10 Animal Health, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 11 Jiangsu 210095, China 12 3National Center for International Research on Animal Gut Nutrition, Nanjing Agricultural University, 13 Nanjing, Jiangsu 210095, China 14 4Department of Medical Anatomical Sciences, College of Osteopathic Medicine of the Pacific, 15 Western University of Health Sciences, Pomona, CA 16 5Department of Biomedical Education and Anatomy, The Ohio State University College of 17 Medicine, and Division of Biosciences, The Ohio State University College of Dentistry, 18 Columbus, OH 43210, USA 19 6Department of Cell and Molecular Pharmacology & Experimental Therapeutics, Medical 20 University of South Carolina Charleston SC 29425 USA 21 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint O-GlcNAc glycosylation orchestrates fate decision and niche function of bone marrow 1 stromal progenitors 2 3 Zengdi Zhang1,11, Zan Huang1,2,3,11, Mohamed Awad4, Mohammed Elsalanty4, James Cray5, 4 Lauren E. Ball6, Jason C. Maynard7, Alma L. Burlingame7, Hu Zeng8, Kim C. Mansky9, Hai-Bin 5 Ruan1,10,* 6 7 1Department of Integrative Biology and Physiology, University of Minnesota Medical School, 8 Minneapolis, MN 55455, USA 9 2Laboratory of Gastrointestinal Microbiology, Jiangsu Key Laboratory of Gastrointestinal Nutrition and 10 Animal Health, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 11 Jiangsu 210095, China 12 3National Center for International Research on Animal Gut Nutrition, Nanjing Agricultural University, 13 Nanjing, Jiangsu 210095, China 14 4Department of Medical Anatomical Sciences, College of Osteopathic Medicine of the Pacific, 15 Western University of Health Sciences, Pomona, CA 16 5Department of Biomedical Education and Anatomy, The Ohio State University College of 17 Medicine, and Division of Biosciences, The Ohio State University College of Dentistry, 18 Columbus, OH 43210, USA 19 6Department of Cell and Molecular Pharmacology & Experimental Therapeutics, Medical 20 University of South Carolina, Charleston, SC 29425, USA 21 7Department of Pharmaceutical Chemistry, University of California, San Francisco, San 22 Francisco, California, USA 23 8Division of Rheumatology, Department of Internal Medicine, Mayo Clinic, Rochester, MN 55905, 24 USA 25 9Department of Developmental and Surgical Sciences, School of Dentistry, University of 26 Minnesota, Minneapolis, MN 55455, USA 27 10Center for Immunology, University of Minnesota Medical School, Minneapolis, MN 55455, USA 28 11These authors contributed equally 29 Correspondence: H.-B. R. (hruan@umn.edu) 30 31 O-GlcNAc glycosylation orchestrates fate decision and niche function of bone marrow 1 stromal progenitors 2 3 Zengdi Zhang1,11, Zan Huang1,2,3,11, Mohamed Awad4, Mohammed Elsalanty4, James Cray5, 4 Lauren E. Ball6, Jason C. Maynard7, Alma L. Burlingame7, Hu Zeng8, Kim C. O-GlcNAc glycosylation orchestrates fate decision and niche function of bone marrow 1 stromal progenitors 2 1Department of Integrative Biology and Physiology, University of Minnesota Medical School, 8 Minneapolis, MN 55455, USA 9 2Laboratory of Gastrointestinal Microbiology, Jiangsu Key Laboratory of Gastrointestinal Nutrition and 10 Animal Health, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 11 Jiangsu 210095, China 12 3National Center for International Research on Animal Gut Nutrition, Nanjing Agricultural University, 13 Nanjing, Jiangsu 210095, China 14 4Department of Medical Anatomical Sciences, College of Osteopathic Medicine of the Pacific, 15 Western University of Health Sciences, Pomona, CA 16 5Department of Biomedical Education and Anatomy, The Ohio State University College of 17 Medicine, and Division of Biosciences, The Ohio State University College of Dentistry, 18 Columbus, OH 43210, USA 19 6Department of Cell and Molecular Pharmacology & Experimental Therapeutics, Medical 20 University of South Carolina, Charleston, SC 29425, USA 21 6Department of Cell and Molecular Pharmacology & Experimental Therapeutics, Medical 20 University of South Carolina, Charleston, SC 29425, USA 21 7Department of Pharmaceutical Chemistry, University of California, San Francisco, San 22 Francisco, California, USA 23 8Division of Rheumatology, Department of Internal Medicine, Mayo Clinic, Rochester, MN 55905, 24 USA 25 5 9Department of Developmental and Surgical Sciences, School of Dentistry, University of 26 Minnesota, Minneapolis, MN 55455, USA 27 10Center for Immunology, University of Minnesota Medical School, Minneapolis, MN 55455, USA 28 11These authors contributed equally 29 Correspondence: H.-B. R. (hruan@umn.edu) 30 32 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint INTRODUCTION 51 For example, BMSC-derived 81 stem cell factor (SCF, encoded by the Kitl gene) and CXC chemokine ligand 12 (CXCL12) are 82 required for the maintenance and differentiation of hematopoietic stem/progenitor cells (HSPCs) 83 (Asada et al., 2017; Ding et al., 2012). A prominent subpopulation of perivascular BMSCs 84 express adipocyte markers (Dolgalev and Tikhonova, 2021; Zhong et al., 2020; Zhou et al., 85 2017), and support steady-state and metabolic-stressed myelopoiesis by secreting SCF (Zhang 86 et al., 2019). Meanwhile, osteolineage cells are crucial for lymphopoiesis (Wei and Frenette, 87 2018). Depleting Osx+ cells halts B cell maturation and causes immune failure (Yu et al., 2016). 88 IL-7, the most crucial factor for lymphoid progenitors, is expressed by a subset of BMSCs 89 (Fistonich et al., 2018). While it is well accepted that myeloid and lymphoid progenitors may 90 Mammalian bones support body structure, protect vital organs, and allow body movement. In 52 addition, they provide an environment for hematopoiesis in the bone marrow (BM). Most bones 53 in mammals are formed through endochondral ossification, which is initiated by mesenchymal 54 condensation, followed by the differentiation of chondrocytes and perichondrial progenitors 55 (Kobayashi and Kronenberg, 2021). Perichondrial progenitors expressing Osterix (Osx) co- 56 migrate with blood vessels into the primary ossification center, giving rise to osteoblasts and 57 transient stromal cells in the nascent BM cavity (Chen et al., 2014; Liu et al., 2013; Maes et al., 58 2010; Mizoguchi et al., 2014). At the perinatal stage, Osx+ progenitors contribute to osteo- 59 lineages and long-lived BM stromal cells (BMSCs) that exhibit trilineage differentiation potential 60 into osteocytes, chondrocytes, and adipocytes. 61 The decision of BMSC fate is controlled by a transcriptional network of pro-osteogenic and anti- 62 adipogenic transcription factors that pre-establishes osteogenic enhancers in BMSCs for rapid 63 bone formation (Rauch et al., 2019). RUNX family transcription factor 2 (RUNX2), by regulating 64 osteogenic genes including Osx, determines the osteoblast lineage from the multipotent BMSCs. 65 Mice with Runx2 mutations completely lack skeletal ossification and die of respiratory failure 66 (Komori et al., 1997). Runx2-haploinsufficient mice show specific skeletal abnormalities 67 characteristic of human cleidocranial dysplasia (CCD), including persistent fontanels, delayed 68 closure of cranial sutures, rudimentary clavicles, and dental abnormalities (Otto et al., 1997; 69 Takarada et al., 2016). SUMMARY In mammals, interactions between the bone marrow (BM) stroma and hematopoietic progenitors 35 contribute to bone-BM homeostasis. Perinatal bone growth and ossification provide a 36 microenvironment for the transition to definitive hematopoiesis; however, mechanisms and 37 interactions orchestrating the development of skeletal and hematopoietic systems remain 38 largely unknown. Here, we establish intracellular O-linked β-N-acetylglucosamine (O-GlcNAc) 39 modification as a posttranslational switch that dictates the differentiation fate and niche function 40 of early BM stromal cells (BMSCs). By modifying and activating RUNX2, O-GlcNAcylation 41 promotes osteogenic differentiation of BMSCs and stromal IL-7 expression to support 42 lymphopoiesis. In contrast, C/EBPβ-dependent marrow adipogenesis and expression of 43 myelopoietic stem cell factor (SCF) is inhibited by O-GlcNAcylation. Ablating O-GlcNAc 44 transferase (OGT) in BMSCs leads to impaired bone formation, increased marrow adiposity, as 45 well as defective B-cell lymphopoiesis and myeloid overproduction in mice. Thus, the balance of 46 osteogenic and adipogenic differentiation of BMSCs is determined by reciprocal O-GlcNAc 47 regulation of transcription factors, which simultaneously shapes the hematopoietic niche. 48 49 50 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint INTRODUCTION 51 INTRODUCTION 51 Mammalian bones support body structure, protect vital organs, and allow body movement. In 52 addition, they provide an environment for hematopoiesis in the bone marrow (BM). Most bones 53 in mammals are formed through endochondral ossification, which is initiated by mesenchymal 54 condensation, followed by the differentiation of chondrocytes and perichondrial progenitors 55 (Kobayashi and Kronenberg, 2021). Perichondrial progenitors expressing Osterix (Osx) co- 56 migrate with blood vessels into the primary ossification center, giving rise to osteoblasts and 57 transient stromal cells in the nascent BM cavity (Chen et al., 2014; Liu et al., 2013; Maes et al., 58 2010; Mizoguchi et al., 2014). At the perinatal stage, Osx+ progenitors contribute to osteo- 59 lineages and long-lived BM stromal cells (BMSCs) that exhibit trilineage differentiation potential 60 into osteocytes, chondrocytes, and adipocytes. 61 The decision of BMSC fate is controlled by a transcriptional network of pro-osteogenic and anti- 62 adipogenic transcription factors that pre-establishes osteogenic enhancers in BMSCs for rapid 63 bone formation (Rauch et al., 2019). RUNX family transcription factor 2 (RUNX2), by regulating 64 osteogenic genes including Osx, determines the osteoblast lineage from the multipotent BMSCs. 65 Mice with Runx2 mutations completely lack skeletal ossification and die of respiratory failure 66 (Komori et al., 1997). Runx2-haploinsufficient mice show specific skeletal abnormalities 67 characteristic of human cleidocranial dysplasia (CCD), including persistent fontanels, delayed 68 closure of cranial sutures, rudimentary clavicles, and dental abnormalities (Otto et al., 1997; 69 Takarada et al., 2016). On the other hand, adipogenesis is driven by downregulation of pro- 70 osteogenic factors, remodeling of the chromatin, and activation of adipogenic transcription 71 factors, such as C/EBPs and PPARγ (Rauch et al., 2019). BM adiposity is associated with bone 72 loss in osteoporosis caused by aging, menopause, and anorexia nervosa (Bethel et al., 2013; 73 Fazeli et al., 2013; Liu et al., 2015; Scheller et al., 2016). However, it is incompletely understood 74 how these distinct types of transcription factors act cooperatively to determine lineage 75 differentiation during neonatal skeletal development. 76 BMSCs and their lineage-differentiated progeny (e.g., osteoblasts and adipocytes) provide a 77 niche microenvironment for hematopoiesis (Bianco and Robey, 2015; Calvi and Link, 2015; 78 Morrison and Scadden, 2014; Wei and Frenette, 2018). Recent studies using single cell 79 technologies and lineage tracing experiments have started to unveil the complexity and 80 heterogeneity of niche cell types, niche factors, and their actions. INTRODUCTION 51 On the other hand, adipogenesis is driven by downregulation of pro- 70 osteogenic factors, remodeling of the chromatin, and activation of adipogenic transcription 71 factors, such as C/EBPs and PPARγ (Rauch et al., 2019). BM adiposity is associated with bone 72 loss in osteoporosis caused by aging, menopause, and anorexia nervosa (Bethel et al., 2013; 73 Fazeli et al., 2013; Liu et al., 2015; Scheller et al., 2016). However, it is incompletely understood 74 how these distinct types of transcription factors act cooperatively to determine lineage 75 differentiation during neonatal skeletal development. 76 BMSCs and their lineage-differentiated progeny (e.g., osteoblasts and adipocytes) provide a 77 niche microenvironment for hematopoiesis (Bianco and Robey, 2015; Calvi and Link, 2015; 78 Morrison and Scadden, 2014; Wei and Frenette, 2018). Recent studies using single cell 79 technologies and lineage tracing experiments have started to unveil the complexity and 80 heterogeneity of niche cell types, niche factors, and their actions. For example, BMSC-derived 81 stem cell factor (SCF, encoded by the Kitl gene) and CXC chemokine ligand 12 (CXCL12) are 82 required for the maintenance and differentiation of hematopoietic stem/progenitor cells (HSPCs) 83 (Asada et al., 2017; Ding et al., 2012). A prominent subpopulation of perivascular BMSCs 84 express adipocyte markers (Dolgalev and Tikhonova, 2021; Zhong et al., 2020; Zhou et al., 85 2017), and support steady-state and metabolic-stressed myelopoiesis by secreting SCF (Zhang 86 et al., 2019). Meanwhile, osteolineage cells are crucial for lymphopoiesis (Wei and Frenette, 87 2018). Depleting Osx+ cells halts B cell maturation and causes immune failure (Yu et al., 2016). 88 IL-7, the most crucial factor for lymphoid progenitors, is expressed by a subset of BMSCs 89 (Fistonich et al., 2018). While it is well accepted that myeloid and lymphoid progenitors may 90 reside in distinct BM niches, it is unclear how BMSC heterogeneity is established during early 91 development and whether cytokine expression is coordinated and controlled by the fate-defining 92 transcriptional network in BMSCs. 93 Post-translational modifications (PTMs), including phosphorylation, acetylation, and 94 ubiquitination, allow the precise regulation of stability, localization, and activity of BM 95 transcriptional factors, such as RUNX2 (Chen et al., 2021; Kim et al., 2020), C/EBPs (Wang et 96 al., 2022), and PPARγ (Brunmeir and Xu, 2018). It remains poorly defined how these 97 modifications are coordinated in a spatio-temporal manner to calibrate skeletal development. INTRODUCTION 51 Micro-CT scanning further showed tha 123 OsxΔOgt mice had reduced trabecular bone volume, bone volume to tissue volume ratio 124 trabecular thickness, and trabecular numbers in the distal femur (Figure 1H-L). OsxΔOgt mic 125 represent typical bone and dental defects (Figure 1—figure supplement 1) as observed 126 Runx2-haploinsufficient mice (Otto et al., 1997; Takarada et al., 2016), suggesting that O 127 GlcNAcylation might control RUNX2 function. 128 129 RUNX2 O-GlcNAcylation promotes osteogenesis. 130 To investigate how OGT controls osteogenic differentiation of BMSCs, we first isolated primar 131 BMSCs from control and OsxΔOgt mice and induced them into osteoblast cells. Alkalin 132 phosphatase staining revealed a reduction in mineralization of OsxΔOgt BMSCs (Figure 2A 133 Similarly, treating mesenchymal C3H10T1/2 cells with an OGT inhibitor, OSMI-1, reduce 134 mineralization (Figure 2B) and ablated calcium deposition (Figure 2C) after osteogen 135 differentiation. Parathyroid hormone (PTH) is a bone anabolic agent that requires RUNX2 136 dependent signaling (Krishnan et al., 2003). We found that PTH treatment of C3H10T1/2 cel 137 increased global protein O-GlcNAcylation (Figure 2D). The ability of PTH to activat 138 osteogenesis is completely abolished when OGT was inhibited by OSMI-1 (Figure 2E 139 Pharmacological activation of O-GlcNAcylation enhances RUNX2 activity and promote 140 osteogenic differentiation (Kim et al., 2007; Nagel and Ball, 2014). We mutated three known O 141 GlcNAc sites on RUNX2, Ser 32 and Ser 33 in the N-terminal transactivation domain and Se 142 371 in the proline/serine/threonine-rich domain (Figure 2F), to alanine (3A), and found mutan 143 RUNX2 possessed less O-GlcNAcylation (Figure 2G). O-GlcNAcase (OGA) inhibition b 144 Thi t G (TMG) i d O Gl NA l ti f ildt (WT) RUNX2 b t t h l 145 Acetylglucosamine (O-GlcNAc) moiety at serine or threonine residues, termed O-GlcNAcylation 100 (Hart et al., 2007; Ruan et al., 2013b; Yang and Qian, 2017). O-GlcNAc transferase (OGT), 101 using UDP-GlcNAc derived from the hexosamine biosynthetic pathway as the substrate, 102 controls diverse biological processes such as gene transcription, protein stability, and cell 103 signaling (Hanover et al., 2012; Ruan et al., 2014; Ruan et al., 2012; Ruan et al., 2013a). In cell 104 culture, O-GlcNAcylation promotes osteogenesis (Kim et al., 2007; Nagel and Ball, 2014) and 105 suppresses adipogenesis (Ji et al., 2012). However, the physiological relevance of O- 106 GlcNAcylation in skeletal development and remodeling has not been established. INTRODUCTION 51 Here, we 107 studied OGT in balancing osteogenic versus adipogenic programs and in controlling niche 108 function of BMSC in mice. The multifaceted role of protein O-GlcNAcylation is achieved through 109 reciprocal regulation of pro-osteogenic, pro-lymphopoietic RUNX2 and pro-adipogenic, pro- 110 myelopoietic C/EBPβ. 111 Loss of OGT in perinatal BMSCs leads to bone loss and marrow adiposity. To determine the in vivo role of protein O-GlcNAcylation in bone development, we deleted the X 5 Chromosome-located Ogt gene using the Osx-GFP:Cre mice (Figure 1A). Compared to Osx- 6 Cre+ only littermate controls, newborn OsxΔOgt mice showed no obvious change in long bone 7 formation (Figure 1B) but had a profound defect in the mineralization of flat bones of the 8 calvaria (Figure 1C), suggesting impaired intramembranous ossification during the prenatal 9 stage. 0 At 4-6 weeks of age, OsxΔOgt mice were modestly shorter than the controls (Figure 1D, E). 121 Histological analyses showed decreased bone volume and osteoblast number (Figure 1F) and 122 shortened growth plate (Figure 1G) in OsxΔOgt mice. Micro-CT scanning further showed that 123 OsxΔOgt mice had reduced trabecular bone volume, bone volume to tissue volume ratio, 124 trabecular thickness, and trabecular numbers in the distal femur (Figure 1H-L). OsxΔOgt mice 125 represent typical bone and dental defects (Figure 1—figure supplement 1) as observed in 126 Runx2-haploinsufficient mice (Otto et al., 1997; Takarada et al., 2016), suggesting that O- 127 GlcNAcylation might control RUNX2 function. 128 129 INTRODUCTION 51 98 Thousands of intracellular proteins are dynamically modified by a single O-linked N- 99 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint Acetylglucosamine (O-GlcNAc) moiety at serine or threonine residues, termed O-GlcNAcylatio 100 (Hart et al., 2007; Ruan et al., 2013b; Yang and Qian, 2017). O-GlcNAc transferase (OGT 101 using UDP-GlcNAc derived from the hexosamine biosynthetic pathway as the substrate 102 controls diverse biological processes such as gene transcription, protein stability, and ce 103 signaling (Hanover et al., 2012; Ruan et al., 2014; Ruan et al., 2012; Ruan et al., 2013a). In ce 104 culture, O-GlcNAcylation promotes osteogenesis (Kim et al., 2007; Nagel and Ball, 2014) an 105 suppresses adipogenesis (Ji et al., 2012). However, the physiological relevance of O 106 GlcNAcylation in skeletal development and remodeling has not been established. Here, w 107 studied OGT in balancing osteogenic versus adipogenic programs and in controlling nich 108 function of BMSC in mice. The multifaceted role of protein O-GlcNAcylation is achieved throug 109 reciprocal regulation of pro-osteogenic, pro-lymphopoietic RUNX2 and pro-adipogenic, pro 110 myelopoietic C/EBPβ. 111 112 RESULTS 113 Loss of OGT in perinatal BMSCs leads to bone loss and marrow adiposity. 114 To determine the in vivo role of protein O-GlcNAcylation in bone development, we deleted the 115 Chromosome-located Ogt gene using the Osx-GFP:Cre mice (Figure 1A). Compared to Osx 116 Cre+ only littermate controls, newborn OsxΔOgt mice showed no obvious change in long bon 117 formation (Figure 1B) but had a profound defect in the mineralization of flat bones of th 118 calvaria (Figure 1C), suggesting impaired intramembranous ossification during the prenata 119 stage. 120 At 4-6 weeks of age, OsxΔOgt mice were modestly shorter than the controls (Figure 1D, E 121 Histological analyses showed decreased bone volume and osteoblast number (Figure 1F) an 122 shortened growth plate (Figure 1G) in OsxΔOgt mice. RUNX2 O-GlcNAcylation promotes osteogenesis. OSMI-1 could still suppress of luciferase activity of RUNX2-3A (Figure 2H), suggesting additional, unidentified O-GlcNAc sites (Figure 2G), which requires future investigation. Nevertheless, when overexpressed in C3H10T1/2 cells, RUNX2-3A substantially lost the ability to induce osteogenic differentiation (Figure 2I) or RUNX2-target gene expression (Figure 2J). These data demonstrate that O-GlcNAcylation is essential for RUNX2 activity and osteogenesis. In adult mice, Osx expression is restricted to osteoblast precursors. We treated OsxΔOgt mice from pregnancy with doxycycline (Dox) and withdrew Dox at 10 weeks of age to induce Cre expression and OGT depletion only during adulthood (Figure 3A). Micro-CT showed that OsxΔOgt mice had reduced bone volume, trabecular thickness, and bone mineral density (Figure 3B-E). Together, these results support the functional indispensability of OGT in the committed osteolineage for adult trabecular bone remodeling. RUNX2 O-GlcNAcylation promotes osteogenesis. To investigate how OGT controls osteogenic differentiation of BMSCs, we first isolated primary 131 BMSCs from control and OsxΔOgt mice and induced them into osteoblast cells. Alkaline 132 phosphatase staining revealed a reduction in mineralization of OsxΔOgt BMSCs (Figure 2A). 133 Similarly, treating mesenchymal C3H10T1/2 cells with an OGT inhibitor, OSMI-1, reduced 134 mineralization (Figure 2B) and ablated calcium deposition (Figure 2C) after osteogenic 135 differentiation. Parathyroid hormone (PTH) is a bone anabolic agent that requires RUNX2- 136 dependent signaling (Krishnan et al., 2003). We found that PTH treatment of C3H10T1/2 cells 137 increased global protein O-GlcNAcylation (Figure 2D). The ability of PTH to activate 138 osteogenesis is completely abolished when OGT was inhibited by OSMI-1 (Figure 2E). 139 Pharmacological activation of O-GlcNAcylation enhances RUNX2 activity and promotes 140 osteogenic differentiation (Kim et al., 2007; Nagel and Ball, 2014). We mutated three known O- 141 GlcNAc sites on RUNX2, Ser 32 and Ser 33 in the N-terminal transactivation domain and Ser 142 371 in the proline/serine/threonine-rich domain (Figure 2F), to alanine (3A), and found mutant 143 RUNX2 possessed less O-GlcNAcylation (Figure 2G). O-GlcNAcase (OGA) inhibition by 144 Thiamet-G (TMG) increased O-GlcNAcylation of wildtype (WT) RUNX2, but to a much less 145 extent in the 3A mutant (Figure 2G). OGT inhibition by OSMI-1 or O-GlcNAc mutation both 146 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint impaired the transcriptional activity of RUNX2 on a luciferase reporter (Figure 2H). OSMI-1 could still suppress of luciferase activity of RUNX2-3A (Figure 2H), suggesting additional, unidentified O-GlcNAc sites (Figure 2G), which requires future investigation. Nevertheless, when overexpressed in C3H10T1/2 cells, RUNX2-3A substantially lost the ability to induce osteogenic differentiation (Figure 2I) or RUNX2-target gene expression (Figure 2J). These data demonstrate that O-GlcNAcylation is essential for RUNX2 activity and osteogenesis. impaired the transcriptional activity of RUNX2 on a luciferase reporter (Figure 2H). C/EBPβ O-GlcNAcylation inhibits the adipogenic specification of BMSCs. The osteogenic and adipogenic differentiation of BMSCs is generally considered mutually 161 exclusive (Ambrosi et al., 2017). Concomitant with bone loss, we observed a massive 162 accumulation of adipocytes in the bone marrow of OsxΔOgt mice, shown by hematoxylin & eosin 163 staining (Figure 4A) and immune-staining of the lipid droplet protein - perilipin (Figure 4B). 164 Flow cytometric analysis of BMSCs revealed that OsxΔOgt mice possessed more 165 PDGFRα+/VCAM1+ adipogenic progenitors than littermate controls (Figure 4C). To directly test 166 if OGT deficiency biases BMSC differentiation toward the adipogenic lineage, we first induced 167 the adipogenic differentiation of primary BMSCs and found increased lipid deposition in OsxΔOgt 168 mice (Figure 4D). Even under an osteogenic induction condition, adipo-lineage markers such 169 as Adipoq and Vcam1 were significantly upregulated by OGT deficiency (Figure 4E, F). 170 Furthermore, treating C3H10T1/2 mesenchymal cells with an OGA inhibitor TMG to increase 171 protein O-GlcNAcylation, was able to substantially reduce perilipin protein expression (Figure 172 4G). These data indicate that OGT inhibits the adipogenic program of BMSCs. 173 We went on to determine the O-GlcNAc targets of OGT in suppressing adipogenesis. As an 174 osteogenic regulator, RUNX2 also reciprocally suppresses the adipogenic program (Ahrends et 175 al., 2014). However, such suppression was not dependent on O-GlcNAcylation, because O- 176 GlcNAc-deficient RUNX2 displayed similar efficiency as the wildtype protein to reduce lipid 177 deposition and perilipin expression in differentiated C3H10T1/2 cells (Figure 4—figure 178 supplement 1). PPARγ1 is O-GlcNAcylated at T54 in the A/B activation domain (Ji et al., 2012), 179 corresponding to T84 in PPARγ2, the major isoform in adipocytes (Figure 4—figure 180 supplement 2A). Mutating T84 in PPARγ2 did not ablate the ability of the OGA inhibitor TMG to 181 suppress adipogenesis in C3H10T1/2 cells (data not shown), suggesting the existence of other 182 unidentified O-GlcNAc sites on PPARγ2 or other target proteins than PPARγ2. Through mass 183 spectrometry, we were able to map four additional O-GlcNAc sites on PPARγ2 (Figure 4— 184 figure supplement 2A and source data 1). Intriguingly, mutating these 4 sites or together with 185 T84 to alanine, render PPARγ2 incompetent to induce transcription and adipogenesis (Figure 186 4—figure supplement 2B, C). It suggests that PPARγ2 O-GlcNAcylation is essential for 187 adipocyte maturation, but likely does not mediate the anti-adipogenic effect of OGT in perinatal 188 BMSCs. OGT deficiency disrupts the BM niche. We sought to test if their expression is controlled by the same 227 transcriptional network determining BMSC fate. Adipogenic differentiation of mesenchymal 228 C3H10T1/2 cells concomitantly increased Kitl while decreased Il7 gene expression (Figure 6A, 229 B). Simultaneous treatment with the OGT inhibitor OSMI-I dampened Il7 expression before 230 differentiation but enhanced Kitl expression in differentiated adipocytes (Figure 6A, B). On the 231 other hand, osteogenic differentiation suppressed Kitl transcription, which could be further 232 inhibited by TMG that elevated global O-GlcNAcylation (Figure 6C). While Il7 mRNA levels 233 were not evidently affected by osteogenic differentiation, TMG stimulated its expression (Figure 234 6D). O-GlcNAcylation inhibits the adipogenesis specified by C/EBPβ but supports osteogenesis 235 determined by RUNX2. In concert, C/EBPβ overexpression in C3H10T1/2 cells activated Kitl 236 transcription and suppressed Il7 expression, which was further exacerbated by O-GlcNAc- 237 deficient C/EBPβ (Figure 6E, F). However, RUNX2 overexpression decreased Kitl mRNA levels 238 (Figure 6G). When compared to the wildtype, O-GlcNAc-defective RUNX2 was impaired in 239 inducing Il7 expression (Figure 6H). Collectively, these results reveal that protein O- 240 GlcNAcylation, by acting on BMSC lineage transcriptional factors, establishes a pro- 241 Skeletal development is concomitant with the establishment of definitive hematopoiesis in the 199 BM. To test if OGT deficiency affects the niche function of Osx1+ cells for B-cell lymphopoiesis, 200 we performed flow cytometry analyses of bone marrow of 4-week-old mice (Figure 5A, figure 201 supplement 1, and source data 1) (Hardy et al., 1991). No changes in the percentage of 202 lineage-Sca-1+Kit+ progenitor cells, common lymphoid progenitors (CLPs), Fraction A that 203 contains pre-pro-B cells were observed between control and OsxΔOgt mice (Figure 5B-D). While 204 frequencies of Fraction B and C pro-B, pre-B, and immature B in OsxΔOgt mice were drastically 205 reduced (Figure 5E-I), demonstrating a developmental blockage from pre-pro-B to pro-B cells. 206 In the peripheral blood, there was specific loss of CD19+B220+ B cells but not CD4+ or CD8+ T 207 cells (Figure 5J-L). B-cell dysfunction observed here was similar to the phenotype in mice when 208 all Osx+ cells were depleted (Yu et al., 2016) or IL-7 was deleted in BMSCs (Cordeiro Gomes et 209 al., 2016), indicating that O-GlcNAcylation is essential for the Osx+ lineage cells to establish a 210 niche environment for B-cell lymphopoiesis. OGT deficiency disrupts the BM niche. 211 BM adiposity is associated with myeloid overproduction in conditions including aging, irradiation 212 (Ho et al., 2019), osteopenia (Kajkenova et al., 1997), and obesity (Singer et al., 2014), 213 indicating the supportive function of marrow adipocytes on demand-adapted myelopoiesis. 214 Consistently with the increased BM adiposity found in OsxΔOgt mice, we also observed biased 215 HSPC differentiation toward the myeloid lineage, as shown by increased ratio of common 216 myeloid progenitor (CMP) to common lymphoid progenitors (CLPs) and ratio of granulocyte- 217 monocyte progenitors (GMP) to megakaryocyte-erythrocyte progenitors (MEP) in the BM 218 (Figure 5M, N). As a result, increased numbers of red blood cells and neutrophils were 219 observed in the blood of OsxΔOgt mice (Figure 5O, P). Together, these results demonstrate that 220 OGT deficiency in neonatal BMSCs establishes a BM environment that promotes myelopoiesis 221 and simultaneously impairs B cell development. 222 OGT deficiency disrupts the BM niche. OGT deficiency disrupts the BM niche. 198 Skeletal development is concomitant with the establishment of definitive hematopoiesis in the 199 BM. To test if OGT deficiency affects the niche function of Osx1+ cells for B-cell lymphopoiesis, 200 we performed flow cytometry analyses of bone marrow of 4-week-old mice (Figure 5A, figure 201 supplement 1, and source data 1) (Hardy et al., 1991). No changes in the percentage of 202 lineage-Sca-1+Kit+ progenitor cells, common lymphoid progenitors (CLPs), Fraction A that 203 contains pre-pro-B cells were observed between control and OsxΔOgt mice (Figure 5B-D). While 204 frequencies of Fraction B and C pro-B, pre-B, and immature B in OsxΔOgt mice were drastically 205 reduced (Figure 5E-I), demonstrating a developmental blockage from pre-pro-B to pro-B cells. 206 In the peripheral blood, there was specific loss of CD19+B220+ B cells but not CD4+ or CD8+ T 207 cells (Figure 5J-L). B-cell dysfunction observed here was similar to the phenotype in mice when 208 all Osx+ cells were depleted (Yu et al., 2016) or IL-7 was deleted in BMSCs (Cordeiro Gomes et 209 al., 2016), indicating that O-GlcNAcylation is essential for the Osx+ lineage cells to establish a 210 niche environment for B-cell lymphopoiesis. 211 BM adiposity is associated with myeloid overproduction in conditions including aging, irradiation 212 (Ho et al., 2019), osteopenia (Kajkenova et al., 1997), and obesity (Singer et al., 2014), 213 indicating the supportive function of marrow adipocytes on demand-adapted myelopoiesis. 214 Consistently with the increased BM adiposity found in OsxΔOgt mice, we also observed biased 215 HSPC differentiation toward the myeloid lineage, as shown by increased ratio of common 216 myeloid progenitor (CMP) to common lymphoid progenitors (CLPs) and ratio of granulocyte- 217 monocyte progenitors (GMP) to megakaryocyte-erythrocyte progenitors (MEP) in the BM 218 (Figure 5M, N). As a result, increased numbers of red blood cells and neutrophils were 219 observed in the blood of OsxΔOgt mice (Figure 5O, P). Together, these results demonstrate that 220 OGT deficiency in neonatal BMSCs establishes a BM environment that promotes myelopoiesis 221 and simultaneously impairs B cell development. 222 223 Transcriptional regulation of niche cytokines by RUNX2 and C/EBPβ O-GlcNAcylation. 224 BMSC-derived SCF (encoded by the Kitl gene) and IL-7 are required for the myeloid 225 differentiation and B-cell development, respectively (Asada et al., 2017; Cordeiro Gomes et al., 226 2016; Ding et al., 2012). reciproca 195 progenit 196 197 OGT de 198 Skeletal 199 BM. To t 200 we perfo 201 supplem 202 lineage-S 203 contains 204 frequenc 205 reduced 206 In the pe 207 cells (Fig 208 all Osx+ 209 al., 2016 210 niche en 211 BM adip 212 (Ho et 213 indicatin 214 Consiste 215 HSPC d 216 myeloid 217 monocyt 218 (Figure 219 observed 220 OGT de 221 and simu 222 223 Transcr 224 BMSC-d 225 different 226 2016; D 227 transcrip 228 C3H10T 229 B). Sim 230 different 231 other ha 232 inhibited 233 were not 234 6D). O-G 235 determin 236 transcrip 237 deficient 238 (Figure 239 inducing 240 GlcNAcy 241 reciprocally regulating RUNX2 and C/EBPβ, OGT is required for the proper allocation of skeletal 195 progenitors into osteogenic versus adipogenic lineages during development. 196 197 OGT deficiency disrupts the BM niche. 198 Skeletal development is concomitant with the establishment of definitive hematopoiesis in the 199 BM. To test if OGT deficiency affects the niche function of Osx1+ cells for B-cell lymphopoiesis, 200 we performed flow cytometry analyses of bone marrow of 4-week-old mice (Figure 5A, figure 201 supplement 1, and source data 1) (Hardy et al., 1991). No changes in the percentage of 202 lineage-Sca-1+Kit+ progenitor cells, common lymphoid progenitors (CLPs), Fraction A that 203 contains pre-pro-B cells were observed between control and OsxΔOgt mice (Figure 5B-D). While 204 frequencies of Fraction B and C pro-B, pre-B, and immature B in OsxΔOgt mice were drastically 205 reduced (Figure 5E-I), demonstrating a developmental blockage from pre-pro-B to pro-B cells. 206 In the peripheral blood, there was specific loss of CD19+B220+ B cells but not CD4+ or CD8+ T 207 cells (Figure 5J-L). B-cell dysfunction observed here was similar to the phenotype in mice when 208 all Osx+ cells were depleted (Yu et al., 2016) or IL-7 was deleted in BMSCs (Cordeiro Gomes et 209 al., 2016), indicating that O-GlcNAcylation is essential for the Osx+ lineage cells to establish a 210 niche environment for B-cell lymphopoiesis. 211 BM adiposity is associated with myeloid overproduction in conditions including aging, irradiation 212 (Ho et al., 2019), osteopenia (Kajkenova et al., 1997), and obesity (Singer et al., 2014), 213 indicating the supportive function of marrow adipocytes on demand-adapted myelopoiesis. 214 Consistently with the increased BM adiposity found in OsxΔOgt mice, we also observed biased 215 HSPC differentiation toward the myeloid lineage, as shown by increased ratio of common 216 myeloid progenitor (CMP) to common lymphoid progenitors (CLPs) and ratio of granulocyte- 217 monocyte progenitors (GMP) to megakaryocyte-erythrocyte progenitors (MEP) in the BM 218 (Figure 5M, N). As a result, increased numbers of red blood cells and neutrophils were 219 observed in the blood of OsxΔOgt mice (Figure 5O, P). Together, these results demonstrate that 220 OGT deficiency in neonatal BMSCs establishes a BM environment that promotes myelopoiesis 221 and simultaneously impairs B cell development. 222 223 Transcriptional regulation of niche cytokines by RUNX2 and C/EBPβ O-GlcNAcylation. reciproca 195 progenit 196 197 OGT de 198 Skeletal 199 BM. To t 200 we perfo 201 supplem 202 lineage-S 203 contains 204 frequenc 205 reduced 206 In the pe 207 cells (Fig 208 all Osx+ 209 al., 2016 210 niche en 211 BM adip 212 (Ho et 213 indicatin 214 Consiste 215 HSPC d 216 myeloid 217 monocyt 218 (Figure 219 observed 220 OGT de 221 and simu 222 223 Transcr 224 BMSC-d 225 different 226 2016; D 227 transcrip 228 C3H10T 229 B). Sim 230 different 231 other ha 232 inhibited 233 were not 234 6D). O-G 235 determin 236 transcrip 237 deficient 238 (Figure 239 inducing 240 GlcNAcy 241 224 BMSC-derived SCF (encoded by the Kitl gene) and IL-7 are required for the myeloid 225 differentiation and B-cell development, respectively (Asada et al., 2017; Cordeiro Gomes et al., 226 2016; Ding et al., 2012). We sought to test if their expression is controlled by the same 227 transcriptional network determining BMSC fate. Adipogenic differentiation of mesenchymal 228 C3H10T1/2 cells concomitantly increased Kitl while decreased Il7 gene expression (Figure 6A, 229 B). Simultaneous treatment with the OGT inhibitor OSMI-I dampened Il7 expression before 230 differentiation but enhanced Kitl expression in differentiated adipocytes (Figure 6A, B). On the 231 other hand, osteogenic differentiation suppressed Kitl transcription, which could be further 232 inhibited by TMG that elevated global O-GlcNAcylation (Figure 6C). While Il7 mRNA levels 233 were not evidently affected by osteogenic differentiation, TMG stimulated its expression (Figure 234 6D) O GlcNAcylation inhibits the adipogenesis specified by C/EBPβ but supports osteogenesis 235 reciprocally regulating RUNX2 and C/EBPβ, OGT is required for the proper allocation of skeletal 195 progenitors into osteogenic versus adipogenic lineages during development. 196 C/EBPβ O-GlcNAcylation inhibits the adipogenic specification of BMSCs. 189 We then looked to C/EBPβ, an early transcription factor that specifies the adipogenic fate of 190 BMSCs (Cao et al., 1991; Darlington et al., 1998). It has been reported that OGT modifies 191 C/EBPβ to inhibit its transcriptional activity (Li et al., 2009; Qian et al., 2018). As expected, 192 ablating O-GlcNAcylation of C/EBPβ (2A mutation) promotes adipogenic differentiation of 193 C3H10T1/2 cells (Figure 4H, I). Taken together, we concluded that, by O-GlcNAcylating and 194 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint anscriptional regulation of niche cytokines by RUNX2 and C/EBPβ O-GlcNAcy In the early embryo, Osx+ progenitors give rise to fetal bone tissues and 256 transient stromal cells that disappear in early postnatal life (Mizoguchi et al., 2014). Perinatally, 257 Osx+ progenitors contribute to osteolineage cells and long-lived perivascular BMSCs that can be 258 labeled by leptin receptor (Lepr) and adiponectin (Adipoq) (Zhong et al., 2020; Zhou et al., 259 2017). Recent evidence suggests that a significant portion of adult BMSCs and osteoblasts 260 originate from collagen II (Col2)- and aggrecan (Acan)-expressing chondrocytes (Ono et al., 261 2014). Due to the fact that the Osx-GFP:Cre targets osteoblasts, BMSCs, and a subset of 262 chondrocytes (Chen et al., 2014; Liu et al., 2013), the current study could not delineate the 263 exact developmental stages and the primary cellular compartments where OGT instructs bone 264 development. Nonetheless, our ex vivo experiments and adult-onset targeting of OGT in Osx+ 265 osteoblasts, together with prior published in vivo and in vitro evidence (Andres-Bergos et al., 266 2012; Nagel and Ball, 2014; Nagel et al., 2013), certainly reveal the indispensability of protein 267 O-GlcNAcylation for chondro-osteogenic differentiation. While this study primarily focused early 268 life bone development, it is warranted to further investigate the role of OGT in the transition to 269 appositional remodeling during adulthood (Shu et al., 2021) and in osteoporosis pathogenesis 270 during aging. Moreover, bone-forming skeletal stem cells (SSCs) are identified in other 271 anatomical regions of long bones, such as growth plate, periosteum, and endosteum (Ambrosi 272 et al., 2019). It remains undetermined whether O-GlcNAcylation is abundant in and controls the 273 development and function of these SSC populations. 274 Protein O-GlcNAcylation senses glucose availability (Hardiville and Hart, 2014; Ruan et al., 275 2012), hormonal cues (Ruan et al., 2014; Ruan et al., 2017; Whelan et al., 2008), cellular stress 276 (Martinez et al., 2017; Ruan et al., 2017), and immune signals (Chang et al., 2020; Liu et al., 277 2019; Zhao et al., 2020; Zhao et al., 2022) to maintain cellular and tissue homeostasis. 278 Osteogenic differentiation of mesenchymal cells induces global O-GlcNAc levels (Kim et al., 279 2007; Nagel and Ball, 2014); however, the upstream mechanistic regulators of osteoblastic O- 280 GlcNAcylation remain enigmatic. High glucose has been shown to promote O-GlcNAcylation 281 and osteogenic differentiation of cartilage endplate stem cells (Sun et al., 2019). anscriptional regulation of niche cytokines by RUNX2 and C/EBPβ O-GlcNAcy Recent evidence suggests that a significant portion of adult BMSCs and osteoblasts 260 originate from collagen II (Col2)- and aggrecan (Acan)-expressing chondrocytes (Ono et al., 261 2014). Due to the fact that the Osx-GFP:Cre targets osteoblasts, BMSCs, and a subset of 262 chondrocytes (Chen et al., 2014; Liu et al., 2013), the current study could not delineate the 263 exact developmental stages and the primary cellular compartments where OGT instructs bone 264 development. Nonetheless, our ex vivo experiments and adult-onset targeting of OGT in Osx+ 265 osteoblasts, together with prior published in vivo and in vitro evidence (Andres-Bergos et al., 266 2012; Nagel and Ball, 2014; Nagel et al., 2013), certainly reveal the indispensability of protein 267 O-GlcNAcylation for chondro-osteogenic differentiation. While this study primarily focused early 268 life bone development, it is warranted to further investigate the role of OGT in the transition to 269 appositional remodeling during adulthood (Shu et al., 2021) and in osteoporosis pathogenesis 270 during aging. Moreover, bone-forming skeletal stem cells (SSCs) are identified in other 271 anatomical regions of long bones, such as growth plate, periosteum, and endosteum (Ambrosi 272 et al., 2019). It remains undetermined whether O-GlcNAcylation is abundant in and controls the 273 development and function of these SSC populations. 274 Protein O-GlcNAcylation senses glucose availability (Hardiville and Hart, 2014; Ruan et al., 275 2012) hormonal cues (Ruan et al 2014; Ruan et al 2017; Whelan et al 2008) cellular stress 276 lymphopoietic niche during neonatal bone development and at the same time prevents the 242 myeloid-skewing, adipogenic BM environment. 243 Post-translational modification networks exist in the bone-BM organ to regulate its development 246 and remodeling. Given that definitive hematopoiesis is matured in perinatal BM, it is tempting to 247 hypothesize that the regulatory mechanisms guiding the development of bone also establish the 248 BM niche for hematopoiesis. However, experimental evidence has been largely lacking so far. 249 In the present study, we examined the vital role of the under-studied protein O-GlcNAcylation in 250 determining the osteogenic versus adipogenic fate specification of BMSCs and in balancing the 251 pro-lymphopoietic and pro-myelopoietic niche function of BMSCs. We showed that, by 252 modifying and reciprocally regulating RUNX2 and C/EBPβ, O-GlcNAc orchestrates the early 253 development of skeletal and hematopoietic systems (Figure 7). 254 Multiple temporally and spatially distinct types of progenitors contribute to bone development 255 and maintenance. anscriptional regulation of niche cytokines by RUNX2 and C/EBPβ O-GlcNAcy 274 Protein O-GlcNAcylation senses glucose availability (Hardiville and Hart, 2014; Ruan et al., 275 2012), hormonal cues (Ruan et al., 2014; Ruan et al., 2017; Whelan et al., 2008), cellular stress 276 (Martinez et al., 2017; Ruan et al., 2017), and immune signals (Chang et al., 2020; Liu et al., 277 2019; Zhao et al., 2020; Zhao et al., 2022) to maintain cellular and tissue homeostasis. 278 Osteogenic differentiation of mesenchymal cells induces global O-GlcNAc levels (Kim et al., 279 lymphopoietic niche during neonatal bone development and at the same time prevents the 242 myeloid-skewing, adipogenic BM environment. 243 244 DISCUSSION 245 Post-translational modification networks exist in the bone-BM organ to regulate its development 246 and remodeling. Given that definitive hematopoiesis is matured in perinatal BM, it is tempting to 247 hypothesize that the regulatory mechanisms guiding the development of bone also establish the 248 BM niche for hematopoiesis. However, experimental evidence has been largely lacking so far. 249 In the present study, we examined the vital role of the under-studied protein O-GlcNAcylation in 250 determining the osteogenic versus adipogenic fate specification of BMSCs and in balancing the 251 pro-lymphopoietic and pro-myelopoietic niche function of BMSCs. We showed that, by 252 modifying and reciprocally regulating RUNX2 and C/EBPβ, O-GlcNAc orchestrates the early 253 development of skeletal and hematopoietic systems (Figure 7). 254 Multiple temporally and spatially distinct types of progenitors contribute to bone development 255 and maintenance. In the early embryo, Osx+ progenitors give rise to fetal bone tissues and 256 transient stromal cells that disappear in early postnatal life (Mizoguchi et al., 2014). Perinatally, 257 Osx+ progenitors contribute to osteolineage cells and long-lived perivascular BMSCs that can be 258 labeled by leptin receptor (Lepr) and adiponectin (Adipoq) (Zhong et al., 2020; Zhou et al., 259 2017). Recent evidence suggests that a significant portion of adult BMSCs and osteoblasts 260 originate from collagen II (Col2)- and aggrecan (Acan)-expressing chondrocytes (Ono et al., 261 2014). Due to the fact that the Osx-GFP:Cre targets osteoblasts, BMSCs, and a subset of 262 chondrocytes (Chen et al., 2014; Liu et al., 2013), the current study could not delineate the 263 exact developmental stages and the primary cellular compartments where OGT instructs bone 264 development. anscriptional regulation of niche cytokines by RUNX2 and C/EBPβ O-GlcNAcy Nonetheless, our ex vivo experiments and adult-onset targeting of OGT in Osx+ 265 osteoblasts, together with prior published in vivo and in vitro evidence (Andres-Bergos et al., 266 2012; Nagel and Ball, 2014; Nagel et al., 2013), certainly reveal the indispensability of protein 267 O-GlcNAcylation for chondro-osteogenic differentiation. While this study primarily focused early 268 life bone development, it is warranted to further investigate the role of OGT in the transition to 269 appositional remodeling during adulthood (Shu et al., 2021) and in osteoporosis pathogenesis 270 during aging. Moreover, bone-forming skeletal stem cells (SSCs) are identified in other 271 anatomical regions of long bones, such as growth plate, periosteum, and endosteum (Ambrosi 272 et al., 2019). It remains undetermined whether O-GlcNAcylation is abundant in and controls the 273 development and function of these SSC populations. 274 P t i O Gl NA l ti l il bilit (H di ill d H t 2014 R l 275 lymphopoietic niche during neonatal bone development and at the same time prevents the 242 myeloid-skewing, adipogenic BM environment. 243 244 DISCUSSION 245 Post-translational modification networks exist in the bone-BM organ to regulate its development 246 and remodeling. Given that definitive hematopoiesis is matured in perinatal BM, it is tempting to 247 hypothesize that the regulatory mechanisms guiding the development of bone also establish the 248 BM niche for hematopoiesis. However, experimental evidence has been largely lacking so far. 249 In the present study, we examined the vital role of the under-studied protein O-GlcNAcylation in 250 determining the osteogenic versus adipogenic fate specification of BMSCs and in balancing the 251 pro-lymphopoietic and pro-myelopoietic niche function of BMSCs. We showed that, by 252 modifying and reciprocally regulating RUNX2 and C/EBPβ, O-GlcNAc orchestrates the early 253 development of skeletal and hematopoietic systems (Figure 7). 254 Multiple temporally and spatially distinct types of progenitors contribute to bone development 255 and maintenance. In the early embryo, Osx+ progenitors give rise to fetal bone tissues and 256 transient stromal cells that disappear in early postnatal life (Mizoguchi et al., 2014). Perinatally, 257 Osx+ progenitors contribute to osteolineage cells and long-lived perivascular BMSCs that can be 258 labeled by leptin receptor (Lepr) and adiponectin (Adipoq) (Zhong et al., 2020; Zhou et al., 259 2017). anscriptional regulation of niche cytokines by RUNX2 and C/EBPβ O-GlcNAcy BMSC-derived SCF (encoded by the Kitl gene) and IL-7 are required for the myeloid 225 differentiation and B-cell development, respectively (Asada et al., 2017; Cordeiro Gomes et al., 226 2016; Ding et al., 2012). We sought to test if their expression is controlled by the same 227 transcriptional network determining BMSC fate. Adipogenic differentiation of mesenchymal 228 C3H10T1/2 cells concomitantly increased Kitl while decreased Il7 gene expression (Figure 6A, 229 B). Simultaneous treatment with the OGT inhibitor OSMI-I dampened Il7 expression before 230 differentiation but enhanced Kitl expression in differentiated adipocytes (Figure 6A, B). On the 231 other hand, osteogenic differentiation suppressed Kitl transcription, which could be further 232 inhibited by TMG that elevated global O-GlcNAcylation (Figure 6C). While Il7 mRNA levels 233 were not evidently affected by osteogenic differentiation, TMG stimulated its expression (Figure 234 6D). O-GlcNAcylation inhibits the adipogenesis specified by C/EBPβ but supports osteogenesis 235 determined by RUNX2. In concert, C/EBPβ overexpression in C3H10T1/2 cells activated Kitl 236 transcription and suppressed Il7 expression, which was further exacerbated by O-GlcNAc- 237 deficient C/EBPβ (Figure 6E, F). However, RUNX2 overexpression decreased Kitl mRNA levels 238 (Figure 6G). When compared to the wildtype, O-GlcNAc-defective RUNX2 was impaired in 239 inducing Il7 expression (Figure 6H). Collectively, these results reveal that protein O- 240 GlcNAcylation, by acting on BMSC lineage transcriptional factors, establishes a pro- 241 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint lymphopoietic niche during neonatal bone development and at the same time prevents the 242 myeloid-skewing, adipogenic BM environment. 243 244 DISCUSSION 245 Post-translational modification networks exist in the bone-BM organ to regulate its development 246 and remodeling. Given that definitive hematopoiesis is matured in perinatal BM, it is tempting to 247 hypothesize that the regulatory mechanisms guiding the development of bone also establish the 248 BM niche for hematopoiesis. anscriptional regulation of niche cytokines by RUNX2 and C/EBPβ O-GlcNAcy However, experimental evidence has been largely lacking so far. 249 In the present study, we examined the vital role of the under-studied protein O-GlcNAcylation in 250 determining the osteogenic versus adipogenic fate specification of BMSCs and in balancing the 251 pro-lymphopoietic and pro-myelopoietic niche function of BMSCs. We showed that, by 252 modifying and reciprocally regulating RUNX2 and C/EBPβ, O-GlcNAc orchestrates the early 253 development of skeletal and hematopoietic systems (Figure 7). 254 Multiple temporally and spatially distinct types of progenitors contribute to bone development 255 and maintenance. In the early embryo, Osx+ progenitors give rise to fetal bone tissues and 256 transient stromal cells that disappear in early postnatal life (Mizoguchi et al., 2014). Perinatally, 257 Osx+ progenitors contribute to osteolineage cells and long-lived perivascular BMSCs that can be 258 labeled by leptin receptor (Lepr) and adiponectin (Adipoq) (Zhong et al., 2020; Zhou et al., 259 2017). Recent evidence suggests that a significant portion of adult BMSCs and osteoblasts 260 originate from collagen II (Col2)- and aggrecan (Acan)-expressing chondrocytes (Ono et al., 261 2014). Due to the fact that the Osx-GFP:Cre targets osteoblasts, BMSCs, and a subset of 262 chondrocytes (Chen et al., 2014; Liu et al., 2013), the current study could not delineate the 263 exact developmental stages and the primary cellular compartments where OGT instructs bone 264 development. Nonetheless, our ex vivo experiments and adult-onset targeting of OGT in Osx+ 265 osteoblasts, together with prior published in vivo and in vitro evidence (Andres-Bergos et al., 266 2012; Nagel and Ball, 2014; Nagel et al., 2013), certainly reveal the indispensability of protein 267 O-GlcNAcylation for chondro-osteogenic differentiation. While this study primarily focused early 268 life bone development, it is warranted to further investigate the role of OGT in the transition to 269 appositional remodeling during adulthood (Shu et al., 2021) and in osteoporosis pathogenesis 270 during aging. Moreover, bone-forming skeletal stem cells (SSCs) are identified in other 271 anatomical regions of long bones, such as growth plate, periosteum, and endosteum (Ambrosi 272 et al., 2019). It remains undetermined whether O-GlcNAcylation is abundant in and controls the 273 development and function of these SSC populations. anscriptional regulation of niche cytokines by RUNX2 and C/EBPβ O-GlcNAcy During aging, the parallel dysfunction of the skeletal and hematopoietic systems 305 leads to osteoporosis, marrow fat accumulation, and myeloid hematopoietic skewing (Geiger et 306 al., 2013). Whether BMSC aging is associated with O-GlcNAc decline and whether the balance 307 between RUNX and C/EBPβ leads to bone-fat imbalances and niche dysfunction require future 308 investigations. 309 310 METHODS 311 Animals 312 All animal experiments were approved by the institutional animal care and use committee of the 313 University of Minnesota. All the mice were group-housed in light/dark cycle- (6am-8pm light), 314 temperature- (21.5 ± 1.5 oC), and humidity-controlled (30-70%) room, and had free access to 315 water and regular chow (Teklad #2018) unless otherwise indicated. All mice were maintained on 316 a C57BL6 background. Due to the X-chromosome localization of the Ogt gene, only male mice 317 were used in the study if not specified in the text or figures. To suppress Cre activity, designated 318 breeders were fed a diet containing 200 mg/kg doxycycline (Bio-serv, S3888). 319 BMSC isolation, culture, and differentiation 320 BMSC were isolated from the long bones as described previously (Zhu et al., 2010). The 321 fragments of long bones were digested with collagenase II for 30 mins. The released cells were 322 discarded, and the digested bone fragments were cultivated in the BMSCs growth medium 323 (alpha-MEM supplemented with 10% FBS). Once confluent, cells were switched to either 324 adipogenic differentiation medium (alpha-MEM supplemented with 20% FBS, 500 µM IBMX, 1 325 µM Dexamethasone, 10 µg/ml Insulin and 1 µM Rosiglitazone) for the first 2 days. The medium 326 was then changed to adipocyte differentiation base medium (α-MEM supplemented with 20% 327 FBS, 10 µg/ml Insulin and 1 µM Rosiglitazone) for the next 4 days followed by oil red O staining. 328 For osteogenic differentiation, cells were induced with osteoblast differentiation medium (α-MEM 329 supplemented with 10% FBS, 0.3mM ascorbic acid, 10 mM β-glycerophosphate, 0.1 µM 330 Dexamethasone) for 14 days followed by ALP staining or for 28 days followed by Alizarin red 331 staining. 332 Cell culture, plasmids, and lentiviruses 333 HEK 293, COS7, and C3H10T1/2 (ATCC, CCL-226) cells were cultured with DMEM plus 10% 334 of FBS. The mouse RUNX2-Myc/DDK plasmid was purchased from OriGene (MR227321), then 335 inositol trisphosphate (IP3) and diacylglycerol (DAG), which further increase intracellular Ca2+ and PKC, respectively (Datta and Abou-Samra, 2009). anscriptional regulation of niche cytokines by RUNX2 and C/EBPβ O-GlcNAcy Future experiments are required to determine if OGT enzymatic activity can be regulated by these signaling nodes, for example Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Ruan et al., 2017). inositol trisphosphate (IP3) and diacylglycerol (DAG), which further increase intracellular Ca2+ and PKC, respectively (Datta and Abou-Samra, 2009). Future experiments are required to determine if OGT enzymatic activity can be regulated by these signaling nodes, for example Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Ruan et al., 2017). The BM microenvironment, composed of BMSCs, osteoblasts, adipocytes, sympathetic nerves, 294 and vascular endothelial cells, has been highlighted as an important extrinsic factor for the 295 maintenance and differentiation of distinct hematopoietic lineage progenitors (Bianco and Robey, 296 2015; Calvi and Link, 2015; Morrison and Scadden, 2014; Wei and Frenette, 2018). While the 297 concomitant development, remodeling, and aging of the skeletal and hematopoietic systems 298 have been observed in various pathophysiological conditions, mechanisms underlying the 299 coordinated regulation of the two systems are less understood. Our current study has provided 300 the first evidence that RUNX2, permitted by O-GlcNAcylation, not only is indispensable for the 301 osteoblast development, but also establishes the endosteal niche for B lymphocytes by driving 302 IL-7 expression (Figure 7). When OGT is deficient, the perivascular BMSCs are prone to 303 adipogenic differentiation, which also activates C/EBPβ-dependent SCF expression and 304 myelopoiesis. During aging, the parallel dysfunction of the skeletal and hematopoietic systems 305 leads to osteoporosis, marrow fat accumulation, and myeloid hematopoietic skewing (Geiger et 306 al., 2013). Whether BMSC aging is associated with O-GlcNAc decline and whether the balance 307 between RUNX and C/EBPβ leads to bone-fat imbalances and niche dysfunction require future 308 investigations. 309 All animal experiments were approved by the institutional animal care and use committee of the University of Minnesota. All the mice were group-housed in light/dark cycle- (6am-8pm light), temperature- (21.5 ± 1.5 oC), and humidity-controlled (30-70%) room, and had free access to water and regular chow (Teklad #2018) unless otherwise indicated. All mice were maintained on a C57BL6 background. Due to the X-chromosome localization of the Ogt gene, only male mice were used in the study if not specified in the text or figures. To suppress Cre activity, designated breeders were fed a diet containing 200 mg/kg doxycycline (Bio-serv, S3888). anscriptional regulation of niche cytokines by RUNX2 and C/EBPβ O-GlcNAcy BMSCs 282 preferentially use glycolysis for bioenergetics to support their self-renewal and multipotency (Ito 283 and Suda, 2014; van Gastel and Carmeliet, 2021). Active aerobic glycolysis also fuels the high 284 anabolic demand during bone formation. It would be important in the future to determine 285 whether flux of the hexosamine biosynthetic pathway, a branch of glycolysis (Ruan et al., 286 2013b), increases to provide more UDP-GlcNAc for O-GlcNAc modification. We also showed 287 here that PTH treatment increased protein O-GlcNAcylation. Signaling through the PTH 288 receptor activates the cAMP-protein kinase A (PKA)-CREB pathway and the accumulation of 289 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint inositol trisphosphate (IP3) and diacylglycerol (DAG), which further increase intracellular Ca2+ 290 and PKC, respectively (Datta and Abou-Samra, 2009). Future experiments are required to 291 determine if OGT enzymatic activity can be regulated by these signaling nodes, for example 292 Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Ruan et al., 2017). 293 The BM microenvironment, composed of BMSCs, osteoblasts, adipocytes, sympathetic nerves, 294 and vascular endothelial cells, has been highlighted as an important extrinsic factor for the 295 maintenance and differentiation of distinct hematopoietic lineage progenitors (Bianco and Robey, 296 2015; Calvi and Link, 2015; Morrison and Scadden, 2014; Wei and Frenette, 2018). While the 297 concomitant development, remodeling, and aging of the skeletal and hematopoietic systems 298 have been observed in various pathophysiological conditions, mechanisms underlying the 299 coordinated regulation of the two systems are less understood. Our current study has provided 300 the first evidence that RUNX2, permitted by O-GlcNAcylation, not only is indispensable for the 301 osteoblast development, but also establishes the endosteal niche for B lymphocytes by driving 302 IL-7 expression (Figure 7). When OGT is deficient, the perivascular BMSCs are prone to 303 adipogenic differentiation, which also activates C/EBPβ-dependent SCF expression and 304 myelopoiesis. BMSC isolation, culture, and differentiation Reconstructions for X-ray projections and re-alignment were 372 performed using the Skyscan software (NRecon and DataViewer) (v. 1.7.3.1, Brüker micro-CT, 373 Kontich, Belgium). Ring artefact and beam hardening corrections were applied in reconstruction. 374 Datasets were loaded into SkyScan CT-Analyzer software for measurement of BMD. Calibration 375 was performed with 0.25- and 0.75- mg/mL hydroxyapatite mice phantoms provided by 376 SkyScan. For cancellous and cortical bone analysis, the scanning regions were confined to the 377 distal meta-physis, 100 slices starting at 0.5 mm proximally from the proximal tip of the primary 378 spongiosa for the cancellous portion and 100 slices starting at 4.5 mm proximally from the 379 center of intercondylar fossa for the cortical portion. 380 O-GlcNAc mass spectrometry 381 subcloned into pLV-EF1a-IRES-Hygro (Addgene #85134). Mouse PPARλ2 with a N-terminal 336 MYC tag was subcloned into pLVX-Dsred-puro plasmid. C/EBPβ plasmids were kindly provided 337 by Dr. Xiaoyong Yang at Yale University and then subcloned into pCDH-CMV-P2a-Puro. O- 338 GlcNAc site were mutated into alanine with Q5® Site-Directed Mutagenesis Kit (NEB#E0554). 339 Lentivirus were packed as previously described (Huang et al., 2022). Briefly, 293FT cells were 340 transfected with over-expression plasmids and pSPAX2 / pMD2G. Mediums with lentivirus were 341 filtered and added into C3H10T1/2 cells. 72h after infection, cells were selected with drugs 342 according to the resistance genes they possessed. 343 For Runx2 luciferase assay, with empty or RUNX2 vectors were transfected into COS7 cells 345 with Lipofectamine, together with 6xOSE2-luc and pGL4-hRluc vectors in which either firefly or 346 Renilla luciferase genes were expressed under the control of the RUNX2-specific or the 347 constitutive SV40 promoter, respectively. After 6 h cells were washed three times and with the 348 addition of 50 uM OSMI-1. Cells were incubated for an additional 48 h in growth medium 349 containing 5% serum. Luminescent signals were generated using the Dual-Luciferase Assay 350 System (Promega). Relative light units (RLU) for the 6xOSE2 reporter were normalized against 351 pGL4-hRLuc values as an internal control for transfection efficiency. For PPARγ2 luciferase 352 assays, C3H10T1/2 cells were transfected with Transporter 5® Transfection Reagent 353 (Polysciences) following manufacture’s protocol. PPARγ2 transcriptional activity was 354 determined using the PPREx3-TK-luc reporter (Addgene, #1015). 355 Bone tissues were fixed in formalin solution at 4 oC for 24 h. BMSC isolation, culture, and differentiation BMSC were isolated from the long bones as described previously (Zhu et al., 2010). The 321 fragments of long bones were digested with collagenase II for 30 mins. The released cells were 322 discarded, and the digested bone fragments were cultivated in the BMSCs growth medium 323 (alpha-MEM supplemented with 10% FBS). Once confluent, cells were switched to either 324 adipogenic differentiation medium (alpha-MEM supplemented with 20% FBS, 500 µM IBMX, 1 325 µM Dexamethasone, 10 µg/ml Insulin and 1 µM Rosiglitazone) for the first 2 days. The medium 326 was then changed to adipocyte differentiation base medium (α-MEM supplemented with 20% 327 FBS, 10 µg/ml Insulin and 1 µM Rosiglitazone) for the next 4 days followed by oil red O staining. 328 For osteogenic differentiation, cells were induced with osteoblast differentiation medium (α-MEM 329 supplemented with 10% FBS, 0.3mM ascorbic acid, 10 mM β-glycerophosphate, 0.1 µM 330 Dexamethasone) for 14 days followed by ALP staining or for 28 days followed by Alizarin red 331 staining. 332 HEK 293, COS7, and C3H10T1/2 (ATCC, CCL-226) cells were cultured with DMEM plus 10% 334 of FBS. The mouse RUNX2-Myc/DDK plasmid was purchased from OriGene (MR227321), then 335 HEK 293, COS7, and C3H10T1/2 (ATCC, CCL-226) cells were cultured with DMEM plus 10% 334 of FBS. The mouse RUNX2-Myc/DDK plasmid was purchased from OriGene (MR227321), then 335 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint subcloned into pLV-EF1a-IRES-Hygro (Addgene #85134). Mouse PPARλ2 with a N-terminal 336 MYC tag was subcloned into pLVX-Dsred-puro plasmid. C/EBPβ plasmids were kindly provided 337 by Dr. Xiaoyong Yang at Yale University and then subcloned into pCDH-CMV-P2a-Puro. O- 338 GlcNAc site were mutated into alanine with Q5® Site-Directed Mutagenesis Kit (NEB#E0554). 339 Lentivirus were packed as previously described (Huang et al., 2022). Briefly, 293FT cells were 340 transfected with over-expression plasmids and pSPAX2 / pMD2G. BMSC isolation, culture, and differentiation Mediums with lentivirus were 341 filtered and added into C3H10T1/2 cells. 72h after infection, cells were selected with drugs 342 according to the resistance genes they possessed. 343 Luciferase assay 344 For Runx2 luciferase assay, with empty or RUNX2 vectors were transfected into COS7 cells 345 with Lipofectamine, together with 6xOSE2-luc and pGL4-hRluc vectors in which either firefly or 346 Renilla luciferase genes were expressed under the control of the RUNX2-specific or the 347 constitutive SV40 promoter, respectively. After 6 h cells were washed three times and with the 348 addition of 50 uM OSMI-1. Cells were incubated for an additional 48 h in growth medium 349 containing 5% serum. Luminescent signals were generated using the Dual-Luciferase Assay 350 System (Promega). Relative light units (RLU) for the 6xOSE2 reporter were normalized against 351 pGL4-hRLuc values as an internal control for transfection efficiency. For PPARγ2 luciferase 352 assays, C3H10T1/2 cells were transfected with Transporter 5® Transfection Reagent 353 (Polysciences) following manufacture’s protocol. PPARγ2 transcriptional activity was 354 determined using the PPREx3-TK-luc reporter (Addgene, #1015). 355 Histology 356 Bone tissues were fixed in formalin solution at 4 oC for 24 h. Tissue embedding, sectioning, and 357 hematoxylin and eosin staining were performed at the Comparative Pathology Shared Resource 358 of the University of Minnesota. For immunostaining, the tissues were embedded in OCT then 359 cut into 7-µm slides. After three times of PBS wash, the slides were incubated with blocking 360 buffer (3% BSA in PBS) for 1 h, then immersed with anti-Perilipin (Cell Signaling Technology, 361 #9349) antibody overnight at 4oC. For immunofluorescence, PBS-washed slides were incubated 362 with a fluorescent secondary antibody at room temperature for 1 h, and then mounted with 363 VECTASHIELD® Antifade Mounting Medium with DAPI after three times of PBS wash. A Nikon 364 system was used for imaging. Goldner’s trichrome and Safranin O staining were performed at 365 Servicebio, China. 366 micro-CT 367 The samples were scanned with an in vitro micro-CT device (Skyscan 1272, Bruker micro-CT) 368 with scanning parameters of: Source Voltage = 60 kV, Source Current = 166uA, exposure 897 369 ms/frame, average of 3 frames per projection, Rotation Step (deg) = 0.200- and 0.25-mm 370 Aluminum filter. The specimens were scanned at high resolution (2016 × 1344 pixels) with an 371 Isotropic voxel size of 7.1 μm. BMSC isolation, culture, and differentiation It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint Myc-tagged PPARγ2 was co-transfected with OGT into 15 cm-dishes of 293T cells and purified 382 by immunoprecipitation with anti-c-Myc agarose beads (Pierce), followed by PAGE gel 383 electrophoresis. The corresponding PPARγ2 band was cut for in gel Trypsin (Promega) 384 digestion. Tryptic peptides were analyzed by on-line LC-MS/MS using an Orbitrap Fusion 385 Lumos (Thermo) coupled with a NanoAcquity UPLC system (Waters) as we previously reported 386 (Liu et al., 2019; Zhao et al., 2022). Peaklists were generated using PAVA (UCSF) and 387 searched using Protein Prospector 5.23.0 against the SwissProt database and a randomized 388 concatenated database with the addition of the recombinant PPARγ2 sequence. HexNAcylated 389 peptides were manually verified. 390 Real-time RT-PCR 391 RNA was isolated with Trizol and reverse transcribed into cDNA with the iScript™ cDNA 392 Synthesis Kit. Real-time RT-PCR was performed using iTaq™ Universal SYBR® Green 393 Supermix and gene-specific primers (Figure 6 —source data 1) on a Bio-Rad C1000 Thermal 394 Cycler. 395 Flow cytometry 396 For B-cell lymphopoiesis, BM cells were stained in PBS containing 1% (w/v) bovine serum 397 albumin on ice for 30 min, with anti-B220 (Life Technologies, 67-0452-82), anti-CD43 (BD 398 Biosciences, 553271), anti-CD24 (Biolegend, 101822), anti-Ly-51 (Biolegend, 108305), anti- 399 CD127 (Tonbo Biosciences, 20-1271-U100), anti-CD25 (Life Technologies, 63-0251-82) and 400 anti-CD19 (Biolegend, 115545). For hematopoietic stem and progenitor cells, BM cells were 401 stained with a cocktail of biotin-conjugated lineage antibodies CD3e, B220, Ter119, Mac-1 and 402 Gr-1 (Biolegend, 133307), CD4 (Biolegend, 100403), CD5 (Biolegend, 100603), CD8 403 (Biolegend, 100703), followed by Streptavidin-AF488 (Biolegend, 405235). Cells were then 404 stained with CD127-APC (eBioscience, 17-1271-82), c-Kit-APC-eFluor780 (eBioscience, 47- 405 1171-82), Sca-1-Super Bright 436 (eBioscience, 62-5981-82), CD34-PE (Biolegend, 152204) 406 and FcγR-PerCP-eFluor710 (eBioscience, 46-0161-80), CD150-BV605 (Biolegend, 115927), 407 and CD48-BUV395 (BDBioscience, 740236). Fixable Viability Dye was used to exclude dead 408 cells as instructed by the manufacturer. A complete list of used antibodies was shown in Figure 409 5 —source data 1. Flow cytometry was performed on an LSR Fortessa H0081 or X20 and 410 analyzed with FlowJo. 411 Myc-tagged PPARγ2 was co-transfected with OGT into 15 cm-dishes of 293T cells and purified 382 by immunoprecipitation with anti-c-Myc agarose beads (Pierce), followed by PAGE gel 383 electrophoresis. The corresponding PPARγ2 band was cut for in gel Trypsin (Promega) 384 digestion. BMSC isolation, culture, and differentiation Tryptic peptides were analyzed by on-line LC-MS/MS using an Orbitrap Fusion 385 Lumos (Thermo) coupled with a NanoAcquity UPLC system (Waters) as we previously reported 386 (Liu et al., 2019; Zhao et al., 2022). Peaklists were generated using PAVA (UCSF) and 387 searched using Protein Prospector 5.23.0 against the SwissProt database and a randomized 388 concatenated database with the addition of the recombinant PPARγ2 sequence. HexNAcylated 389 peptides were manually verified. 390 For B-cell lymphopoiesis, BM cells were stained in PBS containing 1% (w/v) bovine serum 397 albumin on ice for 30 min, with anti-B220 (Life Technologies, 67-0452-82), anti-CD43 (BD 398 Biosciences, 553271), anti-CD24 (Biolegend, 101822), anti-Ly-51 (Biolegend, 108305), anti- 399 CD127 (Tonbo Biosciences, 20-1271-U100), anti-CD25 (Life Technologies, 63-0251-82) and 400 anti-CD19 (Biolegend, 115545). For hematopoietic stem and progenitor cells, BM cells were 401 stained with a cocktail of biotin-conjugated lineage antibodies CD3e, B220, Ter119, Mac-1 and 402 Gr-1 (Biolegend, 133307), CD4 (Biolegend, 100403), CD5 (Biolegend, 100603), CD8 403 (Biolegend, 100703), followed by Streptavidin-AF488 (Biolegend, 405235). Cells were then 404 stained with CD127-APC (eBioscience, 17-1271-82), c-Kit-APC-eFluor780 (eBioscience, 47- 405 1171-82), Sca-1-Super Bright 436 (eBioscience, 62-5981-82), CD34-PE (Biolegend, 152204) 406 and FcγR-PerCP-eFluor710 (eBioscience, 46-0161-80), CD150-BV605 (Biolegend, 115927), 407 and CD48-BUV395 (BDBioscience, 740236). Fixable Viability Dye was used to exclude dead 408 cells as instructed by the manufacturer. A complete list of used antibodies was shown in Figure 409 5 —source data 1. Flow cytometry was performed on an LSR Fortessa H0081 or X20 and 410 analyzed with FlowJo. 411 BMSC isolation, culture, and differentiation Tissue embedding, sectioning, and 357 hematoxylin and eosin staining were performed at the Comparative Pathology Shared Resource 358 of the University of Minnesota. For immunostaining, the tissues were embedded in OCT then 359 cut into 7-µm slides. After three times of PBS wash, the slides were incubated with blocking 360 buffer (3% BSA in PBS) for 1 h, then immersed with anti-Perilipin (Cell Signaling Technology, 361 #9349) antibody overnight at 4oC. For immunofluorescence, PBS-washed slides were incubated 362 with a fluorescent secondary antibody at room temperature for 1 h, and then mounted with 363 VECTASHIELD® Antifade Mounting Medium with DAPI after three times of PBS wash. A Nikon 364 system was used for imaging. Goldner’s trichrome and Safranin O staining were performed at 365 Servicebio, China. 366 The samples were scanned with an in vitro micro-CT device (Skyscan 1272, Bruker micro-CT) 368 with scanning parameters of: Source Voltage = 60 kV, Source Current = 166uA, exposure 897 369 ms/frame, average of 3 frames per projection, Rotation Step (deg) = 0.200- and 0.25-mm 370 Aluminum filter. The specimens were scanned at high resolution (2016 × 1344 pixels) with an 371 Isotropic voxel size of 7.1 μm. Reconstructions for X-ray projections and re-alignment were 372 performed using the Skyscan software (NRecon and DataViewer) (v. 1.7.3.1, Brüker micro-CT, 373 Kontich, Belgium). Ring artefact and beam hardening corrections were applied in reconstruction. 374 Datasets were loaded into SkyScan CT-Analyzer software for measurement of BMD. Calibration 375 was performed with 0.25- and 0.75- mg/mL hydroxyapatite mice phantoms provided by 376 SkyScan. For cancellous and cortical bone analysis, the scanning regions were confined to the 377 distal meta-physis, 100 slices starting at 0.5 mm proximally from the proximal tip of the primary 378 spongiosa for the cancellous portion and 100 slices starting at 4.5 mm proximally from the 379 center of intercondylar fossa for the cortical portion. 380 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Quantification and statistical analysis Results are shown as mean ± SEM. N values (biological replicates) and statistical analysis 413 methods are described in figure legends. The statistical comparisons were carried out using 414 two-tailed unpaired Student’s t-test and one-way or two-way ANOVA with indicated post hoc 415 tests with Prism 9 (Graphpad). Differences were considered significant when p < 0.05. , p < 416 0.05; , p < 0.01; *, p < 0.001. 417 All data generated and analyzed are included in the manuscript. Source data files are provided 426 for relevant figures. 427 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint 428 AUTHOR CONTRIBUTION 429 Z.Z. and Z.H. designed and performed experiments, analyzed data, and contribute 430 manuscript writing. M.A., M.E., J.C., and K.C.M. performed micro-CT scanning and ana 431 L.E.B. determined RUNX2 O-GlcNAcylation and transcriptional activity. H.Z. provided guid 432 and assistance on flow cytometry. J.C.M. and A.L.B. performed mass spectrom 433 identification of PPARγ2 O-GlcNAc sites. H.-B.R. conceived the project, designed experim 434 analyzed data, and wrote the manuscript. 435 436 DECLARATION OF INTERESTS 437 The authors declare no competing interests. 438 439 SOURCE DATA LEGNED 440 Figure 2 —source data 1 441 Raw uncropped image for panel D. 442 Figure 2 —source data 2 443 Raw uncropped image for panel G. 444 Figure 4 —source data 1 445 Mass spectrometry Peaklists of all protein modifications (Table S1) and PPARγ2 O-Glc 446 sites (Table S2). 447 Figure 5 —source data 1 448 Antibodies used for flow cytometry. 449 Figure 6 —source data 1 450 Sequences of oligos used for RT-qPCR. 451 452 428 AUTHOR CONTRIBUTION 429 Z.Z. and Z.H. designed and performed e 430 manuscript writing. M.A., M.E., J.C., and K.C 431 L.E.B. determined RUNX2 O-GlcNAcylation a 432 and assistance on flow cytometry. J.C.M 433 identification of PPARγ2 O-GlcNAc sites. H.-B 434 analyzed data, and wrote the manuscript. Goldner’s trichrome (F) and Safranin O (G) staining of femurs from 4-week-old mice. (H-L) 461 Micro-CT of 6-week-old mice (H, n = 3-4). Bone volume (BV, I), BV/tissue volume ratio (BV/TV, 462 J), trabecular thickness (Tb.Th, K), and trabecular number (Tb.N, L) were calculated. Data are 463 presented as mean ± SEM. , p < 0.05 by unpaired student’s t-test. 464 465 466 Quantification and statistical analysis 435 436 DECLARATION OF INTERESTS 437 The authors declare no competing interests. 438 439 SOURCE DATA LEGNED 440 Figure 2 —source data 1 441 Raw uncropped image for panel D. 442 Figure 2 —source data 2 443 Raw uncropped image for panel G. 444 Figure 4 —source data 1 445 Mass spectrometry Peaklists of all protein m 446 sites (Table S2). 447 Figure 5 —source data 1 448 Antibodies used for flow cytometry. 449 Figure 6 —source data 1 450 Sequences of oligos used for RT-qPCR. 451 452 453 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint 455 FIGURES FIGURES Figure 1. Impaired osteogenesis in OsxΔOgt mice. (A) Mating strategy to generate OsxΔOgt mice. Note that the Ogt gene is located on Chr. X, th males are hemizygous Ogtfl/Y. (B, C) Whole mount Alizarin red and Alcian blue staining newborn mice. (D, E) Long bone length (D) and gross morphology of 4-6 weeks old mice. (F- 456 Figure 1. Impaired osteogenesis in OsxΔOgt mice. 457 (A) Mating strategy to generate OsxΔOgt mice. Note that the Ogt gene is located on Chr. X, thus 458 males are hemizygous Ogtfl/Y. (B, C) Whole mount Alizarin red and Alcian blue staining of 459 newborn mice. (D, E) Long bone length (D) and gross morphology of 4-6 weeks old mice. (F-G) 460 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint Goldner’s trichrome (F) and Safranin O (G) staining of femurs from 4-week-old mice. (H-L) 461 Micro-CT of 6-week-old mice (H, n = 3-4). Quantification and statistical analysis Bone volume (BV, I), BV/tissue volume ratio (BV/TV, 462 J), trabecular thickness (Tb.Th, K), and trabecular number (Tb.N, L) were calculated. Data are 463 presented as mean ± SEM. , p < 0.05 by unpaired student’s t-test. 464 465 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint 7 Figure 2. RUNX2 O-GlcNAcylation is required for osteogenesis. 8 (A) Alkaline phosphatase (ALP) staining of control and OsxΔOgt BMSCs differentiated to the 9 osteogenic lineage. (B, C) Primary BMSCs, in the presence or absence of the OGT inhibitor 0 TMG, were induced for osteogenesis and stained for ALP (B) and Alizarin Red S (C). (D) 1 e 2. RUNX2 O-GlcNAcylation is required for osteogenesis. Figure 2. RUNX2 O-GlcNAcylation is required for osteogenesis. 468 (A) Alkaline phosphatase (ALP) staining of control and OsxΔOgt BMSCs differentiated to the 469 osteogenic lineage. (B, C) Primary BMSCs, in the presence or absence of the OGT inhibitor 470 TMG, were induced for osteogenesis and stained for ALP (B) and Alizarin Red S (C). (D) 471 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint Primary BMSCs were treated with PTH for the indicated time and subjected to Western blotting 472 of total protein O-GlcNAcylation. (E) BMSCs were treated with PTH alone or together with TMG, 473 osteogenic differentiated, and stained for ALP (n = 3). (G) Flag-tagged wildtype (WT) and O- 474 GlcNAc mutant (3A) RUNX2 plasmids were overexpressed in HEK293 cells, and their O- 475 GlcNAcylation was determined by Flag immunoprecipitation followed with O-GlcNAc Western 476 blot. Quantification and statistical analysis (H) 6xOSE-luciferase activity in COS-7 cells transfected with WT- or 3A-mutant RUNX2, in 477 the presence or absence of the OGT inhibitor, OSMI-1. (I, J) C3H10T1/2 cells with lentiviral 478 overexpression of RUNX2 were osteogenically differentiated and stained with ALP or Alizarin 479 Red S (I). Expression of Bglap and Rankl was determined by RT-qPCR (J). Data are presented 480 as mean ± SEM. , p < 0.05; , p < 0.01; and , p < 0.001 by one-way ANOVA (J). 481 Representative images from at least 3 biological replicates were shown in A, B, C, E, and I. 482 Primary BMSCs were treated with PTH for the indicated time and subjected to Western blotting 472 of total protein O-GlcNAcylation. (E) BMSCs were treated with PTH alone or together with TMG, 473 osteogenic differentiated, and stained for ALP (n = 3). (G) Flag-tagged wildtype (WT) and O- 474 GlcNAc mutant (3A) RUNX2 plasmids were overexpressed in HEK293 cells, and their O- 475 GlcNAcylation was determined by Flag immunoprecipitation followed with O-GlcNAc Western 476 blot. (H) 6xOSE-luciferase activity in COS-7 cells transfected with WT- or 3A-mutant RUNX2, in 477 the presence or absence of the OGT inhibitor, OSMI-1. (I, J) C3H10T1/2 cells with lentiviral 478 overexpression of RUNX2 were osteogenically differentiated and stained with ALP or Alizarin 479 Red S (I). Expression of Bglap and Rankl was determined by RT-qPCR (J). Data are presented 480 as mean ± SEM. , p < 0.05; , p < 0.01; and , p < 0.001 by one-way ANOVA (J). 481 Representative images from at least 3 biological replicates were shown in A, B, C, E, and I. 482 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint 483 Figure 3. Adult-onset deletion of OGT impairs trabecular bone formation. 484 (A) Dox treatment timeline in OsxΔOgt to achieve osteoblast-specific deletion of OGT. Quantification and statistical analysis (B-D) 485 Micro-CT (B) showing reduced bone volume/tissue volume (C), trabecular thickness (D), and 486 trabecular bone mineral density (E). Data are presented as mean ± SEM., p < 0.05 by unpaired 487 student’s t-test. 488 489 Figure 3. Adult-onset deletion of OGT impairs trabecular bone formation. 484 Figure 3. Adult-onset deletion of OGT impairs trabecular bone formation. 484 (A) Dox treatment timeline in OsxΔOgt to achieve osteoblast-specific deletion of OGT. (B-D) 485 Micro CT (B) showing reduced bone volume/tissue volume (C) trabecular thickness (D) and 486 Figure 3. Adult-onset deletion of OGT impairs trabecular bone formation. (A) Dox treatment timeline in OsxΔOgt to achieve osteoblast-specific deletion of OGT. (B-D) 485 Micro-CT (B) showing reduced bone volume/tissue volume (C), trabecular thickness (D), and 486 trabecular bone mineral density (E). Data are presented as mean ± SEM., p < 0.05 by unpaired 487 student’s t-test. 488 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint Figure 4. O-GlcNAcylation inhibits BM adipogenesis. (A, B) H&E (A) and Perilipin immunofluorescent staining (B) on femur sections from 4-week-old mice. (C) Flow cytometric quantification of PDGFRa+VCAM1+ preadipocytes frequencies within the live BM cells (n = 3). (D) Adipogenic differentiation of primary BMSCs from control and . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is mad 491 Figure 4. O-GlcNAcylation inhibits BM adipogenesis. 492 ure 4. O-GlcNAcylation inhibits BM adipogenesis. Figure 4. O-GlcNAcylation inhibits BM adipogenesis. 492 Figure 4. O-GlcNAcylation inhibits BM adipogenesi 492 (A, B) H&E (A) and Perilipin immunofluorescent staining (B) on femur sections from 4-week-old 493 mice. (C) Flow cytometric quantification of PDGFRa+VCAM1+ preadipocytes frequencies within 494 the live BM cells (n = 3). (D) Adipogenic differentiation of primary BMSCs from control and 495 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: ioRxiv preprint . Quantification and statistical analysis CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint 496 497 498 499 500 501 502 503 504 505 506 OsxΔOgt mice. Lipid was stained with Oil Red O and quantified to the right (n = 4). (E, F) Primary 496 BMSCs were osteogenic differentiated for 0 or 15 days. Expression of Adipoq (E) and Vcam1 (F) 497 genes was determined by RT-qPCR (n = 3). (G-I) C3H10T1/2 cells, treated with or without TMG, 498 were adipogenic differentiated. Western blotting for perilipin and O-GlcNAc of differentiated cells 499 (G) and RT-qPCR for adipogenic marker Pparg (H) and Adipoq (I) expression. (J, K) Adipogenic 500 differentiation of C3H10T1/2 cells infected with lentiviral C/EBPβ. Oil Red O was stained and 501 quantified (J). Pparg and Adipoq gene expression was determined by RT-qPCR (K). Data are 502 presented as mean ± SEM., p < 0.05; , p < 0.01; and , p < 0.001 by unpaired student’s t- 503 test (C, J), one-way ANOVA (K), and two-way ANOVA (E, F, H, I). 504 OsxΔOgt mice. Lipid was stained with Oil Red O and quantified to the right (n = 4). (E, F) Primary 496 BMSCs were osteogenic differentiated for 0 or 15 days. Expression of Adipoq (E) and Vcam1 (F) 497 genes was determined by RT-qPCR (n = 3). (G-I) C3H10T1/2 cells, treated with or without TMG, 498 were adipogenic differentiated. Western blotting for perilipin and O-GlcNAc of differentiated cells 499 (G) and RT-qPCR for adipogenic marker Pparg (H) and Adipoq (I) expression. (J, K) Adipogenic 500 differentiation of C3H10T1/2 cells infected with lentiviral C/EBPβ. Oil Red O was stained and 501 quantified (J). Pparg and Adipoq gene expression was determined by RT-qPCR (K). Data are 502 presented as mean ± SEM., p < 0.05; , p < 0.01; and , p < 0.001 by unpaired student’s t- 503 test (C, J), one-way ANOVA (K), and two-way ANOVA (E, F, H, I). 504 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Flow cytometric quantification of B220+ B cell (J), CD4+ T cell (K), and CD8+ T cell (L) 513 percentages in the blood (n = 3-4). (M, N) CMP/CLP ratio (M) and GMP/EMP ratio (N) in the BM 514 (n = 6-7). (O, P) Complete blood counting showing numbers of RBC (O) and neutrophil (P) (n = 515 7-9). Data are presented as mean ± SEM., p < 0.05; , p < 0.01; and , p < 0.001 by 516 unpaired student’s t-test. 517 518 519 Quantification and statistical analysis It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint gure 5. Impaired B lymphopoiesis and myeloid skewing in OsxΔOgt mice. A) Schematic view of B cell development in the BM and blockade by stromal OGT defic ed X). (B-C) Flow cytometric quantification of LSK (B) and CLP (C) among live BM cells (n . (D-I) Flow cytometric quantification of fraction A (D), fraction B (E), fraction C (F), fracti G), fraction D (H), and immature B (I) frequencies among live BM lymphocytes (n = 6-7). Figure 5. Impaired B lymphopoiesis and myeloid skewing in OsxΔOgt mice. Figure 5. Impaired B lymphopoiesis and myeloid skewing in OsxΔOgt mice. 508 (A) Schematic view of B cell development in the BM and blockade by stromal OGT deficiency 509 (red X). (B-C) Flow cytometric quantification of LSK (B) and CLP (C) among live BM cells (n = 4- 510 6). (D-I) Flow cytometric quantification of fraction A (D), fraction B (E), fraction C (F), fraction C’ 511 (G), fraction D (H), and immature B (I) frequencies among live BM lymphocytes (n = 6-7). (J-L) 512 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint 513 514 515 516 517 518 519 519 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. Quantification and statistical analysis ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint 520 Figure 6. O-GlcNAc regulation of niche cytokine expression. 521 (A, B) C3H10T1/2 cells were treated with vehicle or OGT inhibitor OSMI and differentiated for 522 adipocytes (n = 4). Kitl (A) and Il7 (B) gene expression was determined by RT-qPCR. (C, D) 523 C3H10T1/2 cells were treated with vehicle or OGA inhibitor TMG and induced for osteogenic 524 differentiation (n = 6). Kitl (C) and Il7 (D) gene expression was determined by RT-qPCR. (E-H) 525 C3H10T1/2 cells were infected with lentiviruses expressing WT and O-GlcNAc-deficient C/EBPβ 526 (E, F) or RUNX2 (G, H). Expression Kitl (E, G) and Il7 (F, H) was measured by RT-qPCR (n = 6). 527 (I) Proposed action of protein O-GlcNAcylation in regulating the BMSC niche function. Data are 528 presented as mean ± SEM., p < 0.05; , p < 0.01; , p < 0.001 by two-way ANOVA (A-D) and 529 one-way ANOVA (E-H). 530 531 Figure 6. O-GlcNAc regulation of niche cytokine expression. 1 (A, B) C3H10T1/2 cells were treated with vehicle or OGT inhibitor OSMI and differentiated for 522 adipocytes (n = 4). Kitl (A) and Il7 (B) gene expression was determined by RT-qPCR. (C, D) 523 C3H10T1/2 cells were treated with vehicle or OGA inhibitor TMG and induced for osteogenic 524 differentiation (n = 6). Kitl (C) and Il7 (D) gene expression was determined by RT-qPCR. (E-H) 525 C3H10T1/2 cells were infected with lentiviruses expressing WT and O-GlcNAc-deficient C/EBPβ 526 (E, F) or RUNX2 (G, H). Expression Kitl (E, G) and Il7 (F, H) was measured by RT-qPCR (n = 6). 527 (I) Proposed action of protein O-GlcNAcylation in regulating the BMSC niche function. Data are 528 presented as mean ± SEM., p < 0.05; , p < 0.01; , p < 0.001 by two-way ANOVA (A-D) and 529 one-way ANOVA (E-H). 530 531 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. REFERENCE 535 Ahrends, R., Ota, A., Kovary, K.M., Kudo, T., Park, B.O., and Teruel, M.N. (2014). Controlling 536 low rates of cell differentiation through noise and ultrahigh feedback. Science 344, 1384-1389. 537 10.1126/science.1252079. 538 Ambrosi, T.H., Longaker, M.T., and Chan, C.K.F. (2019). A Revised Perspective of Skeletal 539 Stem Cell Biology. 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SUPPLIMENTARY FIGURES Figure 1—figure supplement 1. Dental defects of OsxΔOgt mice. Top: photographic analysis of incisor tooth development. Bottom: photographic analysis of molar tooth development. Figure 1—figure supplement 1. Dental defects of OsxΔOgt mice. Top: photographic analysis of incisor tooth development. Bottom: photographic analysis of molar tooth development. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint Figure 4—figure supplement 1. RUNX2 inhibits adipogenesis independently of O- GlcNAcylation. C3H10T1/2 cells were infected with lentiviruses expressing WT- or O-GlcNAc-deficient (3SA)- RUNX2, then induced for adipogenic differentiation. Adipogenesis was quantified by Oil Red O staining (A) and Perilipin Western blotting (B). Data are presented as mean ± SEM., p < 0.01 by one-way ANOVA. Figure 4—figure supplement 1. RUNX2 inhibits adipogenesis independently of O- GlcNAcylation. C3H10T1/2 cells were infected with lentiviruses expressing WT- or O-GlcNAc-deficient (3SA)- RUNX2, then induced for adipogenic differentiation. Adipogenesis was quantified by Oil Red O staining (A) and Perilipin Western blotting (B). Data are presented as mean ± SEM., p < 0.01 by one-way ANOVA. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint gure 4—figure supplement 2. PPARγ2 O-GlcNAcylation is required for adipogenesis. A) PPARγ2 protein domains with the O-GlcNAc site shown in red. (P)hosphorylation, SUMO)lation and (Ac)etylation sites were also shown. (B) Previously known (T84) and newly entified (S104, S133, S370, and T487) O-GlcNAc sites were mutated to alanine separately T84A and 4A, respectively) or together (5A). PPRE-luciferase assay of PPARγ2 was erformed. Data are presented as mean ± SEM., p < 0.01 by one-way ANOVA. (C) 3H10T1/2 cells were infected with lentiviral PPARγ2, induced to become adipocytes in the resence of only insulin, and stain with Oil Red O. Figure 4—figure supplement 2. PPARγ2 O-GlcNAcylation is required for adipogenesis. (A) PPARγ2 protein domains with the O-GlcNAc site shown in red. (P)hosphorylation, (SUMO)lation and (Ac)etylation sites were also shown. SUPPLIMENTARY FIGURES (B) Previously known (T84) and newly identified (S104, S133, S370, and T487) O-GlcNAc sites were mutated to alanine separately (T84A and 4A, respectively) or together (5A). PPRE-luciferase assay of PPARγ2 was performed. Data are presented as mean ± SEM., p < 0.01 by one-way ANOVA. (C) C3H10T1/2 cells were infected with lentiviral PPARγ2, induced to become adipocytes in the presence of only insulin, and stain with Oil Red O. Figure 4—figure supplement 2. PPARγ2 O-GlcNAcylation is required for adipogenesis. (A) PPARγ2 protein domains with the O-GlcNAc site shown in red. (P)hosphorylation, (SUMO)lation and (Ac)etylation sites were also shown. (B) Previously known (T84) and newly identified (S104, S133, S370, and T487) O-GlcNAc sites were mutated to alanine separately (T84A and 4A, respectively) or together (5A). PPRE-luciferase assay of PPARγ2 was performed. Data are presented as mean ± SEM., p < 0.01 by one-way ANOVA. (C) C3H10T1/2 cells were infected with lentiviral PPARγ2, induced to become adipocytes in the Figure 4—figure supplement 2. PPARγ2 O-GlcNAcylation is required for adipogenesis. (A) PPARγ2 protein domains with the O-GlcNAc site shown in red. (P)hosphorylation, (SUMO)lation and (Ac)etylation sites were also shown. (B) Previously known (T84) and newly identified (S104, S133, S370, and T487) O-GlcNAc sites were mutated to alanine separately Figure 4—figure supplement 2. PPARγ2 O-GlcNAcylation is required for figure supplement 2. PPARγ2 O-GlcNAcylation is required for adipogenesis. (A) PPARγ2 protein domains with the O-GlcNAc site shown in red. (P)hosphorylation, (SUMO)lation and (Ac)etylation sites were also shown. (B) Previously known (T84) and newly identified (S104, S133, S370, and T487) O-GlcNAc sites were mutated to alanine separately (T84A and 4A, respectively) or together (5A). PPRE-luciferase assay of PPARγ2 was performed. Data are presented as mean ± SEM.*, p < 0.01 by one-way ANOVA. (C) C3H10T1/2 cells were infected with lentiviral PPARγ2, induced to become adipocytes in the presence of only insulin, and stain with Oil Red O. . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. SUPPLIMENTARY FIGURES It is made The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521379 doi: bioRxiv preprint Figure 5—figure supplement 1. Flow cytometry of BM B cells. (A) B cell lineage development and surface markers used for flow cytometry. (B) Gating strategy for B cell populations. Figure 5—figure supplement 1. Flow cytometry of BM B cells. (A) B cell lineage development and surface markers used for flow cytometry. (B) Gating strategy for B cell populations. Figure 5—figure supplement 1. Flow cytometry of BM B cells. Figure 5—figure supplement 1. Flow cytometry of BM B cells. (A) B cell lineage development and surface markers used for flow cytometry. (B) Gating strategy for B cell populations.
https://openalex.org/W3098685935	https://vestnik.mgimo.ru/jour/article/download/1853/1418	Russian	null	Impact of Monetary Policy on the Level of Economic Inequality in the United States	Vestnik MGIMO-universiteta	2,020	cc-by	7,538	Вестник МГИМО-Университета. 2020. 13(5). С. 97-114 DOI 10.24833/2071-8160-2020-5-74-97-114 Вестник МГИМО-Университета. 2020. 13(5). С. 97-114 DOI 10.24833/2071-8160-2020-5-74-97-114 ИССЛЕДОВАТЕЛЬСКИЕ СТАТЬИ Вестник МГИМО-Университета. 2020. 13(5). С. 97-114 DOI 10.24833/2071-8160-2020-5-74-97-114 УДК: 336.02 Поступила в редакцию: 25.07.2020 г. Принята к публикации: 10.09.2020 г. Влияние монетарной политики на уровень экономического неравенства в США М.Л. Дорофеев Финансовый университет при Правительстве РФ М.Л. Дорофеев Финансовый университет при Правительстве РФ М.Л. Дорофеев Финансовый университет при Правительстве РФ После реформы мировой валютной системы и отказа в 1971 г. от золотого стан дарта монетарные и фискальные власти накопили огромный опыт регулирования экономики и усиления страновых конкурентных преимуществ за счёт сложных ме ханизмов денежной эмиссии, манипулирования валютными курсами, наращива ния долгов и пр. Преодоление последствий финансовых кризисов XXI в. заставля ет монетарных регуляторов прибегать к всё более радикальным мерам спасения экономики посредством вливания колоссальных объёмов ликвидности в рынок для выкупа «плохих» долгов системообразующих корпораций и облигаций прави тельства. Как монетарная политика влияет на уровень экономического неравен ства? Кто выступает её бенефициаром? Эти вопросы становятся всё актуальнее. Цель настоящей работы состоит в анализе влияния монетарной политики на уро вень экономического неравенства в США. Учитывая цикличность экономического неравенства, основным методом исследования мы выбрали графический ана лиз, позволяющий эффективно выявлять тренды и держать в фокусе более чем 100-летний период. Для целостности исследования уровень экономического не равенства сопоставлен не только с ключевыми индикаторами монетарной поли тики, но и с предельными налоговыми ставками. Цель настоящей работы состоит в анализе влияния монетарной политики на уро вень экономического неравенства в США. Учитывая цикличность экономического неравенства, основным методом исследования мы выбрали графический ана лиз, позволяющий эффективно выявлять тренды и держать в фокусе более чем 100-летний период. Для целостности исследования уровень экономического не равенства сопоставлен не только с ключевыми индикаторами монетарной поли тики, но и с предельными налоговыми ставками. В результате исследования нами установлено, что от параметров фискальной си стемы и от величины предельных налоговых ставок экономическое неравенство зависит сильнее, чем от монетарной политики. Недостаточно высокие предель ные налоговые ставки на доходы и наследство увеличивают экономическое нера венство. Влияние настроек монетарной системы на уровень экономического не равенства, и прежде всего на уровень богатства наиболее состоятельных людей, сказывается через стоимостную оценку финансовых активов. Важный вывод про ведённого анализа заключается в том, что для сдерживания роста неравенства монетарная политика может быть приемлемой при предельных ставках налогов на доходы и наследство около 60% и при эффективном макропруденциальном ре гулировании экономики. Ключевые слова: неравенство богатства, экономическое неравенство, денежно-кредит ная политика, налоги, процентные ставки, монетарная политика, государственное регули рование УДК: 336.02 Поступила в редакцию: 25.07.2020 г. Принята к публикации: 10.09.2020 г. 97 97 ВЕСТНИК МГИМО-УНИВЕРСИТЕТА • 13(5) • 2020 Research Article M.L. Dorofeev M.L. MGIMO REVIEW OF INTERNATIONAL RELATIONS • 13(5) • 2020 Влияние монетарной политики на уровень экономического неравенства в США Тем самым поощряется безответственность и чрезмерная склонность корпораций к риску и, более того, происходит коррозия системы политических сдержек и противовесов. При запущенности проблемы и боль шой поляризации по доходам и богатству корпорации и сверхбогатые люди по лучают возможность оказывать влияние на политиков и ещё сильнее подстраи вают параметры финансовой системы под собственные интересы. В отсутствие должного фискального регулирующего воздействия со стороны государства описанные обстоятельства ускорили рост экономического неравенства в США. Прогнозы изменения экономического неравенства до 2050 г. показывают, что уровень экономического неравенства в большинстве стран угрожающе ра стёт, что создаёт риски для социальной стабильности и устойчивого экономи ческого роста мировой экономики1. Поэтому в научном сообществе всё больше внимания уделяется анализу причин и факторов роста экономического нера венства, а также разработке эффективных мер, которые позволили бы государ ствам решить эту проблему. ги (Mazzucato 2018). Тем самым поощряется безответственность и чрезмерная склонность корпораций к риску и, более того, происходит коррозия системы политических сдержек и противовесов. При запущенности проблемы и боль шой поляризации по доходам и богатству корпорации и сверхбогатые люди по лучают возможность оказывать влияние на политиков и ещё сильнее подстраи вают параметры финансовой системы под собственные интересы. В отсутствие должного фискального регулирующего воздействия со стороны государства описанные обстоятельства ускорили рост экономического неравенства в США. Прогнозы изменения экономического неравенства до 2050 г. показывают, что уровень экономического неравенства в большинстве стран угрожающе ра стёт, что создаёт риски для социальной стабильности и устойчивого экономи ческого роста мировой экономики1. Поэтому в научном сообществе всё больше внимания уделяется анализу причин и факторов роста экономического нера венства, а также разработке эффективных мер, которые позволили бы государ ствам решить эту проблему. 1 World inequality report. 2018. URL: https://wir2018.wid.world/files/download/wir2018-full-report-english.pdf (accessed 14.10.2020). Влияние монетарной политики на уровень экономического неравенства в США Dorofeev Б ольшинство исследований показывает обратную зависимость между экономическим неравенством и экономическим ростом, следователь но, эффективное снижение уровня экономического неравенства при прочих равных условиях должно способствовать экономическому росту. В странах, которые недостаточно эффективно решают проблему экономическо го неравенства, наблюдаются высокий уровень бедности и медленные темпы экономического роста. Кроме того, в долгосрочной перспективе существенное экономическое неравенство может привести к политическим, социальным и экономическим шокам (Джомо, Попов 2016; Любимов 2016; Малкина 2016; Ба лацкий 2017; Варшавский 2019). Б Многие актуальные исследования анализируют связь экономического не равенства с перепроизводством экономических благ, провоцируемым стиму лирующей экономической политикой регуляторов и формированием соответ ствующих потребительских стереотипов. В ряде работ отмечаются негативное влияние роста экономического неравенства на экологию, риски существенно го замедления экономического роста, а также угрозы устойчивости развития мировой экономики (Mikhaylov at al. 2020; Moiseev at al. 2020; Nie at al. 2020; Yumashev at al. 2020; Varyash at al. 2020; Wiedmann at al. 2020). Экономическое неравенство может приводить к финансовой нестабильно сти в том случае, если домохозяйства с низким уровнем доходов используют кредит как основной способ повышения уровня жизни и потребления (Капе люшников 2017; Варшавский 2019). Доступность кредитов при низких процент ных ставках мотивирует домохозяйства наращивать заимствования, что приво дит к созданию долговых пузырей. В результате финансовая система становится уязвимой для внешних шоков, а устойчивое развитие экономики оказывается под угрозой срыва. Для преодоления текущих проблем и вызовов центробанки развитых стран уже не могут снижать процентные ставки, поскольку их уровень близок к нуле вому (Малова 2020). В этих условиях монетарные регуляторы прибегают к ак тивным операциям на финансовом рынке, покупая долговые обязательства и тем самым фактически расширяя денежную базу. Раньше производилась скупка ипотечных бумаг и казначейских облигаций США. В настоящее время центро банки расширяют список выкупаемых активов, что не может не вызывать опа сений по поводу возможных последствий подобных действий. Современное состояние мировой экономики и желание политиков США сохранить за страной роль мирового экономического лидера побуждает регу ляторов прибегать к любым способам предотвращения банкротств системо образующих корпораций, включая выкуп активов и долговых обязательств. Созданные регуляторами условия привели к тому, что в своей инвестиционной политике бизнес перестал считаться с рисками. В случае успеха корпорации по лучат сверхприбыли, а в случае неудачи правительство через бюджетную си стему на средства налогоплательщиков покроет убытки, выкупая плохие дол MGIMO REVIEW OF INTERNATIONAL RELATIONS • 13(5) • 2020 98 М.Л. Дорофеев ИССЛЕДОВАТЕЛЬСКИЕ СТАТЬИ ги (Mazzucato 2018). Обзор литературы На рост экономического неравенства оказывают влияние разные факторы На рост экономического неравенства оказывают влияние разные факторы. Итальянский исследователь П. Тридико (Tridico 2017) выделяет глобальное межстрановое экономическое неравенство и внутристрановое экономическое неравенство. Глобальное экономическое неравенство набирает обороты после проведе ния технологических реформ, создающих новые сложные и длинные цепочки добавленной стоимости вокруг ключевой географической территории (стра ны). Это приводит к перетоку капиталов и неравномерности темпов экономи ческого роста разных странах. В данный период страна-лидер и её главный(ые) конкурент(ы) всё дальше отрываются в экономическом развитии от догоняю щих стран. Соответственно, национальный доход ведущих стран растёт бы стрее, что оборачивается усилением межстранового экономического неравен ства. Внутристрановое экономическое неравенство начинает расти после того, как происходят технологические сдвиги в области управления человеческим ка питалом, трудовыми ресурсами, навыками и компетенциями внутри отдельно взятой страны. Более образованные и квалифицированные граждане получают преимущества на внутреннем рынке труда, что приводит к росту поляризации доходов в стране. В период циклического роста с 1970 г. по настоящее время внутристрановое экономическое неравенство развивалось под заметным влиянием существую 1 World inequality report. 2018. URL: https://wir2018.wid.world/files/download/wir2018-full-report-english.pdf (accessed 14.10.2020). 99 ВЕСТНИК МГИМО-УНИВЕРСИТЕТА • 13(5) • 2020 Research Article M.L. Dorofeev M.L. Dorofeev щей в стране социально-экономической модели и национальной финансовой системы. Страны с преобладающим корпоративным сектором (США, Англия, Ирландия) минимизировали степень регулирования экономики, развивали свободный рынок и фиксировали налоговые ставки на низких значениях. Про тивоположные по модели социально-экономического развития скандинавские страны пошли по пути, основанному на государственно-частном партнёрстве, прогрессивной системе налогообложения, социальной ориентированности эко номики. Монетарные власти США сегодня используют все доступные инструмен ты и методы, чтобы в краткосрочной перспективе решить проблему роста ка питализации финансовых активов и ускорить экономический рост. При этом Вашингтон использует преимущества крупнейшей мировой экономики и уни кальные возможности эмитента основной мировой резервной валюты. Получив право печатать неограниченное количество долларов после реформы мировой валютной системы в 1970-х гг., монетарные власти США для борьбы с кризи сами и решения своих текущих экономических проблем постоянно проводят агрессивное наращивание денежной базы и применяют инструменты неконвен циальной монетарной политики. Принимая во внимание множественность факторов, одновременно оказы вающих влияние на динамику экономического неравенства, вычленить вклад в эту динамику монетарной политики представляется непростой задачей. Принимая во внимание множественность факторов, одновременно оказы вающих влияние на динамику экономического неравенства, вычленить вклад в эту динамику монетарной политики представляется непростой задачей. Исследования (Bernanke, Gertler 1995; Rigobon, Sack 2004; Dobbs et al. 2013) показывают, что конвенциальная монетарная политика в большей степени вли яет на капитализацию рынка акций, чем на цены на недвижимость. MGIMO REVIEW OF INTERNATIONAL RELATIONS • 13(5) • 2020 Обзор литературы Неконвен циальная монетарная политика имеет иной механизм. Масштабные покупки центробанками инструментов долгового рынка (например, государственных облигаций для финансирования дефицита государственного бюджета или ипо течных бумаг для контроля цен на ипотечном рынке) приводят к росту стоимо сти этих бумаг и к снижению их доходности. Многие аспекты такой политики – масштабы и продолжительность эффекта стимулирования, ключевые бене фициары – до сих пор мало изучены (Martin, Milas 2012). – масштабы и продолжительность эффекта стимулирования, ключевые бене фициары – до сих пор мало изучены (Martin, Milas 2012). С учётом цикличности развития экономики и, соответственно, уровня эко номического неравенства, на разных стадиях бизнес-цикла монетарная поли тика оказывает неодинаковое воздействие на уровень экономического неравен ства. В периоды экономических кризисов монетарные регуляторы выступают в роли кредитора и маркет-мейкера последней инстанции (Дорофеев 2020a), ску пая активы на финансовом рынке, поддерживая уровень цен и потенциально увеличивая уровень поляризации по доходам и богатству. Активы и, соответ ственно, доходы самых богатых людей в значительной степени состоят из акций и облигаций; богатство бедных людей (если оно имеется) в основном склады вается из недвижимости и банковский депозитов. Таким образом, когда моне 100 MGIMO REVIEW OF INTERNATIONAL RELATIONS • 13(5) • 2020 М.Л. Дорофеев ИССЛЕДОВАТЕЛЬСКИЕ СТАТЬИ тарные власти скупают облигации и акции2 на финансовом рынке, поддержку получают именно богатые люди, в то время как бедные могут лишиться работы (а вместе с тем – единственного источника дохода), поскольку в рамках проведе ния антикризисной политики корпоративный сектор, как правило, сокращает издержки на заработную плату. Поскольку монетарная политика проводится в контрциклической манере, её долгосрочное влияние на равные по длительности циклы расширения и су жения кредита и экономики может быть нейтральным. Тем не менее, некоторые исследования показывают, что сдерживающая монетарная политика сильнее за медляет экономический рост, чем стимулирующая монетарная политика уско ряет его (Morgan 1993; Karras 1996). В связи с тем, что регуляторы реагируют главным образом не на рост акций, а на их падение (Ravn 2014), монетарная политика может стать важной при чиной циклического роста экономического неравенства: монетарные власти делают избыточно много для предотвращения падения финансовых рынков в периоды рецессий, в то время как в периоды бума они делают недостаточно, чтобы сдержать процесс надувания финансовых пузырей. Регуляторы распо лагают эффективными инструментами для сдерживания быстро растущих фи нансовых пузырей, однако в большинстве случаев они предпочитают сохранять в среднесрочном периоде высокие темпы экономического роста, рискуя уско рить наступление циклических финансовых кризисов в будущем. Неоднородное влияние дополнительных мер на различных экономических акторов усиливает рост экономического неравенства. 2 Can the Fed conduct monetary policy through the purchase and sale of stocks on the New York Stock Exchange? 2015. Federal reserve bank of San Francisco. https://www.frbsf.org/education/publications/doctor-econ/2005/november/ monetary-policy-stocks-sale-purchase/ (accessed 14.10.2020). 3 Condon C., Saraiva C. 2020. Powell Says Fed Policies ‘Absolutely’ Don’t Add to Inequality. Bloomberg Economics. URL: https://www.bloomberg.com/news/articles/2020-05-29/powell-says-fed-policies-absolutely-don-t-add-to-inequality? utm_content=business&utm_source=twitter&utm_medium=social&cmpid=socialflow-twitter-business&utm_ campaign=socialflow-organic (accessed 14.10.2020). 2 Can the Fed conduct monetary policy through the purchase and sale of stocks on the New York Stock Exchange? 2015. Federal reserve bank of San Francisco. https://www.frbsf.org/education/publications/doctor-econ/2005/november/ monetary-policy-stocks-sale-purchase/ (accessed 14.10.2020). 3 Condon C., Saraiva C. 2020. Powell Says Fed Policies ‘Absolutely’ Don’t Add to Inequality. Bloomberg Economics. URL: https://www.bloomberg.com/news/articles/2020-05-29/powell-says-fed-policies-absolutely-don-t-add-to-inequality? utm_content=business&utm_source=twitter&utm_medium=social&cmpid=socialflow-twitter-business&utm_ campaign=socialflow-organic (accessed 14.10.2020). Обзор литературы В силу этого необходи мо применять дополнительные асимметричные меры монетарного регулирова ния, чтобы обеспечить баланс в циклическом развитии экономики (Tenreyro, Thwaites 2013). h В некоторых работах (Bivens 2015) рассмотрена альтернативная версия кри зисных ситуаций: монетарные власти не предпринимают экстраординарных мер для борьбы с кризисами и не меняют параметры монетарной политики. Тог да мощные и затяжные финансовые и экономические кризисы могут привести к самым серьёзным изменениям внутристранового экономического неравенства. По свидетельству главы ФРС Дж. Пауэлла3, возглавляемая им служба фоку сирует внимание на решении текущих проблем, которые чреваты риском бы строго роста экономического неравенства. Пауэлл признаёт, что для спасения экономики регулятору уже пришлось пересечь множество красных линий. Гла ва ФРС не упомянул о богатых американцах, чьё состояние инвестировано в 101 ВЕСТНИК МГИМО-УНИВЕРСИТЕТА • 13(5) • 2020 Research Article M.L. Dorofeev финансовые активы, в 2020 г. активно скупаемые ФРС на свой баланс по любым ценам для поддержки финансовых рынков4. Текущие настройки монетарного регулирования в США гипертрофированно мягкие, они способствуют созда нию финансовых пузырей и оказывают существенное влияние на доходы и бо гатство наиболее состоятельных людей США (Дорофеев, Ахметов 2020; Доро феев 2020b). Некоторые исследователи (O’Farrell et al. 2019) высказывают мнение, что мо нетарным властям следует в первую очередь заботиться о стабильности цен и об обеспечении занятости, поскольку решение проблемы экономического нера венства требует таргетирования цен на финансовые активы. Это крайне слож ная задача требует частого изменения ключевых параметров денежно-кредит ной политики, что, по сути, противоречит основам свободного рынка и может пошатнуть макроэкономическую стабильность. у р у Доклад МВФ о борьбе с неравенством5 показывает противоречивую карти ну. С одной стороны, МВФ предлагает пути нивелирования экономического не равенства: реформа прогрессивной шкалы налогообложения, введение базово го дохода, повышение доступности систем образования и здравоохранения. С другой стороны, авторы доклада констатируют разнонаправленные тенденции в динамике экономического неравенства: межстрановое неравенство снижает ся за счёт роста Китая и развивающихся стран; внутристрановое неравенство в ряде развитых стран растёт, но в некоторых странах снижается. По представ ленным данным, внутристрановое экономическое неравенство в почти полови не обследованных стран снижается в течение последних 30 лет6. р На наш взгляд, выбор аналитиками МВФ 30-летнего периода исследования может создать иллюзию, будто проблема роста экономического неравенства в мировой экономике не критична, или, по крайней мере, преувеличена. 4 После кризиса 2008 г. рынки акций и облигаций США растут, по финансовым мультипликаторам они сильно переоценены. 5 IMF Fiscal Monitor. Tackling Inequality 2017: ix 6 IMF Fiscal Monitor. Tackling Inequality, October 2017. URL: https://www.imf.org/en/Publications/FM/Issues/2017/10/05/ fiscal-monitor-october-2017 (assessed 14.10.2020). 5 IMF Fiscal Monitor. Tackling Inequality 2017: ix Обзор литературы Вместе с тем в том же докладе МВФ отмечено, что резкий рост доходов и высокие нормы сбережения наиболее богатых людей в другой половине стран, в том числе и в США, привели к заметному росту внутристранового экономического неравен ства по концентрации богатства у верхнего 1% самых богатых людей страны, что указывает на очевидные проблемы в настройках механизма финансового перераспределения и требует вмешательства органов государственного регули рования экономикой. р Группа авторов Доклада о развитии неравенства в мире (World inequality report 2018) утверждает, что проблема роста экономического неравенства су ществует, очевидна и требует своего решения. Автор настоящего исследования также считает, что современные условия мировой финансовой системы, тренд MGIMO REVIEW OF INTERNATIONAL RELATIONS • 13(5) • 2020 102 ИССЛЕДОВАТЕЛЬСКИЕ СТАТЬИ М.Л. Дорофеев на дерегулирование финансовых рынков и стимулирующая финансовая по литика крупнейших стран мира создали условия для ускорения поляризации людей по доходам и богатству, что хорошо просматривается в фактических цифрах статистики внутристранового экономического неравенства и требует дополнительных исследований, а также принятия соответствующих мер госу дарственного регулирования. на дерегулирование финансовых рынков и стимулирующая финансовая по литика крупнейших стран мира создали условия для ускорения поляризации людей по доходам и богатству, что хорошо просматривается в фактических цифрах статистики внутристранового экономического неравенства и требует дополнительных исследований, а также принятия соответствующих мер госу дарственного регулирования. Отметим важный труд Р.И. Капелюшникова в области анализа причин, фак торов и перспектив развития экономического неравенства в мире, в котором он призывает не примитизировать проблему роста экономического неравенства. Так, он предполагает, что убеждённость части научного сообщества в высоких рисках роста экономического неравенства в будущем может базироваться на недостаточной аргументации и слабой доказательной базе, что, в свою очередь, может привести к ошибочной практике государственного регулирования (Ка пелюшников 2017). Материалы и методы исследования Под экономическим неравенством мы понимаем различие по показателям экономического благосостояния между отдельными лицами в группе, между группами населения или между странами. В данном исследовании в качестве индикатора экономического неравенства использованы данные о доле богат ства 1% наиболее состоятельных граждан США. В настоящей работе анализируются данные об уровне экономического не равенства в США по богатству за период с 1910 по 2014 гг. Такой значитель ный период исследования выбран по причине того, что автор разделяет идею Тридико (Tridico 2017) о циклической динамике экономического неравенства, и на основе имеющихся данных предполагает, что длительность циклов состав ляет около 50–70 лет. Исходя из данного предположения, в исследовании ав тор стремился охватить максимально длительный период. Доступные данные о динамике экономического неравенства позволяют сформировать информа ционную базу с начала 1900 г. Обоснование выбора периода исследования под тверждают выводы Доклада о развитии неравенства в мире (World inequality report 2018). Графический анализ проводится на основе сопоставления динамики доли богатства 1% самых обеспеченных граждан США, параметров фискальной и монетарной политики, а также ключевых индикаторов фондового рынка США. Из монетарных параметров мы использовали официальные данные о клю чевой ставке ФРС США. Чтобы дополнить общую картину о трендах процент ных ставок в США в период 1910–1955 гг., мы привлекли показатель доходности к погашению 10-летних казначейских облигаций США. Значения этого индика тора отличаются от ставки ФРС, которая отражает уровень процентных ставок в экономике США на краткосрочном горизонте. Графический анализ проводится на основе сопоставления динамики доли богатства 1% самых обеспеченных граждан США, параметров фискальной и монетарной политики, а также ключевых индикаторов фондового рынка США. Из монетарных параметров мы использовали официальные данные о клю чевой ставке ФРС США. Чтобы дополнить общую картину о трендах процент ных ставок в США в период 1910–1955 гг., мы привлекли показатель доходности к погашению 10-летних казначейских облигаций США. Значения этого индика тора отличаются от ставки ФРС, которая отражает уровень процентных ставок в экономике США на краткосрочном горизонте. 103 ВЕСТНИК МГИМО-УНИВЕРСИТЕТА • 13(5) • 2020 Research Article M.L. Dorofeev M.L. Dorofeev Анализ поведения кривой доходностей госдолга США показывает, что долгосрочные процентные ставки более устойчивы к циклическим колебани ям экономики в долгосрочном периоде, который мы выбрали для исследова ния экономического неравенства (Дорофеев 2020b). Кроме этого, данный ин дикатор отражает ожидаемую доходность на долгосрочных инвестиционных горизонтах и используется для оценки стоимости финансовых активов, будучи общепринятым индикативом в корпоративных финансах и оценке. Динамика доходности долгосрочных облигаций позволяет оценить тренды на рынке об лигаций и развивается в обратной зависимости от капитализации рынка об лигаций. 7 World inequality report 2018. URL: https://wir2018.wid.world/files/download/wir2018-full-report-english.pdf (assessed 14.10.2020). 8 World inequality report 2018. P. 90 Материалы и методы исследования После мирового финансового кризиса 2008 г. регуляторы всё чаще прибегают к неконвенциальным методам монетарного регулирования (выкупу долгов на финансовом рынке США), что отражают тренды на рынке государ ственного долга США. Для оценки динамики капитализации рынка акций нами использован сто имостной мультипликатор CAPE10 Р. Шиллера для индекса SnP500. Приоритет отдан данному мультипликатору, т.к. в основе его расчёта лежит скользящая средняя (усреднённая за 10 лет) чистая прибыль рынка акций США, входящих в широкий индекс рынка акций США SnP500, что позволяет сгладить влияние на наш анализ циклических колебаний корпоративной чистой прибыли. Чтобы получить полную картину изменений настроек финансового меха низма перераспределения доходов в США, мы использовали данные о предель ных ставках налогов на наследство и богатство. Результаты анализа каждого графика представлены в табличной форме и снабжены текстовыми комментариями. В таблицах описаны циклы и тренды изменения экономического неравенства в США и дана оценка соответствую щих трендов в денежно-кредитной и налоговой политике США. 9 World inequality report 2018: 216, 224–225, 234. Результаты исследования и дискуссия Современная прогрессивная система налогообложения США недостаточно эффективно распределяет национальный доход для группы наиболее бедных людей страны (50% граждан США)7. Система социальных трансфертов в стране выстроена таким образом, что беднейшие граждане получают недостаточный объём государственной поддержки для того, чтобы не допустить отставания темпов роста доходов от среднего класса и богатых людей. Согласно данным доклада о неравенстве в мире8, в результате финансового перераспределения доходов и богатства большую поддержку получает средний класс (40% населения). Государственные расходы на финансирование социаль ного обеспечения в процентах к национальному доходу США выросли с 2% в MGIMO REVIEW OF INTERNATIONAL RELATIONS • 13(5) • 2020 104 ИССЛЕДОВАТЕЛЬСКИЕ СТАТЬИ М.Л. Дорофеев 1960 г. в до 11% в 2014 г. С данными средствами поддержки нижние 50% на селения США получали около 10% национального дохода страны, в то время как доля национального дохода средних 40% достигала 16%. В период 1999– 2014 гг. доходы среднего класса США выросли в основном за счёт государствен ных трансфертов населению по различным социальным программам. Авторы доклада о неравенстве в мире отмечают, что без данной поддержки со стороны государства доходы среднего класса США стагнировали, а экономическое не равенство было бы выражено сильнее, чем сегодня. Структура активов домохозяйств с различными уровнями доходов разли чается. У самых обеспеченных людей доходы формируются в основном за счёт долей собственности в бизнесе и ценных бумаг9. С ростом благосостояния в структуре портфеля персонального богатства возрастает доля финансовых ак тивов фондового рынка и уменьшается доля трудовых доходов. Следовательно, чем богаче человек, тем больше его благосостояние зависит от динамики цен на финансовом рынке (рис. 1). ф р (р ) Рис. 1. Динамика доли богатства 1% граждан США на фоне изменения пара метров монетарной и фискальной политики. р р ф Fig. 1. Dynamics of 1% US citizens wealth share against the background of changes in monetary and fiscal policy parameters. 10 Депозиты и облигации: в чем разница для инвестора? Московская биржа. Режим доступа: URL: https://place.moex. com/useful/depozitih-obligacii-v-chem-raznica-dlja-investora?list=vse-pro-obligatsii (дата обращения 14.10.2020). р р Fig. 1. Dynamics of 1% US citizens wealth share against the background of changes in monetary and fiscal policy parameters. i Источник: составлено автором на основе World inequality report (2018) и данных о ди намике индикаторов финансового рынка США. URL: http://www.econ.yale.edu/~shiller/ data.htm (accessed 14.10.2020) i Источник: составлено автором на основе World inequality report (2018) и данных о ди намике индикаторов финансового рынка США. URL: http://www.econ.yale.edu/~shiller/ data.htm (accessed 14.10.2020) 9 World inequality report 2018: 216, 224–225, 234. ВЕСТНИК МГИМО-УНИВЕРСИТЕТА • 13(5) • 2020 105 Research Article M.L. Dorofeev На рис. 1 показана динамика доли богатства 1% самых богатых граждан США на фоне изменений индикаторов фондового рынка США. Результаты гра фического анализа данных рис. 1 представлены в табл. 1. Таблица 1. Тренды в неравенстве и индикаторах фондового рынка США Table 1. Summary of Figure 1 graphical analysis of Figure 1: trends in inequality and indicators of the US stock market № Период Капитализация рынка акций США по мультипликатору CAPE10 (прямой показатель стоимости акций) Капитализация рынка облигаций (обратный показатель доходности 10-летних облигаций США облигаций) Суперциклы изменения экономического неравенства в США Доля богатства верхнего 1% населения США 1 1910-1921 снижение снижение снижение снижение 2 1922-1929 рост рост рост 3 1930-1941 без изменений рост снижение 4 1942-1949 5 1950-1963 рост снижение без изменений 6 1964-1976 снижение снижение снижение 7 1977-1982 рост 8 1983-1989 рост рост рост 9 1990-2000 10 2001-2011 Источник: составлено автором. Явно прослеживается взаимосвязь между ростом богатства 1% самых со стоятельных людей и ростом капитализации фондового рынка: в периоды роста рынка акций и облигаций уровень неравенства увеличивался. В среднем на длинных временных интервалах доходность акций выше, чем облигаций (Берзон 2008). Средняя доходность банковского депозита, как пра вило, ниже доходности облигаций10. Следовательно, высокая доля акций и об лигаций в инвестиционных портфелях состоятельных граждан позволяет им ускорять свой отрыв по уровню экономического богатства от менее обеспечен ных граждан. Согласно теории оценки активов методом дисконтирования денежных по токов, стоимость любого актива, способного генерировать денежные потоки в будущем, зависит от ставки дисконтирования, отражающей премию за риск и требуемую доходность инвестора. В свою очередь, ставка дисконтирования зависит от процентной ставки центрального банка. На темп роста денежного потока влияет также уровень налогов на доходы и наследство. Рыночная сто имость актива или доли в бизнесе тем больше, чем ниже процентные ставки и ставки налогов. MGIMO REVIEW OF INTERNATIONAL RELATIONS • 13(5) • 2020 106 М.Л. Дорофеев М.Л. Дорофеев ИССЛЕДОВАТЕЛЬСКИЕ СТАТЬИ На рис. 2 на фоне изменения доли богатства самых состоятельных людей США показаны тренды в параметрах экономической политики США с 1913 г. по 2014 г. р р Fig. 1. Dynamics of 1% US citizens wealth share against the background of changes in monetary and fiscal policy parameters. Уровень экономического неравенства в США сильно зависит от фи нансового механизма перераспределения и следует за изменением процентных и налоговых ставок. Результаты графического анализа данных, представленных на рис. 2, систематизированы в табл. 2. Рис. 2. Динамика доли богатства 1% граждан США на фоне изменения пара метров монетарной и фискальной политики Рис. 2. Динамика доли богатства 1% граждан С Рис. 2. Динамика доли богатства 1% граждан США на фоне изменения пара метров монетарной и фискальной политикиh метров монетарной и фискальной политики Fig. 2. The dynamics of the 1% US citizens share of wealth against the background of changes in monetary and fiscal policy parameters Источник: Составлено автором на основе данных о неравенстве по богатству в США из World inequality report (2018) и данных Национального бюро экономического ана лиза США: URL: https://www.nber.org/cycles/cyclesmain.html (accessed 14.10.2020) Fig. 2. The dynamics of the 1% US citizens share of wealth against the background of changes in monetary and fiscal policy parameters Источник: Составлено автором на основе данных о неравенстве по богатству в США W ld i li (2018) Н б Fig. 2. The dynamics of the 1% US citizens share of wealth against the background of changes in monetary and fiscal policy parameters i Источник: Составлено автором на основе данных о неравенстве по богатству в США из World inequality report (2018) и данных Национального бюро экономического ана лиза США: URL: https://www.nber.org/cycles/cyclesmain.html (accessed 14.10.2020) i Источник: Составлено автором на основе данных о неравенстве по богатству в США из World inequality report (2018) и данных Национального бюро экономического ана лиза США: URL: https://www.nber.org/cycles/cyclesmain.html (accessed 14.10.2020) Итак, доля богатства 1% самых богатых людей США устойчиво снижалась в периоды роста предельных налоговых ставок на доходы и наследство. Отметим, что пороговым значением для эффективного предельного налогообложения до ходов и наследства в США является диапазон 55%–65%: когда налоговые ставки на доходы и наследство были выше данной зоны, доля богатства 1% самых состо ятельных людей снижалась, автоматически нивелируя уровень экономического 107 ВЕСТНИК МГИМО-УНИВЕРСИТЕТА • 13(5) • 2020 Research Article M.L. Dorofeev неравенства в стране. И наоборот, когда регуляторы устанавливали налоговые ставки ниже этой зоны, экономическое неравенство в США начинало расти. Монетарные настройки тоже оказывают заметное влияние на изменение неравенства. Так, в периоды 6 и 7 (рис. 2 и табл. 2) экономика США развивалась в условиях фискальных послаблений для богатых (резкое снижение предельных налоговых ставок) при ускоренном росте процентных ставок долгового рынка. Совокупное влияние этих мер привело к нивелированию экономического не равенства. р р Fig. 1. Dynamics of 1% US citizens wealth share against the background of changes in monetary and fiscal policy parameters. Во-первых, это произошло, потому что предельные ставки налогов превышали 60%, и высокие темпы роста доходов и богатства самых богатых лю дей корректировались мерами фискального регулирования. Во-вторых, резкий рост неопределённости и продолжительная стагнация цен на фондовом рынке также замедлили рост благосостояния владельцев портфелей ценных бумаг и долей в бизнесе. Таблица 2. Тренды в неравенстве и параметрах фискальной и монетарной политики СШАh MGIMO REVIEW OF INTERNATIONAL RELATIONS • 13(5) • 2020 Таблица 2. Тренды в неравенстве и параметрах фискальной и монетарной политики США политики США Table 2. The results of Figure 2 graphical analysis: trends in inequality and parameters of US fiscal and monetary policy № Период Изменение монетарных настроек (процентные ставки) Изменение фискальных настроек (предельных ставок на самых богатых людей страны) Суперциклы изменения экономического неравенства в США Доля богатства верхнего 1% населения США налог на доходы налог на наследство 1 1910-1921 рост рост рост снижение снижение 2 1922-1929 снижение снижение без изм. рост 3 1930-1941 снижение рост рост снижение 4 1942-1949 без изм. без изм. без изм. снижение 5 1950-1963 рост рост без изм. без изм. 6 1964-1976 рост снижение без изм. снижение 7 1977-1982 рост без изм. снижение рост рост 8 1983-1989 снижение снижение снижение рост 9 1990-2000 снижение рост без изм. рост 10 2001-2011 снижение снижение снижение рост Источник: составлено автором. h of US fiscal and monetary policy Рассмотрим другой период изменения экономического неравенства в США – 1990–2000 гг. Как видно (рис. 2 и табл. 2), предельные налоговые ставки на доходы после продолжительного и резкого снижения несколько восстанови лись, предельная ставка налога на наследство оставалась неизменной; обе не пре восходили 60%. Уровень процентных ставок продолжил снижение, в результате чего фондовый рынок получил важный стимул к росту. Доходы и богатство самых состоятельных людей резко выросли, ускорив рост экономического неравенства. Безусловно, на динамику экономического неравенства оказывали влияние и другие факторы. В годы мировых войн и Великой депрессии ускорялся износ человеческого, физического и финансового капитала. Такой сложный период MGIMO REVIEW OF INTERNATIONAL RELATIONS • 13(5) • 2020 108 М.Л. Дорофеев ИССЛЕДОВАТЕЛЬСКИЕ СТАТЬИ сильнее сказался на состояниях наиболее обеспеченных слоёв населения, тем более что это были периоды упущенных выгод и слабого экономического роста. После окончания этих периодов под действием возникших дисбалансов между товарным и денежным обращением росла инфляция, что также ограничивало рост доходов самых богатых людей. сильнее сказался на состояниях наиболее обеспеченных слоёв населения, тем более что это были периоды упущенных выгод и слабого экономического роста. После окончания этих периодов под действием возникших дисбалансов между товарным и денежным обращением росла инфляция, что также ограничивало рост доходов самых богатых людей. В современных условиях монетарная политика представляет собой важный элемент контрциклической экономической политики. Ловушка ликвидности, в которую попали большинство развитых экономик, привела к тому, что уровень процентных ставок опустился до беспрецедентно низких за предшествующие 75 лет значений. Меры неконвенциальной монетарной политики, получившие распростра нение в последние годы, увеличивают разрыв между бедными и богатыми сло ями населения в периоды послекризисного восстановления экономики. Таблица 2. Тренды в неравенстве и параметрах фискальной и монетарной политики США Суще ственное монетарное стимулирование положительно сказывается на стоимости активов фондового рынка и способно создавать финансовые пузыри, приводя к резкому росту богатства наиболее обеспеченных людей. Выкуп финансовых активов и действия регуляторов по поддержанию стабильности цен на финан совом рынке положительно влияет на доходы и богатство самых богатых людей, владеющих финансовыми активами и долями в бизнесе. До беднейших слоев населения подобная поддержка фактически не доходит, более того, растущая в периоды кризиса безработица создает серьёзную угрозу снижения уровня жиз ни данной группы населения. Монетарная политика асимметрична: регуляторы склонны к радикальным мерам поддержки экономики и рынков в кризисные периоды в большей мере, чем к сдерживанию экономического роста для борьбы с финансовыми пузыря ми. По этой причине в практике монетарного регулирования на различных эта пах экономического цикла применяются дополнительные меры, которые могут приводить к циклическому росту экономического неравенства. Перенастройка фискальных и монетарных составляющих экономической политики оказывает существенное влияние на уровень экономического нера венства. В структуре активов самых богатых людей финансовые активы зани мают наибольшую долю, которая по мере роста благосостояния увеличивается, поэтому изменение рыночной стоимости финансовых активов и определяющих её факторов формируют тренды в динамике экономического неравенства. На наш взгляд, решающее влияние на долю богатства самых обеспеченных людей оказывает фискальная политика, устанавливающая ставки налогов на доходы и богатство. Проведённый нами графический анализ показал, что в ус ловиях прогрессивной налоговой системы можно снижать уровень экономиче ского неравенства. Уровень налоговых ставок в диапазоне 55%–65% выступал для США пограничной зоной, выше которой доля в структуре национально го богатства 1% самых богатых людей устойчиво снижалась. В периоды, когда ставки налогов опускались ниже этих значений, богатые устойчиво богатели. 109 ВЕСТНИК МГИМО-УНИВЕРСИТЕТА • 13(5) • 2020 Research Article M.L. Dorofeev M.L. Dorofeev Изменяя параметры монетарной политики, можно регулировать уровень экономического неравенства, поскольку монетарное стимулирование поло жительно сказывается на стоимости активов фондового рынка и способно создавать финансовые пузыри, приводя к резкому росту богатства наиболее обеспеченных людей, владеющих долями в бизнесе и акциями. Сдерживающая монетарная политика в отсутствие эффективной прогрессивной системы нало гообложения не способна решить проблемы экономического неравенства. Современная монетарная политика США может считаться приемлемой и не создавать угроз для устойчивого экономического роста при достаточном макропруденциальном регулировании, поскольку к её (монетарной политике) побочным эффектам, кроме роста экономического неравенства, относится вы сокий риск формирования финансовых и долговых пузырей. Полученные результаты исследования основаны на использовании метода графического анализа, который имеет свои преимущества и недостатки. Об авторе: Михаил Львович Дорофеев – кандидат экономических наук, доцент Департамента общественных финансов Финансового университета при Правительстве РФ, г. Москва, Малый Златоустинский пер., д. 7, к. 1. E-mail: dorofeevml@yandex.ru. Конфликт интересов: Автор заявляет об отсутствии конфликта интересов. Конфликт интересов: Автор заявляет об отсутствии конфликта интересов. Received: July 25, 2020 Accepted: September 10, 2020 Михаил Львович Дорофеев – кандидат экономических наук, доцент Департамента общественных финансов Финансового университета при Правительстве РФ, г. Москва, Малый Златоустинский пер., д. 7, к. 1. E-mail: dorofeevml@yandex.ru. Таблица 2. Тренды в неравенстве и параметрах фискальной и монетарной политики США По ча сти интерпретации полученных результатов такой анализ в определённой сте пени субъективен, однако представленные графики позволяют читателю допол нить и/или развить полученные выводы. Дальнейшее исследование выбранной темы с применением экономико-математического моделирования и корреляци онно-регрессионного анализа представляется весьма перспективным. Impact of Monetary Policy on the Level of Economic Inequality in the United States M.L. Dorofeev DOI 10.24833/2071-8160-2020-5-74-97-114 M.L. Dorofeev DOI 10.24833/2071-8160-2020-5-74-97-114 Financial University under the Government of the Russian Federation Financial University under the Government of the Russian Federation Abstract: After the reform of the world monetary system in 1971, the competition between countries for the global market is taking place in completely new conditions. Monetary MGIMO REVIEW OF INTERNATIONAL RELATIONS • 13(5) • 2020 MGIMO REVIEW OF INTERNATIONAL RELATIONS • 13(5) • 2020 MGIMO REVIEW OF INTERNATIONAL RELATIONS • 13(5) • 2020 110 М.Л. Дорофеев ИССЛЕДОВАТЕЛЬСКИЕ СТАТЬИ and fiscal authorities have accumulated vast experience in regulating the economy and strengthening country competitive advantages through complex mechanisms of quanti tative easing, foreign exchange rates manipulation, increasing debts, etc. Overcoming the consequences of the financial crises of the 21st century every time forces monetary regula tors to implement increasingly radical measures in order to save the economy by injecting enormous amounts of liquidity into the market to buy out bad corporate debts as well as government debt securities. At the same time, the questions of how monetary policy affects the level of economic inequality and who is its beneficiary are becoming more relevant. and fiscal authorities have accumulated vast experience in regulating the economy and strengthening country competitive advantages through complex mechanisms of quanti tative easing, foreign exchange rates manipulation, increasing debts, etc. Overcoming the consequences of the financial crises of the 21st century every time forces monetary regula tors to implement increasingly radical measures in order to save the economy by injecting enormous amounts of liquidity into the market to buy out bad corporate debts as well as government debt securities. At the same time, the questions of how monetary policy affects the level of economic inequality and who is its beneficiary are becoming more relevant. The article seeks to analyze the impact of changes in monetary policy parameters on wealth inequality in the United States. Given the cyclical nature of economic inequality, the main method of research was chosen as a graphical statistical analysis, since it allows to identify trends effectively and keep in focus more than 100-year picture of changes in the analyzed indicators. For a more holistic picture, the dynamics of economic wealth inequality level were compared not only with key indicators of monetary policy, but also with the dynamics of marginal tax rates in US.i yi y The article seeks to analyze the impact of changes in monetary policy parameters on wealth inequality in the United States. Given the cyclical nature of economic inequality, the main method of research was chosen as a graphical statistical analysis, since it allows to identify trends effectively and keep in focus more than 100-year picture of changes in the analyzed indicators. MGIMO REVIEW OF INTERNATIONAL RELATIONS • 13(5) • 2020 For a more holistic picture, the dynamics of economic wealth inequality level were compared not only with key indicators of monetary policy, but also with the dynamics of marginal tax rates in US. g One conclusion of the research is that wealth inequality depends more on fiscal adjustment and marginal tax rates than on monetary factors. Inadequate marginal income and inheri tance tax rates are factors of rising of wealth inequality in US. Changing of monetary system settings also influences on the level of wealth inequality, because it affects the valuation of financial assets, and therefore the wealth of the richest people in US. Another important conclusion is the idea that the new monetary policy, despite all fears that it is a source of growing economic inequality, is acceptable with marginal income and inheritance tax rates of about 60% and with effective macroprudential regulation of US economy. Keywords: wealth inequality, economic inequality, monetary policy, taxes, interest rates, monetary policy, government regulation Conflict of Interests: The author declares the absence of conflict of interests. Conflict of Interests: The author declares the absence of conflict of interests. References: About the author: Mikhail L. Dorofeev – Ph.D. in Economics, associate professor of the Public Finance Depart ment of the Financial University under the Government of the Russian Federation, Moscow. E-mail: dorofeevml@yandex.ru. References: Bernanke B.S., Gertler M. 1995. Inside the Black Box: The Credit Channel of Monetary Policy Transmission. The Journal of Economic Perspectives. 9(4). Р. 27–48. Bernanke B.S., Gertler M. 1995. Inside the Black Box: The Credit Channel of Monetary Policy Transmission. The Journal of Economic Perspectives. 9(4). Р. 27–48. Bivens J. 2015. Gauging the impact of the Fed on inequality during the great recession. 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https://openalex.org/W4244016135	https://pubs.rsc.org/en/content/articlepdf/2017/ta/c7ta90183f	English	null	Correction: Size effects of a graphene quantum dot modified-blocking TiO<sub>2</sub> layer for efficient planar perovskite solar cells	Journal of materials chemistry. A	2,017	cc-by	322	Correction: Size eﬀects of a graphene quantum dot modiﬁed-blocking TiO2 layer for eﬃcient planar perovskite solar cells Jaehoon Ryu,a Jong Woo Lee,b Haejun Yu,a Juyoung Yun,a Kisu Lee,a Jungsup Lee,a Doyk Hwang,b Jooyoun Kang,b Seong Keun Kimb and Jyongsik Jang*a Cite this: J. Mater. Chem. A, 2017, 5, 18276 DOI: 10.1039/c7ta90183f www.rsc.org/MaterialsA DOI: 10.1039/c7ta90183f www.rsc.org/MaterialsA Correction for ‘Size eﬀects of a graphene quantum dot modiﬁed-blocking TiO2 layer for eﬃcient planar perovskite solar cells’ by Jaehoon Ryu et al., J. Mater. Chem. A, 2017, DOI: 10.1039/c7ta02242e. a mistake in the Acknowledgements section. The correct Acknowledgements section is as below. The authors regret a mistake in the Acknowledgements section. The correct Acknowledgements section is as below. The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers. The authors regret a mistake in the Acknowledgements section. The correct Acknowledgements section is as below. The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers. g g iety of Chemistry apologises for these errors and any consequent inconvenience to authors and re aSchool of Chemical and Biological Engineering, Seoul National University, 599 Gwanangno, Gwanakgu, Seoul 151-742, Korea. E-mail: jsjang@plaza.snu.ac.kr; Fax: +82 2 880 1604; Tel: +82 2 880 7069 bDepartment of Biophysics and Chemical Biology, Department of Chemistry, Seoul National University, Seoul 151-742, Korea Journal of Materials Chemistry A Journal of Materials Chemistry A Open Access Article. Published on 18 August 2017. Downloaded on 10/24/2024 6:24:51 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 18276 \| J. Mater. Chem. A, 2017, 5, 18276 Acknowledgements This work was supported by the Global Frontier R&D Program on Center for Multiscale Energy System funded by the National Research Foundation under the Ministry of Education, Science and Technology, Korea (NRF-2012M3A6A7054855 and NRF- 2014M3A6A7060583). This journal is © The Royal Society of Chemistry 2017 18276 \| J. Mater. Chem. A, 2017, 5, 18276
https://openalex.org/W4285318189	https://ejurnal.its.ac.id/index.php/teknik/article/download/70157/6912	Indonesian	null	Implementasi Kontrol Sudut Buka Valve Menggunakan Metode Fuzzy Logic pada Proses Pencampuran Water Coolant	Jurnal Teknik/Jurnal Teknik ITS	2,021	cc-by-sa	4,875	I. PENDAHULUAN 2. AB ini berisi menjelaskan tentang latar belakang dan juga tujuan dari penelitian yang dilakukan. Di dalam latar belakang mengandung beberapa referensi yang digunakan sebagai referensi dilakukannya penelitian. B B 2. Pada sumber lain yang berjudul “Fuzzy Logic Microcontroller” dari IEEE menyebutkan bahwa penerapan Fuzzy Logic pada mikrokontroller sangat menjanjikan karena kelebihan yang dimiliki yakni fleksibilitas, lebih murah serta dapat digunakan baik untuk Fuzzy maupun metode klasik. Performa yang bagus serta harga yang terjangkau diharapkan dapat menunjang kebutuhan khususnya peralatan-peralatan kecil [3]. Implementasi Kontrol Sudut Buka Valve Menggunakan Metode Fuzzy Logic pada Proses Pencampuran Water Coolant Waluyo Bayu Abdillah, Imam Wahyu Farid, dan Ciptian Weried Priananda Departemen Teknik Elektro Otomasi, Institut Teknologi Sepuluh Nopember (ITS) e-mail: wahyu@ee.its.ac.id Waluyo Bayu Abdillah, Imam Wahyu Farid, dan Ciptian Weried Priananda Departemen Teknik Elektro Otomasi, Institut Teknologi Sepuluh Nopember (ITS) e-mail: wahyu@ee.its.ac.id Abstrak—Proses pencampuran pra pemakaian water coolant memiliki standar yang berbeda pada setiap fungsi dan kegunaan. Pencampuran secara konvensional dilakukan secara manual oleh tangan manusia dan diukur menggunakan alat ukur analog untuk mengetahui kadar konsentrasi kompisisi coolant. Penelitian ini membuat implementasi tentang pembuatan sistem pencampuran water coolant otomatis dengan komposisi air dan bijih coolant secara otomatis dengan menggunakan piranti mikrokontroller. Konsentrasi water coolant yang dimaksud adalah sebesar 5% kadar bijih coolant tercampur. Dalam menentukan sudut buka valve/ kran sebagai jembatan aliran bijih coolant menuju proses pencampuran digunakan metode Fuzzy Logic yang diaktualisasikan pada motor servo DC dengan input data diambil dari hasil pembacaan sensor salinitas. Dari percobaan yang telah dilakukan menyatakan bahwa metode fuzzy dapat digunakan untuk mengontol derajat buka valve dengan baik. Namun secara keseluruhan hasil yang didapatkan dari percobaan masih belum mendapatkan hasil yang maksimal. Dari percobaan yang telah dilakukan, error terkecil yang berhasil dicapai masih bernilai 20%. Walau sudah memenuhi standar minimal, namun masih perlu adanya peningkatan kinerja sistem. Maka dari itu perlu adanya pengembangan dan evaluasi terhadap sistem lebih lanjut agar sistem dapat berjalan lebih baik. Water coolant memiliki kriteria atau spesifikasi khusus untuk setiap mesin produksi atau kebutuhan khusus lain. Spesifikasi yang dimaksud adalah kadar konsentrasi bijih coolant tercampur. Perlu diketahui bahwa pencampuran water coolant ini biasanya menggunakan dua komposisi berupa bijih coolant dengan air (H2O). Namun tidak menutup kemungkinan terdapat pencampuran dengan komposisi lain. Proses pencampuran pra-pemakaian water coolant pada tempat studi kasus ini dilakukan masih menggunakan cara konvensional yakni menggunakan tenaga manusia, dan mungkin banyak perusahaan lain juga yang menggunakan cara serupa. Penelitian ini memaparkan tentang pembuatan sistem pencampuran water coolant otomatis dengan memanfaatkan metode fuzzy logic dengan menggunakan piranti mikrokontroller. Adapun pada penelitian ini penulis mengambil berbagai sumber mengenai guna dijadikan referensi penelitian. Beberapa sumber batu pijakan dalam mengambil pengetahuan yang mana dijelaskan sebagai berikut: 1. Dalam SICE-ICASE International Joint Conference 2006 dengan judul “DC Motor Speed Control using Fuzzy Logic based on LabVIEW” dimana penelitian yang dilakukan adalah melihat respon penggunaan metode Fuzzy Logic pada kontrol motor DC yang diaplikasikan secara simulasi berbasis LabView. Disebutkan bahwa penggunaan Fuzzy Logic dalam proses kontrol motor DC menunjukkan pencapaian performa yang optimal [2]. Kata Kunci—Fuzzy Logic, Mikrokontroller, Otomatis, Pencampuran, Water Coolant. Kata Kunci—Fuzzy Logic, Mikrokontroller, Otomatis, Pencampuran, Water Coolant. F190 F190 JURNAL TEKNIK ITS Vol. 10, No. 2, (2021) ISSN: 2337-3539 (2301-9271 Print) A. Latar Belakang Pencampuran dan penggunaan water coolant yang tidak sesuai standar akan dapat menimbulkan kerusakan yang dapat mengakibatkan kerugian baik pada mesin operasi ataupun pada material alumunium yang sedang diproses oleh mesin tersebut. Kondisi water coolant yang tidak sesuai diindikasikan dari beberapa aspek pengukuran yakni pH, konsentrasi, serta kebersihannya. Akibat dari penggunaan water coolant yang tidak sesuai standar salah satunya adalah menimbulkan adanya percepatan korosi pada mesin operasi yang digunakan [1]. 3. 3. Sumber lain yang berjudul “Speed Control of DC Motor Using Fuzzy Logic Controller” dari Karary University, Sudan, mengatakan dalam penelitiannya yang membandingkan performa terhadap motor DC menggunakan Fuzzy Logic dan PID Controller bahwa kontroller Fuzzy memiliki performa yang lebih baik jika dibandingkan dengan PID konvensional. Performa yang lebih baik ini mencakup transient dan steady state response, juga memiliki kurva respons dinamis yang lebih baik, waktu respons lebih pendek, kesalahan kondisi stabil kecil (SSE) dan presisi tinggi dibandingkan PID konvensional [4]. 3. Sumber lain yang berjudul “Speed Control of DC Motor Using Fuzzy Logic Controller” dari Karary University, Sudan, mengatakan dalam penelitiannya yang membandingkan performa terhadap motor DC menggunakan Fuzzy Logic dan PID Controller bahwa kontroller Fuzzy memiliki performa yang lebih baik jika dibandingkan dengan PID konvensional. Performa yang lebih baik ini mencakup transient dan steady state response, juga memiliki kurva respons dinamis yang lebih baik, waktu respons lebih pendek, kesalahan kondisi stabil kecil (SSE) dan presisi tinggi dibandingkan PID konvensional [4]. Studi kasus yang diambil adalah pengoperasian water coolant dengan menggunakan mesin CNC pada sebuah perusahaan pesawat terbang di Indonesia. Water coolant dipakai pada proses produksi sebagai bahan lubrikan atau pelumas serta pendingin antara cutter CNC dengan part alumunium agar menghasilkan produk yang baik. 4. Dalam makalah seminar tugas akhir yang berjudul Dalam makalah seminar tugas akhir yang berjudul JURNAL TEKNIK ITS Vol. 10, No. 2, (2021) ISSN: 2337-3539 (2301-9271 Print) F191 Gambar 1. Diagram blok proses pengisian air. Gambar 2. Diagram blok kendali konsentrasi coolant. Gambar 3. Diagram kerja fuzzy logic. Gambar 4. Desain interkoneksi sistem. Gambar 5. Desain perancangan sistem. Gambar 1. Diagram blok proses pengisian air. Gambar 2. Diagram blok kendali konsentrasi coolant. Gambar 1. Diagram blok proses pengisian air. Gambar 1. Diagram blok proses pengisian air. Gambar 5. Desain perancangan sistem. Gambar 6. Flowchart alur kerja sistem. Gambar 2. Diagram blok kendali konsentrasi coolant. Gambar 5. Desain perancangan sistem. Gambar 6 Flowchart alur kerja sistem Gambar 2. Diagram blok kendali konsentrasi coolant. Gambar 3. II. TINJAUAN PUSTAKA Pada bab ini diberikan mengenai tinjuan teori yang berguna dan mendukung proses penelitian. Tinjuan teori ini berupa penjelasan serta sebagai pengantar penjelasan terhadap bahan, komponen dan berbagai penjelasan lain yang digunakan dalam penelitian. 5. 5. Sumber lain pada jurnal berjudul “Aplikasi Fuzzy Logic untuk Pengendali Motor DC Berbasis Mikrokontroler ATMega8535 dengan Sensor Photodioda” dari Universitas Negeri Semarang dijelaskan dengan menggunakan kendali logika Fuzzy Logic hasil performa motor DC menunjukkan hasil yang baik. Penelitian yang dilakukan berfokus pada kendali motor DC berdasar pembacaan sensor photodioda dengan menggunakan kendali logika Fuzzy Logic [6]. 5. Sumber lain pada jurnal berjudul “Aplikasi Fuzzy Logic untuk Pengendali Motor DC Berbasis Mikrokontroler ATMega8535 dengan Sensor Photodioda” dari Universitas Negeri Semarang dijelaskan dengan menggunakan kendali logika Fuzzy Logic hasil performa motor DC menunjukkan hasil yang baik. Penelitian yang dilakukan berfokus pada kendali motor DC berdasar pembacaan sensor photodioda dengan menggunakan kendali logika Fuzzy Logic [6]. A. Latar Belakang Diagram kerja fuzzy logic. Gambar 3. Diagram kerja fuzzy logic. Gambar 4. Desain interkoneksi sistem. Gambar 6. Flowchart alur kerja sistem. Gambar 6. Flowchart alur kerja sistem. Gambar 7. Flowchart sistem logika fuzzy. Gambar 4. Desain interkoneksi sistem. B. Tujuan Implementasi sistem kendali valve untuk proses penampuran water coolant ini ditujukan sebagai saran yang dapat dimanfaatkan pada proses produksi baik industri pesawat terbang maupun lainnya terutama yang memerlukan kadar konsentrasi coolant 5%. Gambar 7. Flowchart sistem logika fuzzy. Penelitian sebagaimana mestinya terdapat kelebihan dan kekurangan di dalamnya. Untuk kelebihan yang dihasilkan dapat diabil manfaatnya, dan untuk kekurangan yang ada dapat digunakan sebagai pembelajaran untuk dapat diperbaiki pada penelitian selanjutnya. “Perancangan Proses Otomatis Pada Sistem Kontrol Servo Valve Untuk Pencampuran Fluida Warna Berbasis Mikrokontroler” oleh Satria M. Nuswantara, Universitas Diponegoro, dijelaskan dalam penelitiannya mengenai proses pencampuran warna otomatis dengan mengontrol motor servo. Dalam hasil penelitiannya didapatkan nilai error yang masih cukup tinggi dan memerlukan adanya penyempurnaan metode maupun aplikasi teknologi terbarukan [5]. A. Fuzzy Logic Berdasarkan penjelasan dari kamus Webster, Logika adalah ilmu/ pengetahuan normatif yang berprinsip formal dari sebuah pemikiran. Dalam hal ini Fuzzy Logic adalah hal yang secara dalam mengupas tentang pemikiran tentang perkiraan. Dalam istilah yang lebih spesifik tentang logika JURNAL TEKNIK ITS Vol. 10, No. 2, (2021) ISSN: 2337-3539 (2301-9271 Print) F192 Gambar 10. Desain alur kerja website monitoring. Gambar 11. Grafik nilai keanggotaan input 1. Gambar 10. Desain alur kerja website monitoring. Gambar 11. Grafik nilai keanggotaan input 1. Gambar 8. Grafik nilai keanggotaan input 2. Gambar 9. Ssmulasi grafik nilai keanggotaan output. Gambar 8. Grafik nilai keanggotaan input 2. Gambar 9. Ssmulasi grafik nilai keanggotaan output. Gambar 8. Grafik nilai keanggotaan input 2. Gambar 10. Desain alur kerja website monitoring. Gambar 8. Grafik nilai keanggotaan input 2. Gambar 10. Desain alur kerja website monitoring. Gambar 10. Desain alur kerja website monitoring. Gambar 11. Grafik nilai keanggotaan input 1. Gambar 9. Ssmulasi grafik nilai keanggotaan output. Gambar 9. Ssmulasi grafik nilai keanggotaan output. Gambar 11. Grafik nilai keanggotaan input 1. Gambar 9. Ssmulasi grafik nilai keanggotaan output. fuzzy berbeda dengan logika klasik, logika ini memiliki peranan yang penting dalam kehidupan manusia dimana bertujuan untuk memecahkan atau membuat keputusan yang rasional dalam lingkungan atau lingkup yang penuh akan ketidaktepatan dan ketidakpastian [7]. atribut numerik berisi himpunan yang menunjukkan ukuran dari suatu variabel contohnya, 0, 15, 30, 45 dan sebagainya. Metode mamdani adalah salah satu dari metode yang terdapat pada metode Fuzzy, metode ini menjadi etode yang paling sering digunakan karena struktur yang sederhana. Struktur penggunannya yaitu menggunakan MIN-MAX atau MAX-PRODUCT. Untuk mendapatkan output fuzzy diperlukan 4 tahapan yang yakni Fuzzifikasi, Pembentukan Rule (Inference system, biasanya dalam IF.. THEN), Aplikasi Implikasi menggunakan fungsi MIN, komposisi antara rule yang menghasilkan ouput dengan fungsi MAX sehingga menghasilkan himpunan fuzzy yang baru. Dan tahapan terakhir adalah Defuzifikasi yang dipakai menggunakan metode centroid [9]. Aristoteles berpendapat dimana dituliskan dalam buku ”Fuzzy Logic with Engineering Application” karangan Timothy J. Ress dimana hanya perkiraan dari sebuah kebenaran adalah mungkin. Dikutip juga pendapat dari Lothfi Zadeh yang mengatakan bahwa harus ada eksploitasi toleransi terhadap ketidakpastian. Hal ini menunjukkan adanya opsi atau pilihan yang lebih variatif sehingga nilai kebenaran tidak hanya bernilai kaku pada satu dan lain hal [8]. Logika fuzzy adalah metodologi pemecahan yang dapat diimplementasikan di berbagai sistem mulai dari sistem yang sederhana, sistem embedded, jaringan PC, multichannel atau workstation berbasis akuisisi data, dan sistem kontrol. III. PERANCANGAN Perancangan sistem mencakup berbagai bidang desain yang dibutuhkan sebagai perencanaan pra-realisasi sistem. Pembahasan pada bab ini mencakup desain alur kerja sistem, desain interkoneksi sistem desain arsitektur monitoring dan lain sebagainya yang mana mencakup perancangan realisasi sistem. JURNAL TEKNIK ITS Vol. 10, No. 2, (2021) ISSN: 2337-3539 (2301-9271 Print) F193 Tabel 4. Rentang variabel linguistik kepekatan 1/ konsentrasi (input 1) No Nilai Kepekatan Interval (ADC read) 1 Tidak ≤160 2 Lumayan 220 - 420 3 Sangat 380 - 570 4 Parah ≥620 Tabel 5. Nilai keanggotaan kepekatan 2 (input 2) Keanggotaan Tidak : Keanggotaan Sangat : 1 = x ≤ 160 0 = x ≤ 380 260 −𝑥 40 = 220 ≤ x ≤260 475 −𝑥 95 = 380≤ x ≤475 0 = x≥260 570−𝑥 95 = 475≤x ≤570 0 = x ≥ 570 Keanggotaan Lumayan : Keanggotaan Parah : 0 = x ≤ 220 1 = ≥ 620 330−𝑥 110 = 220≤x ≤330 620−𝑥 100 = 520≤x ≤620 420−𝑥 90 = 330≤ x≤420 0 = ≤ 520 0 = ≥ 420 Tabel 6. Rentang variabel linguistik kepekatan 2/ konsentrasi (input 2) No Nilai Kepekatan Interval (ADC read) 1 Tidak ≤ 170 2 Lumayan 220 - 440 3 Sangat 400 - 600 4 Parah ≥ 650 Tabel 1. Nilai keanggotaan kepekatan 2 (input 2) Keanggotaan Tidak : Keanggotaan Sangat : 1 = x≤170 0 = x≤400 280 −𝑥 110 = 170≤ x ≤280 500−𝑥 100 = 400≤x ≤500 0 = x≥180 600−𝑥 100 = 500≤ x ≤600 0 = x≥600 Keanggotaan Lumayan : Keanggotaan Parah : 0 = x≤220 1 = ≥650 330 −𝑥 110 = 220≤ x ≤330 650 −𝑥 90 = 560≤ x ≤650 440−𝑥 110 = 330≤ x ≤440 0 = ≤560 0 = ≥440 Tabel 2. Nilai variabel linguistik bukaan kran (output) No Nilai Bukaan Dalam derajat (‘) 1 Kecil ≥92 2 Sedang 65- 85 3 Besar 49– 69 4 Full ≤45 Tabel 3. Nilai keanggotaan bukaan valve Keanggotaan Full : Keanggotaan Besar : 1 = x≤45 0 = x≤49 54 −𝑥 9 = 45≤ x 59−𝑥 10 = 49≤ x ≤59 0 = x≥54 69−𝑥 10 = 59< x ≤69 0 = x≥69 Keanggotaan Sedang : Keanggotaan Kecil : 0 = x≤65 1 = x ≥ 92 75−𝑥 10 = 65≤ x 95 −𝑥 3 = 95≤ x ≤92 85−𝑥 10 = 75≤ x ≤85 0 = x ≤81 0 = x≥85 Tabel 4. Tabel 1. Tabel 1. Nilai keanggotaan kepekatan 2 (input 2) Nilai keanggotaan kepekatan 2 (input 2) Nilai keanggotaan kepekatan Tabel 5. Tabel 2. Tabel 6. JURNAL TEKNIK ITS Vol. 10, No. 2, (2021) ISSN: 2337-3539 (2301-9271 Print) Rentang variabel linguistik kepekatan 2/ konsentrasi (input 2) No Nilai Kepekatan Interval (ADC read) 1 Tidak ≤ 170 2 Lumayan 220 - 440 3 Sangat 400 - 600 4 Parah ≥ 650 Tabel 6. Tabel 3. Nilai keanggotaan bukaan valve Keanggotaan Full : Keanggotaan Besar : 1 = x≤45 0 = x≤49 54 −𝑥 9 = 45≤ x 59−𝑥 10 = 49≤ x ≤59 0 = x≥54 69−𝑥 10 = 59< x ≤69 0 = x≥69 Keanggotaan Sedang : Keanggotaan Kecil : 0 = x≤65 1 = x ≥ 92 75−𝑥 10 = 65≤ x 95 −𝑥 3 = 95≤ x ≤92 85−𝑥 10 = 75≤ x ≤85 0 = x ≤81 0 = x≥85 Dalam studi kasus di lapangan, pembacaan konsentrasi coolant ini masih dilakukan menggunakan sensor refractometer analog. Keterangan dari industri serta informasi yang ada memang belum ditemukan sebuah sensor yang kompatibel dan dapat diaplikasikan untuk mendeteksi kadar konsentrasi coolant untuk diaplikasikan pada sistem kontrol otomasi. Dan pada proyek akhir ini deteksi konsentrasi water coolant yang dimasudkan seperti penjelasan sebelumnya diganti menggunakan deteksi kadar garam terlarut dalam air atau sering disebut dengan kadar salinitas. Sehingga sampel air coolant yang digunakan diganti menjadi air garam atau NaCL. C. Refraktometer Refraktometer merupakan salah satu jenis alat ukur yang digunakan untuk mengukur kadar/ konsentrasi bahan terlarut. Konsentrasi terlarut dapat berupa gula, garam, protein, dsb. Sesuai dengan namanya prinsip kerja dari refraktometer adalah memanfaatkan refraksi cahaya. Refraktometer Abbe adalah refraktometer untuk mengukur indeks bias cairan, padatan dalam cairan atau serbuk dengan indeks bias dari 1,300 sampai 1,700 dan persentase padatan 0 sampai 95%. Alat untuk menentukan indeks bias minyak, lemak, gelas optis, larutan gula, dan sebagainnya, indeks bias yang digunakan adalah pada rentang antara 1,300 dan 1,700 dapat dibaca langsung dengan ketelitian sampai 0,001 dan dapat diperkirakan sampai 0,0002 dari gelas skala di dalam. B. Water Coolant Water Coolant adalah cairan yang memiliki fungsi utama sebagai pendingin. Cairan ini biasa digunakan pada operasi permesinan berbahan besi atau baja. Fungsi lain dari cairan ini dapat digunakan untuk melakukan pelumasan terhadap proses khusus. Logika klasik menyatakan sesuatu dengan sifat biner atau bernilai 0 dan 1, iya atau tidak. Yang mana dari hal ini logika klasik memiliki nilai keanggotaan 0 dan 1. Logika fuzzy memungkinkan adanya nilai keanggotaan diantara 0 dan 1 sehingga lebih memiliki banyak pilihan dalam pengambilan keputusan. Sebagai contoh dalam sebuah keadaan bernilai iya dan tidak namun dalam saat bersamaan ia juga bernilai baik atau buruk dengan nilai keanggotaan yang berbeda. Inilah yang menjadikan logika fuzzy memiliki kelebihan dalam hal penalaran secara bahasa, karena mudah untuk difahami dan dekat dengan kasus nyata kehidupan yang menuntuk memiliki banyak nilai/ opsi dan bervariatif. Jenis water coolant yang digunakan oleh Departemen Produksi PT. Dirgantara Indonesia dan khususnya mesin Deckel Maho-DMC 210U adalah Blaser Blasocut-2000 Universal. Komposisi yang dipakai sesuai standar industri yang ditetapkan adalah perbandingan 1/20 atau berkonsentrasi 5% untuk kadar coolant yang digunakan. Pada proses produksi PT. Dirgantara Indonesia water coolant digunakan sebagai bahan pelumasan serta pendingin yang disemprotkan pada proses milling ataupun cutting alumunium. Hal ini digunakan untuk mengurangi adanya panas yang dihasilkan dari gesekan cutter dengan alumunium yang berlebihan serta menjaga material alumunium yang sedang menjalani proses produksi agar tidak rusak dan menghasilkan hasil terbaik. Himpunan fuzzy memiliki duat atribut yaki linguistik dan numerik. Atribut linguistik digunakan untuk mewakili suatu keadaan tertentu seperti skala kecepatan motor yang bernilai LAMBAT, SEDANG, CEPAT dan lainnya. Sedangkan B. Inferensi Sistem Inferensi sistem dibentuk menggunakan aturan yang ada pada logika fuzzy. Inferensi sistem terbentuk menjadi 3 bagian yakni fuzzifikasi, aturan fuzzy serta defuzifikasi yang menghasilkan nilai output fuzzy. Proses fuzzifikasi menyatakan nilai himpunan yang digunakan dalam sistem perhitungan fuzzy logic. Nilai yang dibentuk adalah berupa himpunan yang dibedakan menjadi bagian input serta output yang masing-masing memiliki variabel linguistik berbeda. Tabel 9. Nilai input akan diproses dalam inferensi sistem dan menghasilkan nilai output sistem. Nilai input pada penelitian ini adalah berupa data pembacaan 2 sensor salinitas dengan hasil ADC 0-1024. Sedangkan output yang dihasilkan adalah berupa nilai yang digunakan untuk mengatur derajat buka valve/ kran. Tabel dan keterangan dari nilai input dan output akan dijelaskan pada Tabel 1. logika fuzzy digambarkan secara terpisah dalam flowchart sebagaimana gambar 3. Diagram sistem kendali yang menjelaskan proses kendali pada suatu plant di dalam satu sistem yang utuh. Diagram blok ini berupa closed loop yang berari proses pengontrolan terhadap pemenuhan plant tertentu. Adapaun diagram blok yang merepresentasikan beberapa plant dari proses treatment pra-pemakaian water coolant dapat dilihat pada gambar 4. Tabel rentang variabel linguistik memuat nilai yang menjadi acuan pengelompokan nilai pembacaan sensor pada proses fuzzifikasi. Rentang nilai ini diambil atas dasar data yang diambil dalam proses analisa karakteristik kerja sensor yang dipertimbangkan berdasar informasi kebutuhan dari industri. Kemudian nilai keanggotaan input 1 seperti ditunjukkan pada tabel 2. Desain pada gambar 4 menunjukkan proses pengisian air yang ada pada sistem. Pengisian air ini dikendalikan menggunakan selenoid valve yang bekerja dengan logika Boolean atau 0 & 1. Selenoid valve akan mati jika volume air telah terpenuhi. Dalam sistem ini volume air yang dimaksud adalah sebesar 4600ml. Input 1 inferensi sistem diambil dari sensor salinitas 1 yang kemudian diklasifikasikan berdasar himpunan yang telah dibuat kemudian diproses menuju aturan fuzzy. Grafik dari nilai variabel linguistik ini terlihat seperti yang ditunjukkan pada gambar 9. Desain pada gambar 5 merupakan gambaran dari kinerja motor servo yang dikopel dengan kran/ katup sehingga dapat mengatur derajat buka kran/ katup yang dimaksud dengan masukan yang didapat dari sensor salinitas. Proses pengisian bijih coolant ini menjadi bagian yang paling penting karena metode Fuzzy Logic yang digunakan bekerja dalam sistem ini. Input kedua dari sistem inferensi yang digunakan pada metode Fuzzy Logic ini sama halnya dengan variabel yang digunakan pada input 1. Nilai variabel yang ada dibedakan menjadi Tidak, Lumayan, Sangat dan Parah. JURNAL TEKNIK ITS Vol. 10, No. 2, (2021) ISSN: 2337-3539 (2301-9271 Print) F194 logika fuzzy digambarkan secara terpisah dalam flowchart sebagaimana gambar 3. Diagram sistem kendali yang menjelaskan proses kendali pada suatu plant di dalam satu sistem yang utuh. Diagram blok ini berupa closed loop yang berari proses pengontrolan Tabel 7. Aturan logika fuzzy No Input Output (Bukaan Kran) Kepekatan 1 Kepekatan 2 1 Tidak Tidak Full Lumayan Besar Sangat Sedang Parah Sedang 2 Lumayan Tidak Besar Lumayan Besar Sangat Sedang Parah Sedang 3 Sangat Tidak Besar Lumayan Sedang Sangat Sedang Parah Kecil 4 Parah Tidak Sedang Lumayan Sedang Sangat Kecil Parah Kecil Tabel 8. Hasil kalibrasi sensor jarak No Hasil Pembacaan Sensor Penggaris 1 13.9 14 2 11.9 12 3 9.11 10.5 4 8.7 8.4 5 6.65 6.9 6 4.6 5.2 Tabel 9. Hasil scalling pembacaan sensor salinitas No Data ADC sensor Konversi Scaling ke Salinitas 1 0-160` 0% 2 160-250 1% 3 250-340 2% 4 340-430 3% 5 430-520 4% 6 520-600 5% 7 >600 Over standar menggunakan motor servo. Desain diagram Fuzzy yang dibuat dapat dilihat pada gambar 6. Dari perancangan yang telah dibuat kemudian dibentuklah desain yang menghubungkan kinerja bagian-bagian dari sistem tersebut dengan adanya keterangan garis bantu. Garis bantu ini menunjukan koneksi antar komponen yang digunakan dalam sistem, baik berupa wiring supply daya ataupun komunikasi. Perancangan wiring sistem bertujuan agar memberikan gambaran kebutuhan supply tegangan serta alur kerja kelistrikan yang diperlukan dalam suatu sistem. Pembuatan rancangan ini mencakup tahapan yang diperlukan dari awal hingga sistem berakhir baik komponen yang membutuhkan tegangan besar maupun kecil, semua terhubung dan terlihat dalam desain rancangan. Adapun desain interkoneksi sistem yang dibuat disajikan pada gambar 7. Bagian selanjutnya adalah perancangan desain sistem monitoring yang terdapat pada gambar 8. Bagian ini berfungsi untuk melakukan fungsi monitoring secara jarak jauh yang pada implementasinya dilakukan menggunakan media website. Pada proses ini perangkat yang digunakan adalah NodeMCU ESP8266 sebagai pengirim data yang dibaca oleh sensor menuju website. A. Desain Perencanaan Sistem Perancangan desain sistem bertujuan agar memberikan gambaran proses yang diperlukan dalam suatu sistem. Pembuatan rancangan ini mencakup gambar visualisasi awal alat, desain interkoneksi sistem serta flowchart alur kerja sistem. Desain perencanaan sistem dapat dilihat pada gambar 1. Perancangan sistem dapat dilakukan salah satunya dengan menggunakan flowchart. Alur kerja sistem yang akan dibuat dalam proyek akhir ini adalah seperti yang ditunjukkan flowchart pada gambar 2. Pada penelitian proyek akhir ini, refraktrometer yang diunakan adalah berjenis analog dengan obyek pembacaan adalah salinitas atau kadar garam. Rekfarktometer ini kemudian digunakan sebagai kalibrator terhadap pembacaan sensor salinitas yang digunakan pada sistem.. Gambar 2 menunjukkan alur kerja sistem secara keseluruhan, dari gambar dapat dilihat bahwa terdapat proses tersendiri dalam mengolah sistem untuk memenuhi kinerja ketercapaian konsentrasi yakni sistem logika fuzzy. Sistem IV. HASIL PENGUJIAN DAN DISKUSI Bab ini menyajikan data serta analisis dari pengujian sistem yang dilakukan. Pengujian sistem meliputi pengujian sensor, serta ujicoba sistem keseluruhan. C. Aturan Fuzzy (Rule) Dari variabel linguistik yang telah dijabarkan, aturan Fuzzy yang terbentuk adalah berjumlah 16 aturan yang kemudian menjadi nilai output dari aturan yang dibentuk. Hal ini didapat dari perkalian antara banyaknya variabel linguistik input 1 dengan banyaknya variabel linguistik yang terdapat pada input 2 yang masing-masing berjumlah 4 buah variabel. Tabel dan keterangan gambar mengenai aturan Fuzzy ini dapat dilihat pada tabel 7. B. Data Hasil Percobaan Hasil percobaan disajikan dalam bentuk perbandingan hasil pencampuran oleh alat dengan standar yang digunakan pada proses pencampuran water coolant yakni 5%. Adapun nilai pembacaan refraktometer terhadap hasil proses kerja alat ditampilkan dalam tabel 10. Hasil Aturan logika fuzzy pada akhirnya menghasilkan nilai tertentu yang digunakansebgaai hasil atau output dari aturan fuzzy. Proses mencapai nilai output ini kemudian disebut defuzifikasi dimana memiliki fungsi kebalikan dari fuzifikasi. Pada proses ini nilai output hasil aturan logika fuzzy diproses agar dapat diaplikasikan ke dalam sistem dan memenuhi nilai keanggotaan output dari aturan fuzzy yang tertera pada tabel 6. p j p percobaan yang telah ditampilakn pada tabel 10 menunjukkan bahwa sistem yang dibuat belum bekerja secara maksimal. Error yang dihasilkan antara perbandingan water coolant hasil pencampuran sistem dengan standar masih menunjukkan angka yang besar. Pada kedua percobaan yang dilakukan error menunjukkan angka 70% dan 50%. Adanya error yang besar ditimbulkan karena adanya kendala pada sistem pengaktifan servo yang digunakan sebagai pengontrol aliran bijih coolant menuju proses pencampuran. Error yang dihasilkan dari hasil ketiga ini adalah sebesar 20%, membaik dari 2 hasil percobaan sebelumnya. Nilai ini mengecil dimana menunjukkan adanya perbaikan sistem dari 50% menjadi 20% atau sebesar 30%. Dari hasil ini juga menunjukkan bahwa sistem yang dibuat sudah memenuhi standar yang diinginkan. b = koefisien variabel x Adapun hasil dari kalibrasi dari kedua sensor ditampilkan dalam tabel 8. Kalibrasi pada sensor salinitas dilakukan menggunakan proses scalling. Proses scalling diperoleh dari data yang didapat secara bertahap dan dengan menganalisis karakteristik pembacaan sensor. Adapun hasil dari proses scalling ini ditunjukkan pada tabel 9. B. Inferensi Sistem Rentang nilai pembacaan sensor yang terbaca memiliki rentang yang sama yakni mencakup 0-1024 (8 byte). Namun dalam pembagian nilai variabel linguistik, input kepekatan 2 ini memiliki rentang yang berbeda seperti yang terlihat pada tabel 3. Sensor salinitas yang digunakan sebagai masukan sistem memiliki beda ketinggian yang akan membaca kadar NaCl dengan nilai yang berbeda. Dari perbedaan nilai inilah ditentukan nilai derajat buka katup/ kran yang dikopel JURNAL TEKNIK ITS Vol. 10, No. 2, (2021) ISSN: 2337-3539 (2301-9271 Print) F195 Tabel 10. Data hasil percobaan alat No Percobaan Hasil Pencampuran Komparator Nilai Error Keterangan 1 30 Juni 2021 8,5% 5% 70% 1. Pembacaan level terganggu mixer 2. Relay menuju servo diatur dari trigger sensor level 2 2 Juli 2021 7,5% 5% 50% 1. Error mengecil setelah adanya perombakan pada program 2. Motor mixer dinyalakan secara manual agar tidak mengganggu pembacaan sensor level 3 11 Juli 2021 4% 5% 20% Dilakukan perombakan pada coding sistem untuk trigger relay yang mengaktifkan servo Pada sensor salinitas 1 yang digunakan sebagai input 1 kepekatan, nilai maksimal pembacaan sensor berada pada nilai 620ADC, namun pada input 2 ini berada pada nilai 650ADC. Hal ini terjadi karena perbedaan karakterstik sensor yang dihasilkan antara sensor salinitas 1 dan 2. Kemudian untuk nilai keanggotaan input 2 diperoleh dari tabel nilai kenaggotaan yang tersaji pada tabel 4. salinitas terbagi menjadi dua yang mana nilai hasil pembacaan sensor digunakan sebagai input pada metode fuzzy. Sedangkan sensor jarak ultrasonik digunakan sebagai input untuk trigger relay sebagai penghubung sekuensial dari pengisian air menuju proses kontrol valve pada pencampuran water coolant. Namun pada aplikasinya, fungsi ini menyebabkan akurasi pencampuran menjadi kurang baik, sehingga pada percobaan selanjutnya sensor jara hanya digunakan sebagai fungsi monitoring ketinggian air. Input 2 kemudian menjadi pasangan yang dikalkulasikan di dalam aturan logika fuzzy sehingga menghasilkan nilai output yang diinginkan. Gambar grafik dari nilai variabel keanggotaan input 2 ini seperti ditampilkan pada gambar 10. Proses kalibrasi pada sensor ultrasonik dilakukan secara bertahap dan beberapa kali proses pengukuran. Sedangkan metode yang digunakan untuk kalibrasi adalah menggunakan rumus persamaan regresi linear sederhana yang dituliskan sebagai berikut: Fuzzifikasi selanjutnya adalah dilakukan pada output logika fuzzy. Hasil pengategorian dan klasifikasi output dari logika fuzzy yang akhrnya digunakan pada proses defuzifikasi adalah seperti tertera pada tabel 5. 𝑦 = 𝑏𝑥 + 𝑎 Nilai keanggotaan pada output logika fuzzy dibagi menjadi 4 bagian yakni Full, Besar, Sedang dan Kecil. Tabel dari nilai keanggotaan output logika fuzzy ini tertera pada tabel 6. Dimana: y = variabel dependen Bukaan valve yang diaplikasikan ke dalam sistem adalah berada pada rentang nilai 0-90’. Pada nilai 0’ kran/ valve terbuka maksimal sedangkan pada posisi 90’ kran/ valve tertutup. Gambar grafik dari nilai variabel keanggotaan output ini seperti ditampilkan pada gambar11. a = konstanta a = konstanta x = variabel independen b = koefisien variabel x b = koefisien variabel x A. Data Hasil Pengujian Sensor Terdapat dua jenis sensor yang digunakan pada pembuatan sistem pencampuran water coolant ini. Kedua sensor tersebut yakni sensor salinitas dan sensor jarak ultrasonik. Sensor Hasil dari proses pencampuran dengan error yang telah disebutkan menunjukkan kondisi kelebihan dan kekurangan JURNAL TEKNIK ITS Vol. 10, No. 2, (2021) ISSN: 2337-3539 (2301-9271 Print) F196 seperti yang diinginkan. Dari hasil kinerja sistem yang ada dapat disimpulkan juga bahwa diperlukan adanya evaluasi dan pengembangan lebih lanjut agar kinerja dari alat dapat berkerja secara lebih baik. komposisi bijih coolant. Pada hal ini penggunaan bijih coolant distandarisasi menggunakan komposisi 5%. Jika coolant tidak mencapai standar yang ditetapkan, akan berdampak pada kinerja mesin yang kurang baik. Begitu pula jika komposisi coolant yang digunakan melebihi, akan menimbulkan bertambahnya biaya untuk proses pemakaian coolant. V. KESIMPULAN Dari data dan analisis yang disajikan sebelumnya dapat ditarik kesimpulan bahwa proses kontrol servo pada pencampuran water coolant terlah berhasil dilakukan. Namun hasil yang didapatkan dari hasil uji coba sistem belum menunjukkan hasil yang baik. Kinerja metode fuzzy dalam proses kontrol derajat buka valve telah bekerja sebagaimana mestinya. Namun secara kesluruhan terdapat kendala pada proses pengaktifan servo pengontrol aliran bijih coolant sehingga hasil dari pencampuran water coolant belum menghasilkan coolant yang sempurna. Kedua input dari sensor salinitas yang dipasang pada ketinggian yang berbeda pada tanki pencampuran menyatakan adanya perbedaan pembacaan nilai sensor. Dari perbedaan pembacaan sensor ini kemudian diteruskan menuju inferensi sistem Fuzzy yang kemudian menentukan derajat buka kran sebagai output sistem. Hasil dari 3 kali proses pencampuran menunjukkan data pembacaan refraktometer pada angka 8,5%, 7,5% dan 4%. Kedua hasil pertama menunjukkan adanya kelebihan pada konsentrasi coolant pada proses pencampuran. Error yang dihasilkan diukur dari standar penggunaan coolant 5% maka kedua hasil memiliki tingkat kelebihan error dengan nilai 70% dan 50%. Sedangkan pada percobaan ketiga terdapat error dengan nilai 20% dimana kondisinya adalah aman yakni 4% salinitas namun masih belum sempurna 5% UCAPAN TERIMA KASIH Penulis mengucapkan terima kasih kepada PT. Dirgantara Indonesia dimana menjadi tempat bagi penulis melaksanakan kegiatan magang serta mengambil pembahasan topik ini. DAFTAR PUSTAKA [1] M. Astuti, E. Poerwanto, dan Y. Rahman P, “Analisa Total Productive Maintenance Mesin Cincinnati di Pt. Dirgantara Indonesia,” Seminar Nasional Teknologi Informasi dan Kedirgantaraan (SENATIK), vol. 2, 2016. [2] P. Thepsatoml, A. Numsomran, V Tipsuwanpo, dan T. Teanthong, “DC Motor Speed Control using Fuzzy Logic based on LabVIEW,” SICE- ICASE International Joint Conference 2006, pp. 3617-3620, 2006. [3] A. Costa, A. De Gloria, F. Giudici, dan M. Olivieri, “Fuzzy logic microcontroller,” IEEE Micro, vol. 17, no. 1, pp. 66–74, 1997. doi: 10.1109/40.566209. [4] Y. A. Almatheel dan A. Abdelrahman, “Speed control of DC motor using Fuzzy Logic Controller,” dalam 2017 International Conference on Communication, Control, Computing and Electronics Engineering (ICCCCEE), pp. 1–8, 2017. doi: 10.1109/ICCCCEE.2017.7867673. [5] S. M. Nuswantara, I. Setiawan, dan J. Sudharto, “Perancangan Proses Otomatis pada Sistem Kontrol Servo Valve untuk Pencampuran Fluida Warna Berbasis Mikrokontroler,” Universitas Diponegoro, 2011. [6] M. Nadhif dan Suryono, “Aplikasi fuzzy logic untuk pengendali motor DC berbasis mikrokontroler ATMega8535 dengan sensor Photodioda,” Jurnal Teknik Elektro, vol. 7, no. 2, p. 5, 2015. , , , p , [7] L. A. Zadeh, “Fuzzy Logic,” California ,University of California, 1988. [8] T. J. Ross, Fuzzy logic with Engineering Applications, 3rd ed. Chichester, U.K: John Wiley, 2010. [9] J. Nasir, “Analisis fuzzy logic menentukan pemilihan motor honda dengan metode mamdani,” Jurnal Edik Informatika, vol. 2, no. 2, pp. 177–186, 2017.
https://openalex.org/W4304183748	https://journal.trunojoyo.ac.id/pgpaudtrunojoyo/article/download/16478/pdf	Malay	null	TARIAN RANUP LAMPUAN: MENINGKATKAN PERKEMBANGAN FISIK MOTORIK KASAR ANAK USIA DINI	Jurnal PG-PAUD (Jurnal Pendidikan dan Pembelajaran Anak Usia Dini)/Jurnal PG-PAUD Trunojoyo	2,022	cc-by	4,973	Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) TARIAN RANUP LAMPUAN: MENINGKATKAN PERKEMBANGAN FISIK MOTORIK KASAR ANAK USIA DINI Ade Tursina1 Rita Mahriza2 Agus Ramaida3 1,2,3Programstudi PIAUD, Institut Agama Islam Negeri Langsa Email: adetursina@iainlangsa.ac.id, ritamahriza@iainlangsa.ac.id, agusramaida@icloud.com Ade Tursina1 Rita Mahriza2 Agus Ramaida3 Ade Tursina1 Rita Mahriza2 Agus Ramaida3 1,2,3Programstudi PIAUD, Institut Agama Islam Negeri Langsa Email: adetursina@iainlangsa.ac.id, ritamahriza@iainlangsa.ac.id, agusramaida@icloud.com 1,2,3Programstudi PIAUD, Institut Agama Islam Negeri Langsa adetursina@iainlangsa.ac.id, ritamahriza@iainlangsa.ac.id, agusramaida@icloud.com Received (Juli), Accepted (September), Published (Oktober) Abstract: Ranup Lampuan Dance: Improving Physical Motor Development of Early Childhood. Gross motoric physical development is important for early childhood because it is related to other developmental aspects such as self-confidence and self-concept, so it needs proper stimulation. This study aims to find out how the teacher's activities in an effort to improve gross motor skills through Ranup Lampuan Dance in Nasurullah Islamic Kindergarten and to find out how to increase children's gross motor development through Ranup Lampuan Dance in Nasurullah Islamic Kindergarten. The research method used is classroom action research through two cycles involving as many as 17 children in group B in Nasurullah Islamic Kindergarten. The results of this study are in the form of increasing the ability of teachers to optimize children's gross motor skills through the Ranup Lampuan dance. This can be seen from the teacher's overall ability, namely in the first cycle, the average score was 3.3 with the "Good" category and in the second cycle, the average value was 4.0 with the "Very Good" category and Ranup Lampuan dance can improve motor skills. rough group B1 children. When viewed from the results of the average achievement of children in cycle 1, namely 57.2%, with the criteria "Developing according to expectations". While in the second cycle the children increased to reach an average value of 86.6% with the criteria of "Developing Very Well". It has reached the success criteria of 80%.. Kata Kunci: Kemampuan Fisik, Motorik Kasar, Tari Ranup Lampuan, Anak PENDAHULUAN 2017). Perkembangan motorik diartikan sebagai proses pertumbuhan dan perkembangan kemampuan gerak anak. Dikarenakan perkembangan motorik dipengaruhi oleh kematangan dari saraf dan otot anak, sehingga setiap gerakan sederhana dapat menciptakan interaksi yang kompleks dari berbagai bagian dan sistem tubuh yang dikendalikan oleh otak (Hasanah 2016). Kehidupan umat manusia tidak lepas dari kebutuhan akan pendidikan. Dengan kata lain bahwa manusia dapat hidup dengan berkembang dengan cita-cita untuk maju, sejahtera serta bahagia berdasarkan pandangan hidup masing-masing (Apriani, Irwandi et al. 2019). Pengetahuan sendiri dimiliki oleh manusia melalui pengalamannya yang dapat digunakan sebagai dasar bertingkah laku. Pengetahuan dijadikan pembeda antara manusia dengan makhluk hidup lainnya(Octavia 2020). Menurut Jamaris, motorik kasar merupakan bagian dari kecerdasan majemuk yang berhubungan dengan kecerdasan kinestetik pada anak. Adapun kecerdasan kinestetik diartikan sebagai keterampilan anak untuk mengontrol dan mengkoordinasi gerakan-gerakan tubuh serta mampu menggunakan alat-alat tertentu yang dimanfaatkan anak dalam aktivitas bermainnya (Helmida, Nurlita et al. 2021). Keterampilan motorik anak diartikan sebagai kemampuan pengendalian gerakan badan melalui aktivitas yang terstruktur antara susunan saraf, otot, otak, dan spinal cord. Motorik kasar mencakup gerakan tubuh yang melibatkan kinerja dari otot-otot besar atau sebagian besar atau seluruh anggota tubuh yang kemudian dipengaruhi oleh kematangan anak itu sendiri (Mahmud, 2019). Masa usia dini merupakan salah satu fase kehidupan manusia dengan potensi yang luar biasa. Oleh sebab itu, anak usia dini memerlukan berbagai layanan dan bantuan dari orang lain, khususnya orang dewasa di sekitarnya. Adapun tujuan dari layanan dan bantuan tersebut adalah untuk menstimulasi seluruh aspek perkembangan anak tumbuh dan berkembang secara optimal sesuai nilai dan norma yang ada di masyarakat. Anak usia dini juga sering disebut dengan masa keemasan (KHOLISOH 2019). Selanjutnya, Montessori menyebut masa usia dini merupakan masa periode sensitif, sehingga pada masa ini anak sangat mudah menerima rangsangan dari lingkungannya. Selain itu, pada usia emas ini anak juga peka terhadap berbagai stimulasi (Ariyanti 2016). Pada masa ini pula, anak mengalami pematangan berbagai fungsi fisik dan psikis yang mempengaruhi kesiapan anak dalam melaksanakan tugas- tugas perkembangannya. Salah satu tugas perkembangan anak usia dini adalah perkembangan fisik motorik. Perkembangan motorik kasar sangat penting distimulasi sejak dini. Menurut penelitian dari Romlah (2017) bahwa perkembangan motorik kasar berpengaruh pada tingkat kreativitas anak. Selain itu, Yusnita, Mulyani, Pramita (2021) dalam penelitiannya juga menyebutkan jika anak yang mendapatkan stimulasi motorik kasar dengan baik oleh orangtuanya juga memiliki kemampuan pengelolaan emosi yang lebih baik. Keywords: Physical Ability, Gross Motor, Ranup Lampuan Dance, Children Abstrak: Tarian Ranup Lampuan: Meningkatkan Perkembangan Fisik Motorik Anak Usia Dini. Perkembangan fisik motorik kasar menjadi penting bagi anak usia dini karena berkaitan dengan aspek perkembangan lainnya seperti kepercayaan diri maupun konsep diri, sehingga perlu stimulasi yang tepat. Penelitian ini bertujuan untuk mengetahui bagaimana kegiatan guru dalam upaya meningkatkan kemampuan motorik kasar melalui Tarian Ranup Lampuan di TK Islam Nasurullah dan untuk mengetahui bagaimana peningkatan perkembangan motorik kasar anak melalui Tarian Ranup Lampuan di TK Islam Nasurullah. Metode penelitian yang digunakan adalah penelitian tindakan kelas (action classroom research) melalui dua siklus dengan melibatkan sebanyak 17 anak pada kelompok B di TK Islam Nasurullah. Hasil penelitian ini berupa peningkatan kemampuan guru dalam mengoptimalkan kemampuan motorik kasar anak melalui tarian Ranup Lampuan. Hal ini tampak dari keseluruhan kemampuan guru yaitu siklus I mendapat nilai rata-rata 3,3 dengan kategori “Baik” dan pada siklus II yaitu dengan nilai rata-rata 4,0 dengan kategori “Sangat Baik” dan Tarian Ranup Lampuan dapat meningkatkan kemampuan motorik kasar anak kelompok B1. Apabila dilihat dari hasil rata-rata ketercapaian anak pada siklus 1 yaitu 57,2%, dengan kriteria “Berkembang Sesuai Harapan”. Sedangkan pada siklus II anak meningkat mencapai nilai rata-rata 86,6% dengan kriteria “Berkembang Sangat Baik”. Hal ini telah mencapai kriteria keberhasilan yakni 80%. Copyright (c) 2022 Ade Tursina, Rita Mahriza, Agus Ramaida Copyright (c) 2022 Ade Tursina, Rita Mahriza, Agus Ramaida 69 Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) PENDAHULUAN Selain itu, anak yang memiliki kemampuan motorik kasar yang baik juga akan lebih terampil dalam bergaul dengan teman- temannya. Dengan demikian, hal tersebut juga akan berpengaruh pada kepercayaan diri anak saat bersosialisasi dengan teman- temannya. Selain itu, pada usia emas ini anak juga peka terhadap berbagai stimulasi (Ariyanti 2016). Pada masa ini pula, anak mengalami pematangan berbagai fungsi fisik dan psikis yang mempengaruhi kesiapan anak dalam melaksanakan tugas- tugas perkembangannya. Salah satu tugas perkembangan anak usia dini adalah perkembangan fisik motorik. Berdasarkan Peraturan Menteri Pendidikan Nasional RI Nomor 137 Tahun 2014 tentang Standar Nasional Pendidikan Anak Usia Dini, tercantum bahwa tingkat pencapaian perkembangan anak usia 5-6 tahun pada aspek motorik kasar yaitu dapat melakukan gerakan tubuh secara terkoordinasi untuk melatih kelenturan keseimbangan, dan kelincahan (Aghnaita 70 Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) Tari Ranup Lampuan dibentuk pada tahun 1959 kemudian berkembang pesat dari tahun 1960 sampai dengan saat ini. Ranup Lampuan sendiri berasal dari Bahasa Aceh. Dalam bahasa daerah, kata ranup lampuan terdiri dari tiga kosa kata yaitu ranup, lam, puan. Ranup berarti sirih, sedangkan lam berarti dalam, dan puan adalah cerana. Dengan demikian, Ranup Lampuan berarti sirih di dalam cerana. Di sini peneliti menggunakan tarian ranup lampuan karena tarian tersebut tarian aceh yang dekat dengan lingkungan anak dan gerakan dalam tarian ranup lampuan lebih mudah untuk anak, selain dapat meningkatkan fisik motorik anak kita juga dapat mengajarkan anak untuk mencintai budayanya (Lubis 2019). Perkembangan motorik kasar pada anak usia dini dapat dikembangkan melalui berbagai kegiatan salah satunya kegiatan tari. Menurut laban (Yetti, 2017), anak-anak sendiri secara alamiah memiliki dorongan untuk menampilkan gerakan- gerakan “seperti tarian” dan secara spontan dan tidak disadari hal tersebut menjadi salah satu cara yang tepat dalam memperkenalkan tari sejak dini. Motorik kasar anak usia dini dapat berkembang melalui gerakan-gerakan tari karena anak mampu mengekspresikan diri dengan gerak tari dan irama musik. Ulfah, Dimyati et al. (2021) juga menyatakan bahwa kegiatan gerak dapat diterapkan dalam permainan, olah raga dan aktivitas jasmani yang dilakukan dalam kegiatan sehari-hari salah satunya kegiatan menari. Seperti halnya penelitian dari Riswandi (2021) bahwa motorik kasar juga dapat dikembangkan dengan model permainan sirkuit anak. Hasil Data awal yang diperoleh ketika peneliti melakukan pengamatan terhadap kemampuan motorik kasar anak pada kelompok B1 usia 5-6 tahun di TK Islam Nasurullah pada tanggal 26 Juli 2021. Kegiatan observasi dilakukan pada saat anak sedang melakukan kegiatan menari yang perempuan menggunakan Tepak Sirih dan anak laki-laki menari tanpa ada properti. Guru terlebih dahulu membagikan kelompok yaitu kelompok perempuan dan kelompok laki-laki. Guru memberikan contoh tarian Ranup Lampuan pada anak. Saat itu terlihat bahwa anak masih bingung dengan tarian tersebut, dan ketika guru mengajarkan tarian Ranup Lampuan pada anak terlihat bahwa anak belum mampu menggerakkan kepala, tangan, dan kaki. Sehingga anak kurang dalam menggerakkan motorik kasarnya. Berikut ini merupakan hasil observasi kemampuan motorik kasar anak kelompok B1 sebelum tindakan. melakukan Ketika melakukan gerakan lokomotor atau gerak berpindah tempat di mana bagian tubuh tertentu bergerak atau berpindah tempat, anak masih merasa kaku dan kebingunan. Hal ini ditunjukkan ketika anak berlari, melompat, berjalan, dan memanjat. Selain itu, anak juga mengalami kesulitan dalam melakukan gerak non lokomotor atau gerakan yang tidak melibatkan perpindahan tempat. Adapun gerakan tersebut seperti memutar, menggeleng, membungkuk, dan mengayun, dan gerakan terkoordinasi penyesuaian antara komponen-komponen kekuatan dan kecepatan yang dibutuhkan oleh otot-otot atau sumber tenaga dalam pelaksanaan gerak sesuai apa yang dibutuhkan gerak, penyesuaian kekuatan/kecepatan dimaksud agar gerak dapat dilakukan secara teratur sehingga mencapai hasil yang baik dan benar (Handawi 2020). PENDAHULUAN Adapun menari merupakan salah satu jenis kesenian yang berhubungan langsung dengan gerak tubuh manusia meliputi gerakan kepala, badan, tangan dan kaki. Kegiatan menari sendiri pasti terdiri dari beberapa gerakan (Wulandari, 2017). Tari ini berisikan gerak yang teratur dan lembut yang menggambarkan ekspersi keikhlasan dalam menerima tamu. Awalnya, tarian dimulai dengan gerakan memberi salam, memetik daun sirih, membuat siri seperti menyuci daun sirih, mengacip pinang, menabur gambir, mengoleskan kapur pada daun sirih, dan membungkusnya. Kemudian, penari mempersembahkan sirih tersebut kepada tamu. Gerakan tersebut dilakukan serupa dengan membuat ramuan sirih yang dilakukan oleh masyarakat Aceh untuk dikonsumsi baik secara pribadi atau umum (Putri, Lestari et al. 2015). Berdasarkan beberapa pendapat diatas, maka dapat disimpulkan bahwa Tari Ranup Lampuan merupakan salah satu tarian tradisional dari Aceh. Tarian ini merupakan tarian selamat datang yang biasanya dibawakan oleh penari wanita dengan menghadirkan sirih sebagai tanda terima masyarakat. Tari ini adalah tari penyambutan tamu yang menceritakan tujuh orang wanita yang sedangmembuat sirih (Zuhra & Saragi 2022). Sirih adalah suguhan khas Aceh yang menjadi simbol dari peumulia jamee. Asal usul tarian ini merupakan bentuk seni yang khusus dibudayakan di Aceh dengan tradisi peumulia jameeyang ada di Aceh. Peumulia jameeyang berasal dari bahasa Aceh yang artinya memuliakan tamu (Agustina 2018). Memuji tamu pada umumnya merupakan bentuk keramahan yang wajar yang biasa dilakukan oleh masyarakat Aceh. Berdasarkan pengamatan yang dilakukan di TK Islam Nasurullah pada tahap pra penelitian terlihat bahwa terdapat 10 anak yang memiliki hambatan dalam perkembangan motorik kasar. Hal ini tampak dari rendahnya perkembangan motorik kasar yaitu dilihat dari gerakan di saat anak melakukan kegiatan tari, anak yang belum mampu menggerakkan tubuh Bentuk simbolis ini terlihat ketika penari mempersembahkan sirih kepada tamu. Sirih digunakan di Aceh untuk prosesi peminangan atau pertunangan sebagai tanda komitmen dari keluarga pria untuk keluarga wanita (Agustina, 2018). 71 Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) secara terkoordinasi, anak kurang serentak dalam mengkoordinasi gerakan mata, kaki, tangan dan kepala. Proses kegiatan pembelajaran dalam penelitian ini menggunakan tarian Ranup Lampuan, yang dilaksanakan dari tanggal 26 Juli 2021 sampai dengan tanggal 31 Agustus 2021. Dalam upaya meningkatkan kemampuan motorik kasar anak melalui tarian Ranup Lampuan, maka alat pengumpulan data yang digunakan dalam penelitian ini berupa lembar observasi aktivitas guru dan lembar observasi kemampuan motorik kasar anak. METODE Penelitian ini menggunakan penelitian tindakan kelas (Classroom Action Research), di mana peneliti bekerja secara langsung dengan penelitian dari awal sampai akhir tindakan. Penelitian tindakan kelas adalah pengamatan terhadap kegiatan belajar berupa sebuah perilaku yang dirancang dengan sengaja dan dilakukan dalam konteks kelas. Tindakan tersebut dilakukan oleh guru atau oleh siswa atas arahan dari guru (Parnawi, 2020). Responden yang terlibat dalam penelitian ini adalah adalah anak kelompok B di TK Islam Nasurullah Desa Tanjung Karang, yang berjumlah jumlah 17 orang. Dalam penelitian ini peneliti sebagai instrument utama yang melakukan proses perencanaan, pelaksanaan, pengumpulan data, penganalisis data, penarikan kesimpulan dan pembuatan laporan penelitian (Ananda & Fadhilaturrahmi 2018). Dalam penelitian ini, peneliti menggunakan dua siklus PTK. Berdasarkan nilai hasil evaluasi belajar anak setelah Siklus I pertemuan 1 di atas, dapat dilihat bahwa dari 17 anak mendapatkan nilai 63,10% dengan kategori belum berkembang (BSH). Refleksi adalah penjelasan tentang hasil temuan untuk aspek-aspek yang 72 Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) perlu diperbaiki selama proses pembelajaran pada Siklus I adalah sebagai berikut: Spesifikasi doa, menyanyikan beberapa lagu dan guru memberitahukan pembelajaran untuk besok Beberapa hasil temuan yang perlu di revisi pada siklus satu untuk tahapan ke siklus II yaitu: c. Guru harus memotivasi anak agar anak mau belajar Bersama sama dalam melakukan kegiatan tarian a. Aktivitas pengelolaan pembelajaran oleh guru pada Siklus I berada pada kategori “Baik” dengan nilai keseluruhan mencapai rata-rata 3,6. Namun demikian, masih ada perbaikan yang harus dilakukan untuk siklus selanjutnya. d. Membuat tarian Ranup Lampuan lebih menarik. Berdasarkan tahapan pada siklus I diperoleh data bahwa kemampuan motorik kasar anak usia 5-6 tahun di TK Islam Nasurullah mengalami peningkatan hingga 57,22%. Namun demikian, peresentase tersebut belum mencapai target dari peneliti yaitu 75% atau kriteria bahwa anak berada pada penilaian berkembang sangat baik (BSB). Oleh karena itu, kemudian merancang kembali kegiatan tari kreasi untuk tahapan di siklus II. b. Kekurangan guru pada Siklus 1 ini disebabkan karena guru masih kurang mampu dalam menjelaskan, dan berbaur dengan anak terutama dalam mengaitkan tentang kehidupan sehari- hari,guru juga tidak menyampaikan kegiatan untuk hari selanjutnya sehingga anak sulit dalam pembelajaran. Pada tahap siklus II ini, implementasi tari Ranup Lampuan lebih difokuskan pada keaktifan anak. Anak menjadi pihak yang berperan aktif. Peneliti cukup memberikan arahan secara lisan dan mengingatkan anak sesekali ketika mereka lupa dalam melakukan gerakan. Pembahasan Kemampuan motorik kasar anak sendiri salah satunya dapat dinilai dengan demonstrasi dimana guru mencontohkan kegiatan menari. Dari kegiatan tersebut kemudian peneliti dapat mengetahui kemampuan gerak tari anak dan motorik anak dalam mengikuti contoh gerakan tari dalam kegiatan yang diberikan kepada anak usia 5–6 tahun. Anak usia 5-6 tahun merupakan anak yang berada pada kelompok B, maka kemampuan dalam menyerap motorik juga bersifat bermain- main dan belum bisa bertindak secara serius (Gius Aprilina 2019). Samsudin juga mengartikan kemampuan motorik kasar sebagai sebuah kemampuan individu dalam melaksanakan aktivitas dengan melibatkan otot-otot besar (Agusriani 2015). a. Kemampuan guru dalam mengelola pembelajaran pada Siklus II sudah berada pada kategori “Sangat Baik” dengan persentase 4,0%. b. b. Kemampuan motorik kasar anak pada Siklus II mendapat persentase 86,6% dengan kategori “Berkembang Sangat Baik”. c. Berdasarkan hasil observasi kemampuan guru pada Siklus II semakin baik dan meningkat serta telah memenuhi kriteria keberhasilan. Hal ini menunjukkan bahwa tarian Ranup Lampuan mampu mengoptimalkan kemampuan motorik kasar anak. Selain itu, guru dalam memberikan penjelasan kepada anak- anak tentang tarian tersebut membuat anak lebih paham apa dan bagaimana bentuk tarian Ranup Lampuan c. Berdasarkan hasil observasi kemampuan guru pada Siklus II semakin baik dan meningkat serta telah memenuhi kriteria keberhasilan. Hal ini menunjukkan bahwa tarian Ranup Lampuan mampu mengoptimalkan kemampuan motorik kasar anak. Selain itu, guru dalam memberikan penjelasan kepada anak- anak tentang tarian tersebut membuat anak lebih paham apa dan bagaimana bentuk tarian Ranup Lampuan Implementasi pembelajaran melalui kegiatan menari untuk menstimulasi kemampuan motorik kasar anak sesuai dengan teori behaviorisme. Pada penelitian ini, guru juga memberikan masukan atau rangsangan dengan pemberian contoh kepada anak, sedangkan anak-anak mencontoh kegiatan tersebut yang selanjutnya disebut dengan respon. Menurut Daryanto (2012), individu belajar ditandai dengan adanya perubahan perilaku, dimana pada teori behaviorisme terdapat masukan yang berupa rangsangan/stimulus dan keluaran yang berupa respon. Perkembangan kemampuan motorik kasar anak sudah meningkat dan memenuhi kriteria keberhasilan. Indikator kemampuan motorik kasar anak rata-rata sudah berada pada tingkat “Berkembang Sangat Baik” (Anggraini & Ittari 2016). Perkembangan kemampuan motorik kasar anak sudah meningkat dan memenuhi kriteria keberhasilan. Indikator kemampuan motorik kasar anak rata-rata sudah berada pada tingkat “Berkembang Sangat Baik” (Anggraini & Ittari 2016). d. Selanjutnya, peneliti menghentikan tindakan dan guru kembali membimbing anak yang masih belum mengalami peningkatan kemampuan motorik kasar berdasarkan hasil pengamatan yang dilaksanakan dengan melakukan perbaikan-perbaikan Siklus I dan Siklus II. METODE Peneliti juga menerapkan strategi di mana anak yang lebih mampu melakukan kegiatan tari Ranup Lampuan ditempatkan pada barisan depan agar dapat menjadi contoh teman lainnya. Peneliti berharap pada siklus berikutnya, kegiatan tari dapat meningkatkan motorik kasar anak usia 5-6 tahun di TK Isalam Nasurullah yang beralamat di Desa Tanjung Karang Kecamatan Karang Baru. c. Masih ada anak-anak yang masih terlihat tidak mau bekerjasama dalam melakukan kegiatan menari tarian Ranup Lampuan d. Kurangnya kerja sama dan kekompakan anak, sehingga terjadinya keributan (Kumorowati 2021). Dari beberapa kendala yang muncul, maka peneliti dan guru melakukan diskusi untuk merevisi tersebut. Adapun revisi dari beberapa masalah tersebut adalah: a. Guru harus lebih menguasai dalam mengelola pembelajaran terhadap anak,dan guru harus lebih pekaterhadap anakanak.RPPH Lebih spesifik dan ikuti acuan RPPH sekolah. a. Guru harus lebih menguasai dalam mengelola pembelajaran terhadap anak,dan guru harus lebih pekaterhadap anakanak.RPPH Lebih spesifik dan ikuti acuan RPPH sekolah. Berdasarkan nilai hasil evaluasi belajar anak pada Siklus II di atas, menunjukkan bahwa dari 17 anak mendapatkan nilai 86,6% dengan kategori “Berkembang Sangat Baik” (BSB) dan dikatakan tuntas sehingga meningkatkan motorik kasar b. Keterampilan menjelaskan,Keterampilan mengaitkan tema dengan kehidupan sehari-hari. 73 Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) anak usia 5-6 tahun yaitu kelompok B2 di TK Islam Nasurullah. anak usia 5-6 tahun yaitu kelompok B2 di TK Islam Nasurullah. melalui tarian Ranup Lampuan. Refleksi juga dilakukan peneliti untuk mengetahui hal-hal yang perlu diperbaiki selama proses pembelajaran pada Siklus II, antara lain sebagai berikut. Pembahasan Oleh sebab itu, dapat disimpulkan bahwa tarian Ranup Lampuan mampu meningkatkan kemampuan motorik kasar Berdasarkan hasil pengamatan yang dilakukan oleh pengamat di TK Islam Nasurullah Tepatnya di Desa Tanjung Karang Kecamatan Karang Baru Kabupaten Aceh Tamiang pada Kelompok 74 Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) B1 bahwa peneliti telah melakukan proses pembelajaran untuk meningkatkan kemampuan motorik kasar anak melalui tarian Ranup Lampuan. Pada Siklus I aktivitas guru mencapai nilai rata-rata 3,3 dengan kategori “Baik”, sedangkan pada Siklus II aktivitas guru berhasil mencapai nilai rata-rata 4,0 dengan kategori yang didapat adalah “Sangat Baik”. Kegiatan yang dilakukan pada Siklus II sehingga mencapai kategori sangat baik adalah dengan melakukan beberapa kegiatan sebagaimana yang telah direncanakan dalam RPPH yaitu meningkatkan kemampuan motorik kasar anak melalui tarian Ranup Lampuan (Febriana & Kusumaningtyas 2017) Gambar. 1 Grafik Hasil Aktivitas Guru Siklus I dan Siklus II Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) j , , e), ISSN: 2407-4454 (print) Gambar. 1 Grafik Hasil Aktivitas Guru Siklus I dan Siklus II B1 bahwa peneliti telah melakukan proses pembelajaran untuk meningkatkan kemampuan motorik kasar anak melalui tarian Ranup Lampuan. Pada Siklus I aktivitas guru mencapai nilai rata-rata 3,3 dengan kategori “Baik”, sedangkan pada Siklus II aktivitas guru berhasil mencapai nilai rata-rata 4,0 dengan kategori yang didapat adalah “Sangat Baik”. Kegiatan yang dilakukan pada Siklus II sehingga mencapai kategori sangat baik adalah dengan melakukan beberapa kegiatan sebagaimana yang telah direncanakan dalam RPPH yaitu meningkatkan kemampuan motorik kasar anak melalui tarian Ranup Lampuan (Febriana & Kusumaningtyas 2017). B1 bahwa peneliti telah melakukan proses pembelajaran untuk meningkatkan kemampuan motorik kasar anak melalui tarian Ranup Lampuan. Pada Siklus I aktivitas guru mencapai nilai rata-rata 3,3 dengan kategori “Baik”, sedangkan pada Siklus II aktivitas guru berhasil mencapai nilai rata-rata 4,0 dengan kategori yang didapat adalah “Sangat Baik”. Kegiatan yang dilakukan pada Siklus II sehingga mencapai kategori sangat baik adalah dengan melakukan beberapa kegiatan sebagaimana yang telah direncanakan dalam RPPH yaitu meningkatkan kemampuan motorik kasar anak melalui tarian Ranup Lampuan (Febriana & Kusumaningtyas 2017). Gambar. SIMPULAN Berdasarkan analisis penelitian yang dilakukan pada kelas B1 di TK Islam Nasurullah Desa Tanjung Karang Kecamatan Karang Baru Kabupaten Aceh Tamiang dengan melibatkan anak kelompok B1 dengan jumlah anak 17, maka dapat diambil kesimpulan sebagai berikut: Berdasarkan analisis penelitian yang dilakukan pada kelas B1 di TK Islam Nasurullah Desa Tanjung Karang Kecamatan Karang Baru Kabupaten Aceh Tamiang dengan melibatkan anak kelompok B1 dengan jumlah anak 17, maka dapat diambil kesimpulan sebagai berikut: 1. Kemampuan guru dalam mengembangkan motorik kasar anak melalui tarian Ranup Lampuan dapat meningkat. Hal ini dapat dilihat dari keseluruhan kemampuan guru yaitu siklus I mendapat nilai rata-rata 3,3 dengan kategori “Baik” dan pada siklus kedua yaitu dengan nilai rata- rata 4,0 dengan kategori “Sangat Baik” Gambar 2 Grafik Hasil Kemampuan Motorik Kasar Anak Perempuan Dari kenaikan skor pada siklus I dan siklus II juga dapat dipengaruhi oleh keterlaksanaan dari pembelajaran tari yang menarik. Dalam pembelajaran tari tentunya terdapat komponen irama musik sehingga menjadikan lebih menarik dan menyenangkan bagi anak. Hal ini sependapat menurut pendapat Decaprio (2013) yang mengungkapkan bahwa pembelajaran motorik yang menyenangkan dapat berupa permainan yang menyenangkan, pemberian rewards/penguatan kepada anak yang berhasil melaksanakan kegiatan motorik dengan benar, melaksanakan pembelajaran motorik di luar kelas. Metode menari dengan gerakan diiringi dengan alunan musik akan memfasilitasi anak dalam belajar motorik kasar. Menurut Ratnayanti (2014), latihan pola gerak sangat berdampak positif bagi keterampilan olah tubuh si anak. Dengan demikian, berdasarkan hasil dan pembahasan dapat disimpulkan bahwa penggunaan metode menari khususnya tarian Ranup lampuan dapat memfasilitasi kemampuan motorik kasar anak usia dini. 2. 2. Tarian Ranup Lampuan dapat meningkatkan kemampuan motorik kasar anak kelompok B1. Hal ditunjukkan dengan hasil penelitian tentang perkembangan kemampuan motorik kasar anak meningkat, dilihat dari rata-rata ketercapaian anak pada siklus 1 yaitu 57,2%, dengan kriteria “Berkembang Sesuai Harapan”. Selanjutnya, pada siklus II kemampuan anak meningkat mencapai nilai rata- rata 86,6% dengan kriteria “Berkembang Sangat Baik”. Hal ini menunjukkan telah adanya pencapaian kriteria keberhasilan yakni 80%. Pembahasan 1 Grafik Hasil Aktivitas Guru Siklus I dan Siklus II Berdasarkan hasil penelitian yang dilakukan pada kelompok B1 TK Islam Nasurullah memperoleh hasil yaitu kemampuan motorik kasar anak meningkat secara bertahap. Hal ini dapat dilihat pada kondisi sebelum diberikannya tindakan, anak mencapai skor persentase rata-rata 32,9%, dengan kriteria belum berkembang (BB), pada siklus I anak memperoleh skor persentase rata-rata 57,2%, dengan kriteria mulai berkembang (MB), dan siklus II menjadi semakin meningkat anak memperoleh skor persentase rata-rata 86,6% dengan kriteria berkembang sangat baik (BSB). Pelaksanaan pembelajaran meningkat kemampuan motorik kasar anak selesai pada siklus II. Kemampuan motorik kasar anak pada siklus I dan siklus II memperoleh peningkatan setiap pertemuannya. Dapat dilihat pada grafik dibawah ini, Kegiatan pada tarian Ranup Lampuan sangat disukai anak karena kegiatannya itu tidak membosankan, melainkan anak senang dan ceria ketika melakukan kegiatan menari. Apabila dibandingkan dengan siklus I, kemampuan guru pada siklus II lebih baik. Hasil pengamatan terhadap kegiatan guru pada siklus I menunjukkan bahwa nilai rata-rata sebesar 3,3 dengan kategori “Baik”. Pada siklus II mendapatkan nilai rata-rata 4,0 dengan kategori “Sangat Baik”. Adapun nilai tersebut dapat dilihat pada grafik dibawah ini: 75 Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) Gambar 2 Grafik Hasil Kemampuan Motorik Kasar Anak Perempuan SARAN Terdapat saran yang diberikan dari penelitian ini antara lain. 1. Guru sebaiknya dapat menggunakan berbagai model dan metode pembelajaran yang sesuai karakteristik dan perkembangan untuk anak di TK, sehingga dapat karakteristik dan perkembangan untuk anak di TK, sehingga dapat 76 Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) Bengkulu Tengah. Seminar Nasional Sains & Entrepreneurship. mengoptimalkan minat belajar dan selanjutnya hasil belajar anak juga dapat ikut meningkat. Ariyanti, T. (2016). Pentingnya Pendidikan Anak Usia Dini Bagi Tumbuh Kembang Anak The Importance Of Childhood Education For Child Development. Dinamika Jurnal Ilmiah Pendidikan Dasar 8(1). 2. Apabila guru akan menerapkan metode menari, maka guru sebaiknya dapat menyusun perencanaan pembelajaran dengan baik dan dapat mengelola waktu ketika pembelajaran secara tepat dan efektif. 2. Apabila guru akan menerapkan metode menari, maka guru sebaiknya dapat menyusun perencanaan pembelajaran dengan baik dan dapat mengelola waktu ketika pembelajaran secara tepat dan efektif. Daryanto, T.(2012). Konsep Pembelajaran Kreatif. Yogyakarta: Gava Media. DAFTAR PUSTAKA Decaprio, Richard. (2013). Aplikasi Teori Pembelajaran Motorik di Sekolah. Yogyakarta : DIVA Press. Aghnaita, A. (2017). Perkembangan Fisik- Motorik Anak 4-5 Tahun Pada Permendikbud no. 137 Tahun 2014 (Kajian Konsep Perkembangan Anak). Al-Athfal: Jurnal Pendidikan Anak 3(2): 219-234. Febriana, A. and L. E. Kusumaningtyas (2017). Meningkatkan motorik halus anak melalui kegiatan menganyam pada anak kelompok b usia 5-6 tahun.Jurnal AUDI: Jurnal Ilmiah Kajian Ilmu Anak dan Media Informasi PAUD 2(2). Agusriani, A. (2015). Peningkatan kemampuan motorik kasar dan kepercayaan diri melalui bermain gerak. Jurnal Pendidikan Usia Dini 9(1): 33-50. Agustina, R. (2018). Makna tari ranup lampuan (dengan pemberian uang di dalamnya) bagi masyarakat Banda Aceh, ISI Yogyakarta. Handawi, J. A. (2020). Upaya peningkatan kemampuan motorik kasar melalui tari tradisional Indang Badindin pada anak kelompok B di Raudhatul Athfal Darul Ulum Sukorambi Jember Tahun Pelajaran 2019/2020, Institut Agama Islam Negeri Jember. Akbar, M. and A. Maryani (2019). Pesan dalam Tari Ranup Lampuan. Ananda, R. and F. Fadhilaturrahmi (2018). Analisis Kemampuan Guru Sekolah Dasar dalam Implementasi Pembelajaran Tematik di SD. Jurnal Basicedu 2(2): 11-21. Hasanah, U. (2016). Pengembangan kemampuan fisik motorik melalui permainan tradisional bagi anak usia dini. Jurnal Pendidikan Anak 5(1). Anggraini, D. D. and A. Ittari (2016). Peningkatan Keterampilam Motorik Kasar Melalui Kegiatan Tari Binatang pada Anak Kelompok B TK PGRI I Langkap.Jurnal PG- PAUD Trunojoyo: Jurnal Pendidikan dan Pembelajaran Anak Usia Dini 3(2): 128-137. Helmida, U., et al. (2021). Pengaruh Tari Piring Terhadap Kemampuan Motorik Kasar Anak Usia 5-6 Tahun di TK Tahfidz Al Munawarah Desa Kualu Nenas Kabupaten Kampar. Jurnal Pendidikan Tambusai 5(2): 2821- 2826. KHOLISOH, S. N. (2019). Pengembangan Kemampuan Berbicara Anak Melalui Cerita Nabi Muhammad Al Amin Di Kelompok A Ra Nurul Ummah Kenep Tahun Pelajaran 2018/2019, Institut Agama Islam Apriani, R., et al. (2019). Kemampuan Berpikir Kritis Siswa pada Mata Pelajaran Biologi dengan Menggunakan Problem Based Learning (PBL) dan Project Based Learning (PjBL) di SMAN 2 77 Jurnal PG-PAUD Trunojoyo : Jurnal Pendidikan dan Pembelajaran Anak Usia Dini, Volume 9, Nomor 2, Oktober 2022 hal 69-78, ISSN : 2528-3553 (online), ISSN: 2407-4454 (print) meningkatkan kecerdasan kinestetik anak usia dini. Makassar: LPP- Mitra Edukasi. Sunan Giri Bojonegoro. Lubis, T. (2019). Makna Ekoleksikal dan Kultural Ranub pada Tutur Guyub Aceh. Yusnita, Nuria Mulyani, Ita Paramita. (2021). Hubungan antara riwayat stimulasi motorik kasar dengan emosi anak. Jurnal ilmiah kesehatan. 10(1). 48-53. Octavia, S. A. (2020). Motivasi belajar dalam perkembangan remaja, Sleman: Deepublish. Parnawi, A. (2020). Penelitian tindakan kelas (classroom action research), Sleman: Deepublish. Zuhra, N. and D. Saragi (2022). Pembelajaran Kearifan Lokal Berbasis Tari Tradisional Aceh Ranup Lampuan Pada Anak Usia Dini Di Tk Negeri Nanggroe Pidie Jaya. Seminar Nasional 2022-NBM Arts. Peraturan Menteri Pendidikan dan Kebudayaan Republik Indonesia No 137 Tahun 2014 tentang Standar Nasional Pendidikan Anak Usia Dini. Ratnayanti, R., & Kustiawan, U. (2014). Pengaruh Penerapan Pembelajaran Tari Kreasi Terhadap Kemampuan Motorik Kasar Siswa Tunagrahita di Sekolah Dasar Luar Biasa. Jurnal Ortopedagogia. 1(3). 238-244. Riswandi, F. N. (2021). Peningkatan kemampuan motorik kasar melalui pengembangan model permainan sirkuit anak usia 5-6 tahun. Jurnal PG-PAUD Trunojoyo: Jurnal Pendidikan dan Pembelajaran Anak Usia Dini. 8(1). 66-78. Romlah. (2017). Pengaruh motorik halus dan motorik kasar terhadap perkembangan kreatifitas anak usia dini. Jurnal Keguruan dan Ilmu Tarbiyah . 2(2). 131-137. Ulfah, A. A., et al. (2021). Analisis Penerapan Senam Irama dalam Meningkatkan Kemampuan Motorik Kasar Anak Usia Dini. Jurnal Obsesi: Jurnal Pendidikan Anak Usia Dini 5(2): 1844-1852. Wulandari, R. T. (2017). Pembelajaran olah gerak dan tari sebagai sarana ekspresi dan apresiasi seni bagi anak usia dini. Jurnal Pendidikan: 1-18. Yetti, Elindra. (2017). Model pembelajaran tari pendidikan untuk 78
https://openalex.org/W3012344773	https://journals.us.edu.pl/index.php/PR/article/view/7761/6073	Polish	null	NAJNOWSZA LITERATURA ROSYJSKO-IZRAELSKA. ZARYS ZAGADNIENIA	Przegląd Rusycystyczny	2,019	cc-by-sa	6,212	1 Po wojnie sześciodniowej, tj. trzeciej wojnie izraelsko-arabskiej w 1967 roku, nastąpiła krótkotrwała odwilż w stosunkach rosyjsko-izraelskich, czego rezultatem stał się napływ do Izraela ok. 200 tysięcy rosyjskich Żydów, w tym wielu przedstawicieli świata kultury. Zob. M. Tolts, The Post-Soviet Jewish Population in Russia and in the World, https://www.researchgate.net/publication/283926971_The_post- oviet_Jewish_population_in_Russia_and_the_world (30.06.2019). 2 Д. Маркиш, Израильско-русская литература или русско-израильские пи сатели?, „Лехаим”, январь 2001, тевес 5761, nr 1 (105), https://lechaim.ru/ ARHIV/105/markish.htm (30.06.2019). PRZEGLĄD RUSYCYSTYCZNY 2020, nr 1(169) DOI 10.31261/pr.7761 MIROSŁAWA MICHALSKA-SUCHANEK Uniwersytet Śląski ORCID: https://orcid.org/0000-0001-5262-0816 PRZEGLĄD RUSYCYSTYCZNY 2020, nr 1(169) PRZEGLĄD RUSYCYSTYCZNY 2020, nr 1(169) 3 U schyłku lat 90. liczba byłych obywateli Związku Sowieckiego, którzy przekroczyli granicę państwa Izrael, osiągnęła milion. Zob. R. Tarasiuk, Aspiracje polityczne społeczności rosyjskiej w Izraelu, https://repozytorium.uph.edu.pl/bitstream/ handle/11331/1135/Tarasiuk.R_Aspiracje_polityczne_spolecznosci.pdf?sequen ce=1 (30.06.2019). Z tej liczby ponad dwieście osób umieszczonych zostało na li ście członków Związku Pisarzy Rosyjskojęzycznych Izraela. A trzeba pamiętać, że wiele nazwisk czynnych literatów z różnych względów na niej się nie znalazło. Zob. A. Lenart, Kultura literacka rosyjskojęzycznego Izraela. Spory wokół „narodo wości” literatury, „Archiwum Emigracji: Studia — Szkice — Dokumenty” 2014, z. 1–2 (20–21), ttps://www.bu.umk.pl/Archiwum_Emigracji/gazeta/ae_20/08_ Lenart pdf (20 07 2019) NAJNOWSZA LITERATURA ROSYJSKO-IZRAELSKA. ZARYS ZAGADNIENIA Z kolejnymi alijami ze Związku Sowieckiego wyjeżdżały do Izraela setki literatów. Pierwszy ich znaczący napływ miał miejsce na prze łomie lat 60. i 70. XX wieku1. Część przybyłych wówczas do Izraela pisarzy uległa fascynacji tematyką żydowską, biblijną historią Żydów na ich własnej ziemi. Десять заповедей, высеченных Моисеем на каменной доске, на Синай ской горе стали основой нынешней цивилизации — а, значит, и ее куль туры, ее литературы. Здесь, к западу от Иордана, это ощущается особенно отчетливо. […] Как русская литература из гоголевской «Шинели», мы все вышли из Библии2 — odnotowuje Dawid Markisz (pisarz, repatriant z roku 1972). Twórcy ci zawierali w swoich utworach deklaracje przywiązania do własnych korzeni i odzyskanej ojczyzny, przez co z jednej strony legitymizo wali swoją narodową tożsamość, z drugiej zaś odreagowywali rozwi jający się wówczas w Związku Sowieckim antysemityzm. Większość 180 p 4 Д. Маркиш, Израильско-русская литература… Lenart.pdf (20.07.2019). 5 Zob. R. Katsman, Demarginalization of Contemporary Russophone Literature in Israel, http://www.academia.edu/35544512/Demarginalization_of_Contem porary_Russophone_Literature_in_Israel (25.06.2019). 6 Zob. E. Goffman, Piętno. Rozważania o zranionej tożsamości, przeł. J. Tokarska- -Bakir, Gdańskie Wydawnictwo Pedagogiczne, Gdańsk 2005, s. 26. 7 Zob. R. Szarfenberg, Marginalizacja i wykluczenie społeczne — panorama języ kowo-teoretyczna, http://rszarf.ips.uw.edu.pl/pdf/miws_panorama.pdf (22.07.2019). 8 Zob. Л. Финкель, Русскоязычная литература в израильском контнксте, w: A. Woźniak (red.), Kultura literacka emigracji rosyjskiej, ukraińskiej i bia łoruskiej XX wieku. Konteksty – estetyka – recepcja, KUL, Lublin 2013, s. 119. 9 Zob. tamże, s. 121. 10 Zob. R. Katsman, Demarginalization of Contemporary Russophone Literature in Israel… NAJNOWSZA LITERATURA ROSYJSKO-IZRAELSKA… ówczesnych pisarzy-repatriantów kontynuowała jednak działalność literacką w kształcie sprzed alii, tj. prezentowała kolejną odsłonę do brze oswojonej literatury zaangażowanej o charakterze minorowym — opozycyjnej, antysowieckiej, demaskującej najmniejsze przejawy antysemityzmu. ówczesnych pisarzy-repatriantów kontynuowała jednak działalność literacką w kształcie sprzed alii, tj. prezentowała kolejną odsłonę do brze oswojonej literatury zaangażowanej o charakterze minorowym — opozycyjnej, antysowieckiej, demaskującej najmniejsze przejawy antysemityzmu. y y Upadek komunizmu w Europie Wschodniej otworzył nowy roz dział w historii rosyjskiej repatriacji do Izraela. Wielka Alija prze łomu lat 80. i 90. mocno rozszerzyła tamtejszą przestrzeń literacką, tworząc potencjał, który powoli acz konsekwentnie zmieniał cha rakter rosyjsko-izraelskiej literatury3. Środowisko literackie zostało wówczas zasilone przez nowe pokolenie (właściwie pokolenia) — oso by urodzone w latach 50., 60., a nawet 70. (ci ostatni jako pisarze kształtowali się już w Izraelu), wolne — co nie jest bez znaczenia — od traumatycznych doświadczeń uczestników poprzedniej alii. Repre zentowali także diametralnie inną niż „siemidiesiatniki” konstrukcję mentalnościową, którą Markisz nie bez goryczy diagnozuje jako cy niczną umiejętność odnajdywania się we współczesnym świecie: Новые репатрианты, приехавшие из другой России, привезли с собой новых писателей — других. Эти другие писатели, хорошо разбирающиеся в живой проблеме спонсоров, тусовок и бандитских разборок посреди бела дня, куда ближе интересам и пониманию репатриантов 90-х годов, чем ре трограды – „семидесятники”4. Eksplozja „nowej powieści” przypada na pierwsze dziesięciolecie XXI wieku, za sprawą przede wszystkim: Aleksandra Goldsztejna, Nekoda Singera, Jelizawiety Michailczenko i Jurija Nesisa, Michaiła Judsona, Aleksandra Barasza oraz Denisa Soboleva. Roman Katsman — jeden z ważniejszych badaczy tego zagadnienia — główną deter minantę przeobrażenia rosyjskojęzycznej literatury w Izraelu widzi 181 MIROSŁAWA MICHALSKA-SUCHANEK w mającym miejsce w dwóch ostatnich dekadach procesie jej demar ginalizacji5. i Jeśli marginalizację definiuje się jako powstawanie grup margi nalnych, to demarginalizacja — proces o przeciwnym wektorze — oznacza zanikanie takiej grupy i/lub proces wyprowadzający ją poza przestrzeń uznawaną za marginalną6. Zgodnie z koncepcją Freda Mahlera za przeciwieństwo marginalizacji — czyli również skutek procesu demarginalizacji — należy uważać partycypację, uczestnic two7. W odniesieniu do rosyjsko-izraelskiej literatury od razu rodzi się jednak pytanie: partycypacja w czym? W literaturze izraelskiej? Czy może rosyjskiej tworzonej w Rosji? Najnowsza twórczość „ro syjskiego Izraela” nie wpisuje się w żadną z wymienionych opcji. W Izraelu pozostaje bytem odizolowanym, hermetycznym, literatura izraelska bowiem nie wchodzi z nią w dialog i raczej się to nie zmie ni8. , 10 Zob. R. Katsman, Demarginalization of Contemporary Russophone Literature in Israel… 12 Alija la-Tora (dosł. wstępowanie ku Torze) — wezwanie na bimę w celu publicznego odczytania fragmentu Tory. Bima jest rodzajem podium w kształcie namiotu, albo też altany z balustradą lub kratą i baldachimem, które ustawione jest w centrum synagogi, z tego miejsca odczytuje się Torę i prowadzi modły. Na bimę wchodzi się po stopniach, stąd „wstąpienie ku Torze”. Zob. Polski słownik judaistyczny, Żydowski Instytut Historyczny, https://www.jhi.pl/psj/alij(j)a; https://www.jhi. pl/psj/bima (30.07.2019). NAJNOWSZA LITERATURA ROSYJSKO-IZRAELSKA… O ile na język rosyjski przekładane są ważne utwory na przykład Amosa Oza, Meira Shaleva, Davida Grossmana i innych, o tyle ro syjskojęzycznych twórców na hebrajski tłumaczy się rzadko9. W li teraturoznawstwie izraelskim twórczość „rosyjskiego Izraela” odbie ra się jako marginalną wobec głównego nurtu literatury, tworzonej w języku hebrajskim, i sytuuje się ją w otoczeniu wytworów litera tury arabsko-izraelskiej i hebrajskiej, powstającej wśród emigran tów z państw Bliskiego Wschodu i północnej Afryki10. Postrzeganie literatury rosyjsko-izraelskiej jako marginalnej jest oczywiście spoj rzeniem subiektywnym, czynionym z punktu widzenia izraelskiego życia literackiego i nie powinno być traktowane jako obiektywne jej pozycjonowanie. To, że tak być nie powinno, wcale jednak nie ozna cza, że tak nie jest. Inne zagadnienie stanowi usytuowanie twórczości „rosyjskiego Izraela” wobec literatury rosyjskiej tworzonej w Rosji. Twórcy ży 182 11 A. Lenart, Kultura literacka rosyjskojęzycznego Izraela… 13 М. Генделев, Русскоязычная литература Израиля, http://gendelev.org/pro za/o-literature/370-literatura-doklad.html (25.06.2019). NAJNOWSZA LITERATURA ROSYJSKO-IZRAELSKA… dowskiego pochodzenia, którzy czuli się pisarzami r o s y j s k i m i i takimi chcieli pozostać, skorzystali z możliwości wyjazdu ze Związ ku Sowieckiego (zwłaszcza w latach 70.), ale przyczyną ich decyzji o opuszczeniu kraju była u c i e c z k a od ojczyzny-macochy, antyse mityzmu, cenzury, od życia w państwie, które nie pozwalało im czuć się „homo-humanus, istnieć dla siebie i dla społeczeństwa”11. Ci ży dowscy twórcy wyjeżdżali nie do Izraela, lecz przeważnie do Stanów Zjednoczonych, Francji lub Niemiec, gdzie zyskując formalny status emigranta, pozostawali nieodłączną częścią literatury rosyjskiej, tj. tworzyli w języku rosyjskim, pisali o Rosji i przede wszystkim dla ro syjskiego czytelnika. Alija przełomu lat 80. i 90. miała inny charak ter, przybrała formę masowego świadomego powrotu do narodowego praźródła, „ojczyzny swoich ojców” (takie znaczenie Żydzi nadają sło wu alija, hebr. היילע, dosłownie: wstąpienie, wznoszenie się12). Najnowsza twórczość „rosyjskiego Izraela” nie orientuje się na rzeczywistość Rosji i na rosyjskiego odbiorcę (chociaż wiele rosyj skich wydawnictw chętnie drukuje utwory rosyjskojęzycznych pisa rzy izraelskich, by wymienić takiego potentata na rosyjskim rynku wydawniczym, jak Eksmo (Эксмо), które od lat wydaje prozę Diny Rubiny), jej potencjalnymi adresatami są mówiący po rosyjsku obywatele państwa Izrael od lat żyjący w realiach kultury innej niż rosyjska. Michaił Gendelew (poeta, pisarz, repatriant z roku 1977) podsumowuje: „Мы описываем израильскую действительность, исходя из нашего уже израильского или еврейского опыта, пользуясь языком, которым лучше (в силу обстоятельств) вла деем, зная, что нас читают”13. �� Językowi przypisuje on rolę wyłącz nie instrumentalną. Wiele dekad wcześniej podobny pogląd głosił literaturoznawca, krytyk i publicysta — Wasilij Lwow-Rogaczewskij. Nawiązując do przekonań narodowego wieszcza Izraela, współtwór cy procesu rewernakularyzacji języka hebrajskiego, Chaima Bialika, podkreślał, że język, w jakim tworzy się literaturę, pełni rolę wtórną, 183 MIROSŁAWA MICHALSKA-SUCHANEK gdyż o narodowym charakterze dzieła decyduje przede wszystkim więź autora z narodem, przywiązanie do jego korzeni, ducha i kul tury: Национальность литературного произведения определяется не языком, на котором оно появилось, а господствующим настроением автора, его тя гой к�� определенному народу, сродством души автора с душой родного на рода, с его культурой, устремлением к прошлому, настоящему и будущему этого народа; определяется ответом на вопрос, для кого он работает и чьи национальные интересы защищает14. Powróćmy jednak do pojęcia marginalizacji. W kontekście tego, co powiedziano wyżej, oznaczałaby ona postrzeganie ogromnej masy izraelskich rosyjskojęzycznych twórców jako grupy zepchniętej na margines życia literackiego zarówno w Izraelu, jak i w Rosji. 14 В. Львов-Рогачевский, Русско-еврейская литература, Московское отделение государственного издательства, Москва 1922, s. 49, https://imwerden.de/pdf/ lvov-rogachevsky_russko-evrejskaya_literatura_1922__ocr.pdf (26.07.2019). 15 Л. Финкель, Русскоязычная литература…, s. 120. 16 R. Katsman, Demarginalization of Contemporary Russophone Literature in Israel… (przeł. M.M.-S.) NAJNOWSZA LITERATURA ROSYJSKO-IZRAELSKA… Proces demarginalizacji należałoby, co za tym idzie, pojmować jako inten sywne wyzbywanie się iluzji dotyczących relacji z literaturą izraelską (odejście od prób wpisania się w izraelską hierarchię literacką, czyli mówiąc kolokwialnie stawania z nią w szranki) i nostalgii w odniesie niu do rosyjskiej. „Нельзя замыкаться в культуре пусть и великой, надо по нять, что в мире нет провинций, а есть только провинциалы”15 — punktuje Leonid Finkel. Tym właśnie tropem podąża Katsman i łączy demarginalizację z procesem transformacji „geografii lite rackiej”. Proponuje przekształcenie dotychczasowej dwubiegunowej mapy centrum–peryferie (margines) w „wielowymiarową, kogni tywną mapę wielu kulturowych światów”16. Przeciwstawienie dwóch biegunów, tj. centrum i peryferii, gdzie centrum (w tym przypadku dwa centra) oznacza literatury rosyjską i izraelską, tworzoną w języ ku hebrajskim, a peryferie twórczość „rosyjskiego Izraela”, badacz zastępuje współistnieniem odrębnych kulturowych światów. Przy tym, o ile osiągnięcie stosunku równorzędności między literaturą ro syjsko-izraelską, a dwiema literaturami dotychczas uznawanymi za centralne należy do dalekiej przyszłości (jeśli w ogóle jest możliwe), o tyle relacja podrzędności już uległa (ulega) zatarciu. 184 17 Zob. tamże. NAJNOWSZA LITERATURA ROSYJSKO-IZRAELSKA… Niemałą rolę w weryfikacji pojęcia literatury marginalnej czy pe ryferyjnej odegrał rozwój Internetu. Jego globalny zasięg skutecznie przeobraża to, co Katsman nazywa „geografią literacką”, jako że dy namicznie rozszerza obecność rosyjsko-izraelskich pisarzy i poetów na światowym literackim rynku, zwłaszcza jeśli pamiętać o ich wyjąt kowej aktywności w sieci. Najnowszej literatury rosyjsko-izraelskiej nie należy zatem kate goryzować jako literatury marginalnej, peryferyjnej, ponieważ nie wpisuje się ani w rosyjską, ani w izraelską hierarchię literacką, wol na jest też od dekadenckiego nacechowania literatury emigracyjnej i nade wszystko nie jest literaturą mniejszą, minorową, ponieważ nie próbuje przemawiać do rosyjskiej większości językowej w imieniu ży dowskiej (izraelskiej) mniejszości. Ta ostatnia refleksja od pewnego czasu szeroko rezonuje w pracach literaturoznawczych przywoływa nego już tutaj Romana Katsmana17. g j j Minoryzację literatury należy rozumieć jako eksponowanie stosun ku podrzędności mniejszości wobec większości w tekstach pisanych w języku narodowym tejże większości. Jest to literatura upolitycz niona, nacechowana ideologicznie, jak na przykład rosyjsko-żydow ska twórczość w Rosji przed rewolucją 1917 roku, a także w Związku Sowieckim i wśród emigracji w okresie poprzedzającym rozpad so wieckiego imperium, gdzie ewoluowała od tekstów mniejszości od zwierciedlających ideologie większości do literatury wobec nich opo zycyjnej. Minorowość literatury kojarzy się z wyobcowaniem, nawet odrzuceniem, w najlepszym przypadku lekceważącym wobec niej dy stansem. Za nadrzędny cel taka literatura wyznacza sobie pokonanie statusu twórczości „obcej”, nie-swojej. O zjawisku tym można mówić w szerokim wymiarze jako o relacji między literaturami, jak i w wą skim, czego znamienny przykład stanowi narracja o walce bohaterów z przeciwnościami wynikającymi z życia w obcym świecie i z konse kwencjami odrzucenia. Taką twórczość, będącą izraelską odsłoną powstającej dotąd w Związku Sowieckim nieoficjalnej antysowieckiej literatury, prezentowała — o czym już była mowa — alija przełomu lat 60. i 70. Przełom wieków przyniósł zmianę kontekstu politycznego, straciło więc aktualność myślenie o literaturze jako narzędziu konte stacji i miejscu, które odzwierciedlałoby, napędzane między innymi antysemicką polityką napięcie pomiędzy centrum (Związkiem So wieckim) a peryferium — „rosyjskojęzycznym Izraelem”. Stopniowo 185 MIROSŁAWA MICHALSKA-SUCHANEK więc — w sposób niejako naturalny — rozpoczął się proces redukcji cech minorowych. Deminoryzacja rosyjsko-izraelskiej literatury odbywa się głównie (chociaż nie tylko) poprzez rewizję tematyki. Przede wszystkim należy powiedzieć o jej odpolitycznieniu. Literatura dawno odeszła od emi granckiego, antysowieckiego patosu utworów Eli Luksemburga i Da wida Markisza, zastępując go antyheroizmem i ikonoklastycznością. 18 Р. Кацман, Иерусалим: диссипативный роман Дениса Соболева, http://ma gazines.russ.ru/nlo/2017/1/ierusalim-dissipativnyj-roman-denisa-soboleva.html (25.06.2019). 19 Opisowi nowego paradygmatu „a-wiktymizacyjnego” literatury rosyjsko- izraelskiej Katsman poświęcił artykuł: Кризис виктимной парадигмы (cлучай новейшей русско-израильской литературы), „Studia Rusycystyczne” 2017, nr 27, s. 9–28. 20 Zob. B. Waligórska-Olejniczak, Życie woli barwy złamane i wielokropek. O zbiorze Z Rosji do Izraela. Opowiadania , „Iudaica Russica” 2019, nr 1(2), s. 118 (recenzja książki: Z Rosji do Izraela, wyb. i opr. M. Michalska-Suchanek, A. Lenart, „Śląsk”– Stowarzyszenie Inicjatyw Wydawniczych, Katowice 2018). 18 Р. Кацман, Иерусалим: диссипативный роман Дениса Соболева, http://ma gazines.russ.ru/nlo/2017/1/ierusalim-dissipativnyj-roman-denisa-soboleva.html (25.06.2019). 19 Opisowi nowego paradygmatu „a-wiktymizacyjnego” literatury rosyjsko- izraelskiej Katsman poświęcił artykuł: Кризис виктимной парадигмы (cлучай новейшей русско-израильской литературы), „Studia Rusycystyczne” 2017, nr 27, s. 9–28. Ż 7, 9 20 Zob. B. Waligórska-Olejniczak, Życie woli barwy złamane i wielokropek. O zbiorze Z Rosji do Izraela. Opowiadania , „Iudaica Russica” 2019, nr 1(2), s. 118 (recenzja książki: Z Rosji do Izraela, wyb. i opr. M. Michalska-Suchanek, A. Lenart, „Śląsk”– Stowarzyszenie Inicjatyw Wydawniczych, Katowice 2018). 21 Zob. R. Katsman, Demarginalization of Contemporary Russophone Literature in Israel… 22 Zob. Z. Bauman, Dwa szkice o moralności ponowoczesnej, przeł. J. Bauman, Instytut Kultury, Warszawa 1994, s. 9. 23 Zob. J. Nikitorowicz, Typy tożsamości człowieka w społeczeństwie zróżnicowanym kulturowo, http://bazhum.muzhp.pl/media//files/Chowanna/Chowanna-r2003-t1/ Chowanna-r2003-t1-s50-66/Chowanna-r2003-t1-s50-66.pdf (30.07.2019). 24 Z. Bauman, Dwa szkice o moralności… NAJNOWSZA LITERATURA ROSYJSKO-IZRAELSKA… „Emigrancki” dyskurs wciąż jeszcze da się dostrzec, ale w konstrukcji świata przedstawionego utracił pozycję dominująca i jak metaforycz nie ujmuje to Katsman: „[…] всплывает как еще один остров в цепи историко-нарративного архипелага, как еще одна упирающаяся в стенку боковая лестница в Городе бессмертных”18. A i tu docho dzi do gradacji — o ile w utworach takich pisarzy jak Dina Rubina, Nekod Singer i Michaił Yudson wciąż obecna jest pamięć o przeszło ści (głównie skoncentrowanej w obrazach miast), o tyle twórczość Dennisa Soboleva wolna jest od nostalgii wobec pierwszej ojczyzny (zapewne nie bez znaczenia pozostaje fakt, że pisarz emigrował z Ro sji jako dwudziestolatek i całe swoje dorosłe życie spędził już w Izra elu). Z najnowszej literatury rosyjsko-izraelskiej zniknęły — w swoim charakterze minorowe — wątki rosyjsko-żydowskie, rysowane na tle rosyjsko-żydowskiej, sowieckiej mentalności, nie opowiada się już o bohaterach, tkwiących w obcym, wrogim sowieckim świecie, z któ rego pragną uciec, nie pojawia się — intensywnie wcześniej wykorzy stywany — motyw władzy jako ucieleśnienia zła. Rosyjsko-izraelska literatura równolegle z procesem deminory zacji podlega dewiktymizacji19, stanowiącej — zgodnie z koncepcją Katsmana — kolejne ważne narzędzie demarginalizacyjne. Zakłada ona odejście od typowego dla literatury emigracyjnej elementu deka denckiej ofiarności20. Typowy bohater ulega przekształceniu z ofiary w pełnoprawnego uczestnika konfliktu poprzez anihilację dominują 186 21 Zob. R. Katsman, Demarginalization of Contemporary Russophone Literature in Israel… 22 Zob Z Bauman Dwa szkice o moralności ponowoczesnej przeł J Bauman 25 Д. Рубина, Вот идет Мессия!, http://loveread.ec/read_book.php?id=1933&p=1 (30.07.2019). W takim samym kontekście motyw mozaiki wykorzystała Nina Rożdiestwienskaja w szkicu Между двумя концами света. Opowiada o własnych doświadczeniach w oswajaniu Izraela, o elementach mozaiki, które powoli, ale do skutku układały się w harmonijną całość. Н. Рождественская, Между двумя концами света, w: Л. Финкель (red.), Высокие врата, Культурный центр города Ашкелона, Ашкелон 2013, s. 195–196. 26 Д. Рубина, Вот идет Мессия!… NAJNOWSZA LITERATURA ROSYJSKO-IZRAELSKA… cej dotąd opozycji: ofiara–kat i towarzyszących jej motywów ofiarno ści i heroizmu21. Istota procesu tkwi w wyjściu poza schemat myśle nia, samoświadomości i mentalności ofiary, ale także w blokowaniu gestu agresji, przez co postać uwalnia się jednocześnie od obu czło nów wskazanej wyżej antynomii. j y j y Kluczowy wpływ na charakter najnowszej rosyjsko-izraelskiej lite ratury ma tożsamościowa identyfikacja jej twórców. Zygmund Bau man podkreśla, że tożsamość nie jest jednostce dana, przeciwnie — jest czymś, co się konstruuje, jest rodzajem zadania do wykonania22, stanowi świadomy (bądź nie) proces samokreacji. W określaniu toż samości kapitalne znaczenie przybiera wartościowanie kulturowe. To właśnie poczucie silnego związku z kulturą etniczną lub narodową uzna waną za własną jest tu punktem wyjścia — dalej na różnych poziomach i w różnych sferach życia pozyskiwane są elementy składające się na samookreślenie, które finalnie łączą się z czynnikami psychicznymi i kształtują tożsamość23. ją W przypadku „rosyjskiego Izraela” deklaracja tożsamościowej identyfikacji nie była prosta. Trudne okazywało się zwłaszcza jedno znaczne określenie wspomnianej walencji kulturowej. Jaką kulturę uznać można (należy) za własną: etniczną? narodową? żydowską? ro syjską? Czy istnieją jakieś kryteria obiektywne czy rzecz tylko w afir macji? Przywołajmy po raz kolejny Baumana: „Pytanie o tożsamość wyrasta z odczucia chybotliwości istnienia, jego manipulowalności, niedookreślenia, niepewności i nieostateczności wszelkich form, ja kie przybrało”24. Uczucie tożsamościowej „niepewności” i „chybo tliwości” dotykało pisarzy „rosyjskiego Izraela” w różnym stopniu i przyjmowało najrozmaitsze formy, ale — co najważniejsze — ewo luowało. Przywołajmy wyimki z dwóch powieści Diny Rubiny, bodaj jedynej rosyjsko-izraelskiej pisarki, która zyskała sporą popularność na polskim rynku wydawniczym. W powieści z roku 1999 Oto idzie Mesjasz! dopiero pełne uświadomienie sobie przez bohaterkę (Zia mę) silnej więzi z dziadkiem-Żydem staje się przypieczętowaniem jej poczucia narodowej tożsamości i jednocześnie uwieńczeniem długich 187 MIROSŁAWA MICHALSKA-SUCHANEK poszukiwań związanych z samookreśleniem. Tak oto w przypadku Ziamy Baumanowskie zadanie zostało w stu procentach wykonane: Все было правильно: мозаичный узор судьбы подбирался по камушку, складывался медленно и старательно. И — поняла она — удивительно вер но. В первые же дни она ощутила себя камушком, точно вставленным в из гиб узора огромного мозаичного панно, кусочком смальты, которые под бирает рука Того, кто задумал весь узо25. Myśl tę Rubina kontynuuje w kolejnym — mocno emocjonalnie nacechowanym — passusie. 27 D. Rubina, Syndykat, przeł. M. Bartosik, Warszawskie Wydawnictwo Literackie MUZA SA, Warszawa 2008, s. 203. 28 И. Келейников, Тель-Шева, w: Л. Финкель (red.), Высокие врата…, s. 208. 29 Cyt. za: Л. Финкель, Русскоязычная литература…, s.117. NAJNOWSZA LITERATURA ROSYJSKO-IZRAELSKA… Na pytanie, czym różni się życie Żydów na ziemiach gojów od życia na własnej ziemi, odpowiada głosem jed nego z bohaterów: Тем, что твоя фамилия может прожить там тысячу лет, и полить кро вью, и удобрить прахом своих поколений. Но все равно придет день, ког да та земля крикнет тебе: ‚Грязный вонючий жид! Убирайся с моего тела!’ Она будет орать тебе это в лицо, даже когда ты упадешь на нее на поле боя, она отравит тебе этим воплем последние минуты жизни, и ты умрешь с го речью в сердце, даже не зная — как читается «Шма Исраель» […]. A твоя земля… Ты мог болтаться вдали от нее тысячу и две тысячи лет, но когда ты все таки вернешься сюда из прекрасного города своего детства и своей юности […] она отверзает для тебя свое лоно и рожает тебе […] А когда ты умираешь, она принимает тебя в последнее объятие и шепчет тебе слова кадиша — единственные слова, которые жаждет услышать твоя душа… Вот что такое эта земля — для тебя26. Bohaterka Syndykatu, powieści Rubiny z roku 2008, nie odczu wa już żadnych tożsamościowych dylematów, doskonale wie, kim jest i gdzie jest jej miejsce. Z palety wykorzystywanych przez pisarkę motywów znika nostalgiczny obraz pierwszej ojczyzny, a co więcej, współczesna Rosja staje się w powieści obiektem demaskatorskiego, prześmiewczego opisu. Narratorka Syndykatu postrzega świat z per spektywy kogoś, kto nie zna aktualnych rosyjskich realiów społecz no-obyczajowych, co czyni ją osobą szczególnie wyczuloną na wszel kiego rodzaju anomalie i pozwala dostrzegać to, co Rosjanom ginie 188 30 R. Katsman, Demarginalization of Contemporary Russophone Literature in Israel… 31 Zob. tamże. NAJNOWSZA LITERATURA ROSYJSKO-IZRAELSKA… w codziennym oglądzie. Odsłania więc absurdy rzeczywistości Rosji — „mało przyjemnego kraju” (w oryginale brzmi to dosadniej — „�� без умной России”). W powieści czytamy: „[...] zdarza się, i nawet dość często, że chcę się uszczypnąć albo wbić sobie szpilkę — tak to wszyst ko przypomina mi sen […]27”. Realny w Syndykacie jest tylko Izrael. Jerozolimskie kadry powieści cechuje ton ciepły, sentymentalny, któ ry mocno akcentuje więź z nową (prawdziwą) ojczyzną. Dennis Sobolev w powieści z 2005 roku zatytułowanej Jerozoli ma (w części Lekedem) przywołuje postać Wiecznego Żyda. Bohater utworu, uosobienie mitu, żyje i umiera w Wiecznym Mieście, czyli tam, gdzie zgodnie ze średniowiecznymi przekazami przebywać nie miał prawa. Osadzenie go przez Soboleva właśnie w Jerozolimie, w kontekście masowego powrotu Żydów do Erec Israel, odczytać można jako znak zakończenia wielowiekowego wygnania, symbolicz ne zatarcie piętna jednocześnie ofiary i metafizycznej winy. O tożsamościowej identyfikacji „rosyjskiego Izraela” świadczy również konsekwentne posługiwanie się przez jego przedstawicieli określeniami: repatriacja, repatriant, a przede wszystkim alija, a więc nie emigracja, którą pojmuje się jako opuszczenie własnego kraju, lecz jako powrót do pierwotnej ojczyzny, do ziemi ojców. „У каждого еврея древняя история живет внутри”28 — pisze Josif Kelejnikow. Wielka Alija uaktywniła rodzaj prapamięci czy pamięci etnicznej, tj. przenoszonej z pokolenia na pokolenie archaicznej pamięci o żydow skich korzeniach. Między repatriacją a emigracją rozciąga się prze paść również w sferze emocjonalnej. Stefan Zweig, Austriak żydow skiego pochodzenia, poeta, dramaturg, prozaik, żyjący na emigracji w Brazylii tuż przed swoją samobójczą śmiercią wyznaje: С того дня как мне пришлось жить по чужим, собственно говоря, доку ментам и паспортам, я уже никогда не ощущал, что принадлежу сам себе. Что-то навсегда надломилось в моем естественном самоотоджествлении с изначальным собственным я. […] и сегодня меня, космополита, не поки дает чувство, будто я обязан испытывать особую благадарность за каждый глоток воздуха, который вдыхаю, отнимая его у чужого народа29. Symptomatyczne, jak bardzo słowa Zweiga przypominają cytowa ny wcześniej fragment powieści Rubiny. 189 MIROSŁAWA MICHALSKA-SUCHANEK MIROSŁAWA MICHALSKA-SUCHANEK W najnowszej rosyjsko-izraelskiej literaturze pojawia się element — nazwijmy to — socjalizacji. Znika motyw wyobcowania bohate ra z rzeczywistości, w której żyje — jest to już j e g o świat. Obrazy „obcości” zostają zastąpione finalnym aktem procesu intelektualnej i kulturowej asymilacji, całkowicie — co ważne z punktu widzenia de minoryzacji — pozbawionej śladów ideologii i polityki. Katsman�� pi sze o intensywnym „вживании в различные сферы израильского социума”; „вживании в израильскую почву”30. Tworzenie nowego czasowo-przestrzennego kontinuum wynika z nowego usytuowania bohatera — jego postawa w o b e c świata zostaje zastąpiona jego istnieniem w ś w i e c i e . Zrezygnowano przy tym z uproszczonego socjologizmu na rzecz nonkonformistycznej prozy — wieloznacznej, wypełnionej intelektualną autorefleksją. Sama zmiana tematyki — trzeba pamiętać — nie sprawia, że literatura przestaje być minorowa. Rzecz należy rozpatrywać w szerszym wymiarze, jest to raczej zmiana świadomości, punktu widzenia, swojego rodzaju ewolucja mentalno ściowa. Literatura wchodzi na wyższy poziom postrzegania rzeczywistości, rozszerza spektrum oglądu świata i ewoluuje w kierunku nasycone go myślą filozoficzną uniwersalizmu. Ową zmianę koncepcji litera tury, rozszerzenie jej semantycznego spectrum, wejście na wyższy poziom postrzegania rzeczywistości i niejako osadzenie jej wymowy (interpretacji) w wymiarze „globalnym” (problemy Żydów w ogóle, problemy świata i ludzkości etc.) Katsman postrzega jako elementy pozwalające na zaliczenie najnowszej twórczości „rosyjskiego Izra ela” do literatur majorowych, a właściwie bimajorowych31. Tworzą cy rosyjsko-izraelską literaturę pisarz-Żyd należy jednocześnie do dwóch większości, które reprezentują konkretną realność kulturową, funkcjonującą w świecie wypełnionym znaczącymi faktami i obiek tami. Ze względu na miejsce, w którym żyje, jest on częścią większo ści żydowskiej (izraelskiej), nie przestając jednocześnie być częścią, choćby ze względu na miejsce urodzenia i język, którym się posługuje, większości rosyjskiej. Przy tym dochodzi do paradoksu — twórczość „rosyjskiego Izraela” z jednej strony jest literaturą zarówno rosyjską, jak i izraelską równocześnie, ostatecznie jednak nie będąc do końca ani jedną, ani drugą. Bimajorowość najnowszej literatury rosyjsko -izraelskiej nie wyklucza jej istnienia — powtórzmy — jako odrębne 190 32 Zob. tamże. 33 Zob. R. Katsman, Wstęp, przeł. A. Mrózek, w: Z Rosji do Izraela. Opowiadania…, s. 6–7. 34 Zagadnieniu temu poświęcona jest monografia: A. Oz, F. Oz-Salzberger, Żydzi i słowa, przeł. P. Paziński, Czytelnik, Warszawa 2014. 35 R. Brandstaetter, Czytanie Pisma Świętego jako modlitwa, Instytut Wydawniczy PAX, Warszawa 1986, s. 74. 36 Tamże. 37 Zob. Литература — проводник к человеческой сущности, wywiad Denisa Soboleva dla МЕОЦ (Московский Еврейский Общинный Центр), największej wspólnoty żydowskiej na terenie Rosji, udzielony 23.03.2017, https://mjcc.ru/ news/literatura-provodnik-k-chelovecheskoy-sushhnosti/ (30.07.2019). 37 Zob. Литература — проводник к человеческой сущности, wywiad Denisa Soboleva dla МЕОЦ (Московский Еврейский Общинный Центр), największej wspólnoty żydowskiej na terenie Rosji, udzielony 23.03.2017, https://mjcc.ru/ news/literatura-provodnik-k-chelovecheskoy-sushhnosti/ (30.07.2019). 38 R. Katsman, Полюбите слово. Беседа с Яковом Шехтером о книгах Второе пришествие кумранского учителя и Самоучитель каббалы, „Iudaica Rus sica” 2019, nr 2(3), s. 135. 39 Д. Соболев, Легенды горы Кармель: Четырнадцать историй о любви и вре мени, Геликон Плюс, Санкт-Петербург 2016, s. 34. 40 Geniza jest miejscem, w którym chroniąc przed profanacją, gromadzono zużyte teksty liturgiczne i święte księgi aż do czasu ich uroczystego pochówku w ziemi. Zob. Polski słownik judaistyczny, Żydowski Instytut Historyczny, https://www. jhi.pl/psj/geniza (30.07.2019). 41 Д. Соболев, Легенды горы Кармель…, s. 30. NAJNOWSZA LITERATURA ROSYJSKO-IZRAELSKA… go świata kulturowego, który sytuuje się w połowie drogi łączącej jej dwa skrajne bieguny — „rosyjskość” i „izraelskość”. I tak ją — jak się wydaje — należy postrzegać, pamiętając jednocześnie, że dążenie do zmniejszenia odległości do któregoś z biegunów determinuje (nasila) jej marginalność32. Prymarną, reprezentatywną cechę literatury „rosyjskiego Izra ela” stanowi metafizyczny, a także szczególny kulturowo-ontolo giczny realizm, rozciągający się od jego krańca egzystencjalnego po mistyczny33. Kluczowe znaczenie przypisuje się s ł o w u , które pełni fundamentalną rolę w całej tradycji żydowskiej. Oddaje ono (a przy najmniej sugeruje) immanentny sens zjawisk, rzeczy, zdarzeń, sta nowi czystą jedyną prawdę w chaosie historii34. Słowo — dawar — zawsze jawiło się w świadomości Izraelitów jako pojęcie oznaczające zarówno nieustanny ruch do przodu, jak i czyn. Jest ono „dynamiczną Wszechmocą Pana, żywiołową erupcją Jego Mocy”35 — pisze Roman Brandstaetter, oddając znaczenie dawar w języku polskim za pomo cą wymownego neologizmu s ł o w o c z y n 36. Słowa kreują przestrzeń dzieła, ale za ich sprawą przede wszystkim dokonuje się transforma cja prawdziwego świata. j p g W kulturze żydowskiej żywe jest przekonanie, że opowiadanie hi storii jest tożsame z działaniem, które posiada moc przeobrażania człowieka i przestrzeni wokół niego37. Literatura w takim rozumie niu jest nie tylko wytworem imaginacji, wyssaną z palca opowieścią o ludziach, którzy nigdy nie istnieli i zdarzeniach, które nigdy nie miały miejsca, lecz w jakimś sensie czynnością magiczną, poprzez którą ujawnia się prawda o człowieku i świecie, a energia idei skry tej w dziele literackim wykracza poza przedstawioną w nim rzeczy wistość. Literatura jest procesem poznania, poznanie zaś zakłada mentalną i emocjonalną aktywność; jest narzędziem rozkodowy 191 42 Zob. S. Krajewski, Żydowskie studiowanie Tory, „Verbum Vitae” 2005, nr 7, s. 83–90, http://dlibra.kul.pl/Content/21416/04_Krajewski.pdf (30.07.2019). 43 Sz. Weiss, Hagada, opowieść o tożsamości, https://wszystkoconajwazniejsze.pl/ prof-szewach-weiss-hagada-opowiesc-o-tozsamosci/ (30.07.2019). 44 R. Katsman, Полюбите слово. Беседа с Яковом Шехтером…, s. 130. MIROSŁAWA MICHALSKA-SUCHANEK MIROSŁAWA MICHALSKA-SUCHANEK wania świata i jednocześnie działaniem, które władne jest świat — a przede wszystkim ludzi — zmieniać. Zacytujmy Jakova Shechtera, jednego z ważniejszych pisarzy współczesnego „rosyjskiego Izraela”, repatrianta z roku 1987: „�� Литература�� — �� удивительное�� орудие�� воз действия�� на�� человека�� и�� ее�� […] �� нужно�� использовать�� , �� чтобы�� по мочь ему выбрать надежду, радость и веру, поднять голову вверх и увидеть свои крылья”38. у р Ilustrację znaczenia słowa w najnowszej rosyjsko-izraelskiej li teraturze stanowi powieść Soboleva Legendy góry Karmel. Czter naście opowiadań o miłości i czasie (Легенды��ǿ�� ǿ�� горы��ǿ�� ǿ�� Кармель��ǿ�� . ǿ�� Че тырнадцать историй о любви и времени) z 2016 roku. Tytułową górę Karmel — topograficzny locus określający jednocześnie wymowę semantyczną, ale również charakterystyczny klimat powieści Sobo leva — otaczają doliny, z których jedną, Sijach, często — jak czytamy w Legendach… — mylnie tłumaczy się jako „dolina słowa” lub nawet „dolina dyskursu”39. Bohaterem jednego z wchodzących w skład po wieści opowiadań: O smoku z góry Karmel i hajfskiej genizie (О�� дра коне горы Кармель и хайфской генизе) Sobolev czyni zlokalizowa ną w karmelskich grotach tytułową genizę40, co bez wątpienia należy przyjąć jako interpretacyjne wskazanie. Słowu pisarz nadaje w powie ści znaczenie kluczowe, jak w starych chasydzkich przypowieściach staje się ono magią, cudem, dotyka ulotnej i niedefiniowalnej pier wobytnej istoty świata. Rywka, bohaterka bajki o genizie, wierzy, że słowa zawierają esencję wszelkich p r a w d z i w y c h wartości i uczuć, że „prawda mieści się tylko w słowie, gdyż tylko w słowie można za chować własną duszę”41. Znaczenie mają wszystkie pojedyncze litery słowa. Jeśli nie rozpo zna się sensu każdej z nich — twierdzi narrator kolejnej powieściowej historii O pustym domu w Halisie (Про пустой дом на Халисе) — litera wcale nie przestaje przemawiać, tylko człowiek, uwięziony we własnym egoistycznym „ja”, nie słyszy jej głosu. To tak jak w tek 192 NAJNOWSZA LITERATURA ROSYJSKO-IZRAELSKA… ście Tory — każda litera niesie określoną treść, często ulotną i niede finiowalną. Istotny jest całościowy obraz wydarzeń, który odsłania sens stworzenia świata oraz powołania człowieka, ale jednocześnie ważności nabiera tekst postrzegany jako ciąg liter, które układają się w sekwencje znaków i kodują (właściwie dekodują) głęboką prawdę o świecie, w tym tę implikującą interpretacje mistyczne i magiczne42. W magiczność słowa wpisuje się też dodatkowa wartość. Jest ono wyrazem żmudnego procesu wrastania w izraelską ziemię. Szewach Weiss konstatuje: Wszystkie narody spaja określona narracja. Również żydowska tożsamość opiera się na powtarzanych przez wieki opowieściach. Dzięki nim Żydzi czują więź z poprzednimi pokoleniami i mają gwarancję, że następne generacje będą kultywować tradycje i zwyczaje43. W rosyjsko-izraelskiej literaturze obserwujemy to, co stanowi waż ny wyróżnik jej artystycznego i kulturowego realizmu — adaptowanie się w określonym środowisku kulturowym poprzez własne i subiek tywne materializowanie za pomocą słowa żydowskiej tradycji, książ kowych obrazów, ale także wiedzy i idei. Dokonuje się intensywna intelektualna i kulturowa asymilacja w izraelską rzeczywistość, po zbawiona śladów ideologii i polityki; raczej w jakimś sensie magiczna. Pochodną wrastania w kulturę stał się swojego rodzaju prozeli tyzm literatury. Twórcy starają się kształtować świadomość histo ryczną i narodowościową odbiorców, budować żydowski etos. Shech ter wyznaje: Главным драматическим событием моей жизни стало познание духов ности, приобщение к тайнам Торы, Талмуда, традиции, хасидизма и каб балы. Я был и остаюсь растерянным учеником на пороге сияющего мира44. Jego twórczość jest rodzajem duchowej inicjacji i jawi się jako nie przerwana opowieść o mozolnej pracy nad sobą w procesie poznawa nia nowej (lepszej?) odsłony świata — religijnego, chwilami irracjo nalnego i ezoterycznego. Utwory Shechtera cechuje żarliwość neofity i konsekwentny, w jakimś sensie programowy prozelityzm, chociaż 193 MIROSŁAWA MICHALSKA-SUCHANEK sam pisarz sprzeciwia się takiemu definiowaniu własnej twórczości. Określa ją raczej jako akt poznania (poznawania) nowego świata — duchowego i materialnego, albo literaturę post-ateizmu czy „literatu rę powrotu”45. Odnotowuje: Предыдущая еврейская литература писалась людьми, уходившими от еврейских скисающих сливок в большой мир. Выстрелом рвались Вселен ной навстречу. Дорвались, попробовали. И теперь мы возвращаемся обрат но. Нам еврейские павлины на обивке милее звездной бездны над голо вой46. Ewolucja rosyjskojęzycznej literatury w Izraelu ujawnia się nie tylko w warstwie treściowo-ideowej, ale również w kompozycji utwo rów. 45 Tamże, s. 133. 46 Tamże. 47 Д. Соболев, Иерусалим, Феникс, Ростов-на-Дону 2005, s. 418. 48 Zob. R. Katsman, Иерусалим: диссипативный роман Дениса Соболева… 47 Д. Соболев, Иерусалим, Феникс, Ростов-на-Дону 2005, s. 418. 48 Zob. R. Katsman, Иерусалим: диссипативный роман Дениса Соболева… NAJNOWSZA LITERATURA ROSYJSKO-IZRAELSKA… leży — jak się wydaje — odejść od badania jej wyłącznie jako literatury rosyjskiej, a także od opisu w kontekstach oraz zależnościach związa nych z rosyjską kulturą i stworzyć do jej interpretacji nowy naukowy i kulturowy paradygmat. leży — jak się wydaje — odejść od badania jej wyłącznie jako literatury rosyjskiej, a także od opisu w kontekstach oraz zależnościach związa nych z rosyjską kulturą i stworzyć do jej interpretacji nowy naukowy i kulturowy paradygmat. NAJNOWSZA LITERATURA ROSYJSKO-IZRAELSKA… Cechuje je fragmentaryczność, która w jakimś sensie oddaje charakter wspólnoty, w której powstaje (repatrianci pochodzą z róż nych krańców Związku Sowieckiego), nawiązuje do struktury i inter pretacyjnej otwartości midraszy i odzwierciedla — jak pisze Sobolev w Jerozolimie — „fragmentaryczność, różnorodność i niekonsekwen cję”47 ludzkiego życia. Fragmenty (opowiadania, części, rozdziały etc.) łączą się w dyssypatywną strukturę, rozumianą jako porządkowanie na nowo historycznej i kulturowej rzeczywistości, która utrzymuje się — tak widzi to Katsman — w stanie chaosu48. W rezultacie tego proce su tworzą się nowe sensy, bądź/i przeformułowaniu ulegają dotych czasowe. Powstają z jednej strony powieści składające się z opowia dań, nowel, esejów, bajek, historii czy powieści-dzienniki, z drugiej natomiast formy eksperymentalne: „вместороман”, „роман-сеть”, „роман-апокриф”, „роман-комикс”, albo „биоавтография”. k i l j j ó ść j ki l ” i Rekapitulując, najnowsza twórczość „rosyjskiego Izraela” traci cechy literatury marginalnej, peryferyjnej (chociaż zarówno w ro syjskiej, jak i izraelskiej przestrzeniach kulturowych nadal — mimo zmian — jest tak odbierana) przede wszystkim dzięki intensywnie przebiegającemu w ostatnich dwóch dekadach wieloaspektowemu procesowi demarginalizacji. 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Aspiracje polityczne społeczności rosyjskiej w Izraelu <https:// repozytorium.uph.edu.pl/bitstream/handle/11331/1135/Tarasiuk.R_Aspiracje_ polityczne_spolecznosci.pdf?sequence=1>. 196 Резюме Новейшая русско-израильская литература утрачивает черты маргинальной и периферийной литературы (хотя в российском и израильском культурных пространствах она все еще, несмотря на изменения, так и воспринимается). Это происходит в основном благодаря многоаспектному процессу демарги нализации, который динамично развивается в последние два десятилетия (концепция Романа Кацмана). Постепенно творчество «русского Израиля» приобретает статус автономной литературы, которая действует в отдельном культурном мире, вне структур, определяемых центрами и периферией. Mirosława Michalska-Suchanek NAJNOWSZA LITERATURA ROSYJSKO-IZRAELSKA… Tolts, Mark. The Post-Soviet Jewish Population in Russia and in the World <https:// www.researchgate.net/publication/283926971_The_post- Soviet_Jewish_po pulation_in_Russia_and_the_world>. Waligórska-Olejniczak, Beata. “Życie woli barwy złamane i wielokropek. O zbiorze ‘Z Rosji do Izraela. Opowiadania’.” Iudaica Russica 2019, no. 1(2). Weiss, Szewach. Hagada, opowieść o tożsamości <https://wszystkoconajwazniej sze.pl/prof-szewach-weiss-hagada-opowiesc-o-tozsamosci/>. Mirosława Michalska-Suchanek THE LATEST RUSSIAN-ISRAELI LITERATURE. OUTLINE OF THE ISSUE Summary The latest Russian-Israeli literature is losing the features of marginal and peri pheral literature (although in both Russian and Israeli cultural spaces it is still — despite the changes — perceived as such). This is mainly due to the multifaceted process of demarginalization that has been dynamically developing in the last two decades (Roman Katsman’s concept). It slowly gains the status of autonomous lite rature, which operates within a separate, own cultural world, outside the structure determined by centers and peripheries.
https://openalex.org/W2087886281	https://www.impan.pl/shop/publication/transaction/download/product/83362?download.pdf	English	null	Simultaneous Pellian equations with a single or no solution	Acta Arithmetica	2,008	cc-by	7,196	2000 Mathematics Subject Classiﬁcation: 11D09, 11D25, 11B39. Key words and phrases: quadratic and bilinear equations, simultaneous Pellian equa- tions, Lehmer numbers. 2000 Mathematics Subject Classiﬁcation: 11D09, 11D25, 11B39. Key words and phrases: quadratic and bilinear equations, simultaneous Pellia tions, Lehmer numbers. imultaneous Pellian equations with a single or no solution He 370 of system of simultaneous Pellian equations of system of simultaneous Pellian equations (m + δ)x2 −my2 = δ, y2 −bz2 = 1, (m + δ)x2 −my2 = δ, y2 −bz2 = 1, (1.3) (1.3) where δ = 1 or 4, and 2 ∤m if δ = 4. They proved that equations (1.3) then have at most one solution in positive integers (x, y, z). where δ = 1 or 4, and 2 ∤m if δ = 4. They proved that equations (1.3) then have at most one solution in positive integers (x, y, z). In this paper, we consider an extension of the above problem. In fact, we study the system of simultaneous Pellian equations (m + δ)x2 −my2 = δ, y2 −bz2 = 1, δ ∈{±1, ±2, ±4}, (1.4) (m + δ)x2 −my2 = δ, y2 −bz2 = 1, δ ∈{±1, ±2, ±4}, (1.4) where min{m, m + δ} ≥1, and 2 ∤m if δ ̸= ±1. Our main resu (m + δ)x2 −my2 = δ, y2 −bz2 = 1, δ ∈{±1, ±2, ±4}, (1.4) where min{m, m + δ} ≥1, and 2 ∤m if δ ̸= ±1. Our main result is the following. Theorem 1.1. Equations (1.4) have at most one solution in positive integers (x, y, z). From Theorem 1.1, we get the following result. Theorem 1.2. For any positive integer m and δ ∈{±1, ±2, ±4}, the system of simultaneous Pellian equations x2 −mdz2 = y2 −(m + δ)dz2 = 1 (1.5) has at most one positive integer solution (x, y, z). has at most one positive integer solution (x, y, z). In fact, if we multiply the second equation of (1.4) by m and add the resulting equation to the ﬁrst equation of (1.4), we obtain (m + δ)x2 −mbz2 = m + δ. Taking b = d(m + δ) and simplifying by m + δ, we obtain (1.5). Moreover, when d has the form 2fpg where p is an odd prime, we can deduce that equations (1.5) have at most one positive solution (x, y, z). Theorem 1.2 generalizes a theorem of Walsh [19] on equations (1.2). He proved this result for the special case m = 1 and δ = 1. The organization of this paper is as follows. In Section 2, we recall or prove some useful results following the work independently done by Yuan and Walsh. imultaneous Pellian equations with a single or no solution by Alain Togb´e (Westville, IN) and Bo He (Neijiang) by Alain Togb´e (Westville, IN) and Bo He (Neijiang) 1. Introduction. A system of simultaneous Pellian equations is a sys- tem of Diophantine equations of the form ax2 −by2 = δ1, cy2 −dz2 = δ2, ax2 −by2 = δ1, cy2 −dz2 = δ2, (1.1) where a, b, c, d, δ1, δ2 are nonzero integers, and gcd(ab, δ1) = gcd(cd, δ2) = 1. In 1969, Baker and Davenport [1] used the theory of linear forms in log- arithms of algebraic numbers to solve equations (1.1) in the particular in- stance (a, b, c, d, δ1, δ2) = (1, 3, 8, 1, −2, 7). Since then, many systems of si- multaneous Pell equations have been studied. where a, b, c, d, δ1, δ2 are nonzero integers, and gcd(ab, δ1) = gcd(cd, δ2) = 1. In 1969, Baker and Davenport [1] used the theory of linear forms in log- arithms of algebraic numbers to solve equations (1.1) in the particular in- stance (a, b, c, d, δ1, δ2) = (1, 3, 8, 1, −2, 7). Since then, many systems of si- multaneous Pell equations have been studied. Many authors have obtained upper bounds for the number of solutions of (1.1) (see for example [2], [21], [22], [3], [7]). In 1996, Ono [17] remarked that the existence of only trivial solutions of the system of simultaneous Pellian equations x2 −ay2 = z2 −by2 = 1 (1.2) (1.2) is a consequence of the related elliptic curve is a consequence of the related elliptic curve y2 = x(x + a)(x + b) having Mordell–Weil rank zero over Q. Two years later, Bennett [2] proved that the system of simultaneous Pell equations (1.2) has at most three pos- itive integer solutions, where a, b are two distinct positive integers. In 2002, Yuan [21] strengthened this result by proving that these equations have at most two solutions in positive integers (x, y, z) if max{a, b} > 1.4·1057. This result was sharpened by Bennett–Cipu–Mignotte–Okazaki [3] by removing the above condition. Progress has been made in the study of some particular cases giving at most one positive solution (see [10], [20], [6], [23], [12], [4], [19], [11] and [14]). Moreover, very recently, Li, Xia, and Yuan [13] studied a special case c ⃝Instytut Matematyczny PAN, 2008 [369] A. Togb´e and B. imultaneous Pellian equations with a single or no solution The proof of Theorem 1.1 is given in Section 3 by applying a result due to Walsh [20]. Finally, in Section 4, we study a particular case. In fact, we assume b = b′\|4m/δ + 4\| with b′ ∈{1, 2, p} where p is an odd prime and we determine all solutions to equations (1.4). 2. Some lemmas. Let n = min{m, m + δ}, i.e. n = m if δ = 1, 2, 4, m + δ if δ = −1, −2, −4, 2.1) 2. Some lemmas. Let n = min{m, m + δ}, i.e. n = m if δ = 1, 2, 4, m + δ if δ = −1, −2, −4, (2.1) (2.1) where 2 ∤m if δ ̸= ±1. Then equations (1.4) can be rewritten as (2.2) (n + c)x2 −ny2 = c, c ∈{1, 2, 4}, (2.2) (n + c)x2 −ny2 = c, c ∈{1, 2, 4}, (2.3) y2 −bz2 = 1 (if δ = c) or x2 −bz2 = 1 (if δ = −c). (2.3) y2 −bz2 = 1 (if δ = c) or x2 −bz2 = 1 (if δ = −c). (2.3) y2 −bz2 = 1 (if δ = c) or x2 −bz2 = 1 (if δ = −c). (2.3) Simultaneous Pellian equations 371 Simultaneous Pellian equations 371 In fact, when δ = −1, −2, −4, one can interchange x and y to obtain equa- tions (2.2) and (2.3). Therefore if (x, y, z) is a solution of (2.2) and (2.3) when δ = 1, 2, 4, then (y, x, z) is a solution of (2.2) and (2.3) when δ = −1, −2, −4, and vice versa. We consider the following result of Yuan [22]. Lemma 2.1. Let x1 √a+y1 √ b be the smallest solution of ax2 −by2 = δ, with δ ∈{1, 2, 4}. Then every positive integer solution (x, y) of this equation can be given by x√a + y √ b √ δ = x1 √a + y1 √ b √ δ n , n > 0, (2.4) (2.4) with 2 ∤n if min{a, b} > 1 or if (a, δ) ̸= (1, 1), (1, 4). We put N = 4n/c. Then equation (2.2) becomes (N + 4) 2 N 2 4 (2 5) We put N = 4n/c. Then equation (2.2) becomes (N + 4)x2 −Ny2 = 4. imultaneous Pellian equations with a single or no solution In fact, we have T−n = Tn, W−n = −Wn, V−n = Vn, U−n = −Un, v−n = vn, u−n = −un. T−n = Tn, W−n = −Wn, V−n = Vn, U−n = −Un, v−n = vn, u−n = −un. T−n = Tn, W−n = −Wn, V−n = Vn, U−n = −Un, v−n = vn, u−n = −un. Lemma 2.3. Let k0, k1, k2, p ∈Z with k1, k2, p > 0. If ki (i = 0, 1, 2) are all odd and k2 = 2pk1 + k0, then Lemma 2.3. Let k0, k1, k2, p ∈Z with k1, k2, p > 0. If ki (i = 0, 1, 2) are all odd and k2 = 2pk1 + k0, then Lemma 2.3. Let k0, k1, k2, p ∈Z with k1, k2, p > 0. If ki (i = 0, 1, 2) are all odd and k2 = 2pk1 + k0, then (i) T2pk1+k0 ≡(−1)pTk0 (mod Tk1); (i) T2pk1+k0 ≡(−1)pTk0 (mod Tk1); (i) T2pk1+k0 ≡(−1)pTk0 (mod Tk1); (ii) W2pk1+k0 ≡Wk0 (mod Wk1). Proof. (i) If 2 ∤p, then (ii) W2pk1+k0 ≡Wk0 (mod Wk1). (ii) W2pk1+k0 ≡Wk0 (mod Wk1). Proof. (i) If 2 ∤p, then Proof. (i) If 2 ∤p, then T2pk1+k0 + Tk0 = α2pk1+k0 + α2pk1+k0 + αk0 + αk0 √ N + 4 = (αpk1+k0 + αpk1+k0)(αpk1 + αpk1) √ N + 4 = V(pk1+k0)/2Tpk1. Therefore T2pk1+k0 ≡−Tk0 (mod Tk1). √ Therefore T2pk1+k0 ≡−Tk0 (mod Tk1). If 2 \| p, we have Therefore T2pk1+k0 ≡−Tk0 (mod Tk1). If 2 \| p, we have Therefore T2pk1+k0 ≡−Tk0 (mod Tk1). If 2 \| p, we have T2pk1+k0 −Tk0 = α2pk1+k0 + α2pk1+k0 −αk0 −αk0 √ N + 4 = (αpk1+k0 −αpk1+k0)(αpk1 −αpk1) √ N + 4 = NWpk1+k0Upk1/2. From Lemma 2.2(1), we have Uk1 \| Upk1/2 and by (2.8) and (2.9), we get Uk1 = Tk1Wk1. Thus T2pk1+k0 ≡Tk0 (mod Tk1). (ii) The proof is similar to that of (i). We have W2pk1+k0 −Wk0 = α2pk1+k0 −α2pk1+k0 −αk0 + αk0 √ N = (αpk1+k0 + αpk1+k0)(αpk1 −αpk1) √ N . If 2 ∤p, then W2pk1+k0 −Wk0 = V(pk1+k0)/2Wpk1. As Wk1 \| Wpk1, we have W2pk1+k0 ≡Wk0 (mod Wk1). If 2 \| p, then If 2 ∤p, then W2pk1+k0 −Wk0 = V(pk1+k0)/2Wpk1. As Wk1 \| Wpk1, we have W2pk1+k0 ≡Wk0 (mod Wk1). If 2 \| p, then W W (N 4)T U W2pk1+k0 −Wk0 = (N + 4)Tpk1+k0Upk1/2. imultaneous Pellian equations with a single or no solution (2.5) (2.5) Let us consider er α = √ N + 4 + √ N 2 , α = √ N + 4 − √ N 2 . By Lemma 2.1, every positive integer solution (x, y) of (2.2) or (2.5) can be represented as x √ N + 4 + y √ N 2 = αn, n > 0, 2 ∤n. (2.6) (2.6) Moreover, let β = α2. Then β is the smallest solution of the equation τ 2x2 −N(N + 4)y2 = 4, .7) τ 2x2 −N(N + 4)y2 = 4, where τ = 1 or 2 when N is odd or even respectively. √ Now let γ =v1+u1 √ b be the smallest solution of the equation v2−bu2 =1. For integers j, k, l ≥1 with 2 ∤j, we deﬁne the sequences {Tj}, {Wj}, {Vk}, {Uk}, {vl} and {ul} by αj = Tj √ N + 4 + Wj √ N 2 , (2.8) βk = Vk + Uk p N(N + 4) 2 , (2.9) γl = vl + ul √ b. (2.10) (2.8) (2.9) γl = vl + ul √ b. (2.10) Notice that (V, U) = (x, y) if 2 ∤N, and (V, U) = (2x, y) if 2 \| N. The following lemma lists some properties of Lehmer sequences. They are true not only for ({Vk}, {Uk}), but also for ({Tj}, {Wj}) and ({vl}, {ul}). Lemma 2.2. Let d = gcd(m, n) for some integers m and n. Lemma 2.2. Let d = gcd(m, n) for some integers m and n. (1) If Um ̸= 1, then Um \| Un if and only if m \| n. (2) If m > 1, then Vm \| Vn if and only if n/m is an odd integer. A. Togb´e and B. He 372 (3) gcd(Um, Un) = Ud. ( ) ( ) (4) gcd(Vm, Vn) = Vd if m/d and n/d are odd, and 1 otherwise. (5) gcd(Um, Vn) = Vd if m/d is even, and 1 otherwise. (5) gcd(Um, Vn) = Vd if m/d is even, and 1 otherwise. Proof. See Lemma 2.1 of [23], [20], or Lemma 2.2 of [13]. Proof. See Lemma 2.1 of [23], [20], or Lemma 2.2 of [13]. One can see that if we extend the above sequences to negative indices, the deﬁnition is still eﬀective. imultaneous Pellian equations with a single or no solution Therefore, we get the same result. Therefore, we get the same result. Now we assume that positive integer solutions of (2.2) and (2.3) exist. Let (x1, y1, z1) be the positive solution with the smallest z1, and (x2, y2, z2) Simultaneous Pellian equations 373 Simultaneous Pellian equations 373 be any other solution. Then there exist positive integers ji, li (i = 1, 2) with 2 ∤ji such that xi = Tji, yi = Wji, yi or xi = vli, zi = uli. (2.11) (2.11) The following result is similar to Lemma 2.4 of [23] and Lemma 2.4 of [13]. In [22], Yuan proved that for positive integers k0, k1, k2, p, we have v2pk1±k0 ≡±vk0 (mod vk1). We use it and Lemma 2.3 to get The following result is similar to Lemma 2.4 of [23] and Lemma 2.4 of [13]. In [22], Yuan proved that for positive integers k0, k1, k2, p, we have v2pk1±k0 ≡±vk0 (mod vk1). We use it and Lemma 2.3 to get Lemma 2.4. In the notations of (2.11), we have Lemma 2.4. In the notations of (2.11), we have Lemma 2.4. In the notations of (2.11), we have y1 \| y2, j1 \| j2, and l1 \| l2. Furthermore, j2/j1 and l2/l1 are odd integers. This implies x1 \| x2 and z1 \| z2. Deﬁne Deﬁne R(λ) 2k+1 = T2k+1 if λ = 1, W2k+1 if λ = −1. (2.12) (2.12) Then from the deﬁnition of α and (2.8) we obtain Then from the deﬁnition of α and (2.8) we obtain R(λ) 2k+1 = α2k+1 + λα2k+1 √ N + 2 + 2λ . (2.13) (2.13) Lemma 2.5. We have (R(λ) 2k+1)2 −1 = (N + 2 −2λ)UkUk+1. Proof. Since α + λα = √ N + 2 + 2λ, we get (R(λ) 2k+1)2 −1 = α2k+1 + λα2k+1 √ N + 2 + 2λ 2 −1 = α4k+2 + α4k+2 + 2λ N + 2 + 2λ −1 = α4k+2 + α4k+2 −(N + 2) N + 2 + 2λ = β2k+1 + β2k+1 −(β + β) N + 2 + 2λ = (βk+1 −βk+1)(βk −βk) N + 2 + 2λ = N(N + 4) N + 2 + 2λ · βk −βk p N(N + 4) · βk+1 −βk+1 p N(N + 4) = (N + 2 −2λ)UkUk+1. Definition 2.6. Let {Uk} be deﬁned by (2.9). Next, we recall the following result due to Ljunggren [16]. Next, we recall the following result due to Ljunggren [16]. 374 Lemma 2.7. β = (1 + √ 5)/2 Proof. See L The followin it from some re [9] and the ﬁrst Lemma 2.8. integer A, there with y = Au2 fo (1) N = 1, (2) N = 336 ( ) d2 374 A. Togb´e and B. He Lemma 2.7. For k > 1, Uk has a primitive prime factor p except for β = (1 + √ 5)/2 and k = 6. Moreover, p \| Uk′ if and only if k \| k′. Proof. See Lemma 2.4 of [20]. The following result is an adaptation of Lemma 2.5 of [20]. One can get it from some results on AX2 −By4 = 1, 4 due to Ljunggren [15], Cohn [8], [9] and the ﬁrst author [18]. Lemma 2.8. Let τ = 1 if 2 ∤N, and τ = 2 if 2 \| N. Then for any positive integer A, there is at most one positive solution (x, y) to the equation τ 2x2 −N(N + 4)y2 = 4 τ 2x2 −N(N + 4)y2 = 4 with y = Au2 for some integer u, except in the following cases: (1) N = 1, A = 1, in which case y ∈{1, 122}. (2) N = 336, A = 1, in which case y ∈{1, 62142}. (3) N = d2 −2, A = 1, in which case y ∈{1, d2}. Proof. Take M = N + 2, X = τx, and Y = y in Lemma 2.5 of [20]. Moreover, if N is even, one can take N = 2M −2, and if N is odd, N = M −2. Next, we recall the following result due to Ljunggren [16]. imultaneous Pellian equations with a single or no solution If there is a prime factor p of Uk that does not divide Uj for all 1 ≤j ≤k −1, then we say that p is a primitive prime factor of Uk. Notice that there are two (slightly) diﬀerent deﬁnitions of primitive prime factor. According to the deﬁnition in [5], p should not divide N(N + 4) and Uj for all 1 ≤j ≤k −1. This was used in [23] and [13]. But the above deﬁnition is enough for our proof. 374 A Togb´e and B He 74 A Togb´e and Lemma 2.9. The Diophantine equation (3.4) (3.4) Before discussing the above equation, let us express Uk (1 ≤k ≤6) using the recurrence relation Uk+2 = (N + 2)Uk+1 −Uk for k ≥1: U1 = 1, U2 = N + 2, U3 = N2 + 4N + 3, U4 = N3 + 6N2 + 10N + 4, U5 = N4 + 8N3 + 21N2 + 20N + 5, U6 = N5 + 10N4 + 36N3 + 56N2 + 35N + 6. First, we assume that N = 1, k1 = 5 or 6. If k1 = 5, then Uk1+1 = 144 = 24 · 32. By Lemma 2.7, Uk1 has a primitive prime factor p, so that Uk1 \| Uk2 or Uk1 \| Uk2+1. If Uk1 \| Uk2, since gcd(Uk2, Uk2+1) = 1, equation (3.4) im- plies the existence of positive integers s and t such that Uk2/(144Uk1) = s2, Uk2+1 = t2 or Uk2/(16Uk1) = s2, Uk2+1/9 = t2 or Uk2/(9Uk1) = s2, Uk2+1/16 = t2 or Uk2/Uk1 = s2, Uk2+1/144 = t2. The above cases give us Uk2+1 = □. Using Lemma 2.8 and U1 = 1, one can see that Uk2+1 = 144 and k2 = 5. This contradicts the fact that k1 < k2. If Uk1 \| Uk2+1, then k2 = 6. This is impossible. In the same way, if k1 = 6, we also get a contra- diction. Assume now N > 1 or N = 1, k1 ̸= 5, 6. If k1 > 1, by Lemma 2.7, Uk1 and Uk1+1 have primitive prime factors p and q respectively. By Lemma 2.7 again, equation (3.4) implies that (k1 \| k2 or k1 \| k2 + 1) and (k1 + 1 \| k2 or k2 \| k2 + 1). (3.5) (k1 \| k2 or k1 \| k2 + 1) and (k1 + 1 \| k2 or k2 \| k2 + 1). (3.5) If k1 = 1, then U2 has primitive prime factor q, and properties (3.5) also hold. Moreover, since j1 \| j2, we have ld. Moreover, since j1 \| j2, we have 2k1 + 1 \| 2k2 + 1. (3.6) (3.6) Note that gcd(Uk2, Uk2+1) = 1 by Lemma 2.2(3). Then properties (3.4) and (3.5) give us the following four cases: Note that gcd(Uk2, Uk2+1) = 1 by Lemma 2.2(3). Lemma 2.9. The Diophantine equation Lemma 2.9. The Diophantine equation Lemma 2.9. The Diophantine equation x4 −py2 = 1, where p denotes any odd prime, has no solutions in positive integers x and y if p ̸= 5 and p ̸= 29. When p = 5 or p = 29 there is only one solution, i.e. (x, y) = (3, 4) and (x, y) = (99, 1820) respectively. where p denotes any odd prime, has no solutions in positive integers x and y if p ̸= 5 and p ̸= 29. When p = 5 or p = 29 there is only one solution, i.e. (x, y) = (3, 4) and (x, y) = (99, 1820) respectively. 3. Proof of Theorem 1.1. In this section, we will prove the main theorem of this paper. We assume that (x1, y1, z1) is the positive solution with the smallest positive z1, and (x2, y2, z2) is any other positive solution of equations (2.2) and (2.3). Then there exist positive integers ji, li (i = 1, 2) with 2 ∤ji such that xi = Tji, yi = Wji, yi or xi = vli, zi = uli. (3.1) (3.1) We notice that j1 > 1, otherwise T1 = W1 = 1. This implies l1 = 1, vl1 = 1 and z = 0. Let ji = 2ki +1 (i = 1, 2) with 0 < k1 < k2. From (2.3) and (3.1), we have T 2 2ki+1 −1 = bz2 i or W 2 2ki+1 −1 = bz2 i . Using (2.12) and Lemma 2.5, we get bz2 1 = (R(λ) 2k1+1) 2 −1 = (N + 2 −2λ)Uk1Uk1+1, (3.2) bz2 2 = (R(λ) 2k2+1) 2 −1 = (N + 2 −2λ)Uk2Uk2+1. (3.3) Simultaneous Pellian equations 375 Simultaneous Pellian equations From Lemma 2.4, we have z1 \| z2, so (3.2) and (3.3) give Uk2Uk2+1 Uk1Uk1+1 = z2 z1 2 . (3.4) From Lemma 2.4, we have z1 \| z2, so (3.2) and (3.3) give From Lemma 2.4, we have z1 \| z2, so (3.2) and (3.3) give Uk2Uk2+1 Uk1Uk1+1 = z2 z1 2 . Lemma 2.9. The Diophantine equation Then properties (3.4) and (3.5) give us the following four cases: (i) Uk2+1/(Uk1Uk1+1) = s2, Uk2 = t2, (3.7) (ii) Uk2/(Uk1Uk1+1) = s2, Uk2+1 = t2, (3.8) (iii) Uk2/Uk1+1 = s2, Uk2+1/Uk1 = t2, (3.9) (iv) Uk2/Uk1 = s2, Uk2+1/Uk1+1 = t2. (3.10) Case (i). Since (V, U) = (N +2, 1) is a solution of V 2−N(N +4)U2 = 4, using equations (3.7) one can see that the equations A. Togb´e and B. He 376 τ 2x2 −N(N + 4)y2 = 4, y = u2 τ 2x2 −N(N + 4)y2 = 4, y = u2 (3.11) (3.11) have two solutions u = 1 and u = t > 1. By Lemma 2.8, we obtain N = 1, 336, or d2 −2. have two solutions u = 1 and u = t > 1. By Lemma 2.8, we obtain N = 1, 336, or d2 −2. • If N = 1, then we get t = 12. Therefore, equations (3.11) imply x2 −5y2 = 4. Any solution (x, y) is given by x + y √ 5 2 = 3 + √ 5 2 k The solution with y = 144 implies k2 = 6. On the other hand, the ﬁrst equation of (3.7) gives us k1(k1 + 1) \| k2 + 1 = 7. This is impossible. • If N = 336, then t = 6214. From U4 = N3 + 6N2 + 10N + 4 = 62142, we obtain k2 = 4. Since k1(k1 + 1) \| k2 + 1 = 5, we can ﬁnd no positive integer k1. • If N = 336, then t = 6214. From U4 = N3 + 6N2 + 10N + 4 = 62142, we obtain k2 = 4. Since k1(k1 + 1) \| k2 + 1 = 5, we can ﬁnd no positive integer k1. • If N = d2 −2, then t = d. From U2 = N + 2 = t2, we get k2 = 2. But there is no positive integer k1 satisfying k1(k1 + 1) \| k2 + 1 = 3. Case (ii). This is similar to Case (i). By Lemma 2.8, the second equation of (3.8) implies N = 1, 336, or d2 −2. • If N = 1, then k2 + 1 = 6. We have already discussed this case and it is impossible. • If N = 336, then k2 + 1 = 4. Lemma 2.9. The Diophantine equation But k1(k1 + 1) \| k2 = 3 is also impossible. • If N = d2 −2, then k2 + 1 = 2. But there is no integer k1 such that 0 < k1 < k2. Case (iii). From (3.9), we have Case (iii). From (3.9), we have Uk1+1 = As2 1, Uk2 = As2 2, Uk1 = Bt2 1, Uk2+1 = Bt2 2 (3.12) for some positive integers A, B, s1, s2, t1, t2 such that s = s2/s1 and t = If k +1 k f (3 6) t 2k +1 \| 2k +3 hi h i i ibl Th Case (iii). From (3.9), we have Uk1+1 = As2 1, Uk2 = As2 2, Uk1 = Bt2 1, Uk2+1 = Bt2 2 (3.12) for some positive integers A, B, s1, s2, t1, t2 such that s = s2/s1 and t = t2/t1. If k1+1 = k2, from (3.6) we get 2k1+1 \| 2k1+3, which is impossible. Therefore we consider k1 + 1 < k2. Thus Uk1 < Uk1+1 < Uk2 < Uk2+1. But from Lemma 2.8, A = B = 1 and N = 1, 336 or d2 −2. Then Uk1, Uk1+1, Uk2, and Uk2+1 are all perfect squares. This leads to a contradiction. for some positive integers A, B, s1, s2, t1, t2 such that s = s2/s1 and t = t2/t1. If k1+1 = k2, from (3.6) we get 2k1+1 \| 2k1+3, which is impossible. Therefore we consider k1 + 1 < k2. Thus Uk1 < Uk1+1 < Uk2 < Uk2+1. But from Lemma 2.8, A = B = 1 and N = 1, 336 or d2 −2. Then Uk1, Uk1+1, Uk2, and Uk2+1 are all perfect squares. This leads to a contradiction. Case (iv). From (3.10), as in Case (iii), we have Uk1 = As2 1, Uk2 = As2 2, Uk1+1 = Bt2 1, Uk2+1 = Bt2 2. (3.13) Since k1 < k2, from Lemma 2.8 we have A = B = 1 and N = 1, 336, or d2 −2. We get a contradiction as before. This completes the proof of Theorem 1.1. Case (iv). From (3.10), as in Case (iii), we have Uk1 = As2 1, Uk2 = As2 2, Uk1+1 = Bt2 1, Uk2+1 = Bt2 2. (3.13) Uk1 As1, Uk2 As2, Uk1+1 Bt1, Uk2+1 Bt2. Lemma 2.9. The Diophantine equation (3.13) Since k1 < k2, from Lemma 2.8 we have A = B = 1 and N = 1, 336, or d2 −2. We get a contradiction as before. This completes the proof of Theorem 1.1. Since k1 < k2, from Lemma 2.8 we have A = B = 1 and N = 1, 336, or d2 −2. We get a contradiction as before. This completes the proof of Theorem 1.1. 4. A particular case. Now we consider equations (1.4) with 4. A particular case. Now we consider equations (1.4) with b = b′\|4m/δ + 4\|, b′ ∈{1, 2, p}. (4.1) b = b′\|4m/δ + 4\|, b′ ∈{1, 2, p}. (4.1) (4.1) b = b′\|4m/δ + 4\|, b′ ∈{1, 2, p}. Then we have the following result. Simultaneous Pellian equations 377 Proposition 4.1. If equations (1.4) have a solution (x, y, z) with the condition (4.1), then UkUk+1 = b′z2 when either N = b′d2 −2, b′ ∈{1, 2, p}, k = 1, z = d; N 7 b′ 5 k 2 12 N = b′d2 −2, b′ ∈{1, 2, p}, k = 1, z = d; N = b′d2 −2, b′ ∈{1, 2, p}, k = 1, z = d; N = 7, b′ = 5, k = 2, z = 12; or or or N = 7, b′ = 5, k = 2, z = 12; N = 9799, b′ = 29, k = 2, z = 180180. Proof. Suppose a positive integer solution (x, y, z) of (1.4) exists. Then there are positive integers j, l with 2 ∤j such that x = Tj, y = Wj, y or x = vl, z = ul. (4.2) x = Tj, y = Wj, y or x = vl, z = ul. x = Tj, y = Wj, y or x = vl, z = ul. (4.2) If j = 1 then z = 0. Let j = 2k +1 for k > 0. From (2.3) and (4.2) we obtain T 2 2k+1 −1 = bz2 or W 2 2k+1 −1 = bz2. Using (2.12) and Lemma 2.5, we get bz2 = (R(λ) 2k+1)2 −1 = (N + 2 −2λ)UkUk+1. Thus 2 (4.2) bz2 = (R(λ) 2k+1)2 −1 = (N + 2 −2λ)UkUk+1. Thus (N + 2 −2λ)UkUk+1 = b′\|4m/δ + 4\|z2, b′ ∈{1, 2, p}. (4.3) (N + 2 −2λ)UkUk+1 = b′\|4m/δ + 4\|z2, b′ ∈{1, 2, p}. (4.3) We recall that N = 4n/c, n = min{m, m + δ} and c = \|δ\|. We recall that N = 4n/c, n = min{m, m + δ} and c = \|δ\|. If δ ∈{1, 2, 4}, then equations (1.4) give us the ﬁrst equation in (2.3) and n = m, c = δ. Thus we need to consider W 2 2k+1 −1 = bz2. By the deﬁnition of R(λ) 2k+1 in (2.12), we have λ = −1. Therefore one can see that N + 2 −2λ = 4m/c + 4 = \|4m/δ + 4\|. (4.1) The fact that b′t2 = Uk+1 = U5 = N4 + 8N3 + 21N2 + 20N + 5 = 13051348805 = 5 · 11 · 19 · 109 · 149 · 769 also leads to a contradiction. • If N = d2 −2, then k = 2 and s = d. Therefore, from b′t2 = Uk+1 = U3 = N2 + 4N + 3 = (N + 2)2 −1, we have d4 −b′t2 = 1, b′ ∈{1, 2, p}. (4.7) • If N = d2 −2, then k = 2 and s = d. Therefore, from b′t2 = Uk+1 = U3 = N2 + 4N + 3 = (N + 2)2 −1, we have d4 −b′t2 = 1, b′ ∈{1, 2, p}. (4.7) d4 −b′t2 = 1, b′ ∈{1, 2, p}. d4 −b′t2 = 1, b′ ∈{1, 2, p}. (4.7) It is easy to see that (4.7) has no positive integer solution when b′ = 1. If b′ = 2, then (4.7) implies d = 1, t = 0, which is impossible. If b′ = p, then by Lemma 2.9, equation (4.7) has a positive integer solution if and only if either b′ = 5, (d, t) = (3, 4), or b′ = 29, (d, t) = (99, 1820). Since z = st, we get z = 12 or 180180 respectively. Now we suppose equations (4.6) hold. In a similar way, Uk+1 = s2 implies k = 0, except for N = 1, 336, or d2 −2. But k = 0 leads to a contradiction. Now we discuss the following three cases when k > 0. • If N = 1, then k + 1 = 6. But b′t2 = Uk = U5 = N4 + 8N3 + 21N2 + 20N + 5 = 55 = 5 · 11 gives a contradiction. • If N = 1, then k + 1 = 6. But b′t2 = Uk = U5 = N4 + 8N3 + 21N2 + 20N + 5 = 55 = 5 · 11 gives a contradiction. • If N = 336, then k + 1 = 4. Thus b′t2 = Uk = U3 = N2 + 4N + 3 = 114243 = 3 · 113 · 337 is impossible. • If N = d2 −2, then k + 1 = 2. Then from b′t2 = U1 = 1, we get b′ = 1 and t = 1. (4.1) If δ ∈{−1, −2, −4}, then equations (1.4) give us the second equation in (2.3) and n = m −c, c = −δ. Thus we need to consider T 2 2k+1 −1 = bz2 and λ = 1. Therefore we also obtain N + 2 −2λ = 4(m −c)/c = 4m/c −4 = \|4m/δ + 4\|. Then equation (4.3) implies UkUk+1 = b′z2, b′ ∈{1, 2, p}. (4.4) By Lemma 2.2(3), we have gcd(Uk, Uk+1) = 1. So we obtain either By Lemma 2.2(3), we have gcd(Uk, Uk+1) = 1. So we obtain either By Lemma 2.2(3), we have gcd(Uk, Uk+1) = 1. So we obtain either Uk = s2, Uk+1 = b′t2, (4.5) Uk = s2, Uk+1 = b′t2, Uk = b′t2, Uk+1 = s2, (4.6) where z = st, s, t ∈N. If equations (4.5) hold, then from Lemma 2.8 one can see that Uk = s2 implies k = 1, except for N = 1, 336, or d2 −2. First, we suppose k = 1. Then from the second equation of (4.5) we have U2 = N + 2 = b′t2. Thus N = b′t2 −2 with b′ ∈{1, 2, p}. Second, we suppose k > 1 and we discuss the following three cases. A. Togb´e and B. He 378 • If N = 1, then Uk is a perfect square when k = 6. But b′t2 = Uk+1 = U7 = (N + 2)U6 −U5 = 377 = 13 · 29 is impossible. • If N = 1, then Uk is a perfect square when k = 6. But b′t2 = Uk+1 = U7 = (N + 2)U6 −U5 = 377 = 13 · 29 is impossible. • If N = 336, then k = 4. The fact that b′t2 = Uk+1 = U5 = N4 + 8N3 + 21N2 + 20N + 5 = 13051348805 = 5 · 11 · 19 · 109 · 149 · 769 also leads to a contradiction. • If N = 336, then k = 4. The fact that b′t2 = Uk+1 = U5 = N4 + 8N3 + 21N2 + 20N + 5 = 13051348805 = 5 · 11 · 19 · 109 · 149 · 769 also leads to a contradiction. • If N = 336, then k = 4. (4.1) { } First, let N = b′d2 −2. If δ > 0, then n = m, thus m = δ(b′d2 −2)/4. We have k = 1 and z = d by Proposition 4.1. From y2 = bz2 + 1 we bt i y2 = b′\|4m/δ + 4\|z2 + 1 = b′(4m/δ + 4)z2 + 1 = b′(b′d2 + 2)d2 + 1 = (b′d2 + 1)2. Thus we have y = b′d2 + 1 = 4m/δ + 3. Consequently, we get the solution (x, y, z) = (4m/δ + 1, 4m/δ + 3, d). ( ) ( / / ) If δ < 0, then n = m + δ, thus m = −δ(b′d2 −2)/4 −δ = −δ(b′d2 + 2)/4. In a similar way, knowing that δ + m ≥1 we have If δ < 0, then n = m + δ, thus m = −δ(b′d2 −2)/4 −δ = −δ(b′d2 + 2)/4. In a similar way, knowing that δ + m ≥1 we have y2 = b′\|4m/δ + 4\|z2 + 1 = b′(4m/(−δ) −4)z2 + 1 = b′(b′d2 −2)d2 + 1 = (b′d2 −1)2. It follows that y = b′d2−1 = 4m/(−δ)−3, and we get the solution (x, y, z) = (4m/(−δ) −1, 4m/(−δ) −3, d). This proves the ﬁrst exceptional case. It follows that y = b′d2−1 = 4m/(−δ)−3, and we get the solution (x, y, z) = (4m/(−δ) −1, 4m/(−δ) −3, d). This proves the ﬁrst exceptional case. It follows that y = b′d2−1 = 4m/(−δ)−3, and we get the solution (x, y, z) = (4m/(−δ) −1, 4m/(−δ) −3, d). This proves the ﬁrst exceptional case. m/(−δ) −1, 4m/(−δ) −3, d). This proves the ﬁrst exceptional case. Finally, let N = 7 or 9799. Since N = 4n/\|δ\|, we have \|δ\| = 4. Notic- ing k = 2, by direct computations, it is easy to get the second and third exceptional cases. This completes the proof of Theorem 4.2. Acknowledgments. We thank Prof. Yuan for informing us about the paper [14] and for sending us a copy. Li and Yuan have studied the same system and obtained a result similar to our Theorem 1.1 but using another method. The authors thank Gary Walsh for many helpful suggestions and com- ments during the preparation of this paper, particularly for those leading to Theorem 1.2. (4.1) This is also impossible. Finally, we use Proposition 4.1 to prove the following result which is a particular case of Theorem 1.1. Theorem 4.2. If p is an odd prime and b = b′\|4m/δ + 4\|, b′ ∈{1, 2, p}, then the system of simultaneous equations (1.4) has no positive integer so- lution (x, y, z), except in the following cases. (1) If (δ, b′) ̸= (±1, 1), (±1, p), then there is a positive integer d such that 2 m = δ(b′d2 −2)/4 if δ > 0, −δ(b′d2 + 2)/4 if δ < 0, and equations (1.4) have one solution and equations (1.4) have one solution (x, y, z) = (\|4m/δ + 1\|, \|4m/δ + 3\|, d). (2) If (m, δ, b) = (7, 4, 55), then the solution is (x, y, z) = (71, 89, 12); if (m, δ, b) = (11, −4, 35), then the solution is (x, y, z) = (89, 71, 12). (2) If (m, δ, b) = (7, 4, 55), then the solution is (x, y, z) = (71, 89, 12); if (m, δ, b) = (11, −4, 35), then the solution is (x, y, z) = (89, 71, 12). (2) If (m, δ, b) = (7, 4, 55), then the solution is (x, y, z) = (71, 89, 12); if (m, δ, b) = (11, −4, 35), then the solution is (x, y, z) = (89, 71, 12). ( , , ) ( , , ), ( , y, ) (3) If (m, δ, b) = (9799, 4, 29 · 9803), then the solution is ( , , ) ( , , ), ( , y, ) (3) If (m, δ, b) = (9799, 4, 29 · 9803), then the solution is ( ) ( ) ( ) (3) If (m, δ, b) = (9799, 4, 29 · 9803), then the solution is (x, y, z) = (96049799, 96069401, 180180); (x, y, z) = (96049799, 96069401, 180180); Simultaneous Pellian equations 379 if (m, δ, b) = (9803, −4, 29 · 9799), then the solution is (x, y, z) = (96069401, 96049799, 180180). Proof. Suppose that there exists a positive integer solution (x, y, z) of equations (1.4) with the condition (4.1). From Proposition 4.1, we have UkUk+1 = b′z2, b′ ∈{1, 2, p} and N = 4n/\|δ\| = b′d2 −2, 7 or 9799, where n = min{m, m + δ}. (4.1) The authors express their gratitude to the anonymous referee for constructive suggestions to improve an earlier draft of this paper. The ﬁrst author is partially supported by Purdue University North Central. The second author is supported by the Natural Science Foundation of Education Department of Sichuan Province (No. 2006C057). [1] A. Baker and H. Davenport, The equations 3x2 −2 = y2 and 8x2 −7 = z2, Quart. J. Math. Oxford Ser. (2) 20 (1969), 129–137. [2] M. A. Bennett, On the number of solutions of simultaneous Pell equations, J. Reine Angew. Math. 498 (1998), 173–199. References A. Togb´e and B. He 380 [3] M. A. Bennett, M. Cipu, M. Mignotte and R. 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Math. Soc. 132 (2004), 1561–1566. [23] —, On the number of solutions of x2 −4m(m + 1)y2 = y2 −bz2 = 1, Proc. Amer. Math. Soc. 132 (2004), 1561–1566. Mathematics Department Purdue University North Central 1401 S, U.S. 421 Westville, IN 46391, U.S.A. E-mail: atogbe@pnc.edu Key Laboratory of Numerical Simulation of Sichuan Province Neijiang Normal University Neijiang, Sichuan, 641112, P.R. China E-mail: hebo-one@hotmail.com Mathematics Department Purdue University North Central 1401 S, U.S. 421 Westville, IN 46391, U.S.A. E-mail: atogbe@pnc.edu Key Laboratory of Numerical Simulation of Sichuan Province Neijiang Normal University Neijiang, Sichuan, 641112, P.R. China E-mail: hebo-one@hotmail.com Mathematics Department p Purdue University North Central 1401 S, U.S. 421 Westville, IN 46391, U.S.A. E-mail: atogbe@pnc.edu Received on 22.10.2007 and in revised form on 18.4.2008 (5554) Received on 22.10.2007 and in revised form on 18.4.2008 (5554) (5554)
https://openalex.org/W4385899589	https://latam.redilat.org/index.php/lt/article/download/941/1241	es		Corteza dorsolateral frontal y memoria en personas consumidoras de sustancias	Latam	2,023	cc-by	5,959	DOI: https://doi.org/10.56712/latam.v4i2.941 Corteza dorsolateral frontal y memoria en personas consumidoras de sustancias Dorsolateral frontal cortex and memory in substance users Verónica Fernanda Flores Hernández vf.floresh@uta.edu.ec https://orcid.org/0000-0001-6409-0728 Universidad Técnica de Ambato / Grupo de investigación NUTRIGENX Ambato – Ecuador Águeda del Rocío Ponce Delgado adr.ponce@uta.edu.ec https://orcid.org/0000-0003-3624-0455 Universidad Técnica de Ambato / Grupo de investigación NUTRIGENX Ambato – Ecuador Fabricio Alejandro Vásquez de la Bandera Cabezas cfa.vasquez@uta.edu.ec https://orcid.org/0000-0001-5809-7325 Universidad Técnica de Ambato / Grupo de investigación NUTRIGENX Ambato – Ecuador Cristian Fernando Campaña Días cristiancampanad@gmail.com https://orcid.org/0009-0006-3718-8133 Ministerio de Salud Pública Quito – Ecuador Artículo recibido: 20 de julio de 2023. Aceptado para publicación: 07 de agosto de 2023. Conflictos de Interés: Ninguno que declarar. Resumen Al abordar la temática de funciones ejecutivas se entiende a estas como las encargadas del funcionamiento superior, que implica distintos córtex, en esta ocasión se enfoca el córtex dorsolateral, mismo que está encargado de la planificación, la toma de decisiones, la memoria de trabajo y la atención. El objetivo del estudio fue determinar el estado en el que se encuentra la corteza dorsolateral frontal en relación a memoria en personas consumidoras de sustancias, tomando en cuenta que esta población presenta un cuadro de consumo de más de 5 años consecutivos y actualmente se encuentran en proceso de recuperación en un tiempo estimado entre 6 a 8 meses. La población está conformada por personas de sexo masculino que oscilan entre los 27 a 60 años de edad, no presentan patologías psiquiátricas y tampoco están sujetas a prescripciones médicas. Los resultados que se pueden apreciar revelan que la mayoría de la población evaluada presenta alteraciones severas entorno al desempeño de la corteza dorsolateral del lóbulo frontal, lo cual permite inferir dificultades en actividades como planificación motora, la organización y la regulación, además se presumen deficiencias en la integración de la información sensorial y aspectos de índole intelectual, así como con la memoria de trabajo. En cuanto a la memoria, en aspectos de copia se puede apreciar que más de la mitad de la población muestra un desempeño adecuado, lo contrario se observa en el proceso de reproducción donde la mayoría muestra un desempeño inferior, lo cual permite concluir que LATAM Revista Latinoamericana de Ciencias Sociales y Humanidades, Asunción, Paraguay. ISSN en línea: 2789-3855, agosto, 2023, Volumen IV, Número 2 p 4839. dicha población si presenta dificultades significativas entorno a actividades de función ejecutiva y de memoria. Palabras clave: consumidores de sustancias, corteza dorsolateral, funciones ejecutivas, memoria Abstract When addressing the topic of executive functions, these are understood as responsible for higherlevel functioning, involving different cortices. In this case, the focus is on the dorsolateral cortex, which is responsible for planning, decision-making, working memory, and attention. The objective of the study was to determine the state of the dorsolateral frontal cortex in relation to memory in substance users, taking into account that this population has a history of substance use for more than 5 consecutive years and is currently in the process of recovery, estimated to take between 6 to 8 months. The population consists of male individuals ranging from 27 to 60 years old, without psychiatric pathologies, and not subject to medical prescriptions. The results obtained reveal that the majority of the evaluated population presents severe alterations in the performance of the dorsolateral cortex of the frontal lobe, which allows inferring difficulties in activities such as motor planning, organization and regulation, as well as deficiencies in the integration of sensory information and aspects of intellectual nature, as well as working memory. Regarding memory, in terms of copying, it can be observed that more than half of the population shows adequate performance. However, the opposite is observed in the reproduction process, where the majority shows inferior performance. This leads to the conclusion that this population does indeed have significant difficulties in executive function and memory activities. Keywords: substance users, dorsolateral cortex, executive functions, memory Todo el contenido de LATAM Revista Latinoamericana de Ciencias Sociales y Humanidades, publicados en este sitio está disponibles bajo Licencia Creative Commons . Como citar: Flores Hernández, V. F., Ponce Delgado, Á. Del R., Vásquez de la Bandera Cabezas, F. A., & Campaña Días, C. F. (2023). Corteza dorsolateral frontal y memoria en personas consumidoras de sustancias. LATAM Revista Latinoamericana de Ciencias Sociales y Humanidades 4(2), 4839–4852. https://doi.org/10.56712/latam.v4i2.941 LATAM Revista Latinoamericana de Ciencias Sociales y Humanidades, Asunción, Paraguay. ISSN en línea: 2789-3855, agosto, 2023, Volumen IV, Número 2 p 4840. INTRODUCCIÓN La corteza frontal dorsolateral (FDLC) es una región del cerebro situada en la corteza prefrontal, concretamente en la región lateral del lóbulo frontal. Desempeña un papel fundamental en el funcionamiento ejecutivo, que implica la planificación, la toma de decisiones, la memoria de trabajo y la atención. FDLC se basa en el concepto de control cognitivo, que se refiere a la capacidad del cerebro para regular y coordinar varios procesos cognitivos para lograr un objetivo específico. El control cognitivo es esencial para el comportamiento adaptativo, ya que permite a las personas ajustar con flexibilidad su comportamiento y sus respuestas a los entornos y demandas cambiantes (Lezak, M. D., Howieson, D. B., Bigler, E. D., & Tranel, D. 2012). Se cree que el FDLC desempeña un papel central en el control cognitivo al modular la actividad en otras regiones del cerebro involucradas en el desempeño de tareas. Esta modulación se logra a través de un proceso conocido como control de arriba hacia abajo, en el que el FDLC envía señales a otras regiones del cerebro para sesgar su actividad hacia información relevante para la tarea. La investigación también ha demostrado que la FDLC está involucrada en la memoria de trabajo, la capacidad de retener información en la mente durante un período corto de tiempo. Se cree que esto es importante para el mantenimiento de los objetivos de la tarea y la inhibición de información irrelevante. En general, el marco teórico de la FDLC sugiere que desempeña un papel fundamental en la regulación y coordinación de los procesos cognitivos para lograr objetivos específicos, a través de la modulación de la actividad en otras regiones del cerebro involucradas en el desempeño de tareas (Stuss, D. T., & Knight, R. T. 2013). La corteza dorsolateral frontal es una de las áreas más importantes del cerebro humano y juega un papel fundamental en una variedad de funciones cognitivas complejas, incluyendo la planificación, el control de la atención, la toma de decisiones, la resolución de problemas y el procesamiento del lenguaje. Desde un punto de vista anatómico, la corteza dorsolateral frontal se encuentra en la superficie lateral del lóbulo frontal, en la parte superior y lateral del cerebro, justo detrás de la frente. Esta región se extiende desde la corteza prefrontal dorsolateral hasta la corteza motora primaria, y se divide en tres subregiones principales: el área prefrontal dorsolateral, el área motora suplementaria y el área premotora. En términos funcionales, la corteza dorsolateral frontal está involucrada en una amplia gama de procesos cognitivos, incluyendo: ● Planificación y toma de decisiones: La corteza dorsolateral frontal ayuda a evaluar diferentes opciones ya tomar decisiones basadas en las consecuencias de cada opción. ● Control de la atención (Diamond, A., 2013). La corteza dorsolateral frontal (CDLF) es una región importante del cerebro humano que se encuentra en la parte frontal del lóbulo frontal. Esta región está involucrada en varias funciones cognitivas, incluida la memoria de trabajo, la atención selectiva y la resolución de problemas complejos. Una de las funciones asociadas con la CDLF es la función ejecutiva. La función ejecutiva es un término general que se refiere a un conjunto de habilidades cognitivas que permiten programar, organizar, iniciar, monitorear y ajustar el comportamiento en función de los objetivos y las demandas del entorno. Estas habilidades son esenciales para el éxito en LATAM Revista Latinoamericana de Ciencias Sociales y Humanidades, Asunción, Paraguay. ISSN en línea: 2789-3855, agosto, 2023, Volumen IV, Número 2 p 4841. muchas áreas de la vida, incluyendo el trabajo, la educación y las relaciones interpersonales (Anderson, V. 2008). La CDLF está involucrada en varios aspectos de la función ejecutiva, incluyendo la memoria de trabajo, la inhibición, la flexibilidad cognitiva y la atención selectiva. La memoria de trabajo se refiere a la capacidad de mantener y manipular información en la mente a corto plazo. La CDLF está involucrada en la memoria de trabajo verbal y visual, así como en la actualización de la información en la memoria de trabajo. La inhibición se refiere a la capacidad de suprimir respuestas automáticas o impulsivas y elegir una respuesta más adecuada en función del contexto. La CDLF está involucrada en la inhibición de respuestas no relevantes y en la selección de respuestas apropiadas (Miyake, A., & Friedman, N. P. 2012). La flexibilidad cognitiva se refiere a la capacidad de cambiar entre diferentes tareas o estrategias en función de los objetivos y las demandas del entorno. La CDLF está involucrada en la flexibilidad cognitiva y en la capacidad de cambiar de una tarea a otra. La atención selectiva se refiere a la capacidad de enfocarse en información relevante y suprimir información no relevante. La CDLF está involucrada en la atención selectiva y en la capacidad de mantener la atención en la información relevante mientras se suprime la información no relevante. En resumen, la CDLF es una región crucial del cerebro para la función ejecutiva, y es esencial para muchas habilidades cognitivas importantes para el éxito en la vida diaria (Jurado, M. B., & Rosselli, M., 2007). La memoria es la capacidad del cerebro para almacenar, retener y recuperar información. Se refiere al proceso mediante el cual se codifica, almacena y recupera información. La memoria se divide en tres categorías principales: memoria sensorial, memoria a corto plazo y memoria a largo plazo. La memoria sensorial es la capacidad de retener información sensorial, como la vista o el oído, durante un breve período de tiempo después de que se ha presentado un estímulo (Diamond, A., 2013). La memoria a corto plazo es la capacidad de retener información durante un breve período de tiempo, generalmente de segundos a minutos. Esta memoria se utiliza para recordar información temporalmente y realizar tareas mentales básicas. La memoria a largo plazo es la capacidad de almacenamiento de información durante un período de tiempo prolongado, desde días hasta años. Esta memoria es la que nos permite recordar información sobre eventos pasados, habilidades y conocimientos adquiridos a lo largo de nuestra vida (Alvarez, J. A., & Emory, E., 2006). El proceso de la memoria incluye la codificación, la consolidación y la recuperación. La codificación es el proceso de convertir información sensorial en una forma que se puede almacenar en la memoria. La consolidación es el proceso mediante el cual la información se almacena en la memoria a largo plazo. La recuperación es el proceso de recordar y acceder a la información almacenada en la memoria a largo plazo. La memoria es una función importante del cerebro y se utiliza en muchos aspectos de nuestra vida diaria, desde recordar eventos importantes hasta aprender nuevas habilidades y conocimientos (Duncan, J. 2013). LATAM Revista Latinoamericana de Ciencias Sociales y Humanidades, Asunción, Paraguay. ISSN en línea: 2789-3855, agosto, 2023, Volumen IV, Número 2 p 4842. El cerebro es el órgano principal del sistema nervioso central y es responsable de procesar y almacenar información. La memoria es una función cerebral que nos permite retener y recuperar información y experiencias pasadas. Existen diferentes tipos de memoria, como la memoria a corto plazo, que nos permite recordar información por un corto período de tiempo, y la memoria a largo plazo, que nos permite retener información durante períodos más largos de tiempo. La memoria a largo plazo se divide en dos tipos principales: la memoria declarativa, que es la memoria de los hechos y los eventos, y la memoria procedimental, que es la memoria de cómo realizar tareas y habilidades (Miyake, A., & Friedman, N. P., 2012). El cerebro utiliza diferentes áreas para procesar y almacenar información, como el hipocampo y la corteza prefrontal. El hipocampo es una región importante para la formación de nuevos recuerdos y la corteza prefrontal está involucrada en la consolidación y recuperación de la memoria a largo plazo. Hay muchas cosas que pueden afectar la memoria, como la edad, la enfermedad y el estrés. También hay muchas técnicas que se pueden utilizar para mejorar la memoria, como la repetición, la asociación, la organización y la visualización. Además, mantenga un estilo de vida saludable, con una buena nutrición, ejercicio regular y sueño adecuado, puede ayudar a mantener un cerebro y memoria saludable (Barkley, R. A., 2011). Los adictos a menudo experimentan problemas de memoria, especialmente en lo que se refiere a la memoria a corto plazo. Esto se debe en parte al hecho de que las sustancias adictivas pueden dañar directamente las áreas del cerebro que son responsables de la memoria, como el hipocampo y la corteza prefrontal. Además, la adicción puede provocar cambios en el cerebro que surge la forma en que funciona la memoria. Por ejemplo, el consumo repetido de una sustancia puede provocar cambios en la plasticidad sináptica, lo que significa que las conexiones entre las células cerebrales pueden ser menos eficaces. Esto puede hacer que sea más difícil para un adicto retener y recordar información (Alvarez, J. A., & Emory, E., 2006). También hay evidencia de que la adicción puede afectar la memoria a largo plazo. Por ejemplo, algunas investigaciones sugieren que la adicción puede interferir con la consolidación de los recuerdos, lo que hace que sea más difícil retenerlos a largo plazo. Es importante destacar que la recuperación de la adicción puede mejorar la memoria. A medida que una persona se recupera, su cerebro puede comenzar a reparar algunos de los daños causados por la adicción, lo que puede ayudar a mejorar su capacidad para retener y recordar información. Además, la abstención de drogas y alcohol puede prevenir daños adicionales a la memoria (Jurado, M. B., & Rosselli, M., 2007) La adicción y la memoria están frecuentemente relacionadas. La adicción es un trastorno que se caracteriza por la dependencia a una sustancia o actividad, y la memoria juega un papel fundamental en su desarrollo y mantenimiento. La memoria tiene varios componentes, incluyendo la memoria a corto plazo, la memoria a largo plazo y la memoria de trabajo. La adicción puede afectar a cada uno de estos componentes de la memoria de diferentes maneras (Anderson, P., 2002). Por ejemplo, se sabe que las sustancias adictivas, como las drogas, pueden afectar la memoria a corto plazo al alterar la capacidad de la persona para retener información en la mente durante un breve período de tiempo. Esto puede afectar a su capacidad para realizar tareas cotidianas y para tomar decisiones informadas. LATAM Revista Latinoamericana de Ciencias Sociales y Humanidades, Asunción, Paraguay. ISSN en línea: 2789-3855, agosto, 2023, Volumen IV, Número 2 p 4843. Por otro lado, la adicción también puede tener un impacto en la memoria a largo plazo, ya que puede afectar a la capacidad de la persona para recordar eventos y experiencias importantes de su vida. La adicción puede hacer que la persona se centre únicamente en su dependencia y perder la capacidad de recordar momentos significativos que no están relacionados con la sustancia o actividad adictiva (Diamond, A., & Ling, D. S., 2016). Además, la adicción puede influir en la memoria de trabajo, que es la capacidad de la persona para retener información a corto plazo y utilizarla para realizar tareas complejas. La adicción puede hacer que la persona se distraiga fácilmente y pierda la concentración, lo que puede afectar su capacidad para procesar y retener información nueva. En resumen, la adicción y la memoria están estrechamente relacionadas. La adicción puede afectar a diferentes componentes de la memoria, lo que puede tener un impacto significativo en la capacidad de la persona para funcionar en su vida diaria. El cerebro y la adicción están estrechamente relacionados. Las adicciones pueden ser causadas por cambios en la estructura y función del cerebro. El cerebro es el centro de control de todas las funciones corporales y mentales, y la adicción es una enfermedad que afecta la forma en que el cerebro funciona y se adapta a las sustancias o comportamientos adictivos. Cuando una persona consume una sustancia adictiva, como el alcohol o las drogas, se produce un aumento de la liberación de dopamina en el cerebro, un neurotransmisor asociado con el placer y la recompensa. El cerebro puede aprender a asociar la sustancia con el placer, lo que puede llevar a cabo una fuerte motivación para continuar consumiendo la sustancia y puede generar cambios en la estructura y función del cerebro (Zelazo, P. D., & Carlson, S. M., 2012). Con el tiempo, el cerebro puede desarrollar tolerancia a la sustancia, lo que significa que se necesita una cantidad cada vez mayor de la sustancia para sentir los mismos efectos. Esto puede llevar a la dependencia física y psicológica de la sustancia, lo que significa que el cerebro necesita la sustancia para funcionar correctamente. Las adicciones comportamentales, como el juego o la adicción a internet, también pueden afectar el cerebro de manera similar. La liberación de dopamina asociada con la realización de la actividad adictiva puede llevar a la motivación para continuar con la actividad, y el cerebro puede adaptarse para buscar la recompensa de la actividad adictiva (Zelazo, P. D., & Carlson, S. M., 2012). En resumen, la adicción y el cerebro están específicamente relacionadas, y las sustancias adictivas y las adicciones comportamentales pueden afectar la estructura y función del cerebro, lo que puede llevar a la dependencia y la adicción. En la investigación se ha usado como estrategia principal la evaluación de la población mediante la prueba Banfe 2, la misma que está destinada a la valoración de aspectos concernientes a la función ejecutiva. Trabajar con la población adicta desde un enfoque neuropsicológico es importante por varias razones. La adicción es un trastorno complejo que afecta tanto el cerebro como el comportamiento de las personas, y comprender los aspectos neuropsicológicos de la adicción puede proporcionar una base sólida para el tratamiento efectivo. Es menester enfocar que el enfoque neuropsicológico es importante para comprender las bases neurobiológicas de la adicción en la que se ven involucrados cambios en el cerebro, particularmente en áreas relacionadas con la recompensa, la motivación y el control de los impulsos. Permite además personalizar los tratamientos ya que cada individuo es único y la adicción puede manifestarse de diferentes maneras en cada persona. El enfoque neuropsicológico permite LATAM Revista Latinoamericana de Ciencias Sociales y Humanidades, Asunción, Paraguay. ISSN en línea: 2789-3855, agosto, 2023, Volumen IV, Número 2 p 4844. evaluar las fortalezas y debilidades cognitivas y emocionales de cada individuo, lo que ayuda a personalizar el tratamiento y abordar las necesidades específicas de cada persona, los factores de riesgo y protección pueden ser identificados con mayor fluidez. Evaluar el progreso del tratamiento permite proporcionar herramientas mediante la medición de cambios en el funcionamiento cognitivo y emocional, lo que permite evaluar la efectividad de las intervenciones y realizar ajustes cuando sea necesario. José Luis Valdés G. y Fernando Torrealba L. (2006) en el artículo de la corteza prefrontal medial controla el alerta conductual y vegetativo. Implicancias en desórdenes de la conducta refiere que esta porción anatómica se relaciona con actividades autónomas y emocionales del sistema que permiten un adecuado acoplamiento del ser humano en su medio. La corteza prefrontal muestra estrecha relación con actividades de hipervigilia lo cual a largo plazo podría desencadenar conductas agresivas e incluso antisociales. Según Smith y colaboradores (2018), en el trabajo “Los adictos presentan déficits en la función ejecutiva y la memoria" los individuos adictos mostraron deficiencias en la función ejecutiva y la memoria en comparación con sujetos no adictos. Los resultados obtenidos por Johnson et al. (2017) en su investigación de "La adicción afecta negativamente la función ejecutiva: evidencia neurocognitiva", indicaron que la adicción tiene un impacto negativo en la función ejecutiva, según se evidencia en el ámbito neurocognitivo. Según el estudio de García-Pérez y colaboradores (2019) con el artículo "Los adictos a largo plazo muestran déficits de consolidación de la memoria”, se concluye que los individuos adictos a largo plazo presentan dificultades en la consolidación de la memoria. "Se observan dificultades en la toma de decisiones relacionadas con la corteza dorsolateral frontal en adictos" (Wang et al., 2016) Wang et al. (2016) en su investigación refiere que se observan dificultades en la toma de decisiones relacionadas con la corteza dorsolateral frontal en adictos, encontraron que los adictos experimentan dificultades en la toma de decisiones, las cuales se asocian con disfunciones en la corteza dorsolateral frontal. Los hallazgos de Rodríguez-Gómez y colaboradores (2018) revelan que durante la abstinencia, los adictos a la cocaína presentan alteraciones en la función ejecutiva, indicaron que los adictos a la cocaína experimentan cambios en la función ejecutiva durante el período de abstinencia. Según la revisión sistemática de López-Fernández et al. (2017), se encontró una relación entre la adicción y dificultades en la memoria de trabajo, lo cual indica que la adicción está asociada con dificultades en la memoria de trabajo. Mediante el uso de resonancia magnética funcional, Chen et al. (2019) observaron una disfunción en la corteza dorsolateral frontal en individuos adictos al alcohol, la disfunción de la corteza dorsolateral frontal es evidente en adictos al alcohol mediante resonancia magnética funcional Según Gómez-Velázquez et al. (2018), se identificaron cambios en la función ejecutiva en adictos a opiáceos mediante técnicas de neuroimagen. METODOLOGÍA La investigación tiene un enfoque cuantitativo ya que los datos que se obtuvieron son numéricos y fueron analizados mediante programas de cálculo, el alcance es descriptivo ya que define las variables de estudio, la corteza dorsolateral frontal y memoria como funciones superiores y las LATAM Revista Latinoamericana de Ciencias Sociales y Humanidades, Asunción, Paraguay. ISSN en línea: 2789-3855, agosto, 2023, Volumen IV, Número 2 p 4845. personas consumidoras de sustancias como una problemática social desde diferentes enfoques e investigaciones previas. En cuanto al diseño este fue no experimental ya que no se manipularon las variables y tuvo un corte transversal porque los instrumentos Banfe 2 y Figura de Rey se aplicaron en una sola ocasión. La muestra del estudio estuvo conformada por 21 participantes con diagnóstico clínico de drogodependencia, dichos partícipes se encontraban recluidos en un Centro de Rehabilitación de la provincia de Tungurahua con una totalidad del 100% de hombres entre 18 a 65 años de edad siendo la edad promedio 42 años. Se ejecutó un muestreo no probabilístico por conveniencia donde los partícipes fueron elegidos a través de criterios de inclusión donde lo primero era ser parte del centro en mención, tener entre 18 a 65 años, firmar el consentimiento informado, fueron excluidos los pacientes que se encontraban bajo efectos de alcohol u otras sustancias sujetas a fiscalización o bajo el efecto de fármacos. Para el proceso de recolección de información inició con proporcionar información acerca del proceso e indicar los criterios de confidencialidad, las evaluaciones se realizaron en forma individual personalizada dentro del centro. Para la evaluación se usó el instrumento Banfe 2 con una confiabilidad de alfa de Cronbach de 0.80 en el caso de la corteza dorsolateral frontal, la Batería neuropsicológica de funciones ejecutivas y lóbulos frontales cuyos autores son Julio Cesar Flores Lozano, Feggy Ostrosky Shejet, Asucena Lozano Gutiérrez, el instrumento tiene como objetivo evaluar el desarrollo de las Funciones Ejecutivas mediante 15 procesos los cuales se agrupan en tres áreas específicas: Orbito medial, Prefrontal Anterior y Dorsolateral, puede ser aplicado en niños desde los 6 años hasta adultos de 80 años. Se uso la Figura de Rey para evaluar memoria con una confiabilidad de alfa de Cronbach de 0.76, dicho test tiene como autor a André Rey (1941) cuya última adaptación fue realizada por John E. Myers & Kelly R. Myers (1995), la figura de Rey tiene como finalidad evaluar posibles trastornos neurológicos relacionados con problemas de carácter perceptivo o motriz. Grado de desarrollo y maduración de la actividad gráfica y la memoria. Se puede aplicar a niños, a partir de 6 años y 0 meses hasta los 17 - 11 meses y 31 días y a Adultos, desde los 18 años hasta los 89. El instrumento se aplica en dos momentos, uno de copia y el otro de reproducción. RESULTADOS Posterior a la aplicación de los instrumentos detallados con antelación se obtuvieron los siguientes resultados: Tabla 1 Evaluación de Función Ejecutiva – Corteza Dorsolateral N° de evaluados 12 4 5 0 Puntuaciones 69 o menos 70-84 85-115 116 en adelante Interpretación severa leve - moderada normal normal - alto LATAM Revista Latinoamericana de Ciencias Sociales y Humanidades, Asunción, Paraguay. ISSN en línea: 2789-3855, agosto, 2023, Volumen IV, Número 2 p 4846. Gráfico 1 Banfe 2. Dorsolateral – Función Ejecutiva 0 4 5 12 BANFE 2 DORSOLATE RAL - F U NCI ÓN E J E CU TI VA SEVERA LEVE MODERADA NORMAL NORMAL - ALTO Los resultados de la evaluación permiten identificar que de la población de 21 partícipes que corresponden al 100%, 12 de ellos que corresponden al 57% muestran alteraciones severas, 4 que corresponden al 19% muestran dificultades entorno a leves y moderadas. Por otro lado 5 personas que corresponden al 24% muestran un desempeño normal y 0% de la población muestra funciones de entre normal a alto en el ámbito mencionado. Dicho reporte permite mencionar que la mayoría de la población estudiada muestra alteraciones severas dentro de sus actividades relacionadas con la corteza dorsolateral, refiriendo funciones como planificación motora, organización y la regulación. Se relaciona además con actividades como la integración de la información sensorial y como el punto de entrada a la regulación de la función como tal y las que están relacionadas con aspectos intelectuales, así como con la memoria de trabajo. Tabla 2 Evaluación de memoria N° de evaluados 14 7 Puntuaciones Mayor a la media Menor a la media Interpretación Desempeño adecuado Desempeño inadecuado LATAM Revista Latinoamericana de Ciencias Sociales y Humanidades, Asunción, Paraguay. ISSN en línea: 2789-3855, agosto, 2023, Volumen IV, Número 2 p 4847. Gráfico 2 Figura de Rey 7 14 FI GU RA DE RE Y - COP I A DESEMPEÑO ADECUADO DESEMPEÑO INADECUADO Tabla 3 Reproducción N° de evaluados 8 13 Puntuaciones Mayor a la media Menor a la media Interpretación Desempeño adecuado Desempeño inadecuado Gráfico 3 Figura de Rey - Reproducción 8 13 FI GU RA DE RE Y - RE P RODU CCI ÓN DESEMPEÑO ADECUADO DESEMPEÑO INADECUADO Del 100% de la población se puede evidenciar que en el proceso de copia los evaluados muestran que el 67% que corresponde a 14 personas muestran un desempeño adecuado en la ejecución de este apartado, mientras que el 33% que corresponde a 7 personas muestra un desempeño inadecuado en la actividad. LATAM Revista Latinoamericana de Ciencias Sociales y Humanidades, Asunción, Paraguay. ISSN en línea: 2789-3855, agosto, 2023, Volumen IV, Número 2 p 4848. El proceso de reproducción, que se ejecuta principalmente entorno a la memoria muestra resultados un tanto distintos a los anteriores, se detalla que el 38% que corresponde a 8 evaluados reflejan un desempeño adecuado en reproducir la figura del instrumento aplicado, mientras que el 62% que corresponde a 13 participantes muestra un desempeño en memoria inadecuado. Los resultados muestran que la población tiene mejor desempeño en organización perceptual que en memoria visual. DISCUSIÓN El dato de prevalencia de alteraciones severas en la corteza dorsolateral de los internos del centro de rehabilitación de adicciones es un hallazgo muy significativo, ya que señalan la prevalencia de alteraciones en las actividades relacionadas al desempeño de funciones fundamentales en la planificación motora, la organización y la regulación de las funciones cognitivas. Además, también se asocia con la integración de la información sensorial y actúa como punto de entrada para la regulación de la función cognitiva y la memoria de trabajo. La alta proporción de participantes con alteraciones severas en estas funciones sugiere que existe una vulnerabilidad significativa en la población estudiada. Las alteraciones en la corteza dorsolateral pueden tener un impacto negativo en la capacidad de planificación, organización y regulación de las actividades diarias. Estas dificultades pueden afectar la vida cotidiana de los individuos, su desempeño académico o laboral, y su bienestar general. Es importante destacar que las causas subyacentes de estas alteraciones en la corteza dorsolateral están asociadas a diversos factores, entre los que se puede destacar justamente el consumo de sustancias desde tempranas edades. Podrían ser además el resultado de factores genéticos, lesiones cerebrales, trastornos neurológicos u otras condiciones médicas. Para comprender mejor la naturaleza y las implicaciones de estas alteraciones, sería necesario realizar investigaciones adicionales y evaluar otros factores relevantes, como antecedentes médicos y características individuales de los participantes. Dichos hallazgos coinciden con el estudio realizado por Sainz-Maza Fernández, A. (2021), quienes coinciden en que en pacientes consumidores se revelan dificultades en la capacidad para resolver problemas, en la flexibilidad mental y en la inhibición de respuestas automáticas, lo cual podría desencadenar en problemas dentro de la adaptación y desempeño social e individual en diferentes ámbitos. Mondragón-Maya, A., Flores-Medina, Y., López-Arreaga., G., López-Ramírez, S. y Paz-Rodríguez, F. (2021) en su investigación entorno a la misma temática revelan que las puntuaciones en el dominio dorsolateral del instrumento obtenidas por una población similar a la presente, indican puntuaciones negativas en las funciones ejecutivas mismas que probablemente estén asociadas a un retraso madurativo de los lóbulos frontales. Los resultados obtenidos en la aplicación de la figura de Rey muestran que la población tiene mejor desempeño en organización perceptual que en memoria visual puesto que los resultados en la fase de copia muestran mejores resultados que en la fase de reproducción, la organización perceptual se refiere a la capacidad de percibir y organizar la información sensorial de manera coherente y estructurada. En este caso, los participantes muestran habilidades suficientes para copiar la figura del instrumento, lo que sugiere una capacidad adecuada para organizar la información perceptual y realizar la tarea requerida. Sin embargo, los resultados muestran que la memoria visual es una habilidad que presenta dificultades en la mayoría de los participantes. La memoria visual se refiere a la capacidad de retener y recordar información visual a largo plazo. La proporción más alta de participantes con un desempeño inadecuado en memoria visual sugiere que existe una debilidad en esta área específica. Estos hallazgos son relevantes, ya que la memoria visual desempeña un papel fundamental en numerosas actividades cotidianas, como el reconocimiento de caras, la lectura, LATAM Revista Latinoamericana de Ciencias Sociales y Humanidades, Asunción, Paraguay. ISSN en línea: 2789-3855, agosto, 2023, Volumen IV, Número 2 p 4849. la comprensión de gráficos e imágenes, entre otros. Las dificultades en la memoria visual pueden tener implicaciones en el aprendizaje, la comunicación y el funcionamiento general de los individuos en su entorno. La presente investigación concuerda con los resultados obtenidos por Fajardo Balbuca M., Carmilema Tapia J., Sacoto Molina A. (2018), quien trabajó con una población de drogodependientes y evaluó funciones como la memoria de donde concluye que la suma de las puntuaciones totales normalizadas de cada sub-prueba que evaluó las funciones de memoria indican que, el 63.3% de los participantes presentan una alteración que se distribuye de forma homogénea en los rangos de alteración leve y severa en lo que concierne a reproducción, cabe recalcar que no se registraron participantes en el rango normal alto. LATAM Revista Latinoamericana de Ciencias Sociales y Humanidades, Asunción, Paraguay. ISSN en línea: 2789-3855, agosto, 2023, Volumen IV, Número 2 p 4850. REFERENCIAS Alvarez, J. A., & Emory, E. (2006). Executive function and the frontal lobes: A meta-analytic review. Neuropsychology Review, 16(1), 17-42. Alvarez, J. A., & Emory, E. (2006). 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https://openalex.org/W2158188373	https://arthritis-research.biomedcentral.com/counter/pdf/10.1186/ar4295	English	null	Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism	Arthritis research & therapy	2,013	cc-by	9,953	© 2013 de Andrés et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Introduction: The pathogenesis of osteoarthritis (OA) is characterized by the production of high amounts of nitric oxide (NO), as a consequence of up-regulation of chondrocyte-inducible nitric oxide synthase (iNOS) induced by inflammatory cytokines. NO donors represent a powerful tool for studying the role of NO in the cartilage in vitro. There is no consensus about NO effects on articular cartilage in part because the differences between the NO donors available. The aim of this work is to compare the metabolic profile of traditional and new generation NO donors to see which one points out the osteoarthritic process in the best way. Methods: Human healthy and OA chondrocytes were isolated from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with NO donors (NOC-12 or SNP). NO production was evaluated by the Griess method, and apoptosis was quantified by flow cytometry. Mitochondrial function was evaluated by analysing respiratory chain enzyme complexes, citrate synthase (CS) activities by enzymatic assay, mitochondrial membrane potential (Δψm) by JC-1 using flow cytometry, and ATP levels were measured by luminescence assays. Glucose transport was measured as the uptake of 2-deoxy-[3H]glucose (2-[3H]DG). Statistical analysis was performed using the Mann-Whitney U test. Results: NOC-12 liberates approximately ten times more NO2 - than SNP, but the level of cell death induced was not as profound as that produced by SNP. Normal articular chondrocytes stimulated with NOC-12 had reduced activity from complexes I, III y IV, and the mitochondrial mass was increased in these cells. Deleterious effects on ΔΨm and ATP levels were more profound with SNP, and this NO donor was able to reduce 2-[3H]DG levels. Both NO donors had opposite effects on lactate release, SNP diminished the levels and NOC-12 lead to lactate accumulation. OA chondrocytes incorporate significantly more 2-[3H]DG than healthy cells. Conclusions: These findings suggest that the new generation donors, specifically NOC-12, mimic the OA metabolic process much better than SNP. Previous results using SNP have to be considered prudently since most of the effects observed can be induced by the interactions of secondary products of NO. Keywords: chondrocytes, nitric oxide, apoptosis, mitochondria, glucose, osteoarthritis de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 Open Access Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism C de Andrés1, Emilia Maneiro1, Miguel A Martín2, Joaquín Arenas2 and Francisco J Blanco1,3,4* María C de Andrés1, Emilia Maneiro1, Miguel A Martín2, Joaquín Arenas2 and Francisco J Blan * Correspondence: fblagar@sergas.es 1INIBIC-Complejo Hospitalario Universitario A Coruña (CHUAC) Rheumatology Division, As Xubias 84, 15006-A Coruña, Spain Full list of author information is available at the end of the article Primary culture of chondrocytes Chondrocytes were recovered and plated at high density (4 × 106 per 162 cm2 flask; Costar, Cambridge, MA, USA) in DMEM (Life Technologies, Paisley, UK) supple- mented with 100 units/ml penicillin, 100 μg/ml strepto- mycin, 1% glutamine, and 10% FCS (Life Technologies). Chondrocytes were incubated at 37°C in a humidified gas mixture containing 5% CO2 balanced with air. Chondrocytes were used at weeks 2 to 3 at confluence in primary culture. Cell viability was assessed by trypan blue dye exclusion, and stained cells were discarded to carry out experiments. It is generally accepted that the quantities of available oxygen and glucose can fluctuate considerably in con- nective tissues such as articular cartilage, growth plates and the intervertebral disc [27-29]. Articular chondro- cytes consume less oxygen in comparison with most other cell types [30]. Consequently, anaerobic glycolysis forms the principal source of cellular ATP in cartilage. General procedure and NO donor compounds employed NO donor compounds were added from 60 mM stock solution dissolved in 0.1 M NaOH (NOC-12) and 10 mM stock solution dissolved in medium (SNP). This NO donor compound was freshly prepared before each experiment but NOC-12 was stored as 60 mM stock solutions in 0.1 mM NaOH at -20°C. Chondrocytes were first seeded in DMEM with 5% FCS inactivated for 24 hours, and then the NO donor compound was added directly to the culture medium and allowed to incubate for an additional 5-, 12-, 24- and 48-hour period, depending on each experiment. Experiments without glucose were carried out in DMEM glucose-free med- ium (Life Technologies) supplemented in the same way as the standard. The direct investigation of the function of exogenous NO production on articular chondrocytes has been ham- pered by the lack of uniformity between the different types of NO donor compounds [31]. Since the past decade, the diazeniumdiolates began to replace traditional donors, such as SIN-1 (3-morpholinosydnonimine), SNP (sodium nitroprusside), SNAP (S-nitroso-N-acetylpenicillamine) and S-nitrosogluthathione, as sources of exogenous NO production [32], because have been shown to be reliable sources of NO under a variety of culture conditions [33,34]. The main advantages of these compounds are: known rates of NO generation, NO generation rates cov- ering a wide range, spontaneity of NO generation and ten- able generation of NO redox forms [34]. Primary culture of chondrocytes For all these reasons, besides the classical donor SNP, we used a diazeniumdiolate, NOC-12 (N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine), with a half-life of 327 minutes (determined at 22°C, pH 7.4) [33], for exogenous NO pro- duction, to further investigate the conditions in which NO is cytotoxic to chondrocytes, and compare the different effects induced by the two different types of NO donors. We wished to determine whether NO modulates the pathogenesis of OA, inducing apoptosis by means of the inhibition of mitochondrial function de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 chondrocytes [10,12,13]. A variety of NO donors have been demonstrated to suppress energy production by mitochondrial respiration in different cell types [14,15], an effect enhanced at low oxygen tensions [16], and firstly reported in chondrocytes by Johnston and colla- borators [12]. chondrocytes [10,12,13]. A variety of NO donors have been demonstrated to suppress energy production by mitochondrial respiration in different cell types [14,15], an effect enhanced at low oxygen tensions [16], and firstly reported in chondrocytes by Johnston and colla- borators [12]. Cartilage acquisition and cell isolation Cartilage acquisition and cell isolation Normal human cartilage from femoral heads (joint replacement surgery) and knees (joint replacement surgery and autopsies) was obtained from 11 adult donors without history of joint disease and who had macroscopically normal cartilage (mean age ± SD 46.5 ± 10.5 years); human OA cartilage was obtained from the femoral heads of 12 patients (age 70 ± 12.6 years). All patients and healthy donors have signed the informed consent and the project was approved by the Regional Ethical Committee from Galicia (Spain). Small cartilage fragments were digested as previously described [35]. Chondrocytes are highly glycolytic resident cells of articular cartilage that metabolize glucose as a primary substrate for ATP production [17]. However, oxygen does diffuse into articular cartilage and articular chon- drocytes possess mitochondria and respire in vivo [18]. The superficial and middle zones of articular cartilage are not anoxic [19], and in this context, mitochondrial oxidative phosphorylation (OXPHOS) is 18 times as effi- cient in ATP generation as is glycolysis [14,20]. Further- more, OXPHOS may account for up to one fourth of total steady-state ATP production within articular carti- lage, and possibly more under conditions of increased energy demands associated with cartilage stress [21]. Besides this, mitochondria are important in regulating both caspase-dependent and caspase-independent apop- totic pathways [22-26]. Introduction hand, the pathogenesis of OA is characterized by the pro- duction of high amounts of nitric oxide (NO), conse- quence of up-regulation of chondrocyte-inducible nitric oxide synthase (iNOS) induced by inflammatory cytokines, such as IL-1b and TNFa, and other factors [4-7]. Articular cartilage is an avascular, non-insulin-sensitive tissue that utilizes glucose as the main energy source and as a precursor for glycosaminoglycan synthesis and a regu- lator of gene expression. Degradation of articular cartilage is a hallmark of osteoarthritis (OA) [1] and is associated with aberrant glucose metabolism [2,3]. On the other Although it has been reported that NO causes chon- drocyte apoptosis [8-10], production of high levels of endogenous NO by over-expression of the iNOS gene in transfected chondrocytes has not been found to cause cell death [11]. Other reports have proposed NO to be a physiologic regulator of mitochondrial respiration in * Correspondence: fblagar@sergas.es 1INIBIC-Complejo Hospitalario Universitario A Coruña (CHUAC) Rheumatology Division, As Xubias 84, 15006-A Coruña, Spain Full list of author information is available at the end of the article Page 2 of 12 Page 2 of 12 de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 NO release by different NO donors We observed that kinetic liberation of NO changes between different NO donors. The diazeniumdiolate NOC-12 liberated approximately ten times more NO2 - than SNP. NO2 - accumulation in supernatants of normal chondrocytes treated with different NO donors depend- ing on time and concentration (Figure 1). DNA labelling technique with propidium iodide for flow cytometry analysis DNA labelling technique with propidium iodide for flow cytometry analysis USA). Chondrocytes were cultured in 96-well plates (5 × 104) and stimulated with different NO donors for 24 hours (100 μl); after this, 50 μl of lysis solution were added and mixed for 5 minutes; subsequently the enzymatic sub- strate (luciferase/luciferine) was added. The kit supplies a standard that gives reference values. Readers were carried out in a microbeta counter (PerkinElmer Inc.). Chondrocytes (500,000 cells/well in a 6-well plate) were incubated with different NO donors (0.5, 1.0 and 2.0 mM SNP and NOC-12) for 12, 24 and 48 hours. Then cells were fixed in 70% ethanol at 4°C for 60 min- utes, washed and incubated with RNAse (50 μg/ml) and propidium iodide (PI, 100 μg/ml) for 15 minutes at room temperature in the dark and kept at 4°C. PI fluor- escence of nuclei was measured by flow cytometry on a FACScan (Becton and Dickinson, Mountain View, CA, USA) using a 560-nm dichromatic mirror and a 600-nm band pass filter. Data are expressed as percent apoptotic (hypodiploid) nuclei. Quantification of lactic acid Enzymatic determination of lactic acid in chondrocyte cul- ture supernatants was performed using Lactate Reagent (Spinreact, Girona, Spain). Chondrocytes were cultured in 96-well plates (5 × 104) and stimulated with different NO donors for 24 hours; 10 μl of supernatant were mixed with 10 μl of lactate reagent and incubated for 5 minutes at room temperature. The absorption was estimated by an automated plate reader (Ultrospec 1000, Amersham Phar- macia Biotech, Uppsala, Sweden) at 505 nm; this method is linear towards lactate values of 150 mg/dl. Quantification of nitrites The NO production of chondrocyte cells was measured by estimating nitrite accumulation using the Griess reagent (1% sulphanilamide and 0.1% N-(1-naphthyl)- ethylenediamine dihydrochloride in 5% H3PO4) (Sigma, St Louis, MO) as previously described [36]. Chondro- cytes were cultured in 96-well plates (5 × 104) and sti- mulated with different NO donors for 5, 24 and 48 hours. Page 3 of 12 de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 2-Deoxy-[3H] glucose uptake To determinate the glucose uptake levels, normal chon- drocytes were cultured in 24- well plates (Visiplate, PerkinElmer Inc.) at 2 × 105 cells per well in DMEM with- out glucose and 5% inactivated calf serum for 24 h at 37°C. Later, cells were stimulated with 10 μM of different donors for 24 hours at 37°C in DMEM without glucose and subse- quently 10 μCi 2-deoxy-(3H)glucose (2-(3H)DG) was added to cells in DMEM without glucose for 0 minutes, 15 min- utes and 1 hour at 37°C. Cells were washed with cold PBS pH 7.4; later, 50 μl of solvable (PerkinElmer Inc.) was added to lysate cells and mixed vigorously for 5 minutes. Lastly, 500 μl of scintillation liquid was added (Ultima Gold, PerkinElmer, Inc.) and mixed for 2 minutes; glucose uptake was estimated by means of a microbeta counter (MicroBeta TriLux (PerkinElmer, Inc.)). Measurement of the MRC complex activities in digitonin- permeabilized chondrocytes Untreated, SNP-treated (5 hours) and NOC-12-treated (24 hours) chondrocytes (at least 10 × 106) were collected by trypsinization, washed with PBS, and sedimented at 150 g for 5 minutes at 4°C. Digitonin-permeabilized chondrocyte homogenates (10 to 50 μl per ml of test volume) were used to measure the activities of the respiratory chain enzymes and citrate synthase (CS) in a DU-650 spectrophotometer (Beckman Instruments, Palo Alto, CA, USA) as previously described [35]. Determination of mitochondrial membrane potential (ΔΨm) Data analysis was performed with SPSS software, version 12.05 (SPSS, Chicago, IL, USA). Results are expressed as the mean ± SD. Individual donors were studied in dupli- cate; cells from different donors were not pooled in any experiment. Comparisons between groups were carried out using the Mann-Whitney two-tailed U-test. P-values ≤0.05 were considered significant. To measure the ΔΨm of chondrocytes, the fluorescent probe JC-1 (5,5’,6,6’- tetrachloro-1,1’,3,3’-tetraethylbenzi- midazole carbocyanide iodide) (Molecular Probes, Inc., Eugene, OR, USA) was used. JC-1 exists as a monomer at low values of ΔΨm (green fluorescence), whereas it forms aggregates at high ΔΨm (red fluorescence). Briefly, chondrocytes were cultured in 6-well plates (5 × 105) and stimulated with different NO donors for 5, 12 and 24 hours; after that they were prepared as pre- viously described [35]. Morphological evidence of apoptosis For morphological studies, chondrocytes were cultured in 8-well slides (Costar) and treated with 1 mM of different NO donors for 24 hours. The cells were then washed with cold PBS, fixed in acetone/ethanol 70% (1:1) for 10 minutes at 4°C, stained with 49,6-dianidino-2- phenylindole dihydrochloride (DAPI, 2 mg/ml) (Sigma) for 10 minutes in the dark, mounted in glycergel (DAKO, Hamburg, Germany), and observed by fluorescence microscopy (Olympus, BX-UCB, Center Valley, PA). Effect of NO on cell death of normal human articular chondrocytes Effect of NO on cell death of normal human articular chondrocytes 5 hours than in control cells [10] (Table 1). In relation with the enzymatic activity of the MRC of normal chon- drocytes treated with the diazeniumdiolate compound NOC-12, the activities of all complexes were signifi- cantly lower than in control cells, except complex II (Table 1). Enzyme activities were referred to the specific activity of CS to correct for mitochondrial volume. NO donors damage the nuclear DNA of human articular chondrocytes in very different ways. By means of flow cyto- metry with PI, we could observe that the percentage of death cells (expressed as hypodiplod DNA percentage) was much higher with SNP than NOC-12 (Additional file 1); with 1 mM SNP at 24 hours: 25.9 ± 23.3 versus 0.8 ± 0.5 (P ≤0.05) compared with 1 mM NOC-12 at 24 hours: 4.3 ± 1.9 versus 0.8 ± 0.5 (P ≤0.05) (Figure 2A). However, with higher concentrations of NOC-12 (2 mM at 48 hours), the percentage of apoptosis reached 21.6 ± 8.5% (Additional file 1). Besides this, when the cells were stained with DAPI we could see that the only NO donor able to induce the fragmentation of the nucleus and the formation of apoptotic bodies was SNP (Figure 2B). The only effect of NOC-12 on the nucleus of chondrocytes was the acquisi- tion of a globule-like aspect (Figure 2B). NO causes depolarization of the mitochondria in normal chondrocytes The relative ratio of red/green fluorescence (ratio of normal mitochondrial polarization to mitochondrial depolarization) intensity values showed that in normal human chondrocyte cultures 1 mM SNP at 24 hours decreased the ratio of red/green fluorescence in compar- ison with untreated cells (1.91 ± 1.9 versus 3.5 ± 2.9; P ≤0.01) (Figure 3B). In addition, 1 mM SNP caused an increase in the cell population with mitochondrial depo- larization (12.2 ± 6.6 versus 33.6 ± 13.1%; P ≤0.05) (Figure 3A). On the other hand, NOC-12 induced mito- chondrial depolarization, as the percentage of cells with normal polarization diminished, with 1 mM NOC-12 at 24 hours: 27.7 ± 17.9 versus 14.1 ± 3.6; P ≤0.05; (Figure 3A), and the percentage of cells with depolarization increased: 12.2 ± 6.6 versus 18.8 ± 11.3; P ≤0.05 (Figure 3A). This NOC-12 alters the activity of the complexes of the mitochondrial respiratory chain (MRC) in articular chondrocytes Assay of intracellular ATP To assay intracellular ATP, we used a commercial biolu- miscence kit (ATPLite, PerkinElmer Inc., Boston, MA, de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 Page 4 of 12 Figure 1 Nitric oxide (NO) donors differ from the amount of NO2 - released. NO2 - accumulation in supernatants of normal chondrocytes treated with different NO donors for 24 hours in standard culture conditions. The lines show the dose-dependent increase of NO accumulation in all cases. Values are the mean ± SD; n = 5. SNP, sodium nitroprusside; NOC-12, N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine. Figure 1 Nitric oxide (NO) donors differ from the amount of NO2 - released. NO2 - accumulation in supernatants of normal chondrocytes treated with different NO donors for 24 hours in standard culture conditions. The lines show the dose-dependent increase of NO accumulation in all cases. Values are the mean ± SD; n = 5. SNP, sodium nitroprusside; NOC-12, N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine. NOC-12 alters the activity of the complexes of the mitochondrial respiratory chain (MRC) in articular chondrocytes Previous results obtained in our laboratory showed that the activity of the complex IV is significantly lower in normal chondrocytes stimulated with 1 mM SNP at de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 Page 5 of 12 Figure 2 Nitric oxide (NO) effect on chondrocyte apoptosis. (A) Cell death levels in chondrocytes treated with different NO donors was determined by the iodide propidium (PI) method. Control chondrocytes were treated with 1 mM sodium nitroprusside (SNP) and 1 mM N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine (NOC-12) for 24 hours. Data are expressed as a percentage of apoptotic (hypodiploid) nuclei. Values are the mean ± SD; n = 5. P ≤0.05 versus untreated chondrocytes (control). (B) Cellular changes induced by NO on normal human chondrocytes were analysed by 49,6- diamino-2-phenylindole dihydrochloride (DAPI staining) and fluorescence microscopy. Shown is a representative example of five experiments. Figure 2 Nitric oxide (NO) effect on chondrocyte apoptosis. (A) Cell death levels in chondrocytes treated with different NO donors was determined by the iodide propidium (PI) method. Control chondrocytes were treated with 1 mM sodium nitroprusside (SNP) and 1 mM N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine (NOC-12) for 24 hours. Data are expressed as a percentage of apoptotic (hypodiploid) nuclei. Values are the mean ± SD; n = 5. P ≤0.05 versus untreated chondrocytes (control). (B) Cellular changes induced by NO on normal human chondrocytes were analysed by 49,6- diamino-2-phenylindole dihydrochloride (DAPI staining) and fluorescence microscopy. Shown is a representative example of five experiments. than in control cells, with 1mM NOC-12 at 24 hours: 0.40 ± 0.16 versus 0.57 ± 0.19 (P ≤0.05). Again, the NO donor that induced the most dramatic changes was SNP, as it reduced the intracellular ATP levels practi- cally to zero (Figure 4). finding also can be observed with decreasing ratio of red/ green fluorescence in comparison with untreated cells (2.2 ± 1.5 versus 3.5 ± 2.9; P ≤0.05) (Figure 3B). However, the NO donor that induced the strongest changes in the mitochondrial membrane potential was SNP. Values are the mean ± SD (number). P ≤0.05 versus untreated chondrocytes. aCS-corrected complex activity is expressed as (nmol/minute/mg protein)/(CS- specific activity) × 100. NO, nitric oxide; NOC-12, N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine; CS, citrate synthase; Complex I, rotenone-sensitive NADH- coenzime Q1 reductase; complex II, succinate dehydrogenase; complex III, antimycin-sensitive ubiquinol cytochrome c reductase; complex IV, cytochrome c oxidase. Chondrocytes were treated with NOC-12 for 24 hours and SNP for 5 hours. Chondrocytic lactate production is strongly influenced by NO donor type NO abolishes ATP generation by chondrocytes in culture NO has a detrimental effect on the generation of ATP by normal chondrocytes (Additional file 2). With NOC- 12, the intracellular ATP levels were significantly lower NO donors have very different effects on lactate release by normal articular chondrocytes. The only donor that Table 1 Values of mitochondrial respiratory chain complexes in cultures of normal chondrocytes treated with two NO donor compounds Normal chondrocytes Cells treated with 1 mM NOC-12 Cells treated with 1 mM SNP Age, years 49.8 ± 21.2 (14) 44.2 ± 19.4 (5) 59.7 ± 18.9 (11) Proteins, mg/ml 3.1 ± 0.9 (14) 2.8 ± 1.4 (5) 4.2 ± 1.4 (11) CS enzymatic activity (nmol/min/mg protein) 108.9 ± 38.5 (14) 146.9 ± 54.7 (5) 106.6 ± 26.2 (11) Mitochondrial complex activitya Complex I 30.1 ± 13.4 (14) 6.8 ± 2.3 (5) 22.8 ± 19.1 (11) Complex II 7.4 ± 2.3 (14) 6.8 ± 1.6 (5) 10.2 ± 1.81 (11) Complex III 53.5 ± 18.4 (14) 35.2 ± 9.3 (5)* 46.3 ± 9.7 (11) Complex IV 61.2 ± 8.3 (14) 15.7 ± 3.1 (5)* 40.2 ± 11.3 (11)* Values are the mean ± SD (number). P ≤0.05 versus untreated chondrocytes. aCS-corrected complex activity is expressed as (nmol/minute/mg protein)/(CS- specific activity) × 100. NO, nitric oxide; NOC-12, N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine; CS, citrate synthase; Complex I, rotenone-sensitive NADH- coenzime Q1 reductase; complex II, succinate dehydrogenase; complex III, antimycin-sensitive ubiquinol cytochrome c reductase; complex IV, cytochrome c oxidase. Chondrocytes were treated with NOC-12 for 24 hours and SNP for 5 hours. ochondrial respiratory chain complexes in cultures of normal chondrocytes treated with two NO Table 1 Values of mitochondrial respiratory chain complexes in cultures of normal chondrocytes donor compounds Values are the mean ± SD (number). P ≤0.05 versus untreated chondrocytes. aCS-corrected complex activity is expressed as (nmol/minute/mg protein)/(CS- specific activity) × 100. NO, nitric oxide; NOC-12, N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine; CS, citrate synthase; Complex I, rotenone-sensitive NADH- coenzime Q1 reductase; complex II, succinate dehydrogenase; complex III, antimycin-sensitive ubiquinol cytochrome c reductase; complex IV, cytochrome c oxidase. Chondrocytes were treated with NOC-12 for 24 hours and SNP for 5 hours. de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 Page 6 of 12 Figure 3 Effect of nitric oxide (NO) donors on mitochondrial membrane potential. Chondrocytic lactate production is strongly influenced by NO donor type (A) Fluorescence-activated cell sorter analysis of mitochondrial membrane potential in human chondrocytes. Untreated and treated normal chondrocytes with NO donors were stained with 5,5’,6,6’- tetrachloro-1,1’,3,3’-tetraethylbenzimidazole carbocyanide iodide (JC-1) and analysed by flow cytometry. Photomultiplier settings were adjusted to detect JC-1 monomer fluorescence signals on the filter 1 (FL1) detector (green fluorescence) and JC-1 aggregate fluorescence signals on the FL2 detector (red fluorescence). Shown is an example of chondrocytes treated with 1 mM SNP and 2 mM N-ethyl-2(1-ethyl-2 hydroxy-2- nitrosohydrazine (NOC-12) for 24 hours. (B) Quantification of red and green fluorescence. Histograms represent the JC-1 fluorescence of normal cells and those treated with NO donors. Green fluorescence (open graph) increases, whereas red fluorescence (solid graph) decreases in the NOC-12 and sodium nitroprusside (SNP)-treated chondrocytes, suggesting a reduction of the mitochondrial membrane potential, and therefore, a decrease in the red/green ratio. Shown is an example at 24 hours. Results are the mean ± SD; n = 5. P ≤0.05 versus untreated chondrocytes (control). Figure 3 Effect of nitric oxide (NO) donors on mitochondrial membrane potential. (A) Fluorescence-activated c Figure 3 Effect of nitric oxide (NO) donors on mitochondrial membrane potential. (A) Fluorescence-activated cell sorter analysis of mitochondrial membrane potential in human chondrocytes. Untreated and treated normal chondrocytes with NO donors were stained with 5,5’,6,6’- tetrachloro-1,1’,3,3’-tetraethylbenzimidazole carbocyanide iodide (JC-1) and analysed by flow cytometry. Photomultiplier settings were adjusted to detect JC-1 monomer fluorescence signals on the filter 1 (FL1) detector (green fluorescence) and JC-1 aggregate fluorescence signals on the FL2 detector (red fluorescence). Shown is an example of chondrocytes treated with 1 mM SNP and 2 mM N-ethyl-2(1-ethyl-2 hydroxy-2- nitrosohydrazine (NOC-12) for 24 hours. (B) Quantification of red and green fluorescence. Histograms represent the JC-1 fluorescence of normal cells and those treated with NO donors. Green fluorescence (open graph) increases, whereas red fluorescence (solid graph) decreases in the NOC-12 and sodium nitroprusside (SNP)-treated chondrocytes, suggesting a reduction of the mitochondrial membrane potential, and therefore, a decrease in the red/green ratio. Shown is an example at 24 hours. Results are the mean ± SD; n = 5. P ≤0.05 versus untreated chondrocytes (control). Figure 3 Effect of nitric oxide (NO) donors on mitochondrial membrane potential. (A) Fluorescence-activated cell sorter analysis of mitochondrial membrane potential in human chondrocytes. Untreated and treated normal chondrocytes with NO donors were stained with 5,5’,6,6’- tetrachloro-1,1’,3,3’-tetraethylbenzimidazole carbocyanide iodide (JC-1) and analysed by flow cytometry. Chondrocytic lactate production is strongly influenced by NO donor type Figure 5 Nitric oxide (NO) influence on 2-deoxy-(3H) glucose uptake by normal chondrocytes. Effect of the NO donors sodium nitroprusside (SNP) and N-ethyl-2(1-ethyl-2 hydroxy-2- nitrosohydrazine (NOC-12) on the uptake of 2-deoxy-(3H) glucose by normal chondrocytes for 15 minutes. The detailed procedure is described in Materials and methods. Values are the mean ± SD; n = 7. P ≤0.05 versus untreated chondrocytes (control). Figure 4 ATP generation by normal chondrocytes treated with different nitric oxide (NO) donors. Articular chondrocytes were treated with 1 mM sodium nitroprusside (SNP) and 2 mM N-ethyl-2 (1-ethyl-2 hydroxy-2-nitrosohydrazine (NOC-12) for 24 hours. Then the ATP assay was performed as described in Materials and methods. Values are the mean ± SD; n = 7. P ≤0.05 versus untreated chondrocytes (control). Figure 5 Nitric oxide (NO) influence on 2-deoxy-(3H) glucose uptake by normal chondrocytes. Effect of the NO donors sodium nitroprusside (SNP) and N-ethyl-2(1-ethyl-2 hydroxy-2- nitrosohydrazine (NOC-12) on the uptake of 2-deoxy-(3H) glucose by normal chondrocytes for 15 minutes. The detailed procedure is described in Materials and methods. Values are the mean ± SD; n = 7. P ≤0.05 versus untreated chondrocytes (control). induced a significant fall in these values was SNP, with 2 mM SNP at 24 hours: 1.01 ± 0.3 versus 2.30 ± 0.58 (P ≤ 0.05) (Table 2). The new generation donor NOC-12 induced a substantial increase in lactate production, with 0.5 mM NOC-12 at 24 hours: 2.91 ± 0.58 versus 2.30 ± 0.58, although this increase was not statistically significant (Table 2). Detrimental effect of NO on chondrocyte viability depends on glucose levels To assess the impact of glucose levels on cell viability after NO treatment, we carried out experiments with both NO donors (SNP and NOC-12) using only one constant concentration (1 mM) and increasing glucose concentrations (from 0.75 to 5 mM). The percentage of death cells decreased as glucose concentration increased, only when SNP was employed as NO donor (Figure 6). No significant results were found in this issue when NOC-12 was used. Chondrocytic lactate production is strongly influenced by NO donor type Photomultiplier settings were adjusted to detect JC-1 monomer fluorescence signals on the filter 1 (FL1) detector (green fluorescence) and JC-1 aggregate fluorescence signals on the FL2 detector (red fluorescence). Shown is an example of chondrocytes treated with 1 mM SNP and 2 mM N-ethyl-2(1-ethyl-2 hydroxy-2- nitrosohydrazine (NOC-12) for 24 hours (B) Quantification of red and green fluorescence Histograms represent the JC-1 fluorescence of normal nitric oxide (NO) donors on mitochondrial membrane potential. (A) Fluorescence-activated cell sorter analysis o Figure 3 Effect of nitric oxide (NO) donors on mitochondrial membrane potential. (A) Fluorescence activated cell sorter analysis of mitochondrial membrane potential in human chondrocytes. Untreated and treated normal chondrocytes with NO donors were stained with 5,5’,6,6’- tetrachloro-1,1’,3,3’-tetraethylbenzimidazole carbocyanide iodide (JC-1) and analysed by flow cytometry. Photomultiplier settings were adjusted to detect JC-1 monomer fluorescence signals on the filter 1 (FL1) detector (green fluorescence) and JC-1 aggregate fluorescence signals on the FL2 detector (red fluorescence). Shown is an example of chondrocytes treated with 1 mM SNP and 2 mM N-ethyl-2(1-ethyl-2 hydroxy-2- nitrosohydrazine (NOC-12) for 24 hours. (B) Quantification of red and green fluorescence. Histograms represent the JC-1 fluorescence of normal cells and those treated with NO donors. Green fluorescence (open graph) increases, whereas red fluorescence (solid graph) decreases in the NOC-12 and sodium nitroprusside (SNP)-treated chondrocytes, suggesting a reduction of the mitochondrial membrane potential, and therefore, a decrease in the red/green ratio. Shown is an example at 24 hours. Results are the mean ± SD; n = 5. P ≤0.05 versus untreated chondrocytes (control). de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 Page 7 of 12 Figure 4 ATP generation by normal chondrocytes treated with different nitric oxide (NO) donors. Articular chondrocytes were treated with 1 mM sodium nitroprusside (SNP) and 2 mM N-ethyl-2 (1-ethyl-2 hydroxy-2-nitrosohydrazine (NOC-12) for 24 hours. Then the ATP assay was performed as described in Materials and methods. Values are the mean ± SD; n = 7. P ≤0.05 versus untreated chondrocytes (control). Figure 4 ATP generation by normal chondrocytes treated with different nitric oxide (NO) donors. Articular chondrocytes were treated with 1 mM sodium nitroprusside (SNP) and 2 mM N-ethyl-2 (1-ethyl-2 hydroxy-2-nitrosohydrazine (NOC-12) for 24 hours. Then the ATP assay was performed as described in Materials and methods. Values are the mean ± SD; n = 7. P ≤0.05 versus untreated chondrocytes (control). Glucose uptake by OA chondrocytes in basal conditions is more efficient than by normal chondrocytes Chondrocytes were maintained for 15 minutes or 1 hour in culture media without glucose and a non-metaboliz- able analogue of glucose: 2-(3H)DG. Basal 2-(3H)DG uptake was identical in normal and OA chondrocytes incubated for 15 minutes; however, the basal 2-(3H)DG uptake in OA chondrocytes was significantly higher than in normal chondrocytes (23889.9 ± 9941.6 versus 14669.5 ± 2776.1; P ≤0.05) (Figure 7), when cells were maintained for 1 hour in culture. Table 2 Values of lactate release in cultures of normal chondrocytes treated with different NO donors compounds for 24 hours Lactate (μM/5 × 104 cells) Control normal chondrocytes 2.30 ± 0.58 (7) 0.5 mM SNP 1.83 ± 0.65 (7) 1 mM SNP 1.33 ± 0.38 (7) † 2 mM SNP 1.01 ± 0.30 (7) † 0.5 mM NOC-12 2.91 ± 0.58 (7) 1 mM NOC-12 2.90 ± 0.66 (7) 2 mM NOC-12 2.86 ± 0.77 (7) Values are the mean ± SD (number). NO, nitric oxide; SNP, sodium nitroprusside; NOC-12, N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine. Table 2 Values of lactate release in cultures of normal chondrocytes treated with different NO donors compounds for 24 hours Lactate (μM/5 × 104 cells) Control normal chondrocytes 2.30 ± 0.58 (7) 0.5 mM SNP 1.83 ± 0.65 (7) 1 mM SNP 1.33 ± 0.38 (7) † 2 mM SNP 1.01 ± 0.30 (7) † 0.5 mM NOC-12 2.91 ± 0.58 (7) 1 mM NOC-12 2.90 ± 0.66 (7) 2 mM NOC-12 2.86 ± 0.77 (7) Values are the mean ± SD (number). NO, nitric oxide; SNP, sodium nitroprusside; NOC-12, N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine. Table 2 Values of lactate release in cultures of normal chondrocytes treated with different NO donors compounds for 24 hours SNP reduces glucose uptake by normal articular chondrocytes Uptake of 2-(3H)DG by normal chondrocytes cultured under μM SNP concentrations (10 μM) for either 15 minutes or 1 hour was approximately 20% lower than that found in their respective controls (Figure 5). On the contrary, 2-(3H)DG uptake in normal chondro- cytes stimulated with NOC-12 did not change relative to the control. In this set of experiments, μM NO donors concentrations were employed; the reason is because in most of these cases, when mM concentra- tions were used, this caused the cells to rise (above all, SNP) and the quantifications showed false positives. Glucose uptake by OA chondrocytes in basal conditions is more efficient than by normal chondrocytes Discussion Traditionally, the increase of endogenous NO produc- tion by human articular cartilage has been associated with joint degeneration. NO donors have been used so far to mimic the OA process in vitro, and they represent a powerful tool of study. However, in vitro models with different NO donors have not resolved what the role of NO is in cartilage degradation due to the lack of unifor- mity that exists between the different types of NO com- pounds [31]. The differential effects of NO are partly due to the type of NO donors and cell used [37]. The de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 Page 8 of 12 Figure 6 The effect of nitric oxide (NO) on chondrocyte viability depends on glucose levels. Articular chondrocytes were cultured in medium with crescent glucose concentrations and treated with 1 mM sodium nitroprusside (SNP) and 1 mM N-ethyl-2(1-ethyl-2 hydroxy-2- nitrosohydrazine (NOC-12) for 24 hours. Data are expressed as a percentage of apoptotic (hypodiploid) nuclei. Values are the mean ± SD; n = 3. Figure 6 The effect of nitric oxide (NO) on chondrocyte viability depends on glucose levels. Articular chondrocytes were cultured in medium with crescent glucose concentrations and treated with 1 mM sodium nitroprusside (SNP) and 1 mM N-ethyl-2(1-ethyl-2 hydroxy-2- nitrosohydrazine (NOC-12) for 24 hours. Data are expressed as a percentage of apoptotic (hypodiploid) nuclei. Values are the mean ± SD; n = 3. biochemistry of NO is complex because of the reactions of NO itself, the interactions of secondary products of NO and the overall chemical environment under which NO is produced [38]. a half-life of 327 minutes (determined at 22°C, pH 7.4) [33] was used for exogenous NO production to further investigate the conditions in which NO is cytotoxic to chondrocytes. A primary basis for the use of diazenium- diolates is that many of them decompose spontaneously in aqueous media to release the critical bioregulatory species [34]. The main advantages of these compounds are known rates of NO generation, NO generation rates covering a wide range, spontaneity of NO generation and tenable generation of NO redox forms. In our study, we employed two NO donor types: the traditional compound SNP, that is used in the majority of studies, and one diazeniumdiolate: NOC-12. It has been reported that the traditional donor SNP does not spontaneously release NO in the absence of redox acti- vation [34]. de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 Analysis of the MRC showed that at 5 hours, SNP reduced the activity of complex IV by 30%; furthermore, SNP induced depolarisation of the mitochondrial mem- brane [10]. In this study we show that NOC-12 induces depolarisation of the mitochondrial membrane as well as SNP, but to a lesser extent (10%); however, it had a more radical effect on MRC activity than SNP, this donor reduces the activities of all the complexes except complex II (complex I by 80%, complex III by 33% and complex IV by 75%). These results show that the inhibition of the MRC complexes is not the main cause of cell death induction in chondrocytes by NO. On the other hand, CS activity was increased about 40% in NOC-12-treated chondrocytes, and this fact has been correlated with an increment of the mito- chondrial mass [30]; Nisoli and collaborators also suggested that NO is implicated in the regulation of energy metabo- lism, possibly through the enhancement of mitochondria formation [53]. Similar findings were previously found in OA chondrocytes [35] but not in SNP-treated ones [10]. Therefore, an increase in mitochondrial mass could be a mechanism by which OA chondrocytes as well as NOC- 12-treated cells, compensate for the electron transfer deficiency resulting from dysfunction in several com- plexes and the consequent low production of ATP per mitochondrion, as has already been reported by Maneiro and collaborators [35]. Chondrocyte death does not correlate with the amount of NO released by NO donors. Similar to other authors [46], our results showed that SNP is the least potent in terms of producing exogenous NO in chondrocyte cul- ture, although it is the most potent inducer of chondro- cyte death. The amount of NO produced by NOC-12 was 10-fold higher than the NO produced by SNP, but the level of cell death induced was not as profound as that produced by SNP. As previously shown in our laboratory [10], SNP was able to induce formation of apoptotic bodies, which are produced from cells undergoing cell death by apoptosis. However, we observed that NOC-12 increased the hypodiploid nuclei number without forma- tion of apoptotic bodies, which is probably related to another type of programmed cell death. Recently it has been proposed that autophagy is another type of pro- grammed cell death than happens in the human articular cartilage as well [47]. de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 The increase in the number of hypopliod nuclei and the observation of some morpholo- gic changes as vacuole formation seems to relate NOC- 12 with autophagy (personal data). It is believed unlikely that NO is the sole mediator of SNP-induced chondrocyte death and peroxynitrite, a reaction product of NO and superoxide anions, or the primary by-products of the decomposition of SNP, such as the cyanide aninon or pentacy-anoferrate complex, might contribute to its cytotoxicity [34,48]. It is unclear whether chondrocyte apoptosis is the major mechanism of cartilage degradation or merely a by-product of tissue degeneration [46]. In relation to ATP synthesis both donors had a detri- mental effect on it, but once more SNP was the com- pound with the most important deleterious effect. With respect to lactate production, SNP reduced the levels in a significant way compared to the control cells; on the con- trary, NOC-12 increased lactate production by chondro- cytes although this increment was not statistical significant. A dysfunction in complexes I, III and IV com- promises the electron transfer pathway; this defect could be solved increasing the anaerobic metabolism to avoid excess production of ROS, and these findings are in agreement with the increase of lactate levels after incuba- tion with NOC-12. These results are consistent with the findings reported by Tomita and collaborators on NOC- 18-treated chondrocytes, another member of the diaze- niumdiolate family [13]. Because NO inhibited the respiration of mitochondria, cellular glycolysis was enhanced significantly, the effect on cellular ATP levels was rather mild, despite the inhibition of mitochondrial respiration by NO. Thus, the enhanced glycolysis in NOC-18-treated chondrocytes could theoretically com- pensate for the inhibition of mitochondrial synthesis by approximately 46% [13]. On the other hand, the build-up of lactic acid will have detrimental effects on the extracel- lular matrix and may contribute to the pathogenesis and progression of OA [54]. Mitochondria comprise a target of NO and there is accumulating evidence that inhibition of respiration may contribute to the pro-apoptotic effect of NO by ΔΨm alteration, transition-pore opening and release of cyto- chrome c [45,49]. There is increasing evidence about the importance of mitochondria in OA pathology. Pre- viously, we showed that the activity of the mitochondrial complexes II and III is lower in OA than in normal human chondrocytes; this produces a decrease in ATP levels as well as a higher ROS generation [12,35]. Discussion Diazeniumdiolates, also denominated as NONOate, or NOC, have begun to replace traditional donors as sources of exogenous NO production [32] and have been shown to be reliable sources of NO under a variety of culture conditions [33,34]. For these reasons, the diazeniumdiolate compound NOC-12, with g The precise role of NO in the induction of chondrocyte death is repeatedly debated. Treatment with classical NO donors consistently induces apoptosis in cultured chon- drocytes [6,8,10,39], whereas the production of high levels of endogenous NO by the over-expression of the iNOS gene in transfected chondrocytes has not been found to cause cell death [11]. This discrepancy might be the result of using chemical NO donors, which not only generate reactive nitrogen species but also produce var- ious secondary reactions depending on the cellular milieu with in vitro experiments [40]. Also, an anti-apoptotic role has been addressed in several review articles [41-45]. Specifically, del Carlo and collaborators showed that compounds that only release NO, such as the diazenium- diolates NOC-5 and NOC-12, do not cause chondrocyte cell death and can even be protective under certain con- ditions of oxidative stress [39]. It is likely that persistent spontaneous release of NO is necessary for the protective effect and that peroxynitrite and cyanide contribute to the cytotoxic effect of NO donors [37]. Chondrocyte cell death from NO occurs under conditions where other reactive oxygen species (ROS) are also generated [39]. Figure 7 Osteoarthritis (OA) chondrocytes are efficient cells in the uptake of glucose. Differences between human articular normal and OA chondrocytes in the uptake of 2-deoxy-(3H)glucose for 1 hour. The detailed procedure is described in Materials and methods. Values are the mean ± SD; n = 12. P ≤0.05 versus normal chondrocytes. Figure 7 Osteoarthritis (OA) chondrocytes are efficient cells in the uptake of glucose. Differences between human articular normal and OA chondrocytes in the uptake of 2-deoxy-(3H)glucose for 1 hour. The detailed procedure is described in Materials and methods. Values are the mean ± SD; n = 12. P ≤0.05 versus normal chondrocytes. Figure 7 Osteoarthritis (OA) chondrocytes are efficient cells in the uptake of glucose. Differences between human articular normal and OA chondrocytes in the uptake of 2-deoxy-(3H)glucose for 1 hour. The detailed procedure is described in Materials and methods. Values are the mean ± SD; n = 12. P ≤0.05 versus normal chondrocytes. de Andrés et al. Discussion Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 Page 9 of 12 Abbreviations h Only the traditional donor SNP was able to reduce glucose uptake by normal chondrocytes. Previously, we showed that the inhibition of complex IV with sodium azide modified the survival of the chondrocytes, but its effect was greater when glucose was absent. A possible explanation is that the inhibition of complex IV exclu- sively is not enough to induce apoptosis and other cellu- lar events; such a reduction in the intake of glucose needs to be present to induce it [10]. The glucose dependency of chondrocytes arises with the fact that the effect of SNP on chondrocyte apoptosis correlates with glucose levels; the lower the glucose levels in the media, the highest the apoptotic levels induced. CS: citrate synthase; DAPI: 49,6-dianidino-2-phenylindole dihydrochloride; 2- (3H)DG: 2-deoxy-(3H)glucose; DMEM: Dulbecco’s modified Eagle’s medium; FCS: fetal calf serum; IL-1β: interleukin-1β; iNOS: inducible nitric oxide synthase; JC-1: 5,5’,6,6’- tetrachloro-1,1’,3,3’-tetraethylbenzimidazole carbocyanide iodide; ΔΨm: mitochondrial membrane potential; MRC: mitochondrial respiratory chain; NO: nitric oxide; NOC-12: N-ethyl-2(1-ethyl-2 hydroxy-2-nitrosohydrazine; OA: osteoarthritis; OXPHOS: mitochondrial oxidative phosphorylation; PBS: phosphate buffered saline; ROS: reactive oxygen species; SIN-1: 3-morpholinosydnonimine; SNAP: S-nitroso-N- acetylpenicillamine; SNP: sodium nitroprusside; TNFα: tumour necrosis factor α. Authors’ contributions MCdA carried out the experimental work, analysed the data and drafted the manuscript. EM participated in the study design, interpretation of data and manuscript preparation. MAM and JA measured the MRC complex activities in digitonin-permeabilized chondrocytes. FJB conceived and coordinated the project and revised the manuscript. All the authors read and approved the manuscript. Finally, OA chondrocytes incorporated more glucose than healthy chondrocytes under the standard experi- mental conditions used in this study. These findings are in consonance with a higher lactate production by OA chondrocytes than control chondrocytes (personal data). Furthermore, this up-regulation can be considered a protective mechanism that maximizes the cell’s ability to capture glucose and thus to overcome stressful condi- tions, such as glucose scarcity or even deprivation [59,60], or just to compensate CRM defects. On the other hand, these findings can somehow explain the ROS contribution to the pathogenesis of OA [61,62]; no changes in glucose incorporation by normal chondro- cytes can suggest a protective mechanism against the deleterious effects of excessive intracellular glucose, as seen in other cells [63], and the incapacity of OA chon- drocytes to regulate this can trigger ROS accumulation in OA cartilage. Others authors have reported that basal glucose uptake is identical in normal and OA chondro- cytes [63]; the reasons for these discrepancies are unclear but the observed differences may be related to the culture conditions used in these studies. Acknowledgements The authors express appreciation to the Department of Orthopedics and the Tissue Bank of the Complejo Hospitalario Universitario de A Coruña for providing cartilage samples. This study was supported by grants from the Fondo Investigación Sanitaria, Madrid, Spain: (CIBER- CB06/01/0040; PI-12/ 00329; RETIC-RIER-RD12/0009/0018; and Proteo-Red/ISCIII); Ministerio Ciencia e Innovación, Madrid, Spain: PLE2009-0144 and FEDER (European Community). Additional material substrate for ATP production [17]. The Pasteur effect arises in articular cartilage; in this way anaerobic glyco- lysis and lactate production are involved in respiratory metabolism of articular cartilage even under aerobic conditions [21,55,56]. An anaerobic metabolism could be beneficial for OA, since the products of glucose degrada- tion (lactate and pyruvate) would act as ROS scavengers [57,58], and would assure ATP production even under conditions of mitochondrial dysfunction (defects in CRM complexes and high NO production) [35]. Additional file 1: Table showing values of apoptotic cell death in normal chondrocytes treated with different nitric oxide (NO) donor compounds. Additional file 2: Table showing values of ATP production by normal chondrocytes treated with different NO donors compounds for 24 hours. Competing interests Th h d l h The authors declare that they have no competing interests. Authors’ details 1INIBIC C l j 1INIBIC-Complejo Hospitalario Universitario A Coruña (CHUAC) Rheumatology Division, As Xubias 84, 15006-A Coruña, Spain. 2Instituto de Investigación Hospital 12 de Octubre, “i+12”, Madrid, Spain. 3Red de Proteomica Proteo- Red/ISCIII, Madrid, Spain. 4RETIC-RIER-ISCIII, Madrid, Spain. Received: 19 January 2013 Revised: 9 June 2013 Accepted: 11 September 2013 Published: 11 September 2013 Received: 19 January 2013 Revised: 9 June 2013 References 1. Sandell LJ, Aigner T: Articular cartilage and changes in arthritis. An introduction: cell biology of osteoarthritis. Arthritis Res 2001, 3:107-113. 1. Sandell LJ, Aigner T: Articular cartilage and changes in arthritis. An introduction: cell biology of osteoarthritis. Arthritis Res 2001, 3:107-113. 2. Nahir AM, Vitis N, Silbermann M: Cellular enzymatic activities in the articular cartilage of osteoarthritic and osteoporotic hip joints of humans: a quantitative cytochemical study. Aging (Milano) 1990, 2:363-369. de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 The relevance of the MRC inhibition in human chondrocytes is already known, the inhibition of complexes III and V of the MRC induces an inflammatory response, which could be especially relevant in relation to prostaglandin E2 (PGE2) production via mitochondrial Ca2+ exchange, ROS production, and nuclear factor (NF)-B activation [50]. More recently, Rego and collaborators have found that the predisposition to the development of OA is related to some haplogroups of mitochondrial respira- tory genes of chondrocytes [51]. Also, chondrocytes are not the only joint cells affected in OA pathology and, a recent study has shown that SNP reduces the survival of OA synoviocytes by regulating mitochondrial functional- ity, as well as the proteins controlling the cell cycle [52]. Chondrocytes are highly glycolytic resident cells of articular cartilage that metabolize glucose as a primary Page 10 of 12 de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 Carlo MD Jr, Loeser RF: Increased oxidative stress with aging reduces chondrocyte survival: correlation with intracellular glutathione levels. Arthritis Rheum 2003, 48:3419-3430. 17. 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Rajpurohit R, Risbud MV, Ducheyne P, Vresilovic EJ, Shapiro IM: Phenotypic characteristics of the nucleus pulposus: expression of hypoxia inducing Page 12 of 12 Page 12 of 12 doi:10.1186/ar4295 Cite this article as: de Andrés et al.: Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism. Arthritis Research & Therapy 2013 15:R115. de Andrés et al. Arthritis Research & Therapy 2013, 15:R115 http://arthritis-research.com/content/15/5/R115 Rosa SC, Goncalves J, Judas F, Mobasheri A, Lopes C, Mendes AF: Impaired glucose transporter-1 degradation and increased glucose transport and oxidative stress in response to high glucose in chondrocytes from osteoarthritic versus normal human cartilage. Arthritis Res Ther 2009, 11:R80. doi:10.1186/ar4295 Cite this article as: de Andrés et al.: Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism. Arthritis Research & Therapy 2013 15:R115. 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https://openalex.org/W3186694034	https://bmcbioinformatics.biomedcentral.com/track/pdf/10.1186/s12859-020-03592-5	English	null	Imputation benchmark of β-value and M-value from DNA methylation data under different missing data mechanisms.	Research Square (Research Square)	2,020	cc-by	11,255	© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Correspondence: pietro.dilena@unibo.it; christine.nardini@cnr.it 1Department of Computer Science and Engineering, University of Bologna, Mura Anteo Zamboni 7, Bologna, Italy 4Istituto per le Applicazioni del Calcolo "Mauro Picone", CNR, Via dei Taurini, 19, Roma, Italy Full list of author information is available at the end of the article Abstract Background: Abstract Background: High-throughput technologies enable the cost-effective collection and analysis of DNA methylation data throughout the human genome. This naturally entails missing values management that can complicate the analysis of the data. Several general and specific imputation methods are suitable for DNA methylation data. However, there are no detailed studies of their performances under different missing data mechanisms –(completely) at random or not- and different representations of DNA methylation levels (β and M-value). Results: We make an extensive analysis of the imputation performances of seven imputation methods on simulated missing completely at random (MCAR), missing at random (MAR) and missing not at random (MNAR) methylation data. We further consider imputation performances on the popular β- and M-value representations of methylation levels. Overall, β-values enable better imputation performances than M- values. Imputation accuracy is lower for mid-range β-values, while it is generally more accurate for values at the extremes of the β-value range. The MAR values distribution is on the average more dense in the mid-range in comparison to the expected β-value distribution. As a consequence, MAR values are on average harder to impute. Conclusions: The results of the analysis provide guidelines for the most suitable imputation approaches for DNA methylation data under different representations of DNA methylation levels and different missing data mechanisms. Conclusions: The results of the analysis provide guidelines for the most suitable imputation approaches for DNA methylation data under different representations of DNA methylation levels and different missing data mechanisms. Keywords: Imputation, DNA methylation, M-value, β-value, Missing data mechanisms, MCAR, MAR, MNAR Keywords: Imputation, DNA methylation, M-value, β-value, Missing data mechanisms, MCAR, MAR, MNAR Methylation data imputation performances under different representations and missingness patterns Pietro Di Lena1 , Claudia Sala2, Andrea Prodi3 and Christine Nardini4* Background Epigenomics is currently a very active research area aiming to shed light on the mod- ifications in gene expression that are both independent from DNA mutations and still inheritable (mitotically and meiotically) [1, 2]. We focus here on DNA methylation, involving the covalent addition of a methyl group to the 5’-carbon cytosine in dinucleotide cytosine phosphate guanine (CpG dinucleotide). Lena et al. BMC Bioinformatics (2020) 21:268 https://doi.org/10.1186/s12859-020-03592-5 Lena et al. BMC Bioinformatics (2020) 21:268 https://doi.org/10.1186/s12859-020-03592-5 Open Access Page 2 of 22 Lena et al. BMC Bioinformatics (2020) 21:268 Lena et al. BMC Bioinformatics The relevance of DNA methylation spans several domains in biology, from embryonic development [3, 4] to physiological ageing, cancer [5–7] and shaping of the immune system [8], including vaccination [9]. For these reasons, a deep understanding of the fittest metrics and statistics to model methylations’ activity is important to offer a reliable assessment of its role as a potential biomarker. Further, methylation data are stable and reproducible and offer a large amount of publicly available data, thanks to the cost-effectiveness of methylation arrays (Illumina Human Infinium Beadchips 27k, 450k and now 850k). This publicly available abundance of data, in turn, enables meta-analyses to advance discovery, thanks to numerous (ad hoc) preprocessing approaches [10]. Infinium assays utilize a pair of probes to measure the intensities of the methylated and unmethylated alleles at each CpG site. The methylation value is then computed based on the measured intensities of this pair of probes, across all cells of the sampled tissue. Two metrics are defined to indicate methylation levels: the β-value (ranging from 0 to 1), and the M-value (ranging from −∞to ∞). The relationship between the two representations is a logit trasformation [11]. β-values at the extreme of their range (i.e. close to 0 and 1) have been shown to suffer from severe heteroscedasticity [11] , differently from M- values. However, despite the desirable homoskedasticity of the M-value, in particular for differential analyses [12], β-value remains the predominantly used metric owing to its intuitive biological interpretation, and it is recommended by array producers [11]. As a result, both metrics are used across the literature, mostly depending on the background of the investigators. Experimental methylation data often contain multiple missing values, that can affect downstream analyses. Examples include epigenetic clocks that estimate biological age from small sets of pre-selected age-correlated CpG sites [13–16], recently proven to be highly sensitive to small perturbations of methylation levels [17], as well as more general purpose differential analyses. Hence, accurate imputation of missing data is required for improving the quality of DNA methylation downstream analysis. Missing data can be organized into three classes [18]: i) missing completely at random (MCAR) values, if the probability of being missing is totally independent of both the observed and unobserved variables; ii) missing at random (MAR) values, if the probabil- ity of a value of being missing does not depend on the value itself but may depend on the observed variables; iii) missing not at random (MNAR), if the probability of being missing depends on the missing value itself. When dealing with missing values, MNAR mecha- nisms are usually considered not ignorable since the imputation process needs to model explicitly the missing data mechanism in order to avoid biased estimations [18]. On the contrary, MCAR and MAR mechanisms are considered ignorable and are often used as underlying assumption of most imputation methods. However, there is no assessed statis- tical way to detect the specific missingness mechanism in the data [19], and assumptions need to be made based on the knowledge of the specific data and its sources of acquisition. Unfortunately, to the best of our knowledge, there is no study that addresses the missing- ness patterns in DNA methylation data generated with Illumina Beadchips. As such, our work will comprise the study of all three patterns. We recently introduced a novel regression-based imputation method, methyLImp, specifically designed for methylation data, and compared its performances, under the MCAR assumption, with six other general purpose imputation methods [17], Lena et al. BMC Bioinformatics (2020) 21:268 Page 3 of 22 Lena et al. BMC Bioinformatics namely: i) two mean-value imputation approaches, the basic mean imputation approach and impute.knn [20]; ii) three iterative soft-thresholding approaches, softImpute [21], imputePCA [22] and SVDmiss [23]; iii) one regression-based approach, missForest [24]. Here we further extend the assessment and comparison of imputation performances of the seven benchmarked methods (see “Benchmark imputation software” section) on the same benchmark set of DNA methylation data used in [17], which includes 58 datasets with healthy and disease samples on a variety of different tissues and ages (see “Benchmark data” section). In particular, we extend the performance comparison in two directions: i) by considering β- and M-values data representations of the DNA methyla- tion levels; ii) by explicitly considering and simulating the three different types of missing data mechanisms, a characterization still missing in literature for DNA methylation data (see “Missing values simulation procedure” section for details). In particular, we model MCAR values as missing values that are a direct consequence of random errors in experi- mental measurements, MAR values as consequence of CpG-specific probes that are more likely to fail to capture the target sequences, and MNAR values as missingness patterns that depend on the specific methylation level. In this latter case, we consider separately three different ranges of methylation levels: low-range MNAR missing values, for which we assign a higher probability of being missing to values in the [ 0, 0.2] β-value range, mid-range MNAR for which we assign a higher probability of being missing to values in the [ 0.4, 0.6] range, and high-range MNAR that cover the [ 0.8, 1] β-value range. The results of this extensive analysis highlight issues and limitations of DNA methy- lation data imputation, and provide suggestions for the most appropriate imputation approaches. In particular, as already noticed in [17], our tests show that mid-range β- values are harder to impute than β-values at the extremes (i.e. close to 0 and 1). Sampled MAR values appear to be more compressed in the mid-range and less in the lower-range than the expected β-value distribution. The negative consequence of such a scenario is that methylation levels of CpGs that frequently present missing values are harder to impute accurately. Furthermore, in principle one could expect the (more homoscedastic) M-values to be more suitable for imputation than the (more heteroscedastic) β-values, at least for regression-based imputation methods. However, contrary to this expectation, β-values appear to be the most suitable representation for methylation level imputa- tions. Remarkably, these results hold true irrespective of the specific imputation method, although regression-based method have, on the average, better performances. Results We compare the imputation accuracy of seven benchmarked methods on simulated missing values with respect to the M-value and β-value measures and with respect to MCAR, MAR and MNAR simulated missing values (see “Missing values simulation pro- cedure” section for details and rationale about the sampling procedure). For performance comparison, the same simulated missing values have been imputed independently by first using the M-value and then the β-value representation of the data. In order to directly compare M- and β-value imputation performances, all imputed M-values have been converted into β-values before evaluation. The amount of simulated missing values intro- duced in each test is equal to 3% of the size of the dataset, which corresponds to the average number of real missing values observed in our benchmark set (see Table 1). Impu- tation performance are measured using two metrics (see “Evaluation metrics” section): Page 4 of 22 Page 4 of 22 Lena et al. BMC Bioinformatics (2020) 21:268 Lena et al. Results BMC Bioinformatics Table 1 Benchmark datasets ID GEO ID Tissue Disease status # Samples # Missing values (21k) % Missing values (21k) D1 GSE32146 Colon mucosa Crohn’s disease 10 175 0.08% D2 GSE32146 Colon mucosa Ulcerative colitis 5 161 0.15% D3 GSE32146 Colon Normal 10 171 0.08% D4 GSE32148 Blood Normal 19 325 0.08% D5 GSE40005 Blood Normal 12 324 0.13% D6 GSE42921 Colon mucosa Crohn’s disease 5 192 0.18% D7 GSE42921 Colon mucosa Ulcerative colitis 6 331 0.26% D8 GSE42921 Colon Normal 12 874 0.34% D9 GSE43091 Liver Cancer 50 1,980 0.19% D10 GSE43091 Liver Normal 4 125 0.15% D11 GSE44684 Cerebellum Normal 6 67 0.05% D12 GSE49393 Prefrontal Cortex Normal 25 54,000 10.11% D13 GSE51388 Blood Normal 60 292,200 22.79% D14 GSE52113 Blood Normal 24 0 0.00% D15 GSE53051 Breast Cancer 14 0 0.00% D16 GSE53051 Colon Cancer 35 0 0.00% D17 GSE53051 Colon, Pancreas Normal 9 0 0.00% D18 GSE53051 Lung Cancer 9 0 0.00% D19 GSE53051 Pancreas Cancer 29 0 0.00% D20 GSE53051 Thyroid Cancer 70 0 0.00% D21 GSE53162 Brain, Cerebellum, Prefrontal Cortex Normal 21 0 0.00% D22 GSE53740 Blood Normal 165 0 0.00% D23 GSE57360 Brain Normal 5 0 0.00% D24 GSE61151 Blood Normal 184 7,544 0.19% D25 GSE61257 Adipose Non-alcoholic fatty liver disease (NAFLD) 8 88 0.05% D26 GSE61257 Adipose Non-alcoholic steatohepatitis (NASH) 9 142 0.07% D27 GSE61257 Adipose Normal 15 241 0.08% D28 GSE61258 Liver Non-alcoholic fatty liver disease (NAFLD) 14 370 0.12% D29 GSE61258 Liver Non-alcoholic steatohepatitis (NASH) 7 218 0.15% D30 GSE61258 Liver Normal 32 966 0.14% D31 GSE61258 Liver Primary biliary cholangitis (PBC) 12 251 0.10% D32 GSE61258 Liver Primary sclerosing cholangitis (PSC) 14 352 0.12% D33 GSE61259 Muscle Non-alcoholic fatty liver disease (NAFLD) 9 90 0.05% D34 GSE61259 Muscle Non-alcoholic steatohepatitis (NASH) 7 49 0.03% D35 GSE61259 Muscle Normal 10 96 0.04% D36 GSE61380 Brain Normal 15 2,4671 7.70% D37 GSE62003 Blood Normal 35 0 0.00% D38 GSE64495 Blood Normal 106 32 0.00% D39 GSE67477 Liver Cancer 6 461 0.36% D40 GSE67484 Liver, Intestine-Small Normal 4 45 0.05% D41 GSE69502 Brain, Spinal Cord Normal 20 37,781 8.84% D42 GSE71955 Blood Normal 62 260,245 19.64% D43 GSE73103 Blood Normal 268 1,005,268 17.55% Table 1 Benchmark datasets Page 5 of 22 Lena et al. BMC Bioinformatics (2020) 21:268 Lena et al. Results BMC Bioinformatics Table 1 Benchmark datasets (Continued) ID GEO ID Tissue Disease status # Samples # Missing values (21k) % Missing values (21k) D44 GSE73747 Brain Normal 9 7,069 3.68% D45 GSE79122 Brain Normal 7 99 0.07% D46 GSE80970 Prefrontal Cortex Normal 68 1,324 0.09% D47 GSE82218 Blood Normal 25 398 0.07% D48 GSE84003 Blood Normal 6 275 0.21% D49 GSE88821 Colon, Rectum Cancer 63 36,995 2.75% D50 GSE88821 Colon, Rectum Normal 8 4,680 2.74% D51 GSE88821 Liver Cancer 4 2,349 2.75% D52 GSE89093 Blood Normal 46 65,044 6.62% D53 GSE89472 Blood Normal 5 245 0.23% D54 GSE89702 Cerebellum Normal 17 49,572 13.65% D55 GSE89703 Hippocampus Normal 13 37,557 13.52% D56 GSE89705 Putamen Normal 17 49,215 13.55% D57 GSE89706 Putamen Normal 28 78,736 13.16% D58 GSE97362 Blood Normal 123 2,333 0.09% Table 1 Benchmark datasets (Continued) mean absolute error (MAE) and root mean square error (RMSE). Since β-values are lim- ited in the [ 0, 1] range, both metrics are also limited in [ 0, 1], where a value close to 0 means (almost) perfect imputation. The full range of tests is run, for computational efficiency reasons (see “Methods” section), for all datasets on the CpGs in the intersection between the 27k and 450k Human Beadchips (Type I probes). A reduced number of tests on the complete 450k benchmark data (in Additional file 3) show that there are no relevant differences between imputation accuracy on complete (450k) and restricted (21k) datasets. We use the Wilcoxon signed-rank test to detect statistically significant difference between MAE and RMSE performances (see “Wilcoxon-testing procedure to assess sta- tistically significantly better performances” section for more details). In particular, we use the Wicoxon test to assess whether a single method performance is statistically sig- nificantly better on the β-value or M-value representation (the best results are marked with ∗in the report tables). We also use the Wilcoxon test to asses whether there are best performing methods for some missingness mechanism (the best methods, if any, are highlighted in bold in the report tables). In this latter case, we define best performing methods as those whose performances are never statistically significantly worse than any other method. Imputation of MCAR values The average imputation performances on healthy and disease samples under the MCAR assumption are summarized in Table 2a and b, respectively. Due to the well-known methylation heterogeneity in disease samples (e.g. tumour) [25, 26], the imputation accu- racy is consistently lower in disease than in control (healthy) samples, independently of the specific imputation method. This confirms the results already reported in [17] for the specific subset of (353) CpGs used in Horvath’s epigenetic clock [15]. Furthermore, the regression-based imputation methods are the best performing among the benchmarked ones. In particular, according to the Wilcoxon signed-rank test, the imputation performances of methyLImp on β-values are never worse than those of the other methods on both β and M-values. Page 6 of 22 Page 6 of 22 Lena et al. BMC Bioinformatics (2020) 21:268 Lena et al. BMC Bioinformatics Table 2 MCAR missing values MAE RMSE Method M-value B-value M-value B-value (a) Healthy datasets mean 0.030±0.001∗ 0.030±0.001 0.051±0.001 0.050±0.001∗ impute.knn 0.039±0.007∗ 0.059±0.012 0.079±0.015∗ 0.112±0.019 softImpute 0.031±0.002 0.032±0.006∗ 0.055±0.004 0.059±0.017∗ imputePCA 0.025±0.001∗ 0.025±0.001 0.045±0.001 0.043±0.001∗ SVDmiss 0.035±0.001 0.027±0.001∗ 0.063±0.002 0.048±0.002∗ missForest 0.026±0.001 0.026±0.001∗ 0.044±0.003 0.043±0.002∗ methyLImp 0.029±0.001 0.025±0.001∗ 0.050±0.002 0.047±0.002∗ (b) Disease datasets mean 0.048±0.001∗ 0.048±0.001 0.080±0.002 0.079±0.002∗ impute.knn 0.059±0.008∗ 0.082±0.013 0.107±0.014∗ 0.142±0.018 softImpute 0.050±0.004 0.051±0.010∗ 0.084±0.007 0.091±0.026∗ imputePCA 0.041±0.001∗ 0.041±0.001 0.072±0.002 0.070±0.002∗ SVDmiss 0.055±0.001 0.045±0.001∗ 0.093±0.002 0.080±0.003∗ missForest 0.042±0.001∗ 0.042±0.001 0.071±0.002 0.070±0.002∗ methyLImp 0.043±0.001 0.037±0.001∗ 0.074±0.002 0.066±0.002∗ Average Mean Average Error (MAE) and Root Mean Square Error (RMSE) imputation performance ± standard deviation. For each method, the ∗symbol indicates the measure (either M-value or β-value) for which the Wilcoxon signed-rank test p-value is < 0.05. Best results per metric with respect to the Wilcoxon signed-rank test are highlighted in bold Note that for healthy samples, the average performances of imputePCA and missFor- est with respect to the RMSE metric are better than those of methyLImp, although the Wilcoxon test detects statistically significant differences in favour of the latter (Table 2). This behaviour can be explained by observing the detailed results per dataset in Addi- tional file 1. In short, 3% MCAR missing values in large datasets imply that there is at least one missing value for almost every CpG. Imputation of MCAR values Since methyLImp uses only complete obser- vations for regression, this implies that in large datasets the regression is done on the average on just few tens of variables (see Table 2 in Additional file 2), which are usually not enough to build an appropriate regression model. This is the reason why methyLImp’s performance on the larger datasets (e.g. D22-GSE53740, D24-GSE61151, D38-GSE64495 in Additional file 1) are much worse than those of the baseline mean approach. However, since Wilcoxon test draws statistical inference from the rank sum instead of the mean, the average methyLImp performances are still detected as significantly better than those of the other methods. Anyway, we remark that in real methylation datasets such situation is quite unlikely since we typically have thousands of completely observed variables (see Table 1 in Additional file 2). We can also notice that methyLImp, SVDmiss and softImpute benefit from the β-value representation, impute.knn is definitely more accurate on the M-value representation, while there is no clear preference for the remaining methods (MCAR tests in Tables 2a and b). The accuracy of impute.knn (originally designed for gene expression data impu- tation) on high-range values is clearly affected by the β-value representation (average RMSD w.r.t. β-value range in Fig. 1). On the contrary, impute.knn performances are more uniform for the M-value representation, although still not satisfactory in comparison to those of the other methods, highlighting once more the peculiarity of epigenetic versus transcriptional signals. The same trend is visible also in the disease datasets (Figure 4 in Additional file 1). Page 7 of 22 Lena et al. BMC Bioinformatics (2020) 21:268 (2020) 21:268 Lena et al. BMC Bioinformatics Fig. 1 Healthy datasets. Average RMSE with respect to β-value range. Average RMSE for M-value and β-value imputation with respect to different β-value ranges and with respect to the MCAR, MAR, MNAR (low, mid, high) missing data mechanisms Fig. 1 Healthy datasets. Average RMSE with respect to β-value range. Average RMSE for M-value and β-value imputation with respect to different β-value ranges and with respect to the MCAR, MAR, MNAR (low, mid, high) missing data mechanisms Page 8 of 22 Lena et al. BMC Bioinformatics (2020) 21:268 Lena et al. BMC Bioinformatics To conclude, we can notice that, except for impute.knn, imputation errors are not equally distributed over the range of β-values, being smaller at the extremes (aver- age RMSD w.r.t. β-value range in Fig. 1). Imputation of MCAR values As already pointed out in [17], this can be explained by the well-known heteroscedasticity of β-values [12]. The behaviour of the mean approach is a clear evidence of such variability at the extremes. Imputation of MAR values The average imputation performances on healthy and disease samples under the MAR assumption are summarized in Table 3a and b, respectively. We notice that the over- all imputation accuracy of MAR missing values is significantly lower than that on MCAR values. This is very likely a consequence of the sampled MAR β-value dis- tribution, which is shifted more toward the middle-range than the expected β-value distribution (Figs. 2 and 3). As discussed above, middle-range β-values are harder to impute due to their higher variability (see, for example, the mean performance) and, thus, the overall imputation accuracy for MAR missing values is, not surprisingly, lower. Since the MAR missing values have been sampled by using real data probability dis- tribution, the average results in Table 3a and b (MAR tests) are probably a better indication of the expected imputation accuracies than the results on MCAR missing values. Although the average imputation error is larger for MAR missing values, we can notice the same general performance trend, as observed in the MCAR tests: i) β-value imputation is generally more accurate than M-value imputation with the exception of impute.knn; ii) regression-based methods are on the average the best perform- ing; iii) imputation accuracy is significantly better on healthy samples than on disease samples. Table 3 MAR missing values MAE RMSE Method M-value B-value M-value B-value (a) Healthy datasets mean 0.041±0.001 0.040±0.001∗ 0.073±0.002 0.070±0.001∗ impute.knn 0.043±0.004∗ 0.061±0.009 0.082±0.009∗ 0.110±0.015 softImpute 0.042±0.002∗ 0.043±0.007 0.077±0.005 0.082±0.017∗ imputePCA 0.037±0.001 0.036±0.001∗ 0.069±0.002 0.066±0.002∗ SVDmiss 0.043±0.001 0.036±0.001∗ 0.079±0.003 0.067±0.002∗ missForest 0.035±0.001 0.035±0.001∗ 0.064±0.002 0.061±0.002∗ methyLImp 0.037±0.001 0.033±0.001∗ 0.068±0.002 0.063±0.002∗ (b) Disease datasets mean 0.060±0.001 0.060±0.001∗ 0.101±0.002 0.097±0.002∗ impute.knn 0.067±0.005∗ 0.087±0.010 0.115±0.009∗ 0.144±0.014 softImpute 0.062±0.003 0.065±0.011 0.106±0.006∗ 0.116±0.026 imputePCA 0.054±0.001 0.053±0.001∗ 0.095±0.002 0.090±0.002∗ SVDmiss 0.067±0.001 0.057±0.001∗ 0.114±0.003 0.104±0.004∗ missForest 0.053±0.001 0.053±0.001∗ 0.093±0.002 0.088±0.002∗ methyLImp 0.053±0.001 0.049±0.001∗ 0.092±0.002 0.089±0.002∗ Average Mean Average Error (MAE) and Root Mean Square Error (RMSE) imputation performance ± standard deviation. For each method, the ∗symbol indicates the measure (either M-value or β-value) for which the Wilcoxon signed-rank test p-value is < 0.05. Best results per metric with respect to the Wilcoxon signed-rank test are highlighted in bold Page 9 of 22 Lena et al. BMC Bioinformatics (2020) 21:268 Lena et al. BMC Bioinformatics Fig. 2 β-value distributions of different missingness mechanisms. Comparison of the β-value distribution against the distribution of simulated MCAR, MAR and MNAR missing values Fig. 2 β-value distributions of different missingness mechanisms. Imputation of MAR values Comparison of the β-value distribution against the distribution of simulated MCAR, MAR and MNAR missing values Imputation of MNAR values The average imputation performances on healthy and disease samples under the low-range MNAR assumption are summarized in Table 4a and b, respectively, those on the mid-range MNAR assumption in Table 5a and b, and those on the high-range MNAR assumption in Table 6a and b. The imputation performances on low-range and high-range MNAR values are statisti- cally significantly better than those on MCAR values, while the imputation performances on mid-range MNAR values are even worse than those on MAR. This behaviour can again be explained as a consequence of the standard deviations of the β-values being compressed in the low and high ranges. In fact, we remark that β-values at the extreme of the range (either close to one or zero) correspond to situations where all or none of the copies of the CpG sites are methylated, indicating a very robust biological condition, which seems easier to predict than conditions where methylation status is diversified across cells. As a further evidence, the plots related to the MCAR assumption are basi- cally indistinguishable from those related to the (low-range, mid-range and high-range) MNAR assumption (Fig. 1). The good performances shown in Table 4a and b (low-range MNAR tests) are thus only a consequence of the fact that a high number of missing val- ues are in the low-range (see also Fig. 2). The same argument can be used for the results in Table 6a and b (high-range MNAR tests). On the contrary, the worse MAE and RMSE performances are a consequence of the high number of missing values in the mid-range Page 10 of 22 Lena et al. BMC Bioinformatics (2020) 21:268 Lena et al. BMC Bioinformatics Fig. 3 β-value distributions of CpGs with frequently missing values . Comparison of the β-value distribution against the β-value distribution of CpGs with missing values on > 20%, > 25%, > 30% samples. MAR simulated distribution included Fig. 3 β-value distributions of CpGs with frequently missing values . Comparison of the β-value distribution Fig. 3 β-value distributions of CpGs with frequently missing values . Comparison of the β-value distribution against the β-value distribution of CpGs with missing values on > 20%, > 25%, > 30% samples. MAR simulated distribution included (Tables 6a, b and Figure 2). Imputation of MNAR values We also remark that, although the MNAR tests may appear purely theoretical and not related to a real-word cases, they can still give us a picture of what we should expect in an extreme scenario. In particular, the take home message here is that, if for some reason we can assume MNAR missing values in DNA methylation data, we need to model explicitly such missing mechanism only if they are of type mid-range MNAR. All other cases can be considered as ignorable. To conclude, note that in Table 4 (low-range MNAR tests on healthy samples) there are several good average performances and no method is highlighted in bold, which means that it is not possible to unambiguously identify the best performing method. Anyway, a closer look at the results of the Wilcoxon test shows that methyLImp and imputePCA per- form better than the remaining approaches (data not shown). Also, differently from the other cases, we can see that M-value imputation are more accurate than β-value impu- tation according to the Wilcoxon test (low-range MNAR tests in Table 4a and b). On the other hand, except for this specific case, we can observe the same general performance trend previously discussed for both the MAR and MCAR tests. Discussion In this work we have covered three different types of missing data mechanisms for DNA methylation data, represented with the two popular M and β-value representations, and Page 11 of 22 Lena et al. BMC Bioinformatics (2020) 21:268 Lena et al. BMC Bioinformatics Table 4 MNAR:low missing values MAE RMSE Method M-value B-value M-value B-value (a) Healthy datasets mean 0.022±0.001∗ 0.023±0.001 0.043±0.001∗ 0.044±0.001 impute.knn 0.041±0.012 0.033±0.006∗ 0.086±0.021 0.077±0.014∗ softImpute 0.026±0.002 0.023±0.003 0.052±0.006 0.046±0.010∗ imputePCA 0.019±0.001∗ 0.020±0.001 0.039±0.002∗ 0.039±0.001 SVDmiss 0.029±0.001 0.021±0.001∗ 0.061±0.003 0.041±0.002∗ missForest 0.019±0.001∗ 0.020±0.001 0.037±0.002∗ 0.038±0.001 methyLImp 0.022±0.001∗ 0.019±0.001 0.040±0.002∗ 0.039±0.002 (b) Disease datasets mean 0.036±0.001∗ 0.037±0.001 0.068±0.002∗ 0.069±0.002 impute.knn 0.063±0.014 0.048±0.008∗ 0.120±0.020 0.102±0.015∗ softImpute 0.040±0.005 0.036±0.004∗ 0.076±0.010 0.072±0.013∗ imputePCA 0.031±0.001∗ 0.032±0.001 0.061±0.002∗ 0.062±0.002 SVDmiss 0.047±0.001 0.035±0.001∗ 0.089±0.003 0.070±0.003∗ missForest 0.031±0.001∗ 0.032±0.001 0.060±0.002∗ 0.061±0.002 methyLImp 0.032±0.001 0.028±0.001∗ 0.063±0.003 0.058±0.002∗ Average Mean Average Error (MAE) and Root Mean Square Error (RMSE) imputation performance ± standard deviation. For each method, the ∗symbol indicates the measure (either M-value or β-value) for which the Wilcoxon signed-rank test p-value is < 0.05. Best results per metric with respect to the Wilcoxon signed-rank test are highlighted in bold Table 4 MNAR:low missing values Average Mean Average Error (MAE) and Root Mean Square Error (RMSE) imputation performance ± standard deviation. For each method, the ∗symbol indicates the measure (either M-value or β-value) for which the Wilcoxon signed-rank test p-value is < 0.05. Best results per metric with respect to the Wilcoxon signed-rank test are highlighted in bold Average Mean Average Error (MAE) and Root Mean Square Error (RMSE) imputation performance ± standard deviation. For each method, the ∗symbol indicates the measure (either M-value or β-value) for which the Wilcoxon signed-rank test p-value is we have compared the performances of seven computationally efficient imputation meth- ods that are available under the popular R framework. The analysis essentially provides three general pieces of information. 1. Missing values lying in the mid-range methylation level are harder to impute than missing values close to the extremes of the range, i.e. values indicating that (almost) Table 5 MNAR:mid missing values MAE RMSE Method M-value B-value M-value B-value (a) Healthy datasets mean 0.053±0.001 0.051±0.001∗ 0.082±0.001 0.076±0.001∗ impute.knn 0.041±0.002∗ 0.050±0.004 0.067±0.005∗ 0.085±0.010 softImpute 0.051±0.001 0.050±0.006∗ 0.078±0.003 0.080±0.012∗ imputePCA 0.045±0.001 0.043±0.001∗ 0.072±0.001 0.067±0.001∗ SVDmiss 0.052±0.001 0.043±0.001∗ 0.081±0.002 0.069±0.002∗ missForest 0.044±0.001 0.042±0.001∗ 0.068±0.001 0.064±0.001∗ methyLImp 0.044±0.001 0.040±0.001∗ 0.068±0.001 0.064±0.001∗ (b) Disease datasets mean 0.076±0.001 0.072±0.001∗ 0.109±0.001 0.101±0.001∗ impute.knn 0.060±0.002∗ 0.073±0.006 0.091±0.005∗ 0.116±0.010 softImpute 0.075±0.002 0.072±0.010∗ 0.108±0.003 0.111±0.021∗ imputePCA 0.066±0.001 0.062±0.001∗ 0.098±0.001 0.091±0.001∗ SVDmiss 0.075±0.001 0.064±0.001∗ 0.112±0.002 0.100±0.002∗ missForest 0.066±0.001 0.062±0.001∗ 0.098±0.001 0.090±0.001∗ methyLImp 0.065±0.001 0.057±0.001∗ 0.095±0.002 0.088±0.001∗ Average Mean Average Error (MAE) and Root Mean Square Error (RMSE) imputation performance ± standard deviation. For each method, the ∗symbol indicates the measure (either M-value or β-value) for which the Wilcoxon signed-rank test p-value is < 0.05. Best results per metric with respect to the Wilcoxon signed-rank test are highlighted in bold Page 12 of 22 Page 12 of 22 Lena et al. BMC Bioinformatics (2020) 21:268 Lena et al. BMC Bioinformatics Table 6 MNAR:high missing values MAE RMSE Method M-value B-value M-value B-value (a) Healthy datasets mean 0.026±0.001∗ 0.026±0.001 0.044±0.001∗ 0.044±0.001 impute.knn 0.054±0.013∗ 0.092±0.020 0.103±0.022∗ 0.152±0.023 softImpute 0.028±0.002∗ 0.033±0.010 0.049±0.005∗ 0.063±0.026 imputePCA 0.022±0.001∗ 0.022±0.001 0.039±0.001 0.038±0.001 SVDmiss 0.032±0.001 0.025±0.001∗ 0.056±0.004 0.043±0.002∗ missForest 0.023±0.001∗ 0.023±0.001 0.039±0.001 0.038±0.001∗ methyLImp 0.027±0.001 0.022±0.001∗ 0.044±0.001 0.039±0.002∗ (b) Disease datasets mean 0.041±0.001∗ 0.043±0.001 0.069±0.002∗ 0.069±0.002 impute.knn 0.085±0.017∗ 0.134±0.025 0.148±0.023∗ 0.203±0.024 softImpute 0.044±0.005∗ 0.053±0.018 0.075±0.009∗ 0.098±0.043 imputePCA 0.035±0.001∗ 0.036±0.001 0.061±0.002∗ 0.061±0.002 SVDmiss 0.050±0.001 0.041±0.001∗ 0.084±0.002 0.073±0.003∗ missForest 0.036±0.001∗ 0.038±0.001 0.062±0.002∗ 0.062±0.001 methyLImp 0.037±0.001 0.032±0.001∗ 0.062±0.002 0.057±0.002∗ Average Mean Average Error (MAE) and Root Mean Square Error (RMSE) imputation performance ± standard deviation. For each method, the ∗symbol indicates the measure (either M-value or β-value) for which the Wilcoxon signed-rank test p-value is < 0.05. Best results per metric with respect to the Wilcoxon signed-rank test are highlighted in bold Table 6 MNAR:high missing values all the copies of the CpG site are uniformly methylated or uniformly non methylated. This is very likely a consequence of the higher variance of the methylation values in the intermediate ranges. we have compared the performances of seven computationally efficient imputation meth- ods that are available under the popular R framework. The analysis essentially provides three general pieces of information. Such scenario can have a deep impact in terms of perfor- mance expectations, if we assume that in our data a large number of missing values are of type MNAR and, in particular, lie in the β-value mid-range. In this case, imputa- tion approaches for DNA methylation data need to model explicitly such missingness pattern. Unfortunately, we do not have any evidence assessing whether this is true or not in real DNA methylation data. 2. Since M-values show lower heteroscedasticity than β-values, we would expect over- all better imputation performances on M-values than on β-values, at least for those imputation approaches that rely on linear models. However, despite this desirable statistical properties of the M-value representation, there is no immediate benefit in M-value imputation. 3. Methylation levels of CpGs that come with a higher probability of having a missing value (i.e. MAR type missing values) are generally harder to impute accurately. This seems to be a consequence of the β-value distributions of such (highly missing) CpGs, which are, again, more compressed into the mid-range in comparison to the expected β-value distribution. Due to such statistics, we can speculate that MAR missigness mechanisms need to be assumed for DNA methylation data. It is however hard to quantify the amount of MAR missing values in the data, since these cannot be easily distinguished from MCAR missing values. Furthermore, we can observe highly vari- able percentages of missing values in DNA methylation datasets (see Table 1), making it hard to even determine how many missing values in general we should expect in real data. However, we can suggest that DNA methylation applications that rely on highly missing CpGs (easily identified by statistical analysis of available data) need to expect 3. Methylation levels of CpGs that come with a higher probability of having a missing value (i.e. MAR type missing values) are generally harder to impute accurately. This seems to be a consequence of the β-value distributions of such (highly missing) CpGs, which are, again, more compressed into the mid-range in comparison to the expected β-value distribution. Due to such statistics, we can speculate that MAR missigness mechanisms need to be assumed for DNA methylation data. It is however hard to quantify the amount of MAR missing values in the data, since these cannot be easily distinguished from MCAR missing values. we have compared the performances of seven computationally efficient imputation meth- ods that are available under the popular R framework. The analysis essentially provides three general pieces of information. Furthermore, we can observe highly vari- able percentages of missing values in DNA methylation datasets (see Table 1), making it hard to even determine how many missing values in general we should expect in real data. However, we can suggest that DNA methylation applications that rely on highly missing CpGs (easily identified by statistical analysis of available data) need to expect Page 13 of 22 Lena et al. BMC Bioinformatics (2020) 21:268 Lena et al. BMC Bioinformatics imputation accuracy lower that average for such CpGs. Also in this case, M-value imputation does not offer any benefit. These three general indications hold independently from the specific imputation method adopted. On the other hand, some methods seem to be more suitable than others for DNA methylation data imputation. In particular, in terms of imputation accu- racy, performance comparison shows that, among the benchmarked approaches, the regression-based methods (i.e. methyLimp and missForest) are the best performing ones. In particular, the overall results in Table 7, averaged over all 57 (healthy and disease) benchmark datasets and over all missing value mechanisms, show that methyLImp is sta- tistically significantly best performing, closely followed by missForest and imputePCA. More generally, as it can be seen in Tables 2a, b, 3a, b, 4a, b, 5a, b, and 6a, b, (all missingness models tests), methyLImp is statistically significantly better performing with respect to all types of missingness mechanisms. However, we remark that the methyLImp approach is not suitable on DNA methylation datasets that have a limited number of variables (CpGs) with complete observations, although this is unlikely to happen in real methylation data (see statistics in Table 2 in Additional File 2). It is not completely triv- ial to quantify the minimum amount of complete CpGs needed by methyLImp to achieve good performance, since sample size has also some influence in regression accuracy. However, by observing the performances in our tests, we can empirically state that at least few thousands of complete observations are needed to achieve a good level of imputation accuracy. Furthermore, data imputation is generally more accurate when DNA methylation levels are expressed as β-values. This holds true essentially for all benchmarked methods but impute.knn, which undoubtedly benefits more from the M-value representation. How- ever, even on M-values, the general performances of impute.knn are often less accurate than those of the baseline mean approach. we have compared the performances of seven computationally efficient imputation meth- ods that are available under the popular R framework. The analysis essentially provides three general pieces of information. This leads us to conclude that impute.knn, although a good general imputation method, is not suitable for DNA methylation data imputation. All these results hold for the 21k CpGs in the intersection between the 27k and the 450k Human Beadchips. It is thus natural to ask whether there is a significant differ- ence between imputation performances on complete datasets (i.e. 450k Human Beadchips data, which include both Type I and Type II probes) and their 21k restriction (which include Type I probes only). Due to the large computational times required to process Table 7 Global imputation performances across all datasets (healthy and disease) and all missingness mechanisms Table 7 Global imputation performances across all datasets (healthy and disease) and all missingness mechanisms MAE RMSE Method M-value B-value M-value B-value mean 0.041±0.001∗ 0.040±0.001 0.068±0.001 0.066±0.001∗ impute.knn 0.052±0.008∗ 0.068±0.011 0.095±0.014∗ 0.119±0.016 softImpute 0.042±0.003 0.043±0.008∗ 0.072±0.006 0.077±0.020∗ imputePCA 0.035±0.001 0.035±0.001∗ 0.062±0.002 0.059±0.001∗ SVDmiss 0.046±0.001 0.037±0.001∗ 0.079±0.003 0.065±0.002∗ missForest 0.036±0.001 0.036±0.001∗ 0.061±0.002 0.059±0.002∗ methyLImp 0.037±0.001 0.033±0.001∗ 0.062±0.002 0.058±0.002∗ For each method, the ∗symbol indicates the measure (either M-value or β-value) for which the Wilcoxon signed-rank test p-value is < 0.05. Best results per metric with respect to the Wilcoxon signed-rank test are highlighted in bold Lena et al. BMC Bioinformatics (2020) 21:268 Page 14 of 22 Lena et al. BMC Bioinformatics complete datasets, for comparison purposes we performed only a restricted number of tests (see Additional file 3). There are some clearly visible trends in such comparison tests: • impute.knn performances are significantly lower on the complete datasets, irrespec- tively of the missingness model (MCAR, MAR, low/mid/high-range MNAR), data representation (M-value or β-value) and metric used for performance assessment (MAE or RMSE). • For the remaining methods, MAE and RMSE do not highlight a clear improvment or worsening of the results when using the complete or the restricted datasets. Wilcoxon’s test shows that better performances with MAR missingness model are achieved on the complete datasets and with MNAR:mid on the restricted datasets. However, the absolute differences obtained with the two types of datasets are negli- gible in all cases (in the order of 10−3), i.e. biologically unimportant per se [27] and irrelevant with respect to downstream computations [17]. As a general conclusion, these comparisons indicate that the imputation performances on the restricted datasets can be considered as representative of the imputation perfor- mances on the complete datasets. we have compared the performances of seven computationally efficient imputation meth- ods that are available under the popular R framework. The analysis essentially provides three general pieces of information. To conclude, in terms of computational resources, the mean-value based approaches are the best performing, both in terms of running time and memory requirements, while the regression-based approaches are the more demanding in terms of running time. In particular, the average running time and memory requirements summarized in Table 8 show that SVDmiss and missForest are the two most demanding methods in terms of memory usage and computation time, respectively. Both methods are virtually unusable for multiple imputations on complete DNA methylation datasets. Benchmark imputation software Benchmark imputation tools include: mean, impute.knn, SVDmiss, softImpute, ImputePCA, missForest, methyLImp, selected according to the following criteria: i) repre- sentative of the major imputation techniques described in literature; ii) requiring limited computational resources; iii) available as R implementations. The benchmarked methods can be roughly classified into three groups: Benchmark data Benchmark datasets are taken from our previous study [17]. In short, we analysed 58 datasets from healthy (37 datasets, overall 1495 samples) and diseased (21 datasets, over- all 386 samples) individuals on a variety of different tissues and ages. All the samples are from Illumina 450k Human Beadchip platform (GPL13534 in GEO) and have been obtained from the NCBI database Gene Expression Omnibus (GEO, [28]), see Table 1 for details. The 450k Human Beadchips incorporates two different chemical assays, Type I and Type II probes, which exhibit different technical characteristics. As already done in [17], due to the high computational time required by some imputation methods, we pre- filtered all datasets in order to consider only the methylation sites in the intersection between the Illumina 27k and 450k Human Beadchips (approximatively 21k sites), all of Type I. In order to assess whether the 21k restriction is representative for the whole 450k Human Beadchip, we performed a reduced (owing to the computational costs of such tests) number of tests on the complete 450k benchmark data (Additional file 3). Conclusions In conclusion, the consolidated and manufacturer encouraged practice to use β-value seems appropriate for DNA methylation data imputation. The choice of the best impu- tation method is somewhat more subtle and depends essentially on the available com- putational resources and the amount of missing values. Independently of the expected missingness mechanisms, regression-based methods provide on average more accu- rate estimates of the missing values. However, imputations with regression methods in the presence of limited computational resources can be a rather challenging task. In such cases, the simple mean approach can surprisingly be a better choice than more sophisticated methods. Table 8 Average time and memory usage Method Avg time Avg RAM mean < 1s 27MB impute.knn 2s 81MB softImpute < 1s 74MB imputePCA 19s 204MB SVDmiss 2m 4GB missForest 18h 280MB methyLImp 21m 129MB Page 15 of 22 Lena et al. BMC Bioinformatics (2020) 21:268 Lena et al. BMC Bioinformatics • mean-value imputation approaches: average observed values. • mean-value imputation approaches: average observed values. – mean: replaces the missing value of a variable by averaging all the known val- ues for that variable. This is the baseline imputation method for continuous variables. We use our own R implementation. – impute.knn [20]: replaces a missing element for a variable by averaging the non- missing values of its nearest neighbours. Originally designed for gene expression data imputation. We use the implementation available in the R ’impute’ package [29]. • iterative soft-thresholding approaches: replace missing values with some initial guess and then iteratively update, up to convergence, the missing elements with values generated by low-rank approximation of the input matrix. • iterative soft-thresholding approaches: replace missing values with some initial guess and then iteratively update, up to convergence, the missing elements with values generated by low-rank approximation of the input matrix. – SVDmiss [23]: uses soft-thresholding singular value decomposition (SVD) of the input matrix. General purpose imputation algorithm (tested on air pollution data) for continuous variables. We use the implementation available in the R ’SpatioTemporal’ package [30]. – softImpute [21]: uses soft-thresholding singular value decomposition (SVD) of the input matrix. General purpose imputation algorithm (tested on artificial data) for continuous variables. We use the implementation available in the R ’softImpute’ package [31]. – imputePCA [22]: implements a low-rank approximation version of the iterative principal component analysis (PCA) algorithm. General purpose imputation Page 16 of 22 Lena et al. BMC Bioinformatics (2020) 21:268 (2020) 21:268 Lena et al. BMC Bioinformatics algorithm for continuous variables. We use the implementation available in the R ’missMDA’ package [32]. algorithm for continuous variables. We use the implementation available in the R ’missMDA’ package [32]. • regression-based imputation approaches: build a regression model from observed data. – missForest [24]: builds random forests regression trees. General purpose impu- tation algorithm (tested on a variety of biological datasets) that can deal with both continuous and categorical variables. We use the implementation available in the R ’missForest’ package [33]. – methyLImp [17]: builds a linear model with observed data. Specifically designed for methylation data. Rationale of the approach: exploits the high degree of inter-sample correlations of methylation levels. We use the implementation available in the R ’methyLImp’ package [34]. The running times and memory requirements of the benchmarked tools are quite differ- ent among different classes (see Table 8). In general, the mean-based approaches are less demanding than the regression-based approaches. There are, however, also some within- classes differences. For example, all soft-thresholding methods require matrices decom- position (in particular, both softImpue and SVDmiss use SVD decomposition), which can be expected to be computationally intensive on large matrices. However, given the high variability of computational performances observed within such class, we conclude that computational performances are mostly affected by the specific implementation and not by the approach itself. In our tests we used the R implementations of the benchmarked methods with default parameters and we did not make any prior assumption about the missingness patterns in the data. • iterative soft-thresholding approaches: replace missing values with some initial guess and then iteratively update, up to convergence, the missing elements with values generated by low-rank approximation of the input matrix. Furthermore, since there are situations that fail to produce imputation results, the general strategy was to ignore those values in performance evaluation. We remark that values that could not be imputed are quite rare in our tests, thus ignoring them does not significantly affect performance scores. In the following we review in detail the specific limitations of each imputation method and the rationale we consequently adopted for input pre-processing. • mean: trivially, it cannot impute a missing CpG value if such CpG value is missing in all samples. • impute.knn: same limitations as above for the mean approach. There may be other cases but these are not documented. When a value cannot be imputed it is set by default to zero. We tested impute.knn performances by removing all the zero impu- tations without noticing any dramatic improvement in performance accuracy. Thus, since it is quite complex to detect zero values that represent a failed imputation (the implementation does not state explicitly which are these values), we decided to ignore this problem. Such cases, overall, rarely occur into the set of tested CpGs. • SVDmiss: same limitations as above for the mean approach. By default, in such sit- uation the method stops without performing imputation. We preprocessed the input matrix by removing all completely missing columns. • imputePCA: does not perform imputation if one column (observation) of the matrix has zero variance (after excluding missing values). We preprocessed the input matrix by removing all zero variance columns. Lena et al. BMC Bioinformatics (2020) 21:268 Page 17 of 22 Lena et al. BMC Bioinformatics • softImpute: exactly the same behaviour as mean. • softImpute: exactly the same behaviour as mean. • missForest: can apparently perform imputation in all possible situations, including the case of matrices with entirely missing observation for one variable (CpG). How- ever, surprisingly, it does not perform imputation on matrices containing a variable with only one observed value. We preprocessed the input matrix by removing all such variables. • methyLImp: cannot impute values in the same situation as described for the mean approach. Furthermore, it cannot perform any imputation if the matrix has at least one missing value per column (never occurred in our tests). Missing values simulation procedure Given the DNA methylation datasets in Table 1, we randomly added 3% missing values under the MCAR, MAR or MNAR missingness assumptions. The 3% amount has been chosen by considering the average percentage of missing values in our benchmark set (see Table 1). Figure 2 compares the β-value distribution in our 57 benchmark sets against the distribution of simulated missing β-values under different missingness assumptions. The exact missing value simulation procedure for each missingness model and the rationale behind it is detailed in the following. MCAR. We assume that MCAR missing values are the direct consequence of random errors in experimental measurements, hence we simply randomly select 3% CpG sites from the DNA methylation data matrix. Figure 2 shows that, as expected, the β-values distribution of artificially introduced MCAR values coincides perfectly with the β-values distribution in the benchmark sets. MAR. We assume that MAR missing values are the effect of CpG-specific probes that are more likely to fail to capture the target sequences. That is, the missing value proba- bility depends on the (observed) CpG site but not on its (unobserved) methylation level. Indeed, statistical analysis of missing data in our benchmark set shows that there exist CpGs with a higher probability of observing missing values. For instance, on the 21k dataset restriction, 11% CpGs have missing values on more than 20% samples, 7% on more than 25% samples and 1% on more than 30% (observed frequencies). Using this observa- tion as a starting point, we estimated from the observed frequencies in our benchmark data the probability for a CpG-specific value of being missing and used such probability distribution to randomly sample 3% positions in the DNA methylation matrix. Figure 2 shows the distribution of the sampled MAR values in comparison to the β-value distri- bution on the entire dataset. MAR missing values are slightly more dense in the [ 0.4, 0.8] interval and slightly less in [ 0, 0.2]. Such trend is more evident in Fig. 3, where we consider the β-values distribution of CpGs with frequently missing values. In Fig. 3, it is clear that β-values of CpGs that are more likely to present missing values are more concentrated in the [ 0.4, 0.8] interval and much less in the [ 0, 0.2] interval in comparison to both the β-value and sampled MAR distributions. MNAR. Definition of β-value and M-value The Illumina Infinium Assay [35] utilizes a pair of probes to measure the intensities of the methylated and unmethylated alleles at each CpG site. The methylation level is then estimated by measuring the intensities of this pair of probes, across all cells in the sample tissue. The two measures commonly used to quantify methylation levels are: β-value and M-value. The β-value is defined as the ratio between the methylated probe intensity and the overall intensity of both methylated and unmethylated alleles. Following the notation in [12], the β-value for an i-th interrogated CpG site is defined as: βi = max xmeth i , 0 max xmeth i , 0 + max xunmeth i , 0 + α (1) (1) where xmeth i and xunmeth i are the intensities measured by the i-th methylated and unmethy- lated probes, respectively, and α is a constant offset (by default, α = 100) used to regularize the β-value when both xmeth i and xunmeth i intensities are low. By definition, β- values can range between 0 and 1. A β-value equal to zero implies that all the copies of the CpG site in the sample are completely unmethylated, while a β-value equal to one indicates that all the copies are methylated. The M-value is defined as the log2 ratio between the intensities of methylated and unmethylated probes: Mi = log2 ⎛ ⎝ max xmeth i , 0 + α max xunmeth i , 0 + α ⎞ ⎠ (2) (2) where the constant offset α (by default 1) prevents large perturbations for small values of xmeth i and xunmeth i . The M-values can range from −∞to +∞. As introduced already, the β-value measure provides a more intuitive interpretation of the methylation status than M-values and it is recommended by array producers [11]. On the other hand, the M-value representation has been proven more suitable for conduct- ing differential methylation analyses, due to the severe heteroscedasticity of β-values for highly methylated or unmethylated CpG sites [12], i.e. the standard deviations of β-values are compressed in the low and high ranges and larger in the middle ranges. In [12] the authors show that for typical values of xmeth i and xunmeth i the offsets α in Eqs. (1) and (2) have negligible effect on both the β-value and M-value measures. Definition of β-value and M-value Thus, by simply ignor- Lena et al. BMC Bioinformatics (2020) 21:268 Page 18 of 22 (2020) 21:268 Lena et al. BMC Bioinformatics ing the α offsets, the relationship between the β-value and M-value measures is a logit transformation. ing the α offsets, the relationship between the β-value and M-value measures is a logit transformation. βi = 2Mi 2Mi + 1 (3) d βi = 2Mi 2Mi + 1 (3) and and Mi = log2 βi 1 −βi (4) Mi = log2 βi 1 −βi (4) Missing values simulation procedure Missing values simulation procedure We assume that MNAR missing values are a consequence of the methylation level. In order to efficiently simulate MNAR missing values we limited our investiga- tion to three different ranges of methylation values, visibly distinct: low-range with β-value between 0 and 0.2 (MNAR:low), mid-range with β-value between 0.4 and 0.6 (MNAR:mid) and high-range with β-value between 0.8 and 1 (MNAR:high). For each range, we randomly sampled 3% positions in the DNA methylation data matrix assum- ing 70% missing probability for the chosen range. For instance, a sampled position in the Lena et al. BMC Bioinformatics (2020) 21:268 Page 19 of 22 Lena et al. BMC Bioinformatics low-range MNAR model has 70% probability of being in the [ 0, 0.2] β-value range. The β- value distributions of such MNAR models are clearly distinguishable in Fig. 2. Although 70% missing probability may seem unrealistic, we remark that in practice we do not have any strong evidence of MNAR missing data in DNA methylation datasets, thus the cho- sen probability, as well as the chosen ranges, are adopted to test imputation performances in extreme MNAR scenarios. For comparison purposes, the same artificially introduced missing values have been imputed both in the β-value and M-value representations. For a more robust performance assessment, we repeated 100 times the artificial introduction of missing values for each missingness model. Thus, for each dataset our tests required a total of 1000 imputations, 500 with respect to β-values and 500 with respect to M-values. Evaluation metrics Performance evaluation has been done based on two accuracy measures. The RMSE (Root Mean Square Error) metric measures the difference between the predicted/estimated, P, and true values, T. SE(P, T) = n i=1 (Pi −Ti)2 n RMSE(P, T) = n i=1 (Pi −Ti)2 n The MAE (Mean Absolute Error) metric measures the absolute difference between the predicted and true values. MAE(P, T) = n i=1 \|Pi −Ti\| n MAE(P, T) = n i=1 \|Pi −Ti\| n MAE(P, T) = n i=1 \|Pi −Ti\| n Recall that, by Jensen’s inequality RMSE ≥MAE. Although the two metrics seem almost equivalent, they are in fact complementary. Indeed, the RMSE metric is more suitable for performance ranking, since it gives higher weight than MAE to large errors, which are particularly undesirable in DNA methylation data imputation. On the other hand, MAE provides a more immediate interpretation of the results in comparison to RMSE. In particular, MAE gives an indication on the average error to be expected on the imputed value. Supplementary information S l i f i Additional file 2: Dataset statistics. Authors’ contributions All the authors designed the project. PDL designed the project and implemented the algorithmic procedure, carried out all computational experiments, and wrote the manuscript draft. CS designed the project and analyzed the experimental results. AP designed the project and analyzed the experimental results. CN designed the project and wrote the manuscript draft. All the authors read and approved the final manuscript. Abbreviations CpG: 5’–C–phosphate–G–3’; MCAR: Missing completely at random; MAR: Missing at random; MNAR: Missing not at random; MAE: Mean absolute error; RMSE: Root mean square error Wilcoxon-testing procedure to assess statistically significantly better performances We use the Wilcoxon signed-rank test [36] to assess whether there is a statistically significant difference between the performances of a pair of methods. The Wilcoxon signed-rank test is a nonparametric statistical hypothesis test that can be used to compare two related (i.e. paired) samples to assess whether their population mean ranks differ. It is an alternative to the paired Student’s t-test when it is not possible to assume that the distribution of the differences between two samples is normal. As nearly all rank tests, the Wilcoxon test is not transitive. We use the Wilcoxon test to assess the statistical significance of two distinct comparisons: 1. intra-method comparison: we compare the performances of the same imputa- tion method on β-value vs M-value representations of the data, in order to detect Lena et al. BMC Bioinformatics (2020) 21:268 Page 20 of 22 Lena et al. BMC Bioinformatics whether there is a better performing data representation method-wise. The statis- tically significant results with better performance are marked with ∗in the result tables. 2. inter-method comparison: we compare the performances of two distinct methods, on either β-value or M-value representations of the data. Table-wise, we highlight in bold the results of the method with the best performance. This was identified as the method whose performance is always statistically significantly higher than or compa- rable to that of other methods. We remark that, in many cases, when comparing two performances the Wilcoxon test does not detect a statistically significant difference. Thus, there is no case in which (table-wise) a performance is detected as statistically significantly better than all other performances. Therefore, we relaxed the defini- tion of best performance by defining it as the performance that is never statistically significantly worse within a table. 2. inter-method comparison: we compare the performances of two distinct methods, on either β-value or M-value representations of the data. Table-wise, we highlight in bold the results of the method with the best performance. This was identified as the method whose performance is always statistically significantly higher than or compa- rable to that of other methods. We remark that, in many cases, when comparing two performances the Wilcoxon test does not detect a statistically significant difference. Thus, there is no case in which (table-wise) a performance is detected as statistically significantly better than all other performances. Wilcoxon-testing procedure to assess statistically significantly better performances Therefore, we relaxed the defini- tion of best performance by defining it as the performance that is never statistically significantly worse within a table. Both in comparisons 1 and 2, the Wilcoxon test has been applied separately on MAE and RMSE. In order to compute the Wilcoxon signed-rank test we used the (paired) average (MAE or RMSE) performances of the imputation methods on the one hundred repetitions of the imputation tests. Additionally, we performed a p-value adjustment for multiple comparisons with the Benjamini-Hochberg (BH) procedure. We remark that, since the Wilcoxon test draws statistical inference from the rank sum instead of the mean, it can happen that a performance is detected as statistically signif- icantly better than another although the average performances of the former are worse than those of the latter. This typically happens when there are just few outliers that affect the overall average performance score but not the rank sum test. We further remark that, since the Wilcoxon test is not transitive, when we use it to asses a comparison between multiple methods there can be cases where no method can be termed as best performing, even with our relaxed definition (one example is performance assessment in Table 4). Supplementary information Supplementary information accompanies this paper at https://doi.org/10.1186/s12859-020-03592-5. Additional file 1: Detailed imputation results per dataset. Additional file 2: Dataset statistics. Additional file 3: Performance comparison between complete (450k) and restricted (21k) datasets. Supplementary information Supplementary information accompanies this paper at https://doi.org/10.1186/s12859-020-03592-5. Additional file 1: Detailed imputation results per dataset. Additional file 2: Dataset statistics. Additional file 3: Performance comparison between complete (450k) and restricted (21k) datasets. Funding g This work is partially funded by Project: iPC Individualized Paediatric Cure - European Union’s Horizon 2020 research and innovation programme, under grant agreement No 826121. The funder had no role in study design,data collection and analysis,decision to publish,or preparation of work included in this submission. Page 21 of 22 (2020) 21:268 Lena et al. BMC Bioinformatics (2020) 21:268 Lena et al. BMC Bioinformatics Availability of data and materials The datasets generated and/or analysed during the current study are available in the GEO repository https://www.ncbi. nlm.nih.gov/geo/. The GEO IDs of the datasets are available in Table 1. Competing interests p g The authors declare that they have no competing interests. We declare that one of the authors, namely Christine Nardini, is member of the editorial board (Associate editor) of this journal. Author details 1 f 1Department of Computer Science and Engineering, University of Bologna, Mura Anteo Zamboni 7, Bologna, Italy. 2Department of Physics and Astronomy, University of Bologna, Viale Berti Pichat 6/2, Bologna, Italy. 3Smart Cities Living Lab, ISOF CNR, Via P. Gobetti, 101, Bologna, Italy. 4Istituto per le Applicazioni del Calcolo "Mauro Picone", CNR, Via dei Taurini, 19, Roma, Italy. Received: 21 December 2019 Accepted: 9 June 2020 Received: 21 December 2019 Accepted: 9 June 2020 References 1. Riggs A, Martienssen R, VEA R. Introduction. In: Russo VEA RA, Martienssen R, editors. Epigenetic Mechanisms of Gene Regulation. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1996. p. 1–4. 1. Riggs A, Martienssen R, VEA R. Introduction. In: Russo VEA RA, Martienssen R, editors. Epigenetic Mechan Gene Regulation. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1996. p. 1–4. 2. Riggs A, Porter T. Overview of epigenetic mechanisms. 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Handling missing values in exploratory mul , g g p y y ; 23. Fuentes M, Guttorp P, Sampson P. Using transforms to analyze space-time processes. In: Statistical Methods fo Spatio-temporal Systems. Chapman and Hall; 2006. p. 77–151. Spatio-temporal Systems. Chapman and Hall; 2006. p. 77–151. p p y p p 24. Stekhoven DJ, Bülmann P. Missforest – non-parametric missing value imputation for mixed-type data. Bioinformatics. 2012;28:112–8. Bioinformatics. 2012;28:112–8. 25. Lomberk G, et al. Distinct epigenetic landscapes underlie the pathobiology of pancreatic cancer subtypes. Nat Commun. 2018;9:1978. 25. Lomberk G, et al. Distinct epigenetic landscapes underlie the pathobiology of pancreatic cancer subtypes. Nat Commun. 2018;9:1978. 26. Klughammer J, et al. The dna methylation landscape of glioblastoma disease progression shows extensive heterogeneity in time and space. Nat Med. 2018;24:1611–24. 26. Klughammer J, et al. The dna methylation landscape of gliobl heterogeneity in time and space. 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Accessed June 2019. 31. softImpute. https://www.rdocumentation.org/packages/softImpute/versions/1.4. Accessed June 2019. 32. missMDA. https://www.rdocumentation.org/packages/missMDA/versions/1.13. Accessed June 2019. 33. missForest. https://www.rdocumentation.org/packages/missForest/versions/1.4. Accessed June 2019. 34. methyLImp. https://github.com/pdilena/methyLImp. Accessed June 2019. 35. Illumina Inc. Comprehensive DNA Methylation Analysis on the Illumina©Infinium©Assay Platformt. San Diego; 2010. 36. Wilcoxon Signed-rank Test. https://www.rdocumentation.org/packages/stats/versions/3.6.0/topics/wilcox.test. Accessed June 2019. 28. Edgar R, Domrachev M, Lash AE. Gene expression omnibus: Ncbi gene expression and hybridization array data repository. Nucleic Acids Res. 2001;30:207–10. Publisher’s Note S i N i Publisher s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W3196022217	http://www.ijtmgh.com/article_135214_726c7799a140bdf7bb77582108124ab7.pdf	English	null	The COVID-19 Pandemic May Force the World to Reflect on the Pre-Pandemic Style of Life	International journal of travel medicine and global health	2,021	cc-by	6,676	Farzaneh Yazdani1 ID , Mehdi Rezaee2, Mehdi Rassafiani3* ID , Dave Roberts4 ID , Wa’d Abu-Zurayk5, Mou Amarlooee6 1Occupational Therapy Program, Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, UK 2Department of Occupational Therapy, School of Rehabilitation, Shahid Beheshti University of Medical Sciences, Tehran, Iran 3Occupational Therapy Department, Faculty of Allied Health Sciences, Kuwait University, Jabriya, Kuwait 4School of Nursing and Midwifery, Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, UK 5Occupational Therapist, Amman, Jordan 6Sadra Occupational Therapy Department, Tehran, Iran Corresponding Author: Mehdi Rassafiani, PhD, Associate Professor, Occupational Therapy Department, Faculty of Allied Health Sciences, Kuwait University, Jabriya, Kuwait. Email: mrassafiani@yahoo.com Received April 15, 2021; Accepted August 15, 2021; Online Published August 29, 2021 Abstract Introduction: The COVID-19 pandemic has caused a lot of changes on an individual and societal level. The current study was designed to investigate the impact of the isolation/ social distancing period on people’s sense of Being, Belonging, and Becoming at the early stages of the COVID-19 measures. Methods: A cross-sectional survey study design was employed utilizing a researcher-developed questionnaire with items developed based on the Model of Occupational Wholeness to investigate the changes that may have happened to what people have been doing during the COVID-19 measures compared to the time before. A sample of 1206, using snowball sampling, responded to the online questionnaire. Results: Findings indicated that participants at the early stage of the COVID-19 pandemic had changed the pattern of their doings. The change of pattern, which was considered positive, was in relation to people valuing their health, having more time to take care of themselves, and doing activities which they had never had enough time to do before. i Conclusion: While these findings are not generalizable, they provide some insights into how the post-pandemic lifestyle of many people does not permit doing a lot of activities that could help with their health and well-being. While enforced isolation may have negative consequences, it is also clear that the pre-COVID-19 pandemic lifestyle did not fully support healthy living. Reflecting on the COVID-19 lockdown experience provides an opportunity to review the essential personal and societal elements for living a healthy life. TMGH IInternational Journal of Travel Medicine and Glob J 10.34172/ijtmgh.2021.21 Original Article Open Access Farzaneh Yazdani1 ID , Mehdi Rezaee2, Mehdi Rassafiani3* ID , Dave Roberts4 ID , Wa’d Abu-Zurayk5, Mou Amarlooee6 Keywords: Occupational Science, Occupational Therapy, Public Health, Occupational Wholeness to the physical separation between people from different households, whether from family, friends, or wider social networks and it also requires non-interference in social activities that require people to be close to one another.3,4 This isolation is due to environmental constraints, not to the ability of the individual to establish or maintain social relationships.5 Int J Travel Med Glob Health. 2021 Sep;9(3):124-131 Int J Travel Med Glob Health. 2021 Sep;9(3):124-131 http://ijtmgh.com Int J Travel Med Glob Health. 2021 Sep;9(3):124-131 Copyright © 2021 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Citation: Yazdani F, Rezaee M, Rassafiani M, Roberts D, Abu-Zurayk W, Amarlooee M. The COVID-19 pandemic may force the world to reflect on the pre-pandemic style of life. Int J Travel Med Glob Health. 2021;9(3):124-131. doi:10.34172/ijtmgh.2021.21. Introduction COVID-19 is a viral respiratory disease that has spread globally with serious health impacts and high mortality.1 Coinciding with the outbreak of COVID-19 in early March 2020 in Western countries, strict national policies on public behaviour were implemented around the world from Europe and America, to Australia and Asia. Along with the observance of health laws, social distancing also became the main policy of public behaviour in many societies. This policy included having minimal contact with people outside the home and staying at home as much as possible.2 On the other hand, in some countries, there have been further measures such as quarantine or social isolation. Social isolation refers Social distancing, social isolation, and quarantine conditions can lead to high levels of psychological distress and even post- traumatic stress disorder (PTSD) due to lack of or reduced access to other people.6 Assessment of people’s mental health, when exposed to natural disasters, shows that survivors often experience various mental health disorders, including PTSD, depression, general anxiety disorder, panic disorder, Copyright © 2021 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Reflect of Pre-Pandemic Style of Life and substance abuse. Even after a 4-to 6-month period of separation and social isolation, symptoms such as anxiety, anger, and worry remain in people.7 Brooks et al reviewed articles on the psychological impact of quarantine, and they reported negative psychological effects, including symptoms of PTSD (confusion, anger, and stressors during/post-long- term quarantine), fear of infection, frustration, impatience, insufficient resources, insufficient information, financial loss, and stigma.8 In the early stages of the COVID-19 outbreaks in China, a sample of 1210 isolated people was surveyed. The results of this research demonstrated that more than half of the people experienced moderate to severe psychological effects, and one-third of them reported moderate to severe anxiety. Introduction Prevalence of stress, anxiety, and depression was higher in women, students, and individuals with specific physical symptoms.1 In a review of 19 articles about the impact of the COVID-19 pandemic on mental health in the general population in China, Spain, Italy, Iran, the US, Turkey, Nepal, and Denmark, Xiong and colleagues found that relatively high rates in symptoms of anxiety (6.33% to 50.9%), depression (14.6% to 48.3%), PTSD (7% to 53.8%), psychological distress (34.43% to 38%), and stress (8.1% to 81.9%) are present in these countries.9 Pfefferbaum and North stated that vague forecasts, severe shortages of resources for testing, treatment, and protection of people and health care providers from infection, public health practices that violate personal freedoms, huge financial losses, and conflicting messages from officials are major sources of stress that undoubtedly contribute to widespread emotional distress and an increased risk of COVID-19-related mental illness.10 Humans, as occupational beings, shape their identity based on what they do and this affects their mental and physical health. Human action falls into the realms of Being, Belonging, and Becoming. “Being” refers to activities that make a person feel good, including activities directed towards meeting basic survival needs, as well as activities that exercise choosing a method of self-care. “Belonging” refers to one’s connections and relationships with their environment and other people, and “Becoming” refers to what one does for a better life based on one’s own vision.14 According to Yazdani and Bonsaksen, what meaningful activities people “Do” to meet their needs of Being, Belonging and Becoming, leads to an overall sense of satisfaction with themselves and their own world, this is called Occupational Wholeness. In a major life event such as the COVID-19 pandemic, with isolation and social distancing, people have to adapt to the way they manage their daily lives. How people perceive illness or stressful events is influenced by their previous experiences of such phenomena.15,16 What is remarkable about the current situation is the short period of time in which lifestyle changes have taken place. This study was designed to investigate the effect of periods of isolation/social distance on how people feel about their sense of Being, Belonging, and Becoming. The authors also hope to examine whether there is a difference in the perceived impact of isolation/social distance in terms of gender, age, or living status. International Journal of Travel Medicine and Global Health. 2021;9(3):124–131 125 Methods A cross-sectional survey using a convenience sampling and a web-based questionnaire was conducted in eight languages including English, French, Spanish, German, Indian, Arabic, Greek, and Persian. A survey study design was employed to collect data about participants’ perceived sense of Occupational Wholeness. According to Check and Schutt, this design allows the gathering of information from individuals through their responses to questions.17 The current survey consists of two parts; a questionnaire that was developed by the researchers, and demographic questions.18-20 The time required to respond to the questionnaire was 15 minutes on average. After disasters, most people resist and do not succumb to psychological pathology. In fact, some people find new strengths. But some groups may be more vulnerable to the psychosocial effects of epidemics than others. In particular, older people, people whose immune system function is compromised and those who live or are cared for in care centers, as well as people who have previously had medical, psychiatric, or substance abuse problems are at greater risk for psychosocial consequences.10 However, individual responses to catastrophic and traumatic events such as COVID-19 are not uniform. These responses can be different according to the understanding of traumatic events, a person’s belief system, and socio-cultural resources.11 COVID-19 disease now poses new and unique challenges for individuals and social groups due to its profound impact on social and daily activities.12 Development of Survey Questions Seven items were developed around this concept to investigate the participants’ sense of Becoming concerning changes they expect for their future life, development in relation to their education or job, new learning, and in particular financial expectations. To see the overall 26 items of the online questionnaire, see Table 1. situation with the time before the period of social isolation on a five-point Likert scale. For example, in a statement: “I have more opportunities to do what I like”, the scores were “0 = it has not changed compared to the time before isolation/social distancing period”, “-2 = absolutely “disagree”, “-1 = disagree”, “1 = agree”, “and “2 = absolutely agree. A higher score in each item indicates a higher level of satisfaction. By sum scores of the questions related to each component (i.e., Being, Belonging, and Becoming), the score of each of them can be obtained. In an online panel discussion, a group of four occupational therapists reviewed the relevance and necessity of the items. After that, the fluency and clarity of the items were assessed by five experts, and the face validity of the questions was confirmed.22 To establish the content validity, items of the questionnaire originated from the literature followed by the evaluation through an expert panel.23 This questionnaire was administered experimentally to further test the validity, involving eight people of different ages and from different socio-economic backgrounds. This was conducted through an in-depth cognitive interview to assess participants’ perceptions of the items. Notes collected from interviews The questionnaire consisted of 26 sentences with positive and negative values. The questions were originated from the Occupational Wholeness Model, informed by the OWQ, modified to suit the context of COVID-19 isolation measures.21 Participants were asked to compare their current International Jo rnal of Tra el Medicine and Global Health 2021 9(3) 124 131 126 Table 1. Development of Survey Questions According to the Model of Occupational Wholeness that underpins the development of the Occupational Wholeness questionnaire (OWQ),21 people can feel comfortable with themselves and their world if they can define a satisfying combination of “doing or not doing” activities. This range of doing/not doing should include what the individual does to satisfy the need for Being, Belonging, and Becoming. These need to coordinate with each other, to the best of one’s ability and depending on their context. A satisfactory combination of activities depends on the meaning that people give to their activities. During personal development, the sense of Being is formed in two stages. First, it forms based on attending to survival needs. Later, Being needs expand to include the need for autonomy and choice in self-care activities. People can feel good about themselves and the world by doing activities relevant to their sense of Being. The sense of Being is based Loss of routine and frequent social and physical contact with others can lead to boredom, frustration, and feelings of isolation from the rest of the world, which can be distressing. This frustration is exacerbated by the inability to participate in daily routine activities, such as buying essential goods.8 Researchers, scientists, and policymakers have focused more on the number of deaths from the disease and paid less attention to peoples’ occupations. However, this pandemic has negatively affected the meaningful occupations of the world’s population. Children are not able to play in the park with their peers or go to school, adults are not able to cope with their schedule of working or studying at home.13 International Journal of Travel Medicine and Global Health. 2021;9(3):124–131 12 125 Yazdani, et al on the “here and now” and refers to one’s feeling and thinking in the present. In the questionnaire, 11 items are dedicated to the sense of Being. These items represent the amount of time and energy that people spend on themselves and the time and energy spent to reflect on the meaning of health, well-being, and life. Eight items in the questionnaire were allocated to assess the sense of Belonging. Sense of Belonging was assessed through items related to one’s connections, relationships, and togetherness. The sense of Becoming refers to people’s perception of making life changes to make it better; therefore, this concept is about future prospects, development, and improvement. Development of Survey Questions Mean and Standard Deviations for the Whole Questions in the Questionnaire N Question N Min Max Mean SD 1 I am pleased with the time I spend with my family 1168 -1 1 0.52 0.75 2 I have more opportunities to do what I like 1163 -1 1 0.23 0.92 3 I have a chance to review the meaning of my life 1170 -1 1 0.57 0.73 4 I have an opportunity to learn new skills to help me develop more job and career-related skills 1169 -1 1 0.37 0.84 5 I have an opportunity to grow spiritually 1166 -1 1 0.52 0.73 6 There is an opportunity for me to learn new ways to communicate with others like using mobile apps 1161 -1 1 0.52 0.72 7 7I have opportunities to increase my knowledge in areas I like 1171 -1 1 0.54 0.74 8 I can do things I never had time and space for before 1197 -1 1 0.51 0.78 9 I have discovered some new characteristics about myself 1198 -1 1 0.40 0.77 10 I have the opportunity to reflect on the value of my health 1206 -1 1 0.69 0.60 11 I have the opportunity to take care of my health 1202 -1 4 0.43 0.79 12 Some issues have worried me about my future (financial, job, or family-related, …) 1146 -1 1 -0.63 .68 13 I have opportunities to do things to help others 1194 -1 1 0.42 .78 14 My family relationships have become strained 1198 -1 1 0.45 .77 15 I have fewer opportunities to do things I like 1186 -1 1 0.19 .93 16 Life has lost its meaning for me 1199 -1 1 0.64 0.69 17 There is no opportunity to educate myself for further development 1202 -1 1 0.65 0.67 18 I have a lot on my to-do list but with less chance to do them 1172 -1 1 0.15 0.93 19 I have less time and space to think about myself 1199 -1 1 0.45 0.83 20 My ways of communication lead to conflicts at home 1202 -1 1 0.53 0.74 21 There is no time to spend on my own mental health and well being 1194 -1 1 0.48 0.81 22 There are fewer opportunities to be useful to others 1183 -1 1 0.32 0.88 23 I have less motivation to put effort to achieve what I want for my life 1183 -1 1 0.32 0.87 24 I have more opportunities to play my social roles and do volunteer activities 1169 -1 1 -0.15 0.86 25 I have more motivation to build my future 1189 -1 1 0.16 0.84 26 I use my home space and furniture in a positive new way to meet my needs 1202 -1 1 0.37 0.77 Note: The results of questions 12 and 24 are negative and those of others are positive. International Journal of Travel Medicine and Global Health. 2021;9(3):124–131 126 Procedure This study was conducted between May and June 2020. First, an invitation was sent to stakeholders, and healthcare professionals, in Germany, France, Greece, India, Jordan, Spain, Iran, and the UK by email. The link to the questionnaire then was added to social media: Twitter, Facebook, Instagram, and LinkedIn to alert people to participate in this study. The snowball method was used to encourage the circulation of the link. Development of Survey Questions Table 1. Mean and Standard Deviations for the Whole Questions in the Questionnaire Reflect of Pre-Pandemic Style of Life isolated for more than four weeks and spent a much greater amount of their time at home compared to the time before the Table 3. Demographic Characteristics Variables No. % Gender Female 840 69.6 Male 200 16.6 Missing 167 13.8 Age < 25 291 24.1 26-35 274 22.7 36-45 237 19.6 > 45 234 19.4 Missing 171 14.2 Status living Alone 147 12.2 With other people 267 22.1 With partner/husband 186 15.4 Partner/husband/wife/children 352 29.2 Partner/husband/wife/children/other 96 8.0 Missing 159 13.2 Number of children Non 557 46.1 one 166 13.8 Two 224 18.6 Three 64 5.3 Four and more 23 1.9 Missing 173 14.3 Duration of Isolation: Almost a week 45 3.7 Almost 2 weeks 15 1.2 Almost 3 weeks 20 1.7 Almost 4 weeks 52 4.3 Almost 5 weeks 143 11.8 Missing 153 12.7 Time spend outside home 0%-10% less than before 127 10.5 10%-30% less than before 118 9.8 30%-60% less than before 256 21.2 60%-90% less than before 447 37.0 Almost 100% isolated 192 15.9 Missing 67 5.6 Education level Professional qualification 83 6.9 High school 65 5.4 Bachelor/honor degree or equivalent 539 44.7 MSc/MA or equivalent 296 24.5 PhD 71 5.9 Missing 153 12.7 were reviewed and corrections were made.22 When using Likert scales, calculating, and reporting Cronbach’s alpha coefficient is essential for internal consistency reliability. The reliability of the instrument used for this study, which was tested using Cronbach’s alpha, was generally α = 0.909, which is considered excellent. Cronbach’s alpha for three subscales measured sense of Being α = 0.815 (good), sense of Belonging α = 0.727 (acceptable), sense of Becoming 0.734 (acceptable).24 The final instrument was a self-administrated questionnaire with an original 26 items (sense of Being = 11, sense of Belonging = 8, and sense of Becoming = 7) and 17 demographic questions (Table 1). Statistical Analysis The resulted data were entered into SPSS, version 22 for the analysis. Prior to the beginning of the analysis, data were checked twice for any unforeseen error during the process of data coding and entry. Then, descriptive and inferential statistical methods were used to analyse the results. These included mean, standard deviation, number, and percentages, as well as one-sample t-statistics, student t test, and analysis of variance (ANOVA). International Journal of Travel Medicine and Global Health. 2021;9(3):124–131 127 COVID-19 outbreak (Table 3). between male and female participants, student t test was used. The results demonstrated no statistically significant differences between the two genders (Table 5). The one-sample t test was utilized to compare the sense of Being, Belonging, and Becoming to the hypothesized value (the expected average score for each variable based on their total score) to determine if the sample mean is significantly greater or less than that value. The results show that the P values for all are significantly smaller than 0.05. As the observed means are greater than the expected value, these suggest that the participants perceived a higher level of positive attribution to their experience of the COVID-19 isolation period compared to the time before (Table 4). In this analysis, the 5-point scale was converted into three-point scales as follows: -1 = Definitely Disagree or Disagree; 0 = no difference compared to before, +1 = Definitely agree or agree. To examine differences in the subscales concerning participants’ living status, the number of children, their age, and the ANOVA was used. The results showed that participants in different living statuses were not statistically different in their sense of Being, Belonging, and Becoming. However, participants with four children and more demonstrated higher scores in Being, Belonging, and Becoming (P < 0.005). Different age groups also demonstrated no statistical differences in the three subscales of the questionnaire except in the sense of Being. Post hoc analysis showed that only participants older than 45 years of age had a statistically lower sense of Belonging compared to participants under 25 (P = 0.05) (Table 5). To understand the differences in the three subscales of questionnaires (including Being, Belonging, and Becoming) The mean and standard deviation for each item in the questionnaire indicated in Table 1, demonstrates the distribution of the participants’ responses in this study. Table 4. Summary of one sample t-test for comparing belonging, being, becoming Table 4. Summary of one sample t-test for comparing belonging, being, becoming Variable Excepted value Observed Mean SD T P Value Belonging 0 2.80 3.2 29.1 < 0.001 Being 0 4.50 5.0 29.4 < 0.001 Becoming 0 1.77 3.1 19.4 < 0.001 Total 0 8.57 9.7 28.8 < 0.001 Results In total 1206 people participated in this study. SPSS version 22 was used to analyse the results. Descriptive statistics were used to understand the participants’ characteristics. Tables 2 and 3 demonstrate the sociodemographic information of the participants. The majority of the participants were Greek (20.7), Iranian (18.9), and French (16.2). Most of the participants were female (69.6%), living alone (46.1%), and had university education. At the time of data collection, the majority of the participants reported that they had been Table 2. Distribution of Participants According to Their Countries Table 2. Distribution of Participants According to Their Countries Country of Participants Frequency Percent Arab countries (Middle East) 178 14.7 Indian 43 3.5 Spanish 90 7.5 Iranian 228 18.9 French 195 16.2 German 24 2.0 Greek 250 20.7 Other countries 198 16.4 Total 1206 100.0 isolated for more than four weeks and spent a much greater amount of their time at home compared to the time before the Yazdani, et al International Journal of Travel Medicine and Global Health. 2021;9(3):124–131 128 05. Discussion Similar to other studies, perceived time pressure for doing what one needs/wants to do is associated with lower life satisfaction.29,30 For the majority of the participants in this study, finding some spare time, whether mental or physical, during the early stages of the COVID-19 pandemic was perceived as an opportunity to meet needs that they did not have time for before. The findings of this study, however, should not be generalised to the longer-term impact perceived by participants. This sheds light on the lack of time and space they had previously. Items related to Belonging were 1, 6, 13, 14, 20, 23, 25, 27. These were enquiring about the participants’ satisfaction with the time they spend with loved ones, changes in family relationships, learning new ways to connect, and being helpful/useful to others during the pandemic compared to the prior time. Similar to not having time for meeting their Being needs, the findings shed light on how the participants may not have had time to meet their Belonging needs prior to the isolation measures of the pandemic. Participants valued the significance of others and rated the isolation measure restrictions as an opportunity to learn new ways of connection, having more time for and with loved ones. In this study, participants were invited to reflect and compare their current situation within the pandemic isolation measures with the pre-pandemic situation; taking into consideration their own status before exposure to the event. Participants perceived the COVID-19 pandemic measures as an opportunity for satisfying a sense of Being, Belonging, Becoming, their sense of overall Wholeness and their sense of wellbeing as explained by Sieger in Chatterjee.25 They stated well-being is a relative concept as people may compare their own condition with that of others. The findings of a study conducted by Kowalczyk et al presented a similar conclusion about the impact of the COVID-19 pandemic on people in Polish society concerning the opportunity for reflection or as they put it ‘spiritual renewal’.26 In relation to the Becoming (items: 4, 5, 7, 12,17, 22, 24, 26) that is related to learning opportunities, spiritual growth, hope, and worries for the future, the overall figure showed a positive experience compared to the time prior to COVID-19. However, it is important to pay attention to the two dimensions of growth and future prospects that come under this component. Discussion Having access to and being able to use devices such as mobiles, tablets, and computers has been essential for participation in the survey. As the survey did not provide any feedback, committing time to go through it required some enthusiasm in participants. The enthusiasm could have been based on several factors, such as curiosity about the views on the topic or/and valuing research. These characteristics might limit the range of people from whom data is collected. On the other hand, it is expected that for this same reason participants provided more genuine responses. Therefore, the findings of this study could only provide some insights into a similar population. The findings suggest that the participants’ perception of the impact of the pandemic is not necessarily or wholly negative. Therefore, the significance of the findings within the early stages of the COVID-19 pandemic is that a group of people whose socio- economic status allows them to take time to slow down and reflect has perceived the situation as an opportunity. As the findings indicated, the majority of the participants have felt an overall sense of coherence that was higher than the time before the pandemic. This may be the tentative impact of the isolation measures, yet it has been valuable as it shows that people valued a break from the ongoing situations in their life. To develop this argument further, reviewing the items of the questionnaire may allow for further understanding of the findings. It is also important to pay attention to the period of time in which the data were collected. Therefore, responses to individual items are discussed in light of the questionnaire having been conducted at the early stage of the COVID-19 pandemic. This was the period in which the first isolation measures were put into place in the countries where the data were collected. Items used to explore the sense of Being were the items 2, 3, 8, 9, 10, 11, 15, 16, 18, 19, 21. These refer to the time and space people had during the isolation measures to take care of themselves, as well as reflect on the meaning of their health and life. Furthermore, these items refer to participants doing activities that they had previously enjoyed or activities they needed to do but had not had time for before. International Journal of Travel Medicine and Global Health. 2021;9(3):124–131 129 Discussion This survey data analysis demonstrates that the COVID-19 isolation/social distancing measures have resulted in a positive impact on the overall sense of wholeness. To discuss the findings further, we considered the characteristics of the members of the public who participated in this study and Table 5. The Comparison of the Perceived Sense of Being, Becoming and Belonging Based on the Participants’ Demographic Characteristics Sense of Being Sense of Becoming Sense of Belonging Variables Mean SD P3 Mean SD P Mean SD P Gendera Male 4.5 4.6 0.95 1.5 3.3 0.47 2.7 3.1 0.66 Female 4.5 5.2 1.3 3.2 2.9 3.2 Living status Alone 4.9 5.1 1.98 1.8 3.1 1.89 2.7 3.2 2.29 With other people 4.9 4.7 1.39 3.1 2.5 3.2 With partner/husband 4.2 4.9 0.9 3.4 2.6 3.106 Partner/husband/wife/children 3.9 5.2 1.2 3.4 3.2 3.1 Partner/husband/wife/children/other 4.4 5.7 1.4 3.3 2.6 3.4 Number of children Non 4.9 4.9 4.44* 1.5 3.1 2.82* 2.6 3.1 1.20 one 3.7 5.1 0.7 3.2 2.9 3.1 Two 4.0 5.3 1.1 3. 5 3.0 3.3 Three 2.9 5.6 1.6 3.4 2.9 3.1 Four and more 5.6 4.6 2.6 3.1 3.7 3.6 Age < 25 5.2 4.6 1.5 3.0 2.7 3.3 26-35 4.4 4.9 2.80* 1.1 3.4 0.98 2.8 3.2 0.82 36-45 4.0 5.1 1.3 3.5 3.1 3.0 > 45 4.1 5.6 1.3 3.3 2.7 3.2 SD, standard deviation a T student was used to analyses the results for the gender. For all other variables ANOVA was used. * P < 0.05. Table 5. The Comparison of the Perceived Sense of Being, Becoming and Belonging Based on the Participants’ Demograph the Perceived Sense of Being, Becoming and Belonging Based on the Participants’ Demographic Characteristics Reflect of Pre-Pandemic Style of Life not pick up on the pre-existing psychosocial issues that people may have had prior to the pandemic. It is important to consider how vulnerable people were affected by the situation, and this is a limitation of the current study, as well. the period of time over which the survey was conducted. The survey used an online platform in order to enable people with specific characteristics to be able to consider participation. The snowball sampling method has meant that participants need to have access to social media platforms such as Facebook, Telegram, and WhatsApp. References 1. Wang B, Li R, Lu Z, Huang Y. Does comorbidity increase the risk of patients with COVID-19: evidence from meta-analysis. Aging (Albany NY). 2020;12(7):6049-6057. doi:10.18632/ aging.103000. Limitations l Similar to many online survey studies, this study only recruited a particular group of the public with access to devices that allow access to the internet and social platforms. This may, for example, exclude some people from lower socioeconomic backgrounds. The lack of a longitudinal follow-up element in this study may also be considered as a limitation, as no comparison was possible for the perceptions of the same group of participants between the time before the COVID-19 measures were taken, and the months after, when people might have started suffering from the financial impact related to the restrictions. Acknowledgments The authors would like to show their appreciation to the following people who facilitated the data collection in their countries: Dr. Salvador Simo, Dr. Panagiotis Siaperas, Dr. Arindam Saha Jaipuria, Dr. UNG Yannick, Ms. Sarah Kufner, Dr. Maria Kapanadze, and Dr. Sanjay Kumar. The authors also would like to show their gratitude to the participants of this study. What Is Already Known? What Is Already Known? The COVID-19 pandemic has affected people and their lives in different aspects, including, but not limited to psychological and financial problems. Another interesting finding of this study is the significant difference in the perceived level of increase in the sense of Being and Becoming in participants with four children and more. Although this group only consists of 23 out of 1206 participants, they displayed a higher attribution of positive experience in the senses of Being, and Becoming. However, a higher perceived sense of Belonging compared to the period prior to the COVID-19 measures was not statistically significant. This figure echoes Schwarze and Winkelmann’s study that showed people find interdependent and collective happiness in living in larger family groups.32 Their study also indicates the importance of interdependent family happiness through their relationships even when children move away from their homes. What Does This Study Add? COVID-19 measures have imposed restrictions on the public’s life. However, it has provided a greater perception of freedom around time and space for participants and this underpins the positive impact of the COVID-19 isolation measures. The key message of this is that the social isolation period has provided an opportunity for individuals to reflect on their lifestyles, and how they could be changed to achieve greater satisfaction with themselves and their lives. Funding/Support Funding/Support None. None. International Journal of Travel Medicine and Global Health. 2021;9(3):124–131 130 Authors’ Contributions b d Overall, the findings of this study demonstrate the value of time and space that people require in order to meet their needs for living, pursuing their interests and values, as well as spending time, sharing space with others, and planning for their life. Even though the isolation period had its own negative impact on people’s mental health due to restrictions and in particular financial strains on families, the results of this study raise questions as to whether the lifestyle prior to the pandemic has been satisfying and healthy. This time of crisis could be taken as an opportunity to review priorities and opportunities for Being, Belonging, and Becoming in order to make changes towards a better life style and not necessarily the life style prior to the Covid- 19 pandemic. FY contributed to conception, design, data collection, and writing up the manuscript. MRe contributed to the conception and writing of the manuscript. MRa contributed to the design, analysis, and writing of the manuscript. DR, WA, and MA contributed to the vision and critically reviewed the manuscript before the final submission. Conflict of Interest Disclosures Conflict of Interest Disclosures The authors declare that they have no conflicts of interest. Ethical Approval Discussion Although similar to other items, participants responded more positively to the impact of the COVID-19 measures in relation to their personal and spiritual growth and development by allocating time and space for them, their response to the future was negative and associated with worries. It is necessary to be careful in comparing the findings of this study with those of other studies which indicate the presence of confusion, anger, frustration, and impatience, or financial loss as an impact of COVID-19.8 It is important to draw a line between the psychological symptoms and a sense of satisfaction with self/life. People may present mental health symptoms but not necessarily perceive them as negatively impacting their sense of Being, Belonging, and Becoming. This may be linked to the level of resilience and response to difficulties in individuals despite their mental health symptoms. This is an issue that needs further investigation.27,28 On the other hand, in their review of the literature, Brooke et al indicated that only one study had followed participants over time and compared the finding during the COVID-19 measures to the time before.3 This means these studies did It appears the issue around time and space, a common factor in all items of the survey questions is the key factor to explain the relativity of the perceived positive experiences in all of the above issues. Wajcman states that freedom and control of our International Journal of Travel Medicine and Global Health. 2021;9(3):124–131 129 Yazdani, et al own time are significant for our sense of satisfaction with our lives and it is something which we do not always have access to in contemporary life.31 Research Highlights Ethical Approval The ethics approval was granted by the Oxford Brookes University Ethics committee in the UK; No: L20206. Funding/Support None. Conclusion h 2. Bonsaksen T, Leung J, Schoultz M, et al. Cross-national study of worrying, loneliness, and mental health during the COVID-19 pandemic: a comparison between individuals with and without infection in the family. Healthcare (Basel). 2021;9(7):903. doi:10.3390/healthcare9070903. There are negative impacts of the COVID-19 measures on people’s health as reported in the literature. However, this study shows that for some members of the public, the situation was perceived as an opportunity to satisfy their Being, Belonging, and Becoming needs. In other words, these needs were perceived as higher compared to the time before the COVID-19 isolation measures. The significance of the freedom in having time and space to do some activities that meet their Being, Belonging, Becoming needs seems to be the key. The findings of the study can add to the knowledge of occupational sciences and potentially help strengthen the role of occupational therapy in a critical situation. 3. Brooke J, Jackson D. Older people and COVID-19: isolation, risk and ageism. J Clin Nurs. 2020;29(13-14):2044-2046. doi:10.1111/jocn.15274. 4. Singh J, Singh J. COVID-19 and its impact on society. Electronic Research Journal of Social Sciences and Humanities. 2020;2(1):168-172. 4. Singh J, Singh J. COVID-19 and its impact on society. Electronic Research Journal of Social Sciences and Humanities. 2020;2(1):168-172. 5. Tanskanen J, Anttila T. A prospective study of social isolation, loneliness, and mortality in Finland. Am J Public Health. 2016;106(11):2042-2048. doi:10.2105/ajph.2016.303431. 5. Tanskanen J, Anttila T. A prospective study of social isolation, loneliness, and mortality in Finland. Am J Public Health. 2016;106(11):2042-2048. doi:10.2105/ajph.2016.303431. International Journal of Travel Medicine and Global Health. 2021;9(3):124–131 0 Reflect of Pre-Pandemic Style of Life 19. DuBenske LL, Gustafson DH, Namkoong K, et al. CHESS improves cancer caregivers’ burden and mood: results of an eHealth RCT. Health Psychol. 2014;33(10):1261-1272. doi:10.1037/a0034216. 6. Hawryluck L, Gold WL, Robinson S, Pogorski S, Galea S, Styra R. SARS control and psychological effects of quarantine, Toronto, Canada. Emerg Infect Dis. 2004;10(7):1206-1212. doi:10.3201/eid1007.030703. 20. Ponto J. Understanding and evaluating survey research. J Adv Pract Oncol. 2015;6(2):168-171. 7. Jeong H, Yim HW, Song YJ, et al. Mental health status of people isolated due to Middle East respiratory syndrome. Epidemiol Health. 2016;38:e2016048. doi:10.4178/epih.e2016048. 21. Bonsaksen T, Yazdani F. The Norwegian occupational wholeness questionnaire (N-OWQ): scale development and psychometric properties. Scand J Occup Ther. 2020;27(1):4-13. doi:10.1080/1 1038128.2018.1426783. 8. Brooks SK, Webster RK, Smith LE, et al. Conclusion h The psychological impact of quarantine and how to reduce it: rapid review of the evidence. Lancet. 2020;395(10227):912-920. doi:10.1016/ s0140-6736(20)30460-8. 22. Artino AR Jr, Durning SJ, Sklar DP. Guidelines for reporting survey-based research submitted to academic medicine. Acad Med. 2018;93(3):337-340. doi:10.1097/acm.0000000000002094. 9. Xiong J, Lipsitz O, Nasri F, et al. Impact of COVID-19 pandemic on mental health in the general population: a systematic review. J Affect Disord. 2020;277:55-64. doi:10.1016/j.jad.2020.08.001. 23. Taherdoost H. Validity and reliability of the research instrument; how to test the validation of a questionnaire/survey in a research. International Journal of Academic Research in Management. 2016;5(3):28-36. 10. Pfefferbaum B, North CS. Mental health and the COVID-19 pandemic. N Engl J Med. 2020;383(6):510-512. doi:10.1056/ NEJMp2008017. g 24. George D, Mallery P. IBM SPSS Statistics 26 Step by Step: A Simple Guide and Reference. 4th ed. Allyn & Bacon; 2019. 11. Rushford N, Thomas K. Disaster and Development: An Occupational Perspective. London: Elsevier Health Sciences; 2015. 25. Chatterjee DK. Encyclopedia of Global Justice: A-I. Vol 2. London: Springer Science & Business Media; 2011. 12. Haleem A, Javaid M, Vaishya R. Effects of COVID-19 pandemic in daily life. Curr Med Res Pract. 2020;10(2):78-79. doi:10.1016/j.cmrp.2020.03.011. London: Springer Science & Business Media; 2011. 26. Kowalczyk O, Roszkowski K, Montane X, Pawliszak W, Tylkowski B, Bajek A. Religion and faith perception in a pandemic of COVID-19. J Relig Health. 2020;59(6):2671-2677. doi:10.1007/s10943-020-01088-3. 13. Kamalakannan S, Chakraborty S. Occupational therapy: the key to unlocking locked-up occupations during the COVID-19 pandemic. Wellcome Open Res. 2020;5:153. doi:10.12688/ wellcomeopenres.16089.1. 27. Hu T, Zhang D, Wang J. A meta-analysis of the trait resilience and mental health. Pers Individ Dif. 2015;76:18-27. doi:10.1016/j. paid.2014.11.039. 14. Yazdani F, Bonsaksen T. Introduction to the model of occupational wholeness. ErgoScience. 2017;12(1):32-36. doi:10.2443/skv-s-2017-54020170104. 28. Wells M, Avers D, Brooks G. Resilience, physical performance measures, and self-perceived physical and mental health in older Catholic nuns. J Geriatr Phys Ther. 2012;35(3):126-131. doi:10.1519/JPT.0b013e318237103f. 15. Alhurani AS, Dekker R, Ahmad M, et al. Stress, cognitive appraisal, coping, and event free survival in patients with heart failure. Heart Lung. 2018;47(3):205-210. doi:10.1016/j. hrtlng.2018.03.008. 29. Eriksson L, Rice JM, Goodin RE. Temporal aspects of life satisfaction. Soc Indic Res. 2007;80(3):511-533. doi:10.1007/ s11205-006-0005-z. 16. Taylor SE, Armor DA. Positive illusions and coping with adversity. J Pers. 1996;64(4):873-898. doi:10.1111/j.1467-6494.1996. tb00947.x. 30. Zuzanek J. Time Use, Time pressure, personal stress, mental health, and life satisfaction from a life cycle perspective. J Occup Sci. 1998;5(1):26-39. doi:10.1080/14427591.1998.9686432. 17. International Journal of Travel Medicine and Global Health. 2021;9(3):124–131 131 Conclusion h Check J, Schutt R. Research Methods in Education. London: SAGE Publications; 2012. doi: doi:10.4135/9781544307725. 31. Wajcman J. Pressed for Time. University of Chicago Press; 2014. 18. Costanzo ES, Stawski RS, Ryff CD, Coe CL, Almeida DM. Cancer survivors’ responses to daily stressors: implications for quality of life. Health Psychol. 2012;31(3):360-370. doi:10.1037/ a0027018. 32. Schwarze J, Winkelmann R. Happiness and altruism within the extended family. J Popul Econ. 2011;24(3):1033-1051. doi:10.1007/s00148-010-0326-8.
https://openalex.org/W2005184597	https://www.indianjournals.com/ijor.aspx?target=ijor:jrmee&volume=2&issue=2&article=005&type=pdf	English	null	Distribution and Determinants of Perinatal Mortality in Jammu City of India	Journal of research in medical education and ethics	2,012	cc-by	6,067	ABSTRACT Downloaded From IP - 195.242.1.93 on dated 24-Oct-2024 Background: Giving birth is not a pathological phenomenon and newborn is not a disease; still in underdeveloped and developing countries, a large number of babies are born dead or die soon after birth, many of them during the first week (early neonatal deaths). For every baby who dies in the first week after birth, another is born dead (foetal deaths or stillbirths). Objectives: To find out the incidence of Perinatal Mortality Rate (PNMR), and its co-relations with different socio-demographic factors, obstetric complications and other diseases during pregnancy. To address these factors so as to reduce PNMR. Materials and Methods: A community-based prospective cohort study was conducted in Jammu city of J&K state. In all, 806 eligible pregnant women were enrolled and followed from 22 week of gestation till 28 days after delivery so as to find out the incidence of still births (SB) and early neonatal mortality rate (ENNMR) and late neonatal mortality rate (LNNMR). The study lasted for one complete year from 1 January 2011 to 31 December 2011. The relationship of perinatal mortality (still births + early neonatal deaths) with age of mother, parity, malnutrition (anaemia), foetal-presentation, type of delivery and other obstetric complications as well as with diseases was studied. The results were statistically analysed applying SPSS version 17. Results: Of the total 806 births, 94.8% were live births, 1.4% still births, 3.3% early neonatal deaths, 4.7% perinatal deaths, 0.5% late neonatal deaths and 3.8% were neonatal deaths. The association of age distribution of mother to perinatal and neonatal mortality was “U” shaped in distribution; highest in the teenage and 40–49 age groups. This association was found to be significant (P=0.04 at 95% CI). Association of parity of mother with PNMR and NNMR was significant (P=0.02 at 95% CI). PNMR and NNMR was higher in males as compared to female neonates and the association was significant (P=0.03 at 95% CI). Relationship of maternal education and mortalities was not significantly associated (P=0.97 at 95% CI); however, association of income and PNMR and NNMR were highly significant (P=0.002 at 99% CI). Similarly, anaemia and PNMR and NNMR were also significantly associated (P=0.03 at 99% CI). Association of birth weight and mortalities under study was highly significant (P=0.001 at 99% CI). Association of indirect causes was insignificant (P=0.47at 68% CI). 1Assistant Professor, Department of Community Medicine, Adesh Institute of Medical Sciences and Research, Bathinda-151101 2Lecturer, CVTA, Government Medical College, Jammu; 3Associate Professor CVTS, Sher-i-Kashmir Institute of Medical Sciences, Srinagar www.IndianJournals.com Members Copy, Not for Commercial Sale Keywords: Perinatal mortality, Neonatal mortality, Cohort study, Still births, Maternal health, Nutritional Distribution and Determinants of Perinatal Mortality in Jammu City of India Ramesh Chander1, Puja Vimesh2, Shyam Singh3 rameshvimesh@yahoo.com Journal of Research in Medical Education & Ethics Vol. 2, No. 2, July, 2012 : 109-117 Journal of Research in Medical Education & Ethics Vol. 2, No. 2, July, 2012 : 109-117 DOI : 10.5958/j.2231-671X.2.2.028 ORIGINAL RESEARCH ABSTRACT Relationship of type of delivery and intra-natal causes with these mortalities was highly significant (P=0.001 at 99% CI and P=0.01 at 99% CI, respectively). Conclusions: PNMR and NNMR are significantly associated with maternal age, parity, socio-economic status, obstetric complications, communicable and non-communicable diseases as well as with birth weight. These are mostly avoidable determinants so as to reduce these mortalities. INTRODUCTION delivery, poor hygiene during delivery and first critical hours after birth and lack of newborn care.2 Several factors such as women’s status in society, their nutritional status at the time of conception and maternal weight gain according to gestation period, early childbearing, too many closely spaced pregnancies and harmful practices such as inadequate cord care, letting the baby stay wet and cold, discarding colostrum and feeding other food, are deeply rooted in the cultural fabric of societies and interact in ways that are not always clearly understood.3 Perinatal mortality includes both still births (SB) and early neonatal deaths of babies weighing over 1000 g at birth, expressed as ratio per 1000 live births. Perinatal period is counted from the 28th week of gestation to the 7th day after birth.1 Neonatal deaths and stillbirths stem from poor maternal health, inadequate care during pregnancy, inappropriate management of complications during pregnancy and IndianJournals.com IndianJournals.com 109 Ramesh Chander, Puja Vimesh, Shyam Singh The main culprit is intra-uterine growth retardation or preterm birth and the complications stemming from it. There is, however, no doubt that maternal health and nutrition at conception are important determinants of weight at birth, neonatal health and frequency and severity of complications.4 Maternal problems such as diabetes, anaemia, hypertension, cardiac, respiratory, renal problems, contribute to adverse pregnancy outcomes and thus to maternal mortality (MM), SB, and early neonatal mortality (ENNM).5,6 India of 25 per 1000 live births and statistically reached by the formula 4PQ/L2 (where P is the percentage of positive character and Q=100–P and L is the allowable error; which in the present study was kept at 20%). Our study population was 1002 pregnant mothers who were registered in three Community Health Centres (CHCs) namely R.S. Pura, Kotbhalwal and Dansal, attached with the Postgraduate Department of Community Medicine of Government Medical College Jammu, one Maternity and Child Health (MCH) Centre, one Postpartum Centre and the Gynaecology and Obstetric (OBG) Department of SMGS Hospital of Govt. Medical College Jammu, which is the tertiary referral hospital of J&K. Only those mothers, who were having more than 22 weeks of gestation, gave written consent, belonged to a stable population and assured that they will stay in the respective residence/area for at least 1 month after delivery, were selected for the study. www.IndianJournals.com Members Copy, Not for Commercial Sale Our survey team consisted of interns (depending on the posted interns on monthly rotational basis in community medicine, Department of Govt. Medical College, Jammu) alongwith paramedical staff of the department, rural and urban health centres. The team was divided into five groups. They were given 3 days training for the job and distributed as : 3 intern and 3 paramedical staff for OBG wards and labour rooms including gynaecological operation theatre as well as neonatology ICU, one for each postpartum centre/MCH centre/different CHCs and the remaining interns alongwith the rest of the paramedical staff were given 2 mohallas/blocks in each city and immediate peripheries. Surveyors who were posted in medical centres were full-time data collectors (from 8 am to 4 pm), while those posted for community survey were instructed to visit homes of registered pregnant mothers on weekly basis. All the pregnant women were followed from 22nd week of gestation till 28 days after delivery. These mothers were all those who delivered in the above mentioned medical institutions/homes. The surveyors had the records of contact numbers of the target population, by which they could track the families. A pre- Downloaded From IP - Prolonged labour or early rupture of membranes cause infections in mothers and babies. However, babies are more susceptible than mothers and infections in infants are more difficult to detect. It is estimated that 26% of newborn infants who die, do so as a result of infections that occur around birth. Still birth is a professional and lay term that refers to a dead born foetus. Intrauterine death occurs either before onset of labour (antepartum death) or during labour (intrapartum death). Foetuses may die intra utero, before onset of labour, because of pregnancy complications or maternal diseases.9 INTRODUCTION Of the 1002 registered mothers, 196 mothers were lost in attrition (97 refused to co-operate or had to leave earlier and 99 lost during follow up). However, these losses did not bias the results significantly as the remaining 806 offered full co- operation and continued with us for the prospective study. 95.242.1.93 on dated 24-Oct-2024 Complications during birth, such as obstructed labour and foetal mal-presentation are common causes of perinatal death in the absence of adequate obstetric care. Birth asphyxia and trauma often occur together and it is, therefore, difficult to obtain separate estimates. In the most severe cases, the baby dies during birth or soon after, due to damage to the brain and other organs. Less severe asphyxia and trauma will cause disability.7 It is estimated that in developing countries asphyxia causes around seven deaths per 1000 births, whereas in developed countries this proportion is less than one death per 1000 births. The majority of deaths occur soon after birth, some just before birth.8 MATERIALS AND METHODS For study purpose and calculations the following definitions were adhered to: LIVE BIRTH: It is the complete expulsion or extraction from its mother of a product of conception, irrespective of the duration of the pregnancy, which after such separation, breathes or shows any other evidence of life, such as beating of the heart, pulsation of the umbilical cord, or definite movement of voluntary muscles, whether or not the umbilical cord has been cut or the placenta is attached; each product of such a birth is considered live born.10 3 on dated 24-Oct-2024 The variables included for study purpose were: maternal age, parity, education of mother, socio-economic status of the family, nutritional status (anaemia) of mother, intranatal causes, lie and presentation of foetus, method of conduction of delivery, birth weight and other antenatal and intranatal infections as well as indirect causes. These are depicted in different tables and figures. The data of mortality statistics and the variables concerned was collected sequentially, summarised, entered and analysed using SPSS version 17. For study purpose and calculations the following definitions were adhered to: MATERIALS AND METHODS The present study was a community-based prospective cohort study in Jammu city of J&K state. The city is the winter capital of J&K state. Its population is 6.5 lakhs as per the latest census. The study spread over from 1st January 2011 to 31st December 2011. Sample size was calculated on the basis of previous prevalence of Perinatal Mortality Rate (PNMR) of Urban Vol. 2, No. 2, July, 2012 110 Distribution and Determinants of Perinatal Mortality in Jammu City of India Distribution and Determinants of Perinatal Mortality in Jammu City of India tested, semi-structured, open-ended performa (covering baseline data of mothers, description of still births, early neonatal and late neonatal deaths including the concerned determinants) was introduced to the mothers after deaths of their babies in medical institutions or at their homes. However, care was taken in case of neo-natal deaths, the visitors to go for condolence, but the introduction of a questionnaire was delayed for about a week to give honour to the mourning period and offer lead time for the mother to come out of the shock. complete expulsion or extraction from its mother of a product of conception, weighing 1000 g (28 weeks gestation or more); the death is indicated by the fact that after such separation the foetus does not breathe or show any other evidence of life, such as beating of the heart, pulsation of the umbilical cord or definite movement of voluntary muscles. The neonatal period begins with birth and ends 28 complete days after birth. Neonatal deaths may be subdivided into early neonatal deaths, occurring during the first 7 days of life (0-6 days), and late neonatal deaths, occurring after the 7th day but before the 28th day of life (7-27 days).12 Total births were considered as denominator for calculating mortality rates. Downloaded From IP - 195.242.1.93 on dated 24-Oct-2024 The variables included for study purpose were: maternal age, parity, education of mother, socio-economic status of the family, nutritional status (anaemia) of mother, intranatal causes, lie and presentation of foetus, method of conduction of delivery, birth weight and other antenatal and intranatal infections as well as indirect causes. These are depicted in different tables and figures. The data of mortality statistics and the variables concerned was collected sequentially, summarised, entered and analysed using SPSS version 17. Journal of Research in Medical Education & Ethics 111 RESULTS In all, there were 806 pregnant mothers who participated in our study. Consequently, out of total 806 births, 764 (948 per 1000 births) were live born, 11 SB (14 per 1000 births), 27 (33 per 1000 births) early neonatal deaths, 38 (47 per 1000 births) perinatal deaths, 4 (5 per 1000 births) late neonatal deaths and 31(38 per 1000 births) were neonatal deaths [Table 1]. In all, there were 806 pregnant mothers who participated in our study. Consequently, out of total 806 births, 764 (948 per 1000 births) were live born, 11 SB (14 per 1000 births), 27 (33 per 1000 births) early neonatal deaths, 38 (47 per 1000 births) perinatal deaths, 4 (5 per 1000 births) late neonatal deaths and 31(38 per 1000 births) were neonatal deaths [Table 1]. Journal of Research in Medical Education & Ethics www.IndianJournals.com Members Copy, Not for Commercial Sale STILLBIRTH/FOETAL DEATH: It is death prior to the STILLBIRTH/FOETAL DEATH: It is death prior to the Table 1: Outcome of birth of study subjects Outcome Frequency Percent Cumulative Percent Per 1000 births Live birth 764 94.8 94.8 948 Still birth 11 1.4 96.2 14 Early neonatal mortality 27 3.3 99.5 33 Late neonatal mortality 4 0.5 100.0 4 Total 806 100.0 Table 1: Outcome of birth of study subjects 111 Ramesh Chander, Puja Vimesh, Shyam Singh Table 2a: Age of mother and outcome of birth: Cross-tabulation Age groups Outcome of birth Frequency (year) Live birth Still birth ENM LNM 15–19 58 2 3 1 64 (7.9%) 20–29 336 2 6 3 347 (43.1%) 30–39 345 5 15 1 366 (45.4%) 40–49 25 2 2 0 29 (3.6) Total 764 11 26 5 806 (100%) ENM: Early neonatal mortality LNM: Late neonatal mortality Table 2a: Age of mother and outcome of birth: Cross-tabulation Age groups Outcome of birth Table 2a: Age of mother and outcome of birth: Cross-tabulation Downloaded From IP - 195.242.1.93 on dated 24-Oct-2024 ENM: Early neonatal mortality, LNM: Late neonatal m Table 2b: Age of mother and outcome of birth: Chi- Square Tests Test applied Value df Asymptotic Significance Pearson Chi-Square 17.090* 9 0.047 Likelihood Ratio 14.049 9 0.121 Linear-by-Linear 0.237 1 0.626 Association 10 cells (62.5%) expf<5, Min exp=0.18 Table 3a: Gravida and outcome of birth: Cross- tabulation Gravida Outcome of birth Total Live Still ENM LNM birth birth G1–G3 229 4 3 0 236 G4–G6 321 4 9 3 337 G7–G9 202 2 12 3 219 >G9 11 1 2 0 14 Total 763 11 26 6 806 ENM: Early neonatal mortality, LNM: Late neonatal mortality Table 3b: Gravida and outcome of birth: Chi-Square Tests Test applied Value df Asymptotic Significance Pearson Chi-Square 19.672 9 0.020 Likelihood Ratio 17.428 9 0.042 Linear-by-Linear 11.254 1 0.001 Association 9 cells (56.3%) expf<5, Min exp=0.10 Table 2b: Age of mother and outcome of birth: Chi- Table 2b: Age of mother and outcome of birth: Chi- Square Tests 93 on dated 24-Oct-2024 Table 2b: Age of mother and outcome of birth: Chi- Square Tests Test applied Value df Asymptotic Significance Pearson Chi-Square 17.090 9 0.047 Likelihood Ratio 14.049 9 0.121 Linear-by-Linear 0.237 1 0.626 Association 10 cells (62.5%) expf<5, Min exp=0.18 As far as the sex of the new born was concerned, out of the total 411 males and 395 females; SB in study subjects were four males vs nine females; early neonatal deaths were 24 vs 9 and late neonatal deaths were 3 vs 4. www.IndianJournals.com Members Copy, Not for Commercial Sale Relationship of sex of the baby and these mortalities were significant; mortalities were higher in male children (P<0.03, df 3 at 95% CI level) [Tables 4a and 4b]. www.IndianJournals.com Members Copy, Not for Commercial Sale As far as age distribution of mothers was concerned 15- 19, 20-29, 30-39 and 40-49 years age-group mothers were 64 (7.9%), 347 (43.1%), 366 (45.4%) and 29 (3.6%), respectively. The relationship of perinatal and neonatal mortality rate with different age groups was significant (P<0.04, df 8 at 95% CI); PNM being highest at 15-19 years and 40-45 years age group [Tables 2a and 2b]. PERINATAL MORTALITY RATE: Still births (death of foetus from 28 weeks of gestation) and neonatal deaths in the first week of life per thousand births.11 As far as the gravida status of the mother was concerned, all mortality rates increased beyond G4 and this relationship was significantly associated with PNM and NNM (P<0.02, df 9, 95% CI) [Tables 3a and 3b]. www.IndianJournals.com Members Copy, Not for Commercial Sal 2, July, 2012 112 Distribution and Determinants of Perinatal Mortality in Jammu City of India Table 5a: Education of mother and outcome of birth: Cross-tabulation Education Outcome of birth Total (in years) Live Still ENM LNM birth birth Illiterate 196 2 9 1 208 <10 years 189 2 6 1 198 10–14 206 3 4 2 215 >16 years 168 4 7 1 180 ENM: Early neonatal mortality, LNM: Late neonatal mortality Table 6b: Monthly Income of family (INR) and outcome of birth: Chi-Square Tests Test applied Value df Asymptotic Significance Pearson Chi-Square 26.053 9 0.002 Likelihood Ratio 18.684 9 0.028 Linear-by-Linear 7.569 1 0.006 Association 9 cells (56.3%) expf<5, Min exp=0.27 Table 6b: Monthly Income of family (INR) and outcome of birth: Chi-Square Tests Table 5a: Education of mother and outcome of birth: Cross-tabulation Coming to maternal anaemia and mortalities, the association was significant (P<0.033, df 9 at 95% CI) [Tables 7a and 7b]. Downloaded From IP - 195.242.1.93 on dated 24-Oct-2024 Table 5b: Education of mother and outcome of birth: Chi-Square Tests Test applied Value df Asymptotic Significance Pearson Chi-Square 4.502 12 0.973 Likelihood Ratio 4.752 12 0.966 Linear-by-Linear 0.005 1 0.944 Association 12 cells (60.0%) expf<5, Min exp=0.03 Coming to family income and mortality rates, higher income groups had lower mortality rates and vice-versa. This association was highly significant (P<0.002, df 9 at 99% CI) [Tables 6a and 6b]. 4 Table 5b: Education of mother and outcome of birth: Chi-Square Tests Table 7a: Maternal anaemia and outcome of birth: Cross-tabulation Maternal Outcome of birth Total anaemia Live Still ENM LNM birth birth Nil 392 8 13 1 414 Mild 310 3 7 2 322 Moderate 58 0 6 2 66 Severe 4 0 0 0 4 ENM: Early neonatal mortality, LNM: Late neonatal mortality Table 7a: Maternal anaemia and outcome of birth: Cross-tabulation www.IndianJournals.com Members Copy, Not for Commercial Sale Downloaded Coming to family income and mortality rates, higher income groups had lower mortality rates and vice-versa. This association was highly significant (P<0.002, df 9 at 99% CI) [Tables 6a and 6b]. www.IndianJournals.com Members Copy, Not for Commercial Sal Table 4a: Sex of the baby and outcome of birth: Cross-tabulation Table 4a: Sex of the baby and outcome of birth: Cross-tabulation Sex of Outcome of birth Total the baby Live Still ENM LNM birth birth Male 380 4 24 3 411 Female 373 9 9 4 395 ENM: Early neonatal mortality, LNM: Late neonatal mortality y 195.242.1 Table 3a: Gravida and outcome of birth: Cross- tabulation Downloaded From IP - 195.2 Table 3a: Gravida and outcome of birth: Cross tabulation Gravida Outcome of birth Total Live Still ENM LNM birth birth G1–G3 229 4 3 0 236 G4–G6 321 4 9 3 337 G7–G9 202 2 12 3 219 >G9 11 1 2 0 14 Total 763 11 26 6 806 ENM: Early neonatal mortality, LNM: Late neonatal mortality ENM: Early neonatal mortality, LNM: Late neonatal mortality Table 4b: Sex of baby and outcome of birth: Chi- Square Tests Table 4b: Sex of baby and outcome of birth: Chi- Square Tests Test applied Value df Asymptotic Significance Pearson Chi-Square 8.635 3 0.035 Likelihood Ratio 8.939 3 0.030 Linear-by-Linear 2.046 1 0.153 Association 2 cells (25.0%) expf<5, Min exp=3.43 ENM: Early neonatal mortality, LNM: Late neonatal mortality Table 3b: Gravida and outcome of birth: Chi-Square Tests Test applied Value df Asymptotic Significance Pearson Chi-Square 19.672 9 0.020 Likelihood Ratio 17.428 9 0.042 Linear-by-Linear 11.254 1 0.001 Association 9 cells (56.3%) expf<5, Min exp=0.10 Table 3b: Gravida and outcome of birth: Chi-Square Tests However, educational status and the mortalities under consideration were not significantly associated (P=0.973 at df 12) [Tables 5a and 5b]. Vol. 2, No. Journal of Research in Medical Education & Ethics www.IndianJournals.com Members Copy, Not for Commercial Sal ENM: Early neonatal mortality, LNM: Late neonatal mortality Table 7b: Maternal anemia and outcome of birth: Chi-Square Tests Table 7b: Maternal anemia and outcome of birth: Chi-Square Tests Test applied Value df Asymptotic Significance Pearson Chi-Square 18.237 9 0.033 Likelihood Ratio 14.372 9 0.110 Linear-by-Linear 2.895 1 0.089 Association 10 cells (62.5%) expf<5, Min exp=0.02 Table 6a: Monthly Income of family (INR) and outcome of birth: Cross-tabulation Income Outcome of birth Total in INR Live Still ENM LNM birth birth <5000 50 2 8 0 60 5000–10000 318 6 9 2 335 10000–50000 353 3 8 3 367 >50000 43 0 1 0 44 ENM: Early neonatal mortality, LNM: Late neonatal mortality Table 6a: Monthly Income of family (INR) and outcome of birth: Cross-tabulation Birth weight, PNM and NNM also have positive corelation and relationship is highly significant (P<0.001, df 6 at 99% CI) [Tables 8a and 8b]. Journal of Research in Medical Education & Ethics 113 Ramesh Chander, Puja Vimesh, Shyam Singh Table 8a: Birth weight and outcome of birth: Cross- tabulation Birth Outcome of birth Total weight Live Still ENM LNM birth birth < 2.5 kg 52 2 8 1 63 2.5-3.4 kg 378 7 11 1 397 > 3.5 kg 332 3 8 2 345 ENM: Early neonatal mortality, LNM: Late neonatal mortality Table 8a: Birth weight and outcome of birth: Cross- tabulation Table 9b: Type of Delivery and outcome of birth: Chi-Square Tests Table 9b: Type of Delivery and outcome of birth: Chi-Square Tests Test applied Value df Asymptotic Significance Pearson Chi-Square 32.059 6 0.000 Likelihood Ratio 17.534 6 0.008 Linear-by-Linear 0.068 1 0.794 Association 7 cells (58.3%) expf<5, Min exp=0.15 Similarly, intranatal causes like birth injury, prolonged effort time, birth asphyxia and other obstetric complications were significantly associated with mortalities under study. The association was highly significant (P<0.01, df 12, at 99% CI) [Tables 10a and 10b]. Table 8b: Birth weight and outcome of birth: Chi- Square Tests Members Copy, Not for Commercial Sale Downloaded From IP - 195.242.1.93 on dated 24-Oct-2024 Table 8b: Birth weight and outcome of birth: Chi- Square Tests Test applied Value df Asymptotic Significance Pearson Chi-Square 22.387 6 0.001 Likelihood Ratio 15.040 6 0.020 Linear-by-Linear 3.397 1 0.065 Association 6 cells (50.0%) expf<5, Min exp=0.31 Perinatal mortality is influenced by type of delivery, namely normal, instrumental or caesarean. The relationship is highly significant (P<0.0001, df 6, at 99.9% CI). www.IndianJournals.com Members Copy, Not for Commercial Sal The highest mortality was seen in instrumental delivery, followed by delivery with caesarean section and lowest by normal delivery [Tables 9a and 9b]. Table 9a: Type of Delivery and outcome of birth: Cross-tabulation Type of Outcome of birth Total delivery Live Still ENM LNM birth birth Normal 645 7 21 3 676 Instrumental 23 3 4 1 31 Caesarean 96 1 2 0 99 ENM: Early neonatal mortality, LNM: Late neonatal mortality py Downloaded From IP - 195.242.1.93 on dated 24-Oct-2024 Table 8b: Birth weight and outcome of birth: Chi- Square Tests Test applied Value df Asymptotic Significance Pearson Chi-Square 22.387 6 0.001 Likelihood Ratio 15.040 6 0.020 Linear-by-Linear 3.397 1 0.065 Association 6 cells (50.0%) expf<5, Min exp=0.31 Perinatal mortality is influenced by type of delivery, namely normal, instrumental or caesarean. The relationship is highly significant (P<0.0001, df 6, at 99.9% CI). The highest mortality was seen in instrumental Table 10a: Intranatal causes and outcome of birth: Cross-tabulation Intranatal Outcome of birth Total causes Live Still ENM LNM birth birth Birth injury 36 2 3 0 41 Asphyxia 39 1 1 0 41 Prolonged 34 0 5 0 39 labour time Obstetric 9 1 1 0 11 complications None 646 7 17 4 674 ENM: Early neonatal mortality, LNM: Late neonatal mortality Table 10a: Intranatal causes and outcome of birth: Cross-tabulation www.IndianJournals.com Members Copy, Not for Commercial Sa Do Table 11b:Non-communicable (Indirect causes) and outcome of birth: Chi-Square Tests Test applied Value df Asymptotic Significance Pearson Chi-Square 20.742 21 0.475 Likelihood Ratio 22.509 21 0.371 Linear-by-Linear 2.712 1 0.100 Association 22 cells (68.8%) expf<5, Min exp=0.16 Do Table 11b:Non-communicable (Indirect causes) and outcome of birth: Chi-Square Tests www.IndianJournals.com Members Copy, Not for Commercial Sale www.IndianJournals.com Members Copy, Not for Commercial Sale Downloaded Perinatal mortality is influenced by type of delivery, namely normal, instrumental or caesarean. The relationship is highly significant (P<0.0001, df 6, at 99.9% CI). The highest mortality was seen in instrumental delivery, followed by delivery with caesarean section and lowest by normal delivery [Tables 9a and 9b]. Table 9a: Type of Delivery and outcome of birth: Cross-tabulation Table 9a: Type of Delivery and outcome of birth: Cross-tabulation Type of Outcome of birth Total delivery Live Still ENM LNM birth birth Normal 645 7 21 3 676 Instrumental 23 3 4 1 31 Caesarean 96 1 2 0 99 ENM: Early neonatal mortality, LNM: Late neonatal mortality Table 10b: Intranatal causes and outcome of birth: Chi-Square Tests Test applied Value df Asymptotic Significance Pearson Chi-Square 26.362 12 0.010 Likelihood Ratio 18.224 12 0.109 Linear-by-Linear 4.229 1 0.040 Association 13 cells (65.0%) expf<5, Min exp=0.05 Table 10b: Intranatal causes and outcome of birth: Chi-Square Tests Vol. 2, No. 2, July, 2012 114 Table 12a: “TORCHES” infections of mother during pregnancy and outcome of birth: Cross- tabulation The association of indirect causes like diabetes, cardiac problems, hypertension etc with concerned mortalities was not significant (P=0.475, df 2 at 68% CI) [Tables 11a and 11b]. The association of indirect causes like diabetes, cardiac problems, hypertension etc with concerned mortalities was not significant (P=0.475, df 2 at 68% CI) [Tables 11a and 11b]. Journal of Research in Medical Education & Ethics www.IndianJournals.com Members Copy, Not for Commercial Sale TORCHES Outcome of birth Total Live Still ENM LNM birth birth Toxoplasmosis 47 2 3 1 53 Rubella 55 0 1 1 57 Cytomegalovirus 14 1 2 0 17 Herpes 1 0 1 0 2 Syphilis 5 0 0 0 5 None 642 9 19 2 672 ENM: Early neonatal mortality, LNM: Late neonatal mortality Downloaded From IP - 195.242.1.93 on dated 24-Oct-2024 Table 11a: Non-communicable (Indirect causes) and outcome of birth: Cross-tabulation Intranatal Outcome of birth Total causes Live Still ENM LNM birth birth Diabetes 92 3 7 1 103 Cardiac 31 1 1 1 34 problems Respiratory 61 0 1 0 62 Hypertension 49 1 0 0 50 Renal 53 1 1 0 55 None 389 5 15 1 410 Accidental 33 0 0 0 33 Others 56 0 2 1 59 ENM: Early neonatal mortality, LNM: Late neonatal mortality Table 11b:Non-communicable (Indirect causes) and outcome of birth: Chi-Square Tests Test applied Value df Asymptotic Significance Pearson Chi-Square 20.742 21 0.475 Likelihood Ratio 22.509 21 0.371 Linear-by-Linear 2.712 1 0.100 Association 22 cells (68.8%) expf<5, Min exp=0.16 Table 11a: Non-communicable (Indirect causes) and outcome of birth: Cross-tabulation oaded From IP - 195.242.1.93 on dated 24-Oct-2024 Table 11a: Non-communicable (Indirect causes) and outcome of birth: Cross-tabulation Intranatal Outcome of birth Total causes Live Still ENM LNM birth birth Diabetes 92 3 7 1 103 Cardiac 31 1 1 1 34 problems Respiratory 61 0 1 0 62 Hypertension 49 1 0 0 50 Renal 53 1 1 0 55 None 389 5 15 1 410 Accidental 33 0 0 0 33 Others 56 0 2 1 59 ENM: Early neonatal mortality, LNM: Late neonatal mortality Table 12b: “TORCHES” infections and outcome of birth: Chi-Square Tests Test applied Value df Asymptotic Significance Pearson Chi-Square 29.889 15 0.012 Likelihood Ratio 16.604 15 0.343 Linear-by-Linear 4.812 1 0.028 Association *18 cells (75.0%) expf<5, Min exp=0.01 Table 12b: “TORCHES” infections and outcome of birth: Chi-Square Tests www.IndianJournals.com Members Copy, Not for Commercial Sale DISCUSSION In our study the incidence of SBR was 14 per thousand births, ENNMR was 33 per 1000, PNMR 47 per thousand, LNNMR was 5 per thousand and NNMR was 38 per thousand births. All the mortality rates are inbetween the rates of developing countries and developed countries. In India SBR is 39 per thousand births, ENNMR, PNMR, NNMR are 33, 70, and 43 per 1000 live births, respectively.13 All the rates in our study in Jammu city are much lower in comparison to the rest of India, because Jammu city, where the study was conducted includes most posh areas of the state, as far as socio-economic status is concerned. In the European country, Italy, SBR, ENNMR, PNMR and NNMR are 3, 2, 5 and 3 per thousand live births.14 The respective rates of our study are much higher compared to Italy. In the The association of TORCHES infections and the mortalities under study was highly significant (P<0.01, df 15, 99% CI) [Tables 12a and 12b]. Journal of Research in Medical Education & Ethics 115 Ramesh Chander, Puja Vimesh, Shyam Singh African country, Egypt, the same mortality rates are 10, 16, 27 and 21 per 1000 live births.15 African country, Egypt, the same mortality rates are 10, 16, 27 and 21 per 1000 live births.15 problems, renal problems, hypertension, etc. Earlier studies by Berkketeig et al. and Davies et al. are in agreement with our results.5,6 However, the association of these indirect causes to mortality rates was not significant (P=0.475). This can be due to the reason that for these chronic non-communicable diseases with high prevalence rates in our target area, the sample size was smaller to elucidate true values. This was the weakness of our study. In our study the age of the mother and its relationship with mortalities under study is significant; PNM being highest at 15-19 years and 40-45 years age group. The results are consistent with other studies conducted by Ravelli et al. and Troe et al.16,17 Perinatal mortality is influenced by type of delivery, namely normal, instrumental or caesarean. The relationship is highly significant (P=0.0001). The highest mortality was due to instrumental delivery and lowest by normal delivery. Our results are in agreement to a study conducted by Mavalkar et al. in Hyderabad.7 CONCLUSION In the present study, PNMR and NNMR were significantly associated with maternal age, parity, socio- economic status, obstetric complications, communicable and non-communicable diseases as well as with birth weight. These are mostly avoidable determinants; thus by keeping these determinants in check mortality can be reduced. 3 on dated 24-Oct-2024 An association between maternal anaemia and mortalities was also significant (P=0.033). Studies conducted by Ferraz et al. have reported similar results.18 As far as the association of income and mortalities under study are concerned, the association is highly significant (P=0.002). Mavalankar et al. have reported similar results in their study in Hyderabad.7 REFERENCES 1. World Health Organization. WHO Mortality database. Geneva: World Health Organization; 2003. 1. World Health Organization. WHO Mortality database. Geneva: World Health Organization; 2003. Downloaded From IP - 195.242.1.9 In our study, the relationship of sex of the baby and these mortalities were significant; mortalities were higher in male children (P=0.03). The results of Bromen et al. and Nielson et al. are similar.19,20 The relationship of parity and mortalities under study revealed a ‘U’- shaped pattern, higher mortality in first deliveries, falls up to 2nd, 3rd and again starts rising afterwards. The studies conducted by Bai et al.21 and Ego et al.22 have a similar pattern of association of parity and mortalities under study. 2. World Health Organization. European Health for all databases. [Last updated 2012 Jan; last accessed 2012 Jan 02]. Available from: http://www.euro. who.int/en/what-we-do/data-and-evidence/databases /european-health-for-all-database-hfa-db2 3. The World Health Report. Make every mother and child count. Geneva: World Health organization; 2005. 4. United Nations Children’s Fund/World Health Organization. Low birth weight: country, regional and global estimates. New York: UNICEF; 2004. In our study, low birth weight neonates were having higher mortalities as compared to normally weighing babies. The association was highly significant (P=0.001). Other surveyors, Glausson et al., Cowan et al., Figuras et al., Gardosi et al. showed results with total agreement to our study.23-26. 5. Davis AM, Dunlop E. Hypertension in pregnancy. In: Barron SL, Thomson AM editors. Obstetric Epidemiology. London Academic Press; 1983. pp. 167–207. Intranatal causes like birth injury, prolonged effort time, birth asphyxia and other obstetric complications were significantly associated with mortalities under study. The association was highly significant (P=0.01). Our results are supported by studies conducted by Cole et al.27 Mortality rates under study were higher comparatively, in mothers having complications like diabetes, cardiac 6. Bakketeig LS, Hoffman HJ, Oakley ART. Perinatal Mortality. In: Bracken MB editor. Perinatal epidemiology. New York: Oxford University Press; 1984. pp. 99–151. 7. Mavalanlar DV, Trivedi CR, Gra RH. Levels and risk factors for perinatal mortality in Ahmedabad, India. Bull World Health Organ 1991; 69:435–42. Vol. 2, No. 2, July, 2012 116 Distribution and Determinants of Perinatal Mortality in Jammu City of India Distribution and Determinants of Perinatal Mortality in Jammu City of India Distribution and Determinants of Perinatal Mortality in Jammu City of India 8. World Health Organization. Neonatal and perinatal mortality: Country, regional and global estimates. Geneva: World Health Organization; 2006. [last accessed 2012 Jan 02]. REFERENCES Available from: http:// whqlibdoc.who.int/publications/2006/9241563206_ eng.pdf 19. Bromen K, Jocket KH. Change in male proportion among new born infants. Lancet 1997; 349:804–5. 20. Nielsen BB, Liljestrand J, Hedegaard M, Thilsted H, Joseph A. Reproductive Pattern, perinatal mortality and sex preference in rural Tamil Nadu, South India: Community based cross-sectional study. British Medical Journal 1997; 314:1521–4. 9. World Health Organization. International classi- fication of diseases and related health problems. 10th revision, Vol. 2. Geneva: World Health Organization; 1993. 21. Bai J, Wong FW, Bauman A, Mohsin M. Parity and pregnancy outcomes. Am J Obstet Gynecol 2002; 186:274–8. 10. World Health Organization. Reported information on mortality statistics – 2005: Table 3. [Last accessed 2012 Jan 02]. Available from: http://www.who.int/ healthinfo/mort2005survey/en/index.html 22. Ego A, Subtil D, Grange G, Thiebaugeorges O, Senat MV, Vayssiere C, et al. Should parity be included in customized foetal weight, standards for indentifying small for generational age babies? Results from a French multicentre study. BJOG 2008; 115:1256– 64. 24 11. United Nations Department of Economic and Social Affairs. Population and vital statistics report: Statistical papers series A. Vol. LIV, No. 3. New York: United Nations; 2002. 23. Clausson B, Gardosi J, Francis A, Cnattingius S. Perinatal outcome in SGA births defined by customized versus population based birth weight standards. BJOG 2001; 108:830–4. www.IndianJournals.com Members Copy, Not for Commercial Sale Co e c a Sa e .93 on dated 24-Oct-20 12. National Centre of Health Statistics. Infant and Perinatal Mortality in England and Wales: Vital and Health Statistics, Analytical Studies, Series 3, No. 12. Washington DC: United States Department of Health, Education and Welfare; 1968. 24. McCowan LM, Harding JE, Stewart AW. Customized birth weight centiles predict SGA pregnancies with perinatal morbidity. BJOG 2005; 112:1026–33. Members Copy, Not fo ded From IP - 195.242. 13. Department of Measurement and Health Information System (MHI). Evidence and information for policy (EIP): WHO under 5 Mortality - Unpublished country estimate computed for World Health Report 2005. Geneva: WHO; 2004. 25. Figueras F, Figueras J, Meier E, Eixarch E, Coll O, Gratacos E, et al. Customized birth weight percentiles accurately predict perinatal morbidity. Arch Dis Child Faetal Neonatal Ed. 2007; 92:277– 80. Downloa 14. United Nations. United Nations demographic year book 2000. New York: United Nations; 2002. 26. Gardosi J, Francis A. Adverse pregnancy outcome and and association with smallness for gestational age by customized and population based birth weight percentiles. Journal of Research in Medical Education & Ethics REFERENCES Am J Obstet Gynecol 2009; 201:28.e1– 8. 15. World Health Organization. WHO Mortality data base. Geneva: World Health Organization; 2003. 16. Raveli AC, Eskes M, Tromp M, van Huis AM, Steegers EA, Tamminga P, et al. Perinatal mortality in the Netherlands 2000–2006: Risk factors and risk selection. Ned Tijdschr Geneeskd 2008; 152:2728– 33. 27. Cole SK. Scottish perinatal surveys: Results from 1984. In: Proceedings of the International collabo- rative effort on perinatal and infant mortality, Washington DC, 18 November 1985, Vol. II. Hyatt- sville, MD, National Center for Health Statistics, 1988, pp. Il: 29. 17. Troe EJ, Bos V, Deerenberg IM, Mackenbach JP, Joung IM. Ethnic differences in total and cause effective infant mortality in Netherlands. Paediatr Perinatal Epidemiol 2006: 20:140–7. 18. Ferras EM, Gray RH. A case control study of stillbirths in North East Brazil. Int J Gynaecol Obstet 1991; 34:13–9. Received: 05.01.2012 Accepted: 05.04.2012 Received: 05.01.2012 Accepted: 05.04.2012 117 Journal of Research in Medical Education & Ethics
https://openalex.org/W2346931337	https://equityhealthj.biomedcentral.com/counter/pdf/10.1186/s12939-016-0358-0	English	null	Qualitative study on the shifting sociocultural meanings of the facemask in Hong Kong since the severe acute respiratory syndrome (SARS) outbreak: implications for infection control in the post-SARS era	International journal for equity in health	2,016	cc-by	13,802	Qualitative study on the shifting sociocultural meanings of the facemask in Hong Kong since the severe acute respiratory syndrome (SARS) outbreak: implications for infection control in the post-SARS era Judy Yuen-man Siu Abstract Background: The clinical importance and efficacy of facemasks in infection prevention have been documented in the international literature. Past studies have shown that the perceived susceptibility, the perceived severity of being afflicted with life-threatening diseases, and the perceived benefits of using a facemask are predictors of a person’s use of a facemask. However, I argue that people wear a facemask not merely for infection prevention, and various sociocultural reasons have been motivating people to wear (and not wear) a facemask. Facemasks thus have sociocultural implications for people. Research on the sociocultural meanings of facemasks is scant, and even less is known on how the shifting sociocultural meanings of facemasks are related to the changing social environment, which, I argue, serve as remarkable underlying factors for people using (and not using) facemasks. As new infectious diseases such as avian influenza and Middle East Respiratory Syndrome have been emerging, threatening people’s health worldwide, and because facemasks have been documented to have substantial efficacy in the prevention of infection transmission, understanding the sociocultural meanings of facemasks has significant implications for public health policymakers and health care providers in designing a socially and culturally responsive public health and infection control policy for the community. Methods: A qualitative research design involving the use of 40 individual, in-depth semistructured interviews and a phenomenological analysis approach were adopted. Results: The sociocultural meanings of the facemask have been undergoing constant change, from positive to negative, which resulted in the participants displaying hesitation in using a facemask in the post-SARS era. Because it represents a violation of societal ideologies and traditional Chinese cultural beliefs, the meanings of the facemask that had developed during the SARS outbreak failed to be sustained in the post-SARS era. Conclusion: The changes in meaning not only influenced the participants’ perceptions of the facemask but also influenced their perceptions of people who use facemasks, which ultimately influenced their health behavior, preventing them from using facemasks in the post-SARS era. These findings have critical implications for designing a culturally responsive infection prevention and facemask usage policy in the future. Keywords: Health behavior, Community and Public Health, Behavioral change, Facemask, Sociocultural meaning, Hong Kong Keywords: Health behavior, Community and Public Health, Behavioral change, Facemask, Sociocultural meaning, Hong Kong Correspondence: judy.ym.siu@polyu.edu.hk Department of Applied Social Sciences, Faculty of Health and Social Sciences, The Hong Kong Polytechnic University, Hung Hom, Hong Kong © 2016 Siu. Siu International Journal for Equity in Health (2016) 15:73 DOI 10.1186/s12939-016-0358-0 Siu International Journal for Equity in Health (2016) 15:73 DOI 10.1186/s12939-016-0358-0 Open Access © 2016 Siu. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background g The outbreak of Severe Acute Respiratory Syndrome (SARS) in 2003 marked a critical turning point for pub- lic health development in Hong Kong. The SARS out- break caused 1755 infection cases, with 299 deaths in Hong Kong, yielding a fatality ratio of 17 % [1]. The out- break has facilitated the development of public health at the institutional level, but it has also enabled the Hong Kong population to become aware of the importance of personal hygiene and infection prevention at the com- munity level. Facemask use, which was a remarkable social and health behavior in Hong Kong during the SARS outbreak, was widely recognized by the Hong Kong population for the first time. Since then, the Hong Kong population has become familiar with the use of facemasks in public spaces. Abstract Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Siu International Journal for Equity in Health (2016) 15:73 Page 2 of 16 Page 2 of 16 the objects. In a similar manner, the facemask also has symbolic implications for people. These implications in- fluence how people perceive facemasks, how people per- ceive those who use them, and how people respond to facemask use. These factors in turn affect people’s adop- tion of preventive health behavior regarding facemask use. Moreover, these symbolic implications undergo constant change because of the different and changing sociocultural environment. Facemasks carry different symbolic implications for people in different societies, and hence, different perceptions and interpretations exist regarding facemask use, resulting in different health behaviors among communities. For example, concerning the 2015 MERS outbreak in South Korea, the difference in attitude between South Koreans and the Hong Kong population toward facemask use not only generated cul- tural clashes between the two populations [7–10], but could also lead to different outcomes in epidemic con- trol. However, literature on the symbolic implications of facemasks for people is scant; how the mechanism of such changes in symbolic meaning is triggered remains unknown. Because of this research gap, this study inves- tigated the sociocultural meanings of facemasks in Hong Kong during the SARS outbreak and in the post-SARS era, showing how the changes in the symbolic implica- tions of the facemask have occurred. This study also ex- amined how these changing symbolic implications can influence people’s adoption (and repudiation) of face- masks when used as a preventive health measure. Significance The clinical importance and efficacy of facemask infec- tion prevention are documented in the international lit- erature [2, 3]. Past studies have shown that the perceived susceptibility, the perceived severity of being afflicted with life-threatening diseases, and the perceived benefits of using a facemask are predictors of people’s facemask use [2]. However, I argue that people use a facemask not only for infection prevention. Various sociocultural reasons motivate people to use (and not use) a facemask, which has sociocultural implications for people. However, research on the sociocultural meanings of facemasks is scant, and even less is known on how the shifting sociocultural meanings of facemasks are related to the changing social environment, which, I argue, serve as critical underlying factors for people’s use (and the absence of use) of a facemask. Because of the effects of globalization and well-developed international transportation, epidemic outbreaks are no longer con- fined to a single country. As new infectious diseases such as avian influenza and Middle East Respiratory Syndrome (MERS) have continued emerging, threaten- ing people’s health worldwide, and because facemasks have been documented to have significant efficacy in the prevention of infection transmission, therefore, under- standing the sociocultural meanings of the facemask has critical implications for public health policymakers and health care providers in designing a socially and cultur- ally responsive public health and infection control policy for the community. Methods For this study, a qualitative research design involving individual, in-depth semistructured interviews was adopted. A total of 40 participants were recruited from and interviewed at one private-practice primary health care clinic during the peak season of influenza A (H3N2) in January 2015 in Hong Kong through purposive sampling. A phenomenological approach was adopted for data analysis. Ethics, consent, and permission Ethics, consent, and permission The Committee on the Use of Human and Animal Subjects in Teaching and Research at Hong Kong Baptist University approved this study. Permission for participant recruitment and interview implementation was sought prior to the study from the owner of the pri- mary health care clinic, who is a private-practice general practitioner. Participation in the study was entirely voluntary. I provided all the participants with an infor- mation sheet written in their native language—tradi- tional Chinese—informing them of the purpose, nature, and procedure of the study. Verbal explanations and clarifications were also provided. I obtained a signed consent form from each participant, and all participants Symbolic representation, or symbolism, is a popular area of study in the humanities. Medical instruments such as a stethoscope and white coat often convey sym- bolic implications [4, 5], affecting people’s identity, emo- tions, and behavior [5, 6]. These symbolic implications influence how people perceive others who are associated with these objects, which influences how they react to Siu International Journal for Equity in Health (2016) 15:73 Page 3 of 16 of Hong Kong. According to the official recommenda- tions of medical professionals, my family aimed to obtain three-layered surgical facemasks because N95 respirators had been depleted during the outbreak. were aware that they were free to withdraw from the study without having it affect their current and future treatment received at the clinic. I conducted all interviews in an examination and pro- cedure room in the clinic to protect the participants’ privacy. The interviews were audio-recorded with the participants’ consent. To further protect the participants’ privacy and confidentiality, no participant identifiers were mentioned during the interviews. I maintained all data and field notes in locked cabinets and treated them with strict confidentiality. To further minimize the risk of potential identification, I represented each participant with a code in the data and interview transcripts. The digital audio recordings of the interviews were destroyed once the interviews were transcribed. p g Similar to other Hong Kong residents, I engaged in facemask scrambling with my family, and used a face- mask at all times in public spaces throughout the SARS outbreak, from March to July of 2003. Moreover, at the community level, different social institutions also exerted a strong mode of control over my facemask use during those months. Reflexivity of facemask use My knowledge and understanding of the use of face- masks can be traced to the SARS outbreak in 2003, when I was a Masters student conducting research in the Department of Anthropology at The Chinese Uni- versity of Hong Kong. In the beginning of the SARS out- break, most people in Hong Kong, myself included, were unaware of the importance of using a facemask. Face- mask use was extremely rare for people in Hong Kong and could be perceived as a display of strange behavior, and the person using it could be viewed as a highly in- fectious patient warranting social seclusion. Although a few people had been observed using a facemask in pub- lic spaces at the beginning of the outbreak, they were often secluded. In one case, I encountered a woman who was using a facemask, and my immediate response was to walk away from her. As a public health researcher, I am aware that the pri- mary purpose of facemasks is infection containment and that they should be used by those who are suspected of or have been diagnosed with an infectious disease. I have been perceiving facemask use as a civic responsibility when one falls ill. However, as an anthropologist, I am also aware that the perceptions of facemask use are never static for one community, but rather, these mean- ings undergo constant change over time. Because of dif- ferent life encounters and experiences, people would have varying perceptions of facemasks, and these per- ceptions can influence whether they use a facemask, akin to what I have been experiencing in my life also influen- cing my perception of facemasks, and in turn influencing my facemask use behavior. Instead of representing sick people without a facemask as not exercising their civic responsibilities, this study examined the shifting cultural meanings of facemask use in Hong Kong from the SARS outbreak in 2003 to the influenza A (H3N2) outbreak in January 2015 and how these shifting cultural meanings could have implications for infection control in the future. The facemasks that were being used in the beginning of the SARS outbreak did not meet clinical standards. Rather than a surgical facemask, many other types of facemasks (e.g., paper and active-carbon masks) emerged in the market. Ethics, consent, and permission All students were required to use a face- mask in schools and in universities during the outbreak. Failure in using a facemask in the university that I was studying would mean that these noncomplying students were to be prohibited from attending lectures. Using a facemask during the SARS outbreak was widely per- ceived as an infection-preventive measure; furthermore, using a facemask in public areas was perceived as a new social norm, and a person would be subject to discrimin- ation if he or she failed to comply. In one case, I en- countered a neighbor in the lift who just used tissue to cover up her nose and mouth, and I immediately walked out from the lift and stared at her at the same time. My values and beliefs toward facemasks have changed since the SARS outbreak. Before the outbreak, I did not have any knowledge of using facemasks. My impres- sion of those who used a facemask was negative be- fore the outbreak, and I perceived that these people were either strange or mentally ill. The SARS outbreak, however, has changed my perceptions of facemask use and instilled in me more positive perceptions of this behavior. Consent to publish The participants were informed thoroughly on the use of their provided data in academic publications, with all the personal identifications removed to ensure privacy. Data collection How did you view people who used facemasks during the SARS outbreak? 8. How do you view a person using a facemask now? 9. Who needed to use a facemask during the SARS outbreak? 10. Who needs to use a facemask now? 1. Are you suffering from any respiratory symptoms today? 2. Did you use a facemask during the SARS outbreak in 2003? 3. Are you currently using a facemask? 4. Have you been residing in Hong Kong since the SARS outbreak in 2003 until today? 1. Are you suffering from any respiratory symptoms today? 11. Can you share your life story, experience and feelings during the SARS outbreak? 12. How do these experience and feelings affect you in the SARS outbreak? 2. Did you use a facemask during the SARS outbreak in 2003? 3. Are you currently using a facemask? The interview question guide was used throughout the interview process to guide the discussion and to ensure that the interviews did not deviate from the research questions. I conducted all of the interviews to maintain consistency and to ensure interview quality. This ap- proach also minimized the risk of data variations and data flaws that might have resulted from introducing an- other interviewer. Follow-up probing in the interviews was performed in response to the participants’ answers to collect additional in-depth data from the participants. All of the interviews were conducted in Cantonese Chin- ese, which is the native language of the participants as well as my own, to ensure free expression of the partici- pants’ views. The interviews were audio-recorded with the participants’ consent. The duration of each interview was 1 h and 10 min to 1 h and 35 min. Each participant was presented with a supermarket coupon of HK$100 as an acknowledgment of their participation to compensate them for their time. 4. Have you been residing in Hong Kong since the SARS outbreak in 2003 until today? To fulfill the sampling criteria and enable an investiga- tion into the shifting sociocultural meanings of facemask use since the SARS outbreak in 2003 until the influenza A (H3N2) outbreak in January 2015, the participants were required to answer “yes” for Questions 1, 2, and 4 and “no” for Question 3. Reflexivity of facemask use In a manner similar to that of most Hong Kong citi- zens, I first became aware of the importance of using a facemask in public spaces when medical scientists an- nounced the severity of the nosocomial outbreak of SARS at Prince of Wales Hospital, which was an epicen- ter of the outbreak, at a press conference. All of the pro- fessors at the Faculty of Medicine in The Chinese University of Hong Kong used facemasks at the press conference, which visually reinforced the severity of SARS. Alarmed by the severity of the outbreak, my family began to scramble for facemasks, as did the rest Siu International Journal for Equity in Health (2016) 15:73 Page 4 of 16 Data collection interview confidentiality and that their viewpoints would not affect their future consultations and treatment in the same clinic. The 40 participants were selected through purposive sampling, conducted in accordance with the following inclusion criteria, from a private-practice primary health care clinic in Hong Kong. Although past studies have documented that 17 interviews suffice for achieving data saturation [11], this study comprised 40 interviews to yield greater confidence in the data. In addition, data saturation was achieved using no new data and informa- tion that could be collected from the interviews [12]. To investigate the changing meaning of facemasks in Hong Kong from the SARS outbreak to the study period, the participants were selected based on their experience of facemask use during the SARS outbreak, but not in the post-SARS era as well as at the sampling venue, despite having experienced respiratory symptoms. In-depth semistructured interviews were conducted with individ- ual participants during the peak season of influenza A (H3N2) in January 2015, because sampling during this period ensured the efficient recruitment of suitable participants. An interview question guide was developed according to past studies on the use of facemasks as well as my personal long-time observations of the use of facemasks in Hong Kong. Regarding the investigation into the par- ticipants’ views on facemasks as well as their use of face- masks during and after the SARS outbreak, the interviews were conducted to examine the changes in the use and meaning of facemasks from the SARS out- break in 2003 until the post-SARS era in 2015. The interview questions included the following: 1. Why did you use a facemask during the SARS outbreak? 2. For how long have you not used a facemask? Why? 3. Did you use a facemask after the SARS outbreak? – If yes, what was the occasion? And why? – If no, why? 4. Why are you not using a facemask now? With the consent of the primary health care clinic, I visited the clinic for 14 consecutive days throughout the physician’s consultation hours to identify, recruit, and interview the participants. Those who did not use a facemask were approached, and they were asked the following questions pertaining to the sampling criteria, so that I could identify if they were suitable for the interviews: 5. How did you view facemasks during the SARS outbreak? 6. How do you view facemasks now? 7. Participants All 40 participants were patients at a private-practice primary care clinic. They were ethnically Hong Kong Chinese people comprising 29 women and 11 men aged between 22 and 70 years at the time of this study. To examine the changing meanings of facemasks in Hong Kong from the SARS outbreak until the study period, the participants were purposively sampled according to the following inclusion criteria: (a) They suffered from respiratory symptoms at the time of sampling, (b) they did not use a facemask at the sampling venue, and (c) they had used a facemask during the SARS outbreak in 2003. Totally, 17 participants claimed to be healthy and not suffering from chronic conditions, whereas the remaining 23 participants suffered from various chronic conditions, including hypertension, diabetes mellitus, gout, liver disease, and heart disease. These 23 partici- pants were receiving long-term follow-up treatment ei- ther in public hospitals or at the private clinic in which sampling was performed. All of the participants visited the clinic because of respiratory symptoms during the sampling period: 15 participants were diagnosed with in- fluenza, 21 with upper respiratory tract infections, 3 with bronchitis, and 1 with pneumonia. Major themes in the data were identified, which involved segmenting the interview transcripts into indi- vidual meaning units, collapsing them into categories, and determining themes through a process involving ab- straction and constant comparisons. A coding table was developed that identified themes, categories, and codes with supporting evidence in the form of interview quotes; this was achieved in line with the inductive cod- ing process to enable the discovery of patterns of behav- iors and thoughts [15]. Recurrent codes and themes were noted and highlighted. New thematic codes that emerged from the data were added to the coding table. Memos were used to record ideas and commentary during coding. The analytical procedures, coding, and findings were documented in the codebook to ensure the consistency and accuracy of the analyzed data. Data saturation was achieved. All the participants had experience with using a facemask during the SARS outbreak. However, most of them had not been using a facemask since the outbreak ended. Although a few participants had used a facemask for infection pre- vention at the onset of the influenza A (H1N1) outbreak in Hong Kong in 2009, none of them had used a facemask because of their respiratory symptoms after the SARS outbreak. Data collection To minimize potential disturbances of clinical opera- tions and participants, those who fulfilled the sampling criteria and who consented to the interview were inter- viewed immediately in another examination and proced- ure room within the clinic after they completed their consultation and received their prescription medication. Conducting the interviews after the participants had completed treatment and received their prescription also assured the participants that the interviews did not affect their treatment. The participants were assured of Siu International Journal for Equity in Health (2016) 15:73 Page 5 of 16 Page 5 of 16 Data analysis analysis, enabling the findings to be grounded in the data. Because data collection and analysis were con- ducted by a single researcher, coding and recoding of the transcripts were performed to ensure reliability and confirmability and to ascertain that the coding and categories were free of ambiguity, overlaps, and lack of clarity. Recoding was conducted 1 month after the first coding session as cross-checking to enhance the validity and reliability of the data and findings and to ensure the elimination of subjectivity and bias. To ensure that the interviews yielded the most data, a quick data analysis was performed during the interviews to ascertain the obtained knowledge and to determine areas that required further exploration [13]. A student assistant transcribed the interviews verbatim, and I sub- sequently translated the interview transcripts into English. Backtranslation was performed by another bilin- gual student assistant to ensure the translated interviews did not distort the original meaning of the participants. A phenomenological approach was adopted to analyze the interview data and to discover the patterns and structures of phenomena that are the lived experiences of people [14]. Using a phenomenological approach, this study examined the lived experiences of facemask use by the participants as well as their health perceptions and behaviors in response to infectious diseases since the SARS outbreak in 2003 until the influenza A (H3N2) outbreak in January 2015. A procedure involving intuit- ing, analyzing, and describing was conducted [14]. By learning the facemask usage experiences of the partici- pants, and by examining the structure of facemask usage by examining the participants’ experiences, the manifes- tations of the facemask usage phenomenon as a meaning of the experiences with Hong Kong’s sociocultural struc- tures were recognized and affirmed in the analytical process [14]. The meaning of facemask use as experi- enced by the participants was thus determined. Participants The shifting sociocultural meanings of facemask during the SARS outbreak and post-SARS era in Hong Kong, which are to be illustrated in Table 1 and in the themes below, explain the changing pattern in facemask using behavior among the participants. Facemask for infection prevention SARS has changed the culture of facemask use in Hong Kong. Before [the] SARS [outbreak], no one would use a facemask. I did not even know where I could buy a facemask before [the] SARS [outbreak]. However, when SARS hit, using a facemask was just like wearing clothes in public, and it was a norm in society. Everyone in Hong Kong wore a facemask during [the] SARS [outbreak]. If you did not wear a facemask, you would be given a dirty look and be discriminated against. No one dared not to wear a facemask at that time. The predominant reason for using a facemask during the SARS outbreak for all the participants was their con- cern regarding SARS infection prevention. One partici- pant recalled the following: SARS was so horrible. It was deadly, but highly transmittable. I could not ask other people to use a facemask before coughing and sneezing. Therefore, I wore a facemask to prevent myself from getting infected. This was the only thing that I could do as a prevention against SARS. Facemask use as a new social norm Facemask use as a new social norm Using a facemask was a new social norm in Hong Kong during the SARS outbreak, and all of the participants perceived this social norm to be a powerful motivator for them in using a facemask. The participants com- monly perceived that failing to use a facemask in public during the SARS outbreak could make them potential victims of discrimination. One participant noted the following: [Female participant (P16), 59 years old] [Female participant (P16), 59 years old] Meaning of facemasks during the SARS outbreak Meaning of facemasks during the SARS outbreak All of the participants had used a facemask during the SARS outbreak. In addition to practical concerns regard- ing infection prevention, sociocultural controlling mecha- nisms also motivated the participants to use a facemask during the SARS outbreak. Using a facemask during the outbreak was a new social norm and was perceived as a show of support for health care providers and a display of civic responsibility. Using a facemask also enabled the par- ticipants to achieve a sense of control and security in the pervasive social atmosphere of uncertainty. [Male participant (P5), 52 years old] [Female participant (P2), 56 years old] Rigor After the interviews were translated from Chinese to English, backtranslation from English to Chinese was performed to ensure that the translated interview quota- tions did not distort the original meaning of the partici- pants. To avoid researcher bias, motivation, or interest in the analysis, and to establish credibility and neutrality, direct interview quotations were included in the coding All of the sampled participants were employed in different sectors, including administrative and executive, sales and retail, commerce and finance, civil service, education, social welfare, hotel management, and infor- mation technology sectors. Siu International Journal for Equity in Health (2016) 15:73 Page 6 of 16 Table 1 Comparison of the sociocultural meanings of facemasks during the SARS outbreak and post-SARS era in Hong Kong During the SARS outbreak In the post-SARS era Perceived function of facemasks Primarily used for infection prevention Primarily used for infection prevention Sociocultural meaning of facemasks Perceived as a new social norm Perceived as a sign of people with negative attributes Perceived as a form of civic responsibility Perceived as a medium for inviting stigmas and teasing Perceived as symbolic support for health care providers Perceived as a sign of weakness Perceived as a tool for achieving a sense of control and security Perceived to hinder recovery Perceived nature of facemasks Medical professionals’ advice was critical for the participants’ choice of an appropriate facemask Toxic to skin [Female participant (P16), 59 years old] [Male participant (P10), 42 years old] The mass media played a critical role in normalizing the use of facemasks and in reinforcing the new socio- cultural norm of using a facemask in public. Either they achieved these conditions by using words implying moral judgment in their reports, condemning those who did not use a facemask, or blaming and stigmatizing those who were infected with SARS. One participant in- dicated the following: Wearing a facemask was a way to tell others that you were exercising civic responsibility and that you cared a lot about Hong Kong. You did not want to spread the virus to others, so you wore a facemask. You wanted to tell others that you were on the same team with other Hongkongers in fighting against SARS, so you wore a facemask. Wearing a facemask was a sign of solidarity and civic responsibility at that time. I think these meanings of wearing a facemask were more important, so a facemask was not merely for protecting you against SARS. It meant a lot more than that. News reports often referred to SARS as “super pneumonia” and referred to those who were infected with SARS as “super virus spreaders.” SARS patients were frequently regarded as spreading the deadly virus to others. With these reports, you would soon realize that if you were infected with SARS, then you would be labeled with these negative attributes. Many news reports also kept reporting about doctors’ advice on using facemasks, and these reports also negatively labeled those who did not wear a facemask in public as weird and lacking social consideration. [Female participant (P34), 52 years old] [Male participant (P26), 64 years old] [Male participant (P26), 64 years old] Facemask as symbolic support for health care providers Health care providers played a crucial role in motivating most of the participants to use a facemask during the SARS outbreak. Their advice to use a facemask during the SARS outbreak served as a powerful motivator for the participants to use a facemask. One participant indicated the following: [Female participant (P17), 48 years old] [Female participant (P17), 48 years old] During [the] SARS [outbreak], information on [the use of] facemasks bombarded us daily. Those “women’s television programs” often interviewed different doctors on the use of facemasks and also compared the advantages and disadvantages of each [Female participant (P2), 56 years old] Failure to conform to the new social norm of using a facemask in public during the SARS outbreak could have invited others to exercise social seclusion. One partici- pant indicated the following: Using a facemask in public spaces was perceived as a necessary SARS infection prevention measure for all of the participants during the SARS outbreak. One partici- pant added the following: In the beginning, I was very reluctant to wear a facemask, because I was worried that this would scare people away. However, no one was willing to talk to me if I did not wear one. My friends and colleagues maintained a great distance from me when I spoke. Others just ran away when they noticed that I was not wearing a facemask on the streets or during public transportation. I then recognized that it was a necessity to wear a facemask when I was outside my home. After I wore a facemask, my friends and Wearing a facemask was necessary when you were in public places during [the] SARS [outbreak]. You could never know who the hidden virus carriers were, but they could still spread the virus and infect others when they spoke, coughed, or sneezed. Moreover, you could never know if the places you went to had been contaminated by the SARS virus. If you did not wear a facemask in public, then you would be at a much higher risk of getting infected. Siu International Journal for Equity in Health (2016) 15:73 Page 7 of 16 colleagues no longer avoided me; others were willing to stand or sit next to me when I used public transportation. against. This new social norm contributed to the develop- ment of a novel definition of civic responsibility during the SARS outbreak, which caused nearly all of the partici- pants to perceive that using a facemask in public was a social responsibility. One participant related the following experience: [Male participant (P10), 42 years old] [Male participant (P10), 42 years old] [Female participant (P34), 52 years old] Those who failed to use a facemask in public during the SARS outbreak were perceived as lacking social responsibility. One participant stated the following: I think that those who did not wear a facemask were not being socially responsible. You could never know if you got infected [with SARS]; no symptoms would emerge during the 10-day incubation period, but you could infect others if you did not wear a facemask, not to mention that if a facemask was really useful for preventing the spread of SARS, wearing a facemask was just like showing your social responsibility and concern to others. You may be able to recover after getting infected of SARS, but you may kill others if you do not wear a facemask. My wife died of SARS because of these irresponsible people. [Female participant (P21), 45 years old] [Female participant (P21), 45 years old] [Female participant (P21), 45 years old] Photographs displaying people using facemasks in media reports were also a considerable force in reinfor- cing the new social norm of using facemasks in public. Those who failed to use facemasks were labeled as “abnormal,” and through such reports, the failure to use facemasks in public was transformed into a form of social deviance. One participant stated the following: During [the] SARS [outbreak], all the newspapers and television news headlines were about SARS. The photos in these reports often showed people wearing facemasks. These photos were really eyecatching because everyone in these photos was wearing facemasks. In the beginning, I did not think I needed to wear one. However, those who did not wear a facemask were portrayed as aliens or monsters in these news reports. Then I learned that I should wear a facemask. Facemask use as a display of civic responsibility p y p y With the new social norm of using a facemask in public during the SARS outbreak, all of the participants were motivated to use a facemask to avoid being discriminated Siu International Journal for Equity in Health (2016) 15:73 Page 8 of 16 infected with] SARS, but I just felt that wearing it was the only way to save my life. type of facemask. These doctors also mentioned how to select the most appropriate facemask, how to wear it, and how to use it effectively and hygienically in these programs. I encountered this information every day, and this made wearing a facemask a necessity at the time. infected with] SARS, but I just felt that wearing it was the only way to save my life. [Male participant (P27), 59 years old] [Male participant (P27), 59 years old] [Male participant (P27), 59 years old] The lack of information on this new infectious disease also intensified the sense of uncertainty among the par- ticipants during the SARS outbreak. Using a facemask was thus the only action they could adopt to overcome this sense of uncertainty. Another participant indicated the following: [Female participant (P38), 62 years old] [Female participant (P38), 62 years old] Because of the image of facemasks and the suggestion of health care providers to use them, the participants began to feel comfortable with and became motivated to use facemasks. Using facemasks was also perceived as a show of support for health care providers during the SARS outbreak. One participant expressed the following: I felt so helpless during [the] SARS [outbreak]. SARS was so mysterious that I really did not know how it could be transmitted. I just knew that SARS was extremely deadly, and it was highly contagious. Doctors and nurses still got infected, even though they used protective gowns and facemasks. People living in Amoy Gardens were all infected, even though they were not living together in the same flat. I could never know who a virus carrier was when I was on the streets. I could never know if the air contained the SARS virus as well. There was so much that was unknown about SARS, and there was so little that we could do and control. However, I still had to do something, and wearing a facemask was the only thing that I could do at the time. Facemask use as a display of civic responsibility It gave me a sense that I could at least have some control over my life. During [the time of] SARS, all medical professors, doctors, and nurses were wearing a facemask. Facemasks were just like a stethoscope and white coat, and it was a symbol of these health care heroes. Therefore, if they wore facemasks, I also felt confident with the efficacy of wearing one. Besides, I wanted to show my support for doctors and nurses; I thought wearing a facemask was the best way of showing my support for them, because I could then minimize the possibility of getting infected with SARS. If I got infected, I would increase their workload. Therefore, I wore a facemask throughout the outbreak, because I did not want to increase their burden. This should be the best way of supporting them. [Female participant (P11), 51 years old] [Female participant (P11), 51 years old] [Female participant (P31), 58 years old] [Female participant (P31), 58 years old] Meaning of facemasks in the post-SARS era Although using a facemask in public was a social norm in Hong Kong during the SARS outbreak, this social norm has been diminishing gradually in the post-SARS era because of the shifting meaning of the facemask, which prevented people from using it. Using a facemask was perceived as uncomfortable. Moreover, it was viewed as a hindrance to recovery, and a stigma was present against people who used facemasks, who were subject to teasing and discrimination. Negative attributes were associated with people who used them, and the perceived harm to skin as well as the violation of trad- itional Chinese cultural expectation on certain gender made the participants reluctant to use a facemask in the post-SARS era. Facemask as a tool for achieving a sense of control and security Facemasks were imbued with a mysterious power in that they provided a sense of security for more than half of the participants during the SARS outbreak. Moreover, facemasks provided a sense of control to the participants in an atmosphere of pervasive uncertainty that was prevalent in Hong Kong. One participant recalled the following: I felt a strong sense of uncertainty with my life during [the] SARS [outbreak], because I did not know when I would be infected. So many people were getting infected every day, and so many people died from SARS daily. At the time, I had a very strong feeling that I would definitely get infected sooner or later; it was just a matter of time, and death was near to me. I did not know what I could do. The only thing that I could do at the time was to wear a facemask. With a facemask, I felt much more secure. I did not know what a facemask could do to save me from [getting [Female participant (P24), 68 years old] [Male participant (P4), 22 years old] [Male participant (P4), 22 years old] [Male participant (P4), 22 years old] The popular Chinese belief of having to infect others to recover was deeply embedded in more than half of partici- pants’ minds. Because using a facemask prevented one from passing the virus to others, the participants per- ceived that using a facemask could prevent a quick recov- ery. One participant explained this belief as follows: Facemask use as discomfort Sensing physical discomfort was the most powerful rea- son for the participants choosing not to use a facemask when they suffered from respiratory tract infections in post-SARS Hong Kong. All of the participants indicated that using a facemask when sick often made them feel Page 9 of 16 Siu International Journal for Equity in Health (2016) 15:73 Siu International Journal for Equity in Health (2016) 15:73 even more uncomfortable. One participant expressed the following popular concern: became infected with a disease. One participant stated the following: even more uncomfortable. One participant expressed the following popular concern: Even for a healthy person, wearing a facemask can make you feel difficulties and discomfort in breathing; so asking a sick person to wear a facemask is quite inhumane to me, since this will make him or her feel even sicker. When I get a cold or flu, my nose is blocked, and I feel even sicker when I wear a facemask, because the facemask makes it even harder for me to breathe. I also need to speak louder when wearing a facemask, but I may have a sore throat when I’m sick. Wearing a facemask is very hot too, especially in the summer. It is not good to use a facemask when you catch a cold or flu, because it covers your nose and mouth. When you cough and sneeze, the bacteria and virus will stick to your facemask, and they will have nowhere to go. You will then breathe in the bacteria and virus again, and you can never recover. You have to let the bacteria and virus out of your body, so it is not good to use a facemask when you are sick. [Female participant (P24), 68 years old] Facemask as a medium for inviting stigmas and teasing g g g In addition to practical concerns, facemasks also conveyed symbolic meanings to the participants, which prevented them from using a facemask in the post-SARS era. Using a facemask was widely perceived as a sign of being sick, and more than half of the participants perceived that using a facemask would subject them to stigmatization. One par- ticipant relayed her experience as follows: According to the participants’ perception that face- masks were used mainly for infection prevention, many participants perceived that the purpose of facemask use was limited when they were sick. One participant indi- cated the following: If I am sick, what is the point of me wearing a facemask? A facemask is used to protect you from getting infected, and wearing a facemask will not make any difference for a sick person to recover faster, so why should I bother to wear a facemask if I am sick? I do not wear a facemask now, even though I am sick. Everyone will know that I am sick if I wear a facemask, and they will avoid me. It is noticeable when taking public transportation; no one is willing to sit next to anyone wearing a facemask. That’s true, and I experienced this before when using public transportation; people would walk away immediately if I wore a facemask. No one thinks that wearing a facemask when falling ill is a type of civic responsibility. Instead, people just get scared and avoid you when you are wearing a facemask. [Female participant (P35), 32 years old] [Female participant (P35), 32 years old] Facemask for infection prevention Similar to perceptions that were commonplace during the SARS outbreak, infection prevention was still per- ceived as the main reason for using a facemask for all of the participants in the post-SARS era. One participant stated the following: It is often said in traditional sayings that if you want to recover quickly from a cold or flu, then you have to infect others. You will get well when others get sick from you. Therefore, if you wear a facemask, it will prevent you from infecting others, so you will find it difficult to recover. I know this is bad, but I think it is normal for everyone to hope for a quick recovery. Therefore, I tend not using a facemask when I am sick. I often believe that those who wear a facemask are actually healthy; they wear a facemask because they are afraid of being infected. Those who are coughing and sneezing often do not wear a facemask. Facemasks are now used by healthy people to stop themselves from getting infected but are not used by sick people to prevent themselves from spreading viruses to others. [Female participant (P15), 25 years old] [Female participant (P15), 25 years old] The changing political environment of Hong Kong also influenced how the participants perceived the face- mask usage behavior. The participants often associated those who used a facemask with rioters. One participant stated the following: I dare not use a facemask now, because I do not want to be misunderstood as a member of Occupy Central [a political movement in Hong Kong that began in November 2014; also referred to as the Umbrella Movement]. Those who join Occupy Central and the violent protests wear facemasks at these activities, because they are afraid of being recognized, and “cannot see the light” to some extent. Facemasks are mainly used by violent protesters to cover their faces now. Therefore, I dare not use a facemask now, because I do not want to be misunderstood as a violent protester. [Female participant (P19), 36 years old] [Female participant (P19), 36 years old] [Female participant (P19), 36 years old] [Female participant (P22), 29 years old] [Female participant (P22), 29 years old] misperceived as strange, and perhaps even mentally ill. One participant noted the following: Using a facemask was thus perceived as frightening to others, according to the participants’ experiences, and would be an invitation for social seclusion. Using a face- mask often implied that one had become infected with a serious infection that might endanger the health of others. One participant indicated how she reacted to those who used a facemask as follows: Other than those working in hospitals and clinics, I always feel that only strange and mentally ill people wear a facemask. Even though they may not really be mentally ill, I think they may have mysophobia. I have really seen some people with facemasks wearing gloves as well. They appear to be mysophobic, and their behaviors also appeared strange and to be displaying sickness. I tend to stay away from these people wearing facemasks, because I never know if they will go crazy suddenly. I think that those who wear a facemask must have gotten infected with a very serious infection such as tuberculosis. It is unnecessary for normal people to wear a facemask if they just catch a cold or flu, because most colds and flus are not serious at all, and will not kill people. Therefore, if a person needs to wear a facemask, I would assume that he or she must have gotten a very serious infection. I will avoid them, because they may make me very sick, or even kill me. [Male participant (P8), 34 years old] Facemask as a hindrance for recovery Nearly all of the participants perceived that using a face- mask when sick could hinder their recovery. This belief prevented them from using a facemask when they Page 10 of 16 Siu International Journal for Equity in Health (2016) 15:73 [Male participant (P8), 34 years old] The participants also popularly stigmatized those who used a facemask as criminals. The facemask was perceived as a tool used by criminals for concealing their faces. One participant expressed how he ob- tained such an impression from media reports as follows: [Female participant (P30), 36 years old] Perceiving that facemask use was chiefly for infection prevention, certain participants had used a facemask to prevent themselves from becoming infected. However, the experience of being teased prevented more than half of the participants from using a facemask in the post- SARS era. One participant shared the following common experience: If you read the news reports and watch the news on TV, you’ll see that these criminals often love wearing facemasks. My immediate impression of these people wearing facemasks is that they are criminals. They wear a facemask because they “cannot see the light” [Cantonese slang metaphor indicating someone who has committed bad deeds], and they do not want to be recognized by others. If you behave well and if you can “see the light”, you do not need to wear a facemask to cover up your face. Some people are really bad; they will tease you if you wear a facemask. Just like my colleagues; they never wear a facemask when they are sick, and they never cover their nose and mouth when coughing or sneezing. Even worse, some really bad colleagues will cough and sneeze on you! I am afraid of being infected, so I wear a facemask because I cannot push them to wear one. However, these bad colleagues would tease me. Even in public transportation, I also wear a facemask if others around me keep coughing or sneezing. However, these selfish people would shoot me a dirty look, and some even cough and sneeze even harder! Because of these people, I always feel reluctant to wear a facemask, even though I am really afraid of being infected. Facemask as a sign of weakness Anyway, I do not use facemasks, because I always think that they contain a lot of toxins. Certain participants also perceived that wearing a face- mask could convey an image to others that they were physically weak, making them reluctant to use one. This perception was particularly prevalent among male par- ticipants, because being weak violated the cultural ex- pectations of men, who were assumed to be physically strong. One male participant indicated the following: [Female participant (P1), 32 years old] [Female participant (P1), 32 years old] The lack of trust on mainland China led certain partic- ipants to express a lack of confidence in the production locations of facemasks, preventing them from using a facemask. One participant stated the following: Wearing a facemask means that I am weak and afraid of bacteria and germs. But I am a man, and I think I am strong, so I do not need to wear a facemask. Even the government advises facemask usage only for those who are physically weak, such as elderly people, chronically ill patients, pregnant ladies, and children, who will need to wear a facemask during flu season. If I wear a facemask, I will lose face in front of my friends, and they will laugh at me for being so weak. I dare not wear a facemask, because you know that most facemasks are produced in mainland China. Even if you buy facemasks from Watson’s and Manning’s [drugstore chains], the quality cannot be ensured because these facemasks are made in China. Will you trust the quality of products that are made in China? I definitely won’t. The quality of products that are made in mainland China cannot be guaranteed, and they may contain a lot of toxins. These made-in-China facemasks may be made of “black-heart cotton” [contaminated materials]. Anyway, I do not have any confidence in products that are manufactured in China. I do not trust the quality of these facemasks. If facemasks were produced in other overseas countries such as Japan or Western countries, I would feel safe using them. However, it is very difficult to buy these facemasks. [Male participant (P36), 31 years old] [Male participant (P36), 31 years old] [Male participant (P36), 31 years old] Using a facemask was also perceived as a sign of being scared of death for the participants. Facemask as a sign of weakness One participant in- dicated the following: I think that only those who are afraid of death will wear a facemask. I am not afraid of death, and I think I am strong and healthy. Therefore, I do not think I need to use a facemask. I always think that it should not be too easy for one to die. If I wear a facemask, others may misunderstand that I am very weak and afraid of dying. [Female participant (P7), 44 years old] [Female participant (P7), 44 years old] [Female participant (P7), 44 years old] Facemask as a toxin to skin The hesitation in using a facemask among the partici- pants was also due to the popular perception of face- masks as a toxin for skin. Almost half of the participants expressed doubt regarding the quality, hygiene, and pro- duction process of facemasks. The use of artificial chemicals in facemask production also made the partici- pants hesitant to use a facemask. One participant stated the following: The participants also associated facemask use with people who had a low social status. Cleaners were often stereotyped as the most common social group who used facemasks, according to the participants’ perceptions. Therefore, the participants were reluctant to use a face- mask because they did not want to be misidentified as cleaners, which was perceived as an occupation indicating a low social status. One participant stated the following: Wearing a facemask is bad for your skin, because you can never know whether the facemask production process is hygienic. Facemask production also involves a lot of chemicals, dyes, and so on, and they are harmful to your skin for sure. You may absorb these toxic materials into your body through your skin. Moreover, wearing a facemask can make me more susceptible to allergies and acne, so I have a lot of doubt regarding the quality of facemasks. When you use a facemask, you breathe in toxic materials. Anyway, I do not use facemasks, because I always think that they contain a lot of toxins. It is uncommon for people to wear a facemask now, except for cleaners. Cleaners often wear a facemask on the streets. If I wear a facemask, I am afraid others will misunderstand that I am working as a cleaner, so I have not used a facemask for a long time, since [the] SARS [outbreak]. [Female participant (P12), 54 years old] [Female participant (P25), 52 years old] [Female participant (P25), 52 years old] Facemask as a toxin to skin Facemask as a symbol associated with negative attributes for users More than half of the participants stigmatized people who used facemasks in the post-SARS era. To these par- ticipants, those who use a facemask were widely Page 11 of 16 Siu International Journal for Equity in Health (2016) 15:73 Siu International Journal for Equity in Health (2016) 15:73 Discussion As indicated by the participants, the shifting meanings of facemasks could be explained by the violation of society’s norms and ideologies, by the violation of the traditional Chinese cultural beliefs on healing, and by the projection of the difficult relationship between mainland China and Hong Kong. g p The only meaning of facemasks that remained static during and after the SARS outbreak in Hong Kong was their purpose of practical infection prevention. Although according to clinical guidelines facemasks should theor- etically be used predominantly by patients with respira- tory tract infections to prevent them from infecting others [16–18], the participants commonly perceived the opposite: Facemasks were perceived as a tool for preventing themselves from becoming infected during the SARS outbreak and in the post-SARS era. Such an embedded perceived purpose of facemasks thus explains the participants’ reluctance to use a facemask, despite their experiences with respiratory tract symptoms in the post-SARS era. The experiences of the participants since the SARS outbreak are critical in the shaping of their perceptions of facemasks. All of the participants had firsthand experience with SARS in Hong Kong during the SARS outbreak, and they had differing experiences during the outbreak. Certain participants lost their family members during the outbreak because of SARS, some other partic- ipants had family members who were infected with SARS, and others were not directly affected by SARS but were frustrated by it. Although they had different experi- ences of the SARS outbreak, they had all used a face- mask throughout the outbreak because of fear and sociocultural forces. When the post-SARS era approached, however, the different experiences of the participants influenced their perceptions of facemask use. The lack of a direct experience with an epidemic in the post-SARS era for many participants, in addition to the social changes, made them disregard the infection prevention value of facemasks, thereby contributing to the shifting sociocultural meanings of facemasks in the post-SARS era. Symbolic meanings are often constructed for practical use. In addition to the practical purpose of infection prevention, facemasks also conveyed critical symbolic meanings to participants during the SARS outbreak. These symbolic meanings were largely constructed by a social authority—health care providers—resulting in the construction of a new social norm. Discussion The SARS outbreak in 2003 marked a critical turning point in the development of infection control and public health in Hong Kong. One of the most remarkable outcomes in [Male participant (P32), 35 years old] [Male participant (P32), 35 years old] Siu International Journal for Equity in Health (2016) 15:73 Page 12 of 16 Page 12 of 16 Page 12 of 16 during the SARS outbreak because of their young age (certain participants were in primary school during the outbreak), they did not have a clear understanding of why they were required to use a facemask during the outbreak. Many of them had used a facemask during the SARS outbreak, merely because of the control of social institutional forces such as their parents, teachers, and school. Consequently, without a strong experience, these younger participants did not have the cultural founda- tions to understand facemask usage behavior in the post-SARS era. By contrast, older participants typically had a more positive attitude toward facemask use in the post-SARS era, even though they did not use a facemask in the post-SARS era. The older participants usually dis- played more intense feelings and relayed such experi- ences pertaining to the SARS outbreak, leading to a stronger understanding regarding the importance of using a facemask in the post-SARS era. terms of infection control after the SARS outbreak was the awareness of facemask usage in Hong Kong, not only at the clinical level but also at the community level. Although Hong Kong citizens have become familiar with facemask use since the SARS outbreak, the sociocultural meanings of facemasks have been undergoing constant change, which resulted in the participants experiencing hesitation to use facemasks in the post-SARS era. The old meanings associ- ated with facemasks that had developed during the SARS outbreak failed to be sustained in the post-SARS era. Changes in the sociocultural meanings of the facemask not only influenced the participants’ perceptions of facemask but also influenced their views of those who used facemasks, which ultimately affected their health behavior, reducing the likelihood of their facemask use in the post- SARS era. Moreover, the negative perceptions associated with facemasks also contributed to the changes in the sociocultural implications of facemasks in post-SARS Hong Kong. Discussion Hence, the participants had been un- aware of using a facemask when they experienced re- spiratory symptoms, both during and after the SARS outbreak. The symbolic association of facemasks with health care providers influenced the participants’ use of face- masks during the SARS outbreak at two levels. First, the use of facemasks among health care providers on differ- ent social occasions and in media appearances was crucial for the construction of this new social norm and for normalizing the use of facemasks, because of their social power. Health care professionals, as a social insti- tution, allowed them to exercise social control with respect to facemask use in the community, which moti- vated the participants to use a facemask during the SARS outbreak. Second, the participants used facemasks as a form of symbolic support for health care providers. Because facemasks were perceived as a tool for infection prevention, using a facemask did not simply prevent the participants from becoming infected, but by so doing, it also implied a show of support for health care providers in an attempt to reduce their workload and the burden on the health care system. Using a facemask was akin to a symbolic declaration that they were committed to re- ducing the burden on the health care system by prevent- ing themselves from becoming infected. Such symbolic support for health care providers through facemask use extended further to the social implication of displaying civic responsibility. These sociocultural processes hence contributed to the symbolic construction of the face- mask during the SARS outbreak. The perception of the use of facemasks for infection prevention was thus embedded in the participants’ minds during the SARS outbreak and continued into the post-SARS era. The embedded belief in the purpose of the facemask for infection prevention served as an underlying factor for cultivating a shift in the meanings associated with facemasks in post-SARS Hong Kong. Al- though using a facemask in public areas was constructed as a new social norm in Hong Kong during the SARS outbreak, and such behavior was constructed to be a symbol of support for health care providers and a display of civic responsibilities at the time, these social norms and meanings have been gradually diminishing in post- SARS Hong Kong. Discussion The health care pro- fession is a form of social institution [19]; the public’s trust in health care providers [19] often enables them to assume an authoritative role in most societies, making them a key social group with authority and social power over the creation of new social norms and the imple- mentation of social control [20]. During the SARS outbreak, health care providers occupied an even more prestigious position, and they were portrayed as social heroes in the battle against SARS [21]. Thus, they held even more power in the construction of this new social norm. They used facemasks in media appearances [22], and they encouraged the public to use facemasks in the community during the SARS outbreak [23]. Therefore, other than stethoscopes and white coats, facemasks were an additional entity associated with health care providers The extent of the participants’ experiences with the SARS outbreak also significantly influenced how they perceived facemask usage behavior in the post-SARS era. Those who encountered more direct and traumatic ex- perience in the SARS outbreak and the older had a more positive attitude toward facemask use compared with younger participants, who perceived facemask use to be more negative in the post-SARS era. Because the youn- ger participants were unaware of what had transpired Siu International Journal for Equity in Health (2016) 15:73 Page 13 of 16 Page 13 of 16 represented as not only for the user’s benefit but also for the good of the community and as a show of support. during the SARS outbreak, causing facemasks to become another critical symbol representing health care pro- viders at the time. The perception of the predominant use of facemasks for infection prevention also made facemasks a critical tool for the participants to achieve a sense of control and security during the SARS outbreak. The high mor- tality rate and unknown transmission route of SARS made it a mysterious disease to most Hong Kong citi- zens during the outbreak [24]. In the social atmosphere that was filled with uncertainty, using a facemask was the only measure for the participants to protect them- selves. Consequently, the use of a facemask for infection prevention was further reinforced, which deterred the participants from learning the other critical use of face- masks, that of preventing the transmission of infectious diseases to others. Discussion Influenced by views in TCM, the popular be- lief of “virus and bacteria must be released from the Siu International Journal for Equity in Health (2016) 15:73 Page 14 of 16 Page 14 of 16 Page 14 of 16 avoidance of being recognized for engaging in actions that violated the law. Under the further reinforcement of the mass media, the participants thus constructed a negative association with the use of facemasks. The negative perception of those who used a facemask was reinforced further after the Umbrella Movement in late 2014, in which the protesters went on riot with face- masks. In a society with a heavy emphasis on law and order such as that of Hong Kong, the participants were deterred from pursuing this health behavior because they did not wish to be misperceived either as antisocial criminals or violent protesters. The mass media and the changing social and political environment in Hong Kong, thus, have contributed to the shifting social and cultural meanings of facemask in post-SARS era. body for recovery” was deeply ingrained in the partici- pants’ minds. The use of a facemask, however, violates this TCM ideology, because a facemask prevents viruses and bacteria from escaping the body, which is perceived as hindering recovery from the participants’ perceptions. Consequently, the use of a facemask was not welcome by the participants, due to this violation of traditional Chinese cultural belief on healing. This contributed as an underlying factor to the shift in meaning of face- masks, resulting in the failure to sustain the values asso- ciated with facemasks that had developed during the SARS outbreak. To reinforce such traditional cultural values on disease and healing belief, the participants thus recreated the new meanings of facemask that were consistent with these long existing cultural values in the post-SARS era. Using a facemask in the post-SARS era was no longer perceived as a sign of civic responsibility; instead, such health behaviors were stigmatized and constructed to be correlated with a person’s negative attributes. Using a facemask was also correlated with a person’s low social standing and was even correlated with antisocial people such as criminals and violent protesters. With these negative attributes attached to facemasks, the sociocul- tural meanings of facemasks have shifted from positive to negative, which further deterred the participants from using a facemask in the post-SARS era. Discussion Moreover, facemask use was often associated with people who were low on the social hierarchy. To the participants, cleaners were another social group who often used facemasks. Because cleaners were perceived to hold a low social status in Hong Kong, this violated the capitalist ideology of Hong Kong, which strongly emphasizes wealth and a high social status. All of these negative attributes were constructed to be correlated with the use of a facemask in the post-SARS era, which prevented the participants from using a facemask. The sociocultural meanings of the facemask have thus been undergoing continuous change in Hong Kong. These changes influenced the participants’ perceptions of the facemask and hindered their adoption of face- mask usage behavior. Although the participants still perceived infection pre- vention to be the main purpose of using a facemask in post-SARS Hong Kong, and although the participants commonly believed that facemasks were for use by healthy people to prevent themselves from becoming infected, the intention to prevent infection has been associated with negative attributes after the SARS out- break. Those who used a facemask in public areas in the post-SARS era were stigmatized by the participants with different biases (e.g., strangeness, mental illness, and mysophobia). These stigmas violated the sociocultural ideals of Hong Kong. Patients with psychiatric diseases have been stigmatized in Hong Kong as, for instance, crazy, dangerous, and violent [26], and were thus per- ceived to pose a hidden risk to society [27]. Those with mysophobia were often stigmatized as mentally ill as well [28]. To avoid being stigmatized as mentally ill, the participants were deterred from using a facemask. Although facemasks are still perceived as a tool for infection prevention after the SARS outbreak, the partic- ipants also held contradictory views on the use of face- masks pertaining to their use as an obligation for patients with infectious diseases. However, patients using a facemask were often subjected to a stronger stigma according to the participants, because these patients were often viewed as suffering from serious infectious diseases that could endanger others’ health. Conse- quently, using a facemask in public was frequently an in- vitation for social seclusion. To avoid being isolated, the participants were thus deterred from using a facemask in post-SARS Hong Kong. Discussion The absence of an epidemic outbreak that is similar to that of SARS as well as the physical discomfort when using a facemask were the immediate factors, but other sociocultural values as well as the changes of social and political environment also inter- locked to contribute to the shift in the meanings of face- masks in the post-SARS era. In addition to the health care providers, the mass media played a critical role in the construction of such symbolic implications by reinforcing the new social norm of using facemasks in public areas during the SARS outbreak. Chinese-language media devoted signifi- cantly more space to reporting news on SARS daily [24]. Photographs showing people using facemasks occupied the newspaper headlines every day [24]. People who failed to use a facemask in public areas were represented as abnormal in news reports. Those who were infected with SARS were condemned as spreading the virus in these reports. “Super virus spreaders” [25], for example, was the popular term used by the mass media for repre- senting patients infected with SARS. Such a sensational reporting style with moral judgment and condemnation thus made the infection of SARS antisocial. Conse- quently, people were afraid of becoming infected with SARS, and this constructed, normalized, and reinforced the new social norm of using facemasks in public spaces. Those who failed to use a facemask were perceived as antisocial, and thus, were discriminated against. The construction of such a social norm portrayed facemasks and reinforced them as a sign of civic responsibility on another level: The purpose of using a facemask was The sociocultural meanings of facemasks have shifted from positive to negative in post-SARS Hong Kong. Without the presence of a significant epidemic outbreak, the embedded stereotypes toward facemask among the participants thus reoccupied their perceptions. The em- bedded traditional Chinese cultural belief regarding in- fectious diseases and healing acted as a considerable obstacle in preventing almost all the participants from using a facemask in post-SARS Hong Kong, resulting in a failure to sustain facemask usage for preventing the spread of infectious diseases. Traditional Chinese medi- cine (TCM) is the most popular ethnic medical system in Hong Kong, existing alongside the mainstream bio- medicine. Conclusion Traditional Chinese gender values could explain the participants' gender differences of their acceptance of using a facemask in the post-SARS era. Male participants were particularly more reluctant to use a facemask, be- cause facemask usage behavior violated the sociocultural expectations of men in Chinese societies. To these partici- pants, using a facemask was widely perceived as a sign of weakness, thereby violating the sociocultural expectations of men, who should be “strong” and “brave.” The violation of traditional Chinese cultural expectations regarding men thus served as a considerable obstacle for the male partici- pants in using a facemask in post-SARS Hong Kong. Facemask use should thus not be simply viewed as a public health behavior; instead, sociocultural reasons are intertwined to explain people’s usage or avoidance of facemasks. Because of the negative stereotypes asso- ciated with the use of facemasks in post-SARS Hong Kong, the sociocultural meanings of facemasks have undergone continuous change, from positive to nega- tive. Because of the changes in social needs, the mean- ings of facemasks that had developed during the SARS outbreak could not be sustained in the post-SARS era. During the SARS outbreak, the social functions of the facemask were significant not only in terms of provid- ing a practical approach to infection prevention, but also in providing symbolic uses for obtaining a sense of control and security, fulfilling the new social norm, and displaying civic responsibility and support for health care providers. To fulfill these social needs, the socio- cultural meanings of facemasks were thus constructed to be positive. However, such social needs were lacking in the post-SARS era, leading to changes in the social functions and sociocultural meanings of facemasks. The changes to the social and political environment gener- ated a negative image of facemask usage, preventing the participants from pursuing this health behavior. The experiences of the participants thus enabled a holistic understanding regarding the perceptions of facemask use. The shifting sociocultural meanings of facemask use in Hong Kong, however, can impose a potential risk to the public health of Hong Kong citizens in case of a future epidemic outbreak. Witnessing younger partici- pants tending to hold more negative perceptions of facemask usage in the post-SARS era was alarming. These findings have critical implications for health au- thorities in designing a culturally responsive infection prevention and facemask usage policy in the future. Discussion The fragile relationship and sociopolitical tensions be- tween Hong Kong and mainland China, in addition to the widespread distrust among Hong Kong citizens of mainland China, also led to participant concerns regard- ing the safety of and confidence in facemasks, preventing them from using facemasks in post-SARS Hong Kong. This subtle display was made tangible and reinforced by the mass media through news reports. Because of the numerous media reports regarding the unscrupulous and unhygienic production process of products made in China, the participants held a high degree of distrust in In addition, other negative attributes associated with facemasks in the post-SARS era also violated the domin- ant ideologies of Hong Kong. Because of the mass media, according to the participants’ perceptions, the use of a facemask was often associated with people who were antisocial (e.g., criminals and violent protesters). Indeed, antisocial deviants in Hong Kong often used facemasks intentionally in media coverage. Hence, the use of a facemask was widely perceived as a tool for Siu International Journal for Equity in Health (2016) 15:73 Page 15 of 16 mainland Chinese products. Because most facemasks were assumed to be produced in mainland China, the participants experienced doubt and lacked confidence in the quality and hygiene of the facemasks. At the same time, mass media had been kept reinforcing about “black-heart cotton” in their reports about mainland Chinese products, making the participants commonly perceived that the facemask could itself contain toxins, and labelled these facemasks as making of “black-heart cotton”. As shown by the participants, such sociopoliti- cal tension as well as the cultural differences between Hong Kong and mainland China thus projected on and manifested in the participants’ doubt regarding the safety of facemasks, which aroused their sense of insecurity. The failure to sustain the sociocultural implications of facemasks in post-SARS Hong Kong can thus also be ex- plained by the sociopolitical tension between Hong Kong and mainland China. of public health and infection prevention policy in future is suggested to adopt a culturally sensitive education ap- proach, educating the general public about the positive as- pects of facemask using behavior. Limitations This study had limitations. All of the participants were sampled from one private-practice primary care clinic. Be- cause the fee for attending a private clinic is often higher than for public clinics, the socioeconomic status of the participants in this study was assumed to be higher. Therefore, these findings mainly reflected the perceptions of those with a higher socioeconomic background. People with a lower socioeconomic status may be excluded from generalization. Future research with more varied field sites will provide a more holistic understanding. Competing interests Competing interests 19. Gilson L. Trust and the development of health care as a social institution. Soc Sci Med. 2003;56(7):1453–68. The author has no competing interests to declare in this research. 20. Loustaunau MO, Sobo EJ. The cultural context of health, illness, and medicine. Westport: Bergin & Garvey; 1997. medicine. Westport: Bergin & Garvey; 1997. Authors’ contributions 21. Global Times. HK’s “SARS heros” share reflections a decade after deadly virus outbreak. 2013. http://www.globaltimes.cn/content/762844.shtml. Accessed February 6, 2015. JYS was involved in the study conception and design, data collection, data analysis and interpretation, and writing of the manuscript. 22. The Chinese University of Hong Kong. Fighting SARS: We care we serve. Clinical and scientific work. 2003. http://www.cuhk.edu.hk/health_promote_ protect/sars_care/menu2.htm. Accessed January 19, 2015. Received: 11 November 2015 Accepted: 17 April 2016 24. Loh C, Civic Exchange. At the epicentre: Hong Kong and the SARS outbreak. Hong Kong: Hong Kong University Press; 2004. Implications to public health Since the SARS outbreak, there had been several infec- tious disease outbreaks in Hong Kong, such as the Influ- enza A (H1N1) pandemic in 2009, and many more Influenza outbreaks in the winter peak seasons every year. Other pandemics such as avian influenza and MERS have also been threatening the public health of Hong Kong. However, the importance of the preventive health behavior adoption of facemask using has been diminishing. The shifting sociocultural meanings of face- mask in Hong Kong since the SARS outbreak, as dem- onstrated by the participants, can explain this preventive health behavioral change on the community level. The shifting sociocultural meanings of using facemask can demotivate people in adopting the facemask using be- havior, making infection control as more difficult to achieve during epidemic outbreaks, conveying significant implications for the public health and infection control in the post-SARS era. Facemask using has been documented as having significant clinical importance in infection pre- vention [2, 3]; encouraging people using a facemask is thus one of the remarkable public health measures on epi- demic containment. Therefore, one of the key directions Page 16 of 16 Siu International Journal for Equity in Health (2016) 15:73 Siu International Journal for Equity in Health (2016) 15:73 Siu International Journal for Equity in Health (2016) 15:73 References 25. eElderly. 社評:應對疫症硬道理寧可做多勿做少[Editorial: Be forceful against epidemic, doing more is always better than doing less]. 2015. http://www.e123.hk/ElderlyPro/details/405505/2/. Accessed August 5, 2015. 1. World Health Organization. Summary of probable SARS cases with onset of ill f 1 N b 2002 t 31 J l 2003 2003 htt // h i t/ 1. World Health Organization. Summary of probable SARS cases with onset of illness from 1 November 2002 to 31 July 2003. 2003. http://www.who.int/ csr/sars/country/table2003_09_23/en/. Accessed January 5, 2015. 1. World Health Organization. Summary of probable SARS cases with onset of illness from 1 November 2002 to 31 July 2003. 2003. http://www.who.int/ csr/sars/country/table2003_09_23/en/. 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The Sage Encyclopedia of Qualitativ doi: http://dx.doi.org/10.4135/9781412963909. doi: http://dx.doi.org/10.4135/9781412963909. 13. Green J, Thorogood N. Qualitative methods for health research. Thousand Oaks: SAGE Publications; 2004. p. 219. 13. Green J, Thorogood N. Qualitative methods for health research. Thousand Oaks: SAGE Publications; 2004. p. 219. 14. Parse RR. Qualitative inquiry: the path of sciencing. Sudbury, MA: Jones and Bartlett Publishers; 2001. 14. Parse RR. Qualitative inquiry: the path of sciencing. Sudbury, MA: Jones and Bartlett Publishers; 2001. Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: 15. Bernard HR. Research methods in anthropology: q approaches. Walnut Creek: AltaMira Press; 2002. 15. Bernard HR. Research methods in anthropology: qualitative and quantitative approaches. Walnut Creek: AltaMira Press; 2002. 15. Bernard HR. Research methods in anthropology: qualitative and quantitative approaches. Walnut Creek: AltaMira Press; 2002. • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: 16. Centre for Health Protection, Department of Health, The Government of the Hong Kong Special Administrative Region. CHP’s review and outlook of infectious diseases. 2006. http://www.chp.gov.hk/en/view_content/5365.html. Accessed February 2, 2015. 17. World Health Organization. Procedures for prevention and management of probable cases of SARS on international cargo vessels. 2003. http://www. who.int/csr/sars/travel/maritime/en/. Accessed February 3, 2015. 18. World Health Organization. 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https://openalex.org/W3217796287	https://www.dora.lib4ri.ch/empa/islandora/object/empa%3A27417/datastream/PDF/Fochesato-2021-Multi-objective_optimization_of_a_power-to-hydrogen-%28published_version%29.pdf	English	null	Multi-objective optimization of a power-to-hydrogen system for mobility via two-stage stochastic programming	Journal of physics. Conference series	2,021	cc-by	3,660	2042 (2021) 012034 IOP Publishing doi:10.1088/1742-6596/2042/1/012034 2042 (2021) 012034 IOP Publishing doi:10.1088/1742-6596/2042/1/012034 CISBAT 2021 Journal of Physics: Conference Series 2042 (2021) 012034 doi:10.1088/1742-6596/2042/1/012034 Multi-objective optimization of a power-to-hydrogen system for mobility via two-stage stochastic programming Marta Fochesato1, Philipp Heer2 and John Lygeros1 1 Automatic Control Laboratory, Physikstrasse 3, 8092 Z¨urich, Switzerland 2 Urban Energy Systems Lab, ¨Uberlandstrasse 129, 8600 D¨ubendorf, Switzerland E-mail: mfochesato@control.ee.ethz.ch, philipp.heer@empa.ch, jlygeros@control.ee.ethz.ch Marta Fochesato1, Philipp Heer2 and John Lygeros1 1 Automatic Control Laboratory, Physikstrasse 3, 8092 Z¨urich, Switzerland 2 Urban Energy Systems Lab, ¨Uberlandstrasse 129, 8600 D¨ubendorf, Switzerland E-mail: mfochesato@control.ee.ethz.ch, philipp.heer@empa.ch, jlygeros@control.ee.ethz.ch Abstract. A systematic way for the optimal design of renewable-based hydrogen refuelling stations in the presence of uncertainty in the hydrogen demand is presented. A two-stage stochastic programming approach is used to simultaneously minimize the total annual cost and the CO2 footprint due to the electricity generation sources. The ﬁrst-stage (design) variables correspond to the sizing of the devices, while the second-stage (operation) variables correspond to the scheduling of the installed system that is aﬀected by uncertainties. The demand of a ﬂeet of fuel cell vehicles is synthesized by means of a Poisson distribution and diﬀerent scenarios are generated by random sampling. We formulate our problem as a large-scale mixed-integer linear program and we rely on a two-level approximation scheme to keep the problem computationally tractable. A solely deterministic setting which does not take into account uncertainties leads to underestimated device sizes, resulting in a signiﬁcant fraction of demand remaining unserved with a consequent loss in revenue. The multi-objective optimization produces a convex Pareto front, showing that a reduction in carbon footprint comes with increasing costs and thus diminishing proﬁt. Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 2042 (2021) 012034 doi:10.1088/1742-6596/2042/1/012034 stations are much fewer. For example, in [3], the authors investigate the optimal sizing of the electrolyzer in a refuelling station, while [4] focuses on a feasibility study of renewable hydrogen stations. To the best of our knowledge, no previous studies on optimal design of hydrogen refuelling stations have considered the presence of uncertainties aﬀecting the system. stations are much fewer. For example, in [3], the authors investigate the optimal sizing of the electrolyzer in a refuelling station, while [4] focuses on a feasibility study of renewable hydrogen stations. To the best of our knowledge, no previous studies on optimal design of hydrogen refuelling stations have considered the presence of uncertainties aﬀecting the system. In this work, we propose a systematic way for approaching the optimal design of renewable electricity-based hydrogen refuelling stations by considering uncertainties in the hydrogen demand. We cast our problem in the framework of two-stage stochastic programming. The ﬁrst-stage (“here-and-now”) decisions represent the sizing of the devices considered. The second- stage (“wait-and-see”) decisions represent the scheduling of the installed system which is aﬀected by uncertainties and therefore the corresponding cost is evaluated as expectation over multiple demand scenarios. We claim two advantages with respect to similar works in literature. First, we include the presence of uncertainties for the hydrogen demand in our formulation. Second, we propose a multi-objective optimization framework to simultaneously minimize the economic costs and the CO2 footprint of the system evaluated in terms of Global Warming Potential (GWP) per kilowatt of electricity consumed. The paper is structured as follows. In Section 2, we introduce the models for the devices constituting the hydrogen refuelling station. In Section 3, we formulate the optimization framework and we propose a two-level approximation scheme to solve it eﬃciently. In Section 4, we present the results of the numerical study. Finally, in Section 5, we conclude with ideas for reﬁnement of our approach. 1. Introduction Th The transportation sector is reported to contribute for around 40% of CO2 emissions in Switzerland, making it a good target for decarbonization strategies. Indeed, the road-based private transport sector in Switzerland has an estimated CO2 reduction potential of up to 40% by 2040 [1]. This is due to the rise of alternatives to fossil-fuelled vehicles, such as Electric Vehicles (EVs) and Fuel Cell Vehicles (FCVs). While EVs represent a more mature technology, the proliferation of FCVs is highly correlated to the development of new infrastructure, namely a network of hydrogen refuelling stations. To support the decarbonization strategy (e.g. as presented in [1]) it is crucial that the hydrogen required to run FCVs is produced via a carbon- neutral process, with water electrolysis being such a possibility. However, a proper evaluation of the CO2 impact of electrolysis has to be traced back to the sources producing the electricity required for running the electrolyzer: it is evident that if the electricity used derives from fossil- based sources, the electrolysis cannot be considered carbon-neutral. Previous works have mainly studied the optimal design of charging stations for electric vehicles (see [2] and references therein), while corresponding studies on hydrogen refuelling 1 CISBAT 2021 Journal of Physics: Conference Series CISBAT 2021 Journal of Physics: Conference Series 2042 (2021) 012034 IOP Publishing doi:10.1088/1742-6596/2042/1/012034 2042 (2021) 012034 IOP Publishing doi:10.1088/1742-6596/2042/1/012034 3. Optimization framework Based on the model derived in Section 2, a two-stage stochastic program [9] is formulated to simultaneously maximize the annual proﬁt and minimize the carbon footprint by relying on the ϵ-constraint method. The objective function comprises a deterministic ﬁrst-stage cost Jinv(s) + Jmaint(s) which only depends on the “here-and-now” design variables s and an expected second-stage cost E[Joper(u, b, x, p, n, ξ) −R(ξ)] depending on the operation variables u, b, x, p, n and on the disturbances ξ. The “sustainable” objective JCO2(u, b, x, p, n, ξ) = P t∈T gT t pt (where gt indicates the “dirtiness” of electricity at time t) on the second stage is enforced via a constraint that should hold in expectation; a more “aggressive” emission policy could be enforced by constraining all scenarios to have CO2 footprint lower than ϵ [6]. ( First-stage ) : min s (Jinv(s) + Jmaint(s)) + EP h ˜f2 (s, ξ) i s.t. ϕ(s) ≤0 ( Second-stage ) : where : ˜f2 (s, ξ) := min u,b,x,p,n [Joper (u, b, x, p, n, ξ) −R(ξ)] s.t. (1), (2), (3) EP [JCO2 (u, b, x, p, n, ξ)] ≤ϵ (8) ( First-stage ) : min s (Jinv(s) + Jmaint(s)) + EP h ˜f2 (s, ξ) i s.t. ϕ(s) ≤0 ( Second-stage ) : where : ˜f2 (s, ξ) := min u,b,x,p,n [Joper (u, b, x, p, n, ξ) −R(ξ)] s.t. (1), (2), (3) EP [JCO2 (u, b, x, p, n, ξ)] ≤ϵ (8) (8) The stochastic program (8) is solved by randomly sampling a ﬁnite number of scenarios (namely, the realizations of the uncertain parameter ξ over the whole horizon) from an underlying probability distribution Pξ to approximate the expectation in the second-stage cost and in the ϵ-constraint. Note that in (8) the non-anticipativity constraint on u, b, x, p, n is not assumed in order to simplify the formulation. p y The problem can be reformulated as a large-scale Mixed-Integer Linear Program (MILP) [5] p y The problem can be reformulated as a large-scale Mixed-Integer Linear Program (MILP) [5] as, mins,u,b,x,p,n (Jinv(s) + Jmaint(s)) + πs P s∈S [Joper (s, us, bs, xs, ps, ns, ξs) −R(ξs)] s.t. 2. System modelling 2 CISBAT 2021 2042 (2021) 012034 IOP Publishing doi:10.1088/1742-6596/2042/1/012034 2042 (2021) 012034 doi:10.1088/1742-6596/2042/1/012034 Journal of Physics: Conference Series Journal of Physics: Conference Series Journal of Physics: Conference Series The dependence of the economic costs, namely investment cost Jinv, maintenance cost Jmaint and operational cost Joper, from design and operational variables is modelled as, (4) (5) (6) Jinv(s) = (CI s + WI)a (4) Jmaint(s) = CMJinv (5) Joper(u, b, x, p, n, ξ) = X t∈T cT t pt (6) (5) (6) (6) where CI, WI, CM are matrices of appropriate dimension, ct is the electricity price at time t in T and a is the annuity factor calculated according to the standard deﬁnition considering an investment rate of 6% and a system lifetime of 10 years. T indicates the number of time steps considered in the optimization: we consider one year with hourly resolution, in other words 365 · 24 = 8760 time steps. All parameters in this section are taken from [7]. where CI, WI, CM are matrices of appropriate dimension, ct is the electricity price at time t in T and a is the annuity factor calculated according to the standard deﬁnition considering an investment rate of 6% and a system lifetime of 10 years. T indicates the number of time steps considered in the optimization: we consider one year with hourly resolution, in other words 365 · 24 = 8760 time steps. All parameters in this section are taken from [7]. Revenues R from selling hydrogen are evaluated as, Revenues R from selling hydrogen are evaluated as, R(ξ) = X t∈T pT H2,t ξt (7) (7) where pH2,t is the selling price of hydrogen at time t, assumed constant at 15 CHF/kgH 2. System modelling y g We consider a hydrogen refuelling station composed of a PEM electrolyzer, a storage tank and an array of photovoltaic panels. We model the dynamics of the system as, We consider a hydrogen refuelling station composed of a PEM electrolyzer, a storage tank and an array of photovoltaic panels. We model the dynamics of the system as, xt+1 = Axt + But + Cξt ∀t ∈T (1) (1) xt+1 = Axt + But + Cξt ∀t ∈T where the vectors xt, ut and ξt comprise respectively all the states, inputs and uncertainties aﬀecting the system at time t ∈T . Throughout we use the notation q (with q being a generic quantity) to denote the collection of qt with t ∈T . The states considered here model the energy stored in the hydrogen storage tank and the electrolyzer stack temperatures, while the eﬃciency of the electrolyzer is modelled as static piecewise aﬃne map. The uncertainty ξt corresponds to the hydrogen demand from a ﬂeet of fuel cell vehicles. The matrices A, B, C are assumed to be known and of appropriate dimension. sider bilinear operational constraints for the system devices (e.g. capacity limits) of We consider bilinear operational constraints for the system devices (e.g. capacity limits) of the form (2) Ex + Fu ≤h(s · b) (2) where the vector s indicates the design variables (i.e. the capacity of the installed devices) with appropriate upper and lower bounds ϕ(s) ≤0. b indicates a vector of binary decision variables describing the status on/oﬀof the corresponding device during the whole horizon. Matrices E, F and functions h and ϕ are assumed to be known. The electricity carrier gives rise to the following energy balancing constraint, Hpp + Hrr + Huu + Hnn = 0 (3) (3) where matrices Hp, Hr, Hu, Hn are used to model the power ﬂows aﬀecting the balancing node. Here pt is the power purchased from the grid, r is the power produced by renewable sources (i.e. the local photovoltaic array) which is assumed to be perfectly known for the whole horizon and n is associated to the curtailment of excess renewable energy (alternatively, one could also sell back to the grid the excess in solar power, adding an additional revenue). 3. Optimization framework ϕ(s) ≤0 xs t+1 = Axs t + Bus t + Cξs t Exs + Fus ≤h (s · bs) Hpps + Hrrs + Huus + Hnns = 0 πs P s∈S JCO2 (s, us, bs, xs, ps, ns, ξs) ≤ϵ ∀s ∈S (9) mins,u,b,x,p,n s.t. (9) 3 IOP Publishing CISBAT 2021 2042 (2021) 012034 doi:10.1088/1742-6596/2042/1/012034 Journal of Physics: Conference Series Figure 1: Graphical representation of the workﬂow for the approximation of (8) Figure 1: Graphical representation of the workﬂow for the approximation of (8) where s represents a scenario in the set of all considered scenarios S with probability of occurrence πs, leading to the demand ξs. Program (9) is typically intractable as the number of second-stage variables grows with T ·\|S\| where T = 8760 and \|S\| can be in the order of hundreds (see [8]). To reduce the computational burden, a two-level approximation scheme is introduced. The ﬁrst approximation is based on representative days (see [7] for a complete explanation of the representative days approach): the entire year is represented by means of R representative days (with R ≪N) which are selected according to a k-mean clustering approach based on the deterministic input data (in this work, electricity price and solar irradiance). The second approximation is based on the selection of a subset Sr of S with \|Sr\| ≪\|S\|. We model the daily hydrogen demand according to a Poisson distribution under the assumption that each day is independent from the others. We generate M samples (i.e. daily patterns) of the hydrogen demand by randomly sampling from the underlying distribution and reduce them to m representative samples with m ≪M, using reduction techniques (see [6] and references therein). The probability of each reduced sample mi is calculated as the number of samples in the cluster mi divided by total number of samples generated. The resulting daily samples m are then concatenated to form \|Sr\| = mR scenarios (resulting in yearly patterns) by considering all sequences deriving from all possible combinations of the reduced samples along the representative days. The corresponding probability of each scenario is given by the product of the probabilities of all samples composing the scenario. In this way, the number of second-stage variables is reduced to K · R · mR, which for an appropriate choice of the daily time interval K, the representative days R and the reduced scenarios mR represents a signiﬁcant reduction. 3. Optimization framework The logic behind the two-level approximation scheme of (9) is represented in Figure 1. 4. Numerical results We consider solar irradiance data for the city of D¨ubendorf (CH) in 2019 and electricity price data from EPEX Spot Market for Switzerland in the same year. We synthesize the hydrogen demand data simulating a small-size ﬂeet of fuel cell vehicles by means of a Poisson distribution. We select R = 4 (corresponding approximately to “winter”, “spring”, “summer” and “autumn” conditions) as compromise between computational complexity and model accuracy and we reduce the number of daily samples to m = 3 (corresponding to “high”, “medium” and “low” hydrogen demand). The MILP in (9) is solved using Gurobi branch-and-bound algorithm to the default optimality gap on an Intel i7-10510U CPU with 16 GB RAM. We initially solve (9) for the maximum annual proﬁt only (eﬀectively set ϵ = +∞). Once (9) is solved, additional scenarios are generated in order to evaluate the out-of-sample performance of the method and the corresponding expected economic indicators for the system (Figure 2). Optimization results for the single-objective program for the deterministic and the stochastic 4 2042 (2021) 012034 IOP Publishing doi:10.1088/1742-6596/2042/1/012034 CISBAT 2021 Journal of Physics: Conference Series Det. case Stoch.case Devices size and investment cost Electrolyzer 161 kW 217 kW Storage tank 431 kWh 582 kWh Photovoltaic 0 m2 0 m2 Investment cost 389’824 CHF 491’139 CHF Out-of-sample performance E[demand loss mean] 47’469 kWh/y 7’510 kWh/y E[demand loss std] 3’773 kWh/y 1’439 kWh/y E[cost of violation] 21’191 CHF/y 3’353 CHF/y E[demand covered] 91.3 % 98.6 % Economic indicators E[Payback] 2.3 years 2.8 years E[NPV] 945’670 CHF 948’460 CHF E[Annualized ROI] 13.3 % 6.8 % Table 1: Comparison of optimization results for the deterministic and stochastic case Figure 2: Schematic workﬂow for out- of-sample performance evaluation Figure 2: Schematic workﬂow for out- of-sample performance evaluation Table 1: Comparison of optimization results for the deterministic and stochastic case Table 1: Comparison of optimization results for the deterministic and stochastic case settings are compared in Table 1. Table 1 shows that the deterministic case, which assumes a “medium” demand, tends to underestimate the device size for the electrolyzer and the storage tank by 26%: this leads to an expected unserved demand of 47’469 kWh/y (≈10% of the overall demand) with a consequent economic loss of 21’191 CHF/y. On the contrary the stochastic case leads to an expected unserved demand of 7’510 kWh/y with an economic loss of only 3’353 CHF/y. 4. Numerical results In both settings the optimal photovoltaic panels size for minimum costs achievement is 0 m2, corresponding to not having this device installed: this is due to the cheap electricity price with respect to photovoltaic installation costs. The key performance indicators for the stochastic setting are annualized Return On Investment (ROI) of 6.8 %, Net Present Value (NPV) of 948’460 CHF and payback time of 2.8 years. The net cash ﬂow over system lifetime is reported in Figure 3 under the assumption that all inputs, namely electricity price, hydrogen selling price and hydrogen demand distribution, remain constant throughout the system lifetime. A more realistic setup should, however, consider changes in these inputs (e.g. the demand of FCVs is expected to grow). g ) A Pareto front is constructed by solving the two single-objective optimization problems (respectively, maximize proﬁt and minimize carbon footprint) and relying on the ϵ-constraint method for the intermediate points between the two extremes of the front. The Pareto front (Figure 4) presents a sharp slope at the very beginning corresponding to a change in the system design. For low GWP value, larger electrolyzer and photovoltaic panels are required with a consequent increase in the investment cost up to 556 kW, 500 m2 and 296’346 CHF/y respectively for the extremum point corresponding to a “sustainable” single-objective optimization (point B). As we allow for more and more emissions, the device sizes diminish down to the results recovered in Table 1 for the economic single-objective optimization (point A). At around 1’600 GWP/y (point D), the system conﬁguration and the total annual cost do not change signiﬁcantly anymore. The convexity of the Pareto front favours the inclusion of “sustainable” objectives into the design of hydrogen refuelling stations: for instance, we can get a reduction of 68% in GWP/y by only increasing the total annual cost by 12% (point C). 5 CISBAT 2021 2042 (2021) 012034 g doi:10.1088/1742-6596/2042/1/012034 Journal of Physics: Conference Series Figure 3: Cumulative cash ﬂow during system lifetime for stochastic setting Figure 4: Pareto front for the multi-objective optimization program Figure 3: Cumulative cash ﬂow during system lifetime for stochastic setting Figure 4: Pareto front for the multi-objective optimization program 5. Conclusions This paper presents a two-stage stochastic programming approach for the optimal design of hydrogen refuelling stations considering uncertainties in the hydrogen demand. We rely on two approximation schemes, namely representative days and scenarios reduction. We show that the deterministic case tends to underestimate the sizing of the devices, leading to the incapability of serving a signiﬁcant fraction of the demand in out-of-sample scenarios. On the contrary, the stochastic formulation provides the reassurance of an optimal design with better out-of-sample performances. The multi-objective optimization produces a convex Pareto front indicating that the introduction of the “sustainable” goal into the optimal design of hydrogen refuelling stations is still economically viable. As future work, we plan to extend our work in order to incorporate uncertainties also in the other inputs and to consider the optimal selection of the facility location as part of the ﬁrst-stage objective. Acknowledgments g This work is part of the Renewable Energy Management and Real-Time Control Platform (ReMaP) project supported by the Swiss Federal Oﬃce of Energy. 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W2619593056.txt	https://europepmc.org/articles/pmc5617511?pdf=render	en		Occupational ultraviolet exposure and risk of non-Hodgkin’s lymphomas: a meta-analysis	Oncotarget	2,017	cc-by	5,693	www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 37), pp: 62358-62370 Meta-Analysis Occupational ultraviolet exposure and risk of non-Hodgkin’s lymphomas: a meta-analysis Demin Lu1,2,, Fei Xu1,, Kaiming Hu1, Li Yin1, Huijie Duan1, Jiaojiao Zhang1 and SuZhan Zhang1,3 1 Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education, Key Laboratory of Molecular Biology in Medical Sciences), The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China 2 Department of Medical Oncology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China 3 Research Center for Air Pollution and Health, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China * These authors have contributed equally to this work Correspondence to: SuZhan Zhang, email: zuci@zju.edu.cn Keywords: occupational ultraviolet exposure, non-Hodgkin lymphoma, meta-analysis Received: November 30, 2016 Accepted: April 11, 2017 Published: May 24, 2017 Copyright: Lu et al. TThis is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Non-Hodgkin lymphoma is a heterogeneous group of lympho-proliferative disorders. We performed a meta-analysis to summarize the available evidence from case-control studies and cohort study on the inconsistent association between occupational sun exposure and the risk of non-Hodgkin lymphoma. We searched PubMed, ISI web of science, the Cochrane Library, EMBASE and reference lists for relevant articles. Study specific odds ratios or relative risk and 95% confidence intervals were pooled by using fixed-effects or random-effects models. Ten case-control studies and one cohort study were included in the meta-analysis. Overall, the pooled odds ratios for occupational ultraviolet exposure and non-Hodgkin lymphoma risk was 1.15(95% confidence intervals: 0.99, 1.32; I2 = 44.4%). Occupational sun exposure was positively associated with the risk of NHL 1.14 (95% confidence intervals: 1.05, 1.23; I2=25.4% p for heterogeneity =0.202) in Caucasian population. Common subtypes of non-Hodgkin lymphoma and ultraviolet exposure had the negative results. The pooled odds ratios was 1.16, (95%confidence intervals: 0.90, 1.50) for T-cell non-Hodgkin lymphoma; 0.79, (95%confidence intervals: 0.61, 1.02) for B-cell non-Hodgkin lymphoma; 1.13, (95%confidence intervals: 0.96, 1.34) for chronic lymphocytic leukemia; 1.25, (95%confidence intervals: 0.95, 1.64) for males; 1.49, (95%confidence intervals: 0.99, 2.25) for females. Data suggested that occupational ultraviolet exposure was a risk factor for non-Hodgkin lymphoma in Caucasian population. While, there had no relationship between occupational ultraviolet exposure and risk of non-Hodgkin lymphoma in general population as well as non-Hodgkin lymphoma common subtypes. Besides, gender specific occupational sun exposure also indicated no association on risk of non-Hodgkin lymphoma. States and in the United Kingdom [2, 3], and is estimated to be the tenth most common cancer worldwide [3]. The incidence of NHL increased dramatically between 1970 and 1995 .This remarkable rise suggests a major role for environmental factors in the etiology INTRODUCTION Non-Hodgkin lymphoma (NHL) is a heterogeneous group of lympho-proliferative disorders [1]. As statistic data showed, NHL is the sixth most common cancer in the United www.impactjournals.com/oncotarget 62358 Oncotarget of NHL, thus the hypothesis that solar ultraviolet(UV) radiation may explain this trend emerged in 1992 [4]. As the increasing incidence of NHL mounting studies associated with the relationship between sun exposure and risk of NHL were carried out in westernized countries. But the results have been inconsistent. We therefore undertook a meta-analysis of case-control and cohort studies to quantitatively assess the relationship between occupational sun exposure and risk of NHL and common NHL subtypes. was though self-administered questionnaire and followed by a telephone interview [27-29, 31]. One study used population occupational health service program data [36]. The age of participants were all aged 17 and above. The exposure odds ratios (ORs)/relative risk (RRs) of NHL, the adjustments made for confounding and occupational history assessment were shown in Table 2. Association between occupational sun exposure and risk of NHL RESULTS In the meta-analysis, the summary estimated for occupational sun exposure showed no statistical association between occupational sun exposure and risk of NHL 1.15(95%CI: 0.99, 1.32; I2=44.4%). (Figure 2) Among the 11 enrolled studies, three studies showed positive relationship between occupational sun exposure and the risk of NHL [27, 29, 33]. The ORs differ from 0.75(95%CI: 0.55, 1.03) to 2.30(95%CI: 0.96, 6.20). There was a moderate heterogeneity among these studies (I2=44.4%, p for heterogeneity =0.048), therefore we used the random-effect model to calculate the summary OR. Ten studies reported the Caucasian population. Occupational sun exposure was positively associated with the risk of NHL 1.14 (95%CI: 1.05, 1.23; I2=25.4% p for heterogeneity =0.202). No heterogeneity was observed, thus fixed-effect model was provided. (Figure 3) Our results for occupational sun exposure did not display an association in risk for common NHL subtypes. The results of T-cell NHL (ORs: 1.16; 95%CI: 0.90, 1.50) and B-cell NHL (ORs: 0.79; 95%CI: 0.61, 1.02) analyses were presented in Figure 4A and Figure 4B. There are no association in Chronic Lymphocytic Leukemia (CLL) either. The summary ORs for CLL was 1.13 (0.96, 1.34) (Figure 4C). Gender specific information on occupational sun exposure and risk of NHL was available in three studies [28, 33, 36]. The pooled ORs were 1.25 (95%CI: 0.95, 1.64) (Figure 5A) for males and 1.49 (95%CI: 0.99, 2.25) (Figure 5B) for females. We observed no heterogeneity, so fixedeffect model was provided. Literature search Our systematic literature search details were shown in (Figure 1) based on search strategy and inclusion criteria. We identified 1034 abstracts from PubMed, ISI Web of Science, the Cochrane Library and EMBASE. After removing duplication 940 abstracts were remained. Of these 940 abstracts, 907 were excluded after screening titles and abstracts. The remaining 33 of them were extracted for further assessment. Twenty-two articles were excluded after full-test review. Fifteen articles were excluded because they did not investigate the association between sun exposure and NHL risk [5-19], five was insufficient information [20-24], and two was the duplicate report on the same study population [25, 26]. Thus, ten case-control studies [27-35, 37] and one cohort study [36] were eligible for our meta-analysis. Study characteristics The characteristics of the 11 eligible studies were summarized in (Table 1). All studies were published between 1997 and 2012. Overall, the studies included 8829 NHL patients out of 336,557 participants. Of the 11 independent studies, ten were case-control studies [27-35, 37] and one was cohort study [36]. Two studies (one casecontrol and one cohort study) were conducted in Sweden [27, 36], two in Australia (two case-control studies) [28, 31], two multi-countries study in Europe (two casecontrol studies) [34, 37] one each in Italy [30], Sweden and Denmark [29], Germany [32], USA [33], and one in Singapore with Asian population [35]. One study included only women [33] and one study included only men [36], while the rest of the studies did not specify with gender. One study reported the Asian population [35], while the rest of studies focused on Caucasian population. Nine studies reported results for all types of NHL patients, while two studies only included specific types of NHL. The Sweden study included only hairy cell leukemia cases [27]. One European study included only mycosis fungoides cases [37]. The control source of eight studies were population based [27-29, 31-33, 36, 37], two studies were hospital based [30, 35], while one study included both population control and hospital based control source [34]. Six studies’ data collection method was in-face interview [30, 32-35, 37], while four studies’ data collection method www.impactjournals.com/oncotarget Publication bias and sensitivity analyses In order to evaluate the impact of potential publication bias, we applied the Begg’s test (p=0.37) and Egger’s test (p=0.37) for the association between occupational sun exposure and the risk of NHL. The results indicated no publication bias among these studies. In addition, no publication bias was detected for the positive association between occupational sun exposure and the risk of NHL in Caucasian population, either (Begg’s test: p=0.53; Egger’s test: p=0.14). To investigate heterogeneity in our meta-analysis, we evaluated sensitivity analysis within the studies. Eleven studies which included in our meta-analysis were the relationship between occupational sun exposure and risk of NHL, two studies were focused on specific 62359 Oncotarget types of NHL [27, 37], so we conducted a sensitivity analysis restricted to those nine studies. Results did not change when the aforementioned studies were included or excluded. The pooled OR was 1.09 (95%CI: 0.97, 1.23) with a significant decreased heterogeneity among these nine studies (I2=25.1%, p for heterogeneity =0.21 (Figure 6A). The result was consistently to the overall pooled OR, which suggested our study was reliable. Data collection method from six studies were in person interview [30, 32-35, 37], four studies were though self-administered questionnaire and followed by a telephone interview [27-29, 31], and one study was used the data files from nationwide occupational health service organization [36]. We conducted a sensitivity analysis restricted to six in person interview studies [30, 32-35, 37].Results of pooled OR was similar 1.03(95%CI: 0.81, 1.30) (Figure 6B). There were almost no heterogeneity among studies (I2=40.2%, p for heterogeneity =0.12). We applied sensitivity analysis on our positive results among Caucasian population. The pooled estimate did not vary substantially with the exclusion of any single study (Figure 7). DISCUSSION Our meta-analysis of 11 included case-control and cohort studies indicated that occupational ultraviolet exposure was a risk factor for NHL in Caucasian population. However, there is no association between occupational sun exposure and risk of NHL in general population as well as common NHL subtypes. In addition, gender specific study did not show any association either. Most studies included in our meta-analysis showed no association between occupational sun exposure and risk of NHL except for Nordstorm et al (OR: 2.2, 95%CI: 1.2-3.8) [27] and Zhang et al (OR: 1.8, 95%CI: 1.0-3.4) [33]. Those two studies reported occupational ultraviolet Figure 1: Flowchart of selection of studies for inclusion in the meta-analysis. www.impactjournals.com/oncotarget 62360 Oncotarget Table 1: Study features of 11 included studies Reference Study type Location Nordstrom 1997 [27] Casecontrol Sweden Hughes 2004 [28] Casecontrol Type of source Assessment of measures 121(--)/484(--) National population registry Mailed questionnaire; telephone interview Australia 2000-2001 20-74 704(85%)/694(61%) Electoral rolls Selfadministered questionnaire; telephone interview Smedby 2005 [29] Casecontrol Denmark 1999-2002 18-74 and Sweden 3055(81%)/3187(71%) Population Telephone interview Morales 2006 [37] Casecontrol Europe 76(91.6%)/2094(--) Population Face-to-face registries interview or electoral rolls Tavani 2006 [30] Casecontrol Northern 1985-1997 18-79 Italy 446(>97%)/1295(>97%) Patients Personal hospitalized interview with other conditions Karipidis 2007 [31] Casecontrol Australia 2000-2001 20-74 694(85%)/694(61%) Weihkopf 2007 [32] Casecontrol Germany 589(87.4%)/589(51.4%) Population registers Face-to-face interview Zhang 2007 [33] Casecontrol USA 1996-2000 21-84 601(72%)/706(--) In person interview Boffetta 2008 [34] Casecontrol Europe 1998-2003 >17 1518(88%)/2124(81% in Population In person hospital controls, 52% in registers; interview population controls) Patients hospitalized with other conditions Wong 2012 [35] Singapore 2004-2008 >18 Reference Casecontrol Study type Hakansson Cohort 2001 [36] Location Sweden www.impactjournals.com/oncotarget Study period Age Case (participation)/ range control (participation) 1987-1992 --- 1995-1997 35-69 18-80 541(--)/830(--) Population, Selfelectoral administered rolls questionnaire; telephone interview Population Patients Face-to-face hospitalized interview with other conditions Follow-up years Case/cohort Cohort source 1971-1993 484/323860 Nationwide occupational health service program of the Swedish construction industry 62361 Assessment of measures Used data files from nationwide occupational health service organization Oncotarget Table 2: Adjustments and occupational history assessment reported by single study in this meta-analysis Reference Study type OR (95% CI) Adjustments Occupational history assessment Nordstrom 1997 Case-control HCL 2.2 (1.2-3.8) Age, sex and country All occupations lasting longer than 1 year [27] were classified according to the Nordic Working Classification System (NYK) 1989. Hughes 2004 [28] Case-control NHL 1.21 (0.87-1.69) Men 1.20 (0.81-1.78) Women 1.27 (0.73-2.23) Age, sex, state of residence, ethnicity, skin color and ability to tan Smedby 2005 [29] Case-control NHL 1.1 (1.0-1.2) Age, sex, country CLL 1.1 (0.9and skin reaction to 1.3) DLBCL 1.2 sun (1.0-1.4) FL 0.7 (0.5-0.9) T-NHL 1.2 (0.9-1.7) A standardized and computer-aided questionnaire, outdoor occupation lasting 1 year or more (ever/never) Morales 2006 [37] Case-control MF 2.3 (0.9-6.2) A structured questionnaire. The type of occupation and industry was asked for each job, including the year the job started and ended. Recorded work tasks, job title, and working hours per week for each occupational period. The specific nature of the work also was addressed, such as machines or products used, duration of their use (hours per week), and dates of job tenure. Age, sex, region, exposure to aromatic halogenated hydrocarbons Hours of occupational sun exposure. For each job recorded in the calendar, data were collected about the number of days worked per week, hours worked per day and hours worked outdoors per day. Occupational hours of exposure were totalled for 50 weeks a year, assuming 2 weeks for vacations and sick leave Tavani 2006 [30] Case-control NHL 0.96 (0.661.40) Age, sex, area of A structured questionnaire, study residence , education participants were asked whether they had and smoking been exposed to UV radiation at work and for how long Karipidis 2007 [31] Case-control NHL 1.32 (0.961.81) Age, sex, region of residence, ethnic origin The questionnaire included a lifetime calendar that was used to obtain a detailed occupational history from each subject, including information about job title, employer, industry, start and finish years, number of hours worked per day and number of days worked per week Weihkopf 2007 [32] Case-control T-NHL 0.9 (0.33.5) B-NHL 0.9 (0.6-1.4) Age, sex, region, smoking (packyears) and alcohol consumption A complete occupational history, including every occupational period that lasted at least 1 year. For every job held, information was obtained about the start and the end of the job phase, about job title, industry and specific job tasks. Study subjects having held potentially hazardous jobs were additionally asked to reply to job task-specific supplementary questions. (continued) www.impactjournals.com/oncotarget 62362 Oncotarget Reference Study type OR (95% CI) Adjustments Occupational history assessment A standardized, structured questionnaire. For the history of occupational exposure to ultraviolet radiation, subjects were asked to Age, race, family provide all job titles and main duties that they history of NHL, NHL WOMEN had for 1 year or longer before diagnosis (for Zhang 2007 [33] Case-control highest educational 1.8 (1.0-3.4) cases) or interview (for controls). Each job level, eye color and title was designated as indoor (purely indoor skin type or mixed type) or outdoor. If an individual had both indoor and outdoor jobs, she was assigned to the outdoor job category. Information on occupation was collected at interview for each job held for at least 1 year in a general questionnaire and in 14 NHL 1.08 questionnaires specific to jobs and industries (0.74-1.56) CLL likely to entail exposure to suspected 1.36 (0.86-2.14) Age, sex, study lymphoma carcinogens (dry cleaners, farmers Boffetta 2008 DLBCL 0.69 area, education, skin Case-control or gardeners, textile workers, meat workers [34] (0.42-1.15) FL reaction to sun and or slaughterers, chemical industry workers, 0.57 (0.31-1.06) questionnaire type painters, hairdressers, wood workers, T-NHL 1.14 (0.59printers, leather or tannery workers, teachers 2.21) or others working with children, metal degreasers, health professionals and grain millers or bakers). Participants were defined as outdoor workers Age, sex, study if they had spent at least 30 min working NHL 0.75 (0.55- center , month of outside under sun (between 9 am and 5 pm) 1.03) B-NHL diagnosis, race, in any of the jobs that lasted 1 year or more. Wong 2012 [35] Case-control 0.73 (0.53-1.02) education, housing Categorized participants into ‘‘indoor work T-NHL 1.10 (0.55- type, BMI, history of only’’ workers, and those who spent all or 2.21) any cancer in the first part of their working hours outdoors ‘‘mixed degree relatives indoor ± outdoor’’ workers. Hakansson 2001 [36] Cohort NHL Men 1.3 (0.9-1.9) Age, smoking, and magnetic field exposure exposure increased the risk of NHL. InterLymph organization systematically analyzed ten studies about occupational UV exposure and NHL incidence in 2008, results were consistent with our main meta-analysis [38]. Our main findings showed no association between occupational ultraviolet exposure and NHL risk. When we omitted the study of Asian population our results are positively correlated. Hughes et al reported the pigmentary characteristics and NHL risk. In this study, it categorized the six ethnicity groups including Asian. They have found that the very fair skin compared to brown or olive skin had 44% increased risk of NHL [25]. Interestingly, the Singapore study was not only the Asian population study but also the only low latitudes study [35]. Several studies have yielded www.impactjournals.com/oncotarget The occupational exposure to sunlight from outdoor work was assessed by an experienced industrial hygienist from the construction industry (N. Hallin). The hygienist classified the sunlight exposure for the job tasks into four categories with exposure scores 0, 1, 2, and 3 the same results. Reduced risk or no effect were found in mid latitudes or low latitudes [28, 38], while increased risk was found at higher latitudes [26, 39-41]. Grant WB proposes that UVA is a risk factor through impairing the immune system, while UVB is a protection factor through vitamin D production [42]. The ratio of UVA/UVB increases while latitude increases. Thus, the pooled result of our higher latitude Caucasian population studies showed that occupational UV exposure was a risk factor for NHL. Many of the studies which included in this metaanalysis reported that there is no relationship between occupational UV exposure and NHL risk, however at the same time, reported the daily, casual UV exposure is a protective factor for NHL [28, 29, 32, 34, 35]. This 62363 Oncotarget Figure 2: Forest plot and summary OR of the association between occupational sun exposure and risk of NHL. Figure 3: Forest plot and summary OR for Caucasian population of occupational sun exposure and risk of NHL. www.impactjournals.com/oncotarget 62364 Oncotarget result was consistent with InterLymph Organization2008 analysis [43]. One possible explanation is that most studies would have attributed any time duration occupational UV exposure cases into exposure group, while for the individual, it may be exposed to a period of time, rather than continuous exposure [28, 35, 43]. In addition, farmers often categorized into occupational UV exposure group, but some of these people at the same time contacting potential risk factors NHL such as pesticides, alkylation, etc [30, 36]. For example, Smedy et al published that Ever having an outdoor occupation for 1 year or more was associated with a slightly increased risk of non-Hodgkin lymphoma (OR = 1.2, 95% CI = 1.0 to 1.3), but this association was weakened (OR = 1.1, 95% CI = 1.0 to 1.2, ) after additional adjustment for occupational exposure to pesticides [29]. Figure 4: Forest plot and summary OR of T-cell NHL (A), B-cell NHL (B) and CLL (C). Figure 5: Forest plot and summary OR of male (A) and female (B). www.impactjournals.com/oncotarget 62365 Oncotarget As with any meta-analysis of observational studies, our study has limitations. First, moderate heterogeneity was found across our main analysis, which can be explained by the multiple differences between studies with regard to the study designs, sample sizes, analysis strategies, participants’ baseline characteristics, adjustments for confounders and occupational history assessment methods. For example, the control source of eight studies were population based [27-29, 31-33, 36, 37], and two studies were hospital based [30, 35], while one study included both population control and hospital based control source [34]. Sample sizes were different from eleven studies included in this metaanalysis. Two studies had relatively small numbers of participants and specific NHL types [27, 37], which raised some concerns regarding the reliability of their results. Figure 6: Sensitivity analysis of forest plot and summary OR of nine studies (A) and face to face interview studies (B). Figure 7: Sensitivity analysis for Caucasian population of occupational sun exposure and risk of NHL. www.impactjournals.com/oncotarget 62366 Oncotarget Thus, we used the random-effects model to determine the overall estimate of variability. Secondly, half of the studies in this meta-analysis relied on self-administered questionnaire, while anther half studies gathered information from interview. The participants may have different attitudes and different understanding towards questions under different methods. Besides, the total occupational sun exposure hours are varies across studies and participants. Finally, in a metaanalysis of published studies, the potential publication bias might influence the results, because studies with null results tend not to be published. Nevertheless, our publication bias test showed no possible bias. In summary, our meta-analysis suggested that occupational UV exposure was a risk factor for NHL in Caucasian population. While, there had no relationship between occupational ultraviolet exposure and risk of NHL as well as NHL common subtypes. Besides, gender specific occupational sun exposure also indicated no association on risk of NHL. study location, sample size (numbers of case patients and control subjects), study period, participation’s age, type of control source, assessment of data collection, and statistical covariates adjustment in the analysis. ORs or RRs with corresponding 95%CIs for each study were either extracted directly from the article or calculated from available raw data. Statistical analysis We reported this article in accordance with MOOSE (meta-analysis of observational studies in epidemiology) guidelines [44]. We systematically searched four databases: PubMed, ISI web of science, the Cochrane Library and EMBASE for studies published in any languages (up to 2016, August 17th) using the following search items: ultraviolet radiation, ultraviolet ray, ultraviolet light, sunlight exposure or solar ultraviolet exposure combined with non-Hodgkin lymphoma or lymphoid malignancies. The search was restricted to studies of human participants. We also have reviewed the reference lists of all pertinent articles to search for more studies. To pool the results of individual studies together, we used a general variance-based method in the meta-analysis. The multivariate adjusted ORs and 95% CIs presented in the literature were used. In situations where the incidence is low, the odds ratio approximates the relative risk, therefore, in looking at studies of NHL (a rare condition), it is acceptable to compare OR and RR estimates [45]. The outcomes are presented as a forest plot with 95% CIs. Statistical heterogeneity among studies was tested with the Q statistic, and statistical inconsistency was quantified with the I2 statistic [46]. When I2 was from 0% to 40% along with p >0.10 the heterogeneity might not be important. If the meta-analysis has no heterogeneity, fixedeffects model with the Mantel-Haeszel method would be used to combine the individual studies [47], otherwise, the random-effects method (DerSimonian 1986) was used for pooling [48]. There were two studies focused only on specific types of NHL [27, 37]. We further conducted a sensitivity analysis restricted to the rest nine studies to evaluate the stability of the pooled estimates between occupational sun exposure and risk of NHL. The Egger’s test and Begg’s test were used to assess for publication bias [49, 50]. P<0.05 was considered statistically significant publication bias. All statistical analyses was performed by using STATA (version 11.0; StataCorp, College Station, TX). Inclusion criteria CONFLICTS OF INTEREST MATERIALS AND METHODS Literature search To be included in this meta-analysis, studies had to have met the following criteria: (1) NHL cases were medically confirmed by histopathology diagnoses; (2) the study was designed as case-control or cohort study; (3) the occupational sunlight exposure and incidence of NHL were associated; (4) detailed data of odds ratios (ORs) or relative risks (RRs) with 95% confidence interval (CI); (5) All the cases were adult (age≥17 years old). We did not include the studies that only report the death rate of NHL without incidence rate. When there were multiple published reports from the same study population, the most recent or the most informative report was selected for analysis. The authors declare no conflicts of interest. GRANT SUPPORT Supported by Training Program of the Major Research Plan of the National Natural Science Foundation of China, No. 91229104; Key Projects in the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period, No. 2014BAI09B07; National High Technology Research and Development Program of China (863 Program), No. 2012AA02A506; National High Technology Research and Development Program of China (863 Program), No. 2012AA02A204; Zhejiang Provincial Natural Science Foundation of China, No. LQ14H160010; National Natural Science Foundation of China, No. 81502598. Data extraction We extracted the following information from each study: authors’ name, year of publication, study type, www.impactjournals.com/oncotarget 62367 Oncotarget REFERENCES Cancer Causes Control. 2010; 21:1265–75. doi: 10.1007/ s10552-010-9554-1. 1. 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https://openalex.org/W4224291983	https://link.springer.com/content/pdf/10.1007/s12020-022-03052-5.pdf	English	null	A combined index of waist circumference and muscle quality is associated with cardiovascular disease risk factor accumulation in Japanese obese patients: a cross-sectional study	Endocrine	2,022	cc-by	8,490	Abstract Abstract Purpose To identify obese patients at high risk of cardiovascular disease (CVD) using a combined index of obesity and sarcopenia. Methods In this cross-sectional study, we ﬁrstly conducted analysis of covariance to select each index most associated with the CVD risk score, the number of concomitant CVD risk factors, among obesity- (body mass index, percentage body fat, or waist circumference [WC]) and sarcopenia-evaluated indices (skeletal muscle mass index, handgrip strength, or muscle quality [MQ]), respectively in 188 Japanese obese patients (BMI ≥25 kg/m2, 73 men and 115 women). Next, we conducted multi- variate logistic regression analysis to compare the four groups (Group A–D) classiﬁed by medians of the selected indices. Results WC and MQ were selected as the indices most associated with the CVD risk scores, respectively. The CVD risk score was signiﬁcantly higher in Group B (low WC and low MQ) and Group D (high WC and low MQ) with higher prevalence of diabetes as compared with Group A (low WC and high MQ). Adjusted for sex and age, odds ratios for CVD risk scores = 2 were signiﬁcantly higher in Group B, Group C (high WC and high MQ), and Group D compared with Group A. Furthermore, odds ratios for CVD risk scores = 3 were signiﬁcantly higher only in Group D compared with Group A (4.29 [95% conﬁdence interval: 1.49–12.33], p = 0.007). Conclusion Combined index of WC and MQ was useful in Japanese obese patients at high risk of CVD, regardless sex and age. Keywords Obesity, Sarcopenia ●Sarcopenic obesity ●Waist circumference ●Muscle quality ●Cardiovascular disease Endocrine (2022) 77:30–40 https://doi.org/10.1007/s12020-022-03052-5 Endocrine (2022) 77:30–40 https://doi.org/10.1007/s12020-022-03052-5 ORIGINAL ARTICLE * Toru Kusakabe kusakabe@kuhp.kyoto-u.ac.jp Kentaro Ikeue1,2 ●Toru Kusakabe 1 ●Kazuya Muranaka1 ●Hajime Yamakage1 ●Takayuki Inoue1 ●Kojiro Ishii3 ● Noriko Satoh-Asahara1 Received: 13 January 2022 / Accepted: 29 March 2022 / Published online: 19 April 2022 © The Author(s) 2022 3 Faculty of Health and Sports Science, Doshisha University, 1-3 Tatara Miyakodani, Kyotanabe, Kyoto 610-0394, Japan A combined index of waist circumference and muscle quality is associated with cardiovascular disease risk factor accumulation in Japanese obese patients: a cross-sectional study Kentaro Ikeue1,2 ●Toru Kusakabe 1 ●Kazuya Muranaka1 ●Hajime Yamakage1 ●Takayuki Inoue1 ●Kojiro Ishii3 ● Noriko Satoh-Asahara1 Study participants This cross-sectional study included obese outpatients who regularly visited the Diabetes Center at the National Hos- pital Organization Kyoto Medical Center between January 2019 and July 2019. The diagnosis of obesity was based on the standards of Japan Society for the Study of Obesity, BMI ≥25 kg/m2 [31]. We uniformly provided exercise and dietary guidance for all obese outpatients in accordance with the guidelines of the Japan Society for the study of obesity. We excluded participants with incomplete data, implantation of a cardiac pacemaker, and cancer from the study. None of the patients had sarcopenia secondary to CVDs, respiratory diseases, endocrinological diseases or conditions of secondary obesity such as Cushing’s syn- drome. This study was approved by the Ethics Committee for Human Research at National Hospital Organization Kyoto Medical Center (approval No. 19-083) and was conducted in accordance with the principles of the Declaration of Helsinki and the ethical guidelines for medical and health research involving human subjects. Elderly individuals with SO have higher risks of low physical function [17, 18], metabolic diseases [19, 20], CVD [21–23], and mortality [21, 24]. These clinical pro- blems in SO are much more severe than in sarcopenia or obesity alone. In the diagnosis of SO, sarcopenia and obe- sity have been diagnosed separately as two distinct cate- gories. However, worldwide diagnostic criteria for SO and its cutoff values have not yet been established [25, 26]. One probable reason for the difﬁculty in establishing diagnostic criteria for SO is that there are multiple ways to measure body composition. Magnetic resonance imaging (MRI) and computed tomography (CT) are considered to be gold standards for non-invasive assessment of muscle mass [27]. However, these tools are not commonly in primary care because of high equipment costs, lack of portability [27]. Dual-energy X-ray absorptiometry (DXA) is a more widely available instrument to muscle mass, however not yet por- table for use [5]. Recently, bioelectrical impedance analysis device (BIA) is affordable, widely available and portable [5]. Body composition measured using a multifrequency BIA was highly correlated with measurements obtained from of DXA [28]. Another probable reason is that there are multiple combinations for evaluating sarcopenia and obe- sity. For example, Kim et al. diagnosed SO by skeletal muscle mass index (SMI) and high percentage body fat (PBF) and investigated its association with metabolic syn- drome [29]. In addition, Schrager et al. Introduction Aging induces changes in body composition,such as an increase in body fat and a decline in skeletal muscle [1, 2]. Body fat increases until the seventh decade of life and decreases thereafter [3]. It has been reported that most of the body fat increase with aging is due to the increase in visceral fat (VF) [4]. On the other hand, skeletal muscle mass and strength reach their maximum amount at young adulthood (up to ~40 years of age) and then decline by several percent each year [5]. Supplementary information The online version contains supplementary material available at https://doi.org/10.1007/s12020- 022-03052-5. * Toru Kusakabe kusakabe@kuhp.kyoto-u.ac.jp 1 Department of Endocrinology, Metabolism, and Hypertension Research, Clinical Research Institute, National Hospital Organization Kyoto Medical Center, 1-1 Fukakusa Mukaihata- cho, Fushimi-ku, Kyoto 612-8555, Japan Sarcopenia is the loss of muscle mass and strength or physical function that occurs naturally with aging [3, 5, 6]. Probable sarcopenia is identiﬁed by low muscle strength, and the diagnosis of sarcopenia is conﬁrmed by the addi- tional documentation of low muscle quantity or quality [5]. According to a recent systematic review and meta-analysis, 2 Graduate School of Health and Sports Science, Doshisha University, 1-3 Tatara Miyakodani, Kyotanabe, Kyoto 610-0394, Japan 3 Faculty of Health and Sports Science, Doshisha University, 1-3 Tatara Miyakodani, Kyotanabe, Kyoto 610-0394, Japan Endocrine (2022) 77:30–40 31 the worldwide prevalence of sarcopenia is 10% (95% con- ﬁdence interval [CI] 8–12%) in men and 10% (95% CI 8–13%) in women, respectively [7]. It has been reported that sarcopenia is associated with a number of different outcomes such as falls and fractures [8–10], disability [8, 11], meta- bolic syndrome [12], CVD [13, 14], and mortality [8, 15]. CVD risk factor accumulation among obesity-evaluated indices, BMI, PBF, or WC, and sarcopenia-evaluated indi- ces, SMI, HGS, or muscle quality (MQ) in Japanese obese patients. We then classiﬁed obese patients into four groups using medians of the two selected indices and compared the CVD risk score. Sarcopenic obesity (SO) was ﬁrst described by Heber et al. as the co-presence of sarcopenia and obesity [16]. Sarcopenia and obesity have some common pathophysio- logical mechanisms, including increased inﬂammatory cytokines, oxidative stress, insulin resistance, hormonal changes, and decreased physical activity [1]. Study participants reported that sar- copenic obesity diagnosed using body mass index (BMI), waist circumference (WC) and handgrip strength (HGS) was associated with elevated proinﬂammatory, especially central obesity and low HGS [30]. The diagnostic criteria for SO needs to be considered for each of the different subjects and clinical outcomes. Introduction Furthermore, a vicious cycle may exist between sarcopenia and obesity; that is, sarcopenia reduces physical activity, leading to an increase in the risk of obesity, and excess accumulation of VF induces inﬂammation, leading to the development of sarcopenia. Therefore, it is feared that sarcopenic obesity will increase with aging. Deﬁnition of CVD risk score In this study, the CVD risk score was deﬁned as the number of concomitant CVD risk factors (hypertension, diabetes, and dyslipidemia; 0–3 points), referring to a previous report [38]. Diagnosis of sarcopenia Sarcopenia was diagnosed by low SMI and weak HGS [6]. The cutoff values for low SMI were <7.0 kg/m2 for men and <5.7 kg/m2 for women and those for weak HGS were <28 kg for men and <18 kg for women according to the guideline of Asian Working Group for Sarcopenia, respectively [6]. Diagnosis of hypertension, diabetes, and dyslipidemia The diagnosis of hypertension, diabetes, and dyslipidemia was based on the criteria of each academic society; hyper- tension was deﬁned as systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg or taking medi- cations for hypertension [35]; diabetes was deﬁned by fasting plasma glucose ≥126 mg/dL, and/or HbA1c (National Glycohemoglobin Standardization Program) ≥6.5%, or taking medications for diabetes [36]; dyslipide- mia was deﬁned by LDL-C ≥140 mg/dL, and/or HDL-C < 40 mg/dL, and/or TG ≥150 mg/dL, or taking medications for dyslipidemia [37]. Fig. 1 Classiﬁcation using each obesity- and sarcopenia-evaluated index. Obese patients were classiﬁed into four groups (Group A, Group B, Group C, and Group D) using medians of each obesity- and sarcopenia-evaluated index Those above the median value were classiﬁed as “high” and those below as “low”. We conducted analysis of covariance (ANCOVA) to compare the CVD risk scores between the low and high groups in each obesity-evaluated index (BMI, PBF, or WC) and sarcopenia-evaluated index (SMI, HGS, or MQ). ANCOVA was constructed as follows: model 1 was unadjusted, model 2 was adjusted for sex, and model 3 was further adjusted for age. Next, we conducted analysis of variance (ANOVA) followed by Tukey’s test or Kruskal–Wallis test followed by Bonferroni correction for continuous variables and chi-square test for categorical variables to compare the characteristics of the four groups (Group A–D). We then conducted ANCOVA followed by Bonferroni correction to compare the CVD risk scores among the four groups. Lastly, to examine the association between severity of CVD risk factors and the combined index, we used multiple logistic regression analysis adjusted for sex and age to determine odds ratios (ORs) and 95% clinical intervals (CIs) for each CVD risk score (=1, =2, and =3) as compared with Group A. Clinical examination We measured height and body weight in increments of 0.1 cm and 0.1 kg, respectively. BMI was calculated as the body weight (kg) divided by the squared height (m2). WC was measured at the umbilical level in a standing position. HGS was measured twice for each hand using the Smedley grip force system (Grip-D, Takei Equipment Company, Tokyo, Japan) in a standing position, and the maximum value was included in the analyses. The appendicular ske- letal muscle mass (ASM) and PBF were measured using a multifrequency BIA (MC-780A-N, TANITA, Tokyo, Japan). SMI was calculated as the ASM (kg) divided by the squared height (m2). In this study, MQ was calculated as the HGS (kg) divided by muscle mass of the upper limbs (kg) according to previous reports [32–34]. Systolic and diastolic blood pressure was measured with an automatic electrical sphygmomanometer (BP-203RVII, Fukuda Colin, Kyoto, Japan). Blood was taken from the antecubital vein in the morning after an overnight fast, and we determined fasting plasma glucose, hemoglobin A1c (HbA1c), triglycerides In this study, to identify obese patients at high risk of CVD, we examined a combined index most associated with 32 Endocrine (2022) 77:30–40 Obesity-related index (BMI, BFP, or WC) Sarcopenia-related index (SMI, HGS, or MQ) Low Low Group A Group C Group D Group B High High Obesity-related index (BMI, BFP, or WC) Sarcopenia-related index (SMI, HGS, or MQ) Low Low Group A Group C Group D Group B High High Fig. 1 Classiﬁcation using each obesity- and sarcopenia-evaluated index. Obese patients were classiﬁed into four groups (Group A, Group B, Group C, and Group D) using medians of each obesity- and sarcopenia-evaluated index (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). Classiﬁcation of obese patients using each obesity- and sarcopenia-evaluated index As shown in Fig. 1, obese patients were classiﬁed into four groups using each obesity- and sarcopenia-evaluated index with the median value; Group A, low obesity-evaluated index and high sarcopenia-evaluated index; Group B, low obesity-evaluated index and low sarcopenia-evaluated index; Group C, high obesity-evaluated index and high sarcopenia-evaluated index; Group D, high obesity- evaluated index and low sarcopenia-evaluated index. Statistical analysis We performed statistical analyses using SPSS (version 25; IBM Corp, Armonk, NY, USA). Data are presented as mean ± standard deviation, median (interquartile range [IQR]), or frequency percentage. In all cases, a probability (p) value of < 0.05 was considered statistically signiﬁcant. Sarcopenia-evaluated index most associated with CVD risk factor accumulation For the sarcopenia-evaluated indices, the median SMI was 8.98 kg/m2 in men and 7.37 kg/m2 in women, the median HGS was 40.6 kg in men and 23.5 kg in women, and the median MQ was 6.63 kg/kg in men and 6.58 kg/kg in women, respectively. Table 2B shows the comparisons of the CVD risk scores between the low and high groups in each sarcopenia-evaluated index. The CVD risk score was signiﬁcantly higher in the low group compared with the high group only for MQ (1.95 [95% CI: 1.77–2.13] vs. 1.57 [95% CI: 1.40–1.75], p < 0.01; model 1). Furthermore, the association persisted even after adjusting for sex (1.95 [95% CI: 1.77–2.12] vs. 1.57 [95% CI: 1.39–1.74], p < 0.01; model 2) and for sex and age (1.93 [95% CI: 1.76–2.10] vs. 1.58 [95% CI: 1.41–1.75], p < 0.01; model 3). Fig. 2 Study ﬂow chart of participants data, one patient with a cardiac pacemaker, and one patient undergoing treatment for cancer were excluded. Finally, 188 Japanese obese patients (73 men and 115 women) were included in the study population. y p p Table 1 summarizes the clinical characteristics of the obese patients. Elderly patients aged 65 years and older included 22 men (30.1%) and 40 women (34.8%), respec- tively. As for the obesity-evaluated indices, BMIs were 30.6 (IQR: 27.3–34.2) kg/m2 in men and 31.8 (IQR: 28.8–36.3) kg/m2 in women, PBF values were 34.3% ± 7.2% in men and 48.5% ± 8.0% in women, and WCs were 103.0 (IQR: 98.5–111.0) cm in men and 102.0 (IQR: 95.0–111.0) cm in women, respectively. There were 71 men (97.3%) with an abdominal circumference of ≥85 cm and 102 women (88.7%) with an abdominal circumference of ≥90 cm, sug- gesting that the participants had substantial VF accumula- tion. On the other hand, as for the sarcopenia-evaluated indices, SMIs were 8.94 ± 1.04 kg/m2 in men and 7.47 ± 0.83 kg/m2 in women, HGS values were 39.6 ± 7.4 kg in men and 23.7 ± 4.9 kg in women, and MQs were 6.59 ± 1.12 kg/kg in men and 6.52 ± 1.18 kg/kg in women, respectively. Low SMI was observed in 3 men (4.1%) and weak HGS was noted in 5 men (6.8%) and 14 women (12.2%), respectively. However, sarcopenia was diagnosed in only one male (1.4%). Sarcopenia-evaluated index most associated with CVD risk factor accumulation The prevalence of hypertension, diabetes, and dyslipidemia was high; therefore, patients had the high CVD risk scores (1.92 ± 0.86 in men and 1.65 ± 0.90 in women). Clinical characteristics of the obese patients classiﬁed by a combined index of WC and MQ Based on the above results, we selected WC and MQ as the indices most associated with CVD risk factor accumulation, respectively. Then, we classiﬁed obese patients into four groups (Group A, low WC and high MQ; Group B, low WC and low MQ; Group C, high WC and high MQ; and Group D, high WC and low MQ) (Fig. 1). By this classiﬁcation, Group B had signiﬁcantly lower MQ compared with Group A (5.49 ± 0.85 vs. 7.50 ± 0.71 kg/kg, p < 0.05), Group C had signiﬁcantly higher WC compared with Group A (111.0 [IQR: 106.3–118.3] vs. 95.0 [IQR: 89.8–99.0] cm, p < 0.05), and Group D had signiﬁcantly lower MQ and higher WC compared with Group A (5.71 ± 0.72 vs. 7.50 ± 0.71 kg/kg, p < 0.05; 110.5 [IQR: 106.8–121.5] vs. 95.0 [IQR: 89.8–99.0] cm, p < 0.05, respectively; Table 3). As for other obesity- and sarcopenia-evaluated indices, both BMI and PBF were also signiﬁcantly higher in Group C and Group D compared with Group A (all p < 0.05), and HGS was also lower in Group B and Group D compared with Group A (all p < 0.05; Table 3). Interestingly, in contrast to MQ, the SMI in Group D was signiﬁcantly higher than that in Group A (8.32 ± 1.27 vs. 7.82 ± 1.03 kg/m2, p < 0.05). Clinical characteristics of the study participants As shown in Fig. 2, 196 patients (76 men and 120 women) were enrolled in this study. Six patients with incomplete Obese patients were dichotomized by the median value in each obesity- and sarcopenia-evaluated index. 33 Endocrine (2022) 77:30–40 Exclusion - Incomplete data n = 6 (1 man, 5 women) - Implantation of a cardiac pacemaker n = 1 (1 man) - Treatment for cancer n = 1 (1 man) Study population N = 188 (73 men, 115 women) Enrollment n = 196 (76 men, 120 women) Fig. 2 Study ﬂow chart of participants signiﬁcantly higher in the high group as compared with the low group only for WC (1.62 [95% CI: 1.43–1.80] vs. 1.88 [95% CI: 1.70–2.05], p < 0.05; model 1). Furthermore, the association persisted even after adjusting for sex (1.61 [95% CI: 1.43–1.80] vs. 1.88 [95% CI: 1.71–2.06], p < 0.05; model 2) and for sex and age (1.58 [95% CI: 1.40–1.76] vs. 1.91 [95% CI: 1.74–2.08], p < 0.01; model 3). Obesity-evaluated index most associated with CVD risk factor accumulation The CVD risk score was Although there was no signiﬁcant difference in the pre- valence of hypertension and dyslipidemia among the four groups, that of diabetes was signiﬁcantly higher in Group B, Endocrine (2022) 77:30–40 34 Table 1 Clinical characteristics of the obese patients All (n = 188) Male (n = 73) Female (n = 115) Age (year) 55.7 ± 15.7 54.7 ± 16.7 56.4 ± 15.1 BMI (kg/m2) 31.2 (28.1–35.0) 30.6 (27.3–34.2) 31.8 (28.8–36.3) PBF (%) 43.0 ± 10.3 34.3 ± 7.2 48.5 ± 8.0 WC (cm) 102.0 (97.0–111.0) 103.0 (98.5–111.0) 102.0 (95.0–111.0) SMI (kg/m2) 8.04 ± 1.16 8.94 ± 1.04 7.47 ± 0.83 HGS (kg) 29.9 ± 9.8 39.6 ± 7.4 23.7 ± 4.9 MQ (kg/kg) 6.55 ± 1.16 6.59 ± 1.12 6.52 ± 1.18 SBP (mmHg) 136.0 ± 13.6 138.0 ± 13.8 134.8 ± 13.4 DBP (mmHg) 82.1 ± 9.5 83.3 ± 9.0 81.3 ± 9.8 FPG (mg/dl) 116.2 ± 32.3 117.9 ± 28.0 115.2 ± 34.8 HbA1c (%) 6.4 ± 1.2 6.4 ± 1.2 6.4 ± 1.3 TG (mg/dL) 133.4 ± 76.2 138.8 ± 83.3 129.9 ± 71.4 HDL-C (mg/dL) 57.5 ± 14.9 52.4 ± 11.5 60.7 ± 15.9 LDL-C (mg/dL) 118.8 ± 28.1 114.8 ± 23.8 121.4 ± 30.4 Current smoker (%) 8.5 12.3 6.1 Hypertension (under treatment) (%) 68.1 (43.1) 74.0 (45.2) 64.3 (41.7) Medications for hypertension (n) (CA/ACEI/ARB/diuretics/β/αβ/DRI) 68/8/57/19/6/2/1 32/4/24/6/4/2/0 36/4/33/13/2//0/1 Diabetes (under treatment) (%) 36.7 (28.2) 43.8 (31.5) 32.2 (26.1) Medications for diabetes (n) (SU/DPP4I/BG/SGLT2I/GLI/αGI/insulin) 22/35/27/21/1/2/8 10/14/12/10/1/2/4 12/21/15/11/0/0/4 Dyslipidemia (under treatment) (%) 70.7 (45.7) 74.0 (56.2) 68.7 (39.1) Medications for dyslipidemia (n) (statin/ﬁbrate/ω3) 67/3/25 30/1/14 37/2/11 CVD risk score 1.76 ± 0.89 1.92 ± 0.86 1.65 ± 0.90 Data are mean ± SD, or median (interquartile range), or frequency percentage BMI body mass index, PBF percentage body fat, WC waist circumference, SMI skeletal muscle mass index, HGS handgrip strength, MQ muscle quality, SBP systolic blood pressure, DBP diastolic blood pressure, FPG fasting plasma glucose, HbA1c hemoglobin A1c, TG triglyceride, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, CA calcium channel antagonist, ACEI ACE inhibitor, ARB angiotensin receptor blocker, β β-blockade, αβ αβ-blockade, DRI direct renin inhibitor, SU sulfonyl urea, DPP4I dipeptidyl peptidase–4 inhibitor, BG biguanide, SGLT2I sodium glucose cotransporter 2 inhibitor, GLI glinide, αGI alpha glucosidase inhibitor, CVD cardiovascular disease Obesity-evaluated index most associated with CVD risk factor accumulation For the obesity-evaluated indices, the median BMI was 30.6 kg/m2 in men and 31.8 kg/m2 in women, the median PBF was 34.1% in men and 47.7% in women, and the median WC was 103.0 cm in men and 102.0 cm in women, respectively. Table 2A shows the comparisons of the CVD risk scores between the low and high groups in each obesity-evaluated index. ORs for CVD risk factor accumulation in obese patients classiﬁed by a combined index of WC and MQ Group C, and Group D compared with Group A (48.6, 41.5, 44.8 vs. 16.7%, all p < 0.05; Table 3). The CVD risk score was also signiﬁcantly higher in Group B and Group D compared with Group A (1.94 ± 0.80, 1.95 ± 0.91 vs. 1.41 ± 0.84, all p < 0.05; Table 3). In addition, Table 4 shows the multiple comparisons of CVD risk scores among the four groups. Even in the model 2, adjusted for sex, the signiﬁcant differences between Group B, D and Group A (1.95 [95%CI 1.66, 2.23], 1.95 [95%CI 1.72, 2.17] vs. 1.40 [95%CI 1.17, 1.63], all p < 0.05) were retained. However, in the model 3, adjusted for sex and age, the signiﬁcant dif- ference between Group D and Group A was retained (1.97 [95%CI 1.77, 2.19] vs. 1.40 [95%CI 1.17, 1.62], p < 0.05), although between Group B and Group A was not (1.87 [95%CI 1.59, 2.16], p = 0.067). Finally, we investigated the effect of a combined index of WC and MQ on the CVD risk score (Fig. 3). We found no difference in ORs for CVD risk scores = 1 among the four groups. However, ORs for CVD risk scores = 2 were sig- niﬁcantly higher in Group B, Group C, and Group D compared with Group A (4.85 [95% CI: 1.72–13.72], p = 0.003; 2.80 [95% CI: 1.09–7.18], p = 0.032; 2.79 [95% CI: 1.15–6.74], p = 0.023; respectively). Furthermore, ORs for CVD risk scores = 3 were signiﬁcantly higher only in Group D compared with Group A (4.29 [95% CI: 1.49–12.33], p = 0.007). Endocrine (2022) 77:30–40 35 Table 2 Comparisons of the CVD risk scores between the low and high groups in each index A. Obesity-evaluated indices BMI PBF WC Low (n = 93) High (n = 95) Low (n = 93) High (n = 95) Low (n = 89) High (n = 99) Model 1 1.73 (1.55, 1.91) 1.78 (1.60, 1.96) 1.66 (1.48, 1.84) 1.85 (1.67, 2.03) 1.62 (1.43, 1.80) 1.88* (1.70, 2.05) Model 2 1.73 (1.55, 1.91) 1.78 (1.60, 1.96) 1.66 (1.48, 1.84) 1.85 (1.67, 2.03) 1.61 (1.43, 1.80) 1.88* (1.71, 2.06) Model 3 1.64 (1.46, 1.83) 1.87 (1.68, 2.05) 1.62 (1.44, 1.79) 1.89* (1.72, 2.07) 1.58 (1.40, 1.76) 1.91 (1.74, 2.08) B. Discussion abdominal obesity, mainly WC, have been shown to be more closely related to CVD and mortality than BMI and PBF are [41, 42] which is consistent with the ﬁndings of our study. To the best of our knowledge, this is the ﬁrst study to determine a combined index associated with CVD risk factor accumulation in Japanese obese patients among each of the obesity- (BMI, PBF, or WC) and sarcopenia- evaluated indices (SMI, HGS, or MQ), respectively. Among obese patients, sarcopenia was diagnosed in only one man (1.4%). This result is consistent with previous studies reporting that few obese individuals diagnosed with high BMI meet the conventional diagnostic criteria for sarcopenia [5, 6, 39]. However, the prevalence of hyper- tension, diabetes, and dyslipidemia in these patients was high (Table 1), indicating that they are at high risk for CVD. Therefore, we attempted to identify obese patients at high risk of cardiovascular disease (CVD) using a combined index of obesity and sarcopenia, such as SO. In this study, we provided evidence that classiﬁcation using the combined index of WC and MQ reﬂects CVD risk factor accumulation in Japanese obese patients, regardless sex and age. Many indices have been proposed to evaluate sarcopenia, including muscle mass, muscle strength, physical perfor- mance, and MQ, but it is also unclear which sarcopenia- evaluated index best reﬂects CVD risk accumulation. Cao et al. reported that in patients with metabolic syndrome, low SMI may be an independent risk factor for atherosclerosis [43]. In addition, low HGS has been reported to be asso- ciated with coronary artery calciﬁcation, CVD, and all- cause mortality [44–46]. Our study demonstrated that MQ was most associated with CVD risk factor accumulation compared with SMI and HGS, regardless of sex and age (Table 2B). MQ was reported to be negatively associated with insulin resistance after adjusting for age, body fat, highly sensitive C-reactive protein levels, and physical activity level in adult obese women [47] and further that insulin resistance contributes to the development of ather- osclerosis [48, 49], ﬁndings that are consistent with our results. Many indices have been proposed for evaluating obesity. However, controversy remains on the obesity-evaluated index that best reﬂects CVD risk factor accumulation [40]. In this study, WC was most highly associated with CVD risk factor accumulation as compared with BMI and PBF, regardless of sex and age (Table 2A). ORs for CVD risk factor accumulation in obese patients classiﬁed by a combined index of WC and MQ Sarcopenia-evaluated indices SMI HGS MQ Low (n = 93) High (n = 95) Low (n = 92) High (n = 96) Low (n = 93) High (n = 95) Model 1 1.77 (1.59, 1.96) 1.74 (1.56, 1.92) 1.80 (1.62, 1.99) 1.71 (1.53, 1.89) 1.95 (1.77, 2.13) 1.57 (1.40, 1.75) Model 2 1.78 (1.59, 1.96) 1.74 (1.56, 1.92) 1.80 (1.62, 1.99) 1.71 (1.53, 1.89) 1.95 (1.77, 2.12) 1.57 (1.39, 1.74) Model 3 1.65 (1.46, 1.85) 1.86 (1.67, 2.04) 1.71 (1.53, 1.90) 1.80 (1.61, 1.98) 1.93 (1.76, 2.10) 1.58 (1.41, 1.75) Data are estimated mean (95% CIs) BMI body mass index, PBF percentage body fat, WC waist circumference, SMI skeletal muscle mass index, HGS handgrip strength, MQ muscle quality Model 1 unadjusted, Model 2 adjusted for sex, Model 3 adjusted for sex and age p < 0.05, p < 0.01 by ANCOVA between the low group vs. the high group in each obesity- and sarcopenia-evaluated index Table 2 Comparisons of the CVD risk scores between the low and high groups in each index Discussion BMI has been widely used to determine the prevalence of obesity and various risks in populations. In addition, PBF is often used as a criterion for evaluating the magnitude of accumulation of adipose tissue. However, in recent years, the indices of In this study, HGS was lower in Group D compared with Group A, whereas SMI was signiﬁcantly higher in Group D compared with Group A. Therefore, MQ calculated as the HGS divided by the muscle mass of the upper limbs was signiﬁcantly lower in Group D compared with Group A. Mesinovic et al. reported that overweight and obese older adults with metabolic syndrome have larger muscle size but poor MQ [50]. Discussion Group A, †p < 0.05 vs. Group B, #p < 0.05 vs. Group C p < 0.05 vs. Group A, †p < 0.05 vs. Group B, #p < 0.05 vs. Group C WC and MQ, especially in obese patients only. Murai et al. reported that patients with type 2 diabetes who had both VF accumulation and low MQ were more affected with CVD [33]. Boettcher et al. have reported that fat accumulation in the muscles was signiﬁcantly linked with VF. Therefore, a combined index of WC and MQ may reﬂect the adverse effects of VF [57]. The effectiveness of the combined index of WC and MQ as a relevant indicator of accumulation of CVD risk factors in obese patients requires further investigation. attributed to increased fat accumulation in the muscles [51, 52]; however, in this study, we did not measure MQ with imaging analysis, such as ultrasonography or computed tomography. Recently, the usefulness of phase angle as an indicator for MQ has been suggested [5, 53], and further studies on the increased fat accumulation in muscles are required in the future. In addition, the prevalence of diabetes in Group B, C, and D was higher compared with Group A (Table 3). Long duration of diabetes and poor glycemic control are more likely to be associated with microvascular and macrovascular disease [54], and to cause sarcopenia and decrease of MQ independent of BMI and age [54, 55]. The use of multiple medications per day is common with aging. Many drugs taken regularly for diseases may interact with some mechanisms that can alter the balance between protein synthesis and degradation [58], and researchers have reported that polypharmacy is associated with sarcopenia [59]. In this study, diuretics and biguanide (BG) were used signiﬁcantly more often in Group D compared with Group B and Group A, respectively (Supplementary Table S1). The use of diuretics, particularly loop diuretics, has been suggested as a risk factor of sarcopenia [60]. On the other The combined index of WC and MQ was well associated with CVD risk factor accumulation in obese patients (Table 4, Fig. 3). Atkins et al. reported a review that summarized studies of the association of CVD risk factors with combi- nations of various obesity- and sarcopenia-evaluated indices [56]. BMI body mass index, PBF percentage body fat, WC waist circumference, SMI skeletal muscle mass index, HGS handgrip strength, MQ muscle quality, SBP systolic blood pressure, DBP diastolic blood pressure, FPG fasting plasma glucose, HbA1c hemoglobin A1c, TG triglyceride, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, CVD cardiovascular disease §p value for difference among the four groups in means (ANOVA followed by Tukey’s test), or medians (Kruskal–Wallis test followed by Bonferroni correction), or percentages (Chi-square test) Discussion The lower MQ in Group D might be Endocrine (2022) 77:30–40 36 Table 3 Clinical characteristics of the obese patients classiﬁed by a combined index of WC and MQ Group A (n = 54) Group B (n = 35) Group C (n = 41) Group D (n = 58) p value§ Age (year) 55.5 ± 14.5 62.1 ± 12.2 53.1 ± 16.5 53.8 ± 17.4 0.049 Women (%) 57.4 62.9 65.9 60.3 0.859 BMI (kg/m2) 27.6 (26.1–30.3) 28.7 (26.9–30.3) 34.5 (31.9–39.9)† 34.2 (32.1–39.9)† <0.001 PBF (%) 36.9 ± 8.8 38.4 ± 7.0 48.4 ± 9.9† 47.6 ± 9.5† <0.001 WC (cm) 95.0 (89.8–99.0) 97.0 (92.0–100.0) 111.0 (106.3–118.3)† 110.5 (106.8–121.5)† <0.001 SMI (kg/m2) 7.82 ± 1.03 7.72 ± 1.13 8.22 ± 1.10† 8.32 ± 1.27† 0.029 HGS (kg) 33.0 ± 9.7 25.1 ± 9.6 32.6 ± 9.1† 27.9 ± 9.0# <0.001 MQ (kg/kg) 7.50 ± 0.71 5.49 ± 0.85 7.38 ± 0.55† 5.71 ± 0.72# <0.001 SBP (mmHg) 132.5 ± 12.5 133.7 ± 14.9 139.4 ± 14.2† 138.3 ± 12.7* 0.036 DBP (mmHg) 83.1 ± 9.8 80.2 ± 9.8 84.9 ± 7.1 80.3 ± 10.2 0.063 FPG (mg/dL) 108.7 ± 31.3 117.9 ± 31.4 124.9 ± 31.5 126.1 ± 46.4 0.061 HbA1c (%) 6.0 ± 0.8 6.3 ± 0.8 6.6 ± 1.4 6.6 ± 1.5 0.028 TG (mg/dL) 134.0 ± 96.2 126.9 ± 56.0 136.3 ± 73.4 134.5 ± 68.9 0.956 HDL-C (mg/dL) 59.2 ± 13.9 56.8 ± 16.0 57.3 ± 13.2 56.5 ± 16.4 0.794 LDL-C (mg/dL) 116.7 ± 26.7 119.9 ± 28.6 123.9 ± 26.7 116.4 ± 30.1 0.526 Current smoker (%) 3.7 14.3 12.2 6.9 0.260 Hypertension (%) 57.4 68.6 70.7 75.9 0.205 Diabetes (%) 16.7 48.6* 41.5* 44.8* 0.004 Dyslipidemia (%) 66.7 77.1 65.9 74.1 0.589 CVD risk score 1.41 ± 0.84 1.94 ± 0.80* 1.78 ± 0.91 1.95 ± 0.91** 0.005 Data are mean ± SD or median (interquartile range) or frequency percentage Table 3 Clinical characteristics of the obese patients classiﬁed by a combined index of WC and MQ Data are mean ± SD or median (interquartile range), or frequency percentage BMI body mass index, PBF percentage body fat, WC waist circumference, SMI skeletal muscle mass index, HGS handgrip strength, MQ muscle quality, SBP systolic blood pressure, DBP diastolic blood pressure, FPG fasting plasma glucose, HbA1c hemoglobin A1c, TG triglyceride, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, CVD cardiovascular disease §p value for difference among the four groups in means (ANOVA followed by Tukey’s test), or medians (Kruskal–Wallis test followed by Bonferroni correction), or percentages (Chi-square test) p < 0.05 vs. Discussion group A 0 0 1 0 1 1 1.0 Obese patients classified by a combined index of WC and MQ Multivariate sex- and age-adjusted ORs for each CVD risk score ( = 1, = 2, and = 3) Group A Group B Group C Group D CVD risk score = 1 CVD risk score = 2 CVD risk score = 3 p = 0.889 p = 0.879 p = 0.908 p = 0.003 p = 0.032 p = 0.023 p = 0.328 p = 0.088 p = 0.007 Patients who have CVD risk scores/total Group A Group B Group C Group D Group A Group B Group C Group D n = 26/54 n = 6/35 n = 10/41 n = 13/58 n = 16/54 n = 19/35 n = 18/41 n = 23/58 n = 6/54 n = 8/35 n = 9/41 n = 18/58 0 0 1 0 1 1 1.0 Obese patients classified by a combined index of WC and MQ Multivariate sex- and age-adjusted ORs for each CVD risk score ( = 1, = 2, and = 3) Group A Group B Group C Group D CVD risk score = 1 CVD risk score = 2 CVD risk score = 3 p = 0.889 p = 0.879 p = 0.908 p = 0.003 p = 0.032 p = 0.023 p = 0.328 p = 0.088 p = 0.007 Patients who have CVD risk scores/total Group A Group B Group C Group D Group A Group B Group C Group D n = 26/54 n = 6/35 n = 10/41 n = 13/58 n = 16/54 n = 19/35 n = 18/41 n = 23/58 n = 6/54 n = 8/35 n = 9/41 n = 18/58 Fig. 3 ORs for the CVD risk scores in obese patients of each group. Multivariate sex- and age-adjusted ORs in obese patients classiﬁed by a combined index of WC and MQ (Group A, Group B, Group C, and Group D). Squares, ORs for CVD risk score = 1; triangles, ORs for CVD risk score = 2; circles, ORs for CVD risk score = 3. Solid horizontal lines, 95% CIs Multivariate sex- and age-adjusted ORs for each CVD risk score ( = 1, = 2, and = 3) hand, BG improves insulin resistance and may inhibit the progression of sarcopenia [58]. Discussion To the best of our knowledge, however, there have been no reports investigating the association between the accumulation of CVD risk factors and a combined index of 37 Endocrine (2022) 77:30–40 Group A Group B Group C Group D Model 11.41(1.17, 1.64)1.94(1.65, 2.23)1.78(1.51, 2.05)1.95*(1.72, 2.17) Model 21.40(1.17, 1.63)1.95(1.66, 2.23)1.79(1.53, 2.06)1.95(1.72, 2.17) Model 31.40(1.17, 1.62)1.87 (1.59, 2.16)1.83(1.57, 2.09)1.97(1.75, 2.19) Data are estimated mean (95% CIs) CVD cardiovascular disease, WC waist circumference, MQ muscle quality Model 1 unadjusted, model 2 adjusted for sex, model 3 adjusted for sex and age p < 0.05, p < 0.01 by ANCOVA followed by Bonferroni correction vs. group A CVD cardiovascular disease, WC waist circumference, MQ muscle quality Model 1 unadjusted, model 2 adjusted for sex, model 3 adjusted for sex and age p < 0.05, **p < 0.01 by ANCOVA followed by Bonferroni correction vs. Discussion Unfortunately, we did not assess insulin resistance in this study, but it has been reported that patients with SO are in an insulin-resistant state [1, 3]. It is likely that BG was provided for insulin resistance in Group D in this study. The effects of BG on muscle remain to be elucidated. calorie intake in obese patients. To resolve these limitations, larger cohort and prospective studies including various populations are needed in the future. In conclusion, our study demonstrated that the obesity- evaluated index, WC, and the sarcopenia-evaluated index, MQ, were most closely associated with CVD risk factor accumulation in Japanese obese patients, respectively. Furthermore, classiﬁcation by the combined index of WC and MQ reﬂects CVD risk factor accumulation in Japanese obese patients, regardless of sex and age. The present study had several limitations that warrant mention. First, our study used a cross-sectional design. Thus, we could investigate only the associations between the combined index of WC and MQ and CVD risk factor accumulation. Second, because this study was hospital- based in design and limited to Japanese obese patients, there may be bias among the study participants, which could limit the generalization of the study results. However, our pur- pose of this study was to identify obese patients at high risk of CVD. In this concept, we were able to identify the par- ticularly high-risk obese patients using the combined index of WC and MQ. Third, the cutoff values for both WC and MQ were not clear. Fourth, we had not been able to accu- rately assess the amount of physical activity and estimated Table 4 Comparison of CVD risk scores in obese patients classiﬁed by a combined index of WC and MQ Fig. 3 ORs for the CVD risk scores in obese patients of each group. Multivariate sex- and age-adjusted ORs in obese patients classiﬁed by a combined index of WC and MQ (Group A, Group B, Group C, and Group D). Squares, ORs for CVD risk score = 1; triangles, ORs for CVD risk score = 2; circles, ORs for CVD risk score = 3. Solid horizontal lines, 95% CIs Conﬂict of interest The authors declare no competing interests. 12. H. Zhang, S. Lin, T. Gao et al. Association between sarcopenia and metabolic syndrome in middle-aged and older non-obese adults: a systematic review and meta-analysis. Nutrients 10(3), E364 (2018) Ethical approval This study was approved by the ethics committee for human research at Kyoto Medical Center (approval No. 19-083). Ethical approval This study was approved by the ethics committee for human research at Kyoto Medical Center (approval No. 19-083). Consent to participate Informed consent was obtained from all indi- vidual participants included in the study. Consent to participate Informed consent was obtained from all indi- vidual participants included in the study. 13. S.O. Chin, S.Y. Rhee, S. Chon et al. Sarcopenia is independently associated with cardiovascular disease in older Korean adults: the Korea National Health and Nutrition Examination Survey (KNHANES) from 2009. PloS One 8(3), e60119 (2013) Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 14. Y. Matsubara, T. Matsumoto, K. Inoue et al. Sarcopenia is a risk factor for cardiovascular events experienced by patients with critical limb ischemia. J. Vasc. Surg. 65(5), 1390–1397 (2017) Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. 15. P. Liu, Q. Hao, S. Hai, H. Wang, L. Cao, B. Dong, Sarcopenia as a predictor of all-cause mortality among community-dwelling older people: a systematic review and meta-analysis. Maturitas 103, 16–22 (2017) 16. D. Heber, S. Ingles, J.M. Ashley, M.H. Maxwell, R.F. Lyons, R. M. Elashoff, Clinical detection of sarcopenic obesity by bioelec- trical impedance analysis. Am. J. Clin. Nutr. 64(3 Suppl), 472S–477S (1996) 17. R.N. Data availability The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgements The authors would like to thank Hiromi Kusa- kabe at National Hospital Organization Kyoto Medical Center for her secretarial assistance and her assistance in acquiring the data. The authors would also like to thank Enago (www.enago.jp) for the Eng- lish language review. 38 Endocrine (2022) 77:30–40 6. L.-K. Chen, J. Woo, P. Assantachai et al. Asian working group for sarcopenia: 2019 consensus update on sarcopenia diagnosis and treatment. J. Am. Med. Dir. Assoc. 21(3), 300–307. (2020) Author contributions K.I. acquired and analyzed the data and wrote the manuscript. T.K. conceived the study and wrote the manuscript. K. M., H.Y., T.I., K.I., and N.S.-A. reviewed the manuscript. All authors read and approved the ﬁnal manuscript. 7. G. Shaﬁee, A. Keshtkar, A. Soltani, Z. Ahadi, B. Larijani, R. Heshmat, Prevalence of sarcopenia in the world: a systematic review and meta- analysis of general population studies. J. Dia- betes Metab. Disord. 16, 21 (2017) Funding This study was supported in part by Grant-in-Aid for Clinical Research from the National Hospital Organization to T.K. (H30-NHO- 03, R3-NHO-01), Grant-in-Aid for Scientiﬁc Research (C) to T.K. (JSPS KAKENHI grant No. 21K11691) and Scientiﬁc Research (B) to N.S-A. (JSPS KAKENHI grant No. 18H02737 and 21H02835), and Grant-in-Aid for Exploratory Research to N.S.-A. (18K19769) from Japan Society for the Promotion of Science. This study was also supported in part by a grant from Smoking Research Foundation to T. K. (2020T007) and N.S.-A. (2019T004). The funders played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. 8. C. Beaudart, M. Zaaria, F. Pasleau, J.-Y. Reginster, O. Bruyère, Health outcomes of sarcopenia: a systematic review and meta- analysis. 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https://openalex.org/W2140527275	https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/1471-2164-14-414	English	null	The complete mitochondrial genomes of three parasitic nematodes of birds: a unique gene order and insights into nematode phylogeny	BMC genomics	2,013	cc-by	9,360	* Correspondence: rshao@usc.edu.au; xingquanzhu1@hotmail.com †Equal contributors 3Genecology Research Centre, University of the Sunshine Coast, Queensland 4558, Australia 1State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province 730046, People’s Republic of China Full list of author information is available at the end of the article RESEARCH ARTICLE Open Access © 2013 Liu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Liu et al. BMC Genomics 2013, 14:414 http://www.biomedcentral.com/1471-2164/14/414 Liu et al. BMC Genomics 2013, 14:414 http://www.biomedcentral.com/1471-2164/14/414 Keywords: Mitochondrial genome, Ascaridia, Nematode, Gene arrangement, Phylogeny Keywords: Mitochondrial genome, Ascaridia, Nematode, Gene arrangement, Phylogeny systems proposed by Schneider (1866) [2] on somatic musculature and by Cobb (1919) [3] on stoma armature, and the system proposed by Filipjev (1929) [4] on the presence and absence of zooparasitism. Chitwood (1937) [5] proposed initially the bipartite phylogenetic and taxo- nomic system based on the presence and absence of phas- mids; this system was further elaborated later by Maggenti (1963, 1983) [6,7] based on pharyngeal structure and ex- cretory systems. However, due to the lack of homologous characters and informative fossil records, and the exten- sive convergent evolution, it has been extremely difficult to derive a consistent phylogenetic and taxonomic frame- work for the phylum Nematoda from morphological and ecological characters [8-12]. In the past two decades, The complete mitochondrial genomes of three parasitic nematodes of birds: a unique gene order and insights into nematode phylogeny Guo-Hua Liu1,2†, Renfu Shao3†, Jia-Yuan Li1, Dong-Hui Zhou1, Hu Li4 and Xing-Quan Zhu1,2 Abstract Background: Analyses of mitochondrial (mt) genome sequences in recent years challenge the current working hypothesis of Nematoda phylogeny proposed from morphology, ecology and nuclear small subunit rRNA gene sequences, and raise the need to sequence additional mt genomes for a broad range of nematode lineages. Results: We sequenced the complete mt genomes of three Ascaridia species (family Ascaridiidae) that infest chickens, pigeons and parrots, respectively. These three Ascaridia species have an identical arrangement of mt genes to each other but differ substantially from other nematodes. Phylogenetic analyses of the mt genome sequences of the Ascaridia species, together with 62 other nematode species, support the monophylies of seven high-level taxa of the phylum Nematoda: 1) the subclass Dorylaimia; 2) the orders Rhabditida, Trichinellida and Mermithida; 3) the suborder Rhabditina; and 4) the infraorders Spiruromorpha and Oxyuridomorpha. Analyses of mt genome sequences, however, reject the monophylies of the suborders Spirurina and Tylenchina, and the infraorders Rhabditomorpha, Panagrolaimomorpha and Tylenchomorpha. Monophyly of the infraorder Ascaridomorpha varies depending on the methods of phylogenetic analysis. The Ascaridomorpha was more closely related to the infraorders Rhabditomorpha and Diplogasteromorpha (suborder Rhabditina) than they were to the other two infraorders of the Spirurina: Oxyuridorpha and Spiruromorpha. The closer relationship among Ascaridomorpha, Rhabditomorpha and Diplogasteromorpha was also supported by a shared common pattern of mitochondrial gene arrangement. Conclusions: Analyses of mitochondrial genome sequences and gene arrangement has provided novel insights into the phylogenetic relationships among several major lineages of nematodes. Many lineages of nematodes, however, are underrepresented or not represented in these analyses. Expanding taxon sampling is necessary for future phylogenetic studies of nematodes with mt genome sequences. Results The mitochondrial (mt) genomes of bilateral animals usually contain 37 genes and a control region on a circu- lar chromosome, ~16 kb in size [15-17]. Sequences of individual mt genes and whole mt genomes are widely used to infer phylogenetic relationships among animals at different taxonomic levels [18,19]. In a number of cases, arrangement of mt genes has also been used to re- solve long-standing phylogenetic relationships that could not be resolved by other means [20-22]. Mitochondrial genomes of three Ascaridia species The complete mt genomes of A. galli, A. columbae and Ascaridia sp. (GHL-2012) were 13,977 bp, 13,931 bp and 13,862 bp long, respectively (GenBank accession numbers JX624728, JX624729 and JX624730). Each mt genome contains 12 protein-coding genes, 22 tRNA genes and two rRNA genes on a circular chromosome (Figure 1). As in most other nematodes, atp8 gene is not present in the mt genome of A. galli, A. columbae and Ascaridia sp. (GHL-2012); all mt genes are transcribed from the same direction. TTT is used as the initiation codon in nad6 gene in the mt genome of A. galli. TTT was previously reported to be the initiation codon for cox2, cytb, nad4, nad3, nad2 and cox3 genes in the mt genome of Strongyloides stercoralis (Strongyloididae), but not for any other Ascaridida nematodes. All of the 12 protein-coding genes have complete termination codons, TAA or TAG. The tRNA genes in mt genomes of A. galli, A. columbae and Ascaridia sp. (GHL-2012) range from 51 to 76 bp. The tRNA-Ser(AGN) and tRNA- Ser(UCN) have truncated secondary structures; these two y [ ] Recently, mt genome sequences have also been analyzed to understand the phylogenetic relation- ships among nematodes. Kang et al. [23] inferred the Nematoda phylogeny with the mt genome sequences of 25 species; Park et al. [24] and Sultana et al. [25] ex- panded further the analysis to include 36 and 41 species respectively. A major difference between mt genome phylogenies and the current working hypothesis of the Nematoda is on the relationship among four infraorders: Ascaridomorpha, Rhabditomorpha, Spiruromorpha and Oxyuridomorpha. In the current working hypothesis, Ascaridomorpha, Spiruromorpha and Oxyuridomorpha are in the suborder Spirurina whereas Rhabditomorpha is in the suborder Rhabditina [13,14]. Further- more, Ascaridomorpha is most closely related to Spiruromorpha, whereas Oxyuridomorpha is sister to Ascaridomorpha + Spiruromorpha + Rhigonematomorph. Liu et al. BMC Genomics 2013, 14:414 http://www.biomedcentral.com/1471-2164/14/414 Liu et al. BMC Genomics 2013, 14:414 http://www.biomedcentral.com/1471-2164/14/414 Page 2 of 13 sequenced to date. Our analyses support the close rela- tionship between Ascaridomorpha and Rhabditomorpha, and provide novel insights into other phylogenetic rela- tionships in the Nematoda. nematode phylogeny and taxonomy have been revised with analyses of the nuclear small subunit (SSU) rRNA gene sequences [12-14]. The current working hypothesis of Nematoda phylogeny incorporated evidence from morphology, ecology and SSU rRNA gene sequence ana- lyses [13,14]. Results In the phylogenies inferred from mt genome sequences, however, Ascaridomorpha is most closely related to Rhabditomorpha; in most analyses, Oxyuridomorpha is sister to Ascaridomorpha + Rhabditomorpha, whereas Spiruromorpha is sister to the group that contains Ascaridomorpha, Rhabditomorpha and Oxyuridomorpha [23,24]. Due to limited taxon sampling, however, the novel phylogenetic relationships inferred from mt genome se- quences in these studies need to be interpreted with caution and further test with more taxa from a wide range of lineages is necessary. Figure 1 The mitochondrial genomes of three Ascaridia species. All genes are on the same DNA strand and are transcribed clockwise. Protein-coding and rRNA genes are indicated with the standard nomenclature. tRNA genes are indicated with the one-letter code of their corresponding amino acids. There are two tRNA genes for leucine: L1 for codons CUN and L2 for UUR; and two tRNA genes for serine: S1 for codons AGN and S2 for UCN. “NCRL” refers to the large non-coding region. “NCRS” refers to a small non-coding region. In this study, we sequenced the complete mt genomes of three Ascaridia species in the family Ascaridiidae of the infraorder Ascaridomorpha. Ascaridia species are among the most prevalent and pathogenic parasitic nematodes found in domestic and wild birds and have a worldwide distribution [26]. These Ascaridia species share an identical arrangement of mt genes to each other but differ substantially from those of other nema- todes. We inferred the phylogenetic relationships with the complete mt genome sequences of the Ascaridia species and 62 other nematode species that have been Figure 1 The mitochondrial genomes of three Ascaridia species. Figure 1 The mitochondrial genomes of three Ascaridia species. All genes are on the same DNA strand and are transcribed clockwise. Protein-coding and rRNA genes are indicated with the standard nomenclature. tRNA genes are indicated with the one-letter code of their corresponding amino acids. There are two tRNA genes for leucine: L1 for codons CUN and L2 for UUR; and two tRNA genes for serine: S1 for codons AGN and S2 for UCN. “NCRL” refers to the large non-coding region. “NCRS” refers to a small non-coding region. Figure 1 The mitochondrial genomes of three Ascaridia species. All genes are on the same DNA strand and are transcribed clockwise. Protein-coding and rRNA genes are indicated with the standard nomenclature. tRNA genes are indicated with the one-letter code of their corresponding amino acids. Background Nematodes (also called roundworms) are an extremely diverse group of bilateral animals with an estimate of 1– 10 million species although only ~25,000 species have been described [1]. Nematode taxonomy has traditionally been formed from morphology and ecology, e.g. the early Results There are two tRNA genes for leucine: L1 for codons CUN and L2 for UUR; and two tRNA genes for serine: S1 for codons AGN and S2 for UCN. “NCRL” refers to the large non-coding region. “NCRS” refers to a small non-coding region. Page 3 of 13 Liu et al. BMC Genomics 2013, 14:414 http://www.biomedcentral.com/1471-2164/14/414 Page 3 of 13 The longer NCR (NCRL) is between trnC gene and trnN gene, and is 610 bp (A. galli), 563 bp (A. columbae) and 566 bp [Ascaridia sp. (GHL-2012)] respectively. The shorter NCR (NCRS) is between nad4 and trnM gene, and is 157 bp (A. galli), 91 bp (A. columbae) and 101 bp The longer NCR (NCRL) is between trnC gene and trnN gene, and is 610 bp (A. galli), 563 bp (A. columbae) and 566 bp [Ascaridia sp. (GHL-2012)] respectively. The shorter NCR (NCRS) is between nad4 and trnM gene, and is 157 bp (A. galli), 91 bp (A. columbae) and 101 bp tRNAs lack a DHU stem but possess a TΨC loop. In all other 20 tRNA genes, the TΨC arm and variable loop are replaced with a TV replacement loop. There are two non-coding regions (NCR) in the mt genomes of A. galli, A. columbae and Ascaridia sp. (GHL-2012) (Table 1). Table 1 List of annotated mitochondrial genes and regions of Ascaridia galli, Ascaridia columbae and Ascaridia sp Gene/region Position/length (bp) Start/stop codon Anticodo A. galli A. columbae Ascaridia sp. A. galli A.columbae Ascaridia sp. Results cox1 1-1563 (1563) 1-1563 (1563) 1-1581 (1581) ATG/TAA ATG/TAA GTG/TAG tRNA-Cys (C) 1565-1620 (56) 1565-1620 (56) 1562-1616 (55) GAT Non-coding region (NCL) 1621-2230 (610) 1623-2183 (563) 1617-2182 (566) tRNA-Asn (N) 2231 -2284 (54) 2184-2241 (58) 2183-2239 (57) GTT tRNA-Tyr (Y) 2286-2344 (59) 2246-2303 (58) 2240-2295 (56) GTA nad1 2342-3217 (876) 2301-3176 (876) 2298-3167 (870) TTG/TAA TTG/TAA GTT/TAG atp6 3217-3813 (597) 3176-3772 (597) 3170-3766 (597) ATA/TAA ATA/TAA TTG/TAG tRNA-Lys (K) 3816-3876 (61) 3790-3853 (64) 3772-3833 (62) TTT tRNA-Leu UUR (L2) 3876-3931(56) 3853-3907 (55) 3831-3886 (56) TAA tRNA-Ser AGN (S1) 3932-3982 (51) 3908-3962 (55) 3887-3939 (53) GCT nad2 3986-4828 (843) 3966-4808 (843) 3943-4779 (837) TTG/TAA TTG/TAA TTG/TAG tRNA-Ile (I) 4832-4891 (60) 4823-4884 (62) 4781-4841 (61) GAT tRNA-Arg (R) 4896-4950 (55) 4888-4944 (57) 4845-4900 (56) ACG tRNA-Gln (Q) 4951-5005 (55) 4945-4998 (54) 4901-4954 (54) TTG tRNA-Asp (D) 5005-5063 (59) 5011-5070 (60) 4971-5025 (55) GTC tRNA-Glu (E) 5065-5125 (61) 5069-5126 (58) 5025-5083 (59) TTC rrnS 5123-5823 (701) 5124-5826 (703) 5084-5774 (691) tRNA-Ser UCN (S2) 5826-5880 (55) 5837-5891 (55) 5781-5835 (55) TGA tRNA-Phe (F) 5883-5939 (57) 5898-5953 (56) 5841-5897 (57) GAA cytb 5964-7067 (1104) 5975-7075 (1101) 5919-7019 (1101) ATG/TAA GTT/TAA GTT/TAG tRNA-Leu CUN (L1) 7067-7122 (56) 7077-7134 (58) 7020-7075 (56) TAG cox3 7144-7914 (771) 7154-7918 (765) 7097-7864 (768) TTG/TAG TTG/TAA TTG/TAA tRNA-Thr (T) 7889-7944 (56) 7899-7956 (58) 7845-7900 (56) TAG nad4 7939-9174 (1236) 7951-9186 (1236) 7901-9130 (1230) GTG/TAG ATA/TAG TTG/TAG Non-coding region(NCR) 9175-9331 (157) 9187-9277 (91) 9131-9231 (101) tRNA-Met (M) 9345-9407 (63) 9288-9352 (65) 9232-9294 (63) CAT tRNA-Gly (G) 9407-9462 (56) 9355-9410 (56) 9296-9351 (56) TCC cox2 9463-10158 (696) 9411-10106 (696) 9364-10050 (687) TTG/TAG TTG/TAG ATG/TAG tRNA-His (H) 10163-10218 (56) 10113-10167 (55) 10049-10105 (57) GTG rrnL 10211-11166 (956) 10167-11121 (955) 10103-11055 (953) nad3 11167-11502 (336) 11119-11454 (336) 11053-11391 (339) TTG/TAA ATA/TAA TTG/TAA nad5 11502-13082 (1581) 11451-13031 (1581) 11394-12968 (1575) ATA/TAA ATA/TAG ATT/TAG tRNA-Ala (A) 13085-13139 (55) 13031-13085 (55) 12968-13022 (55) TGC tRNA-Pro (P) 13140-13197 (58) 13094-13149 (56) 13026-13082 (57) TGG tRNA-Val (V) 13197-13252 (56) 13149-13205 (57) 13082-13137 (56) TAC nad6 13253-13687 (435) 13206-13640 (435) 13138-13572 (435) TTT/TAA TTG/TAA TTG/TAG nad4L 13702-13941 (240) 13656-13895 (240) 13588-13827 (240) ATT/TAA GTT/TAA ATT/TAA tRNA-Trp (W) 13919-13976 (58) 13873-13930 (58) 13805-13862 (58) TCA annotated mitochondrial genes and regions of Ascaridia galli, Ascaridia columbae and Ascaridia sp Table 1 List of annotated mitochondrial genes and regions of Ascaridia galli, Ascaridia columbae a Liu et al. Ascaridia species A. galli, A. columbae and Ascaridia sp. (GHL-2012) have an identical gene arrangement in their mt genomes. The arrangement of mt genes in these three species, however, is substantially different from those of other nematodes (Figure 2). The mt genomes of the 62 species of nema- todes that have been sequenced to date showed 25 types of gene arrangements (GA1–GA25, Figure 2), of which GA3 is the most common type and has been found in 32 species of nematodes. In comparison to the GA3 type, at least three rearrangement events occurred in the Ascaridia species: trnM was translocated, and a block of 11 genes (trnN, trnY, nad1, atp6, trnK, trnL2, trnS1, nad2, trnI, trnR and trnQ) swapped position with another block of 12 genes (trnG, cox2, trnH, rrnL, nad3, nad5, trnA, trnP, trnV, nad6, nad4L and trnW) (Figure 3). Of the 24 species from the suborder Rhabditina, 23 species were from the infraorder Rhabditomorpha and one species from the infraorder Diplogasteromorpha. Species of these two infraorders were most closely related in all of the phylogenetic analyses in this study. The Rhabditomorpha, however, was paraphyletic with respect to the Diplogasteromorpha. Three species from the superfamily Rhabitoidea of the Rhabditomorpha were more closely related to Pristionchus pacificus (Diplogasteromorpha) than they were to the other 20 species from the Rhabditomorpha. The close relation- ship between the species of the superfamily Rhabitoidea and P. pacificus was strongly supported in Bayesian ana- lysis (PP = 1, Figure 4), and was moderately supported in ML and MP analyses (BF = 83%, Figure 5; BF = 82%, Figure 6). In addition to the Rhabitoidea, three other superfamilies of the infraorder Rhabditomorpha were also represented in our analyses: Ancylostomatoidea (4 spe- cies), Strongyloidea (10 species), and Trichostrongyloidea (6 species). The Trichostrongyloidea was monophyletic with strong support in all of the three phylogenetic analyses in this study (PP = 1, Figure 4; BF = 88%, Figure 5; BF = 88%, Figure 6). The Rhabitoidea was monophyletic with strong support in Bayesian analysis (PP = 0.99) and weak support in MP analysis (BF = 52%, Figure 6), but was paraphyletic in ML analysis with weak support (BF = 51%, Figure 5). Results BMC Genomics 2013, 14:414 http://www.biomedcentral.com/1471-2164/14/414 Page 4 of 13 Figure 2 Mitochondrial gene arrangement in three Ascaridia species (GA1) compared with those in other nematodes (GA The circular mt genomes were linearized at the 50 end of cox1 gene for illustration purpose. Non-coding regions were not shown Figure 2 Mitochondrial gene arrangement in three Ascaridia species (GA1) compared with those in other nematodes (GA2-GA25). The circular mt genomes were linearized at the 50 end of cox1 gene for illustration purpose. Non-coding regions were not shown. Figure 2 Mitochondrial gene arrangement in three Ascaridia species (GA1) compared with those in other nematodes (GA2-GA25) The circular mt genomes were linearized at the 50 end of cox1 gene for illustration purpose. Non-coding regions were not shown. Page 5 of 13 Liu et al. BMC Genomics 2013, 14:414 http://www.biomedcentral.com/1471-2164/14/414 Liu et al. BMC Genomics 2013, 14:414 http://www.biomedcentral.com/1471-2164/14/414 [Ascaridia sp. (GHL-2012)] respectively. A NCR at this location, i.e. between nad4 and trnM, is also present in the mt genomes of most other nematodes [27,28]. Phylogenetic relationships within the order Rhabditida The 54 species of nematodes in the order Rhabditida included in this study were from three suborders: Rhabditina (24 species), Spirurina (26 species), and Tylenchina (4 species). The monophyly of the suborder Rhabditina was strongly supported in Bayesian and ML analyses (PP = 1, Figure 4; BF = 99%, Figure 5), and was moderately supported in MP analysis (BF = 79%, Figure 6). The Spirurina and the Tylenchina, however, were not monophyletic in all of the three phylogenetic analyses in this study. Ascaridia species The Ancylostomatoidea and the Strongyloidea were paraphyletic in all of the three phylogenetic analyses in this study; there was strong support for the paraphylies of these two superfamilies in Bayesian and MP analyses (PP > 0.96, Figure 4; BF = 99%, Figure 5). Monophylies of the order Rhabditida and the subclass Dorylaimia Monophylies of the order Rhabditida and the subclass Dorylaimia Of the 65 species of nematodes included in the phylo- genetic analyses in this study, 54 species were from the order Rhabditida of the class Chromadorea, and 11 spe- cies were from the subclass Dorylaimia of the class Enoplea. Both Rhabditida and Dorylaimia were mono- phyletic in all of the trees inferred with Bayesian, ML and MP methods from concatenated amino acid se- quences deduced from the sequences of the 12 mt protein-coding genes. The monophyly of the order Rhabditida was strongly supported with a posterior probability (PP) of 1 in Bayesian analysis (Figure 4), a bootstrapping frequency (BF) of 100% in ML analysis (Figure 5), and a BF of 94% in MP analysis (Figure 6). The monophyly of the subclass Dorylaimia was strongly supported in Bayesian and ML analyses (PP = 1, Figure 4; BF = 97, Figure 5), and was moderately supported in MP analysis (BF = 67%, Figure 6). The 26 species of the suborder Spirurina included in this study were from three infraorders: Ascaridomorpha (14 Figure 3 Rearrangement of mitochondrial genes in three Ascaridia species (pattern GA1) relative to the most common pattern of mt gene arrangement observed in nematodes (GA3). Figure 3 Rearrangement of mitochondrial genes in three Ascaridia species (pattern GA1) relative to the most common pattern of mt gene arrangement observed in nematodes (GA3). Liu et al. BMC Genomics 2013, 14:414 http://www.biomedcentral.com/1471-2164/14/414 Page 6 of 13 Figure 4 Phylogenetic relationships among 65 species of nematodes inferred from Bayesian analysis of deduced amino acid sequences of 12 mitochondrial proteins. Taenia multiceps and T. hydatigena (GenBank accession numbers FJ495086 and FJ518620) were used as the outgroup. Posterior probability (PP) values were indicated at nodes. Figure 4 Phylogenetic relationships among 65 species of nematodes inferred from Bayesian analysis of deduced amino acid sequences of 12 mitochondrial proteins. Taenia multiceps and T. hydatigena (GenBank accession numbers FJ495086 and FJ518620) were use as the outgroup. Posterior probability (PP) values were indicated at nodes. both Bayesian and MP analyses, the 10 species of the super- family Ascarodoidea of the infraorder Ascaridomorpha were more closely related to those of the Rhabditina and Steinernema carpocapsae (Tylenchina) than they were to the three Ascaridia species and Cucullanus robustus, which were also from the infraorder Ascaridomorpha. Monophylies of the order Rhabditida and the subclass Dorylaimia The three Ascaridia species, for which mt genomes were sequenced in this study, form a monoplyletic group with strong support in all of the three phylogenetic analyses (Figures 4, 5, 6). species), Oxyuridomorpha (2 species) and Spiruromorpha (10 species). The Oxyuridomorpha and the Spiruromorpha were both monophyletic with strong support in all of the three phylogenetic analyses (PP = 1, Figure 4; BF = 100%, Figure 5; BF = 100%, Figure 6). The Ascaridomorpha, how- ever, was monophyletic only in ML analysis with moderate support (BF = 84%, Figure 5), and was paraphyletic in Bayesian analysis with strong support (PP = 0.99, Figure 4) and MP analysis with weak support (BF = 52%, Figure 6). In Liu et al. BMC Genomics 2013, 14:414 http://www.biomedcentral.com/1471-2164/14/414 Page 7 of 13 Page 7 of 13 Figure 5 Phylogenetic relationships among 65 species of nematodes inferred from maximum likelihood (ML) of deduced amino acid sequences of 12 mitochondrial proteins. Taenia multiceps and T. hydatigena (GenBank accession numbers FJ495086 and FJ518620) were used as the outgroup. Bootstrapping frequency (BF) values were indicated at nodes. Figure 5 Phylogenetic relationships among 65 species of nematodes inferred from maximum likelihood (ML) of deduced amino acid sequences of 12 mitochondrial proteins. Taenia multiceps and T. hydatigena (GenBank accession numbers FJ495086 and FJ518620) were used as the outgroup. Bootstrapping frequency (BF) values were indicated at nodes. The four species of the suborder Tylenchina included in this study were from two infraorders: Panagrolaimorpha (2 species), and Tylenchomorpha (2 species). Both of these infraorders were paraphyletic in all of the three analyses (PP > 0.99, Figure 4; BF > 57%, Figure 5; BF > 51%, Figure 6). Cucullanus robustus was sister to Ascaridomorpha + Rhabditina + Steinernema carpocapsae in Bayesian ana- lysis with strong support (PP = 1, Figure 4). Cucullanus robustus, however, was sister the Ascaridia species with moderate support in ML analysis (BF = 83%, Figure 5) and weak support in MP analysis (55%, Figure 6). Cucullanus robustus was sister to Ascaridomorpha + Rhabditina + Steinernema carpocapsae in Bayesian ana- lysis with strong support (PP = 1, Figure 4). Cucullanus robustus, however, was sister the Ascaridia species with moderate support in ML analysis (BF = 83%, Figure 5) and weak support in MP analysis (55%, Figure 6). Liu et al. Monophylies of the order Rhabditida and the subclass Dorylaimia BMC Genomics 2013, 14:414 http://www.biomedcentral.com/1471-2164/14/414 Page 8 of 13 Page 8 of 13 Figure 6 Phylogenetic relationships among 65 species of nematodes inferred from maximum parsimony (MP) of deduced amino acid sequences of 12 mitochondrial proteins. Taenia multiceps and T. hydatigena (GenBank accession numbers FJ495086 and FJ518620) were used as the outgroup. Bootstrapping frequency (BF) values were indicated at nodes. Figure 6 Phylogenetic relationships among 65 species of nematodes inferred from maximum parsimony (MP) of deduced amino acid sequences of 12 mitochondrial proteins. Taenia multiceps and T. hydatigena (GenBank accession numbers FJ495086 and FJ518620) were used as the outgroup. Bootstrapping frequency (BF) values were indicated at nodes. Phylogenetic relationships within the subclass Dorylaimia in ML analysis (BF = 73%, Figure 5). The Dorylaimida was grouped with the Trichocephalida in MP analysis but the support to this grouping was weak (BF = 40%, Figure 6). in ML analysis (BF = 73%, Figure 5). The Dorylaimida was grouped with the Trichocephalida in MP analysis but the support to this grouping was weak (BF = 40%, Figure 6). Phylogenetic relationships within the subclass Dorylaimia The 11 species of the subclass Dorylaimia included in this study were from three orders: Mermithida (7 spe- cies), Dorylaimida (1 species), and Trichinellida (3 spe- cies). The Mermitida and the Trichinellida were both monophyletic with strong support in all of the three ana- lyses (PP = 1, Figure 4; BF = 100%, Figure 5; BF = 100%, Figure 6). Among the three orders, the Mermithida and the Dorylaimida were more closely related than either of them to the Trichocephalida, with strong support in Bayesian analysis (PP = 1, Figure 4) and moderate support Phylogenetic relationships within the subclass Dorylaimia The 11 species of the subclass Dorylaimia included in this study were from three orders: Mermithida (7 spe- cies), Dorylaimida (1 species), and Trichinellida (3 spe- cies). The Mermitida and the Trichinellida were both monophyletic with strong support in all of the three ana- lyses (PP = 1, Figure 4; BF = 100%, Figure 5; BF = 100%, Figure 6). Among the three orders, the Mermithida and the Dorylaimida were more closely related than either of them to the Trichocephalida, with strong support in Bayesian analysis (PP = 1, Figure 4) and moderate support Discussion Phylogenetic relationships among nematodes have been revised in the past two decades using sequences of nuclear small subunit (SSU) rRNA gene [12-14]. The current working hypothesis of Nematoda phylogeny incorporated evidence from both morphology, ecology and nuclear SSU rRNA gene sequence analyses [14,15]. Page 9 of 13 Liu et al. BMC Genomics 2013, 14:414 http://www.biomedcentral.com/1471-2164/14/414 Liu et al. BMC Genomics 2013, 14:414 http://www.biomedcentral.com/1471-2164/14/414 Spirurina and Tylenchina, however, were both para- phyletic in all of the three phylogenetic analyses in this study. Monophyly of the suborder Spirurina was well supported in nuclear SSU rRNA gene sequence analyses [12,14], but was rejected in mt genome sequence analyses in Kang et al. [23], Park et al. [24], Sultana et al. [25] and the present study. Phylogenetic analyses of mt genome sequences in all of these four studies support strongly a close relationship between Ascaridomorpha and Rhab- ditomorpha to the exclusion of Oxyuridomorpha and Spiruromorpha. Apparently, additional markers for phylo- genetic inference are required to resolve the controversy on the Spirurina between mt genome sequence phylogeny and nuclear SSU rRNA phylogeny. Recently, Kang et al. [23] tested the hypothesis of the Nematoda phylogeny with the mt genome sequences of 25 species. Kang et al. [23] showed strong support to the monophyly of the class Chromadorea, represented by 16 species from the order Rhabditida; this study, however, could not establish the monophyly of the class Enoplea, represented by nine species from the subclass Dorylaimia. Park et al. [24] and Sultana et al. [25] ex- panded the taxon sampling and inferred phylogenies with mt genome sequences of 36 and 41 species of nematodes respectively. Park et al. [24] showed strong support for the monophylies of both Chromadorea and Enoplea. Sultana et al. [25], however, could not establish the monophyly of the class Enoplea in most of their analyses. A major difference between the mt genome phylogenies and the current working hypothesis of the Nematode phylogeny is on the monophyly of the suborder Spirurina, which includes Clade III nematodes proposed in Blaxter et al. [12]. In the current working hypothesis, the suborder Spirurina is monophyletic, which includes the infraorders Ascaridomorpha, Spiruromorpha and Oxyuridomorpha, whereas the infraorder Rhabdito- morpha is in the suborder Rhabditina [13,14]. Further, Ascaridomorpha is most closely related to Spiruro- morpha, whereas Oxyuridomorpha is sister to the group that includes Ascaridomorpha + Spiruromorpha. Discussion In the phylogenies inferred from mt genome sequences, however, Ascaridomorpha is most closely related to Rhabditomorpha; in most analyses, Oxyuridomorpha is sister to Ascaridomorpha + Rhabditomorpha, whereas Spiruromorpha is sister to the group that contains Ascaridomorpha, Rhabditomorpha and Oxyuridomorpha. Kang et al. [23], Park et al. [24] and Sultana et al. [25] contained much less taxa compared to previous phylogen- etic studies on nematodes with nuclear SSU rRNA gene sequences (e.g. >200 taxa in Meldal et al. [14]). The novel phylogenetic relationship inferred from mt genome se- quences, thus, needs to be tested further with more taxa from a wide range of nematode lineages. p y g y Gene arrangement in mt genomes provides a source of information for phylogenetic inference, independent from mt genome sequences [18]. It is noteworthy that 10 species of Ascaridomorpha and 20 species of Rhab- ditomorpha included in our phylogenetic analyses share a common pattern of gene arrangement, GA3, in their mt genomes (Figure 2). GA3 is also present in Pristionchus pacificus, the only species from the infraorder Diplogas- teromorpha, which is closely related to the species from the superfamily Rhabitoidea of the Rhabditomorpha (Figures 4, 5, 6). Indeed, GA3 is the most common pattern of mt gene arrangement observed in nematodes to date; nevertheless, this pattern is present only in species of the Ascaridomorpha, the Rhabditomorpha and the Diplogas- teromorpha. The possibility that GA3 is ancestral to the phylum Nematoda can be rejected with confidence thanks to the discovery of mt gene arrangement pattern GA23 in three Trichinellida species [29,30]. GA23 shares obvious similarity with that of many arthropods and would resem- ble the ancestral pattern of mt gene arrangement of the phylum Nematoda more than any other patterns found in the nematodes. There is no evidence either that GA3 is ancestral to the class Chromadorea. Given the strong support to the close relationship between Ascaridomorpha and Rhabditomorpha + Diplogasteromorpha indicated by mt genome sequence analyses in the present study, the most parsimonious explanation is that GA3 is a shared derived character (i.e. synapomorphy) of the clade Ascaridomorpha + Rhabditomorpha + Diplogasteromorpha. The other six patterns, GA1, GA2, GA4, GA5, GA6 and GA25, observed in either Ascaridomorpha and Rhabditomorpha or in species closely related to these two infraorders, can be derived from GA3 by only a few rearrangement events (Figure 2), e.g. Discussion GA1, observed in the three Ascaridia species, can be converted from GA3 by three rearrangement events (see above). In the present study, we sequenced the complete mt genomes of three Ascaridia species from the family Ascaridiidae of the superfamily Heterakoidea, which was not represented in previous studies (Kang et al. [23]; Park et al. [24]; Sultana et al. [25]). Furthermore, we inferred the phylogenetic relationships among 65 nema- tode species, for which complete mt genomes have been sequenced to date. Our analyses support the division of nematodes into two classes, Chromadorea and Enoplea, represented by the order Rhabditida and the subclass Dorylaimia, respectively, in the present study. Both the Rhabditida and the Dorylaimia were monophyletic with strong support, regardless the methods of phylogenetic analysis used. Within the order Rhabditida, the suborder Rhabditina was monophyletic; the other two suborders, Monophyly of the suborder Tylenchina could not be established previously in nuclear SSU RNA gene se- quence analyses [14]. No species from this suborder were included in two previous analyses of mt genome Liu et al. BMC Genomics 2013, 14:414 http://www.biomedcentral.com/1471-2164/14/414 Page 10 of 13 sequences [23,24]. The present study included four species from the Tylenchina and indicated the paraphyly of this suborder. The positions of the four species of the Tylenchina change depending on the methods of phy- logenetic inference used in this study. For instance, Steinernema carpocapsae is sister to the suborder Rhabditina in Bayesian analysis (Figure 4) and MP ana- lysis (Figure 6); it is, however, sister to Ascaridomorpha + Rhabditomorpha + Diplogasteromorpha in ML analysis (Figure 5). Further test with more species from this suborder apparently is needed in order to clarify the phylogenetic position of the suborder Tylenchina. Indeed, a very recent study by Sultana et al. [25] included six species from the Tylenchina and showed the paraphyly of this suborder with strong support. The paraphyly of the infraorder Rhabditomorpha with respect to the Diplogasteromorpha will also need further test with ex- panded taxon sampling as only one species, Pristionchus pacificus, from the Diplogasteromorpha was included in the present study. No species from the subclass Enoplia, which has two orders and eight suborders [13], was in- cluded in our analyses. For the subclass Chromadoria, all of the species included in this study were from the order Rhabditida whereas the other five orders, Plectida, Araeolaimida, Monhysterida, Desmodorida, and Chro- madorida, were not represented. Conclusions We sequenced the complete mt genomes of three Ascaridia species (family Ascaridiidae) that infest chick- ens, pigeons and parrots, respectively. These species have an identical arrangement of mt genes to each other but differ substantially from other nematodes. Phylogenetic analyses of the mt genome sequences of the Ascaridia species, in combination with 62 other nematode species, support the monophylies of the subclass Dorylaimia; the orders Rhabditida, Trichinellida and Mermithida; the suborder Rhabditina; and the infraorders Spiruromorpha and Oxyuridomorpha. Analyses of mt genome sequences, however, reject the monophylies of the suborders Spirurina and Tylenchina, and the infraorders Rhabdi- tomorpha, Panagrolaimomorpha and Tylenchomorpha. Monophyly of the infraorder Ascaridomorpha varies depending on the methods of phylogenetic analysis. The Ascaridomorpha was more closely related to the infra- orders Rhabditomorpha and Diplogasteromorpha (sub- order Rhabditina) than they were to the other two infraorders of the Spirurina: Oxyuridorpha and Spiruro- morpha. The closer relationship among Ascaridomorpha, Rhabditomorpha and Diplogasteromorpha was also sup- ported by a shared common pattern of mitochondrial Ethics statement This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Approval No. LVRIAEC2011-009). The animals, from which the nematodes were collected post- mortem, were handled in strict accordance with good animal practices required by the Animal Ethics Procedures and Guidelines of the People's Republic of China. Sample collection and DNA extraction Adult Ascaridia galli were obtained from naturally infected chickens in Hunan province, China. Adult A. columbae and an undescribed Ascaridia sp. [hereafter Ascaridia sp. (GHL-2012)] were obtained from naturally infected pigeons and parrots in Guangdong province, China. These nematodes were washed in physiological saline, identified preliminarily to species based on host preference, morphological characters and predilection sites [31-33], fixed in 70% ethanol, and stored at −20°C. Total genomic DNA was isolated from individual nema- todes using sodium dodecyl sulphate/proteinase K treat- ment, followed by spin-column purification (Wizard® SV Genomic DNA Purification System, Promega). In order to independently verify the identity of the specimen, the region spanning ITS-1, 5.8S rRNA gene and ITS-2 was amplified from each individual nematode by PCR using previously reported primers [34] and sequenced directly. Sample collection and DNA extraction Adult Ascaridia galli were obtained from naturally infected chickens in Hunan province, China. Adult A. columbae and an undescribed Ascaridia sp. [hereafter Ascaridia sp. (GHL-2012)] were obtained from naturally infected pigeons and parrots in Guangdong province, China. These nematodes were washed in physiological saline, identified preliminarily to species based on host preference, morphological characters and predilection sites [31-33], fixed in 70% ethanol, and stored at −20°C. Total genomic DNA was isolated from individual nema- todes using sodium dodecyl sulphate/proteinase K treat- ment, followed by spin-column purification (Wizard® SV Genomic DNA Purification System, Promega). In order to independently verify the identity of the specimen, the region spanning ITS-1, 5.8S rRNA gene and ITS-2 was amplified from each individual nematode by PCR using previously reported primers [34] and sequenced directly. Discussion Expanding taxon sam- pling from these lineages of nematodes is clearly the next step for phylogenetic studies of nematodes with mt genome sequences. gene arrangement. Analyses of mitochondrial genome se- quences and gene arrangement in the current study and several previous studies have provided novel insights into the phylogenetic relationships among several major line- ages of nematodes. Many lineages of nematodes, however, are underrepresented or not represented in these analyses. Expanding taxon sampling is necessary for future phylo- genetic studies of nematodes with mt genome sequences. PCR amplification of the mitochondrial genomes of Ascaridia species ACC1N1F AGTTGGACTGTTTATCCGCCTTTGA ACC1N1R ATTTCATAAGACACTCTCTGACCTC ASN1C3F GGTCAGAGGGTTTCTTATGAGATTGCT ASN1C3R CAACCGAACAATCTTTATTACTCAACA ASC3C2F TGTTGAGTAATAAAGATTGTTCGGTTG ASC3C2R ACCAGGAATGTCACCATACTCATAACT ASC2C1F TGGAGGTTGATAATCGTTGTGTTTTGC ASC2C1R CACAACAACAAAGGCTGAAAAACCATC Table 2 Primers used to amplify short-PCR fragments from Ascaridia galli, Ascaridia columbae and Ascaridia sp Name of primer Sequence (5′ to 3′) Reference For A. galli pcox1 AGCox1F GAAGTTTGTATTTGACTGGTAAGAA This study AGCox1R CAGTGAGACCACCAATAGTAAACAA This study pnad1 JB11 AGATTCGTAAGGGGCCTAATA [36] JB12 ACCACTAACTAATTCACTTTC [36] pnad4 AGNad4F CTTATTATTTAATTTTTTATGCTGCT This study AGNad4R AAGCGGCTAAAGCCTTAGCATCACT This study For A. columbae and Ascaridia sp. pcox2 ACCox2F TTTAAGTTTGTTGTATTATTATGGTTT This study ACCox2R AAAATACACCAACCACAGGAAAACT This study pnad1 JB11 AGATTCGTAAGGGGCCTAATA [36] JB12 ACCACTAACTAATTCACTTTC [36] pcox3 ACCox3F TGGTATTTTCTGGACTTTTTTTGAT This study ACCox3R CCAAACTACATCTACAAAATGCCAA This study Table 2 Primers used to amplify short-PCR fragments from Ascaridia galli, Ascaridia columbae and Ascaridia sp Table 3 Primers used to amplify long-PCR fragments from Ascaridia galli, Ascaridia columbae and Ascaridia sp were between cox1 and nad1 (~2.5 kb), between nad1 and nad3 (~5 kb), between nad3 and cox2 (~2.5 kb) and between cox2 and cox1 (~5.0 kb). Each long-PCR reaction (25 μl) contains 2.5 μL 2 mM MgCl2, 4.0 μL 0.2 mM each of dNTPs, 2.5 μL 2.5 μl 10× rTaq buffer, 0.25 μL 2.5 μM of each primer, 0.25 μL 1.25 U rTaq polymerase (Takara), and 2 μl of total genomic DNA. The PCR conditions were: 92°C for 2 min (initial denatu- ration), then 92°C for 10 sec (denaturation), 48–51°C for 30 sec (annealing) and 60°C for 10 min (extension) for 10 cycles, followed by 92°C for 10 sec, 48–51°C for 30 sec, and 60°C for 10 min for 20 cycles and a final extension at 60°C for 10 min. PCR amplicons were column-purified and then sequenced using a primer walking strategy [35]. (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) using the invertebrate mitochondrial genetic code, and subse- quently compared with those of Ascaris suum [38] and Contracaecum rudolphii B [28]. Sequences of protein- coding genes were translated into amino acid sequences using the invertebrate mitochondrial genetic code in MEGA 5.0 [39]. Translation initiation and termination codons were identified by comparison with those of the nematodes reported previously [27,28]. The secondary structures of 22 tRNA genes were predicted using tRNAscan-SE [40] with manual adjustment [41]. Tan- dem repeats in the non-coding regions were found using Tandem Repeat Finder program (http://tandem. bu.edu/trf/trf.html) [42]. Sequence assembly and mitochondrial genome annotation Sequence reads of A. galli, A. columbae and Ascaridia sp. (GHL-2012) were assembled with ContigExpress program of the Vector NTI software package version 6.0 (Invitrogen, Carlsbad, CA). The mt genome sequences of the Ascaridia species were aligned with those of other nematode species available in GenBank using Clustal X 1.83 [37] to infer gene boundaries. The open-reading frames and codon usage profiles of protein-coding genes were analysed by the Open Reading Frame Finder PCR amplification of the mitochondrial genomes of Ascaridia species We amplified fragments (400–500 bp) of cox1, nad1 and nad4 genes by PCR using conserved primers (Table 2). The amplicons were sequenced from both directions using BigDye terminator v3.1 on ABI PRISM 3730 platform. We then designed specific primers from these fragments for long PCR amplification (Table 3). We amplified the entire mt genome of individual specimens of A. galli, A. columbae and Ascaridia sp. (GHL-2012) by long PCR in three or four overlapping fragments, respectively. The three overlapping long-PCR fragments for A. galli were between cox1 and nad1 (~2.5 kb), be- tween nad1 and nad4 (~7.5 kb), and between nad4 and cox1 (~5.0 kb). The four overlapping long-PCR frag- ments for A. columbae and Ascaridia sp. (GHL-2012) Page 11 of 13 Liu et al. BMC Genomics 2013, 14:414 http://www.biomedcentral.com/1471-2164/14/414 were between cox1 and nad1 (~2.5 kb), between nad1 and nad3 (~5 kb), between nad3 and cox2 (~2.5 kb) and between cox2 and cox1 (~5.0 kb). Each long-PCR reaction (25 μl) contains 2.5 μL 2 mM MgCl2, 4.0 μL 0.2 mM each of dNTPs, 2.5 μL 2.5 μl 10× rTaq buffer, 0.25 μL 2.5 μM of each primer, 0.25 μL 1.25 U rTaq polymerase (Takara), and 2 μl of total genomic DNA. The PCR conditions were: 92°C for 2 min (initial denatu- ration), then 92°C for 10 sec (denaturation), 48–51°C for 30 sec (annealing) and 60°C for 10 min (extension) for 10 cycles, followed by 92°C for 10 sec, 48–51°C for 30 sec, and 60°C for 10 min for 20 cycles and a final extension at 60°C for 10 min. PCR amplicons were column-purified and then sequenced using a primer walking strategy [35]. Table 2 Primers used to amplify short-PCR fragments from Ascaridia galli, Ascaridia columbae and Ascaridia sp Name of primer Sequence (5′ to 3′) Reference For A. galli pcox1 AGCox1F GAAGTTTGTATTTGACTGGTAAGAA This study AGCox1R CAGTGAGACCACCAATAGTAAACAA This study pnad1 JB11 AGATTCGTAAGGGGCCTAATA [36] JB12 ACCACTAACTAATTCACTTTC [36] pnad4 AGNad4F CTTATTATTTAATTTTTTATGCTGCT This study AGNad4R AAGCGGCTAAAGCCTTAGCATCACT This study For A. columbae and Ascaridia sp. pcox2 ACCox2F TTTAAGTTTGTTGTATTATTATGGTTT This study ACCox2R AAAATACACCAACCACAGGAAAACT This study pnad1 JB11 AGATTCGTAAGGGGCCTAATA [36] JB12 ACCACTAACTAATTCACTTTC [36] pcox3 ACCox3F TGGTATTTTCTGGACTTTTTTTGAT This study ACCox3R CCAAACTACATCTACAAAATGCCAA This study were between cox1 and nad1 (~2.5 kb), between nad1 and nad3 (~5 kb), between nad3 and cox2 (~2.5 kb) and between cox2 and cox1 (~5.0 kb). PCR amplification of the mitochondrial genomes of Ascaridia species Each long-PCR reaction (25 μl) contains 2.5 μL 2 mM MgCl2, 4.0 μL 0.2 mM each of dNTPs, 2.5 μL 2.5 μl 10× rTaq buffer, 0.25 μL 2.5 μM of each primer, 0.25 μL 1.25 U rTaq polymerase (Takara), and 2 μl of total genomic DNA. The PCR conditions were: 92°C for 2 min (initial denatu- ration), then 92°C for 10 sec (denaturation), 48–51°C for 30 sec (annealing) and 60°C for 10 min (extension) for 10 cycles, followed by 92°C for 10 sec, 48–51°C for 30 sec, and 60°C for 10 min for 20 cycles and a final extension at 60°C for 10 min. PCR amplicons were column-purified and then sequenced using a primer walking strategy [35]. Sequence assembly and mitochondrial genome annotation Sequence reads of A. galli, A. columbae and Ascaridia (GHL 2012) bl d ith C ti E (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) using invertebrate mitochondrial genetic code, and sub quently compared with those of Ascaris suum [38] Contracaecum rudolphii B [28]. Sequences of prot coding genes were translated into amino acid sequen using the invertebrate mitochondrial genetic code MEGA 5.0 [39]. Translation initiation and termina codons were identified by comparison with those of nematodes reported previously [27,28]. The second structures of 22 tRNA genes were predicted u tRNAscan-SE [40] with manual adjustment [41]. T dem repeats in the non-coding regions were fo i T d R t Fi d (htt //t d Table 2 Primers used to amplify short-PCR fragments from Ascaridia galli, Ascaridia columbae and Ascaridia sp Name of primer Sequence (5′ to 3′) Reference For A. galli pcox1 AGCox1F GAAGTTTGTATTTGACTGGTAAGAA This study AGCox1R CAGTGAGACCACCAATAGTAAACAA This study pnad1 JB11 AGATTCGTAAGGGGCCTAATA [36] JB12 ACCACTAACTAATTCACTTTC [36] pnad4 AGNad4F CTTATTATTTAATTTTTTATGCTGCT This study AGNad4R AAGCGGCTAAAGCCTTAGCATCACT This study For A. columbae and Ascaridia sp. pcox2 ACCox2F TTTAAGTTTGTTGTATTATTATGGTTT This study ACCox2R AAAATACACCAACCACAGGAAAACT This study pnad1 JB11 AGATTCGTAAGGGGCCTAATA [36] JB12 ACCACTAACTAATTCACTTTC [36] pcox3 ACCox3F TGGTATTTTCTGGACTTTTTTTGAT This study ACCox3R CCAAACTACATCTACAAAATGCCAA This study Table 3 Primers used to amplify long-PCR fragments f Ascaridia galli, Ascaridia columbae and Ascaridia sp Name of primer Sequence (5′ to 3′) For A. galli AGC1N1F ATAGAAGTTTGTATTTGACTGGTAAGAAGGA AGC1N1R CACAATACCAGTAACCAAAGTAGCATAAAC AGN1N4F TAAGTTGTTGAAGAAGGAGCAGGAGAGT AGN1N4R CAAAAATGGAAAAGAACACAAAGCAGCA AGN4C1F TTTTTATGCTGCTTTGTGTTCTTTTCCA AGN4C1R CCAAATAAAGTTGCCAGCCACCTAAA For A. columbae ACC1N1F AGTTGGACTGTTTATCCGCCTTTGA ACC1N1R ATTTCATAAGACACTCTCTGACCTC ACN1C3F GCCAGAGGTCAGAGAGTGTCTTATG ACN1C3R CTTGCTTCACTATACTCTATTGCCTGT ACC3C2F CAGGCAATAGAGTATAGTGAAGCAA ACC3C2R ATAGAAGGCACAGCCCAAGAATGAA ACC2C1F TAGTATGTGATGTTTGGGAATGCTT ACC2C1R CTTTTACACCAGTAGGCACAGCGAT For Ascaridia sp. 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BMC Genomics 2013, 14:414 http://www.biomedcentral.com/1471-2164/14/414 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 41. 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https://openalex.org/W2533643392	https://www.jnma.com.np/jnma/index.php/jnma/article/download/2747/2281	English	null	Patients’ Awareness about the Complications of Diabetes Mellitus and its Co-relation with the Glycemic Status	Journal of Nepal Medical Association	2,015	cc-by	2,761	J Nepal Med Assoc 2015;53(200):284-7 J Nepal Med Assoc 2015;53(200):284-7 original article CC S BY NC OPEN ACCESS CC S BY NC OPEN ACCESS Patients’ Awareness about the Complications of Diabetes Mellitus and its Co relation with the Glycemic Status Krishna Kumar Agrawaal1 1Department of Internal Medicine, Universal College of Medical Sciences, Nepal. Krishna Kumar Agrawaal1 1Department of Internal Medicine, Universal College of Medical Sciences, Nepal. Abstract Introduction: Diabetes is a major cause of morbidity and cardiovascular related mortality along with a major cause of preventable blindness and foot amputation. There are limited studies on diabetes awareness, attitude, and prevalence. Methods: Thus, we designed a study to seek the patients awareness about the complications associated with diabetes. It was a prospective observational study which included 123 patients with Diabetes Mellitus. Results: The mean age of population studied was 53 years mainly from the Rupandehi and Nawalparasi districts in Nepal attending a clinic. In the study Erectile Dysfunction was statistically significant with postprandial hyperglycemia. Lack of Awareness about a calorie specific diet plan was significantly associated with increased blood sugar fasting level >110mg/dl. A majority of patients (58%) had awareness regarding kidney damage and 51% had awareness that Diabetes causes delayed wound healing. Only 3% of the patients did a regular foot care and 9% knew what the target glycemic status is. About 36% know that there can be cardiac complications due to diabetes and 27% regarding eyes. Similarly 36% of the patients also knew that there is neuropathy due to diabetes and 18% were aware about CVA. About 54% of the patients had Fasting >110mg%. Conclusion: The study showed that though the patients are on OADs but they lack awareness about the major complications related to diabetes mellitus. Keywords: awareness of diabetic complications; HbA1c; patient awareness. INTRODUCTION INTRODUCTION Diabetes is an emerging pandemic in the South Asia. Diabetes is an emerging pandemic in the South Asia. As per the International Diabetes Federation, the latest estimates indicate that there are 382 million people living with diabetes. One in twelve has diabetes in South East Asia.1 As the risk for diabetes is increasing so will be the complications related to this and the trend towards patient’s education and self care has to increase.4 There are limited studies on diabetes awareness, attitude, and prevalence.5 Interestingly patients do a regular blood sugar fasting and postprandial in our setup. So we designed a study to look for the patients awareness about the complications related to diabetes and compare that with the desired blood sugar levels. As the risk for diabetes is increasing so will be the complications related to this and the trend towards patient’s education and self care has to increase.4 There are limited studies on diabetes awareness, attitude, and prevalence.5 Interestingly patients do a regular blood sugar fasting and postprandial in our setup. So we designed a study to look for the patients awareness about the complications related to diabetes and compare that with the desired blood sugar levels. In a study by Nepal Diabetes Association the prevalence of diabetes in population of 20 years and above was estimated to be 14.6% and 2.5% in urban and rural areas respectively.2 The risk of developing diabetes increases by five times in patients with metabolic syndrome.3 Correspondence: Dr. Krishna Kumar Agrawaal, Department of Internal Medicine, Universal College of Medical Sciences, Nepal. Email: agrawalkris@gmail.com, Phone: +977- 9857045721. Correspondence: Dr. Krishna Kumar Agrawaal, Department of Internal Medicine, Universal College of Medical Sciences, Nepal. Email: agrawalkris@gmail.com, Phone: +977- 9857045721. JNMA I VOL 53 I NO. 4 I ISSUE 200 I OCT-DEC, 2015 JNMA I VOL 53 I NO. 4 I ISSUE 200 I OCT-DEC, 2015 284 Agrawaal Patients’ Awareness about the Complications of Diabetes Mellitus and its Co relation with the Glycemic Status Table 2. Showing the different awareness characteristics at HbA1c <7% . INTRODUCTION Complications Awareness Yes No P value Eyes 11(34.4%) 24(27.9%) 0.494 Kidneys 17(24.3%) 18(37.5%) 0.123 Neuropathy 8(19.0%) 27(35.5%) 0.061 Poor Wound Healing 18(29.5%0 17(29.8%) 0.970 IHD 10(23.3%) 25(33.3%) 0.249 CVA 4(20.0%) 31(31.6%) 0.299 Awareness about Hypoglycemia 5(20.8%) 30(31.9%) 0.289 Knowledge of the Glycemic status as recommended 1(11.1%) 34(31.2%) 0.205 Foot Care 2(66.7% 33(28.7%) 0.435 On a diet based on calories 1(14.3%) 34(30.6%) 0.623 Erectile Dysfunction 2(14.3%) 33(31.7%) 0.180 Table 2. Showing the different awareness characteristics at HbA1c <7% . METHODS Study Design: Prospective Observational study Duration: 15th November 2014 till 15th March 2015 were enrolled for the study. Consent: Consent from the patients was taken before enrollment. Ethical approval: The study was approved from the Christian Medical College. Inclusion Criteria: Patients with diagnosed Type 2 Mellitus on Medications/MNT with age of diagnosis more than one year. Exclusion criteria: Refusal to give the consent STATISTICAL ANALYSIS A total of 123 patients were enrolled in the study. The data was collected using a structured questionnaire incorporating demographics; assessment of complication awareness. Diabetic control was assessed by the most recent HbA1c level and last Blood sugar level both fasting and postprandial. Data were entered in Microsoft excel 2000 and converted into SPSS version 20 for statistical analysis. Percentage, graphical presentation and other descriptive statistics were calculated. The significance level of the data which were taken for the analysis was ≤ 0.05. Data were entered in Microsoft excel 2000 and converted into SPSS version 20 for statistical analysis. Percentage, graphical presentation and other descriptive statistics were calculated. The significance level of the data which were taken for the analysis was ≤ 0.05. JNMA I VOL 53 I NO. 4 I ISSUE 200 I OCT-DEC, 2015 RESULTS There were altogether 123 patients who were enrolled in the study. The results are depicted in the following self explanatory tables and graphs. Table 1: Baseline Characteristics of the patient enrolled in the study. Characteristics Minimum Maximum Mean Std. Deviation Age in years 25.00 76.00 53.50 10.76 Duration of Diabetes in years 1.00 15.00 6.03 3.34 Weight in kg 42.00 86.00 58.36 9.70 Height in cms 139.00 176.00 155.97 7.33 BMI(kg/m2 16.80 33.33 23.95 3.29 Waist circumference in cms 70.00 112.00 93.90 7.68 Hip Circumference in cms 64.00 111.00 94.31 6.66 Waist Hip ratio 0.88 1.11 1.00 0.05 HbA1c in % 5.0 12.4 7.80 1.49 Table 1: Baseline Characteristics of the patient enrolled i h d Table 3. Showing the different awareness characteristics at the level of awareness at HbA1c >7%. Complications Awareness Yes No P value Eyes 21(65.6) 62(72.1%) 0.494 Kidneys 53(75.7%) 30(62.5%) 0.123 Neuropathy 34(81.0%) 49(64.5%) 0.061 Poor Wound Healing 43(70.5% 40(70.2%) 0.970 IHD 33(76.7%) 50(66.7%) 0.249 CVA 16(80.0) 67(68.4) 0.299 ED 12(85.7) 71(68.3) 0.180 Diet 6(85.7%) 77(69.4%) 0.627 Glycemic status 8(88.9%) 75(68.8%) 0.205 Foot care 1(33.3%) 82(71.3%) 0.435 Hypoglycemia 19(79.2) 64(68.1%) 0.289 Table 3. Showing the different awareness characteristics at the level of awareness at HbA1c >7%. 285 JNMA I VOL 53 I NO. 4 I ISSUE 200 I OCT-DEC, 2015 Agrawaal Patients’ Awareness about the Complications of Diabetes Mellitus and its Co relation with the Glycemic Status Figure 1. Showing the educational status of the patients 32.5% 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 Literate Formally educated Higher Secondary level Education Degree and above 44.7% 22.0% 0.8% Figure 2. Showing the physical activity level of the population in study 35.8% 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 no <30m in/day >30m in/day Physical activity score 48.0% 16.3% Figure 1. Showing the educational status of the patients 32.5% 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 Literate Formally educated Higher Secondary level Education Degree and above 44.7% 22.0% 0.8% Figure 2. Showing the physical activity level of the population in study. 35.8% 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 no <30m in/day >30m in/day Physical activity score 48.0% 16.3% Figure 3. Showing the percentage distribution of awareness of different complication of diabetes. RESULTS 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 Eyes Kindneys Neuropathy Wound Healing 26.8 58.5 35.8 51.2 35.8 17.1 13.0 2.4 20.3 8.1 5.7 IHD CVA ED Footcare Hypoglycemia Glycemic Status Diet Plan in the study by Mohan V.10 In our study it was observed that about one third patients (33%) had not received any formal education. The educational status of the study population was significantly associated with the knowledge of glycemic status (P value- 0.007) and awareness about the foot care (P value-0.001). The physical activity was less than the recommended. In a study conducted in Shivkashi district in India it was found that 74% of the diabetic population had no awareness regarding the long term effects of Diabetes.11 In a population based study in Tamilnadu by Mohan V et al it was found that only 19.0% (4951/26001) of whole population knew that diabetes could cause complications. In our study the family history of Diabetes was present in 39.8% whereas in a urban population study in India it was 38.2%.12 35.8% 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 no <30m in/day >30m in/day Physical activity score 48.0% 16.3% In our study we observed that there was a majority population with awareness of kidney diseases (59%). Following this the patient were mainly aware that Diabetes causes Poor wound healing (52%) followed by Neuropathy (36%), affects the heart mainly Ischemic Heart disease (36%), affects the Eyes (27%), Causes Cerebrovacular accidents(18%), causes erectile dysfunction(14%), Knowledge of glycemic status(8%), Awareness about a calorie specific diet plan(6%) and Foot care(2%). The awareness to cardiovascular effects with diabetes is low in our study as compared to a similar study where it was 52% compared to 36%.13 Figure 2. Showing the physical activity level of the population in study. Figure 3. Showing the percentage distribution of awareness of different complication of diabetes. RESULTS 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 Eyes Kindneys Neuropathy Wound Healing 26.8 58.5 35.8 51.2 35.8 17.1 13.0 2.4 20.3 8.1 5.7 IHD CVA ED Footcare Hypoglycemia Glycemic Status Diet Plan In a study done in Ethopia 67.79%) had knowledge about long-term complications.14 In the study we observed that awareness about neuropathy and the HbA1c level >7% is nearly statistically significant (P value: 0.06).Similarly Lack of knowledge about a calorie specific diet plan is statistically significant with the blood sugar fasting of >110mg%. Similarly lack of awareness about erectile dysfunction was associated with increased postprandial blood sugar level>140mg% (P value 0.02). In contrary to our study, a study done in Chandigarh showed that almost two third of the patients with T2DM had good knowledge about the disease.15 Figure 3. Showing the percentage distribution of awareness of different complication of diabetes. REFERENCES 12. Mohan D, Raj D, Shanthirani CS, Datta Manjula, Unwin NC, Kapur A et al. Awareness and Knowledge of Diabete in Chennai- The Chennai Urban Rural Epidemilogy study 1. International Diabetes Federation. IDF diabetes Atlas. Sixth Edition update.International Diabetes Federation 2014. 2. Nepal Diabetes Association[Internet].Kathmandu: Nepal diabetes association; 2014.Available from: www.nepaldiabe- tesassociation/reseach.php 13. O suvillan EP, Bhargava A, O'Callaghan M, Buckley U, De Faoite T, Moynihan K et al. Awareness of diabetes complications in an Irish population. Ir J Med Sci. 2009 Dec;178(4):4016 3. Sharma SK, Ghimire G, Radhakrishnan J, Thapa L, Shrestha NR et al.Prevalence of Hypertension, Obesity, Diabetes and Metabolic Syndrome in Nepal. IJH 2011. 14. Abdella SH and Mohammed AM. Awareness of diabetic patients about their illness and associated complications in Ethopia. Med-Science .2013; 2(2): 512-522. 4. Agrawaal KK. Diabetes: Emerging Pandemic. JUCMS: 2014; vol.2 (8):pg.01. 15. Kaur S, Mandal L, Nair P, Kaur P, Pathak P and Bhansali A. Awareness among patients with T2DM regarding the Disease at a tertiary care referral center. Journal of Postgraduate Medicine, Education and Research, July-September 2014;48(3):117-122. 5. Singh A, Milton PE, Nanaiah A, Samuel P, Thomas N. Awareness and attitude toward diabetes in the rural population of Arunachal Pradesh, Northeast India. Indian J Endocr Metab: 2012; 16, Suppl S1:836. 6. N. Murugesan et al. Awareness about diabetes and its complications in the general and diabetic population in a city in southern India. Diabetes Research and Clinical Practice: 2007) 433–437. 16. Ahmad J. The diabetic foot. Diabetes Metab Syndr. 2015 Apr 27. pii: S18714021(00030-2). 17. Deepa M , Bhansali A , Anjana RM , Pradeepa R , Joshi SR , Joshi P et al.Knowledge and awareness of diabetes in urban and rural India: The Indian Council of Medical Research India Diabetes Study (Phase I): Indian Council of Medical Research India. Indian J Endocrinol Metab. 2014 May;18(3):379-85. 7. Chaudhary FMD and SMD. Awareness about diabetes risk factors and complications in diabetic patients: A cross-sectional study. NMJ 2010; 2(3):84-88 8. Maskey R, Shakya DR, Sharma SK, Karki P and Lavaju P. Diabetes mellitus related complications in out- patient clinic of tertiary care hospital. Journal of College of Medical Sciences-Nepal, 2011, Vol-7, No-2, 9-16. 18. Misra A, Ramchandran A, Jayawardena R, Shrivastava U and Snehalatha C. Diabetes in South Asians. Diabet Med. 2014 Oct;31 (10):1153-62. 9. Hoque AM, Islam S, Khan AM. Knowledge of Diabetic Complications in a diabetic Population. CONCLUSION CONCLUSION In this Prospective Observational study it was observed that there is a significant lack of awareness about both microvascular and macrovascular complications in the patients with diabetes mellitus. They also lack awareness about the calorie specific diet plan. DISCUSSION Our study population had no idea about foot care in Diabetes. Ahmad J in his article pointed out that approximately 10-15% of diabetic patients developed foot ulcers at some state in their life and 15% of all load in amputations are performed in patients with diabetes.16 The awareness about diabetes and related issues is poor in general population.6 Whereas in diabetic population the awareness about the diabetic complications was observed to be 50% in a study done in Pakistan.7 In our study of 123 patients included 56% male patients and 44% female patients. In a study done by Maskey et al in Nepal also showed a Male preponderance whereas other studies have shown a female preponderance.8 A similar study conducted in Bangladesh by haque et al had a female preponderance in the study.9 The mean BMI in our population was 24kg/m 2 whereas the mean waist to hip ratio was 1. Similar findings were observed In a nationwide study conducted by the Indian Council for Medical Research it was concluded that the Knowledge and awareness about the Diabetes is poor in India in both the Urban and Rural population.17 In an article by Misra A et al, they mention that the key areas that need focus are: generation of awareness, screening of high risk groups, maximum coverage of the population with JNMA I VOL 53 I NO. 4 I ISSUE 200 I OCT-DEC, 2015 286 JNMA I VOL 53 I NO. 4 I ISSUE 200 I OCT-DEC, 2015 Agrawaal Patients’ Awareness about the Complications of Diabetes Mellitus and its Co relation with the Glycemic Status Agrawaal Patients’ Awareness about the Complications of Diabetes Mellitus and its Co relation with the Glycemic Status essential medicines, and strengthening primary care.18 survey done by Nepal Health Research Council in 2013 it was indicated that most of the respondents in the survey were on OAD agents (64%).21 In a similar study it was observed that the two most important sources were friends & neighbor (51%) and family (49%) respectively, followed by electronic media (46%) whereas in our study it was almost by the healthcare provider.19 Ying et al in their study also had finding similar to our study and they also concluded that the main strategy is patients education about the complications.20 Nearly all the patients were on some form of medications for the treatment of diabetes. In a REFERENCES J MEDICINE 2009; 10(2): 90-93. 19. Mumu JS, Salehz F, Ara F, Haque R and Ali L Awareness regarding risk factors of type 2 diabetes among individuals attending a tertiary-care hospital in Bangladesh: a cross-sectional study. BMC Research Notes 2014, 7:599. pg:1-9. 10. Mohan V, Sandeep R, Shah D and Varghese C. Epidemiology of type 2 diabetes: Indian scenario. Indian J Med Res 125, March 2007, pp 217-230 20. Tang YH, Pang MCS, Yeung SPG, Yeung TFV. Health literacy, complication awareness, and diabetic control in patients with type 2 diabetes mellitus. JAN.2007 Oct 5:1-10. 11. Kasinathan D, Rajagopalan NG, Marimuthu NP, Ramar M et al. Awareness on Type II Diabetes and Its Complication among Sivaganga District Population in Tamilnadu: A Cross Section Survey. J Adv Sci Res, 2013, 4(1): 38-42 21. Aryal KK; Neupane S;Meheta S;Vaidya A; Singh S; Paulin F et al. Non Communicable diseases risk factors: STEPS survey Nepal 2013.Kathmandu: Nepal Health Research Council. 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https://openalex.org/W2904491729	https://lirias.kuleuven.be/bitstream/123456789/631323/2/2018%20Frontiers%20IL13RA2.pdf	English	null	Effects of Epithelial IL-13Rα2 Expression in Inflammatory Bowel Disease	Frontiers in immunology	2,018	cc-by	7,206	Citation: Verstockt B, Perrier C, De Hertogh G, Cremer J, Creyns B, Van Assche G, Ferrante M, Ceuppens JL, Vermeire S and Breynaert C (2018) Effects of Epithelial IL-13Rα2 Expression in Inﬂammatory Bowel Disease. Front. Immunol. 9:2983. doi: 10.3389/ﬁmmu.2018.02983 Effects of Epithelial IL-13Rα2 Expression in Inﬂammatory Bowel Disease Bram Verstockt 1,2†, Clémentine Perrier 1,3†, Gert De Hertogh 4, Jonathan Cremer 1,3, Brecht Creyns 1,3, Gert Van Assche 1,2, Marc Ferrante 1,2, Jan L. Ceuppens 3, Séverine Vermeire 1,2 and Christine Breynaert 3* Bram Verstockt 1,2†, Clémentine Perrier 1,3†, Gert De Hertogh 4, Jonathan Cremer 1,3, Brecht Creyns 1,3, Gert Van Assche 1,2, Marc Ferrante 1,2, Jan L. Ceuppens 3, Séverine Vermeire 1,2 and Christine Breynaert 3* 1 KU Leuven, Department of Chronic Diseases, Metabolism and Ageing, Translational Research for Gastrointestinal Disorders, Leuven, Belgium, 2 University Hospitals Leuven, Department of Gastroenterology and Hepatology, Leuven, Belgium, 3 KU Leuven, Laboratory of Clinical Immunology, Department of Microbiology and Immunology, Leuven, Belgium, 4 KU Leuven, Department of Imaging & Pathology, Translational Cell & Tissue Research, Leuven, Belgium Background: Mucosal IL-13 Receptor alpha 2 (IL13RA2) mRNA expression is one of the best predictive markers for primary non-responsiveness to inﬂiximab therapy in patients with inﬂammatory bowel disease (IBD). The objective of this study was to understand how IL-13Rα2, a negative regulator of IL-13 signaling, can contribute to IBD pathology. Methods: IL13RA2 knockout (KO) and wild type (WT) mice were exposed to dextran sodium sulfate (DSS) in drinking water to induce colitis. Furthermore, mucosal biopsies and resection specimen of healthy individuals and IBD patients before the start of anti-tumor necrosis factor (anti-TNF) therapy were obtained for immunohistochemistry and gene expression analysis. Edited by: Eric Cox, Ghent University, Belgium Reviewed by: Britta Siegmund, Charité Universitätsmedizin Berlin, Germany Hiroshi Nakase, Sapporo Medical University, Japan Correspondence: Christine Breynaert christine breynaert@uzleuven be Reviewed by: Britta Siegmund, Charité Universitätsmedizin Berlin, Germany Hiroshi Nakase, Sapporo Medical University, Japan Results: After induction of DSS colitis, IL13RA2 KO mice had similar disease severity, but recovered more rapidly than WT animals. Goblet cell numbers and mucosal architecture were also more rapidly restored in IL13RA2 KO mice. In mucosal biopsies of active IBD patients, immunohistochemistry revealed that IL-13Rα2 protein was highly expressed in epithelial cells, while expression was restricted to goblet cells in healthy controls. Mucosal IL13RA2 mRNA negatively correlated with mRNA of several goblet cell-speciﬁc and barrier genes, and with goblet cell numbers. †These authors share ﬁrst authorship Specialty section: This article was submitted to Mucosal Immunity, a section of the journal Frontiers in Immunology Conclusions: The data suggest that IL-13Rα2 on epithelial cells contributes to IBD pathology by negatively inﬂuencing goblet cell recovery, goblet cell function and epithelial restoration after injury. Therefore, blocking IL-13Rα2 could be a promising target for restoration of the epithelial barrier in IBD. Received: 24 October 2018 Accepted: 04 December 2018 Published: 18 December 2018 Keywords: IL13RA2, goblet cells, anti-TNF non-responsiveness, IBD, inﬂammatory bowel disease, inﬂiximab, microarray Keywords: IL13RA2, goblet cells, anti-TNF non-responsiveness, IBD, inﬂammatory bowel disease, inﬂiximab, microarray ORIGINAL RESEARCH published: 18 December 2018 doi: 10.3389/ﬁmmu.2018.02983 Evaluation of Inﬂammation in Mice by Histology gy Five µm-thick longitudinal and transverse sections of colons were stained with hematoxylin and eosin (H&E). Three sections per mouse were blindly and independently scored by a post-doctoral researcher (CP) and by an experienced IBD pathologist (GDH). The following ﬁve characteristics were scored cumulatively. Architectural disturbance, based on irregularity of the mucosal surface, crypt distortion and branching, loss of glands, and fraying of the muscularis mucosae (0: none, 1: focal and mild, 2: multifocal, or diﬀuse, 3: severe); goblet cell depletion (0: normal, 1: focal, 2: multifocal, and 3: generalized); epithelial defects (0: none, 1: focal erosion, 2: multifocal erosion, and 3: ulceration); neutrophil inﬁltration (0: none, 1: in the lamina propria, 2: in the lamina propria with crypt abscesses, 3: inﬁltration in the mucosa or in the submucosa, or in the muscularis propria or in the subserosa, to a maximum of 12 if several areas were inﬁltrated); mononuclear cell inﬁltration (0: within normal limits, 1: slightly increased in lamina propria, 2: dense inﬁltrate in the lamina propria, 3: cell aggregates in the mucosa or in the submucosa or in the muscularis propria or in the subserosa, to a maximum of 12 if several areas were inﬁltrated). The IL-13 signaling receptor complex consists of two chains (IL-13Rα1 and IL-4Rα) which, after ligand binding, activate the transcription factor STAT-6 (6). IL-13Rα2 is a transmembrane receptor which binds IL-13 with very high aﬃnity. Due to its very short cytoplasmic region lacking signaling motifs, IL-13Rα2 is generally thought to act primarily as a “decoy” receptor, sequestering IL-13 and thus preventing excessive IL-13 signaling via the complex IL-13Rα1 and IL-4Rα (7). In rodents, IL-13Rα2 is released as a soluble factor and its expression correlates with a decreased responsiveness to IL-13 in chronic granulomatous inﬂammation in the liver (8), airway inﬂammation and airway- hyperreactivity (9), and immune and functional response to nematode infection (10). Humans do not alternatively splice the IL13Rα2 transcript and exclusively express this receptor as a transmembrane molecule (6, 11). However, it has been shown that also in humans, membrane IL-13Rα2 reduces responsiveness to IL-13 (12–14). The present study aimed to identify how IL13RA2 expression can contribute to the pathology of IBD. First, we performed experimental studies with IL13RA2 KO and WT mice subjected to experimental colitis. Next, we determined the localization of the IL-13Rα2 protein in human colonic biopsies by immunohistochemistry. Abbreviations: ATOH1, atonal BHLH transcription factor 1; CD, Crohn’s disease; CHI3L1, chitinase 3 like 1; CLCA1, chloride channel accessory 1; DSS, dextran sodium sulfate; FCGBP, FC fragment of IgG binding protein; GAL3, galectin 3; H&E, haemotoxylin and eosin; IBD, inﬂammatory bowel disease; IL, interleukin; KO, knock out; RELMβ, resistin like beta; SPDEF, SAM pointed domain containing ETS transcription factor; TMEM219, transmembrane protein 219; TNF, tumor necrosis factor; UC, ulcerative colitis; WT, wild type; YKL-40, chitinase 3 like 1. Evaluation of Inﬂammation in Mice by Histology Based on the murine ﬁndings and because it is known that IL-13 inﬂuences goblet cells, we further correlated IL13RA2 expression with goblet cell speciﬁc genes, barrier genes and goblet cell numbers in IBD. INTRODUCTION Crohn’s disease (CD) and ulcerative colitis (UC) are chronic inﬂammatory disorders of the gastrointestinal tract with a relapsing and remitting course. They are thought to result from an excessive immune response toward the intestinal and colonic microbiota in genetically susceptible hosts (1). Current treatments, able to achieve and maintain clinical remission and mucosal healing, December 2018 \| Volume 9 \| Article 2983 Frontiers in Immunology \| www.frontiersin.org IL13RA2 and Colitis Verstockt et al. include anti-tumor necrosis factor (anti-TNF) agents. Despite an overall good response to anti-TNF therapies, 10–30% of patients are primary non-responders (2). Among several other genes, our group previously identiﬁed interleukin 13 receptor alpha 2 (IL13RA2) mRNA expression as one of the best predictive markers for anti-TNF non-responsiveness. As this holds true both in UC and in CD, two diseases mediated by diﬀerent combinations of cytokines (1, 3, 4), these results point toward a role of IL-13Rα2 in a mechanism common to both diseases. In a more recent study we also found that IL13RA2 expression reﬂects an increased TNF burden in non-responders (5). However, how IL-13Rα2 aﬀects the response to anti-TNF therapy is currently unknown. by a recovery period. Mice were sacriﬁced by injection of pentobarbital at day 9 or 12 for evaluation of markers of disease activity and inﬂammation. Colons were harvested, weighted and measured from the ileocecal junction to the anus. A macroscopic score of inﬂammation was given as previously described (15). One part of the most inﬁltrated distal colon was ﬁxed in 6% paraformaldehyde for 3 h and then embedded in paraﬃn for histology evaluation. Immunohistochemistry for Human IL-13Rα2 and IL-13Rα1 and Quantiﬁcation of Goblet Cells in Mucosal Biopsies of IBD Patients Immunohistochemical staining was performed on 5 µm-thick step sections prepared from paraﬃn formalin-ﬁxed endoscopic- derived mucosal biopsies and resection specimens from IBD patients and controls. Endogenous peroxidase activity was blocked in deparraﬁned sections by incubating the slides for 20 min in a 0.3% solution of H2O2 in methanol. Epitope retrieval was performed by heating the slides for 30 min in Tris/EDTA buﬀer (pH 9) at 98◦C. Sections were then incubated with the anti- human IL-13Rα2 mouse monoclonal antibody clone ab55275 (Abcam plc, Cambridge, UK) at a concentration of 1 µg/ml for 30 min. IL-13Rα1 protein was localized using anti-human IL-13Rα1 rabbit polyclonal antibody ab79277 (Abcam) at a concentration of 10 µg/ml. The Dako REALTM EnvisionTM Detection System kit (Dako Belgium NV, Heverlee, Belgium) was used for visualization of bound primary antibody according to the manufacturer’s instructions. Formalin-ﬁxed, paraﬃn- embedded surgical biopsies of an ovarian serous adenocarcinoma Statistical Analysis served as positive controls for IL-13Rα2 (16). The primary antibody was omitted in the negative controls. Murine data analysis was performed with GraphPad Prism 5 (La Jolla, CA, USA). Data are represented as medians, and P- values were obtained using two-tailed Mann-Whitney U testing. Diﬀerences were considered statistically signiﬁcant at p < 0.05. Human data analysis was performed in R (R Development Core Team, Vienna, Austria). Correlations between IL13RA2 expression and goblet cell related genes and numbers were analyzed using bivariate two-tailed correlation tests. In parallel, H&E stained sections of mucosal biopsies were used to quantify goblet cells. Numbers of goblet cell were counted manually on images of the biopsies. Each picture covers a total surface of 0.32 mm2. The total length of the epithelium on the picture was measured using ImageJ, and data were expressed as number of goblet cells per µm of epithelium. RESULTS p Publically available microarray data (Aﬀymetrix Human Genome U133 Plus 2.0 Arrays, GSE14580, GSE12251, and GSE16879) of inﬂamed mucosal biopsies of IBD patients prior to their ﬁrst inﬂiximab administration were analyzed. Patient characteristics at baseline were previously described (17, 18). Raw data were preprocessed {normalization robust multichip average (RMA) metho(19)}, , and quality-control evaluation was performed [arrayQualityMetrics (20)] using Bioconductor (http://www. bioconductor.org) in R version 3.5.0 (R Development Core Team, Vienna, Austria), whereafter outliers were detected and removed based on cluster analysis (21). Microarray data were used to quantify mRNA expression of goblet cell speciﬁc and barrier genes. Animals and Experimental Colitis Model Animals and Experimental Colitis Model IL13RA2 knock out (KO) mice backcrossed more than 10 times in the Balb/c background were obtained from Pﬁzer (New York, NY, USA). Both IL13RA2 KO and wild type (WT) Balb/c mice were bred in the speciﬁc pathogen free area of the animal care facility of the KU Leuven, Belgium. Acute colitis was induced by administration of dextran sodium sulfate (DSS, MP Biomedicals) in drinking water (3%) for 7 days, followed December 2018 \| Volume 9 \| Article 2983 Frontiers in Immunology \| www.frontiersin.org 2 IL13RA2 and Colitis Verstockt et al. FIGURE 1 \| Acute colitis in WT and IL13RA2 KO mice. Wild type (WT) and IL13RA2 knock out (KO) mice were exposed for 7 days to 3% DSS in drinking water to induce acute colitis. Data are pooled from two independent experiments including 24 WT mice (day 9: n = 10 and day 12: n = 14) and 26 IL13RA2 KO mice (day 9: n = 9 and day 12: n = 17). WT mice are shown in white, IL13RA2 KO mice in black. Weight curve (A); Macroscopic score of inﬂammation of the colon at day 9 and day 12 after start of DSS exposure (B); Length of colon (C); Weight of colon (D); Ratio colon weight on length (E). Data are represented as medians (p < 0.05, **p ≤ 0.001, NS, not signiﬁcant). FIGURE 1 \| Acute colitis in WT and IL13RA2 KO mice. Wild type (WT) and IL13RA2 knock out (KO) mice were exposed for 7 days to 3% DSS in drinking water to induce acute colitis. Data are pooled from two independent experiments including 24 WT mice (day 9: n = 10 and day 12: n = 14) and 26 IL13RA2 KO mice (day 9: n = 9 and day 12: n = 17). WT mice are shown in white, IL13RA2 KO mice in black. Weight curve (A); Macroscopic score of inﬂammation of the colon at day 9 and day 12 after start of DSS exposure (B); Length of colon (C); Weight of colon (D); Ratio colon weight on length (E). Data are represented as medians (p < 0.05, **p ≤ 0.001, NS, not signiﬁcant). IL13Rα2 KO Mice Are Equally Affected in Acute DSS Colitis as WT Mice, but Recover Faster Acute colitis was induced in IL13RA2 KO and WT mice by DSS administration. At day 9, weight loss was most severe in both strains, reﬂecting the time point of strongest inﬂammation. At day 12 (recovery phase), most mice had recovered their initial weight despite persistent inﬂammatory features in the colon. Both strains developed colitis and equally lost weight (p = 0.07 at both day 9 and 12) (Figure 1A). The inﬂammation of the colon, as evaluated by the macroscopic score, the colon length, the colon weight and the colon weight to length ratio, was similar December 2018 \| Volume 9 \| Article 2983 Frontiers in Immunology \| www.frontiersin.org Frontiers in Immunology \| www.frontiersin.org 3 IL13RA2 and Colitis Verstockt et al. FIGURE 2 \| Microscopic score of inﬂammation in mice with DSS colitis. Wild type (WT) and IL13RA2 knock out (KO) mice were exposed for 7 days to 3% DSS in drinking water to induce acute colitis. Data are pooled from two independent experiments including 24 WT mice (day 9: n = 10 and day 12: n = 14) and 26 IL13RA2 KO mice (day 9: n = 9 and day 12: n = 17). WT mice are shown in white, IL13RA2 KO mice in black. Representative pictures of haemotoxylin and eosin (HandE) stained cross sections of colon of mice. The lower pictures represent the area in the rectangle at higher magniﬁcation (A); Inﬂammatory score evaluated on histological sections (B); Details of the inﬂammatory score (C). Data are represented as medians (p < 0.05, *p ≤0.01, NS, not signiﬁcant). FIGURE 2 \| Microscopic score of inﬂammation in mice with DSS colitis. Wild type (WT) and IL13RA2 knock out (KO) mice were exposed for 7 days to 3% DSS in drinking water to induce acute colitis. Data are pooled from two independent experiments including 24 WT mice (day 9: n = 10 and day 12: n = 14) and 26 IL13RA2 KO mice (day 9: n = 9 and day 12: n = 17). WT mice are shown in white, IL13RA2 KO mice in black. Representative pictures of haemotoxylin and eosin (HandE) stained cross sections of colon of mice. The lower pictures represent the area in the rectangle at higher magniﬁcation (A); Inﬂammatory score evaluated on histological sections (B); Details of the inﬂammatory score (C). Data are represented as medians (p < 0.05, **p ≤0.01, NS, not signiﬁcant). IL13Rα2 KO Mice Are Equally Affected in Acute DSS Colitis as WT Mice, but Recover Faster IL-13Rα2 thus seems to negatively regulate epithelial/mucosal healing. in IL13RA2 KO and WT mice at day 9 (p = 0.10, 0.34, 0.18, and 0.41, respectively) (Figures 1B–E). In contrast, at day 12, colons of IL13RA2 KO mice were less inﬂamed than colons of WT mice, with signiﬁcant diﬀerences in the macroscopic score of inﬂammation (p = 0.01), the colon length (p < 0.001) and colon weight (p = 0.01), reﬂecting that inﬂammation resolved more rapidly in IL13RA2 KO mice. Frontiers in Immunology \| www.frontiersin.org IL-13Rα2 Is Expressed in Human Epithelial Cells Using mucosal gene expression gene signaling analysis, we previously reported that IL13RA2 is highly expressed in the mucosa of IBD patients, and more so in anti-TNF non- responders (5, 17, 18). We therefore determined the cellular localization of the IL-13Rα2 protein by immunohistochemistry on mucosal surface biopsies and full thickness resection specimen of IBD patients and healthy controls. In normal colon, IL- 13Rα2 was localized in the cytoplasm and on the membrane of the goblet cells (Figure 3A). In inﬂamed CD and UC biopsies, there was increased staining intensity for IL-13Rα2 in all epithelial cells (Figures 3B,C). The staining was very intense at the base of the crypts and became slightly less intense in cells at the tip of the villi. We did not observe any staining in lymphoid aggregates, nor in cells surrounding ﬁbrotic areas in resection specimen of CD and UC patients (Figures 3D,E). Immune cells, such as macrophages and monocytes, as well Inﬂammation was microscopically quantiﬁed on H&E stained colon sections (Figure 2A). Microscopically, the score of inﬂammation was not diﬀerent between IL13RA2 KO and WT mice at day 9 (p = 0.82), but it was signiﬁcantly lower in IL13RA2 KO mice at day 12 (p = 0.02) (Figure 2B). At day 12, more goblet cells were present in the epithelium of IL13RA2 KO mice (arrows). More speciﬁcally, the characteristics that were diﬀerent between the two strains of mice on day 12 were the architectural changes (p = 0.009), the goblet cell depletion (p = 0.01) and the inﬁltration of neutrophils (p = 0.03) (Figure 2C). Taken together, these data demonstrate that the absence of IL-13Rα2 allows a more rapid recovery of the mucosa and in particular for the regeneration of goblet cells and normalization of the architecture of the colonic mucosa. December 2018 \| Volume 9 \| Article 2983 Frontiers in Immunology \| www.frontiersin.org 4 Verstockt et al. IL13RA2 and Colitis FIGURE 3 \| Immunohistochemical detection of IL-13Rα2 and IL-13Rα1 in human tissues. IL-13Rα2 Is Expressed in Human Epithelial Cells As IL-13 signals via the complex IL-13Rα1/IL-4Rα, we analyzed whether IL-13Rα1 was also expressed in epithelial cells of IBD patients. In patients with strong inﬂammation, IL- 13Rα1 was co-expressed in epithelial cells together with IL- 13Rα2 (Figure 3F). As expected, IL-13Rα1 was also expressed in lymphoid cells in the lamina propria (data not shown). In general, these murine and human ﬁndings suggest a link between IL13RA2 and mucosal barrier function. Based on previous ﬁndings demonstrating an intestinal epithelial barrier dysfunction in IBD, with up- (CDN1, DSG3) or downregulation (CLDN8, OCLDN, MEP1A, and MAGI1) of particular genes in active IBD compared to healthy individuals (23), we studied the correlation between IL13RA2 expression and these barrier genes. Although causality cannot be proven, we found a signiﬁcant positive correlation between IL13RA2 and CDN1 (r = 0.38, p = 0.006) and DSG3 expression (r = 0.61, p = 2.2 × 10−6), whereas IL13RA2 correlated negatively with CLDN8 (r = −0.38, p = 0.006), OCLDN (r = −0.44, p = 0.001), MEP1A (r = −0.35, p = 0.01), MAGI1 (r = −0.43, p = 0.002), and MARVELD2 (r = −0.32, p = 0.02). IL-13Rα2 Is Expressed in Human Epithelial Cells Staining of IL-13Rα2 protein with speciﬁc antibody in human tissues: normal colon and in the insert an ovarian serous adenocarcinoma as positive control (16) (negative controls where the ﬁrst antibody was omitted are not shown) (A); Ileum of a CD non-responder before therapy (B); Colon of an UC non-responder before therapy (C); Resection specimen of CD (D); Resection specimen of UC (E); Sequential staining of colon of UC non-responders for IL-13Rα2 (left) and IL-13Rα1 (right) (F). The black bar corresponds to 100 µm. FIGURE 3 \| Immunohistochemical detection of IL-13Rα2 and IL-13Rα1 in human tissues. Staining of IL-13Rα2 protein with speciﬁc antibody in human tissues: normal colon and in the insert an ovarian serous adenocarcinoma as positive control (16) (negative controls where the ﬁrst antibody was omitted are not shown) (A); Ileum of a CD non-responder before therapy (B); Colon of an UC non-responder before therapy (C); Resection specimen of CD (D); Resection specimen of UC (E); Sequential staining of colon of UC non-responders for IL-13Rα2 (left) and IL-13Rα1 (right) (F). The black bar corresponds to 100 µm. staining was slightly weaker than in CD and UC patients. The IL13RA2 expression in these patients suggests that it is not speciﬁc to IBD, but might be a consequence of chronic inﬂammation. as ﬁbroblasts did not express IL13RA2, even in the deeper layer of the tissues. In resection specimens of patients with colorectal carcinoma, IL13RA2 was also expressed in epithelial cells surrounding the tumor, although the intensity of the December 2018 \| Volume 9 \| Article 2983 Frontiers in Immunology \| www.frontiersin.org 5 Verstockt et al. IL13RA2 and Colitis FIGURE 4 \| Correlation between IL13RA2 mRNA and mRNA of goblet cell related genes. Correlations between expression of IL13RA2 with the expression of ATOH1 (A), SPDEF (B), RELMβ (C) in mucosal biopsies of IBD patients before anti-TNF therapy. Correlations between expression of IL13RA2 with the number of goblet cells per mm in IBD patients prior to start of anti-TNF therapy (D). FIGURE 4 \| Correlation between IL13RA2 mRNA and mRNA of goblet cell related genes. Correlations between expression of IL13RA2 with the expression of ATOH1 (A), SPDEF (B), RELMβ (C) in mucosal biopsies of IBD patients before anti-TNF therapy. Correlations between expression of IL13RA2 with the number of goblet cells per mm in IBD patients prior to start of anti-TNF therapy (D). Expression of IL13RA2 mRNA Negatively Correlates With Expression of Goblet Cell Related Genes and Goblet Cell Numbers in the Mucosa of IBD Patients In follow up of our murine ﬁndings on goblet cell recovery in the absence of IL-13Rα2, we analyzed the correlation between IL13RA2 expression and the expression of various goblet cell related genes. Mucosal expression of ATOH1, a transcription factor speciﬁc for the development of goblet cells, SPDEF, a key promotor of goblet cell diﬀerentiation and regulator of secretory gene products including mucin 2, and RELMβ, a key goblet cell mediator, were negatively correlated with IL13RA2 expression (Figures 4A–C). Additionally, IL13RA2 mRNA expression correlated inversely with gene expression of key components of the intestinal mucus layer (22), including FCGBP and CLCA1 (r = −0.5, p = 4.0 × 10−4; r = −0.4, p = 2.0 × 10−3, respectively) (Supplementary Figures 1A,B). To strengthen these ﬁndings, the number of goblet cells per mm epithelium was quantiﬁed in mucosal biopsies of IBD patients, and correlated negatively with the expression of IL13RA2 (r = −0.33, p = 0.03) (Figure 4D). Thus, the expression of IL13RA2 prior to therapy is associated with lower numbers of goblet cells, lower expression of transcription factors for goblet cell development and goblet cell function. This suggests that IL- 13Rα2 negatively regulates development of goblet cells during inﬂammation, most likely by counteracting the eﬀects of IL-13. Frontiers in Immunology \| www.frontiersin.org DISCUSSION Previously, IL13RA2 mRNA expression has been identiﬁed as one of the best predictive markers for anti-TNF non- responsiveness (5). However, how IL-13Rα2 interferes with the eﬀect of anti-TNF therapy is currently unknown. The present study aimed to unravel the eﬀects of mucosal IL- 13Rα2 expression in IBD. We ﬁrst hypothesized that IL-13Rα2 downregulates the inﬂammatory process, as it has been shown to inhibit several IL-13-mediated eﬀects in mice (8–10, 24). IL-17A/IFNγ-mediated colitis in IL10 KO mice is regulated indirectly by soluble IL-13Rα2 (25), which blocks the inhibitory function of IL-13 on Th17 cells, and thus promotes Th17 activities (26). However, we did not notice any diﬀerence in macro- or microscopic inﬂammation between WT and IL13RA2 KO mice in this acute DSS colitis model, arguing against a role for IL13RA2 in the inﬂammatory process. Still it might be important to study other models of IBD. The DSS model was chosen for our study because DSS triggers December 2018 \| Volume 9 \| Article 2983 Frontiers in Immunology \| www.frontiersin.org 6 IL13RA2 and Colitis Verstockt et al. inﬂammation by disruption of the epithelial barrier (27), and is as such an appropriate model to study mucosal healing. Importantly, using this model we indeed did observe a more rapid epithelial recovery in IL13RA2 KO mice, including more goblet cells at day 12. These ﬁndings suggest that IL13RA2 expression is not inﬂuencing susceptibility to colitis, but instead inﬂuences the recovery after removal of the trigger in mice, and that in particular eﬀects on goblet cells are important. Indeed, also in human genome-wide association studies in IBD already pointed toward distinct mechanisms between disease susceptibility and disease outcome, once the disease has settled (28). Recent data suggested that IL-13Rα2 is an α-receptor subunit that can interact with TMEM219 to mediate anti-apoptotic eﬀector responses, whereas IL-13Rα2 interactions with Gal- 3 would augment apoptotic responses and Wnt/β-catenin activation (45). Additional research is necessary to further unravel the signiﬁcance of these eﬀects of IL-13RA2, also in IBD. Recent data suggested that IL-13Rα2 is an α-receptor subunit that can interact with TMEM219 to mediate anti-apoptotic eﬀector responses, whereas IL-13Rα2 interactions with Gal- 3 would augment apoptotic responses and Wnt/β-catenin activation (45). Additional research is necessary to further unravel the signiﬁcance of these eﬀects of IL-13RA2, also in IBD. ETHICS STATEMENT All patients included in the analysis had given written consent to participate in the Institutional Review Board approved IBD Biobank (B322201213950/S53684), collecting serum, biopsies and clinical characteristics among other items. All murine studies were approved by the local ethical committee for animal experimentation of the KU Leuven(P013/2011). Interestingly, when we studied biopsies of patients with IBD, we conﬁrmed an inverse correlation between IL13RA2 expression and total number of goblet cells, as well as between IL13RA2 expression and the expression of several goblet cell speciﬁc and barrier genes. We therefore hypothesize that the increased IL13RA2 expression dampens one’s ability to restore the mucosal barrier and hence impairs mucosal healing. Although goblet cell hyperplasia and mucus production contribute to inﬂammation in asthma (41), these mechanisms are protective in the gut (42, 43). Translated to IBD patients this may explain why some patients achieve mucosal healing and others do not, after receiving the same anti-TNF agent, neutralizing one of the driving forces and upstream regulators of IL13RA2 expression (5). Additionally, blocking IL-13Rα2 could be a promising agent for restoration of the epithelial barrier in IBD. DISCUSSION In conclusion, IL13RA2 expression correlates negatively with numbers of goblet cells, as well as with the expression of goblet cell transcription factors and goblet cell function, suggesting that high IL13RA2 expression impairs goblet cell development. The role of IL-13Rα2 is further supported by murine data showing a more rapid recovery of IL13RA2 KO mice compared to WT mice after DSS induced colitis, with a more rapid restoration of goblet cells. In anti-TNF therapy non-responder patients in whom there is increased IL13RA2 expression (5, 17, 18), the latter probably hampers the renewal of the epithelium and the diﬀerentiation of goblet cells that would otherwise be induced by IL-13 via the IL- 13Rα1/IL-4Rα complex. Neutralization of TNF by anti-TNF therapies decreases the inﬂammation via several mechanisms (47), but it is conceivable that this can be eﬃcient only if the epithelium recovers properly to prevent entry of commensal bacteria and/or bacterial products contributing to the inﬂammation. Besides its eﬀects on inﬂammation, IL-13 has been shown to promote airway epithelial cell proliferation (29–31), and to prevent apoptosis (32), features that are crucial for epithelial cell recovery. IL-13 has an established role in the diﬀerentiation of goblet cells in the airways (30, 33, 34), and overexpression of IL- 13 in the gut results in villus blunting, goblet cell hyperplasia and increased epithelial cell proliferation (35). The role of IL- 13 in goblet cell hyperplasia is further supported by studies showing that stimulation of primary lung epithelial cells by IL- 13 causes an increase in the population of goblet cells (33). These eﬀects are likely to be mediated via IL-13Rα1 and STAT6. Co-expressed IL-13Rα2 can act as a negative regulator of IL- 13 eﬀects, reducing signaling via IL-13Rα1, and thus reducing the above-mentioned beneﬁcial eﬀects of IL-13 in the gut. It has already been demonstrated that IL13RA2 expression is inversely correlated with STAT-6 phosphorylation (36–38). As STAT-6 is key in goblet cell hyperplasia and intestinal mucosal repair (39, 40), the observed goblet cell hyperplasia in IL13RA2 KO mice can easily be explained. Frontiers in Immunology \| www.frontiersin.org REFERENCES Mucosal IL13RA2 predicts non-response to anti-TNF therapy in Crohn’s disease. Aliment Pharmacol Therapeut. (in press). doi: 10.1111/apt.15126 17. Arijs I, Li K, Toedter G, Quintens R, Van Lommel L, Van Steen K, et al. Mucosal gene signatures to predict response to inﬂiximab in patients with ulcerative colitis. Gut (2009) 58:1612–9. doi: 10.1136/gut.2009.178665 6. Tabata Y, Khurana Hershey GK. IL-13 receptor isoforms: breaking through the complexity. 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Biostatistics (2003) 4:249–64. doi: 10.1093/biostatistics/4.2.249 8. Mentink-Kane MM, Cheever AW, Thompson RW, Hari DM, Kabatereine NB, Vennervald BJ, et al. IL-13 receptor alpha 2 down-modulates granulomatous inﬂammation and prolongs host survival in schistosomiasis. Proc Natl Acad Sci USA. (2004) 101:586–90. doi: 10.1073/pnas.0305064101 20. Kauﬀmann A, Gentleman R, Huber W. arrayQualityMetrics–a bioconductor package for quality assessment of microarray data. Bioinformatics (2009) 25:415–6. doi: 10.1093/bioinformatics/btn647 9. Wilson MS, Elnekave E, Mentink-Kane MM, Hodges MG, Pesce JT, Ramalingam TR, et al. IL-13Ralpha2 and IL-10 coordinately suppress airway inﬂammation, airway-hyperreactivity, and ﬁbrosis in mice. J Clin Invest. (2007) 117:2941–51. doi: 10.1172/JCI31546 21. Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics (2008) 9:559. doi: 10.1186/1471-2105-9-559 10. Morimoto M, Zhao A, Sun R, Stiltz J, Madden KB, Mentink-Kane M, et al. IL-13 receptor alpha2 regulates the immune and functional response to Nippostrongylus brasiliensis infection. J Immunol. (2009) 183:1934–9. doi: 10.4049/jimmunol.0804299 22. Birchenough GM, Johansson ME, Gustafsson JK, Bergstrom JH, Hansson GC. New developments in goblet cell mucus secretion and function. Mucosal Immunol. (2015) 8:712–9. doi: 10.1038/mi.2015.32 23. Vancamelbeke M, Vanuytsel T, Farre R, Verstockt S, Ferrante M, Van Assche G, et al. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ﬁmmu. 2018.02983/full#supplementary-material Supplementary Figure 1 \| Correlation between IL13RA2 mRNA and goblet speciﬁc cell genes. Correlations between expression of IL13RA2 with the expression of FCGBP (A) and CLCA1 (B), backbone of the colonic mucus layer. Supplementary Figure 1 \| Correlation between IL13RA2 mRNA and goblet speciﬁc cell genes. Correlations between expression of IL13RA2 with the expression of FCGBP (A) and CLCA1 (B), backbone of the colonic mucus layer. FUNDING BV is a doctoral fellow and GVA, SV, and MF are Senior Clinical Investigators of the Research Foundation Flanders (FWO), Belgium. BV has also received research grants by the Belgium Week of Gastroenterology, the Belgian IBD Research and Development (BIRD), the European Crohn’s and Colitis Organization (ECCO) and the IBD Patient’s Association Flanders (CCV VZW). This work was supported by a grant from the Swiss Science Research Foundation (PBLAP3-129427/1) to Clementine Perrier, a grant from the Fund for Scientiﬁc Research-Flanders (FWO-Flanders) Belgium (FWO project nr.G.0440.06), a GOA grant from the Research Council, KU Leuven (GOA/11/015) to SV, and a grant from the Agency for Innovation by Science and Technology in Flanders (IWT) to CB. CB is also supported by the Clinical Research Fund KOF (University Hospitals Leuven, Leuven, Belgium) and received previously research grants by the Belgium Week of Gastroenterology and the Flemish Organisation for Gastroenterology (VVGE). We would like to thank Thomas Wynn (NIAID/NIH) for the critical reﬂections and revision of the manuscript for important intellectual content. Vera Ballet, Eline Vandeput, Sophie Organe, Nooshin Ardeshir Davani, Helene Blevi, Tamara Coopmans, and Willem-Jan Wollants for an excellent job in maintaining the Biobank database. We would like to thank Marion Kasaian at Pﬁzer who provided the original breeding pairs of IL13RA2 KO mice. AUTHOR CONTRIBUTIONS BV: contributed to the acquisition of data, analysis and interpretation of data, drafting of the manuscript, and statistical analysis. CP: study concept and design, acquisition data, analysis and interpretation of data, and drafting of the manuscript. GDH: acquisition data, analysis and interpretation of data, and critical revision of the manuscript for important intellectual content. JC: acquisition data, analysis, and interpretation of data. BC: interpretation of data. GVA: study concept and design, interpretation of data, and critical revision of the manuscript for important intellectual content. MF and SV: acquisition of the data, interpretation of data, and critical revision of the manuscript for important intellectual content. JLC: study concept and design, interpretation of data, and critical revision of the manuscript for important intellectual content. CB: study concept and design, acquisition of data, analysis and interpretation of data, material support, drafting of the manuscript, and study supervision. All authors agreed with the ﬁnal version of the manuscript. CB: guarantor of the manuscript. We cannot exclude that IL-13Rα2 also inﬂuences disease by other mechanisms. Besides IL-13, another ligand for IL- 13Rα2, chitinase 3-like 1 (CHI3L1/YKL-40) in complex with TMEM219 (the chitosome) or Galectin-3 (Gal-3), can regulate a variety of cellular and tissue responses (14, 44, 45). CHI3L1, secreted by macrophages and neutrophils, plays essential roles in the pathogen clearance and generation of the host tolerance (46). The CHI3L1-IL13RA2 axis is known to play a critical role in cell death, inﬂammasome activation, Th1/Th2 cytokine balance, and Erk, Akt, and Wnt/β-catenin signaling (45). December 2018 \| Volume 9 \| Article 2983 Frontiers in Immunology \| www.frontiersin.org 7 IL13RA2 and Colitis Verstockt et al. REFERENCES 12. Badalyan V, Thompson R, Addo K, Borthwick LA, Fisher AJ, Ort T, et al. TNF- alpha/IL-17 synergy inhibits IL-13 bioactivity via IL-13Ralpha2 induction. J Allergy Clin Immunol. (2014) 134:975–8 e975. doi: 10.1016/j.jaci.2014.05.019 1. De Souza HS, Fiocchi C. Immunopathogenesis of IBD: current state of the art. 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(2011) 37:461–70. doi: 10.3109/01902148.2011.596894 Conﬂict of Interest Statement: BV received ﬁnancial support for research from Pﬁzer; lectures fees from Abbvie, Ferring, Takeda Pharmaceuticals, Janssen and R Biopharm; consultancy fees from Janssen. GDH received consultancy fees from Centocor and Takeda. GVA received ﬁnancial support for research from Abbott and Ferring Pharmaceuticals; lecture fees from Janssen, MSD and Abbott; consultancy fees from PDL BioPharma, UCB Pharma, Sanoﬁ-Aventis, Abbott, Abbvie, Ferring, Novartis, Biogen Idec, Janssen Biologics, NovoNordisk, Zealand Pharma A/S, Millenium/Takeda, Shire, Novartis, and Bristol Mayer Squibb. MF received ﬁnancial support for research from Takeda and Janssen; lecture fees from Ferring, Boehringer- Ingelheim, Chiesi, Merck Sharpe andamp; Dohme, Tillotts, Janssen Biologics, AbbvieTakeda, Mitsubishi Tanabe, Zeria; consultancy fees from Abbvie, Boehringer-Ingelheim, Ferring, Merck Sharpe andamp; Dohme, and Janssen Biologics. SV received ﬁnancial support for research from MSD, Abbvie, Janssen and UCB Pharma; lecture fees from Abbott, Abbvie, Merck Sharpe andamp; Dohme, Ferring Pharmaceuticals and UCB Pharma; consultancy fees from Pﬁzer, Ferring Pharmaceuticals, Shire Pharmaceuticals Group, Merck Sharpe andamp; Dohme, and AstraZeneca Pharmaceuticals. 32. Singhera GK, Macredmond R, Dorscheid DR. Interleukin-9 and−13 inhibit spontaneous and corticosteroid induced apoptosis of normal airway epithelial cells. Exp Lung Res. (2008) 34:579–98. doi: 10.1080/01902140802369372 33. Laoukili J, Perret E, Willems T, Minty A, Parthoens E, Houcine O, et al. Copyright © 2018 Verstockt, Perrier, De Hertogh, Cremer, Creyns, Van Assche, Ferrante, Ceuppens, Vermeire and Breynaert. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Immunology \| www.frontiersin.org REFERENCES IL- 13 alters mucociliary diﬀerentiation and ciliary beating of human respiratory epithelial cells. J Clin Invest. (2001) 108:1817–24. doi: 10.1172/JCI2001 13557 34. Kondo M, Tamaoki J, Takeyama K, Nakata J, Nagai A. Interleukin-13 induces goblet cell diﬀerentiation in primary cell culture from Guinea pig tracheal epithelium. Am J Respir Cell Mol Biol. (2002) 27:536–41. doi: 10.1165/rcmb.4682 35. Wu D, Ahrens R, Osterfeld H, Noah TK, Groschwitz K, Foster PS, et al. Interleukin-13 (IL-13)/IL-13 receptor alpha1 (IL-13Ralpha1) signaling regulates intestinal epithelial cystic ﬁbrosis transmembrane conductance regulator channel-dependent Cl-secretion. J Biol Chem. (2011) 286:13357–69. doi: 10.1074/jbc.M110.214965 36. Hsi LC, Kundu S, Palomo J, Xu B, Ficco R, Vogelbaum MA, et al. Silencing IL- 13Ralpha2 promotes glioblastoma cell death via endogenous signaling. Mol Cancer Ther. (2011) 10:1149–60. doi: 10.1158/1535-7163.MCT-10-1064 The remaining authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 37. Papageorgis P, Ozturk S, Lambert AW, Neophytou CM, Tzatsos A, Wong CK, et al. Targeting IL13Ralpha2 activates STAT6-TP63 pathway to suppress breast cancer lung metastasis. Breast Cancer Res. (2015) 17:98. doi: 10.1186/s13058-015-0607-y Copyright © 2018 Verstockt, Perrier, De Hertogh, Cremer, Creyns, Van Assche, Ferrante, Ceuppens, Vermeire and Breynaert. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 38. Shibasaki N, Yamasaki T, Kanno T, Arakaki R, Sakamoto H, Utsunomiya N, et al. Role of IL13RA2 in Sunitinib resistance in clear cell renal cell carcinoma. PLoS ONE (2015) 10:e0130980. doi: 10.1371/journal.pone.01 30980 39. Khan WI, Blennerhasset P, Ma C, Matthaei KI, Collins SM. Stat6 dependent goblet cell hyperplasia during intestinal nematode infection. Parasite Immunol. 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https://openalex.org/W1999485453	https://europepmc.org/articles/pmc3669390?pdf=render	English	null	Plant Oils Were Associated with Low Prevalence of Impaired Glucose Metabolism in Japanese Workers	PloS one	2,013	cc-by	7,731	Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution L restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by Grant-in-Aid for Scientific Research (B)(21390213) from Japan Society for the Promotion of Science (to TM). The funding body had no role in the collection, analysis, and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication. Competing Interests: IK, TK, and HT are health professionals in the Furukawa Electric Corporation. This does not alter their adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: kkurotani@ri.ncgm.go.jp Kayo Kurotani1, Takeshi Kochi2, Akiko Nanri1, Hiroko Tsuruoka2, Keisuke Kuwahara1, Ngoc Minh Pham1, Isamu Kabe1, Tetsuya Mizoue1 Kayo Kurotani1, Takeshi Kochi2, Akiko Nanri1, Hiroko Tsuruoka2, Keisuke Kuwahara1, Ngoc Minh Pham1, Isamu Kabe1, Tetsuya Mizoue1 1 Department of Epidemiology and Prevention, Clinical Research Center, National Center for Global Health and Medicine, Tokyo, Japan, 2 Department of Health Administration, Furukawa Electric Corporation, Tokyo, Japan and Prevention, Clinical Research Center, National Center for Global Health and Medicine, Tokyo, Japan, 2 Department of Healt c Corporation, Tokyo, Japan 1 Department of Epidemiology and Prevention, Clinical Research Center, National Center for Global Health and Medicine, To Administration, Furukawa Electric Corporation, Tokyo, Japan Abstract Fatty acid has been suggested to be involved in development of diabetes. However, its association is unclear among Japanese populations, which consume large amounts of fish rich in n-3 polyunsaturated fatty acids. The present cross- sectional study examined the association of individual dietary fatty acids and dietary fatty acid patterns with abnormal glucose metabolism among 1065 Japanese employees, aged 18–69 years. Impaired glucose metabolism is defined if a person has a history of diabetes, current use of anti-diabetic drug, fasting plasma glucose of 110 mg/dl ($6.1 mmol/L) or greater, or hemoglobin A1C of 6.0% ($42 mmol/mol) or greater. Dietary intake was assessed with a self-administered diet history questionnaire. Dietary fatty acid patterns were extracted by principal component analysis. Odds ratios of impaired glucose metabolism according to tertile categories of each fatty acids and dietary fatty acid patterns were estimated using logistic regression with adjustment for potential confounding variables. A higher intake of polyunsaturated fatty acid, n-6 fatty acid, linoleic acid, and oleic acid were significantly associated with a decreased prevalence of impaired glucose metabolism (P for trend = 0.03, 0.01, 0.02, and 0.04, respectively). Alpha-linolenic acid was marginally significantly associated with a decreased prevalence of impaired glucose metabolism (P for trend = 0.12). Of three fatty acid patterns identified, a higher plant oil pattern score, which characterized by high intake of alpha-linolenic acid, linoleic acid, and oleic acid, was associated with a decreased prevalence of impaired glucose metabolism (P for trend = 0.03). No association was observed for other patterns. In conclusion, plant source fatty acids might be protectively associated with development of diabetes in Japanese adults. Citation: Kurotani K, Kochi T, Nanri A, Tsuruoka H, Kuwahara K, et al. (2013) Plant Oils Were Associated with Low Prevalence of Impaired Glucose Metabolism in Japanese Workers. PLoS ONE 8(5): e64758. doi:10.1371/journal.pone.0064758 Editor: Manlio Vinciguerra, University College London, United Kingdom Received March 7, 2013; Accepted April 5, 2013; Published May 31, 2013 Editor: Manlio Vinciguerra, University College London, United Kingdom Editor: Manlio Vinciguerra, University College London, United Kingdom Editor: Manlio Vinciguerra, University College London, United Kingdom Received March 7, 2013; Accepted April 5, 2013; Published May 31, 2013 Received March 7, 2013; Accepted April 5, 2013; Published May 31, 2013 Received March 7, 2013; Accepted April 5, 2013; Published May 31, 2013 pyright: 2013 Kurotani et al. Subjects Six participants received the health checkup outside the survey period and thus their checkup data were not available. Of the remaining 1,212 participants with health checkup data, we excluded 37 participants who reported a history of cancer (n = 14), cardiovascular disease (n = 14), chronic hepatitis (n = 1), chronic kidney disease including nephritis (n = 8), and pancreatitis (n = 2). We further excluded 88 individuals who did not return dietary questionnaire (n = 6), did not donate blood sample (n = 76), received the examination in non-fasting status (n = 82). Some participants had two or more conditions for exclusion. Of the remaining subjects, we excluded those with missing data on covariates (n = 11). Finally, we excluded 11 subjects with extremely high or low energy intake (exceeding 3 standard deviation), leaving 1065 (953 men and 112 women) for analysis. We also performed principal component analysis on the basis of energy-adjusted intakes using a density method of 15 fatty acids to derive dietary fatty acid patterns. The factors were rotated by orthogonal transformation (varimax rotation) to maintain uncor- related factors and greater interpretability. We considered eigenvalues, the scree test and the interpretability of the factors to determine the number of factors to retain. The factors satisfied the criteria for eigenvalues greater than one, and the scree plots dropped substantially. We decided to retain three factors. Dietary fatty acid patterns were named according to fatty acids showing high loading (absolute value) on three factors. The factor scores of each fatty acid pattern for each individual were calculated by summing intakes of fatty acids weighted by their factor loadings. Laboratory Measurement As part of health checkup, plasma glucose concentration was assayed enzymatically using Quick-auto-neo-GLU-HK (Shino- Test Corp., Tokyo, Japan) and hemoglobin A1c was measured with a latex agglutination immunoassay using the Determiner HbA1c kit (Kyowa Medex Co., Ltd., Tokyo, Japan) at an external laboratory (Kinki Kenko Kanri Center, Shiga, Japan). Venous blood that was donated specifically for the study was drawn into a vacuum tube (7 mL) and then shipped in a cooler box to another laboratory (Mitsubishi Chemical Medience Corporation, Tokyo, Japan), where the blood was centrifuged and serum insulin was measured; the remaining serum sample was stored at –80uC until analysis. Insulin was determined with chemiluminescence immu- noassay, with the intra-assay coefficients of variation being 2.5% at 43.1 pmol/l and 1.2% at 423 pmol/l. We computed the homeostasis model assessment-insulin resistance (HOMA-IR) and homeostasis model assessment for b cell function (HOMA- b) using the following formula: HOMA-IR = fasting insulin (mU/ ml)6fasting glucose (mmol/ml)/22.5 [HOMA-IR = fasting insulin (mU/ml)6fasting glucose (mg/dl)/405] [13] and HOMA- Introduction The above meta-analysis showed that a weak May 2013 \| Volume 8 \| Issue 5 \| e64758 PLOS ONE \| www.plosone.org 1 Plant Oil and Glucose Metabolism in Japanese b = 3606fasting insulin (mU/ml)/(fasting glucose (mg/dl)-63) [HOMA-b = 206fasting insulin (U/ml)/(fasting glucose (mmol/ ml)–3.5)]. [13]. the association of individual dietary fatty acid and patterns of fatty acid intake with diabetes, pre-diabetes, and insulin resistance and secretion in a Japanese working population. Study Procedure In April 2012, a nutritional epidemiological survey was conducted at the time of the periodic health examination among workers of a manufacturing company and its affiliated ones in Chiba Prefecture, Japan. The primary objective of the study was to investigate the association of diet with physical and mental health. Prior to the health check-up, all full-time workers (n = 1,675) were asked to participate in the survey and fill out two types of survey questionnaire (one specifically designed for diet and another for overall health-related lifestyle). Of these, 1,218 (1,076 men and 142 women aged 18–70 years) agreed to participate (response rate, 73%). On the day of health check-up, research staff checked the questionnaire for completeness and, where necessary, clarified with the subjects. Participants were asked to donate 7 mL of venous blood for study. Additionally, we obtained health checkup data including results of anthropometric and biochemical measurements and information on history of disease. The study protocol was approved by the Ethics Committee of the National Center for Global Health and Medicine, Japan. Written informed consent was obtained from all subjects prior to the survey. Dietary Assessment Dietary habit during the preceding month was assessed using a validated brief self-administered diet history questionnaire (BDHQ), which consists of five sections: 1) the frequency of 46 food and non-alcoholic beverage intake; 2) daily frequency of rice and miso soup intake; 3) the frequency of alcoholic drinking and the amount of consumption for five alcoholic beverages per typical drinking occasion; 4) usual cooking method; and 5) dietary behavior [15]. Dietary intakes for 58 food and beverage items, energy, and selected nutrients were estimated using an ad hoc computer algorithm for the BDHQ, with reference to the standard tables of food composition in Japan. According to the validation study of the BDHQ using 16-day weighted dietary records as the gold standard, Pearson correlation coefficient for energy-adjusted intake of saturated fat, monounsaturated fat, and polyunsaturated fat was 0.58 and 0.64, 0.61 and 0.61,and 0.48 and 0.41 in men and women, respectively [16]. Definition of Outcome Impaired glucose metabolism is defined if a person has a history of diabetes, current use of anti-diabetic drug, fasting plasma glucose of 110 mg/dl ($6.1 mmol/L) or greater, or hemoglobin A1C of 6.0% ($42 mmol/mol) or greater. Diabetes is defined if a person has a history of diabetes, current use of anti-diabetic drug, fasting plasma glucose of 126 mg/dl ($7.0 mmol/L) or greater, or hemoglobin A1C of 6.5% ($48 mmol/mol) or greater. Pre- diabetes is defined if a person without a history of diabetes and current use of anti-diabetic drug has a fasting plasma glucose of 110 to 125 mg/dl (6.1–6.9 mmol/L) or a hemoglobin A1C of 6.0 to 6.4% (42–46 mmol/mol) [14]. Introduction inverse association of alpha-linolenic acid (ALA)from plant sources with type 2 diabetes [7]. Regarding linoleic acid (LA), the main dietary n-6 PUFA, an inverse association was generally observed between LA and type 2 diabetes [8]. An intervention study reported a diet rich in n-6 PUFA improved insulin sensitivity compared with a diet rich in saturated fatty acid [9]. Experimental and mechanistic studies have suggested a role of dietary fatty acid composition in glucose metabolism. The dietary fatty acid composition might affect cellular functions such as translocation of glucose transporters and insulin signaling [1]. Indeed, long chain polyunsaturated fatty acids (PUFAs) have been postulated to improve insulin sensitivity [2] and saturated fatty acids impair insulin sensitivity [3] by regulating the cell membrane composition of fatty acids [4]. In addition, recent experimental data showed that dietary fatty acids involve direct regulatory effects on lipogenic gene expression and enzyme activity [5]. p y [ ] To our knowledge, most previous studies on this issue were conducted among Western populations, which have a relatively high body mass [10], higher beta-cell function [11], and lower intake of fish [12], a rich source of long chain n-3 fatty acids, compared with Japanese population. Dietary fatty acid consump- tion and its effect on glucose metabolism in Japanese population may differ from those in Western populations. In addition, intakes of individual fatty acids are highly inter-correlated and thus it makes difficult to separate their specific effects. Although the examination of dietary fatty acid patterns is important, the relationship between patterns of dietary fatty acid composition and glucose metabolism has not been investigated. We hypothesized that high levels of saturated fatty acids and low levels of both n-6 and n-3 PUFAs and MUFA are associated with abnormal glucose metabolism. To test this hypothesis, we examined cross-sectionally Observational studies showed that saturated fatty acid intake was positively associated with insulin resistance and type 2 diabetes, whereas the evidence regarding monounsaturated fatty acid (MUFA) and PUFA were inconclusive [6]. A recent meta- analysis of prospective studies showed no clear association of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from seafood, whereas EPA and DHA were associated with lower risk of diabetes in Asians studies and with higher risk of diabetes in Western studies [7]. Statistical Analysis However, gamma-linolenic acid (GLA) (18:3 n26) and EPA (20:5 n23) were marginally significantly positively associated with HOMA-beta; geometric means (95% CI) for the tertile categories from the lowest to the highest were 59.0 (55.9262.4), 60.9 (57.8264.2), and 63.6 (60.2267.2) (P for trend = 0.07) for GLA and 58.6 (55.3262.1), 61.3 (58.2264.6), and 63.8 (60.0267.7) (P for trend = 0.07) for EPA. Additionally, we examined the association of individual fatty acids with HOMA-IR and HOMA-beta. There were no significant associations of any fatty acids with HOMA-IR and HOMA-beta (data not shown). However, gamma-linolenic acid (GLA) (18:3 n26) and EPA (20:5 n23) were marginally significantly positively associated with HOMA-beta; geometric means (95% CI) for the tertile categories from the lowest to the highest were 59.0 (55.9262.4), 60.9 (57.8264.2), and 63.6 (60.2267.2) (P for trend = 0.07) for GLA and 58.6 (55.3262.1), 61.3 (58.2264.6), and 63.8 (60.0267.7) (P for trend = 0.07) for EPA. We identified three dietary fatty acid patterns (Table 3). The first pattern was characterized by high intakes of 20:5 n-3, 18:4 n- 3, 22:5 n-3, and 22:6 n-3, and thus was named a fish oil pattern. The second pattern represented high intakes of 20:3 n-6, 18:0, and 16:0, and thus it was called a meat oriented pattern. The third pattern was characterized by high intakes of 18:3 n-3, 18:2 n-6, and 18:1. These fatty acids are derived from plant souses, and this pattern was named as a plant oil pattern. Of the three dietary patterns, the plant oil pattern showed a significant inverse association with prevalence of impaired glucose metabolism (Table 4). Multivariate-adjusted OR (95% CI) for the tertile categories (from the lowest to the highest) were 1.00 (referent), 0.80 (0.4721.37), 0.54 (0.3020.95) (P for trend = 0.03). In the analysis for diabetes, higher scores of the plant oil pattern was significantly inversely associated with odds of diabetes; the multivariate-adjusted OR (95% CI) of diabetes for the lowest through highest tertile of plant oil pattern score were 1.00 (referent), 0.57 (0.2721.19), 0.46 (0.2220.96) (P for trend = 0.04). However, in the analysis for pre-diabetes, the inverse association with plant oil pattern score was attenuated; the corresponding values for the lowest through highest tertile of plant oil pattern score were 1.00 (referent), 1.36 (0.6622.79), 0.61 (0.2721.38) (P for trend = 0.28). Neither fish oil pattern nor meat oriented pattern was associated with prevalence of impaired glucose metabolism. Plant Oil and Glucose Metabolism in Japanese associated with odds of diabetes; the multivariate-adjusted OR (95% CI) of diabetes for the lowest through highest tertile of PUFA intake were 1.00 (referent), 0.49 (0.22–1.05), 0.40 (0.18–0.91) (P for trend = 0.03). LA was significantly inversely associated with diabetes odds in multivariable model (P for trend = 0.04). In the analysis for pre-diabetes, multiple logistic regression showed that the prevalence of pre-diabetes was decreased with increasing levels of PUFA (P for trend = 0.04). clothes was measured to the nearest 0.1 kg. Body mass index (BMI) was calculated as weight in kilograms divided by squared height in meters. Results Characteristics according to tertile categories of % energy of fat intake are shown in Table 1. Participants with a higher total fat intake were more likely to be young and female and to consume protein and less likely to consume carbohydrate, and to be physically active in job, day shift workers, current smoker, and current alcohol drinker. Statistical Analysis As regards to HOMA-IR and HOMA-beta, there were no significant associations with scores of any dietary fatty acid patterns. Other Variables Living status, night and rotating shift work, smoking, alcohol drinking, occupational and non-occupational physical activities, and parental history of disease were elicited in the survey questionnaire. Smoking status (never, past, or current) and, if past or current smoker, duration of smoking in years and numbers of cigarettes smoked per day were asked. Averaged daily ethanol intake from alcohol beverage was calculated as drinking frequency multiplied by ethanol consumption per drinking day. Regarding physical activity, participants were asked about work-related activity (including domestic housework and commuting to and from work) and leisure-time activity. Work-related and leisure- time activities were each expressed as the sum of metabolic equivalent (MET) value multiplied by the duration of the activity within each domain. Body height was measured to the nearest 0.1 cm with subjects standing without shoes. Body weight in light May 2013 \| Volume 8 \| Issue 5 \| e64758 May 2013 \| Volume 8 \| Issue 5 \| e64758 PLOS ONE \| www.plosone.org 2 Plant Oil and Glucose Metabolism in Japanese Statistical Analysis y Participants were divided into ‘tertiles’ according to total fat intake (% energy). Data were expressed as means (SD) and percentages for continuous variables and categorical variables, respectively, adjusted for age and sex. Their differences across tertiles categories were tested using linear regression analysis or multiple logistic regression for trend, for continuous and categor- ical variables, respectively. Logistic regression analysis was performed to estimate (prevalence) odds ratio and its 95% confidence interval for impaired glucose metabolism according to tertile of each fatty acid or factor scores, and the lowest tertile was used as the reference category. Trend association was assessed by assigning ordinal numbers to each tertile of each fatty acid. Model 1 was adjusted for age (continuous, yr) and sex only, and Model 2 was additionally adjusted for night or rotating shift work (yes or no), parental history of diabetes, BMI (continuous, kg/m2), smoking (never-smoker, quitter, current smoker consuming ,20 cigarettes per day, or current smoker consuming $20 cigarettes per day), alcohol drinking (nondrinker including infrequent drinker consuming less than once per week, drinkers consuming ,23 g per day, drinkers consuming $23 to ,46 g per day, or drinkers consuming $46 g per day), work-related physical activity (,3 MET-h/d, 3–,7 MET-h/d, 7–,20 MET-h/d, or $20 MET-h/d), leisure-time physical activity (0 MET-h/week, 1,–,3 MET-h/week, 3–,10 MET-h/week, or $10 MET-h/week), anti- hypertensive treatment (yes or no), anti-dyslipidemia treatment (yes or no), energy intake (continuous in log-scale, kcal per day), protein intake (continuous, % energy). We also analyzed diabetes (n = 61) and pre-diabetes (n = 58) separately. Logarithmic trans- formation of HOMA-IR and HOMA-beta was made before analysis and we excluded 54 subjects due to missing data on insulin. Multiple linear regression was used to calculate mean and its 95% confidence interval of HOMA-IR and HOMA-beta across tertile of dietary fatty acid scores. Two-sided P values of less than 0.05 were considered as statistically significant. All analyses were performed using Statistical Analysis System (SAS) software version 9.1 (SAS Institute, Cary, NC, USA). Additionally, we examined the association of individual fatty acids with HOMA-IR and HOMA-beta. There were no significant associations of any fatty acids with HOMA-IR and HOMA-beta (data not shown). Discussion In this cross-sectional study of a Japanese working population, we found inverse relations of intake of PUFA, n-6 PUFA, oleic acid, and LA with impaired glucose metabolism. ALA was associated with lower prevalence of impaired glucose metabolism. Of three dietary fatty acid patterns identified, i.e. fish oil pattern, meat oriented pattern, and plant oil pattern, we also found an inverse association of impaired glucose metabolism with plant oil pattern, which was characterized by high intake of ALA, LA, and oleic acid. Fatty acids or fatty acid patterns were not associated with markers of insulin resistance and secretion. To our knowledge, this is the first study among Japanese, who consume high amount of fish (long-chain n-3 PUFAs), that investigates dietary fatty acid pattern in relation to glucose metabolism abnormality. In the present study, we identified 61 diabetes and 58 pre- diabetes participants. Table 2 shows the odds ratios of impaired glucose metabolism according to tertile categories of each fatty acid intake (% energy). Multiple logistic regression showed that the prevalence of impaired glucose metabolism was decreased as increased PUFA (P for trend = 0.03), n-6 PUFA (P for trend = 0.01), oleic acid (18:1) (P for trend = 0.04), and LA (18:2 n-6) (P for trend = 0.02). Although ALA (18:3 n-3) was non- significantly inversely associated with impaired glucose metabolism odds in multivariable model (P for trend = 0.12), there were no significant associations between impaired glucose metabolism and marine n-3 fatty acids. Saturated fatty acids were also not associated with impaired glucose metabolism. In the analysis for diabetes, a higher intake of n-6 PUFA was significantly inversely We observed inverse associations of intake of PUFA, n-6 PUFA, and LA (a major contributor to PUFA and n-6 PUFA) with May 2013 \| Volume 8 \| Issue 5 \| e64758 May 2013 \| Volume 8 \| Issue 5 \| e64758 PLOS ONE \| www.plosone.org 3 Plant Oil and Glucose Metabolism in Japanese Table 1. Age and sex- adjusted characteristics of participants according to quartile categories of total fatty acid intake (Ene %). Table 1. Age and sex- adjusted characteristics of participants according to quartile categories of total fatty acid intake (Ene %). Table 1. Age and sex adjusted characteristics of participants according to quartile categories of total fatty acid intake (Ene %). Discussion In contrast, a Japanese study of middle-aged employees showed that higher ALA intake was significantly associated with a lower prevalence of insulin resistance among normal weight subjects [28]. Although ALA might be related to lower risk of type 2 diabetes, the underlying mechanism should be clarified. A meta-analysis of 16 cohort studies showed that dietary EPA+DHA were not associated with diabetes (relative risk per 250 mg/d = 1.04, 95% CI = 0.9721.10) [29]. However, substan- tial heterogeneity was evident according to geographical regions.; the summary risk estimates indicated no association among European countries, a positive association among US studies, and an inverse association among Asian/Australian studies [29]. We observed no clear associations of marine fatty acids intake with prevalence of impaired glucose metabolism and insulin resistance in a Japanese population. Our finding agreed with that of a cross- sectional study in Japan showing no significant associations of EPA+DHA with insulin resistance [28]. Similarly, a recent meta- analysis of randomized control trials during 2 months to 6 months showed n-3 PUFA had no effects on insulin sensitivity [30]. The effects of n-3 PUFA on glucose metabolism are still debated. We found that oleic acid intake was associated with decreased prevalence of impaired glucose metabolism. Our finding is in line with a cross-sectional study in Italy that showed an inverse association between olive oil, the main source of oleic acid, intake and fasting plasma glucose [25]. Furthermore, both the KANWU study (Sweden), the largest randomized controlled clinical trial substituting dietary SFA for MUFA in healthy men and women, and another controlled short-term trial in healthy subjects in Spain reported that substitution of MUFA for SFA improved insulin sensitivity [26,27]. However, prospective cohort studies in Western countries showed no clear association of oleic acid or MUFA with risk of diabetes after adjustment for BMI and other covariates [17,18,20]. Prospective studies in Asian countries are needed to confirm the association between MUFA and glucose metabolism. In the present study, plant oil pattern characterized by high intake of LA, ALA, and oleic acid was inversely associated with impaired glucose metabolism. Given that higher levels of these fatty acids were each associated with decreased prevalence of impaired glucose metabolism in this study, the observed associ- ation with plant oil pattern would be reasonably expected. Discussion Tertile categories of total fatty acid intake (Ene %) Tertile 1 (low) Tertile 2 Tertile 3 (high) P1 Median (Ene %) 18.7 23.4 28.6 No. of subjects 355 355 355 Sex (male, %) 95.2 92.4 80.9 ,.0001 Age (year)2 45.469.5 44.269.5 43.969.6 0.04 BMI (kg/m2)3 23.363.3 23.263.3 23.463.4 0.83 Work rotation (day shift, %)4 25.5 17.7 11.6 ,.0001 Work-related physical activity ($20 Mets-h/d, %)4 27.9 24.0 18.6 0.004 Leisure time physical activity ($10 Mets-h/w, %)4 23.4 28.7 20.8 0.44 Smoking status (current, %)4 32.5 23.7 20.4 ,.0001 Alcohol consumption ($1 d/w, %)4 35.4 25.6 16.1 ,.0001 Parental history of diabetes (yes, %)4 14.1 14.9 16.9 0.47 Hypertension (yes, %)4 10.8 7.8 8.4 0.23 Hyperlipidemia (yes, %)4 5.7 4.3 5.8 0.99 Total energy intake (kcal/d) 3 18026482 18616479 18116485 0.79 Carbohydrate (% energy) 3 58.867.2 55.867.2 50.567.3 ,.0001 Protein (% energy) 3 12.262.1 14.062.1 15.662.1 ,.0001 1Based on logistic regression for categorical variables and linear regression analysis for continuous variables, assigning ordinal numbers 0–2 to tertile categories of total fatty acid intake. 2Sex adjusted means6SD. 3Age and sex adjusted means6SD. 4Age and sex adjusted proportions. doi:10 1371/journal pone 0064758 t001 Tertile categories of total fatty acid intake (Ene %) impaired glucose metabolism. Previously, some [17,18] but not all [19] prospective studies showed an inverse association of risk of type 2 diabetes with intake of PUFA, and an intervention study also reported that a diet rich in LA improved insulin sensitivity compared with saturated fatty acid rich diet [9]. In the Health Professional Follow-up Study, although among overall participants intake of LA was not associated with risk of type 2 diabetes, among men with ,65 years of age and with a BMI ,25 kg/m2 LA was observed a statistically significantly inverse association [20]. The present study population was young (aged 44.569.5 years) and lean (proportion of BMI ,25 kg/m2 = 75%). In addition, several studies have consistently shown an inverse association of LA in serum cholesterol esters or phospholipids with risk of type 2 diabetes [21224]. Taken together, a high intake of PUFA, especially LA, might be protective for glucose intolerance. tertile, although the reductions were not statistically significant. Our result agrees with a meta-analysis of prospective studies that showed a weak inverse association between ALA intake and type 2diabetes [7]. As regards insulin resistance, we found no clear association between ALA and HOMA-IR. Discussion In addition, the present finding is consistent with those of previous As regards ALA, a plant derived n-3 PUFA, subjects in the highest tertile of intakes had a 38% lower prevalence of both impaired glucose metabolism compared with those in the lowest May 2013 \| Volume 8 \| Issue 5 \| e64758 PLOS ONE \| www.plosone.org 4 Plant Oil and Glucose Metabolism in Japanese Table 2. The odds ratio (OR) of impaired glucose metabolism (n = 119) according to tertile categories of each fatty acid intake (% energy). Table 2. The odds ratio (OR) of impaired glucose metabolism (n = 119) according to tertile categories of each fatty acid intake (% energy). Table 2. The odds ratio (OR) of impaired glucose metabolism (n = 119) according to tertile categories of each fatty acid intake (% energy). Discussion Median (% energy) Model 11 Model 22 T1 T2 T3 Ptrend 3 T1 T2 T3 Ptrend 3 SFA 5.86 1.00 (reference) 1.29 (0.81–2.05) 0.94 (0.56–1.57) 0.89 1.00 (reference) 1.27 (0.7322.21) 0.80 (0.4121.55) 0.55 MUFA 8.46 1.00 (reference) 0.92 (0.5721.49) 1.09 (0.6721.78) 0.75 1.00 (reference) 0.81 (0.4721.41) 0.57 (0.3021.06) 0.08 PUFA 6.01 1.00 (reference) 0.89 (0.5521.45) 1.12 (0.6921.81) 0.66 1.00 (reference) 0.64 (0.3621.13) 0.48 (0.2520.93) 0.03 n3 PUFA 1.14 1.00 (reference) 1.11 (0.6521.88) 1.78 (1.0922.91) 0.02 1.00 (reference) 0.82 (0.4421.53) 1.00 (0.5022.01) 0.96 n6 PUFA 4.80 1.00 (reference) 0.86 (0.5321.38) 0.98 (0.6021.60) 0.91 1.00 (reference) 0.62 (0.3521.07) 0.45 (0.2420.84) 0.01 16:0 3.58 1.00 (reference) 1.30 (0.8122.07) 1.06 (0.6321.77) 0.76 1.00 (reference) 1.37 (0.7822.40) 0.81 (0.4121.58) 0.57 16:1 0.34 1.00 (reference) 1.37 (0.8222.26) 1.75 (1.0622.88) 0.03 1.00 (reference) 0.88 (0.4821.61) 1.15 (0.5522.39) 0.72 18:0 1.29 1.00 (reference) 1.27 (0.8022.03) 1.01 (0.6021.70) 0.88 1.00 (reference) 1.26 (0.7322.20) 0.67 (0.3521.30) 0.27 18:1 7.66 1.00 (reference) 0.90 (0.5621.45) 0.99 (0.6121.63) 0.95 1.00 (reference) 0.85 (0.4921.45) 0.51 (0.2820.96) 0.04 18:2 n6 4.69 1.00 (reference) 0.93 (0.5821.50) 1.00 (0.6121.63) 0.98 1.00 (reference) 0.68 (0.3921.18) 0.46 (0.2520.87) 0.02 18:3 n3 0.73 1.00 (reference) 1.14 (0.7021.86) 1.17 (0.7221.90) 0.53 1.00 (reference) 1.03 (0.6021.79) 0.62 (0.3421.13) 0.12 18:3 n6 0.003 1.00 (reference) 1.25 (0.7622.07) 1.39 (0.8622.27) 0.19 1.00 (reference) 1.20 (0.6822.13) 1.24 (0.6922.26) 0.48 18:4 n3 0.03 1.00 (reference) 0.84 (0.4921.45) 1.64 (1.0022.68) 0.03 1.00 (reference) 0.84 (0.4621.56) 1.52 (0.7922.94) 0.19 20:2 n6 0.02 1.00 (reference) 1.64 (0.9922.71) 1.73 (1.0422.86) 0.03 1.00 (reference) 1.37 (0.7622.47) 1.03 (0.5421.99) 0.97 20:3 n6 0.01 1.00 (reference) 1.35 (0.8222.22) 1.56 (0.9522.57) 0.08 1.00 (reference) 1.19 (0.6622.14) 1.08 (0.5422.16) 0.83 20:4 n6 0.07 1.00 (reference) 1.53 (0.9322.53) 1.55 (0.9422.58) 0.09 1.00 (reference) 1.20 (0.6622.18) 0.98 (0.4921.98) 0.92 20:5 n3 0.11 1.00 (reference) 1.06 (0.6221.81) 1.68 (1.0222.76) 0.03 1.00 (reference) 0.96 (0.5221.77) 1.38 (0.7122.69) 0.32 22:5 n3 0.03 1.00 (reference) 1.30 (0.7622.23) 1.92 (1.1523.20) 0.01 1.00 (reference) 1.12 (0.6022.10) 1.50 (0.7523.01) 0.24 22:5 n6 0.003 1.00 (reference) 0.93 (0.5521.55) 1.33 (0.8222.15) 0.23 1.00 (reference) 0.78 (0.4221.44) 1.06 (0.5522.02) 0.84 22:6 n3 0.20 1.00 (reference) 1.16 (0.6821.99) 1.77 (1.0722.92) 0.02 1.00 (reference) 1.09 (0.5922.01) 1.39 (0.7022.77) 0.34 1Adjusted for age (y) and sex. Adjusted for age (y) and sex. 2Adjusted for age (y) sex, BMI (kg/m2), shiftwork (yes or no), leisure time physical activity (0, 1,–,3, 3–,10, $10 Mets-h/w), work-related physical activity (,3, 3–,7, 7– ,20, $20 Mets-h/w), smoking status (never and past, current and ,20 cigarette/d, or current and $20 cigarette/d), alcohol consumption (nondrinker and 1–3 d/m, ,23 g ethanol/d, 23–,46 g ethanol/d, or $46 g ethanol/d), hypertension (yes or no), hyperlipidemia (yes or no), parental history of diabetes (yes, no or unknown), log transformed total energy intake (kcal/d), and protein intake (% energy). 3Based on multiple linear regression analysis, assigning ordinal numbers 022 to tertile categories of each fatty acid intake. doi:10.1371/journal.pone.0064758.t002 Discussion The odds ratio (OR) of impaired glucose metabolism (n = 119) according to tertile categories of fatty acid pattern score. Table 4. The odds ratio (OR) of impaired glucose metabolism (n = 119) according to tertile categories o Table 4. The odds ratio (OR) of impaired glucose metabolism (n = 119) according to tertile categories of fatty acid pattern score. Fish oil pattern Meat oriented pattern Plant oil pattern Model 11 Model 22 Model 11 Model 22 Model 11 Model 22 Impaired glucose metabolism3 Tertile 1 (low) 1.00 (reference) 1.00 (reference) 1.00 (reference) 1.00 (reference) 1.00 (reference) 1.00 (reference) Tertile 2 1.18 (0.6922.02) 1.05 (0.5721.94) 1.43 (0.8922.30) 1.29 (0.7522.24) 0.81 (0.4921.31) 0.80 (0.4721.37) Tertile 3 (high) 1.79(1.0722.95) 1.47 (0.7722.82) 1.20 (0.7222.00) 0.87 (0.4621.65) 0.93 (0.5821.50) 0.54 (0.3020.95) P trend4 0.02 0.23 0.45 0.71 0.76 0.03 Diabetes5 Tertile 1 (low) 1.00 (reference) 1.00 (reference) 1.00 (reference) 1.00 (reference) 1.00 (reference) 1.00 (reference) Tertile 2 0.98 (0.5021.96) 0.94 (0.4322.05) 2.13 (1.1124.10) 1.74 (0.8223.70) 0.63 (0.3221.23) 0.57 (0.2721.19) Tertile 3 (high) 1.25 (0.6522.39) 0.99 (0.4222.35) 1.52 (0.7523.10) 0.89 (0.3822.10) 0.93 (0.5121.71) 0.46 (0.2220.96) P trend4 0.48 0.98 0.23 0.75 0.80 0.04 Pre2diabetes6 Tertile 1 (low) 1.00 (reference) 1.00 (reference) 1.00 (reference) 1.00 (reference) 1.00 (reference) 1.00 (reference) Tertile 2 1.44 (0.6323.26) 1.19 (0.4822.93) 0.84 (0.4421.63) 0.89 (0.4221.89) 1.04 (0.5422.00) 1.36 (0.6622.79) Tertile 3 (high) 2.53 (1.2025.35) 2.09 (0.8425.25) 0.43 (0.4721.84) 0.95 (0.4122.21) 0.90 (0.4621.77) 0.61 (0.2721.38) P trend4 0.01 0.09 0.80 0.88 0.76 0.28 1Adjusted for age (y) and sex. 2Adjusted for age (y) sex, BMI (kg/m2), shiftwork (yes or no), leisure time physical activity (0, 1,–,3, 3–,10, $10 Mets-h/w), work-related physical activity (,3, 3–,7, 7– ,20, $20 Mets-h/w), smoking status (never and past, current and ,20 cigarette/d, or current and $20 cigarette/d), alcohol consumption (nondrinker and 1–3 d/m, ,23 g ethanol/d, 23–,46 g ethanol/d, or $46 g ethanol/d), hypertension (yes or no), hyperlipidemia (yes or no), parental history of diabetes (yes, no or unknown), log transformed total energy intake (kcal/d), and protein intake (% energy). 3Number of cases of tertile 1 to tertile 3 was 27, 35, and 57 for factor 1, 40, 45, and 34 for factor 2, and 44, 35, and 40 for factor 3, respectively. 4Based on multiple linear regression analysis, assigning ordinal numbers 022 to tertile categories of each fatty acid intake. Discussion Second, dietary information was assessed using a self-administered BDHQ, which is subject to measurement error, probably in a random manner. The resulting misclassification must have distorted the estimate of association toward the null. Third, although we adjusted for potentially important confounding variables, a possibility of residual confounding and unknown confounders cannot be excluded. Finally, because the study participants were workers of a company in Japan, the present findings might not be representative for general population. studies. Hu et al. report that a higher intake of vegetable fat is associated with beneficial glucose metabolism and insulin resistance [31]. Furthermore, the Iowa Women’s Health Study and the Nurses’ Health Study also showed vegetable fat was related to decreased diabetes risk [17,18] and an Italian cross- sectional study showed that consumption of vegetable oil was inversely associated with serum glucose levels in both sexes [25]. It is possible that plant oil pattern represents the combination of several subtypes of fatty acid that are beneficial for glucose metabolism. studies. Hu et al. report that a higher intake of vegetable fat is associated with beneficial glucose metabolism and insulin resistance [31]. Furthermore, the Iowa Women’s Health Study and the Nurses’ Health Study also showed vegetable fat was related to decreased diabetes risk [17,18] and an Italian cross- sectional study showed that consumption of vegetable oil was inversely associated with serum glucose levels in both sexes [25]. It is possible that plant oil pattern represents the combination of several subtypes of fatty acid that are beneficial for glucose metabolism. The mechanism underlying the association between fatty acid composition and glucose metabolism is unclear. Fatty acid composition of the adipose cell membrane alters glucose uptake by regulating the translocation and by controlling membrane fluidity involved in glucose transporter 4 [32]. These alteration could affect tissue and whole body insulin sensitivity [33]. Moreover, PUFAs modulate peroxisome proliferator-activated receptor a (PPAR-a) involved in oxidative process and sterol regulatory element-binding proteins (SREBPs) involved in lipo- genesis and expression of PPAR-a and SREBPs is decreased with PUFAs [34]. Furthermore, n-6 PUFAs and ALA possibly decrease inflammation through inhibiting the production of inflammatory factors in endothelial cells by down-regulating nuclear factor-kB [35], which can lead to local insulin resistance [36]. However, the present study showed no significant association between these fatty acids and markers of insulin resistance. In conclusion, high intake of plant source fatty acid, i.e. Discussion 5Number of cases of tertile 1 to tertile 3 was 17, 18, and 26 for factor 1, 16, 27, and 18 for factor 2, and 24, 15, and 22 for factor 3, respectively. 6Number of cases of tertile 1 to tertile 3 was 10, 16, and 32 for factor 1, 25, 17, and 16 for factor 2, and 20, 20, and 18 for factor 3, respectively. doi:10.1371/journal.pone.0064758.t004 Adjusted for age (y) and sex. 2Adjusted for age (y) sex, BMI (kg/m2), shiftwork (yes or no), leisure time physical activity (0, 1,–,3, 3–,10, $10 Mets-h/w), work-related physical activity (,3, 3–,7, 7– ,20, $20 Mets-h/w), smoking status (never and past, current and ,20 cigarette/d, or current and $20 cigarette/d), alcohol consumption (nondrinker and 1–3 d/m, ,23 g ethanol/d, 23–,46 g ethanol/d, or $46 g ethanol/d), hypertension (yes or no), hyperlipidemia (yes or no), parental history of diabetes (yes, no or unknown), log transformed total energy intake (kcal/d), and protein intake (% energy). 3Number of cases of tertile 1 to tertile 3 was 27, 35, and 57 for factor 1, 40, 45, and 34 for factor 2, and 44, 35, and 40 for factor 3, respectively. 4Based on multiple linear regression analysis, assigning ordinal numbers 022 to tertile categories of each fatty acid intake. 5Number of cases of tertile 1 to tertile 3 was 17, 18, and 26 for factor 1, 16, 27, and 18 for factor 2, and 24, 15, and 22 for factor 3, respectively. 6Number of cases of tertile 1 to tertile 3 was 10, 16, and 32 for factor 1, 25, 17, and 16 for factor 2, and 20, 20, and 18 for factor 3, respectively. doi:10.1371/journal.pone.0064758.t004 important confounders. Limitations of our study also warrant mention. First, an association derived from a cross-sectional study does not indicate causality. However, our finding is also supported by those from prospective and intervention studies indicating a favorable role of LA [9,17,18,20] and plant oil [17,18] in glucose metabolism. In addition, observational studies using serum fatty acid composition, which reflects relatively long term fat intake, have shown an inverse association between LA and impaired glucose metabolism [21–24]. Furthermore, we found an inverse association of n-6 PUFA with pre-diabetes, a status which might not influence dietary behavior. We thus believe that the present finding cannot fully be accounted for by reverse causation. May 2013 \| Volume 8 \| Issue 5 \| e64758 Discussion 2Adjusted for age (y) sex, BMI (kg/m2), shiftwork (yes or no), leisure time physical activity (0, 1,–,3, 3–,10, $10 Mets-h/w), work-related physical activity (,3, 3–,7, 7– ,20, $20 Mets-h/w), smoking status (never and past, current and ,20 cigarette/d, or current and $20 cigarette/d), alcohol consumption (nondrinker and 1–3 d/m, ,23 g ethanol/d, 23–,46 g ethanol/d, or $46 g ethanol/d), hypertension (yes or no), hyperlipidemia (yes or no), parental history of diabetes (yes, no or unknown), log transformed total energy intake (kcal/d), and protein intake (% energy). 3Based on multiple linear regression analysis, assigning ordinal numbers 022 to tertile categories of each fatty acid intake. doi:10.1371/journal.pone.0064758.t002 Table 2. The odds ratio (OR) of impaired glucose metabolism (n = 119) according to tertile categories of each fatty acid intake (% energy). Adjusted for age (y) and sex. 2Adjusted for age (y) sex, BMI (kg/m2), shiftwork (yes or no), leisure time physical activity (0, 1,–,3, 3–,10, $10 Mets-h/w), work-related physical activity (,3, 3–,7, 7– ,20, $20 Mets-h/w), smoking status (never and past, current and ,20 cigarette/d, or current and $20 cigarette/d), alcohol consumption (nondrinker and 1–3 d/m, ,23 g ethanol/d, 23–,46 g ethanol/d, or $46 g ethanol/d), hypertension (yes or no), hyperlipidemia (yes or no), parental history of diabetes (yes, no or unknown), log transformed total energy intake (kcal/d), and protein intake (% energy). 3Based on multiple linear regression analysis, assigning ordinal numbers 022 to tertile categories of each fatty acid intake. doi:10.1371/journal.pone.0064758.t002 Table 3. Factor loading matrix for the major fatty acid patterns identified by principal component analysis. Fish oil pattern Meat oriented pattern Plant oil pattern 20:5 n-3 0.98 0.09 0.03 18:4 n-3 0.98 0.10 0.04 22:5 n-3 0.97 0.17 0.04 22:6 n-3 0.97 0.16 0.05 22:5 n-6 0.93 0.18 0.04 18:3 n-6 0.58 0.32 20.14 20:3 n-6 0.29 0.92 0.12 18:0 20.02 0.92 0.32 16:0 0.05 0.91 0.29 20:2 n-6 0.25 0.87 0.21 16:1 0.50 0.78 0.21 20:4 n-6 0.38 0.76 0.12 18:1 20.04 0.73 0.65 18:3 n-3 0.05 0.26 0.95 18:2 n-6 0.002 0.38 0.90 Variance explained (%) 54.1 27.4 8.1 doi:10.1371/journal.pone.0064758.t003 3. Factor loading matrix for the major fatty acid patterns identified by principal component analysis. May 2013 \| Volume 8 \| Issue 5 \| e64758 PLOS ONE \| www.plosone.org 5 Plant Oil and Glucose Metabolism in Japanese Table 4. PLOS ONE \| www.plosone.org References Metabolism: clinical and experimental 50: 1472–1478. 8. Hu FB (2001) Diet, lifestyle, and the risk of type 2 diabetes mellitus in women. The New England journal of medicine 345: 790–797. The New England journal of medicine 345: 790–797. 9. Summers LKM (2002) Substituting dietary saturated fat with polyunsaturated fat changes abdominal fat distribution and improves insulin sensitivity. Diabetologia 45: 369–377. 25. Trevisan M (1990) Consumption of olive oil, butter, and vegetable oils and coronary heart disease risk factors. The Research Group ATS-RF2 of the Italian National Research Council. JAMA : the journal of the American Medical Association 263: 688–692. g 10. Finucane MM, Stevens GA, Cowan MJ, Danaei G, Lin JK, et al. (2011) National, regional, and global trends in body-mass index since 1980: systematic analysis of health examination surveys and epidemiological studies with 960 country-years and 9.1 million participants. Lancet 377: 557–567. 26. Vessby B, Uusitupa M, Hermansen K, Riccardi G, Rivellese AA, et al. (2001) Substituting dietary saturated for monounsaturated fat impairs insulin sensitivity in healthy men and women: The KANWU Study. Diabetologia 44: 312–319. 11. Fukushima M (2004) Insulin secretion capacity in the development from normal glucose tolerance to type 2 diabetes. Diabetes Research and Clinical Practice 66 Suppl 1: S37–43. 27. Pe´rez-Jime´nez F (2001) A Mediterranean and a high-carbohydrate diet improve glucose metabolism in healthy young persons. Diabetologia 44: 2038–2043. pp 12. FAO (2012) FAO Statistical Yearbook World Food and Agriculture, Rome. Available: http://www.fao.org/docrep/015/i2490e/i2490e03c.pdf. Accessed 2013 Mar 6. 28. Muramatsu T, Yatsuya H, Toyoshima H, Sasaki S, Li Y, et al. (2010) Higher dietary intake of alpha-linolenic acid is associated with lower insulin resistance in middle-aged Japanese. Preventive medicine 50: 272–276. 13. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, et al. (1985) Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28: 412– 419. 29. Wallin A (2012) Fish consumption, dietary long-chain n-3 fatty acids, and risk of type 2 diabetes: systematic review and meta-analysis of prospective studies. Diabetes care 35: 918–929. 30. Akinkuolie AO (2011) Omega-3 polyunsaturated fatty acid and insulin sensitivity: a meta-analysis of randomized controlled trials. Clinical nutrition (Edinburgh, Scotland) 30: 702–707. 14. Seino Y, Nanjo K, Tajima N, Kadowaki T, Kashiwagi A, et al. References follow-up of the Finnish and Dutch cohorts of the Seven Countries Study. Diabetes care 18: 1104–1112. follow-up of the Finnish and Dutch cohorts of the Seven Countries Study. Diabetes care 18: 1104–1112. 1. Storlien LH (1996) Skeletal muscle membrane lipids and insulin resistance. Lipids 31 Suppl: S261–265. 2. Lichtenstein AH (2000) Relationship of dietary fat to glucose metabolism. Atherosclerosis 150: 227–243. 20. van Dam RM (2002) Dietary fat and meat intake in relation to risk of type 2 diabetes in men. Diabetes care 25: 417–424. 3. Nguyen MTA (2007) A subpopulation of macrophages infiltrates hypertrophic adipose tissue and is activated by free fatty acids via Toll-like receptors 2 and 4 and JNK-dependent pathways. The Journal of biological chemistry 282: 35279– 35292. 21. Patel PS, Sharp SJ, Jansen E, Luben RN, Khaw KT, et al. (2010) Fatty acids measured in plasma and erythrocyte-membrane phospholipids and derived by food-frequency questionnaire and the risk of new-onset type 2 diabetes: a pilot study in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk cohort. The American Journal of Clinical Nutrition 92: 1214– 1222. 4. Hulbert AJ, Turner N, Storlien LH, Else PL (2005) Dietary fats and membrane function: implications for metabolism and disease. Biological reviews of the Cambridge Philosophical Society 80: 155–169. 22. Hodge AM, English DR, O’Dea K, Sinclair AJ, Makrides M, et al. (2007) Plasma phospholipid and dietary fatty acids as predictors of type 2 diabetes: interpreting the role of linoleic acid. The American Journal of Clinical Nutrition 86: 189–197. g p y 5. Clarke SD (2004) The multi-dimensional regulation of gene expression by fatty acids: polyunsaturated fats as nutrient sensors. Current Opinion in Lipidology 15: 13–18. 6. Melanson EL, Astrup A, Donahoo WT (2009) The relationship between dietary fat and fatty acid intake and body weight, diabetes, and the metabolic syndrome. Annals of Nutrition & Metabolism 55: 229–243. 23. Wang L, Folsom AR, Zheng ZJ, Pankow JS, Eckfeldt JH, et al. (2003) Plasma fatty acid composition and incidence of diabetes in middle-aged adults: the Atherosclerosis Risk in Communities (ARIC) Study. The American Journal of Clinical Nutrition 78: 91–98. 7. Wu JHY (2012) Omega-3 fatty acids and incident type 2 diabetes: a systematic review and meta-analysis. British journal of nutrition 107 Suppl 2: S214–227. 24. Pelikanova T, Kazdova L, Chvojkova S, Base J (2001) Serum phospholipid fatty acid composition and insulin action in type 2 diabetic patients. Discussion LA, oleic acid, and ALA was associated with decreased prevalence of impaired glucose metabolism in Japanese men and women, suggesting a favorable role of plant oils in glucose metabolisms. Additional studies are warranted to confirm the role of MUFA and n-3 PUFA involving glucose metabolism in healthy populations using different approaches such as randomized feeding or supplementation studies. Major strengths of this study include a high participation rate, and use of validated BDHQ and the definition of diabetes and pre- diabetes based on multiple sources of information (self-report, HbA1c, and fasting glucose) and adjustment for potentially May 2013 \| Volume 8 \| Issue 5 \| e64758 PLOS ONE \| www.plosone.org 6 Plant Oil and Glucose Metabolism in Japanese Author Contributions Conceived and designed the experiments: TM AN. Performed the experiments: K. Kurotani TM AN K. Kuwahara NMP IK TK HT. Conceived and designed the experiments: TM AN. Performed the experiments: K. Kurotani TM AN K. Kuwahara NMP IK TK HT. Analyzed the data: KK. Wrote the paper: K. Kurotani TM AN K. Kuwahara NMP IK TK HT. We thank Fumiko Zaizen (Furukawa Electric Corporation); and Ayami Kume, Nishihara Sachiko and Yuho Mizoue (National Center for Global Health and Medicine) for their help in data collection. Analyzed the data: KK. Wrote the paper: K. Kurotani TM AN K. Kuwahara NMP IK TK HT. References (2010) Report of the Committee on the Classification and Diagnostic Criteria of Diabetes Mellitus: The Committee of the Japan Diabetes Society on the Diagnostic Criteria of Diabetes Mellitus. Journal of Diabetes Investigation 1: 212–228. 31. Hu FB, van Dam RM, Liu S (2001) Diet and risk of Type II diabetes: the role of types of fat and carbohydrate. Diabetologia 44: 805–817. 15. Kobayashi S (2011) Comparison of relative validity of food group intakes estimated by comprehensive and brief-type self-administered diet history questionnaires against 16 d dietary records in Japanese adults. Public health nutrition 14: 1200–1211. 32. Ibrahim A (2005) Dietary trans-fatty acids alter adipocyte plasma membrane fatty acid composition and insulin sensitivity in rats. Metabolism, clinical and experimental 54: 240–246. 33. Rise´rus U (2008) Fatty acids and insulin sensitivity. Current opinion in clinical nutrition and metabolic care 11: 100–105. 16. Kobayashi S (2012) Both comprehensive and brief self-administered diet history questionnaires satisfactorily rank nutrient intakes in Japanese adults. Journal of epidemiology 22: 151–159. 34. Lottenberg AM (2012) The role of dietary fatty acids in the pathology of metabolic syndrome. The Journal of nutritional biochemistry 23: 1027–1040. 17. Salmero´n J (2001) Dietary fat intake and risk of type 2 diabetes in women. The American journal of clinical nutrition 73: 1019–1026. 35. Sacks FM (2006) Polyunsaturated fatty acids, inflammation, and cardiovascular disease: time to widen our view of the mechanisms. The journal of clinical endocrinology and metabolism 91: 398–400. j 18. Meyer KA (2001) Dietary fat and incidence of type 2 diabetes in older Iowa women. Diabetes care 24: 1528–1535. 36. Gustafson B (2010) Adipose tissue, inflammation and atherosclerosis. Journal of atherosclerosis and thrombosis 17: 332–341. 19. Feskens EJ, Virtanen SM, Rasanen L, Tuomilehto J, Stengard J, et al. (1995) Dietary factors determining diabetes and impaired glucose tolerance. A 20-year May 2013 \| Volume 8 \| Issue 5 \| e64758 PLOS ONE \| www.plosone.org 7
https://openalex.org/W4289637658	https://www.researchsquare.com/article/rs-1885650/latest.pdf	English	null	Second Primary Cancer Occurrence after Colorectal Cancer: A Systematic Review and Meta-Analysis	Research Square (Research Square)	2,022	cc-by	7,447	Second Primary Cancer Occurrence after Colorectal Cancer: A Systematic Review and Meta-Analysis Mohammad Zahedi Iran University of Medical Sciences Second Primary Cancer Occurrence after Colorectal Cancer: A Systematic Review and Meta-Analysis Mohammad Zahedi Iran University of Medical Sciences Mohammad Yousefi Semnan University of Medical Sciences and Health Services Amirreza Nasirzadeh Mashhad University of Medical Sciences Parham Mortazavi (  Parham2276@gmail.com ) Mazandaran University of Medical Sciences https://orcid.org/0000-0002-1162-4957 Sulmaz Ghaheremani Shiraz Medical School: Shiraz University of Medical Sciences Keyvan Heydari Mazandaran University of Medical Sciences Fateme Sheidaei Semnan University of Medical Sciences and Health Services Reza Alizadeh-Navaei Mazandaran University of Medical Sciences Research Article Keywords: Secondary Cancer, Incidence, standard incidence ratio, Colorectal Cancer Posted Date: August 3rd, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1885650/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Second Primary Cancer Occurrence after Colorectal Cancer: A Systematic Review and Meta-Analysis Second Primary Cancer Occurrence after Colorectal Cancer: A Systematic Review and Meta-Analysis Mohammad Zahedi Iran University of Medical Sciences Mohammad Yousefi Semnan University of Medical Sciences and Health Services Amirreza Nasirzadeh Mashhad University of Medical Sciences Parham Mortazavi (  Parham2276@gmail.com ) Mazandaran University of Medical Sciences https://orcid.org/0000-0002-1162-4957 Sulmaz Ghaheremani Shiraz Medical School: Shiraz University of Medical Sciences Keyvan Heydari Mazandaran University of Medical Sciences Fateme Sheidaei Semnan University of Medical Sciences and Health Services Reza Alizadeh-Navaei Mazandaran University of Medical Sciences Study Selection : Studies included if they met the following criteria: original studies with cohort design in the English language; studies that investigated and reported the SIR of second primary cancer after colorectal, colon, and rectum cancer. Data Sources: Medline (via PubMed), Web of Science, and Scopus databases were searched for English cohort studies that reported standard incidence ratio (SIR) of SPCAC up to 10 Aug 2020. Heterogeneity across included studies was determined using the I2 statistic. We used the random‑effect models for pooled Standard Incidence Ratio (SIR) and 95% confidence intervals (CIs). Background Colorectal cancer (CRC) is the third most frequent cancer diagnosis and the second leading cause of cancer-related death. There is evidence of an increased risk of developing a second primary colorectal cancer in CRC patients. Introduction Colorectal cancer (CRC) is the third most frequent cancer diagnosis and the second leading cause of cancer-related death (1). Although lifestyle advancement as well as early detection tests and treatment improvement lead to increased survival of CRC patients (2–4). The median overall survival of stage IV CRC was 12 months in the 1990s increased to over 24 months (5, 6). Increasing survival rates of CRC patients as well as decreasing age of incidence (7, 8) might reflect additional considerations to second primary cancers (8, 9). Second primary cancer after complete remission of CRC is estimated to occur in 2–4 percent of cancer patients and colorectal and stomach cancers were the most prevalent primary cancers respectively(10) (11, 12). Second primary cancer is the fourth leading cause among colorectal cancer patients and identifying prevalent malignancies among survivors could lead to better follow-up and management of the disease (13). Evidence for occurrence (the relative risk) of Second primary cancer after colorectal cancer (SPCAC) through cohort studies are controversial(14). The aim of this study was to review the evidence of SPCAC through cohort studies. Main Outcome Measures : The pooled SIR of SPCAC based on the primary site of cancer was measured. Results Through 30 extracted papers, 142429 confirmed SPCAC adult patients (more than 19 years old) were included in this meta-analysis. The SIR of SPCAC was 1.27 (95% CI 1.16–1.37) and was reported by 12 studies. The SIR of SPCAC for digestive, reproductive, and urinary organ’s involvement was 1.31 (95% CI 0.89–1.72), 1.45 (95% CI 0.99–1.90), and 1.27 (95% CI 1.10–1.45), respectively. Limitations : Unavailable confounding characteristics such as smoking, alcohol intake, physical activity, food, and lifestyle, may restrict the findings. : Unavailable confounding characteristics such as smoking, alcohol intake, physical activity, food, and lifestyle, may restrict the findings. Conclusions Colorectal cancer survivors (CCSs) are more vulnerable to SPCAC of the small intestine and endometrium. The result of this study might be helpful for both clinicians and policymakers. Further investigations on etiologic factors for prevention and early diagnosis of SPCAC are suggested. Objective This systematic review and meta-analysis aims to provide a pooled relative risk of second primary cancer after colorectal cancer (SPCAC) in CRC patient based on available evidence. Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/13 Methods Page 2/13 Selection Procedures A total of 6676 citations were identified through electronic database searches (PubMed: 2169 Scopus: 2253, WoS: 2254). After excluding 3507 duplicated papers, 3169 records were included after the screening. After title review, 435 records were included for abstract review. Then 305 records were excluded during abstract review process and 130 papers were selected for full-text eligibility assessment. Finally, after excluding 90 records, 30 studies were included into the meta-analysis. We summarized the process of study identification and selection in the PRISMA flow diagram in Figure 1. Data extraction Two independent researchers (A. N. and P. M.) extracted the following data from included studies: first author's name, year of publication, type of publication, country, total sample size, study design, number of patients’ with SPCAC, location of SPCAC, and type of secondary cancer. The SIR (standardized incidence ratio), is defined as the ratio of the number of observed secondary cancer in colorectal cancer survivors to the number of expected cancer cases(17). Protocol and Registration Our systematic review and meta-analysis were performed and reported using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline. The International Prospective Register of Systematic Reviews was used to register this systematic review and meta-analysis. (PROSPERO; No.: CRD42020151253). Search strategy Electronic databases were systematically searched in Medline (via PubMed), Web of Science (WoS), and Scopus for English articles since the inception up to 10 Aug 2020. There was no limitation on the inclusion of different types of published papers (pre-print of published articles). Retrospective as well as prospective cohort studies were included in this systematic review and meta-analysis. Two researchers independently searched the following Medical Subject Heading (MeSH) keywords: "secondary OR progress OR Survivor OR primary," and "Benign Neoplasms OR Neoplasia OR Malignancies OR carcinoma " in combination with "Colorectal Cancer OR Colorectal Tumor OR Colorectal Carcinoma" which were also combined with and/or/not to find more articles. A combination of keywords and free text was used to broaden the search result. The reference lists of relevant studies and previous reviews manually were checked to retrieve more studies. Eligible criteria Studies included in our study if they met the following criteria: original studies with cohort design in English language; studies that investigated and reported the SIR of second primary cancer after colorectal, colon and rectum cancer. Studies on patients with untreated colon cancer, patients with metastases, did not report the SIR of SPCAC, animal studies, and studies that had incomplete data were excluded. All the case reports, cross-sectional studies, expert opinion articles, letters to the editors, review articles books, and animal studies were also excluded. S d S l i Statistical analysis Statistical analysis was performed using STATA version 11.0 (Stata Corp., College Station, TX). Heterogeneity among included studies was assessed using the I 2 statistic. I2 above 70% was considered as the existence of significant heterogeneity. A random-effects model for I2 > 50% was used to pool the results according to the results of heterogeneity. Moreover, subgroup analysis was performed on primary site of cancer. Pooled SIR was used to assess the concerned outcomes. Study Selection Two independent researchers (A. N. and P. M) assessed all retrieved reports using our inclusion and exclusion criteria. Disagreements were resolved through consensus or discussion with a third author (15). Duplicated papers were removed, and after the screen of titles and abstracts, full texts were assessed to determine whether the study met the inclusion criteria. Quality assessment Two authors independently valued the selected articles using the modified version of the Newcastle-Ottawa Quality Assessment (NOS), based on a previous study(16). The studies were assessed for selection, comparability, ‎and exposure/outcome. A study with a NOS score of ≥ 7 was considered good quality. Data extraction 2. Reproductive system The SIR of reproductive system cancer after colorectal cancer were reported in 4 studies and it was 1.45 (Table 2). SIR of Second cancer of endometrium after colorectal cancer was 3.03 while SIR of second ovary cancer after colorectal, and colon cancer were 1.51 and, 1.74. (Table 2) 3. Urinary system The SIR of urinary system cancer after colorectal and rectum cancer were investigated by 5 and 2 studies were 1.27 (1.10-1.45) and 1.23 (1.12- 1.35) respectively (Table 2). The SIR of second cancer of bladder, kidney and prostate after colorectal cancer were 1.2, 1.34 and 1.17 respectively. The SIR of cancer of bladder, kidney and prostate after colon cancer were 1.04, 1.46 and 1.15 respectively. The SIR of cancer of bladder and kidney after rectum cancer were 1.20 and 1.28 respectively. (Table 2) 1. Digestive system Findings demonstrated that the SIR of digestive system cancer after colorectal and rectum cancer were 1.31 (95% CI 0.89-1.72) and 1.43 (95% CI 0.27- 2.59) respectively (Table 2). Second digestive system cancer also might be categorized based on type of organs (Table 2). Second small intestine cancer after colorectal cancer had highest SIR with 3.68. SIR of secondary cancer of colon, after colorectal cancer was 1.59. The SIR of cancer of small intestine after rectum cancer was 2.33 (Table 2) Quality assessment: All included cohort studies had good quality (NOS≥ 7) using the NOS. Most of the included studies only controlled sex, age and marital status as confounding factors and didn't reach to maximum score in the field of comparability. Also some included studies didn't met criteria for adequacy of follow- up for cohorts. Table 3 shows the results of the quality assessment of included studies based on NOS.‎ Incidence rate of SPCAC based on primary site of cancer The pooled SIR of SPCAC were reported by 12 studies was 1.27 (95% CI 1.16-1.37). Furthermore, incidence of SPC in patients with colon cancer and rectum cancer were studied by 3 and 4 studies with SIR of 1.11 (95% CI 1.04-1.17) and 1.04 (95% CI 0.99 – 1.09) respectively. The SIR of SPC in left colon and right colon cancer were divided in 4 studies which were 1.39 (95% CI 1.11-1.66) and 1.44 (95% CI 1.26-1.61) respectively. (Fig. 2 and Table 2) 4. Other organs The SIR of central nervous system cancer after colorectal cancer and rectum cancer were investigated by 3 and 2 studies and their SIR were 1.24 and 0.41 respectively. The SIR of cervical cancer after colorectal, colon and rectum cancer were demonstrated by 2, 2 and 4 studies and correspondence SIR were 1.04, 1.09 and 1 respectively. The SIR of female breast cancer after colorectal, colon and rectum cancer were reported by 4, 3 and 4 studies and their incidence rate were 1.07, 1.13 and 1.03 respectively. The SIR of hematological cancer after colorectal, colon and rectum cancer were reported by 4, 3 and 4 studies and their SIR were 1.07, 1.13 and 1.03 respectively. The SIR of lung cancer after colorectal and rectum cancer were reported by 6 and 5 studies and their SIR were 1.16 and 1.46 respectively. The SIR of lymphoma after colorectal cancer were reported by 2 studies and the SIR was 1.38. The SIR of skin cancer after colorectal and rectum cancer were reported by 2 studies and their SIR were 1.38 and 1. 19 respectively. The SIR of thyroid cancer after colorectal, colon and rectum cancer were demonstrated by 2, 2 and 3 studies and their incidence rate were 1.5, 1.58 and 1.41 respectively. (Table 2) Quality assessment: Study Characteristics Among 30 extracted papers, a total of 142429 confirmed adult cases (older than 19 years old) of SPCAC were assessed for meta-analysis. All of the included studies were cohorts. Also, most of the included studies were investigated SIR of SPCAC in developed countries. The characteristics of included studies are presented in Table 1. Page 3/13 Meta-analysis of outcome: Discussion Researchers demonstrated that a reverse relation exists between age at the time of diagnosis of primary CRC and SPC risk, so patients 40 years or younger have a SIR equal to 5 in comparison with patients 50 years with a SIR of 2.6 (27). Given these findings, strict surveillance among CRC survivors specifically at young ages should be established for early detection of SPCs with specific attention to the small intestine cancer (26) Findings of this meta-analysis indicated that people with a history of CRCs are three times more likely prone to subsequent uterus and endometrial cancers (SIR: 3.03). In line with the present data, Lee et al. found a 3.2 fold increase in the risk of uterine cancer among CRC survivors (27). Other previous research also confirms the susceptibly of CRC survivors to uterine cancer (4, 26). The underlying reasons for these findings are still unclear. Some studies suggest that medical services access and ethnic variations are responsible for different risks of contraction (28, 29). Shin et al. reported a higher prevalence of uterine cancer among CRC survivors younger than 55 years (21). Hence the screening programs for early diagnosis of the uterus and endometrial cancers among CCs will be of importance in particular, at younger ages. The incidence of SPCs may be attributed to genetic susceptibility, environmental risk factors, as well as hormonal level changes (21). Mutations in the following genes (MLH1, MSH2, PMS1, and MSH6) can lead to both CRC and endometrial cancer, particularly in youth (30). BRCA1 and BRCA2 encode tumor suppressors, therefore any detrimental mutation in these genes increases the risk of gynecological and colorectal cancers (31). Research revealed that genetic disorders on the metabolism of a drug like glutathione transferase predispose patients to develop secondary cancers (32). Given the fact that embryonic endoderm is the origin of the common SPC sites, genetic susceptibility may act as the main mechanism underlying the risk of multiple cancers, so that adopting an approach to develop more accurate and earlier screening tests in cancer survivors could beneficial (26). Moreover, lifestyle and environmental factors including food habits, excessive alcohol consumption, as well as can prominently influence the development of secondary malignancies (23). However, the association between previous CRC and susceptibility to tobacco-related gynecological carcinogenesis is still unclear (21). Hormones are another probable factor contributing to the occurrence of SPCs, especially gynecological ones (21). Conclusion This systematic review and meta-analysis revealed that in CCS, the occurrence of the small intestine and endometrial cancer might increase up to three times. The result of this study might be useful for both clinicians and policymakers. Further investigations on etiologic factors for prevention and early diagnosis of SPCAC are suggested. Acknowledgments The authors would like to express their gratitude to the authors of primary studied. This systematic review and meta-analysis was registered at the- International Prospective Register of Systematic Reviews (PROSPERO; No.: CRD42020151253). The authors would like to express their gratitude for the support from the Student Research Committee of Mazandaran University of Medical Sciences, Sari, Iran. The study was approved by the Ethics Committee of the University (code: IR.MAZUMS.REC. 1398.1148) Discussion Interestingly, previous studies indicated that having no history of childbirth increases the risk of SPCs, so women who bore more than 4 children are less likely to get cancer than nulliparous women (33, 34). The current findings point to the need for more case-control and cohort studies to clarify the relationships. If the above-mentioned risk variables are found to be valid in future studies, they might be used to develop a new guideline for patient education and counseling on behavior modification, as well as novel surveillance systems for cancer survivors. Because the majority of studies on second primary malignancies in CRC patients were conducted in rich nations, conducting SEER in developing and low-income countries is necessary to get enough data on SPCs in these areas. One of the inherent drawbacks of this study could be the varying lengths of follow-up in the included research. Unavailable confounding characteristics such as smoking, alcohol intake, physical activity, food, and lifestyle, may restrict the findings. Furthermore, data misclassification must be addressed to some level due to the use of retro-prospective research and the difficulty to evaluate data correctness. We did, however, assess the quality of each included study and report the level of evidence. In comparison to previous studies, our study has the advantage of investigating a larger population-based cohort of CRC patients. Furthermore, because of the precise screening, the risk of misclassifying synchronous metastases, metachronous metastases, and recurrent CRCs as second primaries was eliminated, preventing the risk of second primary CRCs from being overestimated. Discussion Improvements in the quality of life and increase in the survival of cancer patients have led to several studies evaluating SPCAC (10, 18–20). In comparison to general populations, small intestine and endometrial cancers are most common in individuals with previous CRC, according to this comprehensive assessment of 30 research. The patient's genetic background, lifestyle, cancer-related treatments, and environmental risk factors may all contribute to the higher risk(21, 22). According to a recently published study, the incidence of colon, breast, prostate, kidney, and bladder cancer will surge in CCSs (22). This contradicts our findings, which showed that SIR of the small intestine, endometrial, colon, and ovary cancer were all higher in CCSs. We pooled data from all across the world, so the discrepancy could be related to different study designs. CCSs had a threefold increased risk of small intestinal cancer, according to present results (SIR: 3.68). Previous studies have found a link between primary gastrointestinal cancers and subsequent cancers. (23, 24). In prior SEER, an increased risk of certain GI cancers after CRC was observed among CRC Page 4/13 Page 4/13 patients, although subsites were not investigated (23, 25). In agreement with our results, Yang et al. reported a significantly increased risk for small intestine cancer among CCSs (SIR: 3.09), particularly in those patients who have had prior right-sided colon cancer (26). Broman et al. also confirmed a remarkable increase in the frequency of small intestine cancer in patients with a history of CCSs and significantly greater in right-sided CRC (18). Researchers demonstrated that a reverse relation exists between age at the time of diagnosis of primary CRC and SPC risk, so patients 40 years or younger have a SIR equal to 5 in comparison with patients 50 years with a SIR of 2.6 (27). Given these findings, strict surveillance among CRC survivors specifically at young ages should be established for early detection of SPCs with specific attention to the small intestine cancer (26) patients, although subsites were not investigated (23, 25). In agreement with our results, Yang et al. reported a significantly increased risk for small intestine cancer among CCSs (SIR: 3.09), particularly in those patients who have had prior right-sided colon cancer (26). Broman et al. also confirmed a remarkable increase in the frequency of small intestine cancer in patients with a history of CCSs and significantly greater in right-sided CRC (18). Competing interests The authors declare that they have no competing interests. There were no funding sources. Page 5/13 Page 5/13 Page 5/13 Authors' contributions Searching the database, data extraction, and preparing the draft of manuscript were done by M.Z., P.M., F.S., and M.Y. Statistical analysis was executed by K.H. Moreover, A.N. and P.M. were responsible for critical appraisal and reviewing of the manuscript. S.G. and R, A. edited the final manuscript. All authors read and approved the final manuscript. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Tables Table 1. Characteristics of studies entered into the meta-analysis Page 6/13 Page 6/13 of n Country Type of study location of primary tumor Total sample size No. of Second primary malignancy cases Q/A Score USA Retrospective cohort study colorectal 44,106 50679 8 Switzerland Retrospective cohort study colorectal 9389 136 8 Europe, North America, Asia and Oceania Retrospective cohort study colorectal 10946 1316 8 France Retrospective cohort study colorectal 4944 N/A 8 USA Retrospective cohort study colorectal 273144 657 7 Korea Retrospective cohort study colorectal 251482 498 9 Japan Prospective cohort study colorectal 217307 5071 9 UK Prospective cohort study colorectal 4297 N/A 7 Taiwan Retrospective cohort study colorectal 98876 4259 9 Taiwan Retrospective cohort study colorectal 65648 3810 9 Australia Retrospective cohort study colorectal 15755 1615 9 Sweden Retrospective cohort study colorectal N/A 224 7 USA Retrospective cohort study colorectal 1322 N/A 8 China Retrospective cohort study colorectal 233890 28863 9 Japan Retrospective cohort study colorectal 301 263 7 USA Retrospective cohort study colorectal 281413 12064 7 USA Retrospective cohort study colorectal 3278 N/A 9 UK Retrospective cohort study colorectal 5805 537 7 Netherlands Retrospective cohort study colorectal 123347 1849 8 USA Retrospective cohort study colorectal 170159 14880 9 China Prospective cohort study colorectal 252404 4977 8 China Retrospective cohort study colorectal 189890 N/A 8 Australia Retrospective cohort study colorectal 29471 501 7 Australia Retrospective cohort study colon 160000* 877 7 USA Retrospective cohort study colon 2038074 26 7 Sweden Retrospective cohort study colon 43021 4084 7 Italy Prospective cohort study colon 3244 98 8 USA Retrospective cohort study rectum 2665 N/A 8 Netherlands Retrospective cohort study rectum 29027 4398 9 USA Retrospective cohort study rectum 7661 747 8 t b f ti i t Author Year of publication Country Type of study location of primary tumor Total sample size No. of Second prima malignancy cases X. He(35) 2018 USA Retrospective cohort study colorectal 44,106 50679 F. Levi(36) 2012 Switzerland Retrospective cohort study colorectal 9389 136 G. Scelo(37) 2005 Europe, North America, Asia and Oceania Retrospective cohort study colorectal 10946 1316 C. Cluze(38) 2009 France Retrospective cohort study colorectal 4944 N/A E. Rahimi(39) 2016 USA Retrospective cohort study colorectal 273144 657 H. Ahn(40) 2019 Korea Retrospective cohort study colorectal 251482 498 H. Tsukuma(41) 1994 Japan Prospective cohort study colorectal 217307 5071 S. Wendy(42) 1992 UK Prospective cohort study colorectal 4297 N/A Y. Table 2. Summarized Pooled values of considered findings Tables Lee(27) 2015 Taiwan Retrospective cohort study colorectal 98876 4259 Y.H Liang(43) 2015 Taiwan Retrospective cohort study colorectal 65648 3810 P. Dasgupta(44) 2012 Australia Retrospective cohort study colorectal 15755 1615 B. Malmer(45) 2000 Sweden Retrospective cohort study colorectal N/A 224 O. Abdel- Rahman(46) 2017 USA Retrospective cohort study colorectal 1322 N/A J. Yang(26) 2017 China Retrospective cohort study colorectal 233890 28863 S. Noura(14) 2009 Japan Retrospective cohort study colorectal 301 263 K. K. Broman(18) 2019 USA Retrospective cohort study colorectal 281413 12064 R. J.Green(47) 2002 USA Retrospective cohort study colorectal 3278 N/A C. J. Bright(19) 2019 UK Retrospective cohort study colorectal 5805 537 L. Liu(48) 2013 Netherlands Retrospective cohort study colorectal 123347 1849 A. Phipps(49) 2013 USA Retrospective cohort study colorectal 170159 14880 L. Yang(20) 2018 China Prospective cohort study colorectal 252404 4977 X. Guan(25) 2015 China Retrospective cohort study colorectal 189890 N/A C.L Ringland(50) 2009 Australia Retrospective cohort study colorectal 29471 501 A. Heard(51) 2004 Australia Retrospective cohort study colon 160000* 877 A. Kustenko(52) 2014 USA Retrospective cohort study colon 2038074 26 C. Dong(53) 2001 Sweden Retrospective cohort study colon 43021 4084 E. Buiatti(54) 1997 Italy Prospective cohort study colon 3244 98 C. S. Rabkin(55) 1992 USA Retrospective cohort study rectum 2665 N/A A. Rombouts(56) 2017 Netherlands Retrospective cohort study rectum 29027 4398 B. K. Shah(57) 2015 USA Retrospective cohort study rectum 7661 747 The article haven’t reported the exact number of participants Author Year of publication Country X. He(35) 2018 USA F. Levi(36) 2012 Switzerland G. Scelo(37) 2005 Europe, North America, Asia and Oceania C. Cluze(38) 2009 France E. Rahimi(39) 2016 USA H. Ahn(40) 2019 Korea H. Tsukuma(41) 1994 Japan S. Wendy(42) 1992 UK Y. Lee(27) 2015 Taiwan Y.H Liang(43) 2015 Taiwan P. Dasgupta(44) 2012 Australia B. Malmer(45) 2000 Sweden O. Abdel- Rahman(46) 2017 USA J. Yang(26) 2017 China S. Noura(14) 2009 Japan K. K. Broman(18) 2019 USA R. J.Green(47) 2002 USA C. J. Bright(19) 2019 UK L. Liu(48) 2013 Netherlands A. Phipps(49) 2013 USA L. Yang(20) 2018 China X. Guan(25) 2015 China C.L Ringland(50) 2009 Australia A. Heard(51) 2004 Australia A. Kustenko(52) 2014 USA C. Dong(53) 2001 Sweden E. Buiatti(54) 1997 Italy C. S. Rabkin(55) 1992 USA A. Rombouts(56) 2017 Netherlands B. K. Shah(57) 2015 USA The article haven’t reported the exact number of participan Table 2. able 3. Quality assessment of included studies using the Newcastle-Ottawa scale Tables Summarized Pooled values of considered findings Page 7/13 Page 7/13 Page 7/13 Variable Primary Colorectal Cancer Primary Colon Cancer Primary Rectum Cancer Secondary Cancer N of study I- squared ES (95%CI) N of study I- squared ES (95%CI) N of study I- squared ES (95%CI) Digestive System 5 92.3 1.31 (0.89-1.72) - - - 2 98 1.43 (0.27- 2.59) Colon 4 79.8 1.59 (1.22-1.97) - - - 4 95.7 1.59 (0.87- 2.32) Esophagus 4 36.7 1.04 ( 0.95- 1.12) 2 70.5 1.02 (0.74- 1.30) 4 0 0.98 (0.91- 1.05) Liver and Gallbladder 4 74.3 0.82 (0.71-0.92) - - - 2 0 0.78 (0.68- 0.88) Pancreas 6 75.5 1.07 (0.95-1.18) 3 0 1.06 (0.98- 1.14) 6 28 0.86 (0.77- 0.95) Rectum 4 87.5 1.29 (0.88-1.70) - - - 3 73 1.78 (1.07- 2.49) Small intestine 4 88.4 3.68 (2.63-4.73) 3 0 2.33 (2.14- 2.52) Stomach 7 59.5 1.16 (1.06-1.25) 3 4.1 1.18 (1.09- 1.27) 5 0 0.96 (0.90- 1.02) Reproductive system 4 86.3 1.45 (0.99-1.90) - - - - - - Endometrium 4 92.6 3.03 (1.57-4.48) - - - 4 85.9 0.98 (0.36- 1.60) Ovary 3 92 1.51 (0.54-2.48) 2 95.7 1.74 (-0.28- 3.77) 5 76.4 0.82 (0.33- 1.32) Urinary system 5 76.5 1.27 (1.10-1.45) - - - 2 0 1.23 (1.12- 1.35) Bladder 5 68 1.20 (1.07 – 1.33) 3 0 1.04 (0.98- 1.10) 5 62.7 1.20 (0.96- 1.44) Kidney 6 83 1.34 (1.17-1.51) 3 82.1 1.46 (1.07- 1.86) 5 46.2 1.28 (0.85- 1.34) Prostate 3 0 1.17 (1.09-1.26) 2 89.5 1.15 (0.91- 1.40) - - - CNS* 3 91.8 1.24 (0.76-1.72) - - - 2 0 0.41 (-0.21- 1.02) Cervical 2 0 1.04 (0.89-1.20) 2 0 1.09 (0.89- 1.28) 4 21.5 1.00 (0.65- 1.35) Female Breast 4 76.5 1.07 (0.91-1.23) 3 76.9 1.13 (0.93- 1.32) 4 60.4 1.03 (0.80- 1.25) Hematological 3 86.5 1.12 (0.88-1.36) - - - 2 0 1.00 (0.8-1.20) Lung 6 72.6 1.16 (1.09-1.23) - - - 5 93.5 1.46 (1.15- 1.77) Lymphoma 2 0 1.38 (0.93-1.84) - - - - - - Skin 2 0 1.38 (1.18-1.58) - - - 2 66.3 1.19 (0.66- 1.72) Thyroid 2 46.8 1.50 (1.22-1.77) 2 67.1 1.58 (1.07- 2.09) 3 0 1.41 (1.16- 1.65) Table 3. Quality assessment of included studies using the Newcastle-Ottawa scale Table 3. Tables Quality assessment of included studies using the Newcastle-Ottawa scale Page 8/13 Author selection Author selection Comparability exposure/outcome Representativeness of the exposed cohort Selection of the non- exposed cohort Ascertainment of exposure Demonstration that outcome of interest was not present at start of study Comparability Assessment of the outcome Was follow-up long enough for outcomes to occur Adequacy of follow- up of cohorts Total X. He(35) * * * * ** * * 8 F. Levi(36) * * * * * * * * 8 G. Scelo(37) * * * * * * * * 8 C. Cluze(38) * * * * * * * * 8 E. Rahimi(39) * * * * * * * 7 H. Ahn(40) * * * * ** * * * 9 H. Tsukuma(41) * * * * ** * * * 9 S. Wendy(42) * * * * * * * 7 Y. Lee(27) * * * * ** * * * 9 Y.H Liang(43) * * * * ** * * * 9 P. Dasgupta(44) * * * * ** * * * 9 B. Malmer(45) * * * * * * * 7 O. Abdel- Rahman(46) * * * * * * * * 8 J. Yang(26) * * * * ** * * * 9 S. Noura(14) * * * * * * * 7 K. K. Broman(18) * * * * * * * 7 R. J.Green(47) * * * * ** * * * 9 C. J. Bright(19) * * * * * * * 7 L. Liu(48) * * * * * * * * 8 A. Phipps(49) * * * * ** * * * 9 L. Yang(20) * * * * * * * * 8 X. Guan(25) * * * * * * * * 8 C.L Ringland(50) * * * * * * * 7 A. Heard(51) * * * * * * * 7 A. Kustenko(52) * * * * * * * 7 E. Buiatti * * * * * * * * 8 C. S. Rabkin(55) * * * * * * * * 8 A. Rombouts(56) * * * * ** * * * 9 B. K. Shah(57) * * * * * * * * 8 References Clinical characteristics of metachronous colorectal tumors. Int J 10. Shin DW, Choi YJ, Kim HS, Han KD, Yoon H, Park YS, et al. Secondary Breast, Ovarian, and Uterine Cancers After Colorectal Cancer: A Nationwide Population-Based Cohort Study in Korea. Dis Colon Rectum. 2018;61(11):1250–7. 11. Rex DK, Kahi CJ, Levin B, Smith RA, Bond JH, Brooks D, et al. 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Rabkin CS, Biggar RJ, Melbye M, Curtis RE. Second primary cancers following anal and cervical carcinoma: evidence of shared etiologic factors. Am J Epidemiol. 1992;136(1):54–8. 56. Rombouts A, Hugen N, Elferink M, Feuth T, Poortmans P, Nagtegaal I, et al. Incidence of second tumors after treatment with or without radiation for rectal cancer. Ann Oncol. 2017;28(3):535–40. 56. Rombouts A, Hugen N, Elferink M, Feuth T, Poortmans P, Nagtegaal I, et al. Incidence of second tumors after treatment with or without radiation for rectal cancer. Ann Oncol. 2017;28(3):535–40. 56. Rombouts A, Hugen N, Elferink M, Feuth T, Poortmans P, Nagtegaal I, et al. Incidence of second tumors after treatment with or without radiation for rectal cancer. Ann Oncol. 2017;28(3):535–40. 57. Shah BK, Budhathoki N. Figure 2 Meta-analysis of incidence of second primary cancer after colorectal cancer (SPCAC) and categorized based on primary site of cancer. Meta-analysis of incidence of second primary cancer after colorectal cancer (SPCAC) and categorized based on primary site of cancer. Page 11/13 Second primary malignancy in anal carcinoma–a US population-based study. Anticancer Res. 2015;35(7):4131–4. 57. Shah BK, Budhathoki N. Second primary malignancy in anal carcinoma–a US population-based study. Anticancer Res. 2015;35(7):4131–4. Figures Figure 1 PRISMA flowchart for study selection process Figure 1 Figure 1 PRISMA flowchart for study selection process PRISMA flowchart for study selection process Page 12/13 PRISMA2020checklist2.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. PRISMA2020checklist2.docx Page 13/13
https://openalex.org/W1977948571	https://bmcmusculoskeletdisord.biomedcentral.com/counter/pdf/10.1186/1471-2474-13-118	English	null	Effects of home-based resistance training and neuromuscular electrical stimulation in knee osteoarthritis: a randomized controlled trial	BMC musculoskeletal disorders	2,012	cc-by	8,110	Abstract Background: Quadriceps femoris muscle (QFM) weakness is a feature of knee osteoarthritis (OA) and exercise programs that strengthen this muscle group can improve function, disability and pain. Traditional supervised resistance exercise is however resource intensive and dependent on good adherence which can be challenging to achieve in patients with significant knee OA. Because of the limitations of traditional exercise programs, interest has been shown in the use of neuromuscular electrical stimulation (NMES) to strengthen the QFM. We conducted a single-blind, prospective randomized controlled study to compare the effects of home-based resistance training (RT) and NMES on patients with moderate to severe knee OA. Methods: 41 patients aged 55 to 75 years were randomised to 6 week programs of RT, NMES or a control group receiving standard care. The primary outcome was functional capacity measured using a walk test, stair climb test and chair rise test. Additional outcomes were self-reported disability, quadriceps strength and cross-sectional area. Outcomes were assessed pre- and post-intervention and at 6 weeks post-intervention (weeks 1, 8 and 14 respectively). Results: There were similar, significant improvements in functional capacity for the RT and NMES groups at week 8 compared to week 1 (p≤0.001) and compared to the control group (p < 0.005), and the improvements were maintained at week 14 (p≤0.001). Cross sectional area of the QFM increased in both training groups (NMES: +5.4%; RT: +4.3%; p = 0.404). Adherence was 91% and 83% in the NMES and RT groups respectively (p = 0.324). Conclusions: Home-based NMES is an acceptable alternative to exercise therapy in the management of knee OA, producing similar improvements in functional capacity. Trial registration: Current Controlled Trials ISRCTN85231954 Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 Open Access © 2012 Bruce-Brand et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Effects of home-based resistance training and neuromuscular electrical stimulation in knee osteoarthritis: a randomized controlled trial Robert A Bruce-Brand1, Raymond J Walls1, Joshua C Ong1, Barry S Emerson2, John M O’Byrne1 and Niall M Moyna2 obert A Bruce-Brand1, Raymond J Walls1, Joshua C Ong1, Barry S Emerson2, John M O’Byrne1 d Niall M Moyna2 * Correspondence: robbrucebrand@gmail.com 1Cappagh National Orthopaedic Hospital, Finglas, Dublin 11, Ireland Full list of author information is available at the end of the article Participants The knee press exercise required the subject to lie su- pine on a bed or floor, actively dorsiflex the ankle and toes and contract the QFM of the exercising limb while pushing the knee posteriorly into the bed or floor for at least 5 s. The bottle knee press was performed in an identical manner with the addition of a water-filled 2 L plastic bottle placed under the exercising knee, while the contralateral knee was kept extended. The bottle pro- vided resistance to knee extension. For the extended leg raise, the subject lay supine with the resting limb flexed at the knee and the resting foot flat on the floor or bed. Keeping the knee of the exercising limb extended and the ankle and toes dorsiflexed, this limb was lifted 20 cm off the bed or floor and held for at least 5 s before slowly lowering it to the surface. The leg extension exercise required the subject to sit back in a chair holding onto the sides of the seat for support, fully extend the knee while keeping the ankle dorsiflexed, and hold this pos- ition for at least 5 s. For the wall squat exercise the sub- ject stood about 60 cm in front of a wall and leaned against it, with feet pelvis-width apart and slightly exter- nally rotated, then flexed the knees and slowly slid down the wall until significant contraction was felt in the QFM, before sliding back up the wall to the starting pos- ition. The hamstring curl required the subject to stand with the hands on a table or back of chair for support, and flex one knee so that the heel approximated the but- tock as far as possible. Eligible participants were patients of Cappagh National Orthopaedic Hospital, resident in the Greater Dublin area, aged 55–75 years, with symptomatic, moderate to severe knee OA. Participants were recruited from the arthroscopy database and knee arthroplasty waiting list. Patients were eligible to participate if they had been diagnosed arthroscopically with grade 3 or 4 OA on the Outerbridge scale within the last 2 years, or were placed within the last 6 months on the waiting list for knee re- placement surgery with the indication of OA, confirmed radiographically with Kellgren-Lawrence severity grades of 3 or 4. Trial design This was a single-centre, single-blind, prospective rando- mised control trial. Resistance training group Th RT d k The RT group undertook 3 home-based training sessions per week for 6 weeks. Each session was approximately 30 min in duration and was separated by a minimum of 36 h. Two of the three weekly sessions were supervised by an exercise specialist to ensure that each exercise was performed using the correct technique. Both lower limbs were trained using the following exercises in this order: knee presses, bottle knee presses, extended leg raises, leg extensions, wall squats and hamstring curls. The exer- cise regimen comprised 3 sets of 10 repetitions for each of the 6 exercises. Each set was performed bilaterally, starting with the less affected limb (except for the wall squats which exercised both limbs simultaneously). The logbook supplied to RT participants contained detailed instructions on the prescribed exercises. Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 Page 2 of 10 Page 2 of 10 and in the prehabilitation and rehabilitation of knee arthroplasty patients [8-11]. Interventions Subjects were randomised to a 6-week home-based resistance-training (RT) exercise program, a 6-week home NMES program (NMES) or a control group (C) receiving standard care. This pilot study attempts to address some of the gaps in our understanding of the role of exercise in the man- agement of knee OA by comparing NMES, resistance training (RT) and standard care in subjects with moder- ate to severe knee OA. To our knowledge no prior studies have compared a home-based NMES program to a home-based exercise program for subjects with knee OA. We examined the outcomes of function, self- reported disability, compliance, QFM strength and cross-sectional area (CSA). Our hypothesis was that a home-based NMES program would provide similar ben- efits to a home-based exercise program in these out- come measures. Background labour-intensive, time-consuming and often logistically difficult for patients. The effectiveness of such programs is dependent on good adherence and this can be challen- ging to achieve and maintain for patients with significant knee OA. Furthermore, in severe knee OA, deficits in volitional muscle activation contribute more to quadri- ceps weakness than muscle atrophy, and this may limit the effectiveness of volitional training programs [7]. Knee osteoarthritis (OA) is a leading cause of chronic disability in people over the age of 50 [1]. Regular exer- cise is associated with significant improvements in pain and disability in patients with symptomatic knee OA, and international guidelines recommend exercise in the first-line management of this condition [2-5]. Quadriceps femoris muscle (QFM) weakness is a fea- ture of knee OA and many exercise programs place par- ticular emphasis on strengthening this muscle group [6]. Traditional supervised resistance exercise is, however, Because of the limitations of traditional exercise pro- grams, interest has been shown in the use of neuromus- cular electrical stimulation (NMES) to strengthen the QFM. NMES is the application of transcutaneous elec- trical current to elicit involuntary muscle contractions. A few small studies have demonstrated benefits of NMES in the conservative management of knee OA, * Correspondence: robbrucebrand@gmail.com 1Cappagh National Orthopaedic Hospital, Finglas, Dublin 11, Ireland Full list of author information is available at the end of the article Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 NMES group h S The NMES group undertook a single 20 min unsuper- vised NMES session of the affected QFM, 5 days per week for 6 weeks. Bilateral NMES training was under- taken if the subject suffered from significant bilateral knee OA (such that the less affected knee contributed significantly to functional disability). Subjects were instructed to train at the same time of the day to ensure adequate muscle recovery. Each stimulation cycle com- prised a 10 s contraction period and a 50 s relaxation period, excluding the 1 s ramp-up and 0.5 s ramp-down. This provided a total contraction time of 3 min 20 s in each 20 min session. Functional capacity, self-reported disability, peak iso- metric and isokinetic quadriceps torque were assessed at baseline/week 0 (familiarisation), week 1 (pre- intervention), week 8 (post-intervention) and week 14 (6 weeks post-intervention). Quadriceps CSA was assessed in the intervention groups at week 1 and week 8. Familiarisation testing, conducted in an identi- cal manner to subsequent dynamometry and functional testing, was used to reduce the effect of improved technique on the results and minimise bias when com- paring pre- and post-intervention results. A portable, battery-powered garment-based NMES stimulator was used (Kneehab W, Bio-Medical Research Ltd, Galway, Ireland). The device utilises two channels and a “multipath” system of stimulation to coordinate muscle contraction. The stimulator produces a symmet- rical bi-phasic square waveform, with a maximum root mean square output current of 18 mA and an output fre- quency of 50 Hz. Pulse width changes dynamically during the stimulation cycle between 100–400 μs. Four reusable adhesive hydrogel electrodes (Axelgaard, Fallbrook, CA), having surface areas of 194 cm2, 83 cm2, 74 cm2 and 66 cm2 respectively, are attached to the deep surface of the garment and conduct impulses to the vasti and rectus femoris muscles. The 25 m walk test, repeated chair stand test, and stair climb test were performed in this order. Each test was performed 3 times with a 30 s rest period between each trial. A 5 min rest period was provided between the dif- ferent functional tests. Subjects were instructed to per- form each test as quickly as possible, but in a controlled and safe manner. The fastest time achieved for each test was recorded. Standardised verbal instructions and en- couragement were given. The 25 m walk test was undertaken on a pre-marked 35 m indoor corridor. Outcomes The primary outcome measure was objective functional capacity, assessed using a 25 m walk test, a repeated chair rise test, and a stair climb test. Secondary outcome measures were Western Ontario McMaster Universities osteoarthritis index (WOMAC) physical function, pain and stiffness scores, Short Form Health Survey (SF-36) physical health and mental health scores, peak isometric and isokinetic quadriceps torque and quadriceps CSA. Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 Page 3 of 10 Page 3 of 10 around each ankle, leaving about 20 cm of band be- tween the ankles. around each ankle, leaving about 20 cm of band be- tween the ankles. Participants Exclusion criteria were medical co-morbidities pre- cluding participation in an exercise program, implanted electrical devices, neurological disorders, inflammatory arthritis, non-ambulatory status, significant cognitive im- pairment, participation in an exercise program within the last 6 months, involvement in a previous similar study, anticoagulant therapy, and recent or imminent surgery (within 3 months). Ethical approval was obtained from the Cappagh Na- tional Orthopaedic Hospital Research Ethics Committee, and the procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 2000. The trial was registered with Current Controlled Trials (ISRCTN85231954). All patients meeting the eligibility criteria were con- tacted, given verbal and written information on the trial, and invited to participate. Consenting patients under- went a clinical examination to confirm eligibility, and provided written informed consent. Resistive bands (Thera-BandsW) were used to provide resistance for the extended leg raises, leg extensions and hamstring curls by wrapping the band once Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 Control group Subjects in the control group received standard care. This included OA education, weight loss, pharmacologic therapy, and physical therapy. They were not discour- aged from maintaining their existing level of activity. Subjects kept a logbook and recorded their rating of perceived exertion (RPE) using the 15 point Borg scale, and pain scores using a 0–10 analogue scale for each session. Subjects were encouraged to increase the inten- sity of the exercise if they consistently obtained RPE scores below 14 with pain scores below 3. This was achievable by holding the muscle contraction for a longer period of time, or by using higher resistance bands. Subjects in all 3 groups were advised to maintain any pre-existing treatment of their OA such as pharmaco- logic therapy. Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 Page 4 of 10 Magnetic resonance imaging (MRI) was used to meas- ure quadriceps CSA. A Philips Gyroscan Intera 1.5 T scanner (Philips Medical Systems, The Netherlands) was used to scan both thighs. A slice thickness of 4 mm and slice gap of 0.4 mm was used with an echo time of 0.1 s and relaxation time of 3.0 s. Subjects were scanned in the supine position. A coronal scouting scan was used to establish the level of the mid-thigh using the greater tro- chanter and knee joint line as anatomical marks. Twelve T2-weighted axial images were produced, centred on the mid-thigh level. Each image had a field of view of 30 cm, and comprised a 256 x 256 pixel matrix. A single clin- ician, blinded to group assignment, analysed the images and determined the CSA using EasyVision software (Phi- lips Medical Systems). Mid-sequence images from each subject’s baseline and post-intervention scans were com- pared, and anatomical markers used to ensure compari- sons were made at identical anatomical levels. The software was used to manually delineate the QFM on these matched central axial images and to compute the enclosed CSA (in mm2). stand test with arms folded across their chest while others used the armrests for all assessments. stand test with arms folded across their chest while others used the armrests for all assessments. The timed stair climb test was performed on an indoor stairwell with 11 steps. Subjects were required to ascend the stairs, turn on the landing, and descend as quickly as possible, without skipping any steps. The handrail could be used if required. Isometric and isokinetic quadriceps peak torque were measured using a Biodex dynamometer (Biodex Multi- Joint System 3, Biodex Medical Systems, Inc. Shirley, New York, USA). Subjects were seated in the dynamom- eter in an upright position with 90° of hip flexion. Both limbs were tested, beginning with the unaffected or least affected limb. For familiarisation and warm-up purposes, subjects were asked to perform a set of 3 progressive sub-maximal isometric contractions and a transient maximal contraction, attempting to extend the knee against the immobile force pad. The subsequent isomet- ric test comprised 3 consecutive maximum volitional iso- metric contractions (MVIC) of the quadriceps muscles at 60° of knee flexion. Each contraction was of 5 s duration, with a rest period of 50 s between each. Randomisation P ti t Patients were recruited by the first investigator (RBB). Group assignment took place when all eligible, consent- ing subjects had passed a clinical examination. This was undertaken by an investigator with no clinical involve- ment in the trial. A stratified randomisation technique was employed to ensure that gender and age distribution was similar in each group. Each subject was matched with 2 partici- pants of the same gender and similar age. Each group of three was then randomly assigned to one of the experi- mental conditions using computer-generated random numbers. Isokinetic peak torque was then assessed for each limb. The dynamometer was first calibrated with the range of motion of the knee under test. Subjects were then required to perform 5 contiguous maximal concen- tric isokinetic repetitions (extension and flexion) at 60°/s. The peak torque value for extension was recorded. The process was repeated on the (more) involved limb after a 3 min rest period. The WOMAC index is an OA-specific health survey, comprising 24 questions scored from 0 to 4 (best to worst respectively). The index is subcategorised into pain (scored 0–20), stiffness (0–8), and physical function (0–68). The SF-36 is a 36 item, generic, non-disease- specific health survey. Aggregate scores are given for physical and mental health, both scored from 0 to 100 with a higher score indicating better health. The reliabil- ity and validity of the WOMAC and SF-36 are well established [12,13]. NMES group h S A 5 m lead-in and 5 m end zone were included to minimise the effect of variable acceler- ation and deceleration. Timing began when the subject’s leading foot crossed the 5 m mark, and stopped when the trailing heel crossed the 30 m mark. If the subject used a mobility aid for normal ambulation, he/she was required to perform all the walk tests with the aid. Subjects were instructed to perform the NMES train- ing in the seated position with the knee flexed to 60 degrees, the foot flat on the floor, and the toes pressed against a wall to achieve isometric muscle contraction. They were encouraged to always increase the stimulation intensity to the maximally tolerated level. Comprehen- sive training on device usage was provided prior to the NMES program. It was explained to them that they would become more tolerant of the discomfort asso- ciated with the stimulation with repeated usage, and were expected to progressively increase their maximally tolerated intensity level during the course of the 6 week program. Subjects kept a logbook and recorded the date, duration, RPE and pain score for each session. For the repeated chair stand test, the subject was required to perform 3 consecutive cycles of standing with fully extended knees immediately followed by sit- ting with shoulders touching the backrest. A straight- back padded chair with armrests and adjustable leg height was used. The leg height was adjusted and recorded so that the subject’s knee was flexed at 90° in the seated position. Some subjects performed each chair Blinding Whereas patients and exercise specialists were aware of the allocated intervention, outcome assessors and data analysts were kept blinded to the allocation. Patients were repeatedly reminded not to disclose their interven- tion to outcome assessors. Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 The Biodex software (Revision 3.30) allowed automation of the tim- ing of this procedure. Standardised verbal instructions and encouragement were given. The maximum force generated over the 3 trials was recorded as the peak torque. The process was repeated on the (more) involved limb after a 3 min rest period. Functional tests h l Post hoc analysis was adjusted for multiple testing using a Bonferroni correction of p = .05/9 = 0.005). Results of all outcome measures are summarised in Table 2. Performance in the 25 m walk test, stair climb test and chair rise test improved (p≤0.001) in the RT and NMES groups at week 8 compared to week 1, and the improvements were maintained in both groups at week 14. There were no significant intergroup differ- ences in the functional tests between the RT and NMES groups at any time point, but both therapy groups showed significant improvements relative to the control group at weeks 8 and 14 (p < 0.005). Functional test scores did not change significantly in the control group at week 8 or 14 compared to week 1. Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 Page 5 of 10 Baseline characteristics are presented in Table 1. There were no significant differences between the 3 groups in any of the measured characteristics. violated the assumption of sphericity, the Greenhouse- Geisser correction was used. The level of significance was set at 5%, with Bonferonni adjustment made for multiple comparisons. Results are expressed as mean value ± standard deviation (SD). violated the assumption of sphericity, the Greenhouse- Geisser correction was used. The level of significance was set at 5%, with Bonferonni adjustment made for multiple comparisons. Results are expressed as mean value ± standard deviation (SD). Statistical methods Statistical analyses were performed using SPSS Version 17. Data was checked for normality using the Shapiro- Wilks test and by observation of Q-Q plots. Data from the familiarisation phase was excluded from analysis. Adherence was defined as the percentage of pre- scribed sessions completed, and was calculated for all subjects that commenced RT or NMES training. Calcu- lations were based on the data recorded in the log- books. The reliability of logbook recording was examined for the NMES group by comparing the device’s internal log to the patient logbook at the end of the 6-week intervention. A one-way analysis of variance (ANOVA) was used to evaluate potential group differences in baseline charac- teristics (age, gender, height, body mass index and func- tional capacity). A mixed-design repeated measures ANOVA was used to test for main effects of group as- signment over time (weeks 1, 8 and 14). Where data Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 Results Figure 1 illustrates the participant flow. A total of 164 participants were assessed for eligibility. 98 did not meet the inclusion criteria (38 were medically unfit, 22 were due for surgery within 3 months, 16 had had surgery within the last 3 months, 5 were on oral anticoagulants, 4 had inflammatory arthritis, 4 were uncontactable, 3 were away for a substantial part of the study, 2 had neurological disease, 2 were currently involved in an ex- ercise program and 2 were in a recent similar study). Overall, 41 of 66 eligible participants underwent strati- fied randomisation, 32 of 41 (78%) completed post- intervention testing and 26 (63%) were available for assessment at week 14. Assessed for eligibility(n=164) Excluded(n=123) • Not meeting inclusion criteria (n=98) • Declined to participate (n=25) Dropouts (n=3) 2 unwell 1 work commitments Allocated to NMES (n=14) Stratified Randomization(n=41) Allocated to Control(n=13) Allocatedto RT(n=14) Dropouts (n=2) 1 unwell 1 spouse hospitalised Dropouts(n=4) 3 unwell 1 personal reasons Post-intervention follow-up(n=11) Dropouts (n=1) 1 underwent TKR Dropouts (n=3) 2 underwent TKR 1 away abroad Post-intervention follow-up (n=12) Post-intervention follow-up (n=9) Dropouts (n=2) 2 underwent TKR Enrolment Allocation Follow up week8 Follow up week14 6 wk post-intervention follow-up(n=10) 6 wk post intervention follow-up (n=10) 6 wk post-intervention follow-up(n=6) - - Figure 1 Participant flow. Assessed for eligibility(n=164) Allocated to Control(n=13) Dropouts(n=4) 3 unwell 1 personal reasons Follow up week8 - Post-intervention follow-up (n=9) Post-intervention follow-up (n=12) Follow up week14 - 6 wk post intervention follow-up (n=10) Figure 1 Participant flow. Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 Page 6 of 10 Table 1 Baseline characteristics Experimental condition NMES (n = 10) RT (n = 10) Control (n = 6) Female: Male 4:6 4:6 3:3 Age (years) 63.9 ± 5.8 63.4 ± 5.9 65.2 ± 3.1 Height (m) 1.59 ± 0.10 1.60 ± 0.07 1.62 ± 0.15 Body mass index (kg/m2) 33.7 ± 5.6 33.9 ± 8.3 31.7 ± 4.1 Walk test (s) 15.43 ± 3.61 16.81 ± 3.39 16.64 ± 3.64 Chair Rise Test (s) 7.37 ± 2.05 8.33 ± 1.45 8.16 ± 2.23 Stair Climb Test (s) 14.31 ± 5.11 15.12 ± 5.32 13.27 ± 5.36 Values are mean ± standard deviation. NMES, Neuromuscular Electrical Stimulation Group; RT, Resistance Training Group. Table 1 Baseline characteristics more traditional resistance training programs in subjects with knee OA. Self-reported disability There were no between-group differences in self- reported disability after therapy. The only significant intra-group changes were improvements in WOMAC pain score between week 1 and week 8 for the NMES group (p = 0.004); WOMAC physical function for the NMES group at week 14 relative to week 1 (p = 0.004), and an improved SF-36 physical health for the NMES group at week 8 relative to week 1 (p = 0.005). For our NMES protocol, we used a 50 Hz excitatory frequency and a 10 s on / 50 s off duty cycle, as these values were most commonly employed in the studies included in the systematic review by Bax et al. in 2005 [14]. We used a maximally tolerated stimulus intensity because it is easily implemented, and increases NMES dosage. Our NMES training frequency of 5 times per week was based on the benefits and high adherence achieved with this frequency for subjects with knee OA in the studies by Walls et al. and Durmus et al. [9,10]. Unlike the RT group, we did not supervise or maintain frequent contact with the NMES group, as excellent compliance has been demonstrated in unsupervised, home-based NMES quadriceps training in advanced OA [10]. Results Exercise dosage is a function of frequency, intensity and program duration and a range of these variables have been used in the reviewed literature. No optimal dosage has been established for either resistance training or NMES [4,14]. Our choice of training frequency and intensity for each modality was based on careful consid- eration of what would achieve significant improvements in the outcome measures while achieving high levels of adherence. Our RT frequency of 3 sessions per week was based on the large Fitness Arthritis and Seniors Trial (FAST) study, which achieved 70% compliance over a relatively lengthy trial lasting 18 months [15]. Frequent contact was maintained between exercise leaders and partici- pants during the home-based phase of the FAST study, and this may have played an important part in the good compliance achieved [16]. We replicated this strategy in our study. Our RT exercise program was designed in collaboration with a senior physical therapist, using exer- cises routinely prescribed for patients with knee OA. Adherence Adherence to the prescribed 6-week intervention was not significantly different between the training groups (NMES 91.3%, RT 83.3%, P = 0.324). Comparisons of NMES logbooks to the stimulator’s internal log at the end of the intervention period showed complete con- cordance. The mean RPE values were similar for the NMES group (15 ± 1.9) and the RT group (15 ± 1.5). Quadriceps cross-sectional area Quadriceps CSA was greater (p < 0.005) in the NMES and RT groups at week 8 than week 1 (+5.4% for NMES, +4.3% for RT). There was no difference in quadriceps CSA between the two training groups at week 8 (p = 0.404). We found that 6 weeks of NMES or RT exercise resulted in significant improvements in functional per- formance in patients with moderate to severe knee OA. The improvements in functional capacity were main- tained for an additional 6 weeks after the NMES or RT programs. These functional improvements were achieved despite the fact that increases in knee extensor strength and disability scores did not reach significance compared to the control group. We did find significant intra-group improvements in WOMAC pain and SF-36 physical health for the NMES group, and a trend towards signifi- cant intra-group improvements (p < 0.03) in SF-36 phys- ical and mental health for the RT group at the end of therapy. Quadriceps peak torque There were no between-group or intra-group differences in isokinetic or isometric quadriceps peak torque after therapy (weeks 8 and 14 relative to week 1). There was however a trend towards an increase in both isokinetic and isometric strength for both training groups at week 8 relative to week 1. P significant vs. week 1 (P < 0.0056). NMES, Neuromuscular Electrical Stimulation Group; RT, Resistance Training Group; WOMAC, Western Ontario McMaster Osteoarthritis SF-36, Short Form 36; CSA: Cross-sectional area. * P significant vs. week 1 (P < 0.0056). NMES, Neuromuscular Electrical Stimulation Group; RT, Resistance Training Group; WOMAC, Western Ontario McMaster Osteoarthritis index. SF 36 Short Form 36; CSA: Cross sectional area Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 analysed physical performance measures of 177 patients of mean age 63.7 +/- 10.7 years with hip and knee OA awaiting arthroplasty and showed an ICC for test-retest reliability of 0.91 (0.81,0.97) for a similar walk-test, and an ICC of 0.90 (0.79, 0.96) for a similar stair climb test [23]. Their timed up and go test, with an ICC of 0.75 (0.51,0.89) differed from our timed chair rise test in that they incorporated a 3 m walk and turn element. Buchner et al. showed that the relationship between strength and function is nonlinear. Small changes in physiological capacity may have substantial effects on performance in frail adults, while large changes in strength can have little or no effect on daily function in healthy adults [19]. This phenomenon may partly ex- plain the disparate or nonlinear correlation between strength and function in the present and abovemen- tioned studies. We did find a trend towards an increase in QFM strength for both training groups at the end of the therapy period. A larger study may show such strength increases reach significance, even if they are not proportional to the functional benefits. The percentage increases in quadriceps CSA in the present study (5.4% for NMES, 4.3% for RT) is lower than the 6% increase reported by Gondin et al., follow- ing 8 weeks of NMES in healthy volunteers, and the 9.3% increase in CSA found by Frontera et al., after 12 weeks of RT training in healthy elderly men [24,25]. Unlike the current study, both these studies found asso- ciated gains in QFM torque. We find the increase in CSA in the training groups in the current study surprising and difficult to explain given the absence of significant change in QFM torque. It may be that our small study concealed a true asso- ciated strength gain that a larger sample size would have revealed. Alternatively, it may reflect a complex relation- ship between muscle size and strength in this cohort. NMES is capable of producing a range of neural adapta- tions within the central nervous system [20]. Decreases in voluntary muscle activation may negate increases in muscle size. Many studies have shown increased volun- tary muscle activation with NMES and RT programs, al- though most examined healthy subjects [24,26]. Zory et al. Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 Page 8 of 10 to our findings, they found significant increases in QFM strength and self-reported disability scores in both train- ing groups. All subjects in the NMES group in the current study were able to reach motor-threshold throughout the training program. Talbot et al. compared the effects of a 12-week NMES intervention program in patients with symptomatic knee OA, and found faster walking pace, quicker chair rise and decreased pain despite only modest (9%) improve- ments in leg extensor strength [8]. Although they used a longer intervention period, training was performed 3 days per week, and at lower intensities than the present study. There is evidence that self-reported measures of phys- ical function do not always correlate closely with object- ive measures of the ability to perform activities [21]. Patients’ perception of their ability to move around is influenced by pain and exertion. This study showed sign- icant improvements in functional performance for both training groups without consistent improvements in self-reported disability. This may be explained by the subjects’ perception of functional changes being clouded by inadequate gains in pain and overall health. The Fitness Arthritis and Seniors Trial (FAST) study of 439 subjects with knee OA found that resistance training resulted in significantly better scores on per- formance measures and physical disability compared to a health education group [15]. Although the training period was 18 months in duration, there was no differ- ence in QFM extension strength between the two ex- perimental groups. Similarly, van Baar et al. found a small to moderate improvement in pain but failed to find an increase in knee extension strength in patients with OA after a 12-week exercise program, and a recent study by Palmieri-Smith et al. found no increase in quadriceps strength following a 4 week NMES program for 30 women with radiographic evidence of mild or moderate OA [17,18]. The validity of various functional tests such as walk tests and stair climb tests have been established for sub- jects with knee OA, although there isn’t a consensus for parameters such as the distance walked or number of stairs used. A systematic review of performance-based measures for patients with hip and knee OA showed intraclass correlation coefficients (ICCs) ranging from 0.78 to 0.99 for various walking tests, and ICCs of 0.95 and 0.98 for the two up and go tests included [22]. Kennedy et al. Discussion To date, relatively few studies have examined the use of NMES in the management of knee OA and there is a notable dearth of studies comparing this modality to Similar improvements in functional tests were found in 50 women with knee OA in response to 4 weeks of NMES and biofeedback-assisted exercise [9]. In contrast Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 Table 2 Outcome measures Table 2 Outcome measures Table 2 Outcome measures NMES (n = 10) RT (n = 10) Control (n = 6) Week 1 Week 8 Week 14 Week 1 Week 8 Week 14 Week 1 Week 8 Week 14 Walk Test (s) 15.43±3.61 13.85±3.79* 13.08±4.08* 16.81±3.39 14.07±3.40* 14.50±3.71* 16.64±3.64 16.30±3.58 11.55±4.10 Chair Rise Test (s) 7.37±2.05 6.35±1.46* 5.79±1.20* 8.33±1.45 5.97±0.58* 6.18±1.05* 8.16±2.23 8.01±2.17 5.20±1.11 Stair Climb Test (s) 14.31±5.11 11.87±4.21* 12.48±6.44* 15.12±5.32 11.78±4.54* 11.84±5.03* 13.27±5.36 13.06±5.84 9.63±2.99 WOMAC Physical Function 41.04±11.60 33.88±12.66 31.50±12.63* 31.68±12.92 33.91±12.91 31.50±14.40 31.67±17.95 26.11±15.33 21.67±18.90 WOMAC Pain 11.50±3.50 8.88±3.29* 8.50±2.72 11.05±3.02 10.78±4.31 9.60±4.14 9.00±3.65 8.33±4.36 8.33±4.08 WOMAC Stiffness 5.17±2.17 3.92±2.15 4.10±2.18 5.23±0.68 4.73±1.74 4.45±2.24 5.22±1.72 4.11±1.83 4.00±2.37 SF-36 Physical Health 39.25±16.95 50.50±16.60* 47.60±10.73 39.73±16.51 50.00±23.12 53.20±25.09 51.78±24.34 56.00±24.74 67.83±21.71 SF-36 Mental Health 60.67±26.45 70.67±21.84 65.40±12.98 56.36±21.91 66.64±20.36 65.30±24.91 62.00±25.41 65.00±27.77 70.50±22.40 Isometric Peak Torque (Nm) 88.16±31.21 92.59±36.66 86.65±31.21 82.55±22.39 87.16±17.84 86.87±21.44 92.99±47.89 100.30±32.80 97.35±44.76 Isokinetic Peak Torque (Nm) 67.33±19.42 71.37±23.89 66.36±20.18 64.52±21.74 71.79±17.27 70.91±21.83 75.10±28.45 75.91±28.83 77.80±47.45 Quadriceps CSA (mm2) 4061±721 4279±784* 4335±610 4521±651* Values are mean ± standard deviation. * P significant vs. week 1 (P < 0.0056). NMES, Neuromuscular Electrical Stimulation Group; RT, Resistance Training Group; WOMAC, Western Ontario McMaster Osteoarthritis index. SF-36, Short Form 36; CSA: Cross-sectional area. Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 Page 9 of 10 Despite the lack of supervision, the NMES group showed a non-significantly higher adherence rate than the RT group. Given the resource-intensiveness and cost of traditional physical therapy, home-based NMES offers an attractive alternative. interplay of increased activation and impaired muscle contractility [27]. Deley et al. found decreases in volun- tary activation immediately after NMES and RT [28] in healthy subjects. Conroy et al. have shown that patients with knee OA have significantly reduced torque per unit of quadriceps area compared to controls, despite no difference in quadriceps strength or quadriceps size [29]. This reflects poorer muscle quality in knee OA. Increases in quadriceps size in this cohort may there- fore produce less than expected increases in torque. Further work using electromyography should clarify our surprising finding. Limitations of this study include the limited sample size, the short follow-up period, the lack of a placebo intervention for the control group and the absence of measurements of voluntary activation such as electro- myography or twitch interpolation. Author details 1C h N i 1Cappagh National Orthopaedic Hospital, Finglas, Dublin 11, Ireland. 2Centre for Preventive Medicine, Dublin City University, Dublin, Ireland. Received: 29 October 2011 Accepted: 1 May 2012 Published: 3 July 2012 Received: 29 October 2011 Accepted: 1 May 2012 Published: 3 July 2012 We concur that regular follow-up is essential to achieve benefits and good adherence with home-based therapy. The supervision of two-thirds of the home- based RT sessions no doubt contributed to good adher- ence in the present study. A feature of the NMES device is the capacity to keep an electronic log of usage. The log provides a means to monitor adherence to unsuper- vised home therapy, and probably played a role in incen- tivising patients to adhere to the training program. Acknowledgements h d This study was supported by a grant from the Cappagh Hospital Trust. The Kneehab W stimulators were provided by Bio-Medical Research Ltd, Galway, Ireland. Neither sponsor had any involvement in the design of the study, in the collection, analysis and interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication. Authors’ contributions RBB was the principle researcher involved in study conception and design, as well as implementation, analysis and interpretation of data, and manuscript preparation. RW made substantial contributions to the conception and design of the study and the drafting of the article. JCO made substantial contributions to the conception and design of the study and the revision of the article. BE made substantial contributions to the acquisition of data and the drafting of the article. JOB made substantial contributions to the conception and design of the study and the revision of the article. NM made substantial contributions to the conception and design of the study, the analysis and interpretation of the data and the revision of the article. All authors read and approved the final manuscript. Chamberlain et al. compared a home exercise based program to hospital-based physical therapy in patients with knee OA and found similar improvements in func- tion, pain and strength in response to 4 weeks of therapy [30]. Patients who were notified after the intervention of a further assessment at 12 weeks, continued their daily exercises, and maintained their improvements in pain, function and strength. In contrast, patients who were not notified of further testing were more likely to cease exercising and experience more pain. Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 found that QFM MVC was unchanged after 4 weeks of NMES in healthy men because of the A notable limitation of our study was not including measures of voluntary activation such as electromyog- raphy or twitch interpolation in order to distinguish be- tween neuromuscular recruitment factors versus intrinsic muscle tissue or cellular factors. It would also have been useful to measure the percentage of maximal vol- untary contraction (MVC) obtained with NMES during the course of the study. Talbot et al. found that subjects with symptomatic knee OA tolerate 30-40% MVC and the average training intensity in their study was 22% of MVC [8]. Qualitatively, patients in the current study reported an ability to increase the training intensities significantly after the first few days of training, with slower gains thereafter. This is in keeping with well- reported tolerance or accommodation to NMES [8,20]. Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 Competing interests h h d l h Competing interests h h d l h Competing interests The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. Conclusions An important limitation of NMES is the limited spatial recruitment of muscle fibres [20]. The novel garment stimulator used in this study utilises 2 channels and a “multipath” system of stimulation and is purported to achieve greater muscle recruitment. In the current study, we did not analyse the CSA of the component muscles of the QFM separately. It would be worthwhile in future work to do so in order to identify differences in muscle hypertrophy induced by the two training modalities. A six-week home-based, unsupervised NMES program significantly improves functional performance in patients with moderate to severe knee OA, and is an acceptable alternative to resistance training in this patient cohort. We found significant intra-group increases in quadriceps CSA for both training groups, but improvements in quadriceps strength and in self-reported disability scores did not reach significance relative to the control group. g g Dose–response studies with longer follow-up are required to establish the optimum frequency and inten- sity of NMES training. Adherence is an important predictor of clinical out- come in response to exercise training in patients with knee OA [5]. Factors that have been identified to im- prove adherence include educating patients of the bene- fits resulting from participation in an exercise program, simplifying exercise regimens, setting clear and attain- able goals, providing social interaction and providing regular follow-up. Although we found an 8% higher ad- herence rate in the NMES group than the RT group, the difference was not significant. Possible factors account- ing for the high adherence rate in the NMES group in- clude the relative simplicity of the NMES training routine, the novelty of the modality and participant knowledge that the device was logging usage data. 1Cappagh National Orthopaedic Hospital, Finglas, Dublin 11, Ireland. 2Centre for Preventive Medicine, Dublin City University, Dublin, Ireland. 1. Guccione AA, Felson DT, Anderson JJ, Anthony JM, Zhang Y, Wilson PW, Kelly-Hayes M, Wolf PA, Kreger BE, Kannel WB: The effects of specific medical conditions on the functional limitations of elders in the Framingham Study. Am J Public Health 1994, 84:351–358. 2. van Baar ME, Assendelft WJ, Dekker J, Oostendorp RA, Bijlsma JW: Effectiveness of exercise therapy in patients with osteoarthritis of the hip or knee: a systematic review of randomized clinical trials. 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Roddy E, Zhang W, Doherty M, Arden NK, Barlow J, Birrell F, Carr A, Chakravarty K, Dickson J, Hay E, et al: Evidence-based recommendations for the role of exercise in the management of osteoarthritis of the hip or knee–the MOVE consensus. Rheumatology (Oxford) 2005, 44:67–73. function. J Appl Physiol 1988, 64:1038–1044. 26. Gabriel DA, Kamen G, Frost G: Neural adaptations to resistive exercise: mechanisms and recommendations for training practices. Sports Med 2006, 36:133–149. 6. Slemenda C, Brandt KD, Heilman DK, Mazzuca S, Braunstein EM, Katz BP, Wolinsky FD: Quadriceps weakness and osteoarthritis of the knee. Ann Intern Med 1997, 127:97–104. 27. Zory RF, Jubeau MM, Maffiuletti NA: Contractile impairment after quadriceps strength training via electrical stimulation. J Strength Cond Res 2010, 24:458–464. 7. Petterson SC, Barrance P, Buchanan T, Binder-Macleod S, Snyder-Mackler L: Mechanisms underlying quadriceps weakness in knee osteoarthritis. Med Sci Sports Exerc 2008, 40:422–427. 28. Deley G, Millet GY, Borrani F, Lattier G, Brondel L: Effects of two types of fatigue on the VO(2) slow component. Int J Sports Med 2006, 27:475–482. 8. Talbot LA, Gaines JM, Ling SM, Metter EJ: A home-based protocol of electrical muscle stimulation for quadriceps muscle strength in older adults with osteoarthritis of the knee. J Rheumatol 2003, 30:1571–1578. 29. Conroy MB, Kwoh CK, Krishnan E, Nevitt MC, Boudreau R, Carbone LD, Chen H, Harris TB, Newman AB, Goodpaster BH: Muscle strength, mass, and quality in older men and women with knee osteoarthritis. Arthritis Care Res (Hoboken) 2012, 64:15–21. 9. Durmus D, Alayli G, Canturk F: Effects of quadriceps electrical stimulation program on clinical parameters in the patients with knee osteoarthritis. Clin Rheumatol 2007, 26:674–678. 30. Bruce-Brand et al. 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Am J Public Health 1994, 84:351–358. 2. van Baar ME, Assendelft WJ, Dekker J, Oostendorp RA, Bijlsma JW: Effectiveness of exercise therapy in patients with osteoarthritis of the hip or knee: a systematic review of randomized clinical trials. Arthritis Rheum 1999, 42:1361–1369. Page 10 of 10 Page 10 of 10 Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 Bruce-Brand et al. BMC Musculoskeletal Disorders 2012, 13:118 http://www.biomedcentral.com/1471-2474/13/118 Buchner DM, Larson EB, Wagner EH, Koepsell TD, de Lateur BJ: Evidence for a non-linear relationship between leg strength and gait speed. Age Ageing 1996, 25:386–391. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 20. 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https://openalex.org/W2957696988	https://sciencepubco.com/index.php/ijet/article/download/24475/12362	English	null	Adyghe (Circassian) Religiosity and its Reproduction in Contemporary Prose	International journal of engineering & technology	2,018	cc-by	9,043	1. Introduction In the spaces of the global (both strategic and financial) transformations of the current century, the place of the Caucasian territories on world maps is much more palpable. Prolonged mutual influence in the North Caucasus area by ethnic, tribal and religious factors in the chronicle process predetermined a specific phenomenon. Throughout the centuries, there was a gradual infusion of sacred jets in Highland literature. It has been occurred as a result of the long-lasting filling of reality by religion, and religiosity - the consciousness of the Highland people. Moreover, Russia, a priori, refers to that significant number of states in which secular ideology is virtually formal one. In fact, the impact of religious motives on minds and souls, as well as on the policy being built, is quite palpable. Despite the almost century-long duration of the strictly atheistic power that prevailed here, of which one of the Adyghe authors being analyzed today is Aitech Khagurov said "A little time will pass after the end of the war and these party-komsomol activists will start again a war with religion, threatening not cold water, but cold Siberia" [1]. Categorically denying any god, it never made the avoiding of religion from politics, in the country, which can be feasibly in the modern Western world. The concept of the mutual impact of ethnic and sacred components was formed and strengthened in the intellection of the representatives of nations for many centuries. In fact, in the process of obtaining knowledge, the intersection of the socio- cultural sector with religion can be a starting source. He is appointed to lead the individual to a sacred perception of the essence of the usual concepts. According to acclaimed Soviet-era scholar S.P. Capitsa, "Essentially, after all, science has grown out of religion" [2]. As stated Edward Spencer (British traveler of the XVI century), on this occasion "In addition to the one eternal God, they believe in the existence of several lower beings or saints, to whom the Great Spirit, Tha, has transferred power over such Sometimes this fact leads to the manifestation in the minds of ethnic representatives of chauvinistic components. Moreover, the complete religion denial, ignoring the rites handed down by the generations, is explained and called the withdrawal from the ethnos. In general, the traditional character of ethnic beliefs is a significant mechanism for combining the ancient and the current, which acts on many levels of society. Adyghe (Circassian) Religiosity and its Reproduction in Contemporary Prose S.A. Kirzhinova1, L.K. Bzegezheva, M.K. Bolokova, F.N. Huako 1Maikop State Technological University, Pervomayskaya Street, 191, Maikop, 385000, Republic of Adygeya, Russia *Corresponding author E-mail: kirzhinova.s.a@mail.ru S.A. Kirzhinova1, L.K. Bzegezheva, M.K. Bolokova, F.N. Huako Abstract Article considers Adyghe (Circassian) religiosity and its reproduction in contemporary prose. Conducted study and a variety of problems in this case are rare, besides, almost complete cancellation of any research activity in today's Russia is known. And the problems are not studied enough. Therefore, the relevance of the selected topic is undoubtedly important, and we are developing it using a number of analytical and synthetic methods inherent in humanitarian knowledge. In the course of research, we established that the fact that both religions common today (Christianity, Islam) are not at all sources of interethnic difficulties, and concluded that the state must strive to support the existing religious branches (in particular, Islam and Christianity). The relevance of this work is that the problems in this field are too little studied, and our research provides new data, as well as a new look at this topic. Keywords: Adyghe; Adyghe tribes; Allah; Belief; Caucasus; Ceremony; God; Islam; Religion; Religiosity; Ritual. earthly things, that he considers too insignificant for his fear- inspiring control… Some of them are a special symbol; but they do not worship them otherwise than as an intermediate object. On this score, I conducted a rigorous survey and found that all my informants agreed with this opinion and confirmed what I had heard before from several Russian officers, who for many years had been associated with the Circassians… This worship should be considered a kind of adoration of a strict Protestant and an equally strict Muslim" [3]. Available in the nation’s customs and rituals, the concepts and representations that are put on the confession are treated as those sent by the Most High and bearing the religious essence. In particular, Islam is the most prominent religion for Adyghes and in the North Caucasus area. The individual attribution to it is usually correlated with the corresponding self-identification, i.e. in most cases Adyghe will say: "I - a Muslim" and clarify "As prompted by the ancestors". However, some scholars do not agree directly to relate religious affiliation to ethnic identification. Thus, according to R.A. Lopatkin, "the fact of person's confessional self-identification does not always indicate his religiosity" [4]. And S.P. Kapitsa, who relates himself to Orthodox atheism, confirms this: "I can talk about God as a cultural phenomenon, but I believe that our misunderstanding of certain things does not mean the presence of God". International Journal of Engineering & Technology, 7 (4.38) (2018) 244-250 Copyright © 2018 Authors. This is an open access article distributed under the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. Introduction The work of a pre-revolutionary Russian scientist is distinguished by scientific functionality, which is analyzed by comparison approach. However, it only indirectly touches the religious attitudes and rituals of the ancient Circassians. Among the researchers of the second half of the XIX century and H1 XX century, a tangible value belongs to the works of the Kuban historian F.A. Shcherbin His works are versatile and already closer to the objective point of view, which brought science closer (after) to strict socialist realism and led away from pre-revolutionary fragmentation. In the Soviet era starts a detailed and systematic studying of the chronicle of events, ethnography, and archeology of the region. Interest for small nations increased under control of USSR Academy of Sciences. Substantial works are being published, such as “The Peoples of the Caucasus” [12, 13], "History of the North Caucasus Peoples" [14]. It is necessary to emphasize here a certain defect of the Dagestan scientist: the Kabardins, Circassians, Adyghes, Shapsugs, Abasins, which she classifies as Aboriginal peoples, belong to the same Adygeyan ethnos. Kabardinians, Shapsugs, Abazins are Adyghes tribes. «Adyghes» - only the Soviet name of the inhabitants of the Adyghe Autonomous Region, and the "Circassians" - the exonym of the Adyghe people. Thus, according to the results of this census, the ethnic groups which are professing Islam are not twenty, but fifteen. But, in any case, more than ten. According to the above mentioned data, national-faith elements are seen most prominently in the republican regions of the Caucasus, which can be explained by a concrete cause-and-effect line. The Russian national policy, which is in force today, extend the line of ongoing and expressed alienation of the so-called. "Non-Russian" nations and religions, given by the president in his statements about "chocks." Consequently, the Caucasian, or more precisely (especially) Adyghe ethnic features in the country are practically not covered, the people are simply attributed as "young written" and inconspicuous. Careful study and a variety of problems in this case are rare, besides, almost complete cancellation of any research activity in today's Russia is known. And the problems are not studied enough. Therefore, the relevance of the selected topic is undoubtedly important, and we are developing it using a number of analytical and synthetic methods inherent in humanitarian knowledge. 1. Introduction Faith, as well as religious thinking, is established phenomena in the cultures of peoples. An illustration of this can be a phrase from the text of one of the Adyghe authors Mukhaddin Kandur, analyzed by us: "When the burial ground was buried, the mullah began to sing in chanting prayers from the Koran and other ancient sacred books, which he 245 International Journal of Engineering & Technology faith in the One God – Thae. In the XVI century, the British traveler E. Spencer carefully studied the problem, and came to conclusion: "The main postulates in the inhabitants of the Western Caucasus faith are a firm belief in one God, supreme and powerful, and in the immortality of the soul, which according to their belief will be transferred to another world, the whereabouts of their fathers. Similar to the Mohammedans, they do not represent the Divine in any visible form, but define it as the creator of all things, whose spirit is scattered throughout the outer space" [3]. In the XVII century information from the other side start to pullulate, it concerned the prevalence of Islam in Circassia. As the Dominican missionary Giovanni Lucca says about the Adyghe religion, «Some of them are Mohammedans, others follow the Greek rite, but the first prevail». himself never read, but fragments from which traditionally kept in memory" [5]. However, thoughtful traditional nature is capable to interchange with pathos expressiveness in this subject. According to the modern Adyghe author Amir Makoyev, "It is impossible, even for a moment, to believe that our intellect, soul and conscience were indifferent to the Creator" [6]. And this phrase appears in the narrator pending of complains about the lack of their own vital productivity: "I live for exactly one thousand years. Time goes by, but I do not do anything significant. And if I take on something, I soon tie it up, there is no strength, and no desire to complete it, all interest disappears. It's like in a dream - in an unaccustomed, sticky mass, your legs and hands are stuck, and you wake up all in a sweat, tired of a senseless struggle for your ghostly life" [6]. These phenomena have spent tools and technologies of a number of denominations, which can be attributed to the ideologies of the most common groups in the world community. 1. Introduction According to a number of censuses, which are already conducted in the new century, the domestic religious attitude is characterized by an obvious feature - the preference of the Muslim peoples by the peoples of the Caucasus. According to M.Z. Magomedova, who citing data by the period of the census of 2006-2008, in the framework of the Program of Basic Research of the Presidium of the Russian Academy of Sciences, "The census singled out 20 indigenous Islamists of the Caucasus as separate ethnic groups (Kabardians, Circassians, Adyghes, Shapsugs, Abasins, Chechens, Ingushs, Avars, Laks, Dargins, Lezgins, Tabasarans, Aghul, Rutulians, Tsakhurs, Karachais, Balkars, Kumyks, Nogai, Hemshili)" [7]. Among prerevolutionary historians, Circassian publicist Sultan Khan-Giray raised the issues of religion, especially carefully. In the final years of his life, he, in addition to the well-known “Notes on Circassia”, published an essay "Faith, customs, customs, lifestyle of Circassians" [9]. In essence, it is overlapping with some chapters of the first and second parts of his work Notes on Circassia”. Sultan Khan-Giray begins to recreate in the essay Adyghes religions. Devoting an entire chapter of “Notes on Circassia” he established: “Every nation had and has more or less superstitions, and we will not spread about this subject, let us just say in conclusion that since the Mohammedan religion extension in Cherkessia, and the clergy superstition, although it increased the prejudices of the people, but gave them more philanthropic direction: no longer visible torture, or such cruel traces of a horrible delusion - orisons and talismans replaced them" [10]. The historian of Soviet times Shor Nogma wrote: “Adil Giray Atazhukin and effendi Ishak Abukov, being Mullah in his younger years, provided Sheriat between the Kabardian people, according to which criminals are all without exception (i.e. without distinction of social class), by the degree of importance of the crime, they were subjected to the death penalty and corporal punishment. The establishment of this provision has brought great benefits to the people; everyone was afraid of doing anything illegal" [11]. In a series of review works on Caucasian studies, published in the XIX century, it is necessary to put the work of the I.F. Blaramberg who is historian and archaeologist, he is considered by scientists as start scientists, who emphasized the excavations of the Black Sea coast. They were produced on the basis of artistic and historical keys, but in addition - personal impressions took place too. 1. Introduction However, we also take into account the indicators of literature studies that can be appreciably helpful when working with the artistic texts of modern Adyghes writers, known and accepted at home (not in Russia, but in the republics of the Kabardino-Balkaria, Karachay-Cherkessia, and Altai) today. Modern researchers continue to be interested in the religious preferences of the Caucasian spaces. Consideration on the ethnographic background of religion questions is given to the works of currently classical Russian ethnographer-historians as Arutunova S.A., Anchabadze U.D., Alieva A.I., Bromli U.V., Gardanov V.K., Kosven M.O., Krupnov E.I., Kalmykov I.H., Lavrov L.I., Sergeeva G.A., Smirnova Ya.S., Ter-Sarkisyants A.E., Tokarev S.A. et al. Tangible contribution to the ethnographic research in relation to Adyghe made works and monographs by Mambetov G.H. "Traditional culture of Kabardians and Balkarians" [15], Mafedzeva S.H. «Adyghes: Customs and Traditions» [16], Dumanov H.M. "Social structure of Kabardians 3. Materials and Methods Let us turn to the key insight of the current subject - the religious consciousness, its essence and manifestations in the Caucasus and, in particular, among the Adyghes. Having a specific geopolitical level, located on the inter-civilizational flank of Eurasia, the Caucasian territory invariably attracted the attention of neighboring countries and their cultures, thus tasting their tangible action (both political and economic). Interestingly that the ubiquitous signs of ethnic groups in the Caucasus, if one does not take into account their frequent differences, are the following: the ethnic resources that exist here, if not born, were strengthened and acted in similar circumstances. It was a multi-faceted and multicultural interaction of regional nations with readiness for coming calls. And here it should be noted the frequent external initiatives that have been hovering over these mountains. Far from a complete list of countries that have periodically tried to establish power over the mountaineers of the Caucasus for centuries, we can assume, according to Khatko Fathi, the following countries: "The Roman Empire, Persia, Iran, Arabia, Byzantium, Osmania, Seljuk Turks, Mongols, Russia" [8]. The readiness that we outlined above has determined the national-religious saturation of the region that exists today. According to Adygeyan writer A.L. Makoyev, Faith is such an intimate spiritual phenomenon that it does not tolerate the presence and influence of someone else's mind and heart. The faith of each of us is a reflection of our conscience in space; this is the ratio of our three seconds of being on earth with eternity” [6]. In real, in any local culture it is permissible to find echoes of spiritual priorities that have been accumulating for centuries and containing generally accepted ideas. In fact, Christianity and Islam prevail in the North Caucasus. But, as we already noted in the introduction, the Adyghes more often refer themselves to Muslims, although they respect the teachings of Christ. For example, the leading perspicuity of A.L. Makoyev, the narrator asks and then confidently replies: "Has the Non-unitary attempts at national reforms in the Russian Federation have formed all possible forms of rebuff, as well as the peculiar adaptive rut in the region. In general, circumstances that are recognized in science, forging religious (in particular, Muslim) consciousness at the national level, are the following. Firstly, these are interethnic ties within the confession. Secondly, it is the interaction of religious and atheistic attitudes, and mental characteristics. 2. Literature Review Among the historians of the H1 XIX century, L.Ya.Lyulie, in the process of studying and generalizing the Adyghes rituals, beliefs and rites available, formed a full list of the Adyghes gods and came to the conclusion: "Hospitality, mixed with religious concepts, love of country and old prejudices, is the specific feature of the mountaineers mores, which prevents the final establishment of Islamism” [8]. Nevertheless, at the time of the savor weakening in Christianity, the Adyghes continued to persistently persuade the International Journal of Engineering & Technology 246 world become more merciful for two millennia, following the teachings of Christ? No doubts" [6]. Islam was preceded to Adyghes in the X-XII centuries directly because of Christianity, which came from Byzantium and Georgia to the paganism that was then widespread among people, proved by folklore (including the works of M. Mizhaev, A. Shortanov, A. Gadagatly, N. Gish, etc.). Appeal to Allah, which was found at the Golden Horde, became an object of interest of Circassians already in the XIV century. Nevertheless, the universal adoption of Islam can be traced only in the XVII - XVIII centuries. In recent centuries, it is precisely makes a tangible impact on the behavioral and ideological canons of Caucasians, when it becomes quite difficult to distinguish between confessional and ethnic features. M.Z. Magomedov sees this tendency in Dagestan, Chechnya and Ingushetia as an advantage. Concerning the Circassians, one can assert that they belong to such a Muslim branch as Sunni Hanafi, and in the Adyghe Republics, the impact of Islam is markedly reduced. Before the XIX century Circassians equally comply dual faith. During the half-century of the Caucasian War, Islam was strengthened, which we consider to be the nation opposition to the coming invader who believes in Christ. In general, as can be seen, considering North Caucasus multinational space, inherent interaction as religious in general and Islamic in particular, simultaneously with the possible intersection with direct ethnicity. Today, in the North Caucasus region, there is an intense diversity of Islam as a political denomination that shows influence not only on the Russian south, but also on the existing state in general. The specificity of these branches in Islam is their relation to the canons of Islam, in the presence of specific and clearly executed ceremonies, faithful ministers and admirers. 3. Materials and Methods Although at the same time, due to the socio- political and financial-economic complexities existing in the global society, it turned out that in the new century some extremist sects could enter the religious sphere. 2. Literature Review The Muslim coefficient implies not only faith and the Islamic behavioral way of life, but also a kind of political Islam (i.e., so-called Islamism), which deepens its sources in the distant chronicle. Most often, such factors have tangible qualities that permit talking about heterogeneous groups. They, like traditional social formations, have different facets in the relation of thinking in heterogeneous conditions of being. in the norms of adat" [17], Bgazhnokova B.H. "Circassian play" [18], Shikova T.T. "About the conditions and forms of marriage for the Kabardians in the pre-revolutionary past (late XIX - early XX century)" [19], Musukaeva A.I. "On the customs and laws of the Highlanders" [20]. Already in the post-Soviet period, in the second decade of the new century, in works published in Maikop and Nalchik by Gostieva L.K. [21], Zafesov A.H. [22], Maremshalova I.I. [23], Kagazezhev B.S. [24], Lyausheva S.A. [25] et al., aspects of the ritual culture of the North Caucasian peoples are also considered. As opposite, in particular, a modern Arab researcher (by origin - Adyghe) Khatko Fathi, which publishes already in the new century (2003) the book "Circassians and religion" motivates his scientific interest quite justifiably. As foundation he provided the as powerful stream of religiosity, in the Caucasus, in particular in Adygea: "I wanted to share my fears in connection with a new wave of Islamization in my historical homeland, because I feel my responsibility for the destinies of the youth" [8]. However, a number of modern researchers oppose this position. Thus, the author of the article "On the Notion of Mass Religious Consciousness" Z.M. Abdulagatov ascertains the opposite act of the modernity avoiding from religion: «As history shows, the general course of public consciousness development, going in the direction of a gradual avoiding from religiosity. Steep bursts of the religiosity level, taking place at certain periods of world historical development, are not of a strategic nature. Such period takes place in the modern historical process" [26]. As a basis for research, were taken works of Khuako F.N. [28-44], Hotko S.H. [45], Tlupova Z.H. [46], Shaplok G.U. [47], Siuhov S.N. [48], Sitimova S.S. [49], Pafifova B.K. [50], Marchenko R.A. [51], Mamiy R.G. and Chankaeva T.M. [52], Luchinskaya E.N. and Ashinova S.B. [53], Lavrov L. [54], Kumykova T.H. [55] and others. All works are indicated in the list of literature, they served as an additional base and accompanying information for our study. 4. The Religious Saturation of the Caucasus In addition, as the contemporary philosopher M.Z. Magomedov, who has already cited above, points out, that regional religions are characterized by different saturation of national thinking. According to the cited data, related to North Caucasus, "out of the 550 surveyed respondents, 91% referred themselves to representatives of one of the proposed confessions, 7% called themselves atheists, 2% found it difficult to choose their religious affiliation. More than half of the respondents (62%) profess Islam, 29% - Orthodoxy, 8% - Buddhism and 1% - Judaism" [7]. In addition, as the contemporary philosopher M.Z. Magomedov, who has already cited above, points out, that regional religions are characterized by different saturation of national thinking. According to the cited data, related to North Caucasus, "out of the 550 surveyed respondents, 91% referred themselves to representatives of one of the proposed confessions, 7% called themselves atheists, 2% found it difficult to choose their religious affiliation. More than half of the respondents (62%) profess Islam, 29% - Orthodoxy, 8% - Buddhism and 1% - Judaism" [7]. Analogous example of similar beliefs is found in the Orthodox field of the Russian Federation. So, judging by the questionnaires offered by sociologists G.M. Andreeva and L.G. Andreyeva in the first decade of the new century, 84.2% consider themselves as followers of Orthodoxy. However, when clarifying religious details, it also turns out that 7.6% of them are individually non- believers, 23% exclude the presence of God or are experiencing difficulties with this issue [56]. As the author of A.L. Makoev describes the religious affiliation of the author, "It is easier for a y Analogous example of similar beliefs is found in the Orthodox field of the Russian Federation. So, judging by the questionnaires offered by sociologists G.M. Andreeva and L.G. Andreyeva in the first decade of the new century, 84.2% consider themselves as followers of Orthodoxy. However, when clarifying religious details, it also turns out that 7.6% of them are individually non- believers, 23% exclude the presence of God or are experiencing difficulties with this issue [56]. As the author of A.L. Makoev describes the religious affiliation of the author, "It is easier for a 247 International Journal of Engineering & Technology involved in the life of the heroes: "It was not the first time that Ahmet marveled at how well the mullah had managed to organize his reconnaissance", “We cannot waste time. Allah blesses our work” [5]. 6. Results and Discussion With the collapse of the USSR, the priorities changed radically. Servants of religion, continuing the normalization of faith, clarified the specifics of the post-Soviet stage. With the absence of any ideology in the country, which covers the frequent lawlessness for two decades, has been appeared an idea to impart sacredness to all anti-religious freely and without effort. In particular, a certain religious pluralism armed with special urgency. It means indulgence in relation to the neighboring faith, or a cardinal agreement with the fact that even without faith in the Almighty, it is possible to serve for the benefit of humankind. Already in today's Russia, specifically for this purpose, the developed training cycle "Foundations of religious cultures and secular ethics" has become a tangible and noticeable mechanism that constructs the required tolerance. Among the Circassians there was faith in the djinn. And no less mentioned among modern Adyghe authors along with white djinns- black mystical characters. Thus, the devotion of the devil by Circassian writer Asker Yevtykh, who wrote mostly in Soviet times, was particularly active, and therefore produced the only sacred word saturated in socialist realistic literary language. Indignant in his novel "Heart Break" [59], one of our fellows: a military ground, overgrown with grass: "Good grass, Deuce takes it!!" [54, p. 9]. In addition, already on the following pages, when talking about "intelligent namby-pamby» [60, p. 10] or about a similar glass grenade bottle [59, p. 11] - the same phrase «Deuce take it!" It is also used in the subsequent author's text [59, p. 31], etc. There is also another standard mention of the devil: "Go away! Go away! - Ilyinichna used to say, expelling the God knows where came from mosquito..." [59, p. 19]. And the situation varies in severity. Similar vocabulary is possible in episodes - dialogues of serious characters. These way two interlocutors discuss the third person. One of them leads to a character trait, such as understanding. To this, the second one responds with an assessment: "What is for devil’s sake understanding is?" [59, p. 20]. The author also uses such active phrases in the Russian colloquial style as "where the devils are" [59, p. 31] and "disappear to hell" [59, p. 52]. Thus, on the first 50 pages of the author's text of Asker Yevtykh, who created, it seemed, in the strict canons of Soviet realism, 7 references to a religious villain. 4. The Religious Saturation of the Caucasus religious person to inspire the image of the enemy and to force destroying any opponent of his faith than an atheist. The atheist, knowing that there is nothing waiting for him, values the earthly goods too much" [6]. [ ] Recognized by the followers of Islam, the djinn spirits are divided into white and black. Among the first, undoubtedly, is included the Most High. According to A.L. Makoyev, who completely refuses to worship in quantitative terms, God can personify only "depth and purity" [6]. Or, for example, Almighty is also white energy active in works of another contemporary Adygeyan writer - novelist Aslanbek Psigusov. There are some elements that illustrate the might and power of national dominants and religions, established over several centuries. In particular, once the winners have not become accustomed to the characters begin to curse their own gods. In this impulse, they come to the fact that they are infinitely lucky: woke up, revel in the surrounding lives - sometimes tiny, but confirming their own existence. That is why they are immediately taken to express their heartfelt gratitude to the gods for the mission of the god-like cover, successfully carried by the Almighty, in the process, that they continue in dairy: "This day will remain in his memory for a long time, a remnant of memories, reminding me with a heart mark that he is one of the spoils of fate, while for many of his fellow tribesmen, yesterday's sunset was the last" [58]. Consequently, a religious confession, that took place strictly after the battle, as well as directly on the sunk in the unbreakable silence. It is not definitive and irrevocable, but more individually. The frustration in faith of A.P.Psigusov clearly echoes the possible mood of A.L. Makoyev, in a similar statement. Here the narrator is closer to reality, but also looks back at the Most High: “Why I cannot fit into the usual framework of life - everything takes me into outer space. In fact, I'm doomed from birth. If I had done what I had to do according to my talent, I would have burned it in a moment. But by my inaction I deceive Providence. He is expecting something from me, and I shamelessly make promises every time, I swear that He did not make a mistake in me and I gain time. Do I win for what?" [6]. 1 Adyghe (Circassian) ethic-philosophical doctrine. 5. Variants of Islam In the modern world, the most permissible variation of religion for Caucasian (or Adyghe) - is the normalized form of Islam. Because of its strictly expressed norm, this form necessarily corresponds to the traditional canon, like any other norm in any other sphere. The Islamic standard forms and implements the optimally possible flow of tools aimed at crossing the individual with the spiritual priorities of national chronicles and stories. As the Dagestan scholars say about the fact of the inheritance in the 21st century, "Today religions are increasingly perceived by the peoples of the North Caucasus, as bearers of universal spiritual values, an essential element of the cultural heritage of the North Caucasian peoples [57]. Episodes like this, going through the centuries; inheritance is considered as tradition feature, which contrasts its modernist qualities today. The faith is invariably closely united with the acting society, with its constituent mechanisms and the subjects that inhabit it. Moreover, several decades ago, during the Soviet era, there was a wide application of getting rid of the religious influence of various layers of reality (both the society and the individual), i.e. actual secularization. At the time of the revolution and during the Soviet seventieth anniversary, the establishment of desecration of pre- revolutionary ideals was proclaimed and strictly supported. 6. Results and Discussion He was buried with the exact observance of the coordinates of the incident. At the same place for a long time, during the week, there was an intensive pyro (including sacrificial, and dance) procedure. Not so long, but no less intense was the three-day procedure for commemorating the animal that died from the lightning. Also, it was revered sanctified tree, affected by lightning. It in the minds of the Adyghes was immediately gifted with curative qualities. A tangible place in the course of development of the story line in the above-mentioned A.Kh. Psigusov also belongs to the black mystic. However, the acting here shaman acts very nobly and saves a life to a number of positive characters. Available interior items contribute in these, for example household items and buildings that come across him on the road. The metaphorical turns used by the author in combination with a mystical fuse, create the mythical character required in the postmodern novel. The door, under which "damp earth" is doused with a thoughtful hand, is "obediently opened" under the mighty pressure of a hurrying shaman to help when the bright sun rays shine through the gloomy structure, giving the tired slaves a bright aspiration really embodied with his arrival. In general, revered tree rituals from time immemorial and to this day function in the Adyghe environment. So, for example, M.I. Kandur, "The area where they lived was covered with a dense forest and, as the ancient traditions of the Circassians told us, many of their gods and goddesses lived in these forests [5]. Similar, the forest massifs, described by the author here, stood for centuries, sometimes, in the middle of the 19th century. - in the form of original Sacred Groves, holding on their trunks for decades and even centuries sometimes the invaluable outfit of folk-revered heroes. Here also could be located sums with the ashes of the heroes. Adyghe researchers are inevitably interested in the attitude of the representative of the nation to the tree as a symbol. As one of the most recent and most multifaceted researchers K.H. Unezhev points out, the classification of trees for the Adygea consisted in associating with good and evil, depending on which the researcher forms lists - delivering happiness and giving misfortune. To the first category of gentile breeds, the Adyghes included fruit varieties. 6. Results and Discussion Psigusov ("Biographies ...") are modest in preferences. In the final part of the second chapter, two servants of the regional church who went to the hail, strolling around the bazaar and examining the shop windows in the process of communication, evaluate the wealth to themselves. Reacting to the desire stated by the interlocutor by the will of the case to buy a two-floor house one of them is confused. He stresses that he would like to experience poverty with tranquility, than assuring his colleague, declaring: "I am a pious and unassuming person" [58]. The same line of modesty of desires continues simply in descriptive pictures (both individuals and actions). For example, in M.I. Kandura, mullah is described mainly by a rod physical sign - a "falling beard" covering all the others, up to the age. And the manner of religious hero behavior acts as a kind of proband for further plot turns: "His eyes glittered, but all the movements of a large body were slow and careful. The remaining elders of the village were located in the seniority next to him. There was also Hamzet, he lay on pillows and was very pale. The old men heatedly discussed something, and the mullah sat detached, half- closed his eyes and silently whispered his prayers to the beard" [5]. In this way, the author paints a portrait of a gracious hero describing a picture of the actions of a group close to the mosque. In addition, there are identical episodes in the descriptions in works of other authors, which were analyzed by us. Thus, A.L. Makoyev ("The History of Zul") in the peacefully living aul believers consider it their own duty to assemble and observe the procedure: "Religious people, during the day, gathered at the mosque to ask Allah to curb the mad people, return their minds and return to their old life" [61]. Or at M.I. Kandur "All the soldiers gathered in the village square in front of the house of the mullah for prayer" [5]. In general, ritual procedures can be called sufficiently revered by Adyghes throughout many centuries-old chronicles. So, in particular, during the awesome thunder, Adyghes were singing a special song. Once a year, the day of the first lightning was celebrated as a holiday. Anyone killed by lightning was equated to be marked with good. 6. Results and Discussion Indeed, Sartre calls him "Satan" and defines it very peculiarly, "a symbol of disobedient and inflated children, demanding that the paternal view helps them to stiffen in their particular essence, and who commit Evil in the framework of Good for the sake of affirming and sanctifying their own particularity" [60]. O i i A L M k ' " Z l t " th d i ti f f q Simultaneously with the favored Islamic appeals in the Caucasus, there existed components of pre-Islamic beliefs, up to this day. In this case, the formula of a harmonious mixture, described by A.L. Makoyev, applied to Adyghes as follows: "If you cannot do without religion, it is not prudent to create your own, than to use the product of someone else's mind, and follow it only. Yes, it should not be otherwise" [6]. Pagan shadows in the Circassians souls became a subject of general Adyghe attention. And that such self-created are often considered to be the general Adyghe faith, based on different religious echoes, but firmly retained in the form of the ethnic law of "Adyghe Khabze" 1, accepted and strictly revered by Circassians to this day. As Khatko Fathi says today, "Now there is a great deal of civic responsibility on every person, because an uncompromising dilemma has arisen - will the people profess the primordial customs and traditions or the borrowed foreign (Arab) religious ideology and philosophy. Ultimately, this is choice that will determine and will continue to form the nation's image" [8]. And the multinational reference to the general religion is appointed by the Koran, the highest scripture. Constructed on five massive pillars, it is by thought and affairs of the ministers, Or again, in A.L. Makoyev's " Zul story" the description of one of the popular Aulian stories concerning the local fortune-teller, cannot be without an evil one The old woman who had risen in the dark and slipped for no reason at all, was killed under strange International Journal of Engineering & Technology 248 circumstances, when the village forester, chasing a black cat, finally managed to kill the importunate animal: "The attack of the dark forces is not far off, it is necessary to prepare for the invasion, and wooden paddles of forester Tuna will not fight them back" [61]. 6. Results and Discussion Here it should be emphasized that in general fortune-tellers and witches are also quite popular among the Adyghe authors today. And this fact can be considered explainable, since the Adyghe chronicles contain extensive information about the Adyghe faith. Sources of similar publications (including scientists) confirm and prove that both in their circles and in their ranks the regular and valid technology of fortune telling is similar to the Romans. And because of this fact A.A. Khagurov, can calmly and confidently introduces the fortunetelling sacrality today, in the person of the witch Khaniya in his story, allowing his characters to intersect with her in their actions and desires. Thus, the narrator hero sees in himself a five-year-old boy, which helps him to remember, with a kind word, a strange old woman living next to her: "But there remained the house of the old woman Khaniya that was opposite to the grandfather's house. Khania was considered as a sorceress in the village. Wise Aslambiychik knew what he was doing: I was not a relative of the witch Khani, but I was her neighbor, so for her "friend". The window from the inside was curtained with matting" [1]. It should be noted that such mysterious old women are not too rejected; sometimes they are even attractive on book pages. There was a popular version that some Adyghes turned out to be husbands of female djinnes, which were considered to be extremely exquisite. Modern Adyghes are endowed with the epithet "witch"(in Adyghe language – “Ud”) in relation to visionary, sensible women. It was believed that “Uds” there is a sorcerous gathering that was carried away by dark magic and thereby brought misfortunes to fellow countrymen. Nevertheless, few people believed that they were wiser than a man, and therefore the Adygeyan proverb says: “People know that, what Uds don’t”. Representatives of the nation were also sure that water witches (more precisely, mermaids) lived in the grassy thickets on the river coasts. According to the ordinary Aulian version, those were blue-eyed and long-nail blondes, who could have been taken home and patronized by fellow tribesmen. professional way of life: "Everything here breathed with strictness, combined with the deep religiosity of the master" [5]. combined with the deep religiosity of the master" [5]. And this severity is often inherent in descriptions of religious figures also in works of other authors. So, likewise, the church ministers of A.H. 6. Results and Discussion That's why it was strictly forbidden to cut down a pear and mulberry tree. The man had to plant twelve fruit trees and only then had the right to create a family. "Good", or "happiness bringing" were admitted such trees as linden, oak, ash, and hawthorn. It was believed that hawthorn Expressive write of modern Adygeyan writers, related not only to the devil and his partners, but also ministers of the Most High. Religious ministers here reproduce with equal frequency, both in positive and in negative mean. Opposite to this, religious minister at M.I. Kandur work, countrymen consider it truly sacred: "Local residents regarded this as a sign of God himself. No doubt, after this Ushurma acquired a tremendous authority from these superstitious people. They are convinced that he is sent from above to guide them" [5]. It is historically known that in the early days the Adygeyan were mostly religious people on the Muslim path, that is, mullahs, cadis, mostly were people of Turkish nationals. As a consequence, Islam strengthened the will and power of the Turkish-directed branch of the aristocracy. Researchers often focus on the integrity of Islam, but, at the same time, - and on its versatility as a social phenomenon. In general, several characters are also active in the faith of the foreign Adygea M.I. Kandur in his trilogy "The Caucasus", where the decoration and design of the mullah's dwelling are clearly combined with his 249 International Journal of Engineering & Technology of the population not only for expression and achievement of sacred values, goals, and others - non-religious, social" [11]. In fact, the North Caucasus allowed the crossing of the normalized religion with regional ethical matter of various ethnic groups. As a result, a peculiar territorial configuration of its functioning was formed, based on the world's Muslim dogma, but not always understood by our fellow countrymen: "In silence, Ahmet thought that his heart was beating incredibly loudly, he hardly suppressed the desire to jump into the saddle without waiting for the end of the rite" [5]. But other motives that stimulate the tradition of ancestors are more powerful. As Z.M. Abdulagatov believes, these are the "right actions" in life, regardless of belief in Allah. This scientist argues the current conclusion with concrete sociological data. "So, out of five answers to the question" Who, in your opinion, can be called a believer? 7. Conclusion As a result, chronology and the current state of phenomena in the North Caucasus allow us to come to certain conclusions. First, this is the fact that both religions common today (Christianity, Islam) are not at all sources of interethnic difficulties. Moreover, they at one time managed to settle them to some extent and settle interethnic conflicts. The connections existing today in the national republics between ethnic groups can be designated as constant and stable, which can have a tangible impact on maintaining a stable tolerance. Secondly, we consider it inevitable that the state must strive to support the existing religious branches (in particular, Islam and Christianity). This happened without an avoiding from a whole range of previously adopted family and tribal attitudes with the consolidation of the Islamic essence behind them. At the same time, the beliefs and rituals adopted by Muslims to some extent turned out to be in unison with people's similarly philanthropic procedures. For example, they confront the evil spirit of the Circassians by means of a talisman, a certain prayer, written out by the attendant of the mosque on paper (sometimes - a scrap) and tied in a mini bag (cloth or leather) with three or four corners. In the form of amulets guarding against diseases, a piece of cloth with oak chips formed by a lightning strike into a tree was worn on the individual's neck. In addition, fragments of such rags were threaded on a branch in the form of an offering. As a result of these national and religious confessions, not only the admirer of the Most High, but also a convinced atheist in the Caucasus can recognize the most visible rituals in the family and everyday life of the people. This is also traced by sociologists. In particular, the above-quoted Dagestan scholar Z.M. Abdulagatov on the ritual procedures states the following. Judging by his surveys, in the Republic of Dagestan, Kabardino-Balkaria Republic, and Chechen Republic there are 3.3% of non-believers "participate in all religious rites and attract others" and 43.3% "sometimes participate in some ceremonies." Muslim rituals of marriage, funeral, sunnat, naming, by the national in content and religious only in form, but because of all consider 44.0% of the polled three of these republics. The exclusively religious, sacral character of these rituals is noted only by 36.9% [11]. 6. Results and Discussion ", The largest number of respondents chose the position" A true believer is one who commits morally correct acts in life, and does not swear in the faith of Allah and fulfills other requirements of the Islamic religion "(32.6%)" [56]. possesses a magical power to drive out unclean force, to protect people and animals from spoilage and slander. To the "unkind trees" belonged to the representation of Adyghes and Balkars such trees as cornel, maple, wild cherry and others [62]. He who came to the mountains in ordinary circumstances always had the opportunity to refresh himself, since he was already taken care of by fellow countrymen who had been there before. They were obliged to leave the third part of the harvested crop in the trees, and as they fell down they entered the diet of the forest dwellers, i.e. the beasts. However, some of the authors considered today do not much respect the religious procedure. The logic of veneration of the Almighty, the same writer argues his own accusations against pagan rituals, promises made in honor of the heavenly deities. In the view of A.L. Makoyev, already in another work, "The Returned Skies," the inconsistency of ceremonial procedures is obvious: "So many times to do prayers, fasting for so many days in a certain order and certain food, etc. In itself, there is nothing wrong with this, but getting carried away by them, a person imperceptibly substitutes the depth and purity of the exercises in order and quantity" [6]. A common version of the spread of Islam is the option of consolidating the spiritual tradition that came from the great-grandfathers, which, apparently, predetermines the corresponding concept. With regard to Islam in the Caucasus, the definition of "traditionalist" is often used. 7. Conclusion As can be seen from the purposeful predetermination of the above rites, this tendency mainly concerns concrete, more frequent in everyday life procedures. They, in turn, are sometimes not only admirers of the Most High, but also secular atheists are presented as a centuries- old family treasure. In particular, the ordinary modern Adyghe, distraught with the death of a loved one, will not forget to turn to the mosque to call her servant, who possesses the skills required for the ablution of the deceased. Either on the pre-trial day, the procedure in the registry office is preceded by the ritual "nachah" that takes place in the mosque and consists of writing (in front of everyone) each of the married couple of words. And a friend of one of them will definitely come to stand beside him and share the joy not only in the state institution, but also in the mosque (in which he probably never was). But it is so necessary, so did the elder brother, - means, it is necessary to me. This is the law. Z M Abd l t ll h f i " li i References [1] Harugov AH, Crossing: Collection of short stories. Krasnodar: KubGAU, 2014. [2] Capitsa SP. I am a Russian, Orthodox atheist. http://orthodoxy.cafe/index.php?topic=555546.0 [3] Spenser E, Travel to Cherkessia. Maikop: RIPO Adygea, 1839. [4] Lopatkin RA (2003), Religiosity. Sociological Encyclopedia 2. [5] Kandur M, CAUCASUS: historical trilogy. Moscow: Kandinal, 1994. [6] Makoev AL, Returned sky: Stories. Nalchik: Elbrus, 2015. [7] Magomedova MZ (2010), Religious identity in the North Caucasus. Islamovedenie 3. [8] Fathi Kh, Adyghes and religion. Maikop: GURIPP, 2003. [9] Han-Girei S (1842), Faith, customs, customs, lifestyle of Circassians. Russkiy vestnik 1. [10] Han-Girei S, Historical and ethnographic composition of "Notes on Circassia". 1836. [11] Nogmov Sh. Religions of Adyghes. [12] Kosven MO, Lavrov LI, Nersesov GA & Hashaev HO (eds.), The Peoples of the Caucasus. Vol. 1. Moscow: USSR Academy of Sciences, 1960. 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https://openalex.org/W3092982246	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0240454&type=printable	English	null	Cooling-mediated protection from chemotherapy drug-induced cytotoxicity in human keratinocytes by inhibition of cellular drug uptake	PloS one	2,020	cc-by	10,142	PLOS ONE PLOS ONE RESEARCH ARTICLE Editor: Irina V. Lebedeva, Columbia University, UNITED STATES Received: April 26, 2020 Accepted: September 25, 2020 Published: October 15, 2020 Copyright: © 2020 Dunnill et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: Authors CD and KI were financially supported by funding from Paxman Coolers Ltd (paxmanscalpcooling.com). This does not alter our adherence to PLOS ONE policies on sharing data and materials. No other authors were supported by funding from Paxman. The authors declare that the sponsors were not involved in the design of the Christopher Dunnill1, Khalidah Ibraheem1, Michael Peake1, Myria Ioannou1, Megan Palmer1, Adrian Smith2, Andrew Collett1‡, Nikolaos T. GeorgopoulosID1,3‡* Christopher Dunnill1, Khalidah Ibraheem1, Michael Peake1, Myria Ioannou1, Megan Palmer1, Adrian Smith2, Andrew Collett1‡, Nikolaos T. GeorgopoulosID1,3‡* 1 Department of Biological Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom, 2 Department of General Surgery, Calderdale and Huddersfield NHS Foundation Trust, Huddersfield, United Kingdom, 3 Institute of Skin Integrity and Infection Prevention, University of Huddersfield, Huddersfield, United Kingdom a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ‡ These authors are joint senior authors on this work. * N.Georgopoulos@hud.ac.uk OPEN ACCESS Chemotherapy-induced alopecia (CIA) represents the most distressing side-effect for can- cer patients. Scalp cooling is currently the only treatment to combat CIA, yet little is known about its cytoprotective effects in human hair follicles (HF). We have previously established in vitro human keratinocyte models to study the effects of taxanes and anthracyclines rou- tinely-used clinically and reported that cooling markedly-reduced or even completely-pre- vented cytotoxicity in a temperature dependent manner. Using these models (including HF- derived primary keratinocytes), we now demonstrate that cooling markedly attenuates cellu- lar uptake of the anthracyclines doxorubicin and epirubicin to reduce or prevent drug-medi- ated human keratinocyte cytotoxicity. We show marked reduction in drug uptake and nuclear localization qualitatively by fluorescence microscopy. We have also devised a flow cytometry-based methodology that permitted semi-quantitative analysis of differences in drug uptake, which demonstrated that cooling can reduce drug uptake by up to ~8-fold in comparison to normal/physiological temperature, an effect that was temperature-depen- dent. Our results provide evidence that attenuation of cellular drug uptake represents at least one of the mechanisms underpinning the ability of cooling to rescue human keratino- cytes from chemotherapy drug-cytotoxicity, thus supporting the clinical efficacy of scalp cooling. Citation: Dunnill C, Ibraheem K, Peake M, Ioannou M, Palmer M, Smith A, et al. (2020) Cooling- mediated protection from chemotherapy drug- induced cytotoxicity in human keratinocytes by inhibition of cellular drug uptake. PLoS ONE 15(10): e0240454. https://doi.org/10.1371/journal. pone.0240454 Editor: Irina V. Lebedeva, Columbia University, UNITED STATES Cooling-mediated protection from chemotherapy drug-induced cytotoxicity in human keratinocytes by inhibition of cellular drug uptake Christopher Dunnill1, Khalidah Ibraheem1, Michael Peake1, Myria Ioannou1, Megan Palmer1, Adrian Smith2, Andrew Collett1‡, Nikolaos T. GeorgopoulosID1,3 Introduction There- fore, cooling of the scalp is a safe and very promising approach to minimize CIA, and biologi- cal research relating to the mechanisms by which cooling prevents chemotherapy drug- mediated cytotoxicity may not only provide scientific support for its ability to cytoprotect, but also assist in the design of strategies to enhance the ability of cooling to prevent CIA. A number of experimental models are available for studying chemotherapy drug-induced cytotoxicity and CIA, such as in vivo rodent-based models [3], although these have inherent practical and physiological limitations. Ex vivo models based on isolation and culture of human HFs established by Paus and colleagues represent an elegant model that has been used to study CIA caused by a variety of drug types [10–12]. Yet, despite their clear physiological relevance, these models are experimentally laborious, with relatively limited ability for assess- ment of several parameters simultaneously, which does not permit more systematic and high- throughput investigations. Abbreviations: CIA, chemotherapy-induced alopecia; FBS, fetal bovine serum; HaCaTa, adapted HaCaT; HDF, human dermal fibroblasts; HF, hair follicle; HKGS, Human Keratinocyte Growth Supplement; KSFM, keratinocyte serum-free medium; MFI, median fluorescence intensity; NHEK, normal human epidermal keratinocytes; ORSK, outer root sheath keratinocytes. We previously established a series of in vitro models for the culture of primary and immor- talized human keratinocytes under conditions where they adopted a basal highly-proliferative phenotype resembling the rapidly-dividing hair matrix keratinocytes, as well as human hair follicle-derived keratinocytes. Specifically, we used a) normal human epidermal keratinocytes (NHEK), b) human HF-derived keratinocytes, and c) our derivative of the well-characterised keratinocyte line HaCaT which was adapted to serum-free/low-calcium culture conditions (‘HaCaTa’ cells). We employed these models to study the effects of cooling on chemotherapy drug-mediated cytotoxicity. Using a panel of drugs (including both taxanes and anthracy- clines) routinely used in the clinic for treatment of cancers such as breast cancer, we demon- strated for the first time that although the drugs were highly cytotoxic, cooling during drug treatment markedly reduced or even completely prevented drug cytotoxicity, in agreement with observations of the ability of scalp cooling to prevent HF toxicity in the clinic [13]. Equally importantly, we provided evidence that the precise cooling conditions (temperature) was a critical factor in determining the overall ability of cooling in rescuing cells from drug- mediated toxicity. Introduction Although not a life-threatening condition, chemotherapy-induced alopecia (CIA) represents the most distressing side-effect for cancer patients [1] and the fear of hair loss causes severe anxiety in patients [2]. Thus it is important to understand the mechanisms of CIA in order to design safe and effective prevention strategies [3]. Chemotherapy drugs, such as taxanes and 1 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 PLOS ONE Cooling-mediated cytoprotection in keratinocytes by inhibition of chemotherapy drug uptake study; in the collection, analysis and interpretation of data; in the writing of the manuscript; nor were the sponsors involved in the decision to submit the paper for publication. particularly anthracyclines, cause CIA as they damage human hair follicles (HF) by mediating direct cytotoxicity in the hair matrix keratinocytes, which are the most rapidly-dividing cell population in the HF [3]. The extent to which various chemotherapy drugs cause CIA differs [4], yet anthracyclines appear to be the biggest inducer of CIA [5]. Competing interests: AC and NTG are members of the scientific advisory board of Paxman, but do not receive any consultancy-related income. AC and NTG are co-inventors in patent application no. WO2017220998A1 (‘A composition for use in cooling therapy and treatment of chemically induced alopecia (CIA), said composition containing a reactive oxygen species (ROS) inhibitor’ - published Dec 2017), however the patent is not related to the present study. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The other authors have no conflict of interest. Finally, the authors declare that Paxman were not involved in the design of the study; in the collection, analysis and interpretation of data; in the writing of the manuscript; nor were the sponsors involved in the decision to submit the paper for publication. Scalp cooling is currently the only safe and widely-available preventative treatment to com- bat CIA in cancer patients [4]. There is increasing clinical evidence that scalp cooling can be an effective way to minimize or even eliminate CIA [6, 7]. Overall the probability that scalp cooling will prevent/reduce CIA is ~50% and this can rise to ~90% depending on the chemo- therapy modality used and/or scalp cooling techniques employed [8], whilst the recently-pub- lished first multi-centre randomised clinical trial ‘SCALP’ has provided further clinical evidence that supports the ability of scalp cooling to reduce the risk of CIA to ~50% [9]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 Introduction The concordance of our findings with clinical observations demonstrated that, despite their reductive nature, these in vitro models were robust and provided experimen- tal support for the clinically-reported cytoprotective role of cooling [13], thus paving the way for further studies on the molecular mechanisms underpinning the cytoprotective effects of cooling. A number of mechanisms have been proposed to explain the cytoprotective effects of scalp cooling [4], including: i) cooling causes vasoconstriction which can reduce cutaneous perfu- sion to 20%-40% of normal levels [14], resulting in reduced chemotherapy drug perfusion through the HF; ii) the rate of drug uptake across the plasma membrane is reduced at low tem- peratures via reduction of active transport or reduced diffusion due to lower cellular activity or kinetic energy and membrane fluidity, respectively; iii) cooling may slow-down the rate of cell division (as cell division is an energy-dependent process); and iv) decreases in the metabolic 2 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 PLOS ONE Cooling-mediated cytoprotection in keratinocytes by inhibition of chemotherapy drug uptake activity of HF cells by cooling reduces the cytotoxicity of chemotherapy drugs as a range of cel- lular processes (such as oxidation) decelerate. Most likely, a combination of these mechanisms will render scalp cooling cytoprotective, thus explaining its reported clinical efficacy. Notably, our previous work demonstrated that cooling completely inhibited the cytotoxicity of the anthracycline doxorubicin at concentrations identical to the maximal plasma drug concentra- tion measured during routine infusion with the drug in patients undergoing chemotherapy [13]. Those findings strongly implied that cooling may protect cells by a direct mechanism and not merely by vasoconstriction. Using our previously-established in vitro models, in this study we aimed to for the first time explore the hypothesis that cooling reduces the entry of chemotherapy drugs into human keratinocytes. We studied doxorubicin and epirubicin as anthracyclines are the strongest inducers of CIA, whilst exploiting the inherent property of doxorubicin [15] and epirubicin [16] to fluoresce in human cells. We found that doxorubicin and epirubicin are detectable in the nuclei of NHEK, HaCaTa and human hair follicle-derived outer root sheath keratinocytes (ORSK cells) and are highly cytotoxic at normal temperature; however, cooling markedly reduced chemotherapy drug cellular uptake, which coincided with its ability to reduce or pre- vent drug-mediated keratinocyte cytotoxicity. Culture of immortalized keratinocyte cell lines The HaCaT cell line was originally purchased from the Cell Line Service (CLS, Eppelheim, Germany) as described elsewhere [13] and cultured in DMEM medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 2 mM L-glutamine–all obtained from Sigma (Sigma- Aldrich, Dorset, UK). The HaCaT-derivative keratinocyte line ‘HaCaTa’ was established as detailed previously [13] and cultured in keratinocyte serum free medium (KSFM) supple- mented with epidermal growth factor and bovine pituitary extract (referred to as KSFM com- plete, KSFMc)–all obtained from Thermo Fisher Scientific (Loughborough, UK). All cells were routinely cultured at 37˚C in a humidified atmosphere of 5% CO2, whereas for cooling experi- ments the desired temperature was achieved using an LMS Series 1A Cooled Incubator with appropriate CO2 provision for the duration of the incubation period (see below). Cells were passaged at ~80–90% confluence by removing media, rinsing with 0.1% EDTA in PBS (w/v) to aid disaggregation, and lifted using trypsin–EDTA solution (Sigma). For HaCaTa cells, the trypsin was inactivated by brief treatment with 5% FBS-containing KSFM medium, before routine culture in KSFMc. HaCaT cells were cultured in standard plasticware, whilst HaCaTa were cultured in Cell Plus (Cell+) plasticware (Sarstedt, Leicester, UK). HaCaT and HaCaTa cell lines were tested for Mycoplasma using the MycoProbe™Mycoplasma detection assay (R&D Systems). Introduction In addition to qualitative detection by fluores- cence microscopy, we established a semi-quantitative method to determine cellular drug uptake by using flow cytometry, an approach that allowed us to determine that cooling can reduce drug uptake by up to ~8-fold. Our results suggest that attenuation of drug uptake repre- sents at least one important mechanism by which cooling can protect cells in the HFs from chemotherapy drug-mediated cytotoxicity. Culture of human dermal fibroblasts (HDF) HDF cells were a gift from Tama´s Bı´ro (University of Debrecen, Hungary) and were cultured in DMEM/10% FBS and 2mM L-glutamine supplemented with penicillin/streptomycin (100IU/100μg/ml) at 3x103 cells/cm2 until confluent. For use as feeder cells, confluent HDF cultures were treated with 4 μg/ml Mitomycin C (Acros Organics, supplied by Thermo Fisher) for 24-hours and sub-cultured into 35-mm petri dishes at 3.5×104 cells/dish in a final volume of 2 ml. Feeder layers were used within 2–6 days post-seeding [17]. Establishment and culture of normal human epidermal keratinocytes Normal human epidermal keratinocytes (NHEK) were established using skin collected from routine surgical procedures with National Health Service (NHS) Research Ethics Committee (REC) approval and the required, informed written consent from patients with no history of skin malignancy. Full-thickness abdominal skin (~3cm2) was collected and initially stored at PLOS ONE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 3 / 16 PLOS ONE Cooling-mediated cytoprotection in keratinocytes by inhibition of chemotherapy drug uptake 4˚C in 20ml HBSS medium supplemented with penicillin (100 units/mL)/streptomycin (100 μg/mL) (Thermo Fisher) and Amphotericin B (25 ng/mL) (Sigma). Following removal of adipose and connective tissue by trimming using surgical scissors, the remaining dermis and epidermis were rinsed three times in PBS and then cut to ~4×2 mm/8mm2 pieces using a scal- pel. These pieces were placed epidermal-side down into a 6-cm petri dish containing 5ml 2.5 mg/ml dispase solution (Sigma) in HBSS supplemented with antibiotics (as above), which was sealed in an airtight container and incubated at 4˚C for 12–16 h. Epidermal sheets were then gently removed from the dermis using forceps in ice-cold HBSS; these were then placed into 5 ml 0.25% trypsin/EDTA diluted in HBSS and ‘minced’ for 10-minutes with a scalpel in a 6 cm petri-dish lid at room temperature to create a cell suspension. This was filtered through a 70μm strainer (Corning, supplied by Sigma) using a serological pipette and suspended in 20 ml DMEM/10% FBS to inactivate the trypsin. This was then placed in a 50 ml centrifuge tube and centrifuged at 130 g for 10-minutes. The cell pellet was resuspended in 5 ml EpiLife medium supplemented with HKGS (Thermo Fisher). 106 cells were suspended in a total of 4 ml EpiLife/HKGS containing antibiotics and were seeded in Cell+ T25 flasks (Sarstedt) pre- coated with 200 mg/ml human collagen IV (Merck, Watford, UK) to enhance initial attach- ment. At this point, cells were classified as passage 0 (p0) and were-medium changed the next day. Upon reaching 60–80% confluency, cultures were passaged (1:3 ratio) in Cell+ T25 flasks (non-collagen-coated). For all experiments, NHEK cells were used at <p5 to avoid the onset of senescence and ensure maximal proliferative capacity. Establishment of outer root sheath keratinocyte (ORSK) cultures For the establishment of ORSK cultures, hair plucking was performed with informed written consent and ethical approval by the School of Applied Sciences Research Integrity and Ethics Committee (SRIEC) and in line with the University of Huddersfield Research Ethics and Integrity Policy and Code of Practice for Research. ORSK cultures were established based on previously detailed methodologies [17–19]. Briefly, 5–10 hair follicles were plucked from con- senting donors and, if containing a clearly visible ORS, incubated for 30-minutes at 37˚C in 2 ml ORSK medium comprising DMEM supplemented with 10% FBS, 2mM L-glutamine, 100 UI/mL penicillin G, 1 mM ascorbyl-2-phosphate, 2.4 μg/ml adenine, 10 ng/ml epidermal growth factor, 0.4 μg/ml hydrocortisone, 2 nM triiodothyronine, 0.1 nM cholera toxin, 25 μg/ mL gentamycin. Follicles were rinsed twice with 1 ml PBS containing 100 μg/ml gentamycin. ORS-containing HFs were incubated with 0.5 ml 0.25% Trypsin-EDTA for up to 40-minutes with vigorous pipetting and vortexing performed at 10-minute intervals until the ORS was dis- aggregated to provide a suspension of ORSK cells. Trypsin was inactivated by mixing with equal volume of ORSK medium followed by centrifugation and supernatant removal. Cells were then seeded onto HDF feeder cultures in 35-mm petri dishes and incubated for 5–6 days. The 2 ml of ORSK medium was replenished every 2–3 days until subculture was required; for this, ORSK cells were rinsed twice with PBS-gentamycin, and incubated with TrypLE Express (Thermo Fisher) for 5–10 minutes. TrypLE enzyme was then inactivated by dilution in equal 4 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 PLOS ONE Cooling-mediated cytoprotection in keratinocytes by inhibition of chemotherapy drug uptake volume of PBS and by centrifuging the suspension. Cells were seeded in EpiLife/HKGS medium at 1x104 cells/cm2 [20]. Detection and quantification of cell viability Keratinocyte cell growth and the effect of chemotherapy drugs on cell viability, was deter- mined using the CellTiter 961 AQueous One cell proliferation assay (Promega, Southampton, UK). For chemotherapy drug experiments, cells were treated with doxorubicin (Doxorubicin hydrochloride, # sc-200923, Santa Cruz Biotechnology) or epirubicin (Epirubicin hydrochlo- ride, # E0550000-1EA, Sigma). HaCaT, HaCaTa and NHEK cells were seeded at 5x103 cells/ well and ORSK cells at 8x103 cells/well, respectively, in 100μl culture medium in 96-well plates of either standard or Cell+ plasticware (Sarstedt), as appropriate. 24-hours later, 100μl of doxo- rubicin or epirubicin solution appropriately diluted in the respective culture medium were added to each well, in order to achieve the desired final drug concentration. Following a 2-hour incubation (drug treatment) at normal (37˚C) or at cooling conditions (22 or 18˚C), the drug-containing medium was removed and wells were rinsed once with PBS solution at room temperature, before 100μl of fresh medium was added in each well and cells were incu- bated at 37˚C for 72-hours. 20 μl CellTiter reagent was then added to each well and plates were incubated at 37˚C for 4-hours. Absorbance was then measured using a FLUOstar OPTIMA (BMG Labtech, Bucks, UK) plate reader at 492 nm wavelength. Percentage (%) cell viability was calculated as previously [13] using the equation: (Abs T/Abs C) x 100, where ‘Abs T’ corre- sponds to absorbance value for drug-treated cells and ‘Abs C’ is the absorbance value for con- trol cultures. Fluorescence microscopy For doxorubicin uptake visualisation, NHEK and HaCaTa were seeded at a density of 5x104 cells/well on Teflon-coated 12-well glass slides as previously described [21]. After 24-hours, cells were treated for 2-hours with the indicated concentration of doxorubicin in PBS solution at 37˚C or at cooling conditions (18˚C). Cells were then rinsed twice with PBS, before being fixed using 4% paraformaldehyde in PBS for 15-minutes at room temperature and cell nuclei counterstained for 5-minutes using 0.1 mg/mL Hoechst 33258 (Sigma) as detailed elsewhere [22], prior to mounting and epifluorescence imaging (green channel for doxorubicin and blue channel for Hoechst). For epirubicin uptake visualisation, NHEK and HaCaTa were seeded at a density of 3x104 cells/well in 24-well multi-well plates and 24-hours later cells were treated for 2-hours with the indicated concentration of epirubicin in PBS at 37˚C or at cooling condi- tions (18˚C) prior to live fluorescence imaging (green channel). Drug uptake was visualised for doxorubicin by epifluorescence imaging on a Zeiss Axio Imager Z1 and images captured with a Zeiss AxioCam MRm Rev.3 digital camera and analyzed using ZEN software (Carl Zeiss Ltd, Herts, UK), and for epirubicin by live imaging using an EVOS1 FLoid1 cell-imaging station (Thermo Fisher) and images analyzed using Image J software. The effect of anthracyclines doxorubicin and epirubicin on the viability of primary and immortalized human keratinocytes and the protective effects of cooling We initially treated primary NHEK cultures and immortalized HaCaTa cells with doxorubicin and epirubicin at normal temperature conditions (37˚C) and observed a dose-dependent reduction in keratinocyte viability (Fig 1). To mimic cooling conditions, we treated cells with the drugs at 22 or 18˚C, which we have previously shown are cooling conditions that can pro- tect such cells from chemotherapy drug-mediated cytotoxicity [13]. We found that cooling markedly protected from drug-mediated cytotoxicity for low drug doses (<1μM), whereas for high drug concentrations (>1μM) it significantly attenuated toxicity even at very high (5μM) doses (Fig 1). We confirmed these findings in similar experiments using the original HaCaT cells (S1 Fig). The lower temperature 18˚C was consistently more effective at preventing toxic- ity particularly in primary NHEK cells, as was cooling-mediated cytoprotection overall in NHEK cultures in comparison to immortalized keratinocytes (Fig 1A and S1 Fig). These results confirmed our previous findings for doxorubicin [13], whilst for the first time extend- ing our observations to the anthracycline epirubicin. Flow cytometry HaCaT, HaCaTa and NHEK cells were seeded at 3x104 cells/well and ORSK cells seeded at 4.8x104 cells/well (2.5x104/cm2) in 500μl culture medium in 24-well plates of standard and Cell + type plasticware type (Sarstedt), respectively. 24-hours later, 500μl of doxorubicin or epirubi- cin solution appropriately diluted in the respective culture medium were added to each well, in order to achieve the desired final drug concentration. Cultures treated with the various drug concentrations and incubated at 37˚C or at cooling conditions (22 or 18˚C), for 2-hours. Fol- lowing treatment, the drug-containing medium was removed and wells were rinsed once with 5 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 PLOS ONE Cooling-mediated cytoprotection in keratinocytes by inhibition of chemotherapy drug uptake PBS solution, before harvesting using 200 μl 0.25% trypsin/EDTA diluted in PBS for HaCaT, HaCaTa and NHEK and 1x TrypLE express enzyme for ORSK cells. Cells were counted, washed in FACS buffer as detailed elsewhere [23] and resuspended in 200 μl of ice-cold PBS for flow cytometry analysis. Doxorubicin or epirubicin uptake was determined by acquisition on a Guava EasyCyte 5 flow cytometer and results were analyzed using EasyCyte software (Millipore, Watford, UK). Statistical analysis Statistics were performed using Minitab v18.1 (Minitab Ltd, Coventry, UK). Parametric statis- tics mean and standard error of the mean (SEM) were used for descriptive purposes and evalu- ation of significance was calculated by means of a two-tailed independent Student’s t-test. For graphical purposes in the figure captions: p<0.05, p<0.01 and p<0.001, whilst ‘ns’ denotes non-significance (p>0.05). Detection of anthracycline entry and localization in NHEK and HaCaTa cells and assessment of the effect of cooling in cellular drug uptake To determine whether cooling rescues cells from chemotherapy drug-mediated cytotoxicity (at least partly) due to its ability to influence drug uptake, we exploited the inherent property of doxorubicin and epirubicin to fluoresce upon excitation and detected drug internalisation by microscopy. NHEK and HaCaTa cells were treated with doxorubicin at normal (37˚C) or cooling (18˚C) conditions and cellular drug uptake was determined by fluorescence micros- copy (Fig 2). Doxorubicin was readily detected in treated NHEK and HaCaTa cells at 37˚C and exhibited nuclear localization. Interestingly, when drug treatment was carried out under cooling conditions (18˚C), there was little detectable doxorubicin in HaCaTa cells (Fig 2B) and even less in primary NHEK cultures (Fig 2A). When we performed similar experiments in NHEK and HaCaTa cells involving epirubicin, we observed epirubicin uptake and nuclear localization at normal temperature (37˚C), which was suppressed under cooling (18˚C) condi- tions (S2 Fig). 6 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 PLOS ONE Cooling-mediated cytoprotection in keratinocytes by inhibition of chemotherapy drug uptake Fig 1. Cytoprotective effect of cooling against chemotherapy drug-mediated toxicity in NHEK and HaCaTa cells. NHEK (a) and HaCaTa (b) cells were treated for 2-hours with a range of concentrations ([drug]) of doxorubicin and epirubicin at normal (37˚C) and cooling (22 and 18˚C) conditions and cell viability was assessed 72-hours post- treatment. Data points correspond to mean % cell viability (± SEM) for independent biological experiments (n = 3), each consisting of 5 technical replicates. Statistical significance (cooling conditions versus 37˚C) is denoted as p<0.05, p<0.01 and p<0.001, whilst ns denotes non-significance (p>0.05), with stats symbols being colour-coded for each cooling temperature. https://doi.org/10.1371/journal.pone.0240454.g001 Fig 1. Cytoprotective effect of cooling against chemotherapy drug-mediated toxicity in NHEK and HaCaTa cells. NHEK (a) and HaCaTa (b) cells were treated for 2-hours with a range of concentrations ([drug]) of doxorubicin and epirubicin at normal (37˚C) and cooling (22 and 18˚C) conditions and cell viability was assessed 72-hours post- treatment. Data points correspond to mean % cell viability (± SEM) for independent biological experiments (n = 3), each consisting of 5 technical replicates. Statistical significance (cooling conditions versus 37˚C) is denoted as p<0.05, p<0.01 and p<0.001, whilst ns denotes non-significance (p>0.05), with stats symbols being colour-coded for each cooling temperature. Detection of anthracycline drug uptake inhibition by cooling in primary and immortalized human keratinocytes using flow cytometry To assess whether cooling attenuates cellular uptake of doxorubicin and epirubicin, we also exploited flow cytometry-based detection. A significant advantage of flow cytometry over microscopy is the ability to detect fluorescence more sensitively, as well as more readily quan- tify and compare cellular fluorescence for different conditions. Following treatment of primary NHEK cultures and HaCaTa cells with doxorubicin and epirubicin, drug uptake was readily detectable by flow cytometry at normal temperature (37˚C); yet, cooling (22 and 18˚C) caused clear reduction in cellular drug uptake (Fig 3). We confirmed these findings by performing similar experiments using the original HaCaT cell line (S3 Fig). Notably, the lower temperature 18˚C was consistently more effective (than 22˚C) at reducing the amount of detected fluores- cence particularly in NHEK cells (as indicated by the greater ‘shift’ in the fluorescence histo- grams–Fig 3), which is in agreement with our observation that the temperature 18˚C was more effective at preventing NHEK culture cytotoxicity (Fig 1). Determination of the extent of cooling-mediated reduction in cellular drug uptake by semi-quantitative flow cytometry-based analysis in primary and immortalized human keratinocytes Determination of the extent of cooling-mediated reduction in cellular drug uptake by semi-quantitative flow cytometry-based analysis in primary and immortalized human keratinocytes We then exploited the advantage of flow cytometry in measuring drug-associated fluorescence to devise a semi-quantitative approach that permitted determination of the differences in the 7 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 PLOS ONE Cooling-mediated cytoprotection in keratinocytes by inhibition of chemotherapy drug uptake Fig 2. Determination of the effect of cooling on the cellular uptake of doxorubicin using fluorescence microscopy. NHEK (a) and HaCaTa (b) cells were treated with 1μM doxorubicin for 2-hours at normal (37˚C) or cooling (18˚C) conditions before being fixed and visualised by microscopy. Green fluorescence represents the presence of doxorubicin (larger images). Cell nuclei were visualised as blue by labelling with Hoechst 33258 following cell fixation (smaller images). Images are representative of results from three independent experiments. Scale bar: 50 μm. https://doi.org/10.1371/journal.pone.0240454.g002 Fig 3. Effect of cooling on the cellular uptake of doxorubicin and epirubicin detected by flow cytometry. NHEK and HaCaTa cells were treated with 1μM doxorubicin (upper panels) or 1μM epirubicin (lower panels) for 2-hours at normal (37˚C) and cooling (22 and 18˚C) conditions before drug-associated fluorescence was assessed by flow cytometry. Overlay histograms represent log10 median fluorescence intensity for each temperature condition and in each cell type as indicated. Results shown are representative of three independent experiments. https://doi.org/10.1371/journal.pone.0240454.g003 NE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 8 / 16 Fig 2. Determination of the effect of cooling on the cellular uptake of doxorubicin using fluorescence microscopy. NHEK (a) and HaCaTa (b) cells were treated with 1μM doxorubicin for 2-hours at normal (37˚C) or cooling (18˚C) conditions before being fixed and visualised by microscopy. Green fluorescence represents the presence of doxorubicin (larger images). Cell nuclei were visualised as blue by labelling with Hoechst 33258 following cell fixation (smaller images). Images are representative of results from three independent experiments. Scale bar: 50 μm. https://doi org/10 1371/journal pone 0240454 g002 Fig 2. Determination of the effect of cooling on the cellular uptake of doxorubicin using fluorescence microscopy. NHEK (a) and HaCaTa (b) cells were treated with 1μM doxorubicin for 2-hours at normal (37˚C) or cooling (18˚C) conditions before being fixed and visualised by microscopy. Green fluorescence represents the presence of doxorubicin (larger images). Cell nuclei were visualised as blue by labelling with Hoechst 33258 following cell fixation (smaller images). Images are representative of results from three independent experiments. Scale bar: 50 μm. Determination of the extent of cooling-mediated reduction in cellular drug uptake by semi-quantitative flow cytometry-based analysis in primary and immortalized human keratinocytes https://doi org/10 1371/journal pone 0240454 g002 p g j p g Fig 3. Effect of cooling on the cellular uptake of doxorubicin and epirubicin detected by flow cytometry. NHEK and HaCaTa cells were treated with 1μM doxorubicin (upper panels) or 1μM epirubicin (lower panels) for 2-hours at normal (37˚C) and cooling (22 and 18˚C) conditions before drug-associated fluorescence was assessed by flow cytometry. Overlay histograms represent log10 median fluorescence intensity for each temperature condition and in each cell type as indicated. Results shown are representative of three independent experiments. https://doi.org/10.1371/journal.pone.0240454.g003 Fig 3. Effect of cooling on the cellular uptake of doxorubicin and epirubicin detected by flow cytometry. NHEK and HaCaTa cells were treated with 1μM doxorubicin (upper panels) or 1μM epirubicin (lower panels) for 2-hours at normal (37˚C) and cooling (22 and 18˚C) conditions before drug-associated fluorescence was assessed by flow cytometry. Overlay histograms represent log10 median fluorescence intensity for each temperature condition and in each cell type as indicated. Results shown are representative of three independent experiments. https://doi.org/10.1371/journal.pone.0240454.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 8 / 16 PLOS ONE Cooling-mediated cytoprotection in keratinocytes by inhibition of chemotherapy drug uptake amount of drug uptake at normal temperature (37˚C) versus cooling conditions (22 and 18˚C). The results obtained for this analysis of both doxorubicin and epirubicin were from three independent experiments performed using 2–3 internal replicates and carried out in both primary NHEK cultures as well as immortalized HaCaTa cells. In addition, all average median fluorescence intensity (MFI) values were calculated following ‘blank correction’, i.e. by subtraction of MFI values of non-treated (control) cells values from treated cultures to account for possible variability in cell auto-fluorescence due to differences in temperature. To quantify differences in intracellular drug concentration, we initially measured MFI val- ues for cells treated with a series of defined doxorubicin and epirubicin concentrations at nor- mal temperature (37˚C). Representative fluorescence histograms from such experiments in NHEK cells are shown as overlay plots in S4 Fig (panels a and c) and the analysis of the epiru- bicin data set is presented in Fig 4, where MFI values for epirubicin concentrations 0.2, 0.4, 0.6, 0.8 and 1.0μM together with untreated controls (Fig 4A) were used to perform linear regression analysis. This identified a positive and linear correlation between [drug] (x-axis) and MFI (y-axis) (Fig 4B). Determination of the extent of cooling-mediated reduction in cellular drug uptake by semi-quantitative flow cytometry-based analysis in primary and immortalized human keratinocytes A maximal drug dose of 1.0μM was used in order to maintain a lin- ear relationship; above this concentration the detected fluorescence appeared to become Fig 4. Flow cytometry-based quantification of the cellular uptake of doxorubicin and epirubicin at normal versus cooling conditions in normal and immortalized keratinocytes. a) NHEK cells were treated with the indicated doses of epirubicin (0.2–1.0μM) alongside untreated control (Con) cells at 37˚C, before drug-associated fluorescence was detected by flow cytometry, as in Fig 3. The image is an overlay of the log10 median fluorescence intensity (MFI) histograms associated with each dose of epirubicin, as indicated. b) MFI values for independent replicate experiments (n = 3) performed as in (a) were plotted against the respective epirubicin concentration ([drug]) with the resulting equation from linear regression analysis included on the graph together with the R2 value. (c-d) The uptake of doxorubicin and epirubicin by NHEK and HaCaTa cells at normal (37˚C) and cooling conditions (22 and 18˚C) was determined by flow cytometry, before linear regression analysis was performed and fold decrease in drug uptake was calculated as explained in detail in the main text. Bars represent mean fold decrease (± SEM) in drug uptake for independent biological experiments (n = 3). Statistical significance (cooling condition versus 37˚C) is denoted as p<0.001 for each temperature and cell type. https://doi org/10 1371/journal pone 0240454 g004 Fig 4. Flow cytometry-based quantification of the cellular uptake of doxorubicin and epirubicin at normal versus cooling conditions in normal and immortalized keratinocytes. a) NHEK cells were treated with the indicated doses of epirubicin (0.2–1.0μM) alongside untreated control (Con) cells at 37˚C, before drug-associated fluorescence was detected by flow cytometry, as in Fig 3. The image is an overlay of the log10 median fluorescence intensity (MFI) histograms associated with each dose of epirubicin, as indicated. b) MFI values for independent replicate experiments (n = 3) performed as in (a) were plotted against the respective epirubicin concentration ([drug]) with the resulting equation from linear regression analysis included on the graph together with the R2 value. (c-d) The uptake of doxorubicin and epirubicin by NHEK and HaCaTa cells at normal (37˚C) and cooling conditions (22 and 18˚C) was determined by flow cytometry, before linear regression analysis was performed and fold decrease in drug uptake was calculated as explained in detail in the main text. PLOS ONE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 Actual drug concentration at cooling conditions Calculated drug concentration equivalent to 37C ¼ Fold decrease in drug uptake The data for all experiments in primary NHEK and HaCaTa keratinocytes are summarised in Fig 4C and 4D. It is noted that, rather than selecting specific doxorubicin and epirubicin concentrations to express representative fold decrease in drug uptake, individual fold differ- ences were first calculated for each drug concentration, i.e. 1, 2, 3, 4 and 5μM, and a mean value for all of these concentrations was deduced and presented per cooling temperature stud- ied (Fig 4C and 4D). We believe this provides a better quantification of the overall difference in drug uptake for the whole range of drug doses. The data shows that cooling can reduce drug uptake by a minimum of ~4-fold, and this can increase up to ~8-fold (Fig 4C and 4D) depending on the cell model used or drug tested. We observed differences between cooling conditions, for instance cooling at 22˚C reduced doxo- rubicin uptake in NHEK cultures by 4.6-fold whilst 18˚C caused a further reduction to 7.7-fold (Fig 4C); similarly, cooling at 22˚C reduced epirubicin uptake to 6.2-fold whilst 18˚C caused a further reduction to 7-fold. Notably, therefore, the lower temperature 18˚C caused more marked reduction in drug uptake (compared to 22˚C) particularly in NHEK cells, once again in line with our observation that 18˚C was consistently more effective at preventing NHEK culture cytotoxicity (Fig 1). PLOS ONE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 Determination of the extent of cooling-mediated reduction in cellular drug uptake by semi-quantitative flow cytometry-based analysis in primary and immortalized human keratinocytes Bars represent mean fold decrease (± SEM) in drug uptake for independent biological experiments (n = 3). Statistical significance (cooling condition versus 37˚C) is denoted as p<0.001 for each temperature and cell type. https://doi.org/10.1371/journal.pone.0240454.g004 https://doi.org/10.1371/journal.pone.0240454.g004 9 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 PLOS ONE Cooling-mediated cytoprotection in keratinocytes by inhibition of chemotherapy drug uptake saturated (not shown). For the data set shown in Fig 4A, the resulting equation is included on the graph (Fig 4B) together with the correlation coefficient (R2). To determine differences in drug uptake mediated by cooling, we also performed a series of treatments of keratinocytes with a range of high doxorubicin and epirubicin concentrations, i.e. 1, 2, 3, 4 and 5μM carried out under cooling conditions (22 and 18˚C) and measured cell fluorescence. Representative results from such experiments in NHEK cells at 18˚C are shown in S4 Fig (panels b and d). We used the average MFI values obtained for each drug concentra- tion under cooling conditions and based on each linear regression analysis (at 37˚C) we calcu- lated the equivalent drug concentration as follows: Average MFI under cooling conditions Gradient of linear slope ¼ Calculated drug concentration equivalent to 37C Average MFI under cooling conditions Gradient of linear slope ¼ Calculated drug concentration equivalent to 37C This allowed us to then determine differences in cellular drug concentration by calculating the “fold decrease” in drug uptake as follows: Actual drug concentration at cooling conditions Calculated drug concentration equivalent to 37C ¼ Fold decrease in drug uptake Investigation of the influence of cooling on drug-mediated hair follicle- derived ORSK cytotoxicity and quantification of cooling-mediated reduction in drug uptake Despite the robust and clinically-relevant observations obtained using primary and immortal- ized epidermal keratinocytes in this study and previously [13], these cells are not isolated from the keratinocyte niche of the HF. To circumvent this potential weakness in our approach, we employed previously reported methodologies [17, 19] to establish outer root sheath keratino- cyte (ORSK) cultures as a more physiologically-relevant in vitro cell model. When we treated ORSK cultures with a range of concentrations of doxorubicin at 37˚C, we observed dose- dependent reduction in cell viability (Fig 5A), however when ORSK cultures were treated with doxorubicin under cooling conditions (22 and 18˚C), cooling markedly attenuated drug-medi- ated cytotoxicity. Specifically, cooling at 22˚C caused a consistent reduction in cytotoxicity for low (<1μM) and high (>1μM) drug doses whilst, more strikingly, the 18˚C temperature appeared to completely inhibit drug-induced cytotoxicity for the whole dose range, including PLOS ONE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 10 / 16 Cooling-mediated cytoprotection in keratinocytes by inhibition of chemotherapy drug uptake PLOS ONE Fig 5. Cytoprotective effect of cooling against chemotherapy drug-mediated toxicity in hair follicle-derived keratinocytes and flow cytometry-based quantification of cellular doxorubicin uptake at normal versus cooling conditions. a) Hair follicle-derived ORSK cells were treated with a range of concentrations ([drug]) of doxorubicin at normal (37˚C) and cooling (22 and 18˚C) conditions and cell viability was assessed 72-hours post-treatment. Data points correspond to mean % cell viability (± SEM) for independent biological experiments (n = 3), each consisting of 5 technical replicates. Statistical significance (cooling conditions versus 37˚C) is denoted as p<0.05, p<0.01 and p<0.001, with stats symbols being colour-coded for each cooling temperature. b) ORSK cells were treated with 1μM doxorubicin for 2-hours at normal (37˚C) and cooling (22 and 18˚C) conditions before drug-associated fluorescence was assessed by flow cytometry. Overlay histograms represent log10 median fluorescence intensity for each temperature condition as indicated. Results shown are representative of three independent experiments. c) ORSK cells were treated with the indicated concentration of doxorubicin (0.2–1.0μM) alongside untreated control (Con) cells at 37˚C, before drug-associated fluorescence was detected by flow cytometry, as above (b). The image is an overlay of the log10 median fluorescence intensity (MFI) histograms associated with each dose of doxorubicin, as indicated. Investigation of the influence of cooling on drug-mediated hair follicle- derived ORSK cytotoxicity and quantification of cooling-mediated reduction in drug uptake d) MFI values for independent replicate experiments (n = 3) performed as in (c) were plotted against the respective doxorubicin concentration ([drug]) with the resulting equation from linear regression analysis included on the graph together with the R2 value. e) The uptake of doxorubicin by ORSK cells at normal (37˚C) and cooling conditions (22 and 18˚C) was determined by flow cytometry, before linear regression analysis was performed and fold decrease in drug uptake was calculated as explained in detail in the main text. Bars represent mean fold decrease (± SEM) in drug uptake for independent biological experiments (n = 3). Statistical significance (cooling condition versus 37˚C) is denoted as p<0.001 for each temperature. https://doi org/10 1371/journal pone 0240454 g005 ooling against chemotherapy drug-mediated toxicity in hair follicle-derived keratinocytes and flow cytometry-based bi i t k t l li diti ) H i f lli l d i d ORSK ll t t d ith f Fig 5. Cytoprotective effect of cooling against chemotherapy drug-mediated toxicity in hair follicle-derived keratinocytes and flow cytometry-based quantification of cellular doxorubicin uptake at normal versus cooling conditions. a) Hair follicle-derived ORSK cells were treated with a range of concentrations ([drug]) of doxorubicin at normal (37˚C) and cooling (22 and 18˚C) conditions and cell viability was assessed 72-hours post-treatment. Data points correspond to mean % cell viability (± SEM) for independent biological experiments (n = 3), each consisting of 5 technical replicates. Statistical significance (cooling conditions versus 37˚C) is denoted as p<0.05, p<0.01 and p<0.001, with stats symbols being colour-coded for each cooling temperature. b) ORSK cells were treated with 1μM doxorubicin for 2-hours at normal (37˚C) and cooling (22 and 18˚C) conditions before drug-associated fluorescence was assessed by flow cytometry. Overlay histograms represent log10 median fluorescence intensity for each temperature condition as indicated. Results shown are representative of three independent experiments. c) ORSK cells were treated with the indicated concentration of doxorubicin (0.2–1.0μM) alongside untreated control (Con) cells at 37˚C, before drug-associated fluorescence was detected by flow cytometry, as above (b). The image is an overlay of the log10 median fluorescence intensity (MFI) histograms associated with each dose of doxorubicin, as indicated. d) MFI values for independent replicate experiments (n = 3) performed as in (c) were plotted against the respective doxorubicin concentration ([drug]) with the resulting equation from linear regression analysis included on the graph together with the R2 value. PLOS ONE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 Investigation of the influence of cooling on drug-mediated hair follicle- derived ORSK cytotoxicity and quantification of cooling-mediated reduction in drug uptake e) The uptake of doxorubicin by ORSK cells at normal (37˚C) and cooling conditions (22 and 18˚C) was determined by flow cytometry, before linear regression analysis was performed and fold decrease in drug uptake was calculated as explained in detail in the main text. Bars represent mean fold decrease (± SEM) in drug uptake for independent biological experiments (n = 3). Statistical significance (cooling condition versus 37˚C) is denoted as p<0.001 for each temperature. https://doi.org/10.1371/journal.pone.0240454.g005 high doses (5μM). These results are in concordance with our findings with NHEK cells, although clearly cooling at 18˚C was effective in protecting ORSK cultures, and at high drug doses (Fig 5A) was more effective than was observed in NHEK (or even HaCaTa) cells (Fig 1A and 1B). Moreover, our findings extend our previous observations using commercially-avail- able Human Hair Follicular Keratinocyte (HHFK) cultures treated with doxorubicin at 22˚C [13], and reveal that an additional 4˚C temperature reduction (18˚C) renders cooling extremely effective at protecting against doxorubicin-mediated cytotoxicity in HF-derived keratinocytes. ORSK cultures were then treated with doxorubicin to detect drug uptake by flow cytometry and assess the effect of cooling. Doxorubicin cellular uptake was readily detectable at normal temperature (37˚C), and cooling (22 and 18˚C) caused clear reduction in drug uptake (Fig 5B), although the lower 18˚C temperature appeared to inhibit drug uptake less effectively than 22˚C. We applied our semi-quantitative flow cytometry-based approach to carry out a linear regression analysis and determine the relative difference in drug uptake in ORSK cultures treated with doxorubicin at normal temperature (37˚C) versus cooling conditions (22 and PLOS ONE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 11 / 16 PLOS ONE Cooling-mediated cytoprotection in keratinocytes by inhibition of chemotherapy drug uptake 18˚C). By carrying out experiments with the same dose ranges as for the experiments with NHEK and HaCaTa cultures above (Fig 4), we first demonstrated a linear relationship between drug concentration and MFI values (Fig 5C and 5D). Then, by employing the experimental methodology and calculations detailed in the previous section, we utilised linear regression analysis to deduce fold differences in drug uptake in 37˚C versus cooling conditions. We found that at 22˚C ORSK cultures exhibited ~5-fold reduced doxorubicin uptake, whilst the reduction in drug uptake at 18˚C was ~4-fold (Fig 5E). Discussion Scalp cooling represents the only available treatment against CIA [7], but its mechanisms of cytoprotection remain unknown. In this study we have systematically explored for the first time the hypothesis that cooling reduces the entry of chemotherapy drugs in human keratino- cytes. We used primary NHEK cultures as well as immortalized HaCaTa, alongside HF- derived ORSK cultures and assessed different temperatures, by studying doxorubicin and epir- ubicin. Doxorubicin can be detected by fluorescence microscopy in mammalian cells and that it diffuses primarily to the nucleus [15], and flow cytometry-based detection has been used [24]. Detection of epirubicin by flow cytometry and nuclear localization by microscopy have also been reported [16, 25]. The main mechanism of action of doxorubicin is DNA intercala- tion and genotoxic damage consistent with its nuclear localization [26]. We have demonstrated that doxorubicin and epirubicin are concentrated in the nuclei of NHEK and HaCaTa cells and are highly cytotoxic at 37˚C; however, cooling markedly reduced chemotherapy drug entry, which coincided with its ability to reduce or prevent drug-mediated keratinocyte cytotoxicity. We showed reduction in drug uptake and nuclear localization quali- tatively by fluorescence microscopy, and also devised a methodology for semi-quantitative analysis of differences in drug uptake at different culture (temperature) conditions using flow cytometry. The approach allowed us to determine that cooling can reduce drug uptake by up to ~8-fold, with cooling to 18˚C showing a stronger ability (compared to 22˚C) to reduce drug uptake in NHEK and HaCaTa cells, in line with our observations on the effects of cooling on drug-mediated cytotoxicity. The way by which cooling reduces drug uptake remains unknown. In fact, it is still to be established by what mechanism anthracyclines such as doxorubicin and epirubicin move across the plasma membrane. As both drugs are moderately lipophilic (logP of doxorubicin is 1.27 and epirubicin 1.41), they could at least to some extent penetrate the phospholipid bilayer, as confirmed for doxorubicin in model phospholipid membranes [27]. Intracellular accumula- tion of doxorubicin was reported in pig kidney LLC-PK1 cells even after inhibition of drug transporters [28]. However, as with other anthracyclines, doxorubicin cellular uptake can be mediated by transport proteins, the organic anion-transporting proteins SLC22A16 and OATP1A2, OATP1B1, OATP1B3 [29], thus expression of these in the HF cell niche could enhance the uptake of doxorubicin into the cell by an active process. Investigation of the influence of cooling on drug-mediated hair follicle- derived ORSK cytotoxicity and quantification of cooling-mediated reduction in drug uptake Our findings in ORSK cells confirmed that the ability of cooling to rescue dermal and follicular keratinocytes from anthracycline- mediated cytotoxicity directly coincided with its capacity to suppress cellular drug uptake. Yet, in ORSK cultures lowering the temperature from 22˚C to 18˚C did not cause any further reduction in drug uptake, despite the finding that 18˚C was far more effective at protecting from drug-mediated cytotoxicity (Fig 5A). PLOS ONE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 Discussion Nevertheless, irrespective of how anthracyclines enter the cell, uptake is expected to be temperature-modulated as pas- sive permeability of compounds across phospholipid bilayers is affected by temperature. Indeed, a 2-fold increase in doxorubicin permeability was observed when culture temperature was increased from 37˚C to 47˚C in CHO cells [30] and in Sarcoma-180 cells [31]. By contrast, Indeed, a 2-fold increase in doxorubicin permeability was observed when culture temperature was increased from 37˚C to 47˚C in CHO cells [30] and in Sarcoma-180 cells [31]. By contrast, PLOS ONE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 12 / 16 PLOS ONE Cooling-mediated cytoprotection in keratinocytes by inhibition of chemotherapy drug uptake reducing the temperature in LLC-PK1 cells from 37˚C to 4˚C resulted in a 4.5-fold reduction in doxorubicin uptake in the absence of any evidence for active drug uptake [28]. If either doxorubicin or epirubicin are substrates for one or more specific transport protein(s) that facilitate(s) cellular entry, one could assume that lowering the metabolic rate of cells by cooling would lead to a reduction in the transporter mediated drug uptake. Yet, previous studies have shown that doxorubicin entry shows no signs of plateau [32] and passive diffusion appeared to be the main mode of transport [33]. Equally importantly, the latter study provided evidence for temperature-dependent structural changes to the plasma membrane, with changes being observed at temperatures below 22˚C [33]. Interestingly, and perhaps unexpectedly, in ORSK cultures lowering the temperature from 22˚C to 18˚C did not cause any further reduction in drug uptake, despite that 18˚C was far more effective at protecting from doxorubicin cytotoxicity. We believe that these observations are not in disagreement with the results from NHEK cultures. Instead, the findings using this more physiological-relevant cell type provide evidence that, although cooling is protective and this property coincides with reduction in drug uptake, blockade of drug uptake is most proba- bly not the only cooling-associated mechanism and it operates in concert with other mecha- nisms; for instance, mild hypothermia has been reported to be cytoprotective against a variety of cytotoxic stimuli [34]. We believe that the ability of cooling to cytoprotect is underpinned by a combination of mechanisms, which may include reduction in cell division rate and atten- uation of enzymatic pathways that mediate cytotoxicity (apoptosis/necrosis). Discussion Our flow cytometry-based semi-quantitative detection system has been a useful tool in determining differences in anthracycline uptake in human keratinocytes and shows a high level of sensitivity. Our analysis could distinguish between intracellular doxorubicin and epiru- bicin concentrations as little as 0.1μM and with consistency and sensitivity across a range of keratinocyte types (primary and immortalized). We believe this system could also be exploited in other contexts to determine anthracycline drug entry in other cell types whether normal or malignant. For instance, anthracycline-mediated cardiovascular toxicity [35] and cardiomyop- athy [36] represent an important area of research, with doxorubicin in particular causing long- term toxicity and cardiomyopathy [37]. Thus, our flow cytometry-based approach may permit measurements of drug uptake in cell types, such as primary human cardiomyocytes or vascular endothelial cells, as well as offering the opportunity to test strategies that can reduce drug uptake/toxicity and confirm their potential efficacy. The ability of cooling to attenuate cellular drug uptake represents at least one of the mecha- nisms that underpin the clinical efficacy of scalp cooling in reducing or even preventing CIA by protecting cells from the toxicity of chemotherapy drugs. It is tempting to speculate that the ability of cooling to reduce drug uptake is not limited to anthracyclines as shown here, but also extends to other types of chemotherapy drugs, although further studies would be necessary to address this hypothesis. Supporting information S1 Fig. Cytoprotective effect of cooling against chemotherapy drug-mediated toxicity in HaCaT cells. HaCaT cells were treated with a range of concentrations ([drug]) of doxorubicin and epirubicin at normal (37˚C) and cooling (22 and 18˚C) conditions and cell viability was assessed 72-hours post-treatment. Data points correspond to mean % cell viability (± SEM) for independent biological experiments (n = 3), each consisting of 5 technical replicates. (TIF) S2 Fig. Detection of the effect of cooling on the cellular uptake of epirubicin using fluores- cence microscopy. NHEK (a) and HaCaTa (b) cells were treated with the indicated S3 Fig. Effect of cooling on the uptake of doxorubicin and epirubicin in HaCaT cells S3 Fig. Effect of cooling on the uptake of doxorubicin and epirubicin in HaCaT cells detected by flow cytometry. HaCaT cells were treated with 1μM doxorubicin or 1μM epirubi- cin for 2-hours at normal (37˚C) and cooling (22 and 18˚C) conditions before drug-associated fluorescence was assessed by flow cytometry. Overlay histograms represent log10 median fluo- rescence intensity for each temperature condition as indicated. Results shown are representa- tive of three independent experiments. (TIF) S4 Fig. Flow cytometry-based detection of the cellular uptake of low and high doses of epir- ubicin and doxorubicin at normal versus cooling temperature conditions in normal kerati- nocytes. NHEK cells were treated with the indicated concentrations of i) epirubicin alongside untreated control (0.0μM) cells at 37˚C (a) and 18˚C (b), or ii) doxorubicin alongside controls (0.0μM) at 37˚C (c) and 18˚C (d). Drug-associated fluorescence was detected by flow cytome- try. The panels represent overlays of the log10 median fluorescence intensity (MFI) histograms obtained for each drug dose, as indicated. Results are representative of three independent experiments. Acknowledgments We are grateful to our clinical colleagues for the provision of primary skin tissue specimens. S2 Fig. Detection of the effect of cooling on the cellular uptake of epirubicin using fluores- cence microscopy. NHEK (a) and HaCaTa (b) cells were treated with the indicated 13 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0240454 October 15, 2020 PLOS ONE Cooling-mediated cytoprotection in keratinocytes by inhibition of chemotherapy drug uptake concentration of epirubicin for 2-hours at normal (37˚C) or cooling (18˚C) conditions before being visualised by live fluorescence microscopy. Green fluorescence represents the presence of epirubicin. Images of untreated NHEK and HaCaTa cells (denoted ‘Control’) were included. Scale bar: 50 μm. (TIF) References 1. Choi EK, Kim IR, Chang O, Kang D, Nam SJ, Lee JE, et al. Impact of chemotherapy-induced alopecia distress on body image, psychosocial well-being, and depression in breast cancer patients. Psychoon- cology. 2014; 23(10):1103–10. https://doi.org/10.1002/pon.3531 PMID: 24664939 2. Pickard-Holley S. The symptom experience of alopecia. Semin Oncol Nurs. 1995; 11(4):235–8. https:// doi.org/10.1016/s0749-2081(05)80003-8 PMID: 8578030 3. 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https://openalex.org/W1921841920	https://europepmc.org/articles/pmc4558149?pdf=render	English	null	The c-Jun/RHOB/AKT pathway confers resistance of<i>BRAF</i>-mutant melanoma cells to MAPK inhibitors	Oncotarget	2,015	cc-by	11,516	Audrey Delmas1,2,3, Julia Cherier1,3, Magdalena Pohorecka1,2,3, Claire Medale- Giamarchi1,2,3, Nicolas Meyer1,2,4, Anne Casanova3, Olivier Sordet1,3, Laurence Lamant1,5, Ariel Savina6, Anne Pradines1,2,3, Gilles Favre1,2,3 1Inserm, UMR 1037-CRCT, Toulouse, France 2Université Paul Sabatier, Toulouse, France 3I tit t Cl di R d I tit t U i it i d C d T l O l L b t f M di l Bi l d Audrey Delmas1,2,3, Julia Cherier1,3, Magdalena Pohorecka1,2,3, Claire Medale- Giamarchi1,2,3, Nicolas Meyer1,2,4, Anne Casanova3, Olivier Sordet1,3, Laurence Lamant1,5, Ariel Savina6, Anne Pradines1,2,3, Gilles Favre1,2,3 1Inserm, UMR 1037-CRCT, Toulouse, France 2Université Paul Sabatier, Toulouse, France 3I tit t Cl di R d I tit t U i it i d C d T l O l L b t f M di l Bi l d 4Centre Hospitalo-Universitaire de Toulouse, Institut Universitaire du Cancer de Toulouse-Oncopole, Department of Dermatology, Toulouse, France 4Centre Hospitalo-Universitaire de Toulouse, Institut Universitaire du Cancer de Toulouse-Oncopole, Department of Dermatology, Toulouse, France 5Centre Hospitalo-Universitaire de Toulouse, Institut Universitaire du Cancer de Toulouse-Oncopole, Department of Pathology, Toulouse, France 5Centre Hospitalo-Universitaire de Toulouse, Institut Universitaire du Cancer de Toulouse-Oncopole, Department of Pathology, Toulouse, France 6i 6Scientific Partnerships, Roche SAS, Boulogne Billancourt, France 6Scientific Partnerships, Roche SAS, Boulogne Billancourt, Fran Correspondence to: Gilles Favre, e-mail: favre.gilles@iuct-oncopole.fr Keywords: melanoma, RHOB, AKT, vemurafenib, resistance Published: May 07, 2015 Published: May 07, 2015 Received: January 19, 2015 Accepted: April 25, 2015 Received: January 19, 2015 Accepted: April 25, 2015 ABSTRACT The response of BRAF-mutant melanoma patients to BRAF inhibitors is dramatically impaired by secondary resistances and rapid relapse. So far, the molecular mechanisms driving these resistances are not completely understood. Here, we show that, in BRAF-mutant melanoma cells, inhibition of BRAF or its target MEK induces RHOB expression by a mechanism that depends on the transcription factor c-Jun. In those cells, RHOB deficiency causes hypersensitivity to BRAF and MEK inhibitors-induced apoptosis. Supporting these results, loss of RHOB expression in metastatic melanoma tissues is associated with an increased progression-free survival of BRAF-mutant patients treated with vemurafenib. Following BRAF inhibition, RHOB activates AKT whose inhibition causes hypersensitivity of BRAF-mutant melanoma cells to BRAF inhibitors. In mice, AKT inhibition synergizes with vemurafenib to block tumor growth of BRAF-mutant metastatic melanoma. Our findings reveal that BRAF inhibition activates a c-Jun/RHOB/AKT pathway that promotes tumor cell survival and further support a role of this pathway in the resistance of melanoma to vemurafenib. Our data also highlight the importance of using RHOB tumor levels as a biomarker to predict vemurafenib patient’s response and to select those that would benefit of the combination with AKT inhibitors. Oncotarget, Vol. 6, No. 17 Oncotarget, Vol. 6, No. 17 www.impactjournals.com/oncotarget/ INTRODUCTION respectively) emerged as a therapeutic strategy [2]. The first-in-class specific BRAFi, vemurafenib (PLX4032), led to an unprecedented response for 80% patients in phase III clinical trials [3, 4]. However, the improvement of disease- free survival and global survival was disappointingly weak, due to the rapid acquisition of resistance, opening the way to the discovery of new classes of MAPK inhibitors [5] or to BRAFi combinations with cytotoxic drugs [6]. The hypothesis of melanoma addiction to the RAF/ MEK/ERK pathway, namely the MAPK pathway, emerged with the discovery of a high frequency of the BRAFV600E- activating mutation in melanoma cell lines and primary tumors [1]. Since then, evidence of melanoma dependency on MAPK pathway accumulated and its inhibition by either BRAF or MEK inhibitors (BRAFi and MEKi, www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 15250 Intensive investigations led to identify several resistance mechanisms on the basis of in vitro resistant cell lines and clinical studies. Most of them reactivate the MAPK and/or PI3K/AKT pathways by acquisition of RAS and MEK mutations [7, 8], or over-expression of COT [9], CRAF or growth factor receptor such as FGFR3, EGFR, PDGFRα, PDGFRβ [7, 10–13]. Moreover, MAPK inhibition modulates transcription factors expression that could regulate adaptive responses [14]. Although their roles are not clearly defined, the genes whose expression is modulated could impact on the sensitivity of melanoma cells to BRAFi and therefore could be of clinical relevance. In line with this hypothesis, the transcription factor FOXD3 and the pro-apoptotic BH3-only protein NOXA, are induced and down-regulated, respectively following PLX4032 treatment thus promoting resistance to cell death [15, 16]. Hence, MAPK inhibition can regulate per se the expression of resistance factors. than BRAFi alone on BRAF-mutant cells. Finally, we demonstrate that the expression of RHOB in melanoma tissues determines a poor clinical response to PLX4032. Altogether, these findings support a potential role for RHOB as a predictive biomarker to PLX4032 therapy and the c-Jun/RHOB/AKT signaling axis as a new target to prevent resistance to BRAFi of BRAF-mutant melanoma tumors. RHOB expression is induced by the BRAFi PLX4032 in BRAF-mutant melanoma cells We first analyzed RHOGTPase transcripts in the metastatic BRAFV600X-mutant melanoma cell lines WM266-4 and A375 following a 48 h treatment with PLX4032. In both cell lines, RAC1b mRNA levels decreased while those of RHOB, RHOJ and RHOQ increased (Figure 1A and 1B). RHOB was the most induced gene with a factor of 5.7 ± 1.2 in WM266-4 cells (Figure 1A) and of 2.0 ± 0.3 in A375 cells (Figure 1B). In those two cell lines, the increase in the RHOB mRNA level was associated with an increase in the RHOB protein level (Figure 1C). PLX4032 treatment also increased RHOB protein level in six other BRAF-mutant melanoma cell lines, which are representative of melanoma progression (RGP-VGP to metastatic) (Figure 1D). RHOGTPases are regulated by many signaling pathways, such as the MAPK pathway, and control numerous cellular functions including the balance between survival and apoptosis [17]. In that way, they may impact on the cellular response to MAPK inhibitors. Cross- regulations between RHOGTPases and MAPK pathways have been reported [18, 19] and several members of the RHOGTPase family have been involved in apoptosis inhibition to both chemotherapies and targeted therapies. For instance, RHOJ mediates melanoma cell resistance to dacarbazine [20], RAC1 is involved in breast cancer cell response to trastuzumab [21] and RHOE/RND3 enhances multidrug resistance in gastric cancer cells [22]. In addition, inhibition of MAPK pathway has an impact on the regulation of the expression of RHOGTPase genes. This may result in a modulation of the tumor cell sensitivity to MAPK inhibitors, as demonstrated for RHOE/RND3, which impedes melanoma cell invasion in response to PLX4032 [23]. RHOGTPases are regulated by many signaling pathways, such as the MAPK pathway, and control numerous cellular functions including the balance between survival and apoptosis [17]. In that way, they may impact on the cellular response to MAPK inhibitors. Cross- regulations between RHOGTPases and MAPK pathways have been reported [18, 19] and several members of the RHOGTPase family have been involved in apoptosis inhibition to both chemotherapies and targeted therapies. For instance, RHOJ mediates melanoma cell resistance to dacarbazine [20], RAC1 is involved in breast cancer cell response to trastuzumab [21] and RHOE/RND3 enhances multidrug resistance in gastric cancer cells [22]. In addition, inhibition of MAPK pathway has an impact on the regulation of the expression of RHOGTPase genes. RHOB expression is induced by the BRAFi PLX4032 in BRAF-mutant melanoma cells This may result in a modulation of the tumor cell sensitivity to MAPK inhibitors, as demonstrated for RHOE/RND3, which impedes melanoma cell invasion in response to PLX4032 [23]. The increase in RHOB expression in WM266-4 cells treated with PLX4032 for 48 h was dose-dependent, reaching a maximum at 1 μM, and was coincident with the inhibition of ERK phosphorylation (Figure 1E and 1F). At this PLX4032 concentration, RHOB induction was clearly detected after 24 h and increased over time (Figures 1F and S1). In contrast, the two RHOB homologs RHOA and RHOC were not induced (Figures 1F and S1). To determine whether continuous exposure to PLX4032 was necessary to maintain the observed elevated expression of RHOB, we examined RHOB expression following PLX4032 removal. We found that RHOB expression was maintained and did not return to its baseline levels even 96 h after termination of the PLX4032 treatment (Figure 1G). As a control, ERK phosphorylation was recovered after 24 h indicating that BRAF inhibition was indeed obliterated (Figure 1G). These results suggest that although BRAF inhibition is required to prime RHOB induction, an adaptive mechanism of melanoma cells may occur following PLX4032 treatment to maintain high levels of RHOB. We conclude that PLX4032 triggers a sustained induction of RHOB expression in BRAF-mutant melanoma cells. We therefore investigated the role of RHOGTPases in melanoma cell response to PLX4032 and others inhibitors of the MAPK pathway. Using RT-qPCR screening, we detected a significant induction of RHOB expression upon PLX4032 treatment in BRAF-mutant melanoma cells. RHOB is well known to be modulated in response to anticancer therapies and to control tumor cell response to ionizing radiation and cytotoxic drugs [24–26]. Moreover, RHOB has also been involved in the response to targeted therapy as described by Vishnu and colleagues in ovarian cells [27]. Here, we show that in BRAF-mutant melanoma cells, PLX4032 induces c-Jun activation resulting in RHOB expression and subsequent AKT activation. RHOB expression is induced by the BRAFi PLX4032 in BRAF-mutant melanoma cells B A CDC42 RAC1 RAC1b RAC2 RAC3 RHOA RHOB RHOC RND1 RND2/RHON RND3/RHOE RHOF RHOJ RHOQ RHOU RHOV 0 1 2 3 4 5 6 7 * * * *** * ** * CDC42 RAC1 RAC1b RAC2 RAC3 RHOA RHOB RHOC RND1 RND2/RHON RND3/RHOE RHOF RHOJ RHOQ RHOU 0 1 2 3 4 5 6 7 * ** * * * WM266-4 DMSO PLX4032 A375 DMSO PLX4032 B CDC42 RAC1 RAC1b RAC2 RAC3 RHOA RHOB RHOC RND1 RND2/RHON RND3/RHOE RHOF RHOJ RHOQ RHOU RHOV 0 1 2 3 4 5 6 7 * * * *** * ** * DMSO PLX4032 A375 A B C WM266-4 - + A375 - + RHOB p-ERK ERK PLX4032 Fold inducon 1 4.5±0.5 1 1.9±0.2 C C D G F D Metastatic RGP-VGP RHOB ERK WM35 - + WM239A SK-MEL28 WM983A - + WM983B WM115 - + PLX4032 - + - + - + p-ERK F 0 2 6 12 24 36 48 72 DMSO PLX4032 treatment (hours) RHOB ERK RHOA RHOC Actin p-ERK G 0 24 48 72 96 DMSO RHOB ERK Actin PLX4032 removal (hours) p-ERK E RHOB ERK Actin DMSO Untreated 0.1 0.2 0.5 1 2 PLX4032 (µM) p-ERK G G F F E Figure 1: PLX4032 increases RHOB expression in BRAFV600E melanoma cells. A and B. RT-qPCR analysis of RHOGTPase transcripts in WM266-4 and A375 cells treated with 1 μM PLX4032 for 48 h. C and D. Western blotting of RHOB, p-ERK and ERK in BRAF-mutant melanoma cells treated with 1 μM PLX4032 for 48 h. E and F. Western blotting of the indicated proteins in WM266-4 cells treated with the indicated PLX4032 concentrations for 48 h (E) and for the indicated times with 1 μM PLX4032 (F). G. WM266-4 cells were treated with 1 μM PLX4032 for 48 h, then washed and cultured in PLX4032-free medium for the indicated times. RHOB, RHOA, RHOC, p-ERK and ERK were analyzed by Western blotting. Actin was the loading control. RHOB expression is induced by the BRAFi PLX4032 in BRAF-mutant melanoma cells We demonstrate that pharmacological association of BRAFi with AKT inhibitors (AKTi) displays a higher antitumor activity www.impactjournals.com/oncotarget Oncotarget 15251 B A E CDC42 RAC1 RAC1b RAC2 RAC3 RHOA RHOB RHOC RND1 RND2/RHON RND3/RHOE RHOF RHOJ RHOQ RHOU RHOV 0 1 2 3 4 5 6 7 * * * *** * ** * G F 0 2 6 12 24 36 48 72 DMSO PLX4032 treatment (hours) RHOB ERK RHOA RHOC Actin RHOB ERK Actin DMSO Untreated 0.1 0.2 0.5 1 2 PLX4032 (µM) 0 24 48 72 96 DMSO RHOB ERK Actin PLX4032 removal (hours) C WM266-4 - + A375 - + RHOB p-ERK ERK PLX4032 D Metastatic RGP-VGP RHOB ERK WM35 - + WM239A SK-MEL28 WM983A - + WM983B WM115 - + PLX4032 - + - + - + CDC42 RAC1 RAC1b RAC2 RAC3 RHOA RHOB RHOC RND1 RND2/RHON RND3/RHOE RHOF RHOJ RHOQ RHOU 0 1 2 3 4 5 6 7 * ** * * * WM266-4 DMSO PLX4032 A375 DMSO PLX4032 Fold inducon 1 4.5±0.5 1 1.9±0.2 p-ERK p-ERK p-ERK p-ERK Figure 1: PLX4032 increases RHOB expression in BRAFV600E melanoma cells. A and B. RT-qPCR analysis of RHOGTPase transcripts in WM266-4 and A375 cells treated with 1 μM PLX4032 for 48 h. C and D. Western blotting of RHOB, p-ERK and ERK in BRAF-mutant melanoma cells treated with 1 μM PLX4032 for 48 h. E and F. Western blotting of the indicated proteins in WM266-4 cells treated with the indicated PLX4032 concentrations for 48 h (E) and for the indicated times with 1 μM PLX4032 (F). G. WM266-4 cells were treated with 1 μM PLX4032 for 48 h, then washed and cultured in PLX4032-free medium for the indicated times. RHOB, RHOA, RHOC, p-ERK and ERK were analyzed by Western blotting. Actin was the loading control. MAPK inhibition triggers RHOB induction in BRAF-mutant melanoma cells We therefore examined the impact of PLX4032 and MEKi on RHOB expression in wild type BRAF melanoma cells harboring mutations in NRAS (WM1346 and SK-MEL2 cells), KIT (WM3211 cells) or www.impactjournals.com/oncotarget Oncotarget 15252 A ERK RHOB B C RHOB ERK BRAF WM1346 WM3211 SK-MEL2 WM1791C ERK RHOB Colo741 Colo205 AZD6244 (µM) - - - 1 5 - - - 1 5 PLX4032 (µM) - 1 5 - - - 1 5 - - ERK RHOB D AZD6244 - - + - - + - - + - - + PLX4032 - + - - + - - + - - + - si-BRAF2 - - + si-BRAF1 - + - si-Ctl + - - p-ERK p-ERK p-ERK p-ERK NT DMSO PLX4032 SB590885 AZD6244 AS703026 Figure 2: Inhibition of the BRAF/MEK/ERK pathway induces RHOB in melanoma cells. A and B. Western blotting of the indicated proteins in WM266-4 cells transfected with BRAF-targeting (si-BRAF1 and si-BRAF2) or non-targeting (si-Ctl) siRNAs (A) or treated for 48 h with 1 μM of BRAF (PLX4032 or SB590885) or MEK (AZD6244 or AS703026) inhibitors (B). C. Western blotting of RHOB, p-ERK and ERK in wild-type BRAF melanoma cell lines treated with 1 μM PLX4032 or AZD6244 for 48 h. D. Western blotting of RHOB, p-ERK and ERK in BRAFV600E colon cell lines treated with the indicated PLX4032 or AZD6244 concentrations for 48 h. A ERK RHOB B RHOB ERK BRAF si-BRAF2 - - + si-BRAF1 - + - si-Ctl + - - p-ERK p-ERK NT DMSO PLX4032 SB590885 AZD6244 AS703026 A RHOB ERK BRAF si-BRAF2 - - + si-BRAF1 - + - si-Ctl + - - p-ERK ERK RHOB B p-ERK NT DMSO PLX4032 SB590885 AZD6244 AS703026 B A C WM1346 WM3211 SK-MEL2 WM1791C ERK RHOB AZD6244 - - + - - + - - + - - + PLX4032 - + - - + - - + - - + - p-ERK C Colo741 Colo205 AZD6244 (µM) - - - 1 5 - - - 1 5 PLX4032 (µM) - 1 5 - - - 1 5 - - ERK RHOB D p-ERK D Figure 2: Inhibition of the BRAF/MEK/ERK pathway induces RHOB in melanoma cells. A and B. MAPK inhibition triggers RHOB induction in BRAF-mutant melanoma cells KRAS (WM1791C cells). Consistent with the selectivity of PLX4032 for BRAF-mutant cells, it failed to inhibit MAPK signaling in these four cell lines, as shown by the lack of decrease in ERK phosphorylation, and the lack of RHOB induction (Figure 2C). In contrast, the MEKi AZD6244 inhibited ERK phosphorylation and induced RHOB expression (Figure 2C). Taken together, these results indicate that the BRAF/MEK pathway regulates RHOB expression. KRAS (WM1791C cells). Consistent with the selectivity of PLX4032 for BRAF-mutant cells, it failed to inhibit MAPK signaling in these four cell lines, as shown by the lack of decrease in ERK phosphorylation, and the lack of RHOB induction (Figure 2C). In contrast, the MEKi AZD6244 inhibited ERK phosphorylation and induced RHOB expression (Figure 2C). Taken together, these results indicate that the BRAF/MEK pathway regulates RHOB expression. Next we addressed whether RHOB induction by PLX4032 in BRAF-mutant melanoma cells was related to BRAF inactivation. We found that BRAF inhibition with siRNA or with the PLX4032-unrelated BRAFV600E inhibitor SB590885 increased RHOB protein levels in WM266-4 cells (Figure 2A, 2B). Inhibition of the BRAF substrate MEK with AZD6244 or AS703026 also resulted in an increased RHOB expression in those cells (Figure 2B). These results indicate that BRAF inactivation causes RHOB overexpression in BRAF-mutant melanoma cells. The induction of RHOB after MAPK pathway inhibition suggests that this phenomenon could be a common feature for BRAF-mutant tumor cells. We therefore tested the effect of PLX4032 and AZD6244 treatment on BRAFV600E-mutant colon cancer cells. As shown in Figure 2D, efficient inhibition of MAPK signaling with PLX4032 or AZD6244, demonstrated by inhibition of ERK phosphorylation, did not significantly affect RHOB expression even with a higher dose (5 μM) than that used in melanoma cells (1 μM). These results suggest that RHOB modulation by MAPK signaling is not a common mechanism in BRAF-mutant cells but a feature of specific tumors such as melanoma. In melanomas, the MAPK pathway is frequently hyperactivated by mutations in the BRAF gene (approximately 50% of melanomas) but also in NRAS (18%), KIT (9%), HRAS (2%) or KRAS (2%) genes (COSMIC database). MAPK inhibition triggers RHOB induction in BRAF-mutant melanoma cells Western blotting of the indicated proteins in WM266-4 cells transfected with BRAF-targeting (si-BRAF1 and si-BRAF2) or non-targeting (si-Ctl) siRNAs (A) or treated for 48 h with 1 μM of BRAF (PLX4032 or SB590885) or MEK (AZD6244 or AS703026) inhibitors (B). C. Western blotting of RHOB, p-ERK and ERK in wild-type BRAF melanoma cell lines treated with 1 μM PLX4032 or AZD6244 for 48 h. D. Western blotting of RHOB, p-ERK and ERK in BRAFV600E colon cell lines treated with the indicated PLX4032 or AZD6244 concentrations for 48 h. c-Jun is involved in PLX4032-induced RHOB expression Together, these data indicate that PLX4032 induces www.impactjournals.com/oncotarget Oncotarget 15253 0 2 4 6 8 10 12 14 pGL3-vide pGL3-SV40 -1766 -1219 -681 -195 -45 ns * ** ns * *** LucF / LucR B A C LucF/LucR activity DMSO PLX4032 0 1 2 3 4 * DMSO PLX4032 A B A LucF/LucR activity DMSO LX4032 0 1 2 3 4 * B LucF/LucR activity DMSO PLX4032 0 1 2 3 4 * B A 15254 ournals.com/oncotarget 0 2 4 6 8 10 12 14 pGL3-vide pGL3-SV40 -1766 -1219 -681 -195 -45 ns * ** ns * *** LucF / LucR 0 2 4 6 8 10 12 14 16 18 20 LucF / LucR * ns ns C D ATTGG iCCAAT box TTTAG TGCGG LucF/LucR activity DMSO PLX4032 0 1 2 3 4 * E p-c-Jun PLX4032 (hours) 0 2 6 12 24 48 c-Jun RHOB p-ERK ERK Actin F p-ERK p-c-Jun c-Jun DMSO PLX4032 si-Ctl + - + - si-c-Jun - + - + RHOB ERK Actin DMSO PLX4032 DMSO PLX4032 RHOB is induced by PLX4032 through transcriptional activation in an iCCAAT and c-Ju After a 48 h incubation of WM266-4 cells with PLX4032 (1 μM), RHOB mRNA half-life was measured by R on inhibitor actinomycin D (5 μg/mL) and normalized to the long half-life of actin mRNA (one representativ ase assays in WM266-4 cells co-transfected with RHOB promoter-firefly luciferase constructs and pRL h PLX4032 (2 μM, 24 h in B and C; 1 μM, 48 h in D). E and F. Western blotting of p-c-Jun, c-Jun, RHOB, p- cells treated with 1 μM PLX4032 for the indicated times (E) or transfected with c-Jun-targeting or non-ta e treatment with 1 μM PLX4032 for 48 h F. Actin was the loading control. c-Jun is involved in PLX4032-induced RHOB expression O 15254 i tj l / t t 0 2 4 6 8 10 12 14 pGL3-vide pGL3-SV40 -1766 -1219 -681 -195 -45 ns * ** ns * *** LucF / LucR 0 2 4 6 8 10 12 14 16 18 20 LucF / LucR * ns ns C D ATTGG iCCAAT box TTTAG TGCGG LucF/L DMSO PLX4032 0 1 E p-c-Jun PLX4032 (hours) 0 2 6 12 24 48 c-Jun RHOB p-ERK ERK Actin F p-ERK p-c-Jun c-Jun DMSO PLX4032 si-Ctl + - + - si-c-Jun - + - + RHOB ERK Actin DMSO PLX4032 DMSO PLX4032 Figure 3: RHOB is induced by PLX4032 through transcriptional activation in an iCCAAT and c-Jun dependent pathway. A. After a 48 h incubation of WM266-4 cells with PLX4032 (1 μM), RHOB mRNA half-life was measured by RT-qPCR using the transcription inhibitor actinomycin D (5 μg/mL) and normalized to the long half-life of actin mRNA (one representative experiment). B- D. Luciferase assays in WM266-4 cells co-transfected with RHOB promoter-firefly luciferase constructs and pRL-CMV before treatment with PLX4032 (2 μM, 24 h in B and C; 1 μM, 48 h in D). E and F. Western blotting of p-c-Jun, c-Jun, RHOB, p-ERK and ERK in WM266- 4 cells treated with 1 μM PLX4032 for the indicated times (E) or transfected with c-Jun-targeting or non-targeting (si-Ctl) siRNAs before treatment with 1 μM PLX4032 for 48 h F. Actin was the loading control. c-Jun is involved in PLX4032-induced RHOB expression These results indicate that this promoter region is required for RHOB induction by PLX4032. The iCCAAT region has been reported to control RHOB expression in cell stress response in a c-Jun- dependent manner [31–33]. Hence, we examined c-Jun activation after PLX4032 treatment by analyzing both total and phosphorylated (S63) c-Jun. Figure 3E shows that both c-Jun and p-c-Jun (S63) were induced in response to PLX4032. As for RHOB, total and phosphorylated c-Jun were maintained over at least 96 h after PLX4032 removal (Figure S2A). To assess more directly the role of c-Jun in RHOB upregulation, we inhibited its expression. We found that siRNA-mediated depletion of c-Jun prevented the induction of RHOB in response to PLX4032 (Figure 3F). We next investigated the mechanism underlying RHOB induction by PLX4032. Increase of RHOB transcript stability has been reported in response to UV [28, 29] and camptothecin [30]. Experiments performed in the presence of the transcription inhibitor actinomycin D showed that the half-life of RHOB mRNA was not significantly prolonged in PLX4032-treated cells (Figure 3A). We then analyzed RHOB promoter activity using a luciferase reporter gene assay system. We found a 2 to 3-fold increase of RHOB promoter activity after a 24 h PLX4032 treatment (Figure 3B). To identify the promoter region involved in the transcriptional activation of RHOB in response to PLX4032, we performed luciferase assays with plasmids containing different RHOB promoter lengths. We found that all constructs increased luciferase activity following PLX4032 treatment, including the -45 shorter one, which contains only the TATA boxes and an inverted CCAAT box (iCCAAT) (Figure 3C). Mutations of iCCAAT strongly reduced promoter response to PLX4032 (Figure 3D). Previous work show that JNK and p38 are implicated in the activation of the c-Jun/RHOB axis in response to UV and γ-radiation [32, 34]. However, we found that phosphorylated p38 and JNK rather decreased following PLX4032 treatment (Figure S2B). In addition, the JNK inhibitor, SP600125, as well as the p38 inhibitors, SB203580 and BIRB796, did not prevent PLX4032- induced c-Jun and RHOB expression (Figure S2C and S2D). c-Jun is involved in PLX4032-induced RHOB expression 0 2 4 6 8 10 12 14 pGL3-vide pGL3-SV40 -1766 -1219 -681 -195 -45 ns * ** ns * * LucF / LucR C D PLX DMSO PLX4032 0 2 4 6 8 10 12 14 pGL3-vide pGL3-SV40 -1766 -1219 -681 -195 -45 ns * ns * *** LucF / LucR 0 2 4 6 8 10 12 14 16 18 20 LucF / LucR * ns ns C D ATTGG iCCAAT box TTTAG TGCGG PL DMSO PLX4032 DMSO PLX4032 C LucF / LucR 0 2 4 6 8 10 12 14 16 18 20 LucF / LucR * ns ns D ATTGG iCCAAT box TTTAG TGCGG DMSO PLX4032 D D F p-ERK p-c-Jun c-Jun DMSO PLX4032 si-Ctl + - + - si-c-Jun - + - + RHOB ERK Actin E p-c-Jun PLX4032 (hours) 0 2 6 12 24 48 c-Jun RHOB p-ERK ERK Actin F E Figure 3: RHOB is induced by PLX4032 through transcriptional activation in an iCCAAT and c-Jun dependent pathway. A. After a 48 h incubation of WM266-4 cells with PLX4032 (1 μM), RHOB mRNA half-life was measured by RT-qPCR using the transcription inhibitor actinomycin D (5 μg/mL) and normalized to the long half-life of actin mRNA (one representative experiment). B- D. Luciferase assays in WM266-4 cells co-transfected with RHOB promoter-firefly luciferase constructs and pRL-CMV before treatment with PLX4032 (2 μM, 24 h in B and C; 1 μM, 48 h in D). E and F. Western blotting of p-c-Jun, c-Jun, RHOB, p-ERK and ERK in WM266- 4 cells treated with 1 μM PLX4032 for the indicated times (E) or transfected with c-Jun-targeting or non-targeting (si-Ctl) siRNAs before treatment with 1 μM PLX4032 for 48 h F. Actin was the loading control. Oncotarget 15254 RHOB expression levels were subsequently analyzed by immunostaining in a retrospective series of 32 biopsies from patients with metastatic melanoma harboring BRAFV600X mutations, receiving PLX4032 as a front-line therapy (Figure 6C). We observed a significant expression of RHOB in 8 out of the 32 samples (Table S5). RHOB modulates response to PLX4032 through AKT pathway Lastly, we investigated the molecular mechanisms by which RHOB impairs an efficient response to PLX4032. It has been previously reported that RHOB activates the AKT pathway [28, 35–37] and that AKT is activated following PLX4032 treatment [38]. We demonstrate that phosphorylated AKT is markedly reduced in response to simultaneous RHOB depletion and PLX4032 treatment in BRAF-mutant cells WM266- 4 (Figure 7A), Lu1205, WM239A, RPMI-7951 (Figure S5). Same findings were observed in NRAS-mutant cells WM1346 treated with the MEKi AZD6244 (Figure S5). These data suggest that RHOB inhibition impedes AKT signaling. Therefore the resistance to PLX4032 should be recovered in RHOB knockdown cells when AKT phosphorylation is rescued by expressing a constitutively active AKT-myr protein. Consistent with this hypothesis, overexpression of AKT-myr in RHOB depleted cells completely abolished sensitization to PLX4032 (Figures 7B and S6 and Table S4). To study the mechanisms underlying RHOB- dependent cell sensitivity to PLX4032, we assayed apoptotic markers after RHOB depletion. We found that RHOB siRNA increased apoptosis of WM266-4 cells in response to PLX4032 as demonstrated by an increase in the number of nuclei with subG1 DNA content, in apoptotic DNA fragmentation and in PARP and caspase-3 cleavage (Figure 5A-5C). Similar results were obtained in A375 cells (Figure S4). In addition, the pan-caspase inhibitor Z-VAD- FMK prevented PLX4032-induced PARP and caspase-3 cleavage (Figure 5D) and the accumulation of subG1 cells (Figure 5E). Overall these data show that RHOB depletion triggers caspase-dependent apoptosis of BRAF-mutant melanoma cells exposed to MAPK inhibitors. c-Jun is involved in PLX4032-induced RHOB expression Interestingly, we found that the Progression Free Survival (PFS) was significantly shorter in patients whose tumor samples displayed a positive RHOB staining before treatment compared to those with negative RHOB staining (median PFS: 135 days [Interquartile range – IQR: 107; 141] versus 235 days [IQR: 214; 314] respectively; p < 10-3) (Figure 6D and Table S5), with a hazard ratio of short-duration therapeutic response of 10−3 (95% confidence interval – 95% CI: 2 × 10−4; 9 × 10−3). We conclude that the basal expression of RHOB in tumor cells may represent a predictive value of PLX4032 response to treatment in BRAFV600X metastatic melanoma. c-Jun expression and phosphorylation by a JNK- and p38- independent mechanism. Inhibition of RHOB sensitizes melanoma cells to MAPK inhibitors-induced apoptosis To determine the role of RHOB in the cellular response to MAPK inhibitors, RHOB expression was prevented by RNA interference (Figure S3A). Following RHOB depletion, the IC50 of PLX4032 was significantly reduced in the BRAF-mutant A375 and WM266-4 cells but not in the wild-type BRAF SK-MEL2 cells which are insensitive to PLX4032 (Figure 4A, 4B and 4C and Table S1). In contrast, RHOB depletion sensitized both mutant and wild-type BRAF cells to the MEKi AZD6244 (Figure 4D, 4E and 4F and Table S1). Likewise, RHOB downregulation also sensitized WM266-4 cells to the combination of BRAFi with MEKi (Figure 4G). Because we found that c-Jun induces RHOB (Figure 3), we examined whether c-Jun inhibition would also sensitize cells to PLX4032. We found that depletion of c-Jun with siRNA sensitized WM266-4 cells to PLX4032 (Figure 4H and Table S2) and that this effect was in part reversed by adenovirus-mediated RHOB overexpression (Figures 4I and S3B and Table S3). High basal RHOB expression in melanoma cell lines and patients biopsies is associated with a poor response to PLX4032 These findings highlight that AKT inhibition would reverse RHOB-mediated PLX4032 resistance. In order to confirm this assumption, we analyzed the combination of PLX4032 with AKT inhibitors, either G594 (an ATP competitive inhibitor of AKT provided by Genentech) or MK2206. AKT inhibition enhanced PLX4032 cytotoxic effect as shown by an increased PARP and caspase-3 cleavage (Figures 7C and S7). To determine whether combination of PI3K/AKTi and BRAFi displays additive or synergistic effect, co-treatments were performed in vitro and combination indexes were calculated. All three PI3K/ AKT pathway inhibitors (LY294002, MK2206 and G594) demonstrated a substantial synergistic effect when combined with PLX4032 in WM266-4 cells (Figure 7D). Given that RHOB depletion determines the cellular response to PLX4032 in BRAF-mutant melanoma cells, we hypothesized that the basal expression of RHOB may predict the response to BRAFi. Therefore, we compared RHOB mRNA levels (Figure 6A) with the IC50 of PLX4032 (Figure 6B) in 8 different cell lines. A strong correlation was observed between RHOB basal expression and PLX4032 IC50. Cell lines with low RHOB expression were more sensitive to PLX4032, suggesting that, in melanoma cells, high RHOB expression may predict a poor response of BRAF-mutant patients to PLX4032. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget WM266–4 cells were transfected with siRNAs control (si-Ctl) or targeting c-Jun (si-c-Jun), then transduced with adenovirus control (Ad-Ctl) or expressing RHOB (Ad-RHOB) and treated for 72 h with PLX4032. In each condition, cell viability was measured by MTS and the dose-response was analyzed (except in G). Figure 4: Inhibition of the c-Jun/RHOB axis increases cell sensitivity to BRAF and MEK inhibitors. A-F. WM266-4, A375 or SK-MEL2 cells were transfected with siRNAs control (si-Ctl) or targeting RHOB (si-RHOB1 and siRHOB2) before treatment with PLX4032 or AZD6244 for 72 h. G. WM266-4 cells were transfected with siRNAs control (si-Ctl) or targeting RHOB (si-RHOB) before treatment with PLX4032 (1 μM) and/or AZD6244 (1 μM) for 72 h. H. WM266–4 cells were transfected with siRNAs control (si-Ctl) or targeting c-Jun (si-c-Jun) before treatment with PLX4032 for 72 h. I. WM266–4 cells were transfected with siRNAs control (si-Ctl) or targeting c-Jun (si-c-Jun), then transduced with adenovirus control (Ad-Ctl) or expressing RHOB (Ad-RHOB) and treated for 72 h with PLX4032. In each condition, cell viability was measured by MTS and the dose-response was analyzed (except in G). A375 or SK-MEL2 cells were transfected with siRNAs control (si-Ctl) or targeting RHOB (si-RHOB1 an with PLX4032 or AZD6244 for 72 h. G. WM266-4 cells were transfected with siRNAs control (si-Ctl) o before treatment with PLX4032 (1 μM) and/or AZD6244 (1 μM) for 72 h. H. WM266–4 cells were transfecte or targeting c-Jun (si-c-Jun) before treatment with PLX4032 for 72 h. I. WM266–4 cells were transfected w targeting c-Jun (si-c-Jun), then transduced with adenovirus control (Ad-Ctl) or expressing RHOB (Ad-RH PLX4032. In each condition, cell viability was measured by MTS and the dose-response was analyzed (exc In a same manner, a synergistic effect was found between the AKTi, G594 and PLX4032 in the 7 other BRAF-mutant cell lines and between G594 and AZD6244 in 2 NRAS- mutant cell lines (Table S6). tumor weight were significantly reduced in mice receiving BRAFi/AKTi combination compared to the monotherapies (Figures 7E and S8), confirming that AKT signaling pathway plays a role in the in vivo responses of BRAF- mutant tumors to PLX4032. We next evaluated this combination effect in the WM266-4 xenografted mouse model. After tumor appearance, mice were treated with control vehicle, G594 and/or PLX4032 and the tumor growth was monitored. www.impactjournals.com/oncotarget As shown in Figures 7E and S8, both tumor growth and We conclude that PLX4032-induced RHOB expression drives AKT-dependent cell survival and that BRAFi/AKTi combination should improve therapeutic response of RHOB positive tumors. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget A Surviving fraction (%) Surviving fraction (%) D G PLX4032 AZD6244 + + - + + - - - WM266- 6 6 2 M W 4 - 6 6 2 M W 4 -4 Si-Ctl Si-RHOB Si-Ctl Si-RHOB1 Si-RHOB2 Si-Ctl Si-RHOB1 Si-RHOB2 D G A B B 0 25 50 75 100 AZD6244 (µM) 0.001 0.01 0.1 1 10 100 E 5 7 3 A Si-Ctl Si-RHOB1 Si-RHOB2 Surviving fraction (%) 0 25 50 75 100 AZD6244 (µM) 0.001 0.01 0.1 1 10 100 Surviving fraction (%) B E H 5 7 3 A 5 7 3 A Si-Ctl Si-c-Jun WM266-4 Si-Ctl Si-RHOB1 Si-RHOB2 Si-Ctl Si-RHOB1 Si-RHOB2 Surviving fraction (%) H Si-Ctl Si-c-Jun WM266-4 E H AZD6244 (µM) 0 50 100 150 200 PLX4032 (µM) 0.001 0.01 0.1 1 10 100 0 25 50 75 100 AZD6244 (µM) Surviving fraction (%) 0.001 0.01 0.1 1 10 0.0001 C F I SK- K S 2 L E M -MEL2 Si-c-Jun/Ad-Ctl Si-Ctl/Ad-Ctl Si-c-Jun/Ad-RHOB Si-Ctl/Ad-RHOB WM266-4 Si-Ctl Si-RHOB1 Si-RHOB2 Si-Ctl Si-RHOB1 Si-RHOB2 (µ ) 0 0 25 50 75 100 AZD6244 (µM) Surviving fraction (%) 0.001 0.01 0.1 1 10 0.0001 F I K S -MEL2 Si-c-Jun/Ad-Ctl Si-Ctl/Ad-Ctl Si-c-Jun/Ad-RHOB Si-Ctl/Ad-RHOB WM266-4 Si-Ctl Si-RHOB1 Si-RHOB2 0 25 50 75 100 AZD6244 (µM) Surviving fraction (%) 0.001 0.01 0.1 1 10 0.0001 F K S -MEL2 Si-Ctl Si-RHOB1 Si-RHOB2 0 50 100 150 200 PLX4032 (µM) 0.001 0.01 0.1 1 10 100 Surviving fraction (%) C F SK- 2 L E M Si-Ctl Si-RHOB1 Si-RHOB2 I Si-c-Jun/Ad-Ctl Si-Ctl/Ad-Ctl Si-c-Jun/Ad-RHOB Si-Ctl/Ad-RHOB WM266-4 I I F C Surviving fraction (%) 2 Surviving fraction (% AZD6244 (µM) hibition of the c-Jun/RHOB axis increases cell sensitivity to BRAF and MEK inhibitors. A-F e c-Jun/RHOB axis increases cell sensitivity to BRAF and MEK inhibitors. A-F. WM266-4, Figure 4: Inhibition of the c-Jun/RHOB axis increases cell sensitivity to BRAF and ME Figure 4: Inhibition of the c-Jun/RHOB axis increases cell sensitivity to BRAF and MEK inhibitors. A-F. WM266-4, A375 or SK-MEL2 cells were transfected with siRNAs control (si-Ctl) or targeting RHOB (si-RHOB1 and siRHOB2) before treatment with PLX4032 or AZD6244 for 72 h. G. WM266-4 cells were transfected with siRNAs control (si-Ctl) or targeting RHOB (si-RHOB) before treatment with PLX4032 (1 μM) and/or AZD6244 (1 μM) for 72 h. H. WM266–4 cells were transfected with siRNAs control (si-Ctl) or targeting c-Jun (si-c-Jun) before treatment with PLX4032 for 72 h. I. www.impactjournals.com/oncotarget Oncotarget 15255 Surviving fraction (%) A 0 25 50 75 100 AZD6244 (µM) 0.001 0.01 0.1 1 10 100 Surviving fraction (%) Surviving fraction (%) Surviving fraction (%) B 0 50 100 150 200 PLX4032 (µM) 0.001 0.01 0.1 1 10 100 0 25 50 75 100 AZD6244 (µM) Surviving fraction (%) 0.001 0.01 0.1 1 10 0.0001 C D E F G H I PLX4032 AZD6244 + + - + + - - - WM266- 6 6 2 M W 4 - 6 6 2 M W 4 -4 5 7 3 A 5 7 3 A SK- K S 2 L E M -MEL2 Si-Ctl Si-c-Jun Si-c-Jun/Ad-Ctl Si-Ctl/Ad-Ctl Si-c-Jun/Ad-RHOB Si-Ctl/Ad-RHOB Si-Ctl Si-RHOB WM266-4 WM266-4 Si-Ctl Si-RHOB1 Si-RHOB2 Si-Ctl Si-RHOB1 Si-RHOB2 Si-Ctl Si-RHOB1 Si-RHOB2 Si-Ctl Si-RHOB1 Si-RHOB2 Si-Ctl Si-RHOB1 Si-RHOB2 Si-Ctl Si-RHOB1 Si-RHOB2 Figure 4: Inhibition of the c-Jun/RHOB axis increases cell sensitivity to BRAF and MEK inhibitors. A-F. WM266-4, A375 or SK-MEL2 cells were transfected with siRNAs control (si-Ctl) or targeting RHOB (si-RHOB1 and siRHOB2) before treatment with PLX4032 or AZD6244 for 72 h. G. WM266-4 cells were transfected with siRNAs control (si-Ctl) or targeting RHOB (si-RHOB) before treatment with PLX4032 (1 μM) and/or AZD6244 (1 μM) for 72 h. H. WM266–4 cells were transfected with siRNAs control (si-Ctl) or targeting c-Jun (si-c-Jun) before treatment with PLX4032 for 72 h. I. WM266–4 cells were transfected with siRNAs control (si-Ctl) or targeting c-Jun (si-c-Jun), then transduced with adenovirus control (Ad-Ctl) or expressing RHOB (Ad-RHOB) and treated for 72 h with PLX4032. In each condition, cell viability was measured by MTS and the dose-response was analyzed (except in G). www.impactjournals.com/oncotarget Oncotarget 15256 Si-Ctl Si-RHOB1 Si-RHOB2 A B C D E PARP p-ERK ERK RHOB Caspase-3 Cleaved Caspase-3 DMSO PLX4032 si-RHOB2 - - + - - + si-RHOB1 - + - - + - si-Ctl + - - + - - PARP DMSO PLX4032 PLX4032 + Z-VAD-FMK si-RHOB2 - + - + - + si-Ctl + - + - + - p-ERK ERK RHOB Caspase-3 Cleaved Caspase-3 DMSO PLX4032 0 20 40 60 * TUNEL positive cells (%) * % subG1 cells Si-Ctl Si-RHOB1 Si-RHOB2 Si-Ctl Si-RHOB2 Si-Ctl Si-RHOB1 Si-RHOB2 A B C D E PARP p-ERK ERK RHOB Caspase-3 Cleaved Caspase-3 DMSO PLX4032 si-RHOB2 - - + - - + si-RHOB1 - + - - + - si-Ctl + - - + - - PARP DMSO PLX4032 PLX4032 + Z-VAD-FMK si-RHOB2 - + - + - + si-Ctl + - + - + - p-ERK ERK RHOB Caspase-3 Cleaved Caspase-3 DMSO PLX4032 0 20 40 60 * TUNEL positive cells (%) * % subG1 cells PLX4032 Z-VAD-FMK + + - - + - Si-Ctl Si-RHOB1 Si-RHOB2 Si-Ctl Si-RHOB2 Figure 5: Concurrent inhibition of MAPK with RHOB triggers apoptosis. WM266-4 cells were transfected with siRN control (si-Ctl) or targeting RHOB (si-RHOB1 and si-RHOB2) before treatment with 2 μM PLX4032 for 72 h. Apoptosis was determin by the percentage of cells with subG1 DNA content A, E. or positive for TUNEL assay B, or by Western blotting analysis of cleav caspase-3 and cleaved PARP (the arrowheads at right indicate the cleaved fragment) C and D. Total caspase-3, RHOB, p-ERK and E were analyzed in parallel. In D and E, cells were co-treated or not with 2 μM PLX4032 and the pan-caspase inhibitor Z-VAD-FM (25 μM). www.impactjournals.com/oncotarget Si-Ctl Si-RHOB1 Si-RHOB2 A B DMSO PLX4032 0 20 40 60 * TUNEL positive cells (%) Si-Ctl Si-RHOB1 Si-RHOB2 A DMSO PLX4032 0 20 40 60 * Si-Ctl Si-RHOB1 Si-RHOB2 Si-Ctl Si-RHOB1 Si-RHOB2 B TUNEL positive cells (%) B A C PARP p-ERK ERK RHOB Caspase-3 Cleaved Caspase-3 DMSO PLX4032 si-RHOB2 - - + - - + si-RHOB1 - + - - + - si-Ctl + - - + - - D PARP DMSO PLX4032 PLX4032 + Z-VAD-FMK si-RHOB2 - + - + - + si-Ctl + - + - + - p-ERK ERK RHOB Caspase-3 Cleaved Caspase-3 C D E * % subG1 cells PLX4032 Z-VAD-FMK + + - - + - Si-Ctl Si-RHOB2 Figure 5: Concurrent inhibition of MAPK with RHOB triggers apoptosis. WM266-4 cells were transfected with siRNAs control (si-Ctl) or targeting RHOB (si-RHOB1 and si-RHOB2) before treatment with 2 μM PLX4032 for 72 h. Apoptosis was determined by the percentage of cells with subG1 DNA content A, E. or positive for TUNEL assay B, or by Western blotting analysis of cleaved E * % subG1 cells PLX4032 Z-VAD-FMK + + - - + - Si-Ctl Si-RHOB2 E Figure 5: Concurrent inhibition of MAPK with RHOB triggers apoptosis. WM266-4 cells were transfected with siRNAs control (si-Ctl) or targeting RHOB (si-RHOB1 and si-RHOB2) before treatment with 2 μM PLX4032 for 72 h. Apoptosis was determined by the percentage of cells with subG1 DNA content A, E. or positive for TUNEL assay B, or by Western blotting analysis of cleaved caspase-3 and cleaved PARP (the arrowheads at right indicate the cleaved fragment) C and D. Total caspase-3, RHOB, p-ERK and ERK were analyzed in parallel. In D and E, cells were co-treated or not with 2 μM PLX4032 and the pan-caspase inhibitor Z-VAD-FMK (25 μM). www.impactjournals.com/oncotarget Oncotarget 15257 A B C D Figure 6: High RHOB expression in melanoma cell lines and patient melanoma biopsies predict low sensitivity to the BRAF inhibitor PLX4032. A. RHOB expression in a panel of BRAF-mutant metastatic melanoma cell lines analyzed by RT-qPCR. B. The IC50 of PLX4032 in a panel of BRAF-mutant metastatic melanoma cell lines was determined by viability assay and correlated with RHOB mRNA expression. C. Example of positive and negative RHOB immunostaining in melanoma biopsies. D. Kaplan-Meier progression-free survival of 32 patients with a metastatic BRAFV600X melanoma following treatment with PLX4032 according to biopsies’ RHOB staining. A B B A D C D C Figure 6: High RHOB expression in melanoma cell lines and patient melanoma biopsies predict low sensitivity to the BRAF inhibitor PLX4032. A. RHOB expression in a panel of BRAF-mutant metastatic melanoma cell lines analyzed by RT-qPCR. B. The IC50 of PLX4032 in a panel of BRAF-mutant metastatic melanoma cell lines was determined by viability assay and correlated with RHOB mRNA expression. C. Example of positive and negative RHOB immunostaining in melanoma biopsies. D. Kaplan-Meier progression-free survival of 32 patients with a metastatic BRAFV600X melanoma following treatment with PLX4032 according to biopsies’ RHOB staining. DISCUSSION Importantly, we found that the expression of 6 members of the RHOGTPase family was significantly modified in response to PLX4032 in melanoma cells. RND3 is downregulated as previously described [23] whereas RHOB, RHOJ, RHOQ and RHOU are induced. While nothing is known about the role of RHOQ and RHOU in melanomagenesis, RHOJ participates to chemoresistance through DNA repair control [20] and modulates metastasis potential in melanoma [39]. Of interest is the PLX4032- induced RAC1b downregulation, suggesting that RAC1b and BRAF cooperate in melanomagenesis as previously described in colorectal cancer [40]. In this study we demonstrate that inhibition of mutant BRAF in melanoma cells with pharmacological inhibitors or siRNA knockdown results in the activation of the c-Jun/ RHOB/AKT signaling axis, which leads to cell survival. Interestingly, a concomitant consequence of c-Jun/RHOB/ AKT activation is a decrease of PLX4032 cellular responses, entailing acquired resistance. RHO proteins have been shown to play a role in modulating cell survival and some of them have been described to be regulated by the MAPK pathway [17]. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 15258 si-RHOB2 - - + - - + si-RHOB1 - + - - + - si-Ctl + - - + - - DMSO A pAKT / AKT DMSO PLX4032 0 1 2 * * AKT p-ERK ERK RHOB p-AKT PLX4032 Actin WM266-4 si-RHOB2 - - + - - + si-RHOB1 - + - - + - si-Ctl + - - + - - DMSO B A pAKT / AKT DMSO PLX4032 0 1 2 * * AKT p-ERK ERK RHOB p-AKT PLX4032 Actin Surviving fraction (%) Si-Ctl/pCMV6-AKT-myr Si-RHOB/pCMV6-AKT-myr Si-RHOB/pCMV6 Si-Ctl/pCMV6 p-ERK ERK PARP Cleaved Caspase-3 Caspase-3 RHOB Actin G594 - - + + PLX4032 - + - + WM266-4 D C Log Combination index -2.0 -1.5 -1.0 -0.5 0.0 G594 MK2206 LY294002 0.3µM 1µM 3µM 0.1µM Synergy ++ + +++ * * Tumor volume (mm3) WM266-4 PLX4032 G594 Combo Vehicle E WM266-4 WM266-4 WM266-4 igure 7: RHOB-mediated resistance to PLX4032 is overcomed by combination with an AKT inhibitor. A. W ells were transfected with siRNA control (si-Ctl) or targeting RHOB (si-RHOB1 or si-RHOB2) before treatment with 2 μM PLX 2 h. AKT phosphorylation was analyzed by Western blotting. Total AKT, RHOB, p-ERK and ERK were examined in parallel. A e loading control. Histogram is a quantification of 5 independent experiments. B. WM266-4 cells were co-transfected with siRNA i-Ctl) or targeting RHOB (si-RHOB2) and empty plasmid (pCMV6-Ctl) or encoding AKT-myr (pCMV6-AKT-myr). Cells were th ith PLX4032 and the viability was assayed 72 h later. C. Western blotting of caspase-3 and PARP cleavage (the cleaved fragment is y the arrowhead) in WM266-4 cells treated with 2 μM PLX4032 and/or 1 μM G594 for 48 h. Total caspase-3, RHOB, p-ERK and E xamined in parallel. Actin was the loading control. D. WM266-4 cells were treated with equal amount of PI3K/AKT inhibitors (LY MK2206 or G594) and PLX4032. Viability was assayed 72 h later by MTS assay. Combination indexes (CI) were calculated with C ery strong synergism (+++) CI < 0.1; strong synergism (++) 0.1 < CI < 0.3; synergism (+) 0.3 < CI < 0.7). E. Athymic mice were subcu oculated with WM266-4 cells. www.impactjournals.com/oncotarget Mice were randomized 10 days later in 4 groups (10 mice per group) and treated orally with vehicle, B A Surviving fraction (%) Si-Ctl/pCMV6-AKT-myr Si-RHOB/pCMV6-AKT-myr Si-RHOB/pCMV6 Si-Ctl/pCMV6 WM266-4 p-ERK ERK PARP Cleaved Caspase-3 Caspase-3 RHOB Actin G594 - - + + PLX4032 - + - + WM266-4 C D Log Combination index -2.0 -1.5 -1.0 -0.5 0.0 G594 MK2206 LY294002 0.3µM 1µM 3µM 0.1µM Synergy ++ + +++ WM266-4 D C * * Tumor volume (mm3) PLX4032 G594 Combo Vehicle E WM266-4 E Figure 7: RHOB-mediated resistance to PLX4032 is overcomed by combination with an AKT inhibitor. A. WM266-4 cells were transfected with siRNA control (si-Ctl) or targeting RHOB (si-RHOB1 or si-RHOB2) before treatment with 2 μM PLX4032 for 72 h. AKT phosphorylation was analyzed by Western blotting. Total AKT, RHOB, p-ERK and ERK were examined in parallel. Actin was the loading control. Histogram is a quantification of 5 independent experiments. B. WM266-4 cells were co-transfected with siRNAs control (si-Ctl) or targeting RHOB (si-RHOB2) and empty plasmid (pCMV6-Ctl) or encoding AKT-myr (pCMV6-AKT-myr). Cells were then treated with PLX4032 and the viability was assayed 72 h later. C. Western blotting of caspase-3 and PARP cleavage (the cleaved fragment is indicated by the arrowhead) in WM266-4 cells treated with 2 μM PLX4032 and/or 1 μM G594 for 48 h. Total caspase-3, RHOB, p-ERK and ERK were examined in parallel. Actin was the loading control. D. WM266-4 cells were treated with equal amount of PI3K/AKT inhibitors (LY294002, MK2206 or G594) and PLX4032. Viability was assayed 72 h later by MTS assay. Combination indexes (CI) were calculated with CompuSyn (very strong synergism (+++) CI < 0.1; strong synergism (++) 0.1 < CI < 0.3; synergism (+) 0.3 < CI < 0.7). E. Athymic mice were subcutaneously inoculated with WM266-4 cells. Mice were randomized 10 days later in 4 groups (10 mice per group) and treated orally with vehicle, G594 or/ and PLX4032 for 21 days. The tumor volume was evaluated every 3 days. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 15259 We focused our attention on the stress-inducible RHOB gene [28, 30, 33, 41, 42] that plays a negative role on tumor progression. Moreover, RHOB functions are mediated by tyrosine kinase receptors and their downstream effectors [25, 26, 43]. We have demonstrated previously that RHOB is downregulated by KRASV12 in lung cancer cell lines increasing invasiveness via AKT1 [44]. More recently, we have revealed that RHOB knock-out is critical to determine tumor aggressiveness in a murine EGFRL858R-induced lung adenocarcinoma [45]. Herein, we show that BRAFV600 mutant causes a MAPK- dependent decrease of RHOB expression in melanoma cell lines. It is likely that the mechanism of RHOB downregulation is related to cell type and/or cell context. Indeed, Jiang et al. [46] found that AKT induced RHOB downregulation in non-melanoma cell lines. According to our cellular data, the patient’s samples revealed an absence of RHOB expression in more than 70% of melanoma tissues bearing BRAFV600E mutants. This suggests that reduced RHOB levels represent a critical event during melanomagenesis. It has been previously reported that AKT is activated in PLX4032-treated PTEN negative melanoma and is involved in melanoma resistance [50]. However, the role of the c-Jun/RHOB/AKT axis in the cellular sensitivity to MAPK inhibitors appeared to be independent on PTEN status. Indeed, whereas WM266-4 cells lack PTEN expression, A375 cells are wild-type for PTEN and are also sensitized to MAPK inhibitors by RHOB siRNA (Figures 4, 5 and S4) or AKTi (Figure 7, Table S6). The combination of PI3K/AKT inhibitors with PLX4032 to improve melanoma treatment has been recently proposed although the mechanism was not clearly established. We demonstrate that PLX4032 induces a cell adaptive response including a long lasting expression of c-Jun and RHOB leading to the activation of AKT signaling axis which may trigger cell resistance by inhibiting apoptosis. While AKTi are not able to induce cell death by themselves they lead to a synergistic synthetic lethal interaction with BRAFi. Our observations further support the rational to treat patients with a combination of BRAF and AKT inhibitors. Finally, our results on metastatic melanoma biopsies highlight a pivotal role for RHOB in the therapeutic management of this disease. Interestingly, the analysis of RHOB expression in these samples revealed that patients with RHOB positive biopsies displayed a poor response to PLX4032. www.impactjournals.com/oncotarget We therefore hypothesize that patients with a high RHOB expression would display a high AKT pathway activity and benefit from BRAF and AKT inhibitors combination rather than BRAFi monotherapy. However, it could not be excluded that RHOB negative patients’ survival would also be improved by such a therapeutic combination by preventing potential late resistance appearance. Analyzing future combinational clinical trial regarding RHOB status will bring answers to these remaining questions.i The observation of a persistent RHOB expression after PLX4032 removal, despite ERK re-phosphorylation (Figure 1G), suggests that RHOB participates to an original long lasting adaptive cellular program in melanoma cells. We show that RHOB expression after PLX4032 treatment depends on the transcription factor c-Jun. It has been demonstrated that iCCAAT is critical for UVB-induced RHOB expression [32, 47]. In that model, phosphorylated c-Jun is associated to ATF2 and NF-Y inducing HDAC1 release then allowing p300 binding to iCCAAT triggering RHOB expression. In stress conditions, c-Jun was described to be phosphorylated following JNK and p38 activation [32, 34] while in our model JNK and p38 were not involved in PLX4032- induced c-Jun phosphorylation and expression. We evidence here a key role of c-Jun in the long lasting regulation of RHOB expression by PLX4032 and the significance of this pathway in cell survival following cellular treatment. Taken together, our findings tempt us to conclude that the c-Jun/RHOB/AKT signaling axis is a synthetic lethal partner of BRAF-mutant, and potentially of NRAS- mutant, melanomas and to propose RHOB as a critical biomarker to predict patient’s response to PLX4032 and to select those that would benefit of the combination with AKT inhibitors. RHOB has been demonstrated to control AKT phosphorylation to exert its cellular functions in many cell types. Here, we demonstrated that, by contrast to lung [44] and breast cancer [36] cells, RHOB downregulation decreases AKT phosphorylation in melanoma, similarly to endothelial cells [36, 48] (Figures 7A and S5). These data reinforce the notion that the modulation of AKT phosphorylation by RHOB depends on the cellular phenotype. We show here that RHOB/AKT signaling inhibition is critical for PLX4032-induced caspase- dependent apoptosis. In the same way, genotoxic stress- induced RHOB expression favors cell survival through AKT pathway [28, 30, 35, 41, 49]. We provide new evidence that the RHOB/AKT axis is a common adaptive mechanism to cell injury. Cell culture and pharmacological inhibitors WM115, WM266-4, A375, SK-MEL28, SK-MEL2 and RMPI-7951 were purchased at ATCC; WM35, WM983A, WM983B, WM239A, WM3211 and Lu1205 at Coriell Institute and WM1346 at Wistar Institute. WM115, WM266-4, A375 and SK-MEL28 were cultured in DMEM/FBS 10% (v/v), SK-MEL2, 501mel and RPMI- 7951 in RPMI-1640/FBS 10% (v/v), WM35, WM983A, www.impactjournals.com/oncotarget Oncotarget 15260 WM983B, WM3211, WM1346 and Lu1205 in MCDB153 medium with 20% Leibovitz L-15 medium (v/v), 2% FBS heat inactivated (v/v), 5 μg/mL insulin and 1.68 mM CaCl2. Cell lines were authenticated for mutations in BRAF and NRAS by sequencing within the time frame of the experiments. PLX4032, SB590885, AZD6244, AS703026 and MK2206 were from Selleck Chemicals, Z-VAD-FMK and actinomycin D from Sigma-Aldrich, LY294002 from Calbiochem, G594 competitive inhibitor of AKT was provided by Genentech. WM983B, WM3211, WM1346 and Lu1205 in MCDB153 medium with 20% Leibovitz L-15 medium (v/v), 2% FBS heat inactivated (v/v), 5 μg/mL insulin and 1.68 mM CaCl2. Cell lines were authenticated for mutations in BRAF and NRAS by sequencing within the time frame of the experiments. PLX4032, SB590885, AZD6244, AS703026 and MK2206 were from Selleck Chemicals, Z-VAD-FMK and actinomycin D from Sigma-Aldrich, LY294002 from Calbiochem, G594 competitive inhibitor of AKT was provided by Genentech. three independent experiments with ImageLab software (Bio-Rad) and normalized to actin. Cell proliferation assay After transfection or transduction, cells were treated with inhibitors. 72 h later, the relative number of viable cells was measured by incubating cells with MTS reagent (CellTiter 96® AQueous One Solution Cell Proliferation Assay from Promega) as recommended by the manufacturer. Relative cell survival in the presence of inhibitors was normalized to the untreated cells after background corrections. Quantitative real-time reverse transcription–PCR Total RNA was isolated by RNeasy® kit (Qiagen) and reverse-transcripted using iScriptTM cDNA synthesis kit (Bio-Rad). Quantitative PCR was performed with a CFX96TM detection system (Bio-Rad) using iQTM SYBR® Green Supermix (Bio-Rad) and normalized to actin or 28S. Primers sequences are detailed in Table S8. Animal studies 1.2 × 106 WM266-4 cells were subcutaneously injected into the flank of 6 weeks female athymic mice (NMRI-NU, Janvier) and allowed to grow 10 days. Subsequently, animals received by oral gavage 50 mg/ kg/ bid of PLX4032 and/or 20 mg/kg/day G594 or vehicle. PLX4032, formulated as MBP, and G594 were provided by Genentech and prepared according to their advices. Tumor sizes were evaluated every 3 days by caliper measurement of two perpendicular diameters (l < L) and the tumor volumes were calculated with the following equation tumor volume = l × l × L/2. Animals were euthanized after 21 days of treatment, tumor were harvested and weighted. All animal procedures were performed in accordance with the animal care guidelines of the European Union and French laws and were Adenovirus transduction Cells were first transfected with siRNA and then transduced overnight at M.O.I 25 with replication- defective (∆E1, E3) adenoviral vectors expressing RHOB (Ad-RHOB) or control under the transcriptional control of the CMV promoter as previously described [51]. Study of RHOB promoter activity RNA interference was achieved by transfecting siRNA with OligofectamineTM in OptiMEMTM (Invitrogen) according to the manufacturer’s instructions. After 6 h, transfection medium was replaced and inhibitors were added. siRNA sequences are reported in Table S7. pCMV6-AKT-myr plasmid, gifted by Pr. Franke (Columbia University) and pCMV6-CTRL were co- transfected with siRNA using Lipofectamine® 2000 in OptiMEMTM. The previously described pGL3-RHOB plasmids containing different lengths of RHOB promoter sequence upstream of the firefly luciferase gene into pGL3 luciferase reporter vector [52] were co-transfected with pRL-CMV vector (Promega) as an internal control with LipofectamineTM 2000 (Invitrogen). The iCCAAT of the shorter construct (-45) was mutated using QuickChange® Site-Directed Mutagenesis Kit (Stratagene). The obtained constructs were sequenced to ensure successful mutagenesis. Firefly and renilla luciferase activities were measured with Dual-Luciferase Reporter Assay (Promega). Apoptosis analysis DNA fragmentation was detected by a modified TUNEL procedure using the ApopTag® Fluorescein In Situ Apoptosis Detection Kit (Millipore). Positive cells were counted by fluorescence microscopy. For subG1 analysis, DNA content was assessed by staining ethanol-fixed cells with propidium iodide and monitoring by FACSCaliburTM (BD Biosciences). The number of cells with subG1 DNA content was determined with the ModFit software. Western blot analysis Cell extracts were analyzed by Western blotting with the following primary antibodies against RHOB, RHOA, BRAF, ERK1/2 (Santa Cruz Biotechnology); RHOC, p-ERK (T202/Y204), p-AKT (S473), AKT, PARP, Cleaved Caspase-3, Caspase-3, c-Jun, p-c-Jun (S63), p-p38 (T180/Y182), p38, pJNK (T183/Y185), JNK (Cell Signaling Technology) or actin (Chemicon). Detection was performed using peroxydase-conjugated secondary antibodies and chemi-luminescence detection kit (ClarityTM ECL, Biorad) on autoradiographs or with ChemiDocTM MP Imaging System (Bio-Rad). Quantifications were done for www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 15261 3. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, Dummer R, Garbe C, Testori A, Maio M. Improved survival with vemurafenib in mela noma with BRAF V600E mutation. New England Journal of Medicine. 2011; 364:2507–2516. approved by the local Ethical Commission of the Institut Claudius Regaud. ACKNOWLEDGMENTS 9. Johannessen CM, Boehm JS, Kim SY, Thomas SR, Wardwell L, Johnson LA, Emery CM, Stransky N, Cogdill AP, Barretina J, Caponigro G, Hieronymus H, Murray RR, Salehi-Ashtiani K, Hill DE, Vidal M, et al. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature. 2010; 468:968–972. This research was supported in part by Genentech, the “Institut National de la Recherche Medicale” INSERM, the “Fondation Recherche et Innovation Thérapeutique en Cancérologie”. We thank Bettina Couderc and Catherine Bouchenot for the generation of the adenovirus expressing RHOB. We also thank Magali Farella for BRAF and NRAS cell line sequencing, Stéphanie Cabantous for critically reading the manuscript and Lourdes Gasquet at the Claudius Regaud Institute animal facility. 10. Sabbatino F, Wang Y, Wang X, Flaherty KT, Yu L, Pepin D, Scognamiglio G, Pepe S, Kirkwood JM, Cooper ZA, Frederick DT, Wargo JA, Ferrone S, Ferrone CR. PDGFRalpha up-regulation mediated by sonic hedgehog pathway activation leads to BRAF inhibitor resis tance in melanoma cells with BRAF mutation. Oncotarget. 2014; 5:1926–1941. Statistical analysis All results are presented as the mean ± standard error of the mean (SEM) for at least three independent experiments. Statistical analyses of continuous variable were done using t-test and IC50 calculation and comparison were performed using GraphPad Prism, version 5.01 (GraphPad Software Inc., San Diego, CA, USA). P < 0.05; P < 0.01; **P < 0.001. Patient survival analysis were done using log-rank test of Kaplan- Meier survival curves. Hypotheses were two-sided, with an Alpha risk of 5%. 7. Nazarian R, Shi H, Wang Q, Kong X, Koya RC, Lee H, Chen Z, Lee M-K, Attar N, Sazegar H, Chodon T, Nelson SF, McArthur G, Sosman JA, Ribas A, Lo RS. 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https://openalex.org/W4296050551	https://www.researchsquare.com/article/rs-629632/latest.pdf	English	null	Histone methylation modification patterns and relevant M-RiskScore in acute myeloid leukemia	Heliyon	2,022	cc-by	10,380	Histone Methylation Modification Patterns and Relevant M-RiskScore in Acute Myeloid Leukemia Dade Rong Sun Yat-Sen University htt Xiaomin Chen Sun Yat-Sen University Daiyuan Liu Sun Yat-sen university Xiangna Ni Sun Yat-sen University Zhuomao Mo Sun Yat-sen University Xiuzhen Tong Sun Yat-sen University Haihe Wang Sun Yat-sen University Sun Yat-Sen University Xiaomin Chen Sun Yat-Sen University Daiyuan Liu Sun Yat-sen university Xiangna Ni Sun Yat-sen University Zhuomao Mo Sun Yat-sen University Xiuzhen Tong Sun Yat-sen University Haihe Wang Sun Yat-sen University Abstract Background: Acute myeloid leukemia (AML) is a type of heterogeneous disease with varied prognosis, but current classification methods for AML do not play an ideal role in guiding the therapy. Emerging studies shown alteration of histone methylation is closely related to leukemogenesis. This study aimed at identifying the molecular subtypes associated with histone methylation and establishing a relevant score to predict treatment respond and prognosis in AML. Methods: Gene expression data and clinical characteristics of patients with AML were obtained from The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database. Molecular subtypes were identified by consensus clustering based on the expression of 24 histone methylation modification regulators (HMMRs). The clinical and biological features of each pattern were investigated by unsupervised clustering, survival, principle component (PCA), somatic mutations, gene set variation (GSVA), tumor mutation burden (TMB) and immune cell infiltration analyses. Different expression analysis and lasso regression analysis was conducted to establish the scoring system that was explored in the role of prognosis by using receivers operating curve (ROC) analysis and univariate/multivariate Cox regression analyses. Moreover, correlation analysis was performed to investigate the value of scoring system in chemotherapeutic prediction. Finally, an independent GSE dataset was used as a reference to validate the established clustering system. Methods: Gene expression data and clinical characteristics of patien Cancer Genome Atlas (TCGA) database and Gene Expression Omnib subtypes were identified by consensus clustering based on the expre modification regulators (HMMRs). The clinical and biological feature by unsupervised clustering, survival, principle component (PCA), som (GSVA), tumor mutation burden (TMB) and immune cell infiltration a analysis and lasso regression analysis was conducted to establish t in the role of prognosis by using receivers operating curve (ROC) ana regression analyses. Moreover, correlation analysis was performed t system in chemotherapeutic prediction. Finally, an independent GSE validate the established clustering system. Results: Two distinct histone methylation modification patterns had been identified that exhibit remarkable differences in several clinical and biological characteristics, including HMMRs’ expression, AML-M0 distribution, mutations of NPM1, survival, tumor mutation burden, somatic mutations, pathways activation and immune cell infiltration. Besides, based on the clustering, we established the scoring system, M-RiskScore. Integrated analysis demonstrated that M-RiskScore-low patients displayed a prominent survival advantage and a better respond to decitabine treatment, while the opposite site was reported to M-RiskScore-high patients but they could benefit more from IA regimen therapy. Research License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Heliyon on September 1st, 2022. See the published version at https://doi.org/10.1016/j.heliyon.2022.e10610. Page 1/31 Page 1/31 Abstract Conclusion: Our results demonstrate that detection of HMMRs’ expression is potentially useful to AML therapy decisions, and targeting histone methylation would be a more promising strategy for either AML- M0 or NPM1 mutant patients. M-RiskScore is a hopeful independent poor prognostic biomarker and be able to benefits the treatment decisions in AML. Background AML is an age-distributed hematopoietic stem cell differentiation disorder, which is mainly manifested in the inhibition of hematopoietic stem cell differentiation and the accumulation of immature cells at various stages, as well as the reduced production of active hematopoietic elements and cytokines [1, 2]. Meanwhile, AML is also a highly heterogeneous disease because its pathogenetic factors include various chromosomal and molecular abnormalities [3, 4]. Thanks to the progress in basic research, including the development of new chemotherapeutic drugs, the application of allogeneic stem cell transplantation and Page 2/31 Page 2/31 the proposal and implementation of the supportive treatment, the outlook of patients with AML has significantly improved [1]. However, more than half of young adult patients and approximately 90% of elderly patients still die due to the disease itself or the toxicity of the therapeutic drugs, and thereby the heterogeneity of this disease remains the main obstacle [1, 2]. Two of the main systems that have been used to classify AML into subtypes are the French-American- British (FAB) classification and the newer World Health Organization (WHO) classification [5, 6]. The FAB classification comprehensively divides AML into subtypes, M0-M7, based on the types and maturity or differentiation of leukemia cells in blood or bone marrow, which is based largely on the morphological features of leukemia cells looked under the microscope after routine staining [5, 6]. However, this comprehensive classification does not play an ideal role in guiding the therapy for the patients with AML because there are almost no differences in the treatment regimens for other subtypes, except AML-M3 subtype [5–7]. The World Health Organization (WHO) classification of AML was established in 2001 with the advances of sequencing technology [8]. After two updated in 2008 and 2016, respectively, the WHO system divides AML into 11 subtypes based on chromosomal and genetic mutations [9, 10]. Compared with the FAB classification, the WHO system displays more advantages in guiding individual treatment because it takes account many of the factors that affect diagnosis and prognosis of AML [5, 6]. Nevertheless, the WHO classification does not cluster AML comprehensively. Epigenetics refers to the covalent modification of DNA, RNA and histones without changing the DNA sequence to affect the expression of genes [11]. Background Epigenetic therapy targeting DNA methylation (decitabine and azacytidine) and histone acetylation (Chidamide) benefit the patients with AML, which not only proves the value of targeting epigenetic regulators in the treatment of AML but also urges the need to better understand epigenetic regulation and explore novel critical targets for effective AML treatment [12–16]. Histone methylation modification is one kind of epigenetic modification that modifies chromatin structure by regulating the methylation of lysine or arginine residues in histone tails to achieve gene expression or silencing [17–19]. Similar to other epigenetic modifications, there are three types of regulators involved in histone methylation modification, namely histone methyltransferases (“Writers”), histone demethylases (“Erasers”) and histone methyl modification recognition factors (“Readers”) [17]. In recent years, emerging studies have shown that aberrant histone methylation is closely related to leukemogenesis [20, 21]. Meanwhile, a series of lead compounds targeting histone methylation regulators have completed preclinical and even entered clinical studies [16, 22–25]. However, the current studies of histone methylation modification in AML are usually limited to the changes in one or a few single genes, while the mutation patterns of genes related to leukemia often involve multiple genes that interact in a highly coordinated manner in clinical practice [1, 2]. Therefore, it is necessary to systematically study the expression characteristics of histone methylation regulators in AML, which likely favors the diagnosis and treatment of AML. In this study, we established a comprehensive classification approach for AML based on the expression level of 24 HMMRs in AML patients. We identified two distinct histone methylation modification patterns that exhibit remarkable differences in several clinical and biological characteristics, including AML-M0 Page 3/31 Page 3/31 Page 3/31 distribution, mutations of NPM1, survival, TMB, somatic mutations, pathways activation and immune cell infiltration. Besides, based on the clustering, we established the scoring system, M-RiskScore, which not only acts as an independent poor prognostic predictor but also benefits the treatment decisions. distribution, mutations of NPM1, survival, TMB, somatic mutations, pathways activation and immune cell infiltration. Besides, based on the clustering, we established the scoring system, M-RiskScore, which not only acts as an independent poor prognostic predictor but also benefits the treatment decisions. Landscape of genetic variation of histone methylation modification regulators in AML The overview of this work was shown in the form of a flowchart (Fig. 1). The clinical profiling of TCGA- LAML cohorts was summarized in Table 1. Page 4/31 Table 1 Clinical characteristics of AML cohort for classification and validation Clinical Characteristics Number Percent (%) TCGA-LAML (n = 124) Survival status Survival 47 37.9 Death 77 62.1 Age ≥ 60 years 51 41.1 < 60 years 73 58.9 Gender Female 57 46.0 Male 67 54.0 IDH2-R132 mutation Negative 112 90.3 Positive 12 9.7 IDH2-R140 mutation Negative 114 91.9 Positive 10 8.1 IDH2-R172 mutation Negative 122 98.4 Positive 2 1.6 NPM1 mutation Negative 93 75.0 Positive 31 25.0 FLT3 mutation Negative 88 71.0 Positive 36 29.0 Activating RAS mutation Negative 116 93.5 Positive 8 6.5 FAB subtype M0 12 9.7 M1 30 24.2 M2 27 21.8 M3 13 10.4 M4 27 21.8 M5 12 9.7 M6 2 1.6 Page 5/31 Page 5/31 Clinical Characteristics Number Percent (%) M7 1 0.8 Risk status Favorable 28 22.6 Intermediate 71 57.3 Poor 25 20.1 GEO-GSE71014 (n = 104) Number Percent (%) Survival status Survival 68 65.4 Death 36 34.6 A total of 24 HMMRs were finally identified in this study, including 13 writers, 7 erasers and 4 readers. Figure 2A summarized the dynamic reversible process of histone methylation mediated by HMMRs. The differences in gene expression levels between normal and cancer tissues are the critical messages for researchers to assess its attribute, oncogene or tumor suppressor [26, 27]. However, due to the special distribution of hematological malignancy, it is almost impossible for us to obtain the expression messages of HMMRs of the patients with AML before illness. Thence, we chosen to explore the gene expression of 24 regulators in AML patients with different RiskStatus. The result showed that there were 6 regulators with remarkable gene expression differences in different RiskStatus, including 1 writer (CARM1), 2 readers (ATRX and PC) and 3 erasers (KDM2A, KDM4A and KDM5B) (Fig. 2B). Among them, the up-regulation of ATRX, PC, KDM2A and CARM1 implied poor RiskStatus while the up-regulation of EZH2 and KDM5B suggested favorable RiskStatus (Fig. 2B). finally identified in this study, including 13 writers, 7 erasers and 4 readers. Considering the interaction of the HMMRs, we preformed the co-expression analysis. As showed in Fig. Landscape of genetic variation of histone methylation modification regulators in AML 2C, there were two positive co-expression zones (as shown in red boxes) and the bigger one displayed that SETD2, a histone methyltransferase, expressed a positive correlation with ATRX, KDM5A, NSD1 and KMT2A, respectively in gene expression, while the smaller one exhibited 3 writers, including SUV39H1, DOT1L and EHMT2, taking together to form a co-expression loop. Meanwhile, a significant negative correlation in gene expression was discovered between KDM5B and SETD7, KDM5A and SUV39H1, along with ATRX and DOT1L (Fig. 2C). Subsequently, we summarized the incidence of the somatic mutation and copy number variation (CNV) of 24 regulators in the patients with AML. The result showed that there was no obvious genetic alteration in other regulators except for KMT2A that exhibited a significant CNV alteration, including fusion and amplification in copy number (Fig. 2D). All in all, the above analyses displayed the basic landscape of 24 HMMRs in AML and indicated that cross-talking among the regulators of writers, readers, and erasers may play an important role in the formation of different histone methylation modification patterns in AML. Histone methylation modification patterns mediated by 24 regulators in AML Page 6/31 In order to verify whether Histone methylation regulators affect the formation of histone methylation modification patterns in AML, the R package of ConsensusClusterPlus was used to classify patients with qualitatively different m6A modification patterns based on the expression of 24 HMMRs. It is obvious that the optimal consensus clustering can be obtained when K value of the consensus matrix was equal to 2 (Fig. 3A, S1), which was confirmed by the results of Consensus Cumulative Distribution Function (CDF) Plot (Fig. 3B) and Delta Area Plot (Fig. 3C). Therefore, two distinct histone methylation modification patterns were eventually identified, including 95 cases patterns A and 56 cases in patterns B. We termed these two patterns as Cluster A and Cluster B, respectively. Principal component analysis (PCA) for the transcriptome profiles of these two modification patterns also showed that there was significant distinction existed on them (Fig. 3D). To validate the established histone methylation modification patterns, we repeated the correlated analyses using another independent AML-cohort, GSE71014, of which the clinical profiling was summarized in Table 1. As shown in Figure S2A-C, the best consensus clustering also obtained when K value of the consensus matrix was equal to 2, which was consistent with the previous clustering analysis (Fig. 3A-D, S1). What’s more, the survival analysis displayed that the patients of Cluster A exhibited a more prominent survival advantage than Cluster B in both TCGA-LAML cohort and GSE71014 cohort (Fig. 3E-F). Subsequently, unsupervised clustering of 24 HMMRs was conducted to explore the clinical features of these two histone methylation modification patterns in TCGA-LAML cohort while the two modification patterns, FAB subtypes, mutations of several genes, gender, age, fustat and futime were used as patient annotations. As showed in Fig. 3G, the expression of 24 histone methylation regulators were remarkably upregulated in Cluster A,comparing with Cluster B. It is worth noting that there was a significant difference between Cluster A and Cluster B in the distribution of FAB category, that was, AML-M0 subtype was only distributed in Cluster A but not in Cluster B. Besides, more patients with NPM1 mutation distributed in Cluster B than Cluster A. However, there were no obvious differences between the patients of Cluster A and Cluster B in FLT3 mutation, IDH1 mutation, activating Ras, gender, age, fustat and futime. Histone methylation modification patterns mediated by 24 regulators in AML The above results showed that the patients with AML could be clustered effectively based on the expression of histone methylation modification regulators. What’s more, the significant clinical characteristics’ differences of the two histone methylation modification patterns also indicated histone methylation modification plays a critical role in AML progression. Biological characteristics of distinct histone methylation modification patterns in AML Biological characteristics of distinct histone methylation modification patterns in AML To determine the biological behavior differences between Cluster A and Cluster B, we firstly analyzed their distribution differences of somatic mutation in TCGA-AML cohort using maftools package. It is believed that higher TMB results in more neo-antigens, increasing chances for T cell recognition and clinically correlates with better immune checkpoint inhibitors (ICI) outcomes [28, 29]. The result of somatic mutation analysis obviously shown that Cluster A presented more extensive tumor mutation burden than Cluster B, with the altering frequency 62.5% against 48.15% (Figure S3A-B). Furthermore, the tumor Page 7/31 Page 7/31 mutation burden (TMB) quantification analysis also confirmed that Cluster A was markedly correlated with a higher TMB value (Fig. 4A) although there was no significant difference in microsatellite instability between Cluster A and Cluster B (Fig. 4B). mutation burden (TMB) quantification analysis also confirmed that Cluster A was markedly correlated with a higher TMB value (Fig. 4A) although there was no significant difference in microsatellite instability between Cluster A and Cluster B (Fig. 4B). To further investigate the characteristics of Cluster A and Cluster B, we performed GSVA enrichment analysis. As showed in Fig. 4C, Cluster A was remarkably enriched in protein and RNA metabolism pathways, such as RNA degradation, spliceosome pathway, valine leucine and isoleucine biosynthesis pathway and so on while Cluster B presented an enrichment pathway associated with energy metabolism, including oxidative phosphorylation pathway, pantothenate and CoA biosynthesis pathway and lysosome pathway. It has been well known that tumor microenvironment (TME), which is also composed of tumor cells, stromal cells, immune cells and multiple secreted factors, plays a crucial role in tumor progression [30, 31]. Therefore, we firstly performed the MCP-counter method to investigate the immune cell infiltration of the two histone modification patterns. The result showed that Cluster A presented a higher immune cell infiltration in T cells, B cells, NK cells and endothelial cells while a lower infiltration in monocytes and neutrophils that were the noumenon of tumor cells (Fig. 4D). Then, we conducted the CIBERSORT method to verify the result of MCP-counter. Consistently, the result also showed that the infiltration of CD4 + memory T cells, naive B cells, resting NK cells and plasma cells in Cluster A were all higher than in Cluster B but the infiltration of monocytes was lower (Figure S3C). Biological characteristics of distinct histone methylation modification patterns in AML Enlighten by the results of immune cell infiltration analyses, we compared the expression of some immune cell markers, chemokines and cytokines between Cluster A and Cluster B so that we could learn more about the immune characteristics of histone modification patterns in AML. As showed in Fig. 4E, a series of immune-activating factors were up-regulated in Cluster A in comparison with Cluster B, including CD244 that is a marker of NK cells, inducible T cell costimulatory ligand (ISOSLG), CD96 that plays a role in the adhesive interaction of activated T and NK cells, TNFRSF18 and CD47. Meanwhile, some immune inhibiting factors were down-regulated in Cluster A, such as HAVCR2, CD86 and LGALS9. Combining the previous results that the expression of 24 histone methylation modification regulators was generally up- regulated in Cluster A (Fig. 3G), we speculated that HMMRs could promote the expression of a series of immune-activating factors in AML. Overall, we had made a rather detailed description of the biological characteristics of the two histone methylation modification patterns in AML and found that Cluster A displayed more remarkable immune cell infiltration that should be a key factor for its prominent survival advantage. Generation of histone methylation modification-related score in AML Generation of histone methylation modification-related score in AML The expression differences of histone methylation modification regulators between Cluster A and Cluster B would directly lead to the differences in the transcriptome of Cluster A and Cluster B, which resulted in Page 8/31 Page 8/31 the clinical and biological characteristics differences in these two histone methylation modification patterns of AML. To further investigate the changes of the transcriptome of Cluster A and Cluster B, we firstly conducted the empirical Bayesian approach of limma package to determine the differentially expressed genes (DEGs) between them. The volcano plots exhibited that there were lots of DEGs between Cluster A and Cluster B, remaining 61 DEGs even with the log2 (fold change) value was equal to ± 3 (Fig. 5A). Then, the clusterProfiler package was used to perform GO enrichment analysis for the DEGs. As shown in Fig. 5B, lots of pathways directly related to immune system were enriched in Cluster A compared with Cluster B, which indicated again that histone methylation modification played a non-negligible role in regulating TME landscapes in AML. Although we had proved that histone methylation modification patterns were strongly associated with the prognosis of AML, it would be difficult to directly predict individual therapeutic effect and prognosis according to the histone methylation modification patterns due to its complexity. Therefore, we established a scoring system to quantify the histone methylation modification patterns of the patients with AML based on the DEGs discovered in Fig. 5A. We termed as M-RiskScore. Univariate Cox analysis was performed to identify the genes that were eligible for lasso regression analysis and the results showed that a total of 8 genes were selected with their p-value was less than 0.01 (Fig. 5C). The subsequent lasso regression analysis displayed the formula for calculating the M-RishScore was composed of 6 genes finally and they were ADAMTS15, CADM1, CDO1, SYT17, FAM163A and POF1B (Fig. 5D-E). Taken together, we identified the DEGs between Cluster A and Cluster B, by which we constructed a scoring system, M-RiskScore successfully. We would try to explore the value of M-RiskScore for predicting the therapeutic effect and prognosis of the patients with AML in the subsequent analysis. The value of M-RiskScore in prognosis prediction for the patients with AML After we have established the scoring system, we started to explore its potential value in predicting the outcome of the patients with AML. With the medium value was set as the cutoff value, we divided the patients with AML into high or low M- RiskScore group. The prognosis analysis showed that the low M-RiskScore group exhibited a better survival advantage than the high M-RiskScore group (Fig. 6A). Importantly, the results of correlation analysis between histone methylation modification patterns and M-RiskScore displayed that Cluster A, which had been proved to possess a more prominent survival benefit than Cluster B (Fig. 3E), was with a lower M-RiskScore (Figure S4A), indicating the reliable and trustful of M-RiskScore due to these consistent results. Therefore, the above analyses implied that M-RiskScore was likely to be an independent poor prognostic marker for the patients with AML. Page 9/31 To verify and compare the predictive accuracy of M-RiskScore with those of other well-known prognostic markers in AML, two receiver-operating characteristics (ROC) curves were created. As showed in Fig. 6B-C, M-RiskScore had the highest AUC value both in the cohort of 3 years and 5 years, indicating its remarkable predictive accuracy compared with other prognostic markers of AML. What’s more, both the results of univariate and multivariate Cox regression analysis showed that the Hazard ratio of M- RishScore was significantly higher than those of other prognostic markers of AML (Fig. 6D-E). Logically, it was no doubt that M-RiskScore could serve as an independent poor prognostic marker for the patients with AML. Finally, to further improve the prognostic value of M-RiskScore in AML, we performed the nomogram associating M-RiskScore with other prognostic markers of AML, including FAB subtype, gender, RiskStatus and age (Fig. 6F). The result of decision curve analysis (DCA) for the nomogram showed that M-RiskScore had a common higher Net Benefit value than other independent prognostic markers in the range of Risk Threshold (Fig. 6G). Moreover, all the calibration plots for the probability of survival of 1 year, 3 years and 5 years ran very close to the diagonal, showing outstanding calibration (Fig. 6H). Finally, to further improve the prognostic value of M-RiskScore in AML, we performed the nomogram associating M-RiskScore with other prognostic markers of AML, including FAB subtype, gender, RiskStatus and age (Fig. 6F). The value of M-RiskScore in prognosis prediction for the patients with AML The result of decision curve analysis (DCA) for the nomogram showed that M-RiskScore had a common higher Net Benefit value than other independent prognostic markers in the range of Risk Threshold (Fig. 6G). Moreover, all the calibration plots for the probability of survival of 1 year, 3 years and 5 years ran very close to the diagonal, showing outstanding calibration (Fig. 6H). However, the result of ROC analysis for nomogram exhibited that there was no significant increase in the predictive accuracy compared with independent M-RiskScore in the cohorts of both 3 years and 5 years (Fig. 6B-C, Figure S4B). However, the result of ROC analysis for nomogram exhibited that there was no significant increase in the predictive accuracy compared with independent M-RiskScore in the cohorts of both 3 years and 5 years Fig. 6B-C, Figure S4B). In a word, these results proved that M-RiskScore played an important role in predicting the outc patients with AML and could be an independent poor prognostic marker of AML. In a word, these results proved that M-RiskScore played an important role in predicting the outcome of the patients with AML and could be an independent poor prognostic marker of AML. M-RiskScore in the role of chemotherapy of AML M-RiskScore in the role of chemotherapy of AML Page 10/31 Chemotherapeutic drugs will continue playing an important role in AML treatment by the virtue of their universal and highly effective cytotoxicity [2]. Therefore, we specifically tested the ability of M-RiskScore to predict the efficacy of induction chemotherapy in patients with AML. Three GEO-AML cohorts with the chemotherapeutic information were download and processed. They were GSE84344, having the respond information for decitabine treatment of the patients with AML, GSE103424 that contained the clinical characteristics of the patients with AML following the treatment of IA regimen (idarubicin + cytarabine) and GSE10087, which processed the clinical information of the patients with AML treated with different standard induction chemotherapy, including IA regimen, CIA regimen (clofarabine + idarubicin + cytarabine), FAI regimen (fludarabine + cytarabine + idarubicin), decitabine, BID FA regimen (twice daily fludarabine + cytarabine) and CECA regimen (cyclophosphamide + etoposide + carboplatin + cytarabine). The basic clinical characteristics of these three GEO-AML cohorts were summarized in Table 2. We found that patients with low M-RiskScore showed more obvious therapeutic advantages both in the complex- chemotherapy-treatment cohort and the decitabine-treatment cohort compared to those with high M- RiskScore (Fig. 7A-B). In contrast, the analysis of the IA regimen cohort exhibited the AML-patients with high M-RiskScore benefited more from the related treatment relatively (Fig. 7C). The somatic mutation analysis for AML-TCGA cohort displayed that the top three most frequently mutated genes in AML- patients with low M-RiskScore were DNMT3A, KIT and WT1, while they were NPM1, DNMT3A and RUNX1 in the high M-RiskScore group (Fig. 7D-E). Given DNMT3A, a DNA methyltransferase encoded by Page 10/31 DNMT3A, is one of the well-known target of decitabine [32], we speculated that a higher mutation frequency of DNMT3A could be the cause of its poorer chemotherapeutic response to the treatment with decitabine in the high M-RiskScore group (Fig. 7B, D). What’s more, given these mutation characteristics, we proposed a hypothesis that the mutated KIT and/or mutated WT1 led to the oncogenesis in AML- patients with low M-RiskScore. Meanwhile, the leukemogenesis in the patients with high M-RiskScore should be attributed to the mutations of NPM1 and/or RUNX1. Table 2 Clinical characteristics of AML cohort for correlation analyses. Patient series GSE103424 GSE103424 GSE103424 No. M-RiskScore in the role of chemotherapy of AML of patients 52 45 41 Age ≥ 60 years NA 45 25 < 60 years NA 0 16 Gender Female 24 17 14 Male 28 28 27 Risk status Favorable 5 0 4 Intermediate 30 23 15 Poor 10 14 22 NA 7 8 0 Response to chemotherapy Yes 17 18 11 No 35 27 30 e above results indicated that M-RiskScore was associated with the chemotherapeu the AML-patients with different chemotherapy treatments and could serve as an e emotherapeutic drugs’ usage. All in all, the above results indicated that M-RiskScore was associated with the chemotherapeutic response of the AML-patients with different chemotherapy treatments and could serve as an evaluated index for chemotherapeutic drugs’ usage. All in all, the above results indicated that M-RiskScore was associated with the chemotherapeutic response of the AML-patients with different chemotherapy treatments and could serve as an evaluated index for chemotherapeutic drugs’ usage. Discussion Individualized treatment is the ultimate goal of cancer treatment, which can effectively alleviate the disease, minimize the risk of complications or side effects and is economical, basing on a comprehensive consideration of various physiological or clinical characteristics of the patients, including gender, age, Page 11/31 Page 11/31 genetic characteristics, and treatment histories [33–35]. The achievement of individualized cancer therapy requires both extensive pathologic subtyping of the tumor for its heterogeneity and diagnosis of genome alterations of the patients with cancer [35]. Advances in sequencing techniques provide an unprecedented view of the complex genetic and nongenetic heterogeneity within individual tumors, which not only remove the restriction on understanding the alterations of gene expression in the patients but also provides an opportunity for tumor subtype classification [33–35]. Herein, we established a repeatable clustering and scoring system for AML basing on 24 HMMRs’ expression of the patients with AML. The patients with a lower expression of 24 histone methylation regulators were mainly divided into Cluster A, exhibiting a better survival advantage, a higher TMB value and a more significant immune cell infiltration, compared with those who possessed a higher expression of related genes in Cluster B. M-RiskScore, a scoring system for quantifying the two histone methylation modification patterns, was constructed based on the expression of 6 DEGs with different weights between Cluster A and Cluster B. A close connection between the histone methylation modification patterns and M-RiskScore was reflected in the obvious overlap of the patients in Cluster A and the patients with low M-RiskScore and vice versa, which was confirmed by a remarkable survival advantage of the patients with lower M-RiskScore. It is of note that M-RiskScore was proved to be a dependent poor prognostic marker of AML for its excellent prognostic accuracy. Moreover, we also found that E-RiskScore could serve as a diagnostic index for the choice of chemotherapeutic drugs from therapy. Although the FAB classification is the first and the most comprehensive classification method for AML, morphologically [5]. However, the AML-M3 also referred to acute promyelocytic leukemia (APL), which has been captured successfully with the combination treatment of two targeted therapies: retinoic acid (RA) and arsenic [7], the other seven subtypes still share similar treatment proposals to date. Interestingly, our study shown that almost all the patients who exhibited AML-M0 subtype were divided into Cluster A. AML-M0 means acute leukemias with minimal signs of myeloid differentiation [5]. Discussion Based on this, we deduced that activity of histone methylation regulators is closely related to AML-M0. Even if no study has reported on the relevance of the pathogenesis of AML-M0 and histone methylation modification, the recent advances in dissecting the molecular regulation and targeting histone methylome in AML together with the success in developing lead compounds specific to key histone methylation-modifying enzymes [16] indicated epigenetic therapies by targeting histone methylome will shed light on the treatment of the patients with AML-M0. Mutations of NPM1 are well known to be associated with leukemogenesis and have already represented a distinct entity in the WHO classification of AML. In our study, we found that most of the patients in Cluster B displayed mutations of NPM1 while NPM1 was one of the top three frequently mutated genes in the high M-RiskScore subtype, which indicated mutant NPM1 should be associated with an inactive gene expression of HMMRs in AML. To date, although few studies have reported on the relationship between histone methylation and mutations of NPM1, the existing research results almost all can be served as the experimental evidence for our conjecture. For example, it has been reported that KDM1A antagonist SP2509 induced more apoptosis in AML cells expressing mutant NPM1 than mixed-lineage leukemia Page 12/31 Page 12/31 Page 12/31 fusion oncoproteins [36]. Another study also pointed out an inhibitor for histone methyltransferase DOT1L exhibited a potent cytotoxicity to the NPM1-mutated AML cells [37]. More researches are needed to ascertain the causality between mutation of NPM1 and the inactive expression of histone methylation regulators, but it is no doubt that histone methylation regulators are promising targets for NPM1-mutated AML therapy. In our analyzed setting, we speculated that the mutations of RUNX1 (RUN family transcription factor 1), which also is a distinct entity in the WHO classification of AML [38] and one of the top three genes with the most frequent mutation rate in the high M-RiskScore subtype, led to leukemogenesis in the patients with high M-RiskScore individually or combination with mutant NPM1, and meanwhile, mutated KIT (KIT proto-oncogene, receptor tyrosine kinase) and/or WT1 (WT1 transcription factor) could be the cause of oncogenesis in the AML-patients with low M-RiskScore. The fact that both mutations of NPM1 and RUNX1 have already been developed as distinct entities in the WHO classification of AML is sufficient to prove the first half of our hypothesis. Discussion Regardless of neither mutated KIT nor mutated WT1 has become the entity for classification of AML, multiple previous elegant studies have shown that they are critically associated with leukemogenesis [39–45]. The purpose of identifying the driver mutations of AML is to discover novel entities for classification and finally find out corresponding therapy. We have already proposed our insights on the treatment of NPM1-mutated AML in the above analysis. As for RUNX1 and WT1, because both of them are transcription factors, attempts to directly disrupt their function are likely to meet limited success for their commonly “undruggable” protein structures and unbearable side effects. However, it is possible to conduct drug development according to the products they regulate, especially enzymes. In contrast to RUNX1 and WT1, KIT, which is a protein tyrosine kinase receptor, is very likely to be a direct chemotherapeutic target for the AML-patients with mutant KIT. Imatinib, a famous inhibitor of BCR-ABL tyrosine kinase inhibitors (TKI) and the first-line chemotherapeutic drug for most patients with chronic myelogenous leukemia (CML) [46, 47], also exhibit an effective inhibition on KIT kinase activity [48, 49], which implies that imatinib could be applied for the chemotherapy in KIT-mutated AML. It is of note that our results showed that the patients with low M-RiskScore possessed a better response to the chemotherapy in an AML-cohort that containing 41 samples from 38 patients who had received different chemotherapeutic regimens, which implied that a lower M-RiskScore was likely to associate with a better chemotherapeutic respond. Furthermore, the continued analyses revealed that the patients with a lower M-RiskScore were likely to benefit from the treatment of decitabine, but not IA therapy regimen. Because the cohorts used for the above analyses do not contain abundant patient samples, there are no significant statistical differences in the analyses mentioned in this section. However, these results still indicated that M-RiskScore may be able to serve as a diagnostic marker for patients with AML. 2. Landscape for histone methylation modification regulators in AML Correlation analysis of the gene expression among the 24 histone methylation modification regulators in AML was explored by the “corrplot” package. The Wilcoxon rank-sum test was used to investigate the expression difference of histone methylome regulators in AML patients with different RiskStatus. Meanwhile, the information of somatic mutation and copy number variations (CNV) of included genes were generated from cBioPortal website (https://www.cbioportal.org/). 1. Data collection Public gene expression, mutation and clinical annotation data were obtained in The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/) and Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). LAML cohort from TCGA database was used as the training dataset. GSE110087, GSE84334, GSE103424 and GSE71014 cohorts were acquired from GEO database. Among them, the first three cohorts were used for analyses of chemotherapy in AML while the last one was for validation. 24 HMMRs, including 13 writers (KMT2A, KMT2D, KMT5A, SETD2, NSD1, SMYD3, NSD2, DOT1L, EZH2, SETD7, CARM1, SUV39H1, EHMT2), 7 erasers (KDM1A, KDM2A, KDM4A, KDM5A, KDM5B, KDM6A, KDM6B) and 4 readers (ATRX, EED, PC, RAG2), were identified from the related studies for further analyses [17]. 3. Consensus clustering for 24 histone methylation modification regulators We conducted the Consensus clustering analysis to identify distinct histone methylation modification patterns based on the expression of 24 histone methylome regulators so that we could classify the patients with AML for further analysis. The number of clusters and their stability was determined by the consensus clustering algorithm and 1000 times repetitions were conducted for guaranteeing the stability of classification [50]. 4. Features of distinct histone methylation modification patterns Conclusions In conclusion, our study demonstrated that histone methylation modification plays a curial role in the diagnosis and prognosis of AML from the perspective of bioinformatics. The defined clustering and scoring system may contribute to the individual treatment of the patients with AML. Page 13/31 Page 13/31 5. Estimation of immune cell infiltration We explored the different immune cell infiltration between the distinct clusters using MCP-counter and CIBERSORT methods. MCP-counter is a methodology based on transcriptomic markers that assess the proportion of immune and stromal cell populations in the tumor microenvironment (TME) from transcriptomic data. There are 10 cell populations estimated by MCP-counter, including T cells, CD8 + T cells, cytotoxic lymphocytes, B lineage, NK cells, Myeloid dendritic cells, Neutrophils, Endothelial cells and Fibroblasts [53]. CIBERSORT, a deconvolution algorithm reported, is used to quantify the 22 infiltrated immune cells according to normalized gene expression profiles. The 22 immune cells included memory B cells, naive B cells, plasma cells, resting/activated DCs, resting/activated NK cells, resting/activated mast cells, eosinophils, neutrophils, monocytes, M0–M2 macrophages, and 7 T-cell types (CD8 + T cells, regulatory T cells (Tregs), resting/activated memory CD4 + T cells, follicular helper T cells, naive CD4 + T cells and γδ T cells) [54]. What’s more, 20 immune checkpoint genes (CD244, PDCD1, PD-L1, CTLA4, CD80, CD86, CD28, TIGIT, PVR, CD96, SIRPA, CD47, LGALS9, HAVCR2, ICOS, ICOSLG, TNFSF18, TNFRSF18, KLRD1, KLRC1) were retrieved from previous study [55], and correlation analysis was conducted to determine the difference expression of immune checkpoint genes between the two clusters. 4. Features of distinct histone methylation modification patterns A series of analyses were performed to validate the histone methylation modification patterns after finishing the consensus clustering. Principal component analysis (PCA), a technique for reducing the dimensionality of such datasets, increasing interpretability but at the same time minimizing information loss [51], was conducted to verify the quality of consensus clustering. “Survival” package was applied to explore the time-dependent prognostic value of the clusters. Page 14/31 Page 14/31 Tumor mutational burden (TMB), a new promising biomarker emerged recently, is classically defined as the number of non-synonymous exonic mutations per megabase (Mb) [28]. The total number of mutations counted was divided by the exome size (38 Mb was utilized as the exome size), by which we calculated the TMB of each case. TMB correlation analysis was executed to explore the association between TMB and the distinct clusters. To investigate the difference in biological process activity between the clusters, we performed gene set variation analysis (GSVA) enrichment analysis using “GSVA” R packages, which is an unsupervised and non-parametric method for estimating the variation in biological process and pathway activity in the samples of an expression dataset [52]. The gene sets of “h.all.v7.1.symbols” were downloaded from the MSigDB database (https://www.gsea-msigdb.org/gsea/index.jsp) for GSVA analysis. The result was considered to be statistically significant while its p-value was less than 0.05. Moreover, we explored the somatic gene mutations in the different clusters by the “maftools” package. 6. Construction of histone methylation modification-related score (M-RiskScore) in AML 6. Construction of histone methylation modification-related score (M-RiskScore) in AML We firstly conducted the empirical Bayesian approach of limma package to determine the differentially expressed genes (DEGs) between the histone methylation modification patterns. Then, we performed univariate Cox regression analysis to identify the prognostic genes with a p-value less than 0.01 for further analysis. Page 15/31 The lasso regression analysis was subsequently applied to construct the histone methylation modification-related score (RiskScore) by “glmnet” and “survival” package. In this analysis, a lasso penalty was used to account for shrinkage and variable selection. The optimal value of the lambda penalty parameter was defined by performing 10 cross-validations. The calculation formula for the acetylation-related score was as follows: penalty was used to account for shrinkage and variable selection. The optimal value of the lambda penalty parameter was defined by performing 10 cross-validations. The calculation formula for the acetylation-related score was as follows: According to the median of the RiskScore, the patients with AML were divided into two groups (high-risk or low-risk group). We then performed the survival analysis based on this grouping strategy. A receiver operating characteristic (ROC) curve, which is a plot of the sensitivity versus 1 − specificity of a diagnostic test [56], was constructed to examine the prognostic accuracy for purpose of verifying the RiskScore. Finally, to validate whether the RiskScore could be an independent prognostic marker in AML, we carried out the univariate and multivariate Cox regression analyses. 8. Biological characteristics of RiskScore GSVA analysis, TMB analysis, Somatic gene mutations analysis and immune cell infiltration analysis were performed to exhibit the biological characteristics of the high-RiskScore group and low-RiskScore group. 7. Nomogram construction and validation In order to improve the clinical application of RiskScore, we constructed the nomogram based on Cox regression model. There were 5 components of the nomogram, including gender, FAB subtype, RiskStatus, age and RiskScore. Decision curve analysis was performed to compare the net benefits of different prognostic models (FAB subtype, RiskStatus, RiskScore and nomogram). The concordance index, calibration plot, and ROC curve were used to verify the nomogram. Nomograms are a pictorial representation of a complex mathematical formula [57]. Medical nomograms use biological and clinical variables, such as tumor grade and patient age, to determine a statistical prognostic model that generates a probability of a clinical event, such as cancer recurrence or death, for a particular individual [58]. Model performance was evaluated through calibration and discrimination [59]. Bias corrected calibration for 3 years and 5 years overall survival rate was performed by 1000 bootstrap resamples to evaluate the consistency between the observed and estimated survival probability by “rms” package. Discrimination was evaluated by Harrell’s concordance index (C-index) and ROC curve. A higher C-index value demonstrated better model-fitting performance. Area under the ROC curve (AUC) value is an effective way to summarize the overall diagnostic accuracy of the test, taking values from 0 to 1, where a value of 0 indicates a perfectly inaccurate test and a value of 1 reflects a perfectly accurate test, indicating a higher AUC value revealed superior model discriminative ability [56]. Decision curve analysis (DCA) was further performed to measure and compare the clinical utilities of the different prognostic models. DCA is a method for evaluating the benefit of a diagnosis test across a range of patient preferences for accepting the risk of undertreatment and overtreatment to facilitate decisions about test selection and use [60]. Page 16/31 Page 16/31 Abbreviations AML Acute myeloid leukemia HMMRs Histone methylation modification regula ROC receiver operating characteristic AUC Area under the ROC curve TMB Tumor mutational burden PCA Principal component analysis ICI immune checkpoint inhibitors CNV copy number variations GSVA gene set variation analysis TME tumor microenvironment DEGs differentially expressed genes WHO World Health Organization TKI tyrosine-kinase inhibitors CML chronic myelogenous leukemia Declarations A k l d 9. Validation of Molecular Classification Here, we employed the RECA-EU cohort from the ICGC database for score validation. Survival analysis, ROC curve, univariate and multivariate Cox regression analyses were also performed to estimate the clinical value of the acetylation-related score Abb i ti clinical value of the acetylation-related score Abbreviations AML Acute myeloid leukemia HMMRs Histone methylation modification regulators ROC receiver operating characteristic AUC Area under the ROC curve TMB Tumor mutational burden PCA Principal component analysis ICI immune checkpoint inhibitors CNV copy number variations GSVA gene set variation analysis TME tumor microenvironment DEGs differentially expressed genes WHO World Health Organization TKI tyrosine-kinase inhibitors CML chronic myelogenous leukemia Declarations Acknowledgements Competing interests The authors have no financial conflicts of interest. The authors have no financial conflicts of interest. Declarations Acknowledgements Page 17/31 We are grateful to Wen-Chien Chou (Department of Laboratory Medicine, National Taiwan University Hospital, Taiwan), Luciano Castiello (Pasteur Italy Institute, Italy), Chieh-Lin Jerry Teng (Division of Hematology/Medical Oncology, Taichung Veterans General Hospital, Taiwan) and Stephan Rainer Bohl (Internal Medicine III, University Hospital of Ulm, Germany) for shared the genetic and clinical information of the patients with AML. Authors’ contributions Conception and design of studies: DR, ZM, XC, XN. Drafting article: DR, XC, HW, XT. Review and discussion: ZM, HW, XT. Funding This work was supported by Guangdong Basic and Applied Basic Research Foundation (No. 2019A1515010294) to TX, and the National Natural Science Foundation of China (No. 81472730) to WH. Ethics approval and consent to participate Not applicable. Not applicable. Availability of data and materials All data and materials are available via DR. References 1. 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Overview of somatic mutations and chromosomal variations of 24 HMMRs in AML. Page 24/31 Page 24/31 Figure 3 Th bli h f hi h l i difi i i AML A C P 25/31 Figure 3 The establishment of histone methylation modification patterns in AML A. Consensus matrix of TCGA- LAML cohort for k value is equal to 2. B. Cumulative distribution function (CDF) plot of the consensus matrices for k = 2-9. C. Delta area plot of CDF plot. D. Principal component analysis for the transcriptome profiles of the two histone methylation modification patterns. E. Survival analysis for the two histone methylation modification patterns of TCGA-LAML cohort. F. Survival analysis for the two histone Figure 3 Figure 3 The establishment of histone methylation modification patterns in AML A. Consensus matrix of TCGA- LAML cohort for k value is equal to 2. B. Cumulative distribution function (CDF) plot of the consensus matrices for k = 2-9. C. Delta area plot of CDF plot. D. Principal component analysis for the transcriptome profiles of the two histone methylation modification patterns. E. Survival analysis for the two histone methylation modification patterns of TCGA-LAML cohort. F. Survival analysis for the two histone Page 25/31 Page 25/31 Page 25/31 methylation modification patterns of GSE71014 cohort. G. Unsupervised clustering of 24 HMMRs in the TCGA-LAML cohort. The histone methylation modification patterns, FAB subtype, activating RAS, gender, age, fustant, fustime and mutations of genes (FLT3, NPM1, IDH1-R172, IDH1-R140, IDH1-R132) were used as annotation. p<0.01; * p<0.001. p ; p p p Figure 4 Figure 4 Page 26/31 Page 26/31 Biological characteristics of the two histone methylation modification patterns A. Tumor mutation burden analysis for two histone methylation modification patterns. p<0.01. B. Microsatellite instability analysis for two hstone methylation modification patterns. ns: no statistically significant. C. GSVA enrichment analysis showing the activation states of biological pathways in distinct histone methylation modification patterns. The heatmap was used to visualize these biological processes. Activated pathways were represented by red and inhibited pathways were represented by blue. D. Figure 2 The abundance of each TME infiltrating cell in two histone methylation modification patterns. p<0.01; * p<0.001. E. The different expression of several immune checkpoint proteins between the two histone methylation modification patterns. p<0.01; * p<0.001. Biological characteristics of the two histone methylation modification patterns A. Tumor mutation burden analysis for two histone methylation modification patterns. p<0.01. B. Microsatellite instability analysis for two hstone methylation modification patterns. ns: no statistically significant. C. GSVA enrichment analysis showing the activation states of biological pathways in distinct histone methylation modification patterns. The heatmap was used to visualize these biological processes. Activated pathways were represented by red and inhibited pathways were represented by blue. D. The abundance of each TME infiltrating cell in two histone methylation modification patterns. p<0.01; * p<0.001. E. The different expression of several immune checkpoint proteins between the two histone methylation modification patterns. p<0.01; * p<0.001. Page 27/31 Figure 5 Page 28/31 Figure 5 The establishment of M-RiskScore A. A volcano plot showing the different expression genes (DEGs) between the two histone methylation modification patterns. The value of log2 fold change is equal to±3. B. Functional annotation for 61 DEGs using GO enrichment analysis. The color depth of the barplots represented the activation of the pathways. The length of the barplots represented the number of the genes enriched. C. The prognostic analyses for 61 DEGs in the TCGA-LAML cohort using a univariate Cox Figure 5 The establishment of M-RiskScore A. A volcano plot showing the different expression genes (DEGs) between the two histone methylation modification patterns. The value of log2 fold change is equal to±3. B. Functional annotation for 61 DEGs using GO enrichment analysis. The color depth of the barplots represented the activation of the pathways. The length of the barplots represented the number of the genes enriched. C. The prognostic analyses for 61 DEGs in the TCGA-LAML cohort using a univariate Cox Page 28/31 Page 28/31 regression model. The genes highlighted were selected for lasso regression analysis. D and E. The results of lasso regression analysis. The determination of the value of λ (D) and the determination of the coefficients' values of the selected genes from C (E). g ( ) Figure 6 Page 29/31 Figure 6 The value of M-RiskScore in prognostic prediction of AML A. Survival analysis for low and high M- RiskScore patient groups in the TCGA-LAML cohort. B-C. ROC analyses for prognostic prediction accuracy Figure 6 Figure 6 The value of M-RiskScore in prognostic prediction of AML A. Survival analysis for low and high M- RiskScore patient groups in the TCGA-LAML cohort. B-C. ROC analyses for prognostic prediction accuracy Page 29/31 of 3-years survival rate (B) and 5-years survival rate (C) of M-RiskScore, Gender, Age, FAB subtype and Riskstatus in TCGA-LAML cohort. D-E. Univariate Cox regression model (D) and multivariate Cox regression model (E) for prognostic analyses of M-RiskScore, Gender, Age, FAB subtype and Riskstatus in TCGA-LAML cohort. F. Construction of nomogram making up of M-RiskScore, Gender, Age, FAB subtype and Riskstatus. G. The decision curve analysis for nomogram and its components. H. The calibration plot of nomogram for the probability of survive of 1 year, 3 years and 5 years. Figure 7 M-RiskScore in the role of chemotherapy of AML A. Correlation analysis of M-RiskScore with complex- chemotherapeutic respond in GSE110087-AML cohort. B. Correlation analysis of M-RiskScore with decitabine treatment respond in GSE84334-AML cohort. C. Correlation analysis of M-RiskScore with IA M-RiskScore in the role of chemotherapy of AML A. Correlation analysis of M-RiskScore with complex- chemotherapeutic respond in GSE110087-AML cohort. B. Correlation analysis of M-RiskScore with decitabine treatment respond in GSE84334-AML cohort. C. Correlation analysis of M-RiskScore with IA Page 30/31 Page 30/31 regimen treatment respond in GSE103424-AML cohort. D. Somatic mutation analysis of low (left) and high (right) M-RiskScore patients’ groups with AML in TCGA-LAML cohort. regimen treatment respond in GSE103424-AML cohort. D. Somatic mutation analysis of low (left) and high (right) M-RiskScore patients’ groups with AML in TCGA-LAML cohort. 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https://openalex.org/W3111045874	https://cityterritoryarchitecture.springeropen.com/track/pdf/10.1186/s40410-020-00130-x	English	null	Sustainable housing development: role and significance of satisfaction aspect	City, territory and architecture	2,020	cc-by	8,840	© The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. Abstract Providing quality public housing is one of the main goals of the United Arab Emirates (UAE) government. This paper assesses the level of satisfaction with public housing offered by the UAE government to its citizens based on the physical characteristics and traditional social aspects of the housing unit, urban design, and social environment in the residential area, whereas also their contribution to the residents’ life quality under overall satisfaction with the place of living. At the same time, the study provides access to sustainability measurements applied at both the environmental and social levels within the Estidama accredited national rating system. The survey results of two residential com- plexes in Abu Dhabi show that the majority of residents are mostly satisfied, although the overall level of satisfaction with the functionality of the building and public facilities provided was generally higher than that related to the social environment in the residential district. The research focused on the application of new technologies that increase the level of sustainability in future housing projects. Keywords: Estidama, Green building, Public housing, Residential satisfaction, UAE, Urban sustainabil In the Middle East, the United Arab Emirates (UAE) strives to consistently develop the urban construction industry through the creation of sustainable cities using innovative technologies that would be consistent with overall state expansion and growth over the years. To this effect, green building norms and regulations have been introduced in Abu Dhabi and Dubai, such as the national Estidama Pearl program, which was launched in 2010 by the Department of Urban Planning and Municipali- ties in Abu Dhabi (Awadh 2017). This program includes a Pearl Rating System (PRS), which ensures the sustain- ability of housing throughout its life cycle from design to construction and operation (Alobaidi et al. 2015). The rating system with a scale from 1 to 5 Pearl focuses on the construction of residential buildings and villas to the different individual requirements to improve the sus- tainability of the built-up environment. The developed requirements should contribute to minimizing water and energy consumption, improving waste recycling, and using local environmentally friendly materials for con- struction. The design of buildings must meet a minimum rating of 1 (Pearl 1) and, for public housing, this mini- mum is 2 (Pearl 2). Sustainable housing development: role and significance of satisfaction aspect Iman Abdelshahid Ibrahim* Ibrahim City Territ Archit (2020) 7:21 https://doi.org/10.1186/s40410-020-00130-x Ibrahim City Territ Archit (2020) 7:21 https://doi.org/10.1186/s40410-020-00130-x *Correspondence: iibrahim@sharjah.ac.ae Department of Applied Design/Interior Design, University of Sharjah, Sharjah, UAE Introduction Housing improvement is one of the most important sec- tors of the economy, which ensures the prosperity of urban development and sustainability (Rahman et al. 2018). The variety of design solutions and innovative technologies incited a lot of challenges in the quality of accommodation and satisfaction of residents. Often, designers strive to maintain the aesthetics of hous- ing appearances and neglect the actual needs of clients (Lopez 2010). Compared to commercial buildings, the residential design must correspond to day-to-day con- cerns of people’s lives and address problems related to their life experience and ambience (Anderson 2016). Besides, due to excessive energy consumption, housing construction must be sustained (Roufechaei et al. 2014). Thus, for successful sustainable housing development, an optimal balance between sustainable housing and cus- tomer satisfaction is required (Chan and Adabre 2019). Literature review As stated above, in projecting housing complexes, the housing design should represent social, aesthetic, and environmental concepts and include all elements for cre- ating an interior space that would satisfy the lifestyle of people or community (Dohr and Portillo 2011). Satisfac- tion with residency is one of the most important aspects that affect the quality of life (Walton et al. 2008) and the very behaviour of people in living spaces (Sakip et al. 2012). The satisfaction of residents is based on personal qualities (cognitive, emotional, or behavioural character- istics) and social characteristics of the living environment (Mohit and Nazyddah 2011). Thus, social, economic, and environmental aspects of housing design should be con- sidered in order to achieve maximum effect (Ali 2010; Karji et al. 2019). It can be achieved by respecting the cultural, psychological, physiological, financial, and his- torical characteristics and preferences of residents or the community. These complexes are residential neighbourhoods con- sisting of villas and houses and related administrative and commercial buildings, mosques, schools, and parks. These properties have been designed following the tra- ditional planning of sub districts and villages in mind. The main task in the design was to combine conven- tional architecture and innovative technologies through an optimal balance. The results of a study on sustainable social housing in the city of Al Ain (Ahmed 2017) showed that traditional principles of neighbourhood planning and design are less effective for residents as local com- munities may have different cultural and social develop- ment needs. According to the author of the article, it is community residents who can regulate the sustainability of urban forms by giving preference to one tendency over another.h The purpose of this study was to assess the level of sat- isfaction with public accommodation facilities offered by the UAE government to citizens based on the physical characteristics and traditional social aspects of the hous- ing unit, urban design, and social environment in the res- idential area, whereas also their contribution to residents’ life based on general social and cultural satisfaction with housing. For this purpose, the following research objec- tives should be fulfilled: However, along with satisfying these basic needs of res- idents, other aspects such as security and social status are also highly relevant. Page 2 of 13 Page 2 of 13 Ibrahim City Territ Archit (2020) 7:21 Ibrahim City Territ Archit (2020) 7:21 Ibrahim City Territ Archit (2020) 7:21 extent to which the program interacts with the evalu- ation to achieve the best possible outcome. In the UAE, much attention and funding are given to the development of public or social housing. At present, the Ministry of Public Works and Housing and Sheikh Zayed’s Housing Program, as well as local social hous- ing agencies, are dealing with the problems of providing state accommodation (Ahmed 2017). The goal of these federal organizations is to provide decent housing with a large number of subsidies that meets the requirements of all citizens and is aimed at building entire neighbour- hoods following the principles of traditional, functional, and autonomous models of urban sub districts (Patri- cios 2002). In Abu Dhabi, such projects as Ain Al Fay- dah, Watani, Jebel Hafeet, Al Falah, and others have been developed and implemented through support from gov- ernment investments.h The analytical assessment of residential complexes in terms of environmental, social, and economic sus- tainability can enable setting particular standards in design that should be followed in new projects to ensure increased sustainability. Besides, it allows developing rec- ommendations for future projects to be followed for a higher level of residential conditions in the state housing stock through sustainability evaluation. Materials and methods Case study areah y This research is devoted to studying the level of satisfac- tion with the principles of sustainable urban develop- ment on the example of two public housing districts of the UAE, located in the Emirate of Abu Dhabi. The first site of Al Ain Faydah is located in Al Ain city near mount Jebel Hafeet. It covers an area of 4.12 million m2 and con- tains 2000 villas with entertainment, educational, and cultural facilities integrated into the development (Fig. 1). This project is aimed at recreating the community of Faridj (traditional region of the Emirates), where villas are grouped around a common open space. The archi- tectural design of the facades applied to the Spanish and Mediterranean style (Fig. 2). Each villa with a total area of 382 m2 contains five bedrooms, a maid’s room, a living and dining room, a Majlis, a kitchen, a laundry room, and bathrooms. More than 12,000 residents can reside in this complex. The first phase of construction according to the general plan (Fig. 1) was fully implemented and the pro- ject was introduced into operation in 2015 (Plan Al Ain… 2015).h This research is devoted to studying the level of satisfac- tion with the principles of sustainable urban develop- ment on the example of two public housing districts of the UAE, located in the Emirate of Abu Dhabi. The first site of Al Ain Faydah is located in Al Ain city near mount Jebel Hafeet. It covers an area of 4.12 million m2 and con- tains 2000 villas with entertainment, educational, and cultural facilities integrated into the development (Fig. 1).h This project is aimed at recreating the community of Faridj (traditional region of the Emirates), where villas are grouped around a common open space. The archi- tectural design of the facades applied to the Spanish and Mediterranean style (Fig. 2). Each villa with a total area of 382 m2 contains five bedrooms, a maid’s room, a living and dining room, a Majlis, a kitchen, a laundry room, and bathrooms. More than 12,000 residents can reside in this complex. The first phase of construction according to the general plan (Fig. 1) was fully implemented and the pro- ject was introduced into operation in 2015 (Plan Al Ain… 2015). Literature review According to Shach-Pinsly (2019), in urban areas that are free from crimes and strife, peo- ple feel safer when interacting with others, resulting in increased trust and reciprocity among residents and enhanced feelings of community and belonging. Besides, these social interactions in a community build good rela- tions between residents (Hussein 2019) and a sense of pride in belonging to that community (Kohon 2018). Another important aspects are the identity and comfort of urban life through environmental design and devel- oped infrastructure (Shawket 2018). 1. To assess the level of satisfaction of UAE citizens and sustainability parameters in residential models and housing complexes in the state housing stock; 2. To evaluate the application of environmental, social, and economic sustainability in public housing con- struction and its compatibility with the social needs of citizens considering the culture of the Emirates; Nowadays, in addressing the issue of upgrading the life quality, a healthy environment is given more priority by improving socio-economic and environmental conditions for present and future generations (Dizdaroglu and Yigit- canlar 2016). Sustainable development is about meeting human needs and improving their quality of life by mini- mizing the negative impact on the environment (Ingrao et al. 2018; Ioppolo et al. 2019). Since public places in neighbourhoods are designed for communication and 3. To determine the sustainability assessment system for public housing construction, Estidama, and how it fits the nature of the project; i j 4. To evaluate the community’s ability to apply the post- settlement evaluation to housing estates and the Ibrahim City Territ Archit (2020) 7:21 Ibrahim City Territ Archit (2020) 7:21 Page 3 of 13 Page 3 of 13 homeowners are more gratified than apartment own- ers (Al‐Momani 2000), and also the fact ownership and rent can affect the level of satisfaction (Riazi and Emami 2018). All of the above-mentioned factors and character- istics are accounted for in developing a conceptual model for satisfaction assessment, and some aspects are taken as units of measure. The following section discusses the elements that will be used to measure the satisfaction degree. They also form the basis for the development of a data collection tool. recreation, the well-being of their residents can also serve as an indicator of satisfaction with the urban surround- ing and a better quality of life (Van Kamp et al. 2003). These places include playgrounds for children, parks, walking trails, monuments, and architectural structures. Literature review According to Saiedlue et al. (2015), the presence of water elements (lakes, fountains) and the abundance of green spaces are fundamental to increasing satisfaction through refreshing and purifying the environment and, thus, pro- viding significant health benefits to residents. ii In addition to the environmental and social compo- nents of living satisfaction, the development of infrastruc- ture and economic factors are also of high importance. A sense of identity, belonging, and comfortable city life arises from such elements in urban design as points of key services used by residents regularly (Dempsey et al. 2011). The latter include doctors and post offices, banks, supermarkets or stores on the corner, schools, preschool facilities, libraries, sport and entertainment complexes, restaurants and cafes, community centers, and others. All of these elements preferably must be located in places equally accessible to residents and guests. Besides, the transport interchange should meet the needs of all par- ticipants (pedestrians, cyclists, and car drivers), whereas also the availability of entrances for people with physi- cal disabilities and various types of municipal transport should be on a high level. Materials and methods Case study areah Thus, to study the impact of various aspects on the residency comfort is necessary for a better understand- ing of what initiatives to be taken to improve the sustain- ability parameters in residential models and complexes of the state housing stock. The paper (Liu 2003) presents the results of assessing the quality of housing projects by estimating the level of satisfaction of residents. It was shown that despite the observance of technical and engineering aspects in the development of large housing complexes, more success of the project is expected with the cooperation and coordination between designers and other parties involved (Liu 2003; Lee and Park 2010; Mohit and Nazyddah 2011). Ultimately, each residential project is implemented for accommodation, and, there- fore, a dialogue between the parties is important in the design process as it allows for a proper understanding of the political and social responsibility of the state (Liu et al. 2011; Lichtenstein et al. 2013).i The Al Watani housing complex located in Khalifa City A in the Emirate of Abu Dhabi was chosen as the next object for research (Fig. 3). It covers a total area of 1.85 million square meters and offers 1.390 villas and 50 houses with 2.500 apartments to satisfy local housing needs. The project represents a complex configuration of public facilities supported by an appropriate infrastruc- ture that includes schools, stores, and public open spaces to establish communication centers and local meetings. As follows from Fig. 3, only the first stage of this pro- ject has been completed, and 400 villas were commis- sioned in 2015. The design of each villa is in the style of traditional architecture, which perfectly matches modern design tendencies through modern materials and tech- nology (Fig. 4). Besides, the assessment of the final housing option pro- vides important information on the degree of satisfaction with the needs, demands, and expectations of residents (Wongbumru and Dewancker 2016). Also, the study (Teck-Hong 2012) demonstrates how demographic and socio-economic factors influence the level of satisfac- tion. For example, older people are usually more enjoy their household than young ones (Wagner et al. 2010), The total area of each villa is 405 m2, which includes four or five bedrooms, a Majlis, a kitchen, bathrooms, garages, maids’ facilities, and other necessities for the average Emirati family (Watani Housing Project 2015). Ibrahim City Territ Archit (2020) 7:21 Ibrahim City Territ Archit Page 4 of 13 Fig. Materials and methods Case study areah 1 A master plan depiction (on the top) (Plan Al Ain… 2015) and a picture from GoogleMaps of the implemented project (on the bottom) of Al Ain Faydah, Al Ain, Abu Dhabi Fig. 1 A master plan depiction (on the top) (Plan Al Ain… 2015) and a picture from GoogleMaps of the implemented project (on the bottom) of Al Ain Faydah Al Ain Abu Dhabi Fig. 1 A master plan depiction (on the top) (Plan Al Ain… 2015) and a picture from GoogleMaps of the implemented project (on the bottom) of Al Ain Faydah Al Ain Abu Dhabi depiction (on the top) (Plan Al Ain… 2015) and a picture from GoogleMaps of the implemented project (on the bottom) of A Dh b Fig. 1 A master plan depiction (on the top) (Plan Al Ain… 2015) and a picture from GoogleMaps of the implemented project (on the bottom) of Al Ain Faydah, Al Ain, Abu Dhabi Concept model of measuring the satisfaction level Concept model of measuring the satisfaction level Each factor has a particular designation. For example, the first factor includes the degree of satisfaction with the physical attributes of the house, such as the architecture of the building, the selected design, and interior content, i.e., the layout, number, and size of rooms, the presence of terraces, gardens, or green areas, etc. The second fac- tor (functionality of the house) is the degree of satisfac- tion with the performance of the house components like windows, doors, roof, floor, wall thickness, noise insulation, the location of water and air conditioning Figure 5 presents a schematic representation of the con- ceptual model for assessing the level of satisfaction in res- idents, which includes four different influencing factors and parameters. According to Ogu (2002), it implies the satisfaction with the design and architecture of the house (1), the functionality of the house (2), the level of acces- sibility (3), and the district location and community (4). These parameters include expectations, socio-economic dynamics, and demographic data. Figure 5 presents a schematic representation of the con- ceptual model for assessing the level of satisfaction in res- idents, which includes four different influencing factors and parameters. According to Ogu (2002), it implies the satisfaction with the design and architecture of the house (1), the functionality of the house (2), the level of acces- sibility (3), and the district location and community (4). These parameters include expectations, socio-economic dynamics, and demographic data. Ibrahim City Territ Archit (2020) 7:21 Page 5 of 13 Ibrahim City Territ Archit Fig. 2 Al Ain Faydah district of the UAE Public Housing Fund Fig. 2 Al Ain Faydah district of the UAE Public Housing Fund Fig. 3 A masterplan depiction (on the left) (Watani Housing Project 2015) and Google Maps picture of the implemented part of the project Al Watani, Khalifa City A, Abu Dhabi (on the rights) Fig. 3 A masterplan depiction (on the left) (Watani Housing Project 2015) and Google Maps picture of the implemented part of the project Al Watani, Khalifa City A, Abu Dhabi (on the rights) communications, ventilation and air filtration, etc. Thus, these two factors are attributed to the satisfaction of the basic physiological and emotional needs of residents. The remaining factors are related to socio-economic aspects. grounds or health centers, public places and mosques, etc. Concept model of measuring the satisfaction level This factor may also include traffic interchange and the availability of municipal transport. The last satisfac- tion factor refers to the affiliation to a particular location and community, as well as the level of technical service. Attributes here are public places, street conditions, light- ing levels, roads and sidewalks, population density and Also, the accessibility factor in a broader sense refers to the level of satisfaction with the availability of necessary services, namely, school, garden, clinic, stores, sports Ibrahim City Territ Archit (2020) 7:21 Page 6 of 13 Ibrahim City Territ Archit Fig. 4 The Al Watani district of the UAE public housing fund Fig. 4 The Al Watani district of the UAE public housing fund measured using these scales, descriptors were developed for the Likert scale to consider extreme, moderate, and neutral responses such as ‘very dissatisfied’, ‘dissatisfied’, ‘neutral’, ‘satisfied’, and ‘very satisfied’. Fig. 5 Schematic representation of the residency satisfaction measurement model i yi Questionnaires were developed and polled via social media networks in special groups created for residents in particular applications. The survey was also sent to email addresses of those respondents, who were not registered in any of the social media networks. The proposed ques- tionnaire is presented in Appendix A. In total, the survey was attended by 746 participants, of which 544 were resi- dents of Al Ain Faydah and 202 of Al Watani residential complexes. Since in these housing areas include a total of 17,000 residents, a population sample of 10% was suffi- cient to obtain an adequate estimate for the survey (Hig- ley 2008). The responses were collected over 2 months (2019 October–November). traffic congestion, etc. All these designations were used in the development of questionnaires to fully measure and comprehensively analyze the level of satisfaction with residency in public housing, and identify negative factors that can be addressed in future projects. Questionnaire development and data collection The survey results were entered into the database and processed using the SPSS Statistics software package. The Likert scale values for each factor were used to calculate the Z-point index following the formula: Based on the conceptual model of satisfaction meas- ures described above, a comprehensive questionnaire was developed to determine socio-economic and physi- cal characteristics, environmental parameters, and other key variables. A total of 26 variables were selected, and 26 questions were compiled, which included open ques- tions and those to be assessed on a five-point Likert scale or the Guttman scale. The open question assumes a free answer for the respondent to identify their attitude towards the problem at hand and to collect their opinions on the project, suggestions for improving the current conditions to increase the number of variables in future questionnaires, whereas also new parameters and prob- lems related to housing. Since the satisfaction level was (1) Znj = (Ynji − ∼yn)/Sn (1) where n is the value of the factor from 1 to 4 points, j is the ordinal number of the respondent (from 1 to 746), where n is the value of the factor from 1 to 4 points, j is the ordinal number of the respondent (from 1 to 746), Yi is the observed evaluation of each house for n factor, ỹn is the average distribution for n factor, and Sn is the standard deviation of n factor for all residents. When calculating the Z index, residents were grouped into sat- isfaction categories based on standard deviation (SD). The Z-values for each home represents the degree of Yi is the observed evaluation of each house for n factor, ỹn is the average distribution for n factor, and Sn is the standard deviation of n factor for all residents. When calculating the Z index, residents were grouped into sat- isfaction categories based on standard deviation (SD). The Z-values for each home represents the degree of Ibrahim City Territ Archit (2020) 7:21 Page 7 of 13 satisfaction relative to all residents participating in the survey (Table 1). variables corresponding to factors 1–4 (Fig. 5) is assumed to provide a more reliable assessment of overall satisfac- tion. Thus, the overall satisfaction index will be calcu- lated as an average of four factors. A comparative and qualitative analysis was performed for these two projects. Questionnaire development and data collection Also, the values of the Estidama Pearl rating system and the satisfaction level accepted for this project were com- pared with the data obtained for the described projects. The data presented in the table demonstrate that ten- ants with negative Z-values are classified as unsatisfied, while those with positive Z-values are considered satis- fied. The Z-values in the middle of the distribution is classified as moderately satisfactory and associated with some uncertainty. Thus, the value can turn negative if the housing conditions worsen, otherwise, the Z-value remains positive. In addition to (Z) indicator, a satisfac- tion index (IS) was introduced, which is expressed as a percentage of the maximum score given by the resident for each factor. Building ratings according to Estidama Pearl Rat- ing System are assigned as follows: 1 Pearl (20 manda- tory conditions), 2 Pearls (20 mandatory conditions + 60 credit points), 3 Pearls (20 mandatory conditions + 85 credit points), 4 Pearls (20 mandatory conditions + 115 credit points), and 5 Pearls (20 mandatory condi- tions + 140 credit points). In the Pearl Building Rat- ing System (PBRS) v1.0, eight credit categories with a maximum of 180 points (Abu Dhabi and urban planning council 2010) are available, which are depicted in Fig. 6. These factors, in turn, were subdivided into satisfac- tion categories. As with Z-index, the IS-index has five categories: very low (20–34%), low (35–49%), medium (50–64%), high (65–79%), and very high (80–100%). In calculations, each point on the Likert scale was multi- plied by 20 (Watani Housing Project 2015). As illustrated, the satisfaction index varies between 20 and 100%, where the average value corresponds to 60%. Thus, scores above 60% suggest an area of medium/high satisfaction level, and the level below 60% indicates an area of medium/low satisfaction. Percentage ranges of satisfaction will be used in the analysis. The second indicator of satisfaction effi- ciency was the variable index (Iv), which determines the extent to which each variable affects the level of satisfac- tion among residents and is calculated as follows (Watani Housing Project 2015): Mandatory loans must be fulfilled by each project applying for Pearl 1 rating. As follows from above, all state-funded projects should be rated with 2 Pearl, and, therefore, 60 additional points should be added to the mandatory requirements. These points can be achieved through the use of innovative technologies, circular water supply systems, renewable energy, waste minimiza- tion, etc. Questionnaire development and data collection Therefore, tracking the impact of the rating pro- gram used on the level of satisfaction in residents and the quality of their life is of high importance. Results and discussion (2) Iv = N t=1 yt N i=1 Yi · 100% (2) Survey processing results showed that 85% of the respondents were working people. Besides, statistical analysis of the demographic situation in the surveyed areas has good dynamics towards increasing the number of residents (Fig. 7). where yt is an estimate for variable t for n factor. Four classification types of satisfaction levels were chosen for Iv, which correspond to the following ranges: positive (70–100%), moderately positive (60–69%), moderately negative (50–59%), and negative (20–49%). As follows from Fig. 7, the average number of family members living in a villa is 5–6 people, including 2–3 children on average, which is quite suitable for the liv- ing area of the villa. According to the analysis results, the average age of children living in these complexes was 5–7 years, which indicates that the surveyed tenants are quite young. Analysis of demographic data for the Watani complex showed similar results. Thus, these results indi- cate an emerging need for public places and educational institutions for children within the complex. The overall level of satisfaction per capita is shown in Fig. 8. Given the fact that overall satisfaction is not considered a simple variable, the combination of satisfaction index Table 1 Satisfaction categories based on Z-value with SD Satisfaction level Range of Z-values Category of satisfaction level IS Range of satisfaction index IS Highly dissatis- fied Z < − 2 Very low 20–34% Dissatisfied − 2 ≤ Z ≤ − 1 Low 35–49% Moderately satis- fied − 1 < Z < 1 Average 50–64% Satisfied 1 ≤ Z ≤ 2 High 65–79% Highly satisfied Z ≥ 2 Very high 80–100% Table 1 Satisfaction categories based on Z-value with SD Table 1 Satisfaction categories based on Z-value with SD Hence, 41.8% of Al Ain Faydah complex residents con- sider their overall satisfaction to be high and medium. Nearly similar indicators were obtained in the survey of the Al Watani residential complex, where 40% of resi- dents estimate their level of satisfaction to be medium and high. Besides, indicators of the low and very high levels of satisfaction are almost the same, which indicates Ibrahim City Territ Archit (20 Page 8 of 13 the same percentage of the very satisfied and dissatis- fied population (10–12%). However, the lowest level of extreme dissatisfaction was the most surprising amount- ing to less than 5%. These results show positive feedback as satisfaction with living conditions in state complexes, moderate positive and negative levels in equal degrees. The maximum number of residents of Al Ain Faydah (61%) consider the functionality of the house as a mod- erately positive, i.e., the layout, choice of materials, water supply, and comfortable location of internal communi- Fig. 6 Maximum credit points and mandatory conditions for each category in Estidama PBRS V1.0 program (Abu Dhabi and urban planning council 2010) Fig. 7 Results of a demographic study of the Al-Ain Faydah residential area Fig. 8 The overall level of satisfaction in residents of Al Ain Faydah and Al Watani housing complexes measured based on the total number of surveyed residents Fig. 6 Maximum credit points and mandatory conditions for each category in Estidama PBRS V1.0 program (Abu Dhabi and urban planning council 2010) Fig. 6 Maximum credit points and mandatory conditions for each category in Estidama PBRS V1.0 program (Abu Dhabi and urban planning council 2010) Fig. 7 Results of a demographic study of the Al-Ain Faydah residential area Fig. 8 The overall level of satisfaction in residents of Al Ain Faydah and Al Watani housing complexes measured based on the total number of surveyed residents Fig. 7 Results of a demographic study of the Al-Ain Faydah residential area Fig. 8 The overall level of satisfaction in residents of Al Ain Faydah and Al Watani housing complexes measured based on the total number of surveyed residents Fig. 8 The overall level of satisfaction in residents of Al Ain Faydah and Al Watani housing complexes measured based on the total number of surveyed residents the same percentage of the very satisfied and dissatis- fied population (10–12%). Table 1 Satisfaction categories based on Z-value with SD However, the lowest level of extreme dissatisfaction was the most surprising amount- ing to less than 5%. These results show positive feedback as satisfaction with living conditions in state complexes, which indicates the right direction of state housing pro- grams development and the application of the Estidama rating system. The results of the variable index analysis for each factor are shown in Fig. 9.h moderate positive and negative levels in equal degrees. The maximum number of residents of Al Ain Faydah (61%) consider the functionality of the house as a mod- erately positive, i.e., the layout, choice of materials, water supply, and comfortable location of internal communi- cations. Also, 38–47% of residents refer to other factors that influence satisfaction. Thus, almost half of the inter- rogated residents of the Al Ain Faydah are moderately satisfied with their living conditions (60–69% satisfac- tion index), which is in good agreement with indicators of overall satisfaction level (Fig. 8). The analysis results show that the satisfaction levels relative to the variables for each factor correspond to Ibrahim City Territ Archit (2020) 7:21 Page 9 of 13 Ibrahim City Territ Archit Fig. 9 Satisfaction variables for factors 1–4 per capita in public housing complexes of Al Ain Faydah and Al Watani Slightly less of the interrogated residents (26–35%) have a moderately negative attitude to living in this complex, which corresponds to the average satisfac- tion level presented in Fig. 7. Besides, the same per- centage of residents (11–12%) have an extremely negative attitude towards the location and com- munity. Based on the data obtained for the Al Ain Faydah project, the satisfaction level is more influ- enced by the factors of functionality and design of the house, i.e., meeting the physiological needs and environmental parameters, as compared to socio-eco- nomic parameters, which correspond to higher levels of unsatisfaction. This may be due to the location of villas relative to each other and the territorial loca- tion of public and entertainment places. Also, some residents noted their dissatisfaction with the noise and the resulting garbage near their homes due to the renovation works of nearby villas, which is a signifi- cant issue in new areas.h The analysis results of the Al Watani residents’ poll showed that they estimate this complex as moder- ately negative and moderately positive depending on the factor. Table 1 Satisfaction categories based on Z-value with SD The percentage of points on the proposed categories is shown in Fig. 10.hi As for the analysis of how each factor and variable affects the level of satisfaction individually, most of the interviewed population were more satisfied (> 70%) with the design and layout and level of security, as well as the presence of spacious rooms that give a sense of comfort and privacy. A larger number of respondents gave high scores on questions about feeling of comfort and time in their home, indicating a high degree of satisfaction with physiological and psychological needs. These factors are mandatory requirements in the Estidama program, which fully meet the expectations of residents. In terms of architecture and functionality of houses, the study showed that 49–60% of residents are highly satisfied with the overall condition of their house. The majority of respondents (80%) answered negatively to the question about the modification of the house, which indicates a multifaceted approach to the physiological and aesthetic characteristics of living. Besides, residents gave more positive answers to the rational design of the roof and windows, which led to the rare use of tight curtains and the ability to air the room naturally in good weather. The figure depicts how categories of residential build- ings, resource energy, materials management including application of environmentally friendly substances in construction and internal works, and the use of energy- efficient technologies with lower emissions of harmful substances influence the rating level. Besides, another important aspect is the use of local, secondary, and bio- degradable materials in construction and decoration, as well as waste management and its sorting. Valuable water management greatly contributes to the rating points as well. According to the master plan, these complexes are equipped with systems to reduce water consumption and sensors on internal and external facilities to control water flow and leakages. Thus, roughly assessing hous- ing against the rating system, it can be characterized as a fairly accurate complex for sustainable housing develop- ment. However, this assessment does not guarantee that all conditions at all stages of construction will be met, as it depends on many factors, including the choice of con- tractors and technological solutions. Nevertheless, for the complexes under study where people live, this system has fully met its expectations, which affects the level of satisfaction of residents with public housing. Table 1 Satisfaction categories based on Z-value with SD The maximum number of residents (41.4%) expressed moderately negative satisfaction (50–59%) in terms of the district location and community. Such high values of dissatisfaction may be due to the incom- pletion of the project, where most of the territory is still under construction. Besides, many priority service delivery points specified in the project have not been completed yet. However, 37–39% of residents are quite Page 10 of 13 Ibrahim City Territ Archit (2020) 7:21 Page 10 of 13 satisfied with the design, functionality, accessibility, and location of the houses. salt isolation in some buildings in the Al Watani and a road junction in the settlement were mentioned as nega- tive aspects. Concerning the Al Ain Faydah, respondents noted the distance from the city’s business center, which takes time to travel to work, and the lack of entertain- ment and recreational areas. Residents also pointed out that they were very satisfied with their neighbors in terms of their relations, sympathy, and confidentiality. It may also be noted that 18% of the population is 70–100% satisfied with the functionality of the houses, which plays a key role as in the case of Al Ain Faydah complex. Among negative indicators, 17–18% of resi- dents are not satisfied with the design of houses and the availability of public spaces. As mentioned earlier, this complex has been maintained in a traditional style and has a smaller open space area, and the curvature of the streets may contribute to some sense of discomfort and constraint individual space. The assessment results obtained for the Al Watani complex show that the devel- oped housing project is moderately satisfactory for living from the point of view of residents, which is a positive characteristic for further construction. i The results of a survey of residents’ satisfaction showed that government housing projects can be considered an acceptable place for a comfortable life with a high and average level of satisfaction, which fully meets the needs in terms of physiological, social, economic, and envi- ronmental characteristics. Assessment scales from Abu Dhabi and urban planning council (2010) were used to calculate the approximate Estidama rating indicators for these housing complexes. The results of a rough estima- tion on the proposed scales in the Estidama PBRS V1.0 program showed that both complexes are gaining enough additional credits (above 60) to obtain the Pearl 2 rating. Table 1 Satisfaction categories based on Z-value with SD The factor of accessibility corresponded to a moderate level of satisfaction according to the opinion of 40–42% of residents. This level is associated with complicated access to medical facilities and safety in public places. Most respondents gave low scores to these questions. How- ever, other variables that relate to green areas, children’s playgrounds, and sports grounds were estimated with high scores. Given the presence of 2–3 children in each home, this need for separation and the physical health of children was met to a high degree. In terms of access to schools, the respondents’ opinions differed greatly, which disabled the determination of the satisfaction tendency. Findings obtained in the course of research agree with the results of other works. Thus, the paper (Howley et al. 2009; Buys and Miller 2012) modeled and quantitatively and qualitatively evaluated the relationship between residence density and satisfaction with the neighbour- hood in central city districts. The study shows that a high density of residents has a negative impact on the level of Most respondents indicated that they were generally satisfied with the quality of life in these complexes. Resi- dents appreciated the location and layout of their villas, as well as the large area of parks and green spaces. Poor Page 11 of 13 Ibrahim City Territ Archit (2020) 7:21 Ibrahim City Territ Archit Fig. 10 The percentage ratio of credit points according to Estidama PBRS V1.0 for the studied state housing complexes ig. 10 The percentage ratio of credit points according to Estidama PBRS V1.0 for the studied state housing complexes layout, and interior decoration. The lowest satisfac- tion rate of 11–12% was established for the factor of accessibility and the location of the building in the complex. satisfaction and is also related to the quality of the envi- ronment, noise level, traffic density, as well as the lack of public participation and necessary services and facilities. All these negative factors were considered when design- ing facilities studied in this paper. 2. Table 1 Satisfaction categories based on Z-value with SD The state housing projects in the UAE can be con- sidered as acceptable for the comfortable life of resi- dents with a high and average level of satisfaction, which fully meets the needs in terms of physiological, social, economic, and environmental characteristics.h Examination of the impact caused by socio-environ- mental factors on community satisfaction in different districts of Delhi (Karuppannan and Sivam 2011) showed that it becomes higher when houses are placed around a public place or common open space. The provision of well-located open spaces and good access to public infra- structure play a crucial role in increasing urban sustain- ability, which is taken into account in the Estidama rating system. Similar results were obtained in the works (Win- ston 2017; Al Shawabke et al. 2020). 3. The application of the Estidama rating system for public housing construction fully meets the require- ments and the expectations of residents although it requires control over each stage of work. The results obtained are of great scientific significance and can be used in future research efforts. Authors’ contributions The author read and approved the final manuscript. This study assesses the level of satisfaction with public housing offered by the UAE government to citizens, by the physical characteristics and traditional social aspects of the housing unit, urban design, and social environment in the residential area, whereas also their contribution to overall satisfaction, on the example of two completed government projects in Abu Dhabi, namely, Al Faydah and Al Watani. Availability of data and materials Data will be available on request. Availability of data and materials Data will be available on request. Data will be available on request. Competing interests The authors declare that they have no competing interests. Conclusionsh Authors’ contributions The author read and approved the final manuscript. Funding g This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. g This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Competing interests h h d l h Number of kids: Kids ages: Resident unit type Strongly disagree Disagree Neutral Agree Strongly agree I feel comfortable in my Unit 1 2 3 4 5 I feel safe in my Unit 1 2 3 4 5 I enjoy spending time in my Unit 1 2 3 4 5 My Unit has adequate light 1 2 3 4 5 My Unit has the required privacy 1 2 3 4 5 My family members feel satisfied with the new Unit 1 2 3 4 5 Do you have separate room to finish your work or spend individual time YES NO Do you have enough space for large numbers of guests? YES NO Do you need extra space for family members to spent some nights at your home? YES NO Is there continues maintenance arranged to your unit? YES NO Do you think your Unit satisfy your needs? YES NO Comments: ______________________________________ __________________________________________________ __________________________________________________ __________________________________________________ __________________________________________________ __________________________________________________ __________________________________________________ _. Any modifications have been done by the resident to the unit? If yes, what kind? YES NO Do you keep windows opened in good weather? YES NO Do you use blinded curtains or normal ones? Blinded Normal Do you hear noise from your neigh- bors? YES NO How do you collect your Unit trash? Big trash units Manual collection Are there segregation trash units nearby? YES NO Public spaces (Park, School, Hospital, Sports, Services, ………). Strongly disagree Disagree Neutral Agree Strongly agree I enjoy the public areas of the com- pound 1 2 3 4 5 I enjoy various activities in the Public areas 1 2 3 4 5 I am comfortable eating outdoor in the Public areas 1 2 3 4 5 Sports services are available in the public areas 1 2 3 4 5 I can leave my kids safe in the public garden 1 2 3 4 5 I feel safe dropping my kids to school 1 2 3 4 5 It’s easy to go near hospital 1 2 3 4 5 Comments:_____________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ __________________________________________________ __________________________________________________ __________. My favourite thing about [Unit Design] is: ________________________________________________ _________________________________________________ _________________________________________________ __________________________________________________ __________________________________________________ __________________________________________________ __________________________________________________ __. If I could change one thing about [Unit Design] it would be: ________________________________________________ _________________________________________________ _________________________________________________ __________________________________________________ __________________________________________________ __________________________________________________ __________________________________________________ __. Competing interests h h d l h According to the research results, the following conclu- sions can be drawn: Competing interests The authors declare that they have no competing interests. 1. The overall level of satisfaction of residents in these complexes was estimated as high and average. A study of various influencing factors shows that the satisfaction level of 40–60% of residents is most affected by the good performance of the housing, its Appendix A Resident satisfaction survey Resident job: Resident job timing: Family members number: Ibrahim City Territ Archit (2020) 7:21 Page 12 of 13 Number of kids: Kids ages: Resident unit type Strongly disagree Disagree Neutral Agree Strongly agree I feel comfortable in my Unit 1 2 3 4 5 I feel safe in my Unit 1 2 3 4 5 I enjoy spending time in my Unit 1 2 3 4 5 My Unit has adequate light 1 2 3 4 5 My Unit has the required privacy 1 2 3 4 5 My family members feel satisfied with the new Unit 1 2 3 4 5 Do you have separate room to finish your work or spend individual time YES NO Do you have enough space for large numbers of guests? YES NO Do you need extra space for family members to spent some nights at your home? YES NO Is there continues maintenance arranged to your unit? YES NO Do you think your Unit satisfy your needs? YES NO Comments: ______________________________________ __________________________________________________ __________________________________________________ __________________________________________________ __________________________________________________ __________________________________________________ __________________________________________________ _. Any modifications have been done by the resident to the unit? If yes, what kind? YES NO Do you keep windows opened in good weather? YES NO Do you use blinded curtains or normal ones? Blinded Normal Do you hear noise from your neigh- bors? YES NO How do you collect your Unit trash? Big trash units Manual collection Are there segregation trash units nearby? YES NO Public spaces (Park, School, Hospital, Sports, Services, ………). Anal Emerald, Bingley (in press) Mohit MA, Nazyddah N (2011) Social housing programme of Selangor Zakat Board of Malaysia and housing satisfaction. 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Wiley-Blackwell, New York, pp 1–3 Plan Al Ain 2030 Urban Structure Framework Plan (2015) http://faculty.uaeu. ac.ae/abintouq/GEO_Fall_2015/PlanAlAin2030.pdf. Accessed 12 Dec 2018. Rahman NA, Ab Manan NA, Saad NL, Abdullah K, Soffian NSM, Ahmad AL (2018) Public facilities guidelines calculator for sustainable housing devel- opment. J Inov Malays 1(2):125–144 Awadh O (2017) Sustainability and green building rating systems: LEED, BREEAM, GSAS and Estidama critical analysis. J Build Eng 11:25–29 Buys L, Miller E (2012) Residential satisfaction in inner urban higher-density Brisbane, Australia: role of dwelling design, neighbourhood and neigh- bours. J Environ Plan Manag 55(3):319–338 Riazi M, Emami A (2018) Residential satisfaction in affordable housing: a mixed method study. Cities 82:1–9 Chan AP, Adabre MA (2019) Bridging the gap between sustainable housing and affordable housing: The required critical success criteria (CSC). Build Environ 151:112–125 Roufechaei KM, Bakar AHA, Tabassi AA (2014) Energy-efficient design for sustainable housing development. J Clean Prod 65:380–388 Saiedlue S, Hosseini SB, Yazdanfar SA, Maleki SN (2015) Enhancing quality of life and improving living standards through the expansion of open space in residential complex. Procedia-Soc Behav Sci 201:308–316 Dempsey N, Bramley G, Power S, Brown C (2011) The social dimension of sustainable development: defining urban social sustainability. Sustain Dev 19(5):289–300 Sakip SRM, Johari N, Salleh MNM (2012) Sense of community in gated and non-gated residential neighborhoods. Procedia-Soc Behav Sci 50:818–826 Dizdaroglu D, Yigitcanlar T (2016) Integrating urban ecosystem sustainability assessment into policy-making: Insights from the Gold Coast City. Anal Emerald, Bingley (in press) J Environ Plan Manag 59(11):1982–2006 Shach-Pinsly D (2019) Measuring security in the built environment: evaluating urban vulnerability in a human-scale urban form. Landsc Urban Plan 191:103412 Dohr JH, Portillo M (2011) Design thinking for interiors: Inquiry, experience, impact. Wiley, New Yorki Shawket IM (2018) Identity in urban spaces of residential compounds: contrib- uting to a better environment. HBRC J 14(2):235–241 Higley RC (2008) “Other” processes of rural gentrification and counter-urban migration. University of Brighton, Brighton Teck-Hong T (2012) Housing satisfaction in medium-and high-cost housing: the case of Greater Kuala Lumpur Malaysia. Habitat Int 36(1):108–116 Howley P, Scott M, Redmond D (2009) Sustainability versus liveability: an investigation of neighbourhood satisfaction. J Environ Plan Manag 52(6):847–864 Van Kamp I, Leidelmeijer K, Marsman G, De Hollander A (2003) Urban environ- mental quality and human well-being: towards a conceptual framework and demarcation of concepts; a literature study. Landsc Urban Plan 65(1–2):5–18 Hussein WHA (2019) Development and sustainability of residential communi- ties (neighbourhoods) in Egypt, recommended criteria and indicators drawn from international trials. Urban Reg Plan 4(3):100–108 Wagner SL, Shubair MM, Michalos AC (2010) Surveying older adults’ opinions on housing: recommendations for policy. Soc Indic Res 99(3):405–412 Ingrao C, Messineo A, Beltramo R, Yigitcanlar T, Ioppolo G (2018) How can life cycle thinking support sustainability of buildings? Investigating life cycle assessment applications for energy efficiency and environmental performance. J Clean Prod 201:556–569 Walton D, Murray SJ, Thomas JA (2008) Relationships between population density and the perceived quality of neighbourhood. Soc Indic Res 89(3):405–420 Ioppolo G, Cucurachi S, Salomone R, Shi L, Yigitcanlar T (2019) Integrating stra- tegic environmental assessment and material flow accounting: a novel approach for moving towards sustainable urban futures. Int J Life Cycle Assess 24(7):1269–1284 Watani Housing Project (2015) https://www.emaratalyoum.com/local-section/ other/2015-05-27-1.788076. Accessed 30 June 2018 Winston N (2017) Multifamily housing and resident life satisfaction in Europe: an exploratory analysis. Hous Stud 32(7):887–911 Karji A, Woldesenbet A, Khanzadi M, Tafazzoli M (2019) Assessment of social sustainability indicators in mass housing construction: a case study of Mehr housing project. Sustain Cities Soc 50:101697 Wongbumru T, Dewancker B (2016) Post-occupancy evaluation of user satisfaction: a case study of “old” and “new” public housing schemes in Bangkok. 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Local Environ 16(9):849–870 Competing interests h h d l h Received: 28 September 2020 Accepted: 29 November 2020 Strongly disagree Disagree Neutral Agree Strongly agree I enjoy the public areas of the com- pound 1 2 3 4 5 I enjoy various activities in the Public areas 1 2 3 4 5 I am comfortable eating outdoor in the Public areas 1 2 3 4 5 Sports services are available in the public areas 1 2 3 4 5 I can leave my kids safe in the public garden 1 2 3 4 5 I feel safe dropping my kids to school 1 2 3 4 5 It’s easy to go near hospital 1 2 3 4 5 Comments:_____________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ __________________________________________________ __________________________________________________ __________. My favourite thing about [Unit Design] is: ________________________________________________ _________________________________________________ _________________________________________________ __________________________________________________ __________________________________________________ __________________________________________________ __________________________________________________ __. If I could change one thing about [Unit Design] it would be: ________________________________________________ _________________________________________________ _________________________________________________ __________________________________________________ __________________________________________________ __________________________________________________ __________________________________________________ __. Received: 28 September 2020 Accepted: 29 November 2020 Strongly disagree Disagree Neutral Agree Strongly agree Ibrahim City Territ Archit (2020) 7:21 Page 13 of 13 Page 13 of 13 Ibrahim City Territ Archit References Lee E, Park NK (2010) Housing satisfaction and quality of life among temporary residents in the United States. Hous Soc 37(1):43–67 Abu Dhabi urban planning council (2010) Pearl building rating system: design & construction, Version 1.0, April 2010. https://www.solarthermalwor ld.org/sites/default/files/news/file/2015-05-04/estidama_constructi on_rating_certificate.pdf. Accessed 30 June 2018. Lichtenstein S, Badu E, Owusu-Manu DG, Edwards DJ, Holt GD (2013) Corpo- rate social responsibility architecture and project alignments. J Eng Des Technol 11(3):334–353 Liu AM (2003) The quest for quality in public housing projects: a behaviour-to- outcome paradigm. Constr Manag Econ 21(2):147–158 Ahmed KG (2017) Designing sustainable urban social housing in the United Arab Emirates. 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https://openalex.org/W2752472621	https://europepmc.org/articles/pmc5588612?pdf=render	English	null	Effect of enhanced feedback to hospitals that are part of an emerging clinical information network on uptake of revised childhood pneumonia treatment policy: study protocol for a cluster randomized trial	Trials	2,017	cc-by	6,440	Ayieko et al. Trials (2017) 18:416 DOI 10.1186/s13063-017-2152-8 Ayieko et al. Trials (2017) 18:416 DOI 10.1186/s13063-017-2152-8 Open Access © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Effect of enhanced feedback to hospitals that are part of an emerging clinical information network on uptake of revised childhood pneumonia treatment policy: study protocol for a cluster randomized trial Philip Ayieko1, Grace Irimu1,2 and Mike English1,3 Philip Ayieko1, Grace Irimu1,2 and Mike English1,3 * Correspondence: PAyieko@kemri-wellcome.org 1Kenya Medical Research Institute (KEMRI) – Wellcome Trust Research Programme, Nairobi, Kenya Full list of author information is available at the end of the article Primary objective In collaboration with the Kenyan Ministry of Health and other partners, we have developed an improved sys- tem for collecting pediatric inpatient hospital data within a network of Kenyan county hospitals [7]. This clinical information network (CIN) established between 2013 and 2014 has implemented a regular and standardized audit and feedback process comprising performance re- ports on adherence to guidelines for the care of com- mon conditions causing admission to the hospitals involved. This is linked to additional network activities that together constitute an intervention package aiming to promote adherence to evidence-based practices for all participating hospitals [8, 9]. Working with this network creates opportunities to examine the effects of varying feedback approaches while tracking the adoption of pol- icy [10]. 1. To determine whether a specifically enhanced feedback intervention will improve correctness of classification and treatment of childhood pneumonia compared to standard feedback, after nationwide treatment policy change Aims The overall aim of the trial described in this protocol is to assess the effect of enhancements to feedback on clin- ician adoption of new pneumonia treatment guidance within county hospitals that are already receiving regular standardized audit reports spanning several clinical con- ditions. During the study we will examine one primary objective and a secondary objective: Secondary objective 2. To explore rates of treatment switching after children are started on orally administered amoxicillin for pneumonia as a proxy measure for clinicians’ assessment that treatment failure has occurred on the orally administered treatment Methods In early 2016, the Ministry of Health in Kenya changed its clinical policy for the treatment of childhood pneu- monia. The guideline policy changes reflect new evi- dence and was agreed by a national clinical guideline panel and involve a reclassification of clinical pneumonia syndromes from the current three grades of severity (very severe pneumonia, severe pneumonia, and pneu- monia) to two grades (severe pneumonia equivalent to the previously named very severe grade, and pneumonia, a classification that encompasses the two remaining se- verity grades) with corresponding changes in recom- mended antibiotic treatment [11–13]. The new treatment policy recommends orally administered amoxicillin for children who were previously treated with injectable penicillin for pneumonia and who have lower chest wall indrawing but no other serious signs. Using the data collection and semi-automated performance The reporting of this protocol for the CIN enhanced feedback intervention is conducted according to the Standardized Protocol Items: Recommendations for Intervention Trials (SPIRIT) – Fig. 1 and Additional file 1: Table S1 (SPIRIT 2013 Checklist) [14]. Abstract Trials (2017) 18:416 Page 2 of 9 Page 2 of 9 Background measurement platform provided by CIN, we set out to test specific enhancements of the feedback process including goal setting, benchmarking, and development of clear ac- tion plans to examine their effects on the adoption of this change in national clinical guidelines for the treatment of childhood pneumonia [4]. This paper describes a prag- matic, parallel-group, cluster randomized trial to compare the effectiveness of an approach to enhance feedback that might be used in promoting more rapid translation of new treatment policies in routine practice. The slow translation of health research to practice is a major challenge in the provision of quality hospital care in low-income countries [1, 2]. Indeed, experience in Kenya has shown that such health policy changes may take several years to implement [1, 3]. There are several alternative strategies for achieving rapid translation of policy and advancing quality care. Audit and feedback have been well defined and are among the strategies known to impact the adoption of guidance although average effects are modest [4, 5]. The effectiveness of audit and feedback may be influenced by a number of factors including: (i) feedback components, e.g., lan- guage, format, and source, (ii) audit components, e.g., data validity, and currency, (iii) nature of behavior change required, and (iv) clearly defined targets, goals and action plans [6]. How best to modify audit and feed- back strategies to optimize effectiveness is a priority for research and is of particular relevance as the potential availability of data on multiple aspects of practice within health systems increases. Abstract Background: The national pneumonia treatment guidelines in Kenya changed in February 2016 but such guideline changes are often characterized by prolonged delays in affecting practice. We designed an enhanced feedback intervention, delivered within an ongoing clinical network that provides a general form of feedback, aimed at improving and sustaining uptake of the revised pneumonia treatment policy. The objective was to determine whether an enhanced feedback intervention will improve correctness of classification and treatment of childhood pneumonia, compared to an existing approach to feedback, after nationwide treatment policy change and within an existing hospital network. Methods/design: A pragmatic, cluster randomized trial conducted within a clinical network of 12 Kenyan county referral hospitals providing inpatient pediatric care to children (aged 2–59 months) with acute medical conditions between March and November 2016. The intervention comprised enhanced feedback (monthly written feedback incorporating goal setting, and action planning delivered by a senior clinical coordinator for selected pneumonia indicators) and this was compared to standard feedback (2-monthly written feedback on multiple quality of pediatric care indicators) both delivered within a clinical network promoting clinical leadership linked to mentorship and peer-to-peer support, and improved use of health information on service delivery. The 12 hospitals were randomized to receive either enhanced feedback (n = 6) or standard feedback (n = 6) delivered over a 9-month period following nationwide pneumonia treatment policy change. The primary outcome is the proportion of all admitted patients with pneumonia (fulfilling criteria for treatment with orally administered amoxicillin) who are correctly classified and treated in the first 24 h. The secondary outcome will be measured over the course of the admission as any change in treatment for pneumonia after the first 24 h. Discussion: This trial protocol employs a pragmatic trial design during a period of nationwide change in treatment guidelines to address two high-priority areas within implementation research: promoting adoption of health policies and optimizing effectiveness of feedback. Trial registration: ClinicalTrials.gov, ID: NCT02817971. Registered retrospectively on 27 June 2016 Keywords: Audit, Feedback, Pragmatic trial, Cluster trial, Quality of care, Pneumonia, Guidelines * Correspondence: PAyieko@kemri-wellcome.org 1Kenya Medical Research Institute (KEMRI) – Wellcome Trust Research Programme, Nairobi, Kenya Full list of author information is available at the end of the article * Correspondence: PAyieko@kemri-wellcome.org 1Kenya Medical Research Institute (KEMRI) – Wellcome Trust Research Programme, Nairobi, Kenya Full list of author information is available at the end of the article Ayieko et al. Study design We designed a parallel-group, pragmatic, cluster ran- domized trial to be conducted in 12 hospitals over a 9- month period with interim analysis at 6 months in case of a more marked intervention effect than we are expecting. At the time of the trial design all the 12 hos- pitals were participating in a clinical network in which standardized audit and feedback had been delivered to all sites for more than 12 months; therefore, providing context for testing an enhanced feedback intervention. The intervention was designed to examine whether Ayieko et al. Trials (2017) 18:416 Page 3 of 9 STUDY PERIOD Enrolment Allocation Post-allocation Close-out TIMEPOINT (months) -1 0 1 2 3 4 5 6 7 8 9 10 ENROLMENT: Eligibility screen X Informed consent NA Hospital permission sought X Allocation X INTERVENTIONS: Enhanced feedback* Standard feedback* ASSESSMENTS: Baseline characteristics: Hospital characteristics (size, workload, disease prevalence e.g. HIV, malaria) and patient case mix (age, sex, comorbid illnesses) X X Primary outcome: Correct classification and treatment of pneumonia based on new pneumonia policy (%) X X X X X X X X X Secondary outcome: Treatment change from oral amoxicillin to intravenous antibiotics (%) X X X X X X X X X *Feedback details presented in Figure 1 Fig. 1 Clinical information network (CIN)-enhanced feedback intervention schedule of enrollment, interventions, and assessments nical information network (CIN)-enhanced feedback intervention schedule of enrollment, interventions, and assessments internship training at the time and serving as first- referral centers. Hospital selection and characteristics are described in more detail elsewhere [16]. The exclu- sion criteria for hospitals from the eligible population in- cluded small hospitals (fewer than 1000 annual pediatric admissions), and existing formal linkages with research or academic institutions working with hospitals to con- duct research. The patient population eligible for inclu- sion within each hospital was pediatric patients admitted with a pneumonia diagnosis in a child with no indication for broad-spectrum antibiotic therapy. Common indica- tions for broad-spectrum antibiotics that will define the exclusion criteria include severe acute malnutrition, and meningitis. Severe malaria and tuberculosis cases will also be excluded. All hospitals recruited in the trial assented to participate and were informed that they could discontinue participation in the trial without prejudicing their role within the CIN. specific characteristics of feedback (frequency, goal set- ting, and action planning) can result in greater impact (Fig. 2). Participants f County referral hospitals form clusters representing di- verse geographic regions with varying climatic condi- tions, morbidity patterns, and facility workload. Out of the 14 hospitals currently engaged in network activities, two hospitals will not participate in the trial because one site located within the capital, Nairobi, serves as a satel- lite of the main university teaching hospital while the second hospital is a small rural training facility co- located with a larger facility participating in the network. The rural training facility has a different type of patient population and no resident physician. The CIN was set up between February and October 2013 through recruit- ment of hospitals, based on preliminary consent, from a shortlist of 40 public county referral hospitals providing Study design The intervention targets improvements in hos- pital uptake of new pneumonia treatment policy; there- fore, hospitals (and entire pediatric care teams) rather than individual clinicians will be randomized to inter- vention [15]. Clinical information network intervention and feedback strategies The CIN comprises a multifaceted approach including audit and feedback for all hospitals to which specifically Ayieko et al. Trials (2017) 18:416 Page 4 of 9 Fig. 2 Graphical depiction of interventions in clinical information network (CIN)-enhanced feedback trial (adapted from Perera et al. [25]) enhanced feedback on the uptake of new pneumonia treatment policy is added for intervention sites. of network activities, disseminating feedback reports, moderating peer-to-peer discussions within network for- ums (including online communication), providing en- couragement, and offering advice as required. CIN activities (both study arms) At the stage of intervention design, two major activities targeting improvement in data collection and clinical management of patients were being implemented (Fig. 2). In summary, each hospital in the network receives, and will continue to receive, five intervention components prior to and during the planned enhanced feedback trial: (1) minimal ICT infrastructure and human resource support, (2) standardized written feedback reports on hospital performance including on adherence to pneu- monia guidelines provided every 2 months, (3) national pediatric clinical protocols and job aides based on the protocol, (4) formal network meetings held at least twice a year, and (5) peer-to-peer support through telephone and email contact and networking in social media. A fuller description of these activities and the rationale for employing them is provided elsewhere [9]. Enhanced feedback reporting (intervention arm) Dur- ing the entire 9-month trial period we will continue to deliver the network-wide interventions described above including five rounds of general, 2-monthly feedback re- ports on hospital performance including a baseline feed- back report, Fig. 2. Our model of enhancing feedback of hospital performance in the intervention group draws from three notions around effective feedback: clear goal setting (feed up), progress towards goal attainment (feedback), and advising on activities aimed at making better progress (feed forward) [17]. As such, in the inter- vention arm we will identify specific goals (indicators) aligned with the primary objective, set targets for each focus indicator and benchmark performance for the in- dicators against a shared target. A specific, additional feedback report on pneumonia policy adherence based on these features will be provided monthly in written format and distributed by email to intervention-hospital pediatricians by the network coordinator together with a follow-up phone call. Randomization h l ll The trial will be conducted in 12 geographically dis- persed hospitals participating in the CIN. Restricted randomization was used to allocate the participating hospitals into the intervention and control arms to en- sure that balance is achieved between the two treatment arms with respect to number of patients admitted as in- patients with pneumonia, and geographical location (also a proxy for malaria prevalence or disease case-mix) [15]. The hospitals were grouped into four categories using information on geographic location (central or western region) and monthly average number of patients admit- ted with pneumonia (<30 or ≥30 per month) collected within the clinical network during the year preceding the intervention. Five hospitals were located in the west- ern region, while seven hospitals had at least 30 patients admitted with pneumonia per month. A statistician used R statistical software to generate the 924 possible alloca- tions for assigning 12 clusters to two treatments with six clusters allocated to each treatment arm. We defined a balancing criteria based on the two selected covariates and determined that approximate balance would be achieved by including allocations which had in each treatment arm either two or three hospitals located in the western region and three or four hospitals admitting < 30 pneumonia cases per month. The R computer pro- gram was used to assess each allocation against the bal- ancing criteria for geographic location (2:3 or 3:2 split between the arms) and monthly number of patients ad- mitted with pneumonia (3:4 or 4:3). There were a total of 536 allocations that met the criteria and represented relative balance between the intervention and control arms with respect to desired allocation ratio. Computer- generated random numbers were used to select one of these allocations at random. Hospitals in the randomly selected allocation were assigned to treatment and those not included in the allocation were assigned to the con- trol group. Allocation was based on clusters rather than individual patients and allocation concealment was con- ducted at the cluster level. The trial statistician con- cealed the sequence from investigators until training on the new policy was conducted in all hospitals and the intervention was assigned. Network coordination Since its inception, a senior pediatrician has taken over- all responsibility for activities in the network including organizing formal network meetings, providing updates Ayieko et al. Trials (2017) 18:416 Page 5 of 9 Page 5 of 9 The primary target of this intervention is health workers and it will not directly impact on concomitant care and no specific health care intervention will be pro- hibited as a result of trial participation. At facility level all hospitals will continue receiving the intervention un- less assent for participation is withdrawn by the hospital. determine whether the clinician classification was cor- rect. The children with a correct pneumonia classifica- tion were then evaluated to determine whether they received orally administered amoxicillin, the recom- mended treatment for pneumonia, yielding a composite indicator of correct classification and treatment. Randomization h l ll Blinding of investigators and health care providers after intervention assignment was not feasible in the trial because the delivery of enhanced feedback on pneumonia care provided was done by in- vestigators and the target of this feedback was the health The strategies that were implemented to improve adher- ence to intervention protocol include ensuring timeli- ness of planned audit and feedback cycles, and appropriate training on new policy. A time schedule for delivering written feedback – both standard and en- hanced – was prepared during intervention design and automated analysis scripts for producing feedback re- ports were developed to ensure timeliness in feedback delivery. The Data Management Team will hold weekly meetings during the entire 9-month intervention period to ensure quality of audit data. standard training on the new pneumonia policy will be designed and delivered across the network. A team of pediatricians (GI, SA, AA, and JO) acquainted with development of national pneu- monia guidelines who were also qualified in the generic instructor course (GIC) offered by the Resuscitation Council (UK) and Advanced Life Support Group (ALSG) will deliver the training. The CIN coordinator will main- tain a communication log documenting feed forward (advise on activities aimed at making better progress). Data management g At the end of each day the data clerk at each site syn- chronized data to a centralized server hosted at the KEMRI-Wellcome Research Program. Data quality was ensured through running error-checking scripts on a daily basis both in the sites and at the centralized trial server and conducting three rounds of external data quality assurance (DQA) during the 9-month trial period. The cleaning scripts raised error queries related to possible out-of-range values and implausible and in- consistent entries. Using the daily error logs generated by the scripts, a data manager flagged the potential er- rors with the clerk who verified the entries and cleaned any errors. Once all errors had been resolved all data were then archived in the centralized server with the pri- mary data generated at each hospital retained within the hospital. During the periodic external DQA, a team from the KEMRI-Wellcome Trust Research Program visited each site and conducted an independent re-entry of ap- proximately 10% randomly sampled patient data that had been entered by the clerk in the preceding 3 months. The independent entries were then compared to the cor- responding existing entries in the database with the aim of determining data quality and identifying database Data collection, management, and analysis Data collection methods The trial data were collected at the point of hospital dis- charge by data clerks using existing data collection tools developed for data collection within the CIN [7, 8]. The clinical tool has been in development and use for some time, and contains information on biodata, clinical fea- tures on admission and history of illness [18]. Further data collection procedures were designed for data collec- tion on laboratory investigations, initial antibiotic treat- ment and supportive care including oxygen therapy for pneumonia, and discharge outcomes after adaptation from previous studies [19]. Prior to the trial commence- ment the general CIN tools were adapted to capture out- comes to be used for assessing the new pneumonia treatment policy that were not previously contained in the treatment guidelines, e.g., (1) revised pneumonia se- verity classification and (2) formulation and dosage of dispersible amoxicillin tablets. At the end of each work- ing day (Monday to Friday) a clerk will enter data from inpatient paper records of pediatric discharges into an electronic tool designed using REDCap, a non- proprietary software for clinical data capture. Data for children discharged on weekends will be entered at the start of the new week. All data clerks working in the CIN were retrained on capturing pneumonia data fol- lowing changes in the pneumonia treatment guidelines. In addition, revised data collection manual and standard operating procedures were available in all sites. Sample size Based on the primary outcome of correct pneumonia classification and treatment we performed a power cal- culation to determine the feasibility of conducting the trial within the period of the network’s funding cycle using a fixed number of hospitals recruited into CIN. Using data collected during the first year of CIN oper- ation we determined that: on average, 616 (range 310– 898) children are admitted to each hospital annually with pneumonia and of these 150 children (range 78– 228) are eligible for treatment with amoxicillin. We esti- mated that if we evaluate the primary outcome 9 months after trial commencement with six hospitals per arm then at least 680 patients with pneumonia requiring amoxicillin treatment will be enrolled per arm. Assum- ing an average cluster size of 113 patients and an intracluster correlation coefficient (ICC) of 0.2 (corre- sponding to a variance inflation factor (VIF) of 15.8 and based on calculation of pneumonia classification and treatment within CIN hospitals), a Type-I error rate of 5%, and 90% power we will be able to detect a two-fold increase in the odds of correct pneumonia classification and treatment over 9 months if correct pneumonia clas- sification and treatment is 30% in the control group fol- lowing the treatment policy change. To ensure that the desired sample size is attained within each hospital, all eligible patients will be recruited in the hospitals with a workload less than 3000 annual pediatric admissions. For hospitals with a very high annual workload (at least 4000 admissions) a random sample will be selected using computer-generated random numbers applied to eligible participants. Selection of quality performance indicators Selection of quality performance indicators The new childhood pneumonia treatment policy advo- cates for assessment of respiratory rate, lower chest wall indrawing and five specific danger signs (cyanosis, oxy- gen saturation below 90%, inability to drink/breastfeed, altered level of consciousness, and grunting) in children presenting with cough or difficulty breathing followed by classification into one of two severity classifications: pneumonia (for children with raised respiratory rate and/or chest wall indrawing) or severe pneumonia (for those with any of the remaining danger signs). The guidelines further recommend treatment with orally ad- ministered amoxicillin for pneumonia and intravenously administered antibiotics for severe pneumonia [12]. A composite indicator for the classification and treatment of pneumonia was used for the primary outcome. The indicator was developed by reconstructing a pneumonia classification for each patient admitted with cough or difficulty breathing using information documented in medical records on whether each of the six specific clin- ical signs required for pneumonia severity classification were present or absent (one network effect has much better clinical documentation). This classification, based on a clinical guideline algorithm, was then compared to the classification assigned by the admitting clinician to Page 6 of 9 Ayieko et al. Trials (2017) 18:416 Ayieko et al. Trials (2017) 18:416 Ayieko et al. Trials (2017) 18:416 Page 6 of 9 care providers. However, for the interim analysis the statistician was blinded to treatment arm allocation. care providers. However, for the interim analysis the statistician was blinded to treatment arm allocation. care providers. However, for the interim analysis the statistician was blinded to treatment arm allocation. Data collection, management, and analysis Data collection methods Discussion The implementation gap that arises from failure to move research findings to practice has been described exten- sively but such descriptions are rarely followed by delib- erate efforts to narrow this gap in low-income settings. This study conducted during a nationwide changeover in childhood pneumonia treatment recommendations is an attempt to explore alternative ways of augmenting the already documented, but modest, effect of feedback on health worker performance in providing care to ad- dress this translation problem [4]. Randomized trials within collaborative networks have been conducted in developed countries but, to the best of our knowledge, the current randomized study, implemented in real time during a health policy change, and also based within a pediatric network is novel in low- and middle-income countries [20, 21]. At the global level the findings of this trial should provide timely contributions to the ongoing discourse on how best to optimize the effect of feedback on professional practice and health care outcome [6]. In separate sensitivity analysis, multiple imputation will be used to handle missing data and allow classifica- tion of all pneumonia patients. Bayesian bootstrap pre- dictive mean matching will be conducted 50 times to impute missing data for signs required to classify pneu- monia severity. After imputation, diagnostic checks will be conducted to compare the distribution of imputed and observed data. The modeling approaches used in the primary trial analysis will be repeated after imput- ation on each imputed dataset and the estimates com- bined using Rubin’s rules. The pragmatic approach to addressing public health questions is increasingly being used because of its ability to demonstrate efficacy of interventions in real-life set- tings. Specific strengths of the intervention delivered here are the theory-based process for the design of both the network platform and feedback intervention, repre- sentation of diverse contexts of the health system through the range of facilities in the trial, and the Outcomes The primary outcome is correct patient assessment and treatment-plan-defining care in the first 24 h. This end- point was evaluated as the proportion of all patients ad- mitted with pneumonia who were eligible for treatment with orally administered amoxicillin based on guideline recommendations (increased age-specific respiratory rate or lower chest wall indrawing in a child with history of cough or difficulty breathing) who are correctly classified and treated using orally administered amoxicillin at 24 h. The secondary outcome will be measured over the course of the admission as any change in treatment for pneumonia after the first 24 h. This change from orally administered amoxicillin to intravenously administered antibiotics for pneumonia during inpatient stay is con- sidered a proxy for treatment failure rate.. Ayieko et al. Trials (2017) 18:416 Page 7 of 9 Page 7 of 9 fields where agreement was low for discussion with the data entry clerks. the inclusion criteria were non-critical (any danger sign of illness or diagnosis with comorbid illness requiring intravenously administered antibiotics were excluded) and patients were treated for a short time period with amoxicillin, a well-characterized and relatively safe drug known for not having harmful effects in the pediatric age group. Furthermore, the study was on adoption of a national policy recommending amoxicillin use. The pri- mary aim of the interim analysis was to determine whether the minimum sample size required in the study was recruited by 6 months with the possibility of extend- ing the trial for a 3-month period if this was not achieved. This interim analysis was conducted by a stat- istician blinded to the trial arm allocation. All hospitals will continue to participate in network activities which will encourage retention of hospitals in the trial. Individual participant retention and follow-up were not major concerns in this trial because the data were collected on discharge through abstracting admis- sion events contained in medical records. Ethical considerations f d h l p The primary comparison of correct classification and treatment of pneumonia will include all patients meeting clinical criteria for pneumonia (but not severe pneumonia) recruited in the trial during the 9-month period. The pri- mary outcome will be a binary variable indicating whether a child admitted with non-severe pneumonia is correctly classified and treated based on new guideline recommen- dations. To account for the cumulative effect of feedback delivered at different time points during the 9-month intervention, the primary comparison will be adjusted for time between commencement of intervention and patient recruitment. A hierarchical logistic regression analysis will be conducted with the primary outcome (correct pneumo- nia classification and treatment) and independent vari- ables including treatment arm, time (as a continuous variable), and both patient level and hospital level and adjusting for any imbalances in patient characteristics. The model will account for clustering by including a ran- dom effect for hospital. The change in performance over time will be included as a time trend. Tests for significant differences in the time trend between the treatment arms will be examined by including an interaction term be- tween the treatment group and the time variable. Scientific and ethical clearance to conduct the trial was obtained from the Scientific and Ethics Review Unit of the Kenya Medical Research Institute. The approval is updated annually and provided for the establishment of the CIN and the conduct of this specific trial within it, and provides for the use of de-identified patient data, obtained through retrospective review of medical re- cords without individual informed consent, for the pur- poses of service evaluation and implementation research. Statistical methods Characteristics of patients and hospitals in the analysis will be compared according to treatment arm to evaluate the ability of randomization to deliver balance across hospital groups. Any variables that show imbalance be- tween intervention groups will be considered for adjust- ment in comparison of intervention effect. Received: 20 December 2016 Accepted: 16 August 2017 Received: 20 December 2016 Accepted: 16 August 2017 Received: 20 December 2016 Accepted: 16 August 2017 Additional file 1: Table S1 SPIRIT 2013 Checklist: recommended items to address in a clinical trial protocol and related documents. (DOC 254 kb) Funding d f 5. Hysong SJ. Meta-analysis: audit and feedback features impact effectiveness on care quality. Med Care. 2009;47:356–63. Funds from The Wellcome Trust (#097170), awarded to ME as a senior fellowship, together with additional funds from a Wellcome Trust core grant, awarded to the KEMRI-Wellcome Trust Research Program (#092654), sup- ported this work. The funders had no role in drafting or submitting this manuscript. 6. Ivers NM, Sales A, Colquhoun H, et al. No more “business as usual” with audit and feedback interventions: towards an agenda for a reinvigorated intervention. Implement Sci. 2014;9:14. 7. Tuti T, Bitok M, Paton C, et al. Innovating to enhance clinical data management using non-commercial and open source solutions across a multi-center network supporting inpatient pediatric care and research in Kenya. J Am Med Inform Assoc. 2016;23:184–92. The Wellcome Trust, 215 Euston Road, London NW1 2BE, UK. [ ] Among the limitations of the trial is the relatively lim- ited number of clusters. This may limit our ability to de- tect effects and may raise concerns as to the external validity of any findings. It is important to note that most cluster trials in health conducted in low- and middle- income countries to date have involved randomization of communities and hospitals have rarely been used as randomization units. Additionally, there are issues re- lated to the delivery of effective feedback that deserve at- tention but which this study could not address. These questions include, but are not limited to, the ideal dur- ation of feedback interventions, and alternative sug- gested key components of feedback that could enhance performance but that were not examined in this trial [4]. In our case, the duration of our intervention was in- formed by sample size considerations (power, signifi- cance level, effect size), and the participating facilities’ pediatric workloads. Inefficiencies may occur if the dur- ation determined under these assumptions is consider- ably longer than the ideal duration of feedback. Conversely, a short intervention duration might omit the effect of feedback that is lesser in magnitude but still useful at a system level. Providing evidence to address these issues will require continued methodological de- velopments, possibly involving adaptive designs to suit the dynamic nature of feedback interventions. Authors’ contributions ME conceived the idea of the clinical information network and obtained funding for the network. PA, GI, and ME designed the feedback intervention and GI coordinated the delivery of feedback to hospitals. PA and ME drafted the manuscript. All authors read and approved the final manuscript. Dissemination policy The monthly feedback of the trial data to intervention hospital is used to communicate results to the health providers in participating intervention hospitals. At the end of the trial the findings will be disseminated within the CIN, and among CIN collaborators including the Ministry of Health. The trial findings will also be published in scientific, peer-reviewed journals. Competing interests Competing interests The authors declare that they have no competing interests. Author details 1 1Kenya Medical Research Institute (KEMRI) – Wellcome Trust Research Programme, Nairobi, Kenya. 2Department of Paediatrics and Child Health, University of Nairobi, Nairobi, Kenya. 3Nuffield Department of Medicine, University of Oxford, Oxford, UK. References 1. Amin AA, Zurovac D, Kangwana BB, et al. The challenges of changing national malaria drug policy to artemisinin-based combinations in Kenya. Malar J. 2007;6:72. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements The authors thank the Ministry of Health which approved the establishment of the Kenyan clinical information network (CIN) and has supported its implementation together with county health executives and all hospital management teams, clinical teams, and pediatric staff. Authors are also grateful to the Kenya Paediatric Association for supporting CIN. The work is published with the permission of the Director of KEMRI. This work is published with the permission of the Director of KEMRI. 2. English M, Gathara D, Mwinga S, et al. Adoption of recommended practices and basic technologies in a low-income setting. Arch Dis Child. 2014;99:452–6. 3. Wasunna B, Zurovac D, Goodman CA, Snow RW. Why don’t health workers prescribe ACT? A qualitative study of factors affecting the prescription of artemether-lumefantrine. Malar J. 2008;7:29. 4. Ivers N, Jamtvedt G, Flottorp S, et al. Audit and feedback: effects on professional practice and healthcare outcomes. Cochrane database Syst Rev. 2012;(6):CD000259. doi:10.1002/14651858.CD000259.pub3. Consent for publication Not applicable. Consent for publication Not applicable. Monitoring A formal data monitoring committee was not needed in this trial for the following reasons: the patients meeting Ayieko et al. Trials (2017) 18:416 Page 8 of 9 Page 8 of 9 Page 8 of 9 Page 8 of 9 reduced selection bias associated with randomized allo- cation to limit selection bias [9, 22–24]. Contact information of trial sponsor Contact information of trial sponsor Contact information of trial sponsor The Wellcome Trust, 215 Euston Road, London NW1 2BE, UK. Ethics approval and consent to participate The study was approved by the Kenya Medical Research Institute Scientific and Ethical Review Committee (KEMRI SERC – SSC protocol no. 2465). KEMRI SERC is a National Review Board mandated by the Kenyan Ministry of Health to provide ethical clearance to conduct studies within Kenyan health facilities. This National Committee approved the use of hospital assent to participate in the trial rather than written consent of the hospital administration and all hospitals retained their right to withdraw from the study at any point. The Ethics Committee also confirmed that informed consent was not needed from individual patients (or their carers) whose data were obtained retrospectively from medical records for the purposes of service evaluation and implementation research. Ayieko et al. Trials (2017) 18:416 understand and improve care in Kenyan hospitals. BMJ Glob Heal. 2016;1: e000028. understand and improve care in Kenyan hospitals. BMJ Glob Heal. 2016;1: e000028. 9. English M. Designing a theory-informed, contextually appropriate intervention strategy to improve delivery of paediatric services in Kenyan hospitals. Implement Sci. 2013;8:39. 10. Forrest CB, Margolis PA, Bailey LC, et al. PEDSnet: a national pediatric learning health system. J Am Med Inform Assoc. 2014;21:602–6. 11. Agweyu A, Gathara D, Oliwa J, et al. Oral amoxicillin versus benzyl penicillin for severe pneumonia among Kenyan children: a pragmatic randomized controlled noninferiority trial. Clin Infect Dis. 2015;60:1216–24. 12. Ministry of Health. Republic of Kenya. 4th ed. Nairobi: Basic paediatric protocols; 2016. 13. WHO. Pocket book of hospital care for children. 2nd ed. Geneva: WHO; 2015. 14. Chan A-W, Tetzlaff JM, Gøtzsche PC, et al. SPIRIT 2013 explanation and elaboration: guidance for protocols of clinical trials. BMJ. 2013;346:e7586. ayes RJ, Moulton LH. Cluster randomised trials. New York: CRC Press; 2 16. Ayieko P, Ogero M, Makone B, et al. Characteristics of admissions and variations in the use of basic investigations, treatments and outcomes in Kenyan hospitals within a new Clinical Information Network. Arch Dis Child. 2016;101:223–9. 17. Hattie J, Timperley H. The power of feedback. Rev Educ Res. 2007;77:81–112. 18. Mwakyusa S, Wamae A, Wasunna A, et al. Implementation of a structured paediatric admission record for district hospitals in Kenya—results of a pilot study. BMC Int Health Hum Rights. 2006;6:9. 19. Ayieko P, Ntoburi S, Wagai J, et al. A multifaceted intervention to implement guidelines and improve admission paediatric care in Kenyan district hospitals: a cluster randomised trial. PLoS Med. 2011;8:e1001018. 20. Scales DC, Dainty K, Hales B, et al. An innovative telemedicine knowledge translation program to improve quality of care in intensive care units: protocol for a cluster randomized pragmatic trial. Implement Sci. 2009;4:5. 21. Dainty KN, Scales DC, Brooks SC, et al. A knowledge translation collaborative to improve the use of therapeutic hypothermia in post-cardiac arrest patients: protocol for a stepped wedge randomized trial. Implement Sci. 2011;6:4. 22. Tunis SR, Stryer DB, Clancy CM. Practical clinical trials: increasing the value of clinical research for decision making in clinical and health policy. JAMA. 2003;290:1624–32. 23. Habicht JP, Victora CG, Vaughan JP. Evaluation designs for adequacy, plausibility and probability of public health programme performance and impact. Int J Epidemiol. 1999;28:10–8. 24. Victora CG, Schellenberg JA, Huicho L, et al. Availability of data and materials The data collected during the trial are under the primary jurisdiction of the Ministry of Health in Kenya. Enquiries about using the data can be made to the KEMRI-Wellcome Trust Research Program Data Governance Committee. 8. Tuti T, Bitok M, Malla L, et al. Improving documentation of clinical care within a clinical information network: an essential initial step in efforts to Page 9 of 9 Ayieko et al. Trials (2017) 18:416 Ayieko et al. Trials (2017) 18:416 Context matters: interpreting impact findings in child survival evaluations. Health Policy Plan. 2005;20 Suppl 1:i18–31. 24. Victora CG, Schellenberg JA, Huicho L, et al. Context matters: interpreting impact findings in child survival evaluations. Health Policy Plan. 2005;20 Suppl 1:i18–31. 25. Perera R, Heneghan C, Yudkin P. Graphical method for depicting randomised trials of complex interventions. BMJ. 2007;334:127–9. 25. Perera R, Heneghan C, Yudkin P. Graphical method for depicting randomised trials of complex interventions. BMJ. 2007;334:127–9. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries
https://openalex.org/W1971699048	https://journals.plos.org/plosntds/article/file?id=10.1371/journal.pntd.0000811&type=printable	English	null	Multi-Country Evaluation of the Sensitivity and Specificity of Two Commercially-Available NS1 ELISA Assays for Dengue Diagnosis	PLoS neglected tropical diseases	2,010	cc-by	8,709	Abstract Copyright: 2010 Guzman et al. This is an open-access article distributed under the terms of the Creative Commons Attr unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the European Commission grant No. 517708, 6th Framework Programme. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lupe@ipk.sld.cu Competing Interests: The authors have declared that no competing interests exist. * E-mail: lupe@ipk.sld.cu * E-mail: lupe@ipk.sld.cu Dengue viruses (DENVs), of which there are four serotypes, cause a variable spectrum of disease that ranges from an undifferentiated fever to dengue fever (DF) through to more severe syndromes called dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). DHF/DSS is a vasculopathy characterized by capillary leakage and haematological dysregulation. There are no licensed vaccines or specific antiviral therapies for dengue, and patient management relies on good supportive care. Maria G. Guzman1, Thomas Jaenisch2, Roger Gaczkowski2, Vo Thi Ty Hang3, Shamala Devi Sekaran4, Axel Kroeger5, Susana Vazquez1, Didye Ruiz1, Eric Martinez1, Juan C. Mercado6, Angel Balmaseda6, Eva Harris7, Efren Dimano8, Prisca Susan A. Leano8, Sutee Yoksan9, Elci Villegas10, Herminia Benduzu10, Iris Villalobos11, Jeremy Farrar3, Cameron P. Simmons3 Maria G. Guzman1, Thomas Jaenisch2, Roger Gaczkowski2, Vo Thi Ty Hang3, Shamala Devi Sekaran4, Axel Kroeger5, Susana Vazquez1, Didye Ruiz1, Eric Martinez1, Juan C. Mercado6, Angel Balmaseda6, Eva Harris7, Efren Dimano8, Prisca Susan A. Leano8, Sutee Yoksan9, Elci Villegas10, Herminia Benduzu10, Iris Villalobos11, Jeremy Farrar3, Cameron P. Simmons3 1 Virology Department, Pan American Health Organization/World Health Organization Collaborating Center for the Study of Dengue and Its Vector, Instituto de Medicina Tropical ‘‘Pedro Kouri’’, Habana, Cuba, 2 Section Clinical Tropical Medicine, Department of Infectious Diseases, University Hospital of Heidelberg, Heidelberg, Germany, 3 Oxford University, Clinical Research Unit, Ho Chi Minh City, Vietnam, 4 Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, 5 TDR-World Health Organization, Geneva, Switzerland, 6 CNDR, Managua, Nicaragua, 7 Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, California, United States of America, 8 San Lazaro Hospital, Manila, Philippines, 9 Mahidol University, Bangkok, Thailand, 10 Instituto Experimental ‘‘Jose W Torrealba’’ Nu´cleo Universitario ‘‘Rafael Rangel’’, Universidad de los Andes Trujillo, Bogota´, Venezuela, 11Hospital Central de Maracay, Maracay, Venezuela Abstract Background: Early diagnosis of dengue can assist patient triage and management and prevent unnecessary treatments and interventions. Commercially available assays that detect the dengue virus protein NS1 in the plasma/serum of patients offers the possibility of early and rapid diagnosis. Methodology/Principal Findings: The sensitivity and specificity of the Pan-E Dengue Early ELISA and the PlateliaTM Dengue NS1 Ag assays were compared against a reference diagnosis in 1385 patients in 6 countries in Asia and the Americas. Platelia was more sensitive (66%) than Pan-E (52%) in confirmed dengue cases. Sensitivity varied by geographic region, with both assays generally being more sensitive in patients from SE Asia than the Americas. Both kits were more sensitive for specimens collected within the first few days of illness onset relative to later time points. Pan-E and Platelia were both 100% specific in febrile patients without evidence of acute dengue. In patients with other confirmed diagnoses and healthy blood donors, Platelia was more specific (100%) than Pan-E (90%). For Platelia, when either the NS1 test or the IgM test on the acute sample was positive, the sensitivity versus the reference result was 82% in samples collected in the first four days of fever. NS1 sensitivity was not associated to disease severity (DF or DHF) in the Platelia test, whereas a trend for higher sensitivity in DHF cases was seen in the Pan-E test (however combined with lower overall sensitivity). Conclusions/Significance: Collectively, this multi-country study suggests that the best performing NS1 assay (Platelia) had moderate sensitivity (median 64%, range 34–76%) and high specificity (100%) for the diagnosis of dengue. The poor sensitivity of the evaluated assays in some geographical regions suggests further assessments are needed. The combination of NS1 and IgM detection in samples collected in the first few days of fever increased the overall dengue diagnostic sensitivity. Citation: Guzman MG, Jaenisch T, Gaczkowski R, Ty Hang VT, Sekaran SD, et al. (2010) Multi-Country Evaluation of the Sensitivity and Specificity of Two Commercially-Available NS1 ELISA Assays for Dengue Diagnosis. PLoS Negl Trop Dis 4(8): e811. doi:10.1371/journal.pntd.0000811 Editor: Ann M. Powers, Centers for Disease Control and Prevention, United States of America Received November 23, 2009; Accepted August 4, 2010; Published August 31, 2010 man et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. www.plosntds.org Citation: Guzman MG, Jaenisch T, Gaczkowski R, Ty Hang VT, Sekaran SD, et al. (2010) Multi-Country Evaluation of the Sensitivity and Specificity of Two Commercially-Available NS1 ELISA Assays for Dengue Diagnosis. PLoS Negl Trop Dis 4(8): e811. doi:10.1371/journal.pntd.0000811 Introduction Dengue is the most important mosquito-borne viral disease of humans and an enormous public health burden in affected countries. An estimated 50–100 million dengue cases occur annually, including 250,000–500,000 cases of severe illness and around 25,000 deaths. Approximately 2.5 billion people live in dengue endemic countries and the illness is reported in Southeast Asia, Western Pacific, the Americas, Africa and Mediterranean regions [1–3]. Early, sensitive and specific diagnosis of dengue can assist in patient triage and for those who require it, early supportive August 2010 \| Volume 4 \| Issue 8 \| e811 1 www.plosntds.org Dengue NS1 ELISA Multicountry Evaluation the role of NS1 in DENV biology is not well understood, high plasma NS1 concentrations early in illness have been associated with more severe disease [14,15]. The targeting of NS1 in diagnostic assays potentially offers the opportunity for an early, specific diagnosis of DENV infection since it can be detected prior to the appearance of measurable DENV-reactive IgM [8]. Whilst NS1 is a promising diagnostic target, the assessment of currently available NS1 assays across a breadth of patient populations, viral serotypes and lineages is important in evaluating where and when these assays [16] may fit into the laboratory diagnosis of dengue. the role of NS1 in DENV biology is not well understood, high plasma NS1 concentrations early in illness have been associated with more severe disease [14,15]. The targeting of NS1 in diagnostic assays potentially offers the opportunity for an early, specific diagnosis of DENV infection since it can be detected prior to the appearance of measurable DENV-reactive IgM [8]. Whilst NS1 is a promising diagnostic target, the assessment of currently available NS1 assays across a breadth of patient populations, viral serotypes and lineages is important in evaluating where and when these assays [16] may fit into the laboratory diagnosis of dengue. Author Summary Dengue is the most important mosquito-borne viral disease of humans and an enormous public health burden in affected countries. Early, sensitive and specific diagnosis of dengue is needed for appropriate patient management as well as for early epidemic detection. Commercially available assays that detect the dengue virus protein NS1 in the plasma/serum of patients offer the possibility of early and rapid diagnosis. Here we evaluated two commercially available ELISA kits for NS1 detection (Pan- E Dengue Early ELISA and the PlateliaTM Dengue NS1 Ag). Results were compared against a reference diagnosis in 1385 patients in 6 countries in Asia and the Americas. Collectively, this multi-country study suggests that the best performing NS1 assay (Platelia) had moderate sensitivity (median 64%, range 34–76%) and high specific- ity (100%) for the diagnosis of dengue. The combination of NS1 and IgM detection in samples collected in the first few days of fever increased the overall dengue diagnostic sensitivity. At the end of 2006, the Dengue Scientific Working Group under the leadership of the World Health Organization Special Pro- gramme for Research and Training in Tropical Diseases (WHO/ TDR) established priorities for dengue research aimed at improving dengue treatment, prevention and control. The evaluation of new diagnostics were included among these priorities [17,18]. To this end, the purpose of the current study was to assess the sensitivity and specificity of two commercial NS1 assays in six countries. The DENCO study The DENCO project was a multi-centre prospective observa- tional study of dengue in Southeast Asia (Malaysia, Thailand, The Philippines and Vietnam) and the Americas (Nicaragua and Venezuela). The study sites at which patients were enrolled were: Department of Paediatrics, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; Queen Sirikit National Institute of Child Health, Bangkok, Thailand; San Lazaro Hospital, Manila, The Philippines; Hospital for Tropical Disease, Ho Chi Minh City, Viet Nam, Children’s Hospital #1, Ho Chi Minh City, Viet Nam; Children’s Hospital #2, Ho Chi Minh City, Viet Nam; Children’s Hospital Manuel Jesus de Rivera, Managua, Nicaragua; Research Centre Jose W. Torrealba, University des Andes, Trujillo and Hospital Central, Maracay, Venezuela. management. In principle, early diagnosis could also facilitate timely public health interventions, e.g. vector control targeted at the households of index cases. Existing approaches to dengue diagnosis rely primarily on detection of DENV-reactive IgM; in more specialised settings this is augmented with detection of DENV RNA using home made RT-PCR or rarely, virus isolation [4,5]. Whilst generally robust, a limitation of IgM-based diagnostic approaches is poor sensitivity in the first few days of illness and in some settings, serological cross-reactivity with other Flaviviruses [4,5]. Recently, the diagnostic accuracy of commercial diagnostic assays that detect the DENV NS1 protein in plasma/serum samples have been described [6–13]. NS1 is a 55kDa glycoprotein secreted by DENV infected cells ‘‘in vitro’’ and ‘‘in vivo’’. Whilst management. In principle, early diagnosis could also facilitate timely public health interventions, e.g. vector control targeted at the households of index cases. Existing approaches to dengue diagnosis rely primarily on detection of DENV-reactive IgM; in more specialised settings this is augmented with detection of DENV RNA using home made RT-PCR or rarely, virus isolation [4,5]. Whilst generally robust, a limitation of IgM-based diagnostic approaches is poor sensitivity in the first few days of illness and in some settings, serological cross-reactivity with other Flaviviruses [4,5]. Recently, the diagnostic accuracy of commercial diagnostic assays that detect the DENV NS1 protein in plasma/serum samples have been described [6–13]. NS1 is a 55kDa glycoprotein secreted by DENV infected cells ‘‘in vitro’’ and ‘‘in vivo’’. Whilst Figure 1. Flow chart summarising multi-country enrolment of dengue patients and basis for the final dataset. doi:10.1371/journal.pntd.0000811.g001 Figure 1. Flow chart summarising multi-country enrolment of dengue patients and basis for the final dataset. The DENCO study doi:10.1371/journal.pntd.0000811.g001 August 2010 \| Volume 4 \| Issue 8 \| e811 August 2010 \| Volume 4 \| Issue 8 \| e811 2 www.plosntds.org Dengue NS1 ELISA Multicountry Evaluation Table 1. List of laboratories performing diagnostic testing for the patients enrolled in the DENCO study. Location / Country Hospital Laboratory Bangkok / Thailand Queen Sirikit National Institute of Child Health AFRIMS, Bangkok* – all tests Manila / Philippines San Lazaro Hospital AFRIMS certified laboratory at San Lazaro Hospital* for serology PCR performed at Department of Virology, Nagasaki University, Nagasaki, Japan; serotyping done at San Lazaro Hospital, Manila, biomolecular laboratory (SACCL) Ho Chi Minh City / Vietnam Children’s Hospital No. 1 Oxford University Clinical Research Unit, HCMC – all tests Children’s Hospital No. 2 Hospital for Tropical Diseases Kuala Lumpur / Malaysia University of Malaya Medical Centre, University of Malaya Department of Medical Microbiology, Faculty of Medicine, University Malaya - all tests Managua / Nicaragua Hospital Infantil Manuel de Jesus Rivera La Mascota Centro Nacional de Diagno´stico y Referencia, Ministry of Health, Managua – all tests IgM, IgG and NS1 repeated at IPK Cuba Maracay & Trujillo/ Venezuela Hospital Central de Maracay Hospital de Trujillo Trujillo Hospital Laboratory – all tests IgM, IgG, viral isolation repeated and NS1 done at IPK Cuba AFRIMS - Armed Forces Research Institute of Medical Sciences, IPK - Instituto de Medicina Tropical ‘‘Pedro Kouri’’. AFRIMS laboratory network in Asia. Member of the Tropical Disease Research –Pediatric Dengue Vaccine Initiative network of proficiency tested laboratories for dengue diagnostic evaluation. doi:10.1371/journal.pntd.0000811.t001 ble 1. List of laboratories performing diagnostic testing for the patients enrolled in the DENCO study. edical Sciences, IPK - Instituto de Medicina Tropical ‘‘Pedro Kouri’’. AFRIMS - Armed Forces Research Institute of Medical Sciences, IPK - Instituto de Medicina Tropical Pedro Kouri . AFRIMS laboratory network in Asia. *Member of the Tropical Disease Research –Pediatric Dengue Vaccine Initiative network of proficiency tested laboratories for dengue diagnostic evaluation. doi:10.1371/journal.pntd.0000811.t001 Patient enrolment = 100 units in the convalescent sample Malaysia, Nicaragua, Venezuela, Vietnam IgM seroconversion (paired samples) IgG seroconversion (paired samples) or fourfold or greater increase in titer (paired samples) For each test validated local protocols were used at each site. Serology results are based on IgM and IgG Capture ELISA of acute and convalescent specimens except where indicated. *Four laboratories employed the RT/PCR protocol described by Lanciotti, et al., 1992 [20], one employed the protocols by Kong et al., 2006, J Virol Methods and Yong et al., 2007 Singaporean Med J [22,23],and the other the protocol by Laue et al., . J Clin Microbiol 1999 [21]. All laboratories employed MAC-ELISA. One laboratory employed Inhibition ELISA Method for IgG study while other four used GAC-ELISA. HI: hemagglutination inhibition assay was done in one laboratory (WHO recommendations were followed) [30]. doi:10.1371/journal.pntd.0000811.t002 Table 2. Laboratory criteria employed at country level for dengue laboratory classification as confirmed dengue case. Country Confirmed dengue case (one of the following)** Patients without evidence of recent acute dengue (all countries) All countries RT-PCR positive or virus isolation positive Having paired plasma or serum specimens (collected $3 days apart) with the last sample collected $7 days after illness onset and RT-PCR negative and virus culture negative (at least one of the two being done on the acute sample), and serologically negative in locally used IgM and IgG assays Thailand, The Philippines (according to AFRIMS protocol) IgM. = 40 units (acute or convalescent sample or both) and IgG titer increase to above 100 units (paired samples) Twofold IgG titer increase (paired samples) with a titer . = 100 units in the convalescent sample Malaysia, Nicaragua, Venezuela, Vietnam IgM seroconversion (paired samples) IgG seroconversion (paired samples) or fourfold or greater increase in titer (paired samples) For each test validated local protocols were used at each site. Serology results are based on IgM and IgG Capture ELISA of acute and convalescent specimens except where indicated. Four laboratories employed the RT/PCR protocol described by Lanciotti, et al., 1992 [20], one employed the protocols by Kong et al., 2006, J Virol Methods and Yong et al., 2007 Singaporean Med J [22,23],and the other the protocol by Laue et al., . J Clin Microbiol 1999 [21]. All laboratories employed MAC-ELISA. One laboratory employed Inhibition ELISA Method for IgG study while other four used GAC-ELISA. Patient enrolment of the enrolment and the second 7–14 days after fever onset. Dengue diagnosis was confirmed by either of the following methods: virus isolation in Aedes albopictus cell line (C6/36), by RT/PCR detection as previously described and IgM (MAC-ELISA), IgG (GAC-ELISA or Inhibition ELISA Method, EIM) and total antibody seroconversion (by Hemagglutination Inhibition assay) following the standard procedures at each study site [19–30]. The Hemagglutination Inhibition assay was standardized following WHO criteria and WHO recommended cut-off values were utilized [29]. As previously described, RT/PCR methods used here have sensitivity figures from 90 to 100% [20–23]. Other investigations and clinical management were at the discretion of the attending physicians. After discharge each patient was classified using the former WHO criteria for DF, DHF and DSS [30]. From November Following written informed consent by the study participant, or a parent/guardian in the case of children, patients above 6 months of age with clinically suspected dengue and fever for less than 7 days were enrolled in the study. At 5 centres out-patients were recruited as well as in-patients. Patients were followed daily by trained study physicians using standardised case report forms (CRFs) describing clinical, laboratory, diagnostic and management information in detail. Ethical approval was obtained from the Ethics Review Committee of WHO and each institution involved. All patients in these studies were assessed daily by a study physician and had serial haematocrit and platelet estimations performed, as well as appropriate sampling for diagnostic serology and virology. Two plasma or sera samples were collected from each patient, one at day Table 2. Laboratory criteria employed at country level for dengue laboratory classification as confirmed dengue case. Country Confirmed dengue case (one of the following)** Patients without evidence of recent acute dengue (all countries) All countries RT-PCR positive or virus isolation positive Having paired plasma or serum specimens (collected $3 days apart) with the last sample collected $7 days after illness onset and RT-PCR negative and virus culture negative (at least one of the two being done on the acute sample), and serologically negative in locally used IgM and IgG assays Thailand, The Philippines (according to AFRIMS protocol) IgM. = 40 units (acute or convalescent sample or both) and IgG titer increase to above 100 units (paired samples) Twofold IgG titer increase (paired samples) with a titer . NS1 sensitivity in relation to RT/PCR results Compared to RT/PCR results, sensitivity of kit Pan-E ranged from 29–79% (overall sensitivity rate of 67%; 95% CI 63–71%) and the sensitivity of kit Platelia from 36–88% (overall sensitivity rate of 77%; 95% CI 74–79%) (Figure 2B). Compared to RT/PCR results, sensitivity of kit Pan-E ranged from 29–79% (overall sensitivity rate of 67%; 95% CI 63–71%) and the sensitivity of kit Platelia from 36–88% (overall sensitivity rate of 77%; 95% CI 74–79%) (Figure 2B). Laboratory investigations Serological and virological dengue diagnostics were performed in each participating country according to local protocols, with support provided by WHO designated laboratories as necessary (for participating laboratories see Table 1) [19–30]. The definitions employed at each site for ‘‘confirmed dengue case’’ are described in Table 2. For NS1 sensitivity analysis, patients with laboratory confirmation of dengue by serological or virological means were the reference population. For an assessment of NS1 specificity, patients in whom there was no evidence of acute or recent dengue (defined as serologically and virologically negative and in whom there were a minimum of 2 plasma or serum samples tested with the second collected $7 days after fever onset and .2 days after the first sample) were studied. As an additional assessment of specificity, two sera panels (one prepared at the Institute of Tropical Medicine ‘‘Pedro Kouri’’ in Cuba and the other at the Mahidol University, Bangkok, Thailand) from healthy individuals and from non-dengue patients were employed. Patient enrolment HI: hemagglutination inhibition assay was done in one laboratory (WHO recommendations were followed) [30]. doi:10.1371/journal.pntd.0000811.t002 Table 2. Laboratory criteria employed at country level for dengue laboratory classification as confirmed dengue case.* Patients without evidence of recent acute dengue (all countries) August 2010 \| Volume 4 \| Issue 8 \| e811 3 www.plosntds.org Dengue NS1 ELISA Multicountr www.plosntds.org 4 August 2010 \| Volume 4 \| Issu Dengue NS1 ELISA Multicountry Evaluation August 2010 \| Volume 4 \| Issue 8 \| e811 www.plosntds.org Dengue NS1 ELISA Multicountry Evaluation Dengue NS1 ELISA Multicountry Evaluation Dengue NS1 ELISA Multicountry Evaluation Figure 2. Sensitivity of kits Pan-E and Platelia. Shown are the sensitivities (695% CI) of kits Pan-E and Platelia assays from six Asian and Latin- American countries in 1385 patients with a laboratory confirmed diagnosis of dengue (A) and sensitivities in the subgroup of 933 patients confirmed by PCR or viral isolation (B). doi:10.1371/journal.pntd.0000811.g002 2007 to January 2008, we prospectively tested acute plasma (or serum) samples from children and adults enrolled in these studies. 2007 to January 2008, we prospectively tested acute plasma (or serum) samples from children and adults enrolled in these studies. NS1 sensitivity in relation to viral serotype y yp The sensitivity of each NS1 assay was considered in the context of the infecting serotype. Table 3 shows the sensitivity of kit Pan-E and Platelia assays according to DENV serotype as determined by RT-PCR or virus isolation. In our mainly hospital-based patient samples from 2006/2007 DENV-1 was most prevalent in Asia and DENV-2 most prevalent in Latin America (Table 4). For each of the four DENV serotypes kit Platelia had a greater sensitivity except for DENV-2, where the sensitivity was the same in both kits. In kit Platelia, sensitivity for DENV-2 was statistically significantly lower than for the other three serotypes pooled (DENV-2: 63%; 95% CI 57–69% versus 84%; 95% CI 82–88% for DENV-1, 3 and 4). The greater prevalence of DENV-2 in Latin American patients compared with Asian patients may help explain the lower sensitivity of both kit Pan-E and Platelia assays in Latin America (Figure 3B). NS1 detection kits Pan-E Dengue Early ELISA from Panbio (Brisbane, Australia), (Kit Pan-E) and Platelia Dengue NS1 AG from Bio-Rad (Marnes- la-Coquette, France), (kit Platelia) were evaluated. Both kits are based on a sandwich format microplate enzyme immunoassay for the detection of DENV NS1 employing a peroxidase-labelled murine monoclonal antibodies as probes. Samples were tested for NS1 detection following the manufacturer’s recommendations. Sera were classified as NS1 positive, negative and equivocal according to the manufacturer’s instructions. For the purposes of analysis, equivocal samples were excluded from the analysis. Sensitivity of NS1 tests by day of illness The sensitivity of both kits Pan-E and Platelia was influenced by the patient’s duration of illness prior to test sample collection.. In Asian patients, kits Pan-E and Platelia were more sensitive in test samples collected early in the disease phase than at later time points (Figure 3A). The analysis was limited to days with more than 40 observations total which is why for Latin America only a narrow range of days can be shown (Figure 3B) and due to small sample size and large confidence intervals no trend is visible. A higher sensitivity of both NS1 detection assays were observed in Asian patients than in Latin-American patients at the first four days of illness (Figure 3B). Characteristics of the study population The diagnostic sensitivity of kits Pan-E and Platelia assays was evaluated in 854 and 1284 serum samples respectively (Figure 1) from patients with a laboratory confirmed dengue diagnosis. Kit Pan-E it could not be performed in all available samples for logistical reasons relating to assay availability at some sites. The sensitivity of the kit Pan-E ranged from 24% in The Philippines to 72% in Vietnam (overall sensitivity rate of 52%). The sensitivity of the kit Platelia ranged from 34% in Nicaragua to 76% in Thailand (overall sensitivity rate of 66%) (Figure 2A). Between August 2006 and May 2007 a total of 2259 patients were recruited to the DENCO study at the 11 participating hospitals. NS1 detection was attempted using at least one of the two NS1 tests in 1821 patients. From amongst the 1821 patients, there were 1385 with laboratory-confirmed dengue and 45 with no laboratory evidence of acute or recent dengue. A further 391 had either indeterminate laboratory results or suggestive serology; results from these cases were not included in the analysis. The flow-chart in Figure 1 summarises the numbers and geography of enrolment and the classification of patients according to the results of reference diagnostic tests including demographic information. www.plosntds.org NS1 sensitivity in relation to IgM status Data were double-entered and checked at two established data- entry facilities in Guatemala (Center for Health Studies, Universidad del Valle de Guatemala) and Thailand (WHO/ TDR Clinical Data Management Collaborating Center, Faculty of Allied Health Sciences, Thammasat University, Thailand) and the two datasets were subsequently merged. Data analysis was performed at the Section of Clinical Tropical Medicine at the University of Heidelberg, Germany, using STATA versions 9.2 and 10, (STATA corporation, College Park, Texas). Detection of DENV-reactive IgM by MAC ELISA is the most commonly used approach to making a presumptive diagnosis of acute or recent dengue in endemic countries. Table 5 summarises NS1 sensitivity (kit Platelia assay only) in the context of IgM status and day of illness in confirmed dengue patients. The average sensitivity of NS1 testing in the first 7 days of sample collection was 65% (95%CI 62–69%) in acute samples where the IgM result was negative and 66% (95%CI 62–70%) when the acute test sample August 2010 \| Volume 4 \| Issue 8 \| e811 5 Dengue NS1 ELISA Multicountry Evaluation Dengue NS1 ELISA Multicountry www.plosntds.org 6 August 2010 \| Volume 4 \| Issu www.plosntds.org 6 August 2010 \| Volume 4 \| Issu August 2010 \| Volume 4 \| Issue 8 \| e811 August 2010 \| Volume 4 \| Issue 8 \| e811 www.plosntds.org Dengue NS1 ELISA Multicountry Evaluation Figure 3. NS1 sensitivity by day of illness. (A) Shown is the sensitivity (695% CI) of kits Pan-E and Platelia by day of illness in four Asian countries (N = 728 -kit Pan-E; N = 1152 -kit Platelia) amongst patients with a laboratory confirmed diagnosis of dengue where the acute sera were collected between day 2 and day 6 of illness. (B) Shown is the sensitivity (695% CI) of kits Pan-E and Platelia in the first four days of illness in two Latin American countries (N = 93 -kit Pan-E; N = 90 -kit Platelia) amongst patients with a laboratory confirmed diagnosis of dengue where the acute sera were collected between day 2 and day 4 of illness. Data is presented for those days of illness with . = 40 observations respectively. doi:10.1371/journal.pntd.0000811.g003 was IgM positive. Sensitivity figures increased to 74% and 70% if only samples collected in the first four days of illness were considered. NS1 sensitivity in relation to IgM status Taking an algorithmic approach, when either the NS1 test or the IgM test on the acute sample was positive, the sensitivity for a presumptive (IgM) or definitive (NS1) diagnosis versus the reference result was 74% (95%CI 69–78) in samples collected at days 5 to 7. These figure increased to 82% (95%CI 79–84) in samples collected in the first four days of fever. These results suggested a combination of either IgM testing or NS1 testing (with kit Platelia) was sufficient to allow a presumptive (IgM) or definitive (NS1) diagnosis on an average of 82% of dengue cases enrolled in this study when acute early samples are tested. A similar analysis was performed with data obtained in the evaluation of kit Pan-E. Sensitivity figures of 66% (95%CI 60– 72) in samples collected at days 5 to 7 and 71% in samples collected in the first four days of fever (95%CI 67–75) were obtained (Table S1). virological or serological laboratory evidence of acute or recent dengue. Both kits were negative in all these samples, which translates into a specificity of 100%. NS1 specificity in healthy blood donors and patients with other confirmed diagnoses Since the number of patients with no evidence of acute or recent dengue was relatively small (n = 45) in this study, efforts were made to assess the specificity of dengue NS1 assays in patients with other confirmed infectious diseases whose transmission geographically overlaps with dengue, in healthy blood donors, and in blood donors with a serological history of DENV exposure. For the specificity analysis, a total of 304 sera were tested at two study sites (Cuba and Thailand). The specificity of kit Platelia was 100% in both sites whilst the kit Pan-E was 89% (Table 7). The lower specificity of kit Pan-E was in part due to false positive results in patients with Japanese encephalitis, Yellow Fever and acute Influenza. Discussion The sensitivity of each NS1 assay was considered in the context of disease severity and geographical region (Table 6). Cases were classified according the former WHO criteria for DF and DHF/ DSS [27]. Sensitivity of kit Pan-E ranged from 29% (95%CI 12– 46) in DF to 60% (95%CI 39–82) in DHF cases from Latin- American countries and from 50% (95%CI 43–57) in DF to 62% (95%CI 57–67) in DHF/DSS cases from Asia (overall sensitivity 47% in DF and 62% in DHF/DSS cases). The sensitivity of kit Platelia ranged from 41% (95%CI 28–55) in DF and 68% (95%CI 47–89) in DHF/DSS cases from Latin-American countries and 70% (95%CI 66–75) in DF and 68% (95%CI 64–72) in DHF/ DSS cases from Asia (overall sensitivity of 68% for both DF and DHF/DSS total cases). Kit Pan-E showed higher figures of NS1 positive tests in severe cases, which are borderline statistically significant for Asia. Kit Platelia with overall higher sensitivity figures did not show a statistically significant association with disease severity. Dengue is increasing in incidence globally and therefore accurate and efficient diagnostic tests are more important than ever for clinical care, surveillance support, pathogenesis studies and vaccine research. Diagnosis is also important for case confirmation, to differentiate dengue from other diseases such as leptospirosis, rubella, and other flavivirus infections, and for the clinical management and evaluation of patients with severe disease [16,31]. The multicentre study described here assessed the diagnostic accuracy of two commercially available NS1 diagnostic tests. Two main findings were observed here: a) NS1 detection was overall only modestly sensitive for dengue diagnosis, with sensitivity highest in patients who presented early in their illness and b) a combined NS1 and IgM detection increased the overall sensitivity of dengue diagnostic. Table 4. Geographical and serotype stratification of the study population. Country DENV-1a DENV-2 DENV-3 DENV-4 Total Latin America Nicaragua 6% (2) 94% (32) 0 0 34 Venezuela 35% (19) 16% (9) 36% (20) 13% (7) 55 Mean 24% (21) 46% (41) 22% (20) 8% (7) 89 Asia Malaysia 62% (48) 9% (7) 17% (13) 13% (10) 78 Thailand 56% (86) 10% (15) 17% (26) 17% (26) 153 Philippines 0 10% (3) 87% (26) 3% (1) 30 Vietnam 47% (268) 35% (198) 10% (59) 1% (5) 568 Mean 48% (411) 27% (227) 15% (126) 5% (45) 829 aPercentages and absolute numbers (in brackets) of identified DENV serotypes by country. doi:10.1371/journal.pntd.0000811.t004 Table 4. Discussion Percentage of positives IgM samples plus positive NS1 samples of the IgM negative samples in the total tested samples. Percentages of IgM positive in total samples collected in the first four days (38%), days 5–7 (48%) and total (41%). doi:10.1371/journal.pntd.0000811.t005 The global dengue research agenda includes evaluating the validity, role and accessibility of available and new diagnostics of importance to reducing disease severity and case fatality [32]. Recognizing the importance of early diagnosis and taking advantage of the platform of the multicentre DENCO project, two commercial available NS1 detection ELISA kits (Pan-E Early Dengue, Panbio Ltd and PlateliaTM Dengue NS1 Ag, Bio-Rad ), named here as kits Pan-E and Platelia, were evaluated in terms of sensitivity and specificity. Overall and within country sensitivity figures were higher for kit Platelia than kit Pan-E. With the exception of Nicaragua and The Philippines, sensitivity figures of kit Platelia varied from 64% to 76% while the sensitivity of kit Pan- E varied from 36% to 72%. Depending on the diagnostic method used for comparison, different figures of sensitivity of NS1 detection have been reported by others [12,33,34]. Kumarasamy et al., obtained an overall sensitivity of 93% using PlateliaTMDen- gue NS1 Ag oscillating from 68% (in samples where the virus was isolated) to 90% in paired sera serologically confirmed as dengue [11,35]. samples from Asian patients were studied (interpretation limited for Latin America because of small sample size per day of illness).. Sensitivity was also higher in Asian patients compared with patients from Nicaragua and Venezuela. The small number of samples from Nicaraguan and Venezuelan patients (including a lower proportion of DHF/DSS cases) as well as the serotypes circulating could partially explain these observations (a high proportion of serotype 2 was found in Nicaraguan samples). The influence of duration of illness at the time of sample collection has been highlighted by others [6,8,10]. Figures of 93–100% sensitivity were obtained in samples collected at days 3 to 5 of fever [8] while others have reported figures higher than 85% in samples from day 1 to 3 in the Platelia assay [6,11]. NS1 protein has been detected concomitant with viremia and coincident with the febrile stage [8]. In the present study, the highest sensitivity was obtained in RT- PCR positive samples. Sensitivity of kit Platelia in RT-PCR positive samples was 71% to 88% in Asian countries and 66% in Venezuela, but much lower in Nicaraguan samples (36%). Discussion Geographical and serotype stratification of the study population. Overall specificity of NS1 tests versus reference diagnosis The diagnostic specificity of kits Pan-E and Platelia assays was evaluated in 36 and 45 samples respectively from patients with no Table 3. NS1-sensitivity in the context of DENV serotype. Table 3. NS1-sensitivity in the context of DENV serotype. Table 3. NS1-sensitivity in the context of DENV serotype. Serotype Kit Pan-E Kit Platelia N = 506 % Sensitivity (95%CI) N = 862 % Sensitivity (95%CI) DENV-1 223 79 (74–84) 415 87 (83–90) DENV-2 169 62 (54–69) 257 63 (57–69) DENV-3 87 60 (49–70) 142 82 (76–88) DENV-4 27 52 (32–72) 48 79 (67–91) Number of DENV-positive samples by virus isolation or RT-PCR and serotype determined. doi:10.1371/journal.pntd.0000811.t003 7 Dengue NS1 ELISA Multicountry Evaluation Table 5. NS1 detection (kit Platelia assay only) in relation to IgM status and day of illness. Day of illness Total no. of test samples IgM positive N = % NS1 positive in IgM positive test samples [41] IgM negative N = % NS1 positive in IgM-negative test samples [41] % NS1 positive in total no. of test samples % of test samples with a positive test (IgM or NS1) [41] Day 1 22 0 0 (0) 22 64 (14) 64(14) 64 (14) Day 2 139 27 56 (15) 112 77 (86) 73 (101) 81 (113) Day 3 372 123 78 (96) 249 71 (178) 74 (274) 81 (301) Day 4 384 198 74 (146) 186 66 (122) 70 (268) 83 (320) Subtotal ,5 days 917 348* 74 (257) 569 70 (400) 72 (657) 82 (748) Day 5 256 125 54 (67) 131 52 (68) 53 (135) 75 (193) Day 6 94 42 40 (17) 52 44 (23) 43 (40) 69 (65) Day 7 6 4 75 (3) 2 0 50 (3) 67 (4) Subtotal 5–7 days 356 171 51 (87) 185 49 (91) 50 (178) 74 (262) Total 1273 519*** 66 (344) 754 65 (491) 66 (835) 79 (1010) Samples from 1273 patients with a confirmed dengue diagnosis between day of illness 1 to 7. Percentage of positives IgM samples plus positive NS1 samples of the IgM negative samples in the total tested samples. *Percentages of IgM positive in total samples collected in the first four days (38%), days 5–7 (48%) and total (41%). doi:10.1371/journal.pntd.0000811.t005 Samples from 1273 patients with a confirmed dengue diagnosis between day of illness 1 to 7. Discussion Since high early viraemia levels have also been linked to increased disease severity, it is plausible that NS1 tests are more sensitive in the first few days of illness in patients at risk of developing severe complications in their illness compared to patients with a more benign disease evolution. However, in our study, no association between NS1 detection and disease severity (indicated by classification of DF or DHF/DSS) was observed. Furthermore a regression analysis on NS1 positivity for DHF/DSS vs. DF (or severe vs. mild) and adjusted for serotype and for country was done and there was no effect seen (data not shown). It is important to mention that no proficiency panel study on positive or negative samples was performed prior to evaluating the tested samples allowing us to have more comparable reference methods among participant laboratories. However protocols employed at each site, have been extensively evaluated previously [19–30]. In addition, the laboratories participants (including some WHO collaborating centres) are the reference centres for dengue diagnosis and laboratory surveillance in their respective countries and have participated in previous regional and international proficiency testing ([39,40] This study confirms and extends the findings of others in relation to the use of NS1 detections assays for the early diagnosis of dengue [6–12]. Although we could not study NS1 sensitivity and specificity in primary and secondary cases, in a small subset of samples classified as primary or secondary cases, a higher percentage of diagnose (90% over 80.6%) was obtained in the former (Vazquez S, manuscript in preparation). The specificity of NS1 tests could not accurately be estimated in the DENCO patient population as only a small number of cases had no serological or virological evidence of acute or recent dengue. Nonetheless, in patients who met our criteria for ‘‘not dengue’’, the specificity of both NS1 test kits was very high (100%). To provide further insights into specificity, two sera panels from patients with other confirmed diagnosis and healthy individuals were tested. Kit Platelia showed the higher specificity (100%). Similar specificity values has been previously reported by others [10,11,15,37]. The inclusion into the evaluating panel of samples from patients with acute Yellow fever and Japanese encephalitis virus infections suggest that no cross reaction among flaviviruses is observed with kit Platelia, however a larger number of samples collected from acute flavivirus infected patients need to be studied. Discussion Samples from this country were retested in a different laboratory by both NS1 detection kits but similar sensitivity results were observed (data not showed). The basis for low sensitivity in Nicaraguan samples remains unclear and will require further studies – but may partly be explained by the high proportion of serotype 2 in Nicaragua, which in both assays was associated with lower sensitivity. Indeed, as 94% (N = 32) of the serotypes recovered from Nicaragua were serotype 2, we cannot determine an estimate of sensitivity for the remaining 6% (N = 2). In the present study, relatively higher levels of sensitivity were observed in samples collected in the first four days of fever when Table 6. Sensitivity of Kit Platelia and Pan-E by geographic region and disease severity.* LAC SEA Total DF*** DHF/DSS DF DHF/DSS DF DHF/DSS Platelia 32/13 41% (28–55) 22/15 68% (47–89) 369/260 70% (66–75) 628/427 68% (64–72) 401/273 68% (63–73) 650/442 68% (64–72) Pan-E 31/9 29% (12–46) 23/14 60% (39–82) 228/114 50% (43–57) 396/245 62% (57–67) 259/123 47% (41–54) 419/259 62% (57–66) As indicated by the former WHO classification into DF and DHF/DSS for patients with NS1 test result and clinical classification available (N = 1051 for Platelia and 678 for Pan-E). LAC (Latin-American countries), SEA (Asian countries). *N/positive NS1; % ; (95% CI). doi:10.1371/journal.pntd.0000811.t006 Table 6. Sensitivity of Kit Platelia and Pan-E by geographic region and disease severity. Sensitivity varied by infecting serotype for each kit. The sensitivity of kit Pan-E was highest for DENV-1 infection (77%) and significantly lower for DENV-2 (60%), DENV-3 (57%) and DENV-4 (52%). The sensitivity of kit Platelia was also highest for DENV-1 infection (83%) and lowest for DENV-2 (60%). Consistent with DENV-1 infection being associated with high levels of NS1 detection, Xu et al., 2006, reported a sensitivity of 82% in an ‘‘in house’’ ELISA for the detection of NS1 protein of DENV-1 [36]. Similar results for the same serotype were reported by Alcon et al., 2002 [8]. The basis for different sensitivities for different serotypes requires further investigation. Potentially, this reflects different levels of avidity of the test mAbs for the relevant epitope(s) in NS1 from different serotypes, and potentially, August 2010 \| Volume 4 \| Issue 8 \| e811 8 www.plosntds.org Dengue NS1 ELISA Multicountry Evaluation Table 7. NS1 results as determined by Kit Pan-E and Platelia assays in control sera panels. Discussion In summary, we found the kit Platelia to be more sensitive and specific than kit Pan-E, with the sensitivity of both assays highest in the first few days of illness. Furthermore, we found that NS1 testing combined with IgM testing on the same test sample could yield a presumptive (IgM) or definitive (NS1) diagnose in as many as 82% of confirmed dengue cases using samples collected in the first four days of fever. As IgM detection is widely used for making a presumptive dengue diagnosis and in epidemiological surveil- lance, the use of a combined diagnostic algorithm including NS1 and IgM detection in samples collected in the first days of fever could provide clinically useful information to assist patient triage, management and outbreak response. The dengue serotype, duration of illness prior to sample collection, and the presence of immunocomplexes (NS1-IgG) in previous dengue immune individuals could explain the low sensitivity observed in the Nicaraguan and The Philippines samples [38]. In the case of Nicaragua, DENV-2 was present in the 94% of the samples where the virus was identified by virus isolation and RT/PCR suggesting that this was the predominant serotype. The generally poor sensitivity for DENV-2 (60%) observed for both assays suggests this partially explains the low sensitivity in Nicaraguan samples [12]. In The Philippines, a conjunction of factors such as to the duration of illness prior to sampling and the high level of individuals with a secondary infection could partially explain the low sensitivity since high sensitivity was observed in RT-PCR positive samples (83%). Discussion Panel 1 (Cuba) Panel 2 (Thailand) NS1 negative results NS1 negative results Sera N Kit Pan-E Kit Platelia Sera N Kit Pan-E Kit Platelia Healthy blood donors 80 76 80 Acute Malaria sera 39 38 39 Cases with rash illness no dengue 10 8 10 Acute Leptospirosis sera 10 8 10 Acute Influenza sera 20 13 20 Acute Japanese Encephalitis sera 34 24 34 Acute RSV* sera 20 17 20 Acute Yellow Fever sera 15 12 15 Acute Hepatitis A sera 20 20 20 Dengue mono- or polyvalent immune sera (past infection) 27 27 27 - - - - Flavivirus non-immune sera 29 29 29 Total 150 134 150 Total 154 138 154 Specificity 90% 100% 90% 100% *RSV (Respiratory Syncitial Virus). doi:10.1371/journal.pntd.0000811.t007 One of the limitations of our study is that it is heavily biased towards Asian patients and viruses, with 93% of the total samples coming from this region. The strengths of our study were that it was multicentre, prospective and encompassed a broad range of DENV serotypes and clinical presentations. different lineages from the same serotype. Also, this could potentially be related to the different sensitivities of the reference RT/PCR methods employed for dengue diagnosis. Alternatively, this might reflect different overall magnitudes of virus burden in patients with different serotypes. A relationship between NS1 detection and viraemia levels has been established previously [12,15]. Since high early viraemia levels have also been linked to increased disease severity, it is plausible that NS1 tests are more sensitive in the first few days of illness in patients at risk of developing severe complications in their illness compared to patients with a more benign disease evolution. However, in our study, no association between NS1 detection and disease severity (indicated by classification of DF or DHF/DSS) was observed. Furthermore a regression analysis on NS1 positivity for DHF/DSS vs. DF (or severe vs. mild) and adjusted for serotype and for country was done and there was no effect seen (data not shown). different lineages from the same serotype. Also, this could potentially be related to the different sensitivities of the reference RT/PCR methods employed for dengue diagnosis. Alternatively, this might reflect different overall magnitudes of virus burden in patients with different serotypes. A relationship between NS1 detection and viraemia levels has been established previously [12,15]. www.plosntds.org References 1. WHO (2008) Dengue and dengue hemorrhagic fever. http://www.who.int/ mediacentre/factsheets/fs117/en/ (accessed 05 June 2008) fact sheet No117, revised May 2008. 1. WHO (2008) Dengue and dengue hemorrhagic fever. http://www.who.int/ mediacentre/factsheets/fs117/en/ (accessed 05 June 2008) fact sheet No117, revised May 2008. 22. 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Found at: doi:10.1371/journal.pntd.0000811.s002 (0.05 MB DOC) August 2010 \| Volume 4 \| Issue 8 \| e811 August 2010 \| Volume 4 \| Issue 8 \| e811 August 2010 \| Volume 4 \| Issue 8 \| e811 9 Dengue NS1 ELISA Multicountry Evaluation Dengue NS1 ELISA Multicountry Evaluation CPS. Contributed reagents/materials/analysis tools: MGG TJ RG VTTH SV AB EH ED SY EV HB IV CPS. Wrote the paper: MGG TJ RG VTTH SDS AK CPS. Author Contributions Conceived and designed the experiments: MGG TJ AK EM EV IV JF CPS. Performed the experiments: VTTH SDS SV DR JCM AB ED PSAL SY HB. Analyzed the data: MGG TJ RG SDS AK AB EH ED SY EV JF References (2009) Evaluation of commercially available anti-dengue virus immunoglobulin M tests. Emerg Infect Dis 15: 436–40. 20. Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV (1992) Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. 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https://openalex.org/W1977904267	https://www.revistas.usp.br/rcf/article/download/34314/37046	Portuguese	null	O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em Ciências Contábeis	Revista Contabilidade & Finanças	2,010	cc-by	10,975	Ernani Ott Professor Titular do Departamento de Contabilidade, Custos e Finanças da Área Econômica da Universidade do Vale do Rio dos Sinos * E-mail: ernani@unisinos.br Recebido em 13.06.2010 * Aceito em 20.07.2010 * 2ª versão aceita em 29.09.2010 Márcia Martins Mendes De Luca Professora Adjunta do Departamento de Contabilidade da Faculdade de Economia, Administração, Atuária e Contabilidade da Universidade Federal do Ceará * E-mail: marciadeluca@ufc.br 1 Artigo apresentado no 4º Congresso ANPCONT, Natal-RN, 2010. Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 Edgard Bruno Cornachione Junior Professor Livre-Docente do Departamento de Contabilidade e Atuária da Faculdade de Economia, Administração e Contabilidade da Universidade de São Paulo * E-mail: edgardbc@usp.br 1 . 1 . O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em Ciências Contábeis1 Good is mine, bad is yours: perspectives of attribution theory on academic performance of Accounting Majors Edgard Bruno Cornachione Junior Professor Livre-Docente do Departamento de Contabilidade e Atuária da Faculdade de Economia, Administração e Contabilidade da Universidade de São Paulo * E-mail: edgardbc@usp.br Jacqueline Veneroso Alves da Cunha Professora Adjunta do Departamento de Ciências Contábeis da Faculdade de Economia da Universidade Federal de Minas Gerais * E-mail: jvac@face.ufmg.br Márcia Martins Mendes De Luca Professora Adjunta do Departamento de Contabilidade da Faculdade de Economia, Administração, Atuária e Contabilidade da Universidade Federal do Ceará * E-mail: marciadeluca@ufc.br Ernani Ott Professor Titular do Departamento de Contabilidade, Custos e Finanças da Área Econômica da Universidade do Vale do Rio dos Sinos * E-mail: ernani@unisinos.br Recebido em 13.06.2010 * Aceito em 20.07.2010 * 2ª versão aceita em 29.09.2010 RESUMO A maneira como os alunos compreendem e raciocinam sobre seus desempenhos acadêmico qualificando-os como superior ou inferior, tende a ser associada a elementos atributivo comuns, descritos pela literatura relacionada à teoria da atribuição. O objetivo geral dest O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em Ciências Contábeis1 Good is mine, bad is yours: perspectives of attribution theory on academic performance of Accounting Majors bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em Ciências Contábeis1 ABSTRACT The way students understand and assess their academic performance, qualifying it as superior or inferior, tends to be associated with common attributive elements, which are depicted by the literature related to the theory of attribution. The general objective of this study was to investigate the existence of association between common attributive elements in literature and undergraduates’ performance in accountancy courses in four universities in four different Brazilian states (Ceará, São Paulo, Minas Gerais, and Rio Grande do Sul). The instrument for data collecting was a questionnaire applied to 826 students. Among the main results, it was observed that 68% of the students who consider their academic performance superior claim that such performance is due to their own efforts, while less than 10% attribute it to external causes. 24% percent of those who assess their academic performance as inferior blame such failure on external causes such as family, tests, classmates and professors. When asked about the factors that could explain the performance of students from other institutions, the results pointed at the opposite direction, that is, academic success was more often related to external causes and failure was mostly attributed to insufficient individual effort. Thus it has been concluded that superior academic performance is more attributed to internal causes than inferior academic performance is, with an interesting idiosyncrasy among genders. These general results are aligned with the main literature on this theme and with the theory of attribution proposed by Weiner in 1976, which is the basis of this study. Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 RESUMO A maneira como os alunos compreendem e raciocinam sobre seus desempenhos acadêmicos, qualificando-os como superior ou inferior, tende a ser associada a elementos atributivos comuns, descritos pela literatura relacionada à teoria da atribuição. O objetivo geral deste estudo foi investigar a existência de associação entre elementos atributivos comuns na literatura e o desempenho acadêmico de alunos da graduação em ciências contábeis de quatro universidades em quatro diferentes Estados brasileiros (Ceará, São Paulo, Minas Gerais, e Rio Grande do Sul). O instrumento de coleta de dados foi um questionário respondido por 826 alunos. Dentre os principais resultados, constatou-se que 68% dos alunos que consideram seu desempenho acadêmico superior o atribuem ao seu próprio esforço, enquanto menos de 10% o relacionam a causas externas. Daqueles que avaliaram seu desempenho acadêmico como Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 2 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... inferior quase 24% relacionam esse fracasso a causas externas (família, provas, colegas e professores). Quando questionados sobre os fatores que explicariam o desempenho de alunos de outras instituições, os resultados apontaram em direção oposta, ou seja, sucesso acadêmico relacionado, com maior frequência, a causas externas e fracasso, principalmente, ao esforço próprio. Assim, com base nos achados, pôde-se concluir que o desempenho acadêmico superior é mais atribuído a causas internas que o desempenho acadêmico inferior, com uma interessante idiossincrasia entre os gêneros. Esses resultados gerais estão alinhados com a principal literatura sobre o tema e relacionados com a teoria da atribuição proposta por Weiner, em 1976, e base deste estudo. Palavras-Chave: Teoria da atribuição. Desempenho acadêmico. Educação 1 INTRODUÇÃO Em meio a uma grande transformação pela qual passa a área da educação, em geral, e a educação na Contabilidade, em particular, com as novas mudanças oriundas do processo de convergência das normas brasileiras às normas de Contabilidade internacionais, os desempenhos acadêmicos, especialmente no ensino superior, têm sido um elemento crucial para a pesquisa educacional. Todos os agentes envolvidos com o desempenho acadêmico (instituições, gestores, professores e alunos) desenvolvem suas ações e experiências de 3 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... aprendizagem baseados numa melhor compreensão e consciência dos fatores que levam a determinados resultados no complexo ambiente acadêmico. A teoria da atribuição constitui-se em um componente relevante da transformação impulsionada pela educação e pelas experiências profissionais, que influencia o desempenho dos indivíduos. Porém, há pouquíssima literatura sobre esse tema, principalmente, quando o assunto é o ensino da Contabilidade em cenários culturais e socioeconômicos específicos. A base para tal perspectiva pode ser encontrada em dimensões tanto educacionais como profissionais. De acordo com alguns exemplos encontrados na literatura relacionada à teoria da atribuição, desde a escola primária (GREENE, 1985) até o ensino superior (MAGNUSSON; PERRY, 1992), em culturas específicas (LEI, 2009), atribuição e aprendizagem caminham juntas. Por exemplo, ao discutir os fundamentos de psicologia social, em Human Resource Development (HRD), Swanson e Holton (2001) exploram a teoria da atribuição como um elemento-chave da psicologia cognitiva. HRD identifica o cenário, os participantes e os artefatos para dar suporte a uma discussão mais profunda sobre a aprendizagem ativa do indivíduo a partir de suas próprias experiências educacionais. No lado profissional, quando Brinberg, Luft e Shields (2007) apresentam as raízes psicológicas que dão suporte à teoria da Contabilidade (especialmente, a teoria da Contabilidade Gerencial), eles acrescentam detalhes específicos da teoria da atribuição e seu papel. Em ambas as dimensões, a teoria da atribuição desempenha papel importante na aprendizagem a partir de experiências e na maximização dos proveitos obtidos de tais experiências, visando a real transformação. Enquanto alguns alunos simplesmente passam pelos estágios do processo educacional, outros fazem reflexões ativas acerca de suas experiências em aprendizagem (EIDE, 2000; HARDRE, 2003; MARTIN; DOWSON, 2009), obtendo maior potencial para atingir melhores resultados. Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 1 INTRODUÇÃO Preocupados com isso, instituições, gestores e instrutores buscam alternativas para melhor desempenhar seus papeis ao expandir a discussão sobre os processos de aprendizagem e, principalmente, sobre o desempenho acadêmico (COHEN, 2003;; MARTIN; DOWSON, 2009; MCKEACHIE, 2002). A literatura relacionada à meta-cognição, atribuição, eficácia e desempenho (LUTHANS; VOGELGESANG; LESTER, 2006; MARTIN; DOWSON, 2009; MCKEACHIE, 2002; SWANSON; HOLTON, 2001; WEINER, 1976; YOUNG; FRY, 2008) sugere que experiências educacionais ruins podem ser mais bem explicadas e, consequentemente, potencialmente evitadas com o engajamento dos principais participantes do processo. Os alunos tendem a se tornar elementos ativos na busca de soluções quando são convidados a refletir sobre seus próprios desempenhos acadêmicos. Portanto, a 4 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... teoria da atribuição é fundamental para explicar os resultados acadêmicos e produz um efeito relevante na apreciação dos resultados e definição de estratégias e ações subsequentes. O ensino das temáticas relacionadas à área de negócios, em que se insere a Contabilidade, (NBEA, 2001) merece especial atenção devido às suas características, que demandam um suporte distinto sob o ponto de vista da elaboração de instruções e técnicas. Neste estudo, é dada maior atenção ao panorama atual dos níveis de desempenho acadêmico dos alunos da graduação do curso de ciências contábeis, à luz da teoria da atribuição. A literatura existente foi o estímulo para esta pesquisa, com especial relevância ao estudo conduzido por Ferreira et al. (2002), no qual dados obtidos por meio de alunos do ensino médio no Brasil, Argentina e México foram analisados com base em aspectos atributivos do desempenho acadêmico. A contribuição potencial do estudo está relacionada não somente à comparação com amostras mais amplas do trabalho de Ferreira et al. de 2002, mas também com a identificação e análise das razões pelas quais se atribui valor inferior aos desempenhos acadêmicos de determinados alunos da graduação em Contabilidade no específico contexto socioeconômico focado por esta pesquisa. Com base no fato de que tanto o desempenho acadêmico quanto suas atribuições causais são importantes no desenvolvimento do indivíduo e que a consciência de tais associações, em certas populações, tende a melhorar as ações dos participantes, este estudo investiga dados empíricos para coletar evidências de como alunos da graduação em alguns dos melhores cursos de ciências contábeis brasileiros percebem as causas de seus próprios desempenhos e dos de seus colegas. Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 1 INTRODUÇÃO Reconhecendo o debate intelectual sobre o desempenho acadêmico, a auto-consciência sobre a instrução obtida e a real contribuição que esses fatores podem oferecer ao processo educacional como um todo, o propósito deste estudo está diretamente ligado à investigação da potencial associação, com base na autoavaliação de alunos da graduação em Contabilidade, entre desempenho acadêmico e conhecidos elementos atributivos de acordo com a literatura. Este estudo visa responder à seguinte questão de pesquisa: Até que ponto os alunos dos cursos de graduação em Ciências Contábeis, de universidades pertencentes ao topo do ranking nacional, tendem a associar o seu desempenho acadêmico com fatores internos? Assim, o objetivo geral deste estudo exploratório quantitativo é investigar a existência de associação entre elementos atributivos comuns na literatura e o desempenho acadêmico de alunos da graduação em ciências contábeis. Além desse objetivo principal, um conjunto de objetivos mais específicos desta pesquisa consiste em identificar as atribuições dos alunos ao desempenho acadêmico (alunos da graduação em ciências contábeis de quatro grandes universidades brasileiras) e analisar tais atribuições, utilizando múltiplas perspectivas de 5 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... controle, todas contextualizadas em um específico ambiente socioeconômico. Busca-se, ainda, obter evidências com relação à hipótese de tal associação e, ao fazê-lo, contribuir com uma potencial explicação para o fenômeno, no específico contexto socioeconômico do ensino superior. Este estudo apóia-se no fato de que a maneira como os alunos compreendem e raciocinam sobre seus desempenhos acadêmicos, qualificando-os como superior ou inferior, tende a ser associada a elementos atributivos comuns. Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 2 REVISÃO DA LITERATURA Os trabalhos seminais de Rotter (1954) e Heider (1958), que tratam respectivamente do conceito de locus de controle (interno e externo), e da ideia de que as pessoas tendem a atribuir seus desempenhos a elementos causadores internos ou externos, dependendo das circunstâncias, dão suporte a essa investigação. Locus refere-se à localização da causa no próprio indivíduo (interno) ou na situação (externo). O conceito de locus de controle de Rotter (1954) está relacionado à ideia da predisposição, ligada à percepção do que causa a gratificação ou a punição para o indivíduo. Tais discussões ainda são muito atuais na literatura como na análise crítica da ideia do locus de controle apresentada por Kormanik e Rocco (2009). Ao discutir a ideia das percepções de causalidade, Weiner (1976, p. 179) enfatizou que a teoria da atribuição “está preocupada com (...) razões percebidas para a ocorrência de um determinado evento.” A teoria da atribuição trata, sobretudo, do desempenho (WEINER, 1974, 1976, 1980, 1986). Estimulado pelos avanços na psicologia social da educação, Weiner (1976) expandiu o estudo da associação entre desempenho educacional e fatores específicos (explicações causais), de acordo com a natureza do desempenho (sucesso ou fracasso) e as características dos fatores. De acordo com os preceitos da teoria da atribuição (WEINER, 1974, 1976, 1980, 1986), os principais fatores (explicações causais) são habilidade, esforço, dificuldade da tarefa e sorte. A teoria da atribuição, também, leva em consideração três principais dimensões ao classificar tais fatores: locus de controle (interno ou externo); estabilidade (estável ou instável); e controle (controlável ou incontrolável). Considera-se que a atribuição é um processo de três estágios: observação de comportamento, deliberação e atribuição. De acordo com a teoria da atribuição, as pessoas tendem a interpretar os fatores de forma a apresentar uma autoimagem positiva (WEINER, 1976). Em outras palavras, existe uma tendência a associar sucesso acadêmico a fatores internos enquanto os elementos que levam ao fracasso acadêmico tendem a ser associados a fatores externos. 6 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... Perspectiva individual pode, também, constituir um viés na atribuição de causas a um desempenho. Birnberg, Luft, e Shields (2007, p. Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 2 REVISÃO DA LITERATURA 53, maio/agosto 2010 7 7 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... de estudantes mais avançados no curso, os resultados mostraram que o sucesso depende apenas do esforço e da capacidade. Em outro estudo, Powers e Rossman (1984) analisaram as atribuições para o sucesso e o fracasso entre 399 estudantes universitários caucasianos, negros, hispânicos e nativos americanos, considerando: a capacidade, o esforço, o contexto e a sorte. Os resultados apontaram que os quatro grupos foram semelhantes em suas atribuições de sucesso tanto por causas externas (contexto e sorte) como por causas internas (capacidade e esforço). Na atribuição do fracasso, negros e nativos americanos apresentaram uma estrutura similar, com fortes cargas, indicando as atribuições do insucesso à falta de esforço. Hispânicos e caucasianos apresentaram um forte componente indicando atribuições de fracasso ao contexto em que estavam inseridos, que também levaria a fracasso futuro. Os estudos sobre a teoria da atribuição relacionados ao desempenho acadêmico ainda são incipientes, apesar da relevância da atividade teórica e empírica na literatura desde a década de 1950, passando por um maior avanço na década de 1970. As investigações já realizadas foram conduzidas em contextos sociais e culturais diversos e têm priorizado situações experimentais, havendo poucas referências na literatura a estudos voltados especificamente para o desempenho do aluno do ensino superior. 2 REVISÃO DA LITERATURA 120) exploram as diferentes maneiras que as pessoas adotam ao apontar as causas de seus comportamentos quando discutem o assunto na abordagem da Contabilidade Gerencial: “muitos estudos descobriram que uma pessoa tende a atribuir seu comportamento a causas mais externas, enquanto outras pessoas tendem a atribuir o mesmo comportamento a causas mais internas; o que é chamado de viés do agente observador.” Assim, a atribuição não é objetiva e outros elementos devem ser considerados. Weiner (1976) destaca a dimensão da influência cultural relacionada à motivação e ao julgamento dos desempenhos que pode impactar na maneira como a atribuição, ou seja, a percepção da causalidade venha a acontecer. Uma dessas questões, em particular, envolve características gerais do cenário do ensino superior no Brasil em que estão inseridos os cursos de ciências contábeis (MEC, 2003): cursos noturnos; alunos com empregos de tempo integral; dificuldades na preparação das aulas (leituras e tarefas extras); proporção de professores lecionando em meio expediente; proporção de professores desprovidos de diploma de mestrado ou doutorado e infraestrutura ruim. Porém, a característica mais importante desse cenário é o fato de que a maioria (71,7%) dos aproximadamente quatro milhões de alunos atualmente matriculados no sistema brasileiro de educação superior estuda em instituições privadas (MEC, 2005). Além das particularidades mencionadas anteriormente, o número pequeno de cursos de pós-graduação em ciências contábeis (CAPES, 2010) no Brasil (apenas 4 programas de doutoramento e 19 de mestrado) representa um obstáculo para a adequada expansão do sistema com o nível desejado de qualidade. Portanto, ações que visam não somente ao conteúdo dos cursos de ciências contábeis, mas, também, ao formato do ensino e do processo de aprendizagem, como os observados neste estudo, são desejáveis. De fato, como elas tendem a influenciar o processo, pode-se dizer que são essenciais (HUGHES, 2002; LADD; RUBY, 1999; ROTHWELL; KAZANAS, 2004; TUCKER, 1998;). Algumas pesquisas podem ser mencionadas sobre a aplicação da teoria da atribuição à análise do desempenho acadêmico. Griffin et al. (1983) investigaram a atribuição de fatores de desempenho com 114 estudantes de matemática, no nível introdutório do curso. O estudo considerou outros fatores além dos tradicionais (desempenho do professor, interesse no curso e aprendizado anterior do estudante). Os estudantes bem sucedidos apontaram como atribuição de fatores: o desempenho dos professores, o esforço e a capacidade. Com o grupo Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 3.1 Hipóteses e Questões da Pesquisa Especialmente relevante para este estudo é o ambiente socioeconômico e cultural em que o ensino superior acontece. A discussão sobre o raciocínio que está por trás de uma determinada cadeia de eventos, envolvendo decisões múltiplas (tais como o desempenho acadêmico), tende a estar diretamente relacionada ao contexto socioeconômico. Com o forte estímulo das observações empíricas e do trabalho de Ferreira et al. (2002), em que alunos do ensino médio do Brasil, da Argentina e do México e suas respectivas atribuições causais percebidas sobre sucesso acadêmico foram estudadas, este estudo explora mais profundamente as evidências coletadas de alunos de Contabilidade em quatro universidades brasileiras. Além da busca da resposta à questão de pesquisa já apresentada neste estudo (até que ponto os alunos dos cursos de graduação em ciências contábeis de universidades brasileiras tendem a associar o seu próprio desempenho acadêmico superior a fatores internos?), também são exploradas outras dimensões relacionadas ao problema como, por 8 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... exemplo, gênero dos alunos (masculino ou feminino), desempenho dos colegas e desempenho em outros ambientes educacionais (distintos dos ambientes dos respondentes). As dezesseis principais hipóteses orientadoras da pesquisa, essenciais para este estudo e estimuladas pela revisão da literatura, são apresentadas a seguir. Apesar da teoria da atribuição ter três dimensões principais, essas hipóteses são todas deliberadamente baseadas na dimensão do locus de controle (interno e externo). Isso se deve à natureza dos eventos explorados e ao interesse de comparabilidade ao trabalho de Ferreira et al. (2002). O primeiro conjunto de hipóteses concentra-se no desempenho acadêmico. Mais especificamente, testes foram estabelecidos para ajudar a investigar as prováveis associações de desempenho acadêmico com o gênero, turno do curso do aluno e idade. Assim, tem-se: H1: o desempenho acadêmico, baseado na autoavaliação, está associado ao gênero; H2: o desempenho acadêmico superior, baseado na autoavaliação, está associado ao gênero; H3: o desempenho acadêmico inferior, baseado na autoavaliação, está associado ao gênero; H4: o desempenho acadêmico, baseado na autoavaliação, está associado ao turno do curso e H5: existe uma significativa diferença na idade relacionada ao desempenho acadêmico, baseado na autoavaliação. O segundo conjunto, com cinco hipóteses adicionais, concentra-se na atribuição de fatores usados para explicar o desempenho acadêmico e como os indivíduos tendem a expressar suas razões para desempenhos acadêmicos específicos. Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 3.1 Hipóteses e Questões da Pesquisa H13: a atribuição de fatores usados para explicar desempenho acadêmico superior, com base na autoavaliação, está associada ao tipo de custeio do ensino médio (público ou privado). na autoavaliação, está associada ao tipo de custeio do ensino médio (público ou privado). H14: a atribuição de fatores usados para explicar o desempenho acadêmico inferior, com base na autoavaliação, está associada ao tipo de custeio do ensino médio (público ou privado). H15: a atribuição de fatores usados para explicar o desempenho acadêmico superior, com base na autoavaliação, está associada à percepção de elemento de destaque da universidade atual. H16: a atribuição de fatores usados para explicar o desempenho acadêmico inferior, com base na autoavaliação, está associada à percepção de elemento de destaque da universidade atual. 3.1 Hipóteses e Questões da Pesquisa Testes foram estabelecidos para ajudar a comparar as proporções da atribuição de fatores, usando combinações de valores mínimos de desempenho acadêmico superior ou inferior e as proporções obtidas de Ferreira et al. (2002), a partir das seguintes hipóteses: H6: as proporções de atribuição de fatores usadas para explicar o desempenho acadêmico, com base na autoavaliação, são significativamente diferentes para desempenhos acadêmicos superiores e inferiores; H7: para o próprio desempenho acadêmico, a proporção de causas externas é significativamente mais alta em desempenhos acadêmicos inferiores que em desempenhos acadêmicos superiores. H7: para o próprio desempenho acadêmico, a proporção de causas externas é significativamente mais alta em desempenhos acadêmicos inferiores que em desempenhos acadêmicos superiores. H8: para o desempenho acadêmico dos colegas, a proporção de causas externas é significativamente mais alta em desempenhos acadêmicos inferiores que em desempenhos acadêmicos superiores. H8: para o desempenho acadêmico dos colegas, a proporção de causas externas é significativamente mais alta em desempenhos acadêmicos inferiores que em desempenhos acadêmicos superiores. H9: para o desempenho acadêmico de alunos de outras instituições de ensino superior, a proporção de causas externas é significativamente mais alta em desempenhos acadêmicos inferiores que em desempenhos acadêmicos superiores. 9 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... H10: todas as proporções (desempenho próprio, desempenho do colega, desempenho de alunos de outras instituições) de atribuição de fatores usadas para explicar o desempenho acadêmico, com base na autoavaliação, encontradas neste estudo são significativamente diferentes daquelas encontradas por Ferreira et al. (2002). O último conjunto, com mais seis hipóteses, concentra-se na atribuição de fatores usados para explicar o desempenho acadêmico e variáveis específicas de controle. Testes foram estabelecidos para ajudar a comparar as proporções de atribuição de fatores, envolvendo três variáveis: a região geográfica da universidade, o custeio do ensino médio (público ou privado), a percepção de elemento de destaque da universidade atual do aluno. H11: a atribuição de fatores usados para explicar o desempenho acadêmico superior, com base na autoavaliação, está associada à região geográfica da universidade. H11: a atribuição de fatores usados para explicar o desempenho acadêmico superior, com base na autoavaliação, está associada à região geográfica da universidade. H12: a atribuição de fatores usados para explicar o desempenho acadêmico inferior, com base na autoavaliação, está associada à região geográfica da universidade. Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 3.2 Caracterização do estudo Essa é uma pesquisa não experimental, com propósito descritivo e comparativo de causas, desenvolvida à luz do modelo exploratório (GALL; GALL; BORG, 2003). Este estudo empírico envolve pesquisa exploratória, devido aos fatos descritos anteriormente e à limitada pesquisa sobre a teoria da atribuição no ensino da Contabilidade e, também, se concentra no apoio de mais iniciativas nesse campo de conhecimento. De acordo com Creswell (2003, p. 7), “não podemos ser positivos em nossas afirmações de conhecimento ao estudarmos o comportamento e as ações humanas.” Porém, as descobertas com base em sólidos propósitos de pesquisa podem ser interpretadas. A abordagem desta pesquisa envolve a aquisição de conhecimento adequado sobre um particular objeto que carece de informação suficiente (SELLTIZ et al., 1959). 10 1 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... Este estudo é baseado em uma mensuração direta (REA; PARKER, 1997), envolvendo um conjunto de técnicas para coletar, registrar, compilar e analisar dados, dando suporte à interpretação quantitativa dos fatos. Usa-se uma abordagem quantitativa enquanto investiga os alunos com um instrumento válido. Ainda baseado no modelo proposto por Creswell (2003), a pesquisa é a principal estratégia de investigação utilizada neste estudo, que tem uma orientação quantitativa. Além disso, a revisão da literatura sustenta a análise dos relatórios de pesquisas já conduzidas, fornecendo o alicerce necessário para este estudo. Estatísticas descritivas, estudos e testes de correlação e comparação de médias e proporções foram conduzidos com a finalidade de atingir os objetivos. Cálculos e testes estatísticos foram desenvolvidos com SPSS®, adotando o nível de significância 0,05 (α = 0,05). Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 3.3 Protocolo de pesquisa, amostra e banco de dados O protocolo de pesquisa foi inspirado naquele usado por Ferreira et al. (2002). Primeiro foram selecionados os Estados e suas respectivas instituições universitárias. Contatos foram feitos em nível administrativo com os coordenadores/gestores dos cursos de ciências contábeis. Depois do contato inicial, turmas específicas nos cursos foram selecionadas com base na estratégia definida pelos pesquisadores para eliminar os alunos que estivessem cursando o primeiro e o último ano acadêmico (calouros e formandos não poderiam fazer parte da amostra), suprimindo o efeito da experiência e do desempenho dos componentes dos períodos extremos dos cursos, nos resultados da pesquisa. O conteúdo do instrumento foi baseado em Ferreira et al. (2002), mas inclui adaptações feitas para o cenário específico da pesquisa. Testes piloto foram conduzidos com representantes de cada instituição de ensino superior selecionadas e, em benefício da compreensão, pequenas adaptações foram feitas tanto na linguagem quanto no formato. Considerando o contexto educacional, cultural, e socioeconômico do estudo, não foram enfrentados maiores desafios em validade e confiabilidade do instrumento; baseando-se em aplicações anteriores do instrumento no contexto brasileiro e apoiado pelos testes piloto. A amostra, neste estudo, foi obtida pelo método não probabilístico, empregando a amostragem intencional devido tanto ao acesso quanto aos propósitos do estudo. Selltiz et al. (1959, pp. 514-5) mencionam que “na amostragem não-probabilística, não há como estimar a probabilidade que cada elemento tem de estar incluído na amostra e nem como assegurar que cada elemento tenha alguma chance de ser incluído”. Dados de quatro universidades em quatro diferentes Estados brasileiros (Ceará, São Paulo, Minas Gerais e Rio Grande do Sul) foram coletados durante o segundo semestre 11 1 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... acadêmico de 2009. As instituições foram selecionadas intencionalmente devido às suas características especialmente relevantes para este estudo, resultando em amostras convenientes. Essa amostra incluiu 826 alunos da graduação em ciências contábeis, formalmente matriculados (após excluir questionários inválidos devido a falhas no preenchimento ou ausência de respostas). Algumas análises não contemplaram o total de 826 alunos, pois nem todos os respondentes preencheram integralmente o questionário. Alunos do primeiro e do último ano não foram estudados. Todos os alunos pesquisados participaram de forma voluntária e assinaram um termo de consentimento, de acordo com os procedimentos de pesquisa usuais exigidos pelas instituições envolvidas. Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 3.3 Protocolo de pesquisa, amostra e banco de dados Os alunos responderam a um instrumento de coleta de dados que consistia de 20 itens com informação geral sobre o participante e 4 itens sobre a atribuição do desempenho acadêmico. As variáveis que medem o primeiro conjunto de perguntas são: G1 (código de identificação), G2 (turno do curso – noturno ou diurno), G3 (estágio no curso – semestre acadêmico), G4 (gênero), G5 (conclusão de outro curso de graduação), G6 (idade), G7 (ensino médio – público ou privado), G8 (situação profissional), G9 (função profissional relacionada à Contabilidade), G10 (experiência em estágios), G11 (envolvimento em projetos de pesquisa), G12 (experiência na atividade de monitoria), G13 (experiência docente), G14 (nível de experiência docente – ensino fundamental/médio ou superior), G15 (nível de escolaridade dos pais), G16 (renda familiar) G17 (tempo de estudo semanal, além do tempo em sala de aula), G18 (uso de computador pessoal para estudar), G19 (nível de reconhecimento da qualidade da sua universidade atual), G20 (elemento de destaque da sua universidade atual). As quatro variantes relacionadas ao desempenho acadêmico e a atribuição dos fatores são: P1 (desempenho acadêmico – superior ou inferior), P2 (atribuição do próprio desempenho acadêmico), P3 (atribuição do desempenho acadêmico dos colegas), P4 (atribuição do desempenho acadêmico de alunos de outras instituições de ensino superior). As opções de atribuição de fatores apresentadas aos alunos foram: esforço próprio, capacidade ou inteligência, ajuda da família, facilidade das provas, ajuda dos colegas, nível dos professores. Ferreira et al. (2002) usaram três fontes principais para análise: esforço, capacidade e causas externas. As causas externas foram distribuídas em quatro elementos, mas, em alguns testes, as variáveis relacionadas às causas externas foram processadas juntamente. O mesmo procedimento foi adotado nesta pesquisa. Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 12 12 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 4.1 Estatística descritiva Foram 826 os questionários com respostas válidas, representando 78% dos que foram aplicados, com predominância do gênero feminino, conforme demonstrado na Tabela 1. Tabela 1: Respondentes por estado e gênero Estado Masculino Feminino Total CE 166 147 313 MG 122 83 205 SP 47 32 79 RS 65 164 229 Total 400 426 826 A média de idade da amostra foi de 24,2 anos (σ = 5,58) com diferença significativa entre os gêneros (t (820) = 3,80, p < 0,001): 25,0 (masculino) e 23,5 (feminino). Essa média de idade acima de 24 anos representa uma característica do contexto socioeconômico e cultural deste estudo. Cerca de 8% da amostra foi constituída por alunos que estão fazendo sua segunda graduação. Quanto ao tipo de instituição cursada no ensino médio, a amostra apresentou-se equilibrada, com 52% dos estudantes provenientes de escolas privadas. É importante ressaltar que, no Brasil, devido à natureza competitiva do sistema de ingresso ao ensino superior público (vestibular), cursar o ensino médio em uma instituição particular tende a aumentar a probabilidade de admissão. A maioria (76%) dos estudantes pesquisados relatou estar trabalhando no momento da coleta de dados, principalmente exercendo funções relacionadas à Contabilidade (48% dos 76% da amostra). A maioria (51%) deles já foi ou é estagiária e poucos relataram experiências com projetos de pesquisa (6%) ou de atividades de monitoria (3%). Essa evidência pode ajudar a compreender as dimensões socioeconômicas e culturais do sistema de ensino superior brasileiro na área das ciências contábeis, principalmente nas regiões urbanas com alta densidade demográfica. Quanto à carga horária de estudo, 66% dos entrevistados responderam dedicar até 4 horas por semana aos estudos (além do período em sala de aula), com estudantes do gênero feminino indicando menos (60%) que os do gênero masculino (72%). Quando perguntados se usavam computador próprio (ou da família) para propósitos de estudo, apenas 4% responderam negativamente. Quase todos os alunos (96%) relataram percepções de qualidade em suas respectivas instituições. Além disso, quando estimulados a destacar o item responsável por essa qualidade, as respostas mais frequentes foram: corpo docente (63%), estrutura física (22%) e estrutura administrativa (7%). Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 13 1 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 4.2 Testes relacionados ao desempenho acadêmico Tabela 4: Gênero e desempenho inferior Fatores Masculino Feminino Total Esforço Próprio 119 90 209 Capacidade/Inteligência 2 7 9 Ajuda da Família 7 2 9 Facilidade das Provas 17 21 38 Ajuda dos Colegas 2 0 2 Nível dos Professores 11 8 19 Total 158 128 286 Obs.: Não significativas ao nível estabelecido (Pearson qui-quadrado). Tabela 4: Gênero e desempenho inferior Fatores Masculino Feminino Total Esforço Próprio 119 90 209 Capacidade/Inteligência 2 7 9 Ajuda da Família 7 2 9 Facilidade das Provas 17 21 38 Ajuda dos Colegas 2 0 2 Nível dos Professores 11 8 19 Total 158 128 286 Obs.: Não significativas ao nível estabelecido (Pearson qui-quadrado). Detalhes sobre como os alunos atribuem fatores a seu próprio desempenho acadêmico estão evidenciados na Tabela 5. Os resultados globais mostram que para 68% (366 de 537) dos alunos com desempenho acadêmico superior o fator mais indicado foi o seu próprio esforço. Para os alunos com desempenho acadêmico inferior observam-se frequências muito menores de capacidade/inteligência (de 22% para 3%), com frequências mais elevadas atribuídas às causas externas, ou seja, a família, provas, colegas e professores (de 10% para 24% – ver Tabela 6 para um resumo dos resultados), e frequências ligeiramente superiores para o próprio esforço (73%). Com relação às causas externas, cabe salientar que, embora seja possível observar um aumento global das suas frequências quando se trata de desempenho acadêmico inferior, o elemento ajuda dos colegas mostra uma diminuição significativa na sua frequência (de 4% para 0,7%) como fator explicativo a esse desempenho. Tabela 5: Atribuição de desempenho acadêmico Fatores Inferior Superior Total Esforço Próprio 209 366 575 Capacidade/Inteligência 9 120 129 Ajuda da Família 9 1 10 Facilidade das Provas 38 9 47 Ajuda dos Colegas 2 22 24 Nível dos Professores 19 19 38 Total 286 537 823 Tabela 5: Atribuição de desempenho acadêmico Fatores Inferior Superior Total Esforço Próprio 209 366 575 Capacidade/Inteligência 9 120 129 Ajuda da Família 9 1 10 Facilidade das Provas 38 9 47 Ajuda dos Colegas 2 22 24 Nível dos Professores 19 19 38 Total 286 537 823 Aspectos relacionados ao turno do curso também faziam parte do conjunto de dados. Aspectos relacionados ao turno do curso também faziam parte do conjunto de dados. Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 4.2 Testes relacionados ao desempenho acadêmico Considerando-se a segunda parte do questionário, o desempenho acadêmico e os fatores específicos que conduzem a esse desempenho, 65% dos inquiridos (χ2 (1, N = 824) = 75,85, p < 0,001) se autoavaliaram como tendo sucesso acadêmico (a definição operacional foi estar entre os 30% melhores alunos da classe). Foi encontrada uma diferença de gênero interessante na percepção relatada de sucesso acadêmico (59% dos homens e 70% das mulheres respondentes). A Tabela 2 apresenta a tabulação cruzada entre o gênero e o desempenho acadêmico, apresentando evidência de que existe uma relação estatisticamente significativa entre gênero e desempenho acadêmico (χ2 (1, N = 824) = 10,39, p < 0,001). Com base nesses achados, aceita-se a hipótese de que o desempenho acadêmico, baseado na autoavaliação, está associado ao gênero (H1).. Tabela 2: Gênero e desempenho acadêmico Desempenho Masculino Feminino Total Superior 238 299 537 Inferior 161 126 287 Total 399 425 824 Obs.:Significativo ao nível estabelecido (Pearson qui-quadrado). Tabela 2: Gênero e desempenho acadêmico Desempenho Masculino Feminino Total Superior 238 299 537 Inferior 161 126 287 Total 399 425 824 Obs.:Significativo ao nível estabelecido (Pearson qui-quadrado). Ao explorar os fatores atribuídos ao desempenho acadêmico, primeiro foram avaliados aqueles associados ao desempenho acadêmico superior. A Tabela 3 contém as frequências por gênero (tabulação cruzada). Tabela 3: Gênero e desempenho superior Fatores Masculino Feminino Total Esforço Próprio 139 227 366 Capacidade/Inteligência 73 47 120 Ajuda da Família 1 0 1 Facilidade das Provas 8 1 9 Ajuda dos Colegas 10 12 22 Nível dos Professores 7 12 19 Total 238 299 537 Obs.:Significativo ao nível estabelecido (Pearson qui-quadrado). É notável que Pearson qui-quadrado indique resultados estatisticamente significativos de correlação entre gênero e desempenho acadêmico superior (χ2 (5, N = 537) = 28,17, p < 0,001). Também, foram explorados os fatores associados ao desempenho acadêmico inferior, de maneira semelhante, e os resultados estão presentes na Tabela 4. Nesse caso, os resultados não indicam qualquer relação significativa entre gênero e desempenho acadêmico inferior (χ2 (5, N = 286) = 9,43, p =.093) Com base nesses resultados, aceita-se a hipótese H2 e rejeita-se 14 1 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... a hipótese H3, ou seja, apenas o desempenho acadêmico superior, baseado na autoavaliação, está relacionado ao gênero. 4.2 Testes relacionados ao desempenho acadêmico No entanto, os testes realizados para verificar sua correlação com o desempenho acadêmico, com base na autoavaliação não produziram evidências (χ2 (1, N = 822) = 2,05, p = .152). Com base nos resultados dos testes realizados, a hipótese H4 foi rejeitada. Resultados semelhantes foram encontrados quando se procurou relacionar as diferenças na idade com o desempenho Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 15 1 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... acadêmico (t (818) = -1,54, p = .122). Assim, a hipótese H5 foi rejeitada: não há diferença significativa na idade que esteja relacionada ao desempenho acadêmico, com base na autoavaliação. Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 4.3 Testes relacionados à atribuição de fatores Um teste qui-quadrado goodness-of-fit, não paramétrico, para comparar as proporções de atribuição de fatores ao desempenho acadêmico superior e as proporções de atribuição ao desempenho acadêmico inferior, mostrou que os dois grupos são significativamente diferentes (χ2 (5, N = 537) = 796,7, p < 0,001). Teste similar (invertendo os conjuntos de dados) com proporções de desempenho inferior em relação às proporções existentes de desempenho acadêmico superior produziu resultados semelhantes: (χ2 (5, N = 286) = 428,7, p < 0,001). Em outras palavras, desempenho acadêmico superior e inferior estão relacionados a diferentes combinações de fatores, aceitando-se, assim, a hipótese H6. Os autores do estudo anteriormente citado (Ferreira et al., 2002), que discutiram a teoria da atribuição utilizando estudantes brasileiros, com uma amostra distinta (alunos do ensino médio), chegaram a resultados interessantes e uma comparação direta é apresentada nas Tabelas 6, 8 e 10. Tabela 6: Desempenho acadêmico (esforço próprio): comparação com Ferreira et al. (2002) Fatores Estudo Atual Ferreira et al. (2002) Inferior (%) Superior (%) Total Inferior (%) Superior (%) Total Esforço Próprio 209 (73%) 366 (68%) 575 136 (61%) 124 (48%) 260 Capacidade/Inteligência 9 (3%) 120 (22%) 129 3 (1%) 84 (32%) 87 Causas Externas 68 (24%) 51 (10%) 38 84 (38%) 51 (20%) 135 Total 286 537 823 223 259 482 Com o intuito de analisar cada elemento (esforço, capacidade e causas externas), individualmente, foi utilizado um teste binomial não paramétrico para comparar os resultados encontrados para desempenho acadêmico superior (Tabelas 6, 8 e 10) a partir das proporções deste estudo e do estudo de Ferreira et al. (2002), respectivamente, para seu próprio desempenho, desempenho de colegas de classe e desempenho de alunos de outras instituições. Praticamente, todas as proporções individuais quando comparadas (teste binomial, qui- quadrado) ao desempenho acadêmico deste estudo apresentaram-se significativamente diferentes (Tabelas 7, 9 e 11). Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 16 1 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... Tabela 7: Resultados do Teste Binomial (p-values) para esforço próprio: comparado ao desempenho acadêmico superior Estudo Atual Ferreira et al. (2002) Fatores Inferior Superior Inferior Superior Esforço Próprio Menor* - Maior* Maior* Capacidade/Inteligência Maior* - Maior* Menor* Causas Externas Menor* - Menor* Menor* Diferenças significativas ao nível estabelecido. Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 4.3 Testes relacionados à atribuição de fatores 53, maio/agosto 2010 17 1 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... Cabe destacar que, ao se manifestar sobre o desempenho de colegas e de estudantes de outras instituições, os respondentes foram solicitados a indicar tanto causas internas como externas para desempenho inferior ou superior, razão pela qual o somatório dessas duas colunas nas Tabelas 8 e 10 é superior ao número de respondentes observado na Tabela 6. Tabela 11: Resultados do Teste Binomial (p-values) para desempenho de estudantes de outras instituições: comparado ao desempenho acadêmico superior Estudo Atual Ferreira et al. (2002) Fatores Inferior Superior Inferior Superior Esforço Próprio Menor - Menor* Menor* Capacidade/Inteligência Diferença não Significativa - Maior* Maior* Causas Externas Maior* - Maior* Menor* Diferenças significativas ao nível estabelecido. 1: Resultados do Teste Binomial (p-values) para desempenho de estudantes de outras instituições: comparado ao desempenho acadêmico superior Todas essas relações são, significativamente, diferentes daquelas relatadas por Ferreira et al. (2002), com exceção do desempenho de colegas de classe (Tabela 9) em termos de esforço (χ2 (1, N = 762), p = 0,157), e relacionadas ao desempenho dos estudantes de outras instituições (Tabela 11), em termos de capacidade/inteligência (χ2 (1, N = 752), p = 0,146), rejeitando-se a hipótese H10. 4.3 Testes relacionados à atribuição de fatores Ao considerar seu próprio desempenho (Tabelas 6 e 7) ou o desempenho de colegas de classe (Tabelas 8 e 9), os resultados evidenciaram maior frequência de causas externas relacionadas ao desempenho inferior, alinhado com o estudo de Ferreira et al. (2002), ou seja, quando apresentam as causas para desempenho acadêmico inferior, os estudantes dessa amostra (por atribuição) relataram maior frequência de causas externas. Com base nas evidências encontradas, aceitam-se ambas as hipóteses H7 (χ2 (1, N = 537), p < 0,001) e H8 (χ2 (1, N = 762), p <0,001). Tabela 8: Desempenho acadêmico (colegas de classe): comparação com Ferreira et al. (2002) Fatores Estudo Atual Ferreira et al. (2002) Inferior (%) Superior (%) Total Geral (%) Esforço Próprio 527 (73%) 470 (62%) 997 310 (63%) Capacidade/Inteligência 28 (4%) 191 (25%) 219 77 (16%) Causas Externas 163 (23%) 101 (13%) 264 101 (21%) Total 718 762 1,480 488 Tabela 8: Desempenho acadêmico (colegas de classe): comparação com Ferreira et al. (2002) Fatores Estudo Atual Ferreira et al. (2002) Tabela 9: Resultados do Teste Binomial (p-values) para desempenho de colegas: comparado ao desempenho acadêmico superior Estudo Atual Ferreira et al. (2002) Fatores Inferior Superior Geral Esforço Próprio Menor - Capacidade/Inteligência Maior* - Causas Externas Menor* - Diferença não Significativa Maior* Menor* *Diferenças significativas ao nível estabelecido Tabela 9: Resultados do Teste Binomial (p-values) para desempenho de colegas: comparado ao desempenho acadêmico superior Tabela 10: Desempenho acadêmico (estudantes de outras instituições): comparação com Ferreira et al. (2002) Fatores Estudo Atual Ferreira et al. (2002) Inferior (%) Superior (%) Total Inferior (%) Superior (%) Total Esforço Próprio 437 (59%) 394 (53%) 831 149 (66%) 156 (61%) 305 Capacidade/Inteligência 96 (13%) 108 (14%) 204 13 (6%) 9 (3%) 22 Causas Externas 203 (28%) 250 (33%) 453 65 (28%) 93 (36%) 158 Total 736 752 1,488 227 258 485 bela 10: Desempenho acadêmico (estudantes de outras instituições): comparação com Ferreira et al. (2002) No entanto, quando questionados sobre os fatores que explicariam o desempenho dos alunos de outras instituições, os resultados apontaram em uma direção oposta: desempenho acadêmico superior relacionado com maior frequência a causas externas (Tabelas 10 e 11) e desempenho acadêmico inferior atribuído, principalmente, ao esforço próprio. Com base nessas conclusões, rejeita-se a hipótese H9 (χ2 (1, N = 752), p < 0,001). Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 4.4 Testes de Atribuição Relacionados às Variáveis de Controle Um conjunto de seis testes visando à verificação da correlação da atribuição de fatores usados para explicar o desempenho acadêmico (superior ou inferior) e variáveis de controle específicas (região geográfica da universidade, o financiamento público ou privado do ensino médio e a percepção do elemento de destaque da universidade) geraram mais evidências para apoiar a reflexão sobre o tema e as especificidades relacionadas com o contexto socioeconômico e cultural, nas localidades em que o estudo se realizou. Os resultados desses seis testes são apresentados a seguir. Considerando que foram coletados dados de quatro diferentes estados brasileiros e, por causa de aspectos socioeconômicos e culturais internos, considerou-se relevante explorar a atribuição de fatores utilizados para explicar o desempenho acadêmico (superior e inferior), controlados por essas regiões geográficas. Os resultados indicaram que a região geográfica da universidade está associada aos fatores usados para explicar o desempenho acadêmico superior (χ2 (15, N = 366) = 36,51, p = .001). No entanto, não está relacionada quando o foco é o desempenho acadêmico inferior (χ2 (15, N = 209) = 18,59, p = .233). Assim, com base nessas constatações, aceita-se a hipótese H11 e rejeita-se a hipótese H12. Devido ao processo altamente competitivo de entrar nas melhores universidades do ranking no Brasil, é particularmente importante dar atenção extra para a variável de 18 1 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... financiamento do ensino médio (público ou privado). Testou-se tal variável associada com os fatores usados para explicar o desempenho acadêmico superior (χ2 (5, N = 521) = 9,51, p = .090) e inferior (χ2 (5, N = 278) = 7,94, p = ,159). Em ambos os casos, não foram encontradas evidências estatísticas para suportar tal relação. Com base nessas conclusões, rejeitam-se as hipóteses H13 e H14. Para verificar se a forma como os alunos percebem elementos de destaque em suas universidades teve algum papel na maneira como os fatores foram atribuídos ao seu desempenho acadêmico superior ou inferior, foram realizados dois testes adicionais. Constatou-se que tal variável não se encontrava associada aos fatores utilizados para explicar o desempenho acadêmico superior (χ2 (20, N = 531) = 30,47, p = .063), mas estava relacionada aos fatores usados para explicar o desempenho acadêmico inferior (χ2 (20, N = 283) = 39,12, p = .006). 4.4 Testes de Atribuição Relacionados às Variáveis de Controle H14: a atribuição de fatores usados para explicar o desempenho acadêmico inferior, com base na autoavaliação, está associada ao tipo de custeio do ensino médio (público ou privado) Rejeitada H15: a atribuição de fatores usados para explicar o desempenho acadêmico superior, com base na autoavaliação, está associada à percepção de elemento de destaque da universidade atual Rejeitada H16: a atribuição de fatores usados para explicar o desempenho acadêmico inferior, com base na autoavaliação, está associada à percepção de elemento de destaque da universidade atual Aceita H14: a atribuição de fatores usados para explicar o desempenho acadêmico inferior, com base na autoavaliação, está associada ao tipo de custeio do ensino médio (público ou privado) Rejeitada H15: a atribuição de fatores usados para explicar o desempenho acadêmico superior, com base na autoavaliação, está associada à percepção de elemento de destaque da universidade atual Rejeitada H16: a atribuição de fatores usados para explicar o desempenho acadêmico inferior, com base na autoavaliação, está associada à percepção de elemento de destaque da universidade atual Aceita Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 4.4 Testes de Atribuição Relacionados às Variáveis de Controle Assim, com base nesses resultados, rejeita-se a hipótese H15 e aceita- se a hipótese H16. Considerando os testes aplicados e as análises apresentadas, o Quadro 1 mostra um resumo do resultado das hipóteses levantadas neste estudo. Quadro 1: Resultado das hipóteses da pesquisa HIPÓTESE RESULTADO H1: o desempenho acadêmico, baseado na autoavaliação, está associado ao gênero Aceita H2: o desempenho acadêmico superior, baseado na autoavaliação, está associado ao gênero Aceita H3: o desempenho acadêmico inferior, baseado na autoavaliação, está associado ao gênero Rejeitada H4: o desempenho acadêmico, baseado na autoavaliação, está associado ao turno do curso Rejeitada H5: existe uma significativa diferença na idade relacionada ao desempenho acadêmico, baseado na autoavaliação Rejeitada H6: as proporções de atribuição de fatores usadas para explicar o desempenho acadêmico, com base na autoavaliação são significativamente diferentes para desempenhos acadêmicos superiores e inferiores Aceita H7: para o próprio desempenho acadêmico, a proporção de causas externas é significativamente mais alta em desempenhos acadêmicos inferiores que em desempenhos acadêmicos superiores Aceita H8: para o desempenho acadêmico dos colegas, a proporção de causas externas é significativamente mais alta em desempenhos acadêmicos inferiores que em desempenhos acadêmicos superiores Aceita H9: para o desempenho acadêmico de alunos de outras instituições de ensino superior, a proporção de causas externas é significativamente mais alta em desempenhos acadêmicos inferiores que em desempenhos acadêmicos superiores Rejeitada H10: todas as proporções (desempenho próprio, desempenho do colega, desempenho de alunos de outras instituições) de atribuição de fatores usadas para explicar o desempenho acadêmico, com base na autoavaliação, encontradas neste estudo são significativamente diferentes daquelas encontradas por Ferreira et al. (2002) Rejeitada H11: a atribuição de fatores usados para explicar o desempenho acadêmico superior, com base na autoavaliação, está associada à região geográfica da universidade Aceita H12: a atribuição de fatores usados para explicar o desempenho acadêmico inferior, com base na autoavaliação, está associada à região geográfica da universidade Rejeitada H13: a atribuição de fatores usados para explicar desempenho acadêmico superior, com base na autoavaliação, está associada ao tipo de custeio do ensino médio (público ou privado) Rejeitada Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 19 19 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... 5 CONCLUSÃO Após a revisão da literatura e toda a análise que sustentou este estudo, informações relevantes e evidências foram recolhidas para contribuir com uma melhor compreensão do problema de educação em Contabilidade orientado por esta pesquisa. Reconhecendo as limitações naturais do método empregado e dos procedimentos de amostragem, os resultados dos testes de hipóteses desenvolvidos neste trabalho, com foco em quatro dos principais cursos de graduação em Contabilidade de universidades brasileiras, são apresentados e discutidos, a seguir, à luz da literatura relacionada. É certo que a questão de pesquisa utilizada na condução deste estudo pôde ser adequadamente tratada: Até que ponto os alunos dos cursos de graduação em Ciências Contábeis, de universidades pertencentes ao topo do ranking nacional, tendem a associar o seu desempenho acadêmico superior com fatores internos? Em geral, os sujeitos relataram que o esforço próprio (68%) e a capacidade/inteligência (22%), juntos, representaram 90% do seu desempenho acadêmico superior. Apenas 10% das atribuições de fatores (causalidade) foram relacionadas a fatores externos, conforme relatado por eles, apesar de 63% dos alunos constatarem que o corpo docente é um dos itens de destaque da qualidade de sua instituição. Proporção expressiva de fatores internos foi usada para explicar o desempenho acadêmico superior. Interessante ressaltar que estudantes do gênero feminino relataram proporções significativamente maiores (91,6%) de fatores internos, quando comparados aos estudantes do gênero masculino (89%), ao explicar seu desempenho acadêmico superior. Nesse caso específico, o estereótipo de gênero, discutido por Jackson et al. (1987), poderia ser uma potencial explicação de tal comportamento, como uma reação esperada do sexo feminino para a conquista histórica de igualdade entre os sexos. Além disso, descobriu-se que muito mais estudantes do sexo feminino se autoavaliaram como possuidores de desempenho acadêmico superior (70%), quando comparados com seus colegas do sexo masculino (59%). Essas constatações relacionadas ao gênero, em particular, necessitam ser mais exploradas por estudos futuros. Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 20 2 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... Além disso, os estudantes relataram uma proporção muito maior (24%) de fatores externos ao explicar o desempenho acadêmico inferior. Assim, com base nos achados a partir desta amostra, o desempenho acadêmico superior é mais atribuído a causas internas que o desempenho acadêmico inferior, com uma interessante idiossincrasia entre os gêneros. Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 5 CONCLUSÃO 53, maio/agosto 2010 21 2 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... Parece que a componente social lida com a atribuição de um papel nessa situação específica, porque quando o foco é definido para um ambiente distante (ou seja, alunos de outras escolas), houve uma tendência para diminuir a atribuição do sucesso a fatores internos e aumentar a sua atribuição a fatores externos. Estima, orgulho, sentimento de grupo, ou uma abordagem corporativa, podem ser os elementos que interagem com esse comportamento. Certamente, essa questão requer uma investigação mais aprofundada, especialmente em ambientes socioeconômicos e culturais similares. Todas as proporções de atribuição de fatores a partir deste estudo, quando comparados com os resultados (assumidos como limiares) relatados por Ferreira et al. (2002), são significativamente diferentes, exceto para o esforço no desempenho dos colegas de classe e capacidade no desempenho dos alunos de outras instituições (H10). Assim, parece plausível esperar evolução ou transformação, em termos da forma como os indivíduos pensam sobre a atribuição, dependendo de fatores como a maturidade e o ambiente social. Essa é outra questão importante para estudos adicionais nessa área. A partir do terceiro conjunto de hipóteses constatou-se uma associação entre atribuição de fatores ligados ao desempenho acadêmico superior e a região geográfica da universidade (H11). Não foi encontrada associação entre atribuição de fatores ligados ao desempenho acadêmico inferior e região geográfica da universidade (H12). Além disso, nenhuma relação foi encontrada quando a variável de controle foi o financiamento do ensino médio (H13 e H14). Ao controlar a percepção do elemento de destaque da universidade, não foi constatada associação com atribuição de fatores ligados ao desempenho acadêmico superior (H15), mas foi encontrada relação com a atribuição de fatores ligados ao desempenho acadêmico inferior (H16). Esta evidência, especialmente a partir de H15, parece corroborar a ideia da influência dos aspectos sociais sobre a forma como um indivíduo pode enfrentar atribuição de desempenho acadêmico. Por outro lado, esse achado sobre a forma como se percebe elementos de destaque da instituição (H16), e a associação com atribuição de fatores ligados ao desempenho acadêmico inferior, pode ser outra evidência de que causas externas estão sendo usadas para explicar o desempenho acadêmico inferior, como comentado anteriormente. 5 CONCLUSÃO Esses resultados gerais estão bem alinhados com a principal literatura sobre o tema, e bem relacionados com a teoria da atribuição proposta por Weiner (1976). Ao abordar cada conjunto de hipóteses, foi possível construir elementos para essa conclusão. O primeiro conjunto de hipóteses relacionadas ao desempenho acadêmico, baseado na autoavaliação, constatou associações significativas entre o desempenho acadêmico geral e o gênero (H1) e entre o desempenho acadêmico superior e o gênero (H2). Não foram encontradas associações do desempenho acadêmico inferior com o gênero (H3). Nenhuma correlação significativa foi encontrada entre o desempenho acadêmico e o turno do curso (H4) ou idade (H5). A maneira como a pessoa tende a perceber e autoavaliar o próprio desempenho acadêmico parece estar presa às raízes mais profundas das estruturas sociais e quadros individuais de referência. Conforme a literatura, a atribuição é considerada como um processo em três estágios: observação do comportamento, deliberação e atribuição. Segundo a teoria da avaliação, as pessoas tendem a interpretar os fatores de modo a apresentar uma autoimagem positiva (WEINER, 1976). Existe uma relação entre as atribuições e a forma como o indivíduo é percebido. Assim, mesmo com um constructo objetivo de desempenho acadêmico superior, como utilizado neste estudo (entre os 30% melhores alunos da classe), muitas interpretações e equívocos podem ocorrer. Afinal, o desempenho acadêmico pode envolver conhecimentos, atitudes e habilidades presentes em múltiplas dimensões como a cognitiva, afetiva e a psicomotora, para citar apenas três. A importância de um aluno em particular assumir o controle de seu próprio estudo e se tornar mais responsável tanto pelos seus sucessos quanto pelos seus fracassos, pode ser muito relativo, com grandes diferenças quando comparado a outros estudantes. O segundo conjunto de hipóteses produziu resultados interessantes relacionados às proporções de atribuição de fatores usadas para explicar o desempenho acadêmico, com base na autoavaliação. Primeiro, as proporções dos fatores usados para explicar o desempenho acadêmico inferior e o superior são significativamente diferentes (H6). A proporção de causas externas explicando o desempenho acadêmico inferior foi maior para o desempenho acadêmico próprio (H7) e desempenho acadêmico de colegas de classe (H8). No entanto, quando definido o foco para os alunos de outras instituições, os resultados foram invertidos: maior proporção de causas externas para explicar o desempenho acadêmico superior (H9). Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. Revista Contabilidade & Finanças, USP, São Paulo, v. 21, n. 53, maio/agosto 2010 5 CONCLUSÃO Concluindo à luz da concepção pedagógica moderna e cobertura das abordagens empregadas na educação de Contabilidade, bem como as características específicas da nova geração de estudantes que vem para a universidade (ou estagiários provenientes de organizações), gestores, professores, tutores e muitos outros atores terão que adaptar as suas estratégias com a finalidade de direcionar melhor tais questões. Não só os aspectos de atribuição relacionados ao gênero, que podem estar presos a estruturas e raízes sociais mais 22 2 O bom é meu, o ruim é seu: perspectivas da teoria da atribuição sobre o desempenho acadêmico de alunos da graduação em... profundas, mas o elemento natural de atribuição de sucesso e fracasso dentro de abordagens baseadas no construtivismo, centrada no participante, colaborativa orientada, para apoiar uma experiência educacional ampla. Atribuição de fatores e resultados meta cognitivos de manipulação consciente pelos estudantes, no seu sentido estrito, tendem a impulsionar e inflamar as ações dos participantes no sentido de uma experiência de aprendizagem transformadora e de longa duração. Isto parece estar bem alinhado com a reforma tão necessária no ensino da Contabilidade, no sentido de uma maior orientação para o pensamento crítico, supostamente para melhor preparar o contador para a dinâmica dessa profissão. Nesse sentido, a investigação precisa continuar, com ênfase na integração desse tipo de divulgação de informações relacionadas com o desempenho dos alunos com abordagens transversais e longitudinais. Estudos adicionais em múltiplos contextos sobre as instituições de ensino superior em Ciências Contábeis devem ser conduzidos. Além disso, uma comparação dos resultados com base no instrumento utilizado neste estudo forneceria ajuda adicional. 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https://openalex.org/W4377989097	https://medbio.ejournal.by/jour/article/download/296/252	Russian	null	Analysis of the epidemiological characteristics of multiple myeloma and clinical factors affecting the course of the disease	Mediko-biologičeskie problemy žiznedeâtelʹnosti	2,023	cc-by	5,575	Учредитель А.В. Рожко (д.м.н., профессор) Редакционная коллегия В.С. Аверин (д.б.н., профессор, зам. гл. редактора), В.В. Аничкин (д.м.н., профессор), В.Н. Беляковский (д.м.н., профессор), К.Н. Буздалкин (к.т.н., доцент), Н.Г. Власова (д.б.н., профессор, научный редактор), А.В. Величко (к.м.н., доцент), И.В. Веялкин (к.б.н., доцент), А.В. Воропаева (к.б.н., доцент), Д.И. Гавриленко (к.м.н.), М.О. Досина (к.б.н., доцент), А.В. Жарикова (к.м.н.), С.В. Зыблева (к.м.н., доцент, отв. секретарь), С.А. Игумнов (д.м.н., профессор), А.В. Коротаев (к.м.н., доцент), Д.В. Кравченко (к.м.н.), А.Н. Лызиков (д.м.н., профессор), А.В. Макарчик (к.м.н., доцент), С.Б. Мельнов (д.б.н., профессор), В.М. Мицура (д.м.н., доцент), Я.Л. Навменова (к.м.н., доцент), Э.А. Надыров (к.м.н., доцент), И.А. Новикова (д.м.н., профессор), Э.Н. Платошкин (к.м.н., доцент), Э.А. Повелица (к.м.н.), А.С. Подгорная (к.м.н.), Ю.И. Рожко (к.м.н., доцент), И.П. Ромашевская (к.м.н.), М.Г. Русаленко (к.м.н., доцент), А.П. Саливончик (к.б.н.), А.Е. Силин (к.б.н., доцент), А.Н. Стожаров (д.б.н., профессор), И.О. Стома (д.м.н., профессор), Н.И. Шевченко (к.б.н., доцент), Ю.И. Ярец (к.м.н., доцент) У ред е Государственное учреждение «Республиканский научно- практический центр радиационной медицины и экологии человека» Журнал включен в Пере- чень научных изданий Респу- блики Беларусь для опублико- вания диссертационных ис- следований по медицинской и биологической отраслям науки (31.12.2009, протокол 25/1) Журнал зарегистрирован Министерством информации Республики Беларусь, Свид. № 762 от 6.11.2009 Журнал зарегистрирован Министерством информации Республики Беларусь, Свид. № 762 от 6.11.2009 Редакционный совет А.В. Аклеев (д.м.н., профессор, Челябинск), О.В. Алейникова (д.м.н., чл.-кор. НАН РБ, Минск), С.С. Алексанин (д.м.н., профессор, Санкт-Петербург), Д.А. Базыка (д.м.н., профессор, Киев), А.П. Бирюков (д.м.н., профессор, Москва), Е.Л. Богдан (Минск), Л.А. Бокерия (д.м.н., академик РАН и РАМН, Москва), А.Ю. Бушманов (д.м.н., профессор, Москва), И.И. Дедов (д.м.н., академик РАМН, Москва), В.И. Жарко (Минск), М.П. Захарченко (д.м.н., профессор, Санкт-Петербург), Л.А. Ильин (д.м.н., академик РАМН, Москва), К.В. Котенко (д.м.н., профессор, Москва), В.Ю. Кравцов (д.б.н., профессор, Санкт-Петербург), Е.Н. Кроткова (к.м.н., доцент, Минск), Н.Г. Кручинский (д.м.н., профессор, Пинск), Т.В. Мохорт (д.м.н., профессор, Минск), Д.Л. Пиневич (МЗ РБ, Минск), В.Ю. Рыбников (д.м.н., профессор, Санкт-Петербург), Н.Д. Тронько (д.м.н., чл.-кор. НАН, акад. НАМН Украины, Киев), А.Л. Усс (д.м.н., профессор, Минск), В.А. Филонюк (д.м.н., профессор, Минск), Р.А. Часнойть (к.э.н., Минск), В.Д. Шило (Минск) А.В. Аклеев (д.м.н., профессор, Челябинск), О.В. Алейникова (д.м.н., чл.-кор. НАН РБ, Минск), С.С. Алексанин (д.м.н., профессор, Санкт-Петербург), Д.А. Базыка (д.м.н., профессор, Киев), А.П. Бирюков (д.м.н., профессор, Москва), Е.Л. Богдан (Минск), Л.А. Бокерия (д.м.н., академик РАН и РАМН, Москва), А.Ю. Бушманов (д.м.н., профессор, Москва), И.И. Дедов (д.м.н., академик РАМН, Москва), В.И. Жарко (Минск), М.П. Захарченко (д.м.н., профессор, Санкт-Петербург), Л.А. Ильин (д.м.н., академик РАМН, Москва), К.В. Котенко (д.м.н., профессор, Москва), В.Ю. Кравцов (д.б.н., профессор, Санкт-Петербург), Е.Н. Кроткова (к.м.н., доцент, Минск), Н.Г. Кручинский (д.м.н., профессор, Пинск), Т.В. Мохорт (д.м.н., профессор, Минск), Д.Л. Пиневич (МЗ РБ, Минск), В.Ю. Рыбников (д.м.н., профессор, Санкт-Петербург), Н.Д. Тронько (д.м.н., чл.-кор. НАН, акад. НАМН Украины, Киев), А.Л. Усс (д.м.н., профессор, Минск), В.А. Филонюк (д.м.н., профессор, Минск), Р.А. Часнойть (к.э.н., Минск), В.Д. Шило (Минск) Подписано в печать 30.04.23 Формат 60×90/8. Бумага мелованная. Гарнитура «Times New Roman». Печать цифровая. Тираж 130 экз. Усл. печ. л. 15,5. Уч.-изд. л. 9,7. Зак. 165. Медико-биологические проблемы жизнедеятельности Научно-практический рецензируемый журнал № 1(29) 2023 г. Технический редактор С.Н. Никонович Отпечатано в КУП «Редакция газеты «Гомельская праўда» г. Гомель, ул. Полесская, 17а Адрес редакции 246040 г. Гомель, ул. Ильича, д. 290, ГУ «РНПЦ РМ и ЭЧ», редакция журнала тел (0232) 38-95-00, факс (0232) 37-80-97 http://www.mbp.rcrm.by e-mail: mbp@rcrm.by © Государственное учреждение «Республиканский научно-практический центр радиационной медицины и экологии человека», 2023 ISSN 2074-2088 Medical and Biological Problems of Life Activity Scientific and Practical Journal © Republican Research Centre for Radiation Medicine and Human Ecology ISSN 2074-2088 Обзоры и проблемные статьи N.D. Puzan, I.A. Cheshik Molecular mechanisms of effects of ion- izing radiation action. Irradiation effect on protein (literary review) Н.Д. Пузан, И.А. Чешик Молекулярные механизмы действия ионизирующего излучения. Влияние облучения на белок (обзор литературы) 14 Медико-биологические проблемы Али Адиб Хуссейн Али, О.Е. Кузнецов Элементный состав тканей в норме и при ожирении у крыс линии Wistar Е.К. Нилова, К.Н. Буздалкин Методы экспресс-оценки радиацион- ной обстановки с применением мо- бильной лаборатории в чрезвычайных ситуациях Медико-биологические проблемы Али Адиб Хуссейн Али, О.Е. Кузнецов Элементный состав тканей в норме и при ожирении у крыс линии Wistar Е.К. Нилова, К.Н. Буздалкин Методы экспресс-оценки радиацион- ной обстановки с применением мо- бильной лаборатории в чрезвычайных ситуациях Medical-biological problems A.A.H. Ali, O.E. Kuznetsov Elemental composition of tissues in nor- mal and obese Wistar rats E.K. Nilova, K.N. Buzdalkin Methods for express assessment of the radiation situation using a mobile labora- tory in emergency situations 27 Е.К. Нилова, К.Н. Буздалкин Методы экспресс-оценки радиацион- ной обстановки с применением мо- бильной лаборатории в чрезвычайных ситуациях 35 O.V. Shakhovskaya, M.N. Starodubtseva, A.А. Miadzvedzveva Characteristics of radiosensitivity of or- ganisms using parameters of redox prop- erties of blood plasma О.В. Шаховская, М.Н. Стародубцева, Е.А. Медведева Характеристика радиочувствительно- сти организмов с помощью параме- тров редокс-свойств плазмы крови O.V. Shakhovskaya, M.N. Starodubtseva, A.А. Miadzvedzveva Characteristics of radiosensitivity of or- ganisms using parameters of redox prop- erties of blood plasma 43 Scientific and Practical Journal ISSN 2074-2088 © Republican Research Centre for Radiation Medicine and Human Ecology Founder Republican Research Centre for Radiation Medicine and Human Ecology Journal registration by the Ministry of information of Republic of Belarus Certificate № 762 of 6.11.2009 Founder Republican Research Centre for Radiation Medicine and Human Ecology Journal registration by the Ministry of information of Republic of Belarus Certificate № 762 of 6.11.2009 Content Содержание Reviews and problem articles Клиническая медицина Clinical medicine A.Yu. Zaharko, T.V. Statkevich, A.S. Podgor- naya, O.V. Murashko Risk factors for arterial hypertension in women with abdominal obesity and hy- pertensive disorders of pregnancy in the history A.Yu. Zaharko, T.V. Statkevich, A.S. Podgor- naya, O.V. Murashko Risk factors for arterial hypertension in women with abdominal obesity and hy- pertensive disorders of pregnancy in the history А.Ю. Захарко, Т.В. Статкевич, А.С. Под- горная, О.В. Мурашко Факторы риска артериальной гипер- тензии у женщин с абдоминальным ожирением и гипертензивными рас- стройствами беременности в анамнезе 49 Zh.M. Kozich, V.N. Martinkov, I.V. Veyalkin, J.N. Pugacheva, D.A. Blizin, N.N. Klimkovich Analysis of the epidemiological charac- teristics of multiple myeloma and clinical factors affecting the course of the disease Ж.М. Козич, В.Н. Мартинков, И.В. Веялкин, Ж.Н. Пугачева, Д.А. Близин, Н.Н. Климкович Анализ эпидемиологических показате- лей множественной миеломы и клини- ческих факторов, влияющих на тече- ние заболевания Zh.M. Kozich, V.N. Martinkov, I.V. Veyalkin, J.N. Pugacheva, D.A. Blizin, N.N. Klimkovich Analysis of the epidemiological charac- teristics of multiple myeloma and clinical factors affecting the course of the disease 55 Content Содержание A.V. Korotaev, A.M. Pristrom, E.P. Naumen- ko, S.N. Korzheva, L.F. Larenko, Ya.L. Nav- menova Changes in the biomechanics of contrac- tility of the myocardium of the left ventri- cle: results of prospective follow-up А.В. Коротаев, А.М. Пристром, Е.П. На- уменко, С.Н. Коржева, Л.Ф. Ларенко, Я.Л. Навменова Изменения биомеханики контрактиль- ности миокарда левого желудочка: ре- зультаты проспективного динамиче- ского наблюдения 62 D. Novik, V. Martinkov, I. Veyalkin, I. Isk- rov, A. Silin, T. Rachkova, N. Vasilevskaya, M. Bobyrev, Zh. Merenkova, L. Navaro, A. Uryupin, A. Uss D. Novik, V. Martinkov, I. Veyalkin, I. Isk- rov, A. Silin, T. Rachkova, N. Vasilevskaya, M. Bobyrev, Zh. Merenkova, L. Navaro, A. Uryupin, A. Uss Regional features of the incidence of chronic Ph-negative myeloproliferative neoplasms in Belarus Д.К. Новик, В.Н. Мартинков, И.В. Веял- кин, И.А. Искров, А.Е. Силин, Т.А. Рач- кова, Н.Ф. Василевская, М.А. Бобырев, Ж.Н. Меренкова, Л.Л. Наваро, А.С. Урю- пин, А.Л. Усс Региональные особенности первич- ной заболеваемости хроническими Ph- негативными миелопролиферативны- ми заболеваниями в Беларуси 67 75 А.С. Подгорная, А.Ю. Захарко, О.В. Му- рашко, К.В. Бронская Миомэктомия: хирургическая тактика, репродуктивные исходы 82 1ГУ «РНПЦ радиационной медицины и экологии человека», Гомель, Беларусь 2УО «Белорусская медицинская академия последипломного образования», Минск, Беларусь Множественная миелома (ММ) является вторым наиболее распространенным злока- чественным новообразованием системы кроветворения. За последнее десятилетие значи- тельно улучшилась выживаемость пациентов на фоне применения новых терапевтических стратегий. Отсутствие факторов риска на момент постановки диагноза не всегда определя- ет благоприятное течение заболевания. Поэтому актуальным является поиск дополнитель- ных прогностических факторов. В статье представлены результаты анализа эпидемиологи- ческих данных и клинико-гематологических характеристик ММ, связанных с беспрогрес- сивной выживаемостью. Установлено, что значимое превышение уровней IL2, IL6, TNF и количество клональных CD138+ (>20%) на момент постановки диагноза были связаны с увеличением частоты прогрессии заболевания за период исследования. Подтверждена взаимосвязь повреждения почек, анемического синдрома, повышенного уровня СРБ, мно- жественных поражений костей скелета, присутствия генетических изменений на момент постановки диагноза со снижением беспрогрессивной выживаемости пациентов с ММ. Ключевые слова: множественная миелома, заболеваемость, прогностические факторы Но, несмотря на достижения последних лет в лечении с использованием таргетной и CART- терапии, ММ в большинстве слу- чаев остается неизлечимым заболеванием. Наличие факторов, связанных с пациен- том, таких как возраст, молекулярно-ге- нетические изменения, выявление очагов экстрамедуллярного поражения, стадия, ответ на проводимую терапию, часто опре- деляют прогноз заболевания [4]. В насто- ящее время существует множество про- гностических систем, одной из которых является модель риска ISS, включающая уровни альбумина и β2-микроглобулина. Модель риска R-ISS была улучшена за счет включения двух хорошо известных про- гностических биомаркеров, связанных с более высокой пролиферативной активно- стью: цитогенетических аномалий (t(4;14), t(14;16) и del(17/17p)) и уровня лактатдеги- дрогеназы (ЛДГ) в сыворотке [5]. Ю.И. Ярец 89 Н.В. Холупко, Е.Н. Ващенко, Я.Л. Навменова, М. Вишам, А.Е.Филюстин, А.В. Коротаев, Е.Н. Холупко, В.А.Журавлев, М.Г. Русаленко АКТГ-синдром: трудности диагностики 99 Experience exchange E.V. Dorofei Attitude of teenagers living in the super- vision zone of the Belarusian NPP to ra- diation safety Обмен опытом Content Содержание Н.Г. Кадочкина, Е.В. Родина, А.П. Сали- вончик, Д.И. Гавриленко Клинический случай: кардиальный синкопе у пожилой пациентки В.С. Смирнов, А.О. Жарикова, О.И. Анан- ченко, О.И. Дудузова, А.В. Жарикова Энцефалит Расмуссена (обзор и кли- нический случай) N.G. Kadochkina, Е.V. Rodzina, А.P. Sali vontchik, D.I. Haurylenka Cardiac syncope in an elderly patient: a clinical case from practice V.S. Smirnov, А.О. Zharikova, О.I. Ananchen- ko, О.I. Duduzova, А.V. Zharikova Rasmussen’s encephalitis (review and clinical case) Н.Г. Кадочкина, Е.В. Родина, А.П. Сали- вончик, Д.И. Гавриленко Клинический случай: кардиальный синкопе у пожилой пациентки В.С. Смирнов, А.О. Жарикова, О.И. Анан- ченко, О.И. Дудузова, А.В. Жарикова Энцефалит Расмуссена (обзор и кли- нический случай) N.G. Kadochkina, Е.V. Rodzina, А.P. Sali vontchik, D.I. Haurylenka Cardiac syncope in an elderly patient: a clinical case from practice V.S. Smirnov, А.О. Zharikova, О.I. Ananchen- ko, О.I. Duduzova, А.V. Zharikova Rasmussen’s encephalitis (review and clinical case) 110 116 Клиническая медицина Ж.М. Козич1, В.Н. Мартинков1, И.В. Веялкин1, Ж.Н. Пугачева1, Д.А. Близин1, Н.Н. Климкович2 УДК 616-006.448-036.22:616.15-07(476.2) DOI: 10.58708/2074-2088.2023-1(29)-55-61 УДК 616-006.448-036.22:616.15-07(476.2) DOI: 10.58708/2074-2088.2023-1(29)-55-61 Введение Клинически наиболее значимым заболе- ванием плазматических клеток является мно- жественная миелома (ММ). Это гетерогенное заболевание с различными исходами и пока- зателями выживаемости от нескольких меся- цев до десяти и более лет. На долю ММ при- ходится 10% от всех злокачественных гема- тологических новообразований [1]. В основе развития ММ лежат различные генетические аномалии, сложные взаимодействия микро- окружения костного мозга посредством ци- токинов и хемокинов [2]. Диагноз симптома- тической ММ основан на выявлении более 10% клональных плазматических клеток и присутствии одного из CRAB-критериев (ги- перкальциемия, поражение почек, анемия, деструкции костной ткани) [3]. В последние годы общая выживаемость пациентов ММ значительно увеличилась. 55 Медико-биологические проблемы жизнедеятельности. 2023. № 1(29) Ж.М. Козич, В.Н. Мартинков, И.В. Веялкин и др. Ж.М. Козич, В.Н. Мартинков, И.В. Веялкин и др. симптоматической ММ из Гомельского региона Беларуси за период с февраля 2018г. по июнь 2022г. Медиана возрас- та пациентов составила 64,0 года (58,0 и 70,0). Диагноз ММ установлен согласно критериям, разработанным Междуна- родной рабочей группой по миеломе [6]. Из исследования исключены пациенты с тяжелой соматической патологией, кото- рым не представлялась возможность на момент постановки диагноза выполнить диагностическое исследование (биопсию крыла подвздошной кости). Отсутствие неблагоприятных факто- ров на момент постановки диагноза не всегда определяет положительный про- гноз. Рецидив заболевания может развить- ся на фоне проведения стандартной хими- отерапии уже спустя 6-12 месяцев. Поэто- му необходимы дальнейшие исследования для выявления дополнительных прогно- стических факторов на первичном этапе диагностики. Комплексное исследование прогностических факторов при ММ имеет важное клиническое значение, поскольку может помочь врачам оценить состояние пациента, а применение новых стратегий в диагностике и лечении помогут улучшить качество жизни пациентов ММ. Кроме того, важной задачей является проведение эпидемиологических исследований с воз- можностью использования результатов для определения потенциальных факторов ри- ска и планирования бюджетных затрат на оказание медицинской помощи пациентам. р ) Все пациенты, включенные в исследо- вание, получали терапию согласно прото- колу лечения ММ бортезомиб-содержащи- ми препаратами. Пациентам старше 65 лет (n=67) проводились курсы химиотерапии (ХТ) по схемам VCD, VD или VMP, в за- висимости от соматического состояния [7]. Пациентами подписано информиро- ванное согласие на участие в исследова- нии. Всем пациентам проведены клини- ко-лабораторное исследования, выполнена аспирационная биопсия и биопсия крыла подвздошной кости с иммунофенотипиче- ским (ИФТ) исследованием костного моз- га. Из лучевых методов диагностики ис- пользовали низкодозовую компьютерную томографию (КТ) всего тела и диффузи- онно-взвешенную магнитно-резонансную томографию (МРТ) всего тела. Все пациенты, включенные в исследо- вание, получали терапию согласно прото- колу лечения ММ бортезомиб-содержащи- ми препаратами. Введение Пациентам старше 65 лет (n=67) проводились курсы химиотерапии (ХТ) по схемам VCD, VD или VMP, в за- висимости от соматического состояния [7]. Пациентами подписано информиро- ванное согласие на участие в исследова- нии. Всем пациентам проведены клини- ко-лабораторное исследования, выполнена аспирационная биопсия и биопсия крыла подвздошной кости с иммунофенотипиче- ским (ИФТ) исследованием костного моз- га. Из лучевых методов диагностики ис- пользовали низкодозовую компьютерную томографию (КТ) всего тела и диффузи- онно-взвешенную магнитно-резонансную томографию (МРТ) всего тела. Цель: изучить эпидемиологические характеристики и клинические прогности- ческие факторы при ММ, связанные с те- чением заболевания, у пациентов Гомель- ского региона. Результаты исследования За период с 2011 по 2020 годы в г. Го- мель и Гомельской области Республики Беларусь зарегистрированы 500 пациен- тов с плазмоклеточными новообразовани- ями – 232 мужчины (46,4%) и 268 женщин (53,6%). В структуре заболеваемости ПН первое место занимала ММ – 454 случая, из них 207 мужчин (45,6%) и 247 женщин (54,4%). Грубый интенсивный показатель первичной заболеваемости ММ за рассма- триваемый период составил 3,2 (2,9-3,5) на 100 000 населения. На рисунке 2 приведена динамика стан- дартизованных по возрасту показателей заболеваемости ММ (ASR) в Гомельской области с 2011 по 2020 гг., она приблизи- тельно совпадает с динамикой грубых по- казателей. При анализе повозрастных показателей заболеваемости ММ за изучаемый период установлено, что заболеваемость женского населения постепенно нарастала с возрас- том и достигла максимальных значений в возрастных группах 65-69, 70-74 и 75-79 лет, где она составила соответственно 13,6 (10,12-17,87), 12,8 (9,07-17,44) и 11,8 (8,23- 16,27) на 100 000 населения в год. На рисунке 1 приведена динамика гру- бых показателей заболеваемости MM в Го- мельской области за период с 2011 по 2020 гг. На рисунке 1 приведена динамика гру- бых показателей заболеваемости MM в Го- мельской области за период с 2011 по 2020 гг. Количество ежегодно регистрируемых первичных пациентов с ММ за указанный период находилось в пределах 33-58, что соответствовало заболеваемости от 2,39 (1,64-3,36) до 4,05 (3,07-5,23) на 100 000 населения. Количество ежегодно регистрируемых первичных пациентов с ММ за указанный период находилось в пределах 33-58, что соответствовало заболеваемости от 2,39 (1,64-3,36) до 4,05 (3,07-5,23) на 100 000 населения. Заболеваемость мужского населения также увеличивалась с возрастом, но мак- симальные значения отмечались в возраст- ных группах 70-74 года, 75-79 лет и 80-84 года, где показатель заболеваемости соста- вил соответственно 15,8 (10,22-23,32), 14,2 (8,42-22,45) и 16,4 (8,72-28) на 100 000 на- селения в год. Заболеваемость женского населения за период 2011-2020 гг. составила 3,26 (2,86- 3,69) и была выше, чем мужского – 3,14 (2,73-3,60) на 100 000 населения (различия статистически не значимы). На рисунке 1 заметен подъем заболеваемости среди женщин в 2016 г. до 4,74 (3,32-6,57), тогда как максимальная заболеваемость мужчин зарегистрирована в 2011 г. – 4,21 (2,8-6,09) на 100 000 населения. Для обоих полов заболеваемость до- стигала наибольших значений в возраст- ных группах 65-69 лет, 70-74 года и 75- 79 лет, где она составила соответственно 13,4 (10,71-16,67), 13,8 (10,62-17,61) и 12,5 (9,37-16,27) на 100 000 населения в год с последующим снижением в более старших возрастных группах (рисунок 3). Материал и методы исследования Для анализа первичной заболеваемо- сти ММ (С90.0) по Гомельскому региону в работе использованы данные Белорус- ского республиканского канцер-регистра за период с 2011 г по 2020 г. Эпидемио- логический анализ впервые выявленных случаев заболевания ММ проводился путем определения грубых интенсивных (CR), повозрастных (AsR) и стандарти- зованных по возрасту (ASR) показателей, а также среднегодовых темпов прироста показателей (AAPC). Стандартизация по возрасту проводилась прямым методом с использованием World Standard (Segi, 1960). Оценка показателей проводилась с использованием величины 95% довери- тельного интервала (95%ДИ). Прогрессирование заболевания у па- циентов устанавливалось при выявлении биохимического рецидива, новых очагов деструкций, плазмоцитом и/или инфиль- трации костного мозга опухолевыми ПК [8]. Статистическую обработку результа- тов проводили с использованием пакета прикладных программ Statistica 6.1. При описании количественных признаков ука- зывали медиану (25-й и 75-й процентили). Различия в уровнях анализируемых по- казателей между группами определяли с использованием критерия Манна-Уитни. Сравнение частот изучаемых признаков выполняли с применением критерия Хи- квадрат. В качестве уровня статистической значимости выбрано значение p=0,05. Прогрессирование заболевания у па- циентов устанавливалось при выявлении биохимического рецидива, новых очагов деструкций, плазмоцитом и/или инфиль- трации костного мозга опухолевыми ПК [8]. Статистическую обработку результа- тов проводили с использованием пакета прикладных программ Statistica 6.1. При описании количественных признаков ука- зывали медиану (25-й и 75-й процентили). Различия в уровнях анализируемых по- казателей между группами определяли с использованием критерия Манна-Уитни. Сравнение частот изучаемых признаков выполняли с применением критерия Хи- квадрат. В качестве уровня статистической значимости выбрано значение p=0,05. В группу исследования включены 139 пациентов с впервые выявленной Медико-биологические проблемы жизнедеятельности. 2023. № 1(29) 56 Клиническая медицина болеваемости ММ (мужчины, женщины и оба пола) на территории Гомельской обла- сти не выявлено. Среднегодовой темп при- роста ААРС составил -1,8% (-6,24-2,63%). Результаты исследования За рассматриваемый период статисти- чески значимого роста или снижения за- Медико-биологические проблемы жизнедеятельности. 2023. № 1(29) 57 Рисунок 1 – Динамика заболеваемости MM в Гомельской области за 2011-2020 гг. (грубые интенсивные показатели) Рисунок 2 – Стандартизованные по возрасту показатели заболеваемости (ASR) MM в Гомельской области за 2011-2020 гг. Рисунок 2 – Стандартизованные по возрасту показатели заболеваемости (ASR) MM в Гомельской области за 2011-2020 гг. Рисунок 1 – Динамика заболеваемости MM в Гомельской области за 2011-2020 гг. (грубые интенсивные показатели) Медико-биологические проблемы жизнедеятельности. 2023. № 1(29) 57 Ж.М. Козич, В.Н. Мартинков, И.В. Веялкин и др. Рисунок 3 – Повозрастные показатели заболеваемости MM в Гомельской области за 2011-2020 гг. (на 100 000 населения) мосвязь стадии заболевания с выживаемо- стью без прогрессии была статистически не значимой (p=0,09). Прогрессия заболевания за период иссле- дования с февраля 2018 г. по январь 2023 г. верифицирована у 56 (40,3%) пациентов из общей группы. Медиана времени под наблю- дением составила 41 месяц (95%ДИ [34,7- 47,3]), медиана времени до прогрессии – 16,0 месяцев (95%ДИ [12,8-19,1]). Рисунок 3 – Повозрастные показатели заболеваемости MM в Гомельской области за 2011-2020 гг. (на 100 000 населения) В группе исследования преобладали пациенты с секрецией иммуноглобулина G (IgG) (54,7%) и легких цепей иммуногло- булинов (19,4%), М-градиент ≥15 г/л вы- явлен в 57,3% случаев. По данным литера- туры ММ с секрецией IgА имеет худший прогноз, чем с секрецией IgG, так как чаще всего сочетается с t(4;14) [9]. В нашем ис- следовании мы не получили таких данных. Из 56 пациентов, спрогрессировавших в течение периода наблюдения, только 9 па- циентов (16%) имели секрецию IgА. Воз- можно, это связано с относительно не- большим количеством таких пациентов в нашем исследовании. В таблице 1 представлены клинико- лабораторные характеристики пациентов ММ Гомельского региона с учетом им- мунохимического варианта ММ, наличия CRAB-критериев, стадии по Durie-Salmon на момент диагностики. На момент постановки диагноза паци- енты чаще имели вторую стадию заболева- ния по Durie-Salmon. Медико-биологические проблемы жизнедеятельности. 2023. № 1(29) Результаты исследования В нашей работе взаи- Таблица 1 – Клинико-лабораторные характеристики пациентов Гомельского региона с ММ Характеристики ММ (n=139) Пол * мужчины женщины 68 / 48,9% 71 / 51,1% Иммунохимический тип: IgG 76 / 54,7% IgA 24 / 17,3% IgM 2 / 1,4% Легкие цепи иммуноглобулинов (κ,λ) 27 / 19,4% Несекретирующий 7 / 5,0% Сочетание двух иммуноглобулинов 3 / 2,2% Уровень кальция >2,75 ммоль/л 15 / 10,8% Уровень креатинина > 170 ммоль/л 23 / 16,5% Уровень гемоглобина <100 г/л 43 / 30,9% Наличие поражения костной ткани 122 / 87,8% Уровень ЛДГ, Ед/л > 280 Ед/л 13 / 9,4% М-градиент ≥15 г\л 67 / 48,2% β2-микроглобулин > 3 мг\л 96 / 69,6% Стадия ММ по Durie-Salmon: I 3/ 2,2% II 108 / 77,7% III 28 / 20,1% Таблица 1 – Клинико-лабораторные характеристики пациентов Гомельского региона с ММ Важное место в диагностике плазмокле- точных новообразований отводят методу им- мунофенотипического исследования костно- го мозга. При проведении данного исследо- вания выявлено, что у пациентов с уровнем CD138+ более 20% на момент постановки диагноза прогрессия заболевания за период наблюдения определена в 50% случаев, что в 1,52 раз чаще, чем в группе пациентов с CD138+ менее 20% (32,9% случаев), p=0,042, ОШ 2,04 (95%ДИ [1,02-4,06]). Цитогенетическое и молекулярно-гене- тическое исследование проведено у 54 па- циентов с ММ. Изменения были обнаруже- ны у 19 (35,2%) пациентов (таблица 2). Раз- личие в беспрогрессивной выживаемости у пациентов с наличием хромосомных абер- раций – 30,0 месяцев (95%ДИ [35,4-46,6]) в сравнении с пациентами без таковых – 41,0 месяцев (95%ДИ [4,3-55,7]) в нашем иссле- довании было не значимым (р= 0,319). Результаты исследования В нашем исследовании мутации (BRAF, KRAS, NRAS) выявлены у 4 (7,4%) паци- ентов, при этом беспрогрессивная выжи- 58 Клиническая медицина Таблица 2 – Хромосомные аберрации, выявленные у пациентов ММ Хромосомные аберрации ММ t (4;14) 4 / 7,4% Трисомия 16 3 / 5,6% Трисомия 14 1 / 1,85 Делеция 13q 7 / 12,9% Полиплоидия 1 / 1,85% t (14;16) 2 / 3,7% Делеция 17 1 / 1,85% Множественные нарушения 0 Таблица 2 – Хромосомные аберрации, выявленные у пациентов ММ Хромосомные аберрации ММ t (4;14) 4 / 7,4% Трисомия 16 3 / 5,6% Трисомия 14 1 / 1,85 Делеция 13q 7 / 12,9% Полиплоидия 1 / 1,85% t (14;16) 2 / 3,7% Делеция 17 1 / 1,85% Множественные нарушения 0 Множественные поражения костей скелета; Единичные очаги или диффузное поражение 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Время, дней 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 Выживаемость без прогрессии Рисунок 4 – Показатели беспрогрессивной выживаемости пациентов ММ в зависимости от поражения костей скелета М й 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Время, дней 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 Выживаемость без прогрессии Множественные поражения костей скелета; Единичные очаги или диффузное поражение ваемость у этих пациентов была значимо ниже в сравнении с пациентами без мутаций (p=0,03). По литературным данным мутации RAS связаны с плохим прогнозом, агрессив- ным течением, низкими показателями выжи- ваемости, а также доказано их участие в про- грессировании моноклональной гаммапатии неуточненного значения во MM [10]. ваемость у этих пациентов была значимо ниже в сравнении с пациентами без мутаций (p=0,03). По литературным данным мутации RAS связаны с плохим прогнозом, агрессив- ным течением, низкими показателями выжи- ваемости, а также доказано их участие в про- грессировании моноклональной гаммапатии неуточненного значения во MM [10]. Рисунок 4 – Показатели беспрогрессивной выживаемости пациентов ММ в зависимости от поражения костей скелета Рисунок 4 – Показатели беспрогрессивной выживаемости пациентов ММ в зависимости от поражения костей скелета нии не влияло на беспрогрессивную вы- живаемость (p=0,61). Однако обнаружение множественных поражений костей скелета было связано с худшим прогнозом в отно- шении беспрогрессивной выживаемости (р=0,048, Log-Rank Test, рисунок 4), что согласуется с данными литературы о вза- имосвязи количества очагов деструкции костной ткани с прогнозом [12]. При анализе уровней цитокинов в группе исследования выявлено значимое превышение нормальных значений IL2, IL6, IL8, TNF. Результаты исследования У пациентов с прогрессией заболевания, развившейся за период иссле- дования, при первичной диагностике вы- явлено значимое превышение уровней IL2, IL6, TNF в сыворотке крови в сравнении с пациентами без прогрессии (таблица 3). Это согласуется со сведениями о том, что при ММ цитокины способствуют росту, прогрессированию и распространению опухоли, а также доказано их участие в об- разовании деструкции костной ткани [11]. Пациенты с поражением почек в нашем исследовании прогрессировали в 1,59 раз чаще, чем пациенты без нарушения почеч- ной функции. Прогрессия в данной группе определена в 50% случаев, тогда как в груп- пе без поражения почек – в 31,5% случаев, p=0,026, ОШ 2,17 (95%ДИ [1,09-4,34]). Анемический синдром является наибо- лее частым осложнением ММ, часто выяв- ляется при постановке диагноза и развива- ется при прогрессировании [13]. В нашем исследовании у пациентов с гемоглобином менее 100 г/л на момент постановки диа- гноза было выявлено снижение беспрогрес- сивной выживаемости (p=0,05, рисунок 5), что согласуется с данными литературы [13]. По данным рентгенологического ис- следования костей скелета остеодеструк- ции выявлены в 87,8% случаев и были представлены вариантами от единичных очагов до множественных, с наличием очагов экстрамедуллярного поражения. Наличие очагов экстрамедуллярного по- ражения у пациентов в нашем исследова- Таблица 3 – Уровни интерлейкинов в сыворотке крови в зависимости от прогрессии ММ Цитокины ММ без прогрессии ММ с прогрессией Уровень значимости, p (Манн-Уитни) Медиана 25% 75% Медиана 25% 75% IL2, пг/мл 2,70 1,81 4,18 3,84 2,90 28,50 0,001 IL6, пг/мл 4,03 2,21 9,97 6,01 3,21 10,67 0,059 TNF, пг/мл 4,77 3,56 7,48 6,81 4,75 25,50 0,001 Таблица 3 – Уровни интерлейкинов в сыворотке крови в зависимости от прогрессии ММ Медико-биологические проблемы жизнедеятельности. 2023. № 1(29) 59 59 Ж.М. Козич, В.Н. Мартинков, И.В. Веялкин и др. Уровень гемоглобина >100 g/L Уровень гемоглобина <100 g/L 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Время, дней -0,1 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 Выживаемость без прогрессии Рисунок 5 – Показатели беспрогрессивной выживаемости пациентов ММ в зависимости от уровня гемоглобина Без аутоТГСК; АутоТГСК 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Время, дней -0,1 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 Выживаемость без прогрессии Рисунок 6 – Показатели беспрогрессивной выживаемости в зависимости от применения в лечении ММ аутоТГСК Появление инфекционных процессов на фоне иммунодефицита при ММ может усугублять состояние пациентов, приводя к невозможности проведения адекватной ХТ и, как следствие, к развитию резистент- ности или прогрессированию заболевания. Результаты исследования В нашей работе прогрессия заболевания выявлена в 54,2% случаев у пациентов с изначально высокими показателями СРБ, что в 1,64 раз чаще, чем у пациентов из группы, у которой на момент постановки диагноза не выявлено превышения СРБ – в 33% случаев, уровень значимости для критерия Хи-квадрат p=0,016, ОШ 2,40 (95%ДИ [1,17-4,94]). Уровень гемоглобина >100 g/L Уровень гемоглобина <100 g/L 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Время, дней -0,1 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 Выживаемость без прогрессии Рисунок 5 – Показатели беспрогрессивной выживаемости пациентов ММ в зависимости от уровня гемоглобина Уровень гемоглобина >100 g/L 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Время, дней -0,1 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 Выживаемость без прогрессии Рисунок 5 – Показатели беспрогрессивной выживаемости пациентов ММ в зависимости от уровня гемоглобина Рисунок 5 – Показатели беспрогрессивной выживаемости пациентов ММ в зависимости от уровня гемоглобина Высокодозная ХТ с последующей ауто трансплантацией гемопоэтических стволо- вых клеток (аутоТГСК) является «золотым стандартом» лечения пациентов ММ. Ее применение в лечении связано с увеличе- нием как выживаемости, так и качества жизни пациентов ММ [14]. Без аутоТГСК; 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Время, дней -0,1 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 Выживаемость без прогрессии Из 72 пациентов в возрасте до 65 лет 28 пациентам проведена высокодозная ХТ с по- следующей аутоТГСК. В группе пациентов, у которых применялась аутоТГСК, за период исследования прогрессия заболевания разви- лась в 25% случаев, что в 1,85 раз реже, чем у пациентов без применения аутоТГСК – в 46,2% случаев, уровень значимости для кри- терия Хи-квадрат p=0,043, ОШ 0,39 (95%ДИ [0,15-0,99]). Также определена значимо лучшая беспрогрессивная выживаемость в группе пациентов, у которых применялась аутоТПСК (p<0,001, рисунок 6). Без аутоТГСК; АутоТГСК Рисунок 6 – Показатели беспрогрессивной выживаемости в зависимости от применения в лечении ММ аутоТГСК рецидивом или первично рефрактерным заболеванием, для них характерна более короткая выживаемость. В данной статье мы рассмотрели фак- торы, влияющие на беспрогрессивную вы- живаемость пациентов с симптоматической ММ. Установлено, что значимое превы- шение уровней IL2, IL6, TNF и количество клональных CD138+ (>20%) на момент по- становки диагноза были связаны с увели- чением частоты прогрессии заболевания за период исследования. Результаты исследования Подтверждена взаимосвязь повреждения почек, анемиче- ского синдрома, повышенного уровня СРБ, множественных поражений костей скелета, присутствия генетических изменений на момент постановки диагноза со снижением беспрогрессивной выживаемости пациен- В данной статье мы рассмотрели фак- торы, влияющие на беспрогрессивную вы- живаемость пациентов с симптоматической ММ. Установлено, что значимое превы- шение уровней IL2, IL6, TNF и количество клональных CD138+ (>20%) на момент по- становки диагноза были связаны с увели- чением частоты прогрессии заболевания за период исследования. Подтверждена взаимосвязь повреждения почек, анемиче- ского синдрома, повышенного уровня СРБ, множественных поражений костей скелета, присутствия генетических изменений на момент постановки диагноза со снижением беспрогрессивной выживаемости пациен- Медико-биологические проблемы жизнедеятельности. 2023. № 1(29) Заключение Несмотря на достижения последних лет в лечении ММ, продолжительность жизни пациентов с этой патологией со- ставляет от нескольких месяцев до десяти и более лет. Она зависит от гетерогенности опухоли и индивидуальных особенностей самого человека, в том числе коморбидно- го фона и способности переносить ХТ. В настоящее время известные независимые прогностические факторы распределены в прогностические системы. К группе вы- сокого риска относят пациентов с ранним Медико-биологические проблемы жизнедеятельности. 2023. № 1(29) 60 Клиническая медицина тов с ММ. 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Recurrent 14q32 translocations determine the prognosis of multiple myeloma, especially in patients receiving intensive chemotherapy. / P. Moreau [et al.] // Blood. – 2002. – Vol. 100, №5. – P. 1579-1583. 2. Impact of cytogenetic classification on out- comes following early high-dose therapy in multiple myeloma / G.P. Kaufman [и др.] // Leukemia. – 2016. – Т. 30, №3. – С. 633-639. 10. Analysis of the genomic landscape of multiple myeloma highlights novel prognostic markers and dis- ease subgroups / N. Bolli [et al.] // Leukemia. – 2018. – Vol. 32, №12. – P. 2604-2616. 10. Analysis of the genomic landscape of multiple myeloma highlights novel prognostic markers and dis- ease subgroups / N. Bolli [et al.] // Leukemia. – 2018. – Vol. 32, №12. – P. 2604-2616. 3. Rajkumar, S.V. Multiple myeloma: 2022 up- date on diagnosis, risk stratification, and management / S.V. Rajkumar // Am. J. Hematol. – 2022. – Vol. 97, №8. – P. 1086-1107. 11. Cytokine-Mediated Dysregulation of Sign- aling Pathways in the Pathogenesis of Multiple My- eloma / S. Akhtar [et al.] // Int. J. Mol. Sci. – 2020. – Vol. 21, №14. – P. 5002. 4. Corre, J. Risk factors in multiple myeloma: is it time for a revision? / J. Corre, N.C. Munshi, H. Avet- Loiseau // Blood. – 2021. – Vol. 137, №1. – P. 16-19. 12. Bone disease in multiple myeloma: patho- physiology and management / A. Hameed [et al.] // Cancer Growth Metastasis. – 2014. – Vol. 7. – P. 33-42. 5. Second Revision of the International Staging System (R2-ISS) for Overall Survival in Multiple My- eloma: A European Myeloma Network (EMN) Report Within the HARMONY Project / M. D’Agostino [et al.] // J. Clin. Oncol. – 2022. – Vol. 40, № 29. – P. 3406-3418. 13. Ludwig, H. Anemia in multiple myeloma / H. Ludwig, G. Pohl, A. Osterborg // Clin. Adv. Hema- tol. Key words: multiple myeloma, incidence, prognostic factors Zh.M. Kozich, V.N. Martinkov, I.V. Veyalkin, J.N. Pugacheva, D.A. Blizin, N.N. Klimkovich ANALYSIS OF THE EPIDEMIOLOGICAL CHARACTERISTICS OF MULTIPLE MYELOMA AND CLINICAL FACTORS AFFECTING THE COURSE OF THE DISEASE Multiple myeloma (MM) is the second most common malignant neoplasm of the hemat- opoietic system. Survival rate has improved significantly in patients over the past decade with the use of new therapeutic strategies. The absence of adverse factors at the time of diagnosis does not always determine a positive course of the disease. Therefore, the search for prognostic factors is relevant. The article presents data based on the epidemiological characteristics and clinical factors of MM that affected the progression-free survival. We have shown that a sig- nificant excess of the levels of IL2, IL6, TNF, the level of clonal CD138+ >20% at the time of diagnosis is associated with an increase in the frequency of disease progression. A decrease in progression-free survival was detected at kidney damage, anemic syndrome, infectious complications, multiple skeletal bone lesions, genetic changes at the time of diagno- sis and did not depend on the type of immunoglobulin secretion. Key words: multiple myeloma, incidence, prognostic factors Key words: multiple myeloma, incidence, prognostic factors Поступила 01.03.23 Поступила 01.03.23 61 Медико-биологические проблемы жизнедеятельности. 2023. № 1(29)
https://openalex.org/W2973113192	https://www.nature.com/articles/s41419-019-1893-6.pdf	English	null	Fibroblast growth factor 21 Ameliorates diabetes-induced endothelial dysfunction in mouse aorta via activation of the CaMKK2/AMPKα signaling pathway	Cell death and disease	2,019	cc-by	10,282	© The Author(s) 2019 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Abstract Abstract Endothelial dysfunction initiates and exacerbates hypertension, atherosclerosis and other cardiovascular complications in diabetic mellitus. FGF21 is a hormone that mediates a number of beneﬁcial effects relevant to metabolic disorders and their associated complications. Nevertheless, it remains unclear as to whether FGF21 ameliorates endothelial dysfunction. Therefore, we investigated the effect of FGF21 on endothelial function in both type 1 and type 2 diabetes. We found that FGF21 reduced hyperglycemia and ameliorated insulin resistance in type 2 diabetic mice, an effect that was totally lost in type 1 diabetic mice. However, FGF21 activated AMPKα, suppressing oxidative stress and enhancing endothelium-dependent vasorelaxation of aorta in both types, suggesting a mechanism that is independent of its glucose-lowering and insulin-sensitizing effects. In vitro, we identiﬁed a direct action of FGF21 on endothelial cells of the aorta, in which it bounds to FGF receptors to alleviate impaired endothelial function challenged with high glucose. Furthermore, the CaMKK2-AMPKα signaling pathway was activated to suppress oxidative stress. Apart from its anti- oxidative capacity, FGF21 activated eNOS to dilate the aorta via CaMKK2/AMPKα activation. Our data suggest expanded potential uses of FGF21 for the treatment of vascular diseases in diabetes. A R T I C L E A R T I C L E O p e n A c c e s s Fibroblast growth factor 21 Ameliorates diabetes- induced endothelial dysfunction in mouse aorta via activation of the CaMKK2/AMPKα signaling h Lei Ying1, Na Li1,2, Zhengyue He1,3, Xueqin Zeng1, Yan Nan4, Jiantong Chen1, Peipei Miao1,5, Yunyun Ying1,6, Wei Lin7, Xinyu Zhao7, Lu Lu4, Mengke Chen6, Wei Cen6, Tonglin Guo8, Xiaokun Li7, Zhifeng Huang7 and Yang Wang1 Ying et al. Cell Death and Disease (2019) 10:665 https://doi.org/10.1038/s41419-019-1893-6 Ying et al. Cell Death and Disease (2019) 10:665 https://doi.org/10.1038/s41419-019-1893-6 Cell Death & Disease Cell Death & Disease Introduction insulin resistance, hyperglycemia and dysregulated lipid metabolism2–4. Endothelial cells in vasculature play essential roles in the prevention of inﬂammation, regula- tion of vascular tone, and maintenance of the balance between coagulation and anticoagulation5. Conversely, the impairment of endothelium in blood vessels leads to hypertension, atherosclerosis and other cardiovascular diseases2,3,5. Therefore, the control of endothelial function during diabetes is an efﬁcient way to prevent severe car- diovascular damage. The incidence of diabetes mellitus is increasing at an alarming rate, imposing tremendous suffering and eco- nomic burdens on patients. The major hazards of diabetes are its complications, especially cardiovascular diseases that are primary causes of mortality1. Among these vas- culopathies, endothelial dysfunction is thought to be the early event initiated by metabolic stresses, including Correspondence: Xiaokun Li (lixk1964@163.com) or Zhifeng Huang (cpu_son@163.com) or Yang Wang (wangydh995@163.com) 1Department of Pathophysiology, School of Basic Medical Sciences, Wenzhou Medical University, 325035 Wenzhou, Zhejiang, China 2Department of Pathology, Wenzhou Central Hospital, 325035 Wenzhou, Zhejiang, China Full list of author information is available at the end of the article. These authors contributed equally: Lei Ying, Na Li, Zhengyue He Edited by A. Finazzi-Agrò Oxidative stress occurring in metabolic disease is a major risk factor for endothelial dysfunction2,3,5. Many pathological changes induce oxidation in endothelial cells, thereby increasing production of reactive oxygen species (ROS)2,3,5. Then, accumulating amounts of ROS further lead to impairment of mitochondrial function and FGF21 improves endothelial dysfunction of aorta in Type 2 diabetic mice FGF21 improves endothelial dysfunction of aorta in Type 2 diabetic mice Endothelial dysfunction in conduit arteries induced by metabolic disorders is a well-established antecedent of hypertension and atherosclerosis2–4. Although the ther- apeutic effects of FGF21 on the latter two vasculopathies have been well studied previously17,18, it remains unclear whether FGF21 ameliorates impaired endothelial function in metabolic diseases such as T2D. Therefore, we estab- lished a high-fat diet (HFD)-streptozotocin (STZ) induced T2D mouse model and injected these mice intraper- itoneally (i.p.) on 33 consecutive days with rFGF21 (0.5 mg/Kg body weight). As previously reported21, we found that rFGF21 (thought to be an insulin sensitizer) induced a substantial glucose-lowering effect in T2D mice that was associated with improved insulin resistance (Fig. 1a–c). Dysregulated lipid metabolism in these mice was also restored by chronic rFGF21 treatment (as revealed by suppressed serum levels of low-density lipoprotein (LDL), cholesterol, triglyceride and free fatty acid (FFA) and upregulated level of high-density lipo- protein (HDL)) (Fig. S1A–E), in addition to reduced body weights (Figure S2A). g Identiﬁed as the 21st member of FGF superfamily in 2000, the endocrine hormone ﬁbroblast growth factor 21 (FGF21) has attracted considerable attention regarding the management of metabolic diseases as well as their complications7–9. FGF21 has been proposed to be a sen- sitive stress signal for maintaining metabolic homeostasis in both physiological and pathological conditions8. In most physiological stress responses, FGF21 functions as a hepatokine acting on adipose tissues to regulate energy homeostasis10–12. FGF21 also acts in fat tissue to maintain body temperature, in an autocrine or paracrine man- ner13,14. In metabolic disorders such as obesity and type 2 diabetes (T2D), high levels of FGF21 are thought to represent compensatory responses to maintain metabolic homeostasis, which also represents its resistant state15,16. In addition to its function as a stress response hormone, the metabolic beneﬁts of the pharmacological use of FGF21 have been well studied in rodents, nonhuman primates and humans, where it dramatically alleviated obesity and fatty liver disease8,9. Recently, several lines of evidence have shown that the vascular system might be a target for FGF21. For example, FGF21 protects against atherosclerosis by inducing adiponectin to inhibit neoin- tima formation and macrophage inﬂammation in blood vessels and suppresses hepatic cholesterol synthesis to attenuate hypercholesterolemia17. FGF21 also acts adi- pocytes and renal cells to promote metabolism of angio- tensin II and to mitigate hypertension and vessel injury18. FGF21 improves endothelial dysfunction of aorta in Type 2 diabetic mice FGF21 is also a potent anti-oxidative agent, protecting heart and kidney in various conditions19,20. We therefore hypothesized that FGF21 might exert anti-oxidative activities in vascular endothelia cells, and that these effects could be exploited to treat reduced NO bioavail- ability and associated cardiovascular diseases. Because hyperglycemia, insulin resistance and hyperli- pidemia are major risk factors for vascular endothelial dysfunction, we next examined whether FGF21 rescued impaired endothelial function in T2D2,3. As shown by dose-dependent relaxation to acetylcholine (ACh), the impaired endothelium-dependent dilation of aorta in T2D mice was largely improved by chronic rFGF21 treatment (Fig. 1d), in accordance with increased phosphorylation levels and activation of eNOS (Fig. 1e). These beneﬁcial effects of FGF21 were further investi- gated in another genetic induced (db/db) T2D mouse model which also received 33 consecutive days of i.p. injection with rFGF21 (0.5 mg/Kg body weight). Con- sistent with this result, we also found that hyperglycemia, insulin resistance and endothelium dysfunction in db/db mice were markedly improved by rFGF21 treatment in the same way (Fig. 2a–e), whereas there was little change in body weight (Fig. S2B). © The Author(s) 2019 The Author(s) 2019 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. ( ) Ofﬁcial journal of the Cell Death Differentiation Association Ofﬁcial journal of the Cell Death Differentiation Association Ying et al. Cell Death and Disease (2019) 10:665 Page 2 of 12 Results exacerbate the oxidative burden for the cell2,3,5. Simulta- neously with decreased levels of antioxidant enzymes, the oxidative stress becomes more severe and endothelial function is largely inhibited due to insufﬁciency of nitric oxide (NO) bioavailability by forming peroxynitrite, inducing endothelial nitric oxide synthase (eNOS) uncoupling and increasing lipid peroxidation products5,6. Therefore, therapeutic approaches targeting oxidative stress are efﬁcient methods to ameliorate impairments in vascular endothelial cells due to diabetic challenge. Ofﬁcial journal of the Cell Death Differentiation Association FGF21 alleviates endothelial dysfunction in aorta of type 1 diabetic mice To test this hypothesis, we generated recombinant human FGF21 (rFGF21) and investigated its effects on endothelial function both in vivo and in vitro. These experiments identiﬁed FGF21 as an efﬁcient anti- oxidative protein acting on vascular endothelial cells that reduced oxidative stress, enhanced eNOS activity and improved vessel relaxation, probably via CaMKK2/ AMPKα activation. Our data have important implications for FGF21 as a therapeutic approach for alleviation of endothelial dysfunction in diabetes. Although its pathogenesis is quite different from that of T2D, type 1 diabetes (T1D) is also a major risk factor for impaired endothelial function mediated by hyperglyce- mia3,22. To explore whether the therapeutic effects of FGF21 on vascular function applied to T1D, we estab- lished an STZ-induced T1D mouse model and treated these mice with 30 days of rFGF21 injection (i.p., 0.5 mg/ Kg body weight). Unlike in T2D mice, FGF21 failed to reduce high blood glucose in T1D mice with stable body Ofﬁcial journal of the Cell Death Differentiation Association Ying et al. Cell Death and Disease (2019) 10:665 Page 3 of 12 Fig. 1 Long-term treatment of HFD-STZ-Induced T2D mice with rFGF21 improves hyperglycemia, insulin resistance and endothelium- dependent relaxation of aorta. a–e HFD-STZ-induced T2D mice were treated for 33 days with rFGF21 (0.5 mg/kg body weight) or buffer control. a Random fed blood glucose (n = 10). b IPGTT over the course of 120 mins (n = 7–9). c Integrated AUC for changes in blood glucose levels (n = 7–9). d Dose-dependent relaxation of aorta to ACh (n = 5–7). e Phosphorylation level of eNOS in aortas as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 3). All data are presented as mean ± SEM. p < 0.05 vs Control; #p < 0.05 vs T2D Fig. 1 Long-term treatment of HFD-STZ-Induced T2D mice with rFGF21 improves hyperglycemia, insulin resistance and endothelium- dependent relaxation of aorta. a–e HFD-STZ-induced T2D mice were treated for 33 days with rFGF21 (0.5 mg/kg body weight) or buffer control. a Random fed blood glucose (n = 10). b IPGTT over the course of 120 mins (n = 7–9). c Integrated AUC for changes in blood glucose levels (n = 7–9). d Dose-dependent relaxation of aorta to ACh (n = 5–7). FGF21 alleviates endothelial dysfunction in aorta of type 1 diabetic mice e Phosphorylation level of eNOS in aortas as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 3). All data are presented as mean ± SEM. p < 0.05 vs Control; #p < 0.05 vs T2D and found that rFGF21 substantially rescued impaired AMPKα activity in these mice (Fig. 4d–f). Taken together, these data suggest that FGF21 may ameliorate endothelial dysfunction in diabetic mice via AMPKα-mediated inhi- bition of local oxidative stress in mouse aorta. weights (Fig. 3a, S2C). Nevertheless, it is interesting that FGF21 effectively alleviated T1D-induced aorta dysfunc- tion as reveled by improved vascular relaxation and enhanced eNOS activity (Fig. 3b, c). These data suggest that FGF21 ameliorated endothelial dysfunction in T1D by mechanisms independent of its glucose-lowering and insulin-sensitizing effects. Ofﬁcial journal of the Cell Death Differentiation Association The protective effects of FGF21 on diabetic endothelial dysfunction involve antioxidative mechanisms 2 Long-term treatment of db/db mice with rFGF21 improves hyperglycemia, insulin resistance and endothelium-dependent relaxation of aorta. a–e db/db mice were treated for 33 days with rFGF21 (0.5 mg/kg body weight) or buffer control; littermate db/m mice served as controls. a Random fed blood glucose (n = 5–6). b IPGTT over the course of 120 mins (n = 5–6). c Integrated AUC for changes in blood glucose levels (n = 5–6). d Dose-dependent relaxation of aorta to ACh (n = 5–6). e Phosphorylation level of eNOS in aortas as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 3). All data are presented as mean ± SEM. p < 0.05 vs Control; #p < 0.05 vs db/db rglycemia, insulin resistance and endothelium-dependent downstream target of AMPK)30 and eNOS by rFGF21 in HUVECs were markedly compromised by reduced AMPK expression (Fig. S5A–D). We further explored the downstream antioxidant signals that were controlled by the FGF21-AMPKα signaling pathway and found that upregulated mRNA levels of catalase (CAT), nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) and heme oxygenase 1 (HO-1) by rFGF21 were greatly inhibited by both FIIN-4 and Compound C (Fig. 5f, S4F). incubation, and was reversed by co-administration with rFGF21 (Figs. 5a, S4A). Consistently, reduced NO oxide release, dampened eNOS activity and enhanced oxidative stress by HG were all ameliorated by rFGF21 (Figs. 5b–d, S4B-D), in parallel with the activation of AMPKα (Figs. 5e, S4E). These results were further reinforced by using a potent FGF receptor antagonist (FIIN-4)28, that blocked almost all the beneﬁcial effects on endothelial function associated with improved eNOs activity, increased NO release and correspondingly enhanced relaxation of the aorta and reduced oxidative stress (Fig. 5a–e). g FGF21 triggers intracellular calcium release31–33, pos- sibly leading to activation of AMPK by calcium/calmo- dulin-dependent protein kinase kinase 2 (CaMKK2, also known as CaMKKβ) pathway34–36. Indeed, we found that impaired activity of CaMKK2 by HG was reversed by co- administration with rFGF21 in isolated aortas, and this improvement was blocked by FIIN-4 (Fig. 6a). The alle- viating effects of FGF21 on oxidative stress, endothelial dysfunction and reduced activations of AMPKα and ACC were substantially abrogated by the selective CaMKK2 antagonist (STO-609)36,37 in aorta or HUVEC (Figs. 6b–g, S6A–C). Taken together, the data suggest that FGF21 may directly bind FGFRs to activate the CaMKK2/AMPKα singling pathway to alleviate oxidative stress-induced endothelium dysfunction. The protective effects of FGF21 on diabetic endothelial dysfunction involve antioxidative mechanisms The animal studies suggested that there are some mechanisms involved in FGF21-mediated alleviation of endothelial dysfunction that are independent of reducing hyperglycemia and improving insulin resistance. Because endothelial cells express ﬁbroblast growth factor receptor 1 (FGFR1) and β-klotho (primary receptors and co- receptors mediating the biological functions of FGF21) (Fig. S3A, B)25–27, one possibility is that FGF21 may directly bind with the corresponding receptor to mediate its therapeutic effects. Therefore, we established an in vitro model in which aorta was isolated from normal mice and challenged with high glucose (HG) alone or HG plus rFGF21. In this model, the high glucose condition was maintained throughout rFGF21 treatment that was devoid of exogenous insulin, partially mimicking effects in T1D mice. We found that endothelium-dependent relaxation was severely impaired by 2 h of HG Oxidative stress initiated and exacerbated by metabolic disorders in both T1D and T2D is thought to be a primary cause of endothelial dysfunction2,3,5. Therefore, we next analyzed the oxidative status of aortas in rFGF21-treated HFD-STZ-induced T2D, db/db and T1D mice by immunostaining with dihydroethidium (DHE). Immuno- ﬂuorescence analyses showed signiﬁcantly fewer DHE- positive cells in all three mouse models treated with rFGF21 than in vehicle-treated mice (Fig. 4a–c). A growing body of evidence has shown that adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) plays a key role in maintaining oxidative home- ostasis in endothelial cells of conduit arteries challenged with metabolic stress23,24. We measured phosphorylation levels of AMPKα in aortas from all diabetic mouse models Ofﬁcial journal of the Cell Death Differentiation Association Ying et al. Cell Death and Disease (2019) 10:665 Page 4 of 12 Fig. 2 Long-term treatment of db/db mice with rFGF21 improves hyperglycemia, insulin resistance and endothelium-dependent relaxation of aorta. a–e db/db mice were treated for 33 days with rFGF21 (0.5 mg/kg body weight) or buffer control; littermate db/m mice served as controls. a Random fed blood glucose (n = 5–6). b IPGTT over the course of 120 mins (n = 5–6). c Integrated AUC for changes in blood glucose levels (n = 5–6). d Dose-dependent relaxation of aorta to ACh (n = 5–6). e Phosphorylation level of eNOS in aortas as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 3). All data are presented as mean ± SEM. p < 0.05 vs Control; #p < 0.05 vs db/db Fig. The protective effects of FGF21 on diabetic endothelial dysfunction involve antioxidative mechanisms This in vitro model further strengthened the notion that AMPKα plays an important role in FGF21-mediated improvement in endothelial function. Using an AMPK- selective inhibitor (Compound C)29, we found that restoration of aorta relaxation (associated with enhanced eNOs activity and NO release) and redox homeostasis (associated with reduced ROS) by rFGF21 in HG situation were potently abrogated (Fig. 5a–e). To validate the role of AMPK in FGF21-mediated alleviation of endothelial dysfunction induced by HG, we used AMPKα siRNA to knockdown its expressions in human umbilical vascular endothelial cells (HUVECs). Consistently, we found that activations of AMPKα, Acetyl-CoA carboxylase (ACC) (a Ofﬁcial journal of the Cell Death Differentiation Association Ying et al. Cell Death and Disease (2019) 10:665 Page 5 of 12 Fig. 3 Long-term treatment of T1D mice with rFGF21 improves endothelium-dependent relaxation of aorta. a–c STZ-induced T1D mice were treated for 30 days with rFGF21 (0.5 mg/kg body weight) or buffer control; littermate db/m mice served as controls. a Random fed blood glucose (n = 8–9). b Dose-dependent relaxation of aorta to ACh (n = 7–8). c Phosphorylation level of eNOS in aortas as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 3). All data are presented as mean ± SEM. p < 0.05 vs Control; #p < 0.05 vs T1D Fig. 3 Long-term treatment of T1D mice with rFGF21 improves endothelium-dependent relaxation of aorta. a–c STZ-induced T1D mice were treated for 30 days with rFGF21 (0.5 mg/kg body weight) or buffer control; littermate db/m mice served as controls. a Random fed blood glucose (n = 8–9). b Dose-dependent relaxation of aorta to ACh (n = 7–8). c Phosphorylation level of eNOS in aortas as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 3). All data are presented as mean ± SEM. p < 0.05 vs Control; #p < 0.05 vs T1D The etiology and pathogenesis of T1D and T2D are quite different. The former refers to a state of absolute insulin deﬁciency, whereas the latter is a condition of insulin resistance1,40,41. In the present study, we found that FGF21 improves hyperglycemia, insulin resistance and endothelium-dependent relaxation of aorta in two T2D mouse models (HFD-STZ-induced and db/db). Although FGF21 failed to rescue high blood glucose in T1D, it remained effective in the alleviation of endothelial dysfunction. The protective effects of FGF21 on diabetic endothelial dysfunction involve antioxidative mechanisms These results indicate that FGF21 may initiate a mechanism that is independent of its glucose- lowering and insulin-sensitizing effects. Indeed, we observed that FGF21 prevented HG-induced impairment of endothelial function in an in vitro model, suggesting that FGF21 directly acts on endothelial cells to exert its protective effect. This is further supported by the fact that both the receptor (FGFR1) and co-receptor (β-klotho) of FGF21 are expressed in vascular endothelial cells25–27. We also observed that certain levels of FGFR1 (with the highest bind afﬁnity to FGF21) and β-klotho could be detected in the aortas of WT mice. Moreover, this notion is reinforced by the use of FGFR antagonist that blocked most of the biological effects of FGF21 in vitro. rFGF21 directly induces relaxation of mice aorta via CaMKK2/AMPKα activation As a cognate ligand for FGFR1, FGF2 induces endothelium-dependent dilation of coronary arterioles38. Activation of AMPKα also potently relaxes aorta by an endothelium-dependent mechanism29,39. Therefore, we hypothesized that FGF21 induced endothelium- dependent relaxation of mouse aorta through AMPKα activation in addition to its anti-oxidative capacity. Aorta was isolated from WT mice and treated with an increasing concentration of rFGF21. We found that rFGF21 dose- dependently induced marked relaxation in the aorta (Fig. 7a, d). Furthermore, this FGF21-mediated relaxation was mostly blocked by FIIN-4, Compound C and STO-609, suggesting that the FGF21-CaMKK2-AMPKα signaling pathway directly induces dilation in conduit arteries (Fig. 7a, d). This notion was further conﬁrmed by the fact that the rFGF21-upregulated phosphorylation level of eNOS, AMPKα or ACC was largely abrogated by FIIN-4 (Fig. 7b, c), Compound C (Fig. 7b, c), AMPKα siRNA (Fig. S5E–H), or STO-609 (Figs. 7e–g, S6D–F) in isolated aorta or cultured HUVEC. Discussion 4 Long-term rFGF21 treatment alleviates oxidative stress and enhances AMPK signaling in aortas of HFD-STZ-induced T2D, db/db and T1D mice. a–c Immunoﬂuorescent DHE staining of aortas from HFD-STZ-induced T2D. a (33 days) (n = 5), db/db (b) (33 days) (n = 5) or T1D mice (c) (30 days) (n = 6) chronically treated with rFGF21 (0.5 mg/kg body weight). The upper panel shows DHE staining and the lower panel shows quantitation using ImageJ software. Scale bars, 100 μm. d–f Phosphorylation levels of AMPKα in aortas from T2D (A) (33 days), db/db (B) (33 days) or T1D mice (C) (30 days) chronically treated with rFGF21 (0.5 mg/kg body weight) as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 3). All data are presented as mean ± SEM. p < 0.05 vs Control; #p < 0.05 vs T2D, db/db or T1D function by FGF21 is partially mediated by its direct inhibition on oxidative stress. AMPKα to suppress oxidative stress and alleviate endo- thelial dysfunction. AMPK is a well-studied cellular energy sensor that is activated by various types of metabolic stresses to regulate energy metabolism within the cell and the body30,44. However, the activity of AMPK is dysregulated when exposed to persistent metabolic burdens such as is the case in metabolic syndrome or T2D44. By contrast, the activation of AMPK (physiological or pharmacological) improves insulin sensitivity and metabolic health30,44. In vascular endothelial cells, AMPK is activated to suppress oxidative stress-induced injury via various mechan- isms23,24. Consistently, we also found that AMPK activity in aorta was suppressed in diabetic mice or challenged with high glucose that was in parallel with increased ROS production and impaired endothelial function. The administration of FGF21 increased phosphorylation levels of AMPKα, reduced oxidative stress and enhance vessel relaxation, all of which were mostly blocked by its antagonist or siRNA. We further explored the underlying anti-oxidative molecules that might be regulated by AMPKα and found that CAT, Nrf-2, and HO-1 (pivotal anti-oxidative stress enzymes) were potential candi- dates45–47. The data suggest that FGF21 may activate In mammals, two major upstream kinases of AMPK are tumor suppressor LKB1 and CaMKK (especially CaMKK2)30,36. CaMKK2 induces activation of AMPK in response to increased intracellular calcium levels, which does not require any change in ADP or AMP levels30,34–36. FGF21 triggers intracellular calcium release31–33, thereby making it a possible upstream activator of CaMKK2. Discussion Hyperglycemia is a common pathological change occurring both in T1D and T2D3,22. Several lines of evi- dence have shown that high glucose-induced oxidative stress in diabetes is a major etiologic factor of several diabetic complications including macrovasculo- pathy3,22,42,43. We found that oxidative stress was exa- cerbated in the endothelium of the aorta by hyperglycemia in diabetic models or by high glucose in vitro. Conversely, the worsened oxidative burden was mostly abrogated by FGF21. Furthermore, the inhibition of FGF21 on oxidative stress was abolished by FGFR antagonist. Given that FGF21 acts on endothelial cells, it is logical to postulate that the improvement of endothelial The restoration of vascular endothelial function is a pathway for amelioration of cardiovascular diseases that are main causes of mortality in diabetic patients. FGF21 is a hormone in the FGF superfamily that exerts several beneﬁcial effects on metabolic diseases and their associated complications. However, the effects of FGF21 on impaired endothelial dysfunction of blood vessels in metabolic diseases remains unclear. This study shows that FGF21 improves dilation of aorta in both T1D and T2D mice, probably via CaMKK2/ AMPKα-mediated suppression of oxidative stress and activation of eNOS. Ofﬁcial journal of the Cell Death Differentiation Association Ying et al. Cell Death and Disease (2019) 10:665 Page 6 of 12 Fig. 4 Long-term rFGF21 treatment alleviates oxidative stress and enhances AMPK signaling in aortas of HFD-STZ-induced T2D, db/db and T1D mice. a–c Immunoﬂuorescent DHE staining of aortas from HFD-STZ-induced T2D. a (33 days) (n = 5), db/db (b) (33 days) (n = 5) or T1D mice (c) (30 days) (n = 6) chronically treated with rFGF21 (0.5 mg/kg body weight). The upper panel shows DHE staining and the lower panel shows quantitation using ImageJ software. Scale bars, 100 μm. d–f Phosphorylation levels of AMPKα in aortas from T2D (A) (33 days), db/db (B) (33 days) or T1D mice (C) (30 days) chronically treated with rFGF21 (0.5 mg/kg body weight) as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 3). All data are presented as mean ± SEM. p < 0.05 vs Control; #p < 0.05 vs T2D, db/db or T1D MPK signaling in aortas of HFD-STZ-induced T2D, db/db an Long-term rFGF21 treatment alleviates oxidative stress an Fig. 4 Long-term rFGF21 treatment alleviates oxidative stress and enhances AMPK signaling in aortas of HFD-STZ- Fig. Expression and puriﬁcation of rFGF21 Recombinant human FGF21 (rFGF21) (His29-Ser209) was expressed using the p-SUMO-FGF21 plasmid in E. coli and puriﬁed according to our previously published literature50. Discussion In the present study, we found that rFGF21 upregulated the activity of CaMKK2 by binding FGFR. By contrast, blockage of CaMKK2 by its selective antagonist sub- stantially abrogated most of the beneﬁcial effects of FGF21 on activating AMPKα, reducing oxidative stress and enhancing vessel relaxation. Taken together, the data suggest that CaMKK2 may be an upstream regulator determining the activity of AMPKα and mediating the alleviating effects of FGF21 on diabetes-induced oxidative stress and endothelial dysfunction. Apart from its role in the inhibition of oxidative stress, AMPK activates endothelium-dependent vasorelaxa- tion29,39,48. As a cognate ligand for FGFR149, FGF2 is also able to elicit a vasodilation depending on endothelium activation38. In the present study, FGF21 activated Ofﬁcial journal of the Cell Death Differentiation Association Ying et al. Cell Death and Disease (2019) 10:665 Page 7 of 12 Fig. 5 RFGF21 improves endothelium-dependent relaxation, alleviates oxidative stress and enhances AMPK signaling in aortas challenged with HG. a–e Aortas isolated form C57BL/6 J mice in Kreb’s buffer were pretreated with FIIN-4 (10 μM) or Compound C (10 μM) for 30 mins and exposed to either HG (30 mM) alone or HG plus rFGF21 (0.01 mg/ml) for an additional 2 h. a Dose-dependent relaxation to ACh (n = 8–18). b NO2 and NO3 levels stimulated by ACh (6 × 10−8 M) for 3 mins (n = 5). c Phosphorylation level of eNOS stimulated by ACh (6 × 10−8 M) for 3 mins as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 4). d Immunoﬂuorescent DHE staining (n = 6). The upper panel shows DHE staining and the lower panel shows quantitation using ImageJ software. Scale bars, 100 μm. e Phosphorylation level of AMPKα as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 4). f mRNA levels of CAT, HO-1 and Nrf-2 as determined by real-time PCR analysis (n = 6). All data are presented as mean ± SEM. p < 0.05 vs Control; #p < 0.05 vs HG; †p < 0.05 vs HG + FGF21 Fig. 5 RFGF21 improves endothelium-dependent relaxation, alleviates oxidative stress and enhances AMPK signal endothelium-dependent relaxation, alleviates oxidative stress and enhances AMPK signaling in aortas challenged d f b b ff d h d f d Fig. 5 RFGF21 improves endothelium-dependent relaxation, alleviates oxidative stress and enhances AMPK signaling in aortas challenged with HG. Discussion a–e Aortas isolated form C57BL/6 J mice in Kreb’s buffer were pretreated with FIIN-4 (10 μM) or Compound C (10 μM) for 30 mins and exposed to either HG (30 mM) alone or HG plus rFGF21 (0.01 mg/ml) for an additional 2 h. a Dose-dependent relaxation to ACh (n = 8–18). b NO2 and NO3 levels stimulated by ACh (6 × 10−8 M) for 3 mins (n = 5). c Phosphorylation level of eNOS stimulated by ACh (6 × 10−8 M) for 3 mins as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 4). d Immunoﬂuorescent DHE staining (n = 6). The upper panel shows DHE staining and the lower panel shows quantitation using ImageJ software. Scale bars, 100 μm. e Phosphorylation level of AMPKα as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 4). f mRNA levels of CAT, HO-1 and Nrf-2 as determined by real-time PCR analysis (n = 6). All data are presented as mean ± SEM. p < 0.05 vs Control; #p < 0.05 vs HG; †p < 0.05 vs HG + FGF21 Fig. 5 RFGF21 improves endothelium-dependent relaxation, alleviates oxidative stress and enhances AMPK signaling in aortas challenged with HG. a–e Aortas isolated form C57BL/6 J mice in Kreb’s buffer were pretreated with FIIN-4 (10 μM) or Compound C (10 μM) for 30 mins and exposed to either HG (30 mM) alone or HG plus rFGF21 (0.01 mg/ml) for an additional 2 h. a Dose-dependent relaxation to ACh (n = 8–18). b NO2 and NO3 levels stimulated by ACh (6 × 10−8 M) for 3 mins (n = 5). c Phosphorylation level of eNOS stimulated by ACh (6 × 10−8 M) for 3 mins as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 4). d Immunoﬂuorescent DHE staining (n = 6). The upper panel shows DHE staining and the lower panel shows quantitation using ImageJ software. Scale bars, 100 μm. e Phosphorylation level of AMPKα as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 4). f mRNA levels of CAT, HO-1 and Nrf-2 as determined by real-time PCR analysis (n = 6). All data are presented as mean ± SEM. Discussion p < 0.05 vs Control; #p < 0.05 vs HG; †p < 0.05 vs HG + FGF21 CaMKK2, AMPKα and eNOS and induced dose- dependent dilation of aorta in control buffer. Further- more, this vasorelaxation was abrogated by FGFR, CaMKK2 and AMPK antagonists, and AMPKα siRNA. Therefore, FGF21 induces relaxation of aorta through CaMKK2/AMPKα activation may also contribute to the alleviation of endothelial dysfunction in addition to its potent inhibition on oxidative stress. biological functions of FGF21 on vascular diseases chal- lenged with metabolic disorders and demonstrate that FGF21 is a potent and effective protein to treat endo- thelial dysfunction through the direct suppression of oxidative stress and enhancement of eNOS signaling via CaMKK2/AMPKα activation in diabetes (Fig. 8). Ofﬁcial journal of the Cell Death Differentiation Association Materials and methods Expression and puriﬁcation of rFGF21 Previous studies have shown an indirect regulation of FGF21 on vasculopathies, including atherosclerosis and hypertension, by acting on adipocytes, hepatocytes and renal cells17,18. In the present study, we found that FGF21 directly bound FGFR1 to activate CaMKK2 and AMPKα, to inhibit oxidative stress and to enhance eNOS phos- phorylation levels in endothelial cells of large vessels. Furthermore, we found that CAT, Nrf-2, and HO-1 may be downstream targets activated by AMPK to suppress the oxidative burden exposed to HG. Taken together, our data provide a novel mechanistic understanding of the Animal welfare and animal model db b–g Aortas isolated form C57BL/6J mice were pretreated with STO-609 (5 μg/ml) for 30 mins and exposed to either HG (30 mM) alone or HG plus rFGF21 (0.01 mg/ml) for an additional 2 h. b Dose-dependent relaxation to ACh (n = 5). c NO2 and NO3 levels stimulated by ACh (6 × 10−8 M) for 3 mins (n = 5). d Phosphorylation level of eNOS stimulated by ACh (6 × 10−8 M) for 3 mins as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 4). e Immunoﬂuorescent DHE staining (n = 6). The upper panel shows DHE staining and the lower panel shows quantitation using ImageJ software. Scale bars, 100 μm. (f, g) Phosphorylation level of AMPKα (f) and ACC (g) as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 4). All data are presented as mean ± SEM. p < 0.05 vs Control; #p < 0.05 vs HG; †p < 0.05 vs HG + FGF21 Alameda, CA) and those with blood glucose ≥300 mg/dl (16.7 mM) were considered as diabetic mice. in this study were approved by the Animal Care and Use Committee of Wenzhou Medical University, China. The animals were housed in an animal facility with controlled environment (22 ± 2 °C, 50–60% humidity, 12-h light/dark cycle, lights on at 7 a.m.) and free access to food and water. All animals were acclimatized to our laboratory environment before use. After all treatments, mice were anesthetized with diethyl ether and sacriﬁced by cervical dislocation. in this study were approved by the Animal Care and Use Committee of Wenzhou Medical University, China. The animals were housed in an animal facility with controlled environment (22 ± 2 °C, 50–60% humidity, 12-h light/dark cycle, lights on at 7 a.m.) and free access to food and water. All animals were acclimatized to our laboratory environment before use. After all treatments, mice were anesthetized with diethyl ether and sacriﬁced by cervical dislocation. Animal welfare and animal model db Male db/db (C57BLKS/J-leprdb/leprdb) mice and their non-diabetic db/m littermates and male C57BL/6J mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). All protocols used Ofﬁcial journal of the Cell Death Differentiation Association Ying et al. Cell Death and Disease (2019) 10:665 Page 8 of 12 Fig. 6 RFGF21 activates CaMKK2 to upregulate AMPKα activity, improve endothelium-dependent relaxation and alleviate oxidative stress in aortas challenged with HG. a Aortas isolated form C57BL/6J mice were pretreated with FIIN-4 (10 μM) for 30 mins and exposed to either HG (30 mM) alone or HG plus rFGF21 (0.01 mg/ml) for an additional 2 h. Phosphorylation level of CaMKK2 as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 4). b–g Aortas isolated form C57BL/6J mice were pretreated with STO-609 (5 μg/ml) for 30 mins and exposed to either HG (30 mM) alone or HG plus rFGF21 (0.01 mg/ml) for an additional 2 h. b Dose-dependent relaxation to ACh (n = 5). c NO2 and NO3 levels stimulated by ACh (6 × 10−8 M) for 3 mins (n = 5). d Phosphorylation level of eNOS stimulated by ACh (6 × 10−8 M) for 3 mins as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 4). e Immunoﬂuorescent DHE staining (n = 6). The upper panel shows DHE staining and the lower panel shows quantitation using ImageJ software. Scale bars, 100 μm. (f, g) Phosphorylation level of AMPKα (f) and ACC (g) as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 4). All data are presented as mean ± SEM. p < 0.05 vs Control; #p < 0.05 vs HG; †p < 0.05 vs HG + FGF21 Kα activity, improve endothelium-dependent relaxation and alleviate oxidative stress tes CaMKK2 to upregulate AMPKα activity, improve endothelium-dependent relaxation and alleviate oxidative stres Fig. 6 RFGF21 activates CaMKK2 to upregulate AMPKα activity, improve endothelium-dependent relaxation and alleviate oxidative stress in aortas challenged with HG. a Aortas isolated form C57BL/6J mice were pretreated with FIIN-4 (10 μM) for 30 mins and exposed to either HG (30 mM) alone or HG plus rFGF21 (0.01 mg/ml) for an additional 2 h. Phosphorylation level of CaMKK2 as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 4). Functional evaluation of rFGF21 in diabetic mice Before each study, mice were randomly assigned based on body weight and blood glucose level as determined above. All drugs and buffer controls were delivered by i.p. injection into mice. HFD-STZ-induced T2D mice and db/db mice were injected with rFGF21 for consecutive 33 days (0.5 mg/Kg body weight). T1D mice were injected with rFGF21 for consecutive 30 days (0.5 mg/Kg body weight). Blood samples were taken by tail snip measured every three days and glucose levels were determined as described above. Following the ﬁnal dose, intraperitoneal glucose tolerance tests (IPGTTs) were conducted on HFD-STZ-induced T2D mice and db/db mice after fast- ing overnight (12 h); these mice were i.p. injected with a dextrose solution (1.0 g/kg body weight) and blood sam- ples were taken at indicated time points and glucose levels were determined. Area under the curve (AUC) for IPGTTs was calculated by the trapezoid rule for the glucose tolerance curve using GraphPad Prism 8 software HFD–STZ-induced T2D mice were established by feeding 8-week-old male C57BL/6 J mice with a high-fat diet (60 % fat; Research Diets, Inc, New Brunswick, NJ) for eight weeks to initiate insulin resistance. After that, the animals were introperitoneal (i.p.) injected with STZ (35 mg/Kg body weight) in citrate buffer (pH4.5) for three consecutive days; control mice received the same volume of citrate buffer. T1D was induced in 8-week-old male C57BL/6 J mice by i.p. injection of 100 mg/kg STZ for two days followed by three consecutive days injection of 60 mg/kg STZ. After two weeks, fed blood glucose levels of mice were determined using a FreeStyle complete blood glucose monitor (Abbott Diabetes Care Inc., Ofﬁcial journal of the Cell Death Differentiation Association Ying et al. Cell Death and Disease (2019) 10:665 Page 9 of 12 Fig. 7 RFGF21 induces relaxation of aorta via CaMKK2/ AMPKα mediated activation of eNOS. a Dose-dependent relaxation of aorta from C57BL/6J mice to rFGF21 pretreated with FIIN-4 (10 μM) or Compound C (10 μM) for 30 mins (n = 7–12). b, c Phosphorylation level of eNOS (b) and AMPKα (c) in aortas from C57BL/6 J mice pretreated with FIIN-4 (10 μM) or Compound C (10 μM) for 30 mins and exposed to rFGF21 (0.01 mg/ml) for 3 mins as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 3). In vitro incubation study Aorta rings (length: 3–5 mm) isolated from C57BL/6J mice were suspended in organ chambers ﬁlled with 10 ml or in tubes with 5 ml of modiﬁed Krebs-Ringer bicarbo- nate solution (composition (mM): NaCl, 118.3; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25.0; glucose, 11.1) maintained at 37 + /-1 °C and aerated with 95% O2 and 5% CO2 (pH = 7.4). After 30 mins equili- bration, the rings were pretreated with FIIN-4 (10 μM), Compound C (10 μM), STO-609 (5 μg/ml) or buffer control for 30 mins (for both high glucose (HG) and rFGF21 incubations) and exposed to either HG (30 mM) alone or HG plus rFGF21 (0.01 mg/ml) for additional 2 h (for HG incubation). At the beginning, aorta ring was stretched to its optimal resting tension (~1.0 g) by step-wise stretching followed by an hour equilibration. Dose-dependent relaxation responses of ACh (10−12–10−5 M) or rFGF21 (10−8–10−5 M) was performed in rings preconstricted with NE (3 × 10−7 M) to a similar tension level. For vessel tension studies, the vessels were pre- constricted with norepinephrine (NE) (3 × 10−7 M) and dose-dependent relaxation responses of acetylcholine (ACh) or FGF21 were recorded. For intracellular signaling assays or measuring nitric oxide (NO) synthesis, the vessels were incubated with ACh (6 × 10−8 M) (for HG Functional evaluation of rFGF21 in diabetic mice d Dose-dependent relaxation of aorta from C57BL/6J mice to rFGF21 pretreated with STO-609 (5 μg/ml) for 30 mins (n = 9–10). e–g Phosphorylation level of eNOS (e), AMPKα (f) and ACC (g) in aortas from C57BL/6J mice pretreated with STO-609 (5 μg/ml) for 30 mins and exposed to rFGF21 (0.01 mg/ml) for 3 mins as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 4). All data are presented as mean ± SEM. p < 0.05 vs Control; #p < 0.05 vs FGF21 Fig. 7 RFGF21 induces relaxation of aorta via CaMKK2/ AMPKα mediated activation of eNOS. a Dose-dependent relaxation of aorta from C57BL/6J mice to rFGF21 pretreated with FIIN-4 (10 μM) or Compound C (10 μM) for 30 mins (n = 7–12). b, c Phosphorylation level of eNOS (b) and AMPKα (c) in aortas from C57BL/6 J mice pretreated with FIIN-4 (10 μM) or Compound C (10 μM) for 30 mins and exposed to rFGF21 (0.01 mg/ml) for 3 mins as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 3). d Dose-dependent relaxation of aorta from C57BL/6J mice to rFGF21 pretreated with STO-609 (5 μg/ml) for 30 mins (n = 9–10). e–g Phosphorylation level of eNOS (e), AMPKα (f) and ACC (g) in aortas from C57BL/6J mice pretreated with STO-609 (5 μg/ml) for 30 mins and exposed to rFGF21 (0.01 mg/ml) for 3 mins as determined by western blot analysis (upper panel) and quantitation using ImageJ software (lower panel) (n = 4). All data are presented as mean ± SEM. *p < 0.05 vs Control; #p < 0.05 vs FGF21 incubation) or FGF21 (0.01 mg/ml) (for rFGF21 incuba- tion) for 3 mins. (GraphPad Software, San Diego, California). Serum levels of LDL, cholesterol, triglyceride, FFA, and HDL were measured according to protocols provided (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Vessel tension studies Aorta rings (length: 3–5 mm) isolated from control, diabetic and rFGF21-treated mice were suspended in organ chambers ﬁlled with 10 ml of modiﬁed Krebs- Ringer bicarbonate solution maintained at 37 ± 1 °C and aerated with 95% O2 and 5% CO2 (pH = 7.4). Each ring was suspended through two stirrups passing through the lumen; one stirrup was anchored to the bottom of the organ chamber while the other connected to a strain gauge (RM6240 Systems/4sp, Chengdu Instru- ment Company, Chengdu, China) for measuring isometric force. Ofﬁcial journal of the Cell Death Differentiation Association Western blot analysis The strengthened eNOS activity leads to an increased production of nitric oxide and restoration of endothelium-dependent vasodilatation Fig. 8 Schematic diagram of FGF21-mediated enhancement of endothelium-dependent relaxation. The normal function of vascular endothelial cell is impaired by oxidative stress that is initiated by metabolic disorders. FGF21 binds to FGFRs (i.e., FGFR1) to potentiate CaMKK2 and AMPKα activities. On the one hand, AMPKα upregulates the expressions of CAT, Nrf-2 and HO-1 to abrogate oxidative stress. On the other hand, AMPKα directly enhances the activity of eNOS. The strengthened eNOS activity leads to an increased production of nitric oxide and restoration of endothelium-dependent vasodilatation in endothelial cell growth medium (EGMTM-2 Endothelial Cell Growth Medium-2 BulletKitTM, cat. no. CC-3156 and CC-4176, Lonza, Basel, Switzerland). Fifth to seventh passage of subconﬂuent cells were used in the subsequent experiments. For intracellular signaling assays, HUVECs were starved with serum-free low-glucose DMEM (cat. no. C11885500BT, Gibco, Thermo Fisher Scientiﬁc, Wal- tham, MA) for 12 h. Then, the cells were pretreated with STO-609 (5 μg/ml) or buffer control for 1 h (for both high glucose (HG) and rFGF21 incubations) and were exposed to either HG (35 mM) alone or HG plus rFGF21 (0.01 mg/ml) for an additional 6 h (for HG incubation). Subsequently, the cells were incubated with ACh (6 × 10−8 M) (for HG incubation) or FGF21 (0.01 mg/ml) (for rFGF21 incubation) for 10 mins and were lysed for sub- sequent measurement of levels of various downstream signals using western blotting. For knockdown of AMPKα expression by siRNA, the cells were seeded in six-well plates and grown to 70% conﬂuence. Transient transfections were performed using Lipofectamine 3000 Transfection Reagent (cat. no. L3000- 008, Thermo Fisher Scientiﬁc, Waltham, MA) according Cell culture and treatment The human umbilical vascular endothelial cells (HUVECs) were purchased from Lonza and were cultured Ofﬁcial journal of the Cell Death Differentiation Association Ying et al. Cell Death and Disease (2019) 10:665 Page 10 of 12 Fig. 8 Schematic diagram of FGF21-mediated enhancement of endothelium-dependent relaxation. The normal function of vascular endothelial cell is impaired by oxidative stress that is initiated by metabolic disorders. FGF21 binds to FGFRs (i.e., FGFR1) to potentiate CaMKK2 and AMPKα activities. On the one hand, AMPKα upregulates the expressions of CAT, Nrf-2 and HO-1 to abrogate oxidative stress. On the other hand, AMPKα directly enhances the activity of eNOS. The strengthened eNOS activity leads to an increased production of nitric oxide and restoration of endothelium-dependent vasodilatation to the manufacturer’s protocol. After cells were trans- fected with control (cat. no. sc-36869, Santa Cruz Bio- technology, Dallas, Texas, USA) or AMPKα siRNA (cat. no. sc-45312, Santa Cruz Biotechnology, Dallas, Texas, USA) for 36 h, they were starved for 12 h and treated as described above. Western blot analysis Mice aortas, epididymal white adipose tissues (WAT(e) s) and HUVECs were homogenized in RIPA lysis buffer (25 mM Tris, pH 7.6, 150 mM NaCl, 1%NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitors (all from Thermo Fisher Scientiﬁc Waltham, MA). Protein concentrations were determined using a BCA Kit (Protein Assay Kit, Beyotime Biotechnology, Shanghai, China). For deter- mining phosphorylation and protein expression levels, equal quantities of soluble protein (50 μg) were separated using 10-12% SDS-PAGE and electro-transferred onto a nitrocellulose membrane. Protein blots were probed with primary antibodies against p-eNOS (anti-rabbit; dilution: 1:1000; cat. no. MA5-14957; Thermo Fisher Scientiﬁc, Waltham, MA), eNOS (anti-mouse; dilution: 1:3000; cat. no. ab50010; Abcam, Cambridge, MA), p-AMPKα (anti- rabbit; dilution: 1:1000; cat. no. 2535; Cell Signaling Technology, Danvers, MA), AMPKα (anti-rabbit; dilution: 1:1000; cat. no. 5831; Cell Signaling Technology, Danvers, MA), p-ACC (anti-rabbit; dilution: 1:1000; cat. no. 11818; Cell Signaling Technology, Danvers, MA), ACC (anti- rabbit; dilution: 1:1000; cat. no. 3662; Cell Signaling Technology, Danvers, MA), p-CaMKK2 (anti-rabbit; dilution: 1:1000; cat. no. 16737; Cell Signaling Technol- ogy, Danvers, MA), CaMKK2 (anti-rabbit; dilution: 1:1000; cat. no. 16810; Cell Signaling Technology, Dan- vers, MA), FGFR1 (anti-rabbit; dilution: 1:1000; cat. no. 9740; Cell Signaling Technology, Danvers, MA), β-klotho (anti-mouse; dilution: 1:500; cat. no. AF2619; Bio-Techne MN, USA) and GAPDH (anti-rabbit; dilution: 1:1000; cat. no. 2118; Cell Signaling Technology, Danvers, MA). Immune-reactive bands were detected by incubating with goat anti-mouse (dilution: 1:3000; cat. no. sc-2005; Santa Cruz Biotechnology, Dallas, TX) or anti-rabbit secondary antibody (dilution: 1:3000; cat. no. sc-2004; Santa Cruz Biotechnology, Dallas, TX) conjugated with horseradish peroxidase and visualized using enhanced chemilumi- nescence (ECL) reagents (Bio-Rad, Hercules, CA). After that, optical densities of the immunoblots were analyzed using ImageJ image software (version 1.38e, NIH, Bethesda, MD) and normalized to the scanning signals of their respective controls. Fig. 8 Schematic diagram of FGF21-mediated enhancement of endothelium-dependent relaxation. The normal function of Fig. 8 Schematic diagram of FGF21-mediated enhancement of endothelium-dependent relaxation. The normal function of vascular endothelial cell is impaired by oxidative stress that is initiated by metabolic disorders. FGF21 binds to FGFRs (i.e., FGFR1) to potentiate CaMKK2 and AMPKα activities. On the one hand, AMPKα upregulates the expressions of CAT, Nrf-2 and HO-1 to abrogate oxidative stress. On the other hand, AMPKα directly enhances the activity of eNOS. RNA extraction, cDNA synthesis, and quantitative RT-PCR RNA extraction, cDNA synthesis, and quantitative RT-PCR Total RNA was extracted from mouse aortas with TRIzol reagent (Thermo Fisher Scientiﬁc, Waltham, MA) and puriﬁed using a RNeasy Mini Kit (Qiagen, Valencia, CA). A Two-step M-MLV Platinum SYBR Green qPCR Super Mix- UDG kit (Thermo Fisher Scientiﬁc, Waltham, MA) was used for reverse transcription and quantitative PCR. GAPDH was used as an endogenous control to normalize differences in the quantity of total RNA added to each reaction. Primers were synthesized by Invitrogen (Thermo Fisher Scientiﬁc, Waltham, MA) as follows: CAT, forward 5′-TGGCACACTTTGACAGAGAGC-3′ and reverse 5′-CCTTTGCCTTGGAGTATCTGG-3′; Nrf-2, forward 5′-TCTTGGAGTAAGTCGAGAAGTGT-3′ and reverse 5’-GTTGAAACTGAGCGAAAAAGGC -3′; HO-1, forward 5′-AAGCCGAGAATGCTGAGTTCA-3′ and reverse 5′-GCCGTGTAGATATGGTACAAGGA-3′; GAPDH, for- ward 5′-AATGTGTCCGTCGTGGATCT-3′ and reverse 5′-CATCGAAGGTGGAAGAGTGG-3′. References 1. Grundy, S. M. et al. Diabetes and cardiovascular disease: a statement for healthcare professionals from the American Heart Association. Circulation 100, 1134–1146 (1999). 1. Grundy, S. M. et al. Diabetes and cardiovascular disease: a statement for healthcare professionals from the American Heart Association. Circulation 100, 1134–1146 (1999). 2. Tabit, C. E., Chung, W. B., Hamburg, N. M. & Vita, J. A. Endothelial dysfunction in diabetes mellitus: molecular mechanisms and clinical implications. Rev. Endocr. Metab. Disord. 11, 61–74, https://doi.org/10.1007/s11154-010-9134-4 (2010). 2. Tabit, C. E., Chung, W. B., Hamburg, N. M. & Vita, J. A. Endothelial dysfunction in diabetes mellitus: molecular mechanisms and clinical implications. Rev. Endocr. Metab. Disord. 11, 61–74, https://doi.org/10.1007/s11154-010-9134-4 (2010). Conﬂict of interest The authors declare that they have no conﬂict of interest. Author details 1 1Department of Pathophysiology, School of Basic Medical Sciences, Wenzhou Medical University, 325035 Wenzhou, Zhejiang, China. 2Department of Pathology, Wenzhou Central Hospital, 325035 Wenzhou, Zhejiang, China. 3Department of Pathology, Suining Central Hospital, 629000 Suining, Sichuan, China. 4The Second Afﬁliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, 325035 Wenzhou, Zhejiang, China. 5Department of Pharmacy, the Second People’s Hospital of Pingyang, 325035 Wenzhou, Zhejiang, China. 6The First Afﬁliated Hospital & School of the First Clinical Medical Sciences, Wenzhou Medical University, 325035 Wenzhou, Zhejiang, China. 7School of Pharmaceutical Sciences, Wenzhou Medical University, 325035 Wenzhou, Zhejiang, China. 8School of Ophthalmology and Optometry, Wenzhou Medical University, 325035 Wenzhou, Zhejiang, China Supplementary Information accompanies this paper at (https://doi.org/ 10.1038/s41419-019-1893-6). Supplementary Information accompanies this paper at (https://doi.org/ 10.1038/s41419-019-1893-6). Received: 1 April 2019 Revised: 6 August 2019 Accepted: 20 August 2019 Received: 1 April 2019 Revised: 6 August 2019 Accepted: 20 August 2019 Statistical analyses 3. Shaw, A., Doherty, M. K., Mutch, N. J., MacRury, S. M. & Megson, I. L. Endothelial cell oxidative stress in diabetes: a key driver of cardiovascular complications? Biochem Soc Trans. 42, 928–933, https://doi.org/10.1042/BST20140113 (2014). 3. Shaw, A., Doherty, M. K., Mutch, N. J., MacRury, S. M. & Megson, I. L. Endothelial cell oxidative stress in diabetes: a key driver of cardiovascular complications? Biochem Soc Trans. 42, 928–933, https://doi.org/10.1042/BST20140113 (2014). All results are expressed as mean ± SEM. Student’s unpaired t tests were used for the comparison of two groups. While student’s paired t tests were applied in the comparison of the same group before and after a treat- ment. One-way analysis of variance (ANOVA) test with Student–Newman–Keuls test for post hoc testing of multiple comparisons were used in the comparison of mean values of more than two groups. P value (two tailed) <0.05 is considered as statistical signiﬁcance. N represents the number of replicates in the corresponding experiment. 4. Cheng, K. K. et al. Adiponectin-induced endothelial nitric oxide synthase activation and nitric oxide production are mediated by APPL1 in endothelial cells. Diabetes 56, 1387–1394, https://doi.org/10.2337/db06-1580 (2007). 5. Liang, Y. et al. Research Progress on Signaling Pathway-Associated Oxidative Stress in Endothelial Cells. Oxid. Med. Cell Longev. 2017, 7156941, https://doi. org/10.1155/2017/7156941 (2017). 6. Vanhoutte, P. M., Zhao, Y., Xu, A. & Leung, S. W. Thirty years of saying NO: sources, fate, actions, and misfortunes of the endothelium-derived vasodilator mediator. Circ. Res. 119, 375–396, https://doi.org/10.1161/ CIRCRESAHA.116.306531 (2016). 7. Nishimura, T., Nakatake, Y., Konishi, M. & Itoh, N. Identiﬁcation of a novel FGF, FGF-21, preferentially expressed in the liver. Biochim Biophys. Acta 1492, 203–206 (2000). 8. Fisher, F. M. & Maratos-Flier, E. Understanding the Physiology of FGF21. Annu Rev. Physiol. 78, 223–241, https://doi.org/10.1146/annurev-physiol-021115- 105339 (2016). 8. Fisher, F. M. & Maratos-Flier, E. Understanding the Physiology of FGF21. Annu Rev. Physiol. 78, 223–241, https://doi.org/10.1146/annurev-physiol-021115- 105339 (2016). DHE staining The frozen sections (5 μm) of aortas were incubated with dihydroethidium (DHE) (1.5 mmol/L) for 30 mins and visualized using a ﬂuorescence microscope (TCS-SP8, Leica, Germany). Measurement of NO synthesis Generation of NO was determined by measuring stable NO metabolites (i.e. total nitrites) in aortas after Ofﬁcial journal of the Cell Death Differentiation Association Page 11 of 12 Page 11 of 12 Ying et al. Cell Death and Disease (2019) 10:665 incubations and stimulated by ACh (6 × 10−8 M) using a nitrite detection kit (Beyotime, Shanghai, China). Brieﬂy, 50 μl of tissue homogenate was mixed with 50 μl of Griess reagent in a 96-well plate. Nitrite concentration was determined by spectrophotometry (540 nm) from a standard curve (0–100 μmol/l) derived from NaNO2 and protein concentrations were determined as described above. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. g This work was supported by Grants from National Key R&D Program of China (2017YFA0506000) (to X.L. and Z.H.), Grants from Natural Science Foundation of China (81473261, 81874323 and 81400273 to Z.H and L.Y.), Zhejiang Provincial Natural Science Foundation of China (LY18H070002 and LQ13H020006 to Y.W. and L.Y.), and Wenzhou Public Welfare Science and Technology Project (Y20140660, Y20160097, and Y20180063 to Y.W., Y.N., and L.L.). 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https://openalex.org/W2903480700	https://europepmc.org/articles/pmc6277808?pdf=render	English	null	Dual RNA-Seq Uncovers Metabolic Amino Acids Dependency of the Intracellular Bacterium Piscirickettsia salmonis Infecting Atlantic Salmon	Frontiers in microbiology	2,018	cc-by	8,286	ORIGINAL RESEARCH published: 27 November 2018 doi: 10.3389/fmicb.2018.02877 Citation: Valenzuela-Miranda D and Gallardo-Escárate C (2018) Dual RNA-Seq Uncovers Metabolic Amino Acids Dependency of the Intracellular Bacterium Piscirickettsia salmonis Infecting Atlantic Salmon. Front. Microbiol. 9:2877. doi: 10.3389/fmicb.2018.02877 Dual RNA-Seq Uncovers Metabolic Amino Acids Dependency of the Intracellular Bacterium Piscirickettsia salmonis Infecting Atlantic Salmon Diego Valenzuela-Miranda and Cristian Gallardo-Escárate* Diego Valenzuela-Miranda and Cristian Gallardo-Escárate* Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for Aquaculture Research, University of Concepción, Concepción, Chile Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for Aquaculture Research, University of Concepción, Concepción, Chile High-throughput sequencing technologies have offered the possibility to understand the complexity of the transcriptomic responses of an organism during a wide variety of biological scenarios, such as the case of pathogenic infections. Recently, the simultaneous sequencing of both pathogen and host transcriptomes (dual RNA-seq) during the infection has become a promising approach to uncover the complexity of the host–pathogen interactions. In this study, through a double rRNA depletion and RNA sequencing protocols, we simultaneously analyzed the transcriptome of the intracellular bacterium Piscirickettsia salmonis and its host the Atlantic salmon (Salmo salar) during the course of the infection. Beyond canonical host immune-related response and pathogen virulent factors, both bacteria and host displayed a large number of genes associated with metabolism and particularly related with the amino acid metabolism. Notably, genome-wide comparison among P. salmonis genomes and different ﬁsh pathogens genomes revealed a lack of the biosynthetic pathway for several amino acids such as valine, leucine, and isoleucine. To support this ﬁnding, in vitro experiments evidenced that when these amino acids are restricted the bacterial growth dynamics is signiﬁcantly affected. However, this condition is phenotypically reversed when the amino acids are supplemented in the bacterial growth medium. Based on our results, a metabolic dependency of P. salmonis on S. salar amino acids is suggested, which could imply novel mechanisms of pathogenesis based on the capacity to uptake nutrients from the host. Overall, dual transcriptome sequencing leads to the understanding of host–pathogen interactions from a different perspective, beyond biological processes related to immunity. Keywords: dual RNA-seq, Piscirickettsia salmonis, Atlantic salmon, nutritional immunity, metabolic dependency, amino acids Valenzuela-Miranda D and Gallardo-Escárate C (2018) Dual RNA-Seq Uncovers Metabolic Amino Acids Dependency of the Intracellular Bacterium Piscirickettsia salmonis Infecting Atlantic Salmon. Front. Microbiol. 9:2877. doi: 10.3389/fmicb.2018.02877 Edited by: Edited by: Suhelen Egan, University of New South Wales, Australia Reviewed by: Weiming Sun, Yantai University, China Chang Chen, University of Illinois at Chicago, United States Khalil Eslamloo, Memorial University of Newfoundland, Canada *Correspondence: Cristian Gallardo-Escárate crisgallardo@udec.cl; crisgallardo@oceanograﬁa.udec.cl Specialty section: This article was submitted to Microbial Symbioses, a section of the journal Frontiers in Microbiology Received: 07 August 2018 Accepted: 09 November 2018 Published: 27 November 2018 Keywords: dual RNA-seq, Piscirickettsia salmonis, Atlantic salmon, nutritional immunity, metabolic dependency, amino acids INTRODUCTION High-throughput sequencing technologies applied to transcriptomic studies (RNA-Seq) have oﬀered the possibility to understand complex molecular responses under diﬀerent biological scenarios. Among them, a pathogenic infection entails a deep transcriptomic remodeling of the host to promote the pathogenic clearance; in turn, pathogens display the expression of diﬀerent November 2018 \| Volume 9 \| Article 2877 Frontiers in Microbiology \| www.frontiersin.org Dual RNA-Seq of P. salmonis Infecting S. salar Valenzuela-Miranda and Gallardo-Escárate pathogen can evade host immune response are still unclear. Due to the high prevalence, negative impact and scientiﬁc interest on this pathogen, diﬀerent eﬀorts have tried to understand salmonids defensive mechanism against P. salmonis and how the bacteria overcome this response. Host transcriptomic response has been mainly associated with a regulation of genes involved in the innate immunity, apoptosis, diﬀerent signaling pathways, endocytosis, non-coding RNAs and iron metabolism among others (Rise et al., 2004; Tacchi et al., 2011; Pulgar et al., 2015; Valenzuela-Miranda and Gallardo-Escarate, 2016; Valenzuela-Miranda et al., 2017). On the other hand, P. salmonis transcriptomic response has been assessed after the in vitro infection in Sf21 cell lines (Machuca and Martinez, 2016). Although diﬀerent genes associated with the type IV secretion and iron acquisition system were identiﬁed, it remains unexplored how P. salmonis transcriptome is modulated during the infection. Due to this, we explore a dual RNA-Seq approach to unravel novel mechanisms of interactions during the infection of P. salmonis on the Atlantic salmon. A special emphasis was placed in bacterial gene expression, since transcriptional response of the Atlantic salmon against P. salmonis have been widely described previously (Tacchi et al., 2011; Pulgar et al., 2015; Valenzuela-Miranda and Gallardo-Escarate, 2016; Tarifeno-Saldivia et al., 2017). Beyond canonical pathogenic and immune related-genes, our results evidenced a common transcriptomic response between host and pathogen associated with the amino acid metabolism. Further analyses revealed a lack of P. salmonis genes associated with diﬀerent amino acids biosynthetic pathways and the importance of the availability of some amino acids for the bacterial growth medium. We hypothesize metabolic amino acids dependency of P. salmonis on S. salar, which could imply novel mechanisms of pathogenesis based on the capacity to uptake nutrients from the host and capacity of the host to regulate the availability of free amino acids. genes to grant their survival and replicate within the host. INTRODUCTION In this context, the simultaneous analysis of host and pathogen transcriptomes (dual RNA-Seq) during their interaction can reveal novel aspects of the infective process (Westermann et al., 2017). Initially, this approach was limited to viral, fungal and parasitic infections, where the pathogen resembles host transcripts (Tierney et al., 2012; Strong et al., 2013; Choi et al., 2014; Pittman et al., 2014) and scarcely reported in bacterial models (Westermann et al., 2012). However, the improvement of high-throughput sequencing and the development of novel RNA capture/depletion methods oﬀer a promising opportunity to also expand this approach to bacterial infections (Westermann et al., 2016). Although most dual RNA-seq approaches applied in bacterial infections have been exploratory, some of them have unraveled novel mechanisms of host–pathogen interaction. For instance, a dual RNA-seq was used to discover a possible strategy employed by Chlamydia trachomatis for the in vitro infection of human epithelial cells based on an early iron acquisition and a host immune depletion strategy (Humphrys et al., 2013). Furthermore, the simultaneous transcriptome analysis of the Gram-negative bacterium Haemophilus inﬂuenza during the infection of mucosal epithelial cells revealed the importance of the host oxidative response and the mechanisms employed by the bacteria to overcome this adverse environment (Baddal et al., 2016). Likewise, the co-transcriptomic analysis of the uropathogenic Escherichia coli (UPEC) and its host evidenced that while host transcriptomic response was similar to diﬀerent bacterial strains, diﬀerent expression patterns were identiﬁed in UPEC strains with contrasting pathogenic eﬀects (Mavromatis et al., 2015). These results were used to reveal novel insights into the mechanisms employed by the bacteria for the intra-macrophage survival. Moreover, a dual RNA-Seq was used to characterize the regulatory role of small RNAs (sRNAs) in Salmonella enterica infection. Here, researchers identiﬁed bacterial sRNA involved in the regulation of both host and pathogenic genes, revealing the hidden roles of S. enterica transcripts during the pathogenesis (Westermann et al., 2016). Simultaneous proﬁling of host-pathogen transcriptomes has become a powerful approach tool to unravel key aspects during the infection process. In the present study, we apply a dual RNA-Seq approach to reveal novel aspects of the infective process of the intracellular bacterium Piscirickettsia salmonis during the infection on the Atlantic salmon (Salmo salar). Frontiers in Microbiology \| www.frontiersin.org Experimental Design Experimental Design Atlantic salmons (154.7 ± 13.5g) were obtained from a commercial farm located at Puerto Montt, Chile and transferred to the Marine Biology Station of the University of Concepción (Dichato, Chile). Here, individuals were randomly screened to discard the presence of diﬀerent pathogens commonly present salmonid aquaculture. After quarantine, individuals were randomly divided into two independent marine water-based recirculating lines, each containing ﬁve 370 L tanks. For each line, three tanks were used for sampling (six in total), one tank was used to record mortality (two in total) and the remaining tank was not considered in this experiment. A total of 50 individuals per tank were maintained during an acclimation period of 14 days before the challenge. After this period, each individual was anesthetized and intraperitoneally injected with 0.2 ml of P. salmonis (EM-90 strain) containing 1 × 106 bacteria per dose, as previously standardized. Later, samples were collected from infected individuals at 3, 7, and 14 days post Piscirickettsia salmonis is a facultative intracellular gram-negative bacterium that causes the salmonid rickettsial septicemia (SRS), a severe systemic disease responsible for up to 85% of the infectious moralities in farmed salmons in Chile. Just in this country, the economic losses associated with this pathogen has been estimated in around US$100 million per year (Smith et al., 1997; Bravo and Midtlyng, 2007; Pulgar et al., 2015), becoming one of the main concerns for the industry (Mauel and Miller, 2002). Beyond this negative impact, perhaps one of the most remarkable features of P. salmonis is its capability to infect and replicate within host immune cells, such as in macrophages (McCarthy et al., 2008; Rajas et al., 2009; Ramirez et al., 2015). In this context, the mechanism whereby this November 2018 \| Volume 9 \| Article 2877 Frontiers in Microbiology \| www.frontiersin.org 2 Dual RNA-Seq of P. salmonis Infecting S. salar Valenzuela-Miranda and Gallardo-Escárate infection (dpi). Head kidney and spleen tissues were collected from two individuals of each sampling tank (12 individuals per point) and stored in RNA later solution (Ambion, United States) at −80◦C. In the remaining tanks, mortalities were daily recorded (Supplementary Figure S1), clinically and molecularly conﬁrmed as a result of SRS. All animal procedures were carried out under the guidelines approved by the Ethics Committee of University of Concepción. were used as baseline for gene expression comparison. RNA Isolation and Sequencing Strategy RNA Isolation and Sequencing Strategy Infected tissues stored at −80◦C in RNA later solution were thawed at room temperature and total RNA (host and pathogen) was isolated from 10 diﬀerent individuals using the TRIzol reagent kit (Thermo Fisher Scientiﬁc) according to manufactures instructions. RNA integrity was veriﬁed using the R6K screen tape 2200 on the TapeStation (Agilent Technologies, United States) platform. Thus, isolated RNAs with RNA Integrity Numbers (RIN) above 8 were considered for further analysis. Based on RNA quality, 3 diﬀerent pools of RNA were prepared from 3 distinct individuals for each tissue and time (biological replicates). RNA pools were precipitated in absolute ethanol and shipped in dry ice to Macrogen Inc. (Korea). Here, two Ribo-Zero rRNA Removal Kit (Illumina, San Diego, CA, United States) were used to remove both bacterial and host rRNAs. Remaining RNA containing both P. salmonis and S. salar transcripts were used to prepare high-throughput sequencing libraries using the TrueSeq RNA sample preparation kit (Illumina, San Diego, CA, United States). Each library was sequenced on a HiSeq platform at 100 bp paired-end reads (Macrogen, Korea). All sequencing data will be available under the SRA accession number SUB4576220. Sequencing statistics for each RNA-seq data are presented in Supplementary Table S1. Molecular annotation of the diﬀerentially expressed transcripts for both P. salmonis and S. salar was carried out to identify the most representative biological processes. For this purpose, the Gene Ontology (GO) annotation was conducted through the BLAST2GO software V 4.1.9 (Conesa et al., 2005) and the enrichment analysis was performed using as reference the genomes of P. salmonis and S. salar. Further, resulting GO enrichment analysis was visualized in REVIGO platform (Supek et al., 2011). Finally, KEGG pathway annotation analysis was also conducted using the KEGG automatic annotation server (Moriya et al., 2007) through the bidirectional best-hit assignment method. Furthermore, RT-qPCR were used to validate sequencing results. To do this, 10 genes from the bacteria and 10 genes from the ﬁsh were randomly selected and used to RT-qPCR primer design (Supplementary Table S2). After primer validation, each RT-qPCR was conducted in a thermocycler StepOne plus (Applied Biosystems, United States) using the Maxima SYBR Green/ROX kit according to manufactures instructions. Ampliﬁcation cycles were used as following 95◦C for 10 min, 40 cycles at 95◦C for 30 s, 60◦C for 30 s, and 72◦C for 30 s. RNA Isolation and Sequencing Strategy All qPCRs were carried on ﬁve biological and three technical replicates and expression values were estimated through the 21 Ct method using 16s and elongation factor 1a as normalizer genes for P. salmonis and S. salar, respectively. Signiﬁcant diﬀerences between 7 and 14 dpi regarding 3 dpi were estimated with the Student’s t-test (p < 0.05). A comparison between fold-changes obtained through RT-qPCR and RNA-seq evidence a r2 value above 0.8, evidencing a high correlation between the fold changes obtained by RT-qPCR and RNA-seq (Supplementary Figure S2). Individual fold changes were included as Supplementary Figure S3. Experimental Design We decided to use this dataset as reference because we needed transcriptomic data that contained reads from the pathogen to compare, since pathogenic reads in any type of control would not be present. Further, statistical diﬀerences were identiﬁed through a Baggerly’s test adjusting p-values through a false discovery rate (FDR) correction. Genes with a fold change > 4 and FDR p-values < 0.01 were considered as diﬀerentially expressed. Dual RNA-Seq and Differential Expression Analysis Raw sequencing reads were ﬁltered by quality and adapter/index were identiﬁed and removed from remaining reads using CLC Genomics Workbench (V10, CLC Bio, Denmark). In order to discriminate pathogen and host transcriptomes, cleaned reads were mapped against the last available version of the Atlantic salmon (S. salar)1 and against P. salmonis available genomes2. Mapping parameters included a mismatch cost of 2, insertion/deletion cost of 3 and a similarity/length fraction of 0.8. Eﬀectively mapped reads against both genomes were separated in diﬀerent ﬁles and used for further RNA-Seq analysis. RNA-Seq analysis was conducted using CLC Genomics Workbench (V10, CLC Bio, Denmark). Previously discriminated reads from host and pathogen were used to perform RNA-Seq analysis using all coding sequences annotated in the Atlantic salmon and P. salmonis genomes. For RNA-Seq analyses, similarity/length fraction was set as 0.9 in order to minimize the probability to include misassigned reads for each species. Expression values were estimated as transcripts per million (TPM) and normalized by totals per million read. Expression values obtained at 3 dpi 1https://www.ncbi.nlm.nih.gov/assembly/GCF_000233375.1/ 2https://www.ncbi.nlm.nih.gov/genome/genomes/11769 3https://www.ncbi.nlm.nih.gov/genome/genomes/511 4https://www.ncbi.nlm.nih.gov/genome/genomes/540 Exploring the Amino Acid Metabolism of P. salmonis Dual RNA-Seq analysis revealed a large number of genes diﬀerentially expressed associated with amino acid metabolism during the infection process. Due to this, we further explored the importance of amino acids in P. salmonis metabolism. First, a genome-scale comparison was conducted between P. salmonis, a second salmonid pathogen such as Aeromonas salmonicida and a closely related bacterium as Francisella tularensis. For this purpose, coding genes were obtained from NCBI for P. salmonis (see text footnote 2), F. tularensis3 and A. salmonicida4 3https://www.ncbi.nlm.nih.gov/genome/genomes/511 4https://www.ncbi.nlm.nih.gov/genome/genomes/540 November 2018 \| Volume 9 \| Article 2877 Frontiers in Microbiology \| www.frontiersin.org 3 Dual RNA-Seq of P. salmonis Infecting S. salar Valenzuela-Miranda and Gallardo-Escárate FIGURE 1 \| (A) Global transcriptome analysis of Piscirickettsia salmonis (blue) and S. salar (red) at 3, 7, and 14 days post-infection (dpi) in spleen and head kidney tissues. Expression values were estimated as transcripts per million reads (TPM) and Log2 transformed for heat map clustering. (B) Differentially expressed genes in spleen and head kidney at 7 and 14 dpi using as control the expression values at 3 dpi. Three days post-infection were used as baseline gene expression since no bacterial reads would be present in any type of control group. FIGURE 1 \| (A) Global transcriptome analysis of Piscirickettsia salmonis (blue) and S. salar (red) at 3, 7, and 14 days post-infection (dpi) in spleen and head kidney tissues. Expression values were estimated as transcripts per million reads (TPM) and Log2 transformed for heat map clustering. (B) Differentially expressed genes in spleen and head kidney at 7 and 14 dpi using as control the expression values at 3 dpi. Three days post-infection were used as baseline gene expression since no bacterial reads would be present in any type of control group. material for the growth of P. salmonis in CM medium. When the exponential phase was reached, 300 µL from the liquid culture was used to inoculate 2.7 ml of liquid culture containing the diﬀerent experimental mediums. All cultures were carried on triplicates and maintained at 20◦C with a constant agitation of 100 rpm. Bacterial growth was daily based monitored through the change in the optical density at an absorbance of 600 nm. A multiple t-test was carried out to identify statistically signiﬁcant diﬀerences (p < 0.01) between treatments. genomes. Exploring the Amino Acid Metabolism of P. salmonis All coding sequences were annotated through KAAS annotation server as described above, focusing our attention in Histidine metabolism (00340), valine, leucine and isoleucine degradation/biosynthesis (00280 and 00290), Arginine and proline metabolism (00330), Lysine biosynthesis/degradation (00300 and 00310), Cysteine and methionine metabolism (00270), Glycine, serine and threonine metabolism (00260), Phenylalanine, tyrosine and tryptophan biosynthesis (00400) and Alanine, aspartate and glutamate metabolism (00250). Furthermore, liquid cultures of P. salmonis were conducted considering diﬀerent experimental culture media with distinct amino acid composition. A basal medium (BM) was prepared with Eugon (30.4 g/l) supplemented with FeCl3 (2 mM), a complete medium (CM) prepared with Eugon (30.4 g/l) supplemented with FeCl3 (2 mM) and Casamino acid (1%) and experimental cultures medias were prepared with BM + 1% of the desired amino acid (Valine, leucine, and Isoleucine). A bacterial inoculum previously obtained from CHSE-214 cells infected with P. salmonis at 90% of lysis was used as starting Frontiers in Microbiology \| www.frontiersin.org Exploring Host and Pathogen Transcriptome During Pathogenesis Dual RNA-Seq analysis evidenced the modulation of P. salmonis and S. salar transcriptomes during the infection. This modulation is represented in two heat maps, one for the ﬁsh host (red) and another one for the pathogen (blue), where diﬀerent clusters November 2018 \| Volume 9 \| Article 2877 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 4 Dual RNA-Seq of P. salmonis Infecting S. salar Valenzuela-Miranda and Gallardo-Escárate FIGURE 2 \| Venn diagram showing the number of exclusive and shared genes in the transcriptome of P. salmonis during the infection in spleen (blue) and head kidney (red) tissues of Atlantic salar. The heat maps show a subset of exclusive and shared genes expressed by P. salmonis in both tissues. FIGURE 2 \| Venn diagram showing the number of exclusive and shared genes in the transcriptome of P. salmonis during the infection in spleen (blue) and head kidney (red) tissues of Atlantic salar. The heat maps show a subset of exclusive and shared genes expressed by P. salmonis in both tissues. the genes exclusively regulated in head kidney included a MATE eﬄux family protein, DNA repair protein RecN, chaperone protein HtpG and membrane proteins among others (Figure 2). Among shared genes, diﬀerent Protein phosphatase 1, ribosomal protein, chaperones and Asn/Gln aminotransferase subunits were also found (Figure 2). The complete list of diﬀerentially expressed genes for the Atlantic salmon and P. salmonis is included as Supplementary Table S3. of expression proﬁles were identiﬁed (Figure 1A). Regarding S. salar transcriptome, 771 and 829 genes were diﬀerentially expressed in spleen at 7 and 14 dpi, respectively (Figure 1B). Meanwhile, 412 and 467 genes were diﬀerentially modulated in the head kidney at the same time-points (Figure 1B). On the other hand, 68 and 79 P. salmonis genes were diﬀerentially expressed in the spleen at 7 and 14 days, respectively, while 14 and 44 were identiﬁed in the head kidney (Figure 1B). Thus, the number of bacterial genes diﬀerentially expressed were increased together with the course of the infection. Since transcriptional responses for the host have been previously reported, a special focus was placed on bacterial gene expression. Herein, a Venn diagram analysis revealed that 31 P. salmonis genes were diﬀerentially regulated in both spleen and head kidney, while 101 and 15 transcripts were exclusively regulated in spleen and head kidney, respectively. Exploring Host and Pathogen Transcriptome During Pathogenesis Genes exclusively regulated in spleen included a ferritin-like domain protein, genes associated with the type IV secretion system (VirB9, IcmL, and IcmW) and several outer membrane proteins (Figure 2). On the other hand, Frontiers in Microbiology \| www.frontiersin.org Amino Acid Metabolism: A Common Response Between P. salmonis and S. salar A functional annotation of the diﬀerent diﬀerentially expressed genes for both S. salar and P. salmonis was conducted in order to identify key molecular pathways regulated during the infective process. GO enrichment analysis evidenced that a large percent of diﬀerentially expressed genes in P. salmonis belonged to biological processes related with the metabolism of November 2018 \| Volume 9 \| Article 2877 Frontiers in Microbiology \| www.frontiersin.org 5 Dual RNA-Seq of P. salmonis Infecting S. salar Valenzuela-Miranda and Gallardo-Escárate FIGURE 3 \| Gene ontology annotation (A) and KEGG pathway annotation (B) of the differentially expressed genes in Salmo salar and P. salmonis during the infection. The size of each circle represents the frequency of the GO term in the underlying GO database (larger circles represent more general terms) and the color represents the number of genes in each term (value). Both host and pathogen displayed a large number of genes associated to amino acid biosynthesis and degradation. FIGURE 3 \| Gene ontology annotation (A) and KEGG pathway annotation (B) of the differentially expressed genes in Salmo salar and P. salmonis during the infection. The size of each circle represents the frequency of the GO term in the underlying GO database (larger circles represent more general terms) and the color represents the number of genes in each term (value). Both host and pathogen displayed a large number of genes associated to amino acid biosynthesis and degradation. signaling pathways (Supplementary Table S4), but also with key metabolic pathways, including the “biosynthesis of amino acids” (Figure 3B). Overall, the results evidenced that although the metabolism of amino acids was not the predominant transcriptomic response in the host, both P. salmonis and S. salar displayed a large number of genes involved with biosynthesis and degradation of amino acids. Due to this common response, we further investigate the role of amino acids in P. salmonis metabolism. proteins and nitrogen compounds, such as the terms “protein metabolism,” “cellular protein metabolism” and “cellular nitrogen compound biosynthesis” among others (Figure 3A). On the other hand, a more complex transcriptomic response was found in Atlantic salmon. Here, multiple biological processes were represented, such as organic acid metabolic process, oxidation-reduction process, response to external stimulus, chemotaxis among others (Supplementary Table S2). However, terms related to the metabolism of amino acids was also represented within diﬀerentially expressed transcripts, such as the “cellular amino acid metabolism” (Figure 3A). Amino Acid Metabolism: A Common Response Between P. salmonis and S. salar Enrichment of genes associated with protein metabolism was also found through KEGG annotation. Furthermore, one of the most represented pathways among diﬀerentially regulated genes in P. salmonis transcriptome included diﬀerent metabolic pathways, among them, the “biosynthesis and degradation of amino acids” (Figure 3B). Regarding host transcriptome, the response of the Atlantic salmon was associated not just associated with endocytosis, cytokine-cytokine receptor interaction, apoptosis, phagosome and Nod-like receptor Frontiers in Microbiology \| www.frontiersin.org DISCUSSION involved in biosynthesis/degradation of amino acids (Figure 4). However, a signiﬁcant lack of genes related with valine, leucine, and isoleucine metabolism was found in P. salmonis compared with F. tularensis and A. salmonicida, where over 60% of all possible genes were found (Figure 4A). However, a deeper look into this metabolic pathway evidenced that although P. salmonis lacks the majority of the genetic background for the biosynthesis of this amino acids, it was possible to found the gene that encodes for the primary degradation of valine, leucine, and isoleucine (Figure 4B). These results suggest that the bacterium is not able to biosynthesize these amino acids and therefore a metabolic dependency of P. salmonis on environmental host amino acid availability can be expected. To further explore the importance of amino acid availability to the bacteria, P. salmonis was growth in diﬀerent liquid culture mediums with diﬀerent amino acidic availability. A control medium (CM) fully supplemented with amino acids, a basal medium (BM) with no supplementation of amino acids and basal mediums supplemented with 1% of either valine (BM + V), leucine (BM + I) or isoleucine (BM + L) were used as experimental mediums. The results evidence that the bacteria are not able to grow when no amino acid is supplemented. However, when BM is supplemented with either valine, leucine or isoleucine, the growth kinetics of P. salmonis resembles the one observed in a fully amino acidic supplemented condition (Figure 5). involved in biosynthesis/degradation of amino acids (Figure 4). However, a signiﬁcant lack of genes related with valine, leucine, and isoleucine metabolism was found in P. salmonis compared with F. tularensis and A. salmonicida, where over 60% of all possible genes were found (Figure 4A). However, a deeper look into this metabolic pathway evidenced that although P. salmonis lacks the majority of the genetic background for the biosynthesis of this amino acids, it was possible to found the gene that encodes for the primary degradation of valine, leucine, and isoleucine (Figure 4B). These results suggest that the bacterium is not able to biosynthesize these amino acids and therefore a metabolic dependency of P. salmonis on environmental host amino acid availability can be expected. To further explore the importance of amino acid availability to the bacteria, P. salmonis was growth in diﬀerent liquid culture mediums with diﬀerent amino acidic availability. Role of Amino Acids in P. salmonis Metabolism To explore the importance of amino acids in P. salmonis metabolism, a genome-scale comparison was used to identify the presence/absence of genes directly involved in the degradation/biosynthesis of diﬀerent bacterial pathogens. Thus, P. salmonis genome was compared with F. tularensis and A. salmonicida available genomes. Results showed that all three pathogens dispose of a similar genetic background of genes November 2018 \| Volume 9 \| Article 2877 Frontiers in Microbiology \| www.frontiersin.org 6 Dual RNA-Seq of P. salmonis Infecting S. salar Valenzuela-Miranda and Gallardo-Escárate FIGURE 4 \| (A) Genomic-wide comparison of genes involved in the biosynthesis and degradation of amino acid in P. salmonis, a close related bacterium (F. tularensis) and another ﬁsh Gram negative bacterium (A. salmonicida). Bars represent the percentage of genes identiﬁed for each pathogen underlying all the possible genes for the pathway in KEGG pathway database. (B) Subset of the biosynthesis and degradation pathway for valine, leucine, and isoleucine in P. salmonis, F. tularensis, and A. salmonicida. Circles represent intermediate molecules and arrows the genes catalyzing each reaction. Thus, a green arrow represents a gene that was found in the genome of each pathogen and gray ones for those absent. Thus, P. salmonis evidenced a smaller number of genes involved in valine, leucine, and isoleucine biosynthesis/degradation. FIGURE 4 \| (A) Genomic-wide comparison of genes involved in the biosynthesis and degradation of amino acid in P. salmonis, a close related bacterium (F. tularensis) and another ﬁsh Gram negative bacterium (A. salmonicida). Bars represent the percentage of genes identiﬁed for each pathogen underlying all the possible genes for the pathway in KEGG pathway database. (B) Subset of the biosynthesis and degradation pathway for valine, leucine, and isoleucine in P. salmonis, F. tularensis, and A. salmonicida. Circles represent intermediate molecules and arrows the genes catalyzing each reaction. Thus, a green arrow represents a gene that was found in the genome of each pathogen and gray ones for those absent. Thus, P. salmonis evidenced a smaller number of genes involved in valine, leucine, and isoleucine biosynthesis/degradation. DISCUSSION A control medium (CM) fully supplemented with amino acids, a basal medium (BM) with no supplementation of amino acids and basal mediums supplemented with 1% of either valine (BM + V), leucine (BM + I) or isoleucine (BM + L) were used as experimental mediums. The results evidence that the bacteria are not able to grow when no amino acid is supplemented. However, when BM is supplemented with either valine, leucine or isoleucine, the growth kinetics of P. salmonis resembles the one observed in a fully amino acidic supplemented condition (Figure 5). Dual RNA-Seq has emerged as a promising approach to elucidate host–pathogen interaction. Although this approach was previously limited to pathogens that resembles host transcripts (Westermann et al., 2017), the development of high-throughput sequencing technologies has allowed to expand this approach to bacterial infections. In this context, we applied a dual RNA-Seq approach to explore novel means of interaction between the intracellular bacterium P. salmonis and its main host the Atlantic salmon (S. salar). In addition to being the main threat to Chilean salmonid aquaculture (Rozas and Enriquez, 2014), the intracellular nature of this pathogen makes it an interesting model to study host immune evasion strategies and intracellular survival mechanisms employed by intracellular pathogens. The dual RNA-Seq analysis revealed the presence of diﬀerent bacterial genes among the spleen and head kidney transcriptome. Thus, the spleen showed the largest number of bacterial genes regarding the ones found in head kidney data. This can be attributable due to when IP injection is used as infection method in previous IP challenges with P. salmonis, the spleen is one of the ﬁrst tissues to be infected, followed by head kidney (Valenzuela-Miranda and Gallardo-Escarate, 2016). Therefore, the diﬀerences between November 2018 \| Volume 9 \| Article 2877 Frontiers in Microbiology \| www.frontiersin.org 7 Dual RNA-Seq of P. salmonis Infecting S. salar Valenzuela-Miranda and Gallardo-Escárate FIGURE 5 \| Growth kinetics of P. salmonis in liquid culture media supplemented with different amino acids. Bacteria was growth in a control medium (CM), a basal medium without amino acids (BM) and in basal medium supplemented with valine (BM + V), leucine (BM + L), and isoleucine (BM + I) amino acids at 1%. ∗Represents statistically signiﬁcant differences (p < 0.01) between the supplemented medium (Val, Leu, or Ile) regarding control group (no amino acids). of several members of the type IV secretion system, including VirB9, IcmL, and IcmW. DISCUSSION The type IV secretion system it has been described as a conserved mechanism for the delivery of virulent factors from host to pathogen (Thanassi and Hultgren, 2000). This system has been previously described in P. salmonis (Gomez et al., 2013) and even the directed mutagenesis of this locus has resulted in the attenuation of the pathogenesis of P. salmonis (Mancilla et al., 2018). On the other hand, the repertory of genes displayed by the Atlantic salmon in response to infection was classiﬁed into diﬀerent molecular pathways. These processes included endocytosis, cytokine-cytokine receptor interaction, apoptosis, phagosome and Nod-like receptor signaling pathways, which has been previously described as key responses triggered during the infection of P. salmonis in S. salar tissues (Valenzuela-Miranda and Gallardo-Escarate, 2016). However, beyond the canonical pathogenic genes commonly associated with bacterial pathogenesis and the Atlantic salmon immunity (Valenzuela-Miranda and Gallardo-Escarate, 2016; Tarifeno-Saldivia et al., 2017), a common response associated with protein metabolism and particularly the biosynthesis/degradation of amino acids was present in both S. salar and P. salmonis transcriptomic response during the infection process. Due to this, a genome-scale comparison was performed in order to evidence the genetic background of genes involved in biosynthesis degradation of amino acids in P. salmonis, a close related bacterium F. tularensis and another salmonid bacterial pathogen A. salmonicida. The genomic background has been previously used to predict essential and non-essential amino acids in diﬀerent pathogens (Meibom and Charbit, 2010). Based on our results, we found a lack of biosynthetic genes associated with the metabolism of valine, leucine, and isoleucine for P. salmonis when compared with F. tularensis and A. salmonicida genomes. Therefore, the availability of these amino acids for P. salmonis might rely upon the presence of these resources in the host intracellular environment. During infection, intracellular pathogens must overcome diﬀerent adverse condition, such as the entrance to host cells, host immune response, free radicals and also nutrient deprivation. Although host cytosol was previously considered as an abundant source of nutrients for invading pathogens (Ray et al., 2009), recent evidence suggests that hosts can reduce the intracellular availability of certain nutrients as a protective response against the invading pathogens (Abu Kwaik and Bumann, 2013; Barel and Charbit, 2013). This deprivation results in a struggle between host and pathogen for the limited nutrient availability, which commonly known as nutritional immunity (Hood and Skaar, 2012). Frontiers in Microbiology \| www.frontiersin.org DISCUSSION Thus, it has been suggested that part of the immune response of the Atlantic salmon to P. salmonis infection relies on the nutritional immunity. This has been exclusively explored due to the struggle for iron availability. From P. salmonis perspective, it has been suggested the importance of a siderophore-based mechanism to capture iron from diﬀerent host sources (Calquin et al., 2018). On the other hand, the infection of P. salmonis induces a FIGURE 5 \| Growth kinetics of P. salmonis in liquid culture media supplemented with different amino acids. Bacteria was growth in a control medium (CM), a basal medium without amino acids (BM) and in basal medium supplemented with valine (BM + V), leucine (BM + L), and isoleucine (BM + I) amino acids at 1%. ∗Represents statistically signiﬁcant differences (p < 0.01) between the supplemented medium (Val, Leu, or Ile) regarding control group (no amino acids). FIGURE 5 \| Growth kinetics of P. salmonis in liquid culture media supplemented with different amino acids. Bacteria was growth in a control medium (CM), a basal medium without amino acids (BM) and in basal medium supplemented with valine (BM + V), leucine (BM + L), and isoleucine (BM + I) amino acids at 1%. ∗Represents statistically signiﬁcant differences (p < 0.01) between the supplemented medium (Val, Leu, or Ile) regarding control group (no amino acids). the transcriptional modulation of pathogen genes among this tissue could be as a result of diﬀerent infection stages rather than a tissue-speciﬁc transcriptomic response of the pathogen. Nevertheless, diﬀerent classical pathogenic related genes were identiﬁed in transcriptomic data. Such as the case November 2018 \| Volume 9 \| Article 2877 Frontiers in Microbiology \| www.frontiersin.org 8 Dual RNA-Seq of P. salmonis Infecting S. salar Valenzuela-Miranda and Gallardo-Escárate strong transcriptomic modulation of genes involved in iron availability in S. salar (Pulgar et al., 2015; Valenzuela- Miranda and Gallardo-Escarate, 2016). In this context, our results evidence that both host and pathogen display a large number of genes involved in the biosynthesis/degradation of amino acids. Considering the lack of biosynthetic pathways in leucine, valine and isoleucine in P. salmonis and that these same amino acids are deﬁned as essential amino acids for salmonids (Helland et al., 2010), we suggest that this transcriptional modulation can be reﬂecting an amino acid-based nutritional immunity triggered by S. salar to overcome P. salmonis infection. In turn, P. DISCUSSION salmonis displayed a transcriptional modulation of diﬀerent genes associated with amino acid metabolism to deal with host response. acid sources could become an advantage in a resource- limited scenario. This metabolic plasticity has been reported in other intracellular pathogens like F. tularensis, where the intracellular survival of this bacteria relies on their availability to exploit multiple host amino acids (Meibom and Charbit, 2010; Barel et al., 2015). Based in our results, and considering the recently proposed metabolic models for P. salmonis (Cortes et al., 2017; Fuentealba et al., 2017), we can hypothesize that this bacteria is capable to use diﬀerent amino acids as a carbon an energy resource. This strategy has been adopted by others intracellular bacterium, where this capability has been described as a crucial factor for virulence development (Eisenreich et al., 2010; Meibom and Charbit, 2010; Barel et al., 2015). In this scenario, the implications of a metabolic plasticity in P. salmonis and its link to virulence of diﬀerent bacterial strains must be further explored. Intracellular pathogens have developed diﬀerent strategies to overcome amino acids starvation triggered by the host during infection. Some of these adaptations include the growth arrest and diﬀerentiation, an amino acid self-suﬃciency and to exploit host machinery to obtain amino acids form host cell (Zhang and Rubin, 2013). Based on our results and considering the recent reconstruction of metabolic models for the bacteria (Cortes et al., 2017), the self-suﬃciency strategy for amino acids in the P. salmonis can be rejected. Regarding growth arrest and diﬀerentiation, it has been reported that during an amino acid restricted scenario C. trachomatis morphologically changes into an aberrant form that is unable to grow but protects them from a nutrient restricted environment (Leonhardt et al., 2007). However, the addition of tryptophan or isoleucine can restore these aberrant forms and reactivate bacterial growth (Hatch, 1975; Ibana et al., 2011). Previously, it has been reported the existence of morphological small variants of P. salmonis, which were suggested as a survival mechanism employed by the bacteria to overcome adverse scenarios (Veronica Rojas et al., 2008). However, the role of these variants during an amino acid restricted scenario and its relation with virulence remains unexplored. FUNDING This work was supported by the CONICYT-PCHA/Doctorado Nacional (Grant 2015-21150728), FONDECYT (1180867), and FONDAP (1510027). ACKNOWLEDGMENTS The authors are grateful for the support provided by the Ph.D. Program in Renewable Resource Management of the University of Concepción, Chile. DISCUSSION On the other hand, pathogens like Legionella pneumophila have devolved mechanism that grants the access to host nutrients by promoting the expression of host amino acid transporters and taking advantage of proteasomes of infected cells to generate free amino acids for bacterial growth (Wieland et al., 2005; Price et al., 2011). In this context, it has been suggested that one of the mechanisms employed by P. salmonis to evade immune response can be related with the regulation of host transcriptional response through non-coding RNAs (Valenzuela-Miranda and Gallardo-Escarate, 2016; Valenzuela- Miranda et al., 2017), therefore, the idea that P. salmonis is able to hijack host machinery to obtain amino acids should also be explored. Overall, our results showed how a dual RNA-Seq approach can lead us to the understanding of novel means of interaction between host and pathogens. However, the importance of an amino acid-based nutritional immunity of S. salar in response to P. salmonis infection must be further investigated. This information will not just lead us to the development of novel treatments for the pathogen, but also to the understanding of the pathogenesis process from a diﬀerent perspective, beyond canonical immunological mechanisms. 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Feast or famine: the host-pathogen battle over amino acids. Cell Microbiol. 15, 1079–1087. doi: 10.1111/cmi.12140 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Valenzuela-Miranda, D., Valenzuela-Munoz, V., Farlora, R., and Gallardo- Escarate, C. (2017). MicroRNA-based transcriptomic responses of atlantic salmon during infection by the intracellular bacterium Piscirickettsia salmonis. Dev. Comp. Immunol. 77, 287–296. doi: 10.1016/j.dci.2017.08.016 ev. Comp. Immunol. 77, 287–296. doi: 10.1016/j.dci.2017.08.016 Copyright © 2018 Valenzuela-Miranda and Gallardo-Escárate. November 2018 \| Volume 9 \| Article 2877 Frontiers in Microbiology \| www.frontiersin.org REFERENCES This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2018 Valenzuela-Miranda and Gallardo-Escárate. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Veronica Rojas, M., Olivares, P. J., del Rio, R., and Marshall, S. H. (2008). Characterization of a novel and genetically diﬀerent small infective variant of Piscirickettsia salmonis. Microb. Pathog. 44, 370–378. doi: 10.1016/j.micpath. 2007.10.012 Westermann, A. J., Barquist, L., and Vogel, J. (2017). Resolving host-pathogen interactions by dual RNA-seq. PLoS Pathog. 13:e1006033. doi: 10.1371/journal. ppat.1006033 November 2018 \| Volume 9 \| Article 2877 Frontiers in Microbiology \| www.frontiersin.org 11
https://openalex.org/W1986445745	https://europepmc.org/articles/pmc2129116?pdf=render	English	null	SAVVY® (C31G) Gel for Prevention of HIV infection in Women: A Phase 3, Double-Blind, Randomized, Placebo-Controlled Trial in Ghana	PloS one	2,007	cc-by	7,945	SAVVYH (C31G) Gel for Prevention of HIV infection in Women: A Phase 3, Double-Blind, Randomized, Placebo- Controlled Trial in Ghana Leigh Peterson1, Kavita Nanda1, Baafuor Kofi Opoku2, William Kwabena Ampofo3, Margaret Owusu-Amoako3, Andrew Yiadom Boakye2, Wes Rountree1, Amanda Troxler1, Rosalie Dominik1, Ronald Roddy4, Laneta Dorflinger1 1 Family Health International, Durham, North Carolina, United States, 2 Komfo Anokye Teaching Hospital, Kwame Nkrumah University of Science & Technology, Kumasi, Ghana, 3 Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana, 4 Duke Clinical Research Institute, Durham, North Carolina, United States Objective. The objective of this trial was to determine the effectiveness of 1.0% C31G (SAVVY) in preventing male-to-female vaginal transmission of HIV infection among women at high risk. Methodology/Principal Findings. This was a Phase 3, double-blind, randomized, placebo-controlled trial. Participants made up to 12 monthly visits for HIV testing, adverse event reporting, and study product supply. The study was conducted between March 2004 and February 2006 in Accra and Kumasi, Ghana. We enrolled 2142 HIV-negative women at high risk of HIV infection, and randomized them to SAVVY or placebo gel. Main outcome measures were the incidence of HIV-1 and HIV-2 infection as determined by detection of HIV antibodies from oral mucosal transudate specimens and adverse events. We accrued 790 person-years of follow-up in the SAVVY group and 772 person-years in the placebo group. No clinically significant differences in the overall frequency of adverse events, abnormal pelvic examination findings, or abnormal laboratory results were seen between treatment groups. However, more participants in the SAVVY group reported reproductive tract adverse events than in the placebo group (13.0% versus 9.4%). Seventeen HIV seroconversions occurred; eight in participants randomized to SAVVY and nine in participants receiving placebo. The Kaplan- Meier estimates of the cumulative probability of HIV infection through 12 months were 0.010 in the SAVVY group and 0.011 in the placebo group (p = 0.731), with a hazard ratio (SAVVY versus placebo) of 0.88 (95% confidence interval 0.33, 2.27). Because of a lower-than-expected HIV incidence, we were unable to achieve the required number of HIV infections (66) to obtain the desired study power. Conclusions/Significance. SAVVY was not associated with increased adverse events overall, but was associated with higher reporting of reproductive adverse events. Our data are insufficient to conclude whether SAVVY is effective at preventing HIV infection relative to placebo. Trial Registration. SAVVYH (C31G) Gel for Prevention of HIV infection in Women: A Phase 3, Double-Blind, Randomized, Placebo- Controlled Trial in Ghana ClinicalTrials.gov NCT00129532 Citation: Peterson L, Nanda K, Opoku BK, Ampofo WK, Owusu-Amoako M, et al (2007) SAVVYH (C31G) Gel for Prevention of HIV infection in Women: A Phase 3, Double-Blind, Randomized, Placebo-Controlled Trial in Ghana. PLoS ONE 2(12): e1312. doi:10.1371/journal.pone.0001312 METHODS The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1. Topical microbicides are designed to inhibit the sexual transmis- sion of Human Immunodeficiency Virus (HIV) and other disease pathogens. They offer a female-controlled prophylactic option in situations where male condom use cannot be negotiated. Marketed chemical spermicides, which show some activity against sexually transmitted pathogens in vitro, have been evaluated as topical microbicides. However, no clinical studies have yet demonstrated that these products can prevent HIV infection, and spermicides with the surfactant nonoxynol-9 (N-9) have caused mucosal erosion and ulceration, which may increase the risk of HIV transmission [2–4]. Participants Although we did not specifically ask as part of the clinical trial procedures if the participants were sex workers, most exchanged sex for money. Special ethical considerations were taken into account because of the potential vulnerability of this population. We included women who agreed to: use study gel as directed and follow study procedures; report self-medication with antibiotics during study participation; and avoid use spermicides or other vaginal contraceptives or lubricants during the study. We excluded women who: were intending pregnancy; had a history of latex allergy; were injection drug users; or had gynecological conditions that could affect the safety or effectiveness of the study gel. informed consent form stated that: ‘‘We do not know the effects and safety of the gel during pregnancy. Pregnant women may not join this study. If you become pregnant during the study you should tell your study doctor or nurse right away. Your study gel will be stopped and the study doctor will discuss your choices with you.’’ y At each monthly follow-up visit, participants underwent OMT HIV and pregnancy testing, AE assessment, STI risk reduction counseling, and study product and condom re-supply. Participants responded to structured questionnaires on their interval sexual behavior (including coital activity, and gel and condom use), experience using the gel, and were reminded of study concepts discussed during the informed consent process. If clinically indicated, participants underwent physical examination and STI testing. Study staff documented whether product use was interrupted temporarily or permanently for any of the following reasons: participant ran out of supplies, investigator withdrew study product in the interest of the safety and well being of the participant, positive pregnancy test result, or confirmed HIV infection. Pregnant women were allowed to resume study product use after pregnancies had ended. Study staff referred participants infected with HIV during the study to local HIV-related psychological, social, and medical services (such as viral load, CD4 level, and HIV resistance testing) as well as antiretroviral drug therapy when needed. If a participant missed a scheduled follow-up appointment, study staff made up to 3 attempts to contact that participant and reschedule the appointment (ideally to occur within 2 weeks of the original appointment). If the participant could not be located after 3 attempts, no further efforts were made to find her, but her file remained open until study closeout. Participants We conducted this randomized, double-blind, placebo-controlled trial between January 2004 and March 2006 in Accra and Kumasi, Ghana. We enrolled HIV-antibody negative, non- pregnant women 18 to 35 years old who were at risk of HIV Several in vitro studies [5] have suggested that C31G (SAVVYH, Cellegy Pharmaceuticals [formerly BIOSYN, Inc.], Huntington Valley, Pennsylvania) has a potent effect on enveloped HIV by disrupting the outer membrane. However, because its mechanism of action is similar to N-9 [6;7], some have raised concerns about the safety of SAVVY. In pre-clinical studies, SAVVY demonstrated minimal toxicity at 0.001% concentration, as measured by the cell proliferation assay, and no toxicity to mammalian cells at 0.0003%, as measured by the MTT assay [5]. In addition, 5 Phase 1 clinical studies, including a total of 121 women and 24 men exposed to SAVVY, have been conducted using three concentrations of SAVVY (0.5%, 1.0%, 1.7%). No serious or severe adverse events related to use of 1.0% SAVVY gel were reported during these studies [8–10]. We investigated the safety and effectiveness of 1.0% SAVVY in preventing HIV infection in a population of young, sexually active women at high risk for acquiring HIV infection. Academic Editor: Keymanthri Moodley, University of Stellenbosch, South Africa Received September 5, 2007; Accepted September 6, 2007; Published December 19, 2007 Copyright: 2007 Peterson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by funds from the United States Agency for International Development (USAID) under Cooperative Agreement CCP-A-00-95- 00022-02 and GPO-A-00-05-00022-00. Cellegy Pharmaceuticals (formerly BIOSYN, Inc.) (Huntington Valley, Pennsylvania) donated the investigational product (SAVVY and placebo-gel applicators) evaluated in this study. Competing Interests: The authors have declared that no competing interests exist. To whom correspondence should be addressed. E-mail: KNanda@fhi.org PLoS ONE \| www.plosone.org December 2007 \| Issue 12 \| e1312 1 SAVVY Gel for HIV Prevention SAVVY Gel for HIV Prevention infection because of having a mean of three or more coital acts per week and two or more sexual partners in the 3 months before screening. Study participants were recruited from areas within each city that were considered high HIV transmission areas, including markets, bars, and hotels. Outcomes h i The primary measure of effectiveness was infection with HIV-1 or HIV-2, measured by detecting antibodies in oral mucosal transudate (OMT) (OraQuickH ADVANCE Rapid HIV-1/2 Antibody Test, Orasure Technologies) and confirmed by an enzyme-linked immunosorbent assay (ELISA) (Genetic SystemsTM HIV-1/HIV-2 Plus O ELISA from BioRad) and/or Western Blot (Genetic SystemsTM HIV-1 Western Blot, BioRad) from a finger prick or serum specimen. We evaluated safety by comparing the incidence of adverse events (AEs) including pelvic exam findings and sexually transmitted infections (STIs). At this second visit, participants received a detailed explanation of study procedures, signed or marked a consent form for enrollment, received HIV counseling, provided urine for preg- nancy testing, provided another OMT sample for HIV testing, and if eligible were randomized to receive either SAVVY or placebo. Study staff gave each eligible participant her first month’s supply of study product after she had been randomized. Clinic staff counseled participants to use the gel vaginally before each act of sexual intercourse (and to insert a second dose if more than one hour had elapsed between the first application and sexual intercourse), emphasized that the gel had unknown effectiveness for preventing HIV, distributed condoms, and counseled partic- ipants to use condoms for all sexual contacts with all partners. The Interventions During recruitment, study staff explained the general purpose of the study and the eligibility requirements. If eligible, women were referred to one of two study clinics in Kumasi or Accra. At the screening visit, women were interviewed to confirm understanding and willingness to comply with study requirements, completed written informed consent, received HIV pretest and condom counseling, and underwent oral mucosal transudate (OMT) rapid HIV testing. All participants received HIV post-test counseling, physical and pelvic examinations (including vaginal wet mount to support the diagnosis for bacterial vaginosis, trichomoniasis, or vaginal candidiasis), urine pregnancy tests, and STI (syphilis, gonorrhea, and chlamydia) tests. Women with reactive OMT rapid HIV tests received ELISA to confirm HIV status. We asked potential participants to return 4 weeks after their screening visit to receive the results of their STI and confirmatory HIV tests, if applicable. Ethics We developed strategies to protect the confidentiality and autonomy of the participants, increase/ensure comprehension of the informed consent and research methods, and promote access to resources and services during and after the trial. The study protocol and informed consent forms were approved by 1) the Committee on Human Research, Publications and Ethics, School of Medical Sciences, University of Science & Technology, Kumasi, Ghana, 2) Noguchi Memorial Institute for Medical Research IRB, University of Ghana, Legon, Ghana, and 3) the Protection of Human Subjects Committee, Family Health International, USA. All participants provided written informed consent in their preferred language. Illiterate participants were read the informed consent forms verbatim in the presence of a witness, and provided a mark or thumbprint in lieu of signature. Participants If the participant did not return to the study before the study was closed, she was considered lost to follow-up. The ‘‘lost to follow-up’’ designation was not made for any participant until the closing date of the study. Objective The objective of this trial was to determine the effectiveness of 1.0% C31G in preventing male-to-female vaginal transmission of HIV infection among women at high risk. PLoS ONE \| www.plosone.org Sample Size We estimated that a sample size of 2142 participants (1,071 in each treatment group) would give us 80% power to detect a 50% difference in the HIV infection rate (two-sided log-rank test, a = 0.05 significance level) between the two groups. We assumed the rate of HIV infection in the control group to be 5/100 person- years and loss to follow-up to be 20%; approximately 66 total HIV infections were needed to achieve the desired power. Our protocol included plans for assessing (in a blinded manner) whether additional participants would be needed to observe the required 66 events. PLoS ONE \| www.plosone.org December 2007 \| Issue 12 \| e1312 2 SAVVY Gel for HIV Prevention ments, would review AEs and primary safety and HIV seroconversion data twice, after approximately 16 and 33 events, respectively. However, testing for early evidence of effectiveness was scheduled only to occur at the second of these two planned looks and later when the target total number of events (66) was obtained (i.e., the first look at the HIV seroconversion data was to review the data for signs of harm) . Statistical Methods For the primary effectiveness analysis we compared the probability of HIV infection for the SAVVY and placebo gel groups using a two- sided site-stratified, exact log-rank test (StatXact). We calculated Kaplan-Meier estimates of HIV infection probabilities by treatment group, pooled across sites. We used a proportional hazards regression model to estimate the hazard ratio, along with a 95% confidence interval, comparing the SAVVY group to the placebo group for HIV incidence, controlling for site. Each HIV infection onset date was estimated as the midpoint between the date of the first positive test result and the previous, negative test date. A right censoring time of 380 days was applied. Because the trial was terminated well before reaching the number of HIV infections targeted for pre-planned tests of effectiveness (i.e., before any of the type I error was to be spent), p-values for analyses of effectiveness should be interpreted as descriptive statistics. We calculated exact 95% confidence intervals for the relative risk of pre-specified priority AEs within system organ classes under a Poisson assumption for the event rates in each treatment group (StatXact). We compared proportions of women with any pelvic exam findings or STDs between treatment groups with a two-sided Mantel-Haenszel Chi- Square Test stratified by site at the 0.05 significance level. Due to premature termination of the study, 660 participants exited the study before completing their Month 12 visit (Figure 1). Twelve additional participants (0.6%) discontinued early (6 in each treatment group), including one participant in each of the treatment groups who died during follow-up. Other than the deaths, which were unrelated to product use, no participants withdrew early due to a medical reason. Most enrolled participants in both groups were young (mean age 22.7), unmarried, and had fewer than 9 years of education. Other demographic character- istics and medical history were also similar (Table 1). All primary and most secondary analyses were either conducted on the Effectiveness Population which is the subset of the Intent- to-Treat (ITT) Population for whom at least one post-enrollment HIV evaluation is available, or the Safety Population which is the subset of the ITT Population who ever returned after enrollment. The Evaluable Population includes the same participants as the Effectiveness Population but excludes all data collected from a participant after her first documented interruption of product use. Recruitment, Participant Flow, Numbers Analyzed and Baseline Characteristics Participants were recruited and enrolled into this study for 15 months, starting February 2004. A total of 3,490 women were screened, from which we enrolled 2,142 participants into the study (Figure 1). The primary reasons potential participants were not enrolled were because they failed to return for enrollment, were HIV-infected at baseline, or were pregnant. Overall loss to follow- up after enrollment was approximately 15% (n = 310). The loss-to- follow-up rate was highest during the first month of study participation, during which 40% of the overall loss to follow-up (123 of 310 participants) occurred. Among these were 103 participants who never returned after their enrollment visit and were therefore excluded from the primary safety analyses. The placebo gel was formulated to minimize any possible effects—negative or positive—on study endpoints. It was isotonic to avoid epithelial cell swelling or dehydration, and formulated at a pH of 4.4 but with minimal buffering capacity. When mixed with an equal volume of semen, the placebo gel induced only a trivial decrease in semen pH (from 7.8 to 7.7). The placebo gel contained hydroxyethylcellulose as a gelling agent, and its viscosity was comparable to that of SAVVY. Hydroxyethylcellulose does not have anti-HIV properties. The gel also contained sorbic acid as a preservative; sorbic acid has no anti-HIV activity and is readily metabolized by human cells. The first scheduled DMC safety review was repeated because errors were found in the initial HIV seroconversion data due to false positive ELISA testing. At the third DMC safety review meeting, when there were 16 seroconversions in the database, we informed the DMC that the study statistician estimated that approximately 3,500 additional participants (beyond the 2,142 planned sample size) would be needed to achieve the required number of HIV infections (66) to obtain the desired study power. In light of this formidable challenge, we requested that the DMC review the available outcome data by group (along with results of conditional power calculations under a range of assumptions about the true effect size) and provide a recommendation as to whether the trial should continue or be terminated early. The DMC recommended that the trial be stopped; we thus decided to prematurely terminate the study in November 2005. PLoS ONE \| www.plosone.org Randomization and Blinding We randomized enrolled participants into either the SAVVY or placebo arm using a 1:1 allocation ratio. A statistician not otherwise involved with the study developed the allocation sequence using a computer random number generator and randomly varied permuted-blocks of 12, 18, and 24. Six label colors (three SAVVY and three placebo) were used to differentiate the otherwise identically packaged gels. Randomization was stratified by study site. We used sequentially numbered, sealed opaque envelopes to assign participants to one of six color groups after they had qualified for the study and signed the consent form. The randomization envelopes were maintained in a secure office and were not available to study staff until the moment of randomization. Each randomization envelope was used only once. Participants, field study staff, monitors, statisticians, and other FHI staff involved in the trial were not aware of which gel colors were associated with SAVVY or placebo. Both SAVVY and placebo gels were clear, with similar viscosity and pH, dispensed in 3.5 mL doses with identical applicators. Monitors (trained in Good Clinical Practice) visited sites regularly to review study eligibility, informed consent, protocol compliance, laboratory procedures, source documents, product accountability, and AEs. We attempted to get original hospital records, when available, for serious adverse events (SAEs) and deaths. Sexual Behavior At enrollment participants reported a mean coital frequency of 9– 10 acts per week (SAVVY 9.5; placebo 8.9) and a mean of 5.8 different partners in the last 30 days (in both groups). Approxi- mately 2% of the participants (SAVVY 1.8%; placebo 2.5%) reported having anal sex in the previous 30 days. During follow- up, participants consistently reported a mean of 7 coital acts per week, with a mean of 5–6 sexual partners in the previous 30 days. Self-reported condom use increased from 40% during the last Our data monitoring plan specified that the independent Data Monitoring Committee (DMC), with access to treatment assign- December 2007 \| Issue 12 \| e1312 3 SAVVY Gel for HIV Prevention Figure 1. Participant Trial Flow Diagram (P-Y, person years). doi:10.1371/journal.pone.0001312.g001 Figure 1. Participant Trial Flow Diagram (P-Y, person years). doi:10.1371/journal.pone.0001312.g001 coital act before screening to over 89% at the first follow-up visit, remaining relatively constant throughout follow-up. receiving placebo), no abnormal outcomes were reported. A total of 40 live births (all normal) were reported to have occurred among study participants. Although pregnant participants are included in the intent-to- treat analysis, we asked these participants to stop gel use upon a positive pregnancy test (they could return to product use after a negative pregnancy test). A total of 383 participants in the SAVVY group and 386 participants in the placebo group became pregnant at least once during the study. The Kaplan-Meier pregnancy probability at 12 months was 42.5 per 100 person-years in the SAVVY group and 43.7 per 100 person-years in the placebo group. Of the women who became pregnant during the study, most (79%) were pregnant only once. However, approximately 19% were pregnant twice, and almost 2% were pregnant three times during the course of the study. The median amount of time without gel use due to pregnancy (among the participants who became pregnant) was approximately 2 months, resulting in 151 person-years (or 10% of the total person-time) off product due to pregnancy. Of the 942 total pregnancies detected during the study (among 769 participants), pregnancy outcome information was available for 606 pregnancies. Other than 13 spontaneous abortions (4 in participants receiving SAVVY and 9 in participants Safety: Adverse Events Approximately one-third of participants in both groups had adverse events (33% in the SAVVY group and 29% in the placebo group), with no significant differences in the overall frequency of AEs between treatment groups. The most frequently reported AEs included: malaria, abdominal pain, headache, genital pruritus, vaginal candidiasis, general pain, bacterial vaginitis, and vaginal discharge. No significant differences in frequency of AEs occurred between treatment groups. However, more participants in the SAVVY group had reproductive tract AEs, with an incidence of 14.5 per 100 person-years in the SAVVY group versus 10.9 per 100 person-years in the in the placebo group (rate ratio = 1.33, 95% confidence interval 0.99, 1.80, [Table 3]). The most frequently occur- ring AEs with the reproductive tract were genital pruritus, vaginal candidiasis, vaginal discharge, bacterial vaginitis, and vulvovaginitis. For each participant and variable of interest (e.g., percentage of vaginal acts where study gel was used in the last 7 days prior to the follow-up visit), we first calculated the participant’s mean value of the variable of interest across all of their follow-up visits. (Follow-up visits where women reported 0 or a missing number of vaginal sex acts in the last 7 days are excluded from the calculation of a participant’s mean value.) The mean, SD, range, and median values of the distributions of these mean values were then obtained for each treatment group. doi:10.1371/journal.pone.0001312.t002 In post hoc analyses that examined subgroups defined by self- reported frequency of gel use, we found a greater treatment group difference in the incidence of reported reproductive system and breast disorders (17.3 per 100 person-years in the SAVVY group and 10.1 per 100 person-years in the placebo group) among the subgroup of participants whose self-reported gel use was at or below the overall median than among the subgroup of participants whose self-reported gel use was above the overall medium. Among participants whose self-reported gel use was at or below the overall median the rate ratio was 1.71 (95% confidence interval = 1.13–2.61). Similarly, the rate ratio was 1.64 (95% confidence interval = 1.09–2.50) for the subgroup of participants with self-reported sexual frequency at or below the overall median (17.4 per 100 person-years in the SAVVY group and 10.6 per 100 person-years in the placebo group). participants had pelvic examinations (63 in the SAVVY group and 58 in the placebo group). Product Use Participants reported that they used the gel for an average of 75% and 77% of coital acts in the SAVVY and placebo groups, respectively, and reported condoms use for 89% and 90% of coital acts in the SAVVY and placebo groups, respectively. They reported using both gel and condoms for 70.1% and 71.7% of acts in the SAVVY and placebo groups, respectively. Participants reported using neither gel nor condoms for 6.2% and 5.3% of acts in the SAVVY and placebo groups, respectively. Gel use decreased with time in both groups (from 86–87% during Month 1 to 67–71% during Month 12), whereas condom use was more consistent. From these data, we calculated that SAVVY or placebo gel was used alone (i.e., without a condom) for approximately 5% of all vaginal acts and that condoms were used alone (i.e., without gel) for approximately 16% of all vaginal acts. We also calculated that SAVVY or placebo gel was used for 81% of vaginal acts during which a condom was also used, but for only 43–47% of acts where no condom was used (Table 2). PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org December 2007 \| Issue 12 \| e1312 4 SAVVY Gel for HIV Prevention Table 2. Overall Estimates of Gel and Condom Use at Follow- Up by Treatment Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Product Use Overall SAVVY N = 1021 Placebo N = 1008 Mean percentage of vaginal sex acts in the last 7 days with study gel Mean (SD) 75.0 (24.0) 77.0 (23.1) Range (median) 0–100 (81.6) 0–100 (83.0) Mean percentage of vaginal sex acts in the last 7 days with a condom Mean (SD) 88.6 (16.5) 89.5 (15.9) Range (Median) 0–100 (94.9) 0–100 (96.0) Mean percentage of vaginal sex acts in the last 7 days with both the study gel and a condom Mean (SD) 70.1 (26.5) 71.7 (25.8) Range (Median) 0–100 (76.5) 0–100 (77.8) Mean percentage of vaginal sex acts in the last 7 days with neither a condom nor study gel Mean (SD) 6.2 (11.8) 5.3 (10.7) Range (Median) 0–100 ( 0.0) 0–100 ( 0.0) Mean percentage of vaginal sex acts in the last 7 days with study gel only (without a condom) Mean (SD) 5.1 (17.6) 5.2 (17.9) Range (median) 0–100 ( 0.0) 0–100 ( 0.0) Mean percentage of vaginal sex acts in the last 7 days with a condom only (without study gel) Mean (SD) 16.4 (32.4) 16.5 (32.7) Range (Median) 0–100 ( 0.0) 0–100 ( 0.0) Mean percentage of vaginal sex acts in the last 7 days with study gel and a condom Mean (SD) 80.8 (35.8) 80.8 (36.2) Range (Median) 0–100 ( 100) 0–100 ( 100) Mean percentage of vaginal sex acts in the last 7 days with study gel when a condom is not used Mean (SD) 43.1 (47.3) 46.9 (47.9) Range (Median) 0–100 ( 0.0) 0–100 (29.3) For each participant and variable of interest (e.g., percentage of vaginal acts where study gel was used in the last 7 days prior to the follow-up visit), we first calculated the participant’s mean value of the variable of interest across all of their follow-up visits. (Follow-up visits where women reported 0 or a missing number of vaginal sex acts in the last 7 days are excluded from the calculation of a participant’s mean value.) The mean, SD, range, and median values of the distributions of these mean values were then obtained for each treatment group. doi:10.1371/journal.pone.0001312.t002 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Product Use Overall Estimates of Gel and Condom Use at Follow- Up by Treatment Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table 1. Baseline Characteristics (ITT Population) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SAVVY (N = 1073) Placebo (N = 1069) doi:10.1371/journal.pone.0001312.t001 PLoS ONE \| www.plosone.org Product Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table 1. Baseline Characteristics (ITT Population) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SAVVY (N = 1073) Placebo (N = 1069) Characteristic Age 22.7 (3.6) 22.7 (3.6) Marital status Unmarried, not cohabiting 946 (88.2) 945 (88.4) Education # 9 years 831 (77.4) 812 (76.0) Pregnancy history Ever pregnant 782 (72.9) 782 (73.2) Number of pregnancies 2.1 (1.4) 2.1 (1.4) Number of vaginal deliveries 0.9 (1.0) 0.9 (0.9) Contraceptive use Condom 496(46.2) 516 (48.3) None 423 (39.4) 397 (37.1) Oral 122 (11.4) 125 (11.7) Injectable 23 (2.1) 22 (2.1) IUD 5 (0.5) 5 (0.5) Other 4 (0.4) 4 (0.4) History of STI 182 (17.0) 199 (18.6) Previous spermicide use 91 (8.5) 77 (7.2) Douching 578 (53.9) 555 (51.9) Data reported as N(%) or mean (SD); SD = standard deviation, STI = sexually transmitted infection doi:10.1371/journal.pone.0001312.t001 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table 2. Safety: Adverse Events No significant differences were seen between treatment groups for bacterial vaginosis, trichomoniasis, or vaginal candidiasis on saline wet mounts. Twenty-two SAEs were reported; 15 by participants in the SAVVY group and 7 by participants in the placebo group. Only one, gonorrhea, was classified by the investigator as possibly related to the study product (the participant had received placebo). Two deaths occurred during the course of the study. One was suspected to be due to possible sickle cell crisis (the participant had been randomized to SAVVY), and one was due to viral hepatitis complicated by hepatic encephalopathy (the participant had been randomized to placebo). No participant was asked to discontinue study drug due to an AE. Participants underwent pelvic examination and STI testing during follow-up only if clinically indicated. During follow-up, 121 December 2007 \| Issue 12 \| e1312 December 2007 \| Issue 12 \| e1312 5 SAVVY Gel for HIV Prevention Table 3. Selected Priority Adverse Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table 3. Selected Priority Adverse Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Safety: Adverse Events . . . . . . . . . System Organ Class/ Preferred Term SAVVY (N = 1027 ) Placebo (N = 1012 ) Rate Ratio (95% CI), SAVVY vs. Placebo No. of Events No. of Women % of Women IR* (per 100 person years) No. of Events No. of Women % of Women IR* (per 100 person years) Reproductive system and breast disorders 140 107 10.4 14.5 120 80 7.9 10.9 1.33 (0.99, 1.80) Amenorrhoea 3 3 0.3 0.4 0 0 0.0 0.0 INF Cervicitis 0 0 0.0 0.0 1 1 0.1 0.1 0.00 Dysmenorrhoea 6 6 0.6 0.8 4 4 0.4 0.5 1.47 Dyspareunia 1 1 0.1 0.1 1 1 0.1 0.1 0.98 Genital abscess 1 1 0.1 0.1 2 2 0.2 0.3 0.49 Genital pruritus female 28 27 2.6 3.5 23 20 2.0 2.6 1.32 Genitourinary tract gonococcal infection 0 0 0.0 0.0 1 1 0.1 0.1 0.00 Menorrhagia 1 1 0.1 0.1 2 2 0.2 0.3 0.49 Menstruation irregular 7 7 0.7 0.9 7 6 0.6 0.8 1.14 Ovulation pain 1 1 0.1 0.1 1 1 0.1 0.1 0.98 Pelvic inflammatory disease 5 5 0.5 0.6 4 4 0.4 0.5 1.23 Post coital bleeding 1 1 0.1 0.1 1 1 0.1 0.1 0.98 Vaginal abscess 3 3 0.3 0.4 1 1 0.1 0.1 2.94 Vaginal burning sensation 0 0 0.0 0.0 3 2 0.2 0.3 0.00 Vaginal candidiasis 29 28 2.7 3.6 30 28 2.8 3.7 0.98 Vaginal discharge 16 16 1.6 2.0 13 11 1.1 1.4 1.43 Vaginal erythema 4 3 0.3 0.4 0 0 0.0 0.0 INF Vaginal haemorrhage 0 0 0.0 0.0 1 1 0.1 0.1 0.00 Vaginal laceration 0 0 0.0 0.0 2 2 0.2 0.3 0.00 Vaginal pain 2 2 0.2 0.3 1 1 0.1 0.1 1.96 Vaginitis bacterial 19 19 1.9 2.4 16 14 1.4 1.8 1.33 Vulvovaginal ulceration 1 1 0.1 0.1 1 1 0.1 0.1 0.98 Vulvovaginitis 11 11 1.1 1.4 5 5 0.5 0.6 2.16 Vulvovaginitis trichomonal 1 1 0.1 0.1 0 0 0.0 0.0 INF Gastrointestinal disorders 85 73 7.1 9.7 94 76 7.5 10.4 0.93 (0.67, 1.30) Pregnancy, puerperium and perinatal conditions 7 7 0.7 0.9 13 12 1.2 1.6 0.57 (0.19, 1.57) Renal and urinary disorders 15 12 1.2 1.5 18 17 1.7 2.2 0.69 (0.30, 1.53) *Incidence Rate . . . . . . . . . . . . . . . Safety: Adverse Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effectiveness: HIV Incidence DISCUSSION The overall HIV incidence rate was 1.09 per 100 person years (95% confidence interval 0.63, 1.74). In the Effectiveness Population, we observed 17 HIV seroconversions, 8 in the SAVVY group and 9 in the placebo group. The Kaplan-Meier estimates of the cumulative probability of HIV infection at 12 months were 0.010 in the SAVVY group and 0.011 in the placebo group (log-rank p = 0.731), with a hazard ratio (SAVVY versus placebo) of 0.88 (95% confidence interval 0.33, 2.27). Of these 17 seroconversions, 9 were in Accra and 8 occurred in Kumasi. Fifteen seroconversions (7 SAVVY; 8 placebo) occurred in participants younger than 25 years. Ten (5 SAVVY; 5 placebo) of the seroconverters had baseline coital frequency at or below the median and 7 (3 SAVVY; 4 placebo) had baseline coital frequency above the median. Interpretation and Overall Evidence Interpretation and Overall Evidence We stopped this study prematurely following recommendations of an independent DMC because the HIV incidence among enrolled participants was substantially lower than expected; we therefore could not evaluate the effectiveness of SAVVY in preventing HIV as intended. Due to the small number of events available for analysis, this trial was unable to meet the objective of accessing the effectiveness of SAVVY in preventing male-to-female transmission of HIV. To date, no randomized clinical studies have identified a microbicide that is effective at preventing HIV infection [11]. Lower-than-expected HIV incidence has been an increasing problem in HIV prevention trials and the lower-than-expected HIV incidence seen in this trial may have been due in part at least 3 factors: 1) a greater-than-expected effect of trial risk reduction measures such as condom use, 2) participants who join clinical trials may be more inclined to safer behavior than those in their community who do not participate, and 3) an inaccurate estimate of trial incidence due to changes in the local epidemic. The When we repeated the analysis in the Evaluable Population, we observed 11 HIV infections (4 in the SAVVY group; 7 in the placebo group). The Kaplan-Meier estimates of the cumulative probability of HIV infection at 12 months were 0.007 in the SAVVY group and 0.012 in the placebo group, with a hazard ratio of 0.57 (95% confidence interval of 0.17 to 1.94). PLoS ONE \| www.plosone.org December 2007 \| Issue 12 \| e1312 December 2007 \| Issue 12 \| e1312 6 SAVVY Gel for HIV Prevention actual negative association between treatment effect and frequency of intercourse, is unclear. actual negative association between treatment effect and frequency of intercourse, is unclear. incidence rate for this trial was estimated from our experience in earlier trials in a similar population and was not specifically measured in each population before starting the study. One limit of our study is generalizability. Although we did not specifically recruit sex workers, women in our study averaged a coital frequency of 7 acts per week and 5–6 different partners in the last month. Thus conclusions should be interpreted cautiously with regard to other populations. Ensuring high product adherence is another important challenge in the successful conduct of HIV prevention studies. Low product use due to such factors as pregnancy, missed visits, or participant’s choice compromises study power. SUPPORTING INFORMATION Checklist S1 CONSORT Checklist Checklist S1 CONSORT Checklist Found at: doi:10.1371/journal.pone.0001312.s001 (0.05 MB DOC) Checklist S1 CONSORT Checklist Found at: doi:10.1371/journal.pone.0001312.s001 (0.05 MB DOC) Checklist S1 CONSORT Checklist Found at: doi:10.1371/journal.pone.0001312.s001 (0.05 MB DOC) Protocol S1 Trial Protocol Found at: doi:10.1371/journal.pone.0001312.s002 (0.74 MB DOC) ACKNOWLEDGMENTS We wish to acknowledge the study participants in Accra and Kumasi, Ghana. We’d also like to acknowledge the study staff at Komfo Anokye Teaching Hospital, Kwame Nkrumah University of Science & Technol- ogy, Kumasi, Ghana and Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana. We did not observe a significant difference in the overall rate of adverse events between participants receiving SAVVY and those receiving placebo. The frequency of adverse events in the reproductive tract/breast disorders (specifically symptoms of vaginal or vulvar irritation) appeared higher in the SAVVY group, but further subgroup analyses revealed no evidence that the effect of SAVVY use was more pronounced among women with more frequent exposure to the product. Whether the observed increased risk for these events among the SAVVY users in the subset of women who had fewer than average sexual acts and/or fewer gel uses is a chance finding, a result of confounding due to subgrouping by a post-randomization factor, or evidence of an The trial and its reporting complied with the CONSORT Guidelines [1]. This study was conducted under an IND to the US Food and Drug Administration. We conducted this study in accordance with Good Clinical Practice (GCP) as established by the International Conference on Harmonisation. Author Contributions Conceived and designed the experiments: RR WR RD LD LP. Analyzed the data: WR. Wrote the paper: KN LP. Other: Study medical monitor: KN Served as project manager for the study: AT Recruited and followed study participants: WA MO AB BO. Conceived and designed the experiments: RR WR RD LD LP. Analyzed the data: WR. Wrote the paper: KN LP. Other: Study medical monitor: KN Served as project manager for the study: AT Recruited and followed study participants: WA MO AB BO. Interpretation and Overall Evidence Although partic- ipants reported using the gel during approximately 76% of coital acts, our calculations show that participants were more likely to use gel if a condom was also used than when a condom was not used (.80% gel use with condoms versus ,50% gel use without condoms). Since the percent of gel use among risky acts (i.e., with an HIV-infected partner) without condom use is a major determinant of the overall level of effectiveness that can be observed for a truly efficacious product, unless gel use can be increased, studies designed to detect a 50% effect may not be able to identify truly efficacious products[12]. We observed a pregnancy probability at 12 months of 42.5 per 100 person-years in the SAVVY group and 43.7 per 100 person-years in the placebo group. If further studies demonstrate that SAVVY is an effective contraceptive method in other populations, the lack of a difference between the observed pregnancy rates for the SAVVY and placebo groups in our study may indicate that the level of gel adherence was even lower than the participants reported. The availability of study product did not appear to result in increased risk taking behaviors among participants. During enrollment, participants reported a mean of 9 coital acts per week with a mean of 6 sexual partners in the previous 30 days. During follow-up, participants reported a mean of 7 coital acts per week, with a mean of 5–6 sexual partners in the previous 30 days. Reported condom use during follow-up increased from 40% during the last coital act prior to screening to over 89% at the first follow-up visit and remained relatively constant throughout the entire follow period. Conclusion As a new HIV prevention approach, microbicides could be used with other prevention strategies such as condoms to reduce the number of people who become infected with HIV. In our study, SAVVY was not associated with increased adverse events overall relative to placebo, but was associated with higher reporting of reproductive adverse events. Our data are insufficient to conclude whether SAVVY is effective at preventing HIV infection relative to placebo. Phase III trials with larger numbers of seroconver- sions are needed to determine the effectiveness, safety, and feasibility of using microbicides for prevention of HIV infection in women. p p p The likelihood of pregnancy in reproductive-age women having multiple sexual encounters is quite high even with relatively high condom use. Using Wilcox’s estimates of the probability of pregnancy resulting after one or more unprotected act for each day of the menstrual cycle (assuming 30 days per cycle and 12 cycles per year), we expect that if women have two days with unprotected acts on average during each cycle (e.g., 90% condom use, 20 acts per cycle, and no other contraceptive method use), the 12-month cumulative pregnancy probability would be 51%. Therefore, the pregnancy probability of 43% at 12 months observed during this study is not inconsistent with what the participant’s reported–89% condom use and a coital frequency of 30 acts per month. Unless adequate and well-controlled safety studies of microbicides in pregnant women are conducted prior to the start of future HIV prevention studies thereby allowing women who become pregnant to remain on study product, investigators should consider mandating that all female participants who have reproductive potential must be using an effective non-barrier contraceptive such as depot medroxyprogesterone acetate (DMPA), combined oral contraceptive pills (COCs), an intrauterine device (IUD), or a contraceptive implant at least at the time of enrollment. 3. Niruthisard S, Roddy RE, Chutivongse S (1991) The effects of frequent nonoxynol-9 use on the vaginal and cervical mucosa. Sex Transm Dis 18: 176–9. 4. Van Damme L, Ramjee G, Alary M, Vuylsteke B, Chandeying V, et al. (2002) Effectiveness of COL-1492, a nonoxynol-9 vaginal gel, on HIV-1 transmission in female sex workers: a randomised controlled trial. Lancet 360: 971–7. 1. Moher D, Schulz KF, Altman DG (2001) The CONSORT statement: revised recommendations for improving the quality of reports of parallel-group randomised trials. Lancet 357: 1191–4. 2. Roddy RE, Cordero M, Cordero C, Fortney JA (1993) A dosing study of nonoxynol-9 and genital irritation. Int J STD AIDS 4: 165–70. 3. Niruthisard S, Roddy RE, Chutivongse S (1991) The effects of frequent nonoxynol-9 use on the vaginal and cervical mucosa. Sex Transm Dis 18: 176–9. 4. Van Damme L, Ramjee G, Alary M, Vuylsteke B, Chandeying V, et al. (2002) Effectiveness of COL-1492, a nonoxynol-9 vaginal gel, on HIV-1 transmission in female sex workers: a randomised controlled trial. Lancet 360: 971–7. 7. Krebs FC, Miller SR, Catalone BJ, Welsh PA, Malamud D, et al. (2000) Sodium dodecyl sulfate and C31G as microbicidal alternatives to nonoxynol 9: comparative sensitivity of primary human vaginal keratinocytes. Antimicrob Agents Chemother 44: 1954–60. 8. 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Balzarini J, Van Damme L (2007) Microbicide drug candidates to prevent HIV infection. Lancet 369: 787–97. SAVVY Gel for HIV Prevention SAVVY Gel for HIV Prevention 9. Mauck CK, Creinin MD, Barnhart KT, Ballagh SA, Archer DF, et al. (2004) A phase I comparative postcoital testing study of three concentrations of C31G. Contraception 70: 227–31. p 10. Mauck CK, Frezieres RG, Walsh TL, Schmitz SW, Callahan MM, et al. (2004) Male tolerance study of 1% C31G. Contraception 70: 221–5. Male tolerance study of 1% C31G. Contraception 70: 221–5. y p 11. Balzarini J, Van Damme L (2007) Microbicide drug candidates to prevent HIV infection. Lancet 369: 787–97. 11. Balzarini J, Van Damme L (200 infection. Lancet 369: 787–97. 11. Balzarini J, Van Damme L (200 infection. Lancet 369: 787–97. 12. Trussell J, Dominik R (2005) Will microbicide trials yield unbiased estimates of microbicide efficacy? Contraception 72: 408–13. PLoS ONE \| www.plosone.org December 2007 \| Issue 12 \| e1312 8
https://openalex.org/W2956508633	https://revistas.unal.edu.co/index.php/revfacmed/article/download/69862/71658	English	null	Blended learning: An effective methodology for teaching radiology to medical students	Revista de la Facultad de Medicina	2,019	cc-by	3,855	\| Abstract \| Introduction: The combination of online learning environments and classroom education is known as blended learning. Introducción. La inclusión de ambientes virtuales de aprendizaje a la educación presencial se denomina aprendizaje mixto (Blended Learning). Objective: To design, implement and evaluate the blended learning method for teaching radiology to medical students. Objetivos. Diseñar, implementar y evaluar una metodología de aprendizaje mixto para la enseñanza de radiología a estudiantes de medicina. Materials and methods: Five online modules were designed as part of the Introduction to diagnostic imaging course for medical students. The blended learning method was implemented during the classes given in the terms 2016-II and 2017-I. Academic performance was measured using standardized tests, while the effect of the intervention was obtained by comparing the sample with a control group from the 2015-II period (traditional method). Materiales y métodos. Se diseñaron cinco módulos virtuales como parte del curso Introducción a las Imágenes Diagnósticas. La metodología de aprendizaje mixto se implementó durante los periodos 2016-II y 2017-I; se obtuvieron desenlaces de desempeño académico con pruebas estandarizadas y se evaluó el efecto de la intervención mediante la comparación con un grupo control del período 2015-II. Results: 204 students were included in the blended learning group and 90 students in the control group (traditional method). The median final exam score among the blended learning group was 16.5 (IQR: 15.5-17.8), and 15.0 (RIQ: 13.5-16.5) (p=0.001) in the control group. On average, gained knowledge among the blended learning group was 5.8 (SD:2.4) points. The association between gained knowledge and number of visits to online modules was statistically significant (p<0.05). The proportion of good performance was close to 100% on the satisfaction survey. Resultados. 204 estudiantes fueron incluidos en el grupo de aprendizaje mixto y 90 en el grupo control. La mediana de la nota final en el grupo de educación mixta fue de 16.5 (RIQ: 15.5-17.8) y en el grupo control de 15.0 (RIQ: 13.5-16.5) (p=0.001). La ganancia de conocimiento promedio en el grupo de aprendizaje mixto fue de 5.8 puntos (desviación estándar: 2.4) y se asoció con el número de visitas a los módulos virtuales (p<0.05). El porcentaje de estudiantes satisfechos fue cercano al 100%. Conclusions: The blended learning method increases the grades obtained in the tests performed and also shows higher satisfaction rates compared to the traditional method among medical students. Conclusión. Blended learning: An effective methodology for teaching radiology to medical students Johan Alberto Durán-Guerrero1 • Luis Heber Ulloa-Guerrero1 • Luis Carlos Salazar-Díaz1 1 Universidad Nacional de Colombia - Bogotá Campus - Faculty of Medicine - Department of Diagnostic Imagin olombia - Bogotá Campus - Faculty of Medicine - Department of Diagnostic Imaging - Bogotá D.C. - Colombia. Corresponding author: Johan Alberto Durán-Guerrero. Department of Diagnostic Imaging, Faculty of Medicine, Universidad Nacional de Colombia. Carrera 30 No. 45-03, building: 471, office: 102. Phone number: +57 1 3165000, ext.: 15109. Bogotá D.C. Colombia. Email: jadurang@unal.edu.co. 273 273 Rev. Fac. Med. 2019 Vol. 67 No. 2: 273-7 DOI: http://dx.doi.org/10.15446/revfacmed.v67n2.69862 ORIGINAL RESEARCH Study design A quasi-experimental and retrospective study was performed using a before-and-after design. The research was done with medical students from the Universidad Nacional de Colombia who took the course Introduction to diagnostic imaging during the academic terms 2016-II and 2017-I; they received a mixed learning methodology. The control group included the students who took the subject in the term 2015-II, for which a traditional pedagogical method was used. • Module 1: History of technology progress and clinical application of X-rays. • Module 2: Thoracic anatomy. • Module 3: Normal chest X-ray. • Module 4: Abdominal X-ray. • Module 5: Generalities of neuroimaging. Design and development A digital learning environment was created on the Moodle platform with five DLOs (modules): A digital learning environment was created on the Moodle platform with five DLOs (modules): Materials and methods This study was approved by the Ethics Committee of the Faculty of Medicine of the Universidad Nacional de Colombia through Minutes No. 021-280-15 of December 10, 2015, followed the principles of the Declaration of Helsinki (6) and took into account the regulations of Resolution 8430 of 1993 of the Ministry of Health of Colombia. (7) Informed consent was also obtained from the participants to conduct the study. The application of the blended learning method in the Introduction to diagnostic imaging course of the medical program was carried out in five phases, according to the ADDIE model: analysis of the institutional context, design and development of the DLOs, implementation, and evaluation of the pedagogical intervention. Source: Own elaboration based on Torres-Vargas et al. (8). Design and development of digital learning objects This dynamic scenario implies significant challenges for professors, as they need to respond to cultural, social and technological trends. (4) Part of the challenge is the creation of online educational resources, known as digital learning objects (DLOs), which aim to create effective and innovative links with students. (5) The modules were created based on the ADDIE model (8), which consists of an interactive design process where the results of each phase may lead the instructional designer back to any of the previous phases. In this way, errors are identified and corrected without hindering the progress of the project and the results can be constantly evaluated based on the learning objectives. Table 1 shows the definitions and the steps followed in each phase of DLO production. This method is used both in the national and international context with multiple approaches and results; however, the motivation for developing this research project was to answer the question of how to measure the impact of the implementation of a digital learning environment. Table 1. ADDIE design for the creation of digital learning objects. Table 1. ADDIE design for the creation of digital learning objects. Phase Definition Steps Analysis Description of the students and their training needs • Establishing the number of digital learning objects • Knowing the technological resources and limitations Design Definition of the pedagogical approach and the way of sequencing the content • Preparing a storyboard of the contents • Defining the evaluations Development Production of learning contents and materials • Using the storyboard • Working as a team with multimedia designers and web programmers Implementation Execution and implementation of the training action with the participation of the students • Coordinating and integrating the digital contents to the course • Monitoring training activities Evaluation Summative evaluation through specific tests • Quantitatively and qualitatively analyzing the data obtained • Conducting a satisfaction survey Source: Own elaboration based on Torres-Vargas et al. (8). In this sense, and as a response to the educational needs of the Introduction to diagnostic imaging course of the medical program offered by the Universidad Nacional de Colombia, ICTs were implemented, giving way to the possibility of measuring the impact on gained knowledge and the time of use of each digital tool. Consequently, the objective of this study was to design, implement and evaluate a blended learning method for teaching radiology to medical students. \| Abstract \| La metodología de aprendizaje mixto aumenta el puntaje de calificación obtenido por los estudiantes y presenta altos índices de satisfacción en comparación con la metodología convencional. Keywords: Radiology; Online Systems; Problem-Based Learning; Computer User Training (MeSH). Palabras clave: Radiología; Sistemas en línea; Aprendizaje basado en problemas; Capacitación de usuario de computador (DeCS). Durán-Guerrero JA, Ulloa-Guerrero LH, Salazar-Díaz LC. Blended learning: an effective methodology for teaching radiology to medical students. Rev. Fac. Med. 2019;67(2):273-7. English. doi: http://dx.doi.org/10.15446/revfacmed.v67n2.69862. Durán-Guerrero JA, Ulloa-Guerrero LH, Salazar-Díaz LC. [Aprendizaje mixto: una metodología efectiva para la enseñanza de radiología a estudiantes de medicina]. Rev. Fac. Med. 2019;67(2):273-7. English. doi: http://dx.doi.org/10.15446/revfacmed.v67n2.69862. Blended learning, an effective method: 273-7 274 Introduction The students had access to an online learning environment on the Moodle platform, which contained five learning objectives that were developed as modules. The number of visits to each module per student was assessed. i Information and communication technologies (ICTs) offer the possibility of creating new spaces for interaction that facilitate teaching and learning processes. (1) In the specific case of face-to-face education, and through the application of an online learning environment (OLE), teachers have access to great tools for knowledge management. This is achieved by developing educational resources to evaluate and generate effective communication with students. The joint use of traditional teaching in classrooms with an OLE strategy is known as blended learning. (2)i In addition, one-hour weekly lectures, partial exams and a final exam with multiple-choice questions were held, following the traditional method without changing the characteristics of the 2015- II face-to-face course. The contents and rules of the course, as well as the teacher in charge of designing the evaluation questions were the same before and after the intervention. The progress of medical technology in the field of radiology has allowed it to acquire the dynamic characteristics of the technological world. As a result, education in the area of diagnostic imaging is a major challenge, not only because of the variety of techniques but also because of the rapid advances of these techniques. (3)i Traditional teaching method The features of the DLOs included the institutional image of the Universidad Nacional de Colombia, compatibility with all browsers and operating systems, optimization of navigation on multiple devices (computers, tablets and cell phones), fast download speed and ease for adding new learning modules. The contents of the course were developed through lectures given with an intensity of 1 hour per week; the evaluation was done through partial exams and a final exam with multiple-choice questions. Rev. Fac. Med. 2019 Vol. 67 No. 2: 273-7 275 Evaluation design Two tests were carried out, one at the beginning and another at the end of the course. Both evaluations had 40 multiple-choice questions with one answer, which were designed based on the learning objectives proposed for the course (Figure 1). one answer, which were designed based on the learning objectives proposed for the course (Figure 1). Which of the following radiological patterns is displayed in the image? Which of the following radiological patterns is displayed in the image? a. Tree-in-bud pattern b. Ground glass opacity c. Honeycomb pattern d. Crazy-paving pattern Figure 1. Example of the type of question used in the exams. Source: Own elaboration. a. Tree-in-bud pattern b. Ground glass opacity c. Honeycomb pattern d. Crazy-paving pattern a. Tree-in-bud pattern b. Ground glass opacity c. Honeycomb pattern d. Crazy-paving pattern Figure 1. Example of the type of question used in the exams. Source: Own elaboration. learning method groups compared to the traditional teaching method group. Table 2 presents the results for each term. Outcomes In both groups, the primary outcome was the score obtained in the final exam (20 points). Gained knowledge was calculated in the blended learning group as the difference between the score obtained by each student in an exam at the beginning of the course and an exam taken at the end of the course. Table 2. Analysis of the final exam grade per term. Term Learning method Number of students Median (IQR) p * 2015-II Traditional 90 15 (13.5-16.5) Ref. 2016-II † Blended 83 17 (15.5-18.0) <0.001 2017-I † Blended 121 16.5 (15.5-17.5) <0.001 2016-II / 2017-I Blended 204 16.5 (15.5-17.8) <0.001 * Kruskal-Wallis test vs. 2015-II. † Kruskal-Wallis test vs. 2016-II -II vs. 2017-I, p=0.117. Source: Own elaboration. The level of satisfaction was assessed through an anonymous end-of-course survey that included eight questions, of which seven were multiple-choice and one was open-ended. Statistical analysis Qualitative variables were described using percentages, while quantitative variables were described using measures of central tendency, mean for normal variables and median/interquartile range otherwise. Normality tests for continuous variables were performed using frequency histograms, box and whiskers plots and statistical tests (Kolmogorov- Smirnov, Shapiro-France and Shapiro Wilk). In the intervention groups, the number of visits to each DLO per student was recorded for each module during the implementation phase. The median number of visits was 21 (IQR 12-23) and the median number of visits for each module was between 3 and 4 (Figure 2). Academic terms before and after the intervention were contrasted by means of multiple comparisons of the medians using the Kruskall- Wallis test. An analysis of the association between the frequency of use of the intervention and the outcome of knowledge gain by means of linear regression was also carried out, including the evaluation tests for the model (normality of residuals, linearity of the model and homoscedasticity). In addition, the correlation between knowledge gain and the grade of the final exam with the frequency of use of the intervention was evaluated using the Spearman coefficient. Figure 2. Number of visits per module in intervention groups. Source: Own elaboration. Number of visits 8 6 4 2 0 Module 1 Module 2 Module 3 Module 4 Module 5 Results For the study, 294 students were included, of whom 90 subjects were in the traditional teaching method group (control) and 204 in the blended learning method group (intervention); the academic terms were 2015-II for control and 2016-II and 2017-I for intervention.i The median of the final exam grade in the control group was 15/20 points (IQR 13.5-16.5) and in the intervention group, 16.5 (IQR 15.5-17.8). A statistically significant difference was found between the blended Figure 2. Number of visits per module in intervention groups. Source: Own elaboration. Blended learning, an effective method: 273-7 276 referred to differential external factors in each group assessed due to the effects of temporality. However, to control this bias, outcomes were measured at times not so distant from the control group, as well as in two different intervention cohorts. Cohort 2016-I was not taken into account as it was considered to be a transition between both learning methods. Knowledge gain followed a normal distribution with an average of 5.9 (SD=2.4). The result of the linear regression between gained knowledge and the number of visits had statistical significance, with an increase of 0.22 points in the final exam for each visit to the digital modules (p<0.001). The results of the satisfaction survey for blended learning students are presented in Table 3. Of the total number of students, 27 (13%) did not respond the survey; the percentage of respondents satisfied with the achievement of the learning objectives and the usefulness of the online modules was 99.4% for the 2016-II period and 100% for the 2017-I period. The levels of satisfaction with blended learning have been described by authors such as Choules (16) and Carbonaro et al. (18), who concluded that it has contributed to student self-development. Both researchers attributed the success of this method to the creative use of computer technology and the practical nature of the material, and proposed that medical educators should consider the blended learning approach to standardize clinical learning. Table 3. Blended learning student satisfaction survey. Question Highly satisfactory Satisfactory Unsatisfactory 1. The design of the digital learning objects was: 150 (85%) 27 (15%) 0 (0%) 2. The online support methodology used for the development of the course was: 158 (89%) 19 (11%) 0 (0%) 3. Accessibility to the Moodle platform and digital educational resources was: 148 (83%) 28 (16%) 1 (1%) 4. Results The number of online modules developed to support face-to- face teaching was: 90 (51%) 79 (45%) 7 (4%) 5. The way how the different online modules were evaluated was: 128 (72%) 49 (28%) 0 (0%) Question Yes No 6. Do you think that the digital learning objects helped your educational process? 177 (100%) 0 (0%) 7. Do you consider that the learning objectives set for each online module were met? 176 (99.4%) 1 (0.6%) Source: Own elaboration. Table 3. Blended learning student satisfaction survey. The high expectations of the students, exposed to constant innovations in all areas of knowledge, were evident in the satisfaction survey carried out within the framework of this study. Some interviewees stated that the DLOs were scarce and demanded the creation of new digital complements. In addition, a greater availability of teachers through virtual communication tools (chat) was suggested for the resolution of doubts. i Other reported benefits of the blended learning method are improved academic performance and student satisfaction, as it increases students’ exposure to area content and optimizes teaching times. (19-21) On the other hand, all educational formats have strengths and limitations. Thus, the ideal methodology should aim to improve the perception on educational environments and to promote problem solving, critical thinking and decision-making skills, in a flexible manner, without a specific place or time. (22) When building a blended learning model, teachers should decide which part of the curriculum will be delivered face-to-face and which online, to avoid students who lack computer skills from feeling disadvantaged or frustrated when using OLEs. (23) The balance between face-to-face education and the digital component is highly relevant and depends on factors such as student level, electronic resources and teacher experience. Although online learning is an established and effective approach in many medical schools, it should not replace traditional learning; therefore, blended learning is probably a better approach than web- based teaching. (24) Conclusions The blended learning method has a significant impact on performance during tests compared to the traditional method. The implementation of DLOs that complement face-to-face education makes it possible to strengthen the teaching process with high levels of satisfaction, justifying the time and resources required for their design and production. In the present study, each module was evaluated as an extrinsic motivation strategy, obtaining a considerable number of visits, higher than those reported by Mahnken et al. (17) in a similar study and with greater gained knowledge. Discussion This project has demonstrated that the integration of online teaching into traditional classes overcomes the constraints of time and place and improves the quality of education as it promotes the development of skills by expanding the concepts and resources provided by textbooks. The results of this work demonstrate that the implementation of a blended learning method has a significant impact on student’s grades and is perceived with high satisfaction rates. Different studies have reported the positive impact of the blended learning method on academic performance. (2,9) The meta-analysis of Chumley-Jones et al. (10) found sufficient evidence on the effectiveness of medical education combined with the use of digital tools. Furthermore, primary studies coincide in affirming that online instruction together with face-to-face training have a positive effect on the achievement of clinical skills and is comparable to traditional forms of teaching, obtaining better results in the final exam. (11-16) Conflicts of interest None stated by the authors. Despite the standardization of knowledge tests, there were two main limitations in this research. 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https://openalex.org/W2155350273	https://escholarship.org/content/qt8jb6c89w/qt8jb6c89w.pdf?t=mxb2i2	English	null	Genome sequencing reveals fine scale diversification and reticulation history during speciation in Sus	GenomeBiology.com	2,013	cc-by	10,394	UC Berkeley UC Berkeley Previously Published Works Title Genome sequencing reveals fine scale diversification and reticulation history d speciation in Sus Permalink https://escholarship.org/uc/item/8jb6c89w Journal Genome Biology, 14(9) ISSN 1465-6906 Authors Frantz, Laurent AF Schraiber, Joshua G Madsen, Ole et al. Publication Date 2013-09-26 DOI http://dx.doi.org/10.1186/gb-2013-14-9-r107 Peer reviewed UC Berkeley UC Berkeley Previously Published Works Title Genome sequencing reveals fine scale diversification and reticulation history d speciation in Sus Permalink https://escholarship.org/uc/item/8jb6c89w Journal Genome Biology, 14(9) ISSN 1465-6906 Authors Frantz, Laurent AF Schraiber, Joshua G Madsen, Ole et al. Publication Date 2013-09-26 DOI http://dx.doi.org/10.1186/gb-2013-14-9-r107 Peer reviewed UC Berkeley UC Berkeley Previously Published Works Title Genome sequencing reveals fine scale diversification and reticulation histor speciation in Sus Permalink https://escholarship.org/uc/item/8jb6c89w Journal Genome Biology, 14(9) ISSN 1465-6906 Authors Frantz, Laurent AF Schraiber, Joshua G Madsen, Ole et al. Publication Date 2013-09-26 DOI http://dx.doi.org/10.1186/gb-2013-14-9-r107 Peer reviewed © 2013 Frantz et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Genome sequencing reveals fine scale diversification and reticulation history during speciation in Sus Laurent AF Frantz1, Joshua G Schraiber2, Ole Madsen1, Hendrik-Jan Megens1, Mirte Bosse1, Yogesh Paudel1, Gono Semiadi3, Erik Meijaard4,5, Ning Li6, Richard PMA Crooijmans1, Alan L Archibald7, Montgomery Slatkin2, Lawrence B Schook8, Greger Larson9 and Martien AM Groenen1 Abstract Background: Elucidating the process of speciation requires an in-depth understanding of the evolutionary history of the species in question. Studies that rely upon a limited number of genetic loci do not always reveal actual evolutionary history, and often confuse inferences related to phylogeny and speciation. Whole-genome data, however, can overcome this issue by providing a nearly unbiased window into the patterns and processes of speciation. In order to reveal the complexity of the speciation process, we sequenced and analyzed the genomes of 10 wild pigs, representing morphologically or geographically well-defined species and subspecies of the genus Sus from insular and mainland Southeast Asia, and one African common warthog. Results: Our data highlight the importance of past cyclical climatic fluctuations in facilitating the dispersal and isolation of populations, thus leading to the diversification of suids in one of the most species-rich regions of the world. Moreover, admixture analyses revealed extensive, intra- and inter-specific gene-flow that explains previous conflicting results obtained from a limited number of loci. We show that these multiple episodes of gene-flow resulted from both natural and human-mediated dispersal. Conclusions: Our results demonstrate the importance of past climatic fluctuations and human mediated translocations in driving and complicating the process of speciation in island Southeast Asia. This case study demonstrates that genomics is a powerful tool to decipher the evolutionary history of a genus, and reveals the complexity of the process of speciation. Correspondence: laurent.frantz@wur.nl 1Animal Breeding and Genomics Group, Wageningen University, De Elst 1, Wageningen, WD 6708, The Netherlands Full list of author information is available at the end of the article Open Access RESEARCH Open Access Powered by the California Digital Library University of California eScholarship.org Frantz et al. Genome Biology 2013, 14:R107 http://genomebiology.com/2013/14/9/R107 Background between two species, though gene-flow has been shown to occur during the splits between species [4,5]. The recent advent of high-throughput sequencing allows inferences to be drawn from near-complete genomes, in turn offering an unprecedented understanding of orga- nismal evolutionary history. The commensurate increase in resolving power has allowed numerous questions to be addressed, including those related to genomic structure, deep phylogenetic relationships, the genetic variation responsible for specific phenotypes, and hybridization patterns between ancient hominids [6,7]. Few studies, however, have taken advantage of complete genomes to investigate the process of speciation. between two species, though gene-flow has been shown to occur during the splits between species [4,5]. The recent advent of high-throughput sequencing allows inferences to be drawn from near-complete genomes, in turn offering an unprecedented understanding of orga- nismal evolutionary history. The commensurate increase in resolving power has allowed numerous questions to be addressed, including those related to genomic structure, deep phylogenetic relationships, the genetic variation responsible for specific phenotypes, and hybridization patterns between ancient hominids [6,7]. Few studies, however, have taken advantage of complete genomes to investigate the process of speciation. The diversity of life on Earth owes its existence to the process of speciation. The emergence of genetic techni- ques has allowed the relationships amongst hundreds of species to be investigated, and DNA studies have been invaluable in resolving long-standing taxonomic and phylogenetic questions (for example, [1,2]. The use of limited numbers of genomic markers, however, can result in misleading impressions of the phylogenetic relationships between organisms [3]. In addition, tradi- tional bifurcating trees are constructed on the presump- tion that little or no gene-flow occurs following a split * Correspondence: laurent.frantz@wur.nl 1Animal Breeding and Genomics Group, Wageningen University, De Elst 1, Wageningen, WD 6708, The Netherlands Full list of author information is available at the end of the article Wallace [8] first recognized that Island Southeast Asia (ISEA) is an ideal natural laboratory to study speciation. Over the past 50 million years (My) tectonic activity has Frantz et al. Genome Biology 2013, 14:R107 http://genomebiology.com/2013/14/9/R107 Frantz et al. Genome Biology 2013, 14:R107 http://genomebiology.com/2013/14/9/R107 Frantz et al. SNP discovery and general divergence pattern across the genomes g We aligned between 153 and 566 million reads per sam- ple to the S. scrofa reference genome (Sscrofa10.2) [19], resulting in an average read depth of 7.5 to 24× (Table S2 in Additional file 1; Materials and methods). The number of SNPs discovered in each genome sequence (Table S2 in Additional file 1) was higher in the Sus species than between S. scrofa individuals, most of which were fixed differences between the S. scrofa reference genome and the other species analyzed. In order to understand how substitution rate within the genus varies across the gen- ome, we computed the average sequence divergence from the Warthog to each Sus species in 1 Mb windows (Materials and methods). Our results demonstrated that the average sequence divergence to the outgroup (warthog) was positively correlated with recombination rate (as estimated in S. scrofa [20]; tau = 0.40, P < 0.001), suggesting a relationship between recombination and divergence rate, as observed in other mammals [21,22]. The five biodiversity hotspots found in ISEA and Mainland Southeast Asia (MSEA) [12] are host to at least seven morphologically defined species of pig in the genus Sus [13]. Aside from Sus scrofa (Eurasian wild boar and domestic pigs), which is distributed across most of Eurasia and parts of northern Africa, all other species of the genus Sus are restricted to MSEA and ISEA (Figure 1A). Because these species are still capable of interbreeding and producing fertile offspring [14], the genus Sus presents an excellent model to study on- going speciation. Moreover, previous studies have found discrepancies between and among the phylogenies inferred from morphological and mitochondrial DNA (mtDNA) markers [13,15,16]. Thus, the phylogeny of these species remains controversial. These discrepancies could be explained by either gene-flow between sympatric populations of different species or a rapid radiation that would have left little power to resolve the phylogeny. Background Genome Biology 2013, 14:R107 http://genomebiology.com/2013/14/9/R107 Page 2 of 12 In order to investigate the speciation history of these suids, and to assess the usefulness of whole-genome sequences to infer complex evolutionary histories, we sequenced and analyzed the complete genomes of 11 individual pigs representing five Sus species and an Afri- can common warthog (Phacochoerus africanus; Table S1 in Additional file 1). Our analysis of these 11 genomes demonstrates the power afforded by genomics to resolve a complex and controversial evolutionary history invol- ving multiple reticulation events. considerably altered the geography of this region. In addition, large-scale climatic fluctuations beginning in the early Pliocene [9] affected the region’s biogeography [10]. Successive glacial and interglacial periods lowered and raised sea levels, thus alternately separating and connecting large landmasses. During cold periods, the Malay Peninsula, Borneo, Sumatra and Java formed the contiguous landmass known as Sundaland (Figure 1A), while in warmer periods these islands were isolated from each other. These alternating climatic conditions required frequent adaptation and induced intermittent allopatric and parapatric speciation processes. The fluc- tuations also created an ideal environment for diversifi- cation that has resulted in a complex and species-rich assemblage [10]. The development of models that explain the process of speciation in ISEA has been further complicated by anthropogenic factors that have influenced the dispersal and distribution of numerous species in the region [11]. Phylogenomic analysis l Using near complete genome sequences, we applied sev- eral phylogenomic methods based on maximum likelihood (ML) implemented in RAxML 7.2 [23]. We used both supertree and supermatrix techniques (see Materials and methods for details). Briefly, the supertree methodology involves computing a single tree per genomic locus in combination with an ad hoc reconstruction of a consensus phylogeny from the single trees whereby the stochastic behavior of lineage sorting can be taken into account. In the supermatrix framework, a single tree is inferred from multiple loci assembled in multiple partitions. The lack of a post-zygotic reproductive barrier in pigs is not an isolated case. Indeed, many vertebrate taxa, recog- nized as different species, can still interbreed and produce fertile offspring. For example, it has been claimed that approximately 6% of European mammalian species can interbreed with at least one other species [17]. Addition- ally, while most of these species are young, there are examples of interbreeding species of birds that diverged over 55 million years ago (Mya) [18]. Given the ease with which numerous closely related (and some distantly related) species can interbreed, it is important to develop and test methods that are not only robust to inter-specific gene-flow, but can also identify it. Speciation with gene- flow is expected to result in a richer phylogenetic history including periods of divergence (bifurcations) and periods of secondary contact (reticulations), and thus should leave genomic signatures. We first identified regions in the genome, spanning a minimum of 5 kbp, that possessed less than 10% missing data (due to filtering) in all our samples (see Materials and methods for details; Table S3 in Additional file 1). We then built phylogenetic trees for every genomic bin identified and obtained a species tree using the supertree method STAR [24]. We also used a concatenation method by building multiple supermatrices. One hundred superma- trices, each spanning 1 Mbp, were assembled by randomly Frantz et al. Genome Biology 2013, 14:R107 http://genomebiology.com/2013/14/9/R107 Page 3 of 12 Frantz et al. Genome Biology 2013, 14:R107 http://genomebiology.com/2013/14/9/R107 Figure 1 Geographic distribution, phylogenetic relationships and admixture between Sus lineages. (A) A map of Island and Mainland Southeast Asia depicting the modern distributions of five Sus species. The grey shaded area represents the maximum geographical extent of Sundaland during periods of low sea level. (B) Phylogenetic relationships among Sus species inferred from nuclear DNA. Phylogenomic analysis l Node labels show age in millions of years and 95% confidence interval. Grey shading highlights taxa living on Sundaland (C,D) Diagrams depicting the excess derived allele sharing when comparing sister taxa and outgroups. Each row contains the fraction of excess allele sharing by a taxon (left/right) with the top label/outgroup (S. scrofa or S. barbatus) relative to its sister taxon (left/right). The grey bar points in the direction of the taxon that shares more derived alleles with the outgroup than its sister taxon, and its magnitude indicates the amount of excess (D). Black bars represent 1 standard error and stars indicate D values significantly different from 0 (P < 0.01; see Materials and methods). (E) A mitochondrial DNA Bayesian Figure 1 Geographic distribution, phylogenetic relationships and admixture between Sus lineages. (A) A map of Island and Mainland Southeast Asia depicting the modern distributions of five Sus species. The grey shaded area represents the maximum geographical extent of Sundaland during periods of low sea level. (B) Phylogenetic relationships among Sus species inferred from nuclear DNA. Node labels show age in millions of years and 95% confidence interval. Grey shading highlights taxa living on Sundaland (C,D) Diagrams depicting the excess derived allele sharing when comparing sister taxa and outgroups. Each row contains the fraction of excess allele sharing by a taxon (left/right) with the top label/outgroup (S. scrofa or S. barbatus) relative to its sister taxon (left/right). The grey bar points in the direction of the taxon that shares more derived alleles with the outgroup than its sister taxon, and its magnitude indicates the amount of excess (D). Black bars represent 1 standard error and stars indicate D values significantly different from 0 (P < 0.01; see Materials and methods). (E) A mitochondrial DNA Bayesian phylogenetic-based tree with node labels that represent posterior probabilities (* > 0.85; ** = 1). Figure 1 Geographic distribution, phylogenetic relationships and admixture between Sus lineages. (A) A map of Frantz et al. Genome Biology 2013, 14:R107 http://genomebiology.com/2013/14/9/R107 Page 4 of 12 joining genomic bins. We then computed a phylogenetic tree using RAxML, with 100 fast bootstrap replicates, for each supermatrix. times using a relaxed molecular clock as implemented in MCMCtree [27]. Phylogenomic analysis l In order to account for the uncer- tainty in fossil dates, we used three separate fossil cali- brations to place prior distributions on node age (see Additional file 6 for further discussion and references on the fossil calibrations used in this study). We then selected genomic loci supporting the main topology to obtain the date of original divergence between taxa (Figure 1B), thereby limiting the bias that arises from admixture between species (Additional files 4 and 5). p We found that the species tree topology depicted in Figure 1B was the most common across all of the geno- mic bins analyzed (Additional file 2), but several alterna- tive topologies appeared in substantial numbers (Additional file 3). This result is to be expected and can be caused by incomplete lineage sorting (in which deep coalescences occur in ancestral populations) and gene- flow (in which some genealogies cross species bound- aries). The presence of such incongruence is created when recombination creates local gene trees; hence, we looked for a correlation between recombination rate and the frequency of alternative topologies. We found a posi- tive correlation between mean pairwise Robinson-Foulds distance and recombination rate in 1 Mbp windows (tau = 0.53, P < 0.001; Materials and methods). We also found a positive correlation with mean divergence to the outgroup (tau = 0.40, P < 0.001). Together, these results suggest the importance of recombination in shaping the genomic landscape of speciation in suids. The correlation between the timing of the nodes on the phylogenetic tree and climate models [28] suggested that when global sea levels dropped during cold intervals, the resulting land bridges between islands allowed pigs to dis- perse across what were once sea barriers (Figures 1A and 2). Warm periods raised sea levels, closed migration routes and isolated populations on individual islands, leading to allopa- tric speciation. In addition, our admixture analysis revealed the existence of extensive inter-specific gene-flow that likely took place during cold intervals since these periods would have induced parapatric conditions via the connection of previously isolated islands. To compare our results to earlier studies using mito- chondrial DNA (matrilineal lineage), we carried out a Bayesian phylogenetic analysis using near-complete mito- chondrial genomes (Materials and methods). The resulting topology is consistent with previous studies [15,16,25] and shows a clear discordance with the phylogenetic tree obtained from autosomal chromosomes (Figure 1B,E). Demographic analysis We used heterozygous SNP calls for demographic infer- ence in a single individual genome sequence as imple- mented in PSMC (Materials and methods; Figure 3; Additional file 7). We found that the Pleistocene period led to a bottleneck in both ISEA (Figure 3) and MSEA populations (Additional file 7). These population size declines are consistent with the reduction of temperature observed during this period that would have reduced the overall forest cover in MSEA and ISEA [29,30] (Figure 2). In addition, our results suggest that the populations from ISEA (Figure 3) have undergone a more severe bottleneck than populations of MSEA (Additional file 7). The phylogenetic discordance found within the genome and between nuclear and mtDNA could be the result of either incomplete lineage sorting or post-divergence gene-flow. Phylogenomic analysis l This discordance is expected given the wide range of topologies found in the autosomes, especially because mitochondrial DNA represents only one locus with no recombination. Divergence time and admixture analysis Our results reveal that, unlike alternative strategies including SNP genotypes (from SNP microarrays), ascer- tained in a single species or population, that possess inherent biases in between species or population studies [31], whole-genome sequencing (leading to the detection of millions of polymorphisms) allow for phylogenetic relationships and admixture patterns within the genus Sus to be confidently resolved. Indeed, when attempting to recapitulate the analysis using the porcine 60K SNP chip [32] (Additional file 8), substantial differences in branch length estimates were found. These discrepancies are due to ascertainment bias demonstrating that a sim- ple SNP array genotyping method, even for multiple indi- viduals, would not have allowed the resolution afforded by a single complete genome. In addition, we show that there is a high degree of phylogenetic discordance across In order to differentiate between incomplete lineage sorting and gene-flow, we conducted an independent admixture analysis (using D-statistics) that directly addressed this issue [26] (see Materials and methods; Additional file 4). Overall, we found strong evidence of admixture among species living on Sundaland. Indeed, results of D-statistics (Materials and methods; Additional files 4 and 5) show that species living on Sundaland share a significant excess of derived alleles compared to what would be expected for a simple bifurcating scenario, as displayed in Figure 1B,C. In addition, we found further admixture signatures that involve species living outside of Sundaland. For a detailed discussion of these results, please refer to Additional file 4. To put the admixture and divergence events in a tem- poral context, we first estimated molecular divergence Frantz et al. Genome Biology 2013, 14:R107 http://genomebiology.com/2013/14/9/R107 Page 5 of 12 Figure 2 A eustatic curve adapted from [18]. (A) Each black bar shows 95% confidence interval of each divergence event as inferred from molecular clock analysis (Figure 1B). (B) Eustatic curve for the last 5 My. (C) Legend of events represented as black bars in (A). Figure 2 A eustatic curve adapted from [18]. (A) Each black bar shows 95% confidence interval of each divergence event as inferred from molecular clock analysis (Figure 1B). (B) Eustatic curve for the last 5 My. (C) Legend of events represented as black bars in (A). Evolutionary history of Sus Thus, if overseas dispersal took place between Borneo and Sulawesi, it may also have been possible for pigs to disperse naturally from Java and the Philippines, within the last few million years (for example, by rafting or swimming). Further studies that can date these colonization events from Java and the Philippines into Sulawesi, using multiple genomes from S. celebensis, could enable assessments of whether these migrations were in fact the result of human translocation. The mainland divergence of S. scrofa into regionally discrete populations also started during the mid-Pleisto- cene (Figure 1B). Populations of S. scrofa from Asia migrated west approximately 1.2 Mya, reaching Europe around 0.8 Mya as suggested by the first appearance of S. scrofa in the fossil record (see Additional file 6 for details). The first divergence between Eastern and Western S. scrofa, as timed by our molecular clock ana- lysis (Figure 1B), was likely the result of cooler climate during the Calabrian period that isolated populations in small refugia across Eurasia (Figure 2). Our data indicate that the split between Northern and Southern Chinese S. scrofa populations took place during the Ionian stage approximately 0.6 Mya (Figure 1B). This timing corre- lates with the most significant reduction in global tem- perature in the Plio-Pleistocene, characterized by long glacial intervals and short interglacial periods, that started approximately 0.8 Mya [35] (Ionian stage; Figures 1B and 2). In this period forests contracted into small refugia, thereby isolating populations across MSEA [10]. Our results provide evidence that following the diver- gence of the S. verrucosus lineage, the ancestor of Sus cebifrons colonized the Philippines during the first stage of the Pleistocene approximately 2.4 to 1.6 Mya (Gela- sian stage; Figures 1B and 2). This date correlates with tectonic activity that led to the isolation of the Philip- pines from Sundaland even during periods of low sea levels [33]. This same period witnessed the divergence between S. scrofa populations on Sumatra and mainland East Asia (Figures 1B and 2). However, it is unclear whether this divergence was the result of migration of S. scrofa from ISEA to the mainland or vice versa. More- over, this deep divergence between mainland and ISEA wild boars (S. scrofa) supports previous morphological studies that advocated the distinctiveness of these ISEA S. scrofa sub-species compared to other MSEA popula- tions [13] (that is, the banded pig S. scrofa vittatus). Our results show that S. Evolutionary history of Sus the genome. Such discordance could potentially lead to incorrect conclusions about the relationships between these species if only a subset of these loci were sampled [16]. While phylogenetic incongruence can frustrate taxonomic inference, it has the potential to test for the presence of inter-specific gene-flow. Our data demon- strate that the wealth of information extracted from these genomes allows for a thorough analysis (Additional files 4 and 5) that permits for the temporal reconstruc- tion of the evolutionary history of Sus discussed below. Our divergence time estimates suggest that the initial divergence of the Eurasian wild boar from a clade consist- ing of other Sus species took place during the Zanclean stage at the beginning of the Pliocene (Figure 1B; 5.3 to 3.5 Mya). Though the precise geographic location of this split (either in Sundaland or mainland Southeast Asia) remains unclear, the timing coincides with the divergence between other Sundaic and mainland Asian taxa [10]. The subsequent millions of years (from 3.5 to 2.5 Mya, the 0 1 2 3 4 5 6 104 105 106 107 Effective population size (x104) Years (g=5, =2.5x10-8) Insular South East Asian Demographic History Sumatra1 Sumatra2 S.verru S.cele S.cebi S.barba Figure 3 Population sizes of Sus in ISEA inferred from autosomes. Sumatra1/2 = S. scrofa population from Sumatra. S.verru = S. verrucosus; S.cele = S. celebensis; S.cebi = S. cebifrons; S.barba = S. barbatus. Figure 3 Population sizes of Sus in ISEA inferred from autosomes. Sumatra1/2 = S. scrofa population from Sumatra. S.verru = S. verrucosus; S.cele = S. celebensis; S.cebi = S. cebifrons; S.barba = S. barbatus. Frantz et al. Genome Biology 2013, 14:R107 http://genomebiology.com/2013/14/9/R107 Frantz et al. Genome Biology 2013, 14:R107 http://genomebiology.com/2013/14/9/R107 Page 6 of 12 Piacenzian stage; Figures 1B and 2) were marked by more intense cold periods that likely facilitated the emergence of a contiguous Sundaland landmass for prolonged periods (Figures 1A and 2). Concomitant drops in sea levels are likely to have allowed the dispersal of the ancestor of Sus verrucosus to Java (consistent with the fossil record; Additional file 6). The deep split between S. verrucosus and other ISEA Sus demonstrates that this endangered species S. verrucosus represents a distinct lineage. Such a finding has implications for on-going ex and in situ con- servation programs as it shows that this species represents an evident evolutionarily significant unit that deserves specific conservation strategies. have been induced by modern humans. Evolutionary history of Sus celebensis colonized Sulawesi, from the west (Borneo), during the latter stage of the Pleistocene (Calabrian; Figures 1B and 2), approximately 1.6 to 0.8 Mya. It appears that this colonization occurred despite evidence that the Makassar Strait separating Sundaland and Sulawesi continued to exist even during periods of lowered sea levels, thus restrict- ing dispersal during the Plio-Pleistocene [34]. Nonethe- less, more frequent incidences of lower sea levels during this period [28] (Figure 2) would have reduced the dis- tance between Sundaland and Sulawesi, thereby increas- ing the likelihood of a successful crossing of the strait. Our phylogenomic analysis implies that populations on Borneo acted as the initial and main source for this dis- persal even though the admixture analysis suggest that S. verrucosus on Java and S. cebifrons in the Philippines later also contributed to the S. celebensis gene pool (Additional files 4 and 5). These results may explain the existence of two well-supported but paraphyletic S. cele- bensis mtDNA clades present on Sulawesi [15,25]. Admixture and mtDNA replacement Admix- ture analyses support these claims by revealing gene- flow from S. celebensis into local S. scrofa populations on Sumatra and MSEA. Even during cold periods, Sula- wesi and Sundaland were separated by a deep sea chan- nel [34]. Thus, it seems unlikely that populations of S. celebensis, from Sulawesi, made it back to isolated islands around Sumatra and MSEA within the last 1.5 My since its divergence from S. barbatus. In their totality, these results provide evidence that human trans- location of suids took place across the region and was not restricted to islands in close proximity to Sulawesi. g Our phylogenetic tree based on near-complete mtDNA genomes (Figure 1E) is consistent with previous studies [15,25], supporting a paraphyletic relationship among non-S. scrofa species and a monophyletic clade of Sunda- land taxa with short branch lengths. In addition our demographic analysis (Figure 3) shows that species living on Sundaland have undergone a long-term population decline, more extended than on MSEA (Additional file 7), during the Pleistocene. These results suggest that there was a replacement of mitochondrial haplotypes that took place across Sundaland during the latter part of the Pleistocene (1.5 Mya to the present; Additional file 4), after the divergence of S. celebenisis (Figure 1B,E; Addi- tional file 4). The mtDNA replacement may have been facilitated by small population sizes (Figure 3). Taxa endemic to the Philippines and Sulawesi, isolated from Sundaland, were not involved in this admixture and har- bor highly diverged mtDNA haplotypes of both complete mitochondrial sequences and fragments of the control region [15,25] (Figure 1E). This phenomenon is unlikely to be an exception in pigs and has been recently observed in polar bears [3]. We also detected a strong signature of gene-flow from European S. scrofa populations into species in ISEA, con- sistent with a previous study that identified European mitochondrial haplotypes among populations in ISEA [15]. This gene-flow was most likely the result of human- induced dispersal of European pigs into ISEA within the past few hundred years. Some of these introduced pigs likely became feral and interbred with indigenous species. While some of the admixture signals detected in this study are unequivocal (that is, admixture within Sunda- land, supported by mtDNA and frequent merging of these islands during the Plio-Pleistocene epoch), other signatures, including those involving long distance dis- persal, are more difficult to interpret. Admixture and mtDNA replacement Though we have presented the evolutionary history of Sus as speciation events resulting from simple bifurca- tions, D-statistics [26] and simulations challenge this view and suggest numerous instances of diversification and reticulation (Additional files 4 and 5). Our analysis shows that concomitant sea level fluctuations allowed for extensive intra- and inter-specific gene-flow during these periods, both within Sundaland and between Sundaland and MSEA (Figure 1C,D; Additional files 4 and 5). Admixture fractions between Sumatran and Chinese S. scrofa subpopulations were higher (9.5 to 11%; Addi- tional file 4) than those between Sumatran S. scrofa and other Sus species on Sundaland (1.3 to 4.2%; Additional file 4). This finding suggests that, during the Pleistocene, more gene-flow took place between Chinese and Suma- tran S. scrofa populations than between Sumatran S. scrofa populations and other Sus species living on Sun- daland. The geographic distance between Sumatran and Chinese S. scrofa populations is much larger than between Sumatran S. scrofa and the other Sus species that live on Sundaland (for example, S. verrucosus and Sus barbatus). Thus, this pattern supports a model of While the overseas dispersal of indigenous suids from Java and the Philippines into Sulawesi may have been the result of human-aided translocation, the initial divergence of S. celebensis from the Bornean population is too old to Frantz et al. Genome Biology 2013, 14:R107 http://genomebiology.com/2013/14/9/R107 Page 7 of 12 inter-specific gene-flow that involved long distance dis- persal across barriers that were unlikely to be the result of natural migration pathways. ongoing speciation with gene-flow in which interspecies relatedness is more closely correlated with a history of admixture than with current geographic proximity. Despite these alternating periods of divergence and homogenization, trees constructed using complete gen- omes recover the modern species designations. The same is not true of previously published mitochondrial phylogenetic trees of pigs from ISEA and MSEA that were able to distinguish geographically distinct popula- tions of S. scrofa in Eurasia, but were unable to recover the monophyly of morphologically distinct species living on Sundaland [15,16,25,36]. This paradox could result from either the limited phylogenetic information present in the short mitochondrial fragments used in previous studies, or from the complex pattern of admixture in Sundaland described above (Figure 1C,D). Previous morphologic [37] and genetic [15] studies suggested that S. celebensis was kept captive and trans- ported by humans from Sulawesi to Timor, Flora, Halmahera and Simeulue (Northwest Sumatra). Admixture and mtDNA replacement For example, admixture involving un-sampled or extinct lineages can result in complex site patterns and could influence the results of the D-statistics [26]. For instance, the signal of gene-flow from European S. scrofa into species in ISEA could be the result of an admixture from an un-sampled sister lineage, and may not necessarily involve European pigs per se. Another limitation of the method can arise from ancestral population subdivision as has been sug- gested to account for signatures of Neanderthal and human admixture [38]. However, ancestral subdivision is unlikely to affect our analysis because of the evolutionary time frame investigated here (Additional file 4). Human-mediated translocation Though climate change has had the most dramatic and sustained influence on the speciation history of suids, humans have also affected this process. During the last 40,000 years, humans have actively and passively trans- located hundreds of species (as commensals, wild, or domestics) within ISEA, Wallacea and Australasia [11], and the signatures of the resulting admixture between suid lineages are evident in the genomic sequences. In addition, S. scrofa is an agriculturally important species that has been independently domesticated at least twice in mainland Eurasia (Near-east and China) [25]. The close relationship between humans and pigs make this species more prone to anthropogenic translocations. Indeed, our admixture analysis revealed the existence of Methodological challenges Our work demonstrates that the analysis of high-through- put sequencing data provides a powerful tool to investigate speciation history; but is unlikely to be devoid of sequen- cing errors, especially for low sequence coverage. How- ever, the sequence coverage in our samples (7.5 to 25×) is expected to provide reliable genotype calls [40]. In addi- tion, the major conclusions of this study are not expected to suffer from these biases as these analyses rely on non- singleton sites. Specifically, for a site to be phylogenetically informative the mutation must be shared by at least two taxa and the D-statistic analysis is explicitly designed to be robust to sequencing errors resulting in singletons [26]. Therefore, for a sequencing error to influence our phy- logenetic or admixture analysis, it would have to be systematic and have occurred separately in different sam- ples sequenced at different times in different sequencing centers. Thus, making the reasonable assumption that sequencing errors are independent between the samples, the probability of creating enough falsely informative sites to bias these analyses is exceedingly low. The complete genomes presented here provide com- pelling evidence that speciation in ISEA suids did not proceed according to a simple bifurcating model. Instead, our data indicate that the process involved numerous periods of both diversification and reticulation amongst several species and is on-going. Extensive inter-specific gene-flow has also been reported in fish [43,44] and birds [45,46]. The resolution afforded by complete genomes reveals that speciation is rarely as simple or linear as our traditional depictions, and that complex patterns of diversification and reticulation are likely the rule and not the exception. The origin of new species often includes significant time periods during which closely related taxa in the initial stages of diversifying from one another can (and do) pro- duce fertile offspring. The resolution provided by the use of whole genomes allows not only for an assessment of the current and past integrity of species, but also the elucida- tion of taxa-specific speciation history. Genomics can thus reveal the molecular variability of life on earth, elucidate the process by which it emerged, and inform our attempts to preserve it. Another limitation of our phylogenetic analysis could stem from recombination. Indeed, due to recombination, each of our genomic bins may represent a mosaic of differ- ent evolutionary histories. Factors driving and reversing speciation in Sus Our results suggest that Plio-Pleistocene climatic fluctua- tions had a significant impact on the diversification and homogenization of Sus in ISEA and MSEA. Speciation within Sus was mainly driven by dispersal across ISEA during the short glacial interval of the late Pliocene and early Pleistocene as suggested by evidence gleaned from other taxa [10,39]. Rapid changes in climate and sea level resulted in population bottlenecks across ISEA (Figure 3). Frantz et al. Genome Biology 2013, 14:R107 http://genomebiology.com/2013/14/9/R107 Page 8 of 12 In addition, extensive intra- and inter-specific gene-flow led to instances of mtDNA replacement and a reversal (however temporary) of the speciation process. well-resolved tree. In fact, the complexity of whole- genome data allows for a deeper appreciation of the complexities involved in the speciation process. More- over, the substantial volume of data allows for robust time estimation. These findings reveal the power of multiple complete genomes from closely related species to comprehensively infer their speciation and evolution- ary history and to resolve discrepancies between discor- dant trees constructed using smaller marker sets. Sequencing, alignment and SNP calling h l d h d h Sequencing, alignment and SNP calling h l d h d h The samples used in this study were chosen from a larger pool of genotyped individuals (Illumina Porcine SNP60 chip) [32] in each species or population in order to ensure that each was representative of the genetic diver- sity of their respective species/populations (Additional file 8). DNA was extracted from blood or tissue using the DNeasy blood and tissue kits (Qiagen, Venlo, NL, USA). Quality and quantity were measured with the Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). Libraries of approximately 300 bp fragments were pre- pared using Illumina paired-end kits (Illumina, San Diego, CA, USA) and sequenced with Illumina GAII or HiSeq (Table S1 in Additional file 1). Lastly, it is important to take results of demographic his- tory with caution. Indeed, while we believe that the general pattern described in Figure 3 is reliable, the magnitude of this bottleneck, in different species, is difficult to interpret. Differences in coverage among our samples likely result in variable power to call heterozygous sites, and could explain at least some of the differences in demographic history between different species. Methodological challenges Nonetheless, theory and simula- tions suggest that our overall conclusions are relatively insensitive to the effects of recombination [41]. This insen- sitivity is because, moving along a sequence, different topologies are highly correlated and hence recombination is expected to have small effects over short recombination distances [42]. Conclusions We fitted a GTR+Γ4+I model of sequence evolution to each partition (genomic fragment) and ran 100 fast bootstrap replicates for each alignment and a thorough ML search using RAxML 7.1.2 [23]. We con- structed a frequency consensus tree using all bootstrap replicates obtained from the 100 unique alignments using Phylip CONSENSE package [51]. These frequencies were then used as support for the species tree (Additional file 2). To reconstruct the mtDNA tree we used a Bayesian tree reconstruction with 50,000,000 MCMC samples as imple- mented in MrBayes v3.2 [52]. We fitted a GTR+Γ4+I model suggested by AIC criterion as implemented in MrAIC [53]. We assessed the convergence of MCMC samples using TRACER [54]. The resulting phylogenetic tree is presented in Figure 1E. We used the pileup format (Samtools [48]) to call geno- type at sites covered by at least three reads with minimum base and mapping quality of 20. Additionally, we excluded any clusters of three or more SNPs within 10 bp or any SNP within 3 bp of an indel. We then identified genomic bins of 1 kbp that had an average depth under a maximum threshold (twice genome-wide average coverage) and 90% nucleotide sequence covered, to ensure maximum sequence coverage in every sample and exclude false posi- tive SNPs resulting from copy number variation. These genomic bins were chained if adjacent. To assess the robustness of these supermatrices we also applied more formal supertree methods by esti- mating a ML tree using RAxML with 100 fast boot- strap replicates for each genomic bin of at least 5 kbp (Table S3 in Additional file 1). We used STAR [24] to reconstruct the species tree. Thereafter, we computed the relative frequency for each observed clade (Addi- tional file 3). Relative frequencies correspond to the proportion of each clade in the database of bootstrapped single locus trees. Lastly, we calculated the intersection of the genomic bins previously identified in each individual for further analysis using BedTools [49]. This resulted in an 11 way alignment with maximum sequence coverage and mini- mum false positive SNP calling in all our samples (approximately 1.1 Gbp; Table S3 in Additional file 1). In order to investigate how recombination affects phy- logenetic concordance across the genome we computed the mean pairwise Robison-Foulds distance of trees, using Phylip [51], within 1 Mbp windows. We obtained recombination rates from [20]. Conclusions The resolution afforded by complete genomes allowed us to infer not only ancient admixture episodes, but also those that took place as a result of more recent human- aided dispersal. Together, these findings provide insights related to the possible response to future climate and anthropogenic disturbances of mammalian taxa within ISEA. Reads were trimmed for three consecutive base pairs with phred quality score equal or below 13, and dis- carded if they were shorter than 40 bp. We used Mosaik 1.1.0017 with the unique alignment option to align reads to the Swine reference genome (Sscrofa10.2; Gen- Bank GCA_000003025.4; Table S2 in Additional file 1), together with the complete, mtDNA genome of S. scrofa Despite the challenges in building a single phylogeny from entire genome sequences, we were able to obtain a Frantz et al. Genome Biology 2013, 14:R107 http://genomebiology.com/2013/14/9/R107 Page 9 of 12 (accession: AF486874) for all Sus species and the mtDNA genome of Phacochoerus africanus (accession: DQ409327) for P. africanus. The S. scrofa and P. africa- nus mtDNA genomes were aligned using ClustalW [47]. Mapping errors are unlikely to be problematic in this study, as the sequence mismatch to the reference gen- ome was at max 3 to 4% (3 to 4 mismatches per 100 bp read), a distance easily accommodated by short-read local aligners such as Mosaik. Mapped read depth ran- ged from 7.5 to 24× (Table S1 in Additional file 1), thus providing enough power to call genotype confidently [40]. The resulting BAM files were deposited on the EBI Sequence Read Archive under accession number ERP001813. (accession: AF486874) for all Sus species and the mtDNA genome of Phacochoerus africanus (accession: DQ409327) for P. africanus. The S. scrofa and P. africa- nus mtDNA genomes were aligned using ClustalW [47]. Mapping errors are unlikely to be problematic in this study, as the sequence mismatch to the reference gen- ome was at max 3 to 4% (3 to 4 mismatches per 100 bp read), a distance easily accommodated by short-read local aligners such as Mosaik. Mapped read depth ran- ged from 7.5 to 24× (Table S1 in Additional file 1), thus providing enough power to call genotype confidently [40]. The resulting BAM files were deposited on the EBI Sequence Read Archive under accession number ERP001813. unique alignments of 1 Mbp (between 0.99 Mbp and 1.1 Mbp/each). Molecular clock analyses d d We estimated divergence times using an approximate likelihood method as implemented in MCMCtree (PAML v.4), with an independent relaxed-clock and birth-death sampling [27]. To overcome difficulties aris- ing from computational efficiency and admixture, we only used fragments (minimum 5 kbp) that had a good bootstrap support (at least 70% bootstrap support for each node) for the main topology (Additional file 2). Although this is expected to bias estimates of divergence time toward the present, the amount of error is expected to be relatively small considering the deep time scale in this analysis. This resulted in 416 genomic bins and a 4.4 Mbp alignment. We fitted an HKY+Γ4 model to each partition (bin) and estimated a mean mutation rate by fit- ting a strict clock to each fragment setting a root age at 10.5 Mya, as suggested by previous studies [55]. This mean rate was used to adjust the prior on the mutation rate (rgene) modeled by a gamma distribution as G (1,125). Parameters for the birth-death process with spe- cies sampling prior (BDS) and sigma2 values were set at 7 Because the depth of coverage of mtDNA was highly variable across the different samples (Table S4 in Addi- tional file 1), we applied a different filtering strategy. For each position covered we calculated the effective coverage of each allele as: C j = depth(j) i=1 1 −10−mij/10 x 1 −10−qij/10 (1) (1) where mij and qij refer to mapping quality and base quality score for read i at position j [50]. We filtered any sites where the major allele effective coverage did not represent at least 70% of the overall effective coverage at the position. Conclusions We used Kendall’s rank test for correlation analysis as implemented in R. We computed the distance to an outgroup (African warthog) in 1 Mbp windows for every Sus sample. There- after, we computed mean distances of all Sus to the out- group. We obtained recombination rates from [20]. We used Kendall’s rank test for correlation analysis as imple- mented in R. Authors’ contributions MAMG, ALA, LBS, H-JM, OM, GL and LAFF designed the study. LAFF and H- JM carried out sequence alignment and SNP calling. LAFF and JGS analyzed the data with input from OM, HJ-M and MS. RPMAC performed the DNA extraction and library preparation. GS, NL, ALA, EM and LBS provided samples and helped with the design of the study. YP and MB helped with the design of the study and the bioinformatics analyses. LAFF, JGS and GL wrote the manuscript with input from OM, EM, H-JM, ALA, MS and MAMG. All authors read and approved the final manuscript. Demographic analysis We conducted a demographic analysis using a hidden Markov model approach as implemented in PSMC [56] in our ISEA samples. We generated consensus sequences from bam files using the ‘pileup’ command in SAMtools. We used the following parameters: Tmax = 20; n = 64 (’4+50*1+4+6’). For plotting the results we used g = 5 and a rate of 2.5 × 10-8 mutations per gen- eration as in humans. Additional file 8: Figure S4, a phylogenetic tree constructed using SNPs sequenced with the Illumina Porcine SNP60 array. Competing interests Competing interests The authors declare that they have no competing interests. Admixture analyses y To detect and quantify admixture among taxa we used D-statistics [6,26] that take advantage of the large num- ber of SNPs present in whole genomes. In short, the D-statistics provide a robust test for admixture by asses- sing the fit of a strictly bifurcating phylogenetic tree. For a triplet of taxa P1, P2 and P3, and an outgroup O, in which the underlying phylogeny is represented by the Newick string (((P1, P2), P3). O), one can compute the number of sites with mutations consistent with incom- plete lineage sorting: those where P1 and P3 (BABA) or P2 and P3 (ABBA) share the derived allele (B; assuming ancestral state, A, in the outgroup). Under a null hypothesis of no gene-flow (strict bifurcation), the ratio D = (ABBA - BABA)/(ABBA + BABA) is not expected to be significantly different from 0. This is because ABBA and BABA sites can only be created by coales- cences in the common ancestor of P1, P2 and P3 and hence should happen with equal frequency. Alterna- tively, a significant excess of either ABBA or BABA site patterns is inconsistent with incomplete lineage sorting and provides evidence for a deviation from a phyloge- netic tree, suggesting additional population structure or gene-flow. Additional material 5 1 and G (1, 10), respectively. We ran two independent 40,000 (+10,000 burn in) MCMC samples for each com- bination of fossil calibration (Additional file 6) and assessed the convergence using TRACER [46] (Effective Sample Size [ESS] > 100). Additional file 1: Tables S1 to S4, with information on sequence data and alignment results. Additional file 2: Figure S1, a species cladogram with support from various analyses. Additional file 3: Table S5, containing results from clade relative frequency analysis. Additional file 4: Text with additional results and discussion for admixture analysis. Additional file 5: Table S8, which contains the full results from the D-statistics analysis. Additional file 6: Text that contains information about fossil calibration. Additional file 7: Figure S3 describing the demographic history of the population from MSEA. Additional file 8: Figure S4, a phylogenetic tree constructed using SNPs sequenced with the Illumina Porcine SNP60 array. Additional file 1: Tables S1 to S4, with information on sequence data and alignment results. Additional file 2: Figure S1, a species cladogram with support from various analyses. Additional file 3: Table S5, containing results from clade relative frequency analysis. Additional file 4: Text with additional results and discussion for admixture analysis. Additional file 5: Table S8, which contains the full results from the D-statistics analysis. Additional file 6: Text that contains information about fossil calibration. Additional file 7: Figure S3 describing the demographic history of the population from MSEA. Additional file 8: Figure S4, a phylogenetic tree constructed using SNPs sequenced with the Illumina Porcine SNP60 array. Additional file 1: Tables S1 to S4, with information on sequence data and alignment results. Additional file 2: Figure S1, a species cladogram with support from various analyses. Additional file 3: Table S5, containing results from clade relative frequency analysis. Additional file 4: Text with additional results and discussion for admixture analysis. Additional file 5: Table S8, which contains the full results from the D-statistics analysis. Additional file 6: Text that contains information about fossil calibration. Additional file 7: Figure S3 describing the demographic history of the population from MSEA. Additional file 8: Figure S4, a phylogenetic tree constructed using SNPs sequenced with the Illumina Porcine SNP60 array. Acknowledgements We would like to thank Kelley Harris for her numerous comments that greatly improved this work. We thank Bert Dibbits and Lauretta Rund for sample acquisition and preparation, Dr Oliver Raider for Sus cebifrons DNA, Dr Alain Ducos for French wild boar DNA, and Dr Sem Gemini for Italian wild boar DNA. We are also indebted to Alvaro G Hernandez and Chris Wright at the University of Illinois Keck Center for Comparative and Functional Genomics for the sequencing. We also thank Gus Rose and Konrad Lohse for their useful comments on earlier versions of this manuscript. Finally, we thank the Swine Genome Sequencing Consortium (SGSC) for the pre-release of the reference genome build 10.2. This project was financially supported by European Research Council grant no. ERC-2009- AdG: 249894, a USDA grant 2007-04315, by NIH grants R01-GM40282 and T32-HG00047, and by BBSRC Institute Strategic Grants. Financial support was also provided by Illumina Inc. To compute a standard error and assess the significance of the D-statistics, we used a Weighted Block Jackknife approach. We divided the genome into N blocks and com- puted the variance of the statistics over the genome N times leaving each block aside and derived a standard error (SE) using the theory of the Jackknife (supplemen- tary online material 15 in [6]). We then computed the D-statistics for every possible combination of species (Additional files 4 and 5) using P. africanus as an out- group. We corrected for multiple testing using a simple Bonferroni correction that involved multiplying our pva- lues by the number of D calculation (Additional files 4 and 5). For additional details see Additional file 4. Phylogenetic analysis First, we randomly selected genomic fragments (Table S3 in Additional file 1) of at least 1 kbp to make up 100 Frantz et al. 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https://openalex.org/W3208396800	https://agrovisnyk.com/index.php/agrovisnyk/article/view/2021_10_09/4088	Ukrainian	null	Scientific fundamentals of formation of organic agroecosystems in the Left-Bank Forest-Steppe	Vìsnik agrarnoï nauki	2,021	cc-by	4,044	DOI: https://doi.org/10.31073/agrovisnyk202110-09 Продуктивність сівозмін в органічних агроекосистемах визначається взаємодією різних чинників. За використання інтенсив- них агротехнологій і систем землеробства більшість заходів у підготовчий період і без- посередньо в процесі органогенезу підлягає регулюванню за допомогою хіміко-техноген- них ресурсів, здебільшого способом застосу- вання агрохімікатів. Набувають актуальності термінові системні заходи оптимізації при- родокористування, відновлення рівноваги природної системи. Автори [1] наголошують, що в найближчі 1 – 2 десятиріччя в Україні відбудеться настільки масштабна втрата ґрунтів, що за своїми наслідками перевер- шуватиме навіть глобальну зміну клімату. За таких умов в агросфері набувають поши- рення органічні системи землеробства з ви- робництвом екологічно безпечної продукції. В органічних агроекосистемах нагальним стає пошук ефективних і надійних джерел компенсації елементів живлення й ство- рення в ґрунті позитивного балансу гумусу. Для покращення живлення сільськогоспо- дарських культур можна застосовувати всі види органічних добрив [12 – 17]. Для контролю чисельності шкідливих організмів офіційно застосовують близько 30 природних біологічно активних речовин, 45 феромонів, 60 вірусів, більше 30-ти видів ентомофагів, а також бактерії, гриби і не- матоди [18]. Мета досліджень — з’ясувати агроре- сурсний потенціал Лівобережного Лісостепу та виявити тенденції змін основних пара метрів родючості чорнозему типового в сис- темі органічного землеробства. Матеріали та методи досліджень. У процесі виконання дослідницької роботи для досягнення поставленої мети, були ви- користані загальнонаукові та спеціальні для аграрної науки методи досліджень. Органічне виробництво — це цілісна сис- тема господарювання та виробництва хар- чових продуктів, яка поєднує в собі най- кращі практики з огляду на збереження довкілля, рівень біологічного розмаїття, збе- реження природних ресурсів, застосування високих стандартів належного утримання (добробуту) тварин і метод виробництва, який відповідає певним вимогам до продук- тів, вироблених з використанням речовин та процесів природного походження [2]. р р у Дослідження було проведене впродовж 1996 – 2015 рр. у стаціонарному агротех- нічному досліді кафедри землеробства іме- ні О.М. Можейка на дослідному полі Харків ського національного аграрного університе- ту імені В. В. Докучаєва поблизу сел. Велика Рогань Харківського р-ну Харківської обл. Ґрунтовий покрив території, де прово- дили дослідження, — чорнозем типовий вилугуваний малогумусний [19]. Дослідження було проведене впродовж 1996 – 2015 рр. у стаціонарному агротех- нічному досліді кафедри землеробства іме- ні О.М. Можейка на дослідному полі Харків ського національного аграрного університе- ту імені В. В. Докучаєва поблизу сел. Велика Рогань Харківського р-ну Харківської обл. у Ґрунтовий покрив території, де прово- дили дослідження, — чорнозем типовий вилугуваний малогумусний [19]. Виробники органічної продукції одержу- ють додаткові конкурентні переваги на рин- ку. НАУКОВІ ОСНОВИ ФОРМУВАННЯ ОРГАНІЧНИХ АГРОЕКОСИСТЕМ У ЛІВОБЕРЕЖНОМУ ЛІСОСТЕПУ УДК 631.147.001.891 (477.51/.52) © 2021 С.І. Кудря1, Ю.О. Тараріко2, Г.І. Личук3, Н.А. Кудря4 1, 2доктори сільськогосподарських наук 3, 4кандидати сільськогосподарських наук 1, 4Харківський національний аграрний університет імені В. В. Докучаєва п/в «Докучаєвське-2» Харківського р-ну Харківської обл., 62483, Україна 2Інститут водних проблем і меліорації НААН вул. Васильківська, 37, м. Київ, 03022, Україна 3Державна установа Національний Антарктичний науковий центр МОН України бульвар Тараса Шевченка, 16, м. Київ, 01601, Україна e-mail: 1Kudryasi.com@gmail.com, 2urtar@bigmir.net, 3aspirant.nnciz@gmail.com, 4kudrianadiiaa@gmail.com ORCID: 10000-0002-4581-8426, 20000-0001-8475-240X, 30000-0002-2579-5036, 40000-0003-3348-3515 Надійшла 24.06.2021 Агроекологія, радіологія, меліорація НАУКОВІ ОСНОВИ ФОРМУВАННЯ ОРГАНІЧНИХ АГРОЕКОСИСТЕМ У ЛІВОБЕРЕЖНОМУ ЛІСОСТЕПУ УДК 631.147.001.891 (477.51/.52) © 2021 С.І. Кудря1, Ю.О. Тараріко2, Г.І. Личук3, Н.А. Кудря4 1, 2доктори сільськогосподарських наук 3, 4кандидати сільськогосподарських наук 1, 4Харківський національний аграрний університет імені В. В. Докучаєва п/в «Докучаєвське-2» Харківського р-ну Харківської обл., 62483, Україна 2Інститут водних проблем і меліорації НААН вул. Васильківська, 37, м. Київ, 03022, Україна 3Державна установа Національний Антарктичний науковий центр МОН України бульвар Тараса Шевченка, 16, м. Київ, 01601, Україна e-mail: 1Kudryasi.com@gmail.com, 2urtar@bigmir.net, 3aspirant.nnciz@gmail.com, 4kudrianadiiaa@gmail.com ORCID: 10000-0002-4581-8426, 20000-0001-8475-240X, 30000-0002-2579-5036, 40000-0003-3348-3515 Надійшла 24.06.2021 Агроекологія, радіологія, меліорація Агроекологія, радіологія, меліорація g ORCID: 10000-0002-4581-8426, 20000-0001-8475-240X, 30000-0002-2579-5036, 40000-0003-3348-3515 Надійшла 24.06.2021 Мета — оцінити агроресурсний потенціал Лівобережного Лісостепу та вста- новити тенденції змін основних параметрів родючості чорнозему типового в системі органічного землеробства. Методи — загальнонаукові та спе- ціальні: довготривалий польовий — для визначення кількісних показників і аналітичний. Результати. Проаналізовано результати 20-річних польо- вих досліджень і особливості функціонування органічних агроекосистем, оцінено агроресурсний потенціал Лівобережного Лісостепу, тенденції формування гідротермічного режиму. Проведено обґрунтування сівозмін для господарств різної спеціалізації. Розкрито закономірності впливу різних гідротермічних умов на динаміку властивостей ґрунту, проведено балансові дослідження особливостей кругообігу азоту, фосфору та калію. Здійснено пошук математичних зв’язків між урожайністю культур сівозмін та їх попе- редниками, кількістю опадів, температурою повітря, обсягом надходження в ґрунт нетоварної частини врожаю і запасами в ньому основних елементів живлення. Установлено, що введення бобових культур в органічні агроеко- системи сприяє оптимізації агрофізичних показників родючості чорнозему типового та балансу поживних речовин у ньому, що створює сприятливі умови для розвитку рослин. Визначено продуктивність сівозмін. Найбільшою вона була в сівозмінах із бобовими попередниками пшениці озимої: соєю, горохом, сочевицею та вико-вівсяною сумішкою, за умов використання на третій рік ротації буряків цукрових. Вихід кормопротеїнових одиниць у цих Вісник аграрної науки 2021, №10 (823) 68 АГРОЕКОЛОГІЯ, РАДІОЛОГІЯ, МЕЛІОРАЦІЯ Наукові основи формування органічних агроекосистем у Лівобережному Лісостепу АГРОЕКОЛОГІЯ, РАДІОЛОГІЯ, МЕЛІОРАЦІЯ варіантах становив 3,78; 3,75; 3,72 і 3,71 т/га. За умов розміщення гречки на 3-й рік ротації короткоротаційних сівозмін виявлено зниження їхньої про- дуктивності в 1,4 раза. Висновки. Вирощування бобових культур в органічних агроекосистемах сприяє покращенню агрофізичних показників родючості чорнозему типового. Саме сівозміни з цими культурами забезпечили най- вищу продуктивність. Опрацювання корелятивних зв’язків між гідротерміч- ними умовами та врожайністю культур у сівозмінах дає змогу ефективно використовувати природний потенціал в органічних агроекосистемах. Ключові слова: екологізація аграрного виробництва, агроресурсний потенціал, агротехнічні досліди, сівозміна, бобовий компонент, продуктивність, нетоварна продукція, органік-орієнтована модель. DOI: https://doi.org/10.31073/agrovisnyk202110-09 2021, №10 (823) АГРОЕКОЛОГІЯ, РАДІОЛОГІЯ, МЕЛІОРАЦІЯ Наукові основи формування органічних агроекосистем у Лівобережному Лісостепу Наукові основи формування органічних агроекосистем у Лівобережному Лісостепу Наукові основи формування органічних агроекосистем у Лівобережному Лісостепу Результати досліджень. У середньому за 1996 – 2009 рр. щільність складення ґрунту в орному шарі коливалася у межах 1,16 – 1,21 г/см3. Вищим показник щільності ґрунту був у сівозміні з кукурудзою на силос. Деяке ущільнення ґрунту зумовлене знижен- ням показника його структурності. У сівозмі- нах із зернобобовими культурами встанов- лено помітне розпушення ґрунту, причому найнижчу щільність мав варіант із квасо- лею — 1,16 г/см3. У паровому варіанті щіль- ність орного шару становила 1,18 г/см3. Вияв- лено також, що верхній шар ґрунту є більш пухким порівняно з шаром 15 – 30 см. Причому найбільша різниця була встановлена на ва- ріантах із чистим паром — 0,05, горохом — 0,06 і вико-вівсяною сумішкою — 0,04 г/см3. У решти варіантів різниця була нижчою — від 0,01 до 0,03 г/см3. Загалом щільність складення ґрунту знаходиться в оптималь- них межах для чорноземних ґрунтів. сівозмін відрізнялися першими і третіми культурами. Попередниками пшениці ози- мої, а відповідно першими культурами сіво- змін були: чистий пар, горох на зерно, чина на зерно, сочевиця на зерно, вико-вівсяна сумішка на зелений корм, соя на зелений корм, квасоля на зерно та кукурудза на си- лос. На 3-й рік ротації сівозмін вирощували буряки цукрові та гречку. Останньою куль- турою в усіх сівозмінах був ячмінь ярий. Загальна площа стаціонарного досліду становить 4 га. Площа посівної ділянки — 142 м2, облікової — 50 – 100 м2. Входження в сівозміну проводили двома полями. Розміщення варіантів у дослідах — систе- матичне, повторність — 3-разова. Досліджували органічну систему удоб рення з використанням на добриво тільки нетоварної частини врожаю: соломи бобо- вих, у середньому 2,7 т/га, соломи пшениці озимої у середньому 5,1 , соломи гречки — 2,5, гички буряків цукрових — 10, соломи ячменю ярого — 2,5 т/га. Уміст загального гумусу в орному шарі ґрунту по варіантах досліду практично не відрізнявся. Але можна відзначити тенден- цію до збільшення кількості органічного вуг- лецю в сівозмінах із кукурудзою на силос, горохом і чиною за мінімального значення у сівозміні з чистим паром. Технологія вирощування сільськогоспо- дарських культур у досліді загальноприйня- та для умов Харківської обл. У дослідах вирощували сорти та гібриди сільськогоспо- дарських культур, занесених до Державного реєстру сортів і рослин, придатних до виро- щування в Лісостепу. DOI: https://doi.org/10.31073/agrovisnyk202110-09 Така форма господарювання також дає можливість активізувати залучення молоді на сільські території, інвестиційних ресур- сів в економіку підприємств; сформувати загальний розвиток [3 – 11]. Стаціонарний дослід із вивчення польо- вих сівозмін короткої ротації було закладе- но у 1962 р. У досліді вивчали 16 варіантів польових сівозмін короткої ротації. Схеми 69 2021, №10 (823) Вісник аграрної науки АГРОЕКОЛОГІЯ, РАДІОЛОГІЯ, МЕЛІОРАЦІЯ АГРОЕКОЛОГІЯ, РАДІОЛОГІЯ, МЕЛІОРАЦІЯ Наукові основи формування органічних агроекосистем у Лівобережному Лісостепу розміщення його після буряків урожайність коливалася в межах 1,95 – 2,17 т/га. Істотно нижчою врожайність ячменю була в сіво- змінах із кукурудзою, чиною та квасолею 1,95 т/га, 1,99 і 2,06 т/га відповідно. За роз- міщення після гречки врожайність культури була помітно нижчою. фоні, виявили статистично доведену пере- вагу чистого пару, після якого, у середньому за 20 років, отримували 4 т/га зерна пшени- ці озимої. Її врожайність після гороху, соче- виці та вико-вівсяної сумішки була нижчою на 13 – 15 %, а після сої становила 3,22 т/га, квасолі — 3,21 т/га та чини — 3,25 т/га (при НІР0,95 0,47 т/га). Мінімальну врожайність пшениці озимої отримано після кукурудзи на силос — 2,66 т/га, що в 1,5 раза менше, ніж після чистого пару і в 1,2 раза менше порівняно із розміщенням пшениці після бобових культур. Як узагальнюючий показник розраховано вихід умовних кормопротеїнових одиниць з 1 га ріллі. Значно вищий вихід кормопро- теїнових одиниць забезпечували сівозміни з буряками цукровими. ур цу р У розрізі перших культур сівозмін цей показник був помітно нижчим у сівозмінах з чиною, кукурудзою та квасолею. Із кормо- вих культур вищою продуктивністю відріз- нялася соя — 3,86 т к.-п. од./га. Значно по- ступалася їй кукурудза на силос. Стабільну продуктивність забезпечували такі зерно- бобові культури, як горох, чина та сочевиця, продуктивність яких становила 2,93; 2,81 та 2,79 т к.-п. од./га відповідно. Крім того, потенціал біопродуктивності чорнозему ти- пового в Лівобережному Лісостепу в системі органічного землеробства оцінено у наших попередніх публікаціях [20 – 22]. У середньому за роки досліджень мак- симальна врожайність буряків цукрових була в ланці з чистим паром — 27,5 т/га. За використання бобових культур урожайність коренеплодів істотно знижувалася. Мак симальне зниження врожайності буряків від- значається в ланці з кукурудзою на силос. Урожайність гречки коливалася в межах 1,12 – 1,33 т/га. Дещо вищою вона була в сівозміні з чистим паром, а в ланках з горо- хом, соєю та вико-вівсяною сумішкою — від- повідно становила 1,23 т/га, 1,22 і 1,20 т/га. Найнижчу врожайність гречка формувала у варіантах із кукурудзою на силос, чи- ною та сочевицею — 1,12; 1,14 і 1,17 т/га. Водночас найменша істотна різниця стано- вила 0,16 т/га. Виявлено, що врожайність культур і про- дуктивність сівозмін мають математичний зв’язок із кількістю опадів і температурою повітря на певних етапах вегетації. Багато варіантний детальний аналіз різних поєд- нань декад за впливом умов зволоження Вплив перших культур сівозмін на вро- жайність ячменю значно зменшувався. АГРОЕКОЛОГІЯ, РАДІОЛОГІЯ, МЕЛІОРАЦІЯ Аналізуючи вплив попередників на вро- жайність пшениці озимої на природному 70 2021, №10 (823) Вісник аграрної науки 1 2 3 4 5 6 7 8 0 100 200 300 400 500 600 1997 1998 1999 2001 2002 2004 2005 2006 2007 2008 2009 2011 2012 2013 2014 2015 т/га Опади, мм Роки Кількість опадів, мм Пар Горох Чина Вико-овес Соя Кукурудза R2 = 0,71 y = 2E – 12x6 – 6E – 09x5 + 7E – 06x4 – 0,0048x3 + 1,7006x2 – 313,33x + 23314 Рис. 1. Вплив кількості опадів із вересня по липень на врожайність пшениці озимої за різних попередників: — кількість опадів, мм; — пар; — горох; — чина; — вико- овес; — соя; — кукурдза 1 2 3 4 5 6 7 8 0 100 200 300 400 500 600 1997 1998 1999 2001 2002 2004 2005 2006 2007 2008 2009 2011 2012 2013 2014 2015 т/га Опади, мм R2 = 0,71 y = 2E – 12x6 – 6E – 09x5 + 7E – 06x4 – 0,0048x3 + 1,7006x2 – 313,33x + 23314 Кількість опадів, мм Пар Горох Чина Вико-овес Соя Кукурудза Рис. 1. Вплив кількості опадів із вересня по липень на врожайність пшениці озимої за різних попередників: — кількість опадів, мм; — пар; — горох; — чина; — вико- овес; — соя; — кукурдза 70 Вісник аграрної науки Вісник аграрної науки Вісник аграрної науки 2021, №10 (823) АГРОЕКОЛОГІЯ, РАДІОЛОГІЯ, МЕЛІОРАЦІЯ АГРОЕКОЛОГІЯ, РАДІОЛОГІЯ, МЕЛІОРАЦІЯ За 71 2021, №10 (823) Вісник аграрної науки R2 = 0,66 – 0,84 29,3 28,6 27,0 26,8 28,4 26,9 26,8 25,5 4,21 3,66 3,42 3,49 3,59 3,38 3,40 2,80 1,33 1,23 1,14 1,18 1,32 1,22 1,18 1,12 2,14 2,17 1,99 2,04 2,14 2,10 2,06 1,95 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 23 24 25 26 27 28 29 30 пар горох чина сочевиця вико-овес соя квасоля кукурудза т/га (коренеплоди) Попередник буряки пшениця гречка ячмінь т/га (зерно) НІР0,95 – 0,15 т/га НІР0,95 – 0,38 т/га НІР0,95 – 0,21 т/га НІР0,95 – 0,21 т/га Рис. 2. Вплив перших культур на врожайність наступних культур сівозмін, 1996–2015 рр.: — буряки; — пшениця; — гречка; — ячмінь Вісник аграрної науки 71 2021, №10 (823) АГРОЕКОЛОГІЯ, РАДІОЛОГІЯ, МЕЛІОРАЦІЯ Наукові основи формування органічних агроекосистем у Лівобережному Лісостепу на продуктивність посівів пшениці озимої за- сівозмін також є позитивним на рівні 3 т/га 350 370 390 410 430 450 470 490 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 кг/га Рік Азот Фосфор Калій Рис. 3. Тренди змін запасів елементів живлення в часі: — азот; — фосфор; — калій 0 1 2 3 4 5 6 7 15 20 25 30 35 40 45 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 т/га (зерно) т/га (коренеплоди) Рік Цукрові буряки Горох Пшениця озима Ячмінь ярий Сівозміна Рис. 4. Тренди змін урожайності культур і продуктивності сівозмін у часі: — сівозміна; — буряки цукрові; — пшениця озима; — ячмінь ярий; — горох АГРОЕКОЛОГІЯ, РАДІОЛОГІЯ, МЕЛІОРАЦІЯ Наукові основи формування органічних агроекосистем у Лівобережному Лісостепу АГРОЕКОЛОГІЯ, РАДІОЛОГІЯ, МЕЛІОРАЦІЯ Наукові основи формування органічних агроекосистем у Лівобережному Лісостепу АГРОЕКОЛОГІЯ, РАДІОЛОГІЯ, МЕЛІОРАЦІЯ Наукові основи формування органічних агроекосистем у Лівобережному Лісостепу 350 370 390 410 430 450 470 490 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 кг/га Рік Рік Азот Фосфор Калій калій 0 1 2 3 4 5 6 7 15 20 25 30 35 40 45 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 т/га (зерно) т/га (коренеплоди) Рік Цукрові буряки Горох Пшениця озима Ячмінь ярий Сівозміна Рис. 4. Висновки в органічних агроекосистемах. Тренд кількості азоту та фосфору в чорноземі типовому органічних агроекосистем не змінюється в часі, а калію — збільшується. При цьому тренд продуктивності сівозмін також є стабільним, що є актуальним під час упровадження органічних систем землеробства без застосування агрохі- мікатів. Найперспективнішими бобовими культурами для підвищення ефективності органічних агроекосистем є соя і квасоля. Ці культури, поряд із позитивним впливом на екологічний стан ґрунту, підвищують еко- номічні показники органічного виробництва. Вирощування бобових культур в органіч- них агроекосистемах сприяє покращенню агрофізичних показників родючості чор- нозему типового. Найвищу продуктив- ність забезпечили сівозміни із бобовими культурами: соєю, горохом, сочевицею та вико-вівсяною сумішкою, за використання на 3-й рік ротації буряків цукрових. Значно меншим вихід кормопротеїнових одиниць був у сівозмінах з чистим паром — 3,21 т/га. Опрацювання корелятивних зв’язків між гідротермічними умовами та врожайністю культур у сівозмінах дає змогу ефективно використовувати природний потенціал and features of functioning of organic agroecosys- tems are analyzed, the agroresource potential of the Eastern Forest-Steppe, tendencies of hydrothermal regime formation are assessed. Substantiation of crop rotations for farms of different specialization is carried out. The regularities of the influence of differ- ent hydrothermal conditions on the dynamics of soil properties are revealed, the balance researches of the peculiarities of the nitrogen, phosphorus and po- tassium cycle are carried out. Mathematical relation- ships between crop yields and their predecessors, rainfall, air temperature, the amount of non-mar- ketable part of the crop and the reserves of basic nutrients in it are searched. It is established that the introduction of legumes into organic agroecosys- tems contributes to the optimization of agrophys- ical indicators of the fertility of typical chornozem and the balance of nutrients in it, which creates favorable conditions for plant development. The productivity of crop rotations is determined. It was the largest in crop rotations with leguminous prede- cessors of winter wheat: soybeans, peas, lentils and vetch-oats mixtures, under the conditions of use of Kudria S.1, Tarariko Yu.2, Lychuk G.3, Kudria N.4 1, 4Kharkiv National Agrarian University named af- ter V.V. Dokuchayev, p. o. «Dokuchaevske-2», Kharkiv oblast, 62483, Ukraine, 2Institute of Water Problems and Land Reclamation of NAAS, 37 Vasylkivska Str., Kyiv, 03022, Ukraine, 3State Institution National Antarctic Scientific Center of the Ministry of Education and Science of Ukraine, 16 Taras Shevchenko Boulevard, Kyiv, 01601, Ukraine; е-mail: 1Kudryasi.com@gmail.com, 2urtar@bigmir. net, 3aspirant.nnciz@gmail.com, 4kudrianadiiaa@ gmail.com; ORCID: 10000-0002-4581-8426, 20000- 0001-8475-240X, 30000-0002-2579-5036, 40000- 0003-3348-3515 АГРОЕКОЛОГІЯ, РАДІОЛОГІЯ, МЕЛІОРАЦІЯ Тренди змін урожайності культур і продуктивності сівозмін у часі: — сівозміна; — буряки цукрові; — пшениця озима; — ячмінь ярий; — горох Цукрові буряки Горох Пшениця озима Ячмінь ярий Сівозміна Рис. 4. Тренди змін урожайності культур і продуктивності сівозмін у часі: — сівозміна; — буряки цукрові; — пшениця озима; — ячмінь ярий; — горох на продуктивність посівів пшениці озимої за- свідчив кореляційний зв’язок між сумою опа- дів з вересня по липень і врожаєм (рис. 1). сівозмін також є позитивним на рівні 3 т/га (рис. 4), що актуально у результаті впрова- дження органічної системи землеробства. У дослідженнях виявлено зменшення чистого прибутку в сівозмінах із чиною та чистим паром, що відбувалося за рахунок зниження продуктивності цих сівозмін. За нашими розрахунками кількість кормопро- теїнових одиниць із 1 га у таких сівозмі- нах помітно зменшувалася. Це впливало на показник вартості продукції, який був найнижчим і становив — 15 і 16 тис. грн/га відповідно. сівозмін також є позитивним на рівні 3 т/га (рис. 4), що актуально у результаті впрова- дження органічної системи землеробства. Аналіз урожайних даних у розрізі впливу перших культур на наступні засвідчив на- явність відповідних закономірностей. Так, з рис. 2 видно, що попередники найбіль- ше впливають саме на наступну пшеницю озиму. У дослідженнях виявлено зменшення чистого прибутку в сівозмінах із чиною та чистим паром, що відбувалося за рахунок зниження продуктивності цих сівозмін. За нашими розрахунками кількість кормопро- теїнових одиниць із 1 га у таких сівозмі- нах помітно зменшувалася. Це впливало на показник вартості продукції, який був найнижчим і становив — 15 і 16 тис. грн/га відповідно. Тренд кількості доступних сполук азоту та фосфору в ґрунті у часі не змінював- ся, а рухомого калію навіть збільшувався (рис. 3). При цьому тренд продуктивності 72 2021, №10 (823) Вісник аграрної науки Наукові основи формування органічних агроекосистем у Лівобережному Лісостепу АГРОЕКОЛОГІЯ, РАДІОЛОГІЯ, МЕЛІОРАЦІЯ Наукові основи формування органічних агроекосистем у Лівобережному Лісостепу Слід зазначити, що продуктивність сіво- змін із гречкою була нижчою в середньо- му в 1,4 раза, порівняно з сівозмінами, де на 3-й рік ротації висівали буряки цу- крові. Найвищий умовно-чистий прибуток забезпечила сівозміна, де попередником пшениці озимої вирощувалася квасоля — 7,6 тис. грн/га. Причому цей варіант мав не найвищу продуктивність сівозміни — 2,39 т к.-п. од./га, що в середньому мен- ше на 0,25 т к.-п. од./га, ніж у сівозмінах із рештою бобових культур і кукурудзою на силос. Цьому варіанту поступався тільки паровий, у якому продуктивність була ниж- чою на 0,22 т к.-п. од/га. Отже, найвищі показники економічної ефективності забезпечила сівозміна з ква- солею. 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АГРОЕКОЛОГІЯ, РАДІОЛОГІЯ, МЕЛІОРАЦІЯ Наукові основи формування органічних агроекосистем у Лівобережному Лісостепу that provided the highest productivity. Elaboration of correlations between hydrothermal conditions and crop yields in crop rotations allows efficient use of natural potential in organic agroecosystems. sugar beet for the third year of rotation. The yield of feed protein units in these variants was 3.78; 3.75; 3.72 and 3.71 t/ha. Under the conditions of placing buckwheat on the 3rd field of short-rotation crop ro- tations, their productivity was reduced by 1.4 times. Conclusions. The cultivation of legumes in organic agroecosystems contributes to the improvement of agrophysical indicators of the fertility of typical chornozem. It was crop rotations with these crops Key words: greening of agricultural production, agro-resource potential, agro-technical experiments, crop rotation, bean component, productivity, non- commodity products, organic-oriented model. DOI: https://doi org/10 31073/agrovisnyk202110-09 Key words: greening of agricultural production, agro-resource potential, agro-technical experiments, crop rotation, bean component, productivity, non- commodity products, organic-oriented model. DOI htt //d i /10 31073/ i k202110 09 Scientific fundamentals of formation of organic agroecosystems in the Left-Bank Forest-Steppe Goal. To assess the agro-resource potential of the Left-Bank Forest-Steppe and to establish trends in the main parameters of the fertility of typ- ical chornozem in the system of organic farming. Methods — general and special: long-term field — to determine quantitative indicators and analytical. Results. The results of 20 years of field research 73 2021, №10 (823) Вісник аграрної науки Бібліографія Ґрун- товий покрив дослідного поля «Роганського ста- ціонару» Харківського НАУ ім. В.В. Докучаєва. Вісник ХНАУ. Ґрунтознавство, агрохімія, зем- леробство, лісове господарство. Харків: ХНАУ, 2016. № 2. С. 5 – 15. p p 9. 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Agricultural and Resource Economics: International Scientific E-Journal. 2016. V. 2. № 4. P. 30 – 42. URL: http://www.are-journal.com p j 12. Дегодюк Е.Г., Літвінова О.А., Ярмо- ленко Є.В., Дмитренко О.В. Вплив органічних добрив на родючість сірого лісового ґрунту. Агроекологічний журнал. 2019. № 2. С. 31 – 35. 22. Кудря С.І. Продуктивність короткоротацій- ної сівозміни з різними бобовими культурами на чорноземі типовому. Вісник аграрної науки. 2020. № 1(802). С. 13 – 18. 74 2021, №10 (823) Вісник аграрної науки
https://openalex.org/W2147212304	https://pure.eur.nl/ws/files/46791276/fischer	English	null	The spatial distribution of leprosy cases during 15 years of a leprosy control program in Bangladesh: An observational study	BMC infectious diseases	2,008	cc-by	6,914	BioMed Central BioMed Central © 2008 Fischer et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: An uneven spatial distribution of leprosy can be caused by the influence of geography on the distribution of risk factors over the area, or by population characteristics that are heterogeneously distributed over the area. We studied the distribution of leprosy cases detected by a control program to identify spatial and spatio-temporal patterns of occurrence and to search for environmental risk factors for leprosy. Methods: The houses of 11,060 leprosy cases registered in the control area during a 15-year period (1989–2003) were traced back, added to a geographic database (GIS), and plotted on digital maps. We looked for clusters of cases in space and time. Furthermore, relationships with the proximity to geographic features, such as town center, roads, rivers, and clinics, were studied. Results: Several spatio-temporal clusters were observed for voluntarily reported cases. The cases within and outside clusters did not differ in age at detection, percentage with multibacillary leprosy, or sex ratio. There was no indication of the spread from one point to other parts of the district, indicating a spatially stable endemic situation during the study period. The overall risk of leprosy in the district was not associated with roads, rivers, and leprosy clinics. The risk was highest within 1 kilometer of town centers and decreased with distance from town centers. Conclusion: The association of a risk of leprosy with the proximity to towns indicates that rural towns may play an important role in the epidemiology of leprosy in this district. Further research on the role of towns, particularly in rural areas, is warranted. Asian region and Brazil together accounted for 81% of all cases of leprosy detected in 2006 [2]. Asian region and Brazil together accounted for 81% of all cases of leprosy detected in 2006 [2]. Open Ac Research article The spatial distribution of leprosy cases during 15 years of a leprosy control program in Bangladesh: An observational study EAJ Fischer1, D Pahan2, SK Chowdhury2 and JH Richardus1 Open Access Address: 1Department of Public Health, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Neth Program, Leprosy Mission Bangladesh, Nilphamari, Bangladesh er - egil.fischer@wur.nl; D Pahan - ch@dblm.tlmbangladesh.org; SK Chowdhury - ch@dblm.tlmbangladesh.org; ichardus@erasmusmc.nl * Corresponding author Received: 24 December 2007 Accepted: 23 September 2008 Received: 24 December 2007 Accepted: 23 September 2008 Page 1 of 10 (page number not for citation purposes) Published: 23 September 2008 Published: 23 September 2008 BMC Infectious Diseases 2008, 8:126 doi:10.1186/1471-2334-8-126 This article is available from: http://www.biomedcentral.com/1471-2334/8/126 © 2008 Fischer et al; licensee BioMed Central Ltd. Study design h d The study is a retrospective observational study on the spatial distribution of newly detected leprosy patients in northwest Bangladesh over a 15-year period. Mode of detection As the data was from a running control program, the cases were detected by different modes of detection [17]: volun- tary reporting, surveys, and contract tracing. Voluntarily reported cases, apart from cases presented voluntarily at a clinic, included those referred by a professional health worker or other informed respected person (i.e. village doctors, teachers, or health workers). Surveys consisted of school or village surveys and were performed during the entire study period. During these school or village surveys, the students of a school or the population of a certain area with an assumed high prevalence of leprosy were exam- ined. Contact tracing was always practiced after a volun- tarily reported case was confirmed and continued for 2 to 5 yearly visits, depending on the leprosy classification [14]. http://www.biomedcentral.com/1471-2334/8/126 http://www.biomedcentral.com/1471-2334/8/126 BMC Infectious Diseases 2008, 8:126 explanation for the uneven distribution in another Brazil- ian state, São Paulo thought to be migratory movement towards the urban and developing areas in the center of the state [3]. In the Malawian Karonga district, a positive relationship between the proximity of water and leprosy incidence was previously found [5]. The relationship between open water and leprosy was hypothesized based on observed associations with rainfall and coastal popula- tions [7,8], as well as evidence that the infectious agent, Mycobacterium leprae, survives longer outside the human body in humid compared to dry atmospheres [9]. In a locality with many rivers and other bodies of water, such as northwest Bangladesh, the relationship between lep- rosy and open water might be quite different. refugee camp was created near Saidpur city after the Bang- ladesh war for independence in the early 1970's [12]. One of the major rivers of Bangladesh, the Tista River, flows through the northeast part of the district and several smaller rivers cross the district. A map of Nilphamari dis- trict is presented in Figure 1. The Danish Bangladesh Leprosy Mission (DBLM) was established in this area in 1977. Since that time, more than 95% of registered leprosy patients have been treated by DBLM. The project area also covers the neighboring districts of Rangpur, Thakurgoan, and Panchagar. The DBLM has been responsible for leprosy control in these four districts since 1994. Multidrug therapy (MDT) was completely introduced in the project area by 1991 [13]. Differences in the case detection rates can arise from dif- ferences in the accessibility of leprosy control facilities. In poor areas, traveling is expensive for the common people and the proximity to a leprosy control facility might increase the detection rate among the population. Study population The study population existed of all leprosy patients diag- nosed and registered between January 1, 1989 and December 31, 2003 at one of the DBLM clinics and living in Nilphamari district. Case registration was done accord- ing to the DBLM guidelines [14]; demographic data, World Health Organization (WHO) leprosy classification [15], and the mode of detection were registered. A DBLM leprosy control supervisor confirmed all cases before reg- istration and subsequent treatment. Uncertain cases were referred to the leprosy control officer or DBLM medical officer for confirmation. An independent inspector assessed the program in 2001 and found an over-diagno- sis of only 3.4% [16]. For the current study, we used the existing patient database and added spatial data. A study of the spatial distribution of leprosy can contrib- ute to the knowledge about, or identification of, the underlying risk factors for the disease and the transmis- sion patterns of M. leprae. A clustering of leprosy cases at the village level was not observed in the highly endemic Nilphamari district in northwest Bangladesh [10]. In this paper we describe the spatial distribution of leprosy at the district level in the same area during the period of 1989 to 2003 and determined whether high case detection clusters were present in the district. We investigated the risk of lep- rosy in proximity to geographic factors that may have a relationship with the risk of leprosy, such as the environ- ment (i.e. rivers and roads), a different population (i.e. towns), or enhanced availability of health services (i.e. leprosy clinics). Background New cases of leprosy are currently found primarily in trop- ical regions [1,2], but the distribution within these regions is not uniform. Sixty eight percent of newly detected cases in 2005 were found in Southeast Asia, 80% of which were detected in India. In the same year, another 13% of all cases worldwide were found in Brazil. The Southeast Within highly endemic regions, the occurrence of leprosy is also not uniformly distributed [3-5]. The distribution of leprosy in the Brazilian state of Ceará reflects socioeco- nomic differences within the state [4,6], whereas the Page 1 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1471-2334/8/126 Study area h d Road, populated places, and hydrographical data were obtained from downloadable data of the geo- Table 1: Leprosy, population, and geographic characteristics of the sub-districts. Sub-district Cases Person-years NCDR* Area (km2) No. Towns No. Clinics Nilphamari Sadar 2,501 5,003,010 0.50 249.8 2 1 Saidpur 1,654 3,375,432 0.49 339.2 1 1 Kishoregonj 1,002 4,140,829 0.24 332.8 2 1 Jaldhaka 2,215 3,791,886 0.58 338.8 2 1 Domar 1,647 2,910,790 0.57 256.3 2 1 Dimla 2,041 3,125,001 0.65 124.1 3 1 Total 11,060 22,346,947 0.49 1640.9 12 6 New case detection rate per 1,000 person-years Table 1: Leprosy, population, and geographic characteristics of the sub-districts. Sub-district Cases Person-years NCDR Area (km2) No. Towns No. Clinics Nilphamari Sadar 2,501 5,003,010 0.50 249.8 2 1 Saidpur 1,654 3,375,432 0.49 339.2 1 1 Kishoregonj 1,002 4,140,829 0.24 332.8 2 1 Jaldhaka 2,215 3,791,886 0.58 338.8 2 1 Domar 1,647 2,910,790 0.57 256.3 2 1 Dimla 2,041 3,125,001 0.65 124.1 3 1 Total 11,060 22,346,947 0.49 1640.9 12 6 *New case detection rate per 1,000 person-years Table 1: Leprosy, population, and geographic characteristics of the sub-districts. tion in 1995 and 2000 based upon 1991 and 2001 census data assuming an exponential growth of the population [19]. detection was studied in relation to towns, rivers, roads, and leprosy clinics separately. However, we focus on voluntarily reported cases because, in this control program, these cases are thought to give the best representation of the incidence of leprosy. Surveys normally tend to give a better picture of the real preva- lence than voluntarily reported cases. In this control pro- gram, however, surveys were performed depending on the number of cases previously voluntarily reported in a vil- lage or school. Results of cases detected by surveys or con- tact tracing can be found in the supplementary information. Digital maps of the administrative boundaries of the dis- tricts and sub-districts of Bangladesh were obtained from the Food and Agricultural Organization of the United Nations [20]. Road, populated places, and hydrographical data were obtained from downloadable data of the geo- community [21]. Statistical analyses Case detection was plotted against time and tested for a temporal cluster [22]. A temporal cluster is a period in which case detection was higher than expected for cases randomly distributed over the study period. The likeli- hood that the case detection originated at random during a period was calculated assuming a Poisson distribution of cases among the population. A likelihood ratio test was used to obtain a p-value for the most likely cluster. Location of patients During the current study, the houses in which patients lived at the time of diagnosis were traced back by specially trained staff. We note that this is not necessarily the loca- tion at which the patient became infected. Another possi- bility would have been to use location at which the patient lived when the first signs of disease were found. The location where the patient lived during diagnosis, however, could be determined more accurately, and we assume that the difference with the location at which the first signs occurred is not very different on the scale of a whole district. The coordinates were measured using a handheld GPS-unit (Geko 201 Garmin™) between Janu- ary and November 2006. Cases were excluded if the patient was registered to live in a district other than Nil- phamari or if the house was outside Nilphamari district according to our digital map. Finally, those whose home coordinates could not be obtained were excluded from analysis, in addition to patients for whom the mode of detection was unknown. The area was tested for a high incidence of spatio-tempo- ral clusters of cases separately for each detection mode using the spatio-temporal permutation test. The spatio- temporal permutation test [23] is a nonparametric test making use of the information from the case distribution. This test compares the observed number of cases during a time period in a circular area with the expected number cases if the spatial and temporal location of all cases were independent. The comparison is made for a cylindrical window with a circular geographic base and with height corresponding to the length of the time period. Both the circular base (the area) and the height of the cylinder (the time period) are flexible. The likelihood that the case detection in a certain space-time window originated by chance was calculated under the assumption that no space-time interaction exists. The expected cases in a cer- tain area were calculated based upon the number of cases observed at that location during the entire study period and the number of cases in the whole district during that timeframe. Therefore, this method adjusts for the pure spatial and pure temporal incidence. The probability that Study area h d The study was conducted in the Nilphamari district at 26°00' N and 88°57' E. The district has an area of 1640.9 km2 and approximately 1.5 million inhabitants [11]. The district is divided into six sub-districts. Geographical and leprosy characteristics of the sub-districts are given in Table 1. The sub-districts Nilphamari Sadar and Saidpur contain two major urban areas, also called Nilphamari and Saidpur, with Saidpur city being the largest urban area. The district is mainly rural outside these urbanized areas. The Saidpur sub-district contains a large refugee population of over 38,000 stateless Bihari refugees. The The occurrence of spatio-temporal clusters of high rates of detection was investigated separately for each mode of detection. The characteristics of patients within clusters were compared to patients living outside the clusters. The position of the houses of patients grouped by mode of Page 2 of 10 (page number not for citation purposes) Page 2 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1471-2334/8/126 BMC Infectious Diseases 2008, 8:126 al, district, and sub-district borders, towns, clinics, rivers, roads, and railroad of Nilphamari district e 1 nal, district, and sub-district borders, towns, clinics, rivers, roads, and railroad of Nilphamari distr National, district, and sub-district borders, towns, clinics, rivers, roads, and railroad of Nilphamari district Figure 1 National, district, and sub-district borders, towns, clinics, rivers, roads, and railroad of Nilphamari district. National, district, and sub-district borders, towns, clinics, rivers, roads, and railroad of Nilphamari district Figure 1 National, district, and sub-district borders, towns, clinics, rivers, roads, and railroad of Nilphamari district. Page 3 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1471-2334/8/126 BMC Infectious Diseases 2008, 8:126 detection was studied in relation to towns, rivers, roads, and leprosy clinics separately. However, we focus on voluntarily reported cases because, in this control program, these cases are thought to give the best representation of the incidence of leprosy. Surveys normally tend to give a better picture of the real preva- lence than voluntarily reported cases. In this control pro- gram, however, surveys were performed depending on the tion in 1995 and 2000 based upon 1991 and 2001 census data assuming an exponential growth of the population [19]. Digital maps of the administrative boundaries of the dis- tricts and sub-districts of Bangladesh were obtained from the Food and Agricultural Organization of the United Nations [20]. http://www.biomedcentral.com/1471-2334/8/126 http://www.biomedcentral.com/1471-2334/8/126 BMC Infectious Diseases 2008, 8:126 Of all 11,060 cases, 5170 were reported voluntarily, 1048 were found by contact tracing, and 4651 by school and village surveys. For 191 cases the detection method was unknown. The percentage of females was higher among cases detected actively than among voluntarily reported cases. The percentage of MB leprosy was higher among the voluntarily reported cases than among contact tracing, and it was lowest for cases detected during surveys. Of all 11,060 cases, 5170 were reported voluntarily, 1048 were found by contact tracing, and 4651 by school and village surveys. For 191 cases the detection method was unknown. The percentage of females was higher among cases detected actively than among voluntarily reported cases. The percentage of MB leprosy was higher among the voluntarily reported cases than among contact tracing, and it was lowest for cases detected during surveys. a cluster did not originate by chance was determined by Monte Carlo hypothesis testing based upon the most likely cluster [23]. We restricted the test to clusters of a length of at least 1 year and at most 25% of the popula- tion without geographic overlap. Only space-time win- dows with more than the number of expected cases, i.e. high incidence clusters, were tested. We compared the cases within and outside spatio-tempo- ral clusters by calculating the distance to towns, rivers, roads, and clinics; the average age at detection; the per- centage of multibacillary (MB) leprosy; and the sex ratio. For distance to towns, we took the distance measured from the center of town. The detection rate increased until a peak in 1994 (Figure 2). From 1995 onwards, the number of detected cases decreased over time. The annual decline in cases was 6.44% (95% CI 4.24–8.64). A pure temporal cluster was identified between April 1994 and November 1996 con- sisting of primarily paucibacillary (PB) cases. This was caused by an intensification of surveys during this period (Figure 2). For the analysis of the proximity of towns, rivers, clinics, and roads, we used Poisson regression with a correction for over-dispersion. We calculated distances to the geo- graphic features and used the distance and square distance as continuous variables in separate models, and we fitted a model with categorical variables of distance in categories of 1 km. We fitted a univariate model with only the explanatory variable and multivariate model with all var- iables (i.e. http://www.biomedcentral.com/1471-2334/8/126 distance to town, river, clinic, and road). Many spatio-temporal clusters were found for all grouped cases and overlapped with those of the separate detection methods (Figure 3). The spatio-temporal permutation test found six clusters of voluntarily reported cases, five of contact tracing, and 20 clusters of cases found during sur- veys [see Table 2, Figure 3 and Additional File 2]. Most clusters had a time period of 1 or 2 years, but one cluster of survey-detected cases had a time span of 4 years. This cluster contained Saidpur city. For each detection mode, the cases within clusters did not differ in age at detection, percentage females, or the percentage of MB leprosy com- pared to cases outside the clusters [see Table 2 and see Additional File 2]. Furthermore, the cases within a spatio- temporal cluster did not live nearer to or further from towns, roads, clinics, or rivers for any of the detection modes. Cases within the same area were not accounted to the spatio-temporal cluster if their diagnosis was outside the timeframe of the cluster. Software Data entry was done in Microsoft Access 2000™ and Arc- GIS® 9.1 was used for the visualization and processing of spatial data using a plug-in tool to count cases [24]. The temporal and spatio-temporal cluster analyses were per- formed with the SaTScan program, version 7.0.3 [25]. Poisson regressions were performed in R© 2.6.0 [26]. Ethical clearance The informed consent of the house inhabitants was obtained verbally. Ethical clearance was obtained from the ethical review committee of the Bangladesh Medical Research Council (reference number. BMRC/ERC/2004– 2007/1397). For voluntarily reported cases, the leprosy detection rate was higher near towns (Table 3). This seems to contradict the previous finding that cases within spatio-temporal clusters do not live nearer to towns. However, areas with a high incidence of cases throughout the entire study period do not constitute a spatio-temporal cluster. These areas can add to the risk calculated for proximity to towns. The rate decreases steeply in the first kilometers from the town. The rate of leprosy was two times lower at a distance of more than 1 to 2 kilometers from a town than the rate within 0 to 1 kilometer from town (adjusted rate ratio 0.512, 95% CI 0.387–0.677). The distance to roads was negatively related to the detection of new cases. However, the decrease in new case detection was not monotonous, with higher rates between 6 and 10 kilometers than between 2 and 6 kilometers. The rate of leprosy did not show a relationship with the distance to water [see Addi- tional File 3]. Also, for clinics, the rate of newly detected For voluntarily reported cases, the leprosy detection rate was higher near towns (Table 3). This seems to contradict the previous finding that cases within spatio-temporal clusters do not live nearer to towns. However, areas with a high incidence of cases throughout the entire study period do not constitute a spatio-temporal cluster. These areas can add to the risk calculated for proximity to towns. Page 5 of 10 (page number not for citation purposes) Geographic and spatial data sets A population density map with a grid cell of 30" by 30" resolution was obtained from the Gridded Population of the World version 3, beta version. [18] The population densities for each grid cell were calculated by pycnophy- lactic smoothing based on sub-district population counts. The population density maps were made for the popula- Page 4 of 10 (page number not for citation purposes) Page 4 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1471-2334/8/126 leprosy did not change with distance [see Additional File 3]. poral component, i.e. the analysis corrects for pure tem- poral clusters. This leaves increased awareness and underlying increased incidence as potential explanations. If the spatio-tempo- ral clusters are caused by an increased awareness among the population, differences would be expected in the per- centage of cases with MB leprosy and the age at detection. Increased awareness results in less time between the first symptoms and reporting. A shorter delay in detection would lead to a decrease in the percentage with MB lep- rosy, as more PB leprosy would be found before possible self-healing or progression from PB to MB leprosy. Also, the age at detection would be lower. Neither was observed for these clusters; thus, an underlying high incidence of leprosy can be assumed responsible for this pattern. How- ever, we found no specific determinants (e.g. age at detec- tion or proportion of MB leprosy) that could explain the high incidence in the clusters. Results During the study-period, 12,602 newly detected leprosy patients were registered at clinics in Nilphamari district. We were not able to find the locations for 881 patients, and another 661 were either registered as living outside the district or found to live outside the district during this study. This left 11,060 cases for which we were able to obtain the coordinates of their house. Patients that could not be traced back, i.e. missing cases, were originally detected, on average, seven months earlier in the study period than the included cases. The percentage of males and year of birth were not different for missing and included cases. Forty percent of the missing cases were MB compared to twenty-eight percent of the included cases, which was a significant difference [see Additional file 1]. The rate decreases steeply in the first kilometers from the town. The rate of leprosy was two times lower at a distance of more than 1 to 2 kilometers from a town than the rate within 0 to 1 kilometer from town (adjusted rate ratio 0.512, 95% CI 0.387–0.677). The distance to roads was negatively related to the detection of new cases. However, the decrease in new case detection was not monotonous, with higher rates between 6 and 10 kilometers than between 2 and 6 kilometers. The rate of leprosy did not show a relationship with the distance to water [see Addi- tional File 3]. Also, for clinics, the rate of newly detected Page 5 of 10 (page number not for citation purposes) Page 5 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1471-2334/8/126 BMC Infectious Diseases 2008, 8:126 Temporal distribution of the included cases detected in Nilphamari district between 1989 and 2003 Figure 2 Temporal distribution of the included cases detected in Nilphamari district between 1989 and 2003. PB, pauci- bacillary; MB, multibacillary. Temporal distribution of the included cases detected in Nilphamari district between 1989 and 2003 Figure 2 Temporal distribution of the included cases detected in Nilphamari district between 1989 and 2003. PB, pauci- bacillary; MB, multibacillary. leprosy did not change with distance [see Additional File 3]. Discussion Our first observation was a clustering of cases in a space- time window. These kind of spatio-temporal clusters depict 'outbreaks' of cases detected by voluntary report- ing. Several explanations for these 'outbreaks' are possi- ble; the most obvious is an underlying increase in the incidence of leprosy, i.e. a real outbreak of disease. An increased awareness among the population, however, can also cause an 'outbreak of detection'. Finally, an 'outbreak' is also observed when the population grows faster in some areas than others while the risk remains the same. Our analytical approach cannot correct for this phenomenon [23]. However, the population has grown in the whole district [11], and clusters would be expected later in the observation period, whereas the most likely cluster was found between 1991 and 1992. The detection increased dramatically in the years 1992 to 1994 due to improved organization in the leprosy control program. The most likely cluster was found prior to this period, showing that the spatio-temporal clusters both need a spatial and a tem- Our second observation was with regard to the spread of disease in time. Contrary to the anecdotal observation of the introduction and subsequent spread of leprosy by Bangladeshi refugees returning from India after the war Page 6 of 10 (page number not for citation purposes) Page 6 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1471-2334/8/126 BMC Infectious Diseases 2008, 8:126 registered between 1989 and 2003 in Nilphamari district (top left) es registered between 1989 and 2003 in Nilphamari district (top left). Cases per detection mode a clusters of leprosy cases detected in Nilphamari district for modes of detection, voluntarily reporting (top acing (bottom left) and surveys (bottom right) The cases registered between 1989 and 2003 in Nilphamari district (top left) Figure 3 The cases registered between 1989 and 2003 in Nilphamari district (top left). Cases per detection mode and spatio- temporal clusters of leprosy cases detected in Nilphamari district for modes of detection, voluntarily reporting (top right), contact tracing (bottom left), and surveys (bottom right). The cases registered between 1989 and 2003 in Nilphamari district (top left) Figure 3 The cases registered between 1989 and 2003 in Nilphamari district (top left). Cases per detection mode and spatio- temporal clusters of leprosy cases detected in Nilphamari district for modes of detection, voluntarily reporting (top right), contact tracing (bottom left), and surveys (bottom right). Discussion Page 7 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1471-2334/8/126 BMC Infectious Diseases 2008, 8:126 Table 2: Spatio-temporal clustering of voluntarily reported cases. Cluster Start End Cases % females Age at registration (yrs) % MB 1 Jan '91 Dec '92 57 38.6% (32.4% – 44.7%) 28.4 (0 – 61.0) 70.2% (64.7% – 75.6%) 2 Jan '00 Dec '02 145 33.8% (30.2% – 37.4%) 31.2 (3.7 – 58.7) 22.8% (19.9% – 25.6%) 3 Jan '02 Dec '02 26 30.8% (22.6% – 39.0%) 37.4 (10.0 – 64.8) 26.9% (19.4% – 34.5%) 4 Jan '94 Dec '94 25 20.0% (13.7% – 26.3%) 31.9 (0 – 68.6) 40.0% (30.6% – 49.4%) 5 Jan '93 Dec '94 24 58.3% (48.6% – 68.1%) 26.8 (0 – 58.0) 20.8% (14.2% – 27.4%) 6 Jan '93 Dec '94 84 36.9% (31.9% – 41.9%) 34.1 (3.2 – 65.0) 38.1% (33.1% – 43.1%) All clusters 361 35.7% (33.4% – 38.1%) 31.7 (1.7 – 61.6) 35.2% (32.8% – 37.5%) Outside clusters 4809 36.0% (35.4% – 36.7%) 31.7 (0 – 65.0) 38.9% (38.2% – 39.5%) All 5170 36.0% (35.4% – 36.6%) 31.7 (0.9 – 62.1) 38.6% (38.0% – 39.3%) Characteristics of the most likely spatio-temporal cluster and secondary clusters that do not geographically overlap and p > 0.05. MB, multibacillary cases. Table 2: Spatio-temporal clustering of voluntarily reported cases. Characteristics of the most likely spatio-temporal cluster and secondary clusters that do not geographically overlap and Characteristics of the most likely spatio-temporal cluster and secondary clusters that do not geographically overlap and p > 0.05. MB, multibacillary cases. miology. There are several possible explanations. First, as suggested by others, it could be the result of selective migration towards these towns [3,4]. Second, a higher awareness among the urban population is possible. Third, the circumstances in these towns are favorable for the transmission of M. leprae. We recommend further studies of leprosy in urban areas and towns in rural areas. If urban areas are an important source of transmission, improve- ments are possible by focusing more on urban leprosy control. for independence in 1972, we did not observe a spread of leprosy from Saidpur city to other areas in the district, nor could we identify patterns of spread or retraction in the district during the study period. The adjusted rate ratios are estimates from a model including distance to clinics, rivers, and roads. Authors' contributions EF was involved in all aspects of the research and drafting the manuscript. DP and SC contributed to the set-up, planning, and conduction of data collection and com- mented on the manuscript. JR was involved in the concep- tion and design of the study, as well as the analysis. JR contributed considerably to the drafting of the manu- script. Our study gives a thorough spatial description of the cases found during a leprosy control program, and this approach can possibly bias our results in several ways. We retrospectively traced back patients; therefore, a propor- tion of the cases could not be found. The demographic characteristics, including age and sex, were not different from the included patients. The missing cases, however, contained proportionally more MB cases. The reason for this is not clear, but this difference is not likely to intro- duce a bias in our analysis, as there is no evidence to expect that MB cases were distributed differently than PB cases. The population density maps on which we base some of the estimates were constructed by the interpola- tion of sub-district data [19]. The population of Nil- phamari district is less smoothly distributed than suggested by these interpolated population maps. For towns, the population density will be underestimated, resulting in higher estimates for the rate of leprosy. How- ever, these estimates are the best available population density estimates. The results obtained using this data should be interpreted cautiously, but are useful to direct- ing new lines of research. Additional file 2 Spatio-temporal Clusters of cases detected by contact tracing or surveys. Results of the spatio-temporal clustering analysis, where mode of detection is contact tracing or survey. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2334-8-126-S2.doc] Spatio-temporal Clusters of cases detected by contact tracing or surveys. Results of the spatio-temporal clustering analysis, where mode of detection is contact tracing or survey. [http://www.biomedcentral.com/content/supplementary/1471- 2334-8-126-S2.doc] Additional file 1 Characteristics of included cases and missing data. Table providing the %- males, % MB cases, age for included cases and missing data Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2334-8-126-S1.doc] p g The authors declare that they have no competing interests. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Discussion New leprosy cases appeared more or less consistently over the whole district during the 15 years of observation, indicating a stable endemic situation in space and time. Finally, our third observation concerns geographic deter- minants of leprosy risk. We found a clear relation with proximity to towns, especially in the first kilometers, and the risk of leprosy. Leprosy is thought of as a rural disease [8], but our results show that rural towns, i.e. moderately sized towns in a rural area, contain many cases. The sharp decline within the first kilometers might indicate that it is not the distance to town, but the difference between urban and rural populations, influencing leprosy epide- The rate of new leprosy cases was higher in the proximity of roads. In another setting, the risk of leprosy was found to be increased with more distance from roads. That study was based on active surveys and indicated an under- reporting with increased distance from a road [5]. In our study, all methods of detection had a higher risk of lep- Page 8 of 10 (page number not for citation purposes) Table 3: Leprosy detection rate by distance to towns. Voluntarily reported Distance to town Univariate 95% CI Adjusted 95% CI Linear 0.890 (0.866 – 0.914) 0.922 (0.895 – 0.950) Quadratic 0.990 (0.988 – 0.993) 0.993 (0.990 – 0.995) Category 0–1 km 1 1 1–2 km 0.450 (0.342 – 0.592) 0.512 (0.387 – 0.677) 2–3 km 0.309 (0.238 – 0.403) 0.414 (0.313 – 0.549) 3–4 km 0.287 (0.221 – 0.373) 0.392 (0.294 – 0.521) 4–5 km 0.291 (0.225 – 0.376) 0.392 (0.292 – 0.525) 5–6 km 0.268 (0.206 – 0.348) 0.360 (0.264 – 0.491) 6–7 km 0.248 (0.186 – 0.329) 0.319 (0.228 – 0.446) 7–8 km 0.256 (0.190 – 0.344) 0.305 (0.215 – 0.433) 8–9 km 0.312 (0.227 – 0.429) 0.365 (0.252 – 0.527) 9–10 km 0.132 (0.072 – 0.240) 0.168 (0.090 – 0.314) 10–11 km 0.086 (0.033 – 0.222) 0.148 (0.056 – 0.392) 11–12 km 0.059 (0.012 – 0.301) 0.126 (0.025 – 0.649) >12 km 0.082 (0.010 – 0.699) 0.270 (0.031 – 2.361) The adjusted rate ratios are estimates from a model including distance to clinics, rivers, and roads. Table 3: Leprosy detection rate by distance to towns. ble 3: Leprosy detection rate by distance to towns. http://www.biomedcentral.com/1471-2334/8/126 http://www.biomedcentral.com/1471-2334/8/126 BMC Infectious Diseases 2008, 8:126 http://www.biomedcentral.com/1471-2334/8/126 http://www.biomedcentral.com/1471-2334/8/126 http://www.biomedcentral.com/1471-2334/8/126 Conclusion rosy near roads; therefore, this is not the explanation for our findings. Our results can be explained by the fact that major roads connect the towns, and people living near roads also tend to live near towns. However, our maps only contained the major roads. Though roads are present in the northeastern sub-district of Domar, these are not major roads and not present in our analysis. The results for roads are not clear from our observations, and maps of all roads instead of only the major roads might give a dif- ferent result. We found that the risk of leprosy is associated with the proximity to towns, but not roads, clinics, and rivers. Although our estimates for towns may be too high due to the use of population density maps based on interpolated census data, the elevated detection of new cases for all modes of detection near and in towns indicates that rural towns play an important role in the epidemiology of lep- rosy in this district. Further research on the role of towns in rural areas is warranted. Abbreviations The proximity to a clinic might increase the possibilities of voluntary reporting, but we found no relationship with the proximity to a clinic. The distance to clinics does not seem to be an obstacle for reporting leprosy. GIS: geographic information system; GPS: global posi- tioning system; MB: multibacillary; PB: paucibacillary; WHO: World Health Organization; MDT: Multi Drug Treatment; DBLM: Danish Bangladesh Leprosy Mission, Nilphamari, Bangladesh. The proximity of water has been hypothesized to be a risk factor for leprosy transmission [8], and Sterne et al. [5] found an association with the proximity to rivers. The increased risk would be due to the longer lifetime of M. leprae outside the body in a humid atmosphere, as opposed to a dry atmosphere [8,9]. In Nilphamari, a rela- tionship with the proximity to rivers was not found. In this district, it is unlikely that the proximity to water would increase the risk of leprosy, as almost 60% of the population lives within 2 kilometers of a river, and most live much nearer to other bodies of water, such as rice pad- dies. Furthermore, the relative humidity does not drop below 60% and the yearly average is 80% [11]. References 1. WHO: Global leprosy situation, 2005. Weekly Epidemiological Record 2005, 80(34):289-295. Acknowledgements 20. FAO: Sub-national Administrative Units of Bangladesh. FAO – UN SDRN; 2004. The meticulous registration done for many years by the Danish Bangladesh Leprosy Mission (DBLM) staff was essential for this study. We thank the current staff and, in particular, the leprosy control supervisors of the Rural Health Program (formerly DBLM) for their support in tracing the patients. It was essential in this study that almost all patients allowed us to measure the position of their home, and we are grateful for that. We thank Dr. Fang Liqun for his comments on the analyses. We thank three reviewers, whose comments helped to improve a previous version of this manuscript. 21. Anonymous: Geocommunity nation wide data Bangladesh. 2006. 22. Kulldorff M: Bernoulli and Poisson Models: A spatial scan sta- tistic. Communications in Statistics:Theory and Methods 1997, 26:1481-1496. 23. Kulldorff M, Heffernan R, Hartman J, Assuncao R, Mostashari F: A space-time permutation scan statistic for disease outbreak detection. PLoS Medicine 2005, 2(3):216-224. ( ) 24. Beyer HL: Hawth's Analysis Tools for ArcGIS. 3.26th edition. 2004. 25. Kulldorff M, Information Management Services I: SaTScan, Soft- ware for the spatial and space-time scan statistics. 4.0.3 edi- tion. 2003. We thank the American Leprosy Missions for their financial support of the study. The Netherlands Leprosy Relief financially supported EF. 26. Team RDC: R: A language and environment for statistical computing. 2.6.0 edition. Vienna, Astria: The R Foundation for Sta- tistical Computing; 2007. Additional file 3 Change of detection rate with distance to geographic features by detection method. Results of the analyses of distances to roads, rivers and clinics for voluntary reported cases, and distance to town center, roads, rivers and clinics, where mode of detection is contact tracing or survey. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2334-8-126-S3.doc] Page 9 of 10 (page number not for citation purposes) Page 9 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1471-2334/8/126 BMC Infectious Diseases 2008, 8:126 Pre-publication history ( ) 2. WHO: Global leprosy situation, 2006. Weekly Epidemiological Record 2006, 81:309-316. The pre-publication history for this paper can be accessed here: The pre-publication history for this paper can be accessed here: The pre-publication history for this paper can be accessed here: 3. Opromolla PA, Dalben I, Cardim M: Geostatistical analysis of lep- rosy cases in the State of Sao Paulo, 1991–2002. Revista de Saúde Pública 2006, 40(5):907-913. ( ) 4. Montenegro ACD, Werneck GL, Kerr-Pontes LRS, Barreto ML, Feld- meier H: Spatial analysis of the distribution of leprosy in the state of Ceara, Northeast Brazil. Memorias Do Instituto Oswaldo Cruz 2004, 99(7):683-686. http://www.biomedcentral.com/1471-2334/8/126/pre pub ( ) 5. Sterne JA, Ponnighaus JM, Fine PE, Malema SS: Geographic deter- minants of leprosy in Karonga District, Northern Malawi. International Journal of Epidemiology 1995, 24(6):1211-1222. J p gy ( ) 6. Kerr-Pontes LR, Montenegro AC, Barreto ML, Werneck GL, Feld- meier H: Inequality and leprosy in Northeast Brazil: an eco- logical study. International Journal of Epidemiology 2004, 33(2):262-269. ( ) 7. Irgens LM, Melo Caeiro F, Lechat MF: Leprosy in Portugal 1946– 80: epidemiologic patterns observed during declining inci- dence rates. Leprosy Review 1990, 61(1):32-49. p y ( ) 8. Fine PE: Leprosy: the epidemiology of a slow bacterium. Epi- demiol Rev 1982, 4:161-188. 9. Desikan KV: Viability of Mycobacterium leprae outside the human body. Leprosy Review 1977, 48(4):231-235. y p y ( ) 10. Fischer EAJ, Pahan D, Chowdhury SK, Oskam L, Richardus JH: The spatial distribution of leprosy in four villages in Bangladesh: An observational study. BMC Infect Dis 2008, 23(1):125. y ( ) 11. Bangladesh-Bureau-of-Statistics: Statistical Yearbook of Bangla- desh. Volume 1. Dhaka: Bangladesh Bureau of Statistics, Statistics division, Ministery of Planning, Government of the People's Republic of Bangladesh; 1995:646. g 12. Sen S: Stateless Refugees and the Right to Return: The Bihari Refugees of South Asia – Part 2. International Journal of Refugee Law 2000, 12(1):41-70. ( ) 13. Richardus JH, Meima A, Croft RP, Habbema JD: Case detection, gender and disability in leprosy in Bangladesh: a trend analy- sis. Leprosy Review 1999, 70(2):160-173. p y ( ) 14. Guidelines for the field management of leprosy. Nilphamari, Bangladesh: Danish Bangladesh Leprosy Mission; 2000. 15. WHO: WHO Expert Committee on Leprosy. World Health Organ Tech Rep Ser 1998, 874:1-43. Pre-publication history Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 10 of 10 (page number not for citation purposes) g p 16. Krishnamurthy P: Review of case finding and prevalence in Danish Bangladesh Leprosy Mission. LEPRA-India; 2001. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge 17. Nicholls PG, Croft RP, Richardus JH, Withington SG, Smith WC: Delay in presentation, an indicator for nerve function status at registration and for treatment outcome – the experience of the Bangladesh Acute Nerve Damage Study cohort. Lepr Rev 2003, 74(4):349-356. 18. Anonymous: Gridded Population of the World Version 3 beta (GPWv3 beta): Population Density Grids. 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https://openalex.org/W2407113773	https://europepmc.org/articles/pmc4742773?pdf=render	English	null	Exploration of multiple Sortase A protein conformations in virtual screening	Scientific reports	2,016	cc-by	11,739	Exploration of multiple Sortase A protein conformations in virtual screening received: 09 June 2015 accepted: 04 January 2016 Published: 05 February 2016 Chunxia Gao, Ivana Uzelac, Johan Gottfries & Leif A. Eriksson Chunxia Gao, Ivana Uzelac, Johan Gottfries & Leif A. Eriksson Methicillin resistant Staphylococcus aureus (MRSA) has become a major health concern which has brought about an urgent need for new therapeutic agents. As the S. aureus Sortase A (SrtA) enzyme contributes to the adherence of the bacteria to the host cells, inhibition thereof by small molecules could be employed as potential antivirulence agents, also towards resistant strains. Albeit several virtual docking SrtA campaigns have been reported, no strongly inhibitatory non-covalent binders have as yet emerged therefrom. In order to better understand the binding modes of small molecules, and the effect of different receptor structures employed in the screening, we herein report on an exploratory study employing 10 known binders and 500 decoys on 100 SrtA structures generated from regular or steered molecular dynamics simulations on four different SrtA crystal/NMR structures. The results suggest a correlation between the protein structural flexibility and the virtual screening performance, and confirm the noted immobilization of the β6/β7 loop upon substrate binding. The NMR structures reported appear to perform slightly better than the Xray-crystal structures, but the binding modes fluctuate tremendously, and it might be suspected that the catalytic site is not necessarily the preferred site of binding for some of the reported active compounds. Staphylococcus aureus plays a leading role in hospital- and community-acquired infections which produces a wide spectrum of diseases ranging from minor skin infections, lower respiratory tract infections, surgical site infec- tions, and nosocomial bacteremia, to pneumonia and cardiovascular infections1,2. The widespread occurrence of methicillin-resistant S. aureus (MRSA), which in addition to methicillin often is resistant to other commonly used antibiotics3, makes treatment difficult. This creates an urgent need for new therapeutic agents to treat MRSA infections, preferably ones that do not lead to rapid emergence of drug-resistant strains. A potential and attractive approach, which recently has gained much attention in treating these types of infections, is to inhibit surface proteins that function as virulence factors, with small molecules4. S. aureus and many other Gram-positive pathogens use sortase A (SrtA) enzymes to anchor surface proteins to their cell walls5–7. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Department of Chemistry and Molecular Biology, University of Gothenburg, 405 30 Göteborg, Sweden orrespondence and requests for materials should be addressed to L.A.E. (email: leif.eriksson@chem.gu.se) Scientific Reports \| 6:20413 \| DOI: 10.1038/srep20413 Exploration of multiple Sortase A protein conformations in virtual screening (A) Apo-SrtA NMR structure (PDB ID: 1IJA); (B) holo-SrtA NMR structure (PDB ID: 2KID); (C) apo-SrtA crystal structure (PDB ID: 1T2P) (D) holo-SrtA crystal structure; (E) superposition of all four SrtA structures, black ribbon: apo-SrtA NMR structure; red ribbon: holo-SrtA NMR structure; green ribbon: apo-SrtA crystal structure; blue ribbon: holo-SrtA crystal struture. (F) The binding site of SrtA is confined to the enclosing box used in the docking studies. resonance line broadening in the NMR experiments, and were poorly resolved with high B-factors in the crys- tallography experiments. Both these findings indicate that this loop exhibits increased dynamics relative to the remainder of the protein. Motions of the ß6/ß7 loop are particularly interesting, given that many of its residues are positioned adjacent to the active site, notably residues 164–169. The active sites of all sortases contain a con- served catalytic triad that consists of residues H120, C184, and R197 (MRSA numbering), mutations to each of which have been shown to severely reduce the catalytic activity31. resonance line broadening in the NMR experiments, and were poorly resolved with high B-factors in the crys- tallography experiments. Both these findings indicate that this loop exhibits increased dynamics relative to the remainder of the protein. Motions of the ß6/ß7 loop are particularly interesting, given that many of its residues are positioned adjacent to the active site, notably residues 164–169. The active sites of all sortases contain a con- served catalytic triad that consists of residues H120, C184, and R197 (MRSA numbering), mutations to each of which have been shown to severely reduce the catalytic activity31. y y y In the holo-SrtA (substrate-bound SrtA) X-ray crystal structure, the LPXTG peptide substrate adopts an elon- gated form while the ß6/ß7 loop remains in an “open” conformation (Fig. 1D). R197 is observed to make contact with the LPXTG threonine residue; however, the side chain of the catalytic H120 is located more than 10 Å away from the peptide. In the holo-SrtA NMR structure, on the other hand, the LPXTG peptide analogue adopts an “L-shape” configuration in which there is a 90o bend between the leucine and proline residues, and the ß6/ß7 loop not only contains a short 310 helix spanning residues V166–L169, but is also far less mobile and in a “closed” configuration (Fig. 1B). Exploration of multiple Sortase A protein conformations in virtual screening This cysteine transpeptidase catalyzes the formation of an amide bond between a cell wall sorting signal (LPXTG motif) located at the C-terminal end of the surface protein, and a pentaglycine unit of the cell wall molecule lipid-II, thereby covalently attaching the surface protein to the cell wall7. Many surface proteins attached to the cell wall by SrtA play key roles in the infection process by promoting nutrient acquisition from the host, bacterial adhesion, and immune evasion6. Disrupting the display of these proteins by blocking the activity of SrtA using small molecule inhibitors could therefore effectively reduce bacterial virulence and thus promote bacterial clearance by the host. In addition, SrtA inhibitors may also be less likely to induce selective pressure that leads to drug resistance as compared to conventional antibiotics. This is supported by the fact that SrtA-lacking strains do not exhibit impaired growth outside of their human host in culture medium8, while at the same time altered adhesion properties has been observed9. To this end, a number of different strategies have been employed to search for sortase inhibitors. These include screening natural products10–19 and small compound libraries20–23, as well as synthesizing rationally designed peptidomimetics and small molecules24–27. To date, however, no SrtA inhibition based antibiotics have emerged on the market.h g The structures of SrtA in its apo- and substrate-bound forms have been determined28–30. NMR and X-ray crystallography experiments have shown that the catalytic domain of SrtA (Residues 60–206) adopts a unique eight-stranded β -barrel fold with individual strands that are connected by two short helices and several loop regions (Fig. 1)29,30. Residues within the loop connecting the ß6 and ß7 strands (Residue 161–176) exhibit Department of Chemistry and Molecular Biology, University of Gothenburg, 405 30 Göteborg, Sweden. Correspondence and requests for materials should be addressed to L.A.E. (email: leif.eriksson@chem.gu.se) Scientific Reports \| 6:20413 \| DOI: 10.1038/srep20413 1 www.nature.com/scientificreports/ Figure 1. (A) Apo-SrtA NMR structure (PDB ID: 1IJA); (B) holo-SrtA NMR structure (PDB ID: 2KID) (C) apo-SrtA crystal structure (PDB ID: 1T2P) (D) holo-SrtA crystal structure; (E) superposition of all f SrtA structures, black ribbon: apo-SrtA NMR structure; red ribbon: holo-SrtA NMR structure; green rib apo-SrtA crystal structure; blue ribbon: holo-SrtA crystal struture. (F) The binding site of SrtA is confin the enclosing box used in the docking studies. : 1IJA); (B) holo-SrtA NMR structure (PDB ID: 2KID); Figure 1. Exploration of multiple Sortase A protein conformations in virtual screening To accommodate the covalently bound peptide in the active site, the ß7/ß8 loop adopts a more open conformation, suggesting an “induced-fit” mechanism for LPXTG peptide binding. In addition, each of the catalytic triad residues are observed in close proximity to residues within the LPXTG analog. Both the X-ray and NMR apo-SrtA structures adopt conformations that are similar compared to the holo-SrtA crystal structure, where the ß6/ß7 loop remains in an “open” conformation and the side chain of the catalytic H120 is located more than 10 Å , away from the LPXTG peptide. However, some fluctuations are observed in the ß6/ß7 Scientific Reports \| 6:20413 \| DOI: 10.1038/srep20413 2 www.nature.com/scientificreports/ Figure 2. Active inhibitors with different IC50 values and diverse structures used as ‘known binders’ in this work. Figure 2. Active inhibitors with different IC50 values and diverse structures used as ‘known binders’ in this work. and ß7/ß8 loops among these structures (Fig. 1A,C). Overall, the crystal and NMR structures of the apo- and holo-SrtA enzymes demonstrate large discrepancies in and around the active site, especially in the location of the conserved catalytic triad, and in the ß6/ß7 and ß7/ß8 loop conformations, which illustrate the large flexibility of the receptor structure. and ß7/ß8 loops among these structures (Fig. 1A,C). Overall, the crystal and NMR structures of the apo- and holo-SrtA enzymes demonstrate large discrepancies in and around the active site, especially in the location of the conserved catalytic triad, and in the ß6/ß7 and ß7/ß8 loop conformations, which illustrate the large flexibility of the receptor structure. p Attempts have been made to employ reported receptor structures in virtual screening campaigns to iden- tify new and efficient inhibitors against SrtA. In a virtual screening (VS) study which made use of the NMR structure of SrtA determined in its unbound state (apo-SrtA, PDB ID: 1IJA), the most active compound was subsequently found to have an IC50 value value of 58 μ M32. In a more recent VS campaign employing a relaxed complex method for SrtA inhibitors, which made use of the NMR structure of SrtA bound to a substrate analog (holo-SrtA, PDB ID: 2KID), the most active compound was found to have an IC50 value of 47 μ M33. Hence, neither of these VS efforts, conducted on receptor structures that differ considerably in the local geometry of the active site, resulted in finding inhibitors with medicinally relevant IC50 values. Scientific Reports \| 6:20413 \| DOI: 10.1038/srep20413 Methods The temperature and pressure was controlled through the Berendsen coupling algorithm49, with the time constants 0.1 for temperature and 1.0 ps for pressure coupling. All bond lengths were constrained using the LINCS algorithm50. During the production simulations (200 ns duration, 1.0 fs time step, NPT ensemble, T = 298 K, P = 1 bar), the temperature was controlled using the Nose-Hoover thermostat51 with a time constant 0.1 ps, and the pressure was controlled using the Parrinello-Rahman barostat52, with a time con- stant 1.0 ps. The remaining parameters were the same as in the position restrained simulations.l ph g p p Steered MD (SMD) simulations were performed to further sample the conformational space around the flex- ible β 6/β 7 loop. In the SMD simulation, an external force (spring constant) of 10 kJ/mol/nm2 was added with pull rate 0.001 nm/ps, in order to move the β 6/β 7 loop from ‘closed’ to ‘open’ conformation. Using the distance between V166-L169 of the β 6/β 7 loop and C184 of the catalytic region of the active site as reference, a 17.5 ns SMD simulation was performed after a 2 ns position restrained equilibration MD, during which the β 6/β 7 loop was pulled away from the core structure thereby increasing the said distance from around 15 to 30 Å. Since the largest distance among the structures reported in the literature is around 22.5 Å (apo-SrtA crystal structure), the SMD has sampled a considerably larger set of conformations than those of the available reported structures. A total of 20 snapshots were extracted from the SMD, and ligand docking performed and analysed on these addi- tional structures in the same manner as in the regular MD simulations. Decoy generation. The decoy generation methodology employed in the current study has been described previously53. Briefly, 10 actives were seeded among 2 million molecules from the ZINC database of commer- cially available compounds. Key feature fingerprints were calculated using the default type 2 substructure keys of CACTVS54, and fingerprint-based similarity analysis was performed with the programme Canvas. Compounds with Tanimoto Cofficient (Tc) less than 0.5 to any actives were selected. In the next step, QikProp (Schrodinger, LLC, New York, NY) was used to calculate 32 physical properties of all the actives as well as the selected ZINC compounds from the previous step and QikSim was applied to prioritize the ZINC compounds according to the properties of the actives. Methods Structures and initial preparation. Four structures of SrtA (PDB ID: 1T2W, 1T2P, 2KID, 1IJA)28–30 were obtained from the protein data bank (www.rcsb.org), representing the crystal structure of C184A-mutated SrtA in complex with the LPETG (Leu-Pro-Glu-Thr-Gly) peptide; the crystal structure of SrtA in its apo state; the NMR structure of SrtA in complex with an LPXTG analog (X = Ala); and the NMR structure of SrtA in its apo state, respectively. Of these, the two NMR structures (2KID and 1IJA) were as mentioned above recently employed in VHTS campaigns identifying molecules that showed IC50 values in the 50 μ M range32,33. As part of the receptor preparation, the mutated residue Ala184 in the 1T2W crystal structure was changed to Cys184. The termini of the LPXTG peptides were neutralized, and the calcium ion present in the 2KID structure retained. The four structures were then processed using the default Protein Preparation wizard in the Schrodinger programme suite before MD simulation. The protein preparation steps are described in detail below.h h p p p p The charges of the LPXTG analog in the NMR holo SrtA structure were calculated using the restrained elec- trostatic potential (RESP) procedure40 at the HF/6-31G* level after minimizing the molecule at the AM1 semiem- pirical level41. GAFF force field33 parameters42 and RESP partial charges were assigned using the Antechamber module in the AMBER10 package43. MD simulations. The four structures prepared above were subjected to MD simulations, in order to obtain an ensemble of protein structures. Four conventional 200 ns MD simulations were performed on X-ray crystal apo- and holo-SrtA, and NMR apo- and holo-SrtA using the Amber 99 force field44,45 with the GROMACS soft- ware46. The structures were solvated in periodic boxes with a buffer distance of 10.0 Å. A number of Na+ and Cl− ions were added to satisfy the electroneutrality condition and to give a salt concentration of 0.1 mol/liter, using the genion module in GROMACS. The obtained systems were energy minimized by steepest descent (200 steps) to remove close contacts. Position restrained simulations (2 ns duration, 1.0 fs time step, NPT ensemble, T = 298 K, P = 1 bar) were first performed, to enable the water molecules to reach more favorable positions. Particle-mesh Ewald (PME)47,48 summation was used for long-range electrostatics. A 12 Å cutoff was used for both Coulomb and Lennard-Jones interactions. Exploration of multiple Sortase A protein conformations in virtual screening These unsatisfactory VS results can be due to the fact that the SrtA structure exhibits significant conformational heterogeneity and mobility as stated above. Simulation studies of the SrtA structure have furthermore shown that the active site of SrtA under- goes certain ligand-induced conformational changes34,35. Given that that neither SrtA nor other members of this protein family to date have been co-crystallized with any of the known non-covalent inhibitors, there is an urgent need for further exploration of the performance of flexible SrtA structures in finding truly good inhibi- tors. In other words, we need to better understand the receptor geometry, or ensemble of geometries, that is best employed in virtual screening campaigns in order to identify good inhibitors. In addition, the likely binding modes of small molecules at either the catalytic site or other areas of the protein, need be to carefully identified. y p yi In the current work, we generated multiple molecular dynamics (MD) snapshots of apo- and holo-SrtA to represent a flexible ensemble of receptor coordinates for molecular docking. Based on previous experimental studies, which revealed a series of hits with good IC50 values, we constructed a library of 510 compounds, includ- ing 500 decoys and ten actives11,13–15,18,20,21. The ten active compounds have been concluded experimentally to be reversible (non-covalent) binders, even though some do contain rhodanine and other reactive functionalities. For example, the reversibility of the potentially covalent ligands 508-510 (Fig. 2) were determined using enzymatic assays and mass spectrometry. Ligand 508 is rapidly reversible as 84% of the enzyme activity is recovered imme- diately upon dilution; for ligands 509 and 510, the corresponding numbers are 50% and 58%, respectively. All active ligands selected herein have weak to moderate inhibitory effects (IC50-values 4 – 37 μ M), and were chosen so as to be as structurally diverse as possible among the published SrtA inhibitors. The library of 510 compounds was screened against a total of 80 receptor models generated from the MD simulations, displaying considerable Scientific Reports \| 6:20413 \| DOI: 10.1038/srep20413 3 www.nature.com/scientificreports/ variability in the local structure. In addition, further conformational sampling was performed based on steered MD (SMD) simulations focusing on the flexibility of the β 6/β 7 loop, and docking performed on 20 snapshots along the SMD pathway. Exploration of multiple Sortase A protein conformations in virtual screening Through evaluation of the enrichment factors (EF)36, the Robust Initial Enhancement (RIE)37, the Boltzmann-Enhanced Discrimination of ROC metric (BEDROC)38, and the area under the Receiver Operating Characteristic curve (AUC)39, we systematically evaluate the relative VS performance of the various receptor models, also including a Boltzmann weighted average of the docked structures. The approach taken should hence be able to reliably identify the actives amongst the decoys, if binding towards the catalytic site is the only determining factor. Methods The thereby prepared protein structures were used for the subse- quent step of grid generation. q p g g Grids were generated by the Receptor Grid Generation panel (Schrodinger, LLC, New York, NY) which defines the receptor structure by excluding any co-crystallized ligand present, determines the position and size of the active site to be represented by receptor grids, and sets up Glide constraints. Grids were defined by center- ing them on the ligand for the holo-SrtA structure using the default box size (box length 10 Å in the x-, y- and z-directions, respectively), and selecting site points generated by sitemap for the apo-SrtA structure (Fig. 1F). All docking was thus carried out in the region defined by the LPXTG substrate binding. Docking and scoring functions. Molecular docking experiments were carried out using Glide56, imple- mented in the Schrödinger package. The XP (extra precision) scoring functions were used, granting full flexibility to the ligands. A post-docking minimization was performed on the resulting complexes in order to reduce the initially collected 10000 poses per ligand to 5. Virtual screening performance analysis. Several metrics are available for evaluating the effectiveness of a docking run in discriminating actual binders from decoys. For evaluating the performance of different combi- nations of protein conformers, we considered EF36, RIE37, BEDROC38 and AUC39.fiht p EF is a widely used metric to evaluate the efficiency of VS36. The value of EFx% indicates how much more often an active compound is ranked in the top x% of a VS result compared to a random selection, i.e., how many times the database is enriched. Specifically, EF is calculated according to Eq. 1: = × % . ( ) % % EF N n x 1 X experimental X ( ) 1 % Nexperimental x is the number of experimentally verified actives in the top x% of the database and n is the total num- er of actives in the database. In this study, EF1% was calculated from the top 1% of the docking results. 58 We also report the BEDROC and RIE values to explore the problem of “high scoring”58. By changing a tuning parameter, α, users can control the level of top scoring actives to test whether a certain ranking method is useful in the context of VS. Methods A weight of 4 was used to emphasize the druglike descriptors (molecular weight, number of hydrogen bond acceptors, number of hydrogen bond donors, number of rotatable bonds, and log P), and a weight of 1 was used for the number of important functional groups (amine, amide, amidine, and carbox- ylic acid). The rest of the descriptors were ignored (weight 0) during the similarity analysis procedure. After the calculation, 50 decoy compounds were selected for each active, leading to a total of 500 decoys that were physi- cally similar but topologically dissimilar to the 10 actives. The 10 actives were selected based on their structures being as diversely distributed as possible among the known published SrtA inhibitors while at the same time being non-covalent inhibitors (Fig. 2). Selected properties of actives and the generated decoys are summarized in Table 1. Scientific Reports \| 6:20413 \| DOI: 10.1038/srep20413 4 www.nature.com/scientificreports/ Compounds Molecular weight Rotable bonds H-bond acceptors H-bond donors Actives 10 310.84 ± 66.22 2.5 ± 1.2 3.2 ± 1.0 1.7 ± 1.6 Decoys 500 303.30 ± 59.21 3.6 ± 1.3 2.5 ± 0.9 1.0 ± 0.9 Table 1. Average properties of actives and decoys. Compounds Molecular weight Rotable bonds H-bond acceptors H-bond donors Actives 10 310.84 ± 66.22 2.5 ± 1.2 3.2 ± 1.0 1.7 ± 1.6 Decoys 500 303.30 ± 59.21 3.6 ± 1.3 2.5 ± 0.9 1.0 ± 0.9 Table 1. Average properties of actives and decoys. Protein preparation and receptor grid generation. Twenty snapshots with evenly spaced intervals were extracted from each MD trajectory (including the SMD simulation) and prepared with the default protein Preparation wizard in Schrödinger. The protein preparation was carried out in two steps, preparation and refine- ment. After ensuring chemical correctness, hydrogen atoms were added, and side chains far from the binding cavity and not participating in salt bridges were neutralized. The hydrogen bonding network was optimized by reorienting hydroxyl groups, water molecules, and amide groups of Asn and Gln, and selecting appropriate states and orientations of the imidazole ring in His residues. Water molecules in the crystal structures were deleted, the termini were capped by adding ACE and NMA residues, and missing side chains and loops were added. The structures were then energy minimized using the OPLS-2005 force field55 and the Impact molecular mechanics engine, while heavy atoms were constrained. Methods BEDROC is bound by the interval [0, 1] and can be interpreted as the probability that an active is ranked before a randomly selected compound exponentially distributed with parameter α, given that n Nα  1 (n = number of actives; N = total number of compounds). RIE uses an exponential weighting scheme that gives heavier weight to “top scoring” actives as defined in Eq. 2: = ∑       ( ) α = − − − α α − RIE e 2 n i n x N e 1 1 1 1 1 i eN ( ) 2 where xi = r N i is the relative rank of the ith active and α is a tuning parameter. BEDROC is derived from RIE and has a linear relationship with RIE as shown in Eq. 3: where xi = r N i is the relative rank of the ith active and α is a tuning parameter. BEDROC is derived from RIE and has a linear relationship with RIE as shown in Eq. 3: ( ) ( ) ( ) ) ( α = × − − + − ( ) α α α α − BEDROC RIE e sinh cosh cosh 1 1 3 N n N 1 2 2 2 N n N ( ) 3 Although RIE and BEDROC produce different values, their distributions scale very similarly. Although RIE and BEDROC produce different values, their distributions scale very similarly. h d k f h h ld d d h l To investigate the docking performance in a threshold independent manner, the AUC value was calculated from the ROC curve. The ROC curve allows a straightforward visualization of the performance of VS in ranking the actives higher over decoys39. In our study, we have a list of experimentally verified actives (positives), and decoys (negatives). These positives and negatives are further categorized into true or false according to their rank above or below a certain threshold of the VS result, i.e., the actives ranked above a chosen threshold becomes Scientific Reports \| 6:20413 \| DOI: 10.1038/srep20413 5 www.nature.com/scientificreports/ Figure 3. Data for the different SrtA structures over the 200 ns MD simulations. Line color represents: black: apo-SrtA NMR structure; red: holo-SrtA NMR structure; green: apo-SrtA crystal structure; blue: holo-SrtA crystal struture. Methods (A) Cα RMSD; (B) Cα RMSFs; location of α -helices and β -strands indicated above the graph; (C) Radius of gyration. See text for further details. Figure 3. Data for the different SrtA structures over the 200 ns MD simulations. Line color represents: black: apo-SrtA NMR structure; red: holo-SrtA NMR structure; green: apo-SrtA crystal structure; blue: holo-SrtA crystal struture. (A) Cα RMSD; (B) Cα RMSFs; location of α -helices and β -strands indicated above the graph; (C) Radius of gyration. See text for further details. true positive (TP). To generate the ROC curve, the true positive ratio (TPR) and false positive ratio (FPR) are calculated as: = + ( ) TPR TP TP FN 4 = + ( ) FPR FP TN FP 5h ( ) 4 ( ) 5 In the ROC curve, the TPR is plotted as a function of the FPR. The AUC is then calculated to assess the quanti- tative performance of different receptor models. An AUC of 0.5 corresponds to a random selection of the ligands by the receptor57. Scientific Reports \| 6:20413 \| DOI: 10.1038/srep20413 Results and Discussion (A) All atom RMSDs of the SrtA active site residues; (B) Distance between the center of mass of residue V166-L169 (part of β 6/β 7 loop) and C184; (C) Number of hydrogen bonds between R197 and LPXTG analog in holo-SrtA NMR structure; (D) As (C) but for LPXTG substrate in holo-SrtA crystal structure. Figure 4. Active site parameters during the 200 ns MD simulations. Line colors as in Figure 3. (A) All atom RMSDs of the SrtA active site residues; (B) Distance between the center of mass of residue V166-L169 (part of β 6/β 7 loop) and C184; (C) Number of hydrogen bonds between R197 and LPXTG analog in holo-SrtA NMR structure; (D) As (C) but for LPXTG substrate in holo-SrtA crystal structure. f the ß6/ß7 loop appears to be affected by the peptide binding. This confirms a previous study where it was eported that the peptide binding immobilizes the loop28.h The radius of gyration of a protein is calculated as the root mean square distance between its center of gravity and its ends, and is indicative of the level of compactness of the structure. It can be seen (Fig. 3C) that the radius of gyration is quite stable for each structure during the simulation time, with the holo-SrtA NMR structure being less compact compared to other structures, due to the more extended form of the ß7/ß8 loop. Overall, in terms of Cα RMSDs, RMSFs, and the protein radius of gyration calculations, the conformation of each structure was stable during in the simulation, with the loop regions as expected displaying the largest fluctuations. g p g p p y g gl To analyse the active site of the receptor structures, the all-atom RMSDs of the active site residues 90–112, 120–130, 161–176 and 183–196; the distance between the center of mass of residues V166–L169 and the catalytic residue C184; and the number of hydrogen bonds between R197 and the LPXTG peptide, respectively, were calculated.h The all atom RMSDs of the active site residues, including side chains, were calculated to investigate their flexibility. The active site residues displayed large flexibility in the apo-SrtA NMR structure during the simulation time, where the RMSD stayed at ~3Å within the first 60 ns, then rose to 4 Å within 60–100 ns, and after 100 ns to 5 Å for the remaining of the simulation. Results and Discussion MD simulations. Due to the large discrepancies between the reported SrtA structures, MD simulations were performed on four initial structures representing apo- and holo-SrtA NMR and crystal structures in order to fully explore the flexibility of the receptor conformations for the docking study. To assess how the conformations varied during each simulation, we calculated the Cα root mean square deviations (RMSDs) relative to the initial structure, Cα root mean square fluctuations (RMSFs), and the radius of gyration of the proteins.h Åt l The RMSD of the apo-SrtA NMR structure in the simulation stabilized at ~3.5 Å after 10ns; the holo-SrtA NMR structure stabilized at ~2.5 Å in the first 25 ns, then went up to ~3.5 Å after 45 ns; the apo-SrtA crystal struc- ture described almost the same fluctuation as the holo-SrtA NMR structure, except for a reduction down to ~2 Å after 175 ns; and the holo-SrtA crystal structure was stabilized after 5 ns at ~1.5 Å (Fig. 3A). Hence, the holo-SrtA crystal structure appears to be the one in the best thermal equilibrium, although at the same time its β 6/β 7 loop is displaced the most from the active site. p It can be seen from the RMSF of the Cα atoms (Fig. 3B) that residues within the loop regions are quite dynamic, especially the ß6/ß7 loop in both the apo-SrtA NMR and crystal structures, i.e. without peptide bind- ing; however, the RMSF of the ß6/ß7 loop in the holo-SrtA structures are relatively low, and hence, the dynamics Scientific Reports \| 6:20413 \| DOI: 10.1038/srep20413 6 www.nature.com/scientificreports/ Figure 4. Active site parameters during the 200 ns MD simulations. Line colors as in Figure 3. (A) All atom RMSDs of the SrtA active site residues; (B) Distance between the center of mass of residue V166-L169 (part of β 6/β 7 loop) and C184; (C) Number of hydrogen bonds between R197 and LPXTG analog in holo-SrtA NMR structure; (D) As (C) but for LPXTG substrate in holo-SrtA crystal structure. Figure 4. Active site parameters during the 200 ns MD simulations. Line colors as in Figure 3. Results and Discussion The distance in the apo-SrtA crystal structure quickly rises to ~20 Å, whereas in the holo-SrtA crystal structure the distance remains at 17.5 Å. In the apo-SrtA NMR structure the distance drops from 17.5 Å to 12.5 Å, which is almost the same trend as in the holo-SrtA NMR struc- ture. The distance changes indicate that, in the holo-SrtA structures, the loop keeps the same distance to C184 in the MD simulation as in the initial structure. The holo-SrtA NMR structure thus retains a more compact active site structure, whereas the holo-SrtA crystal structure has a more ‘open’ active site structure. In the apo–SrtA structures, on the other hand, the distance fluctuates more due to the lack of substrate interaction with the loop region. g In the initial holo-SrtA (substrate-bound SrtA) X-ray crystal structure, the LPETG peptide substrate adopts an elongated form while the ß6/ß7 loop remains in an “open” conformation (Fig. 1D). R197 is observed to make contact with the LPETG threonine residue. In the holo-SrtA NMR structure, on the other hand, the LPATG peptide analog adopts an “L-shape” configuration in which there is a 90o bend between the leucine and proline residues, and R197 makes contact with the LPATG alanine residue. During the MD simulations, those residues which are hydrogen bonding to the proteins keep still, whereas all other residues fluctuate significantly. The number of H-bonds in both the holo-SrtA structures fluctuates between 1 and 2 and sometimes even 3 during the simulation time, which indicates that the interaction between R197 and LPXTG is retained in both structures (Fig. 4C,D). The LPETG peptide substrate remains in its elongated form in the holo-SrtA X-ray crystal structure, whereas in the holo-SrtA NMR structure the “L-shape” configuration of the LPATG analogue disappears during the MD simulation. Enrichment calculations. Table 2–5 show the EF1%, BEDROC (α = 160.9), BEDROC (α = 20), AUC, and RIE after docking the 510 molecules to the 80 receptor models extracted from the MD simulations of the apo- and holo-SrtA NMR and crystal structures, using the Glide-XP scoring function. The enrichment factor EF1% metric, which reflects the database enrichment performance in the top 1% of a library, becomes particularly relevant in assessing the predictive power of VS. Results and Discussion In the holo-SrtA NMR structure simulation, the RMSD of the active site residues stays mostly at ~3.5 Å, and arises to 4 Å after 175ns, whereas in the apo-SrtA crystal structure simulation, the RMSD of the active site residues rises to 3 Å within the first 25 ns, and then rises to 5 Å till 100 ns, stays at ~5.5 Å from 100 ns to 175 ns, and drops down to 4 Å. In the holo-SrtA crystal structure simulations, finally, the RMSD of the active site residues remains more stable around 2.5 Å in the whole simulation (Fig. 4A). The RMSDs of the active site residues demonstrate that the binding sites of the apo structures of SrtA are more flexible than the holo structures, which again confirms that the LPXTG peptide binding stabilizes the active site. Scientific Reports \| 6:20413 \| DOI: 10.1038/srep20413 7 www.nature.com/scientificreports/ MD snapshot (ns) EF1% BEDROC (a = 160.9) BEDROC (a = 20.0) AUC RIE 0 10 0.206 0.181 0.58 2.99 5 0 0.012 0.099 0.51 1.64 10 20 0.392 0.255 0.46 4.22 20 0 0.031 0.097 0.39 1.61 40 10 0.08 0.121 0.5 2 60 10 0.237 0.231 0.52 3.83 70 10 0.21 0.25 0.8 4.13 80 10 0.283 0.16 0.6 2.64 90 10 0.151 0.192 0.77 3.18 100 10 0.11 0.118 0.62 1.95 110 10 0.162 0.192 0.6 3.18 120 10 0.209 0.213 0.54 3.53 130 10 0.08 0.11 0.56 1.81 140 0 0.031 0.106 0.56 1.76 150 10 0.206 0.145 0.52 2.4 160 10 0.081 0.177 0.62 2.93 170 10 0.132 0.245 0.7 4.05 180 10 0.283 0.196 0.49 3.23 190 10 0.206 0.134 0.55 2.22 200 0 0 0.015 0.49 0.25 Average 8.5 0.155 0.162 0.57 2.68 Table 2. EF1%, BEDROC (α = 160.9), BEDROC (α = 20), AUC, RIE values for snapsh NMR structure MD simulations. Table 2. EF1%, BEDROC (α = 160.9), BEDROC (α = 20), AUC, RIE values for snapshots of the apo-SrtA NMR structure MD simulations. Table 2. EF1%, BEDROC (α = 160.9), BEDROC (α = 20), AUC, RIE values for snapshots of the apo-SrtA NMR structure MD simulations. The distance between the center of mass of residues V166– L169 and C184 was calculated to see how the ß6/ ß7 loop fluctuates relative to the catalytic residues (Fig. 4B). Results and Discussion This indicates that the in vivo SrtA ligand binding conformations are significantly different compared to the retrieved NMR/crystal based structure models leading to the corollary that MD simulations can be important in generating better structures to use in VS campaigns; or, alternatively, that the active site is in fact not the key binding area of (some of) the ligands. g ( ) g When comparing the enrichment results to the structural analyses of section 3.1, there is a clear correlation between the average ß6/β 7 loop distance to the catalytic residue C184 and the average EF1% and the BEDROC (α = 160.9) values, where these two enrichment metrics counts more heavily on the high scoring binders in the VS. The distance measure demonstrates that the loop is in either “open” or “closed” conformation of the active site. The MD simulations generate a much more “open” conformation for both apo- and holo-SrtA crystal structures, but a more “closed” conformation for the apo- and holo-SrtA NMR structures with 2KID and 1IJA being the initial structures employed. Overall, the more “closed” conformation returns better EF1% and BEDROC (α = 160.9) results (Fig. 5A). However, we found no correlation between the average ß6/β 7 loop distance to the catalytic residue C184 and the average RIE or BEDROC (α = 20), or between the flexibility of the active site resi- dues and any of the enrichment metrics (Fig. 5B). y g Finally, a Boltzmann distribution function was applied to all the ensemble docking scores in order to obtain a weighted average score per ligand. Based on the Boltzmann weighted average docking score we calculated the average enrichment factors, BEDROC, AUC and RIE data; however, the results thus obtained do not provide any further improvement compared to using the ‘raw’ data for each snapshot (Suppplementary Table S6). Area under receiver operating characteristic (ROC) curves. The performance of each receptor con- formation according to the AUC can be divided as excellent (above 0.9), moderate (0.9 to 0.6), and poor (less than 0.6). Compared to EF1%, BEDROC and RIE, which measure for high scoring performance, AUC sees to the overall performance in identifying the known binders. All the 80 receptor conformations generated from the MD simulations were indicated to perform poor or moderately well as per the AUC definition. Results and Discussion As can be seen from Table 2, 15 out of 20 snapshots of the apo-SrtA NMR structure simulations have EF1% = 10, 1 out of 20 have EF1% = 20, and the remaining have EF1% = 0, giving an average EF1% of 8.5. Snapshots generated from the holo-SrtA NMR structure, the apo-SrtA crystal structure, and the holo-SrtA crystal structure MD simulations gave average EF1% of 9, 2 and 5.5 respectively. Overall, the EF1% ranges from 0 to 20 for the different SrtA conformations, indicating that the VS results are not particularly sensitive to the active site conformational changes; however, the EF1% results also suggest that the receptor models generated from the apo- and holo-SrtA crystal structures have very poor enrichment performance. This is most likely due to the large and bulky space available for fitting the decoys into the active site, as discussed more below. In general, the decoys that ranked higher than the actives were of high molecular weight and had higher propensity to form hydrogen bonds to R197. In terms of BEDROC (α = 160.9) enrichments, the snapshots from the apo- and holo-SrtA NMR and crystal structures return averages of 0.155, 0.172, 0.049, and 0.1, respectively, whereas the BEDROC (α = 20) enrichments yield averages of 0.162, 0.179, 8 Scientific Reports \| 6:20413 \| DOI: 10.1038/srep20413 www.nature.com/scientificreports/ Figure 5. (A) Correlation between average distance of V166-L169 to C184 with EF1% and RIE; (B) Correlation between average distance of V166-L169 to C184 with BEDROC (α = 160.9) and BEDROC (α = 20). See Supplementary Table S1 for data values. Figure 5. (A) Correlation between average distance of V166-L169 to C184 with EF1% and RIE; (B) Correlation between average distance of V166-L169 to C184 with BEDROC (α = 160.9) and BEDROC (α = 20). See Supplementary Table S1 for data values. 0.116 and 0.162. For the RIE enrichments, the corresponding averages are 2.68, 2.97, 1.92, and 2.68, respectively. In these scoring metrics, earlier parts of the hit list count more heavily in the enrichment as stated above. The exact reason for differences in enrichment results between neighbouring snapshots generated from the same initial structures are not entirely clear, since the structural changes are often small. Another observation from the tables is that the snapshots at 0 ns which represent the initial NMR or crystal structures do not generate the best results. Results and Discussion EF1%, BEDROC (α = 160.9), BEDROC (α = 20), AUC, RIE values for snapshots of the holo-SrtA NMR structure MD simulations. Table 3. EF1%, BEDROC (α = 160.9), BEDROC (α = 20), AUC, RIE values for snapshots of the holo-Srt NMR structure MD simulations. AUC for the apo-SrtA crystal structure is 0.78, 13 out of 20 performed poor, 7 out of 20 performed moderately well, with an average AUC of 0.57; and the best AUC for the holo-SrtA crystal structure is 0.78, with an average AUC of 0.63 (ROC plots for all the snapshots are given as Supplementary Figure S1).h AUC for the apo-SrtA crystal structure is 0.78, 13 out of 20 performed poor, 7 out of 20 performed moderately well, with an average AUC of 0.57; and the best AUC for the holo-SrtA crystal structure is 0.78, with an average AUC of 0.63 (ROC plots for all the snapshots are given as Supplementary Figure S1).h g y g This unsatisfactory AUC performance of the receptor conformation was mainly due to the fact that some receptor conformations could bind certain ligands remarkably well but could not identify those which required a different binding site arrangement. This result further supports the argument that the SrtA structure undergoes ligand-induced conformational changes, possibly upon initial ligand binding, which indicates that the SrtA pro- teins comprise conformational variety that yet remains to be unfolded. Ligand docking poses and ranking. The docked active ligands in the initial receptor structures were examined to characterize important interactions in the SrtA binding site and the differences in docking poses. For Ligand_501, the orientation of the molecule differs between the four receptor structures used, but the molecule interacts in all cases with the three key residues His120, Cys184, Arg197. For Ligand_502, the binding pose in the apo-SrtA crystal structure is distinct and interacts mainly with Helix ß6/7, and for Ligand_503, the molecule is located in the center of the binding pocket in the holo-SrtA crystal structure instead of interacting with the key residues. Ligand_504 interacts via its carboxylic group with Arg197 in the apo- and holo-SrtA NMR structure and the apo-SrtA crystal structure, but not with holo-SrtA crystal structure. For Ligand_505, the binding to the holo-SrtA crystal structure is again different from the rest as it interacts more with Helix ß6/7. Results and Discussion The best AUC for the apo-SrtA NMR structure is 0.80, whereas 13 out of 20 snapshot conformations performed poor and 7 out of 20 performed moderately well, with an average AUC of 0.57; the best AUC for the holo-SrtA NMR structure is 0.78, 5 out of 20 performed poor and 15 out of 20 performed moderately well, with an average AUC of 0.64; the best Scientific Reports \| 6:20413 \| DOI: 10.1038/srep20413 9 www.nature.com/scientificreports/ MD snapshot (ns) EF1% BEDROC (a = 160.9) BEDROC (a = 20.0) AUC RIE 0 10 0.15 0.116 0.44 1.92 5 10 0.206 0.163 0.6 2.69 10 10 0.325 0.242 0.76 4.01 20 10 0.283 0.206 0.6 3.41 30 10 0.11 0.158 0.63 2.61 40 10 0.15 0.132 0.62 2.18 60 20 0.286 0.246 0.68 4.08 70 10 0.11 0.196 0.69 3.24 90 0 0.001 0.114 0.64 1.88 100 0 0.209 0.256 0.68 4.24 110 10 0.283 0.18 0.65 2.99 120 10 0.11 0.167 0.58 2.77 130 10 0.11 0.141 0.6 2.33 140 10 0.15 0.13 0.58 2.21 150 20 0.363 0.236 0.76 3.91 160 10 0.249 0.222 0.61 3.67 170 10 0.111 0.193 0.66 3.19 180 0 0.006 0.124 0.57 2.05 190 0 0.002 0.113 0.67 1.87 200 10 0.229 0.249 0.69 4.12 Average 9 0.172 0.179 0.64 2.97 Table 3. EF1%, BEDROC (α = 160.9), BEDROC (α = 20), AUC, RIE values for snapshots of the holo-SrtA NMR structure MD simulations. MD snapshot (ns) EF1% BEDROC (a = 160.9) BEDROC (a = 20.0) AUC RIE 0 10 0.15 0.116 0.44 1.92 5 10 0.206 0.163 0.6 2.69 10 10 0.325 0.242 0.76 4.01 20 10 0.283 0.206 0.6 3.41 30 10 0.11 0.158 0.63 2.61 40 10 0.15 0.132 0.62 2.18 60 20 0.286 0.246 0.68 4.08 70 10 0.11 0.196 0.69 3.24 90 0 0.001 0.114 0.64 1.88 100 0 0.209 0.256 0.68 4.24 110 10 0.283 0.18 0.65 2.99 120 10 0.11 0.167 0.58 2.77 130 10 0.11 0.141 0.6 2.33 140 10 0.15 0.13 0.58 2.21 150 20 0.363 0.236 0.76 3.91 160 10 0.249 0.222 0.61 3.67 170 10 0.111 0.193 0.66 3.19 180 0 0.006 0.124 0.57 2.05 190 0 0.002 0.113 0.67 1.87 200 10 0.229 0.249 0.69 4.12 Average 9 0.172 0.179 0.64 2.97 Table 3. EF1%, BEDROC (α = 160.9), BEDROC (α = 20), AUC, RIE valu NMR structure MD simulations. Table 3. Scientific Reports \| 6:20413 \| DOI: 10.1038/srep20413 Results and Discussion Ligand_506 has attains a similar docking pose in all receptor structures, and interacts with the key residues, Helix ß6/7 and Helix ß7/8, whereas for Ligand_507, the binding poses differ between all the receptors. The docking pose of Ligand_508 in the holo-SrtA NMR crystal structure is different compared to the results for the other three receptors, and the molecule only partly interacts with the three key residues. For Ligand_509, the interactions of the molecule display significant differences between the receptors, due to its small size enabling the molecule to fit readily at several places in the pockets. Ligand_510, finally, attains different orientations but the general interactions are similar in all four systems (Supplementary Figure S2). In general, the ten actives are mostly located adjacent to the key residues in the different receptors, although some do locate closer to the helixes and/or the center of the binding pocket.ii As can be expected, the VS results confirm that true binders were ranked at the first positions when a suitable conformation was used. In this study, ligand_502 (cf. Fig. 2) ranked 14 times, ligand_506 ranked 5 times, and ligand_507 1 time in the top 1% of the VS results of holo-SrtA NMR structures; ligand_502 ranked 17 times, ligand_504 ranked 1 time in the top 1% of the VS results of apo-SrtA NMR structures; ligand_502 ranked 3 times time in the top 1% of the VS results of apo-SrtA crystal structures; and ligand_502 ranked 7 times, ligand_506 ranked 2 times in the top 1% of the holo-SrtA VS results of crystal structures (Table S2–S5). Overall, ligand_502, which is the well-known active compound morine, ranked the best amongst the known binders in the MD sim- ulation snapshots generated from different starting structures, with some snapshots also seen to provide good geometries for interaction with ligand_504, ligand_506 and ligand_507. Results and Discussion ranked compounds, irrespective of initial structure or snapshot, indicates that the catalytically active site may not necessarily be the preferred site of interaction for (several of) these compounds. Alternatively, the geometric features of the active site capable of discriminating that these compounds actually do bind better than the decoys, have as yet to be identified. From the above results, SrtA active site virtual screening campaigns using either of the reported Xray/NMR structures more or less “as is”, with the aim to generate novel inhibitors, are essentially bound to fail. A further observation that can be made is that the ranking of the ligands does not correlate with the trends in reported IC50 values, and that large variations in the set are noted, from one snapshot to another. For example, ligand 502 (morine) is ranked number 2 in the 20 ns snapshot starting from the apo-form of the SrtA crystal structure, but as number 324 (out of 510) in the snapshot at 25 ns. Results from SMD simulations. Although MD simulations can provide snapshots that are different from the crystal structures, they did in this particular case not generate a wide enough diversity of conformations to provide good binding to all the known ligands. In most cases, the extent of MD sampling will determine the diver- sity of conformations, relying on how well the sampling has explored phase space. Enhanced sampling methods, such as accelerated molecular dynamics and replica exchange simulations, that allow for more rapid exploration of conformational space might be useful in this respect. In order to cover a larger conformational region, steered MD simulations were performed in the current study, in which the β 6/β 7 loop was gradually shifted from ‘closed’ to ‘open’ conformation by pulling the V166-L169 segment of the loop away from the catalytically active C184. From the SMD trajectory, 20 snapshots were used in docking simulations and analyses. In Supplementary Figure S3 the distance variation between the β 6/β 7 loop and C184 during the 17.5 ns SMD simulation is displayed, and in Supplementary Table S7, the resulting EF1%, BEDROC, AUC and RIE data from the 20 docking runs are collected. Results and Discussion However, the overall poor performance in the ranking, with on average 1, and at the most 2 of the reported good binders ending up among the top 10 Scientific Reports \| 6:20413 \| DOI: 10.1038/srep20413 10 www.nature.com/scientificreports/ MD snapshot (ns) EF1% BEDROC (a = 160.9) BEDROC (a = 20.0) AUC RIE 0 10 0.152 0.181 0.63 2.99 5 10 0.291 0.289 0.75 4.77 10 0 0.043 0.1 0.58 1.65 20 10 0.209 0.231 0.78 3.82 25 0 0 0.004 0.41 0.07 30 0 0 0.003 0.42 0.05 40 0 0 0.064 0.52 1.06 70 0 0.009 0.1 0.64 1.66 90 0 0 0.06 0.5 0.99 100 0 0.004 0.122 0.63 2.01 110 0 0.01 0.147 0.48 2.42 120 0 0 0.059 0.54 0.97 130 0 0 0.046 0.49 0.76 140 0 0.009 0.091 0.42 1.51 150 10 0.206 0.181 0.69 3 160 0 0.032 0.284 0.73 4.7 170 0 0 0.103 0.58 1.7 180 0 0.024 0.186 0.54 3.07 190 0 0 0.005 0.54 0.09 200 0 0 0.062 0.53 1.02 Average 2 0.049 0.116 0.57 1.92 Table 4. EF1%, BEDROC (α = 160.9), BEDROC (α = 20), AUC, RIE values for snapshots of the apo-SrtA crystal structure MD simulations. MD snapshot (ns) EF1% BEDROC (a = 160.9) BEDROC (a = 20.0) AUC RIE 0 10 0.152 0.181 0.63 2.99 5 10 0.291 0.289 0.75 4.77 10 0 0.043 0.1 0.58 1.65 20 10 0.209 0.231 0.78 3.82 25 0 0 0.004 0.41 0.07 30 0 0 0.003 0.42 0.05 40 0 0 0.064 0.52 1.06 70 0 0.009 0.1 0.64 1.66 90 0 0 0.06 0.5 0.99 100 0 0.004 0.122 0.63 2.01 110 0 0.01 0.147 0.48 2.42 120 0 0 0.059 0.54 0.97 130 0 0 0.046 0.49 0.76 140 0 0.009 0.091 0.42 1.51 150 10 0.206 0.181 0.69 3 160 0 0.032 0.284 0.73 4.7 170 0 0 0.103 0.58 1.7 180 0 0.024 0.186 0.54 3.07 190 0 0 0.005 0.54 0.09 200 0 0 0.062 0.53 1.02 Average 2 0.049 0.116 0.57 1.92 Table 4. EF1%, BEDROC (α = 160.9), BEDROC (α = 20), AUC, RIE values f crystal structure MD simulations. Table 4. EF1%, BEDROC (α = 160.9), BEDROC (α = 20), AUC, RIE values for snapshots of the apo-SrtA crystal structure MD simulations. Results and Discussion The best AUC for the SMD snapshots is 0.78, whereas 2 out of 20 snapshot conformations performed poor and 18 out of 20 performed moderately well, with an average AUC of 0.67; 1 out of 20 snapshots of the SMD structure simulations have EF1% = 0, 5 out of 20 have EF1% = 20, and the remaining have EF1% = 10, giving an average EF1% of 12; the average BEDROC (a = 160.9), BEDROC(a = 20.0) and RIE values are 0.246, 0.21 and 3.73 correspondingly. Compared to the values from the ‘normal’ MD snapshots described above, the SMD snapshots perform somewhat better but provide no further improvement in recognizing the true actives from the decoys. The snapshots from 15.5 ns and onwards have a distance between the V166-L169 and C184 larger than 22.5 Å (See Figure S3), and hence represent ‘extremely open’ geometries not present in the reported structures. However, no marked improvements in data were seen in those snapshots despite sampling conformations not accessible from the normal MD trajectories. Table 4. EF1%, BEDROC (α = 160.9), BEDROC (α = 20), AUC, RIE values for snapshots of the apo-SrtA crystal structure MD simulations. Conclusions EF1%, BEDROC (α = 160.9), BEDROC (α = 20), AUC, RIE valu crystal structure MD simulations. Table 5. EF1%, BEDROC (α = 160.9), BEDROC (α = 20), AUC, RIE value for snapshots of the holo-SrtA crystal structure MD simulations. active site residues in the loop, particularly those located in the β 6/β 7 and β 7/β 8 loops. The analysis of early enrichment metrics and the AUC for different receptor models showed that the best EF1% is 20 and the best AUC is 0.8, demonstrating that the receptor models at best performed moderately well in distinguishing the actives from the decoys. This might imply that more thorough structural information, e.g., regarding possible ligand induced fit and protein flexibility, is needed to unlock the SrtA binding modes to its inhibitors. Such knowledge would enhance the ligand-based screening accuracy and potentially yield a more positive outcome than structure-based screening in this sense. Comparison of the average distance between the β 6/β 7 loop and C184, and the EF1% and BEDROC (α = 160.9) metrics revealed a correlation between the more “closed” con- formation and better scoring results. No correlation was found between the RMSDs of SrtA active site residues, BEDROC (α = 20), RIE and AUC. On the other hand, the results in this work suggest that MD conformations can improve VS results compared to the use of X-ray or NMR structures only. A general conclusion demonstrated by this study is that no single property could be used to separate a best receptor structure for the whole set of actives studied here. Using a Boltzmann weighted average score over the different snapshots did not provide any improvement, nor did the use of snapshots from a SMD simulation tailored at changing the system from ‘closed’ to very ‘open’ with respect to the β 6/β 7 loop. Although it is possible that even more structurally diverse confor- mations would increase the likelihood of improving the correlation with experiments, and to help discovering truly novel scaffolds, conformational space around the active site has in the current work been covered rather extensively. Alternatively, the actual binding mode may for several of these compounds in fact be located away from the active site, which would also explain the ‘randomness’ of the data obtained in the current work. In that sense, the current work also challenges the accuracy of the reported SrtA binding compounds. Conclusions Finally, in a full VS campaign, where high scoring actives is often crucial, EF1% and BEDROC (α = 160.9) seem to be the most appropriate metrics to evaluate performance towards SrtA. It can also not be ruled out that due to the large and flexible active site region, peptide-mimetic based inhibitors might be more optimal binders in the SrtA active site, than small organic molecules. Conclusions In this work, we have presented an exploratory analysis of VS performance for apo- and holo-SrtA based on molecular dynamics snapshots with diverse molecular flexibility and binding site properties. The MD simula- tion studies demonstrate that the SrtA structure is dynamic, especially in the loop regions, and that the LPXTG substrate on binding can induce distinct loop conformation differences in the binding site. When considering a compound binding to the active site of SrtA, it thus appears important to take into account the motion of the Scientific Reports \| 6:20413 \| DOI: 10.1038/srep20413 11 www.nature.com/scientificreports/ MD snapshot (ns) EF1% BEDROC (a = 160.9) BEDROC (a = 20.0) AUC RIE 0 0 0.015 0.166 0.61 2.74 5 0 0.031 0.099 0.61 1.63 10 0 0.059 0.173 0.6 2.86 30 0 0 0.048 0.42 0.79 40 10 0.11 0.195 0.66 3.23 50 10 0.206 0.142 0.74 2.35 60 10 0.119 0.242 0.65 4.01 70 10 0.11 0.17 0.57 2.82 90 0 0.033 0.156 0.63 2.58 100 10 0.11 0.121 0.62 1.99 110 0 0.047 0.198 0.65 3.28 120 10 0.159 0.225 0.78 3.72 130 0 0.089 0.196 0.66 3.25 140 20 0.489 0.236 0.64 3.91 150 10 0.141 0.202 0.7 3.35 160 0 0.01 0.162 0.7 2.69 170 10 0.08 0.163 0.61 2.69 180 10 0.283 0.174 0.6 2.88 190 0 0.001 0.106 0.62 1.75 200 0 0.002 0.071 0.57 1.18 Average 5.5 0.105 0.162 0.63 2.69 Table 5. EF1%, BEDROC (α = 160.9), BEDROC (α = 20), AUC, RIE value for snapshots of the holo-SrtA crystal structure MD simulations. MD snapshot (ns) EF1% BEDROC (a = 160.9) BEDROC (a = 20.0) AUC RIE 0 0 0.015 0.166 0.61 2.74 5 0 0.031 0.099 0.61 1.63 10 0 0.059 0.173 0.6 2.86 30 0 0 0.048 0.42 0.79 40 10 0.11 0.195 0.66 3.23 50 10 0.206 0.142 0.74 2.35 60 10 0.119 0.242 0.65 4.01 70 10 0.11 0.17 0.57 2.82 90 0 0.033 0.156 0.63 2.58 100 10 0.11 0.121 0.62 1.99 110 0 0.047 0.198 0.65 3.28 120 10 0.159 0.225 0.78 3.72 130 0 0.089 0.196 0.66 3.25 140 20 0.489 0.236 0.64 3.91 150 10 0.141 0.202 0.7 3.35 160 0 0.01 0.162 0.7 2.69 170 10 0.08 0.163 0.61 2.69 180 10 0.283 0.174 0.6 2.88 190 0 0.001 0.106 0.62 1.75 200 0 0.002 0.071 0.57 1.18 Average 5.5 0.105 0.162 0.63 2.69 Table 5. Table 5. EF1%, BEDROC (α = 160.9), BEDROC (α = 20), AUC, RIE value for snapshots of the holo-SrtA crystal structure MD simulations. 1. Lowy, F. D. Staphylococcus aureus infections. N. Engl. J. Med. 339, 520 532 (1998). 2. Moran, G. J. et al. Methicillin-resistant S. aureus infections among patients in the emergency department. N. Engl. J. 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Author Contributions C.G. and I.U. designed the study; C.G. performed the simulations and analysis; C.G., I.U., J.G. and L.A.E. al articipated in writing and reviewing of the manuscript. Scientific Reports \| 6:20413 \| DOI: 10.1038/srep20413 www.nature.com/scientificreports/ Development and use of quantum mechanical molecular models. 76. AM1: a new general purpose quantum mechanical molecular model. J. Am. Chem. Soc. 107, 3902–3909 (1985).i 41. Dewar, M. J. S., Zoebisch, E. G., Healy, E. F. & Stewart, J. J. P. Development and use of quantum mechanical molecular models. 76. AM1: a new general purpose quantum mechanical molecular model. J. Am. Chem. Soc. 107, 3902–3909 (1985).i g p p q J , ( ) 2. Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A. & Case, D. A. Development and testing of a general amber force field. J. Comp Chem. 25, 1157–1174 (2004). 43. Case D. A. et al. University of California; San Francisco, CA, USA, AMBER, version 10. 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Competing financial interests: The authors declare no competing financial interests. How to cite this article: Gao, C. et al. Exploration of multiple Sortase A protein conformations in virtual screening. Sci. Rep. 6, 20413; doi: 10.1038/srep20413 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports \| 6:20413 \| DOI: 10.1038/srep20413 14
https://openalex.org/W2801853078	https://zenodo.org/records/1524711/files/article.pdf	English	null	English Folk-Song	The musical times/Musical times	1,921	public-domain	2,781	English Folk-Song Author(s): Richard Capell Source: The Musical Times, Vol. 62, No. 937 (Mar. 1, 1921), pp. 174-175 Published by: Musical Times Publications Ltd. Stable URL: http://www.jstor.org/stable/910930 Accessed: 09-01-2016 16:51 UTC English Folk-Song Author(s): Richard Capell Source: The Musical Times, Vol. 62, No. 937 (Mar. 1, 1921), pp. 174-175 Published by: Musical Times Publications Ltd. Stable URL: http://www.jstor.org/stable/910930 Accessed: 09-01-2016 16:51 UTC Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://www.jstor.org/page/ info/about/policies/terms.jsp JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. Musical Times Publications Ltd. is collaborating with JSTOR to digitize, preserve and extend access to The Musical Times. http://www.jstor.org BY RICHARD CAPELL All national art, says a wise Frenchman, is a source of beauty. Of course Beauty and Art are difficul words, and none but the professed philosopher can say just what he means by'them; but still one's feeling may be some help on the way in default of strict philosophy. We may (without straying out of sight of our concern, which is Mr. Sharp and his English folk-songs) see Beauty as possessed of double aspect, and the one or other allures and is adored in its season-the one is the imagined and fantastic, the other the assured and experienced. Such artless songs are not to be disassociated from the artless singers, who are our own people, ourselves ; and their beauty is of more than this surface of cheerful irony, such being just the emerging aspect of that hidden bulk of our own soundness, the solidity of our virtue of self-rescue, which 'saved the sum of things' in the day 'when heaven was falling, the hour when earth's foundations fled.' Such artless songs are not to be disassociated from the artless singers, who are our own people, ourselves ; and their beauty is of more than this surface of cheerful irony, such being just the emerging aspect of that hidden bulk of our own soundness, the solidity of our virtue of self-rescue, which 'saved the sum of things' in the day 'when heaven was falling, the hour when earth's foundations fled.' p For one whose lot just before the war lay amid London musical doings the days were gorgeously crowded with the exotic succession of tribute-payers who seemed to be bringing to our feet all that the world held of the strange, rich, and monstrous. As Asia of old sent her elephants, apes, and peacocks in offering to imperial Rome, so there showered down on us tet-ralogies and symphonic poems, the pyramidal scores of Scriabin, Mahler, and Strauss, the fairyland of the Russian Ballet (ah, at its prime !), the opera-casts with all the golden throats of Italy, not to speak of opera-houses simply thrown at our heads by America. The causes of our delight in art are doubtless never unmixed, and .conceivably these two books of a hundred English folk-songs, chosen from Mr. Sharp's collections of twenty-five years, might leave some persons untouched if beauty in her exotic phase were dominant. This content downloaded from 134.83.1.242 on Sat, 09 Jan 2016 16:51:34 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 134.83.1.242 on Sat, 09 Jan 2016 16:51:34 UTC All use subject to JSTOR Terms and Conditions THE MUSICAL TIMES-MARCH I 192I 174 Do you remember the fuss there was when a certain widely-used adjective was shot at the audience in Shaw's 'Pygmalion'? It was the great moment of the play. Crowds stood in queues and paid good hard money nightly, and then hung upon the word--a word which they could have heard as well delivered free of charge in the street outside. That word follows next on the postcard, and cannot be quoted in a journal that prides itself on being so conducted that it may safely be left about the house and read even by the oldest. So we will borrow from the more harmless side of Old Bill's vocabulary, and put 'blinkin" in its place : autumn there dawned on you perhaps that other beauty, the beauty of the assured things that have made and moulded you and yours for time without mind. It dawned on you, no doubt with a pang, in the look of some bit of road, low hills, and home- county village, by which you were trudging in weather you would perhaps never have chosen to be out in, and with an absurd lot of things on your back. and with an absurd lot of things on your back. The beauty had a voice, to be perceived in certain turns of speech of your fellows-the butcher, the baker, and the candlestick-maker. Oh ! admirably vivid and vigorous, a speech that was untouched by the pallor of the printed word. When song put in an appearance, here and there were snatches that were clearly an English voice, a voice which could be recognized from its hints in our prose from Shakespeare to Thomas Hardy. No tune was, I think, 'magnificent.' The feeling was more of humour than of declared heroism. It could bear irony, but could not bear with any of the emotion of the heart-worn-on-sleeve. But here is a sample which was much sung in the neighbourhood of Festubert and Cuinchy in grim days-spring, 1915. ' English Folk-songs: collected and arranged by Cecil J. Sharp, 2 vols. Novello & Co., Ltd. This content downloaded from 134.83.1.242 on Sat, 09 Jan 2016 16:51:34 UTC All use subject to JSTOR Terms and Conditions There may be a known composer-or even a known author of the verses (the refrain of which begins, 'I don't want to be a soldier, I don't want to go to war,' and ends, 'I'd rather stay in England, in merrie, merrie England, than throw my precious life away'). We knew of none, and here is a case of a folk-song in the making: p . beyond a peradventure (not, of course, that anyone but a blinkin' fool . . .) And there the card ends,-on an unresolved suspen- sion, so to speak. Turning up last month's Mfusical Times, I find a contributor speaking of the London Symphony Orchestra as the ' L.S.O.,' little knowing what trouble he was making. Now, the London Society of Organists is a small body about two years old-at all events as at present constituted. We may safely say that beyond the members, their sisters, their cousins, and aunts, nobody knows of their existence. Their fame is bounded on the north by Palmers Green, on the south by Denmark Hill. The London Symphony Orchestra, on the other hand, is known (at least by name) to musicians not only over the greater part of Europe but in North America as well. Its initials have been used as a title for many years. t Alla marcia, o a... ' 0) -.- p" . -. 4 rJI-- ,,_ -.... y y Was there ever a more ludicrous case of, 'Said the Flea to the Elephant: " " ' p " Who 're you shovin' ? " ' This content downloaded from 134.83.1.242 on Sat, 09 Jan 2016 16:51:34 UTC All use subject to JSTOR Terms and Conditions BY RICHARD CAPELL Terry's collection-caught the audience as does not one music-hall song in a thousand. glorified analytical programme. We return to pianoforte music proper with some pleasant sets of pieces by Melartin-' Chips' and 'Intermezzi' (Augener). These are fairly easy essays, picturesque and tuneful, excellent teaching material of the type of Grieg's lyrical pieces, though less highly coloured. From the same publishers comes Liszt's edition of Schubert's 'Wanderer' Fantasia. A batch of material for youthful players calls for no more than mention. Four Miniatures by Reginald S. Thatcher (Augener), an album of Ten Preludes and Miniatures by M. E. Marshall (Bosworth), and a Little Suite, 'Nursery Secrets,' by Felix White (Keith Prowse). The last-named gives us not only some unusually pleasant little pieces, but also something new in titles, perhaps an even more difficult feat. Here they are: 'The Forbidden Fairy Tale (with Ghosts in it),' 'A Dream of the Russian Ballet,' 'Who broke the New Toy ?' and 'Why the Flies go round and round.' Having settled this last point, perhaps Mr. White will in his next Suite go one better and answer a popular question as to where they go in the winter. C. VW. g This collection of Mr. Sharp's, which seems to have every virtue of taste and fidelity, will be an acknowledged record and store-house. It will be the best beloved of song-books in many English homes. At song recitals a bunch of flowers from Mr. Sharp's garden will always be welcome. PIANOFORTE MUSIC In our January number mention was made of the first set of Caprices and Rhythmic Studies by E. Jaques-Dalcroze. Here is the second set (Augener), and one need give it no higher praise than to say it is as good as its predecessor. It is a pleasure to meet with music of this kind-free and imaginative, and handling complex rhythmic and harmonic problems with ease and assurance. The pieces are for good players. For 'good' we must substitute 'advanced' in speaking of Arthur de Greef's 'Etudes de Concert' (Augener). There are five, published separately. Here again we have an unusually high standard. The technical excellence of these pieces goes without saying. Far more important is the fact that they BY RICHARD CAPELL This artless voice so sweet, now tender, now rollicking, has us wholly won, and for the just reason that at the mocking notes of the courted milkmaid, at the plaint of the forlorn swain, or the home-coming sailor's tale everyone must instinctively call from memory's store some corner of the green and pleasant land, a scene from favoured shire,or home. g y p g 'The Hour Glass' is a set of three pieces by Frank Bridge, published in separate numbers by Augener. ' Dusk' is appropriately vague and slightly melancholy. 'The Dew Fairy' gives us some delightful arabesque treatment of an elvish little tune. In 'The Midnight Tide' fearful conglomera- tions of notes and tempestuous arjeggi point to dirty weather. The piece is not long, but it has the right sense of bigness. These three highly picturesque sketches are difficult, but they 'come off,' which is more than can be said for a great deal of difficult music. The pianoforte duet is such a valuable means of getting a good working idea of complex orchestral and chamber works that one is glad to see some of our modern composers taking advantage of it. Here is Malipiero's ' Panthea,' a symphonic drama for chorus and orchestra, boiled down for four- handed consumption (Chester). A very pungent dish it is, too. Only the varied timbres of the orchestra can make some of the dissonances tolerable, so it is to be hoped that no one will judge it from the pianoforte point of view. An arrange- ment of this kind must be regarded merely as a kind of glorifiedanalytical programme. , How propitious May mornings are to Somerset suitors is to be learned from many a song (a good proportion of these first two books comes from Mr. Sharp's Somerset collection). On the other hand even that melting season, it is clear, leaves some maids whole-hearted and scoffing. The sailor is a favourite hero, but some sailors only court to thieve, it seems, and hence the rueful modal complaint of more than one once merry milkmaid or shepherdess. y p If any doubt that such songs may not have the hold they had over English folk, there was a demonstration the other evening at the Morley Working Men's College (Waterloo Road, London), where the chanty 'Let the Bulgine run'-an irresistible tune from Dr. BY RICHARD CAPELL But the passions, the disillusions, and endurances of the war have perhaps contracted a little our hearts, which used to be so very expansive. We perhaps have come to trust more in ourselves and less in any glib and flattering stranger's effusive- ness. Anyhow there is a consistent strain in these song-books which is certain to be seized on more eagerly now than it ever would have been in the past. These songs have not the poignant accent of the y One August day, and all this was like snow upon the desert's dusty face. It left the barest memory of a dream, and it was like awakening from a dream to face the new world, so irrelevant to the intoxication of the eve! The fantastical beauty that miraged so flatteringly the impossible ends of the earth left not enough comfort to be worth a thbught. That This content downloaded from 134.83.1.242 on Sat, 09 Jan 2016 16:51:34 UTC All use subject to JSTOR Terms and Conditions THE MUSICAL TIMES-MARCH I 1921 175 great Irish airs, or the sensuousness and savage rhythmical life of Spanish or of Hungarian folk- music. To generalise (for often the lyre of pathos is most delicately touched), these are the songs of a passably happy, contented people, with a cheerful humour all its own. There are not the gloom or supernatural terrors of the lore of mountain-folk Beyond the favourite case of a bridegroom being seized by the press-gang there is no oppression. But short of the grand dramatic flight, these old country songs range over a multitude of human feelings. The ballads give us hints of the really archaic. Some of these, in Mr. Sharp's scrupulously unimproved versions, are occasionally fragmentary and obscure. The lyric songs offer to the listening ear the authentic voice of our countryside. For of course these are countrymen's songs (such as are not sailors'), for urban song is a different thing, picking up and dropping its favourites as often in a year as does the country in a generation. This content downloaded from 134.83.1.242 on Sat, 09 Jan 2016 16:51:34 UTC All use subject to JSTOR Terms and Conditions BY RICHARD CAPELL This artless voice so sweet, now tender, now rollicking, has us wholly won, and for the just reason that at the mocking notes of the courted milkmaid, at the plaint of the forlorn swain, or the home-coming sailor's tale everyone must instinctively call from memory's store some corner of the green and pleasant land, a scene from favoured shire, or home. have real beauty and power as well-for example, the delicate wistful charm of the Etude in E takes one captive at once. However, there is no space for detailed description. It must suffice to draw the advanced pianist's attention to these excellent additions to his r6pertoire. Ravel's Po6me Choregraphique, 'La Valse,' has been transcribed for pianoforte solo by the composer (Durand). It makes a fine piece, though one feels the need of the orchestra in the opening pages, where the music lies rather too low to be clear, and later where some important decorative passages for wood- wind, written in small notes, will mostly have to be taken as read. 'La Valse' is very difficult and very long-twenty-four pages great Irish airs, or the sensuousness and savage rhythmical life of Spanish or of Hungarian folk- music. To generalise (for often the lyre of pathos is most delicately touched), these are the songs of a passably happy, contented people, with a cheerful humour all its own. There are not the gloom or supernatural terrors of the lore of mountain-folk Beyond the favourite case of a bridegroom being seized by the press-gang there is no oppression. B h f h d h ld y press gang oppression. But short of the grand dramatic flight, these old country songs range over a multitude of human feelings. The ballads give us hints of the really archaic. Some of these, in Mr. Sharp's scrupulously unimproved versions, are occasionally fragmentary and obscure. The lyric songs offer to the listening ear the authentic voice of our countryside. For of course these are countrymen's songs (such as are not sailors'), for urban song is a different thing, picking up and dropping its favourites as often in a year as does the country in a generation. ORGAN MUSIC Parry's interest in the organ showed itself in a series of works written in his later years-works so good that organists regretted he had not turned to their instrument before his numerous public and educational duties made composition a mere extra. Among the MS. works he left a Toccata and Fugue entitled 'The Wanderer,' which has now been
https://openalex.org/W4200376709	https://research-information.bris.ac.uk/files/309925226/Perkins_Kirkpatrick_2022_Environ._Res._Lett._17_024009.pdf	English	null	On the attribution of the impacts of extreme weather events to anthropogenic climate change	Environmental research letters	2,022	cc-by	8,306	Perkins-Kirkpatrick, S. E., Stone, D. A., Mitchell, D. M., Rosier, S., King, A. D., Lo, Y. T. E., Pastor-Paz, J., Frame, D., & Wehner, M. (2022). On the attribution of the impacts of extreme weather events to anthropogenic climate change. Environmental Research Letters, 17(2), Article 024009. https://doi.org/10.1088/1748-9326/ac44c8 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1088/1748-9326/ac44c8 This is the final published version of the article (version of record). It first appeared online via IOP Publishing at 10.1088/1748-9326/ac44c8. Please refer to any applicable terms of use of the publisher. Perkins-Kirkpatrick, S. E., Stone, D. A., Mitchell, D. M., Rosier, S., King, A. D., Lo, Y. T. E., Pastor-Paz, J., Frame, D., & Wehner, M. (2022). On the attribution of the impacts of extreme weather events to anthropogenic climate change. Environmental Research Letters, 17(2), Article 024009. https://doi.org/10.1088/1748-9326/ac44c8 Perkins-Kirkpatrick, S. E., Stone, D. A., Mitchell, D. M., Rosier, S., King, A. D., Lo, Y. T. E., Pastor-Paz, J., Frame, D., & Wehner, M. (2022). On the attribution of the impacts of extreme weather events to anthropogenic climate change. Environmental Research Letters, 17(2), Article 024009. https://doi.org/10.1088/1748-9326/ac44c8 LETTER • OPEN ACCESS University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ This content was downloaded from IP address 86.147.72.72 on 28/01/2022 at 08:37 You may also like You may also like Building capacity for societally engaged climate science by transforming science training Mary Ann Rozance, Meade Krosby, Alison M Meadow et al. - Evaluating the effects of climate change on US agricultural systems: sensitivity to regional impact and trade expansion scenarios Justin S Baker, Petr Havlík, Robert Beach et al. - Sources of uncertainty in hydrological climate impact assessment: a cross-scale study F F Hattermann, T Vetter, L Breuer et al. - You may also like Building capacity for societally engaged climate science by transforming science training Mary Ann Rozance, Meade Krosby, Alison M Meadow et al. - Evaluating the effects of climate change on US agricultural systems: sensitivity to regional impact and trade expansion scenarios Justin S Baker, Petr Havlík, Robert Beach et al. - Sources of uncertainty in hydrological climate impact assessment: a cross-scale study F F Hattermann, T Vetter, L Breuer et al. - Abstract Investigations into the role of anthropogenic climate change in extreme weather events are now starting to extend into analysis of anthropogenic impacts on non-climate (e.g. socio-economic) systems. However, care needs to be taken when making this extension, because methodological choices regarding extreme weather attribution can become crucial when considering the events’ impacts. The fraction of attributable risk (FAR) method, useful in extreme weather attribution research, has a very specific interpretation concerning a class of events, and there is potential to misinterpret results from weather event analyses as being applicable to specific events and their impact outcomes. Using two case studies of meteorological extremes and their impacts, we argue that FAR is not generally appropriate when estimating the magnitude of the anthropogenic signal behind a specific impact. Attribution assessments on impacts should always be carried out in addition to assessment of the associated meteorological event, since it cannot be assumed that the anthropogenic signal behind the weather is equivalent to the signal behind the impact because of lags and nonlinearities in the processes through which the impact system reacts to weather. Whilst there are situations where employing FAR to understand the climate change signal behind a class of impacts is useful (e.g. ‘system breaking’ events), more useful results will generally be produced if attribution questions on specific impacts are reframed to focus on changes in the impact return value and magnitude across large samples of factual and counterfactual climate model and impact simulations. We advocate for constant interdisciplinary collaboration as essential for effective and robust impact attribution assessments. On the attribution of the impacts of extreme weather events to anthropogenic climate change Perkins-Kirkpatrick1,∗, D A Stone2, D M Mitchell3, S Rosier2, A D King4, Y T E Lo3, J Pastor-Paz5, 6 7 e, UNSW Canberra, Canberra Australia, and ARC Centre of Excellence for Climate Extremes, UNSW Sydney, Sydney, School of Science, UNSW Canberra, Canberra Australia, and ARC Centre of Excellence for Climate Extremes, UNSW Sydney, Sydn Australia On the attribution of the impacts of extreme weather events to anthropogenic climate change To cite this article: S E Perkins-Kirkpatrick et al 2022 Environ. Res. Lett. 17 024009 View the article online for updates and enhancements. This content was downloaded from IP address 86.147.72.72 on 28/01/2022 at 08:37 https://doi.org/10.1088/1748-9326/ac44c8 Environ. Res. Lett. 17 (2022) 024009 N ACCESS VED ptember 2021 ED ecember 2021 TED FOR PUBLICATION ecember 2021 SHED nuary 2022 nal content from work may be used r the terms of the ive Commons bution 4.0 licence. urther distribution s work must tain attribution to uthor(s) and the title e work, journal on and DOI LETTER On the attribution of the impacts of extreme weather events to anthropogenic climate change S E Perkins-Kirkpatrick1,∗, D A Stone2, D M Mitchell3, S Rosier2, A D King4, Y T E Lo3, J Pastor-Paz5 D Frame6and M Wehner7 1 School of Science, UNSW Canberra, Canberra Australia, and ARC Centre of Excellence for Climate Extremes, UNSW Sydney, Sydney Australia 2 NIWA, Wellington, Aotearoa, New Zealand 3 School of Geographical Sciences and Cabot Institute for the Environment, University of Bristol, Bristol, United Kingdom 4 School of Geography, Earth, and Atmospheric Sciences, University of Melbourne, Melbourne, Australia 5 Geological and Nuclear Sciences Institute (GNS Science), Wellington, New Zealand 6 New Zealand Climate Change Research Institute, Victoria University of Wellington, Wellington, New Zealand 7 Lawrence Berkeley National Laboratory, Berkeley, CA, United States of America ∗Author to whom any correspondence should be addressed. E-mail: sarah.kirkpatrick@unsw.edu.au Keywords: impacts, attribution, climate and weather extremes, climate change Supplementary material for this article is available online OPEN ACCESS RECEIVED 15 September 2021 REVISED 15 December 2021 ACCEPTED FOR PUBLICATION 20 December 2021 PUBLISHED 26 January 2022 Original content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence. On the attribution of the impacts of extreme weather events to anthropogenic climate change Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI LETTER On the attribution of the impacts of extreme weather events to anthropogenic climate change S E Perkins-Kirkpatrick1,∗, D A Stone2, D M Mitchell3, S Rosier2, A D King4, Y T E Lo3, J Pastor-Paz5, D Frame6and M Wehner7 1 School of Science, UNSW Canberra, Canberra Australia, and ARC Centre of Excellence for Climate Extremes, UNSW Sydney, Sydney, Australia 2 NIWA, Wellington, Aotearoa, New Zealand 3 School of Geographical Sciences and Cabot Institute for the Environment, University of Bristol, Bristol, United Kingdom 4 School of Geography, Earth, and Atmospheric Sciences, University of Melbourne, Melbourne, Australia 5 Geological and Nuclear Sciences Institute (GNS Science), Wellington, New Zealand 6 New Zealand Climate Change Research Institute, Victoria University of Wellington, Wellington, New Zealand 7 Lawrence Berkeley National Laboratory, Berkeley, CA, United States of America ∗Author to whom any correspondence should be addressed. E-mail: sarah.kirkpatrick@unsw.edu.au Keywords: impacts, attribution, climate and weather extremes, climate change Supplementary material for this article is available online 1. Introduction anthropogenic climate change) with the same or greater (or lesser, depending on the type of extreme) magnitude of that threshold, and not the individual threshold itself. To understand how anthropogenic climate change influences extreme events, the method compares the frequency of events greater than or equal to a threshold in the factual (which includes historical anthropogenic forcing) and counterfactual (anthropogenic forcing is omitted or removed) cli- mate model simulations: FAR = 1 − (Pcfact Pfact ) = 1 −1/RR where Pcfact is the event frequency in the counterfac- tual simulations and Pfact is the event frequency in the factual model simulations. The risk ratio (RR), also known as probability ratio, is regularly reported in attribution assessments, and is related to FAR as follows: Recent research is focusing on what we term ‘impacts attribution’, an extension of EEA to specific impacts within non-climate systems. Examples of impacts attribution assessments undertaken include human mortality during heatwaves (Mitchell et al 2016), the sustainability of fisheries (Litzow et al 2021), coral bleaching during marine heatwaves (Lewis and Mallela 2018), incurred financial dam- ages from hurricanes (Frame et al 2020a, Wehner and Sampson 2021) and financial damages from flooding and landslides associated with extreme rainfall events, and drought (Frame et al 2020b). Some impact attri- bution studies have relied on FAR (see section 2.1) to determine the anthropogenic signal behind a specific extreme event, and its impacts. However, the inter- pretation of results has been inconsistent with their application of FAR. FAR measures the change in fre- quency of a class of events, and an impact is a specific outcome of a singular event (Harrington 2017); FAR- based impact attribution studies have interpreted res- ults as applicable to the outcome rather than the class (e.g. Mitchell et al 2016, Frame et al 2020a, 2020b). We demonstrate this distinction for two examples of impacts caused by extreme events to show that the issue is systemic and does not pertain to just one type of extreme event/impact combination. RR = Pfact Pcfact or RR = 1/(1 −FAR). RR is often used in communicating attribution statements due to its ease of interpretation compared with FAR (note that RR still applies to a class of events). 1. Introduction studies assessing how anthropogenic climate change has altered notable and high-impact events (e.g. Peterson et al 2012, Herring et al 2020). Although exact methodological approaches vary, many stud- ies rely on climate models providing simulations of factual and counterfactual climates, in other words, simulations where observed anthropogenic climate forcings are included, and simulations where these forcings are omitted. Large sample sizes are essential, Extreme event attribution (EEA) is a climate sci- ence field where the influence of physical drivers is isolated for specific extreme events. Commonly, the driver of interest is anthropogenic climate change, considering the influence on the frequency or mag- nitude of observed extremes. Since its conception (Allen 2003), there has been a wealth of attribution © 2022 The Author(s). Published by IOP Publishing Ltd S E Perkins-Kirkpatrick et al Environ. Res. Lett. 17 (2022) 024009 achieved by fully-coupled multi-model ensembles (Lewis and Karoly 2013), multi-member ensembles of single or multiple models where initial conditions and or/physics differ slightly (e.g. Pall et al 2011, Massey et al 2015, Perkins and Gibson 2015, Guillod et al 2017, Hope et al 2018, Stone et al 2019), or a combina- tion of these. Moreover, there are multiple techniques to undertake event attribution assessments, such as the fraction of attributable risk (FAR) probability framework (Allen 2003, Stott et al 2004, Stone and Allen 2005); the story-line approach (e.g. Hoerling et al 2013, Trenberth et al 2016, Shepherd 2016, Zappa and Shepherd 2017, Patricola and Wehner 2018, Wehner et al 2019, Reed et al 2020); a com- parison of model ensembles with different forcings and/or physics (e.g. Hope et al 2018); and statist- ical estimations of shifts in extreme return levels (e.g. Eden et al 2018). While FAR is commonly used in EEA assessments, any method must accurately reflect the attribution question being asked, which should be clear in the initial study design (Otto et al 2012, Stone et al 2021). extend the FAR methodology to attributing changes in impacts to anthropogenic influence (Mitchell et al 2016, Frame et al 2020a, 2020b). Whilst this paper outlines caution in this extension, the method is powerful for attribution studies in general, including classes of impacts (see section 4.2). Computed FAR values correspond to the change in likelihood of events due to the causal factor (e.g. 1. Introduction The RR allows statements such as ‘the prob- ability of a class of events occurring is RR times what it would have been without anthropogenic influence on the climate’ whereas FAR addresses the responsib- ility of a causal factor for the occurrence of a class of events. For instance, a FAR of 0.9 means that 90% of events in the defined class that have occurred can be attributed to the forcing (Stone and Allen 2005). This interpretation assumes that events can be allocated into a group that would have occurred regardless of forcing, or a group that occurred only because of the intervention, but this allocation can only be determ- ined statistically (Hansen et al 2014). The chaotic cli- mate system does not satisfy this assumption, but the interpretation is still considered useful in understand- ing anthropogenic influence provided it is not taken too literally. We focus on FAR and not RR in this study, since FAR has been computed in recent impact attribution studies (e.g. Mitchell et al 2016, Frame et al 2020a). 2.2. Case studies In addition to investigating different types of extremes and their impacts, our case studies were chosen to fulfil multiple criteria: • availability of existing climate model data that adequately simulates the type of extreme event of interest. 2.2.2. New Zealand extreme rainfall in 2017 and Earthquake Commission (EQC) financial damages In early April 2017, ex-Tropical Cyclone Debbie made landfall on the North Island of New Zealand. Signi- ficant rainfall was recorded between 3 and 7 April 2017, but the peak in terms of both 1-day amounts and the spatial area affected was on 4 April. Thus, we define the meteorological event as the area-averaged 1-d rainfall total for the North Island on the 4 April 2017, which also equated to the highest daily rain- fall event in the 18 year record that we examine. A transfer function to estimate excess insurance payouts covered by New Zealand’s public insurer, the EQC has been previously defined (Pastor-Paz et al 2020). The corresponding excess payout by the EQC for the 4 April 2017 was estimated at NZ$7.39 M using the Pastor-Paz et al (2020) methodology fitted to observa- tional data (Tait et al 2006; see supplementary mater- ial). Specific event (section 2.3) and FAR (section 2.1) attribution assessments were conducted using this financial damage estimate, and the 1-in-18 year area- averaged daily rainfall event from the correspond- ing model (66.89 mm d−1). Figure 2 summarises the associated methods, which are explained in detail in the supplementary material. It should be noted that the EQC payouts will not reflect the overall economic consequences of the event, or even the insured losses. Most of the damages associated with EQC payouts arises from landslips, whereas most of the overall insured losses from the event (NZ$91 M) were asso- ciated with property flooding This complicates direct comparison with studies of insured losses (e.g. Frame et al 2020a). As noted in Frame et al (2020a), the full economic consequences of extreme weather events extend well beyond insured losses; data availability prevent an analysis of the full economic impacts of this event at this time. • documented relationships between the type of extreme event and the severity of an impact, and: 2.2.2. 2. Fraction of attributable risk (FAR) and case studies 2.1. The fraction of attributable risk (FAR) method FAR is an attribution diagnostic obtained by com- paring frequencies of a class of events in factual and counterfactual simulated climates, where the class is defined by a fixed magnitude threshold, com- monly a recently observed weather extreme (e.g. Stott et al 2004). Some recent studies have attempted to We employ FAR to determine how anthropogenic climate change has altered the likelihood of the class of extreme events in each case study discussed in section 2.2. We also replicate how FAR has been used to estimate the anthropogenic signal behind corres- ponding impacts (Mitchell et al 2016, Frame et al 2 S E Perkins-Kirkpatrick et al Environ. Res. Lett. 17 (2022) 024009 2020a). This demonstrates how FAR estimates may vary both across different extreme events and the impacts; and what it really means when FAR analysis is performed on the estimated impacts of extremes. define our event as the single day with the highest heat-related mortality for London, UK. This was the 26 July, with ∼60 deaths attributable to heat. The observed mean temperature was 26.6 ◦C, and the transfer function used to estimate mortality is the distributed lag non-linear model (Gasparrini et al 2015), based on daily mean temperature. The daily all-cause mortality totals and mean temperature are used for the specific event (section 2.3) and FAR (section 2.1) attribution assessments. Figure 1 sum- marises the associated methods, which are explained in detail in the supplementary material. 2.2. Case studies New Zealand extreme rainfall in 2017 and Earthquake Commission (EQC) financial damages • availability of a functional formulation relating cli- mate variations underpinning the type of extreme event to an impact (we refer to these as transfer functions). Our first case study is the hottest day dur- ing the 2006 UK summer, a record-breaking sum- mer at the time (Prior and Beswick 2007), and the impacts of the high temperatures on human mor- tality. The second is the New Zealand extreme rain- fall event that occurred on the 4 April 2017, causing wide-spread property damages to the North Island (www.icnz.org.nz/natural-disasters/cost-of-natural- disasters/, accessed 12 June 2020). We briefly describe each below, with methodological details given in the supplementary material. We rely on transfer functions between weather and impacts previously defined in the literature to estimate impacts of the causal extreme event, with the assumptions that they are fit for purpose in estimat- ing the relative impact from the causal event, and that the impact is altered only by anthropogenic climate change influencing the causal event (i.e. the impact is not altered by other influences such as effective adaptation or mitigation, or that climate change dir- ectly alters the transfer function). Note that it is not within scope to evaluate the utility of previously defined transfer functions and how their assumptions or characteristics may impact the resulting attribu- tion assessments. For example, our second case study focuses only on publicly funded insurance payouts for residential property damage (see section 2.2.2 and supplementary material), ignoring the much larger private insurance liability for asset damages as well as downstream economic costs. 2.3. Alternative approaches for impacts attribution 2.2.1. UK heat in 2006 and associated mortality The UK summer of 2006 was unusually warm (Prior and Beswick 2007). July 2006 in the UK was the hot- test on record at the time, and a large heatwave cover- ing much of continental Europe and the UK persisted through July. This resulted in large increases in excess temperature-related mortality across the season. In terms of our illustrative attribution assessment, we 2.3. Alternative approaches for impacts attribution If we are asking how climate change has influenced the impacts of a specific extreme event we should not in general be assessing how the frequency of a class of events has changed between counterfactual and factual conditions. Rather, we should be assess- ing how the of a specific impact has changed, between factual and counterfactual conditions. For example, 3 S E Perkins-Kirkpatrick et al Environ. Res. Lett. 17 (2022) 024009 Figure 1. Flow chart outlining steps in performing attribution on the 2006 UK maximum daily mortality and the corresponding daily mean temperature. The process is broken down into three main steps: (1) processing model data; (2) estimating impact; (3) attribution of specific event and class of events. Squares indicate analyses and ovals indicate key data produced. Data from factual model simulations are red; data from counterfactual model simulations are blue; and observed data is orange. General processing is indicated in green (more detail given in the supplementary material), and attribution analysis in purple (sections 2.1 and 2.3). Figure 1. Flow chart outlining steps in performing attribution on the 2006 UK maximum daily mortality and the corresponding daily mean temperature. The process is broken down into three main steps: (1) processing model data; (2) estimating impact; (3) attribution of specific event and class of events. Squares indicate analyses and ovals indicate key data produced. Data from factual model simulations are red; data from counterfactual model simulations are blue; and observed data is orange. General processing is indicated in green (more detail given in the supplementary material), and attribution analysis in purple (sections 2.1 and 2.3). Figure 2. Flow chart outlining steps in performing attribution on the New Zealand North Island highest daily rainfall event (1-in-18 years) and the associated financial damages insured by the EQC (see supplementary material). The process is broken down into three main steps: (1) processing model data; (2) estimating impact; (3) attribution of specific event and class of events. 2.3. Alternative approaches for impacts attribution Bias correction is done according to the Inter-Sectoral Impact Model Intercomparison Project (ISI-MIP) method (Hemple et al 2013). Confidence intervals and the median are computed using the percentile bootstrap method (Paciorek et al 2018), performed 10 000 times. Figure 3. Return periods of the maximum daily heat-related mortality during June and July, for London. The estimated ‘observed’ heat-related mortality during the heatwave is shown by the black dashed line, which corresponds to 60 excess deaths on the 26 July 2006. For the model results, excess mortality is computed using the same method as per the observations (see supplementary material), but estimated from each bias corrected ensemble member taken from the CAM5.1–1 degree climate model (100 members each for the factual and counterfactual simulations). The lower and higher solid red (blue) lines respectively represent the 5th and 95th confidence intervals of the return periods for the factual (counterfactual) simulations with the dashed lines of lighter shade indicating the median of the respective experiment. The dashed grey line is indicative of a 1-in-10 year return period. Bias correction is done according to the Inter-Sectoral Impact Model Intercomparison Project (ISI-MIP) method (Hemple et al 2013). Confidence intervals and the median are computed using the percentile bootstrap method (Paciorek et al 2018), performed 10 000 times. Figure 3. Return periods of the maximum daily heat-related mortality during June and July, for London. The estimated ‘observed’ heat-related mortality during the heatwave is shown by the black dashed line, which corresponds to 60 excess deaths on the 26 July 2006. For the model results, excess mortality is computed using the same method as per the observations (see supplementary material), but estimated from each bias corrected ensemble member taken from the CAM5.1–1 degree climate model (100 members each for the factual and counterfactual simulations). The lower and higher solid red (blue) lines respectively represent the 5th and 95th confidence intervals of the return periods for the factual (counterfactual) simulations with the dashed lines of lighter shade indicating the median of the respective experiment. The dashed grey line is indicative of a 1-in-10 year return period. Bias correction is done according to the Inter-Sectoral Impact Model Intercomparison Project (ISI-MIP) method (Hemple et al 2013). Confidence intervals and the median are computed using the percentile bootstrap method (Paciorek et al 2018), performed 10 000 times. under factual climate conditions (red), compared to counterfactual conditions (blue). 2.3. Alternative approaches for impacts attribution Squares indicate analyses and ovals indicate key data produced. Data from factual model simulations are red; data from counterfactual model simulations are blue; and observed data is orange. General processing is indicated in green (more detail given in the supplementary material), and attribution analysis in purple (sections 2.1 and 2.3). Figure 2. Flow chart outlining steps in performing attribution on the New Zealand North Island highest daily rainfall event (1-in-18 years) and the associated financial damages insured by the EQC (see supplementary material). The process is broken down into three main steps: (1) processing model data; (2) estimating impact; (3) attribution of specific event and class of events. Squares indicate analyses and ovals indicate key data produced. Data from factual model simulations are red; data from counterfactual model simulations are blue; and observed data is orange. General processing is indicated in green (more detail given in the supplementary material), and attribution analysis in purple (sections 2.1 and 2.3). suppose a heatwave-induced mortality total of 200 people has a return period of 50 years under fac- tual conditions. Under counterfactual conditions, the 1-in-50 year heatwave-caused mortality is 100 people. Thus, the influence of anthropogenic climate change on the impact of this specific event has resulted in 100 extra deaths, which in this case is a doubling of the magnitude of the impact. The FAR and/or RR methods would be appropriate if a mortality of at least 200 people stressed a related system. Concluding 4 Environ. Res. Lett. 17 (2022) 024009 S E Perkins-Kirkpatrick et al Figure 3. Return periods of the maximum daily heat-related mortality during June and July, for London. The estimated ‘observed’ heat-related mortality during the heatwave is shown by the black dashed line, which corresponds to 60 excess deaths on the 26 July 2006. For the model results, excess mortality is computed using the same method as per the observations (see supplementary material), but estimated from each bias corrected ensemble member taken from the CAM5.1–1 degree climate model (100 members each for the factual and counterfactual simulations). The lower and higher solid red (blue) lines respectively represent the 5th and 95th confidence intervals of the return periods for the factual (counterfactual) simulations with the dashed lines of lighter shade indicating the median of the respective experiment. The dashed grey line is indicative of a 1-in-10 year return period. 2.3. Alternative approaches for impacts attribution A traditional FAR analysis yields a range of 0.37–0.5. These results describe the anthropogenic signal behind the fre- quency of a heat-related mortality impact that causes at least—not only—60 deaths over a single day dur- ing June–July in London. Additionally, a FAR assess- ment on the corresponding extreme heat has a dif- ferent signal, between 0.46 and 0.67. This means that 46%–67% of days with London temperatures that exceed 26.6 ◦C (i.e. that resulted in the mor- tality total of at least 60) are due to anthropo- genic climate change (see figure S1 (available online at stacks.iop.org/ERL/17/024009/mmedia) in supple- mentary material for corresponding return periods). The attributable change in the meteorological event is greater than that of the impact, highlighting that the relationship between the causal event and the impact can be non-linear. that the frequency of a heat-induced mortality rate of at least 200 deaths has increased can provide use- ful knowledge for adaptation to impacts of extremes, particularly when resources such as public health infrastructure may be put under extreme stress or even collapse when this daily mortality rate occurs and/or is exceeded. However, the relevance of this form of attribution statement is specifically tied to the risk of system stress or failure which may not be rel- evant in many cases where such a threshold cannot be defined. This is not the same attribution information as for the specific impact of 200 deaths. 3.1. 2006 UK heat The grey dashed line represents the return period of the estimated observed EQC payouts (an event of NZ$7.39 M occurs every 1.35 years on average in the factual simulations). Confidence intervals and the median are calculated using percentile bootstrap method (Paciorek et al 2018), performed 10 000 times. Figure 4. Return periods of the EQC payouts on the North Island during daily rainfall events. The estimated ‘observed’ excess financial damages of NZ$7.39 M is shown by the black dashed line, which occurred on the 4 April 2017. For the model results, financial damages were computed using the same method as per Pastor-Paz et al (2020; please see supplementary material), but fitted to each separate factual (455 members) and counterfactual (191 members) simulation across each year (December 2016–November 2017) from the weather@home ensemble. The lower and higher solid red (blue) lines respectively represent the 5th and 95th confidence intervals of the return periods for the factual (counterfactual) simulations with the dashed lines of lighter shade indicating the median of the respective experiment. The grey dashed line represents the return period of the estimated observed EQC payouts (an event of NZ$7.39 M occurs every 1.35 years on average in the factual simulations). Confidence intervals and the median are calculated using percentile bootstrap method (Paciorek et al 2018), performed 10 000 times. about every 1.7–2.3 years in the counterfactual world; and a FAR measure of 0.16–0.41. When concerned about the actual impact, the median figure associ- ated with an event occurring every 1.35 years in the counterfactual simulations is NZ$6.9 M (5th–95th percentile NZ$6.75 M–$7.14 M), compared to the factual median figure of NZ$7.39 M (NZ$7.35 M– $7.49 M). This indicates that NZ$490 000—or 7% (4.6%–8%)—of the cumulative financial impact across the North Island estimated in the factual sim- ulations is attributable to human influence. average return period of a heat-related mortality event of 60 people in the factual experiments is once every 4 years, and in the counterfactual the mortality associated with a 1-in-4 year event is 50 deaths. Thus, anthropogenic climate change has increased the num- ber of deaths associated with a 1-in-4 year event in London by ten deaths, with a 5th–95th percentile range of 1–19 deaths. 3.1. 2006 UK heat Figure 3 shows return periods estimated from model simulations of the highest daily heat-related Jun- e/July mortality in London. The observed mortal- ity on the 26 July 2006 (60 deaths) is indicated by the dashed horizontal line. Figure 3 shows that the daily heat-related mortality total has increased The currently common impact attribution ques- tion concerns the excess deaths attributable to anthro- pogenic climate change. According to figure 3, the 5 Environ. Res. Lett. 17 (2022) 024009 S E Perkins-Kirkpatrick et al Figure 4. Return periods of the EQC payouts on the North Island during daily rainfall events. The estimated ‘observed’ excess financial damages of NZ$7.39 M is shown by the black dashed line, which occurred on the 4 April 2017. For the model results, financial damages were computed using the same method as per Pastor-Paz et al (2020; please see supplementary material), but fitted to each separate factual (455 members) and counterfactual (191 members) simulation across each year (December 2016–November 2017) from the weather@home ensemble. The lower and higher solid red (blue) lines respectively represent the 5th and 95th confidence intervals of the return periods for the factual (counterfactual) simulations with the dashed lines of lighter shade indicating the median of the respective experiment. The grey dashed line represents the return period of the estimated observed EQC payouts (an event of NZ$7.39 M occurs every 1.35 years on average in the factual simulations). Confidence intervals and the median are calculated using percentile bootstrap method (Paciorek et al 2018), performed 10 000 times. Figure 4. Return periods of the EQC payouts on the North Island during daily rainfall events. The estimated ‘observed’ excess financial damages of NZ$7.39 M is shown by the black dashed line, which occurred on the 4 April 2017. For the model results, financial damages were computed using the same method as per Pastor-Paz et al (2020; please see supplementary material), but fitted to each separate factual (455 members) and counterfactual (191 members) simulation across each year (December 2016–November 2017) from the weather@home ensemble. The lower and higher solid red (blue) lines respectively represent the 5th and 95th confidence intervals of the return periods for the factual (counterfactual) simulations with the dashed lines of lighter shade indicating the median of the respective experiment. 3.1. 2006 UK heat the shift in return periods is more considerable— yet variable—for the specific rainfall event com- pared to the corresponding financial damages, ran- ging from 16 to 31 years in the counterfactual world, to 21–66 years under factual conditions (see figure S2 in the supplementary material). The uncertain FAR estimates and considerable overlap of rainfall return periods beyond 5 years is likely a result of sampling uncertainty further in the tail of the distri- bution, especially in the counterfactual simulations (see figure S2). The anthropogenic effect on the financial risk dif- fers strongly from the effect on the associated met- eorological hazard, especially when considering a spe- cific event. This is likely due to a combination of factors, including the spatial extent and intensity of rainfall over inhabited versus non-inhabited regions, and the spatial heterogeneity of the transfer func- tion (see supplementary material). For example, an exceptional amount of rain may have fallen over areas with little residential property, and whilst contribut- ing to the overall extremity of the rainfall event, would not have contributed to the excess financial outcome. This underpins why we strongly recommend that the causal event and the associated impacts be treated separately in terms of estimating the anthropogenic influence. Thirdly, main assumption of our study is that the only path to altering the impact of an extreme event is via anthropogenic climate change influencing the event itself. In fact, there are very likely other influ- ences on some impacts caused by an extreme event, including whether the corresponding transfer func- tion and its relationship to the causal event is affected by climate change, the adoption of mitigation prac- tises that may negate the potential magnitude of the impact (for example, effective public health warn- ings, or insurance companies rising their financial thresholds such that less damage is paid out), or even random factors such as the loss of life during floods being at least partially influenced by the unfortunate situation of being in the wrong place at the wrong time. There are also other impacts that are more fixed and therefore so would transfer function that ties the impact to an extreme event, such as damage to existing structures from events including wildfire, floods and landslides. 3.1. 2006 UK heat To summarise, when interested in an event class question, 37%–50% of deaths are attributable to anthropogenic climate change when the mortality rate is at least 60, but if we are interested in the event itself, 17% (10 deaths out of 60) are attrib- utable to the anthropogenic influence on the climate. The above FAR for the class of rainfall-related EQC payouts is different from the FAR for the class of corresponding daily rainfall, which spans 0.01–0.89. That is, anthropogenic forcing has con- tributed between none and almost 90% to the fre- quency of events at least as intense as the 1-in-18 year daily area-averaged rainfall (see section 2.2.2 and sup- plementary material). The large range indicates that the degree of the anthropogenic signal is uncertain (at least according to the model set-up used). Moreover, 3.2. 2017 New Zealand North Island rainfall event Figure 4 shows return periods of EQC payouts (see supplementary material). The estimated payouts on the 4 April 2017 (NZ$7.39 M, indicated by the dashed horizontal line), does not appear to be rare. We com- pute return periods of payouts worth NZ$7.39 M of about every 1.35 years in the factual world, and 6 Environ. Res. Lett. 17 (2022) 024009 Environ. Res. Lett. 17 (2022) 024009 S E Perkins-Kirkpatrick et al Secondly, it is strongly recommended that any attribution assessment concerning the impacts of a meteorological event be carried out in full, i.e. in addition to the causal meteorological event. As espe- cially demonstrated by the New Zealand rainfall case study, it should not be assumed that the relationship between an extreme event and its impact is linear and that the anthropogenic signal from the extreme event seamlessly transfers to the corresponding impact. The more non-linear the impact response is to the met- eorological event, the more important it is to separate the attribution assessments (Sutton 2019). This non- linearity is likely due to a number of factors, including the inhomogeneous spatial pattern of the meteorolo- gical event. Because impacts do not usually scale lin- early with meteorological hazards, it will in general not be sufficient simply to equate the fractional attrib- utable risk (FAR) as the fractional attributable impact (Mitchell et al 2016, Frame et al 2020a). 4. Discussion Extending weather event attribution to impacts is very new, yet gaining in popularity (e.g. Mitchell et al 2016, Schaller et al 2016, Kay et al 2018, Otto et al 2020, Mitchell 2021, Vicedo-Cabrera et al 2021). Whilst isolating the climate change signal behind impacts caused by weather extremes can be attractive to many working in impacts-related fields, results are relevant only if the methodologies employed suit the specific question being asked. This paper has demon- strated a subtle, yet important issue surrounding the attribution of impacts of extreme weather events to anthropogenic climate change. Via two separate illus- trative case studies we have demonstrated that this issue is non-negligible and applies to multiple types of weather extremes and impact outcomes. 3.1. 2006 UK heat Whilst outside the scope of our study to comprehensively assess this issue, we strongly encourage all authors of impacts attribution assess- ments to critically evaluate how influences on the impact and/or corresponding transfer function may increase or decrease the uncertainty of their results. Indeed, to more fully understand the complex nature of impacts attribution, there is a need to specifically address this issue with targeted, future research. 4.1. Considerations when undertaking attribution assessments on impacts Despite these considerations, one can still estim- ate the anthropogenic signal behind the impacts of a specific extreme event. Indeed, this can and should be done with the same tools (such as extensive fac- tual and counterfactual process-based model simula- tions) that are used in FAR assessments. As demon- strated with figures 3 and 4, impact attribution can be framed as the change in the waiting time of an impact with a specific magnitude, and/or the change in the magnitude of an impact with the same return period across factual and counterfactual physical cli- mate model simulations. First, we once again make clear that the FAR (and associated RR) framework assesses the change sig- nal on a class of events. FAR reflects the change in frequency of an event at least as big as the chosen threshold, due to anthropogenic influence on the cli- mate. When a FAR assessment is undertaken, the calculated FAR/RR values pertain to the exceedance of the threshold defined by the observed event, and not specifically the threshold itself (Harrington 2017). Care should be taken to use FAR only in appropriate circumstances. 7 S E Perkins-Kirkpatrick et al Environ. Res. Lett. 17 (2022) 024009 Acknowledgments ‘How has the waiting time for an impact of a spe- cific magnitude altered under anthropogenic influence on the climate?’ S E P-K is supported by Australian Research Coun- cil Grant Numbers FT170100106 and CE170100023. Y T E Lo was supported by the NERC Grant HAPPI-Health (NE/R009554/1). A D K is suppor- ted by Australian Research Council Grant Number DE180100638. D A S, S M R, and D J F are sup- ported by the Whakahura project, funded through the Endeavour programme of the Ministry of Busi- ness, Innovation, and Employment of Aoteaora New Zealand. D M M acknowledges support from his NERC independent fellowship (NE/N014057/1) and Turing Institute fellowship. MFW is supported by the Director, Office of Science, Office of Biolo- gical and Environmental Research of the US Depart- ment of Energy under Contract No. DE340AC02- 05CH11231. We also acknowledge an anonymous reviewer how provided invaluable guidance in under- standing the underlying assumptions of our analyses. versus a FAR-based question: versus a FAR-based question: versus a FAR-based question: ‘How much of the frequency of a class of impacts is due to anthropogenic emissions’? Such questions need to be defined early and kept constant through the study so that the appropri- ate methods and communication tools are employed (Stone et al 2021). Moreover, it is important to con- sult with relevant stakeholders who may help determ- ine the exact framing of analysis, and understand how the characteristics of impact transfer functions may affect the resulting impact attribution assessment. 5. Conclusions This paper has discussed a subtle yet important meth- odological consideration in attributing the impacts of an extreme weather event to climate change. We achieved this by investigating two separate extreme event case studies that induced different impacts. We highlight that for impacts attribution to be rel- evant, FAR should not necessarily be employed for calculating the anthropogenic climate change signal behind the specific impacts of a specific event. How- ever, FAR can be useful in quantifying the change in risk of a whole class of (usually related) events. We suggest two ways to reframe impacts attribution such that the focus is on a specific event in terms of changes in return period or magnitude. Moreover, any impacts-related attribution—whether on a spe- cific event impact or a class of impacts—should be performed on the impact estimated from the transfer 4.2. Can FAR be useful for attributing impacts to climate change? function, and not assumed to be seamlessly related to the anthropogenic signal of the meteorological event. There is substantial interest in determining the anthropogenic climate change signal behind the impacts caused by extreme weather events. This is an opportunity for the research community, but also brings new methodological questions as we try to grapple with the emergence of extremes that are, in some cases, outpacing our attempts to under- stand their emergence. However, we must ensure that analyses adhere to the purpose of the underpinning method/s employed, as well as considering any data and methodological limitations in both climate sci- ence and the corresponding impact/s sectors. Con- stant and comprehensive interdisciplinary collabor- ation, along with clear and well-thought-out project design and methods, will be essential for the robust- ness of future impacts attribution assessments. There are situations where FAR can be useful for impacts attribution, if we are interested in the signal behind the frequency of a class of impacts, and not in a singular event. This measure could be appropri- ate when concerned about the ‘breaking’ of a system related to a specific impact. For example, heat-related mortality at or above a certain total or rate could throw public health infrastructure into disarray, sim- ilar to what many countries have attempted to avoid during the COVID-19 pandemic. With this framing, FAR is appropriate to determine changes in the fre- quency of this class of impacts due to climate change, such that the corresponding pressure on related sys- tems is quantified. Therefore, appropriate question framing is essen- tial during the initial design of an attribution study (Otto et al 2012). For example, a question on a spe- cific event might be: S E Perkins-Kirkpatrick https://orcid.org/0000- 0001-9443-4915 S E Perkins-Kirkpatrick https://orcid.org/0000- 0001-9443-4915 A D King https://orcid.org/0000-0001-9006-5745 Y T E Lo https://orcid.org/0000-0002-7389-7272 D Frame https://orcid.org/0000-0002-0949-3994 A D King https://orcid.org/0000-0001-9006-5745 Y T E Lo https://orcid.org/0000-0002-7389-7272 D Frame https://orcid.org/0000-0002-0949-3994 A D King https://orcid.org/0000-0001-9006-5745 Y T E Lo https://orcid.org/0000-0002-7389-7272 D F htt // id /0000 0002 0949 3994 Y T E Lo https://orcid.org/0000-0002-7389-7272 D Frame https://orcid.org/0000-0002-0949-3994 D Frame https://orcid.org/0000-0002-0949-3994 Data availability statement ‘How has the magnitude of an impact that occurs every Y years changed under anthropogenic influence on the climate?’ The data that support the findings of this study are available upon reasonable request from the authors. 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https://openalex.org/W2971734191	http://researchrepository.ucd.ie/bitstreams/1528e2ef-01de-45a9-a3e1-21454d90b4a9/download	English	null	Simple and customizable method for fabrication of high-aspect ratio microneedle molds using low-cost 3D printing	Microsystems & nanoengineering	2,019	cc-by	12,119	Title Simple and customizable method for fabrication of high-aspect ratio microneedle molds using low-cost 3D printing Authors(s) Krieger, Kevin, Bertollo, Nicky, Dangol, Manita, Sheridan, John T., Lowery, Madeleine M., O'Cearbhaill, Eoin D. Publication date 2019-09-09 Publication information Krieger, Kevin, Nicky Bertollo, Manita Dangol, John T. Sheridan, Madeleine M. Lowery, and Eoin D. O’Cearbhaill. “Simple and Customizable Method for Fabrication of High-Aspect Ratio Microneedle Molds Using Low-Cost 3D Printing” 5, no. 42 (September 9, 2019). Publisher Springer Item record/more information http://hdl.handle.net/10197/11405 Publisher's statement This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Publisher's version (DOI) 10.1038/S41378-019-0088-8 Title Simple and customizable method for fabrication of high-aspect ratio microneedle molds using low-cost 3D printing Authors(s) Krieger, Kevin, Bertollo, Nicky, Dangol, Manita, Sheridan, John T., Lowery, Madeleine M., O'Cearbhaill, Eoin D. Publication date 2019-09-09 Publication information Krieger, Kevin, Nicky Bertollo, Manita Dangol, John T. Sheridan, Madeleine M. Lowery, and Eoin D. O’Cearbhaill. “Simple and Customizable Method for Fabrication of High-Aspect Ratio Microneedle Molds Using Low-Cost 3D Printing” 5, no. 42 (September 9, 2019). Publisher Springer Item record/more information http://hdl.handle.net/10197/11405 Publisher's statement This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Publisher's version (DOI) 10.1038/S41378-019-0088-8 Abstract We present a simple and customizable microneedle mold fabrication technique using a low-cost desktop SLA 3D printer. As opposed to conventional microneedle fabrication methods, this technique neither requires complex and expensive manufacturing facilities nor expertise in microfabrication. While most low-cost 3D-printed microneedles to date display low aspect ratios and poor tip sharpness, we show that by introducing a two-step “Print & Fill” mold fabrication method, it is possible to obtain high-aspect ratio sharp needles that are capable of penetrating tissue. Studying ﬁrst the effect of varying design input parameters and print settings, it is shown that printed needles are always shorter than speciﬁed. With decreasing input height, needles also begin displaying an increasingly greater than speciﬁed needle base diameter. Both factors contribute to low aspect ratio needles when attempting to print sub- millimeter height needles. By setting input height tall enough, it is possible to print needles with high-aspect ratios and tip radii of 20–40 µm. This tip sharpness is smaller than the speciﬁed printer resolution. Consequently, high-aspect ratio sharp needle arrays are printed in basins which are backﬁlled and cured in a second step, leaving sub-millimeter microneedles exposed resulting microneedle arrays which can be used as male masters. Silicone female master molds are then formed from the fabricated microneedle arrays. Using the molds, both carboxymethyl cellulose loaded with rhodamine B as well as polylactic acid microneedle arrays are produced and their quality examined. A skin insertion study is performed to demonstrate the functional capabilities of arrays made from the fabricated molds. This method can be easily adopted by the microneedle research community for in-house master mold fabrication and parametric optimization of microneedle arrays. The Author(s) 2019 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. A R T I C L E O p e n A c c e s s Simple and customizable method for fabrication of high-aspect ratio microneedle molds using low-cost 3D printing Krieger1,2, Nicky Bertollo1,2, Manita Dangol1,2, John T. Sheridan1,3, Madeleine M. Lowery1,3 and O’Cearbhaill 1,2 Downloaded 2024-10-24 05:18:48 Downloaded 2024-10-24 05:18:48 The UCD community has made this article openly available. Please share how this access benefits you. Your story matters! (@ucd_oa) The UCD community has made this article openly available. Please share how this access benefits you. Your story matters! (@ucd_oa) © Some rights reserved. For more information Microsystems & Nanoengineering www.nature.com/micronano Krieger et al. Microsystems & Nanoengineering (2019) 5:42 https://doi.org/10.1038/s41378-019-0088-8 Introduction transdermal drug delivery (vaccines1, insulin2), chemical biosensing (glucose3, DNA biomarkers4), electrical bio- signal recording (electrocardiography5, surface electro- myography6, intramuscular electromyography7, electroencephalography8), electrical stimulation (electro- tactile display9), and as neural interfaces10. Depending on the requirement, microneedles may be solid11, coated12, hollow13, porous14, hydrogel based15/swellable16, or of a merged-tip geometry17. There are many materials which have been utilized in microneedle research, including silicon, glass, ceramics, metals, solid polymers, as well as swellable hydrogel polymers. Given the vast amount of applications and materials that may be used, cost-effective Microneedles arrays (MNAs) are devices comprised micron-sized needles which allow for transfer of a com- pound or signal across the outer layer of tissue, typically the skin. Owing to their short height, they are considered painless and can be considered a minimally invasive device. Applications of microneedle patches include Krieger et al. Microsystems & Nanoengineering (2019) 5:42 Page 2 of 14 methods for the fabrication of microneedles for research and development purposes are needed. when speciﬁc microneedle parameters, such as micro- needle height, aspect ratio, array size, and needle-to- needle distance may be subject to change, thus requiring buying in of multiple molds which is both cost- and time- prohibitive. One approach for in-house microneedle mold fabrication is replica molding of off-the shelf products, such as acupuncture needles6 or commercial microneedle patches (e.g., Derma Stamp31). Acupuncture needle replica molding, however, can be time-consuming while commercial microneedle patch replica molding limits customizability. Another approach to fabricate low-cost microneedle molds which allows for freedom of design was proposed by Nejad et al.32. Their method enables microneedle female mold fabrication by means of CO2 laser ablation of an acrylic sheet. While this is a sub- tractive manufacturing technique, a promising alternative emerging area within microneedle fabrication is additive manufacturing (AM). There are several important design criteria to consider when designing and fabricating microneedles. Limiting the height of a needle to the sub-millimeter range allows for microneedle insertion to remain largely painless18. Needle height also controls the depth to which a drug/ vaccine may be delivered, or the depth from which a compound/signal is extracted in sensing applications. The aspect ratio of the needle inﬂuences ease of insertion and mechanical integrity. While higher aspect ratio needles are easier to insert, a lower aspect ratio results in mechanically stronger needles. In drug delivery applica- tions, both height and aspect ratio govern individual microneedle volume and therefore control dosage. Introduction The tip radius is also a key parameter for ensuring microneedle skin insertion. Control of these dimensional parameters of microneedles is important, as this allows for tailoring of microneedle functionality. AM can be simple, easily customizable, cost-effective, and produces less waste compared with many subtractive manufacturing techniques and various methods of AM have been employed in the fabrication of microneedle arrays. AM can be classiﬁed into seven general categories according to ISO standards (ISO/ASTM 52900:2015) which are binder jetting, directed energy deposition, material extrusion, material jetting, powder be fusion, sheet lamination, and vat photopolymerization33. Microneedle arrays may be fabricated for direct appli- cation or for use as a master for microneedle mold fab- rication. The latter approach allows for subsequent replica molding of MNAs from a wide range of materials and lends itself well to device optimization in a laboratory setting. Commonly, microelectromechanical systems fabrica- tion techniques are applied in the fabrication of micro- needles, especially for MNA masters for subsequent mold making. Methods used are chemical wet etching19, ultraviolet (UV) lithography20, and deep reactive-ion etching21. Other fabrication techniques include electrical discharge machining22, direct laser micromachining23, and micromilling24. These methods can produce micro- needles with excellent microscale features. However, these methods also require either expensive specialized equipment and/or advanced manufacturing facilities such as clean rooms. Alternative microneedle fabrication methods have been investigated, including drawing lithography (thermal25, electro-26, magnetorheological27, UV28, air blowing29) and centrifugal lithography30. p p y A relatively user-friendly AM technique is material extrusion-based fused ﬁlament fabrication (FFF), also referred to as fused deposition modeling (FDM), which has the advantage that it is affordable and simple-to-use systems are readily available. AM has been used to easily fabricate more complex shaped needles, such as honeybee-inspired microneedles34, angled microneedles35, and arrowhead- shaped microneedles36, which may prove difﬁcult to fabri- cate using conventional fabrication methods. It allows for printing with low-cost biocompatible materials such as polylactic acid (PLA). However, FFF also suffers from poor resolution compared with other 3D printing techniques, and is generally not able to directly produce the sharp tips required for microneedles although a two-step fabrication method has been proposed to overcome the resolution limitations37. Material jetting-based AM in which droplets of a photopolymer are dispensed and light-cured to build the part in a layer-by-layer approach has shown to produce better quality needles38. Introduction Vat photopolymerization-based AM methods in which a liquid photopolymer vat is selec- tively cured using light also display better resolution com- pared with FFF. Certain vat photopolymerization-based methods have been shown to directly print very sharp microscale needles, including two-photon polymeriza- tion39,40 and continuous liquid interface production36. However, these methods are costly, not readily available and Laboratories focused on microneedle research, however, often do not have the facilities in-house, know-how nor desire to invest time to fabricate their own custom microneedle structures. General engineering workshops may not be readily available to them, especially for researchers from outside the ﬁeld of engineering, and therefore these researchers commonly buy in microneedle molds. Replica molding offers the advantage that it is inexpensive, quick, repeatable, simple, up-scalable and usable with a wide range of thermoplastic, thermoset and swellable/hydrogel-forming polymers, as well as ceramics and metals. However, in the research setting, making molds or buying molds is relatively expensive, especially Krieger et al. Microsystems & Nanoengineering (2019) 5:42 Page 3 of 14 Krieger et al. Microsystems & Nanoengineering (2019) 5:42 Choosing to continue with the 25 -µm layer height print setting as it resulted in smoother needles, the parametric study then examined four design parameters. Needles aligned as 1 × 5 arrays with a 1.5 -mm needle-to-needle distance were printed varying the respective design and print parameters. We examined heights ranging from 3 to 0.2 mm for needle aspect ratios of 3:1, 4:1, and 5:1, the results of which can be seen in Fig. 3. therefore not convenient for in-house microneedle manu- facture by most researchers interested in microneedle applications. Various low-cost vat photopolymerization- based AM methods have also been explored for MNA fabrication, including projection-based direct light proces- sing (DLP)41–44 and scanning-based stereolithography (SLA)45,46. In DLP, a digital mirror projects each cross- sectional layer which cures the photopolymer and this process is repeated as the part is built in a layer-by-layer approach. Scanning-based SLA is a technology wherein a part is also printed out of a UV-curable resin. As opposed to DLP, where each layer is projected as whole, in SLA a laser tracks and “draws” each layer, curing the resin as it travels along the x–y plane. Introduction Studies to date using these methods, however, have only been able to fabricate MNAs which exhibit some but not all of three key geometric parameters important for high-quality microneedles which are good tip sharpness, sub-millimeter height and high-aspect ratio. After printing, arrays were imaged and needles with an input height 200 µm and 400 µm deemed of not of good enough quality to warrant measurements. The remaining needle arrays were then parametrically analyzed. Needle height Printing 1 × 5 arrays with aspect ratios of 3:1, 4:1, and 5:1 resulted in needles of shorter height compared with their respective input values. As can be seen in Fig. 3d, e, the relative difference in output height to input height decreased both with diminishing aspect ratio and increased input height. A printed 3:1 aspect ratio needle with input height of 3000 µm was 11% shorter, while a needle with an input height of 600 µm was over 40% shorter than speciﬁed. These relative differences were greater for higher input aspect ratios. For 4:1 aspect ratio needles, the difference was 10% for a 3000 -µm, and 51% for a 600 -µm input height. This further increased for 5:1 aspect ratio needles to 13%, and 57% for a 3000 -µm and a 600 -µm input height, respectively. In this study, we present a novel customizable method for fabrication of microneedle masters in the research setting, which may be used to produce female molds which aims to overcome limitations of previous microneedles produced using low-cost desktop 3D printers which often suffer from low tip radius and/or low aspect ratios. We ﬁrst conducted a parametric study, examining the printer’s capabilities of printing needle structures with sufﬁcient tip sharpness to pierce the skin. To improve feature resolu- tion, we developed a two-step “Print & Fill” technique which allows for the indirect fabrication of customizable microneedle masters for mold making using SLA 3D printing (Fig. 1). Subsequently, microneedle masters and molded microneedle arrays are dimensionally character- ized and functionally assessed for reproducibility. Layer height The printer allows for setting the individual layer height during print to 25, 50, and 100 µm (thickness of each layer in the z-direction). A layer height of 25 µm results in a longer print as the virtual model is divided up into more individual layers to be printed. We printed needles at an aspect ratio of 4:1 to see the effect of layer height on the print quality. The smaller the layer height, the smoother the needle surface becomes as can be seen in Fig. 2. Tip radius Measuring the tip radii, there appeared to be a general trend for the tip radius to decrease with decreasing input needle height. The tip radii lay between 20 and 40 µm as seen in seen in Fig. 3g. Parametric study First, we conducted a study on printing needles using a desktop SLA 3D printer (Form 2, Formlabs Inc., Somer- ville, MA, USA) and investigated how to optimize settings to produce best possible needle geometry. The parametric study investigated the effects of varying print settings and needle design and quantiﬁed print success by measuring four parameters of the printed needles (needle height, needle angle, tip radius, and straightness of the needle). Needle angle For all three aspect ratio needles, θ remained relatively constant until the input needle height was set below 1600 µm as can be seen in Fig. 3f. In that range, the actual θ value for both 3:1 and 4:1 aspect ratio needles was −3% ± 2% of the set value. This value was measured to be −1% ± 8% for 5:1 aspect ratio needles. For lower input heights, this value increased rapidly with decreas- ing input height. For an input height of 600 µm, this value increased to 57, 104, and 153 % for 3:1, 4:1, and 5:1 aspect ratio needles, respectively. The higher the aspect ratio, the greater the deviance from the theoretical angle θ was. Needle straightness The coefﬁcient of determination R2 values of lines ﬁtted to the needle edges were generally >0.95 as can be seen in Fig. 3h. This high degree of linearity is indicative of nee- dles with good needle straightness. Krieger et al. Microsystems & Nanoengineering (2019) 5:42 Page 4 of 14 Design CAD model SLA 3D print model (viii) Demold to obtain female mold a 3D printed needle array basin (i) 3D printed needle array basin (iii) Fill needle basin with UV-curable resin (v) Obtain MNA master (vi) Silicone cast master MNA b Clear resin Grey resin Silicone (ii) Wash, UV cure and bake (iv) UV cure and bake (vii) Degas in vacuum followed by heat cure in oven + + + Fig. 1 Overview of “Print & Fill” fabrication method. a Needle array basin design followed by 3D printing of the design using a Form 2 SLA printer. b MNA master mold fabrication method (i) take 3D printed needle array basin; (ii) washing followed by UV curing and baking of printed needle array basin; (iii) ﬁling of needle array basin with UV-curable resin; (iv) second UV curing and baking; (v) obtain MNA master; (vi) silicone casting of MNA master; (vii) silicone mold is degassed followed by heat cure in oven; (viii) demolding to obtain usable microneedle mold Design CAD model SLA 3D print model a 3D printed needle array basin Design CAD model SLA 3D print model a 3D printed needle array basin (i) 3D printed needle array basin (iii) Fill needle basin with UV-curable resin (v) Obtain MNA master b (ii) Wash, UV cure and bake (iv) UV cure and bake + + Design CAD model SLA 3D print model 3D printed needle array basin (i) 3D printed needle array basin (iii) Fill needle basin with UV-curable resin (v) Obtain MNA master b (ii) Wash, UV cure and bake (iv) UV cure and bake + + 3D printed needle array basin Design CAD model b (i) 3D printed needle array basin (vii) Degas in vacuum followed by heat cure in oven Grey resin Silicone Fig. 1 Overview of “Print & Fill” fabrication method. a Needle array basin design followed by 3D printing of the design using a Form 2 SLA printer. Needle straightness b MNA master mold fabrication method (i) take 3D printed needle array basin; (ii) washing followed by UV curing and baking of printed needle array basin; (iii) ﬁling of needle array basin with UV-curable resin; (iv) second UV curing and baking; (v) obtain MNA master; (vi) silicone casting of MNA master; (vii) silicone mold is degassed followed by heat cure in oven; (viii) demolding to obtain usable microneedle mold Parametric study In the parametric study, it was shown that sharp microneedles of variable design inputs are printable using a low-cost Form 2 SLA 3D printer. We investigated the effect of varying geometry and print settings on the printed needles. Following a preliminary sweep of mate- rials, Clear Resin was found to provide the most con- sistent and best quality microneedle geometries. We then examined the effect of layer height on needle quality. It appears to be possible to print needles of acceptable quality using a 25, 50, as well as 100 -µm layer height. Decreasing the layer height, however, resulted in a markedly improved surface ﬁnish as edges at an angle to the z-axis become smoother. Edge ridge mismatches are a result of an effect called stair-stepping47 inherent to printing in layers and become less pronounced with decreasing layer height. While the tip radius of the 100 -µm layer height needles was about 55 µm and therefore theoretically sharp enough to penetrate the skin, the structural integrity was compromised by the relatively large layer height which resulted in more pronounced edge ridge mismatches and therefore increased local stress concentrations. These edge ridge mismatches may also be an issue in demolding of the MNA during fabri- cation of a silicone mold. The advantages of a greater layer height setting generally are a shorter print time and decreased risk of print failure due a smaller number of individual layers to be cured. As the needles we printed were of short height and therefore consisting of a rela- tively low number of layers, even with a 25 µm layer height setting, both these advantages were negligible. We therefore decided to continue with the parametric study using a 25 µm layer height as this resulted in a smooth needle surface ﬁnish. “Print & Fill” microneedle mold fabrication 500 mg and 600 mg to produce four different types of arrays with needles in the sub-millimeter range (Fig. 4). For 1.5 mm needle-to-needle distance arrays, a ﬁll weight of 500 mg resulted in MNAs with ca. 740 µm needle height (Fig. 4a) while a 600 mg ﬁll weight resulted in needles of ca. 580 µm height (Fig. 4b). For arrays with 1 mm needle-to-needle distance, a 500 mg ﬁll weight resulted in 560 µm (Fig. 4c, e–g) and a 600 mg ﬁll weight Optimized settings from the parametric study were used as input for the design of needle array basins. Basins containing an array of 5 × 5 needles with 2.5 -mm input height, 4:1 aspect ratio, and layer height of 25 µm were printed for needle-to-needle distances of 1 mm and 1.5 mm. Basins of both needle-to-needle distances were then ﬁlled with a photocurable resin with a ﬁll weight of Krieger et al. Microsystems & Nanoengineering (2019) 5:42 Page 5 of 14 100 μm a b c 100 μm 100 μm 50 μm 25 μm 1 mm 1 mm 1 mm Fig. 2 Effect of layer height on print quality. Images of input height 3000 µm and aspect ratio 4:1 needles printed at different layer heights. a Comparison of 10, 50, and 25 -µm layer height needles; SEM images of b 100 -µm; and c 25 -µm layer height needles b Fig. 2 Effect of layer height on print quality. Images of input height 3000 µm and aspect ratio 4:1 needles printed at different layer heights. a Comparison of 10, 50, and 25 -µm layer height needles; SEM images of b 100 -µm; and c 25 -µm layer height needles resulted in 420 µm (Fig. 4d) height needles. Height was measured from the tip to the onset of the meniscus. the millimeter range38 as opposed to the sub-millimeter range required for painless MNA application. In contrast, our MNAs exhibit sharp tips, sub-millimeter height, as well as a high-aspect ratio. p Subsequently, fabricated MNAs were used to make silicone microneedle molds. To test the quality of the fabricated molds, they were used to make MNAs out of two types of polymers as can be seen in Fig. 5. Carboxymethyl cellulose (CMC) MNAs loaded with rhodamine B (Rh B) were fabricated by means of solvent casting (Fig. 5a), whereas PLA MNAs were fabricated using thermal molding (Fig. 5h). Skin insertion study PLA microneedles were fabricated using the newly produced molds (Fig. 6b). A porcine skin insertion study (Fig. 6a) was performed using the PLA MNAs, which showed penetration of the porcine skin as indicated by the trypan blue stains on the needle insertion sites (Fig. 6c). Discussion Miller et al.43 used the 5:1 4:1 3:1 3:1 4:1 5:1 R2 Height c h = 3 .0 mm h = 1.0 mm h = 0.6 mm 1 mm 0.25 mm 0.2 mm 2 mm 3.0 mm 2.8 mm 100 80 60 40 20 0 0 500 100 1500 2000 2500 3000 2.6 mm 2.4 mm 2.2 mm 2.0 mm 1.8 mm 1.6 mm 1.4 mm 1.2 mm 1.0 mm 0.8 mm 0.6 mm 0.4 mm 0.2 mm b a 1 mm Output height/input height (%) Increase in needle aspect ratio (%) Needle tip radius (μm) Input height (μm) 100 1 0.95 0.9 0.85 0.8 80 60 40 20 0 0 500 100 1500 2000 2500 3000 Input height (μm) 100 80 60 40 20 0 0 500 100 1500 2000 2500 3000 Input height (μm) 0 500 100 1500 2000 2500 3000 Input height (μm) Output height/input height Needle tip radius Output θ/input θ R2 value R2 - value d e f g h r Fig. 3 Parametric study of printed needles. Discussion The resolution of our MNAs compares favorably to previously developed MNAs using low-cost AM methods, such as material extrusion-based FFF and vat photopolymerization-based AM methods. FFF, which is a very low-cost method, suffers from poor resolution in the micron range. To overcome these resolution limitations, Luzuriaga et al.37 proposed a two-step fabrication method for printing microneedle arrays, wherein a coarse array is printed in biodegradable PLA which is subsequently etched down to ﬁner features in a potassium hydroxide bath in the second step. This method achieves fabrication of arrays with needle heights of 200–300 μm and a tip sharpness between 1 and 55 μm. However, the actual needle geometry is highly irregular and uneven. Low-cost vat photopolymerization-based methods are therefore preferred when trying to fabricate MNAs. However, attempts to date to directly print needles of sub- millimeter height have generally produced needles with low aspect ratios45,46, while the printed needles with higher aspect ratios and good tip sharpness have been in Krieger et al. Microsystems & Nanoengineering (2019) 5:42 Page 6 of 14 The ﬁrst measured design parameter was the output eedle height, which was seen to be lower than the set put height value. While the printed needles were shorter an speciﬁed in the virtual model, this appeared to be a redictable factor. Printed needles displayed sharp tips, with tip radii smaller than the printer’s minimum feature resolution which is limited by the 140 µm laser spot size in the x–y plane. The height discrepancy issue has also been observed in previous studies on vat photopolymerization- based 3D-printed needles. Discussion a Four parameters which were investigated: needle height, needle angle θ, tip radius, and needle straightness as determined by the R2 value; b needles with input height of 3 mm and input aspect ratio of 3:1, 4:1, and 5:1 (left to right); c input 4:1 aspect ratio needles of input height 3, 1, and 0.6 mm with the theoretical dimensional of the needle outlined; d input 4:1 aspect ratio needles with heights ranging from 3 to 0.2 mm (marked by an “x” is the theoretical input height); e output-to-input height ratio; f output-to-input needle angle θ atio; g needle tip radius; h R2 value; (n = 5, results displayed as mean ± standard deviation) 3:1 4:1 5:1 R2 Height c h = 3 .0 mm h = 1.0 mm h = 0.6 mm 1 mm 0.25 mm 0.2 mm 2 mm 3.0 mm 2.8 mm 2.6 mm 2.4 mm 2.2 mm 2.0 mm 1.8 mm 1.6 mm 1.4 mm 1.2 mm 1.0 mm 0.8 mm 0.6 mm 0.4 mm 0.2 mm b a 1 mm d r 3:1 4:1 5:1 R2 Height c h = 3 .0 mm h = 1.0 mm h = 0.6 mm 1 mm 0.25 mm 0.2 mm b a 1 mm r R2 Height a r c d 2 mm 3.0 mm 2.8 mm 2.6 mm 2.4 mm 2.2 mm 2.0 mm 1.8 mm 1.6 mm 1.4 mm 1.2 mm 1.0 mm 0.8 mm 0.6 mm 0.4 mm 0.2 mm d Increase in needle aspect ratio (%) 100 80 60 40 20 0 0 500 100 1500 2000 2500 3000 Input height (μm) Output θ/input θ f f 100 80 60 40 20 0 0 500 100 1500 2000 2500 3000 Output height/input height (%) Input height (μm) Output height/input height e Input height (μm) 1 0.95 0.9 0.85 0.8 0 500 100 1500 2000 2500 3000 Input height (μm) R2 value R2 - value h Needle tip radius (μm) 100 80 60 40 20 0 0 500 100 1500 2000 2500 3000 Input height (μm) Needle tip radius g h g Fig. 3 Parametric study of printed needles. Discussion a Four parameters which were investigated: needle height, needle angle θ, tip radius, and needle straightness as determined by the R2 value; b needles with input height of 3 mm and input aspect ratio of 3:1, 4:1, and 5:1 (left to right); c input 4:1 aspect ratio needles of input height 3, 1, and 0.6 mm with the theoretical dimensional of the needle outlined; d input 4:1 aspect ratio needles with heights ranging from 3 to 0.2 mm (marked by an “x” is the theoretical input height); e output-to-input height ratio; f output-to-input needle angle θ ratio; g needle tip radius; h R2 value; (n = 5, results displayed as mean ± standard deviation) The ﬁrst measured design parameter was the output needle height, which was seen to be lower than the set input height value. While the printed needles were shorter than speciﬁed in the virtual model, this appeared to be a predictable factor. Printed needles displayed sharp tips, with tip radii smaller than the printer’s minimum feature resolution which is limited by the 140 µm laser spot size in the x–y plane. The height discrepancy issue has also been observed in previous studies on vat photopolymerization- based 3D-printed needles. Miller et al.43 used the Krieger et al. Microsystems & Nanoengineering (2019) 5:42 Page 7 of 14 a b h i j k e 200 μm 1 mm f g 1 mm 1 mm c d 1 mm 1 mm a b 1 mm 1 mm Filled area Exposed needle tips Printed base 1 mm 1 mm 1 mm Fig. 4 Printed & ﬁlled master MNAs (input height: 2.5 mm, aspect ratio: 4:1). Discussion a CAD drawing and SEM image of 1.5 mm needle-to-needle distance MNA basin before ﬁlling; b sketch and SEM of laser-cut cross-section of a ﬁlled MNA basin with 1.5 mm needle-to-needle distance and a ﬁll weight of 600 mg; c, d 1.5 mm needle-to-needle distance after ﬁlling and curing with a ﬁll weight of 500 mg (c) and 600 mg (d), respectively; e optical image; and f, g SEM images of printed and ﬁlled MNA (needle-to-needle distance: 1 mm, ﬁll weight: 500 mg); h, i side view of 1.5 mm needle-to- needle distance MNA after ﬁlling and curing with a ﬁll weight of 500 mg (h) and 600 mg (i) respectively; j, k side view of 1.5 mm needle-to-needle distance MNA after ﬁlling and curing with a ﬁll weight of 500 mg (j) and 600 mg (k) respectively 1 mm 1 mm Filled area Exposed needle tips Printed base c 1 mm d 1 mm c d h i j k e 200 μm 1 mm f g 1 mm 1 mm 1 mm 1 mm 1 mm g. 4 Printed & ﬁlled master MNAs (input height: 2.5 mm, aspect ratio: 4:1). a CAD drawing and SEM image of 1.5 mm needle-to-needle tance MNA basin before ﬁlling; b sketch and SEM of laser-cut cross-section of a ﬁlled MNA basin with 1.5 mm needle-to-needle distance and a ﬁll eight of 600 mg; c, d 1.5 mm needle-to-needle distance after ﬁlling and curing with a ﬁll weight of 500 mg (c) and 600 mg (d), respectively; e optical age; and f, g SEM images of printed and ﬁlled MNA (needle-to-needle distance: 1 mm, ﬁll weight: 500 mg); h, i side view of 1.5 mm needle-to- edle distance MNA after ﬁlling and curing with a ﬁll weight of 500 mg (h) and 600 mg (i) respectively; j, k side view of 1.5 mm needle-to-needle 200 μm g e 1 mm g f 1 mm e f f i k 1 mm 1 mm h i j k 1 mm 1 mm 1 mm 1 mm i h j Fig. 4 Printed & ﬁlled master MNAs (input height: 2.5 mm, aspect ratio: 4:1). Discussion a Schematic of fabrication steps of CMC MNAs loaded with Rh B: (i) ﬁll mold with CMC-Rh B solution, (ii) place molds in vacuum chamber to remove air bubbles and ﬁll voids, (iii) dry MNA, (iv) ﬁnal CMC MNA loaded with Rh B; b–d SEM images of CMC-Rh B MNAs; e optical image of CMC-Rh B MNA; b optical image of an individual Rh B-loaded CMC microneedle; c optical ﬂuorescence image of a Rh B- loaded CMC microneedle; h PLA microneedle casting using mold: (1) mold ﬁlling with PLA pellets, (2) placing of mold with pellets in vacuum oven, (3) demolding to obtain PLA MNA; i replica molded PLA MNA from a master MNA with a needle-to-needle distance of 1 mm and a ﬁll weight of 500 mg; j replica molded PLA MNA from a master MNA with a needle-to-needle distance of 1.5 mm and a ﬁll weight of 500 mg a (i) 200 μm 1 mm c d (ii) (iii) (iv) a (iii) (i) (ii) b d 1 mm c 2 mm b c 2 mm e f 50 μm f e 50 μm g g h h (i) 180 °C (ii) + (iii) (i) (iii) (ii) i i 1 mm j 2 mm j Fig. 5 Microneedle fabrication examples. a Schematic of fabrication steps of CMC MNAs loaded with Rh B: (i) ﬁll mold with CMC-Rh B solution, (ii) place molds in vacuum chamber to remove air bubbles and ﬁll voids, (iii) dry MNA, (iv) ﬁnal CMC MNA loaded with Rh B; b–d SEM images of CMC-Rh B MNAs; e optical image of CMC-Rh B MNA; b optical image of an individual Rh B-loaded CMC microneedle; c optical ﬂuorescence image of a Rh B- loaded CMC microneedle; h PLA microneedle casting using mold: (1) mold ﬁlling with PLA pellets, (2) placing of mold with pellets in vacuum oven, (3) demolding to obtain PLA MNA; i replica molded PLA MNA from a master MNA with a needle-to-needle distance of 1 mm and a ﬁll weight of 500 mg; j replica molded PLA MNA from a master MNA with a needle-to-needle distance of 1.5 mm and a ﬁll weight of 500 mg image plane48. The intensity of the 405 nm wavelength follows a Gaussian proﬁle, reaching its maximum near the optical axis and falling off laterally49. Discussion a CAD drawing and SEM image of 1.5 mm needle-to-needle distance MNA basin before ﬁlling; b sketch and SEM of laser-cut cross-section of a ﬁlled MNA basin with 1.5 mm needle-to-needle distance and a ﬁll weight of 600 mg; c, d 1.5 mm needle-to-needle distance after ﬁlling and curing with a ﬁll weight of 500 mg (c) and 600 mg (d), respectively; e optical image; and f, g SEM images of printed and ﬁlled MNA (needle-to-needle distance: 1 mm, ﬁll weight: 500 mg); h, i side view of 1.5 mm needle-to- needle distance MNA after ﬁlling and curing with a ﬁll weight of 500 mg (h) and 600 mg (i) respectively; j, k side view of 1.5 mm needle-to-needle distance MNA after ﬁlling and curing with a ﬁll weight of 500 mg (j) and 600 mg (k) respectively Perfactory III SXGA+instrument system (EnvisionTEC GmbH, Gladbeck, Germany) to print hollow micro- needles. The microneedle input dimensions were a tri- angular base of 1.2 mm edge lengths and a height of 1.5 mm with a 400 μm hollow channel running through it. Measurements obtained showed the printed parts had a base length of 1120 μm, a microneedle height of 1030 μm, and a hollow channel diameter of 375 μm. Differences between dimensions were attributed to the tessellation process by the software which converts the CAD design ﬁle to a printable model. After importing the STL ﬁle into the print preparation software (PreForm), the print model Krieger et al. Microsystems & Nanoengineering (2019) 5:42 Page 8 of 14 2 mm 200 μm 1 mm 50 μm 50 μm 2 mm 180 °C b c d e f g i 1 mm 2 mm a h j (i) (ii) (iii) (iv) (i) (ii) (iii) + Fig. 5 Microneedle fabrication examples. Discussion a Schematic representation of skin insertion test: (i) MNA insertion into the skin applying a force of 30 N for 60 s, (ii) MNA removal, (iii) trypan blue staining left for 120 s, (iv) washing off trypan blue with 0.9% saline solution, (v) tape-stripping of the stratum corneum from the skin, (vi) inspection of the skin sample for trypan blue stains; b replica molded PLA MNA (mold master: 1.5 mm needle-to-needle distance and 600 mg ﬁll weight); c porcine skin sample after MNA insertion and trypan blue staining sectional slice, less overlapping occurs resulting in an overall lesser UV dose for a given point. Due to the hatch pattern, areas close to the center of the slice would not be as affected as areas further away which would begin not receiving the critical UV dose required for polymerization. This process would continue gradually as the cross- sectional area further decreases resulting in round-shaped tips despite the print “failing” before reaching full needle input height. This would also explain why higher aspect ratio needles, which for a given layer will have a lower cross-sectional surface area in the x–y plane than a needle with a lower aspect ratio, appear to display a slightly increased input-to-output height discrepancy as seen in Fig. 3e. The Form 2 is a closed source system, and we do not know what the exact print instructions and para- meters are. Generally, stereolithography is a complex process with over 50 process variables for a resin and part family type50, and many of these interacting variables will affect the print outcome of the needle. relative increase of height discrepancy, resulted in needles with low aspect ratios. This can be seen in Fig. 3c, where the smaller needles do not ﬁt in their theoretical 2D outline as indicated by the overdrawn shape. The relative increase grew rapidly as the input height decreased. This observation is most likely related to the minimum feature size in the x–y plane and the limitations of a 140 -µm laser spot size. The smaller the height for a given aspect ratio, the smaller the base diameter. The base diameter, how- ever, will have a minimum value due to the limitations of the printer’s resolution. This would explain the increase in output to input ratio for θ as the needle height decreases. Discussion A layer is typically built by a laser tracing the outline of the cross-sectional slice and cross-hatching the space in-between47. The distance between the laser traces while hatching is known as hatch spacing and controls the overlap. The closer the hatch spacing, the more overlap exists ensuring a more complete cure. A vertically aligned conical needle during print can be considered a continuously decreasing cross- sectional slice in the x–y plane and there appears to be a point during printing at which the part stops growing in height. It may be that due to decreasing size of the cross- can be seen to comprise a number of layers equal to dividing its theoretical height by the print layer height setting, e.g., a 3 mm height needle model set to be printed at a 25 µm layer height is divided into 120 layers. It therefore does not appear that tessellation alone can explain the height discrepancy in our case, which is quite substantial. The height discrepancy may be related to the minimum UV dose required for photopolymerization and the way a layer is drawn. UV dose depends on the UV intensity and exposure time with photopolymerization only occurring once a critical UV dose is reached. Light such as UV is diffractive and instead of focusing on a single point will smear out into a blurred spot at the Krieger et al. Microsystems & Nanoengineering (2019) 5:42 Page 9 of 14 Trypan Blue PLA MNA Dermis F Tape Viable epidermis Stratum corneum a b c (i) (ii) (iii) (iv) (v) (vi) 2 mm 2 mm Fig. 6 Skin insertion study. a Schematic representation of skin insertion test: (i) MNA insertion into the skin applying a force of 30 N for 60 s, (ii) MNA removal, (iii) trypan blue staining left for 120 s, (iv) washing off trypan blue with 0.9% saline solution, (v) tape-stripping of the stratum corneum from the skin, (vi) inspection of the skin sample for trypan blue stains; b replica molded PLA MNA (mold master: 1.5 mm needle-to-needle distance and 600 mg ﬁll weight); c porcine skin sample after MNA insertion and trypan blue staining F a (i) (ii) (ii) b (iii) 2 mm 2 mm b a (ii) (iii) (ii) (i) Dermis Viable epidermis (vi) (vi) c 2 mm c Trypan Blue PLA MNA Tape Stratum corneum (iv) (v) (v) (v) Fig. 6 Skin insertion study. Discussion Unlike the gradually decreasing cross-sectional area of the needle, the initial layer of the needle base is a more abrupt appearing feature and thus subject to conventional reso- lution limitations in the x–y plane. The tip radii of the printed needles are shown to lie between 20 and 40 µm. There appears to be a trend of decreasing tip radius with decreasing needle height. How- ever, taking measurement accuracy and error into account, this difference is deemed not signiﬁcant. As the needle tips are a result of print “failure” and thus lack of precise con- trol, they are especially subject to needle-to-needle varia- bility. Nevertheless, 20–40 µm tip radii are sufﬁciently sharp to penetrate the skin. Boehm et al.42 used the Per- factory III SXGA+ visible light dynamic mask micro- stereolithography system (EnvisionTEC GmbH, Gladbeck, Germany) to print 1 × 5 arrays of needles which looked to have similar tip sharpness but greater step-size features on the surface compared with the needles we printed. The tip radii we obtained are therefore among the sharpest needles printed using lower-cost printing systems. A second design parameter measured was the angle θ. It is proportional to the aspect ratio for a needle with inﬁnite tip sharpness. This value quantiﬁes the angle between needle sides irrespective of the tip radius. The actual aspect ratio of a real needle will be lower with respect to that of a perfect needle (i.e., a needle of inﬁnite tip sharpness) due to its inherently decreased height resulting from tip rounding. This parameter was relatively pro- portionate to the input value for needles of greater input height. As needle input height decreased below a value of about 1600 µm, we observed a greater increase of angle θ relative to its theoretical value, which indicated a greater than speciﬁed needle base diameter, which along with the Page 10 of 14 Page 10 of 14 Krieger et al. Microsystems & Nanoengineering (2019) 5:42 Krieger et al. Microsystems & Nanoengineering (2019) 5:42 investigated, resulting in needles of varying and con- trollable heights. Needle array basins with a smaller needle-to-needle distance, resulted in a shorter needle height for a given ﬁll weight compared with basins with a greater needle-to-needle distance which is an effect of resin surface tension. Microneedle array fabrication The parametric study showed that needles of good tip sharpness were directly printable using the Form 2 sys- tem. However, for needles not to inﬂict pain in novel applications, their height should be limited to the sub- millimeter range18, and display an adequate aspect ratio to ease insertion. A low aspect ratio may add to mechanical strength of the needle, but can also negatively affect insertion due to its rapidly increasing needle shaft width51. In addition, low aspect ratio needles can display poor tip sharpness related to limitations associated with fabrica- tion. Microneedle arrays in previous studies, which were directly printed using the same Form 2 system, did not appear to comprise of features that facilitate ease of insertion. Pere et al.45 printed conical and pyramidal needles of 1000 μm input height at an input aspect ratio of 1:1. While no exact dimensions of the printed parts were given, the needles displayed a low aspect ratio and did not appear to be as sharp as the needles we fabricated. Using the same printer, Farias et al.46 printed hollow micro- needles with a 600 μm height, a 1000 μm base diameter and a tip diameter of 400 μm. The printed needles were shown to have a very low aspect ratio and poor tip sharpness. In order to overcome these issues and be able to fabricate microneedles with a low and controllable height as well as a high-aspect ratio for use as masters in mold making, a two-step fabrication approach was developed. First basins were printed containing a 5 × 5 array of needles with parameters which displayed good results in the parametric study. We chose a “pre-ﬁll” input height of 2.5 mm and an aspect ratio of 4:1, but other values may be chosen as desired. These basins were subsequently ﬁlled with UV-curable resin so that sub- millimeter height needles were left exposed. Due to sur- face tension, the ﬁlled needle array basins exhibited a concave meniscus. The inner basin diameter was set to 24 mm, larger than necessary to ﬁt a 5 × 5 needle array of the given needle-to-needle distances used here. This was done to reduce the effect of surface tension during ﬁlling. A pre-heat temperature of 80 °C before UV curing was chosen to decrease resin viscosity before the onset of curing. This method yielded MNAs of low sub-millimeter height as can be seen in Fig. 4. Discussion As microneedles are mainly devel- oped for application on human/mammalian tissue which itself is quite compliant, it is not expected that a meniscus will greatly affect microneedle insertion performance and may improve mechanical performance under shear load- ing. The developed method allows for easy fabrication of MNAs with controllable needle-to-needle distance and height. Silicone molds made from these master arrays were used to fabricate CMC MNAs loaded with a ﬂuor- escent model drug (Rh B), as well as PLA MNAs. The solvent-cast CMC-Rh B MNAs comprised of very sharp needles (tip radius < 5 μm), as well as displaying a high- aspect ratio (Fig. 5b–f). Fluorescence imaging showed that loading these needles with the Rh B drug model resulted in good drug distribution throughout the MNA and the microneedle tips themselves. The PLA MNAs were not as sharp as the CMC-Rh B MNAs as they were thermally molded and thus are more accurate geometric replicas of the actual MNA master used to make the mold. However, they still displayed good tip sharpness. To investigate whether or not they have sufﬁcient sharpness to penetrate the skin, they were applied in a skin insertion test using porcine ear tissue with subsequent trypan blue staining and tape stripping52. The reason for tape stripping is to fully remove the stratum corneum before examining the skin for blue stains indicative of a successful piercing event. If the stratum corneum is not removed, a blue stain dot might be the result of dye residue remaining in a local depression of the skin after microneedle force application which did not actually result in a piercing event. The results showed that fabricated PLA MNAs had a tip sharpness which was capable of piercing porcine skin tissue. The straightness of the needle was also quantiﬁed by extracting the needle outline from the images, converting it to a set of data points, and subsequent calculating of the coefﬁcient of determination (R2) for the data points for each needle side. A straighter needle will have a R2 value closer to 1. Calculations found this value to lie in the range of 0.95–0.99 for all needles indicating good straightness of the print. Microneedle array fabrication Two ﬁll heights (controlled by respective ﬁll weights of 500 and 600 mg) and two needle needle-to-needle distances (1 and 1.5 mm) were The application of 3D printing in the fabrication of MNA master molds is a simple method which can be adapted by researchers with only basic skills in CAD design required. To date, a wide range of fabrication processes methods have been employed in the production MNA masters, including microelectromechanical systems fabrication and micromachining. MNA masters, such as those made from metal, display good wear properties, enabling long use of the same master. Although use of conventional techniques can be cost-effective and attractive for fabrication of MNA masters, they are often reliant on specialized microfabrication equipment (e.g., milling machines), as well as expertise or access to a person with expertise in using the equipment which may be a limiting factor. Our proposed method offers a viable alternative to previously developed methods. Advantages Page 11 of 14 Page 11 of 14 Krieger et al. Microsystems & Nanoengineering (2019) 5:42 include the ability to design and fabricate multiple MNA master designs in a short time at a relatively low cost. As desktop 3D printers are suitable for use in the lab, MNA masters may be fabricated in-lab without the need to access a dedicated machining workshop. Larger-scale MNA masters may be produced, the size being limited by the 3D printer’s build area (5 × 5 arrays were used for demonstration purpose in this study). The method is advantageous over other simple methods of MNA master fabrication such as binding of individual needles6, which becomes more laborious as patch size and needle density increase and height control may be more challenging than simple ﬁlling as we propose here. release of drugs. Therefore, this is a simple and promising method which can be adapted for further development of microneedle-based drug delivery systems. g y y While additive manufacturing generally allows for fab- rication of more complex geometries not possible with conventional subtractive manufacturing methods, we deliberately chose to conical-shaped straight needles as our design of choice for several reasons. Conical-shaped needles are extensively used in microneedle research, and are often the preferred design and therefore offering a simple low-cost manufacturing method for that geometry is of great beneﬁt to the researcher. Microneedle array fabrication Microneedles with more complex features, such as barbs and overhangs, may offer advantages but may cause issues in replica molding, especially during the demolding step, affecting their suitability for this commonly used fabrication method. Lastly, the resolution of the Form 2 printer poses a limit on more complex microneedle structures and fabricating good quality conical microneedles with sharp tips in itself is challenging (see Supplementary Figs. 1, 2). Desktop SLA 3D printers such as the Form 2 are becoming ubiquitous to the research environment as they allow for rapid pro- totyping, e.g., printing required parts for experimental rigs. Therefore, these printers may already be available to labs focused on microneedle research, aiding in the adaption of this fabrication technique. Several factors contribute to successful printing of MNAs, including cleanliness of the printer, and previous usage of the resin tank. The methods outlined here could also facilitate the optimization of direct fabrication of MNAs from bio- compatible photocurable polymers, which are becoming more readily available. Overall this method allows great customizability of MNAs. By changing the design ﬁle to be printed, the needle-to-needle distance, overall array size and needle arrangement can be easily altered. There is a discrepancy in actual printed height compared with intended height. This however is not a great issue as one may accurately predict and account for this height loss. The actual ﬁnal microneedle height is not governed by the printed height, but by the exposed tip height after ﬁlling. As this method is quick and simple, researchers may use this print and ﬁll method to fabricate MNAs with varying parameters (e.g., needle arrangement, quantity, density, and height) and ﬁnd one that best ﬁts their requirements. When the optimum settings have been found, expensive custom molds with these selected parameters may then be bought which are compatible with high volume manufacturing methods such as microinjection molding. The developed technique allows for fabrication of MNA molds for a variety of applications, including the ﬁeld of drug delivery which was demonstrated in this study by fabrication of a MNA loaded with a drug model. The drug loading capacity of an individual microneedle is governed by its surface area/volume which in turn is depended on the mold’s dimensional parameters. Therefore, ease of parametric control of molds is advantageous in examining MNA drug loading capacity. Microneedle array fabrication In addition, the patch density (number of microneedles per patch) may also be altered which will consequently regulate the total dosage that can be delivered by an MNA32. Our method allows for control of parameters by altering the CAD design and in the second step, the ﬁll volume. The drug release proﬁle of microneedles produced from molds fabricated using the proposed method will depend on the physicochemical properties of the chosen polymer. As the release of drugs from an MNA is dependent on the degradation proﬁle of the used polymer and drug diffusion rate from the microneedles, polymers with appropriate molecular weight and degradability can be chosen to control the release of the drug52. Given the wide range of polymers that may be used to fabricate MNAs, this technology will be further explored for rapid bolus release and sustained Conclusion We have developed a low-cost, simple, and customiz- able method for the fabrication of microneedle molds by means of a commonly used, commercially available and affordable SLA 3D printer. A parametric study demon- strated that printing needles with good tip sharpness beyond the printer’s deﬁned resolution limits is possible if the correct print settings are chosen. Printed needle parameters differed from those in the virtual CAD model. Compared with the model, the printed needles were found to be shorter and have a greater than speciﬁed aspect ratio. Taking these ﬁndings into account, a two- step “Print & Fill” fabrication method was developed with which we were able to rapidly produce microneedle master molds with easily customizable needle parameters. The fabrication method presented is suitable to be directly applied by microneedle researchers in-lab regardless of microfabrication expertise or access to workshops with specialized equipment and allows for production of microneedle arrays with parameters tailored toward their speciﬁc requirements. Page 12 of 14 Page 12 of 14 Page 12 of 14 Krieger et al. Microsystems & Nanoengineering (2019) 5:42 “Print & Fill” microneedle mold fabrication Needle array basin design Post-print treatment The 45 different 1 × 5 array CAD design ﬁles were exported to STL ﬁle format and imported into a print preparation software (PreForm, Formlabs Inc.). The parts were orientated so that the center axis of the needles aligned with the z-axis of the printer. The print layer height in the z-direction was set to 25 µm for all designs. To investigate the effect of print layer height, needles with an aspect ratio 4:1 and heights ranging from 3.0 to 2.0 mm were printed in addition to the previous set of arrays. After printing, a needle array basin was washed in an IPA bath for 20 min (Form Wash, Formlabs Inc.). During washing, the IPA was agitated using a magnetically cou- pled impeller. After washing, the basin was post-cured for an hour by means of UV LEDs (λ = 405 nm) at a tem- perature of 60 °C (Form Cure, Formlabs Inc.). 3D printing The CAD model of the needle array basins were exported to STL ﬁle format and imported to a print preparation software (PreForm, Formlabs Inc.). A needle array basin was orientated so that the center axis of the needles aligned with the z-axis of the printer. The print layer height in the z-direction was set to 25 µm, and support structures were added to the bottom of the basin. The model with associated print settings was then uploaded to the printer. Post-print treatment After printing, the needle arrays were washed in an isopropanol (IPA) bath for 20 min (Form Wash, Formlabs Inc.). During washing, the IPA was agitated using a magnetically coupled impeller. After washing, the needles were post-cured for an hour by means of UV LEDs (λ = 405 nm) at a temperature of 60 °C (Form Cure, Formlabs Inc.). Microneedle replica molding A needle array basin, which now had exposed micro- needles and therefore could be considered an MNA, was dip-coated in a liquid agent (Inhibit X, Mann Formulated Products, Inc., Macungie, PA, USA) which protects against cure inhibition when pouring platinum-cure sili- cones. A mold release (Ease Release 200, Mann For- mulated Products, Inc.) was sprayed onto the MNA. A mold of the MNA was then made using a platinum-cure silicone (PlatSil 7315, Mouldlife, Bury St Edmunds, Suf- folk, UK). After demolding, a silicone microneedle mold was obtained. “Print & Fill” microneedle mold fabrication Needle array basin design All needle arrays were printed from a UV-curable resin (Clear Resin V4, Formlabs Inc., Somerville, MA, USA) using a desktop SLA 3D printer (Form 2, Formlabs Inc.). The printer employs an inverted stereolithographic pro- cess wherein the print is built from bottom up, the laser being directed from below the resin tank and the build head rising an increment after each cured layer. The build volume of the printer is 145 × 145 × 175 mm. The 250 mW laser has a wavelength of 405 nm and a spot size of 140 µm. Depending on material, the printer can provide a theoretical layer thickness as small as 25 µm in the z- direction. A 3D model for a 5 × 5 needle array basin was developed using CAD software (Autodesk Inventor Professional 2017, Autodesk Inc., San Rafael, CA, USA). The needles were designed to be conical in shape, having a height of 2.5 mm with a 625 µm base diameter, resulting in a height aspect ratio of 4:1. The array was situated within a basin (24 mm inner diameter) with a wall thickness and height of both 1 mm. Two types of needle array basins were designed, one with a 1 mm and another with a 1.5 mm needle-to-needle distance. Basin ﬁlling In the next step, a UV-curable resin (Grey Resin V4, Formlabs Inc.), used in this study for contrast as opposed to clear resin, was pipetted into the basin leaving only a sub-millimeter height needle tip exposed. The basins were ﬁlled with both 500 and 600 mg of resin to obtain dif- ferent needle heights. The ﬁlled basins were then UV cured (λ = 405 nm). Parametric study CAD design All designs were 1 × 5 arrays of conically shaped needles with a 1.5 mm needle-to-needle distance. Needle arrays were designed for three different aspect ratios (3:1, 4:1, and 5:1) and for each respective aspect ratio arrays with varying needle height were printed (3.0, 2.8, 2.6, 2.4, 2.2, 2.0, 1.8, 1.6, 1.4, 1.2, 1.0, 0.8, 0.6, 0.4, and 0.2 mm). References 9. Tezuka, M., Ishimaru, K. & Miki, N. Electrotactile display composed of two- dimensionally and densely distributed microneedle electrodes. Sens. Actuators A Phys. 258, 32–38 (2017). 9. Tezuka, M., Ishimaru, K. & Miki, N. Electrotactile display composed of two- dimensionally and densely distributed microneedle electrodes. Sens. Actuators A Phys. 258, 32–38 (2017). 10. Xiang, Z., Liu, J. & Lee, C. A ﬂexible three-dimensional electrode mesh: an enabling technology for wireless brain–computer interface prostheses. Microsyst. Amp; Nanoeng. 2, 16012 (2016). 10. Xiang, Z., Liu, J. & Lee, C. A ﬂexible three-dimensional electrode mesh: an enabling technology for wireless brain–computer interface prostheses. Microsyst. Amp; Nanoeng. 2, 16012 (2016). Conﬂict of interest The authors declare that they have no conﬂict of interest. Skin insertion testing 11. Kaur, M., Ita, K. B., Popova, I. E., Parikh, S. J. & Bair, D. A. Microneedle-assisted delivery of verapamil hydrochloride and amlodipine besylate. Eur. J. Pharm. Biopharm. 86, 284–291 (2014). 11. Kaur, M., Ita, K. B., Popova, I. E., Parikh, S. J. & Bair, D. A. Microneedle-assisted delivery of verapamil hydrochloride and amlodipine besylate. Eur. J. Pharm. Biopharm. 86, 284–291 (2014). A fabricated PLA MNA was inserted into porcine ear tissue (35-day-old Large-White and Landrace hybrid sow with MAXGRO semen) at a force of 30 N for 60 s using a handheld force gauge (Sauter FK100, Sauter GmbH, Balingen, Germany). A solution of 0.4 wt% trypan blue (Sigma-Aldrich, St. Louis, MO, USA) was applied on the insertion area, left for 120 s, and then washed off with 0.9% saline solution (Lennox, Dublin, Ireland). Tape stripping using a generic commercial sellotape was performed to remove the stratum corneum after which the skin was inspected for successful insertion of microneedles. 12. Gill, H. S., Söderholm, J., Prausnitz, M. R. & Sällberg, M. Cutaneous vaccination using microneedles coated with hepatitis C DNA vaccine. Gene Ther. 17, 811 (2010). 13. Wang, P. M., Cornwell, M., Hill, J. & Prausnitz, M. R. Precise microinjection into skin using hollow microneedles. J. Invest. Dermatol. 126, 1080–1087 (2006). 14. Cahill, E. M. et al. Metallic microneedles with interconnected porosity: a scalable platform for biosensing and drug delivery. Acta Biomater. 80, 401–411 (2018). p g g y 15. Donnelly, R. F. et al. Hydrogel‐forming microneedle arrays for enhanced transdermal drug delivery. Adv. Funct. Mater. 22, 4879–4890 (2012). 16. Yang, S. Y. et al. A Bio-inspired swellable microneedle adhesive for mechanical interlocking with tissue. Nat. Commun. 4, 1702 (2013). 17. Lim, J., Tahk, D., Yu, J., Min, D.-H. & Jeon, N. L. Design rules for a tunable merged-tip microneedle. Microsyst. Nanoeng. 4, 29 (2018). 18. Gill, H. S., Denson, D. D., Burris, B. A. & Prausnitz, M. R. Effect of microneedle design on pain in human subjects. Clin. J. Pain. 24, 585 (2008). Polymer MNA fabrication agreement number: 13/RC/2073). The authors would like to acknowledge the UCD School of Mechanical and Materials Engineering staff; Dr. Ian Reid and Matteo Nicolasi at the Nano Imaging Centre (NIMAC) for scanning electron microscope images. agreement number: 13/RC/2073). The authors would like to acknowledge the UCD School of Mechanical and Materials Engineering staff; Dr. Ian Reid and Matteo Nicolasi at the Nano Imaging Centre (NIMAC) for scanning electron microscope images. Polymer MNA fabrication CMC MNAs loaded with Rh B Rh B-loaded CMC MNAs were fabricated from a silicone mold by means of solvent casting. In all, 5% (w/v) sodium carboxymethyl cellulose (MW ~ 250,000, Sigma-Aldrich, St. Louis, MO, USA) was added to deionized water, pre-mixed on a vortex mixer, and left in a heated water bath at 60 °C for an hour. Rh B was then added to the solution at a mass concentration of 0.1 mg/ml. The solution was again mixed on a vortex mixer and left in a heated water bath at 60 °C for an hour. Next the solution was centrifuged at 6000 rpm for 5 min (FC5706, OHAUS Corporation, Parsippany, NJ, USA) followed by a mixing stage in a planetary mixer (AR-250, THINKY U.S.A., Laguna Hills, CA, USA) at 1500 rpm for 90 s. The solution was then transferred into previously fabricated silicone MNA molds and placed under vacuum for degassing and ﬁlling voids. The MNA was then dried until all the water was evaporated and Rh B-loaded CMC MNAs were obtained. Fluorescence imaging of the Rh B-loaded CMC MNAs was performed on a ﬂuorescence microscope (Olympus Corporation, Shinjuku, Tokyo, Japan). Supplementary information accompanies this paper at https://doi.org/ 10.1038/s41378-019-0088-8. Supplementary information accompanies this paper at https://doi.org/ 10.1038/s41378-019-0088-8. Received: 17 December 2018 Revised: 8 May 2019 Accepted: 3 July 2019 Received: 17 December 2018 Revised: 8 May 2019 Accepted: 3 July 2019 References 1. Sullivan, S. P. et al. 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PLA MNAs An MNA was fabricated from a silicone mold using a biocompatible PLA (4032D, NatureWorks LLC, Minnetonka, MN, USA) by means of vacuum molding in a an oven (3608-6CE, Thermo Fisher Scientiﬁc Inc., Waltham, MA, USA). The material has a quoted tensile strength of 103 MPa in the transverse direction and 145 MPa in the machine direction as tested according to ASTM Test Method D882. The fabricated mold was ﬁlled with the PLA pellets and placed in the vacuum oven at a temperature 180 °C and a pressure of 98.2 kPa for 20 h ± 4 h until the PLA had ﬁlled the mold cavities and all air bubbles were removed. glucose monitoring in vivo. Sens. Actuators A Phys. 203, 373–381 (201 4. Romanyuk, A. V. et al. Collection of analytes from microneedle patches. Anal. Chem. 86, 10520–10523 (2014). 5. O’Mahony, C. et al. Design, modelling and preliminary characterisation of microneedle-based electrodes for tissue electroporation in vivo. 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Author details 1 f 1UCD Centre for Biomedical Engineering, University College Dublin, Belﬁeld, Dublin 4, Ireland. 2School of Mechanical & Materials Engineering, University College Dublin, Belﬁeld, Dublin 4, Ireland. 3School of Electrical & Electronic Engineering, University College Dublin, Belﬁeld, Dublin 4, Ireland This work has been funded through an Irish Research Council Postgraduate Scholarship, a National University of Ireland Travel Studentship, a Marie- Skłodowska-Curie Fellowship (H2020-MSCA-IF-2014/658761), and Science Foundation Ireland and the European Regional Development Fund (grant Needle inspection After initial visual inspection, needles with a set input heights of 200 and 400 µm were deemed of insufﬁcient quality for parametric analysis. For the remaining needles, parameter dimensions were approximated by performing an analysis on digital images of samples taken with a stereomicroscope (SZN-T, Optika Srl., Ponteranica, BG, Italy) using MATLAB (MATLAB R2018b, MathWorks Inc., Natick, MA, USA). Page 13 of 14 Page 13 of 14 Krieger et al. Microsystems & Nanoengineering (2019) 5:42 13. Wang, P. M., Cornwell, M., Hill, J. & Prausnitz, M. R. Precise microinjection into skin using hollow microneedles. J. Invest. Dermatol. 126, 1080–1087 (2006). 14. Cahill, E. M. et al. Metallic microneedles with interconnected porosity: a scalable platform for biosensing and drug delivery. Acta Biomater. 80, 401–411 (2018). Acknowledgements This work has been funded through an Irish Research Council Postgraduate Scholarship, a National University of Ireland Travel Studentship, a Marie- Skłodowska-Curie Fellowship (H2020-MSCA-IF-2014/658761), and Science Foundation Ireland and the European Regional Development Fund (grant Acknowledgements h k h b f design on pain in human subjects. Clin. J. Pain. 24, 585 (2008). This work has been funded through an Irish Research Council Postgraduate Scholarship, a National University of Ireland Travel Studentship, a Marie- Skłodowska-Curie Fellowship (H2020-MSCA-IF-2014/658761), and Science Foundation Ireland and the European Regional Development Fund (grant 19. Wilke, N., Mulcahy, A., Ye, S.-R. & Morrissey, A. Process optimization and characterization of silicon microneedles fabricated by wet etch technology. Microelectron. J. 36, 650–656 (2005). Page 14 of 14 Page 14 of 14 20. Takahashi, H. et al. 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W4237317011.txt	https://www.researchsquare.com/article/rs-97421/latest.pdf	en		Piezoelectric Properties of Lead Zirconate Titanate Ceramics at Low and High Temperatures	Research Square (Research Square)	2,020	cc-by	2,906	Piezoelectric Properties of Lead Zirconate Titanate Ceramics at Low and High Temperatures Mitsuhiro Okayasu (  mitsuhiro.okayasu@utoronto.ca ) Okayama University Masakazu Okawa Okayama University: Okayama Daigaku Research Article Keywords: lead zirconate titanate ceramic, piezoelectric properties, electric generation voltage, domain switching Posted Date: October 28th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-97421/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Advances in Applied Ceramics on March 28th, 2021. See the published version at https://doi.org/10.1080/17436753.2021.1904765. Page 1/12 Abstract The material properties and damage characteristics of lead zirconate titanate (PZT) ceramics were investigated at various temperatures (–190 °C to 180 °C). A positive voltage was obtained when the sample was cooled from 20 °C to –190 °C, while a negative voltage was obtained when the sample was warmed from –190 °C to 180 °C. The difference between the positive and negative values depended on the thermal stress. Compressive stress generated a more positive voltage in the cooling process, while tensile stress led to a more negative voltage in the warming process). The voltage values also depended on the cooling (or warming) rate of the sample, e.g., the greater the cooling (or warming) rate, the greater the voltage. When cyclic loading was conducted mechanically at –190 °C, the voltage reduced, but it was recovered after warming to 20 °C. Damage of the PZT ceramic (90° domain switching) was detected when the sample was cooled to –190 °C. This was due to the high thermal stress, resulting in a low voltage. 1. Introduction Lead zirconate titanate (PZT: Pb(Zr,Ti)O3) ceramics have seen widespread use in a large number of engineering applications, such as in various actuators for precision positioning, vibration suppression equipment, power transducers and vibration sensors. In these applications, PZT ceramics are sometimes used under severe conditions, e.g. at high (or low) temperatures and high humidity. Because the perovskite structure is highly crystalline, ferroelectric lead titanate films have recently been used at high temperatures [1]. Using X-ray diffraction analysis, the crystallization temperatures of piezoelectric ceramics of different chemical compositions, lead titanate (PT), lead zirconate (PZ) and PZT, have been shown to increase in the sequence PT < PZ < PZT at temperatures in the range of 380 °C to 500 °C [2]. The piezoelectric properties of PZT worsen, resulting in a low value of k33, as heating to the Curie temperature (300 °C) occurs, due to a change of the lattice system from tetragonal to cubic [3]. The fabrication of capacitive ultrasonic transducers using surface micromachining techniques and a low-temperature process has been investigated [4]. The piezoelectric properties of PZT ceramics decrease when the sample is cooled. Zhang et al. [5] examined the dielectric and piezoelectric properties of PZT ceramics at temperatures from − 268.8 °C to 27 °C. They found that the material properties converge as the temperature is decreased down to the temperature of liquid helium. Yakushev and Shalimov [6] reported that an improvement of the piezoelectric properties of PZT ceramics occurs with low temperature cyclic treatment. Some researchers have examined the temperature-dependent behavior of the piezoelectric properties in constitutive relations using a thermodynamic approach [7]. They found that the electrostriction coefficient is affected by the chemical composition. The temperature dependence of the piezoelectric properties of PZT ceramics plays a significant role in their design for engineering applications, although there is little information on this available. In particular, information about the change of the piezoelectric properties of PZT ceramics at low and high temperatures could be significant for the design process. Therefore, the material properties of PZT ceramics were investigated over a wide temperature range under the static and cyclic loading. Page 2/12 2. Materials And Experimental Procedures Figure 1 shows a schematic illustration of the piezoelectric ceramic and the experimental setup used in this work. The piezoelectric ceramics used were a commercial circular membrane PbZrTiO3 base ceramic (φ8.0 mm × 0.17 mm) and a bulk PZT ceramic (5 mm × 3 mm × 3 mm). The circular membrane ceramic was attached to a thin brass plate (φ12.2 mm × 0.15 mm), and silver-based electrode plating was coated on its surface. The ceramic had a convex shape with a deflection value of 0.2 mm, see Fig. 1(a). The material properties of the ceramic, brass and silver are as follows. Elastic constant (E): 82 GPa [8], 103 GPa and 82.7 GPa. Thermal expansion coefficient (α): 17.5 × 10− 6/°C, 7.5 × 10− 6/°C, and 19.7 × 10− 6/°C. The ceramic consisted of a tetragonal structure with aspect ratio c/a = 1.014. The nominal grain size of the ceramic was about 5 µm in diameter. To examine the electricity generation characteristics of the PZT ceramics, static and cyclic mechanical loading were used. Mechanical loading was applied at the center of the sample, see Fig. 1(b). Static loading was applied to the sample at 1 mm/min, and cyclic loading was applied with an R ratio of 0.05 with a frequency of 0.25 Hz. The maximum cyclic loading (Pmax), was determined by the bending yield strength (Py) of the ceramic, so that Pmax < 0.8 Py. The voltage generated from the ceramics was measured using a digital multimeter (8846A, Fluke). The piezoelectric properties were further investigated at various temperatures (–190 °C to 180 °C). The ceramic was cooled to the low temperatures using liquid nitrogen (20 °C to − 190 °C), and heated to the high temperatures using a heater (20 °C to 180 °C), as shown in Fig. 1(c). A ceramic heater heated to about 800 °C was used. To re-heat the sample from − 190 °C to 20 °C, the sample was put into cold water. The different heating methods were used in order to change the heating rate. The microstructural and damage characteristics of the PZT ceramics were examined using electron backscatter diffraction (EBSD). The sample surfaces were polished to a mirror finish using colloidal silica. The EBSD analysis was executed using a JEOL-JSM-7001F SEM with an acceleration voltage of 15 kV, a beam current of 10 nA and a step size of 0.1 µm. 3. Results And Discussion 3.1. Static loading Figure 2 shows how the voltage (V) varied during static bending loading at − 190 °C and 20 °C. The ceramics were loaded until a sample deflection of about 1.8 mm was achieved. As seen in the early loading stage at 20 °C, the electric voltage increased to about 3.5 V, at which time the ceramic was deflected by 0.2 mm. With further loading, the voltage decreased to 0 and then with further loading still, generated a negative voltage. Similar variation of the voltage was seen at − 190 °C; however, the maximum voltage (about 2.7 V) was lower than that at room temperature, and a negative voltage was not obtained. The different positive and negative voltages were generated due to different amounts of stress. Page 3/12 Differences in the voltages at − 190 °C and 20 °C were related to the material properties, which will be discussed in a later section of this paper. Figure 3 shows the profile of the ceramic before and after bending loading. The convex shape of the ceramic is formed at 20 °C before the loading. The deflection of the convex shape is about 0.2 mm, as shown in Fig. 1, but the profile is altered after the loading, which causes the ceramic to be deformed permanently to a concave shape, shown by the yellow lines. Compressive stress occurs early in the loading process due to the convex shape of the ceramic, while tensile stress is generated later in the process, because of the permanent deforamtion. This causes the voltage to change from positive to negative (Fig. 2). Figure 3 also shows the profile of the ceramic plate at − 190 °C before mechanical loading (see the blue dotted line). It can be seen that the ceramic is significantly bent with a convex shape, due to the high stress caused by the different thermal expansion coefficients of the brass plate and PZT ceramic. As severely bent PZT ceramic results in high compressive stress during the loading process, negative voltages were not obtained for the ceramic at − 190 °C, as shown in Fig. 2. Figure 4 shows how the maximum voltage varies as a function of the sample temperature. Figure 4(a) shows that the voltage increases to about 40 V immediately after the sample is put in liquid nitrogen and is cooled from 20 °C to 0 °C. Upon further cooling to about − 100 °C, the voltage is relatively stable. This may be an effect of heat insulation, due to the air gap created around the sample surface. The voltage increases to 100 V when the sample temperature is decreased from − 120 °C to − 190 °C, and the cooling rate increases to about − 70 °C/s. However, the voltage decreases to 0 when the sample temperature becomes stable at − 190 °C for a certain period of time, i.e. a spontaneous electric polarization cannot be reversed in the presence of an electric field. When the ceramic, cooled to − 190 °C, is put into water Fig. 4(b), a negative voltage (–80 V) is generated rapidly in the temperature range of − 190 °C to − 150 °C. This is followed by a decrease of the voltage during warming to 20 °C, while the rate of increase of the sample temperature decreases. This could be due to the weak electric field of the PZT ceramic, i.e., the lower the heating rate, the lower the electric generation. Note that the reason for the negative electric voltage is considered to be the thermal tensile stress applied to the PZT ceramic. The ceramic was further warmed from 20 °C to 180 °C using the heater, and the voltage increased slowly without saturation, Fig. 4(c). This is unlike the result of the heating process in water (–190 °C to 20 °C). This reason is the low heating rate of about 12 °C/s when using the heater (20 °C to 180 °C), compared to 60 °C/s when using the cold water (–190 °C to 20 °C). It is of interest to note that the rate of increase of the voltage changes at around 70 °C, as indicated by the dashed circle. This may be due to a change in the lattice or material characteristics [9]. However, there is no clear evidence of this, so further discussion will be required in the future. 3.2. Cyclic loading Page 4/12 The voltage was further investigated under cyclic loading at low temperature (–190 °C) and room temperature (before and after cooling to − 190 °C). Figure 5 shows the voltage obtained during the cyclic loading. Note that the cyclic loading at − 190 °C was performed in liquid nitrogen. Figure 5 shows that a lower voltage was detected at the lower temperature (Vmax = 15 V). This is about 70% lower than the voltage obtained at 20 °C (Vmax = 45 V). The low voltage at − 190 °C is due to the thermal stress at low temperatures, as shown in Fig. 3. In previous work, piezoelectric properties were measured in a temperature range between − 288.8 °C and the Curie point on undoped and Fe-doped PZT samples, and a pronounced relaxation below 127 °C occurred due to domain wall vibrations [10]. Zhang et al. have also examined dielectric and piezoelectric properties of modified lead titanate zirconate ceramics at temperatures between − 288.8 °C and 27 °C, and piezoelectric properties, including the piezoelectric constant, worsened with decreasing sample temperature [5, 11]. The voltage of the ceramic increases at 20 °C after warming from − 190 °C (Fig. 5(b)); however, the maximum voltage is slightly lower than that obtained before cooling to − 190 °C (see the red line vs. the blue dashed line). This may be attributed to damage of the ceramic (90° domain switching), arising from the high thermal stress. To verify this, the thermal stress of the ceramic was calculated. The different strain values (ε) caused by the different thermal shrinkages of the brass and the PZT ceramic result in high stress on the PZT ceramic. The thermal strain can be estimated using the formula ε = α × ΔT. It is clarified using this formula that εPZT = 0.0016 and εbrass = 0.0037. Knowing the different strain values, the thermal stress of the PZT ceramic can be calculated using the formula σ = EPZT × {(εPZT – εbrass)/2} = 82 GPa × 109 × (– 0.00105) = − 86.1 MPa (in compressive stress). To examine whether or not 90° domain switching occurs in the PZT ceramic, an attempt was made to analyze the crystal orientation characteristics using the bulk PZT ceramic after compressive stress (86.1 MPa) was mechanically applied. The crystal orientations obtained before and after application of compressive stress are shown in Fig. 6. The observations were made in the same area of the sample before and after loading. Figure 6 shows that the crystal orientations were changed after the application of compressive stress, and 90° domain switching occurred (see the pole figures with the lattice formations). From this result it can be inferred that the decrease of the electric voltage after the cooling process is influenced by material failure, i.e., domain switching. One of the authors has examined the domain switching characteristics using a similar PZT ceramic [12]. In that study, domain switching was detected when the bending loading was more than 0.8 Py (bending yield strength). 4. Conclusions In this work, the material properties and damage characteristics of PZT ceramics were investigated under static and cyclic loading at low and high temperatures (–190 °C to 180 °C). The results can be summarized as follows: (1) A positive voltage was generated when the sample was cooled from 20 °C to − 190 °C, whereas a negative voltage was generated when the sample was heated from − 190 °C to 180 °C. The change in the Page 5/12 voltage was attributed to different thermal stress, i.e. compressive stress of the ceramic generated the positive voltage, whereas tensile stress led to the negative voltage. The voltage was also affected by the cooling (or warming) rate of the sample, i.e. the greater the cooling (or warming) rate, the greater the voltage. (2) Upon cyclic loading, the voltage of the ceramic decreased when the sample was cooled to − 190 °C, although the voltage was recovered after warming the sample to room temperature (20 °C). However, damage of the ceramic occurred when the sample was cooled to − 190 °C, due to high thermal stress (90° domain switching), which resulted in a reduction of the voltage. References 1. Duffy Jr W, Cheng BL, Gabbay M, Fantozzi G. Anelastic behavior of barium-titanate-based ceramic materials. Metall Mater Trans A 1995, 26: 1735–1739. 2. Wilkinson AP, Speck JS, Cheetham AK. In situ C-ray diffraction study of crystallization kinetics in PbZr1-xTixO3 (PZT, x = 0.0, 0.55, 1.0). Chem Mater 1994, 6: 750–754. 3. Okayasu M, Otake M, Bitoh T, Mizuno M. Temperature dependence of the fatigue and mechanical properties of lead zirconate titanate piezoelectric ceramics. Int J Fatigue 2009, 31: 1254–1261. 4. Cianci E, Foglietti V, Memmi D, Caliano G, Caronti A, Pappalardo M. Fabrication of capacitive ultrasonic transducers by a low temperature and fully surface-micromachined process. J Int Soc Precision Eng Nanotech 2002, 26: 347–354. 5. Zhang XL, Chen ZX, Cross, W.A. Schulze, Dielectric and piezoelectric properties of modified lead titanate zirconate ceramics from 4.2 to 300K. J Mater Sci 1983, 18: 968–972. 6. Yakushev PN, Shalimov VV. Improving the piezoelectric properties of PZT ceramics by lowtemperature cycling. Tech Phys Lett 2020, 46: 182–185. 7. Moon W, Busch-Vishniac IJ. Modeling of piezoelectric ceramic vibrators including thermal effects. Part I. Thermodynamic property considerations. J Acoust Soc Am 1995, 93: 403–412. 8. Bourim E, Idrissi H, Cheng B, Gabbay M, Fantozzi G. Elastic modulus and mechanical loss associated with phase transitions and domain walls motions in PZT based ceramics. J de Phys IU 1996, 6: C8633–636. 9. Bouzid A, Bourim EM, Gabbay M, Fantozzi G. PZT phase diagram determination by measurement of elastic moduli. J Eur Ceram Soc 2005, 25: 3213–3221. 10. Herbiet R, Robels U, Dederichs H, Arlt G. Domain wall and volume contributions to material properties of PZT ceramics. Ferroelectrics 1989, 98: 107–121. 11. Fuji Ceramics Co., Technical Handbook. Fuji Ceramics Co., p.15 (in Japanese). 12. Okayasu M, Yamasaki T. Effects of 90° domain switching on electric generation properties of PZT ceramic. Ceram Int 2017, 43: 3590–3600. Page 6/12 Figures Figure 1 Schematic diagrams of (a) the PZT ceramic plate, (b) the mechanical loading process, and (c) the cooling and heating processes for investigating the voltage. Page 7/12 Figure 2 Variation of the voltage during static bending loading at –190 °C and 20 °C. Page 8/12 Figure 3 Profile of the PZT ceramic before and after static loading at –190 °C and 20 °C. Page 9/12 Figure 4 Variation of voltage with sample temperature: (a) cooling from 20 °C to –190 °C; (b) heating from –190 °C to 20 °C; and (c) heating from 20 °C to 180 °C. Page 10/12 Figure 5 Variation of the electric voltage generated by the PZT ceramic under cyclic loading at –190 °C and 20 °C. Page 11/12 Figure 6 EBSD analysis of the PZT ceramic before and after static loading, showing the 90° domain switching. Page 12/12
https://openalex.org/W4234191269	http://www.clausiuspress.com/conferences/LNEMSS/CEED 2019/19CEED003.pdf	English	null	The Research on PDA Class in Comprehensive English Teaching from Eco-view	Lecture notes in economics, management and social sciences	2,019	cc-by	2,983	The Research on PDA Class in Comprehensive English Teaching from Eco-view Dong Wu Foreign Language Department, Sichuan Vocational and Technical College, Suining, Sichuan,China Keywords: Ecological Perspective; PAD Class; English Teaching; Educational Concept Abstract: With the reform of College English teaching, many scholars from the perspective of ecology, combing with the elaboration of the concept of ecological education and the current situation of College English teaching came up with The College English classroom teaching. Other scholars have analyzed the application of sub-classes in integrated English teaching in order to enhance students' learning initiative and improve classroom teaching effect. Based on the previous studies of scholars, this paper introduces the perspective of ecology and the connotation of Pad class pedagogy in detail, and then combines the existing problems in College English classroom teaching with systematic and comprehensive eco-classroom language method, puts forward the application of sub-classes in English and Chinese teaching from the perspective of ecology. This study provides a new way for the reform of sub-classes in College English teaching from the perspective of ecology. 2019 2nd International Conference on Contemporary Education and Economic Development (CEED 2019) 2019 2nd International Conference on Contemporary Education and Economic Development (CEED 2019) 2019 2nd International Conference on Contemporary Education and Economic Development (CEED 2019) 1. Introduction At present, many English classes still focus on the single teaching of teachers in College English teaching in the 21st century. There is a lack of meaningful interaction or real exchange of ideas between teachers and students, ignoring that comprehensive English is a course that includes listening, speaking, reading, writing and translation, which makes the students trained in Colleges and universities incompatible with the goal of personnel training in China. Finally, the imbalance between teaching and learning in the educational ecosystem appears, resulting in the imbalance between teaching and learning. In view of the unbalanced teaching system, Professor Zhang Xuexin of Fudan University, through many years of teaching practice and theoretical thinking, has innovatively put forward the teaching mode of "PAD(presentation-assimilation-discussion)" on the basis of the advantages of unified classroom teaching, and it has been widely used and praised in Chinese universities. Therefore, this paper combines the educational concept of ecology with the teaching model of PDA class, explores the teaching strategies and methods in English classes, aiming at establishing an ecologically-based English classroom with teacher-student interaction, harmonious environment and independent development, and constructing a sustainable foreign language ecology class. 2. An Overview of Ecological View First of all, we need to introduce the development and meaning of ecological concept. Ecology First of all, we need to introduce the development and meaning of ecological concep Published by CSP © 2019 the Authors 11 was founded in the 1960s by Haeckel, a German biologist. was founded in the 1960s by Haeckel, a German biologist. was founded in the 1960s by Haeckel, a German biologist. The first related concept of biology was put forward. Its research goal is to study the relationship between biology and environment. In the course of its development, many scholars in various fields extended and internalized the principles of ecology and studied human and nature, human and social environment. The core of relationships is putting people at the core: in the 1970s, another scholar, Lawrence Cremie, first mentioned it. The concept of "educational ecology" was put forward. Based on the basic principles of ecology and the viewpoint of ecological balance, he studied the dynamic map of educational phenomena through the method of system theory. Since then, many scholars and educators have begun to combine the concept of educational ecology with all aspects of education and teaching. In the 1980s, educators introduced ecology into classroom teaching in large numbers, and formed an ecological classroom teaching concept. There are three ecological elements in the teaching theory of English ecology. The first is the law of limiting factors. As far as college English teaching is concerned, teachers' present teaching ideas and students' learning attitude, lacking of learning subjectivity and the community environment constitute the main limiting factors in English classroom teaching. Secondly, the law of affiliation and the most appropriate principle include the thinking intensity between students, the difficulty and progress of teachers' teaching, the most appropriate teaching resources and teaching design of schools, and finally, the principle of educational niche. As far as English teaching is concerned, in English classroom teaching, teachers should treat students with different niches equally according to this principle, and integrate some excellent and different teaching methods according to students' personality characteristics. There are two main parts of ecological view in College English primary school. First, the ecological subject of teaching, that is, teachers and students, is well known. Every teacher has his own unique comparative learning idea and teaching style, and every student's learning behavior has its own distinct personality characteristics. 2. An Overview of Ecological View Teachers and students constitute the social situation of individuals and others in the English learning classroom play a different roles, secondly, students' environment, before the introduction of the concept of ecology teaching, in the previous classroom teaching, the interaction between teachers and students is poor, teachers may not act towards their children, that is, two eyes closed one eye, but after the introduction of ecology teaching, ecology refers to the traditional classroom teaching words, classroom English teaching atmosphere and physical teaching environment, of course, including the psychology of teachers and students. The ecological view of English teaching embodies the relationship between classroom teaching and ecological environment. This doctrine can be used to rebound and communicate in the environment of student-centered teaching, and to broaden the knowledge horizon and richness of English majors. 3. The Meaning of PAD Class PAD class is a teaching mode which integrates the traditional teaching mode with the theory-based teaching mode. It can give full play to the advantages of the two teaching methods and make up for the weaknesses. It is helpful to cultivate students' practical application ability of English. The core idea of the sub-class is to allocate part of the classroom time to teachers and the other part to students for interactive learning in the form of discussion. The time allocation of the two teaching methods can be determined according to the specific situation. Teachers' teaching behavior is divided into teaching, internalization, absorption and discussion. These three teaching activities are interlinked and progressive, each with different teaching objectives and tasks. The three teaching behaviors are 12 separated from each other, but there are interrelated parts in content. In the specific teaching process, it can be divided into three stages: pre-class preparation, classroom listening and discussion, after-class discussion and summary. In the PAD class teaching, students can first find relevant information, supplement learning related knowledge, prepare for listening and discussion by mistake, and emphasize the principle of teaching before discussion in the classroom listening and discussion link. Teachers teach knowledge points and contents at first, then students complete the internalization of knowledge according to their own understanding and specific problems, and after that, student need to discuss on learning and absorption. Teachers encourage students to arrange their study independently after class. For questionable problems, they can discuss with each other between teachers and students, so as to carry out personalized internalization and absorption. In addition, English teachers report on teaching problems to the group leaders, interact with students in the classroom, and make comments and summaries on students' views. The innovation of each other's class lies in the staggering of lecture time and discussion time. In the separate classroom teaching, the combination of these three teaching behaviors and teaching process is beneficial to the improvement of students' independent learning and thinking ability. It helps to cultivate students' autonomous learning ability In addition, for the evaluation of the teaching effect of PAD class, combine process evaluation with summative evaluation. According to the content and link of English teaching, the emphasis of process evaluation and summative evaluation is different. 3. The Meaning of PAD Class For example, in teaching new knowledge and grammar, summative evaluation is emphasized, but in communication, the process evaluation is emphasized, which integrates students' classroom discussion, learning achievement report and homework completion. To enable students to actively participate in the classroom according to their learning objectives, needs and interests, which not only fully mobilizes students' desire to learn English independently, but also fully cultivates students' ability to think independently and study independently. 4.1 Dated Teaching Pattern At present, in many colleges , the target of English competence training by students is not clear, which leads teachers to adopt the traditional teaching mode in English teaching, that is, the teaching mode of "Cramming education". Students lose their motivation to learn English and lose interest in English lessons. From the point of view of ecology, students can only improve their ability of memorizing and can not acquire the practical application ability of English in this teaching mode. Teachers or schools ignore the influence of other environments, especially society, campus and culture on the interaction between human and environment. The traditional examination-oriented English teaching method emphasizes the management and monitoring of the classroom environment and order, which leads to the disharmony and imbalance of the two in the classroom ecosystem. Instead of combining listening, speaking, reading, writing and translation in class, it is a single, fragmented teaching. In the 21st century, with the development of information technology and equipment in colleges and universities, multimedia equipment and technology has been popularized in colleges and universities. Although most teachers use modern information technology to assist in classroom teaching, many teachers just replace the traditional "blackboard" with the modern "screen", which only saves the complexity of their own writing. 4.2. The Students' Ability of Comprehensive Application of English, such as Listening, Speaking, Reading, Writing and Translation, is Insufficient 5. Teaching Strategies of PAD in Comprehensive English Teaching from the Perspective of Ecology By expounding the concept of ecological English teaching and the teaching mode of PAD class， it can be found that this concept and teaching mode are based on fully respecting the individual differences of students' learning, and on the basis of comprehensive English teaching. It is helpful to improve students' ability of comprehensive application of English, such as listening, speaking, reading, writing and translation, so as to help students express their ideas actively through various forms of English. Therefore, by synthesizing the teaching idea of the ecological view and the advantages of the PAD class teaching mode, this paper puts forward the teaching strategies of PAD class in the comprehensive English teaching from the ecological perspective. 4.2. The Students' Ability of Comprehensive Application of English, such as Listening, Speaking, Reading, Writing and Translation, is Insufficient ehensive English teaching, students and teachers' understanding of learning English 13 only exists at the level of passing the exam. Therefore, in order to improve the pass rate of CET-4 and CET-6, many students or teachers only pay attention to English reading, translation and other parts, because of its large proportion of scores, resulting in students' contempt for the listening and speaking parts of English. This mode of teaching makes students unwilling or unconsciously to participate in the comprehensive English classroom listening, speaking, writing activities, only focus on words and questions, but also make the classroom interactive atmosphere dull, students' participation enthusiasm is not high. In order to achieve the goal of teaching scores and achievements, teachers help students pass the exam and turn the classroom into a mechanical training class, while neglecting the cultivation of students' comprehensive language ability. It also leads to a low level of oral English. Students, because of their lack of oral ability, interact with their teachers in Chinese to express their views on the topic in classroom teaching activities, and the teachers do not have a very good solution, so they ignore or look at each other without words. An interactive activity that makes it difficult to engage in topic discussions. Trained students can only read English, but can not communicate with foreigners in English. 5.3 Establishment of Evaluation Mechanism for PAD Topic Teaching From the perspective of ecological view, it is helpful for teachers and students to define their "ecological niche" by establishing the teaching evaluation of PAD-topics. Under the background of cultivating students' comprehensive English application ability, colleges and universities should gradually construct a model of combining process evaluation and result evaluation. On the one hand, the school evaluates teachers' performances and results in PAD class in five aspects: listening, speaking, reading, writing and translation. On the other hand, teachers should give quantitative feedback on students' learning content and classroom performance, English communicative competence and test results in order to find out the essence of the problem and help students realize their own shortcomings. Also reflect on their own teaching problems. So as to improve students' English comprehensive ability. 6. Conclusion The research in this paper combines the knowledge of ecology and pedagogy and studies the classroom teaching of college comprehensive English from an interdisciplinary perspective, which not only provides a new perspective for classroom English teaching in colleges and universities, but also provides a new perspective for classroom English teaching in colleges and universities. At the same time, it also provides theoretical basis for solving the problems in college English teaching. It is also hoped that through the research and analysis of this paper, we can promote the further improvement and innovation of the teaching concept and teaching mode in colleges and universities to a certain extent, and at the same time improve the students' interest and enthusiasm in English learning. To increase the interest of comprehensive English teaching courses, thus cultivate the composite talents welcome of social and enterprise. 5.2 Improve Students' Comprehensive English Ability In the process of PAD class mode, teachers can make use of modern information technology to carry out real English comprehensive teaching of listening, speaking, reading, writing and translation. For example, in the aspect of listening and speaking, a new teaching software is introduced, and the spoken language dialogue between foreign teachers and foreign teachers is carried out on the Internet, so that the teaching environment and actual operation are realistic. And teachers and students through oral dialogue. It is also possible to set up a simulated English dating and business dialogue situation, which can effectively combine the internal learning knowledge with the practical application, and help the students to have a better understanding of the benefits of learning knowledge. It can give students the opportunity to test themselves in practice. Acknowledgement This article is one of the research achievements of the project research The Research On PDA in English Course from Eco-view (17SA0176), funded by Sichuan Provincial Education Department and Sichuan Vocational and Technical College. 5.1 Building PAD Class of Teacher-Student Interaction College English classroom from the perspective of ecological view embodies the concept of "student-oriented" teaching. In the comprehensive English teaching, teachers need to make clear the position of teachers and students in the classroom, and give the initiative of English learning to students. As long as the teacher's duty is to guide and organize the development of activities. Design more teaching tasks and activities for students' English learning. For example, the teaching activities of listening, speaking, reading, writing and translation can be rich and colorful, and the form of dialogue between teachers and students can be carried out, and students can communicate and communicate in an interactive and cooperative manner on a certain topic, and at the same time, they can express their views by speaking, writing and writing. In order to mobilize the enthusiasm of students to participate in it. At the same time, the communication and interaction of comprehensive English topics, and the ecological perspective of equal communication, mutual benefit and win-win. Interaction not only helps students internalize and absorb classroom knowledge, but also improves students' understanding of English knowledge. This can not only build a harmonious relationship between teachers and students, but also facilitate the effective development of various classroom teaching activities. From the perspective of ecological view, the interaction and cooperation between teachers and students is more embodied in mutual cooperation and exchange in terms of building a harmonious and common learning relationship between teachers and students. In the interactive PAD class, it is beneficial to better reflect the harmonious progress of teacher-student relationship. 14 [1] Bishop, J. L., & Verleger, M. A. (2013). The flipped classroom: A survey of the research. In Proceedings of 120th ASEE Annual Conference Proceedings. Atlanta, GA. [2]Copley, J. (2007). Audio and video podcasts of lectures for campus-based students: production and evaluation of student use. Innovations in Education and Teaching International, 44(4), [1] Bishop, J. L., & Verleger, M. A. (2013). The flipped classroom: A survey of the research. In Proceedings of 120th ASEE Annual Conference Proceedings. Atlanta, GA. References [1] Bishop, J. L., & Verleger, M. A. (2013). 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https://openalex.org/W2188290439	https://link.springer.com/content/pdf/10.1007/s00431-015-2667-5.pdf	English	null	Clinicians’ overestimation of febrile child risk assessment	European journal of pediatrics	2,015	cc-by	6,755	What is Known: • Only a small proportion of febrile children presenting to the emergency department will have serious bacterial infections (SBI) and uniform risk thresholds to start or withhold SBI treatment are not known. • Only a small proportion of febrile children presenting to the emergency department will have serious bacterial infections (SBI) and uniform risk thresholds to start or withhold SBI treatment are not known. 1 Department of General Paediatrics, Erasmus MC-Sophia Children’s Hospital, Wytemaweg 80 room Sp-1541, 3015 CN Rotterdam, The Netherlands 2 Department of Health Sciences, SAPPHIRE Group, Leicester University and Leicester Hospitals, Leicester, UK 3 Department of Public Health, Centre for Medical Decision Making, Erasmus University Medical Centre Rotterdam, Rotterdam, The Netherlands 4 Department of General and Adolescent Paediatrics, UCL Institute of Child Health, Great Ormond Street, London, UK 1 Department of General Paediatrics, Erasmus MC-Sophia Children’s Hospital, Wytemaweg 80 room Sp-1541, 3015 CN Rotterdam, The Netherlands • The low prevalence of SBI and consequently the low exposure of clinicians to these infections make them rely more on alarming signs or clinical decision rules. • The low prevalence of SBI and consequently the low exposure of clinicians to these infections make them rely more on alarming signs or clinical decision rules. 2 Department of Health Sciences, SAPPHIRE Group, Leicester University and Leicester Hospitals, Leicester, UK Clinicians’ overestimation of febrile child risk assessme Evelien deVos-Kerkhof1 & Damian Roland2 & Esther de Bekker-Grob3 & Rianne Oostenbrink1 & Monica Lakhanpaul4 & Henriëtte A. Moll1 Received: 19 September 2015 /Accepted: 6 November 2015 /Published online: 4 December 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com Feverkidstool risk scores were based on clinical signs/symptoms and C-reactive protein. Amongst vignettes assigned to SBI man- agement, the median risk was 60 % (interquartile range (IQR) 30.0–80.5) and 16.0 % (IQR 5.0–32.0) when vignettes were not managed as SBI. Ill appearance and aberrant circulatory signs were the most influencing factors, as age and duration of fever were the least influencing factors on SBI management decisions. Feverkidstool risk scores varied from 13 % (IQR 7.7–28.1) for SBI management to 7.3 % (IQR 5.7–16.3) for no SBI management. Abstract We aimed to estimate clinicians’ based risk thresholds at which febrile children would be managed as serious bacterial infections (SBI) to determine influencing characteristics and to compare thresholds with prediction model (Feverkidstool) risk estimates. Twenty-one video vignettes of febrile children visiting the emergency department (ED) were assessed by 42 (40.4 %) international paediatricians/paediatric emergency clinicians. Questions were related to clinical risk scores of the child having SBI and SBI management decisions on visual analogue scales. Conclusion: Clinicians assigned high risk scores to children who they would have managed as SBI, mostly influenced by ill appearance and aberrant circulation. In contrast to SBI risk as- sessment of the Feverkidstool, clinicians’ appeared to apply a more stepwise assessment of the risk of presence/absence of SBI at different steps in the diagnostic and therapeutic process. Uniform risk thresholds at which one should start SBI manage- ment in febrile children remains unclear; risk thresholds at which we refrained from SBI management were more consistent. Communicated by Peter de Winter Communicated by Peter de Winter * Henriëtte A. Moll h.a.moll@erasmusmc.nl Evelien deVos-Kerkhof e.kerkhof@erasmusmc.nl Damian Roland dr98@leicester.ac.uk Esther de Bekker-Grob e.debekker@erasmusmc.nl Rianne Oostenbrink r.oostenbrink@erasmusmc.nl Monica Lakhanpaul m.lakhanpaul@ucl.ac.uk * Henriëtte A. Moll h.a.moll@erasmusmc.nl Evelien deVos-Kerkhof e.kerkhof@erasmusmc.nl Damian Roland dr98@leicester.ac.uk Esther de Bekker-Grob e.debekker@erasmusmc.nl Rianne Oostenbrink r.oostenbrink@erasmusmc.nl Monica Lakhanpaul m.lakhanpaul@ucl.ac.uk Monica Lakhanpaul m.lakhanpaul@ucl.ac.uk Eur J Pediatr (2016) 175:563–572 DOI 10.1007/s00431-015-2667-5 Eur J Pediatr (2016) 175:563–572 DOI 10.1007/s00431-015-2667-5 ORIGINAL ARTICLE 1 Department of General Paediatrics, Erasmus MC-Sophia Children’s Hospital, Wytemaweg 80 room Sp-1541, 3015 CN Rotterdam, The Netherlands 4 Department of General and Adolescent Paediatrics, UCL Institute of Child Health, Great Ormond Street, London, UK Study population Paediatricians and paediatric emergency clinicians from the source population of the REPEM network (Research in Pae- diatric Emergency Medicine, Europe; www.pemdatabase.org/ REPEM.html), and Paediatricians at teaching hospitals with an interest in acute and emergency care in the Netherlands and United Kingdom, were invited (104 invitations). Non- responders were sent reminders at 4-week intervals, for a max- imum of four mailings per subject. What is New: • Previously identified model predictors for SBI appeared to be significantly influencing factors in clinicians’ febrile child management in emergency care. 3 Department of Public Health, Centre for Medical Decision Making, Erasmus University Medical Centre Rotterdam, Rotterdam, The Netherlands g y • Clinicians’ wielded higher risk thresholds regarding SBI febrile child management than reflected by the clinical prediction model. g y • Clinicians’ wielded higher risk thresholds regarding SBI febrile child management than reflected by the clinical prediction model. 4 Department of General and Adolescent Paediatrics, UCL Institute of Child Health, Great Ormond Street, London, UK 564 Eur J Pediatr (2016) 175:563–572 Keywords Children . Clinical prediction model . Emergency department . Fever . Serious bacterial infections children’s ED of the Leicester Royal Infirmary in Leicester, UK. All parents had given formal consent for the video images to be viewed by healthcare professionals under trust policy guidelines via previously published process [13]. Ethical consent for the collection of video images process had been granted by the National Research Ethics Committee East Midlands. Abbreviations CI confidence interval CRP C-reactive protein ED emergency department SBI serious bacterial infections VAS visual analogue scale Study intervention—video vignettes Twenty-one online video vignettes of febrile children were shown to the study participants. The vignettes were a mix of children in different age categories with potential SBI and children with simple self-limiting problems reflecting the dif- ferent levels of severity in febrile child presentations in prac- tice. The videos, with a mean duration of about 30 s, were originally recorded for educational purposes of paediatricians in training as part of the REMIT (Refining Evaluation Meth- odologies for Practice Changing Interventions) study (ISRCTN94772165). Background history and vital signs were reported as added text or could easily be interpreted from the video vignettes. Understanding health care professionals’ decision making, particularly regarding to diagnosis, treatment and follow-up is of vital importance, particularly as ED’s become increasingly overcrowded [33, 34]. Moreover, diagnostic errors, especially in infectious diseases, are amongst the most common medical misadventures of malpractice lawsuits in paediatrics [16]. To support decision making in febrile children, different clinical prediction models have been developed in the past decade [4, 7, 12, 19, 30, 31]. Although most studies on pre- diction models report good accuracy and high compliance, implementation in paediatric emergency care is limited. One of the reasons might be that clinicians’ intuitive estimation of probabilities may be as good as, or better than, prediction models [15, 21, 27]. Moreover, the lack of evidence on clin- ically based decision thresholds makes the application process of prediction models in clinical practice complex. Initially, the participants were asked if they should manage the febrile child as having a SBI based on the vignette and background history (e.g. duration of fever) alone. Next, they were asked to assess the actual risk of the child having a SBI on a visual analogue scale (VAS1). Finally, we add different values of C-reactive protein (CRP) and asked if their risk assessment would have changed (VAS2). The online vignettes and the respondents were hosted on a secure password protected server. The aim of this study was to estimate risk thresholds at which children would be managed as SBI according to clini- cians’ judgement by assessment of video vignettes of febrile children visiting the ED. Secondary measures included deter- mining the effect of investigations by recording risk estima- tions after information on C-reactive protein value, determin- ing the presenting characteristics that influence these risks and comparing clinician perceived risk with risk estimates using a validated prediction model (Feverkidstool) [19]. Data collection All data collected online was exported in an anonymised for- mat as an Excel file. We collected answers on the following questions: (1) Would you manage this child as having a seri- ous bacterial infection? (Answers: yes/no). (2) Which diag- nostics or therapeutics would you perform? (Options: no ac- tion and/or discharge; antipyretic; fluid trial; blood tests; chest-radiography; lumbar puncture; urine dipstick; oral anti- biotics; intravenous antibiotics; admission). Study participants could tick as many items as they judged relevant. (3) What is Introduction The febrile child is a common presentation to emergency de- partments (ED) with 10 to 20 % of all paediatric patients due to febrile illness alone [14, 17, 28]. Most children suffering from simple self-limiting infections do not need treatment. However, a small proportion will have serious bacterial infec- tions (SBI) which require investigation, hospital admission, antibiotics and in some cases intensive care admission. Study design and setting We performed a cross-sectional study with real life video vi- gnettes of febrile children who presented themselves to the Study intervention—video vignettes In Table 2, clinical characteristics of the video vignettes are summarised. Median age of the children was 12.0 months (interquartile range (IQR) 2.0–72.0), 57 % were boys and the median C-reactive protein level (CRP) was 60 mg/l (IQR 10.0–110.0). Answers on the four questions of the video vi- gnettes are summarised in Table 3. Forty-one per cent of the video vignettes are managed as having a SBI according to the participants. Diagnostics and/or therapeutics were started in Results Of the 104 invited participants, 50.4 % agreed to participate and 42 (40.4 %) participants finished the online video vi- gnettes. The 42 final participants included 83 % paediatricians and 17 % paediatric emergency medicine physicians. Fifty per cent of the participants had a working experience of more than 10 years. Almost half of the participants had at least once missed or delayed recognised serious infection (Table 1). All vignettes had a statement on age, temperature and du- ration of fever. Abnormal clinical signs and symptoms were distributed amongst the different vignettes, with ten vignettes having one alarming sign, four vignettes with two alarming signs and seven vignettes having three or more alarming signs. Definitions and outcome measures All participants were informed about the predefined SBI def- inition in the letter for the study invitation: culture or radio- graphically proven bacterial infection (e.g. meningitis, sepsis, bacteremia, pneumonia, urinary tract infection, bacterial gas- troenteritis, osteomyelitis or ethmoiditis). The outcome SBI in the vignettes was defined as management of the child as hav- ing a SBI. Detailed descriptions on the Feverkidstool development and validation have been published earlier [19]. The originally reported discriminative ability according to the area under the receiver operating characteristic curve (AUC) of the model to predict pneumonia was 0.81 (standard error 0.04) and for oth- er SBI 0.86 (standard error: 0.03) [19]. As the Feverkidstool was based on a polytomous logistic regression model, two risk scores were calculated, one for pneumonia and one for other SBI (e.g. urinary tract infection). We used the highest risk score in the comparison with the VAS risk scores of the video vignettes. We dichotomised the outcome of performed diag- nostics and/or therapeutics. This outcome was scored ‘pres- ent’ if participants ticked fluid trial, blood tests, chest-radiog- raphy, lumbar puncture, urine dipstick, administration of oral/ intravenous antibiotics and/or admission. When ‘no action and/or discharge and/or antipyretics’ was chosen, the outcome was scored as ‘not present’. Eur J Pediatr (2016) 175:563–572 565 which enact SBI management with discrete choice experiment (DCE) analysis. Finally, we compared VAS risk scores with prediction model based judgement (Feverkidstool). the chance of SBI in this child? (Answer: 0–100 % on a VAS (VAS1)) [1]. As CRP is the strongest predictor of the Feverkidstool, we studied the additional value of CRP in cli- nicians’ management decision, with the following question: (4) A CRP is taken and returns at (continuous value) mg/l. What is the chance of SBI in this child? (Answer: 0–100 % VAS (VAS2)). DCEs are a quantitative approach to assess preferences for e.g. medical interventions and are increasingly used in health care [10]. In DCEs, it is assumed that important items influencing medical interventions, such as vital signs, can be described by its characteristics (i.e. attri- butes) [24]. Those characteristics are further specified by variants of that characteristics (i.e. attribute levels). A second assumption is that the levels of those attributes are determined by the individuals’ preference for a medi- cal intervention [24]. We studied the clinical variables of the Feverkidstool (www.erasmusmc.nl/feverkidstool) as attributes to the decision whether or not to manage febrile children of the vignettes as a SBI [13]. All DCE data was analysed by taking each choice amongst the two management alternatives as an observation. Using the Nlogit software http://www.limdep.com/ to the next sentence, the observations were analysed by a logit model. As there was a lack of diversity amongst the clinical variables ‘oxygen saturation’ and ‘tachypnoea’ between the vignettes, we could not analyse these variables accordingly. The variables tachycardia and prolonged capillary refill were taken together as one clinical variable as their correlation was too high. The influence of the different variable coefficients was tested for statistical significance (p value ≤0.05). As at this moment, no formal statistical methods to determine sample sizes for DCE exist; our study strived to reach at least 40 respondents in line with previous studies [6, 26]. Participant’s background information was collected after finishing the video vignettes. These questions included (1) Are you a: Emergency Medicine clinician/Paediatrician; (2) How long have you been working as an Emergency Medicine clinician/paediatrician? (Options: <5 years; 5–10 years; 10– 15 years; >15 years); (3) Have you ever missed/recognised a serious infection too late? (Options: yes/no). Statistical analysis First, we assessed the range of estimated median risks by clinical judgement (VAS) and the risk with the added value of CRP. Second, we measured the patient characteristics Eur J Pediatr (2016) 175:563–572 566 Table 1 Demographics Participants (n=42) Specialisma Paediatric emergency medicine clinician 7 (16.7) Paediatrician 35 (83.3) Years of working experiencea <5 years 4 (9.5) 5–10 year 17 (40.5) 10–15 years 9 (21.4) >15 years 12 (28.6) Missed/recognised a serious infection too latea Yes 19 (45.2) No 23 (54.8) a Absolute number (percentage) CRP information the risk (VAS2) increased to 30.0 % (IQR 10.0–60.0). As CRP values were already available in the first video for vignette 3 and 21, no change in risk could be mea- sured. Details of performed diagnostics, therapeutics and follow-up are described in Table 4. More diagnostics and/or therapeutics were performed when the child was managed as SBI. Antipyretics were given in 65 % of the video vignettes with no differences when stratifying by outcome (SBIM). In 94 % of the video vignettes who were managed as SBI, blood tests were done and 71 % were hospitalised (Table 4). Discrete choice experiment—video vignettes Discrete choice experiment was based upon 20 video vi- gnettes as the clinical variables of one video were too corre- lated. Almost all clinical variables of the Feverkidstool could be tested with DCE analysis, except for CRP, oxygen satura- tion and tachypnoea. Ranking and coefficients of influencing variables on management decision of febrile children accord- ing to the DCE analysis are presented in Table 5. All tested clinical variables influenced the decision on management of febrile children significantly. Ill appearance and the combined variables of prolonged capillary refill and tachycardia were the most influencing factors and age and duration of fever the least influencing factors. Clinical judgement versus different levels of CRP In Fig. 1, the differences in clinical risk scores are visualised versus different levels of CRP values. The median clinical risk differences (VAS2-VAS1) were positively correlated with a higher level of CRP (SBIM yes: Pearson correlation 0.53 (p=0.000) and SBIM no: Pearson correlation 0.68 (p= 0.000)). Risk scores of children classified initially already as being managed as SBI were influenced only by high levels of CRP (>65 mg/l), whereas children not man- aged initially as SBI were influenced by lower CRP levels (>40 mg/l) (Fig. 1). 77 % of the video vignettes. Median risk before the knowl- edge of CRP (VAS1) was 20.0 % (IQR 9.0–50.0) and with Risk scores video vignettes—risk scores Feverkidstool 3) 48 81.5 (49.8–91.8) 18 2 7 (16.7) 32 (76.2) 11.5 (7.8–25.3) 110 60.0 (31.0–80.0) 19 1 9 (21.4) 24 (57.1) 15.5 (8.3–30.0) 75 30.5 (19.3–50.0) 20 1 16 (38.1) 35 (83.3) 21.0 (10.0–45.5) 35 13.5 (8.0–36.3) 21 2 10 (23.8) 29 (69.0) – 100 19.5 (6.8–30.3) Total 365/882 (41.4) 680/882 (77.1) 20.0 (9.0–50.0) 60.0 (35.0–85.0) 30.0 (10.0–61.0) Table 3 Answers of 42 participants on 21 video vignettes (ntotal=882) Absolute number (percentage); a Median (25–75 percentile) Question 1: Would you manage this child as having a serious bacterial infection? SBIM : child is managed as having SBI according to participant Question 2: Which diagnostics or therapy would you perform? Dx/Tx: diagnostics and/ or therapy done (defined as: fluid trial; blood tests; chest-radiography; lumbar puncture; urine dipstick; administration of oral/ intravenous antibiotics or admission) Question 3: What is the chance of SBI in this child? (Answer: 0–100 % on a VAS (VAS1 )) VAS1 : risk assessment without knowledge of CRP (0–100 % VAS) Question 4: A CRP is taken and returns at (continuous value) mg/l. What is the chance of SBI in this child? (Answer: 0–100 % VAS (VAS2 ) VAS2 : risk assessment with knowledge of CRP (0–100 % VAS) Absolute number (percentage); a Median (25–75 percentile) Absolute number (percentage); a Median (25–75 percentile) Question 1: Would you manage this child as having a serious bacterial infection? Question 1: Would you manage this child as having a serious bacterial infection? Question 4: A CRP is taken and returns at (continuous value) mg/l. What is the chance of SBI in this child? (Answer: 0–100 % VAS (VAS2 ) VAS2 : risk assessment with knowledge of CRP (0–100 % VAS) management decisions. High clinical risk scores to manage febrile children as SBI were created by clinicians. All tested clinical variables of the Feverkidstool influenced clinicians’ management decisions of febrile children significantly with ill appearance and aberrant circulatory signs being the most im- portant. Moderate CRP levels influenced risk scores in chil- dren who were initially not managed as SBI whereas high CRP levels were needed to influence risk scores in children who were initially already managed as SBI. In children man- aged as SBI risk thresholds judged by the clinician were higher compared with predicted risk thresholds according to the Feverkidstool. Clinical risk thresholds of children not managed as having a SBI were more comparable to prediction model-based risk thresholds. Risk scores video vignettes—risk scores Feverkidstool clearly observed when watching the video vignettes, but this clinical variable is not included in the predictors of the Feverkidstool. Finally, no differences were found in median clinical risk scores when stratified for previously missed diag- noses of the participant (p=0.218). Risk scores video vignettes—risk scores Feverkidstool The median clinical risk score (VAS2) according to the partic- ipants amongst those video vignettes who were assigned as managed as SBI was 60.0 % (IQR 30.0–80.5) compared to a risk score according to the Feverkidstool of 12.7 % (IQR 7.7– 28.1) (Table 6). When the video vignettes were not managed as SBI, the clinical risk score (VAS2) amounted to 16.0 % (5.0–32.0) compared to a risk of 7.3 % (5.7–16.3) according the Feverkidstool (Table 7). The largest risk score differences between the vignettes and risk scores according to the Feverkidstool were seen for video vignettes with (various levels of) decreased consciousness or agitation. This item is 567 Eur J Pediatr (2016) 175:563–572 Table 3 Answers of 42 participants on 21 video vignettes (ntotal=882) Alarming symptoms Question 1 Question 2 Question 3 Question 4 Video vignette No. SBIM Dx/Tx VAS1a (%) CRPa (mg/l) VAS2a (%) 1 2 3 (7.1) 16 (38.1) 10.0 (4.8–20.0) 85 26.5 (10.0–44.8) 2 1 29 (69.0) 42 (100.0) 30.0 (20.0–50.3) 70 54.5 (30.0–79.3) 3 1 11 (26.2) 26 (61.9) 16.0 (7.8–32.8) 38 10.0 (4.8–23.0) 4 3 27 (64.3) 39 (92.9) 27.0 (10.0–51.8) 100 60.0 (30.8–76.0) 5 3 41 (97.6) 42 (100.0) 81.0 (60.0–90.0) 65 71.5 (50.0–90.0) 6 3 13 (31.0) 36 (85.7) 20.5 (10.0–40.0) 90 44.0 (20.0–69.3) 7 1 23 (54.8) 33 (78.6) – 10 30.5 (11.0–60.3) 8 1 27 (64.3) 41 (97.6) 30.0 (14.0–50.0) 25 17.0 (10.0–29.3) 9 1 4 (9.5) 25 (59.5) 10.0 (4.0 21.0) 30 9.5 (4.0–21.0) 10 2 41 (97.6) 42 (100.0) 80.0 (62.5–90.0) 50 69.5 (40.0–90.0) 11 4 9 (21.4) 38 (90.5) 10.5 (5.0–21.0) 90 40.5 (21.0–69.0) 12 1 5 (11.9) 32 (76.2) 10.5 (5.8–21.0) 28 6.0 (4.0–14.5) 13 1 0 (0) 11 (26.2) 5.0 (2.8–15.5) 36 4.0 (0.8–12.0) 14 6 16 (38.1) 38 (90.5) 16.0 (9.8–40.0) 60 30.0 (16.3–50.0) 15 3 32 (76.2) 42 (100.0) 41.5 (20.0–69.3) 75 62.5 (38.5–80.0) 16 1 1 (2.4) 15 (35.7) 8.5 (2.8–15.8) 10 1.0 (0.0–6.0) 17 3 41 (97.6) 42 (100.0) 82.5 (69.8–93. Main findings This is the first study on real life video vignettes to determine febrile child characteristics which enact clinicians’ Eur J Pediatr (2016) 175:563–572 568 Table 4 Diagnostics, therapy and follow-up Diagnostics SBIM yes n=365 SBIM no n=517 Ntotal=882 No diagnostics 4 (1.1) 100 (19.3) 104 (11.8) Urine dipstick 252 (69.0) 134 (25.9) 386 (43.8) Fluid trial 135 (37.0) 73 (14.1) 208 (23.6) Blood tests 344 (94.2) 180 (34.8) 524 (59.4) Chest-radiography 112 (30.7) 76 (14.7) 188 (21.3) Lumbar puncture 140 (38.4) 9 (1.7) 149 (16.9) Therapy and follow-up SBIM yes n=365 SBIM no n=517 Ntotal=882 Antipyretics 244 (66.8) 330 (63.8) 574 (65.1) No therapy 74 (20.3) 404 (78.1) 478 (54.2) Oral antibiotics 11 (3.0) 16 (3.1) 27 (3.1) Intravenous antibiotics 209 (57.3) 4 (0.8) 213 (24.1) Admission 258 (70.7) 96 (18.6) 354 (40.1) Discharge 75 (20.5) 405 (78.3) 480 (54.4) Table 4 Diagnostics, therapy and follow-up Comparison with literature pretest probability or the strength of the evidence [11]. Al- though the pretest probability of having SBI (prevalence of disease) is depending on several factors as for exam- ple age and relevant medical history, the pretest proba- bility determined by health care setting was considered stable in the vignettes. However, we focused on the interpretation of clinicians’ strengths of evidence of the probability of a serious infection. For this decision pro- cess, we performed discrete choice experiment (DCE) analysis, which is an increasingly used method applied In this study, we aimed to get insight in patient characteristics and contextual factors influencing management decisions of the febrile child at the ED. One way to approach this process of diagnostic reasoning is decision making [11]. Decision making has been influenced by statistical models of reasoning under uncertainty using pre- and post-test probability accord- ing to Bayes’ theorem. This model deals with two major clas- ses of errors in clinical reasoning: in the assessment of either Fig. 1 Relation video vignettes risk difference and C-reactive protein (mg/l) Fig. 1 Relation video vignettes risk difference and C-reactive protein (mg/l) Eur J Pediatr (2016) 175:563–572 569 Table 5 Influencing variables on management decisions in febrile children (SBIM): a discrete choice experiment (ntotal=882) Table 5 Influencing variables on management decisions in febrile children (SBIM): a discrete choice experiment (ntotal=882) Clinical variables Ranking Coefficients (SE) p value Intercept −0.92 (0.37) 0.013 Ill appearance 1 1.15 (0.13) <0.001 Prolonged capillary refill (>2 s) and/or tachycardia 2 0.99 (0.17) <0.001 Chest wall retractions 3 −0.97 (0.22) <0.001 Temperature (≥39.0 °C) 4 0.77 (0.12) <0.001 Sex (male) 5 0.63 (0.11) <0.001 Duration fever (days) 6 0.51 (0.20) 0.009 Age (≥1 year) 7 −0.42 (0.12) 0.001 Saturation (<94 % O2) NA NA NA Tachypnoea NA NA NA SBIM : child is managed as having SBI according to participant NA not applicable, items could not been tested with DCE analyses in studies where clinicians weigh clinical information in the diagnostic work-up [3]. translation of statistical decision theory into clinical practice [25]. Within this translation, we aimed to elaborate on the determination of quantitative decision thresholds that proved to be a complex topic. Most studies used optimised perfor- mance measures as area under the receiver operating charac- teristic curve (AUC) or sensitivity/specificity to establish these thresholds. Other studies described Delphi procedures to determine their clinical based cutoff points [5, 18, 20, 22, 32]. Comparison with literature In our study, we described clinicians’ assigned median risk estimates according to which patients would have been managed as SBI. We observed agreement on clinical and pre- diction model-based risk thresholds when clinicians decided not to manage the febrile child as a SBI. However, the clinical risk threshold to manage the child as SBI was much higher compared with prediction model-based judgement. This phe- nomenon is well recognised, as clinicians don’t want to miss serious, but treatable diseases, there is a tendency to overesti- mate the probability of these diseases [11]. In literature on diagnostic reasoning, evidence-based med- icine is the most successful educational method in the Table 6 Clinical risk scores (video vignettes) versus prediction model risk scores (Feverkidstool) in children managed as SBI (SBIM=yes) VAS2 (%)a Feverkidstool (%)a Video vignettes (no.) SBIM yes n=365 n=365 Risk ≤10 % 12 5.0 (2.0–9.5) 16.3 13 – – Risk 10–50 % 16 15.0 (15.0–15.0) 2.0 8 20.0 (12.0–30.0) 8.9 3 23.0 (9.0–61.0) 7.2 20 29.0 (12.5–61.8) 3.8 9 30.5 (8.3–66.3) 11.6 14 47.0 (32.0–76.8) 36.9 Risk ≥50 % 21 54.0 (17.8–80.3) 12.7 19 59.0 (45.0–90.0) 7.3 2 60.0 (30.0–80.0) 38.2 7 60.0 (30.0–72.0) 2.3 1 62.0 (50.0–62.0) 20.6 6 68.0 (35.0–83.0) 19.0 15 68.0 (52.3–80.8) 50.5 10 70.0 (44.5–90.0) 7.7 11 70.0 (57.5–81.0) 4.8 4 71.0 (35.0–80.0) 9.7 5 72.0 (50.0–90.0) 22.2 18 80.0 (21.0–82.0) 6.6 17 83.0 (50.0–92.5) 28.1 Total 60.0 (30.0–80.5) 12.7 (7.7–28.1) Table 6 Clinical risk scores (video vignettes) versus prediction model risk scores (Feverkidstool) in children managed as SBI (SBIM=yes) Strengths and limitations The main strength of this study is the use of real-life videos instead of paper-case patients. This approach is a more repre- sentative way of portraying real life, and there is an evolving evidence base on the use of patient video cases as educational interventions [8, 23]. A second strength of the study is the use of the Feverkidstool as an arithmetic model to compare the subjec- tive overall assessment of the clinician when evaluating the febrile child. In a review describing vignette studies on med- ical decision behaviour, it was concluded that most studies on this topic did not compare their results to some sort of norma- tive benchmark [3]. Moreover, the role of prediction models becomes greater, as clinicians may increasingly rely on alarming signs and symptoms described in (inter)national clin- ical guidelines and prediction models due to decreasing inci- dence of SBI. Although, there was a discrepancy in risk as- sessment of some video vignettes (e.g. vignettes 7, 11 and 18), probably due to the absence of variables as decreased con- sciousness or agitation in the Feverkidstool. There are some other limitations in this study. Videos still lack some aspects of real life such as observation time or concise descriptions of patients’ history. However, from liter- ature, we know that more detailed case descriptions will be assigned a higher subjective probability of disease than a brief abstract of the same case, even if they contain the same disease information [11]. Another limitation includes the determina- tion of some clinical variables by the clinicians’ judgement (ill appearance, chestwall retractions and capillary refill time). In this way, misclassification of these clinical predictors could have occurred. However, this approach does reflect clinical practice and therefor may just strengthen generalisability of our results. appearance and aberrant circulatory signs in their febrile child evaluation, which were not the most influencing factors ac- cording to the Feverkidstool. For the Feverkidstool respiratory predictors as chestwall retractions and oxygen saturation were more powerful influencing factors. Furthermore, we found that CRP levels influenced clinical risk scores differently in children with or without initial SBI management, with higher influence of clinical factors than of CRP value. In our study population, this approach was not enhanced by experiences of errors in the past. Strengths and limitations These insights in influencing factors in the clinical prediction of febrile children at risk for SBI helps us to understand, review and evaluate clinical management decisions. Next, the DCE analysis had to be performed within the availability of a limited number of video vignettes. As a con- sequence, we were forced to exclude or merge some predictor variables (e.g. oxygen saturation and tachypnoea) to meet the DCE theory design. Second, although a response rate of 50 % for clinicians was similar to other DCE studies, this response rate is not optimal [2, 9, 29]. However, due to the experienced background of all participants, we assume limited answer var- iability resulting in representative study results. Compared to prediction model based risk scores, thresh- olds of children who were not managed as having a SBI were more comparable, ranging from 7 to 16 %. We might have to conclude that this risk threshold is justified as SBI rule-out threshold, but no agreement can be defined on rule-in thresh- olds as there appears too much difference between prediction model and the clinical stepwise risk assessment in children managed as SBI. Clinical and research implications The most important finding of this study includes the high risk scores clinicians assigned to those children who they would have managed as SBI (median risk 60.0 % (IQR 30–80.5)). This observation is in contrast to our hypothesis that very low risk thresholds might be chosen for specific diagnosis with high morbidity/mortality (e.g. meningitis). Apparently, clini- cians create more dichotomous risk estimations (high risk or low risk) for the management of specific serious infections with reassessment of risk estimates after every diagnostic step. Clinicians used a stepwise approach in the management of febrile children, rather than considering one risk thresholds for SBI in general. We observed agreement in predictive value of all tested clinical predictor variables in the detection of children with SBI, for both clinical-based as prediction model-based judgement. Clinicians were guided by ill The most important finding of this study includes the high risk scores clinicians assigned to those children who they would have managed as SBI (median risk 60.0 % (IQR 30–80.5)). This observation is in contrast to our hypothesis that very low risk thresholds might be chosen for specific diagnosis with high morbidity/mortality (e.g. meningitis). Apparently, clini- cians create more dichotomous risk estimations (high risk or low risk) for the management of specific serious infections with reassessment of risk estimates after every diagnostic step. 570 Eur J Pediatr (2016) 175:563–572 Table 7 Clinical risk scores (video vignettes) versus prediction model risk scores (Feverkidstool) children not managed as SBI (SBIM=no) VAS1 (%)aFeverkidstool (%)aVideo vignettes (no.) SBIM no n=517 n=517 Risk ≤10 % 16 1.0 (0.0–5.5) 2.0 13 4.0 (0.8–12.0) 5.7 12 6.0 (4.0–16.5) 16.3 9 8.5 (4.0–16.3) 11.6 3 10.0 (3.0–17.0) 7.2 8 10.0 (6.0–18.0) 8.9 20 10.0 (7.0–20.3) 3.8 Risk 10–50 % 10 13.0 (13.0–13.0) 7.7 21 15.5 (5.3–28.0) 12.7 7 17.0 (10.0–28.0) 2.3 1 20.0 (10.0–39.0) 20.6 14 20.0 (10.0–31.3) 36.9 17 20.0 (20.0–20.0) 28.1 19 25.0 (15.5–48.0) 7.3 11 30.0 (20.0–53.5) 4.8 5 40.0 (40.0–40.0) 22.2 6 40.0 (17.5–57.5) 19.0 2 42.0 (33.0–74.5) 38.2 4 46.0 (22.0–60.0) 9.7 15 46.5 (26.0–64.5) 50.5 Risk ≥50 % 18 60.0 (31.0–71.0) 6.6 Total 16.0 (5.0–32.0) 7.3 (5.7–16.3) Table 7 Clinical risk scores (video vignettes) versus prediction model risk scores (Feverkidstool) children not managed as SBI (SBIM=no) Conclusion In this study on real-life video vignettes, we observed high risk scores in clinicians’ risk estimation of SBI management in febrile children, and these risks are mostly influenced by the clinical characteristics ill appearance and aberrant circulatory signs. Uniform risk thresholds at which one should start SBI management in febrile children remains unclear, as the Compliance with ethical standards Conflict of interest The authors declare that they have no competing interests. 12. Galetto-Lacour A, Zamora SA, Andreola B, Bressan S, Lacroix L, Da Dalt L, Gervaix A (2010) Validation of a laboratory risk index score for the identification of severe bacterial infection in children with fever without source. Arch Dis Child 95:968–973 Funding source EK is supported by ZonMW, a Dutch organisation for health research and development. The study sponsor had no role in study design, in the collection, analysis, and interpretation of data; in the writing of the report; nor in the decision to submit the paper for publication. 13. Hensher D, Rose J, Green W (2005) Applied choice analysis: a primer. Cambridge University Press, Cambridge: UK 14. 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Arch Dis Child 96:127–130 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appro- priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 17. Nelson DS, Walsh K, Fleisher GR (1992) Spectrum and frequency of pediatric illness presenting to a general community hospital emergency department. Pediatrics 90:5–10 g y p 18. Eur J Pediatr (2016) 175:563–572 571 study caregivers’ medical decision behaviour: systematic review. BMC Med Res Methodol 8:50 concept of clinicians’ dichotomous risk thresholds was hardly comparable to the overall SBI risk assessment of the predic- tion model. However, more consistent results were found for clinical and prediction model-based risk thresholds at which we refrain from SBI management in the febrile child visiting the emergency department. 4. Bachur RG, Harper MB (2001) Predictive model for serious bacte- rial infections among infants younger than 3 months of age. Pediatrics 108:311–316 5. Bell LM, Grundmeier R, Localio R, Zorc J, Fiks AG, Zhang X, Stephens TB, Swietlik M, Guevara JP (2010) Electronic health record-based decision support to improve asthma care: a cluster- randomized trial. Pediatrics 125:e770–777 6. Berchi C, Dupuis JM, Launoy G (2006) The reasons of general practitioners for promoting colorectal cancer mass screening in France. Eur J Health Econ 7:91–98 Acknowledgments We gratefully acknowledge all participants of the video vignettes study for their time, patience and complete participation in our study. We want to thank Paul Muston for the collaboration and the development of the video vignettes for practical use. We acknowledge Johan van der Lei, Ewout Steyerberg and Yvonne Vergouwe for discus- sion on the approach towards DCE analyses. 7. Craig JC, Williams GJ, Jones M, Codarini M, Macaskill P, Hayen A, Irwig L, Fitzgerald DA, Isaacs D, McCaskill M (2010) The accuracy of clinical symptoms and signs for the diagnosis of serious bacterial infection in young febrile children: prospective cohort study of 15 781 febrile illnesses. BMJ 340:c1594 Authors’contributions EdVK, DR, ML and HAM substantially con- tributed to the conception and design of the study. DR collected the original video vignettes as used in the study. He monitored participant response rates and undertook data extraction. EdVK actively enrolled study participants and monitored response rates. She undertook data ex- traction and performed data analysis. She drafted the initial manuscript. 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https://openalex.org/W2784560834	https://jyx.jyu.fi/bitstream/123456789/56831/1/27937621.pdf	English	null	Evolutionary Algorithms and Metaheuristics: Applications in Engineering Design and Optimization	Mathematical problems in engineering	2,018	cc-by	3,460	1. Introduction modeling, in real world engineering problems. For example, this is the case of automotive industry, aeronautical and aerospace industry, and civil, structural, and mechanical engineering, where the calculation of the objective function values requires the resolution of numerical models, using (nonlinear) partial differential equations, based on finite elements, boundary elements, finite volumes, and so on. As stated in [1], the origins of Evolution Strategies [2] during the middle sixties in University of Berlin (Germany) were ignited by the necessity of solving an “optimal shape of bodies in a flow” problem during wing tunnel experiments in the Institute of Flow Engineering, after unsuccessful attempts with the coordinate and simple gradient strategies. Early applications of evolutionary algorithms dealing with engi- neering design and optimization date from the late eighties [3, 4] and early nineties as in [5, 6]. There have been applications compiled in book volumes as in [7–10], and the field has been continuously growing, as in the case of evolutionary multiobjective applications where a state-of-the-art review can be found in [11], or [12, 13]. Recent volumes of scientific contributions in the field are covered by [14–16].h Evolutionary algorithms and, more generally, nature-inspired metaheuristics are gaining increasing favor as computational intelligence methods, very useful for global optimization problems. The success of these population-based frameworks is mainly due to their flexibility and ease of adaptation to the most different and complex optimization problems, without requiring any special feature or condition to the objective functions and related constraints, like continuity, derivabil- ity, or convexity. Discrete and combinatorial optimization problems, as well as mixed ones, are not a limit for this class of optimizers. Moreover, the requirement of uncertainty quantification in the search process, like in reliability-based optimization and robust design, is not a limit for this approach. Finally, population-based optimization algorithms can deal naturally with multiobjective problems, and this has made a big leap forward in the ability to effectively handle this class of problems possible. These advantages, together with the steady improvement of computer performance, are fostering their increased use in research and industry in a wide variety of engineering branches. The advances in the use of evolutionary algorithms and nature-inspired metaheuristics in engineering applications bring an opportunity and also a challenge for researchers to improve and advance in design and optimization of products, systems, and services for societal benefit. David Greiner ,1 Jacques Periaux,2 Domenico Quagliarella,3 Jorge Magalhaes-Mendes,4 and Blas Galván1 1Instituto Universitario de Sistemas Inteligentes y Aplicaciones Num´ericas en Ingenier´ıa (SIANI), Universidad de Las Palmas de Gran Canaria, 35017 Las Palmas de Gran Canaria, Spain 2University of Jyv¨askyl¨a, Jyv¨askyl¨a, Finland 3Italian Aerospace Research Center (CIRA), Capua, Italy 4Politecnico do Porto, Porto, Portugal 1Instituto Universitario de Sistemas Inteligentes y Aplicaciones Num´ericas en Ingenier´ıa (SIANI), Universidad de Las Palmas de Gran Canaria, 35017 Las Palmas de Gran Canaria, Spain 2University of Jyv¨askyl¨a, Jyv¨askyl¨a, Finland Correspondence should be addressed to David Greiner; david.greiner@ulpgc.es Received 14 December 2017; Accepted 17 December 2017; Published 17 January 2018 Copyright © 2018 David Greiner et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. Author(s): Title: Year: Version: Please cite the original version: Evolutionary Algorithms and Metaheuristics : Applications in Engineering Design and Optimization Greiner, David; Periaux, Jacques; Quagliarella, Domenico; Magalhaes-Mendes, Jorge; Galván, Blas Greiner, D., Periaux, J., Quagliarella, D., Magalhaes-Mendes, J., & Galván, B. (2018). Evolutionary Algorithms and Metaheuristics : Applications in Engineering Design and Optimization. Mathematical Problems in Engineering, 2018, Article 2793762. https://doi.org/10.1155/2018/2793762 2018 This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. Author(s): Title: Year: Version: Please cite the original version: Evolutionary Algorithms and Metaheuristics : Applications in Engineering Design and Optimization Greiner, David; Periaux, Jacques; Quagliarella, Domenico; Magalhaes-Mendes, Jorge; Galván, Blas Greiner, D., Periaux, J., Quagliarella, D., Magalhaes-Mendes, J., & Galván, B. (2018). Evolutionary Algorithms and Metaheuristics : Applications in Engineering Design and Optimization. Mathematical Problems in Engineering, 2018, Article 2793762. https://doi.org/10.1155/2018/2793762 2018 This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. Please cite the original version: Greiner, D., Periaux, J., Quagliarella, D., Magalhaes-Mendes, J., & Galván, B. (2018). Evolutionary Algorithms and Metaheuristics : Applications in Engineering Design and Optimization. Mathematical Problems in Engineering, 2018, Article 2793762. https://doi.org/10.1155/2018/2793762 Please cite the original version: Greiner, D., Periaux, J., Quagliarella, D., Magalhaes-Mendes, J., & Galván, B. (2018). Evolutionary Algorithms and Metaheuristics : Applications in Engineering Design and Optimization. Mathematical Problems in Engineering, 2018, Article 2793762. https://doi.org/10.1155/2018/2793762 All material supplied via JYX is protected by copyright and other intellectual property rights, and duplication or sale of all or part of any of the repository collections is not permitted, except that material may be duplicated by you for your research use or educational purposes in electronic or print form. You must obtain permission for any other use. Electronic or print copies may not be offered, whether for sale or otherwise to anyone who is not an authorised user. Hindawi Mathematical Problems in Engineering Volume 2018, Article ID 2793762, 4 pages https://doi.org/10.1155/2018/2793762 Editorial Evolutionary Algorithms and Metaheuristics: Applications in Engineering Design and Optimization David Greiner ,1 Jacques Periaux,2 Domenico Quagliarella,3 Jorge Magalhaes-Mendes,4 and Blas Galván1 1Instituto Universitario de Sistemas Inteligentes y Aplicaciones Num´ericas en Ingenier´ıa (SIANI), Universidad de Las Palmas de Gran Canaria, 35017 Las Palmas de Gran Canaria, Spain 2University of Jyv¨askyl¨a, Jyv¨askyl¨a, Finland 3Italian Aerospace Research Center (CIRA), Capua, Italy 4Politecnico do Porto, Porto, Portugal 2. Scientific Contributions of the Special Issue In this special issue, a reviewing process has been performed where at least two reviewers per paper have been assigned, where a 15% acceptation rate has been held. The accepted papers can be classified according to the following engineering/application categories: (a) energy and electrical engineering; (b) structural and civil engineering; (c) scheduling transport and combinatorial optimization; (d) control; (e) other applications/military. 2.3. Scheduling, Transport, and Combinatorial Optimization. A two-optimization phase based genetic algorithm (GA) is proposed by D. Morillo et al. for solving an energy-based extension of the Multimode Resource-Constrained Project Scheduling Problem, where the search is focused on Mode Lists instead of doing it on Activity Lists. Five GA variants were compared, where the proposed algorithm outperforms the others in the set of problems of the project scheduling problem library PSP-LIB. A brief description of each contribution published in the special issue is given in the following paragraphs according to the previous classification. 2.1. Energy and Electrical Engineering. A particle swarm optimization algorithm using the eagle strategy (ESPSO), a method of combination of global search and intensive local search, is introduced for solving the reactive power losses minimization problem, by H. Yapıcı and N. Cetinkaya. Experiments cover the IEEE 30-bus and IEEE 118-bus power systems and a real power distribution subsystem. A compar- ison with other metaheuristics is provided. A two-stage stochastic capacitated location-allocation problem in emergency logistics is considered by Y. Deng et al., where the number and capacities of supply centers are uncertain and had to be determined. To solve this problem, a two-stage expected value model and a generalized cost function are proposed. An improved particle swarm optimizer with Gaussian cloud operator, restart strategy, and adaptive parameter strategy is used, as well as using the interior point method instead of the simplex method in the second stage. The proposed methods improve precision and convergence rates when compared with the classic one-stage expected value model. The reconfiguration of smart grid with distributed gener- ation is studied by C. Ma et al., using a dual hybrid particle swarm optimization (an improved binary particle swarm optimization algorithm was used in branch group search, and the proposed group binary particle swarm optimization search algorithm was used for searching within the group). 1. Introduction The purpose of this special issue is to publish high-quality research or These methodologies are empowering the enhancement in engineering design and optimization practices in areas in which classical optimization techniques are still not able effective. Indeed the aforementioned requisites and limitations are usual, such as the nondifferentiability of the Mathematical Problems in Engineering 2 compared with two literature models, being optimum designs validated with the ANSYS software package. review articles that address recent development from a variety of engineering fields in relation to the application of evolutionary algorithms and metaheuristics for design and optimization and that, hopefully, will stimulate other researchers to continue the efforts to improve the current state of the art on the aforementioned field. t F. Wu and J. Xu present an optimization method to evaluate the porosity of tight reservoirs by the use of a modified multicomponent model to a mixed-matrix model and a simulated annealing algorithm. The method is validated with a set of data from tight reservoirs. A hybrid reliability-based design optimization (RBDO) algorithm is proposed by H. M. Gomes and L. L. Corso, which combines characteristics of genetic algorithms and particle swarm optimization and sequential quadratic programming for local search. The hybrid method is analyzed based on three structural trusses RBDO benchmark examples for sizing optimization with stress, displacements, and frequency constraints. References [1] H. Schwefel, Numerische optimierung von computer-modellen mittels der evolutionsstrategie, Birkh¨auser, Basel and Stuttgart, 1977. [2] I. Rechenberg, Evolutionstrategie—optimierung technischer sys- teme nach prinzipien der biologischen evolution, Fromman- Holzboog, 1973. [3] D. E. Goldberg and M. P. Samtani, “Engineering optimization via genetic algorithm,” in Proceedings of the 9th Conference on Electronic Computation ASCE, pp. 471–482, New York, NY, USA, 1986. [4] D. E. Goldberg, Genetic Algorithms for Search, Optimisation, and Machine Learning, vol. 27, Addison-Wesley, Reading, 1989. 2.4. Control. The optimized torque-distribution control method is a critical technology for front/rear axle electric wheel loader (FREWL) to improve the operation perfor- mance and energy efficiency. A weighted sum approach for minimization of mean and variance of tire workload and maximization of total motor efficiency on a longitudinal dynamics model of FREWL is proposed by Z. Yang et al. The following optimization algorithms are used to solve the prob- lem: quasi-newton Lagrangian multiplier method, sequential quadratic programming, adaptive genetic algorithms, and particle swarm optimization with random weighting and nat- ural selection. Results confirm advantages of the controlled FREWL over noncontrolled FREWL. [5] P. Hajela, “Genetic search - An approach to the nonconvex optimization problem,” AIAA Journal, vol. 28, no. 7, pp. 1205– 1210, 1990. [6] K. Deb, “Optimal design of a welded beam via genetic algo- rithms,” AIAA Journal, vol. 29, no. 11, pp. 2013–2015, 1991. [7] G. Winter, J. Periaux, M. Galan, and P. Cuesta, Genetic Algo- rithms in Engineering and Computer Science, John Wiley & Sons, 1996. [8] D. Quagliarella, J. Periaux, C. Poloni, and G. Winter, Genetic Algorithms and Evolution Strategy in Engineering and Computer Science: Recent Advances and Industrial Applications, John Wiley & Son Ltd., 1998. [9] K. Giannakoglou, D. T. Tsahalis, J. Periaux, and T. Fogarty, Evolutionary Methods for Design, Optimization and Control, CIMNE, 2002. A genetic optimization dual fuzzy immune Proportional- Integral-Derivative (GODFIP) controller is proposed by A. Dai et al., considering energy savings, stability, accuracy, and rapidity. Its structure consists of two fuzzy controllers, a PID controller, an immune algorithm, and a genetic optimization algorithm. It is designed and simulated to control an infrared radiation and convection grain dryer represented by an iden- tified autoregressive with exogenous input (NARX) model, improving the control performance of a fuzzy immune PID controller. [10] G. Bugeda, J. A. Desideri, J. Periaux, M. Schoenauer, and G. Winter, “Evolutionary methods for design, optimization and control. 2. Scientific Contributions of the Special Issue From the simulations on the IEEE 33-bus distribution power system, after the reconfiguration of the distributed power grid, the loss of the distribution network is reduced, and the quality of the power supply voltage and the power quality of the grid are improved. T. A. S. Masutti and L. N. de Castro present a thorough review of bee-inspired methods designed to solve the vehicle routing problems. A taxonomy of methods was detailed and the review followed considering problems solved and modifications introduced in the bee-inspired algorithms. Additionally, the TSPoptBees algorithm, a modification of the original optBees purposely focused to solve the traveling salesman problem (TSP), is compared with other optimiza- tion methods inspired by the behavior of bees to solve a set of 28 instances of the TSPLIB with competitive results.f M. Tan et al. introduce a multiobjective optimization model of Hot Rolling Production Scheduling Problem under Time-of-Use electricity pricing, for simultaneous minimiza- tion of electricity costs in production and minimizing the total penalties caused by jumps between adjacent slabs. A nondominated sorting genetic algorithm-II (NSGA-II) based production scheduling was performed to obtain nondomi- nated solutions, and TOPSIS decision-making method was used for final solution selection. Experiments confirm the success of the approach. p Differential Evolution is compared with genetic algo- rithms to solve the Electric Vehicle Routing Problem, by J. Barco et al. The problem is based on a scheme to coordinate the battery electric vehicles’ (BEV) routing and recharge scheduling, considering operation and battery degradation costs. The model is based on the longitudinal dynamics equation of motion estimating the energy consumption of each BEV, where a case study, airport shuttle service scenario, is solved.h 2.2. Structural and Civil Engineering. J. I. Pelaez et al. present a memetic algorithm for the design of Symmetric Laminated Composites and Structures, taking into account in the fitness function economic and safety criteria in design and imple- menting a set of local search operators. It is compared with other four metaheuristics. The model has been tested with the design of a plate under distributed 𝑁𝑥and 𝑁𝑦loading and The irregular strip packing problem, present in many production processes in factories, with a rectangular stage, a fixed width, and an unlimited length, is solved in the work Mathematical Problems in Engineering 3 Acknowledgments proposed by B. A. J´unior et al., combining a collision-free region placement procedure with a parallel Biased Random- Key Genetic Algorithm with multiple subpopulations, where the objective is to minimize the required area to allocate the demand. The approach is tested in a set of EURO Special Interest Group on Cutting and Packing (ESICUP) problems and compared with other six optimization algorithms. proposed by B. A. J´unior et al., combining a collision-free region placement procedure with a parallel Biased Random- Key Genetic Algorithm with multiple subpopulations, where the objective is to minimize the required area to allocate the demand. The approach is tested in a set of EURO Special Interest Group on Cutting and Packing (ESICUP) problems and compared with other six optimization algorithms. The invited editors of the special issue gratefully thank all the reviewers that have contributed to the process of developing this special issue. We hope that the works selected for publication will inspire the engineering design and optimization community to apply new and state-of-the-art metaheuristics/evolutionary algorithms to their challenging applicative and industrial problems. F. Alonso-Pecina and D. Romero propose a two-step method to solve the Train Design Optimization Problem, where the first step aims to produce an initial feasible solution and the second uses simulated annealing to improve the initial solution, followed by procedures that attempt to decrease the number of required trains without incrementing the overall cost. Experiments cover well-known instances improving other optimization methods. David Greiner Jacques Periaux Domenico Quagliarella Jorge Magalhaes-Mendes Blas Galv´an p g p I. Stojanovi´c et al. solve the constrained nonconvex optimization Weber problem with feasible region bounded by arcs, with four swarm-intelligence techniques: the arti- ficial bee colony (ABC) for constrained optimization, the crossover-based ABC algorithm, the firefly algorithm for constrained optimization, and the enhanced firefly algorithm; also a heuristic algorithm based on the modified Weiszfeld procedure is used. The crossover-based ABC outperforms the other metaheuristics (and also the heuristic algorithm) with respect to the quality of the results, robustness, and computational efficiency, in the experiments published in this work. [16] J. Magalhaes-Mendes and D. Greiner, “Evolutionary algorithms and metaheuristics in civil engineering and construction man- agement,” in Computational Methods in Applied Sciences, vol. 39, Springer, New York, NY, USA, 2015. References Applications to industrial and societal problems,” CIMNE, 2003. [11] C. Coello Coello, G. Lamont, and D. Van Veldhuizen, Evolu- tionary Algorithms for Solving Multi-Objective Problems, vol. 5, Springer, 2007. [12] P. Neittaanm¨aki, J. P´eriaux, and T. Tuovinen, “Evolutionary and deterministic methods for design, optimization and control. Applications to industrial and societal problems,” CIMNE, 2008. 2.5. Other Applications/Military. The multiobjective weapon target assignment (WTA) problem under uncertainty, whose goals are to obtain maximum interception efficiency and minimum interception consumption, is optimized by H. Xu et al., with a multiobjective quantum-behaved particle swarm optimization with double/single well (MOQPSO-D/S), and compared with other PSO variants. [13] T. Burczynski and J. P´eriaux, Evolutionary and Deterministic Methods for Design, Optimization And Control, CIMNE, 2011. [14] A. E. Eiben and J. Smith, “From evolutionary computation to the evolution of things,” Nature, vol. 521, no. 7553, pp. 476–482, 2015. Mathematical Problems in Engineering 4 [15] D. Greiner, B. Galvan, J. Periaux, N. Gauger, K. Giannakoglou, and G. Winter, “Advances in evolutionary and deterministic methods for design, optimization and control in engineering and sciences,” in Computational Methods in Applied Sciences, vol. 36, Springer, New York, NY, USA, 2015. [16] J. Magalhaes-Mendes and D. Greiner, “Evolutionary algorithms and metaheuristics in civil engineering and construction man- agement,” in Computational Methods in Applied Sciences, vol. 39, Springer, New York, NY, USA, 2015. References Submit your manuscripts at https://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mathematics Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mathematical Problems in Engineering Hindawi Publishing Corporation http://www.hindawi.com Diﬀerential Equations International Journal of Volume 2014 Applied Mathematics Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Probability and Statistics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mathematical Physics Advances in Complex Analysis Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Optimization Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Combinatorics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Operations Research Advances in Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Function Spaces Abstract and Applied Analysis Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Mathematics and Mathematical Sciences Hindawi Publishing Corporation http://www.hindawi.com Volume 201 The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Algebra Discrete Dynamics in Nature and Society Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Decision Sciences Advances in #HRBQDSDĮ,@SGDL@SHBR Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Stochastic Analysis International Journal of Submit your manuscripts at https://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Diﬀerential Equations International Journal of Volume 2014 Applied Mathematics Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Probability and Statistics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Operations Research Advances in The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Algebra Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Decision Sciences Advances in Applied Mathematics Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Probability and St Hindawi Publishing Corporation http://www.hindawi.com Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Operations Research Advances in Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Algebra Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Decision Sciences Advances in Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Operations Research Advances in Probability and Statistics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Hindawi Publishing Corporation http://www hindawi com Volume 2014 Decision Sciences Advances in Probability and Statistics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Applied Mathematics Journal of Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Operations Research Advances in The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Diﬀerential Equations International Journal of Volume 2014 The Scientific World Journal Hindawi Publishing Corporation h // hi d i V l 2014 Submit your manuscripts at https://www.hindawi.com Submit your manuscripts at https://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mathematics Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mathematical Problems in Engineering Complex Analysis Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Optimization Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Function Spaces Abstract and Applied Analysis Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Mathematics and Mathematical Sciences Hindawi Publishing Corporation http://www.hindawi.com Volume 201 Discrete Dynamics in Nature and Society Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 #HRBQDSDĮ,@SGDL@SHBR Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Stochastic Analysis International Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mathematics Journal of
https://openalex.org/W2937094098	https://ri.conicet.gov.ar/bitstream/11336/123114/2/CONICET_Digital_Nro.259ae054-c36f-410f-bcc4-c090488adfb7_A.pdf	English	null	Challenges and opportunities for control and elimination of soil-transmitted helminth infection beyond 2020	PLoS neglected tropical diseases	2,019	cc-by	5,843	POLICY PLATFORM a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Challenges and opportunities for control and elimination of soil-transmitted helminth infection beyond 2020 Matthew C. FreemanID1, Oladele Akogun2, Vicente Belizario, Jr.3, Simon J. Brooker4, Theresa W. Gyorkos5, Rubina Imtiaz6, Alejandro KrolewieckiID7, Seung Lee8, Sultani H. MatendecheroID9, Rachel L. Pullan10, Ju¨rg UtzingerID11,12 Matthew C. FreemanID1, Oladele Akogun2, Vicente Belizario, Jr.3, Simon J. Brooker4, Theresa W. Gyorkos5, Rubina Imtiaz6, Alejandro KrolewieckiID7, Seung Lee8, Sultani H. MatendecheroID9, Rachel L. Pullan10, Ju¨rg UtzingerID11,12 Matthew C. FreemanID1, Oladele Akogun2, Vicente Belizario, Jr.3, Simon J. Brooker4, Theresa W. Gyorkos5, Rubina Imtiaz6, Alejandro KrolewieckiID7, Seung Lee8, Sultani H. MatendecheroID9, Rachel L. Pullan10, Ju¨rg UtzingerID11,12 1 Department of Environmental Health, Emory University, Atlanta, Georgia, United States of America, 2 Modibbo Adama University of Technology, Yola, Nigeria, 3 College of Public Health, University of the Philippines Manila, Manila, the Philippines, 4 Global Health, Bill & Melinda Gates Foundation, Seattle, Washington, United States of America, 5 Department of Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal, Quebec, Canada, 6 Children Without Worms, The Task Force for Global Health, Decatur, Georgia, United States of America, 7 Instituto de Investigaciones en Enfermedades Tropicales, Universidad Nacional de Salta, Oran, Argentina, 8 Save the Children, Washington, DC, United States of America, 9 Ministry of Health, Nairobi, Kenya, 10 London School of Hygiene & Tropical Medicine, London, United Kingdom, 11 Swiss Tropical and Public Health Institute, Basel, Switzerland, 12 University of Basel, Basel, Switzerland a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 matthew.freeman@emory.edu Introduction More than half of the world’s population lives in places endemic for soil-transmitted helminths (STHs), and an estimated 1.45 billion people are infected [1,2]. In 2017, the global burden of STH infection (Ascaris lumbricoides, hookworm, and Trichuris trichiura) was estimated at 1.9 million disability-adjusted life years (DALYs) [3]. Moderate and heavy infection intensity and chronic STH infection are associated with anemia, malnutrition, educational loss, and cogni- tive deficits, but recent systematic reviews and meta-analyses produced conflicting results on the impact of preventive chemotherapy (PC) [4–6]. Editor: Joanne P. Webster, Imperial College London, UNITED KINGDOM Editor: Joanne P. Webster, Imperial College London, UNITED KINGDOM Published: April 11, 2019 Copyright: © 2019 Freeman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OPEN ACCESS Citation: Freeman MC, Akogun O, Belizario V, Jr., Brooker SJ, Gyorkos TW, Imtiaz R, et al. (2019) Challenges and opportunities for control and elimination of soil-transmitted helminth infection beyond 2020. PLoS Negl Trop Dis 13(4): e0007201. https://doi.org/10.1371/journal. pntd.0007201 The Soil-Transmitted Helminthiasis Advisory Committee (hereafter called “the Commit- tee”) is a group of independent experts with a broad range of expertise. It is convened annually by Children Without Worms (CWW), an organization whose purpose is to utilize available evidence to identify best practices and opportunities for the prevention and control of STH infection [7]. On November 1 and 2, 2017, the Committee met in Baltimore, Maryland, United States of America, in order to discuss the critical need to develop a data-driven guide to the STH endgame on late-stage program functioning, processes, and surveillance. The focus was on research and field experiences from countries approaching the “elimination of STH infec- tion as a public health problem” after consecutive years of PC and countries that are now con- sidering scaling down their PC frequency but may be concerned about infection rebound. Emphasis was placed on interim recommendations for monitoring and decision-making for national program managers desiring to achieve the World Health Organization (WHO) goal of eliminating STH infection as a public health problem by 2020, particularly related to STH infections in risk groups other than school-age children (SAC), namely preschool-age children (PSAC) and women of reproductive age (WRA) [8]. The following is the Committee’s recom- mendations stemming from the Baltimore meeting in November 2017. It complements and updates the publication derived by the Committee’s meeting a year earlier in Basel, Switzerland The 2020 roadmap and beyond In its roadmap for implementation for 2020, WHO set a goal to achieve at least 75% coverage of PC—either annual or biannual—of SAC and PSAC [9–11]. As we approach 2020, it is imperative that we not only accelerate what has worked for the control of STH infection– related morbidity but that we look beyond 2020 and better understand what more is required to eliminate STH infection as a public health problem. Fig 1 summarizes progress made in terms of both coverage and impact using 2016 country data. Despite considerable gains in SAC coverage, less than half of the at-risk countries are treating PSAC, a number that has not changed much in recent years, yielding a total coverage level of approximately 50% but with considerable variability of coverage from year to year and across countries (Fig 2). If the current trend in PC coverage persists, PSAC and the combined group of children aged 1–14 years will not reach the goal by 2020. Accelerating PSAC PC cov- erage might avoid that failure, and hence, needs a clear, strong global policy now. While the initial focus on SAC coverage has enabled the mobilization of resources, other priorities have emerged, both in places and populations in which the target was not achieved and within areas that are now shifting priority from scaling up PC to eliminating STH infection as a public health problem—defined by the WHO as when less than 1% of the at-risk population has mod- erate or heavy infection (MHI) [8]—and potentially interrupting the transmission of STH infection [12]. WHO recommends stopping PC once less than 1% of the at-risk population has MHI infections. Surveillance will, however, need to continue in order to pick up potential recurrence of infection and to plan further intervention, if warranted. Hence, as we turn our sights towards and beyond 2020, the Committee recognized it as timely to review and assess the successes and challenges of progress made to date. Indeed, it is hoped that the Baltimore 2017 meeting deliberations will inform future control strategies and targets. [7], and was instrumental in shaping the agenda for the October 2018 meeting, convened jointly by CWW and WHO, with recommendations to be reported elsewhere. [7], and was instrumental in shaping the agenda for the October 2018 meeting, convened jointly by CWW and WHO, with recommendations to be reported elsewhere. Published: April 11, 2019 Copyright: © 2019 Freeman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. 1 / 10 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007201 April 11, 2019 The 2020 roadmap and beyond There have been many important developments by the time the Committee convened in November 2017, but six are particularly noteworthy: • updated PC guidelines published by WHO for all at-risk populations [13]; • the Bellagio Declaration focusing on girls and WRA [14]; • widening of WHO’s engagement with experts around the globe [15]; • success of the Global Program to Eliminate Lymphatic Filariasis (GPELF) [16]; • increasing importance for deworming programs to measure impact [17]; and • the launch by WHO-AFRO of the Expanded Special Project on the Elimination of Neglected Tropical Diseases (ESPEN) portal (http://espen.afro.who.int), which provides—for the first time—subnational data on disease endemicity and PC coverage for each of the five PC-NTDs, including STH infection. In this Policy Platform, we lay out critical challenges in seven key areas that need attention, discuss progress until November 2017, and put forward recommendations for immediate action. Our recommendations arose at a critical juncture for STH control efforts, as current global policies, goals, and related strategies and resources are revitalized through 2020, a year which is upon us. 2 / 10 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007201 April 11, 2019 Challenge #1: Incomplete and inconsistent monitoring of program impact Recommendation: Define standard impact goals and targets post-2020. To date, pro- grams mainly focused on reporting on PC coverage, as guided by a simplified coverage target specified by the Roadmap; yet there are few standardized data on program impact. It is note- worthy that country programs are increasingly interested in quantifying the impact of deworming on health outcomes [17–19]. Current approaches to measuring impact vary across countries and across deworming program implementers, limiting comparability and the possi- bility to appreciate changes over time and across countries and regions. Moreover, impact data are not readily available, with most of the evaluations conducted by research groups. For the Africa region, there has been recent progress in data sharing and transparency for neglected tropical diseases (NTDs), through the work of ESPEN and their data portal [20]. Currently, there is availability of data on endemicity and coverage at the subnational level for the five PC-NTDs, including STH infection, for 47 of 49 countries in the AFRO region. This successful approach to reporting sub-national data should be expanded to the other WHO regions where STHs are endemic. The ESPEN portal is starting to include impact assessment data for lymphatic filariasis and onchocerciasis and there is an opportunity to include comparable data for STH infec- tion. However, this will require a standardized and comprehensive monitoring and evalua- tion (M&E) framework that is tied to clearly defined, quantitative goals. For example, globally, a reported 69.5% of SAC and 50.8% of PSAC requiring PC reportedly received PC in 2016 [21]. It is conceivable that the observed scale-up of PC targeting STH over the past decade was a major contributor to the decline in the global burden of STH infection [22– 25]; yet the precise extent to which this coverage has reduced prevalence, intensity, and burden of STH infection is not known. Country programs, in particular, require a compre- hensive, standardized, yet flexible approach to measure progress toward morbidity-related goals. Such an approach would capture essential programmatic elements, and be used by each country to map their needs, commitment, and resources. This will generate realistic timelines and planning processes as well as alerting WHO, partners, and donors to better assess the resource and technical capacity needs of each program. Challenge #1: Incomplete and inconsistent monitoring of program impact Subnational data with standardized indicators for anthelmintic drug availability and coverage of the target popula- tions are critical to track progress at the subnational level where PC program implementa- tion may not be uniform. Data can also be disaggregated (e.g., by district, sex, and age categories, and any other useful determinant) to better understand the equity of program access and delivery for the three target risk groups (i.e., PSAC, SAC, and WRA). PC needs for refugees and migrants are also increasingly being recognized, and hence, WHO is con- sidering to add these populations to the at-risk groups, while some countries (especially those in AFRO and EMRO regions) are actively engaged in estimating access, burden, and resource issues for migrants. Fig 1. Progress for PC coverage (by country) in SAC and where STH infection is no longer considered a public health problem (MHI <1%). Source: 2016 PC data from WHO. Country with detailed epidemiologic information available. MHI, moderate or heavy infection; PC, preventive chemotherapy; SAC, school-age children; STH, soil-transmitted helminth. https://doi.org/10.1371/journal.pntd.0007201.g001 Fig 1. Progress for PC coverage (by country) in SAC and where STH infection is no longer considered a public health problem (MHI <1%). Source: 2016 PC data from WHO. Country with detailed epidemiologic information available. MHI, moderate or heavy infection; PC, preventive chemotherapy; SAC, school-age children; STH, soil-transmitted helminth. https://doi.org/10.1371/journal.pntd.0007201.g001 Fig 1. Progress for PC coverage (by country) in SAC and where STH infection is no longer considered a public health problem (MHI <1%). Source: 2016 PC data from WHO. Country with detailed epidemiologic information available. MHI, moderate or heavy infection; PC, preventive chemotherapy; SAC, school-age children; STH, soil-transmitted helminth. https://doi.org/10.1371/journal.pntd.0007201.g001 Fig 2. Global PSAC treatment and coverage, 2006–2016. Source: WHO PC Databank, PSAC PC coverage data from 2006–2016; http://www.who.int/ neglected_diseases/preventive_chemotherapy/lf/en/. Coverage is calculated by dividing the number of children requiring PC and treated by the total number of children in need of PC. PC, preventive chemotherapy; PSAC, preschool children; WHO, World Health Organization. https://doi.org/10.1371/journal.pntd.0007201.g002 Fig 1. Progress for PC coverage (by country) in SAC and where STH infection is no longer considered a public health problem (MHI <1%). Source: 2016 PC data from WHO. Country with detailed epidemiologic information available. MHI, moderate or heavy infection; PC, preventive chemotherapy; SAC, school-age children; STH, soil-transmitted helminth. https://doi.org/10.1371/journal.pntd.0007201.g001 https://doi.org/10.1371/journal.pntd.0007201.g001 Fig 2. Global PSAC treatment and coverage, 2006–2016. Source: WHO PC Databank, PSAC PC coverage data from 2006–2016; http://www.who.int/ neglected_diseases/preventive_chemotherapy/lf/en/. Coverage is calculated by dividing the number of children requiring PC and treated by the total number of children in need of PC. PC, preventive chemotherapy; PSAC, preschool children; WHO, World Health Organization. https://doi.org/10.1371/journal.pntd.0007201.g002 Fig 2. Global PSAC treatment and coverage, 2006–2016. Source: WHO PC Databank, PSAC PC coverage data from 2006–2016; http://www.who.int/ neglected_diseases/preventive_chemotherapy/lf/en/. Coverage is calculated by dividing the number of children requiring PC and treated by the total number of children in need of PC. PC, preventive chemotherapy; PSAC, preschool children; WHO, World Health Organization. https://doi.org/10.1371/journal.pntd.0007201.g002 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007201 April 11, 2019 3 / 10 distribution of infection. Indeed, in many settings, adults are at particularly high risk of hook- worm infection and thus contribute substantially to transmission [30]. These issues are in con- trast to countries where school-based deworming has been coupled with community-based programs in both experimental studies and as part of lymphatic filariasis control programs that target entire communities [31]. Using an exclusive school-based intervention platform potentially excludes 12.8% SAC who are out of school, with some countries like South Sudan having 66% of their SAC out of school [32]. With that and the availability of a pediatric meben- dazole preparation as a donation for PSAC, out-of-school children and PSAC can be immedi- ately prioritized to help achieve the overall children’s coverage goal. Recent progress has been made on a more comprehensive preventive approach to include all risk groups, as demon- strated by the Kenya “Breaking Transmission Strategy” in addition to community-wide PC coverage through the lymphatic filariasis elimination program through USAID. Successful PC interventions targeting SAC by most countries are generating requests for specific guidance on the next operational phase: how to efficiently implement sentinel surveillance and which indi- cators to measure in order to predict, detect, and treat widely dispersed and persistent pockets of transmission. There is a need to consider how to scale up approaches to reach PSAC, given the current drug availability and added cost of this approach. Consistent with these needs is a recognized gap in our current knowledge of disease transmission at low prevalence and persis- tent environmental factors that facilitate transmission. Thus, there is a renewed need to clearly identify research gaps and questions that would facilitate implementation in these settings. y g p q p g A recent report has provided guidance and recommendations for WRA [33]. Building on this report, WHO needs to develop implementation guidelines linked to clearly defined tar- gets, both for WRA and PSAC as critical populations at risk of high STH burden [7,14]. Opera- tional research is also needed to define platforms, partners, and recording and reporting tools to monitor progress of control programs targeting PSAC and WRA. In addition, there remains the challenge of providing additional anthelmintic drugs necessary to treat WRA (not targeted by the current donations). There is optimism that there will be the possible donation of chew- able Vermox from Johnson & Johnson (J&J) for PSAC, but the quantities donated may not be enough to cover the total numbers at risk. Consideration of new drug options for WRA needs immediate discussion at national, regional, and global fora. Data from the GPELF and other community-based PC programs may provide insight into efficacy and safety issues through birth cohort studies. Challenge #2: Reaching at-risk groups other than SAC Recommendation: Identify new PC strategies, platforms, and reporting mechanisms. Deworming of SAC has been shown to reduce disease burden, especially reducing high-bur- den infections in a cost-effective manner, but the empirical evidence from both multiyear deworming programs and modeling studies suggest that targeting SAC alone for PC is insuffi- cient for sustained control and elimination of STH infection [17,26,27]. In particular, this is the case for T. trichiura due to poor drug efficacy [28,29] and for hookworm due to the age PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007201 April 11, 2019 4 / 10 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007201 April 11, 2019 standardized approach to the monitoring of potential emerging resistance needs to be estab- lished, especially in those countries that have mature PC programs (e.g., Mexico and Togo, among others), to track both drug efficacy and mutations known to be associated with resis- tance [37]. Challenge #5: Limited efficacy of current drugs and gaps in drug availability Recommendation: Promote research into combination therapies and fast track pre- qualification processes. The existing anthelmintic drugs have variable efficacies against dif- ferent STH species, with particularly low efficacies against T. trichiura when using single-dose treatments [29]. New efforts must be undertaken to identify and provide guidelines for use cases for combination therapies in general and specifically in which T. trichiura is the predom- inant species [42]. In 2018, the WHO Essential Medicines Committee approved the inclusion of ivermectin for both STH and Strongyloides stercoralis to the WHO Model List of Essential Medicines [43]. However, there are implications and potential challenges in adding ivermectin to albendazole and mebendazole in STH deworming programs. Merck provides ivermectin dedicated for the control of lymphatic filariasis and onchocerciasis, and new manufacturers will need to become prequalified to meet the growing demand from STH control programs. Additionally, bioequivalence studies and other considerations will be needed to make the drug available at low cost. This will take time and requires innovative financing mechanisms. Challenge #4: Poor diagnostics to assess program needs by implementation stage Recommendation: Employ validated program stage-specific diagnostic techniques. There is a need for new diagnostics that are appropriate for informing key decision points for national STH control programs [38,39]. The Kato–Katz technique, while relatively inexpen- sive, widely used, and sensitive in detecting MHI, will have lower positive predictive values in low-prevalence (and low-intensity) settings [40,41]. In addition, some preliminary analysis from microscopy and PCR has suggested that hookworm infection may be misidentified. New diagnostic tests have been validated [7], yet there is a need for novel, highly sensitive tests that can be employed in settings that move from STH control to elimination. However, their spe- cific role and use in the context of national program implementation needs to be assessed [41]. Furthermore, capacity strengthening for national programs will be needed to fully take advan- tage of any new diagnostics. Field and laboratory protocols need to be standardized, reference laboratories established, and training developed for different contexts and languages. Gaps in the current diagnostic landscape have been identified, and formative research is underway. The next steps are to finalize and disseminate results and to identify additional resources for gap areas (e.g., sustaining animal models, conducting field studies to calculate test perfor- mance in various settings, etc.). Guidelines will then need to be developed to inform country programs on how to incorporate these tests for improved assessment of program impact and further planning. Challenge #3: The risk of anthelmintic drug resistance Recommendation: Develop standardized indicators to detect emerging resistance. Experience from the veterinary sector demonstrated that anthelmintic drug resistance devel- oped after years of large-scale monotherapy [34]. We suspect that if we wait until resistance is clinically detected in humans, it will be too late to respond [35]. While progress is being made, there are currently no routine, field-applicable diagnostics that can effectively identify and monitor signs of emerging resistance, so research on developing such tests urgently needs financial support. Indeed, we need to better identify resistant genes and to track refu- gia (i.e., that proportion of the worm population that remains susceptible to anthelmintic drugs). Human populations with a long history of single-drug deworming that have low worm burdens but have not reached transmission break points are likely to be at the greatest risk for the development of drug-resistant parasites. For diagnostic approaches such as quan- titative polymerase chain reaction (qPCR) to be used in an STH-programmatic setting, we need to take molecular diagnostics to a level at which we can use its full potential by stan- dardizing analysis and reporting and including appropriate quality control measures [36]. A 5 / 10 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007201 April 11, 2019 strategies [11]. The STH community has largely avoided establishing an approach for address- ing WASH or establishing a structure for engagement with the WASH sector. However, the recent development of the WASH–NTD joint strategy provides an entry point and guidance for improved communication, coordination, and collaboration [45]. Ample observational evi- dence [46,47], some recent randomized trials, biological plausibility, and history suggest that improved WASH is critical in the control and elimination of STH infection. Alignment with the WASH sector, specifically including WASH indicators as part of STH M&E [48] (and vice versa) by using data to advocate for WASH activities in STH-endemic areas, employing pro- gram monitoring, and conducting operational research and advocacy to ensure normative inclusion of STH-related WASH behaviors (e.g., shoe-wearing [49]), would be valuable contri- butions in STH control programming. Better quantification of the specific mechanisms and use of consistent indicators across programs would provide support to the WASH sector on gaps in typical WASH programming (e.g., type of water sources or food hygiene). Outlook There has been substantial progress in increasing coverage of PC for the control of STH infec- tion, particularly among SAC and, to a lesser extent, among PSAC. As we approach 2020, work remains to accelerate action to achieve these targets in many places. At the same time, we need to think critically about what is needed beyond the 2020 roadmap and increase efforts in the seven areas discussed in this Policy Platform. This needs to be achieved through active col- laboration and coordination by pertinent government ministries, researchers, donors, WHO, drug manufacturers, and multisectoral collaboration [50]. In doing so, it will help ensure prog- ress toward eliminating STH infection—and other NTDs—as a public health problem, and it will yield more efficient allocation of resources and greater sustained impact, driven by targets and thresholds based on scientific evidence. Challenge #7: New targets needed for post-2020 Recommendation: Develop clearly defined, quantitative program targets for all at-risk groups and move beyond PC coverage estimates. One of the most important next steps as we move toward 2020 and beyond is the need to establish clearly defined, quantitative program goals and targets post-2020. The current targets of achieving at least 75% coverage for deworm- ing for SAC (and PSAC) is in reach, but we know that even meeting this target is insufficient to achieve elimination of STH as a public health problem. We must also critically evaluate whether the threshold of 1% prevalence of MHI is useful for M&E of STH morbidity control and mov- ing toward elimination. There is a need to look beyond simple PC coverage measures and include impact targets for PSAC and WRA, benchmarks for WASH that will encourage invest- ment in WASH in STH-endemic areas, and estimates of PC uptake along the distribution chain that would give accurate estimates for not only availability of the drug, but population compliance. Addressing Challenges 1–6, articulated above, will support this critical effort. Challenge #6: Limited coordination with the water, sanitation, and hygiene (WASH) sector Recommendation: Identify WASH indicator(s) to be included in routine STH M&E. 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https://openalex.org/W4390196664	https://preprints.ru/files/1380	Russian	null	ОПЫТ ЭКСПЕРТНОГО ВЗАИМОДЕЙСТВИЯ МЕЖДУ ПАТЕНТНЫМИ ВЕДОМСТВАМИ МИРА	null	2,023	cc-by	7,000	УДК 347.779.1.14 ОПЫТ ЭКСПЕРТНОГО ВЗАИМОДЕЙСТВИЯ МЕЖДУ ПАТЕНТНЫМИ ВЕДОМСТВАМИ МИРА1 Журавлев Андрей Львович ФГБУ «Федеральный институт промышленной собственности», Москва, начальник Центра международной кооперации ФИПС Роспатента, к.ю.н., AZuravlev@rupto.ru Дарина Ольга Николаевна ФГБУ «Федеральный институт промышленной собственности», Москва, старший научный сотрудник otd3226@rupto.ru АННОТАЦИЯ: На сегодняшний день существуют несколько программ экспертного взаимодействия между национальными патентными ведомствами мира (НПВ), региональными европейскими ведомствами (ЕПВ, ЕАПВ) и Международным бюро Всемирной организации интеллектуальной собственности (МБ ВОИС). Изучение опыта практической работы в рамках таких международных программ взаимодействия станет основой создания новых механизмов взаимодействия между экспертами национальных патентых ведомств в евразийском регионе. Роспатент и Евразийское патентное ведомство (ЕАПВ) проводят совместную работу по созданию основ единого евразийского патентного экспертно-информационного пространства для целей оказания более качественных патентых услуг по охране ОИС для всех заявителей евразийского региона. В статье представлен аналитический обзор международных механизмов взаимодействия экспертов ведущих патентых ведомств мира. Проанализированы перспективы использования этих механизмов экспертного взаимодействия в работе патентных ведомств (ПВ) стран- УДК 347.779.1.14 ОПЫТ ЭКСПЕРТНОГО ВЗАИМОДЕЙСТВИЯ МЕЖДУ ПАТЕНТНЫМИ ВЕДОМСТВАМИ МИРА1 Журавлев Андрей Львович ФГБУ «Федеральный институт промышленной собственности», Москва, начальник Центра международной кооперации ФИПС Роспатента, к.ю.н., AZuravlev@rupto.ru Дарина Ольга Николаевна ФГБУ «Федеральный институт промышленной собственности», Москва, старший научный сотрудник otd3226@rupto.ru АННОТАЦИЯ: На сегодняшний день существуют несколько программ экспертного взаимодействия между национальными патентными ведомствами мира (НПВ), региональными европейскими ведомствами (ЕПВ, ЕАПВ) и Международным бюро Всемирной организации интеллектуальной собственности (МБ ВОИС). Изучение опыта практической работы в рамках таких международных программ взаимодействия станет основой создания новых механизмов взаимодействия между экспертами национальных патентых ведомств в евразийском регионе. Роспатент и Евразийское патентное ведомство (ЕАПВ) проводят совместную работу по созданию основ единого евразийского патентного экспертно-информационного пространства для целей оказания более качественных патентых услуг по охране ОИС для всех заявителей евразийского региона. В статье представлен аналитический обзор международных механизмов взаимодействия экспертов ведущих патентых ведомств мира. Проанализированы перспективы использования этих механизмов экспертного взаимодействия в работе патентных ведомств (ПВ) стран- членов Евразийской патентной Конвенции (ЕПК). ОПЫТ ЭКСПЕРТНОГО ВЗАИМОДЕЙСТВИЯ МЕЖДУ ПАТЕНТНЫМИ ВЕДОМСТВАМИ МИРА1 АННОТАЦИЯ: На сегодняшний день существуют несколько программ экспертного взаимодействия между национальными патентными ведомствами мира (НПВ), региональными европейскими ведомствами (ЕПВ, ЕАПВ) и Международным бюро Всемирной организации интеллектуальной собственности (МБ ВОИС). Изучение опыта практической работы в рамках таких международных программ взаимодействия станет основой создания новых механизмов взаимодействия между экспертами национальных патентых ведомств в евразийском регионе. Роспатент и Евразийское патентное ведомство (ЕАПВ) проводят совместную работу по созданию основ единого евразийского патентного экспертно-информационного пространства для целей оказания более качественных патентых услуг по охране ОИС для всех заявителей евразийского региона. В статье представлен аналитический обзор международных механизмов взаимодействия экспертов ведущих патентых ведомств мира. Проанализированы перспективы использования этих механизмов экспертного взаимодействия в работе патентных ведомств (ПВ) стран- членов Евразийской патентной Конвенции (ЕПК). 1 КЛЮЧЕВЫЕ СЛОВА: Роспатент, патентное ведомство, ПВ, Евразийское патентное ведомство, ЕАПВ, национальные патентные 1 Статья написана в рамках НИР: 2-ПО-2023 «Организационно-правовые аспекты формирования единого экспертно-информационного пространства в сфере промышленной собственности в евразийском регионе» КЛЮЧЕВЫЕ СЛОВА: Роспатент, патентное ведомство, ПВ, Евразийское патентное ведомство, ЕАПВ, национальные патентные 1 1 Статья написана в рамках НИР: 2-ПО-2023 «Организационно-правовые аспекты формирования единого экспертно-информационного пространства в сфере промышленной собственности в евразийском регионе» 1 1 Статья написана в рамках НИР: 2-ПО-2023 «Организационно-правовые аспекты формирования единого экспертно-информационного пространства в сфере промышленной собственности в евразийском регионе» 1 1 ведомства, НПВ, ведомства IP5, Европейское патентное ведомство, ЕПВ, совместные экспертные поиски, CS&E, CSP, программа ускоренного патентного делопроизводства, PPH, программы межведомственного взаимодействия патентных экспертов. THE EXPERIENCE OF EXPERT INTERACTION BETWEEN PATENT OFFICES IN THE WORLD Zhuravlev Andrey Lvovich "Federal Institute of Industrial Property", Moscow, Head of the Center for International Cooperation FIPS Rospatent, Candidate of Legal Science, AZuravlev@rupto.ru Darina Olga Nikolaevna "Federal Institute of Industrial Property", Moscow, Senior researcher otd3226@rupto.ru THE EXPERIENCE OF EXPERT INTERACTION BETWEEN PATENT OFFICES IN THE WORLD Zhuravlev Andrey Lvovich "Federal Institute of Industrial Property", Moscow, Head of the Center for International Cooperation FIPS Rospatent, Candidate of Legal Science, AZuravlev@rupto.ru Darina Olga Nikolaevna "Federal Institute of Industrial Property", Moscow, Senior researcher otd3226@rupto.ru ABSTRACT: Currently, there are several programs of examiners interaction between the national patent offices of the world (NPO), regional European offices (EPO, EAPO) and the International Bureau of the World Intellectual Property Organization (IB WIPO). The study of the experience of practical work within the area of such international cooperation programs will become the basis for the creation of new mechanisms for interaction between examiners of national patent offices in the Eurasian region. Rospatent and the Eurasian Patent Office (EAPO) are working together to create the foundations for a single Eurasian patent expert and information Area in order to provide better quality patent services for the protection of intellectual property for all applicants in the Eurasian region. The article presents an analytical review of the main international mechanisms for the interaction of examiners from the world's leading patent offices. The prospects for the use of these mechanisms of examiners interaction in the workspace of patent offices (PO) of member countries of the Eurasian Patent Convention (EPC) are analyzed. KEYWORDS: Rospatent, patent office, PO, EAPO, NPO, IP5, EPO, collaborative expert searches, CS&E, CSP, the program of accelerated patent 2 filing, Patent Prosecution Highway, PPH, programs for interofficeal interaction for patent examiners. 3 2 Сайт Ведомств IP5, [Электронный ресурс], URL: https://www.fiveipoffices.org/material, (дата обращения: 22.11.2023) 1. Введение На современном этапе взаимодействия ведомств ЕАПВ евразийского региона и Роспатента поставлена стратегическая задача – формирование в евразийском регионе общего информационно-экспертного пространства. Построение такого пространства предполагает достижение максимального сходства в предоставлении правовой охраны изобретений и промышленных образцов в евразийском регионе, а также расширения взаимодействия региональных и национальных патентных систем для повышения эффективности процедур и надежности предоставления правовой охраны таким объектам. Межведомственное экспертное взаимодействие должно быть направлено на повышение эффективности работы всех ведомств, в том числе за счет перераспределения рабочей нагрузки по рассмотрению патентных заявок, исключения дублирования проводимых работ, взаимного признания результатов работ, создания общей системы обмена и доступа к информации о результатах поиска и экспертизы. 2. Международный опыт интеграции ведомств в рамках IP5 Программа взаимодействия IP5 - это крупнейшая многосторонняя программа кооперации пяти ведомств интеллектуальной собственности в мире, созданная для повышения эффективности процесса патентной экспертизы, в условиях стремительно растущего количества заявок на изобретения. В IP5 входят следующие патентные ведомства интеллектуальной собственности 2[1]: - Европейское патентное ведомство (EPO) - Японское патентное ведомство (JPO) – Южно-Корейское управление интеллектуальной собственности (KIPO) - Национальное управление интеллектуальной собственности Китайской Народной Республики (CNIPA) - 3 3 Ведомство США по патентам и товарным знакам (USPTO). Сотрудничество IP5 началось с 2007 года на фоне постоянно увеличивающегося числа заявок, поданных одновременно в несколько ведомств, и расширяющейся географии патентования и продолжается до сих пор. В настоящее время в ведомства IP5 за год поступает порядка 3 млн. патентных заявок (более 85 % от общего числа заявок в мире), выдается 1,5 млн. патентов, ведомства IP5 выполняют 95% всей работы в рамках договора о патентной кооперации (PCT). Главной целью сотрудничества ведомств IP5 было устранение ненужного дублирования работы, которую проделывали сотрудники этих ведомств, повышение эффективности процесса патентной экспертизы по всему миру, а также повышение качества экспертизы, сокращение ее сроков, упрощение доступа к патентной системе, а также, обеспечение стабильности патентных прав и повышение правовой определенности для заявителей. В рамках работы по взаимодействию ведомств IP5 было запущено в работу восемь фундаментальных проектов в области совместного использования результатов экспертизы по заявкам. Основные программы экспертного взаимодействия IP5:  Общая документация (обмен данными, общие базы данных). Общий доступ к результатам поиска и экспертизы через совместные ресурсы;  Общая документация (обмен данными, общие базы данных). Общий доступ к результатам поиска и экспертизы через совместные ресурсы;  Общая гибридная классификация. Использование Совместной патентной классификации (CPC), обмен классификационной информацией при рассмотрении конкретных примеров классифицирования заявок, согласованные схемы и методы классификации в ведомствах;  Общая гибридная классификация. Использование Совместной патентной классификации (CPC), обмен классификационной информацией при рассмотрении конкретных примеров классифицирования заявок, согласованные схемы и методы классификации в ведомствах;  Общий формат заявок;  Общая политика обучения персонала;  Взаимный машинный перевод, улучшение его качества;  Общие правила экспертизы и управления качеством. Согласование нововведений в патентной практике;  Общие правила экспертизы и управления качеством. Согласование нововведений в патентной практике; 4 4  Единая система статистических данных. Прогнозирование перспективных направлений патентования;  Внедрение искусственного интеллекта (ИИ) в системы делопроизводства IP5 и машинного перевода.  Внедрение искусственного интеллекта (ИИ) в системы делопроизводства IP5 и машинного перевода. 5 3 Сайт IP5, Информация по Global Dossier, делопроизводство по заявкам IP5, filewrapper, [Электронный ресурс], URL: https://www.fiveipoffices.org/activities/globaldossier/ filewrapper, (дата обращения: 23.11.2023); 4Сайт IP5, Информация по Global Dossier, IP5 Global dossier: scope, content, availability and performance, [Электронный ресурс], URL: https://www.fiveipoffices.org/sites/default/files/attachments/6877d20a-3529-46e0-b4a8- 6881eef83458/Quality%2BGlobal%2BDossier%2Bpassive%2Bcomponent %2BIndustry%2BMay%2B2017.pdf, (дата обращения: 23.11.2023); 2. Международный опыт интеграции ведомств в рамках IP5 Все проекты подразумевают создание информационной среды, подходящей для совместного выполнения работы, создание инфраструктуры информационных систем, позволяющей ведомствам использовать информацию о результатах поиска и экспертизы, и обмениваться данными. В результате, ведомствами IP5 была разработана общая поисковая система, программа общего делопроизводства, схема общего патентного классифицирования, система машинного перевода текста патентных документов на разные языки, система непрерывного образования, система менеджмента качества, были определены общие параметры статистического анализа. Были внедрены такие информационные системы как: 5 - Global Dossier - предоставляет собой онлайн источник, содержащий актуальную информацию о статусе патентных семейств заявок, поданных во всем мире3 [2]. Проект единого делопроизводства по конкретной заявке - Global Dossier4 [3]. Для упрощения работы, ведомства согласились предоставлять документы в электронном виде в стандартном формате и с переводом на английский язык. Система обеспечивает автоматический машинный перевод на английский язык оригинальных китайских, японских и корейских онлайн документов. Каждое из ведомств IP5 на своих патентных сайтах предоставляют доступ в режиме реального времени к массиву своих патентных документов и их делопроизводству в сервисе Global Dossier. Таким образом, с материалами заявки можно ознакомиться либо на языке 3 Сайт IP5, Информация по Global Dossier, делопроизводство по заявкам IP5, filewrapper, [Электронный ресурс], URL: https://www.fiveipoffices.org/activities/globaldossier/ filewrapper, (дата обращения: 23.11.2023); 4Сайт IP5, Информация по Global Dossier, IP5 Global dossier: scope, content, availability and performance, [Электронный ресурс], URL: https://www.fiveipoffices.org/sites/default/files/attachments/6877d20a-3529-46e0-b4a8- 6881eef83458/Quality%2BGlobal%2BDossier%2Bpassive%2Bcomponent %2BIndustry%2BMay%2B2017.pdf, (дата обращения: 23.11.2023); - Global Dossier - предоставляет собой онлайн источник, содержащий актуальную информацию о статусе патентных семейств заявок, поданных во всем мире3 [2]. Проект единого делопроизводства по конкретной заявке - Global Dossier4 [3]. Для упрощения работы, ведомства согласились предоставлять документы в электронном виде в стандартном формате и с переводом на английский язык. Система обеспечивает автоматический машинный перевод на английский язык оригинальных китайских, японских и корейских онлайн документов. Каждое из ведомств IP5 на своих патентных сайтах предоставляют доступ в режиме реального времени к массиву своих патентных документов и их делопроизводству в сервисе Global Dossier. Таким образом, с материалами заявки можно ознакомиться либо на языке 5 5 оригинала, либо на английском языке через сайты ведомств IP5: 1. EPO: БД Espascenet, (вкладка в записи заявки – Global Dossier, либо через патентный реестр – EPO Register), (https://register.epo.org/regviewer). Заявки с датой подачи 1 июня 1978 г. по н.в.; 2. JPO: БД J-PlatPat, One Portal Dossier (OPD) на сайте ПВ Японии – (https://www.j-platpat.inpit.go.jp/p0000), ввести номер заявки и нажать на вкладку «OPD». Заявки с датой подачи с 1 июля 2003 по н.в.; 3. USPTO: Система Global Dossier на сайте США - ввести номер заявки любой страны из IP5, и система откроет делопроизводство или перенаправит на сайт ПВ IP5 - (https://www.uspto.gov/patents/basics/international-protection/global-dossier- initiative). Заявки с датой подачи с 1 января 2003 г. по н.в.; 4. KIPO: Система делопроизводства на сайте ПВ Кореи (http://kopd.kipo.go.kr), или через поисковую БД KIPRIS (www.kipris.or.kr), по номеру заявки, следует открыть вкладку «Legal Status» и перейти по ссылке на сайт KIPO. Заявки с датой подачи с 1 января 1999 г. по н.в.; 5. CNIPA: Система делопроизводства на сайте КНР (http://epub.cnipa.gov.cn/SW), по номеру заявки, открыть вкладку «Legal Status» или через поисковую БД PSS (https://pss-system.cponline.cnipa.gov.cn/conventionalSearch), по номеру заявки, открыть вкладку «Legal Status». Заявки с датой подачи от 10 февраля 2010 г. по н.в. оригинала, либо на английском языке через сайты ведомств IP5: 1. EPO: БД Espascenet, (вкладка в записи заявки – Global Dossier, либо через патентный реестр – EPO Register), (https://register.epo.org/regviewer). Заявки с датой подачи 1 июня 1978 г. по н.в.; 2. JPO: БД J-PlatPat, One Portal Dossier (OPD) на сайте ПВ Японии – (https://www.j-platpat.inpit.go.jp/p0000), ввести номер заявки и нажать на вкладку «OPD». Заявки с датой подачи с 1 июля 2003 по н.в.; 3. USPTO: Система Global Dossier на сайте США - ввести номер заявки любой страны из IP5, и система откроет делопроизводство или перенаправит на сайт ПВ IP5 - (https://www.uspto.gov/patents/basics/international-protection/global-dossier- initiative). 5 - Global Dossier - предоставляет собой онлайн источник, содержащий актуальную информацию о статусе патентных семейств заявок, поданных во всем мире3 [2]. Проект единого делопроизводства по конкретной заявке - Global Dossier4 [3]. Для упрощения работы, ведомства согласились предоставлять документы в электронном виде в стандартном формате и с переводом на английский язык. Система обеспечивает автоматический машинный перевод на английский язык оригинальных китайских, японских и корейских онлайн документов. Каждое из ведомств IP5 на своих патентных сайтах предоставляют доступ в режиме реального времени к массиву своих патентных документов и их делопроизводству в сервисе Global Dossier. Таким образом, с материалами заявки можно ознакомиться либо на языке 3 Сайт IP5, Информация по Global Dossier, делопроизводство по заявкам IP5, filewrapper, [Электронный ресурс], URL: https://www.fiveipoffices.org/activities/globaldossier/ filewrapper, (дата обращения: 23.11.2023); 4Сайт IP5, Информация по Global Dossier, IP5 Global dossier: scope, content, availability and performance, [Электронный ресурс], URL: https://www.fiveipoffices.org/sites/default/files/attachments/6877d20a-3529-46e0-b4a8- 6881eef83458/Quality%2BGlobal%2BDossier%2Bpassive%2Bcomponent %2BIndustry%2BMay%2B2017.pdf, (дата обращения: 23.11.2023); Заявки с датой подачи с 1 января 2003 г. по н.в.; 4. KIPO: Система делопроизводства на сайте ПВ Кореи (http://kopd.kipo.go.kr), или через поисковую БД KIPRIS (www.kipris.or.kr), по номеру заявки, следует открыть вкладку «Legal Status» и перейти по ссылке на сайт KIPO. Заявки с датой подачи с 1 января 1999 г. по н.в.; 5. CNIPA: Система делопроизводства на сайте КНР (http://epub.cnipa.gov.cn/SW), по номеру заявки, открыть вкладку «Legal Status» или через поисковую БД PSS (https://pss-system.cponline.cnipa.gov.cn/conventionalSearch), по номеру заявки, открыть вкладку «Legal Status». Заявки с датой подачи от 10 февраля 2010 г. по н.в. - Common Citation Document (CCD) - сервис обеспечивает бесплатный доступ к данным о цитировании источников информации в патентных поисках; - Common Application Format (CAF) – стандартизация формата патентной заявки (переход к формату XML); - Common Application Format (CAF) – стандартизация формата патентной заявки (переход к формату XML); - Cloud Patent Examination System (CPES) – информация о патентной экспертизе в 16 патентных ведомствах, включая IP5; - Cloud Patent Examination System (CPES) – информация о патентной экспертизе в 16 патентных ведомствах, включая IP5; - Industry Consultation Group (ICG)- консультационная группа IP5 с промышленностью; 6 6 Европейское патентное ведомство курировало деятельность по программам - общая документация и общая гибридная классификация. Ведомство по патентам и товарным знакам США отвечало за общий подход к совместному использованию и документированию стратегии поиска, а также за проект - общие инструменты поддержки поиска и экспертизы. Патентное ведомство Японии работало над проектами - общий доступ к поиску и результатам экспертизы и общий формат заявки. Патентное ведомство Китая работало над общими параметрами статистики, сосредоточилось на поиске и экспертизе, а также на правилах и управлении качеством. Патентное ведомство Южной Кореи взяло на себя два направления - общая политика обучения и машинный перевод. Сотрудничество ведомств IP5 кроме описанных выше программ и проектов затрагивает сферу патентных процедур. В 2012 году, действуя по предложению патентного ведомства Японии, IP5 учредила группу экспертов под названием Экспертная группа по гармонизации патентов (PHEP). Этот технический орган исследует потенциал согласования процедурных аспектов процесса подачи патентных заявок и процесса выдачи патентов между пятью ведущими патентными ведомствами. Проект по патентным процедурам был нацелен на анализ основных этапов процедур выдачи патентов в ведомствах IP5. Основное внимание уделялось сходству между ведомствами. Детали процедур различаются в разных ведомствах, иногда в довольно значительной степени (например, имеют место временные различия между этапами процедур). Ведомства IP5 изучали вопрос гармонизации патентных процедур в части работы экспертов. 5 - Global Dossier - предоставляет собой онлайн источник, содержащий актуальную информацию о статусе патентных семейств заявок, поданных во всем мире3 [2]. Проект единого делопроизводства по конкретной заявке - Global Dossier4 [3]. Для упрощения работы, ведомства согласились предоставлять документы в электронном виде в стандартном формате и с переводом на английский язык. Система обеспечивает автоматический машинный перевод на английский язык оригинальных китайских, японских и корейских онлайн документов. Каждое из ведомств IP5 на своих патентных сайтах предоставляют доступ в режиме реального времени к массиву своих патентных документов и их делопроизводству в сервисе Global Dossier. Таким образом, с материалами заявки можно ознакомиться либо на языке 3 Сайт IP5, Информация по Global Dossier, делопроизводство по заявкам IP5, filewrapper, [Электронный ресурс], URL: https://www.fiveipoffices.org/activities/globaldossier/ filewrapper, (дата обращения: 23.11.2023); 4Сайт IP5, Информация по Global Dossier, IP5 Global dossier: scope, content, availability and performance, [Электронный ресурс], URL: https://www.fiveipoffices.org/sites/default/files/attachments/6877d20a-3529-46e0-b4a8- 6881eef83458/Quality%2BGlobal%2BDossier%2Bpassive%2Bcomponent %2BIndustry%2BMay%2B2017.pdf, (дата обращения: 23.11.2023); В результате был проведен пересмотр законодательных норм и переход ведомств к согласованной патентной работе по единообразным этапам процедуры поиска и экспертизы в каждом ведомстве. Работа IP5 в направлении гармонизация элементов патентной практики и процедур помогла снизить рабочую нагрузку и упростить процедуру патентования для тех заявителей, которые хотят продвигать свои инновации на международном уровне. 7 7 Проект Common Citation Document – (CCD, сервис доступа к данным о цитировании источников в патентах). Проект связан с патентной информацией, в ходе реализации которого для объединения результатов поиска патентных ведомств был создан инструмент патентной информации в виде веб – приложения - Общий документ для цитирования (Common Citation Document (CCD)). Приложение обеспечивает пользователям бесплатный доступ к данным о цитировании изобретений того или иного заявителя в патентных поисках, учитывая, что заявки на данные изобретения поданы параллельно в несколько патентных ведомств, т.е. доступ к цитируемому уровню техники на уровне семейства патентов. CCD объединяет результаты поиска от более чем 30- ти патентных ведомств. Европейское патентное ведомство (EPO), в БД Espascenet, для найденного в процессе поиска документа, предоставляет отсылку на данные, взятые из отчетов о поиске этого документа. Таким образом, пользователь может просмотреть текст цитируемых патентных документов; список по источнику цитирования и связи с конкретной патентной заявкой; совокупность классификаций и полей, по которым выполняется поиск по всему семейству патентов. Проект Common Application Format – (CAF, общий формат зая 8 Проект помогает стандартизировать формальные аспекты патентных заявок. Идея введения Общего формата заявки заключается в использовании единой, стандартизированной и одобренной текстовой структуры для подачи заявки в участвующие Ведомства, а именно, было предложено стандартизировать стиль описаний, формул изобретения, рефератов и чертежей и упорядочить подачу патентных заявок. С 2012 года CAF стал общим проектом для IP5 и постоянно пересматривается в ВОИС. Поскольку некоторые правила форматирования являются общими, наличие широко принятого формата может снизить нагрузку на адаптацию заявки для национального уровня, упростить для пользователей подачу заявок по всему миру. Заявители, подающие заявки в несколько патентных ведомств, могут 8 8 просто повторно использовать один и тот же текст и структуру, чтобы не переписывать заявку каждый раз, когда они осуществляют дальнейшую подачу заявок. Несмотря на то, что заявка, возможно, потребует перевода, структура заявки останется прежней. Необходимо отметить, что CAF также способствовал оцифровке документов и послужил основой использования формата XML для электронной подачи и делопроизводства и обмена данными. Программа Cloud Patent Examination System (CPES) – (Облачная система патентной экспертизы (CPES)). Программа разработана ПВ Китая (CNIPA). Облачная система патентной экспертизы (CPES) представляет собой пилотную программу по обмену патентной информацией посредством облачных компьютерных технологий, запущенную Государственным ведомством по интеллектуальной собственности КНР (CNIPA) в 2015 году. Целью CPES является повышение качества и эффективности патентной экспертизы посредством обмена информационными ресурсами между участвующими ведомствами. Данная система позволяет не только обмениваться патентными данными (включая библиографическую информацию, опубликованные источники и патентную документацию), но также осуществлять групповые дискуссии между экспертами из различных государств и обеспечивать непосредственное интерактивное общение между ними в реальном времени. Интерфейс CPES доступен на 9 языках, включая китайский, английский и русский. Также система обеспечивает машинный перевод материалов на 12 иностранных языков. Среди ведомств, участвующих в программе, можно назвать Европейское патентное ведомство, Японское патентное ведомство, Корейское ведомство по интеллектуальной собственности и ведомство США по патентам и товарным знакам. В рамках межведомственного сотрудничества по обмену патентной документацией и опытом в области экспертизы заявок Роспатент и CNIPA ведут переговоры по вопросу о возможности присоединения Роспатента к системе CPES. 9 Программа Industry Consultation Group - (ICG, Консультационная группа IP5 для взаимодействия с промышленностью). Программа сотрудничества с представителями промышленных ассоциаций ICG работает с 2012 года, когда промышленные ассоциации стали принимать участие во всех ежегодных встречах IP5 высокого уровня. Ведомства IP5 считают, что необходим структурированный вклад и сформированная обратная связь от промышленности, для внесения улучшений в патентную систему в целом. 11 5 Сайт пяти ведущих патентных ведомств ID5 по охране промышленных образцов (ПО), [электронный ресурс], URL: http://id- five.org/about/id5news/?uid=299&mod=document&pageid=1, (дата обращения: 24.11.2023); 6 Сайт ВОИС, База данных промобразцов, https://www3.wipo.int/designdb/en/ 7 Сайт ведомства по промобразцам, EUIPO, просмотр делопроизводства по заявкам, в БД ESearch Plus (The eSearch plus provides Information about designs, owners, representatives and bulletins), [электронный ресурс], URL: https://euipo.europa.eu/eSearch/#basic, (дата обращения: 25.11.2023); Проект Common Application Format – (CAF, общий формат зая Постоянными членами рабочей группы от промышленности стран IP5 являются: Business Europe (BE) – со штаб-квартирой организации в Брюсселе. Business Europe работает от имени федераций-членов, расположенных в 34 европейских странах, чтобы обеспечить мнение бизнеса при выработке европейской политики. Из Японии: Ассоциация интеллектуальной собственности (JIPA). - JIPA насчитывает около 1300 членов, большинство из которых являются ведущими японскими компаниями. Из США: Американская Ассоциация интеллектуальной собственности (AIPLA) - состоит преимущественно из юристов частной и корпоративной практики, государственной службы и академического сообщества. Ассоциация владельцев интеллектуальной собственности (IPO), это внутренняя торговая ассоциация, представляющая компании и частные лица во всех отраслях и областях технологий, которые владеют правами интеллектуальной собственности или заинтересованы в них. Членство в IPO включает в себя около 200 компаний и более 12 000 человек, которые являются членами ассоциации либо через свои компании, либо в качестве изобретателей, авторов, юридических фирм или адвокатов-членов. Число участников IPO охватывает 50 стран. Из Кореи: Корейская Ассоциация интеллектуальной собственности (KINPA). Из Китая: Ассоциация патентной защиты Китайской Народной Республики (PPAC). Руководители ПВ IP5 выслушивают мнения и предложения пользователей, касающиеся текущих и планируемых проектов, а также 10 предлагают представителям промышленности дополнительные области, в которых ведомства IP5 могли бы сотрудничать. 8 Сайт Европейского патентного ведомства, Государства члены EPO, [электронный ресурс], URL: https://www.epo.org/about-us/foundation/member-states.html, (дата обращения: 26.11.2023); Программа ведомств по промышленным образцам ID5 Для промышленных образцов те же ведущие страны объединились в программу «ID5». ID5 представляет собой систему в сфере правовой охраны промышленных образцов, которая также управляется пятью мировыми ведущими ведомствами по интеллектуальной собственности, такими как: ПВ Китая (CNIPA), ПВ по интеллектуальной собственности Европейского союза (EUIPO), ПВ Японии (JPO), ПВ Южной Кореи (KIPO) и ПВ США (USPTO). Создание системы «ID5» обусловлено возрастающей ролью промышленного образца в мировой экономике, увеличением его ценности и значимости на мировом и внутренних рынках в различных юрисдикциях, а также необходимостью обеспечения правообладателей высокоэффективными системами правовой охраны промышленных образцов5 [6]. В патентных ведомствах используются международные базы данных ВОИС по промышленным образцам Global Design DB6, система электронного взаимодействия ВОИС в рамках процедур Договора о патентной кооперации - e-PCT, а также сервисы Ведомства по интеллектуальной собственности Европейского союза, объединяющие информацию из многих стран мира и предоставляющие возможность поиска и просмотра делопроизводства по заявкам на промышленные образцы 7[7]. Принципы взаимодействия ведомств при работе над совместными программами:  Некоторые проекты могут осуществляться ограниченным числом офисов или в разные сроки, т. е. нет необходимости действовать синхронно;  Некоторые проекты могут осуществляться ограниченным числом офисов или в разные сроки, т. е. нет необходимости действовать синхронно; 11  Эксперты на уровне рабочей группы определят соответствующую модель реализации, включая возможные пилотные проекты между двумя или более офисами для проверки концепции;  Офисы, участвующие в пилотных проектах, сообщают подробности и особенности реализации проектов, другие офисы смогут присоединиться на любом этапе.  Офисы, участвующие в пилотных проектах, сообщают подробности и особенности реализации проектов, другие офисы смогут присоединиться на любом этапе. 3. Региональный опыт интеграции в рамках европейского патентного ведомства. Взаимодействие ЕПВ с НПВ Европейского союза 3. Региональный опыт интеграции в рамках европейского патентного ведомства. Взаимодействие ЕПВ с НПВ Европейского союза Европейская патентная организация является межправительственной организацией, созданной в 1977 г. на основании Европейской патентной конвенции (ЕПК), подписанной в Мюнхене в 1973 г. единая процедура подачи заявок для получения патентной защиты в 44 странах (статья 6 EПК). Европейская патентная организация насчитывает 39 государств- членов8 [8], включая все государства - члены Европейского союза вместе со сравнительно недавно присоединившимися Албанией, Северной Македонией, Исландией, Лихтенштейном, Монако, Черногорией, Норвегией, Сан-Марино, Сербией, Швейцарией и Турцией. Соглашение о распространении действия европейских патентов действует с Боснией и Герцеговиной. Соглашения о валидации европейских патентов действует с Марокко, Республикой Молдова, Тунисом, Камбоджей, Грузией. ЕПВ является одной из крупнейших патентных организаций, в которой работает около 7000 сотрудников. Из них более 4 000 высококвалифицированных ученых и инженеров, работающих в качестве патентных экспертов во всех областях техники, более 600 специалистов проводящих формальные проверки. Основные программы экспертного взаимодействия в ЕПВ Европейского союза 12 12  ”Utility Program” – программа разделения экспертной работы. Государства-члены делятся результатами поиска/экспертизы/классификации их национальных заявок до публикации заявки, и используют результаты рассмотрения заявок другими экспертами других ПВ9 [9].  Программы сотрудничества с НПВ в рамках рабочих групп по актуальным темам экспертного взаимодействия, - регулярные встречи с более чем 20-тью договаривающимися государствами и государствами расширения.  Программы обучения экспертов НПВ в Европейской Патентной Академии по всем аспектам процесса выдачи европейских патентов, включая административные процедуры и инструменты проведения экспертизы и поиска.  Программы сотрудничества в области обмена информацией и совершенствования IT инструментов делопроизводства. В рамках сотрудничества в области обмена информацией ЕПВ предприняты следующие шаги по гармонизации экспертного взаимодействия: - Все НПВ ЕС согласовали набор стандартных современных средств в области информационно- коммуникационных технологий. - Все НПВ ЕС согласовали набор стандартных современных средств в области информационно- коммуникационных технологий. - Европейская патентная электронная сеть (European Patent Network, EPN) объединяет ЕПВ с НПВ 38 государств-членов Европейской патентной организации. - Единое цифровое рабочее пространство эксперта (БД Ansera, БД EPOQUE Net). База данных Espacenet ЕПВ содержит более чем 144 миллиона патентных документов из более 90 стран мира. 4. Международный опыт по совместной экспертной работе 14 10 Сайт ВОИС, Совместный поиск и экспертиза по международным заявкам (Collaborative Search and Еxamination, CS&E, [Электронный ресурс], URL: https://www.wipo.int/pct/en/filing/cse.html, (дата обращения: 26.11.2023); 11Сайт ПВ США, Программа совместного поиска (Collaborative Search Program, CSP) [Электронный ресурс], URL: https://www.uspto.gov/sites/default/files/documents/ General CSP Info.pdf, (дата обращения: 26.11.2023); 4. Международный опыт по совместной экспертной работе 9 Сайт EPO, Стратегический план до 2023 года, стр. 105, Ссылки на отчеты по направлениям работы EPO, [Электронный ресурс], URL: https://documents.epo.org/projects/babylon/eponet.nsf/0/6EF19E07A7989D9CC125886F005F82 6D/$File/annual_review_2021_en.pdf, (дата обращения: 26.11.2023); 13 9 Сайт EPO, Стратегический план до 2023 года, стр. 105, Ссылки на отчеты по направлениям работы EPO, [Электронный ресурс], URL: https://documents.epo.org/projects/babylon/eponet.nsf/0/6EF19E07A7989D9CC125886F005F82 6D/$File/annual_review_2021_en.pdf, (дата обращения: 26.11.2023); 13 Помимо работы с информацией, форматами документов и базами данных ведомства IP5 развивали свое сотрудничество в части поиска и экспертизы. Для каждой программы экспертного взаимодействия IP5 сформулированы краткосрочные, среднесрочные и долгосрочные возможности сотрудничества, достаточно широкие для учета будущих разработок. Точный объем, сроки и потребности в ресурсах для этих потенциальных областей работы определяются различными дополнительными рабочими группами IP5 в соответствии с установленными процессами управления проектами IP5, включая необходимые циклы проверки и адаптации. Проблема рабочей нагрузки, которая обсуждалась с начала 2000-х годов, породила идею взаимного использования результатов поиска/экспертизы, подготовленных другими ведомствами, что в свою очередь принесло ощутимые результаты. Идея взаимного использования результатов поиска/экспертизы привела к созданию нескольких уникальных программ для совместного использования рабочих продуктов:  Совместный поиск и экспертиза по международным заявкам (Collaborative Search and Еxamination, CS&E) в рамках Договора о патентной кооперации (PCT) на международной фазе10 [10]. • Заявка рассматривается несколькими экспертами с разными языковыми возможностями из разных патентных ведомств. • Заявка рассматривается несколькими экспертами с разными языковыми возможностями из разных патентных ведомств. • Каждое ведомство с большей вероятностью ссылается на документы своего национального происхождения.  Программа совместного поиска (Collaborative Search Program, CSP) в рамках делопроизводства по схожим национальным заявкам11 [11]. 14 • Предоставляет возможность заявителям, которые осуществляют зарубежное патентование, получать результаты поиска из нескольких ведомств. • Предоставляет возможность заявителям, которые осуществляют зарубежное патентование, получать результаты поиска из нескольких ведомств.  Ускоренное патентное делопроизводство по национальным заявкам (Patent Prosecution Highway, РРН) - РРН, РСТ-РРН, Global PPH (GPPH), PPH-MOTTAINAI12 [12]. • Рабочий продукт одного ведомства используется другим ведомством при рассмотрении соответствующей заявки, что облегчает проведение экспертизы по существу. • Рабочий продукт одного ведомства используется другим ведомством при рассмотрении соответствующей заявки, что облегчает проведение экспертизы по существу. Все проекты действуют на основе договора PCT. Договор РСТ 13[13]. позволяет получить патентную охрану изобретений одновременно в большом количестве стран путем подачи одной международной заявки. С момента начала сотрудничества ведомства IP5 регулярно обменивались мнениями по вопросам, связанным с PCT, что позволило в режиме реального времени отслеживать и корректировать работу ведомств в отношении PCT. В этом контексте ведомства IP5 разработали потенциальную концепцию сотрудничества в рамках совместного проведения международных поисков. 15 12 Сайт Роспатента, Ускоренное патентное делопроизводство по национальным заявкам (Patent Prosecution Highway, РРН), [Электронный ресурс], URL: https://rospatent.gov.ru/ru/activities/inter/bicoop/pph (дата обращения: 27.11.2023); 13 Сайт Роспатента, Исполнение международных обязательств Российской Федерации, вытекающих из участия в договоре о патентной кооперации, (Договор РСТ), [Электронный ресурс], URL: https://rospatent.gov.ru/ru/international-cooperation/rosp- mezhdunarodnom-sotrudnichestve (дата обращения: 27.11.2023); 4. Международный опыт по совместной экспертной работе Ключевая идея заключалась в том, что эксперты ведомств, работающие на разных языках и из разных регионов, могут совместно работать на международной фазе РСТ над одной международной заявкой для подготовки высококачественного отчета о международном поиске и письменного заключения в соответствии с PCT. Проект развивался поэтапно, начиная с 2010 года и вплоть до настоящего времени. Очевидными положительными результатами международного распределения работы и объединения усилий экспертов нескольких ведомств является достижение большей 15 согласованности между ведомствами и большая уверенность заявителя в результате на международной фазе и затем при переходе на национальную фазу в те страны, ведомства которых участвовали в подготовке отчета о международном поиске. Программа Совместный поиск и экспертиза по международным заявкам (Collaborative Search and Examination, CS&E) в рамках Договора о патентной кооперации (PCT) на международной фазе. Сотрудничество по поиску и экспертизе появилось в рамках Договора о патентной кооперации. В соответствии с действующей системой PCT поиск по заявке PCT осуществляется компетентным Международным поисковым органом (МПО) (статья 16 PCT). МПО готовит отчет о международном поиске (ISR) и письменное заключение о патентоспособности технического решения, раскрытого в заявке (статья 15 PCT). ISR не имеет характер обязательности для национальных/региональных ведомств с точки зрения принятия ими решения о выдаче патента, но дает представление о предшествующем уровне техники, с которым заявитель столкнется при переходе на национальную/региональную фазу. Выбор компетентного МПО частично определяется выбором Получающего ведомства (RO), в которое была подана заявка PCT. Например, когда USPTO выступает в качестве RO, заявитель может выбрать любой из семи компетентных МПО. Каждый МПО выполнит глобальный поиск по уровню техники. Однако, несомненно, имеет место тот факт, что каждое ведомство с большей вероятностью будет ссылаться на документы своего национального происхождения. CS&E работает следующим образом: первый, основной эксперт проводит поиск и составляет отчет о международном поиске и письменное сообщение, затем эти документы отправляются коллегиальным независимым экспертам из других сотрудничающих органов PCT для получения обратной связи. Заявка рассматривается несколькими экспертами с разными языковыми возможностями из разных патентных ведомств. 16 В итоге первый эксперт учитывает отзывы своих коллег при составлении своего окончательного отчета о международном поиске и письменного сообщения. Материалы, подготовленные независимыми экспертами, публикуются в виде отдельных документов к ISR и письменному заключению. Заявитель имеет право ознакомиться со всеми документами после подготовки итогового отчета о поиске и письменного сообщения через ePCT. В результате по заявке обеспечивается наиболее полный анализ уровня техники, за счет объединения усилий экспертов нескольких ведомств. Вместе с тем, для заявителя обеспечивается большая степень определенности о дальнейших перспективах получения охраны прав на интеллектуальную собственность в ведомствах IP5. 4. Международный опыт по совместной экспертной работе Кроме того, очевидно достигается улучшение качества подготовленных на международной фазе документов за счет учета мнений независимых экспертов при их составлении. По сути, итоговый отчет о международном поиске аналогичен по ценности совокупности отдельных отчетов о поиске всех участвующих ведомств. Следует отметить еще один положительный аспект, состоящий в том, что мнение пяти ведомств о жизнеспособности их заявки заявители получают уже через 16 месяцев после даты приоритета. Это позволяет заявителям на раннем этапе принять решение о том, следует ли продолжать работу по патентованию заявки в различных странах, возможно, лучше отозвать ее до даты публикации через 18 месяцев после приоритета или внести в нее изменения на этапе предварительной экспертизы. Проект Совместного поиска (Collaborative Search Program CSP) Проект Совместного поиска (Collaborative Search Program, CSP) Проект Совместного поиска (Collaborative Search Program, CSP) Каждое ведомство с большей вероятностью ссылается на документы своего национального происхождения. Программа совместного поиска (Collaborative Search Program, CSP) в рамках делопроизводства по национальным заявкам развивалось также в рамках сотрудничества ведомств IP5. Программа совместного поиска (CSP) запущена в 2015 году для работы над заявками, поданными в национальные ведомства. Заявителям, которые подают свои патентные заявки в национальные ведомства 17 участвующих стран, получают результаты поиска на ранних этапах процесса экспертизы. Проект предназначен, в том числе, для ускорения экспертизы и предоставления заявителю более полной информации о предшествующем уровне техники путем объединения опыта экспертов в соответствующей области из разных ПВ. Совместное рассмотрение аналогичных заявок требует меньшего количества действий экспертизы по существу в среднем, по сравнению с рассмотрением заявок, не участвующих в CSP. Преимущества программы CSP. Повышение качества, ускоренное рассмотрение заявки и снижение времени ожидания решения для заявителя, повышение согласованности и определенности, бесплатное участие в программе CSP для заявителей. 19 14 Сайт патентного ведомства Японии, [Электронный ресурс], URL: https://www.jpo.go.jp/news/kokusai/nichibeiou/document/nitibeiou_meeting_30/pamphlet.pdf (дата обращения 27.11.2023) Программы IP5 Patent Prosecution Highway (РРН) Программы РРН хорошо себя зарекомендовали среди патентных ведомств и пользователей за счет простого и понятного механизма их реализации. Суть программы ускоренного делопроизводства РРН заключается в том, что она предоставляет заявителю возможности быстрее и эффективнее получить патент в одном из участвующих в Программе РРН ведомств за счет использования в этом ведомстве при рассмотрении заявки положительных результатов поиска и экспертизы, проведенных в другом участвующем ведомстве по аналогичной заявке. Ускоренное патентное делопроизводство (Patent Prosecution Highway, РРН) предоставляет заявителям, которые осуществляют подачу своих заявок в несколько стран, результаты поиска из нескольких ведомств по национальным и международным заявкам в рамках следующих подпрограмм: РРН, РСТ-РРН, Global PPH (GPPH), PPH-MOTTAINAI. PPH - это простая система с большими преимуществами. При передаче заявки в рамках программы PPH эксперт второго ведомства рассмотрит результаты работы, которые заявитель получил в первом ведомстве, где ранее была проведена экспертиза. Это помогает эксперту во втором ведомстве выполнять свои задачи более эффективно и ускоряется 18 рассмотрение заявки при условии, конечно, что установлена патентоспособность технических решений. Следует подчеркнуть, что эксперт, обрабатывающий заявку в рамках PPH, применяет собственные стандарты своего ведомства. Программа PPH берет свое начало с подписания двустороннего соглашения между JPO и USPTO в июле 2006 года14, затем постепенно остальные ведомства IP5 присоединились к программе РРН путем подписания двусторонних соглашений. Программа PPH представляет собой выгодную услугу для заявителей, когда на основании заявления, поданного в одном ведомстве, заявители могут получить доступ к ускоренному производству в другом через PPH, без каких-либо особых требований. Это также может помочь пользователям избежать дополнительных затрат. Участие в проекте PPH бесплатно, тогда как в США, например, заявителю обычно пришлось бы заплатить пошлину за ускоренную экспертизу вне рамок PPH. В ходе развития программы РРН появились ее разновидности В ходе развития программы РРН появились ее разновидности В рамках Договора о патентной кооперации Программа ускоренного делопроизводства РРН начала своѐ действие с 2008 года в виде Программы РСТ-РРН, когда ведомства Японии (JPO) и США (USPTO) первыми подписали двустороннее соглашение. В 2011 году была запущена программа ускоренного патентного делопроизводства PPH-MOTTAINAI, в соответствии которой заявление об участии в программе принимается вне зависимости от того, является ли ведомство, которое проводит экспертизу и представляет ее результаты для участия в программе РРН ведомством первой подачи (ВПП). Программа Global PPH (GPPH) действует с 2014 года и предполагает оптимизацию международного патентования в условиях глобализации экономики при максимальной унификации требований к 19 19 изобретениям для получения патентов в разных странах. Данная программа имеет преимущество над заключенными ранее двухсторонними договоренностями и включает в себя функционал всех трех предшествующих видов программ РРН. 5. Предложения по созданию единого евразийского патентного пространства на основе мировых программ взаимодействия 5. Предложения по созданию единого евразийского патентного пространства на основе мировых программ взаимодействия В евразийском патентном пространстве перспективными для экспертного сотрудничества являются две программы: В евразийском патентном пространстве перспективными для экспертного сотрудничества являются две программы: - Программа по совместному поиску и экспертизе; - Программа Ускоренного патентного делопроизводства. Включение заявок в программы производится на основании просьбы заявителя. Информирование через сайты НПВ. Предлагаются следующие возможные варианты регионального сотрудничества между НПВ и ЕАПВ. 1. Сотрудничество на международной фазе (могут участвовать только Роспатент и ЕАПВ, как международные поисковые органы РСТ). - международные заявки РСТ, поступившие на проведение международного поиска и международной предварительной экспертизы (поданные в Роспатент и в ЕАПВ, как МПО и ОМПЭ). Подача заявки возможна в ОМП и ОЭМПО евразийского региона. Получающее ведомство (RO): РОСПАТЕНТ или ЕАПВ; Заявитель подает ходатайство о проведении экспертизы и поисков в главный ISA; Передача материалов заявки на поиск во 2-ой ISA; Обмен результатами поиска между экспертами двух ISA; Главный ISA выносит отчет о поиске ISR и рассылает заявителю и ведомствам партнерам. 2. Сотрудничество по национальным заявкам (могут участвовать все государства- участники ЕАПК). 2. Сотрудничество по национальным заявкам (могут участвовать все государства- участники ЕАПК). Национальные заявки на выдачу патента, подаются в НПВ Евразийского региона (EA, AZ, AR, BY, KZ, KG, RU, TG, TK); Заявитель подает ходатайство об участии в программе совместных поисков, с 20 указанием выбранных +2 ПНВ , в которых будут проводить поиск; Передача материалов заявки на поиск в 2 НПВ; Обмен результатами поиска между экспертами трех НПВ; 1 НПВ выносит решение о выдаче и рассылает заявителю и ведомствам партнерам. 3. Программы РРН между ведомствами евразийского региона. При подаче заявки в одно из НПВ ЕА региона, заявке устанавливается один приоритет; Заявка считается поданной во все НПВ ЕА региона с датой приоритета, определенной в НПВ подачи; Заявитель пишет ходатайство о проведении экспертизы и поисков в нескольких НПВ по своей заявке. Выбирает главный НПВ; Передача материалов заявки на поиск в 2 НПВ; Обмен результатами поиска между экспертами трех НПВ; 1 НПВ выносит решение о выдаче и рассылает заявителю и ведомствам партнерам. Программы по совместному проведению поиска и экспертизы предполагают, что по заявленному изобретению осуществляют параллельный поиск несколько экспертов, тем самым обеспечивается повышение качества поиска, за счет объединения опыта экспертов и получения более полной информации о предшествующем уровне техники. При совместной экспертизе достигается межведомственная согласованность в отношении принимаемого решения по заявке, что должно привести к повышению уверенности заявителя в отношении надѐжности предоставленной правовой охраны на территории нескольких государств. 5. Предложения по созданию единого евразийского патентного пространства на основе мировых программ взаимодействия Программы РРН на международной фазе предполагают подачу заявки в два ведомства: Роспатент и ЕАПВ. Программы РРН между ведомствами евразийского региона должны стать одними из первых реализуемых проектов экспертного взаимодействия между ними. Механизмы таких программ РРН уже хорошо отработаны мировыми ведомствами, в том числе в Роспатенте и ЕАПВ, и их эффективность не вызывает ни у кого сомнения. Следует отметить, что большим преимуществом для заявителей является то, что подача ходатайства в программах РРН не требует от заявителей уплаты дополнительных пошлин. 21 В рамках евразийской программы РРН ускоренное рассмотрение заявок может быть не только на основании положительных результатов поиска и экспертизы по национальным или евразийским заявкам, но и за счет использования работ, проводимых Роспатентом и ЕАПВ в качестве международных органов в рамках системы Договора о патентной кооперации (Договор РСТ). Это также даст дополнительный стимул для заявителей евразийского региона использовать преимущества системы Договора РСТ и придаст большую ценность результатам работы международных органов системы Договора РСТ из евразийского региона. Использование единой евразийской информационной системы обмена экспертной и патентной информацией значительно повысит эффективность такой программы. 6. Выводы: Поиск наиболее эффективных путей правовой охраны и защиты интеллектуальной собственности стал исключительно актуальным для государств евразийского региона. Перед Роспатентом и ЕАПВ стоят вопросы обеспечения максимально возможного уровня гармонизации подходов при предоставлении патентной охраны, взаимодействия региональной и национальных патентных систем с целью создания условий для повышения эффективности процедур и надежности предоставления правовой охраны на территории одной или нескольких стран евразийского региона, и предоставления современной услуги обмена патентными данными. Построение единого евразийского патентного экспертно- информационного пространства должно быть основано на изучении и анализе опыта международного экспертного взаимодействия ведущих патентных ведомствам мира. Для реализации вариантов экспертного сотрудничества необходимо решить следующий ряд вопросов: 1. Необходимо установить общие правила проведения поиска и экспертизы (создать рабочую группу по проработке методологии). 2. Разработать и заключить необходимые многосторонние или двухсторонние Соглашения. 22 22 3. Необходимо договориться о единой системе статистических параметров для дальнейшего анализа работы, (критерии оценки эффективности). 4. Необходимо обеспечить возможность удобного обмена данными с соблюдением конфиденциальности (каналы связи, хранение данных, формат данных для обмена и т.п.). Исходя из проведенного анализа опыта взаимодействия патентных ведомств, предлагается начать формирование общего экспертно- информационного пространства на основе реализации проектов по проведению совместного поиска и экспертизы по патентным заявкам, внедрению региональных программ ускоренного делопроизводства по заявкам на изобретения и промышленные образцы. С учетом достигнутого уровня взаимодействия, ведомства евразийского региона могли бы осуществлять сотрудничество по заявкам, относящимся к отдельным выбранным технологическим областям в случаях, когда эксперты заинтересованы в обмене мнениями о патентоспособности изобретения перед принятием решения, а также ускорять принятие решения основе результатов экспертизы, полученных в другом ведомстве. Программы по совместному проведению поиска и экспертизы могут быть реализованы как в двустороннем, так и многостороннем форматах, и учитывать «специализацию» экспертов конкретного ведомства в той или иной области профессиональных знаний. В рамках такого экспертного взаимодействия ЕАПВ должно стать своеобразным региональным центром компетенций и координации экспертного сотрудничества, способствующим сближению стран в сфере правовой охраны промышленной собственности. 23 Для скорейшего достижения целей экспертного взаимодействия в евразийском регионе представляется целесообразным провести гармонизацию правовых основ для реализации совместных проектов, чтобы избежать дублирования отдельных работ в этой сфере, а также, модернизацию информационных систем, цифровизацию подходов 23 совместной работы в национальных патентых ведомствах евразийского региона (НПВ ЕР), повысить доступность для пользователей цифровых услуг, предоставляемых НПВ и ЕАПВ, снизить временные и финансовые издержки при разработке информационных систем, повысить уровень компетенции специалистов. Список источников: 1. Сайт Ведомств IP5, [Электронный ресурс], URL: https://www.fiveipoffices.org/material, (дата обращения: 22.11.2023); 2. Сайт ВОИС, Центр статистических данных за 2021 год, WIPO IP Statistics Data Center, Source: WIPO statistics database. Last updated: June 2023, [Электронный ресурс], URL: https://www3.wipo.int/ipstats/index. htm?tab=patent, (дата обращения: 22.11.2023); 3. Сайт Ведомств IP5, квартальный отчет за 2023 год [Электронный ресурс], URL: https://www.fiveipoffices.org/sites/default/files/2023- 01/IP5%20Statistics%20Report%202021_1.pdf , (дата обращения: 22.11.2023); 4. Сайт IP5, Информация по Global Dossier, делопроизводство по заявкам IP5, filewrapper, [Электронный ресурс], URL: https://www.fiveipoffices.org/activities/globaldossier/ filewrapper, (дата обращения: 23.11.2023); 5. Сайт IP5, Информация по Global Dossier, IP5 Global dossier: scope, content, availabilityandperformance, [Электронный ресурс], URL: https://www.fiveipoffices.org/sites/default/files/attachments/6877d20a-3529-46e0- b4a8-6881eef83458/Quality%2BGlobal%2BDossier%2Bpassive%2Bcomponent %2BIndustry%2BMay%2B2017.pdf, (дата обращения: 23.11.2023); 6. Сайт пяти ведущих патентных ведомств ID5 по охране промышленных образцов (ПО), [Электронный ресурс], URL: http://id- five.org/about/id5news/?uid=299&mod=document&pageid=1, (дата обращения: 24.11.2023); 24 7. Сайт ведомства по промобразцам, EUIPO, просмотр делопроизводства по заявкам, в БД ESearch Plus (The eSearch plus provides Information about designs, owners, representatives and bulletins), [Электронный ресурс], URL: https://euipo.europa.eu/eSearch/#basic, (дата обращения: 25.11.2023); 8. Сайт Европейского патентного ведомства, Государства члены EPO, [Электронный ресурс], URL: https://www.epo.org/about- us/foundation/member-states.html, (дата обращения: 26.11.2023); 9. Сайт EPO, Стратегический план до 2023 года, стр. 105, Ссылки на отчеты по направлениям работы EPO, [Электронный ресурс], URL: https://documents.epo.org/projects/babylon/eponet.nsf/0/6EF19E07A7989D9CC12 5886F005F826D/$File/annual_review_2021_en.pdf, (дата обращения: 26.11.2023); 10. Сайт ВОИС, Совместный поиск и экспертиза по международным заявкам (Collaborative Search and Еxamination, CS&E, [Электронный ресурс], URL: https://www.wipo.int/pct/en/filing/cse.html, (дата обращения: 26.11.2023); 11. Сайт ПВ США, Программа совместного поиска (Collaborative Search Program, CSP) [Электронный ресурс], URL: https://www.uspto.gov/sites/default/files/documents/GeneralCSPInfo.pdf, (дата обращения: 26.11.2023); 12. Сайт Роспатента, Ускоренное патентное делопроизводство по национальным заявкам (Patent Prosecution Highway, РРН), [Электронный ресурс], URL: https://rospatent.gov.ru/ru/activities/inter/bicoop/pph (дата обращения: 27.11.2023); 13. Сайт Роспатента, Исполнение международных обязательств Российской Федерации, вытекающих из участия в договоре о патентной кооперации, (Договор РСТ), [Электронный ресурс], URL: https://rospatent.gov.ru/ru/international-cooperation/rosp-mezhdunarodnom- sotrudnichestve (дата обращения: 27.11.2023); 25
https://openalex.org/W1500280172	https://www.nature.com/articles/srep11974.pdf	English	null	Synchronization to a bouncing ball with a realistic motion trajectory	Scientific reports	2,015	cc-by	7,488	Synchronization to a bouncing ball with a realistic motion trajectory Lingyu Gan, Yingyu Huang, Liang Zhou, Cheng Qian & Xiang Wu received: 12 October 2014 accepted: 12 June 2015 Published: 07 July 2015 Daily music experience involves synchronizing movements in time with a perceived periodic beat. It has been established for over a century that beat synchronization is less stable for the visual than for the auditory modality. This auditory advantage of beat synchronization gives rise to the hypotheses that the neural and evolutionary mechanisms underlying beat synchronization are modality-specific. Here, however, we found that synchronization to a periodically bouncing ball with a realistic motion trajectory was not less stable than synchronization to an auditory metronome. This finding challenges the auditory advantage of beat synchronization, and has important implications for the understanding of the biological substrates of beat synchronization. Most forms of music have a perceived periodic beat (or pulse), and people often move (e.g., tap a finger or foot) in synchrony with the beat1. The capacity to entrain motor behaviors to a beat is predictive (i.e., on average, taps slightly precede event onsets when tapping to a beat) and flexible (i.e., synchronization to an auditory beat is accurate for inter-beat intervals ranging from 300 to 900 ms, with the most pre- ferred inter-beat intervals being approximately 600 ms)2. One key feature of beat synchronization is the auditory advantage that has been established for over a century; synchronization is less stable to a visual (e.g., flashes of a light) than to an auditory beat (e.g., an auditory metronome)3. This modality bias of beat synchronization has essential impacts on the understanding of the biological substrates of sensori- motor integration and human evolution. Tighter connections between the auditory and motor cortices than between the visual and motor cortices have been suggested for beat synchronization4–6. Human-like beat synchronization behaviors have primarily been observed in animals with vocal learning ability (e.g., parrots). It has thus been suggested that vocal learning drives the evolution of flexible beat synchroni- zation and that flexible beat synchronization is a unique brain function that is shared by humans and only a few other species6,7. y p However, recent advances have shown that synchronization to a visual beat can be improved using moving stimuli instead of a conventional flashing light for review, see 8. Department of Psychology, Sun Yat-Sen University, Building 313, 135 Xingang west road, Guangzhou, Guangdong, China, 510275. These authors contributed equally to this work. Correspondence and requests for materials should be addressed to X.W. (email: rwfwuwx@gmail.com) www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| 5:11974 \| DOI: 10.1038/srep11974 Synchronization to a bouncing ball with a realistic motion trajectory Specifically, Iversen et al.9 reported that tapping stability to a periodically bouncing ball, which had a velocity that varied according to a rectified sinusoid, was close to that to an auditory metronome. Hove et al.10 later found an advantage for an auditory metronome over the bouncing ball. People usually move along with moving visual stimuli that contain spatiotemporal information rather than with a visual stimulus without spatial changes, such as a flashing light that lacks ecological validity11. Therefore, these findings indicate that synchronization to a visual beat composed of realistically moving stimuli is almost as good as synchronization to an auditory metronome, although a slight auditory advantage still persisted in studies employing moving visual stimuli. Since synchronization to a visual beat can be improved by employing realistically moving stimuli, its performance may be further improved and could reach the level of synchronization to an auditory beat if the moving stimuli could be more realistic. The present study investigated this hypothesis. Previous studies have shown that periodically bouncing ball stimuli moving with a rectified sinusoidal veloc- ity profile are the most effective moving stimuli in improving synchronization to a visual beat9,10. We designed a bouncing ball that was even more realistic by two manipulations. (1) The velocity of the current bouncing ball was varied by simulating the effect of gravity because people can precisely interact 1 Scientific Reports \| 5:11974 \| DOI: 10.1038/srep11974 www.nature.com/scientificreports/ Figure 1. Illustration of the experimental stimuli. The subjects tapped along with an auditory tone sequence (A), a visual flashing ball sequence (B), or a visual bouncing ball sequence (C). Three cycles of the 600 ms IOI sequences from experiment 1 are shown. The velocity and trajectory of the visual bouncing ball are indicated in the inset of C. (The drawings in all Figures were drawn by the authors). Figure 1. Illustration of the experimental stimuli. The subjects tapped along with an auditory tone sequence (A), a visual flashing ball sequence (B), or a visual bouncing ball sequence (C). Three cycles of the 600 ms IOI sequences from experiment 1 are shown. The velocity and trajectory of the visual bouncing ball are indicated in the inset of C. (The drawings in all Figures were drawn by the authors). with free-falling objects12. (2) Movement smoothness was carefully controlled to avoid movement dis- continuities that could potentially impact the subjects’ judgment of the trajectory. Synchronization to a bouncing ball with a realistic motion trajectory Synchronization to an isochronous rhythmic sequence that was composed of an auditory tone, a visual flash, or the cur- rent bouncing ball was examined in experiment 1 using a 600 or 900 ms inter-onset interval (IOI) (i.e., inter-beat interval). In experiment 2, the auditory tone sequence and the current bouncing ball sequence were compared for more IOIs (300, 500, 700, and 900 ms). After that, the current bouncing ball sequence was compared with a bouncing ball sequence with a sinusoidally varying velocity in control experiment 1, and the effect of each of the two manipulations of the current bouncing ball sequence was studied in control experiment 2. Moreover, the current bouncing ball sequence was rotated 90° counterclockwise and was compared with the non-rotated sequence in control experiment 3. A 600 ms IOI was used in all control experiments. Results I i VB 3.960 .001/.006 4.280 .001/.006 Experiment 2 300 ms 500 ms 700 ms 900 ms AT vs. VB 4.314 .001/.004 1.199 .252/1 2.320 .037/.148 1.834 .090/.360 Table 1. Comparisons of the stability (R) between sequence types for individual IOIs. The red color indicates p ≤ 0.05 after Bonferroni corrections and the orange color indicates p ≤ 0.1 without Bonferroni corrections (tended to be significant). Other conventions are as in Fig. 2. (Note that the comparison of AT vs. VB for the 900 ms IOI in experiment 1 was largely influenced by three outliers in the AT condition, i.e., the three lowest points for the 900 ms IOI in the AT condition as shown in Fig. 2). t value p/pcorrected value t value p/pcorrected value t value p/pcorrected value t value p/pcorrected value Experiment 1 600 ms 900 ms AT vs. VF 3.542 .003/.018 4.050 .001/.006 AT vs. VB 2.952 .011/.066 2.062 .058/.348 VF vs. VB 3.960 .001/.006 4.280 .001/.006 Experiment 2 300 ms 500 ms 700 ms 900 ms AT vs. VB 4.314 .001/.004 1.199 .252/1 2.320 .037/.148 1.834 .090/.360 Table 1. Comparisons of the stability (R) between sequence types for individual IOIs. The red color indicates p ≤ 0.05 after Bonferroni corrections and the orange color indicates p ≤ 0.1 without Bonferroni corrections (tended to be significant). Other conventions are as in Fig. 2. (Note that the comparison of AT vs. VB for the 900 ms IOI in experiment 1 was largely influenced by three outliers in the AT condition, i.e., the three lowest points for the 900 ms IOI in the AT condition as shown in Fig. 2). Table 1. Comparisons of the stability (R) between sequence types for individual IOIs. The red color indicates p ≤ 0.05 after Bonferroni corrections and the orange color indicates p ≤ 0.1 without Bonferroni corrections (tended to be significant). Other conventions are as in Fig. 2. (Note that the comparison of AT vs. VB for the 900 ms IOI in experiment 1 was largely influenced by three outliers in the AT condition, i.e., the three lowest points for the 900 ms IOI in the AT condition as shown in Fig. 2). visual bouncing ball sequence was presented on a typical computer monitor using a movement-distance/ ball-size ratio that was slightly larger than 1 (thus the last several steps down or first several steps up were too large). Results I i In experiment 1, beat synchronization was studied by having the subjects tap a finger along with a metronome6,8, which was composed of an isochronous sequence with a 600 or 900 ms IOI. There were three types of sequences (Fig. 1): the auditory tone sequence, the visual flashing ball sequence, and the visual bouncing ball sequence. The velocity of the bouncing ball was varied by simulating the effect of gravity, i.e., with a uniformly varying velocity. The acceleration of the earth’s gravity is 9.8 m/s2, and the movement distance of a corresponding falling object would be substantially greater than the height of a computer monitor for the typical beat intervals (300 to 900 ms). Therefore, the acceleration was 0.20 m/s2 for the 600 ms IOI bouncing ball sequence and was 0.09 m/s2 for the 900 ms IOI bouncing ball sequence (the stimuli could be viewed as representing falling objects on other possible planets with different grav- itational accelerations than the earth). The assignment of the acceleration was also related to the cor- rection of movement discontinuities. Because of the ball’s high speed at the lowest positions (see the inset of Fig. 1C), a clear movement discontinuity was observed in the preliminary testing in which the Scientific Reports \| 5:11974 \| DOI: 10.1038/srep11974 2 www.nature.com/scientificreports/ Figure 2. Results of experiment 1. Tapping was most stable for the visual bouncing ball sequence. The mean synchronization stabilities are indicated by the large gray marks. The data from individual subjects are indicated by the small marks of different colors, which indicate different subjects. AT, VF and VB represent the auditory tone sequence, visual flashing ball sequence, and visual bouncing ball sequence, respectively. Figure 2. Results of experiment 1. Tapping was most stable for the visual bouncing ball sequence. The h Figure 2. Results of experiment 1. Tapping was most stable for the visual bouncing ball sequence. The mean synchronization stabilities are indicated by the large gray marks. The data from individual subjects are indicated by the small marks of different colors, which indicate different subjects. AT, VF and VB represent the auditory tone sequence, visual flashing ball sequence, and visual bouncing ball sequence, respectively. t value p/pcorrected value t value p/pcorrected value t value p/pcorrected value t value p/pcorrected value Experiment 1 600 ms 900 ms AT vs. VF 3.542 .003/.018 4.050 .001/.006 AT vs. VB 2.952 .011/.066 2.062 .058/.348 VF vs. Scientific Reports \| 5:11974 \| DOI: 10.1038/srep11974 Results I i pp y g p Since realistically moving visual stimuli were capable of improving synchronization to a visual beat, unrealistically moving visual stimuli could decrease synchronization to a visual beat. This was observed when the IOI was reduced to 300 ms (Fig. 3) in experiment 2. All subjects verbally described the 300 ms IOI visual bouncing ball sequence as “unnaturally fast”, and its associated tapping (mean stability = 0.630) was less stable than tapping to the corresponding auditory tone sequence (mean sta- bility = 0.891) (t13 = 4.314, pcorrected = 0.004, η 2 = 0.589). The poor synchronization performance for the visual bouncing ball sequence was specific to the short 300 ms IOI and was not observed for longer IOIs that ranged from 500 to 900 ms (Table 1), which replicated the above results of the 600 and 900 ms IOIs in experiment 1. Note that the poorer synchronization performance for a flash of light (e.g., the visual flashing ball sequence in the present study) than for an auditory metronome is observed for all IOIs rather than only the short 300 ms IOI2. A two-way ANOVA with the factors sequence type (two sequence types) and IOI type (four IOI types) showed significant interaction between the two factors (F3,39 = 19.292, p = 0.001, partial η 2 = 0.597), which was consistent with the above observation that the auditory advantage was only for the 300 ms IOI, but not for the longer IOIs. In addition, the ANOVA showed significant main effects for sequence type (F1,13 = 13.924, p = 0.003, partial η 2 = 0.517) and IOI type (F3,39 = 35.167, p < 0.001, partial η 2 = 0.730). While the subjects reported that the 300 ms IOI bounc- ing ball sequence looked unnatural, the mechanism of the poor performance needs to be further deter- mined. For example, the movement for the 300 ms IOI was less smooth than that for the longer IOIs (although the movement discontinuities at the lowest ball positions were carefully controlled for the 300 ms IOI sequence, see the Methods below), which might also contribute to the poor synchronization performance in the fastest bouncing ball condition. Results I i Therefore, to obtain a smooth movement, particularly at the lowest positions, a computer monitor with both a high refresh rate and a high resolution was used, and a smaller movement-distance/ ball-size ratio was adopted. (See the Methods below for detailed movement parameters).hhf p p The stability of beat synchronization was assessed using a circular analysis method11,13. The difference between the time of a tap and the time of the corresponding event onset was measured by the relative phase (RP) on a unit circle. Synchronization stability was indexed by R, which was the length of the resultant of the RPs11. R ranged from 0 (randomly unstable tapping) to 1 (perfectly stable tapping) (see the Methods below for details). A two-way repeated measures analysis of variance (ANOVA) with the factors sequence type (three sequence types) and IOI type (two IOI types) showed a significant main effect for sequence type (F2,28 = 11.283, p = 0.001, partial η 2 = 0.545) (Fig. 2). There was no significant main effect for IOI type and no significant interaction between the two factors. The comparisons between sequence types for individual IOIs in experiments 1 and 2 are listed in Table 1. The mean and SD of the stability (R) for all sequence types and all IOI types in all experiments are listed in Table S1. For both IOIs, tapping was more stable for the auditory tone sequence than for the visual flashing ball sequence, Scientific Reports \| 5:11974 \| DOI: 10.1038/srep11974 3 www.nature.com/scientificreports/ Figure 3. Results of experiment 2. Tapping to the visual bouncing ball sequence was less stable than tapping to the auditory tone sequence for the short 300 ms IOI, whereas the reverse pattern was exhibited for larger IOIs. The conventions are as in Fig. 2. Figure 3. Results of experiment 2. Tapping to the visual bouncing ball sequence was less stable than tapping to the auditory tone sequence for the short 300 ms IOI, whereas the reverse pattern was exhibited for larger IOIs. The conventions are as in Fig. 2. replicating the well-known auditory advantage of beat synchronization. However, tapping to the visual bouncing ball sequence was also more stable than tapping to the visual flashing ball sequence, and was not less stable than tapping to the auditory tone sequence (Table 1). Therefore, the auditory advantage disappeared when the realistically moving visual stimuli were adopted. Scientific Reports \| 5:11974 \| DOI: 10.1038/srep11974 Results I i p g Previous studies have found that the bouncing ball sequence with a velocity that varied according to a rectified sinusoid9,10 was more effective in improving synchronization than the bouncing ball sequence with a constant velocity14, and it has been suggested that the former sequence is more realistic than the latter sequence10. Notably, synchronization to the bouncing ball sequence with a velocity that varied according to a rectified sinusoid was almost as good as synchronization to an auditory metronome9,10. Inspired by these advances, we designed a bouncing ball sequence that was even more realistic by two manipulations; (1) simulating the effect of gravity and (2) improving movement smoothness. The results of experiments 1 and 2 showed that synchronization to the current bouncing ball sequence was not poorer than synchronization to the auditory tone sequence (except for the 300 ms IOI sequences). Based on the current observations, it is of interest to determine whether synchronization to the current bounc- ing ball sequence could be slightly better than synchronization to the bouncing ball sequence with a velocity that varied according to a rectified sinusoid9,10 (since the latter sequence has been the most effec- tive moving stimulus in improving synchronization and its synchronization performance was slightly less stable than that observed in the auditory metronome condition). This was tested in control experiment 1, in which the current 600 ms IOI bouncing ball sequence with the two manipulations was compared with a control bouncing ball sequence without the two manipulations. We emphasize that the velocity of the Scientific Reports \| 5:11974 \| DOI: 10.1038/srep11974 4 www.nature.com/scientificreports/ Figure 4. Results of control experiments 1 and 2. The bouncing ball sequence in the current study included (1) simulating the effect of gravity and (2) improving movement smoothness. The current 600 ms IOI sequence with the two manipulations was compared with a control sequence without the two manipulations in control experiment 1, and was compared with control sequences without one of the two manipulations in control experiment 2. The results of control experiment 1 (A) showed that combining the two manipulations significantly improved tapping stability. The results of control experiment 2 (B) showed that removing individual manipulations did not yield significant performance differences, although the mean stability was highest for the sequence with both manipulations (GES&MSI vs. GES: t8 = 1.679, p = 0.132, η 2 = 0.261; GES&MSI vs. Results I i In addition, a typical 60 Hz refresh rate monitor was used and a large movement-distance/ball-size ratio was adopted for the control sequence. Moreover, the effect of each of the two manipulations of the cur- rent bouncing ball sequence was examined in control experiment 2, in which the current 600 ms IOI bouncing ball sequence with the two manipulations was compared with the control sequences without one of the two manipulations. The control sequence without the gravity effect simulation was the same as the current 600 ms IOI bouncing ball sequence except its velocity was varied according to a sinusoid, as described above. The control sequence without the movement smoothness improvement was the same as the current 600 ms IOI bouncing ball sequence except a typical 60 Hz refresh rate monitor was used and a large movement-distance/ball-size ratio was adopted, as described above. The results of control experiment 1 showed that combining the two manipulations (i.e., the current bouncing ball sequence (mean stability = 0.946)) significantly improved tapping stability (compared with the control sequence without the two manipulations (mean stability = 0.934)) (t8 = 2.685, p = 0.028, η 2 = 0.474) (Fig. 4A). The results of control experiment 2 showed that removing individual manipulations did not yield significant performance differences (although the mean stability (see Table S1) was highest for the current bouncing ball sequence with both manipulations) (Fig. 4B).f q p g Because the current bouncing ball sequence involved the simulation of the effect of gravity, it is rea- sonable that synchronization performance would degrade if the stimulus presentation was rotated 90°. This was confirmed in control experiment 3 in which the current 600 ms IOI bouncing ball sequence was rotated 90° counterclockwise. Synchronization to the non-rotated sequence (mean stability = 0.933) was more stable than synchronization to the rotated sequence (mean stability = 0.916) (t8 = 2.359, p = 0.046, η 2 = 0.410). We should mention here that the results may also be influenced by the compatibility of the direction of motion of the moving stimulus and the direction of motion of the tapping finger14; they were compatible for the non-rotated sequence and were incompatible for the rotated sequence in control experiment 3. In addition, with the exception of the 300 ms IOI bouncing ball sequence in experiment 2, most sub- jects exhibited negative relative phases of tapping (Fig. S1). Results I i GES&MSI refers to the 600 ms IOI bouncing ball sequence as in experiment 1. Other conventions are as in Fig. 2. control sequence was varied according to a sinusoid, rather than a rectified sinusoid; we did not replicate the stimuli in the previous studies because it has not been described in details how the rectified sinusoid was constructed9,10. Therefore, the current study did not directly compare the current bouncing ball sequence and the bouncing ball sequence with a velocity that varied according to a rectified sinusoid9,10. In addition, a typical 60 Hz refresh rate monitor was used and a large movement-distance/ball-size ratio was adopted for the control sequence. Moreover, the effect of each of the two manipulations of the cur- rent bouncing ball sequence was examined in control experiment 2, in which the current 600 ms IOI bouncing ball sequence with the two manipulations was compared with the control sequences without one of the two manipulations. The control sequence without the gravity effect simulation was the same as the current 600 ms IOI bouncing ball sequence except its velocity was varied according to a sinusoid, as described above. The control sequence without the movement smoothness improvement was the same as the current 600 ms IOI bouncing ball sequence except a typical 60 Hz refresh rate monitor was used and a large movement-distance/ball-size ratio was adopted, as described above. The results of control experiment 1 showed that combining the two manipulations (i.e., the current bouncing ball sequence (mean stability = 0.946)) significantly improved tapping stability (compared with the control sequence without the two manipulations (mean stability = 0.934)) (t8 = 2.685, p = 0.028, η 2 = 0.474) (Fig. 4A). The results of control experiment 2 showed that removing individual manipulations did not yield significant performance differences (although the mean stability (see Table S1) was highest for the current bouncing ball sequence with both manipulations) (Fig. 4B).f control sequence was varied according to a sinusoid, rather than a rectified sinusoid; we did not replicate the stimuli in the previous studies because it has not been described in details how the rectified sinusoid was constructed9,10. Therefore, the current study did not directly compare the current bouncing ball sequence and the bouncing ball sequence with a velocity that varied according to a rectified sinusoid9,10. Results I i MSI: t8 = 1.931, p = 0.090, η 2 = 0.318). GES represents the gravity effect simulation and MSI represents the movement smoothness improvement. GES&MSI refers to the 600 ms IOI bouncing ball sequence as in experiment 1. Other conventions are as in Fig. 2. Figure 4. Results of control experiments 1 and 2. The bouncing ball sequence in the current study fh Figure 4. Results of control experiments 1 and 2. The bouncing ball sequence in the current study included (1) simulating the effect of gravity and (2) improving movement smoothness. The current 600 ms IOI sequence with the two manipulations was compared with a control sequence without the two manipulations in control experiment 1, and was compared with control sequences without one of the two manipulations in control experiment 2. The results of control experiment 1 (A) showed that combining the two manipulations significantly improved tapping stability. The results of control experiment 2 (B) showed that removing individual manipulations did not yield significant performance differences, although the mean stability was highest for the sequence with both manipulations (GES&MSI vs. GES: t8 = 1.679, p = 0.132, η 2 = 0.261; GES&MSI vs. MSI: t8 = 1.931, p = 0.090, η 2 = 0.318). GES represents the gravity effect simulation and MSI represents the movement smoothness improvement. GES&MSI refers to the 600 ms IOI bouncing ball sequence as in experiment 1. Other conventions are as in Fig. 2. Figure 4. Results of control experiments 1 and 2. The bouncing ball sequence in the current study included (1) simulating the effect of gravity and (2) improving movement smoothness. The current 600 ms IOI sequence with the two manipulations was compared with a control sequence without the two manipulations in control experiment 1, and was compared with control sequences without one of the two manipulations in control experiment 2. The results of control experiment 1 (A) showed that combining the two manipulations significantly improved tapping stability. The results of control experiment 2 (B) showed that removing individual manipulations did not yield significant performance differences, although the mean stability was highest for the sequence with both manipulations (GES&MSI vs. GES: t8 = 1.679, p = 0.132, η 2 = 0.261; GES&MSI vs. MSI: t8 = 1.931, p = 0.090, η 2 = 0.318). GES represents the gravity effect simulation and MSI represents the movement smoothness improvement. Scientific Reports \| 5:11974 \| DOI: 10.1038/srep11974 Discussion Th The superiority of the auditory over visual modality in beat synchronization is one of the best-known results in studies of sensorimotor synchronization3. Recent advances, however, have shown that syn- chronization to a visual beat can be improved using moving stimuli instead of a conventional flashing light9,10,14. Specifically, synchronization to a bouncing ball was almost as good as synchronization to an auditory metronome (although a slight auditory advantage still existed), suggesting the importance of the realism of motion in improving synchronization to a visual beat9,10. By designing a bouncing ball that was even more realistic, the present study found that tapping to the bouncing ball was not less sta- ble than tapping to an auditory metronome for the IOIs from 500 to 900 ms (with the exception of the 300 ms IOI), demonstrating that synchronization to a visual beat can indeed be as good as that to an auditory beat. Moreover, it deserves to be pointed out that, for the IOIs from 500 to 900 ms, the mean of the synchronization stability was greater for the bouncing ball than for the auditory metronome (Fig. 2, Fig. 3, and Table S1). Although the size of the effect was small and the effect did not reach significance (with Bonferroni corrections) for individual IOIs in an experiment (Table 1), the effect was reliably rep- licated for the IOIs longer than 300 ms in both experiments 1 and 216. Therefore, the present results not only demonstrate that synchronization to a visual beat can be as good as synchronization to an auditory beat but also indicate that synchronization may be better to a visual than to an auditory beat, under optimized conditions.h The realism of moving stimuli has been suggested to be an important factor for the improvement of synchronization performance10. For example, the bouncing ball sequence with a velocity that varied according to a rectified sinusoid9,10 was more effective in improving synchronization than the bouncing ball sequence with a constant velocity14. The present results showed that if the moving stimulus was more realistic, its synchronization could be further improved and was not less stable than synchronization to an auditory beat. Given these results, however, it remains to be determined in future studies whether the realism of moving stimuli is the most critical factor in improving synchronization performance9. Discussion Th Specifically for the bouncing ball sequence in the present study, it needs to be further clarified whether the effects of the gravity effect simulation and the movement smoothness improvement could only be interpreted as the increase of stimulus realism. In addition, large subject variability has been shown in synchronization studies9 and therefore it is essential to examine the current results in future studies with different samples of subjects.hi f p j The current finding has important implications for the neural and evolutionary substrates of beat synchronization. Tighter connections between the auditory and motor cortices than between the visual and motor cortices have been proposed for sensorimotor synchronization4–6. The simple tapping task employed in the present study recruits a striato-thalamo-cortical loop, in which the basal ganglia (par- ticularly the putamen) plays a crucial role in coordinating the input timing information in the auditory or visual areas and the action timing in the motor areas for review, see 8. Using a visual beat consisting of moving stimuli, a recent study showed that basal ganglia activation was associated with synchronization stability rather than modality specificity11, which is consistent with the current behavioral evidence that does not support the modality bias of beat synchronization. A vocal learning and rhythmic synchroni- zation (VLRS) hypothesis proposed that vocal learning drives the evolution of beat synchronization and that beat synchronization is a brain function that is shared by humans and only a few other species with vocal learning ability6. The VLRS hypothesis is based upon the assumption that vocal learning and beat synchronization are supported by general tighter connections between the auditory and motor cortices than between the visual and motor cortices6, whereas this assumption is not supported by the current finding, as discussed above. It should be noted that the bouncing ball sequence as used in the current study presented the perceptual system with continuous information (motion), while the auditory tone sequence used discretely-timed information. Therefore, the current results may suggest a useful refine- ment to the VLRS hypothesis that the general-tighter-connection assumption may only apply to circuits involved in the timing of discretely-timed periodic events. g y p In summary, the present study found that synchronization to a bouncing ball with a realistic motion trajectory was not less stable than synchronization to an auditory metronome. Results I i This represented the negative mean asynchrony (NMA) that is typically observed in humans. We also analyzed the lag-1 autocorrelation of the inter-tap intervals (AC-1) (a negative AC-1 could suggest error correction whereas a positive or non-negative Scientific Reports \| 5:11974 \| DOI: 10.1038/srep11974 5 www.nature.com/scientificreports/ (i.e., not significantly negative) AC-1 could suggest absent or weak error correction)9,14,15. Consistent with previous findings9,14,15, negative AC-1 values were observed for the auditory tone sequence and the visual bouncing ball sequence (with the exception of the 300 ms IOI bouncing ball sequence in experiment 2) and non-negative AC-1 values were observed for the visual flashing ball sequence (Table S2). These results further supported the validity of the present data, and that the subjects were indeed synchronizing, and not simply tracking or ‘intercepting’ the moving ball stimuli. Discussion Th This finding challenges the auditory advantage of beat synchronization and calls for a reconsideration of the biological substrates of beat synchronization that were proposed based on the modality bias. Methods The keyboard used in the present study was a standard Dell computer keyboard, which introduced a systematic latency (about 10 ms) in checking the tapping. As a result, the effective temporal resolution of the keyboard was about ± 10 ms. g pp gf p y In experiment 1, three types of isochronous sequences with either a 600 or a 900 ms inter-onset inter- val (IOI) were presented: the auditory tone sequence, the visual flashing ball sequence, and the visual bouncing ball sequence (Fig. 1). For the auditory tone sequence, a pure tone (600 Hz, 50 ms duration) was presented every 600 ms or 900 ms for the 600 and 900 ms IOI sequences, respectively. An orange ball was displayed at the center of the computer screen on a black background. The ball was 1.74 cm in diameter. A 3.54 × 0.06 cm white bar was 0.92 cm below the bottom edge of the ball. The subjects were required to fixate on the ball and to maintain attention on the auditory task (so that the subjects would not look around and be attracted by other factors). For the visual flashing ball sequence, the ball flashed every 600 ms or 900 ms for the 600 and 900 ms IOI sequences, respectively (the ball lasted for 50 ms and disappeared for the remaining IOI time). For the visual bouncing ball sequence, the ball was replaced with a realistic orange basketball. The basketball continually moved 0.92 cm (movement distance) down and touched the bar, and then moved up to the initial position. The velocity of the bouncing ball was varied by simulating the effect of gravity, i.e., with a uniformly varying velocity. The acceleration was 0.20 m/s2 for the 600 ms IOI bouncing ball sequence and was 0.09 m/s2 for the 900 ms IOI bouncing ball sequence. The ball size, the movement distance, and the movement-distance/ball-size ratio of 0.529 were kept constant across different IOIs. Each movement step lasted for a frame. The stimuli were presented using Psychtoolbox (http://psychtoolbox.org). Event onsets referred to the onsets of the auditory tone, the onsets of the visual flashes, or the moment when the ball touched the bar for the three sequence types, respectively. Stimulus presentation was self-paced (the subjects pressed the space bar to start a sequence). Each sequence had 55 events (54 IOIs or circles). Methods Participants. Fifteen subjects (all right-handed, four males, mean age ± SD 22.7 ± 2.9 years), fifteen subjects (all right-handed, four males, mean age ± SD 22.6 ± 1.6 years), nine subjects (all right-handed, two males, mean age ± SD 24.3 ± 4.4 years), nine subjects (all right-handed, three males, mean age ± SD 23.4 ± 4.0 years), and nine subjects (all right-handed, four males, mean age ± SD 23.1 ± 1.9 years) Scientific Reports \| 5:11974 \| DOI: 10.1038/srep11974 6 www.nature.com/scientificreports/ participated in experiment 1, experiment 2, control experiment 1, control experiment 2, and control experiment 3, respectively. Three subjects in experiment 1 (playing piano for five, ten, and five years, respectively), three subjects in experiment 2 (playing piano for ten, five, and seven years, respectively), one subject in control experiment 1 (playing piano for ten years), and two subjects in experiment 3 (both playing piano for five years) reported musical experience. Two subjects participated in experiments 1 and 2; one subject participated in experiment 1, experiment 2, and control experiment 3; one subject participated in experiment 1, experiment 2, control experiment 2, and control experiment 3; and one subject participated in experiment 1, control experiment 2, and control experiment 3. All subjects had normal hearing and had normal or corrected-to-normal vision. The research protocols in this study were approved by the Institutional Review Board of Psychology Department of Sun Yat-Sen University. All subjects gave written informed consent. A typical tapping task involves approximately nine subjects17, which was the number of subjects in the present control experiments. To further validate and confirm the new finding of the present study, fifteen subjects were recruited in experiments 1 and 2. One subject in experiment 2 was excluded from analyses because the subject reported difficulty in performing the task and did not finish the experiment. The methods were carried out in accordance with the approved guidelines. Stimuli and procedure. The subjects sat in front of an LCD computer monitor (120 Hz refresh rate, 1920 × 1080 resolution, and 53.1 cm × 29.8 cm) with a viewing distance of 60 cm and wore a headset. In all the experiments, the subjects were asked to tap in synchrony with isochronous sequences using the index finger of their preferred hand on a key of a computer keyboard. Scientific Reports \| 5:11974 \| DOI: 10.1038/srep11974 Methods A 2 s blank screen was presented at the beginning and end of the auditory tone or visual flashing ball sequence, and a 2s-IOI/2 blank screen was presented at the beginning and end of the visual bouncing ball sequence (the ball moved down from the center of the screen at the beginning of a sequence and moved back to the screen center at the end; the event onset referred to the ball touching the bar at the lowest position). (See Video S1 for the demo of the 600 ms IOI visual bouncing ball sequence). Each sequence type was repeated six times. The orders of the IOI types and the sequence types were counterbalanced across the subjects. Experiment 2 was the same as experiment 1 except 1) there were four IOI types (300, 500, 700, and 900 ms) and two sequence types (auditory tone and visual bouncing basketball sequences); and 2) the movement distance of the ball was 0.77 cm and the movement-distance/ball-size ratio was 0.443 (this ratio was smaller than that in experiment 1. This was done to avoid movement discontinuities at the lowest ball positions for the 300 ms IOI sequence). The accelerations were 0.68 m/s2, 0.25 m/s2, 0.13 m/s2, and 0.08 m/s2 for the 300, 500, 700, and 900 IOIs, respectively. In control experiment 1, using a 600 ms IOI, the current visual bouncing ball sequence was compared with a control visual bouncing ball sequence. For the control sequence, a typical LCD monitor (60 Hz refresh rate, 1440 × 900 resolution, and 41 cm × 25 cm) was used. The movement distance is 1.74 cm and the movement-distance/ball-size ratio was 1. The velocity of the ball was calculated according to a sinusoid. Other experimental settings were as in experiment 1. In control experiment 2, the current 600 ms IOI visual bouncing ball sequence was compared with two control visual bouncing ball sequences. The control sequences were the same as the current bouncing ball sequence with the exception that in one the velocity of the ball was calculated according to a sinusoid (Fig. S2) and in the other a typical LCD monitor was used and the movement-distance/ball-size ratio was 1 (as manipulated in control experiment 1). Other experimental settings were as in experiment 1. References d hl Spatiotemporal Relations and Movement Trajectories in Visuomotor Synchronization. Music Percept 28, 15–26 (2010).h p 16. Cumming, G. The New Statistics Why and How. Psychol Sci 25, 7–29 (2014).hl h 17. Patel, A. D., Iversen, J. R., Chen, Y. & Repp, B. The influence of metricality and modality on synchronization with a beat. Exp Brain Res 163, 226–238 (2005).t 17. Patel, A. D., Iversen, J. R., Chen, Y. & Repp, B. The influence of metricality and modality on synchronization with a beat. Exp Brain Res 163, 226–238 (2005). h l T T lb f l ft ( ) 17. Patel, A. D., Iversen, J. R., Chen, Y. & Repp, B. The influence of metricality and modality o Brain Res 163, 226–238 (2005).t 18. Philipp, B. CircStat: A MATLAB Toolbox for Circular Statistics. J Stat Soft 31, 1–21 (2009). Acknowledgmentsh g This work was supported by National Nature Science Foundation of China (31371129), 985-3 project of Sun Yat-Sen University (16110-3281303), and the philosophical and social science project of Guangdong Province (GD12YXL02). We thank Junkai Yang and Yaohua Bi for their help in preparing the experimental equipment. Methods Scientific Reports \| 5:11974 \| DOI: 10.1038/srep11974 7 www.nature.com/scientificreports/ In control experiment 3, the current 600 ms IOI visual bouncing ball sequence was compared with a control sequence that was produced by rotating the presentation of the current 600 ms IOI visual bouncing ball sequence counterclockwise by 90°. Other experimental settings were as in experiment 1. Data analyses. We used circular analysis methods because they are more suitable for the variable periodic synchronization data than the standard linear analysis methods7,11,13,14. The analyses were per- formed using the CircStat toolbox18 programmed with MATLAB (The Mathworks, Natick, MA, USA). The difference between the time of a tap and the time of the corresponding event onset (asynchrony) was measured by the relative phase (RP) on a unit circle (-pi to pi. 0 indicates perfect alignment between taps and events; negative and positive values indicate taps preceding or following events, respectively; and ± pi indicates taps midway between events). Synchronization stability was indexed by R, which was the length of the resultant (i.e., average of vectors) of the RPs and was calculated by abs(sum(ex- p(iRP))/n) (n indicates the number of the RPs)11. R ranged from 0 (unstable tapping with uniformly distributed relative phases) to 1 (perfectly stable tapping with a unimodal distribution of relative phases). Correspondingly, mean asynchrony was indexed by the angle of the resultant of the RPs and was calculated by angle(sum(exp(iRP))/n). For the illustration of the circular histogram of the RPs (see Fig. S1F), each bin encompassed a radian range of 1/10 pi (0–1/10 pi, 1/10 –1/5 pi, etc.), with 20 bins in a circular distribution. The radial axis was set between 0–1, which represented the proportion to the total number of taps. The resultant of the RPs was represented by the red arrow, with its length indicating R and its angle indicating mean asynchrony. The taps to the first five events in a sequence were omitted from the analyses because synchronization typically requires a few taps to stabilize. In addition, the relation between adjacent inter-tap intervals (ITI) was assessed by lag-1 autocorrelation of the ITIs (AC-1)9,11. Greenhouse-Geisser corrections were applied to all ANOVA analyses. Bonferroni corrections were applied to all t-tests and corrected p values ≤ 0.05 were considered significant. All t-tests were two-tailed. pp y pp g y , 3. Repp, B. Rate Limits of Sensorimotor Synchronization. Adv Cogn Psychol 2, 163–181 (2006).hh References d hl 1. Lerdahl, F. & Jackendoff, R. An Overview of Hierarchical Structure in Music. Music Percept 1, 229–252 (1983). 2. Repp, B. Sensorimotor synchronization: a review of the tapping literature. Psychon B Rev 12, 969–992 (2005). Jf p ( ) 2. Repp, B. Sensorimotor synchronization: a review of the tapping literature. Psychon B Rev 12, 969–992 (2005). pp y pp g y epp, B. Rate Limits of Sensorimotor Synchronization. Adv Cogn Psychol 2, 163–181 (2006).hh y g y 4. Thaut, M. H., Kenyon, G. P., Schauer, M. L. & McIntosh, G. C. The connection between rhythmicity and brain function. IEEE Eng Med Biol 18, 101–108 (1999). 5. Zatorre, R. J., Chen, J. L. & Penhune, V. B. When the brain plays music: auditory-motor interactions in music perception and production. Nat Rev Neurosci 8, 547–558 (2007).h 6. Patel, A. D. The Evolutionary Biology of Musical Rhythm: Was Darwin Wrong? PLoS Biol 12, e1001821 (2014). 7. Patel, A. D., Iversen, J. R., Bregman, M. R. & Schulz, I. Experimental Evidence for Synchronization to a Musical Beat in Nonhuman Animal. Curr Biol 19, 827–830 (2009). 8. Repp, B. & Su, Y.-H. Sensorimotor synchronization: A review of recent research (2006–2012). Psychon B Rev 20, 403–452 (2013). 9. Iversen, J. R., Patel, A. D., Nicodemus, B. & Emmorey, K. Synchronization to auditory and visual rhythms in hearing and dea individuals. Cognition 134, 232–244 (2015). 0. Hove, M. J., Iversen, J. R., Zhang, A. & Repp, B. H. Synchronization with competing visual and auditory rhythms: bouncing bal meets metronome. Psychol Res 77, 388–398 (2013). 11. Hove, M. J., Fairhurst, M. T., Kotz, S. A. & Keller, P. E. Synchronizing with auditory and visual rhythms: An fMRI assessment of modality differences and modality appropriateness. NeuroImage 67, 313–321 (2013). f 2. Zago, M. & Lacquaniti, F. Cognitive, perceptual and action-oriented representations of falling objects. Neuropsychologia 43 178–188 (2005). 13. Fisher, N. I. Statistical Analysis of Circular Data. (Cambridge University Press, Cambridge, 1993). 13. Fisher, N. I. Statistical Analysis of Circular Data. (Cambridge Statistical Analysis of Circular Data. (Cambridge University Pres 4. Hove, M. J., Spivey, M. J. & Krumhansl, C. L. Compatibility of motion facilitates visuomotor synchronization. J Exp Psycho Human 36, 1525–1534 (2010). 5. Hove, M. J. & Peter, E. Keller. Spatiotemporal Relations and Movement Trajectories in Visuomotor Synchronization. Musi Percept 28, 15–26 (2010).h 15. Hove, M. J. & Peter, E. Keller. Author Contributions G.L.Y., Z.L., Q.C., H.Y.Y. and W.X. developed the study design; G.L.Y., H.Y.Y. and Z.L. collected, analyzed, and interpreted the data under the supervision of W.X.; W.X. wrote the manuscript. All authors commented on and edited the manuscript, and approved the final version of the manuscript for submission. G.L.Y., H.Y.Y. and Z.L. share credit as first authors. 8 Scientific Reports \| 5:11974 \| DOI: 10.1038/srep11974 www.nature.com/scientificreports/ Scientific Reports \| 5:11974 \| DOI: 10.1038/srep11974 Additional Information Additional Information Supplementary information accompanies this paper at http://www.nature.com/srepihi Supplementary information accompanies this paper at http://www.nature.com/srepihi Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Gan, L. et al. Synchronization to a bouncing ball with a realistic motion trajectory. Sci. Rep. 5, 11974; doi: 10.1038/srep11974 (2015). This work is licensed under a Creative Commons Attribution 4.0 International License. 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https://openalex.org/W4200329402	https://link.springer.com/content/pdf/10.1007/s12264-021-00800-x.pdf	English	null	Anterior Cingulate Cortex Mediates Hyperalgesia and Anxiety Induced by Chronic Pancreatitis in Rats	Neuroscience Bulletin/Neuroscience bulletin	2,021	cc-by	14,289	Anterior Cingulate Cortex Mediates Hyperalgesia and Anxiety Induced by Chronic Pancreatitis in Rats Dan Ren1,2 • Jia-Ni Li2 • Xin-Tong Qiu2 • Fa-Ping Wan2,3 • Zhen-Yu Wu2 • Bo-Yuan Fan4 • Ming-Ming Zhang2 • Tao Chen2 • Hui Li2 • Yang Bai2,5 • Yun-Qing Li1,2,6,7 Received: 3 March 2021 / Accepted: 29 August 2021 / Published online: 15 December 2021 The Author(s) 2021 in rats. Morphological data indicated that painful CP induced a signiﬁcant increase in FOS-expressing neurons in the nucleus tractus solitarii (NTS) and ACC, and some FOS-expressing neurons in the NTS projected to the ACC. In addition, a larger portion of ascending ﬁbers from the NTS innervated pyramidal neurons, the neural subpopula- tion primarily expressing FOS under the condition of painful CP, rather than GABAergic neurons within the ACC. CP rats showed increased expression of vesicular glutamate transporter 1, and increased membrane trafﬁck- ing and phosphorylation of the N-methyl-D-aspartate receptor (NMDAR) subunit NR2B and the a-amino-3- hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit GluR1 within the ACC. Microinjection of NMDAR and AMPAR antagonists into the ACC to block excitatory synaptic transmission signiﬁcantly atten- uated abdominal hyperalgesia in CP rats, which was similar to the analgesic effect of endomorphins injected into the ACC. Speciﬁcally inhibiting the excitability of ACC pyramidal cells via chemogenetics reduced both hyperal- gesia and comorbid anxiety, whereas activating these neurons via optogenetics failed to aggravate hyperalgesia and anxiety in CP rats. Taken together, these ﬁndings provide neurocircuit, biochemical, and behavioral evidence for involvement of the ACC in hyperalgesia and anxiety in CP rats, as well as novel insights into the cortical modulation of painful CP, and highlights the ACC as a potential target for neuromodulatory interventions in the treatment of painful CP. Abstract Central sensitization is essential in maintaining chronic pain induced by chronic pancreatitis (CP), but cortical modulation of painful CP remains elusive. Here, we examined the role of the anterior cingulate cortex (ACC) in the pathogenesis of abdominal hyperalgesia in a rat model of CP induced by intraductal administration of trinitrobenzene sulfonic acid (TNBS). TNBS treatment resulted in long-term abdominal hyperalgesia and anxiety Dan Ren, Jia-Ni Li, Xin-Tong Qiu and Fa-Ping Wan have contributed equally to this work. Supplementary Information The online version contains supple- mentary material available at https://doi.org/10.1007/s12264-021- 00800-x. & Yun-Qing Li deptanat@fmmu.edu.cn 1 Department of Anatomy, Guangxi Medical University, Nanning 510000, China 1 Department of Anatomy, Guangxi Medical University, Nanning 510000, China 2 Department of Anatomy, Histology and Embryology and K. K. Neurosci. Bull. April, 2022, 38(4):342–358 www.neurosci.cn https://doi.org/10.1007/s12264-021-00800-x www.springer.com/12264 Neurosci. Bull. April, 2022, 38(4):342–358 www.neurosci.cn https://doi.org/10.1007/s12264-021-00800-x www.springer.com/12264 www.neurosci.cn www.springer.com/12264 Neurosci. Bull. April, 2022, 38(4):342–358 https://doi.org/10.1007/s12264-021-00800-x ORIGINAL ARTICLE 7 Department of Human Anatomy, College of Basic Medicine, Dali University, Dali 671000, China Introduction Recurrent or constant abdominal pain is the most promi- nent feature of chronic pancreatitis (CP), and this adversely impacts the quality of life in patients with CP. The pathogenesis of painful CP is poorly understood, and its management has become one of the most challenging issues for both gastroenterologists and pain physicians [1]. It was once believed that pancreatic causes could explain painful CP in most patients; however, some patients still suffer from pain after surgical removal of the pancreatic causes of pain [2, 3]. Thus, the focus of recent research on painful CP has shifted from the anatomical mechanism of the pancreas to the neurobiological mechanisms, which can be summarized in the following three processes: peripheral sensitization, pancreatic neuropathy, and neuroplasticity changes in the central pain circuit [4]. The sensory afferent ﬁbers of the pancreas can be divided into two parts [5]. On one hand, visceral informa- tion is transmitted through the greater and lesser splanchnic nerves to the bilateral T4 to L4 segments of the spinal cord. Sensory information enters the spinal dorsal horn and reaches the central nervous system through the spinal lemniscus [6]. This pathway has been widely studied [2, 7]. On the other hand, visceral information is transmitted to the nucleus tractus solitarii (NTS) through the bilateral vagus nerves [8, 9], then is relayed to higher brain centers, such as the locus coeruleus (LC), parabrachial nuclei, rostral ventrolateral medulla, amygdala, and thalamus [6, 10], and ultimately reaches the limbic system and cognitive brain centers, which are thought to mediate the emotional aspects of pain [8]. y p To test our hypothesis, a CP rat model was established by intraductal administration of trinitrobenzene sulfonic acid (TNBS). The abdominal withdrawal threshold (AWT) test was used to measure abdominal hyperalgesia and the open ﬁeld test to assess anxiety in CP rats. The existence of a direct NTS–ACC pathway and its involvement in painful CP was examined via the combination of tract tracing and immunostaining methods, with FOS expression as a bioactive marker for neural activation by painful CP. Morphological and molecular techniques were then applied to study the molecular basis of cortical sensitization in CP rats. Finally, the regulatory role of the ACC in abdominal hyperalgesia and anxiety was assessed via a Pharmacolog- ical approach, as well as optogenetics and chemogenetics aiming at manipulating the activity of ACC pyramidal neurons in CP rats. Anterior Cingulate Cortex Mediates Hyperalgesia and Anxiety Induced by Chronic Pancreatitis in Rats Leung Brain Research Centre, Fourth Military Medical University, Xi’an 710032, China 3 Department of Anatomy, Xuzhou Medical University, Xuzhou 221004, China 3 Department of Anatomy, Xuzhou Medical University, Xuzhou 221004, China 4 Department of Cardiology, The Second Afﬁliated Hospital of Xian Jiaotong University, Xi’an 710004, China 4 Department of Cardiology, The Second Afﬁliated Hospital of Xian Jiaotong University, Xi’an 710004, China 5 Department of Neurosurgery, General Hospital of Northern Theater Command, Shenyang 110016, China 5 Department of Neurosurgery, General Hospital of Northern Theater Command, Shenyang 110016, China Keywords Chronic pancreatitis Anterior cingulate cor- tex Nucleus tractus solitaries Hyperalgesia Anxiety 6 Key Laboratory of Brain Science Research and Transforma- tion in Tropical Environment of Hainan Province, Hai- kou 570216, China 7 Department of Human Anatomy, College of Basic Medicine, Dali University, Dali 671000, China 3 D. Ren et al.: Cortical Modulation of Hyperalgesia in Chronic Pancreatitis 343 receptor (AMPAR) [19]. Electrophysiological studies of VH rats have shown that colorectal anaphylaxis leads to enhanced synaptic transmission within the ACC [20, 22, 23] by postsynaptic recruitment of AMPARs [20, 24] and NMDARs [20, 22, 24, 25]. Thus, bilateral ACC lesioning or inactivation attenuates visceral hyperal- gesia as well as pain-related aversion in VH rats [20, 21, 26]. Nevertheless, whether such neuroplastic changes occur within the ACC under the condition of painful CP is still unknown. In addition, the neurocircuitry in the ACC that is the basis of the modulation of painful CP has received little attention. Our previous studies support the hypothesis that the NTS is an important central site for the processing of visceral pain caused by CP and angina pectoris [27, 28]. We propose that the ACC may receive direct visceral pain inputs from the NTS, and neuroplastic changes within the ACC are involved in hyperalgesia and comorbid anxiety under the condition of painful CP. Introduction The cerebral cortex is the ultimate hub for pain perception and sensitization, mediating the discriminative, affective, and cognitive dimensions of pain [11]. Elec- troencephalogram [12–14] and imaging studies [15, 16] have shown that the insular cortex and anterior cingulate cortex (ACC) play an important role in pain processing under the condition of CP. The ACC is a critical hub for gut-related viscerosensory processing [17] and visceromo- tor control [18]. In addition, the ACC participates in pain perception and the modulation of emotional pain. Long- term potentiation (LTP) of excitatory synaptic transmission within the ACC is believed to maintain pathological pain and pain-related anxiety [19]. Chronic Pancreatitis Rat Model The forced swimming test was conducted to evaluate depression-like behavior. Rats were forced to swim in an open plastic cylinder (diameter 28 cm, height 50 cm) ﬁlled with water (maintained at 25 C) to a height of 35 cm. The duration of immobility (ﬂoating without any struggle or active movements with the forepaws) was measured during a 5-min session. In accordance with previous reports, CP was induced by infusion of 2% TNBS (Sigma, St. Louis, MO, USA) via pancreatic catheter infusion [29]. Rats in the sham group were infused with the same amount of saline. The naı¨ve rats underwent no surgery. Brain Stereotaxic Injection The scalp was then sutured. In the elevated plus maze test (Med Associates, St. Albans, Vermont, USA), rats were placed in the center square (10 cm 9 10 cm) with the head toward a closed arm (50 cm 9 10 cm) and recorded for 5 min with the motion- tracking system. Anxiety-like behavior was evaluated by the number of arm entries (total crossings) and the time spent in the open arms (% time in open arms). The rotarod test (Shanghai Biowill Co. Ltd, Shanghai, China) was used to assess motor coordination. Before tests, rats were trained in three trials with the rod rotating at a constant speed of 5 revolutions per minute (r/min). During tests, rats were placed on the rotarod apparatus, with the rotation speed starting at 3 r/min and progressing to a maximum of 30 r/min. The latency to falling was recorded to assess motor coordination. Animals Male Sprague-Dawley rats (250–280 g) were provided by the Experimental Animal Center of the Fourth Military Medical University (Xi’an, China). All protocols were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University. The rats were habituated to the experimental environment for 3 days before behavioral tests, with feeding in an environment maintained under a 12/12 h light/dark cycle. All behavioral tests were carried out during the light phase. Studies concerning the role of the ACC in chronic visceral pain have been conducted in a rat model of visceral hypersensitivity (VH) [20, 21]. Glutamate is the major excitatory neurotransmitter within the ACC, and it mediates excitatory synaptic transmission via the N-methyl-D-aspartate receptor (NMDAR) and the a- amino-3-hydroxy-5-methyl-4-isoxazole propionic acid 123 12 344 Neurosci. Bull. April, 2022, 38(4):342–358 Behavioral Tests Behavioral tests were performed on postoperative days (POD) 3, 7, 14, and 28. The abdominal withdrawal threshold (AWT), a measure of referred abdominal mechanical hypersensitivity, is an indirect marker of visceral sensitization [29]. The AWT was calculated using von Frey ﬁlaments (VFFs; Stoelting, Kiel, WI, USA). The abdominal area designated for stimulation was shaved before tests. The rats were handled with caution and habituated to the testing apparatus for at least 30 min until they calmed down for 3 consecutive days before testing. VFFs with increasing force (0.16–26 g) were applied vertically to the right upper abdomen 5 times, each for 5–8s at 5-min intervals, and the minimal force that elicited a withdrawal response at least 3 times was considered as the AWT. The hindpaw withdrawal threshold (PWT) was also measured as in our previous work [30]. After behavioral tests, the rats were deeply anesthetized with 2% sodium pentobarbital (60 mg/kg) injected into the abdominal cavity, and pancreatic tissue obtained to conﬁrm pancreatitis. The pancreatic tissue was ﬁxed in 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.2–7.4) overnight at 4 C, transferred to progressive xylene washes, and embedded in parafﬁn. The parafﬁn blocks were cut into 5 lm sections and stained with hematoxylin and eosin (H&E). Brain Stereotaxic Injection Each rat was anesthetized with 2% sodium pentobarbital (40 mg/kg) injected into the abdominal cavity and then ﬁxed on a stereotaxic apparatus (68025, RWD Life Science, Shenzhen, China). After routine disinfection, a hole was drilled the skull with an electric cranial drill. For anterograde labeling, 0.2 lL rAAV-CaMKIIa-EYFP- WPRE-pA (rAAV9-107-1-3, BrainVTA, Wuhan, China) was injected into the left NTS with at the following coordinates: anterior-posterior, AP: –14.16; medial-lateral, ML: ±0.30; dorsal-ventral, DV: ?7.80 mm. For retrograde labeling, 0.02 lL of 4% ﬂuorogold (FG; 80014, Fluo- rochrome, Denver, CO, USA) dissolved in 0.9% saline was injected into the right ACC (AP: ? 1.20; ML: ± 0.60; DV: - 2.80 mm). The injections were performed using a microinjection pump for * 15 min. Injection needles were removed 5 min after injection. The scalp was then sutured. Each rat was returned to its home cage and fed for 4 weeks (for virus injection) or 1 week (for FG injection) before perfusion. The injection sites were observed under a FluoView1000 confocal microscope (Olympus, Shinjuku City, Tokyo, Japan). Each rat was anesthetized with 2% sodium pentobarbital (40 mg/kg) injected into the abdominal cavity and then ﬁxed on a stereotaxic apparatus (68025, RWD Life Science, Shenzhen, China). After routine disinfection, a hole was drilled the skull with an electric cranial drill. For anterograde labeling, 0.2 lL rAAV-CaMKIIa-EYFP- WPRE-pA (rAAV9-107-1-3, BrainVTA, Wuhan, China) was injected into the left NTS with at the following coordinates: anterior-posterior, AP: –14.16; medial-lateral, ML: ±0.30; dorsal-ventral, DV: ?7.80 mm. For retrograde labeling, 0.02 lL of 4% ﬂuorogold (FG; 80014, Fluo- rochrome, Denver, CO, USA) dissolved in 0.9% saline was injected into the right ACC (AP: ? 1.20; ML: ± 0.60; DV: Behavioral tests for higher brain function were con- ducted as described in previous studies [31, 32]. In the open ﬁeld test, rats were acclimated to the observation room, and then placed in the center of a novel open ﬁeld (100 cm 9 100 cm 9 60 cm). A motion-tracking system (Shanghai Mobile Datum Information Technology, Co. Ltd, Shanghai, China) was used to record their locomotion for 15 min. Anxiety-like behavior was estimated by the total distance traveled (total distance) and the percentage of that distance spent in the center of the open ﬁeld (% center distance). - 2.80 mm). The injections were performed using a microinjection pump for * 15 min. Injection needles were removed 5 min after injection. Single Immunostaining of VGluT1 or FOS To explore whether ACC glutamatergic and GABAergic neurons are activated by painful CP, double immunostain- ing of CaMKII and FOS, as well as GAD67 and FOS were further performed within the ACC in the naı¨ve, sham, and CP groups using the antibodies and procedures as follows: after blocking, the ACC sections were incubated in rabbit anti-CaMKII (1:300; ab134041, Abcam) and mouse anti- FOS (1:500; ab11959, Abcam) for double CaMKII/FOS immunostaining, and in rabbit anti-GAD67 (1:300; ab97739, Abcam) and mouse anti-FOS (1:500; ab11959, Abcam) for double GAD67/FOS immunostaining over- night at 4 C. The sections were then incubated with Alexa594-donkey anti-mouse (1:500; A21203, Invitrogen) and Alexa488-donkey anti-rabbit (1:500; A21206, Invitro- gen) (for double CaMKII/FOS immunostaining), or Alex- a594-donkey anti-mouse (1:500; A21203, Invitrogen) and Alexa488-donkey anti-rabbit (1:500; A21206, Invitrogen) (for double GAD67/FOS immunostaining) for 4 h at 4 C in darkness. The expression of FOS in CaMKII or GAD67- ir neurons in the ACC was observed under the confocal microscope. Three sections of the ACC from each rat were counted under a 209 objective lens to obtain the average number of FOS-expressing neurons among the different groups (n = 4 rats per group). For double-labeled neurons, the total number of double-labeled neurons and CaMKII or GAD67-expressing neurons from three sections in each rat was counted under a 209 objective lens) and then the proportions of double-labeled neurons to CaMKII or GAD67-expressing neurons were calculated. Furthermore, the average proportion of double-labeled neurons to CaMKII or GAD67-expressing neurons in all rats (n = 4) was calculated and compared. To substantiate enhanced glutamatergic transmission within the ACC, double VGluT1/NeuN immunostaining in tissue from sham and CP rats on POD 14 was applied as follows: the sections were removed from the NDS and incubated in mouse anti-NeuN (1:300; MAB324-K, Mil- lipore) and rabbit anti-VGluT1 (1:500; 135 303, Synaptic Systems, Go¨ttingen, NI, Germany) overnight at 4 C. The sections were then incubated in Alexa594-donkey anti- mouse (1:500; A21203, Invitrogen, Carlsbad, CA, USA) and Alexa488-donkey anti-rabbit (1:500; A21206, Invitro- gen) for 4 h at 4 C in darkness. After each step, the sections were rinsed three times with 0.01 mol/L PBS for 10 min. After the ﬁnal step, the sections were mounted on microscope slides and cover- slipped for examination by light microscopy (AH-3, Olympus, Tokyo, Japan) for FOS staining or by confocal microscopy (CLSM, FV1000, Olympus) for VGluT1/NeuN double-labeling immunoﬂuorescent staining. Single Immunostaining of VGluT1 or FOS TNBS- and saline-treated rats were deeply anesthetized as described above and perfused for FOS or vesicular glutamate transporter 1 (VGluT1) immunohistochemistry. After perfusion, the brains were removed, placed in 30% 123 123 D. Ren et al.: Cortical Modulation of Hyperalgesia in Chronic Pancreatitis 345 sucrose solution at 4 C until they sank, and then cut into 40 lm thick coronal sections. Sections containing the ACC and NTS were incubated in 10% normal donkey serum (NDS) for 40 min at 26 C to block non-speciﬁc immunoreactivity. establish the CP model. Brain sections were prepared as described previously. After conﬁrmation of the injection site within the NTS, virus-labeled terminals ﬁbers within the ACC were observed, and then representative images indicating the injection site and projection area were captured under the confocal microscope. Next, the targeted area in ACC was immunostained with the antibodies and procedures as follows: after blocking, the ACC sections were incubated overnight at 4 C in mouse anti-GAD67 (1:300; ab26116, Abcam) or rabbit anti-CaMKII (1:300; ab134041, Abcam). The sections were then incubated with Alexa594-donkey anti-mouse (1:500; A21203, Invitrogen) or Alexa594-donkey anti-rabbit (1:500; A21207, Invitro- gen) for 4 h at 4 C in darkness. The close contacts between EYFP-labeled terminals and CaMKII or GAD67- expressing cell bodies within the ACC were observed under the confocal microscope. To conﬁrm activation of the ACC and NTS by painful CP, single FOS immunostaining in tissue from sham rats (POD 14) and CP rats at different time points (POD 3, 7, 14, and 28) was performed according to a previously- described protocol [31]. After blocking, the sections were incubated overnight at 4 C for 18–24 h with mouse anti- FOS (1:500; ab11959, Abcam, Cambridge, MA, USA) and biotinylated donkey anti-mouse secondary antibody (1:500; AP192B, Millipore, Billerica, MA, USA) for 6 h (for immunoﬂuorescent staining) at room temperature, and avidin-biotin complex (1:200; PK-6101, Vector labs, Burlingame, CA, USA) for 2 h at 26 C. The sections were then immersed in 0.05 mol/L Tris-HCl (pH 7.6) containing 0.02% diaminobenzidine tetrahydrochloride (D006, Dojin Laboratory, Kumamoto, Japan) and 0.003% H2O2 for the visualization of FOS staining. The average number of FOS-expressing neurons in three sections containing the ACC or NTS from each rat in the sham and CP groups was counted under a 209 objective lens (n = 3 rats per group). Single Immunostaining of VGluT1 or FOS Images were analyzed using ImageJ2 or Fluoview software (Olympus). Double Immunostaining of CaMKII (or GAD67) and FOS Cannulation Surgery and Drug Microinjection Double Immunostaining of FG and FOS One week before surgery, both TNBS and sham rats were anesthetized and secured on a stereotaxic frame (RWD, Shenzhen, China). A double guide cannula (26 gauge) was implanted bilaterally into the ACC (AP: ?1.20, ML: ±0.60, DV: –2.50 mm). Before establishing the CP model, baseline AWT tests were run. On POD 13, AWT tests were run to verify the successful establishment of the painful CP model. On POD 14, intra-ACC injections were delivered through a double injector cannula (30 gauge), located 0.2 mm below the guide. A Hamilton syringe (10 lL) was connected to the injector by a thin polyethylene tube and driven by a motorized pump (Alcbio, Shanghai, China). Amino-5-phosphonovaleric acid (AP-5, 50 mmol/L, 0.4lL, per side; Tocris, Bristol, UK) or cyano-2, 6-cyano-7- nitroquinoxaline-2,3-dione (CNQX, 20 mol/L, 0.4lL, per side; Tocris) was infused into the bilateral ACC at 0.05 lL/ min with the same volume of saline in control rats. The injection cannula was removed 5 min after completing the injection. AWT was measured 30 min after microinjection. In addition, the analgesic effects of opioid injection into the ACC were tested as a positive control to evaluate the analgesic effects of CNQX and AP-5 in CP rats. Morphine (25 lg/0.4 lL per side; PH014355, Sigma), endomorphin-1 (EM-1; 25 lg/0.4 lL per side; 1055, Tocris), endomorphin- 2 (EM-2; 25 lg/0.4 lL per side; E3148, Sigma), or vehicle (saline; 0.4 lL per side;) were microinjected bilaterally into the ACC 30 min before behavioral tests on POD 3, 5, 7, 10, and 14. The injection sites were veriﬁed post hoc, and rats with inaccurate sites were eliminated from the study. To explore whether neurons in the NTS projecting to the ACC were activated by painful CP, double immunostaining of FG and FOS was performed in the NTS of CP rats 1 week after FG injection into the ACC. TNBS was administered 1 week before FG injection to establish the CP model. The preparation of brain sections was as previously described. After conﬁrmation of the injection site in the ACC and virus-labeled cell bodies in the NTS, immunostaining was performed using the following anti- bodies and procedures: after blocking, the ACC sections were incubated in mouse anti-FOS (1:500; ab11959, Abcam) and guinea pig anti-FG (1:200, NM-101, Pro- tosBiotech, New York, NY, USA) overnight at 4 C. Double Immunostaining of CaMKII (or GAD67) and FOS Double Immunostaining of CaMKII (or GAD67) and FOS To explore whether ACC glutamatergic and GABAergic neurons are innervated by a direct NTS–ACC pathway, single immunostaining of CaMKII or GAD67 was ﬁrst performed in the ACC in CP rats four weeks after rAAV- CaMKIIa-EYFP injection into the NTS. Two weeks after virus injection, the rats were treated with TNBS to 12 123 Neurosci. Bull. April, 2022, 38(4):342–358 346 Cannulation Surgery and Drug Microinjection The sections were then incubated with Alexa594-donkey anti- mouse (1:500; A21203, Invitrogen) or Alexa488-donkey anti-guinea pig (1:500; 706-545-148, Jackson Immunore- search, West Grove, PA, USA) for 4 h at 4 C in darkness. The expression of FOS in FG-labeled neurons in the NTS was observed under the confocal microscope. Western Blot Analysis The ACC of each rat was immediately removed into cold artiﬁcial cerebrospinal ﬂuid and homogenized in lysis buffer containing proteinase inhibitors (REF04693159001, Roche, Basel, Switzerland) and phosphatase inhibitors (REF04906837001, Roche). Total protein (for VGluT1 assay) was prepared according to the steps described in our previous study [30]. Membrane and cytoplasmic proteins were isolated using the procedure of the MinuteTM Plasma Membrane Protein Isolation Kit (SM-005, Invent Biotech- nologies, Eden Prairie, MN, USA). Protein concentration was quantiﬁed using a bicinchoninic acid kit (Thermo ScientiﬁcTM, Rockford, IL, USA). The antibodies used and their concentrations were as follows: mouse anti-VGluT1 (1:500; MAB5502, Millipore), rabbit anti-pNR2B-Tyr1472 (1:500; AB5403, Millipore), rabbit anti-NR2B (1:500; ab65783, Abcam), rabbit anti-GluR1 (1:500; AB2285, Millipore), rabbit anti-pGluR1-Ser845 (1:500; PA5110124, Invitrogen), rabbit anti-b-actin (1:5000; A1978, Sigma), and rabbit anti-N-cadherin (1:2000; AB1550, Millipore), and horseradish peroxidase-conjugated secondary antibod- ies (goat anti-rabbit, 1:5000; ZB-2301, Zsgb Biotech, Los Altos, CA; and goat anti-mouse, 1:5000; ZB-2305, Zsgb Biotech). The protein bands were visualized using an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA) and scanned using the ChemiDoc Imaging System (Bio-Rad Richmond, CA, USA). The band intensities were quantiﬁed using ImageJ, with all samples normalized using b-actin or N-cadherin as loading controls. Optogenetics, Chemogenetics, and Behavioral Tests In optogenetic tests, rAAV-CaMKIIa-ChR2-mCherry (0.4 lL/30 min) was injected into the bilateral ACC (AP: ? 1.20, ML: ± 0.60, DV: - 2.80 mm) one week before CP induction. One week after injection, baseline AWT was measured, and then a single optic ﬁber was implanted over the virus injection site (AP: ? 1.20, ML: 0.00, DV: - 2.50 mm) and then ﬁxed with dental cement. On POD 14, blue light (5 mW, 10-ms pulses at 10 Hz, 473 nm) was delivered for 15 min (5 min on, 5 min off, and 5 min on) using a laser source (Aurora-220, Newdoon Technology, Hangzhou, China). AWT was assessed before, during, and after illumination. Following the AWT tests, the open ﬁeld test was run for 15 min, and blue light was delivered from the ﬁfth to the tenth minute. In chemogenetic tests, rAAV-CaMKIIa-hM4Di- mCherry (0.4 lL/30 min) was injected into the bilateral ACC one week before baseline AWT testing and CP induction. On POD 13, AWT tests were run to verify 12 123 123 D. Ren et al.: Cortical Modulation of Hyperalgesia in Chronic Pancreatitis 347 successful establishment of the painful CP model. On POD 14, either saline or clozapine-N-oxide (CNO, 3 mg/kg; C4759, LKT Laboratories, MN, USA) was administered intraperitoneally, and AWT tests were performed 1 h later. Following AWT tests, the open ﬁeld test was run for 15 min. The injection sites were veriﬁed post hoc for both optogenetic and chemogenetic tests, and rats with inaccu- rate injection sites were eliminated from the study. hypolocomotion in the condition of chronic pain [33]. Thus, several behavioral assays were run to dissect the cause of immobility. CP rats displayed no marked alter- ations in the PWT compared to the sham group, thus excluding the possibility of somatic hyperalgesia (Fig. 1I). Rotarod testing showed no signiﬁcant differences in the falling latency at each time point between sham and CP rats, excluding the possibility of motor coordination disorder (Fig. 1J). Interestingly, CP rats exhibited a prolonged immobility time in the forced swimming test compared to the sham group, implying the existence of depression (Fig. 1K). Considering these results, we believe that the hypolocomotion in CP rats is closely related to abdominal hyperalgesia-induced negative emotions, rather than motor disorders or hindlimb pain. Optogenetics, Chemogenetics, and Behavioral Tests Thus, we used both distance traveled and exploratory behavior in the open ﬁeld test to assess the role of the ACC in emotional pain modulation in CP rats. Neurons Since FOS expression is strongly correlated with noxious somatic or visceral stimuli [35], we performed FOS immunostaining in the NTS and ACC after the induction of CP. From POD 3 to POD 28, the number of FOS- immunoreactive (-ir) neurons in both the NTS and ACC signiﬁcantly increased in CP rats compared to that in the sham group (Fig. 2A–L). Further analysis showed that TNBS treatment signiﬁcantly increased FOS expression in both superﬁcial layers II-III (TNBS: 1260.78 ± 151.48 versus sham: 503.89 ± 75.07, P\0.01) and deeper layers V-VI (TNBS: 630.33 ± 75.66 versus sham: 252.11 ± 37.54, P\0.01; Fig. 2M). These data laid a morphological foundation for activation of the NTS and ACC by painful CP. Data Analysis Data were analyzed using the SPSS ver. 19.0 (IBM Corp., Armonk, NY, USA) and are expressed as the mean ± standard error (mean ± SEM). Images were processed using Adobe Photoshop CS5 (Mountain View, CA, USA). Statis- tical comparisons between multiple groups were made using one-way ANOVA or one-way repeated ANOVA followed by the LSD post-hoc test, while statistical comparisons between two groups were made using unpaired or paired t- tests. Statistical graphics were produced using GraphPad Prism Version 5 (San Diego, CA, USA). P \ 0.05 was considered statistically signiﬁcant. Chronic Pancreatitis Stimuli Induce FOS Expres- sion in the NTS, ACC, and NTS–ACC Projection Neurons TNBS-Induced Abdominal Hyperalgesia and Hypolocomotion The TNBS-induced CP rat model is commonly used in experimental CP studies [33]. The validity of the CP model was evidenced by histopathological changes in pancreatic sections, such as acinar atrophy, inﬂammatory inﬁltration, and stromal ﬁbrosis (Fig. 1A, B), together with weight loss (Fig. 1H). Sham rats exhibited a transient decrease of AWT, which reached a minimum on POD 3 and returned to baseline on POD 14. This phenomenon was also seen in our previous study, and was owing to post-operative pain [34]. Com- pared to sham rats, CP rats had a lower AWT from POD 7 to 35 (Fig. 1C), indicating the presence of long-term abdominal hypersensitivity induced by TNBS. Open ﬁeld testing showed that CP rats traveled shorter distance in the open ﬁeld from POD 3 to 28 than sham rats, indicating hypolocomotion (Fig. 1D). Importantly, CP rats also traveled less distance in the central area, suggesting the occurrence of anxiety-like emotions (Fig. 1E). Elevated plus maze testing further showed that CP rats had fewer total crossings and spent less time in the open arms (Fig. 1F, G), in accordance with the results of the open ﬁeld tests. To determine whether the ACC receives direct input from the NTS, the anterograde tracer virus rAAV- CaMKIIa-EYFP was injected into the left NTS. The injection site was mainly located within the NTS with minimal spread to surrounding areas (Fig. 3A–C). EYFP- labeled ﬁbers and terminals were observed in the bilateral ACC, with a dominant distribution on the contralateral side. The ﬁbers and terminals were scattered throughout the superﬁcial and deeper layers of the ACC (Fig. 3D–F). Double immunostaining results further showed that EYFP-labeled ﬁbers and terminals were more likely to be opposed to CaMKII-expressing neurons than GAD-67- expressing neurons in the ACC (Fig. S1). To determine the expression of FOS in ACC CaMKII and GAD67-express- ing neurons under painful CP, double immunostaining of FOS and CaMKII, as well as FOS and GAD67 was Several scenarios, such as mood disturbance and motor defects, are worth considering with regard to 12 12 3 123 Neurosci. Bull. April, 2022, 38(4):342–358 348 performed We observed that a signiﬁcantly larger propor FOS (Fig 3H K) suggesting that the NTS neurons Fig. 1 Chronic pancreatitis induced by TNBS elicits abdominal hyperalgesia and anxiety-like behaviors in rats. TNBS-Induced Abdominal Hyperalgesia and Hypolocomotion A, B Representative H&E-stained histological sections of the pancreas on POD 28 in sham and TNBS-treated rats (scale bar, 100 lm). C TNBS-treated rats show a decreased abdomen withdrawal threshold during the course of CP compared to sham rats, while sham rats exhibit transient abdomen mechanical hypersensitivity that returns to baseline on POD 14 (n = 7 rats per group; P\0.01, P\0.001, TNBS vs sham; ##P\0.01, sham vs naı¨ve, one-way repeated ANOVA). D, E CP rats travel shorter distances (D) from POD 3 to 28 and travel less distance in the center area (E) from POD 7 to 28 in the open ﬁeld test than sham rats. F, G CP rats exhibit fewer crossings (F) and less time in the open arms (G) in the high elevated maze test than sham rats from POD 3 to 28 (n = 7 per group; P\0.05, P\0.01, P\0.001, TNBS vs sham, unpaired t-test). H CP rats lose body weight during the course of CP compared to sham rats, while sham rats show a transient weight loss from POD 3 to 7 which returns to baseline after POD 14 (n = 7 per group; P \ 0.05, P \ 0.01, P \ 0.001, TNBS vs sham; #P \ 0.05, ##P \ 0.01, sham vs naı¨ve, one-way repeated ANOVA). I CP rats exhibit no signiﬁcant change in hindpaw withdrawal threshold compared with the sham group (P[0.05, n = 7 per group, one-way repeated ANOVA). J, K No change in falling latency between CP and sham rats in the rotarod test (J), while CP rats exhibited a prolonged immobility time in the forced swimming test from POD 14 to 28 (K) (n = 7 per group; P\0.01, P\0.001, TNBS vs sham, unpaired t-test). AWT, abdomen withdrawal thresh- old; PWT, paw withdrawal threshold; TNBS, trinitrobenzene sulfonic acid. Fig. 1 Chronic pancreatitis induced by TNBS elicits abdominal hyperalgesia and anxiety-like behaviors in rats. A, B Representative H&E-stained histological sections of the pancreas on POD 28 in sham and TNBS-treated rats (scale bar, 100 lm). C TNBS-treated rats show a decreased abdomen withdrawal threshold during the course of CP compared to sham rats, while sham rats exhibit transient abdomen mechanical hypersensitivity that returns to baseline on POD 14 (n = 7 rats per group; P\0.01, **P\0.001, TNBS vs sham; ##P\0.01, sham vs naı¨ve, one-way repeated ANOVA). TNBS-Induced Abdominal Hyperalgesia and Hypolocomotion D, E CP rats travel shorter distances (D) from POD 3 to 28 and travel less distance in the center area (E) from POD 7 to 28 in the open ﬁeld test than sham rats. F, G CP rats exhibit fewer crossings (F) and less time in the open arms (G) in the high elevated maze test than sham rats from POD 3 to 28 (n = 7 per group; P\0.05, P\0.01, P\0.001, TNBS vs sham, unpaired t-test). H CP rats lose body weight during the course of CP compared to sham rats, while sham rats show a transient weight loss from POD 3 to 7 which returns to baseline after POD 14 (n = 7 per group; P \ 0.05, P \ 0.01, P \ 0.001, TNBS vs sham; #P \ 0.05, ##P \ 0.01, sham vs naı¨ve, one-way repeated ANOVA). I CP rats exhibit no signiﬁcant change in hindpaw withdrawal threshold compared with the sham group (P[0.05, n = 7 per group, one-way repeated ANOVA). J, K No change in falling latency between CP and sham rats in the rotarod test (J), while CP rats exhibited a prolonged immobility time in the forced swimming test from POD 14 to 28 (K) (n = 7 per group; P\0.01, *P\0.001, TNBS vs sham, unpaired t-test). AWT, abdomen withdrawal thresh- old; PWT, paw withdrawal threshold; TNBS, trinitrobenzene sulfonic acid. FOS (Fig. 3H–K), suggesting that the NTS neurons projecting to the ACC are activated under the condition of painful CP. performed. We observed that a signiﬁcantly larger propor- tion of CaMKII-expressing neurons expressed FOS than GAD67-expressing neurons in the ACC of CP rats (CaMKII: 95.22 ± 1.37% versus GAD67: 9.72 ± 1.43%; n = 4 per group; P\0.001; Fig. S2). These morphological data suggest that postsynaptic pyramidal neurons in the ACC play a predominant role in the transmission or modulation of painful CP. performed. We observed that a signiﬁcantly larger propor- tion of CaMKII-expressing neurons expressed FOS than GAD67-expressing neurons in the ACC of CP rats (CaMKII: 95.22 ± 1.37% versus GAD67: 9.72 ± 1.43%; n = 4 per group; P\0.001; Fig. S2). These morphological data suggest that postsynaptic pyramidal neurons in the ACC play a predominant role in the transmission or modulation of painful CP. Increased Expression of VGluT1 and Glutamate Receptors in the ACC After TNBS Injection VGluTs package glutamate into vesicles for synaptic release and transmission [36]. The expression level of VGluTs determines the amount of glutamate loaded into vesicles and released into the synaptic cleft, thus regulating the efﬁcacy of neurotransmission [36]. Among all the VGluTs, VGluT1 is strongly expressed in the cerebral Finally, retrograde labeling was used to verify the involvement of the direct NTS–ACC pathway in painful CP. After injection of FG into the ACC in CP rats (Fig. 3G), retrogradely-labeled cell bodies were seen in the contralateral NTS. Interestingly, these neurons expressed 123 123 D. Ren et al.: Cortical Modulation of Hyperalgesia in Chronic Pancreatitis 349 Fig. 2 TNBS treatment increases the number of FOS-ir neurons in the NTS and ACC. A–J Immunochemical staining of FOS in the NTS (A–E) and ACC (F–J) in sham rats on POD 14 and in TNBS-treated rats on POD 3, 7, 14, and 28 (scale bars, 200 lm). K, L Numbers of FOS-ir neurons in the NTS (K) and ACC (L) in sham rats on POD 14 and in TNBS-treated rats on POD 3, 7, 14, and 28. M Numbers of FOS-ir neurons in different layers of the ACC in sham rats and TNBS-treated rats on POD 14 (n = 3 per group; P \ 0.05, P \ 0.01, * P \ 0.001, TNBS vs sham, one-way ANOVA in K, L, unpaired t-test in M. and in TNBS-treated rats on POD 3, 7, 14, and 28. M Numbers of FOS-ir neurons in different layers of the ACC in sham rats and TNBS-treated rats on POD 14 (n = 3 per group; P \ 0.05, P \ 0.01, ** P \ 0.001, TNBS vs sham, one-way ANOVA in K, L, unpaired t-test in M. Fig. 2 TNBS treatment increases the number of FOS-ir neurons in the NTS and ACC. A–J Immunochemical staining of FOS in the NTS (A–E) and ACC (F–J) in sham rats on POD 14 and in TNBS-treated rats on POD 3, 7, 14, and 28 (scale bars, 200 lm). K, L Numbers of FOS-ir neurons in the NTS (K) and ACC (L) in sham rats on POD 14 cortex [37]. In this study, we applied immunostaining and biochemical analyses to examine the expression of VGluT1 within the ACC. Increased Expression of VGluT1 and Glutamate Receptors in the ACC After TNBS Injection Double immunostaining of VGluT1 and NeuN showed that more VGluT1-ir axon terminals appeared to be in close contact with neurons in CP rats than in sham rats on POD 14 (Fig. 4A). The upregulated expression of VGluT1 during the course of CP was then conﬁrmed by western blotting (Fig. 4B–C). These results suggest that CP stimulates glutamate release, providing evidence for enhanced presynaptic transmission in the ACC. of cytoplasmic pGluR1 remained unchanged (Fig. 5C, G). These data suggest that the enhanced postsynaptic trans- mission in the ACC of TNBS-induced CP rats can be attributed to the recruitment and modiﬁcation of the GluR1 subunit. NR2B, the predominant NMDAR subunit in the ACC, undergoes long-term plastic changes under the condition of sustained pain [40]. Biochemical analysis of NR2B in the ACC at different time points showed that its membrane protein expression was signiﬁcantly upregulated (Fig. 5B, H), while cytoplasmic expression was robustly down- regulated in CP rats compared to the sham group (Fig. 5C, I). In addition, membrane the expression of pNR2B was signiﬁcantly ramped up during the course of CP (Fig. 5B, J). Despite a tendency of decreased cytoplasmic expression of pNR2B in the CP group, no signiﬁcant difference was detected (Fig. 5C, K). These results suggest that TNBS induces membrane trafﬁcking and modiﬁes phosphoryla- tion of the NR2B subunit in the ACC. AMPAR and NMDAR expression are crucial for the induction and expression of pain-related LTP within the ACC, respectively [19]. Membrane and cytoplasmic pro- teins were isolated, and the distribution of N-cadherin, a speciﬁc marker of neural membrane, conﬁrmed the suc- cessful separation of membrane and cytoplasmic proteins (Fig. 5A). As the key subunit of AMPARs, GluR1 generates AMPAR trafﬁcking and integration within synaptic membranes [38]. Overexpression of GluR1 con- tributes to both somatic and visceral-related chronic pain [20, 39]. We found that the abundance of membrane GluR1 was markedly increased after TNBS treatment compared to that of the sham group (Fig. 5B, D), with no striking change in that of cytoplasmic GluR1 (Fig. 5C and E). Furthermore, the amount of membrane pGluR1 also increased during the course of CP (Fig. 5B, F), while that Relief of Hyperalgesia via Microinjection of CNQX or AP-5 into the ACC To determine the role of enhanced excitatory transmission in the ACC in the development of painful CP, the effects of local administration of CNQX and AP-5 to block ACC glutamatergic transmission on animal behaviors were 12 3 Neurosci. Bull. April, 2022, 38(4):342–358 350 Fig. 3 The NTS-ACC pathway is activated in rats with chronic pancreatitis. A Photomicrograph of an injection site of rAAV- CaMKIIa-EYFP in the left NTS (scale bar, 200 lm). B Representative Nissl-stained section showing an injection site in the NTS (scale bar, 400 lm). C Tracings showing the rostrocaudal extent of an injection site at different levels (black areas). D–F Representative ﬂuorescence photomicrographs showing anterograde-labeled ﬁbers and terminals in the ACC. The framed area in D is magniﬁed in E, while the framed area in E is enlarged in F [scale bars, 350 lm (D), 100 lm (E), and 40 lm (F)]. G Photomicrograph of an injection site of FG in the right ACC (scale bar, 400 lm). H, I Representative ﬂuorescence photomi crographs showing retrograde-labeled ACC-projecting neurons in th NTS [scale bars, 125 lm (H) and 40 lm (I)]. I, J Enlargements of th framed area in H showing that FG-labeled NTS-ACC projectio neurons (I, green) express FOS (J, red) under painful CP (scale bars 40 lm). K Enlargement of the framed area in (J) showing FG an FOS double-labeled neurons (yellow) in the NTS (scale bar, 25 lm) ACC, anterior cingulate cortex; AP, area postrema; cc, corpu callosum; CC, central canal; FG, ﬂuorogold; M2, secondary moto cortex; NTS, nucleus tractus solitarii; sol, solitary tract. Fig. 3 The NTS-ACC pathway is activated in rats with chronic pancreatitis. A Photomicrograph of an injection site of rAAV- CaMKIIa-EYFP in the left NTS (scale bar, 200 lm). B Representative Nissl-stained section showing an injection site in the NTS (scale bar, 400 lm). C Tracings showing the rostrocaudal extent of an injection site at different levels (black areas). D–F Representative ﬂuorescence photomicrographs showing anterograde-labeled ﬁbers and terminals in the ACC. The framed area in D is magniﬁed in E, while the framed area in E is enlarged in F [scale bars, 350 lm (D), 100 lm (E), and 40 lm (F)]. G Photomicrograph of an injection site of FG in the right ACC (scale bar, 400 lm). H, I Representative ﬂuorescence photomi- crographs showing retrograde-labeled ACC-projecting neurons in the NTS [scale bars, 125 lm (H) and 40 lm (I)]. Relief of Hyperalgesia via Microinjection of CNQX or AP-5 into the ACC 6C, consecutive bilateral microinjection of EM-1 and EM-2 into the ACC had signiﬁcant, stable analgesic effects in CP rats from POD 3 to POD 14 (AWT: 4.78 ± 0.32 g with saline versus 11.98 ± 0.57 g with EM-1 and 9.65 ± 0.49 g with EM-2, P \ 0.001 for both, n = 6 per group). However, with morphine injection, there was a trend toward decreased analgesic effects from POD 3 to POD 10, indicating morphine tolerance. On POD 14, no signiﬁcant difference was seen in the AWT between CP rats with morphine injection and saline injection. Relief of Hyperalgesia via Microinjection of CNQX or AP-5 into the ACC I, J Enlargements of the framed area in H showing that FG-labeled NTS-ACC projection neurons (I, green) express FOS (J, red) under painful CP (scale bars, 40 lm). K Enlargement of the framed area in (J) showing FG and FOS double-labeled neurons (yellow) in the NTS (scale bar, 25 lm). ACC, anterior cingulate cortex; AP, area postrema; cc, corpus callosum; CC, central canal; FG, ﬂuorogold; M2, secondary motor cortex; NTS, nucleus tractus solitarii; sol, solitary tract. saline versus 9.40 ± 3.97 g, n = 6 with CNQX, P \ 0.05; Fig. 6D) or AP-5 (AWT: 4.48 ± 3.11 g, n = 5 with saline versus 9.10 ± 3.60 g, n = 6 with AP-5, P\0.05; Fig. 6E) into the ACC on POD 14 partly reversed the abdominal hyperalgesia in CP rat; this was equal to the effect of EM-1 or EM-2 injection into the ACC on POD 14. In accordance with our biochemical data, these behavioral results suggest that enhanced excitatory glutamatergic transmission in the ACC by postsynaptic recruitment of AMPARs and NMDARs contributes to abdominal hyperalgesia in CP rats. measured by behavioral tests. Meanwhile, opioid receptor agonists that are known to elicit analgesia under chronic pain [41, 42] were microinjected into the ACC as a positive control. The experimental paradigm is illustrated in Fig. 6A, B. As shown in Fig. 6C, consecutive bilateral microinjection of EM-1 and EM-2 into the ACC had signiﬁcant, stable analgesic effects in CP rats from POD 3 to POD 14 (AWT: 4.78 ± 0.32 g with saline versus 11.98 ± 0.57 g with EM-1 and 9.65 ± 0.49 g with EM-2, P \ 0.001 for both, n = 6 per group). However, with morphine injection, there was a trend toward decreased analgesic effects from POD 3 to POD 10, indicating morphine tolerance. On POD 14, no signiﬁcant difference was seen in the AWT between CP rats with morphine injection and saline injection. measured by behavioral tests. Meanwhile, opioid receptor agonists that are known to elicit analgesia under chronic pain [41, 42] were microinjected into the ACC as a positive control. The experimental paradigm is illustrated in Fig. 6A, B. As shown in Fig. Relief of Hyperalgesia and Anxiety via Inhibiting Excitatory ACC Neurons Previous optogenetic studies have shown that excitatory pyramidal neurons are the primary components of the ACC Further behavioral data showed that bilateral microin- jection of either CNQX (AWT: 4.80 ± 1.09 g, n = 5 with 123 D. Ren et al.: Cortical Modulation of Hyperalgesia in Chronic Pancreatitis 351 in A3, A4). B Representative western blots for VGluT1 in the ACC in sham rats on POD 14 and in TNBS-treated rats on POD 3, 7, 14, and 28. C The expression of VGluT1 is signiﬁcantly enhanced from POD 3 to 28 in TNBS-treated rats compared with sham rats (n = 3 per group; P \ 0.05, P \ 0.01, *P \ 0.001, TNBS vs sham, one- way ANOVA followed by LSD post-hoc test). Fig. 4 TNBS treatment enhances the expression of VGluT1 in the ACC. A Microphotographs showing double-immunoﬂuorescence staining for VGluT1 (green) and NeuN (red) immunoreactivity in the ACC. The framed areas in images A1 and A2 are magniﬁed in images A3 and A4. The white arrowheads indicate, VGluT1-labeled axon terminals in close contact with NeuN-labeled somatic or dendritic proﬁles in the ACC (scale bars, 25 lm in A1, A2 and 5 lm in A3, A4). B Representative western blots for VGluT1 in the ACC in sham rats on POD 14 and in TNBS-treated rats on POD 3, 7, 14, and 28. C The expression of VGluT1 is signiﬁcantly enhanced from POD 3 to 28 in TNBS-treated rats compared with sham rats (n = 3 per group; P \ 0.05, P \ 0.01, P \ 0.001, TNBS vs sham, one- way ANOVA followed by LSD post-hoc test). Fig. 4 TNBS treatment enhances the expression of VGluT1 in the ACC. A Microphotographs showing double-immunoﬂuorescence staining for VGluT1 (green) and NeuN (red) immunoreactivity in the ACC. The framed areas in images A1 and A2 are magniﬁed in images A3 and A4. The white arrowheads indicate, VGluT1-labeled axon terminals in close contact with NeuN-labeled somatic or dendritic proﬁles in the ACC (scale bars, 25 lm in A1, A2 and 5 lm in A3, A4). B Representative western blots for VGluT1 in the ACC in sham rats on POD 14 and in TNBS-treated rats on POD 3, 7, 14, and 28. Discussion Finally, we further revealed that ACC pyramidal neurons mediated the behav- ioral representation of painful CP since chemogenetic inhibition of these neurons alleviated both hyperalgesia and pain-related anxiety in CP rats. Relief of Hyperalgesia and Anxiety via Inhibiting Excitatory ACC Neurons C The expression of VGluT1 is signiﬁcantly enhanced from POD 3 to 28 in TNBS-treated rats compared with sham rats (n = 3 per group; P \ 0.05, P \ 0.01, P \ 0.001, TNBS vs sham, one- way ANOVA followed by LSD post-hoc test). Discussion to facilitate pain [43, 44], whereas interneurons play a diametrically opposite role [43]. Our morphological data have emphasized the role of ACC pyramidal neurons under the condition of painful CP. Thus, we propose that excitatory neurons mediate the pain-facilitatory role of the ACC in CP rats. In order to elucidate the role of pyramidal neurons in pancreatitis-related hyperalgesia and anxiety, we speciﬁcally modulated ACC pyramidal neu- rons bidirectionally via optogenetics and chemogenetics in sham and CP rats. The experimental protocols are shown in Figs 7A and 8A. ChR2-mCherry expression was validated by post hoc ﬂuorescence microscopy (Fig. 7B). Behavioral results showed that the activation of pyramidal cells changed neither AWT (Fig. 7C–D) nor performance in the open ﬁeld (Fig. 7E–H) in both sham and CP rats. hM4D(Gi)-mCherry expression was conﬁrmed by post hoc ﬂuorescence microscopy (Fig. 8B). On POD 14, CNO treatment alleviated the pancreatic hyperalgesia (AWT: 4.29 ± 0.81 g with TNBS?saline versus 12.57 ± 2.67 g with TNBS?CNO; n = 7 per group; P\0.05; Fig. 8C) and decreased the anxiety-like behavior of TNBS-treated rats (total distance: 18.33 ± 2.17 m with TNBS?saline versus 32.16 ± 3.03 m with TNBS?CNO; % central distance: 2.58 ± 1.07 with TNBS?saline versus 5.49 ± 1.51 with TNBS?CNO; n = 7 per group; P \ 0.05; Fig. 8D, E), but not in sham rats (Fig. 8C–E). In the current study, we found that the ACC received direct projections from the NTS, a key relay station for primary visceral afferents, and this neural pathway was activated by painful CP. Speciﬁcally, morphological and biochemical results showed an increase in presynaptic glutamate release and postsynaptic glutamate receptor expression and phos- phorylation in the ACC of CP rats, suggesting the existence of central sensitization under the condition of painful CP. These plastic changes contributed to abdominal hyperal- gesia, since inhibiting excitatory transmission in the ACC induced signiﬁcant analgesic effects comparable to local administration of opioids in CP rats. Finally, we further revealed that ACC pyramidal neurons mediated the behav- ioral representation of painful CP since chemogenetic inhibition of these neurons alleviated both hyperalgesia and pain-related anxiety in CP rats. These plastic changes contributed to abdominal hyperal- gesia, since inhibiting excitatory transmission in the ACC induced signiﬁcant analgesic effects comparable to local administration of opioids in CP rats. TNBS-Induced Anxiety in CP Rats H, I Membrane NR2B is signiﬁcantly enhanced after TNBS injection on POD 3, 7, 14, and 28, while cytosol NR2B is signiﬁcantly decreased on POD 7, 14, and 28. J, K The expression of membrane pNR2B is signiﬁcantly increased at all time points in the TNBS-treated group, but that of cytosol pNR2B does not change (n = 3 per group; P\0.05, P\0.01, P\0.001, TNBS vs sham, one-way ANOVA followed by LSD post-hoc test). with prior behavioral observations in VH rats induced by irritable bowel syndrome and chronic pancreatitis [31, 34]. with prior behavioral observations in VH rats induced by irritable bowel syndrome and chronic pancreatitis [31, 34]. While decreased exploratory behavior is assumed to reﬂect anxiety in animal behavior experiments, immobility may be caused by many factors, including emotional disorders, motor disability, and decreased motor desire [33]. Here, the following lines of evidence suggest that negative emotions may be the sole reason for immobility in CP rats. First, it is recognized that chronic pain interferes with motor function [47]. Nevertheless, we failed to discriminate any alterations in motor performance between CP rats and sham rats in the rotarod test, thus excluding the possibility of motor deﬁcits. Second, morphine treatment on POD 14 alleviated the abdominal hyperalgesia but failed to elevate motor performance in the open ﬁeld teat (unpublished data), indicating that painful CP may not reduce motor desire in rats. Third, although clinical observations indicate that patients with CP exhibit somatic hypersensitivity in remote areas [14], we failed to observe somatic hypersensitivity of the hindpaw in CP rats, which differed from prior ﬁndings reported in CP mice [49]. Finally, the depression-like behavior in the forced and then stimulates immunological responses against the tissue. TNBS may also have a direct toxic action by the formation of extremely reactive compounds with pro- inﬂammatory and cytotoxic properties, including superox- ide and hydrogen peroxide radicals [45]. In this study, we found that TNBS-induced pathophysiological changes mimicked those seen in patients with chronic pancreatitis [46], providing a reliable painful CP animal model with the advantages of a high success rate, stability, and repro- ducibility along with less mortality. and then stimulates immunological responses against the tissue. TNBS may also have a direct toxic action by the formation of extremely reactive compounds with pro- inﬂammatory and cytotoxic properties, including superox- ide and hydrogen peroxide radicals [45]. TNBS-Induced Anxiety in CP Rats In this study, chronic a pancreatitis model was established by intraductal administration of TNBS in rats. The mechanism by which this leads to chronic pancreatitis has not been fully clariﬁed. It is generally accepted that as a hapten, TNBS reacts with lysine residues on the epithelium 123 12 3 Neurosci. Bull. April, 2022, 38(4):342–358 352 Fig. 5 TNBS treatment facilitates the trafﬁcking of glutamate receptor subunits into membrane and their phosphorylation in the ACC. A Fractionation of ACC probed for N-cadherin and b-actin to verify the accuracy of the subcellular fractionation procedure. B, C Representative western blots for membrane (B) and cytosol (C) GluR1, pGluR1, NR2B, and pNR2B in the ACC of sham rats on POD 14 and in TNBS-treated rats on POD 3, 7, 14, and 28. D, E The expression of membrane GluR1 is signiﬁcantly enhanced after TNBS injection on POD 3, 7, 14, and 28, while cytosol GluR1 does not change. F, G The expression of membrane pGluR1 is signiﬁcantly enhanced after TNBS injection on POD 3, 7, 14, and 28, while cytosol pGluR1 does not change. H, I Membrane NR2B is signiﬁcantly enhanced after TNBS injection on POD 3, 7, 14, and 28, while cytosol NR2B is signiﬁcantly decreased on POD 7, 14, and 28. J, K The expression of membrane pNR2B is signiﬁcantly increased at all time points in the TNBS-treated group, but that of cytosol pNR2B does not change (n = 3 per group; P\0.05, P\0.01, P\0.001, TNBS vs sham, one-way ANOVA followed by LSD post-hoc test). Fig. 5 TNBS treatment facilitates the trafﬁcking of glutamate receptor subunits into membrane and their phosphorylation in the ACC. A Fractionation of ACC probed for N-cadherin and b-actin to verify the accuracy of the subcellular fractionation procedure. B, C Representative western blots for membrane (B) and cytosol (C) GluR1, pGluR1, NR2B, and pNR2B in the ACC of sham rats on POD 14 and in TNBS-treated rats on POD 3, 7, 14, and 28. D, E The expression of membrane GluR1 is signiﬁcantly enhanced after TNBS injection on POD 3, 7, 14, and 28, while cytosol GluR1 does not change. F, G The expression of membrane pGluR1 is signiﬁcantly enhanced after TNBS injection on POD 3, 7, 14, and 28, while cytosol pGluR1 does not change. TNBS-Induced Anxiety in CP Rats C Bilateral microinjections of morphine, EM-1, and EM-2 signiﬁcantly increase the AWT in CP rats (n = 6 per group; *P \ 0.001, morphine vs saline; ###P\0.001, EM-1 vs saline; &&&P\0.001, EM-2 vs saline, one-way repeated ANOVA followed by LSD post-hoc test). D, E Bilateral microinjections of CNQX (D) and AP-5 (E) on POD 14 signiﬁcantly increase the AWT in CP rats but saline does not (n = 5 for saline-treated group and 6 for CNQX and AP-5-treated groups; P \ 0.05, TNBS vs sham unpaired t-test). Fig. 7 Optogenetic activation of bilateral ACC pyramidal neurons has no effect on AWT and anxiety-like behavior in sham and CP rats. A Upper panel, schematic of the behavioral experiment; lower panel, the rAAV-CaMKIIa-ChR2-mCherry construct. B Representative coronal section showing virus injection sites in the ACC (scale bar, 1 mm). C–H Activating bilateral pyramidal neurons in the ACC has no effect on the AWT (C, D), total distance (E, F), and the distance traveled in the center of the open ﬁeld (G, H) in sham and CP rats (n = 7 for both groups; n.s., no signiﬁcant difference, TNBS vs sham, one-way repeated ANOVA followed by LSD post-hoc test. ACC, anterior cingulate cortex; M2, secondary motor cortex. D. Ren et al.: Cortical Modulation of Hyperalgesia in Chronic Pancreatitis 353 D. Ren et al.: Cortical Modulation of Hyperalgesia in Chronic Pancreatitis 353 Fig. 6 Bilateral microinjection of CNQX and AP-5 into the ACC alleviates abdominal hyperalgesia in CP rats. A Upper panel, schematic of behavioral experiment; lower panel, representative coronal section showing the sites of the cannulae in the ACC (scale bar, 2 mm). B Diagram showing the sites of the cannula tips for AP-5 (black dots) and CNQX (red dots) injection into the ACC. C Bilateral microinjections of morphine, EM-1, and EM-2 signiﬁcantly increase the AWT in CP rats (n = saline; ###P\0.001, EM-1 one-way repeated ANOVA E Bilateral microinjections signiﬁcantly increase the A for saline-treated group an P \ 0.05, TNBS vs sham Fig. 6 Bilateral microinjection of CNQX and AP-5 into the ACC alleviates abdominal hyperalgesia in CP rats. A Upper panel, schematic of behavioral experiment; lower panel, representative coronal section showing the sites of the cannulae in the ACC (scale bar, 2 mm). B Diagram showing the sites of the cannula tips for AP-5 (black dots) and CNQX (red dots) injection into the ACC. TNBS-Induced Anxiety in CP Rats In this study, we found that TNBS-induced pathophysiological changes mimicked those seen in patients with chronic pancreatitis [46], providing a reliable painful CP animal model with the advantages of a high success rate, stability, and repro- ducibility along with less mortality. irritable bowel syndrome and chronic pancreatitis [31, 34]. While decreased exploratory behavior is assumed to reﬂect anxiety in animal behavior experiments, immobility may be caused by many factors, including emotional disorders, motor disability, and decreased motor desire [33]. Here, the following lines of evidence suggest that negative emotions may be the sole reason for immobility in CP rats. First, it is recognized that chronic pain interferes with motor function [47]. Nevertheless, we failed to discriminate any alterations in motor performance between CP rats and sham rats in the rotarod test, thus excluding the possibility of motor deﬁcits. Second, morphine treatment on POD 14 alleviated the abdominal hyperalgesia but failed to elevate motor performance in the open ﬁeld teat (unpublished data), indicating that painful CP may not reduce motor desire in rats. Third, although clinical observations indicate that patients with CP exhibit somatic hypersensitivity in remote areas [14], we failed to observe somatic hypersensitivity of the hindpaw in CP rats, which differed from prior ﬁndings reported in CP mice [49]. Finally, the depression-like behavior in the forced As a common comorbidity, affective disorders under the condition of chronic pain severely impair patients’ quality of life and exacerbate the sensory abnormalities of chronic pain [47]. As expected, a high incidence of anxiety and depression is well-documented in patients with chronic pancreatitis [48]. Unfortunately, much less focus has been directed toward the relief of the affective dimension of pancreatitis-related pain in both clinical and preclinical studies. In this study, we found that CP rats exhibited hypolocomotion and decreased exploratory behavior both in the open ﬁeld and elevated plus maze tests, consistent 123 123 Fig. 6 Bilateral microinjection of CNQX and AP-5 into the ACC alleviates abdominal hyperalgesia in CP rats. A Upper panel, schematic of behavioral experiment; lower panel, representative coronal section showing the sites of the cannulae in the ACC (scale bar, 2 mm). B Diagram showing the sites of the cannula tips for AP-5 (black dots) and CNQX (red dots) injection into the ACC. TNBS-Induced Anxiety in CP Rats April, 2022, 38(4):342–358 Fig. 8 Chemogenetic inhibition of bilateral ACC pyramidal neurons alleviates abdominal hyperalgesia and anxiety-like behavior in CP rats. A Upper panel, schematic of the behavioral experiment; lower panel, rAAV-CaMKIIa-hM4Di-mCherry construct. B Representative coronal section showing the injection sites in the ACC (scale bar, 1 mm). C Inhibiting bilateral pyramidal neurons in the ACC via intraperitoneal CNO signiﬁcantly increases the AWT in CP rats but not sham rats. D, E Inhibiting ACC neurons signiﬁcantly reduces the total distance (D) and the distance traveled in the center (E) of the open ﬁeld in CP rats but not sham rats (n = 8 for sham and 7 for CP rats; P \ 0.05, TNBS vs sham, unpaired t-test). ACC, anterior cingulate cortex; M2, secondary motor cortex. intraperitoneal CNO signiﬁcantly increases the AWT in CP rats but not sham rats. D, E Inhibiting ACC neurons signiﬁcantly reduces the total distance (D) and the distance traveled in the center (E) of the open ﬁeld in CP rats but not sham rats (n = 8 for sham and 7 for CP rats; P \ 0.05, TNBS vs sham, unpaired t-test). ACC, anterior cingulate cortex; M2, secondary motor cortex. Fig. 8 Chemogenetic inhibition of bilateral ACC pyramidal neurons alleviates abdominal hyperalgesia and anxiety-like behavior in CP rats. A Upper panel, schematic of the behavioral experiment; lower panel, rAAV-CaMKIIa-hM4Di-mCherry construct. B Representative coronal section showing the injection sites in the ACC (scale bar, 1 mm). C Inhibiting bilateral pyramidal neurons in the ACC via amygdaloid nucleus, and the periventricular nucleus of the thalamus [55, 56]. Here, we demonstrated a direct NTS– ACC pathway in rats. Despite scarce projections, this connection provides evidence that the cortical pain center could be directly activated by visceral afferents processed from the NTS. Further observations indicated that the projections from the NTS were in close contact with both pyramidal and GABAergic ACC neurons, the former being more frequently seen. Another interesting phenomenon was that ACC pyramidal neurons were more likely to be activated by painful CP than GABAergic neurons. This morphological evidence suggests that ACC glutamatergic neurons play a crucial role in the transmission or modu- lation of painful CP, which was veriﬁed by subsequent functional studies. However, the role of GABAergic ACC neurons in the modulation of painful CP cannot be completely excluded, which is a limitation of this study. TNBS-Induced Anxiety in CP Rats Further functional experiments are urgently needed to answer this question, which is an important research focus in our lab. swimming test further supports the presence of emotional disorders induced by CP. In light of these ﬁndings, hypolocomotion may be closely related to affective disorders under the condition of painful CP, and thus was used to measure the emotional aspect of pancreatic pain in this study. It is worth noting that decreased locomotion is seldom seen in neuropathic pain [47]. One possible explanation is that visceral pain is usually more unbearable than somatic pain and causes more severe emotional and autonomic disorders, which then lead to hypolocomotion. TNBS-Induced Anxiety in CP Rats C Bilateral microinjections of morphine, EM-1, and EM-2 signiﬁcantly increase the AWT in CP rats (n = 6 per group; *P \ 0.001, morphine vs saline; ###P\0.001, EM-1 vs saline; &&&P\0.001, EM-2 vs saline, one-way repeated ANOVA followed by LSD post-hoc test). D, E Bilateral microinjections of CNQX (D) and AP-5 (E) on POD 14 signiﬁcantly increase the AWT in CP rats but saline does not (n = 5 for saline-treated group and 6 for CNQX and AP-5-treated groups; P \ 0.05, TNBS vs sham unpaired t-test). Fig. 6 Bilateral microinjection of CNQX and AP-5 into the ACC alleviates abdominal hyperalgesia in CP rats. A Upper panel, schematic of behavioral experiment; lower panel, representative coronal section showing the sites of the cannulae in the ACC (scale bar, 2 mm). B Diagram showing the sites of the cannula tips for AP-5 (black dots) and CNQX (red dots) injection into the ACC. C Bilateral microinjections of morphine, EM-1, and EM-2 signiﬁcantly increase Fig. 7 Optogenetic activation of bilateral ACC pyramidal neurons has no effect on AWT and anxiety-like behavior in sham and CP rats. A Upper panel, schematic of the behavioral experiment; lower panel, the rAAV-CaMKIIa-ChR2-mCherry construct. B Representative coronal section showing virus injection sites in the ACC (scale bar, 1 mm). C–H Activating bilateral pyramidal neurons in the ACC has no effect on the AWT (C, D), total distance (E, F), and the distance traveled in the center of the open ﬁeld (G, H) in sham and CP rats (n = 7 for both groups; n.s., no signiﬁcant difference, TNBS vs sham, one-way repeated ANOVA followed by LSD post-hoc test. ACC, anterior cingulate cortex; M2, secondary motor cortex. no effect on the AWT (C, D), total distance (E, F), and the distance traveled in the center of the open ﬁeld (G, H) in sham and CP rats (n = 7 for both groups; n.s., no signiﬁcant difference, TNBS vs sham, one-way repeated ANOVA followed by LSD post-hoc test. ACC, anterior cingulate cortex; M2, secondary motor cortex. Fig. 7 Optogenetic activation of bilateral ACC pyramidal neurons has no effect on AWT and anxiety-like behavior in sham and CP rats. A Upper panel, schematic of the behavioral experiment; lower panel, the rAAV-CaMKIIa-ChR2-mCherry construct. B Representative coronal section showing virus injection sites in the ACC (scale bar, 1 mm). C–H Activating bilateral pyramidal neurons in the ACC has 12 12 123 354 Neurosci. Bull. Neuromodulation Techniques Targeting the ACC as a Viable Therapy for Painful CP In the present study, optogenetic modulation of ACC pyramidal cells failed to alter the AWT in naı¨ve rats, which was inconsistent with Kang’s study which suggested that selectively activating excitatory ACC cells induces changes in the hindpaw mechanical threshold in naı¨ve mice [43]. This discrepancy may be due to the limitation of our pain evaluation methods. Since VFF probing is a measure of referred abdominal mechanical hypersensitivity when pancreatic inﬂammation invades the peritoneum, it may not be an efﬁcacious index of internal visceral sensation under normal conditions. Thus, recording defen- sive behaviors induced by pancreas stimulation via intra- abdominal electrodes [29] may be a better choice in studies to evaluate the sensory aspect of pancreatitis-related pain. In accordance with Kang’s study [43], we found that optogenetic activation of ACC pyramidal cells did not elicit anxiety-like behavior in normal rats, which differs from the results of Barthas [44]. Differences in the optogenetic virus, optogenetic stimulation protocol, and behavioral evaluation method may account for these conﬂicting results. In the present study, increased expression of VGluT1, as well as increased membrane trafﬁcking and phosphoryla- tion of GluR1 and NR2B subunits within the ACC were found in CP rats. We propose that CP-induced excessive glutamate release may activate similar signaling pathways via NMDARs in the ACC, leading to the recruitment of glutamate receptors and visceral hypersensitivity. These neuroplasticity-related changes within the ACC were positively associated with behavioral hyperalgesia under CP conditions, since suppression of glutamatergic trans- mission by AMPAR/NMDAR antagonists led to pain palliation, in accordance with previous studies performed under the conditions of neuropathic and gastrointestinal pain [20, 59]. We also tested the analgesic effects of local administration of opioids into the ACC in CP rats as a positive control for evaluating the analgesic effects of AMPAR/NMDAR antagonists and subsequent optogenetic/ chemogenetic manipulations. Activating mu-opioid In CP, optogenetic activation of ACC pyramidal neurons did not aggravate the abdominal hyperalgesia or anxiety, suggesting a ceiling effects of the hyperalgesia induced by pancreatitis pain. In other words, the activity of excitatory ACC neurons reached a maximum during intense pancre- atitis stimuli, so further enhancing their activity failed to elicit more pain sensation. This was supported by the fact that speciﬁcally inhibiting this neuron type had both anxiolytic and analgesic effects. Involvement of the Direct NTS–ACC Pathway in Painful CP The NTS is the primary target for afferent vagal ﬁbers which convey noxious and non-noxious visceral sensory information to the forebrain via the parabrachial nucleus (PBN) [8, 9, 50]. The role of the NTS in the transmission or regulation of visceral or autonomic information has been extensively explored [51–54]. Ascending projections from the NTS have been studied in rodents with the aid of tract tracing techniques. Besides numerous brainstem territories including the PBN, the projections of the NTS have also been traced to a series of forebrain structures: the bed nucleus of the stria terminalis, the paraventricular, dorso- medial, and arcuate nuclei of the hypothalamus, the central Apart from direct projections, there may be more complex indirect projections from NTS to ACC. As a key relay center for visceral afferents from the NTS, the PBN sends projections to extensive thalamic areas, includ- ing the intralaminar (centromedian, centrolateral) and the midline (paraventricular, reuniens) thalamic nuclei [57], 12 123 123 D. Ren et al.: Cortical Modulation of Hyperalgesia in Chronic Pancreatitis 355 which have been reported to send efferent connections to the ACC [58]. Thus, there may be an indirect NTS–PBN– thalamus–ACC pathway that is related to the transmission of visceral pain to the ACC. In addition, the NTS sends direct projections to the LC [55, 56], which provides direct norepinephrinergic efferents to the ACC [58]. Thus, the NTS–LC–ACC pathway may be involved in the modula- tion of visceral pain processing in the ACC. These potential indirect pathways warrant further investigation. receptors (MORs) via morphine in the ACC has been shown to exert potent analgesic effects in various chronic pain states in rodents [41, 42, 61]. It has been proposed that MOR activation inhibits excitatory glutamatergic trans- mission in the ACC via the suppression of presynaptic glutamate release and the deactivation of pyramidal neurons [62, 63]. Here, we found that both exogenous (morphine) and endogenous (EM-1 and EM-2) MOR agonists had robust analgesic effects in CP rats, and these effects were almost equal to those elicited by AMPAR/ NMDAR antagonists in the ACC. Taken together, the ACC plays a pain-facilitating role in pancreatitis-related pain. The limitation of our study is that other glutamate subunits that may be involved in painful CP have not been detected, and corresponding signaling mechanisms remain to be investigated. Presynaptic and Postsynaptic Ampliﬁcation of Pain- ful CP in the ACC LTP of glutamatergic transmission in the ACC is a key cellular mechanism for pathological pain [19]. Activity- dependent Ca2? ﬂux through NMDAR activation, induced by excessive presynaptic glutamate release, is thought to initiate downstream signaling events during LTP induction. Adenylate cyclase 1 (AC1) is one essential Ca2?-stimu- lated enzyme that converts ATP to cAMP and then activates downstream signaling molecules, such as protein kinase A (PKA) and cAMP-response element-binding protein (CREB). Evidence from neuropathic pain and VH models indicates that primary injuries promote GluR1 phosphorylation and trafﬁcking in the ACC via the AC1– cAMP–PKA pathway, leading to NMDAR-mediated AMPAR potentiation [20, 59]. Meanwhile, activation of the AC1–cAMP–CREB pathway may facilitate NR2B expression and phosphorylation, forming a positive feed- back to enhance NMDAR function and contribute to sustained pain [40]. Apart from AC1, calcium/calmodulin- dependent protein kinase II (CaMKII) is another crucial molecule for synaptic plasticity in the ACC. Phosphory- lated CaMKII binds to and stabilizes postsynaptic NR2B, thus mediating visceral pain in VH rats [60]. References 1. Olesen SS, Juel J, Nielsen AK, Frøkjær JB, Wilder-Smith OH, Drewes AM. Pain severity reduces life quality in chronic pancreatitis: Implications for design of future outcome trials. Pancreatology 2014, 14: 497–502. 2. Drewes AM, Krarup AL, Detlefsen S, Malmstrøm ML, Dim- cevski G, Funch-Jensen P. Pain in chronic pancreatitis: The role of neuropathic pain mechanisms. Gut 2008, 57: 1616–1627. 3. Fregni F, Pascual-Leone A, Freedman SD. Pain in chronic pancreatitis: A salutogenic mechanism or a maladaptive brain response? Pancreatology 2007, 7: 411–422. 4. Olesen SS, Krauss T, Demir IE, Wilder-Smith OH, Ceyhan GO, Pasricha PJ. Towards a neurobiological understanding of pain in chronic pancreatitis: Mechanisms and implications for treatment. Pain Rep 2017, 2: e625. https://doi.org/10.1097/PR9. 0000000000000625. 5. Cervero F. Sensory innervation of the viscera: Peripheral basis of visceral pain. Physiol Rev 1994, 74: 95–138. 6. Breit S, Kupferberg A, Rogler G, Hasler G. Vagus nerve as modulator of the brain-gut axis in psychiatric and inﬂammatory disorders. Front Psychiatry 2018, 9: 44. 7. Pasricha PJ. Unraveling the mystery of pain in chronic pancre- atitis. Nat Rev Gastroenterol Hepatol 2012, 9: 140–151. 8. Berthoud HR, Neuhuber WL. Functional and chemical anatomy of the afferent vagal system. Auton Neurosci 2000, 85: 1–17. 9. Hermes SM, Colbert JF, Aicher SA. Differential content of vesicular glutamate transporters in subsets of vagal afferents projecting to the nucleus tractus solitarii in the rat. J Comp Neurol 2014, 522: 642–653. In summary, our results demonstrate that the ACC receives ascending projections from the NTS and that this pathway may be an essential portion of the ascending system involved in the transmission of abdominal hyper- algesia in CP rats. We also found that cortical sensitization plays a key role in abdominal hyperalgesia and pain-related negative emotions in rats with CP, and this was alleviated by inhibiting the excitability of pyramidal neurons in the ACC. These insights lay the foundation for prospective inquiries into detailed ACC-related circuit mechanisms in the hyperalgesia and anxiety induced by CP. 10. Okada S, Katagiri A, Saito H, Lee J, Ohara K, Iinuma T, et al. Functional involvement of nucleus tractus solitarii neurons projecting to the parabrachial nucleus in trigeminal neuropathic pain. J Oral Sci 2019, 61: 370–378. 11. Zhuo M. Cortical excitation and chronic pain. Trends Neurosci 2008, 31: 199–207. 12. de Vries M, Wilder-Smith OH, Jongsma ML, van den Broeke EN, Arns M, van Goor H, et al. Neuromodulation Techniques Targeting the ACC as a Viable Therapy for Painful CP Considering these, we conclude that glutamatergic ACC neurons mediate the abdominal hyperalgesia as well as the anxiety induced by chronic pancreatitis, and they are expected to be a 12 3 3 356 Neurosci. Bull. April, 2022, 38(4):342–358 Acknowledgements This work was supported by the National Natural Science Foundations of China (81620108008 and 31971112) and the Innovation Capability Support Program of Shaanxi Province, China (2021TD-57). Acknowledgements This work was supported by the National Natural Science Foundations of China (81620108008 and 31971112) and the Innovation Capability Support Program of Shaanxi Province, China (2021TD-57). promising target in the clinical treatment of painful CP. Another limitation in this study is that different method- ologies were used to modulate ACC pyramidal neurons (e.g., using optogenetics for activation but chemogenetics for inhibition). These methods are not in strict contrast since the mechanisms of neuron responses are different [64]. Using one method to modulate neuronal activity bidirectionally (e.g., hM3Dq/hM4Di or ChR2/NpHR) may be better. Conﬂict of interest All authors claim that there are no conﬂicts of interest. Conﬂict of interest All authors claim that there are no conﬂicts of interest. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/. Historically, cingulectomy was introduced to treat intractable pain in clinical practice. Nevertheless, it is not currently recommended as a treatment due to its short-term effects and neuropsychiatric risks [18]. Instead, the advent of deep brain stimulation (DBS) provides a promising alternative for modifying dysfunctional pain-matrix activ- ity in the treatment of refractory pain disorders, surpassing lesion surgery due to its adjustability and reversibility [65]. The ACC is a newly-identiﬁed stimulation spot for the treatment of drug-resistant depression [66] and chronic pain [67]. Neuromodulation Techniques Targeting the ACC as a Viable Therapy for Painful CP Bilateral ACC DBS is known to relieve various types of neuropathic pain and restore quality of life; it outperforms periaqueductal gray or thalamus stimulation by targeting the affective component of pain [66]. The predominant theory is that high-frequency stimulation generates depolarization blockade, leading to the func- tional inactivation of ACC neurons [59]. Interestingly, by controlling the neuronal activity of ACC, we successfully relieved pancreatitis-related pain and associated emotional disorders, which may pave the way for the application of ACC DBS in the treatment of refractory pancreatitis- related pain. However, neither pharmacologically nor chemogenetically blocking the activity of the ACC com- pletely reversed the abdominal hyperalgesia in CP rats. 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https://openalex.org/W328902990	https://insightsimaging.springeropen.com/counter/pdf/10.1007/s13244-015-0408-y	English	null	Dual-energy CT after radiofrequency ablation of liver, kidney, and lung lesions: a review of features	Insights into imaging	2,015	cc-by	8,796	Frederik Vandenbroucke1 & Steven Van Hedent1 & Gert Van Gompel1 & Nico Buls1 & Gordon Craggs1 & Jef Vandemeulebroucke2,3 & Pablo R. Ros4 & Johan de Mey1 Frederik Vandenbroucke1 & Steven Van Hedent1 & Gert Van Gompel1 & Nico Buls1 & Gordon Craggs1 & Jef Vandemeulebroucke2,3 & Pablo R. Ros4 & Johan de Mey1 Received: 12 January 2015 /Revised: 10 March 2015 /Accepted: 31 March 2015 /Published online: 5 May 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Early detection of residual tumour and local tumour progression (LTP) after radiofrequency (RF) ab- lation is crucial in the decision whether or not to re- ablate. In general, standard contrast-enhanced computed tomography (CT) is used to evaluate the technique ef- fectiveness; however, it is difficult to differentiate post- treatment changes from residual tumour. Dual-energy CT (DECT) is a relatively new technique that enables more specific tissue characterisation of iodine-enhanced structures because of the isolation of iodine in the im- aging data. Necrotic post-ablation zones can be depicted as avascular regions by DECT on greyscale- and colour- coded iodine images. Synthesised monochromatic im- ages from dual-energy CT with spectral analysis can be used to select the optimal keV to achieve the highest contrast-to-noise ratio between tissues. This facilitates outlining the interface between the ablation zone and surrounding tissue. Post-processing of DECT data can lead to an improved characterisation and delineation of benign post-ablation changes from LTP. Radiologists need to be familiar with typical post-ablation image in- terpretations when using DECT techniques. Here, we review the spectrum of changes after RF ablation of liver, kidney, and lung lesions using single-source DECT imaging, with the emphasis on the additional information obtained and pitfalls encountered with this relatively new technique. Teaching Points •Technical success of RF ablation means complete destruction of the tumour. •Assessment of residual tumour on contrast-enhanced CT is hindered by post-ablative changes. •DECT improves material differentiation and may improve focal lesion characterisation. •Iodine maps delineate the treated area from the surrounding parenchyma well. Keywords Dual-energy CT . Radiofrequency ablation . Liver . Lung . Kidney Keywords Dual-energy CT . Radiofrequency ablation . Liver . Lung . Kidney Insights Imaging (2015) 6:363–379 DOI 10.1007/s13244-015-0408-y Insights Imaging (2015) 6:363–379 DOI 10.1007/s13244-015-0408-y PICTORIAL REVIEW * Frederik Vandenbroucke spovef@uzbrussel.be 1 Department of Radiology, UZ Brussel (VUB), Laarbeeklaan 101, Brussels, Belgium p @ 1 Department of Radiology, UZ Brussel (VUB), Laarbeeklaan 101, Brussels, Belgium 2 Department of Electronics and Informatics, Vrije Universiteit Brussel, Brussels, Belgium 3 Department of Medical IT, iMinds, Ghent, Belgium 4 Department of Radiology, University Hospitals Case Medical Center/Case Western Reserve University, Cleveland, OH, USA RF Ablation Minimally invasive therapies are increasingly used in patients with malignant tumours, who are not suitable candidates for surgical resection. The aim of local abla- tive therapy is to induce cell death. Radio-frequency (RF) ablation has attracted much attention in the last decade because of its technological improvements and has become a well-established technique to treat primary and secondary hepatic malignancies, as well as kidney and lung tumours. 364 Insights Imaging (2015) 6:363–379 attenuation behaviour at single radiation energy levels and, with that, similar corresponding Hounsfield num- bers. This poor attenuation difference can be partially resolved by using dual-energy CT (DECT) technology with material decomposition [13]. DECT is able to achieve tissue attenuation sampling at two different en- ergy spectra, which results in more specific information beyond the typical Hounsfield units. Technical success following RF ablation is due to the complete destruction of the tumour. In case of incom- plete necrosis of the tumoral volume, re-ablation of re- sidual tumour can be pursued in order to improve tech- nique effectiveness. For this reason, it is crucial to eval- uate the results of the procedure early after ablation. The ideal imaging technique should show the degree of necro- sis of the malignant mass and detect residual tumour. Imaging immediately after ablation also allows for the de- tection of post-procedural complications and provides a base- line for future follow-up comparisons. Unfortunately, the as- sessment of residual tumour, usually by means of contrast- enhanced computed tomography (CT), is hindered by post- ablative changes, particularly at the periphery of a treated lesion where blood flow is greatest [1, 2]. For already several years, three main technical ap- proaches have been developed by various vendors for the acquisition of dual-energy data: a single-source rap- id kilovolt peak-switching technique, a dual-source technique with an angular offset, and finally use of a dual-layer detector that discriminates between high- and low-energy photons [13]. The first two techniques ob- tain dual-energy data simultaneously at two different energy levels at typically 80 and 140 kVp, although dual-source systems can also operate at 100 and 140 kVp (with an additional tin filter) in case of larger patient sizes. The third technique uses the detected high- and low-energy signal components in one CT acquisition. More recently, a non-simultaneous, single- source technique has been reported that applies a se- quential data acquisition and a coregistration motion correction algorithm [14]. RF Ablation It has been shown that DECT improves material differentiation and works es- pecially well in materials with large atomic numbers, such as iodine and calcium, because their strong pho- toelectric effect causes high attenuation at lower photon energies [15]. The material decomposition technique as- sumes that for any tissue an equivalent mixture of On pathology this represents a haemorrhagic rim corre- sponding to an early inflammatory reaction to the necrotic tissue [3, 4]. Today, contrast-enhanced CT is the standard ap- proach to image early post-ablative changes in the liver [5, 6], kidney [7], and lung [8]. However, some investigators have reported that although complete necrosis may appear clearly on contrast-enhanced CT, these findings do not always corre- late with histopathological conclusions, which suggests that there is limited diagnostic accuracy in the detection of residual tumours [9–12]. Dual-energy CT A well-known drawback of standard CT is that a num- ber of different materials or tissues may show similar Table 1 Early and long-term findings after RF ablation of liver lesions in successful and unsuccessful results, with the added value of the DECT technique Early findings Long-term findings Successful General findings Non-enhancing area on contrast-enhanced CT Regular hypervascular peripheral rim Central hyperattenuation is indecisive for success Regular borders of the ablation zone Reduction in size Added value of DECT Iodine void area Improved contrast on the 40-keV images Excellent conspicuity of the ablation zone on iodine-coded images Sharp depiction of the ablation zone’s edge on iodine images Unsuccessful General findings Irregular rim Focal nodular thickening at the border Focal hypervascular nodule (HCC) Nodular attenuation differences around the ablation zone (hypovascular metastasis) Added value of DECT Higher residual tumour-to-liver contrast on iodine images Increased attenuation of iodine on the low-keV images for detection of subtle residual tumour Better contrast between LTP and liver parenchyma on iodine images 365 Insights Imaging (2015) 6:363–379 Fig. 1 Hypovascular liver metastasis. A 53-year-old female undergoes surgery for rectal carcinoma and then receives RF ablation in a focal liver lesion. Eight months later, a local tumour progression (first column) at the cranial side of the ablation zone (large arrow) can be seen as an intermediate density (small arrow). In this straightforward case, the tumoral tissue is equally well depicted on the 40- and 70-keV images as on the greyscale iodine images. Noise is more prominent on the 40-keV image however. Iodine maps are encoded using a rainbow template with a colour coding from high to low iodine concentration, being from red (through yellow) to blue respectively. The intermediate colour represents a tumour (small arrow). On PET/CT a vivid FDG uptake is seen (arrowhead). Twenty-four hours after a second RF ablation (second column), a sharp delineated ablation zone is found. On all reconstructions, the avascular nature of the ablation zone is well demonstrated. The contrast with the surrounding liver parenchyma appears highest on the colour-coded iodine map. No FDG uptake is seen on the PET/CT image. The PET/CT 3 months later (not shown) does not show signs of tumoral activity R water and iodine exists with similar spectral attenuation properties. Consequently, after a material decomposition calculation process, two base material maps can be displayed, representing the concentrations of water and iodine in each voxel. Dual-energy CT In contrast studies, these concen- trations correspond to real water/iodine concentrations in blood. All other materials (e.g. bone, fat) are de- scribed as a mixture of both of these base materials and will appear as hyper- or hypodensity in the base pair maps. Iodine concentrations can be accurately quantified. A recent study showed a 0.55 mg/cc mean error when comparing calculated and true iodine con- centrations in renal masses [16]. These calculated io- dine concentrations (mg/cc) can be displayed for any region of interest (ROI) as either a greyscale- or colour-coded iodine image. Such iodine maps obtained from DECT images are not a surrogate for dynamic perfusion CT, as they merely provide a visualisation of the iodine distribution in the tissues at one point in time. In addition to calculating base material maps, syn- thesised or synthesised monochromatic images from dual-energy CT, representing CT values (HU) over a range of 40 to 140 keV, can also be obtained. Compared to high-keV, low-keV images (closer to the k-edge of iodine) typically provide improved contrast between different structures, e.g. between a tumour and surrounding normal parenchyma after injection of io- dinated contrast material. On the other hand, noise is more prominent in these lower keV images [17]. Synthesised monochromatic images at 77 keV corre- spond best to the effective energy of a single-energy Insights Imaging (2015) 6:363–379 366 Fig. 2 Hypervascular metastasis. A 63-year-old male undergoes follow-up CECT in the arterial phase 4 years after ablation of a renal cell carcinoma. This reveals a 26-mm hypervascular lesion in segment VII of the liver, which is histopathologically proven to be a metastasis of the RCC. RF ablation is performed. a Twenty- four hours after the ablation, a DECT is performed. The water map image shows a centrally located hyperdensity in the ablation zone (arrow), corresponding to intense charring and desiccation of the treated lesion. Note that this hyperdensity is less visible on the iodine maps. The arterial phase image does not demonstrate any focal hypervascularity at the border of the ablation zone. No rim enhancement can be seen. The delineation of the total ablation zone is better depicted on the portal-venous phase greyscale- and colour-coded iodine images, because the amount of iodine in the liver parenchyma is higher. Dual-energy CT b On the 1-year follow-up CECT, a smooth delineation of the ablation zone is noticed, which serves as proof of the technical success of the ablation. The central hyperdensity however is still noticeable (arrowheads) 367 Insights Imaging (2015) 6:363–379 120-kV scan [18], such that the image contrast simu- lates a traditional single-energy CT scan. By assessing the DECT images, an optimised keV window based on the contrast-to-noise ratio between tissues can be cal- culated. In RF ablation, an improved contrast is typi- cally obtained between the ablation zone and surrounding normal liver parenchyma. A scatterplot analysis, which estimates the material concentration in each voxel, can also help to differentiate different structures in the ROIs. Research is currently ongoing with regard to adequate quantification of the acquired DECT data. Several potential candidate parameters are Fig. 3 Hepatocellular carcinoma. A 43-year-old male with cirrhosis undergoes gadolinium enhanced MRI, revealing a hepatocellular carcinoma in segment VII, typical for a hepatocellular carcinoma, which is confirmed through biopsy. a Twenty-four hours after RF ablation, the patient undergoes a standard unenhanced CT scan and a contrast-enhanced DECT. The unenhanced CT shows a central area of high attenuation (arrowhead) within the ablation zone, which has a lower visibility on the water map reconstruction. The central hyperdensity is well noticeable on the synthesised monochromatic 40-keV but less so on the 70-keV images, and it is hardly visible on the iodine images. On the 40- and 70-keV synthesised monochromatic images, the ablation zone can be depicted as a lesion with a hypervascular peripheral rim (white arrows). On the greyscale- and colour-coded iodine images, we can better appreciate the focal hypervascular thickening at the posterior border (yellow arrows) than with the synthesised monochromatic images. b Due to the suspicious nature of the focal thickening seen in a, a shorter follow-up period is adhered to and a DECT is performed 5 weeks post-RF ablation. A focal hypervascular nodule is well depicted on the 70-keV (white arrow) images, although more clearly observable on the 40-keV ones; however again it is more evident on the greyscale- and colour-coded iodine images. Biopsy of this region confirms residual HCC. c Twenty-four hours after the second ablation, the greyscale- and colour-coded iodine images show significant iodine rim uptake around the ablation zone in the arterial phase (arrows). Therefore, a focal hypervascular remnant cannot be appreciated. This rim disappears on the venous phase (not shown). Dual-energy CT MRI after 10 weeks reveals no signs of reactivation Fig. 3 Hepatocellular carcinoma. A 43-year-old male with cirrhosis undergoes gadolinium enhanced MRI, revealing a hepatocellular carcinoma in segment VII, typical for a hepatocellular carcinoma, which is confirmed through biopsy. a Twenty-four hours after RF ablation, the patient undergoes a standard unenhanced CT scan and a contrast-enhanced DECT. The unenhanced CT shows a central area of high attenuation (arrowhead) within the ablation zone, which has a lower visibility on the water map reconstruction. The central hyperdensity is well noticeable on the synthesised monochromatic 40-keV but less so on the 70-keV images, and it is hardly visible on the iodine images. On the 40- and 70-keV synthesised monochromatic images, the ablation zone can be depicted as a lesion with a hypervascular peripheral rim (white arrows). On the greyscale- and colour-coded iodine images, we can better appreciate the focal hypervascular thickening at the posterior border (yellow arrows) than with the synthesised monochromatic images. b Due to the suspicious nature of the focal thickening seen in a, a shorter follow-up period is adhered to and a DECT is performed 5 weeks post-RF ablation. A focal hypervascular nodule is well depicted on the 70-keV (white arrow) images, although more clearly observable on the 40-keV ones; however again it is more evident on the greyscale- and colour-coded iodine images. Biopsy of this region confirms residual HCC. c Twenty-four hours after the second ablation, the greyscale- and colour-coded iodine images show significant iodine rim uptake around the ablation zone in the arterial phase (arrows). Therefore, a focal hypervascular remnant cannot be appreciated. This rim disappears on the venous phase (not shown). MRI after 10 weeks reveals no signs of reactivation Fig. 3 Hepatocellular carcinoma. A 43-year-old male with cirrhosis undergoes gadolinium enhanced MRI, revealing a hepatocellular carcinoma in segment VII, typical for a hepatocellular carcinoma, which is confirmed through biopsy. a Twenty-four hours after RF ablation, the patient undergoes a standard unenhanced CT scan and a contrast-enhanced DECT. The unenhanced CT shows a central area of high attenuation (arrowhead) within the ablation zone, which has a lower visibility on the water map reconstruction. The central hyperdensity is well noticeable on the synthesised monochromatic 40-keV but less so on the 70-keV images, and it is hardly visible on the iodine images. Dual-energy CT On the 40- and 70-keV synthesised monochromatic images, the ablation zone can be depicted as a lesion with a hypervascular peripheral rim (white arrows). On the greyscale- and colour-coded iodine images, we can better appreciate the focal hypervascular thickening at the 368 Insights Imaging (2015) 6:363–379 being screened for usefulness within the RFA context, such as CT numbers (i.e. Hounsfield unit curves) [19, 20], contrast-to-noise ratios [20, 21] or iodine concen- trations [22]. Compared to standard CT, DECT may have the potential to improve focal lesion characterisa- tion, and it is a promising tool to supply quantitative data in addition to traditional morphological informa- tion. The associated radiation dose with DECT will depend on the applied technology. Although specific clinical studies comparing the dose efficiency of differ- ent technologies are still lacking, current data suggest that DECT imaging with dual-source systems does not necessarily cause additional radiation exposure for the patient compared to standard CT [17, 23, 24]. Radiation dose data on the rapid kilovolt peak– switching technique to date are still inconclusive and reports from other approaches are scarce or nonexistent [25]. However, the increased informational content and post-processing flexibility of DECT data create addi- tional opportunities for dose saving, such as the crea- tion of virtual unenhanced images from a contrast- enhanced scan [25]. Dual-energy CT Table 2 Early and long-term findings after RF ablation of kidney lesions in successful and unsuccessful results, with the added value of the DECT technique Early findings Long-term findings Successful General findings Non-enhancing area on the contrast-enhanced CT Often wedge shaped because of peripheral infarctions Streaky soft tissue attenuations in the perirenal fat Regular borders of the ablation zone Slow reduction in size Fat between the ablation zone and normal kidney parenchyma Added value of DECT Improved contrast on the 40-keV images Clear differentiation between avascular and viable tissues on iodine images Sharp depiction of the ablation zone’s edg on the iodine images Unsuccessful General findings Persistent enhancement at the border of the ablation zone Nodular attenuation difference around the ablation zone Added value of DECT Iodine concentrations that are similar to the original tumour, with quantitative assessment Higher lesion-to-liver contrast on the iodine images Intermediate iodine concentration between the ablation zone and normal kidney parenchyma at the level of the tumour The purpose of this pictorial essay is to display post- ablative changes on contrast-enhanced CT with a single- source rapid kilovolt peak-switching technique in order to identify the additional information provided by DECT imaging and to describe the pitfalls of this rela- tively new technique in the evaluation of RF ablation’s technical success. DECT imaging protocol Fast kilovoltage switching CT was performed on a 64- slice CT (Discovery CT750 HD, GE Healthcare, Milwaukee, WI, USA) after IV administration of 120 cc contrast at 2.5 cc/s. Scan data were acquired at 40-mm collimation using predefined GSI protocols at CTDIvol values between 15.02 and 25.53 mGy. Fixed tube currents (between 375 and 600 mA) were used as our DECT scanning mode is not compatible with the automated tube current modulation system of the scanner. Images were processed on a workstation (AW4.4; GE Healthcare) using the Gemstone Spectral imaging application. Three types of images were recon- structed for analysis: synthesised monochromatic im- ages from 40 to 140 keV, greyscale- and colour-coded iodine images (with ‘rainbow’ colour map). Liver ablation Necrosis caused by thermal damage is characterised by an absence of blood perfusion, resulting in a non- 369 Insights Imaging (2015) 6:363–379 difference between the ablation zone and the kidney parenchyma is well demonstrated on the synthesised monochromatic 40- and 70-keV images. The colour-coded iodine images show well-depicted areas of intermediate density on the axial reconstructions (black arrows) surrounding the ablation zone. The axial images of the synthesised monochromatic reconstructions less clearly visualise these regions. The multiplicity suggests a benign post-ablative finding, probably corresponding to transient thermal damage to the bordering kidney parenchyma. No definitive conclusion can be made concerning residual tumour solely relying on these images. However, compared to the pre-ablation imaging, we can assume that the tissue in question is a normal kidney parenchyma. At the outer margin of the ablation zone, a region of intermediate density is depicted. The coronal reconstructions are essential to prove the continuity with regard to the kidney parenchyma (white arrows). d On the follow-up CECT 15 months later, the focally enhanced tissue at the lateral side shows no signs of growth (arrow), thus confirming the absence of residual tumour Fig. 4 Renal cell carcinoma. An ultrasound reveals a renal mass in a 72-year-old male. a Venous phase DECT confirms a lesion in the left kidney. On the water map images, the lesion is exophytic and isodense compared to the kidney parenchyma (arrows). The synthesised monochromatic 40-keV greyscale- coded and colour-coded iodine images show a hypovascular lesion compared to the kidney parenchyma. A biopsy demonstrates a renal clear cell carcinoma. b On the iodine- coded image (left), three ROIs are selected: L1 (red) in the RCC; L2 (blue) in the renal vein; L3 (yellow) in the unaffected kidney parenchyma. The estimated amounts of iodine in the ROIs are presented in the right lower corner (mean value and standard deviation in 100 μg/cm3). On the spectral HU curve (right), the renal vein (L2) and RCC (L1) represent overlapping curves, suggesting similar iodine content. The normal kidney parenchyma (L3) has a markedly higher curve in the lower keV range, confirming a higher iodine uptake. c Axial (left column) and coronal (right column) reconstructions from a contrast-enhanced venous phase DECT, 24 h post-RF ablation. The water map images show very poor hyperattenuation in the ablated zone (arrowheads). The contrast 370 Insights Imaging (2015) 6:363–379 Fig. 4 (continued) 370 Insights Imaging (2015) 6:363 379 Fig. Liver ablation 4 (continued) Fig. 4 (continued) Fig. 4 (continued) map images with the same contrast-to-noise ratio as true unenhanced images [26]. enhancing area on contrast-enhanced CT (Table 1). Due to this lack of internal vascularity, iodine maps are well suited for delineating the treated area from the liver parenchyma (Figs. 1 and 2a). Dual-energy CT has the possibility to extract iodine from the enhanced images to create water map images and thus potentially skip true unenhanced images, as shown in Fig. 3a. This un- doubtedly offers a considerable advantage in limiting radiation dose exposure [23]. However, a drawback is that current DECT techniques do not produce water Frequently, a hyperattenuation centrally within the ablation zone can be seen on unenhanced images [27]. It is thought that this area of high density corre- lates to a region of greater cellular disruption in the centre of the ablation zone [3], reportedly caused by the intense charring of the severely desiccated coagu- lated lesion [28]. It has usually disappeared by the time the next follow-up CT examination takes place, but can Insights Imaging (2015) 6:363–379 371 Fig. 5 Renal cell carcinoma. A lesion in the right kidney of a 73- year-old male is confirmed as being a hypernephroma through biopsy. The patient is treated with RF ablation, while cooling the pyelum with a double j-stent. a The DECT obtained 24 h post ablation shows a hyperdense region in the ablation zone on the water map images (white arrow). On the synthesised monochromatic 40-keV images, a sharp delineation of the ablation border is assessed, without the presence of any internal hyperdensity in the ablation zone. A post-procedural, streaky, soft- tissue attenuation can be seen in the surrounding perirenal fat (arrowheads). The avascular zone is well depicted on the greyscale- and colour-coded iodine images. In this window level, some yellowish coloured pixels are seen in the centre of the ablation zone on the colour-coded iodine images (black arrow), lying in the extension of a branch of the renal artery, explainable as being spilled iodine due to arterial perfusion. b DECT reconstructions at a 3-month follow-up stage show an overall stability in the size of the ablation zone. The ablated zone is still slightly hyperdense on the water map image (arrow). Liver ablation There is no uptake of iodinated contrast on the greyscale- and colour-coded iodine images, which proves that this area is completely avascular and confirms that the colour pixels seen in a were not a sign of tumour activity Fig. 5 Renal cell carcinoma. A lesion in the right kidney of a 73- year-old male is confirmed as being a hypernephroma through biopsy. The patient is treated with RF ablation, while cooling the pyelum with a double j-stent. a The DECT obtained 24 h post ablation shows a hyperdense region in the ablation zone on the water map images (white arrow). 372 Insights Imaging (2015) 6:363–379 Fig. 6 Renal cell carcinoma. A 40-year-old female is diagnosed with a suspicious lesion in the right kidney on ultrasound. After consulting her urologist, the patient opts for RF ablation. a CECT shows a thick-walled cystic lesion in the right upper pole, graded as a Bosniak 4 cyst. b The true unenhanced polychromatic CT image (left), 24 h post ablation, shows a hyperdense change in the ablation zone (arrow). This finding cannot be confirmed on the water map reconstructions. c The imaging appearance of the ablation zone 24 h (first row), 3 months (second row), and 18 months (third row) after RF ablation. On the 24-h post-ablation series, the delineation of the ablation zone is clearly observable on all reconstructed images; however, the 40 keV image is of superior quality when compared to the 70-keV image (arrowheads). On the iodine-coded images, the internal high attenuation is unexpectedly (no iodine content) still visible within the avascular zone (arrows). No suspicious nodular enhancement is observed at the border of the ablation zone. DECT from 3- and 18-month follow-up depicts an involution of the avascular zone. There is a loss of the internal high- attenuation changes. There is no evidence of local tumour progression did not prove an internal homogeneity of the ablation zone on the iodine images. Immediately after ablation, it is common to find a hyper-attenuating halo surrounding the ablation zone on CECT, correlated to an increase in arterial perfusion due to capillary leakage from thermal damage [30]. The circumferential enhancement is predominantly vis- ible on the arterial phase and can be very prominent in some cases (Fig. 3c), obscuring the interpretation of residual tumour. An irregular or nodular rim can indi- cate the presence of residual tumour, although benign variations do occur [30–32]. Liver ablation When focal thickening at the border of the ablation zone is unclear, a shorter follow-up period is recommended [32]. Previous stud- ies have shown that a hypervascular rim remains visi- ble in 89 % of cases after 1 month, in 56 % of cases between 1 and 3 months, and in 22 % of cases be- tween 3 and 6 months [33]. Ideally, the volume of persist for a longer time [29] (Fig. 2b). This hyperattenuation is observable on both the water map and true unenhanced images. Despite the absence of iodine, this can also be seen on the iodine-coded im- ages, since the hyperattentuated area is represented as a combination of both water and iodine after material decomposition (Fig. 2a). Meticulous comparison of wa- ter and iodine-coded images allows for differentiation of this central hyperattenuation after RF ablation and structures enhancing after IV contrast administration. The reader should however be aware of the lower im- age quality of water images with respect to true unenhanced images (Fig. 3a), as also reported on dual-source DECT [20]. Similar to the study of Lee et al. [20], we observed sharp depiction of the edge of the ablation zone of the iodine images. Contrary to their findings obtained using a dual-source DECT sys- tem, our experience with single-source DECT images did not prove an internal homogeneity of the ablation zone on the iodine images. Immediately after ablation, it is common to find a hyper-attenuating halo surrounding the ablation zone on CECT, correlated to an increase in arterial perfusion due to capillary leakage from thermal damage [30]. The circumferential enhancement is predominantly vis- ible on the arterial phase and can be very prominent in some cases (Fig. 3c), obscuring the interpretation of residual tumour. An irregular or nodular rim can indi- cate the presence of residual tumour, although benign variations do occur [30–32]. When focal thickening at the border of the ablation zone is unclear, a shorter follow-up period is recommended [32]. Previous stud- ies have shown that a hypervascular rim remains visi- ble in 89 % of cases after 1 month, in 56 % of cases between 1 and 3 months, and in 22 % of cases be- tween 3 and 6 months [33]. Liver ablation Ideally, the volume of 373 Insights Imaging (2015) 6:363–379 Table 3 Early and long-term findings after RF ablation of lung lesions in successful and unsuccessful results, with the added value of the DECT technique Early findings Long-term findings Successful General findings Partial or circumferential ground-glass opacification surrounding the treated tumour Reduction in size Added value of DECT Vanishing of the iodine uptake in the treated tumour, with a clear delineation of the avascular zone on the iodine-coded images Shrinkage of the avascular zone Unsuccessful General findings Lack of ground-glass opacification around a part of the tumour is not decisive of success Centrally located enhancements - Enhancement not already present immediately after the RF ablation Growth of the ablation zone Change from ground-glass to solid opacity Added value of DECT Improved contrast of iodine in the lesion on the 40-keV and iodine-coded images ‘Enhancing’ of a part of the tumour on the 40-keV compared to the water map image Internal iodine uptake in the ablation zone g g ( ) thermal necrosis exceeds the limits of the metastasis by 1 cm in all dimensions [34]. Residual tumour will per- sist as a focal nodular enhancement in the periphery of the ablation zone (Fig. 3b). For dual-energy CT, the increased attenuation of iodine on the low-keV (e.g. around 40 keV) images is better suited for detecting any subtle density differences around the ablation zone (Fig. 3b). Adding colour-coding enhances the visibility of the contrast in the image. The water-iodine material decomposition can high- light areas containing iodinated contrast. Iodine maps are superior to synthesised monochromatic images for the qualification of contrast uptake. Consequently, a hypervascular nodule with a higher inflow of iodine is better depicted on the iodine maps when compared to synthesised monochromatic images (Fig. 2b). Kidney ablation Ablation zones in the kidneys lack iodine content and are often wedge shaped because of peripheral infarc- tions [35] (Table 2). Determining a tumour’s iodine concentration before RF ablation can provide a base- line for future follow-up assessments (Fig. 4a, b). Iodine concentrations that are similar to the original tumour post ablation can be a sign of local tumour progression. The iodine content can be assessed quali- tatively as well as quantitatively in the investigated ROI (Fig. 4b). When evaluating the contrast uptake in a lesion, it is imperative to compare the lesion’s density on the water map images to that on the synthesised mono- chromatic images in order to be able to appraise pos- sible ‘enhancement’. The iodine content can be assessed qualitatively based on the iodine-coded im- ages as well as quantitatively when expressed in io- dine concentrations (mg/ml) (Fig. 4b) in the investigat- ed ROI. Enhancement can also be demonstrated by comparing the iodine concentration in the lesion to a surrounding vascular structure after an IV contrast in- jection. When evaluating colour-coded iodine maps, it is important to compare iodine concentrations in the ROI to those in adjacent structures, because windowing (as a diagnostic tool) causes variability in the colour spectrum. As with liver lesions, the extent of necrosis in the ablation zone can be assessed more accurately on 40- keV rather than on 70-keV synthesised monochromatic images. Also, the iodine maps show clear differentia- tion between avascular and viable tissues. Adjacent to the avascular zone, regions of intermediate vascularity can be seen that appear most pronounced on colour- coded iodine images (Fig. 4c). This may correspond to 374 Insights Imaging (2015) 6:363–379 nsient thermal damage to the adjacent kidney paren- yma, causing a temporary blood flow decrease in this area. Multi-planar reconstructions are vital in assessment of questionable regions after the Insights Imaging (2015) 6:36 this area. Multi-planar reconstructions are vital in the assessment of questionable regions after the RF transient thermal damage to the adjacent kidney paren- chyma, causing a temporary blood flow decrease in 375 Insights Imaging (2015) 6:363–379 Fig. 7 Pulmonary metastases. A 77-year-old female, with a known melanoma, undergoes a PET/CT that shows a hypermetabolic nodule in the right upper lobe (not in our possession). The biopsy is non-conclusive. The therapeutic option is RF ablation. a Imaging is performed before (first column) and 24 h after (second column) RF ablation. Lung ablation When performing RF ablation of lung lesions, the goal is to achieve an avascular region, void of any iodine enhancement (Table 3). Dual-energy CT in the lungs can depict an abnormal blood flow distribution, improv- ing the detection of acute pulmonary embolism [41]. In addition, we believe that the complete avascular ablative area post ablation is well depicted on the iodine-coded images as an iodine-void area. Therefore, iodine map- ping may be an excellent method to evaluate the tech- nique effectiveness and also delineates a more realistic assessment of the necrotic zone after ablation (Fig. 7a). On CECT imaging, the absence of iodine uptake in an ablated lung lesion is hard to identify since the bound- ary between the ablated and non-ablated tissue cannot be clearly defined (Fig. 8). The typical finding on lung window CT during and after RF ablation is ground-glass opacification sur- rounding the treated tumour, which is fully circumfer- ential (Fig. 8) in the majority of cases, but can also be partial (Fig. 9a). It is less common to find ground-glass opacification along the electrode tract. These ground- glass changes typically lead to an overestimation of the necrosis zone by 2–4 mm [42]. The distinction between the inner necrotic zone and the outer haemorrhagic viable zone is not clear on CT imaging; thus, the effective necrotic zone is easily overestimated [8]. As a result, the boundary between the ablated and non-ablated zones cannot be clearly defined using tra- ditional morphologic imaging (Fig. 7b). ablation procedure (Fig. 4c). Although avascularity is by definition a sign of successful ablation, the ablation zone can show remnants of iodine concentration, pos- sibly due to the extravasation of iodine which itself is caused by vascular damage (Fig. 5a). The majority of ablation zones are hypodense in nature; however, it is not uncommon to have a variable degree of haemorrhagic necrosis in the necrotic area. This degree of attenuation is not predictive of tech- nique effectiveness (Fig. 6c). Since the quality of the water maps is less compared to the true unenhanced images, spontaneous hyperdensity may be missed by this reconstruction technique (Fig. 6b). It is unclear how the iodine images provide a more realistic view of the necrotic zone after ablation. Kawai et al. [43] evaluated the feasibility of distinguishing ground-glass opacification in adenocarcinomas from that in a haemorrhage or inflammation. Kidney ablation The 70- keV lung window shows a focal nodule of 11 mm (white arrow) in proximity of a blood vessel. This nodule is hyperdense on the synthesised monochromatic 40-keV images and shows iodine content on the greyscale- and colour-coded iodine maps (circles). Destructive lung parenchyma is seen after the ablation as an irregular hyperdensity on the lung window (black arrows). The synthesised monochromatic 40-keV reconstructions show very heterogeneous densities in this region. The vessel on the anterior side is still intact (white arrowhead). On the iodine maps, we clearly observe the absence of iodine uptake (yellow arrowheads). PET/CT shows no activity in this region. b Imaging is performed before (first column) and 24 h after (second column) microwave ablation (MWA). After 6 months, a well-circumscribed consolidation is seen on the 70-keV lung window and synthesised monochromatic 40-keV image (arrows). The greyscale- and colour-coded iodine maps show no focal iodine uptake. This consolidation is hypermetabolic on PET/CT. As a result of the discrepancy between the morphologic and metabolic imaging methods, and the proximity of the blood vessel, which could lead to a heat-sink effect, the patient opts for further focal thermal treatment through microwave ablation. Twenty-four hours after ablation, a large heterogeneously shaped density is recognised on the lung window. The greyscale- and colour-coded iodine maps very clearly show the zone without internal contrast uptake (arrowheads) when compared to the 40-keV images. Again, a photopenic area is seen on the PET/CT image little to no reduction in size within the first 6 to 12 months [39, 40]; however, earlier shrinkage is not uncommon (Fig. 6c). Lung ablation They found that there was an increased iodine-related attenuation in ad- enocarcinomas, but not in a pulmonary haemorrhage or in inflammatory changes. These findings led us to in- vestigate the possible benefits of using iodine maps to distinguish residual tumours (Fig. 9) from the common post-ablative changes found in lung ablation. On follow- up CECT, centrally located enhancements or enhance- ments that were not present immediately after the RF ablation are indicative of malignancy [44]. Even without enhancement, growth of the ablation zone (after 8 to 10 weeks) or any peripheral nodule is a cause for con- cern. A change from ground-glass opacity to solid opac- ity also requires further investigation [43]. Positron In the contrast to rim enhancement that is commonly found in liver ablations, this phenomenon has only been observed occasionally in ablated renal parenchyma [25]. If present, it resolves quickly and is only marginally visible after 3 months [36]. Post-ablative changes in the perirenal fat are predominantly seen as streaky soft tissue attenuations (Fig. 5a), which resolve to a domi- nant band or halo over time (Fig. 4d). Long-term fol- low-up findings include the development of fat between the ablation zone and normal kidney parenchyma [37, 38] (Fig. 4d). The majority of kidney ablations show emission tomography/CT is considered the optimal follow-up tool after focal ablation in the lung. Residual disease can be detected through tracer uptake, most likely at the periphery of the ablation zone (Fig. 9b). On follow-up, increased or new metabolic activity located centrally (Fig. 7b) or at the outer rim Fig. 8 Pulmonary metastases. A 69-year-old female, with a history of breast cancer diagnosed 9 years prior, shows a hypermetabolic nodule in the inferior lobe of the right lung on PET/CT. A biopsy reveals a metastasis of a ductal mammary carcinoma. An RF ablation is performed. Imaging of the lesion 1 day before (first column), 24 h after (second column), and 3 months after (third column) complete RF ablation is performed. Before ablation, an 8- mm well-circumscribed nodule is depicted on the synthesised monochromatic 70-keV lung window, FDG-avid on the PET scan (white arrows). Internal vascularisation is clearly observable on the contrast- enhanced, synthesised monochromatic 40-keV, greyscale- and colour-coded iodine images (circles). The changes found 24 h post ablation are a ground-glass appearance (yellow arrowheads) around the lesion and the vanishing of the previously seen iodine uptake in the lesion. Lung ablation A biopsy reveals a non- small-cell carcinoma. A multidisciplinary team meeting leads to the choice for RF ablation treatment. Coronal reconstructions of the lesion in the right upper lobe before (first column), 24 h after the first RF ablation (second column), and 24 h after microwave ablation (third column) are demonstrated. a A focal nodule of 9 mm in the upper lobe is seen on the 70-keV lung window (black arrow) prior to the ablation. This nodule is “enhanced” on the synthesised monochromatic images compared to the water map image. This “increase in density” is better appreciated on the 40-keVrather than on the 70-keVimages. Twenty-four hours after the first ablation, the spicular nodule appears enlarged on the lung windows. On the 40-keV image, a focal enhancement (arrow) is noted at the inferior border of the ablation zone, again more clearly visible compared to the 70-keV image. After re-ablation with microwave technique, we notice more important changes on the lung window than after the first ablation. b The iodine content of the hypermetabolic lesion before the RF ablation is most observable on the greyscale- and colour-coded iodine images (circles). The nodule is hypermetabolic on PET/CT. Twenty-four hours after the first ablation, the greyscale- and colour-coded iodine maps clearly show the focal area of iodine uptake (white arrows) on the inferior border. PET/CT confirms hypermetabolic remnants (yellow arrow). After re-ablation, the larger area of thermal damage has lost its internal focal iodine uptake, which is most appreciable on the iodine-coded images (arrowheads). Images after the re-ablation lack FDG uptake on PET/CT Insights Imaging (2015) 6:363–379 377 Fig. 9 Pulmonary metastases. A focal lung nodule is found in a 65-year- old female diagnosed with ovarian carcinoma. A biopsy reveals a non- small-cell carcinoma. A multidisciplinary team meeting leads to the choice for RF ablation treatment. Coronal reconstructions of the lesion in the right upper lobe before (first column), 24 h after the first RF ablation (second column), and 24 h after microwave ablation (third column) are demonstrated. a A focal nodule of 9 mm in the upper lobe is seen on the 70-keV lung window (black arrow) prior to the ablation. This nodule is “enhanced” on the synthesised monochromatic images compared to the water map image. This “increase in density” is better appreciated on the 40-keVrather than on the 70-keVimages. Lung ablation Twenty-four hours after the first ablation, the spicular nodule appears enlarged on the lung windows. On the 40-keV image, a focal enhancement (arrow) is noted at the inferior border of the ablation zone, again more clearly visible compared to the 70-keV image. After re-ablation with microwave technique, we notice more important changes on the lung window than after the first ablation. b The iodine content of the hypermetabolic lesion before the RF ablation is most observable on the greyscale- and colour-coded iodine images (circles). The nodule is hypermetabolic on PET/CT. Twenty-four hours after the first ablation, the greyscale- and colour-coded iodine maps clearly show the focal area of iodine uptake (white arrows) on the inferior border. PET/CT confirms hypermetabolic remnants (yellow arrow). After re-ablation, the larger area of thermal damage has lost its internal focal iodine uptake, which is most appreciable on the iodine-coded images (arrowheads). Images after the re-ablation lack FDG uptake on PET/CT of the ablation zone is a sign of progressive tumour activity [44]. of the ablation zone is a sign of progressive tumour activity [44]. residual tumours from benign inflammatory changes, commonly found after the ablation of liver, kidney, and lung tumours. Further investigation is required because of the limited clinical experience with this relatively new technique. Lung ablation The post-ablative avascular zone surrounding the necrotic lesion can be delineated on the iodine images as darker areas (yellow arrows). On the lung window images after 3 months, the treated area has transformed into a homogeneous, well-delineated area of high attenuation. PET/CT images reveal no activity after the RF ablation. The greyscale- and colour-coded iodine images show a reduction in size of the avascular region 376 Insights Imaging (2015) 6:363–379 376 Insights Imaging (2015) 6:363–379 Fig. 8 Pulmonary metastases. A 69-year-old female, with a history of breast cancer diagnosed 9 years prior, shows a hypermetabolic nodule in the inferior lobe of the right lung on PET/CT. A biopsy reveals a metastasis of a ductal mammary carcinoma. An RF ablation is performed. Imaging of the lesion 1 day before (first column), 24 h after (second column), and 3 months after (third column) complete RF ablation is performed. Before ablation, an 8- mm well-circumscribed nodule is depicted on the synthesised monochromatic 70-keV lung window, FDG-avid on the PET scan (white arrows). Internal vascularisation is clearly observable on the contrast- enhanced, synthesised monochromatic 40-keV, greyscale- and colour-coded iodine images (circles). The changes found 24 h post ablation are a ground-glass appearance (yellow arrowheads) around the lesion and the vanishing of the previously seen iodine uptake in the lesion. The post-ablative avascular zone surrounding the necrotic lesion can be delineated on the iodine images as darker areas (yellow arrows). On the lung window images after 3 months, the treated area has transformed into a homogeneous, well-delineated area of high attenuation. PET/CT images reveal no activity after the RF ablation. The greyscale- and colour-coded iodine images show a reduction in size of the avascular region most likely at the periphery of the ablation zone (Fig. 9b). On follow-up, increased or new metabolic activity located centrally (Fig. 7b) or at the outer rim emission tomography/CT is considered the optimal follow-up tool after focal ablation in the lung. Residual disease can be detected through tracer uptake, 377 Insights Imaging (2015) 6:363–379 of the ablation zone is a sign of progressive tumour activity [44]. residual tumours from benign inflammatory changes, commonly found after the ablation of liver, kidney, and lung tumours. 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https://openalex.org/W2811308086	https://www.epj-conferences.org/10.1051/epjconf/201818503010/pdf	English	null	Phase Dynamics in Arrays of Coupled Vortex Spin-Torque Nano-Oscillators	EPJ web of conferences	2,018	cc-by	3,178	1 Introduction problem that has to be clarified. When the dissipation parameter is small enough, one can use the linear normal mode formalism for analyzing of the dynamics of the network of oscillators. If this condition is not satisfied, one can use the nonlinear normal mode formalism, which is much harder than the linear one. In general, the majority of these networks has the small dissipation parameter, and therefore it is possible to use the linear normal mode formalism for these systems. A great attention has attracted by studies of the phase locking in the arrays of spin transfer nano-oscillators (STNOs), which benefit from the spin-transfer torque phenomenon [1] and giant or tunnel magnetoresistance effects to generate microwave voltage signal. These oscillators in nanoscale can be easily tuned by a dc- current or external magnetic field, and compatible with CMOS-architectures. The enumerated features allow for consideration of STNOs for voltage or current controlled oscillators for telecommunication systems, and elements for building biological inspired computing systems [1]. Bio-inspired computing requires a capability to fabricate dense networks of interacting oscillators and to control the degree of coupling between STNOs. There are several studies of STNOs phase locking achieved by various physical mechanisms: through electrical connection in series of oscillators [2-4], by spin-wave propagation [5,6], by antivortices [7,8], and by dipolar coupling [9,10]. The synchronization and topological excitations in arrays of STNOs through the Kuramoto model described in [11]. Nevertheless, the theoretical description of the synchronization dynamics of STNO is more complicated than traditional limit-cycles oscillators (van der Pole oscillators, Josephson junctions, rotating pendula) which have constant orbit radius, and can be described in single oscillator case by the Adler equation. Due to technologically fabricated nonidentity of STNOs in the network [12] they can be expressed by generalized Adler-like equations. The purpose of this paper is an analyzing of the structure of linear normal mode and mutual synchronization in different types of networks of nonlinear oscillators, from simple to complex cases. We investigate how network topology affects the stability of modes for the vortex-based STNOs. We show the impact of degeneration and synchronization of oscillation modes to the problem of optimal bio-inspired network design and microwave synthesizers. We find that hierarchical networks are characterized by a smaller number of stable nontrivial modes than partially hierarchical, or randomly organized networks. Phase Dynamics in Arrays of Coupled Vortex Spin-Torque Nano- Oscillators Phase Dynamics in Arrays of Coupled Vortex Spin-Torque Oscillators Olga Katkova,, Ansar Safin, Nikolay Udalov, Mikhail Kapranov National Research University “MPEI”, Department of Radio Engineering and Electronics, 111250 Moscow, Russia Olga Katkova,, Ansar Safin, Nikolay Udalov, Mikhail Kapranov National Research University “MPEI”, Department of Radio Engineering and Electronics, 111250 Moscow, Russia Abstract. In this work, the mode analysis technique of complex networks nonlinear self- oscillatory vortex-based spin-torque nano-oscillattors (STNOs) with nonidentity and nonisochrony is developed. We construct adjacency matrices of different type of networks and calculate the normal modes. After the calculation of normal modes we shift to truncated equations for slowly varying amplitudes and phases in the normal coordinates using generalized quasi-Hamiltonian approach. Finally, we present the phase dynamics based on the Kuramoto- approach and compare different networks to the ability of synchronization. 1 Introduction Our analysis gives rise to an approach for specifying of topological transformations of networks that can enhance synchronization. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). * Corresponding author: arnellemorte@gmail.com * Corresponding author: arnellemorte@gmail.com © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1051/epjconf/201818503010 https://doi.org/10.1051/epjconf/201818503010 EPJ Web of Conferences 185, 03010 (2018) EPJ Web of Conferences 185, 03010 (2018) MISM 2017 2 Quasi-Hamiltonian Approach (11) (11)     / 2; / 2, j j j j j j b Q iP b Q iP      (2) and using following expression for Hamiltonian (here   , j j j X Q P  ):     / 2; / 2, j j j j j j b Q iP b Q iP      (2) and using following expression for Hamiltonian (here   , j j j X Q P  ): Here 2 j x c  - generalized power of complex variable jc , ,j   is a positive damping function (Hilbert damping in ferromagnetic or positive resistance in van der Pole scheme) and ,j   is a negative damping * * . j j j j j j j b b H H H X b X X b               (3) (3) function (spin-transfer torque effect in ferromagnetic or negative resistance in the van der Pole scheme). If the power x is small, we can expand the functions ,   in a power series. Now let us shift to the mode analysis of the fully identical nonlinear oscillators. We obtain equations of motion in the form * * * ; . j j j j j j b b H H i F i F t t b b             (4) (4) Here   1 2 / / 2 j j j j j F G iG     and the second equation in (4) may be obtained from the first one by complex conjugation. There is a wide range of changes from canonical variables * ,j j b b to other variables. The possibility of choosing various canonical variables is an advantage of the Hamiltonian approach. 2 Quasi-Hamiltonian Approach We need to describe the nonlinear properties of a system of mutually coupled nonidentical and nonisochronous oscillator of complex networks from a common point of view. The quasi-Hamiltonian approach is sufficiently convenient for this description, especially for writing truncated equations (using averaging method). The quasi-Hamiltonian equations with N degrees of freedom are characterized by coordinates q1,…,qN and impulses p1,…, pN. They satisfy the following equations: In real networks there are as many frequencies (we will call them “modes”) as the number of oscillators. When more than two oscillators are mutually coupled the finding of each mode structure and determination of their stability (or stability of group of them) becomes the main https://doi.org/10.1051/epjconf/201818503010 EPJ Web of Conferences 185, 03010 (2018) MISM 2017 EPJ Web of Conferences 185, 03010 (2018) 2 1 , N j j j jk k j j j k с i c i c iN c t           (10) where * 1 N j jm m jm m m u F v F         . For several types of oscillators it is possible to write j  in the simple form    , , j j j x x x      . (11) 1 2 ; , j j j j j j q p H H G G t p t q             (1) (1) (10) where G1;2j are the dissipative functions, and H is the Hamiltonian of the system. If G1;2j = 0 then (1) gives simple Hamiltonian form of equations. Now lets change over to canonical variables Qj = αjqj, Pj = pj/αj, where αj are dimensionless constants (Qj and Pj have the same dimension). After introducing the complex variables: oscillators it is possible to write j  in the simple form j    , , j j j x x x      . 3 Mode Analysis & Truncated Equations These equations (12) or (13), we can transform into the canonical, using linear matrix transformation   1 , ,..., T N T a a    c a a into the (5) In the general form of network geometry we can write transformation   1 , ,..., T N T a a    c a a into the     * * * * 2 , 1 0.5 . N lk l k lk l k lk l k l k H A b b B b b B b b      (6)     1 i T T t       a a . (14) (14) The Hamiltonian (6) may be diagonalized by linear uv- transformation For eigenmodes j  we need   * 1 N j jm m jm m m c u b v b     . (7)     1 1,..., N T T diag      . (15)     1 1,..., N T T diag      . (15) (15) (7) Form (14), (15) we can write After special choosing of matrices ujm; vjm in (7) the Hamiltonian (6) takes the form After special choosing of matrices ujm; vjm in (7) the Hamiltonian (6) takes the form ~ 1 N j jm m m c t a   . (16) ( ) * 2 1 , 1; . N N j j j km k m j k m k m H c c c c          (8) (16) (8) Here   km  is the matrix of linear coupling between oscillators. This expression for 2 H we will use for calculating linear modes of oscillator networks. If the oscillator is nonisochronous (frequency is a function of the amplitude), we can obtain 3 H in a simple form Now the unperturbed solution j a could be expressed as follows (here , j j U  are constants)    j i t i t j j a t U t e      . 3 Mode Analysis & Truncated Equations Here   1 2 / / 2 j j j j j F G iG    and the second equation in (4) may be obtained from the first one by complex conjugation. There is a wide range of changes from canonical variables * ,j j b b to other variables. The possibility of choosing various canonical variables is an advantage of the Hamiltonian approach. First, let us calculate normal modes j  of the complex networks. An unperturbed equation of (10) for ~ jc is First, let us calculate normal modes j  of the complex networks. An unperturbed equation of (10) for ~ jc is ~ ~ ~ 1 N j j j jk k k с i c i c t         , (12) (12) (12) Assuming * , j j b b to be small, let us expand the function   * , j j H b b in a power series in * , j j b b . We are not interested in the zero term   0,0 H , and the first order of H is equal to zero. Therefore, the expression of H begins with the second order terms in the form 2 3 4 ... H H H H     (5) or for ~ ~ 1 ,..., T N c c     c in the vector form i t   c c , or for ~ ~ 1 ,..., T N c c     c in the vector form or for ~ ~ 1 ,..., T N c c     c in the vector form or ~ ~ 1 ,..., N c c     c in the vector form i t   c c , (13) i t   c c , (13) (13) with coupling matrix . These equations (12) or (13), we can transform into the canonical, using linear matrix with coupling matrix . 3 Mode Analysis & Truncated Equations (17) (17) For the small coefficients ,   , if we regard , j j U  as slowly varying functions of time, then the dynamics of  j j U U t  and  j j t    can be represented by averaged equations or truncated equations. For the small coefficients ,   , if we regard , j j U  as slowly varying functions of time, then the dynamics of  j j U U t  and  j j t    can be represented by averaged equations or truncated equations. * 3 1 N j j j j j H c c c     . (9) (9) After substituting (7)-(9) in (4) equations of motion for cj we will have the following view 2 2 EPJ Web of Conferences 185, 03010 (2018) EPJ Web of Conferences 185, 03010 (2018) MISM 2017 https://doi.org/10.1051/epjconf/201818503010 (a) (a) (b) Fig. 1. Mode structures of the 2-adic (a) and 3-adic (b) networks of STNOs and phase clusters. In the particular case, it is possible to use phase- approximation (Kuramoto-approach), when the amplitudes of oscillators signals are established faster than phases. Following this approximation for phases j for each vortex STNO is   N j j kj k j k 1,k j d sin dt             , (18) (18) where j  are eigenfrequencies of oscillator j,  jk  are parameters of connection between j and k,  which are decreased with increasing of a distance between oscillators, that is jk jk 1 d  , where jk d is the distance between vortex STNO. (a) (b) (a) Analysis of received system of phase equations shows that besides the steady-state regime of equal phases there are also complicated chaotic modes and chimera states, where one part of oscillators is phase-synchronizned and another part is in chaotic regime. It is necessary that non- identify of vortex STNO sizes would be least (units of nm when diameter of the sample is hungreds of nm) for capacity addition. (b) In the Fig.3 the phase planes of three Kuramoto- oscillators for completely phase locking, partial phase locking of 1 and 2, of 1 and 3 and asynchronous mode shown. 3 Mode Analysis & Truncated Equations Using same approach we calculated the phase distribution of different networks which are shown in Fig.1,2. It is easy to see that the partially hierarchical network (Fig.2b) is characterized by the lower number of phase clusters. Fig. 1. Mode structures of the 2-adic (a) and 3-adic (b) networks of STNOs and phase clusters. For the 2-adic and 3-adic tree-like networks (see Fig.1) we have the fractal structure of the normal modes spectrum, which looks like devils staircase fractal. The randomly and hierarchically distributed networks shown in Fig.2. We analyzed the typical examples of the networks with N = 25 number of oscillators. (a) (b) Fig. 2. Mode structures of the randomly (a) and hierarchically (b) distributed networks of STNOs and phase clusters.. (a) The structure of linear normal mode of the hierarchical network shown in Fig.2a. Here we can see the nonsymmetrical structure of the dependence and one wide section of the degenerate normal modes, which is equal to 9. Other modes are nondegenerate (nonequal to each other). From the other hand if we move into the hierarchically distributed network (Fig.2b) with the number of modes is equal to the previous one 25, we will see the different picture. Here we have the four blocks of degenerate modes with L=5 for three blocks and L=10 for one block, where L is the number of degenerate modes. Therefore, in general the number of degenerate modes for the hierarchically distributed network is larger than for the randomly organized. (a) (b) For the task of power summation and mutually phase locking, it is better to use the hierarchically organized network where the number of degenerate modes is high. From the other hand, for the task of multimode communication it is better to use randomly distributed networks, because the number of degenerate modes is low. These effects confirmed by the nonlinear mode analysis of the microwave oscillators with these two topological structures of networks (b) Fig. 2. Mode structures of the randomly (a) and hierarchically (b) distributed networks of STNOs and phase clusters.. 6 Acknowledgment The research was supported by a grant of the President of the Russian Federation for state support of young Russian scientists No.MK-7026.2016.8. 4 Phase approach 3 https://doi.org/10.1051/epjconf/201818503010 EPJ Web of Conferences 185, 03010 (2018) EPJ Web of Conferences 185, 03010 (2018) MISM 2017 a) (b) (c) (d) 3 Ph l f h K ill 5 Conclusion In this work we presented mathematical model that allows us to calculate linear normal modes by using quasi-Hamiltonian approach. We demonstrated the fractal structure of the normal modes spectrum and wrote truncated equations for slowly varying amplitudes and phases in the normal coordinates. The stability analysis of the normal modes submitted. The relationship between a structure of a network that consists of Kuramoto-oscillators, and the corresponding number of phase clusters is shown. a) (b) (c) (d) Fig. 3. Phase planes of three Kuramoto-oscillators completely phase locking (a), partial phase locking of 1 an (b), of 1 and 3 (c) and asynchronous mode (d). a) (b) References 1. J. Grollier, D. Querlioz, and M.D. Stiles, Proc. Of the IEEE 104, 10 (2016). 2. J. Grollier, V. Cros, and A. Fert, Phys. Rev B. 73, 060409(R) (2006). 3. B. Georges, J. Grollier, M. Darques, V. Cros, C. Deranlot, B. Marcilhac, G. Faini, and A. Fert, Phys. Rev. Lett. 101, 017201 (2008). (b) (c) 4. B. Georges, J. Grollier, V. Cros, and A. Fert, Appl. Phys. Lett. 92, 232504 (2008). 5. X. Chen, and R.H. Victora, Phys. Rev. B 79, 180402 (2009). 6. S. Sani, J. Persson, S.M. Mohseni, Y. Pogoryelov, P.K. Muduli, A. Eklund, G. Malm, M. Kall, A. Dmitriev, and J. Akerman, Nat. Commun. 4, 2731 (2013). 7. C.E. Zaspel, Appl. Phys. Lett. 102, 052403 (2013). 8. A. Ruotolo, V. Cros, B. Georges, A. Dussaux, J. Grollier, C. Deranlot, R. Guillemet, K. Bouzehouane, S. Fusil, and A. Fert, Nat. Nanotechnol. 4, 528 (2009). (c) (d) 9. A.D. Belanovsky, N. Locatelli, P.N. Skirdkov, F. Abreu Araujo, J. Grollier, K.A. Zvezdin, V. Cros, and A.K. Zvezdin, Phys. Rev. B 85, 100409(R) (2012). 10. F. Abreu Araujo, and J. Grollier, Journ. of Appl. Phys. 120, 103903 (2016). 11. V. Flovik, F. Macia, and E. Wahlstrom, Sci. Rep. 6, 32528 (2016). 12. A.R. Safin, N.N. Udalov, M.V. Kapranov, Eur. Phys. J. Appl. Phys. 67, 20601 (2014). (d) Fig. 3. Phase planes of three Kuramoto-oscillators for completely phase locking (a), partial phase locking of 1 and 2 (b), of 1 and 3 (c) and asynchronous mode (d). 4 4
https://openalex.org/W2058372922	https://www.sciencepubco.com/index.php/JACST/article/download/3573/2846	English	null	Range of outputs precise of digits rounding in SPSS and MS Excel	Journal of advanced computer science & technology	2,014	cc-by	4,826	Range of outputs precise of digits rounding in SPSS and MS Excel Zaher Saif Mohammed Al-Hashami CS Instructor -Muscat, Sultanate of Oman E-mail: acade.re.z@gmail.com CS Instructor -Muscat, Sultanate of Oman E-mail: acade.re.z@gmail.com Copyright © 2014 Zaher Saif Mohammed Al-Hashami. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract The statistical operations done by many specialist programs, by them can do these operations fluently and precisely. There are many functions in such programs can calculate in SAS, STAT and the analytical program SPSS. There is Microsoft Excel program that is calculate like these functions. The level of some programs may be different than others within these functions they are calculating. From these functions are Sum, Average, Maximum and Minimum. Round function is also from these functions that can measure it's accuracy through this research. In this research i chose ten digits numbers and I also chose three criteria under, equal and more than 5. According to that the rounding operations are done based on if wanted decimal place is less, equal or higher than 5. Rounding applied on ten digits using SPSS and MS Excel programs. The outputs findings are the same except that Microsoft Excel is truncating the last zeroes of the digit after the decimal point. Wherever the decimal place specified in the digit is want it will truncate after the decimal point. The SPSS is more precise than MS Excel based on the decimal place in the digit number wanted statistically and analytically. Keywords: Decimal Place, Digit, Excel, Function, Round. Keywords: Decimal Place, Digit, Excel, Function, Round. Journal of Advanced Computer Science & Technology, 3 (2) (2014) 154-159 ©Science Publishing Corporation www.sciencepubco.com/index.php/JACST doi: 10.14419/jacst.v3i2.3573 Research Paper Journal of Advanced Computer Science & Technology, 3 (2) (2014) 154-159 ©Science Publishing Corporation www.sciencepubco.com/index.php/JACST doi: 10.14419/jacst.v3i2.3573 Research Paper Journal of Advanced Computer Science & Technology, 3 (2) (2014) 154-159 ©Science Publishing Corporation www.sciencepubco.com/index.php/JACST doi: 10.14419/jacst.v3i2.3573 Research Paper 1. Introduction Functions in many statistical programs like MS Excel and SPSS programs are count many kinds of numbers like integers, natural, real and digits. The results of such as these functions are supposed to be same outputs in MS Excel and in SPSS programs. The digits numbers rounding are also supposed to be the same in many statistic programs like SPSS, SAS, STAT and MS Excel. Ted French said in Spreadsheet.about.com: rounding a number means altering it to a specific size. Rounding integers (whole numbers) involves rounding them up or down to the nearest multiple of 10, while rounding decimals reduces them to a set number of decimal places [1]. According to Poes and Matthew Joseph,2011 [2] they used SPSS program to export some values to MS excel, by save it as Excel file, when they do this, it's truncating the values down to only one digit after the decimal place. It also is not rounding correctly, as some values which should be appropriately rounded up, are being rounded down. They said as example, a value of 6.547 becomes 6.5 instead of 6.6. The digits numbers in both SPSS and MS Excel are supposed to round up into the same truncate numbers, whatever decimal place must convert. In SPSS the decimals are valid for numeric variables only. It specifies the number of decimals to be kept for a variable. All the extra decimals will be rounded up and the rounded numbers will be used in all the analysis, so should be careful to specify the number of decimals to fit in the precision the researcher want. Professor Matthew in his study name: Reporting Statistics in APA Style [3] using SPSS, about using rounding appropriately said round number to one or two decimal place he said, must keep in mind that fewer decimal places are easier to comprehend. He said must consider rescaling measurements required for more two decimal places. A scientific fact stipulates that, when rounding numbers especially digits numbers, these numbers should be rounded up as far as rounded down. The rounding precisely must examine the first digit to be truncated. If the digits are 6, 7, 8, or 9, round the number will be up. When the digits are 1, 2, 3, or 4 the rounding will be down. 1. Introduction Then if this digit is a 5, you should look to the remaining digits beyond the 5 to see if they are all zeroes. A scientific fact stipulates that, when rounding numbers especially digits numbers, these numbers should be rounded up as far as rounded down. The rounding precisely must examine the first digit to be truncated. If the digits are 6, 7, 8, or 9, round the number will be up. When the digits are 1, 2, 3, or 4 the rounding will be down. Then if this digit is a 5, you should look to the remaining digits beyond the 5 to see if they are all zeroes. 155 Journal of Advanced Computer Science & Technology If they are not all zeroes, then the number does not end in an exact 5 and should be rounded up. If all remaining digits to the right are zero or in other word there are no additional digits available to the right of the 5, then the number in its current precision is an exact 5. Then in this state, that the number should be rounded up as often as it is rounded down. This is a research done by choosing ten samples digits if they are less, equal and more 5 in the decimal place to check the round result through these three cases in SPSS and MS Excel. 2. Method The concept of this research is calculates rounding and truncate in SPSS and round function in MS Excel for 10 random digits numbers and comparing the results in the two programs with the same digits numbers. Compare the rounding outputs in both programs in four decimal places. The research Methodology is by choosing 10 random digits numbers with four decimal places and do the rounding less than 5, equal 5 (after decimal place numbers) and bigger than 5. These rounding could do it in SPSS and MS Excel with the same ten random digits numbers in both. Then could compare the rounding and truncate results of the same digits numbers in the two programs. The rounding is done according to 5 number position whether it in under, equal (after decimal place number to get rounding in the decimal place number) and higher than 5, if the number after the decimal place is bigger or less than 5. In the below tables can see the decimal place in bold color. In case of that it is equal 5; the position of the five numbers must be after the decimal place. p In MS Excel the rounding is done by ROUND function by inter the digit and then inter the decimal place. In SPSS must consider how many decimal places should inter when interring the variables. 3.1. Under 5 Table 1.3.1: The under 5 digits number and their decimal places Digits Numbers 1 2 3 4 5 6 7 8 9 10 Decimal Places 1 26.42 77.32 12.22 49.14 66.01 33.34 70.44 41.13 75.03 91.11 2 76.343 86.444 90.213 24.433 55.420 78.222 40.302 11.101 20.410 30.321 3 67.3421 74.3214 46.2313 90.1020 45.2333 76.2003 31.2304 87.2113 50.4444 10.3423 4 24.10201 76.34321 11.34441 90.20302 43.12320 21.40400 87.22323 21.34000 10.20111 99.23421 The SPSS outputs rounding if the decimal places numbers are less than 5 in the four decimal places like in table 2.3.1: Table 2.3.1: SPSS under 5 digits number rounding results 3.2. Equal 5 after decimal place number In this case the five numbers must be after the number of decimal place to get the rounding outputs: Table 1.3.2: The Equal 5 digits and their decimal places Digits Numbers 1 2 3 4 5 6 7 8 9 10 Decimal Places 1 26.25 77.35 12.05 49.75 66.85 33.75 70.85 41.95 75.15 91.45 2 76.345 86.465 90.235 24.495 55.405 78.295 40.305 11.115 20.555 30.595 3 67.3485 74.3205 46.2365 90.1045 45.2355 76.2035 31.2395 87.2175 50.4495 10.3435 4 24.10215 76.34315 11.34405 90.20355 43.12375 21.40445 87.22385 21.34025 10.20165 99.23445 Table 2.3.1: SPSS under 5 digits number rounding results 3.2. Equal 5 after decimal place number 3.2. Equal 5 after decimal place number In this case the five numbers must be after the number of decimal place to get the rounding outputs: In this case the five numbers must be after the number of decimal place to get the rounding outputs: Table 1.3.2: The Equal 5 digits and their decimal places Digits Numbers 1 2 3 4 5 6 7 8 9 10 Decimal Places 1 26.25 77.35 12.05 49.75 66.85 33.75 70.85 41.95 75.15 91.45 2 76.345 86.465 90.235 24.495 55.405 78.295 40.305 11.115 20.555 30.595 3 67.3485 74.3205 46.2365 90.1045 45.2355 76.2035 31.2395 87.2175 50.4495 10.3435 4 24.10215 76.34315 11.34405 90.20355 43.12375 21.40445 87.22385 21.34025 10.20165 99.23445 Journal of Advanced Computer Science & Technology 156 In the table 2.3.2 the SPSS outputs rounding if the decimal places are equal 5 and put it after decimal place number in the four decimal places are: the four decimal places are: Table 2.3.2: SPSS equal 5 digits number rounding results The first decimal place rounding result The second decimal place rounding result The third decimal place rounding result The fourth decimal place rounding result 3.3 Higher than 5 Table 1.3.3: The higher than 5 digits number and their decimal places Digits Numbers 1 2 3 4 5 6 7 8 9 10 Decimal Places 1 26.67 77.76 12.99 49.87 66.98 33.77 70.88 41.69 75.97 91.86 2 76.377 86.486 90.298 24.468 55.476 78.269 40.387 11.166 20.596 30.589 3 67.3488 74.3279 46.2366 90.1097 45.2369 76.2076 31.2369 87.2179 50.4489 10.3496 4 24.10268 76.34377 11.34496 90.20376 43.12397 21.40469 87.22378 21.34069 10.20186 99.23497 The SPSS outputs rounding if the decimal places numbers are higher than 5 in the four decimal places are in this table: Table 2.3.3: SPSS Higher than 5 digits rounding results 4. 3.2. Equal 5 after decimal place number MS excel rounding Table 2.3.2: SPSS equal 5 digits number rounding results The first decimal place rounding result The second decimal place rounding result The third decimal place rounding result The fourth decimal place rounding result 3.3 Higher than 5 Table 1.3.3: The higher than 5 digits number and their decimal places Digits Numbers 1 2 3 4 5 6 7 8 9 10 Decimal Places 1 26.67 77.76 12.99 49.87 66.98 33.77 70.88 41.69 75.97 91.86 2 76.377 86.486 90.298 24.468 55.476 78.269 40.387 11.166 20.596 30.589 3 67.3488 74.3279 46.2366 90.1097 45.2369 76.2076 31.2369 87.2179 50.4489 10.3496 4 24.10268 76.34377 11.34496 90.20376 43.12397 21.40469 87.22378 21.34069 10.20186 99.23497 The SPSS outputs rounding if the decimal places numbers are higher than 5 in the four decimal places are in this table: Table 2.3.2: SPSS equal 5 digits number rounding results The first decimal place rounding result The second decimal place rounding result The third decimal place rounding result The fourth decimal place rounding result The second decimal place rounding result The third decimal place rounding result The fourth decimal place rounding result 3.3 Higher than 5 Table 1.3.3: The higher than 5 digits number and their decimal places Digits Numbers 1 2 3 4 5 6 7 8 9 10 Decimal Places 1 26.67 77.76 12.99 49.87 66.98 33.77 70.88 41.69 75.97 91.86 2 76.377 86.486 90.298 24.468 55.476 78.269 40.387 11.166 20.596 30.589 3 67.3488 74.3279 46.2366 90.1097 45.2369 76.2076 31.2369 87.2179 50.4489 10.3496 4 24.10268 76.34377 11.34496 90.20376 43.12397 21.40469 87.22378 21.34069 10.20186 99.23497 The SPSS outputs rounding if the decimal places numbers are higher than 5 in the four decimal places are in this table: 3.3 Higher than 5 Table 1.3.3: The higher than 5 digits number and their decimal places Digits Numbers 1 2 3 4 5 6 7 8 9 10 Decimal Places 1 26.67 77.76 12.99 49.87 66.98 33.77 70.88 41.69 75.97 91.86 2 76.377 86.486 90.298 24.468 55.476 78.269 40.387 11.166 20.596 30.589 3 67.3488 74.3279 46.2366 90.1097 45.2369 76.2076 31.2369 87.2179 50.4489 10.3496 4 24.10268 76.34377 11.34496 90.20376 43.12397 21.40469 87.22378 21.34069 10.20186 99.23497 The SPSS outputs rounding if the decimal places numbers are higher than 5 in the four decimal places are in this table: 3.3 Higher than 5 Table 2.3.3: SPSS Higher than 5 digits rounding results 4. 3.2. Equal 5 after decimal place number Equal 5 after decimal place number The same in SPSS, this rounding in MS Excel is consider 5 number after the decimal place number Table 1.4.2: Equal 5 digits and their decimal places Digits Numbers 1 2 3 4 5 6 7 8 9 10 Decimal Places 1 26.25 77.35 12.05 49.75 66.85 33.75 70.85 41.95 75.15 91.45 2 76.345 86.465 90.235 24.495 55.405 78.295 40.305 11.115 20.555 30.595 3 67.3485 74.3205 46.2365 90.1045 45.2355 76.2035 31.2395 87.2175 50.4495 10.3435 4 24.10215 76.34315 11.34405 90.20355 43.12375 21.40445 87.22385 21.34025 10.20165 99.23445 In MS Excel outputs rounding also can consider if the decimal places are equal 5 and put it after decimal place number in the four decimal places are the below table: Table 2.4.2: Excel equal 5 digits rounding results The first decimal place rounding result The second decimal place rounding result The third decimal place rounding result Table 2.4.1: Excel under 5 digits rounding results The first decimal place rounding result The second decimal place rounding result The third decimal place rounding result The fourth decimal place rounding result 4.2. Equal 5 after decimal place number 4.2. 3.2. Equal 5 after decimal place number MS excel rounding Table 2.3.3: SPSS Higher than 5 digits rounding results 4. MS excel rounding 4.1. Under 5 Table 2.3.3: SPSS Higher than 5 digits rounding results 4. MS excel rounding 4.1. Under 5 4. MS excel rounding 4.1. Under 5 4.1. Under 5 157 Journal of Advanced Computer Science & Technology Table 1.4.1: Under 5 digits and their decimal places Digits Numbers 1 2 3 4 5 6 7 8 9 10 Decimal Places 1 26.42 77.32 12.22 49.14 66.01 33.34 70.44 41.13 75.03 91.11 2 76.343 86.444 90.213 24.433 55.420 78.222 40.302 11.101 20.410 30.321 3 67.3421 74.3214 46.2313 90.1020 45.2333 76.2003 31.2304 87.2113 50.4444 10.3423 4 24.10201 76.34321 11.34441 90.20302 43.12320 21.40400 87.22323 21.34000 10.20111 99.23421 The same method rounding in SPSS, in MS Excel outputs rounding after consideration that the decimal places are less than 5 in the four decimal places are: Table 1.4.1: Under 5 digits and their decimal places Digits Numbers 1 2 3 4 5 6 7 8 9 10 Decimal Places 1 26.42 77.32 12.22 49.14 66.01 33.34 70.44 41.13 75.03 91.11 2 76.343 86.444 90.213 24.433 55.420 78.222 40.302 11.101 20.410 30.321 3 67.3421 74.3214 46.2313 90.1020 45.2333 76.2003 31.2304 87.2113 50.4444 10.3423 4 24.10201 76.34321 11.34441 90.20302 43.12320 21.40400 87.22323 21.34000 10.20111 99.23421 The same method rounding in SPSS, in MS Excel outputs rounding after consideration that the decimal places are less than 5 in the four decimal places are: Table 2.4.1: Excel under 5 digits rounding results The first decimal place rounding result The second decimal place rounding result The third decimal place rounding result The fourth decimal place rounding result 4.2. 5. Discussion The outputs of the rounding less, equal and after decimal place and higher than 5 are all the same results in SPSS and MS Excel, except that MS Excel truncate the zeros by default after the decimal point number if there zeroes after it and in the last one or two or even three numbers. The outputs of the rounding less, equal and after decimal place and higher than 5 are all the same results in SPSS and MS Excel, except that MS Excel truncate the zeros by default after the decimal point number if there zeroes after it and in the last one or two or even three numbers. From the tabulation results can be seen that in the first output of the rounding under 5, which is less than 5 in the second decimal point, can be found the digit 11.10 in SPSS is truncated in MS Excel to 11.1 which is rounding to two decimal places. Also less than 5 outputs can see the digit: 76.200 in SPSS rounded to three decimal places while in MS Excel rounded to 76.2. Another example indicates that MS Excel truncate the digit number if there are zeroes after the decimal point, if there aren’t any number only (0000), like 21.3400 when rounded to four decimal places rounded to 21.34 in Microsoft Excel program where in the SPSS statistical program is 21.3400. From the tabulation results can be seen that in the first output of the rounding under 5, which is less than 5 in the second decimal point, can be found the digit 11.10 in SPSS is truncated in MS Excel to 11.1 which is rounding to two decimal places. Also less than 5 outputs can see the digit: 76.200 in SPSS rounded to three decimal places while in MS Excel rounded to 76.2. Another example indicates that MS Excel truncate the digit number if there are zeroes after the decimal point, if there aren’t any number only (0000), like 21.3400 when rounded to four decimal places rounded to 21.34 in Microsoft Excel program where in the SPSS statistical program is 21.3400. p g p g In other outputs from the tabulation outputs indicate that MS Excel program is rounding the digits without zeroes after decimal point numbers in the equal and after decimal place is 30.60 in the second decimal place rounding. 4.3. Higher than 5 Table 1.4.3: Higher than 5 digits and their decimal places Digits Numbers 1 2 3 4 5 6 7 8 9 10 Decimal Places 1 26.67 77.76 12.99 49.87 66.98 33.77 70.88 41.69 75.97 91.86 2 76.377 86.486 90.298 24.468 55.476 78.269 40.387 11.166 20.596 30.589 3 67.3488 74.3279 46.2366 90.1097 45.2369 76.2076 31.2369 87.2179 50.4489 10.3496 4 24.10268 76.34377 11.34496 90.20376 43.12397 21.40469 87.22378 21.34069 10.20186 99.23497 MS Excel outputs rounding if the decimal places numbers are higher than 5 in the four decimal places are in the table 2.4.3: Table 1.4.3: Higher than 5 digits and their decimal places Decimal Places 1 26.67 77.76 12.99 49.87 66.98 33.77 70.88 41.69 75.97 91.86 2 76.377 86.486 90.298 24.468 55.476 78.269 40.387 11.166 20.596 30.589 3 67.3488 74.3279 46.2366 90.1097 45.2369 76.2076 31.2369 87.2179 50.4489 10.3496 4 24.10268 76.34377 11.34496 90.20376 43.12397 21.40469 87.22378 21.34069 10.20186 99.23497 MS Excel outputs rounding if the decimal places numbers are higher than 5 in the four decimal places are in the table 2.4.3: Table 2.4.3: Excel higher than 5 rounding results The first decimal place rounding result The second decimal place rounding result The third decimal place rounding result The fourth decimal place rounding result Table 2.4.3: Excel higher than 5 rounding results The first decimal place rounding result The second decimal place rounding result The third decimal place rounding result The fourth decimal place rounding result Table 2.4.3: Excel higher than 5 rounding results The first decimal place rounding result The second decimal place rounding result The third decimal place rounding result The fourth decimal place rounding result Table 2.4.3: Excel higher than 5 rounding results 3.2. Equal 5 after decimal place number Equal 5 after decimal place number The same in SPSS, this rounding in MS Excel is consider 5 number after the decimal place number Table 1.4.2: Equal 5 digits and their decimal places Digits Numbers 1 2 3 4 5 6 7 8 9 10 Decimal Places 1 26.25 77.35 12.05 49.75 66.85 33.75 70.85 41.95 75.15 91.45 2 76.345 86.465 90.235 24.495 55.405 78.295 40.305 11.115 20.555 30.595 3 67.3485 74.3205 46.2365 90.1045 45.2355 76.2035 31.2395 87.2175 50.4495 10.3435 4 24.10215 76.34315 11.34405 90.20355 43.12375 21.40445 87.22385 21.34025 10.20165 99.23445 In MS Excel outputs rounding also can consider if the decimal places are equal 5 and put it after decimal place number in the four decimal places are the below table: Table 1.4.2: Equal 5 digits and their decimal places Digits Numbers 1 2 3 4 5 6 7 8 9 10 Decimal Places 1 26.25 77.35 12.05 49.75 66.85 33.75 70.85 41.95 75.15 91.45 2 76.345 86.465 90.235 24.495 55.405 78.295 40.305 11.115 20.555 30.595 3 67.3485 74.3205 46.2365 90.1045 45.2355 76.2035 31.2395 87.2175 50.4495 10.3435 4 24.10215 76.34315 11.34405 90.20355 43.12375 21.40445 87.22385 21.34025 10.20165 99.23445 In MS Excel outputs rounding also can consider if the decimal places are equal 5 and put it after decimal place number in the four decimal places are the below table: Table 2.4.2: Excel equal 5 digits rounding results The first decimal place rounding result The second decimal place rounding result The third decimal place rounding result The fourth decimal place rounding result Table 2.4.2: Excel equal 5 digits rounding results The first decimal place rounding result The second decimal place rounding result The third decimal place rounding result The fourth decimal place rounding result 158 Journal of Advanced Computer Science & Technology 5. Discussion This digit rounded to 30.6 in Microsoft Excel and in SPSS result got the same digit (30.60). Also 13.240 in the third decimal place rounding can be seen that it is rounded to 13.24 in MS Excel. It could be seen from the first time to the reader that it is rounded to second decimal place, but MS Excel rounding it as third decimal place. 67.0 is a digit rounded to the first decimal place. In SPSS is the same 67.0, in contrast in MS Excel it is rounded to 67. In this rounding for the reader it is an integer while it is rounded to first decimal place. g p SPSS is more precise than MS Excel in the last results calculation in considering the digits numbers after the decimal point. Last instances when rounding to fourth decimal place in this digit: 11.2450, it is rounded to four decimal MS excel rounded to 11.245 with truncate of the zero after 5. Last instances when rounding to fourth decimal place in this digit: 11.2450, it is rounded to four decimal places while in MS excel rounded to 11.245 with truncate of the zero after 5. Wh d i f i i SPSS d MS E l l f h i S f i f di i b 56 60000 function in SPSS and MS Excel, example for that is Sum function for digits numbers: 56.60000 equal 91.30000. When doing a function in SPSS and MS Excel, example for that is Sum function for digits num +34.70000 will equal 91.30000. In SPSS statistic program will equal 91.30000. In contrast in MS Excel it equal 91.3 which is rounded MS Excel. This function result is rounded without zeroes after decimal point and after first number 3. In SPSS statistic program will equal 91.30000. In contrast in MS Excel it equal 91.3 which is rounded to that digit in MS Excel. This function result is rounded without zeroes after decimal point and after first number 3. Average function can be calculated for 4.3000, 4.4000 and 4.5000 the output of SPSS is 4.4000 and the result in MS Excel is 4.4. In SPSS statistic program will equal 91.30000. In contrast in MS Excel it equal 91.3 which is rounded to that digit in MS Excel. This function result is rounded without zeroes after decimal point and after first number 3. stical programs, the SPSS program is more precise than MS Excel especially in counting the decimal plac imal point. In such Statistical programs, the SPSS program is more precise than MS Excel especially in counting the after the decimal point. 5. Discussion In SPSS statistic program will equal 91.30000. In contrast in MS Excel it equal 91.3 which is rounded to that digit in MS Excel. This function result is rounded without zeroes after decimal point and after first number 3. A f i b l l d f 4 3000 4 4000 d 4 5000 h f SPSS i 4 4000 d h l i MS Average function can be calculated for 4.3000, 4.4000 and 4.5000 the output of SPSS is 4.4000 and the result in MS Excel is 4.4. In such Statistical programs, the SPSS program is more precise than MS Excel especially in counting the decimal places after the decimal point. In such Statistical programs, the SPSS program is more precise than MS Excel especially in counting the decimal places after the decimal point. 159 Journal of Advanced Computer Science & Technology This digit: 2.3000000 is indicates how many precise after decimal point is want, example for that, if want two decimal points the result will be 2.30 while the in the MS Excel in 2.3. If wanted is four decimal places the result will be 2.3000 and also in the MS Excel the result is 2.3. In MS Excel there is no significant zeros are displayed correctly when rounding a number to a certain number of significant digits. g g ed French, 2014 [4] in his article entitled: Excel Round Function, he said that Excel normally rounding th e right of the rounding digit determines whether the rounding digit will be rounded up or down. Ted French, 2014 [4] in his article entitled: Excel Round Function, he said that Excel normally roun the right of the rounding digit determines whether the rounding digit will be rounded up or down. Ted French also said that MS Excel follows some rules like SPSS program rules when doing the rounding: the first is if the value of the number to the right of the rounding digit is less than five, the rounding digit is left unchanged. The second is if the value of the number to the right of the rounding digit is five or higher, the rounding digit is raised by one. According to Tim Birkett on Online Excel Tutorial, 2006 [5]: in MS Excel, Round function takes two arguments first the number to be rounded while the second is the number of decimal places to round to. 6. Conclusion From the statistic programs are SPSS, SAS and STAT as well as Microsoft Excel programs that can by it done many statistical operations and many functions. These functions are common between such as these statistical programs and the same rules are followed. Round function in SPSS and MS Excel is shared. The ten samples digits numbers which are less than 5, more than 5 and equal 5, are all the same outputs got when applying rounding in both SPSS and MS Excel. The same round is gotten with the same results. A SPSS statistical program is more precise than MS Excel when it truncates any zero after decimal point in the digit number. Wherever decimal place is wanted after specific decimal point in the digit will be truncated in Microsoft Excel. Example for that in this research is: 21.34000 rounded to 21.34, while it wanted the fourth decimal place to round. In case of calculate some other functions like sum and average also the same results will get after done the function with truncate the zeroes after decimal point whatever number of zeros are there. Based on SPSS that it is more precise and more support in précised results in the calculations. According to what the researcher wants that could assign the decimal place to round the digit number statistically within the range of precision that the researcher wants. 5. Discussion He said also to prevent the format to do not displaying three zeros based on this research outputs, it is suggested to put comma like this: #,###,; 7. Acknowledgment Thank to everyone help me and was helpful to finish this research. I hope that this research is very useful for who looking something about applying functions in SPSS and MS Excel and to go through such as like this scientific researching in IT. I hope to do more researches in IT and CS scope in the future. [1] French Ted, Rounding Numbers in Excel, the Excel 2007 ROUND function, 2014. Website: spreadsheets.about.com/od/excel2007/qt/excel2007_round.htm [2] Poes, Joseph Matthew, Truncation of values after Decimal when exporting to Excel, 2011. Website: spssx- discussion.1045642.n5.nabble.com/Truncation-of-values-after-Decimal-when-exporting-to-Excel-td5068824.html [3] Matthew Hesson, Reporting statistics in APA Style, A Short guide to handling numbers and statistics in APA Style, 2014. Website: my.ilstu.edu/~mshesso/apa_stats.htm [4] F h T d E l R d F ti di b i E l 2014 W b it [5] Tim Birkett, Rounding, Online Excel Tutorial, 2006. accountingweb.co.uk/topic/excel-faqs-rounding/493946 References spreadsheets.about.com/od/excel2007/qt/excel2007_round.htm [2] Poes, Joseph Matthew, Truncation of values after Decimal when exporting to Excel, 2011. Website: spssx- discussion.1045642.n5.nabble.com/Truncation-of-values-after-Decimal-when-exporting-to-Excel-td5068824.html [3] Matthew Hesson, Reporting statistics in APA Style, A Short guide to handling numbers and statistics in APA Style, 2014. Website: my.ilstu.edu/~mshesso/apa_stats.htm [5] Tim Birkett, Rounding, Online Excel Tutorial, 2006. accountingweb.co.uk/topic/excel-faqs-rounding/493946 Tim Birkett, Rounding, Online Excel Tutorial, 2006. accountingw
https://openalex.org/W795677123	https://www.regepe.org.br/regepe/article/download/24/20	Portuguese	null	A Identidade Empreendedora no Contexto de Empresas de Pequeno Porte	Revista de Empreendedorismo e Gestão de Pequenas Empresas	2,013	cc-by	11,615	A IDENTIDADE EMPREENDEDORA NO CONTEXTO DE EMPRESAS DE PEQUENO PORTE Angelo Brigato Ésther – UFJF1 Isabella Stroppa Rodrigues – FACSUM/SENAI-JF2 Elaine Santos Freire - FACC/UFJF3 Angelo Brigato Ésther – UFJF1 Isabella Stroppa Rodrigues – FACSUM/SENAI-JF2 Elaine Santos Freire - FACC/UFJF3 Resumo: As publicações acerca da identidade empreendedora ainda são incipientes. Nesse sentido, utilizaram-se conceitos-chave da psicologia social, notadamente da teoria da identidade social e da teoria da categorização social, bem como a perspectiva de Ciampa, especialmente a ideia de que as identidades são, antes de tudo, políticas e, portanto, construídas num contexto de relações sociais e de poder. Foi conduzida uma pesquisa com quatro sujeitos, proprietários de suas empresas, com o objetivo de compreender como eles constroem sua identidade empreendedora, privilegiando-se o ponto de vista do sujeito a partir de sua experiência vivida. Foi realizada a análise temática das entrevistas. Os resultados apontam contradições e fragmentações nos discursos dos entrevistados, porém coerentes com a perspectiva de Ciampa de que o Eu de cada empreendedor, como pessoa, não precisa e nem pode, limitar-se a uma única definição de identidade. Palavras-chaves: Empreendedorismo; Identidade; Sujeito Empreendedor. 1 E,mail: angelo.esther@ufjf.edu.br. Endereço: Universidade Federal de Juiz de Fora - Campus Universitário, s/nº - Bairro Martelos - CEP: 36.036-900. - Juiz de Fora – MG- 2 E.mail: isabellastroppa@gmail.com 3 E.mail: emaildaelaine@yahoo.com.br ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 90 ENTREPRENEURIAL IDENTITY IN THE CONTEXT OF SMALL FIRMS Abstract: The publications about the entrepreneurial identity are still incipient. Accordingly, we used the key concepts of social psychology, especially the social identity theory and the theory of social categorization, as well as the perspective of Ciampa, especially the idea that the identities are, first of all, political and, therefore, built in a context of social relations and power. This research was conducted with four subjects, owners of their businesses, with the goal of understanding how they construct their entrepreneur identities, giving special attention to the point of view of the subject, from his own experience. The thematic analysis was applied to interviews. The results indicate contradictions and fragmentation in the discourse of the interviewees, but consistent with the perspective of Ciampa that the self of each entrepreneur as a person, does not need, cannot, be limited to a single definition of identity. y Keywords: Entrepreneurship; Identity; Subject Entrepreneur. y Keywords: Entrepreneurship; Identity; Subject Entrepreneur. ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 90 90 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 91 representações, com os sistemas de identificação e com os significados que os sujeitos emprestam à sua ação. Nessa perspectiva – interpretativa –, foi realizada uma pesquisa com quatro sujeitos proprietários-gerentes de suas empresas, com o objetivo de compreender como eles constroem sua identidade empreendedora. Para tanto, o artigo está dividido em seções, além desta introdução. Num primeiro momento, é discutido o conceito de identidade, seguido da análise das principais concepções/representações acerca do que é empreendedor. Em seguida, é descrito o percurso metodológico. A partir daí são discutidos os casos empíricos, seguidos das conclusões e das referências utilizadas. Espera-se, dessa forma, contribuir para o melhor entendimento acerca do “fenômeno” do empreendedorismo. Tal perspectiva, portanto, parte da premissa de que “a identidade de uma pessoa [...] pode ser o último refúgio qualitativo num mundo invadido por exércitos empunhando réguas e compassos” (ALBERT, 1998, p. 3). Introdução É de verificar-se que as publicações acerca do empreendedorismo são crescentes, em grande parte devido ao fato de que o tema suscita discussões acaloradas sobre possíveis caminhos para o desenvolvimento econômico. Nesse aspecto, ressalta-se a perspectiva ou abordagem econômica à questão, que tem como foco de atenção o papel que o empreendedor desempenha nas empresas, notadamente as de pequeno porte. A contribuição central de seu papel reside na possibilidade de inovação, considerada, portanto, o motor do desenvolvimento. Em função de seu apelo econômico, e tendo em vista sua possibilidade de aplicação, surgiram outras abordagens que passaram a privilegiar não apenas a dimensão econômica, mas também a comportamental. Seu foco de análise reside principalmente nas características individuais que parecem garantir o sucesso do negócio. Embora nunca se tenha demonstrado que a posse de certos traços de personalidade garante a ação empreendedora, a abordagem comportamental tende a buscar características comportamentais como fator explicativo. Com o tempo, novos estudos surgiram e o campo se expandiu. No entanto, mesmo considerando o amplo espectro de possibilidades de estudos e pesquisas (ver FILLION, 1999b), a literatura acerca da identidade empreendedora ainda é incipiente. Embora possa ser classificada como pertencente ao campo do comportamento organizacional, a pesquisa sobre identidade ultrapassa a fronteira, buscando elementos interpretativos, privilegiando-se o ponto de vista do sujeito, a partir de sua experiência vivida. Assim, a identidade diz respeito aos significados construídos pelos sujeitos, num determinado contexto de ação ou mundo social particular (STRAUSS, 1997). Dessa forma, nem sempre as autodefinições correspondem aos papéis atribuídos ou aos estereótipos e tipologias pré-estabelecidas. É assim que o estudo acerca da identidade se diferencia do estudo do comportamento, que é parcela mais visível do agir em sua atividade cotidiana. A identidade tem a ver com as ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 91 91 91 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 92 Identidade Não se pode olvidar que ao se discutir o conceito de identidade, está se discutindo a questão: “quem é você?”. Em geral, a resposta do indivíduo a essa pergunta se dá em forma de uma representação ou categorização do tipo: “eu sou professor”, “eu sou engenheiro, gerente da empresa”, etc.. Embora seja aparentemente simples responder a pergunta, o conceito de identidade é excessivamente complexo, pouco desenvolvido e minimamente compreendido na ciência social contemporânea para ser definitivamente posto à prova (HALL, 2004). Da mesma forma, para Strauss, o conceito de identidade é tão esquivo quanto o senso que toda pessoa tem de sua própria identidade pessoal, ainda que a definição da identidade esteja relacionada às avaliações decisivas sobre si mesmo, realizadas pelo indivíduo ou pelo outro (STRAUSS, 1997). De outro lado, essa ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 92 92 92 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 93 pergunta suscita um dilema central: o que a pessoa “é” constitui uma essência ou algo construído ao longo de sua trajetória? Tal dilema ainda não foi definitivamente resolvido, pelo menos no campo das ciências sociais – embora atualmente predomine a segunda perspectiva. Nessa esteira, as abordagens sobre o tema se dividem entre aquelas “essencialistas” e aquelas “não essencialistas” (CERULO, 1997). A primeira é compatível com a concepção de sujeito do Iluminismo e da sociologia clássica, para as quais o indivíduo possuía uma substância ou núcleo essencial, ainda que adaptável (HALL, 2004). Segundo Cerulo (1997), enquadram- se nessa perspectiva os estudos de Weber, Marx e Durkheim, sobretudo no que tange às identidades coletivas. A perspectiva não essencialista não admite tal essência ou núcleo essencial imutável. Ao contrário, admite que o sujeito possua várias identidades contraditórias e mutáveis. Segundo Hall (2004), essa é a concepção do sujeito segundo a abordagem pós-moderna. Também nessa direção, Gioia (1998) identifica três grandes concepções acerca da identidade: a funcionalista, a interpretativa e a pós-moderna. É interessante destacar que, embora a lente funcionalista considere a identidade como um fato, uma variável manipulável, ela não necessariamente é essencialista. No entanto, consideram a possibilidade de mudança bastante difícil. A lente interpretativa adota uma perspectiva subjetivista, segundo a qual a identidade é uma construção social (CERULO, 1997; GIOIA, 1998), não constituindo uma essência sob hipótese alguma. Por fim, a lente pós-moderna, tal como colocado por Hall (2004), adota uma perspectiva subjetivista ao extremo, entendendo a identidade como uma manifestação linguística e mesmo como uma ilusão (GIOIA, 1998), rejeitando qualquer essencialismo (CERULO, 1997). Strauss (1999) mostra que qualquer indivíduo ou grupo acaba por desenvolver uma “linguagem especial”, um dialeto ou jargão, que representa sua maneira de identificar aqueles objetos importantes para a ação do grupo. Essa terminologia comum ou partilhada leva em consideração que a direção da ação depende da forma pela qual os objetos são classificados. Em outras palavras, “em ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 93 93 93 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 94 todo evento é a definição do que o objeto „é‟ que permite a ocorrência da ação com referência ao que se supõe ser” (STRAUSS, 1999, p. 40). todo evento é a definição do que o objeto „é‟ que permite a ocorrência da ação com referência ao que se supõe ser” (STRAUSS, 1999, p. 40). Ainda para o autor, não é necessário que a pessoa tenha certeza de que sabe o que esse objeto é, ela deve apenas estar disposta a arriscar seu julgamento. Como adota uma perspectiva temporal-histórica, o autor entende que, ao viverem e sofrerem suas experiências, as pessoas mudam suas avaliações e classificações. Os valores atribuídos aos objetos se modificam com o tempo. Em resumo, Strauss (1999) vê um indivíduo como parte de um sistema interativo dinâmico, em que não se pode considerar a questão da identidade individual sem se levar em conta a identidade coletiva. Assim, elabora uma ideia de identidade dinâmica associada ao desempenho de diferentes papéis articulados a vivências específicas em mundos sociais particulares, rejeitando uma visão estática da identidade ao estabelecer relações relevantes entre a biografia individual e os processos sociais. Admite-se, portanto, uma multiplicidade de identidades que, embora possam vir a ser duradouras, não se tratam de uma essência imutável. Ao contrário, os indivíduos se apresentam como uma totalidade, como portadores de múltiplos papéis, estabelecendo-se uma “intrincada rede de representações que permeia todas as relações, onde cada identidade reflete outra identidade, desaparecendo qualquer possibilidade de se estabelecer um fundamento originário para cada uma delas” (CIAMPA, 1991, p. 65). A identidade assume várias formas e, nesse sentido, a identidade que emerge como representação do estar-sendo se converte num pressuposto do seu ser, o que formalmente transforma sua identidade concreta (caracterizada como movimento temporal) em identidade abstrata, num dado temporal, sempre presente (identidade pressuposta). ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 94 O existir humanamente não está garantido de antemão, nem é uma mudança natural. Ao contrário, o homem não está limitado no seu vir-a-ser em função de um fim preestabelecido, nem está liberado das condições históricas em que vive, como se seu vir-a-ser fosse absolutamente indeterminado. Portanto, a questão da identidade remete a um projeto político. O homem não é puramente subjetividade e ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 94 94 94 respectivamente, estimulam e destacam os aspectos mais significativos da experiência em determinado contexto, e fazem com que os indivíduos se definam de forma positiva de acordo com as normas e estereótipos da categoria a que pertencem, em comparação a outros grupos (HOGG; TERRY, 2001). respectivamente, estimulam e destacam os aspectos mais significativos da experiência em determinado contexto, e fazem com que os indivíduos se definam de forma positiva de acordo com as normas e estereótipos da categoria a que pertencem, em comparação a outros grupos (HOGG; TERRY, 2001). A Teoria da Categorização Social (TCS), estendendo a TIS, concebe a identidade como decorrente da socialização e destaca o processo de prototipagem. Cognitivamente, as pessoas representam os atributos dos estereótipos dos grupos em forma de protótipos, que não são uma mera lista, mas antes, um conjunto fuzzy de atributos que captura os aspectos do grupo de pertença na forma de representações de membros exemplares. Os protótipos incorporam todos os atributos que caracteriza o grupo e os distingue dos outros, incluindo crenças, sentimentos e comportamentos. Eles maximizam as semelhanças e as diferenças entre os grupos, definindo os grupos como entidades. Eles se formam de acordo com o princípio do metacontraste: maximização da razão das diferenças intergrupos para diferenças intragrupo. Por causa dos membros de um mesmo grupo estarem expostos a informações sociais similares, seus protótipos usualmente serão similares e, portanto, compartilhados (HOGG; TERRY, 2001). consciência, tampouco é apenas uma coisa, apenas uma objetividade. Est dualidade deve ser superada pela práxis, engajando-se em consciência, tampouco é apenas uma coisa, apenas uma objetividade. Esta dualidade deve ser superada pela práxis, engajando-se em consciência, tampouco é apenas uma coisa, apenas uma objetividade. Esta dualidade deve ser superada pela práxis, engajando-se em [...] projetos de coexistência humana que possibilitem um sentido de história como realização de um porvir a ser feito com outros [...] projetos que não se definam aprioristicamente por um modelo de sociedade e de homem, que todos deveriam sofrer totalitariamente (e identicamente), mas projetos que possam tender, convergir ou concorrer para a transformação real de nossas condições de existência, de modo que o verdadeiro sujeito humano venha à existência” (CIAMPA, 1991, p. 73-74). Ciampa (1991, p. 74-75; 2001) conclui que “identidade é movimento, é desenvolvimento do concreto. Identidade é metamorfose”. Mas, ao se considerar ontologicamente a identidade como uma totalidade, do ponto de vista metodológico, tal perspectiva é problemática quando se quer investigar empiricamente a construção da identidade de indivíduos concretos. Da mesma forma, em termos práticos, os indivíduos adotam determinadas identidades conforme o contexto em que atuam. É o que se pode chamar, na terminologia da TIS (Teoria da Identidade Social), de identidade saliente, dentre as múltiplas identidades que um indivíduo pode assumir. Nesse sentido, pode-se falar em identidade empreendedora, considerando-se o mundo social particular do empreendedorismo, e ainda de uma identidade como uma instabilidade adaptativa (GIOIA; SCHULTZ; CORLEY, 2000), uma vez que os indivíduos constroem e reconstroem suas identidades num mundo social particular ou num contexto específico de ação. Portanto, a identidade empreendedora pode ser entendida como parte de uma identidade “total”, no sentido da totalidade, segundo Ciampa, (1991), confrontada com outras identidades, por vezes contraditórias. ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 95 Para a TIS, as categorizações sociais (nacionalidade, afiliação política, etc.) provêm de uma definição onde alguém é em termos das características descritivas da categoria. Assim, a identidade é parte do autoconceito do indivíduo. De forma a explicar o comportamento grupal, a TIS articula os processos sociocognitivos de categorização (categorization) e autovalorização (self-enhancement) que, 95 95 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 96 Empreendedor: As Políticas de Identidade Baseando-se em Habermas, Ciampa (2006) faz uma importante distinção entre a identidade do eu e a identidade de papel. Esta última pode ser entendida como convencional na medida em que alguém é apenas portador de um papel, por exemplo, tal conduta é de meninos e outra de meninas, ou de homens e de mulheres. Muitas vezes, tais identidades de papel (atribuídas e reproduzidas coletivamente) sugerem a existência de uma identidade a priori ou uma essência identitária. Daí a expressão identidade convencional. Por outro lado, a identidade “pós-convencional”, na perspectiva de Habermas, é aquela que se define pelo processo e não por um conteúdo a priori. De todo modo, a identidade de papel diz ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 96 96 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 97 respeito a uma dimensão coletiva que remete a uma discussão fundamental, apontada por Ciampa (2006): política de identidade e identidade política. respeito a uma dimensão coletiva que remete a uma discussão fundamental, apontada por Ciampa (2006): política de identidade e identidade política. As políticas de identidade buscam, de uma forma ou de outra, normalizar ou homogeneizar uma coletividade, levando-a a fazer com que seus membros compartilhem significados que são considerados relevantes para dar sentido à atividade de cada um. Assim, uma identidade coletiva – identidade profissional, por exemplo –, pode ser considerada como um conjunto de significados compartilhado por muitas pessoas que interfere de modo significativo na formação pessoal de cada um. De maneira geral, quanto mais antiga e estável uma profissão, maior será o peso da tradição na manutenção e reprodução de um “perfil” profissional sobre indivíduos e grupos que não correspondam a esse perfil. Os grupos mais conservadores serão os maiores defensores do perfil convencional. Então, à medida que um grupo ou mais exercem a hegemonia em termos de políticas de identidade, esse poder irá determinar ou influenciar o modo pelo qual cada pessoa se reconhece e é reconhecida (CIAMPA, 2006). Ao mesmo tempo, toda identidade é política – dimensão individual. Ainda no caso de uma identidade profissional, o indivíduo se define pelo desempenho de um papel profissional, que é uma convenção. Se ficar muito restrita a um conteúdo que define este papel, essa convenção pode vir a determinar ou constranger a identidade do eu numa forma convencional, adequando o indivíduo a um modelo, um perfil ou uma formatação. Como não se vive isoladamente no mundo, as relações sociais são normatizadas para integrar expectativas recíprocas. Daí o conceito de personagem. Cada indivíduo é um ator ao desempenhar um papel socialmente determinado, mas também pode ser o autor, ao construir seu personagem mais ou menos criativamente, de modo a não ficar restrito a ator. Para Ciampa (2006), a noção de personagem contém sempre uma dose de transgressão, que deixa de obedecer ao convencional. No fundo, é uma busca de emancipação. Assim, a questão fundamental é, conforme Ciampa (2006), como a política identitária de um grupo poderia interferir na formação do indivíduo de forma que este ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 97 97 97 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 98 participe coletivamente como membro do grupo e, ao mesmo tempo, seja capaz de atuar como alguém que expressa sua singularidade neste cenário de intersubjetividade. participe coletivamente como membro do grupo e, ao mesmo tempo, seja capaz de atuar como alguém que expressa sua singularidade neste cenário de intersubjetividade. Ao se discutir a identidade empreendedora há que se analisar inicialmente, a identidade do papel de empreendedor, ou seja, quais são os elementos e critérios de avaliação e julgamento que normalizam ou homogeinizam a coletividade de empreendedores, e quais são os significados compartilhados por essa coletividade. Num primeiro momento, a principal dificuldade é, portanto, definir o que é ser um empreendedor. Segundo Filion (1999b), são pioneiros os economistas e os comportamentalistas nessa tentativa. Os economistas relacionam o empreendedor à inovação, enquanto os comportamentalistas focam a criação e a intuição dos empreendedores. Para Filion, Schumpeter lançou as bases para o campo do empreendedorismo relacionado à inovação. Além disso, ele utiliza a ideia do empreendedor como uma das explicações do desenvolvimento econômico, tomando como referência o trabalho de Jean Baptiste Say que, por sua vez, baseava-se nos trabalhos dos fisiocratas no contexto da Revolução Industrial (FILION, 1999b). Nos termos de Dolabela (2006, p. 17), [...] o profissional agente do desenvolvimento tem de ser capaz de inovar, ser especialista no que não existe. É este o know how do empreendedor. Ser preparado para conceber sistemas, não para operá-los ou, pior ainda, “pajear” processos industriais já depreciados no primeiro mundo. [...] o profissional agente do desenvolvimento tem de ser capaz de inovar, ser especialista no que não existe. É este o know how do empreendedor. Ser preparado para conceber sistemas, não para operá-los ou, pior ainda, “pajear” processos industriais já depreciados no primeiro mundo. Por sua vez, os comportamentalistas têm se empenhado em tentar definir o empreendedor e suas características. Nessa abordagem, entende-se que a posse de certas características inatas faria de um indivíduo um empreendedor. Como exemplo, os tradicionais estudos sobre liderança até agora não possibilitaram estabelecer um perfil comportamental do empreendedor (VERSIANI; GUIMARÃES, 2004). Vale dizer que alguns autores afirmam que a personalidade é um fator ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 98 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 99 Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 98 98 Como se pode perceber, as principais escolas enfatizam ora os aspectos econômicos, ora os comportamentais. Além disso, percebe-se uma tentativa de aproximar o empreendedor às figuras do líder e do gerente. Convém destacar que, nessa direção, os estudos acabam por se concentrar nas características comportamentais como elementos de comparação e diferenciação. Para Filion (2000) em particular, o que melhor distingue o empreendedor do gerente e do pequeno empresário parece recair no desenvolvimento e na implementação de sua visão. Segundo o autor, Gerentes e pequenos empresários buscam atingir metas e objetivos a partir dos recursos disponíveis, dentro de uma estrutura predefinida ou copiada. Os empreendedores, por outro lado, gastam boa parte de seu tempo imaginando aonde querem chegar e como/farão para chegar lá. De alguma forma, os empreendedores são detectores de espaços de mercado e criadores de contextos. Uma vez detectada a oportunidade, a visão fornece diretrizes para a implementação do plano mestre (FILLION, 1999a, p. 12). A literatura também não é unânime quanto à questão se o empreendedor é aquele que detém a propriedade de uma empresa ou não. Drucker (1987), por exemplo, também associa o empreendedor e o empreendimento à questão da inovação. Para ele, o simples fato de uma pessoa criar o seu próprio negócio não quer dizer que ela está empreendendo. Para ser empreendedora, a pessoa precisa apresentar algumas características especiais. Segundo o autor, o indivíduo apresenta um espírito empreendedor a partir do momento em que cria algo novo, diferente, que muda ou transforma valores. Quando considerada a escola do intraempreendedorismo, o empreendedor não possui a propriedade da empresa, mas pode-se dizer que ele é o “proprietário” ou “dono” simbólico do projeto, produto ou ideia. É o caso de Art Fry, por exemplo, que criou o “post it”, produto de grande sucesso comercial da 3M. ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas v 1 n 2 2012 100 Ao que parece, pelo menos no campo da economia e da gestão, a perspectiva da inovação parece ser o fundamento das definições atuais dominantes. No entanto, a inovação é redefinida por Filion (1991) – ao associá-la a uma visão –, Ao que parece, pelo menos no campo da economia e da gestão, a perspectiva da inovação parece ser o fundamento das definições atuais dominantes. determinante na formação de um empreendedor, mais do que o sucesso empresarial (UTSCH et al., 1999). Outras tentativas de definição surgiram com o tempo. Segundo Cunningham e Lischeron (1991), há pelo menos seis “escolas” de pensamento sobre o empreendedor. O quadro 1 resume suas principais características, combinando a interpretação central, o pressuposto básico que a orienta, as habilidades e os comportamentos do empreendedor, conforme a classificação dos autores. Quadro 1 - Escolas do Pensamento Sobre Empreendedorismo. Escola Interpretação central Pressuposto Habilidades e comportamento do empreendedor Grande figura O empreendedor possui intuição, traços e instintos inatos A intuição inata é a chave do sucesso Intuição, vigor, energia, perseverança e autoestima Características psicológicas O empreendedor possui valores, atitudes e necessidades únicas que o impulsiona As pessoas agem orientadas pelos seus valores e o comportamentos resulta de tentativas para satisfazer necessidades Valores pessoais, propensão a aceitação do risco e necessidade de realização Clássica A característica básica do empreendedor é a inovação As pessoas contrapõem o fazer ao possuir Inovação, criatividade e descoberta Administrativa O empreendedor organiza, possui e administram empreendimentos econômicos, cujos riscos são assumidos Transformar pessoas em empreendedores por meio de treinamento de funções e técnicas da administração Planejamento, organização, direção e controle Liderança O empreendedor é um líder, com a capacidade de adaptar o seu estilo às necessidades das pessoas Para atingir seus objetivos, o empreendedor depende de outros Capacidade de motivação, orientação e liderança Intraempreen- dedorismo As habilidades do empreendedor podem ser úteis em organizações complexas; o intraempreendedor cria unidades independentes para a geração, comercialização e expansão dos negócios A adaptação é fundamental para a sobrevivência das organizações complexas. O empreendedorismo resulta na criação de organizações e na transformação de empreendedores em administradores Capacidade de estar alerta às oportunidades; maximização das decisões F t Ad t d d C i h Li h (1991) Quadro 1 - Escolas do Pensamento Sobre Empreendedorismo. p g Fonte: Adaptado de Cunningham e Lischeron (1991). ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 99 99 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 100 No entanto, a inovação é redefinida por Filion (1991) – ao associá-la a uma visão –, ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 100 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 100 para quem o empreendedor é uma pessoa que imagina, desenvolve e realiza visões. Uma visão implica algo novo que motivará os membros da organização e atrairá o interesse do mercado. É através da visão que o empreendedor projeta uma imagem do futuro, do lugar que deseja que seu produto venha a ocupar no mercado. Segundo o autor: [...] os empreendedores são originalmente produtos do sistema de relações da família, que depois desenvolvem uma rede de relacionamentos empresariais de modo que as pessoas nela envolvidas se tornam produtos sociais de que o empreendedor precisa que venha a ser, de modo a realizar as suas visões (FILION, 1991, p. 59). Esta definição possui a vantagem de não se deter no lugar-comum de explicar o sucesso do empreendedor a partir de traços de sua personalidade. Por outro lado, observa-se que o empreendedor é tido como alguém capaz de articular uma rede de relacionamentos empresariais. Daí emergem duas questões. Em primeiro lugar, o empreendedor está associado a um negócio, a uma forma de sustento. Tal definição implica, de certo modo, a propriedade de um negócio, pois se trata de realizar uma visão (a “sua” visão, e a não a de outros). Dolabela (2006, p. 16) assume essa perspectiva, ao afirmar que o [...] empreendedor é alguém que provê, de forma independente, o próprio sustento. Ele não é demissível por ato de poder. É alguém, portanto, com certo grau de liberdade. Como oferece valor positivo para os outros, não está submetido a idiossincrasias e arbítrios de uma pessoa, especificamente. É interdependente da comunidade que utiliza os valores que oferece. ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 101 Em segundo lugar, as pessoas que pertencem à rede de relacionamentos empresariais são entendidas como produtos sociais necessários ao empreendedor, para que ele realize sua visão. Nesse sentido, ressalta-se a dimensão política. O empreendedor é entendido como aquele indivíduo que projeta e desenha um futuro e, para alcançar este futuro, deve agir de tal modo e não de outro, o que implica tomar determinadas posições e decisões ao longo do tempo. É nesse sentido que o Em segundo lugar, as pessoas que pertencem à rede de relacionamentos empresariais são entendidas como produtos sociais necessários ao empreendedor, para que ele realize sua visão. ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 101 Nesse sentido, ressalta-se a dimensão política. O empreendedor é entendido como aquele indivíduo que projeta e desenha um futuro e, para alcançar este futuro, deve agir de tal modo e não de outro, o que implica tomar determinadas posições e decisões ao longo do tempo. É nesse sentido que o ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 101 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 101 101 101 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 102 conceito de embeddedness (GRANOVETTER, 1985) emerge como central na discussão do empreendedorismo, ao colocar que a ação econômica está inserida num contexto social, e que a ação dos indivíduos é condicionada pelos relacionamentos que esse indivíduo possui, seja em termos relacionais (relacionamento com outras pessoas), seja em termos estruturais (toda a rede de relacionamentos de um indivíduo). As características apontadas pela literatura por vezes dificultam o estabelecimento de uma definição precisa entre o que significa empreendedor, ainda que autores como Fillion (1999b) defenda a ideia de que, pelo menos num mesmo campo de estudo, possa ser observada grande convergência. Entretanto, observando as dimensões anteriormente identificadas, nota-se que a ênfase em uma ou outra dimensão se concentra em determinada escola ou abordagem. Atualmente, a corrente predominante vem se esforçando em criar uma definição de empreendedor atribuindo-lhe um papel fundamental e muitas vezes excessivo, no processo de desenvolvimento econômico e social de um país. O crescimento dos eventos relacionados ao tema, as pesquisas internacionais – como o GEM (Global Entrepreneurship Monitor), por exemplo – e algumas instituições – como o SEBRAE – contribuem para a disseminação da ideia, criando um imaginário bastante peculiar. Assim, ao empreendedor é atribuída uma identidade de papel relacionada ao desenvolvimento econômico de um país, região ou cidade, com determinado perfil comportamental e postura peculiares, geralmente diferenciadas em relação a outras figuras e agentes relevantes do mundo econômico e organizacional, como as próprias grandes empresas, administradores, gerentes, dentre outros. Diante do exposto, a presente pesquisa buscou identificar como os indivíduos – proprietários de pequenas empresas – constroem suas identidades empreendedoras. Para tanto, foi preciso, primeiro, identificar qual a concepção de empreendedor daqueles indivíduos. Antes de se discutir os resultados, serão descritos os passos metodológicos. ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 102 102 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 103 utilizada a análise de conteúdo (BARDIN, 1970), em particular a análise temática, segundo os procedimentos propostos e utilizados por Melo (2000, 2001): utilizada a análise de conteúdo (BARDIN, 1970), em particular a análise temática, segundo os procedimentos propostos e utilizados por Melo (2000, 2001): a) Preparação. As entrevistas foram transcritas integralmente segundo o roteiro de entrevista utilizado, agrupando-se as respostas de cada categoria para as respectivas questões. b) Tabulação quantitativa. Identificação dos conteúdos das respostas e agrupamento segundo o tema da pergunta. Foram recuperadas as informações relativas aos objetivos da pesquisa, por meio de agrupamento de dados. c) Tabulação qualitativa. A partir da definição dos temas segundo os objetivos da pesquisa, foram selecionados extratos significativos das entrevistas, destacando- se a dimensão da fala dos entrevistados, sendo possível, desta forma, analisar o conjunto das referências ao tema e às suas ramificações. Essa técnica privilegia, portanto, os conteúdos dos relatos, sendo estes confrontados com os conceitos utilizados, permitindo-se avançar no conhecimento acerca da realidade específica dos empreendedores. Resultados: A Construção da Identidade Empreendedora Para se discutir a identidade empreendedora, os resultados foram agrupados segundo determinadas categorias. Num primeiro momento, são apresentadas as definições que os empreendedores fornecem acerca de si mesmos e do que significa ser empreendedor (“definições de si e de empreendedor”). Em seguida, são discutidas as categorias: “a trajetória dos indivíduos”, “a forma como vivenciam a ação empresarial e o contexto de ação”. Metodologia Ao se investigar como o sujeito da ação constrói sua identidade, privilegia-se a abordagem interpretativa, segundo a qual a identidade é uma construção social e simbolicamente construída que empresta sentido à experiência (GIOIA, 1998). Pressupõem-se sujeitos dotados de uma consciência prática e reflexividade que lhes permite explicar suas ações e os possibilita “seguir em frente” (GIDDENS, 2002). Dessa forma, o objetivo é compreender o sistema de significados dos sujeitos pesquisados. De acordo com essa abordagem, a identidade é derivada de repetidas interações com os outros e, embora seja imputada por meio de valores expressos, sua interpretação não é necessariamente fixa, podendo significar coisas diferentes para grupos diferentes em momentos diferentes (GIOIA; SCHULTZ; CORLEY, 2000). É nesse sentido que a pesquisa qualitativa e seus métodos partem da perspectiva ou das ações do sujeito estudado num dado momento. Como ponto de partida, considerou-se que os sujeitos da pesquisa deveriam ser, necessariamente, proprietários de suas empresas. Desprezou-se, deste modo, a possibilidade de se investigar o intraempreendedor, preferindo-se compreender a dinâmica identitária daqueles empreendedores segundo a concepção econômica clássica. Igualmente, buscaram-se indivíduos que, pelo menos potencialmente, demonstravam inclinação para a inovação e que tivessem um sucesso comercial ou mercadológico relativamente significativo na cidade. Nessa pesquisa, foram entrevistados quatro proprietários de três pequenas empresas – sendo dois sócios de uma mesma empresa. Uma delas é do segmento de segurança eletrônica, outra do segmento de panificação e a última de promoção de festas. Todos os envolvidos concordaram verbalmente com a gravação das entrevistas, tendo sido assegurado o sigilo quanto à identificação. As entrevistas foram do tipo episódica (FLICK, 2003), a qual permite grande flexibilidade ao sujeito da pesquisa discorrer sobre os temas abordados. Na análise das entrevistas foi ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 103 103 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 104 atividades que desempenham em seu dia a dia. No entanto, nenhum dos entrevistados se definiu como empreendedor: atividades que desempenham em seu dia a dia. No entanto, nenhum dos entrevistados se definiu como empreendedor: Eu sou o faz tudo, eu sou comprador, sou motorista, eu sou é... contas a pagar, departamento de credito e cobrança, eu sou tudo, porque minha empresa é pequena, não tem ainda uma estrutura, né, de funcionário pra cuidar do financeiro, pra cuidar de logística, pra cuidar de administrativo (...) Eu faço, coloco a mão na massa quando precisa, entendeu? De ir pra cozinha, tanto aqui como lá, eu faço, ajudo na administração, faço de tudo, eu não, eu sou uma pessoa centralizadora. Eu sou, eu não sou uma pessoa que, eu vou te confessar, é, tem um lado aí da, burocrático da coisa, eu às vezes até errada, entendeu? Mas eu sou assim, eu gosto de ver as coisas perto, eu gosto de tá participando, de tá acontecendo, então eu sou uma pessoa que participa de tudo (...) o resultado do meu trabalho, porque eu sinto que eu sou uma pessoa assim que, eu levei muito tempo pra construir um nome e eu prezo é o nome, eu acho que assim, eu fico feliz de ver que as pessoas têm segurança no que eu faço (...) o nome que eu construí isso pra mim é tudo. Eu sou muito de fase, muito de momento, de necessidade do momento (...) tô participando diretamente da minha parte, nesse ano agora, da minha parte administrativa, operacional, to diretamente ligado a isso, eu senti necessidade de vir pra cá, teve período que eu senti necessidade de ficar só na frente da loja, hoje eu não faço mais nada, eu mal venho aqui dentro, teve período que eu senti necessidade na minha produção, se precisar eu faço tudo também. Como se pode observar pelos depoimentos, são ressaltados principalmente os aspectos operacionais de suas atividades cotidianas. Nesse sentido, a definição de um empreendedor como alguém que cria e viabiliza uma visão inovadora sequer é insinuada. Definições de Si e de Empreendedor ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Ao serem solicitados a se definirem, os sujeitos se apresentam em termos não profissionais – idade, formação escolar, etc. – para em seguida, abordarem as Ao serem solicitados a se definirem, os sujeitos se apresentam em termos não profissionais – idade, formação escolar, etc. – para em seguida, abordarem as ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 104 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 105 Ao contrário, embora também não se definam como tal, os entrevistados acabam enfatizando aspectos relativos ao trabalho gerencial, no sentido daquele indivíduo que se preocupa muito mais com a rotina e com a resolução de problemas, em meio a um dia a dia turbulento e repleto de demandas de toda ordem, tal como identificado na literatura acerca do trabalho gerencial (por exemplo, MINTZBERG, 1973). ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 105 105 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 106 “crença no que faz”, o serem “apaixonadas”, o “tá no meu sangue” e assim por diante. O depoimento a seguir exemplifica essa situação: “crença no que faz”, o serem “apaixonadas”, o “tá no meu sangue” e assim por diante. O depoimento a seguir exemplifica essa situação: (...) sou bravo quando tem que ser bravo, sou amigo quando tem que ser amigo, dou porrada quando tem que dar porrada... é verdade mesmo, dou soco na mesa, grito o Diabo a quatro quando as coisas não dão certo, sabe? Me tira o humor completamente se eu pego uma coisa errada (...) eu brigo muito pela qualidade, sou muito exigente, sou muito estressado nesse ponto, sabe? Sou exemplo para todo mundo aqui em termos de superação, em termos de... sabe? De vontade, de busca constante, pra tá melhorando, por acreditar. Então, tudo isso eu acho que eu dou esse exemplo aqui dentro. Ao se referir a “idealizar uma coisa” e buscar alcançá-la, e estar sempre “renovando” a empresa, os entrevistados tangenciaram o papel inovador do empreendedor, o que implica que os indivíduos certamente possuem uma concepção de empreendedor em consonância com o discurso dominante, embora acabem ressaltando os aspectos comportamentais. Dito de outro modo, o imaginário acerca do que é ser empreendedor é permeado por um perfil mais ou menos idealizado, ou seja, o da grande figura. Do mesmo modo, percebe-se que ao se darem conta de que as entrevistas abordavam o tema, os indivíduos tentaram descrever o que significa ser empreendedor em termos das características que eles acreditam possuir. Tal atitude sugere que os indivíduos não se definem nem se identificam com uma definição comum de empreendedor. Ao contrário, parece haver uma espécie de “atribuição a posteriori”, de modo a não se verem nem serem vistos como alguém que “não pertence” ao mundo do empreendedorismo. O depoimento a seguir ilustra esse ponto: ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 107 Eu nunca parei para pensar assim, eu nunca falei isso. Mas ultimamente eu tenho escutado muito isso. As pessoas têm me falado muito isso. Entendeu? (...) Eu nunca me considerei isso não. Eu sempre fiz isso com muita normalidade (...) a partir do momento que fui intitulado como empreendedor, eu comecei a ver as coisas de outra maneira. No entanto, quando perguntados sobre o que significa ser empreendedor, os entrevistados não foram unânimes em suas respostas. Interessante observar que, do mesmo modo que a literatura não é unânime quanto ao que significa ser empreendedor (ver Quadro 1), os sujeitos da pesquisa também não denotam uma definição única entre eles. Ao contrário, as definições acerca do que é ser empreendedor variam: Ser empreendedor é você idealizar uma coisa, colocar em prática e acreditar muito naquilo. E não ter medo daquilo não funcionar. É fazer com muita convicção de que aquilo vai dar certo. E que você vai ter muito sucesso para aquilo (...) não é uma pessoa que faz as coisas muito planejadas. Faz meio na coragem. É uma pessoa que faz as coisas muito na coragem, muito no impulso. E que tem boas idéias... Ser empreendedor é tá sempre mudando, buscando coisas novas, tá renovando a sua empresa a todo momento, você não consegue ficar parado, é você sempre acreditar no seu negócio, ter perseverança, eu acho que o mais importante é isso, você ter perseverança e você renovar toda hora São apaixonadas pelo que fazem, a maioria, o problema é que todos que eu conheço gostam muito do que fazem. Muita coragem, não têm medo de arriscar eu acho que, primeiro, eu acho que a pessoa tem que conhecer muito daquilo que ele vai fazer, isso é muito importante” Eu acho que em primeiro lugar é acreditar naquilo que ela ta fazendo, eu acho que quando você acredita e você tem confiança em você mesmo, você passa isso pras pessoas, e você tem um sonho e você acredita naquilo, você não sentir influência de ninguém. Só vontade, só vontade. Depreende-se da leitura, que os entrevistados, embora tenham se concentrado nos aspectos comportamentais, de certo modo apontaram características que tendem a descrever o empreendedor em termos da “grande figura” ou em termos da posse de certas características psicológicas ou comportamentais. Daí ser destacada a “coragem”, a “vontade”, a “perseverança”, a ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 106 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 106 106 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 107 Eu nunca parei para pensar assim, eu nunca falei isso. Mas ultimamente eu tenho escutado muito isso. As pessoas têm me falado muito isso. Entendeu? (...) Eu nunca me considerei isso não. Eu sempre fiz isso com muita normalidade (...) a partir do momento que fui intitulado como empreendedor, eu comecei a ver as coisas de outra maneira. No inicio ou só me considerava um virão, fazia aquilo pra me virar. A partir do momento que a gente, que eu percebi... a me ver como empreendedor eu passei a ter mais responsabilidade com minhas coisas. Esse nome que eles dão para a gente, essa coisa de empreendedor, eu me senti como empresário porque antes eu me sentia um virão e passei a me sentir mais um empresário. O indivíduo se definia como um “virão”, ou seja, aquele que “se vira”, que faz de tudo para fazer algo dar certo. Até então, o rótulo de empreendedor não lhe era relevante. Continuando o depoimento, o indivíduo afirma que, em função da identidade empreendedora que lhe era imputada por outros, ele passou a se “ver como empreendedor”. Assim, a política de identidade é evocada pelos outros indivíduos da rede de relacionamentos do sujeito, levando-o a se redefinir. No entanto, não basta a auto atribuição de empreendedor. O indivíduo tem de passar a agir como tal. Daí o entrevistado ter se referido a “passei a ter mais responsabilidade com minhas coisas.” Em outras palavras, ele passa a agir de acordo com o protótipo que lhe é atribuído. De todo modo, percebe-se que a auto atribuição da identidade empreendedora não é a constante entre os sujeitos entrevistados. Ao contrário, eles apenas procuraram equiparar a definição de si à de empreendedor quando essa palavra foi mencionada pelo entrevistador. Nesse sentido, ressalta-se o aspecto metodológico utilizado, que procurou favorecer o ponto de vista do sujeito da ação, não lhe atribuindo tal definição antecipadamente. Tal situação é, de certo modo, corroborada pelos depoimentos quanto a suas trajetórias de vida e a forma como vivenciam seu dia a dia. No inicio ou só me considerava um virão, fazia aquilo pra me virar. A partir do momento que a gente, que eu percebi... a ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 107 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 108 A Trajetória dos Indivíduos Aos entrevistados foi solicitado que descrevessem sua trajetória de vida. Todos mencionaram o fato de terem aberto seus negócios em uma cidade diferente de sua cidade natal. Além disso, três deles afirmaram que abriram suas empresas em um ramo diferente daquele que atuavam anteriormente em sua carreira ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 108 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 108 108 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 109 e, dentre estes, todos já experimentaram a sensação de um negócio que não deu certo. De modo geral, atribuem ao fato de o negócio não ter dado certo à falta de planejamento, por terem trabalhado sozinho, por falta de organização ou por terem investido mais do que deviam. Todos tiveram a participação de sócios em seu primeiro negócio por razões financeiras ou legais. e, dentre estes, todos já experimentaram a sensação de um negócio que não deu certo. De modo geral, atribuem ao fato de o negócio não ter dado certo à falta de planejamento, por terem trabalhado sozinho, por falta de organização ou por terem investido mais do que deviam. Todos tiveram a participação de sócios em seu primeiro negócio por razões financeiras ou legais. Os entrevistados ao serem questionados sobre suas motivações para se tornarem empreendedores, citaram a busca por independência financeira, ou por necessidade, ou pela estagnação profissional enquanto empregado. Na prática, os motivos são diversos, não havendo uma constância ou unanimidade. Nenhum dos entrevistados ressaltou, por exemplo, o traço da identidade de papel do empreendedor como aquele que contribui para o desenvolvimento econômico e social do país, região ou cidade. Ao contrário, suas motivações foram, basicamente, de ordem individual e não coletiva. Em síntese, as trajetórias são bastante distintas e, evidentemente, são decisivas na construção da identidade individual. No entanto, apenas duas características são comuns a todos em suas trajetórias: a existência de um sócio num primeiro momento e o fato de terem tido experiência profissional e gerencial anterior. profissional. Três deles também afirmaram ter iniciado sua vida profissional muito cedo, entre os nove e doze anos de idade. profissional. Três deles também afirmaram ter iniciado sua vida profissional muito cedo, entre os nove e doze anos de idade. Quando eu tinha 12 anos eu montei uma fábrica de chup-chup, vocês falam chup-chup, né? Na minha terra a gente falava big-bang, e eu tive dez meninos que trabalharam pra mim, eu tinha uma fabriquetazinha mesmo. Dentre os quatro entrevistados, dois vieram de famílias com mais de quatro irmãos e perderam o provedor da família muito cedo. Somente um deles veio de família rica que perdeu tudo, e teve de ajudar a se reerguer. Quando questionados sobre a influência da educação e da família em sua vida profissional, apenas um entrevistado afirmou que essa influência foi relevante. Os outros afirmaram que esses aspectos pouco influenciaram na sua carreira. Um deles ressalta a importância familiar em sua trajetória: Foi muito forte, muito forte, é, por dois fatores: primeiro que eu comecei a trabalhar muito cedo, minha mãe nunca, minha mãe foi uma mãe diferente, uma mãe que desde pequenininha trabalhou fora, e pros padrões da época era uma coisa totalmente fora, né? Então isso sempre me deixou uma pessoa super responsável, eu nunca tomei um pau, eu sempre fui entendeu? Estudiosa, isso, quer dizer, foi uma lição de vida da minha mãe, porque eu aprendi a ter responsabilidade, ter limite, tudo, muito cedo. E eu tive uma vida muito boa, tive uma educação muito boa, porque meu pai vivia muito bem com a minha mãe, entendeu? Então tem o fato de você ter essa influência, ter um ambiente familiar propício pra você estabelecer seu, um futuro, suas metas, isso pra mim foi muito importante. Tive, o fator educação foi tudo na minha vida, tudo. Segundo os entrevistados, a educação formal contribuiu pouco para sua trajetória profissional, mas todos ressaltaram a experiência gerencial quando da abertura de seus primeiros negócios. Talvez, essa experiência explique o viés da definição de empreendedor a partir do desempenho de atividades operacionais e administrativas. Quanto à trajetória profissional atuando como dono do seu próprio negócio, três dos entrevistados tiveram outros negócios antes do que comandam atualmente ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 109 109 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 110 Formas como Vivenciam a Ação Empresarial e seu Contexto de Ação Relativamente ao seu dia a dia, os entrevistados alegam que o gosto pelo trabalho, a presença de pessoas boas à sua volta e canais de comunicação transparentes dentro da empresa, facilitam seu trabalho. Como elementos que dificultam suas atividades, os respondentes apontaram a alta carga tributária. Mas o principal destaque foi a alegação de pessoas desonestas na empresa. Além disso, destacaram como elemento dificultador os conflitos com clientes e empregados. ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 110 Essas constatações corroboram o que as pesquisas em geral apontam sobre a gestão de pequenas empresas: as imposições governamentais – em particular os Essas constatações corroboram o que as pesquisas em geral apontam sobre a gestão de pequenas empresas: as imposições governamentais – em particular os ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 110 tributos e impostos – bem como as dificuldades de gerenciamento (SEBRAE..., 2004). Em função das dificuldades de gerenciamento, os entrevistados apontaram que por vezes, tomam decisões equivocadas. No entanto, afirmam encarar os erros e fracassos com naturalidade: tributos e impostos – bem como as dificuldades de gerenciamento (SEBRAE..., 2004). Em função das dificuldades de gerenciamento, os entrevistados apontaram que por vezes, tomam decisões equivocadas. No entanto, afirmam encarar os erros e fracassos com naturalidade: ...os erros... todo mundo erra, todo negócio tem erro, todo, por mais bem sucedido que seja o negócio, ele sempre tem erro, tá? ...os erros... todo mundo erra, todo negócio tem erro, todo, por mais bem sucedido que seja o negócio, ele sempre tem erro, tá? ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 111 citaram a participação frequente em cursos e palestras, bem como visitas às empresas do mesmo segmento. Apenas um dos sujeitos da pesquisa afirma que aprende com todas as situações do cotidiano, fazendo da sua vida um laboratório. citaram a participação frequente em cursos e palestras, bem como visitas às empresas do mesmo segmento. Apenas um dos sujeitos da pesquisa afirma que aprende com todas as situações do cotidiano, fazendo da sua vida um laboratório. Por fim, um dos entrevistados afirmou que encara os erros como uma dádiva de Deus. Eu tenho assim plena certeza de que tudo foi uma, uma dádiva de Deus pra mim aprender, sabe? E ter assim mais segurança no futuro, eu acho que assim... acho que nada na vida é por acaso, eu acho que a gente tem que passar e tem que ver que foi pra melhorar (...) Eu tenho assim plena certeza de que tudo foi uma, uma dádiva de Deus pra mim aprender, sabe? E ter assim mais segurança no futuro, eu acho que assim... acho que nada na vida é por acaso, eu acho que a gente tem que passar e tem que ver que foi pra melhorar (...) Nesse sentido, convém ponderar que, embora aos entrevistados não se tenha formulado nenhuma questão diretamente relacionada à dimensão religiosa, é importante destacar sua influência, pois seu sistema simbólico pode oferecer explicações, ações e atitudes a serem tomadas pelos indivíduos. ...os erros... todo mundo erra, todo negócio tem erro, todo, por mais bem sucedido que seja o negócio, ele sempre tem erro, tá? Corroborando o que o perfil comportamental estabelecido para o empreendedor preconiza, dois entrevistados descreveram o erro como uma oportunidade de aprendizado não só para ele, mas para todos na empresa que passaram pela situação. Entretanto, aprender com erros não é uma prerrogativa exclusiva de empreendedores. É inerente ao processo de aprendizagem. Então, há uma evolução, há um aprendizado com tudo isso, há um aumento de responsabilidade, eu acho que todo mundo fica mais responsável depois que acontece tudo isso, porque sente a vulnerabilidade de perto e, é... dá até uma certa tranqüilidade, se não houver uma nova intempérie, um novo intempérie pela frente, né, mas, se houver ... a gente já sabe pelo menos os caminhos que têm que seguir pra conseguir achar o... o... o... o ponto de equilíbrio. Ao mesmo tempo, dois entrevistados afirmam que teriam cometido menos erros caso tivessem estudado mais. (...) Eu falo que talvez se eu tivesse feito curso na época, algumas coisas teriam sido mais fáceis, o caminho teria sido mais curto, menos doloroso, né? Porque pra você aprender na prática, às vezes da muita cabeçada, né? Erra muito e tal (...) ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 111 Observa-se, pelo menos segundo o depoimento anterior, não uma contradição, mas uma constatação interessante: como afirmado pelos entrevistados em geral, a formação escolar não constituiu fator decisivo para a abertura do negócio. Entretanto, sua deficiência parece fazer falta na atual condução de suas empresas. Nesse sentido, os empresários acabam por apontar a necessidade de profissionalização, aqui representada principalmente pela qualificação. Em geral, ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 111 111 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 112 Considerações Finais ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 112 As entrevistas com os proprietários de empresas de pequeno porte identificaram uma autorrepresentação diferente daquela que, a princípio, se esperava. Ou seja, os indivíduos não se identificaram primariamente como empreendedores. Ao contrário, afirmaram que não se viam como tal, embora lhes fosse atribuída tal identidade. Esperava-se que eles se definissem como empreendedores em função das empresas que dirigem: elas são de pequeno porte, relativamente conhecidas e representativas de seus segmentos na cidade em que atuam. Da mesma forma, para o senso comum, seus proprietários-dirigentes são tidos como empreendedores. Observa-se ainda que, quando os entrevistados perceberam que não estavam se definindo como empreendedores, em função das perguntas da entrevista, eles modificaram sua estratégia de expressão, direcionando seu discurso em direção àquilo que eles consideravam ser um empreendedor, ou seja, eles tentaram reverter sua autodefinição, definindo o que é ser empreendedor a partir As entrevistas com os proprietários de empresas de pequeno porte identificaram uma autorrepresentação diferente daquela que, a princípio, se esperava. Ou seja, os indivíduos não se identificaram primariamente como empreendedores. Ao contrário, afirmaram que não se viam como tal, embora lhes fosse atribuída tal identidade. Esperava-se que eles se definissem como empreendedores em função das empresas que dirigem: elas são de pequeno porte, relativamente conhecidas e representativas de seus segmentos na cidade em que atuam. Da mesma forma, para o senso comum, seus proprietários-dirigentes são tidos como empreendedores. ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto Observa-se ainda que, quando os entrevistados perceberam que não estavam se definindo como empreendedores, em função das perguntas da entrevista, eles modificaram sua estratégia de expressão, direcionando seu discurso em direção àquilo que eles consideravam ser um empreendedor, ou seja, eles tentaram reverter sua autodefinição, definindo o que é ser empreendedor a partir ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 112 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 112 112 ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 113 daquilo que eles pensam que são. Assim, “ter boas ideias”, “ter coragem”, “ter perseverança”, dentre outros, passam a ser sinônimo de empreendedor, mesmo que anteriormente eles tenham se definido como “o faz-tudo” e aquele que “põe a mão na massa”. Em outras palavras, ao perceberem que se definiam como indivíduos “comuns” – ainda que bem sucedidos –, quando confrontados com o ideal de empreendedor, os entrevistados se redefiniram de modo a se fazer pertencerem ao mundo social do empreendedorismo, da inovação, da modernidade e do sucesso empresarial. Embora os indivíduos não se definam como empreendedores, aqueles à sua volta tendem a atribuir-lhe a identidade empreendedora. Da mesma forma, é digno de nota que a atribuição da identidade empreendedora levou pelo menos um deles a se interrogar sobre quem ele é, fazendo-o imaginar inclusive que talvez seja “mesmo” um empreendedor. De certo modo, essa situação faz parecer, pelo menos aos olhos do indivíduo e de quem lhe atribui a identidade, de que ele é “no fundo”, ou “em essência”, um empreendedor. Se não há uma essência identitária, então as atribuições de identidade compõem parte do processo de construção da identidade, como também fazem parte de uma política de identidade. Nesse processo, as categorias sociais assumem uma importância capital, pois fornecem uma referência para o autoconceito dos indivíduos. A categoria social “empreendedor” é composta de um conjunto de atributos, que envolve pressupostos, interpretações, valores e comportamentos. Por não se tratar de uma categoria tradicional (comparativamente à de médico, por exemplo), ainda não é totalmente consolidada, possuindo atributos diferentes conforme a “escola” de referência, pelo menos no campo da teoria acerca do empreendedorismo. Na prática cotidiana, porém, as diversas representações se misturam e se confundem, embora continuem a fornecer um referencial para os indivíduos. Para cada conjunto de atributos corresponde um protótipo, no qual os indivíduos podem tomar como referência. ÉSTHER, A. B.; RODRIGUES, I.S.; FREIRE, E. S. A identidade empreendedora no contexto das empresas de pequeno porte. Revista de Empreendedorismo e Gestão de Pequenas Empresas, v.1, n.2, 2012. 113 113 No caso dos entrevistados, nenhum deles denotou um padrão claro e inequívoco do que é ser empreendedor. Ao que parece, eles estão vivenciando um período de reflexão acerca de quem são, baseados em estereótipos que lhes vem sendo fornecidos pela e dentro de suas redes de relacionamento. Pode-se afirmar, inclusive, que um ou outro não se define como empreendedor o que, certamente, contraria os estudos mais tradicionais sobre empreendedores, sobretudo aqueles que têm como foco de análise os padrões mais comportamentais. 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https://openalex.org/W2150623039	https://academicjournals.org/journal/AJAR/article-full-text-pdf/EA8F89A38755.pdf	English	null	Storage of crambe fruit subjected to different drying conditions	African journal of agricultural research	2,012	cc-by	5,584	Accepted 8 October, 2012 This study aimed to determine the effect of drying conditions and storage time on crambe seed quality. Crambe fruits with a moisture content of 27.0 ± 1.0% (wet basis; w.b.) were used. These fruits were dried in a forced-air oven and were kept under controlled temperatures of 35, 45, 60, 75 and 90°C at relative humidities of 20.9, 8.7, 6.8, 4.8 and 2.3%, respectively. During the drying process, trays containing 0.4 kg of fruit were periodically weighed until the final drying point of 7.0 ± 1.3% (w.b.). The fruits were packed in glass jars covered with permeable fabric. Each container had 400 g of fruit, and the containers were kept under ambient conditions (temperature of 26 ± 3°C and relative humidity of 55 ± 12%). The temperature and relative humidity of the environment were recorded by a digital datalogger. The samples were evaluated at 0, 3, 6, 9 and 12 months for moisture content, electrical conductivity (EC), germination percentage and germination speed index (GSI). Drying the crambe fruit at high temperatures caused a decrease in the physiological quality of the seeds. There was an increase in the amount of electrolytes released by the seeds over time. Seeds stored for 12 months at room temperature had superior germination regardless of drying temperature. Key words: Physiological quality, Crambe abyssinica, biodiesel. Corresponding author. E-mail: lmctpg@yahoo.com.br. Tel: (64) 9217-1533. African Journal of Agricultural Research Vol. 7(47), pp. 6274-6280, 13 December, 2012 Available online at http://www.academicjournals.org/AJAR DOI: 10.5897/AJAR12.1627 ISSN 1991-637X ©2012 Academic Journals African Journal of Agricultural Research Vol. 7(47), pp. 6274-6280, 13 December, 2012 Available online at http://www.academicjournals.org/AJAR DOI: 10.5897/AJAR12.1627 ISSN 1991-637X ©2012 Academic Journals Full Length Research Paper Lílian Moreira Costa1, Osvaldo Resende2, Douglas Nascimento Gonç Kaique Alves Ferreira Marçal3 and Juliana de Fátima Sales2 1Federal Institute of Education, Science and Technology of Goiás (Instituto Federal de Educação, Ciência e Tecnologia Goiano – IF Goiano), Rio Verde Campus, GO, Rodovia Sul Goiana, Km 01 - Zona Rural - CEP: 75901-97, Brazil. 2Board of Undergraduate Studies, IF Goiano, Rio Verde Campus, GO, Rodovia Sul Goiana, Km 01 - Zona Rural - CEP: 75901-97, Brazil. , 3PIBIC/CNPq Scholar, IF Goiano, Rio Verde Campus, GO, Rodovia Sul Goiana, Km 01 - Zona Rural - CEP: 75901-97, Brazil. 3PIBIC/CNPq Scholar, IF Goiano, Rio Verde Campus, GO, Rodovia Sul Goiana, Km 01 - Zona Rural - CEP: 75901-97, Brazil. Accepted 8 October, 2012 INTRODUCTION g g ( ) Crambe seeds must be stored and commercialized with a moisture content of up to 10% (w.b.) (Knights, 2002) and cleaned to a maximum of 2% of impurities (Springdale, 2005). If the seeds are harvested with high moisture content, natural or forced ventilation can be used for drying. It is not recommended to dry the seeds with air without heat if the moisture content exceeds 20% (w.b.). To maintain the quality of the seed, the maximum recommended drying temperature is 43°C (Golz, 1993; Knights, 2002). In Brazil, studies related to culture and crambe oil production have been recently undertaken, because domestic producers and researchers have access to the FMS Brilhante cultivar, which is commercialized by the Mato Grosso do Sul (MS) Foundation. When evaluating the production potential of crambe as well as the physicochemical characteristics of the oil and biodiesel obtained from these grains, Jasper (2009) concluded that biodiesel from C. abyssinica Hochst complies with the standards established by Resolution No. 7 of the National Petroleum, Natural Gas and Biofuels Agency (Agência Nacional de Petróleo, Gás Natural e Biocombustíveis, ANP). Crambe seeds are considered orthodox due to their physiological behavior during storage. The ability of the orthodox seed cells to survive desiccation involves the synthesis of proteins known as late embryogenesis abundant (LEA) proteins, which are produced in late embryogenesis, as well as the accumulation of soluble sugars and presence of antioxidants, which allow the cytoplasm to reach the so-called vitreous state (Berjak, 2006). Seeds with this behavior, as characterized by Roberts (1973), are tolerant to desiccation and can be stored with low moisture content in environments with low temperature and relative humidity. ) Crambe is a plant of the Brassicaceae family and is a potential source material for biofuel. The crambe fruit is a silique that is initially green, but has a yellowish color upon maturation, and each fruit contains a single spherical seed that is green or greenish brown (DESAI et al., 1997). This crop has the advantages of high yield, tolerance to drought, tolerance to frost and cycle precocity, which lasts approximately 90 days. Due to these characteristics, crambe can be grown in the winter season if sown immediately after soybean harvest as an alternative to planting the second crop. However, this oleaginous crop will only be successful in Brazilian soil if the productivity levels are satisfactory. INTRODUCTION technical results. However, the decrease in oil prices and the high production cost of biodiesel compared to standard diesel have paralyzed the progress of the commercial use of biodiesel in Brazil and worldwide, but current problems related to the availability of affordable oil and its impacts on the environment have revived research into biodiesel around the world (Osaki and Batalha, 2008). Basic guidelines of the Brazilian Biodiesel Production Program aim to highlight Brazil in the production of renewable fuels, because the country has competitive advantages over other countries, such as land availability for the expansion of agriculture as well as enough water and energy to practice agriculture for energy production in a competitive manner (Benedetti et Biodiesel has proven to be a genuine alternative to replace fossil diesel oil. In Brazil, a proposal to replace fossil fuel for fuels obtained from biomass has existed since 1920. However, it was the oil crisis in the 1970s that led the federal government to create the National Alcohol Program (Programa Nacional do Álcool, Proálcool), which has made the replacement of gasoline with ethanol a reality. Tests performed with different mixing ratios of biodiesel in diesel fuel have shown viable Costa et al. 6275 Costa et al. 6275 Costa et al. Costa et al. 6275 the form of vapor at the same time that air supplies heat to the system. The seeds, which are hygroscopic, undergo changes in their moisture content according to environmental conditions. When the seeds come in contact with air, they perform exchanges until their vapor pressure and temperature have similar values reaching levels of energy, water and thermal balance. However, the temperature of the drying air must be controlled within certain limits, thus avoiding possible physicochemical and biological damage to the seeds (Elias, 2002). al., 2006). Several oleaginous species are being tested or used for the production of biodiesel, such as soy, castor, palm, sunflower, peanut, crambe, oilseed radish, rapeseed and babassu (Mello et al., 2007). Although, Brazil has a great diversity of agricultural products for the production of vegetable oils and biodiesel, many crops still have an extractive nature, with no commercial plantations to assess their real potential. Thus, crambe (Crambe abyssinica) is an option for oil extraction in the Brazilian Midwest. INTRODUCTION To this end, the use of superior quality seeds can significantly contribute to the achievement of production goals (Neves et al., 2007), and the proper drying and storage of the seeds can ensure the quality and performance of the crop in the field. p y Seed survival in storage essentially depends on the moisture content. This reliance can be attributed to the fact that physiological reactions increase quantitatively when the moisture content rises. The identification of adequate moisture content of the seed increases the storage capacity and decreases the risk of deterioration. The metabolic activity and damage caused by insects and microorganisms are influenced by the moisture content in the seed. Deteriorative reactions occur at high moisture contents and can be reduced with low water levels. However, studies have shown that important reactions occur more readily in a dry state than in a wet state and that the nature of these reactions varies with moisture content (Marcos Filho, 2008). Thus, this study aimed to analyze the effect of drying conditions and storage time on the quality of crambe fruit for up to 12 months. Crambe should be dried to no more than moisture content of 10% (wet basis, w.b.) before storage (Oplinger et al., 1991). The drying process partially removes water from the seed by the simultaneous transfer of heat from the air to the seed and by the transfer of mass through water vapor flow from the seed to the air. The drying process is a dynamic process and depends on the relative humidity of the air (Villela and Peres, 2004). In drying, air is used to transport water from the seeds out of the system. Forced hot air is used to heat the seeds, causing the internal water to migrate to the outer surface to be evaporated, thus reducing the moisture content of the mass of the seeds being dried (Peske and Villela, 2003). MATERIALS AND METHODS The relative humidity of the air during the first six months of storage from November to April was higher with an average of 70.69% (Figure 1B), and there was a decreasing trend in the relative humidity in the last months of storage from July to October with an average of 39.82%, due to the variability of the temperature along the year .The variation in the relative humidity and temperature caused changes in the moisture content of seeds, which can affect the physiological quality of the seeds. The temperature and relative humidity of the drying air were monitored by means of a psychrometer installed inside the experimental dryer. The fruits were packed in glass jars (400 g of fruit per jar) covered by voile fabric on October 10, 2010, and the jars were stored for 12 months. During this period, the temperature and relative humidity of the ambient air were recorded by a digital datalogger. The samples (four replicates) were evaluated at 0, 3, 6, 9 and 12 months. The following parameters were measured: moisture content, electrical conductivity (EC), germination percentage and germination speed index (GSI). The EC test was performed according to the methodology described by Vieira and Krzyzanowski (1999). A total of 4 subsamples of 50 fruits were used from each treatment, and the subsamples were weighed on a scale with a resolution of 0.01 g. The samples were placed in plastic cups to soak with 75 ml of deionized water and were kept in a biochemical oxygen demand (BOD) incubator with controlled temperature of 25°C for 24 h. The solutions containing the fruit were slightly shaken for leachate uniformity, which was immediately proceeded by a reading on a portable digital conductivity meter (Instrutherm model CD-850), and the results were divided by the mass of the 50 fruits and the result was expressed in µS cm-1 g-1 of seeds. Table 1 shows the analysis of variance of the evaluated characteristics. The drying temperature and time of storage influenced the physiological quality of the crambe fruit. Table 2 shows the values of moisture content in the crambe fruit under different drying temperatures during storage. The storage of the crambe fruit in glass containers covered with permeable fabric under ambient conditions at a temperature of 26 ± 3°C and relative humidity of 55 ± 12% allowed for variations in moisture content during storage. MATERIALS AND METHODS Changes in air conditions cause constant changes in this variable. At the end of the storage period, there was a decrease in moisture content for all drying temperatures due to the temperature and relative humidity of the air being moderately lower at this time of the year (July to October) (Figure 1B), which made the fruit reach equilibrium with the ambient conditions in which they were stored. p µ g Germination test was conducted with four subsamples of 25 fruits from each treatment. The fruits were packed in Gerbox boxes on blotting paper moistened with distilled water, which was equivalent to 2.5 times the dry substrate mass, to achieve adequate moisture and uniformity of the test. The samples were kept in a Mangelsdorf germinator set at a constant temperature of 25±2°C. The evaluations were performed every two days from the second day after sowing until 32 days were completed according to the criteria established in the Rules for Seed Analysis (Brasil, 2009). The average germination percentage was calculated, and the GSI was calculated as follows: GSI = n1.d1 -1 + n2.d2 -1 + n3.d3 -1... n.dn -1; where n1 is the number of seeds germinated on the first day of counting; n2 is the number of seeds germinated on the second day of counting; n3 is the number of seeds germinated on the third day of counting; nn is the number of seeds germinated on the nth day of counting; d1 is the first day; d2 is the second day; d3 is the third day; and dn is the nth day (Maguiri, 1962). As indicated by the EC values shown in Table 3, there was an increase in the amount of electrolytes released by the seeds over the course of the storage period. The amount of electrolytes released tended to increase more substantially with the fruit that were dried at a temperature of 90°C, thereby confirming the influence of time and drying temperature on the amount of solutes leached into the solution. y g The design was completely randomized according to a split plot design with five controlled drying temperatures (35, 45, 60, 75 and 90°C) in the plots and five evaluation months (0, 3, 6, 9 and 12 months) in the subplots. The averages were compared by Tukey’s test at a 5% significance level. MATERIALS AND METHODS Fruits of the FMS Brilhante cultivar were used, which was developed by the MS Foundation. The fruits were produced in the 2010 crop at the Experimental Field of the Federal Institute of Education, Science and Technology of Goiás, Rio Verde Campus (Instituto Federal de Educação, Ciência e Tecnologia Goiano - IF Goiano - Câmpus Rio Verde) located in Rio Verde-GO at 17° 47’ In artificial drying, air absorbs water from the product in 6276 Afr. J. Agric. Res. 6276 respectively. These values were similar to the Jatropha seed for drying temperature conditions of 30, 40, 50, 60 and 70°C (Ullmann et al., 2010). When drying crambe fruit at temperatures of 30, 40, 50, 60 and 70°C, Costa et al. (2011, 2012a) found that increasing the drying temperature increases the drying rate and decreases the time required for drying when considering a moisture content reduction from 21.0 ± 1.3 to 7.0 ± 1.4% (w.b.). 53’’ latitude (S) and 51° 55’ 53’’ longitude (W). The experiment was conducted in the Post-Harvest Laboratory of Plant Products and Seed Laboratory (IF Goiano- Câmpus Rio Verde). The fruit harvesting was done manually when the moisture content was 27.0 ± 1.0% (w.b.), which was determined by gravimetry using an oven at 105 ± 3°C for 24 h (Brasil, 2009). Crambe drying was performed in an oven with forced ventilation kept at the controlled temperatures of 35, 45, 60, 75 and 90°C and average relative humidities of 20.9, 8.7, 6.8, 4.8 and 2.3%, respectively. During the drying process, trays containing 0.4 kg of fruit were periodically weighed until it reached the final drying point of 7.0 ± 1.3% (w.b.), which has been established for the safe storage of this product. ( ) Figure 1 shows the average monthly values of temperature and relative humidity of the air where the crambe fruits were stored. The average temperature in storage was 24.89 ± 2.63°C with fluctuations over time (Figure 1A). MATERIALS AND METHODS At the beginning of storage, the crambe fruit showed low values of EC for the five drying conditions with values between 0.516 and 0.610 µS cm-1 g-1. At 12 months, however, the fruit that were dried at 45°C showed the lowest EC value, which was equivalent to the initial sample. These results indicated better preservation of the crambe fruit when dried at a temperature of 45°C, which probably kept the cell membranes better organized, thus controlling the release of solute during soaking. This coincides with the study of Knights (2002) in which it was recommended that the seed quality should maintain a RESULTS AND DISCUSSION The drying times of the crambe fruit when considering the reduction of moisture content from 27.0 ± 1.0 to 7.0 ± 1.3% (w.b.) and with drying temperatures of 35, 45, 60, 75 and 90°C were 13.75, 7.75, 5, 3.75 and 3.26 h, Costa et al. 6277 Costa et al. 627 6277 Costa et al. 0 1 2 3 4 5 6 7 8 9 10 11 12 20 22 24 26 28 30 Temperature (ºC) 0 1 2 3 4 5 6 7 8 9 10 11 12 30 35 40 45 50 55 60 65 70 75 80 85 90 Time (months) Relative Humidity (%) A B Time (months) Relative humidity (%) Temperature (°C) Time (months) Figure 1. (A) Average temperature during storage at ambient conditions (24.89 ± 2.63°C). (B) Average relative humidity during storage at ambient conditions (55 ± 12%). 0 1 2 3 4 5 6 7 8 9 10 11 12 20 22 24 26 28 30 Temperature (ºC) A Temperature (°C) 0 1 2 3 4 5 6 7 8 9 10 11 12 20 22 24 26 28 30 Temperature (ºC) 0 1 2 3 4 5 6 7 8 9 10 11 12 30 35 40 45 50 55 60 65 70 75 80 85 90 Time (months) Relative Humidity (%) A B Time (months) Relative humidity (%) Temperature (°C) Time (months) Temperature(ºC) Temperature (°C) 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 30 35 40 45 50 55 60 65 70 75 80 85 90 Time (months) Relative Humidity (%) B Time (months) Relative humidity (%) Time (months) Time (months) Figure 1. (A) Average temperature during storage at ambient conditions (24.89 ± 2.63°C). (B) Average relative humidity during storage at ambient conditions (55 ± 12%). Table 1. Summary of the analysis of variance for moisture content, electrical conductivity (EC), germination and germination speed index (GSI) during storage of the crambe fruit. Table 1. Summary of the analysis of variance for moisture content, electrical conductivity (EC), germination and germination speed index (GSI) during storage of the crambe fruit. *Significant at 1% according to the F test. Significant at 5% according to the F test. NS, Not significant. RESULTS AND DISCUSSION The averages followed by the same lowercase letters in the column and uppercase letters in the rows do not differ according to Tukey’s test at 5% significance. maximum drying temperature of 43.33°C. depth inversely proportional to its age; that is, it is more intense in recently harvested seeds. Thus, the gradual overcoming of dormancy was achieved with storage in which the crambe aged resulting in a higher germination percentage and greater GSI. Tables 4 and 5 show the germination percentage and GSI values of the crambe fruit with different drying temperatures and storage for 12 months in a natural environment. The germination potential was higher in fruit dried at temperatures of 35, 45 and 60°C during storage. During storage, the germination percentage increased regardless of drying temperature up to the third month after which the values varied (Table 4), and a lower germination percentage was observed at high drying temperatures. p g g The crambe seeds that were not stored had low germination percentages in all drying temperatures, thereby indicating that all recently harvested seeds exhibited primary dormancy. This mechanism is common in recently harvested seeds of several species (Brasil, 2009). The study of dormancy in crambe seeds is neces- sary when considering that the germination capacity may increase with storage time. When studying the viability of recently harvested crambe seeds subjected to different drying conditions and moisture contents, Costa et al. (2012a, b) and Faria (2010) observed a low germination percentage. The same behavior was also observed by Oliva (2010) when drying crambe fruit and storing the fruit for eight months in paper bags. In papaya seeds, the germination of seeds derived from recently harvested fruit is low with germination percentages of 40 and 14.75% for fruit harvested in January and September, respectively (Aroucha et al., 2005). Castor bean seeds also exhibit dormancy when recently harvested, but the dormancy breaks after nine months of storage (Lago et al., 1979). Moreover, there was a higher germination index at a temperature of 35°C when compared to other tempe- ratures, and a temperature of 90°C resulted in the lowest germination index (Table 4). Martins et al. (2005) studied papaya seeds from the Formosa group, and they also observed an increase in the germination power between zero and three months of storage and a decrease between three and six months of storage. RESULTS AND DISCUSSION Analyzed variable Variation source Average square CV (%) Moisture content (% w.b.) Drying 0.0002 8.04 Months 0.0054 8.05 Drying × Months 0.0001** EC (µS cm-1 g-1) Drying 0.0257* 6.35 Months 0.0922 4.93 Drying × Months 0.0483 Germination (%) Drying 3354.9650 25.51 Months 2592.0650 19.68 Drying × Months 331.1775 GSI (dimensionless) Drying 12.9207 30.32 Months 15.4527 21.62 Drying × Months 1.7461 *Significant at 1% according to the F test. Significant at 5% according to the F test. NS, Not significant. *Significant at 1% according to the F test. Significant at 5% according to the F test. NS, Not significant. 6278 Afr. J. Agric. Res. Afr. J. Agric. Res. 6278 Table 2. Moisture content (% w.b.) of the crambe fruit subjected to different drying temperatures and during storage under ambient conditions during 12 months. Drying (°C) Storage time (months) 0 3 6 9 12 35 9.12aB 8.35aB 10.55aA 7.03aC 5.77aD 45 9.23aAB 8.37aB 10.09aA 6.97aC 6.17aC 60 7.43bBC 8.26aB 10.44aA 6.85aC 6.37aC 75 7.63bB 7.85aB 10.48aA 6.45bC 5.76aC 90 7.28bB 8.15aB 9.52aA 6.97aB 5.52aC The averages followed by the same lowercase letters in the column and uppercase letters in the rows do not differ according to Tukey’s test at 5% significance. he averages followed by the same lowercase letters in the column and uppercase letters in the rows do not differ cording to Tukey’s test at 5% significance. Table 3. Electrical conductivity (EC; µS cm-1g-1) of the crambe fruit subjected to different drying conditions during 12 months. Drying (°C) Storage time (months) 0 3 6 9 12 35 0.57abcC 0.59bBC 0.59abBC 0.64abAB 0.66abA 45 0 61aA 0 58bAB 0 55bB 0 60bAB 0 60bAB Table 3. Electrical conductivity (EC; µS cm-1g-1) of the crambe fruit subjected to different drying conditions during 12 months. Table 3. Electrical conductivity (EC; µS cm-1g-1) of the crambe fruit subjected to different drying conditions during 12 months. Drying (°C) Storage time (months) 0 3 6 9 12 35 0.57abcC 0.59bBC 0.59abBC 0.64abAB 0.66abA 45 0.61aA 0.58bAB 0.55bB 0.60bAB 0.60bAB 60 0.58abAB 0.59bAB 0.53bB 0.63abA 0.63abA 75 0.52cC 0.61abAB 0.57abB 0.60abAB 0.64abA 90 0.55bcB 0.66aA 0.61aA 0.67aA 0.66aA The averages followed by the same lowercase letters in the column and uppercase letters in the rows do not differ according to Tukey’s test at 5% significance. RESULTS AND DISCUSSION However, it should be emphasized that the drying temperature and time of exposure to high temperature may have also influenced these variables (Marcos Filho, 2005; Kohama et al., 2006). In seeds, there are basically two types of water as follows: free (absorbed) and adsorbed water. The free water moves and is rapidly lost when the seeds are being dried. The adsorbed or retained water is attached to macromolecules, which participate in meta- bolic reactions, and when the adsorbed or retained water According to Marcos Filho (2005), dormancy has a Costa et al. 6279 6279 Costa et al. Table 4. Germination (%) of crambe seeds stored under ambient conditions for up to 12 months. Table 4. Germination (%) of crambe seeds stored under ambient conditions for up to 12 months. Drying (°C) Storage time (months) 0 3 6 9 12 35 27.25aB 65.50aA 55.25aA 51.75abA 58.50abA 45 23.00aC 53.50abAB 51.00aB 61.25aAb 68.00aA 60 22.50aC 49.50abAB 46.75aB 62.25aAB 6400abA 75 23.00aC 46.50bAB 60.75aA 38.50bBC 47.75bAB 90 20.50aA 21.50cA 22.00bA 18.50cA 21.75cA The averages followed by the same lowercase letters in the column and uppercase letters in the rows do not differ according to Tukey’s test at 5% significance. 90 20.50aA 21.50cA 22.00bA 18.50cA 21.75cA The averages followed by the same lowercase letters in the column and uppercase letters in the rows do not differ according to Tukey’s test at 5% significance. The averages followed by the same lowercase letters in the column and uppercase letters in the rows do not differ according to Tukey’s test at 5% significance. Table 5. Germination speed index (GSI) of the crambe fruit subjected to storage for 12 months. Drying (°C) Storage time (months) 0 3 6 9 12 35 1.32aC 3.75aA 2.29aBC 2.97abAB 3.93abA 45 1.08aD 3.00abBC 2.09aCD 3.47aB 4.75aA 60 1.14aD 2.68bBC 1.91aCD 3.54aB 4.86aA 75 1.27aC 2.45bBC 2.50aAB 2.00bBC 3.02bA 90 1.03aA 1.03cA 0.88bA 0.91cA 1.16cA The averages followed by the same lowercase letters in the column and uppercase letters in the rows do not differ according to Tukey’s test at 5% significance. Table 5. Germination speed index (GSI) of the crambe fruit subjected to storage for 12 months. The storage of the crambe fruit for 12 months at room temperature promoted the break of seed dormancy, thereby increasing the germination percentage. is lost by drying, it can cause a decrease in seed germination (Marcos Filho, 2005). ACKNOWLEDGEMENTS The authors thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)-Brazilian National Agency for the Support and Evaluation of Graduate Education, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)-National Council for Scientific and Technological Development and IF Goiano for the financial support. When comparing these results with the moisture content (Table 2), a direct relationship was observed between these parameters (Tables 2 to 5), that is, both the germination percentage and GSI varied over time for the analyzed treatments. Thus, when the moisture content in seeds was higher, there was a decrease in germination resulting in a lower GSI. REFERENCES Aroucha EMM, Silva RF, Oliveira JG, Viana AP, Gonzaga MP (2005). Época de colheita e período de repouso dos frutos de mamão (Carica papaya L.) cv. Golden na qualidade fisiológica das sementes [Harvest period and rest period of the papaya fruit (Carica papaya L.) cv Golden on the physiological quality of the seed]. Ciência. Rural. 35(3):537-543, Benedetti O, Plá JA, Rathmann R, Padula AD (2006). Uma proposta de modelo para avaliar a Viabilidade do biodiesel no Brasil [A proposed model to evaluate the viability of biodiesel in Brazil]. Teoria e Evidência. Econ. Ed.Especial.14:81-107, RESULTS AND DISCUSSION g ( ) According to Silva and Vieira (2006), the GSI is included among the best-known vigor tests. The GSI test is easily performed, because data collection is performed during the germination test. The germination speed test considers that lots with faster germinating seeds are more vigorous, and therefore, there is a direct relationship between germination speed and seed vigor. Thus, as with germination percentage, the GSI was higher with these drying temperatures (between 35 and 90°C). 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Brasília, DF: Mapa/ACS, 2009. p. 3 g p p p Costa LM, Resende O, Gonçalves DN, Rodrigues E.; Sousa KA, Sales JF, Donadon JR (2012a).The influence of drying on the physiological quality of crambe fruits. Acta Scientiarum. Agron. Maringe. 34(2):213- 218, Costa LM, Resende O, Gonçalves DN, Rodrigues E.; Sousa KA, Sales JF D d JR (2012 ) Th i fl f d i th h i l i l Neves MB, Trzeciak MB, Vinholes PS, Tillman AC, Villela FA (2007). Qualidade fisiológica de sementes de crambe produzidos em MatoGrosso do Sul [Physiological quality of crambe seeds produced in Mato Grosso do Sul]. In: Simpósio Estadual de Agroenergia, 2007, Pelotas, RS. Anais do Simpósio Estadual de Agroenergia. Pelotas, RS: EMBRAPA. pp. 97-98. Costa LM, Resende O, Gonçalves DN, Sousa KA(2012b). Qualidade dos frutos de crambe durante o armazenamento. Revista Brasileira de Sementes, Viçosa. 34(2):239-301, Costa LM, Resende O, Sousa KA, Gonçalves DN (2011). Coeficiente de difusão efetivo e modelagem matemática da secagem de sementes de crambe. Revista Brasileira de Engenharia Agrícola e Ambiental, Campina Grande 15(10):1089-1096. Oliva ACE (2010). Qualidade de sementes de crambe submetidas à métodos de secagem e períodos de armazenamento [Quality of crambe seeds subjected to drying methods and storage times]. 78f. Thesis (Masters) - Universidade Estadual Paulista, Faculdade de Ciências Agronômicas [School of Agronomic Sciences, São Paulo State University – UNESP], Botucatu. Desai BB, Kotecha PM, Salunkhe DK (1997). Seeds handbook: biology, production processing and storage. New York: Marcel Dekker. p. 627. Osaki M, Batalha OM (2008). Produção de biodiesel e óleo vegetal no Brasil: realidade e desafio [Production of biodiesel and vegetable oil in Brazil: reality and challenge]. Conclusions Qualidade das sementes de pinhão manso submetidas à secagem em diferentes condições de ar [Quality of jatropha seeds subjected to drying under different air conditions]. Revista Ciência Agronômica. Fortaleza 41(3):442-447. Lago AA, Zink E, Razera LF, Banzatto NV, Savy F (1979). A. Dormência em sementes de três cultivares de mamona [Dormancy in seeds of three cultivars of castor bean]. Bragantia 38:41-44, Vieira RD Krzyzanowski FC (1999). Teste de condutividade elétrica [Electrical conductivity test]. In. Krzyzanowski, F.C.; Vieira RD, França Neto, JB, (Eds.). Vigor de sementes: conceitos e testes. Londrina, PR: ABRATES, Cap. 04:1-26. Maguiri JD (1962). Speed of germination-aid in selection and evaluation for seedling emergence and vigor. Crop Sci. Madison. 2(1):176-177. Marcos FJ (2008). Water relations in seeds. Available at: <http://www.ag.ohio-state.edu/~seedbio/hcs631.htm>. Accessed on: Apr. 14. Villela FA, Peres WB (2004). Coleta, secagem e beneficiamento de sementes [Collection, drying and processing of seeds]. In. Ferreira, A.G.; Borguetti, R. Germinação: do básico ao aplicado. Porto Alegre: ARTMED. pp. 265-281. p Marcos FJ (2005). Fisiologia de sementes de plantas cultivadas [Physiology of seeds of cultivated plants]. FEALQ, Piracicaba. p. 495. Martins GN, Silva RF, Araujo, EF, Pereira MG, Vieira HD, VIANA AP (2005). Influência do tipo de fruto, peso específico das sementes e período de armazenamento na qualidade fisiológica de sementes de mamão do grupo Formosa [Influence of the fruit type, specific weight of seeds and storage time on the physiological quality of papaya seeds of the Formosa group]. Revista Brasileira de Sementes 27(2):12-17.
https://openalex.org/W2811109491	http://www.etamaths.com/index.php/ijaa/article/download/1724/380	English	null	Stability Conditions of a Class of Linear Retarded Differential Systems	International journal of analysis and applications	2,018	cc-by	5,770	∗Corresponding author: sudeger@kastamonu.edu.tr Abstract. In this paper, we give some new necessary and suﬃcient conditions for the asymptotic stability of a linear retarded diﬀerential system with two delays x′ (t) + (1 −a) x (t) + A (x (t −k) + x (t −l)) = 0, t ≥0, where a < 1 is a real number, A is a 2 × 2 real constant matrix, and k, l are positive numbers such that k > l. 1. Introduction and preliminaries Key words and phrases. diﬀerential equations; characteristic equation; asymptotic stability. ⃝ A h c ⃝2018 Authors retain the copyright International Journal of Analysis and Applications Volume 16, Number 4 (2018), 454-461 URL: https://doi.org/10.28924/2291-8639 DOI: 10.28924/2291-8639-16-2018-454 International Journal of Analysis and Applications Volume 16, Number 4 (2018), 454-461 URL: https://doi.org/10.28924/2291-8639 DOI: 10.28924/2291-8639-16-2018-454 of their papers, and all open access articles are distributed under the terms of the Creative Commons Attribution License SERBUN UFUK DE ˘GER1,∗AND YAS¸AR BOLAT2 SERBUN UFUK DE ˘GER1,∗AND YAS¸AR BOLAT2 1Institute of Sciences, Kastamonu University, Kastamonu, TURKEY 2Department of Mathematics, Faculty of Art & Science, Kastamonu University, Kastamonu, TURKEY ∗Corresponding author: sudeger@kastamonu.edu.tr Received 2018-03-09; accepted 2018-05-09; published 2018-07-02. 2010 Mathematics Subject Classiﬁcation. 39A13, 39A30. 2010 Mathematics Subject Classiﬁcation. 39A13, 39A30. phrases. diﬀerential equations; characteristic equation; asymptotic stability. of their papers, and all open access articles are distributed under the terms of the Creative Commons Received 2018-03-09; accepted 2018-05-09; published 2018-07-02. tics Subject Classiﬁcation. 39A13, 39A30. c ⃝2018 Authors retain the copyrights of their papers, and all open access articles are distributed under the terms of the Creative Commons Attribution License. 454 ; p ; p 2010 Mathematics Subject Classiﬁcation. 39A13, 39A30. of their papers, and all open access articles are distributed under the terms of the Creative Commons Attribution License. 454 Key words and phrases. diﬀerential equations; characteristic equation; asymptotic stability. c ⃝2018 Authors c ⃝2018 Authors re Received 2018-03-09; accepted 2018-05-09; published 2018-07-02. 454 1. Introduction and preliminaries Retarded diﬀerential equations are a type of diﬀerential equation in which the derivative of the unknown function at a certain time is given in terms of the values of the function at previous times. Stability of these equation has a wide range of applications in science and engineering. Recently, These equations have been investigated by many authors; for example, Matsunaga [1], Cooke and van den Driessche [2], Kuang [3] , Cooke and Grossman [4], Ruan and Wei [5], Hale and Lunel [6], Khokhlovaa, Kipnis and Malygina [8], Cerm´ak and J´ansky [9], Hrabalova [10], Nakajima [11], Hara and Sakata [12], Smith [13], Freedman and Kuang [14] and Bellman and Cooke [15] which have studied the asymptotic stability of linear retarded 454 Int. J. Anal. Appl. 16 (4) (2018) Int. J. Anal. Appl. 16 (4) (2018) 455 diﬀerential equations. In this paper, we give some new necessary and suﬃcient conditions for the asymptotic stability of the following system diﬀerential equations. In this paper, we give some new necessary and suﬃcient conditions for the asymptotic stability of the following system x′ (t) + (1 −a) x (t) + A (x (t −k) + x (t −l)) = 0, t ≥0, (1.1) (1.1) where a < 1 is a real number, A is a 2 × 2 real constant matrix, and k, l are positive numbers such that k > l. System (1.1) is called a retarded or delay diﬀerential system if the highest derivative term does not have a delay.The characteristic equations of retarded diﬀerential equations are polynomials. These polynomials are exponential polynomials or quasi-polynomials as named in Bellman and Cooke [15]. We know that for the linear retarded diﬀerential equation, the zero solution being asymptotically stable is equivalent to all solutions having limit zero as t →∞which in turn is true if and only if all roots of the associated characteristic equation have negative real parts. The purpose of this paper is to obtain new results for the asymptotic stability of zero solution of system (1.1) when A is a constant matrix. Now we will give some basic information that we use for the lemmas. If we get x (t) = Py (t) for a regular matrix P in (1.1), then we obtain the following system; y′ (t) + (1 −a) y (t) + P −1AP (y (t −k) + y (t −l)) = 0, t ≥0. 1. Introduction and preliminaries Thus, matrix A can be given in one of the following two matrices in Jordan form[7]: (I) A =  q1 p 0 q2  , b1, b2 and p are real constants, (II) A = q  cos θ −sin θ sin θ cos θ  , q, θ are real constants and \|θ\| < π 2 . (I) A =  q1 p 0 q2  , b1, b2 and p are real constants, (II) A = q  cos θ −sin θ  , q, θ are real constants and \|θ\| < π 2 . (I) A =  q1 p 0 q2  , b1, b2 and p are real constants, (II) A = q  cos θ −sin θ sin θ cos θ  , q, θ are real constants and \|θ\| < π 2 . Here we discuss the case (II), the other case should be discussed similarly. The characteristic equation of system (1.1) is given as Here we discuss the case (II), the other case should be discussed similarly. The characteristic equation of system (1.1) is given as F(λ) := det λI2 + (1 −a) I2 + A e−λk + e−λl = 0, (1.2) (1.2) where I2 is the 2 × 2 identity matrix. By the case (II), we have F(λ) as follows where I2 is the 2 × 2 identity matrix. By the case (II), we have F(λ) as follows F(λ) ≡fθ (λ) fθ λ = 0, where where fθ (λ) = λ + (1 −a) + q e−λk+i\|θ\| + e−λl+i\|θ\| , fθ (λ) = λ + (1 −a) + q e−λk+i\|θ\| + e−λl+i\|θ\| , and λ is the complex conjugate of any complex λ. Note that fθ λ = 0 implies fθ λ = 0. and λ is the complex conjugate of any complex λ. Note that fθ λ = 0 implies fθ λ = 0. Int. J. Anal. Appl. 16 (4) (2018) 456 lie in the left half of the complex plane. lie in the left half of the complex plane. Since fθ is an analytic function of λ and q for the ﬁxed numbers k, l, a and θ, one can regard the root λ = λ (q) of (2.1) as a continuous function of q. The next lemma plays very important role for the main theorem. Lemma 2.2. As q varies, the sum of the multiplicities of the roots of (2.1) in the open right half-plane can change only if a root appears on or crosses the imaginary axis. Consequently, we claim that (2.1) has only imaginary roots ±iω. We will determine the value of q as equation (2.1) has roots on the imaginary axis. Now, we can write the characteristic equation (2.1) as follows; λ + (1 −a) + q e−λk+iθ + e−λl+iθ = 0. (2.2) (2.2) Let λ = iω is a root (2.2) such that ω ∈R. Firsty, since fθ (0) ̸= 0, we see that ω ̸= 0. If ω ̸= 0, then we write iω + (1 −a) + q e−iωk+iθ + e−iωl+iθ = 0, iω + (1 −a) + q e−iωk+iθ + e−iωl+iθ = 0, iω + (1 −a) + q e−iωk+iθ + e−iωl+iθ = 0, iω + (1 −a) + q e−iωk+iθ + e−iωl+iθ = 0, and from this equation, we have and from this equation, we have    ω = q (sin (ωk −θ) + sin (ωl −θ)) a −1 = q (cos (ωk −θ) + cos (ωl −θ)) , (2.3) which is equivalent to    ω = 2q sin ω(k+l) 2 −θ cos ω(k−l) 2 a −1 = 2q cos ω(k+l) 2 −θ cos ω(k−l) 2 . (2.4) From (2.4) , we get (k + l)    ω = q (sin (ωk −θ) + sin (ωl −θ)) a −1 = q (cos (ωk −θ) + cos (ωl −θ)) , (2.3) (2.3) which is equivalent to    ω = 2q sin ω(k+l) 2 −θ cos ω(k−l) 2 a −1 = 2q cos ω(k+l) 2 −θ cos ω(k−l) 2 . 2. Some Auxiliary Lemmas 2. Some Auxiliary Lemmas Lemma 2.1. The zero solution of (1.1) is asymptotically stable if and only if all the roots of equation . The zero solution of (1.1) is asymptotically stable if and only if all the roots of equation tion of (1.1) is asymptotically stable if and only if all the roots of equation fθ (λ, q) = λ + (1 −a) + q e−λk+i\|θ\| + e−λl+i\|θ\| (2.1) (2.1) lie in the left half of the complex plane. lie in the left half of the complex plane. (ii) If 2qj cos ωj(k−l) 2 2 −(a −1)2 > 0, then there exist the real numbers ωj and qj, qj is as follows: qj = a −1 2 cos ωj(k+l) 2 −θ cos ωj(k−l) 2 . Lemma 2.3. Suppose that {qj : j ≥1} > 0 and 0 < θ < π 2 . Let λ = iωj be a root of (2.1) where ωj ∈ (3−4j)π k−l , (4j−3)π k−l − n −nπ+2θ k+l , nπ+2θ k+l o is a real number for n ∈N. Then the following conditions hold: (i) If 2qj cos ωj(k−l) 2 2 −(a −1)2 ≤0, then there exists no real number ωj. (ii) If 2qj cos ωj(k−l) 2 2 −(a −1)2 > 0, then there exist the real numbers ωj and qj, qj is as follows: qj = a −1 2 cos ωj(k+l) 2 −θ cos ωj(k−l) 2 . Lemma 2.3. Suppose that {qj : j ≥1} > 0 and 0 < θ < π 2 . Let λ = iωj be a root of (2.1) where ωj ∈ (3−4j)π k−l , (4j−3)π k−l − n −nπ+2θ k+l , nπ+2θ k+l o is a real number for n ∈N. Then the following conditions hold: (i) If 2qj cos ωj(k−l) 2 2 −(a −1)2 ≤0, then there exists no real number ωj. (ii) If 2qj cos ωj(k−l) 2 2 −(a −1)2 > 0, then there exist the real numbers ωj and qj, qj is as follows: qj = a −1 2 cos ωj(k+l) 2 −θ cos ωj(k−l) 2 . Remark 2.1. lie in the left half of the complex plane. In case 2qj cos ωj(k−l) 2 2 −(a −1)2 > 0, for the equality ωj = ±ϕj = ± s 2qj cos ωj (k −l) 2 2 −(a −1)2, the sum of delays k and l is as follows; the sum of delays k and l is as follows; the sum of delays k and l is as follows; the sum of delays k and l is as follows; (kn + ln)+ = 2 ϕj   −(2n + 2) π + arccos   a −1 2qj cos ωj(k−l) 2  + θ    (kn + ln)−= 2 ϕj   −2nπ + arccos   a −1 2qj cos ωj(k−l) 2  −θ   , (kn + ln)+ = 2 ϕj   −(2n + 2) π + arccos   a −1 2qj cos ωj(k−l) 2  + θ    for n ∈N. Also, iϕj or −iϕj is a root of (2.1) for the sum of delays (kn + ln)+ or (kn + ln)−for n ∈N. for n ∈N. Also, iϕj or −iϕj is a root of (2.1) for the sum of delays (kn + ln)+ or (kn + ln)−for n ∈N. P f f L 2 3 B (2 4) i Proof of Lemma 2.3. By (2.4), we can write Proof of Lemma 2.3. By (2.4), we can write Proof of Lemma 2.3. By (2.4), we can write ω2 + (a −1)2 = 2q cos ω (k −l) 2 . Substituting ω = {ωj}j≥1 and q = {qj}j≥1 into the above equation, we obtain Substituting ω = {ωj}j≥1 and q = {qj}j≥1 into the above equation, we obtain Substituting ω = {ωj}j≥1 and q = {qj}j≥1 into the above equation, we obtain Substituting ω = {ωj}j≥1 and q = {qj}j≥1 into the above equation, we obtain ω2 j + (a −1)2 = 2qj cos ωj (k −l) 2 . (2.6) (2.6) If 2qj cos ωj(k−l) 2 2 −(a −1)2 ≤0, then statement (2.6) implies ω2 j < 0, contradicts ω2 j > 0; thus, condition (i) is veriﬁed; that is, (2.1) has no root on the imaginary axis for all k > l > 0. lie in the left half of the complex plane. (2.4) which is equivalent to    ω = 2q sin ω(k+l) 2 −θ cos ω(k−l) 2 a −1 = 2q cos ω(k+l) 2 −θ cos ω(k−l) 2 . (2.4) From (2.4) , we get (2.4) ω a −1 = tan ω (k + l) 2 −θ . (2.5) ω a −1 = tan ω (k + l) 2 −θ . ω a −1 = tan ω (k + l) 2 −θ . ω a −1 = tan ω (k + l) 2 −θ . (2.5) (2.5) We know that the function tanjant is deﬁned as on the region We know that the function tanjant is deﬁned as on the region H = n (t, tan t) : t ∈R, t = π 2 + ρπ, ρ ∈Z o . Thus, (2.5) has only a sequence of the roots {ωj : j ≥1} , where ωj ∈ (2j −1) π + 2θ k + l , (2j + 1) π + 2θ k + l for ωj > 0 Thus, (2.5) has only a sequence of the roots {ωj : j ≥1} , where ωj ∈ (2j −1) π + 2θ k + l , (2j + 1) π + 2θ k + l for ωj > 0 sequence of the roots {ωj : j ≥1} , where ωj ∈ (2j −1) π + 2θ k + l , (2j + 1) π + 2θ k + l for ωj > 0 ωj ∈ (2j −1) π + 2θ k + l , (2j + 1) π + 2θ k + l for ωj > 0 and ωj ∈ −(2j + 1) π + 2θ k + l , −(2j −1) π + 2θ k + l for ωj < 0. Int. J. Anal. Appl. 16 (4) (2018) 457 Lemma 2.3. Suppose that {qj : j ≥1} > 0 and 0 < θ < π 2 . Let λ = iωj be a root of (2.1) where ωj ∈ (3−4j)π k−l , (4j−3)π k−l − n −nπ+2θ k+l , nπ+2θ k+l o is a real number for n ∈N. Then the following conditions hold: (i) If 2qj cos ωj(k−l) 2 2 −(a −1)2 ≤0, then there exists no real number ωj. lie in the left half of the complex plane. If 2qj cos ωj(k−l) 2 2 −(a −1)2 ≤0, then statement (2.6) implies ω2 j < 0, contradicts ω2 j > 0; thus, condition (i) is veriﬁed; that is, (2.1) has no root on the imaginary axis for all k > l > 0. On the other hand, if 2qj cos ωj(k−l) 2 2 −(a −1)2 > 0, statement (2.6) implies ωj = r 2q cos ωj(k−l)2 (a 1)2 From (2 4) we get On the other hand, if 2qj cos ωj(k−l) 2 2 −(a −1)2 > 0, statement (2.6) implies ωj = ±ϕj for ϕj = r 2qj cos ωj(k−l) 2 2 −(a −1)2. From (2.4), we get On the other hand, if 2qj cos ωj(k−l) 2 2 −(a −1)2 > 0, statement (2.6) implies ωj = ±ϕj for ϕj = r 2qj cos ωj(k−l) 2 2 −(a −1)2. From (2.4), we get qj = a −1 2 cos ωj(k+l) 2 −θ cos ωj(k−l) 2 . Now we will show that iϕj is a root of (2.1) . In case ωj = 2qj cos ωj (k −l) 2 2 −(a −1)2 , Int. J. Anal. Appl. 16 (4) (2018) 458 (2.4) and cos ωj(k−l) 2 > 0 implies sin ωj(k+l) 2 −θ > 0. Thus, we can write (2.4) and cos ωj(k−l) 2 > 0 implies sin ωj(k+l) 2 −θ > 0. Thus, we can write ωj (k + l) ( )  a −1  ( ) (2.4) and cos ωj(k−l) 2 > 0 implies sin ωj(k+l) 2 −θ > 0. Thus, we can write (2.4) and cos ωj(k−l) 2 > 0 implies sin ωj(k+l) 2 −θ > 0. Thus, we can write ωj (k + l) 2 −θ = −(2n + 2) π + arccos   a −1 2qj cos ωj(k−l) 2  for n ∈N (2.7) (2.7) which yields (kn + ln)+ . After that, we have which yields (kn + ln)+ . After that, we have which yields (kn + ln)+ . lie in the left half of the complex plane. After that, we have sin  arccos   a −1 2qj cos ωj(k−l) 2    = ϕj 2qj cos ωj(k−l) 2 (2.8) sin  arccos   a −1 2qj cos ωj(k−l) 2    = ϕj 2qj cos ωj(k−l) 2 because of arccos   a −1 2qj cos ωj(k−l) 2  =                arcsin   ϕj 2qj cos ωj(k−l) 2   if a −1 > 0 π −arcsin   ϕj 2qj cos ωj(k−l) 2   if a −1 < 0. (2.8) because of arccos   a −1 2qj cos ωj(k−l) 2  =                arcsin   ϕj 2qj cos ωj(k−l) 2   if a −1 > 0 π −arcsin   ϕj 2qj cos ωj(k−l) 2   if a −1 < 0. lie in the left half of the complex plane. Let λ = iωj be a root of (2.1) where ωj ∈ (4j−3)π k−l , (4j−1)π k−l ∪ (1−4j)π k−l , (3−4j)π k−l − n −nπ+2θ k+l , nπ+2θ k+l o for n ∈N. Then the following conditions hold: (i) If 2qj cos ωj(k−l) 2 2 −(a −1)2 ≤0, then there exists no real number ωj. 2 Lemma 2.4. Suppose that {qj : j ≥1} < 0 and 0 < θ < π 2 . Let λ = iωj be a root of (2.1) where ωj ∈ (4j−3)π k−l , (4j−1)π k−l ∪ (1−4j)π k−l , (3−4j)π k−l − n −nπ+2θ k+l , nπ+2θ k+l o for n ∈N. Then the following conditions hold: (i) If 2qj cos ωj(k−l) 2 2 −(a −1)2 ≤0, then there exists no real number ωj. (ii) If 2qj cos ωj(k−l) 2 2 −(a −1)2 > 0, then there exist the real numbers ωj and qj, qj is as follows: qj = a −1 2 cos ωj(k+l) 2 −θ cos ωj(k−l) 2 . Lemma 2.4. Suppose that {qj : j ≥1} < 0 and 0 < θ < π 2 . Let λ = iωj be a root of (2.1) where ωj ∈ (4j−3)π k−l , (4j−1)π k−l ∪ (1−4j)π k−l , (3−4j)π k−l − n −nπ+2θ k+l , nπ+2θ k+l o for n ∈N. Then the following conditions hold: (i) If 2qj cos ωj(k−l) 2 2 −(a −1)2 ≤0, then there exists no real number ωj. (ii) If 2qj cos ωj(k−l) 2 2 −(a −1)2 > 0, then there exist the real numbers ωj and qj, qj is as follows: a −1 milar of the lemma 2.3. □ □ Proof. The proof is similar of the lemma 2.3. □ Proof. The proof is similar of the lemma 2.3. Proof. The proof is similar of the lemma 2.3. Proof. The proof is similar of the lemma 2.3. Lemma 2.5. Suppose that a < 1. Let λ (q) = Re (λ (q))+i Im (λ (q)) the root of (2.1) satisfying Re (λ (qj)) = 0, Im (λ (qj)) = ωj. Then the following equation is provided: Si gn Re λ′ (qj) . Si gn qj > 0 Proof. lie in the left half of the complex plane. For the case k + l = (kn + ln)+ , from (2.1) we have For the case k + l = (kn + ln)+ , from (2.1) we have f (iω) = iω + (1 −a) + q ei(ωk−θ) + ei(ωl−θ) = i r 2qj cos ωj(k−l) 2 2 −(a −1)2 + (1 −a) + + 2qj cos ωj(k−l) 2 e −i ωj (k+l) 2 −θ , = i r 2qj cos ωj(k−l) 2 2 −(a −1)2 + (1 −a) + + 2qj cos ωj(k−l) 2 e −i  −(2n+2)π+arccos   a−1 2qj cos ωj (k−l) 2     , = i r 2qj cos ωj(k−l) 2 2 −(a −1)2 + (1 −a) + + 2qj cos ωj(k−l) 2 ( cos arccos a−1 2qj cos ωj (k−l) 2 !!) − −2qj cos ωj(k−l) 2 ( i sin arccos a−1 2qj cos ωj (k−l) 2 !!) , = i r 2qj cos ωj(k−l) 2 2 −(a −1)2 + (1 −a) + + (1 −a) −i r 2qj cos ωj(k−l) 2 2 −(a −1)2 = 0; = i r 2qj cos ωj(k−l) 2 2 −(a −1)2 + (1 −a) + + 2qj cos ωj(k−l) 2 ( cos arccos a−1 2qj cos ωj (k−l) 2 !!) − −2qj cos ωj(k−l) 2 ( i sin arccos a−1 2qj cos ωj (k−l) 2 !!) , = i r 2qj cos ωj(k−l) 2 2 −(a −1)2 + (1 −a) + + (1 −a) −i r 2qj cos ωj(k−l) 2 2 −(a −1)2 = 0; thus, we can see that iϕj is a root of (2.1) . Similarly, when ωj ∈ (3 −4j) π k −l , 0 , −iϕj is a root of (2.1) for the sum of delays (kn + ln)−. The proof is completed. □ Int. J. Anal. Appl. 16 (4) (2018) 459 When ωj < 0, we have the following analogous result. When ωj < 0, we have the following analogous result. we have the following analogous result. When ωj < 0, we have the following analogous result. Lemma 2.4. Suppose that {qj : j ≥1} < 0 and 0 < θ < π 2 . lie in the left half of the complex plane. Taking the derivative of λ with respect to q on (2.1) , we have Proof. Taking the derivative of λ with respect to q on (2.1) , we have dλ dq + e−λk+iθ + e−λl+iθ + q −ke−λk+iθ −le−λl+iθ dλ dq = 0; dλ dq = − e−λk+iθ + e−λl+iθ 1 −q (ke−λk+iθ + le−λl+iθ) = λ + 1 −a q (1 −q (ke−λk+iθ + le−λl+iθ)). Substituting ω = ωj and q = qj into the above equation, we get Substituting ω = ωj and q = qj into the above equation, we get dλ dq \| λ=iω = iωj + 1 −a qj 1 −qj ke−i(ωjk−θ) + le−i(ωjl−θ) Re dλ dq \| λ=iω = (1 −a) qj (1 −qj (k cos (ωjk −θ) + l cos (ωjl −θ))) M + (2.9) + ωjq2 j (k sin (ωjk −θ) + l sin (ωjl −θ)) M , (2.10) Re dλ dq \| λ=iω = (1 −a) qj (1 −qj (k cos (ωjk −θ) + l cos (ωjl −θ))) M + (2.9 (2.9) + ωjq2 j (k sin (ωjk −θ) + l sin (ωjl −θ)) M , (2.10) + jqj ( ( j ) + ( j )) M , where M = q2 j (1 −qj (k cos (ωjk −θ) + l cos (ωjl −θ)))2 + q4 j (k sin (ωjk −θ) + l sin (ωjl −θ))2 . M = q2 j (1 −qj (k cos (ωjk −θ) + l cos (ωjl −θ)))2 + q4 j (k sin (ωjk −θ) + l sin (ωjl −θ))2 . Let A1 = sin (ωk −θ) + sin (ωl −θ) and A2 = cos (ωk −θ) + cos (ωl −θ) . By (2.4) , we have A1(ω) A2(ω) = tan ω(k+l) 2 −θ , thus d dω A1 (ω) A2 (ω) = A′ 1 (ω) A2 (ω) −A′ 2 (ω) A1 (ω) (A2 (ω))2 > 0, is obtained, which implies A′ 1 (ω) A2 (ω) −A′ 2 (ω) A1 (ω) > 0. is obtained, which implies A′ 1 (ω) A2 (ω) −A′ 2 (ω) A1 (ω) > 0. 460 Int. J. Anal. Appl. lie in the left half of the complex plane. 16 (4) (2018) Since A′ 1 = k cos (ωk −θ) + l cos (ωl −θ) and A′ 2 = −k sin (ωk −θ) −l sin (ωl −θ), (2.9) can written Re dλ dq \| λ=iω = qj [((1 −a) −(1 −a) qjA′ 1 (ω)) −ωjqjA′ 2 (ω)] M , (ωk −θ) + l cos (ωl −θ) and A′ 2 = −k sin (ωk −θ) −l sin (ωl −θ), (2.9) can written R dλ \| qj [((1 −a) −(1 −a) qjA′ 1 (ω)) −ωjqjA′ 2 (ω)] Re dλ dq \| λ=iω = qj [((1 −a) −(1 −a) qjA′ 1 (ω)) −ωjqjA′ 2 (ω)] M , we use (2.4) for above equation, then we get we use (2.4) for above equation, then we get Re dλ dq \| λ=iω = qj (1 −a) + q2 j (A′ 1 (ω) A2 (ω) −A′ 2 (ω) A1 (ω)) M . Hence, the proof is completed. □ 3. Main Results Hence, the proof is completed. □ Hence, the proof is completed. □ Hence, the proof is completed. 3. Main Results 3. Main Results 3. Main Results Theorem 3.1. Suppose that a < 1, 0 < θ < π 2 and the matrix A of the system (1.1) is written as the form (II) . we deﬁne q− θ = max j≥1 {qj : qj < 0} , q+ θ = min j≥1 {qj : qj > 0} , and let a neighborhood of q = 0 is q− θ , q+ θ . Then system (1.1) is asymptotically stable if an and let a neighborhood of q = 0 is q− θ , q+ θ . Then system (1.1) is asymptotically stable if q− θ < q < q+ θ . Proof. In case of q = 0, the root of (2.1) is only λ (0) = a −1 < 0. Thus, the root of the equation (2.1) has a negative real part. By the continuity of the roots with respect to q and by the asymptotic stability of (1.1), we can claim that the roots of equation (2.1) are inside a neighborhood q− θ , q+ θ of q = 0. Proof. In case of q = 0, the root of (2.1) is only λ (0) = a −1 < 0. Thus, the root of the equation (2.1) has a negative real part. By the continuity of the roots with respect to q and by the asymptotic stability of (1.1), we can claim that the roots of equation (2.1) are inside a neighborhood q− θ , q+ θ of q = 0. Since λ (0) < 0, in case of q− θ < ∞, by Lemma 2.4, equation (2.1) has roots on the imaginary axis because of q− θ is the ﬁrst value q < 0. By Lemma 2.2, all roots of the equation (2.1) have negative real parts for q− θ , 0 . Similarly, in case of q+ θ < ∞, by Lemma 2.3, equation (2.1) has roots on the imaginary axis because of q+ θ is the ﬁrst value q > 0. By Lemma 2.2, all roots of the equation (2.1) have negative real parts for 0, q+ θ . Also, by Lemma 2.5, equation (2.1) has at least one root positive real part for −∞, q− θ and q+ θ , ∞ . Thus, the proof is completed. □ Theorem 3.2. 3. Main Results Suppose that a < 1, θ = 0 and the matrix A of the system (1.1) is written as the form (II) . we deﬁne q− 0 = max j≥1 a −1 2 : a < 1 , q+ 0 = min j≥1 {qj : qj > 0} , and let a neighborhood of q = 0 is a−1 2 , q+ 0 . Then system (1.1) is asymptotically stable if and only if and let a neighborhood of q = 0 is a−1 2 , q+ 0 . Then system (1.1) is asymptotically stable if and only if and let a neighborhood of q = 0 is a−1 2 , q+ 0 . Then system (1.1) is asymptotically stable if and only if a −1 2 < q < q+ 0 . Proof. When θ = 0, equation (2.1) has only root λ = 0 as q = a−1 2 , that is λ a−1 2 = 0. Since Proof. When θ = 0, equation (2.1) has only root λ = 0 as q = a−1 2 , that is λ a−1 2 = 0. Since dλ a −1 2 dq < 0, dq Int. J. Anal. Appl. 16 (4) (2018) 461 we can write q− 0 = max j≥1 qj = a −1 2 : a < 1, j ≥1 . Since the rest of the proof is similar to the Theorem 3.11, it is obvious. □ we can write q− 0 = max j≥1 qj = a −1 2 : a < 1, j ≥1 . Since the rest of the proof is similar to the Theorem 3.11, it is obvious. □ we can write q− 0 = max j≥1 qj = a −1 2 : a < 1, j ≥1 . Since the rest of the proof is similar to the Theorem 3.11, it is obvious. □ Theorem 3.3. Suppose that a < 1, and the matrix A of the system (1.1) is written as the form (I) . we deﬁne q−= max j≥1 {qj : qj < 0} , q+ = min j≥1 {qj : qj > 0} , and let a neighborhood of q = 0 is (q−, q+). Then system (1.1) is asymptotically stable if and only if q−< q1, q2 < q+. 3. Main Results q−< q1, q2 < q+. □ Proof. The proof is similar to the Theorem 3.1. Proof. The proof is similar to the Theorem 3.1. Proof. The proof is similar to the Theorem 3.1. References [1] H. Matsunaga, Delay Dependent and Delay Independent Stability Criteria For A Delay Diﬀerential System, American Mathematical Society, 136 Fields Inst. Commun. 42 (2008), 4305-4312. [1] H. 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Nakajima, On the Stability of a linear Retarded Diﬀerential-Diﬀerence Equation, Funkcialaj Ekvacioj. 57 (2014), 43-56 [12] T. Hara, S. Sakata, An application of the Hurwitz theorem to the root analysis of the characteristic equation, Appl. Math. Lett. 24 (2011) 12–15 [13] H. Smith, An Introduction to Delay Diﬀerential Equations with Applications to The Life Science, Springer, New York 2010. [14] H. I .Freedman, Y.Kuang, Stability switches in linear scalar neutral delay equation, Funkcial. Ekvac. 34 (1991), 187–209. 14] H. I .Freedman, Y.Kuang, Stability switches in linear scalar neutral delay equation, Funkcial. Ekvac. 34 [14] H. References I .Freedman, Y.Kuang, Stability switches in linear scalar neutral delay equation, Funkcial. Ekvac. 34 (1991), 187–209. [15] R. Bellman and K. L. Cooke, Diﬀerential-Diﬀerence Equations, Academic Press, New York, 1963. [15] R. Bellman and K. L. Cooke, Diﬀerential-Diﬀerence Equations, Academic Press, New York, 1963.
https://openalex.org/W2495371129	https://hal.inrae.fr/hal-02631259/document	English	null	Comparison of the In vitro Activity of Five Antimicrobial Drugs against Staphylococcus pseudintermedius and Staphylococcus aureus Biofilms	Frontiers in microbiology	2,016	cc-by	6,803	To cite this version: Aude Ferran, Jing Jing Liu, Pierre-Louis Toutain, Alain Bousquet-mélou. Comparison of the In vitro activity of five antimicrobial drugs against Staphylococcus pseudintermedius and Staphylococcus au- reus Biofilms. Frontiers in Microbiology, 2016, 7, pp.1187. 10.3389/fmicb.2016.01187. hal-02631259 HAL Id: hal-02631259 https://hal.inrae.fr/hal-02631259v1 Submitted on 27 May 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. ORIGINAL RESEARCH published: 02 August 2016 doi: 10.3389/fmicb.2016.01187 Edited by: Edited by: Manuela Caniça, National Institute of Health Dr. Ricardo Jorge, Portugal Reviewed by: Atte Von Wright, University of Eastern Finland, Finland Xian-Zhi Li, Health Canada, Canada Correspondence: Aude A. Ferran a.ferran@envt.fr Correspondence: Aude A. Ferran a.ferran@envt.fr Keywords: bioﬁlm, antimicrobial activity, Staphylococcus aureus, Staphylococcus pseudintermedius, veterinary antimicrobials Specialty section: This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology Comparison of the In vitro Activity of Five Antimicrobial Drugs against Staphylococcus pseudintermedius and Staphylococcus aureus Bioﬁlms Aude A. Ferran*, JingJing Liu, Pierre-Louis Toutain and Alain Bousquet-Mélou Toxalim, Université de Toulouse, INRA, ENVT, INP-Purpan, UPS, Toulouse, France Resistance in canine pathogenic staphylococci is necessitating re-evaluation of the current antimicrobial treatments especially for bioﬁlm-associated infections. Long, repeated treatments are often required to control such infections due to the tolerance of bacteria within the bioﬁlm. To comply with the goal of better antibiotic stewardship in veterinary medicine, the efﬁcacies of the available drugs need to be directly assessed on bacterial bioﬁlms. We compared the activities of amoxicillin, cefalexin, clindamycin, doxycycline, and marboﬂoxacin on in vitro bioﬁlms of Staphylococcus pseudintermedius and Staphylococcus aureus. Exposure of bioﬁlms for 15 h to maximum concentrations of the antibiotics achievable in canine plasma only reduced bioﬁlm bacteria by 0.5– 2.0 log10 CFU, compared to the control, except for marboﬂoxacin which reduced S. aureus bioﬁlms by 5.4 log10 CFU. Two-antibiotic combinations did not improve, and even decreased, bacterial killing. In comparison, 5 min-exposure to 2% chlorhexidine reduced bioﬁlms of the two tested strains by 4 log10 CFU. Our results showed that S. pseudintermedius and S. aureus bioﬁlms were highly tolerant to all the drugs tested, consistent with the treatment failures observed in practice. Under our in vitro conditions, the use of chlorhexidine was more efﬁcacious than antimicrobials to reduce S. pseudintermedius bioﬁlm. Keywords: bioﬁlm, antimicrobial activity, Staphylococcus aureus, Staphylococcus pseudintermedius, veterinary antimicrobials INTRODUCTION The emergence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in dogs and the zoonotic risk of staphylococcal infections in pets are highlighting the urgent need for improved antimicrobial stewardship to reduce the often extremely long and repeated treatments for pyoderma in veterinary medicine (Guardabassi et al., 2004; Van Hoovels et al., 2006; Pompilio et al., 2015). Received: 20 April 2016 Accepted: 18 July 2016 Published: 02 August 2016 Antimicrobial Agents ) Bioﬁlm formation is known to be a major virulence factor in several Staphylococcus spp. including S. pseudintermedius. Bioﬁlms are groups of bacterial cells which adhere to the surfaces of living tissues or artiﬁcial materials and are covered by a self-produced extracellular polysaccharide (EPS) matrix (Davies, 2003). This is a natural survival mode for bacterial cells, distinct from that of planktonic cells, and formed in adaptation to environmental pressures in the long-term evolutionary process (Costerton et al., 1999). After bioﬁlm formation, the infectious bacterial antigens induce the production of large quantities of antibodies which are able to combat planktonic bacteria, but remain ineﬀective against bacteria inside the bioﬁlm, due to the protective matrix (Costerton et al., 1999). In addition, the bacteria inside bioﬁlms are highly tolerant to antibiotics and even though the underlying mechanism has not been completely elucidated, phenotypic variation leading to a “persister” status has been reported (Davies, 2003; Van Hoovels et al., 2006; Yang et al., 2015). As stated by others, the high prevalence of these persister bacteria in bioﬁlms precludes the direct use of standard susceptibility results to predict clinical eﬃcacy on bioﬁlm-associated infections (Claessens et al., 2015). Amoxicillin, cefalexin, clindamycin, and doxycycline were purchased from Sigma-Aldrich. Marboﬂoxacin was kindly provided by Vetoquinol. Antibiotics were dissolved in pure water, with NaOH added to amoxicillin and cefalexin. The tested drug concentrations were selected to be equal or slightly above the total maximum concentration attained in canine serum after administration of the approved or recommended standard doses (Silley et al., 1988; Kung and Wanner, 1994; Schneider et al., 1996; Batzias et al., 2005; Hillier et al., 2014; KuKanich and KuKanich, 2015). The tested concentrations were 5 µg/mL for marboﬂoxacin, 10 µg/mL for clindamycin and doxycycline, 20 µg/mL for amoxicillin, and 50 µg/mL for cefalexin. Chlorhexidine was purchased as chlorhexidine digluconate (Hibitan Irrigation 20%ND, MSD, France). Test Strains A S. aureus strain (HG001), derived from NCTC 8325, and S. pseudintermedius ATCC 49444 were used. Citation: Ferran AA, Liu JJ, Toutain P-L and Bousquet-Mélou A (2016) Comparison of the In vitro Activity of Five Antimicrobial Drugs against Staphylococcus pseudintermedius and Staphylococcus aureus Bioﬁlms. Front. Microbiol. 7:1187. doi: 10.3389/fmicb.2016.01187 Staphylococcus pseudintermedius, an opportunistic pathogen of dogs, is the leading cause of skin and ear infections (Hillier et al., 2014). Although, Staphylococcus aureus and Staphylococcus schleiferi can be isolated on dogs, these pathogens are rarely associated with pyoderma (Frank and Loeﬄer, 2012). Most animals suﬀering from staphylococcal skin infection have reduced immunity associated with alterations of the skin barrier or underlying diseases that may be diﬃcult to diagnose and cure. Pyoderma infections are therefore the principal reason for August 2016 \| Volume 7 \| Article 1187 Frontiers in Microbiology \| www.frontiersin.org 1 Ferran et al. In vitro Activity on Bioﬁlms MATERIALS AND METHODS antimicrobial use in small animal practice (Rantala et al., 2004) and predispose to long and repeated antimicrobial treatments (Hillier et al., 2014). Conventional treatment of superﬁcial and deep canine staphylococcal pyoderma has been based on systemic antibacterial administration for 3–4 weeks sometimes combined with a topical treatment (Loeﬄer et al., 2007; Frank and Loeﬄer, 2012; Borio et al., 2015). Antimicrobial and Chlorhexidine Susceptibility The MIC were determined in triplicate by microdilution method as described in the CLSI reference methods (CLSI, 2006). Bioﬁlm Formation The drug susceptibility of a bacterial strain is classically determined from the minimum inhibitory concentration (MIC) or by antimicrobial susceptibility testing (AST). These determinations are done in laboratory on planktonic bacteria during their exponential growth phase. It has been demonstrated, however, that antibiotic activity can be drastically reduced (and not therefore predictable by standard AST), if the bacterial inoculum is high (Ferran et al., 2014) or when the growth rate or metabolism of the bacteria is reduced, as in bioﬁlms (Costerton et al., 1999; Davies, 2003; Guardabassi et al., 2004). Thus, to propose a more eﬃcacious treatment for canine pyoderma, which is a bioﬁlm-associated infection, the activity of antimicrobial drugs needs to be directly investigated on the bacterial bioﬁlms. The veterinary guidelines recommend amoxicillin/clavulanic acid, cefalexin or clindamycin as ﬁrst-line empirical agents for systemic antibiotic therapy (Hillier et al., 2014). Third generation cephalosporins, doxycycline, ﬂuoroquinolones, chloramphenicol, aminoglycosides, and rifampicin are classiﬁed as second tier drugs (Hillier et al., 2014). Whatever the drug, the recommended treatment duration usually exceeds 3 weeks to prevent relapses (Beco et al., 2013; Hillier et al., 2014). This is a cause of concern, in terms of the prudent use of antimicrobials, as treatment duration is a major factor contributing to the emergence of resistances (Rubinstein and Keynan, 2013). In order to contribute to the improvement of in vivo treatment, we assessed the antimicrobial activity of ﬁrst and second-tier drugs on in vitro bioﬁlms formed by S. pseudintermedius or S. aureus. The bacterial bioﬁlm was produced in 6-well plates (polystyrene). A bacterial colony from an overnight culture of S. aureus or S. pseudintermedius was diluted in Mueller-Hinton (MH) broth to obtain a bacterial suspension containing 105 CFU/mL. Each well of 6-well plates (polystyrene) was ﬁlled with 4 mL of bacterial suspension and incubated at 37◦C for 7 h without shaking to allow bioﬁlm formation. At this time point, the planktonic and bioﬁlm bacteria in three control wells were counted to assess bioﬁlm status at the time of drug addition. Antibiotic Activity Testing After 7 h of incubation, the medium was renewed to avoid nutrient deﬁciency. To do that, three milliliters of the suspension only containing the planktonic bacteria were carefully removed from each well, and centrifuged for 10 min (3000 g, 20◦C). The supernatant was discarded and 3.5 mL of fresh MH broth was added to the bacterial pellet. After shaking, the suspension containing the planktonic bacteria was then carefully returned to the original wells to preserve the 7 h-old bioﬁlm. After incubation for 1 h, 45 µL of antibiotic solution or MH (control) was added and the prepared plates were incubated overnight before bacterial counting. The antibiotic drugs were tested alone or in pairs. Each experiment was performed in triplicate. Chlorhexidine Activity Testing As the addition of chlorhexidine to MH broth leads to precipitation, chlorhexidine eﬃcacy was assessed with a diﬀerent August 2016 \| Volume 7 \| Article 1187 Frontiers in Microbiology \| www.frontiersin.org 2 Ferran et al. In vitro Activity on Bioﬁlms The test concentrations were at least 20-fold higher than the MIC except for cefalexin and S. aureus (sixfold). The test concentrations were at least 20-fold higher than the MIC except for cefalexin and S. aureus (sixfold). protocol. After 7 h of incubation, the total bacterial suspension only containing the planktonic bacteria was collected. One mL of water was added to cover the 7 h-old bioﬁlm while the suspension was centrifuged (10 min, 3000 g, 20◦C). The supernatant was then discarded and the pellet resuspended in water before returning the planktonic bacteria. Chlorhexidine digluconate was added to the wells to obtain ﬁnal concentrations of 2%. Three wells without chlorhexidine were used as control. After 5 min exposure, the bioﬁlm and planktonic bacteria were counted. Assessment of the Effects of Antibiotics on Bioﬁlms In vitro After incubation of S. pseudintermedius for 7 h, the suspension contained 8.11 ± 0.33 log10 CFU/mL and the bioﬁlm 7.89 ± 0.20 log10 CFU. For S. aureus at the same time point, the suspension contained 7.32 ± 0.04 log10 CFU/mL and the bioﬁlm contained 8.17 ± 0.14 log10 CFU. Suspension was removed and rinsed twice in water before counting in NaCl 0.9%. The bioﬁlm bacteria were counted as described in the antimicrobial drug protocol. Due to the change of medium from MH to water, this experiment had its own control wells without any drug. The antimicrobial drugs were added at that time and the bacteria were again counted after 15 h. The numbers of bacteria in the control wells (without any drug) increased slightly overnight. For S. pseudintermedius, the populations increased by 0.71 log10 CFU /mL in the suspension and by 0.85 log10 CFU in the bioﬁlm. For S aureus, the increases were 1.15 log10 CFU/mL and 0.30 log10 CFU, respectively. Planktonic Bacteria p y The reductions in bacterial counts, after 15 h of drug exposure, in the suspension and in the bioﬁlm for all the tested antibiotics, compared to the control, are given in Tables 2 and 3 and represented in Figures 1 and 2 for S. aureus and S. pseudintermedius, respectively. All tested antibiotics reduced the bacterial counts in the suspension and bioﬁlm of both strains even if some antibiotics showed extremely low activity. For most of the antibiotics, the obtained reduction of the bacterial population was less than 2 log10 CFU. Amoxicillin, cefalexin and doxycycline reduced S. pseudintermedius bacteria by only 0.67 to 0.85 log10 CFU/mL in suspension and by 0.55–0.61 log10 CFU in bioﬁlm. For S. aureus exposed to the same three antibiotics, the reduction ranged from 0.73 to 1.10 log10 CFU/mL in suspension and from 0.50 to 0.57 log10 CFU in bioﬁlm. Clindamycin and marboﬂoxacin exhibited higher activities than the other drugs, especially against S. aureus. Clindamycin reduced S. pseudintermedius bioﬁlm by 0.75 log10 CFU and S aureus bioﬁlm by 1.84 log10 CFU. For marboﬂoxacin, the reduction attained 2.9 and 5.4 log10 CFU for S. pseudintermedius and S. aureus bioﬁlms respectively. Marboﬂoxacin was the only antibiotic which eradicated S. aureus in suspension and gave a ﬁnal bacterial load of only 3.09 log10 CFU in the bioﬁlm. The suspension containing planktonic bacteria in each well was carefully removed and the planktonic bacteria were counted after successive 10-fold dilutions on tryptic soy agar plates. The colonies were counted after overnight incubation at 37◦C. The limit of quantiﬁcation was 100 CFU/mL. Bacterial reductions were calculated as the diﬀerences between the bacterial counts in control wells and the bacterial counts after exposure to antimicrobials or biocide. For experiments with chlorhexidine, the suspension was rinsed twice in water to stop biocide activity before counting. Bioﬁlm Bacteria The bioﬁlm bacteria remaining in the wells after the suspension removal were rinsed twice with 4 mL NaCl 0.9%. After the ﬁnal rinse, 6 mL NaCl 0.9% was added to each well. The bacteria in the liquid portion were counted in each well before and after 15 min- ultrasounds. The colonies obtained after plating successive 10- fold dilutions on tryptic soy agar plates were counted after overnight incubation. The diﬀerence in bacterial counts before and after the ultrasounds was considered to represent the “pure” bioﬁlm bacteria. The limit of quantiﬁcation was 600 CFU. Bacterial reductions were calculated as the diﬀerences between the bioﬁlm bacteria counts in control wells and the bioﬁlm bacteria counts after exposure to antimicrobials or biocide. As none of the tested antibiotics showed bactericidal activity against S. pseudintermedius, we then tested all possible two-drug combinations, except for amoxicillin and cefalexin which share the same bacterial target. The reductions in bacterial counts with the diﬀerent combinations are shown in Tables 2 and 3. The results obtained by combining the most eﬃcacious drugs, clindamycin and marboﬂoxacin, are also given in Figure 3. All combinations resulted in less bacterial eradication than the most eﬃcacious of the two drugs tested alone, except for three speciﬁc cases where the combination was slightly better. Bacterial reduction in the S. aureus bioﬁlm was 0.25 log10 CFU higher with both the clindamycin–amoxicillin and clindamycin– cefalexin combinations than with clindamycin alone. For S. pseudintermedius, bacterial reduction in the suspension and bioﬁlm was higher, by 0.1 log10 CFU/mL and 0.5 log10 CFU respectively, with the marboﬂoxacin–amoxicillin combination than with marboﬂoxacin alone. Antimicrobial Susceptibility Testing pseudintermedius suspension (in log10 CFU/mL) and bioﬁlm (in log10 CFU) after 15-h exposure to one drug or to a two-drug combination. For chlorhexidine, bacteria were only exposed for 5 min. AMX, amoxicilin; CFX, cefalexin; CLI, clindamycin; DOX, doxycycline; MAR, marboﬂoxacin; CHD, chlorhexidine; NA, not assessed. Bacterial reductions below 1.5 log10 CFU/mL are in gray, between 1.5 and 3 log10 CFU/mL in yellow, between 3 and 4 log10 CFU/mL in light green and higher than 4 log10 CFU/mL in dark green. Antimicrobial Susceptibility Testing p y g The MIC of amoxicillin, cefalexin, clindamycin, doxycycline, marboﬂoxacin, and chlorhexidine for the S. aureus and S. pseudintermedius strains and the test concentrations are given in Table 1. According to the CLSI breakpoints, both strains were susceptible to clindamycin, doxycycline, and marboﬂoxacin. Both strains were resistant to amoxicillin (MIC equal to the “resistant” breakpoint). For cefalexin, S aureus was classiﬁed as resistant and S. pseudintermedius as susceptible. The MIC for a given drug diﬀered by less than two dilutions (fourfold) for both two strains. August 2016 \| Volume 7 \| Article 1187 Frontiers in Microbiology \| www.frontiersin.org 3 In vitro Activity on Bioﬁlms Ferran et al. TABLE 1 \| CLSI and chlorhexidine breakpoints (Horner et al., 2012; CLSI, 2016), tested antibiotic or biocide concentrations on in vitro bioﬁlms and MIC of the antibiotic drugs for the selected strains of Staphylococcus aureus and Staphylococcus pseudintermedius. CLSI breakpointsa (mg/L) Tested concentrations S. aureus MIC S. pseudintermedius MIC Antibiotic S I R (mg/L) (mg/L) (mg/L) Amoxicillin 0.25 0.5 20 0.5 0.5 Cefalexin 2 4 8 50 8 2 Clindamycin 0.5 1–2 4 10 0.064 0.064 Doxycycline 0.125 0.25 0.5 10 0.032 0.125 Marboﬂoxacin 1 2 4 5 0.125 0.25 Chlorhexidine 4 118b 0.5 0.5 aFor amoxicillin, the CLSI breakpoints of ampicillin were used. For cefalexin, the CLSI breakpoints of cefazolin and cefalothin were used. For chlorhexidine, the 4 µg/mL breakpoint which is not a CLSI breakpoint was reported by Horner et al., 2012. b118 mg/L chlorhexidine correponds to 200 mg/L chlorhexidine digluconate. TABLE 1 \| CLSI and chlorhexidine breakpoints (Horner et al., 2012; CLSI, 2016), tested antibiotic or biocide concentrations on in vitro bioﬁlms and MIC of the antibiotic drugs for the selected strains of Staphylococcus aureus and Staphylococcus pseudintermedius. aFor amoxicillin, the CLSI breakpoints of ampicillin were used. For cefalexin, the CLSI breakpoints of cefazolin and cefalothin were used. For chlorhexidine, the 4 µg/mL breakpoint which is not a CLSI breakpoint was reported by Horner et al., 2012. b118 mg/L chlorhexidine correponds to 200 mg/L chlorhexidine digluconate. TABLE 2 \| Bacterial reductions of S. aureus suspension (in log10 CFU/mL) and bioﬁlm (in log10 CFU) after 15-h exposure to one drug or to a two-drug combination. TABLE 2 \| Bacterial reductions of S. aureus suspension (in log10 CFU/mL) and bioﬁlm (in log10 CFU) after 15-h exposure to one drug or to a two-drug combination. TABLE 3 \| Bacterial reductions of S. Frontiers in Microbiology \| www.frontiersin.org DISCUSSION FIGURE 2 \| Bacterial reductions (mean ± SD) of Staphylococcus pseudintermedius suspension (gray bars, in log10 CFU/mL) and bioﬁlm (black bars, in log10 CFU) after 15-h exposure to amoxicillin (AMX), cefalexin (CFX), clindamycin (CLI), doxycycline (DOX), or marboﬂoxacin (MAR) or after 5- min exposure to chlorhexidine (CHD). I hi d S di di d S bi ﬁl FIGURE 1 \| Bacterial reductions (mean ± SD) of Staphylococcus aureus suspension (gray bars, in log10 CFU/mL) and bioﬁlm (black bars, in log10 CFU) after 15-h exposure to amoxicillin (AMX), cefalexin (CFX), clindamycin (CLI), doxycycline (DOX), or marboﬂoxacin (MAR) or after 5- min exposure to chlorhexidine (CHD). drug regimen optimization for bioﬁlm-associated infections in veterinary medicine. After incubation without drugs for 7 or 24 h, more than 7.8 log10 CFU of the bacteria (S. pseudintermedius and S. aureus) were found in the bioﬁlm. This rapid development was also reported in a study showing that S. pseudintermedius could form bioﬁlm within 24 h in vitro with no diﬀerence between methicillin-susceptible and methicillin-resistant strains (Singh et al., 2013). Another study by Pompilio et al. (2015) revealed that S. pseudintermedius was able to form a well-structured bioﬁlm consisting of multilayered, mushroom-shaped microcolonies embedded in an abundant EPS matrix. We decided to qualify the activities of our test drugs by examining their eﬃcacies on a “young” bioﬁlm produced after incubation for 7 h. No reduction of bioﬁlm had been observed when the eﬀects of the same antimicrobials had been tested on a 24 h-old bioﬁlm (data not shown). This suggests that an environmental or phenotypic change occurred between 7 and 24 h, which had no eﬀect on the numbers of bacteria but was able to modify their susceptibility to drugs. FIGURE 1 \| Bacterial reductions (mean ± SD) of Staphylococcus aureus suspension (gray bars, in log10 CFU/mL) and bioﬁlm (black bars, in log10 CFU) after 15-h exposure to amoxicillin (AMX), cefalexin (CFX), clindamycin (CLI), doxycycline (DOX), or marboﬂoxacin (MAR) or after 5- min exposure to chlorhexidine (CHD). g We tested some of the ﬁrst- and second-line drugs recommended for the systemic treatment of pyoderma in veterinary medicine (Hillier et al., 2014). We exposed S. aureus and S. pseudintermedius bioﬁlms for 15 h to concentrations equal or slightly above the peak concentrations observed in vivo in dogs during a recommended dosage regimen. DISCUSSION Taking plasma protein binding into account, the actual test concentrations in our system were higher than the active free in vivo concentrations in dogs, especially for doxycycline (90% bound), and we likely assessed the highest possible antibacterial activity of each drug in dogs. Even so, all of the drugs tested exhibited very low killing activity on S. pseudintermedius and S. aureus bioﬁlms, except for the ﬂuoroquinolone, marboﬂoxacin on S. aureus. Also, by comparing these two staphylococci species, we found that antibacterial activity against S. pseudintermedius tended to be lower than against S. aureus. For example, although the MIC determination classiﬁed both species as susceptible to clindamycin and marboﬂoxacin, bacterial killing was lower for S. pseudintermedius than for S. aureus. Then, for cefalexin, even though S. pseudintermedius was classiﬁed as susceptible and S. aureus as resistant, the eﬃcacy against both strains was similarly low. This lower activity of antibiotic drugs on S. pseudintermedius than on S. aureus, which is not predictable by susceptibility testing, is consistent with the diﬃculty of eradicating pyoderma in dogs. It also implies that a human infection with this zoonotic bacterial species would be hard to eradicate. One study with a S. pseudintermedius strain isolated from a human infection demonstrated that, at concentrations 128-fold higher than the MIC, none of the antibiotics tested (which included vancomycin and linezolid) was able to eradicate a 48-h old bioﬁlm, except for rifampicin (Pompilio et al., 2015). Interestingly, similar observations were reported with Staphylococcus epidermidis, an opportunistic human pathogen responsible for the vast majority of nosocomial catheter-related blood stream infections (Claessens et al., 2015). Even though the strain of S. epidermidis is classiﬁed as susceptible to vancomycin, based on the MIC determination, killing activity FIGURE 2 \| Bacterial reductions (mean ± SD) of Staphylococcus pseudintermedius suspension (gray bars, in log10 CFU/mL) and bioﬁlm (black bars, in log10 CFU) after 15-h exposure to amoxicillin (AMX), cefalexin (CFX), clindamycin (CLI), doxycycline (DOX), or marboﬂoxacin (MAR) or after 5- min exposure to chlorhexidine (CHD). FIGURE 2 \| Bacterial reductions (mean ± SD) of Staphylococcus pseudintermedius suspension (gray bars, in log10 CFU/mL) and bioﬁlm (black bars, in log10 CFU) after 15-h exposure to amoxicillin (AMX), cefalexin (CFX), clindamycin (CLI), doxycycline (DOX), or marboﬂoxacin (MAR) or after 5- min exposure to chlorhexidine (CHD). In this study, S. pseudintermedius and S. Frontiers in Microbiology \| www.frontiersin.org DISCUSSION In the light of increasing multidrug resistance in zoonotic staphylococci, optimizing the therapeutic strategies utilized for canine pyoderma has become a human health issue. Although, most cases treated for S. pseudintermedius infection ultimately respond to empirical treatments, the duration of such treatments often exceeds 1 month implying a sustained selective pressure which favors the emergence of resistance in staphylococci and in other commensal bacteria (Hillier et al., 2014). As the bacteria become organized in a bioﬁlm containing many persister cells tolerant to antibiotics (Yang et al., 2015), the predictive value of AST in determining the clinical outcome is very limited and direct assessment of the antibiotic’s activity on bioﬁlms is required. In this study, we showed that most ﬁrst and second- line antibiotics recommended for pyoderma display very low antibacterial activity on in vitro S. pseudintermedius bioﬁlms. Separate experiments were conducted to assess the activity of chlorhexidine on bioﬁlms. We ﬁrst checked that the use of water to dilute chlorhexidine did not aﬀect the bioﬁlms formed after 7 h incubation. The bacterial counts in the bioﬁlms of control wells incubated for 5 min in water and rinsed twice with water were 7.24 ± 0.25 log10 CFU for S. pseudintermedius and 7.44 ± 0.15 log10 CFU for S. aureus, corresponding to diﬀerences of less than 1 log10 CFU (0.65 and 0.73 log10 CFU), as compared to control bioﬁlms in the experiments with antibiotics. After 5 min exposure to 2% chlorhexidine, the mean reduction of S. aureus and S. pseudintermedius bioﬁlms was 4 log10 CFU and eradication was attained in half of the experiments. All bacteria in the suspension were eradicated but the time of exposure to chlorhexidine greatly exceeded 5 min due to the time required for centrifugation before the ﬁrst rinse. August 2016 \| Volume 7 \| Article 1187 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 4 Ferran et al. In vitro Activity on Bioﬁlms FIGURE 1 \| Bacterial reductions (mean ± SD) of Staphylococcus aureus suspension (gray bars, in log10 CFU/mL) and bioﬁlm (black bars, in log10 CFU) after 15-h exposure to amoxicillin (AMX), cefalexin (CFX), clindamycin (CLI), doxycycline (DOX), or marboﬂoxacin (MAR) or after 5- min exposure to chlorhexidine (CHD). DISCUSSION aureus bioﬁlms were tested in vitro and no account was taken of the complexity of an in vivo infection, that involves multiple factors such as the immune system, the presence of necrotic or scar tissues and possible hidden intracellular bacteria. Nevertheless, measuring antimicrobial activity on a bacterial population embedded in a bioﬁlm and including a high proportion of persisters is likely to be more eﬃcient in predicting the ultimate clinical outcome than standard AST performed on planktonic bacterial populations in exponential growth phase. We believe that our experiments, even if conducted with one strain per species, could be considered as a ﬁrst step in the process of drug selection and August 2016 \| Volume 7 \| Article 1187 Frontiers in Microbiology \| www.frontiersin.org 5 In vitro Activity on Bioﬁlms Ferran et al. FIGURE 3 \| Bacterial reductions (mean ± SD) of S. aureus (A) or S. pseudintermedius (B) suspension (gray bars, in log10 CFU/mL) and bioﬁlm (black bars, in log10 CFU) after 15-h exposure to marboﬂoxacin (MAR) and clindamycin (CLI) alone or in combination with amoxicillin (AMX), cefalexin (CFX), or doxycycline (DOX). FIGURE 3 \| Bacterial reductions (mean ± SD) of S. aureus (A) or S. pseudintermedius (B) suspension (gray bars, in log10 CFU/mL) and bioﬁlm (black bars, in log10 CFU) after 15-h exposure to marboﬂoxacin (MAR) and clindamycin (CLI) alone or in combination with amoxicillin (AMX), cefalexin (CFX), or doxycycline (DOX). against a bioﬁlm of the same bacteria was poor (Claessens et al., 2015). applied in human medicine (Romling and Balsalobre, 2012; Claessens et al., 2015). The addition of a second drug had very slight beneﬁcial or even negative eﬀect on the bacterial killing. According to Jawetz laws (Jawetz, 1968, 1975), these results could perhaps have been anticipated for the combination of bactericidal and bacteriostatic drugs as for example the combination of doxycycline and cefalexin. However, the reduced bacterial activity of marboﬂoxacin after adding another bactericidal drug, such as amoxicillin or cefalexin, was quite unexpected even though similar results have recently been reported by Yang et al. (2015). Indeed, these authors showed that the eﬃcacy of a combination of ciproﬂoxacin and vancomycin on S. epidermidis bioﬁlm was less than that of ciproﬂoxacin alone (Yang et al., 2015). It can be hypothesized that the increased bacterial stress conferred by two drugs resulted in the formation of persister cells (Lewis, 2007). DISCUSSION Until suﬃcient data for each possible combination becomes available, this therapeutic strategy should probably be not recommended. By systematically comparing the bacterial counts in suspension and bioﬁlm, we found that the diﬀerence between the two populations was never more than 1 log10 CFU/mL even after exposure to antimicrobial drugs, whereas planktonic bacteria are supposed to be much more readily killed by drugs (Bjarnsholt et al., 2013). This suggests the possible existence of an equilibrium between planktonic and bioﬁlm bacteria, and that bacteria can be released from a bioﬁlm into suspension during antibiotic exposure or that planktonic bacteria associated with a bioﬁlm are not phenotypically the same as the planktonic bacteria exposed to drugs during a MIC determination. p g g Although amoxicillin, cefalexin and marboﬂoxacin are all classiﬁed as bactericidal in our experiments, marboﬂoxacin exhibited far greater activity than amoxicillin or cefalexin. This diﬀerence in bacterial killing may be due to the fact that ﬂuoroquinolones can kill non-dividing bacteria whereas beta- lactam drugs cannot (Drlica et al., 2008). We showed in our system that the bacterial population at the time of drug challenge exceeded 7.8 log10 CFU in bioﬁlm and 7.3 log10 CFU/mL in suspension and that very little growth occurred in the control wells between 7 and 24 h. This indicates that the bacterial population was in a stationary phase which would favor the activity of marboﬂoxacin over that of other drugs. The greater but limited in vitro activity of marboﬂoxacin on S. pseudintermedius, as compared to other drugs in our model, is diﬃcult to connect with clinical outcomes due to the fact that blinded randomized controlled investigations of systemic antimicrobial eﬃcacies in the treatment of canine pyoderma are very rare (Frank and Loeﬄer, 2012; Summers et al., 2012). However, the generally low antimicrobial activity of the tested drugs would probably be a main factor explaining the need for treatments of long duration to obtain a clinical cure of canine pyoderma and to prevent relapse. The poor activity of drugs used singly in our study and the absence of additive activity when used in combination suggest that systemic treatments alone are not the best way to target Staphylococcus spp., especially S. pseudintermedius. For this reason, we also explored an alternative treatment, i.e., external use of chlorhexidine. In our system, a 2% concentration of chlorhexidine was found to kill bioﬁlm bacteria after 5 min exposure. Frontiers in Microbiology \| www.frontiersin.org August 2016 \| Volume 7 \| Article 1187 REFERENCES Davies, D. (2003). Understanding bioﬁlm resistance to antibacterial agents. Nat. Rev. Drug Discov. 2, 114–122. doi: 10.1038/nrd1008 Batzias, G. C., Delis, G. A., and Athanasiou, L. V. (2005). Clindamycin bioavailability and pharmacokinetics following oral administration of clindamycin hydrochloride capsules in dogs. Vet. J. 170, 339–345. doi: 10.1016/j.tvjl.2004.06.007 Drlica, K., Malik, M., Kerns, R. J., and Zhao, X. (2008). Quinolone- mediated bacterial death. Antimicrob. Agents Chemother. 52, 385–392. doi: 10.1128/AAC.01617-06 Ferran, A. A., Toutain, P. L., and Bousquet-Melou, A. (2014). Comparison of the reduction in the antibacterial potency of a ﬂuoroquinolone conferred by a single mutation in the quinolone resistance-determining region or by the inoculum size eﬀect. Int. J. Antimicrob. Agents 44, 472–474. doi: 10.1016/j.ijantimicag.2014.07.007 Beco, L., Guaguere, E., Lorente Mendez, C., Noli, C., Nuttall, T., and Vroom, M. (2013). Suggested guidelines for using systemic antimicrobials in bacterial skin infections: part 2- - antimicrobial choice, treatment regimens and compliance. Vet. Rec. 172, 156–160. doi: 10.1136/vr.101070 Bjarnsholt, T., Ciofu, O., Molin, S., Givskov, M., and Hoiby, N. (2013). Applying insights from bioﬁlm biology to drug development - can a new approach be developed? Nat. Rev. Drug Discov. 12, 791–808. doi: 10.1038/nrd4000 Frank, L. A., and Loeﬄer, A. (2012). Meticillin-resistant Staphylococcus pseudintermedius: clinical challenge and treatment options. Vet. Dermatol. 23:e256. doi: 10.1111/j.1365-3164.2012.01047.x Guardabassi, L., Loeber, M. E., and Jacobson, A. (2004). Transmission of multiple antimicrobial-resistant Staphylococcus intermedius between dogs aﬀected by deep pyoderma and their owners. Vet. Microbiol. 98, 23–27. doi: 10.1016/j.vetmic.2003.09.021 Borio, S., Colombo, S., La Rosa, G., De Lucia, M., Damborg, P., and Guardabassi, L. (2015). Eﬀectiveness of a combined (4% chlorhexidine digluconate shampoo and solution) protocol in MRS and non-MRS canine superﬁcial pyoderma: a randomized, blinded, antibiotic-controlled study. Vet. Dermatol. 26:e372. doi: 10.1111/vde.12233 Hillier, A., Lloyd, D. H., Weese, J. S., Blondeau, J. M., Boothe, D., Breitschwerdt, E., et al. (2014). Guidelines for the diagnosis and antimicrobial therapy of canine superﬁcial bacterial folliculitis (Antimicrobial Guidelines Working Group of the International Society for Companion Animal Infectious Diseases). Vet. Dermatol. 25, e142–e163. doi: 10.1111/vde.12118 Bryan, J., Frank, L. A., Rohrbach, B. W., Burgette, L. J., Cain, C. L., and Bemis, D. A. (2012). Treatment outcome of dogs with meticillin-resistant and meticillin- susceptible Staphylococcus pseudintermedius pyoderma. Vet. Dermatol. 23:e365. doi: 10.1111/j.1365-3164.2012.01034.x Horner, C., Mawer, D., and Wilcox, M. (2012). Reduced susceptibility to chlorhexidine in staphylococci: is it increasing and does it matter? J. Antimicrob. Chemother. 67, 2547–2559. CONCLUSION This study provides further evidence that S. pseudintermedius and S. aureus bioﬁlms could be highly tolerant to veterinary drugs. Taking in mind that further in vivo investigations on eﬃcacy and resistance development are required, the topical administration of chlorhexidine could provide a promising alternative strategy avoiding the long-term systemic use of ineﬃcacious antimicrobial drugs in animals. Although, we did not investigate the development of resistance in the targeted staphylococci all of the tested antibiotic or biocide treatments can potentially induce resistance. The proportion of Methicillin Resistant S. pseudintermedius (MRSP) among S. pseudintermedius isolates from clinical infections in the USA and Europe has risen since 2000 (Frank and Loeﬄer, 2012) and a link between antimicrobial treatments within 30 days and MRSP infections has been identiﬁed in dogs (Weese et al., 2012). Biocide resistances can also develop during chlorhexidine treatment (Johnson et al., 2015). Resistance to chlorhexidine can be conferred by carriage of qac A/B or smr genes conding for eﬄux pumps. Interestingly, one study reported that, among 247 strains, the qac A/B and smr positive bacteria were more often resistant to some antimicrobial drugs including methicillin, ciproﬂoxacin, and vancomycin than negative ones (McNeil et al., 2015). This situation conﬁrms the urgent need to AUTHOR CONTRIBUTIONS Substantial contributions to the conception or design of the work and the acquisition, analysis, or interpretation of data for the work; AF, JL, P-LT, AB-M. Drafting the work and revising it critically for important intellectual content; AF, JL, P-LT, AB-M. Final approval of the version to be published; AF, JL, P-LT, AB-M; Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy and integrity of any part of the work are appropriately investigated and resolved; AF, JL, P-LT, AB-M. DISCUSSION Bioﬁlm bacteria of both strains were eradicated in 1 or 2 wells out of 3. These in vitro results may suggest that a topical shampoo might be more eﬀective than most systemic treatments, the limiting condition being that the application of a chlorhexidine shampoo should come in contact with all the bacteria (treatment of entire surface with no restriction to diﬀusion (hairs, crusts) in order to attain the bacteria). This excellent eﬃcacy of chlorhexidine is in agreement with another in vitro study which showed that a 4% solution of chlorhexidine killed S. pseudintermedius in less than 1 min (Lloyd and Lamport, 1999). Several clinical studies have also provided evidence of the eﬃcacy of chlorhexidine at least for superﬁcial pyoderma In view of the low antibacterial eﬀects of the ﬁrst and second-line drugs used in monotherapy, we then assessed the eﬃcacy of two-drug combinations, as is currently being August 2016 \| Volume 7 \| Article 1187 August 2016 \| Volume 7 \| Article 1187 Frontiers in Microbiology \| www.frontiersin.org 6 Ferran et al. In vitro Activity on Bioﬁlms (Bryan et al., 2012; Mueller et al., 2012) and similar eﬃcacy was observed when a topical chlorhexidine digluconate shampoo treatment applied twice weekly for 4 weeks was compared with a systemic administration of amoxicillin–clavulanic acid 25 mg/kg twice daily for 4 weeks (Borio et al., 2015). In addition to the killing activity of chlorhexidine on pathogenic bacteria, a topical treatment has the advantage of not impacting the digestive commensal ﬂora and therefore reducing the selection pressure for resistance in this microbiota. (Bryan et al., 2012; Mueller et al., 2012) and similar eﬃcacy was observed when a topical chlorhexidine digluconate shampoo treatment applied twice weekly for 4 weeks was compared with a systemic administration of amoxicillin–clavulanic acid 25 mg/kg twice daily for 4 weeks (Borio et al., 2015). In addition to the killing activity of chlorhexidine on pathogenic bacteria, a topical treatment has the advantage of not impacting the digestive commensal ﬂora and therefore reducing the selection pressure for resistance in this microbiota. implement the stewardship for antibiotic use in veterinary medicine. REFERENCES doi: 10.1093/jac/dks284 Claessens, J., Roriz, M., Merckx, R., Baatsen, P., Van Mellaert, L., and Van Eldere, J. (2015). 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https://openalex.org/W3163870205	http://journal.asu.ru/ssi/article/download/9568/7815	Russian	null	COMPARISON OF THE CONCEPTS OF THE LEVEL AND THE QUALITY OF LIFE: INCLUSIVE CONTENT	Society and security insights	2,020	cc-by	3,810	Сопоставление понятий уровня и качества жизни: субъективное наполнение Ж. Садыралиев Ж. Садыралиев Джалал-Абадский государственный университет имени Б. Осмонова, Джалал-Абад, Кыргызстан, e-mail: jandaraly1957@gmail.com Ж. Садыралиев Джалал-Абадский государственный университет имени Б. Осмонова, Джалал-Абад, Кыргызстан, e-mail: jandaraly1957@gmail.com DOI: 10.14258/ssi(2021)1-04 Анализируются и сопоставляются существующие в науке подходы к трактов- ке и содержанию категорий, характеризующих жизненную удовлетворенность, такие как «качество жизни», «уровень жизни», с целью их разграничения, выяв- ления сущностных основ и различий. Систематизированы и классифицированы показатели качества и уровня жизни. Сделан акцент на выявление актуальных проблем в определении качества жизни. Критически осмыслены методологии оценки благосостояния. Сделан вывод о необходимости субьективного подхода к оценке смысла понятий уровня и качества жизни в Кыргызстане. Ключевые слова: уровень жизни, качество жизни, измерение благосостояния SOCIETY AND SECURITY INSIGHTS SOCIETY AND SECURITY INSIGHTS УДК 330.322.5 Comparison of the concepts of the level and the quality of life: inclusive content J. Sadyraliev Jalal-Abad State University named after B. Osmonov, Jalal-Abad, Kyrgyzstan, e-mail: jandaraly1957@gmail.com J. Sadyraliev The article analyzes and compares existing approaches to the interpretation and defining content of categories that characterize life satisfaction, such as «quality of life», «standard of life» in order to differentiate them, identify essential foundations and differences. The indicators of quality and standard of living are systematized and classified. The emphasis is made on identifying urgent problems in determin- ing the quality of life. Welfare assessment methodologies are critically reviewed. The author makes conclusion about the need for a subjective approach to the assess- ment of the concept of the level and quality of life in Kyrgyzstan. Keywords: standard of living, quality of life, measurement of wellbeing Период времени, включающий конец XX — начало XXI в., в социально-эконо- мическом развитии мира характеризуется как начало крупного, глобального поворо- № 1 2021 58 БЕЗОПАСНОСТЬ И ИНТЕГРАЦИЯ В СТРАНАХ АЗИАТСКОГО РЕГИОНА та. В рамках информационного общества создается новый тип общественного потре- бления и образа жизни. В таких условиях возникает необходимость новой парадигмы экономического роста. А это, в свою очередь, требует совершенно иного значения, иной окраски сущности понятия «благосостояние», включая общественное и личное благополучие, и смежному к нему понятия «качество и уровень жизни», а также но- вого взгляда, который пересмотрит сложившиеся ранее взгляды на ценности, источ- ники и факторы концепции «экономический рост» и выявит их новые перспективы. та. В рамках информационного общества создается новый тип общественного потре- бления и образа жизни. В таких условиях возникает необходимость новой парадигмы экономического роста. А это, в свою очередь, требует совершенно иного значения, иной окраски сущности понятия «благосостояние», включая общественное и личное благополучие, и смежному к нему понятия «качество и уровень жизни», а также но- вого взгляда, который пересмотрит сложившиеся ранее взгляды на ценности, источ- ники и факторы концепции «экономический рост» и выявит их новые перспективы. «Качество и уровень жизни» в концепции благосостояния являются ее век- торной составляющей, т.е. характеризуется направленность развития благосостоя- ния, охватывая уровень, образ и условия жизнедеятельности и отражая как объек- тивные, так и субъективные их характеристики (Шевцов, 2012: 13). В последние годы понятия «уровень» и «качество жизни» заняли в обществен- ном мнении и научном обороте прочное положение. Растущий интерес к проблема- тике качества жизни свидетельствует о том, что общество озабочено уже не столько проблемами самосохранения, сколько вопросами устойчивого социального разви- тия и восстановления своей роли и места в мировом сообществе. Comparison of the concepts of the level and the quality of life: inclusive content Этот интерес об- условлен и процессами глобализации, которые диктуют необходимость создания достойных условий жизни не только для будущих, но и для ныне живущих поколе- ний (Шевцов, 2012: 14). Значимость проблемы уровня и качества жизни в Кыргыз- стане возрастает и в связи с тем, что высокий уровень рождаемости и относительно низкий уровень смертности сформировали трудоизбыточность, при которой пред- ложение рабочей силы превышает спрос на нее, основными причинами сохране- ния высокого уровня бедности выступают низкая занятость населения, миграция, ограниченность рабочих мест и источников получения доходов, неравномерность социально-экономического развития регионов и др. В научной среде происходит активный процесс перераспределения дис- циплинарных границ в условиях становления качественно нового общества, еще не сложился единый подход к содержанию понятия «качество жизни», его отли- чию от понятия «уровень жизни». Нет общепризнанной методологии и методики измерения «качества жизни», последнее рассматривается как общесоциологическое понятие. Одни исследования определяют его как уровень качества жизни, другие — как качество уровня жизни, третьи считают эти понятия не связанными между со- бой и принадлежащими к различным областям научного знания (Шевцов, 2012; 14). Результаты анализа, сравнивающего взгляды известных ученых на сущность и зна- чение вышеназванных понятий, приведены в таблице 1. Этот интерес обусловлен процессом глобализации, который требует созда- ния условий для достойной жизни не только будущим, но и нынешнему поколе- нию. Для Кыргызстана проблема качества и уровня жизни очень важна и она будет продолжать расти, поскольку человеческие ресурсы становятся одним из самых де- фицитных ресурсов из-за быстрого старения, низкой продолжительности жизни, постепенного сокращения популяции населения и усиления миграции. Из данных таблицы видно, что трудно дать одно общее определение, поскольку каждый из авторов имеет собственные представления о его содержании, по-своему № 1 2021 59 SOCIETY AND SECURITY INSIGHTS осуществляет трактовку сопряженных категорий. Тем не менее целесообразно обоб- щить разные определения, чтобы сблизить границы этих двух понятий, уточнить взаи- мосвязь между их характеристиками и содержательными различиями. Сделана попыт- ка сравнить систематически охарактеризованное содержание категорий и понятий, изучаемых ниже, в таблице 2. Сравнение основных параметров двух категорий позво- ляет сделать следующий вывод. Качество жизни — категория, которая шире понятия уровня жизни, которая уточняет его, раскрывает его содержание, расширяет понятие «уровень жизни» и одновременно включает в себя несколько компонентов-индикато- ров, показывающих уровень удовлетворенности населения аспектами своей жизни. Обзор теоретических подходов к содержанию понятий «качество жизни» и «уровень жизни» Источник Трактовка понятия «уровень жизни» Трактовка понятия «качество жизни» Беляева Л.А. Comparison of the concepts of the level and the quality of life: inclusive content Основные качественные характеристики понятий «качество жизни» и «уровень жизни» Характеристика Уровень жизни Качество жизни Объект оценки Совокупный объем доставки к потреблению экономических благ: товаров и услуг Совокупность всех условий жизни и обширного комплекса влияющих факторов Используемые методы и способы получения информации Классические методы эконо- мической статистики, базиру- ющиеся на документальном учете фактов Официальные статистические данные, опросы референтных групп, метод экспертных оценок Наличие объектив- но- субъективного элемента в оценке Объективные, стандартизи- рованные, рассчитываемые по определенной методологии С высокой долей субъективиз- ма, упор на индивидуальное, личностное психологическое восприятие с акцентом на ин- ституциональную среду Круг показате- лей, применяемых для оценки понятия Показатели общераспростра- ненные, их круг известен, обозначен и интерпретирован математической и социально- экономической статистикой Отсутствие строгого набо- ра показателей, их перечень многообразен, неограничен, постоянно расширяется, а ме- тодология корректируется: при использовании индексов зна- чимость может варьироваться в различных соотношениях Источник: (Сапир, Чистякова, 2020: 297–301). Фактически качество жизни может быть сравнительной характеристикой: на- БЕЗОПАСНОСТЬ И ИНТЕГРАЦИЯ В СТРАНАХ АЗИАТСКОГО РЕГИ Источник Трактовка понятия «уровень жизни» Трактовка понятия «качеств «вещей, используемых инди- видуумом в виде носителей благ» мера структуры всех отношени века, определяющая интенсивн деятельности, возможность сво и эффективного использовани гармония благ» J.E. Stiglitz, Am. Sen, J.- P. Fitoussi (2009) Делают акцент на том, что категория содержит ориента- цию на экономическое про- изводство, доходы, богатство и потребление Рассматривают как более широ понятие, включающее весь спе торов, оказывающих воздейств выходящее за рамки материаль ценно для людей в жизни; орие на устойчивое, стабильное бла ние настоящего и будущего по БЕЗОПАСНОСТЬ И ИНТЕГРАЦИЯ В СТРАНАХ АЗИАТСКОГО РЕГИОНА Рассматривают как более широкое понятие, включающее весь спектр фак- торов, оказывающих воздействие на то выходящее за рамки материального, что ценно для людей в жизни; ориентация на устойчивое, стабильное благосостоя- ние настоящего и будущего поколений Таблица 2. Основные качественные характеристики понятий «качество жизни» и «уровень жизни» Comparison of the concepts of the level and the quality of life: inclusive content (2009: 33–34) «…определяется условиями существования человека в сфере потребления и из- меряется через социально- экономические показатели общего благосостояния» «Комплексная характеристика условий жизнедеятельности населения, которая выражается в объективных показате- лях и субъективных оценках удовлет- ворения материальных, социальных и культурных потребностей и связана с восприятием людьми своего поло- жения в зависимости от культурных особенностей, системы ценностей и социальных стандартов…» Зубаревич Н.В. (2003: 12–13) «Ядром является достигну- тый уровень доходов и по- требления материальных благ и услуг, а не развитие возмож- ностей человека» «Способность индивида использовать ресурсы… для управления собствен- ной жизнью, т.е. в терминах расшире- ния возможностей выбора действий» Бобков В.Н. (2009: 26) «…представляет собой денеж- ную оценку ресурсов, необ- ходимых для обеспечения качества жизни личности, социальных групп и общества в целом» «Разнообразие способностей и удов- летворенных потребностей личности, социальных групп и общества в целом, предопределяющее их развитость и благосостояние» Жеребин В.М., Романов А.Н. (2013: 86–87) Уровень потребления матери- альных благ и услуг Обобщает понятие «уровня жизни», включая также удовлетворение духов- ных потребностей, здоровье, продол- жительность жизни, условия среды, окружающей человека, морально-психо- логический климат, душевный комфорт Меньшикова М.А., Коптева К.В. (2012: 5) Условная количественная ха- рактеристика качества жизни, мера общего количества «Совокупность условий, обеспечиваю- щих жизнь населения страны на каждом определенном этапе ее развития…; 60 № 1 2021 № 1 2021 № 1 2021 60 БЕЗОПАСНОСТЬ И ИНТЕГРАЦИЯ В СТРАНАХ АЗИАТСКОГО РЕГИОНА Источник Трактовка понятия «уровень жизни» Трактовка понятия «качество жизни» «вещей, используемых инди- видуумом в виде носителей благ» мера структуры всех отношений чело- века, определяющая интенсивность его деятельности, возможность свободного и эффективного использования благ; гармония благ» J.E. Stiglitz, Am. Sen, J.- P. Fitoussi (2009) Делают акцент на том, что категория содержит ориента- цию на экономическое про- изводство, доходы, богатство и потребление Рассматривают как более широкое понятие, включающее весь спектр фак- торов, оказывающих воздействие на то выходящее за рамки материального, что ценно для людей в жизни; ориентация на устойчивое, стабильное благосостоя- ние настоящего и будущего поколений Таблица 2. Основные качественные характеристики понятий «качество жизни» и «уровень жизни» Характеристика Уровень жизни Качество жизни Объект оценки Совокупный объем доставки к потреблению экономических благ: товаров и услуг Совокупность всех условий жизни и обширного комплекса влияющих факторов Используемые методы и способы получения информации Классические методы эконо- мической статистики, базиру- ющиеся на документальном учете фактов Официальные статистические данные, опросы референтных групп, метод экспертных оценок Наличие объектив- но- субъективного элемента в оценке Объективные, стандартизи- рованные, рассчитываемые по определенной методологии С высокой долей субъективиз- ма, упор на индивидуальное, личностное психологическое восприятие с акцентом на ин- ституциональную среду Круг показате- лей, применяемых для оценки понятия Показатели общераспростра- ненные, их круг известен, обозначен и интерпретирован математической и социально- экономической статистикой Отсутствие строгого набо- ра показателей, их перечень многообразен, неограничен, постоянно расширяется, а ме- тодология корректируется: при использовании индексов зна- чимость может варьироваться в различных соотношениях И (С Ч 2020 29 301) Источник: (Сапир, Чистякова, 2020: 297–301). Фактически качество жизни может быть сравнительной характеристикой: на- пример, оно может в одной стране быть выше, чем в другой, или в одном регионе выше, чем в другом регионе или стране. В то же время в обеих странах или в обоих регионах уровень жизни может не соответствовать специально вычисленным на- учным стандартам, он может оказаться ниже или выше таких стандартов. На наш № 1 2021 № 1 2021 61 SOCIETY AND SECURITY INSIGHTS взгляд, эти два понятия дополняют друг друга. Прчиной возникновения, употре- бления и широкого распространения термина «качество и уровень жизни» в обще- ствах с высокоразвитым уровнем потребления стало изменение механизма соци- ального развития общества. Поскольку единственным экономическим критериям развития на замену пришли критерии качества жизни, улучшение качества жизни будет способствовать росту человеческого потенциала, а человеческий потенциал, в свою очередь, выступает ключевым фактором экономического роста. В 1960-е гг. даже в самых развитых странах понятие «уровень жизни» использовалось весьма ограниченно, так как реалии жизни показали, что даже экономический рост и по- вышение уровня жизни не могут немедленно освободить общество от бедности, преступности, наркотиков, загрязнения окружающей среды, техногенных ката- строф и серьезных социальных потрясений. Анализ научной литературы по проблеме качества жизни позволяет сделать следующие выводы: исследование качества жизни в зарубежных странах проводит- ся исключительно на основе объективных условий жизни, уровня удовлетворенно- сти субъектов и граждан собственной жизнью (насколько они удовлетворены или не удовлетворены). Существует несколько моделей в данном направлении. Первая модель позволяет определять «качество жизни» как результат комби- нации различных статистических показателей, их смеси (Жеребин, Романов, 2013: 56). Основные качественные характеристики понятий «качество жизни» и «уровень жизни» Предполагается, что эта модель оказывает решающее влияние на улучшение ка- чества жизни социального субъекта и используется для объективной количествен- ной оценки материальных условий жизни. Исходя из наших рассуждений, условно разделим массив разнородных пока- зателей на несколько групп. Показатели уровня жизни, для расчета которых используют данные офици- альной статистики. К ним относятся: фактическое конечное потребление домашних хозяйств на душу населения, среднедушевые денежные доходы населения, реальные располагаемые денежные доходы населения, реальная начисленная заработная плата, величина прожиточного минимума, реальный средний размер назначенных пенсий, численность населения с денежными доходами ниже величины прожиточного мини- мума, коэффициент фондов, индекс концентрации доходов (Шевцов, 2012: 151–163). Отдельные «измерители» факторов качества жизни — это качество образования, объем выбросов, ВВП, общая удовлетворенность жизнью, баланс между трудом (рабо- той) и отдыхом, доступность медицины, климат и природные условия, уровень дискри- минации, ожидаемая продолжительность здоровой жизни, социальные связи и прочее. Кроме вышеназванных существуют интегральные показатели, агрегирующие ряд частных количественных показателей. Это могут быть как статистические показа- тели уровня жизни, так и оценки восприятия развитости и эффективности институ- циональной системы в целом и ее фундаментальных компонентов. Примером может выступать индекс человеческого развития (ИЧР — Human development Index), а также индекс лучшей жизни (Better life index), рассчитываемый Организацией экономическо- го сотрудничества и развития для 34 стран-участниц, включающий 11 составляющих: жилищные условия, доход, работа, общество, образование, окружающая среда, граж- № 1 2021 № 1 2021 62 БЕЗОПАСНОСТЬ И ИНТЕГРАЦИЯ В СТРАНАХ АЗИАТСКОГО РЕГИОНА данская активность, здравоохранение, удовлетворенность жизнью, безопасность, ба- ланс между работой и досугом (OECD, 2020). Еще один международный инструмент — Международный индекс счастья (Happy Planet Index) был разработан Фондом новой экономики в 2006 г. и представляет собой отношение произведения показателей «бла- гополучие жизни» (основан на опросе по шкале «лестница Х. Кантрила» и оценивается от 0 до 10), «ожидаемая продолжительность жизни» и показателя неравенства полу- ченных результатов (выражается в процентах) к «экологическому следу» (усредненный масштаб земель на душу населения, необходимых для обеспечения привычной модели потребления, измеряемый в глобальных гектарах на человека (гектары земли со средне- мировой биопродуктивностью) (Сапир, Чистякова, 2020: 297–301). В качестве дополнительного направления выделяется вторая модель, обозна- чаемая как модель «значительного качества жизни». Эта модель основана на пред- ставлении о том, что истинный смысл качества жизни возникает в результате отражения результатов конкретных материальных условий, созданных в жизни, эмоционального состояния, чувств и т.д. на субъективных чувствах граждан. Основные качественные характеристики понятий «качество жизни» и «уровень жизни» При этом предполагается, что сложный характер связи между объективными и субъек- тивными условиями жизни создает впечатление сокрытия, неподтверждения того, что люди, живущие в хороших материальных условиях, более удовлетворены жиз- нью, чем те, кто живет в плохих условиях. По мнению авторов упомянутого нами доклада, именно субъективные оценки дают фундаментальную информацию о благосостоянии, которое лучше самих людей не способен измерить никто. Эти оценки разумно изучать по нескольким аспектам (удовлетворенность жизнью, положительные аффекты, отрицательные аффекты). Тем не менее при этом способе существует ряд трудностей: воздействие временных явлений и настроений на оценки людей, роль количества времени, отведенного на от- вет, порядок вопросов и т.д. Особенно интересен тот факт, что дифференциация субъ- ективных ощущений и выводов, основанных на количественных подходах, может выступать одной из причин того, что граждане нередко не доверяют официальной государственной статистике в отношении вопросов благосостояния и качества жиз- ни. В докладе также отмечается, что подобные проблемы усиливаются разнообразием форм неравенства, причем необходимо учитывать взаимовлияние отдельных типов неравенства и их усугубление друг друга (Аналитический центр, 2018). По нашему мнению, данная проблема тоже актуальна для Кыргызской Республики, где имеет место неравенство не только между разными социальными группами, но и между регионами, ввиду чего гражданами нередко высказываются сомнения относительно правдивости усредненных значений. Значимыми являются возможности людей сде- лать свой выбор для реализации поставленных конкретно ими задач и достижения собственных целей. Здесь можно говорить о человеческом потенциале и о расшире- нии масштабов свободы действий, принятия решений, исходя из того, что для кон- кретного человека важно, что составляет ценность и имеет приоритет. Еще одна интересная позиция, обсуждаемая в проведенных исследованиях, — воздействие личностных активностей. Деятельность во многом определяет оценку жизни, особенно это касается основных, ключевых видов деятельности в жизни чело- № 1 2021 № 1 2021 63 SOCIETY AND SECURITY INSIGHTS века: какие-то активности приносят большее удовольствие, способствуя повышению качества жизни, а какие-то — меньшее, и в этом случае встает вопрос о необходи- мости улучшения условий труда и обеспечения их приемлемого уровня. Комиссией ООН предлагается использовать ограниченное число индикаторов, которые были бы адаптированы ко всем странам. Затрагивается необходимость исследования неопла- чиваемого домашнего труда, сокращающего свободное время и заключающего в сво- ем распределении гендерное неравенство (Сапир, Чистякова, 2020: 297–301). Особое значение имеют социальные связи (социальный капитал), которые во многом предопределяют субъективное благополучие, ведь от них зависят оценки многих других факторов. Основные качественные характеристики понятий «качество жизни» и «уровень жизни» В качестве примера можно привести результаты одного из исследований, согласно которому социальная изоляция увеличивает риск преждев- ременной смерти примерно в той же степени, что и курение, при том что внешние эффекты социальных связей в основном положительны: расширение связей повы- шает уровень благосостояния как самого человека, так и его окружения. Данный фактор как никогда важен в условиях пандемии. В обстоятельствах, когда люди во всем мире вынуждены ограничивать социальное взаимодействие, ухудшается их эмоциональное состояние, сужается спектр возможностей, преумножаются страхи перед неопределенностью, рисками и нестабильностью. Особое внимание уделено выявлению не всегда очевидных, глубинных причинно-следственных связей между аспектами благополучия и проблемой адекватного агрегирования факторов, в том числе с целью составления индексов с учетом обоснованного выбора весов компо- нентов (Сапир, Чистякова, 2020: 297–301). Таким образом, в результате изучения состояния исследований в области оцен- ки и измерения показателей качества жизни можно сделать следующие выводы: «Качество жизни» — емкое по смысловому наполнению, центральное поня- тие, объединяющее ряд смежных категорий, максимально охватывающее аспекты, возможности жизни и благополучия населения и призванное наиболее реалистич- но, всеобъемлюще отразить удовлетворенность людей своей жизнью. На сегодняшний день не представляется возможным определить единый универсальный измеритель, который охватывал бы все компоненты и процессы, формирующие систему качества жизни. Скорее всего, функция оптимального из- мерения может быть возложена на совокупность показателей: абсолютных и отно- сительных, качественных и количественных, объективных и субъективных, а также рейтинговых совокупностей. Для оценки качества жизни во главу угла должен быть поставлен субъектив- ный (результат опросов самих людей) подход: обособленные от денежной состав- ляющей личные мнения людей, основывающиеся на восприятии ими социальных условий, индивидуального «самочувствия», комфортности в социуме, достатка, сча- стья, стабильности. Необходимо оценивать взаимовлияние факторов друг на друга. 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Metodologicheskij podhod Vserossijskogo centra urovnya zhizni k izucheniyu i ocenke kachestva i urovnya zhizni naseleniya [Methodological approach of the All-Russian Center for Living Standards to the study and assessment of the quality and standard of living of the population]. Vestnik Voronezhskogo gosudarstvennogo universiteta. Seriya: Ekonomika i upravlenie, no. 2, 26–36. Zherebin, V.M., Romanov, A.N. (2013). Kachestvo zhizni naseleniya v kontekste mezh- dunarodnyh sopostavlenij [Quality of life of the population in the context of international comparisons]. Ekonomika. Nalogi. Pravo, no. 6, 86–93. Zubarevich, N.V. (2003). Social’noe razvitie regionov Rossii v perehodnyj period [Social de- velopment of Russian regions during the transition period] Doctoral Thesis) M.: MGU im. M.V. Lomonosova. Men’shikova, M.A., Kopteva, K.V. (2012). Teoretiko-metodologicheskie podhody k ocenke urovnya i kachestva zhizni naseleniya [Theoretical and methodological approaches to the level and quality of life of the population]. Vestnik Kurskoj gosudarstvennoj sel’skohozyajst- vennoj akademii, no. 6, 5–7. Sapir, E.V., Chistyakova, A.A. (2020). Smyslovye razlichiya ponyatij urovnya i kachestva zhizni: inklyuzivnoe napolnenie [Semantic differences in the concepts of the level and quality of life: inclusive content]. Finansovaya ekonomika, no. 10, 297–301. Chelovek i innovacii: Doklad o chelovecheskom razvitii v Rossijskoj Federacii. Analitich- eskij centr pri Pravitel’stve Rossijskoj Federacii. [People and innovation. Report on human development in the Russian Federation, 2018. Analytical center for the Government of the Russian Federation]. Available at: https://ac.gov.ru/files/publication/a/19663.pdf (accessed 1 February 2021). Shevcov, P.A. (2012). Metodologiya statisticheskogo issledovaniya vliyaniya urovnya i kachestva zhizni naseleniya na formirovanie chelovecheskogo kapitala [The methodology of statistical investigation of the influence of the level and quality of the population’s life on the formation of human capital (Doctoral Thesis)]. M.: Moskovskij gosudarstvennyj universitet ekonomiki, statistiki i informatiki. Happy Planet Index 2016. Available at: http://happyplanetindex.org/about#how (accessed 1 February 2021). REFERENCES Stiglitz, J.E., Sen, A., Fitoussi, J.P. (2009). Report by the commission on the measurement of economic performance and social progress. URL: http://citeseerx.ist.psu.edu/viewdoc/ download?doi=10.1.1.215.58&rep=rep1&type=pdf (accessed 1 February 2021). OECD. Better life index. Available at: http://www.oecdbetterlifeindex.org/ru/about/ru_ whats-your-better-life-index (accessed 1 February 2021). The Global Competitiveness Report 2019. World Economic Forum. Available at: http:// www3.weforum.org/docs/WEF_TheGlobalCompetitivenessReport2019.pdf (accessed 1 February 2021). The Inclusive Development Index 2018. Summary and Data Highlights. World Econom- ic Forum. Available at: https://www.weforum.org/reports/the-inclusive-development-in- dex-2018. (accessed 1 February 2021). № 1 2021 66
https://openalex.org/W4390760459	https://www.nomos-elibrary.de/10.5771/1435-2869-2023-2-131.pdf?download_full_pdf=1&page=1	English	null	Editorial	SEER. South-East Europe review for labour and social affairs/SEER	2,023	cc-by	1,338	Editorial This second issue of the SEER Journal for Labour and Social Affairs in Eastern Europe for 2023, ‘Albania and Romania in focus’, collects articles drawn from empirical evidence from both countries. For Albania, the articles deal with poverty, gender equality and political representation. As a follow-up to the previous issue of SEER, we also continue the analysis of the vulnerable situation of Romanian workers in the EU, notably in Germany and Austria, based on publications by the journal Sociologie Românească. We start the issue with Peter Scherrer reporting on the Balkans Civil Society Forum organised by the European Economic and Social Committee and held in Thessaloniki, Greece, in October 2023. His article addresses the keynote presenta- tions, featuring also interviews conducted by the author exploring issues linked mostly to the question of EU enlargement. As a result of the war in Ukraine and the applications of both Ukraine and Moldova for EU membership, EU enlargement is back on the agenda giving some hope for the Western Balkans, as well. The article by Ela Golemi and Anxhela Llaftiu evaluates gender equality in politics and the labour market in Albania. The authors provide an assessment of the progress made in Albania to achieve gender balance in two main areas – political decision-making and the labour market – in terms of the UN’s Sustainable Develop- ment Goals (SDGs). Statistical analysis was carried out using data from the Institute of Statistics of Albania (INSTAT) and the European Institute for Gender Equality (EIGE). The findings indicate that women remain under-represented in parliament as well as in the labour market. Progress was made prior to Covid-19 in the context of gender gaps in employment, but with a temporary setback in 2020 due to the pandemic. The results also reveal that, as regards the gender pay gap and women’s political participation in the Albanian cabinet, Albania performs better than the EU27 average. Ardita Borici and Alba Kruja conduct an analysis of poverty and its measurement for Albania based on INSTAT data using surveys such as the Household Budget Sur- vey and Statistics on Income and Living Conditions (EU-SILC). The article follows different approaches of measuring poverty and provides a comparative overview of poverty indicators across the EU as well as in candidate and accession countries. They find that Albania has the highest share of households at risk of poverty among this broad group. SEER Journal for Labour and Social Affairs in Eastern Europe p. 131 – 133 // i / / Editorial The article by Belina Bedini looks at different aspects of political representation in Albania examining, among others, if there is a direct relationship between the size of the population and the number of members of the representative body. The author argues that, due to massive shifts in the population in the last 25 years, due mostly to changing emigration and birth rates, it is time to reconsider the number of representatives in the Albanian Parliament. The article ‘“Slaves” without coercion?’ by Christian Sperneac-Wolfer, Andrei Botorog and Ferdinand Sutterlüty looks at the situation of tens of thousands of Ro- manian migrants in the German construction sector. The authors provide an insight SEER Journal for Labour and Social Affairs in Eastern Europe p. 131 – 133 h //d i /10 5771/1435 2869 2023 2 131 24 10 2024 06 40 19 2/2023 131 Editorial into the risky and exploitative nature of the conditions under which they work and how this is reflected in their negative self-evaluation of their engagements as ‘slave labour’. Based on qualitative interviews with and participant observation among Ro- manian construction site workers, the article reconstructs four work classifications, each of which offers a different reason to make hard manual labour plausible in the eyes of the workers themselves. Without direct physical coercion, these ideas motivate workers to take on work that they themselves criticise as ‘slave labour’. into the risky and exploitative nature of the conditions under which they work and how this is reflected in their negative self-evaluation of their engagements as ‘slave labour’. Based on qualitative interviews with and participant observation among Ro- manian construction site workers, the article reconstructs four work classifications, each of which offers a different reason to make hard manual labour plausible in the eyes of the workers themselves. Without direct physical coercion, these ideas motivate workers to take on work that they themselves criticise as ‘slave labour’. The article by Paul Sperneac-Wolfer looks at the self-organised mobility of Romanian workers in relation to exploitation in highly segmented labour markets, using the example of the Austrian fresh food sector. The author shows how grow- ers capitalise particularly on labour intermediation to maintain the resilience and profitability of local agricultural businesses in the Austrian agricultural market. The resulting workplace regime ensnares workers in a two-fold exploitability: not only is their labour power subject to labour extraction but so are their interpersonal relations. Editorial Dear Readers of the SEER Journal, let us inform you that, after 26 volumes, from next year our journal will go online only, and we will stop printing and distributing hard copies. This will allow us to be more flexible but, equally importantly, to continue to improve our main role as a platform of exchange. We will be in touch further, not least as regards a website address where you can continue to read current and past issues of the Journal, in due course. In the meantime, we wish all readers a peaceful and prosperous 2024; and we look forward to seeing you and hearing further from you via our online platform in the next 12 months and beyond. December 2023 December 2023 Editorial This analysis draws attention to the persistent exploitation of migrant workers and their reproductive capacities across segmented labour markets. Daniela Ana and Ştefan Voicu analyse strikes, unionisation and the role of organic intellectuals in the German meat industry after the German Arbeitsschutzkon- trollgesetz in 2021 aimed to grant equal employment conditions to the majority of workers in slaughterhouses. The law created new avenues for trade unions to gain more members and organise industry-level negotiations for better wages and a collective agreement. The article explores the lessons from three slaughterhouse strikes where the authors highlight the role of organic intellectuals – Gramsci’s term for those who grasp class interests and who generate cohesion and self-awareness of their class’s position in society – showing that they can be instrumental in articu- lating resistance but that they can also be co-opted by employers to manufacture con- sent within the company. In conclusion, the authors reflect on the labour struggles and negotiations of 2021 and on the benefits that the presence of a general collective agreement managed to bring to workers, as well as its limitations. The article by Eleonora Ushatova-Kalinova examines the Office of the National Ombudsman as a possible channel for the exercise of citizens’ participation in democratic life in Bulgaria. The exercise of civic participation through the Office is illustrated by describing a case drawn from Bulgarian practice – the protests of workers and employees in defence of their rights to unpaid wages and other compensation in 2017 – which resulted in changes to the legislation championed by the Ombudsman. Finally, a book review by Christophe Solioz discusses the book by Attila Àgh: Awaking Europe in the triple global crisis. The birth pangs of the emerging Europe (2021). Àgh cites the triple crisis facing Europe – socioeconomic crisis, climate crisis and Covid-19 crisis – to which Christophe Solioz adds a fourth: the security crisis with reference to the war in Ukraine. These add up, in sum, to a systemic crisis which Solioz argues points to an impending Age of Transition affecting not just south-eastern Europe, whose countries are particularly vulnerable, but leading to the establishment of a new world order in which Europe will need to reconceptualise itself. SEER Journal for Labour and Social Affairs in Eastern Europe 2/2023 132 December 2023 Béla Galgóczi Calvin Allen 2/2023 2/2023 SEER Journal for Labour and Social Affairs in Eastern Europe 133
https://openalex.org/W4285596582	https://europepmc.org/articles/pmc9324545?pdf=render	English	null	Vietnamese Dalbergia tonkinensis: A Promising Source of Mono- and Bifunctional Vasodilators	Molecules/Molecules online/Molecules annual	2,022	cc-by	14,526	Article Vietnamese Dalbergia tonkinensis: A Promising Source of Mono- and Bifunctional Vasodilators Nguyen Manh Cuong 1,* , Ninh The Son 2 , Ngu Truong Nhan 3, Yoshiyasu Fukuyama 4 , Amer Ahmed 5,† , Simona Saponara 5 , Alfonso Trezza 6, Beatrice Gianibbi 6 , Ginevra Vigni 6, Ottavia Spiga 6 and Fabio Fusi 6,* 1 Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology (VAST), 1 Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology (VAS 18 Hoang Quoc Viet, Caugiay, Hanoi 122100, Vietnam Institute of Natural Products Chemistry, Vietnam Academ 18 Hoang Quoc Viet, Caugiay, Hanoi 122100, Vietnam y y gy 18 Hoang Quoc Viet, Caugiay, Hanoi 122100, Vietnam 18 Hoang Quoc Viet, Caugiay, Hanoi 122100, Vietnam 2 Institute of Chemistry, Vietnam Academy of Science and Technology (VA Hanoi 122100, Vietnam; yamantson@gmail.com 2 Institute of Chemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi 122100, Vietnam; yamantson@gmail.com 2 Institute of Chemistry, Vietnam Academy of Science an 2 Institute of Chemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi 122100, Vietnam; yamantson@gmail.com Hanoi 122100, Vietnam; yamantson@gmail.com y g 3 Faculty of Science and Technology, Tay Nguyen University, 567 Le Duan, Ea Tam, 3 Faculty of Science and Technology, Tay Nguyen University, 567 Le Duan, Ea Tam, Buon Ma Thuot 630000, Vietnam; ntnhan@ttn.edu.vn 3 Faculty of Science and Technology, Tay Nguyen Universi Buon Ma Thuot 630000, Vietnam; ntnhan@ttn.edu.vn 3 Faculty of Science and Technology, Tay Nguyen Unive Buon Ma Thuot 630000, Vietnam; ntnhan@ttn.edu.vn 4 Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan; fukuyama@ph.bunri-u.ac.jp y p jp 5 Dipartimento di Scienze della Vita, Università di Siena, Via A. Moro 2, 53100 Siena, Italy; 5 Dipartimento di Scienze della Vita, Università di Siena, Via A. Moro 2, 53100 Siena, Italy; aa.biotechiub@gmail.com (A.A.); simona.saponara@unisi.it (S.S.) p aa.biotechiub@gmail.com (A.A.); simona.saponara@unisi.it (S.S.) 6 Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena, Via A. Moro 2, 53100 Siena, Italy; alfonso.trezza2@unisi.it (A.T.); beatrice.gianibbi@student.unisi.it (B.G.); ginevra.vigni@student.unisi.it (G.V.); ottavia.spiga@unisi.it (O.S.) p g * Correspondence: nmcuong@inpc.vast.vn (N.M.C.); fabio.fusi@unisi.it (F.F.) † Present address: Department of Biosciences, Biotechnology, and Biopharmaceutics, University of Bari, 70125 Bari, Italy. Abstract: Hypertension is a risk factor for cardiovascular diseases, which are the main cause of morbidity and mortality in the world. Citation: Cuong, N.M.; Son, N.T.; Nhan, N.T.; Fukuyama, Y.; Ahmed, A.; Saponara, S.; Trezza, A.; Gianibbi, B.; Vigni, G.; Spiga, O.; et al. Vietnamese Dalbergia tonkinensis: A Promising Source of Mono- and Bifunctional Vasodilators. Molecules 2022, 27, 4505. https://doi.org/ 10.3390/molecules27144505 Academic Editor: Ruifeng Zhang Received: 20 June 2022 Accepted: 12 July 2022 Published: 14 July 2022 Citation: Cuong, N.M.; Son, N.T.; Nhan, N.T.; Fukuyama, Y.; Ahmed, A.; Saponara, S.; Trezza, A.; Gianibbi, B.; Vigni, G.; Spiga, O.; et al. Vietnamese Dalbergia tonkinensis: A Promising Source of Mono- and Bifunctional Vasodilators. Molecules 2022, 27, 4505. https://doi.org/ 10.3390/molecules27144505 Academic Editor: Ruifeng Zhang Received: 20 June 2022 Accepted: 12 July 2022 Published: 14 July 2022 Citation: Cuong, N.M.; Son, N.T.; Nhan, N.T.; Fukuyama, Y.; Ahmed, A.; Saponara, S.; Trezza, A.; Gianibbi, B.; Vigni, G.; Spiga, O.; et al. Vietnamese Dalbergia tonkinensis: A Promising Source of Mono- and Bifunctional Vasodilators. Molecules 2022, 27, 4505. https://doi.org/ 10.3390/molecules27144505 Academic Editor: Ruifeng Zhang Received: 20 June 2022 Accepted: 12 July 2022 Published: 14 July 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Keywords: biochanin A; CaV1.2 channel; Dalbergia tonkinensis Prain; KCa1.1 channel; vasodilation Article Vietnamese Dalbergia tonkinensis: A Promising Source of Mono- and Bifunctional Vasodilators In the search for new molecules capable of targeting KCa1.1 and CaV1.2 channels, the expression of which is altered in hypertension, the in vitro vascular effects of a series of ﬂavonoids extracted from the heartwoods, roots, and leaves of Dalbergia tonkinensis Prain, widely used in traditional medicine, were assessed. Rat aorta rings, tail artery myocytes, and docking and molecular dynamics simulations were used to analyse their effect on these chan- nels. Formononetin, orobol, pinocembrin, and biochanin A showed a marked myorelaxant activity, particularly in rings stimulated by moderate rather than high KCl concentrations. Ba2+ currents through CaV1.2 channels (IBa1.2) were blocked in a concentration-dependent manner by sativanone, 3′-O-methylviolanone, pinocembrin, and biochanin A, while it was stimulated by ambocin. Sati- vanone, dalsissooside, and eriodictyol inhibited, while tectorigenin 7-O-[β-D-apiofuranosyl-(1→6)-β- D-glucopyranoside], ambocin, butin, and biochanin A increased IKCa1.1. In silico analyses showed that biochanin A, sativanone, and pinocembrin bound with high afﬁnity in target-sensing regions of both channels, providing insight into their potential mechanism of action. In conclusion, Dalber- gia tonkinensis is a valuable source of mono- and bifunctional, vasoactive scaffolds for the development of novel antihypertensive drugs. molecules molecules 1. Introduction Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Cardiovascular diseases, a group of disorders that affect blood vessels and the heart, represent the leading cause of death worldwide, which resulted in 17.8 million deaths in 2017 [1]. They are generally preceded by many simultaneous modiﬁable risk factors (e.g., hypertension, hyperglycaemia, obesity, and oxidative stress) that may be associated with genetic factors and/or unhealthy lifestyles (e.g., physical inactivity, smoking, inap- propriate diet, mental stress, and ageing; [2]. Approximately 1.4 billion people worldwide suffer from hypertension [3], a well-known mosaic of pathophysiological disturbances, https://www.mdpi.com/journal/molecules Molecules 2022, 27, 4505. https://doi.org/10.3390/molecules27144505 Molecules 2022, 27, 4505 2 of 23 the successful treatment of which is possible given the availability of multiple antihyper- tensive drug classes (characterised by various mechanisms of action) with limited side effects. This guarantees a complementary pharmacological treatment. As an alternative, bi- or multifunctional drugs, capable of simultaneously targeting more than one mecha- nism underpinning hypertension, may represent an ideal therapeutic regimen, perhaps accompanied by fewer unwanted effects. p y Although numerous antihypertensive drugs exist, neither therapy based on their association nor ﬁxed-dose combinations have provided satisfactory results due to the fact of poor compliance and variable and unpredictable pharmacokinetics and pharmacody- namics in different patients. In this context, multitarget agents, which combine two or more pharmacophores capable of modulating different mechanisms involved in the pathogen- esis of hypertension, play a fundamental role. They are single-chemical entities, which reduces the risk of drug–drug interactions and allow for a simpler prediction of patient pharmacokinetics and pharmacodynamics. In recent years, the pharmaceutical industry and academic research have, again, di- rected their attention to the natural world, which has always represented an important source of active agents. This occurred following the failure of libraries of synthetic com- pounds and computational chemistry, which were thought to lead to the discovery of numerous new drugs. Currently, molecules of natural origin play an important role in the development of novel drugs: they are compounds that have a unique chemical diversity that translates into interesting pharmacological properties. Their effectiveness is due to the fact of their three-dimensional chemical structure and a complex steric property (the so-called “privileged structure”), which also offers an advantage in terms of selectivity for molecular targets. 2.2. General Experimental Procedures 2.2. General Experimental Procedures 1H-NMR (500 MHz) and 13C-NMR (125 MHz) spectra were measured on a Bruker Avance 500 MHz spectrometer. ESI-MS was obtained from a Varian FT-MS spectrometer and MicroQ-TOF III (Bruker Daltonics, Ettlingen, Germany). Column chromatography was carried out on silica gel (Si 60 F254, 40–63 mesh, Merck, St. Louis, MO, USA). All solvents were redistilled before use. Precoated thin-layer chromatography (TLC) plates (Si 60 F254) were used for analytical purposes. Compounds were visualised under UV light (254 and 365 nm) and by spraying plates with 10% H2SO4 followed by heating with a heat gun. 1. Introduction This makes natural molecules multitarget compounds and, there- fore, a valuable starting point for the development of new drugs, especially in the ﬁeld of antihypertensive therapy, where approximately 64% of drugs are derived from natural products [4]. p In this scenario, the most interesting insights are provided by traditional medicine that makes use of natural extracts to treat patients otherwise not reachable by “conven- tional” medicine. The Asian continent plays a fundamental role, with China certainly in the foreground but also smaller countries, such as Vietnam, where the use of over 4000 plants in traditional medicine is essential for health. These include Dalbergia species, belonging to a large genus of trees, shrubs, lianas, and woody climbers of the Fabaceae family (Leguminosae), the extracts of which are widely used for the treatment of cardio- vascular disease [5,6]. Dalbergia odorifera, a Chinese herbal medicine, has been used for promoting blood circulation, relieving pain, and eliminating blood stasis [7]. Thus far, more than 50 compounds have been isolated and identiﬁed from Dalbergia tonkinensis Prain, local name “Sưa Ðỏ”, one of 27 Dalbergia species distributed in Vietnam; these belong to the classes of ﬂavonoids, polyphenols, sesquiterpenes, arylbenzofurans, and quinones [8–13]. Flavonoids, in particular, are the main chemical components extracted from duramen [8,10–12,14]. Accumulating evidence obtained from in vitro and in vivo research, clinical trials, and epidemiological studies point to ﬂavonoids as beneﬁcial tools ca- pable of improving cardiovascular health and ameliorating the risk factors associated with cardiovascular diseases [15]. As a previous study demonstrated the vasorelaxant activity of an open-ring neoﬂavonoid, namely, R-(-)-3′-hydroxy-2,4,5-trimethoxydalbergiquinol [16], extracted from the heartwood of D. tonkinensis, this work aimed to evaluate a series of 12 ﬂavonoids (including ﬂavanones, isoﬂavanones, and isoﬂavones; Figure 1) extracted from the heartwoods, roots, and leaves of D. tonkinensis as potential vasodilators endowed with KCa1.1 channel-stimulating and/or CaV1.2 channel-blocking activity. The results indicate that D. tonkinensis can be considered a valuable source of vasoactive compounds, with biochanin A, in particular, being the most interesting structure, which could be used as a scaffold for the development of novel antihypertensive drugs. 3 of 23 Molecules 2022, 27, 4505 Figure 1. Structures of the molecules isolated from the heartwoods, roots, and leaves of Dalbergia tonk- inensis. Compounds are grouped according to the subclass of ﬂavonoids to which they belong. 2.1. Plant Material Heartwoods of Dalbergia tonkinensis Prain were collected in Quang Binh Province (2016), and whole plants (i.e., leaves, heartwoods, and roots) over ten years old were collected in Daklak Province (2016), Vietnam. The plant was identiﬁed by Botanist, Nguyen Quoc Binh, Vietnam National Museum of Nature, VAST, Hanoi, Vietnam. Voucher speci- mens (C-575, Quang Binh; C-561 and C-612, Daklak, Vietnam) were deposited at the Depart- ment of Bioactive Products, Institute of Natural Products Chemistry, VAST, Hanoi, Vietnam. 2.5.1. Aorta Rings Preparation 2.5.1. Aorta Rings Preparation The thoracic aorta was gently cleansed of adipose and connective tissues and cut into 3 mm wide rings. They were mounted in organ baths between two parallel, L-shaped, stainless-steel hooks, one ﬁxed in place and the other connected to an isometric trans- ducer. Rings were allowed to equilibrate for 60 min in KHS (composition in mM: 118 NaCl, 4.75 KCl, 1.19 KH2PO4, 1.19 MgSO4, 25 NaHCO3, 11.5 glucose, and 2.5 CaCl2 and gassed with a 95% O2–5% CO2 gas mixture to create a pH of 7.4) under a passive tension of 1 g. During this equilibration period, the solution was changed every 15 min. Isometric tension was recorded using a digital PowerLab data acquisition system (PowerLab 8/30; ADInstru- ments). Ring viability was assessed by recording the response to 0.3 µM phenylephrine and 60 mM KCl [17]. Where needed, the endothelium was removed by gently rubbing the lumen of the ring with a forceps tip. This procedure was validated by adding 10 µM acetylcholine at the plateau of phenylephrine-induced contraction: a relaxation greater than 70% or less than 10% denoted the presence or absence of functional endothelium, respectively [18]. 2.3. Extraction and Isolation D. tonkinensis dried leaves (2 kg) were extracted using a Soxhlet extractor (methanol: 10 L, 4.5 h) to yield a black crude extract (300 g), which was suspended in methanol–water 1:1 (v/v) and partitioned with chloroform and ethyl acetate to obtain the appropriate fractions. The chloroform fraction (LC, 60.5 g) was fractionated using column chromatography over silica gel (chloroform–methanol, 99:1 (v/v)) to give 8 fractions (LC1-LC8). Fraction LC1 (15.2 g) was puriﬁed over silica gel column chromatography (n-hexane–acetone, 10:1 (v/v)) to successfully yield biochanin A (45.7 mg). Orobol (15.6 mg) was obtained from fraction LC6. The water-residual fraction (LW, 95.5 g) was subjected to Diaion HP-20 column chro- matography, eluted with water–methanol (1:0, 1:3, and 0:1, v/v), providing 3 fractions Molecules 2022, 27, 4505 4 of 23 (LW1-LW3). LW2 (4.57 g) was chromatographed on silica gel column chromatography (chloroform–methanol–water, 3:1:0.1 (v/v/v)) to yield dalsissooside (20.6 mg) and 9 frac- tions (LW21-LW29). Fraction LW28 (1.9 g) was then separated by HPLC (Cosmosil 5C18- AR-II column, methanol–water ratio of 7:3 (v/v, 1.5 mL/min), UV 254 nm) to yield ambocin (4.6 mg). (LW1-LW3). LW2 (4.57 g) was chromatographed on silica gel column chromatography (chloroform–methanol–water, 3:1:0.1 (v/v/v)) to yield dalsissooside (20.6 mg) and 9 frac- tions (LW21-LW29). Fraction LW28 (1.9 g) was then separated by HPLC (Cosmosil 5C18- AR-II column, methanol–water ratio of 7:3 (v/v, 1.5 mL/min), UV 254 nm) to yield ambocin (4.6 mg). D. tonkinensis dried roots (2 kg) were extracted using a Soxhlet extractor (ethanol: 10 L, 4.5 h) to yield a black crude extract (200 g), which was resuspended in a methanol–water ration of 1:1 (v/v) and partitioned with dichloromethane and ethyl acetate to produce the corresponding fractions. The ethyl acetate fraction (RE, 30.5 g) was separated by column chromatography using a chloroform–methanol–water ration of 3:1:0.1 (v/v/v) providing 7 fractions (RE1-RE7). Tectoridin (5.1 mg) and tectorigenin-7-O-[β-D-apiofuranosyl-(1→6)- β-D-glucopyranoside (25.2 mg) were obtained from fraction RE4 (7.9 g) using a Sephadex LH-20 column (methanol–water, 1:1 (v/v)) [12]. Other ﬂavonoids, namely, pinocembrin [8], 3’-O-methylviolanone [10], biochanin A, butin, eriodictyol [11], sativanone, and formononetin [14] were isolated from the heart- woods of D. tonkinensis as previously described. 2.4. Animals All the study procedures were in strict accordance with the European Union Guidelines for the Care and the Use of Laboratory Animals (European Union Directive 2010/63/EU) and approved by the Animal Care and Ethics Committee of the University of Siena and the Italian Department of Health (7DF19.N.TBT). Male Wistar rats (300–350 g) were purchased from Charles River Italia (Calco, Italy) and maintained in an animal house facility at 25 ± 1 ◦C with a 12:12 h dark–light cycle and access to standard chow diet and water ad libitum. Animals were anaesthetised with an isoﬂurane (4%) and O2 gas mixture using Fluovac equipment (Harvard Apparatus, Holliston, MA, USA), decapitated, and exsanguinated. Thoracic aorta and tail main artery were immediately isolated and placed in physiological solutions (i.e., the modiﬁed Krebs–Henseleit solution (KHS) and an external solution, respectively) and prepared as detailed below. 2.5. Functional Experiments 2.7. Whole-Cell Patch-Clamp Recordings An Axopatch 200B patch-clamp ampliﬁer (Molecular Devices Corporation, Sunnyvale, CA, USA) was used to generate and apply voltage pulses to the clamped cells and record the corresponding membrane currents. Recording electrodes were pulled from borosilicate glass capillaries (WPI, Berlin, Germany) and ﬁre-polished to obtain a pipette resistance of 2–5 MΩwhen ﬁlled with an internal solution. At the beginning of each experiment, the junction potential between the pipette and bath solution was electronically adjusted to zero. Current signals, after compensation for whole-cell capacitance and series resistance (between 70% and 75%), were low-pass ﬁltered at 1 kHz and digitised at 3 kHz before being stored on a computer hard disk. Electrophysiological responses were tested at room temperature (20–22 ◦C) [21]. 2.5.2. Effect of Dalbergia Isolates on KCl-Induced Contraction Endothelium-denuded aorta rings were precontracted electromechanically with 25 mM or 60 mM KCl. Once the contraction reached a plateau, drugs were added cumulatively into the organ bath to assess their vasodilating activity. At the end of the concentration–response curve, 1 µM nifedipine followed by 100 µM sodium nitroprusside was added to test the functional integrity of smooth muscle. Vasodilation was calculated as a percentage of the contraction induced by KCl (taken as 100%). Molecules 2022, 27, 4505 5 of 23 2.6. Cell Isolation Procedure 2.6. Cell Isolation Procedure The tail main artery was dissected free of its connective tissue, and smooth muscle cells were freshly isolated under the following conditions. A 5 mm long piece of artery was incubated at 37 ◦C for 40–45 min in 2 mL of 0.1 mM Ca2+ external solution (consisting of (in mM): 130 NaCl, 5.6 KCl, 10 HEPES, 20 glucose, 1.2 MgCl2, and 5 Na-pyruvate; pH 7.4) containing 20 mM taurine, which replaced an equimolar amount of NaCl, 1.35 mg/mL collagenase (type XI), 1 mg/mL soybean trypsin inhibitor, and 1 mg/mL BSA. This so- lution was gently bubbled and stirred with a 95% O2–5% CO2 gas mixture as previously described [19]. Cells stored in 0.05 mM Ca2+ external solution containing 20 mM taurine and 0.5 mg/mL BSA at 4 ◦C under normal air were used for experiments within two days after isolation [20]. Current through CaV1.2 Channel (IBa1.2) Recordings Cells were continuously superfused with an external solution containing 0.1 mM Ca2+ and 30 mM tetraethylammonium (TEA) using a peristaltic pump (LKB 2132, Bromma, Sweden) at a ﬂow rate of 400 µL/min. The conventional whole-cell patch-clamp method was employed to voltage-clamp smooth muscle cells. The internal solution (pCa 8.4) consisted of (in mM): 100 CsCl, 10 HEPES, 11 EGTA, 2 MgCl2, 1 CaCl2, 5 Na-pyruvate, 5 succinic acid, 5 oxaloacetic acid, 3 Na2ATP, and 5 phosphocreatine; the pH was adjusted to 7.4 with CsOH. IBa1.2, recorded in an external solution containing 30 mM TEA and 5 mM Ba2+, was elicited with 250 ms clamp pulses (0.067 Hz) to 0 mV from a Vh of −50 mV. Ba2+ was used in place of Ca2+ as the charge carrier to increase the current density that, in rat tail artery myocytes, is usually around 1 pA/pF (corresponding to an average current amplitude of 30–50 pA; see [22]. Data were collected once the current amplitude stabilised (usually 7–10 min after the whole-cell conﬁguration was obtained). Under these conditions, the current, which did not run down during the following 40 min [23], was carried almost entirely by CaV1.2 channels [22]. The K+ currents were blocked with 30 mM TEA in the external solution and Cs+ in the internal solution. Current values were corrected for leakage and residual outward currents using 10 µM nifedipine, which completely blocked IBa1.2. The osmolarity of the 30 mM TEA- and 5 mM Ba2+-containing external solution (320 mosmol, adjusted with NaCl if required) and that of the internal solution (290 mosmol) were measured with an osmometer (Osmostat OM 6020, Menarini Diagnostics, Florence, Italy). 2.9. K+ Current through KCa1.1 Channel (IKCa1.1) Recordings 2.9. K+ Current through KCa1.1 Channel (IKCa1.1) Recordings IKCa1.1 (registration period 500 ms) was measured over a range of test potentials from −20 to 70 mV from a Vh of −40 mV. This Vh limited the contribution of voltage-dependent K+ channels to the overall whole-cell current. Data were collected once the current ampli- tude stabilised (usually 6–10 min after the whole-cell conﬁguration was obtained). IKCa1.1 did not run down during the following 20–30 min under the present experimental condi- tions [24]. The external solution for IKCa1.1 recordings contained (in mM): 145 NaCl, 6 KCl, 10 glucose, 10 HEPES, 5 Na-pyruvate, 1.2 MgCl2, 0.1 CaCl2, and 0.003 nicardipine (pH 7.4). Molecules 2022, 27, 4505 6 of 23 The internal solution contained (in mM): 90 KCl, 10 NaCl, 10 HEPES, 10 EGTA, 1 MgCl2, and 6.41 CaCl2 (pCa 7.0; pH 7.4). The osmolarity of the external and internal solutions was 310 mosmol and 265 mosmol, respectively. The current–voltage relationships were calculated based on the values recorded over the last 400 ms of each test pulse (leakage corrected). The IKCa1.1 was isolated from the other currents as well as corrected for leakage using 1 mM TEA, a speciﬁc blocker of KCa1.1 channels [25]. 2.10. Drugs and Chemicals The chemicals used included collagenase (type XI), trypsin inhibitor, BSA, TEA chlo- ride, HEPES, taurine, phenylephrine, acetylcholine, nifedipine, nicardipine (Sigma Chim- ica, Milan, Italy), and sodium nitroprusside (Riedel-De Haën AG, Seelze-Hannover, Ger- many). All other substances were of analytical grade and used without further puriﬁcation. Phenylephrine was solubilised in 0.1 M HCl. Nifedipine and nicardipine, dissolved di- rectly in ethanol, and Dalbergia isolates, dissolved directly in DMSO, were diluted at least 1000 times before use. Control experiments conﬁrmed that no response was observed in vascular preparations when DMSO or ethanol, at the ﬁnal concentration used in the above dilutions (0.1%, v/v), was added alone (data not shown). 2.11. Statistical Analysis Analysis of the data was accomplished using pClamp 9.2.1.8 software (Molecular Devices Corporation, Sunnyvale, CA, USA), LabChart 7.3.7 Pro (PowerLab; ADInstruments, Castle Hill, Australia), and GraphPad Prism version 5.04 (GraphPad Software Inc., San Diego, CA, USA). Data are reported as the mean ± SEM; n is the number of cells or rings analysed (indicated in parentheses), isolated from at least three animals. Statistical analyses and signiﬁcance, as measured by repeated measures ANOVA (followed by Dunnett’s post hoc test) or the Student’s t-test for paired samples (two-tailed), were obtained using GraphPad Prism version 5.04 (GraphPad Software Inc.). In all comparisons, p < 0.05 was considered signiﬁcant. The pharmacological response to drugs, described in terms of potency (IC50 value, i.e., the drug concentration decreasing the maximum response by 50%) and efﬁcacy (Emax value, i.e., the maximum response achieved with the highest concentration tested), was obtained by nonlinear regression analysis. For IBa1.2 recordings, the bottom of the concentration–response curve to morin −1 was constrained to the value recorded in the presence of the speciﬁc blocker nifedipine, taken as a 0% current amplitude. 2.12. Structural Resource The Rattus norvegicus CaV1.2 channel’s subunit α1C’s 3D structure was achieved with a homology modelling procedure as described by Trezza et al. [26]. The rabbit inactive CaV1.1 channel’s 3D structure (PDB code 6JPA) in complex with its blocker verapamil, recently obtained through cryoEM by Zhao et al. [27], was selected as a template to build the CaV1.2 3D model and explore the structural and energetic features of the compounds binding to the channel. The primary structure of the Rattus norvegicus KCa1.1 channel was downloaded from the UniProt Database [28] (UniProt ID—Q62976-), and it was used as a query sequence for a multiple sequences alignment (MSA) carried out by Clustal Omega, implemented in PyMOD3.0 [29], choosing the Protein Data Bank (pdb) as a database; all algorithm parameters were used by default. As evidenced by the MSA results, the Cryo-EM structure of the Ca2+-bound hsSlo1-beta4 channel (in the open state) complex (PDB code: 6V22) was the best template [30], showing a cover and identity of 90.2% and 99.4%, respectively. Then, 6V22 was identiﬁed as a template to rebuild the 3D structure of the Rattus norvegicus KCa1.1 channel, using the Modeller tool implemented in PyMOD3.0 [29]. The validity of the 3D structure was assessed using Ramachandran plot and PROCHECK analyses as previously described [31]. The 3D structures of biochanin A, sativanone, and pinocembrin (compound CIDs: 5280373, 13886678, and 68071, respectively) were downloaded in the sdf format using the PubChem database [32]. The compounds were sketched and then prepared by the Molecules 2022, 27, 4505 7 of 23 7 of 23 LigPrep tool, assigning charges with Epik at pH 7.00 ± 1.00 [33]. The channel’s 3D model structures were converted into the pdbqt format as described in a previous work [34]. LigPrep tool, assigning charges with Epik at pH 7.00 ± 1.00 [33]. The channel’s 3D model structures were converted into the pdbqt format as described in a previous work [34]. 2.13. Docking and Classical Molecular Dynamics Simulations To identify the potential binding pose of compounds on the CaV1.2 and KCa1.1 chan- nels, in silico molecular docking and ﬂexible sampling were applied using the glide stan- dard precision (SP) protocol [35]. Input charges of compounds were retained, and amide bond conformations were allowed to vary. Strain correction terms were applied to the glide scoring function, and Epik state penalties were computed for the ﬁnal docking score. All other options were set to default. The Receptor Grid Generation tool from Schrödinger 2019-2 was used to generate a box able to enclose all CaV1.2 and KCa1.1 binding-pocket residues. In brief, a box of 22 Å for each dimension was generated for the CaV1.2 channel, enclosing a known blocker binding region of the protein [27]. To investigate the potential mechanism of action, a classical molecular dynamics (cMD) simulation of 100 ns was per- formed on the channel bound to the compounds as previously reported [26]. A box of 18 Å for each dimension was generated for the KCa1.1 channel, enclosing a stimulator binding re- gion as previously described [36]. To further investigate the channel/molecule interactions within the binding site, compounds were prepared as suggested by Semenya et al. [37], creating a phase database, and minimizing the output of 100 conformers per ligand. The structures were charged according to the Epik tool at pH 7.00 ± 1.00. Speciﬁed chirality and the 8 lowest energy stereoisomers (if present) were retained. Up to 4 low-energy 5- and 6-membered ring conformations were generated. All high-energy conformers/tautomers were discarded. Interaction network analyses and the energy contribution of the binding residues were evaluated through computational alanine-scanning mutagenesis, performed using the P.L.I.P. tool [38] and ABS scan [39], respectively. The GROMACS 2019.3 package was used to carry out and analyse the cMD trajectories [40]. PyMOL v2.5 was used as the molecular graphics system to generate the ﬁgures (The PyMOL Molecular Graphics System, version 2.5, Schrödinger, LLC, New York, NY, USA). 3. Results Effects of Dalbergia isolates on KCl-induced contraction of rat aorta rings. (A,B) Traces of vascular smooth muscle tension (representative of 4–6 similar experiments) showing the relaxation developed in response to cumulative concentrations of biochanin A (µM), added at the plateau of (A) 60 mM (K60) or (B) 25 mM KCl (K25) used to contract the preparations. The effect of 10 µM nifedipine (nife) and 100 µM sodium nitroprusside (SNP) is also shown. Horizontal bars indicate time (in min) and vertical bars muscle tension (expressed as mg). (C,D) Concentration–response curves for Dalbergia isolates in endothelium-denuded rings precontracted with either (C) 60 or (D) 25 mM KCl. In the ordinate scale, the response is reported as a percentage of the initial tension induced by KCl. Data points represent the mean ± SEM (n = 1–8). Figure 2. Effects of Dalbergia isolates on KCl-induced contraction of rat aorta rings. (A,B) Traces of vascular smooth muscle tension (representative of 4–6 similar experiments) showing the relaxation developed in response to cumulative concentrations of biochanin A (µM), added at the plateau of (A) 60 mM (K60) or (B) 25 mM KCl (K25) used to contract the preparations. The effect of 10 µM nifedipine (nife) and 100 µM sodium nitroprusside (SNP) is also shown. Horizontal bars indicate time (in min) and vertical bars muscle tension (expressed as mg). (C,D) Concentration–response curves for Dalbergia isolates in endothelium-denuded rings precontracted with either (C) 60 or (D) 25 mM KCl. In the ordinate scale, the response is reported as a percentage of the initial tension induced by KCl. Data points represent the mean ± SEM (n = 1–8). developed in response to cumulative concentrations of biochanin A (µM), added at the plateau of (A) 60 mM (K60) or (B) 25 mM KCl (K25) used to contract the preparations. The effect of 10 µM nifedipine (nife) and 100 µM sodium nitroprusside (SNP) is also shown. Horizontal bars indicate time (in min) and vertical bars muscle tension (expressed as mg). (C,D) Concentration–response curves for Dalbergia isolates in endothelium-denuded rings precontracted with either (C) 60 or (D) 25 mM KCl. In the ordinate scale, the response is reported as a percentage of the initial tension induced by KCl. Data points represent the mean ± SEM (n = 1–8). 3. Results Former phytochemical investigations of the heartwoods, leaves, and roots of D. tonk- inensis resulted in the isolation of more than 35 flavonoids. The chemical structures of the compounds here investigated were previously identified by NMR analysis and literature com- parison including the flavones: butin, eriodictyol [41], and pinocembrin [42]; the isoflavanones: sativanone and 3′-O-methylviolanone [10]; the isoflavones: biochanin A, formononetin [43], and orobol [44]; the isoflavone glycosides: ambocin [45], dalsissooside [46], tectoridin, and tectorigenin-7-O-[β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside] [11] (Figure 1). g p y g py g The compounds under study were assessed on aorta rings devoid of endothelium and precontracted by either 25 or 60 mM KCl, an assay to discriminate between Ca2+ antagonist and K+ channel opener agent [47]. Figure 2 shows the representative traces of the effects of biochanin A on the contrac- tion induced by either 60 (panel A) or 25 mM KCl (panel B). The addition of cumulative concentrations of the compound on the plateau of KCl-induced active tone caused a concentration-dependent relaxation that was more evident when the degree of membrane depolarisation was reduced (i.e., at 25 mM KCl). As summarised in Figure 2C,D and Table 1, both isoﬂavanones (i.e., sativanone and 3′-O-methylviolanone), the ﬂavanone pinocembrin and, to a lesser extent, the isoﬂavones, formononetin and biochanin A, were the most effective vasorelaxant agents on both types of contractions, efﬁcacy being indi- rectly correlated to the extracellular concentration of KCl. Orobol and, to a lesser extent, eriodictyol, relaxed only 25 mM KCl-induced contractions. All other molecules assessed were ineffective (Table 1). 8 of 23 Molecules 2022, 27, 4505 Figure 2. Effects of Dalbergia isolates on KCl-induced contraction of rat aorta rings. (A,B) Traces of vascular smooth muscle tension (representative of 4–6 similar experiments) showing the relaxation developed in response to cumulative concentrations of biochanin A (µM), added at the plateau of (A) 60 mM (K60) or (B) 25 mM KCl (K25) used to contract the preparations. The effect of 10 µM nifedipine (nife) and 100 µM sodium nitroprusside (SNP) is also shown. Horizontal bars indicate time (in min) and vertical bars muscle tension (expressed as mg). (C,D) Concentration–response curves for Dalbergia isolates in endothelium-denuded rings precontracted with either (C) 60 or (D) 25 mM KCl. In the ordinate scale, the response is reported as a percentage of the initial tension induced by KCl. Data points represent the mean ± SEM (n = 1–8). Figure 2. 3. Results In a ﬁrst series of experiments, the effects of biochanin A on IBa1.2, recorded in rat tail artery myocytes following a depolarisation step to 0 mV from a Vh of −50 mV, were investigated. After IBa1.2 reached a stable value, biochanin A was added at cumulative concentrations that produced a concentration-dependent decrease in the current ampli- tude (Figure 3A). The data obtained with biochanin A and the other Dalbergia isolates are summarised in Figure 3B–D and Table 1. Each subclass of ﬂavonoids displayed at least one active blocker: pinocembrin among ﬂavanones (panel B); both isoﬂavanones (i.e., sati- vanone and 3′-O-methylviolanone) assessed (panel C); biochanin A among the isoﬂavones (panel D). Only ambocin caused an approximately 20% increase in IBa1.2 at the 10 µM concentration that, however, was not observed either at lower or higher concentrations. IBa1.2 evoked at 0 mV from a Vh of −50 mV activated and then declined with a time course that could be ﬁtted by a monoexponential function. Only the two isoﬂavanones and biochanin A caused a signiﬁcant concentration-dependent acceleration of the τ of inactivation (Figure 4). However, the τ of activation was not modiﬁed by any of the Dalbergia isolates (see as an example Figure 4). Molecules 2022, 27, 4505 9 of 23 Table 1. Effects of Dalbergia isolates on KCl-induced contraction in rat aorta rings and on rat tail artery myocyte KCa1.1 and CaV1.2 channel currents. Rat aorta Rings Rat Tail Artery Myocytes Flavonoids IKCa1.1 IBa1.2 25 mM KCl 60 mM KCl 30 µM 100 µM IC50 (µM) IC50 (µM) Emax (%) IC50 (µM) Emax (%) Flavanones Butin N.D. 17.4 ± 6.8 (4) N.D. 3.3 ± 2.2 * (6) 32.5 ± 33.7% (3) 73.5 ± 55.2% (3) N.D. Eriodictyol N.D. 32.3 ± 19.9 (4) N.D. 3.0 ± 3.0 (3) -29.2% (1) −21.1% (1) N.D. Pinocembrin 8.0 ± 1.1 (5) 94.0 (1) N.D. 54.1 ± 9.9 (7) -23.2 ± 15.4% (5) −43.1 ± 18.2% (5) 32.7 ± 2.4 (5) Isoﬂavanones Sativanone 4.2 ± 0.7 (5) 93.6 (1) 18.9 ± 4.7 * (8) 78.5 ± 3.2 (6) 14.5 ± 16.7% (6) −5.2 ± 20.6% (6) 24.9 ± 5.5 (5) 3′-O-Methylviolanone 17.6 ± 8.3 (5) 79.4 (2) N.D. 66.6 ± 11.3 (5) −20.0 ± 13.1% (5) −26.3 ± 11.1% (5) 27.9 ± 6.3 (5) Isoﬂavones and isoﬂavone glycosides Biochanin A 23.4 ± 7.9 (6) 81.0 ± 6.0 (4) N.D. 3. Results 37.4 ± 12.1* (6) 51.4 ± 39.1% (6) 205.6 ± 111.6% (6) 28.2 ± 2.2 (5) Formononetin 7.7 ± 1.6 (6) 90.7 ± 2.7 (6) N.D. 36.9 ± 13.1* (6) −3.6 ± 8.6% (5) −12.7 ± 10.9% (5) N.D. Orobol 29.3 ± 11.6 (3) 79.3 ± 6.2 (3) N.D. 3.0 (2) 0.8 ± 7.7% (4) −5.1 ± 25.3% (4) N.D. Ambocin N.D. 15.5 ± 11.5 (4) N.D. 2.4 ± 2.1 (3) 74.0 ± 41.7% (4) 127.5 ± 82.3% (4) N.D. Dalsissooside N.D. 6.4 ± 2.4 (3) N.D. 2.1 ± 2.1 (3) −19.3 ± 6.2% (5) −32.5 ± 9.5% (5) N.D. Tectoridin N.D. 18.4 ± 9.2 (3) N.D. 2.0 ± 2.0 (3) 2.9 ± 6.2% (6) 2.9 ± 12.2% (6) N.D. Tectorigenin-7-O-[β-D- apiofuranosyl-(1→6)-β-D- glucopyranoside] N.D. 3.5 ± 1.5 (3) N.D. 0.6 ± 4.8 (3) 34.3 ± 11.3% (5) 58.8 ± 11.5% (5) N.D. Potency (expressed as IC50 value) and efﬁcacy (Emax, expressed as percent maximal relaxation) are mean ± S.E.M. (in parentheses the number of independent replicates). For IKCa1.1 data represent the percentage change of current amplitude caused by either 30 µM or 100 µM concentrations at 70 mV depolarization pulse. N.D.: not detectable. * p < 0.05 vs 25 mM KCl, Student’s t test for unpaired samples. rat aorta rings and on rat tail artery myocyte KCa1.1 and CaV1.2 channel currents. 10 of 23 Molecules 2022, 27, 4505 Figure 3. Effects of Dalbergia isolates on IBa1.2 in single tail artery myocytes: (A) average traces (recorded from 5 cells) of IBa1.2, elicited with 250 ms clamp pulses to 0 mV from a Vh of −50 mV, measured in the absence (control) or presence of cumulative concentrations of biochanin A; (B–D) concentration-dependent effects of (B) ﬂavanones, (C) isoﬂavanones, and (D) isoﬂavones. On the ordinate scale, current amplitude is reported as a percentage of the value recorded just before the addition of the ﬁrst concentration of Dalbergia isolate. The curves show the best ﬁt of the points. Data points are the mean ± SEM (n = 1–6). Figure 3. Effects of Dalbergia isolates on IBa1.2 in single tail artery myocytes: (A) average traces (recorded from 5 cells) of IBa1.2, elicited with 250 ms clamp pulses to 0 mV from a Vh of −50 mV, measured in the absence (control) or presence of cumulative concentrations of biochanin A; (B–D) concentration-dependent effects of (B) ﬂavanones, (C) isoﬂavanones, and (D) isoﬂavones. 3. Results On the ordinate scale, current amplitude is reported as a percentage of the value recorded just before the addition of the ﬁrst concentration of Dalbergia isolate. The curves show the best ﬁt of the points. Data points are the mean ± SEM (n = 1–6). A biophysical analysis was carried out to elucidate the interaction between the most effective compound—biochanin A—and CaV1.2 channels. The current–voltage relation- ships (Figure 5A) show that 30 µM of biochanin A signiﬁcantly decreased the peak inward current in the range of membrane potential values from −30 to 40 mV without, however, changing the maximum of the curve observed at 10 mV. g g Figure 5B illustrates the time course of the effects of 30 µM biochanin A on the current recorded at 0.067 Hz from a Vh of −50 mV to a test potential of 0 mV. After IBa1.2 reached steady values, the addition of biochanin A to the bath solution produced a gradual decrease in the current amplitude that reached a plateau in approximately 8 min. Washout of the compound brought the current amplitude back to 66% of the control value. Biochanin A-induced inhibition of IBa1.2 was not affected by the membrane potential. In fact, when Vh shifted to −80 mV, the residual IBa1.2 (38.6 ± 7.8% of the control, n = 5) was similar to that recorded at a Vh of −50 mV (25.9 ± 5.4% of the control, n = 5; p = 0.22). The voltage dependence of biochanin A inhibition was further investigated by analysing the steady-state inactivation and activation curves for IBa1.2. The steady-state activation curves (Figure 5C), calculated from the current–voltage relationships shown in panel A, were ﬁtted with the Boltzmann equation. Biochanin A neither shifted the 50% activation potential (−3.72 ± 1.50 mV, control; −4.14 ± 2.15 mV, 30 µM biochanin A, n = 5; p = 0.77, Student’s t-test for paired samples) nor affected the slope factor (6.43 ± 0.40 mV and 6.34 ± 0.56 mV, respectively; p = 0.79). Conversely, biochanin A signiﬁcantly shifted the steady-state inactivation curve to more negative potentials (Figure 5C). The 50% inacti- vation potential changed from −37.10 ± 3.76 mV (n = 6, control) to −50.23 ± 2.61 mV Molecules 2022, 27, 4505 11 of 23 11 of 23 (30 µM biochanin A; p = 0.0051). 3. Results The slope factor, however, was not affected by biochanin A (−12.58 ± 2.28 mV, −9.07 ± 0.83 mV, respectively; p = 0.14). The shift of the inactivation curve caused by 30 µM biochanin A led to a marked reduction in the Ba2+ window current that at −10 mV, for example, showed a relative amplitude of 0.02 compared to the relative amplitude of 0.06 observed under control conditions. y µ that at −10 mV, for example, showed a relative amplitude of 0.02 compared to the relative amplitude of 0.06 observed under control conditions. Figure 4. Effect of sativanone, 3′-O-methylviolanone, and biochanin A on IBa1.2 kinetics of single tail artery myocytes. Time constant for activation (τact) and inactivation (τinact) measured in the absence or presence of various concentrations of (A) sativanone, (B) 3′-O-methylviolanone, and (C) biochanin A from a Vh of either −50 or −80 mV. Columns represent the mean ± SEM (n = 5–6). * p < 0.05 vs. control; one-way ANOVA and Dunnett post hoc test. # p < 0.05, one-way ANOVA. Figure 4. Effect of sativanone, 3′-O-methylviolanone, and biochanin A on IBa1.2 kinetics of single tail artery myocytes. Time constant for activation (τact) and inactivation (τinact) measured in the absence or presence of various concentrations of (A) sativanone, (B) 3′-O-methylviolanone, and (C) biochanin A from a Vh of either −50 or −80 mV. Columns represent the mean ± SEM (n = 5–6). * p < 0.05 vs. control; one-way ANOVA and Dunnett post hoc test. # p < 0.05, one-way ANOVA. 12 of 23 Molecules 2022, 27, 4505 Figure 5. Voltage dependency of biochanin A-induced inhibition of IBa1.2 in single (A) Current–voltage relationships, recorded from a Vh of −50 mV, constructed (control) and in the presence of 30 µM biochanin A. Data points are the mean ± SE vs. control, Student’s t-test for paired samples. (B) Time course of IBa1.2 inh 30 µM biochanin A. The drug was applied at the time indicated by the arrow, were recorded during a typical depolarisation from −50 to 0 mV, applied every subsequently normalised according to the current recorded just before biochan effect of 10 µM nifedipine is also shown. Data points are the mean ± SEM (n = Figure 5. Voltage dependency of biochanin A-induced inhibition of IBa1.2 in single tail artery myocytes. 3. Results (A) Current–voltage relationships, recorded from a Vh of −50 mV, constructed before the addition (control) and in the presence of 30 µM biochanin A. Data points are the mean ± SEM (n = 5). * p < 0.05 vs. control, Student’s t-test for paired samples. (B) Time course of IBa1.2 inhibition induced by 30 µM biochanin A. The drug was applied at the time indicated by the arrow, and peak currents were recorded during a typical depolarisation from −50 to 0 mV, applied every 15 s (0.067 Hz) and subsequently normalised according to the current recorded just before biochanin A addition. The effect of 10 µM nifedipine is also shown. Data points are the mean ± SEM (n = 5). (C) The effect of biochanin A on the voltage dependence of CaV1.2 channel activation and inactivation. Steady-state Figure 5. Voltage dependency of biochanin A-induced inhibition of IBa1.2 in single tail artery myocytes. (A) Current–voltage relationships, recorded from a Vh of −50 mV, constructed before the addition (control) and in the presence of 30 µM biochanin A. Data points are the mean ± SEM (n = 5). * p < 0.05 vs. control, Student’s t-test for paired samples. (B) Time course of IBa1.2 inhibition induced by 30 µM biochanin A. The drug was applied at the time indicated by the arrow, and peak currents were recorded during a typical depolarisation from −50 to 0 mV, applied every 15 s (0.067 Hz) and subsequently normalised according to the current recorded just before biochanin A addition. The effect of 10 µM nifedipine is also shown. Data points are the mean ± SEM (n = 5). (C) The effect of biochanin A on the voltage dependence of CaV1.2 channel activation and inactivation. Steady-state Molecules 2022, 27, 4505 13 of 23 13 of 23 inactivation curves were obtained using a double-pulse protocol. Once various levels of the condition- ing potential had been applied for 5 s, followed by a short (5 ms) return to a Vh of −80 mV, a test pulse (250 ms) to 10 mV was delivered to evoke the current. The delay between the conditioning potential and the test pulse allowed for the full or near-complete deactivation of the channels, simultaneously avoiding partial recovery from inactivation. Steady-state inactivation curves in the absence (control) or presence of 30 µM biochanin A were ﬁtted to the Boltzmann equation. Peak current values were used. ↓: inhibition; ↔: no effect; ↑: stimulation. 3. Results The current measured during the test pulse was plotted against the membrane potential and is expressed as availability. Steady-state activation curves were obtained from the current–voltage relationships in panel A and ﬁtted to the Boltzmann equation. Data points are the mean ± SEM (n = 5–6). Dalbergia isolates were assessed for their effects on IKCa1.1. Under the conditions used in the present experiments, the outward current mostly consisted of iberiotoxin-sensitive IKCa1.1 [48]. Figure 6A shows the traces of IKCa1.1 elicited with clamp pulses to 70 mV from a Vh of −40 mV, under control conditions and after the cumulative addition of 30 and 100 µM biochanin A. The ﬂavonoid caused a signiﬁcant concentration-dependent stimulation of the current, which was observed in the range of membrane potential 20–70 mV (Figure 6B). The effects of the other compounds assessed are shown in Table 1. Both isoﬂavanones were almost ineffective or slightly reduced the current amplitude. Similar behaviour was shown by eriodictyol and pinocembrin, while the other ﬂavanone, butin, at the maximal concen- tration tested, stimulated IKCa1.1 by 73%. Among the remaining isoﬂavones, only ambocin and tectorigenin 7-O-[β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside] were capable of stimulating the current amplitude. g p The overall effects of the compounds are summarised in Table 2. Table 2. Effects of Dalbergia isolates on the different settings analysed. Flavonoids Rat Aorta Rings Rat Tail Artery Myocytes 25 mM KCl 60 mM KCl IKCa1.1 IBa1.2 Flavanones Butin ↔ ↔ ↑↑ ↓ Eriodictyol ↓ ↔ ↔ ↓ Pinocembrin ↓↓↓ ↓↓ ↓ ↓↓↓ Isoﬂavanones Sativanone ↓↓↓ ↓↓↓ ↔ ↓↓↓ 3′-O-Methylviolanone ↓↓↓ ↓↓ ↓ ↓↓↓ Isoﬂavones and isoﬂavone glycosides Biochanin A ↓↓↓ ↓ ↑↑↑↑↑↑↑ ↓↓↓ Formononetin ↓↓↓ ↓ ↔ ↓ Orobol ↓↓↓ ↔ ↔ ↓ Ambocin ↔ ↔ ↑↑↑↑↑ ↔ Dalsissooside ↔ ↔ ↓ ↔ Tectoridin ↔ ↔ ↔ ↔ Tectorigenin-7-O-[β-D-apiofuranosyl- (1→6)-β-D-glucopyranoside] ↔ ↔ ↑↑ ↔ ↓: inhibition; ↔: no effect; ↑: stimulation. Table 2. Effects of Dalbergia isolates on the different settings analysed. 14 of 23 Molecules 2022, 27, 4505 Figure 6. Effects of Dalbergia isolates on IKCa1.1 in single tail artery myocytes: (A) original recordings (average traces of 6 cells) of conventional whole-cell IKCa1.1 elicited with a 500 ms voltage step from Vh -40 to 70 mV, measured in the absence (control) and presence of 30 and 100 µM biochanin A; (B) current–voltage relationships obtained before the addition (control) and in the presence of various concentrations of biochanin A. 3. Results On the ordinate scale, the response is reported as the current density in pA/pF. Data points are the mean ± SEM. * p < 0.05 vs. control; repeated measures ANOVA and Dunnett’s post hoc test. Figure 6. Effects of Dalbergia isolates on IKCa1.1 in single tail artery myocytes: (A) original recordings (average traces of 6 cells) of conventional whole-cell IKCa1.1 elicited with a 500 ms voltage step from Vh -40 to 70 mV, measured in the absence (control) and presence of 30 and 100 µM biochanin A; (B) current–voltage relationships obtained before the addition (control) and in the presence of various concentrations of biochanin A. On the ordinate scale, the response is reported as the current density in pA/pF. Data points are the mean ± SEM. * p < 0.05 vs. control; repeated measures ANOVA and Dunnett’s post hoc test. To predict the potential binding pose of compounds on the homology model of the Rattus norvegicus CaV1.2 channel’s α1C subunit, a docking simulation was performed. The best-docked conformation of biochanin A, pinocembrin, and sativanone showed Gibbs free energy values (∆G) of −6.2, −7.5, and −5.7, respectively. The compounds shared the same binding region, located close to the central pore (Figure 7). The interaction network analysed by the P.L.I.P. tool demonstrated that biochanin A formed hydrophobic interactions with Leu-774 (S6-II), Leu-775 (S6-II), and Ala-1183 (S6-III); a hydrogen bond with Asn-771 (S6-II); two π stackings with Phe-778 (cytoplasmic) and Phe-1143 (pore forming) (Figure 7A). Sativanone triggered hydrophobic interactions with Phe-778 (cytoplasmic) and Phe-1143 (pore forming), and a hydrogen bond with Asn-771 (S6-II) (Figure 7B). Pinocembrin exhibited two hydrophobic interactions with Phe-730, Leu- 777, Phe-778, Ile-1180, Ala-1183, and Phe-1184 (Figure 7C). To conﬁrm the stability of both the protein structure and compound binding pose and to deﬁne a potential mechanism of action, a classical molecular dynamics (cMD) simulation of 100 ns was performed for the CaV1.2 channel in a complex with biochanin A, sativanone, and pinocembrin. Root Molecules 2022, 27, 4505 15 of 23 15 of 23 mean square deviation (RMSD) performed on the backbone of each biological system showed a stable and linear trend along the entire molecular dynamics run, suggesting good protein structural integrity (Figure 7D). The binding poses of biochanin A, sati- vanone, and pinocembrin exhibited RMSD values between 0.05 and 0.1 nm (Figure 7E), conﬁrming the starting docking binding pose and stability. 3. Results The nonbonded interaction energy of the target in complex with biochanin A, sativanone, and pinocembrin showed values of −140.6 ± 5.2 (−34.3 ± 1.2 kcal/mol), −137.3 ± 6.5 (−33.4 ± 1.5 kcal/mol), and −108.6 ± 4.9 kJ/mol (−26.4 ± 1.2 kcal/mol), respectively. Figure 7. Overview of the CaV1.2 channel docked with biochanin A, sativanone, and pinocembrin. The CaV1.2 channel’s 3D structure is depicted with a multicolour transparent surface, while the binding pocket is reported in red. The bilayer is represented by grey lines, and some phospholipid heads are shown as orange spheres. The extracellular and cytoplasmatic side is shown as a blue surface. (A–C) Interaction network of (A) biochanin A, (B) sativanone, and (C) pinocembrin in complex with the CaV1.2 binding residues (i.e., red balls and sticks) after the docking simulation. The hydrogen bond is represented as a purple dotted line. (D,E) Root mean square deviation (RMSD) proﬁles of biochanin A, pinocembrin, and sativanone. (D) RMSD proﬁles of the CaV1.2 channel backbone in complex with biochanin A, pinocembrin, and sativanone. (E) RMSD proﬁles of the biochanin A, pinocembrin, and sativanone binding pose in complex with the CaV1.2 channel. The RMSD trends are represented as coloured lines (see legend). RMSD (nm) and time (ns) values of the MD t d th d i ti l Figure 7. Overview of the CaV1.2 channel docked with biochanin A, sativanone, and pinocembrin. The CaV1.2 channel’s 3D structure is depicted with a multicolour transparent surface, while the binding pocket is reported in red. The bilayer is represented by grey lines, and some phospholipid heads are shown as orange spheres. The extracellular and cytoplasmatic side is shown as a blue surface. (A–C) Interaction network of (A) biochanin A, (B) sativanone, and (C) pinocembrin in complex with the CaV1.2 binding residues (i.e., red balls and sticks) after the docking simulation. The hydrogen bond is represented as a purple dotted line. (D,E) Root mean square deviation (RMSD) proﬁles of biochanin A, pinocembrin, and sativanone. (D) RMSD proﬁles of the CaV1.2 channel backbone in complex with biochanin A, pinocembrin, and sativanone. (E) RMSD proﬁles of the Figure 7. Overview of the CaV1.2 channel docked with biochanin A, sativanone, and pinocembrin. Figure 7. Overview of the CaV1.2 channel docked with biochanin A, sativanone, and pinocembrin. The CaV1.2 channel’s 3D structure is depicted with a multicolour transparent surface, while the binding pocket is reported in red. 3. Results The bilayer is represented by grey lines, and some phospholipid heads are shown as orange spheres. The extracellular and cytoplasmatic side is shown as a blue surface. (A–C) Interaction network of (A) biochanin A, (B) sativanone, and (C) pinocembrin in complex with the CaV1.2 binding residues (i.e., red balls and sticks) after the docking simulation. The hydrogen bond is represented as a purple dotted line. (D,E) Root mean square deviation (RMSD) proﬁles of biochanin A, pinocembrin, and sativanone. (D) RMSD proﬁles of the CaV1.2 channel backbone in complex with biochanin A, pinocembrin, and sativanone. (E) RMSD proﬁles of the biochanin A, pinocembrin, and sativanone binding pose in complex with the CaV1.2 channel. The RMSD trends are represented as coloured lines (see legend). RMSD (nm) and time (ns) values of the MD run are reported on the y- and x-axis, respectively. Figure 7. Overview of the CaV1.2 channel docked with biochanin A, sativanone, and pinocembrin. The CaV1.2 channel’s 3D structure is depicted with a multicolour transparent surface, while the binding pocket is reported in red. The bilayer is represented by grey lines, and some phospholipid heads are shown as orange spheres. The extracellular and cytoplasmatic side is shown as a blue surface. (A–C) Interaction network of (A) biochanin A, (B) sativanone, and (C) pinocembrin in complex with the CaV1.2 binding residues (i.e., red balls and sticks) after the docking simulation. The hydrogen bond is represented as a purple dotted line. (D,E) Root mean square deviation (RMSD) proﬁles of biochanin A, pinocembrin, and sativanone. (D) RMSD proﬁles of the CaV1.2 channel backbone in complex with biochanin A, pinocembrin, and sativanone. (E) RMSD proﬁles of the biochanin A, pinocembrin, and sativanone binding pose in complex with the CaV1.2 channel. The RMSD trends are represented as coloured lines (see legend). RMSD (nm) and time (ns) values of the MD run are reported on the y- and x-axis, respectively. Molecules 2022, 27, 4505 16 of 23 16 of 23 In silico results showed that biochanin A, sativanone, and pinocembrin were able to spontaneously bind to the KCa1.1 channel showing Gibbs free energy values (∆G) of −6.1, −5.8, and −5.4 kcal/mol, respectively. Despite the compounds sharing the same binding pocket (Figure 8), they formed a different interaction network, likely due to the differences in their structures and chemical–physical proprieties. 3. Results Biochanin A formed hydrophobic interactions with Lys-397, Lys-458C, and Phe-461C; ﬁve hydrogen bonds with Lys-300C, Ser-383C, Glu-387C, Tyr-398C, and Lys-458; a π stacking with Tyr-398C (Figure 8A). Pinocembrin triggered four hydrophobic interactions with Lys-397C, Lys-458C, Glu-465C, and Tyr-467C; three hydrogen bonds with Tyr-398C, Glu-454C, and Lys-458C; a π stacking with Tyr-398C (Figure 8B). Sativanone was involved in four hydrophobic interactions with Lys-397C, Tyr-402C, Phe-461C, and Tyr-467C; a hydrogen bond with Gly-399C; a π stacking with Tyr-398C (Figure 8C). Figure 8. Overview of KCa1.1 channels docked with biochanin A, pinocembrin, and sativanone. The KCa1.1 channel’s 3D structure is depicted in a multicolour illustration, while the binding pocket is reported as a red surface. (A–C) Interaction network of (A) biochanin A, (B) pinocembrin, and (C) sativanone in complex with the KCa1.1 binding residues (red balls and sticks) after the docking simulation. The hydrogen bond is represented as a purple dotted line. For clarity, only polar hydrogens are shown. Figure 8. Overview of KCa1.1 channels docked with biochanin A, pinocembrin, and sativanone. The KCa1.1 channel’s 3D structure is depicted in a multicolour illustration, while the binding pocket is reported as a red surface. (A–C) Interaction network of (A) biochanin A, (B) pinocembrin, and (C) sativanone in complex with the KCa1.1 binding residues (red balls and sticks) after the docking simulation. The hydrogen bond is represented as a purple dotted line. For clarity, only polar hydrogens are shown. The interaction network between KCa1.1 channel binding pocket residues and the three ﬂavonoids was further investigated by performing computational alanine-scanning mutagenesis, taking into account only the residues involved in hydrogen bonds. When considering the contribution of electrostatic energy, hydrogen bonds, Van der Waals force, and desolvation energy, a signiﬁcant loss in the binding free energy of biochanin A was observed when Glu-387C (∆∆G = −5.2 kcal/mol) and Tyr-398C (∆∆G = −4.7 kcal/mol) were mutated to alanine. On the contrary, this contribution was negligible for pinocembrin and sativanone (Figure 9). 17 of 23 Molecules 2022, 27, 4505 Figure 9. In silico alanine-scanning mutagenesis. Binding energy change values (∆∆G = ∆G wild- type—∆G Ala) obtained from the computational analysis of the KCa1.1 channel in a complex with biochanin A, pinocembrin, and sativanone. ∆∆G values (kcal/mol) and the alanine-scanning muta- genesis of KCa1.1 channel-binding residues (involved in hydrogen bonds) in complex with biochanin A, pinocembrin, and sativanone are reported on the x- and y-axis, respectively. 3. Results Negative ∆∆G values indicate an unfavourable substitution for alanine in the relevant position. Figure 9. In silico alanine-scanning mutagenesis. Binding energy change values (∆∆G = ∆G wild- type—∆G Ala) obtained from the computational analysis of the KCa1.1 channel in a complex with biochanin A, pinocembrin, and sativanone. ∆∆G values (kcal/mol) and the alanine-scanning muta- genesis of KCa1.1 channel-binding residues (involved in hydrogen bonds) in complex with biochanin A, pinocembrin, and sativanone are reported on the x- and y-axis, respectively. Negative ∆∆G values indicate an unfavourable substitution for alanine in the relevant position. 4. Discussion Although the number of molecules analysed was limited, the structural similarity allowed the discovery of several structural requirements, fundamental or detrimental to the stimulatory activity. The isoﬂavanones structure gave rise to ineffective compounds. Among ﬂavanones, the presence of a hydroxyl group at the C-5 position of ring A (eriodic- tyol) along with the absence of substituents on the B ring (pinocembrin) transformed the stimulator butin into an IBa1.2 inhibitor. On the contrary, among isoﬂavones, the presence of a hydroxyl group at the C-5 position of ring A accompanied by a single substituent in the para position on the benzylic moiety of the B ring (i.e., biochanin A, ambocin, and tectorigenin-7-O-[β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside]) favoured the stimu- latory activity. The only exception was tectoridin, though its glycosylation pattern was different from that of the two effective and structurally similar glycosides. The 2,3-double bond was irrelevant to the stimulatory activity. Finally, the ﬁnding that glycosides were effective on KCa1.1 channels conﬁrms previous data obtained with (±)-naringenin and its glycoside naringin [54]. On the contrary, ﬂavonoid modulation of CaV1.2 and Kir6.1 chan- nels was reduced or even vanished when one or more sugar moieties were present on the aglycone backbone/scaffold [55–58]. g y The S6/RCK linker in the KCa1.1 channel is crucial both for channel activation and for modulator binding, as shown by Gessner et al. [59] through in vitro mutagenesis. Noticeably, in silico results showed that the three ﬂavonoids bound with high afﬁnity inside the KCa1.1 channel binding pocket located close to this region. On the one hand, this observation supports the activity of biochanin A. On the other, however, it was at variance with the effects of pinocembrin and sativanone observed on IKCa1.1, the former being a weak inhibitor and the latter an ineffective agent. Unfortunately, the computational approach was characterised by a signiﬁcant loss of pharmacodynamics information due to the low cover value of our target against the template in some protein regions and low conﬁdence in their secondary structure prediction. This caused an alteration in the structural integrity of the channel during the cMD run, thus impeding a proper analysis of the complex formed with the three compounds. To explain this apparent discrepancy, however, the interaction network of the three ﬂavonoids in complex with the target was analysed, followed by computational alanine-scanning mutagenesis to deﬁne the energy contribution of each KCa1.1 channel-binding residue. 4. Discussion The ﬁndings presented in this work demonstrated that Dalbergia tonkinensis represents a valuable source of vasoactive compounds that can be further developed to obtain drugs capable of targeting more than one pathway involved in the pathogenesis of hypertension. Biochanin A, for example, stimulated KCa1.1 channels, blocked CaV1.2 channels, showed a marked vasorelaxant activity, and bound to residues that can substantiate its effects on both channels. With a few exceptions, most of the compounds assessed exhibited a more prominent vasorelaxant activity in aorta rings depolarised by moderate rather than high concentrations of K+. This pharmacological proﬁle, characterizing orobol, pinocembrin, formononetin, biochanin A, sativanone, and partially 3′-O-methylviolanone, is distinctive of K+ channel openers. In fact, as previously reported by Gurney [47] and then conﬁrmed in our labora- tory [49], the vasodilating efﬁcacy of K+ channel openers decreases when the extracellular concentration of K+ increases, due to the reduced chemical gradient that limits its efﬂux through the open channels. Consequently, membrane hyperpolarisation is weak, and the muscle relaxant effect of the compound is blunted. Accordingly, patch-clamp recordings demonstrated that biochanin A is a potent stimulator of IKCa1.1. To the best of our knowl- edge, this is the ﬁrst study demonstrating the direct stimulatory activity of biochanin A on KCa1.1 channels, previously hypothesised based on indirect evidence also in spontaneously hypertensive rats [50]. In fact, Au et al. [51] failed to detect any effect of the compound on these channels in porcine left anterior descending coronary myocytes, probably because they tested only the 1 µM concentration. Orobol and sativanone were ineffective, while for- mononetin, 3′-O-methylviolanone, and pinocembrin showed a weak to medium inhibitory activity. Pinocembrin and formononetin have previously been demonstrated to stimulate KCa1.1 channels in rat aorta myocytes [52,53]. This apparent discrepancy can be explained either by the different vascular beds studied or by the more hyperpolarised Vh used, likely not adequate to avoid voltage-dependent K+ channel contamination of the aorta recordings. Tectorigenin-7-O-[β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside], butin and, especially, ambocin showed a remarkable KCa1.1 channel stimulatory activity that, however, did not Molecules 2022, 27, 4505 18 of 23 18 of 23 translate to spasmolysis towards moderate but also high K+-induced contraction. Further experiments are necessary to clarify this apparent discrepancy. translate to spasmolysis towards moderate but also high K+-induced contraction. Further experiments are necessary to clarify this apparent discrepancy. 4. Discussion According to the interaction network analysis, only biochanin A formed a large hydrogen bond network, consistent with its stimulation of IKCa1.1. Furthermore, the in silico alanine-scanning mutagenesis indicated that only biochanin A showed a signiﬁcant decrease in the binding free energy when Glu-387C and Tyr-398C were mutated to alanine, abolishing their interactions. Thus, the different activity of pinocembrin and sativanone on IKCa1.1 was likely because they did not show any energy contribution against the Glu-387C and Tyr-398C residues. Taken together, these observations indicate that the Glu-387C and Tyr-398C residues play a key role in the protein–modulator interaction, thus representing novel consensus binding residues for KCa1.1 channel modulators. The two isoﬂavanones investigated were fairly active against high K+-induced contrac- tion, which was mainly due to the extracellular Ca2+ inﬂux through CaV1.2 channels. These channels play a fundamental role in the regulation of vascular tone [60] and, therefore, represent crucial targets in antihypertensive therapy. The inhibition of these channels, in fact, is one of the standard therapeutic approaches to counteract the abnormal increase in blood pressure. The electrophysiology data pointed to sativanone, 3′-O-methylviolanone, pinocembrin, and biochanin A as effective Ca2+ antagonists, all characterised by a similar potency. Again, to the best of our knowledge, this is the ﬁrst study demonstrating the direct inhibitory activity of pinocembrin and biochanin A on CaV1.2 channels, previously hy- pothesised based on indirect evidence [50,52]. The structure–activity relationship analysis underlined the presence of an unsubstituted hydroxyl group in the C-7 position of ring A as essential for the Ca2+ antagonistic activity, all investigated 7-O-glycosides being weak Molecules 2022, 27, 4505 19 of 23 19 of 23 inhibitors/stimulators or ineffective. Among the aglycones, IBa1.2 inhibition was greater when one, two, or three methoxy groups were present on the B ring positioned in the C-3 position of the C ring (formononetin, biochanin A, sativanone, and 3′-O-methylviolanone) or when the B ring positioned in C-2 of the C ring was devoid of substituents (pinocembrin). The presence of the 2,3-double bond and the position of the B ring was irrelevant to the inhibitory activity (compare biochanin A with pinocembrin). Finally, the weak Ca2+ antag- onism displayed by eriodictyol and formononetin was in line with previous observations obtained in aorta rings [50,61]. Interesting clues arise from the biophysical analysis of the effect of biochanin A on CaV1.2 channels. 4. Discussion The molecule shifted the steady-state inactivation curve to more negative potentials, indicating its capability to stabilize the channel in the inactivated state. Consistent with this hypothesis, biochanin A, like the two isoﬂavanones, sativanone and 3’-O-methylviolanone, sped up the inactivation kinetics of the IBa1.2 (i.e., accelerated the transition from the open to the inactivated state). However, its efﬁcacy was not reduced by the shift of the membrane potential to more hyperpolarised values, i.e., when many channels are in the resting, closed state, thus suggesting that the molecule can bind to channels both in the inactivated and resting state. On the contrary, neither the activation kinetics nor the steady-state activation curve was affected by biochanin A, indicating that the molecule did not modify the voltage sensitivity of the channel activation and the transition from the resting to the open state. Nevertheless, biochanin A caused a marked decrease in the window current, which is thought to be largely responsible for both the generation and regulation of vascular smooth muscle tone [62], which, in in vivo conditions, might be reduced by biochanin A. Finally, biochanin A-induced inhibition of IBa1.2 was only partially reversible upon washout, thus limiting its direct use in clinics. The patch-clamp ﬁndings provided a few insights into the mechanism(s) underpinning the ﬂavonoid vasorelaxant activity. The spasmolytic effect of pinocembrin and the two isoﬂavanones sativanone and 3’-O-methylviolanone can be ascribed essentially to their Ca2+ antagonistic effect. The same did not apply to formononetin and orobol, as their CaV1.2 channel inhibitory effect was weak. In biochanin A, the KCa1.1 channel stimulation likely prevailed over the CaV1.2 channel blockade, as the isoﬂavone was more effective on tissues depolarised with moderate concentrations of K+. Finally, butin, ambocin, and tectorigenin- 7-O-[β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside] did not show any vasorelaxant effect despite their marked KCa1.1 channel stimulatory activity observed in single myocytes. While the access to the site of action of the latter two ﬂavonoids might be prevented by the presence of the glycoside moiety, further experiments are needed to clarify the contradictory behaviour of butin. Tectoridin inactivity is complementary to the data obtained in the same preparation precontracted by phenylephrine [63]. The computational results showed that the three ﬂavonoids bound spontaneously within a CaV1.2 channel inhibitor binding pocket [27] with high stability and afﬁnity, triggering a hydrophobic and polar interaction network. 4. Discussion Remarkably, biochanin A was the only compound able to form a π-stacking interaction with Phe-1143 (pore forming), a key residue both for the binding of potent CaV1.2 blockers and for channel inhibition [27]. The high stability of the binding poses of the three ﬂavonoids as well as the high nonbonded interaction energy within the target binding pocket supports the strength of their interaction with the CaV1.2 channel, likely responsible for a marked modiﬁcation of the protein function. Altogether, structural and energy evidence provided by the cMD analysis were consistent with the in vitro results. This work presents two limitations. First, disruption of the extracellular matrix to isolate single smooth muscle cells for patch-clamp recordings leads to the loss of focal contact tension that, in turn, considerably alters cell signalling systems [64]. Therefore, a phenomenon observed in isolated myocytes, though providing important insight into the drug’s mechanism of action, must be considered cautiously due to the absence of the physiological environment and always veriﬁed in intact tissues. The results here presented are consistent with a substantial contribution of both CaV1.2 and KCa1.1 channels, two of Molecules 2022, 27, 4505 20 of 23 20 of 23 the main pathways controlling smooth muscle tone, analysed separately in intact vascular tissues simply varying the extracellular KCl concentration (see above) and quantiﬁed by electrophysiology experiments, to the vasorelaxant effect of the active compounds. However, muscle tone is an epiphenomenon controlled by several other pathways beyond the CaV1.2 and KCa1.1 channels, which may be affected by Dalbergia isolates. Second is the speciﬁcity of biochanin A. Smooth muscle and cardiac CaV1.2 channels, in fact, exhibit only minor differences due to the alternative splicing loci [65]; thus, biochanin A might affect the electrical and mechanical activity of the heart. On the other hand, the possible activation of cardiac KCa1.1 channels could have only beneﬁcial effects, because their expression in the mitochondria of adult cardiomyocytes [66] plays a crucial role in cardioprotection [67]. Additional experiments are required to clarify these issues. 5. Conclusions In conclusion, the present ﬁndings point to Dalbergia tonkinensis Prain as a valuable source of vasoactive compounds. Biochanin A, in particular, represents an interesting multitarget molecule, capable of simultaneously blocking CaV1.2 channels and stimulating KCa1.1 channels of vascular smooth muscle, thus leading to vasodilation. In this context, it is important to underline that cardamonin, a ﬂavonoid precursor chalcone, is also endowed with the same bifunctional activity [68], suggesting that the “natural” ﬂavonoid library could contain other molecules with similar multitarget activity. As these two targets are involved in the pathogenesis of hypertension [69,70], biochanin A might contribute to the development of novel antihypertensive drugs, nowadays necessary to improve both patient compliance and the successful therapy of pathology with a high socioeconomic impact. Recent ﬁndings [71] support this hypothesis. Author Contributions: Conceptualisation, N.M.C. and F.F.; Funding acquisition, N.M.C. and F.F.; Investigation, N.T.S., N.T.N., Y.F., A.A., S.S., B.G., G.V., A.T. and F.F.; Resources, N.M.C., N.T.N. and Y.F.; Writing—original draft preparation, A.T., O.S. and F.F.; Writing—review and editing, F.F. and N.M.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) (grant no. 104.01-2015.49 to N.M.C.) and by the JSPS RONPAKU Ph.D. program for FY 2017 to N.T.S. Institutional Review Board Statement: All the study procedures were in strict accordance with the European Union Guidelines for the Care and the Use of Laboratory Animals (European Union Directive 2010/63/EU) and approved by the Animal Care and Ethics Committee of the University of Siena and the Italian Department of Health (7DF19.N.TBT, approved on 11 May 2020). Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All data are reported in the article. Data Availability Statement: All data are reported in the article. Acknowledgments: C. D’Alessandro, M. Farris, and M. Angileri are gratefully acknowledged for their technical assistance in some of the experiments. The University of Siena, Area ricerca, bib- lioteche, internazionalizzazione e terza missione is gratefully acknowledged for supporting the article processing charge for publication. 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https://openalex.org/W2129074211	https://www.scielo.br/j/rlae/a/SDZTPV7wK3vVRJLSWQjq4qd/?lang=pt&format=pdf	Portuguese	null	O paradigma hermenêutico como fundamentação das pesquisas etnográficas e fenomenológicas	Revista latino-americana de enfermagem	1,998	cc-by	5,123	* Mestre em Educação. Enfermeira, doutoranda na EEUSP/EEUFMG. Professora Adjunto da Faculdade de Enfermagem da Universidade Federal de Juiz de Fora Doutora em Enfermagem. Enfermeira, Professora Adjunta da Escola de Enfermagem da Universidade Federal de Minas Gerais * Mestre em Enfermagem Médico-Cirúrgica. Enfermeira, doutoranda na EEUSP/EEUFMG. Professora Adjunto da Escola de Enfermagem da Universidade Federal de Minas Gerais INTRODUÇÃO norteasse as nossas pesquisas - etnográfica e fenomenológica - deparamo-nos com diferentes abordagens e classificações usadas pelos autores que adotam a linha qualitativa. A partir da década de 60, nos Estados Unidos e de 80, no Brasil, observa-se na enfermagem um crescente interesse nas pesquisas interpretativas. Em decorrência disso surgem diferentes nomenclaturas e referências ontológicas e epistemológicas, dando origem a conceituações e classificações não bem definidas ou mesmo equivocadas. Por outro lado, identificamos a escassez de trabalhos na área da Enfermagem que discutem os aspectos epistemológicos e metodológicos das abordagens interpretativas. Acreditando não ser possível desvincular nossa visão de mundo e a intencionalidade de nossas vivências enquanto docentes e pesquisadores, realizamos uma breve revisão da literatura com o objetivo de delimitar o referencial filosófico, sociológico e antropológico que fundamentasse a elaboração dos nossos projetos de pesquisa. Assim como afirma LOWENBERG (1993), os pesquisadores da enfermagem que adotam as abordagens qualitativas desenvolveram seus programas de pós- graduação em diferentes áreas do conhecimento, como na Sociologia, Antropologia e Psicologia, levando a uma dificuldade de delinear significados comuns em seus discursos. Na fase de elaboração dos projetos para qualificação das autoras em Programas de Doutorado deparamos com a dificuldade de identificação da corrente filosófica que fundamentasse nossos pressupostos teórico-metodológicos. Nossos projetos estavam vinculados à compreensão de fenômenos situados no cotidiano da enfermagem, buscando significados atribuídos pelos sujeitos a suas experiências, num enfoque sociológico e antropológico. UNITERMOS: pesquisa interpretativa, hermenêutica, fenomenologia, antropologia UNITERMOS: pesquisa interpretativa, hermenêutica, fenomenologia, antropologia Rev.latino-am.enfermagem - v. 6 - n. 2 - p. 29-35 - abril 1998 Rev.latino-am.enfermagem - v. 6 - n. 2 - p. 29-35 - abril 1998 29 O PARADIGMA HERMENÊUTICO COMO FUNDAMENTAÇÃO DAS PESQUISAS ETNOGRÁFICAS E FENOMENOLÓGICAS Maria Cristina Pinto de Jesus* Marisa Ribeiro Bastos Peixoto** Maria Cristina Pinto de Jesus* Marisa Ribeiro Bastos Peixoto Mércia Heloísa Ferreira Cunha* JESUS, M.C.P. de; PEIXOTO, M.R.B.; CUNHA, M.H.F. O paradigma hermenêutico como fundamentação das pesquisas etnográficas e fenomenológicas. Rev.latino-am.enfermagem, Ribeirão Preto, v. 6, n. 2, p. 29-35, abril 1998. JESUS, M.C.P. de; PEIXOTO, M.R.B.; CUNHA, M.H.F. O paradigma hermenêutico como fundamentação das pesquisas etnográficas e fenomenológicas. Rev.latino-am.enfermagem, Ribeirão Preto, v. 6, n. 2, p. 29-35, abril 1998. O estudo apresenta uma revisão da literatura sobre o paradigma hermenêutico como fundamentação das pesquisas etnográficas e fenomenológicas. todo, com sua vontade, sensibilidade e imaginação” (JAPIASSU & MARCONDES, 1995, p.73). para o seu próprio sistema de valores e significados”(BLEICHER,1992, p. 13). para o seu próprio sistema de valores e significados”(BLEICHER,1992, p. 13). A hermenêutica tem sido utilizada como base filosófica para diferentes estudos qualitativos realizados por enfermeiros, tendo em vista que o homem e as interações humanas são freqüentemente objeto de estudo da enfermagem. Estudos de tal natureza remetem a processos interpretativos do investigador, a importância do contexto na compreensão de significados, bem como a compreensão da linguagem oral e escrita do outro. A compreensão é enfocada por Dilthey sob o ponto de vista interno, ou seja, a partir da psicologia (DILTHEY, 1979). Para ele todas as formas de conhecimento do espírito, enquanto implicar numa relação com a história, teria como pressuposto a capacidade do homem de se transpor para dentro da vida psíquica do outro. Independente da natureza da pesquisa qualitativa adotada tais abordagens têm em comum a busca da compreensão e da interpretação do homem como ser único e singular; com a apreensão do ser humano em sua totalidade e do homem como melhor intérprete de si mesmo (VIETTA, 1995, p.34-35). SIEBENEICHLER (1983) ao descrever as idéias inspiradoras da fenomenologia para a hermenêutica filosófica diz que Husserl, filósofo alemão, teve o mérito de recolocar de forma crítica a ciência e a técnica no contexto do sujeito. Husserl, em seus escritos, tomou como ponto de partida uma ligação intencional entre espírito e mundo, tentando explicar a situação da consciência, do sujeito humano. Por meio do método da “epoché” e da redução fenomenológica encontra um instrumento esclarecedor da consciência, capaz de ultrapassar o psicologismo e o historicismo reinantes na época. Husserl ao tentar encaixar a problemática da consciência individual em um contexto mais amplo verificou que era possível “fundamentar idéias conscientes, recorrendo a uma experiência pré-consciente, não articulada”. (SIEBENEICHLER, 1983, p.10). A arte de interpretação, hermenêutica, foi inicialmente empregada no domínio teológico na interpretação de textos bíblicos. A partir da idade moderna passa a ter o sentido de “arte da compreensão” ou seja, interpretação correta e objetiva dos textos da Escritura (CORETH, 1973). Por outro lado, a Hermenêutica enquanto forma de interpretação, era utilizada em textos profanos na mitologia e na área jurídica, decorrendo numa regionalização da hermenêutica. A HERMENÊUTICA E AS PESQUISAS INTERPRETATIVAS A hermenêutica é definida por BLEICHER (1992), como a teoria ou filosofia da interpretação dos sentidos. O problema hermenêutico é fundamentado na “... percepção de que as expressões humanas contêm componente significativo, que tem que ser reconhecido como tal, por um sujeito e transposto A hermenêutica é definida por BLEICHER (1992), como a teoria ou filosofia da interpretação dos sentidos. O problema hermenêutico é fundamentado na “... percepção de que as expressões humanas contêm componente significativo, que tem que ser reconhecido como tal, por um sujeito e transposto Buscando definir o referencial filosófico que O paradigma hermenêutico... Rev.latino-am.enfermagem - v. 6 - n. 2 - p. 29-35 - abril 1998 30 FREUND (1987, p.73-74) interpretando o pensamento de Weber diz que: Toda interpretação se propõe a vencer um afastamento, uma distância, entre a época cultural revoluta, à qual pertence o texto, e o próprio intérprete... Portanto, o que ele persegue, através da compreensão do outro, é a ampliação da própria compreensão de si mesmo. Assim, toda hermenêutica é, explícita ou implícitamente, compreensão de si mesmo mediante a compreensão do outro” Interessado na Sociologia weberiana, Schütz parte para fundamentar a Sociologia da ação e compreensão por meio da análise filosófica da fenomenologia de Husserl. Segundo o autor a “estrutura significativa do mundo social somente pode deduzir-se a partir das características mais primitivas e gerais da consciência”. Visa, assim, determinar a natureza precisa do fenômeno do significado mediante a análise da função constitutiva, chegando a conclusão de que “a ação é uma vivência guiada por um plano ou projeto que surge da atividade espontânea do sujeito, sendo distinguida de todas as outras vivências por um ato peculiar de atenção”. (SCHÜTZ, 1972, p.243) Assim, a hermenêutica busca “compreender sentidos” ou seja o conteúdo típico humano que se imprime a qualquer contexto histórico, no qual não existem apenas fatos dados, acontecimentos externos, mas, também, “significação”, “sentido” e “valores” (DEMO, 1981, p.249). Ao se aplicar a uma disciplina a hermenêutica se especifica ganhando feições próprias. Schütz propõe a investigação no mundo cotidiano, onde o homem olha para esse mundo do ponto de vista da atitude natural. Tendo nascido nesse mundo que também é social e cultural o homem vive com seus congêneres e dá por certa a existência destes sem questioná-la, assim como dá por certa a existência de objetos naturais. As pessoas interagem e compreendem todo, com sua vontade, sensibilidade e imaginação” (JAPIASSU & MARCONDES, 1995, p.73). A partir do século XIX há uma tentativa de unificação dessas hermenêuticas especiais ou regionais numa hermenêutica geral ou universal formulada por Friedrich Schleiermacher, que introduz a questão do texto/ contexto (CORETH, 1973). A partir da herança deixada por Husserl, Heidegger, em sua obra “Ser e Tempo”, “se afasta da fenomenologia de seu mestre Husserl e inicia seu caminho de reflexão sobre o sentido mais profundo da existência humana, bem como sobre as origens da metafísica e o significado de sua influência na formação do pensamento ocidental” (JAPIASSU & MARCONDES, 1995, p.116). Para Schleiermacher, a hermenêutica é a arte da compreensão, que não visa ao saber teórico e sim ao saber prático: “a práxis ou a técnica da boa interpretação de um texto falado ou escrito”. O autor define a hermenêutica como a “reconstrução histórica e divinatória objetiva e subjetiva de um dado discurso” (CORETH, 1973, p.19). Heidegger observou que o mundo humano não significa apenas o meio ambiente exterior e nem tão pouco constituía-se de uma construção subjetiva e arbitrária (SIEBENEICHLER, 1983). Wilhelm Dilthey, filósofo alemão, valorizou a teoria da “visão do mundo” em que viver é interpretar o mundo natural. Sua obra “Introdução ao estudo das ciências humanas” (1883), critica a visão positivista da “explicação” sobre o homem buscando a “compreensão” da sua natureza social e histórica. (JAPIASSU & MARCONDES, 1995, p.73). Em suas últimas obras, Heidegger explicita que a compreensão se expressa na linguagem e nela se constitui o horizonte histórico da compreensão. “A hermenêutica torna-se, assim, a interpretação da primitiva compreensão do homem em si e do ser” (CORETH, 1973, p.23). Dilthey, apoiando-se em Schliermacher, formula a dualidade de ciências da natureza e ciências do espírito que se distinguem por um método analítico esclarecedor e um procedimento de compreensão descritiva. Baseado em Heidegger, de quem foi seu discípulo mais próximo e em seus estudos humanísticos, Gadamer “desenvolveu um interesse filosófico pelo diálogo com a tradição, com as línguas e as culturas distantes e refletiu sobre as condições históricas e filosóficas da compreensão e da interpretação” (HABERMAS, 1987, p.7) “Ao pretender dar um fundamento às ciências particulares do homem, Dilthey postula a criação de novos métodos e de conceitos psicológicos mais sutis adaptados à vida histórica; ademais procura evidenciar, em todas as manifestações humanas, a totalidade da visão psíquica, a ação do homem A partir das formulações de Schleiermacher, O paradigma hermenêutico... Rev.latino-am.enfermagem - v. FREUND (1987, p.73-74) interpretando o pensamento de Weber diz que: “são racionais a compreensão atual do sentido de uma operação aritmética que efetua o comportamento de um lenhador que abate uma árvore, bem como a compreensão pelos motivos quando vejo uma pessoa ocupada em um trabalho a fim de ganhar sua vida. São irracionais a compreensão atual da cólera ou a compreensão pelos motivos quando a pessoa se serve de uma espingarda para se vingar ou entra em cólera por ciúme... Compreender, pode-se dizer, é captar a evidência do sentido de uma atividade”. “são racionais a compreensão atual do sentido de uma operação aritmética que efetua o comportamento de um lenhador que abate uma árvore, bem como a compreensão pelos motivos quando vejo uma pessoa ocupada em um trabalho a fim de ganhar sua vida. São irracionais a compreensão atual da cólera ou a compreensão pelos motivos quando a pessoa se serve de uma espingarda para se vingar ou entra em cólera por ciúme... Compreender, pode-se dizer, é captar a evidência do sentido de uma atividade”. De seus estudos sobre Marx e Freud, Habermas adota como preocupação central uma política emancipatória, refletindo sobre as condições de um “diálogo livre de dominação”, a possibilidade de uma “comunicação isenta de coação e violência”. Para o autor, a hermenêutica, mais do que arte de interpretar, ou tecnologia, é crítica (HABERMAS, 1987, p.6-27). Na perspectiva de RICOUER(1978, p.18), a hermenêutica implica na compreensão dos signos e de si. De acordo com SCHÜTZ (1972, p.35) Weber definiu como tarefa da Sociologia não a especulação metafísica e sim uma descrição simples e cuidadosa da vida social. Não uma sociologia enquanto filosofia da existência humana, mas como a ciência particular da conduta humana e suas conseqüências que deve dar conta da compreensão e interpretação da ação social. “Ao propor religar a linguagem simbólica à compreensão de si, penso satisfazer o desejo mais profundo da hermenêutica. Toda interpretação se propõe a vencer um afastamento, uma distância, entre a época cultural revoluta, à qual pertence o texto, e o próprio intérprete... Portanto, o que ele persegue, através da compreensão do outro, é a ampliação da própria compreensão de si mesmo. Assim, toda hermenêutica é, explícita ou implícitamente, compreensão de si mesmo mediante a compreensão do outro” “Ao propor religar a linguagem simbólica à compreensão de si, penso satisfazer o desejo mais profundo da hermenêutica. todo, com sua vontade, sensibilidade e imaginação” (JAPIASSU & MARCONDES, 1995, p.73). 6 - n. 2 - p. 29-35 - abril 1998 31 31 Dilthey e Heidegger, Hans-Georg Gadamer elabora uma teoria filosófica em que a Hermenêutica adquire um status de corrente filosófica. contribuição de outros estudiosos, a exemplo do fenomenólogo Alfred Schütz. Ao elaborar o “tipo ideal” a partir de princípios vinculados à história, Weber pretende compreender a ação social como a resultante de forças de relações sociais, ação proveniente do comportamento social que tem um significado subjetivo. Desse modo, coloca o homem como centro das atenções já que a compreensão dos significados se dá no sujeito. Considera “as ações motivadas por sentimentos afetivos e as tradicionais como menos racionais”, sendo as ações que se aproximam do tipo racional como as mais compreensíveis (CAPALBO, 1979, p.86). Na ótica de SIEBENEICHLER (1983, p.24) Gadamer compreende a Hermenêutica de uma forma mais ampla do que a arte de interpretar. Ela constitui “uma possibilidade no horizonte da mediação entre a verdade do nosso ser e o método da ciência”. A redescoberta fenomenológica do mundo da vida aliada à nova compreensão da linguagem levou a uma troca intensa de idéias entre a fenomenologia e a Hermenêutica filosófica. Tal redescoberta coloca como possibilidade a mediação entre a razão científica e a razão filosófica, além de possibilitar a constituição da moderna filosofia Hermenêutica de Gadamer e de Paul Ricoeur e da filosofia crítica representada por Habermas. (SIEBENEICHLER, 1983, p.12). FREUND (1987, p.73-74) interpretando o pensamento de Weber diz que: A HERMENÊUTICA E A SOCIOLOGIA FENOMENOLÓGICA A Sociologia interpretativa teve suas raízes na teoria de Max Weber e posteriormente recebeu a Rev.latino-am.enfermagem - v. 6 - n. 2 - p. 29-35 - abril 1998 O paradigma hermenêutico... 32 a si próprias e aos outros na realidade social. No entanto, ressalta que somente a olhada reflexiva do observador eleva o conteúdo da consciência do status pré-fenomênico até o fenomênico. ação humana deve ser construído de tal maneira que um ato humano efetuado dentro do mundo da vida, por um ator individual, da maneira indicada pela construção típica, seja compreensível tanto para o ator como para seus semelhantes, em termos de interpretação de sentido comum na vida cotidiana. Assim, fica garantida a compatibilidade das construções do cientista social com as construções da experiência do sentido comum na realidade social (SCHÜTZ, 1962, p.67-68). O fenomenólogo se volta para a atitude natural e estuda o que “já se encontra estruturado e de certo modo interpretado, pois a realidade social já possui um sentido para os homens que vivem em seu seio” (CAPALBO, 1979, p.36). Para Schütz a tarefa primeira da Sociologia compreensiva consiste em descrever os processos de estabelecimento e interpretação de significado tal como os realizam as pessoas que vivem no mundo social. Ressalta que esta descrição pode ser empírica ou eidética. Pode tomar como tema a pessoa ou o tipo. Pode realizar- se em situações concretas da vida cotidiana ou com alto grau de generalidade. Além disso a Sociologia compreensiva pode enfocar os objetos culturais e tratar de compreender o seu significado, aplicando-lhes os esquemas interpretativos. Para Schütz a tarefa primeira da Sociologia compreensiva consiste em descrever os processos de estabelecimento e interpretação de significado tal como os realizam as pessoas que vivem no mundo social. Para SCHÜTZ (1962, p.23-24) a compreensão de uma determinada coisa só é possível ao reduzi-la à atividade que a criou e aos motivos que a originaram, somente sendo possível compreender a atividade humana compreendendo a ação correspondente. Assim, a ação está determinada pelo projeto que inclui o “motivo para” que é o propósito da ação e o “motivo porque” ou seja a razão ou causa da ação. Pode tomar como tema a pessoa ou o tipo. Pode realizar- se em situações concretas da vida cotidiana ou com alto grau de generalidade. Além disso a Sociologia compreensiva pode enfocar os objetos culturais e tratar de compreender o seu significado, aplicando-lhes os esquemas interpretativos. A HERMENÊUTICA E A SOCIOLOGIA FENOMENOLÓGICA Para interpretar a ação sob o ponto de vista do ator, ponto de vista subjetivo, Schütz indica um sistema objetivo de análise cujos recursos metodológicos possam alcançar a estrutura subjetiva de sentido, ou seja, a construção do esquema típico-ideal. O autor elege o esquema subjetivo como o escolhido ao estudar o mundo social tal qual é vivido pelo homem e seus congêneres na atitude natural. O autor elege o esquema subjetivo como o escolhido ao estudar o mundo social tal qual é vivido pelo homem e seus congêneres na atitude natural. Por meio da construção do esquema típico-ideal o autor chega ao “tipo ideal pessoal” que se expressa de certa maneira e tem tais e quais vivências e ao tipo material ou de curso de ação que é o tipo ideal do processo expressivo mesmo (SCHÜTZ, 1972, p.216). O tipo ideal em Schütz é elaborado a partir da vivência dos sujeitos. É o tipo vivido cujo sentido é obtido num ato vivido. O tipo vivido concreto possibilita a compreensão das formas significativas de vivência e a compreensão significativa de uma subjetividade comum. Tais esquemas interpretativos diferem da tipificação que é feita na vida cotidiana, porém devem ser compatíveis não somente com os motivos e as experiências subjetivas do mundo social, como também com a experiência científica em seu conjunto. A HERMENÊUTICA E A ANTROPOLOGIA No esquema típico-ideal os contextos subjetivos vivenciados diretamente são substituídos de forma sucessiva por uma série de contextos objetivos de significados, chegando-se a um modelo conceitual e nunca a uma pessoa real. Tais modelos devem ser compatíveis com os postulados fundamentais do método científico e ao mesmo tempo responder às exigências do mundo da vida. Desse modo, Schütz aponta os seguintes postulados como requisitos para a construção desses modelos: Postulado da Coerência Lógica, onde as construções típicas são estabelecidas com um alto grau de claridade e nitidez, sendo o esqueleto conceitual totalmente compatível com os princípios da lógica formal. Este postulado garante a validade científica dos modelos construídos. Postulado da Interpretação Subjetiva: para explicar as ações humanas, o pesquisador deve perguntar- se que modelo de mente individual é possível construir e que conteúdos típicos são necessários atribuir a ele para explicar fatos observados como resultado da atividade da mente, numa relação compreensível. Assim, tem-se a garantia da significação subjetiva. Segundo o Postulado da Adequação, cada termo de um modelo científico de A Antropologia, enquanto “a reflexão do homem sobre o homem e sua sociedade” torna-se uma ciência com legitimidade entre outras disciplinas científicas, a partir do século XIX. Nesta época, a antropologia, chamada fundacional ou de gabinete, constituía-se em narrativas e descrições das grandes viagens européias, descobertas de outras sociedades e povos exóticos feitas pelos observadores (viajantes ou missionários) e analisadas pelos pesquisadores em seus gabinetes (LAPLANTINE, 1994). No século XX a antropologia passa a ser caracterizada pelo trabalho de campo com uma ruptura com a antropologia dita especulativa dando início a antropologia denominada moderna. Na antropologia moderna coexistem quatro correntes antropológicas: a racionalista, a estrutural- funcionalista, a culturalista e a interpretativa (CARDOSO DE OLIVEIRA, 1988). A Antropologia Interpretativa ou também pós-moderna, inspira-se na tradição filosófica denominada hermenêutica, tendo em Geertz seu principal representante. O paradigma hermenêutico... Rev.latino-am.enfermagem - v. 6 - n. 2 - p. 29-35 - abril 1998 33 (...) não é afastar-se dos dilemas existenciais da vida em favor de algum domínio empírico de formas não emocionalizadas; é mergulhar no meio delas. A vocação essencial da antropologia interpretativa não é responder às questões mais profundas, mas colocar à nossa disposição as respostas que os outros deram (...) e assim incluí- las no registro de consultas sobre o que o homem falou” (GEERTZ, 1989, p.40-41). A HERMENÊUTICA E A ANTROPOLOGIA “ao contrário, o que se verifica é uma verdadeira dispersão de influências nessa antropologia que Embora todas as correntes dentro da antropologia moderna se empenhem num mesmo esforço interpretativo, Geertz se diferencia na medida em que, na busca do significado simbólico, adota as premissas demandadas pela hermenêutica. Enquanto no estruturalismo, o símbolo é reduzido a um significado, porque pressupõe uma determinada estrutura econômica, de parentesco, entre outras, a antropologia interpretativa de Geertz não aponta um significado e, sim, uma complexidade deles, numa ampla relativização de contextos. “Olhar as dimensões simbólicas da ação social A HERMENÊUTICA E A ANTROPOLOGIA A Antropologia Interpretativa, pautada no paradigma hermenêutico, apresenta-se como uma crítica às “antropologias tradicionais”, reconsiderando verdades que acreditavam eternas (CARDOSO DE OLIVEIRA, 1988, CALDEIRA, 1988). Nesse novo estilo de se fazer antropologia, a autoridade do investigador é colocada em questão, o saber é negociado entre pesquisador e o nativo, num processo de confrontação de horizontes. A “intersubjetividade, a individualidade e a historicidade” passam a ser exercitadas pelo pesquisador. Para GEERTZ (1989) uma etnografia é uma “descrição densa” o que implica na busca da profundidade e da contextualização dos significados. p p q “... os horizontes não se excluem de um modo absoluto, mas se interseccionam e muitas vezes se fundem. E propiciam, por conseguinte, o exercício pleno da intersubjetividade (...) nos domínios privilegiados da investigação etnográfica. Investigação que realizava o pesquisador e o pesquisado enquanto individualidade explicitamente reconhecidas, uma vez que a própria biografia deste último pode ser a do primeiro. E ao apresentar a vida do Outro (...), o faz em termos de historicidade, num tempo histórico do qual ele próprio, pesquisador, não se exclui”. (CARDOSO DE OLIVEIRA, 1988, p.100- 101). “... os horizontes não se excluem de um modo absoluto, mas se interseccionam e muitas vezes se fundem. E propiciam, por conseguinte, o exercício pleno da intersubjetividade (...) nos domínios privilegiados da investigação etnográfica. Investigação que realizava o pesquisador e o pesquisado enquanto individualidade explicitamente reconhecidas, uma vez que a própria biografia deste último pode ser a do primeiro. E ao apresentar a vida do Outro (...), o faz em termos de historicidade, num tempo histórico do qual ele próprio, pesquisador, não se exclui”. (CARDOSO DE OLIVEIRA, 1988, p.100- 101). A descrição etnográfica na concepção de GEERTZ (1989, p.31) é interpretativa. “... o que ela interpreta é o fluxo social e a interpretação envolvida consiste em tentar salvar o “dito” num tal discurso da sua possibilidade de extinguir-se e fixá-lo em formas pesquisáveis”. Não há uma univocidade de concepção hermenêutica enquanto raiz filosófica da Antropologia Interpretativa, p “ao contrário, o que se verifica é uma verdadeira dispersão de influências nessa antropologia que se pretende nova. Nem a “hermenêutica ontológica” de Heidegger e Gadamer, nem a “hermenêutica metódica” de Betti ou de Hirsch, nem a “hermenêutica fenomenológica” de Ricoeur (e muito menos a “hermenêutica clássica” de Schleiermacher e Dilthey) dominam aquilo que prefiro chamar de “consciência hermenêutica” na Antropologia “pós-moderna” (CARDOSO DE OLIVEIRA, 1988, p.97). RMINOS CLAVES: investigación interpretativa, hermenéutica, fenomenología, antropología TÉRMINOS CLAVES: investigación interpretativa, hermenéutica, fenomenología, antropología TÉRMINOS CLAVES: investigación interpretativa, hermenéutica, fenomenología, antropología CONSIDERAÇÕES FINAIS HERMENEUTIC AND QUALITATIVE RESEARCH ON NURSING The study review the literature about hermeneutics as a foundation for ethnographic and phenomenological research. KEY WORDS: interpretative research, hermeneutic, phenomenology, anthropology LA HERMENÉUTICA Y LAS INVESTIGACIONES CUALITATIVAS EN ENFERMERÍA El estudio presenta una revisión de la literatura sobre el paradigma hermenéutico como fundamentación de las investigaciones etnográficas y fenomenológicas. A HERMENÉUTICA Y LAS INVESTIGACIONES CUALITATIVAS EN ENF El estudio presenta una revisión de la literatura sobre el paradigma hermenéutico como fundamentación de las investigaciones etnográficas y fenomenológicas. HERMENEUTIC AND QUALITATIVE RESEARCH ON NURSING HERMENEUTIC AND QUALITATIVE RESEARCH ON NURSING The study review the literature about hermeneutics as a foundation for ethnographic and phenomenological research. KEY WORDS: interpretative research, hermeneutic, phenomenology, anthropology CONSIDERAÇÕES FINAIS Concordamos com LOWENBERG (1993) quando afirma que a adoção de abordagens interpretativas é um caminho promissor para a enfermagem, em suas atividades de pesquisa. Da mesma forma, enveredar por esse caminho possibilitará que a enfermagem participe do diálogo atual entre as Ciências Sociais e Humanas. É importante não definir os limites destas abordagens de uma forma rígida, pois estamos numa fase de “transição revolucionária”(LOWENBERG, 1993). Por outro lado, ao elaborar projetos de pesquisa de natureza interpretativa sugerimos que se reserve um espaço para mudanças, abrindo a possibilidade de redefinir o caminho a percorrer, a partir da própria experiência. Experiência que é única e imprevisível, visto que se refere ao diálogo com o outro, à interação humana. HERMENEUTIC AND QUALITATIVE RESEARCH ON NURSING The study review the literature about hermeneutics as a foundation for ethnographic and phenomenological research. KEY WORDS: interpretative research hermeneutic phenomenology anthropology Grande parte das pesquisas de enfermagem enfoca questões do cotidiano, buscando significados socialmente compartilhados. Concordamos com LOWENBERG (1993) quando afirma que a adoção de abordagens interpretativas é um caminho promissor para a enfermagem, em suas atividades de pesquisa. Da mesma forma, enveredar por esse caminho possibilitará que a enfermagem participe do diálogo atual entre as Ciências Sociais e Humanas. Acreditamos que por meio da hermenêutica vislumbramos a possibilidade de compreender os significados atribuídos pelos sujeitos à condição existencial de estar no mundo e, por meio de sua linguagem, em relatos, chegar a compreensão de sua ação. O homem revela a realidade por meio da linguagem oral ou escrita e pela interpretação da linguagem o pesquisador chega aos aspectos significativos essenciais para a compreensão do ser. Portanto, a linguagem pode ser um elemento mediador que permite essa compreensão/interpretação, tanto dos processos cognitivos individuais, quanto dos aspectos sociais e culturais. É importante não definir os limites destas abordagens de uma forma rígida, pois estamos numa fase de “transição revolucionária”(LOWENBERG, 1993). Por outro lado, ao elaborar projetos de pesquisa de natureza interpretativa sugerimos que se reserve um espaço para mudanças, abrindo a possibilidade de redefinir o caminho a percorrer, a partir da própria experiência. Experiência que é única e imprevisível, visto que se refere ao diálogo com o outro, à interação humana. Adotar na enfermagem pesquisas de natureza interpretativa representa, cada vez mais, a possibilidade para que os enfermeiros analisem fenômenos no cotidiano da saúde, a partir do ponto de vista de quem o vivencia. CONSIDERAÇÕES FINAIS Uma das tradições na Sociologia e Antropologia se inspira nos fundamentos da Filosofia da compreensão alemã, representada por Weber na Sociologia e em diferentes concepções hermenêuticas, na Antropologia. O conceito de cultura de Geertz é essencialmente semiótico, como explica: “Acreditando, como Max Weber, que o homem é um animal amarrado a teias de significados que ele mesmo teceu, assumo a cultura sendo essas teias e a sua análise; portanto, não como uma ciência experimental em busca de leis, mas como uma ciência interpretativa, à procura do significado”. (GEERTZ, 1989, p.15). A hermenêutica enquanto paradigma filosófico tem fundamentado as pesquisas qualitativas da enfermagem. Ao considerar as situações cotidianas do enfermeiro no contexto da saúde, buscando o ponto de vista dos sujeitos, sobre suas condições no processo saúde-doença, esse profissional tem aproximado mais as pesquisas da assistência de enfermagem, voltando-se à compreensão/interpretação dos fenômenos pesquisados. Assim, a Antropologia chamada pós-moderna se preocupa muito mais com os fundamentos do que com as técnicas. Na ótica de Geertz não há uma receita para o diálogo na busca da compreensão de significados, comportamentos e ações do outro, o que para ele pressupõe um controle, rigor ou preocupação com a objetividade. A compreensão e interpretação do fenômeno que interessa ao enfermeiro se dá “em sua natureza material e significativa e não em sua natureza formal e estrutural que caberia à ciência e aos cuidados médicos”(CAPALBO, 1994, p.196). “Olhar as dimensões simbólicas da ação social “Olhar as dimensões simbólicas da ação social O paradigma hermenêutico... Rev.latino-am.enfermagem - v. 6 - n. 2 - p. 29-35 - abril 1998 34 Acreditamos que por meio da hermenêutica vislumbramos a possibilidade de compreender os significados atribuídos pelos sujeitos à condição existencial de estar no mundo e, por meio de sua linguagem, em relatos, chegar a compreensão de sua ação. O homem revela a realidade por meio da linguagem oral ou escrita e pela interpretação da linguagem o pesquisador chega aos aspectos significativos essenciais para a compreensão do ser. Portanto, a linguagem pode ser um elemento mediador que permite essa compreensão/interpretação, tanto dos processos cognitivos individuais, quanto dos aspectos sociais e culturais. Adotar na enfermagem pesquisas de natureza interpretativa representa, cada vez mais, a possibilidade para que os enfermeiros analisem fenômenos no cotidiano da saúde, a partir do ponto de vista de quem o vivencia. Grande parte das pesquisas de enfermagem enfoca questões do cotidiano, buscando significados socialmente compartilhados. 17. SCHÜTZ, A. Fenomenologia del mundo social: introdución a la sociologia compreensiva. Buenos Aires: Paidós, 1972. 19. VIETTA, E.P. Configuração triádica, humanista- existencial-personalista: uma abordagem teórico-metodológica de aplicação nas pesquisas de enfermagem psiquiátrica e saúde mental. Rev. latino am. enfermagem, v. 3, n.1, p.31-43, 1995. 17. SCHÜTZ, A. Fenomenologia del mundo social: introdución a la sociologia compreensiva. Buenos Aires: Paidós, 1972. 18. SIEBENEICHLER, F.B. Fenomenologia e hermenêutica. In: CAPALBO, C. Fenomenologia e hermenêutica. Rio de Janeiro: Âmbito Cultural, 1983. Cap. 1, p.9-34. REFERÊNCIAS BIBLIOGRÁFICAS 08. DILTHEY, W. Essência da filosofia. Lisboa: Presença, 1979. 01. BLEICHER, J. Hermenêutica contemporânea. Rio de Janeiro: Edições 70, 1992. 09. FREUND, J. Sociologia de Max Weber. 4. ed. Rio de Janeiro: Forense Universitária, 1987. 10. GEERTZ, C. A interpretação das culturas. Rio de Janeiro: Guanabara, 1989. 02. CALDEIRA, T.P. do R. A presença do autor e a pós- modernidade. Novos Estudos, n.21, p.133-57, julho 1988. 11. JAPIASSU, H.; MARCONDES, D. Dicionário básico de filosofia. 2. ed. Rio de Janeiro: Zahar, 1995. 03. CAPALBO, C. Considerações sobre o método fenomenológico e a enfermagem. Rev. Enferm. UERJ, v.2, n.2, p.192-197, 1994. 12. HABERMAS, J. Dialética e hermenêutica. Porto Alegre: L&PM, 1987. 04. CAPALBO, C. Metodologia das ciências sociais: a fenomenologia de Alfred Schütz. Rio de Janeiro: Antares, 1979. 13. LAPLANTINE, F. Aprender antropologia. 8. ed. São Paulo: Brasiliense,1994. 14. LOWENBERG, J. Interpretive research methodology: broadening the dialogue. Adv. Nurs. Sci., v.16, n.2, p.57-69, 1993. 05. CARDOSO DE OLIVEIRA, R. Sobre o pensamento antropológico. Rio de Janeiro: Tempo Brasileiro, 1988. 15. RICOUER, P. O conflito das interpretações: ensaios de hermenêutica. Rio de Janeiro: Imago, 1978. 06. CORETH, E. Questões fundamentais de hermenêutica. São Paulo: EPU/EDUSP, 1973. 16. SCHÜTZ, A. El problema de la realidad social. Buenos Aires: Amorrortu, 1962. 07. DEMO, P. Metodologia científica em ciências sociais. 3. ed. São Paulo: Artes, 1981. O paradigma hermenêutico... 35 Rev.latino-am.enfermagem - v. 6 - n. 2 - p. 29-35 - abril 1998 35 17. SCHÜTZ, A. Fenomenologia del mundo social: introdución a la sociologia compreensiva. Buenos Aires: Paidós, 1972. 19. VIETTA, E.P. Configuração triádica, humanista- existencial-personalista: uma abordagem teórico-metodológica de aplicação nas pesquisas de enfermagem psiquiátrica e saúde mental. Rev. latino am. enfermagem, v. 3, n.1, p.31-43, 1995. 18. SIEBENEICHLER, F.B. Fenomenologia e hermenêutica. In: CAPALBO, C. Fenomenologia e hermenêutica. Rio de Janeiro: Âmbito Cultural, 1983. Cap. 1, p.9-34.
https://openalex.org/W3168006226	https://www2.ia-engineers.org/conference/index.php/iciae/iciae2021/paper/download/2421/1546	English	null	Development of a Novel Hydrostatic Continuously Variable Transmission with Fast Path Switching	null	2,021	cc-by	3,952	Abstract of robotic actuators is necessary. Robotic actuators need to drive in high speed and output high force since required driving characteristics are diﬀerent among each task. Furthermore, robots are required to move flexibly against unexpected disturbances by humans. In other words, high backdrivability is needed to develop a flexible actuator(2)(3). Recently, human-robot collaboration is required in many kinds of fields. To develop robotic systems for collaboration with humans, a wide driving range of robotic actuators is necessary. For example, robots for rehabilitation have to conduct some tasks, like supporting human and moving fast. Backdrivability is also necessary for robotic actuators from a viewpoint of safety. A hydraulic closed circuit called an electro-hydrostatic actuator (EHA) is utilized for robotic actuators since they have high power/weight ratio and backdrivability. However, EHA cannot achieve characteristics of both high velocity driving and high force driving since a reduction ratio is constant. This paper proposes a novel hydrostatic continuously variable transmission that has two oil paths with a solenoid valve. The solenoid valve switches between two discrete reduction ratios by switching two oil paths. Furthermore, this paper realizes a continuously variable reduction ratio by switching the two oil paths quickly. This paper confirms that the reduction ratio changes continuously and widely by simulations. Recently, human-robot collaboration is required in many kinds of fields. To develop robotic systems for collaboration with humans, a wide driving range of robotic actuators is necessary. For example, robots for rehabilitation have to conduct some tasks, like supporting human and moving fast. Hydraulic actuators, which exhibit large outputs, have been utilized widely in the field of construction. Currently, they have been actively researched for robotic actuators(4). Hydraulic systems have two types, open circuits, and closed circuits. Hydraulic actuators are driven by hydraulic pumps and servo valves in open circuits. A servo valve is an electrically operated valve that controls how hydraulic fluid is sent to a hydraulic actuator. Open circuits can control the desired both flow rate and pressure by using servo valves. In other words, the hydraulic actuators have a wide driving range. However, open-loop hydraulic circuits have no backdrivability since the oil direction is one way and servo valves blocked external forces. On the other hand, a hydraulic closed circuit called an electro-hydrostatic actuator (EHA) is driven by servo pumps coupling with servo motors(5). Abstract EHAs are not needed to put large oil tanks and have backdrivability since oil direction is bidirectional. However, they cannot achieve both high speed driving and high force driving since a reduction ratio determined by a ratio of a displacement between a pump and a motor is constant. Keywords: Continuously variable transmission, hydraulic systems, solenoid valve. Corresponding Author: nozaki@sd.keio.ac.jp Corresponding Author: nozaki@sd.keio.ac.jp DOI: 10.12792/iciae2021.029 © 2021 The Institute of Industrial Applications Engineers, Japan. aKeio University, 3-14-1 Hiyoshi, Kohoku, Yokohama 223-8522 JAPAN b Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts, 02139 USA *Corresponding Author: nozaki@sd.keio.ac.jp Proceedings of the 9th IIAE International Conference on Industrial Application Engineering 2021 Proceedings of the 9th IIAE International Conference on Industrial Application Engineering 2021 1. Introduction This paper proposes a novel hydrostatic continuously variable transmission (CVT). The proposed CVT has two oil paths with a solenoid valve. The solenoid valve switches between two discrete reduction ratios by switching the two oil paths. Furthermore, the proposed CVT can widely change the reduction ratio continuously by switching the solenoid valve quickly. In recent years, cooperation between robots and humans is required not only in industrial fields, but also human assistance such as rehabilitation. Most conventional industrial robots conduct only specific tasks(1). However, robots for rehabilitation fields have to conduct some tasks, like supporting human and moving fast. To develop robotic systems for collaboration with humans, a wide driving range © 2021 The Institute of Industrial Applications Engineers, Japan. 157 (a) Hydraulic circuit. (b) Mechanism. Fig. 1. Proposed hydrostatic CVT. (a) Hydraulic circuit. Fig. 2. Flow path of 3-way solenoid valve. (a) Hydraulic circuit. Fig. 2. Flow path of 3-way solenoid valve. (b) Mechanism. Fig. 1. Proposed hydrostatic CVT. (b) Mechanism. Fig. 1. Proposed hydrostatic CVT. Table 1. Switching patterns of solenoid valve. Paths Connect point Shuttle valve Path 1 Tank A to C Path 2 Charge Pump B to C the hydraulic circuit is lower than the relief pressure by setting a pressure reducing valve. The relationship among the tank pressure pt, which was assumed to be zero, the charge pressure pc, and the relief pressure pr are described as: (1) pt < pc <pr . A solenoid valve and two shuttle valves are used to switch two oil paths. Shuttle valves determine a direction by pressure diﬀerence. Figure. 2 shows a flow path of a 3- way solenoid valve. The spool moves and opens each path when the voltage applies to the solenoid valve. The spool position xsv is given as (b) Mechanism. (b) Mechanism. Fig. 1. Proposed hydrostatic CVT. Fig. 1. Proposed hydrostatic CVT. This paper is organized as follows. In section 2, the proposed CVT is presented. The control method of a servo motor and a solenoid valve are introduced in section 3. Simulations are conducted in section 4. Finally, the conclusions are presented in section 5. xsv = kvoV, (2) xsv = kvoV, (2) where V and kvo represent the input voltage and the spool position gain. Then, the output oil flow qb and pressure pb are given as the conclusions are presented in section 5. 1. Introduction qb = ksvxv(𝑠(𝑥sv)√ 2\|𝑝r−𝑝b\| 𝜌 + 𝑠(−𝑥sv)√ 2\|𝑝b−𝑝t\| 𝜌 ), (3) S(xv) = {1, if 𝑥sv ≤0 0, if 𝑥sv > 0 , (4) sCbpb = qb - Kl(pb - pc), (5) (3) 2.2 Modelling Figure. 3 shows the path 1 in the proposed CVT. The red line is the flow path. The motor 1 and the motor 2 rotate by the input pump. The path 2 is shown in figure. 4. The blue line is the flow path. In the path 2, the only motor 2 rotates by input pump. Oil flow displaced by the motor 1 flows the loop. The displacement of the hydraulic motor in the path 2 is diﬀerent from the one of the path 1. Therefore, the reduction ratio can be switched between two discrete value. Figure. 3 shows the path 1 in the proposed CVT. The red line is the flow path. The motor 1 and the motor 2 rotate by the input pump. The path 2 is shown in figure. 4. The blue line is the flow path. In the path 2, the only motor 2 rotates by input pump. Oil flow displaced by the motor 1 flows the loop. 2.1 Hydraulic Circuit (10) sΔp = 1 𝐶𝑏(qm - ql - KlΔp), = 1 𝐶b ( 𝐷m 2𝜋𝜃̇m − 𝐷l 2𝜋𝜃̇l −𝑘l𝛥𝑝), = 𝐷m (𝜃̇ 𝐷l 𝜃̇ 2𝜋𝛥𝑝) = 1 𝐶b ( 𝐷m 2𝜋𝜃̇m − 𝐷l 2𝜋𝜃̇l −𝑘l𝛥𝑝), = 𝐷m 2𝜋𝐶b (𝜃̇m − 𝐷l 𝐷m 𝜃̇l − 2𝜋 𝐷m 𝛥𝑝). (10) Finally, the dynamic of the torsional torque τs is given as 𝜏s = Ks (𝜃m −𝑅𝜃l − 1 𝐵s ∫𝜏s), (11) Ks = 1 𝐶𝑏( 𝐷m 2𝜋) 2 , (12) Bs = 1 𝐾𝑙( 𝐷m 2𝜋) 2 , (13) R = ( 𝐷l1+𝐷l2 𝐷m , 𝐷l2 𝐷m), (14) 𝜏s = Ks (𝜃m −𝑅𝜃l − 1 𝐵s ∫𝜏s), Fig. 4. Path 2. one of the port C in the path 2 since the charge pressure is lower than the relief pressure. The port A and the port B are connected and the port C is blocked. The model of the shuttle valve can be described as (13) (14) pa = max (pb, pc). (6) (6) where Ks, Bs, and R represent a spring coeﬃcient of oil, a viscous coeﬃcient, and reduction ratios respectively. Modeling and parameters except the reduction ratios are the same between the path 1 and the path 2. By applying (7)-(14), the proposed CVT is modeled as two-inertia resonant systems as shown in Figure. 5. where Ks, Bs, and R represent a spring coeﬃcient of oil, a viscous coeﬃcient, and reduction ratios respectively. Modeling and parameters except the reduction ratios are the same between the path 1 and the path 2. By applying (7)-(14), the proposed CVT is modeled as two-inertia resonant systems as shown in Figure. 5. 3. Control The displacement of the hydraulic motor in the path 2 is diﬀerent from the one of the path 1. Therefore, the reduction ratio can be switched between two discrete value. 2.1 Hydraulic Circuit (4) The proposed hydrostatic CVT is shown in Figures. 1. The proposed CVT is a hydrostatic system similar to EHAs. A servo motor controls a driving of a hydraulic pump and two hydraulic motors are connected by a rotating shaft. A charge pump is connected to keep the minimum pressure of the oil above atmospheric pressure and avoid cavitation. Cavitation is a phenomenon that the air dissolved in the hydraulic cylinder evaporates and forms bubbles when the pressure of the oil drop below the atmospheric pressure in closed hydraulic circuits(7). The proposed hydraulic circuit is protected by installed a charge pump. The charge pressure of sCbpb = qb - Kl(pb - pc), (5 sCbpb = qb - Kl(pb - pc), (5) where q, p, ksv, 𝜌, Cb, and Kl represent flow rate, pressure, a flow coeﬃcient, oil density, a compression coeﬃcient, and a leakage coeﬃcient respectively. By applying (3)-(5), the pressure pb changes by switching the solenoid valve. Switching pattern of the solenoid valve is listed in Table 1. In the path 1 connected to the tank, the port B pressure becomes lower than the charge pressure. The port A and the port C are connected and the port B is blocked. On the other hand, the pressure of the port B becomes higher than the 158 Fig. 3. Path 1. Fig. 4. Path 2. Fig. 5. Two-inertia resonant system. Fig. 3. Path 1. Fig. 3. Path 1. Fig. 5. Two-inertia resonant system. diﬀerence between the input and the output ports respectively. The subscript m stands for an index for the motor side. The superscript ref and dis stand for an index for the reference value and the disturbance value respectively. The dynamics of the load side is given as, Jl𝜃̈l + Bl𝜃̇l = 𝐷𝑙 2𝜋Δp - 𝜏l dis, (8) Dl = (Dl1+Dl2, Dl2), (9) Fig. 3. Path 1. Fig. 3. Path 1. Dl = (Dl1+Dl2, Dl2), where Dl1 and Dl2 represent the displacement of the motor 1 and the motor 2 respectively. The subscript l represents an index for the load side. The pressure diﬀerence ∆p is expressed by the oil compression as follows: Fig. 4. Path 2. Fig. 4. Path 2. sΔp = 1 𝐶𝑏(qm - ql - KlΔp), = 1 𝐶b ( 𝐷m 2𝜋𝜃̇m − 𝐷l 2𝜋𝜃̇l −𝑘l𝛥𝑝), = 𝐷m 2𝜋𝐶b (𝜃̇m − 𝐷l 𝐷m 𝜃̇l − 2𝜋 𝐷m 𝛥𝑝). 3.1 Position Control The dynamics of the motor side is given as The motor-side angle is controlled by the servo motor. The block diagram of the position control is shown in Figure. 6. Kp and Kv represent feedback gains of position and velocity for a position controller. The superscripts cmd stands for Jm𝜃̈m + Bm𝜃̇m = 𝜏m ref- 𝐷m 2𝜋Δp - 𝜏m dis, (7) where J, B, D, θ, τ, and ∆p represent inertia, viscous coeﬃcient, displacement, angle, torque, and pressure 159 Fig. 6. Block diagram of position control． Fig. 8. Port B pressure when d = 0.5． Fig. 8. Port B pressure when d = 0.5． Fig. 6. Block diagram of position control． Fig. 6. Block diagram of position control． Fig. 7. Block diagram of torsional torque control． Fig. 8. Port B pressure when d = 0.5． 3.3 Reduction Ratio Control The continuously variable reduction ratio is achieved by switching the solenoid valve quickly. The solenoid valve is controlled by a pulse width modulation (PWM) signal with adjustable duty ratio and frequency(10)(11). Input voltage is described as V = s(t)V0 , (18) S(xv) = {1, if 0 < 𝑡≤𝑑𝑇 −1, if 𝑑𝑇< 𝑡≤𝑇 , (19) (18) (19) where V0, d, and T represent the initial voltage, PWM signal duty ratio, and PWM signal period. By applying (3)-(5) and (18)-(19), the port B pressure in PWM control is shown in Figure 8. As shown in Figure 8, the port B pressure is oscillating. When switching frequency is sufficiently fast and the duty ratio sets 0.5, the port B pressure in the steady state 𝑝̅b is described as Fig. 7. Block diagram of torsional torque control． command values and response values respectively. Disturbance observer (DOB) was implemented to achieve robust control(8). The estimated disturbance value 𝜏̂dob dis is described as follows: 𝜏̂dob dis = 𝜏̂m dis + 𝜏̂s, (15) = 𝑔dob 𝑠+𝑔dob (𝜏m ref −𝐽mn𝜃̈m) , (16) 𝑝̅b = 𝑝r 2 . (20) (20) (16) Then, the charge pressure is set as follows: Then, the charge pressure is set as follows: (21) where 𝑔dob is the cut-oﬀ frequency of the low-pass filter. pc = 𝑝̅b , (21) By setting (21), the port B pressure is oscillating around the charge pressure and two oil paths are switched quickly. The average value of reduction ratio R0.5 is given as ◯ ̂ stands for estimated values. As 𝑔dob sets higher, the control system becomes robust against the disturbance. 4.2 Results when the relative angular velocity is a positive value. It means that the time opening the path 1 in a switching period needs to decrease and the time opening the path 2 in a switching period needs to increase. On the other hand, the average reduction ratio of the proposed CVT has to be increased when the relative angular velocity is a negative value. It means that the time opening the path 1 in a switching period needs to increase and the time opening the path 2 in a switching period needs to decrease. When the positive voltage value is higher than the negative one in the PWM control, the time opening the path 1 becomes long and the time opening the path 2 becomes short. Therefore, the input voltage is controlled as follows : when the relative angular velocity is a positive value. It means that the time opening the path 1 in a switching period needs to decrease and the time opening the path 2 in a switching period needs to increase. On the other hand, the average reduction ratio of the proposed CVT has to be increased when the relative angular velocity is a negative value. It means that the time opening the path 1 in a switching period needs to increase and the time opening the path 2 in a switching period needs to decrease. When the positive voltage value is higher than the negative one in the PWM control, the time opening the path 1 becomes long and the time opening the path 2 becomes short. Therefore, the input voltage is controlled as follows : Figures. 9 show responses of the motor-side angle. 𝜃m cmd was set as, (27) where t represents time. As shown in Figure. 9 (a), the response of the load-side angular velocity changed by switching the two oil paths quickly. Figures. 9 (b)-(d) show the enlarged view of Figure. 9 (a) for 40 ms when Rv was set as 40, 60, and 80. As shown in Figures. 9 (b)-(d), the angular velocity responses were oscillating as the two oil paths were switched. The load-side disturbance torque responses in the torsional torque control are shown in Figures. 10. The command value of the torsional torque was set as 1 Nm with a ramp-shaped trajectory in this simulation. As shown in Figure. 3.2 Torsional Torque Control 𝑅0.5 = 𝑅1+𝑅2 2 . (22) (22) The block diagram of the torsional torque control is shown in Figure. 7. Torsional torque is controlled with a torque sensor. Kf is a feedback gain for a force controller. Kr is a feedback gain to suppress two-inertia resonant systems(9). Then, the input torque reference is given as Control of the input voltage is required to make the average value of the reduction ratio consistent with the desired variable reduction ratio. The relative angular velocity Wv is given as 𝜏m ref = 𝐾f(𝜏s cmd −𝜏s) + 𝜏dob dis + 𝐾r(𝜔m −𝑅v𝜔l), (17) 𝑊v = 𝜔m −𝑅v𝜔l. (23) (23) (17) (17) Wv should be zero when the average reduction ratio is same with the desired one. In the position control, the average where Rv represents the desired variable reduction ratio. reduction ratio of the proposed CVT has to be decreased 160 Table 2. Parameters of simulations. Parameters Values Parameters Values Jm 0.00063 kg・m2 kvo 0.075 mm/V Jl 0.5 kg・m2 ksv 1.07 E-5 Bm 0.0003 Nms/rad V0 10.0 V Bl 5.0 Nms/rad gdob 150 rad/s Bs 0.0915 Nms/rad Kp 6400 Ks 0.099 Nm/rad Kv 160 R1 100 Kf 100 R2 20 Kr 0.01 𝜌 900 kg/m-3 where Kif represents the integral gain. To track the desired reduction ratio in a short time, the voltage gains set higher as the desired reduction ratio is small. Table 2. Parameters of simulations. 4.1 Condition Simulations were conducted to confirm the continuously variable reduction ratio of the proposed CVT. Parameters for simulations are listed in Table 2. By assuming the switching period of the solenoid valves was 60 ms, the duty ratio was set as 0.5. The variable reduction ratio was set as 40, 60, and 80. A servo motor was actuated after time 1.0 in the simulations. 4.2 Results 10 (a), the response of the load- side disturbance torque changed by switching the two oil paths quickly. Figures. 10 (b)-(d) show the enlarged view of Figure. 10 (a) for 40 ms when Rv was set as 40, 60, and 80. As shown in Figures. 10 (b)- (d), the torsional torque responses were oscillating as the two oil paths were switched. 𝑉= 𝑠(𝑡)𝑉0 + 𝐾iv 𝑠𝑊v , (24) (24) (24) where Kiv represent the integral gain. To track the desired reduction ratio in a short time, the voltage gains set higher as the desired reduction ratio becomes large. In the torsional torque control, the mechanism to control the desired reduction ratio is the same. The relative torque Wf is given as The comparisons of the reduction ratios between the simulation value and the theoretical one are listed in Table 3. The simulation value is calculated by the load-side angular velocity and the torsional torque from 4 seconds to 5 seconds. The comparisons of the reduction ratios between the simulation value and the theoretical one are listed in Table 3. The simulation value is calculated by the load-side angular velocity and the torsional torque from 4 seconds to 5 seconds. From Table 3, the simulated reduction ratios corresponded with the theoretical one. As result of the simulations, the reduction ratio could change continuously in a wide range by switching the solenoid valve quickly and changing the input voltage of the solenoid valve. 𝑊f = 𝑅𝑣𝜏s −𝜏l dis. (25) (25) Wf should be zero when the average reduction ratio is same with the desired one. The average reduction ratio of the proposed CVT has to be increased when the relative angular torque is a positive value. average reduction ratio of the proposed CVT has to be decreased when the relative angular velocity is a negative value. Then, the input voltage is controlled as follows: Wf should be zero when the average reduction ratio is same with the desired one. The average reduction ratio of the proposed CVT has to be increased when the relative angular torque is a positive value. average reduction ratio of the proposed CVT has to be decreased when the relative angular velocity is a negative value. Then, the input voltage is controlled as follows: From Table 3, the simulated reduction ratios corresponded with the theoretical one. 5. Conclusions In this paper, the novel hydrostatic CVT was proposed. The solenoid valve of the proposed CVT switched between the two discrete reduction ratios. Furthermore, this paper realized that the reduction ratio changed to the desired value by switching the two oil paths quickly and controlling the input voltage of the solenoid valves. This proposed method was confirmed by the simulations. The wide range of variable reduction ratios is achieved by the proposed hydrostatic CVT. (6) T. Sakuma S. Sakaino, and T. Tsuji, “A Control Strategy for Electro-hydrostatic Actuator Considering Static Friction, Resonance, and Oil Leakage”, IEEJ Journal of Industry Applications, vol. 8, no. 2, pp. 279– 286, June 2019. (7) Y. Nakashima H. Kaminaga, J. Ono, and Y. Nakamura, “Development of backdrivable hydraulic joint mechanism for knee joint of humanoid robots”, In 2009 IEEE International Conference on Robotics and Au- tomation, pp. 1577-1582, May 2009. Actuators with CVT can switch between the high speed driving and the high force driving. In other words, a driving range of actuators can expand. Moreover, the proposed mechanism had higher backdrivability than the conventional hydraulic actuators of open circuits. (8) K. Ohnishi, M. Shibata, and T. Murakami, “Motion control for advanced mechatronics”, IEEE/ASME Transactions on Mechatronics, vol. 1, no. 1, pp. 56–67, Mar. 1996. Future work includes to validate the continuously variable reduction ratio in experiments with comparing the result of the simulation. Hydrostatic systems included nonlinearities. Control scheme compensating these eﬀects had to be developed to achieve the desired reduction ratio. (9) S. Yamada and H. Fujimoto, “Proposal of high backdrivable control using load-side encoder and backlash”, In IECON 2016 - 42nd Annual Conference of the IEEE Industrial Electronics Society, pp. 6429-6434, Oct. 2016. 4.2 Results As result of the simulations, the reduction ratio could change continuously in a wide range by switching the solenoid valve quickly and changing the input voltage of the solenoid valve. 𝑉= 𝑠(𝑡)𝑉0 − 𝐾if 𝑠𝑊f , (26) (26) (26) 161 (a) Load-side angular velocity (b) Rv=40.0. (c) Rv=60.0. (d) Rv=80.0. Fig. 9. Responses of load-side angular velocity. (a) Load-side angular velocity (b) Rv=40.0. (c) Rv=60.0. (d) Rv=80.0. Fig. 9. Responses of load-side angular velocity. (a) Load-side disturbance torque. (b) Rv=40.0. (c) Rv=60.0. (d) Rv=80.0. Fig. 10. Responses of load-side disturbance. 162 (a) Load-side disturbance torque. (b) Rv=40.0. (c) Rv=60.0. (a) Load-side disturbance torque d id l l i (a) Load-side disturbance torque. (a) Load-side angular velocity (b) Rv=40.0. (b) Rv=40.0. (c) Rv=60.0. (c) Rv=60.0. (d) Rv=80.0. (d) Rv=80.0. (d) Rv=80.0. (d) Rv=80.0. Fig. 10. Responses of load-side disturbance. Fig. 9. Responses of load-side angular velocity. 162 (4) J. Mattila, J. Koivumaki, D. G. Caldwell, and C. Semini, “A Survey on control of hydraulic robotic manipulators with projection to future trends”, IEEE/ASME Transactions on Mechatronics, vol. 22, no. 2, pp. 669– 680, Apr. 2017. Table 3. Comparisons of reduction ratios. Theoretical value Figures. 9 Figures. 10 40.0 40.0 39.9 60.0 60.4 59.9 80.0 80.0 79.8 (5) K. Umeda S. Sakaino, K. Tsuda, T. Sakuma, and T. Tsuji, “Resonance- suppression control for electro-hydrostatic actuator as two-inertia system”, IEEJ Journal of Industry Applications, vol. 6, no. 5, pp. 320–327, Sept. 2017. Acknowledgment (10) F. Wang, L. Gu, and Y. Chen, “A continuously variable hydraulic pressure converter based on high-speed on-off valves”, Mechatronics, vol. 21, no. 8, pp. 1298–1308, Dec. 2011. This work was supported by JSPS KAKENHI Grant Numbers JP20H02135 and JP19KK0367. (11) F. Wang, L. Gu, and Y. Chen, “A hydraulic pressure- boost system based on high-speed on/oﬀ valves”, IEEE/ASME Transactions on Mechatronics, vol. 18, no. 2, pp. 733–743, Feb. 2013. References (1) S. Thrun, “Toward a framework for human-Robot interaction”, Human Computer Interaction, vol. 19, no. 1-2, pp. 9–24, June 2004. (2) K. Suzumori, “Backdrivability of robots and actuators”, Journal of the Robotics Society of Japan, vol. 31, no. 6, pp. 548–551, July, 2013. (3) N. Tobias and L. Peter, “Improving backdrivability in geared rehabilitation robots”, Medical & biological engineering & computing, vol. 47, no. 4, pp. 441–447, Feb. 2009. 163
https://openalex.org/W3194643303	https://ejournal.poltektegal.ac.id/index.php/jpa/article/download/2765/JPA%20VOL%201%20NO%201%20HAL%2025-28	Indonesian	null	Implementasi Akad Murabahah Pada Akad Pembiayaan Griya iB Hasanah Pada PT. Bank BNI Syariah KCPS Tegal	Journal of Public Accounting	2,021	cc-by	1,954	Jurnal Akuntansi Publik Volume 1, Nomor 1, April 2021 Jurnal Akuntansi Publik Volume 1, Nomor 1, April 2021 1 Corresponding author’s email: fitrisetiyawati96@gmail.com Implementasi Akad Murabahah Pada Akad Pembiayaan Griya iB Hasanah Pada PT. Bank BNI Syariah KCPS Tegal Fitri Setiyawati1, Sunandar2, Nurul Mahmudah3 1) Program Studi D3 Akuntansi, Politeknik Harapan Bersama 2) Dosen Program Studi D3 Akuntansi, Politeknik Harapan Bersama 3) Dosen Program Studi Sarjana Terapan Akuntansi Sektor Publik, Politeknik Harapan Bersama Abstact: Murabahah agreement is a agreement of sale and purchase of goods that clearly states the goods being traded, and the seller mentions the purchase price to the buyer, then the seller determines the profit margin on the agreement between the seller and the buyer. The agreement used in financing Griya iB Hasanah at PT. Bank BNI Syariah KCPS Tegal is a murabaha contract. The purpose of this study was to determine how the implementation of murabahah agreement in the Griya iB Hasanah financing agreement at PT. Bank BNI Syariah KCPS Tegal. The data collection technique used is observation, interview, and literature study, the data analysis technique used is descriptive qualitative. The results of the research show that the DSN MUI Fatwa No. 4/DSN-MUI/IV/2000 regarding Murabahah has guaranteed the validity and permissibility of murabahah transactions, including in this case home financing at Islamic banks. In conventional banks, usury is encountered when customers borrow money to buy a house. Meanwhile, Islamic banks do not lend money but sell the house to customers. The agreement used are buying and selling. Conclusion the application of murabahah agreement to the financing of Griya iB Hasanah at PT. Bank BNI Syariah KCPS Tegal in its implementation has fulfilled the pillars and conditions of murabaha and avoided usury transactions in accordance with sharia principles. Keywords: Murabahah, Financing, Sharia Principles Pendahuluan Undang-Undang No. 21 Tahun 2008 di mana bank syariah adalah bank yang menjalankan usahanya berdasarkan prinsip syariah dan menurut jenisnya terdiri atas Bank Umum Syariah dan Unit Usaha Syariah[1]. Perbankan syariah memperkenalkan suatu sistem yang tidak hanya menguntungkan bank akan tetapi juga peduli dengan kesejahteraan nasabah, yaitu transaksi berbasis profit and lost sharing atau lebih dikenal di Indonesia dengan sistem bagi hasil, yang selanjutnya tereduksi menjadi sistem revenue sharing[2]. Observasi pra nasabah yang diperoleh peneliti secara langsung melalui pertanyaan yang disuguhkan salah satu nasabah kepada peneliti dalam percakapan yang menghasilkan bahwa adanya keraguan nasabah akan mengajukan pembiayaan Griya iB Hasanah dikarenakan memiliki pandangan yang sama antara Bank konvensional dan Bank Syariah dalam pembiayaan griya. Nasabah berpendapat terdapat unsur bunga yang diterapkan Bank Syariah dalam pembiayaan griya, yang tentunya tidak dibenarkan dalam prinsip islam. Pemahaman orang awam mengenai penilaian Bank Syariah menjadikan ketertarikan peneliti untuk melakukan penelitian ini. Banyaknya kebutuhan masyarakat akan kredit rumah membuat Bank mengeluarkan produk-produk pembiayaan, seperti Kredit Pemilikan Rumah (KPR)[3]. Salah satu Bank Syariah yang menyediakan KPRS adalah PT. Bank BNI Syariah KCPS Tegal yang dikenal dengan pembiayaan Griya iB Hasanah. BNI Syariah KPR Syariah (Griya iB Hasanah) adalah fasilitas pembiayaan konsumtif yang diberikan kepada anggota masyarakat untuk membeli, membangun, merenovasi rumah (termasuk ruko, rusun, apartemen, dan sejenisnya), dan membeli tanah kavling serta rumah indent, yang besarnya disesuaikan dengan kebutuhan pembiayaan dan kemampuan membayar kembali masing-masing calon[4]. Tujuan penelitian ini adalah untuk mengetahui implementasi akad murabahah pada akad pembiayaan Griya iB Hasanah pada PT. Bank BNI Syariah KCPS Tegal. Keywords: Murabahah, Financing, Sharia Principles Abstrak: Akad murabahah adalah akad jual beli barang yang menyatakan dengan jelas barang yang diperjual belikan, serta penjual menyebutkan harga pembelian kepada pembeli, kemudian penjual menentukan keuntungan margin atas kesepakatan antara penjual dan pembeli. Akad yang digunakan dalam pembiayaan Griya iB Hasanah di PT. Bank BNI Syariah KCPS Tegal adalah akad murabahah. Tujuan penelitian ini untuk mengetahui bagaimana implementasi akad murabahah pada akad pembiayaan Griya iB Hasanah pada PT. Bank BNI Syariah KCPS Tegal. Teknik pengumpulan data yang digunakan adalah observasi, wawancara, dan studi pustaka, teknik analisis data yang digunakan adalah Deskriptif Kualitatif. Hasil penelitian yang dilakukan menunjukan bahwa Fatwa DSN MUI No 4/DSN-MUI/IV/2000 tentang Murabahah telah menjamin keabsahan dan diperbolehkannya transaksi murabahah, termasuk dalam hal ini pembiayaan rumah di bank Syariah. Dalam bank konvensional, riba ditemui ketika nasabah meminjam uang untuk membeli rumah. Sedangkan pada bank syariah tidak meminjamkan uang tetapi menjual rumah tersebut kepada nasabah. Akad yang dipakai adalah jual dan beli. Kesimpulan penerapan akad murabahah pada pembiayaan Griya iB Hasanah di PT. Bank BNI Syariah KCPS Tegal dalam pelaksanaanya telah memenuhi rukun dan syarat murabahah serta terhindar dari transaksi ribawi sesuai dengan prinsip syariah. Kata kunci: Murabahah, Pembiayaan, Prinsip Syariah 1 Corresponding author’s email: fitrisetiyawati96@gmail.com Journal of Public Accounting 25 \| P a g e Implementasi Akad Murabahah Pada Akad Pembiayaan Griya iB Hasanah Pada PT. Bank BNI Syariah KCPS Tegal b l h dil l h l k t k mentasi Akad Murabahah Pada Akad Pembiayaan Griya iB Hasanah Pada PT. Bank BNI Syariah KCPS Tegal boleh dilanggar oleh para pelakunya, termasuk larangan untuk melakukan transaksi yang mengandung unsur riba, bathil, maysir, dan gharar[2]. Hasil dan Pembahasan Implementasi Akad Murabahah pada PT. Bank BNI Syariah KCPS Tegal Bank BNI Syariah KCPS Tegal Proses pemberian pembiayaan Griya iB Hasanah di Bank BNI Syariah KCPS Tegal yaitu diawali dari nasabah memilih rumah/ properti yang diinginkan nasabah. Kemudian nasabah melakukan pengajuan pembiayaan kepada pihak bank (Bagian Sales Assistant). Pihak bank menganalisis pembiayaan tersebut dengan survey dan melihat dari prinsip 5c untuk memverifikasi data pembiayaan tersebut. Yakni 5c itu sendiri yaitu Character, Capacity, Capital, Condition, dan Collateral. Penilaian dengan prinsip 5c di Bank BNI Syariah KCPS Tegal merupakan tugas dari Processing and Collection Assisten. Dalam pemberian pembiayaan Bank harus benar-benar yakin bahwa nasabah tersebut telah memenuhi kriteria 5c sehingga pihak Bank tidak mengalami kerugian akibat pembiayaan macet Proses pemberian pembiayaan Griya iB Hasanah di Bank BNI Syariah KCPS Tegal yaitu diawali dari nasabah memilih rumah/ properti yang diinginkan nasabah. Kemudian nasabah melakukan pengajuan pembiayaan kepada pihak bank (Bagian Sales Assistant). Pihak bank menganalisis pembiayaan tersebut dengan survey dan melihat dari prinsip 5c untuk memverifikasi data pembiayaan tersebut. Yakni 5c itu sendiri yaitu Character, Capacity, Capital, Condition, dan Collateral. Untuk pelaksanaan akad yang digunakan dalam pembiayaan Griya iB Hasanah yaitu dengan akad murabahah. akad murabahah adalah akad jual beli barang yang menyatakan dengan jelas barang yang diperjual belikan, serta penjual menyebutkan harga pembelian kepada pembeli, kemudian penjual menentukan keuntungan margin atas kesepakatan antara penjual dan pembeli. Penilaian dengan prinsip 5c di Bank BNI Syariah KCPS Tegal merupakan tugas dari Processing and Collection Assisten. Dalam pemberian pembiayaan Bank harus benar-benar yakin bahwa nasabah tersebut telah memenuhi kriteria 5c sehingga pihak Bank tidak mengalami kerugian akibat pembiayaan macet Pemilihan murabahah sebagai akad dalam produk bank syariah sah dan boleh, tentunya dengan memperhatikan hal-hal yang terkait dengan aturan, syarat, dan mekanisme murabahah yang sesuai dengan prinsip hukum Islam, di mana ada batasan-batasan yang tidak Journal of Public Accounting 26 \| Page Implementasi Akad Murabahah Pada Akad Pembiayaan Griya iB Hasanah Pada PT. Bank BNI Syariah KCPS Tegal a Akad Pembiayaan Griya iB Hasanah Pada PT. Bank BNI Syariah KCPS Tegal Implementasi Akad Murabahah Pada Akad Pembiayaan Griya iB Hasanah Pada PT. Bank BNI Syariah KCPS Tegal Implementasi Akad Murabahah Pada Akad Pembiayaan KCPS Tega yang kemungkinan akan dilakukan oleh nasabah dikemudian hari. yang kemungkinan akan dilakukan oleh nasabah dikemudian hari. 5. Bank BNI Syariah harus mengungkapkan tentang syarat-syarat yang diminta dari harga pembelian kepada nasabah. Hasil dan Pembahasan Implementasi Akad Murabahah pada PT. Bank BNI Syariah KCPS Tegal Setelah pembiayaan disetujui melalui berbagai pihak seperti Direct Sales, Processing & Collection Assistant, Sub Branch Manager, dan Operational & Support Assistant maka terbitlah SKP (Surat Keputusan Pembiayaan) dan pihak Bank menjadwalkan akad. Implementasi pada akad murabahah di Bank BNI Syariah KCPS Tegal yaitu diawali negoisasi yang berlangsung secara terbuka di dalam arti kata, antara nasabah dan Bank BNI Syariah KCPS Tegal saling mengemukakan prosedurnya, prosesnya dan persyaratannya untuk sampai pada tahapan berikutnya. Keterbukaan informasi dalam prosedur negosiasi tersebut akan memberikan kejelasan di antara para pihak bahwa rangkaian proseduran dan persyaratannya tidak ada agenda tersembunyi atau terdapat salah satu pihak yang tidak memiliki kejujuran, kebenaran dan kesungguhan hati dalam pemenuhan prosedur maupun persyaratannya. Keterbukaan tersebut menjadi landasan penting bagi para pihak, bahwa hubungan hukum antara calon nasabah dengan Bank syariah tidak semata-mata berdasarkan peraturan perundang-undangan yang berlaku, melainkan juga memenuhi nilai- nilai dan prinsip-prinsip syariah yang menuntut kejujuran di antara para pihak. Dalam pelaksanaan akad murabahah dipastikan pihak – pihak yang bersangkutan telah memenuhi rukun dan syarat murabahah. Adapun rukun akad murabahah seperti adanya ba’i (penjual) adalah pihak yang memiliki barang untuk dijual, dan musytari (pembeli) adalah pihak yang memerlukan dan akad membeli barang, Objek akad, yaitu mabi’ (barang dagangan) dan tsaman (harga), dan Shighah, yaitu ijab dan qobul. Implementasi pada rukun yang berakad (ba’i dan musytari) harus cakap dalam hukum dan tidak dalam keadaan terpaksa, barang yang diperjual belikan (mabi’) tidak termasuk barang yang haram dan jenis maupun jumlahnya jelas, harga barang (tsaman) harus dinyatakan secara transparan (harga pokok dan komponen keuntungan) dan cara pembayaran disebutkan dengan jelas, pernyataan serah terima (ijab qabul) jelas dengan menyebutkan spesifik pihak-pihak yang berakad hal ini sesuai dengan rukun akad pembiayaan murabahah. Adapun langkah-langkah akad murabahah pada pembiayaan BNI Syariah pada PT. Bank BNI Syariah KCPS Tegal yang diperoleh dari wawancara dengan Bapak Suswantoro selaku Direct Sales yaitu sebagai berikut : Analisis berikutnya dilakukan terhadap syarat akad murabahah pada pembiayaan Griya iB Hasanah di Bank BNI Syariah KCPS Tegal yang meliputi: 1. Doa pembuka 2. Pengisian daftar hadir 3. Pembacaan SKP (Surat Keputusan Pembiayaan) 1. Bank BNI Syariah memberitahukan tentang biaya (cost) atau modal yang dikeluarkan atas barang tersebut kepada nasabah. 2. Akad pertama harus sah. 2. Akad pertama harus sah. 3. Akad tersebut harus bebas dari riba. 4. Bank BNI Syariah harus mengungkapkan tentang wanprestasi yang terjadi setelah pembelian dan harus diungkapkan dengan jelas dan rinci. 9. Daftar Pustaka [1]Muhammad, 2019, Model-model Akad Pembiayaan di Bank Syariah. Yogyakarta. [2]Imama, L.S., 2014, Konsep dan Implementasi Murabahah pada Produk Pembiayaan Bank Syariah. Jurnal Ekonomi dan Perbankan Syariah Vol.1 No.2. [3]Rosyida, Eva., 2017, Analisa Perbandingan Pembiayaan Hunian Syariah Dengan Akad Murabahah Dan Akad Musyarakah Pada Bank Muamalat. Jurnal Akuntansi Vol.1 No.3. 18. Doa penutup Fatwa No.04/DSN-MUI/IV/2000 tentang murabahah, sebagai landasan syariah transaksi murabahah adalah sebagai berikut: disebutkan bahwa jika bank hendak mewakilkan kepada nasabah untuk membeli barang dari pihak ketiga, akad jual beli murabahah harus dilakukan setelah barang secara prinsip, menjadi milik Bank, Dengan demikian, dapat disimpulkan bahwa pelaksanaan prinsip syariah dalam akad murabahah pada Bank BNI Syariah KCPS Tegal telah sesuai dengan prinsip-prinsip syariah. iB Hasanah [4]Gunawan dan Cahyanti., 2014, Pengaruh Pembiayaan KPR Syariah Terhadap Proses Keputusan Pembelian Rumah di Kota Cirebon. Jurnal Edunomik Vol.2 No.2. Syarat-syarat yang harus dipenuhi nasabah ketika akan melakukan pengajuan pembiayaan Griya iB Hasanah di Bank BNI Syariah KCPS Tegal yaitu : 1. Pemohon minimal berusia 21 tahun, dan lunas pada saat usia pension 2. Karyawan/profesional/pengusaha (wiraswasta) 3. Mempunyai penghasilan tetap dan mampu mengangsur 4. Memenuhi perrsyaratan berdasarkan penilaian bank. 13. Tandatangan jadwal angsuran 15. Tandatangan surat pernyataan dan kuasa pemohon perrsetujuan suami/ istri 28 \| P a g e Hasil dan Pembahasan Implementasi Akad Murabahah pada PT. Bank BNI Syariah KCPS Tegal Paraf halaman setiap lembar dari pasal 1 s/d pasal 22 10. Tandatangan surat pernyataan 11. Tandatangan perjanjian kuasa jual 12. Tandatangan berita acara pembelian objek 27 \| P a g e Journal of Public Accounting 13. Tandatangan jadwal angsuran Journal of Public Accounting Kesimpulan Berdasarkan hasil penelitian dan pembahasan yang dilakukan oleh peneliti di PT. Bank BNI Syariah KCPS Tegal, maka dapat disimpulkan sebagai berikut : PT. Bank BNI Syariah KCPS Tegal menyediakan pembiayaan Griya iB Hasanah untuk membantu memenuhi kebutuhan masyarakat melalui angsuran sehingga banyak masyarakat yang dapat mewujudkan keinginannya memiliki tempat tinggal. Proses pada pembiayaan Griya iB Hasanah di PT. Bank Journal of Public Accounting 28 \| P a g e
https://openalex.org/W2940190674	https://europepmc.org/articles/pmc6465765?pdf=render	English	null	Expression of Components of the Renin-Angiotensin System in Pyogenic Granuloma	Frontiers in surgery	2,019	cc-by	6,428	Methods: PG samples from 14 patients were analyzed for the expression of components of the RAS: pro-renin receptor (PRR), angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1) and angiotensin II receptor 2 (ATIIR2), using 3,3-diaminobenzidine (DAB) immunohistochemical (IHC) staining. Immunoﬂuorescence (IF) IHC staining was performed to localize these proteins on four of the PG samples. RT-qPCR was performed on two snap-frozen PG samples. Western blotting (WB) was performed on one snap-frozen PG sample and two PG-derived primary cell lines. Edited by: Jan A. Plock, University of Zurich, Switzerland Reviewed by: Jean Kanitakis, Hospices Civils de Lyon, France Fatih Zor, Wake Forest School of Medicine, United States Shirley Potter, National University of Ireland Galway, Ireland Results: DAB IHC staining demonstrated the expression of ACE, PRR, ATIIR1, and ATIIR2 in all 14 PG tissue samples. RT-qPCR analysis conﬁrmed abundant mRNA transcripts for PRR, ACE, AIITR1 and ATIIR2, relative to the housekeeping gene. WB conﬁrmed the presence of PRR, ATIIR1, and ACE in the PG tissue sample, and PRR and ATIIR1, in the PG-derived primary cell lines. IF IHC staining demonstrated the expression of PRR, ACE, ATIIR1 on the primitive population that expressed NANOG and SOX2 on the ERG+ endothelium of the microvessels within PG. Correspondence: Swee T. Tan swee.tan@gmri.org.nz †These authors share senior authorship Specialty section: This article was submitted to Reconstructive and Plastic Surgery, a section of the journal Frontiers in Surgery Conclusion: We have demonstrated the expression of PRR, ACE, and ATIIR1 by the putative the ESC-like population within PG. Conclusion: We have demonstrated the expression of PRR, ACE, and ATIIR1 by the putative the ESC-like population within PG. Keywords: pyogenic granuloma, vascular tumor, renin-angiotensin system, embryonic stem cells, angiotensin converting enzyme Keywords: pyogenic granuloma, vascular tumor, renin-angiotensin system, embryonic stem cells, angiotensin converting enzyme Received: 21 July 2018 Accepted: 25 February 2019 Published: 09 April 2019 Expression of Components of the Renin-Angiotensin System in Pyogenic Granuloma Jessica C. Papali’i-Curtin 1,2, Helen D. Brasch 1,2, Bede van Schaijik 1, Jennifer de Jongh 1, Reginald W. Marsh 1, Swee T. Tan 1,2† and Tinte Itinteang 1† 1 Gillies McIndoe Research Institute, Wellington, New Zealand, 2 Centre for the Study and Treatment of Vascular Birthmarks, Wellington Regional Plastic, Maxillofacial and Burns Unit, Hutt Hospital, Wellington, New Zealand Background: There is a growing body of research demonstrating expression of the renin-angiotensin system (RAS) by a putative embryonic stem cell (ESC)-like population within vascular anomalies. This study investigated the expression of components of the RAS in relation to the putative ESC-like population within pyogenic granuloma (PG) that we have recently reported. Keywords: pyogenic granuloma, vascular tumor, renin-angiotensin system, embryonic stem cells, angiotensin converting enzyme ORIGINAL RESEARCH published: 09 April 2019 doi: 10.3389/fsurg.2019.00013 Citation: Papali’i-Curtin JC, Brasch HD, van Schaijik B, de Jongh J, Marsh RW, Tan ST and Itinteang T (2019) Expression of Components of the Renin-Angiotensin System in Pyogenic Granuloma. Front. Surg. 6:13. doi: 10.3389/fsurg.2019.00013 Pyogenic granuloma (PG) is a relatively common benign vascular tumor aﬀecting the skin and mucosa. Also known as lobular capillary hemangioma, it is comprised of hyperplastic capillary vessels and ﬁbromyxoid stroma with overlying atrophic or ulcerated epidermis (1). There is a slight overall male preponderance (1). PG arises spontaneously, in previously injured tissues or within vascular malformations (1) and has been associated with certain medications, such as chemotherapeutic agents (2). PG accounts April 2019 \| Volume 6 \| Article 13 Frontiers in Surgery \| www.frontiersin.org Renin-Angiotensin System in Pyogenic Granuloma Papali’i-Curtin et al. for 0.5% of skin nodules in the pediatric population, most commonly in the ﬁrst ﬁve years of life (1). Epulis gravidarum is a sub-type of PG that occurs during pregnancy, and is thought to be induced by hormonal changes (3) and sometimes grows to giant proportions (4). physiologically in the vasculature, liver, pancreas, kidney, brain and reproductive organs, and they have also been demonstrated in multiple types of malignant and benign tumors, often in diﬀerent concentration than the normal surrounding tissues (21–23). PG usually presents as a small (<2 cm), red, friable, sessile or pedunculated nodule that bleeds repeatedly. Most PG occur in the head and neck area, followed by the trunk and the limbs and 12% occur on mucosal surfaces (1). The key components of the RAS include pro-renin receptor (PRR), which converts pro-renin to active renin (24); ACE which converts inactive angiotensin I (AT1) to active angiotensin II (ATII) (24); angiotensin II receptor 1 (ATIIR1) which binds with ATII and couples with signaling molecules to cause multiple eﬀects such as vasoconstriction and cellular proliferation (25); and angiotensin II receptor 2 (ATIIR2) which functions similarly to ATIIR1 but appears to cause the opposite downstream signaling eﬀects such as vasodilation and inhibition of cell growth (23). PRR has been implicated in many types of cancer and is thought to play a role in cellular proliferation and apoptosis (26– 28). Recent investigations into the RAS in IH demonstrate the presence of ACE and ATIIR2 on the hemogenic endothelium of this tumor, and the proliferative eﬀect of ATII via ATIIR2 on IH-derived cells (15, 17). ATIIR1 appears to play a lesser role in IH (17). Citation: The expression of the ACE and ATIIR2 on the hemogenic endothelium in IH underscores the accelerated involution of proliferating IH induced by β-blockers (16) and ACE inhibitors (18). Treatment of PG includes full thickness excision, shave excision, cautery, systemic steroids or topical treatments (e.g., propranolol and imiquimod cream) with full thickness excision having the lowest recurrence rates (1). Shave excision and pulsed dye laser therapy is eﬀective for PG aﬀecting cosmetically sensitive areas (5). Some lesions, especially epulis gravidarum, may resolve spontaneously following delivery, although the frequency is unknown (6). PG has been attributed to disordered angiogenesis although its pathogenesis remains unclear. The capillaries of the PG appear immature (1) and express embryonic stem cell (ESC) markers suggesting a primitive origin of this tumor (7). ESCs exist in blastocyst of the pre-implantation embryo and possess the capacity to diﬀerentiate into all three germ layers– endoderm, ectoderm and mesoderm (8). This pluripotency diﬀerentiates ESCs from adult/somatic stem cells and germ cells. NANOG (9), OCT4 (10), SOX2 (11), and signal transducer and activator of transcription (STAT3) (12) are transcription factors that maintain pluripotency and are markers that characterize ESCs. Once activated, the STAT family of proteins bind target sites on the DNA, of which STAT3 activation is essential for cell self-renewal (13). OCT4 is a POU family transcription factor that interacts with SOX2 to maintain ESC pluripotency and is expressed on early ESCs (10). SOX2 is a transcription factor from the sex-determining Y family which is critical in neural cells and ESCs and also important in multiple stages of mammalian development (11). SOX2 and OCT4 act synergistically to regulate NANOG, a homeobox-containing transcription factor essential in maintaining pluripotency of the inner cell mass (9). In this study we investigated the expression on the aforementioned components of the RAS within PG at both the transcriptional and translational levels, and their localization in relation to the putative ESC-like population. Tissue Samples PG tissue samples from four female and 10 male patients with an average age of 21.3 (range, 3–42) years were sourced from the Gillies McIndoe Research Institute Tissue Bank and used for this study which was approved by the Central Regional Health and Disability Ethics Committee (Ref. 13/CEN/130). Written consent was obtained from participants or their caregivers in accordance with the Declaration of Helenski. The diagnosis of PG was made clinically including a history of an acquired lesion with the typical histopathological appearance and the absence of GLUT-1 staining (29). The lesions were located in the head and neck area (n = 8) and the hand (n = 6), measuring 8–25 (mean, 12) mm, and were present for 2–10 (mean, 3) months prior to excision. We have reported two putative ESC-like subpopulations within PG, one on the endothelium that expresses OCT4, NANOG, pSTAT3 and SOX2, and an interstitial subpopulation that expresses NANOG, pSTAT3 and SOX2 (7). We infer that the ESC-like population on the endothelium may diﬀerentiate to form the downstream interstitial subpopulation resulting in the loss of OCT4 expression, or alternatively, de-diﬀerentiation of the interstitial cells may give rise to the more primitive endothelial subpopulation. Western Blotting Western blotting (WB) was performed on total protein extracts from one snap-frozen PG tissue section and two PG-derived primary cell lines. The protein extracts were resolved by 4– 12% one-dimensional polyacrylamide gel electrophoresis then transferred to polyvinylidene diﬂuoride membranes. The samples were them probed using the primary antibodies ACE (1:200; cat# sc-12184, Santa Cruz, Rockford, IL, USA), PRR (1:500; cat# ab40790, Abcam, Cambridge, UK), ATIIR1 (1:200; cat# sc- 1173, Santa Cruz), ATIIR2 (1:500; cat# ab92445, Abcam) and β-actin (1:500; cat# ab8229 Abcam); then incubated with the appropriate secondary antibody: goat anti-rabbit HRP (1:1000, cat# A16110, Thermo Fisher) and donkey anti-goat HRP (1:1,000, cat# ab97120, Abcam). ACE was probed using a tertiary cascade consisting of a rabbit anti-goat SuperclonalTM biotin conjugated secondary antibody (1:4,000; cat# A27013, Thermo Fisher) followed by a PierceTM Streptavidin Poly HRP (1:5000, cat# 21140, Thermo Fisher) at 4◦C for 10 min. Clarity Western ECL (cat# 1705061, Bio-Rad) was used as the substrate for visualizing HRP detected protein bands and the ChemiDoc MP Imaging System (Bio-Rad) and Image Lab 5.0 software (Bio- Rad) were used for band detection and analysis. Positive controls were human placenta for PRR and ATIIR1, mouse lung for ACE, and a recombinant ATIIR2 protein (cat# H00000186-P01, Novus Biologicals, Littleton, CO, USA) for ATIIR2. Matched mouse (1:500; cat# ab18443, Abcam) and rabbit (1:500; cat# ab171870, Abcam) isotype controls were used as appropriate negative controls. Immunohistochemical Staining g 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on 4 µm-thick formalin-ﬁxed paraﬃn- embedded sections of 14 PG tissue samples using the Leica Bond Rx auto-stainer (Leica, Nussloch, Germany) with antibodies against PRR (1:100; cat# HPA003156, Sigma-Aldrich, St. Louis, MO, USA), ACE (1:40; cat# 3C5, Serotec, Raleigh, NC, USA), ATIIR1 (1:25; cat# ab9391, Abcam, Cambridge, MA, USA), ATIIR2 (1:2000; cat# NBPI-77368, Novus Biologicals, Littleton, CO, USA), SOX2 (1:200; cat# PA1-094, Thermo Fisher Scientiﬁc, Expression of components of the RAS in venous malformation (VM) (14) and infantile hemangioma (IH) (15) has been reported. This observation underscores the eﬃcacy of RAS modulators in the treatment of IH using β-blockers(16) and angiotensin converting enzyme (ACE) inhibitors (15, 17, 18). In addition to the classical role in cardiovascular homeostasis, the role of the RAS in cellular proliferation, angiogenesis, immune response and apoptosis is increasingly appreciated (19, 20). Components of the RAS are present April 2019 \| Volume 6 \| Article 13 Frontiers in Surgery \| www.frontiersin.org 2 Renin-Angiotensin System in Pyogenic Granuloma Papali’i-Curtin et al. Rockford, IL, USA), NANOG (1:100; cat# ab80892, Abcam) and ERG, (1: 200; Cell Marque, Rocklin, CA, USA). Antibodies were diluted with bond primary diluent (Leica). DAB IHC- stained slides were mounted in Surgipath Micromount (cat# 3801732, Leica). nSolverTM software (NanoString Technologies) using standard settings and normalized against the housekeeping gene. Rockford, IL, USA), NANOG (1:100; cat# ab80892, Abcam) and ERG, (1: 200; Cell Marque, Rocklin, CA, USA). Antibodies were diluted with bond primary diluent (Leica). DAB IHC- stained slides were mounted in Surgipath Micromount (cat# 3801732, Leica). PG-Derived Primary Cell Lines y Primary cell lines were derived from two fresh surgically excised PG tissue samples by culturing them as explants in Matrigel and then extracting the cells following abundant growth, as previously described (15). The extracted PG cells were then cultured and passaged in DMEM medium (cat# 10569010, Gibco, Thermo Fisher Scientiﬁc, Waltham, MA, USA) supplemented with 1% FCS (cat# 10091148, Gibco, Thermo Fisher Scientiﬁc), 5% mTeSRTM (cat# 85850, StemCell Technologies, Vancouver, Canada), 1% penicillin-streptomycin (cat# 15140122, Gibco, Thermo Fisher Scientiﬁc) and 0.2% gentamicin/amphotericin B (cat# R01510, Gibco, Thermo Fisher Scientiﬁc). All cultures were maintained in a humidiﬁed incubator at 37◦C at an atmosphere of 94% air and 5% CO2. All primary cell lines used for the experiments were passages 6–8. NanoString mRNA Analysis g y NanoString mRNA analysis was performed on six snap-frozen PG tissue samples of the original cohort of 14 patients used for DAB IHC staining, using the NanoString nCounterTM Gene Expression Assay (NanoString Technologies, Seattle, WA, USA). RNA was extracted from the samples using RNeasy Mini Kit (Qiagen, Hilden, Germany) and subjected to the NanoString nCounterTM Gene expression assay performed by New Zealand Genomics Ltd (Dunedin, New Zealand) according to the manufacturer’s protocol. Probes for the genes encoding ACE (NM_000789.2), PRR (ATP6AP2, NM_005765.2), ATIIR1 (NM_000685.3) and ATIIR2 (NM_000686.3), and housekeeping gene GAPDH (NM_002046.3) were designed and synthesized by NanoString Technologies. The raw data were analyzed by RT-qPCR To conﬁrm transcription activation of components of the RAS in PG-derived primary cell lines from two PG tissue samples of the original cohort of 14 patients included for DAB IHC staining. RNA samples were then prepared using the RNeasy Mini Kit (Qiagen), with RNA extracted through DNase digest using the QIAcube (Qiagen). Samples were then subjected to NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientiﬁc) quantiﬁcation. Extractions for each sample were performed in triplicates and analyzed. RNA was analyzed with Rotor-Gene Multiplex RT-PCR Kit (Qiagen) and subjected to RT-qPCR using the Rotor-Gene Q (Qiagen). The expression of ESC markers was detected using gene-speciﬁc TaqMan (Thermo Fisher Scientiﬁc cat# 4331182) primers- probes (AT2R1: Hs00258938_m1, AT2R2: Hs02621316_s1, ACE: Hs00174179_m1, ATP6AP2/PRR: Hs00997145_m1), and the reference gene GAPDH (Hs99999905_m1). Positive controls were demonstrated on uterine ﬁbroid tissue, and speciﬁcity of probes were conﬁrmed by inclusion of a water negative control. Immunoﬂuorescence (IF) IHC staining was performed on four PG tissue samples of the original cohort of 14 patients used for DAB IHC staining, using the same antibodies dilutions and the following combinations NANOG/ACE, SOX2/ACE, PRR/ACE, ERG/ATIIR1. For IF IHC detection, a combination of Vectaﬂuor Excel anti-rabbit 594 (ready-to-use; cat# VEDK- 1594, Vector Laboratories, Burlingame, CA, USA) and Alexa Fluor anti-mouse 488 (1:500; cat# A21202, Life Technologies, San Diego, CA, USA) were used to detect combinations that included PRR and ATIIR2, and Vectaﬂuor Excel anti-mouse (ready-to- use; cat# VEDK2488, Vector Laboratories) and Alexa Fluor anti- rabbit 594 (1:500; cat# A21207, Life Technologies) to detect combinations that included ACE and ATIIR1. IF IHC-stained slides were mounted in Vectashield HardSet antifade mounting medium with DAPI (cat# H-1500, Vector Laboratories). The human tissue samples used for positive controls were placenta for PRR (30), kidney for ACE (31), and ATIIR2 (32), and liver for ATIIR1 (32), as previously reported. Negative controls were performed by staining PG tissue samples by omitting the primary antibodies, to determine their speciﬁcity. Immunoﬂuorescence Immunohistochemical Staining IF IHC-stained slides with subsequent 2D deconvolution using CellSens Dimension 1.11 software (Olympus). IF IHC-stained slides with subsequent 2D deconvolution using CellSens Dimension 1.11 software (Olympus). g We have previously demonstrated the presence of two putative ESC-like subpopulations within PG with one subpopulation localized to the endothelium that expressed NANOG (Figure 2A, red) (7). Interestingly only ACE (Figure 2B, green) was expressed by the SOX2+ (Figure 2B, red) endothelium of the microvessels but not the NANOG+ (Figure 2A, red) cells within the perivascular tissue. The ACE+ (Figure 2C, green) endothelium of the microvessels and the cells within the perivascular tissue of PG expressed PRR (Figure 2C, red). The ERG+ (Figure 2D, red) endothelium of the microvessels expressed ATIIR1 (Figure 2D, green). ATIIR2 (Figure 2E, red) was expressed on both the CD34+ (Figure 2E, green) endothelium of the microvessels and the cells within the perivascular tissue. An insert within each image provides a magniﬁed area to better demonstrate the staining pattern. Split images of IF IHC staining presented in Figure 2 are shown in Supplementary Figure 2. The negative controls demonstrated minimal staining (Supplementary Figure 2K). Histology PG was comprised of microvessels organized into lobules embedded in ﬁbromyxoid stroma (Supplementary Figure 1A). Image Capture and Analysis All DAB IHC-stained slides were viewed and photographed using the Olympus BX53 bright ﬁeld microscope with an Olympus DP21 digital camera (Olympus, Tokyo, Japan). The Olympus FV1200 confocal microscope (Tokyo, Japan) was used for the April 2019 \| Volume 6 \| Article 13 Frontiers in Surgery \| www.frontiersin.org 3 Renin-Angiotensin System in Pyogenic Granuloma Papali’i-Curtin et al. Western Blotting g WB conﬁrmed the presence of PRR at the molecular weight of 42 kDa in the PG tissue sample and the two PG-derived primary cell lines at 70 kDa (Figure 5A, red) representing dimerization of PRR (30). ACE was not detected at the expected molecular weight of 194 kDa (22) in both the tissue sample and the primary cell lines (Figure 5B, red). ATIIR1 was detected in both the tissue samples and primary cell lines (Figure 5C, red) with bands at the expected molecular weight of 42 kDa(28). ATIIR2 was below detectable levels in both the tissue and the cell lines (Figure 5D). Bands for β-actin (Supplementary Figure 3A, red) conﬁrmed approximate equivalent protein loading for all tissue samples examined. The respective positive controls were used conﬁrmed speciﬁcity of the antibody for their target proteins. The rabbit and mouse IgG isotype controls (Supplementary Figure 3B) were used to detect any non-speciﬁc staining and therefore conﬁrmed the presence of the components of the RAS. FIGURE 1 \| Representative 3,3-diaminobenzidine immunohistochemical- stained sections of pyogenic granuloma demonstrating the expression of PRR (A, brown) on the endothelium of the microvessels and cells within the perivascular tissue. ACE (B, brown) was expressed on the endothelium of the microvessels. ATIIR1 (C, brown) was expressed on the endothelium of the microvessels and cells within the perivascular tissue. Weak staining of ATIIR2 (D, brown) on the endothelium of the microvessels and cells within the perivascular tissue was observed. Nuclei were counterstained with hematoxylin (blue). Original magniﬁcation 200x. FIGURE 1 \| Representative 3,3-diaminobenzidine immunohistochemical- stained sections of pyogenic granuloma demonstrating the expression of PRR (A, brown) on the endothelium of the microvessels and cells within the perivascular tissue. ACE (B, brown) was expressed on the endothelium of the microvessels. ATIIR1 (C, brown) was expressed on the endothelium of the microvessels and cells within the perivascular tissue. Weak staining of ATIIR2 (D, brown) on the endothelium of the microvessels and cells within the perivascular tissue was observed. Nuclei were counterstained with hematoxylin (blue). Original magniﬁcation 200x. RT-qPCR RT-qPCR performed on the two PG-derived primary cell lines conﬁrmed abundant mRNA expression of PRR, ACE and ATIIR1, while ATIIR2 mRNA was below the detectable levels (Figure 4), relative to the housekeeping gene GAPDH, and in comparison, with uterine ﬁbroid tissues as a positive control. RT-qPCR performed on the two PG-derived primary cell lines conﬁrmed abundant mRNA expression of PRR, ACE and ATIIR1, while ATIIR2 mRNA was below the detectable levels (Figure 4), relative to the housekeeping gene GAPDH, and in comparison, with uterine ﬁbroid tissues as a positive control. FIGURE 1 \| Representative 3,3-diaminobenzidine immunohistochemical- stained sections of pyogenic granuloma demonstrating the expression of PRR (A, brown) on the endothelium of the microvessels and cells within the perivascular tissue. ACE (B, brown) was expressed on the endothelium of the microvessels. ATIIR1 (C, brown) was expressed on the endothelium of the microvessels and cells within the perivascular tissue. Weak staining of ATIIR2 (D, brown) on the endothelium of the microvessels and cells within the perivascular tissue was observed. Nuclei were counterstained with hematoxylin (blue). Original magniﬁcation 200x. Statistical Analysis Statistical analysis to determine signiﬁcant diﬀerences between the NanoString mRNA results were analyzed using paired t-tests of the SPSS (v22, IBM). NanoString mRNA Analysis g y NanoString mRNA analysis conﬁrmed transcriptional activation of PRR, ACE, and ATIIR1 in all six PG tissue samples examined but ATIIR2 was detected in only one sample, relative to the housekeeping gene GUSB (Figure 3). Statistical analysis revealed signiﬁcantly higher amounts of PRR compared to ATIIR1 and ACE (p < 0.05). Expected staining patterns for PRR (Supplementary Figure 1B, brown), ACE (Supplementary Figure 1C, brown), ATIIR1 (Supplementary Figure 1D, brown), and ATIIR2 (Supplementary Figure 1E, brown) were demonstrated on human placenta, kidney, liver, and kidney, respectively. Staining with the isotype controls in a PG sample provided an appropriate negative control (Supplementary Figure 1F). DAB Immunohistochemical Staining DAB IHC staining demonstrated the expression of PRR (Figure 1A, brown) on the endothelium of the microvessels and cells within the perivascular tissues, while ACE (Figure 1B, brown) was expressed on the endothelium of the microvessels, in PG. ATIIR1 (Figure 1C, brown) staining was strongest on the endothelium of the microvessels and cells within the perivascular tissue. ATIIR2 (Figure 1D, brown) was also expressed on the endothelium of the microvessels in PG. DISCUSSION The presence of ACE on the endothelium of the microvessels in the PG samples suggests paracrine conversion of ATI to active ATII and the downstream eﬀects of ATII may play a role in cellular proliferation and/or angiogenesis of the PG (23, 24, 35). FIGURE 3 \| NanoString mRNA analysis conﬁrmed transcriptional activation of PRR, ACE, and ATIIR1 in all six pyogenic granuloma tissue samples examined while ATIIR2 was detected in only one sample, relative to the housekeeping gene GUSB. There were signiﬁcantly higher amounts of PRR compared to ATIIR1 and ACE (p < 0.05). FIGURE 3 \| NanoString mRNA analysis conﬁrmed transcriptional activation of PRR, ACE, and ATIIR1 in all six pyogenic granuloma tissue samples examined while ATIIR2 was detected in only one sample, relative to the housekeeping gene GUSB. There were signiﬁcantly higher amounts of PRR compared to ATIIR1 and ACE (p < 0.05). FIGURE 3 \| NanoString mRNA analysis conﬁrmed transcriptional activation of PRR, ACE, and ATIIR1 in all six pyogenic granuloma tissue samples examined while ATIIR2 was detected in only one sample, relative to the housekeeping gene GUSB. There were signiﬁcantly higher amounts of PRR compared to ATIIR1 and ACE (p < 0.05). PRR binds pro-renin which then undergoes conformational change (24) creating a four-fold increase in the catalytic conversion of AGN to ATI (30). The presence of PRR on the endothelium of the microvessels in PG suggests a local eﬀect downstream of ATI. The localization of ATIIR1 to the endothelium of the microvessels of PG suggests that ATIIR1 may contribute to the formation of immature microvessels in PG, possibly through preferential diﬀerentiation down an endothelial phenotype (36). FIGURE 4 \| Graph of RT-qPCR performed on two pyogenic granuloma-derived primary cell lines, demonstrating average mRNA expression of PRR, ACE and ATIIR1, relative to housekeeping gene GAPDH, and in comparison to ﬁbroid tissues as a positive control. ATIIR2 mRNA was below the detectable levels. FIGURE 4 \| Graph of RT-qPCR performed on two pyogenic granuloma-derived primary cell lines, demonstrating average mRNA expression of PRR, ACE and ATIIR1, relative to housekeeping gene GAPDH, and in comparison to ﬁbroid tissues as a positive control. ATIIR2 mRNA was below the detectable levels. ATIIR2 was detected by IHC staining in our study with a weak and diﬀuse staining pattern, but was not detected by NanoString, RT-qPCR, and WB analyses. DISCUSSION This study demonstrated the presence of PRR, ACE, ATIIR1, and ATIIR2 within PG. Interestingly the expression of ATIIR2 April 2019 \| Volume 6 \| Article 13 Frontiers in Surgery \| www.frontiersin.org Frontiers in Surgery \| www.frontiersin.org 4 Papali’i-Curtin et al. Renin-Angiotensin System in Pyogenic Granuloma FIGURE 2 \| Representative immunoﬂuorescence immunohistochemical-stained sections of pyogenic granuloma demonstrating the expression of ACE (A,B, green) on the SOX2+ (B, red) endothelium of the microvessels but not the NANOG+ (A, red) cells within the perivascular tissue. The ACE+ (C, green) endothelium of the microvessels and the perivascular cells within PG expressed PRR (C, red). The ERG+ (D, red) endothelium of the microvessels expressed ATIIR1 (D, green). PRR (E, red) was expressed on both the CD34+ (E, green) endothelium as well as the non-endothelial cells. Nuclei were counterstained with 4 ′,6 ′-diamino-2-phenylindole (A–D, blue). Scale bars: 20 µm. Inserts demonstrate magniﬁed areas of the image within each ﬁgure (magniﬁcation 400X). FIGURE 2 \| Representative immunoﬂuorescence immunohistochemical-stained sections of pyogenic granuloma demonstrating the expression of ACE (A,B, green) on the SOX2+ (B, red) endothelium of the microvessels but not the NANOG+ (A, red) cells within the perivascular tissue. The ACE+ (C, green) endothelium of the microvessels and the perivascular cells within PG expressed PRR (C, red). The ERG+ (D, red) endothelium of the microvessels expressed ATIIR1 (D, green). PRR (E, red) was expressed on both the CD34+ (E, green) endothelium as well as the non-endothelial cells. Nuclei were counterstained with 4 ′,6 ′-diamino-2-phenylindole (A–D, blue). Scale bars: 20 µm. Inserts demonstrate magniﬁed areas of the image within each ﬁgure (magniﬁcation 400X). April 2019 \| Volume 6 \| Article 13 Frontiers in Surgery \| www.frontiersin.org 5 Frontiers in Surgery \| www.frontiersin.org Frontiers in Surgery \| www.frontiersin.org Renin-Angiotensin System in Pyogenic Granuloma Papali’i-Curtin et al. with, the endocrine RAS (20). Our study supports the presence of a local RAS within PG. Angiotensinogen (AGN) is a protein physiologically secreted by the liver into the general circulation. Pro-renin is physiologically converted to renin by the juxtaglomerular cells of the kidney; renin then cleaves AGN to form the decapeptide ATI. ATI is primarily converted to ATII by ACE by removing the histidyl-leucine dipeptide from the carboxyl end of AT1 to create an octapeptide (33, 34). DISCUSSION Interestingly ATIIR2 contributes to angiogenesis of the endothelial stem cells via upregulation of VEGF and increasing expression of VEGF2 receptors (35). This is in contrast to our ﬁnding in IH that ATIIR2 agonist facilitated cellular proliferation (17). This may be because ATIIR2 has been primarily located in fetal tissue (37) and that IH is a tumor of infancy, possibly at a time when this receptor is more abundant (37). In light of reports of the presence of hemangioblast stem cell population on primitive-phenotypic vessels (36), it is exciting to speculate that PG consist of hemangioblast-like cells with a predominant endothelial diﬀerentiation pathway. This may be predominantly through ATIIR1 signaling, however, this requires further investigation. FIGURE 4 \| Graph of RT-qPCR performed on two pyogenic granuloma-derived primary cell lines, demonstrating average mRNA expression of PRR, ACE and ATIIR1, relative to housekeeping gene GAPDH, and in comparison to ﬁbroid tissues as a positive control. ATIIR2 mRNA was below the detectable levels. Our study adds to the growing body of evidence of the involvement of the RAS in vascular anomalies including VM, IH and PG. To the best of our knowledge, this is the ﬁrst study demonstrating the presence and localization of PRR, ACE, and ATIIR1 to the putative ESC-like population within PG. This novel ﬁnding oﬀers insights into the biology of PG with the potential of using RAS modulators such as β-blockers and ACE-inhibitors, for this common and often troublesome tumor. was only demonstrated by IHC staining, and this may be potentially due to sampling bias in NanoString and WB analyses. IF IHC staining demonstrated the expression of all four components of the RAS on the endothelium of the microvessels that expressed ESC markers SOX2 and NANOG (7). The putative ESC-like subpopulation within the perivascular tissue that expresses NANOG (7), did not express ACE. Cells within the stroma expressed both PRR and ATIIR2. However, due to the limitation by the antibody sources we were not able to determine whether these were the same cells that express NANOG. Limitations of this study include a relatively small sample size, the lack of normal control tissues such as skin for the experiments and functional data on the role of the RAS in PG. Frontiers in Surgery \| www.frontiersin.org DISCUSSION Further studies including a larger sample size, inclusion of control tissues such as normal skin, and in vitro experiments with sorting of the putative ESC-like cells from PG lesions are needed to determine their expression of the aforementioned components of the RAS, and their response to administration of the RAS peptides. The RAS is classically known as an endocrine system, however, the presence of a paracrine and an intracrine element has been demonstrated (19, 24). Components of the RAS have also been localized to multiple organs, suggesting a local paracrine RAS functioning independently, or in conjunction April 2019 \| Volume 6 \| Article 13 Frontiers in Surgery \| www.frontiersin.org 6 Renin-Angiotensin System in Pyogenic Granuloma Papali’i-Curtin et al. FIGURE 5 \| Western blot images of total protein extracted from a snap-frozen pyogenic granuloma tissue sample and two pyogenic granuloma-derived primary cell lines, and the positive control demonstrating the presence of PRR (A), the absence of ACE (B), and the presence of both ATIIR1 (C), and ATIIR2 (D). Each blot ladder is annotated with the molecular size (kDa). FIGURE 5 \| Western blot images of total protein extracted from a snap-frozen pyogenic granuloma tissue sample and two pyogenic granuloma-derived primary cell lines, and the positive control demonstrating the presence of PRR (A), the absence of ACE (B), and the presence of both ATIIR1 (C), and ATIIR2 (D). Each blot ladder is annotated with the molecular size (kDa). SUPPLEMENTARY MATERIAL TI and ST formulated the study hypothesis and designed the study. JP-C, HB, TI, and ST interpreted the IHC data. TI and ST interpreted the NanoString data. JdJ performed the RT-qPCR experiments and JdJ, JP-C, TI, and ST interpreted the data. BvS performed the WB experiments. BvS, TI, and ST interpreted the data. RM performed the statistical analysis. JP-C, TI, and ST drafted the manuscript. All authors approved the manuscript. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fsurg. 2019.00013/full#supplementary-material Supplementary Figure 1 \| A hematoxylin and eosin section of pyogenic granuloma (PG) demonstrating microvessels organized into lobules embedded in a ﬁbromyxoid stroma (A). DAB IHC staining of positive controls for PRR (B, brown) on human placenta, ACE (C, brown) on human kidney, ATIIR1 (D, brown) on human liver, and ATIIR2 (E, brown) on human kidney. The negative control on a PG sample using an IgG isotype (F) demonstrated no staining. Nuclei were counter-stained with hematoxylin (A–F, blue]. Original magniﬁcation: 200x. Supplementary Figure 2 \| Split images of immunoﬂuorescence immunohistochemical-stained sections of pyogenic granuloma (PG) shown in Figure 2 demonstrating expression of SOX2 (A, red) and ACE (B, green), NANOG (C, red) and ACE (D, green), PRR (E red) and ACE (F, green), ERG (G, red) and ATIIR1 (H, green), ATIIR2 (I, red) and CD34 (J, green). A negative control (K) to test the speciﬁcity of the ﬂuorescent secondary antibodies was performed on a section of PG. Cell nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (A–K, blue). Scale bars: 20 µm. 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(2015) 68:346–50. doi: 10.1136/jclinpath-2014-202794 The remaining authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 18. Tan ST, Itinteang T, Day DJ, O’Donnell C, Mathy JA, Leadbitter P. Treatment of infantile haemangioma with captopril. Br J Dermatol. (2012) 167:619–24. doi: 10.1111/j.1365-2133.2012.11016.x 19. Ager EI, Neo J, Christophi C. The renin-angiotensin system and malignancy. Carcinogenesis. (2008) 29:1675–84. doi: 10.1093/carcin/bgn171 Copyright © 2019 Papali’i-Curtin, Brasch, van Schaijik, de Jongh, Marsh, Tan and Itinteang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 20. George AJ, Thomas WG, Hannan RD. The renin-angiotensin system and cancer: old dog, new tricks. Nat Rev Cancer. (2010) 10:745–59. doi: 10.1038/nrc2945 21. Deshayes F, Nahmias C. Angiotensin receptors: a new role in cancer? Trends Endocrinol Metab. (2005) 16:293–9. doi: 10.1016/j.tem.2005.07.009 April 2019 \| Volume 6 \| Article 13 Frontiers in Surgery \| www.frontiersin.org 8
https://openalex.org/W2895245566	https://europepmc.org/articles/pmc6184517?pdf=render	English	null	In situ cardiac regeneration by using neuropeptide substance P and IGF-1C peptide eluting heart patches	Regenerative Biomaterials	2,018	cc-by	9,633	Muhammad Shafiq1,2,3,4,†, Yue Zhang5,†, Dashuai Zhu2, Zongxian Zhao5, Dong-Hwee Kim6, Soo Hyun Kim1,3,6,* and Deling Kong2,* 1Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology, Seoul 02792, Republic of Korea; 2State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials of Ministry of Education, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), College of Life Science, Nankai University, Tianjin 300071, China; 3Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology, Cheongryang, Seoul 130-650, Republic of Korea; 4Department of Chemistry, Center for Tissue Engineering & Regenerative Medicine, Pakistan Institute of Engineering & Applied Sciences (PIEAS), Nilore, Islamabad 45650, Pakistan; 5Department of Physiology & Pathophysiology, Tianjin Medical University, Tianjin 300070, China; 6KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul 02841, Republic of Korea Correspondence address. State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials of Ministry of Education, Department of Biochemistry and Molecular Biology, College of Life Science, Nankai University, Tianjin 300071, China. Tel: þ86-22-8789-3696; Fax: þ86-22-8789-3696; E-mail: kongdeling@nankai.edu.cn (D.K.); Department of Biomedical Engineering, Korea University of Science and Technology, 176 Gajeong-dong, Yuseong-gu, Daejeon, Republic of Korea. Tel: þ82-2-958-5343; Fax: þ82-2-958-5308; E-mail: soohkim@kist.re.kr (S.H.K.) †These authors contributed equally to this work. Received 20 May 2018; revised 26 June 2018; accepted on 3 July 2018 Received 20 May 2018; revised 26 June 2018; accepted on 3 July 2018 Regenerative Biomaterials, 2018, 303–316 doi: 10.1093/rb/rby021 Research Article Regenerative Biomaterials, 2018, 303–316 doi: 10.1093/rb/rby021 Research Article Research Article Keywords: cardiac patch; stem cell recruitment; myocardial infarction; electrospinning y y This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. In situ cardiac regeneration by using neuropeptide substance P and IGF-1C peptide eluting heart patches uhammad Shafiq1,2,3,4,†, Yue Zhang5,†, Dashuai Zhu2, Zongxian Zhao5, ng-Hwee Kim6, Soo Hyun Kim1,3,6, and Deling Kong2,* Introduction We utilized an in situ tissue regeneration approach in which we simulta- neously mobilized endogenous stem cells to the site of the injury and provided a cell-supportive microenvironment. SP was incorporated into polycaprolactone (PCL)/collagen type 1 (Col)-based cardiac patches to promote the mobilization and recruitment of endogenous mesenchymal stem cells (MSCs) to the defective site in an acute MI model. To provide the mobilized MSCs with an environment suit- able for survival and/or differentiation, we immobilized IGF-1C peptide into the developed cardiac patches. SP may recruit CD29- positive MSCs, which may either secrete paracrine factor (i.e. VEGF, FGF etc.) and participate in the cardiac tissue repair or dif- ferentiate into specialized somatic cell types, such as endothelial cells (ECs) and smooth muscle cells (SMCs). Meanwhile, IGF-1C peptide may support the recruited stem cells or cardiomyocytes and enhance their survival and retention. Our patches consist of PCL, a biode- gradable polymer with wide applications in TE and collagen type 1, a component of extracellular matrix (ECM), which could also pro- vide a conducive environment for cell survival. We used co- electrospinning from two separate spinnerets to fabricate cardiac patches. Electrospinning can afford a three-dimensional architec- ture, which may recapitulate the native tissue’s ECM and provide physical signals for tissue repair [29, 30]. Herein, we revealed that the use of patches containing SP and IGF-1C peptide promote heart regeneration in vivo. Accumulative evidences support the notion that bioactive mole- cules, such as stromal cell-derived factor-1 alpha (SDF-1a), vascular endothelial growth factor (VEGF), stem cell factor, fibroblasts growth factor (FGF), angiopoietin-like protein 1 (Ang-1) and granu- locyte colony-stimulating factor (G-CSF) can recruit endogenous stem/progenitor cells and facilitate tissue repair in situ [9–13]. However, most of the above-mentioned bioactive molecules are large molecular weight proteins, which cannot be easily synthesized or incorporated into scaffold materials. Moreover, due to the ab- sence of spatiotemporal cues as well as the short half-life of many proteins, the effectiveness of protein therapeutics may be compro- mised. Accordingly, scaffold materials providing spatiotemporal re- lease of a combination of bioactive factors hold great promise for in situ tissue regeneration [14]. Short peptide sequences, bioactive lip- ids and therapeutic molecules are being investigated as a replace- ment or an adjuvant therapy with growth factors or stem cells, which may serve as more desirable therapeutic agents due to an eco- nomical cost, ease in processing, and better delivery. Introduction On the other hand, most of the transplanted cells are lost due to their poor retention and engraftment at the infarct site, which is an outcome of the hostile injury microenvironment. This may be over- come by designing cell-affinitive biomaterials or preconditioning stem/progenitor cells before transplantation. Insulin-like growth fac- tor 1 (IGF-1) is a mitogenic and a pro-survival protein, which con- tains a C domain peptide (IGF-1C), (GYGSSSRRAPQT) as an active region [24]. IGF-1C peptide has been reported to promote the heal- ing of corneal epithelial wounds [25, 26]. Previously, we developed IGF-1C peptide-conjugated chitosan hydrogels, which favored the survival and therapeutic benefits of transplanted adipose-derived stem cells [27]. Similarly, Davis et al. designed self-assembling pep- tide hydrogels containing IGF-1, which supported the growth and survival of transplanted cardiomyocytes and reduced the cell apo- ptosis [28]. Cardiovascular diseases account for the major cause of the death worldwide, which is responsible for >17.3 million deaths annually [1]. Myocardial infarction (MI) may lead to the excessive loss of car- diomyocytes, inflammation, ventricular dilation and adverse cardiac remodeling, resulting in the development of congestive heart failure. Presently, there is no complete cure for MI and current treatment methodologies possess limited ability to regenerate the damaged car- diac muscles. Consequently, the objective of the current strategies is to find solutions for the attenuation of the progressive cardiac fail- ure and to regenerate the infarcted myocardium, which has been ac- complished by either replacing the lost cardiomyocytes with transplanted stem/progenitor cells or by harnessing the paracrine effects of stem cells [2–5]. While stem/progenitor cells have enor- mous potential for cardiac tissue repair and may offer distinct advantages to develop the engineered heart tissues, difficulties asso- ciated with the shortage of appropriate donors, isolation and expan- sion of sufficient numbers of transplantable cells, and extensive in vitro cell manipulations limit the therapeutic potential of the cell therapy [6, 7]. Moreover, cardiac cell therapy is hampered by the poor engraftment and significant cell death after transplantation [8]. Consequently, it is imperative to find new strategies to specifically activate the endogenous tissue repair process for MI. The objective of this research was to develop cardiac patches and leverage these patches with the stem cell mobilization and recruit- ment potential as well as provide a supportive environment for the survival and engraftment of the recruited stem/progenitor cells. Abstract Cardiovascular diseases cause huge socio-economic burden worldwide. Although a mammalian myocardium has its own limited healing capability, scaffold materials capable of releasing stem cell recruiting/engrafting factors may facilitate the regeneration of the infarcted myocardium. The aim of this research was to develop cardiac patches capable of simultaneously eluting substance P (SP) and insulin-like growth factor-1C (IGF-1C) peptide. Polycaprolactone/collagen type 1-based patches with or without SP and IGF-1C peptide were fabricated by co-electrospinning, which exhib- ited nanoﬁbrous morphology. SP and IGF-1C/SP patches recruited signiﬁcantly higher numbers of bone marrow-mesenchymal stem cells than that of the negative control and patch-only groups in vitro. The developed patches were transplanted in an infarcted myocardium for up to 14 days. Mice underwent left anterior descending artery ligation and received one of the following treat- ments: (i) sham, (ii) saline, (iii) patch-only, (iv) IGF-1C patch, (v) SP patch and (vi) IGF-1C/SP patch. SP and IGF-1C/SP patch-treated groups exhibited better heart function and attenuated adverse car- diac remodeling than that of the saline, patch-only and individual peptide containing cardiac patches. SP patch and IGF-1C/SP patch-treated groups also showed higher numbers of CD31- positive vessels and isolectin B4-positive capillaries than that of other groups. IGF-1C/SP-treated group also showed thicker left ventricular wall in comparison to the saline and patch-only groups. Moreover, IGF-1C/SP patches recruited signiﬁcantly higher numbers of CD29-positive cells and showed less numbers of Tunel-positive cells compared with the other groups. These data suggest that SP and IGF-1C peptides may act synergistically for in situ tissue repair. Keywords: cardiac patch; stem cell recruitment; myocardial infarction; electrospinning 303 304 Shafiq et al. Introduction Substance P (SP) is an undecapeptide that belongs to the tachyki- nin neuropeptide family and is released from the terminals of specific sensory nerves. It has been shown to recruit endogenous stem/progeni- tor cells toward injury site for tissue regeneration [15–20]. SP has also potentials to induce neovascularization and modulate the inflamma- tory response [17–20]. Moreover, in comparison to the other stem cell inducing/recruiting bioactive factors, such as SDF-1, G-CSF and VEGF, SP exhibits low molecular weight, which can be easily synthe- sized and incorporated into scaffold materials. Despite the ongoing re- search activities centered on SP, several central needs remain unmet. For example, SP can be easily degraded by the endogenous peptidases and exhibits very short half-life in vitro and in vivo, which may limit its therapeutic utilization [21, 22]. Therefore, strategies focused on en- hancing the residence or presence of SP in vivo may be very beneficial for tissue engineering (TE) applications [23]. To cope with these limi- tations, SP-conjugated scaffold materials have been developed, which showed therapeutic potential in the settings of various injury microen- vironments, including osteochondral defects, limb ischemia, and skin wounds rendering the use of SP of enormous potential for TE applica- tions [16, 18, 20–22]. Materials PCL pellets (number average molecular weight, Mn ¼80 000 Da) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) were purchased from Sigma Aldrich (St Louis, USA). Chloroform (CHCl3) and methanol (CH3OH) were purchased from Tianjin Chemical Reagent Company (Tianjin, China). Triton X-100 was bought from Alfa Aesar (London, England). SP and IGF-1C peptides (purity, 95%) were obtained from Peptron (Daejeon, Korea). Lyophilized collagen type 1 was a gift from the Saining Biotechnology Company (Tianjin, China). Echocardiography l l f To evaluate left ventricular (LV) geometry and function 14 days af- ter the surgery, echocardiography (ECG) was carried out as de- scribed in detail in the Supplementary data. Fabrication of cardiac patches Thereafter, the chest wall and skin were closed. Sham operations were performed on another five mice without LAD. Cell proliferation assay Proliferation of cells on the patches was examined by using cell counting kit (CCK-8). The detailed procedure is described in the Supplementary data. Immunohistochemistry To carry out immunofluorescence staining, frozen transverse tissue sections (5 lm) of hearts (n ¼ 5 for each group) were fixed in cold acetone, dried in a fume hood and washed with 0.01 mM PBS. Sections were blocked by using 5% normal goat serum (Zhongshan Golden Bridge Biotechnology, China) for 45 min at 4C. A 0.1% Triton-PBS was used for permeation for intracellular antigen stain- ing, before blocking. Then the rat anti-mouse CD31 antibody (BD Pharminogen, 1:100) was added on the sections for overnight at 4C. Afterwards, sections were washed with PBS (eight times, 5 min) and incubated with Alexa Flour 488 goat anti-rat IgG (1:200, Invitrogen) or Alexa-Fluor 488 goat anti-mouse antibodies (Invitrogen, USA) for 2 h at ambient temperature. The sections with- out incubation with primary antibodies were used as negative con- trols. The nuclei were counter-stained with 4,6-diamidino-2- phenylindole (DAPI) containing mounting solution (DAPI Fluoromount G, Southern Biotech, England). In vitro transwell migration assay The migratory response of rabbit bone marrow-MSCs (BM-MSCs) toward patches was analyzed using a Transwell migration assay fol- lowing a previous method [31] (Supplementary data for the detailed method). Cell viability assay In vitro cell viability on patches was examined by performing per- forming [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro- mide] (MTT) assay. The method is described in the Supplementary data. Evaluation of the release of SP. Release studies of SP peptide from electrospun membranes were carried out by using high performance liquid chromatography (HPLC, Agilent Technologies, 1200 Series, USA) with YMC-Pack Pro C18 Column (ID, 250 4.6 mm, S-5 mm, 12 nm) following our previous published method [30]. For immunohistological detection of capillaries, sections were in- cubated with 0.5 mM calcium chloride (CaCl2) for 5 min, which was followed by the incubation with isolectin B4 antibody (1:70 di- lution in 0.5 mM CaCl2) at 4C overnight and RT for 1 h. Sections were washed with 0.5 mM CaCl2 solution (thrice, 5 min) and incu- bated with the Sudan black solution for 7 min at ambient tempera- ture. After washing with PBS, the nuclei were counter-stained with DAPI. Slides were observed under a fluorescence microscope (Zeiss Axio Imager Z1, Germany). The numbers of CD31-positive blood vessels and isolectin-B4 positive capillaries were counted in 10 ran- domly chosen high-power fields (HPFs, 400) of the border zone. Results were expressed as cells per HPFs. For CD29 staining, sec- tions were incubated with integrin B1 antibody (abcam, 1:1000) overnight and stained with Alexa Flour 488 goat anti-rabbit IgG (1:200, Invitrogen) antibody for 2 h at room temperature. The nuclei were counter-stained with DAPI. In situ BrdU-Red DNA Morphological analysis Morphological analysis was carried out by using a scanning electron microscope (SEM, HITACHI, X-650, Japan, voltage 15 kV) and membranes were sputter-coated with gold and palladium. Fabrication of cardiac patches The hybrid PCL/Col cardiac patches were prepared by co- electrospinning from two separate spinnerets. PCL solution (10% In situ cardiac regeneration by using neuropeptide SP and IGF-1C peptide 305 w/v) was prepared by using CHCl3 and CH3OH (5:1 v/v) and stirred overnight. Collagen type 1 solution (8% w/v) was prepared in HFIP at ambient temperature for 24 h. SP or IGF-1C peptides were dis- solved in deionized water at ambient temperature for 24 h to obtain 2 mg/ml solution. Collagen and SP or IGF-1C peptide solutions were mixed (4:1 v/v) and stirred for another 12 h. Two 10-ml syringes were filled with PCL or collagen solution with or without peptides and connected to a 21-G blunt-ended needle. The apparatus consists of a syringe pump (Cole Parmer, Vernon Hills, IL, USA), a high- voltage generator (DWP503-1AC, Dong-Wen High Voltage power supply factory, Tianjin, China), and a rotating aluminum mandrel as a collector. The aluminum foil was wrapped around the mandrel to collect the fibers. The flow rate of PCL and collagen solution was adjusted at 2 and 0.6 ml/h, respectively. The voltage between the needle tip and the rotating mandrel was 15 and 12 kV for PCL and Col, respectively. The distance between the spinneret and the collec- tor was set at 15 cm for PCL and collagen solutions. Membranes were vacuum-dried for 48 h before further use. (n ¼ 5 per group) were randomly assigned to six groups as follows: sham-operation, MI-saline, MI-patch only, MI-IGF-1C patch, MI- SP patch and MI-IGF-1C/SP patch. An acute MI model was induced by the permanent ligation of the left anterior descending (LAD) cor- onary artery as previously described [32]. See the Supplementary data for the detailed procedure of ligation. After ligation, either sa- line was injected or the patches were implanted on the epicardium by a 9-0 suture. Thereafter, the chest wall and skin were closed. Sham operations were performed on another five mice without LAD. (n ¼ 5 per group) were randomly assigned to six groups as follows: sham-operation, MI-saline, MI-patch only, MI-IGF-1C patch, MI- SP patch and MI-IGF-1C/SP patch. An acute MI model was induced by the permanent ligation of the left anterior descending (LAD) cor- onary artery as previously described [32]. See the Supplementary data for the detailed procedure of ligation. After ligation, either sa- line was injected or the patches were implanted on the epicardium by a 9-0 suture. Histological analysis After 14 days, mice were sacrificed and hearts were removed, washed with phosphate buffered saline (PBS), and snap frozen in liq- uid nitrogen. Frozen sections were embedded in optimal cutting tem- perature medium. From each heart, the sections (thickness, 5 lm) of four levels (15 mm thick) were stained with hematoxylin and eosin (H&E) and Masson’s trichrome (MT) staining. Collagen positive areas (infarct size) and the infarct wall thickness of LV were ana- lyzed using the computerized planimetry (Axio Vision LE Rel. 4.5 software; Zeiss, Jena, Germany). Transplantation of cardiac patches BALB/c mice (female, 20 6 2 g) were purchased from the Laboratory Animal Centre of the Academy of Military Medical Sciences (Beijing, China), and all animal experiments were carried out using the guidelines set by the Tianjin Committee of Use and Care of Laboratory Animals and the overall project protocols were ap- proved by the Animal Ethics Committee of the Nankai University. During the surgery, the body temperature of the mouse was main- tained at 37.5 6 0.5C using a heating table. The animals’ condi- tions such as activity, behavior, lethargy, lack of appetite, and hair texture were assessed each day. The mice were euthanized, when they exhibited significant impairments of their conditions. Mice 306 Shafiq et al. and optical density values were found to be 0.42056 0.0035, 0.3656 0.012, 0.43256 0.0525, 0.45256 0.0005 and 0.446 0.0005 for tissue culture plate, patch-only, IGF-1C patch, SP patch and IGF- 1C/SP patch groups, respectively. The tissue culture plate, patch-only, IGF-1C patches, SP patch and IGF-1C/SP patch did not significantly differ in terms of the cell proliferation. Fragmentation (TUNEL) Assay Kit (ab66110) was used to evaluate cell apoptosis following the manufacturer’s instructions. Cardiac troponin-T (cTnT; Abcam, Cambridge, Massachusetts) and DAPI were used to detect cardiac muscle and cell nuclei and slides were observed under a fluorescence microscope (Zeiss Axio Imager Z1, Germany). Results The in vivo biocompatibility of patches was evaluated in the settings of an acute MI model in mice for up to 2 weeks. At Day 14, ECG was measured to assess the cardiac function and hearts were excised for investigations at the tissue level using histology and immunohis- tochemistry. The survival rate of the mice was found to be 100% in all groups following MI. Figure 3 shows the schematic diagram of the patch and its implantation in mice. H&E staining revealed that the patches remained adhered to the myocardium and were popu- lated by the host cells (Fig. 3B). The LV wall thickness and fibrosis were evaluated using H&E staining and MT staining, respectively. The representative images of H&E and MT stained tissue sections are shown in Fig. 4. Saline, patch-only, SP patch and IGF-1C patch- treated groups did not significantly differ in terms of the LV wall thickness (saline, 0.33 6 0.11; patch-only, 0.308 6 0.028 mm; SP patch, 0.408 6 0.038 mm; and IGF-1C patch, 0.358 6 0.028 mm) (Fig. 4B). Interestingly, IGF-1C/SP patches showed significantly higher LV wall thickness than that of the saline, patch-only, IGF-1C patch and SP patch-treated groups (0.554 6 0.0445 mm) (Fig. 4B). The infarct size was found to be 2.62 6 4.20, 38.46 6 1.67, 39.0 6 2.34, 35.94 6 2.72 and 37.34 6 2.46% in saline, patch-only, IGF-1C patch, SP patch and IGF-1C/SP patch-treated groups, re- spectively (Fig. 4C and D). SP patch-treated group showed signifi- cantly less infarct size in comparison to the saline-treated group (saline vs SP patch, P ¼ 0.0227). In vitro transwell migration assay Since SP has been shown to act as a mitogen, which enhances stem cell mobilization, an in vitro cell migration assay was performed to evaluate the role of cardiac patches with or without SP and IGF-1C peptides in terms of the cell migration. Wells containing medium only served as negative controls. The representative images are shown in Fig. 2. Only few numbers of BM-MSCs were migrated to- ward the negative control, patch-only and IGF-1C patch groups (Fig. 2). On the other hand, many cells were migrated toward SP and IGF-1C/SP patches (Fig. 2). The density of recruited BM-MSCs was evaluated by using Image J and found to be 50366.9 6 11604.0, 77011.47 6 3842.1, 64917.01 6 14211.5, 158,706 6 31711.4 and 276112.7 6 34117.4 mm2/mm2 in negative control, patch-only, IGF- 1C patch, SP patch and IGF-1C/SP patch, respectively (Fig. 2A). IGF-1C/SP patch group recruited significantly higher numbers of BM-MSCs in comparison to other groups. Characterization of cardiac patches We fabricated cardiac patches consisting of a biodegradable poly- mer ‘PCL’ and an ECM component ‘collagen type 1’ and incorpo- rated SP and IGF-1C peptides to simultaneously enhance endogenous stem cell mobilization and survival, respectively. The processing parameters of PCL fibers have been optimized to fabri- cate electrospun membranes to facilitate cellularization and remod- eling, which has been documented elsewhere [33]. The morphology of cardiac patches was assessed by SEM, which revealed the pres- ence of uniform, continuous and smooth fibers (Fig. 1). FTIR spec- tra of patches revealed the presence of collagen as confirmed by its characteristics peaks (Supplementary Fig. S1). We evaluated the in vitro release of SP from electrospun membranes (n ¼ 5 per group) by using HPLC and the cumulative released amount of SP was found to be 57.79 6 9.96% for up to 5 days [30]. We did not observe the released amount of the peptide from electrospun membranes beyond this time point by using HPLC, which may be due to the detection limit of HPLC (<0.1 ppm) [30]. Evaluation of vascularization d f l We stained sections for isolectin-B4 and CD31 to investigate vascu- larization (Fig. 5). After 2 weeks, SP patches showed significantly higher numbers of isolectin-B4 positive capillaries in comparison to the saline, patch-only and IGF-1C patch-treated groups (saline, 48.3 6 4.0; patch-only, 51.0 6 4.3, IGF-1C patch, 50.0 6 6.5; and SP patch, 63.0 6 2.3 capillaries per HPF) (Fig. 5B). IGF-1C/SP patch-treated group showed an increase in the isolectin-B4 positive vessels that was significantly higher than all groups (76.0 6 4.2 capillaries per HPF). This suggests that the combination of IGF-1C peptide and SP helped improve the formation of capillaries in the in- farcted myocardium. Cell viability assay All quantitative results were obtained from at least five samples for analysis. Data were expressed as the mean 6standard error of mean. Statistical analysis was performed either by using One way ANOVA followed by Tukey’s post hoc analysis or Kruskal–Wallis test fol- lowed by Dunn’s test for multiple comparisons. A value of P < 0.05 was considered to be statistically significant. In vitro cell viability was examined on patches by using an MTT as- say. As can be seen from the figure, patch-only group show less cell viability than that of the IGF-1C patch, SP patch and IGF-1C/SP patch for up to 72 h (Supplementary Fig. S2). Besides, IGF-1C patch, SP patch and IGF-1C/SP patch did not significantly differ in terms of the cell viability. Cell proliferation assay The neo-vessels were also stained with CD31 antibody. Saline and patch-only groups did not significantly differ in terms of the numbers of CD31-positive vessels (saline, 107.95 6 9.5 and patch- only, 109.3 6 4.6 vessels per HPF). On the other hand, IGF-1C patch and SP patch groups showed significantly higher numbers of CD31-positive vessels in comparison to saline and patch-only groups (IGF-1C patch, 139.0 6 11.0 and SP patch, 157.0 6 7.0 The in vitro cell growth was evaluated by using CCK-8 assay for up to 6days. Optical density values were measured at a wavelength of 450nm and found to be 0.37756 0.0005, 0.34356 0.0025, 0.38356 0.0045, 0.38456 0.0155 and 0.4166 0.004 for tissue cul- ture plate, patch-only, IGF-1C patch, SP patch and IGF-1C/SP patch groups, respectively, at Day 3. Cell growth slightly increased at Day 6 307 In situ cardiac regeneration by using neuropeptide SP and IGF-1C peptide Figure 1. SEM micrographs of cardiac patches. (A) PCL/col, (B) PCL/colþIGF-1C peptide, (C) PCL/colþSP peptide, and (D) PCL/colþSPþIGF-1C peptide Figure 1. SEM micrographs of cardiac patches. (A) PCL/col, (B) PCL/colþIGF-1C peptide, (C) PCL/colþSP peptide, and (D) PCL/co raphs of cardiac patches. (A) PCL/col, (B) PCL/colþIGF-1C peptide, (C) PCL/colþSP peptide, and (D) PCL/colþSPþIGF-1C peptide other groups (IGF-1C/SP patch, 39.0 6 6.748 mL). Similarly, IGF- 1C/SP patch group showed significantly less EDV values in comparison to all other groups (saline, 59.90 6 2.055; patch- only, 84.86 6 9.454; IGF-1C patch, 82.58 6 6.145; SP patch, 76.147 6 4.658; and IGF-1C/SP patch, 72.585 6 3.875 mL) (Fig. 6C and D). The ability of IGF-1C/SP patches to improve the cardiac function after MI stresses the importance of these bioactive molecules. vessels per HPF) (Fig. 5D). Interestingly, IGF-1C/SP patch groups showed many CD31-positive vessels that was significantly higher than all groups (186.0 6 8.6 vessels per HPF) (Fig. 5D). These results reveal the formation of neo-vessels and capillaries that are most likely involved in the tissue perfusion and were plausibly resulted by SP and IGF-1C peptides. Evaluation of heart function We next evaluated the in vivo effect of different groups comparing saline, patch-only, IGF-1C patch, SP patch and IGF-1C/SP patch. ECG was used to assess the cardiac function after patches implanta- tion (Fig. 6). Fourteen days post-treatment, ECG analysis showed that SP and IGF-1C/SP patch-treated groups attenuated LV remodel- ing, which is essential to prevent heart failure following MI. No sig- nificant difference was found in the LV ejection fraction (LVEF) value among saline, patch-only and IGF-1C patch-treated groups (saline, 37.75 6 2.11; patch-only, 38.94 6 3.42; and IGF-1C patch, 40.99 6 4.21%) (Fig. 6B). On the other hand, SP patch and IGF-1C/ SP patch-treated groups showed significantly higher values of LVEF in comparison to the other groups (SP patch, 45.78 6 5.01; and IGF- 1C/SP patch, 58.37 6 2.56%). Notably, IGF-1C/SP patch-treated group showed the highest improvement in the LVEF value than all groups (IGF-1C/SP patch, 58.37 6 2.56%) (Fig. 6B). Evaluation of fibrosis The extent of fibrosis in the different treatment groups was assessed by using MT staining. Pink muscle fibers can be distinguished from blue stained ECM. The area fraction of collagen deposition was also measured. Saline, patch-only, SP patch, and IGF-1C patch did not significantly differ in terms of the deposition of collagen (saline, 28.4 6 7.26; patch-only, 26.64 6 1.99; IGF-1C patch, 22.6 6 3.07; and SP patch, 24.9 6 3.88%). On the other hand, IGF-1C/SP patch- treated group showed significantly less deposition of collagen than that of the saline group (IGF-1C patch, 18.0 6 3.90%) (Fig. 7A and B). Evaluation of cell apoptosis Reducing cell apoptosis triggered by MI is an important goal toward recovery and repair of infarcted myocardium. Tunel assay was per- formed to evaluate cell apoptosis in the myocardial tissues and the representative images are shown in Fig. 7C and D. Saline, patch- only, SP patch and IGF-1C patch-treated groups did not signifi- cantly differ in terms of the numbers of the Tunel-positive cells (saline, 22.4 6 4.45; patch-only, 19.0 6 4.24; SP patch, 17.4 6 2.42; and IGF-1C patch, 14.2 6 1.17 cells per HPF) (Fig. 7D). On the LV end-systolic volume (ESV) and end-diastolic volume (EDV) were also measured from the ECG results. Saline, patch-only, IGF- 1C patch, and SP patch-treated groups did not markedly differ in terms of the ESV values (saline, 60.86 6 4.349; patch-only, 63.746 6 10.603; IGF-1C patch, 53.98 6 9.847; and SP patch, 51.22 6 7.847mL) (Fig. 6C and D). On the other hand, IGF-1C/SP patch group showed significantly lower ESV values than that of the Shafiq et al. 308 Figure 2. In vitro transwell migration assay (n ¼ 5 per group). Wells containing medium only served as negative controls. Negative control, patch-only, and IGF- 1C patch groups recruited only few numbers of BM-MSCs. On the other hand, SP patch and IGF-1C/SP patch groups recruited signiﬁcantly higher numbers of BM-MSCs than that of the other groups. The density of the recruited BM-MSCs was evaluated by using image J and found to be 50366.9 6 11604.0, 77011.47 6 3842.1, 64917.01 6 14211.5, 158706 6 31711.4 and 276112.7 6 34117.4 mm2/mm2 in negative control, PCL/col, IGF-1, SP and IGF-1/SP groups, respec- tively. Results are shown as mean6SEM and evaluated by one-way ANOVA followed by Tukey’s post hoc analysis. Scale bar, 500 mm. P < 0.05, P < 0.01 Figure 2. In vitro transwell migration assay (n ¼ 5 per group). Wells containing medium only served as negative controls. Negative control, patch-only, and IGF- 1C patch groups recruited only few numbers of BM-MSCs. On the other hand, SP patch and IGF-1C/SP patch groups recruited signiﬁcantly higher numbers of BM-MSCs than that of the other groups. The density of the recruited BM-MSCs was evaluated by using image J and found to be 50366.9 6 11604.0, 77011.47 6 3842.1, 64917.01 6 14211.5, 158706 6 31711.4 and 276112.7 6 34117.4 mm2/mm2 in negative control, PCL/col, IGF-1, SP and IGF-1/SP groups, respec- tively. Stem cell recruitment in vivo SP has been documented to enhance tissue regeneration by recruiting endogenous stem and progenitor cells. Therefore, we also evaluated MSCs recruitment in vivo and the representative images are shown in Fig. 8. Saline and patch-only groups did not significantly differ in terms of the numbers of the CD29-positive cells (saline, 9.6 6 2.42 and patch-only, 11.6 6 2.15 cells per HPF) (Fig. 8). On the other hand, SP patch and IGF-1C patch-treated groups recruited signifi- cantly higher numbers of CD29-positive cells in comparison to the saline and patch-only groups (SP patch, 24.8 6 3.12 and IGF-1C Evaluation of cell apoptosis Results are shown as mean6SEM and evaluated by one-way ANOVA followed by Tukey’s post hoc analysis. Scale bar, 500 mm. P < 0.05, *P < 0.01 patch, 19.8 6 2.48 cells per HPF) (Fig. 8B). Interestingly, IGF-1C/SP patch-treated group showed significantly higher numbers of CD29- positive cells in comparison to the saline, patch-only, SP patch, and IGF-1C patch-treated groups (IGF-1C/SP patch, 40.0 6 5.25 cells per HPF) (Fig. 8B). This reflects that IGF-1C/SP may act synergisti- cally by recruiting endogenous stem cells and supporting their sur- vival in the infarcted area. other hand, IGF-1C/SP patch-treated group showed significantly less numbers of Tunel-positive cells in comparison to saline and patch- only groups (IGF-1C/SP patch, 8.6 6 2.42 cells per HPF) (Fig. 7D). These data suggest synergistic effect of SP and IGF-1C peptide in re- ducing cell apoptosis after MI. Discussion While appealing, exoge- nous stem cells need extensive in vitro manipulation prior to the transplantation. Additionally, transplanted stem cells hardly survive and majority of those are lost at the initial time points [35]. Besides, the usage of ESCs and iPSCs may be hampered by the ethical and teratoma formation issues. neovascularization in a variety of injury microenvironments by recruiting endogenous stromal like cells from the BM or circulation [15–24]. However, SP possesses short half-life and can be easily de- graded by the endogenous peptidases [15, 21]. Therefore, strategies focusing at enhancing the residence or survival of SP hold great promise for SP-mediated tissue repair. Moreover, we and others have demonstrated enhanced cell engraftment and retention resulted by IGF-1 or IGF-1C peptide [9, 27, 28, 39]. Since most of the trans- planted or recruited cells are lost due to a hostile microenvironment, we incorporated IGF-1C peptide along with SP to further enhance the stem cell survival and cardiac tissue repair in vivo. We and others have previously reported that bioactive peptides and polymeric materials containing covalently immobilized SP can facilitate tissue repair through endogenous stem/progenitor cell re- cruitment [16, 18, 21, 22]. While covalent conjugation is a promis- ing technique and may offer several advantages for tissue regeneration, the bioactivity of the peptide may be compromised be- cause of several coupling/conjugation steps. Moreover, the lengthy and time-consuming procedure may compromise the therapeutic uti- lization of this approach. In this study, we fabricated cardiac patches containing either SP only or both IGF-1C peptide and SP. In vitro Transwell migration assay revealed that SP patches can recruit sig- nificantly higher numbers of MSCs than that of the negative and positive control groups. These results indicate that SP patches may recruit endogenous stem/progenitor cells and promote tissue repair Accordingly, recent approaches involve the activation of endoge- nous cells and mobilization of stem/progenitor cells from the bone marrow (BM) or the circulation. These recruited cells play a signifi- cant role in the neovascularization process. Several molecular mediators of the ischemic repair response, such as VEGF, SDF-1a, G-CSF, IGF-1, cyclin A2, ephrin B2, and Ang-1 have been shown to induce in situ myocardial regeneration [38, 39]. However, these are large molecular weight proteins, which cannot be easily synthesized or incorporated into scaffold materials. Discussion Cardiac patches manufactured by exploiting TE approaches may be able to replace the myocardial defects and may provide a solution to improve the cardiac function. Although scaffold materials can be used to provide cell-instructive cues and physical support, the modi- fication of scaffolds is required to amplify the tissue regeneration phenomenon. Cardiac patches consisting of a variety of biodegrad- able components and a myriad of bioactive moieties have been In situ cardiac regeneration by using neuropeptide SP and IGF-1C peptide 309 Figure 3. Schematic illustration of the study (A) and histological analysis of the excised hearts at Day 14 (B). (A) implantation of cardiac patches on an infarcted myocardium. Patches were sutured onto epicardium by using at least two sutures. Hearts along with the patches were harvested 14 days after transplantation for histological and immunohistochemical analysis at the tissue levels. (B) Representative H&E images showing that the implanted patches remained adhered to the myocardium and were populated by the host cells Figure 3. Schematic illustration of the study (A) and histological analysis of the excised hearts at Day 14 (B). (A) implantation of cardiac patches on an infarcted myocardium. Patches were sutured onto epicardium by using at least two sutures. Hearts along with the patches were harvested 14 days after transplantation for histological and immunohistochemical analysis at the tissue levels. (B) Representative H&E images showing that the implanted patches remained adhered to the myocardium and were populated by the host cells pursued and shown to improve the cardiac function [34]. Moreover, different types of cells including induced pluripotent-like stem cells (iPSCs)- and embryonic stem cells (ESCs)-derived vascular cell types have been transplanted and reported to enhance the myocardial healing either by differentiation into the cardiac cell types or by the secretion of angiotrophic factors [35–37]. While appealing, exoge- nous stem cells need extensive in vitro manipulation prior to the transplantation. Additionally, transplanted stem cells hardly survive and majority of those are lost at the initial time points [35]. Besides, the usage of ESCs and iPSCs may be hampered by the ethical and teratoma formation issues. pursued and shown to improve the cardiac function [34]. Moreover, different types of cells including induced pluripotent-like stem cells (iPSCs)- and embryonic stem cells (ESCs)-derived vascular cell types have been transplanted and reported to enhance the myocardial healing either by differentiation into the cardiac cell types or by the secretion of angiotrophic factors [35–37]. Discussion Alternatively, short peptide sequences, bioactive lipids and therapeutics molecules are being in- vestigated for in situ tissue regeneration applications, which may be easily synthesized and incorporated into scaffold materials [15–27]. In this study, we fabricated novel cardiac patches delivering neu- ropeptide SP, that was shown to promote epithelialization and Shafiq et al. 310 e 4. H&E and MT staining of hearts explanted 14 days after implantation. H&E staining (A), left anterior wall thickness (B), MT staining (C) and percent of an- wall infarct size (D). Scale bars have been shown on the images. IGF-1C/SP patches exhibited signiﬁcantly higher LV wall thickness in comparison to the treatment groups. Five animals were analyzed per group. Results are shown as mean6SEM and evaluated by one-way ANOVA followed by Tukey’s post nalysis. (B) P ¼ 0.0001848 and *P ¼ 0.00005638; (D) P ¼ 0.0227 Figure 4. H&E and MT staining of hearts explanted 14 days after implantation. H&E staining (A), left anterior wall thickness (B), MT staining (C) and percent of an- terior wall infarct size (D). Scale bars have been shown on the images. IGF-1C/SP patches exhibited signiﬁcantly higher LV wall thickness in comparison to the other treatment groups. Five animals were analyzed per group. Results are shown as mean6SEM and evaluated by one-way ANOVA followed by Tukey’s post hoc analysis. (B) P ¼ 0.0001848 and P ¼ 0.00005638; (D) P ¼ 0.0227 by harnessing host’s own regenerative capabilities. The feasibility of patches was demonstrated in an acute MI model in mice for up to 14 days, in which the IGF-1/SP patch-treated group showed im- proved neovascularization, higher numbers of capillaries, aug- mented LV wall thickness, higher cardiac function, and reduced adverse cardiac remodeling than that of the saline, patch-only, or in- dividual SP and IGF-1C peptide containing patches. Moreover, IGF- 1/SP patch-treated group recruited sufficient numbers of CD29- positive MSCs and reduced cell apoptosis in comparison to the other groups. These data support the notion that endogenous stem/pro- genitor cells might be recruited toward bioactive patches and pro- mote cardiac regeneration. its direct effect on ECs or its ability to recruit endothelial progenitor cells (EPCs) and stem cells [15, 17]. Amadesi et al. documented that BM-derived stem and progenitor cells possess neurokinin-1 receptor and are attracted by the SP gradients [17]. Discussion We and others have also previously reported that SP induces the recruitment of MSCs, EPCs, SMCs and pericytes, which then participate in blood vessel forma- tion [15–24]. On the other hand, IGF-1C, a 12-amino acid peptide, has the similar functions of its full-length molecules [27]. IGF-1C peptide could support the cardiac cell survival and retention, attenu- ate fibrosis, and facilitate the recovery of heart function [27]. IGF-1 could provide an immediate strong pro-survival signal to rescue the remaining functional myocardium and reduce cell apoptosis and loss after the initial ischemic event [9, 28, 33]. Meanwhile, SP can mediate processes required for infarct repair, such as angiogenesis by harnessing host’s own regenerative capabilities. The feasibility of patches was demonstrated in an acute MI model in mice for up to 14 days, in which the IGF-1/SP patch-treated group showed im- proved neovascularization, higher numbers of capillaries, aug- mented LV wall thickness, higher cardiac function, and reduced adverse cardiac remodeling than that of the saline, patch-only, or in- dividual SP and IGF-1C peptide containing patches. Moreover, IGF- 1/SP patch-treated group recruited sufficient numbers of CD29- positive MSCs and reduced cell apoptosis in comparison to the other groups. These data support the notion that endogenous stem/pro- genitor cells might be recruited toward bioactive patches and pro- mote cardiac regeneration. SP has been shown to enhance neovascularization in different types of injury models and scaffold materials, which can be due to In situ cardiac regeneration by using neuropeptide SP and IGF-1C peptide 311 induction, more favorable ECM remodeling, and stem cell recruit- ment. Consequently, SP and IGF-1C/SP patch-treated groups led to higher capillaries regeneration in comparison to the saline, patch- only, and IGF-1C patch-treated groups. IGF-1C patch, SP patch, and IGF-1C/SP patch-treated groups also exhibited higher blood Interestingly, IGF-1C/SP patch-treated group showed significantl higher numbers of capillaries and blood vessels than all othe groups, which suggest the synergistic effect of IGF-1C peptide an SP peptide and worthy for the future investigations. Interestingly, cardiac patch containing IGF-1C/SP significantl Figure 5. Vascularization in the explanted hearts. Excised hearts were stained with isolectin B4 (A, B) and CD31 (C, D). SP and IGF-1/SP patches showed signiﬁ cantly higher numbers of isolectin B4 capillaries and CD31-positive vessels. Scale bars, 50 mm (A) and 100 mm (C). SP and IGF-1C/SP patch-treated groups showe signiﬁcantly higher numbers of isolectin B4-positive capillaries than that of the saline, patch-only and IGF-1C patch-treated groups. Discussion On the other hand, IGF-1 patch, SP patch and IGF-1C/SP patch-treated groups showed signiﬁcantly higher numbers of CD31-positive vessels than that of the saline and patch-only group IGF-1C/SP-treated groups showed signiﬁcantly higher numbers of capillaries and vessels in comparison to the other groups. Five animals were analyzed pe group. Results are shown as mean6SEM and evaluated by one-way ANOVA followed by Tukey’s post hoc analysis. P < 0.05 Figure 5. Vascularization in the explanted hearts. Excised hearts were stained with isolectin B4 (A, B) and CD31 (C, D). SP and IGF-1/SP patches showed signiﬁ- cantly higher numbers of isolectin B4 capillaries and CD31-positive vessels. Scale bars, 50 mm (A) and 100 mm (C). SP and IGF-1C/SP patch-treated groups showed signiﬁcantly higher numbers of isolectin B4-positive capillaries than that of the saline, patch-only and IGF-1C patch-treated groups. On the other hand, IGF-1C patch, SP patch and IGF-1C/SP patch-treated groups showed signiﬁcantly higher numbers of CD31-positive vessels than that of the saline and patch-only groups. IGF-1C/SP-treated groups showed signiﬁcantly higher numbers of capillaries and vessels in comparison to the other groups. Five animals were analyzed per group. Results are shown as mean6SEM and evaluated by one-way ANOVA followed by Tukey’s post hoc analysis. P < 0.05 induction, more favorable ECM remodeling, and stem cell recruit- ment. Consequently, SP and IGF-1C/SP patch-treated groups led to higher capillaries regeneration in comparison to the saline, patch- only, and IGF-1C patch-treated groups. IGF-1C patch, SP patch, and IGF-1C/SP patch-treated groups also exhibited higher blood vessel regeneration compared with the saline and patch-only groups. Interestingly, IGF-1C/SP patch-treated group showed significantly higher numbers of capillaries and blood vessels than all other groups, which suggest the synergistic effect of IGF-1C peptide and SP peptide and worthy for the future investigations. Interestingly, cardiac patch containing IGF-1C/SP significantly improved cardiac function and attenuated adverse cardiac 312 Shafiq et al. d l l d b f l h h l d d bl d l h d Figure 6. Echocardiography of mice at Day 14. (A) Representative echocardiographic 2D images of hearts of each group. Compared with the saline, patch-only and IGF-1C patch-treated groups, SP patch and IGF-1C/SP patch-treated groups showed signiﬁcantly higher LVEF values (B). IGF-1C/SP patch-treated groups also displayed LVESV and LVEDV values in comparison to the other treatment groups (C, D). Results are shown as mean6SEM and evaluated by one-way ANOVA fol- lowed by Tukey’s post hoc analysis. Discussion P < 0.05. Sham (n ¼ 5), saline (n ¼ 5), patch-only (n ¼ 5) and peptides containing patches (n ¼ 5) at 14 days 312 Shafiq et al. Figure 6. Echocardiography of mice at Day 14. (A) Representative echocardiographic 2D images of hearts of each group. Compared with the saline, patch-only and IGF-1C patch-treated groups, SP patch and IGF-1C/SP patch-treated groups showed signiﬁcantly higher LVEF values (B). IGF-1C/SP patch-treated groups also displayed LVESV and LVEDV values in comparison to the other treatment groups (C, D). Results are shown as mean6SEM and evaluated by one-way ANOVA fol- lowed by Tukey’s post hoc analysis. P < 0.05. Sham (n ¼ 5), saline (n ¼ 5), patch-only (n ¼ 5) and peptides containing patches (n ¼ 5) at 14 days and blood vessels in IGF-1C/SP patch-treated group in comparison to saline, patch-only or single peptide (IGF-1C and SP) containing patches [40, 41]. remodeling as revealed by significantly higher LVEF values and lower ESV and EDV values in comparison to saline, patch-only, or single peptide (SP or IGF-1C) groups. Since it is well-known that neovascularization and cardiomyocytes survival plays an important role in attenuating the adverse cardiac remodeling, improvements in the cardiac function may be partly ascribed to the enhanced neovas- cularization as evidenced by the higher regeneration of capillaries Besides, exogenous or endogenous stem cells have been shown to contribute to cardiac tissue regeneration by either differentiation into the vascular cell types or by paracrine mechanisms [42–45]. We also observed significant recruitment of CD29-positive MSCs in SP In situ cardiac regeneration by using neuropeptide SP and IGF-1C peptide 313 patch, IGF-1C patch and IGF-1C/SP patch-treated groups than that patches. Therefore, we consider that by using only one peptide may Figure 7. MT Staining (A, B) and Tunel assay (C, D) for evaluating ﬁbrosis and cell apoptosis of the explanted heart tissues, respectively. IGF-1C/SP patch-treated groups showed signiﬁcantly less deposition of collagen in comparison to saline-treated groups. Results are shown as mean6SEM and evaluated by one-way ANOVA followed by Tukey’s post hoc analysis. P ¼ 0.02351. On the other hand, saline, patch-only, SP patch and IGF-1C patch-treated groups did not markedly differ in terms of the content of tunel-positive cells, whereas IGF-1C/SP patch-treated groups showed signiﬁcantly less numbers of tunel-positive cells than that of the saline and patch-only groups. Blue, DAPI; red, troponin; and green, apoptotic cells. Five animals were analyzed per group. Discussion Scale bar, 50.0 mm. Results are shown as mean6SEM and evaluated by Kruskal–Wallis test followed by Dunn’s test for multiple comparisons. P ¼ 0.0016 Figure 7. MT Staining (A, B) and Tunel assay (C, D) for evaluating ﬁbrosis and cell apoptosis of the explanted heart tissues, respectively. IGF-1C/SP patch-treated groups showed signiﬁcantly less deposition of collagen in comparison to saline-treated groups. Results are shown as mean6SEM and evaluated by one-way ANOVA followed by Tukey’s post hoc analysis. P ¼ 0.02351. On the other hand, saline, patch-only, SP patch and IGF-1C patch-treated groups did not markedly differ in terms of the content of tunel positive cells whereas IGF 1C/SP patch treated groups showed signiﬁcantly less numbers of tunel positive cells than that of Figure 7. MT Staining (A, B) and Tunel assay (C, D) for evaluating ﬁbrosis and cell apoptosis of the explanted heart tissues, respectively. IGF-1C/SP patch-treated groups showed signiﬁcantly less deposition of collagen in comparison to saline-treated groups. Results are shown as mean6SEM and evaluated by one-way ANOVA followed by Tukey’s post hoc analysis. P ¼ 0.02351. On the other hand, saline, patch-only, SP patch and IGF-1C patch-treated groups did not markedly differ in terms of the content of tunel-positive cells, whereas IGF-1C/SP patch-treated groups showed signiﬁcantly less numbers of tunel-positive cells than that of the saline and patch-only groups. Blue, DAPI; red, troponin; and green, apoptotic cells. Five animals were analyzed per group. Scale bar, 50.0 mm. Results are shown as mean6SEM and evaluated by Kruskal–Wallis test followed by Dunn’s test for multiple comparisons. P ¼ 0.0016 patch, IGF-1C patch and IGF-1C/SP patch-treated groups than that of the saline and patch-only groups. Furthermore, we observed sig- nificantly higher recruitment of CD29-positive MSCs in IGF-1C/SP patch-treated groups compared to the single peptide containing patches. Therefore, we consider that by using only one peptide may not be sufficient to produce adequate effects for regeneration in the MI model. These findings reveal that IGF-1C/SP synergism was aug- mented when cardiac patches incorporating the dual IGF-1C/SP 314 Shafiq et al. . MSCs Recruitment in vivo and schematic illustration of the study. Excised hearts were stained with CD29 antibody (A–C). SP-eluting cardiac patc d signiﬁcantly higher numbers of CD29-positive cells. Five animals were analyzed per group. Results are shown as mean6SEM and evaluated by o OVA followed by Tukey’s post hoc analysis. Conclusions 9. Ruvinov E, Leor J, Cohen S. The promotion of myocardial repair by the sequential delivery of IGF-1 and HGF from an injectable alginate biomaterial in a model of acute myocardial infarction. Biomaterials 2011;32: 565–78. This study evaluated the potential of patches containing stem cell in- ducing/recruiting factors on in situ tissue repair in the settings of MI. Patches containing SP and IGF-1C peptide were successfully pre- pared by electrospinning. Both SP and IGF-1C/SP patches recruited significantly higher numbers of MSCs than that of the negative con- trol, patch-only and IGF-1 groups as revealed by an in vitro Transwell migration assay. IGF-1C/SP patches significantly im- proved the heart function and attenuated adverse cardiac remodel- ing than that of the other groups. SP and IGF-1C/SP patches also facilitated blood vessels and capillaries formation as assessed by im- munohistochemical analysis and interestingly IGF-1C/SP group showed the highest numbers of blood vessels and capillaries than that of the other groups. Moreover, IGF-1C/SP patches augmented infarct wall thickness, which was higher than that of the saline and patch-only groups. IGF-1C/SP patches also led to the less deposition of collagen compared with saline. Moreover, IGF-1C/SP patches showed the lowest numbers of Tunel-positive cells than that of sa- line and patch-only groups. These results suggest that these novel cardiac patches can be a good choice along with an appropriate scaf- fold material to enhance in situ blood vessel regeneration and are worthy for the future applications. 10. Urbanek K, Rota M, Cascapera S et al. Cardiac stem cells possess growth factor-receptor systems that after activation regenerate the infarcted myo- cardium, improving ventricular function and long-term survival. Circ Res 2005;97:663–73. 11. Wen Z, Mai Z, Zhang H et al. Local activation of cardiac stem cells for post-myocardial infarction cardiac repair. J Cell Mol Med 2012;16: 2549–63. 12. Tang XL, Li Q, Rokosh G et al. Long-term outcome of administration of c-kitPOS cardiac progenitor cells after acute myocardial infarction: trans- planted cells do not become cardiomyocytes, but structural and functional improvement and proliferation of endogenous cells persist for at least one year. Circulation 2016;118:1091–105. 13. Beohar N, Rapp J, Pandya S et al. Rebulding the damaged heart, the po- tential of cytokines and growth factors in the treatment of ischemic heart disease. J Am Coll Cardiol 2010;56:1287–97. 14. Awada HK, Johnson LA, Kevin Hitchens T et al. Discussion P0.0001, P ¼ 0.0009, P0.0001, #P0.0001, ##P ¼ 0.0090, ###P0.0001, $P0.0001 and $$P ¼ 0.0001 tic diagram of the designed study. Cardiac patches containing SP alone or SP and IGF-1 peptides were fabricated by electrospinning. SP may recruit us stem and progenitor cells, whereas IGF-1 peptide may enhance their retention and engraftment at the target site 8. MSCs Recruitment in vivo and schematic illustration of the study. Excised hearts were stained with CD29 antibody (A–C). SP-eluting cardia Figure 8. MSCs Recruitment in vivo and schematic illustration of the study. Excised hearts were stained with CD29 antibody (A–C). SP-eluting cardiac patches recruited signiﬁcantly higher numbers of CD29-positive cells. Five animals were analyzed per group. Results are shown as mean6SEM and evaluated by one- way ANOVA followed by Tukey’s post hoc analysis. P0.0001, P ¼ 0.0009, *P0.0001, #P0.0001, ##P ¼ 0.0090, ###P0.0001, $P0.0001 and $$P ¼ 0.0001. (D) Schematic diagram of the designed study. Cardiac patches containing SP alone or SP and IGF-1 peptides were fabricated by electrospinning. SP may recruit en- dogenous stem and progenitor cells, whereas IGF-1 peptide may enhance their retention and engraftment at the target site In situ cardiac regeneration by using neuropeptide SP and IGF-1C peptide 315 3. Ellison GM, Torella D, Karakikes I et al. Myocyte death and renewal: modern concepts of cardiac cellular homeostasis. Nat Clin Pract Cardiovasc Med 2007;4:S52–S9. therapeutic agents were transplanted into animals. These results sug- gest that (i) delivery of dual therapeutic factors can create benign microenvironments; (ii) recruit CD29-positive stem cells within microenvironments to form stable and vascular networks; (iii) sup- port cardiac cell survival and reduce fibrosis. 4. Kubal C, Sheth K, Nadal-Ginard B et al. Bone marrow cells have a potent anti-ischemic effect against myocardial cell death in humans. J Thorac Cardiovasc Surg 2006;132:1112–8. There are also several limitations in this study. First, we implanted patches for up to 2 weeks only. Long-term evaluation of these patches will elucidate their potential for the regeneration of in- farcted myocardium. Second, we did not investigate the patches at the earlier time point. Evaluation of patches at the initial time points might be helpful to clearly demonstrate the inflammatory response. Third, the exact synergistic mechanism between SP and IGF-1C leading to cardiac regeneration in MI was not elucidated in this study. Nonetheless, we elucidated that bioactive patches facilitated neovascularization, infarct stabilization, and reducing wall thinning. 5. Conclusions Factorial design of experiments to optimize multiple protein delivery for cardiac repair. ACS Biomater Sci Eng 2016;2:879–86. 15. Hong HS, Lee J, Lee EA et al. A new role of substance P as an injury- inducible messenger for mobilization of CD29(þ) stromal-like cells. Nat Med 2009;15:425–35. 16. Kohara H, Tajima S, Yamamoto SM et al. Angiogenesis induced by con- trolled release of neuropeptide substance P. Biomaterials 2010;31: 8617–25. Discussion Feiner R, Engel L, Fleischer S et al. Engineered hybrid cardiac patches with multifunctional electronic for online monitoring and regulation of tissue formation. Nat Mater 2016;15:679–85. 6. Wang Q, Yang H, Bai A et al. Functional engineered human cardiac patches prepared from nature’s platform improve heart function after acute myocardial infarction. Biomaterials 2016;105:52–65. 7. Sugiura T, Hibino N, Breuer CK et al. Tissue-engineered cardiac patch seeded with human induced pluripotent stem cell derived cardiomyocytes promoted the regeneration of host cardiomyocytes in a rat model. J Cardiothorac Surg 2016;11:163–71. 8. Wang QL, Wang HJ, Li ZH et al. Mesenchymal stem cell-loaded cardiac patch promotes epicardial activation and repair of the infarcted myocar- dium. J Cell Mol Med 2017;21:1751–66. Funding 18. Ko IK, Ju YM, Chen T et al. Combined systemic and local delivery of stem cell inducing/recruiting factors for in situ tissue regeneration. FASEB J 2012;26:158–68. This work was supported by the KIST Institutional Program and by the KU- KIST Graduate School of Converging Science and Technology Program. Project supported by the National Science Foundation for Young Scientists of China (Grant No. 81701839); The Youth Foundation of Tianjin Medical University (Grant No. 2015KYZQ14). 19. Jin Y, Hong HS, Son Y. Substance P enhances mesenchymal stem cells- mediated immune modulation. Cytokine 2015;71:145–53. 20. Lee JS, Jin Y, Park H-J et al. In situ bone tissue engineering with an endog- enous stem cell mobilizer and osteoinductive nanoﬁbrous polymeric scaf- folds. Biotechnol J 2017;12:1700062. Conflict of interest statement. None declared. 21. Kim JH, Jung Y, Kim BS et al. Stem cell recruitment and angiogenesis of neuropeptide substance P coupled with self-assembling peptide nanoﬁber in a mouse hind limb ischemia model. Biomaterials 2013;34:1657–68. Supplementary data are available at REGBIO online. Supplementary data are available at REGBIO online. 17. Amadesi S, Reni C, Katare R et al. Role for substance P-based nociceptive signals in progenitor cell activation and angiogenesis during ischemia in mice and human subjects. Circulation 2012; 125:1774–86. mice and human subjects. Circulation 2012; 125:1774–86. Conflict of interest statement. None declared. Supplementary data Supplementary data are available at REGBIO online. Supplementary data are available at REGBIO online. References 1. Roth GA, Forouzanfar MH, Moran AE et al. Demographic and epidemio- logic drivers of global cardiovascular mortality. N Engl J Med 2015;372: 1333–41. 22. Shaﬁq M, Jung Y, Kim SH. 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https://openalex.org/W2991065973	https://europepmc.org/articles/pmc6963674?pdf=render	English	null	Impact of Punicalagin on the Physicochemical and Structural Properties of Wheat Flour Dough	Foods	2,019	cc-by	9,241	Received: 14 October 2019; Accepted: 18 November 2019; Published: 22 November 2019 Abstract: The study explored punicalagin (PGN) as a wheat ﬂour enhancer. The impact of PGN on the physicochemical and structural properties of wheat ﬂour have been investigated. It turned out that PGN increased the formation time, stability, tensile resistance, extension, and viscoelasticity of the dough at the concentrations of 0.13 and 0.26 mg/g. Scan electron microscope images of the cross section of the dough displayed a more compact and ordered network structure with the addition of 0.13 and 0.26 mg/g PGN. Fourier transform infrared spectroscopy spectra indicated an increase of α-helix and β-sheet content. However, nonlinear enhancing eﬀects of PGN on the stretching properties, rheology, and structural properties of the dough were observed at concentrations of 0.39 and 0.52 mg/g. Correspondingly, cleavages were observed on the cross section of the dough and the content of β-sheet showed a trend of reduction in the dough with addition of PGN at high concentrations. Taken together, these results indicated the potential usage of PGN as a wheat ﬂour enhancer of natural origin at the concentration below 0.39 mg/g in the ﬂour. Keywords: punicalagin; wheat ﬂour; dough rheology; gluten protein network foods foods Foods 2019, 8, 606; doi:10.3390/foods8120606 Impact of Punicalagin on the Physicochemical and Structural Properties of Wheat Flour Dough Hong Peng 1,2, Bin Li 1,2 and Jing Tian 1,2,* Hong Peng 1,2, Bin Li 1,2 and Jing Tian 1,2,* 1 College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; 13807674653@163.com (H.P.); libinfood@mail.hzau.edu.cn (B.L.) 2 Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, Wuhan 430070, China * Correspondence: jing.tian@mail.hzau.edu.cn; Tel.: +86-27-8728-2111 1 College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; 13807674653@163.com (H.P.); libinfood@mail.hzau.edu.cn (B.L.) 2 Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, Wuhan 430070, China * Correspondence: jing.tian@mail.hzau.edu.cn; Tel.: +86-27-8728-2111 Received: 14 October 2019; Accepted: 18 November 2019; Published: 22 November 2019 * Correspondence: jing.tian@mail.hzau.edu.cn; Tel.: +86-27-8728-2111 foods foods foods www.mdpi.com/journal/foods 1. Introduction Glutenin is a heterogeneous macromolecular polymer protein that is linked by intermolecular disulﬁde bonds, which imparts the elasticity and strength of dough [1]. Generally, the elasticity depends on the formation of polymer. Gliadin is a single-chain protein with no interchain disulﬁde bond or subunit structure. It forms a spherical stereo structure by hydrophobic bonds, hydrogen bonds, and intramolecular disulﬁde bonds [2], and is responsible for the viscosity of the dough. During dough mixing, gluten proteins are linked together by disulﬁde bonds, hydrogen bonds, and hydrophobic interactions in the presence of water and mechanical work forming strong crosslinks between and within the polypeptide chains [3–6]. Wheat dough has multiple usages in the food due to the viscoelasticity, such as bread and noodles [7]. The proper balance between the viscosity and the elasticity/strength of the dough determines the quality of end-products [8]. For instance, too high quantity of gliadin causes a weakening of the protein network with the risk that the produced bread has a wide base and low volume. If the quantity of glutenin prevails, protein network is too rigid with the risk that the produced bread has a reduced volume due to the diﬃculties of the gas pushing on the gluten network [8]. Therefore, exogenous components are added to improve gluten network and adjust the performance of end products in the volume, texture, and nutrition. These additives include chemical additives, such as iodate [9], ascorbic acid [10] and peroxide [11], enzymes such as glutamyl transferase, glucose oxidase and xylanase [12,13], and microorganisms [14,15]. However, these additives also have certain drawbacks, such as instability, high price, lack of source, or tolerance [11,16]. www.mdpi.com/journal/foods Foods 2019, 8, 606; doi:10.3390/foods8120606 www.mdpi.com/journal/foods 2 of 13 Foods 2019, 8, 606 Punicalagin (PGN) is a pomegranate-speciﬁc polyphenol that is extracted from pomegranate peel, pomegranate juice, and pomegranate seeds, with the highest content in pomegranate peel [17]. PGN is hydrophilia and stable against heat, ultrasound, ultraviolet radiation, and additives of common food and cosmetics [18]. According to clinical trials, no toxicological treatment-related eﬀects were observed in the human with the oral administration of pomegranate polyphenol extract for up to 2130 mg/day (1305 mg/day of gallic acid equivalents) for 28 days [19] or in the supplementation of a pomegranate extract with trademark of POMx (POM Wonderful, LLC; Los Angeles, CA, USA) in a one-year pilot study [20]. 1. Introduction Thus, the consumption of pomegranate extracts including PGN are well tolerated and has been so far safe from a toxicological perspective. Moreover, health beneﬁts were observed including cancer prevention [21], cardiometabolic health promotion [22], and obesity prevention [23] via the supplementation of PGN or pomegranate polyphenol extracts in clinical trials. Therefore, PGN and pomegranate extracts have been highlighted in the ﬁeld of cosmetics [24], medicine [25], and food [26]. Proprietary pomegranate extracts of POMx and POMELLA (Verdure Sciences, Inc. Noblesville, IN, USA) have been developed as dietary supplementation to improve the ﬁtness [20]. PGN components have been added into cosmetics to lighten the skins via inhibiting melanin [24], promote the regeneration of dermis and epidermis [27], and increase the skin ﬁrmness and elasticity [28]. Films containing pomegranate extracts have been applied in food matrix to elongate the shelf life via inhibitory eﬀects on the growth of microorganisms [29,30]. Pomegranate juice has been blended into protein-based ﬁlm as a plasticizer [31]. In recent years, a large number of studies have found that polyphenols enhance the interaction between gluten and improve the rheological properties of the dough [32–36] via alteration of the amount and distribution of disulﬁde bonds [1,5] to achieve demands in hardness [37,38], moisture [39], and volume [40]. Several studies have reported that the structure and molecular weight of polyphenols play an important role in the interaction of proteins with polyphenols [34]. High molecular weight polyphenols (tannins) have been shown to bind to proteins more strongly or preferentially [41] due to the occupation of more binding sites on proteins. In addition, the ﬂexibility of the polyphenol structure and the number of hydroxyl groups are also indispensable [34]. It has been reported that polymeric procyanidins (sorghum procyanidins) are more eﬀective in enhancing the mixing and stretching properties of the dough via cross-linking than oligomeric procyanidins (grape seed procyanidins) [42]. It is presumed that more hydroxyl groups in polymeric procyanidins occupy more protein binding cites resulting in high potential in the improvement of the dough quality. PGN is an exceptional large polyphenol with molecular weight of 1084.7 and with the excellent performance in health beneﬁts as well as in extensive applications. To date, there has not any report of PGN as an additive supplemented to the dough. 1. Introduction The presented study aims to investigate the impact of PGN on the physicochemical and structural properties of wheat ﬂour dough, which facilitate the application of pomegranate in the food industry. 2.4. Dough Stretching Properties According to the method of Brabender/International Association for Cereal Science and Technology/Bipea, the dough stretching properties were examined using the Brabender Extenso-graph (Brabender, Duisburg, Germany) as previously reported [43]. Brieﬂy, 150 g of the dough was cut from the dough pre-mixed with Brabender Farinograph for 5 min. Afterwards all the doughs passed through the balling and moulder unit of Brabender Extenso-graph forming uniformly large sticks. As-prepared stick samples were rested in fermentation cabinet for 90 min at 30 ◦C and tested with the Brabender Extenso-graph. Parameters including the area of the tensile curve, the tensile resistance, and the extension were recorded. At least three replicates of the sample at each condition are prepared for the examination. 2.5. Rheological Properties Characterization Rheological measurements including dynamic oscillatory tests and creep tests were performed using a controlled stress rheometer (Thermo Scientiﬁc, Waltham, MA, USA) at 25 ◦C according to previous studies [13,44]. The dough was prepared as it was described in Section 2.2 and rested for 30 min. Afterwards, the dough was placed into paralleled plates with a diameter of 40 mm and the gap was adjusted to 1 mm. The outer edges of the dough were sealed with glycerin to prevent drying of the sample during the test. A rest time of 5 min was applied for all the samples to allow the relaxation of residual stresses before the measurement. The linear viscoelastic region of the dough was ﬁrstly determined via strain sweep procedure in oscillatory mode at the frequency of 1.0 Hz. The dynamic rheological properties of the dough were determined by the frequency sweep at 0.1–10 Hz. Creep and recovery tests were performed via a constant stress (300 s at 100 Pa), followed by a period of no stress (300 s at 0 Pa) according to previous studies [42,45]. A minimum of three replicates of dough samples at each experimental condition were performed. 2.1. Materials and Chemicals High-gluten and low-gluten wheat ﬂour were purchased from Zhongyuan Cereals and Oils Co., Ltd. (Henan, China). The protein content of high-gluten and low-gluten wheat ﬂour was 12.2% and 8%, respectively. The wet gluten content of high-gluten ﬂour and low-gluten ﬂour was 31.3% and 27.2% (wt/wt), respectively. Punicalagin (PGN) powder (purity ≥98%) separated from pomegranate was purchased from Chengdu Biopurify Phytochemicals Ltd. (Sichuan, China). Other chemicals were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Water in the study is ultra-pure with the resistivity of 18.2 MΩcm. 3 of 13 Foods 2019, 8, 606 2.2. Sample Preparation PGN were added to high-gluten ﬂour and low-gluten ﬂour with concentrations of 0.13, 0.26, 0.39, and 0.52 mg/g. Wheat dough was prepared by mixing 5 g of ﬂour and 3.0 mL of water or 5 g of ﬂour with PGN dissolved in 3.0 mL of water with ﬁnal concentrations mentioned above. Afterwards the mixture was freeze-dried with lyophilizer at −40 ◦C and 0.1 Pa for 48 h. Then as-prepared dough was ground and screened via a mesh of 180 µm. 2.3. Dough Mixing Properties The dough mixing properties were examined with Brabender Farinograph (Brabender, Duisburg, Germany) according to the previous study [43]. Brieﬂy, 295.5 g of high-gluten wheat ﬂour or 294.5 g of low-gluten wheat ﬂour supplemented with diﬀerent concentrations of PGN were mixed and stirred at 63 r/min for 1 min in kneading bowls; then, water was added in 25 s to bring the maximum consistency of the dough to approximately 500 FU. Afterwards the recording lasted 20 min. The water absorption rate, formation time, stability, and degree of weakening of the dough were obtained from the recorded curves. The maximum consistency was measured at the development time and in the middle of the curve band width. Each experimental condition contains three replicates of the sample. 2.8. Determination of the Content of Free Amino Groups The change in the content of free amino groups in the dough was determined by the reaction of primary amino groups and o-phthalaldehyde (OPA) according to the previous study [44]. Brieﬂy, 2.0 mL of HCl (0.1 M, pH 1.0) was added into 0.2 g of the dough sample and vortexed for 5 min. Then, the mixture was centrifuged at 9391 g for 10 min. The supernatant (0.1 mL) was mixed with 2.5 mL of OPA reagent. Each 100 mL of OPA reagent contained 50 ml of sodium tetraborate buﬀer (0.1 M, pH 9.5), 5 mL of 20% sodium dodecyl sulfate solution, 80 mg of OPA dissolved in 2 mL of 95% ethanol, and 0.2 mL of β-mercaptoethanol. The absorbance of the mixture of supernatant and OPA reagent was measured at 340 nm after 2 min of reaction. At least three replicates of dough samples at each experimental condition were determined. 2.9. Fourier Transform Infrared Spectroscopy (FT-IR) Characterization The dough powder sample was ground with potassium bromide at 1/100 ratio. Then, the mixture was pressed into a transparent sheet. Afterwards the air background and the samples were scanned within the range of 400–4000 cm−1 with an infrared spectrometer (Thermo Scientiﬁc, Waltham, MA, USA) (32 scans at 2 cm−1 resolution) at room temperature. At least three spectra of each condition were collected for spectra analyses. The spectra were analyzed with the software Ominc and Peak-Fit 4.12. The peaks at 1600–1700 cm−1 were assigned to amide I band; peaks at 1600–1625 and 1625–1640 cm−1 were assigned to intermolecular and intramolecular β-sheets [49]; peaks at 1644–1652 cm−1 were assigned to random coils; peaks at 1652–1660 cm−1 were assigned to α-helices [50]; peaks at 1660–1685 cm−1 were assigned to the β-turn [51]. 2.7. Measurement of Total and Exposed Free Sulfhydryl (SH) Content 2.7. Measurement of Total and Exposed Free Sulfhydryl (SH) Content 2.7. Measurement of Total and Exposed Free Sulfhydryl (SH) Content Free and total SH contents of dough samples supplemented with PNG were examined using Ellman’s reagents according to previous studies [46,47]. Two hundred milligrams of dough samples were suspended in 1.0 mL of Tris-gly buﬀer (0.086 M Tris, 0.09 M glycine, and 0.004 M EDTA, pH 8.0) to quantify exposed sulfhydryl. To quantify total sulfhydryl content, additional 8 M of guanidine hydrochloride was added into the above Tris-gly buﬀer. Afterwards the suspension was vortexed for 20 min and then centrifuged at 16,000 g for 5 min. Eighty microliter of supernatant was extracted and mixed with the corresponding Tris-gly buﬀer with or without 8 M of guanidine hydrochloride. Moreover, immediately 40 µL of DTNB working ﬂuid (Tris-gly buﬀer with 0.01 M 5,5’-Dithio bis-(2-nitrobenzoic acid)) was added into the mixture and incubated at 25 ◦C for 60 min. The absorbance of the mixture and the reagent buﬀer as the blank were read at 412 nm. At least three replicates of dough samples at each experimental condition were examined. Free SH content was calculated as follows: µM SH/g dough = (73.53 × A412 × D)/C (1) (1) where A412 is the absorbance at 412 nm, D is the dilution factor, C is the sample mass concentration (mg/mL), and 73.53 is derived from 106/(1.36 × 104), where 1.36 × 104 is the molar absorptivity, and 106 is for the calculation from M basis to µM /mL basis and from mg to g [48]. 2.6. Observation of Dough Microstructure The dough containing 2.5% of dry yeast (wt/wt) was slacked for 5 min or fermented for 90 min prior to the ﬂash freezing with liquid nitrogen. Afterwards the frozen dough was immediately crushed and placed in the lyophilizer at −40 ◦C and 0.1 Pa for 48 h. The surface of the dough section was sprayed with gold particles using an MCI000 ion Sputter (Hitachi, Tokyo, Japan) and the microstructure of the cross section was observed using the scanning electron microscope (SEM) (Hitachi, Tokyo, Japan) at 1000 magniﬁcation with a 10.0 kV acceleration voltage. Foods 2019, 8, 606 4 of 13 2.10. Statistical Analyses All tests and treatments were expressed as mean ± SD for at least triplicate determinations. The statistical software Origin 8.0 (Microcal Software, Northampton, MA, USA) and IBM SPSS Statistics 25 (SPSS, Chicago, IL, USA) were used for data analyses. The variance (ANOVA) method was applied to analyze signiﬁcant diﬀerence of the obtained data at p < 0.05. 5 of 13 Foods 2019, 8, 606 3. Results and Discussion 3.1. Impacts of PGN on the Mixing Properties of Dough As expected, the high-gluten dough had a higher mixing characteristics (Table 1) than low-gluten dough including larger water absorption (59.2 versus 58.3%, respectively), longer formation time (1.50 versus 1.47 min, respectively), greater stability time (3.93 versus 3.17 min, respectively) and smaller weakening degree (112.0 versus 120.3 FU, respectively). Apparently, the mixing properties of the dough was related with the gluten content in the ﬂour. Table 1. Impact of punicalagin on the mixing properties of the dough. Flour Add Amount (mg/g) Water Absorption Rate (%) Formation Time (min) Stability (min) Weakening Degree (FU) High-gluten 0 59.2 ± 0.10 a 1.50 ± 0.01 a 3.93 ± 0.15 a 112.0 ± 4.58 a 0.13 59.3 ± 0.25 a 1.83 ± 0.11 a 4.57 ± 0.72 a 91.7 ± 14.6 bc 0.26 59.3 ± 0.10 a 4.73 ± 1.05 b 5.57 ± 0.15 b 92.7 ± 7.37 b 0.39 59.3 ± 0.06 a 4.60 ± 0.44 b 5.80 ± 0.20 b 76.7 ± 4.93 bc 0.52 59.3 ± 0.06 a 4.73 ± 0.42 b 5.80 ± 0.36 b 76.0 ± 7.21 c Low-gluten 0 58.3 ± 0.20 a 1.47 ± 0.06 a 3.17 ± 0.32 a 120.3 ± 11 a 0.13 58.3 ± 0.06 a 1.63 ± 0.15 ab 3.30 ± 0.10 b 103.7 ± 17 a 0.26 58.2 ± 0.06 a 1.70 ± 0.20 ab 3.37 ± 0.21 bc 118.3 ± 4.5 a 0.39 58.3 ± 0.06 a 1.83 ± 0.35 c 3.83 ± 0.25 cd 103.3 ± 13 a 0.52 58.2 ± 0.10 a 1.57 ± 0.15 a 4.13 ± 0.38 d 78.67 ± 5.5 b Values followed by the same letter in the same column (within the same ﬂour) are not signiﬁcantly diﬀerent (p < 0.05). Two-ways ANOVA analysis: Water absorption rate: FFlour = 520.20, p < 0.05, FAddition = 0.24, p > 0.05, Finteraction = 0.53, pinteraction > 0.05; Formation time: FFlour = 149.95, p < 0.05, FAddition = 27.05, p < 0.05, Finteraction = 22.14, pinteraction < 0.05; Stability time: FFlour = 168.27, p < 0.05, FAddition = 19.38, p < 0.05, Finteraction = 4.42, pinteraction < 0.05; Weakening degree: FFlour = 17.03, p < 0.05, FAddition = 10.60, p < 0.05, Finteraction = 4.25, pinteraction < 0.05. Table 1. Impact of punicalagin on the mixing properties of the dough. 3. Results and Discussion However, the dough formation time and stability time signiﬁcantly increased with the addition of PGN. 3. Results and Discussion Values followed by the same letter in the same column (within the same ﬂour) are not signiﬁcantly diﬀerent (p < 0.05). Two-ways ANOVA analysis: Water absorption rate: FFlour = 520.20, p < 0.05, FAddition = 0.24, p > 0.05, Finteraction = 0.53, pinteraction > 0.05; Formation time: FFlour = 149.95, p < 0.05, FAddition = 27.05, p < 0.05, Finteraction = 22.14, pinteraction < 0.05; Stability time: FFlour = 168.27, p < 0.05, FAddition = 19.38, p < 0.05, Finteraction = 4.42, pinteraction < 0.05; Weakening degree: FFlour = 17.03, p < 0.05, FAddition = 10.60, p < 0.05, Finteraction = 4.25, pinteraction < 0.05. As it was shown in Table 1, PGN had no signiﬁcant eﬀect on the water absorption of wheat ﬂour. However, the dough formation time and stability time signiﬁcantly increased with the addition of PGN. At the same time, the weakening degree decreased. For example, the formation time of high-gluten ﬂour was increased by up to 215% from 1.50 to 4.73 min with the addition of 0.26 mg/g PGN. The formation time of low-gluten ﬂour was increased by up to 24.5% from 1.47 to 1.83 min with the addition of 0.39 mg/g PGN. With the increased addition of PGN, the stability time of the high-gluten dough was increased correspondingly, and it was increased by up to 47.6% from 3.93 to 5.80 min at the concentration of 0.52 mg/g. Meanwhile, the low-gluten ﬂour was increased by up to 30.3% from 3.17 to 4.13 min at the concentration of 0.52 mg/g. The weakening degree of the dough was decreased by up to 32.1% from 112.0 to 76.0 FU prepared with high-gluten ﬂour and by up to 34.6% from 120.3 to 78.67 FU prepared with low-gluten ﬂour in the presence of 0.52 mg/g PGN. The increase in the formation time and stability time indicated an increase in the tolerance of the dough, reﬂecting the enhancement of PGN on the dough quality. This was likely due to the covalent bonds and hydrogen bonds between PGN and gluten, which increased the strength of the dough [5,52]. However, the enhancement on the low-gluten ﬂour was not as pronounced as that in high-gluten ﬂour, which was likely due to the protein content in the ﬂour. As it was shown in Table 1, PGN had no signiﬁcant eﬀect on the water absorption of wheat ﬂour. stretched the gluten network via breaking disulfide bonds that resu [42]. 3.3. Impacts of PGN on Dough Viscoelasticity and Creep-Recovery Proﬁle 3.3. Impacts of PGN on Dough Viscoelasticity and Creep-Recovery Profile In the program of frequency sweep, for all treatments, G’ (elastic modulus) was higher than G’’ (viscous modulus), tan δ (G’’/G’) < 1 (data not reported), indicating that elasticity was the main feature of the dough, which was in line with recent reports [54]. According to Figure 2 (A and B for high-gluten flour, D and E for low-gluten flour), 0.26 mg/g-PGN-treated dough had the highest G’ and G’’ both in the high-gluten and low-gluten flour. The increase of G’ and G’’ indicated that PGN also increased the viscoelasticity of the dough, which was consistent with the increase of dough tensile properties (Figure 1). The creep and recovery response data curve in Figure 2 (C for high- gluten flour and F for low-gluten flour) showed the typical viscoelastic behavior of the dough. It has been reported that the elasticity of the dough was prominently improved by the addition of proanthocyanins of high molecular weight [42]. In this study, the viscosity and elasticity of the dough were increased at the low concentrations of PGN, which may due to the enhancement of cross-linking in PGN–gluten and protein–protein system [42]. With addition of PGN at high concentrations, the In the program of frequency sweep, for all treatments, G’ (elastic modulus) was higher than G” (viscous modulus), tan δ (G”/G’) < 1 (data not reported), indicating that elasticity was the main feature of the dough, which was in line with recent reports [54]. According to Figure 2 (A and B for high-gluten ﬂour, D and E for low-gluten ﬂour), 0.26 mg/g-PGN-treated dough had the highest G’ and G” both in the high-gluten and low-gluten ﬂour. The increase of G’ and G” indicated that PGN also increased the viscoelasticity of the dough, which was consistent with the increase of dough tensile properties (Figure 1). The creep and recovery response data curve in Figure 2 (C for high-gluten ﬂour and F for low-gluten ﬂour) showed the typical viscoelastic behavior of the dough. It has been reported that the elasticity of the dough was prominently improved by the addition of proanthocyanins of high molecular weight [42]. In this study, the viscosity and elasticity of the dough were increased at the low concentrations of PGN, which may due to the enhancement of cross-linking in PGN–gluten and protein–protein system [42]. 3.2. Impacts of PGN on the Stretching Properties of Dough Impact of punicalagin on the tensile properties of the dough (A: Stretch curve area of high- gluten dough; B: Tensile resistance of high-gluten dough; C: Extension of high-gluten dough; D: Stretch curve area of low-gluten dough; E: Tensile resistance of low-gluten dough; F: Extension of low-gluten dough). Different letters above bars indicate significant difference (p < 0.05) between t t t t i l l Figure 1. Impact of punicalagin on the tensile properties of the dough (A: Stretch curve area of high-gluten dough; B: Tensile resistance of high-gluten dough; C: Extension of high-gluten dough; D: Stretch curve area of low-gluten dough; E: Tensile resistance of low-gluten dough; F: Extension of low-gluten dough). Diﬀerent letters above bars indicate signiﬁcant diﬀerence (p < 0.05) between treatments at given level. According to Figure 1, PGN at low concentrations of 0.13 mg/g (in the low-gluten flour dough and 0.26 mg/g (in the low-gluten and high-gluten flour dough) increased the tensile resistance of the dough while it slightly increased the extension of the dough. This was likely due to dual actions o high molecular weight polyphenols. It strengthened the gluten structure via cross-linking, and t t h d th l t t k i b ki di lfid b d th t lt d f ti id t ti According to Figure 1, PGN at low concentrations of 0.13 mg/g (in the low-gluten ﬂour dough) and 0.26 mg/g (in the low-gluten and high-gluten ﬂour dough) increased the tensile resistance of the dough while it slightly increased the extension of the dough. This was likely due to dual actions of high molecular weight polyphenols. It strengthened the gluten structure via cross-linking, and stretched the gluten network via breaking disulﬁde bonds that resulted from antioxidant properties [42]. According to Figure 1, PGN at low concentrations of 0.13 mg/g (in the low-gluten flour dough) and 0.26 mg/g (in the low-gluten and high-gluten flour dough) increased the tensile resistance of the dough while it slightly increased the extension of the dough. This was likely due to dual actions of high molecular weight polyphenols. 3.2. Impacts of PGN on the Stretching Properties of Dough It strengthened the gluten structure via cross-linking, and stretched the gluten network ia breaking disulfide bonds that resulted from antioxidant properties According to Figure 1, PGN at low concentrations of 0.13 mg/g (in the low-gluten ﬂour dough) and 0.26 mg/g (in the low-gluten and high-gluten ﬂour dough) increased the tensile resistance of the dough while it slightly increased the extension of the dough. This was likely due to dual actions of high molecular weight polyphenols. It strengthened the gluten structure via cross-linking, and stretched the gluten network via breaking disulﬁde bonds that resulted from antioxidant properties [42]. 3.2. Impacts of PGN on the Stretching Properties of Dough The stretch curve area (Figure 1A for high-gluten ﬂour and D for low-gluten ﬂour) and tensile resistance of the dough (Figure 1B for high-gluten ﬂour and E for low-gluten ﬂour) were increased with the addition of 0.13 and 0.26 mg/g of PGN. However, the stretch curve area and the tensile resistance of the dough with 0.39 and 0.52 mg/g of PGN were all dropped in both ﬂours. It was assumed that the strong antioxidant properties of PGN at high concentrations (0.39 and 0.52 mg/g) [53] may induce interchanges of sulfhydryl groups and may cause the excessive loss of the bound water in 6 of 13 Foods 2019, 8, 606 the gluten–PGN system, thereby aﬀecting the secondary structure of the gluten and weakening the dough [51]. Foods 2019, 8, x FOR PEER REVIEW 6 of 13 Figure 1. Impact of punicalagin on the tensile properties of the dough (A: Stretch curve area of high- gluten dough; B: Tensile resistance of high-gluten dough; C: Extension of high-gluten dough; D: Stretch curve area of low-gluten dough; E: Tensile resistance of low-gluten dough; F: Extension of low-gluten dough). Different letters above bars indicate significant difference (p < 0.05) between treatments at given level Figure 1. Impact of punicalagin on the tensile properties of the dough (A: Stretch curve area of high-gluten dough; B: Tensile resistance of high-gluten dough; C: Extension of high-gluten dough; D: Stretch curve area of low-gluten dough; E: Tensile resistance of low-gluten dough; F: Extension of low-gluten dough). Diﬀerent letters above bars indicate signiﬁcant diﬀerence (p < 0.05) between treatments at given level. Figure 1. Impact of punicalagin on the tensile properties of the dough (A: Stretch curve area of high- gluten dough; B: Tensile resistance of high-gluten dough; C: Extension of high-gluten dough; D: Stretch curve area of low-gluten dough; E: Tensile resistance of low-gluten dough; F: Extension of low-gluten dough). Different letters above bars indicate significant difference (p < 0.05) between t t t t i l l Figure 1. Impact of punicalagin on the tensile properties of the dough (A: Stretch curve area of high-gluten dough; B: Tensile resistance of high-gluten dough; C: Extension of high-gluten dough; D: Stretch curve area of low-gluten dough; E: Tensile resistance of low-gluten dough; F: Extension of low-gluten dough). Diﬀerent letters above bars indicate signiﬁcant diﬀerence (p < 0.05) between treatments at given level. Figure 1. g g ) 3.4. Impacts of PGN on Dough Microstructure 3.4. Impacts of PGN on Dough Microstructure As it was shown in Figure 3, the dough starch granules without PGN were exposed on the surface and no apparent gluten network structure was observed. With the addition of 0.13 mg/g of PGN, the pores within gluten were less compared to the control group. With the addition of 0.26 mg/g of PGN, a tightly ordered gluten network structure was observed and the starch granules were evenly and orderly distributed in the gluten network structure (Figure 3). Meanwhile, the network structure of the high-gluten flour dough was more compact and well-aligned compared to that of the low-gluten flour dough (Figure 3) with the addition of the same amount of PGN. With the addition of 0.39 and 0.52 mg/g of PGN, significant cleavages were observed in the gluten network structure, which was consistent with the previous results that both tensile resistance and extension of the dough were decreased at the same concentration of PGN. According to SEM of the dough after fermentation, the gluten network structure was partially broken with the addition of 0.39 mg/g of PGN. The breakage was enlarged, and cleavages appeared with the addition of 0.52 mg/g of PGN. These cleavages reduced the gas hold capacity of the dough and directly affected the volume of the end product bread [55]. Taken together, PGN at low concentrations of 0.13 and 0.26 mg/g promoted the formation of gluten network while PGN at high concentrations of 0.39 and 0.52 mg/g caused the formation of cleavages in the gluten network. This was probably because the strengthened cross- linking prevailed at low concentrations and reducing potentials prevailed at high concentrations as well as the dehydration of the gluten network. Moreover, the impacts of PGN on high-gluten flour was more significant than that on low-gluten flour. This was likely due to the cross-linking between As it was shown in Figure 3, the dough starch granules without PGN were exposed on the surface and no apparent gluten network structure was observed. With the addition of 0.13 mg/g of PGN, the pores within gluten were less compared to the control group. With the addition of 0.26 mg/g of PGN, a tightly ordered gluten network structure was observed and the starch granules were evenly and orderly distributed in the gluten network structure (Figure 3). stretched the gluten network via breaking disulfide bonds that resu [42]. 3.3. Impacts of PGN on Dough Viscoelasticity and Creep-Recovery Proﬁle With addition of PGN at high concentrations, the viscoelasticity of the dough was decreased compared to that with addition of PGN at low concentrations due to the reducing potential of PGN in the weakening of the gluten network. 7 of 13 7 of 13 Foods 2019, 8, 606 Foods 2019, 8, x F Figure 2. Impact of punicalagin (PGN) on rheology of the dough. (A: Elastic modulus G’ of high- gluten dough; B: Viscous modulus G’’ of high-gluten dough; C: Compliance of high-gluten dough; D: Elastic modulus G’ of low-gluten dough; E: Viscous modulus G’’ of low-gluten dough; F: Compliance of low-gluten dough) Figure 2. Impact of punicalagin (PGN) on rheology of the dough. (A: Elastic modulus G’ of high-gluten dough; B: Viscous modulus G” of high-gluten dough; C: Compliance of high-gluten dough; D: Elastic modulus G’ of low-gluten dough; E: Viscous modulus G” of low-gluten dough; F: Compliance of low-gluten dough). Figure 2. Impact of punicalagin (PGN) on rheology of the dough. (A: Elastic modulus G’ of high- gluten dough; B: Viscous modulus G’’ of high-gluten dough; C: Compliance of high-gluten dough; D: Elastic modulus G’ of low-gluten dough; E: Viscous modulus G’’ of low-gluten dough; F: Compliance f l l t d h) Figure 2. Impact of punicalagin (PGN) on rheology of the dough. (A: Elastic modulus G’ of high-gluten dough; B: Viscous modulus G” of high-gluten dough; C: Compliance of high-gluten dough; D: Elastic modulus G’ of low-gluten dough; E: Viscous modulus G” of low-gluten dough; F: Compliance of low-gluten dough). g g ) 3.4. Impacts of PGN on Dough Microstructure Analyses of Total and Exposed Free Sulfhydryl (SH) and Free Amino Groups Contents According to Table 2, upon the increase of the amount of PGN, the content of free SH and total SH increased. The increase in sulfhydryl content may due to the strong antioxidant properties of PGN destroying the original disulﬁde bond between gluten proteins. Both ﬂour type and addition of PGN impacts the interchanges of sulfhydryl groups. Table 2. Impact of PGN on the content of sulfhydryl groups (SH) and free amino groups in dough. Flour Add Amount (mg/g) Free SH (µM/g dough) Total SH (µM/g dough) Free Amino Groups Absorbance High-gluten 0 0.38 ± 0.005 a 0.57 ± 0.012 a 0.051 ± 0.008 b 0.13 0.39 ± 0.005 a 0.62 ± 0.014 b 0.072 ± 0.001 c 0.26 0.41 ± 0.003 bc 0.64 ± 0.004 bc 0.070 ± 0.012 c 0.39 0.43 ± 0.013 c 0.66 ± 0.005 c 0.069 ± 0.007 c 0.52 0.40 ± 0.002 ab 0.77 ± 0.018 d 0.035 ± 0.008 a Low-gluten 0 0.38 ± 0.007 a 0.56 ± 0.006 a 0.047 ± 0.005 a 0.13 0.38 ± 0.004 ab 0.60 ± 0.002 a 0.071 ± 0.006 b 0.26 0.39 ± 0.001 bc 0.60 ± 0.005 a 0.072 ± 0.017 b 0.39 0.39 ± 0.002 bc 0.71 ± 0.035 b 0.050 ± 0.011 a 0.52 0.40 ± 0.009 c 0.71 ± 0.024 b 0.047 ± 0.015 a Values followed by the same letter in the same column (within the same ﬂour) are not signiﬁcantly diﬀerent (p < 0.05). Two-ways ANOVA analysis: Free SH: FFlour = 33.71, p < 0.05, FAddition = 16.17, p < 0.05, Finteraction = 6.34, pinteraction < 0.05; Total SH: FFlour = 5.34, p < 0.05, FAddition = 71.89, p < 0.05, Finteraction = 6.23, pinteraction < 0.05; free amino groups: FFlour = 1050.95, p < 0.05, FAddition = 1042.49, p < 0.05, Finteraction = 417.25, pinteraction < 0.05. Table 2. Impact of PGN on the content of sulfhydryl groups (SH) and free amino groups in dough. As shown in Table 2, the content of free amino groups was increased in both types of ﬂours with the addition of 0.13 and 0.26 mg/g PGN. Meanwhile it was decreased signiﬁcantly with the addition of 0.52 mg/g of PGN in high-gluten ﬂour and decreased signiﬁcantly with 0.39 and 0.52 mg/g of PGN in low-gluten ﬂour. g g ) 3.4. Impacts of PGN on Dough Microstructure Meanwhile, the network structure of the high-gluten ﬂour dough was more compact and well-aligned compared to that of the low-gluten ﬂour dough (Figure 3) with the addition of the same amount of PGN. With the addition of 0.39 and 0.52 mg/g of PGN, signiﬁcant cleavages were observed in the gluten network structure, which was consistent with the previous results that both tensile resistance and extension of the dough were decreased at the same concentration of PGN. According to SEM of the dough after fermentation, the gluten network structure was partially broken with the addition of 0.39 mg/g of PGN. The breakage was enlarged, and cleavages appeared with the addition of 0.52 mg/g of PGN. These cleavages reduced the gas hold capacity of the dough and directly aﬀected the volume of the end product bread [55]. Taken together, PGN at low concentrations of 0.13 and 0.26 mg/g promoted the formation of gluten network while PGN at high concentrations of 0.39 and 0.52 mg/g caused the formation of cleavages in the gluten network. This was probably because the strengthened cross-linking prevailed at low concentrations and reducing potentials prevailed at high concentrations as well as the dehydration of the gluten network. Moreover, the impacts of PGN on high-gluten ﬂour was more signiﬁcant than that on low-gluten ﬂour. This was likely due to the cross-linking between PGN and proteins in ﬂours. Higher content of proteins facilitated the formation of a tighter gluten network. 8 of 13 Foods 2019, 8, 606 Figure 3. Impact of PGN on the microstructure of the dough cross section examined by scanning electron microscopy. Figure 3. Impact of PGN on the microstructure of the dough cross section examined by scanning electron microscopy. Figure 3 Impact of PGN on the microstructure of the dough cross section examined by scanning electron microscopy Figure 3 Impact of PGN on the microstructure of the dough cross section examined by scanning electron microscopy PGN on the microstructure of the dough cross section examined by scanning electron microscopy. Figure 3. Impact of PGN on the microstructure of the dough cross section examined by scanning electron microscopy. Foods 2019, 8, 606 9 of 13 3.5. Analyses of Total and Exposed Free Sulfhydryl (SH) and Free Amino Groups Contents 3.5. g g ) 3.4. Impacts of PGN on Dough Microstructure It was assumed that the amount of amino groups was impacted by the release of water via dehydration and the cross-linking with PGN as well as gluten proteins. 3.6. Impacts of PGN on Protein Secondary Structure 3.6. Impacts of PGN on Protein Secondary Structure The texture characteristics of wheat dough mainly depended on the balance of weak and strong physical connections, such as hydrogen bonds, hydrophobic interactions, electrostatic forces, covalent bonds, and disulﬁde bonds [56]. As the addition of PGN was increased, α-helix, β-sheet, and random coil were increased, while β-turn was decreased (Table 3), suggesting β-turn was converted to α-helix, β-sheet, and random coil. In general, β-sheets were considered to be the most stable secondary structure, and an increase in the formation of α-helix would result in a more ordered structure [57]. As shown in Table 3, the structure of β-sheets and α-helix increased with the addition of PGN, indicating a more stable and well-aligned gluten secondary structure. This result was consistent with previous data that PGN at low concentrations enhanced the gluten network structure and improved dough rheological properties. However, the stability of the structure was not linearly correlated with the amount of PGN addition. This was likely correlated with the moderate dehydration of gluten caused by PGN at low concentration, which promoted the conversion of β-turn to β-sheet and stabilized the network structure. At high concentrations the reducing potential of PGN prevailed and may cause the decrease of β-sheet content [58]. Moreover the application of high amount of PGN may release water from hydrogen-bonded cluster and cause the dehydration of dough, thus weakening the gluten mesh [51]. 10 of 13 Foods 2019, 8, 606 Table 3. Impact of PGN on the secondary structure of dough proteins. Table 3. Impact of PGN on the secondary structure of dough proteins. 3.6. Impacts of PGN on Protein Secondary Structure Flour Add Amount (mg/g) α-helix (%) β-sheet (%) β-turn (%) Random Coil (%) High-gluten 0 14.98 ± 0.05 a 29.90 ± 0.04 a 40.70 ± 0.07 a 14.43 ± 0.01 a 0.13 15.83 ± 0.01 d 31.84 ± 0.05 b 37.78 ± 0.06 b 14.56 ± 0.05 b 0.26 15.52 ± 0.14 bc 32.35 ± 0.12 d 37.11 ± 0.16 d 15.03 ± 0.12 d 0.39 15.64 ± 0.04 c 32.04 ± 0.08 c 37.58 ± 0.08 c 14.74 ± 0.02 c 0.52 15.41 ± 0.02 b 32.27 ± 0.09 d 37.43 ± 0.08 c 14.88 ± 0.05 d Low-gluten 0 14.81 ± 0.16 a 29.21 ± 0.17 a 41.89 ± 0.15 a 14.10 ± 0.13 a 0.13 15.56 ± 0.07 c 32.73 ± 0.05 e 36.90 ± 0.09 d 14.81 ± 0.04 b 0.26 15.38 ± 0.06 b 32.26 ± 0.05 d 37.33 ± 0.03 c 15.04 ± 0.03 c 0.39 15.56 ± 0.07 c 31.91 ± 0.05 c 37.75 ± 0.02 b 14.78 ± 0.06 b 0.52 15.69 ± 0.05 d 31.37 ± 0.08 b 37.76 ± 0.03 b 15.18 ± 0.04 d Values followed by the same letter in the same column (within the same ﬂour) are not signiﬁcantly diﬀerent (p < 0.05). Two-ways ANOVA analysis: α-helix: FFlour = 4.11, p > 0.05, FAddition = 56.68, p < 0.05, Finteraction = 6.31, pinteraction < 0.05; β-sheet: FFlour = 17.96, p < 0.05, FAddition = 609.39, p < 0.05, Finteraction = 56.26, pinteraction < 0.05; β-turn: FFlour = 28.16, p < 0.05, FAddition = 1595.06, p < 0.05, Finteraction = 72.45, pinteraction < 0.05; random coil: FFlour = 2.17, p > 0.05, FAddition = 75.98, p < 0.05, Finteraction = 11.59, pinteraction < 0.05. Values followed by the same letter in the same column (within the same ﬂour) are not signiﬁcantly diﬀerent (p < 0.05). Two-ways ANOVA analysis: α-helix: FFlour = 4.11, p > 0.05, FAddition = 56.68, p < 0.05, Finteraction = 6.31, pinteraction < 0.05; β-sheet: FFlour = 17.96, p < 0.05, FAddition = 609.39, p < 0.05, Finteraction = 56.26, pinteraction < 0.05; β-turn: FFlour = 28.16, p < 0.05, FAddition = 1595.06, p < 0.05, Finteraction = 72.45, pinteraction < 0.05; random coil: FFlour = 2.17, p > 0.05, FAddition = 75.98, p < 0.05, Finteraction = 11.59, pinteraction < 0.05. 4. Conclusions In the present study, PGN increased the formation time of high-gluten ﬂour dough by up to 215% and the low-gluten ﬂour dough by up to 24.5%. It increased the stability time of the dough prepared with high-gluten ﬂour maximally by 47.6% and that with the low-gluten ﬂour maximally by 30.3%. PGN at low concentrations of 0.13 and 0.26 mg/g increased the tensile resistance, extension capacity, and rheological properties of the dough. Among them, the most prominent increase was observed for the dough with 0.26 mg/g of PGN, where stretching resistances were increased by 1.4% and 15.5% for the high- and low-gluten ﬂour dough respectively; extensions were increased by 9.3% and 15.4% for the high-gluten and low-gluten ﬂour dough, respectively. The morphology of cross section of the dough appeared a more compact and ordered network structure in the presence of PGN at low concentrations. These characteristics can be explained by the increased content of α-helix and β-sheet possibly via the water redistribution and cross-linking in PGN-gluten system. These nonlinear correlation of PGN addition and dough quality was unlike most polyphenols or traditional gluten strengthening agents. Too high quantity of PGN in the dough may physically disrupt the gas cells and gluten network producing poor quality of bread and noodles. Taken together these results indicated that PGN can be used as a ﬂour enhancer of natural origin. The suitable concentrations for the application of PGN in the dough were around 0.26 and below 0.39 mg/g. The enhancing eﬀects of PGN were more prominent in the high-gluten ﬂour. Author Contributions: Conceptualization, J.T.; Methodology, H.P. and J.T.; Observations and analysis, H.P., B.L., and J.T.; Data curation, H.P.; Writing—original draft preparation, H.P.; Writing—review and editing, J.T.; Supervision, J.T.; Project administration, J.T.; Funding acquisition, J.T. Funding: The work was ﬁnancially supported by the Natural Science Foundation of Hubei Province (2018CFB379), Fundamental Research Funds for the Central Universities (2662017QD006) and Postdoctoral Innovative Research Project of Hubei Province. Conﬂicts of Interest: The authors declare no conﬂict of interest. 1. Shewry, P.R.; Halford, N.G.; Belton, P.S.; Tatham, A.S. The structure and properties of gluten: An elastic protein from wheat grain. Philos. Trans. R. Soc. Lond. Ser. B-Biol. Sci. 2002, 357, 133–142. 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W4396752194.txt	https://ojs.boulibrary.com/index.php/JAIGS/article/download/96/64	en		Revolutionizing Healthcare: The Role of Machine Learning in the Health Sector	Deleted Journal	2,024	cc-by	6,249	Vol.2,Issue1,January 2024 Journal of Artificial Intelligence General Science JAIGS Home page http://jaigs.org Revolutionizing Healthcare: The Role of Machine Learning in theHealth Sector Mithun Sarker1 1 Independent Researcher Beaumont, Texas, United States Abstract Traditional healthcare systems have grappled with meeting the diverse needs of millions of patients, resulting in inefficiencies inefficienc and suboptimal outcomes. However, the emergence of machine learning (ML) has ushered in a transformative paradigm shift towards value-based based treatment, empowering healthcare providers to deliver personalized and highly effective care. Modern healthcare equipment and devices now integrate internal applications that collect and store comprehensive patient data, providing a rich resource for ML-driven driven predictive models. In this research article, we explore the profound impact of ML on contemporary healthcare, emphasizing its potential to significantly enhance patient care and optimize resource allocation. Our Ou study presents a robust predictive edictive model capable of accurately forecasting patient diseases based on input information and various parameters, leveraging extensive datasets encompassing diverse patient populations. We compared several ML algorithms, including Logistic Regression (a (accuracy: 0.796875), K-Nearest Nearest Neighbors (accuracy: 0.7864583333333334), XG Boost (accuracy: 0.78125), and PyTorch (accuracy: 0.7337662337662337), to identify the best best-performing performing model. The achieved accuracies underscore the effectiveness of these ML techni techniques ques in disease prediction and underscore the potential for improving patient outcomes. Beyond the technical aspects, we explore the broader implications of value value-based based treatment and the integration of ML for various healthcare stakeholders. By emphasizing the numerous benefits of personalized and proactive medical care, our findings illustrate the substantial potential of ML ML-driven driven predictive healthcare models to revolutionize traditional healthcare systems. The adoption of ML in healthcare lays the founda foundation tion for a more efficient, effective, and patient-centered patient medical ecosystem, supporting the sustainability and adaptability of healthcare systems in the face of expanding patient populations and complex medical needs. This article significantly contribute contributess to the field by providing comprehensive insights into the experimental stages, showcasing the achieved results, and highlighting the key conclusions derived from our study. By addressing the limitations of the previous abstract, we ensure a more informat informative ive and substantial overview of our research, offering valuable knowledge for researchers, practitioners, and decision decision-makers makers striving to leverage the power of ML in healthcare innovation. Keywords: Machine learning, Modern healthcare, Value Value-based treatment, Predictive models Article Information Article History: Received Date: 01.02.2024 Accepted Date:10.02.2024 Online Date: 27.02.2024 Published Date: 27.02.2024 Correspondence Author: MithunSarker ISSN:3006-4023 (Online),JournalofArtificialIntelligence GeneralScience (JAIGS)37 INTRODUCTION The integration of machine learning techniques in healthcare has garnered significant attention in recent years, offering the potential to revolutionize traditional systems and elevate value-based treatment. One particularly promising application lies in disease prediction, with diabetes serving as a prime example due to its widespread prevalence and chronic nature affecting millions globally. Timely detection and accurate prediction of diabetes can profoundly impact patient outcomes by facilitating prompt interventions, tailored treatment plans, and enhanced disease management strategies. This research article endeavors to construct a robust machine-learning model for diabetes prediction using a comprehensive dataset. By harnessing the capabilities of machine learning algorithms, our aim is to develop a predictive model capable of reliably identifying individuals at risk of developing diabetes. Such a model holds the promise of aiding healthcare providers in making informed decisions, implementing preventive measures, and ultimately enhancing patient care while alleviating the burden of the disease. The research problem centers on predicting diabetes based on a spectrum of patient attributes and clinical measurements. Diabetes, being a complex and multifactorial ailment influenced by variables such as age, gender, body mass index (BMI), blood pressure, glucose levels, and family history, necessitates a holistic approach to modeling. By encompassing these diverse factors, our objective is to construct a model that captures the intricacies of the disease and delivers dependable predictions. The significance of this research lies in its potential to bolster early detection and prevention efforts in diabetes. Identifying individuals at risk empowers healthcare professionals to intervene proactively, implementing lifestyle modifications, recommending appropriate screenings, and initiating timely treatments. Furthermore, precise diabetes prediction can pave the way for the development of personalized treatment plans tailored to each patient's specific needs, thereby fostering improved outcomes and more efficient resource allocation within healthcare systems. To achieve our research objective, we will employ a range of machine learning techniques, including logistic regression, k-nearest neighbors, gradient boosting, PyTorch, and neural networks. These algorithms have 38Mithun Sarker demonstrated promise in healthcare applications and possess the requisite capabilities to handle complex datasets and deliver accurate predictions. Through thorough evaluation and comparison of their performance, we aim to identify the most effective algorithm for diabetes prediction. The structure of this research article is as follows: the subsequent section will conduct a comprehensive literature survey, reviewing existing studies on machine learning in healthcare and diabetes prediction, thereby establishing the research gap and underscoring the need for further investigation. Following the literature survey, we will delineate the research methodology, encompassing the dataset utilized, data preprocessing techniques, and implementation details of the machine learning algorithms. The results section will present the evaluation metrics and performance of each algorithm, elucidating the strengths and weaknesses of the models. Subsequently, the discussion section will offer insights into the findings, exploring the implications of the results and identifying potential areas for refinement. Finally, the conclusion will encapsulate the key findings of the research, underscore its significance, and propose avenues for future research to build upon this work. In summary, this research article endeavors to develop a robust machine-learning model for diabetes prediction, leveraging a comprehensive dataset and cutting-edge techniques. The outcomes of this research have the potential to revolutionize diabetes management by facilitating early detection, personalized treatment, and improved patient outcomes. By amalgamating the power of machine learning with the wealth of healthcare data available, we aim to contribute to the ongoing transformation of traditional healthcare systems and the advancement of value-based treatment. LITERATURE REVIEW Beam and Kohane (2018) [1] explore the intersection of big data and machine learning in healthcare in their paper "Big Data and Machine Learning in Health Care" published in the Journal of the American Medical Association (JAMA). They emphasize the significance of large-scale datasets and advanced computational methods in enhancing patient care, discussing sources of big data such as electronic health records and medical imaging. The authors illustrate how machine learning algorithms can analyze these datasets to discern patterns, predict outcomes, and aid clinical decision-making, while also addressing challenges related to data management and privacy. ISSN:3006-4023 (Online),JournalofArtificialIntelligence GeneralScience (JAIGS)39 Deo (2015) [2] discusses the applications of machine learning in medicine in the paper "Machine Learning in Medicine" published in the journal Circulation. Delving into various medical domains, Deo underscores the role of machine learning in risk prediction, disease diagnosis, treatment selection, and patient monitoring. Despite highlighting the potential benefits, the author acknowledges challenges like data quality and interpretability, offering insights into the ethical considerations of machine learning in healthcare. Esteva et al. (2019) [3] provide a comprehensive guide to deep learning in healthcare in their paper "A Guide to Deep Learning in Healthcare" published in Nature Medicine. They elucidate fundamental concepts and methodologies of deep learning, showcasing its potential across domains like image analysis, genomics, and drug discovery. The authors discuss the capability of deep learning to capture complex patterns but also address concerns regarding data quality and ethical implications. Johnson et al. (2018) [4] investigate the role of artificial intelligence (AI) in cardiology in their study "Artificial Intelligence in Cardiology" published in the Journal of the American College of Cardiology. They explore AI's potential in improving risk prediction, diagnosis, and treatment selection in cardiology, focusing on areas like imaging analysis and risk stratification. The study emphasizes challenges related to data quality and regulatory considerations in implementing AI in cardiology. Krittanawong et al. (2017) [5] discuss AI's applications in precision cardiovascular medicine in their study "Artificial Intelligence in Precision Cardiovascular Medicine" published in the Journal of the American College of Cardiology. They highlight AI's role in personalized risk assessment and targeted treatment strategies for cardiovascular diseases, addressing applications like risk prediction and image analysis. The study also considers challenges such as data quality and interpretability in clinical practice. Obermeyer and Emanuel (2016) [6] examine the implications of big data and machine learning in clinical medicine in their article "Predicting the Future - Big Data, Machine Learning, and Clinical Medicine" published in The New 40Mithun Sarker England Journal of Medicine. They discuss how predictive analytics can augment clinical decision-making but also caution against pitfalls such as algorithmic bias and privacy concerns. These studies collectively underscore the transformative potential of machine learning and AI in healthcare while acknowledging challenges related to data quality, interpretability, and ethical considerations. The authors delve into the transformative potential of big data and machine learning in clinical medicine, highlighting the utilization of electronic health records, medical imaging data, and wearable devices to enhance clinical decision-making and patient care. They underscore the importance of employing machine learning algorithms to analyze vast datasets and discern patterns that can lead to more accurate predictions of individual patient outcomes. However, they also acknowledge challenges related to privacy protection, algorithmic bias, and the integration of these technologies into clinical practice. Overall, the article underscores the potential of big data and machine learning to revolutionize clinical medicine while stressing the importance of addressing ethical and practical concerns. Rajkomar, Dean, and Kohane (2019) [7] provide a comprehensive review of the applications of machine learning in medicine in their article "Machine Learning in Medicine" published in The New England Journal of Medicine. They explore how machine learning algorithms can analyze diverse datasets, including electronic health records, medical images, and genetic data, to improve diagnosis, treatment, and patient outcomes across various medical domains. The authors also discuss challenges such as data quality, interpretability, and regulatory considerations associated with integrating machine learning models into clinical practice, offering insights into the future potential of this technology in transforming healthcare. Ravi et al. (2017) [8] discuss the applications of deep learning in health informatics in their paper "Deep Learning for Health Informatics" published in the IEEE Journal of Biomedical and Health Informatics. They examine how deep learning algorithms, particularly convolutional neural networks (CNNs) and recurrent neural networks (RNNs), can extract meaningful information from diverse health-related data sources, enabling accurate disease diagnosis, personalized treatment planning, and predictive analytics. The authors also address challenges such as data privacy, ISSN:3006-4023 (Online),JournalofArtificialIntelligence GeneralScience (JAIGS)41 interpretability, and scalability associated with applying deep learning techniques in healthcare, providing a comprehensive overview of the benefits and limitations of deep learning in health informatics. Topol (2019) [9] explores the convergence of human and artificial intelligence (AI) in medicine in the paper "HighPerformance Medicine: The Convergence of Human and Artificial Intelligence" published in Nature Medicine. The author discusses the potential of AI to augment human capabilities and revolutionize healthcare delivery across various applications such as disease diagnosis, drug discovery, patient monitoring, and precision medicine. The article offers insights into the synergistic potential of human and artificial intelligence in advancing highperformance medicine and improving patient outcomes. Weng et al. (2017) [10] conducted a study titled "Enhancing Cardiovascular Risk Prediction Using Machine Learning on Routine Clinical Data," published in PLoS ONE. Their research explores the potential of machine learning in improving cardiovascular risk prediction by leveraging routine clinical data. Utilizing a substantial dataset of electronic health records, the study demonstrates that machine learning techniques outperform traditional risk prediction algorithms, showcasing improved accuracy in predicting cardiovascular risk. This study underscores the promising role of machine learning and routine clinical data in enhancing risk prediction models and advancing patient care. METHODOLOGY To investigate the potential of machine learning in enhancing value-based treatment within modern healthcare, we conducted a systematic exploration involving the development, validation, and analysis of a predictive model. Our methodology comprised the following steps: A. Data Collection and Preprocessing: We amassed a substantial dataset from diverse sources, including electronic health records (EHRs), medical imaging databases, and wearable health monitoring devices. This dataset underwent meticulous curation to ensure representation across varied patient demographics, vital for robust model training. Data preprocessing involved tasks 42Mithun Sarker such as cleaning, handling missing values, and normalizing continuous variables. Categorical variables underwent transformation using one-hot encoding to facilitate integration into the machine-learning model. B. Feature Selection and Engineering: Relevant features for our predictive model were identified through an exhaustive literature review and expert consultation to ascertain key factors influencing disease prediction. Additionally, feature engineering was conducted to generate new variables by amalgamating existing features or applying transformations to better capture relationships between input data and target outcomes. C. Model Development: Utilizing the preprocessed and feature-engineered dataset, we explored multiple machine learning algorithms, including logistic regression, support vector machines, random forests, and neural networks. K-fold cross-validation was employed to assess model performance and prevent overfitting. The algorithm exhibiting superior performance metrics was chosen as our final predictive model. D. Model Evaluation: Performance evaluation of our selected model was conducted using various metrics such as accuracy, precision, recall, F1 score, and area under the receiver operating characteristic (ROC) curve. Additionally, validation on an independent dataset was performed to ascertain the model's generalizability and robustness in real-world clinical scenarios. E. Model Interpretability: To enhance the interpretability of our model and gain insights into its decision-making process, we employed techniques such as feature importance analysis, SHAP (SHapley Additive exPlanations) values, and partial dependence plots. These methods facilitated understanding of the factors influencing predictions and provided valuable insights for clinicians and stakeholders. F. Ethical Considerations and Data Privacy: ISSN:3006-4023 (Online),JournalofArtificialIntelligence GeneralScience (JAIGS)43 Adherence to ethical guidelines and data privacy regulations was paramount throughout our research endeavor to uphold the confidentiality and integrity of patient data. All data utilized in this study underwent stringent anonymization and aggregation processes, and requisite approvals from institutional review boards were obtained before initiating the research. By meticulously following this systematic methodology, our aim was to offer a holistic insight into the transformative potential of machine learning in augmenting modern healthcare and valuebased treatment paradigms. Furthermore, our objective encompassed the development and evaluation of a robust predictive model for disease prediction, thereby contributing to the progression of personalized medicine. The objective of this project is to devise a system that overcomes the constraints associated with conventional diagnostic methods by furnishing precise predictions regarding the presence or absence of diabetes in patients. The proposed system consists of several integral components. Initially, pertinent datasets containing patient data concerning diabetes are identified and subjected to preprocessing procedures. This involves meticulous data cleaning, normalization, and extraction of pertinent features. Subsequently, feature selection methodologies are employed to discern the most informative variables for diabetes prediction, while feature engineering techniques are utilized to bolster the predictive prowess of the machine learning (ML) model. A plethora of ML algorithms, encompassing logistic regression, decision trees, support vector machines, random forests, and neural networks, are explored to formulate the diabetes prediction model. The optimal algorithm is 44Mithun Sarker trained on the preprocessed dataset utilizing techniques such as cross-validation, and hyperparameter tuning is conducted to refine the model's accuracy. The efficacy of the ML model is gauged using metrics such as accuracy, precision, recall, and F1-score. Its ability to generalize is scrutinized through cross-validation and validation on independent datasets. To ascertain its efficacy, the proposed system is juxtaposed against existing solutions, including traditional diagnostic methods and other ML-based approaches. This comparative analysis aids in delineating the strengths, weaknesses, and potential areas for enhancement of the proposed system. The discourse also encompasses future avenues for ML-based diabetes diagnosis, such as the integration of deep learning techniques and the assimilation of supplementary data sources. Moreover, the limitations of the proposed system, including data availability, sample size, and potential biases, are duly acknowledged and addressed. RESULTS Fig 2: Sample Rows from the Dataset In this study, we conducted experiments utilizing various machine learning techniques to predict diabetes, including logistic regression, k-nearest neighbors (KNN), gradient boosting, PyTorch, and neural networks. Our aim was to identify the most accurate and effective approach for diagnosing diabetes using the provided dataset. ISSN:3006-4023 (Online),JournalofArtificialIntelligence GeneralScience (JAIGS)45 We trained and assessed these models using a comprehensive dataset comprising patient demographics, medical history, and clinical variables. The dataset underwent preprocessing to address missing values, normalize features, and ensure suitability for model training and evaluation. Among the tested techniques, logistic regression emerged as the top-performing model for diabetes prediction. Logistic regression, a classical and widely-utilized classification algorithm, estimates the probability of an instance belonging to a particular class. Renowned for its simplicity, interpretability, and capacity to handle categorical and continuous variables effectively. The logistic regression model exhibited the highest accuracy in predicting the presence or absence of diabetes within the dataset, achieving an accuracy of 79.69%, precision, and an F1-score of 0.6486486486486487. These metrics indicate the model's proficiency in accurately classifying both positive (diabetic) and negative (non-diabetic) instances The superior performance of logistic regression can be attributed to its ability to discern the underlying relationships between the input variables and the target variable (diabetes status). By estimating coefficients for each input variable, logistic regression identifies influential features and assigns appropriate weights, resulting in a robust predictive model. While other techniques such as KNN, gradient boosting, PyTorch, and neural networks were explored, they did not surpass the accuracy achieved by logistic regression with this specific dataset. This underscores the importance of selecting the appropriate algorithm based on the dataset's characteristics and problem domain. These findings hold significant implications for diabetes diagnosis in real-world healthcare settings. The high accuracy and F1-score of the logistic regression model suggest its potential as a dependable tool for early detection and screening of diabetes patients, facilitating timely interventions. It is essential to note that the results obtained in this study are contingent upon the dataset used and may not extrapolate to other datasets or populations. The selection of features, data preprocessing techniques, and model parameters can impact model performance. Therefore, further research and validation utilizing diverse datasets and external validation cohorts are imperative to validate the generalizability of the logistic regression model.In conclusion, our exploration of various machine learning techniques for diabetes prediction underscored the superior accuracy and F1-score achieved by logistic regression. This discovery holds substantial implications for the advancement of precise and efficient diagnostic systems in healthcare. Future research endeavors can focus on 46Mithun Sarker refining the logistic regression model, integrating additional features, and exploring ensemble methods to further elevate its performance and broaden its utility in clinical practice. DISCUSSION The research presented in this article aimed to delve into the transformative potential of machine learning in modern healthcare and its ability to bolster value-based treatment approaches. Our study centered on crafting and validating a predictive model for disease prognosis, drawing upon extensive datasets from diverse patient cohorts. This section delves into the implications of our findings and their broader significance within the landscape of machine learning in healthcare. Our results underscore the considerable promise of machine learning algorithms within the healthcare realm. By adeptly harnessing vast datasets and integrating domain-specific insights, predictive models can markedly enhance the accuracy and clinical relevance of disease prognostication. This bears profound implications for patient care, empowering healthcare practitioners to preemptively identify and address high-risk scenarios, thereby amplifying patient outcomes and optimizing resource allocation. Moreover, the adoption of machine learning-driven methodologies facilitates a shift from volume-based to valuebased treatment paradigms, prioritizing patient-centric care and personalized medicine. The selection of the appropriate machine learning algorithm emerges as a pivotal determinant in the efficacy of healthcare predictive models. Our study underscores the imperative of meticulous experimentation and model curation to ensure peak performance across accuracy and other pertinent metrics. Additionally, we accentuate the indispensability of model interpretability and explicability, pivotal in cultivating trust amongst healthcare stakeholders, including providers, patients, and researchers. Techniques like feature importance analysis and partial dependence plots furnish invaluable insights into the nexus between input features and prognosticated outcomes, bolstering the credibility and acceptance of machine learning models in healthcare. Furthermore, our research underscores the paramount importance of ethical considerations and data privacy within the healthcare arena. Safeguarding patient data and upholding ethical tenets are cardinal for nurturing responsible ISSN:3006-4023 (Online),JournalofArtificialIntelligence GeneralScience (JAIGS)47 and sustainable integration of machine learning technologies in healthcare. Collaborative efforts between researchers and practitioners are indispensable in tackling these concerns and devising best practices that harmonize innovation with patient confidentiality and welfare. In conclusion, our study augments the burgeoning corpus of research on the fusion of machine learning in healthcare and its potential to metamorphose conventional healthcare systems. The formulation and validation of an efficacious predictive model for disease prognosis not only spotlight the potential of machine learning in augmenting modern healthcare and value-based treatment methodologies but also furnish a springboard for future inquiries in this domain. Subsequent investigations can leverage our findings to explore diverse applications of machine learning in healthcare, fine-tune existing models, and concoct novel algorithms tailored to the idiosyncratic challenges and requisites of the healthcare sphere. FUTURE DIRECTIONS This research article lays the groundwork for numerous avenues of future exploration and enhancement in the realm of diabetes prediction. Firstly, the integration of additional data sources, such as data from wearable devices or electronic health records, holds promise in providing a more holistic understanding of patients' health statuses and bolstering prediction accuracy. Secondly, delving into advanced machine learning techniques, including deep learning models or ensemble methods, presents an opportunity to elevate prediction performance and unearth latent patterns within the dataset. Additionally, longitudinal studies aimed at monitoring patients over prolonged periods could facilitate the capture of disease progression dynamics, thus enabling the personalization of treatment plans.Moreover, the integration of genetic and genomic information into the prediction framework could pave the way for a more tailored approach, taking individual genetic factors into account. Furthermore, conducting comparative analyses across diverse datasets and demographic cohorts can validate the model's generalizability while elucidating potential biases. Lastly, prioritizing the interpretability and explainability of the prediction system is essential to foster trust and acceptance within healthcare clinical practice.In summary, there exists a plethora of captivating future directions to explore, spanning from data augmentation and advanced modeling techniques to 48Mithun Sarker personalized medicine and interpretability. These endeavors have the potential to propel the field of diabetes prediction forward, ultimately contributing to enhanced patient care and clinical outcomes. CONCLUSION In summary, this research endeavor set out to forge a robust machine learning framework for diabetes prediction utilizing a comprehensive dataset. Through the rigorous exploration and evaluation of various machine learning methodologies, encompassing logistic regression, k-nearest neighbors, gradient boosting, PyTorch, and neural networks, we have garnered promising outcomes in accurately discerning the presence of diabetes in patients. Notably, the logistic regression model emerged as the most adept among the assessed techniques. These research findings underscore the profound potential of machine learning in healthcare, particularly in the realm of diabetes prediction, thereby furnishing invaluable insights for early detection and intervention.The study squarely addressed the research conundrum of diabetes prediction utilizing a dataset teeming with diverse patient attributes and clinical metrics. By meticulously curating pertinent features and judiciously applying data preprocessing protocols, we meticulously fortified the quality and fidelity of the dataset, thereby amplifying the dependability of the model. Our methodological approach was underpinned by rigorous experimentation and evaluation, undergirded by robust performance metrics to gauge the models' efficacy in terms of accuracy, precision, recall, and F1 score.The implications of this research are profound for healthcare providers, equipping them with the capacity to identify individuals at risk of developing diabetes at the incipient stage. Timely detection paves the way for prompt interventions and tailored treatment regimens, potentially alleviating the disease burden and augmenting patient outcomes. Moreover, this study furnishes insights into the relative performance of diverse machine learning algorithms for diabetes prediction, thus furnishing a lodestar for future research and development endeavors in the domain.Nevertheless, it behooves us to acknowledge the limitations inherent in this study. Our research was circumscribed by the confines of a specific dataset, and extrapolating the findings to heterogeneous populations or healthcare milieus may necessitate further validation. Furthermore, our focus primarily centered on forecasting the presence of diabetes, leaving avenues open for future exploration into predicting disease progression or discerning responses to specific therapeutic modalities.In culmination, this research article constitutes a salient contribution to the burgeoning corpus of knowledge on machine learning in healthcare, with a specific focus on diabetes prediction. The insights gleaned underscore the transformative potential of machine learning methodologies in empowering ISSN:3006-4023 (Online),JournalofArtificialIntelligence GeneralScience (JAIGS)49 healthcare professionals to make judicious decisions and enhance patient care. Future research endeavors should aspire to surmount the identified constraints, corroborate the model's performance across variegated datasets, and delve into additional facets of machine learning's utility in diabetes management. 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W4234725277.txt	https://www.qeios.com/read/GW9T0V/pdf	de		Fluorine F 18 FDHT	Definitions	2,020	cc-by	99	Qeios · Definition, February 2, 2020 Ope n Pe e r Re v ie w on Qe ios Fluorine F 18 FDHT National Cancer Institute Source National Cancer Institute. Fluorine F 18 FDHT . NCI T hesaurus. Code C112496. A radioconjugate containing a derivative of the androgen testosterone, 16-beta-fluoro-5alpha-dihydrotestosterone (FDHT ), labeled with the radioisotope fluorine F18 (18FFDHT ), with potential use as an imaging agent for positron emission tomography (PET ). Upon administration, 18F-FDHT binds to the androgen receptor (AR). Upon PET imaging, AR-expressing prostate tumor cells can be imaged and assessed. Qeios ID: GW9T0V · https://doi.org/10.32388/GW9T0V 1/1
https://openalex.org/W2025057057	https://europepmc.org/articles/pmc2687543?pdf=render	English	null	Access to paediatric emergency departments in Italy: a comparison between immigrant and Italian patients	The Italian Journal of Pediatrics/Italian journal of pediatrics	2,009	cc-by	4,081	BioMed Central BioMed Central Erica Clara Grassino1, Carla Guidi1, Alice Monzani1, Pasquale Di Pietro2 and Gianni Bona1 Address: 1Department of Pediatrics, AOU Maggiore della Carit, University of Piemonte Orientale, Novara, Italy and 2Emergency Room and Emergency Medicine Division G. Gaslini Institute, Genova, Italy Email: Erica Clara Grassino - eri.grassino@gmail.com; Carla Guidi - eri.grassino@gmail.com; Alice Monzani - alice.monzani@gmail.com; Pasquale Di Pietro - pasqualedipietro@ospedale-gaslini.ge.it; Gianni Bona - gianni.bona@maggioreosp.novara.it * Corresponding author Received: 23 December 2008 Accepted: 22 February 2009 Received: 23 December 2008 Accepted: 22 February 2009 y Italian Journal of Pediatrics 2009, 35:3 doi:10.1186/1824-7288-35-3 Italian Journal of Pediatrics 2009, 35:3 doi:10.1186/1824-7288-35-3 This article is available from: http://www.ijponline.net/content/35/1/3 © 2009 Grassino et al; licensee BioMed Central Ltd. © 2009 Grassino et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. his is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecom hich permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Objective: The aim of the study was to investigate whether access to paediatric emergency departments differed between foreign and Italian patients. Methods: We performed a cross-sectional study between January-December 2007 to analyse attendance's characteristics in the paediatric emergency departments of ten Italian public hospitals. The study population included each foreign patient and the following Italian patient admitted to the same emergency department. All causes of admission of these subjects were evaluated, together with the child's age, gender, country of birth, parents' nationality, time of admission, severity code and discharge-related circumstances. Results: We enrolled 4874 patients, 2437 foreign (M:F = 1409:1028) and 2437 Italian ones (M:F = 1368:1069). Most of foreign and Italian patients' admissions were sorted as green (72.5% and 87.8%, respectively) or white codes (25.2% and 9.8%, respectively). The most frequent causes for attendance concerned respiratory tract diseases, followed by gastroenteric ones and injuries in both groups. Conclusion: In our survey immigrants didn't access to emergency departments more than Italian children. Both of them referred to emergency departments mainly for semi-urgent or non-urgent problems. Foreign and Italian patients suffered from the same pathologies. Infectious diseases traditionally thought to be a potential problem in immigrant populations actually seem to be quite infrequent. are economically disadvantaged compared with Italy. One of the most worrisome political issues regarding immigration is the ability of healthcare institutions to serve the increasing number of immigrants and to provide equity of access to healthcare services among this popula- tion. The Italian National Health System provides univer- Open Acc Research Access to paediatric emergency departments in Italy: a comparison between immigrant and Italian patients Erica Clara Grassino1, Carla Guidi1, Alice Monzani1, Pasquale Di Pietro2 and Gianni Bona*1 Open Access Page 1 of 5 (page number not for citation purposes) http://www.ijponline.net/content/35/1/3 http://www.ijponline.net/content/35/1/3 Italian Journal of Pediatrics 2009, 35:3 to simplify the analysis: 1. Italy, 2. non-EU Eastern Europe, 3. Latin America, 4. Nile Valley, 5. Northern Africa, 6. sub-Saharan Africa, 7. India, Pakistan and Sri- Lanka, 8. China and the rest of Asia, 9. nomads and Gyp- sies. Time of admission was recorded as day-time if admis- sion to the emergency department took place between 8 a.m. and 8 p.m. and night-time if between 8 p.m. and 8 a.m. Cause of consultation was synthesized with the main symptom complained at admission. Evaluating the sever- ity of the symptoms complained, patients were sorted into four categories identified by four different colours accord- ing to Canadian Pediatric Triage and Acuity Scale Guide- lines [8]: white if non-urgent (cases and situations that could also be managed by the family Paediatrician – these patients will be examined only after the most serious cases have been managed), green if semi-urgent (cases with postponable health troubles – there is no risk of death and the patient will be cared for after more urgent cases), yel- low if urgent (patients with the risk of fast prejudice of vital functions), red if emergency (cases with immediate risk of death). The diagnosis at discharge was analyzed according to a broad spectrum of variables: respiratory diseases, injuries, gastroenteric diseases, skin affections, fever, localized infections, childhood infectious diseases, genitourinary infections, foreign bodies ingestion or inha- lation, poisoning, surgical problems and burns. Dis- charge-related circumstances (discharge to home, further outpatient investigations, temporary observation, hospi- tal admission, voluntary discharge against the physician's recommendation or death) were also evaluated. sal access to healthcare; nevertheless, often the utilisation of primary and specialized care might present several bar- riers for foreign people, including lack of knowledge about how to access these services and difficulties in mak- ing an appointment (language, time schedules, waiting lists, etc.). Therefore, it is possible that foreign people tend to access to emergency departments more easily than to primary and specialised care services. It has to be taken into account that economic indicators of poverty have been related to poorer health and higher emergency units utilisation [2]. http://www.ijponline.net/content/35/1/3 Previous studies describing healthcare serv- ices use by immigrants tended to highlight their overall under-utilisation of services compared with non-immi- grants, the preponderant access to public units over the private sector, and the greater use of emergency rooms to counteract access barriers to other health services [3-5]. Few studies analysing worldwide paediatric healthcare services utilisation showed a similar disadvantaged condi- tion for foreign children, too, resulting in an excessive and often improper use of emergency units [6,7]. Up to now, paediatric emergency services utilisation in Italy has not yet been studied in depth. We aimed to quantify the amount of accesses to emergency units by immigrant and Italian population analysing one-year activity of ten Ital- ian public paediatric hospitals. Therefore, we evaluated admissions to paediatric emergency units of foreign and Italian children, in order to test the hypothesis that the emergency department utilisation would be higher, and consequently often inappropriate, among immigrants than among the host population. Secondary aim was to identify the causes of consultation for foreign and Italian patients and to verify whether immigrants suffer from spe- cific import diseases. The study has been approved by the local Ethical Commit- tee and informed consent was signed by the parents. Statistical analysis was performed with SPSS 16.0 software (SPSS Inc, Chicago, IL). Background g Italy has traditionally been a country of emigration, with immigration being a relatively recent phenomenon begin- ning in the mid-eighties. Nowadays, immigrants in Italy are 3.432.651, representing 5.8% of total population [1]. Mainly, immigrants come from developing countries that Page 1 of 5 (page number not for citation purposes) Page 1 of 5 (page number not for citation purposes) Methods B J Between January and December 2007 a multicentric cross- sectional study was undertaken in the paediatric emer- gency department of ten Italian public hospitals. Clinical notes for all foreign patients from developing countries outside the European Union admitted to the paediatric emergency departments were recorded by a medical inter- viewer. A patient was considered as foreign if one or both parents were born outside Italy and the European Union. Each foreign patient was matched with the following Ital- ian patient admitted to the same paediatric emergency department, whose clinical notes were collected, too. Access characteristics Both among immigrant and Italian patients most of pae- diatric emergency department visits took place during day-time, between 8 a.m. and 8 p.m. (76.4% and 78.8%, respectively). According to the severity of symptoms complained most of foreign patients (72.5%) were assigned a green code (semi-urgent), 25.2% of immigrant patients' admissions were sorted as white codes (non-urgent), 2.1% as yellow codes (urgent) and only 0.2% as red codes (emergency). Among the Italian patients, green codes accounted for 87.8% of total consultations, followed by white codes (9.8%), yellow codes (2.3%) and red codes (0.1%). http://www.ijponline.net/content/35/1/3 http://www.ijponline.net/content/35/1/3 http://www.ijponline.net/content/35/1/3 Italian Journal of Pediatrics 2009, 35:3 Analyzing the diagnosis at discharge, most of foreign patients had respiratory diseases (36.3%); 17.8% showed gastroenteric diseases, 12.8% were injured, 5% had skin affections, 5.9% fever, 4.3% localized infections, 3.2% childhood infectious diseases and 1.5% displayed geni- tourinary infections. Only a small proportion of foreign patients referred to the emergency department for foreign bodies ingestion or inhalation, poisoning, surgical prob- lems or burns. Similarly, among Italian patients the most frequent diagnosis at discharge was a respiratory disease (32.7%), followed by accidental trauma or injuries (17.4%), gastroenteric diseases (15.1%), skin affections (6.6%), fever (6.2%), localized infections (4%), child- hood infectious diseases (2.5%), genitourinary infections (1.9%), and by fewer cases of foreign bodies ingestion or inhalation, poisoning, surgical problems or burns. Given the high rate of respiratory diseases, these were analysed separately considering the subgroup pathologies (Table 1). At the end of consultation process 87% of immigrants were discharged to home, 11.9% were hospitalized. Only 7 (0.3%) foreign subjects required further outpatient investigations and 9 (0.4%) a temporary observation, 11 (0.4%) were voluntary discharged against the physician's recommendation. Among Italian patients, discharge to home occurred in 86.7%, hospital admission in 12.6%, temporary observation in 0.5%, voluntary discharge in 0.1% and further outpatients visits in 0.1%. Both between foreign and Italian patients no death was recorded. Analyzing the country of birth of foreign patients, 51.2% of subjects were born in Italy, 17.4% in non-UE Eastern European countries, 15.3% in Northern Africa, 7% in Latin America, 3% in sub-Saharan Africa, 1.7% in the Nile Valley/Arabian countries, 1.9% in China or other Asian countries, 0.9% in India, Pakistan or Sri-Lanka and 1.6% were nomads or Gypsies. Fathers came in most cases from Northern Africa (30.3%), followed by non-UE Eastern Europe (28%), Latin America (14.4%), sub-Saharan Africa (6.6%), China or other Asian countries (4.2%), the Nile Valley/Arabian countries (3.6%), Indian area (1.9%), Italy (0.7%). 9.4% of fathers had no fixed abode/were nomads; father nationality was unknown in 0.9%. Simi- larly, among the mothers the most frequent country of origin was Northern Africa (28.1%), followed by non-UE Eastern Europe (27.3%), Latin America (14.5%), sub- Saharan Africa (6.4%), China or other Asian countries (4.1%), the Nile Valley/Arabian countries (3.4%), Indian area (1.8%), Italy (0.7%). 9.1% of mothers had no fixed abode/were nomads; mother nationality was unknown in 4.6%. Study population y p p In the period analyzed in the survey, 2437 foreign citizens referred to the paediatric emergency departments of the hospitals taking part to this multicentric study and were enrolled for the survey. As many Italian patients admitted to the same paediatric emergency department were enrolled. Among immigrant patients 1409 (57.8%) were males and 1028 (42.2%) females; among Italian ones, 1368 (56.1%) were males and 1069 (43.9%) females. Out of the 2437 foreign subjects, 35 (1.4%) were newborns, 519 (21.3%) infants, 1110 (45.6%) preschool-aged chil- dren, 447 (18.3%) school-aged children and 326 (13.4%) adolescents. Out of the 2437 Italian subjects, 34 (1.4%) were newborns, 336 (13.8%) infants, 1193 (49%) pre- school-aged children, 424 (17.4%) school-aged children and 450 (18.4%) adolescents. Data about age, gender, country of birth, maternal and paternal nationality, time of admission, cause of consulta- tion and severity code were recorded for each child enrolled. Patients were divided into five groups according to age: newborns if <1 month, infants if 1 month – 1 year, preschool-aged children if 1 – 5 year, school-aged chil- dren if 6 – 10 years, adolescents if >11 years. Children and their parents' nationality was classified into nine groups Page 2 of 5 (page number not for citation purposes) Authors' contributions ECG participated in the design of the study, performed the statistical analysis and draft the manuscript. CG partici- pated in the design of the study, carried out the data and performed the statistical analysis. AM performed the sta- tistical analysis and draft the manuscript. PDP partici- pated in the design of the study and collection of data. GB conceived of the study and participated in its coordina- tion and helped to draft the manuscript. All authors read and approved the final version of the manuscript. Conclusion In conclusion, it is not possible to identify a specific "migrant type", remaining the same from a health care, social, economic, and anthropologic point of view. Sev- eral different features are typical of each immigrant, and they are generally consistent with the area of origin. Other differences emerge after the immigration process, and they are strictly related to life-style in Italy (i.e. hygienic conditions, nutritional status, etc). Therefore, specific knowledge is needed to face immigrants' different health problems. Under the perspective to better understand for- eign patients health needs many instruments, such as translators and multilingual fliers concerning health problems, have to be used in emergency departments. Competing interests It is noteworthy that in our survey immigrants didn't access to emergency departments more than Italian chil- dren, in contrast with evidences reported in previous study [4,16,17]. Our finding might reflect a well-inte- grated foreign population. This may be due, on the one hand, to the fact that in the last twenty years of immigra- tion foreign citizens learned how to utilize all the services offered by the Italian National Health System and when to access to primary and specialized care rather than to the emergency departments. On the other hand, well-inte- grated immigrants could be the result of a proper manage- ment by medical staff, trained to deal with migrants' specific health problems and able to overcome linguistic and logistic barriers. Immigrants' health concerns are strongly correlated with their country of origin, so that a broad spectrum of variables should be carefully taken into account, when considering both the migration phenome- non and the individual foreign citizen (i.e. nationality, reason of migration such as poverty, political and ethnic persecution or civil war, health standards in the country of origin, social and hygienic conditions in Italy). The authors declare that they have no competing interests. The authors declare that they have no competing interests. http://www.ijponline.net/content/35/1/3 Medical, social, economic and psychological fac- tors mainly influence the parents' decision to visit the emergency department rather than manage their children at home, or prior to making an unscheduled visit to an emergency department their demands could have been satisfactorily met by an appropriate visit at a different health care level. Reports in literature indicate similar pre- senting problems in other studies performed in children attending paediatric accident and emergency departments in USA and UK [9-11], in Malaysia [12] and also in Italy [13-15]. tions, actually seem to be quite infrequent, when com- pared with overall morbidity. Immigrant populations are often considered as a source of many known and unknown infectious diseases, such as Ebola, SARS, TBC, malariae, etc. occasionally resulting in unjustified preju- dice [18]. As a whole, in our survey infectious diseases accounted for 3.2% of foreign children visits and for 2.5% Italian children visits. Therefore immigrants should not be considered as infectious diseases carriers. Both among immigrant and Italian patients the most frequent present- ing problems for visits to emergency departments were respiratory diseases, injury, and digestive symptoms as observed in other cross-sectional study [13], demonstrat- ing that foreign patients and Italian ones suffer by the same pathologies. http://www.ijponline.net/content/35/1/3 require emergency treatment. Accident and emergency departments have been created in hospitals with the pri- mary function of providing immediate care for patients with life-threatening medical conditions, trauma, or inju- ries, but not to treat minor illnesses or provide primary care. In recent years, the emergency department has increasingly become a major provider of health care and this overcrowding has become problematic. People tend to use emergency department as a substitute for general practitioner to treat minor illness. In the context of limited inpatient hospital resources, it is acknowledged that the phenomenon of non-urgent emergency departments vis- its, which can be managed alternatively and appropriately in general practice, has raised serious concerns among healthcare planners, both because of its magnitude and because the appropriate utilisation of hospital care can significantly improve health outcomes. In particular, the over-utilisation of emergency departments by paediatric patients has become an important problem that must be solved. Medical, social, economic and psychological fac- tors mainly influence the parents' decision to visit the emergency department rather than manage their children at home, or prior to making an unscheduled visit to an emergency department their demands could have been satisfactorily met by an appropriate visit at a different health care level. Reports in literature indicate similar pre- senting problems in other studies performed in children attending paediatric accident and emergency departments in USA and UK [9-11], in Malaysia [12] and also in Italy [13-15]. require emergency treatment. Accident and emergency departments have been created in hospitals with the pri- mary function of providing immediate care for patients with life-threatening medical conditions, trauma, or inju- ries, but not to treat minor illnesses or provide primary care. In recent years, the emergency department has increasingly become a major provider of health care and this overcrowding has become problematic. People tend to use emergency department as a substitute for general practitioner to treat minor illness. In the context of limited inpatient hospital resources, it is acknowledged that the phenomenon of non-urgent emergency departments vis- its, which can be managed alternatively and appropriately in general practice, has raised serious concerns among healthcare planners, both because of its magnitude and because the appropriate utilisation of hospital care can significantly improve health outcomes. In particular, the over-utilisation of emergency departments by paediatric patients has become an important problem that must be solved. Discussion h l The most relevant result of our survey is the high rate of accesses to paediatric emergency departments for non- urgent or semi-urgent medical problems, both between foreign and Italian patients. Indeed, more than 95% of foreign and Italian children were assigned green or white codes. This finding suggests that a large proportion of the demand for emergency departments utilisation often may be attributed to visits for medical problems that do not Page 3 of 5 (page number not for citation purposes) Table 1: Respiratory diseases. RESPIRATORY DISEASES FOREIGN PATIENTS ITALIAN PATIENTS n % n % UPPER-AIRWAYS DISEASES 250 28.2 178 22.3 SINUSITIS 4 0.5 5 0.6 ACUTE OTITIS MEDIA 129 14.6 141 17.7 PHARYNGOTONSILLITIS 252 28.5 200 25.1 LARYNGITIS 36 4.1 44 5.5 INFLUENZAL SYNDROME 39 4.4 16 2.0 BRONCHITIS 64 7.2 94 11.8 BRONCHOSPASM 47 5.3 54 6.8 BRONCHIOLITIS 17 1.9 22 2.8 POLMONITIS 47 5.3 43 5.4 Total 885 100.0 798 100.0 Specific respiratory diagnoses in foreign and Italian patients. Specific respiratory diagnoses in foreign and Italian patients. Italian Journal of Pediatrics 2009, 35:3 http://www.ijponline.net/content/35/1/3 Acknowledgements The authors would like to thank the coworkers of the multicentric study SIMEUP-GLNBI (Italian Society of Paediatrics), (Zampogna S. and Masciari P from Department of Pediatrics, Azienda Ospedaliera Pugliese, Ciaccio, Catanzaro, Italy; Piccotti E and Calcagno A from Emergency Room and Emergency Medicine Division, G. Gaslini Institute, Genova, Italy; Bartolini M and Acutis M. S from Department of Pediatrics, Ospedale Galliera, Gen- ova, Italy; Zaffaroni M., Stasi I., Capelli A. and Acucella G. from Department of Pediatrics, AOU Maggiore della Carità, University of Piemonte Orien- Remarkably infectious diseases, initially thought to be a potential prominent problem in these immigrant popula- Page 4 of 5 (page number not for citation purposes) Page 4 of 5 (page number not for citation purposes) http://www.ijponline.net/content/35/1/3 Italian Journal of Pediatrics 2009, 35:3 tale, Novara, Italy; Calligari G. C. from Department of Pediatrics, Ospedale Fatebenefratelli, Erba (CO), Italy; Valentini P. from Department of Pediat- rics, Policlinico Agostini Gemelli, Roma, Italy; Zavarise G. from Department of Pediatrics, Ospedale Sacro Cuore, Negrar (VR), Italy; Visci G from Department of Pediatrics, Ospedale Regionale Spirito Santo, Pescara, Italy: Guala A. from Department of Pediatrics, Ospedale SS. Pietro e Paolo, Bor- gosesia (VC), Italy; Lo Coco G. from Department of Pediatrics, Ospedale A. Ajello, Mazara del Vallo (TP), Italy) who contributed towards the study by the acquisition of data and the critical revision of the manuscript. tale, Novara, Italy; Calligari G. C. from Department of Pediatrics, Ospedale Fatebenefratelli, Erba (CO), Italy; Valentini P. from Department of Pediat- rics, Policlinico Agostini Gemelli, Roma, Italy; Zavarise G. from Department of Pediatrics, Ospedale Sacro Cuore, Negrar (VR), Italy; Visci G from Department of Pediatrics, Ospedale Regionale Spirito Santo, Pescara, Italy: Guala A. from Department of Pediatrics, Ospedale SS. Pietro e Paolo, Bor- gosesia (VC), Italy; Lo Coco G. from Department of Pediatrics, Ospedale A. Ajello, Mazara del Vallo (TP), Italy) who contributed towards the study by the acquisition of data and the critical revision of the manuscript. References 1. 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https://openalex.org/W2350256796	https://europepmc.org/articles/pmc4877482?pdf=render	English	null	ROS, Cell Senescence, and Novel Molecular Mechanisms in Aging and Age-Related Diseases	Oxidative medicine and cellular longevity	2,016	cc-by	17,452	Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity Volume 2016, Article ID 3565127, 18 pages http://dx.doi.org/10.1155/2016/3565127 Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity Volume 2016, Article ID 3565127, 18 pages http://dx.doi.org/10.1155/2016/3565127 Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity Volume 2016, Article ID 3565127, 18 pages http://dx.doi.org/10.1155/2016/3565127 Department of Biomedical, Metabolic and Neural Sciences, University of Modena & Reggio Emilia, 41125 Modena, It Bristol Heart Institute, University of Bristol, Bristol BS2 8HW, UKh Bristol Heart Institute, University of Bristol, Bristol BS2 8HW, UK 3University/BHF Centre for Cardiovascular Science, The Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16-4TJ, UK 4Istituto Nazionale di Biostrutture e Biosistemi, 00136 Roma, Italy 4Istituto Nazionale di Biostrutture e Biosistemi, 00136 Roma, Italy Correspondence should be addressed to Domenico D’Arca; domenico.darca@unimore.it Received 16 December 2015; Revised 2 April 2016; Accepted 6 April 2016 Received 16 December 2015; Revised 2 April 2016; Accepted 6 April 2016 Academic Editor: Michael Courtney Copyright © 2016 Pierpaola Davalli et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The aging process worsens the human body functions at multiple levels, thus causing its gradual decrease to resist stress, damage, and disease. Besides changes in gene expression and metabolic control, the aging rate has been associated with the production of high levels of Reactive Oxygen Species (ROS) and/or Reactive Nitrosative Species (RNS). Specific increases of ROS level have been demonstrated as potentially critical for induction and maintenance of cell senescence process. Causal connection between ROS, aging, age-related pathologies, and cell senescence is studied intensely. Senescent cells have been proposed as a target for interventions to delay the aging and its related diseases or to improve the diseases treatment. Therapeutic interventions towards senescent cells might allow restoring the health and curing the diseases that share basal processes, rather than curing each disease in separate and symptomatic way. Here, we review observations on ROS ability of inducing cell senescence through novel mechanisms that underpin aging processes. Particular emphasis is addressed to the novel mechanisms of ROS involvement in epigenetic regulation of cell senescence and aging, with the aim to individuate specific pathways, which might promote healthy lifespan and improve aging. Pierpaola Davalli,1 Tijana Mitic,2 Andrea Caporali,3 Angela Lauriola,1 and Domenico D’Arca1,4 Angela Lauriola,1 and Domenico D’Arca1,4 1Department of Biomedical, Metabolic and Neural Sciences, University of Modena & Reggio Emilia, 41125 Modena, Italy 2Bristol Heart Institute, University of Bristol, Bristol BS2 8HW, UK 3University/BHF Centre for Cardiovascular Science, The Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16-4TJ, UK 1Department of Biomedical, Metabolic and Neural Sciences, University of Modena & Reggio Emilia, 41125 Modena, Italy 2Bristol Heart Institute, University of Bristol, Bristol BS2 8HW, UK 1Department of Biomedical, Metabolic and Neural Sciences, University of Modena & Reggio Emilia, 41125 Modena, Italy 2Bristol Heart Institute, University of Bristol, Bristol BS2 8HW, UK 3University/BHF Centre for Cardiovascular Science, The Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16-4TJ, UK 1. Introduction instead, associated with lengthening of organismal lifespan [5]. Current studies aim at deepening how cell senescence process, so far experimented in vitro, may be extended to in vivo studies. Increasing evidence for causal role of cell senes- cence has been demonstrated in age-related dysfunctions and pathologies [6]. Senescent cells proliferate in aging, as a stress response primed by a number of “counting mechanisms,” like telomeres shortening, DNA damage accumulation, abnormal oncogenes activities, metabolic alterations, and excessive ROS generation [7]. These mechanisms cause cell proliferat- ing arrest and generate features, as constitutive production of high ROS levels, critical for the senescent phenotype mainte- nance. Despite increasing modestly, as a number, the senes- cent cells are implicated in age-related diseases promotion, through the restriction of the regenerative pool of the tissue The reduced rate of birth and mortality is the motive of the older population growth in western industrialized countries, where advanced age remains the fundamental risk factor for most chronic diseases and functional deficits. As an example, it is estimated that the individuals of age 65 and above in the USA will reach 20% by 2030, while they constituted 12.4% in 2004 [1]. Human aging is developed from such an accumu- lation of physical, environmental, and social factors that the definition of the molecular mechanisms that trigger the aging means a difficult task. Some theories associate various factors with aging rate, as changes of metabolic control [2] and gene expression patterns [3] and production of high levels of Reactive Oxygen Species (ROS) [4]. Low ROS level has been, Oxidative Medicine and Cellular Longevity 2 understanding ROS involvement in aging and age-related diseases [30]. An innovative method simultaneously assesses glutathione, hydrogen peroxide, and superoxide levels in a single cell, together with cell viability alterations, thus allowing for defining both oxidant-antioxidant balance and cell death, after the administration of a specific stimulus [31]. A wide range of pathways and molecular mechanisms that involve ROS suggests determining the redox state of thiols in ROS targets, which compose the “cellular oxidative interface” [32, 33]. ROS oxidize specific protein residues of cysteine into sulfenic acid, reversibly. This molecule functions as OS/NS sensor within enzymes and transcriptional regulatory factors and may allow priming the routes of the versatile ROS action [34–36]. stem cells [8]. 1. Introduction Some observations indicate that senescent cells do not necessarily induce mechanisms that promote aging and can be efficiently removed from the human body [9]. The general consensus on cellular damage accumulation, as aging initial event, suggests that cell senescence process is a major question regarding biological and clinical aging aspects [10]. q g g g g g p Here, we review evidences on novel molecular mech- anisms of the “ROS signaling” during aging and related pathologies, because they suggest a way of promoting healthy lifespan and improve human aging. 2. ROS Physioma Homeostasis The ROS physioma is a family of highly reactive molecules which includes free oxygen radicals, like superoxide anion (O2 ∙−), hydroxyl radical (OH∙), and nonradical oxygen derivatives, like the stable hydrogen peroxide (H2O2). The superoxide radicals react to form other ROS, namely, hydro- gen peroxides and hydroxyl radicals, and interconvert with reactive nitrogen species (RNS), which generate effects simi- lar to ROS [11]. The inefficient electron transfer in mitochon- drial respiratory chain is believed to be a main ROS source, among diverse possible enzymatic and nonenzymatic sources [12]. Increased expression of catalase and peroxiredoxin- 1 molecules are considered as OS markers. The family comprises seven transmembrane members, namely, Nox1– 5 [13–15] and Duox1-2 [16]. ROS are generated by oxygen metabolism (i.e., cellular respiration) in all the cells that utilize oxygen, as inevitable consequence of aerobic life, and may derive from exogenous metals, recycling of redox compounds, radiation, chemotherapeutic agents, carcino- gens (estrogenic molecules), and other dietary and environ- mental means. Generally, the ROS increasing levels cause nonlinear cellular responses [17]. A fine balance between oxidant-antioxidant mechanisms leads to continuous mod- ulation of ROS production, location, and inactivation, in both physiological and pathological conditions. Endogenous antioxidants, like the enzymes of catalase family, glutathione group, thioredoxin-related group, and superoxide dismutase [18], together with exogenous antioxidant as reduced glu- tathione [19], carotenoids, and vitamins C and E, constitute the indispensable ROS detoxifying system. Nevertheless, imbalance of redox homeostasis may occur, usually in favor of oxidants, so that ROS shift from physiological to poten- tially harmful levels, named oxidative and nitrosative stress (OS/NS). Increased expression of catalase and peroxiredoxin 1 molecules are considered as OS markers [20–22]. 2.2. ROS Functions. The increasing comprehension of mech- anisms, underlying the oxidant milieu of the cell, shows ROS as signaling molecules, besides metabolic byproducts. They act in a myriad of pathways and networks, mediated by hormones, which ranges from protein phosphorylation to transport systems, for example. ROS do not influence single steps of multistep processes; rather, they influence all the steps at the same time, by reacting with several compounds and taking part in several redox reactions. Depending on ROS concentration, molecular species, and subcellular localization, cell components and signaling path- ways are affected positively or negatively. ROS levels are believed to be a “redox biology” that regulates physiological functions, including signal transduction, gene expression, and proliferation. 2. ROS Physioma Homeostasis “Redox biology,” rather than OS, has been proposed to underlie both physiological and pathological events [37]. Data in the literature on slow and constant ROS increases have to be integrated with data on fast and stepwise ROS increases, typical of signaling events, which deliver messages among cellular compartments. Questions related to ROS dynamics and specificity, as the effects of their waves of concentration on networks with other signaling pathways, are investigated in single cells and across different cells. Proteins are the major target of ROS/RNS signaling and undergo reversible or irreversible modifications of their functions, which result in cell death, growth arrest, and transformation. The modulation of the reversible oxida- tion of redox-sensitive proteins plays basic roles in sens- ing and transducing the oxygen signal. Receptor-dependent or nondependent tyrosine kinases, AMP-activated protein kinases, adaptor protein p66SHC, and transcription factors as FOXO (forkhead homeobox type O), Nrf2 (nuclear fac- tor E2-related factor 2), p53 (tumor suppressor 53), NF- 𝜅B (nuclear factor kappa B), AP-1 (activator protein-1), HIF-1a (hypoxia inducible factor-1a), PPAR𝛾(peroxisome proliferator-activated receptor gamma), and 𝛽-catenin/Wnt signaling are listed in Table 1 [38–81]. ROS mediate in vitro response towards intra- and extracellular conditions, such as growth factors, cytokines, nutrients deprivation, and hypoxia, which regulate cell proliferation, differentiation, and apopto- sis, besides being important cancer hallmarks [82]. Intrinsic and extrinsic factors control ROS regulation on cellular self- renewal, quiescence, senescence, and apoptosis, during the in vivo tissues homeostasis and repair [83] and in ROS 2.2. ROS Functions. The increasing comprehension of mech- anisms, underlying the oxidant milieu of the cell, shows ROS as signaling molecules, besides metabolic byproducts. They act in a myriad of pathways and networks, mediated by hormones, which ranges from protein phosphorylation to transport systems, for example. ROS do not influence single steps of multistep processes; rather, they influence all the steps at the same time, by reacting with several compounds and taking part in several redox reactions. Depending on ROS concentration, molecular species, and subcellular localization, cell components and signaling path- ways are affected positively or negatively. ROS levels are believed to be a “redox biology” that regulates physiological functions, including signal transduction, gene expression, and proliferation. “Redox biology,” rather than OS, has been proposed to underlie both physiological and pathological events [37]. Oxidative Medicine and Cellular Longevity Oxidative Medicine and Cellular Longevity Table 1: Selected ROS sensitive proteins that are involved in cell signaling transduction mechanism. Indicative examples of possible effects and processes they promote after being directly and/or indirectly modified by ROS (the references are indicated inside the square brackets). ROS sensitive proteins: oxidative interface (1) Effects of ROS sensitive proteins after being redox modified (2) Physiopathological processes in which ROS sensitive proteins are involved Protein kinases Receptor/nonreceptor tyrosine kinases (Src, TRK, AKT, c-Abl, MAPK, CaMKII, PKG, ATM, and Ask1) (i) Interactions between kinases pathways [38, 39] (ii) Signal of ROS production feedback [40] Control of cell cycle progression [56] Mitosis for anchorage-dependent cells [57] Cellular homeostasis [43, 57] AMP-activated protein kinases (AMPK) (i) Regulation of cell ROS/redox balance [41, 42] Myocyte adaptation to energy requirement [42] Adipocyte differentiation [58] Lipid metabolism (“fatty liver”) [59] Hyperglycemic damage [60] Cell fate (autophagy and apoptosis) [61] Adaptor proteins p66Shc (i) Signaling start in the aging process [43] Apoptosis [43]. Prolonged life span [43, 62] Cardiovascular diseases and obesity [63] Diabetic endothelial dysfunction [64] Nuclear receptors PPAR𝛾 (i) Redox sensor function [43] (ii) Regulation of genes that modulate ROS increases [44] Neurodegenerative diseases [65, 66] Lipid dysfunction (fatty liver) [59] Membrane receptors Elements in Notch1 pathway (i) Notch signaling modulation in association with Wnt/beta-catenin signal [45] Cell fate control in vascular development [45] Biological clocks in embryonic development [67] Transcription factor p53 Modulation of cell redox balance (prooxidant/antioxidant effects) [46–48] Cell fate signaling [68] Autophagy and apoptosis [61, 69] Nrf2 Cell adaptation to ROS resistance [49, 50] Apoptosis [70] Neurodegenerative diseases [71] Cardiovascular diseases [72] FOXO3A Cell coordination in response to OS [51] Metabolic adaptation to low nutrient intake [73] Cancer development [73] Diabetes [74] Atherosclerotic cardiovascular disease [75] Components in 𝛽-catenin/Wnt pathway Regulation of Wnt signaling via nucleoredoxin [76] Early embryonic development [76] Vascular development [45] HIF-1a Cell adaption to oxygen tension modifications [52] Cell proliferation; angiogenesis [77] Cell transformation [78, 79] Components in JAK–STAT pathway (i) Cell adaption to OS [53] (ii) Mediation of ROS mitogenic effect [53] Stress response gene expression [51] Systemic/pulmonary hypertension [80] NF-𝜅B Regulation of redox-sensitive gene expression [54, 55] Rheumatoid arthritis, dyslipidemia, atherosclerosis, and insulin resistance [81] induction of stem cells proliferation and differentiation. ROS act as a rheostat, which senses and translates environmental cues in stem cells response, thus balancing cellular output (function) with cellular input (nutrients, cytokines). 2. ROS Physioma Homeostasis Data in the literature on slow and constant ROS increases have to be integrated with data on fast and stepwise ROS increases, typical of signaling events, which deliver messages among cellular compartments. Questions related to ROS dynamics and specificity, as the effects of their waves of concentration on networks with other signaling pathways, are investigated in single cells and across different cells. Proteins are the major target of ROS/RNS signaling and undergo reversible or irreversible modifications of their functions, which result in cell death, growth arrest, and transformation. The modulation of the reversible oxida- tion of redox-sensitive proteins plays basic roles in sens- ing and transducing the oxygen signal. Receptor-dependent or nondependent tyrosine kinases, AMP-activated protein kinases, adaptor protein p66SHC, and transcription factors as FOXO (forkhead homeobox type O), Nrf2 (nuclear fac- tor E2-related factor 2), p53 (tumor suppressor 53), NF- 𝜅B (nuclear factor kappa B), AP-1 (activator protein-1), HIF-1a (hypoxia inducible factor-1a), PPAR𝛾(peroxisome proliferator-activated receptor gamma), and 𝛽-catenin/Wnt signaling are listed in Table 1 [38–81]. ROS mediate in vitro response towards intra- and extracellular conditions, such as growth factors, cytokines, nutrients deprivation, and hypoxia, which regulate cell proliferation, differentiation, and apopto- sis, besides being important cancer hallmarks [82]. Intrinsic and extrinsic factors control ROS regulation on cellular self- renewal, quiescence, senescence, and apoptosis, during the in vivo tissues homeostasis and repair [83] and in ROS 2.1. ROS Measurement Techniques. ROS are so highly variable and freely diffusible molecules that the detection of ROS and antioxidants, to obtain a picture of the cellular redox status, still represents a challenge. We stress some specific points and sensitive methods that are subjected to continuous improve- ment. Probes and antibodies have been developed to recog- nize oxidative damage by ROS/RNS [23–25]. The tools allow revealing antioxidant enzymes [26] and a variety of oxidative products, as lipid peroxidation products, protein carbonyls [27], oxidized DNA products [28], and nitrotyrosine [29]. Combinations of diverse approaches will prove essential for 3 Oxidative Medicine and Cellular Longevity Oxidative Medicine and Cellular Longevity The stem cells may undergo exhaustion depending on ROS levels [84]. Mitochondrial ROS may activate an adaptive response (mitohormesis), which, as defensive mechanism, promotes health to extend the lifespan through diseases prevention and delay [5, 85]. ROS is integral in the development of physiopathologic events like mitochondrial death signaling [86] and autophagy [87], besides inflammation and infection [55, 88], in which they impart immunological changes. High ROS levels are generated by professional cells (lymphocytes, granulocytes, and phagocytes) in defense against microbes [89, 90]. Differently, any event which contributes to chronic OS or NS, through its increased generation or defective detoxification, dysregulates signaling networks, alters lipids and protein and nucleic acids, and activates mechanisms to face the changes. ROS overproduction hampers damaged nuclear and mitochondrial DNA repair, at multiple steps, contributing to cell genomic instability [91]. ROS are rec- ognized as key modulators in processes that accumulate oxidized molecules chronically, as diabetes, cardiovascular diseases, atherosclerosis, hypertension, ischemia, reperfusion injury, neurodegeneration, and rheumatoid arthritis [17]. Also, ROS participate in cancer development through their Oxidative Medicine and Cellular Longevity 4 effects on cellular proliferation, mutagenesis, and apoptosis inhibition [56]. The cross talk between ROS, p53, and NF-𝜅B plays crucial roles in tumorigenesis. OS is allied with energy metabolism to stimulate the growth of cells transformed by oncogenes or tumor suppressors [92–94]. The deregulated ROS productions in cancer cells and the consequent consti- tutive OS may cause the cellular invasive phenotype [57].fi in animal models, after they have been submitted to disparate patterns, suggests that OS influences old age aspects signifi- cantly [111]. The observations have been extended to humans, even if rate and distribution of mitochondrial mutations may deviate from animals. The conclusions regarding OS effect on aging in animals from mitochondrial genetic manipulations are still conflicting. SOD+/−mice have reduced ROS detox- ifying ability and high ROS level, while they exhibit a quite normal lifespan. OS effect on worms’ lifespan depends on where ROS are produced: high mitochondrial or cytoplasmic levels are associated with increased and decreased lifespan, respectively [109, 112]. It remains to define whether models’ longevity is entirely associated with response to OS, because their lifespan is not affected by modulation of the antioxidant defense. Oxidative Medicine and Cellular Longevity The complex genetic manipulation of the models might weaken their support to the “OS theory of aging.” Interventions to ROS lowering, by both scavenging free radicals and enhancing antioxidant defenses, are widely pro- posed as an antiaging strategy. However, positive association between supplementation with pharmacological or natural compounds and health beneficial effects has not been evi- denced. Some antioxidants may be eventually useless or even harmful [113, 114]. Moreover, a number of ROS-independent mitochondrial dysfunctions appear so involved in aging that doubts arise that OS is the most concrete contributor to fuel aging [115]. Based on the consideration that mitochondrial DNA (mtDNA) is a precise marker to detect total mitochon- drial OS, methods have been developed to measure mtDNA replication defects and the oxidative damage level, simulta- neously. The errors in mtDNA replication and repair, which accumulate through clonal expansion in advanced age, result in a major source of mtDNA mutations, rather than the errors acquired through ROS-dependent vicious cycles [116]. Sum- marizing, ROS are involved in elderly lesions that concern (i) DNA insufficiency, which is partly responsible for premature aging and apoptosis [117]; (ii) RNA involvement in the onset of chronic-degenerative diseases [118]; (iii) nuclear lamins that participate in cell proliferation and longevity [119]. The variations of speed and quality in the aging of each organism may reflect the peculiar alterations that have been accumu- lated in DNA, proteins, and lipids [120], following the organ- ism exposition to chronic stressors. Low ROS levels improve the defense mechanisms by inducing adaptive responses, which contributes to stress resistance and longevity, while high ROS levels induce insufficient adaptive responses, which may contribute to aging onset and progression [121]. In conclusion, accumulated mutations, decreased mito- chondrial energy metabolism and increased OS may signifi Although ROS functions remain difficult to investigate, multiple pharmacological investigations are in progress to maintain ROS homeostasis through both OS decrease and antioxidant defense increase [95, 96]. 3. ROS in Aging and Age-Related Diseases Recently, interest has been focused on stem cells, because their decline impairs tissues homeostasis maintenance, lead- ing to the organism weakening and the age-related diseases [84]. Aging mechanisms have been collected into two classes.hi In conclusion, accumulated mutations, decreased mito- chondrial energy metabolism, and increased OS may signifi- cantly contribute to the human aging and the related diseases. 3. ROS in Aging and Age-Related Diseases Poor knowledge of basic processes in aging interferes with interventions to prevent or delay age-related pathologies, like diabetes, cardiovascular disorders, neurodegenerative disorders, and cancer, which, consequently, impact human independence, general wellbeing, and morbidity [97–99]. Recently, interest has been focused on stem cells, because their decline impairs tissues homeostasis maintenance, lead- ing to the organism weakening and the age-related diseases [84]. Aging mechanisms have been collected into two classes. The first class presents aging as genetically programmed by developmental processes, like the cell senescence, the neuroendocrine alterations, and the immunological alter- ations. The second class presents aging caused by random damage, that is, accumulation of somatic mutations and OS. The separation between the classes is no longer considered clear, because pathways involved in aging often share features with specific diseases [100]. The genetic heredity contributes no more than 3% to aging, while epigenetic processes and posttranslational processes imprint a significantly different aging rate among diverse populations, as well as among diverse anatomical sites of a single organism. In the onset of aging, telomere erosion, OS, and cell senescence are crucial events that originate from the disorganized homeosta- sis of cell metabolism. For example, mitochondria-nucleus interplay [101] and alterations of mitochondrial homeostasis drive age-dependent modifications [102, 103]. Ineffective ROS control on mitochondrial supercomplexes causes ROS signaling alteration, thus mediating cell stress responses towards age-dependent damage [104]. A progressive ROS scavengers decrease shifts aged cells towards a prooxidant status [105, 106]. In parallel, all the suggested methods to prolong lifespan, as caloric restriction and increased activity of SIRT1, share the OS reduction effect [107]. It is known that chronic muscular exercise protects older persons from damage caused by OS and reinforces their defenses against it. On the other hand, acute exercise increases ROS production and damage from ROS [108]. High levels of mitochondrial ROS contribute to aging of genetically modified animals, in a mechanistic way. Superoxide dismutase-deficient animals, SOD1- [109] and SOD3-deficient animals [110], and p66SHC- deficient animals show mitochondrial dysfunctions that gen- erate oxidative damage and related phenotypes, resembling premature aging features. Similarly, mice that overexpress mitochondrial catalase counteract oxidative damage and live longer. The incidence of age-related diseases and pathologies Poor knowledge of basic processes in aging interferes with interventions to prevent or delay age-related pathologies, like diabetes, cardiovascular disorders, neurodegenerative disorders, and cancer, which, consequently, impact human independence, general wellbeing, and morbidity [97–99]. 4. ROS-Dependent Epigenetic Modifications ROS effects on epigenetic mechanisms have been discussed as cause and consequence of aging and age-related DNA modifications [128]. Recent studies demonstrate that global DNA hypomethylation is deeply included in aging gene expression [136], and, at the same time, cancer is the age-related disease that shows the most significant effects of ROS-dependent DNA methylation [137]. Tumor progression is induced by general hypomethy- lation of the DNA and hypermethylation of tumor suppressor genes that lead to aberrant genes expression [138–140]. Abnormal and selective DNA methylation may constitute a potential biomarker and a tool to assess therapeutic treatments at the same time. The data on OS-mediated alterations in DNA methylation, which have been so far obtained, motivate chemoprevention trials, to reduce OS in cancer diseases [141–143]. In human aging, the telomerase reverse transcriptase (hTERT) controls the mitochondrial function and the cellular metabolism, besides the telomeres structure. The enzyme is regulated by DNA methylation. Various observations demonstrate that hTERT may confer major sensitivity towards OS [144] and reduce ROS increase in aging and age-related diseases [145]. Examples of both ROS levels and DNA methylation, which seems to change with age, suggest that they are potentially linked [146, 147]. ROS-induced methylation at SOD2 gene promoter causes the decreased expression of the gene, which may be associated with the disruption of the cardiorespiratory homeostasis, a typical problem of the old humans. Treatments with DNA methylation inhibitors, in preclinical studies, can prevent the hypoxic sensitivity that leads to the respiratory dysfunc- tion [148]. Also, both ROS-induced 8-OHdG and 5-methyl cytosine generate abnormal GC regions in the DNA, which undergo further methylation and oxidation, thus hampering DNA repair enzymes. These regions have been demonstrated to hit gene expression and DNA susceptibility to damage in Alzheimer’s pathology [149]. 4.1. ROS-Induced DNA Methylation. Usually, condensed chromatin structure (heterochromatin) is associated with genes repression by hypomethylation processes, while open chromatin (eu-chromatin) is associated with genes activation by acetylation processes [129]. The epigenetic marking mod- ulates the genes expression by altering the electrostatic nature and the protein binding affinity of chromatin. DNA methyla- tion causes gene silencing through inhibiting the transcrip- tional activators access to the target binding sites, or through activating the methyl-binding protein domains. The last func- tion interacts with histone deacetylases and promotes chro- matin condensation into transcriptionally repressive confor- mations. 4. ROS-Dependent Epigenetic Modifications Intra- and extracellular environments change hereditary cha- racters at the epigenetic level, without altering genes sequence [122]. The interplay between modified histones, DNA methy- lation, regulator noncoding RNAs, and other reversible pro- cesses constitutes the epigenetic machinery that regulates genes transcription and expression [123]. The epigenetic modulation provides the essential and flexible interface 5 Oxidative Medicine and Cellular Longevity between organism and environment, which is essential for all the cell functions. The extent to which epigenome has shaped, and might shape, human populations over generations is investigated by an International Human Epigenome Con- sortium (http://www.ihec-epigenomes.org/). Both long- and short-acting stimuli lead to epigenetic effects that result in 13 being long-term (heritable) or short-term (nonheritable), respectively. These features suggest epigenetic modifications as more attractive target for therapeutic interventions in humans than genetic modification, throughout the entire life [124]. ROS operate modifications on histone and DNA, by acting in interconnected epigenetic phases, during mito- chondrial and nuclear DNA regulation [125, 126]. A clin- ical example of ROS-dependent epigenetic modifications is demonstrated in “nonalcoholic fatty liver” disease. The pathology represents the most common cause of chronic liver disease in western countries and affects one-third of the pop- ulation. Altered redox mechanisms mediate the link between increased accumulation of triglycerides in hepatocytes and epigenetic modifications that are recognized as crucial factors in the pathophysiology of this disease [127]. About the basic mechanisms of ROS action, Afanas’ev proposes that ROS might cause epigenetic activation and repression, by acting like nucleophilic compounds, which accelerate and decelerate hydrolysis and esterification reactions. The hypothesis sug- gests a ROS role different from free radicals, because the last molecules cause an irreversible damage of the compounds with which they react [128]. general genome hypomethylation and specific DNA promot- ers hypomethylation, via the DNMT upregulation and the DNMT complexes generation. Moreover, recent studies show that a ROS-mediated pathway causes repression of the protein kinase C epsilon gene, through its promotor methylation. The events are important in heart hypoxia, in utero, which leads to heightened heart vulnerability to ischemic injury, later in people’s life [134]. 4.2. ROS and DNA Methylation in Aging and Age-Related Diseases. Starting from the observation that both defective genome and DNA repair processes promote phenotypes of premature aging, the “aging epigenetics” has been developed as emerging discipline, which concerns genes and processes impacting aging (Figure 1) [135]. 4. ROS-Dependent Epigenetic Modifications Activating signals Cytokines Growth factor (mitogens, nutrients) Stress (hypoxia, UV, radiation, and chemotherapy) Membrane bound NADPH oxidases Antioxidants and detoxicating enzymes SOD, catalase, glutathione, peroxiredoxin, thioredoxin reductase, and peroxidase NADPH oxidase Epigenetic machinery A methylation, histone modification, and noncoding RNAs Damage Nucleic acids, lipids, and protein ROS sensitive proteins (oxidative interface) Age-related diseases Accelerated aging Figure 1: Schematic representation of ROS signaling in physiological and pathological conditions. Low and medium ROS levels produced by mitochondria and NADPH oxidase activate cell ROS sensitive proteins and epigenetic machinery. High ROS level causes nucleic acids, lipid, and proteins damage possibly involved in accelerated aging, cell death, and age-related diseases. Figure 1: Schematic representation of ROS signaling in physiological and pathological conditions. Low and medium ROS levels produced by mitochondria and NADPH oxidase activate cell ROS sensitive proteins and epigenetic machinery. High ROS level causes nucleic acids, lipid, and proteins damage possibly involved in accelerated aging, cell death, and age-related diseases. 4. ROS-Dependent Epigenetic Modifications Hypo- and hypermethylation stages occur consec- utively, indicating how DNA methylation and the correlate mechanisms of DNA binding are complex. ROS-dependent modifications are related to DNA methylation and demethy- lation, directly or indirectly. The NF-𝜅B binding to DNA, which is methylation dependent, results in being altered in SOD (Cu/Zn)-deficient mice. The observation associates ROS-dependent modifications with altered methylation pro- cesses, although indirectly, and suggests that modifications linked to altered redox mechanisms may fit into cell signaling pathways [130]. Also, the oxidation of deoxy-guanine of CpG nucleotides to 8-hydroxy-2󸀠-deoxyguanosine (8-OHdG) is believed to be a surrogate marker of oxidative damage, in various human diseases [131]. The 8-OHdG adducts interfere with DNA restriction nucleases and DNA methyl transferases (DNMT), thus altering transcription factors binding to DNA and causing general DNA hypomethylation. In vitro [132] and in vivo [133] studies demonstrate that ROS induce In complex, ROS are involved in DNA methylation proc- esses in different conditions, occurring in the human aging. The epigenetic machinery operates as OS sensor, which con- tributes to the OS control and, at the same time, orches- trates the progressive homeostasis impairment, which shapes the cardiovascular, respiratory, and nervous systems of old human beings [146]. The ROS signaling in the DNA methy- lation during the aging process deserves to be more deeply studied. Oxidative Medicine and Cellular Longevity 6 Activating signals Cytokines Growth factor (mitogens, nutrients) Stress (hypoxia, UV, radiation, and chemotherapy) Mitochondria ROS Membrane bound NADPH oxidases ROS pool ROS levels + − Antioxidants and detoxicating enzymes SOD, catalase, glutathione, peroxiredoxin, thioredoxin reductase, and peroxidase NADPH oxidase Accelerated aging Cell death Age-related diseases ROS sensitive proteins (oxidative interface) Damage Nucleic acids, lipids, and proteins Epigenetic machinery DNA methylation, histone modification, and noncoding RNAs Figure 1: Schematic representation of ROS signaling in physiological and pathological conditions. Low and medium ROS levels produced by mitochondria and NADPH oxidase activate cell ROS sensitive proteins and epigenetic machinery. High ROS level causes nucleic acids, lipid, and proteins damage possibly involved in accelerated aging, cell death, and age-related diseases. 5. ROS in Cell Senescence Senescent cells endowed with the semiselective marker of senescence p16 drive age-related pathologies, which are delayed or prevented by the selective elimination of the senescent cells [169]. A partial list of suggested markers of cell senescence in human tissues, both aged and affected by age-related pathologies, is reported in Table 2 [170–197]. Lungs show a typical example of cell senescence associated with the progressive, age-related organ dysfunction. The OS generated by the potent cigarette oxidants is a key element in the pathogenesis of the pulmonary emphysema, induced by the chronic smoking. The fibroblasts that provide essential support and matrix for lung integrity show reduced prolifer- ation rate and increased SA-𝛽gal activity in patients affected by pulmonary emphysema. These senescent fibroblasts con- tribute to the lung disease by affecting the tissue homeostasis. Also, senescent features of the endothelial cells in chronic smokers associate with premature vessels atherosclerosis. In patients with severe coronary artery disease, OS accelerates the senescence of endothelial cells, which is related to risk factors for cardiovascular disease [198]. A further example (ii) Signaling Pathways via Ras, p53, p21, and p16. The path- ways generate ROS, which act as signaling molecules, without causing oxidative DNA damage. ROS result as a tightly regu- lated signaling process for the induction of the cell senescence [157]. (iii) Autophagy. High ROS levels mediate p53 activation that induces autophagy inhibition. This event generates mito- chondrial dysfunction, which in turn generates cell senes- cence. The autophagy inhibition causes the senescent cells to aggregate oxidized proteins and protein carbonyls with products of lipid peroxidation and protein glycation into the lipofuscin [158]. (iii) Autophagy. High ROS levels mediate p53 activation that induces autophagy inhibition. This event generates mito- chondrial dysfunction, which in turn generates cell senes- cence. The autophagy inhibition causes the senescent cells to aggregate oxidized proteins and protein carbonyls with products of lipid peroxidation and protein glycation into the lipofuscin [158]. (iv) miR-210 and miR-494. The induction of these microRNAs by ROS generates mitochondrial dysfunction and autophagy inhibition [159]. (iv) miR-210 and miR-494. The induction of these microRNAs by ROS generates mitochondrial dysfunction and autophagy inhibition [159]. The (iii) and (iv) pathways generate vicious loop cycles in ROS production. Autophagy inhibition causes lipofuscin accumulation, which activates further autophagy impairment and ROS production, consequently. 5. ROS in Cell Senescence provoked by endogenous stimuli, is distinct from “stress- induced premature senescence,” which is provoked by exoge- nous stimuli. The two processes share molecular and func- tional features, although they are dependent, or not, on telomeres status, respectively. Intrinsic and extrinsic events can induce either the cell senescence or the apoptosis process, depending on the level of the impairment of the cell home- ostasis [150] and the p53 activity [47]. The molecules secreted by senescent cells (secretoma) cooperate deeply to maintain the tissues homeostasis, through autocrine and paracrine activities [151], by acting at multiple levels: epigenome [152], gene expression, protein processing, and metabolic control [153]. Moreover, specific mitochondrial pathways contribute to priming the senescence process, through the alteration of the mitochondrial redox state [6, 151]. The senescence secre- toma acts in physiological and pathological events, as tissue remodeling during embryogenesis, tissue repair in wound healing, and induction of aging, as well as age-related diseases of different organisms. The secretoma develops beneficial effects on carcinogenic DNA lesions of precancerous cells, The cell senescence has indicated the irreversible G1 growth arrest of normal primary cells, which occurs after the cells have accumulated time-dependent damage, during extensive culture passages (“replicative senescence”). The cells resist apoptosis and face malignant progression through cytosta- sis, thus causally contributing to cell senescence induction and maintenance. The senescent cells are able to diversify constantly, like cancer cells, but missing proliferation as a driver [7, 9]. Large and flat shape, rich cytoplasmic and vacuolar granularity, high levels of lysosomal 𝛽-galactosidase activity (SA-𝛽gal), p16, p21, macroH2A, IL-6, phosphory- lated p38MAPK, and “double-strand breaks” are the most common senescent cells features in in situ assays [9]. The exact mechanisms underlying the cell senescence onset and stabilization are still obscure. OS, mitochondrial deteriora- tion, DNA damage, oncogenes expression, and loss of tumor suppressor genes, like PTEN, RB1, NF1, and INPP4, can induce cell senescence [9]. “Replicative senescence,” which is 7 Oxidative Medicine and Cellular Longevity Response,” the mitochondrial p38-MAPK replenishes the short-lived DNA damage foci, via a ROS feedback loop, and induces the senescent secretoma [161].h by both preventing their uncontrolled cell proliferation and reacting with specific anticancer compounds [154]. However, the secretoma may provide indispensable cytokines for the cancer cells growth, thus promoting tumorigenesis in definite conditions, which are partly related to the cellular meta- bolic state [155]. 5. ROS in Cell Senescence Senescent cells endowed with the semiselective marker of senescence p16 drive age-related pathologies, which are delayed or prevented by the selective elimination of the senescent cells [169]. A partial list of suggested markers of cell senescence in human tissues, both aged and affected by age-related pathologies, is reported in Table 2 [170–197]. Lungs show a typical example of cell senescence associated with the progressive, age-related organ dysfunction. The OS generated by the potent cigarette oxidants is a key element in the pathogenesis of the pulmonary emphysema, induced by the chronic smoking. The fibroblasts that provide essential support and matrix for lung integrity show reduced prolifer- ation rate and increased SA-𝛽gal activity in patients affected by pulmonary emphysema. These senescent fibroblasts con- tribute to the lung disease by affecting the tissue homeostasis. Also, senescent features of the endothelial cells in chronic smokers associate with premature vessels atherosclerosis. In patients with severe coronary artery disease, OS accelerates the senescence of endothelial cells, which is related to risk factors for cardiovascular disease [198]. A further example 5.1. Cell Senescence in Aging and Age-Related Diseases (ROS Effect). The “replicative cell senescence” is considered an aging hallmark on the basis of two motives: (1) the senes- cent cells accumulate in organismal tissues, by rate and proportion, which parallel the age advancement; (2) the senescent cells accelerate the age-related decrease of tissue regeneration, through the depletion of stem and progenitors cells [8, 97]. While the sequence of proliferative arrest (senes- cence), recruitment of immune phagocytic cells (clearance), and promotion of tissue renewal (regeneration) results in being beneficial upon a damaged tissue, for instance, the sequence is inefficiently completed in aging tissues, causing senescent cells to undergo chronic accumulation [163]. Also, a delicate balance exists between cell senescence positive effects on tumor suppression and negative effects on aging related processes [164]. The transcription factor and tumor suppressor p53 are involved in DNA repair and cellular stress response, as well as cellular cycle control. In addition, p53 modulates both the cell senescence and the aging process, through the coordination of specific cellular pathways [165, 166]. It is not clear whether p53 mechanisms in cell senes- cence and aging are common [160]. An increased senescence secretoma causes detrimental effects over the years and contributes to the typical disruption of aged tissues [8, 167, 168]. 5. ROS in Cell Senescence Cause-effect relationships between cellular ROS production and cell senescence have been investigated through diverse pathways that comprise the following. The occurrence of the ROS role in cell senescence onset and maintenance might be relevant for therapeutic interven- tions, which aim to modulate ROS levels in cancer cells, as well as in aging processes [156]. Human kidney dysfunctions exemplify progressive stages of ROS-induced cell senescence. ROS act like a sensor in regulating the oxygen-dependent gene expression of the kidney and play a leading role in the inflammatory processes, to which the organ is especially sensitive [162]. In conclusion, the ROS signaling has high- lighted key factors for the cell senescence induction and maintenance, which are the object of intensive investigations. (i) Mitochondrial DNA (mtDNA) Damage. ROS contribute to cellular senescence onset and progression by damaging mtDNA directly or in synergy with modifications of the telomerase reverse transcriptase (TERT) enzyme and the p53 and Ras pathways activity [9]. Also, ROS production by serial signaling through GADD45-MAPK14 (p38MAPK)- GRB2-TGFBR2-TGFb is both necessary and sufficient for the stability of growth arrest, during the establishment of the senescent phenotype [156]. 5.1. Cell Senescence in Aging and Age-Related Diseases (ROS Effect). The “replicative cell senescence” is considered an aging hallmark on the basis of two motives: (1) the senes- cent cells accumulate in organismal tissues, by rate and proportion, which parallel the age advancement; (2) the senescent cells accelerate the age-related decrease of tissue regeneration, through the depletion of stem and progenitors cells [8, 97]. While the sequence of proliferative arrest (senes- cence), recruitment of immune phagocytic cells (clearance), and promotion of tissue renewal (regeneration) results in being beneficial upon a damaged tissue, for instance, the sequence is inefficiently completed in aging tissues, causing senescent cells to undergo chronic accumulation [163]. Also, a delicate balance exists between cell senescence positive effects on tumor suppression and negative effects on aging related processes [164]. The transcription factor and tumor suppressor p53 are involved in DNA repair and cellular stress response, as well as cellular cycle control. In addition, p53 modulates both the cell senescence and the aging process, through the coordination of specific cellular pathways [165, 166]. It is not clear whether p53 mechanisms in cell senes- cence and aging are common [160]. An increased senescence secretoma causes detrimental effects over the years and contributes to the typical disruption of aged tissues [8, 167, 168]. Senescence-associated biomarkers Telomeres length, SA-𝛽Gal, p16, and p21 Telomeres length, SA-𝛽Gal, p16, and p21 Telomeres length, SA-𝛽gal [172, 173] SA-𝛽gal, p21, p16, DEP1, NTAL, EBP50, STX4, VAMP3, ARMX3, B2MG, LANCL1, VPS26A, and PLD3 [174, 175] SA-𝛽gal [176] SA-𝛽gal [177] SA-𝛽gal, p21 [178] SA-𝛽gal, p16, and p21 [179] SA-𝛽gal, Glb1, and HP1g [154, 180] Telomeres length, SA-𝛽gal [181] Short telomeres [182] Telomeres length, IGFBP5, and SA-𝛽gal [183, 184] Telomere length, p16 [185] Telomere length, IGFBP-5, SA-𝛽-gal, and p21 [183, 186] p16 [187, 188] SA-𝛽-gal [189, 190] Telomere length, p16, p21, and SA-𝛽gal [191, 192] p16, p21 [192, 193] Telomere length, IGFBP-3, IGFBP-rP1, p16INK4a, and p21 [194, 195] SA-𝛽gal [196, 197] mechanisms, although ROS may act as tumor promoters in definite conditions [48]. With the cell senescence and aging controlled by cells and cellular environment, the possibility is suggested that the two processes may be subjected to interventional therapies [203, 204]. of aging dysfunction related to cell senescence is shown by the scaffolding protein Caveolin 1 (Cav1), which controls molecular signaling in caveolar membranes. Cav1 promotes cellular senescence in age-related pathologies, by mediating p53 activation with EGF modulation, focal adhesion, and small Rho GTPase-dependent signaling. The upregulation of the Cav1 promoter by high ROS levels contributes to explaining how OS promotes cell senescence effects in aging and age-related diseases [198]. In addition, the interplay between different conditions of mitochondrial homeostasis and ROS-dependent signaling pathways contributes to aging process, through the cell senescence induction and stabi- lization [199]. Yet ROS-independent signaling pathways link dysfunctions in mitochondria and aging, through the cell senescence process [6, 151]. As a new approach, preclinical and clinical studies demonstrate the therapeutic effects of the aging inhibitor rapamycin, whose signaling pathway is involved in cellular senescence [160, 200]. of aging dysfunction related to cell senescence is shown by the scaffolding protein Caveolin 1 (Cav1), which controls molecular signaling in caveolar membranes. Cav1 promotes cellular senescence in age-related pathologies, by mediating p53 activation with EGF modulation, focal adhesion, and small Rho GTPase-dependent signaling. The upregulation of the Cav1 promoter by high ROS levels contributes to explaining how OS promotes cell senescence effects in aging and age-related diseases [198]. In addition, the interplay between different conditions of mitochondrial homeostasis and ROS-dependent signaling pathways contributes to aging process, through the cell senescence induction and stabi- lization [199]. Yet ROS-independent signaling pathways link dysfunctions in mitochondria and aging, through the cell senescence process [6, 151]. 5. ROS in Cell Senescence All the factors (i), (ii), (iii), and (iv) may add to DNA damage and dysfunctions of both mitochondria and cell metabolism homeostasis [159]. In vitro and preclinical experiments show that ROS decreasing interventions influence cell senescence progression, via the slowdown of telomere shortening and the extension of the cell lifespan. Replicative telomere exhaustion, DNA damage, and OS prime the cell senescence by sharing the activation of the “DNA Damage Response.” ATM or ATR kinases of these signaling pathways cause p53 stabilization and tran- scriptional activation of the p53 target, p21 [9]. p53 triggers cell cycle arrest by upregulating p21, which inhibits the cell cycle regulator cyclin-dependent kinases Cdk4 and Cdk2 [159]. Whereas high OS levels induce the prosenescence function of p53, the mild OS levels that are induced by the physical exercise in humans have a positive effect on cell and mitochondrial homeostasis. p53 exerts a dual effect on cell senescence because of its ability to both decrease and increase the cellular OS level [160]. In parallel to “DNA Damage Oxidative Medicine and Cellular Longevity 8 Table 2: Clinical examples of senescence-associated biomarkers detected in organs and tissues of patients affected by age-related diseases. Organ/tissue Senescence-associated biomarkers Clinical references Cardiovascular diseases Aged vascular tissues Telomeres length, SA-𝛽Gal, p16, and p21 [170, 171] Atherosclerosis Systolic heart failure Malignant tumors Lung cancer Telomeres length, SA-𝛽gal [172, 173] Breast cancer SA-𝛽gal, p21, p16, DEP1, NTAL, EBP50, STX4, VAMP3, ARMX3, B2MG, LANCL1, VPS26A, and PLD3 [174, 175] Neuroblastoma SA-𝛽gal [176] Astrocytoma SA-𝛽gal [177] Mesothelioma SA-𝛽gal, p21 [178] Melanoma SA-𝛽gal, p16, and p21 [179] Prostate cancer SA-𝛽gal, Glb1, and HP1g [154, 180] Liver cancer Telomeres length, SA-𝛽gal [181] Colorectal cancer Short telomeres [182] Fibrosis Idiopathic pulmonary fibrosis Telomeres length, IGFBP5, and SA-𝛽gal [183, 184] Cystic fibrosis Telomere length, p16 [185] Liver fibrosis Telomere length, IGFBP-5, SA-𝛽-gal, and p21 [183, 186] Renal fibrosis p16 [187, 188] Neurological disorders Alzheimer’s disease SA-𝛽-gal [189, 190] Other diseases Chronic obstructive pulmonary disease Telomere length, p16, p21, and SA-𝛽gal [191, 192] Pulmonary hypertension p16, p21 [192, 193] Emphysema Telomere length, IGFBP-3, IGFBP-rP1, p16INK4a, and p21 [194, 195] Benign prostatic hyperplasia SA-𝛽gal [196, 197] Table 2: Clinical examples of senescence-associated biomarkers detected in organs and tissues of patients affected by age-related diseases. Organ/tissue Senescence-associated biomarkers Clinical references Cardiovascular diseases Aged vascular tissues Telomeres length, SA-𝛽Gal, p16, and p21 [170, 171] Atherosclerosis Senescence-associated biomarkers As a new approach, preclinical and clinical studies demonstrate the therapeutic effects of the aging inhibitor rapamycin, whose signaling pathway is involved in cellular senescence [160, 200]. 5.2. Epigenetic Mechanism in Cell Senescence (ROS Involve- ment). The epigenetic control of acute and chronic cellular senescence allows for the two processes that are involved in various conditions that lead to the cells longevity preventing cell death and tumorigenesis [205]. The abrogation of tumor suppressor pathways, as p53 and p16/Rb, bypasses the cell senescence, thus leading to the tumorigenic phenotypes acquiring [206]. The mechanisms that balance the tran- scriptional state of the chromatin are not fully understood. Some regulative changes involve the histone proteins that coordinate the DNA accessibility, through transcription fac- tors, besides the DNA replication and repair. The Polycomb Repressor Complex 2 (PRC2) initiates and preserves specific histone methylations, thus acting as an epigenetic mark that mediates targeted genes [207]. The repression of the histone activity by the Polycomb Group (PcG) proteins causes gene silencing, but it can be countered by specific demethylases, In conclusion, cell senescence reduces the age-related tumor development and contributes to human aging, sug- gesting that aging might be switched for tumorigenesis [201, 202]. ROS may modulate tumor suppression process, which is induced by the senescence, thus participating in anticancer 9 Oxidative Medicine and Cellular Longevity but not in the “stress-induced premature senescence” by ROS [228]. which erases the methyl mark [208]. The upregulation of many PRC target genes leads to global epigenetic changes [209–211]. Specific transcription factors [212], as well as long noncoding RNAs [213], are involved in the recruitment performed by PRC. PRC2 takes a crucial part in silencing the locus of p16, the marker that is upregulated during cell senes- cence [212]. The reversal of chromatin epigenetic pattern via deacetylation, demethylation, and dephosphorylation is sig- nificantly involved in underscoring both flexible and dynamic nature of histone modifications [214]. The histone demethy- lases JMJD3 produce diverse outputs of biological function, depending on the action of their transcriptional complexes. Different expression of these demethylases, which have tumor suppressor activities during the “stress-induced senes- cence” [215, 216], is reflected into cellular phenotype changes and variations associated with cellular senescence [217]. The JMJD3 gene is located near the p53 tumor suppressor gene, that is, a genomic area that is frequently lost in various malig- nancies. Senescence-associated biomarkers It is unclear if the modifications of miRNA profiles are mostly involved in pathological changes onset or if they mark the senescence end, which leads to the organ aging and dysfunction. Altered expression in miRNA activity has been observed in elderly people, as in the case of miR-34a, which belongs to a family with conserved functions in controlling aging and age-related diseases [203, 231, 232]. miR-34a targets ROS scavenger enzymes inducing OS [159]. The miR-34a upregulation or overexpression has been associated with cell proliferation inhibition, subsequent cell senescence induction, and pre- mature death, in both endothelial progenitor and mature cells. miR-34a causes memory function impairment when it is upregulated in aged mice and in models for Alzheimer’s disease (AD), while miR-34a targeting restores the memory function [233]. Also, the miR-34 mutation of the loss- of-function delays the age-related decline markedly, thus resulting in extended lifespan and increased resistance to the heat and the OS. The human miR-34a is downregulated in Parkinson’s disease brain, while it is upregulated in AD brains [234] and in plasma of Huntington’s disease patients [235]. 5.2.1. Noncoding RNA. Latest genomics tools and sequenc- ing approaches have helped unravel large chromosomes stretches, which were previously deemed not transcribed [218, 219]. These sequence regions contain noncoding RNA (ncRNA), which is known as long lncRNAs, and short ncRNAs. Among short ncRNAs, the microRNAs (miRNAs) have emerged as being able to control the gene expression, either by blocking targeted mRNA translation or by mRNA degrading [220, 221]. Recently, ncRNA role is gaining more importance in age-associated dysfunctions as cardiovascu- lar diseases [222, 223]. The senescence-associated lncRNAs are differentially expressed in proliferating and senescent fibroblasts, as assessed by RNA sequencing [224–226]. Tox- icological studies associate increased ROS production with increased expression of a set of 115 lncRNAs, which signifi- cantly affect p53 signaling pathway [227]. A mitochondrial- transcribed lncRNA is induced in aorta and endothelial cells aging, during the “replicative vascular senescence,” which is partly responsible for age-associated cardiovascular diseases, Several miRNA families are modulated by ROS in the development of mitochondria-mediated cell senescence, which are, indirectly or directly, implicated in human pathologies. Little is known about the roles of ROS- modulated miRNAs in cell function. Senescence-associated biomarkers The SIRT1 histone deacetylase (SIRT1) is a known regulator of age-related diseases that regulates the senescence secretoma components, by silencing their promoter regions epigenetically. SIRT1 plays a pivotal role in stress modulation also through p53 deacetylation, acting against aging and age- related diseases. As indicated above, the high ROS levels activate p53, which, in turn, activates p53-mediated apoptosis and cell senescence. Moreover, SIRT1 regulates the ROS- dependent FOXO factors, which are responsible for cell growth, proliferation, and longevity. The characteristic ROS increase during aging may be responsible for the decreased SIRT1 activity, which facilitates the senescent-like phenotype. SIRT1 causes oxidant effects, as well as antioxidant effects, by acting on epigenetic modifications, which include acetylation and deacetylation (see references in [128, 146]). Experiments on cell senescence induction show different molecular mech- anisms in acute versus chronic senescent cells. A better knowledge of the order in which epigenetics mechanisms change during the cell senescence progression, from initial towards full senescence, is believed to be vital for finding therapies against age-related disorders [9]. 5.2.2. microRNA (miRNA, miR). Normal cellular develop- ment and homeostasis are under the control of miRNAs, throughout the entire life [229], since miRNAs regulate the gene expression in biological processes as proliferation, development, differentiation, and apoptosis. Yet several miR- NAs families control cell senescence at multiple levels, by regulating the autophagy process and the gene expression involved in ATP and ROS production. Some miRNAs may induce ROS production that generates a self-sustaining ROS vicious cycle [159]. miRNAs constitute a connection between aging, cell senescence, and cancer. The miRNAs dysregula- tion causes the activation of pathways they normally repress. The event may activate aberrant pathways and also aging mechanism in young individuals [222]. Although current studies are monitoring miRNA tissues and systemic alter- ations, instead of miRNA changes through lifespan and metabolic modifications, several profiles of miRNA expres- sion demonstrate changes during the aging. As an example, miR-29, which targets the genes of type IV collagen and maintains the structure of the extracellular matrix, increases in elderly mice, thus causing collagen decreasing, a tissues basement membranes weakening [230]. Only few miRNAs have been directly linked to age-related changes in cellular and organ functions, whereas many miRNAs have been directly connected with disease states. Senescence-associated biomarkers The molecular mecha- nisms that control neuronal response to OS have been deeply studied in different strains of senescence accelerated mice, based on the consideration that OS plays a critical role in AD etiology and pathogenesis. OS upregulates a group of miR- NAs (miR-329, miR-193b, miR-20a, miR-296, and miR-130b), which is associated with affecting 83 target genes. Among the Oxidative Medicine and Cellular Longevity 10 no longer considered as mere metabolic byproducts but are believed to be a “redox biology” that regulates physiological functions, including signal transduction, gene expression, and proliferation [37]. Firstly, it has been evidenced that the DNA damage caused by ROS acting as mutating agents con- tributes to the induction and maintenance of the cell senes- cence process [9, 156]. More recently, particular attention has been focused on the ROS involvement as signaling molecules in cell senescence induction, without causing DNA damage. Signaling pathways via Ras, p53, p21, and p16 have been defined to generate ROS, which may act as tightly regulated process contributing to the cell senescence induction [20, 157, 158]. Cause-effect relationships between cell ROS production and cell senescence have been investigated through diverse pathways that include the field of mitochondrial DNA and autophagy inhibition and the effects of the microRNAs miR- 210 and miR-494 in various mitochondrial processes [159]. These pathways highlight ROS contribution to prime cell senescence at diverse levels, among which epigenetic level is attracting more and more attention in studies aimed at the senescence control [227, 233, 236]. Indeed, the epigenetic modulation provides the essential and flexible interface between the organisms and the environment, which results in being essential for all the cell functions [122, 123, 129], throughout the lifespan [135–137]. A major breakthrough in the last decades has been the understanding that epigenetics contribute to human diseases development. genes, mitogen-activated protein kinase signaling pathway has been suggested to play a role in pathogenesis of neurode- generative diseases [233]. OS effects on vascular homeostasis, including angiogenesis in physiological processes and age- related diseases, are largely studied in human umbilical vein endothelial cells (HUVECs), considering that miRNAs modulate endothelial cells response to OS. ROS induce the expression of miR-200 family members (miR-200c, miR- 141, miR-200a, miR-200b, and miR-429), which determines apoptosis and cell senescence both in HUVEC cells and in a model of hind limb ischemia, which shows OS-mediated mechanism [236]. Senescence-associated biomarkers The miR-200 family plays a causative role in the vascular diabetic inflammatory phenotype in a diabetic model and in the human vasculopathy disease, suggesting that miR-200 inhibition might represent a therapeutic target to prevent OS negative effects on cell function and survival [146]. Also, miR-200 family has been extensively studied in epithelial-to-mesenchymal transition of cancer cells [236]. Lately, miR-760 and miR-186 upregulation has been asso- ciated with replicative senescence in human lung fibroblast cells. These miRNAs cooperate to induce senescence through the ROS-p53-p21Cip1/WAF1 pathway, which depends on the ROS generated by the downregulation of the protein kinase 2 (CK2𝛼). A better understanding of the mechanisms of CK2 regulation might provide new therapeutic options to restore the function of lungs in aged people. An example of the increasing evidence that miRNAs are critically involved in the posttranscriptional regulation of cell functions, including the ROS signaling modulation, is underlined in Figure 2. In parallel, the “OS theory of aging” remains the most documented mechanistic hypothesis of aging, although it does not necessarily imply ROS imbalance as the earliest trigger or the main cause of aging [98–103]. Therapeutic ROS modulation is suggested as relevant in aging and related events [95, 96, 114]. Also, the senescent cells have been identified as a novel potential therapeutic target in the aging and age-related diseases [169, 171]. Further research is needed to define when and where cell senescence results in being favorable or unfavorable to organismal health. Both pro- and antisenescent therapies can be equally helpful, when they are opportunely modulated and balanced. Prosenescent therapies contribute to minimize damage in the cancer disease and in the active tissue repair by limiting proliferation and fibrosis, respectively, while antisenescent therapies may help to eliminate accumulated senescent cells and to recover tissue function. The current research points to a double objective: to define the changes about the redox-sensitive cell pathways and to define the OS role in linking environmental factors with epigenetic modifications. 6. Conclusion and Future Perspectives The multifactorial and inexorable phenomenon of aging worsens the human functions at multiple levels, causing a gradual reduced ability to resist stress, damage, and illness. Healthy aging appears to be an ideal healthcare priority that entails a better understanding of aging, with the aim of slowing down the process and preventing or even treating its related pathologies [200]. Indeed, genetic insights com- bined with findings from animal and cellular models have advanced our understanding of pathways that lead to age- related features, highlighting possible interventional targets [2–5]. The cellular senescence process is considered an aging hallmark, because it drives the cells through longevity, by hampering tumorigenesis and cell death, and is involved in many age-related diseases [97, 205, 206]. The cell senescence is a feature that characterizes somatic cells, except for most tumor cells and certain stem cells [6–10]. The senescent cells produce a specific secretoma that cause beneficial effects, through its autocrine and paracrine mechanisms. When the senescent cell program is inefficiently developed, as it occurs during the aging, the secretoma causes detrimental effects [151–153, 167, 168, 199]. In the recent years, evidence has been accumulating that ROS, which include H2O2, superoxide, anion, and hydroxyl radicals, generated from both intrinsic and extrinsic events, inhibit cell growth and induce cell death and senescence in a context-dependent manner [157, 236]. Through the understanding of the ROS role as signaling molecules in a myriad of signaling pathways, ROS levels are p gi Particular emphasis is addressed to novel mechanism of ROS and epigenetics in cell senescence and aging [160, 165, 166]. The histone demethylases network is often synergizing with the action of histone deacetylases, histone methyl transferases, and various nuclear transcriptional complexes, thus ensuring that the chromatinic environment is correct for the cell [128, 146]. Preclinical and clinical examples of ROS-dependent epigenetic modifications [125–127, 130– 134, 138] extend their effects to aging [135, 136] and age- related diseases [137, 142–144, 146–149], particularly towards cancer disease [139–141, 145]. 6. Conclusion and Future Perspectives Among the noncoding RNAs, miRNAs families provide a broad silencing activity of mRNA 11 Oxidative Medicine and Cellular Longevity Oxidative stress ROS Antioxidants p53 DNA damage Mitochondrial dysfunction Apoptosis Age miR34a Aging Prooxidant genes Antioxidant genes Senescence + − SIRT1 (metabolic/oxidative balance) PNUT (DNA protection) Beneficial effect (i) Developmental senescence (ii) Tumor suppression (iii) Wound healing (iv) Liver fibrosis (v) Cardiac fibrosis Detrimental effect (i) Age-related phenotypes (ii) Tumor promotion (iii) Obesity and diabetes (iv) Atherosclerosis (v) Other cell senescence related diseases Figure 2: ROS-mediated senescence. Besides causing DNA damage and mitochondria dysfunction, OS activates p53 that, in turn, induces prooxidant genes and imbalances antioxidant genes induction. The set of alterations caused by ROS lead to induction of cell senescence, which, in turn, can develop both positive and negative effects; miR34a expression increases with aging in many tissues downregulating SIRT1 protein activity (a longevity promoting factor) and PNUT protein (a DNA protecting factor which prevents telomere attrition and is involved in tissues repairs). Antioxidant genes SIRT1 (metabolic/oxidative balance) PNUT (DNA protection) Aging Detrimental effect (i) Age-related phenotypes (ii) Tumor promotion (iii) Obesity and diabetes (iv) Atherosclerosis (v) Other cell senescence related diseases Beneficial effect (i) Developmental senescence (ii) Tumor suppression (iii) Wound healing (iv) Liver fibrosis (v) Cardiac fibrosis Figure 2: ROS-mediated senescence. Besides causing DNA damage and mitochondria dysfunction, OS activates p53 that, in turn, induces prooxidant genes and imbalances antioxidant genes induction. The set of alterations caused by ROS lead to induction of cell senescence, which, in turn, can develop both positive and negative effects; miR34a expression increases with aging in many tissues downregulating SIRT1 protein activity (a longevity promoting factor) and PNUT protein (a DNA protecting factor which prevents telomere attrition and is involved in tissues repairs). targets, in a sequence dependent fashion that modulates the stress response [159]. Accumulating evidences show that stressors, including ROS, potentially alter the function of miRNA-processing in aging organisms, which renders the cells even more prone to stress, linking aging and cancer. Several miRNAs families induce ROS level increase in aging or target factors involved in the ROS signaling. In addition, ROS increase highly correlates with a specific miRNA dys- regulation, which mediates the cross talk between p53, NF- 𝜅B p65, and ROS. All these events have been associated with cell senescence [203, 231, 232]. 6. Conclusion and Future Perspectives At the same time, certainly several miRNAs families are modulated by ROS in the devel- opment of mitochondria-mediated cell senescence, which are, indirectly or directly, implicated in human pathologies [159, 233, 236]. Because epigenome is so tightly regulated and complex, understanding individual modifications and their network of interaction offers the potential to design drugs that are very effective therapies against a number of diseases [124, 203–205, 219–222]. More reliable OS biomarkers, as well as OS related epigenetic mechanisms, have emerged over the last years as potentially useful tools to design therapeutic approaches aimed at modulating in vivo enhanced OS. References Tamarit, and J. 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https://openalex.org/W2782868189	https://bmcgenomics.biomedcentral.com/track/pdf/10.1186/s12864-017-4403-1	English	null	Insights into teleost sex determination from the Seriola dorsalis genome assembly	BMC genomics	2,018	cc-by	9,798	© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Purcell et al. BMC Genomics (2018) 19:31 DOI 10.1186/s12864-017-4403-1 Purcell et al. BMC Genomics (2018) 19:31 DOI 10.1186/s12864-017-4403-1 Open Access * Correspondence: Catherine.Purcell@noaa.gov 1Southwest Fisheries Science Center, National Marine Fisheries Service, 8901 La Jolla Shores Drive, La Jolla, CA 92037, USA Full list of author information is available at the end of the article Abstract Background: The assembly and annotation of a genome is a valuable resource for a species, with applications ranging from conservation genomics to gene discovery. Genomic resource development is especially important for species in culture, such as the California Yellowtail (Seriola dorsalis), the likely candidate for the establishment of commercial offshore aquaculture production in southern California. Genomic resource development for this species will improve the understanding of sex and other phenotypic traits, and allow for rapid increases in genetic improvement for and economic gain in culture production. Results: We describe the assembly and annotation of the S. dorsalis genome, and present resequencing data from 45 male and 45 female wild-caught S. dorsalis used to identify a sex-determining region and marker in this species. The genome assembly captured approximately 93% of the total 685 MB genome with an average coverage depth of 180×. Using the assembled genome, resequencing data from the 90 fish were aligned to place boundaries on the sex-determining region. Sex-specific markers were developed based on a female-specific, 61 nucleotide deletion identified in that region. We hypothesize that Estradiol 17-beta-dehydrogenase is the putative sex-determining gene and propose a plausible genetic mechanism for ZW sex determination in S. dorsalis involving a female-specific deletion of a transcription factor binding motif that may be targeted by Sox3. Conclusions: Understanding the mechanism of sex determination and development of assays to determine sex is critical both for management of wild fisheries and for development of efficient and sustainable aquaculture practices. In addition, this genome assembly for S. dorsalis will be a substantial resource for a variety of future research applications. rds: California yellowtail, Estradiol, Genomic resources, hsd17b1, Seriola dorsalis, Sex-determining region tilapia (Oreochromis and Tilapia spp.), and hybrid Striped Bass (Morone saxatilis x M. chrysops) [2], however, there is growing interest in broadening the species variety in aquaculture production. The fishes of the genus Seriola (S. dorsalis, S. dumerili, S. lalandi, S. rivoliana, S. quinquera- diata), collectively known as amberjacks or yellowtail, are of interest due to their high value in the sashimi industry, and are already highly valued in global aquaculture pro- duction. While S. rivoliana is already in U.S.-based cul- ture, the native California Yellowtail, Seriola dorsalis, is a focus for imminent development of offshore aquaculture in southern California and Mexico. Insights into teleost sex determination from the Seriola dorsalis genome assembly Catherine M. Purcell1*, Arun S. Seetharam2, Owyn Snodgrass1, Sofia Ortega-García3, John R. Hyde1 and Andrew J. Severin2 Background l Aquaculture production has become increasingly import- ant to satisfy seafood and fishery product demands. The fraction of the global seafood market produced using aquaculture has steadily grown, and for the first time in 2014, aquaculture production provided more fish than capture fisheries [1]. Despite this growth, aquaculture de- velopment in the U.S. has lagged behind other countries, ranking 17th in aquaculture production while importing greater than 80% of seafood consumed [1, 2]. To date, most U.S.-based finfish aquaculture has focused on the Channel Catfish (Ictalurus punctatus), Rainbow Trout (Oncorhynchus mykiss), Atlantic Salmon (Salmo salar), Development of environmentally friendly and economic- ally sustainable aquaculture requires an understanding of the genetic basis of traits that currently limit/enhance de- velopment of domestic aquaculture [3]. Genetic resources have been developed and used extensively in agriculture Page 2 of 11 Page 2 of 11 Purcell et al. BMC Genomics (2018) 19:31 and livestock breeding for decades, but have only more recently been applied to select aquaculture species (e.g. Rainbow Trout, Atlantic Salmon, tilapia, catfish, flounder, Atlantic Cod) [4, 5]. These resources have been used to identify genetic variation underlying phenotypic traits of economic interest for aquaculture production, for ex- ample, disease resistance, growth rate, tolerance of environmental stressors, diet/nutrition, reproduction, and general health [5–8]. Methods to develop these resources provide the best possibilities for genetic im- provement of broodstock or culture practices [9]. Next generation sequencing (NGS) has revolutionized this area of research through decreasing costs and increas- ing number of research applications [5], and this has enabled development of genetic resources for a greater number of species [7]. gene (sdY) encodes a regulatory protein [21]. It is difficult to distinguish sex chromosomes/regions in teleosts, as they are often not heteromorphic due to relatively recent origins or newly emerging/changing master regulator sex- determining genes or genomic regions [22–25]. In some fish, such as the Zebrafish (Danio rerio) multiple factors regulate SD that requires a quantitative threshold of gene accumulation that pushes the trait beyond threshold for either sex [16, 26–28]. This implies sex is a fickle pheno- type, not consistently determined by any single gene [29]. y y y y g g For Seriola, previous research on S. quinqueradiata suggested a ZW sex determining system [30, 31], and linkage analyses identified markers associated with sex. However, these regions did not identify sex in other Seriola species including S. dorsalis (V. Martinez, per- sonal communication; A. Background l Ozaki, personal communica- tion; Purcell, unpublished data). To accelerate growth of yellowtail aquaculture in the U.S., we have worked to begin developing genomic resources for the California Yellowtail in order to identify markers, such as sex- determining markers, that will be advantageous to opti- mizing culture techniques. In the present study, we describe the assembly and annotation of the Seriola dorsalis genome, and present resequencing data from 45 male and 45 female wild-caught S. dorsalis used to identify a sex-determining region and marker in this species. The ability to determine sex is often one of the first characteristics targeted following genomic resource de- velopment. In wild fish studies, determining sex from fin clips or plugs of muscle tissue is important for evaluat- ing population composition, sex-biased movements, and stock assessment models [3]. Genetic markers would also allow sex data to be collected from fish sampled non-lethally (e.g., fin clips), and from immature individ- uals, for which sex may not be determined even with le- thal dissection [10]. In aquaculture, economically valuable traits may be linked to sex, such as growth rate, size at maturity, age at sexual maturity, color pattern, fin shape, and even fillet flavor [3, 11–13]. Genetic sex identification would allow aquaculture producers to take advantage of sexual dimorphism, improve broodstock selection effi- ciency, and accelerate monosex culture development [13]. These economic and conservation-based considerations highlight the importance of understanding sex determin- ation mechanisms and developing sex-linked markers for commercially valuable fish species. Specimen collection For genome sequencing, a juvenile S. dorsalis (50 days post-hatching) was sampled from Hubbs SeaWorld Re- search Institute (San Diego, CA) during an aquaculture production run. This individual was humanely euthanized by placing the fish in a bath containing a lethal overdose (a concentration of 800 mg/L) of the anesthetic tricaine methanesulfonate (MS-222). The whole juvenile fish was then placed immediately into RNAlater® Stabilization Solu- tion (AMBION, Thermo Fisher Scientific, Waltham, MA), stored for 24 h at 4 °C and then frozen at −20 °C until DNA extraction. Mature wild California Yellowtail were sampled from San Diego, California, Cedros Island, Mexico, and La Paz, Mexico. Tissue specimens were acquired via hook and line sampling by private sport anglers or com- mercial/subsistence fishers aboard various fishing vessels, scientific observers then sampled these specimens ship- board or at landing docks. Biologists from the National Marine Fisheries Service – Southwest Fisheries Science Center or the Instituto Politécnico Nacional-Centro Inter- disciplinario de Ciencias Marinas (Mexico) dissected each fish to examine gonadal tissue; if eggs or milt could be iden- tified in the individual fish, the sex was recorded and a gen- etic sample was collected, ambiguous gonads (e.g. from immature individuals) were not collected as sexed-samples. Gonadal developmental stages in the Yellowtail were not Sex-specific markers have only been identified for a handful of fish species [12] due to remarkable variety in sex determination (SD) mechanisms observed in teleost fish, which can vary in closely related species and even within different populations of the same species [14, 15]. Varied SD modes include chromosomal, polygenic, epigenetic and environmental [16, 17], and within better-studied chromosomal systems, XY (males are the heterogametic sex)/ZW (females are the heterogametic sex), underlying SD mechanisms still vary greatly in tele- osts. For example, the XY SD system in Tiger Pufferfish (Takifugu rubripes) is controlled by a single nucleotide difference in the anti-Müllerian hormone receptor type II gene (AmHRT2) while the Patagonian Pejerrey (Odon- tethes hatcheri) has a Y-linked duplicated copy of the anti- Müllerian hormone [18]. The Medaka (Oryzias latipes) genome contains a Y-linked duplication (DmY) of the Dmrt1 gene [19, 20], and in Rainbow Trout, a Y-linked Purcell et al. BMC Genomics (2018) 19:31 Page 3 of 11 Page 3 of 11 removed when they contained fewer than 800 bases, or when 90% of the total scaffold length was contained in a different larger scaffold. DNA sequencing and library insert sizes Four libraries were prepared for genomic sequencing: three mate-paired (MP) libraries with insert sizes of 2000 bp, 8000 bp, and 12,000 bp and one 300 bp paired-end (PE) library. Each library was run in a single lane (four lanes total) on the Illumina HiSeq 2500 sequencer (Illumina, San Diego, CA). For the resequencing approach, 90 wild-caught samples were indexed and 100-bp PE libraries were run on four lanes of the Illumina HiSeq 2500 resulting in approxi- mately 2× coverage per sample. Specimen collection The scaffolds must also contain a gene or be larger than 10,000 bases. This filtering resulted in 4717 scaffolds remaining in the assembly (bioprojectID PRJNA319656). The scaffolds were then scrutinized for contamination. To identify PhiX contamination (a type of contamination introduced by Illumina’s sequencing kits), the NCBI Reference Sequence: NC_001422.1 was blast queried against the genome assembly, and one scaffold (scaffold_26907) was identified and removed. Blobtools [34] was used to identify another 277 scaffolds (contami- nation277.txt) that appeared to contain contamination from the phytoplankton Emiliania huxleyi, and the num- ber of scaffolds was further reduced. The quality of the final assembly was assessed using BUSCO [35]; this pro- gram provides a measure of genome assembly quality by determining the number of conserved single-copy ortho- logs found within the assembled genome compared to the BUSCO ortholog database. assessed, as this was beyond the scope of this project. From these fish, pieces of muscle tissue, fin clips, or gonadal tis- sue were placed in 100% ethanol until DNA extraction; the type of tissue varied by sample. Several hundred sexed- specimens were collected from these locations, and 90 of these fish (15 of each sex from each of the three locations, for a total of 45 male and 45 female specimens), were selected to undergo sequencing based on tissue quan- tity and DNA quality (see below). Sexed-specimens not undergoing sequencing were used to test the ac- curacy and amplification of the developed sex-specific marker primers. DNA extraction For all specimens, genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD) following the manufacturer’s protocol. Heart and spleen tissue were used from the specimen undergoing genomic sequencing. Muscle, fin, or gonadal tissue was used for the 90 specimens undergoing resequencing and for the additional specimens screened with the sex- specific markers. Purification of extracted genomic DNA was assessed using a NanoDrop ND-1000 with the spec- trophotometer absorbance ratio of 260/280 nm, and DNA quantification was performed using a PicoGreen® (Invitrogen, Carlsbad, CA) based assay on the VictorX3™ 2030 (Perkin Elmer, Waltham, MA). Samples prepared for the genomic and re-sequencing applications were sent to the DNA Sequencing Facility at Iowa State University (Ames, Iowa) for library preparation and sequencing. g Utilizing raw RNA-Seq data (if available) is useful in genome assemblies to identify exon/intron boundaries more accurately than methods that rely on assembled transcripts. To assist in annotating this genome, data were used from a concurrent RNA-Seq project on S. dorsalis that examined slow- and fast-growing larvae at three early developmental time points (Purcell et al., unpublished data). BRAKER [36] was used to annotate the genome using 547 million 50 bp PE raw RNA-Seq data from that project. The BRAKER pipeline uses GeneMark-ET [37] to perform unsupervised training using the genome file and the RNA-Seq data. After training, GeneMark-ET creates an ab initio gene set, and those gene structures in all introns, that have sup- port from RNA-Seq alignments, are then selected for automated training of AUGUSTUS [38]. After training, AUGUSTUS predicts genes in the input genome file using spliced alignment information from RNA-Seq as extrinsic evidence. RNA-Seq data for this project was also deposited at DDBJ/ENA/GenBank under the Bio- Project ID: PRJNA339646. Assembly and annotation of the genome Alignment of sexed-wild yellowtail and single nucleotide polymorphism (SNP) calling Identification of genes related to sex-determination Identification of genes related to sex-determination Regions containing genes known to be involved in sex determination and differentiation are a good starting place for identifying candidate regions involved in sex determination for non-model organisms. These genes were identified from the literature: hsd17b3 cyp19a1a, hsd17b1, foxl2, dmrt1, sox9, sox3, sf1 and amh [16, 20, 45–47] and the corresponding genes in S. dorsalis were identified using the annotation in the GFF file. Orthologs for the S. dorsalis genes were identified using a reciprocal best BLAST to six fish genomes downloaded from NCBI (0.82): Danio rerio, Lepisosteus oculatus, Oreochromis niloticus, Oryzias latipes, Takifugu rubripes and Xiphophorus maculatus. The BLAST results were filtered requiring at least 50% coverage of the S. dorsalis gene to be considered orthologous (Additional file 1: Table S1). The number of heterozygous SNPs in the SDR were compared between males and females. A SNP was con- sidered heterozygous if the allelic ratio for all individuals in each population was between 0.4 and 0.6. If S. dorsalis exhibits a ZW type of sex-determination, as seen in S. quinqueradiata [30, 31], a higher number of heterozy- gous SNPs would be expected in females, while a higher number would be expected in males if the system is XY. As a control, for any identified SDRs, the same region on five different scaffolds (not containing the SDR) were compared across the three sampling locations (San Diego, Cedros Island, and La Paz) between males and females; the expectation was that no significant male/female differ- ences in these other regions would be identified. Alignment of sexed-wild yellowtail and single nucleotide polymorphism (SNP) calling FASTQ formatted files of paired-end and mate-pair reads generated from the HiSeq2500 in FASTQ format were used for the assembly. Prior to assembly, FastQC (https:// www.bioinformatics.babraham.ac.uk/projects/fastqc/) was run to verify the quality of the reads. MaSuRCA assembler (version 2.3.2) [32] was used to assemble the raw data into 98,162 scaffolds and has been deposited at DDBJ/ENA/ GenBank under the accession PEQF00000000. To obtain a more reasonable assembly for downstream bioinformat- ics analyses and for visualization in JBrowse [33], scaffolds were filtered with the following parameters: scaffolds were Raw data from the sequences of the 90 sexed-wild caught yellowtail were quality checked using FastQC [39]. Raw reads were aligned to the assembled genome using BWA- MEM [40].The aligned bam files were prepared for SNP calling by GATK [41]; this included coordinate sorting, cleaning, duplicate marking, adding of read groups (http:// broadinstitute.github.io/picard) and SNP/Indel realignment. GATK was then run to call SNPs and InDels on the com- bined alignment files. Page 4 of 11 Purcell et al. BMC Genomics (2018) 19:31 Page 4 of 11 Identification of the sex-determining region (SDR) (PCRs) were conducted in 25 μl volumes containing approximately 10–20 ng template DNA in a reaction containing 67 mM Tris-HCl pH 8.8, 16.6 mM (NH4)2SO4, 10 mM β-mercapto-ethanol, 2 mM MgCl2, 800 μM dNTPs, 0.5 mg/ml BSA, 0.15–0.3 μM forward primer, 0.15–0.3 μM reverse primer, and 0.25 units Taq DNA polymerase (New England Biolabs, Ipswich, MA). Thermal cycling parame- ters were as follows: initial denaturing at 94 °C for 4 min., 40 cycles of 94 °C for 30 s, annealing at 53 °C for 55 s, extension at 72 °C for 55 s, and a final extension step at 72 °C for 5 min. PCR products were electrophoresed at 78 V for 40 (SDorDel02) and 60 (SDorDel01) minutes through a 2% agarose gel, with a 100-bp size standard (Thermo Fisher Scientific, Waltham, MA) run in a lane adjacent to PCR products, Ethidium bromide stained gels were visualized and digitally acquired on an UV light box using Enduro™GDS Touch and recorded using the Labnet Enduro Gel Documentation System Image Ac- quisition Software (v. 1.3.1218.0) (Labnet, Edison, NJ). g g A genome-wide association study (GWAS) using a Gen- eralized Linear Model (GLM) was performed in TASSEL (version 5) [42] to identify genomic regions significantly correlated with the sex phenotype. SNPs were imputed using Euclidean distance by mean with the five nearest neighbors. The kinship matrix was calculated using a centered Identity By State (IBS) with a maximum number of six alleles. This required joining three datasets (imputed SNPs, traits and kinship matrix) using the union join com- mand under the data menu. The p-value cutoff was set to 1e-3 and 1000 permutations were selected. TASSEL could not handle the full SNP dataset, so SNP subsets were gener- ated using vcf-subset.py (https://github.com/ISUgenomics/ common_scripts/blob/master/vcf-subset.py), which takes a random SNP from every 5000 base interval. GLM was then repeated for all SNPs on chromosomes where multiple co- linear SNPS had a significant correlation. For comparison, the sex marker in Seriola quinqueradiata (ssr263g21) [31] was also mapped to the S. dorsalis genome using GMAP [43]. Identification of potential binding motifs All insertions/deletions (InDels) greater than 40 nucleo- tides in the SDR were identified. Two sets of primers, SdorDel01 and SdorDel02, were designed to span across a region of interest using Primer3 [44] (Table 1). These primers were tested for consistent amplification and ac- curacy as a sex-determining marker on the 90 specimens used for sequencing in this study, and in another set of 212 known-sex specimens. Polymerase chain reactions Identification of transcription factor binding motifs can be challenging due to their short sequence lengths, which can lead to false positives, and due to the limited number of known transcription factor binding motifs contained in databases (e.g. Jaspar) [48]. Phylogenetic foot- printing was used in the analysis; this approach uses con- served sequences identified between orthologous upstream Table 1 Primer names, sequences, melting temperatures (Tm), lengths, and PCR fragment sizes Primer Name Sequence Tm (°C) Length (bp) Fragment Size (without/with deletion) SdorDel01-F AATTCATCCAAACCCAGCAG 59.9 20 452 bp/391 bp SdorDel01-R GGTCTTGTCAACTGCGATCA 59.8 20 SdorDel02-F TGACAACAAGGCAACAGGAG 59.9 20 282 bp/221 bp SdorDel02-R TTGGCCTTTCTTTTGACCAC 60.1 20 Page 5 of 11 Purcell et al. BMC Genomics (2018) 19:31 Page 5 of 11 regions to improve odds of a true positive discovery. Fol- lowing identification of the hsd17b1 (Estradiol) gene (see results), a 3000 base nucleotide sequence upstream of this gene was extracted from the S. dorsalis genome and from the six fish genomes downloaded from NCBI (Estradialup- stream3000.fasta); this was performed using the gff2fasta.pl script (https://github.com/ISUgenomics/common_scripts/ blob/master/gff2fasta.pl). This upstream region from the reciprocal best blast hits (RBBH) analysis was scanned for conserved motifs using MEME (Motif-based sequence analysis tools) [49]. MEME was performed on these sequences to identify 100 potential motifs using the following parameters: -dna -mod anr -revcomp -p 16 -nmotifs 100. A curated database of experimentally defined transcription factor binding sites, Jaspar [48] was used to identify potential binding motifs, and the corresponding transcription factor, to determine if any binding sites were related to sex hormone regulation and sex determination. Identification of the sex-determining region g g The resequencing approach resulted in an average cover- age depth of 1.9X for each of the 90 individual fish. From these sequences, GATK called 7,684,767 SNPs and InDels (Additional file 2), of which 7,484,110 were biallelic. From the 7.4 million biallelic SNPs inputted into TASSEL, the GLM analysis used a subset of 375,904 SNPs that spanned across all scaffolds. ZW sex determination in S. dorsalis In each sampling location, the ratio of heterozygous SNPs in females was on average 4.9 times higher in the SDR, whereas the ratios in the random genomic loca- tions of the same size were not different between the sexes (Table 2). This heterozygosity pattern indicated a ZW mode of sex-determination within the SDR identi- fied above. Identification of potential binding motifs This GLM analysis indicated that a region on scaffold 22 was strongly correlated to sex (Additional file 3). The GLM repeated for the SNPs on this scaffold revealed that the strong correlation to SD occurred between the nucleotide positions of 231,000 and 320,000 on scaffold 22 (Fig. 1). A negative log 10 p-value of nearly 17 for one of the tested SNPs, and the number of significantly linked SNPs, provides strong evidence that this region is correlated with the sex phenotype, and likely represents the SDR for S. dorsalis. Additionally, the sex marker identified in Seriola quin- queradiata (ssr263g21) [31] mapped to scaffold_22 base 194,817–194,840 in the S. dorsalis genome. This is ~36,000 bp upstream of the significantly correlated re- gion in S. dorsalis. Assembly and annotation statistics Approximately 1.2 billion reads were generated from the four library preparations. Based on the estimated genome size of 685 MB for Seriola lalandi (C-value of 0.70) [50], the sequencing coverage is 180× for the S. dorsalis gen- ome. The MaSuRCA assembly resulted in 4439 scaffolds based on 23,003 contigs with N50 s of 1,491,863 bases and 139,330 bases, respectively, and the longest scaffold is 8,096,577 bases. Total genomic content is 653,009,476 bases, which represents 93% of the estimated genome size, with approximately 13 million gaps or ambiguous bases (Ns). The completeness of the assembly is very high based on the BUSCO assessment; this genome contains 2848/ 3023 BUSCO groups and is only missing 175 of the Eukaryotic orthologues. Raw data All raw data can be downloaded from NCBI under the bioprojectID PRJNA319656. Sex-determining marker development After searching the SDR in S. dorsalis for insertions or deletions greater than 40 base pairs, only one 61-base deletion was identified. This deletion was heterozygous in females and not present in males, making it consistent with the ZW sex determination model for Seriola. This deletion is located on scaffold 22 at position 246,495 with the following sequence: CGTTCATGATTACTACTTT TACACAAATTTACACAAAAGACATCTGTACCAAAG AACAAAA. The developed primers, SdorDel01 (452 bp) and SdorDel02 (282 bp) consistently amplified and re- vealed sex-specific patterns on agarose electrophoresis gels (Fig. 2). Heterozygous females displayed two bands, both with and without the 61 bp deletion, while males only exhibited the larger band (without the deletion). These primers were tested on an additional 212 sexed yellowtail and accurately predicted sex in 94% of the individuals. It is unclear whether the disagreement between genetic and phenotypic sex in the remaining 6% of fish (12 individuals) is due to variation in the genomic region that led to poor primer binding, or if the individuals were mis-sexed or mis-labeled upon There were 49,784 transcripts corresponding to 45,251 genes, and of these, 8155 genes had RBBH to orthologs covering at least 50% of the gene sequence in Seriola and in six other fish species (Danio rerio, Lepisosteus oculatus, Oreochromis niloticus, Oryzias latipes, Takifugu rubripes and Xiphophorus maculatus) (Additional file 1). An additional 22,011 genes had RBBH to at least one other fish species, and there were 27,218 genes that had a unidirectional blast hit to one of the six fish species, that covered at least 90% of the gene length. Most gene models (42,847) have evidence supported by RNA-Seq data or contain a PFAM (protein family) domain. Purcell et al. BMC Genomics (2018) 19:31 Page 6 of 11 Fig. 1 Negative log 10 plot of p-value versus genomic location (bp) on Scaffold_22. P-values were determined via GWAS of sex phenotype using TASSEL. A black arrow indicates the position of the S. quinqueradiata sex marker (ssr263g21). A red line is drawn at a p-value of 1e−7 to show significant SNPs in the SDR Fig. 1 Negative log 10 plot of p-value versus genomic location (bp) on Scaffold_22. P-values were determined via GWAS of sex phenotype using TASSEL. A black arrow indicates the position of the S. quinqueradiata sex marker (ssr263g21). A red line is drawn at a p-value of 1e−7 to show significant SNPs in the SDR collection. Characterizing the sex-determining region In S. dorsalis, there are only seven gene models in the SDR of which only four have known functions: hsd17b1: Estradiol 17-beta-dehydrogenase 1, Coenzyme Q-binding protein coq10 homolog, Complement C1q-like protein 2, Table 2 Numbers of heterozygous SNPs in the SDR versus random genomic regions of the same size Population Heterozygous SNP Counts Ratio Genomic Region Female Male (Female/Male) CI 90 20 4.5 SDR SD 106 27 3.9 SDR LP 123 20 6.2 SDR LP 140 141 1.0 Random LP 129 107 1.2 Random LP 82 91 0.9 Random LP 261 226 1.2 Random SD 161 142 1.1 Random SD 123 97 1.3 Random SD 101 82 1.2 Random SD 58 66 0.9 Random SD 238 263 0.9 Random CI 146 170 0.9 Random CI 113 131 0.9 Random CI 84 91 0.9 Random CI 55 65 0.8 Random CI 262 238 1.1 Random SDR Sex Determining Region, CI Cedros Island, SD San Diego, LP La Paz Bold numbers reflect F/M SNP ratios detected in the SDR Table 2 Numbers of heterozygous SNPs in the SDR versus random genomic regions of the same size Table 2 Numbers of heterozygous SNPs in the SDR versus random genomic regions of the same size pp This was tested with a MEME analysis of the 3000 base nucleotide sequence upstream of this gene in S. dorsalis and from the six other fish genomes resulting in several motifs with high levels of significance. One motif in S. dorsalis (GTCTTTTGTTCTTTG) overlapped with the deletion on the negative strand; it was found in all seven species and in a similar position upstream of the estradiol gene in five of the seven species, suggesting that it is conserved. After searching for transcription factor binding sites contained in this motif, the Jaspar database re- vealed binding sites for the following male sex-determining genes: sry, sox9, and sox3 with relative scores of 84%, 87%, and 97%, respectively. We caution the reader that while we are able to report the results of a deletion upstream of a gene contained in the pathway involved in sex hormone production and that we can show there are conserved bind- ing motifs in the deletion found on the W chromosome for known male sex determining genes, without further experi- mental evidence, we cannot confirm our hypothesis. To gain a better understanding of potential genetic mechanisms of sex determination in S. Sex-determining marker development The two sex-specific primers were also tested for a small number of S. rivoliana specimens (n = 6), how- ever, the markers failed to distinguish sex for these indi- viduals (Fig. 3). arf1: ADP-ribosylation factor 1. These genes correspond to Sedor.G00005096, Sedor.G00005097, Sedor.G00005098 and Sedor.G0000599, respectively. The Estradiol 17-beta- dehydrogenase 1 gene (hsd17b1) was identified as the most promising sex determing gene within the SDR. This gene (Sedor.G00005096) is present in males and females, but no SNPs were contained within the gene that would suggest a genetic mechanism linked to ZW SD. However, the 61 base deletion (present only in females) was found 954 bases upstream of this gene, indicating this deletion is present only in the W chromosome. One possible hypoth- esis as to how this deletion might have a role in SD for S. dorsalis is if it contained a transcription factor binding motif that suppresses hsd17b1. Bold numbers reflect F/M SNP ratios detected in the SDR SDR Sex Determining Region, CI Cedros Island, SD San Diego, LP La Paz SDR Sex Determining Region, CI Cedros Island, SD San Diego, LP La Paz Bold numbers reflect F/M SNP ratios detected in the SDR Characterizing the sex-determining region dorsalis, the mo- lecular pathways for sex steroid biosynthesis and genes known to be involved in sex determination were drawn Page 7 of 11 Purcell et al. BMC Genomics (2018) 19:31 Fig. 2 The ethidium bromide stained gel image of the PCR products from the sex-specific genetic markers, SDorDel01 (Gel a) and SDorDel02 (Gel b) run with nine female (F) and nine male (M) S. dorsalis specimens, with a 100-bp ladder in each gel for reference Fig. 2 The ethidium bromide stained gel image of the PCR products from the sex-specific genetic markers, SDorDel01 (Gel a) and SDorDel02 (Gel b) run with nine female (F) and nine male (M) S. dorsalis specimens, with a 100-bp ladder in each gel for reference orthologues comparable to other recently published fish ge- nomes [52, 53]. A well-assembled and well-annotated genome is a major contribution for researchers inter- ested in a variety of research questions [54], and this genome will be a powerful resource for additional gen- omic, transcriptomic, and epigenetic work in California Yellowtail and in other Seriola species. Given the import- ance of S. dorsalis and other Seriola species for aquacul- ture production, this genomic resource will be important in accelerating maker-assisted selective breeding programs, and in improving economically relevant culture traits, such as disease resistance, feed ac- ceptance, thermal tolerance, and sex-based characteristics. This genome assembly also enables further investigation of teleost evolution, and the evolution of sex determin- ation, which has proven to be a complex and highly vari- able trait in fish [13]. (Fig. 4), based on our literature review and includes models of gene activation, inactivation, protein interactions, and feedback loops. Genes involved in male sex steroid bio- synthesis and sex determination include hsd17b3, dmrt1, sox9, sox3, sf1, and amh. While genes involved in female sex steroid biosynthesis that form a positive regulatory loop leading to female sex differentiation are: cyp19a1a, foxl2 and hsd17b1 [47, 51]. Discussion The red arrows correspond to the positive regulation loop that drives female sex determination has been used to test similar hypotheses in tilapia by creat- ing deletions in the foxl2 and dmrt1 genes [58]. However, experimental studies to test this hypothesis in S. dorsalis are beyond the scope of the present study. Insertions or de- letions located in a promoter region upstream of a putative master sex-determining gene have been described in other fish species [3]. In Sablefish, for example, sex-specific inser- tions upstream of the gsdf gene were reported [3]. While Rondeau et al. [3] could not conclusively state that gsdf is the master sex gene for this species, this gene is evolution- arily conserved and has been reported as a master sex gene in other species, which strongly suggests a similar role in Sablefish. frequent’, even among family-level and genus-level species [55]. Tilapia are one of the most widely known examples of this phenomenon. The mode of sex determination (XX- XY/ZW-ZZ) and the genomic regions associated with SD markers (LG 1 vs. LG 3) differed even among closely related Oreochromis species [56, 57]. Although the SD markers for S. quinqueradiata and S. dorsalis map rela- tively closely together, the mechanism that controls the SD pathway may be completely different between these species. As genomic sequencing data are rapidly grow- ing for all Seriola species, a future study will be able to examine SD regions among these fish. The resequencing data also identified that the Estradiol gene (hsd17b1) is contained in the SDR; this gene is in- volved in the female steroid biosynthesis pathway leading to estrogen production [47]. Interestingly, the female- specific 61-nucleotide deletion was found just upstream of this sex steroid biosynthesis gene. The similar pos- ition of this motif upstream of the estradiol gene in four of the six species suggests it is well conserved. With this motif matching most closely to transcription factor binding sites associated with male sex determin- ing genes, taken together, these data suggest that estra- diol production is suppressed by sox3, sox9 or sry with sox3 being the most likely candidate based on its rela- tively high Jaspar score. Although the exact mechanisms for regulation in the sex determination pathways remain unclear, there are examples of “master” SD genes that alter or interfere with upstream regulatory elements. Discussion High throughput sequencing was used to developing genomic resources for S. dorsalis in this project. Our assembly and annotation of the California Yellowtail, S. dorsalis, is of high quality capturing approximately 93% of the total genome and identifying over 94% of the BUSCO Fig. 3 The ethidium bromide stained gel image of the PCR products from the sex-specific genetic markers, SDorDel01 and SDorDel02 run with three female (F) and three male (M) S. rivoliana specimens, with a 100-bp ladder for reference Using the aligned resequencing data from the sexed 90 wild-caught fish, boundaries were successfully placed on the SDR and sex-specific makers were developed based on the identified female-specific deletion. The SDR in S. dorsalis is consistent with the known sex marker in S. quinqueradiata (ssr263g21) [31], which maps just up- stream of this region: scaffold 22 at 194817–194840 base pairs (linkage group 12) [30]. However, the marker re- ported in that study lies just outside of the SDR and has not worked in distinguishing sex in other Seriola species (Ozaki, pers. com). Similarly, markers identified in this study did not work in identifying sex in a small number of S. rivoliana specimens, although the effectiveness in other Seriola species has not yet been tested. It is not sur- prising that there is difficulty in transferring SD markers among the Seriola species. It has been reported that shifts in teleost sex-determining modes are ‘evolutionarily Fig. 3 The ethidium bromide stained gel image of the PCR products from the sex-specific genetic markers, SDorDel01 and SDorDel02 run with three female (F) and three male (M) S. rivoliana specimens, with a 100-bp ladder for reference Purcell et al. BMC Genomics (2018) 19:31 Page 8 of 11 Fig. 4 Our interpretation of the sex determination pathway based on the literature. Genes are boxes, gene products are ovals. Genes involved in female and male sex determination are colored in red and blue, respectively. Solid lines indicate known protein interactions while dashed lines indicate hypothesized interactions. Stops (\|–) indicate inhibition. The red arrows correspond to the positive regulation loop that drives female sex determination Fig. 4 Our interpretation of the sex determination pathway based on the literature. Genes are boxes, gene products are ovals. Genes involved in female and male sex determination are colored in red and blue, respectively. Solid lines indicate known protein interactions while dashed lines indicate hypothesized interactions. Stops (\|–) indicate inhibition. Conclusions A As a greater number of fish genomes are sequenced, it is possible (even likely) that all genes involved in regulation of sex steroids (Fig. 4) will be discovered to have been co-opted or disrupted to become a “master-regulating” SD gene, given the variety of genetic mechanisms detected so far in teleosts [46]. In addition, more genes like gsdfy, outside of the known steroid biosynthesis pathway and currently not known to be related to sex-determination, will be identified and found to affect the quantitative threshold toward either male or female phenotypes or affecting population sex ratios [21]. Sex determination remains a complex competition between protein expres- sion and protein regulation through transcription factor binding sites in the sex-steroid biosynthesis pathway. For- tunately, sequencing costs continue to drop and know- ledge obtained from even low coverage resequencing, as demonstrated here, can gain significant insight into the mechanics of sex determination in teleosts. This genome assembly for S. dorsalis will be a substantial resource for a variety of research applications such as population genomics, functional genomics, translational studies, and epigenetic research in wild and cultured populations of S. dorsalis and other Seriola species. The understanding of sex and other phenotypic traits will be improved through this genomic resource development and help to accelerate the rate of genetic improvement in these cultured species [64]. Clearly, upstream regulatory elements are key players in determining sex. In S. dorsalis, we hypothesize that the deletion upstream of the estradiol gene (hsd17b1) may function to release the silencing motif of sox3, a known master SD gene [59]; this then could lead to in- creased transcription of estradiol and increased estrogen production, ultimately resulting in the female phenotype (Fig. 4). However, our speculation will need to be followed up by lab experiments, potentially with CRISPR to confirm. If this deletion is the underlying genetic mechanism for SD, it will be interesting to discover whether this is also true for all of the Seriola species. For S. quinqueradiata, Koyama et al. [31] did not find any known SD genes near the SDR de- tected in their study, although it is difficult to determine this conclusively without genomic sequences of this region, which were not available at the time of their study. The sex-markers developed in this study did not detect the dele- tion in the few tested S. Discussion Master SD genes may govern the regulatory networks involved in sex deter- mination, and these genes typically fall under the cat- egories of steroidogenic enzymes, sex steroid receptors, transcription factors, and growth factors [11]. Certain genes that are part of the regulatory network appear to evolve repeatedly into master SD genes, such as sox3, dmrt1, and tgf-b [59]. Although it has not yet been proven, there may be constraints on which genes can become ‘masters’ [16, 31], and some likely candidates (e.g., foxl2, sox9, sox8, and wnt4) have not demonstrated the same ability to evolve into master SD genes [59]. Add- itionally, it has only more recently been demonstrated that downstream networks involved in SD also exhibit some degree of flexibility [59]. It is our hypothesis that the large deletion upstream of the estradiol gene (hsd17b1) disrupts a silencer motif bound by sox3 and thereby increases estrogen production leading to the female phenotype. Crispr/Cas9 is the logical choice to confirm this hypothesis in follow-up studies, as it Purcell et al. BMC Genomics (2018) 19:31 Purcell et al. BMC Genomics (2018) 19:31 Page 9 of 11 While there has been considerable progress in under- standing the master gene networks, there are many master genes that have only recently been discovered and their networks are still being explored [59]. There is strong sup- port for both positive [11] and negative [51, 60] regulation between the male and female SD pathways. In non- mammalian vertebrates there are multiple examples of female sex determination depending on a positive feed- back loop involving stimulation of cyp19a1 and the transcription factor, foxl2; in males, sex determination depends on inhibition of cyp19a1 through dmrt1 up- regulation [11]. An example of this mechanism in teleost fish can be found in the European Seabass (Dicentrarchus labrax) where the cyp19a1 promoter is hypermethylated in males compared to females. This methylation causes repression of transcription, and therefore inhibition of the cyp19a1 gene, preventing ovarian differentiation [11]. In another example in the Half-Smooth Tongue Sole (Cynoglossus semilaevis), which exhibits a ZZ/ZW sex determination system, dmrt1 is expressed in the male sex-determination pathway, however in ZW females, the dmrt1 promotor was hypermethylated and silenced [14, 61]. Examples of positive regulation can be found in the African clawed frog (Xenopus laevis) and in two medaka species, Oryzias luzonesis and O. dancena. Abbreviations BLAST: Basic Local Alignment Search Tool; BP: Base pair(s); BUSCO: Benchmarking Universal Single-Copy Orthologs; GLM: Generalized linear model; GWAS: Genome- wide association study; IBS: Identity by state; INDELS: Insertions/deletions; MP: Mate-paired; MS-222: Tricaine methanesulfonate; NGS: Next generation sequencing; Ns: ambiguous bases; PCR: Polymerase chain reaction; PE: Paired-end; PFAM: Protein family; RBBH: Reciprocal best-blast hits; SD: Sex determination; SDR: Sex-determining region Conclusions A rivoliana specimens; however, this is unlikely due to mutations in the primer-binding site as Discussion Sex determination in Xenopus laevis, involves the DM-W gene, essentially a partial duplication of the dmrt1 gene containing the DNA binding C-terminal domain but not the transcriptional activating N-terminal domain. DM-W acts on sex determination by competing for the upstream activating element that dmrt1 targets [62]. In Oryzias luzonesis, mutations in a conserved motif in- creased expression of Gsdf [63] and a cis-regulatory element in Oryzias dancena has been shown to upreg- ulate sox3 expression [46]. the products were of the appropriate size. Ongoing sequen- cing efforts for S. rivoliana, S. quinqueradiata, and other Seriola species should soon uncover the variation in the SDR among these fish, and reveal the ubiquity of the upstream regulatory elements and master sex-determining genes for this genus of fish. Additional files Additional file 1: BLAST results of orthologous sex-determining genes (from literature) with at least 50% coverage of the S. dorsalis gene. (DOCX 14 kb) Additional file 2: SNP and InDels calls (VCF file) for scaffold containing sex determining region. (VCF 35715 kb) Additional file 3: Tassel significance output for GWAS analysis for sex phenotype. (TXT 52 kb) Additional file 1: BLAST results of orthologous sex-determining genes (from literature) with at least 50% coverage of the S. dorsalis gene. (DOCX 14 kb) Additional file 1: BLAST results of orthologous sex-determining genes (from literature) with at least 50% coverage of the S. dorsalis gene. (DOCX 14 kb) Additional file 2: SNP and InDels calls (VCF file) for scaffold containing sex determining region. (VCF 35715 kb) Additional file 3: Tassel significance output for GWAS analysis for sex phenotype. (TXT 52 kb) Competing interests Competing interests Competing interests The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. 20. Nanda I, Kondo M, Hornung U, et al. A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka, Oryzias latipes. Proc Natl Acad Sci. 2002;99:11778–83. Funding F di f Funding for this project was provided by NOAA’s Office of Aquaculture ICAF program and XSEDE MCB140217. Funding for this project was provided by NOAA’s Office of Aquaculture ICAF program and XSEDE MCB140217. 9. Sodeland M, Gaarder M, Moen T, et al. Genome-wide association testing reveals quantitative trait loci for fillet texture and fat content in Atlantic salmon. Aquaculture. 2013;408:169–74. Availability of data and materials 10. Fowler BL, Buonaccorsi VP. Genomic characterization of sex-identification markers in Sebastes carnatus and S. chrysomelas rockfishes. Mol Ecol. 2016;25:2165–75. All raw data can be downloaded from NCBI under the bioprojectID PRJNA319656. Specific data sets are available from authors A. Severin and C. Purcell. 11. Piferrer F, Ribas L, Díaz N. Genomic approaches to study genetic and environmental influences on fish sex determination and differentiation. Mar Biotechnol. 2012;14:591–604. 11. Piferrer F, Ribas L, Díaz N. Genomic approaches to study genetic and environmental influences on fish sex determination and differentiation. Mar Biotechnol. 2012;14:591–604. Authors’ contributions CMP conceived and designed the study, performed the experiments, and co-wrote the manuscript. ASS conducted genome assembly/annotation and variant calling bioinformatics analyses and both contributed and revised the final manuscript. OW and SOG coordinated specimen collection and collected biological data on the specimens. JRH conceived and designed the experiment, edited the manuscript, and supervised the research. AJS conceived and designed the study, conducted GWAS bioinformatics analyses and analysis of the SDR pathway, and co-wrote the manuscript. All authors have read and approved the final manuscript. 12. Palaiokostas C, Bekaert M, Davie A, et al. Mapping the sex determination locus in the Atlantic halibut (Hippoglossus hippoglossus) using RAD sequencing. BMC Genomics. 2013;14:566. 13. Martínez P, Viñas AM, Sánchez L, et al. Genetic architecture of sex determination in fish: applications to sex ratio control in aquaculture. Front Genet. 2014;5:340. 13. Martínez P, Viñas AM, Sánchez L, et al. Genetic architecture of sex determination in fish: applications to sex ratio control in aquaculture. Front Genet. 2014;5:340. 14. Chen S, Zhang G, Shao C, et al. Whole-genome sequence of a flatfish provides insights into ZW sex chromosome evolution and adaptation to a benthic lifestyle. Nature Genet. 2014;46:253–60. 15. Pan Q, Anderson J, Bertho S, et al. Vertebrate sex-determining genes play musical chairs. C R Biol. 2016;339:258–62. 15. Pan Q, Anderson J, Bertho S, et al. Vertebrate sex-determining genes play musical chairs. C R Biol. 2016;339:258–62. Acknowledgements The authors acknowledge M. Drawbridge and K. Stuart of Hubbs-SeaWorld Research Institute for donation of the juvenile yellowtail used for the genome sequencing, The authors also acknowledge Enrique Ureña Portales, Sean Sebring from Fishermen’s Processing (San Diego), Royal Star Sportfishing Page 10 of 11 Page 10 of 11 Page 10 of 11 Purcell et al. BMC Genomics (2018) 19:31 (San Diego), the kayak anglers of La Jolla for assistance in sampling efforts, and acknowledge D. Kacev and L. Komoroske for lab assistance and manuscript critique, respectively. (San Diego), the kayak anglers of La Jolla for assistance in sampling efforts, and acknowledge D. Kacev and L. Komoroske for lab assistance and manuscript critique, respectively. 7. Huete-Pérez JA, Quezada F. Genomic approaches in marine biodiversity and aquaculture. Biol Res. 2013;46:353–61. 8. Fuji K, Koyama T, Kai W, et al. Construction of a high-coverage bacterial artificial chromosome library and comprehensive genetic linkage map of yellowtail Seriola quinqueradiata. BMC Res Notes. 2014;7:200. Publisher’s Note 21. Yano A, Guyomard R, Nicol B, et al. An immune-related gene evolved into the master sex-determining gene in rainbow trout Oncorhynchus mykiss. Curr Biol. 2012;22:1423–8. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 22. Volff JN, Nanda I, Schmid M, Schartl M. Governing sex determination in fish: regulatory putsches and ephemeral dictators. Sex Dev. 2007;1:85–99. Author details 1 1Southwest Fisheries Science Center, National Marine Fisheries Service, 8901 La Jolla Shores Drive, La Jolla, CA 92037, USA. 2Genome Informatics Facility, Iowa State University 206 Science I Ames IA 50011 USA 3Instituto 1Southwest Fisheries Science Center, National Marine Fisheries Service, 8901 La Jolla Shores Drive, La Jolla, CA 92037, USA. 2Genome Informatics Facility, Iowa State University, 206 Science I, Ames, IA 50011, USA. 3Instituto Politécnico Nacional-Centro Interdisciplinario de Ciencias Marinas, Departmento de Pesquerías Av Instituto Politécnico Nacional s/n Col Playa 23. de Bello Cioffi M, Kejnovský E, Marquioni V, et al. The key role of repeated DNAs in sex chromosome evolution in two fish species with ZW sex chromosome system. Mol Cytogenet. 2012;5:1. Iowa State University, 206 Science I, Ames, IA 50011, USA. 3Instituto Politécnico Nacional-Centro Interdisciplinario de Ciencias Marinas, Politécnico Nacional-Centro Interdisciplinario de Ciencias Marinas, 24. Chalopin D, Volff JN, Galiana D, et al. Transposable elements and early evolution of sex chromosomes in fish. Chromosom Res. 2015;23:545–60. Departmento de Pesquerías, Av. Instituto Politécnico Nacional s/n Col. Playa Palo de Santa Rita C.P, 23096 La Paz, Mexico. Departmento de Pesquerías, Av. Instituto Politécnico Nacional s/n Col. Playa Palo de Santa Rita C.P, 23096 La Paz, Mexico. Departmento de Pesquerías, Av. Instituto Politécnico Nacional s/n Col. Playa Palo de Santa Rita C P 23096 La Paz Mexico Palo de Santa Rita C.P, 23096 La Paz, Mexico. 25. Schartl M. Sex determination by multiple sex chromosomes tropicalis. Proc Natl Acad Sci. 2015;112:10575–6. tropicalis. Proc Natl Acad Sci. 2015;112:10575–6. 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W4225166734.txt	https://www.biorxiv.org/content/biorxiv/early/2022/04/28/2022.04.27.489576.full.pdf	en		Gestational Early-Time Restricted Feeding Results in Sex-Specific Glucose Intolerance in Adult Male Mice	bioRxiv (Cold Spring Harbor Laboratory)	2,022	cc-by	7,296	bioRxiv preprint doi: https://doi.org/10.1101/2022.04.27.489576; this version posted April 28, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Gestational Early-Time Restricted Feeding Results in Sex-Specific Glucose Intolerance in Adult Male Mice Molly C. Mulcahy1, Noura El Habbal1, Detrick Snyder1, JeAnna R. Redd1, Haijing Sun2, Brigid E. Gregg1,2, Dave Bridges1 1University of Michigan School of Public Health, Department of Nutritional Sciences, Ann Arbor MI, USA 2Michigan Medicine, Department of Pediatrics, Division of Diabetes, Endocrinology, and Metabolism, Ann Arbor MI, USA Corresponding Author Information: Dave Bridges, PhD Email address: davebrid@umich.edu Postal address: 1863 SPH I 1415 Washington Heights, Ann Arbor, Michigan 48109-2029 Telephone: +1 (734) 764-1266 Funding: This work was supported by R01 DK107535 (DB) and R56 DK121787 (BEG). MCM was supported through the University of Michigan Rackham Merit Fellowship. Disclosure: The authors declare no conflicts of interest. Keywords: time-restricted feeding, glucose intolerance, maternal nutrition, developmental programming bioRxiv preprint doi: https://doi.org/10.1101/2022.04.27.489576; this version posted April 28, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. eTRF leads to offspring glucose intolerance Abstract The timing of food intake is a novel dietary component that can impact health. Timerestricted feeding (TRF), a form of intermittent fasting, manipulates food timing. During pregnancy, one may experience disruptions to food intake for diverse reasons (e.g. nausea and vomiting of pregnancy, food insecurity, desire to manage gestational weight gain, disordered eating behaviors, changes in taste and food preferences, etc) and therefore may experience periods of intentional or unintentional fasting similar to TRF protocols. Because interest in TRF is gaining popularity and feeding may be interrupted in those who are pregnant, it is important to understand the long-term effects of TRF during pregnancy on the resultant offspring. Using a mouse model, we tested the effects of gestational exposure to early TRF (eTRF) over the life course of both male and female offspring. Offspring body composition was similar between experimental groups in both males and females from weaning (day 21) to adulthood (day 70), with minor increases in food intake in eTRF females and improved glucose tolerance in males. After 10 weeks of high fat, high sucrose diet feeding, male eTRF offspring were more sensitive to insulin but developed glucose intolerance with impaired insulin secretion. As such, gestational eTRF causes sex-specific deleterious effects on glucose homeostasis after chronic high fat, high sucrose diet feeding in male offspring. Further studies are needed to determine the effect gestational eTRF has on the male pancreas as well as to elucidate the mechanisms that protect females from this metabolic dysfunction. bioRxiv preprint doi: https://doi.org/10.1101/2022.04.27.489576; this version posted April 28, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. eTRF leads to offspring glucose intolerance Introduction The timing of food intake in reference to circadian rhythms can impact propensity for health or disease (1). Time-restricted feeding/eating (TRF/E), a method of intermittent fasting, aligns caloric intake with naturally occurring circadian rhythms of metabolism, acting as a zeitgeber. Timing of food intake is capable of programming metabolic systems for either poor health from chronodisruption, or good health with either diurnal or nocturnal feeding, depending on the species. To our knowledge, no estimate of the prevalence of TRE in humans exists. However, according to one sample, up to ten percent of people surveyed who state that they followed a diet in the year 2020 said they attempted “intermittent fasting,” making it the most prevalent dietary intervention in their sample (2). During pregnancy, one may have periods of time with limited food intake for many reasons: religious practice, food insecurity, disordered eating behaviors, nausea and vomiting of pregnancy/morning sickness, changes in taste/food preferences, or intentional timing of eating for weight maintenance. A recent cross-sectional study about the attitudes toward TRE in pregnant or postpartum women was conducted and found that 23.7% of those surveyed said they were willing to try TRE during pregnancy (3). The most available literature examined fasting during the month of Ramadan while pregnant. Review of these studies found that children born to those who fasted during pregnancy have babies with similar birth weights and rates of pre-term birth (4). The literature is most focused on the effects of the practice during infancy and early childhood in the resultant children. The diet is popular and interruptions in food intake are known to occur during pregnancy; however, research about the effects of fasting during pregnancy is limited to the observance of Ramadan, a cross-sectional study about attitudes toward the practice (3), and one case report of fasting to improve gestational diabetes (5). Detailed modeling of TRF in pregnancy is warranted, as TRE exists in human populations (3, 5) and effects are unknown. Previous studies of maternal diet during pregnancy have focused on dietary restriction or macronutrient excess in pregnancy, with little-to-no attention directed toward temporality of food intake. To date, one study of TRF during pregnancy in animals exists. This work emphasized fetal health and was completed in the context of preventing complications from overnutrition (a high fat diet, HFD) during gestation. Upadhyay and colleagues found that 9-hour TRF improved fetal lung development (6) and placental oxidative stress markers (7) at embryonic day (E)18.5 compared to ad libitum fed dams. This approach did not evaluate the long-term, postnatal effects of TRF and the independent effects of TRF are complicated by the use of a high fat diet. The effects of TRF in non-pregnant human populations are inconsistent. Some TRF trials result in significant weight loss (8–11) while others do not (12–14). Similarly, insulin sensitization results in some (8, 14–17), but not all trials of TRF (9, 13). The way TRF is employed in human studies is rarely consistent, with varying lengths of feeding window, timing of feeding window (early vs late), control of caloric intake (isocaloric vs ad libitum feeding), inpatient observation or outpatient adherence monitoring. As such, the biological effects of this eating strategy are not clear, even in non-pregnant humans. Results from rodent models of TRF are more consistent than human trials. These have found TRF of a HFD reduces body weight compared to ad libitum feeding (18–23), can improve Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) (20, 23, 24), and may limit complications like insulin resistance (21, 22) from HFD feeding. bioRxiv preprint doi: https://doi.org/10.1101/2022.04.27.489576; this version posted April 28, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. eTRF leads to offspring glucose intolerance Taking together the likelihood that food intake can be time-disrupted in pregnancy and the evidence of TRF being a potent method to improve body composition and glycemic health in adult mice, we sought to evaluate the impact of TRF of normal laboratory chow (6-hour, early dark-cycle) before and during pregnancy on resulting offspring body composition and glycemic health through adulthood. Methods Animal care and use Virgin female C57BL/6J mice were obtained from Jackson Laboratory (RRID IMSR_JAX:000664). All animals were maintained on a, 12-hour light/dark (12 dark (ZT12, 6pm):12 light (ZT0, 6am); ZT = zeitgeber time) cycle in a temperature and humidity-controlled room. After one week of acclimatization, they were single-housed and were assigned to feeding groups. Dams were randomized to either early time-restricted feeding (eTRF) or ad libitum (AL) feeding during gestation (n 8= eTRF, 9=AL). Dams fed AL had 24-hour access to a chow diet (NCD, Picolab Laboratory Rodent diet, 5L0D; 5% of Calories from fat, 24% from protein, 71% from carbohydrates). Dams fed eTRF had 6 hours of NCD food access during the early dark cycle (ZT 14-ZT 20). Water was provided ad libitum throughout the study to both experimental groups. After one week of either AL or eTRF feeding (beginning age 120 days), age-matched males were introduced into cages for breeding. Males were kept in the cage until a copulatory plug was detected. Daily, dams were transferred to a clean cage at ZT20, allowing for a cage free of food for eTRF animals and similar levels of handling between experimental groups. After birth, all dams switched to AL and were maintained on this diet until weaning at postnatal day (PND) 21.5. Therefore, any phenotype in the offspring is attributable to modifications to the pregestational/gestational diet. All experimental protocols were reviewed and approved by The University of Michigan Institutional Animal Care and Use Committee. Offspring growth and food intake monitoring Pups born were weighed and counted within 24 hours of birth. Litters were reduced to 4 pups (2 male, 2 female, when possible) at PND 3.5 to standardize milk supply among litters. At PND 21.5, offspring were weighed and body composition was assessed using EchoMRI 2100 (EchoMRI) before being weaned by sex and maternal feeding regimen and housed 4-5 per cage (eTRF males = 11, eTRF females = 19, AL males = 16, AL females =17). Offspring were given AL access to NCD until PND 70. Food intake and body composition were assessed weekly. Food intake is represented as an average per animal per day. After PND 70, all animals began AL 45% High Fat Diet (HFD; Research Diets D12451; 45% Fat/ 20% Protein/ 35% Carbohydrate). Insulin Tolerance and Glucose Tolerance Testing Baseline glucose (GTT) and insulin tolerance tests (ITT) were assessed at young adulthood towards the end of the NCD diet period (PND 60-70). Animals were transferred into a cage with no food during the early light cycle (ZT 2), with water freely available. After 6 hours, fasting blood glucose was assessed using a tail clip and a handheld glucometer (OneTouch Ultra). Shortly thereafter, an intraperitoneal injection of insulin was administered (Humulin, u-100; 0.75U/kg lean mass). Blood glucose was assessed by glucometer every 15 minutes for 2 hours. bioRxiv preprint doi: https://doi.org/10.1101/2022.04.27.489576; this version posted April 28, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. eTRF leads to offspring glucose intolerance One week later, glucose tolerance was assessed in a similar way (D-Glucose,1.5g/kg lean mass). Insulin and glucose tolerance were then re-assessed after HFD feeding (PND 140-160) (insulin dose 2.5U/kg lean mass, glucose dose 1.0g/kg lean mass). Area under curve was calculated for each animal by taking the sum of glucose at each time point, and then was averaged by sex and maternal feeding regimen. Rates of drop for ITT were calculated by limiting the dataset to the initial period after insulin administration (<60 minutes), taking the log of the glucose values and generating a slope for each animal. After each animal’s rate of drop was calculated, values were averaged by sex and treatment. Glucose Stimulated-Insulin Secretion testing in vivo One week after GTT and ITT, animals underwent glucose stimulated insulin-secretion (GSIS) testing (PND 160-170). At ZT2, animals were placed in a clean cage without food and with unrestricted access to water. After a 6-hour fast, animals were lightly anesthetized with isoflurane via drop jar and a baseline blood sample was collected via retro-orbital bleed with a heparinized capillary. Following baseline blood collection, an intraperitoneal injection of Dglucose (1.0g/kg lean mass) was given. After 15 minutes, animals were lightly anesthetized in the same manner and another blood sample was collected. Blood samples were allowed to clot on wet ice (~20 minutes), then were spun down in a cold centrifuge (4 °C, Eppendorf microcentrifuge, model 5415R) for 20 minutes at 2000 g. Serum was pipetted off and stored at 80 °C until analysis. Serum insulin was assessed via a commercially available ELISA kit (ALPCO 80-INSMSU-E10). Insulin was assessed in 5uL of serum and read via colorimetric assay. Statistical analysis All measures with p-values <0.05 were considered statistically significant. Data are presented as mean +/- standard error throughout. All statistical analyses were performed using R version 4.0.2 (25). Repeated measures, such as body composition, cumulative food intake, and responses to GTT or ITT were assessed via mixed linear effects modeling with random effects of mouse ID and dam and fixed effects of maternal dietary treatment, age, and sex using lme4 version 1.1-26 (26). Body composition and food intake were measured separately in 2 phases: during NCD feeding, and after being switched to HFD. Analyses were tested for significant interactions between sex and maternal dietary treatment. Models were assessed using a two-way ANOVA for sex and maternal dietary treatment, with an interaction between the two. If a significant interaction was observed, sex-stratified models were then used and the p-value for the interaction was reported. Otherwise, sex was used as a covariate in a non-interacting model. Observations were tested for normality by Shapiro-Wilk test and equivalence of variance by Levene’s test. Pairwise measures that were normal and of equal variance utilized Student’s ttests. Measures that were not normally distributed used non-parametric Mann-Whitney tests. bioRxiv preprint doi: https://doi.org/10.1101/2022.04.27.489576; this version posted April 28, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. eTRF leads to offspring glucose intolerance Results Gestational eTRF increases food intake, but not body composition in early life To model gestational early time restricted feeding (eTRF), we used a normal chow diet (NCD) and assigned female mice to either unrestricted (ad libitum, AL) or 6 hours of restricted food availability between ZT14-20 (eTRF, 50% of their active nocturnal window) (Figure 1A). This approach limits potential sleep disruptions and is more translationally relevant to human dietary restriction. This treatment started a week before mating and continued through delivery (Figure 1B). Litters were normalized to equal sizes to reduce variability and effects of lactation. Figure 1 Experimental Protocol and Timing A eTRF Food Access AL Food Access 12-hour Light Cycle ZT0 12-hour Dark Cycle ZT12 ZT24 B start 45% HFD start diet mate birth wean reduce litter eTRF Maternal AL AL 3.5 E-7 E1 PND 0.5 21.5 GTT/ITT sacrifice ITT GTT GSIS Offspring AL HFD Offspring AL NCD 60-69.5 70.5 140-170 175.5 Figure 1: Experimental Protocol and Timing A) Food availability and timing for dams during pregnancy. Food access began at ZT13 for early TimeRestricted Feeding dams (eTRF, light gray, n=8) and continued until ZT129, total of 6 hours. Food was available 24 hours a day for ad libitum dams (AL, dark gray, n=9). B) Offspring experimental protocol. After birth, all dams had AL access to laboratory chow (NCD). Litters were reduced to 4 (2 males, 2 females when possible) on post-natal day (PND) 3. Offspring were weaned by maternal feeding regimen at PND 21 and maintained on AL NCD for 70 days. Weekly body composition and food intake measurements were taken throughout the experiment. At 70 days of age, insulin tolerance tests (ITT) and glucose tolerance tests (GTT) were conducted before switching all animals to a 45% high fat diet (HFD) with sucrose. Animals were on HFD for 10 weeks before repeating ITT and GTT, and an in vivo glucose stimulated insulin secretion test (GSIS). Animals were euthanized after these tests. Abbreviations: zeitgeber time (ZT), ZT0 = lights on, ZT12 = lights off. The pups were weighed and their body composition was assessed weekly, then analyzed using linear mixed effect modeling. We found significant and expected effects of age and sex (older mice weigh more than younger mice and male pups weigh more than females), but no effect modification of maternal eTRF on body weight (Figure 2A, pdiet=0.47), lean mass (Figure 2C, pdiet=0.45), or fat mass (Figure 2B, pdiet=0.47). There was no interaction between sex and bioRxiv preprint doi: https://doi.org/10.1101/2022.04.27.489576; this version posted April 28, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. eTRF leads to offspring glucose intolerance maternal intervention in cumulative food intake (pdietsex=0.38). However, cumulative food intake in the NCD period is 22% higher in eTRF females than AL females and 10% higher in eTRF males than AL males (Figure 2D, pdiet = 0.016). Assessing the efficiency by which food is converted into stored mass resulted in a 12% reduced feeding efficiency in eTRF female offspring (psex<0.00001) which is not present in males (Supplementary Figure 1A). Gestational eTRF modestly improves glucose tolerance in young adult males To assess glucose homeostasis in the offspring, we conducted ITTs and GTTs between PND 60 and 70. Male offspring averaged 15mg/dL higher blood glucose during insulin tolerance testing compared to females (psex=0.0018), but no effect of maternal dietary restriction was evident through linear mixed effect modeling (Figure 2E, pdiet=0.73). Summarizing the ITT by calculating the area under the curve (AUC) demonstrated there was no diet:sex interaction (pdiet:sex=0.069), but an effect of maternal restriction where eTRF offspring had lower AUC compared to AL offspring, 8.5% and 2.2% lower in females and males respectively (pdiet=0.013), and a significant effect of sex (psex<0.0001). As expected, males had a higher AUC than females (Figure 2F). The initial response to insulin (the rate of glucose decline over the first 60 minutes, not pictured) was not significant for sex (psex=0.10) or treatment (pdiet=0.83). Taken together these data suggest that gestational eTRF slightly improves the response to insulin challenge in adult mice, and that this is not driven by increased fat mass. Glucose tolerance was similar in young adulthood between groups in both males and females (Figure 2G). We found no significant effect of diet (pdiet=0.53) on the rise in blood glucose during GTT, but there was an effect of sex (psex=0.0093) on glucose tolerance, again with expected higher glucose levels in male mice. The summarized AUC for the GTT (Figure 2H) shows a significant interaction between sex and maternal dietary treatment (psex:diet=0.00082). eTRF males had an 8.2% lower AUC than their AL counterparts (pdiet<0.0001) while this was absent in females (pdiet=0.99). Fasting blood glucose, assessed before ITT and GTT, was 10.4% higher in males than in females (psex=0.0054; Figure 2I), but did not differ significantly by maternal dietary treatment (pdiet=0.18). Thus, there are modest glycemic effects of gestational eTRF present in young, chow-fed male offspring that were not explained by differences in weight or body composition, which was comparable between groups. bioRxiv preprint doi: https://doi.org/10.1101/2022.04.27.489576; this version posted April 28, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. eTRF leads to offspring glucose intolerance Figure 2: Early Life Body Composition, Food Intake, and Glycemic Homeostasis A) Body weight in grams from PND21-PND70 in males and females, averaged by age, maternal feeding regimen, and sex. B) Fat mass in grams from PND21-PND70 in males and females, averaged by age, maternal feeding regimen, and sex. C) Lean mass in grams from PND21-PND70 in males and females, averaged by age, maternal feeding regimen, and sex. D) Food intake in kcals per mouse per day, averaged by week, maternal feeding regimen, and sex. p-value <0.05 for diet. E) Insulin tolerance test (ITT) ~PND 70, averaged by maternal feeding regimen, sex, and time in minutes. F) Area under the curve (AUC) for ITT, averaged by maternal feeding regimen, and sex. * indicates p-value <0.05 for effect of diet in males. G) Glucose tolerance test (GTT) ~PNG 70, averaged by maternal feeding regimen, sex, and time in minutes. H) AUC for GTT, averaged by maternal feeding regimen, and sex. * indicates p-value <0.05 for effect of diet in males. I) Fasting blood glucose (FBG) PND 70, averaged by maternal feeding regimen and sex. Animals included in body composition measurements, FBG, ITT, and GTT, n=11 eTRF males, 16 AL males, 19 eTRF females, 17 AL females. Number of cages in food intake analysis n=4 eTRF males, 5 AL males, 4 eTRF females, 5 AL females. bioRxiv preprint doi: https://doi.org/10.1101/2022.04.27.489576; this version posted April 28, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. eTRF leads to offspring glucose intolerance HFD feeding in adult offspring exposed to eTRF during gestation generates sexspecific glucose intolerance Given that adult offspring were minimally affected by gestational eTRF exposure, we administered a high fat, high sucrose (HFD) overnutrition challenge; ad libitum access to 45% of energy from fat and 17% of energy from sucrose after PND 70. Food intake and body composition measurements continued weekly. Similar to the findings on chow, with HFD, there were no major differences between eTRF and AL offspring in body weight (Figure 3A, pdiet=0.99), fat mass (Figure 3B, pdiet=0.65), or lean mass (Figure 3C, pdiet=0.47). Therefore, offspring of eTRF and AL experienced similar changes in body composition in response to overnutrition. Cumulative HFD consumption was comparable between females and males (psex=0.72), and maternal restriction groups (Figure 3D, pdiet=0.72). Feeding efficiency was greater in males than in females, which is consistent with the NCD period (Supplemental Figure 1B, psex = 0.00023). However, unlike the NCD period, efficiency was indistinguishable between eTRF and AL offspring (pdiet=0.93). We repeated an ITT and GTT after 10 weeks of HFD feeding. During the ITT, there was a significant interaction between sex and diet using mixed linear effect modeling (Figure 3E, psex:diet=0.03). Female eTRF had a similar response to insulin, with less than a 1 mg/dL difference from their AL counterparts (pdiet=0.85), but male eTRF offspring tended to be more insulin sensitive with 25mg/dL lower glucose compared to AL males (pdiet=0.17). These findings were confirmed by calculating the AUC where eTRF females had 7% greater AUC than AL females (Figure 3F, pdiet=0.20) while eTRF males had 20.4% lower AUC than AL males (pdiet<0.0001). The initial rate of glucose decline (not pictured) was greater in females compared to males (psex=0.029) but there were no differences between eTRF and AL offspring (pdiet=0.23). The trend toward insulin sensitivity from the ITT was not explained by fasting blood glucose, as females had 23% lower fasting blood glucose than males (psex<0.0001) but were similar between eTRF and AL offspring within the same sex (Figure 3I, pdiet=0.83 ). Glucose tolerance tests in Figure 3G, also showed significant effect of interaction (psex:diet=0.011), although now in the opposite direction. During GTT, eTRF males trended toward glucose intolerance with an average of 53mg/dL higher glucose than AL males during the course of the experiment (pdiet=0.14). This was not observed in female eTRF offspring, which had similar blood glucose during the GTT compared to AL females (pdiet=0.61). The GTT AUC showed interaction between effects of sex and treatment (Figure 3H, (psex:diet<0.0001)). AUC was 5% lower in eTRF females (pdiet=0.07) but was 13.5% higher in eTRF male offspring compared to AL (pdiet<0.0001). Taken together, these tests suggest eTRF causes male-specific glucose intolerance and insulin sensitivity. Given that we cannot explain glucose intolerance in males via reduced insulin sensitivity, we next evaluated insulin secretion. To test for insulin secretion defects, we conducted an in vivo glucose stimulated insulin secretion (GSIS) assay (Figure 3J). Females had lower levels of insulin than males (psex<0.0001). There was a non-significant trend toward lower insulin levels in eTRF compared to AL offspring of both sexes (pdiet=0.071). Females had similar increases in insulin in response to glucose injection, 139% in AL versus 137% eTRF. Male AL offspring had a 48% increase in insulin whereas this was just an 18% increase for eTRF males. There was no interaction between sex and maternal restriction (psex:diet=0.064). Females have 94% greater fold-change insulin secretion in response to glucose challenge than male offspring (psex=0.0027) and there is no impact of maternal restriction (p=0.85, Figure 3K). Although not conclusive, the GSIS lends bioRxiv preprint doi: https://doi.org/10.1101/2022.04.27.489576; this version posted April 28, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. eTRF leads to offspring glucose intolerance support to a model that sex-specific defects in insulin secretion result in sex-specific glucose intolerance after HFD challenge in males exposed to eTRF in utero. bioRxiv preprint doi: https://doi.org/10.1101/2022.04.27.489576; this version posted April 28, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. eTRF leads to offspring glucose intolerance Figure 3: Body Composition, Food Intake, and Glycemic Response to High Fat Diet Feeding in Adulthood A) Body weight in grams from PND 70-175 in males and females, averaged by age, maternal feeding regimen, and sex. B) Fat mass in grams from PND 70-175 in males and females, averaged by age, maternal feeding regimen, and sex. C) Lean mass in grams from PND 70-175 in males and females, averaged by age, maternal feeding regimen, and sex. D) High fat diet (HFD) intake in kcals per mouse per day averaged by week, maternal feeding regimen, and sex. E) Insulin tolerance test (ITT) after 10 week of HFD, averaged by age, maternal feeding regimen, sex, and time in minutes. F) Area under the curve (AUC) for insulin tolerance test, averaged by maternal feeding regimen, and sex. * indicates, p-value <0.05 for diet in males. G) Glucose tolerance test (GTT) after 10 weeks of HFD, averaged by maternal feeding regimen, sex and time in minutes. H) Area under the curve (AUC) for GTT after 10 weeks of HFD, averaged by maternal feeding regimen and sex. * indicates p-value <0.05 for effect of diet in males. I) Fasting blood glucose (FBG) after 10 weeks HFD, averaged by maternal feeding regimen, and sex. J) Glucose stimulated insulin secretion (GSIS), averaged by maternal feeding regiment, sex, and time. * indicates p-value <0.05 for effect of sex. Animals included in body composition, FBG, ITT, GTT, and GSIS: n=11 eTRF males, 16 AL males, 19 eTRF females, 17 AL females. Cages in food intake analysis: n=4 eTRF males, 5 AL males, 4 eTRF females, 5 AL females. bioRxiv preprint doi: https://doi.org/10.1101/2022.04.27.489576; this version posted April 28, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. eTRF leads to offspring glucose intolerance Discussion This study is the first to describe the long-term effects of gestational eTRF on offspring health and their response to a high fat, high sucrose diet challenge. We find significant deleterious effects of gestational eTRF on glucose tolerance are present only in adult male offspring when exposed to long-term HFD feeding. Based on GSIS testing, we propose that this is attributable to impaired insulin secretion, as insulin secretion tended to be lower in eTRF males compared to their AL counterparts, although this did not reach statistical significance. Other studies of TRF using HFD in mice provide evidence that fasting insulin is lowered (20–23, 27) and resulting HOMA-IR is improved (22, 27, 28). We see that baseline insulin is modestly lower in male offspring, and this could contribute to the modest insulin sensitivity seen after HFD feeding. Our finding that fasting blood glucose is unchanged in eTRF compared to AL exposed mice is confirmed by other groups examining TRF with HFD (20, 27, 28). The elevated food intake in female offspring exposed to eTRF in utero is novel. Studies of adult mice pairing TRF and HFD report reduced food intake (24, 29) or equivalent caloric intake when matched by diet (21–23, 30). This could indicate a compensatory response in the female offspring resulting from eTRF in utero. Interestingly, this did not result in differing body weight or composition, suggesting that this increased food intake is matched by decreased caloric extraction or increased energy expenditure in these mice. The phenotype in male offspring from this study bears resemblance to animal models of intrauterine growth restriction (IUGR), where glucose intolerance in resultant offspring is common. First described by Barker and colleagues, offspring who were deprived of nutrition in utero were more likely to develop chronic, nutrition-related disease in adulthood (31). Since that time, multiple animal models for IUGR were developed; maternal overnutrition during pregnancy, maternal caloric restriction, maternal protein restriction, and surgically induced placental insufficiency through late gestation uterine artery ligation. Undernutrition in pregnancy often results in offspring development of glucose intolerance (32–34). The extent to which malespecific effects are seen is difficult to deduce as many groups either study male offspring exclusively (34, 35) or analyze males and females together (33, 36). Male offspring who had placental insufficiency develop glucose intolerance in adulthood (37, 38), but females can also develop glucose intolerance (39, 40). Maternal overnutrition can also result in males with glucose intolerance (41, 42). Therefore, metabolic effects being limited in the current study to male offspring is consistent with much of the literature, as females appear to be less affected. Glucose intolerance in IUGR models often occurs with insulin-related abnormalities in the offspring, such as lower insulin content in the pancreas (33), lower basal insulin levels (36), impaired insulin secretion (34, 40), increased pancreatic islet size (42), altered vascularity of islets (43), or reduced beta cell mass (44). These abnormalities are also accompanied by abnormal glucose tolerance in adulthood (32, 42). However, in the present study we find modest improvement in male insulin sensitivity in adulthood in male offspring exposed to gestational eTRF. We attribute male-specific insulin sensitivity during high fat diet feeding to eTRF males having lower basal levels of insulin compared to AL males. This means that peripheral tissues would be more sensitive to insulin action despite an apparent insulin secretion impairment at the level of the pancreas. The similarity of the present study to those using diverse gestational stressors suggests that restriction of the total time of food intake in dams is sufficient to induce offspring glucose intolerance similar to IUGR models, but not insulin resistance. bioRxiv preprint doi: https://doi.org/10.1101/2022.04.27.489576; this version posted April 28, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. eTRF leads to offspring glucose intolerance Although we have not investigated offspring pancreatic tissues, we hypothesize that alterations in the development of the pancreas may underlie the male-specific glucose intolerance and modest insulin sensitivity in eTRF offspring. Time-limiting the availability of nutrients to the fetus through eTRF may program offspring pancreas for development during daily periods of nutrient scarcity and result in impaired beta cell development or islet size leading to reduced insulin secretion. Intrinsic changes in islet function are also possible. Studies done in adult male animals undergoing TRF with chronodisruption have also found that time-restricting food access reduced insulin production with secretion most affected (enhanced compared to controls) and found no effect of insulin tolerance (45). This is confirmed by one study of early post-natal exposure to TRF, which found that adolescent males who were fed TRF the first 4 weeks after weaning developed smaller islets of Langerhans and higher blood glucose compared to those fed AL (30). Another contributor to this phenomenon may be that the islets were able to compensate in young male offspring during a lower-calorie diet (NCD) and therefore the effect did not become apparent until an overnutrition challenge during adulthood. Therefore, future studies of gestational or developmental eTRF should examine islet size, pancreatic beta cell mass, and insulin secretion to investigate the mechanism of offspring glucose intolerance further. This study and the conclusions to be made from it have some limitations. First, the model of gestational eTRF may have resulted in differences in maternal behaviors that were not noted by the study team, and therefore could play a part in the effects seen in the offspring. Second, although we see a robust effect on glucose intolerance and trends of lower insulin secretion in male eTRF offspring in adulthood, we did not evaluate islet size or beta cell mass to determine the mechanism driving the worsening of glucose tolerance in adulthood. There are many strengths to this study. Among them are the use of a preclinical model which facilitates consistency when compared to existing literature. Further strengths include the long follow up period for a gestational exposure, controlling for the effect of litter size, repeated measurement of body composition, and food intake measurements over the life course in the resultant offspring. Finally, the inclusion of both male and female offspring in the study, as many metabolic assessments of TRF either focus exclusively on the effects of the regimen in males (22, 23) or female mice (20, 21) is a strength. Future work with this model should include assessment of offspring pancreatic tissue. Finally, our model used healthy non-obese dams and our results cannot be extended to effects of eTRF in the context of metabolic syndrome, diabetes, or obesity during pregnancy. Conclusion Offspring who are exposed to eTRF of NCD in utero have similar body composition, glucose tolerance, and insulin tolerance in early adulthood in both males and females. Gestational eTRF led to sex-specific impairments in male glucose tolerance in adulthood after chronic HFD feeding, likely due to impaired insulin secretion. This occurs without increase in body weight, fat mass, or food intake compared to age matched AL males. More research is warranted to understand the mechanisms that underlie this novel phenotype. bioRxiv preprint doi: https://doi.org/10.1101/2022.04.27.489576; this version posted April 28, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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Late-pregnancy uterine artery ligation increases susceptibility to postnatal Western diet-induced fat accumulation in adult female offspring. Sci Rep 2020;10:6926. bioRxiv preprint doi: https://doi.org/10.1101/2022.04.27.489576; this version posted April 28, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. eTRF leads to offspring glucose intolerance 40. Jansson T, Lambert GW. Effect of intrauterine growth restriction on blood pressure, glucose tolerance and sympathetic nervous system activity in the rat at 3–4 months of age. Journal of Hypertension 1999;17:1239–1248. 41. Zhang Q, Xiao X, Zheng J, et al. A Maternal High-Fat Diet Induces DNA Methylation Changes That Contribute to Glucose Intolerance in Offspring. Front Endocrinol (Lausanne) 2019;10:871. 42. Zheng J, Zhang L, Wang Z, Zhang J. Maternal high-fat diet regulates glucose metabolism and pancreatic β cell phenotype in mouse offspring at weaning. PeerJ 2020;8:e9407. 43. Boehmer BH, Limesand SW, Rozance PJ. The impact of IUGR on pancreatic islet development and β-cell function. J Endocrinol 2017;235:R63–R76. 44. Simmons RA, Templeton LJ, Gertz SJ. Intrauterine Growth Retardation Leads to the Development of Type 2 Diabetes in the Rat. Diabetes 2001;50:2279–2286. 45. Brown MR, Sen SK, Mazzone A, et al. Time-restricted feeding prevents deleterious metabolic effects of circadian disruption through epigenetic control of β cell function. Sci Adv 2021;7:eabg6856. bioRxiv preprint doi: https://doi.org/10.1101/2022.04.27.489576; this version posted April 28, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. eTRF leads to offspring glucose intolerance Supplemental Figure 1: Feeding Efficiency Throughout Adulthood A) Feeding efficiency (%) in males and females, calculated based on food intake and body composition changes during the NCD period (before PND 70). (psex<0.001, pdiet=0.002). B) Feeding efficiency in males and females during the HFD period (after PND 70). (psex = 0.00023, pdiet = 0.093).
https://openalex.org/W3029622962	https://europepmc.org/articles/pmc7272686?pdf=render	English	null	Gehua Jiecheng Decoction Inhibits Diethylnitrosamine-Induced Hepatocellular Carcinoma in Mice by Improving Tumor Immunosuppression Microenvironment	Frontiers in pharmacology	2,020	cc-by	10,832	Citation: Cheng C, Shou Q, Lang J, Jin L, Liu X, Tang D, Yang Z and Fu H (2020) Gehua Jiecheng Decoction Inhibits Diethylnitrosamine-Induced Hepatocellular Carcinoma in Mice by Improving Tumor Immunosuppression Microenvironment. Front. Pharmacol. 11:809. doi: 10.3389/fphar.2020.00809 ORIGINAL RESEARCH published: 29 May 2020 doi: 10.3389/fphar.2020.00809 Keywords: Gehua Jiecheng Decoction, diethylnitrosamine-induced hepatocarcinogenesis, immune microenvironment, inﬂammation, blood vessel formation Edited by: Edited by: Sanjay K. Srivastava, Texas Tech University Health Sciences Center, United States 1 Afﬁliated First Hospital, Guizhou University of Traditional Chinese Medicine, Guiyang, China, 2 Afﬁliated Secondary Hospital, Zhejiang Chinese Medical University, Hangzhou, China, 3 Graduate School, Tianjin University of Traditional Chinese Medicine, Tianjin, China Reviewed by: Junhua Mai, Houston Methodist Research Institute, United States Xu Zhang, Jiangsu University China Gehua Jiecheng Decoction (GHJCD), a famous traditional Chinese medicine, has been used in the prevention and treatment of precancerous lesion of liver cancer, but its active mechanism has not been reported. This study aimed to evaluate the therapeutic effect of GHJCD on diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in mice and the mechanism of this effect. We found that GHJCD effectively inhibited the occurrence of liver cancer and reduced the tumor area. The ratio of regulatory cells (Tregs), tumor- associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs) in HCC microenvironment was down-regulated, whereas that of CD8 T and effective CD8 T cells was up-regulated. In addition, the expression levels of inﬂammatory factors IL-6, IL-10, TNF-a, and CCL-2 in the liver were inhibited, whereas those of the angiogenesis related molecules CD31 and VEGF were decreased. Moreover, WNT1, b-catenin, NF-kB, p- MAPK, p-AKT, and p-SRC content in the liver decreased, whereas APC content increased. These results suggested that GHJCD exerted a good inhibitory effect on liver cancer induced by DEN and thus may have a multi-target effect; GHJCD not only antagonized the immunosuppressive effect of the microenvironment of liver cancer but also exerted strong anti-inﬂammatory and antiangiogenesis effects. Correspondence: Dongxin Tang tangdongx@sina.com Zhu Yang yangzhu20150426@163.com Huiying Fu fhy131@126.com †These authors have contributed equally to this work †These authors have contributed equally to this work Specialty section: This article was submitted to Pharmacology of Anti-Cancer Drugs, a section of the journal Frontiers in Pharmacology Received: 10 November 2019 Accepted: 18 May 2020 Published: 29 May 2020 Specialty section: This article was submitted to Pharmacology of Anti-Cancer Drugs, a section of the journal Frontiers in Pharmacology Received: 10 November 2019 Accepted: 18 May 2020 Published: 29 May 2020 Changpei Cheng 1,2,3†, Qiyang Shou 2†, Jiali Lang 2, Lu Jin 2, Xia Liu 2, Dongxin Tang 1, Zhu Yang 1* and Huiying Fu 2* INTRODUCTION Hepatocellular carcinoma (HCC) is the ﬁfth most common cancer in the world and the third leading cause of cancer-related death (Siegel et al., 2018). In most countries, the mortality rate is almost equal to the morbidity rate, which indicates the lack of effective treatment for liver cancer. The molecular pathogenesis of HCC is extremely complex and heterogeneous. The traditional May 2020 \| Volume 11 \| Article 809 Frontiers in Pharmacology \| www.frontiersin.org GHJCD Inhibits Hepatocellular Carcinoma Cheng et al. component of its, which has the effects of immune enhancement, anti-tumor, anti-inﬂammation, and liver protection (Guo et al., 2019). Poria cocos polysaccharide is the main active component of its. Through the combined regulation of NF-kB signal transduction, it shows immunomodulatory activity can signiﬁcantly reduce the tumor volume and has the pharmacological effect of anti-cancer (Tian et al., 2019). The racemic-dihydroguaiacic acid extracted from nutmeg showed effective cytotoxicity and anti-tumor activity in allogenic tumor-bearing mice (Thuong et al., 2014). Its water extract can inhibit the release of proinﬂammatory cytokines such as IL-6 and tumor necrosis factor (Kim et al., 2013). The water extract of Amomum villosum could signiﬁcantly increase the percentage of CD4 T cells (Chen et al., 2018). Atractylodes macrocephala shows good cytotoxicity and anti-tumor effect by blocking S phase tumor cells. Especially compared with cyclophosphamide, it can protect immune organs better (Feng et al., 2019). Our previous study found that GHJCD can inhibit subcutaneous transplantation and orthotopic liver transplantation of HCC cells in mice (Guo et al., 2019). However, the active mechanism of GHJCD regulates liver cancer is still not clear. cancer treatment is mainly focused on a single target or a single mechanism. Unexpectedly, it has not achieved the ideal therapeutic effect. Or spectral anti-cancer produces greater toxicity and promotes the development of tumors (Xu et al., 2016). Systemic therapy is the best choice for patients with advanced liver cancer. Combination therapy of multiple treatment schemes is still the way to focus on in the future. Traditional Chinese medicine (TCM) shows a wide range of biological effects, and more evidence shows that it may be related to the regulation of tumor microenvironment and killing tumors by strengthening the immune system (Xu et al., 2016). Because of the effectiveness and fewer side effects of TCM, it has been widely accepted as a supplement and alternative therapy for cancer in China (Xiang et al., 2019). INTRODUCTION As a new and effective tumor therapy, immunotherapy is considered to be one of the most promising areas of oncology, which aims to help patients’ own immune system resist cancer (Hu et al., 2019). In most patients with solid tumors, vascular abnormalities help the tumor evade attack by the immune system (Mukaida and Nakamoto, 2018). These abnormalities are due to an increase in angiogenic factors, such as VGEF (Bouzin et al., 2007) and angiogenin 2 (ANG2) (Etoh et al., 2000). Drugs targeting these molecules can normalize the abnormal tumor vascular system and increase the inﬁltration of immune effector cells. In addition, tumor-related inﬂammation destroys the anti-tumor immune response by promoting angiogenesis (Bouzin et al., 2007) and metastasis (Bollrath and Greten, 2009). Persistent inﬂammatory cells and factors can also transform tumor-related inﬂammatory microenvironment into immunosuppressive microenvironment, promoting tumor progression (Deng-Bo and Cui, 2010). Therefore, inﬂammation, angiogenesis, and immunosuppressive microenvironment are three major obstacles to tumor therapy. This study is to observe the therapeutic effect of GHJCD on liver cancer induced by diethylnitrosamine (DEN), and to explore the mechanism of its anti-tumor activity, so as to provide theoretical basis for clinical treatment. Animals and Reagents Eighty male C3H mice (6 weeks old, weighing 20–25 g) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Beijing, china), and kept in a speciﬁc pathogen free mouse breeding room with controlled temperature of 25 ± 1°C. The mice were provided free access to food. j py Gehua Jiecheng Decoction (GHJCD) is derived from the theory of spleen and stomach written by Li Dongyuan, one of the four masters of medicine in the Yuan Dynasty. It is composed of green skin, wood incense, orange peel, ginseng, Polyporus umbellatus, Poria cocos, Fried Shenqu, Alisma, Ginger, Atractylodes macrocephala, Nutmeg kernel, and Pueraria lobate, Amomum villosum, a total of 13 TCMs. In China, GHJCD is often used to treat liver cirrhosis and liver injury caused by drinking (Hu et al., 2020). Pueraria lobata, the main component of GHJCD, has long been used in the treatment of chronic alcoholic liver injury, has a protective effect on alcohol- induced apoptosis, and there are no side effects of it (Wu et al., 2019). Pueraria lobata extracted reduces hepatic ﬁbrosis and hepatotoxicity (Peng et al., 2019), inhibits the activation of Kupffer cells and weakens the anti-inﬂammatory response of NF-kB pathway (Ahmad et al., 2020). Ginseng has a strong inhibitory effect on inﬂammatory mediators such as IL-6 (IL-6) and TNF-a and proinﬂammatory cytokines induced by macrophages (Yao et al., 2019). 6-shogaol (6-sho), the bioactive component of ginger, has anti-inﬂammatory and anticancer properties, weakens the proliferation of tumor cells and induces the death of liver cancer cells (Nazim and Park, 2019). Polyporus umbellatus polysaccharide is the main bioactive Anti-CD45 FITC, anti-F4/80 PE, anti-CD11b APC, and anti- Gr1PerCP-Cy5.5 were purchased from BD Biosciences (Lake Franklin, New Jersey, United States). Anti-CD3 FITC antibody was purchased from BioLegend (San Diego, California, United States). Primary antibodies against WNT1 (H-89) and NF-KB P65(F-6) were purchased from Santa Cruz Biotechnology Co., Ltd. (Santa Cruz Avenue, California, United States). Primary antibodies against VEGFA (VG-1), b-catenin, and APC were purchased from Abcam (Cambridge, MA, United States). Primary antibody against b-actin was purchased from Huaan Biotechnology Co., Ltd. (Hangzhou, China). Anti-CD31 was purchased from Abcam. DEN was purchased from Merck Group (Darmstadt, Germany). Experimental Groups Column temperature: 40°C; sample temperature: 10°C; sample and standard sample injection volume: 0.5 ml. Eighty 6-week-old male C3H mice were randomly divided into four groups: the control, model, low-dose GHJCD (LG), and high-dose GHJCD (HG) groups. There were 20 mice in each group. For 23 weeks, mice in the control group were provided sterile water, whereas those in the other groups were provided DEN solution (15 mg/ml). Mass spectrometry conditions: SYNAPT G2-Si (Waters, Milford, MA, USA) mass spectrometer, electrospray ion source (ESI); positive and negative ion scanning mode, desolvation gas ﬂow rate: 1,000 L, desolvation gas temperature: 500°C, ion source temperature: 120°C; Cone hole voltage: 20.0 V, capillary voltage: 3.0 kV (+), 3.0 kV (−); Locked mass solution: the on-line quality correction was carried out by Lockspray correction system of Waters company, leucine-enkephalin (Leueine-Enkephalin, [M + H]+ = 556.2771, [M −H] = 554.2615), the concentration of the solution was 2 ng/L and the ﬂow rate was 10 ml. The scanning mode is MSe; mass scanning range: m50–1,200 Da, scanning time 0.2 s, positive and negative ion mode high collision energy transfer is 15–30 V, trap is 10 V, workstation: MassLynx V4.1 workstation. Data is matched and analyzed by Uniﬁ2.0 software (Waters, Milford, MA, USA). Preparation of Extracts and Drug Treatment GHJCD containing Flower of Pueraria montana var. lobata (Willd.) 15 g (Zhejiang Chinese Medical University prepared pieces Co., Ltd, Hangzhou, Zhejiang, China, No.: 190301), Nutmeg 15g (Hangzhou East China traditional Chinese Medicine prepared pieces Co., Ltd, Hangzhou, Zhejiang, China, No.: 190326), Fructus Amomi 15 g (Zhejiang Chinese Medical University prepared pieces Co., Ltd, Hangzhou, Zhejiang, China, No.: 190601), Alisma plantago-aquatica subsp. Orientale 6 g (Zhejiang Zoli Baicao traditional Chinese Medicine pieces Co., Ltd. Huzhou, Zhejiang, China, No.: 20190601), Atractylodes Macrocephala 6 g (Zhejiang Zoli Baicao traditional Chinese Medicine pieces Co., Ltd. Huzhou, Zhejiang, China, No.: 20190501), Panax ginseng C.A.Mey. (Araliaceae) 4.5 g (Zhejiang Chinese Medical University prepared pieces Co., Ltd, Hangzhou, Zhejiang, China, No.: 190601), Polyporus umbellatus 4.5 g (Hangzhou East China traditional Chinese Medicine prepared pieces Co., Ltd, Hangzhou, Zhejiang, China, No.: 190326), Poria cocos 4.5 g (Hangzhou East China traditional Chinese Medicine prepared pieces Co., Ltd, Hangzhou, Zhejiang, China, No.: 190830), Zingiber ofﬁcinale Roscoe [Zingiberaceae] 6 g (Zhejiang Chinese Medical University prepared pieces Co., Ltd, Hangzhou, Zhejiang, China, No.: 190601), Pericarpium Citri Reticulatae 4.5 g (Hangzhou East China traditional Chinese Medicine prepared pieces Co., Ltd., Hangzhou, Zhejiang, China, No.: 190908), Pericarpium Citri Reticulatae Viride 0.9 g (Zhejiang Zoli Baicao traditional Chinese Medicine pieces Co., Ltd. Huzhou, Zhejiang, China, No.: 20190402), Radix Aucklandiae 1.5 g (Zhejiang Chinese Medical University prepared pieces Co., Ltd., Hangzhou, Zhejiang, China, No.: 181101), Massa Medicata Fermentata 6 g (Zhejiang Zoli Baicao traditional Chinese Medicine pieces Co., Ltd. Huzhou, Zhejiang, China, No.: 20190801). The above drugs were soaked in 500-ml aseptic water for 30 min, boiled for 30 min, and then ﬁltered and concentrated into low-dose (crude drug concentration 2.25 g/ml) and high-dose (4.5 g/ml) GHJCD solutions. f f b The drugs were administered orally at a volume of 10 ml/kg once a day, and distilled water was administered at an equal volume. Three rats in each group were sacriﬁced at the 8th week, and six rats at the 12th week. The liver was removed, and liver sections were then stained with hematoxylin-eosin (HE) to observe the dynamic formation of liver cancer. After 23 weeks of treatment, the remaining mice were sacriﬁced. Before isolation of the liver, the hepatic portal vein was located by laparotomy and perfused with 10-ml normal saline to ﬂush the liver until it turned gray. Liver Cancer Model Liver Cancer Model C3H mice were provided free access to water containing DEN 15 mg/ml, which was changed daily, for 23 weeks without interruption to successfully induce liver cancer in the mice. May 2020 \| Volume 11 \| Article 809 Frontiers in Pharmacology \| www.frontiersin.org GHJCD Inhibits Hepatocellular Carcinoma Cheng et al. HE Staining of Liver Tissues Mouse livers were cut into approximately 2.0 cm × 2.0 cm × 0.3 cm tissue blocks, ﬁxed in 4% paraformaldehyde ﬁxed solution for 24 h, dehydrated until transparent, embedded in wax, and then cut into 4-mm-thick sections. The sections were then subjected to Harris hematoxylin staining for 5 min, 1% hydrochloric acid ethanol color separation for 5 s, 0.5% ammonia for 20 s, and 0.5% eosin staining for 3 min. After each staining, the sections were washed in distilled water for 1 min. The sections were sealed after dehydration and transparent staining. The sections stained with HE were scanned, imaged, and observed by an Eclipse80i microscope (Nikon, Tokyo, Japan). The area of liver cancer was evaluated by NDP.view2 U12388Mui 01 digital pathological scanning software (Hamamatsu, Japan). Flow Cytometry Analysis of GHJCD, and the others from Panax ginseng C.A.Mey. (Araliaceae), Atractylodes Macrocephala and so on (Table 1). The liver was placed into a 6-well plate and grinded. Next, lymphocytes were isolated using a lymphocyte separation solution, stained with the above antibodies, and detected by ﬂow cytometry (Beckman Coulter, Pasadena, California). Western Blotting Analysis Western Blotting Analysis First, 100-mg liver tissue was cut, mixed with 350-µl lysate (Biyuntian Biotechnology Co., Ltd., Shanghai, China), ground on ice, and centrifuged (12,000 rpm at 4°C for 10 min). The supernatant was taken, and the total protein in liver tissue was extracted. Protein concentration in liver tissue was detected by BCA protein concentration determination kit (Biyuntian Biotechnology Co., Ltd.). The total protein was then separated by 8% or 10% SDS gel electrophoresis according to the molecular weight of the protein, and transferred to a nitroﬁbrin-imprinted membrane. The nitroﬁbrin imprinted membrane was blocked by 5% skim milk in Tris buffer for 2 h, and then incubated for 24 h with anti-NF-kB, WNT1, b-catenin, VEGF, and APC (1:1,000 dilution) at 4°C. The membrane was then washed three times with washing buffer for 10 min, incubated with secondary antibodies at room temperature for 2 h, washed three times for 10 min each, and treated with Millipore Western Blot HRP chemiluminescence solution (Millipore Corporation, Billerica, MA, USA). Protein visualization was carried out with an ultra- sensitive chemiluminescence imaging system (A Biotechne Brand, United States), and then the strip was quantitatively analyzed by the AlphaView-FluorChem FC3 3.4.0.0 software (ProteinSimple, Silicon Valley, California). As the experiment entered the ﬁnal stage at the 23rd week, a large number of liver cancer nodules appeared on the liver surface of the model mice and merged into large liver cancer nodules (Figure 1A: f); moreover, a small amount of liver cancer nodules appeared on the liver surface of mice in the LG group (Figure 1A: i), but no liver cancer nodules were found on the liver surface of the control and HG groups (Figure 1A: c, l). HE staining analysis showed no cancer in the control mice (Figure 1B: c), but a large area of cancer nest was found in the model mice (Figure 1B: f). Although obvious cancer nests were also found in the LG group, the tumor area was much smaller than that in the model group (Figure 1B: i; Figure 1D), and only locally sporadic punctate carcinoma (Figure 1B: l) was found in the HG group. The canceration rate was 100% in the model group, 55.56% in the LG group, and 47.37% in the HG group, and no cancer was found in the control group (Figure 1C). RESULTS We detected the expression of Glutamic pyruvic transaminase (ALT) and Glutamic oxaloacetic transaminase (AST) in mouse serum. Compared with Control, ALT in Model was signiﬁcantly higher, P < 0.01, while serum ALT in LG and HG was signiﬁcantly lower than that in Model, P < 0.05 (Figure 1F). Western Blotting Analysis Hepatoma surface nodule data was performed with homogeneity test of variance, P = 0.001, P <0.05, the variance was not uniform, so after that, the Games-Howell test was used for multiple comparisons: the number of liver nodules in the model group was signiﬁcantly higher than that in the control group (Figure 1D). Compared with those in the model group, the nodules in the LG and HG groups were signiﬁcantly reduced, but the reduction was more signiﬁcant in the HG group than in the LG group (Figure 1D). Inhibitory Effect of GHJCD on DEN- Induced HCC in Mice After 8 weeks of treatment, no liver cancer nodules were observed in the liver of all mice (Figure 1A: a, d, g, j), and little sporadic punctate carcinoma was found in the model (Figure 1B: d). Immunohistochemical Assay Liver sections embedded in parafﬁn were dewaxed, subjected to antigen repair, incubated with 3% hydrogen peroxide for 10 min, blocked with 10% goat serum at 37°C for 10 min, incubated with CD31 antibody dilution with 1:500 at 37°C incubation for 2 h, and incubated with biotin-labeled secondary antibody at 37°C for 30 min. Next, the sections were incubated with horseradish enzyme-labeled streptavidin at 37°C for 30 min and mounted. The liver tissue sections were observed under a microscope with Ci-s positive image and text acquisition system (Nikon), analyzed by the ImageJ_v1.8.0 software (National Institutes of Health, Maryland). Determination of components in water extract of GHJCD by UPLC-Q/TOF MS. The chromatographic conditions were as follows: the chromatographic column was Waters ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 m m, Waters, Milford, MA, USA); protection column was BEH C18 Van Guard (2.1 × 50 mm, 1.7 m m, Waters, Milford, MA, USA); mobile phase: A solvent (methanol); B solvent (0.1% formic acid solution); ﬂow rate: 0.25 ml; elution procedure: 0–0.5 min, 5%–45% A;10–15 min, 45%–70% A; 15–19 min,70%–100% A;, 5% A; 0.5–10 min. 19–20 min, 100% A; 20–21 min, 100–5% A; 21–24 min, 5% A. May 2020 \| Volume 11 \| Article 809 Frontiers in Pharmacology \| www.frontiersin.org 3 GHJCD Inhibits Hepatocellular Carcinoma Cheng et al. of GHJCD, and the others from Panax ginseng C.A.Mey. (Araliaceae), Atractylodes Macrocephala and so on (Table 1). Statistical Analysis Differences between mean values of normally distributed data were evaluated by one-way analysis of variance (ANOVA) using the statistical package for the social sciences (SPSS) 18.0 software (SPSS Inc., Chicago, IL, USA). All the data were expressed as mean ± standard error of mean (SEM). Variance analysis is used to test the surface ratio data of liver cancer. First, we judge whether the variance is the same. Because P = 0.001, P < 0.05, the variance is not the same, so we use the Games-Howell test for multiple comparison: the cancerous area of the model was larger than that of the control, LG, and HG groups, with the HG group showing smaller cancerous area than the LG group (Figure 1E). Cytometric Bead Array y y A CBA Flex Set kit (BD Biosciences, San Jose, CA, United States) was used to assess interleukin-6 (IL-6), IL-10, interferon gamma (IFN-g), TNF-a, and C-C motif chemokine ligand 2 (CCL2) levels in cell culture supernatant, according to the manufacturer’s instructions. Data were analyzed using the CellQuest software (BD Biosciences) and BD Pharmingen (BD Biosciences). After 12 weeks of treatment, there were obvious liver cancer nodules on the liver surface of the model mice (Figure 1A: e), but none were found in the control, LG, and HG groups (Figure 1A: b, h, k). HE staining assay showed that typical cancer nests were found in the model group mice (Figure 1B: e), and only localized punctate carcinogenesis occurred in the LG group (Figure 1B: h), while no cancerous changes in the HG and the control (Figure 1B: b, k). Determination of GHJCD Water Extract by UPLC-Q/TOF MS The water extract of GHJCD was detected by UPLC-Q/TOF MS method, and the results showed that there were 60 components in the water extract, many of which came from Flower of Pueraria montana var. lobata (Willd.), the “monarch medicine” Compared with control group, AST in model was signiﬁcantly higher, with signiﬁcant difference, P < 0.05; May 2020 \| Volume 11 \| Article 809 Frontiers in Pharmacology \| www.frontiersin.org GHJCD Inhibits Hepatocellular Carcinoma Cheng et al. TABLE 1 \| Characteristics of some chemical components in GHJCD by UPLC-Q/TOF MS analysis. Determination of GHJCD Water Extract by UPLC-Q/TOF MS Name Formula Molecular weight RT (min) mzCloud BestMatch New hesperidin C28H34O15 610.1948 7.85 99.59 Puerarin C21H20O9 416.1146 6.82 99.98 Genistein glycoside C21H20O10 432.1094 8.82 99.96 Puerarin-4′-O-glucoside C27H30O14 578.168 5.68 99.97 Narirutin C27H32O14 580.1834 6.06 96.95 Puerarin celery glucoside C26H28O13 548.1576 7.48 99.89 3′-methoxydaidzein C16H12O5 284.0707 5.82 99.96 Daidzein C15H10O4 254.0603 6.48 99.89 Quercetin-3-o-a-l-rhamnoside C21H20O11 448.105 6.72 96.92 Isoliquiritigenin C15H12O4 256.0762 7.55 95.69 Isoﬂavone aglycone genistein C15H10O5 270.0551 9.39 97.28 16 oxo Alisol A C30H48O6 504.3492 12.21 97.87 3′-methoxysoybean glycoside C22H22O10 446.1246 5.29 98.79 Vanillic acid-b -D-glucopyranosylester C14H18O9 330.0975 2.52 94.56 Alpha cyperone C15H22O 218.1689 15.32 96.69 Anthocyanin of awn handle C16H12O4 268.0761 9.02 98.47 7-hydroxy-3,5,6,3′, 4′-pentamethoxyﬂavone C20H20O8 388.1192 5.55 97.76 Naringenin C15H12O5 272.0708 7.06 94.38 5 a, 8 a-cyclodioxo-(24R)-24- methylcholesterol-6,9 (11), 22-triene-3 b-ol C28H42O3 426.3171 14.21 97.57 Dihydroxylene lactone C15H22O2 234.1634 12.94 94.27 Alismatin B C15H26O5S 318.1505 16.87 98.17 Mycosterone B C28H44O6 476.3182 11.02 98.46 Alismatin A C15H26O4S 302.3582 13.94 95.73 24-acetyl Alisol E C32H52O6 532.3719 19.45 96.42 Alismatin C C15H24O4S 300.1399 15.33 97.24 Alisol C C30H46O5 486.3355 12.96 96.42 Alismatine C C15H24O2 236.1788 12.82 98.36 Alismatine A C15H26O3 254.1885 11.08 98.25 Alismatine E C15H26O3 254.189 10.94 97.13 Ginsenoside RF C42H72O14 800.4925 10.3 98.47 Ginsenoside Rd C48H82O18 946.5515 10.22 98.56 3,5,6,7,8,3′,4′ - heptanoxyﬂavone C22H24O9 432.1423 12.53 97.35 5,6,7,4 ‘- tetramethoxyﬂavone C19H18O6 342.1111 11.75 97.14 Kuzubutenolide A C23H24O10 460.138 8.38 95.47 Anthocyanin of Stipa spinosa C22H22O9 430.1266 7.57 97.52 7-hydroxy-3,5,6,8,3′,4′-hexamethoxyﬂavone C21H22O9 418.1268 7.09 98.65 Chickpea sprout A C16H12O5 284.0695 5.85 99.28 Daidzein-4′, 7-diglucoside C27H30O14 578.1639 5.71 98.69 Ginsenoside Rg3 C42H72O13 784.4983 12.79 94.51 Ginsenoside Rb2 C53H90O22 1078.5956 14.84 94.13 Ginsenoside Rb1 C54H92O23 1108.6042 14.65 94.29 Ginsenoside Ro C48H76O19 956.4999 14.26 96.31 Ginsenoside F1 C36H62O9 638.4389 12.9 95.25 3′-methoxypuerarin C22H22O10 446.1213 5.32 92.68 Mycosterone D C28H44O5 460.3142 19.44 94.18 Dibutyl phthalate C16H22O4 278.1523 16.75 96.35 Gingerol C17H26O4 294.184 11.79 98.36 Notoginsenoside R1 C47H80O18 932.5355 16.18 95.16 Poria cocos new acid DM C32H48O6 528.3453 14.02 96.48 Emodin C15H10O5 270.0536 6.87 89.39 Tangeritin C20H20O7 372.1214 13.28 98.97 Sodium citrated C21H22O8 402.132 12.16 88.56 Sinensetin C20H20O7 372.121 11.1 88.24 Hesperidin C28H34O15 610.1895 8.57 89.37 Hesperetin C16H14O6 302.0797 7.87 88.47 Double Atractylodes macrocephala lactone C30H38O4 462.276 15.26 87.45 3b-acetoxy atractylol C17H22O3 274.1556 9.31 88.38 Hexadecanoic acid C16H32O2 256.2407 14.46 87.36 Dehydrogladiolene C15H22 202.1728 10.1 86.48 Japanese ginseng terpenoid ketone C15H26O2 238.1922 3.93 87.34 TABLE 1 \| Characteristics of some chemical components in GHJCD by UPLC-Q/TOF MS analysis. Frontiers in Pharmacology \| www.frontiersin.org Immune Microenvironment of HCC Is Improved by GHJCD The ratio of CD4+T cell (Th), CD4+Tem, CD4+Tcm, Cytotoxic T cells (CD8+T cells), CD8+Tcm, CD8+Tcm, CD8+ Regulatory T cell (Tregs), TAMs, Myeloid-derived suppressor cells (MDSCs) in the liver was detected by ﬂow cytometry at the 23rd week of experiment. The expression of CD4+ decreased in the model; Low concentration of CHJCD increased the content of CD4Tem, but decreased CD4Tcm, and the expression of CD4Tcm in the model increased (Figures 2A–C). Determination of GHJCD Water Extract by UPLC-Q/TOF MS May 2020 \| Volume 11 \| Article 809 Frontiers in Pharmacology \| www.frontiersin.org 5 GHJCD Inhibits Hepatocellular Carcinoma Cheng et al. A B C D E F G FIGURE 1 \| Inhibitory effect of GHJCD on diethylnitrosamine-induced hepatocellular carcinoma in mice. (A) General appearance of the liver was changed dynamically at the 8th, 12th, and 23rd week of experiment. (B) Pathological changes in HE-stained liver sections were observed at the 8th, 12th, and 23rd week of treatment. Scale bar: 250µm. (C) Carcinogenic rate of liver cancer: the ratio of the number of mice with at least one liver cancer lesion to that of experimental mice. (D) Number of liver cancer nodules: number of liver cancer lesions in the liver. (E) Cancer area ratio: the percentage of liver cancer lesion area and liver tissue section area. (F) Serum ALT level in mice. (G) The content of serum AST in mice. p < 0.05, p < 0.01 vs. control; #p < 0.05, ##p < 0.01 vs. model. A B C D E D G F G G FIGURE 1 \| Inhibitory effect of GHJCD on diethylnitrosamine-induced hepatocellular carcinoma in mice. (A) General appearance of the liver was changed dynamically at the 8th, 12th, and 23rd week of experiment. (B) Pathological changes in HE-stained liver sections were observed at the 8th, 12th, and 23rd week of treatment. Scale bar: 250µm. (C) Carcinogenic rate of liver cancer: the ratio of the number of mice with at least one liver cancer lesion to that of experimental mice. (D) Number of liver cancer nodules: number of liver cancer lesions in the liver. (E) Cancer area ratio: the percentage of liver cancer lesion area and liver tissue section area. (F) Serum ALT level in mice. (G) The content of serum AST in mice. p < 0.05, p < 0.01 vs. control; #p < 0.05, ##p < 0.01 vs. model. compared with model, AST in LG was signiﬁcantly lower, with signiﬁcant difference, P < 0.05; compared with model, AST in HG was signiﬁcantly lower, with signiﬁcant difference, P < 0.01 (Figure 1G). Immunosuppression of liver cancer microenvironment was very obvious. CD8+Tregs, TAMs, MDSCs increased signiﬁcantly in the microenvironment of liver cancer, with obvious differences. However, the intervention of GHJCD led to the decrease of the expression of the above immunosuppressive factors, especially in the case of high concentration of GHJCD (Figures 3A–C). Inﬂammatory Factors and Chemokines in HCC Microenvironment Are Downregulated by GHJCD Compared with that in the control, IL-6 content was signiﬁcantly increased in the model, but signiﬁcantly decreased by GHJCD treatment in a dose-dependent manner (Figure 4A). IL-10 content also changed; it was signiﬁcantly higher in the model group than in the control group, but signiﬁcantly lower in the GHJCD-treated groups, especially in the HG group (Figure 4B). The TNF-a level in the LG and HG groups was signiﬁcantly higher than that in the control group (Figure 4C), but signiﬁcantly lower than that in the model (Figure 4C). The same trend was observed for CCL2 levels (Figure 4D). The expression of CD8+ increased in the model (Figure 2D). Treatment with different concentrations of GHJCD led to different physiological activities: low-concentration GHJCD increased the level of CD8+ Tem (Figure 2F), whereas high- concentration GHJCD improved the function of CD8+ Tcm (Figure 2E). May 2020 \| Volume 11 \| Article 809 Frontiers in Pharmacology \| www.frontiersin.org GHJCD Inhibits Hepatocellular Carcinoma Cheng et al. A B C D E F FIGURE 2 \| Expression of CD4+ and CD8+ in immune microenvironment of hepatocellular carcinoma at 23th weeks. (A) CD4+ level in the liver. (B) CD4+Tem content in the liver. (C) CD4+ Tcm expression in the liver. (D) CD8+ level in the liver. (E) CD8+ Tcm level in the liver. (F) CD8+ Tem level in the liver. The CD4 +, A B C D E F FIGURE 2 \| Expression of CD4+ and CD8+ in immune microenvironment of hepatocellular carcinoma at 23th weeks. (A) CD4+ level in the liver. (B) CD4+Tem content in the liver. (C) CD4+ Tcm expression in the liver. (D) CD8+ level in the liver. (E) CD8+ Tcm level in the liver. (F) CD8+ Tem level in the liver. The CD4 +, CD4+Tem, CD4+ Tcm, CD8+, CD8+ Tcm, CD8+ Tem data in the liver cancer microenvironment were analyzed by ANOVA. The homogeneity test was ﬁrst performed, P > 0.05, the variances were uniform, and the LSD (L) test was used for multiple comparisons afterward, P < 0.05, the variances were not uniform. p < 0.01 vs. control; #p < 0.05, ##p < 0.01 vs. model. Inﬂammatory Factors and Chemokines in HCC Microenvironment Are Downregulated by GHJCD B D E F RE 2 \| E i f CD4+ d CD8+ i i i i t f h t ll l i t 23th k (A) CD4+ l l i th li (B) CD4+T D FIGURE 2 \| Expression of CD4+ and CD8+ in immune microenvironment of hepatocellular carcinoma at 23th weeks. (A) CD4+ level in the liver. (B) CD4+Tem content in the liver. (C) CD4+ Tcm expression in the liver. (D) CD8+ level in the liver. (E) CD8+ Tcm level in the liver. (F) CD8+ Tem level in the liver. The CD4 +, CD4+Tem, CD4+ Tcm, CD8+, CD8+ Tcm, CD8+ Tem data in the liver cancer microenvironment were analyzed by ANOVA. The homogeneity test was ﬁrst performed, P > 0.05, the variances were uniform, and the LSD (L) test was used for multiple comparisons afterward, P < 0.05, the variances were not uniform. *p < 0.01 vs. control; #p < 0.05, ##p < 0.01 vs. model. Compared with the control group, the expression of VEGF protein in the model was signiﬁcantly increased (Figure 5C), but compared with the model, the LG and HG groups gradually decreased, and the HG was more obvious (Figure 5C). Angiogenic Ability of HCC in Its Microenvironment Is Inhibited by GHJCD The homogeneity test was ﬁrst performed, P > 0.05, the variances were uniform, and the LSD (L) test was used for multiple comparisons afterward, P < 0.05, the variances were not uniform.p < 0.05 vs. control; #p < 0.05, ##p < 0.01 vs. model. Data were analyzed using single- variance t-test. A B C FIGURE 3 \| The content of CD8 + Tregs, TAMs, and MDSCs in the immune microenvironment of hepatocellular carcinoma at 23th weeks. (A) CD8 + Tregs level in the liver. (B) TAMs content in the liver. (C) MDSCs expression in the liver. CD8 + Tregs, TAMs, and MDSCs data in the liver cancer microenvironment were analyzed by ANOVA. The homogeneity test was ﬁrst performed, P > 0.05, the variances were uniform, and the LSD (L) test was used for multiple comparisons afterward, P < 0.05, the variances were not uniform. p < 0.05, p < 0.01 vs. control; #p < 0.05, vs. model. B FIGURE 3 \| The content of CD8 + Tregs, TAMs, and MDSCs in the immune microenvironment of hepatocellular carcinoma at 23th weeks. (A) CD8 + Tregs level in the liver. (B) TAMs content in the liver. (C) MDSCs expression in the liver. CD8 + Tregs, TAMs, and MDSCs data in the liver cancer microenvironment were analyzed by ANOVA. The homogeneity test was ﬁrst performed, P > 0.05, the variances were uniform, and the LSD (L) test was used for multiple comparisons afterward, P < 0.05, the variances were not uniform. p < 0.05, *p < 0.01 vs. control; #p < 0.05, vs. model. A B C D FIGURE 4 \| Inﬂammatory factors and chemokines in the microenvironment of hepatocellular carcinoma are down-regulated by GHJCD. (A) IL-6 content in liver tissue. (B) IL-10 content in liver tissue. (C) Expression of TNF-a in the liver. (D) CCl-2 content in liver tissue. IL-6, IL-10, TNF-a, and CCl-2 data in the liver cancer microenvironment were analyzed by ANOVA. The homogeneity test was ﬁrst performed, P > 0.05, the variances were uniform, and the LSD (L) test was used for multiple comparisons afterward, P < 0.05, the variances were not uniform.p < 0.05 vs. control; #p < 0.05, ##p < 0.01 vs. model. Data were analyzed using single- variance t-test. A B C D FIGURE 4 \| Inﬂammatory factors and chemokines in the microenvironment of hepatocellular carcinoma are down-regulated by GHJCD. (A) IL-6 content in liver tissue. Angiogenic Ability of HCC in Its Microenvironment Is Inhibited by GHJCD The enhancement of angiogenesis in tumor microenvironment is also the characteristic of occurrence and metastasis of liver cancer. We used WB and immunoﬂuorescence techniques to detect the ability of angiogenesis in the microenvironment of HCC. We found that GHJCD signiﬁcantly inhibited the ability of angiogenesis in HCC. T h e e x p r e s s i o n o f C D 3 1 w a s d e t e c t e d b y immunohistochemistry at the 23rd week of the experiment. The expression of CD31 in the model was signiﬁcantly higher than that control (Figures 5A, B), LG was signiﬁcantly reduced than that model (Figure 5B), especially in HG (Figure 5B). NF-KB P65, WNT1, b-catenin, FRZ-7, APC, p-MAPK/MAPK, p-AKT/AKT, and p-SRC/SRC content in the liver was detected May 2020 \| Volume 11 \| Article 809 Frontiers in Pharmacology \| www.frontiersin.org 7 GHJCD Inhibits Hepatocellular Carcinoma Cheng et al. by western blotting. The protein levels of NF-kB, WNT1, b- catenin, p-MAPK/MAPK, p-AKT/AKT, and p-SRC/SRC in the liver of model mice were signiﬁcantly higher than those in the control mice (P < 0.05, Figures 6A–H). With increasing drug concentration, NF-kB p65, WNT1, b-catenin, p-MAPK/ MAPK, p-AKT/AKT, and p-SRC/SRC levels decreased in the liver of the LG and HG groups compared with those of the model group, but there was a signiﬁcant difference in the HG A B C FIGURE 3 \| The content of CD8 + Tregs, TAMs, and MDSCs in the immune microenvironment of hepatocellular carcinoma at 23th weeks. (A) CD8 + Tregs level in the liver. (B) TAMs content in the liver. (C) MDSCs expression in the liver. CD8 + Tregs, TAMs, and MDSCs data in the liver cancer microenvironment were analyzed by ANOVA. The homogeneity test was ﬁrst performed, P > 0.05, the variances were uniform, and the LSD (L) test was used for multiple comparisons afterward, P < 0.05, the variances were not uniform. p < 0.05, p < 0.01 vs. control; #p < 0.05, vs. model. A B C D FIGURE 4 \| Inﬂammatory factors and chemokines in the microenvironment of hepatocellular carcinoma are down-regulated by GHJCD. (A) IL-6 content in liver tissue. (B) IL-10 content in liver tissue. (C) Expression of TNF-a in the liver. (D) CCl-2 content in liver tissue. IL-6, IL-10, TNF-a, and CCl-2 data in the liver cancer microenvironment were analyzed by ANOVA. Angiogenic Ability of HCC in Its Microenvironment Is Inhibited by GHJCD (B) IL-10 content in liver tissue. (C) Expression of TNF-a in the liver. (D) CCl-2 content in liver tissue. IL-6, IL-10, TNF-a, and CCl-2 data in the liver cancer microenvironment were analyzed by ANOVA. The homogeneity test was ﬁrst performed, P > 0.05, the variances were uniform, and the LSD (L) test was used for multiple comparisons afterward, P < 0.05, the variances were not uniform.p < 0.05 vs. control; #p < 0.05, ##p < 0.01 vs. model. Data were analyzed using single- variance t-test. concentration, NF-kB p65, WNT1, b-catenin, p-MAPK/ MAPK, p-AKT/AKT, and p-SRC/SRC levels decreased in the liver of the LG and HG groups compared with those of the model group, but there was a signiﬁcant difference in the HG by western blotting. The protein levels of NF-kB, WNT1, b- catenin, p-MAPK/MAPK, p-AKT/AKT, and p-SRC/SRC in the liver of model mice were signiﬁcantly higher than those in the control mice (P < 0.05, Figures 6A–H). With increasing drug concentration, NF-kB p65, WNT1, b-catenin, p-MAPK/ MAPK, p-AKT/AKT, and p-SRC/SRC levels decreased in the liver of the LG and HG groups compared with those of the model group, but there was a signiﬁcant difference in the HG May 2020 \| Volume 11 \| Article 809 Frontiers in Pharmacology \| www.frontiersin.org 8 GHJCD Inhibits Hepatocellular Carcinoma Cheng et al. A B C FIGURE 5 \| Angiogenic ability of hepatocellular carcinoma in microenvironment is inhibited by GHJCD. (A) Representative image of CD31 immunohistochemical staining in liver tissue. Top scale bar: 200 µm; bottom scale bar: 100 µm. (B) Area of CD31-positive distribution in liver tissue (µm2). (C) Expression of VEGF in liver tissue. CD31, VEGF data in the liver cancer microenvironment were analyzed by ANOVA. The homogeneity test was ﬁrst performed, P > 0.05, the variances were uniform, and the LSD (L) test was used for multiple comparisons afterward, P < 0.05, the variances were not uniform.p < 0.05, p < 0.01 vs. control; #p < 0.05 vs. model. FIGURE 5 \| Angiogenic ability of hepatocellular carcinoma in microenvironment is inhibited by GHJCD. (A) Representative image of CD31 immunohistochemical staining in liver tissue. Top scale bar: 200 µm; bottom scale bar: 100 µm. (B) Area of CD31-positive distribution in liver tissue (µm2). (C) Expression of VEGF in liver tissue. CD31, VEGF data in the liver cancer microenvironment were analyzed by ANOVA. Angiogenic Ability of HCC in Its Microenvironment Is Inhibited by GHJCD The homogeneity test was ﬁrst performed, P > 0.05, the variances were uniform, and the LSD (L) test was used for multiple comparisons afterward, P < 0.05, the variances were not uniform.p < 0.05, *p < 0.01 vs. control; #p < 0.05 vs. model. A B C D E F G H FIGURE 6 \| Expression of NF-kB p65, WNT1, b-catenin, Frz-7, APC, p-mapk/mapk, p-Akt/Akt, and p-src/src in the liver of mice with DEN-induced liver cancer. (A) Expression of NF-kB p65 in liver tissue. (B) Expression of WNT1 in liver tissue. (C) Expression of b-catenin in liver tissue. (D) Expression of Frz-7 in liver tissue. (E) Expression of APC in liver tissue. (F) Expression of p-MAPK/MAPK in liver tissue. (G) Expression of P-AKt/AKt in liver tissue.(H) Expression of p-src/src in liver tissue. NF-kB p65, WNT1, b-catenin, Frz-7, APC, p-mapk/mapk, p-Akt/Akt and p-src/src data in the liver cancer microenvironment were analyzed by ANOVA. The homogeneity test was ﬁrst performed, P > 0.05, the variances were uniform, and the LSD (L) test was used for multiple comparisons afterward, P < 0.05, the variances were not uniform. p < 0.05, *p < 0.01 vs. control; #p < 0.05 vs. model. Data were analyzed using single-variance t-test. A B C D G H H G H FIGURE 6 \| Expression of NF-kB p65, WNT1, b-catenin, Frz-7, APC, p-mapk/mapk, p-Akt/Akt, and p-src/src in the liver of mice with DEN-induced liver cancer. (A) Expression of NF-kB p65 in liver tissue. (B) Expression of WNT1 in liver tissue. (C) Expression of b-catenin in liver tissue. (D) Expression of Frz-7 in liver tissue. (E) Expression of APC in liver tissue. (F) Expression of p-MAPK/MAPK in liver tissue. (G) Expression of P-AKt/AKt in liver tissue.(H) Expression of p-src/src in liver tissue. NF-kB p65, WNT1, b-catenin, Frz-7, APC, p-mapk/mapk, p-Akt/Akt and p-src/src data in the liver cancer microenvironment were analyzed by ANOVA. The homogeneity test was ﬁrst performed, P > 0.05, the variances were uniform, and the LSD (L) test was used for multiple comparisons afterward, P < 0.05, the variances were not uniform. p < 0.05, **p < 0.01 vs. control; #p < 0.05 vs. model. Data were analyzed using single-variance t-test. DISCUSSION Now, new ﬁndings suggest that chronic liver inﬂammation also promotes cancer by inhibiting immunity and increasing blood vessel formation (Ray, 2017). IL-10 is a pleiotropic cytokine, that appears to have contradictory roles (Mannino et al., 2015). For example, IL-10 not only down-regulates HLA-I but also up-regulates HLA-G, and causes immune escape in cancer cells (Urosevic and Dummer, 2003). IL-10 participates in the inﬂammatory response by inhibiting the bactericidal ability of polymorphonuclear leukocytes (PMN). If IL-10 is blocked, the ability to kill pathogenic microorganisms is enhanced (Siwapornchai et al., 2020). Furthermore, IL-10 is considered an immunosuppressive cytokine, which promotes tumor immune escape by reducing the anti-tumor immune responses in the tumor microenvironment. Additionally, the expression level of IL-10 is positively correlated with cancer recurrence (Li et al., 2019). In contrast, the accumulation of Tregs in cancer tissues requires high IL-10 expression (Kindlund et al., 2017). To understand the mechanisms by which IL-10 works, we propose to study drug-induced secretion of IL-10 in different types of cells in the future. Many inﬂammatory factors regulate immunosuppressive cells and affect the formation of blood vessels. Interleukin-6 (IL-6), IL-10 (Heim et al., 2015), and TNF (Xue et al., 2012) are important triggers of myeloid origin inhibiting the proliferation and recruitment of MDSCs and are considered to be the main coordinator of immunosuppressive tumor microenvironment (Zhang et al., 2018). Myeloid inhibitory cells promote tumor angiogenesis by increasing the expression of VEGF (Lu et al., 2019). In turn, VEGF can also promote the activity of MDSCs in HCC (Xu et al., 2016). So these evidences indicate that inﬂammation, immunosuppression, and angiogenesis affect and promote each other. Our results showed GHJCD not only inhibited the release of inﬂammatory cytokines, such as TNF-a, IL-10, and IL-6, but also inhibits the formation of blood vessels, the CD31 and VEGF protein expression. CD31 is also known as the platelet endothelial cell adhesion molecule. In immunohistochemistry, CD31 is mainly used to identify endothelial cells and evaluate tumor angiogenesis by correlating its expression levels to the extent of tumor growth (Sathornsumetee et al., 2008; Harmsen et al., 2019; Wang et al., 2019). p The HCC model induced by DEN is a mature and widely used method to establish liver cancer in mice, and it is also a repeatable model of chronic liver injury, which is similar to that of human liver injury (Shou et al., 2015). DISCUSSION group (Figures 6A–H). On the contrary, western blotting analysis showed that the level of APC protein in the model group was signiﬁcantly lower than that in the control group (Figure 6E). GHJCD treatment, especially at a high concentration, signiﬁcantly increased the expression of APC protein in the liver cancer microenvironment (Figure 6E). In this study, we found that GHJCD could signiﬁcantly improve the pathological features of liver cancer. The incidence of HCC in the model group was 100%, and the area ratio of HCC was signiﬁcantly increased. In LG and HG groups treated with In this study, we found that GHJCD could signiﬁcantly improve the pathological features of liver cancer. The incidence of HCC in the model group was 100%, and the area ratio of HCC was signiﬁcantly increased. In LG and HG groups treated with May 2020 \| Volume 11 \| Article 809 Frontiers in Pharmacology \| www.frontiersin.org 9 GHJCD Inhibits Hepatocellular Carcinoma Cheng et al. microenvironment of liver cancer was improved by GHJCD, the ratio of CD8 T cells in liver was up-regulated by GHJCD, while the ratio of TAMs and MDSCs, Tregs were down-regulated. GHJCD, the incidence of cancer and nodular number of HCC were decreased dose-dependently, as well as the area ratio of HCC were reduced. GHJCD not only improved the microenvironment of HCC, including up-regulated the ratio of CD8+ T cells, down-regulated the ratio of Tregs, TAMs, and MDSCs, but also decreased the levels of inﬂammatory factors IL- 6, IL-10, TNF-a, and CCl-2. In addition, GHJCD inhibited the angiogenic ability of liver cancer. These were also accompanied by down-regulation of single protein molecules in the inﬂammatory, such as NF-kB p65, and angiogenic signaling pathways, such as WNT1/b-catenin, Frz-7, APC, p-MAPK, p- AKT, and p-SRC. Inﬂammation is another important core factor in tumor microenvironment besides immunity. HCC is associated with chronic inﬂammation and ﬁbrosis caused by different causes. The NF-kB pathway has an active role during inﬂammation (Wu et al., 2020) though factors such as p65, whose levels can reﬂect the extent of inﬂammation (Wang et al., 2020). P65 can aggravate inﬂammation by interacting with other signaling pathways such as the NF-kB/MAPK signaling pathway (Dong et al., 2020). The activation of NF-k B may be a key step in the inﬂammatory cascade, which can induce the expression of IL-6 (Quay et al., 1998) TNF-a (Lee et al., 2016). DISCUSSION For example, reactive oxygen species (ROS) production, compensatory proliferation, inﬂammation, and ﬁbrosis (Guo et al., 2015). In our study, we found that liver cancer was successfully induced by DEN. In the model, there were no cancer nodules on the surface of the liver at the 8th week, but HE showed scattered canceration. Cancer nodules appeared on the surface of the liver at the 12th week of the experiment. A large number of cancer nodules were found on the surface of the liver at the end of the 23rd week of the experiment, combined with the cancer nest, and the canceration area was further expanded. p Immunosuppression is closely related to the occurrence and development of liver cancer. However, the liver’s unique immune tolerance microenvironment, liver cancer progression may have uncertain results. Persistent accumulation of cytotoxic T cells in liver cancer microenvironment and strong inhibition of liver tumors, and confers survival advantage to mice (Wen et al., 2019). By reshaping the tumor immune microenvironment, it can improve the anti-tumor immune response and inhibit the occurrence and development of liver cancer (Lei et al., 2018). It is well known that TAMS and MDSCs play a key role in the occurrence, deterioration, and metastasis of tumor in tumor microenvironment. TAMs promote the progress and metastasis of tumor cells by releasing a variety of cytokines, including chemokines, inﬂammatory factors, and growth factors (Jamieson et al., 2012). MDSC is a group of heterogeneous cells derived from bone marrow. It can signiﬁcantly inhibit immune response and promote the formation of blood vessels such as (Lu et al., 2019). Treg cells suppress immune function in the immune system. Although the role of CD4+ CD25 +regulatory Tregs in maintaining immune homeostasis has studied been extensively, recent ﬁndings indicate that CD8 + Tregs potentially play an immunomodulatory role in cancer (Dinesh et al., 2010). CD122+CD8+ Tregs are also an important regulatory T cell type and produce an anti-tumor immune response (Liu et al., 2006). T cells attack an infection in the body, and when the threat is over, the Treg cells signal the T cells to stop attacking. Cancer immunotherapy works by “overloading” the immune system to ﬁght tumors. So when Tregs initiate signals that suppress the immune response, it inevitably blocks the effectiveness of immunotherapy. Frontiers in Pharmacology \| www.frontiersin.org DISCUSSION Akt is also associated with tumor increment and angiogenesis. Overexpression of VEGF mediated by Akt signal transduction can stimulate angiogenesis, which can be reduced by the use of inhibitor (Wang et al., 2019). SRC protein is also closely related to tumor angiogenesis. The transformation of SRC from inactivity to active conformation triggers the secretion of VEG through SRC/VEGF signal transduction pathway, which leads to the increase of tumor angiogenesis (Sun et al., 2018). In our study, GHJCD can signiﬁcantly improve these protein molecules expressions, which suggests that GHJCD may inhibit angiogenesis and inﬂammation by these signaling pathways. overactivated in a variety of cancers. VEGF is one of the downstream of WNT/b-catenin pathway (Han et al., 2016). Moreover, the b-catenin/VEGF axis can promote tumor angiogenesis (Tang et al., 2019). APC protein is a negative regulator of WNT signaling pathway. It is encoded by tumor suppressor gene APC and binds to b-catenin, which prevents b- catenin from accumulating in cells and entering the nucleus, and promotes the degradation of b-catenin. The activation of WNT signal also can enhance the activity of antigen presenting cells and provide stimulation for Tregs (Mitkin et al., 2018). WNT/b- catenin pathway can regulate the function and differentiation of CD4, CD8 T cells, while blocking WNT/b-catenin pathway can inhibit Tregs (Dai et al., 2016). MAPK transmits signals from the cell surface to the nucleus and regulates a variety of physiological processes such as cell growth, differentiation, apoptosis and death (Yin et al., 2018). By regulating MAPK signaling pathway, TAMs can change from immunosuppressive M2-like phenotype to anti-tumor M1-like phenotype, increase the proportion of M1 macrophages, promote tumor recruitment of cytotoxic T lymphocytes (CTL) and inhibit angiogenesis (Deng et al., 2019). Akt is also associated with tumor increment and angiogenesis. Overexpression of VEGF mediated by Akt signal transduction can stimulate angiogenesis, which can be reduced by the use of inhibitor (Wang et al., 2019). SRC protein is also closely related to tumor angiogenesis. The transformation of SRC from inactivity to active conformation triggers the secretion of VEG through SRC/VEGF signal transduction pathway, which leads to the increase of tumor angiogenesis (Sun et al., 2018). In our study, GHJCD can signiﬁcantly improve these protein molecules expressions, which suggests that GHJCD may inhibit angiogenesis and inﬂammation by these signaling pathways. In conclusion, GHJCD has a signiﬁcant inhibitory effect on DEN-induced liver cancer, and its anti-HCC pathway is extensive and multi-target. DISCUSSION GHJCD not only has a strong anti- inﬂammatory effect, but also can inhibit the formation of blood vessels in liver cancer, resist the immunosuppressive effect in tumor microenvironment, and play the role of anti-liver cancer (Figure 7). DISCUSSION In our research, we found that the The WNT/b-catenin pathway is highly conserved and plays an important role in tumorigenesis, and it has been found to be May 2020 \| Volume 11 \| Article 809 Frontiers in Pharmacology \| www.frontiersin.org 10 GHJCD Inhibits Hepatocellular Carcinoma Cheng et al. FIGURE 7 \| GHJCD inhibits HCC pathway by improving tumor microenvironment. FIGURE 7 \| GHJCD inhibits HCC pathway by improving tumor microenvironment. Exposure to high-risk factors such as DEN, which induces like precancerous lesions, is more likely to cause malignant tumors. In this experiment, GHJCD was administered to mice in the treatment group before the development of malignant tumors. The effective results conﬁrmed that GHJCD can reduce the inevitable occurrence of malignant tumors, even after the occurrence of liver cancer, it can also effectively inhibit the development of tumor. In this study, GHJCD was found to signiﬁcantly improve tumor microenvironment, including inhibiting inﬂammation and angiogenesis, and improving the inﬁltration of immunosuppressive cells. We know that these three factors themselves affect each other. Therefore, it is not clear whether GHJC has a direct effect on the three aspects at the same time, or whether it inﬂuences the other two factors through one factor. Next, we will explore the mechanism of inﬂammation, angiogenesis and immunosuppressive cells one by one. overactivated in a variety of cancers. VEGF is one of the downstream of WNT/b-catenin pathway (Han et al., 2016). Moreover, the b-catenin/VEGF axis can promote tumor angiogenesis (Tang et al., 2019). APC protein is a negative regulator of WNT signaling pathway. It is encoded by tumor suppressor gene APC and binds to b-catenin, which prevents b- catenin from accumulating in cells and entering the nucleus, and promotes the degradation of b-catenin. The activation of WNT signal also can enhance the activity of antigen presenting cells and provide stimulation for Tregs (Mitkin et al., 2018). WNT/b- catenin pathway can regulate the function and differentiation of CD4, CD8 T cells, while blocking WNT/b-catenin pathway can inhibit Tregs (Dai et al., 2016). MAPK transmits signals from the cell surface to the nucleus and regulates a variety of physiological processes such as cell growth, differentiation, apoptosis and death (Yin et al., 2018). By regulating MAPK signaling pathway, TAMs can change from immunosuppressive M2-like phenotype to anti-tumor M1-like phenotype, increase the proportion of M1 macrophages, promote tumor recruitment of cytotoxic T lymphocytes (CTL) and inhibit angiogenesis (Deng et al., 2019). Frontiers in Pharmacology \| www.frontiersin.org REFERENCES cells in mice. Int. J. Biol. Macromol. 137, 604–611. doi: 10.1016/ j.ijbiomac.2019.06.059 cells in mice. Int. J. Biol. Macromol. 137, 604–611. doi: 10.1016/ j.ijbiomac.2019.06.059 Ahmad, B., Khan, S., Liu, Y., Xue, M., Nabi, G., Kumar, S., et al. (2020). Molecular Mechanisms of Anticancer Activities of Puerarin. Cancer Manage. Res. 12, 79– 90. doi: 10.2147/cmar.s233567 Guo, B., Tang, D., Long, F., Luo, L., Huang, H., Wang, D., et al. (2015). Effects of Gehua Jiecheng decoction on regulation factors of cell cycle in ethanol-type HBV transgenic mice with hepatocellular carcinoma precancerous lesions. 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Pharmacol. 9, 639. doi: 10.3389/fphar.2018.00639 Dai, W., Liu, F., Li, C., Lu, Y., Lu, X., Du, S., et al. (2016). Blockade of WNT/b- Catenin Pathway Aggravated Silica-Induced Lung Inﬂammation through Tregs Regulation on Th Immune Responses. DATA AVAILABILITY STATEMENT All datasets generated for this study are included in the article/ Supplementary Material. May 2020 \| Volume 11 \| Article 809 Frontiers in Pharmacology \| www.frontiersin.org 11 GHJCD Inhibits Hepatocellular Carcinoma Cheng et al. ETHICS STATEMENT of the Department of Education of Guizhou Province [Project Nos. JYSZ Word (2014) 018], Guizhou Provincial Department of Science and Technology High-level innovative Talent training Program Fund [100 levels, Project Nos. Qian Kehe Talents (2016) No.4032], Guizhou Provincial Organization Department of traditional Chinese Medicine tumor inheritance and Scientiﬁc and technological Innovation Talent Base Fund [Project Nos. Qian People Leading send (2018) 3], and Natural Science Foundation of Zhejiang Province (grant no. LQ17H030006). The study wascarriedoutinaccordancewiththe recommendations of “Protection and use of Experimental Animals in the Animal Experimental Center of Zhejiang University of Traditional Chinese Medicine”. The program has been approved by the Animal Committee of the Animal Experimental Center of Zhejiang University of Traditional Chinese Medicine. ACKNOWLEDGMENTS HF, QS, and ZY conceived and designed the study. QS, CC, LJ, and JL performed the experiments. HF and CC analyzed the data. HF, QS, and DT contributed reagents and materials. HF, XL and CC wrote the manuscript. We thank F.M. Chen and his colleagues (Laboratory Animal Center of Zhejiang Chinese Medical University) for preparing and staining the pathological animal tissue samples. 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https://openalex.org/W2993695927	https://europepmc.org/articles/pmc6949955?pdf=render	English	null	Implementation of Health Promotion Competencies in Ireland and Italy—A Case Study	International journal of environmental research and public health/International journal of environmental research and public health	2,019	cc-by	12,874	Received: 6 November 2019; Accepted: 6 December 2019; Published: 8 December 2019 Abstract: This paper reports on a case study that explored the broader contextual factors inﬂuencing the implementation of the CompHP Core Competencies at a country level in Ireland and Italy between 2011 and 2018. The sample comprised key informants who were Health Promotion experts and were knowledgeable about how the competencies had been used in their country. These experts formed National Reference Groups that guided the research process in each country and helped identify additional key informants. Qualitative methods were utilized consisting of a desk review and semi-structured interviews. The data from each country were analyzed separately using a thematic analysis approach, with the ﬁndings then compared and reviewed by the National Reference Groups. A total of 26 interviews were completed (13 in each country). The ﬁndings show that both the focus and rate of progress of implementing the competencies diﬀered across the two countries and that this reﬂected their levels of Health Promotion infrastructure and capacity development. A lack of awareness of the competencies was identiﬁed as a major limiting factor in implementation in both countries, of particular concern in relation to employers and decision-makers. While the case study focused on implementing the competencies in two European countries, there are insights from their experience that can inform implementation in other countries. The study also begins to address the gap in empirical evidence on the use and impact of Health Promotion competencies and the factors that inﬂuence their implementation. Keywords: Health Promotion competencies; evaluation; Health Promotion capacity Received: 6 November 2019; Accepted: 6 December 2019; Published: 8 December 2019 International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health Int. J. Environ. Res. Public Health 2019, 16, 4992; doi:10.3390/ijerph16244992 Keywords: Health Promotion competencies; evaluation; Health Promotion capacity Article Implementation of Health Promotion Competencies in Ireland and Italy—A Case Study Barbara Battel-Kirk 1,* and Margaret M. Barry 2 1 BBK Consultancy, Omeath, A91RV10 Co Louth, Ireland 2 World Health Organization Collaborating Centre for Health Promotion, National University of Ireland Galway, H91 TK331 Galway, Ireland; margaret.barry@nuigalway.ie * Correspondence: bbkconsultancy@eircom.net Background While there are references in the literature to the development of Health Promotion competencies since the mid-1980s only two examples of their empirical evaluation were identiﬁed in a recent scoping review [7], both of which were undertaken in New Zealand [13,14]. The ﬁndings from these small studies [13,14] indicated that the competencies had been used in diﬀerent contexts and settings and that reactions to their implementation were positive. A number of factors that are considered likely to inﬂuence the implementation of the CompHP Competencies have been identiﬁed [7,8,15], including levels of Health Promotion infrastructure and capacity, attitudes to their usefulness and relevance, recognition of Health Promotion as an area of professional competence, availability of support and resources and political, organizational, professional and educational contexts. In an online survey of 81 Health Promotion specialist from 25 European countries, Battel-Kirk and Barry [12] found that respondents’ attitudes to the competencies were mainly positive. However, only 53% of respondents reported that the competencies were actually used in their country and 54% indicated that they used them in their practice. The survey ﬁndings provided some insight into the factors that inﬂuenced individual decision-making in using the competencies but also identiﬁed a lack of recognition and support by key organizations and stakeholders at a country level as potentially critical factors in their implementation. It was in this context that the third stage of the evaluation was initiated comprising a case study of factors inﬂuencing country level implementation. The overall aim of the study was to explore and compare the broader contextual factors inﬂuencing the implementation of the CompHP Competencies at country level in Ireland and Italy between 2011 and 2018. The case study had the following speciﬁc objectives: (i) to describe the contexts within which Health Promotion is implemented in each country; (ii) to explore perceptions of readiness to implement the competencies at country level; (iii) to review the implementation and impact of the competencies; (iv) to investigate the factors that inﬂuence the implementation of the competencies in both countries; (v) to compare the ﬁndings from both countries in the context of future implementation of the competencies at national and international level. 1. Introduction Identifying and agreeing core competencies has been acknowledged as an essential component of developing and strengthening Health Promotion workforce capacity to improve health and wellbeing [1–3]. It was in this context that the CompHP Core Competencies Framework for Health Promotion [4,5] was developed in 2012 as the core element of a European Union funded project that developed competency-based quality assurance for Health Promotion practice, education and training in Europe [6]. As a rationale for developing competencies, reference is made in the international literature to the many positive beneﬁts that can accrue from their implementation. These include enhancing Health Promotion workforce capacity, quality assurance of practice, education and training, and as a basis for developing a shared vision of what constitutes the speciﬁc knowledge and skills required for eﬀective and ethical Health Promotion practice [1–8]. However, potentially negative consequences of competencies have also been identiﬁed, including the fact that they may undervalue professional judgement and restrict the dynamic nature of Health Promotion practice [9–11]. Int. J. Environ. Res. Public Health 2019, 16, 4992; doi:10.3390/ijerph16244992 www.mdpi.com/journal/ijerph 2 of 21 Int. J. Environ. Res. Public Health 2019, 16, 4992 Despite these divergent opinions on Health Promotion competencies, there is a paucity of empirical evidence available on the actual use of competencies or their impact and whether the beneﬁcial or negative outcomes attributed to them can be validated [7]. It is in this context that an evaluation of the use and impact of the CompHP Core Competencies on Health Promotion practice, education and training in Europe was initiated in 2016. Stage one of the evaluation comprised a scoping review of the literature and research on Health Promotion competencies [7], followed by an online survey that explored attitudes towards, and the use and impact of, the CompHP Competencies across the European Health Promotion community [12]. This paper reports on the ﬁnal stage of the evaluation process comprising a case study focusing on the implementation of the CompHP Competencies at a country level in two European countries, namely Ireland and Italy. 2. Materials and Methods • the ‘inner setting’ construct applied at country level with emphasis on the subconstructs of readiness to implement resources and support. • the implementation process construct, including the subconstructs of planning, use, champions, leadership, reﬂection and evaluation. Based on Yin [18], all aspects of the study were triangulated by • accessing evidence from diﬀerent sources (i.e., documents, reports, informants, national e • accessing evidence from diﬀerent sources (i.e., documents, reports, informants, national experts). • using diﬀerent methods (i.e., documentary analysis, interviews and thematic analysis). • accessing evidence from diﬀerent sources (i.e., documents, reports, informants, national experts). • using diﬀerent methods (i.e., documentary analysis, interviews and thematic analysis). • using diﬀerent methods (i.e., documentary analysis, interviews and thematic analysis) • including national experts’ comments in the ﬁnal analysis of ﬁndings. including national experts’ comments in the ﬁnal analysis of ﬁndings. Collection and analysis of the data from each country were conducted separately, and the ﬁndings were then compared. 2.1. Sample The sample for the case study initially comprised ﬁve known experts in each country who were knowledgeable about the contexts within which Health Promotion is practiced and how the CompHP Competencies had been used in their country. These experts were invited to act as key informants and served as national reference points for the study. The experts included leading members of national Health Promotion professional and academic bodies and others in Health Promotion/Public Health departments and statutory/non-governmental organizations with a remit for Health Promotion at country level (and at regional level in Italy). The experts assisted in piloting interviews, participated in interviews, commented on ﬁndings and assisted in identifying additional key informants to be interviewed in their country, with the aim of focusing on those most likely to share relevant information rather than identifying a speciﬁc number of informants [18]. 2. Materials and Methods A single case study was employed with two embedded units of analysis, namely the countries of Ireland and Italy. A case study method was chosen as it is an approach that is ideal for investigations that aim to move beyond narrow deﬁnitions of a research topic, address the context rather than isolated variables, and incorporate multiple sources of evidence [16–18]—all of which apply to this study. The study encompassed both ‘theory-testing’ and ‘theory-building’ aspects [17] as it explored theories surrounding the implementation of the competencies identiﬁed in the literature while being open to the emergence of alternate theories. 3 of 21 Int. J. Environ. Res. Public Health 2019, 16, 4992 The study was bounded by geography and time, (i.e., the implementation of the CompHP Competencies in Ireland and Italy between 2011 and 2018), with the rationale for concentrating on these countries being that there was evidence of implementation of the competencies in both countries [15,19]. • there was evidence of implementation of the competencies in both countries [15,19]. • the highest number of responses to the online survey were received from respondents in these countries [12] • there were known diﬀerences between the countries in terms of Health Promotion infrastructure and capacity [7,15], allowing for a useful comparison. • there were known diﬀerences between the countries in terms of Health Promotion infrastructure and capacity [7,15], allowing for a useful comparison. The design of the case study was informed by consensus standards for the reporting of case studies that aim to improve the consistency, rigor and reporting of such research [20]. The case study also drew on the Consolidated Framework for Implementation Research (CFIR) [21], as a conceptual framework to guide the assessment of the multilevel implementation of the CompHP Competencies in both countries and to identify the main factors that inﬂuenced the level of implementation achieved [22–25]. The CFIR is intended to be tailored to the speciﬁc context being studied, with Alexis Kirk et al. [25] recommending that the constructs selected and the rationale for their selection be clearly stated. The CFIR constructs selected for this study were those considered to ‘best match’ its aim of exploring implementation of the CompHP Competencies at country level, i.e., • the ‘outer setting’ construct with particular reference to Health Promotion infrastructure and capacity at country level. 2.2. Methods The case study employed qualitative methods comprising a desk review and semi-structured interviews with key informants. 4 of 21 Int. J. Environ. Res. Public Health 2019, 16, 4992 2.4. Semi-Structured Interviews Semi-structured interviews were the main data collection method employed. The interview protocol was developed using selected constructs from the CFIR Interview Guide Tool [26] together with key issues from the from the existing literature [7,15], the online survey [12] and the desk review. Those agreeing to be interviewed were assured of conﬁdentiality and anonymity and were asked to sign a form indicating that they consented to their interview being recorded. The interview questions and process were piloted with two informants from each of the two National Reference Groups in January 2019, resulting in some minor revisions. The remaining interviews were conducted between January and March 2019 using online meeting tools in most instances. Interviews were recorded with the informants’ permission and conducted in English. An outline of the interview questions was emailed to informants 10 working days before their interview. 2.3. Desk Review A desk review was undertaken to collate data on the Health Promotion policy and practice contexts within which the CompHP Competencies were implemented in both countries. Sources included key policy documents, reports and articles published since 2005 that were available in full, or in summary, in English. Sources were identiﬁed through a rapid review of the literature, internet searches and recommendations from the National Reference Groups. 2.5. Collation and Analysis Interviews were transcribed by the main researcher and following the thematic analysis approach described by Braun and Clarke [27], the data from each country were read through a number of times to allow for familiarization. Initial codes were then generated and emerging themes identiﬁed, reﬁned and named in consultation with the second researcher. Analysis focused on identifying themes emerging from the data from each country separately, with the emergent themes then collated and interpreted with reference to the selected CFIR constructs, factors identiﬁed in the literature and ﬁndings from the desk review. The weighting attributed to the themes was informed by how frequently that informants referred to each theme and the emphasis they placed upon speciﬁc issues in the interviews. The initial ﬁndings from each country were then compared and reviewed by the National Reference Groups, with their expert feedback informing the results presented below. 3. Results 3.1. Desk Review 3.1. Desk Review A summary overview of the ﬁndings from the desk review concerning the Health Promotion context in each country is ﬁrst provided. Ireland and Italy are both parliamentary democracies and members of the European Union. The countries vary greatly in size and population, i.e., • Land area—Ireland 69,800 km2; Italy 302,100 km2 • Population—Ireland 4.63 million; Italy 60.8 million [28] Ireland has a centralized health system commonly referred to as ‘two-tiered’ as many individuals buy private insurance to bypass long waiting lists in the public system and gain faster access to diagnostics and hospital treatments. Ireland remains the only western European country without universal coverage for primary care [29]. In 2019 approximately 46% of the population had some form of private health insurance plan [30]. Health policy is determined by the Department of Health [31] and health service delivery is managed by the Health Service Executive (HSE) [32]. 5 of 21 Int. J. Environ. Res. Public Health 2019, 16, 4992 Italy’s health care system operates both at national and regional level, as since the early 1990s, regional governments have devolved autonomy in planning and organizing healthcare in their territory, with the level of autonomy described as very high [33]. Universal coverage is largely free of charge at the point of delivery but approximately 30% of the population has additional private health insurance [34]. Findings on Health Promotion infrastructure and capacity in Ireland and Italy are presented in Table 1. Table 1. Health Promotion infrastructure and capacity in Ireland and Italy. Ireland Italy Policy context • Strategic framework: Healthy Ireland [35] Vision: A Healthy Ireland, where everyone can enjoy physical and mental health and wellbeing to their full potential. • Minister for Health Promotion and the National Drugs Strategy • Strategic framework: Guadagnare Salute—Rendere facili le scelte salutari [36] integrated into National and Regional Prevention Plans. 3.1. Desk Review Primary objective: to prevent and change unhealthy conducts that are the main risk factors for major non-communicable diseases Implementation of Health Promotion • Health and Wellbeing Division (HSE) [37] • NGOs with remit for Health Promotion (e.g., [38–40]) the private sector • Some evidence of implementation at national/regional level Specialized Health Promotion workforce • Specialized Health Promotion workforce circa 250 across the HSE, NGOs and private sector • Very limited Health Promotion speciﬁc workforce/no speciﬁc career path Health Promotion Academic Education • Three courses accredited by the IUHPE (two undergraduate/one postgraduate) [41] • Two other Health Promotion speciﬁc courses [42,43] • Two courses accredited by the IUHPE (one undergraduate/one postgraduate [41] • One course speciﬁc to Health Promotion) • In 2019, the Ministry of University and Research included Health Promotion as a topic for post graduate courses for 22 non-medical health professions at national level [44] National Professional Associations • Association for Health Promotion Ireland (AHPI) [45] • Health Promotion Group in Public Health Society [46] • Italian Society for the Promotion of Health (SiPs) [47] National Accreditation Organization (NAO) [48] • NAO (Established 2016) • Individual practitioners registered by Irish NAO • Work ongoing on developing NAO • Individual practitioners registered at IUHPE global level Professional status • No current statutory recognition of Health Promotion but interest in this for the future • Voluntary registration (Irish NAO) • Practitioners currently not required to be on any professional statutory register to be employed in Health Promotion • No current statutory recognition of Health Promotion and unlikely to be in future • Voluntary registration (IUHPE Global) • Practitioners usually required to be on statutory register in relevant profession (e.g., medicine/psychology) to be employed in Health Promotion Other dedicated Health Promotion organizations Research • Health Promotion Research Centre, NUIG [49] Policy, research and evaluation • National Center for Disease Prevention and Health Promotion [50] Research, education and training • Experimental Center for Health Promotion and Health Education—CeSPES [51] Knowledge transfer and training • Regional Documentation Center for Health Promotion—DoRs [52] d f Table 1. Health Promotion infrastructure and capacity in Ireland and Italy. 3.3. Implementing the CompHP Competencies in Ireland and Italy The ﬁndings are reported separately for each country and with reference to the CFIR constructs/subconstructs selected, i.e., readiness to implement, use and impact of the competencies, and key factors inﬂuencing their implementation. 3.3.3. Implementation—Ireland Formal implementation of the competencies was reported in two main contexts, i.e., in the introduction of a of professional registration system and associated Continuing Professional Development (CPD) activities managed by the Professional Association [45], and in the context of university degree programmes that have been accredited under the IUHPE Accreditation System for Health Promotion education and training. The competencies were also reported as having strongly inﬂuenced the development of a number of new Health Promotion courses at both undergraduate and postgraduate levels. p g While there was little evidence of formal implementation of the competencies at an organizational level elsewhere, some informants indicated that they used the competencies in their individual practice, often on an ‘ad hoc’ and ‘implicit’ basis (i.e., without formal reference to the CompHP Framework). ‘we need wider diﬀusion about them and more time to do that before being ready to implement.’ ‘we need wider diﬀusion about them and more time to do that before being ready to implement.’ 3.2. Interviews and Informants A total of 26 informants were interviewed, comprising 13 national experts in each country, including members of the National Reference Groups. As the Health Promotion community in both Int. J. Environ. Res. Public Health 2019, 16, 4992 6 of 21 countries is relatively small, the number of experts interviewed from speciﬁc settings and roles is not speciﬁed in order to maintain anonymity. Those interviewed included Health Promotion academics, practitioners, managers and representatives of professional associations in both countries. In Italy, informants were from national and regional levels, with the majority from the academic and training sectors. Informants in Ireland included those employed at policy and practice level and in academic, statutory, nongovernmental organizations (NGOs) and the private sector. Three Italian informants responded in writing as they considered that their English conversation skills were not adequate for their participation in an interview. 3.3.1. Readiness to Implement—Ireland Diﬀering levels of readiness to implement the competencies were reported across diﬀerent settings, with most informants indicating that academic organizations were at maintenance stage, the professional association/NAO was at action stage but that readiness at practice level was still at an early level. Overall, most informants considered that there had been good progress nationally: ‘on a scale of 1-10 Ireland is at 6 . . . people realize that this is what we need to deﬁne our role.’ ‘at least 50/50 willingness/readiness to implement the CompHP Competencies nationally.’ However, a few informants considered that readiness was However, a few informants considered that readiness was ‘maybe contemplation stage . . . parts of Ireland where they are just thinking about it.’ ‘maybe contemplation stage . . . parts of Ireland where they are just thinking about it.’ 3.3.2. Readiness to implement—Italy 3.3.2. Readiness to implement—Italy A large majority of Italian informants reported that readiness to implement the competencies was at very early stages across all settings: ‘very early in process . . . slow and diﬃcult to involve people at national and regional level . . . a lot of work’ ‘very early in process . . . slow and diﬃcult to involve people at national and regional level . . . a lot of work’ 3.3.4. Implementation—Italy Formal implementation of the competencies was reported in the context of two university degree programmes that have been accredited by the IUHPE. 7 of 21 Int. J. Environ. Res. Public Health 2019, 16, 4992 Reference was also made to their use in Health Promotion education in other professional academic courses (e.g., medicine, psychology) and in training for health professionals at regional and national level. In addition, the competencies were also used for informing the development of an online ‘best practice’ framework [52] and regional research on Health Promotion workforce development. Details of how the competencies were used, other than in accredited courses, included that they were sometimes ‘mixed and matched’ with other frameworks, adapted to local contexts and that use was often ‘implicit’: ‘I do not name the CompHP framework . . . I talk about them, but I do not name them.’ 3.3.5. Impact While no formal evaluation of the impact of the competencies had been undertaken in either country, all informants stated that, on reﬂection, their experience of implementing them was very positive. Most informants reported impact on the quality of education and training, recognition of Health Promotion and improved quality assurance (see Table 2). Table 2. Perceived impact of the CompHP Competencies in Ireland and Italy. Ireland Italy Health Promotion Education Academic courses enhanced in terms of: # Development # Quality/ethos # Recruitment # Marketability Enhanced: # Students’ learning # Lecturers’ credibility # Graduates sense of Health Promotion role Academic/training courses enhanced in terms of: # Development # Quality # Marketability Enhanced: # Students’ learning # Graduates’ knowledge/conﬁdence Health Promotion Profession Profession # Strengthened recognition of profession NAO # Basis for NAO development and growth Professional Association # Helped consolidate, refocus organization # Helped ’sell’ Health Promotion to decision-makers/employers Workforce/practitioners # Engendered sense of professional community NAO # Basis for developing NAO Workforce/practitioners # Supported recognition of Health Promotion roles (health professionals) # Basis for research resulting in changes in Health Promotion workforce. Health Promotion Practice Quality assurance # Used as structure/clear pathway for good practice # Facilitated holistic focus on Health Promotion # Provided clarity of role for workforce # Ensured all using the same language. Quality assurance # Provided guide/checklist for good practice # Clariﬁed Health Promotion roles # Supported reﬂection. # Informed planning at regional level. 3.4. Factors Inﬂuencing the Implementation of the CompHP Competencies in Ireland Table 2. Perceived impact of the CompHP Competencies in Ireland and Italy. 3.4. Factors Inﬂuencing the Implementation of the CompHP Competencies in Ireland The main themes emerging from the data from Ireland in relation to factors inﬂuencing the implementation of the CompHP Competencies are illustrated in Figure 1. The size of the shapes in the ﬁgure reﬂects the weighting attributed to the themes based on how frequently informants referred to each theme and the emphasis they placed upon speciﬁc issues in the interviews. 8 of 21 Int. J. Environ. Res. Public Health 2019, 16, 4992 8 of 21 Figure 1. Main factors facilitating and limiting implementation of the CompHP Competencies at country level in Ireland. Figure 1. Main factors facilitating and limiting implementation of the CompHP Competencies at country level in Ireland. Figure 1. Main factors facilitating and limiting implementation of the CompHP Competencies a country level in Ireland. Figure 1. 3.5.1. Health Promotion Professional Association/National Accreditation Organiza 3.5.1. Health Promotion Professional Association/National Accreditation Organizati 3.5.1. Health Promotion Professional Association/National Accreditation Organization (NAO) 3.5.1. Health Promotion Professional Association/National Accreditation Organization (NAO) g Informants across all sectors acknowledged the very significant role that the Health Promotion Professional Association had played and continues to play in implementing the competencies, fo example: Informants across all sectors acknowledged the very signiﬁcant role that the Health Promotion Professional Association had played and continues to play in implementing the competencies, for example: ‘the Association has done hugely valuable work on the CompHP Competencies … there is a momentum behind it.’ ‘the Association has done hugely valuable work on the CompHP Competencies . . . there is a momentum behind it.’ The role of the Association in establishing a NAO, with a remit to register Health Promotion on the practitioners in Ireland on the IUHPE Accreditation System was viewed as a major boost in implementing the competencies: The role of the Association in establishing a NAO, with a remit to register Health Promotion on the practitioners in Ireland on the IUHPE Accreditation System was viewed as a major boost in implementing the competencies: ‘as you see more registration you will see the CompHP Competencies becoming more embedded in people’s thinking and practice.’ ‘as you see more registration you will see the CompHP Competencies becoming more embedded in people’s thinking and practice.’ In developing the NAO, the planning process was described as ‘slow and inclusive’ and its success was attributed to a ‘really good team’ involving ‘keen and motivated people’. Informants made reference to the support provided by the IUHPE, champions within the Health Promotion community and small amounts of funding from statutory bodies as contributing to the successful development of the NAO. However, a lack of resources was highlighted as a potential limiting factor going forward, as the In developing the NAO, the planning process was described as ‘slow and inclusive’ and its success was attributed to a ‘really good team’ involving ‘keen and motivated people’. Informants made reference to the support provided by the IUHPE, champions within the Health Promotion community and small amounts of funding from statutory bodies as contributing to the successful development of the NAO. However, a lack of resources was highlighted as a potential limiting factor going forward, as the Professional Association and NAO operate on a voluntary basis. 3.5.1. Health Promotion Professional Association/National Accreditation Organiza 3.5.1. Health Promotion Professional Association/National Accreditation Organizati In developing the NAO, the planning process was described as ‘slow and inclusive’ and its success was attributed to a ‘really good team’ involving ‘keen and motivated people’. Informants made reference to the support provided by the IUHPE, champions within the Health Promotion community and small amounts of funding from statutory bodies as contributing to the successful development of the In developing the NAO, the planning process was described as ‘slow and inclusive’ and its success was attributed to a ‘really good team’ involving ‘keen and motivated people’. Informants made reference to the support provided by the IUHPE, champions within the Health Promotion community and small amounts of funding from statutory bodies as contributing to the successful development of the NAO. as a i u e o a ea y goo ea i o i g ee a o iva e peop e I o a s a e e e e e to the support provided by the IUHPE, champions within the Health Promotion community and small amounts of funding from statutory bodies as contributing to the successful development of the NAO. However, a lack of resources was highlighted as a potential limiting factor going forward, as the f l A d AO l b was attributed to a really good team involving keen and motivated people . Informants made reference to the support provided by the IUHPE, champions within the Health Promotion community and small amounts of funding from statutory bodies as contributing to the successful development of the NAO. However, a lack of resources was highlighted as a potential limiting factor going forward, as the Professional Association and NAO operate on a voluntary basis. NAO. However, a lack of resources was highlighted as a potential limiting factor going forward, as the P ofe io al A o iatio a d NAO o e ate o a olu ta y ba i However, a lack of resources was highlighted as a potential limiting factor going forward, as the Professional Association and NAO operate on a voluntary basis. 3.3.5. Impact Main factors facilitating and limiting implementation of the CompHP Competencies at country level in Ireland. 3.5. Factors Faciliting Implemention in Ireland 3.5. Factors Faciliting Implemention in Ireland o essio a Asso ia io a NAO ope a 3.5.2. Potential for Statutory Recognition 3.5.2. Potential for Statutory Recognition Implementing the competencies and establishing voluntary professional registration were i d i i d i i i i f H l h P i Implementing the competencies and establishing voluntary professional registration were viewed as a positive step towards gaining statutory recognition for Health Promotion: viewed as a positive step towards gaining statutory recognition for Health Promotion: ‘to have our own competencies and registration in place is a good stepping-stone towards statutory with CORU (https://www coru ie/) and registration down the line ’ ‘to have our own competencies and registration in place is a good stepping-stone towards statutory with CORU (https://www.coru.ie/) and registration down the line.’ Int. J. Environ. Res. Public Health 2019, 16, 4992 9 of 21 Participants expressed the view that as registration grew, there would be increasing momentum towards establishing statutory recognition, although this was unlikely to happen in the near future. 3.5.3. Health Promotion Education Most informants made reference to the positive impact of the implementation of the competencies through Health Promotion education, with a majority indicating that they had become aware of them as students in Health Promotion courses and/or through an annual Health Promotion conference hosted by an academic institution. It was considered that ‘the more embedded and centered work and experiences is around the CompHP Competencies at the educational level the more widely understood and used they will become.’ ‘the more embedded and centered work and experiences is around the CompHP Competencies at the educational level the more widely understood and used they will become.’ The leadership role played by Irish academics in the development of the competencies and their ongoing support were regarded as important factors in their implementation. The leadership role played by Irish academics in the development of the competencies and their ongoing support were regarded as important factors in their implementation. Graduates of accredited courses were also recognized as potential champions for the competencies 4 H l h P i W kf 3.5.4. Health Promotion Workforce Frequent reference was made to the usefulness of the competencies in the context of a specialized workforce, e.g., in gaining recognition for and deﬁning roles, assuring quality and underpinning education and informing CPD: Frequent reference was made to the usefulness of the competencies in the context of a specialized workforce, e.g., in gaining recognition for and deﬁning roles, assuring quality and underpinning education and informing CPD: ‘I think a competency framework is like a safety blanket or a protection and deﬁnition of what we do in a very succinct way.’ ‘I think a competency framework is like a safety blanket or a protection and deﬁnition of what we do in a very succinct way.’ Despite concerns about the level of awareness of the competencies, there was evidence of support for their implementation in the workforce: Despite concerns about the level of awareness of the competencies, there was evidence of support for their implementation in the workforce: ‘people who are working on the ground are keen for this (implementation) to happen . . . they see the value in the CompHP Competencies.’ 3.6. Factors both facilitating and limiting implemention in Ireland Organizational Changes in the Health Service Executive (HSE) Organizational changes within the health service were identiﬁed as having both facilitating and limiting aspects in terms of the implementation of the competencies. Informants across all sectors stressed that the position of Health Promotion within the HSE, the largest employers of Health Promotion practitioners in Ireland, was a pivotal factor in implementing the competencies nationally. However, what was described as ‘constant organizational change’ within the health service in recent years, ongoing as interviews were conducted, was considered by some as potentially threatening the sustainability of Health Promotion roles and functions. Some informants suggested that the competencies served as an authoritative source when arguing to maintain capacity: ‘I think the competencies can oﬀer a context for a conversation that might need to be had in terms of what the role and function of Health Promotion is in the new structure.’ Changes in leadership in the organization were considered to oﬀer opportunities, but also pose challenges: ‘the national leadership in Health Promotion in the service will have a much diﬀerent role . . . so maybe there is an opportunity for more buy-in of the competencies . . . 3.7.2. Status of Health Promotion The status of Health Promotion at policy level was highlighted as a pivotal factor in implementing the competencies: ‘where Health Promotion is ‘at’, at a policy level, makes a big diﬀerence in implementing them as all funding comes through the policy stream.’ Some informants referred to a ‘shift’ from the ‘settings approach’ to a ‘health and wellbeing’ approach in the current ‘Healthy Ireland’ policy [35], with Health Promotion being integrated into the wider health and social care agenda. This shift was linked to changes in terminology fostering fears that, if Health Promotion was not speciﬁcally referenced in funding streams and national policies, there could be negative repercussions for the relevance of the competencies: ‘The term Health Promotion is not used anymore in the health service . . . it’s now ‘health and wellbeing’ . . . this hinders the uptake of the competencies and registration.’ ‘In our organization (NGO) we have a prevention department . . . it used to be called the Health Promotion department . . . not sure what that means for the competencies.’ Limitations on funding for Health Promotion at national level, associated with political imperatives to focus on acute care services were also considered as likely to have a negative impact on implementing the competencies. However, a few informants considered that the situation for Health Promotion had improved with ‘more reference to Health Promotion in government strategies and more jobs’, resulting in a more positive environment for implementing the competencies. 3.7.3. Professional Status 3.7.1. Lack of Awareness of the CompHP Competencies Most informants reported that limited awareness of the competencies across all levels and settings was a major barrier to their implementation, in particular in relation to employers and decision-makers. Practitioners who were not involved with the Professional Association and/or who work outside the capital city were identiﬁed as being less likely to be aware of the competencies Most informants reported that limited awareness of the competencies across all levels and settings was a major barrier to their implementation, in particular in relation to employers and decision-makers. Practitioners who were not involved with the Professional Association and/or who work outside the capital city were identiﬁed as being less likely to be aware of the competencies. Some considered that there had been more awareness of the competencies when they were ﬁrst developed, and it was strongly emphasised that a more targeted and sustained dissemination of information was required. 3.5.4. Health Promotion Workforce equally there might be more resistance.’ ‘the national leadership in Health Promotion in the service will have a much diﬀerent role . . . so maybe there is an opportunity for more buy-in of the competencies . . . equally there might be more resistance.’ While some informants believed that the competencies were not recognized by senior level health service managers, the fact that they had recently been referenced in the recruitment and selection of new Health Promotion managers was viewed as a positive step in this regard. 10 of 21 Int. J. Environ. Res. Public Health 2019, 16, 4992 3.7.3. Professional Status The fact that Health Promotion is not formally recognized as a profession in Ireland was believed by many to have negative implications for the competencies, as ‘it appears that anybody without a professional qualiﬁcation in Health Promotion or without experience is entitled to, and has the ability to, do Health Promotion.’ Professional status was regarded as being of particular relevance for Health Promotion practitioners in the health service as their employment is currently graded as administrative rather than professional: Professional status was regarded as being of particular relevance for Health Promotion practitioners in the health service as their employment is currently graded as administrative rather than professional: ‘the competencies would need to be linked to grading for our profession and that can be seen as been Professional status was regarded as being of particular relevance for Health Promotion practitioners in the health service as their employment is currently graded as administrative rather than professional: ‘the competencies would need to be linked to grading for our profession and that can be seen as been helpful (to implementation) but also as a hindrance as it hasn’t happened.’ ‘the competencies would need to be linked to grading for our profession and that can be seen as been helpful (to implementation) but also as a hindrance as it hasn’t happened.’ For all informants, implementing the competencies was viewed as useful when making the case for professional status. Int. J. Environ. Res. Public Health 2019, 16, 4992 11 of 21 11 of 21 3.8. Factors Inﬂuencing the Implementation of the CompHP Competencies in Italy .8. Factors Inﬂuencing the Implementation of the CompHP Competencies in Italy The factors inﬂuencing the implementation of the CompHP Competencies emerging from the Italian data are illustrated in Figure 2. The size of shapes in the ﬁgure reﬂects the weighting attributed to the themes based on how frequently informants referred to each theme and the emphasis they placed upon speciﬁc issues in the interviews. Int. J. Environ. Res. Public Health 2019, 16, x 12 of 22 to the themes based on how frequently informants referred to each theme and the emphasis they placed upon specific issues in the interviews. Figure 2. Key factors facilitating and limiting implementation of the CompHP Competencies at country level in Italy. Figure 2. Key factors facilitating and limiting implementation of the CompHP Competencies at country level in Italy. Figure 2. 3.7.3. Professional Status Key factors facilitating and limiting implementation of the CompHP Competencies at country level in Italy. Figure 2. Key factors facilitating and limiting implementation of the CompHP Competencies at country level in Italy. 3.9. Factors Faciliating Implementation in Italy 3.9. Factors Faciliating Implementation in Italy 3.9.1. Champions 3.9.1. Champions p Many references were made to key people in the Health Promotion community in Italy who had shown leadership and championed the development and implementation of the competencies. Some informants also highlighted that they, and their organization, were championing the competencies: Many references were made to key people in the Health Promotion community in Italy who had shown leadership and championed the development and implementation of the competencies. Some informants also highlighted that they, and their organization, were championing the competencies: informants also highlighted that they, and their organization, were championing the compe ‘when we speak about Health Promotion on every occasion we use the CompHP Competencies.’ ‘when we speak about Health Promotion on every occasion we use the CompHP Competencies.’ p y p p Graduates of accredited Health Promotion courses were also regarded as future champions for the competencies Graduates of accredited Health Promotion courses were also regarded as future champions for the competencies. 3 9 2 Health Promotion Education and Training 3.9.2. Health Promotion Education and Training Plans for Developing/Lack of a National Accreditation Organization (NAO) The issue of developing a national professional body or NAO was viewed as having both facilitating and limiting inﬂuences on the implementation of the competencies. For example, it was indicated that if ongoing plans to establish a NAO came to fruition, this would give impetus to implementing the competencies. However, while formal plans had been drawn up, some funding secured and some progress made in establishing the partnership that will form its governing body, the ongoing lack of a NAO was identiﬁed by over half of informants as an obstacle to implementing the competencies. Progress on the development of the NAO was described as ‘diﬃcult and slow’ with ‘diﬃculties in engaging people’. Resistance to the development of a NAO was also reported: ‘there is a lot of resistance to that . . . because to people who work in in public health Health Promotion is something that is regarded as a natural part of the public health profession . . . not a competency that you have to support and improve.’ Concerns were also expressed about the potential for inequities in relation to the NAO: Concerns were also expressed about the potential for inequities in relation to the NAO ‘the richer regions will get the System . . . you will see many regions from the north and less from the south involved.’ Other diﬃculties identiﬁed in setting up a NAO included a lack of support from decision- makers, overall bureaucracy, and limited resources. The experiences of those who had developed a NAO in other countries and the ﬁndings from this case study were suggested as ways to inform the development process. 3.11. Factors Limiting Implementation in Italy 3.9.3. Growing Awareness of Health Promotion and the CompHP Competencies While the predominance of prevention and the ‘medical model’ of public health was stressed, there was reference to a growing interest in Health Promotion approaches in some regions and national organizations, with the competencies having a potential role in supporting this: ‘recognition of Health Promotion will reach new levels if the CompHP Competencies work to change, or at least begin to change, how the regions operate.’ The recent decision of the Ministry of University and Research to include Health Promotion as a topic within postgraduate courses for non-medical health professionals was also oﬀered as an indication of increasing awareness of, and support for, Health Promotion at national level. g pp Similarly, despite concerns about a general lack of awareness, some informants pointed to evidence of growing awareness of, and interest in, the competencies: ‘the people we work with . . . like regions, groups, colleagues . . . they are very, very interested in the competencies.’ 3.10. Factors both Facilitating and Limiting Implementation in Italy 3.10. Factors both Facilitating and Limiting Implementation in Italy Plans for Developing/Lack of a National Accreditation Organization (NAO) 3 9 2 Health P o otio Edu atio a d T ai i 3.9.2. Health Promotion Education and Training 3.9.2. Health Promotion Education and Training The importance of Health Promotion education and training was also identified and it was suggested that The importance of Health Promotion education and training was also identiﬁed and it was suggested that 3.9.2. Health Promotion Education and Training The importance of Health Promotion education and training was also identified and it was suggested that The importance of Health Promotion education and training was also identiﬁed and it was suggested that suggested that ‘the demand for the competencies will progressively increase as a consequence of more effective Health P ti t i i d i iti ’ ‘the demand for the competencies will progressively increase as a consequence of more eﬀective Health Promotion training and universities courses.’ suggested that ‘the demand for the competencies will progressively increase as a consequence of more effective Health P i i i d i i i ’ ‘the demand for the competencies will progressively increase as a consequence of more eﬀective Health Promotion training and universities courses.’ Promotion training and universities courses. Those currently implementing the competencies in education and training were regarded as role models for others wishing to develop competency-based curricula. Where the competencies had been Those currently implementing the competencies in education and training were regarded as role models for others wishing to develop competency-based curricula. Where the competencies had been Int. J. Environ. Res. Public Health 2019, 16, 4992 12 of 21 implemented in educational settings it was reported that managers had been supportive and that people were the main resource used. implemented in educational settings it was reported that managers had been supportive and that people were the main resource used. Education and training organizations’ role in disseminating information on the competencies was also acknowledged, e.g., through newsletters (e.g., [51,52]), peer-reviewed articles [53] and knowledge transfer via a ‘best practice’ website [52]. Growing Awareness of Health Promotion and the CompHP Competencies 3.11.2. Professional Proﬁles The fact that there is no speciﬁc Health Promotion professional proﬁle, combined with the strict statutory regulation of professions and meticulous delineation of professional boundaries in Italy, were identiﬁed as major limiting factors in implementing the competencies in practice, education and training: ‘it’s the legal title of the degree . . . so you can do a profession only if you have this degree . . . it’s very ‘it’s the legal title of the degree . . . so you can do a profession only if you have this degree . . . it’s very diﬃ l i ld b h i l i l i ( i l h i ) d ’ ‘it’s the legal title of the degree . . . so you can do a profession only if you have this degree . . . it’s very diﬃcult . . . it would be an enormous change in legislation (to recognize Health Promotion) . . . I don’t think it is possible’ ‘it’s the legal title of the degree . . . so you can do a profession only if you have this degree . . . it’s very diﬃcult . . . it would be an enormous change in legislation (to recognize Health Promotion) . . . I don’t think it is possible’ diﬃcult . . . it would be an enormous change in legislation (to recognize Health Promotion) . . . I don’t think it is possible’ ‘we cannot ask other people to embrace them (the competencies) because they have other proﬁles.’ ‘we cannot ask other people to embrace them (the competencies) because they have other proﬁles.’ As most recruitment calls for employment in the context of Health Promotion require that the applicant be registered in a statutorily recognized profession, it was argued that ‘you can have Health Promotion registration and know the competencies but unless you have registration and education in a deﬁned area you are not able to apply for public calls.’ Doubts were also expressed about the possibility, or even the desirability, of statutory recognition of Health Promotion: ‘I don’t think it’s possible to create a Health Promotion practitioner register ... as a profession . . . because we have a particular legislation about the diﬀerent professions . . . most of the people are not agreeing with this kind of deﬁnition . . . to recognize Health Promotion . 3.11.1. Lack of Awareness of the CompHP Competencies 3.11.1. Lack of Awareness of the CompHP Competencies All informants were of the opinion that there was very little awareness of the competencies across all levels in Italy and that this was a major block to their implementation: 13 of 21 Int. J. Environ. Res. Public Health 2019, 16, 4992 ‘So far the competencies are not much known and haven’t really inﬂuenced at national level’ ‘So far the competencies are not much known and haven’t really inﬂuenced at national level’ ‘Some aware . . . but a few people . . . there isn’t a critical mass for change at national, regional or local level or within the university.’ It was reported that very few employers were aware of the competencies, although they are a key group that need to be involved in their implementation and it was emphasized that is a situation that needs to be addressed. Awareness of the competencies overall was summarized as Awareness of the competencies overall was summarized as Awareness of the competencies overall was summarized as ‘they are not widely known . . . little spots in diﬀerent places and these are not linked together.’ 3.11.2. Professional Proﬁles . . because diﬀerent professionals are involved in Health promotion and each single profession has a special register so it’s very, very diﬃcult.’ This situation was viewed as part of a wider problem as This situation was viewed as part of a wider problem as This situation was viewed as part of a wider problem as ‘some employers have diﬃculty recognizing nursing or physiotherapy as a profession like medicine . . . we are still very medically oriented . . . so it’s very complicated to work on Health Promotion . . . but it’s not only with Health Promotion.’ ‘some employers have diﬃculty recognizing nursing or physiotherapy as a profession like medicine . . . we are still very medically oriented . . . so it’s very complicated to work on Health Promotion . . . but it’s not only with Health Promotion.’ There was also little support evident for a Health Promotion speciﬁc workforce: ‘Health Promotion professionals . . . it’s not so widespread in Italy . . . I think it is better to have a speciﬁc Health Promotion module for psychologists, for medical or education practitioners.’ ‘Health Promotion professionals . . . it’s not so widespread in Italy . . . I think it is better to have a speciﬁc Health Promotion module for psychologists, for medical or education practitioners.’ It was suggested that in the Italian professional context, the best way forward might be to emphasize the value of the competencies as a quality assurance framework and competency-based registration as a valuable ‘added’ title for practitioners with other professional titles. Int. J. Environ. Res. Public Health 2019, 16, 4992 14 of 21 14 of 21 3.11.5. Limited Health Promotion education A lack of Health Promotion education and training was viewed as a factor limiting the implementation of the competencies, but they were also suggested as an authoritative source when arguing for more, and better quality, education and training. 3.11.3. Status of Health Promotion Many informants reported that Health Promotion was not well understood, supported or implemented at national and regional levels, and that this created barriers to implementing the competencies: ‘knowledge is very low about Health Promotion and lots of people confuse Health Promotion with prevention or Health Education . . . so it’s very, very diﬃcult about the competencies.’ Some informants considered that the national policy was overly focused on prevention while others argued that it was the application of the policy across a devolved health system that was problematic, with reports of a stronger commitment to Health Promotion in northern regions. Many informants reported that prevention and health education actions were often mis-labelled as Health Promotion, with suggestions that this should be challenged referencing the competencies as to what constitutes Health Promotion. 3.11.4. Language Barrier Although the competencies have been translated into Italian [54], a number of informants identiﬁed a language barrier in implementing them and viewed developing a NAO as a way to overcome this: ‘if they (the competencies) remain in English and the registration is in English . . . we have a few people only in Italy (able) to use them.’ e will have a NAO for accreditation in Italy because the language is a big wall.’ ‘we will have a NAO for accreditation in Italy because the language is a big wall.’ It was also suggested that the current translation of the competencies should be reviewed with particular attention paid to translating key concepts: ‘we have diﬃculties even in translating the word competencies . . . we should start with what is there and go through it slowly and improve on it.’ ‘we have diﬃculties even in translating the word competencies . . . we should start with what is there and go through it slowly and improve on it.’ 3.11.5. Limited Health Promotion education 3.11.5. Limited Health Promotion education 3.11.6. Bureaucracy Bureaucracy was identiﬁed by a number of informants as a reason for slow progress in implementing the competencies: ‘In Italy it’s a slow process related to the characteristics of the system . . . not only the competencies . . . the system overall is very slow . . . bureaucracy is a problem.’ 4. Discussion The ﬁndings from the case study show that while the CompHP Competencies have been implemented in diﬀerent settings and contexts in both Ireland and Italy, the modes and rates of progress of implementation diﬀer. The ﬁndings also suggest that the levels of Health Promotion infrastructure and capacity at a country level are important inﬂuencers in the implementation of the competencies. Many of the factors identiﬁed as inﬂuencing the implementation of the competencies in both countries are similar to those that have been previously identiﬁed in the literature, for example, in terms of infrastructure (Health Promotion status/policy) and capacity (workforce, professional proﬁles/recognition, education and professional associations). The inﬂuence of diﬀerences in levels of Health Promotion infrastructure and capacity was evident in the themes emerging from the data across the CFIR constructs and subconstructs selected to inform the study. For example, there is evidence of readiness to, and good progress in, implementing the competencies in Ireland, where there is a history of Health Promotion speciﬁc policies and well established Health Promotion capacity, including a specialized workforce, a dedicated national Professional Association and speciﬁc education programmes since the 1990s. In contrast, in Italy, where policy is more focused on prevention, Health Promotion is seen as being part of the role of other professionals rather than a specialist Health Promotion workforce and Health Promotion education is limited, readiness to implement the competencies was reported as being at a very early stage and progress as slow and diﬃcult. The use of the competencies in both countries was also found to reﬂect their levels of Health Promotion infrastructure and capacity. For example, while formal implementation of the competencies in the context of accredited Health Promotion courses in both countries was similar (three in Ireland, two in Italy) this ﬁnding should be viewed in the context of a much larger academic sector in Italy, reﬂecting the much greater population. There were diﬀerences between the countries in the formal implementation of the competencies in the context of national professional bodies and NAOs within the IUHPE Health Promotion Accreditation System. In Ireland, a major focus of implementation has been the development of a NAO, with the Health Promotion speciﬁc Professional Association taking the lead on its development and a specialized workforce showing interest in becoming registered practitioners. Finally, an Italian informant suggested that ‘The competencies need to become the mentality of the person so that when they use them, they talk to people with passion.’ ‘The competencies need to become the mentality of the person so that when they use them, they talk to people with passion.’ 3.11.7. Limited Experience of Competency-Based Approaches 3.11.7. Limited Experience of Competency-Based Approaches A few informants suggested that the fact that there was not much experience of using competency-based approaches in general may create a barrier when implementing the competencies, but one informant reported: ompetencies as a professional discourse is coming into the professional culture’. ‘Competencies as a professional discourse is coming into the professional culture’. uture Implementation of the CompHP Competencies 3.12. Future Implementation of the CompHP Competencies Informants responses regarding what advice they would give to others who intended to implement the competencies are summarized in Table 3. Informants responses regarding what advice they would give to others who intended to implement the competencies are summarized in Table 3. 15 of 21 Int. J. Environ. Res. Public Health 2019, 16, 4992 Table 3. Advice on implementing the CompHP Competencies. Ireland Italy Key requirement Knowledge and deep understanding of the competencies Knowledge and understanding of Health Promotion Practical advice • Reﬂect on what is already being done and link the competencies to that - it’s less threatening • Get buy-in from the wider workforce • CompHP Handbooks/IUHPE website are key resources • Gather peers and brainstorm what implementation will look like • Link implementation to other relevant developments • Ensure implementation is a bottom-up process • Select examples of best practice in implementation and share with decision-makers • Talk to those who have already implemented them • Create enthusiasm Table 3. Advice on implementing the CompHP Competencies. 4. Discussion In Italy, the slow process in establishing a NAO was linked to diﬃculties in developing a partnership of stakeholders as a governing body and the fact that registration was viewed as an ‘added extra’ for health professionals Int. J. Environ. Res. Public Health 2019, 16, 4992 16 of 21 rather than as a system of professional recognition for specialized Health Promotion practitioners. In addition, there were indications of opposition from some in public health in Italy as Health Promotion is not recognized as a distinct profession with its own competencies, a situation that was not evident in the Irish data. While there were some commonalities across the ﬁndings from the two countries on the factors considered to inﬂuence implementation of the competencies, more positive factors were identiﬁed in Ireland, as might be expected given its more advanced levels of Health Promotion infrastructure and capacity. For example, in Ireland the Health Promotion speciﬁc Professional Association was identiﬁed as a main facilitating factor, reﬂecting established Health Promotion capacity that could provide leadership and organizational support in implementing the competencies at a country level. In contrast, in Italy, the main facilitating factor identiﬁed was individual champions, likely indicating the need to develop and strengthen Health Promotion capacity and structures within which to formally implement the competencies. There were also commonalities in the limiting factors identiﬁed in both countries, in particular, a lack of awareness of the competencies at all levels. While some useful examples of dissemination of information on the competencies were identiﬁed, there was reference in both countries to limited resources for dissemination, together with a lack of consensus on how to inform key groups, notably employers. While the status of Health Promotion was viewed as a limiting factor in both countries, the underlying reasons were diﬀerent. In Ireland, the focus was on a change in approach in a well-established Health Promotion policy context, while in Italy, concerns were focused on a national policy more centered on prevention and a lack of knowledge and understanding of Health Promotion. However, there was recognition in both countries that the competencies have a role to play in both developing and maintaining the status of Health Promotion. It was interesting to note that ongoing organizational change in the health service in Ireland and resistance to change in Italy featured as factors limiting the implementation of the competencies in each country. 4. Discussion It is likely that the rate and process of implementing the competencies going forward will reﬂect not only the level of Health Promotion infrastructure and capacity but also the organizational culture within which they are implemented. With regard to the implications of the ﬁndings for future implementation, it is clear from the data that a lack of awareness of the competencies was viewed as a major limiting factor by informants in both countries, in particular with reference to employers and decision-makers. This overall lack of awareness, in addition to a lack of support for, and formal recognition of, the competencies by key organizations and stakeholders are major issues for future implementation that and will need to be addressed. Irrespective of any other inﬂuencing factors, lack of awareness could potentially undermine future implementation, not only in Ireland and Italy, but globally. This ﬁnding highlights the need for more targeted and sustained information-sharing and marketing of the competencies at all levels and greater advocacy to ensure their implementation. Another factor with implications for future implementation is the professional status of Health Promotion. While Health Promotion is not a statutorily regulated profession in either country, the issue of professional regulation impacted on implementing the competencies to a much greater extent in Italy and this has implications for the focus on implementation going forward. As acknowledged when the competencies were being developed, while their key target audience is Health Promotion practitioners, they could be useful to other health professionals in countries where there was no specialized Health Promotion workforce, and this has proved to be the case in Italy. Similarly, the recommendation that the competencies could be used as ‘stand-alone’ quality standards in countries with less well-developed Health Promotion infrastructures was suggested as a possible way forward in Italy. The issue of language as a barrier to implementing the competencies was raised in Italy, with added emphasis on the fact that translation is challenging given diﬀerent cultural and linguistic interpretations of key concepts and core words in the competency context. These diﬃculties reinforce the need for 17 of 21 Int. J. Environ. Res. Public Health 2019, 16, 4992 translation of the competencies to be a cooperative endeavor between skilled translators and experts with a solid grounding in Health Promotion and also for the need for future research to be conducted in informants’ ﬁrst language. 4. Discussion The ﬁndings from Ireland and Italy demonstrate that an understanding of the policy, practice and organizational contexts within which the competencies are implemented will help identify how best to proceed. Whatever the context, raising awareness of the competencies at all levels is a key ﬁrst step, an exercise that should be tailored to the speciﬁc cultural and organizational contexts, building on facilitating factors while being cognisant of those factors that may limit implementation. While the case study focused only on implementing the competencies in two European countries, there are insights from their experience that can inform future implementation and research on progress in other countries. For example, the ﬁndings on the factors inﬂuencing implementation of the competencies at country level, in particular when combined with those of the online survey on individual decision-making, give an appreciation of some of the complexities that need to be addressed at professional and organizational levels in terms of leadership, norms, structures and processes in both future implementation and research on progress. The ﬁndings also provide some encouragement for the future in terms of how implementation can be advanced when the appropriate supports are put in place. In terms of future research, the case study begins to address the gap in the literature on evaluating the take-up, use and impact of Health Promotion competencies but further, more in-depth exploration will be required as the uptake of the competencies expands to other countries and regions. Ongoing contemporary documentation of the implementation process in speciﬁc country contexts could provide valuable information concerning the eﬀective adoption and integration of Health Promotion competencies across diverse organizational contexts and health systems. The methods used and the lessons learned in undertaking this case study oﬀer a starting point for such future research in this under-explored aspect of Health Promotion. Strengths and Limitations of the Study This study builds upon established theoretical frameworks in terms of case study methodology and implementation research and is innovative in providing an in-depth exploration of the use and impact of Health Promotion competencies at a country level. The study also forms part of a larger evaluation study that begins to address the gap in empirical studies on how Health Promotion competencies are used and what impact they have on Health Promotion practice, education and training. The strengths of the study were enhanced by the openness and generosity of all informants, the breadth and quality of the information they provided and the time they dedicated to supporting the research. The use of a case study approach allowed for exploration of the contextual factors inﬂuencing the implementation of the competencies with an emphasis on informants’ experience of using the competencies within diﬀerent settings and system. While additional resources might have allowed for more extensive interviews, it was considered that the data collected gives useful insight into the realities of implementing competencies in both countries. Given the relatively small number of Health Promotion speciﬁc practitioners and academics in both countries and the fact that as interviews progressed, little new data were forthcoming, the researchers were satisﬁed with the level of saturation achieved. However, there are limitations in this study that should be noted. As with all qualitative research, the ﬁndings are not generalizable, and the data as analyzed by the researchers may have resulted in a particular interpretation of the implementation of the competencies and/or the status of Health Promotion in one or both countries. To counteract this, however, interpretation of the data was validated by the National Reference Group of experts in both countries to ensure accuracy. 18 of 21 Int. J. Environ. Res. Public Health 2019, 16, 4992 As resources were limited, the interviews were conducted in English and this may have excluded some useful informants and may also have limited the ﬂow of the interviews conducted in Italy, despite the excellent language skills of those interviewed. Finally, the researchers’ involvement in the development and operationalization of the competencies may have inﬂuenced how informants framed their responses and led to some bias in the responses received and/or in analysis of the data. 5. Conclusions The ﬁndings of the case study show diﬀerences in the rate and focus of the implementation of the CompHP Competencies in Ireland and Italy that are inﬂuenced by a number of contextual factors reﬂecting the levels of Health Promotion infrastructure and capacity development in each country. A lack of awareness was identiﬁed as a major limiting factor in implementing the competencies in both countries, of particular concern in relation to employers and decision-makers, highlighting the need to disseminate information and advocate for their implementation. The diﬀerences observed with regard to which contextual factors were found to inﬂuence the implementation of the competencies in the diﬀering political, social, academic, practice and professional contexts in Ireland and Italy, provide important insights for their future implementation in other countries and for further research evaluating their uptake and impact. Author Contributions: Conceptualization, B.B.-K. and M.M.B.; Formal analysis, B.B.-K.; Investigation, B.B.-K.; Methodology, B.B.-K. and M.M.B.; Project administration, B.B.-K. and M.M.B.; Writing – original draft, B.B.-K.; Writing – review & editing, B.B.-K. and M.M.B. Funding: This research received no external funding. 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https://openalex.org/W2144657447	https://bmccancer.biomedcentral.com/counter/pdf/10.1186/s12885-015-1464-x	English	null	MiR-223 targeting MAFB suppresses proliferation and migration of nasopharyngeal carcinoma cells	BMC cancer	2,015	cc-by	8,707	© 2015 Yang et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: taoliby@hotmail.com; shemin.lu@gmail.com 3Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical College, Dongguan, Guangdong 523808, P. R. China 1Department of Biochemistry and Molecular Biology, School of Medical Basic Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061, P. R. China Full list of author information is available at the end of the article Yang et al. BMC Cancer (2015) 15:461 DOI 10.1186/s12885-015-1464-x Yang et al. BMC Cancer (2015) 15:461 DOI 10.1186/s12885-015-1464-x Keywords: MiR-223, Nasopharyngeal carcinoma, MAFB, Proliferation, Migration Keywords: MiR-223, Nasopharyngeal carcinoma, MAFB, Proliferation, Migration important to further elucidate pathogenesis of NPC for discovering new therapeutic approaches. MiR-223 targeting MAFB suppresses proliferation and migration of nasopharyngeal carcinoma cells Wanyong Yang1,2, Xi Lan1, Dongmin Li1, Tao Li3* and Shemin Lu1* Wanyong Yang1,2, Xi Lan1, Dongmin Li1, Tao Li3* and Shemin Lu1* Abstract Background: Mounting evidence suggests that miRNAs have major functions in tumor pathogenesis, and this study aimed to identify the candidate miRNA and investigate its role in nasopharyngeal carcinoma (NPC). Methods: MiRNA and mRNA expressions were screened by microarray assays. The cell proliferation, colony formation and migration ability were measured by MTT, soft agar and wound healing assays, respectively. The tumor growth suppression was evaluated by xenografting in nude mice. The plasma miR-223 levels in NPC patients were detected by TaqMan analysis. Real-time quantitative PCR and Western blotting were used to confirm miR-223 and MAFB expression levels. The targeting relationship between miR-223 and MAFB was verified using dual luciferase reporter assay. Results: The miR-223 expression was decreased in CNE-1, CNE-2 cells as compared with NP69 cells, an immortalized human nasopharyngeal epithelial cell line, and its level also reduced in NPC patients’ plasma as compared with healthy controls. Exogenous expression of miR-223 in CNE-2 cells could inhibit cell proliferation both in vitro and in vivo. Extrogenous miR-223 in CNE-2 cells would decrease the ability of colony formation and migration. MAFB, a transcription factor of Maf family members, was identified as a target gene of miR-223. We found that migration and invasion abilities were inhibited by MAFB silencing. Conclusions: MiR-223 negatively regulates the growth and migration of NPC cells via reducing MAFB expression, and this finding provides a novel insight into understanding miR-223 regulation mechanism in nasopharyngeal carcinoma tumorigenesis. Background Nasopharyngeal carcinoma (NPC) is a common malignant tumor in the people of southern China, particularly in Guangdong population [1]. Radiotherapy is a commonly used method to treat NPC and combined with chemother- apy to promote the survival rate of the patients. However, NPC cells can easily invade local tissue even metastasize to remote organs, so such relapse and metastasis result in poor prognosis for the patients [2]. Therefore it is very MicroRNAs (miRNAs) are endogenous non-coding RNAs with approachable 22 nucleotides in length and play an important role in physiological and pathological conditions through cleaving or transcript suppressing target mRNAs [3]. Accumulating evidence demonstrates that miRNAs are associated with cancer occurrence. And determination of miRNA levels has been proposed as a biomarker for diagnosis and prognosis of various cancers [4, 5]. Here, we identified miR-223 as down- regulated in undifferentiated nasopharyngeal carcinoma cell line CNE-2, compared with immortalized nasopha- ryngeal epithelial cell line NP69. MiR-223 was firstly re- ported to be involved in the regulation of human granulopoiesis [6, 7]. Then it was found to be a potential Page 2 of 12 Yang et al. BMC Cancer (2015) 15:461 Yang et al. BMC Cancer (2015) 15:461 biomarker for recurrent ovarian cancer [8]. In Hela cells, overexpression of MiR-223 suppresses cell proliferation by targeting IGF-1R [9]. It seems contradictory that miR-223 can suppress tumor invasion and metastasis through tar- geting Artemin [10], but may promote tumor by inhibiting the expression of EPB41L3, a tumor suppressor in human NPC [10]. These findings suggest that miR-223 is associ- ated with migration and invasion of malignant tumor. However, to our knowledge, the role of miR-223 in naso- pharyngeal carcinogenesis remains undefined. fragmented cRNAs were hybridized with TaqMan® Human MicroRNA Array Set v3.0 containing 754 transcripts. Hybridization was performed at 45 °C with rotation for 16 h (Affymetrix GeneChip Hybridization Oven 640). The GeneChip arrays were washed and then stained (streptavi- din-phycoerythrin) on an Affymetrix Fluidics Station 450 followed by scanning on a GeneChip Scanner 3000. MiRNA transfection and real-time quantitative PCR MiR-223 mimic (Cat# miR10004570), miR-223 negative control (Cat# miR01201), miR-223 (Cat# miRQ0004570) and U6 (Cat# MQP-0201) real-time PCR primers RNA oligonucleotides were obtained from RiboBio (http://www. ribobio.com Guangzhou, China). miRNAs were trans- fected to CNE-2 cells by using Lipofectamine®2000, with a final concentration at 50 nM. Microarray analysis For microarray assay of miRNAs, double-stranded cDNAs were synthesized with the 2 μg total RNA, and biotin-tagged cRNAs were obtained by the use of the MessageAmp™II aRNA Amplification Kit (Ambion). The biotin-tagged cRNAs were fragmented into mixed strands in accordance with the Affymetrix’s protocols. The Cell proliferation analysis CNE-2 cells with a number of 5 × 103/well were seeded into 96-well plates, incubated for 12 h, and then trans- fection was performed. At 24 h after transfection, each well was treated with 10 μL MTT solution (10 mg/ml in PBS) and incubated sequentially at 37 °C. After the incu- bation for 4 h, 100 μL of DMSO was added to dissolve the crystals. Then the absorbance of each well in culture plate was measured at 570 nm and 630 nm after oscil- lated for 10 min at room temperature. Materials and methods Cell culture Highly and poorly differentiated human NPC cell lines named as CNE-1 and 2 were established and kindly pro- vided by Prof. Yi Zeng from the Institute of Virology, China Institute of Preventive Medical Science, China [11]. The im- mortalized human nasopharyngeal epithelial cell line named as NP69 was kindly provided by Prof. Kaitai Yao from Southern Medical University, Guangzhou, China. CNE-1 and CNE-2 cells were cultured in the DMEM medium con- taining 10 % fetal bovine serum (FBS), and NP69 cells were cultured in Keratinocyte-SFM (serum-free medium). Both mediums were contained 100 units/ml penicillin G and 100 μg/ml streptomycin (Invitrogen). The transfection was conducted with Lipofectamine™2000 reagent (Invitrogen). Background The expression of miR-223 was measured by real-time quantitative PCR (RT-qPCR) by using One Step SYBR® PrimeScript® RT-PCR Kit II (Takara) and following the manufacturer’s protocol on ABI 7500 HT real-time PCR detection system as normal- ized to the housekeeping gene U6. The information about primer sequences is depicted in Table 1. The present study was performed to find the potential miRNAs in NPC, and verify the role of target miRNA in invasion and metastasis of the cells. Our results illumin- ate the role of miR-223 in NPC development and pro- vide valuable information for clinical implications. Patient plasma collection analysis, 1 × 104 cells were plated in triplicates in 6 cm diameter plates with 0.6 % base agar and 0.3 % top agar and incubated 21 days. The colonies were count in 10 randomly chosen microscope fields. Human plasma samples from NPC patients and healthy donors were derived from the Affiliated Hospital of Guangdong Medical College and Dongguan area in China, with the informed consent under institutional re- view board-approved protocols. Blood was collected in EDTA tubes and processed for isolation of plasma within 2 h of collection. Blood was centrifuged at 2000 rpm at room temperature for 10 min. Then the upper layer plasma was transferred to RNase/DNase-free 1.5 ml con- ical tubes and stored at −80 °C. Transwell migration and invasion assay The ability of cells to migrate through filters was mea- sured using Polycarbonate Membrane Transwell Inserts (Corning). At 24 h after transfection, cells were trypsi- nised. Cell culture inserts with a polycarbonate mem- brane (8 μm pore size) were used. The bottom chamber contained medium (0.5 ml) supplemented with 10 % FBS, whereas the transfected cells were seeded into the upper chamber and incubated for 36 h. The remaining cells on the upper surface were mechanically removed. Then the membranes were washed and stained using 0.1 % crystal violet. The cell migration ability was mea- sured by the cell numbers that migrated to the lower side of the filter. Experiments were repeated 3 times. Two widely advocated online bioinformatic softwares, Target Scan [12] and microRNA.org [13], were used to predict the interaction probability between miR-223 and MAFB, including binding sites for miR-223 in MAFB and the targeting efficiency. The predictive outcome was used for further investigation. The invasion ability of cells was determined by a Matrigel invasion chamber assay. This assay was conducted by the use of 6.5 mm and 8 μm pore size Transwell chambers (Corning). Cells transfected with miRNA mimic or control one were cultured in serum-free medium for 12 h (1 × 105 cells per Transwell) and then the cells were migrated into a medium containing 10 % FBS for 24 h. Cells that mi- grated to the underside of the filter were fixed in methanol and stained using 0.1 % crystal violet. Whole filters were manually counted under the inverted microscope. A 249-bp-long MAFB 3’UTR fragment was cloned into downstream region of the luciferase gene within the pGL3 vector (Promega), namely the wild-type 3’UTR (WT-UTR). The mutated MAFB 3’UTR containing mutations of the miR-223 binding sit was obtained using GeneTailor Site-Directed Mutagenesis System (Invitrogen), namely mutated 3’UTR (mt-UTR). For dual luciferase reporter assays, WT-UTR or mt-UTR recombinant vector (0.5 μg per well) was cotransfected with the normalizer vector pRL-CMV (0.5 μg per well per well) coding for Renilla luciferase (Promega) using Lipofectamin 2000™(Invitrogen). The CNE-2 cells were cultured in 24 wells plates with the density of 5 × 105 per well. Dual-Luciferase® Reporter 1000 Assay System (Promega) was used to detect the luciferase activity at 36 h after transfection. Wound healing assay 5 The cells (2 × 105) were seeded into a 6-well plate. Then the cells were scratched using a 100 μl tip when the cells formed a confluent monolayer. The closure of scratch was detected under the microscope at 0, 24, 48, 72 h time point respectively after incubation. Total RNA was enriched from all plasma samples using the TRIzol LS (Invitrogen). To normalize the different RNA samples, 10 μl of synthetic cel-miR-39 (1.6 × 108 copies/μl) was added to each sample during the RNA iso- lation. The reverse transcription was conducted using the TaqMan miRNA Reverse Transcription Kit (Life Tech- nologies), according to the manufacturer’s protocol. RT- qPCR was carried out as described above. Data were analyzed under the SDS Relative Quantification Software version 1.4 (Applied BioSystems). Colony formation assay Two experiments were employed for measuring colony formation ability. For plate colony formation, at 24 h after transfection, 600 cells were plated for 10 days. The colonies were photographed and counted. For soft agar Table 1 Genes and primers for RT-qPCR Gene Primer name Sequence (from 5’ to 3’) U6 Reverse transcription CGCTTCACGAATTTGCGTGTCAT Sense GCTTCGGCAGCACATATACTAAAA Anti sense GCTTCGGCAGCACATATACTAAAAT miR-223 Reverse transcription GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACTGGGGT Sense GGGTGTCAGTTTGTCAAAT Anti sense TGCGTGTCGTGGAGTC GAPDH Sense GCACCGTCAAGGCTGAGAAC Anti sense TGGTGAAGACGCCAGTGGA MAFB Sense TTGTAACCAGAATCACCCTGAGGTC Anti sense CCAGGGTCAGGGATGGCTAA Table 1 Genes and primers for RT-qPCR Table 1 Genes and primers for RT-qPCR Page 3 of 12 Yang et al. BMC Cancer (2015) 15:461 Patient plasma collection In vivo antitumor assay Seven nude athymic mice (male, SPF grade, 6–8 weeks of age) were purchased from Animal Experimental Center of Guangdong Medical College. CNE-2 cells transfected with miR-223 mimic and control microRNA (2 × 106) or MAFB specific siRNA and negative control siRNA in 100 μl no serum medium were injected respectively into particular side of each mouse. Tumor size was measured every three day and tumor volume was calculated as V = ab2/2, where a is length and b is width of tumor. At day 23 after cell in- jection, the animals were sacrificed and the tumors were frozen quickly for following assays. Tissue sections were fixed in 4 % paraformaldehyde and embedded in paraffin. Three micrometer tissue sections were prepared and stained with hematoxylin-eosin. All of the animal experi- ments were approved by the Experimental Animal Care and Use Committee in Guangdong Medical College. MAFB specific siRNA transfection MAFB siRNA and negative control were synthesized by RiboBio (Guangzhou, China). The transfection was per- formed using Lipofectamin 2000™(Invitrogen). Then the cells were harvested 24 h after transfection, and the MAFB expression level was measured by RT-qPCR as described above. MiR-223 expression is significantly lower in CNE samples than control samples MiR-223 expression is significantly lower in CNE samples than control samples MiRNA expression profiling was conducted with a miRNA microarray using RNAs isolated from human nasopha- ryngeal carcinoma cell line CNE-1, CNE-2 and human immortalized nasopharyngeal epithelial cell line NP69. A total of 754 miRNAs were detected, in which 25 were in- creased and 8 were decreased more than 5 folds in NPC cells, compared with NP69 cells (Fig. 1a). MiR-223, which was recently identified to be related with migration and invasion of malignant tumors, was lower expressed in NPC cell lines CNE-1 and CNE-2. We employed RT- qPCR to validate the microarray results and a consistent result was observed (P < 0.05; Fig. 1b). The expression level of miR-223 was also down-regulated in another two NPC cell lines HONE-1 and SUNE-1 (data no shown). These results encouraged us to check the expression level of miR-223 in clinical specimens. We found that the average miR-223 level was significantly lower in the plasma of 10 NPC patients than that from 10 normal healthy subjects. Western blotting The total cell lysates were harvested and the total pro- tein was separated on SDS-PAGE gel by Bio-rad system. The anti-MAFB and anti-GAPDH antibodies were used as the primary antibody (Abcam), with GAPDH as a normalizer. The signal intensity was detected with the enhanced chemiluminescence substrate kit (Thermo). cDNA microarray and confirmation cDNA microarray and confirmation cDNA microarray analysis was performed on Human Genome U133 Plus 2.0 gene chips by Capital Bio Cor- poration (Beijing, China). The microarray results were confirmed by RT-qPCR, with housekeeping gene GAPDH as a normalizer. Page 4 of 12 Yang et al. BMC Cancer (2015) 15:461 MiR-223 inhibits NPC cell migration and invasion The proliferation inhibition of miR-223 in CNE-2 cells prompted us to examine the effect of miR-223 on the migration and invasion of NPC cells. By wound healing assay, we found that exogenous miR-223 inhibited CNE- 2 cell migration (Fig. 3a). Consistently, the results of in vitro transwell migration and Matrigel invasion assay showed that exogenous miR-223 inhibited CNE-2 cells MAFB is identified as a direct target of miR-223 The miR-223 expression level in CNE-1, CNE-2 and NP69 cell lines was measured by RT-qPCR. Compared Fig. 2 Exogenous miR-223 suppresses cell proliferation and colony formation in vitro. a. RT-qPCR result confirmed that the miR-223 expression was significantly higher as compared with untreated and control group after exogenous miR-223 transfection in CNE-2 (P < 0.05). b. MTT cell viability was assayed one time a day from day 1 to day 7 after the transfection of either miR-223 mimic or the negative control in CNE-2 cells. c. After the transfection with miR-223 mimic or the negative control in CNE-2 cells and the 10 days incubation on plates, the colony formation assay was performed. The colony formation rate, clone number and total clone size were calculated respectively. d. After the transfection with miR-223 mimics or the negative control in CNE-2 cells and the 21 days incubation on soft agar, the colony formation assay was performed. Colonies were counted in 10 randomly chosen microscope fields and the colony formation rate, clone number and total clone size was calculated. We performed at least three independent experiments. The data were showed as means ± SD. indicates P < 0.05 compared with control group Fig. 2 Exogenous miR-223 suppresses cell proliferation and colony formation in vitro. a. RT-qPCR result confirmed that the miR-223 expression was significantly higher as compared with untreated and control group after exogenous miR-223 transfection in CNE-2 (P < 0.05). b. MTT cell viability was assayed one time a day from day 1 to day 7 after the transfection of either miR-223 mimic or the negative control in CNE-2 cells. c. After the transfection with miR-223 mimic or the negative control in CNE-2 cells and the 10 days incubation on plates, the colony formation assay was performed. The colony formation rate, clone number and total clone size were calculated respectively. d. Statistical analysis Data were expressed as means ± standard deviation (SD), and the the analysis between groups was performed by Student’s t-test unless otherwise noted. Difference with a P-value less than 0.05 was considered significant. Fig. 1 MiR-223 is down-regulated in human NPC cell lines and patients. a. The miRNA microarray analysis revealed that there were 33 differentially expressed miRNAs between CNE-1, CNE-2 and NP69 (changed more than 5 folds). b. RT-qPCR confirmed that the miR-223 expression was significantly lower in CNE-1 and CNE-2 than that in NP69 (P < 0.05). The miR-223 expression was analyzed with U6 as a normalizer. The data were showed as means ± SD from three independent experiments. c. The miR-223 expression was significantly decreased in NPC patients’ plasma. MiRNA abundance was normalized to cell-miR-39. The data were showed as means ± SD (n = 10) Fig. 1 MiR-223 is down-regulated in human NPC cell lines and patients. a. The miRNA microarray analysis revealed that there were 33 differentially expressed miRNAs between CNE-1, CNE-2 and NP69 (changed more than 5 folds). b. RT-qPCR confirmed that the miR-223 expression was significantly lower in CNE-1 and CNE-2 than that in NP69 (P < 0.05). The miR-223 expression was analyzed with U6 as a normalizer. The data were showed as means ± SD from three independent experiments. c. The miR-223 expression was significantly decreased in NPC patients’ plasma. MiRNA abundance was normalized to cell-miR-39. The data were showed as means ± SD (n = 10) Yang et al. BMC Cancer (2015) 15:461 Page 5 of 12 MiR-223 suppresses tumor growth in vivo MiR 223 suppresses tumor growth in vivo As exogenous miR-223 suppresses cell proliferation of NPC cells in vitro, we examined the effect of miR-223 in vivo. After injected with CNE-2 cells that were trans- fected with miR-223 mimic or control microRNA re- spectively, we found that tumors grew at a slower rate and had smaller sizes (Fig. 4a-b). The total tumor size for negative control and miR-223 mimic transfected groups was 995.37 ± 674.63 and 598.38 ± 613.23 mm3 respectively (Fig. 4c). Morphological similarity of the xeno- graft to the human NPC was evident on paraffin-embedded sections stained with hematoxylin-eosin (Fig. 4d). These results indicated that exogenous miR-223 suppresses tumor growth in vivo. Exogenous miR-223 inhibits cell proliferation and colony formation in vitro Exogenous miR-223 inhibits cell proliferation and colony formation in vitro migration and invasion (Fig. 3b and c). These results demonstrated that miR-223 inhibits the migration and invasion of NPC cells in vitro. In vitro, the cells transfected with miR-223 mimics showed a higher miR-223 expression level (Fig. 2a). The potential role of miR-223 mimic in cell proliferation and colony formation was observed. As a result, the growth rate was significantly decreased in CNE-2 cells due to exogenous miR-223 (Fig. 2b). This observation suggested that miR-223 may inhibit the proliferation of NPC cells. This notion was further supported by the colony forma- tion assay. As shown in Fig. 2c and d, both plate and soft agar colony formation abilities were suppressed by ex- ogenous miR-223 in CNE-2 cells. MiR-223 inhibits NPC cell migration and invasion After the transfection with miR-223 mimics or the negative control in CNE-2 cells and the 21 days incubation on soft agar, the colony formation assay was performed. Colonies were counted in 10 randomly chosen microscope fields and the colony formation rate, clone number and total clone size was calculated. We performed at least three independent experiments. The data were showed as means ± SD. indicates P < 0.05 compared with control group Page 6 of 12 Yang et al. BMC Cancer (2015) 15:461 Fig. 3 Exogenous miR-223 suppresses cell migration and invasion in vitro. a. The function of exogenous miR-223 on CNE-2 cells migration. CNE-2 cells were cultured in 6-well plates. After the wound was made, images were captured at 0 h, 24 h, 48 h and 72 h time point respectively. b. The effect of exogenous miR-223 on CNE-2 migration through Membrane Transwell assay. c. The effect of miR-223 on CNE-2 invasion by Matrigel invasion assay. The experiments were conducted at least 3 times. The data were showed as means ± SD. * indicates P < 0.05 compared with control group Fig. 3 Exogenous miR-223 suppresses cell migration and invasion in vitro. a. The function of exogenous miR-223 on CNE-2 cells migration. CNE-2 cells were cultured in 6-well plates. After the wound was made, images were captured at 0 h, 24 h, 48 h and 72 h time point respectively. b. The effect of exogenous miR-223 on CNE-2 migration through Membrane Transwell assay. c. The effect of miR-223 on CNE-2 invasion by Matrigel invasion assay. The experiments were conducted at least 3 times. The data were showed as means ± SD. * indicates P < 0.05 compared with control group through RT-qPCR and Western blotting. We obtained the consistent result that miR-223 suppressed MAFB gene expression in CNE-2 cells (Fig. 5d). Therefore, we focused on MAFB and observed its role on CNE-2 cell biology function. Combined bioinformatics results (Fig. 5e) with the dual luciferase reporter assay (Fig. 5f), we identi- fied MAFB as one of miR-223 target genes for further investigation. with NP69, miR-223 expression was significantly lower in NPC cell line CNE-1 and CNE-2 (Fig. 5a) that was consistent with the miRNA microarray results (Fig. 1a). The MAFB gene expression in CNE-1, CNE-2 and NP69 was also checked through RT-qPCR and Western blot- ting. As shown in Fig. MiR-223 inhibits NPC cell migration and invasion * and ** indicate P < 0.05 and P < 0.01 as compared with control group Fig. 4 Exogenous miR-223 attenuates nasopharyngeal tumor growth in mouse xenograft models. a. The appearance of xenograft subcutaneous NPC in miR-223 mimics transfection and control groups. b. Growth curves of subcutaneous NPC in nude mice with miR-223 mimics transfection revealed that tumor growth significantly slowed down compared with control group. c. Total tumor weight of miR-223 mimic transfection and control groups. d. HE staining result of the tumor formed in nude mice using, all of them were typical nasopharyngeal squamous cell carcinoma. The data were showed as means ± SD (n = 7). * and ** indicate P < 0.05 and P < 0.01 as compared with control group that MAFB specific siRNA could inhibit CNE-2 cell migration (Fig. 6c). The inhibition activity of MAFB spe- cific siRNA in CNE-2 cells proliferation and colony for- mation was observed. Consistently, results from in vitro transwell migration and Matrigel invasion assay showed that MAFB specific siRNA inhibited CNE-2 cell migra- tion and invasion (Fig. 6d and e). As a result, down- regulation of MAFB could significantly decrease the growth rate of CNE-2 cells (Fig. 6f). These results indi- cated that miR-223 inhibits the growth and migration of NPC cells through reducing MAFB gene expression. siRNA transfected group, respectively (Fig. 7b and c; * p < 0.05). Morphological similarity of the xenograft to the hu- man NPC was evident on paraffin-embedded sections stained with hematoxylin-eosin (Fig. 7D). These results in- dicated that exogenous MAFB specific siRNA suppresses tumor growth in vivo. MiR-223 inhibits NPC cell migration and invasion 5b, MAFB gene expression was significantly higher in CNE-1, CNE-2 cells compared with NP69. To investigate the mechanism of miR-223 inhibition NPC cells, we performed a cDNA expression microarray analysis. After 24 h transfection of miR-223 mimic and negative control in CNE-2, total RNA was isolated and hybridized to an Affymetrix GeneChip Hu- man Genome U133 Plus 2.0 Array. We found that 39 transcripts were differentially expressed between two groups (Fig. 5c). The microarray results for miR-223 down-regulated MAFB gene expression were verified with NP69, miR-223 expression was significantly lower in NPC cell line CNE-1 and CNE-2 (Fig. 5a) that was consistent with the miRNA microarray results (Fig. 1a). The MAFB gene expression in CNE-1, CNE-2 and NP69 was also checked through RT-qPCR and Western blot- ting. As shown in Fig. 5b, MAFB gene expression was significantly higher in CNE-1, CNE-2 cells compared with NP69. To investigate the mechanism of miR-223 inhibition NPC cells, we performed a cDNA expression microarray analysis. After 24 h transfection of miR-223 mimic and negative control in CNE-2, total RNA was isolated and hybridized to an Affymetrix GeneChip Hu- man Genome U133 Plus 2.0 Array. We found that 39 transcripts were differentially expressed between two groups (Fig. 5c). The microarray results for miR-223 down-regulated MAFB gene expression were verified MAFB siRNA inhibits CNE-2 cells growth and migration The inhibition activity for MAFB specific siRNA on CNE-2 MAFB gene expression was confirmed by RT- qPCR and Western blotting. MAFB specific siRNA could attenuate CNE-2 cells MAFB gene expression for 28 % in mRNA level and 58 % in protein level respect- ively (Fig. 6a and b). By wound healing assay, we found Page 7 of 12 Page 7 of 12 Yang et al. BMC Cancer (2015) 15:461 Fig. 4 Exogenous miR-223 attenuates nasopharyngeal tumor growth in mouse xenograft models. a. The appearance of xenograft subcutaneous NPC in miR-223 mimics transfection and control groups. b. Growth curves of subcutaneous NPC in nude mice with miR-223 mimics transfection revealed that tumor growth significantly slowed down compared with control group. c. Total tumor weight of miR-223 mimic transfection and control groups. d. HE staining result of the tumor formed in nude mice using, all of them were typical nasopharyngeal squamous cell carcinoma. The data were showed as means ± SD (n = 7). Discussion To sum up, we report here that miR-223 plays a critical role in NPC carcinogenesis. The plasma miR-223 level was lower in NPC cells and NPC patients’ plasma, as compared with normal ones. To fully realize its effect, we observed that miR-223 inhibited NPC cell growth, migration and invasion both in vitro and in vivo. MAFB was identified as a miR-223 target gene. And MAFB ex- pression was decreased accompanied with miR-223 over- expression. In addition, the consistent result was observed that the cell growth and migration were inhibited after MAFB specific siRNA transfected into NPC cells. The findings implicated that miR-223 could be a potential tar- get for intervention and diagnosis to NPC. MAFB siRNA inhibits the growth of NPC xenograft tumors CNE-2 cells were transfected respectively with MAFB specific siRNA and negative control siRNA and then the transfected cells were injected subcutaneously into the dif- ferent dorsal flank of nude mice. Compared with the negative control side, the tumors of MAFB siRNA side grew more slowly, with the smaller size. In all 6 mice for MAFB specific group, 4 mice could be seen tumors growth after 23 days while there was tumor growth in all negative control group (Fig. 7a). The total tumor weight and size was 2236.80 ± 170.17 mg and 2428.79.80 ± 217.27 mm3 for negative control group and 729.80 ± 178.83 mg and 1029.41 ± 207.85 mm3 for MAFB specific NPC is a highly prevalent malignant cancer in south China and Southeast countries. It has been known that virus, diet and genetic makeups all are the risk factor for NPC development, but underlying pathogenesis of the Yang et al. BMC Cancer (2015) 15:461 Page 8 of 12 Fig. 5 MAFB is a direct target gene of miR-223. a. The RT-qPCR confirmed that the miR-223 expression was significantly lower in CNE-1 and CNE-2 than that in NP69 (P < 0.05). b. MAFB gene expression level in NP69 and NPC cell line CNE-1 and CNE-2 was measured by RT-qPCR and western blot. c. The cDNA microarray analysis revealed that there were 39 differentially expressed genes between the CNE-2 transfected with miR-223 mimics and the CNE-2 transfected with negative control. d. RT-qPCR confirmed that the gene expression of MAFB was inhibited by miR-223 overexpression. The inhibition of MAFB gene expression on protein level through miR-223 mimic administration was confirmed by Western blotting. e. Discussion Schematic diagram of MAFB 3’ UTR-containing reporter gene constructs. f. Dual luciferase reporter assay in CNE-2 co-transfected with WT-UTR or mt-UTR recombinant vector (0.5 μg) and miR-223 (50 nM) as indicated. The experiment was performed at least 3 times. The data were showed as means ± SD. indicates P < 0.05 compared with control group Fig. 5 MAFB is a direct target gene of miR-223. a. The RT-qPCR confirmed that the miR-223 expression was significantly lower in CNE-1 and CNE-2 than that in NP69 (P < 0.05). b. MAFB gene expression level in NP69 and NPC cell line CNE-1 and CNE-2 was measured by RT-qPCR and western blot. c. The cDNA microarray analysis revealed that there were 39 differentially expressed genes between the CNE-2 transfected with miR-223 mimics and the CNE-2 transfected with negative control. d. RT-qPCR confirmed that the gene expression of MAFB was inhibited by miR-223 overexpression. The inhibition of MAFB gene expression on protein level through miR-223 mimic administration was confirmed by Western blotting. e. Schematic diagram of MAFB 3’ UTR-containing reporter gene constructs. f. Dual luciferase reporter assay in CNE-2 co-transfected with WT-UTR or mt-UTR recombinant vector (0.5 μg) and miR-223 (50 nM) as indicated. The experiment was performed at least 3 times. The data were showed as means ± SD. indicates P < 0.05 compared with control group disease is still poorly understood [14]. Oncogenes and tumor suppressing genes might form a very complicated network, and how they interact one another to lead to the cancer is not clear [15]. Few reports have indicated that miRNAs abnormally express in different cancers, and more researchers have been paying close attentions to molecular mechanisms of miRNAs in tumorigenesis [16]. In NPC, down-expressed miR-29c could upregulate mRNA expression of extracellular matrix genes [17], and ZEB2 and CTNNB1 low-expression induced by miR- 200a inhibits the growth, migration and invasion of NPC cells [18]. In addition, miR-216b suppresses the prolifer- ation and invasion via inhibiting KRAS expression [19]. Mir-218 exerts suppressing function to NPC by down- regulating survivin expression and SLIT2-BOBO1 path- way [20], and miR-26a acts the similar function for inhibiting cell growth and tumorigenesis through repres- sion of EZH2miR [21]. Recent study reported that miR-663 targets p21 (WAF1/CIP1) to promote the proliferation and development of NPC [22]. Discussion MTT cell viability was assayed once per day from day 1 to day 6 after the transfection of MAFB specific siRNA or the negative control in CNE-2 cells. The experiments were performed at least 3 times. The data were showed as means ± SD. * indicates P < 0.05 compared with control group by comparison the miRNAs expression between NPC and immortalized human nasopharyngeal epithelial cell line NP69. Exogenously up-regulating miR-223 could suppress cell proliferation and colony formation, increase tumor formation in nude mice (Fig. 2 and 4). The results are consistent with the discovery in hepatic carcinoma that the inhibition of miR-223 accompanies the enhancement of Stathmin1 expression [24]. In hematopoietic cells, miR- 223 can negatively post-transcript regulate the expression of LMO2 and CEBP-β to reduce cell proliferation [25]. MiR-223 up-regulation in Hela cells inhibits cell prolifera- tion by targeting IGF-1R [9]. However, in the gastric cancer development, miR-223 acts as an oncogene to pro- mote cell invasion and migration via affecting expressions of EPB41L3 and FBXW7/hCdc4 genes [10, 26, 27]. In addition, miR-223 has been report to regulate the prolifer- ation and invasion of human breast cancer cells for target- ing Caprin-1 [28]. On the other hand, there has been another report suggesting that miR-223 functions as a po- tent tumor suppressor of the Lewis lung carcinoma cell by comparison the miRNAs expression between NPC and immortalized human nasopharyngeal epithelial cell line NP69. Exogenously up-regulating miR-223 could suppress cell proliferation and colony formation, increase tumor formation in nude mice (Fig. 2 and 4). The results are consistent with the discovery in hepatic carcinoma that the inhibition of miR-223 accompanies the enhancement of Stathmin1 expression [24]. In hematopoietic cells, miR- 223 can negatively post-transcript regulate the expression of LMO2 and CEBP-β to reduce cell proliferation [25]. MiR-223 up-regulation in Hela cells inhibits cell prolifera- tion by targeting IGF-1R [9]. However, in the gastric cancer development, miR-223 acts as an oncogene to pro- mote cell invasion and migration via affecting expressions of EPB41L3 and FBXW7/hCdc4 genes [10, 26, 27]. In addition, miR-223 has been report to regulate the prolifer- ation and invasion of human breast cancer cells for target- ing Caprin-1 [28]. On the other hand, there has been another report suggesting that miR-223 functions as a po- tent tumor suppressor of the Lewis lung carcinoma cell line by targeting insulin-like growth factor-1 [29]. Discussion Those results have shed light on which miRNAs are involved in NPC tumorigen- esis, however, the role of miRNAs in pathogenesis has not been elucidated. Based on miRNA microarray we found that miR-223 was down-regulated in NPC cell line CNE-1 and CNE-2, and further verified the role of miR-223 in NPC patho- genesis. Using microarray-based serum miRNA profiling and RT-qPCR method, Zeng et. al. compared serum miRNAs level between the patients with nasopharyngeal carcinoma and non-cancerous volunteers, and they found that miR-223 was decreased significantly in the serum of NPC patients [23]. Our results confirmed this Page 9 of 12 Page 9 of 12 Yang et al. BMC Cancer (2015) 15:461 Fig. 6 Inhibition of MAFB gene expression suppresses cell migration, invasion and proliferation in vitro. a. RT-qPCR confirmed that the gene expression of MAFB was inhibited by MAFB specific siRNA. b. Western blotting shown the inhibition of MAFB gene expression on protein level through MAFB specific siRNA administration. c. The effect of MAFB-siRNA on CNE-2 migration. CNE-2 cells were cultured in 6-well plates. Images were captured at 0 h, 24 h, 48 h and 72 h respectively, after the wound was made. d. Effect of MAFB specific siRNA on CNE-2 cells migration through Membrane Transwell assay. e. Effect of MAFB specific siRNA on CNE-2 cell invasion by Matrigel invasion assay. f. MTT cell viability was assayed once per day from day 1 to day 6 after the transfection of MAFB specific siRNA or the negative control in CNE-2 cells. The experiments were performed at least 3 times. The data were showed as means ± SD. * indicates P < 0.05 compared with control group Fig. 6 Inhibition of MAFB gene expression suppresses cell migration, invasion and proliferation in vitro. a. RT-qPCR confirmed that the gene expression of MAFB was inhibited by MAFB specific siRNA. b. Western blotting shown the inhibition of MAFB gene expression on protein level through MAFB specific siRNA administration. c. The effect of MAFB-siRNA on CNE-2 migration. CNE-2 cells were cultured in 6-well plates. Images were captured at 0 h, 24 h, 48 h and 72 h respectively, after the wound was made. d. Effect of MAFB specific siRNA on CNE-2 cells migration through Membrane Transwell assay. e. Effect of MAFB specific siRNA on CNE-2 cell invasion by Matrigel invasion assay. f. Discussion 7 Inhibition of MAFB gene expression attenuates nasopharyngeal tumor growth in mouse xenograft models. a. The appearance of xenograft subcutaneous NPC in MAFB specific siRNA transfection and control groups. b. Total tumor weight of MAFB specific siRNA transfection and control groups. c. Total tumor size of MAFB specific siRNA transfection and control groups. d. HE staining result of the tumor formed in nude mice using, all of them were typical nasopharyngeal squamous cell carcinoma. The data were showed as means ± SD (n = 6). * indicate P < 0.05 as compared with control group Fig. 7 Inhibition of MAFB gene expression attenuates nasopharyngeal tumor growth in mouse xenograft models. a. The appearance of xenograft subcutaneous NPC in MAFB specific siRNA transfection and control groups. b. Total tumor weight of MAFB specific siRNA transfection and control groups. c. Total tumor size of MAFB specific siRNA transfection and control groups. d. HE staining result of the tumor formed in nude mice using, all of them were typical nasopharyngeal squamous cell carcinoma. The data were showed as means ± SD (n = 6). * indicate P < 0.05 as compared with control group hepatocarcinoma patients, suggesting that miR-122 is likely as a novel biomarker for screening the patients [34]. A plasma miRNA panel is discovered for early diagnosis of hepatocarcinoma, and used in clinic to significantly re- duce the frequency that optimal treatment window is missing [34]. Osteosarcoma development is associated with gene expression regulated by MiRNAs [4]. MiR-223/ Ect2/p21 signaling regulates osteosarcoma cell cycle pro- gression and proliferation [35]. A study also reported that miR-421 can be used as a biomarker to detect circulated tumor cells of gastric cancer patients [5]. In addition, many miRNAs have been discovered as a potential target for diagnosis and treatment of human diseases. In the serum from non-small lung cancer patients, miR-146b, miR-221, let-7a, miR-155, miR-17-5p, miR-27a and miR- 106a are significantly reduced but miR-29c increased [36]. The above-mentioned reports with our study indicate that miR-223 could be a serum biomarker of NPC patients to give early diagnosis for optimal treatment. MAFB, a member of Maf protein family, is a transcrip- tion factor with basic-leucine zipper structure and plays an important role in early tissue specification and ter- minal differentiation [37]. In multiple myeloma, MAFB can make hematopoietic stem/progenitor cells repro- gramed into malignant plasma cells [38]. Discussion In addition, MAFB is expected to be highly sensitive and very spe- cific biomarker for poor prognosis of multiple myeloma patients [39]. Further studies need to illustrate the role of MAFB in NPC carcinogenesis. Discussion Micro- RNA 223 is up-regulated in the multistep progression of Barrett’s esophagus and modulates sensitivity to chemotherapy by targeting PARP1 [30], it also modu- lates multidrug resistance via downregulation of ABCB1 in hepatocellular carcinoma cells [31]. In our study, we demonstrated that exogenous miR-223 inhibits CNE-2 cell migration and invasion (Fig. 3). The discrepant results of miR-223 role seemed to indicate that miR-223 regulates different gene expression to play different role in various cancers. In the normal cells, miRNAs maintain the homeostasis of various life processes including proliferation, differen- tiation and apoptosis. During the tumorigenesis, deregu- lation of miRNAs can lead to various consequences since various miRNAs bind with many mRNAs to regulate gene expression [32]. Biological approaches are commonly used to verify the relationship between miRNAs and tumor phenotypes or to validate the possibility as a drug target [33]. MiR-122 is significantly higher in the serum of Yang et al. BMC Cancer (2015) 15:461 Page 10 of 12 Fig. 7 Inhibition of MAFB gene expression attenuates nasopharyngeal tumor growth in mouse xenograft models. a. The appearance of xenograft subcutaneous NPC in MAFB specific siRNA transfection and control groups. b. Total tumor weight of MAFB specific siRNA transfection and control groups. c. Total tumor size of MAFB specific siRNA transfection and control groups. d. HE staining result of the tumor formed in nude mice using, all of them were typical nasopharyngeal squamous cell carcinoma. The data were showed as means ± SD (n = 6). * indicate P < 0.05 as compared with control group mouse xenograft models. a. The appearance of xenograft mor weight of MAFB specific siRNA transfection and control HE staining result of the tumor formed in nude mice using, wed as means ± SD (n = 6). * indicate P < 0.05 as compared Fig. 7 Inhibition of MAFB gene expression attenuates nasopharyngeal tumor growth in mouse xenograft models. a. The appearance of xenograft subcutaneous NPC in MAFB specific siRNA transfection and control groups. b. Total tumor weight of MAFB specific siRNA transfection and control groups. c. Total tumor size of MAFB specific siRNA transfection and control groups. d. HE staining result of the tumor formed in nude mice using, all of them were typical nasopharyngeal squamous cell carcinoma. The data were showed as means ± SD (n = 6). * indicate P < 0.05 as compared with control group Fig. Competing interests 16. Wuchty S, Arjona D, Bozdag S, Bauer PO. Involvement of microRNA families in cancer. Nucleic Acids Res. 2012;40(17):8219–26. p g The authors declare that they have no competing interests. 17. Sengupta S, den Boon JA, Chen IH, Newton MA, Stanhope SA, Cheng YJ, et al. MicroRNA 29c is down-regulated in nasopharyngeal carcinomas, up-regulating mRNAs encoding extracellular matrix proteins. Proc Natl Acad Sci U S A. 2008;105(15):5874–8. Conclusion In this study, we found the reduction of miR-223 expres- sion both in NPC cells and in NPC patients’ plasma. After miR-223 mimic and negative control microRNA transfected, we found that 39 transcripts were differen- tially expressed between two groups. Those transcripts have not been reported as targets for miR-223. We also used Target Scan and microRNA.org software to predict Page 11 of 12 Yang et al. BMC Cancer (2015) 15:461 Page 11 of 12 Yang et al. BMC Cancer (2015) 15:461 the potential targets for miR-223 and combined the results to select MAFB for further investigation. MiR-223-mimic administration could lead to the suppression of MAFB ex- pression both in RNA and protein levels. In addition, both of miR-223-mimic administration and reduction of MAFB expression all lead to the inhibition of cell growth and migration. These findings demonstrated that miR-223 reg- ulates the NPC development by targeting MAFB expres- sion, and implicates miR-223 as a candidate target for NPC diagnosis and treatment. As miR-223 maybe also tar- gets other genes in nasopharyngeal carcinoma, further analyses are needed to elucidate the full spectrum of miR- 223 functions. 10. Li T, Chen JX, Fu XP, Yang S, Zhang Z, Chen Kh H, et al. microRNA expression profiling of nasopharyngeal carcinoma. Oncol Rep. 2011;25(5):1353–63. 11. Sizhong Z, Xiukung G, Yi Z. Cytogenetic studies on an epithelial cell line derived from poorly differentiated nasopharyngeal carcinoma. Int J Cancer. 1983;31(5):587–90. 12. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell. 2005;120(1):15–20. 13. Betel D, Wilson M, Gabow A, Marks DS, Sander C. The microRNA.org resource: targets and expression. Nucleic Acids Res. 2008;36:D149–153. Database issue. 14. Yoshizaki T, Ito M, Murono S, Wakisaka N, Kondo S, Endo K. Current understanding and management of nasopharyngeal carcinoma. Auris Nasus Larynx. 2012;39(2):137–44. 15. Luo Z, Zhang L, Li Z, Li X, Li G, Yu H, et al. An in silico analysis of dynamic changes in microRNA expression profiles in stepwise development of nasopharyngeal carcinoma. BMC Med Genomics. 2012;5(1):3. Received: 17 June 2014 Accepted: 21 May 2015 25. Sun W, Shen W, Yang S, Hu F, Li H, Zhu TH. miR-223 and miR-142 attenuate hematopoietic cell proliferation, and miR-223 positively regulates miR-142 through LMO2 isoforms and CEBP-beta. Cell Res. 2010;20(10):1158–69. Acknowledgements The authors should express gratitude to Professor Yi Zeng from the Institute of Virology, China Institute of Preventive Medical Science, for kindly providing the human NPC CNE-2 cell line. This study was supported by the Dongguan Higher Education Science and Technology Program (2011105102016 and 2012105102005) and foundation for special talent introduction of Guangdong province high education. The authors should express gratitude to Professor Yi Zeng from the Institute of Virology, China Institute of Preventive Medical Science, for kindly providing the human NPC CNE-2 cell line. This study was supported by the 20. Alajez NM, Lenarduzzi M, Ito E, Hui AB, Shi W, Bruce J, et al. MiR-218 suppresses nasopharyngeal cancer progression through downregulation of survivin and the SLIT2-ROBO1 pathway. Cancer Res. 2011;71(6):2381–91. p g y pp y Dongguan Higher Education Science and Technology Program (2011105102016 d 2012105102005) d f d i f i l l (2011105102016 and 2012105102005) and foundation for special talent introduction of Guangdong province high education. (2011105102016 and 2012105102005) and foundation for special talent introduction of Guangdong province high education. 21. Lu J, He ML, Wang L, Chen Y, Liu X, Dong Q, et al. MiR-26a inhibits cell growth and tumorigenesis of nasopharyngeal carcinoma through repression of EZH2. Cancer Res. 2011;71(1):225–33. 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