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https://openalex.org/W4312115059	https://www.researchsquare.com/article/rs-1524248/latest.pdf	English	null	Effect of the COVID-19 outbreak on emergency transport of children by an emergency medical service system: a population-based, ORION registry study	BMC emergency medicine	2,022	cc-by	6,590	Effect of the COVID-19 outbreak on emergency transport of children by an emergency medical service system: A population-based, ORION registry study Effect of the COVID-19 outbreak on emergenc transport of children by an emergency medica service system: A population-based, ORION re study Koshi Ota (  koshi.ota@ompu.ac.jp ) Osaka Medical and Pharmaceutical University Daisuke Nishioka Osaka Medical and Pharmaceutical University Yusuke Katayama The Working Group to analyze the emergency medical care system in Osaka Prefecture Tetsuhisa Kitamura The Working Group to analyze the emergency medical care system in Osaka Prefecture Jun Masui Osaka Prefectural Government Kanna Ota Osaka Medical and Pharmaceutical University Masahiko Nitta Osaka Medical and Pharmaceutical University Tetsuya Matsuoka The Working Group to analyze the emergency medical care system in Osaka Prefecture Akira Takasu Osaka Medical and Pharmaceutical University y Koshi Ota (  koshi.ota@ompu.ac.jp ) Osaka Medical and Pharmaceutical University Daisuke Nishioka Osaka Medical and Pharmaceutical University Yusuke Katayama The Working Group to analyze the emergency medical care system in Osaka Prefecture Tetsuhisa Kitamura The Working Group to analyze the emergency medical care system in Osaka Prefecture Jun Masui Osaka Prefectural Government Kanna Ota Osaka Medical and Pharmaceutical University Masahiko Nitta Osaka Medical and Pharmaceutical University Tetsuya Matsuoka The Working Group to analyze the emergency medical care system in Osaka Prefecture Akira Takasu Osaka Medical and Pharmaceutical University Research Article Keywords: COVID-19, children, difficult-to-transfer cases, pandemic Posted Date: May 17th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1524248/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International Research Article Page 1/25 Abstract Background: Coronavirus disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2, has spread rapidly around the world. Objective: To assess the effect of the COVID-19 pandemic on the emergency medical service (EMS) and hospital admission course for children transported by ambulance. Methods: This study was a retrospective, descriptive study with a study period from January 1, 2018 to December 31, 2020 using the Osaka Emergency Information Research Intelligent Operation Network (ORION) system. All children who were transported by ambulance in Osaka Prefecture were included. The main outcome of this study was the rate of difficult-to-transfer cases, which was calculated by univariate and multivariate Poisson regression analyses. Results: Over the 3 years between January 1, 2018 and December 31 2020, 1,436,212 patients were transported to hospitals by ambulances in Osaka Prefecture, with children accounting for 102,473 (37,064, 39,590, and 25,819, in 2018, 2019, and 2020, respectively). Poisson regression analysis showed that children were negatively associated with difficult-to-transfer cases (risk ratio (RR) 0.35, 95% CI 0.33 to 0.37). With reference to 2018, 2020 was not significantly associated with difficult-to-transfer cases in children (RR 1.14, 95% CI 0.99 to 1.32, P=0.075), but was significantly related (RR 1.24, 95% CI 1.21 to 1.27, P<0.001) to difficult-to-transfer cases in the general population. Conclusion: Children were consistently associated with a reduced RR for difficult-to-transfer cases, even in the COVID-19 pandemic in 2020. Study design and setting This was a retrospective, descriptive study with a study period from January 1, 2018 to December 31, 2020 using the Osaka Emergency Information Research Intelligent Operation Network (ORION) system.11 Osaka Prefecture is the largest metropolitan community in western Japan, with a population of about 8.8 million and a total area of 1905 km2. The Osaka Prefecture Government has developed and introduced an information system for emergency patients (the ORION system) that uses a smartphone application for hospital selection by on-scene emergency medical service personnel and has been collecting all ambulance records. Medical institutions have obtained information on the diagnoses and outcomes of patients transported to medical institutions, and the ORION system has merged these data with ambulance records, including smartphone application data, since January 2015. To assess the effect of the COVID-19 pandemic on the EMS system, this study focused on children transported by ambulance in Osaka Prefecture (Fig. 1). The children were divided into four age groups: infant (0 years); toddler (1–4 years); childhood (5–9 years); and adolescent (10–14 years). The presumptive diagnosis and the final diagnosis if the patients were admitted, with the International Classification of Diseases, 10th Revision (ICD-10), were used.12 Data were collected using ICD-10 codes: Infectious and parasitic diseases, A00 to B99; Neoplasms, C00 to D48; Endocrine, nutritional and metabolic diseases, E00 to E90; Mental and behavioral disorders, F00 to F99; Diseases of the nervous system, G00 to G99; Diseases of the eye and the ear, H00 to H95; Diseases of the nervous system, I00 to I99; Diseases of the respiratory system, J00 to J99; Diseases of the digestive system, K00 to K93; Diseases of the skin and subcutaneous tissue, L00 to L99; Diseases of the musculoskeletal system and connective tissue, M00 to M99; Diseases of the genitourinary system, N00 to N99; Congenital diseases, Q00 to Q99; Symptoms, signs and abnormal clinical and laboratory findings, R00 to R99; Injury and poisoning, S00 to T98. ICD-10 codes O00 to O99 and P00 to P96 were excluded because these codes were defined as pregnancy-related cases (pregnant patients). Data for ‘COVID-19’ were collected using ICD-10 code U07.1, and data for ‘COVID-19 suspected’ (if the virus was not identified) were collected using ICD-10 code U07.2. This study excluded patients who were not transported to a hospital. Introduction Coronavirus disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), was identified in China in December 2019, and the COVID-19 outbreak spread rapidly around the world.1 On March 11, 2020, the World Health Organization (WHO) declared COVID-19 a pandemic. Children with COVID-19 tend to have a milder clinical course than adults, and the clinical manifestations in children are heterogeneous, with a wide spectrum of clinical features.2345 The WHO recommends testing all suspected cases, but children with COVID-19 may not meet all the criteria of the suspected case definition.67 A previous study in Osaka city showed a similar proportion of children transported by the emergency medical service (EMS) system to that seen in other countries, about 4–13%.8910 Our previous study showed about a 10% reduction in EMS service use in the overall population, including elderly patients, in 2020, possibly due to the COVID-19 pandemic. However, EMS service studies related to the COVID-19 pandemic and children have been scarce. Page 2/25 Page 2/25 Presently, to the best of our knowledge, no clinical studies of the effect of the COVID-19 outbreak and transport of children by the EMS system have been published. Presently, to the best of our knowledge, no clinical studies of the effect of the COVID-19 outbreak and transport of children by the EMS system have been published. This study aimed to assess the effect of the COVID-19 pandemic on the transport by ambulance of children by the EMS system in Osaka Prefecture. Outcomes The primary outcome of this study was the rate of difficulty obtaining hospital acceptance for transfer of a patient (difficult-to-transfer cases). According to the guidelines of the Fire and Disaster Management Agency of the Ministry of Internal Affairs and Communications, “difficult-to-transfer cases” were defined as those in which the time interval from arrival at the scene to departure from the scene was longer than 30 min, and those in which ambulance crews needed to make four or more phone calls to hospitals before obtaining hospital acceptance. In addition, disposition at the emergency department (ED), such as emergency admission, discharged to home, transfer to other hospital, and death at the ED, was also collected. Study design and setting The ambulance records in Osaka Prefecture are considered administrative records, and the need to obtain informed consent from the participants was waived because the data were anonymous. This study was approved by the Ethics Committee of Osaka Medical and Pharmaceutical University (Takatsuki City, Japan) (reference number 2021-006). The Strengthening the Reporting of Observational studies in Epidemiology (STROBE) guidelines were used to design and report the results of this study 13 Page 3/25 Data analysis The number of patients transported by ambulance due to any cause except interhospital transport was calculated for the year from January 1 to December 31, 2020. Patient demographics were compared among the three years using the χ2 test for categorical variables and the Kruskal-Wallis test for continuous variables. For comparison purposes, the numbers of patients transported by ambulance for the same reasons per year from January 1 to December 31, 2018 and January 1 to December 31, 2019 were also collected. In addition, Poisson regression analysis with a robust standard error estimator was used to calculate the rate of difficulty of hospital acceptance of patients, and then the risk ratio (RR) for difficult-to-transfer cases and the 95% confidence interval (CI) were calculated with 2018 as the reference. Then, the adjusted RR and 95% CI of difficult-to-transfer cases by month, time of transportation, day of week, COVID-19 suspected during transportation, and children were estimated using multivariate analyses. All data were analyzed using SPSS version 25.0 software (IBM Corp., Armonk, NY, USA) or STATA (version 6.1; StataCorp., College Station, TX, USA). All tests were two-tailed, and p-values < 0.05 were considered i ifi All data were analyzed using SPSS version 25.0 software (IBM Corp., Armonk, NY, USA) or STATA (version 16.1; StataCorp., College Station, TX, USA). All tests were two-tailed, and p-values < 0.05 were considered significant. Data collection and quality control Data were uniformly collected using specific data collection forms and included the reason for the ambulance call, location of the accident, the time of day and the day of the week, and tools used, in addition to age, sex, and ICD-10 code. The detailed situation and patient information were recorded in text form. These data were completed by EMS personnel and then transferred to the information center in the Osaka Municipal Fire Department (OMFD). To assure the quality of the data, if the data were incomplete, the data sheet was supposed to be returned to the relevant EMS personnel to correct it. Baseline characteristics Baseline characteristics Baseline characteristics Page 4/25 For 3 years between January 1, 2018 and December31, 2020, 1,436,212 patients were transported to hospitals by ambulances in Osaka Prefecture. Of them, 1,346,457 were enrolled in this study. Excluded were 89,755 patients who were transferred to a different hospital. By year, 462,773 patients in 2018, 468,697 patients in 2019, and 414,987 patients in 2020 were transported to hospitals by ambulances (Table 1), with the total number of children (0–14 years old) being 97,558 (35,314, 37,547, and 24,697, in 2018, 2019, and 2020, respectively, P < 0.001). In addition, the total number of infants was 9,090 (3,290, 3,375, and 2,425, in 2018, 2019, and 2020, respectively). Table 1 shows other baseline characteristics of the pediatric patients transported to hospitals by ambulances in Osaka Prefecture. Figure 1 displays the flow diagram of this study. For 3 years between January 1, 2018 and December31, 2020, 1,436,212 patients were transported to hospitals by ambulances in Osaka Prefecture. Of them, 1,346,457 were enrolled in this study. Excluded were 89,755 patients who were transferred to a different hospital. By year, 462,773 patients in 2018, 468,697 patients in 2019, and 414,987 patients in 2020 were transported to hospitals by ambulances (Table 1), with the total number of children (0–14 years old) being 97,558 (35,314, 37,547, and 24,697, in 2018, 2019, and 2020, respectively, P < 0.001). In addition, the total number of infants was 9,090 (3,290, 3,375, and 2,425, in 2018, 2019, and 2020, respectively). Table 1 shows other baseline characteristics of the pediatric patients transported to hospitals by ambulances in Osaka Prefecture. Figure 1 displays the flow diagram of this study. Baseline characteristics Page 5/25 Table 1 Demographic characteristics of transported patients Year 2018 2019 2020 Total P value Number of Patients 462,773 468,697 414,987 1,346,457 < 0.001 Age, median (IQR) 69.0(38) 70.0(38) 71.0(35) < 0.001 Sex (male), % 234,542 (50.7) 236,661 (50.5) 210,334 (50.7) 681,537 (50.6) 0.111 Age, median (IQR) 67.0 (38) 67.0 (38) 69.0 (34) < 0.001 Children, % 35,314 (7.6) 37,547 (8.0) 24,697 (6.0) 97,558 (7.3) < 0.001 Age, median (IQR) 3.0 (7) 3.0 (7) 4.0 (7) < 0.001 Adult, % 166,402 (36.0) 164,722 (35.1) 143,740 (34.6) 474,864 (35.3) < 0.001 Age, median (IQR) 43.0 (26) 44.0 (26) 45.0 (26) < 0.001 Elderly patients, % 261,057 (56.4) 266,428 (56.8) 246,550 (59.4) 774,035 (57.5) < 0.001 Age, median (IQR) 80.0 (12) 80.0 (12) 80.0 (12) < 0.001 Children age category < 0.001 Infant, % 3,661 (9.9) 3,790 (9.6) 2,641 (10.2) 10,092 (9.9) Toddler, % 17,964 (48.5) 19,803 (50.0) 11,873 (46.0) 49,640 (48.4) Childhood, % 8,508 (23.0) 8,879 (22.4) 6,146 (23.8) 23,533 (23.0) Adolescent, % 6,931 (18.7) 7,118 (18.0) 5,159 (20.0) 19,208 (18.7) Children, 0–14 years; Adults, 15–64 years; Elderly patients, ≥ 65 years Infant, 0 years; Toddler, 1–4 years; Childhood, 5–9 years; Adolescent, 10–14 years Determined by the χ2 test for categorical variables and Kruskal–Wallis test for continuous variables Page 6/25 Year 2018 2019 2020 Total P value Reason for transportation (Children) Infectious and parasitic diseases, % 2,009 (5.8) 2,602 (7.0) 1,065 (4.4) 5,676 (5.9) < 0.001 Neoplasms, % 59 (0.2) 56 (0.2) 57 (0.2) 172 (0.2) 0.052 Endocrine, nutritional, and metabolic diseases, % 253 (0.7) 251 (0.7) 167 (0.7) 671 (0.7) 0.713 Mental and behavioral disorders, % 430 (1.3) 389 (1.1) 267 (1.1) 1,086 (1.1) 0.056 Diseases of the nervous system, % 912 (2.7) 1,018 (2.7) 892 (3.7) 2,822 (2.9) < 0.001 Diseases of the eye and the ear, % 152 (0.4) 164 (0.4) 87 (0.4) 403 (0.4) 0.224 Diseases of the circulatory system, % 200 (0.6) 238 (0.6) 190 (0.7) 662 (0.6) 0.199 Diseases of the respiratory system, % 4,224 (12.3) 4,798 (12.9) 2,252 (9.3) 11,274 (11.8) < 0.001 Diseases of the digestive system, % 858 (2.5) 921 (2.5) 712 (2.9) 2,491 (2.6) 0.001 Diseases of the skin and subcutaneous tissue, % 349 (1.0) 380 (1.0) 229 (0.9) 958 (1.0) 0.57 Diseases of the musculoskeletal system and connective tissue, % 404 (1.2) 458 (1.2) 350 (1.4) 1,212 (1.3) 0.011 Diseases of the genitourinary system, % 130 (0.4) 134 (0.4) 145 (0.6) 409 (0.4) < 0.001 Congenital diseases, % 23 (0.1) 32 (0.1) 23 (0.1) 78 (0.1) 0.441 Symptoms, signs, and abnormal clinical and laboratory findings, % 9,925(28.8) 11,171 (30.1) 6,276 (25.8) 27,372 (28.5) < 0.001 Injury and poisoning, % 14,542 (42.2) 14,536 (39.2) 11,676 (47.9) 40,754 (42.5) < 0.001 Children, 0–14 years; Adults, 15–64 years; Elderly patients, ≥ 65 years Infant, 0 years; Toddler, 1–4 years; Childhood, 5–9 years; Adolescent, 10–14 years Determined by the χ2 test for categorical variables and Kruskal–Wallis test for continuous variables. Baseline characteristics Page 7/25 It was difficult to obtain hospital acceptance for transfer of a total of 1,147 pediatric patients (397, 433, and 317, in 2018, 2019, and 2020, respectively, P = 0.179) (Table 2). Table 2 Difficult-to-transfer cases for children and hospital course Year 2018 2019 2020 Total P value All 35,314 37,547 24,697 97,558 Not difficult- to-transfer cases, % 34,917 (98.9) 37,114 (98.9) 24,380 (98.7) 96411 (98.8) 0.179 Difficult-to- transfer cases, % 397 (1.1) 433 (1.2) 317 (1.3) 1147 (1.2) Disposition at the ED < 0.001 Admission, % 5,708 (16.2) 6,186 (16.5) 4,467 (18.1) 16361 (16.8) Home, % 29,385 (83.2) 31,143 (83.0) 20,063 (81.2) 80591 (82.6) Transfer, % 178 (0.5) 172 (0.5) 133 (0.5) 483 (0.5) Death, % 42 (0.1) 45 (0.1) 34 (0.1) 121 (0.1) Determined by the χ2 test for categorical variables Determined by the χ2 test for categorical variables Table 2 also shows the disposition at the ED. Table 2 also shows the disposition at the ED. The number of children who were suspected to have COVID-19 during transportation was 43 (4 infants, 20 toddlers, 8 childhoods, and 11 adolescents). Of these 43, only one adolescent was a difficult-to- transfer case. Outcomes and adjusted analyses The RR of difficult-to-transfer cases for pediatric patients in 2020 was not significant with reference to 2018 (1.16, 95%CI 0.74 to 1.82) (Table 3). The RR of difficult-to-transfer cases for adult patients older than or equal to 15 years in 2020 was significant with reference to 2018 (1.25, 95% CI 1.22 to 1.28) (Table 3). With reference to 2018, 2020 was significant for difficult-to-transfer cases for all patients (RR 1.24, 95% CI 1.21 to 1.27) in Multivariate Poisson regression analysis (Table 4). In Table 4, with reference to Friday, all day, Saturday and Sunday especially had significantly higher RRs for difficult-to-transfer cases (RR 1.26, 95% CI 1.21 to 1.30 and RR 1.38, 95% CI 1.34 to 1.43, respectively). In Table 4, with reference to 9 to 10 am, other times of day were significantly associated with difficult-to-transfer cases. With reference to June, other months were also significantly associated with difficult-to-transfer cases. There was a Page 8/25 significant association of patients suspected to have COVID-19 with difficult-to-transfer cases (RR 2.50, 95% CI 2.27 to 2.75). Table 5 shows the same analysis as Table 4 for children only, and there was no significant association with difficult-to-transfer cases in 2020 (RR 1.14, 95% CI 0.98 to 1.32, P = 0.081). Different from the general population, children were more accepted by hospitals in January and December than in June. With reference to 9 to 10 am, 3 pm to 2 am was significantly associated with difficult-to-transfer cases for children (Table 5). Similar to the general population, with reference to Friday, Saturday and Sunday had significantly higher RRs for difficult-to-transfer cases (RR 1.37, 95% CI 1.11 to 1.70 and RR 1.37, 95% CI 1.11 to 1.70, respectively). There was no significant association between pediatric patients suspected to have COVID-19 and difficult-to-transfer cases (RR 1.72, 95% CI 0.25 to 12.08, P = 0.584). Outcomes and adjusted analyses Page 9/25 Table 3 Table 3 Univariate Poisson regression analysis for difficult-to-transfer cases and disposition at the emergency department Risk Ratio 95% confidence interval P value Difficult-to-transfer cases Children, < 15 years 2018 Reference 2019 1.03 0.90 − 1.17 0.712 2020 1.14 0.99 − 1.32 0.077 Adults, ≥ 15 years 2018 Reference 2019 0.92 0.90 − 0.94 < 0.001 2020 1.25 1.22 − 1.28 < 0.001 Disposition at the Emergency Department Admission Children, < 15 years 2018 Reference 2019 1.02 0.99 − 1.05 0.255 2020 1.12 1.08 − 1.16 < 0.001 Adults, ≥ 15 years 2018 Reference 2019 1.02 1.01 − 1.02 < 0.001 2020 1.08 1.07 − 1.08 < 0.001 Home Children, < 15 years 2018 Reference 2019 1.00 0.99 − 1.00 0.337 2020 0.98 0.97 − 0.98 < 0.001 Adults, ≥ 15 years 2018 Reference Page 10/25 Risk Ratio 95% confidence interval P value 2019 0.99 0.98 − 0.99 < 0.001 2020 0.94 0.94 − 0.95 < 0.001 Death Children, < 15 years 2018 Reference 2019 1.01 0.66 − 1.53 0.971 2020 1.16 0.74 − 1.82 0.526 Adult, ≥ 15 years 2018 Reference 2019 1.00 0.96 − 1.04 0.945 2020 1.22 1.17 − 1.27 < 0.001 Page 11/25 Table 4 Multivariate Poisson regression analysis of difficult to transfer cases in all patients Risk Ratio 95% confidence interval P value Year 2018 Reference 2019 0.93 0.90 − 0.95 < 0.001 2020 1.24 1.21 − 1.27 < 0.001 Month June Reference January 1.93 1.84 − 2.02 < 0.001 February 1.85 1.76 − 1.94 < 0.001 March 1.49 1.41 − 1.57 < 0.001 April 1.59 1.51 − 1.67 < 0.001 May 1.39 1.32 − 1.47 < 0.001 July 1.14 1.08 − 1.20 < 0.001 August 1.41 1.34 − 1.49 < 0.001 September 1.20 1.14 − 1.27 < 0.001 October 1.12 1.06 − 1.18 < 0.001 November 1.28 1.21 − 1.35 < 0.001 December 1.52 1.45 − 1.60 < 0.001 Time of transportation 9 am to 10 am Reference 0 am to 1 am 6.44 5.91 − 7.02 < 0.001 1 am to 2 am 7.42 6.80 − 8.08 < 0.001 2 am to 3 am 7.95 7.29 − 8.67 < 0.001 3 am to 4 am 7.66 7.02 − 8.37 < 0.001 4 am to 5 am 7.61 6.96 − 8.31 < 0.001 5 am to 6 am 6.80 6.21 − 7.44 < 0.001 6 am to 7 am 5.49 5.01 − 6.00 < 0.001 Page 12/25 Page 12/25 Risk Ratio 95% confidence interval P value Risk Ratio 95% confidence interval P value 7 am to 8 am 3.98 3.63 − 4.36 < 0.001 8 am to 9 am 2.16 1.97 − 2.38 < 0.001 10 am to 11 am 1.25 1.13 − 1.38 < 0.001 11 am to 12 pm 1.58 1.44 − 1.75 < 0.001 12 pm to 1 pm 1.92 1.75 − 2.11 < 0.001 1 pm to 2 pm 2.18 1.98 − 2.39 < 0.001 2 pm to 3 pm 2.21 2.01 − 2.42 < 0.001 3 pm to 4 pm 2.41 2.19 − 2.64 < 0.001 4 pm to 5 pm 2.41 2.19 − 2.64 < 0.001 5 pm to 6 pm 3.08 2.82 − 3.37 < 0.001 6 pm to 7 pm 3.85 3.54 − 4.20 < 0.001 7 pm to 8 pm 4.48 4.12 − 4.88 < 0.001 8 pm to 9 pm 4.81 4.42 − 5.23 < 0.001 9 pm to 10 pm 4.86 4.46 − 5.29 < 0.001 10 pm to 11 pm 5.25 4.82 − 5.72 < 0.001 11 pm to 0 am 5.76 5.29 − 6.28 < 0.001 Day of week Friday Reference Monday 1.11 1.07 − 1.15 < 0.001 Tuesday 1.08 1.04 − 1.12 < 0.001 Wednesday 1.09 1.05 − 1.13 < 0.001 Thursday 1.06 1.02 − 1.10 0.004 Saturday 1.26 1.21 − 1.30 < 0.001 Sunday 1.38 1.34 − 1.43 < 0.001 Adults, ≥ 15 years Reference Children 0.35 0.33 − 0.37 < 0.001 Suspected COVID-19 2.50 2.27 − 2.75 < 0.001 Page 14/25 Multivariate Poisson regression analysis of difficult to transfer cases in children Risk Ratio 95% confidence interval P value Year 2018 Reference 2019 1.03 0.90 − 1.18 0.704 2020 1.14 0.98 − 1.32 0.081 Month June Reference January 0.75 0.57 − 0.99 0.041 February 0.82 0.62 − 1.09 0.18 March 1.16 0.89 − 1.50 0.28 April 0.90 0.68 − 1.19 0.446 May 0.92 0.70 − 1.21 0.549 July 0.83 0.63 − 1.09 0.18 August 0.90 0.69 − 1.19 0.466 September 1.02 0.78 − 1.34 0.888 October 1.01 0.77 − 1.33 0.931 November 1.03 0.79 − 1.36 0.806 December 0.72 0.54 − 0.97 0.03 Time of transportation 9 am to 10 am Reference 0 am to 1 am 2.26 1.23 − 4.13 0.008 1 am to 2 am 2.15 1.15 − 4.03 0.017 2 am to 3 am 1.94 0.99 − 3.82 0.053 3 am to 4 am 1.56 0.74 − 3.27 0.238 4 am to 5 am 0.82 0.32 − 2.12 0.689 5 am to 6 am 1.07 0.44 − 2.63 0.874 6 am to 7 am 0.72 0.26 − 1.98 0.528 Page 15/25 Page 15/25 Risk Ratio 95% confidence interval P value Risk Ratio 95% confidence interval P value 7 am to 8 am 0.67 0.26 − 1.73 0.414 8 am to 9 am 1.30 0.66 − 2.57 0.453 10 am to 11 am 1.31 0.68 − 2.51 0.423 11 am to 12 pm 1.35 0.72 − 2.52 0.346 12 pm to 1 pm 2.06 1.16 − 3.67 0.013 1 pm to 2 pm 1.54 0.86 − 2.77 0.147 2 pm to 3 pm 1.31 0.72 − 2.37 0.376 3 pm to 4 pm 2.16 1.24 − 3.79 0.007 4 pm to 5 pm 2.06 1.18 − 3.60 0.011 5 pm to 6 pm 2.76 1.61 − 4.74 < 0.001 6 pm to 7 pm 3.16 1.85 − 5.39 < 0.001 7 pm to 8 pm 3.17 1.85 − 5.41 < 0.001 8 pm to 9 pm 2.54 1.48 − 4.38 0.001 9 pm to 10 pm 3.05 1.78 − 5.24 < 0.001 10 pm to 11 pm 3.71 2.16 − 6.38 < 0.001 11 pm to 0 am 3.08 1.76 − 5.40 < 0.001 Day of week Friday Reference Monday 1.18 0.94 − 1.48 0.156 Tuesday 0.98 0.77 − 1.25 0.895 Wednesday 0.95 0.75 − 1.21 0.687 Thursday 1.18 0.94 − 1.49 0.151 Saturday 1.37 1.11 − 1.70 0.004 Sunday 1.37 1.11 − 1.70 0.003 Suspected COVID-19 1.72 0.25 − 12.08 0.584 male sex was associated with difficult-to-transfer cases (RR 1.20, 95% CI 1.07 to 6). were positively associated with difficult-to-transfer cases (RR 1.66, 95% CI 1.01 to 2.75, P = 0.047 and RR 1.53, 95% CI 1.13 to 2.08, P = 0.006, respectively). Other diseases were inversely or not significantly associated with difficult-to-transfer cases (Table 6). Discussion Children were associated with a reduced RR for difficulty in obtaining hospital acceptance for transfer of a patient (difficult-to-transfer cases) (RR 0.35, 95% CI 0.33 to 0.37, P < 0.001) (Table 4). In contrast, adult patients had a greater RR for difficult-to-transfer cases than the other population (RR 2.87, 95% CI 2.71 to 3.04) (Supplemental Table 1). During the 3-year study period, 1,147 children were difficult-to-transfer cases (397, 433, and 317 in 2018, 2019, and 2020, respectively). However, it is important to reduce difficult-to-transfer cases, with or without the COVID-19 pandemic. There was a strong association of difficult-to-transfer cases with patients suspected to have COVID-19 in all age patients, but, interestingly, it was not applicable in children (Table 5). As a previous study showed, hospitals were most likely to accept patients of all ages in the morning (9 am to 10 am), on Fridays, and in the month of June, but this was not applicable to children (Table 5).** In children, the year 2020 was not significantly associated with difficult-to-transfer cases, but the year was significantly associated with hospital admission at the ED (RR 1.14, 95% CI 1.10 to 1.18, P < 0.001), even though children had a reduced RR for hospital admission from the ED (RR 0.51, 95% CI 0.50 to 0.51, P < 0.001) (Supplemental Table 2 and Supplemental Table 4). The exact reason why children had a greater RR of admission in 2020 than the other two years is unclear. One of the reasons for this finding might be due to withholding the medically unnecessary EMS transports during the COVID-19 pandemic in 2020, as shown in Table 3 and Supplemental Table 2. In fact, COVID-19 was significantly associated with hospital admission from the ED (RR 5.27, 95% CI 4.06 to 6.85, P < 0.001). Table 6 shows an all-children analysis to investigate which diseases contributed to difficult-to-transfer cases. Injury and poisoning, genitourinary diseases and musculoskeletal diseases were associated with difficult-to-transfer cases for three years, and injury and poisoning were strongly associated with difficult-to-transfer cases in 2020, which was not thought to be related to COVID-19 (Table 6 and Supplemental Table 3). However, diseases of the musculoskeletal system and connective tissue was inversely associated with hospital admission from the ED (Supplemental Table 2). Outcomes and adjusted analyses With reference to injury, poisoning and certain other consequences of extern In children, female sex was associated with difficult-to-transfer cases (RR 1.20, 95% CI 1.07 to 1.35, P = 0.002) (Table 6). With reference to injury, poisoning and certain other consequences of external causes, diseases of the genitourinary system and diseases of the musculoskeletal system and connective tissue Page 16/25 Page 16/25 Page 16/25 were positively associated with difficult-to-transfer cases (RR 1.66, 95% CI 1.01 to 2.75, P = 0.047 and RR 1.53, 95% CI 1.13 to 2.08, P = 0.006, respectively). Other diseases were inversely or not significantly associated with difficult-to-transfer cases (Table 6). were positively associated with difficult-to-transfer cases (RR 1.66, 95% CI 1.01 to 2.75, P = 0.047 and RR 1.53, 95% CI 1.13 to 2.08, P = 0.006, respectively). Other diseases were inversely or not significantly associated with difficult-to-transfer cases (Table 6). were positively associated with difficult-to-transfer cases (RR 1.66, 95% CI 1.01 to 2.75, P = 0.047 and RR 1.53, 95% CI 1.13 to 2.08, P = 0.006, respectively). Other diseases were inversely or not significantly associated with difficult-to-transfer cases (Table 6). Page 17/25 Table 6 Multivariate Poisson regression analysis of difficult-to-transfer cases in children Difficult-to-transfer cases Risk Ratio 95% confidence interval P value Year 2018 Reference 2019 1.07 0.93 − 1.22 0.93 2020 1.01 0.87 − 1.17 0.17 Injury and poisoning Reference Infectious and parasitic diseases 0.10 0.06 − 0.17 < 0.001 Neoplasms 0.51 0.13 − 2.03 0.338 Endocrine, nutritional, and metabolic diseases 0.33 0.14 − 0.79 0.013 Mental and behavioral disorders 0.60 0.36 − 1.00 0.049 Diseases of the nervous system 0.16 0.08 − 0.29 < 0.001 Diseases of the eye and the ear 0.88 0.44 − 1.75 0.708 Diseases of the circulatory system 0.16 0.04 − 0.63 0.009 Diseases of the respiratory system 0.08 0.05 − 0.13 < 0.001 Diseases of the digestive system 0.25 0.15 − 0.42 < 0.001 Diseases of the skin and subcutaneous tissue 0.05 0.01 − 0.33 0.002 Diseases of the musculoskeletal system and connective tissue 1.53 1.13 − 2.08 0.006 Diseases of the genitourinary system 1.66 1.01 − 2.75 0.047 Congenital diseases 0.57 0.08 − 3.97 0.569 Symptoms, signs, and abnormal clinical and laboratory findings 0.12 0.09 − 0.15 < 0.001 Male Reference Female 1.20 1.07 − 1.35 0.002 Page 18/25 Availability of data and material: The datasets used in the current study are available from the corresponding author on reasonable request. Conflicts of interest/Competing interests: The authors declare that they have no competing interests. Discussion Neoplasms, diseases of the circulatory system, diseases of the nervous system, and congenital diseases had higher RRs of hospital admission than other diseases (Supplemental Table 2). This result was similar to other studies.141516 Multisystem inflammatory syndrome in children (MIS-C) is characterized by COVID-19-associated Kawasaki disease-like symptoms together with cardiac inflammation and toxic shock syndrome.17 Kawasaki disease data were collected as ICD-10 code with M303, and 43 patients (19 in 2018, 15 in 2019, and 9 in 2020) were found. In addition, only 7 children had confirmed COVID-19 with admission, and all of them were discharged within 21 days. Thus, diseases of the circulatory system appeared to be different from MIS-C, and MIS-C was not a problem in Osaka Prefecture in 2020. Neoplasms, congenital malformations, deformations, and chromosomal abnormalities also contributed to hospital admission from the ED. The caregivers of these patients might delay seeking medical services Page 19/25 Page 19/25 Page 19/25 due to fear of COVID-19 exposure, which resulted in a hospital stay for treatment because most of these patients were immunocompromised.1819 The present results suggest that injury and poisoning were the most common reasons for EMS calls in children and resulted in difficult-to-transfer cases, especially in 2020, but they did contribute to hospital admission from the ED. There are several limitations in this study. First, COVID-19 is a new disease identified in 2020 in Japan, and acute upper respiratory tract infection or gastroenteritis as ICD-10 code U07.2 in which COVID-19 was suspected was included. Second, this was a retrospective, observational study, and there might be some unknown confounding factors due to this study type. Third, this study uniformly defined the difficult-to- transfer cases regardless of the patient’s condition to assess differences by demographic factors or reasons for ambulance calls. Finally, adjustment for various factors such as medical history, medications, and health status could not be performed in the Poisson regression analysis, because this information was not available. Conclusion The present study found that children had a reduced RR for difficult-to-transfer cases, even in 2020 with the COVID-19 outbreak. Children also had a reduced RR for hospital admission from the ED, but it increased in 2020. Injury and poisoning were the most common reasons for EMS calls and difficult-to- transfer cases. Funding: No funding was received for the study. Code availability: All data were statistically analyzed using SPSS version 25.0 software (IBM Corp., Armonk, NY, USA) or STATA (version 16.1; StataCorp, College Station, TX, USA). The name of the Ethical committee The Ethics Committee of Osaka Medical and Pharmaceutical University (Takatsuki City, Japan) (reference number 2021-006) The Ethics Committee of Osaka Medical and Pharmaceutical University (Takatsuki City, Japan) (reference number 2021-006) Authors’ contributions: Page 20/25 K.O. designed the study and wrote the initial draft of the manuscript. Ka.O., D.N., T.K., Y.K., M.N., T.M., and A.T. contributed to analysis and interpretation of the data and assisted in the preparation of the manuscript. All authors contributed to data collection and interpretation and critically reviewed the manuscript. All authors approved the final version of the manuscript and agree to be accountable for all aspects of the work, including ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Consent for publication: Informed consent from the participants was waived because the data were anonymous. Informed consent from the participants was waived because the data were anonymous. Acknowledgements: The authors thank the EMS providers, nurses, emergency physicians and administrators for their cooperation in the ORION. We thank all the members of The Working Group to Analyze the Emergency Medical Care System in Osaka Prefecture for their kind support. Ethics approval: This study was approved by the Ethics Committee of Osaka Medical and Pharmaceutical University (Takatsuki City, Japan)(reference number 2021-006). The Strengthening the Reporting of Observational studies in Epidemiology (STROBE) guidelines were used to design and report the results of this study. The Strengthening the Reporting of Observational studies in Epidemiology (STROBE) guidelines were used to design and report the results of this study. Consent to participate: Informed consent from the participants was waived because the data were anonymous. Informed consent from the participants was waived because the data were anonymous. Consent to Publish NOT APLLICABLE References 1. Wiersinga WJ, Rhodes A, Cheng AC, Peacock SJ, Prescott HC. Pathophysiology, Transmission, Diagnosis, and Treatment of Coronavirus Disease 2019 (COVID-19): A Review. JAMA - J Am Med Assoc. 2020;324(8):782-793. doi:10.1001/jama.2020.12839 1. Wiersinga WJ, Rhodes A, Cheng AC, Peacock SJ, Prescott HC. Pathophysiology, Transmission, Diagnosis, and Treatment of Coronavirus Disease 2019 (COVID-19): A Review. JAMA - J Am Med Assoc. 2020;324(8):782-793. doi:10.1001/jama.2020.12839 Page 21/25 Page 21/25 2. Shekerdemian LS, Mahmood NR, Wolfe KK, et al. Characteristics and outcomes of children with coronavirus disease 2019 (COVID-19) infection admitted to US and Canadian pediatric intensive care units. JAMA Pediatr. 2020;174(9):868-873. doi:10.1001/jamapediatrics.2020.1948 3. Jurado Hernández JL, Álvarez Orozco IF. 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Okamoto J, Katayama Y, Kitamura T, et al. Profile of the ORION (Osaka emergency information Research Intelligent Operation Network system) between 2015 and 2016 in Osaka, Japan: a population-based registry of emergency patients with both ambulance and in-hospital records. Acute Med Surg. 2019;6(1):12-24. doi:10.1002/ams2.371 11. Okamoto J, Katayama Y, Kitamura T, et al. Profile of the ORION (Osaka emergency information Research Intelligent Operation Network system) between 2015 and 2016 in Osaka, Japan: a population-based registry of emergency patients with both ambulance and in-hospital records. Acute Med Surg. 2019;6(1):12-24. doi:10.1002/ams2.371 12. WHO. International Statistical Classification of Diseases and Related Health Problems, 10th Revision ICD-10 : Tabular List. World Heal Organ. 2016;1:332-345. http://www.who.int/classifications/icd/icdonlineversions/en/. 12. WHO. International Statistical Classification of Diseases and Related Health Problems, 10th Revision ICD-10 : Tabular List. World Heal Organ. 2016;1:332-345. http://www.who.int/classifications/icd/icdonlineversions/en/. 13. von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, Vandenbroucke JP. The strengthening the reporting of observational studies in epidemiology (STROBE) statement: Guidelines for reporting observational studies. Int J Surg. 2014;12(12):1495-1499. doi:10.1016/j.ijsu.2014.07.013 13. von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, Vandenbroucke JP. The strengthening the reporting of observational studies in epidemiology (STROBE) statement: Guidelines for reporting observational studies. Int J Surg. 2014;12(12):1495-1499. doi:10.1016/j.ijsu.2014.07.013 14. Graetz D, Agulnik A, Ranadive R, et al. Global effect of the COVID-19 pandemic on paediatric cancer care: a cross-sectional study. 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Loke Y-H, Berul CI, Harahsheh AS. Multisystem inflammatory syndrome in children: Is there a linkage to Kawasaki disease? Trends Cardiovasc Med. 2020;30(7):389-396. doi:10.1016/j.tcm.2020.07.004 17. Loke Y-H, Berul CI, Harahsheh AS. Multisystem inflammatory syndrome in children: Is there a linkage to Kawasaki disease? Trends Cardiovasc Med. 2020;30(7):389-396. doi:10.1016/j.tcm.2020.07.004 18. Page 21/25 Ding YY, Ramakrishna S, Long AH, et al. Delayed cancer diagnoses and high mortality in children during the COVID-19 pandemic. Pediatr Blood Cancer. 2020;67(9):3-5. doi:10.1002/pbc.28427 18. Ding YY, Ramakrishna S, Long AH, et al. Delayed cancer diagnoses and high mortality in children during the COVID-19 pandemic. Pediatr Blood Cancer. 2020;67(9):3-5. doi:10.1002/pbc.28427 19. Lazzerini M, Barbi E, Apicella A, Marchetti F, Cardinale F, Trobia G. Delayed access or provision of care in Italy resulting from fear of COVID-19. Lancet Child Adolesc Heal. 2020;4(5):e10-e11. doi:10.1016/S2352-4642(20)30108-5 19. Lazzerini M, Barbi E, Apicella A, Marchetti F, Cardinale F, Trobia G. Delayed access or provision of care in Italy resulting from fear of COVID-19. Lancet Child Adolesc Heal. 2020;4(5):e10-e11. doi:10.1016/S2352-4642(20)30108-5 Figure 1 Patient flow in this study. All pediatric patients aged less than 15 years were included and then divided into four groups by age. Figures Page 23/25 This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to download. Page 24/25 Page 24/25 SupplementalTablekid.docx Page 25/25
https://openalex.org/W3135760905	https://dspace.mit.edu/bitstream/1721.1/143828/2/s41467-021-21852-7.pdf	English	null	Flexible and high-performance electrochromic devices enabled by self-assembled 2D TiO2/MXene heterostructures	Nature communications	2,021	cc-by	11,481	MIT Open Access Articles The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Version: Final published version: final published article, as it appeared in a journal, conference proceedings, or other formally published context Version: Final published version: final published article, as it appeared in a journal, conference proceedings, or other formally published context Terms of use: Creative Commons Attribution 4.0 International license Terms of use: Creative Commons Attribution 4.0 International license Terms of use: Creative Commons Attribution 4.0 International license ARTICLE 67-021-21852-7 1-21852-7 1 State Key Laboratory for Modiﬁcation of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University, Shanghai, China. 2 Department of Chemistry, Boston University, Boston, MA, USA. 3 Division of Materials Science and Engineering, Boston University, Boston, MA, USA. 4 Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, USA. 5 The Photonics Center, Boston University, Boston, MA, USA. 6Present address: Department of Electrical Engineering and Computer Sciences, University of California, Berkeley, CA, USA. 7These authors contributed equally: Ran Li, Xiaoyuan Ma. ✉email: yxlin@berkeley.edu; wanghz@dhu.edu.cn; xiling@bu.edu NATURE COMMUNICATIONS \| (2021) 12:1587 \| https://doi.org/10.1038/s41467-021-21852-7 \| www.nature.com/naturecommunications Flexible and high-performance electrochromic devices enabled by self-assembled 2D TiO2/MXene heterostructures Ran Li1,2,7, Xiaoyuan Ma 2,7, Jianmin Li1, Jun Cao 2, Hongze Gao3, Tianshu Li 3, Xiaoyu Zhang1, Lichao Wang1, Qinghong Zhang 1, Gang Wang1, Chengyi Hou 1, Yaogang Li1, Tomás Palacios4, Yuxuan Lin 4,6✉, Hongzhi Wang 1✉& Xi Ling 2,3,5✉ Transition metal oxides (TMOs) are promising electrochromic (EC) materials for applications such as smart windows and displays, yet the challenge still exists to achieve good ﬂexibility, high coloration efﬁciency and fast response simultaneously. MXenes (e.g. Ti3C2Tx) and their derived TMOs (e.g. 2D TiO2) are good candidates for high-performance and ﬂexible EC devices because of their 2D nature and the possibility of assembling them into loosely networked structures. Here we demonstrate ﬂexible, fast, and high-coloration-efﬁciency EC devices based on self-assembled 2D TiO2/Ti3C2Tx heterostructures, with the Ti3C2Tx layer as the transparent electrode, and the 2D TiO2 layer as the EC layer. Beneﬁting from the well- balanced porosity and connectivity of these assembled nanometer-thick heterostructures, they present fast and efﬁcient ion and electron transport, as well as superior mechanical and electrochemical stability. We further demonstrate large-area ﬂexible devices which could potentially be integrated onto curved and ﬂexible surfaces for future ubiquitous electronics. 1 State Key Laboratory for Modiﬁcation of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University, Shanghai, China. 2 Department of Chemistry, Boston University, Boston, MA, USA. 3 Division of Materials Science and Engineering, Boston University, Boston, MA, USA. 4 Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, USA. 5 The Photonics Center, Boston University, Boston, MA, USA. 6Present address: Department of Electrical Engineering and Computer Sciences, University of California, Berkeley, CA, USA. 7These authors contributed equally: Ran Li, Xiaoyuan Ma. ✉email: yxlin@berkeley.edu; wanghz@dhu.edu.cn; xiling@bu.edu 1 TURE COMMUNICATIONS \| (2021) 12:1587 \| https://doi.org/10.1038/ ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 E lectrochromic (EC) materials, which can change their optical absorption reversibly due to the electrochemical redoxation with inserted ions under an applied potential1–3, have shown great promise in applications such as displays, smart windows and optical camouﬂage3–5. With the growing interest in bringing electrochromism to the ﬁeld of ﬂexible and wearable electronics in recent years, incorporating the functionality of ﬂexibility to EC devices while maintaining high energy efﬁciency, fast switching speed and long durability has become increasingly critical6,7. Results Preparation of 2D TiO2 via the chemical conversion from Ti3C2Tx. Figure 1 illustrates the fabrication process of the self- assembled TiO2/Ti3C2Tx heterostructure on a ﬂexible structure as the core functional component of EC devices. The fabrication process consists of three steps: First, the Ti3C2Tx nanosheet dis- persion in deionized (DI) water is obtained from selective etching of Ti3AlC2 powder (MAX phase)22, and diluted to 0.2 mg/L, followed by the formation of the Ti3C2Tx aerogel through freeze drying. The Ti3C2Tx aerogel is then annealed in air at 400 °C to fully be converted to the TiO2 aerogel; Second, the Ti3C2Tx (TiO2) aerogels are dispersed into isopropyl alcohol (IPA) to form the Ti3C2Tx (TiO2) solutions, which are then injected into the interface between hexane and water, where the nanosheets self- assemble into uniform nanometer-thick ﬁlms; Last, the self- assembled Ti3C2Tx ﬁlm and TiO2 ﬁlm are transferred sequentially onto a ﬂexible polyethylene terephthalate (PET) substrate to form TiO2/Ti3C2Tx heterostructures, in which the Ti3C2Tx ﬁlm serves as the transparent conductive electrode, and the TiO2 ﬁlm serves as the EC layer in EC devices (more details about ﬁlm assembly and transfer processes are in Supplementary Fig. 1, Supplemen- tary Movie 1 and Supplementary Movie 2 in Supplementary Information). Recently, two-dimensional (2D) transition metal carbides, nitrides, and carbonitrides (referred to as MXenes) have emerged as promising nanoscale electronic materials with good electro- chemical stability8,9. They have also been reported as promising templates for the synthesis of functional micro/nanostructured materials, such as transition metal oxides (TMOs), alkali-metal titanates, and their hybrids10,11. Owing to their unique optical and electrical properties, MXenes and their derivatives have demonstrated their potential in the application of energy storage, catalysis, and electronics12–14. More importantly, 2D nanosheets of both MXenes and their derivatives can be produced in liquid phase scalably, which makes it easy for constructing self- assembled ﬁlms and hybrid structures, thus suitable for device applications in large scale. We propose that self-assembled MXene/TMO hetero- structures, with the MXenes as the ﬂexible transparent electrode, and the TMOs (derived from the MXenes) as the ﬂexible EC layer, are promising for high performance and ﬂexible EC devices, yet it has never been explored previously. Flexible and high-performance electrochromic devices enabled by self-assembled 2D TiO2/MXene heterostructures However, this is an extremely challenging task because: (a) both the EC performance and the mechanical properties are nontrivially correlated with the materials of choice, their nanos- cale formations, and the assembly approach; and (b) the co- optimization of the transparent conductive electrode and the EC material, as well as the way to integrate them, are needed. E the fastest coloration speed, the highest coloration efﬁciency and superior electrochemical stability. We further demonstrate a large-area ﬂexible device which could potentially be integrated with arbitrary curved and ﬂexible surfaces for future ubiquitous electronics. Results In addition to the advantage of the easiness of fabrication and integration with different functional components from MXene derivatives as mentioned above, an even more important reason is that the nanosheets network of TMOs, such as tungsten oxide (WO3) and titanium oxide (TiO2), can potentially realize not only superior EC properties, but also reliable mechanical and chemical stability among various candidates of EC materials. First, these inorganic materials are long-lasting and robust against environmental fac- tors such as high temperature, ultraviolet radiation, and mechanical wear15–17, as compared to their organic counter- parts18. Second, the nanostructured TMO network, especially in the form of 2D nanosheets, improves electrical and ionic trans- port signiﬁcantly, because: (a) the increased surface-to-volume ratio of nanosheets as compared to the bulk forms leads to larger interface areas with the electrolyte and the shorter ion diffusivity pathways within the nanostructured channels;3,15 and (b) the 2D formation of the nanostructures makes the contact area between adjacent nanosheets much larger than that between 1D or 0D structures (nanowires or quantum dots)19, which promotes the internanosheets electron transfer20. Finally, networks of 2D nanostructures also help to redistribute the induced strain evenly, thus improving both the mechanical strength and the ﬂexibility of the ﬁlm, which is fundamentally more advantageous than 1D or 0D nanostructures for ﬂexible devices21. To obtain the anatase phase of TiO2, which has been demonstrated a good EC material23, we anneal the Ti3C2Tx aerogel in air at 400 °C for 50 h. X-ray diffraction (XRD) and Raman spectroscopy of the ﬂakes are measured before and after the thermal annealing process to investigate the compositional and the structural changes. As shown in Fig. 2a, after annealing in air at 400 °C, new peaks at 26°, 38°, 48°, 54°, and 63° appear in the XRD spectra, corresponding to the (101), (112), (200), (105), and (204) planes of the anatase phase of TiO224. Raman spectra of Ti3C2Tx ﬂakes after annealing conﬁrms the conversion from Ti3C2Tx to TiO2 (Fig. 2b), where peaks at 145 cm−1 (Eg), 398 cm−1 (B1g), 518 cm−1 (B1g), and 639 cm−1 (Eg) match well with the Raman features of the anatase phase of TiO225. X-ray photoelectron spectroscopy (XPS) characterization further sug- gests the transformation from Ti3C2Tx to TiO2. It’s clearly seen that the peaks for the Ti element in Ti3C2Tx (i.e., Ti-C 2p3/2 (455.3 eV), Ti(II) 2p3/2 (456.1 eV), Ti(III) 2p3/2 (457.2)) disappear after the annealing (Fig. Results 2c top and Supplementary Fig. 2), while peaks at 458.8 eV and 464.8 eV, corresponding to Ti(IV) 2p3/2 and Ti(IV) 2p1/2 in TiO2, become dominant (Fig. 2c bottom)26. To evaluate the morphology and crystallinity of the obtained TiO2 nanosheets, optical microscopy (OM), transmission electron microscopy (TEM) and atomic force microscopy (AFM) are used to characterize Ti3C2Tx ﬂakes before and after the in situ conversion to TiO2. Supplementary Fig. 3 shows OM images of a typical ﬂake (~7 µm in lateral dimension) undergoing the annealing treatment at 400 °C for 50 h, where the morphology of the ﬂake maintains during the process. AFM images (Fig. 2d) show that the derived TiO2 ﬂake remains the same shape as the Ti3C2Tx precursor. The surface roughness of the ﬂake also does not change much after conversion (root mean square roughness changes from 514.3 to 528.2 pm). The thickness of the ﬂake stays about 2 nm after being converted to TiO2. Typical TEM images (Fig. 2e and Supplementary Fig. 4) and selective area electron diffraction (SAED) patterns (insets of Supplementary Fig. 4) indicate that both Ti3C2Tx and TiO2 are in high crystallinity. To obtain the anatase phase of TiO2, which has been demonstrated a good EC material23, we anneal the Ti3C2Tx aerogel in air at 400 °C for 50 h. X-ray diffraction (XRD) and Raman spectroscopy of the ﬂakes are measured before and after the thermal annealing process to investigate the compositional and the structural changes. As shown in Fig. 2a, after annealing in air at 400 °C, new peaks at 26°, 38°, 48°, 54°, and 63° appear in the XRD spectra, corresponding to the (101), (112), (200), (105), and (204) planes of the anatase phase of TiO224. Raman spectra of Ti3C2Tx ﬂakes after annealing conﬁrms the conversion from Ti3C2Tx to TiO2 (Fig. 2b), where peaks at 145 cm−1 (Eg), 398 cm−1 (B1g), 518 cm−1 (B1g), and 639 cm−1 (Eg) match well with the Raman features of the anatase phase of TiO225. X-ray photoelectron spectroscopy (XPS) characterization further sug- gests the transformation from Ti3C2Tx to TiO2. It’s clearly seen that the peaks for the Ti element in Ti3C2Tx (i.e., Ti-C 2p3/2 (455.3 eV), Ti(II) 2p3/2 (456.1 eV), Ti(III) 2p3/2 (457.2)) disappear after the annealing (Fig. 2c top and Supplementary Fig. 2), while peaks at 458.8 eV and 464.8 eV, corresponding to Ti(IV) 2p3/2 and Ti(IV) 2p1/2 in TiO2, become dominant (Fig. 2c bottom)26. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 Freeze drying MXene aerogel TiO2 aerogel Disperse Disperse IPA solution IPA solution Anneal in air Ti C Tx DI Water 1-Hexene MXene/H2O gel Interface assembly Pipette Single flake layer Flakes Ti3C2Tx Tx: O, OH, F Inject Inject PET film MXene/PET TiO2/MXene/PET Transfer Transfer Step 2: Step 1: Step 3: Fig. 1 Fabrication process of TiO2/Ti3C2Tx heterostructure on a ﬂexible substrate. Step 1: MXene (Ti3C2Tx, crystal structures in the left panel, is drawn by Vesta software) aerogel is obtained by freeze drying of the Ti3C2Tx nanosheets dispersion and the derived TiO2 aerogel is obtained by annealing the MXene aerogel in air at 400 °C. Step 2: Ti3C2Tx and TiO2 2D nanosheets are dispersed, respectively, in IPA, injected into the interface between hexene and DI water, and self-assembled into nanometer-thick Ti3C2Tx and TiO2 ﬁlms. Step 3: the thin ﬁlms are transferred onto a ﬂexible substrate (e.g., PET) in a layer-by-layer fashion to realize the TiO2/MXene heterostructure, where the TiO2 layer serves as the electrochromic component, and the MXene layer serves as the transparent electrode. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 ARTICLE Freeze drying IPA Ti C Tx MXene/H2O gel Ti3C2Tx Tx: O, OH, F Step 2: TiO2 aerogel Disperse Anneal in air MXene aerogel Disperse TiO2 aerogel Freeze drying Anneal in air Disperse Disperse IPA solution DI Water 1-Hexene Interface assembly Pipette Single flake layer Flakes Tx: O, OH, F Inject PET film MXene/PET Transfer Step 2: Step 3: Fig. 1 Fabrication process of TiO2/Ti3C2Tx heterostructure on a ﬂexible substrate. Step 1: M by Vesta software) aerogel is obtained by freeze drying of the Ti3C2Tx nanosheets dispersion MXene aerogel in air at 400 °C. Step 2: Ti3C2Tx and TiO2 2D nanosheets are dispersed, respect DI water, and self-assembled into nanometer-thick Ti3C2Tx and TiO2 ﬁlms. Step 3: the thin ﬁl layer-by-layer fashion to realize the TiO2/MXene heterostructure, where the TiO2 layer serve serves as the transparent electrode. IPA solution Inject TiO2/MXene/PET Transfer IPA solution Inject TiO2/MXene/PET Transfer IPA solution Inject Inject Step 3: Transfer Transfer PET film MXene/PET TiO2/MXene/PET Fig. 1 Fabrication process of TiO2/Ti3C2Tx heterostructure on a ﬂexible substrate. Step 1: MXene (Ti3C2Tx, crystal structures in the left panel, is drawn by Vesta software) aerogel is obtained by freeze drying of the Ti3C2Tx nanosheets dispersion and the derived TiO2 aerogel is obtained by annealing the MXene aerogel in air at 400 °C. Results l h h l d ll f h b d To evaluate the morphology and crystallinity of the obtained TiO2 nanosheets, optical microscopy (OM), transmission electron microscopy (TEM) and atomic force microscopy (AFM) are used to characterize Ti3C2Tx ﬂakes before and after the in situ conversion to TiO2. Supplementary Fig. 3 shows OM images of a typical ﬂake (~7 µm in lateral dimension) undergoing the annealing treatment at 400 °C for 50 h, where the morphology of the ﬂake maintains during the process. AFM images (Fig. 2d) show that the derived TiO2 ﬂake remains the same shape as the Ti3C2Tx precursor. The surface roughness of the ﬂake also does not change much after conversion (root mean square roughness changes from 514.3 to 528.2 pm). The thickness of the ﬂake stays about 2 nm after being converted to TiO2. Typical TEM images (Fig. 2e and Supplementary Fig. 4) and selective area electron diffraction (SAED) patterns (insets of Supplementary Fig. 4) indicate that both Ti3C2Tx and TiO2 are in high crystallinity. As a proof-of-concept demonstration, we successfully assemble Ti3C2Tx (T = O, OH, or F) and the derived TiO2 nanosheets into uniform nanometer-thick ﬁlms at a liquid-liquid interface, and fabricate EC devices based on the TiO2/Ti3C2Tx heterostructures on a ﬂexible substrate. Due to the much improved ion diffusion and electron conductivity of assembled single-ﬂake-layer TiO2 ﬁlm and the 2D nature of both the MXene electrode and the TiO2 EC layer, our devices are among the best ﬂexible EC devices with TURE COMMUNICATIONS \| (2021) 12:1587 \| https://doi.org/10.1038/s41467-021-21852-7 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| (2021) 12:1587 \| https://doi.org/10.1038/s41467-021-21852-7 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 a b c Ti(III) Ti(IV)-TiO2 Ti-C Ti(III) Ti(II) Ti(IV)-TiO2 TiO2 Ti2C3Tx Ti3C2Tx TiO2 A1g Eg Eg Eg A1g D G Eg B1gA1g Eg (101) (112) (200) (105) Ti3C2Tx TiO2 (002) (006) (008) (110) (0010) (204) d 6 nm -7.3 nm 8.2 nm -8.7 nm e Ti3C2Tx Ti3C2Tx TiO2 TiO2 0.262 nm (1 0 0) 0.270 nm (1 -1 0) f Ti3C2Tx (1 0 0) b c a TiO2 (1 -1 0) a c Fig. 2 Conversion of Ti3C2Tx into 2D TiO2 ﬂakes. a XRD patterns and b Raman spectra of the Ti3C2Tx before and after annealing at 400 °C for 50 h. c XPS spectra of the Ti3C2Tx and the derived TiO2; d AFM images and height proﬁles of Ti3C2Tx and a derived TiO2 ﬂake on a SiO2/Si substrate (scale bar: 2 μm); e HRTEM images of Ti3C2Tx and derived TiO2 ﬂakes (scale bar: 1 nm); f Crystal structures of Ti3C2Tx and derived anatase phase of TiO2. b Ti3C2Tx TiO2 A1g Eg Eg Eg A1g D G Eg B1gA1g Eg a (101) (112) (200) (105) Ti3C2Tx TiO2 (002) (006) (008) (110) (0010) (204) c Ti(III) Ti(IV)-TiO2 Ti-C Ti(III) Ti(II) Ti(IV)-TiO2 TiO2 Ti2C3Tx b c a f Ti3C2Tx (1 0 0) b c a d 6 nm -7.3 nm 8.2 nm -8.7 nm e Ti3C2Tx Ti3C2Tx TiO2 TiO2 0.262 nm (1 0 0) 0.270 nm (1 -1 0) f Ti3C2Tx (1 0 0) b c a TiO2 (1 -1 0) a c Fig. 2 Conversion of Ti3C2Tx into 2D TiO2 ﬂakes. a XRD patterns and b Raman spectra of the Ti3C2Tx before and after annealing at 400 °C for 50 h. c XPS spectra of the Ti3C2Tx and the derived TiO2; d AFM images and height proﬁles of Ti3C2Tx and a derived TiO2 ﬂake on a SiO2/Si substrate (scale bar: 2 μm); e HRTEM images of Ti3C2Tx and derived TiO2 ﬂakes (scale bar: 1 nm); f Crystal structures of Ti3C2Tx and derived anatase phase of TiO2. d 8.2 nm -8.7 nm Ti3C2Tx f e Ti3C2Tx 0.262 nm (1 0 0) f d 6 nm -7.3 nm 8.2 nm -8.7 nm e Ti3C2Tx Ti3C2Tx TiO2 TiO2 0.262 nm (1 0 0) 0.270 nm (1 -1 0) f Ti3C2Tx (1 0 0) b c a TiO2 (1 -1 0) a c Fig. 2 Conversion of Ti3C2Tx into 2D TiO2 ﬂakes. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 a XRD patterns and b Raman spectra of the Ti3C2Tx before and after annealing at 400 °C for 50 h. c XPS spectra of the Ti3C2Tx and the derived TiO2; d AFM images and height proﬁles of Ti3C2Tx and a derived TiO2 ﬂake on a SiO2/Si substrate (scale bar: 2 μm); e HRTEM images of Ti3C2Tx and derived TiO2 ﬂakes (scale bar: 1 nm); f Crystal structures of Ti3C2Tx and derived anatase phase of TiO2. d e TiO2 (1 -1 0) a c 6 nm -7.3 nm TiO2 TiO2 0.270 nm (1 -1 0) TiO2 -7.3 nm Fig. 2 Conversion of Ti3C2Tx into 2D TiO2 ﬂakes. a XRD patterns and b Raman spectra of the Ti3C2Tx before and after annealing at 400 °C for 50 h. c XPS spectra of the Ti3C2Tx and the derived TiO2; d AFM images and height proﬁles of Ti3C2Tx and a derived TiO2 ﬂake on a SiO2/Si substrate (scale bar: 2 μm); e HRTEM images of Ti3C2Tx and derived TiO2 ﬂakes (scale bar: 1 nm); f Crystal structures of Ti3C2Tx and derived anatase phase of TiO2. e from the AFM image, use it to calibrate the optical contrast of the OM image to obtain the thickness distribution over the area (Supplementary Fig. 8). The thickness distributions extracted from both the AFM image (small area) and the OM image (large area) are shown in Fig. 3c. It is observed that the peaks for both distributions appear at around 2 nm, which corresponds to the thickness of 1-layer of Ti3C2Tx ﬂake. The second most signiﬁcant peak is at around 3.8 nm, which corresponds to the overlapped area of two adjacent ﬂakes (2 layers). These 1-layer and 2-layer regions are the key functional components for the electrical conductance, which covers 64% and 18% of the ﬁlm, respectively. Because of the uniform thickness distributions and the high coverage of 1-layer and 2-layer, the LLIA Ti3C2Tx ﬁlms show both high optical transparency and high electrical conductance, making them ideal for the transparent electrodes of EC devices. The optical and electronic properties are evaluated by ultraviolet- visible (UV-vis) spectrophotometry and a four-point probe from the AFM image, use it to calibrate the optical contrast of the OM image to obtain the thickness distribution over the area (Supplementary Fig. 8). The thickness distributions extracted from both the AFM image (small area) and the OM image (large area) are shown in Fig. 3c. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 Step 2: Ti3C2Tx and TiO2 2D nanosheets are dispersed, respectively, in IPA, injected into the interface between hexene and DI water, and self-assembled into nanometer-thick Ti3C2Tx and TiO2 ﬁlms. Step 3: the thin ﬁlms are transferred onto a ﬂexible substrate (e.g., PET) in a layer-by-layer fashion to realize the TiO2/MXene heterostructure, where the TiO2 layer serves as the electrochromic component, and the MXene layer serves as the transparent electrode. Ti3C2Tx shows a lattice spacing of 0.26 nm, corresponding to its (100) plane. After annealing at 400 °C, the derived TiO2 ﬂake shows a lattice spacing of 0.27 nm, which refers to the1–10 plane of the anatase phase of TiO2. crystals over micrometer large area with nanometer thin thicknesses. Based on these experimental results and comparing the crystal structures of Ti3C2Tx and the anatase phase TiO2, we propose the following mechanism to interpret the transformation from Ti3C2Tx to TiO2. As shown in Fig. 2f, during the conversion, two O atoms need to replace one C atom sandwiched between two adjacent Ti layers. In this process, due to the gentle gas-solid reaction, the diffusion of Ti atoms is limited, and due to the similarity of the Ti frameworks in the Ti3C2Tx and TiO2 crystals, it does not require large rearrangement of Ti atoms during the conversion27. Thus, the morphology of the ﬂake can be retained. Moreover, when applying the conversion to the Ti3C2Tx aerogels, remained morphology is also observed after annealing at 400 °C to convert it to TiO2 (Supplementary Fig. 5). The pore size distribution in the Ti3C2Tx and TiO2 aerogels are similar (both have peaks at around 2 nm extracted from the nitrogen adsorption isotherm at 77K) (Supplementary Fig. 6). This result further suggests that the conversion reaction is conﬁned on initial 2D Ti3C2Tx ﬂakes, leading to the good separation of each ﬂake in the skeleton of aerogels during the conversion process. Self-assembled ultrathin MXene ﬁlms as high-performance transparent electrodes. To obtain large-area homogeneous thin ﬁlms of the TiO2 or Ti3C2Tx network, a liquid/liquid interfacial self-assembly (LLIA) technique is used28. Speciﬁcally, the 2D ﬂakes/IPA dispersion is injected to the interface between hexane Although the crystal lattice is transformed drastically from hexagonal (Ti3C2Tx) to tetragonal (anatase TiO2), our results clearly demonstrate the successful production of Ti3C2Tx ﬂakes and their derived TiO2 ﬂakes, both of which are high-quality 3 ARTICLE NATURE COMMUNICATIONS \| (2021) 12:1587 \| https://doi.org/10.1038/s41467-021-21852-7 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 Comparing the resistances of materials with the similar transparency, our values are lower than MXene ﬁlms made by other methods like spin coating (201 Ω sq−1 at 87%), dip coating (4300 Ω sq−1 at 94%), and spray coating (7974 Ω sq−1 at 82%), and also lower than other transparent conductive materials such as graphene, and reduced graphene oxide (see Supplementary Table 1 for a summary of different transparent conductive materials)30,31,33–40. Finally, Fig. 3f shows that the LLIA Ti3C2Tx ﬁlm can be transferred onto a ﬂexible substrate (e.g., PET) uniformly in a large area (~25 cm2) with excellent optical transparency, suitable for transparent conductive electrodes for the proposed ﬂexible EC devices. LLIA ﬁlm also connect with each other to form a porous network, with the pore size in the range of nanometers, when observed under TEM and OM measurements (Supplementary Fig. 9 and Supplementary Fig. 10). The morphology of TiO2/Ti3C2Tx het- erostructures are further characterized by OM, SEM, and AFM measurements. As shown in Fig. 4a, the boundary between the TiO2/Ti3C2Tx heterostructure and l-layer TiO2 ﬁlm is clearly seen from the OM image. AFM characterization shows a thickness different of ~2 nm across the boundary, matching well with the thickness of 1-layer Ti3C2Tx, indicating a close contact between the TiO2 and Ti3C2Tx layers. Moreover, Supplementary Fig. 11 shows OM and SEM images of different layers (1, 4, and 7 L) of LLIA TiO2 on Ti3C2Tx ﬁlms, where the porous structures can be still seen in the stacked multiple layers. y The heterostructure provides a metal-semiconductor junction between Ti3C2Tx and TiO2, where Ti3C2Tx serves as the transparent electrode and TiO2 serves as the electrochromic layer. Note that Ti3C2Tx has a low work function (1.6–4.4 eV)41,42, when comparing to other transparent electrodes, e.g. indium tin oxide (ITO, with the work function of 4.73 eV)43. When contacting with TiO2 (work function ~4.2 eV), the Schottky barrier between Ti3C2Tx and TiO2 is much lower or it could even be an Ohmic contact44, compared to that between ITO and TiO2. Thus, the electron transport between the Ti3C2Tx and the TiO2 layer is expected to be greatly improved in the heterostructure. Because of the well-controlled LLIA process, it is possible to regulate the EC properties simply by the thickness of the TiO2 layer. As shown in Figs. 4b, 1–5 layers of the LLIA TiO2 ﬁlm are transferred sequentially onto a PET substrate in a layer-by-layer fashion. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 It is observed that the peaks for both distributions appear at around 2 nm, which corresponds to the thickness of 1-layer of Ti3C2Tx ﬂake. The second most signiﬁcant peak is at around 3.8 nm, which corresponds to the overlapped area of two adjacent ﬂakes (2 layers). These 1-layer and 2-layer regions are the key functional components for the electrical conductance, which covers 64% and 18% of the ﬁlm, respectively. and water. Because of the fast dissolution of IPA into the hexane layer, a surface tension gradient is established along the interface (Marangoni force)29. Driving by this Marangoni force, the 2D ﬂakes spreads out quickly and settles at the edge of the liquid interface, that is, either the sidewall of the container or the edge of an already assembled ﬁlm. By optimizing the amount of the injected 2D ﬂakes/IPA solution, a full coverage is achieved and a continuous 2D thin ﬁlm is obtained. As shown in the OM image of Fig. 3a, the LLIA Ti3C2Tx ﬁlm is homogeneous over a large area. According to the AFM image (Fig. 3b and Supplementary Fig. 7) taken at the labeled area in Fig. 3a, the Ti3C2Tx ﬂakes are all horizontally oriented, and placed next to each other with very small overlaps. This is very different from the ﬁlms produced by the spray coating or spin coating methods where the orientations of the ﬂakes are random, and they are mostly overlapped and bundled30,31. To quantify the uniformity of the LLIA ﬁlm in a large area, we extract the height proﬁles (Supplementary Fig. 7) Because of the uniform thickness distributions and the high coverage of 1-layer and 2-layer, the LLIA Ti3C2Tx ﬁlms show both high optical transparency and high electrical conductance, making them ideal for the transparent electrodes of EC devices. The optical and electronic properties are evaluated by ultraviolet- visible (UV-vis) spectrophotometry and a four-point probe 4 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 a b d e c f Substrate 1 Layer 2 Layers (overlap) AFM OM 0 nm 15.2 nm Spin coating LB coating Dip coating Self assembly Spray coating This work RGO Graphene Fig. 3 Characterization of the self-assembled Ti3C2Tx thin ﬁlm as a transparent electrode. a OM image of the assembled Ti3C2Tx thin ﬁlm (scale bar: 10 μm); b AFM image and the height proﬁle of the assembled Ti3C2Tx thin ﬁlm in corresponding area marked in a (scale bar: 2 μm). c Thickness distributions extracted from the AFM image (top) and the OM image (bottom); the Y-axes are probability density, namely, frequency per unit height; d Transmittance spectra of Ti3C2Tx ﬁlms with various concentrations of injected Ti3C2Tx; e Transmittance at 550 nm as a function of sheet resistance for the Ti3C2Tx ﬁlms and data from previously reported transparent electrode technologies; RGO reduced graphene oxide, LB Langmuir–Blodgett. f Photograph of a Ti3C2Tx ﬁlm deposited on a PET substrate that presents excellent transparency and ﬂexibility. b 0 nm 15.2 nm c Substrate 1 Layer 2 Layers (overlap) AFM OM a b c a d d e Spin coating LB coating Dip coating Self assembly Spray coating This work RGO Graphene f f e e Fig. 3 Characterization of the self-assembled Ti3C2Tx thin ﬁlm as a transparent electrode. a OM image of the assembled Ti3C2Tx thin ﬁlm (scale bar: 10 μm); b AFM image and the height proﬁle of the assembled Ti3C2Tx thin ﬁlm in corresponding area marked in a (scale bar: 2 μm). c Thickness distributions extracted from the AFM image (top) and the OM image (bottom); the Y-axes are probability density, namely, frequency per unit height; d Transmittance spectra of Ti3C2Tx ﬁlms with various concentrations of injected Ti3C2Tx; e Transmittance at 550 nm as a function of sheet resistance for the Ti3C2Tx ﬁlms and data from previously reported transparent electrode technologies; RGO reduced graphene oxide, LB Langmuir–Blodgett. f Photograph of a Ti3C2Tx ﬁlm deposited on a PET substrate that presents excellent transparency and ﬂexibility. resistance measurement technique. The UV-vis spectra of these ﬁlms with different Ti3C2Tx area concentrations are presented in Fig. 3d, where the area concentration is deﬁned as the mass of Ti3C2Tx per square centimeter, and tuned by the volume of 2 mg/ml Ti3C2Tx dispersion injected at the interface during the assembly. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 The transmittance of LLIA Ti3C2Tx at 550 nm reaches 96.8% when the area concentration is 1 µg cm−2, and decreases steadily to 64.3% as the area concentration increases to 60 µg cm−2 32. The transmittance at 550 nm versus the sheet resistance (Rs) of the LLIA ﬁlms, together with the data from previously reported techniques, are plotted in Fig. 3e. The Rs value of the LLIA Ti3C2Tx ﬁlm reaches 1623 Ω sq−1 at a transmittance of 96.9%, 210 Ω sq−1 at 94.1%, and 123 Ω sq−1 at 87.8%. Comparing the resistances of materials with the similar transparency, our values are lower than MXene ﬁlms made by other methods like spin coating (201 Ω sq−1 at 87%), dip coating (4300 Ω sq−1 at 94%), and spray coating (7974 Ω sq−1 at 82%), and also lower than other transparent conductive materials such as graphene, and reduced graphene oxide (see Supplementary Table 1 for a summary of different transparent conductive materials)30,31,33–40. Finally, Fig. 3f shows that the LLIA Ti3C2Tx ﬁlm can be transferred onto a ﬂexible substrate (e.g., PET) uniformly in a large area (~25 cm2) with excellent optical transparency, suitable for transparent conductive electrodes for the proposed ﬂexible EC devices. resistance measurement technique. The UV-vis spectra of these ﬁlms with different Ti3C2Tx area concentrations are presented in Fig. 3d, where the area concentration is deﬁned as the mass of Ti3C2Tx per square centimeter, and tuned by the volume of 2 mg/ml Ti3C2Tx dispersion injected at the interface during the assembly. The transmittance of LLIA Ti3C2Tx at 550 nm reaches 96.8% when the area concentration is 1 µg cm−2, and decreases steadily to 64.3% as the area concentration increases to 60 µg cm−2 32. The transmittance at 550 nm versus the sheet resistance (Rs) of the LLIA ﬁlms, together with the data from previously reported techniques, are plotted in Fig. 3e. The Rs value of the LLIA Ti3C2Tx ﬁlm reaches 1623 Ω sq−1 at a transmittance of 96.9%, 210 Ω sq−1 at 94.1%, and 123 Ω sq−1 at 87.8%. NATURE COMMUNICATIONS \| (2021) 12:1587 \| https://doi.org/10.1038/s41467-021-21852-7 \| www.nature.com/naturecommunicatio NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 h b l d b b l h d ff f h ﬁl ) l l h l h f Fig. 4 Electrochromic properties of the TiO2/Ti3C2Tx heterostructure. a OM image of a junction between a 1-layer TiO2 ﬁlm and the TiO2/Ti3C2Tx heterostructure (Top) (Scale bar: 10 μm), and AFM image and the height proﬁle of across the junction in corresponding area marked in the OM image (Bottom) (Scale bar: 1 μm); b Photographs of the TiO2/Ti3C2Tx/PET ﬁlm with different layers of TiO2 at the bleaching state (no bias) and the colored state (biased at −1.6 V). Inset: structure of TiO2/Ti3C2Tx/PET ﬁlm with different TiO2 layers; c Optical transmittance spectra with 1, 4, and 7 layers of TiO2 at the biases of 0 V (solid) and −1.6 V (dotted); d Optical density change (ΔOD) as a function of the injected charge density for the heterostructures with 1, 4, and 7 layers of TiO2; e Temporal response of the transmittance at 550 nm of the heterostructures with 1, 4, and 7 layers of TiO2; f Diffusion coefﬁcients (D) of the heterostructures with 1−7 layers of TiO2 extracted from the EIS and CV measurements; g Coloration efﬁciency (CE) as a function of coloration time (τc) for the TiO2/Ti3C2Tx heterostructures and previously reported TiO2-based structures; h Schematic of the pathways of electron conduction and ion diffusion within the self-assembled TiO2 thin ﬁlm. ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467 021 21852 7 f the TiO2/Ti3C2Tx heterostructure. a OM image of a junction between a 1-layer TiO2 ﬁlm and the TiO2/Ti3C2Tx Fig. 4 Electrochromic properties of the TiO2/Ti3C2Tx heterostructure. a OM image of a junction between a 1-layer TiO2 ﬁlm and the TiO2/Ti3C2Tx heterostructure (Top) (Scale bar: 10 μm), and AFM image and the height proﬁle of across the junction in corresponding area marked in the OM image (Bottom) (Scale bar: 1 μm); b Photographs of the TiO2/Ti3C2Tx/PET ﬁlm with different layers of TiO2 at the bleaching state (no bias) and the colored state (biased at −1.6 V). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 Inset: structure of TiO2/Ti3C2Tx/PET ﬁlm with different TiO2 layers; c Optical transmittance spectra with 1, 4, and 7 layers of TiO2 at the biases of 0 V (solid) and −1.6 V (dotted); d Optical density change (ΔOD) as a function of the injected charge density for the heterostructures with 1, 4, and 7 layers of TiO2; e Temporal response of the transmittance at 550 nm of the heterostructures with 1, 4, and 7 layers of TiO2; f Diffusion coefﬁcients (D) of the heterostructures with 1−7 layers of TiO2 extracted from the EIS and CV measurements; g Coloration efﬁciency (CE) as a function of coloration time (τc) for the TiO2/Ti3C2Tx heterostructures and previously reported TiO2-based structures; h Schematic of the pathways of electron conduction and ion diffusion within the self-assembled TiO2 thin ﬁlm. of the ﬁlm). Particularly, ΔT at the wavelength of 550 nm increases gradually with the layer numbers of the transferred LLIA TiO2 ﬁlms (7.19%, 12.26%, 21.12%, 25.60%, 32.12%, 38.20%, and 41.09% from 1 to 7 layers) (Supplementary Table 2). ΔT reaches its maximum at around 60% with 15 layers of LLIA TiO2 ﬁlms and then declines as the layers number reaches to 20. This is because the transmittance of the bleach state decreases signiﬁcantly when the ﬁlm becomes thick, resulting in the decrease of the optical window (Supplementary Fig. 13). We further extracted Δα = αc – αb ≈[ln(Tc/Tb)]/L from the transmittance spectra. Compared to the TiO2-based EC ﬁlm prepared using other methods (Supple- mentary Table 3)16,19,46–59, our LLIA TiO2 ﬁlm shows a Δα of 0.12 nm−1, which is about one order of magnitude higher. This when no bias is applied, but become translucent with different opacity at the colored state, when emerged in an electrolyte solution (1 M LiClO4/propylene carbonate (PC)) and biased with a constant negative voltage (−1.6 V). This optical opacity induced by a negative potential is associated with the reduction of TiO2 by Li+ counterions and the formation of LixTiO245. As the in situ UV-Vis-NIR spectroscopy measurements shown (Fig. 4c and Supplementary Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 They are almost indistinguishable at the bleach state, Fast and highly efﬁcient electrochromism in MXene/TiO2 heterostructures. The ﬂexible TiO2/Ti3C2Tx heterostructure electrodes are fabricated by transferring LLIA TiO2 ﬁlms on the Ti3C2Tx/PET substrates. Similar to Ti3C2Tx, TiO2 ﬂakes in the 5 TURE COMMUNICATIONS \| (2021) 12:1587 \| https://doi.org/10.1038/s41467-021-21852-7 \| www.nature.com/naturecommunications ARTICLE ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 the LLIA TiO2 ﬁlm due to its high surface-to-volume ratio, which will be explained later. results suggest that more TiO2 per unit volume in our LLIA TiO2 ﬁlm participate in the EC process. Such high utilization rate of TiO2 in the LLIA ﬁlms is one of the key factors responsible for the improved EC performance. To benchmark the overall EC performance, the values of CE and τc of the LLIA TiO2/Ti3C2Tx heterostructures as well as previously reported EC materials are summarized in Fig. 4g and Supplementary Table 3. It is clearly observed that both the CE and the response time of our proposed structures are several times or even one order of magnitude better than those from both other materials such as WO3, and other nanostructured TiO2, including dense ﬁlms and other 0D or 1D nanostructures, and comparable to organic materials. Moreover, the good cycling stability of our LLIA TiO2/Ti3C2Tx heterostructures could address the ultraviolet/electrochemical decomposition issues in traditional organic materials in practical applications18. To quantify the utilization rates of both the EC material and the pumped charge, the optical density change (ΔOD = log10(Tb/T)) as the function of the pumped charge density (Q, calculated by an integration of the driving current density over time) during the coloration half-cycle is measured and plotted in Fig. 4d. The coloration efﬁciencies (CE)60, deﬁned as the slope of the ΔOD-Q plot when Q is close to 0, for the LLIA TiO2 ﬁlms with 1–7 layer numbers are extracted to be 277, 271, 260, 263, 259, 245, and 243 cm2 C−1, respectively (Fig. 4d and Supplementary Table 2). These high CE values indicate that small amounts of charge insertion/extraction can result in a large optical modulation (normally CE values of TMO-based EC materials are lower than 100 cm2 C−1), which is a results of high utilization rates of both the EC materials and the pumped charges. High CE also guarantees the long-term electrochemical stability. As expected, the LLIA ﬁlms show maintained optical contrast (up to 95% of its original value) after 500 EC cycles (Supplementary Fig. 14 and Supplementary Fig. 15). This high cycling stability is essential for practical applications of the EC devices. We believe that the superiority of the EC performance of the LLIA TiO2/Ti3C2Tx heterostructures mainly results from the 2D nature of the TiO2 ﬂakes as well as the loosely networked structures. ARTICLE Given that τ is proportional to the square of the depth of the diffusion, we speculate that the effective diffusion depth is likely to be determined by the average thickness of individual TiO2 ﬂakes, rather than the thickness of the entire assembled TiO2 ﬁlm. Last, the increasing trend between D and the thickness indicates that it is less likely that the extremely slow internanosheet diffusion is contributed to the measured apparent diffusion coefﬁcient. Instead, D follows a parabolic trend with the thickness (see Supplementary Fig. 18), or the number of layers, which agrees well if considering the contribution from the electrochemical area AEC and assuming a linear increase of AEC with the number of layers, that is, D~(AEC/A)2D0 ∝N2, where A is the geometrical area, D0 is the intrinsic diffusion coefﬁcient, and N is the number of layers. All these experimental observations can be explained well if the performance is governed by the second ion transport pathway, in which all the surfaces of the 2D TiO2 ﬂakes are interfacing well with the electrolyte, and the actual diffusion length is approximately the thickness of individual TiO2 ﬂakes (Fig. 4h). As a comparison, for 1D or 0D nanostructures, although the ideal surface-to-volume might be even larger, the surfaces may not be fully exposed to the electrolyte because of the denser packing of these nanostructures in a ﬁlm. Consequently, the Li+ ions have to undergo the ﬁrst pathway in order to access the deeper EC materials, which tremendously limits the speed and thus reduces the coloration efﬁciency. y Ion diffusion process inside the EC material plays a crucial role in the EC performance. To further explore the Li+ diffusion behavior in the LLIA TiO2 ﬁlms during the EC process, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) are measured (details are listed in the Method section). All LLIA ﬁlms exhibit one pair of redox peaks in their CV curves, corresponding to the reduction and oxidation of TiO2 (Supple- mentary Fig. 16). According to the EIS results shown in Supplementary Fig. 17 and Supplementary Table 4, the equivalent series resistance (ESR) does not change much as the TiO2 thickness increases from 1 to 7 layers, and the charge-transfer resistance (Rct) only raises from 8.7 to 15.7 Ω, indicating that multilayer stacking does not decrease the conductivity and charge-transfer speed much at the interface of the TiO2 ﬁlm and electrolyte. ARTICLE To understand this, we propose two possible ion transport pathways15. The ﬁrst ion transport pathway is that the Li+ ions enters the EC material only on the top surface, and the redoxation deeper into the EC material is accessed by inter-crystal-lattice diffusion of Li+ ions; whereas in the second ion transport pathway (as illustrated in Fig. 4h), Li+ ions could travel to all the surfaces of individual EC nanostructures efﬁciently in the liquid phase, and as a result, the diffusion and the redoxation take place throughout the entire EC nanostructures simultaneously. For dense ﬁlms, only the ﬁrst pathway takes place, resulting in very bad coloration efﬁciency and very slow coloration/bleaching time; while for nanostructured ﬁlms (including our 2D nanostructures and previously reported 0D or 1D nanostructures), the second pathway, which is much more efﬁcient and much faster, starts to play an important role. The higher probability the second pathway takes place in a nanos- tructured ﬁlm, the better EC performance it can achieve. The coloration/bleaching time τc/τb, another important ﬁgure- of-merit for EC materials, deﬁned as the time needed for 90% of the total optical change, is also investigated on the LLIA TiO2/Ti3C2Tx heterostructures. The temporal responses of the transmittance at 550 nm are measured as the bias is switched between −1.6 and 0 V periodically (Fig. 4e). The coloration/ bleaching times for 1–7 layers of the LLIC TiO2 ﬁlms are extracted to be 0.76/0.15 s, 0.73/0.12 s, 1.03/0.20 s, 1.03/0.23 s, 1.08/0.26 s, 1.23/0.29 s, and 1.71/0.36 s (also summarized in Supplementary Table 2), which are much shorter than those of reported TiO2-based EC devices (generally >10 s) and most reported inorganic materials, and comparable to those of organic EC materials (Supplementary Table 3). , p Our experimental results suggest that the second pathway is the dominating Li+ ion transport mechanism in our self- assembled 2D TiO2 ﬁlms. First, the high CE and its small decrease with the thickness suggest that Li+ ions can easily access all the TiO2 ﬂakes regardless of the depth of them from the top surface of the ﬁlm. Second, the color change time τ (τc or τb) is fast and stays almost the same with different thicknesses. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 12), the optical modulation range ΔT changes with the increase of the number of layers of the transferred LLIA TiO2 ﬁlm, where ΔT is deﬁned as ΔT = Tb −Tc = exp(−αbL) −exp(−αcL) (Tb and Tc are the transmittance of the bleach state and the colored state, αb and αc represent absorption coefﬁcient of the bleach and the colored state of the LLIA ﬁlm, and L is the thickness NATURE COMMUNICATIONS \| (2021) 12:1587 \| https://doi.org/10.1038/s41467-021-21852-7 \| www.nature.com/naturecommunications 6 ARTICLE y The apparent ion diffusion coefﬁcient D, which represents the kinetics of ion insertion/extraction in the EC ﬁlm, can be extracted either from the scanning-rate-dependent CV mea- surements, or from the Warburg region of the Bode plot obtained from the EIS measurements (see methods for details about the extraction methods of D)61. As shown in Fig. 4f, the D values extracted from the CV and from the EIS measurements are 5.05 × 10−14, 9.63 × 10−14, 2.68 × 10−13, 1.15 × 10−12, 5.45 × 10−12, 6.29 × 10−12, and 1.10 × 10−11 cm2 s−1, and 2.75 × 10−12, 3.94 × 10−12, 6.59 × 10−12, 1.21 × 10−11, 1.39 × 10−11, 1.88 × 10−11, 2.24 × 10−11 cm2 s−1, respectively, as the number of layer of the TiO2 ﬁlm increases from 1 to 7. The positive correlation between D and the layer number is attributed to the increased effective electrochemical area of 7 TURE COMMUNICATIONS \| (2021) 12:1587 \| https://doi.org/10.1038/s41467-021-21852-7 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 properties of the TiO2/Ti3C2Tx heterostructure and demonstration of a large-area ﬂexible EC device. a Comparison of the sheet e LLIA Ti3C2Tx/PET and the ITO/PET ﬁlms after 1000 bending cycles; b Comparison of the electrochromic switching behaviors of the PET and the ITO/PET ﬁlms after 1000 bending cycles; c Schematic diagram of the ﬂexible solid electrochromic device; d, e As-prepared device with an A4 paper size (about 20 × 30 cm) is d triggered in bending state and e attached on a window. Fig. 5 Mechanical properties of the TiO2/Ti3C2Tx heterostructure and demonstration of a large-area ﬂexible EC device. a Comparison of the sheet resistance (Rs) of the LLIA Ti3C2Tx/PET and the ITO/PET ﬁlms after 1000 bending cycles; b Comparison of the electrochromic switching behaviors of the LLIA TiO2/Ti3C2Tx/PET and the ITO/PET ﬁlms after 1000 bending cycles; c Schematic diagram of the ﬂexible solid electrochromic device; d, e As-prepared solid electrochromic device with an A4 paper size (about 20 × 30 cm) is d triggered in bending state and e attached on a window. Fig. 5 Mechanical properties of the TiO2/Ti3C2Tx heterostructure and demonstration of a large-area ﬂexible EC device. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 Finally, a large-area ﬂexible EC device with a dimension of 30 × 20 cm are demonstrated as an example for practical large-area applications. As shown in Fig. 5c, it is composed of a LLIA TiO2/ Ti3C2Tx heterostructure on a PET substrate as the EC electrode, another LLIA Ti3C2Tx/PET ﬁlm as the counter electrode, and a 1 M LiClO4/PC/Poly(methyl methacrylate) (PMMA) layer as the electrolyte. Beneﬁting from the exceptional optical memory effect of TiO2 based materials, the EC device can maintain its coloration state after disconnecting from the voltage supply (Fig. 5d, e), which is advantageous for energy saving in future applications. The EC device presents a uniform color distribution even in the bending state, which opens up the possibility of scaling up this technology for applications, such as ﬂexible displays, smart windows, and other ubiquitous electronics. In addition, the ﬂexible solid EC device also exhibits excellent cycling stability, demonstrated by the 92% transmittance presentation after 1000 EC cycles between the bias of -3 V and 1 V (S). To assemble the Ti3C2Tx ﬂakes into a continuous ﬁlm, the Ti3C2Tx aerogel was dispersed in an IPA solution (2 mg/ml) by sonicating for 30 min. Hexane was carefully poured on the top of DI water to setup an interface between these two liquids. Then, the Ti3C2Tx/IPA dispersion was slowly injected near the interface to form a continuous ﬁlm. After removing the top hexane layer, the self-assembly ﬁlm was transferred onto a substrate and annealed at 90 °C in vacuum for 50 h (<0.05 MPa). The assembly of the TiO2 ﬁlms is similar to the Ti3C2Tx assembly process. For multilayered TiO2 ﬁlm, the transferred ﬁlms were dried at 60 °C in vacuum for 1 h, and then treated with oxygen plasma (the oxygen gas ﬂow rate is 50 sccm, and the plasma power is 100 W) for 30 s before each transfer cycle. The TiO2/Ti3C2Tx heterostructures were annealed at 90 °C in vacuum (<0.05 MPa) for 50 h before use. Assembly of the Electrochromic device. The electrolyte gel was obtained by mixing 1 M LiClO4 in a PC solution and 20 wt% PMMA (relative to the LiClO4/PC solution). For the large-area (20 × 30 cm) electrode, silver paste line is screen printed around the Ti3C2Tx/PET ﬁlm and then protected by continuous printed UV curing resin to improve the electrical conductivity. Discussion Material characterization. X-ray diffraction (XRD) patterns were measured using an X-ray diffractometer (Bruker AXS X8 Proteum-R instrument) with the radiation at 40 kV and 200 mA. Raman spectroscopy measurements were performed on a Renishaw inVia Raman microscope equipped with a 532 nm laser line (The laser power on the samples is about 1.6 mW). All the spectra in comparison were taken under the same conditions. Transmission electron microscopy (TEM) measure- ments were performed on a FEI Tecnai Osiris TEM. Selected area electron dif- fraction (SAED) was measured on a JEOL 2100 TEM. AFM measurements were conducted on a Bruker Dimension system. X-ray photoelectron spectroscopy (XPS) measurements were performed using a PHI Versaprobe II. The UV-vis spectroscopy was carried out using a Agilent CARY 5000. In summary, high performance and ﬂexible EC devices are rea- lized based on assembled 2D TiO2/Ti3C2Tx heterostructures. This success beneﬁts from several important achievements in this work, including the realization of single crystalline 2D TiO2 ﬂakes with micrometer large lateral sizes and nanometer thin thick- nesses, and the liquid/liquid interfacial self-assembly technique enabled mass production of MXene and 2D TMO ﬁlms with excellent uniformity. We expect our approach to synthesize 2D TiO2 crystals to be developed to a general methodology for the synthesis of high-quality 2D MXene derivatives. Also, the self- assembled MXene ﬁlms could be used as ﬂexible, transparent, and porous electrodes in large-scale electronic, optoelectronic, and energy-harvesting applications. Our performance test clearly shows that exceptional electrochromic efﬁciency, fast coloration speed, and excellent mechanical ﬂexibility are achieved simulta- neously on the self-assembled 2D TMO/MXene heterostructures- based EC devices, thanks to the unique electrical, electrochemical, and mechanical properties of the networked 2D nanostructures. Finally, a large-area ﬂexible EC device is demonstrated, paving the way for integrating EC devices to next-generation ﬂexible and wearable electronics. Electrochemical measurements and parameter extraction. Electrochemical measurements were carried out in a PC solution containing 1 M LiClO4 electrolyte using three-electrode electrochemical cells with the TiO2/Ti3C2Tx/PET ﬁlms as the working electrodes, a platinum plate as the counter electrode, and a freshly pre- pared Ag/Ag+ as the pseudo-reference electrode (calibrated by ferrocene). Elec- trochemical impedance spectroscopy (EIS) and cycle voltammetry (CV) were conducted by an electrochemical workstation (Bio-logic SAS, VSP-300) over a frequency range of 100 mHz–1000 KHz. The measured EIS spectra was ﬁtted with a circuit model illustrated in u. Discussion The Nyquist plot of all ﬁlms is composed of three parts: an equivalent series resistance (ESR) in series with a semicircle consisting of a non-faradaic double layer capacitance (Cdl) and a charge-transfer resistance (Rct) at high frequency; a 45° linear region representing the Warburg element (W), which captures the ion diffusion process at medium frequency; and a line related to the capacitive charging behavior of the pseudo-capacitance (Cp) of TiO2 ﬁlm and its bulk resistance (Rp) at low frequency. The apparent ion diffusion coefﬁcient D can be extracted from both the EIS measurements and the CV measurements. In the ﬁrst method, DEIS can be calculated from the Warburg region according to Eqs. (1) and (2):61,62 Methods Ch i l ip ¼ 2:687 ´ 105 ´ n3=2 ´ D1=2 CV ´ C ´ A ´ v1=2 ð3Þ ip ¼ 2:687 ´ 105 ´ n3=2 ´ D1=2 CV ´ C ´ A ´ v1=2 ð3Þ etching was performed following Gogosti et al. s work to prepare 2D Ti3C2Tx. Brieﬂy, 1.6 g lithium ﬂuoride was dissolved into 5 mL of DI water with 15 mL 12 M hydrochloric acid solution in a plastic bottle as the etchant solution. Then 1 g Ti3AlC2 (MAX phase) powders was gradually added to the etchant solution within 5 min and the reaction is maintained with stirring the reactants at 40 °C in a silicon oil bath for 24 h. Etched MXene was washed with DI water, and separated through centrifugation at 1988 × g for 5 min. Washing was repeated several times until a stable dark where v represents the potential sweeping rate, ip represents the peak current density. where v represents the potential sweeping rate, ip represents the peak current density. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 The EC devices were fab- ricated by hot pressing the electrolyte gel sandwiched betwen the TiO2/Ti3C2Tx/ PET electrode and the Ti3C2Tx /PET counter electrode with a scotch tape spacer (with the thickness of 1 mm) at 60 °C. ARTICLE a Comparison of the sheet resistance (Rs) of the LLIA Ti3C2Tx/PET and the ITO/PET ﬁlms after 1000 bending cycles; b Comparison of the electrochromic switching behaviors of the LLIA TiO2/Ti3C2Tx/PET and the ITO/PET ﬁlms after 1000 bending cycles; c Schematic diagram of the ﬂexible solid electrochromic device; d, e As-prepared solid electrochromic device with an A4 paper size (about 20 × 30 cm) is d triggered in bending state and e attached on a window. MXene/TiO2 heterostructure enabled ﬂexible EC devices for large-area applications. The overall ﬂexibility of an EC device is determined by the most rigid layer. In our TiO2/Ti3C2Tx hetero- structure, both the EC layer and the transparent electrode have superior mechanical bendability. To conﬁrm this, bending test is performed on the as-fabricated LLIA electrode. As shown in Fig. 5a, the resistance of the Ti3C2Tx/PET ﬁlm increases by only 6% after 1000 bending and release cycles at a bending radius of 0.3 cm, whereas the sheet resistance of a commercial ITO/PET ﬁlm increases quickly by three orders of magnitude after 1000 bending cycles even at a much larger bending radius of 1.5 cm. The EC optical switching behaviors of the TiO2/Ti3C2Tx/PET ﬁlm and the TiO2/ITO/PET before and after 1000 bending cycles are also tested (Fig. 5b). ΔT of the TiO2/Ti3C2Tx/PET electrode remains at 94% of its original value after 1000 bending cycles. In contrast, ΔT of the TiO2/ITO/PET electrode drops to about 30% after 1000 cycles. NATURE COMMUNICATIONS \| (2021) 12:1587 \| https://doi.org/10.1038/s41467-021-21852-7 \| www.nature.com/naturecommunications 8 Methods Ch i l Chemicals. All the starting materials are available commercially and used as received. Polyethylene terephthalate (PET) was rinsed sequentially in the baths of deionized water, acetone, and isopropanol. Ti3AlC2 (MAX-Phase, purchased from 11 Technology Co. Ltd), lithium ﬂuoride (LiF, Alfa Aesar, 99.99%), hydrochloric acid (HCl, Fisher Chemical, 6M), 1-hexene (Acros Organics, 99%), isopropyl alcohol (IPA, Fisher Chemical, 99%), propylene carbonate (PC, Alfa Aesar, 99%), lithium perchlorate (LiClO4, Acros Organics, 99+%), and poly(methyl methacry- late) (PMMA, ACROS Organics, M.W. 35000) were utilized without initial treatments. Zw ¼ σω1=2 ð1Þ Zw ¼ σω1=2 ð1Þ DEIS ¼ R2T2=ð2A2n4F4C2σ2Þ ð2Þ ð1Þ Zw σω ð1Þ DEIS ¼ R2T2=ð2A2n4F4C2σ2Þ ð2Þ DEIS ¼ R2T2=ð2A2n4F4C2σ2Þ ð2Þ ð2Þ where σ is the slope of Zw against ω−1/2, R is the gas constant, T is the absolute temperature, A is the effective working area (geometrical area), n is the number of electrons involved in the electrochemical reaction, F and C are the Faraday d h l i f Li+ i onstant and the molar concentration of Li+ ions. y constant and the molar concentration of Li+ ions. In the second method, the apparent ion diffusion coefﬁcients DCV can be calculated by:63 Synthesis and interfacial assembly of Ti3C2Tx and 2D TiO2 ﬁlms. Ti3AlC2 etching was performed following Gogosti et al.’s work22 to prepare 2D Ti3C2Tx. Synthesis and interfacial assembly of Ti3C2Tx and 2D TiO2 ﬁlms. Ti3AlC2 etching was performed following Gogosti et al.’s work22 to prepare 2D Ti3C2Tx. 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Cells 157, 624–635 (2016). 23. Khalifa, Z. ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 Data availability All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Information. Additional data related to this paper may be requested from the authors. The Ti3C2Tx sedimen was diluted to 0.2 mg/ml with DI water, and then, the solution were freeze dried at −47 °C for days to form an aerogel. To obtaine the TiO2 aerogel, the Ti3C2Tx aerogel was put in a ceramic boat and annealed at the temperature of 400 °C for 50 h with the heating rate of 1 °C/min. Received: 19 July 2020; Accepted: 4 February 2021; 9 NATURE COMMUNICATIONS \| (2021) 12:1587 \| https://doi.org/10.1038/s41467-021-21852-7 \| www.nature.com/naturecommunications Acknowledgements d k l d 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/. H.W. and R.L. acknowledge the ﬁnancial support of the Natural Science Foundation of China (Grant No.51972054) and Fundamental Research Funds for the Central Universities (18D110308). X.L., J.C., T.L., and T.P. acknowledge the support from Semiconductor Research Corporation (SRC). X.L., J.C., and T.L. also acknowledge the support from the Department of Energy under Award No. DE-SC0021064. X.L. acknowledge the member- ship of the Photonics Centers at Boston University. R.L. acknowledges the support of Graduate Student Innovation Fund of Donghua University (CUSF-DH-D-2019002). Reprints and permission information is available at http://www.nature.com/reprints 63. Tang, S. B., Lai, M. O. & Lu, L. Study on Li+-ion diffusion in nano-crystalline LiMn2O4 thin ﬁlm cathode grown by pulsed laser deposition using CV, EIS and PITT techniques. Mater. Chem. Phys. 111, 149–153 (2008). 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Transparent, ﬂexible, and conductive 2D titanium carbide (MXene) ﬁlms with high volumetric capacitance. Adv. Mater. 29, 1702678 (2017). 10 NATURE COMMUNICATIONS \| (2021) 12:1587 \| https://doi.org/10.1038/s41467-021-21852-7 \| www.nature.com/naturecommunicatio ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-21852-7 Competing interests 58. Zhang, S. et al. Preparation, characterization, and electrochromic properties of nanocellulose-based polyaniline nanocomposite ﬁlms. ACS Appl. Mater. Inter. 9, 16426–16434 (2017). © The Author(s) 2021 Author contributions R.L. and X.M. performed most of experiments. R.L., X.M., X.Z., and L.W. prepared the samples, J.L., J.C., H.G., and T.L. characterized the samples. G.W., C.H., and Y.L. ana- lyzed the data, Q.Z., Y.Li, Y.L., H.W., T.P., and X.L. designed experiments. All authors discussed the results and commented on the manuscript. 11 NATURE COMMUNICATIONS \| (2021) 12:1587 \| https://doi.org/10.1038/s41467-021-21852-7 \| www.nature.com/naturecommunicatio
https://openalex.org/W4243177809	https://europepmc.org/articles/pmc7805881?pdf=render	English	null	Repetitive robot behavior impacts perception of intentionality and gaze-related attentional orienting.	null	2,021	cc-by	10,644	Repetitive Robot Behavior Impacts Perception of Intentionality and Gaze-Related Attentional Orienting Social Cognition in Human-Robot Interaction (S4HRI) Unit, Istituto Italiano di Tecnologia, Genova, Italy Social Cognition in Human-Robot Interaction (S4HRI) Unit, Istituto Italiano di Tecnologia, Genova, Italy Gaze behavior is an important social signal between humans as it communicates locations of interest. People typically orient their attention to where others look as this informs about others’ intentions and future actions. Studies have shown that humans can engage in similar gaze behavior with robots but presumably more so when they adopt the intentional stance toward them (i.e., believing robot behaviors are intentional). In laboratory settings, the phenomenon of attending toward the direction of others’ gaze has been examined with the use of the gaze-cueing paradigm. While the gaze-cueing paradigm has been successful in investigating the relationship between adopting the intentional stance toward robots and attention orienting to gaze cues, it is unclear if the repetitiveness of the gaze-cueing paradigm inﬂuences adopting the intentional stance. Here, we examined if the duration of exposure to repetitive robot gaze behavior in a gaze-cueing task has a negative impact on subjective attribution of intentionality. Participants performed a short, medium, or long face-to-face gaze-cueing paradigm with an embodied robot while subjective ratings were collected pre and post the interaction. Results show that participants in the long exposure condition had the smallest change in their intention attribution scores, if any, while those in the short exposure condition had a positive change in their intention attribution, indicating that participants attributed more intention to the robot after short interactions. The results also show that attention orienting to robot gaze-cues was positively related to how much intention was attributed to the robot, but this relationship became more negative as the length of exposure increased. In contrast to subjective ratings, the gaze-cueing effects (GCEs) increased as a function of the duration of exposure to repetitive behavior. The data suggest a tradeoff between the desired number of trials needed for observing various mechanisms of social cognition, such as GCEs, and the likelihood of adopting the intentional stance toward a robot. Keywords: social cognition, human robot interaction, gaze cueing, intentional stance, intention attribution, attention orienting Edited by: Astrid Weiss, Vienna University of Technology, Austria Edited by: Astrid Weiss, Vienna University of Technology, Austria Reviewed by: Emilia I. Barakova, Eindhoven University of Technology, Netherlands Barbara Kühnlenz, Hochschule Coburg, Germany Correspondence: Abdulaziz Abubshait abdulaziz.abubshait@iit.it Reviewed by: Emilia I. Barakova, Eindhoven University of Technology, Netherlands Barbara Kühnlenz, Hochschule Coburg, Germany Reviewed by: Emilia I. Barakova, Eindhoven University of Technology, Netherlands Barbara Kühnlenz, Hochschule Coburg, Germany Correspondence: Abdulaziz Abubshait abdulaziz.abubshait@iit.it Specialty section: This article was submitted to Human-Robot Interaction, a section of the journal Frontiers in Robotics and AI Received: 26 May 2020 Accepted: 25 September 2020 Published: 09 November 2020 ORIGINAL RESEARCH published: 09 November 2020 doi: 10.3389/frobt.2020.565825 Citation: Although prior studies (e.g., Marchesi et al., 2019) have uncovered instances where people do adopt the intentional stance toward humanoid robots, here we ask if certain procedures used in experimental design can actually create obstacles for measuring the intentional stance in human–robot interaction (HRI). Several studies do in fact show that people are capable of adopting the intentional stance toward artiﬁcial agents in some cases. For example, when robots, avatars, or computers perform unexpected actions (Morewedge, 2009; Waytz et al., 2010), resemble humans in their physical appearance (Kiesler et al., 2008; Admoni et al., 2011), display emotions (Fussell et al., 2008), react to people’s actions (Terada et al., 2007), exhibit humanlike non-verbal behaviors such as shrugging (Carter et al., 2014), cheat (Short et al., 2010), or engage in eye contact with others (Ito et al., 2004; Yonezawa et al., 2007), humans are more likely to adopt the intentional stance toward them. When humans adopt the intentional stance, it can lead to positive outcomes in HRI1 as robots that are perceived as intentional are seen as more trustworthy (Kiesler et al., 2008; Carter et al., 2014), are able to engage humans better (Yamazaki et al., 2010), and have positive eﬀects on people’s moods (Carter et al., 2014). Adopting the intentional stance toward robots can also have positive eﬀects on human performance in HRI as they can facilitate learning (Brown and Howard, 2013), improve physical interactions where hand– eye coordination is needed (Carter et al., 2014), induce social facilitation eﬀects (Bartneck, 2003; Woods et al., 2005; Looije et al., 2010), and improve interactions in team settings (Breazeal et al., 2005). By the same token, not adopting the intentional stance toward social robots can pose a problem for HRI, as humans might be engaging fewer socio-cognitive mechanisms in social interactions with robots, and thereby less social attunement (i.e., activation of socio-cognitive mechanisms) occurs between the two agents (Wiese et al., 2012, 2018; Wykowska et al., 2014; Özdem et al., 2016; Caruana et al., 2017; Ciardo et al., 2020; for a review, see Perez-Osorio et al., 2015; Wiese et al., 2017; Schellen and Wykowska, 2019). g More recent studies have shown that attention orienting to gaze cues can be top-down modulated (i.e., volitional), depending on the social relevance of the gaze. Citation: Abubshait A and Wykowska A (2020) Repetitive Robot Behavior Impacts Perception of Intentionality and Gaze-Related Attentional Orienting. Front. Robot. AI 7:565825. doi: 10.3389/frobt.2020.565825 Humans rely on non-verbal behavior to interact with one another. For example, when we observing someone looking at an apple, we infer that that person is hungry. This process, in which people make inferences about what others are thinking, is called mentalizing (Baron-Cohen, 1995), and is essential for successful human social interactions (Frith and Frith, 2006). Mentalizing is a highly automatic process that we engage in (Frith and Frith, 2006). Assuming that others’ behavior is based November 2020 \| Volume 7 \| Article 565825 Frontiers in Robotics and AI \| www.frontiersin.org Repetitive Behavior Impacts HRI Abubshait and Wykowska 2011). The gaze-cueing paradigm generally starts with a gazing stimulus (i.e., a face or face-like drawing) that looks directly at an observer (i.e., participant) and then makes a gaze shift to a certain direction (e.g., right or left), which shifts the observer’s attention to the gazed-at location (Friesen and Kingstone, 1998; Driver et al., 1999; Langton and Bruce, 1999; Quadﬂieg et al., 2004), if the observer engages in joint attention with the gazer. After the gaze- shift, a target appears either in the same (i.e., valid trial) or a diﬀerent location (i.e., invalid trail) relative to the gaze cue. Studies have consistently shown that people are faster at responding to valid trials compared to invalid trials, with the magnitude of the diﬀerence between valid reaction times and invalid reaction times indicative of how strongly or how frequently attention was oriented to the gazed-at location (i.e., the gaze-cueing eﬀect: GCE). Studies investigating the GCE have also shown that the eﬀects are observed even when gaze was counter- indicative of the target location, which suggests the reﬂexivity of attention orienting to gaze cues (Ristic and Kingstone, 2005; Kingstone et al., 2019). on their thoughts, emotions, and internal states is called adopting the intentional stance (Dennett and Haugeland, 1987; Dennett, 1989). While it is assumed that when humans interact with one another they adopt the intentional stance (i.e., they explain and predict others’ behaviors with reference to their underlying thoughts, feelings, and intentions), an interesting question remains if (and when) people adopt the intentional stance toward artiﬁcial agents that resemble humans, such as humanoid robots. Citation: For example, attention orienting to gaze cues is stronger when the gazer is of higher social rank (Shepherd et al., 2006; Jones et al., 2010; Ohlsen et al., 2013; Cui et al., 2014; Dalmaso et al., 2014), is physically similar to the observer (Hungr and Hunt, 2012), is perceived as trustworthy (Süßenbach and Schönbrodt, 2014), has the possibility to see (Teufel et al., 2010), or when gaze cues are meaningful to the context of the gaze (Perez-Osorio et al., 2015). Paramount to our study, prior work has also shown that GCEs are enhanced when gaze shifts are thought to originate from an intentional agent (Wiese et al., 2012; Wykowska et al., 2014; Caruana et al., 2017), compared to just a pre-programmed machine, which suggests that adopting the intentional stance toward a robot can inﬂuence how strongly it can induce attentional orienting to its directional cues. Although previous literature showed that, indeed, people are able to socially attune with a robot, and perhaps more strongly so if they adopt the intentional stance toward it, more recent studies have shown that using artiﬁcial agents in gaze-cueing studies can have negative consequences (Abubshait et al., 2020a). Therefore, it is unclear if classical psychological paradigms such as the gaze-cueing paradigm are the best choice to investigate socio-cognitive mechanisms, due to the fact that the gaze-cueing paradigm is quite unnatural in terms of social context. Not only does it often employ 2D stimuli on the screen (which is unlike natural social interactions), but it also uses repetitive movements (i.e., the gaze-cueing paradigm employs many trials of robots gazing in one of two directions). Prior work has shown negative impacts of multiple exposures on subjective perceptions of robots over time (Bergmann et al., 2012), and recent approaches in social neuroscience (Schilbach et al., 2013) highlight the need for more ecologically valid paradigms for studying mechanisms of social cognition. 1While studies have shown positive outcomes of attributing intentionality to robots in HRI, future studies should consider ethical implications of attributing intentionality toward an inanimate object. For similar arguments (see de Graaf, 2016; Lake et al., 2017). November 2020 \| Volume 7 \| Article 565825 Questionnaires The questionnaires used in the study included the Intentional Stance questionnaire measuring people’s tendencies to adopt the intentional stance toward robots (Marchesi et al., 2019); the anthropomorphism questionnaire measuring people’s anthropomorphic and attachment tendencies (Neave et al., 2015); the Body, Heart and Mind questionnaire measuring people’s perceptions of mental life (Weisman et al., 2017); and ﬁnally a simple perceived predictivity question measuring how much subjects can predict iCub’s actions. The gaze-cueing task also included a question regarding people’s subjective perceptions of their engagement toward iCub (see gaze-cueing task section for details). All items were translated and administered in Italian and can be found on osf.io/y6c9b. The Intentional Stance Questionnaire is a set of 34 scenarios. Each scenario contained three pictures of iCub involved in regular daily scenes (e.g., playing cards with another human, sorting objects into containers). Below each scenario, two sentences were presented that described the scenario, one in mentalistic terms (e.g., “iCub pretends to be a gardener”) presented on the right, and the other in mechanistic terms (e.g., “iCub adjusts the force to the weight of the object”) presented on the left. Participants were tasked to move a slider (positioned initially between the two sentences) toward the description that best ﬁt the scenario. The logic behind this questionnaire was that if participants moved the slider toward the mentalistic description, they likely adopted the intentional stance, while if they chose the mechanistic description, they were more likely to adopt the “design stance” (Dennett and Haugeland, 1987) for explaining the robot’s behavior. Based on a split-halves analysis, the questionnaire was split into two sections that were correlated with each other. The ﬁrst half was presented at the beginning of the study, and the second half was presented at the end of the study, with the order of items of each half presented randomly. The items were scored on a 10-point-magnitude scale with an additional decimal point, with 0 indicating adopting the design stance and 10 indicating adopting the intentional stance. The scores were calculated by averaging all items together. Descriptions of the remaining questionnaires can be found in the Supplementary Material. Apparatus The gaze-cueing experiment used the iCub robot along with two 27-in. screens that were rotated sideways (i.e., vertically) for target presentation (see Kompatsiari et al., 2018 for a similar setup). iCub’s movements were controlled by the YARP gaze platform (Metta et al., 2006). iCub’s eye vergence was set to 5◦and remained constant, while the timing trajectories of the eye and neck movements were set to 200 and 400 ms, respectively, to allow for smoother and natural- looking movements. Data collection and target presentation were controlled and programmed using OpenSesame (Mathôt et al., 2012). Responses were collected using a standard keyboard. In the current study, we examined the eﬀect of the duration of exposure to repetitive gaze behavior on both subjective perceptions of intentionality and gaze-induced attentional orienting in a gaze-cuing paradigm. Using a face-to-face gaze- cueing paradigm with a humanoid robot (i.e., iCub: Metta et al., 2008), participants were assigned to either a short exposure (i.e., 96 trials), a medium exposure (i.e., 176 trials), or a long exposure condition (i.e., 256 trials), while we collected pre- and post-ratings of adopting the intentional stance (InStance Questionnaire, Marchesi et al., 2019) and attributing mental capacities (Weisman et al., 2017). If the duration of interaction with a robot does indeed inﬂuence attribution of intentionality toward a robot, we expect that participants assigned to the diﬀerent exposure conditions would experience changes in likelihood to adopt the intentional stance toward iCub after their interaction diﬀerently. To that extent, we expected to observe a diﬀerent impact of interaction with the robot on the adoption of the intentional stance, depending on duration of the interaction. Additionally, we hypothesized that the relationship between adopting the intentional stance and GCEs (i.e., social attunement via attention orienting) would depend on the duration of exposure to repetitive behavior. Citation: Furthermore, the fact that multiple interactions over time might change our perceptions of a robot is in line with Epley et al’s (2007) theory of anthropomorphism, which suggests that repetitive interactions can decrease our likelihood to attribute One successful paradigm to measure social attunement in HRI is the gaze-cueing paradigm (Wiese et al., 2012; Wykowska et al., 2014; Perez-Osorio et al., 2015; Kompatsiari et al., 2018) as prior work has shown that attentional orienting to gaze cues is the foundation of many (often higher-level) social- cognitive processes, such as mentalizing (Baron-Cohen, 1995; Nummenmaa and Calder, 2009; Teufel et al., 2010; Pfeiﬀer et al., November 2020 \| Volume 7 \| Article 565825 Frontiers in Robotics and AI \| www.frontiersin.org 2 Abubshait and Wykowska Repetitive Behavior Impacts HRI correct priors can provide an advantage to testing our hypotheses (McNeish, 2016). mental states (i.e., adopting the intentional stance) toward a robot since we no longer have the motivation to understand its behavior: eﬀectance motivation. While prior studies have shown negative impacts of multiple exposures on subjective ratings, it is unclear if this eﬀect can also be generalized to behavioral measurements, such as the GCEs. Speciﬁcally, it is unclear if the standard procedure of the gaze-cueing paradigm in HRI conceals aspects of social attunement, due to the fact that many trials are needed to establish an accurate and stable GCE (e.g., face-to-face studies include 160 trials; Lachat et al., 2012). Frontiers in Robotics and AI \| www.frontiersin.org Participants Twenty-seven participants were recruited (M = 24.4; range = 19–49; 17 females; 25 right-handed) and were quasi-randomly assigned to one of three exposure conditions (i.e., short, medium, or long exposure). Participants were compensated 15 euros upon completion of the study. The experiment was conducted in accordance with the World Medical Association Declaration of Helsinki ethical principles for medical research involving human subjects and was approved by the local Ethical Committee (Comitato Etico Regione Liguria). An a priori power analysis was conducted using R version 3.6 and the pwr package to determine our sample size. The analysis was based on an f test of three independent groups, an alpha of 0.05, a medium eﬀect size of 0.4 (based on prior research: Kompatsiari et al., 2018) and power set to (1—b = 0.8). The analysis resulted in a sample size of 63 participants (i.e., 21 participants per group). While the a priori power analysis suggested a larger sample (N = 63), due to nationwide COVID-19 lockdowns, we were only able to collect half of the required sample. To alleviate this issue, we used Bayesian models to test our hypotheses as opposed to parametric analyses. While Bayesian inferences are not immune to symptoms related to small sizes, the use of the November 2020 \| Volume 7 \| Article 565825 Frontiers in Robotics and AI \| www.frontiersin.org Frontiers in Robotics and AI \| www.frontiersin.org 3 Repetitive Behavior Impacts HRI Abubshait and Wykowska FIGURE 1 \| Trial sequence of the gaze-cueing paradigm: Each trial started with iCub’s eyes closed. iCub would then open its eyes and ﬁxate on the observer’s eyes to engage in mutual gaze. Next, iCub would make a gaze shift to either the right or left. Next, a target appeared for 200 ms. If the target appeared in the same direction of the cue, the trial was considered valid. Targets appeared in the opposite direction of the cue for invalid trials. The stimulus onset asynchrony (SOA) was set to 500 ms. FIGURE 1 \| Trial sequence of the gaze-cueing paradigm: Each trial started with iCub’s eyes closed. iCub would then open its eyes and ﬁxate on the observer’s eyes to engage in mutual gaze. Next, iCub would make a gaze shift to either the right or left. Next, a target appeared for 200 ms. If the target appeared in the same direction of the cue, the trial was considered valid. Participants Targets appeared in the opposite direction of the cue for invalid trials. The stimulus onset asynchrony (SOA) was set to 500 ms. Main Analyses While classical parametric and null hypothesis signiﬁcance testing (NHST) require larger sample sizes, we opted to use Bayesian inference to alleviate issues with the inability to collect the desired sample size. Bayesian inference can provide three major advantages that directly inﬂuence the present study. First, we alleviate issues that are related to smaller sample sizes due to the fact that the use of prior distributions to estimate the posterior distributions can help ﬁnd better estimates of Gaze-Cueing Protocol participants. The target stimuli were either a “T” or “V” and were 4.5◦and 7◦. Each trial of the gaze-cueing paradigm started with iCub’s eyes closed. 2,000 ms later, iCub opened its eyes and looked directly at the participant for 500 ms; 2,000 ms later, iCub looked at one of the two screens (i.e., at the left screen or the right screen); 500 ms later, a target (i.e., a T or V) appeared on either of the two screens. On valid trials, the target appeared on the same screen that iCub was looking at. On invalid trials, the target appeared on the opposite screen (e.g., iCub looked at the right screen and the target appeared on the left screen). The target appeared for a duration of 200 ms, and iCub remained looking at the screen until participants responded, which ended the trial. iCub’s gaze validity was non-predictive of the target location (i.e., 50% valid trials and 50% invalid trials). Each block consisted of 16 trials, which ended by asking participants about their subjective perception of iCub’s engagement. The short exposure condition consisted of 96 trials (i.e., 6 blocks), the medium exposure condition consisted of 176 trials (i.e., 11 blocks), and the long exposure condition consisted of 256 trials (i.e., 16 blocks). See Figure 1 for the trial sequence of the Gaze-cueing protocol. Next, participants completed the ﬁrst set of questionnaires, which included an anthropomorphism questionnaire (Neave et al., 2015), the Intentional Stance Questionnaire (ISQ, Marchesi et al., 2019), a mental capacity questionnaire (Weisman et al., 2017), and a perceived predictivity question. After completing the questionnaires, which were presented in random order, participants completed a practice session of the gaze-cueing task with iCub. The practice session contained 16 gaze-cueing trials. One half of participants were instructed to press the “T” key with their right index ﬁnger and the “V” key with their left index ﬁnger, while the other half were instructed to do the opposite. Next, participants completed the experimental gaze- cueing task. Upon completion, participants moved on to the post- questionnaire phase, which included the ISQ, the mental capacity questionnaire, and the predictivity question. Finally, participants were debriefed and thanked for their participation. Procedure After participants provided written consent, they were seated at a desk ∼125 cm away from iCub. The two screens were placed laterally on the desk 32.5 cm away from the robot on each side (i.e., 75 cm apart from one another). The screens were tilted back vertically 12.5◦and rotated 76◦toward the participant from the lateral position. The screens were ∼105 cm away from the Frontiers in Robotics and AI \| www.frontiersin.org November 2020 \| Volume 7 \| Article 565825 4 Repetitive Behavior Impacts HRI Abubshait and Wykowska TABLE 1 \| Instance questionnaire scores. Pre ISQ Post ISQ Difference ISQ Exposure Length M SD M SD M SD Short 4.20 2.96 4.77 2.60 0.57 1.27 Medium 3.18 2.11 3.76 1.28 0.58 1.12 Long 3.98 1.66 4.06 0.08 0.08 1.18 Positive ISQ differences indicates a higher rating at post. TABLE 2 \| Reaction time data. Valid Invalid GCE Exposure Length M SD M SD M SD Short 494.99 59.18 502.64 68.00 7.65 11.80 Medium 484.23 125.46 496.86 118.06 12.62 13.86 Long 477.23 50.63 492.49 56.33 15.26 10.19 GCE is the difference between valid and invalid trials. TABLE 1 \| Instance questionnaire scores. the relationships in question (McNeish, 2016). Second, using Bayesian inference provides the advantage of examining the results in terms of probabilities, and not simply whether the null hypothesis is accepted or rejected, which is easily interpreted and can be generalized to future studies. Finally, we are able to use credible intervals that ﬁt our data and not simply ones that are conventional, as researchers suggest that the conventional 95% credible interval band is not appropriate for Bayesian statistics due to the fact that it restricts our posterior distributions (Kruschke, 2014). While some researchers suggest using an 89% credible interval (McElreath, 2020), we opted to use a 90% credible interval for rounding purposes. For both Bayesian models, we used ﬁve Markov Chain Monte Carlo (MCMC) chains with 10,000 samples. The ﬁrst 500 samples were used as warm-up and discarded. All Bayesian models used weakly informed priors based on the observed mean and a wide SD (i.e., SD = 10). To measure changes in questionnaire scores, we ﬁrst scored the pre-items and post-items separately. Next, we calculated a diﬀerence score between pre- and post-scores (i.e., post-score— pre-score). Positive values would indicate higher scores after exposure. Negative values would indicate lower scores after exposure. Exploratory Analyses Exploratory Analyses Additional exploratory analyses were conducted to investigate if attribution of mental capacities (i.e., Body, Heart, and Mind; Weisman et al., 2017) and perceived predictivity were correlated with gaze-cueing eﬀects, if individual diﬀerences in anthropomorphism were correlated to adopting the intentional stance, how adopting the intentional stance related to attribution of mental capacities, if the perceived predictivity and mental capacity ratings changed as a function of exposure, and if subjective ratings of engagement decreased over time, as prior work has shown a correlation between robots that engage in gaze behavior and subjective ratings of engagement (Admoni et al., 2011; Kompatsiari et al., 2018). Due to the exploratory nature of these analyses, we only report descriptive statistics, coeﬃcient estimates, and the 90% conﬁdence level rather than signiﬁcance level, as not to inﬂate alpha levels. Exploratory analyses and results can be found in the Supplementary Material. To examine if adopting the intentional stance aﬀected GCEs as a function of Exposure, we ﬁrst calculated a GCE for each participant. To do so, we excluded trials that were faster than 100 ms and slower than 1,500 ms. Next, we removed incorrect trials and trials that were slower than 2.5 SD away from the individual mean. Afterwards, we calculated the GCE, which is the diﬀerence between valid and invalid reaction times (i.e., invalid RT—valid RT), with larger positive GCE indicating stronger attention orienting in relation to iCub’s gaze. After calculating the GCE, we used a Bayesian regression to regress GCE onto the mean-centered Pre-ISQ scores, Exposure length (i.e., a dummy coded variable: Short = 1, Medium = 2, Long = 3), and their interaction. This regression model would allow us to examine (A) if gaze-cueing eﬀects were diﬀerent depending on the length of exposure to repetitive behavior (i.e., main eﬀects) and (B) if the relationship between GCEs and ISQ scores is dependent on exposure length. Similar to the previous analysis, using dummy coding allows us to follow up on any interactions without the use of post-hoc tests. Mean RTs in valid and invalid conditions across the three exposure duration groups are presented in Table 2. Main Results All participants performed the gaze-cueing task with high accuracy (M = 95.8%, SD = 0.03). One participant was identiﬁed as an outlier and was removed from the analyses due to a GCE score of more than 3 SD away from the mean (SD = −3.19). Data of the remaining 26 participants were analyzed. Frontiers in Robotics and AI \| www.frontiersin.org Procedure A value of 0 would suggest no change after exposure. By creating diﬀerence scores, we were able to examine how participants changed their scores as opposed to only predicting their ﬁnal scores. To examine whether people adopted a diﬀerent stance after exposure, we used a Bayesian regression model to predict participants’ diﬀerence ISQ scores from the exposure length (a dummy coded variable: Short = 0, Medium = 1, Long = 2) and each participant’s Pre-ISQ scores. No interaction term was included between the two variables as we were only interested in the eﬀect of exposure duration. Using this method would allow us to compare each of our conditions to the Short Exposure condition without the need for follow-up tests and control for baseline diﬀerences in ISQ scores. Mean ISQ scores for the three exposure duration groups are presented in Table 1. Gaze Cueing Effects Gaze Cueing Effects 1.32 with 90% PI [0.53, 2.12]. When examining the means, the 90% credible intervals of the posterior sample show that there is a 27% chance that the short and medium conditions overlap in their estimates, which suggests that there is a meaningful diﬀerence in their estimates. This diﬀerence is even smaller for the short and long conditions as there is a 12% chance that the means overlap. In other words, there is a 27 and a 12% chance, respectively, that the medium exposure and long exposure conditions contain a mean of 1.87, respectively. The model also predicts that there is an 83% overlap between the 90% credible intervals of the short and medium posterior distributions and a 70% overlap between the short and long distributions. This suggests that there is a 17% chance that a true diﬀerence between short and medium conditions exists, and a 30% chance that a true diﬀerence between the short and long conditions exists. See Figure 2 for the observed data and Supplementary Figure 1A for the posterior distributions. This suggests a linear relationship between exposure length and adopting the intentional stance. 1.32 with 90% PI [0.53, 2.12]. When examining the means, the 90% credible intervals of the posterior sample show that there is a 27% chance that the short and medium conditions overlap in their estimates, which suggests that there is a meaningful diﬀerence in their estimates. This diﬀerence is even smaller for the short and long conditions as there is a 12% chance that the means overlap. In other words, there is a 27 and a 12% chance, respectively, that the medium exposure and long exposure conditions contain a mean of 1.87, respectively. The model also predicts that there is an 83% overlap between the 90% credible intervals of the short and medium posterior distributions and a 70% overlap between the short and long distributions. This suggests that there is a 17% chance that a true diﬀerence between short and medium conditions exists, and a 30% chance that a true diﬀerence between the short and long conditions exists. See Figure 2 for the observed data and Supplementary Figure 1A for the posterior distributions. This suggests a linear relationship between exposure length and adopting the intentional stance. Frontiers in Robotics and AI \| www.frontiersin.org InStance Scores The Bayesian model did not run into issues with convergence and ﬁt the observed data well (ˆR = 1 and MCSE = 0 for all parameters). It took about 0.92 s for each chain to converge. The posterior distribution of the Bayesian model showed that the short exposure condition had a mean of 1.87 with a 90% PI [1.01, 2.72], the medium condition had a mean of 1.57 with 90% PI [0.77, 2.39], and the long condition had a mean of Frontiers in Robotics and AI \| www.frontiersin.org November 2020 \| Volume 7 \| Article 565825 5 Abubshait and Wykowska Repetitive Behavior Impacts HRI FIGURE 2 \| Change in ISQ ratings after exposure. The graph illustrates the difference in pre- and post-ratings. A positive increase illustrates that the rating changed to a higher rating at post, while a zero value would indicate no changes between pre- and post-ratings. The observed ISQ difference scores show the short and medium exposure conditions show a positive increase in ISQ scores after completing of the gaze-cueing task, while the long exposure condition shows no increase. FIGURE 2 \| Change in ISQ ratings after exposure. The graph illustrates the difference in pre- and post-ratings. A positive increase illustrates that the rating changed to a higher rating at post, while a zero value would indicate no changes between pre- and post-ratings. The observed ISQ difference scores show the short and medium exposure conditions show a positive increase in ISQ scores after completing of the gaze-cueing task, while the long exposure condition shows no increase. DISCUSSION short exposure and medium exposure conditions exist, and a 64% chance that a true diﬀerence between the short and long exposure conditions exist. These data suggest the linearity of the relationship as it was becoming more negative as we move from the short exposure condition to the long exposure condition. However, the evidence is not very strong, which is possibly due to our limited sample size. See Figure 4 for the observed data and Supplementary Figure 1C for the posterior distributions. short exposure and medium exposure conditions exist, and a 64% chance that a true diﬀerence between the short and long exposure conditions exist. These data suggest the linearity of the relationship as it was becoming more negative as we move from the short exposure condition to the long exposure condition. However, the evidence is not very strong, which is possibly due to our limited sample size. See Figure 4 for the observed data and Supplementary Figure 1C for the posterior distributions. The aim of the current study was to examine if the duration of exposure to a humanoid robot’s repetitive behavior in a gaze- cueing task inﬂuences adopting the intentional stance toward it, and if adopting of the intentional stance correlates with the GCE depending on the duration of exposure to repetitive behavior. Since previous work suggests that being exposed to robots for long durations decreases the likelihood of anthropomorphizing robots and is associated with negative aﬀective responses, we hypothesized that participants who were exposed to more repetitive behavior would show a decrease in adopting the intentional stance in comparison to those who are exposed to less repetitive robot behavior. Additionally, we expected that the relationship between ISQ scores and GCEs would depend on exposure of the repetitive behavior of iCub (i.e., the relationship changes as a function of being exposed to repetitive gaze behavior of the robot). Finally, this study took a Bayesian approach to investigate these eﬀects, which allows us to examine our eﬀects in terms of probabilities and not whether an eﬀect is signiﬁcant or not. FIGURE 3 \| Gaze-cueing effects (GCEs) as a function of exposure. The graph illustrates the mean values of GCE, which is the calculated difference between valid and invalid trials, with more positive values indicating stronger attention orienting in response to iCub’s gaze shifts. Gaze Cueing Effects The Bayesian model predicting GCEs did not have issues with convergence (ˆR = 1, MCSEMedium = 0.1, MCSELong = 0.1, MCSEMedium = 0, for the rest of the parameters) and each chain took approximately 1 s to converge. The posterior distribution of the Bayesian model showed that the short exposure condition had a mean of 7.41 with a 90% PI [−0.01, 14.85], the medium exposure condition had a mean of 12.16 with 90% PI [1.88, 22.45], and the long exposure condition had a mean of 15.49 with 90% PI [5.2, 25.63], and the Pre-ISQ scores had a positive slope of 1.27 with 90% PI [−1.45, 3.95]. The interaction term between the medium exposure condition and the Pre-ISQ scores had a slope of −1.92 with 90% PI [−6.34, 2.57], and the interaction term between the long exposure condition and the Pre-ISQ scores showed a slope of −2.99 with 90% PI [−8.29, 2.33]. The 90% credible interval of the posterior distributions of the main eﬀects suggests that there is a 38% chance that a diﬀerence between the short exposure and medium exposure conditions exist and a 60% chance that a diﬀerence between the short exposure and long exposure conditions. See Figure 3 for the observed data and Supplementary Figure 1B for the posterior distributions. Due to a clear descriptive diﬀerence between the medium exposure condition and the long exposure condition in Table 1 and Figure 2, we also examined the probabilities that the two means overlap. The posterior distributions showed that there is a 30% chance that the means of the medium exposure condition and the long exposure condition overlap. In other words, there is a 70% chance that there is a true diﬀerence between the two means. This additional test provides more evidence for the linearity of the relationship between exposure duration and the change in ISQ ratings. Analysis of the interaction eﬀects (i.e., the Pre-ISQ score–GCE relationship as a function of length of exposure), showed that there is a 50% overlap between the 90% credible interval of the short exposure and medium exposure posterior distributions and only a 36% overlap between the 90% credible intervals of the short exposure and long exposure posterior distributions. This suggests that there is a 50% chance that a true diﬀerence between November 2020 \| Volume 7 \| Article 565825 Frontiers in Robotics and AI \| www.frontiersin.org 6 Abubshait and Wykowska Repetitive Behavior Impacts HRI DISCUSSION (2020) examined the eﬀect of repeated exposure to a robotic platform and its eﬀectiveness in improving the joint attention skills of children with ASC. They found that, unlike the suggestion of prior work, learned skills are not maintained over longer periods (Zheng et al., 2020). Similarly, Anzalone et al. (2019) showed a similar pattern where developing children typically did not show improvements in joint attention after prolonged exposures to a robot. These studies show that exposure over longer periods may not have a clear beneﬁt to HRI. The results of our study shed light on this by showing that changes in subjective judgments of intentionality do change when interactions are short-lived. However, after long exposure to iCub’s repetitive behavior, the change in attribution of intentionality diminished. Therefore, roboticists have to carefully investigate how longer durations of exposure can inﬂuence the human interaction partner. The ﬁnding that short exposure durations were likely to have a positive eﬀect on subjective ratings of adopting the intentional stance is in line with prior work that used the gaze-cueing paradigm and found that subjective ratings of the mind status of a robot increases after completing the gaze-cueing paradigm (Abubshait and Wiese, 2017; Abubshait et al., 2020b) and other studies showing that initial impressions inﬂuenced subjective ratings positively, but latter interactions kept the interactions unchanged (Paetzel et al., 2020). Furthermore, the positive impact of short interactions on adopting the intentional stance might be due to interacting with a real embodied humanoid robot, which initially might evoke social attunement, due to its physical presence (Wainer et al., 2007). However, this eﬀect might diminish after medium and long exposure conditions, due to the repetitiveness of its behavior. The current study also illustrates how a Bayesian framework can be a better method in quantifying HRI. HRI studies often have limited sample sizes due to their complex protocols (i.e., sample sizes of about 20 participants; e.g., Mutlu et al., 2006; Terada et al., 2007; Wainer et al., 2007; Wang and Lewis, 2007; Brown and Howard, 2013; de Graaf et al., 2015; Warren et al., 2015; Zheng et al., 2020), which is problematic when analyzing data using parametric statistics. By using a Bayesian framework, we assume that the data are ﬁxed and that the parameter estimates are dynamic, while the opposite is true for parametric methods. DISCUSSION This indicates that shorter interactions with a robot, even if it shows repetitive behavior, can still have a positive inﬂuence on adopting the intentional stance. agents but not humans (Gazzola et al., 2007), which suggests that attention orienting to gaze cues (a phenomenon related to the social brain; Wiese et al., 2018) can be inﬂuenced by being exposed to repetitive behaviors. The results of this study highlight an important issue in the ﬁeld, which is that many of the eﬀects of attributing intentionality to robots that we see are short-lived. Epley et al. (2007) theory of anthropomorphism provide an explanation to why, which is that when people are exposed to repetitive behaviors from an agent, it can reduce their motivation to understand the agent’s behavior, which reduces the propensity to anthropomorphize the agent and adopt the intentional stance toward it. Not only do we ﬁnd this short-lived eﬀect in our data, but other studies also have found similar results. For example, when using entertainment robots, de Graaf et al. (2015) have found that the most important robot characteristic to be perceived as social is that the robot should be able to verbally respond in a free and social manner (i.e., unpredictable) and not a preprogrammed way. Analyses of the GCEs showed that as the length of repetition increased, GCEs were likely to become larger, as short exposure had a high probability of inducing smaller GCEs as compared to the medium exposure condition. This diﬀerence between GCEs was even more likely when comparing the short exposure condition and the long exposure condition, which indicates that more trials (i.e., repetitions) are warranted in order to measure GCEs. The results also showed that, as hypothesized, the relationship between adopting the intentional stance and gaze cueing was likely dependent on the length of exposure to repetitive robot behavior. Initially, this relationship was positive, however, as the duration of exposure increased, the relationship showed a tendency to be more negative. While this eﬀect was not very strong, possibly due to being statistically underpowered, it suggests that longer exposures to repetitive robot behavior can reverse the positive relationship between joint attention (a marker of social attunement) and adoption of intentional stance. This result generalizes to other areas in HRI that use repetitive interactions with a robot as interventions with populations diagnosed with autism spectrum condition (ASC). For example, Zheng et al. DISCUSSION The GCEs show that attention orienting to gaze-cues is enhanced as the length of exposure (i.e., trial numbers) increased. To test our hypotheses, we employed a face-to-face gaze- cueing paradigm with iCub, and used an experimental design where observers completed a pre-questionnaire block, then the gaze-cuing experiment with iCub, then a post-questionnaire block. Results of the experiment indicated that the probability of showing a large and positive ISQ change after interacting with iCub was highest for participants who were exposed to less repetitions of iCub’s gaze behavior and that the probability of showing a large and positive change decreased as a function of duration exposure. Speciﬁcally, the results showed that this probability of showing a positive ISQ diﬀerence score decreased by 73% from those who were exposed to iCub for a short duration (i.e., fewer repetitions) to those who were exposed to iCub for FIGURE 3 \| Gaze-cueing effects (GCEs) as a function of exposure. The graph illustrates the mean values of GCE, which is the calculated difference between valid and invalid trials, with more positive values indicating stronger attention orienting in response to iCub’s gaze shifts. The GCEs show that attention orienting to gaze-cues is enhanced as the length of exposure (i.e., trial numbers) increased. FIGURE 4 \| Interaction between exposure and Pre-ISQ scores in GCE. The graph illustrates that an interaction is evident between the length of exposure and Pre-ISQ scores. The interaction shows a positive correlation between ISQ scores and GCE when exposure length is short; however, this relationship becomes more negative as exposure length increases. FIGURE 4 \| Interaction between exposure and Pre-ISQ scores in GCE. The graph illustrates that an interaction is evident between the length of exposure and Pre-ISQ scores. The interaction shows a positive correlation between ISQ scores and GCE when exposure length is short; however, this relationship becomes more negative as exposure length increases. November 2020 \| Volume 7 \| Article 565825 7 Frontiers in Robotics and AI \| www.frontiersin.org Repetitive Behavior Impacts HRI Abubshait and Wykowska a medium duration. This probability decreased even further to 88% from participants who were exposed to iCub in the short exposure condition in comparison to those who were exposed to iCub for longer (i.e., long exposure condition). Interestingly, participants in the long exposure condition did not change their scores after the interaction in respect to their scores prior to interacting with iCub. Frontiers in Robotics and AI \| www.frontiersin.org DISCUSSION In other words, we assume that our parameter estimates for speciﬁc behaviors (e.g., gaze-cueing eﬀects in reaction times) follow a distribution as opposed to it having a ﬁxed estimate. By doing so, this method allows us to update our prior knowledge from past studies and estimate an updated parameter distribution. Not only does it allow us to update our knowledge from past studies, but it also allows roboticists to use probabilities of an eﬀect in question. Using probabilities to quantify HRI is much more informative than using signiﬁcance thresholds. For example, it is more informative for researchers to know which The ﬁnding that gaze cueing was likely related to adopting the intentional stance depending on exposure is supported by prior work investigating social engagement between children and social robots that shows that initial interactions with robots are paired with high social engagement but that this social engagement is reduced after a while (Komatsubara et al., 2014; Coninx et al., 2016; Ahmad et al., 2017). This suggests that the reduction in social engagement can generalize to other populations and not simply children. This account is also supported by fMRI studies that argue that the action perception network, a network in the brain that is involved in understanding and predicting others’ actions (Decety and Grèzes, 1999), is activated when trying to predict movements from others’ gaze behavior (Ramsey et al., 2012) and unfamiliar robotic behavior (Cross et al., 2012). After viewing repetitive actions performed by humans and robots, this activation is reduced for only robotic November 2020 \| Volume 7 \| Article 565825 Frontiers in Robotics and AI \| www.frontiersin.org Frontiers in Robotics and AI \| www.frontiersin.org 8 Repetitive Behavior Impacts HRI Abubshait and Wykowska robot has a 70% chance of successfully engaging in joint attention with children with ASC as opposed to which robot signiﬁcantly engages in joint attention with children with ASC. the likelihood of adopting the intentional stance toward an agent when it displays repetitive behavior over a longer duration, it poses a challenge on how to maintain suﬃcient number of trials for statistical power without decreasing the reception of the robot as an intentional or a human-like agent. While the gaze-cueing paradigm might be successful in evoking joint attention, it might not decrease the perception of the robot as human-like or intentional. DISCUSSION g g j Despite the advantages of the methods used here, it is also important to discuss some of the limitations associated with the current study. First, the small sample size could render the results unstable. While this could be a major issue when using parametric analyses due to violations of the general linear model’s assumptions, we do not believe that the small sample size dramatically inﬂuenced our results. We believe that our results are stable due to using a Bayesian framework, and more so due to using uninformative priors (i.e., uninformative priors are considered conservative even though we could have used informed priors from prior gaze-cueing studies with iCub: e.g., Kompatsiari et al., 2018). By doing so, we inﬂate our credible intervals, which underestimates the conﬁdence of our results. Another possible limitation is the use of a pre-/post-test design. In this study, participants had to complete a questionnaire that measured their attribution of intentionality to robots prior to completing the gaze-cueing experiment. This design (i.e., participation in the test prior to the experiment) could have biased the participants into attributing intentions to iCub. While this is a limitation that can be addressed by using ecologically valid behavioral measures of intention attribution (e.g., Mwangi et al., 2018), due to our controlled lab setting this method was not feasible. Still, the fact that participants did show group diﬀerences when they completed short, medium, and long gaze-cueing tasks with iCub suggests that the length of exposure does inﬂuence our interactions with robots in general. FUNDING This study provides important contribution regarding implementation of classical experimental protocols in HRI studies by showing two eﬀects that oppose one another. First, our study showed that more trials (i.e., repetitions) were needed in order to measure a social-cognitive process that is associated with social attunement (i.e., gaze cueing), as the gaze-cueing eﬀect was more likely more positive as repetitions increased. On the other hand, subjective ratings of adopting the intentional stance were likely less related to GCEs as repetitions (i.e., trials) increased in the gaze-cueing paradigm, which suggests that studies measuring these two constructs (i.e., social attention and adopting the intentional stance) may sacriﬁce the validity of one measurement over the other. In our case, the validity of social attention was likely to increase with more trials, but the validity of measuring subjective intention attribution was likely to decrease with more repetitions. Since the data suggest that we might not increase This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program, ERC Starting Grant ERC-2016-StG-715058, awarded to AW, titled InStance: Intentional Stance for Social Attunement. The content of this paper is the sole responsibility of the authors. The European Commission or its services cannot be held responsible for any use that may be made of the information it contains. DATA AVAILABILITY STATEMENT The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://osf.io/y6c9b/. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/frobt. 2020.565825/full#supplementary-material ETHICS STATEMENT The studies involving human participants were reviewed and approved by Comitato Etico Regione Liguria. The patients/participants provided their written informed consent to participate in this study. AUTHOR CONTRIBUTIONS AA and AW conceptualized the study, interpreted the results, and wrote the manuscript. AA collected and analyzed the data. All authors contributed to the article and approved the submitted version. Ahmad, M. I., Mubin, O., and Orlando, J. (2017). Adaptive social robot for sustaining social engagement during long-term children-robot interaction. Int. J. Hum. Comput. Interact. 33, 1–21. doi: 10.1080/10447318.2017.1300750 Admoni, H., Bank, C., Tan, J., Toneva, M., and Scassellati, B. 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Psychol. 26, 1–9. doi: 10.1080/20445911.2014.945457 Copyright © 2020 Abubshait and Wykowska. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). REFERENCES The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2020 Abubshait and Wykowska. 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. Terada, K., Shamoto, T., Ito, A., and Mei, H. (2007). “Reactive movements of non- humanoid robots cause intention attribution in humans,” in 2007 IEEE/RSJ International Conference on Intelligent Robots and Systems (San Diego, CA) 3715–3720. doi: 10.1109/IROS.2007.4399429 Teufel, C., Alexis, D. M., Clayton, N. S., and Davis, G. (2010). Mental-state attribution drives rapid, reﬂexive gaze following. Attent. Percept. Psychophys. 72, 695–705. doi: 10.3758/APP.72.3.695 November 2020 \| Volume 7 \| Article 565825 Frontiers in Robotics and AI \| www.frontiersin.org 11
https://openalex.org/W2333012496	https://zenodo.org/records/2288072/files/article.pdf	English	null	Gold, Prices, and Wages.	The economic journal/Economic journal	1,913	public-domain	2,980	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. Wiley and Royal Economic Society are collaborating with JSTOR to digitize, preserve and extend access to The Economic Journal. This content downloaded from 128.235.251.160 on Mon, 19 Jan 2015 08:35:50 AM All use subject to JSTOR Terms and Conditions 1913] HOBSON: GOLD, PRICES, AND WAGES 393 standing. The governing consideration in his mind seems to have been that a permanent difference of monetary standard between India and the chief countries with which she traded was intoler able; and there could be no permanent remedy "except either the general adoption of the system of double legal tender or the exten- sion of the single gold standard." As soon as proposals for the first alternative seemed to be definitely defeated, Sir David Barbour did not delay or hesitate in falling back on the second. To those of us who were in our nurseries when these things happened, it is an interesting speculation whether, living then, the Bimetallic or the Monometallic camp would have contained us. To a cool backward glance, Sir David Barbour's course seems to have had very much to recommend it. The choice of one metallic standard rather than another for any individual country is rightly guided by historical circumstance; it is not a matter on which theory as to what would make the best universal standard should count for much. In the 'eighties bimetallism must have seemed worth fighting for. But the course of events-American politics, South African gold and so forth-defeated it. lThe wise bimetallist should have hastened to acknowledge defeat. The statesmanlike opportunism of Sir David Barbour appears to such a backward glance in a stronger light than the dogmatisms of, for example, Sir Robert Giffen about the impracticability of a gold standard or the immeasurable evils of a managed currency. g This book is of a very rare type-wonderfully full of practica wisdom, yet showing at the same time the intellectual sharpnes and precision of a first-rate academic mind. This content downloaded from 128.235.251.160 on Mon, 19 Jan 2015 08:35:50 AM All use subject to JSTOR Terms and Conditions Happy the country which can discover in its administrators so fine a combinatio of gifts. J. M. KEYNES. Gold, Prices, and Wages. By J. A. HOBSON. (London: Methuen. 1913. Pp. xi + 181. 3s. 6d. net.) ONE comes to a new book by Mr. Hobson with mixed feelings in hope of stimulating ideas and of some fruitful criticisms of orthodoxy from an independent and individual standpoint, but expectant also of much sophistry, misunderstanding, and pervers thought. In some of his books the first elements greatly pre- dominate. In his latest work now before us, the latter prevail almost throughout. The book is a very bad one, made much worse than a really stupid book could be, by exactly those charac- teristics of cleverness and intermittent reasonableness which have E E E E No. 91.-voL xxIII. No. 91.-voL xxIII. 394 THE ECONOMIC JOURNAL [SEPT. borne good fruit in the past. I will endeavour to elucidate his main contentions. Mr. Hobson begins by distinguishing the different sources of the supply of money-new gold from the mines, additional credit from the bankers, and money "derived from prior acts of sale." If all money were derived from prior acts of sale, the aggregate receipts would rise and fall with these acts of sale, and therefore prices must remain stable. This w-ould be the case with a stationary community or with one "growing in such a way that it did not shift the proportion of its demand for different classes of goods." If that is to say no new gold were to be mined and no new credit created by bankers, but we were all to produce, buy, and consume double (say) of what we do now, then prices, according to Mr. Hobson, would remain stable. "The only way in which prices could change in a community where money was entirely derived from previous receipts would be . . . if a larger proportion of money were directed to buying goods whose pro- duction conformed to the so-called law of diminishing returns," and conversely. What is one to say to such an argument? It sounds like a parody of economic reasoning; the words have, in a way, the right sort of jingle to the ear, but the mind is left at a loss. Mr. Hobson seems to be content with this conclusion for its own sake, and does not make much use of it. This content downloaded from 128.235.251.160 on Mon, 19 Jan 2015 08:35:50 AM All use subject to JSTOR Terms and Conditions He passes on to prove that the gold produced during the last fifteen years cannot have had any appreciable influence in raising prices. Since the net income of the British nation is about ?2,000,000,000, we may estimate the gross turnover at ?10,000,000,000. The gross turn- over of this country cannot be more than one-tenth of that of the world. "This would give ?100,000,000,000 as the quantity of money operative for a year. To this sum there has been added (annually) from an extraneous source the gross income of the gold mines, an amount of ?67,000,000. The gross income from gold-mining will have precisely the same amount of influence on general prices as the same gross income got from the textile or metal industries. The effect would be an increase of the aggre- gate quantity of money to the extent of - 'I. The influenc upon prices would thus be considerably less than yl, or -11 per cent. The actual influence of this addition to money in raising prices would, of course, be much less, if allowance were made for the increase of goods which has been going on." It seems almost incredible that Mr. Hobson should have convinced himself by such an argument. It appears to be based on the assumption 1913] HOBSON: GOLD, PRICES, AND WAGES 396 that no new coin can be used more than once; but it depends, I think, in Mr. Hobson's mind on an earlier and rather obscure argument that new money only influences prices the first time it changes hands. g Mr. Hobson turns next to the confutatio n of the theory "that an increased supply of gold somehow will necessarily expand the volume of credit which is said to be based upon it, and therefore enhances proportionately the entire volume of purchasing power." If the theory were true, we should find, now that prices have risen, Mr. Hobson says, a lower average rate of discount than formerly, and a larger accumulation of gold in the reserves of banks. Of course, the average rate of discount has risen and not fallen, but Mr. This content downloaded from 128.235.251.160 on Mon, 19 Jan 2015 08:35:50 AM All use subject to JSTOR Terms and Conditions Hobson must know very well that the adherents of the theory he is disputing maintain, not that the average rate of discount must fall, but that new gold in bank reserves has the temporary effect of making the rate lower than it would otherwise have been-for he quotes the relevant passage from Dr. Marshall's evidence before the Gold and Silver Commission some pages further on, and points out himself that the rise in the average rate of discount is mainly due to the greatly increased demands for capital in new countries. Mr. Hobson goes on to deny the second indication, namely, the accumulation of gold in the reserves of banks, basing his conclusion on the amount of gold in the issue department of the Bank of England (not even on the Bank's reserve, and in spite of his own statement-p. 80-that there have been considerable new accumulations of gold in the reserves of English Joint Stock Banks), and slips on two pages later (p. 43) to apply this conclusion to "other European countries," although, since he himself gives a table setting forth the contrary on p. 46, he must well know that this is not the case. But having thus disposed of the orthodox contention and greatly belittled the part played by gold in the raising of prices and the manufacture of credit, Mr. Hobson seems to come back to a position very little removed from that which he has demolished and hardly consistent with the weapons he has lately used. "Gold is not," he now maintains, "the chief efficient cause or stimulus of the enlarged credit, but it is, or may be, an essential or at least a facilitating condition of its production." "Had there been," he admits on p. 61, "a constriction of the gold supply and bank reserves been low, the price of money would have been higher than it has been, the aggregate amount of borrowing less, the subsequent demand for goods reduced, and the rise of prices correspondingly less." If Mr. Hobson merely wishes to deny E E 2 This content downloaded from 128.235.251.160 on Mon, 19 Jan 2015 08:35:50 AM All use subject to JSTOR Terms and Conditions 396 [SEPT. THE ECONOMIC JOURNAL that the new gold has been "an efficient cause " of higher prices while admitting that it has been a necessary condition of them, his earlier arguments have proved too much. Whether the new gold has been a necessary or only a favourin condition to the manufacture of new credit, Mr.. Hobson sees the main explanation of the increased credit elsewhere, namely, in the greatly increased volume, chiefly arising out of the borrow ings of Governments, municipalities, and railways in new countries of "stocks and shares and other certificates of value." "All such modern saving can furni-sh material for the creation of more credit." The argument he bases -on this is founded on two very old confusions, which always have been and probably always will be made by certain types of mind having, as it were, a natural affinity to this way of thinking. The first is between the volume of credit (measured in terms of goods) and the value of credit (measured in terms of money). If credit, Mr. Hobson argues, is based on actual goods, and every piece of wealth carries with it "a credit-note representing its present value," how can it ever become redundant? "Each specific piece of wealth would have a corresponding token of general wealth attached to it. That token could be used for general purposes of purchase, its recipien holding a claim upon the general wealth into which the specifi piece of wealth will be convertible. The volume of credit would evidently expand or contract with the expansion or contractio of the value of the goods which command it, and the notion of an excess or deficiency of 'money' would be meaningless." This is newly transmuted. - But can we not hear in the distance the voice of Mirabeau urging on the National Assembly the issue of assignats? -"Paper money, we are told, will become super- abundant. Of -what paper do you speak? If of a paper without a solid basis, undoubtedly; if of one based on the firm foundatio of landed property, never. This content downloaded from 128.235.251.160 on Mon, 19 Jan 2015 08:35:50 AM All use subject to JSTOR Terms and Conditions Reabsorbed progressively in the pur- chase of the national domains, this paper money can never become redundant, any more than the humidity of the atmosphere can become excessive, which descends in rills, finds the river, and is at length lost in the mighty ocean." g g y The second confusion, of which I spoke above, is between the two senses of the word "credit," the sense in which it stands for the method by which the control of liquid wealth is temporaril transferred from those who have less need of it to those who have more, and the sense in which it stands for methods of making payments and effecting exchanges without the use of actual coin. There is, of course, no necessary connection 1913] HOESON: GOLD, PRICES, AND WAGES 397 whatever between -these two. If a bank- receives money from depositors and then lends it out again tao borrowers against suit- able security, the use of coin as a nmedium of exchange is in no wise diminished and prices, directly at any rate, in no degree affected. Only when a bank passes to its other function and creates notes, bills, or cheques as a means of effecting exchanrge does the influence on prices come in. Mr. Hobson has muddled up the two senses to the extremest point conceivable, and ascribes to causes which have facilitated the development of the firs function results which could only arise out of the developmen of the second. No doubt the creation of suitable security, due to the opening up of new countries, has greatly developed the mechanism of credit in the first sense. But I doubt whether the development in these new countries of credit in the second sense, though considerable, has kept pace with the growth of business and the demand for media of exchange in these same new countries. If, therefore, gold had been mined at the same rate as in recent years, and new countries had not been developed as they have been, prices would, I expect, have been appreciably higher than they actually are. The development of South America, for example, since credit in the second sense is less perfectly developed there than in Europe, has retarded the rise of prices, not, as Mr. Hobson argues, hastened it, by affording a fresh source of demand for large quantities, of gold. This content downloaded from 128.235.251.160 on Mon, 19 Jan 2015 08:35:50 AM All use subject to JSTOR Terms and Conditions I amn in- clined to think, therefore, that his main practical conclusion is exactly opposite to the truth. There are numerous other points of detail which might be worth dealing with if this review were not already so long. I will catalogue some of them :-The confusion of borrowers, who are mainly the entrepreneur class and the holders of ordinary shares, with the poorer part of the community (p. 116); the view that " the increasing proportion of - the energy of modern nations that is applied to the distributive, as distinguished from the productive trades " tends to raise prices (based on the eternal fallacy that only the latter are 'truly productive); the opinion that a rapid intro- duction of improved industrial methods is socially wasteful because it involves the scrapping of the older plant; and the theory that the rate of interest and the level of prices are but two faces of the same phenomenon, since one is the hire-price and the other the purchase-price of money. This last point deserves a little more attention. "It would be impossible to conceive," Mr. Hobson writes, "the general price for houses to be rising over a period of years while the rents of these houses were falling." This 398 THE ECONOMIG JOURNAL [:SEPT. is so, apparently, because "if motor-cars become cheaper to buy, we know they will become cheaper to hire." Since the rate of interest has risen while the purchasing power of money has fallen we have a paradox to explain. Mr. Hobson is led to the view that "money has not really a sale-price at all." For the extra- ordinary theory he builds on this, the reader must be referred to his last chapter. Belonging to no one race or age more than another, there lives an intellectually solitary race of beings who by some natura prompting of the soul think about monetary theory in certain specific, definite ways, superstitious or delusive, mystically, not materially, true, if true at all. All of these will find their natura instincts expressed here in forms more plausible-topical than they can usually shape themselves. Mr. Hobson has given us the Mythology of Money,-intellectualised, brought up to journalisti date, most subtly interlarded (and this is how it differs from the rest) with temporary concessions to reason. J. M. KEYNES Luxus und Capitalismus and Krieg und Capitalismus. By WERNER SOMBART. (Leipzig: Duncker and Humblot. This content downloaded from 128.235.251.160 on Mon, 19 Jan 2015 08:35:50 AM All use subject to JSTOR Terms and Conditions 1913. Pp. 220 and pp. 232. Each volume 6 marks.)- SOMBART'S studies for a new edition of his Modern Capitalism have led him far. So much material has accumulated, not all strictly economic, that he is publishing it in monographs. The first was his Die Juden und das Wirtschaftsleben. These volumes are of the same class and workmanship. There is the same immense reading and the same cocksureness in all provinces of knowledge the unpruned, slangy style that infects a reviewer; the real, yet slightly over-advertised, originality, and the tendency to drive a good notion further than it will go. g g The titles roughly indicate the theses, and the sub-title to the first volume-Liebe, Luxus und Capitalismus-indicates the part of it that is not strictly economic. A central thesis is common to both volumes, namely, that the fashionable connectioln of capitalism with production for a geographically wide market is not of primary importance in the "early capitalistic" age. The media'val weapon industry had a wide market, but its industria organisation was "HandwerksmAnnig" (Krieg, p. 92); so was that of the Yorkshire clothiers of the eighteenth century, whose goods went far and wide (-Luxus, p. 208); while many of the luxurious industries worked -mainly for the local court or metro polis, yet were- early organised on capitalist lines (Luxus, p. 138).
https://openalex.org/W2129154063	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/AC4F48BC2623B7A29C55A15F7601E4FC/S1758320900003085a.pdf/div-class-title-readmission-after-discharge-from-a-medium-secure-unit-div.pdf	English	null	Readmission after discharge from a medium secure unit	The psychiatrist	2,013	cc-by	4,803	Method The cohort consisted of 595 ﬁrst admissions to Arnold Lodge from its opening in July 1983 to 30 June 2003 - a period of 20 years. Four patients died in the unit following their ﬁrst admission and 41 had not been discharged from their ﬁrst admission at the census date. Therefore, the study sample comprised all 550 ﬁrst admissions discharged from Arnold Lodge during the study period. Declaration of interest None. Declaration of interest None. the community in particular will not have all the protection that a secure environment would provide. p units usually focus on reconviction,1-5 but where reported, readmission to any psychiatric hospital was common.6-8 The previous largest follow-up of a single unit found that of 234 discharged patients, 174 (74%) were readmitted to a psychiatric hospital (mean follow-up 6.6 years), with more previous admissions and an earlier ﬁrst psychiatric contact predicting readmission.6 Other variables such as age, gender and number of convictions were not associated with readmission. That study also found that 50 former patients (21%) spent some of the follow-up period in a high secure hospital, including 27 patients discharged directly to such hospitals.6 A high rate of readmission to any hospital was also shown in another, albeit smaller, medium secure unit cohort of 63 former patients with schizophrenia, with 56 (89%) patients requiring at least one readmission to a psychiatric hospital (mean follow-up 5.4 years), of whom 41 (73%) had multiple readmissions.7 A large survey of all medium secure units in England and Wales found a readmission rate of 27% for women and 21% for men within 1 year.9 Readmission to medium security has also been reported in a follow-up of 70 men with a history of violence or a violent index offence discharged from a medium secure unit to the community (either directly or via lower security).8 Followed up for at least 2 years, 33 patients (47%) were readmitted to the unit, with 16 patients (23%) being formally recalled while on restriction orders. Readmission is a proxy measure of both the risk that these patients pose and the continued psychiatric care that they require, so it is an important outcome to assess. Patients in This study examined the subsequent readmissions to psychiatric hospitals of patients admitted to Arnold Lodge Medium Secure Unit in Leicester, England, over a 20-year period during which it changed substantially. Overviews of outcomes, mortality and gender differences for this cohort are described elsewhere.10-12 A description of the ﬁrst 16 years admission cohort is also available.13 ORIGINAL PAPERS The Psychiatrist (2013), 37, 124-129, doi: 10.1192/pb.bp.112.039289 Data sources Admission characteristics were gathered from medical records at Arnold Lodge. Readmission outcome data were obtained from clinical records at Arnold Lodge and other psychiatric services, the Special Hospitals case register and the Home Ofﬁce Mental Health Unit. Ethical approval was granted by the Trent Multi-centre Research Ethics Committee. The research was conducted under Section 60 of the Health and Social Care Act 2001, allowing for the 124 The Psychiatrist (2013), 37, 124-129, doi: 10.1192/pb.bp.112.039289 1Institute of Mental Health, University of Nottingham; 2University of Nottingham and Partnerships in Care; 3University of Leicester; 4Institute of Mental Health and Nottinghamshire Healthcare NHS Trust; 5Nottinghamshire Healthcare NHS Trust; 6Countywide Mentally Disordered Offender Services, Northamptonshire Healthcare NHS Trust Correspondence to Martin Clarke (drmartinclarke@googlemail.com) First received 8 Mar 2012, ﬁnal revision 12 Sep 2012, accepted 3 Oct 2012 Aims and method We examined readmission to psychiatric hospital of 550 patients discharged from one medium secure unit over 20 years. Multiple sources were used to obtain readmission data. Results Readmission was common, particularly to non-secure psychiatric hospitals. At least 339 patients (61.6%) were readmitted to any psychiatric hospital (mean follow-up 9.5 years), with over a third (37.6%) subsequently being readmitted to medium- or high-security or both. Of those discharged directly to the community, having previous in-patient treatment and a Mental Health Act classiﬁcation of mental illness were associated with shorter time to ﬁrst readmission. Correspondence to Martin Clarke (drmartinclarke@googlemail.com) Clinical implications The long-standing nature of disorders is evident in the high rates of readmission overall and the need for readmission to medium and high secure services, suggesting that these patients require long-term follow-up and support from mental health services. First received 8 Mar 2012, ﬁnal revision 12 Sep 2012, accepted 3 Oct 2012 First received 8 Mar 2012, ﬁnal revision 12 Sep 2012, accepted 3 Oct 2012 Declaration of interest None. 124 https://doi.org/10.1192/pb.bp.112.039289 Published online by Cambridge University Press ORIGINAL PAPERS Clarke et al Readmission after discharge from a medium secure unit collection of follow-up data without the patient’s consent and thereby increasing the completeness of the sample. contact or the census date was 9.5 years (s.d. = 4.8). Women had a longer mean follow-up time than men: 11.5 years (s.d. = 4.1) v. 9.1 years (s.d. = 4.9); t = 4.887, d.f. = 145.6, P50.001. There was no signiﬁcant difference in the mean follow-up times between patients with Mental Health Act 1983 classiﬁcations of psychopathic disorder (9.8 years, s.d. = 5.1) or mental illness (9.4 years, s.d. = 4.6). There were 5246 person-years of follow-up after discharge. Statistical analysis Data were analysed using PASW Statistics 18.0 for Windows. Kaplan-Meier survival analyses were conducted to examine time to readmission. The effects of predictor variables on time to readmission were examined using Cox proportional hazards regression analysis. Predictors of readmission were identiﬁed using logistic regression analysis. P-values less than 0.05 were considered to be statistically signiﬁcant. y g Characteristics of the cohort are shown in Table 1. Two- thirds of patients had a Mental Health Act classiﬁcation of mental illness, whereas a quarter had a Mental Health Act classiﬁcation of psychopathic disorder (Arnold Lodge had a dedicated personality disorder service throughout most of the study period). Men and women had different back- ground characteristics, with 94% of the men and only 65% of the women having an index offence. Women (42%) were also more likely than men (11%) to be admitted under a civil section of the Act. Almost half the sample (45%) were discharged to another psychiatric hospital. These comprise transfers to higher security (7%), transfers to equivalent medium security (3%) and discharges to lower security, principally non-secure psychiatric hospitals (35%). Results Of the 550 patients discharged from their ﬁrst admission to Arnold Lodge, 459 (83.5%) were men and 91 (16.5%) women. The majority of patients were White (78.9%). The median length of stay for this sample was 164 days (range 2-3872) and was longer for women (median 227 days) than for men (median 154 days), although not signiﬁcantly longer. The mean length of follow-up from discharge to death, loss of Table 1 Admission characteristics Men (n = 459) Women (n = 91) Total (n = 550) n (%) n (%) n (%) Type of section Awaiting trial (s.35, s.36, s.48, s.48/49) 69 (15.0) 5 (5.5) 74 (13.5) Awaiting sentence (s.38) 147 (32.0) 24 (26.4) 171 (31.1) Hospital orders (s.37, s.37/41, CPIA) 101 (22.0) 17 (18.7) 118 (21.5) Sentenced prisoners (s.47, s.47/49) 83 (18.1) 5 (5.5) 88 (16.0) Civil sections (s.2, s.3) 50 (10.9) 38 (41.8) 88 (16.0) Informal 9 (2.0) 2 (2.2) 11 (2.0) Mental Health Act 1983 classiﬁcation Mental illness 323 (70.4) 46 (50.5) 369 (67.1) Psychopathic disorder 110 (24.0) 40 (44.0) 150 (27.3) Other (MI & PD; mental impairment) 14 (3.1) 3 (3.3) 17 (3.1) Unclassiﬁed/missing 12 (2.6) 2 (2.2) 14 (2.5) Index offence (Home Ofﬁce classiﬁcation) Grave 171 (37.3) 32 (35.2) 203 (36.9) Standard list 262 (57.1) 27 (29.7) 289 (52.5) No index offence 26 (5.7) 32 (35.2) 58 (10.5) Number of previous admissions (any psychiatric service) None 195 (42.5) 5 (5.5) 200 (36.4) One 77 (16.8) 7 (7.7) 84 (15.3) Two 48 (10.5) 11 (12.1) 59 (10.7) Three or more 114 (28.4) 59 (64.8) 173 (31.5) Missing 25 (5.4) 9 (9.9) 34 (6.2) Admission source Prison/criminal justice system 349 (76.0) 40 (44.0) 389 (70.7) High secure hospital 34 (7.4) 6 (6.6) 40 (7.3) Medium secure unit 13 (2.8) 2 (2.2) 15 (2.7) Non-secure hospital 56 (12.2) 41 (45.1) 97 (17.6) Community (including hostel) 7 (1.5) 2 (2.2) 9 (1.6) Discharge destination Prison/criminal justice system 136 (29.6) 11 (12.1) 147 (26.7) High secure hospital 26 (5.7) 14 (15.4) 40 (7.3) Medium secure unit 14 (3.1) 2 (2.2) 16 (2.9) Low secure unit/PICU 7 (1.5) 0 (0) 7 (1.3) Non-secure hospital 144 (31.4) 39 (42.9) 183 (33.3) Community (including hostel) 126 (27.5) 25 (27.5) 151 (27.5) Missing (e.g. AWOL) 6 (1.3) 0 (0) 6 (1.1) AWOL, absent without leave; CPIA, Criminal Procedure and Investigations Act 1996; MI & PD, mental illness and psychopathic disorder; PICU, psychiatric intensive care unit. Results 125 Overview of readmission readmitted at some point during the follow-up, 13 patients (42%) were readmitted informally at least once while on a restriction order and 11 patients (35%) were recalled to hospital. Of the 151 patients discharged directly to the community, 40 (26%) were readmitted to a medium secure unit during the follow-up; these comprised 30 men (24%) and 10 women (40%). Eight men (6%) were admitted to a high secure hospital at some point following their discharge to the community; ﬁve of these were also readmitted to a medium secure unit and the other three men were admitted to a high secure hospital as a result of a recall to hospital (n = 1) and new offences for which the former patient received a manslaughter conviction (n = 2). In spite of obtaining Section 60 support, there were still considerable problems in accessing data in certain trusts. The main trusts comprised by Arnold Lodge’s catchment area (Derbyshire, Leicestershire, Lincolnshire and Nottinghamshire) were very helpful in facilitating the study. Of the 490 patients for whom readmission data were available, 339 patients (69.2%) were readmitted to a psychiatric hospital at some point prior to the census. At least 239 patients were known to have been readmitted under provisions of the Mental Health Act, of whom 86 also had at least one informal readmission. An additional 86 patients were informally readmitted at least once to a psychiatric hospital and were not readmitted under provisions of the Act. Over the whole study period there were missing readmission data for 60 patients (10.9%). Those with missing readmission data had a shorter length of stay (median 111 days) than those whose readmission data were available (median 170 days; U = 11 794.0, Z = 72.501, P = 0.012). Time to readmission for those discharged to the community was ﬁrst examined using Kaplan-Meier survival analysis, with cases censored on death or at the census at 30 June 2003. Potential predictors of time to readmission were expressed as binary variables (either directly or via median splits): gender; ethnicity (White v. https://doi.org/10.1192/pb.bp.112.039289 Published online by Cambridge University Press https://doi.org/10.1192/pb.bp.112.039289 Published online by Cambridge University Press ORIGINAL PAPERS ORIGINAL PAPERS Clarke et al Readmission after discharge from a medium secure unit Clarke et al Readmission after discharge from a medium secure unit Overview of readmission non-White); Mental Health Act classiﬁcation of mental illness; grave index offence; custodial sentence prior to age 18 years; problematic behaviour at school; contact with child mental health services; alleged childhood sexual abuse; alleged childhood physical abuse; previous in-patient care; previous self-harm; previous attempted suicide; history of ‘severe’ alcohol misuse; history of ‘severe’ drug misuse; age at admission; number of previous convictions; age at ﬁrst conviction; length of stay;14 and discharge on a restriction order. Only two of these variables (previous in-patient care and Mental Health Act classiﬁcation of mental illness) had a signiﬁcant effect on the survival rates. The log rank test (Mantel-Cox) indicated that the survival time to the ﬁrst readmission for those discharged directly to the community was signiﬁcantly shorter for patients with a classiﬁcation of mental illness than for patients with a classiﬁcation of psychopathic disorder: w2(1,n = 141) = 8.1, P50.01. The median time to ﬁrst readmission for patients with a classiﬁcation of mental illness was 1.9 years compared with 5.2 years for those with a classiﬁcation of psychopathic disorder. The survival time to the ﬁrst readmission for those discharged directly to the community was signiﬁcantly shorter for people who had received previous in-patient care: w2(1,n = 140) = 10.7, P50.01. The median times to ﬁrst readmission for patients who had received previous in-patient care and those who had not were 2.0 years and Signiﬁcantly more women (87.5%) than men (65.6%) were readmitted to hospital (w2(1,n = 490) = 15.05, P50.001; odds ratio (OR) 3.67, 95% CI 1.83-7.34, missing 60 cases). Readmission data were available for 462 patients with a Mental Health Act classiﬁcation of either psychopathic disorder or mental illness. Of these readmissions, 320 patients (69.3%) were readmitted to a psychiatric hospital. Signiﬁcantly more patients with a classiﬁcation of mental illness (72.7%) than with a classiﬁcation of psychopathic disorder (61.0%) were readmitted (w2(1,n = 462) = 6.14, P50.05; OR = 1.70, 95% CI 1.12-2.59, missing 57 cases). Readmission frequency data were available for 485 patients (Table 2). In total, 33 patients were readmitted on at least 10 occasions, of whom 12 were readmitted at least 20 times. Readmission of patients discharged to the community More than a quarter of men and women were discharged directly to the community: 126 men (27.5%) and 25 women (27.5%). Of these, a higher percentage of women (79.2%) than men (66.7%) were readmitted to a psychiatric hospital, but this was not signiﬁcantly higher: w2(1,n = 141) = 1.45, P40.05 (missing data for 10 cases). Thirty-one patients (29 men and 2 women) were discharged to the community on a restriction order. Of these, 21 patients (68%) were Table 2 Number of readmissions categorised by gender and Mental Health Act classiﬁcation Recorded readmissions to any hospital (frequency) 0 1 2-5 6-9 10-19 20+ Men (n = 406) 142 100 111 32 15 6 Mental illness (n = 284) 86 68 89 27 11 3 Psychopathic disorder (n = 99) 48 26 15 4 3 3 Women (n = 79) 10 25 21 11 6 6 Mental illness (n = 39) 4 12 11 6 2 4 Psychopathic disorder (n = 36) 5 13 8 5 3 2 All (n = 485) 152 125 132 43 21 12 Mental illness (n = 323) 90 80 100 33 13 7 Psychopathic disorder (n = 135) 53 39 23 9 6 5 Table 2 Number of readmissions categorised by gender and Mental Health Act classiﬁcation 126 https://doi.org/10.1192/pb.bp.112.039289 Published online by Cambridge University Press https://doi.org/10.1192/pb.bp.112.039289 Published online by Cambridge University Press ORIGINAL PAPERS Clarke et al Readmission after discharge from a medium secure unit Clarke et al Readmission after discharge from a medium secure unit Fig 1 Kaplan-Meier curve for readmission of patients discharged directly to the community according to their Mental Health Act classiﬁcation. Fig 2 Kaplan-Meier curve for readmission of patients discharged directly to the community according to previous in-patient care. Fig 1 Kaplan-Meier curve for readmission of patients discharged directly to the community according to their Mental Health Act classiﬁcation. psychopathic disorder (Table 3). Patients who had received previous in-patient treatment had a hazard of readmission 2.2 times higher than those who had not. Also, patients with a classiﬁcation of mental illness had a hazard of readmission 1.8 times higher than those with psychopathic disorder. The model was not signiﬁcantly improved by adding interactions between the predictor variables. psychopathic disorder (Table 3). Patients who had received previous in-patient treatment had a hazard of readmission 2.2 times higher than those who had not. Also, patients with a classiﬁcation of mental illness had a hazard of readmission 1.8 times higher than those with psychopathic disorder. The model was not signiﬁcantly improved by adding interactions between the predictor variables. p A logistic regression analysis was then conducted to examine which of the 19 variables predicted readmission (n = 112, missing data 39). Prediction success overall was 80% (94% for readmission but only 50% for no readmission). Two variables made a signiﬁcant contribution to prediction: previous in-patient treatment and not having a history of self-harm. Those with previous in-patient treatment were 5.7 times more likely to be readmitted, and a history of self-harm was associated with an 88% decrease in the odds of being readmitted. Three variables approached signiﬁcance: classiﬁcation of mental illness (P = 0.055), number of previous convictions (P = 0.055) and age (P = 0.050). Fig 1 Kaplan-Meier curve for readmission of patients discharged directly to the community according to their Mental Health Act classiﬁcation. Fig 2 Kaplan-Meier curve for readmission of patients discharged directly to the community according to previous in-patient care. Readmission to Arnold Lodge and other secure hospitals Of the 550 discharged patients, 113 (20.5%) were readmitted to Arnold Lodge. Of these, 19 (3.5%) were readmitted twice and 7 (1.3%) were readmitted three times. There was no signiﬁcant difference in gender (19.6% men, 25.3% women) or in Mental Health Act classiﬁcation (22.2% mental illness, 16.7% psychopathic disorder) in readmission to Arnold Lodge. A further 43 patients (7.8%)- excluding patients who were discharged directly to another medium secure unit - were readmitted to other medium secure units, resulting in 156 patients (28.4%) being readmitted to medium secure care at some point during the follow-up. Forty-two patients were admitted to a high secure hospital (40 transferred directly) at some point during the follow-up, resulting in 82 patients (14.9%) spending time in high secure care after discharge from Arnold Lodge. Twelve patients (2.2%) had two admissions to a high secure hospital. Experiencing further secure care following discharge was common, with 207 patients (37.6%) subsequently spending some time in medium and/or high security. Fig 2 Kaplan-Meier curve for readmission of patients discharged directly to the community according to previous in-patient care. 6.5 years respectively. Survival curves for these variables are shown in Figs 1 and 2. Discussion Readmission was common for patients discharged from their ﬁrst admission to Arnold Lodge medium secure unit (62%), particularly to non-secure psychiatric hospitals. Women were 3.7 times more likely than men to be readmitted. Many patients had several readmissions over the follow-up period - an outcome noted in a different medium secure unit sample.7 Readmission is not of itself a negative outcome; for example, informal readmission to an open psychiatric unit is a far better outcome than the need to admit to high security following a serious offence. Over a quarter of patients were readmitted at least once to a medium secure unit - a ﬁfth to Arnold Lodge - and this proportion would have been larger if another unit had not opened that readmitted patients from the north of the region. Over a third of those discharged from their ﬁrst admission to Arnold Lodge subsequently spent time in medium or high security, with some patients spending time in both. Over a quarter were discharged directly to the community from their ﬁrst admission; nonetheless, a quarter of these were readmitted to a medium secure unit during the follow-up. Multiple sources were used to corroborate information and to minimise attrition. Missing data for readmission to any psychiatric hospital were fairly low (11%) given the large sample size and long follow-up period. The research was conducted under Section 60 of the Health and Social Care Act 2001. Despite this, delays imposed by one trust made it impractical to collect data from their case ﬁles and made contacting further trusts pointless; this accounts for some missing readmission data. Further limitations were that data were collected retrospectively and that the study is of a single unit; therefore the ﬁndings may not be generalisable to other medium secure units. g Of those discharged directly to the community, only previous in-patient treatment and having a mental illness were associated with shorter time to ﬁrst readmission. Previous in-patient treatment was also a predictor of readmission to any hospital. In addition, a history of self- harm was associated with a decreased risk of readmission. This may be explained by self-harm being more common in those with a classiﬁcation of psychopathic disorder compared with mental illness, a predictor that approached signiﬁcance. It is clinically intuitive that the mentally ill group would have more previous and future hospital admissions. Discussion Indeed, in our cohort two-thirds of men with a Mental Health Act classiﬁcation of mental illness had a previous admission compared with less than half of men with a classiﬁcation of psychopathic disorder (further information available from authors). Conversely, patients with a classiﬁcation of psychopathic disorder had a shorter time to reconviction than those with mental illness.14 Incidentally, the risk of death for patients with a classiﬁcation of either mental illness or psychopathic disorder was higher than that for the general population. However, although the risk of death was higher for patients with mental illness compared with psychopathic disorder, the difference was not signiﬁcant.11 ORIGINAL PAPERS ORIGINAL PAPERS Clarke et al Readmission after discharge from a medium secure unit Using the same exclusion criteria (missing data and those who spent all their ﬁrst year in hospital), the overall percentage of patients readmitted at least once to a psychiatric hospital in the ﬁrst year after discharge (32%) was similar to that of a national survey of medium secure units (28%).9 The percentage of patients readmitted to any psychiatric hospital (69%) approaches that found in the previous largest follow-up of a single medium secure unit (74%).6 However, our study had a longer mean follow-up: 9.5 years v. 6.6 years. A smaller percentage of discharged patients (15%) spent some of the follow-up period in a high secure hospital than that found by Maden et al (21%).6 These rates of readmission have to be placed in a national context as they will have been affected by the contraction in the numbers of high secure beds and the expansion of medium and low secure units. Community treatment has also undergone substantial changes, for example, assertive outreach, community forensic teams, crisis resolution and home treatment services now provide treatment in the community that was not previously available. The legal powers available for community treatment also changed during the study period,15 with the introduction of supervised discharge in 1996 (superseded by supervised community treatment orders in the 2007 amendments to the Mental Health Act). The patients’ diagnoses also have implications as to whether services are likely to accept them. Arnold Lodge has a dedicated personality disorder unit and consequently this cohort has more cases of personality disorder than reported in other medium secure unit studies. Time spent in institutions Some patients remained ‘institutionalised’ in hospital or prison throughout the follow-up. Data were available for 535 discharged patients, of whom 46 (8.6%) remained in a hospital throughout the follow-up. The mean time in hospital after transfer from Arnold Lodge for these 46 patients was 6.3 years (s.d. = 4.7). When time spent in prison was included, 88 (16.5%) of the 534 discharged patients for whom data were available remained either in hospital or in prison throughout the follow-up. The mean time in hospital or prison after discharge for these patients was 5.7 years (s.d. = 4.6). Time of readmission A Cox proportional hazards model, which allows for the effects of several predictor variables, was then developed to investigate the time to readmission for patients discharged directly to the community by taking into account the signiﬁcant predictors. This model examined the time to readmission controlling for previous in-patient care and Mental Health Act classiﬁcation of mental illness or Time of readmission In the ﬁrst year after discharge, 121 patients (22.0%) had at least one readmission to a psychiatric hospital. This group comprised 92 men (20.0%) and 29 women (31.9%). The overall percentage rises to 31.6% when those who spent all their ﬁrst year in hospital (n = 101) or had missing data (n = 66) are excluded. Table 3 Estimates of variables for Cox regression (for readmission) B s.e. Wald d.f. P Exp(B) Hazard ratio (95% CI) Previous in-patient 0.784 0.248 9.991 1 0.002 2.190 (1.347-3.561) Mental illness 0.577 0.219 6.936 1 0.008 1.780 (1.159-2.734) 127 ://doi.org/10.1192/pb.bp.112.039289 Published online by Cambridge University Press Table 3 Estimates of variables for Cox regression (for readmission) B s.e. Wald d.f. P Exp(B) Hazard ratio (95% CI) Previous in-patient 0.784 0.248 9.991 1 0.002 2.190 (1.347-3.561) Mental illness 0.577 0.219 6.936 1 0.008 1.780 (1.159-2.734) Table 3 Estimates of variables for Cox regression (for readmission) 127 Funding The initial description of the cohort and follow-up was funded by Nottinghamshire Healthcare National Health Service (NHS) Trust; further follow-up was funded by the NHS National Forensic Mental Health Research and Development Programme. 6 Maden A, Rutter S, McClintock T, Friendship C, Gunn J. Outcome of admission to a medium secure psychiatric unit. I. Short- and long-term outcome. Br J Psychiatry 1999; 175: 313-6. About the authors 7 Baxter R, Rabe-Hesketh S, Parrott J. Characteristics, needs and reoffending in a group of patients with schizophrenia formerly treated in medium security. J Forensic Psychiatry 1999; 10: 69-83. Martin Clarke (PhD) is a Research Fellow at the Institute of Mental Health, University of Nottingham, UK; Conor Duggan (PhD, MD, FRCPsych) is Emeritus Professor Forensic Mental Health at the Institute of Mental Health, University of Nottingham, UK and Head of Research and Development for Partnerships in Care; Clive R. Hollin (PhD) is Professor of Criminological Psychology in the School of Psychology at the University of Leicester, UK; Nick Huband (PhD) is a Clinical Research Fellow, Section of Forensic Mental Health at the Institute of Mental Health, University of Nottingham, and Nottinghamshire Healthcare NHS Trust, UK; Lucy McCarthy (PhD) is a Senior Research Fellow at Arnold Lodge, Nottinghamshire Healthcare NHS Trust, Leicester, UK; Steffan Davies (FRCPsych) is Consultant Forensic Psychiatrist at Northamptonshire Healthcare NHS Foundation Trust, Northampton, UK. 8 Dolan M, Khawaja A. The HCR-20 and post-discharge outcome in male patients discharged from medium security in the UK. Aggress Behav 2004; 30: 469-83. 9 Maden A, Skapinakis P, Lewis G, Scott F, Burnett R, Jamieson E. Gender differences in reoffending after discharge from medium-secure units. National cohort study in England and Wales. Br J Psychiatry 2006; 189: 168-72. 10 Davies S, Clarke M, Hollin C, Duggan C. Long-term outcomes after discharge from medium secure care: a cause for concern. Br J Psychiatry 2007; 191: 70-4. 11 Clarke M, Davies S, Hollin C, Duggan C. Long-term suicide risk in forensic psychiatric patients. Arch Suicide Res 2011; 15: 16-28. ORIGINAL PAPERS ORIGINAL PAPERS Clarke et al Readmission after discharge from a medium secure unit 5 Gray NS, Snowden RJ, MacCulloch S, Phillips H,Taylor J, MacCulloch MJ. Relative efﬁcacy of criminological, clinical, and personality measures of future risk of offending in mentally disordered offenders: a comparative study of HCR-20, PCL:SV, and OGRS. J Consult Clin Psychol 2004; 72: 523-30. Study implications Patients discharged from medium security have long-term conditions and present long-term risks requiring support from mental health services. Our cohort comprised a group of men and women who were at risk of reconviction of a new offence,10 premature death, particularly by suicide,11 and readmission to secure psychiatric services. All these adverse outcomes are most common in the year following discharge, but the risk remains over many years of follow- up. Those treated in medium security remain at risk of recurrence of their mental disorder and risk events for many years and require careful long-term follow-up, retaining a detailed knowledge of their conditions and risks - a difﬁcult task in modern services with multiple teams and transitions in care. 128 https://doi.org/10.1192/pb.bp.112.039289 Published online by Cambridge University Press https://doi.org/10.1192/pb.bp.112.039289 Published online by Cambridge University Press https://doi.org/10.1192/pb.bp.112.039289 Published online by Cambridge University Press References 12 Sahota S, Davies S, Duggan C, Clarke M, Huband N, Owen V. Women admitted to medium secure care: their admission characteristics and outcome as compared with men. Int J Forensic Ment Health 2010; 9: 110-7. 1 Friendship C, McClintock T, Rutter S, Maden A. Re-offending: patients discharged from a Regional Secure Unit. Crim Behav Ment Health 1999; 9: 226-36. 13 Ricketts D, Carnell H, Davies S, Kaul A, Duggan C. First admissions to a regional secure unit over a 16-year period: changes in demographic and service characteristics. J Forensic Psychiatry 2001; 12: 78-89. 2 Edwards J, Steed P, Murray K. Clinical and forensic outcome 2 years and 5 years after admission to a medium secure unit. J Forensic Psychiatry 2002; 13: 68-87. 14 Gibbon S, Huband N, Bujkiewicz S, Hollin C, Clarke M, Davies S, et al. The inﬂuence of admission characteristics on outcome: evidence from a medium secure forensic cohort. Personal Ment Health 2013; 7: 1-10. 3 Falla S, Sugarman P, Roberts L. Reconviction after discharge from a regional secure unit. Med Sci Law 2000; 40: 156-7. 4 Maden A, Scott F, Burnett R, Lewis GH, Skapinakis P. Offending in psychiatric patients after discharge from medium secure units: prospective national cohort study. BMJ 2004; 328: 1534. 15 Davies S. Compulsory treatment in the community: current legal powers. Adv Psychiatr Treat 2002; 8: 180-8. 129 https://doi.org/10.1192/pb.bp.112.039289 Published online by Cambridge University Press
https://openalex.org/W4205966075	http://pdf.blucher.com.br/engineeringproceedings/siintec2021/208672.pdf	English	null	BIOBUTANOL AS AN ALTERNATIVE AND SUSTAINABLE FUEL: AN INTEGRATIVE REVIEW	Blucher Engineering Proceedings	2,021	cc-by	3,822	BIOBUTANOL AS AN ALTERNATIVE AND SUSTAINABLE FUEL: AN INTEGRATIVE REVIEW Ana Carolina Araújo dos Santos a, Ana Caroline Sobral Loureiro b ,Ana Lúcia Barbosa de Souza c; Natália Barbosa da Silva d, Reinaldo Coelho Mirre e; Fernando Luiz Pellegrini Pessoa f a Graduanda em Engenharia Química; PRH 27.1 – ANP/FINEP; ana.santos@aln.senaicimatec.edu.br bCentro Universitário SENAI CIMATEC; Salvador-BA; carolsobral.loureiro@gmail.com c Centro Universitário SENAI CIMATEC; Salvador-BA; ana.lbs@fieb.org.br dCentro Universitário SENAI CIMATEC; Salvador-BA; nataliabarbosasv@gmail.com e Centro Universitário SENAI CIMATEC; Salvador-BA; reinaldo.mirre@fbter.org.br f Centro Universitário SENAI CIMATEC; Salvador-BA; fernando.pessoa@fieb.org.br a Graduanda em Engenharia Química; PRH 27.1 – ANP/FINEP; ana.santos@aln.senaicimatec bCentro Universitário SENAI CIMATEC; Salvador-BA; carolsobral.loureiro@gmail.com c Centro Universitário SENAI CIMATEC; Salvador-BA; ana.lbs@fieb.org.br dCentro Universitário SENAI CIMATEC; Salvador-BA; nataliabarbosasv@gmail.com e Centro Universitário SENAI CIMATEC; Salvador-BA; reinaldo.mirre@fbter.org.br f Centro Universitário SENAI CIMATEC; Salvador-BA; fernando.pessoa@fieb.org.br c Centro Universitário SENAI CIMATEC; Salvador-BA; ana.lbs@fieb.org.br dCentro Universitário SENAI CIMATEC; Salvador-BA; nataliabarbosasv@gmail.com e Centro Universitário SENAI CIMATEC; Salvador-BA; reinaldo.mirre@fbter.org.br f Centro Universitário SENAI CIMATEC; Salvador-BA; fernando.pessoa@fieb.org.br Abstract: It is necessary to invest in cleaner, renewable and sustainable energy sources to meet global fuel demand. Biobutanol is produced by biotechnological route, by ABE fermentation process. Biobutanol as biofuel has gasoline-like properties and its energy efficiency is 25% higher than ethanol. The objective of this work was to conduct an integrativ review on the production of biobutanol, in order to collect data on the market of this biofuel in order to understand the challenges involved in the production of biobutanol. The systematic review was carried out using the inclusion method. It was possible to analyze the world scenario of biobutanol and evaluate that so far in Brazil its demand is of greater applicability as a solvent in industries. Keywords: Biofuel; Biobutanol; ABE Fermentation; n-Butanol; Integrative Review Keywords: Biofuel; Biobutanol; ABE Fermentation; n-Butanol; Integrative Review Resumo: É cada vez mais necessário investir em fontes de energias mais limpas, renováveis e sustentáveis para atender a demanda global de combustível. O biobutanol é produzido por rota biotecnológica, pelo processo de fermentação ABE. O biobutanol, como biocombustível, possui propriedades semelhantes às da gasolina e sua eficiência energética é 25% maior que a do etanol. O objetivo deste trabalho foi realizar uma revisão integrativa acerca da produção de biobutanol, com o intuito de levantar dados sobre o mercado deste biocombustível a fim de compreender os desafios envolvidos na sua produção. A revisão sistemática foi feita utilizando o método de inclusão e exclusão. VII INTERNATIONAL SYMPOSIUM ON INNOVATION AND TECHNOLOGY (SIINTEC) One Planet, one Ocean and one Health - 2021 VII INTERNATIONAL SYMPOSIUM ON INNOVATION AND TECHNOLOGY (SIINTEC) One Planet, one Ocean and one Health - 2021 VII INTERNATIONAL SYMPOSIUM ON INNOVATION AND TECHNOLOGY (SIINTEC) One Planet, one Ocean and one Health - 2021 Palavras-chave: Biocombustíveis; Biobutanol; Fermentação ABE; n-butanol; Revisão Integrada. 1. INTRODUCTION The growing environmental need is to turn to cleaner, renewable and sustainable energy sources to meet the ever-increasing demand for fuel. Renewable energy will be the world's fastest growing energy source, expected to double from 2015 to 2030. [1] Biofuel, produced through biological process, has drawn great attention from the world due to its environment-friendly feature. [2] Biobutanol refers to the butanol produced from the natural or organic or biodegradable or renewable biomass.[3] Butanol is a higher alcohol whose chemical formula is represented as C4H10O and which has four structural isomers: n-butanol, isobutanol, tert-butanol and sec-butanol.[4] n-Butanol is a chemical compound that falls within the alcohol reagent family. Due to their increasing use as additives, solvents and fuels, alcohols have found their position in the market. Biobutanol as a fuel derived from biomass feedstock produced using ABE fermentation turns out to be an extremely clean and sustainable fuel with a high energy density comparable to gasoline. [3] Biobutanol is considered the gasoline of the future, and a good substitute for gasoline due to its physical properties such as high boiling point, economy and safety [4,5]. In the United States (USA) perspective, ASTM D7862 - 21 standard allows butanol intended to be blended with gasoline at 1% to 12.5% by volume for use as automotive spark ignition engine fuel [6], while ASTM D787533 provides a method for determining the butanol and acetone content in butanol by gas chromatography technique, intended for blending with gasoline [4,7]. Brazil has a large availability of fermentable raw material, especially sugarcane and corn, and well-established industrial facilities for alcoholic fermentation, so it has great potential to become a reference for the export of biobutanol [8]. Most industrial initiatives in the field of n-butanol, however, are aimed at the biofuels market because of n-butanol's better properties compared to ethanol, as it has 25% more energy than ethanol, lower water miscibility and less corrosive properties. Butanol can be blended with gasoline and diesel in higher proportions, it can replace the use of gasoline, while ethanol can only be used as an additive [5,9]. Therefore, the objective of this work was to conduct an integrative review on the production of biobutanol, in order to collect data on the foreign and internal market of this biofuel, as well as to understand the challenges involved in the production of biobutanol. BIOBUTANOL AS AN ALTERNATIVE AND SUSTAINABLE FUEL: AN INTEGRATIVE REVIEW O cenário mundial de biobutanol foi analisado e, constatou-se que, no Brasil, até o momento, a sua demanda é de maior aplicabilidade como solvente em indústrias. ABE; Palavras-chave: Biocombustíveis; Biobutanol; Fermentação ABE; n-b Revisão Integrada. Palavras-chave: Biocombustíveis; Biobutanol; Fermentação ABE; n-butanol; Revisão Integrada. ISSN: 2357-7592 VII INTERNATIONAL SYMPOSIUM ON INNOVATION AND TECHNOLOGY (SIINTEC) One Planet, one Ocean and one Health - 2021 VII INTERNATIONAL SYMPOSIUM ON INNOVATION AND TECHNOLOGY (SIINTEC) One Planet, one Ocean and one Health - 2021 VII INTERNATIONAL SYMPOSIUM ON INNOVATION AND TECHNOLOGY (SIINTEC) One Planet, one Ocean and one Health - 2021 This review was divided into four stages, as shown in Figure 1, according to a previously established protocol [10]. This review was divided into four stages, as shown in Figure 1, according to a previously established protocol [10]. previously established protocol . Figure 1: Stages of the integrative review that will be addressed at work. Source: Authors (2021). 3. INTEGRATIVE REVIEW 3.1 Stage 1: Preparation of a guide question In this stage are defined the questions that will be guide the research that will be developed. For the elaboration of this work, the following questions were used: 1- What are the applications of Biobutanol? 2- Is there a possibility that biobutanol is a biofuel used in Brazil? 3- What is the market forecast for biobutanol by 2030? 3.2. Stage 2 Research or Sampling in literature Figure 1: Stages of the integrative review that will be addressed at work. Figure 1: Stages of the integrative review that will be addressed at work Figure 1: Stages of the integrative review that will be addressed at work. Source: Authors (2021). Source: Authors (2021). 3. INTEGRATIVE REVIEW 3.1 Stage 1: Preparation of a guide question In this stage are defined the questions that will be guide the research that will be developed. For the elaboration of this work, the following questions were used: 1- What are the applications of Biobutanol? 1- What are the applications of Biobutanol? 2- Is there a possibility that biobutanol is a biofuel used in 3- What is the market forecast for biobutanol by 2030? 3.2. Stage 2 Research or Sampling in literature 2. METHODOLOGY The present work sought with the review integrativ, to show the main points related to the production process of biobutanol, commercial prospection in the Brazilian scenario. The main databases that provide scientific articles were consulted, such as: SciELO, ScienceDirect and Google Academic. The search was for articles written between 2013 and 2021, preferably considering articles available in its full version. Inclusion and exclusion criteria were also used in which more than 60 articles were found throughout the research. Throughout the research, 10 sources were evaluated, considering some themes: Renewable energies; Production of Biobutanol, economic and market analysis. After the selection of the sources, according to the inclusion criteria, the following steps. ISSN: 2357-7592 VII INTERNATIONAL SYMPOSIUM ON INNOVATION AND TECHNOLOGY (SIINTEC) One Planet, one Ocean and one Health - 2021 VII INTERNATIONAL SYMPOSIUM ON INNOVATION AND TECHNOLOGY (SIINTEC) One Planet, one Ocean and one Health - 2021 ISSN: 2357-7592 3.2. Stage 2 Research or Sampling in literature In this step, the strings used to search the literature and the databases to be searched are defined. The data were obtained using the following platforms: SciELO, Google Scholar and Science Direct. Figure 2 shows the steps followed for the bibliographic survey. Figure 2: Steps for bibliographic survey. Source: Authors (2021). Stages 1 and 2 were performed in order to obtain information regarding the amount of research conducted on biofuels and biobutanol specifically. In step 3, we were raised on the biobutanol production processes and their production prospects. In step 4, it reflects the crossover between biobutanol production and its market prospects. At this stage, the inclusion and exclusion criteria were applied, considering the exclusion criteria for duplicate articles and non-eligibility for the proposed theme. Figure 2: Steps for bibliographic survey. Source: Authors (2021). Figure 2: Steps for bibliographic survey. Source: Authors (2021). Figure 2: Steps for bibliographic survey. Source: Authors (2021). Stages 1 and 2 were performed in order to obtain information regarding the amount of research conducted on biofuels and biobutanol specifically. In step 3, we were raised on the biobutanol production processes and their production prospects. In step 4, it reflects the crossover between biobutanol production and its market prospects. At this stage, the inclusion and exclusion criteria were applied, considering the exclusion criteria for duplicate articles and non-eligibility for the proposed theme. While the inclusion criteria were selected articles, dissertations and technical reports published in English or Portuguese. ISSN: 2357-7592 VII INTERNATIONAL SYMPOSIUM ON INNOVATION AND TECHNOLOGY (SIINTEC) One Planet, one Ocean and one Health - 2021 present the production of n-butanol by biochemical route through ABE fermentation using clostridium bacteria as the main microorganism for the process. present the production of n-butanol by biochemical route through ABE fermentation using clostridium bacteria as the main microorganism for the process. The solvent separation technologies obtained by ABE fermentation are presented in most articles as impact agents in the economy of biochemical processes, because the separation processes directly influence the production price of biobutanol. The prospect of biobutanol by 2030 was optimistic according to the technical reports prepared between 2019 and 2021[1,11,12]. It also points out that countries such as China, India and the United States have great interest in investing in biobutanol as fuel due [11], mainly the ability of biobutanol to be mixed and have squealing gasoline properties and have greater efficiency than ethanol. In the United States the mixture of biobutanol in gasoline is regulated by ASTM D7862 [6] and ASTM D7875 [7]. The Brazilian market is promising for biofuels, since the second largest consumer in the domestic energy matrix is the transportation sector, responsible for 31.2% of domestic energy consumption in 2020 (BEN, 2020) [13,14], but in Brazil, biobutanol as a biofuel, since there is a large investment in the ethanol production market and since cars are well adapted to this biofuel [4], at the moment,the largest market in Brazil for biobutanol to be used is as a solvent in the paint and varnish producers' industry. 3.3. Stage 3: Data Collection g In the third stage, the articles are synthesized and organized according to the reference to the theme addressed. Figure 3 shows the number of publications found in the integrative review steps. Figure 3: Number of searches found at each stage. Figure 3: Number of searches found at each stage. Source: Authors (2021). Source: Authors (2021). Research on biofuels has great relevance in the market. A total of 60 articles addressing the theme of biobutanol were selected. 10 articles were evaluated, considering some themes: Renewable energies; Production of Biobutanol, economic and market analysis. Besides,2 international standard and 6 technical reports without full access were consulted in order to understand the projection of the biobutanol market by 2030. y 3.4. Step 4: Critical Analysis of Studies Inclued In this stage it is performed after the application of the inclusion and exclusion criteria of papers in order to make a critical analysis of the selected bibliography. Figure 4 shows the quantity of the bibliographic survey corresponding to 67% of articles, 22% of technical reports and 11% of international standards. Figure 4: Quantitative bibliography. Source: Authors (2021). Figure 4: Quantitative bibliography. Source: Authors (2021). udies address the scope of the insertion of biofuels in the energy matrix Figure 4: Quantitative bibliography. Figure 4: Quantitative bibliography. Source: Authors (2021). The studies address the scope of the insertion of biofuels in the energy matrix as a relevant factor to combat global warming. The applications of biobutanol as solvent in the chemical, petrochemical and also biofuel industries. They specifically Source: Authors (2021). Source: Authors (2021). The studies address the scope of the insertion of biofuels in the energy matrix as a relevant factor to combat global warming. The applications of biobutanol as solvent in the chemical, petrochemical and also biofuel industries. They specifically ISSN: 2357-7592 VII INTERNATIONAL SYMPOSIUM ON INNOVATION AND TECHNOLOGY (SIINTEC) One Planet, one Ocean and one Health - 2021 has 25% more energy than ethanol, lower water miscibility and less corrosive properties. Butanol can be blended with gasoline and diesel in higher proportions, it can replace the use of gasoline, while ethanol can only be used as an additive. [5,9] has 25% more energy than ethanol, lower water miscibility and less corrosive properties. Butanol can be blended with gasoline and diesel in higher proportions, it can replace the use of gasoline, while ethanol can only be used as an additive. [5,9] The biobutanol market is expected to register a CAGR (Compound Annual Growth Rate) of over 7%, during the forecast period. The major factors driving the market studied are largely carbon emission reduction and gaining prominence as a foundation for chemical manufacturing. [11] In 2020, the worldwide n-butanol market volume was more than 5.1 million metric tons. The market volume of this organic compound is forecast to grow to around 6.2 million metric tons worldwide by the year 2026 [18]. In Figure 6, we can analyze the n-butanol market by 2026. g y y Figure 6: Global n-butanol market volume from 2015 to 2020, with forecast from 2021 to 2026 (in million metric tons). Source: Statista (2020). The biobutanol market is in an incipient and apparently consolidated phase. The main biobutanol producing industries are Cathay Industrial Biotech, Gevo inc, Butamax Advanced Biofuels and Cobalt Technologies.[11] Cobalt Technologies has developed several technological innovations to produce biobutanol to reduce production costs by 30% to 60% and radically reduce the impact of carbon compared to petroleum-derived butanol, which finds applications in various chemicals and fuels, including 1-butylene, butadiene, lubricating oil and poly-alpha-olefins. [12] In Brazil, according to the Brazilian Chemical Industry Association (ABIQUIM), Oxiteno and Elekeiroz are currently producing butanol isomers. Figure 6: Global n-butanol market volume from 2015 to 2020, with forecast from 2021 to 2026 (in million metric tons). S S i (2020) Figure 6: Global n-butanol market volume from 2015 to 2020, with forecast from 2021 to 2026 (in million metric tons). Source: Statista (2020). The biobutanol market is in an incipient and apparently consolidated phase. VII INTERNATIONAL SYMPOSIUM ON INNOVATION AND TECHNOLOGY (SIINTEC) One Planet, one Ocean and one Health - 2021 The main biobutanol producing industries are Cathay Industrial Biotech, Gevo inc, Butamax Advanced Biofuels and Cobalt Technologies.[11] Cobalt Technologies has developed several technological innovations to produce biobutanol to reduce production costs by 30% to 60% and radically reduce the impact of carbon compared to petroleum-derived butanol, which finds applications in various chemicals and fuels, including 1-butylene, butadiene, lubricating oil and poly-alpha-olefins. [12] In Brazil, according to the Brazilian Chemical Industry Association (ABIQUIM), Oxiteno and Elekeiroz are currently producing butanol isomers. ISSN: 2357-7592 3.5. Step 5: Discussion of results In this stage, the interpretation and sinthesis of the information obtained from the results of the research on the theme of biobutanol is made. The research covered bibliography from 2011 to 2021. it is possible to observe in Figure 5 the distribution of material used in this work per year. p y Figure 5: Distribution of publications on the theme of research by year. Source: Authors (2021). According to The Energy, Information Administration (EIA) the increase in world energy consumption would be around 56% in 2040 compared to 2010 [15, 16]. Shenbagamuthuraman (2021) showed that gasoline and other liquid fuels are the dominant energy sources for the transportation sector, although there is a slight decline in total transport energy consumption from 96% in 2012 to 88% in 2040. [17] Countries such as the US, China and Brazil have a goal of achieving 15-27% of biofuel mixture with conventional fuel by 2020-2022. [1] Source: Authors (2021). Source: Authors (2021). Source: Authors (2021). According to The Energy, Information Administration (EIA) the increase in world energy consumption would be around 56% in 2040 compared to 2010 [15, 16]. Shenbagamuthuraman (2021) showed that gasoline and other liquid fuels are the dominant energy sources for the transportation sector, although there is a slight decline in total transport energy consumption from 96% in 2012 to 88% in 2040. [17] Countries such as the US, China and Brazil have a goal of achieving 15-27% of biofuel mixture with conventional fuel by 2020-2022. [1] Most industrial initiatives in the field of n-butanol, however, are aimed at the biofuels market because of n-butanol's better properties compared to ethanol, as it ISSN: 2357-7592 VII INTERNATIONAL SYMPOSIUM ON INNOVATION AND TECHNOLOGY (SIINTEC) One Planet, one Ocean and one Health - 2021 inserting biobutanol as biofuels in its energy matrix, mainly in countries such as China and the U.S. For Brazil, biobutanol may be a great possibility for insertion as a biofuel due to the large supply of raw material from sugarcane close to the areas where the plants will be implemented. inserting biobutanol as biofuels in its energy matrix, mainly in countries such as China and the U.S. For Brazil, biobutanol may be a great possibility for insertion as a biofuel due to the large supply of raw material from sugarcane close to the areas where the plants will be implemented. However, to date, greater use of n-butanol is as a solvent applied in the production of paints and coatings. In addition, another obstacle to the inclusion of biobutanol as biofuel in the energy matrix is regulation, since this biofuel is currently can be considered an experimental fuel by the Brazilian National Agency of Petroleum, Natural Gas and Biofuels (ANP), because at present Brazil does not have technical standards to designate the use or mixture of this biofuel with gasoline or diesel. It was also possible to highlight the scarcity of publications about the biobutanol market in Brazil, although the country has great potential to be a producer of this biofuel. Acknowledgments To the ANP Human Resources Program, through PRH 27.1 ANP/FINEP/SENAI CIMATEC, for the scholarship granted and financial support to this work. To the ANP Human Resources Program, through PRH 27.1 ANP/FINEP/SENAI CIMATEC, for the scholarship granted and financial support to this work. 4. CONCLUSION In this work, 60 more articles related to the theme were collected using the databases of the Academic, Science Direct and SciELO. Six technical reports and 2 international standards were considered to complement the information. This review demonstrated that biobutanol holds a promise as a renewable biofuel, given its ability to be a substitute for fossil fuels and its property of being blended with gasoline and diesel. In addition, the world market is totally open to the possibility of ISSN: 2357-7592 5. REFERENCES 1MORDOR INTELLIGENCE. Biofuels Market. Available at: < Biofuels Market \| 2021 - 26 \| Industry Share, Size, Growth - Mordor Intelligence >. Accessed on: 18 Aug. 2021. 2 Y. Li. et al. Potential of acetone-butanol-ethanol (ABE) as a biofuel, 242 (2019), pp. 673 – 686. Available at: <https://doi.org/10.1016/j.fuel.2019.01.063>. Accessed on: 30 Mar. 2021. 3INDUSTRYARC. n-Butanol Market- Forecast (2020-2025). Available at:<https://www.industryarc.com/Report/15518/n-butanol-market.html>. Accessed on: 18 Aug. 2021. 4 BRANDÃO, L.F.P. Estudo do 1-butanol e 2-metil-1- propanol em misturas com a gasolina e o diesel: uma análise sob a perspectiva da especificação brasileira. 2017. Tese (Doutorado em Química) - Instituto de Química, Universidade de Brasília, Brasília, Distrito Federal, 2017. 5 Pugazhendhi et al., 2019. Bio-butanol as a promising liquid fuel for the future- recent updates and perspectives. Fuel, 253 (2019), pp. 637-646. 6 Norma ASTM D7862 – Standard Specification for Butanol for Blending with Gasoline for Use as Automotive Spark-Ignition Engine Fuel.< https://www.abntcatalogo.com.br/norma.aspx?ID=466168 >. Accessed on: 27 Apr. 2021. 7 Norma ASTM D7875 - Standard Test Method for Determination of Butanol and Acetone Content of Butanol for Blending with Gasoline by Gas Chromatography. ISSN: 2357-7592 VII INTERNATIONAL SYMPOSIUM ON INNOVATION AND TECHNOLOGY (SIINTEC) One Planet, one Ocean and one Health - 2021 VII INTERNATIONAL SYMPOSIUM ON INNOVATION AND TECHNOLOGY (SIINTEC) One Planet, one Ocean and one Health - 2021 VII INTERNATIONAL SYMPOSIUM ON INNOVATION AND TECHNOLOGY (SIINTEC) One Planet, one Ocean and one Health - 2021 8 NATALENSE, J.; ZOUAIN, D. Technology roadmapping for renewable fuels: Case of biobutanol in Brazil. Journal of Technology Management and Innovation, v. 8, n. 4, p. 143–152, 2013. 9 MATURANA, M.G.V. DILEMAS ESTRATÉGICOS NA DIFUSÃO DE INOVAÇÕES EM BIOPRODUTOS. 2019. Dissertação (Mestre em Ciências) - Escola de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 2019. 10 EVANGELISTA, A.T. et al. Princípios da química verde e a produção de ferro- gusa: uma revisão integrativa. In: V Simpósio Internacional de Inovação e Tecnologia, Anais do V SIINTEC, 1-9, 2019. 11Mordor Intelligence. Bio-butanol Market. Available at: <https://www.mordorintelligence.com/industry-reports/bio-butanol-market>. Accessed on: 18 Aug. 2021. 12 GRAND VIEW RESEARCH. n-Butanol Market Size, Share & Trends Analysis Report By Application, Regional Outlook, Competitive Strategies, And Segment Forecasts, 2019 To 2025. Available at:< https://www.grandviewresearch.com/industry-analysis/n-butanol-market>. Accessed on: 18 Aug. 2021. 13 BEN. Balanço Energético Nacional 2021. Disponível em:< https://www.epe.gov.br/pt/publicacoes-dados-abertos/publicacoes/balanco- energetico-nacional-2021>. Accessed on: 15 Aug. 2021. 14 FERREIRA, Marinalvo, L. Importância dos biocombustíveis na bioeconomia. PPV 688 – CULTURAS ENERGÉTICAS, 2020. 5. REFERENCES Available at:< http://site.ufvjm.edu.br/ica/files/2020/07/1-Import%C3%A2ncia-dos- biocombust%C3%ADveis-na-bioeconomia.pdf>. Accessed on: 27 Apr. 2021. EIA. International energy outlook. U.S.: Energy Information Administration; 2015 16 Awad, Omar I.. et.al. Overview of the oxygenated fuels in spark ignition engine: Environmental and performance. Renovar. Sustentar. Rev. Energia , 91 ( 2018 ) , pp. 394 – 408. Available at: https://doi.org/10.1016/j.rser.2018.03.107. Accessed on: 27 Apr. 2021. 17 Shenbagamuthuraman V.et al. State of art of valorising of diverse potential feedstocks for the production of alcohols and ethers: Current changes and perspectives. Chemosphere, Volume 286, 2022. Available at: https://doi.org/10.1016/j.chemosphere.2021.131587. Accessed on:15 Jun.2021. 18 STATISTA. Market volume of n-Butanol worldwide from 2015 to 2020. Available at: <https://www.statista.com/statistics/1245211/n-butanol-market-volume- worldwide/>. Accessed on: 18 Aug. 2021. 19 RIBEIRO, L.C.P. Produção de butanol por Clostridium beijerinckii NRRLB 598 a partir de matérias primas agroindustriais. 2019. Dissertação (Mestre em Ciências) - Escola de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 2019. ISSN: 2357-7592
https://openalex.org/W4395047617	https://ojs.library.queensu.ca/index.php/pocus/article/download/16761/11428	English	null	Point of Care Ultrasound Identification and Aspiration of a Neck Lymph Node	POCUS journal	2,024	cc-by	1,985	Point of Care Ultrasound Identification and Aspiration of a Neck Lymph Node Andrew Moore, DO1; Ali Mrad, MD1; Leonard Riley, MD1,2; Sonia M. Castillo MD2 (1) The University of Kansas Medical Center, Division of Pulmonary and Critical Care, Kansas City, KS, USA (2) Kansas City VA Medical Center, Division of Pulmonary and Critical Care, Kansas City, KS, USA Abstract The tissue diagnosis and staging of all types of lung cancer is foundational for prognosis and establishing the optimal treatment plan. In order to appropriately stage lung cancer, the highest stage should be established using the 8th edition TNM criteria, where tumor size (T), nodal involvement (N), and metastasis (M) are all taken into account. Establishing a tissue diagnosis may involve the use of CT guided biopsy, navigational bronchoscopy, endobronchial biopsy, endobronchial ultrasound, percutaneous lymph node biopsy and/or excisional biopsy of supraclavicular nodes. It is recommended to proceed with the method that is considered least invasive and provides the highest staging. We present a case of recurrent lung adenocarcinoma diagnosed with real time ultrasound-guided fine needle aspiration of a neck lymph node. Case Presentation needle aspiration (TBNA) for both diagnostic and staging purposes. However, his scans were reviewed and notable for an enlarged right-sided supraclavicular lymph node, which was not palpable on exam (Figure 2). A point of care ultrasound (POCUS) assessment of his right supraclavicular region with a linear probe demonstrated the findings in Figure 3 and Videos S1 and S2. Based on the patient’s clinical history and findings from the images and videos, we proceeded with ultrasound-guided fine needle aspiration (FNA). This provided a diagnosis of lung cancer and provided staging in a safer and less invasive way than EBUS. A 68-year-old man with a history of orthotopic liver transplant maintained on immunosuppression, right upper lobe adenocarcinoma status post chemotherapy and right upper lobectomy eight years prior, and 45-pack-year history of tobacco disorder presented to the clinic for consultation of his chronic cough. As part of his investigation, he underwent a computed tomography (CT) of the chest that was notable for mediastinal lymphadenopathy in the subcarinal and paratracheal regions (Figure 1A and 1B). There was no reported axillary or supraclavicular adenopathy. Case File Case File 11 \| POCUS J \| APR 2024 vol. 09 iss. 01 Discussion He was subsequently referred to pulmonary medicine for endobronchial ultrasound (EBUS) and transbronchial The tissue diagnosis and staging of all types of lung Corresponding Author: Dr. Sonia Castillo (email: sonia.castillovega@va.gov) This work is licensed under a Creative Commons Attribution 4.0 International License. Author(s) retain the copyright for their work. At the time of submission to POCUS Journal the author(s) grant the journal a limited and non revokable right to publish in the Journal under a Creative Commons Attribution 4.0 International License. DOI: https://doi.org/10.24908/pocusv9i1.16761 Figure 1. CT scan of the chest with contrast in the mediastinal window and transverse plane showing an enlarged (A) subcarinal and (B) lower right paratracheal lymph node. Figure 1. CT scan of the chest with contrast in the mediastinal window and transverse plane showing an enlarged (A) subcarinal and (B) lower right paratracheal lymph node. Corresponding Author: Dr. Sonia Castillo (email: sonia.castillovega@va.gov) This work is licensed under a Creative Commons Attribution 4.0 International License. Author(s) retain the copyright for their work. At the time of submission to POCUS Journal the author(s) grant the journal a limited and non revokable right to publish in the Journal under a Creative Commons Attribution 4.0 International License. DOI: https://doi.org/10.24908/pocusv9i1.16761 *Corresponding Author: Dr. Sonia Castillo (email: sonia.castillovega@va.gov) This work is licensed under a Creative Commons Attribution 4.0 International License. Author(s) retain the copyright for their work. At the time of submission to POCUS Journal the author(s) grant the journal a limited and non revokable right to publish in the Journal under a Creative Commons Attribution 4.0 International License. DOI: https://doi.org/10.24908/pocusv9i1.16761 APR 2024 vol. 09 iss. 01 \| POCUS J \| 12 Figure 2. CT scan of the chest with contrast in the mediastinal window and coronal plane showing an enlarged right supraclavicular lymph node. evaluation is needed through either percutaneous needle aspiration or open surgical biopsy. Factors limiting the use of surgical sampling include the need for an incision, bleeding, infection, potential need for sedation and missing the node of interest. All of these factors can be either reduced or eliminated with the use of percutaneous needle aspiration, which has demonstrated its utility in the literature [6,7]. While needle aspiration may have risk of bleeding because of its close proximity to the large neck vessels, it is exceedingly rare when done by a trained provider [8-10]. Discussion In a study by El- Shaarawy and colleagues, a neck ultrasound in subjects with evidence of intrathoracic lymphadenopathy found abnormal neck lymph nodes in more than one third of patients [7]. Additionally, they performed neck lymph node biopsies in eligible patients, which had a diagnostic yield of 92%, similar to previous reports [6,7]. Importantly, tissue sampling can be carried out by pulmonary physicians and avoids more imaging studies, procedures, and potential adverse effects from sedation and anesthesia [11-12]. Figure 2. CT scan of the chest with contrast in the mediastinal window and coronal plane showing an enlarged right supraclavicular lymph node. In our case, we were able to review the suspicious CT and noted the enlarged supraclavicular lymph node that failed to be reported on the formal read. This is a documented blind spot that has been demonstrated by Hassan et al., to miss 18% of cases of abnormally enlarged supraclavicular lymph nodes, with 55% of those being positive for malignancy. A critically important consideration for ensuring proper staging [8,13]. Our initial plan was to pursue EBUS-TBNA to provide tissue sampling and mediastinal staging. However, upon further investigating our patient’s concerning CT findings with POCUS, a suspicious 15 mm right supraclavicular lymph cancer is foundational for prognosis and establishing an optimal treatment plan. In order to appropriately stage lung cancer, the highest stage should be established using the 8th edition TNM criteria where tumor size (T), nodal involvement (N), and metastasis (M) are all taken into account [1]. Current guidelines for non-small cell lung cancer defines N0 disease as no regional lymph node involvement, N1 disease as involvement of ipsilateral peribronchial and/or ipsilateral hilar lymph nodes, N2 disease as involvement of the ipsilateral mediastinal and/or subcarinal lymph nodes, and N3 involvement of any of the following lymph node groups: contralateral mediastinal, contralateral hilar, ipsilateral or contralateral scalene, or supraclavicular nodes [1,2]. In order to establish tissue diagnosis, sampling is needed and can involve the use of CT guided biopsy, navigational bronchoscopy, endobronchial biopsy, EBUS, percutaneous lymph node biopsy, and/or excisional biopsy of supraclavicular nodes. All methods come with their own safety and efficacy profile that include, but are not limited to, pneumothorax, bleeding, infection, bronchospasm, laryngospasm, hypoxemia, hypercarbia, and aspiration [3,4]. These risks are first mediated by proceeding with the method that is considered least invasive and provides the highest staging. Figure 3. References 1. Goldstraw P, Chansky K, Crowley J, et al. The IASLC Lung Cancer Staging Project: Proposals for Revision of the TNM Stage Groupings in the Forthcoming (Eighth) Edition of the TNM Classification for Lung Cancer. Journal of Thoracic Oncology. 2016;11(1):39-51. 2. El-Sherief AH, Lau CT, Wu CC, Drake RL, Abbott GF, Rice TW. International Association for the Study of Lung Cancer (IASLC) Lymph Node Map: Radiologic Review with CT Illustration. RadioGraphics. 2014;34(6):1680-1691. 3. Eapen GA, Shah AM, Lei X, Jimenez CA, Morice RC, Yarmus L, Filner J, Ray C, Michaud G, Greenhill SR, Sarkiss M, Casal R, Rice D, Ost DE; American College of Chest Physicians Quality Improvement Registry, Education, and Evaluation (AQuIRE) Participants. Complications, consequences, and practice patterns of endobronchial ultrasound-guided transbronchial needle aspiration: Results of the AQuIRE registry. Chest. 2013 Apr;143(4):1044-1053. doi: 10.1378/ chest.12-0350. PMID: 23117878; PMCID: PMC3616680 4. Stahl DL, Richard KM, Papadimos TJ. Complications of bronchoscopy: A concise synopsis. Int J Crit Illn Inj Sci. 2015 Jul-Sep;5 (3):189-95. doi: 10.4103/2229-5151.164995. PMID: 26557489; PMCID: PMC4613418. Figure 4. Soft tissue ultrasound image showing an the enlarged right supraclavicular lymph node with the needle visible in its entire length. 5. Satterwhite, Lewis G. MD; Berkowitz, David M. MD; Parks, Christopher S. MD; Bechara, Rabih I. MD. Central Intranodal Vessels to Predict Cytology During Endobronchial Ultrasound Transbronchial Needle Aspiration. Journal of Bronchology & Interventional Pulmonology. 18(4):p 322-328, October 2011. \| DOI: 10.1097/ LBR.0b013e31823577d1 node was found. After discussing the risks, benefits, and alternatives, a percutaneous lymph node biopsy was pursued. The results of our lymph node aspiration were consistent with the patient’s prior adenocarcinoma of the lung. He was referred to oncology and started on systemic chemotherapy. 6. Kumaran M, Benamore RE, Vaidhyanath R, et al. Ultrasound guided cytological aspiration of supraclavicular lymph nodes in patients with suspected lung cancer. Thorax. 2005;60(3):229-233. 7. Prosch H, Strasser G, Sonka C, et al. Cervical Ultrasound (US) and US-guided Lymph Node Biopsy as a Routine Procedure for Staging of Lung Cancer. Ultraschall Med. 2007;28(06):598-603. 8. Hassan M, Nicholson T, Taylor L, et al. Focused Neck Ultrasound and Lymph Node Sampling by Respiratory Physicians in Suspected Lung Cancer. Respiration. 2022;101(1):57-62. Practically, suspicious neck lymph nodes are identified using a linear transducer. References Lymph nodes are characterized as echodense structures surrounded by a clearly defined hyperechoic capsule that are not collapsible, may have a fatty central hilum, and do not show evidence of blood flow on color or spectral Doppler [5]. Once location is confirmed, the site is cleaned, a local anesthetic is applied, and if needed, additional sedation mirroring other routine subcutaneous procedures is provided. In our practice, sampling of the identified lymph is done under real-time ultrasound guidance and an in-plane needle approach with 3-5 passes using an 18, 21, or 22 gauge needle and 10 cc syringe assembled in a needle gun (Figure 4, Video S3). This is akin to a version of the traditional view seen with EBUS-TBNA sampling. Each pass is evaluated with rapid on-site examination by the cytopathology team in the procedure room. 9. Hoosein MM, Barnes D, Khan AN, et al. The importance of ultrasound in staging and gaining a pathological diagnosis in patients with lung cancer--a two year single centre experience. Thorax. 2011;66 (5):414-417. 10. Ahmed M, Daneshvar C, Breen D. Routine neck ultrasound by respiratory physicians in the diagnosis and staging of patients with lung cancer and mediastinal lymphadenopathy: a prospective pilot study. ERJ Open Res. 2020 Feb 10;6(1):00180-2019. 11. El-Shaarawy B, Gharraf HS, El-Hefny A, Hassan M. Prospective Study of Focused Neck US by Respiratory Physicians in Patients With Pulmonary Disease and Intrathoracic Lymphadenopathy. Chest. 2023;163(4):994-996. 12. Ahmed M, Flannery A, Daneshvar C, Breen D. PET and Neck Ultrasound for the Detection of Cervical Lymphadenopathy in Patients with Lung Cancer and Mediastinal Lymphadenopathy. Respiration. 2018;96(2):138-143. 13. Wu CC, Khorashadi L, Abbott GF, Shepard JA. Common blind spots on chest CT: where are they all hiding? Part 2, Extrapulmonary structures. AJR Am J Roentgenol. 2013;201(5):W671-W677. Discussion Soft tissue ultrasound image showing an enlarged right supraclavicular lymph node. To evaluate the neck region, CT or ultrasound can be used. When evaluating lymph nodes, ultrasonographic characteristics that are more suggestive of malignancy include larger size (>5 mm), rounded shape (as opposed to oval or reniform), irregular borders, and lack of visible hilum [5]. If suspicious nodes are found, further Figure 3. Soft tissue ultrasound image showing an enlarged right supraclavicular lymph node. 13 \| POCUS J \| APR 2024 vol. 09 iss. 01 Figure 4. Soft tissue ultrasound image showing an the enlarged right supraclavicular lymph node with the needle visible in its entire length. Disclosures The authors have no conflict of interest to disclose. 13. Wu CC, Khorashadi L, Abbott GF, Shepard JA. Common blind spots on chest CT: where are they all hiding? Part 2, Extrapulmonary structures. AJR Am J Roentgenol. 2013;201(5):W671-W677. Patient Consent Statement Written consent was obtained from the patient to publish the details of this case, including images and videos.
https://openalex.org/W2111047324	https://www.scielo.br/j/bjmbr/a/FqynmbR6KrRnngMWsz8Ffkv/?lang=en&format=pdf	English	null	Serum laminin, type IV collagen and hyaluronan as fibrosis markers in non-alcoholic fatty liver disease	Brazilian Journal of Medical and Biological Research	2,005	cc-by	4,633	Serum laminin and hyaluronan in NAFLD Brazilian Journal of Medical and Biological Research (2005) 38: 747-753 ISSN 0100-879X Serum laminin and hyaluronan in NAFLD Brazilian Journal of Medical and Biological Research (2005) 38: 747-753 ISSN 0100-879X Abstract Hepatic fibrosis in patients with non-alcoholic fatty liver disease is associated with progression of the disease. In the present study, we analyzed the discriminative ability of serum laminin, type IV collagen and hyaluronan levels to predict the presence of fibrosis in these patients. In this preliminary report, we studied 30 overweight patients divided into two groups according to the absence (group I, N = 19) or presence (group II, N = 11) of fibrosis in a liver biopsy. Triglycerides, aspartate aminotransferase, alanine aminotransferase, γ-glutamyl- transpeptidade, hyaluronan (noncompetitive fluoroassay), type IV collagen, and laminin (ELISA) were determined. Group II presented significantly higher mean laminin, hyaluronan, type IV collagen, and aspartate aminotransferase values, which were due to the correlation between these parameters and the stage of fibrosis in the biopsy (Spearman’s correlation coefficient, rS = 0.65, 0.62, 0.53, and 0.49, respectively). Analysis of the ROC curve showed that laminin values >282 ng/ml were those with the best diagnostic performance, with 87% accuracy. Association of laminin with type IV collagen showed improvement in the positive predictive value (100%), but with reduc- tion in diagnostic sensitivity (64%). When compared with the criteria of Ratziu et al. [Gastroenterology (2000) 118: 1117-1123] for the diagnosis of septal fibrosis, laminin values presented a better diagnos- tic accuracy (83 vs 70%). Determination of extracellular matrix com- ponents in serum, especially of laminin, may identify patients with non-alcoholic fatty liver disease and fibrosis and these components may be used as indicators for liver biopsy in these patients. Key words • Hepatic fibrosis • Hyaluronan • Laminin • Non-alcoholic fatty liver disease • Non-alcoholic steatohepatitis • Type IV collagen Serum laminin, type IV collagen and hyaluronan as fibrosis markers in non-alcoholic fatty liver disease V.N. dos Santos1, M.M.B. Leite-Mór1, M. Kondo1, J.R. Martins2, H. Nader2, V.P. Lanzoni3 and E.R. Parise1 1Seção de Gastroenterologia, Departamento de Medicina, 2Divisão de Biologia Molecular, Departamento de Bioquímica, 3Departamento de Patologia, Universidade Federal de São Paulo, São Paulo, SP, Brasil 1Seção de Gastroenterologia, Departamento de Medicina, 2Divisão de Biologia Molecular, Departamento de Bioquímica, 3Departamento de Patologia, Universidade Federal de São Paulo, São Paulo, SP, Brasil Key words • Hepatic fibrosis • Hyaluronan • Laminin • Non-alcoholic fatty liver disease • Non-alcoholic steatohepatitis • Type IV collagen Correspondence E.R. Parise Rua Botucatu, 740, 2º andar 03411-900 São Paulo, SP Brasil Fax: +55-11-5549-1711 E-mail: parise@gastro.epm.br Publication supported by FAPESP. Received August 27, 2004 Accepted March 9, 2005 Received August 27, 2004 Accepted March 9, 2005 Introduction tive or fibrotic alterations. The follow-up of these patients shows that the disease is stable (3,4). However, in about 2-3% of the popula- tion steatosis will be accompanied by bal- looning degeneration, Mallory’s hyaline bod- ies and fibrosis, characterizing non-alcoholic steatohepatitis (NASH) which may progress Non-alcoholic fatty liver disease (NAFLD) affects approximately 20% of the general population (1,2). Most of these patients pres- ent steatosis alone or associated only with an inflammatory infiltrate, without degenera- Braz J Med Biol Res 38(5) 2005 Braz J Med Biol Res 38(5) 2005 748 V.N. dos Santos et al. to cirrhosis and liver failure (4-6). to cirrhosis and liver failure (4-6). and/or ultrasound signs of liver failure and/ or portal hypertension were also excluded. Differentiation between these NAFLD forms can be safely performed only by liver biopsy (4,7-9). However, due to the high prevalence of the disease, it would be neces- sary to find noninvasive parameters which would allow the selection of patients at a higher risk of developing cirrhosis to be submitted to a biopsy, despite the slight risk of mortality and morbidity involved. Serum COL-IV and LM concentrations were determined by ELISA with mouse monoclonal antibodies against human LM and COL-IV using commercial Fuji Chemi- cal Ind. (Takaoka, Tokyo, Japan) kits. HA determinations were performed us- ing a noncompetitive fluorescence-based assay with specific binding proteins from bovine cartilage which were later used in solid phase on ELISA plates and with biotin- ylated probes (14,15). The presence of fibrosis in the biopsy is accompanied by progression of the disease to cirrhosis and liver failure in 15 to 26% of cases (3-6), thus constituting a criterion of NAFLD severity. On the other hand, extracellular ma- trix components, especially type III and IV collagens (COL-IV), laminin (LM) and hyalu- ronic acid (HA), have been used as indirect fibrogenesis markers in many chronic liver diseases, such as hemochromatosis, viral hepa- titis and alcoholic liver disease (10-13). Biopsy fragments were stained with he- matoxylin-eosin, Masson’s trichrome, Prus- sian blue (Perls’ test), and reticulum impreg- nation with silver (Gomori). NAFLD grading of liver tissue samples was performed according to Lee (16), Brunt et al. (17), and Dixon et al. (18). Introduction Liver steato- sis was classified according to the percent- age of fatty vacuole-containing hepatocytes: grade 0 (absent), grade 1 (<5% of the hepa- tocytes), grade 2 (5-25% of the hepatocytes), grade 3 (25-75% of the hepatocytes), grade 4 (>75% of the hepatocytes). Lobular inflam- mation, portal inflammation, hepatocyte bal- looning, and Mallory’s hyaline bodies were also evaluated. Fibrosis was classified into 0 = absent; stage 1 = perisinusoidal/pericellu- lar fibrosis in zone 3; stage 2 = perisinusoi- dal/pericellular fibrosis in zone 3, with focal or diffuse periportal fibrosis; stage 3 = peri- sinusoidal/pericellular fibrosis and focal por- tal fibrosis with focal or diffuse bridging fibrosis; stage 4 = cirrhosis. Patients with cirrhosis and portal fibrous expansion with emission of septa of any intensity were clas- sified as patients with septal fibrosis. In the present study, we assessed the discriminative ability of serum LM and COL- IV to predict the presence of fibrosis in NAFLD in patients with compensated dis- ease, without clinical or ultrasound signs of cirrhosis or portal hypertension. Patients and Methods Thirty patients with body mass index (BMI) >25, with an ultrasound diagnosis of liver steatosis and with a persistent increase of at least 1.5 times the upper limit of nor- mality of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and/or γ- glutamyltranspeptidase (γ-GT) levels for a minimum period of three months were stud- ied. All patients were submitted to a percuta- neous liver biopsy. NAFLD was classified on the basis of histological data using the classification of Matteoni et al. (4): type 1 (isolated liver steatosis); type 2 (liver steatosis and lobular inflammation); type 3 (presence of hepato- cyte ballooning); type 4 (presence of Mallory’s hyaline bodies and/or fibrosis). Patients ingesting more than 20 g alcohol per day, with decompensated non-insulin- dependent diabetes mellitus (NIDDM), tak- ing hepatotoxic drugs, with systemic dis- eases or infections, those presenting hepati- tis B or C virus or other concomitant liver disease were excluded. Patients with clinical After evaluation of the histological pa- Braz J Med Biol Res 38(5) 2005 Braz J Med Biol Res 38(5) 2005 749 Serum laminin and hyaluronan in NAFLD rameters, the patients included in the study were divided into 2 groups: group I (patients with type 1, 2 and 3 NAFLD) and group II (patients with type 4 NAFLD, with fibrosis). tween steatosis grade and severity of NAFLD classification was observed. Fibrosis was found in 11 (36.7%) of the 30 patients evalu- ated. The groups, separated according to the presence or absence of fibrosis, did not dif- fer regarding age, gender or the presence of signs and symptoms on the occasion of diag- nosis. They also did not differ regarding γ- GT, ALT levels and AST/ALT ratio. Mean AST levels as well as serum COL-IV, LM and HA levels were significantly higher in group II (Table 1). BMI, age, ALT, and triglyceride concen- trations (BAAT criteria) were considered for the evaluation of parameters indicating fibrosis in overweight patients according to the criteria proposed by Ratziu et al. (19). The Mann-Whitney test and the Spear- man nonparametric correlation coefficient were used for statistical analysis. The dis- criminative power of each variable was as- sessed on the basis of positive predicting value, negative predicting value and accu- racy, with determination of the cut-off of the area under the ROC curve. The level of significance was set at 5% in all analyses (20,21). Results Parameter Group I (N = 19) Group II (N = 11) Gender (male/female) 11/8 7/4 Age (years) 41.6 ± 2.90 50.5 ± 4.10 BMI 31.1 ± 1.00 31 ± 2.40 ALT (IU/l) 60.6 ± 11.5 105.8 ± 34.3 AST (IU/l) 36.4 ± 5.90 47.6 ± 3.10* AST/ALT ratio 0.7 ± 0.1 0.7 ± 0.1 γ-GT (IU/l) 84.9 ± 12.5 118.4 ± 30.4 Type IV collagen (ng/ml) 116.7 ± 4.6 149.3 ± 12* Laminin (ng/ml) 244.7 ± 11.9 381.4 ± 38.9* Hyaluronan (ng/ml) 25.6 ± 7.0 73.5 ± 23.5* Data are reported as means ± SEM. BMI = body mass index; ALT = alanine aminotransferase; AST = aspartate aminotransferase; γ-GT = γ-glutamyltranspepti- dase. P < 0.05 compared to group I (Mann-Whitney test). A total of 186 patients were originally evaluated, 156 of whom were excluded for various reasons, the main cause being the ingestion of more than 20 g/day alcohol. Re- fusal to submit to a biopsy, use of hepatotoxic drugs, and signs of advanced hepatopathy were other frequent causes of exclusion. Of the 30 patients included, 18 were males. Mean age was 44.9 years. All patients presented BMI >25, with a mean of 31. Eight patients (26.7%) presented hepatomegaly with no signs suggestive of hepatocellular failure. Data are reported as means ± SEM. BMI = body mass index; ALT = alanine aminotransferase; AST = aspartate aminotransferase; γ-GT = γ-glutamyltranspepti- dase. dase P < 0.05 compared to group I (Mann-Whitney test). Besides obesity, 7 patients (23.3%) pre- sented NIDDM and dyslipidemia, without identification of any other risk factor for NAFLD development. Braz J Med Biol Res 38(5) 2005 Figure 1. Correlation between serum laminin concentrations and grade of fibrosis in liver bi- opsies. Correlation between these parameters was calcu- lated by the Spearman non- parametric correlation coeffi- cient (rS). Figure 1. Correlation between serum laminin concentrations and grade of fibrosis in liver bi- opsies. Correlation between these parameters was calcu- lated by the Spearman non- parametric correlation coeffi- cient (rS). Figure 1. Correlation between serum laminin concentrations and grade of fibrosis in liver bi- opsies. Correlation between these parameters was calcu- lated by the Spearman non- parametric correlation coeffi- cient (rS). Figure 1. Correlation between serum laminin concentrations and grade of fibrosis in liver bi- opsies. Correlation between these parameters was calcu- lated by the Spearman non- parametric correlation coeffi- cient (rS). Patients and Methods Serum LM levels correlated significantly with fibrosis grade (P = 0.65), Mallory’s bodies (P = 0.51) and lobular inflammation (P = 0.50), the same being observed for HA (P = 0.62), COL-IV (0.53), and AST (0.49) levels. Figure 1 shows the correlation be- Table 1. Biochemical (serum) and demographic parameters of patients with non- alcoholic fatty liver disease with (group I) and without (group II) fibrosis. Table 1. Biochemical (serum) and demographic parameters of patients with non- alcoholic fatty liver disease with (group I) and without (group II) fibrosis. Discussion NAFLD is currently the most prevalent liver disease worldwide. Since it may be associated with different risk factors, we included in this study only overweight indi- viduals who did not present significant varia- tion in body weight during the last three months. These patients presented a high NAFLD prevalence (23-25), often associ- ated with the presence of fibrosis (7). No differences were observed between these groups regarding clinical or demo- graphic characteristics (gender, age, BMI, present signs and symptoms), or ALT and γ- GT levels and the AST/ALT ratio. Several studies have shown that serum ALT and γ- GT and the AST/ALT ratio may represent indicators of advanced liver disease and fi- brosis (3,8,19,26,28). In particular, an AST/ ALT ratio >1 would be observed in patients with a more advanced grade of liver fibrosis, such as cirrhosis and septal fibrosis (4,7). In our patients, the AST/ALT ratio was less than 1 in both groups, certainly reflecting the selection criterion by which patients with signs of liver failure and with manifest portal hypertension were excluded from this study. Table 2. Discriminative serum concentrations of extracellular matrix components and of aspartate aminotransferase alone or associated with laminin values for the identifi- cation of fibrosis in patients with non-alcoholic fatty liver disease. Table 2. Discriminative serum concentrations of extracellular matrix components and of aspartate aminotransferase alone or associated with laminin values for the identifi- cation of fibrosis in patients with non-alcoholic fatty liver disease. Table 2. Discriminative serum concentrations of extracellular matrix components and of aspartate aminotransferase alone or associated with laminin values for the identifi- cation of fibrosis in patients with non-alcoholic fatty liver disease. of aspartate aminotransferase alone or associated with laminin values for the identifi- cation of fibrosis in patients with non-alcoholic fatty liver disease. SEN SP PPV NPV Accuracy Laminin (>282 ng/ml) 82 89 82 89 87 Type IV collagen (>145 ng/ml) 64 89 78 81 80 Hyaluronan (>24.6 ng/ml) 82 68 60 87 73 AST (>44 IU/l) 64 79 70 75 73 Laminin + hyaluronan 73 95 89 86 87 Laminin + type IV collagen 64 100 100 83 87 Laminin + AST 55 95 86 78 80 Data are reported as percent. SEN = sensitivity; SP = specificity; PPV = positive predictive value; NPV = negative predictive value; AST = aspartate aminotrans- ferase. Results Based on the histological data obtained, 6 patients were classified as type 1, 10 as type 2, 3 as type 3, and 11 as type 4 NAFLD according to the criteria proposed by Matteoni et al. (4). Upon histological analysis all patients presented steatosis, with 43.3% of the cases being grades 1 and 2. No correlation be- Braz J Med Biol Res 38(5) 2005 Braz J Med Biol Res 38(5) 2005 750 V.N. dos Santos et al. tween serum LM values and grade of fibrosis in liver biopsy. With the potential progress of NAFLD to cirrhosis, an adequate evaluation of these patients is required in order to define the prognosis of the disease. Retrospective stud- ies have indicated that, if steatosis associ- ated or not with an inflammatory infiltrate is detected in a biopsy, no progression of the disease is observed even during follow-up of the patients for a long period of time (3,4). On the other hand, in patients with associ- ated ballooning degeneration and/or Mallo- ry’s bodies with or without fibrosis, the de- velopment of cirrhosis can be observed in up to 30% of cases (4,26,27). In addition, the presence of liver fibrosis, even in its initial form, seems to be a good marker for the evolution of the disease, as observed by Matteoni et al. (4). Once the discriminative values of each parameter were evaluated, we observed that, based on the ROC curve, the best diagnostic accuracy in the identification of patients with NAFLD and fibrosis was obtained with se- rum LM levels, followed by AST and HA values. Association of serum LM values with HA, COL-IV or AST determinations showed improvement in the positive predictive value (specially for the combination of LM with COL-IV values), but with an important re- duction in diagnostic sensitivity (Table 2). Serum LM values were then compared with BAAT criteria (22) considering the di- agnosis of septal fibrosis (stages 2 to 4). In such situation, LM determination presented a better diagnostic performance than the BAAT score (Table 3). Therefore, based on a liver biopsy, pa- tients with liver fibrosis (group II) were sepa- rated from the other NAFLD patients (group I) in the expectation that this increase in fibrogenic activity could be detected by se- rum determination of extracellular matrix components. Discussion However, AST levels were significantly higher in group II patients, as also reported by others (4,7,29). Such difference seems to be due to the significant correlation observed between this parameter and the grade of Braz J Med Biol Res 38(5) 2005 751 751 Serum laminin and hyaluronan in NAFLD fibrosis in the biopsy. It is believed that AST clearance by endothelial liver cells is de- creased in individuals with advanced fibro- sis, possibly explaining the correlation ob- served (30). a higher fibrogenic activity of the disease (4, 18). Type IV collagen, instead, was the only parameter correlated with hepatocyte bal- looning and steatosis grade, suggesting a relationship between this basement mem- brane protein and lipid metabolism. How- ever, the mechanism of this interaction is still unknown. The extracellular matrix components stud- ied presented significantly higher values in patients with liver fibrosis than in those with- out this histological finding. Among the ex- tracellular matrix components, we selected for this study those with the highest correla- tion with parenchymatous and perisinusoidal fibrosis as it occurs in NAFLD (31-33). LM is the largest protein of the basement mem- brane and in chronic liver diseases it is pro- gressively deposited along Disse’s space ac- companying sinusoid capillarization (34,35). In these patients, its serum values have been related to the degree of portal hypertension (13,36,37) and, together with HA determi- nation, to the diagnosis of fibrosis (31). COL- IV is the main collagen component of the basement membrane, being co-distributed with LM in the perisinusoidal space (32,35). Studies on patients with chronic liver dis- ease have demonstrated a relationship be- tween serum COL-IV values and severity of liver fibrosis (22,32). HA is a non-sulfated glycosaminoglycan synthesized in the liver by stellate cells. Its serum concentration sig- nificantly increases in chronic diseases of the liver of different etiologies, due not only to a greater hepatic production but also to a decrease in its degradation by endothelial liver cells (33,38). In these patients, espe- cially those with disease of viral and alco- holic etiology, increased levels of this gly- cosaminoglycan have been detected and were shown to be related to the degree of fibrosis in liver tissue (38,39). In attempts to obtain a noninvasive diag- nosis of fibrosis, several parameters have been proposed (7,18,19). Since our study only included overweight patients, it was closer to that by Ratziu et al. Discussion (19) who pro- posed an index of fibrosis predictors based on BAAT criteria. Comparing the discrimi- native ability of blood LM concentrations with these BAAT criteria, it can be observed that LM values presented a better diagnostic performance for the diagnosis of septal fi- brosis. It should be emphasized that the val- ues observed here were similar to those de- scribed in the original paper by the cited authors. It is important to note that the patients in the present study did not present any clinical or ultrasound sign of chronic liver disease, exactly because our intention was to assess patients who did not present the stigmata of liver failure or indirect signs of liver cirrho- sis, in need of a biopsy to define the diagno- sis and prognosis of the disease. Thus, in this type of patient, the finding of high LM val- ues would be associated with liver fibrosis in 82% of the cases and would present a false- Table 3. Discriminative values of laminin and of BAAT criteria (body mass index, age, alanine ami- notransferase and triglyceride concentrations) for the diagnosis of septal fibrosis and cirrhosis in over- weight patients with non-alcoholic fatty liver dis- ease. Parameter PPV NPV Accuracy Laminin 64 95 83 BAAT 47 93 70 Data are reported as percent. PPV = positive pre- dictive value; NPV = negative predictive value. We confirmed this association between serum concentration of these extracellular matrix components and the degree of fibro- sis in patients with NAFLD. In addition, we also observed a significant correlation of such parameters with the presence of Mallory’s bodies which are associated with Braz J Med Biol Res 38(5) 2005 Braz J Med Biol Res 38(5) 2005 752 V.N. dos Santos et al. negative result in about 10% of the cases. Therefore, LM determination could be used for the screening of patients to be submitted to a liver biopsy, since it presents a better performance than the criteria currently used to indicate a biopsy for these patients (7,19). since it is an easily obtained biochemical parameter, it is especially attractive for the selection of patients for biopsy. References predictive value of serum laminin for the risk of variceal bleeding related to portal pressure levels. Hepatogastroenterology, 42: 542- 545. 1. Clark JM, Brancati FL & Diehl AM (2002). Nonalcoholic fatty liver disease. Gastroenterology, 122: 1649-1657. 2. Yu AS & Keeffe EB (2002). Nonalcoholic fatty liver disease. Re- views in Gastroenterological Disorders, 2: 11-19. 14. Hardingham TE & Adams P (1976). A method for the determination of hyaluronate in presence of other glycosaminoglycans and its application to human intervertebral disc. Biochemical Journal, 159: 143-147. 3. Teli MR, James OFW, Burt AD, Bennett MK & Day CP (1995). The natural history of nonalcoholic fatty liver: A follow-up study. Hepatol- ogy, 22: 1714-1719. 15. Martins JRM, Passerotti CC, Maciel RMB, Sampaio LO, Dietrich CP & Nader HBN (2003). Practical determination of hyaluronan by a new noncompetitive fluorescence-based assay on serum of normal and cirrhotic patients. Analytical Biochemistry, 319: 65-72. 4. Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC & McCullough AJ (1999). Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. 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Angulo P, Keach JC, Batts KP & Lindor KD (1999). Independent predictors of liver fibrosis in patients with nonalcoholic steatohepati- tis. Hepatology, 30: 1356-1362. 19. Ratziu V, Giral P, Charlotte F, Bruckert E, Thibault V, Theodorou I, Khalil L, Turpin G & Opolon P (2000). Liver fibrosis in overweight patients. Gastroenterology, 118: 1117-1123. 8. Neuschwander-Tetri BA (2002). Evolving pathophysiologic concepts in nonalcoholic steatohepatitis. Liver, 4: 31-36. 20. Zweig MH & Campbell G (1993). Receiver-operating characteristics (ROC) plots: a fundamental evaluation tool in clinical medicine (review). Clinical Chemistry, 39: 561-577. Discussion The results of the present study show the important contribution made by the determi- nation of extracellular matrix components, especially LM, and also of AST levels, to the selection of patients with NAFLD for a liver biopsy, in order to identify patients with the progressive forms of the disease. Studies involving a larger number of patients and repeated determinations of these parameters should be performed for a better evaluation of their prognostic value in NAFLD. On the other hand, the good performance observed for serum AST levels should be emphasized. Differentiation of advanced NAFLD forms according to AST values has also been reported by other authors (4,18), who, however, did not evaluate the discrimi- native ability of this determination. Since the observed cut-off value represents a 1.5-fold increase in the upper limit of normality and References 9. Garcia-Tsao G & Boyer JL (1993). Outpatient liver biopsy: how safe is it? Annals of Internal Medicine, 118: 150-153. 10. Luo R, Yang S, Xie J, Zhao Z, He Y & Yao J (2001). Diagnostic value of five serum markers for liver fibrosis. Zhonghua Ganzangbing Zazhi, 9: 148-150. 21. Glantz AG (1992). Primer of Bio-Statistics. 4th edn. McGraw-Hill, Inc., New York. 11. Gibson PR, Fraser JRE, Brown TJ, Finch CF & Jones PA (1992). Hemodynamic and liver function predictors of serum hyaluronan in alcoholic liver disease. Hepatology, 15: 1054-1059. 22. Takamatsu S, Nakabayashi H, Okamoto Y & Nakano H (1997). Noninvasive determination of liver collagen content in chronic hepa- titis. Multivariate regression modeling with blood chemical param- eters as variables. Journal of Gastroenterology, 32: 355-360. 12. Niemelä O, Risteli J, Blake JE, Risteli L, Compton KV & Orrego H (1990). Markers of fibrogenesis and basement membrane formation in alcoholic liver disease. Relation to severity, presence of hepatitis and alcohol intake. Gastroenterology, 98: 1612-1619. 23. Andersen T, Christoffersen P & Gluud C (1984). The liver in con- secutive patients with morbid obesity: a clinical, morphological, and biochemical study. International Journal of Obesity, 8: 107-115. 13. Kondo M, Miszputen S, Leite-Mor MMB & Parise ER (1995). The 24. Hautekeete ML, Degott C & Benhamou JP (1990). Microvesicular Braz J Med Biol Res 38(5) 2005 Serum laminin and hyaluronan in NAFLD 753 steatosis of the liver. Acta Clinica Belgica, 5: 311-326. 32. Hahn E, Wick G, Pencev D & Timpl R (1980). Distribution of base- ment membrane proteins in normal and fibrotic human liver: col- lagen type IV, laminin, and fibronectin. Gut, 21: 63-71. 25. Willner IR, Waters B, Patil SR, Reuben A, Morelli J & Riely CA (2001). Ninety patients with nonalcoholic steatohepatitis: insulin resistance, familial tendency, and severity of disease. American Journal of Gastroenterology, 96: 2957-2961. 33. Fraser JRE, Laurent TC & Laurent UBG (1997). Hyaluronan: its nature, distribution, functions and turnover. Journal of Internal Medi- cine, 242: 27-33. 26. Powell EE, Cooksley WGE, Hanson R, Searle J, Halliday JW & Powell LW (1990). The natural history of nonalcoholic steatohepati- tis: a follow-up study of forty-two patients for up to 21 years. Hepatol- ogy, 11: 74-80. 34. Orrego H, Medline A, Blendis LM, Rankin JG & Kreaden DA (1979). Collagenization of the Disse space in alcoholic liver disease. Gut, 20: 673-679. 27. Braz J Med Biol Res 38(5) 2005 References Pinto HC, Baptista A, Camilo ME, Valente A, Saragoça A & Moura MC (1996). Nonalcoholic steatohepatitis. Clinicopathological com- parison with alcoholic hepatitis in ambulatory and hospitalized pa- tients. Digestive Diseases and Sciences, 41: 172-179. 35. Martinez-Hernandez A, Delgado FM & Amenta PS (1991). The extracellular matrix in hepatic regeneration. Localization of collagen types I, III, IV, laminin and fibronectin. Laboratory Investigation, 64: 157-166. 36. Parise ER & Rosa H (1992). Serum laminin in hepatic schistosomia- sis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 86: 179-181. 28. Marchesini G, Brizi M, Bianchi G, Tomassetti S, Bugianesi E, Lenzi M, McCulloug AJ, Natale S, Forlani G & Melchionda N (2001). Nonalcoholic fatty liver disease. 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https://openalex.org/W4200234848	https://www.degruyter.com/document/doi/10.1515/9783110716559-202/pdf	English	null	Acknowledgements	De Gruyter eBooks	2,021	cc-by	411	Open Access. © 2021 Elodie Paillard and Silvia Milanezi, published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. https://doi.org/10.1515/9783110716559-202 Acknowledgements The major part of the work required for the publication of this book took place during the 2020–2021 Covid pandemic. Without the strength, patience, generos- ity, and immense effort of all the contributors to this volume, its co-editors would have been left empty-handed. Our heartfelt thanks therefore goes out first to all our colleagues and friends who have embarked on this unexpectedly difficult adventure with us and who have courageously surmounted all the obs- tacles raised by the virus in order to make their contributions to this book. The outstanding help of Vanessa Monteventi, who did the major part of the editorial work required to prepare the manuscript of this collective volume, must be underscored. We are deeply grateful to her and thank her for her pa- tience, precision, reliability, and amazingly-constant good mood even when faced with the most frustrating tasks sometimes required in such work. The team at De Gruyter, in particular Katharina Legutke and Torben Behm, have been very helpful and patient throughout the publication process: we should also like to extend our warmest thanks to them all, and to add that we have greatly appreciated the fact that communication with the press has been so consistent and so easy. For this co-edited volume, where less than half of the contributors (and none of the co-editors) are native English speakers, the help of Carson Bay, who relentlessly tracked all our infelicities, strange turns of phrases, plain mistakes, and so on, has been fundamental. We thank him for his work and apologize to him (and all our readers) for the remaining errors (no doubt added after his final revision!). The two anonymous reviewers helped us identify residual weaknesses in the volume, and their comments and suggestions allowed many of the contribu- tors to strengthen their chapters and take their reflections further. They both deserve appropriate thanks, all the more because they have accepted the un- grateful task of anonymously reviewing an entire book manuscript in the mid- dle of what has been for no academic an easy and quiet year. Finally, and fundamentally, we should like to thank the Swiss National Sci- ence Foundation for providing the financial means to publish this volume and to ensure that it could be shared with as many as possible thanks to an open access format.
https://openalex.org/W1986659133	https://www.zora.uzh.ch/id/eprint/105763/1/Mueller_Dissemination%20Bias_PLoS%20ONE_2014_Neo_USZ.pdf	English	null	Dissemination Bias in Systematic Reviews of Animal Research: A Systematic Review	PloS one	2,014	cc-by	7,681	Zurich Open Repository and Archive Zurich Open Repository and Archive University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Katharina F. Mueller1, Matthias Briel2,3, Daniel Strech4, Joerg J. Meerpohl5, Britta Lang5, Edith Motschall6, Viktoria Gloy2, Francois Lamontagne7, Dirk Bassler8* 1. Center for Pediatric Clinical Studies, University Children’s Hospital Tuebingen, Tuebingen, Germany, 2. Basel Institute for Clinical Epidemiology and Biostatistics, University Hospital Basel, Basel, Switzerland, 3. Department of Clinical Epidemiology and Biostatistics, McMaster University, Hamilton, Canada, 4. CELLS – Center for Ethics and Law in Life Sciences, University of Hannover, Hannover, Germany, 5. German Cochrane Centre, Medical Center - University of Freiburg, Freiburg, Germany, 6. Center for Medical Biometry and Medical Informatics, Medical Center – University of Freiburg, Freiburg, Germany, 7. Clinical Research Centre Clinique du Centre Hospitalier Universitaire de Sherbrooke, Universite´ de Sherbrooke, Sherbrooke, Canada, 8. Department of Neonatology, University Hospital Zuerich, Zuerich, Switzerland dirk.bassler@usz.ch dirk.bassler@usz.ch Abstract Background: Systematic reviews of preclinical studies, in vivo animal experiments in particular, can influence clinical research and thus even clinical care. Dissemination bias, selective dissemination of positive or significant results, is one of the major threats to validity in systematic reviews also in the realm of animal studies. We conducted a systematic review to determine the number of published systematic reviews of animal studies until present, to investigate their methodological features especially with respect to assessment of dissemination bias, and to investigate the citation of preclinical systematic reviews on clinical research. RESEARCH ARTICLE Dissemination Bias in Systematic Reviews of Animal Research: A Systematic Review Katharina F. Mueller1, Matthias Briel2,3, Daniel Strech4, Joerg J. Meerpohl5, Britta Lang5, Edith Motschall6, Viktoria Gloy2, Francois Lamontagne7, Dirk Bassler8* Citation: Mueller KF, Briel M, Strech D, Meerpohl JJ, Lang B, et al. (2014) Dissemination Bias in Systematic Reviews of Animal Research: A Systematic Review. PLoS ONE 9(12): e116016. doi:10.1371/journal.pone.0116016 Citation: Mueller KF, Briel M, Strech D, Meerpohl JJ, Lang B, et al. (2014) Dissemination Bias in Systematic Reviews of Animal Research: A Systematic Review. PLoS ONE 9(12): e116016. doi:10.1371/journal.pone.0116016 Citation: Mueller KF, Briel M, Strech D, Meerpohl JJ, Lang B, et al. (2014) Dissemination Bias in Systematic Reviews of Animal Research: A Systematic Review. PLoS ONE 9(12): e116016. doi:10.1371/journal.pone.0116016 Editor: Lamberto Manzoli, University of Chieti, Italy semination bias in systematic reviews of animal research: A systematic review Mueller, Katharina F ; Briel, Matthias ; Strech, Daniel ; Meerpohl, Joerg J ; Lang, Britta ; Motschall, Edith ; Gloy, Viktoria ; Lamontagne, Francois ; Bassler, Dirk DOI: https://doi.org/10.1371/journal.pone.0116016 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-105763 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-105763 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 Int Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-105763 Journal Article Published Version ollowing work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at: Mueller, Katharina F; Briel, Matthias; Strech, Daniel; Meerpohl, Joerg J; Lang, Britta; Motschall, Edith; Gloy, Vik- toria; Lamontagne, Francois; Bassler, Dirk (2014). Dissemination bias in systematic reviews of animal research: A systematic review. PLoS ONE, 9(12):e116016. DOI: https://doi.org/10.1371/journal.pone.0116016 OPEN-Dissemination Bias in Animal Research Discussion: Preclinical systematic reviews may have an influence on clinical research but their methodological quality frequently remains low. Therefore, systematic reviews of animal research should be critically appraised before translating them to a clinical context. PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 Editor: Lamberto Manzoli, University of Chieti, Italy Received: August 6, 2014 Accepted: November 30, 2014 Published: December 26, 2014 Methods: Eligible studies for this systematic review constitute systematic reviews that summarize in vivo animal experiments whose results could be interpreted as applicable to clinical care. We systematically searched Ovid Medline, Embase, ToxNet, and ScienceDirect from 1st January 2009 to 9th January 2013 for eligible systematic reviews without language restrictions. Furthermore we included articles from two previous systematic reviews by Peters et al. and Korevaar et al. Results: The literature search and screening process resulted in 512 included full text articles. We found an increasing number of published preclinical systematic reviews over time. The methodological quality of preclinical systematic reviews was low. The majority of preclinical systematic reviews did not assess methodological quality of the included studies (71%), nor did they assess heterogeneity (81%) or dissemination bias (87%). Statistics quantifying the importance of clinical research citing systematic reviews of animal studies showed that clinical studies referred to the preclinical research mainly to justify their study or a future study (76%). Copyright: 2014 Mueller et al. This is an open- access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and repro- duction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding: The OPEN Project (www.open-project. eu) is funded by the European Union Seventh Framework Programme (FP7 – HEALTH.2011.4.1- 2) under grant agreement n˚ 285453. 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. 1 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 OPEN-Dissemination Bias in Animal Research selective publication and dissemination of results [12, 13]. In this situation, published studies are no longer a random sample of all studies that have been conducted, but constitute a biased sample leading to spurious conclusions. A recently published survey conducted in animal laboratories in the Netherlands reported that researchers (n5454) thought that just about 50% of animal experiments are published and employees (n521) of for-profit organizations estimated that only 10% are published [14]. Lack of statistical significance was discussed as one of several important reasons for non-publication. Since the number of systematic reviews of preclinical research is growing, also the problem of dissemination bias in systematic reviews of preclinical research is getting more important [15–17]. But still dissemination bias is rarely considered in preclinical reviews. Peters et al. showed that only 37% (17/46) of meta-analyses considered dissemination bias [5], likewise, Mignini et al. reported that it has been considered only in 16% (5/30) [4]. Korevaar et al. reported that between 2005 and 2010 the proportion of meta-analyses of in vivo animal studies that assessed dissemination bias increased to 60% (21/35) [6]. Korevaar et al. completed their search for systematic reviews of animal experiments in 2009/10 [6]. Since systematic reviews of preclinical research are only now becoming more prevalent and new guidelines and support, such as CAMARADES are only recently becoming available, an update of the previous research to assess the development of systematic reviews of preclinical studies of the last years is crucial. Especially if one considers that preclinical systematic reviews may also influence clinical care it is indispensable to assess their methodological rigor not only to prevent unnecessary studies on animals but also on humans and eventually even unnecessary or in the worst case dangerous treatment of patients. Until today, the influence of preclinical systematic reviews on studies with human participants has not been evaluated. In this systematic review we will do a first step by analyzing the citation profiles of preclinical systematic reviews as a measure of the influence on clinical research. This systematic review is part of the OPEN Project (To Overcome failure to Publish nEgative fiNdings), which was designed with the goal of elucidating the scope of dissemination bias and non-publication of studies through a series of systematic reviews and policy evaluations (www.open-project.eu). PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 Introduction Preclinical research has its main purpose in enhancing our understanding of physiologic and pathologic processes. However, preclinical studies, in vivo animal experiments in particular, also influence clinical research and might thus even influence clinical care by i) informing the design of clinical studies, ii) informing clinical guidelines that consider preclinical evidence when clinical evidence is lacking, or iii) directly guiding clinical practice. But the benefit of animal research on humans has been questioned [1, 2]. Systematic reviews offer a systematic and transparent way to comprehensively identify, evaluate, and critically appraise available evidence on a specific topic. Meta-analyses increase precision and generalizability of effect estimates by quantitatively summarizing the results of individual studies included in a systematic review in order to provide a single best estimate with maximal statistical power [3]. Systematic reviews and meta-analyses of preclinical studies are still relatively rare in the medical literature: Mignini et al. identified 30 systematic reviews of laboratory animal experiments in 2006 and Peters et al. found 86 using a more sensitive search strategy and a broader definition of laboratory animal experiments [4, 5]. But preclinical systematic reviews are getting more prevelant over the last years, as shown by Korevaar et al. in 2011 [6]. y y Methodological quality of primary animal studies is often not satisfying [7]. The Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines for reporting animal research have been compiled to help improve the reporting of in vivo animal experiments [8, 9]. Apart from the poor methodological quality of primary studies, also the often low methodological quality of systematic reviews and meta-analyses of preclinical research can be problematic. While principles of critically appraising in systematic reviews of clinical research are well established [10], their application to systematic reviews of preclinical studies appears variable. Since 2004 the Collaborative Approach to Meta-Analysis and Review of Animal Data in Experimental Studies (CAMARADES) provides support for groups conducting systematic reviews and meta-analyses of data from experimental animal studies [11]. Some of their focuses include identifying potential sources of bias in animal work, developing recommendations for improvements in the design and reporting of animal studies, and developing better methodologies for meta-analysis of animal studies. One of the major threats to systematic reviews is dissemination bias. Dissemination bias, often also referred to as publication bias, describes the PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 2 / 15 OPEN-Dissemination Bias in Animal Research Inclusion criteria We included systematic reviews and meta-analyses with a potential for being interpreted as applicable to humans. The potential for being interpreted as applicable to humans was defined by the use of in vivo models and a focus on one of the following: i) the efficacy of a medical or surgical intervention, ii) the side-effects or toxicity of a medical intervention, iii) the mechanisms of action of a medical intervention, iv) risk factors for a human illness, v) the effects of an exposure to a chemical substance, vi) overview of animal models for disease, vii) the accuracy of diagnostic tests [6]. We defined systematic reviews as publications that described the source(s) searched for evidence as well as one of the following: i) the search terms used, ii) any limitation placed on the search, iii) explicit inclusion and exclusion criteria [5]. An article was included if it fulfilled one of these criteria of a broad definition of systematic review. Additional to this definition, as it has been used by Peters et al. [5] and Korevaar et al. [6], we used a second more stringent definition of systematic reviews. For the more stringent definition systematic reviews had to incorporate: An article was included if it fulfilled one of these criteria of a broad definition of systematic review. Additional to this definition, as it has been used by Peters et al. [5] and Korevaar et al. [6], we used a second more stringent definition of systematic reviews. For the more stringent definition systematic reviews had to incorporate: N a systematic search (statement on the search strategy, including more than one database, and ‘‘search terms’’ mentioned), N explicit inclusion and exclusion criteria (statement of inclusion and exclusion criteria in the methods section), N a focused research question (according to PICO (a technique used in evidence based practice to frame and answer a clinical question, or to develop literature search strategies, the acronym stands for Patient/Population Intervention Comparison/Control Outcome)) [19], N a systematic evaluation of the risk of bias in included studies [5, 6, 10]. We define meta-analyses as publications incorporating a quantitative synthesis of results from animal experiments. We define meta-analyses as publications incorporating a quantitative synthesis of results from animal experiments. Eligibility criteria We used the same criteria as Peters et al. and Korevaar et al. [5, 6] and combined the results of our literature search (2009–2013) to the list of systematic reviews included in these previous works. Methods A detailed protocol of our methods has been published [18]. In brief, the following methods were used for the systematic literature search. PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 Objectives The specific goals of the present systematic review of animal studies are: N To determine the number of published systematic reviews of animal studies until present. N To investigate methodological features of systematic reviews of animal studies especially with respect to assessment of dissemination bias. N To investigate the influence of systematic reviews of animal studies on clinical research by examining citations of systematic reviews by clinical studies. 3 / 15 OPEN-Dissemination Bias in Animal Research behavior and not to inform human health-care. We did not exclude publications that incorporated results of clinical studies [6]. behavior and not to inform human health-care. We did not exclude publications that incorporated results of clinical studies [6]. Search strategy We updated the search of Peters et al. and Korevaar et al. and therefore systematically searched electronic databases, Ovid Medline, Embase, Toxnet (http://toxnet.nlm.nih.gov/; including Toxline, DART, and HSDB) and ScienceDirect, all from 1st January 2009 to 9th January 2013 (the full search strategy is displayed in the study protocol [18]) [5, 6]. In addition, the bibliographies of any eligible articles identified were checked for additional references. No language restrictions were applied. We did not search any grey literature (eg literature that has not been formally published as journal articles). Study selection Two reviewers, independently and in duplicate, screened titles and abstracts of search results. If a title and abstract could not be rejected with certainty by both reviewers, the full text of the paper was retrieved and assessed for eligibility. Any disagreement among reviewers was resolved by discussion and consensus or, if needed, third party arbitration. Data extraction Working in teams of two, we independently extracted the following information from each eligible article (from this literature search and from the included articles by Peters et al. and Korevaar et al.): search strategy (database, language restriction, search of grey literature), clearly defined inclusion and exclusion criteria, list of included an excluded articles, formal assessment of methodological quality of included studies (by score (eg Jadad), by dimension (eg allocation concealment, blinding etc.), funding sources from included studies, report of a meta-analysis (report of effect estimates of individual studies, method for data synthesis), assessment of heterogeneity (Cochrane Q, I2, Tau2, other), assessment of dissemination bias (Funnel plot, Begg’s or Egger’s test, Fail-Safe Number, Trim and fill method, other). Any disagreement was resolved by discussion and consensus or, if needed, third party arbitration. Exclusion criteria We excluded genome-wide association studies and animal experiments with the main purpose to learn more about fundamental biology, physical functioning or 4 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 Investigation of the citation of systematic reviews of animal studies on clinical research We used the Web of Science Internet-based citation database to identify clinical publications citing included systematic reviews and meta-analyses. We conducted this analysis in two randomly selected samples of included studies published between 2005 and 2009 to allow a minimum of 4 years to elapse between publication of the review and our analysis: we included 25 systematic reviews with a meta-analysis out of 29 and a random sample of 25 systematic reviews without a meta-analysis out of 57. We searched Web of Science on 11 August 2013 for clinical human studies or guidelines citing the selected animal reviews. All included studies were reviewed independently and in duplicate. The reviewers determined how the review of preclinical studies has been cited by the clinical study by allocating each citation to one of the following categories: i) used citation unrelated to animal studies in review, ii) used citation to provide at least partial justification for the study or a future study, iii) used citation to support or explain their findings, iv) used citation to discuss physiological pathways, and v) used citation to justify the measurement etc. their findings, iv) used citation to discuss physiological pathways, and v) used citation to justify the measurement etc. PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 Appraisal of methodological quality of included reviews pp g q y We assessed the methodological quality of the included systematic reviews by focusing on various methodological features, such as clearly defined inclusion criteria, assessment of heterogeneity, assessment of dissemination bias, report according to guidelines. We did not use a scoring approach to assess the methodological quality. 5 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 OPEN-Dissemination Bias in Animal Research Data analysis and reporting Data synthesis involved a descriptive summary of included studies. Citation of Systematic reviews and meta-analyses of animal studies on clinical research Our search on Web of Science retrieved 337 articles, which cited the included 50 systematic reviews. Of these we excluded a total of 56 articles, because they could either not be classified as clinical studies and involved only animals (16/56), or were no original studies, but reviews (37/56), or letters (3/56). Thus, we included 281 articles reporting on 281 studies. Most of the included studies were randomized controlled trials or prospective cohort studies. The clinical studies referred to the preclinical research mainly to justify the current study or a future study (76%) (Table 4). Systematic reviews, which also included a meta-analysis have been cited more often (3 (0–73) (Mean (Min-Max))), than systematic reviews without meta-analysis by clinical studies (1 (0–32) (Mean (Min-Max))). Results of included Meta-Analyses In 31% of all the included studies a meta-analysis is reported. Most of the reported meta-analyses evaluated a medical intervention (73%) and were preceded by a systematic review (83%). Only 54% of the meta-analyses reported also effect estimates of individual studies. Mostly (48%) a random effects model was chosen for data synthesis (Table 3). OPEN-Dissemination Bias in Animal Research Methodological features of included systematic reviews and assessment of dissemination bias Methodological features of included systematic reviews and assessment of dissemination bias Only 59% of all the included systematic reviews clearly defined inclusion and exclusion criteria, and only just over half (51%) of the included studies displayed a list or flow diagram of the included studies, as suggested by the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) reporting guideline [20]. 24% of all the included studies did not report how many studies they included in their systematic review or meta-analysis. The majority of the included systematic reviews and meta-analyses of animal studies did not assess methodological quality of included studies (71%), nor did they asses heterogeneity (81%), or dissemination bias (87%). For more details, see Table 2. PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 Study selection and characteristics Study selection and characteristics The literature search identified 3019 records. After screening titles and abstracts, we retrieved 375 full text articles and ultimately included 246 publications. Furthermore we augmented the list of included publications with the previous work by Peters et al. [5] (103 studies) and Korevaar et al. [6] (163 studies) (see Fig. 1). This shows an increasing number of published systematic reviews and meta-analyses on animal studies, a trend that had already been found in the two previous systematic reviews by Peters et al. [5] and Korevaar et al. [6]. 512 of the included articles fulfilled the broad definition of a systematic review, but only 126 matched the more stringent definition of a systematic review. Most articles were excluded, because they did not qualify as systematic review or meta- analysis (50%). The objectives of the included systematic reviews and meta- analyses were mainly to investigate the efficacy of a medical or surgical intervention (41%). Additional characteristics of the included 512 systematic reviews (combined results from our literature search and the results of Peters et al. and Korevaar et al.) are summarized in Table 1. 6 / 15 OPEN-Dissemination Bias in Animal Research Fig. 1. Flow chart for selection of systematic reviews included in the systematic review. doi:10.1371/journal.pone.0116016.g001 one.0116016 December 26, 2014 Fig. 1. Flow chart for selection of systematic reviews included in the systematic review. Fig. 1. Flow chart for selection of systematic reviews included in the systematic review PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 7 / 15 Comparison of included studies by Peters et al. and Korevaar et al. and studies included from this literature search Updating the previous work of Peters et al. [5] and Korevaar et al. [6] we found a growing number of systematic reviews of animal studies. We compared the group of included systematic reviews by Peters et al. and Korevaar et al., which have been published between 1963 and 2010 to the systematic reviews included from our literature search published between 2009 and 2013 (Table 2 and Table 3). Looking at the methodological quality of the systematic reviews in the two groups the assessment of methodological quality and of heterogeneity remained similar, but dissemination bias is mentioned and assessed less often in the group by Peters et PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 8 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 0116016 December 26, 2014 9 / 15 OPEN-Dissemination Bias in Animal Research Table 1. Cont. l d K t l th i th f t ti i i l d d f thi Table 1. Cont. Results from Peters et al. and Korevaar et al. Results from this literature search All results Number absolute Number in percentage* Number absolute Number in percentage Number absolute Number in percentage Funding Funding source extracted from included studies and reported 8/266 3% 6/246 2% 14/512 3% Funding of the systematic review Not reported 168/266 63% 147/246 60% 315/512 62% Governmental/public 56/266 21% 42/246 17% 98/512 19% Industry/private for profit 11/266 4% 15/246 6% 26/512 5% Charity/private not for profit 5/266 2% 13/246 5% 18/512 4% Not funded/only in house source 26/266 10% 29/246 12% 55/512 11% all percentages rounded to integral numbers. multiple selection possible. doi:10.1371/journal.pone.0116016.t001 al. and Korevaar et al. than in the group of systematic reviews included from this literature search published between 2009 and 2013. al. and Korevaar et al. than in the group of systematic reviews included from this literature search published between 2009 and 2013. al. and Korevaar et al. than in the group of systematic reviews included from this literature search published between 2009 and 2013. OPEN-Dissemination Bias in Animal Research Table 1. Characteristics of included systematic reviews. Results from Peters et al. and Korevaar et al. Results from this literature search All results Number absolute Number in percentage Number absolute Number in percentage Number absolute Number in percentage Inclusion criteria Broad definition of systematic review applied 266/266 100% 246/246 100% 512/512 100% Narrow definition of systematic review applied 59/266 22% 67/246 27% 126/512 25% Objectives i) investigation of the efficacy of a medical or surgical intervention 103/266 39% 108/246 44% 211/512 41% ii) investigation of the side-effects or toxicity of a medical intervention 22/266 8% 20/246 8% 42/512 8% iii) investigation of the mechanisms of action of a medical intervention 56/266 21% 46/246 19% 102/512 20% iv) investigation of risk factors (epi- demiological associations or mechanisms of action of disease) 8/266 3% 8/246 3% 16/512 3% v) investigation of effects of an exposure to a chemical substance 20/266 8% 8/246 3% 28/512 6% vi) overview of animal models for disease 50/266 19% 51/246 21% 101/512 20% vii) investigation of diagnostic test accuracy 7/266 3% 5/246 2% 12/512 2% Type of Article (as presented in title or abstract) Article presents itself as systematic review 194/266 73% 179/246 73% 373/512 73% Article presents itself as meta-ana- lysis 44/266 17% 20/246 8% 64/512 13% Article presents itself as both 26/266 10% 31/246 13% 57/512 11% Article presents itself neither as systematic review nor as meta- analysis 2/266 1% 16/246 7% 18/512 4% Literature search within review Databases searched** Embase 76/266 29% 96/246 39% 172/512 34% Toxnet 9/266 3% 6/246 2% 15/512 3% Web of Science 8/266 3% 44/246 18% 52/512 10% Medline 237/266 89% 239/246 97% 476/512 93% Other 117/266 44% 137/246 56% 254/512 50% Language restrictions applied Yes 92/266 35% 110/246 45% 202/512 39% No 35/266 13% 43/246 18% 78/512 15% Not reported 139/266 52% 93/246 38% 232/512 45% Any attempt to search grey literature Yes 76/266 29% 63/246 26% 139/512 27% No 24/266 9% 60/246 24% 84/512 16% Not reported 166/266 62% 123/246 50% 289/512 56% Table 1. Characteristics of included systematic reviews. PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 9 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 doi:10.1371/journal.pone.0116016.t001 Discussion Korevaar et al. [6] found 163 systematic reviews of animal studies published between 2005 and 2010. We identified 246 systematic reviews between 2009 and January 2013. So the number of systematic reviews of animal studies roughly doubled in the last five years, similar to the trend already asserted by Korevaar et al. [6]. With the growing number of preclinical systematic reviews also the problem of low methodological quality and dissemination bias in systematic reviews of preclinical research is getting more attention [21, 22] and new efforts have been made to improve methodological quality, such as the CAMARADES initiative, or a newly developed RoB tool for animal intervention studies (SYRCLE’s RoB tool) [11, 23]. Still, the methodological quality of preclinical systematic reviews and meta-analyses, especially the assessment of dissemination bias remains poor. But it seems that methodological quality improved, as PRISMA or Quality of Reporting of Meta-analyses (QUOROM) (14% vs. 3% (results from this literature search vs. results from included studies by Peters et al. and Korevaar et al.)) are more often mentioned and also seem to be followed more consequently as clear inclusion and exclusion criteria (65% vs. 56%), and the number of included studies (82% vs. 70%) is reported more often, and a list or flow diagram of the included studies (62% vs. 42%) is shown more often. The assessment of the methodological quality of included studies as well as the consideration and assessment of heterogeneity remained more or less unchanged. Over the last five years, the problem of dissemination bias has been recognized more widely, this we also showed in our data set, as dissemination bias has been 10 / 15 doi:10.1371/journal.pone.0116016.t002 all percentages rounded to integral numbers. all percentages rounded to integral numbers. *multiple selection possible. OPEN-Dissemination Bias in Animal Research Table 2. Methodological features of included systematic reviews. Results from Peters et al. and Korevaar et al. Results from this literature search All results Number absolute Number in percentage Number absolute Number in percentage Number absolute Number in percentage Inclusion Clearly defined eligibility cri- teria 145/266 56% 159/246 65% 304/512 59% Number of included studies Not reported 79/266 30% 45/246 18% 124/512 24% ,10 23/266 9% 28/246 11% 51/512 10% 10–50 97/266 37% 103/246 42% 200/512 39% 51–100 36/266 14% 40/246 16% 76/512 15% .100 31/266 12% 30/246 12% 61/512 12% List/flow diagram of screened and included studies 111/266 42% 152/246 62% 263/512 51% Assessment of methodological quality Not assessed 192/266 72% 171/246 70% 363/512 71% Assessed by dimension 14/74 19% 22/75 29% 36/149 24% Assessed by score 18/74 24% 20/75 27% 38/149 25% Assessed differently 42/74 57% 33/75 44% 75/149 50% Assessment of heterogeneity Not assessed 219/266 82% 197/246 80% 416/512 81% Assessed by Cochrane Q 4/47 9% 19/49 39% 24/96 25% I2 8/47 17% 30/49 8% 38/96 40% Tau2 2/47 4% 4/49 8% 6/96 6% Other 29/47 62% 19/49 39% 48/96 50% Assessed, but not described how 8/47 17% 3/49 6% 11/96 11% Dissemination Bias Dissemination Bias men- tioned 53/266 20% 60/246 24% 113/512 22% Dissemination Bias assessed with 29/266 11% 35/246 14% 64/512 13% Funnel plot only 5/29 17% 12/35 34% 17/64 27% Funnel plot and statistical test 4/29 14% 9/35 26% 13/64 20% Begg’s or Egger’s test 13/29 45% 12/35 34% 25/64 39% Trim and fill 2/29 7% 6/35 17% 8/64 13% Fail Safe Number 3/29 10% 2/35 6% 5/64 8% Other 2/29 7% 1/35 3% 3/64 5% Evidence for dissemination bias 18/29 62% 14/35 40% 32/64 50% Reporting Guidelines (PRISMA, QUOROM) men- tioned 7/266 3% 34/246 14% 41/512 8% Table 2. Methodological features of included systematic reviews. PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 all percentages rounded to integral numbers. 10–50 97/266 37% 103/246 42% 200/512 39% 51–100 36/266 14% 40/246 16% 76/512 15% .100 31/266 12% 30/246 12% 61/512 12% List/flow diagram of screened and included studies 111/266 42% 152/246 62% 263/512 51% Assessment of methodological quality Not assessed 192/266 72% 171/246 70% 363/512 71% Assessed by dimension 14/74 19% 22/75 29% 36/149 24% Assessed by score 18/74 24% 20/75 27% 38/149 25% Assessed differently 42/74 57% 33/75 44% 75/149 50% Assessment of heterogeneity Not assessed 219/266 82% 197/246 80% 416/512 81% Assessed by * Cochrane Q 4/47 9% 19/49 39% 24/96 25% I2 8/47 17% 30/49 8% 38/96 40% Tau2 2/47 4% 4/49 8% 6/96 6% Other 29/47 62% 19/49 39% 48/96 50% Assessed, but not described how 8/47 17% 3/49 6% 11/96 11% Dissemination Bias Dissemination Bias men- tioned 53/266 20% 60/246 24% 113/512 22% Dissemination Bias assessed with 29/266 11% 35/246 14% 64/512 13% Funnel plot only 5/29 17% 12/35 34% 17/64 27% Funnel plot and statistical test 4/29 14% 9/35 26% 13/64 20% Begg’s or Egger’s test 13/29 45% 12/35 34% 25/64 39% Trim and fill 2/29 7% 6/35 17% 8/64 13% and included studies Assessment of methodological quality Not assessed 192/266 72% 171/246 70% 363/512 71% Assessed by dimension 14/74 19% 22/75 29% 36/149 24% Assessed by score 18/74 24% 20/75 27% 38/149 25% Assessed differently 42/74 57% 33/75 44% 75/149 50% Assessment of heterogeneity Not assessed 219/266 82% 197/246 80% 416/512 81% Assessed by Cochrane Q 4/47 9% 19/49 39% 24/96 25% I2 8/47 17% 30/49 8% 38/96 40% Tau2 2/47 4% 4/49 8% 6/96 6% Other 29/47 62% 19/49 39% 48/96 50% Assessed, but not described how 8/47 17% 3/49 6% 11/96 11% Dissemination Bias Dissemination Bias men- tioned 53/266 20% 60/246 24% 113/512 22% Dissemination Bias assessed with ** 29/266 11% 35/246 14% 64/512 13% Funnel plot only 5/29 17% 12/35 34% 17/64 27% Funnel plot and statistical test 4/29 14% 9/35 26% 13/64 20% Begg’s or Egger’s test 13/29 45% 12/35 34% 25/64 39% Trim and fill 2/29 7% 6/35 17% 8/64 13% Fail Safe Number 3/29 10% 2/35 6% 5/64 8% Other 2/29 7% 1/35 3% 3/64 5% Evidence for dissemination bias 18/29 62% 14/35 40% 32/64 50% Reporting Guidelines (PRISMA, QUOROM) men- tioned 7/266 3% 34/246 14% 41/512 8% Assessment of methodological quality PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 11 / 15 doi:10.1371/journal.pone.0116016.t003 OPEN-Dissemination Bias in Animal Research considered (24% vs. 20%) and assessed (14% vs. 11%) more often [14–16]. A shift to more valid methods [24] such as funnel plot and statistical test (26% vs. 14%) can be noticed. Funnel plot is one of the simplest and most common used methods to detect dissemination bias in systematic reviews. It is a graphical method and its visual interpretation is subjective, and often there may be other reasons for funnel plot asymmetry than dissemination bias. Therefore, it is recommended to also run statistical test for funnel plot asymmetry to assess dissemination bias [10]. Preclinical research might even influence clinical research by informing the design of clinical studies. In this systematic review we showed that systematic reviews of animal studies are cited especially by clinical randomized controlled trials, which are considered the gold standard of clinical trials. Mostly, the citations are used to justify the conduct of the clinical trial (76%), but also to support or explain the findings (37%). Our study has strengths and limitations. The strengths are that we used a comprehensive approach to identify systematic reviews of in vivo animal studies through a sensitive search strategy and inclusion of previously identified articles. We updated information on preclinical summaries. We also incorporated citation Table 3. Results of included meta-analyses. Table 3. Results of included meta-analyses. Results from Peters et al. and Korevaar et al. 0116016 December 26, 2014 12 / 15 all percentages rounded to integral numbers. doi:10.1371/journal.pone.0116016.t004 OPEN-Dissemination Bias in Animal Research PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 13 / 15 Table 4. Influence of systematic reviews of preclinical research on clinical research. Number absolute Number in percentage Type of clinical study Randomized controlled trials 87/281 31% Non-randomized controlled trials 25/281 9% Cross-over trials 2/281 1% Uncontrolled prospective trials 6/281 2% Retrospective cohort studies 42/281 15% Prospective cohort studies 87/281 31% Laboratory experiments with healthy human volunteers 6/281 2% Cross-sectional surveys 1/281 0% Case report/case series 26/281 9% Guidelines 2/281 1% Other 0/281 0% Use of citation Use of citation unrelated to animal studies 8/281 3% Use of citation to provide at least partial justification for the study or a future study 213/281 76% Use of citation to support or explain their findings 103/281 37% Other 12/281 4% all percentages rounded to integral numbers. multiple selection possible. doi:10 1371/journal pone 0116016 t004 Table 4. Influence of systematic reviews of preclinical research on clinical research. doi:10.1371/journal.pone.0116016.t004 profiles to show the influence of animal research on clinical research. A limitation of our study is that our results might be affected by dissemination bias because we did not search any grey literature. Thus, this systematic review might miss a number of systematic reviews of preclinical research. We are therefore limiting the generalizability of our results about methodological quality and dissemination bias to the published systematic reviews of in vivo animal studies. Regarding the quality assessment one can assume that non-published systematic reviews might be of even lower quality and our results might be too positive. Regarding the influence on clinical studies non-published systematic reviews of animal studies might have less impact on clinical research, since they are not easily accessible. Furthermore, we arbitrarily selected only 50 systematic reviews and meta-analyses to assess their influence on clinical research. Thus, the citation rate might just show a trend of the influence of preclinical systematic reviews on clinical, above all since we did not measure the influence directly. This trend could be proven in further studies by assessing whether the objectives or the study design of the clinical studies is similar to the one of the preclinical study cited. Research synthesis depends on high methodological quality of primary research. ARRIVE guidelines are helping to improve methodological quality and reporting of animal research [8]. Results from this literature search All results Number absolute Number in percentage Number absolute Number in percentage Number absolute Number in percentage Meta-Analysis included 93/266 35% 63/246 26% 156/512 31% Meta-analysis evaluating a medical intervention 65/93 70% 49/63 78% 114/156 73% Meta-analysis preceded by a systematic review 71/93 76% 59/63 94% 130/156 83% Meta-analysis combines animal and human data 20/93 22% 8/63 13% 28/156 18% Number of included studies Not reported 18/93 19% 16/63 25% 34/156 22% ,10 16/93 17% 11/63 18% 27/156 17% 10–50 46/93 50% 19/63 30% 65/156 42% 51–100 7/93 8% 9/63 14% 16/156 10% .100 6/93 6% 8/63 13% 14/156 9% Methods for data synthesis Not reported 37/93 40% 11/63 18% 48/156 31% Fixed effects model 4/93 4% 1/63 2% 5/156 3% Random effects model 38/93 41% 37/63 59% 75/156 48% Both 14/93 15% 14/63 22% 28/156 18% Effect estimates of indi- vidual studies reported 45/93 48% 39/63 62% 84/156 54% * ll t d d t i t l b PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 12 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 OPEN-Dissemination Bias in Animal Research profiles to show the influence of animal research on clinical research. A limitation of our study is that our results might be affected by dissemination bias because we did not search any grey literature. Thus, this systematic review might miss a number of systematic reviews of preclinical research. We are therefore limiting the generalizability of our results about methodological quality and dissemination bias to the published systematic reviews of in vivo animal studies. Regarding the quality assessment one can assume that non-published systematic reviews might be of even lower quality and our results might be too positive. Regarding the influence on clinical studies non-published systematic reviews of animal studies might have less impact on clinical research, since they are not easily accessible. Furthermore, we arbitrarily selected only 50 systematic reviews and meta-analyses to assess their influence on clinical research. Thus, the citation rate might just show a trend of the influence of preclinical systematic reviews on clinical, above all since we did not measure the influence directly. This trend could be proven in further studies by assessing whether the objectives or the study design of the clinical studies is similar to the one of the preclinical study cited. Research synthesis depends on high methodological quality of primary research. ARRIVE guidelines are helping to improve methodological quality and reporting of animal research [8]. Furthermore, it is important that all research results are accessible for systematic reviews in order to allow valid synthesis. Unfortunately, it has been shown that animal research often does not get published, and that the direction of results might be a reason for non-publication Table 4. Influence of systematic reviews of preclinical research on clinical research. Number absolute Number in percentage* Type of clinical study Randomized controlled trials 87/281 31% Non-randomized controlled trials 25/281 9% Cross-over trials 2/281 1% Uncontrolled prospective trials 6/281 2% Retrospective cohort studies 42/281 15% Prospective cohort studies 87/281 31% Laboratory experiments with healthy human volunteers 6/281 2% Cross-sectional surveys 1/281 0% Case report/case series 26/281 9% Guidelines 2/281 1% Other 0/281 0% Use of citation Use of citation unrelated to animal studies 8/281 3% Use of citation to provide at least partial justification for the study or a future study 213/281 76% Use of citation to support or explain their findings 103/281 37% Other 12/281 4% all percentages rounded to integral numbers. *multiple selection possible. Conclusions and Implications Over the years, the number of systematic reviews and meta-analyses of preclinical research has increased. In this systematic review, we showed that preclinical systematic reviews and meta-analyses influence clinical research and thus might influence even clinical care. Unfortunately, according to our data the quality of systematic reviews and meta-analyses of animal research still remains poor. Therefore, we strongly encourage every effort made to improve the methodology of systematic reviews and meta-analyses on preclinical research, such as CAMARADES or the registration of animal studies before inception [11]. Author Contributions Conceived and designed the experiments: MB JJM DB. Analyzed the data: KFM VG MB DS FL. Wrote the paper: KFM DB. Designed search strategies: MB EM. Critically reviewed the manuscript for important intellectual content: MB DS JJM BL EM VG FL. Read and approved the final version: KFM MB DS JJM BL EM VG FL DB. Guarantors: KFM DB. 1. Pound P, Bracken MB (2014) Is animal research sufficiently evidence based to be a cornerstone of biomedical research? BMJ 348: g3387. OPEN-Dissemination Bias in Animal Research [14, 25]. In this study, there has been evidence for dissemination bias in 50% of systematic reviews, which assessed dissemination bias. Similarly Sena et al. also showed the presence and the impact of dissemination bias in systematic reviews of animal studies [25]. Thus if we agree that dissemination bias has an influence on the results of systematic reviews particularly of animal research, it can not only result in erroneous conclusions but might also lead to unsafe and unnecessary clinical research. [14, 25]. In this study, there has been evidence for dissemination bias in 50% of systematic reviews, which assessed dissemination bias. Similarly Sena et al. also showed the presence and the impact of dissemination bias in systematic reviews of animal studies [25]. Thus if we agree that dissemination bias has an influence on the results of systematic reviews particularly of animal research, it can not only result in erroneous conclusions but might also lead to unsafe and unnecessary clinical research. CAMARADES has already made a major step in improving systematic reviews of clinical research [11]. But in order to allow valid research synthesis, the availability of all research results is crucial. Therefore, the registration of animal studies before inception seems to be necessary [25, 26]. The registration of a clinical study, before the first participant has been included is required for publication; this should be applied to animal studies too. Since all animaly experiments must pass Institutional Animal Care and Use Committee or similar organizations for ethics approval they could play a crucial in the registration of animal studies and thus in the prevention of dissemination bias, as already suggested by ter Riet et al. [14]. 2. 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(2010) Dissemination and publication of research findings: an updated review of related biases. Health Technol Assess 14: iii, ix–xi, 1–193. 14. ter Riet G, Korevaar DA, Leenaars M, Sterk PJ, Van Noorden CJF, et al. (2012) Publication Bias in Laboratory Animal Research: A Survey on Magnitude, Drivers, Consequences and Potential Solutions. Plos One 7. 15. Begley CG, Ellis LM (2012) Drug development: Raise standards for preclinical cancer research. Nature 483: 531–533. 16. PLOS ONE \| DOI:10.1371/journal.pone.0116016 December 26, 2014 References Liu S (2009) Dealing with publication bias in translational stroke research. J Exp Stroke Transl Med 2: 16–21. 17. Tsilidis KK, Panagiotou OA, Sena ES, Aretouli E, Evangelou E, et al. (2013) Evaluation of excess significance bias in animal studies of neurological diseases. PLoS Biol 11: e1001609. 18. Briel M, Muller KF, Meerpohl JJ, von Elm E, Lang B, et al. (2013) Publication bias in animal research: a systematic review protocol. 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https://openalex.org/W4310782382	https://www.researchsquare.com/article/rs-1640412/latest.pdf	English	null	Castor1 overexpression regulates microglia M1/M2 polarization via inhibiting mTOR pathway	Metabolic brain disease	2,022	cc-by	6,165	Castor1 overexpression regulates microglia polarization via inhibiting mTOR pathway Huiling Hu (  huhling5@mail.sysu.edu.cn ) Sun Yat-Sen University 2nd Affiliated Hospital: Sun Yat-Sen Memorial Hospital Xiaoxia Lu Sun Yat-Sen University 2nd Affiliated Hospital: Sun Yat-Sen Memorial Hospital Xiuli Liu Sun Yat-Sen University 2nd Affiliated Hospital: Sun Yat-Sen Memorial Hospital Yuqing He Southern Medical University Chaohui Duan Sun Yat-Sen University 2nd Affiliated Hospital: Sun Yat-Sen Memorial Hospital Castor1 overexpression regulates microglia polarization via inhibiting mTOR pathway Huiling Hu (  huhling5@mail.sysu.edu.cn ) Sun Yat-Sen University 2nd Affiliated Hospital: Sun Yat-Sen Memorial Hospital Xiaoxia Lu Sun Yat-Sen University 2nd Affiliated Hospital: Sun Yat-Sen Memorial Hospital Xiuli Liu Sun Yat-Sen University 2nd Affiliated Hospital: Sun Yat-Sen Memorial Hospital Yuqing He Southern Medical University Chaohui Duan Sun Yat-Sen University 2nd Affiliated Hospital: Sun Yat-Sen Memorial Hospital Research Article DOI: https://doi.org/10.21203/rs.3.rs-1640412/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 Metabolic Brain Disease on December 1st, 2022. See the published version at https://doi.org/10.1007/s11011-022-01135-w. Page 1/16 Page 1/16 Abstract Microglia are resident immune cells in the brain and are closely associated with central nervous system inflammation and neurodegenerative diseases. It is known that mTOR pathway plays an important role in the polarization of microglia. Castor1 has been identified as the cytosolic arginine sensor for the mTORC1 pathway, but the role of Castor1 in microglial polarization is still unknown. The purpose of this study was to explore the regulatory effect of Castor1 on microglial polarization and the underlying mechanism. The results demonstrated that Castor1 expression was significantly decreased in LPS and IFNγ treated microglia. Castor1 overexpression inhibited the microglia M1 polarization by reducing the expression of M1 related markers. However, microglia M2 polarization was promoted when Castor1 was overexpressed in IL-4 treated microglia. Mechanistically, Castor1 overexpression inhibited the activation of mTOR signaling pathway. In addition, after treatment with the mTOR activator MHY1485, the inhibitory effect of Castor1 overexpression on M1 polarization was attenuated, indicating that the regulation effects of Castor1 on M1 polarization was dependent on its inhibition of mTOR pathway. We propose that Castor1-mTOR signaling pathway could be considered as a potential target for treatment and intervention of central nervous system-related diseases by regulating microglia polarization. Castor1-mTOR signaling pathway could be considered as a potential target for treatment and intervention of central nervous system-related diseases by regulating microglia polarization. Introduction Castor1, the GATOR2-interacting cellular arginine sensor of mTORC1, is located on human chromosome 2, which acting in parallel with SLC38A9 to regulate arginine sensing through mTORC1 (Chantranupong et al., 2016). By studying the crystal structure of Castor1, it was found that a fine clipping pocket was carved between the NTD and CTD domains of Castor1 to recognize arginine, revealing the molecular mechanism by which Castor1 senses arginine (Gai et al., 2016). Previous studies have shown that Castor1 is involved in the development of various diseases through the mTOR pathway. Zhou et al. found that Castor1 regulates the progression of lung adenocarcinoma by inhibiting phosphorylation of mTOR and its downstream S6K (Zhou et al., 2018a). Li et al. demonstrated that Castor1 overexpression attenuated mTORC1 activation and thus inhibited cell proliferation of KSHV-transformed cells (Li et al., 2019). However, no studies have been reported in investigating the regulatory effect of Castor1 on microglia polarization. Mammalian target of rapamycin (mTOR) is an important regulator of cell growth, metabolism and proliferation, and is a catalytic subunit of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (Saxton and Sabatini, 2017). The mTOR signaling pathway regulates innate and adaptive immune responses, and one of the main functions of mTORC1 is to regulate downstream p70S6 Kinase(p70S6K) and eukaryotic initiation factor 4E binding protein (4EBPs) to regulate mRNA translation, thus regulating immune responses and microglial inflammatory responses (Keane et al., 2021). Many studies have shown that mTOR signaling pathway is involved in the regulation of microglia polarization, thus affecting the occurrence and development of central nervous system-related diseases. Zhuang et al. found that hydrogen treatment inhibited M1 polarization of microglia and promoted M2 polarization by inhibiting the mTOR pathway, playing a protective role in the brain (Zhuang et al., 2020). Wang et al. 's study showed that Salidroside treatment of microglia reduced the phosphorylation of mTOR and P70S6K, thus participating in the polarization regulation of microglia (Wang et al., 2018). Zhao et al showed that the loss of mTOR in microglia reduced the expression of M1-related inflammatory cytokines, such as TNF-α and IL-1β, and exacerbated the loss of neurons (Zhao et al., 2020). However, the functional role of Castor1 in mTOR mediated microglial polarization remains unclear. In our study, we found that the expression of Castor1 was reduced in M1-polarized microglia induced by LPS and IFN-γ treatment. Introduction Microglia, the residential cells of the brain, has been proven to be the central nervous system (CNS) sentinels, maintaining normal brain functionality (Colonna and Butovsky, 2017). In brain, microglia presents as different morphologies: radially branched, compact circular and longitudinally branched (Lawson et al., 1990). Microglia participate in two essential functions involving homeostasis and host defense mechanisms (Hickman et al., 2018). The first function of microglia is screening environmental stimuli and protecting against it, including pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) (Glass et al., 2010; Hickman et al., 2013; Stephenson et al., 2018). The second function refers to the physiological house-guarding function, including migrating towards impaired sites, reshaping synapses, and preserving myelin homeostasis (Hickman et al., 2018; Zhan et al., 2014). The microglia functions above are achieved through the heterogeneous activation of microglia in CNS, and the activation can be categorized into M1 and M2 phenotype, which present totally different toxicity on CNS (Tang and Le, 2016). Under the stimulation of LPS and IFN-γ, microglia were activated as M1 phenotype, releasing M1-related markers CD86, iNOS, and M1-related inflammatory cytokines IL-1β, IL-6, TNF-α and MCP-1. In response to the stimulation of IL-4, IL-10 and IL-13, microglia activate to the M2 phenotype, releasing M2-related markers ARG-1 and CD206 as well as m2-related anti- inflammatory cytokines TGF-β and IL-10 (Li et al., 2021a). The functional differences between M1 and M2 microglias are closely related to central nervous system diseases, so it is very important to regulate the M1/M2 balance of microglia. Du et al. found that Ki20227 improved neurobehavioral function, reduced cerebral infarction size and played a neuroprotective role by inhibiting M1 polarization of microglia and activating M2 polarization inhibited by NLRP3 pathway (Du et al., 2020). Yang et al. found that Exo-miR-124 treatment promoted M2 polarization of microglia, Page 2/16 Page 2/16 Page 2/16 enhanced neurogenesis in the hippocampus, and improved functional recovery after traumatic brain injury (Yang et al., 2019). It has also been suggested that chitinase 1 May play a protective role in Alzheimer's disease by promoting the polarization of microglia into the M2 phenotype (Xiao et al., 2017). Therefore, studying the mechanism of regulating M1 and M2 balance of microglia is of great significance for seeking treatment and improving central nervous system injury and related diseases. Introduction In addition, Castor1 overexpression inhibited M1 polarization and promoted M2 polarization of microglia. Further studies demonstrated that Castor1 overexpression regulates microglia M1/M2 polarization via inhibiting mTOR pathway. Taken together, this study provides a new method for regulating the polarization characteristics of microglia, and also provides a potential therapeutic target for diseases caused by abnormal polarization of microglia. Page 3/16 Page 3/16 Quantitative real-time PCR Total RNA was extracted from cells using Trizol reagent (TaKaRa). According to the manufacturer's instructions, Total RNA was reverse transcribed into cDNA using the First-Strand cDNA Synthesis Kit (Applied Biological Materials). SYBR Green Master Mix (Roche) was used for the real-time PCR reactions of the synthesized cDNA in LightCycler 480 system (Roche). The primers used in this study were shown in Supplementary Table 1. Relative gene expression was analyzed by normalization of the cycling threshold (CT) values against the β-actin using the 2−ΔΔCT method. Immunofluorescence staining After been fixed with 4% PFA for 30 min at room temperature (RT), BV2 cells were washed with PBS three times and subsequently blocked with PBS containing 0.1% Triton X-100 and 3% BSA for 1 h at RT. Then the cells were incubated with primary antibody of iNOS (18985-1-AP, Proteintech), CD86 (13395-1-AP, Proteintech), CD206 (18704-1-AP, Proteintech) and Arg1 (16001-1-AP, Proteintech) overnight at 4°C. The cells were then washed three times with PBS and incubated with Alexa Fluor 488-conjugated donkey anti- rabbit secondary antibody (A-21206, Life technologies) or Alexa Fluor 594-conjugated donkey anti-rabbit secondary antibody (A-21207, Life technologies) for additional 24 h at 4℃. The fluorescence images were taken with Leica DMI 8 microscope. Methods BV2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium containing 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin/ streptomycin (Gibco) in a 5% CO2 incubator at 37°C. For M1 induction, cells were treated with 100 ng/mL LPS (PeproTech) and 10 ng/mL IFN-γ (PeproTech) for 12 hours. For M2 induction, cells were treated with 20 ng/mL IL-4 (PeproTech) for 12 h. For Castor1 overexpression, the plasmids of pcDNA3.1-Castor1-Flag were synthesized from Tsingke Biotechnology (Guangzhou). The transfection were performed with Lipofectamine™ 3000 reagent according to the manufacturer’s instructions. Transfected BV2 cells were used for the following experiments 12 h after transfection Western blot BV2 cells were lysed in RIPA lysis buffer (HANGZHOU FUDE Biotechnology) containing protease and phosphatase inhibitors. The homogenates were centrifuged at 12,000 g at 4 ℃ for 5 min, and the protein concentrations were determined using the BCA Protein Assay kit (Beyotime Biotechnology). Total proteins were separated by 12% SDS-PAGE and then electroblotted onto polyvinylidene fluoride membranes (PVDF) (Millipore). The membranes were blocked with 5% skim milk at room temperature (RT) for 1 h, then incubated overnight with the following primary antibodies: anti-iNOS antibody (18985-1-AP, Proteintech), anti-CD86 antibody (13395-1-AP, Proteintech), anti-CD206 antibody (18704-1-AP, Page 4/16 Page 4/16 Proteintech), anti-Arg1 antibody (16001-1-AP, Proteintech), anti-Castor1 antibody (sc-377385, Santa cruz) anti-β-actin antibody (bs-0061R, Bioss), anti-Flag antibody (14793S, CST), anti-mTOR antibody (2983S, CST), anti-p-mTOR antibody (2971S, CST), anti-p70s6k antibody (9202S, CST), anti-p-p70s6k antibody (9205S, CST), anti-4EBP1 antibody (9644S, CST), and anti- p-4EBP1 antibody (2855S, CST). The membranes were washed three times with TBST, then incubated with goat anti-rabbit HRP-conjugated secondary antibody (bs-0295G, Bioss) or goat anti-mouse HRP-conjugated secondary antibody (bs- 0296G, Bioss) at RT for 2 h. The membranes were scanned on a MP ChemiDoc Imaging System (Bio- Rad). Densitometric values of the western blot bands were analyzed using ImageJ software. Proteintech), anti-Arg1 antibody (16001-1-AP, Proteintech), anti-Castor1 antibody (sc-377385, Santa cruz) anti-β-actin antibody (bs-0061R, Bioss), anti-Flag antibody (14793S, CST), anti-mTOR antibody (2983S, CST), anti-p-mTOR antibody (2971S, CST), anti-p70s6k antibody (9202S, CST), anti-p-p70s6k antibody (9205S, CST), anti-4EBP1 antibody (9644S, CST), and anti- p-4EBP1 antibody (2855S, CST). The membranes were washed three times with TBST, then incubated with goat anti-rabbit HRP-conjugated secondary antibody (bs-0295G, Bioss) or goat anti-mouse HRP-conjugated secondary antibody (bs- 0296G, Bioss) at RT for 2 h. The membranes were scanned on a MP ChemiDoc Imaging System (Bio- Rad). Densitometric values of the western blot bands were analyzed using ImageJ software. Statistical analysis All experimental data were presented as the mean ± SEM. An unpaired two-sided Student’s t test was used to analyze the significance between two experimental groups. GraphPad Prism (GraphPad Software) was used to process the data, and P values < 0.05 were considered statistically significant. The Expression of Castor1 was Downregulated in M1 microglia polarization To investigate the expression and the role of Castor1 in M1 microglia polarization, BV2 cells were treated with 100 ng/mL LPS and 10 ng/mL IFN-γ for 12 h. The expression of M1-related markers and Castor1 was detected by quantitative real-time PCR and western blotting. As expected, compared with the control group, the expression of M1-related markers such as CD86 (P < 0.05), iNOS (P < 0.01), IL-β (P < 0.01), IL-6 (P < 0.01), TNF-α (P < 0.001), MCP-1 (P < 0.001), were significantly increased in the LPS/IFN-γ group. And the the expression of Castor1 (P < 0.001) was downregulated in M1 microglia polarization conditions (Fig. 1). These results suggested that Castor1 had a potential role in regulating microglia polarization. Castor1 overexpression suppressed M1 microglia polarization Since the expression level of Castor1 was obviously reduced in LPS/IFN-γ induced microglia, we further investigated whether overexpression of Castor1 affects M1 polarization. Castor1 overexpression in BV2 cells was carried out by pcDNA3.1 plasmid transfection. In brief, cells were transfected with pcDNA3.1- vector or pcDNA3.1-Castor1 plasmid for 12 h and then treated with LPS/IFN-γ for 12 h. The results demonstrated that Castor1 protein levels were obviously increased after transfected with pcDNA3.1- Castor1 plasmid (Fig. 2B). To verify whether Castor1 overexpression further regulates M1 polarization, the expression of M1 polarization markers was detected. We found that Castor1 overexpression inhibited the microglia M1 polarization by reducing the expression of M1 related markers, including CD86 (P < 0.05), iNOS (P < 0.001), IL-β (P < 0.001), IL-6 (P < 0.05), TNF-α (P < 0.001), MCP-1 (P < 0.001) (Fig. 2A, B). Furthermore, immunofluorescence staining results also confirmed that the expression of CD86 and iNOS Furthermore, immunofluorescence staining results also confirmed that the expression of CD86 and iNOS Page 5/16 was inhibited after Castor1 was overexpressed (Fig. 2C). In conclusion, Castor1 overexpression could suppress M1 microglia polarization. was inhibited after Castor1 was overexpressed (Fig. 2C). In conclusion, Castor1 overexpression could suppress M1 microglia polarization. Castor1 overexpression promoted microglia polarization to the M2 phenotype Microglia will polarize into M1 type or M2 type when confronted with stimulation. These two activated subtypes have different morphology and opposite functions. Since Castor1 overexpression could inhibit M1 polarization, what is the effect of Castor1 overexpression on M2 polarization? To investigate the effect of Castor1 overexpression on M2 polarization, BV2 cells were treated with 20 ng/mL IL-4 for 12 h after they were transfected with pcDNA3.1-vector or pcDNA3.1-Castor1 plasmid for 12 h. Compared with the pcDNA3.1-vecor group, the microglia M2 polarization was promoted when Castor1 was overexpressed, as the expression of Arg1 (P < 0.01), CD206 (P < 0.05) and IL-10 (P < 0.05) was significantly increased (Fig. 3A, B). And the immunofluorescence staining results further confirmed that Castor1 overexpression promoted the expression of Arg1 and CD206 (Fig. 3C). These results suggested that Castor1 overexpression promoted microglia polarization to the M2 phenotype Castor1 overexpression inhibited mTOR pathway Previous studies have shown that Castor1, as an upstream regulatory molecule of the mTOR signaling pathway, plays an important role in the pathogenesis of disease. To investigate the effect of Castor1 overexpression on mTOR pathway, BV2 cells were transfected with pcDNA3.1-vecor or pcDNA3.1-Castor1 for 12 h and then treated with LPS/IFN-γ for 12 h. And then expression of some key proteins involved in the mTOR pathway was detected, such as mTOR, P70S6K and 4E-BP1 and their respective phosphorylated forms. Results demonstrated that the phosphorylation levels of mTOR (P < 0.05), 4E-BP1 (P < 0.05) and P70S6K (P < 0.05) were significantly reduced after overexpression of Castor1 (Fig. 4). The overall results indicated that Castor1 overexpression inhibited the phosphorylation of key proteins involved in the mTOR signaling pathway, thus inhibiting the activity of the mTOR pathway activation. Discussion Microglia polarize into different phenotypes (mainly M1 type and M2 type) in response to various stimulus and participate in the occurrence and development of diverse central nervous system diseases (Crain et al., 2013; Subhramanyam et al., 2019). It has been found that the mTOR pathway plays an important regulatory role in microglia polarization (Srivastava et al., 2016). However, the role of Castor1 as an upstream regulatory molecule of mTOR signaling pathway in microglia polarization has not been reported. In this experiment, we found that Castor1 overexpression can inhibit M1 polarization and promote M2 polarization, and Castor1's regulation of M1 polarization depends on the inhibition of mTOR signaling pathway. Microglia, as inherent immune cells in the central nervous system, play an important role in the occurrence and development of a series of neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease (Zhou et al., 2018b), multiple sclerosis (Benveniste, 1997). In addition, they are also closely related to central nervous system injury, ischemia, inflammation (Wang et al., 2015). Microglia can be divided into inflammatory microglia (M1 type) and anti-inflammatory microglia (M2 type) under different states of activation. M1 microglia mainly express INOS, CD86 and other surface markers, which often induce inflammation and nerve injury (Nguyen et al., 2017). M2 microglia mainly express ARG1, CD206 and other surface markers mainly play the role of nerve repair, induction and anti-inflammatory (Colonna and Butovsky, 2017). Previous studies have shown that M1/M2 balance of microglia plays an important role in maintaining central nervous system homeostasis and preventing neurodegenerative diseases (Song et al., 2016). It has been proved that α -synuclein induced polarization of microglia M1 could amplify neuronal damage and aggravate motor deficits in Parkinson's disease (Du et al., 2018; Tang and Le, 2016). Mirren et al. showed that in the early stage of multiple sclerosis, microglia polarize into the M1 phenotype, leading to neuronal demyelination and nerve damage. In the later stage of multiple sclerosis, microglia polarize into the M2 phenotype, releasing anti-inflammatory factors and promoting myelin regeneration (Miron et al., 2013). Hu X et al. reported that microglia M1 polarization increased during inflammation after ischemic stroke, which expanded the scope of ischemic infarction and further worsened nerve injury (Hu et al., 2015; Hu et al., 2012). Therefore, inhibiting M1 polarization of microglia and promoting M2 polarization of microglia may be a new therapeutic target. The regulation of Castor1 on M1 polarization depends on the suppression of the mTOR signaling pathway The regulation of Castor1 on M1 polarization depends on the suppression of the mTOR signaling pathway Since Castor1 overexpression inhibits the polarization of M1 and also inhibits the activity of the mTOR signaling pathway, we speculated that the regulation of Castor1 on M1 polarization depends on the suppression of the mTOR signaling pathway. To confirm this hypothesis, an experimental group was added on the basis of the previous experiment, namely, microglia were transfected with pcDNA3.1- Castor1 for 12 h and treated with mTOR activator (MHY1485, 5 uM) at the same time as being treated with LPS + IFN-γ for 12 h. The results shown that after treatment with the mTOR activator MHY1485, the inhibitory effect of Castor1 overexpression on M1 polarization was attenuated, as the expression of M1- related markers such as CD86 (P < 0.05), iNOS (P < 0.001), IL-β (P < 0.001), IL-6 (P < 0.05), TNF-α (P < 0.001) and MCP-1 (P < 0.01) were significantly increased compared with pcDNA3.1-Castor1 group Page 6/16 Page 6/16 (Fig. 5). All these results indicated that the regulation effects of Castor1 on M1 polarization was dependent on its inhibition of mTOR pathway. Discussion Results demonstrated that the phosphorylation levels of mTOR, 4E-BP1 and P70S6K were significantly reduced after overexpression of Castor1. This suggests that Castor1 overexpression inhibits the activity of mTOR signaling pathway in M1 polarization of microglia. In order to verify that Castor1 overexpression inhibits mTOR signaling pathway and thus inhibits M1 polarization of microglia, we treated microglia with mTOR activator MHY1485 and found that after the mTOR signaling pathway was activated, the inhibitory effect of Castor1 overexpression on M1 polarization was attenuated, as the expression of M1-related markers such as CD86, iNOS, IL-β, IL-6, TNF- α, and MCP-1 were significantly increased. This indicates that the activation of mTOR signaling pathway antagonizes the inhibitory effect of Castor1 overexpression on microglia M1 polarization, suggesting that the inhibition of Castor1 overexpression on microglia M1 polarization depends on the inhibition of mTOR signaling pathway Discussion Our study induced M1 polarization of microglia using LPS + IFN-γ and found a decrease in Castor1 expression, which has not been previously reported. In order to investigate the effect of Castor1 on microglia polarization, we constructed a pcDNA3.1-Castor1 overexpression plasmid vector. After LPS + IFN-γ treatment, Castor1 overexpression inhibited the expression of M1-related markers such as CD86 and INOS. Under The treatment of IL-4, Castor1 overexpression promoted the expression of M2-related markers Arg1 and CD206. This suggested that Castor1 overexpression inhibited M1 polarization and promoted M2 polarization of microglia. Page 7/16 Page 7/16 Castor1 is a cytoplasmic arginine sensor consisting of four tandem ACT domains that dissociate from GATOR2 after binding to arginine, thereby activating the mTORC1 signaling pathway and playing an important regulatory role in the occurrence of a variety of diseases (Saxton et al., 2016; Zhuang et al., 2019). Li et al. first reported that Castor1 could inhibit cell proliferation and transformation and induce cell cycle arrest (Li et al., 2019). Another study have shown that Castor1 has a tumor suppressor function on breast cancer cells, and the activation of Castor1 can be considered as a potential treatment for some cancers (Li et al., 2021b). However, the regulatory effect of Castor1 on microglia has not been reported. mTOR is a serine/threonine protein kinase, belonging to the PI3K-related kinase family, which is regulated by a variety of cell signals and plays a key role in cell proliferation, metabolism, immune response and other processes, and is also closely related to the occurrence of many diseases (Saxton and Sabatini, 2017). It has been reported that mTOR signaling pathway plays a very important role in regulating M1/M2 polarization of microglia. The study of Hu et al. showed that LPS could induce rapid activation of mTOR signaling pathway in microglia. On the contrary, inhibition of the mTOR signaling pathway reduces LPS-induced microglial proinflammatory cytokines (Hu et al., 2020). Li et al. reported that specific blocking of mTORC1 signaling pathway can reduce LPS-induced M1 Maker expression and increase M2 Maker expression (Li et al., 2016). Therefore, we speculated that the inhibition of M1 polarization in microglia by Castor1 overexpression may depend on its inhibition of mTOR signaling pathway. Thus, the expression of some key proteins involved in the mTOR pathway was detected, such as mTOR, P70S6K and 4E-BP1 and their respective phosphorylated forms. Code availability Not applicable Conclusions In summary, our study demonstrated that the expression of Castor1 was significantly decreased in LPS/IFN-γ treated microglia, and the overexpression of Castor1 inhibited LPS + IFN-γ-induced M1 polarization and promoted IL-4 Induced microglial M2 polarization. The regulatory effect of Castor1 on M1 polarization depends on its inhibition of mTOR signaling pathway and can be antagonized by mTOR signaling pathway activator MHY1485. Therefore, Castor1's regulation of M1/M2 polarization of microglia may make it a potential therapeutic target for neurodegenerative and neuroinflammation- related diseases. Page 8/16 Conflicts of interest The authors have no conflict or competing interests. Authors' contributions Huiling Hu and Chaohui Duan contributed to the study conception and design. Material preparation, data collection and analysis were performed by Huiling Hu, Xiaoxia Lu and Yuqing He. The first draft of the manuscript was written by Huiling Hu, Xiaoxia Lu and Xiuli Liu. All authors read and approved the final manuscript. Funding This work was supported by National Natural Science Foundation of China (82000803, 82100921), Guangdong Basic and Applied Basic Research Foundation (2019A1515010980), the Fundamental Research Funds for the Central Universities (20ykpy96), Guangzhou Science and Technology Plan Project (202102021099). 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Zhuang Y, Wang XX, He J, He S, Yin Y (2019) Recent advances in understanding of amino acid signaling to mTORC1 activation. Frontiers in bioscience (Landmark edition). 24:971–982 Figures Figure 1 The Expression of Castor1 was downregulated in M1 microglia polarization. (A) The relative mRNA expression of Castor1 and M1 related makers detected by qPCR. (B) Representative Western blots results showing the expression of Castor1, iNOS, CD86. Data are expressed as mean ± SEM, P<0.05, P<0.01, *P<0.01 compared to the CON group. The Expression of Castor1 was downregulated in M1 microglia polarization. (A) The relative mRNA expression of Castor1 and M1 related makers detected by qPCR. (B) Representative Western blots results showing the expression of Castor1, iNOS, CD86. Data are expressed as mean ± SEM, P<0.05, P<0.01, P<0.01 compared to the CON group. ( ) p y q ( ) Representative Western blots results showing the expression of Castor1, iNOS, CD86. Data are expressed as mean ± SEM, P<0.05, P<0.01, P<0.01 compared to the CON group. Page 12/16 Castor1 overexpression suppressed M1 microglia polarization. Castor1 overexpression suppressed M1 microglia polarization. (A) The relative mRNA expression of M1 related makers detected by qPCR. (B) Representative Western blots results showing the expression of Castor1, iNOS, CD86. (C) Representative images of the immunofluorescence of of iNOS and CD86. Data are expressed as SEM, P<0.05, **P<0.001 compared to the pcDNA3.1-vector group. Scale bars, 25 μm. Page 13/16 mTOR pathway was involved in Castor1-mediated microglia M1 polarization. (A) The relative mRNA expression of M1 related makers detected by qPCR. (B) Representative Western blots results showing the expression of iNOS and CD86. Data are expressed as SEM, P<0.05, P<0.01, P<0.01 compared to the pcDNA3.1-vector+LPS+IFNγ group. (A) The relative mRNA expression of M1 related makers detected by qPCR. (B) Representative Western blots results showing the expression of iNOS and CD86. Data are expressed as SEM, P<0.05, P<0.01, P<0.01 compared to the pcDNA3.1-vector+LPS+IFNγ group. Castor1 overexpression promoted microglia polarization to the M2 phenotype. Castor1 overexpression promoted microglia polarization to the M2 phenotype. (A) The relative mRNA expression of M2 related makers detected by qPCR. (B) Representative Western blots results showing the expression of CD206 and Arg1. (C) Representative images of the immunofluorescence of of CD206 and Arg1. Data are expressed as SEM, P<0.05, *P<0.01 compared to the pcDNA3.1-vector+IL4 group. Scale bars, 25 μm. Page 14/16 Figure 4 Castor1 overexpression inhibited mTOR pathway. A) Representative Western blots results showing the expression of key proteins involved in the mTOR pathway. (B) Quantitative analysis of Western blot of key proteins involved in the mTOR pathway. Data are expressed as SEM, P<0.05 compared to the pcDNA3.1-vector+LPS+IFNγ group. Castor1 overexpression inhibited mTOR pathway. A) Representative Western blots results showing the expression of key proteins involved in the mTOR pathway. (B) Quantitative analysis of Western blot of key proteins involved in the mTOR pathway. Data are expressed as SEM, P<0.05 compared to the pcDNA3.1-vector+LPS+IFNγ group. Page 15/16 Page 15/16 Figure 5 mTOR pathway was involved in Castor1-mediated microglia M1 polarization. (A) The relative mRNA expression of M1 related makers detected by qPCR. (B) Representative Western blots results showing the expression of iNOS and CD86. Data are expressed as SEM, P<0.05, P<0.01, *P<0.01 compared to the pcDNA3.1-vector+LPS+IFNγ group. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryTables.docx Figure 5 SupplementaryTables.docx Figure 5 mTOR pathway was involved in Castor1-mediated microglia M1 polarization. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryTables.docx Page 16/16
https://openalex.org/W2792171409	https://hal.inria.fr/hal-01736600/file/peerj-4492%20%281%29.pdf	English	null	Electroencephalographic modulations during an open- or closed-eyes motor task	PeerJ	2,018	cc-by	8,103	To cite this version: Sébastien Rimbert, Rahaf Al-Chwa, Manuel Zaepffel, Laurent Bougrain. Electroencephalographic modulations during an open- or closed-eyes motor task. PeerJ, 2018, 6, pp.1-18. 10.7717/peerj.4492. hal-01736600 HAL Id: hal-01736600 https://inria.hal.science/hal-01736600v1 Submitted on 18 Mar 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. Electroencephalographic modulations during an open- or closed-eyes motor task Sébastien Rimbert1,2,3, Rahaf Al-Chwa1,2, Manuel Zaepffel4 and Laurent Bougrain1,2,3 1 Neurosys team, Inria, Villers-lès-Nancy, France 2 Artificial Intelligence and Complex Systems, Université de Lorraine, LORIA, Vandœuvre-lès-Nancy, France 3 Neurosys team, CNRS, LORIA, Vandœuvre-lès-Nancy, France 4 Unaffiliated, Dambach-la-ville, France 1 Neurosys team, Inria, Villers-lès-Nancy, France ABSTRACT There is fundamental knowledge that during the resting state cerebral activity recorded by electroencephalography (EEG) is strongly modulated by the eyes-closed condition compared to the eyes-open condition, especially in the occipital lobe. However, little research has demonstrated the influence of the eyes-closed condition on the motor cortex, particularly during a self-paced movement. This prompted the question: How does the motor cortex activity change between the eyes-closed and eyes-open conditions? To answer this question, we recorded EEG signals from 15 voluntary healthy subjects who performed a simple motor task (i.e., a voluntary isometric flexion of the right-hand index) under two conditions: eyes-closed and eyes-open. Our results confirmed strong modulation in the mu rhythm (7–13 Hz) with a large event-related desynchronisation. However, no significant differences have been observed in the beta band (15–30 Hz). Furthermore, evidence suggests that the eyes-closed condition influences the behaviour of subjects. This study gives us greater insight into the motor cortex and could also be useful in the brain-computer interface (BCI) domain. Submitted 21 November 2017 Accepted 20 February 2018 Published 15 March 2018 Corresponding author Sébastien Rimbert, sebastien.rimbert@inria.fr Academic editor Tjeerd Boonstra Additional Information and Declarations can be found on page 14 DOI 10.7717/peerj.4492 Copyright 2018 Rimbert et al. Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS Submitted 21 November 2017 Accepted 20 February 2018 Published 15 March 2018 Corresponding author Sébastien Rimbert, sebastien.rimbert@inria.fr Academic editor Tjeerd Boonstra Additional Information and Declarations can be found on page 14 DOI 10.7717/peerj.4492 Subjects Neuroscience, Human–Computer Interaction, Computational Science Keywords Electroencephalography, Motor cortex, Event-relatedsynchronization (ERS), Event- related desynchronization (ERD), Eyes-closed, Eyes-open, Voluntary movement, Real movement Subjects Neuroscience, Human–Computer Interaction, Computational Science Keywords Electroencephalography, Motor cortex, Event-relatedsynchronization (ERS), Event- related desynchronization (ERD), Eyes-closed, Eyes-open, Voluntary movement, Real movement How to cite this article Rimbert et al. (2018), Electroencephalographic modulations during an open- or closed-eyes motor task. PeerJ 6:e4492; DOI 10.7717/peerj.4492 1 Neurosys team, Inria, Villers-lès-Nancy, France 2 Artificial Intelligence and Complex Systems, Université de Lorraine, LORIA, Vandœuvre-lès-Nancy, France 3 Neurosys team, CNRS, LORIA, Vandœuvre-lès-Nancy, France 4 Unaffiliated, Dambach-la-ville, France INTRODUCTION Every day we accomplish voluntary movements in our environment, and yet the simplicity with which we perform these movements contrasts with the high complexity of their underlying physiological and neuronal processes. Voluntary movements, also called self-paced movements or self-initiated movements, can be subdivided into two categories: internal and external voluntary movements. For an internal voluntary movement, the motion is selected according to a defined goal, based on internal cognitive processes relative to our previous experience. This is contrary to a reflex response which is an immediate and stereotyped reaction to a stimulus from the environment (Haggard et al., 2005). For an external voluntary movement, the motor preparation phase is absent, but the phases of stimulus processing and anticipation are maintained (Brass & Haggard, 2008). Copyright 2018 Rimbert et al. Distributed under Creative Commons CC-BY 4.0 Various specific areas are associated with the motor control. For example, the pre- supplementary motor area is associated with selective motor initiation, and the medial pre-motor system is associated with internal timing (Hoffstaedter et al., 2013). In addition, a voluntary movement is part of a sensorimotor loop composed of a preparation, an execution, and an evaluation phase, each modulating the activity of different motor areas (Deiber et al., 2012). A self-paced movement is characterised by three distinct phases which are easily identifiable in the recorded EEG signal. Initially, compared to a resting state, the movement preparation phase shows a gradual decrease of power in the mu (7–13 Hz) and the beta (15–30 Hz) bands (Pfurtscheller & Neuper, 1997). This is referred to as an event-related desynchronisation (ERD) (Pfurtscheller & Aranibar, 1979; Pfurtscheller & Lopes da Silva, 1999). During the movement, a minimal power level is maintained in both bands (Pfurtscheller & Lopes da Silva, 1999; Cheyne, 2013). Finally, 300 to 500 ms after the end of the movement, there is an increase of power referred to as an event- related-synchronisation (ERS) in the beta band, also known as post-movement beta rebound, lasting approximately one second (Salenius et al., 1997; Cheyne, 2013; Kilavik et al., 2013). Concurrently, in the mu band, the power returns to a baseline after several seconds. Typically, these patterns occur in the motor cortex, in the contralateral position (Pfurtscheller & Aranibar, 1977; Pfurtscheller & Neuper, 2001; Bai et al., 2005) but can appear bilaterally (Fok et al., 2011; Pfurtscheller, Stancák & Neuper, 1996; Formaggio et al., 2013). Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 INTRODUCTION ERD and ERS patterns are observed also during a passive movement (Müller et al., 2003), an observed movement (Avanzini et al., 2012), a kinesthetic illusion (Keinrath et al., 2006), a median nerve stimulation (Salenius et al., 1997) and a motor imagery (Pfurtscheller & Solis-Escalante, 2009). A self-paced movement affects not only the power in the mu and the beta frequency bands, but also the event-related potentials in the spatio-temporal domain (Kornhuber & Deecke, 1965; Shibasaki & Hallett, 2006). Voluntary movements, such as grasping and manipulation of objects in our environment, require sensory inputs and feedback (Desmurget et al., 1998; Jeannerod, 1995). Indeed, directed movements towards an object are the result of transforming the visual properties of this object into a well-defined sequence of muscular contractions. Several studies (Berger, 1929; Compston, 2010; Jasper, 1936; Barry et al., 2007; Barry et al., 2009) show differences in resting state EEG signals recorded during eyes-closed (EC) and eyes-open (EO) conditions. These differences include an increase in alpha activity during an EC resting condition with respect to an EO condition with a visual stimulation (Berger, 1929). Moreover, the EC condition modulates the occipital, parietal and frontal alpha activities (Li, 2010) and the effect of the EO condition is most pronounced in posterior regions (Chapman, Armington & Bragdon, 1962; Volavka, Matouek & Roubek, 1967; Legewie, Simonova & Creutzfeldt, 1969). Very few studies have investigated the influence of the EC condition on the motor cortex during a voluntary movement. One of these studies carried out by Westphal in 1993, found no difference between the EC and EO conditions compared to the Bereitschaftpotential (Westphal et al., 1993). However, to the best of our knowledge, no study has investigated the effect of the EC condition on the ERD and ERS patterns reflecting excitation and Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 2/18 inhibition occurring on the motor cortex. This raises the question of how the EEG power over the motor cortex are modulated before and after a voluntary movement during an EC condition. In this work we analyse and compare the modulation of mu rhythm and beta band activities for a voluntary movement during EO and EC conditions. To this end, we recruited 15 healthy subjects and computed time-frequency maps, ERD and ERS modulation, and EEG power during resting state to show differences occurring on the motor cortex in the EEG signal. Experimental environment The experiments took place in a confined and calm room and were conducted by the same researcher. Subjects were seated comfortably on a chair with their right arm at their side with their hand placed on the computer mouse on a table without any tension of the muscles. Experimental task The task was an isometric flexion of the right index finger in order to click on a computer mouse during two different conditions: EC and EO. Although simple to execute, this kind of finger movement has been found already to activate the motor cortex (Shibasaki et al., 1993). To ensure that this movement was a voluntary one, the protocol dictated that the subject performed the task at their own pace, without receiving a triggering signal. INTRODUCTION Our results indicate that the EC condition increased the ERD in the mu rhythm but had no effect on the ERD and ERS in the beta band. Participants Fifteen right-handed healthy volunteer subjects (nine females; from 19 to 40 years-old; 24.1 years ±3.2) were recruited for this study. This experiment followed the statements of the WMA declaration of Helsinki on ethical principles for medical research involving human subjects (World Medical Association, 2002). In addition, participants signed an informed consent which was approved by the ethical committee of Inria (COERLE, approval number: 2016-011/01) as it satisfied the ethical rules and principles of the institute. The subjects had normal or corrected-to normal visual acuity and had no medical history which could have influenced the task, such as diabetes, antidepressant treatment or neurological disorders. Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 Experimental design The protocol contained two conditions (EC and EO) split in different runs that were completed by the subject on the same day. Condition 1 and condition 2 corresponded to the voluntary movement in EC and EO conditions respectively (Fig. 1). For each condition, 3 runs of 5 min were performed and a randomisation of the runs was applied to avoid that fatigue, gel drying, or other confounding factors that might have caused possible biases in the results. During a run, the subject had to perform a real movement task approximately once every ten seconds. At the beginning of each run, the subject remained relaxed for 10 s. Breaks of a few minutes were taken between runs to prevent fatigue of the subject. Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 3/18 3/18 Figure 1 Paradigm scheme representing the two different conditions: EC (Condition 1) and EO (Condition 2). Each condition was composed by three runs of 5 min and randomisation of the order was applied. Full-size DOI: 10.7717/peerj.4492/fig-1 Figure 2 Timing scheme for each run. The subject performed a right-hand index isometric flexion on the mouse when they wished while providing sufficient time (>8 s) between each movement. Full-size DOI: 10.7717/peerj.4492/fig-2 Before the experiment, the task was described and the subject was trained to execute it over 10 min. During the training period the subject learned to estimate 10 s in the two given conditions and for the open-eyes condition the subject had to perform the task while they fixate their gaze on a target placed at eye-level to avoid random eye movements (Fig. 2). The subjects were instructed to avoid swallowing or any other movements while performing the task, particularly all eye-movement. Figure 1 Paradigm scheme representing the two different conditions: EC (Condition 1) and EO (Condition 2). Each condition was composed by three runs of 5 min and randomisation of the order was applied. Full-size DOI: 10.7717/peerj.4492/fig-1 Figure 1 Paradigm scheme representing the two different conditions: EC (Condition 1) and EO (Condition 2). Each condition was composed by three runs of 5 min and randomisation of the order was applied. Full-size DOI: 10.7717/peerj.4492/fig-1 Figure 2 Timing scheme for each run. The subject performed a right-hand index isometric flexion on the mouse when they wished while providing sufficient time (>8 s) between each movement. Experimental design Full-size DOI: 10.7717/peerj.4492/fig-2 Before the experiment, the task was described and the subject was trained to execute it over 10 min. During the training period the subject learned to estimate 10 s in the two given conditions and for the open-eyes condition the subject had to perform the task while they fixate their gaze on a target placed at eye-level to avoid random eye movements (Fig. 2). The subjects were instructed to avoid swallowing or any other movements while performing the task, particularly all eye-movement. Figure 2 Timing scheme for each run. The subject performed a right-hand index isometric flexion on the mouse when they wished while providing sufficient time (>8 s) between each movement. Full-size DOI: 10.7717/peerj.4492/fig-2 Figure 2 Timing scheme for each run. The subject performed a right-hand index isometric flexion on the mouse when they wished while providing sufficient time (>8 s) between each movement. Full-size DOI: 10.7717/peerj.4492/fig-2 Before the experiment, the task was described and the subject was trained to execute it over 10 min. During the training period the subject learned to estimate 10 s in the two given conditions and for the open-eyes condition the subject had to perform the task while they fixate their gaze on a target placed at eye-level to avoid random eye movements (Fig. 2). The subjects were instructed to avoid swallowing or any other movements while performing the task, particularly all eye-movement. 4/18 Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 4/18 Figure 3 Electrode positions localised around the primary motor cortex, the motor cortex, the so- matosensory cortex and the occipital cortex in accordance with the international 10–20 system. Full-size DOI: 10.7717/peerj.4492/fig-3 Figure 3 Electrode positions localised around the primary motor cortex, the motor cortex, the so- matosensory cortex and the occipital cortex in accordance with the international 10–20 system. Full-size DOI: 10.7717/peerj.4492/fig-3 Figure 3 Electrode positions localised around the primary motor cortex, the motor cortex, the so- matosensory cortex and the occipital cortex in accordance with the international 10–20 system. Full-size DOI: 10.7717/peerj.4492/fig-3 Physiological recordings A custom-written scenario for the open source software OpenViBE (Renard et al., 2010) was designed to automate the recording of EEG signals and was triggered during the task. EEG signals were recorded with a BiosemiTM Active Two 32-channel EEG system at 2,048 Hz. In the BiosemiTM system the ground electrodes used were two separate electrodes: Common Mode Sense (CMS) active electrode and Driven Right Leg (DRL) passive electrode. The EEG was recorded from 32 sites in accordance with the international 10–20 system: FC5, FC3, FC1, FCz, FC2, FC4, FC6, C5, C3, C1, Cz, C2, C4, C6, CP5, CP3, CP1, CPz, CP2, CP4, CP6, P3, P1, Pz, P2, P4, PO3, POz, PO4, O1, Oz, O2 (Fig. 3). These sites are localised around the primary motor cortex, the motor cortex, the somatosensory cortex and the occipital cortex, which allowed us to observe the physiological changes due to the task for the open eyes condition. The setup of the EEG channels was the same in both EC and EO conditions. An external electromyogram (EMG) electrode was added in order to measure the extensor indicis muscle activity. For some subjects the measured EMG activity was less reliable than the trigger obtained by mouse click, which is why we used this trigger for the analysis. Impedance was kept below 10k for all electrodes to ensure that the background noise in the acquired signal was low. No additional filtering was used during the recording. Spectrum The power spectrum was computed with EEGLAB (Fig. 4A) and showed the signal power distribution along a range of frequencies spanning (8–35 Hz). Topographies Brain topography allowed us to display the possible changes over different electrodes on the scalp in order to localise which part of the brain was involved when the subject performed the requested task. Two bands of frequencies were used to compute ERSPs: alpha/mu (10–13 Hz, Fig. 5A) and beta (15–30 Hz, Fig. 5B) for EC and EO conditions. Time-frequency analysis To analyse the differences between both conditions, we performed an event-related spectral perturbation (ERSP) analysis between 8–35 Hz with a resolution of 0.5 Hz at 13.68 ms intervals. We used a 256 point sliding fast Fourier transform (FFT) window with a padratio of 4 and we computed the mean ERSP 1.5 s before the task to 3.5 s after the task. ERSP allows to visualise event-related changes in the average power spectrum relative to a baseline (2 s) interval taken 2 s before each trial (Brunner, Delorme & Makeig, 2013). Time-frequency analysis is very useful to know precisely which frequency band will be used to analyse the results. As we can see in Fig. 4B, mu rhythm (10–13 Hz) and beta band (15–30 Hz) are modulated strongly, therefore, we used these frequency bands for plots and other figures such as the time frequency representation or the topographic maps of the brain. Signal pre-processing All offline analyses were performed using the EEGLAB toolbox (Delorme & Makeig, 2004) and Matlab2016a (The MathWorks Inc. Natick, MA, USA). The data were processed in General Data Format (GDF). First, raw EEG data were filtered, transformed, and Laplacian processing was applied to minimise the noisy EEG signals (Perrin et al., 1989). Then, EEG 5/18 Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 5/18 signals were resampled at 256 Hz and divided into 6 s epochs corresponding to 2 s before and 4 s after the mouse click for each run. Trials contaminated by excessive noise or movement artefacts were removed. Baseline was defined 2 s before each trial, therefore a specific baseline was chosen for the two conditions. Finally all runs were merged for a given condition for each subject. In this article, we chose the C3 electrode because it corresponds to the motor area of the right hand control. These electrodes are commonly analyzed in motor control studies since they are located above cortical regions known to be involved in movement preparation and execution Spectrum Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 ERD/ERS quantiﬁcation q We computed the ERD/ERS% using the ‘‘band power method’’ (Pfurtscheller & Lopes da Silva, 1999). ERD/ERS% = x2 −BL2 BL2 ×100, ERD/ERS% = x2 −BL2 BL2 ×100, (1) (1) where x2 is the average of the squared signal smoothed using a 250-millisecond sliding window with a 100 ms shifting step, BL2 is the mean of a baseline segment taken at the beginning of the corresponding trial, and ERD/ERS% is the percentage of the oscillatory power estimated for each step of the sliding window. A positive ERD/ERS% indicates a synchronisation whereas a negative ERD/ERS% indicates a desynchronisation. This percentage was computed separately for all EEG channels. The EEG signal was filtered in the mu rhythm (10–13 Hz) and in the beta band (15–30 Hz) for all subjects using a 4th-order Butterworth band-pass filter. Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 6/18 Figure 4 (A) Spectrum analysis grand average (n = 15) difference between Condition 1 (EC) and Con- dition 2 (EO) for electrode C3. (B) Time-frequency grand average analysis (ERSP) for Condition 1 (EC) and for Condition 2 (EO) for electrode C3. A red colour corresponds to strong modulations in the band of interest. Significant difference (p < 0.01) are shown in the final part of the figure. Full-size DOI: 10.7717/peerj.4492/fig-4 Figure 4 (A) Spectrum analysis grand average (n = 15) difference between Condition 1 (EC) and Con- dition 2 (EO) for electrode C3. (B) Time-frequency grand average analysis (ERSP) for Condition 1 (EC) and for Condition 2 (EO) for electrode C3. A red colour corresponds to strong modulations in the band of interest. Significant difference (p < 0.01) are shown in the final part of the figure. Figure 4 (A) Spectrum analysis grand average (n = 15) difference between Condition 1 (EC) and Con- dition 2 (EO) for electrode C3. (B) Time-frequency grand average analysis (ERSP) for Condition 1 (EC) and for Condition 2 (EO) for electrode C3. A red colour corresponds to strong modulations in the band of interest. Significant difference (p < 0.01) are shown in the final part of the figure. Full-size DOI: 10.7717/peerj.4492/fig-4 ERD and ERS are difficult to observe from the raw EEG signal. Indeed, an EEG signal expresses the combination of activities from many neuronal sources. We used the averaging technique to represent the modulation of power of the mu and beta rhythms during both conditions (Fig. ERD/ERS quantiﬁcation 6) since it is considered one of the most effective and accurate techniques used to extract events (Quiroga & Garcia, 2003; Pfurtscheller, 2003). Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 Statistical analysis (2018), PeerJ, DOI 10.7717/peerj.4492 8/18 Figure 6 Grand average (n = 15) ERD/ERS% curves in the mu (A) and the beta (B) bands for EO (in blue) and EC (in orange) conditions for electrode C3. The green bar at 0 s corresponds to the voluntary movement performed. Full-size DOI: 10.7717/peerj.4492/fig-6 Figure 7 Individual and average results of number of voluntary movements in EO (in blue) and EC (in orange) conditions. Full-size DOI: 10.7717/peerj.4492/fig-7 Figure 6 Grand average (n = 15) ERD/ERS% curves in the mu (A) and the beta (B) bands for EO (in blue) and EC (in orange) conditions for electrode C3. The green bar at 0 s corresponds to the voluntary movement performed. Full-size DOI: 10.7717/peerj.4492/fig-6 Figure 6 Grand average (n = 15) ERD/ERS% curves in the mu (A) and the beta (B) bands for EO (in blue) and EC (in orange) conditions for electrode C3. The green bar at 0 s corresponds to the voluntary movement performed. Full-size DOI: 10.7717/peerj.4492/fig-6 Figure 7 Individual and average results of number of voluntary movements in EO (in blue) and EC (in orange) conditions. Full-size DOI: 10.7717/peerj.4492/fig-7 Figure 7 Individual and average results of number of voluntary movements in EO (in blue) and EC (in orange) conditions. Full-size DOI: 10.7717/peerj.4492/fig-7 Figure 7 Individual and average results of number of voluntary movements in EO (in blue) and EC (in orange) conditions. Full-size DOI: 10.7717/peerj.4492/fig-7 Statistical analysis We chose to apply a paired t-test (two-sided) to show the significant difference about number of clicks performed shown on Fig. 7 (p-value < 0.01). The same t-test was applied on three chosen parts of the EEG signal, pre-movement phase (pre-M) (−2,000 ms; 0 ms), movement phase (M) (0 ms; 500 ms), and post-movement phase (post-M) (500 ms; 7/18 7/18 Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 Figure 5 Topographic map of ERD/ERS% (grand average, n = 15) in the Alpha/mu band (A, 10–13 Hz) and in the beta band (B, 15–30 Hz) during two conditions: eyes-closed (EC) and eyes-open (EO). A red colour corresponds to a strong ERS and a blue one to a strong ERD. A black line indicates when the iso- metric flexion started. This figure is an extrapolation through 32 electrodes. Red electrodes indicate a sig- nificant difference (p < 0.01). Full-size DOI: 10.7717/peerj.4492/fig-5 Figure 5 Topographic map of ERD/ERS% (grand average, n = 15) in the Alpha/mu band (A, 10–13 Hz) and in the beta band (B, 15–30 Hz) during two conditions: eyes-closed (EC) and eyes-open (EO). A red colour corresponds to a strong ERS and a blue one to a strong ERD. A black line indicates when the iso- metric flexion started. This figure is an extrapolation through 32 electrodes. Red electrodes indicate a sig- nificant difference (p < 0.01). Full-size DOI: 10.7717/peerj.4492/fig-5 2,000 ms) shown on Fig. 8 (p-value < 0.01). These parts were selected specifically by using the literature and our results for EO and EC conditions (Pfurtscheller & Solis-Escalante, 2009; Pfurtscheller, 2003; Avanzini et al., 2012; Kilavik et al., 2013). A surrogate permutation test (p < 0.01; 2,000 permutations) from the EEGLAB toolbox was used to validate differences in terms of time-frequency and localisation of this ERSPs (Figs. 4 and 5). In addition to this treatment, we applied a false discovery rate (FDR) correction test in order to clarify how the false discovery rate was controlled for multiple comparisons. This test consisted of repetitively shuffling values between conditions and recomputing the measure of interest using the shuffled data. This test performed the drawing of data samples without replacement and is thought to be appropriate to show the difference between EC and EO conditions (Manly, 2006). Rimbert et al. Spectrum and time frequency Spectrum and time frequency With the EC condition, significantly higher power values (, p < 0.01) of the spectrum were measured on C3 for a real movement in the upper mu rhythm (10–13 Hz) and low beta band (15–25 Hz) (Fig. 4A). In Fig. 4B, before the movement, a decrease of ERSP (in blue) appeared in the mu rhythm, which started 400 ms before the execution of the motor task. From 0 to 500 ms, this desynchronisation remained present in the mu and beta frequency bands but was Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 9/18 Figure 8 EEG signal power (grand average, n = 15) in the alpha band (A and B, 7–13 Hz) and in the beta band (C and D, 15–30 Hz) for both conditions: EC (in orange) and EO (in blue) for the electrode C3 in the three phases of voluntary movement: pre-movement (pre-M), movement (M) and post-movement (post-M). * corresponds to a p-valu e < 0.001. Full-size DOI: 10.7717/peerj.4492/fig-8 Figure 8 EEG signal power (grand average, n = 15) in the alpha band (A and B, 7–13 Hz) and in the beta band (C and D, 15–30 Hz) for both conditions: EC (in orange) and EO (in blue) for the electrode C3 in the three phases of voluntary movement: pre-movement (pre-M), movement (M) and post-movement (post-M). *** corresponds to a p-valu e < 0.001. Full-size DOI: 10.7717/peerj.4492/fig-8 stronger in the mu rhythm. In this time interval, Fig. 4B shows on the right side a significant difference with a p-value < 0.01 for the EO condition vs the EC condition comparison on the electrode C3. In the beta frequency band, at 1,000 ms, both conditions showed a similar increase (in red) but in the EO condition a residual rebound is maintained in the mu frequency bands. This rebound was absent in the EC condition. These differences seem not to be significant at p-value < 0.01 (Fig. 4B; right-side). Topography The time-frequency analysis of ERSPs (Fig. 4B) showed powerful differences in the upper mu band (10–13 Hz) between EO and EC conditions. This allowed us to select two specific frequency bands for the topographic figures. For the higher mu band (10–13 Hz), before the movement, there was no detectable difference between the EC and EO condition (Fig. 5A). Interestingly, 500 ms after the Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 10/18 voluntary movement, a significant difference of ERSP was detected on several bilateral electrodes, specially close to the motor cortex and the somatosensory motor cortex. Moreover, a stronger desynchronisation can be observed in the EO condition located in the motor cortex on the contralateral side with these changes also appearing in the occipital area. Conversely, a synchronisation was observed in the EC condition located on the somatosensory contralateral cortex. At 1,000 ms, a transient generalised synchronisation of the motor cortex was observed bilaterally and centrally for the closed eyes condition. At this time, for the EO condition, the synchronisation was located only on the contralateral side of the motor cortex; therefore, it was significantly different from the EC condition on the central and ipsilateral side. From 1,500 ms to 4,000 ms no significant changes were observed, but the synchronisation appeared to be stronger in the EO condition on C3 which represents, contralaterally, the area of the hand on the motor cortex. No significant difference was observed in the beta frequency band (15–30 Hz) between both conditions (Fig. 5B) and confirmed results showed in Fig. 4B. A contralateral synchronisation first occurred and it spread out over the central area near by the C3 and C5 electrodes. This synchronisation started around 500 ms and maintained itself until 1,500 ms and then slowly disappeared while still remaining very visible at 4,000 ms. ERD and ERS modulations ERD and ERS modulations were computed in the mu band (7–13 Hz) and the beta band (15–30 Hz) for all subjects. Figure 6 shows the grand average of ERD/ERS% curves over 15 subjects on electrode C3. In the mu band, the desynchronisation described in the literature appeared one second before the voluntary movement (i.e., isometric flexion). An interesting synchronisation started at 500 ms to reach a maximum of 50% for the EC condition or 60% for the EO condition (Fig. 6A). A total of 500 ms after the self-paced movement in EO condition, there was a desynchronisation which was absent for EC condition. This difference was observed already on the spectrogram (Fig. 4B) and topographic map (Fig. 5A). In the beta band, there was a desynchronisation which appeared one second before the voluntary movement and remained present 500 ms afterwards (Fig. 6B). One second after the voluntary movement, the power in the beta band increased by around 60%, reached its maximum and returned to the baseline after 4 s. The evolution from ERD to ERS was rapid (less than one second) and should be linked to the type of movement (isometric flexion of right index). For the EC condition the synchronisation seems to be slightly higher than the opposite condition but is not statistically significant. Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 The number of movements performed decreases with eyes-closed The number of movements performed decreases with eyes-closed While all subjects received the same instructions, were trained for both conditions (EO and EC), and performed all runs in randomised order, a significant behavioural difference was observed. Indeed, the numbers of isometric flexions performed in both conditions were different (Fig. 7, p-value < 0.01). With some exceptions (S5 and S15), all subjects performed more voluntary movements when they had to keep their eyes open. In average, 66.8 clicks were executed for the EO condition versus 61.7 for the EC condition. Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 11/18 DISCUSSION Diﬀerences observed between eyes-closed and eyes-open condition during the voluntary movement Variations of EEG power during pre-movement, movement and post-movement phases In order to better understand the influence of the EC condition on the motor cortex, we computed EEG power in the mu band (7–13 Hz) and beta band (15–30 Hz) for the electrode C3 during three phases: Pre-movement (−2,000; 0 ms), Movement (0; 500 ms) and Post-Movement (500; 2,000 ms). Figure 8A shows that, for the EO condition, there was a significant decrease of power (p < 0.01) in the mu band between pre-M and M phases. There was an increase of power between M and post-M phases which was also significant compared to pre-M phase. Figure 8B shows for the EC condition that there was also a significant decrease of power between pre-M and M phases but the power returned back to the pre-M phase level and did not increase significantly under this condition. The same pattern was reflected significantly (p-value < 0.01) in Figs. 8C–8D in the beta band, namely a decrease between pre-M and M phases and an increase in post-M phase but here, the decrease of power was more obvious for the EC condition. Finally, it is interesting to note that the power was higher for the EC condition in both frequency bands, so the decrease of power was more obvious in this condition. Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 Diﬀerences observed between eyes-closed and eyes-open condition during the voluntary movement In this study, the differences observed between EC and EO conditions for a voluntary movement task prompted the question of the sources of these differences. Two possibly complementary hypotheses explain the differences obtained in terms of ERD and ERS (Fig. 4B) and behaviour (Fig. 7): • The EC condition creates a strong modulation of the mu rhythm (7–13 Hz) and disturbs • The EC condition creates a strong modulation of the mu rhythm (7–13 Hz) and disturbs the EEG signal over the whole cortex; the EEG signal over the whole cortex; • The EC condition involves a behavioral change during a voluntary movement and modulates the activation/deactivation of the motor cortex. The first hypothesis suggests that results described in this study were a consequence of global disruption generated by EC condition in the EEG signal. Moreover, the major difference observed was the large ERD in the mu rhythm for the EO condition (Fig. 4B). Several studies (Barry et al., 2007; Barry et al., 2009; Westphal et al., 1993; Legewie, Simonova & Creutzfeldt, 1969) have described differences occurring only in the alpha band and support this hypothesis. The second hypothesis can be complementary to the first hypothesis, suggests that when a voluntary movement is performed, the preparation, the execution and the feedback phases linked to this movement can change according to EC and EO conditions. Our results showed that the number of voluntary movements performed is different for both conditions (Fig. 7). This shows that the internal representation of time is not similar between EC and EO conditions. This has been confirmed also by several studies showing that opening and closing eyes are fundamentally different behaviours (Marx et al., 2004; Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 12/18 Liang et al., 2014). More specifically, there is an ‘‘interoceptive’’ mental activity identified by imagination and multisensory activity during the EC condition (Miraglia et al., 2016; Marx et al., 2004). The post-testimonies of subjects support this idea. Therefore, in EC condition, preparation and feedback phases could be different and that will have consequences on the activation/deactivation of the motor cortex. In the literature, modulations of both mu and beta bands are involved in a motor task (Kilavik et al., 2013; Pfurtscheller & Lopes da Silva, 1999; Avanzini et al., 2012; Neuper & Pfurtscheller, 2001). Although only changes in the mu band were observed (Fig. 4), and support the first hypothesis, other results (Fig. ERD and ERS modulations for EO and EC conditions RD and ERS modulations for EO and EC conditions The results are coherent with previous studies describing ERD/ERS% modulations in the mu and the beta bands during motor actions (Pfurtscheller & Aranibar, 1979; Kilavik et al., 2013; Avanzini et al., 2012; Shibasaki et al., 1993). The low power of the ERD (Fig. 6) can be explained because subjects were instructed to focused more on the precision rather than the speed of the movement (Pastötter, Berchtold & Bäuml, 2012). However, although subjects were consciously making an effort to carry out a voluntary movement, we must consider that clicking on a mouse could be somewhat of a reflex due to habit. This could have an impact on the low ERD amplitude. The desynchronisation in the mu rhythm (Fig. 6) started 2 s before the voluntary movement and was bilateral and not predominant on the ipsilateral position (Pfurtscheller & Aranibar, 1979). This result suggests a bilateral activation of the motor cortex during an unilateral movement (Formaggio et al., 2013). Interestingly, the stronger desynchronization was found between 10–13 Hz and corresponds to the mu rhythm (Pfurtscheller, 2001). This shows that this results are due from the combination of a change in the alpha band (over the occipital region) under to EC condition and a change in the mu (over the motor cortex) due to the movement performed in EC condition. No major differences were observed in the beta band between EO and EC conditions. This result confirms that the beta band is not modulated, in term of ERDs and ERSs, depending on the experimental conditions (Kilavik et al., 2013). Diﬀerences observed between eyes-closed and eyes-open condition during the voluntary movement 8) showed clearly differences in term of EEG power for movement phases (Pre-M, M and Post-M) between both conditions. These results suggest that the mechanism on the motor cortex is different between the two conditions, particularly in EC condition because there was no synchronisation in the mu rhythm but a synchronisation in the beta band (Fig. 8). This hypothesis is supported by a recent paper published by Cambieri et al. (2017) showing that the excitability of the motor cortex is the same for EC and EO. Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 Competing Interests Competing Interests The authors declare there are no competing interests. The authors declare there are no competing interests. Author Contributions • Sébastien Rimbert and Rahaf Al-Chwa conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. • Manuel Zaepffel and Laurent Bougrain analyzed the data, contributed reagents/mate- rials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. Human Ethics The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): The ethical committee of Inria (COERLE) granted approval to carry out the study within its facilities (approval number: 2016-011/01). The ethical committee of Inria (COERLE) granted approval to carry out the study within its facilities (approval number: 2016-011/01). Data Availability Funding The authors received no funding for this work. The authors received no funding for this work. Role in the BCI domain The results obtained could be helpful for the Brain–Computer Interface (BCI) domain. Next step in this direction would be to verify that modulations observed for the EC condition are confirmed during an attempted movement or a motor imagery. Similarly, this finding could be used for motor rehabilitation after a stroke with a BCI (Cincotti et al., 2012). These specific BCIs normally use visual feedback to inform the user Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 about system’s decisions after a motor imagery task. However, performing the motor task with closed-eyes with a tactile (Jeunet et al., 2015) or an auditory feedback (Nijboer et al., 2008) could be considered to improve concentration and attention during a motor task (Egeth & Yantis, 1997; Kim & Cruz, 2011). In addition, the EC condition would solve the problems of gaze fixation and artifacts caused by the EO condition. In order to progress towards this hypothesis, it would be necessary to carry out further experiments and establish a link between learning, cerebral plasticity and the EC condition. CONCLUSION In this article, we compared modulations in the EEG signal over the motor cortex correlated with a voluntary movement for two conditions: eyes-closed and eyes-open. We showed that a greater desynchronisation appeared 500 ms during the voluntary movement in the mu rhythm (10–13 Hz) for the EC condition. We found also that there was no significant difference in the beta band (15–30 Hz). Furthermore, the number of voluntary movements was significantly different between the two conditions and showed that the closed eye condition influenced behaviour of the subjects. This study gives us greater insight into the motor cortex and could also be useful in the BCI domain. Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 Supplemental Information Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.4492#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.4492#supplemental-information. Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 Data Availability The following information was supplied regarding data availability: The following information was supplied regarding data availability: The raw data for Figs. 7 and 8 have been provided as Supplemental Information 1. The raw data for Figs. 7 and 8 have been provided as Supplemental Information 1. Rimbert et al. (2018), PeerJ, DOI 10.7717/peerj.4492 14/18 REFERENCES Avanzini P, Fabbri-Destro M, Dalla Volta R, Daprati E, Rizzolatti G, Cantalupo G. 2012. The dynamics of sensorimotor cortical oscillations during the observation of hand movements: an EEG study. PLOS ONE 7(5):e37534 DOI 10.1371/journal.pone.0037534. Bai O, Mari Z, Vorbach S, Hallett M. 2005. 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https://openalex.org/W3191599602	http://izvestia-soigsi.ru/izvestia/2017/24/g-i-tsibirov.pdf	Russian	null	THE PROBLEMS OF THE HISTORY OF OSSETIA AND THE PEOPLES OF THE NORTH CAUCASUS IN THE A.K. DZHANAEV’S WORKS	Izvestiâ SOIGSI	2,017	cc-by-sa	7,056	СОИГСИ СОИГСИ История. Этнология Keywords: history of Ossetia, A. K. Dzhanaev, feudal landowning, revolutionary movement in the North Caucasus, Ossetian historiography, source studying. ПРОБЛЕМЫ ИСТОРИИ ОСЕТИИ И НАРОДОВ СЕВЕРНОГО КАВКАЗА В ТРУДАХ А. К. ДЖАНАЕВА Г. И. Цибиров В статье прослеживается жизненный и творческий путь Акима Казбековича Джана- ева — одного из видных представителей поколения осетинской научной интеллигенции, чья творческая биография складывалась в период глубоких общественных трансформа- ций первой половины XX в. А. К. Джанаев рано проявил интерес к гуманитарному знанию, который со временем превратился в осознанное желание стать профессиональным исто- риком. Этому способствовала и общественная атмосфера 1920-х — 1930-х гг., когда об- разованная часть общества активно вовлекалась в обсуждение вопросов идеологического обоснования строительства социализма в СССР. Джанаев много лет преподавал в разных учебных заведениях. Но его главным призванием стало занятие наукой. В статье отмеча- ется большой вклад ученого в развитие исторического осетиноведения и кавказоведения. Автор характеризует основные направления научно-исследовательской деятельности Джанаева. Обширность его научных интересов подчеркивает перечень тем, к которым исследователь обращался на протяжении своей долгой жизни в науке. Среди них вопросы феодального землевладения в Дигории, развития революционного движения и социалисти- ческого строительства на Северном Кавказе, проблемы осетинской историографии, ар- хеографические и источниковедческие изыскания и т.д. Представлен библиографический список трудов А. К. Джанаева, в котором значится 84 названия, включающих монографии, статьи, а также сборники научных статей и документов, в подготовке которых уча- ствовал исследователь. Ключевые слова: история Осетии, А. К. Джанаев, феодальное землевладение, револю- ционное движение на Северном Кавказе, осетинская историография, источниковедение. Аким Казбеко- вич переехал в Москву с намерением поступить на историко-философский факультет МГУ. На руках он имел ре- комендательное письмо от руководства Политехнического института с прось- бой о принятии его в студенты МГУ «в порядке перевода». Однако А. Джана- еву было отказано в зачислении с мо- тивировкой: «отсутствие свободных мест». В результате юноша поступил в Московский плановый институт на фа- культет планирования научно-исследо- вательской работы, который успешно окончил в 1934 г. ич Джанаев родился 8 марта 1909 г. в кре Нарского прихода Владикавказского округ ывавшая острое малоземелье в горах семья лась в селении Кадгарон. В 1918 г. ее пос б Аким Казбекович Джанаев родился 8 марта 1909 г. в крестьянской семье в горном селении Сындзысар Нарского прихода Владикавказского округа. ович, и семья осталась без кормильца. В го адгаронскую сельскую школу. Проучивши вий он вынужден был оставить учебу ям и большое трудолюбие подростка оказа которое время он продолжил учебу. В 192 ени, Аким Джанаев поступил в Северо-О етыре года он уехал в г. Закатала Азербай очистительный завод. В свободное от я к поступлению в вуз. Казбекович стал студентом Казанского по специальность не приносила удовлетворе к общественным и гуманитарным наукам агогическом техникуме во Владикавказе ф В 1911 г. испытывавшая острое ма- лоземелье в горах семья Джанаевых пе- реселилась на равнину и обосновалась в селении Кадгарон. В 1918 г. ее постиг- ло большое горе — умер отец, Казбек Бесланович, и семья осталась без кор- мильца. В год смерти отца девятилет- ний Аким поступил в Кадгаронскую сельскую школу. Проучившись три года, из-за тяжелых материальных ус- ловий он вынужден был оставить учебу и наняться общественным пастухом в селении. ния. В юноше все больше который проявился еще в ɗтому способствовала и чение научной, творческой лософии, идеологического намерением поступить на комендательное письмо от ии его в студенты МГɍ «в числении с мотивировкой: М й й Педагогическую деятельность А. К. Джанаев начал еще будучи студен- том. Он преподавал политическую эко- номию в Институте золота, в Мясном институте. По окончании Московского планового института Аким Казбекович некоторое время работал в плановой комиссии ЦИК Юго-Осетинской авто- номной области. сфера в стране в целом, и активное вовле бсуждение вопросов политэкономии, фи строительства социализма в СССР. г. Аким Казбекович переехал в Москву с кий факультет МГɍ. На руках он имел ре ехнического института с просьбой о приня Однако А. Джанаеву было отказано в за ных мест». В результате юноша поступи ультет планирования научно-исследовате 1934 г. деятельность А.К. Ключевые слова: история Осетии, А. К. Джанаев, феодальное землевладение, револю- ционное движение на Северном Кавказе, осетинская историография, источниковедение. Ключевые слова: история Осетии, А. К. Джанаев, феодальное землевладение, револю- ционное движение на Северном Кавказе, осетинская историография, источниковедение. The article traces the course of life and creativity of Akim Kazbekovich Dzhanaev — one of the prominent representatives of the generation of Ossetian scientific intelligentsia, whose creative biography was being formed during the period of profound social transformations in the first half of the XX c. A. K. Dzhanaev developed interest in the Humanities at a very early age, and this interest eventually turned into conscious desire to become a professional historian. The social atmosphere of 1920-1930 facilitated this, when the educated layer of the society was actively engaged in discussing issues of ideological foundation for the socialism construction in USSR. Dzhanaev taught for many years in different educational organizations. But scientific research was his main calling. The article marks the scientist’s important contribution into development of the Ossetian studies and Caucasology. The author characterized the main directions of Dzhanaev’s research activities. The list of themes the researcher treated during his long life in science outlines breadth of his scientific interests. Among them there are problems of feudal landowning in Digoria, development of the revolutionary movement and socialist construction in the North Caucasus, as well as the questions of the Ossetian historiography, archeographical and source studying, etc. The bibliographical list of the Dzhanaev’s works is presented. It contains 84 titles including the monographs, articles, as well the collected scientific articles and documents, in elaborating which the researcher participated. Keywords: history of Ossetia, A. K. Dzhanaev, feudal landowning, revolutionary movement in the North Caucasus, Ossetian historiography, source studying. ИЗВЕСТИЯ СОИГСИ 24 (63) 2017 70 СОИГСИ История. Этнология В 1930 г. Аким Казбекович стал сту- дентом Казанского политехнического института. Однако избранная специ- альность не приносила удовлетворе- ния. В юноше все больше укреплялся интерес к общественным и гуманитар- ным наукам, который проявился еще в годы учебы в педагогическом техни- куме во Владикавказе. Этому способ- ствовала и общественная атмосфера в стране в целом, и активное вовлечение научной, творческой интеллигенции в обсуждение вопросов политэкономии, философии, идеологического обосно- вания теории строительства социализ- ма в СССР. стьянской семье в горном а. Джанаевых переселилась на игло большое горе – умер смерти отца девятилетний сь три года, из-за тяжелых и наняться общественным ись сильнее материальных 5 г. окончив Кадгаронскую сетинский педагогический жанской ССР и устроился аботы время штудировал литехнического института. ния В юноше все больше В декабре 1930 г. Ключевые слова: история Осетии, А. К. Джанаев, феодальное землевладение, револю- ционное движение на Северном Кавказе, осетинская историография, источниковедение. Джанаев начал ещ ческую экономию в Институте золота, кого планового института Аким Казбекови и ɐИК ɘго-Осетинской автономной облас Джанаев был призван на действительну Но тяга к знаниям и большое тру- долюбие подростка оказались сильнее материальных невзгод, и через некото- рое время он продолжил учебу. В 1925 г. окончив Кадгаронскую школу первой ступени, Аким Джанаев поступил в Северо-Осетинский педагогический техникум. Спустя четыре года он уехал в г. Закатала Азербайджанской ССР и устроился работать на Орехоочисти- тельный завод. В свободное от работы время штудировал учебники, готовил- ся к поступлению в вуз. л в Московский плановый льской работы, который будучи студентом. Он в Мясном институте. По ч некоторое время работал и. Осенью 1934 г. Джанаев был при- зван на действительную службу в РККА. После демобилизации, в де- кабре 1935 г. ему предложили занять должность экономиста-исследователя в Грозненском нефтяном научно-ис- ИЗВЕСТИЯ СОИГСИ 24 (63) 2017 71 у у должность экономиста- ИЗВЕСТИЯ СОИГСИ 24 (63) 2017 71 у у должность экономиста- 71 ИЗВЕСТИЯ СОИГСИ 24 (63) 2017 должность экономиста- СОИГСИ История. Этнология следовательском институте. Одновре- менно по совместительству он препо- давал политэкономию в Грозненском плановом техникуме. 1943 и до конца 1944 г. — Аким Казбе- кович работал заместителем начальни- ка Управления по делам искусств при Совете Народных Комиссаров Севе- ро-Осетинской АССР. К этому времени Джанаев окон- чательно утвердился в желании стать профессиональным историком. Осоз- нанное стремление заниматься наукой привело его в Северо-Кавказский (Гор- ский) научно-исследовательский ин- ститут. В сентябре 1936 г. он был зачис- лен в аспирантуру по специальности «История народов Северного Кавказа» с тем условием, что вместе с выполне- нием кандидатского минимума он будет сдавать вузовские курсы по историче- ским дисциплинам. В 1937 г. в связи ре- организацией Северо-Кавказского края Горский НИИ был упразднен, и Джа- наев был переведен в аспирантуру при Северо-Осетинском научно-исследова- тельском институте. После окончания аспирантуры в 1939 г. его оставили в институте в качестве ученого секретаря СОНИИ. Одновременно он был принят ассистентом на кафедру истории СССР Северо-Осетинского государственного пединститута. Джанаев возобновил прерванную войной работу над диссертацией по теме «Феодальное землевладение в Стыр Дигории». Успешная защита дис- сертации на соискание ученой степени кандидата исторических наук состоя- лась 12 января 1945 г. в Азербайджан- ском государственном университете. у р у р После реэвакуации Северо-Осетин- ского педагогического института Аким Казбекович вернулся к педагогической деятельности. В 1947 г. он был утвер- жден в звании доцента по кафедре исто- рии СССР. С 1952 по 1955 гг. Ключевые слова: история Осетии, А. К. Джанаев, феодальное землевладение, револю- ционное движение на Северном Кавказе, осетинская историография, источниковедение. являлся заведующим кафедрой истории СССР Северо-Осетинского госпединститута и одновременно исполнял обязанно- сти заместителя директора Учитель- ского института. Джанаев продолжал заниматься и научной деятельностью. Начиная с 1946 г. он работал по совме- стительству старшим научным сотруд- ником Северо-Осетинского научно-ис- следовательского института. В 1955 г. по решению бюро Северо-Осетинского обкома КПСС был переведен в Севе- ро-Осетинский НИИ на должность за- местителя директора по научной рабо- те. В этой должности он проработал до 1975 г. В последующее время вплоть до ухода на заслуженный отдых работал старшим научным сотрудником отдела истории СОНИИ. В мае 1941 г. Аким Казбекович как офицер запаса был призван на перепод- готовку в город Днепропетровск, где его и застала Великая Отечественная война. С самого начала войны Джана- ев находился в действующей армии. Участвовал в боях на Западном фрон- те, был командиром топографического взвода и начальником штаба артдиви- зиона 249-ой стрелковой дивизии, поз- же преобразованной в 16-ую гвардей- скую дивизию. Идейно-политическую закалку Джанаев получил в рядах ленинского комсомола, членом которого он состо- ял с 1924 по 1939 гг. В 1939 г. он всту- пил в ряды членов ВКП (б). За время пребывания в рядах комсомола и Ком- мунистической партии он принимал активное участие в общественной 3 апреля 1942 г. Джанаев был тяже- ло ранен в боях под городом Велиж, в связи с чем 13 августа того же года был временно снят с военного учета, а затем уволен в запас. После возвращения до- мой в течение полутора лет — с июня ИЗВЕСТИЯ СОИГСИ 24 (63) 2017 72 История. Этнология СОИГСИ жизни. Так, в 1928-1929 гг. являлся се- кретарем комсомольской организации Оспедтехникума, в 1933 г. избирал- ся членом вузовского бюро ВЛКСМ Московского планового института. В 1937 г. состоял членом Северо-Кавказ- ского краевого комитета профсоюзов высшей школы. Накануне и в период Великой Отечественной войны был членом бюро партийной организации Всесоюзных курсов комсостава артил- лерии, парторгом управления дивизи- она в действующей армии. Возглавлял партийную организацию Северо-Осе- тинского научно-исследовательского института, избирался в состав партбю- ро пединститута, депутатом Орджони- кидзевского городского Совета депу- татов трудящихся VI, VII, VIII созы- вов. Он был награжден орденом Крас- ной Звезды, медалями «За отвагу», «За победу над фашистской Германией», «За доблестный труд в Великой Оте- чественной Войне», «За трудовое от- личие», «К 100-летию со дня рождения Ленина». А. К. Джанаев скончался 26 июня 1990 г. Ключевые слова: история Осетии, А. К. Джанаев, феодальное землевладение, револю- ционное движение на Северном Кавказе, осетинская историография, источниковедение. Эксплуатация осетинско- го крестьянства местными и русскими помещиками и буржуазией, острота аграрного вопроса, тяжесть налогов и повинностей, гнет царизма были пред- посылками крестьянского движения в Осетии, причиной борьбы против самодержавия. Ученый отмечает, что в формировании революционных на- строений в Северной Осетии большую роль сыграли революционно-демокра- тические идеи Коста Хетагурова. И в поэзии, и в публицистических статьях Хетагуров давал глубокий анализ тяже- лого политического и экономического положения народов Кавказа под вла- стью царизма и феодалов, выступал с революционной страстью против само- державия, местных и русских помещи- ков и буржуазии. ХХ в. В Северной Осетии, также как и в других районах страны, в результате развития товарно-денежных отноше- ний шли активные процессы расслое- ния крестьянства, усиливалась эксплу- атация деревенской бедноты. «В насто- ящее время, — писал в 1899 гг. М. Ца- голов, — среди аульного населения наблюдается страшная хозяйственная дифференциация. С одной стороны, мы видим буржуазию, сельских «богатеев», имеющих иногда довольно солидные средства, с другой — аульную голытьбу, в лучшем случае, имеющих одну лоша- денку или пару еле-еле двигающих ноги быков» [1]. Эксплуатация осетинско- го крестьянства местными и русскими помещиками и буржуазией, острота аграрного вопроса, тяжесть налогов и повинностей, гнет царизма были пред- посылками крестьянского движения в Осетии, причиной борьбы против самодержавия. Ученый отмечает, что в формировании революционных на- строений в Северной Осетии большую роль сыграли революционно-демокра- тические идеи Коста Хетагурова. И в поэзии, и в публицистических статьях Хетагуров давал глубокий анализ тяже- лого политического и экономического положения народов Кавказа под вла- стью царизма и феодалов, выступал с революционной страстью против само- державия, местных и русских помещи- ков и буржуазии. и искровских организаций в России, в том числе Донкома и Кавказского со- юзного комитета РСДРП, способство- вала распространению марксистских идей на Кубани, на Тереке, в Ставропо- ле. Идейные, организационные прин- ципы «Искры» подготовили почву для зарождения социал-демократических групп и организаций на Северном Кав- казе. На протяжении многих лет научные интересы Джанаева были сосредото- чены на проблеме участия Северно- го Кавказа в революции 1905-1907 гг. Приступая к исследованию сложной и неразработанной проблемы, Аким Каз- бекович осознавал, что ее нельзя осве- тить без глубокого освоения и анализа относящихся к теме документов и мате- риалов. С этой целью он скрупулезно и тщательно формировал источниковую базу исследования, собирал архивные документы, материалы периодической печати и т.д. Работа была выполнена совместно с Асламбеком Ахметовичем Тедтоевым и издана в 1955 г. Ключевые слова: история Осетии, А. К. Джанаев, феодальное землевладение, револю- ционное движение на Северном Кавказе, осетинская историография, источниковедение. * * * Имя Акима Казбековича Джана- ева — видного осетинского ученого, представителя советской исторической школы, исследователя истории осетин- ского народа и народов Северного Кав- каза — широко известно как в нашей республике, так и за ее пределами. Его работы по истории феодализма в Осе- тии, истории рабочего класса Северной Осетии и другим проблемам истории народов Северного Кавказа получили признание научной общественности. «Восстановительный период в Север- ной Осетии. 1921-1925 гг.», «Борьба за Советскую власть в Северной Осетии», «Съезды народов Терека», «Революция 1905-1907 гг. на Тереке» в 2-х томах и др. Много внимания ученый уделял ис- следованию проблемы «Ленин и наро- ды Кавказа». Одному из аспектов этой обширной темы была посвящена ста- тья «Ленинская газета «Искра» о Доне и Северном Кавказе», опубликованная в «Известиях Северо-Осетинского на- учно-исследовательского института». В ней, в частности, прослеживались пути распространения ленинской газеты и ее влияние на формирование революци- онных настроений в крае. Постановка этих вопросов и их разработка имели непосредственное отношение к исто- рии зарождения и деятельности соци- ал-демократических групп и организа- ций на Тереке. При этом следует отме- тить, что проблема решалась в рамках целого региона, а не только на примере одной Северной Осетии. Ученый внес заметный вклад в раз- работку таких вопросов, как изучение трудов классиков марксизма-лениниз- ма в Осетии, революционных событий 1905-1907 гг. в Северной Осетии и на Тереке, особенностей развития фео- дализм в Северной Осетии, вопросов историографии Осетии, истории Се- верной Осетии в восстановительный период и др. Несомненным вкладом в развитие исторического осетиноведения стала публикация Джанаевым источников по различным проблемам истории Осе- тии. При его активном участии были изданы сборники документов: «Север- ная Осетия в революции 1905-1907 гг.», Приведя большое количество при- меров и фактов пропаганды материа- лов газеты «Искра» на Кавказе, автор утверждал, что деятельность «Искры» ИЗВЕСТИЯ СОИГСИ 24 (63) 2017 73 ГСИ 24 (63) 2017 73 73 ИЗВЕСТИЯ СОИГСИ 24 (63) 2017 СОИГСИ История. Этнология ХХ в. В Северной Осетии, также как и в других районах страны, в результате развития товарно-денежных отноше- ний шли активные процессы расслое- ния крестьянства, усиливалась эксплу- атация деревенской бедноты. «В насто- ящее время, — писал в 1899 гг. М. Ца- голов, — среди аульного населения наблюдается страшная хозяйственная дифференциация. С одной стороны, мы видим буржуазию, сельских «богатеев», имеющих иногда довольно солидные средства, с другой — аульную голытьбу, в лучшем случае, имеющих одну лоша- денку или пару еле-еле двигающих ноги быков» [1]. Ключевые слова: история Осетии, А. К. Джанаев, феодальное землевладение, револю- ционное движение на Северном Кавказе, осетинская историография, источниковедение. Основное содержание работ Джана- ева, посвященных революционным со- бытиям начала XX в., сводится к тому, чтобы как можно полнее показать, что в основе революции 1905-1907 гг. на Кавказе, в том числе и в Северной Осе- тии, несмотря на местную специфику, лежали те же причины, что и в России в целом. И революционная борьба ра- бочих и крестьян Северного Кавказа за свое социальное и национальное освобождение в эти годы являлась со- ставной частью первой русской буржу- азно-демократической революции. Подробно осветив ход революци- онной борьбы трудящихся масс Север- ной Осетии в революции 1905-1907 гг., Джанаев в заключении указывал, что революция потерпела поражение, но она явилась для рабочих и крестьян ве- ликой школой политической борьбы, генеральной репетицией Великой Ок- тябрьской социалистической револю- ции 1917 г. Рабочие и крестьяне Север- ной Осетии, «ведя в годы революции В работе «Из истории революции 1905-1907 гг. в Северной Осетии» автор на основе изучения внушительного мас- сива архивных источников дает харак- теристику социально-экономического развития Северной Осетии в начале 74 ИЗВЕСТИЯ СОИГСИ 24 (63) 2017 4 История. Этнология СОИГСИ Осетии. Она во многом дополнила ис- следование Б. В. Скитского «К вопросу о феодализме в Дигории». под руководством русского пролета- риата освободительную борьбу против царизма, еще больше сблизились с ве- ликим русским народом; они убедились в том, что осетины, как и другие наро- ды окраин царской России, могут быть освобождены от социального и коло- ниального гнета царизма только при условии совместной борьбы с русским пролетариатом под руководством пар- тии большевиков» [2, 121]. под руководством русского пролета- риата освободительную борьбу против царизма, еще больше сблизились с ве- ликим русским народом; они убедились в том, что осетины, как и другие наро- ды окраин царской России, могут быть освобождены от социального и коло- ниального гнета царизма только при условии совместной борьбы с русским пролетариатом под руководством пар- тии большевиков» [2, 121]. И в последующие годы проблема развития феодализма в Осетии не вы- падала из поля зрения ученого. Он вни- мательно следил за состоянием ее изу- чения в отечественной историографии. В 1964 г., принимая участие в рабо- те научного совета «Закономерности исторического развития общества и перехода от одной социально-эконо- мической формации к другой», создан- ного при Институте истории АН СССР, Аким Казбекович высказал весьма ин- тересные мысли, актуальность которых сохраняется и в наши дни. «Нашими историками, научными учреждениями, — указывал он, — проведена извест- ная работа по изучению общественного строя северокавказских народов, в том числе по разработке проблемы феода- лизма у горских народов. Ключевые слова: история Осетии, А. К. Джанаев, феодальное землевладение, револю- ционное движение на Северном Кавказе, осетинская историография, источниковедение. Выявлено и опубликовано много источников, на- писаны монографии по феодализму у адыгских народов, в Дагестане, в Осе- тии. Но имеется еще много нерешен- ных вопросов. В дальнейшем предстоит всестороннее изучение таких сложных вопросов, как генезис феодализма, как особенности горской экономики, на основе которой зародилось феодаль- ное хозяйство, влияние общественных отношений соседских феодальных об- ществ на классообразование, степень развития феодализма у них, народные движения горцев в период феодализма, вопрос об особенностях феодализма в горских обществах Северного Кавказа» [3, 220]. Ряд работ Джанаев посвятил отдель- ным эпизодам истории классовой борь- бы осетинского крестьянства накануне и в период первой русской революции. Из числа этих работ особый интерес представляет статья «Крестьянское восстание в с. Гизель», опубликованная в журнале «Мах дуг» («Наша эпоха»). ур у Одной из важнейших задач исто- рической науки является всестороннее изучение основных закономерностей развития человеческого общества. Сре- ди конкретных теоретических вопро- сов особое место занимает проблема перехода от одной социально-эконо- мической формации к другой, станов- ление формации, когда новый способ производства не стал еще господству- ющим и определяющим, а старый, ухо- дящий способ производства еще суще- ствует. В этой связи интересны работы Джанаева, посвященные разработке вопросов развития феодализма в Осе- тии. Свою научно-исследовательскую деятельность Аким Казбекович начал именно с изучения феодализма в Осе- тии, точнее, в ее западной части — в Дигории. Монографическое исследова- ние «Феодальное землепользование в Стыр-Дигории» было представлено им на соискание ученой степени кандидата исторических наук. Работа эта, выпол- ненная под руководством профессора Бориса Васильевича Скитского, стала крупным вкладом в изучение истории Большой интерес для осетиноведов представляет статья Акима Казбеко- вича «Осетинская советская истори- ография». Обращение ученого к этой проблеме было не случайным. Исто- риография — наука сложная и требует ИЗВЕСТИЯ СОИГСИ 24 (63) 2017 75 ИЗВЕСТИЯ СОИГСИ 24 (63) 2017 75 75 ИЗВЕСТИЯ СОИГСИ 24 (63) 2017 История. Этнология СОИГСИ СОИГСИ к себе пристального внимания. Чтобы дать объективную оценку деятельно- сти ученого-историка, исследователь должен быть подготовлен не менее того автора, научное наследие которо- го он изучает, представлять его науч- ную лабораторию и уметь в ней ориен- тироваться, проявлять интерес к тому кругу вопросов и проблем, которыми занимался исследователь. Только при этих условиях можно объективно оце- нить деятельность как отдельного ис- следователя, так и целого коллектива. В этой статье Джанаев много внимания уделил освещению деятельности Осе- тинского историко-филологического общества. Если внимательно просле- дить за исторической литературой, то можно увидеть, что деятельность это- го общества в разные периоды совет- ской истории оценивалась по-разно- му. Ключевые слова: история Осетии, А. К. Джанаев, феодальное землевладение, револю- ционное движение на Северном Кавказе, осетинская историография, источниковедение. Иной раз его деятельность просто игнорировалась. Все это происходило потому, что никто никогда не пытал- ся серьезно проанализировать работу Историко-филологического общества, хотя материалы этой научной органи- зации были общедоступны. русских. Уставом общества не делалось ограничений по национальным и дру- гим признакам… До окончательной победы Совет- ской власти в Осетии деятельность об- щества ограничивалась узким кругом вопросов культурно-просветитель- ского характера. Начиная с 1920 г., при поддержке местных органов власти, а также Главнауки Наркомпроса РСФСР, оно участвовало в культурном строи- тельстве, занималось вопросами осе- тинского языка, обычного права, архе- ологии, театра, музыкального фолькло- ра, организацией научно-популярных чтений… Наибольшее значение в деятельно- сти общества имело собирание мате- риалов осетинского народного творче- ства, которое оно считало «своей удар- ной задачей», собирание рукописного наследия К. Хетагурова, Т. Мамсурова и других писателей. Собирание текстов осетинского народного творчества, ру- кописей писателей, книжного фонда литературы о народах Кавказа явилось в известной мере подготовкой базы для научной работы по филологии и исто- рии Осетии в будущем… Как же решает и оценивает деятель- ность Осетинского историко-филоло- гического общества сам Джанаев? В своей статье он писал: «В первые годы Советской власти в Осетии, естествен- но, не было научных учреждений, кото- рые могли бы заниматься глубокой раз- работкой истории народа. Для изучения культуры, филологии, истории в начале 20-х гг. некоторую работу провело Осе- тинское историко-филологическое об- щество. Возникшее осенью 1919 г. во Владикавказе, это общество ставило своей целью, как говорилось в его уста- ве, изучение «памятников осетинской старины». Кроме филологов, истори- ков Северной Осетии в обществе было немало лиц из Южной Осетии, а также Разносторонний характер занятий Осетинского историко-филологиче- ского общества, первого у горцев Се- верного Кавказа, в условиях, когда в Осетии еще не было самостоятельных учреждений культуры, науки, искус- ства, естественно, не дал возможности определить ведущее направление сво- ей работы. Но общество искало пути организации изучения широкого кру- га вопросов культуры и науки Осетии и сыграло в этом деле положительную роль» [4, 125]. Таким образом, Джанаев рассматривает многогранную деятель- ность Осетинского историко-филоло- гического общества как подготовитель- ный этап для будущего развития науки 76 ИЗВЕСТИЯ СОИГСИ 24 (63) 2017 История. Этнология СОИГСИ периодической печати. Немалый вклад он внес и в изучение наследия великого Коста, видных революционных деяте- лей Осетии. и культуры Осетии. В фокусе внимания ученого находится весь процесс раз- вития исторической науки в Северной Осетии. Хотя работа эта и не претен- дует на исчерпывающую полноту, она служит хорошим помощником для тех, кто интересуется вопросами истории развития исторической науки в Осетии. 1. Цаголов Г. М. Заметки из горской жизни // Терские ведомости. 1899, 1 дека- бря. 2. Джанаев А. К. Из истории революции 1905-1907 гг. в Северной Осетии // Из- вестия СОНИИ. 1956. Т. 17. С. 9-120. 3. Джанаев А. К. О развитии феодализма у горских народов Северного Кавказа // Проблемы феодализма у народов СССР. М., 1969. С. 219-221. 4. Джанаев А. К. Осетинская советская историография // Известия СОНИИ. 1971. Т. 28. С. 121-140. 1. Цаголов Г. М. Заметки из горской жизни // Терские ведомости. 1899, 1 дека- бря. 4. Джанаев А. К. Осетинская советская историография // Известия СОНИИ. 1971. Т. 28. С. 121-140. 2. Джанаев А. К. Из истории революции 1905-1907 гг. в Северной Осетии // Из- вестия СОНИИ. 1956. Т. 17. С. 9-120. 3. Джанаев А. К. О развитии феодализма у горских народов Северного Кавказа / Проблемы феодализма у народов СССР. М., 1969. С. 219-221. бря. 2. Джанаев А. К. Из истории революции 1905-1907 гг. в Северной Осетии // Из- вестия СОНИИ. 1956. Т. 17. С. 9-120. 3. Джанаев А. К. О развитии феодализма у горских народов Северного Кавказа // Проблемы феодализма у народов СССР. М., 1969. С. 219-221. 4. Джанаев А. К. Осетинская советская историография // Известия СОНИИ. 1971 Т 28 С 121 140 Ключевые слова: история Осетии, А. К. Джанаев, феодальное землевладение, револю- ционное движение на Северном Кавказе, осетинская историография, источниковедение. Кропотливую работу провел А. К. Джанаев по выявлению и публика- ции архивных материалов, существен- но обогатив тем самым источниковую базу последующих исследований раз- личных аспектов истории Осетии в пе- риоды глубоких общественных транс- формаций. Аким Казбекович также много сде- лал для освещения вопросов истории восстановительного периода в Север- ной Осетии, истории рабочего класса, ИЗВЕСТИЯ СОИГСИ 24 (63) 2017 77 ИЗВЕСТИЯ СОИГСИ 24 (63) 2017 77 СОИГСИ История. Этнология Библиография работ А. К. Джанаева 1. Ной Буачидзе // Мах дуг. 1940. № 3. С.10-13 (в соавт. с А. А. Тедтоевым). 2. О книге Б. В. Скитского «Очерки по истории осетинского народа» // Социа- истическая Осетия. 1948, 16 января (в соавт. с М. С. Тотоевым). 2. О книге Б. В. Скитского «Очерки по истории осетинского народа» // Социа- листическая Осетия. 1948, 16 января (в соавт. с М. С. Тотоевым). 3. Об очерках истории осетинского народа Б. В. Скитского // Мах дуг. 1948. № 12. С. 41-46. (в соавт. с М. С. Тотоевым). 3. Об очерках истории осетинского народа Б. В. Скитского // Мах дуг. 1948. № 12. С. 41-46. (в соавт. с М. С. Тотоевым). 4. Феодальное землепользование в Стыр Дигории // Известия СОНИИ. Т. 15 Вып. 3. 100с. 4. Феодальное землепользование в Стыр Дигории // Известия СОНИИ. Т. 15. Вып. 3. 100с. 5. Е. Ц. Бритаев и революция 1905-1907 гг. // Мах дуг. 1949. № 5. С. 41-44. 5. Е. Ц. Бритаев и революция 1905-1907 гг. // Мах дуг. 1949. № 5. С. 41-44. 6. Северная Осетия в период столыпинской реакции // Ученые записки СОГПИ. 1949. Т. 18. С. 36-46. 6. Северная Осетия в период столыпинской реакции // Ученые записки СОГПИ. 1949. Т. 18. С. 36-46. 7. Большое явление в развитии осетинской культуры. О труде В. И. Абаева «Язык и фольклор» // Рæстдзинад. 1949, 13 декабря. (соавт. Б. В. Скитский) 7. Большое явление в развитии осетинской культуры. О труде В. И. Абаева «Язык и фольклор» // Рæстдзинад. 1949, 13 декабря. (соавт. Б. В. Скитский) 8. Почему победила Советская власть // Рæстдзинад. 18 октября 1949 г. №205. 8. Почему победила Советская власть // Рæстдзинад. 18 октября 1949 г. №205. б 9. О состоянии и задачах разработки истории Осетии // Социалистическая Осетия. 1952, 11 мая; Рæстдзинад. 1952, 17 мая; Молодой коммунист. 1952, 15 мая. 9. О состоянии и задачах разработки истории Осетии // Социалистическая Осетия. 1952, 11 мая; Рæстдзинад. 1952, 17 мая; Молодой коммунист. 1952, 15 мая. 10. Реакционные происки Турции на Северном Кавказе // Социалистическая Осетия. 1953, 6 июня. 10. Реакционные происки Турции на Северном Кавказе // Социалистическая Осетия. 1953, 6 июня. 11. Общность исторических судеб (К воссоединению Украины с Россией) // Молодой коммунист. 1954, 23 мая. 11. Общность исторических судеб (К воссоединению Украины с Россией) // Молодой коммунист. 1954, 23 мая. 12. Празднование 300-летия присоединения Украины к России // Мах дуг. 1954. № 5. С. 4-12. 12. Библиография работ А. К. Джанаева Крестьянское движение в Северной Осетии в революции 1905-1907 гг. // Социалистическая Осетия. 1965, 2 декабря. 30. Крестьянское движение в Северной Осетии в революции 1905-1907 гг. // Социалистическая Осетия. 1965, 2 декабря. 31. Восстановительный период в Северной Осетии. 1921-1925 гг.: Сборник до- кументов / Сост. А. К. Джанаев, Е. П. Кричина, Т. Купчая, Е. А. Кияницкая. Орджо- никидзе, 1965. 371 с. 31. Восстановительный период в Северной Осетии. 1921-1925 гг.: Сборник до- кументов / Сост. А. К. Джанаев, Е. П. Кричина, Т. Купчая, Е. А. Кияницкая. Орджо- никидзе, 1965. 371 с. 32. Рецензия на кн.: Г. Гойгова. Народы Чечено-Ингушетии в борьбе против Де- никина // История СССР. 1965. № 5. С. 147-148. 32. Рецензия на кн.: Г. Гойгова. Народы Чечено-Ингушетии в борьбе против Де- никина // История СССР. 1965. № 5. С. 147-148. 33. Б. В. Скитский. 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https://openalex.org/W4396660726	https://link.springer.com/content/pdf/10.1007/s40279-024-02026-z.pdf	English	null	Influence of Preterm Birth and Low Birthweight on Physical Fitness: A Systematic Review, Meta-Analysis, and Meta-Regression	Sports medicine	2,024	cc-by	9,675	Influence of Preterm Birth and Low Birthweight on Physical Fitness: A Systematic Review, Meta‑Analysis, and Meta‑Regression Accepted: 25 March 2024 / Published online: 6 May 2024 © The Author(s) 2024 * Óscar Martínez‑de‑Quel odequel@ucm.es; o.martinezdequel@upm.es Óscar Martínez-de-Quel and Pedro L. Valenzuela share senior authorship. Abstract Background Preterm birth and low birthweight (LBW) might be associated with reduced physical fitness, although evidence remains inconclusive. Objective To examine the influence of preterm birth and LBW on physical fitness, as well as to assess whether variables such as gestational age, birthweight, or age at assessment moderate these effects. f Methods PubMed, Scopus, and PsycINFO were systematically searched from inception to 7 December 2023 for case–control and cohort studies analyzing the association between preterm birth or LBW (or gestational age or birthweight as continuous variables) with at least one physical fitness-related outcome (i.e., cardiorespiratory fitness (CRF), muscle strength, flex- ibility, speed, agility). Random-effects meta-analysis and meta-regression models were used to estimate the pooled effect size, as well as to examine potential associations between the magnitude of the effect and gestational age, birthweight, or age at assessment. Results Fifty-two studies (n = 920,603 participants, average age ranging from 4.7 to 34.4 years) were included. Pre- term birth was associated with reduced CRF (standardized mean difference (SMD) = −0.38, 95% confidence interval (CI) = −0.51 to −0.25) and muscle strength (SMD = −0.44, 95% CI = −0.79 to −0.08). LBW was associated with reduced CRF (SMD = −0.40, 95% CI = −0.64 to −0.17), muscle strength (SMD = −0.18, 95% CI = −0.24 to −0.13), flexibility (SMD = −0.11, 95% CI = −0.22 to −0.01), and agility (SMD = −0.99, 95% CI = −1.91 to −0.07). Meta-regression analyses showed that a lower gestational age or birthweight were associated with larger reductions in physical fitness, whereas no consistent association was found for the age at assessment.i Conclusion Both preterm birth and LBW seem associated with reduced physical fitness regardless of age, with larger reductions overall observed in individuals with lower gestational age or birthweight. These findings might support the implementation of preventive strategies (e.g., fitness monitoring and physical exercise interventions) in these populations through the life course. PROSPERO registration: CRD42021231845. Óscar Martínez-de-Quel and Pedro L. Valenzuela share senior authorship. * Óscar Martínez‑de‑Quel odequel@ucm.es; o.martinezdequel@upm.es * Pedro L. Sports Medicine (2024) 54:1907–1919 https://doi.org/10.1007/s40279-024-02026-z Sports Medicine (2024) 54:1907–1919 https://doi.org/10.1007/s40279-024-02026-z SYSTEMATIC REVIEW * Pedro L. Valenzuela pedrol.valenzuela@uah.es 4 Physical Activity and Health Research Group (PaHerg), Instituto de Investigación Hospital 12 de Octubre (‘imas12’), Centro de Actividades Ambulatorias, 7ª Planta, Bloque D, Av. de Córdoba s/n, 28041 Madrid, Spain 1 Faculty of Education, Complutense University of Madrid, Madrid, Spain 2 Faculty of Sciences for Physical Activity and Sport (INEF), Polytechnic University of Madrid, C/Martín Fierro, 7, 28040 Madrid, Spain Abstract For a full list of declaraons, including funding and author disclosure statements, and copyright informaon, please see the full text online. [Abbreviaons: CRF, Cardiorespiratory ﬁtness; LBW, Low birth weight ] This graphical abstract represents the opinions of the authors. For a full list -regression Óscar Mar nez-de-Quel; Pedro [Abbreviaons: CRF, Cardiorespiratory ﬁtness; LBW, Low birth weight ] Preterm birth Low birth weight Associated with reduced CRF and muscle strength. Not enough evidence available for other outcomes Associated with reduced CRF, muscle strength, ﬂexibility and agility Conclusions: Both preterm and LBW seem associated with reduced physical ﬁtness regardless of age, with greater reducons observed in individuals with lower gestaonal age or birth weight 52 studies n=920,603 (age: 4.7-34.4 years) 26 studies n=455,135 27 studies n=466,506 Inﬂuence of preterm birth and low birth weight on physical ﬁtness: a systemac review, meta-analysis and meta Marcos D. Mar nez-Zamora; Carlos Mar n Mar nez; L. Valenzuela SPORTS MEDICINE of declaraons, including funding and author disclosure statements, and copyright informaon, please see the full text online. 1908 M. D. Martínez‑Zamora et al. Graphical Abstract Abstract Valenzuela pedrol.valenzuela@uah.es 1 Faculty of Education, Complutense University of Madrid, Madrid, Spain 2 Faculty of Sciences for Physical Activity and Sport (INEF), Polytechnic University of Madrid, C/Martín Fierro, 7, 28040 Madrid, Spain 3 Department of Systems Biology, University of Alcalá, Madrid, Spain 4 Physical Activity and Health Research Group (PaHerg), Instituto de Investigación Hospital 12 de Octubre (‘imas12’), Centro de Actividades Ambulatorias, 7ª Planta, Bloque D, Av. de Córdoba s/n, 28041 Madrid, Spain Óscar Martínez-de-Quel and Pedro L. Valenzuela share senior authorship. * Óscar Martínez‑de‑Quel odequel@ucm.es; o.martinezdequel@upm.es * Pedro L. Valenzuela pedrol.valenzuela@uah.es 1 Faculty of Education, Complutense University of Madrid, Madrid, Spain 2 Faculty of Sciences for Physical Activity and Sport (INEF), Polytechnic University of Madrid, C/Martín Fierro, 7, 28040 Madrid, Spain 3 Department of Systems Biology, University of Alcalá, Madrid, Spain 4 Physical Activity and Health Research Group (PaHerg), Instituto de Investigación Hospital 12 de Octubre (‘imas12’), Centro de Actividades Ambulatorias, 7ª Planta, Bloque D, Av. de Córdoba s/n, 28041 Madrid, Spain 2 Faculty of Sciences for Physical Activity and Sport (INEF), Polytechnic University of Madrid, C/Martín Fierro, 7, 28040 Madrid, Spain Óscar Martínez-de-Quel and Pedro L. Valenzuela share senior authorship. 3 Department of Systems Biology, University of Alcalá, Madrid, Spain * Pedro L. Valenzuela pedrol.valenzuela@uah.es 4 Physical Activity and Health Research Group (PaHerg), Instituto de Investigación Hospital 12 de Octubre (‘imas12’), Centro de Actividades Ambulatorias, 7ª Planta, Bloque D, Av. de Córdoba s/n, 28041 Madrid, Spain 1 Faculty of Education, Complutense University of Madrid, Madrid, Spain M. D This graphical abstract represents the opinions of the authors. For a full list -regression Óscar Mar nez-de-Quel; Pedro [Abbreviaons: CRF, Cardiorespiratory ﬁtness; LBW, Low birth weight ] Preterm birth Low birth weight Associated with reduced CRF and muscle strength. Not enough evidence available for other outcomes Associated with reduced CRF, muscle strength, ﬂexibility and agility Conclusions: Both preterm and LBW seem associated with reduced physical ﬁtness regardless of age, with greater reducons observed in individuals with lower gestaonal age or birth weight 52 studies n=920,603 (age: 4.7-34.4 years) 26 studies n=455,135 27 studies n=466,506 Inﬂuence of preterm birth and low birth weight on physical ﬁtness: a systemac review, meta-analysis and meta-regression Marcos D. Mar nez-Zamora; Carlos Mar n Mar nez; Óscar Mar nez-de-Quel; Pedro L. Valenzuela SPORTS MEDICINE This graphical abstract represents the opinions of the authors. Key Points In this systematic review and meta-analysis, which included 52 studies (n = 920,603 participants, aver- age age ranging from 4.7 to 34.4 years), both preterm birth and low birthweight were associated with reduced physical fitness (e.g., cardiorespiratory fitness, muscle strength). A reduced physical fitness might play a role in the adverse effects associated with preterm birth and LBW. Growing evidence does indeed suggest that individuals born preterm [23, 24] or with LBW [25] present a reduced physical fitness, although meta-analytical evidence is scarce. In a meta-analysis of 22 studies, Edwards et al. [26] also found that preterm par- ticipants aged between 5 and 21 years had a lower maximum oxygen uptake (VO2max) compared with their peers born at term. Moreover, Dodds et al. [27] meta-analyzed 19 studies and found a positive association between birthweight and mus- cle strength, which was maintained across the life course. In a meta-analysis of ten studies, Poole et al. recently reported that participants aged over 18 years with LBW had a reduced CRF (as assessed by VO2max) compared with their term-born peers [28]. However, to our knowledge no meta-analytical evidence exists on the effect of preterm birth on muscle strength, nor for the effects of these conditions on other physical fitness out- comes such as flexibility, agility, or speed. Moreover, whether a lower birthweight or gestational age might be associated with greater reductions in physical fitness remains unclear, as well as whether the magnitude of these reductions might vary depending on the individuals’ age at assessment (e.g., with these differences decreasing at older ages). Meta-regression analyses revealed that a lower gesta- tional age or birthweight were overall associated with larger reductions in physical fitness, whereas no consist- ent associations were found between participants’ age at assessment and the magnitude of the reductions in physical fitness (i.e., similar reduction from childhood to adulthood). These findings might support the implementation of preventive strategies (e.g., fitness monitoring, physical exercise interventions) in these populations through the life course. SPORTS MEDICINE SPORTS MEDICINE 52 studies n=920,603 (age: 4.7-34.4 years) 52 studies n=920,603 (age: 4.7-34.4 years) Associated with reduced CRF and muscle strength. Not enough evidence available for other outcomes Associated with reduced CRF and muscle strength. Not enough evidence available for other outcomes Associated with reduced CRF, muscle strength, ﬂexibility and agility Associated with reduced CRF, muscle strength, ﬂexibility and agility Conclusions: Both preterm and LBW seem associated with reduced physical ﬁtness regardless of age, with greater reducons observed in individuals with lower gestaonal age or birth weight Conclusions: Both preterm and LBW seem associated with reduced physical ﬁtness regardless of age, with greater reducons observed in individuals with lower gestaonal age or birth weight 1909 Preterm Birth, Low Birthweight, and Physical Fitness predictor of both short- and long-term morbidity and mor- tality risk in children and adolescents [17]. For instance, a reduced CRF during childhood is associated with a higher risk of developing conditions such as obesity and cardio- metabolic diseases later in life [18, 19], and similar findings have been reported for muscle strength [20]. Moreover, CRF and muscle strength are strong predictors of morbidity and mortality in adults [21, 22].i 2 Methods The present systematic review was registered in PROSPERO (CRD42021231845), and is reported according to the Pre- ferred Reporting Items for Systematic Reviews and Meta- analyses (PRISMA) [29]. 1 Introduction Preterm birth (conventionally defined as < 37 weeks of gesta- tion) and low birthweight (LBW) (birthweight < 2.5 kg) are highly prevalent conditions [1, 2]. It is estimated that ~ 15 million children are born preterm every year, representing 10% of births worldwide [3], whereas approximately 20 mil- lion children are born with LBW, representing 15.5% of all births [4]—of note, although prematurity and LBW are two different conditions, they are highly interrelated, as many preterm individuals are born with LBW. Although advances in neonatal medicine have improved survival rates in preterm children and in those with LBW [5], these conditions are still linked to a higher risk of morbidity and mortality [6–8]. Indeed, one-quarter of all early deaths in newborns that are not produced by congenital malformations are due to preterm birth [9]. It is worth noting, however, that these conditions are not only associated with a higher morbidity risk during early childhood [10, 11], but also later in life. For instance, children with LBW have a 40-fold higher risk of mortality during the first month of life compared to those born with normal birthweight, with both preterm birth and LBW being linked to, for example, a higher risk of respiratory and car- diovascular conditions during adulthood [11–16].if f The present systematic review and meta-analysis aimed to determine the effects of preterm birth and LBW on different physical fitness indicators, as well as to examine whether different variables (i.e., gestational age, birthweight, age at assessment) moderate these effects. Of note, although preterm birth and LBW are two interrelated conditions, we aimed to study them separately, which could help to under- stand the similarities and differences between them. 2.4 Statistical Analysis Two authors (MDMZ and CMM) independently per- formed the systematic search for relevant articles in Pub- Med, Scopus, and PsycINFO from inception up to 7 Decem- ber 2023. The search included terms in titles and abstracts related to both the populations (i.e., preterm birth and LBW) and the outcomes of interest (i.e., physical fitness compo- nents). The search was limited to peer-reviewed articles pub- lished in English and Spanish. Additional search filters were not applied. The search strategies are shown as Table S1 in the Online Supplementary Material (OSM). The electronic search was supplemented with a manual review of reference lists from relevant publications and other reviews related to the topic [26–28] to locate additional studies.i Pooled analyses were performed using a random-effects model (DerSimonian and Laird method) when at least three studies assessed a given outcome. The pooled standardized mean difference (SMD) between groups was computed along with 95% CI, and if the studies reported the same out- come using the same assessment method and measurement units (e.g., VO2max in ml/kg/min, handgrip strength in kg), the absolute mean difference (MD) was computed. When a study assessed a given outcome at several time points, the longest follow-up was used for analyses. In the same line, when two studies shared some of the same partici- pants, the study with the longest follow-up was analyzed. When two studies shared some of the same participants and had the same follow-up, the study with the largest sample size was used for analyses. For an initial general analysis, in those cases in which one study assessed different indica- tors related to the same outcome, we selected the outcome most commonly assessed (e.g., VO2max over distance in the shuttle run test for the analysis of CRF, or handgrip strength over curl ups or horizontal jump distance for the analysis of muscle strength). However, sub-analyses were performed for the different indicators when possible. As recommended elsewhere, when a study included more than two groups of cases (e.g., extremely preterm and preterm individuals) in comparison with a control group, we halved the number of participants in the control group for each of the compari- sons [30]. Sensitivity analyses were conducted by testing significance when removing one study at a time (leave-one- out method) to check if findings were mostly driven by an individual study. 2.2 Data Extraction Two authors (MDMZ and CMM) independently extracted the relevant information from each study (i.e., participants’ characteristics, outcomes assessed, and main results). Data for quantitative analyses were extracted, when available, as mean and standard deviation (SD). When data were pro- vided as the median and/or using other measures of disper- sion (e.g., standard error, range, 95% confidence interval (CI)), the required information was estimated as explained elsewhere [30]. When available, we used the most adjusted model (e.g., adjusting for covariates such as sex, age at assessment, or socioeconomic status) for analyses. We had to contact the authors of 14 studies because the required data were not reported. Of these, the authors of eight studies provided the required information [31–38]. 2.4 Statistical Analysis Meta-regression analyses were performed using a random effects model (method of moments) to assess the association between birthweight, gestational age, or age at assessment with the magnitude of the differences between groups. Meta-regression analyses were only performed for those outcomes assessed by ten or more studies [30]. Begg’s test was used to determine the presence of publication bias (small-study effects), and the I2 statistic was used to assess heterogeneity across studies. I2 values > 25%, 50%, and 75% were considered indicative of low, moderate, and high het- erogeneity, respectively. The level of significance was set at 0.05. All statistical analyses were performed using the statistical software package Comprehensive Meta-analysis 2.0 (Biostat; Englewood, NJ, USA). Citations were first retrieved and preliminarily screened by title and abstract, and duplicates were removed manually. Full-texts of those studies that met the inclusion criteria were assessed. Each author provided a separate list with the stud- ies selected at each stage, as well as with those to be finally included. Potential disagreements were resolved through discussion with two other authors (OMDQ and PLV). 2.1 Study Selection and Search Strategy Case–control and cohort studies analyzing the association between preterm birth or LBW (or gestational age or birth- weight as a continuous variable) with at least one physical Physical fitness (including different components such as cardiorespiratory fitness (CRF), muscle strength, speed, flexibility, or agility) is an important health indicator and a 1910 M. D. Martínez‑Zamora et al. be classified as having good (≥ 8), fair (7), or poor quality (≤ 6). be classified as having good (≥ 8), fair (7), or poor quality (≤ 6). fitness-related outcome (i.e., CRF, muscle strength, flexibil- ity, speed, or agility) were included. Studies were excluded if they were solely focused on specific populations such as individuals with overweight, respiratory conditions, or func- tional disabilities. 3.3.1 Cardiorespiratory Fitness (CRF) Twenty-three studies [24, 37, 38, 40, 45, 49–66] assessed the influence of preterm birth on CRF-related measures such as VO2max, maximal power output (Wmax) on a cycle ergom- eter, distance covered on an incremental treadmill test, or the 6-min walking test. Of these, 17 studies including 24 group comparisons (n = 89,230 participants, weighted aver- age age at assessment = 19 years) could be meta-analyzed. Pooled analyses revealed a reduced CRF in preterm individ- uals compared to controls (SMD = −0.38, 95% CI = −0.51 to −0.25, p < 0.001, Fig. S1), with no signs of risk of bias (Begg’s p = 0.185) but with large heterogeneity (I2 = 77.4%). Sensitivity analyses by removing one study at a time con- firmed these differences, as well as when replacing the data from the 6-min walking test with data from the shuttle run test in the study by Cheong et al. [55], which analyzed both tests. Sub-analysis of those studies assessing VO2max (11 studies, 15 group comparisons, n = 688 participants) also confirmed significantly lower values in individuals born pre- term compared to controls (MD = −3.47 ml/kg/min, 95% CI = −5.04 to −1.89, p < 0.001; equivalent to SMD = −0.45, 95% CI = −0.61 to −0.28, I2 = 34.9%, Begg’s p = 0.444). Five studies could not be meta-analyzed [23, 46, 50, 58, 59], but only one of them did not find differences in CRF between preterm individuals and controls [59]. The studies focused on preterm birth included between 21 and 218,802 participants with an average age ranging from 4.7 to 28 years (weighted average age 17.5 years), whereas those focused on LBW included between 30 and 144,369 participants with an average age ranging between 5 and 34.4 years (weighted average age 17.5 years). Most studies analyzed both male and female participants except for five studies that included solely male participants [23, 33, 34, 44, 45]. Most studies followed a cross-sectional case–control design, except for four cross-sectional cohort studies [33, 34, 46, 47]. Only three studies followed a longitudinal case–con- trol design, in which the last measurement was considered for analyses [48–50]. Of the included case–control studies, eight divided the participants in sub-groups according to their gestational age or birthweight (e.g., very low birth- weight and extremely low birthweight, extremely preterm and very preterm), whereas 40 of them combined them in one single group (i.e., controls vs. preterm or LBW). 2.3 Study Quality Assessment Study quality was determined using the Newcastle–Ottawa Scale (NOS) [39], which assesses the risk of bias consid- ering three domains: selection of participants, comparabil- ity, and outcomes (Table S2, OSM). Two authors (MDMZ and CMM) independently scored the studies, and disagree- ments were resolved through discussion with a third author (OMDQ). A 0–10 total score was determined by counting the number of criteria satisfied by each study, which could Preterm Birth, Low Birthweight, and Physical Fitness 1911 described appropriately the statistical test performed, and 82% employed an independent blind assessment. described appropriately the statistical test performed, and 82% employed an independent blind assessment. 3.2 Quality Assessment The quality of the included studies was overall good (aver- age score of 8 out of 10, Table S5 (OSM)). Most studies adequately described the representativeness of the sample and justified the sample size (72% and 79%, respectively), although only 41% reported a satisfactory rate of response. All studies described the assessment tool and used a vali- dated tool. Most studies (~ 93%) adjusted for the main demographic variables such as age and sex, and others also adjusted for socioeconomic status, physical activity levels, or body composition, but most of the studies did not adjust for potential confounding variables such as delivery mode or body mass index at the time of assessment. All studies 3.3.1 Cardiorespiratory Fitness (CRF) Most studies (n = 7) included participants from the USA, followed by Australia (n = 6), Norway (n = 4), and Sweden (n = 4). Most studies were conducted in medium- and high-income countries except for one [44] conducted in Mozambique. p Seventeen [25, 34–36, 40–43, 47, 60, 67–73] studies assessed the influence of LBW on CRF-related measures, of which 14 including 18 group comparisons (n = 1758 partici- pants, weighted average age at assessment = 18 years) could be meta-analyzed. Their pooled analysis revealed a reduced CRF in individuals born with LBW compared to controls (SMD = −0.40, 95% CI = −0.64 to −0.17, p = 0.001, Fig. S2 (OSM)), albeit with signs of heterogeneity (I2 = 76.1%). Sensitivity analyses confirmed these differences. All of them analyzed VO2max, and differences corresponded to a MD = −2.81 ml/kg/min (95% CI = −4.45 to −1.17, p = 0.001). Only two studies could not be meta-analyzed [34, 73], but both of them reported a significantly lower CRF among individuals with LBW. A summary of the pooled results is shown in Table 1. A summary of the pooled results is shown in Table 1. 3.3 Synthesis A flowchart of the literature search is shown in Fig. 1. A total of 52 studies (n = 920,603 participants) were eventually included, of which 26 (n = 455,135 participants) assessed the influence of preterm birth and 27 (n = 466,506 participants) were focused on the influence of LBW. In one study [40] all participants had both conditions. Some studies shared part of the same sample [41–43], and only the largest one was used for the computation of the total sample size to avoid duplication of participants. The characteristics of the included studies are summarized in Tables S3 and S4 (SM) for preterm and low birthweight participants, respectively. 3.3.2 Muscle Strength Nine studies [24, 38, 46, 50, 58, 59, 74–76] assessed differ- ent muscle strength indicators such as standing long jump test, vertical jump, or handgrip strength with a dynamometer 1912 M. D. Martínez‑Zamora et al. Fig. 1 Flow diagram of the literature search Fig. 1 Flow diagram of the literature search in preterm individuals. Of these, eight studies including ten group comparisons (n = 2845 participants, weighted average age at assessment = 17 years) could be meta-analyzed. The initial general analysis revealed a reduced muscle strength in preterm individuals compared to controls (SMD = −0.44, 95% CI = −0.79 to −0.08, p = 0.016, Fig. S3 (OSM)), with no signs of publication bias (Begg’s p = 0.059) but large het- erogeneity (I2 = 89.1%). Sensitivity analyses revealed a sig- nificant or quasi-significant trend (all p ≤ 0.06) when remov- ing each individual study. Sub-analysis of the five studies (n = 1843 participants) that assessed handgrip strength (in kg) confirmed significantly lower values in preterm indi- viduals compared to controls in standardized (SMD = −0.19, 95% CI = −0.33 to −0.06, p = 0.004) and absolute units (MD = −0.64 kg, 95% CI = −1.01 to −0.27, p = 0.001), with no heterogeneity (I2 = 0%) and no signs of publication bias (Begg’s p = 0.226). One study could not be meta-analyzed [50], but found lower scores in extremely preterm partici- pants compared to controls. assessing handgrip strength also confirmed significantly lower values in individuals with LBW compared to con- trols (MD = −1.37 kg, 95% CI = −1.81 to −0.93, p < 0.001; equivalent to SMD = −0.23, 95% CI = −0.29 to −0.16), again with large heterogeneity (I2 = 86.4%) and no signs of publication bias (p = 0.296). Sub-analysis of the four studies (n = 129,112 participants) assessing lower-limb strength indicators such as vertical or horizontal jump tests also revealed significantly lower values in indi- viduals with LBW compared to controls (SMD = −0.23, 95% CI = −0.38 to −0.08, p = 0.002), and the separate analysis of the three studies (n = 596 participants) that assessed vertical jump ability confirmed these differences (MD = −6.66 cm, 95% CI = −10.24 to −3.08, p < 0.001, equivalent to SMD = −0.74, 95% CI = −1.19 to −0.30), in this case with no heterogeneity (I2 = 0%) and no publica- tion bias (p = 0.154). 3.3.3 Flexibility Ten studies [33, 35, 44, 47, 48, 69, 73, 77–79] including 16 group comparisons (n = 274,100 participants, weighted average age at assessment = 18 years) assessed the influ- ence of LBW on muscle strength, and all of them could be meta-analyzed. A reduced muscle strength was overall observed in individuals with LBW compared to controls (SMD = −0.18, 95% CI = −0.24 to −0.13, p < 0.001, Fig. S4 (OSM)), albeit with signs of heterogeneity (I2 = 86.6%). Sensitivity analyses confirmed these differences. Sub- analysis of the seven studies (n = 145,299 participants) Only data from two studies [24, 38] were available for the analysis of flexibility in preterm individuals, and therefore this outcome could not be meta-analyzed. However, only one of them found differences in flexibility related to preterm birth, with term individuals showing better scores [24]. Four studies [35, 44, 73, 78] including nine group compari- sons (n = 29,779 participants, weighted average age at assess- ment = 7 years) assessed flexibility in individuals with LBW, Preterm Birth, Low Birthweight, and Physical Fitness 1913 Table 1 Summary of pooled results Results are shown as standardized mean difference (SMD) along with 95% confidence intervals (CIs) Significant p-values are shown in bold font CRF cardiorespiratory fitness, NA not applicable, NOS Newcastle Ottawa Scale Outcome Studies/groups (participants) SMD (95% CI) p-Value I2 Begg’s p-value Quality (mean NOS score, range) Preterm birth CRF 17/24 (n = 89,230) −0.38 (−0.51, −0.25) < 0.001 77.4% 0.185 8 (6–10) Muscle strength 8/10 (n = 2845) −0.44 (−0.79, −0.08) 0.016 89.1% 0.036 8 (6–10) Flexibility 2/2 (n = 216) NA NA NA NA 7 (7–7) Agility 2/2 (n = 229) NA NA NA NA 6 (6–6) Speed 1/1 (n = 60) NA NA NA NA 6 (6–6) Low birthweight CRF 14/18 (n = 1758) −0.40 (−0.64, −0.17) 0.001 76.1% 0.279 8 (6–10) Muscle strength 10/16 (n = 274,100) −0.18 (−0.24, −0.13) < 0.001 86.6% 0.083 9 (7–10) Flexibility 4/9 (n = 29,779) −0.11 (−0.22, −0.01) 0.031 70.3% 0.038 9 (7–10) Agility 3/7 (n = 29,695) 0.99 (−1.91, −0.07) 0.036 99.7% 0.500 9 (8–10) Speed 2/2 (n = 506) NA NA NA NA 9 (8–10) These studies found a reduced speed among preterm indi- viduals compared to controls, but no differences when ana- lyzing LBW. all of them using the sit and reach test. 3.4 Meta‑Regressions Meta-regression analyses could be performed on the influ- ence of preterm birth and LBW on CRF and muscle strength. Analyses revealed a significant or quasi-significant trend towards a greater reduction of CRF in those individuals with a lower gestational age (p = 0.028, Fig. S7 (OSM)) or birth- weight (p = 0.058, Fig. S8 (OSM)). Similarly, both a lower gestational age (p = 0.024, Fig. S9 (OSM)) and a lower birth- weight (p < 0.001, Fig. S10 (OSM)) were associated with greater reductions in muscle strength. No significant associa- tions were found in any case between the magnitude of the reductions in CRF or muscle strength and age at assessment (all p > 0.05, Figs. S11–S14 (OSM)). 4 Discussion The present systematic review and meta-analysis [including 52 studies and 920,603 participants with a wide age range (from 4.7 to 34.4 years)] shows that both preterm birth and LBW are associated with a reduced physical fitness, as reflected by a lower CRF and muscle strength. Although the number of studies available precluded drawing strong conclusions on other fitness outcomes, our results also sug- gest that these conditions might also be associated with reductions in other fitness components, such as flexibility, 3.3.3 Flexibility Their pooled analy- sis revealed a reduced flexibility in individuals with LBW compared to controls (MD = −0.83 cm, 95% CI = −1.59 to −0.08, p = 0.031; equivalent to SMD = −0.11, 95% CI = −0.22 to −0.01, Fig. S5 (OSM)), albeit with large heterogeneity (I2 = 70.3%) and signs of publication bias (p = 0.038). Results remained significant in sensitivity analyses. all of them using the sit and reach test. Their pooled analy- sis revealed a reduced flexibility in individuals with LBW compared to controls (MD = −0.83 cm, 95% CI = −1.59 to −0.08, p = 0.031; equivalent to SMD = −0.11, 95% CI = −0.22 to −0.01, Fig. S5 (OSM)), albeit with large heterogeneity (I2 = 70.3%) and signs of publication bias (p = 0.038). Results remained significant in sensitivity analyses. 3.3.4 Agility Only data from two studies [24, 58] were available for the meta-analysis of agility in preterm individuals, and therefore this outcome could not be analyzed. However, these studies reported that preterm birth is related to a reduced agility, as assessed by the 4 × 10 test. On the other hand, three studies [35, 44, 78] including seven group comparisons (n = 29,695, weighted average age at assessment = 7 years) assessed agility in individuals with LBW using the 10 × 5 m shuttle test or the 4 × 4 m square test. Their pooled analysis revealed a reduced agility in indi- viduals with LBW compared to controls (SMD = −0.99, 95% CI = −1.91 to −0.07, p = 0.036; Fig. S6 (OSM)), albeit with signs of large heterogeneity (I2 = 99.7%). 3.3.5 Speed Only data from one [58] and two studies [35, 44] were avail- able for the meta-analysis of speed (assessed by 20-m or 50-m running tests) in preterm and LBW individuals, respec- tively, and therefore this outcome could not be analyzed. 1914 M. D. Martínez‑Zamora et al. Moreover, our results show reductions in other outcomes that to the best of our knowledge had not been previously meta-analyzed such as flexibility. Further research is, how- ever, warranted to confirm whether other physical fitness components (e.g., agility, speed) are also reduced in these populations, as well as the mechanisms involved.i or agility (which were reduced in individuals with LBW). Of note, a lower gestational age or birthweight seemed to be associated with greater reductions in physical fitness outcomes (at least for CRF and muscle strength, for which meta-regressions could be performed). No consistent asso- ciations were found between age at assessment and the magnitude of the reductions (see graphical summary of the results in Fig. 2).i p p Another major finding of the present study derives from the meta-regression analyses. Similar to what has been reported for the risk of medical conditions [85], in the pre- sent study we found a trend toward greater reductions in physical fitness (specifically, CRF and muscle strength) with lower gestational age or birthweight. Confirming these trends, large cohort studies have also reported that birth- weight is inversely associated with CRF and muscle strength [33, 34, 46]. Interestingly, our meta-regression analyses also suggest that the reductions in physical fitness associated with preterm birth or LBW seem not to be ameliorated at an older age, which is in line with the few longitudinal studies avail- able on this topic. For instance, Pikel et al. [50] reported reductions in physical fitness components in preterm indi- viduals after the period of childhood, some of which were maintained into early adulthood. Morrison et al. [48] found that individuals with LBW showed a similar change in grip strength from their mid-20s to their mid-30s, leading to con- sistently low levels in the former. Similarly, another longi- tudinal study reported that extremely preterm individuals showed a consistently lower physical fitness compared with those born at term through the school age, albeit only if they suffered from bronchopulmonary dysplasia [49]. Thus, pre- term individuals not suffering from this condition progres- sively improved their fitness level through the school age, eventually reaching ‘normal’ values [49]. 3.3.5 Speed More research is therefore warranted to confirm these findings. g ) The present findings are of potential clinical relevance due to the importance of physical fitness for both short- and long-term health. For instance, our results show that preterm individuals and those with LBW present a reduced CRF compared to controls (average differences in VO2max of 4.29 and 3.10 ml/kg/min, respectively). A lower CRF dur- ing youth is associated with a higher risk of obesity and cardiometabolic diseases in later years [19], and indeed CRF has proven to be a major prognostic factor of mortality in individuals of all ages [22]. Interestingly, each metabolic equivalent reduction in CRF (i.e., 3.5 ml/kg/min, which is approximately the difference observed in the present study) has been associated with a 11.6%, 16.1%, and 14.0% increase in all-cause, cardiovascular, and cancer mortality, respectively [80], which further supports the potential rel- evance of our findings. Similar findings were observed for muscle strength levels in the present study, which were also reduced in individuals born preterm or with LBW. Low mus- cle strength levels during childhood are likely to be main- tained into adulthood [81, 82], and are associated with a greater cardiometabolic risk later in life [20]. Indeed, muscle strength has been inversely associated with mortality risk in young [83] and adult populations [21]. Therefore, although further research is warranted to confirm the long-term clini- cal relevance of the observed differences, preventive strate- gies are needed to counteract the reduction of physical fit- ness observed in individuals born preterm or with LBW. In this regard, physical exercise might be an effective option, as shown by a recent study that reported increases in VO2max in preterm individuals after 16 weeks of training [84].i ii g The present results might be at least partly explained by biological processes during pregnancy, which could be still evident at older ages. For instance, the association between gestational age and CRF might be explained by the late development of the lungs and cardiac chambers dur- ing pregnancy, which can lead to interrupted lung growth [86] in very preterm individuals. Dysanapsis might also be present in preterm individuals, that is, normal lung volumes and total cardiac size but smaller cardiac chambers and lung airways, which might impair expiratory airflow limitation [87]. i Fig. 2 Graphical summary of the study findings could potentially confound our findings, as most included studies did not adjust their analyses for these variables. Moreover, the inclusion of individuals with extremely low gestational age or birthweight could overestimate the observed results. Another limitation that should be consid- ered is the fact that in the present review we did not assess the effects of being born small for gestational age, which should be addressed in future research. levels usually seen in preterm individuals might also be a confounding factor [92]. However, a recent study by our research group found reductions in several fitness compo- nents such as CRF, muscle strength, flexibility, and agility among preterm individuals compared to individuals born at term despite performing similar physical activity levels [24]. Thus, whether increasing physical activity levels can counteract the reduced fitness observed in individuals born preterm or with LBW remains to be elucidated. On the other hand, the major strengths of this study are having analyzed two highly prevalent conditions such as pre- term birth and LBW, which could help in understanding the similarities between them. In this regard, it is worth noting that these conditions are highly interrelated, and many indi- viduals may present both of them concomitantly. Indeed, we observed rather similar estimates for outcomes such as CRF (SMD = −0.38 and −0.40 for preterm and LBW, respec- tively), although research is warranted to confirm whether there can be differences for other outcomes such as muscle strength. Moreover, the wide variety of fitness-related out- comes included and the inclusion of meta-regression analy- ses can also be considered strengths of our study. The large sample size, including participants with a wide age range 3.3.5 Speed Similarly, the reduction observed for other fitness out- comes such as muscle strength might also be related with essential processes in brain development that occur during the last weeks of gestation [88–90]. Moreover, although body composition was not analyzed in the present study, pre- term individuals might also have a lower muscle mass and body mass index than their peers born at term [91], which might negatively influence fitness parameters such as muscle strength. It must be noted, nonetheless, that other behavioral or environmental factors such as the lower physical activity The present findings expand on those from previous meta-analyses that provided preliminary evidence of reductions of physical fitness in individuals with LBW or preterm birth. Specifically, Edwards et al. found lower VO2max values (−2.20 ml/kg/min) in preterm individu- als compared to their counterparts aged between 5 and 21 years [26]. Poole et al. also reported that participants aged over 18 years with very low birthweight had lower VO2max (−3.35 ml/kg/min) compared with their term-born peers [28]. In addition to confirming these findings, the present work suggests that preterm and LBW individu- als might also show reductions in other major fitness out- comes such as muscle strength. This is in line with the meta-analysis by Dodds et al. [27], who reported a posi- tive association between birthweight and muscle strength. 1915 Preterm Birth, Low Birthweight, and Physical Fitness Fig 2 G hi l f th t d fi di 5 Conclusions 1. Howson C, Kinney M, Lawn J. Born too soon: the global action report on preterm birth. World Heal Organ. 2012;29:283–7. 2. Goldenberg RL, Culhane JF. Low birthweight in the United States. Am J Clin Nutr. 2007;85:584–90. Both preterm birth and LBW seem to be associated with a reduced physical fitness, as reflected by lower values for outcomes such as CRF and muscle strength in an overall dose–response manner. Of note, the magnitude of these reductions seems overall independent of participants’ age at assessment, which suggests that these conditions are asso- ciated with reductions in physical fitness components not only during childhood, but also later in life. However, the magnitude of these reductions was inversely associated with both gestational age and birthweight. These findings might support the implementation of preventive strategies (e.g., fitness monitoring and lifelong exercise training) in these populations. 3. Chawanpaiboon S, Vogel JP, Moller AB, Lumbiganon P, Petzold M, Hogan D, et al. Global, regional, and national estimates of levels of preterm birth in 2014: a systematic review and modelling analysis. Lancet Glob Heal. 2019;7:e37-46. 4. WHO. Global nutrition targets. Low birth weight policy brief. World Heal Organ. 2025;2002(287):270. 5. Perin J, Mulick A, Yeung D, Villavicencio F, Lopez G, Strong K, et al. Global, regional, and national causes of under-5 mortality in 2000–19: an updated systematic analysis with implications for the sustainable development goals. Lancet Child Adolesc Heal. 2022;6:106–15. https://doi.org/10.1016/S2352-4642(21)00311-4. 6. Risnes K, Bilsteen J, Brown P, Pulakka A, Andersen A, Opdahl S, et al. Mortality among young adults born preterm and early term in 4 Nordic nations. JAMA Netw Open. 2021;4:1–12. 7. 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Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. 10. 4.1 Limitations and Strengths Some limitations of the present study should be acknowl- edged. The limited number of available studies for some outcomes such as speed or agility hindered performing meta- analyses. Moreover, meta-regression analyses could not be performed for all outcomes, as ten or more studies were required [30]. The required data from some studies could not be obtained despite asking the corresponding authors, which made it impossible to include these studies in quantitative synthesis. In addition, variables such as physical activity, body mass index, delivery mode, or socioeconomic status 1916 M. D. Martínez‑Zamora et al. need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. from several cohorts of different countries, can also be con- sidered a strength of the study, although the present findings might not be necessarily applicable to all populations, as only one study came from a low-income country. 5 Conclusions Nugent JT, Lu Y, Deng Y. Effect measure modification by birth weight on the association between overweight or obesity and hypertension in children and adolescents. JAMA J Am Med Assoc. 2023;7:735–7. Declarations Authors’ Contributions MMZ, OMDQ, and PLV designed the original idea. MMZ and CMM performed the systematic search and extracted the data. MMZ, CMM, and OMDQ performed the quality assessment. PLV performed the statistical analyses. MMZ and PLV drafted the original version of the manuscript. OMDQ and PLV supervised the study. All authors revised the manuscript critically for important intel- lectual content and approved the final version. 11. 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https://openalex.org/W3211369773	https://www.e3s-conferences.org/articles/e3sconf/pdf/2021/93/e3sconf_icenis2021_05025.pdf	English	null	Geospatial Modeling to Assess The Land Suitability of Mangrove Restoration at Bantul Regency, Yogyakarta, Indonesia	E3S web of conferences	2,021	cc-by	4,180	* Corresponding author: kantisuraningsih@students.undip.ac.id Geospatial Modeling to Assess The Land Suitability of Mangrove Restoration at Bantul Regency, Yogyakarta, Indonesia Abstract. The geo-position of Bantul Regency, which has open seas directly facing the Indian Ocean, is very vulnerable to tsunami and abrasion hazards, therefore disaster mitigation is needed in the form of restoring mangrove forest ecosystems. The purpose of this study was to assess the land suitability of mangrove restoration. The method used in this research is geospatial modelling that utilizes field surveys and interviews. The parameters tested consisted of maximum inundation, inundation frequency and land use. The results showed the potential for mangrove restoration as disaster mitigation at the Opak river estuary in Tirtohargo Village Kretek District and Rejosari Village in Sanden District. The Progo River estuary is also a mangrove restoration area in Poncosari Village, Srandakan District. After doing the geospatial analysis, it was found that the potential for mangrove forest restoration with a very suitable class was 168 ha and a suitable class was 181 ha. This mangrove forest is highly recommended because it physically has a coastal defence, protecting beaches and river banks, and prevents seawater abrasion. © 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/). E3S Web of Conferences 317, 05025 (2021) ICENIS 2021 E3S Web of Conferences 317, 05025 (2021) ICENIS 2021 https://doi.org/10.1051/e3sconf /202131705025 1 Introduction The total area of mangroves in 2000 was 137,760 km2; it was located in 118 countries and regions in tropical and subtropical regions of the world. Almost 75% of the world's mangroves are found only in 15 countries, and only 6.9% are protected under the existing network of protected areas (IUCN I -IV) [1]. At the same time, mangrove forests have great potential in supporting the development of SDGs (Sustainable Development Goals). SDGs is a global and national commitment to improve the welfare of society. 1 of 17 SDGs contributed by mangrove forests is Addressing Climate Change [2]. The restoration of mangroves in the Indonesian archipelago with carbon-rich should be a component of a high © 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/). E3S Web of Conferences 317, 05025 (2021) ICENIS 2021 https://doi.org/10.1051/e3sconf /202131705025 priority strategy for climate change mitigation [3]. Mangroves can deal with climate change because mangroves can store higher carbon than tropical forests. Mangrove forests typically contain 1,023 Mg of carbon per hectare. The organic-rich soil ranges from 0.5 m to more than 3 m deep and accounts for 49–98% of carbon storage in the system [4]. priority strategy for climate change mitigation [3]. Mangroves can deal with climate change because mangroves can store higher carbon than tropical forests. Mangrove forests typically contain 1,023 Mg of carbon per hectare. The organic-rich soil ranges from 0.5 m to more than 3 m deep and accounts for 49–98% of carbon storage in the system [4]. Indonesia has the largest mangrove forest in the world. The area is about 3.2 million ha which is 22.6% of the world's total mangrove [5]. However, in the last two to three decades, almost 50% of the total mangroves in Indonesia have been lost, from around 6.7 million ha to about 3.2 million ha [6]. The islands of Java and Bali were the islands with the most significant damage, around 88%. Previously, these two islands had about 171,500 ha, but currently, there is only 19,577 ha [7]. It is regrettable if the mangroves area was converted into commercial areas such as settlements, aquaculture, rice fields, etc. When mangroves area is lost, it will cause the threat of erosion, abrasion, tsunami, land subsidence, etc. 1 Introduction The mangrove forest in Bantul Regency is located at Baros Hamlet, Tirtohargo Village, Kretek District, with about 4 ha [8]. The suitability of mangrove land in Bantul has been studied, but only limited to Baros hamlet [9]. Meanwhile, the Bantul coast is an open sea area that directly faces the Indian Ocean. The Bantul coast, precisely in the estuary waters of the Opak River and the Progo River, has a significant impact in the event of a tsunami [10, 11]. Therefore, mitigation efforts are needed to prevent this condition. Mangrove forests are an effort that can be made to reduce the impact of disasters. Therefore, this study aims to analyze the suitability of mangrove restoration land in the Bantul Regency. 2.1 Study dan Sampling Area The soil sampling and observations were taken from April to May 2021. The determination of the sampling point was carried out with the help of the Ach GIS 10.6.1 software. There were 39 sample points from the mouth of the Opak River in Tirtohargo village, Kretek district and Rejosari village at Sanden district, also the estuary area on the Progo River in Poncosari village, Srandakan district. The research sites can be seen in figure 1. The measurements of the study were made by direct observation in the field. Fig. 1. Research sites. Fig. 1. Research sites. 2 2 E3S Web of Conferences 317, 05025 (2021) ICENIS 2021 https://doi.org/10.1051/e3sconf /202131705025 2.2 Analysis of Mangrove Land Suitability 3 3 https://doi.org/10.1051/e3sconf /202131705025 E3S Web of Conferences 317, 05025 (2021) ICENIS 2021 E3S Web of Conferences 317, 05025 (2021) 3 Land use Mangrove, forest, open land 3 1 Mining, aquaculture 2 Residential, industrial 1 3 Land use Mangrove, forest, open land 3 1 Mining, aquaculture 2 Residential, industrial 1 2.3 Geospatial Modelling The progress of geospatial analysis is currently very fantastic; world researchers in various fields use it. In the agriculture sector, it is used to determine the distribution of insecticide residues [13,14]. For example, in the marine sector, it is used by marine experts at marine biodiversity conservation and coastal blue carbon ecosystems [15–17]. In the environment and anti-disaster management, it is applied to earthquake intensity assessment and making tsunami vulnerability index maps [18]. The spatial analysis method can be combined with other analysis methods such as Hierarchy Component Analysis (HCA) and Principal Component Analysis (PCA), which is then called multivariate statistical analysis [19] because the technique is proven to be flexible and practical in many ways. Geospatial modelling in this research is used to determine a suitable area for mangroves. The main thing in the modelling geospatial mapping process, it can also be done using remote sensing from a satellite platform [20]. The research of the suitability of mangrove land was taken from coordinate points that previously used Microsoft Excel. It was converted into a mapping system using ArcGIS 16.1. ArcGIS software was essential in determining geographic information both inland and water, such as length of the coast, level of abrasion and changes in shoreline [21]. GIS technology integrates a joint database operation, such as querying and statistical analysis, with mapping a unique visualization and analyzing capabilities. This ability is what distinguishes Geospatial Information Systems from other Information Systems [22]. This mangrove land suitability modelling also uses the Bantul Regency Spatial Plan and ESRI's Basemap World Imagery. Table 2. The average value of maximum inundation height and inundation frequency at each location point. 2.2 Analysis of Mangrove Land Suitability The obtained data were then analyzed using the scoring method on physical parameters. Therefore it could evaluate the mangrove land. Land suitability classification is carried out by compiling a suitability matrix to assess the feasibility based on scoring on the mangrove boundary parameters (Table 1). Each parameter was divided into three classes in this study, namely very suitable, suitable, and conditionally suitable. The very suitable class is given a value of 3, the suitable class is given a value of 2, and the conditionally suitable class is given a value of 1. Furthermore, every single parameter in this study is weighted based on a literature study to assess or determine the level of land suitability. Parameters that have a more substantial effect are given a higher weight than those with a weaker effect. The total score from the multiplication of the parameter values and their weights is then used as a formula to determine the suitability class for mangrove land [12]. (1) ∑ (1) Information : Y = Total land suitability value ai = weighting factor Xn = Value of land suitability The land suitability class interval is obtained based on the Equal Interval method [12] Y = Total land suitability value Y = Total land suitability value ai = weighting factor Xn = Value of land suitability The land suitability class interval is obtained based on the Equal Interval method [12] Xn Value of land suitability The land suitability class interval is obtained based on the Equal Interval method [12] (2) Information : = Land suitability class interval i = Land suitability class interval i = Land suitability class interval k = Number of land suitability classes desired Based on the formula and calculations above, the class interval and value (score) of land suitability are obtained as follows: S1 = very suitable, with a value of 11-15 S2 = suitable, with a value of 6 – 10 S3 = conditionally suitable, with a value of 1 - 5 Table 1. Matrix of Criteria Mangrove Land Suitability. NO PARAMETER CLASS VALUE WEIGHT 1 maximum inundation height (m) < 0,5 3 2 0,5 – 1 2 >1 1 2 Inundation frequency (Day / month) 20 3 2 10 – 19 2 <10 atau >20 1 Table 1. Matrix of Criteria Mangrove Land Suitability. 3 Results and discussions The maximum inundation height (MIH) yields the highest yield in the Srandakan district, namely 155 cm (Table 2) (Fig. 3). Meanwhile, the lowest maximum inundation height was at stations Sa6, Sa8, and Sa9 in Sanden District (Fig. 2). The tides influence inundation height in the region. The tides in the Bantul regency are at a high level of vulnerability. The tidal value of the Bantul coast has an average of 1.3 m [23]. The tides in the sea significantly affect the inundation height in estuary waters. Meanwhile, the tides in the Bantul regency have caused erosion. This is because the movement of geomorphological power from west to east occurs due to the strengthening of the coast in the Kulonprogo Regency concerning YIA (Yogyakarta International Airport) [24]. It is also coupled with an angle between the direction of the waves and the coastline, which is reflected by littoral drift. 4 https://doi.org/10.1051/e3sconf /202131705025 E3S Web of Conferences 317, 05025 (2021) ICENIS 2021 E3S Web of Conferences 317, 05025 (2021) Station Kretek MIH (cm) FI (day/ month) Station Sanden MIH (cm) FI (day/ month) Station Srandaka n MIH (cm) FI (day/ month) Kr1 110 20 Sa1 70 15 Sr1 150 26 Kr2 100 20 Sa2 80 20 Sr2 145 26 Kr3 90 20 Sa3 90 18 Sr3 155 26 Kr4 80 18 Sa4 100 16 Sr4 70 20 Kr5 70 20 Sa5 60 15 Sr5 80 22 Kr6 70 20 Sa6 50 16 Sr6 85 22 Kr7 80 18 Sa7 60 18 Sr7 90 20 Kr8 100 17 Sa8 50 22 Sr8 75 20 Kr9 120 20 Sa9 50 20 Sr9 125 23 Kr10 100 21 Sa10 70 23 Sr10 80 20 Kr11 110 24 Sa11 70 24 Kr12 120 25 Sa12 120 21 Kr13 70 21 Kr14 80 22 Kr15 60 23 Kr16 60 20 Kr17 70 21 This is different when compared to research that was conducted on the north coast of Java. Because many factors influence inundation include sea-level rise and land subsidence. 3 Results and discussions Th l ti l hi h l l i d l d b id lt i tid b i l h th Station Kretek MIH (cm) FI (day/ month) Station Sanden MIH (cm) FI (day/ month) Station Srandaka n MIH (cm) FI (day/ month) Kr1 110 20 Sa1 70 15 Sr1 150 26 Kr2 100 20 Sa2 80 20 Sr2 145 26 Kr3 90 20 Sa3 90 18 Sr3 155 26 Kr4 80 18 Sa4 100 16 Sr4 70 20 Kr5 70 20 Sa5 60 15 Sr5 80 22 Kr6 70 20 Sa6 50 16 Sr6 85 22 Kr7 80 18 Sa7 60 18 Sr7 90 20 Kr8 100 17 Sa8 50 22 Sr8 75 20 Kr9 120 20 Sa9 50 20 Sr9 125 23 Kr10 100 21 Sa10 70 23 Sr10 80 20 Kr11 110 24 Sa11 70 24 Kr12 120 25 Sa12 120 21 This is different when compared to research that was conducted on the north coast of Java. Because many factors influence inundation include sea-level rise and land subsidence. The relatively high sea-level rise and land subsidence can result in tides, obviously where the sea level reaches a height that exceeds normal conditions [25]. Whereas in mangrove planting, the best maximum inundation height is below 0.5 m. 5 5 E3S Web of Conferences 317, 05025 (2021) ICENIS 2021 https://doi.org/10.1051/e3sconf /202131705025 Fig. 2. Map of maximum inundation height in Kretek and Sanden Districts, Bantul Regency, Indonesia. Fig. 2. Map of maximum inundation height in Kretek and Sanden Districts, Bantul Regency, Indonesia. Fig. 2. Map of maximum inundation height in Kretek and Sanden Districts, Bantul Regency, Indonesia Fig. 3. Map of maximum inundation height in Srandakan Districts, Bantul Regency, Indonesia. Fig. 3. Map of maximum inundation height in Srandakan Districts, Bantul Regency, Indonesia. 6 6 E3S Web of Conferences 317, 05025 (2021) ICENIS 2021 https://doi.org/10.1051/e3sconf /202131705025 Results the highest frequency inundation (FI) occurred at stations Sr1, Sr2, and Sr3 in the district of Srandakan at 30 days/month. Meanwhile, the lowest inundation frequency was found at the Sa1 and Sa5 stations in the Sanden district (15 days/month). This is different from research in Tugu district, Semarang City; the research obtained inundation frequency results of 30-31 days per month [12]. This is due to the selection of Semarang mangrove land on the north coast so that it has a high frequency of inundation. 3 Results and discussions It is also supported because the north coast is not directly facing the ocean. Meanwhile, the selection of mangrove land on the south coast is more optimal in the estuary area. The statement confirms that mangrove ecosystems will live optimally in areas protected from large currents and waves, such as coastal areas with large river estuaries and deltas, where water flow contains much mud. Mangroves are challenging to grow in coastal areas that are steep and wavy with strong tidal currents. These conditions do not allow for siltation to occur as a substrate for growth [26]. The inundation frequency parameter was also strengthened by research that almost all of the coasts in Bantul Regency, including sandy beaches [27]. It also causes shoreline changes, coastal erosion and sedimentation, and frequent changes in the inundation frequency in the estuary. Fig. 4. Map of frequency inundation at Kretek and Sanden Districts, Bantul Regency, Indonesia Fig. 4. Map of frequency inundation at Kretek and Sanden Districts, Bantul Regency, Indonesia 7 8 8 https://doi.org/10.1051/e3sconf /202131705025 E3S Web of Conferences 317, 05025 (2021) ICENIS 2021 Kr6 Mangroves / open land Sa6 Mangroves / open land Sr6 Mangroves / open land Kr7 Mangroves / open land Sa7 Mangroves / open land Sr7 Mangroves / open land Kr8 Mangroves / open land Sa8 Mangroves / open land Sr8 Mangroves / open land Kr9 Mangroves / open land Sa9 Mangroves / open land Sr9 Mangroves / open land Kr10 Mangroves / open land Sa10 Mangroves / open land Sr10 Mangroves / open land Kr11 Mangroves / open land Sa11 Mangroves / open land Kr12 Mangroves / open land Sa12 Mangroves / open land Kr13 Mangroves / open land Kr14 Mangroves / open land Kr15 Mangroves / open land Kr16 Mangroves / open land Kr17 Mangroves / open land Kr6 Mangroves / open land Sa6 Mangroves / open land Sr6 Mangroves / open land Kr7 Mangroves / open land Sa7 Mangroves / open land Sr7 Mangroves / open land Kr8 Mangroves / open land Sa8 Mangroves / open land Sr8 Mangroves / open land Kr9 Mangroves / open land Sa9 Mangroves / open land Sr9 Mangroves / open land Kr10 Mangroves / open land Sa10 Mangroves / open land Sr10 Mangroves / open land Kr11 Mangroves / open land Sa11 Mangroves / open land Kr12 Mangroves / open land Sa12 Mangroves / open land Kr13 Mangroves / open land Kr14 Mangroves / open land Kr15 Mangroves / open land Kr16 Mangroves / open land Kr17 Mangroves / open land Fig. 6. Map of Land Use at the Research Site. The results of the land suitability analysis using the equal interval show that the stations in the Kretek district obtained the very suitable assessment criteria for nine stations (LSV 11- 13) and the assessment criteria suitable to 8 stations (LSV 7-9). Likewise, the stations in Fig. 6. Map of Land Use at the Research Site. Fig. 6. Map of Land Use at the Research Site. The results of the land suitability analysis using the equal interval show that the stations in the Kretek district obtained the very suitable assessment criteria for nine stations (LSV 11- 13) and the assessment criteria suitable to 8 stations (LSV 7-9). 7 7 7 E3S Web of Conferences 317, 05025 (2021) ICENIS 2021 https://doi.org/10.1051/e3sconf /202131705025 Fig. 5. Map of frequency inundation in Srandakan Districts, Bantul Regency, Indonesia. Fig. 5. Map of frequency inundation in Srandakan Districts, Bantul Regency, Indonesia. The results show that the estuary area and open land are on average in the land use parameter. However, there are also areas where sand mining is utilised in the stations of Sr1, Sr2 and Sr3, Srandakan district. Even though they are included in the appropriate land criteria at these three stations, the score they have is only 6. It shows that the three stations are at the minimum threshold for suitable land criteria to mangroves. Based on research, sand mining has caused soil change and damage, increasing bulk density, decreasing organic C, decreasing and destroying soil microorganisms, also changes in soil physical properties, soil chemical properties and soil biology [28]. Sand mining activities have caused the loss of organic matter and biomass of mangrove forests, thus endangering sustainable forest management. Table 3. The land use at each location point. Station Kretek Land Use Station Sanden Land Use Station Srandakan Land Use Kr1 Mangroves / open land Sa1 Mangroves / open land Sr1 mining Kr2 Mangroves / open land Sa2 Mangroves / open land Sr2 mining Kr3 Mangroves / open land Sa3 Mangroves / open land Sr3 mining Kr4 Mangroves / open land Sa4 Mangroves / open land Sr4 Mangroves / open land Kr5 Mangroves / open land Sa5 Mangroves / open land Sr5 Mangroves / open land Table 3. The land use at each location point. 7 Likewise, the stations in The results of the land suitability analysis using the equal interval show that the stations in the Kretek district obtained the very suitable assessment criteria for nine stations (LSV 11- 13) and the assessment criteria suitable to 8 stations (LSV 7-9). Likewise, the stations in 9 E3S Web of Conferences 317, 05025 (2021) ICENIS 2021 https://doi.org/10.1051/e3sconf /202131705025 Sanden and Srandakan districts can be observed in table 2. If the land suitability value (LSV) is averaged at the research location, the result in Kretek district is 10.29 (very suitable), Sanden district is 10, 83 (very suitable), and Srandakan district is 9, 5 (suitable). Table 4. Value of Mangrove Land Suitability at Each Research Station. Table 4. Value of Mangrove Land Suitability at Each Research Station. Station Kretek LSV Class Station Sanden LSV Class Station Srandakan LSV Class Kr1 11 S1 Sa1 11 S1 Sr1 6 S2 Kr2 11 S1 Sa2 13 S1 Sr2 6 S2 Kr3 13 S1 Sa3 11 S1 Sr3 6 S2 Kr4 11 S1 Sa4 9 S2 Sr4 13 S1 Kr5 13 S1 Sa5 11 S1 Sr5 9 S2 Kr6 13 S1 Sa6 13 S1 Sr6 9 S2 Kr7 11 S1 Sa7 11 S1 Sr7 13 S1 Kr8 9 S2 Sa8 11 S1 Sr8 13 S1 Kr9 11 S1 Sa9 15 S1 Sr9 7 S2 Kr10 9 S2 Sa10 9 S2 Sr10 13 S1 Kr11 7 S2 Sa11 9 S2 Kr12 7 S2 Sa12 7 S2 Kr13 9 S2 Kr14 9 S2 Kr15 9 S2 Kr16 13 S1 Kr17 9 S2 Information : S1 = very suitable, S2 = suitable, S3 = conditionally suitable. Information : S1 = very suitable, S2 = suitable, S3 = conditionally suitable. 10 10 E3S Web of Conferences 317, 05025 (2021) ICENIS 2021 https://doi.org/10.1051/e3sconf /202131705025 E3S Web of Conferences 317, 05025 (2021) Fig. 7. map of mangrove land suitability at Kretek and Sanden Districts, Bantul Regency, Indonesia Fig. 7. map of mangrove land suitability at Kretek and Sanden Districts, Bantul Regency, Indonesi Fig. 8. map of mangrove land suitability at Srandakan Districts, Bantul Regency, Indonesia. After analyzing the geospatial method, it was found that the very suitable class area for mangrove restoration land was 168 ha and the suitable class was 181 ha. However, some of Fig. 8. map of mangrove land suitability at Srandakan Districts, Bantul Regency, Indonesia. 4 Conclusions Based on this research, it can be concluded that the suitability of mangrove restoration land can be carried out at locations based on priority parameters in Kretek district, Sanden district (Opak Estuary River) and Srandakan District (Progo Estuary River). After analyzing the geospatial method, it was found that the very suitable class area for mangrove restoration land was 168 ha and the suitable class was 181 ha. 7 After analyzing the geospatial method, it was found that the very suitable class area for mangrove restoration land was 168 ha and the suitable class was 181 ha. However, some of 11 E3S Web of Conferences 317, 05025 (2021) ICENIS 2021 https://doi.org/10.1051/e3sconf /202131705025 the physical parameters analyzed in this study are insufficient to develop disaster management efforts or sustain climate change mitigation efforts. It still requires a community development approach from planning, planting, maintenance, and management to achieve mangrove cultivation [29]. Acknowledgement Thank you to the head of the Mangrove group in Sanden district, Bantul regency, CV. Maheswari Bumi for assisting in completion of this research. References 1. C. Giri, E. Ochieng, L. L. Tieszen, Z. Zhu, A. Singh, T. Loveland, J. Masek, N. Duke, Glob. Ecol. Biogeogr., 20, 154 (2011) 2. J. Chow, J. Sustain. For., 37, 139 (2018) 3. D. Murdiyarso, J. Purbopuspito, J. B. Kauffman, M. W. Warren, S. D. Sasmito, D. C. Donato, S. Manuri, H. Krisnawati, S. Taberima, S. Kurnianto, Nat. Clim. Chang., 5, 1089 (2015) 4. D. C. Donato, J. B. Kauffman, D. Murdiyarso, S. Kurnianto, M. Stidham, M. Kanninen, Nat. Geosci., 4, 293 (2011) 5. R. DasGupta, R. Shaw, J. Ecosyst., 2013, 1 (2013) 6. R. Fitri, K. Anwar, J. FISIP, 1, 1 (2014) 7. S. Eddy, A. Mulyana, M. S. Dr. Moh. Rasyid Ridho, M. S. Prof. Dr. Iskhaq Iskandar, 1, 240 (2017) 8. A. N. Indah, Domistic Case Study, 9, (2019) 9. N. P. D. M. N. Setiawan, Analisis Kesesuaian Habitat Mangrove Pada Kawasan Mangrove Buatan Baros Bantul, Daerah Istimewa Yogyakarta, (Universitas Gadjah Mada Yogyakarta, 2013) 10. P. Subardjo, R. Ario, J. Kelaut. Trop., 18, 82 (2016) 11. H. Y. L. Totok Wahyu Wibowo, E. A. W. Putri, Pros. Semin. Nas. Geogr., UMS, 8, 343-355 (2017) 12. Zaky et al, J. Mar. Res., 1, 88 (2012) 13. Indratin, M. A. Budihardjo, M. Helmi, E3S Web Conf., 202, (2020) 14. Poniman, T. R. Soeprobowowati, M. Helmi, E3S Web Conf., 202, (2020) 15. Iswandi, M. Helmi, Hadiyanto, IOP Conf. Ser. Earth Environ. Sci., 448, (2020) 16. N. Sudirman, M. Helmi, N. S. Adi, E3S Web Conf., 73, (2018) 17. H. S. H. M.Helmi, A. Satriadi, A. A. Dwi Suryoputro, J. Marwoto, Int. J. Civ. Eng. Technol., 9, 2949 (2018) 12 E3S Web of Conferences 317, 05025 (2021) ICENIS 2021 https://doi.org/10.1051/e3sconf /202131705025 18. R. W. M. Helmi, Y. H. Pholandani, H. Setiyono, A. Wirasatriya, W. Atmodjo, A. A. D. S. I, Int. J. Sci. Technol. Res., 9, (2020) 19. T. T. Putranto, T. Amanah, B. Warsito, H. Purnaweni, M. Helmi, Int. J. Geomate, 18, 124 (2020) 20. B. W. Mutaqin, M. A. Marfai, M. Helmi, M. G. Rindarjono, R. Windayati, Sunarto, IOP Conf. Ser. Earth Environ. Sci., 451, (2020) 21. M. M. Helmi, A. Aysira, R. W., A. Wirasatria, R. Ario, Indones. J. Oceanogr., 2, 8 (2020) 22. A. D. P. C. P. S. Budhi, N. Latifah, Semin. Nas. Penginderaan Jauh Ke 6, 8, 439 (2019) 23. A. T. M. A. D. Savitri, Junianto, J. Perikan. References Dan Kelaut., 3, 301 (2012) 24. F. I. D. S. Wahyuningsih, M. D. Putra, Th. R. Wulan, S. P. Anggara, E. Maulana, Semin. Nas. Geogr., UMS 8, (2016) 25. R. V. S. E. Mlcleod, Managing Mangroves for Resilience to Climate Change, (2006) 26. Dahuri, Keanekaragaman Hayati Laut: Aset Pembangunan Berkelanjutan Indonesia (PT. Gramedia Pustaka, 2003) 27. E. M. G.B. Saputro, M.I.C. Marschiavelli, F.Ibrahim, IOP Conf. Ser. Earth Environ. Sci., 54, 1 (2017) 28. B. Wasis, Dep. 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https://openalex.org/W3125601656	https://www.detstom.ru/jour/article/download/308/277	Russian	null	Dentist’s approach to management of oral mucosa lesions in children with Behcet’s disease	Stomatologiâ detskogo vozrasta i profilaktika	2,021	cc-by	6,728	Оригинальная статья Оригинальная статья DOI: 10.33925/1683-3031-2020-20-4-288-295 Резюме При дополнительном обследовании детей с ББ, только с тяжелой формой ХРАС, выявляли наличие анемии, увеличение СОЭ и изменение иммунологических показателей: снижение общего количества Т-лимфоцитов и Т-хелперов, нарастание уровня циркулирующих иммунных комплексов (ЦИК), повышение отдельных классов иммуноглобулинов (особенно IgA), хемотаксической активности нейтрофилов, снижение в слюне секреторного IgA. 288 р р g Выводы. Таким образом, ББ у детей недостаточно изучена, в связи с чем отмечается поздняя диагностика заболевания. В течение 5,40 ± 0,04 лет у девочек с ББ, а у мальчиков 4,10 ± 0,09 лет заболевание протека- ет как неполный синдром Бехчета, проявляясь только хроническим рецидивирующим афтозным стоматитом в 100% случаев, который является первым диагностическим симптомом заболевания. Разработанный нами комплексный план обследования предложен для врачей-стоматологов, которые наблюдают детей с заболе- ваниями слизистой оболочки полости рта. Диагностика и лечение ББ у детей требует междисциплинарного подхода врачей различной специализации и обязательное участие врача-стоматолога. р р у р Ключевые слова: дети, болезнь Бехчета, хронический рецидивирующий афтозный стоматит, неполный син- дром Бехчета Для цитирования: Скакодуб А.А., Адмакин О.И., Геппе Н.А. Тактика врача стоматолога при поражении слизистой оболочки полости рта у детей с болезнью Бехчета. Стоматология детского возраста и профилакти- ка.2020;20(4):288-295. DOI: 10.33925/1683-3031-2020-20-4-288-295. Для цитирования: Скакодуб А.А., Адмакин О.И., Геппе Н.А. Тактика врача стоматолога при поражении слизистой оболочки полости рта у детей с болезнью Бехчета. Стоматология детского возраста и профилакти- ка.2020;20(4):288-295. DOI: 10.33925/1683-3031-2020-20-4-288-295. Резюме Резюме Актуальность. Болезнь Бехчета (ББ) – редкая патология детского возраста, среди всех пациентов с болезнью Бехчета дети составляют 2-3%. Стоматологи первые сталкиваются с этой тяжелой патологией, длительно наблюдая детей с хроническим рецидивирующим афтозным стоматитом (ХРАС), который имеет длительное течение, частые рецидивы и резистентность к проводимому лечению. Это потребовало разработки плана про- ведения совместного обследования и лечения детей у стоматологов, ревматологов и педиатров. Материалы и методы. Нами было проведено обследование детей, находящихся на стационарном лечении в отделении ревматологии УДКБ Первого МГМУ им. И.М. Сеченова, в количестве 21 ребенка в возрасте от 6 до 17 лет, с диагнозом «болезнь Бехчета», которых направляли на полное совместное обследование всего организма к стоматологу, педиатру и ревматологу. Был разработан план обследования, он включал в себя: установление зональности заболевания и генетической предрасположенности, выявление провоцирующего фактора и срока появления первичного диагностического признака ХРАСа, а также сроков присоединения последующих клинических признаков заболевания, проведение лабораторных исследований и гистоморфо- логических исследований пораженных тканей слизистой оболочки. р Результаты. При применении предложенного нами плана обследования у детей с болезнью Бехчета было установлено, что 18 детей (85,7%) имеют национальную принадлежность «шелкового пути» данного забо- левания. У 100% детей первичный симптом этого заболевания ХРАС, и он протекает у 14 детей (66,67%) в тяжелой форме, у 7 детей (33,34%) с ежемесячными рецидивами и у 6 детей (28,57%) с постоянными реци- дивами. Из других симптомов ББ у детей наиболее часто – у 90,9% – поражаются органы зрения, у 90,9% наблюдается поражение органов ЖКТ, у 72,7% поражены органы выделительной системы, и у 45,4% встре- чается суставной синдром. При дополнительном обследовании детей с ББ, только с тяжелой формой ХРАС, выявляли наличие анемии, увеличение СОЭ и изменение иммунологических показателей: снижение общего количества Т-лимфоцитов и Т-хелперов, нарастание уровня циркулирующих иммунных комплексов (ЦИК), повышение отдельных классов иммуноглобулинов (особенно IgA), хемотаксической активности нейтрофилов, снижение в слюне секреторного IgA. р Результаты. При применении предложенного нами плана обследования у детей с болезнью Бехчета было установлено, что 18 детей (85,7%) имеют национальную принадлежность «шелкового пути» данного забо- левания. У 100% детей первичный симптом этого заболевания ХРАС, и он протекает у 14 детей (66,67%) в тяжелой форме, у 7 детей (33,34%) с ежемесячными рецидивами и у 6 детей (28,57%) с постоянными реци- дивами. Из других симптомов ББ у детей наиболее часто – у 90,9% – поражаются органы зрения, у 90,9% наблюдается поражение органов ЖКТ, у 72,7% поражены органы выделительной системы, и у 45,4% встре- чается суставной синдром. Тактика врача-стоматолога при поражении слизистой оболочки полости рта у детей с болезнью Бехчета Скакодуб А.А., Адмакин О.И., Геппе Н.А. Первый МГМУ им. И.М. Сеченова (Сеченовский университет), Москва, Российская Федерация Оригинальная статья Оригинальная статья Materials and methods. We examined children hospitalized to rheumatology department of University Children’s Clinical Hospital of Sechenov First Moscow State Medical University. There were 21 children aged between 6 and 17 with Behcet’s disease who were referred to a dentist, pediatrician and rheumatologist for a comprehensive examination of the whole body. The developed plan included assessment of disease localization and genetic predisposition, detection of a trigger and time of first manifestations of the primary diagnostic sign of RAS and manifestation time of subsequent clinical signs of the disease, laboratory tests and histomorphologic evaluation of damaged mucosa. g Results. Upon application of the suggested plan of examination in children with Behcet’s disease it was found out that 18 (85.7%) children had nationality of the Silk Road. In 100% of children chronic recurrent aphtous stomatitis was the first symptom of the disease with severe course in 14 (66.67%) children, monthly relapses in 7 (33.34%) children and chronic relapses in 6 (28.57%) children. Among other symptoms of Behcet’s disease in children eye lesions are the most frequent (90.9%), the digestive system is affected in 90.9%, excretory organs are affected in 72.7%, and articular syndrome is frequently encountered (45.4%). Anemia, elevated ESR, immune index changes, decrease of total number of T-lymphocytes and T-helpers, elevated level of circulating immune complex (CIC), elevation of specific classes of immunoglobulins (especially IgA), hemotoxic activity of neutrophils and decrease of secretory IgA in saliva were detected on additional examination of children with Behcet’s disease (only severe RAS). Conclusions. Thus, Behcet’s disease has not been sufficiently studied yet, that’s why late diagnosis is observed. The disease proceeds as incomplete Behcet’s syndrome during 5.4+0.04 years in girls and 4.1+0.09 years in boys, manifesting itself only by chronic recurrent aphtous stomatitis in 100% of cases which is the first diagnostic symptom of the disease. The comprehensive diagnostic plan, developed by us, is offered to dentists who observe children with oral mucosa diseases. Diagnosis and treatment of Behcet’s disease in children require an interdisciplinary approach by doctors of different specialties and an obligatory involvement of a dentist. Key words: children, Behcet’s disease, chronic recurrent aphtous stomatitis, incomplete Behcet’s syndrome For citation: Skakodub, A.A., Admakin, O.I., Geppe, N.A. Dentist’s approach to management of oral mucosa lesions in children with Behcet’s disease. Pediatric dentistry and dental prophylaxis.2020;20(4):288-295. DOI: 10.33925/1683- 3031-2020-20-4-288-295. 289 Заболевания слизистой полости рта занимают важ- ное место в практике врача-стоматолога детского. Оригинальная статья Спектр их довольно широк – от изолированных мел- ких поражений до тяжелых проявлений системных заболеваний, к которым и относится болезнь Бехчета (ББ), представляющих угрозу жизни ребенка [2, 3, 5, 12, 14, 18, 19], что требует серьезных знаний в области стоматологии, педиатрии и ревматологии. и, связываясь между собой, образуют иммунные ком- плексы. Последние, оседая на стенках сосудов, вызы- вают повреждения их эндотелия [1, 4, 18, 19]. Несмотря на многообразие исследований, этио- логия и патогенез заболевания остаются предметом многочисленных дискуссий. Окончательно не установлено, какие факторы до- минируют в патогенезе хронического рецидивирую- щего афтозного стоматита, являясь первичным сим- птомом этого заболевания, а какие предрасполагают к заболеванию [1, 8, 9, 11, 14, 15]. ББ – своеобразная клиническая форма системного васкулита, поражающего преимущественно артерии и вены среднего и мелкого калибра. Характеризуется рецидивирующим эрозивно-язвенным поражением слизистых оболочек полости рта, половых органов, ча- стым поражением глаз, кожи, с вовлечением суставов, нервной системы, внутренних органов [1, 3-5, 11, 12, 14, 18, 19]. ББ – редкая патология детского возраста, среди всех пациентов с ББ дети составляют 2-3%. ББ возникает преимущественно у молодых мужчин в воз- расте 20-40 лет; клинические признаки этого заболе- вания формируются у детей в возрасте до 7-13 лет. За- болевание чаще возникает у мужчин, они поражаются в два-три раза чаще, чем женщины. Наиболее часто ББ встречается в Турции (2-42:10 000) и Японии (1:10 000), сведений о распространенности в России нет [1, 5, 12, 19]. Этиология ББ неизвестна. Предполагается, но не доказана окончательна роль вирусной инфекции, в частности вируса простого герпеса и стрептококко- вой инфекции, а также токсических агентов, которые служат провокатором аутоиммунного механизма при наличии генетической предрасположенности. Извест- но, что иммуногенетическим маркером ББ являются антигены HLA-B5, -B51 и DRw52, особенно велик риск развития болезни при одновременном наличии анти- генов -B51 и DRw52. Полагают, что нефиксированные антитела и антигенны встречаются в кровяном русле Стоматологи первые сталкиваются с этой тяжелой патологией, наблюдая течение хронического рециди- вирующего афтозного стоматита, и выявляют, что для него свойственно длительное течение, частые рециди- вы и резистентность к проводимому лечению [1, 2, 7, 8, 10, 13, 16]. В настоящее время, несмотря на многообразие раз- личных методов лечения хронического рецидивирую- щего афтозного стоматита при ББ, проблема излече- ния данного заболевания остается достаточно острой. Цель исследования – повышение уровня диагно- стики и лечения ББ у детей за счет междисциплинар- ного подхода в реабилитации этого заболевания. Abstract Abstract Relevance. Behcet’s diasease (BD) is a rare pediatric pathology, children compose 2-3% of all patients with Behcet’s disease. Dentists are the first to encounter this severe disease, they provide continuous observation of children with chronic recurrent aphtous stomatitis (RAS), which has a prolonged course, frequent recurrences and resistance to the administered treatment. Elaboration of a program of collaborative examination and treatment of children by dentists, rheumatologists and pediatricians was required. Purpose – to increase the level of diagnosis and treatment of Behcet’s disease in children through the interdisciplinary approach to rehabilitation of the disease. 020 (76) Стоматология детского возраста и профилактика РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ И ИХ ОБСУЖДЕНИЕ РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ И ИХ ОБСУЖДЕНИЕ ми такой редкой патологии позволил нам проанализи- ровать особенности течения, клиники, диагностики и лечения ББ у детей. За эти годы мы оказали стоматологическую помощь 21 ребенку с ББ. Из них девочек было 9 (42,8%), маль- чиков 12 (57,2%). Начало этого заболевания может длительно проявляться только в виде хронического ре- цидивирующего афтозного стоматита, а остальные ди- агностические симптомы заболевания присоединяют- ся гораздо позже, поэтому детям в основном ставится диагноз «вероятная» или «неполная» ББ. Поэтому чрез- вычайно важно было применять составленный нами очень подробный план обследования, основанный на жалобах, симптоматических проявлениях, характере рецидивов, генетических и национальных аспектах, до- полнительных лабораторных исследованиях. Под диспансерное наблюдение и лечение мы взяли 21 ребенка с ББ, из них 9 девочек и 12 мальчиков, в возрасте от 6-17 лет (средний возраст 12,5 лет). Пер- вичный симптом этого заболевания – хронический рецидивирующий афтозный стоматит (ХРАС), поэтому ребенок первично обращался к врачу-стоматологу, а мы соответственно направляли на полное совмест- ное обследование всего организма к врачу-педиатру и ревматологу. у При проведении совместного обследования со- ставляли план, который включал в себя: В результате обследования наших пациентов по со- ставленному плану мы: 1. Установление зональности заболевания. 1. Установление зональности заболевания. 2. Выявление провоцирующего фактора. 1. Установили зональность заболевания и нацио- нальную принадлежность детей с ББ (табл. 1). 1. Установили зональность заболевания и нацио- нальную принадлежность детей с ББ (табл. 1). 3. Определение генетической предрасположенности. 4. Выявление срока появления первичного диагно- стического признака ХРАС. Из данной таблицы, видно, что у детей с ХРАС по- сле полного обследования был поставлен диагноз ББ у 21 ребенка. Проанализировав их национальную принадлежность, мы установили, что восемь детей (38,09%) – выходцы с Кавказа и Закавказья, четве- ро детей (19,04%) – с татарскими корнями, четверо детей (19,04%) – выходцы из Якутии и Бурятии, двое детей (9,52%) – с еврейскими корнями и трое детей (14,29%) – славянской национальности. Такие данные, возможно, подтверждают теорию о «шелковом пути» распространения этого заболевания и генетической предрасположенности. Поэтому врачам-стоматоло- гам при обращаемости детей с ХРАС, нужно уделять внимание этим факторам и обязательно проводить скрининг на наличие иммуногенетических маркеров ББ, что позволит на ранних этапах заболевания прово- дить адекватное противоревматическое лечение. 5. Установление периодичности, тяжести и длитель- ности течения ХРАС. 6. РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ И ИХ ОБСУЖДЕНИЕ Выявление сроков присоединения последующих клинических признаков заболевания: – поражения половых органов; – поражение кожи; – поражение глаз; – поражение суставов; – поражение сосудов; 290 – поражение органов пищеварения; – поражение нервной системы; – поражение сердца; – поражение легких; – поражение почек. 7. При проведении лабораторных исследований учитывали: 2. Тщательно собирали анамнез и выявили основ- ные провоцирующие факторы (табл. 2). – наличие анемии, увеличение СОЭ, появление рев- матоидных показателей; – иммунологических показателей: снижение обще- го количества Т-лимфоцитов и Т-хелперов, нарастание уровня циркулирующих иммунных комплексов (ЦИК), повышение отдельных классов иммуноглобулинов (особенно IgA), хемотаксической активности нейтро- филов, снижение в слюне секреторного IgA. По данным этой таблицы, мы делам выводы, что врачам-стоматологам при обращаемости детей с ХРАС требуется уделять особое внимание при сборе анамнеза провоцирующих факторов, особенно если у ребенка частые ангины, перенесенные вирусные герпетические инфекции (I, II, IV типа) и хронические заболевания желудочно-кишечного тракта и бронхо- легочной системы, так как это основные провоциру- ющие факторы в преморбидном периоде ББ у детей. 8. Проведение гистоморфологических исследований пораженных тканей слизистой оболочки и выявление: – наличие васкулитов: в стенках капилляров ин- фильтрация лимфоцитами, плазматическими клетка- ми, моноцитами, нейтрофилами, наличие внутрисосу- дистых конгломератов из нейтрофилов; 3. По данным скрининга на наличие иммуногенетиче- ских маркеров ББ, проведенного у 12 детей, были обна- ружены статистически значимые ассоциации ББ и анти- гены гистосовместимости HLA-B5, -B51 у девяти детей с «неполной формой» ББ, а у трех детей отсутствовали эти маркеры, хотя у них была выявлена триада больших симптомов этого заболевания, что дало право назна- чать базисную глюкокортикостероидную терапию. 3. По данным скрининга на наличие иммуногенетиче- ских маркеров ББ, проведенного у 12 детей, были обна- ружены статистически значимые ассоциации ББ и анти- гены гистосовместимости HLA-B5, -B51 у девяти детей с «неполной формой» ББ, а у трех детей отсутствовали эти маркеры, хотя у них была выявлена триада больших симптомов этого заболевания, что дало право назна- чать базисную глюкокортикостероидную терапию. – наличие пролиферации эндотелия, сужение или облитерация сосуда, фибриноидный некроз стенки. 9. Проведение консультации других специалистов, в зависимости от вовлечения органов и систем в пато- логический процесс (лора, окулиста, гинеколога, уро- лога, гастроэнтеролога, невропатолога и т. д.). Такие полученные данные говорят нам еще раз о том, что недостаточно одного метода исследования для по- становки такого тяжелого диагноза, особенно у детей. Диагноз у всех исследуемых был поставлен в соответ- ствии с данными нашего обследования и международны- ми диагностическими критериями ББ (ISBD1990 г.). 4. Оригинальная статья Оригинальная статья МАТЕРИАЛЫ И МЕТОДЫ ИССЛЕДОВАНИЯ На кафедре стоматологии детского возраста и ор- тодонтии Первого МГМУ им. И.М. Сеченова на протя- жении 15 лет оказывали стоматологическую помощь детям с системными васкулитами, находившимся на лечении в ревматологическом отделении универси- тетской детской клинической больницы клинического института детского здоровья им. Н.Ф. Филатова Пер- вого МГМУ им И.М Сеченова. Этот опыт работы с деть- РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ И ИХ ОБСУЖДЕНИЕ Выявили сроки появления первичного диагности- ческого признака ХРАС и возраст постановки основ- ного диагноза (табл. 3). 4. Выявили сроки появления первичного диагности- ческого признака ХРАС и возраст постановки основ- ного диагноза (табл. 3). Данное научное исследование прошло локальный комитет по этике ФГАОУ ВО им. М.М. Сеченова Пер- вого Московского государственного медицинского университета Министерства здравоохранения Россий- ской Федерации (Сеченовский университет). У обсле- дуемых были получены информированные согласия на диагностику, лечение и проведение фотопротоко- лов в рамках проводимого исследования. Данный анализ наглядно показывает, что в среднем от появления первичного симптома ХРАС до постанов- ки основного диагноза ББ у девочек проходит 5,40 ± 0,04 лет, а у мальчиков 4,10 ± 0,09 лет, и ребенок дли- тельно остается без базисного лечения, что приводит к тяжелым необратимым последствиям поражения вну- Оригинальная статья № Регион проживания и национальность Region of residence and nationality Девочки / Girls (n = 9) Мальчики / Boys (n = 12) 1 Северный Кавказ (чеченка, осетин, дагестанцы) / North Caucasus (chechen girl, ossetian, 2 dagestani) 1 3 2 Москва (грузинка, два армянина, сириец, еврей) / Moscow (georgian girl, 2 armenians, syrian, jew) 1 4 3 Московская область (еврей, татарин) / Moscow region (jew, tatar) – 2 4 Пермский край (русские) / Perm region (russians) 2 – 5 Оренбургская область (татарин) / Orenburg region (tatar) – 1 6 Читинская область (бурятка) / Chita region (buriat) 1 – 7 Якутск (якутка) / Yakutsk (yakut) 1 – 8 Новосибирская область (русская) / Novosibirsk region (russian) 1 – 9 Казань (татарин) / Kazan (tatar) – 1 10 Южно-Сахалинск (якут) / Yuzhno-Sakhalinsk (yakut) – 1 11 Улан-Удэ (бурятка) / Ulan-Ude (buriat) 1 – 12 Пензенская область (татарка) / Penza region (tatar) 1 – Таблица 1. Место проживания и национальная принадлежность детей с ББ Table 1. РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ И ИХ ОБСУЖДЕНИЕ Region of Residence and Nationality of Children with Behçet's Disease Предшествующие патологические состояния, n (%) Preexisting pathologic conditions n (%) Девочки / Girls (n = 9) Мальчики / Boys (n = 12) Частые ангины (хронический тонзиллит) – 8 (38,09) / Frequent quinsy (chronic tonsillitis) – 8 (38.09) 4 4 Перенесли ветряную оспу – 5 (23,81) / Past chicken-pox – 5 (23.81) 3 2 Острый герпетический стоматит перенесен – 6 (28,57) / Past acute herpetic stomatitis – 6 (28.57) 2 4 Бронхолегочные инфекции – 5 (23,81) / Bronchopulmonary infections – 5 (23.81) 2 3 Геморрагический васкулит – 1 (4,76) / Hemorrhagic purpura – 1 (4.76) 1 – Заболевания органов пищеварения – 8 (38,09) / Diseases of the digestive system – 8 (38.09) 3 5 Таблица 2. Провоцирующие факторы преморбидном периоде ББ у детей Table 2. Provoking factors in children during premorbid period of Behcet's disease Показатели / Parameters Девочки / Girls (n = 9) Мальчики / Boys (n = 12) Среднее значение показателей возрастного дебюта первичного симптома ХРАС Mean age of the onset of the primary symptom of CRAS (years) 5,80 ± 0,09 7,90 ± 0,07 Среднее значение возрастных показателей постановки основного диагноза ББ Diagnosis of Behcet’s disease, mean age (years) 11,30 ± 0,03 12,10 ± 0,06 Среднее значение возрастного периода течения ББ от дебюта первичного симптома ХРАС до постановки основного диагноза Mean time from the onset of the primary symptom of chronic recurrent aphthous stomatitis to diagnosis of the main disease (Behcet’s disease) (years) 5,40 ± 0,04 4,10 ± 0,09 Таблица 3. Анализ среднего значение возрастного периода течения ББ от дебюта первичного симптома ХРАС до постановки основного диагноза (лет) у детей при ББ Table 3. Analysis of the mean age period of Behcet's disease course from the onset of the primary symptom of CRAS to the diagnosis (years) in children with Behcet's disease Таблица 1. Место проживания и национальная принадлежность детей с ББ Table 1. Region of Residence and Nationality of Children with Behçet's Disease 291 элемент поражения – это афты, которые представ- ляли собой эрозии различной формы и размером от 1-5 мм до 1,5-2 см, покрытые фибринозным налетом и окруженные гиперемированным ободком воспаления. Они локализовались на слизистой: преддверия рта, верхней и нижней губы, боковых поверхностях языка, ретромолярной области и щечной по линии смыкания зубов. Мы провели анализ периодичности и степени тяжести течения ХРАС (табл. Рис. 3. Ребенок 8 лет, ББ. Тяжелый хронический рецидивирующий афтозный стоматит, обширные афты и язвы, с последующим грубым рубцеванием и деформацией органов полости рта (собственное наблюдение) Fig. 3. 8-year-old child, Behcet’s disease. Severe chronic recurrent aphthous stomatitis, major and minor aphthous ulcers with subsequent significant scars and deformed oral organs (self-assessment) РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ И ИХ ОБСУЖДЕНИЕ 4) и выявили, что у двух детей легкое течение ХРАС, в полости рта наблюдали единичные эрозии размером 1-5 мм, рецидивирующие тренних органов и систем. Поэтому врач-стоматолог должен иметь настороженность при длительном тече- нии ХРАС у ребенка и его утяжелении, для этого требо- валось обследование слизистой полости рта. элемент поражения – это афты, которые представ- ляли собой эрозии различной формы и размером от 1-5 мм до 1,5-2 см, покрытые фибринозным налетом и окруженные гиперемированным ободком воспаления. р 5. Проведено тщательное клиническое обследова- ние слизистой полости рта. у Они локализовались на слизистой: преддверия рта, верхней и нижней губы, боковых поверхностях языка, ретромолярной области и щечной по линии смыкания зубов. Мы провели анализ периодичности и степени тяжести течения ХРАС (табл. 4) и выявили, что у двух детей легкое течение ХРАС, в полости рта наблюдали единичные эрозии размером 1-5 мм, рецидивирующие Наблюдали большие, малые и герпетиформные афты. Они были одиночные и множественные, в виде язв с последующим грубым рубцеванием слизистой, часто инфицировались. В полости рта наблюдается разнообразная клиническая картина ХРАС. Основной 4/2020 (76) Стоматология детского возраста и профилактик Оригинальная статья Рис. 2. Ребенок 14 лет, ББ. Среднетяжелый хронический рецидивирующий афтозный стоматит, множественные афты размером 0,5-1 см, рецидивирующие ежемесячно (до лечения) (собственное наблюдение) Fig. 2. 14-year-old child, Behcet’s disease. Moderate chronic recurrent aphthous stomatitis, multiple aphthous ulcers, 0.5-1 cm in size, monthly recurrence (before treatment) (self-assessment) Рис. 2. Ребенок 14 лет, ББ. Среднетяжелый хронический рецидивирующий афтозный стоматит, множественные афты размером 0,5-1 см, рецидивирующие ежемесячно (до лечения) (собственное наблюдение) Рис. 1. Ребенок 12 лет, ББ. Легкий хронический рецидивирующий афтозный стоматит, единичные эрозии размером 1-5 мм, рецидивирующие два раза в год (до лечения) (собственное наблюдение) Fig. 1. 12-year-old child, Behcet’s disease. Mild chronic recurrent aphthous stomatitis, single erosions, 1-5 mm in size, securrent twice a year (before treatment) (self-assessment) Рис. 1. Ребенок 12 лет, ББ. Легкий хронический рецидивирующий афтозный стоматит, единичные эрозии размером 1-5 мм, рецидивирующие два раза в год (до лечения) (собственное наблюдение) Fig. 1. 12-year-old child, Behcet’s disease. Mild chronic recurrent aphthous stomatitis, single erosions, 1-5 mm in size, securrent twice a year (before treatment) (self-assessment) Рис. 1. Ребенок 12 лет, ББ. Легкий хронический рецидивирующий афтозный стоматит, единичные эрозии размером 1-5 мм, рецидивирующие два раза в год (до лечения) (собственное наблюдение) Рис. 3. Ребенок 8 лет, ББ. Тяжелый хронический рецидивирующий афтозный стоматит, обширные афты и язвы, с последующим грубым рубцеванием и деформацией органов полости рта (собственное наблюдение) Fig. 3. 8-year-old child, Behcet’s disease. Рис. 2. Ребенок 14 лет, ББ. Среднетяжелый хронический рецидивирующий афтозный стоматит, множественные афты размером 0,5-1 см, рецидивирующие ежемесячно (до лечения) (собственное наблюдение) Fig. 2. 14-year-old child, Behcet’s disease. Moderate chronic recurrent aphthous stomatitis, multiple aphthous ulcers, 0.5-1 cm in size, monthly recurrence (before treatment) (self-assessment) РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ И ИХ ОБСУЖДЕНИЕ Severe chronic recurrent aphthous stomatitis, major and minor aphthous ulcers with subsequent significant scars and deformed oral organs (self-assessment) 292 два-четыре раза в год, эпителизирующиеся четыре- пять дней, без рубцов (рис. 1). при легкой степени течения ХРАС надо тщательно соби- рать анамнез, и наличие хотя бы оного симптома требу- ет проведения обследования ребенка по предложенно- му плану. Кроме клинических обследований большое значение уделяется и дополнительным методам. У пяти детей среднетяжелое течение ХРАСа в по- лости рта выявляли множественные афты размером 0,5-1 см, рецидивирующие ежемесячно, эпителизиру- ющиеся 7-10 дней, без грубой деформации (рис. 2). 7. Были проведены лабораторные исследование и выявлено следующее (табл. 6). У 14 детей тяжелое течение ХРАС: наблюдали обшир- ные афты и язвы размером 1-2,5 см, постоянно рецидиви- рующие, эпителизирующиеся 10-14 дней, с последующим грубым рубцеванием и деформацией слизистой (рис. 3). У детей при ББ с тяжелой формой ХРАС выявляли наличие анемии, увеличение СОЭ, появление ревма- тоидных показателей. По данным иммунологических показателей, у детей при ББ с тяжелой формой ХРАС выявляли снижение общего количества Т-лимфацитов и Т-хелперов, нарастание уровня ЦИК, повышение от- дельных классов иммуноглобулинов (особенно IgA), хемотаксической активности нейтрофилов, снижение в слюне секреторного sIgA (табл. 6). При сборе анамнеза большое значения уделяли пе- риодичности рецидивов. Проведенное тщательное клиническое обследо- вание слизистой полости рта и выявление периодич- ности рецидивов показали, что при ББ у детей ХРАС протекает у 14 детей (66,67%) в тяжелой форме, у 7 де- тей (33,34%) с ежемесячными рецидивами и у 6 детей (28,57%) с постоянными рецидивами. 8. У пяти больных с ББ с тяжелой формой постоянно рецидивирующего течения ХРАС была взята биопсия пораженных тканей слизистой оболочки (онконасто- роженность) и проведено гистоморфологическое ис- следование биоптатов. Такое длительное течение, частые рецидивы и рези- стентность к проводимому лечению должно насторо- жить врача-стоматолога как в сторону ревматической патологии, так и онконастороженности. Было выявлено: Было выявлено: Чтобы установить диагноз ББ, нужно наличие сле- дующих критериев: рецидивирующие афты в полости рта, на гениталиях, поражения глаз, поражения кожи и других органов [1, 3-5], поэтому выявляли сроки и на- личие последующих симптомов заболевания. – наличие васкулитов: в стенках капилляров ин- фильтрация лимфацитами, плазматическими клетка- ми, моноцитами, нейтрофилами, наличие внутрисосу- дистых конгломератов из нейтрофилов; – наличие васкулитов: в стенках капилляров ин- фильтрация лимфацитами, плазматическими клетка- ми, моноцитами, нейтрофилами, наличие внутрисосу- дистых конгломератов из нейтрофилов; – наличие пролиферации эндотелия, сужение или облитерация сосуда, фибриноидный некроз стенки. 6. У наших пациентов в процессе наблюдения и об- следования были выявлены следующие клинические признаки ББ (табл. 5). Рис. 1. Ребенок 12 лет, ББ. Легкий хронический рецидивирующий афтозный стоматит, единичные эрозии размером 1-5 мм, рецидивирующие два раза в год (до лечения) (собственное наблюдение) Fig. 1. 12-year-old child, Behcet’s disease. Mild chronic recurrent aphthous stomatitis, single erosions, 1-5 mm in size, securrent twice a year (before treatment) (self-assessment) РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ И ИХ ОБСУЖДЕНИЕ Recurrence rate of various forms of chronic recurrent aphthous stomatitis in children with Behcet’s disease Клинические признаки ББ Clinical signs of Behcet’s disease Девочки / Girls (n = 9) Мальчики / Boys (n = 12) Всего / Total (n = 21) Хронический рецидивирующий афтозный стоматит / Chronic recurrent aphthous stomatitis 100 100 100 Поражения половых и выделительных органов / Genital ulcers and urinary organ lesions 75 71,4 72,7 Поражение кожи / Skin lesions 50 28,5 36,6 Нарушение зрения / Ocular lesions 75 100 90,9 Поражение суставов / Joint involvement 50 42,8 45,4 Поражение органов пищеварения / GI manifestations 100 85,7 90,9 Поражение нервной системы / Nervous system involvement 9 14,2 9,1 Поражение сердца / Heart involvement 25 28,5 27,2 Поражение легких / Lung involvement – 28,5 18,1 Таблица 5. Клинические признаки ББ (%) Table 5. Clinical signs of Behcet's disease (%) Лабораторные показатели Легкая форма ХРАС. Единичные эрозии (n = 2) Mild CRAS Среднетяжелая форма ХРАС. Множественные афты (n = 5) Тяжелая форма ХРАС. Обширные афты и язвы, с последу- ющим грубым рубцеванием (n = 14) Severe CRAS Таблица 6. Лабораторные показатели состояния организма при различных формах хронического рецидивирующего афтозного стоматита у детей с ББ Table 6. Laboratory values of body condition in various forms of chronic recurrent aphthous stomatitis in children with Behcet’s disease Таблица 5. Клинические признаки ББ (%) Table 5. Clinical signs of Behcet's disease (%) Таблица 5. Клинические признаки ББ (%) Table 5. Clinical signs of Behcet's disease (%) Клинические признаки ББ Clinical signs of Behcet’s disease Девочки / Girls (n = 9) Мальчики / Boys (n = 12) Всего / Total (n = 21) Хронический рецидивирующий афтозный стоматит / Chronic recurrent aphthous stomatitis 100 100 100 Поражения половых и выделительных органов / Genital ulcers and urinary organ lesions 75 71,4 72,7 Поражение кожи / Skin lesions 50 28,5 36,6 Нарушение зрения / Ocular lesions 75 100 90,9 Поражение суставов / Joint involvement 50 42,8 45,4 Поражение органов пищеварения / GI manifestations 100 85,7 90,9 Поражение нервной системы / Nervous system involvement 9 14,2 9,1 Поражение сердца / Heart involvement 25 28,5 27,2 Поражение легких / Lung involvement – 28,5 18,1 g ( ) 293 Лабораторные показатели Laboratory values Легкая форма ХРАС. Единичные эрозии (n = 2) Mild CRAS. Single aphthae (n = 2) Среднетяжелая форма ХРАС. Множественные афты (n = 5) Moderate CRAS. Multiple apthae (n = 5) Тяжелая форма ХРАС. РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ И ИХ ОБСУЖДЕНИЕ Обширные афты и язвы, с последу- ющим грубым рубцеванием (n = 14) Severe CRAS. Minor and major aphthous ulcers with subsequent significant scarring (n = 14) НВ (норма 120-140) НВ (ref. range 120-140) 120-140 100-120 80-110 СОЭ (норма 2-5 мм/ч) ESR (ref. range 2-5 mm/h) 10-25 25-35 35-50 Уровень IgA (норма 0,4-3,5 г/л) IgA Level (ref range 0.4-3.5 g/l) 1,2-3,5 3,6 -4,5 4,46-9,71 ЦИК (норма 0-4,5 г/л) CIC (ref. range 0- 4.5 g/l) 0,05-5 4,5- 6,5 6,50-9,07 Таблица 6. Лабораторные показатели состояния организма при различных формах хронического рецидивирующего афтозного стоматита у детей с ББ Table 6. Laboratory values of body condition in various forms of chronic recurrent aphthous stomatitis in children with Behcet’s disease ления первичного диагностического признака ХРАС и сроков присоединения последующих клинических признаков заболевания, проведение лабораторных исследований и гистоморфологических исследований пораженных тканей слизистой оболочки, предложен для врачей-стоматологов, которые наблюдают детей с заболеваниями слизистой оболочки полости рта. РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ И ИХ ОБСУЖДЕНИЕ 6. У наших пациентов в процессе наблюдения и об- следования были выявлены следующие клинические признаки ББ (табл. 5). 9. Картина многоорганной редкой ревматической патологии требует лечения и наблюдения у ревмато- лога и многих специалистов, в том числе и стоматоло- га, что представляется непростой задачей. При анализе клинических симптомов ББ у наших об- следуемых в 100% случаев установлен ХРАС, наиболее часто, у 90,9%, поражаются органы зрения, у 90,9% на- блюдается поражение органов ЖКТ, у 72,7% поражены органы выделительной системы и часто, у 45,4%, встре- чается суставной синдром. Эти данные должны насто- рожить врачей-стоматологов, потому что даже у детей Больным с ББ назначалось комплексное лечение, в зависимости от иммунологической активности забо- левания, длительности течения и степени вовлечения различных органов и систем в этот патологический процесс. 2020 (76) Стоматология детского возраста и профилактика Оригинальная статья Оригинальная статья Периодичность появления ХРАС в полости рта Recurrence rate of chronic recurrent aphthous stomatitis Легкая форма. Единичные эрозии (n = 2) Mild. Single minor aphthae (n = 2) Среднетяжелая форма. Множественные афты (n = 5) Moderate. Multiple aphthae (n = 5) Тяжелая форма. Обширные афты и язвы с последующим грубым рубцеванием (n = 14) Severe. Minor and major aphthous ulcers with subsequent significant scarring (n = 14) 3-4 раза в год / 3-4 times a year 2 3 1 Каждый месяц / Every month - 1 7 Постоянные рецидивы / Chronic recurrence - 1 6 Таблица 4. Периодичность появления хронического рецидивирующего афтозного стоматита в полости рта при различных формах, у детей с ББ Table 4. Recurrence rate of various forms of chronic recurrent aphthous stomatitis in children with Behcet’s disease Периодичность появления ХРАС в полости рта Recurrence rate of chronic recurrent aphthous stomatitis Легкая форма. Единичные эрозии (n = 2) Mild. Single minor aphthae (n = 2) Среднетяжелая форма. Множественные афты (n = 5) Moderate. Multiple aphthae (n = 5) Тяжелая форма. Обширные афты и язвы с последующим грубым рубцеванием (n = 14) Severe. 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American Journal of Ophthalmology. 2003; 136 (6): 1114–1119. Available at: http://doi.org/10.1016/s0002- 9394(03)00791-8. 9. Успенская О.А., Казарина Л.Н., Шевченко Е.А. Изменения местного иммунитета полости рта у па- циенток с хроническим рецидивирующим афтозным стоматитом на фоне урогенитальной инфекции. Со- 9. Успенская О.А., Казарина Л.Н., Шевченко Е.А. Изменения местного иммунитета полости рта у па- циенток с хроническим рецидивирующим афтозным стоматитом на фоне урогенитальной инфекции. Со- ВЫВОДЫ 1. Таким образом, ББ у детей недостаточно изучена, в связи с чем отмечается поздняя диагностика заболевания. 2. В течение 5,40 ± 0,04 лет у девочек и 4,10 ± 0,09 лет у мальчиков с ББ заболевание протекает как неполный синдром Бехчета, проявляясь только первым диагно- стическим симптомом заболевания – хроническим ре- цидивирующим афтозным стоматитом в 100% случаев. 4. Диагностика и лечение ББ у детей требует меж- дисциплинарного подхода врачей: стоматолога, пе- диатра, ревматолога, окулиста, гастроэнтеролога и более узких специалистов, в зависимости от присо- единившихся симптомов заболевания. 4. Диагностика и лечение ББ у детей требует меж- дисциплинарного подхода врачей: стоматолога, пе- диатра, ревматолога, окулиста, гастроэнтеролога и более узких специалистов, в зависимости от присо- единившихся симптомов заболевания. 3. Разработанный нами комплексный план обследо- вания, включающий в себя: установление зональности заболевания и генетической предрасположенности, выявление провоцирующего фактора и срока появ- Оригинальная статья Gabriel Riera Matute, Elena Riera Alonso. La aftosis oral recurrente en Reumatologia. Reumatologia Clinica. 295 Оригинальная статья https://journaldoctor.ru/catalog/pediatriya/khronicheskiy- retsidiviruyushchiy/#tab4. 2011; 7 (5): 323-328. Available at: http://doi.org/10/1016/j. reuma.2011.05.003. 2011; 7 (5): 323-328. Available at: http://doi.org/10/1016/j. reuma.2011.05.003. https://journaldoctor.ru/catalog/pediatriya/khronicheskiy- retsidiviruyushchiy/#tab4. https://journaldoctor.ru/catalog/pediatriya/khronicheskiy- retsidiviruyushchiy/#tab4. y y 9. Uspensky, O.A., Kazarina, L.N., Shevchenko, E.A. Changes in local oral immunity in patients with chronic re- current aphtous stomatitis against the background of uro- genital infection. Modern problems of science and educa- tion. 2015; 1(1): 1051-1056. (In Russ.). Available at: http:// www.science-education.ru/ru/article/view?id=18177. y y 9. Uspensky, O.A., Kazarina, L.N., Shevchenko, E.A. Changes in local oral immunity in patients with chronic re- current aphtous stomatitis against the background of uro- genital infection. Modern problems of science and educa- tion. 2015; 1(1): 1051-1056. (In Russ.). Available at: http:// www.science-education.ru/ru/article/view?id=18177. 15. Ioan_Buraga, Roxana Martin, Adrian Dobrescu, Mag- da Buraga. Behcet's disease – case presentation. Roma- nian Journal of Neurology. Revista Romana de Neurologie. 2010; 9(3): 145-150. Available at: http://doi.org/10.37897/ RJN.2010.3.7. 16. Laskaris G. Treatment of Oral diseases. A concise text- book. Thiema Stuttgart. New York. 2011: 47-49. Available at: https://books.google.ru/books?id=GRmfM9dv0vYC&redir_ esc=y. 10. Chernysheva, N.D. Use of the drug "Viferon" in the complex therapy of chronic recurrent aphtosis stomatitis. Ural Medical Journal. 2010; 8; 11-13. (In Russ.). Available at: http://elib.fesmu.ru/elib/Article.aspx?id=228707. 17. Stephen J. Challacombe, Surab Alsahaf, Anwar Tap- puni. Recurrent aphthous stomatitis: Towards evidence- based treatment? Curr Oral Health Rep. 2015; 2: 158-167. Available at: http://doi.org/ 10.1007/s40496-015-0054-y. 11. Shamov, I.A., Abdulpatakhov, D.D., Gitinova, S.H., Abdulpatakhov, E.E., Dadayev, B.N., Alygadzhiev, S.S. Bekh- chet disease in Dagestan. Scientific and Practical Rheu- matology. 2004; 4: 39. (In Russ.). Available at: https://rsp. mediar-press.net/rsp/article/download/937/629. 11. Shamov, I.A., Abdulpatakhov, D.D., Gitinova, S.H., Abdulpatakhov, E.E., Dadayev, B.N., Alygadzhiev, S.S. Bekh- chet disease in Dagestan. Scientific and Practical Rheu- matology. 2004; 4: 39. (In Russ.). Available at: https://rsp. mediar-press.net/rsp/article/download/937/629. 18. Tugal-Tutkin J., Urgancioglu M. Childhood-onset uveitis in Behcet's disease: a descriptive study of 36 cas- es. American Journal of Ophthalmology. 2003; 136 (6): 1114–1119. Available at: http://doi.org/10.1016/s0002- 9394(03)00791-8. 12. Shevchenko, E.A., Reshetin, A.V. Developing a new treatment regimen for chronic recurrent aphtosis stoma- titis. Modern problems of science and education. 2016: 4. (In Russ.). Available at: https://science-education.ru/ru/ article/view?id=24804. Конфликт интересов: Авторы декларируют отсутствие конфликта интересов/ Сonflict of interests: The authors declare no conflict of interests Поступила / Article received 03.06.2020 Поступила после рецензирования / Revised 23. 06.2020 Принята к публикации / Accepted 29.06.2020 13. Shilkina, N.P. Systemic vasculitides: Some debatable aspects of the problem. Therapeutic archive). 2015;8 7(5): 100-105. (In Russ.). Available at: http://doi.org/10.17116/ terarkh2015875100-105. 14. Сведения об авторах / Information about the authors Geppe Natalia A., MD, Head of the of Department of Childhood diseases – FSAEI of HE I.M. Sechenov First Mos- cow State Medical University of the Ministry of Health of the Russian Federation (Sechenov University), Moscow, Rus- sian Federation Скакодуб Алла Анатольевна, к.м.н., доцент кафедры стоматологии детского возраста и ортодонтии Институ- та стоматологии Первого МГМУ им. И.М. Сеченова Скакодуб Алла Анатольевна, к.м.н., доцент кафедры стоматологии детского возраста и ортодонтии Институ- та стоматологии Первого МГМУ им. И.М. Сеченова skalla71@mail.ru ORCID: https://orcid.org/0000-0003-0735-0583 skalla71@mail.ru ORCID: https://orcid.org/0000-0003-0735-0583 Адмакин Олег Иванович, д.м.н., профессор, зав. ка- федрой профилактики и коммунальной стоматологии Института стоматологии Первого МГМУ им. И.М. Се- ченова Skakodub Alla A., PhD, assistant professor of dentistry in the Department of Pediatric Dentistry and Orthodon- tics — Institute of Dentistry FSAEI of HE I.M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation (Sechenov University), Moscow, Russian Federation References 1. Alekberova, Z.S. Behchet's disease in children. Cur- rent Pediatrics. 2009; 8(6); 63-70. (In Russ.). Available at: https://cyberleninka.ru/article/n/bolezn-behcheta-u-detey. 2011; 13-22; 587-595. (In Russ.). Available at: http://king- med.info/knigi/Revmatologiya/book_4108/Praktiches- koe_rukovodstvo_po_detskoy_revmatologii-Geppe_NA_ Podchernyaeva_NS_Liskina_GA-2011-pdf. 2. Vinogradova, T.F. Dentistry for pediatricians. MED- press-Inform. M. 2014; 187-191. (In Russ.). Available at: https://rusneb.ru/catalog/000200_000018_RU_NLR_ BIBL_A_010192541/. 6. Sevostyanov, V.K., Zholobova, E.S., Baranova, O.V., Po- emshina, K.S., Polukhina, A.I., Novikov, A.S., Balashov, S.L. Pediatric rheumatology care in the Central Federal District of the Russian Federation. Clinical Practice in Pediatrics. 2019; 14 (3); 90–96. (In Russ.). Available at: http://doi.org/ 10.20953/1817-7646-2019-5-95-101. 6. Sevostyanov, V.K., Zholobova, E.S., Baranova, O.V., Po- emshina, K.S., Polukhina, A.I., Novikov, A.S., Balashov, S.L. Pediatric rheumatology care in the Central Federal District of the Russian Federation. Clinical Practice in Pediatrics. 2019; 14 (3); 90–96. (In Russ.). Available at: http://doi.org/ 10.20953/1817-7646-2019-5-95-101. 3. Lyskina, G.A. Systemic vasculitis in children. Clin- ic, diagnosis, treatment. Autoref. Dics. D.M.S. M. 1994; 23-24. (In Russ.). Available at: https://rusneb.ru/cata- log/000199_000009_000105817/. 3. Lyskina, G.A. Systemic vasculitis in children. Clin- ic, diagnosis, treatment. Autoref. Dics. D.M.S. M. 1994; 23-24. (In Russ.). Available at: https://rusneb.ru/cata- log/000199_000009_000105817/. 7. Skakodub, A.A. Oral organ condition in children with diffusic connective tissue diseases // Autoref. Dics. PhD. M. 2000; 10-17. (In Russ.). Available at: https://rusneb.ru/ catalog/000199_000009_000796021/. 4. Rheumatology: National Guide / Edited by E.L. Na- sonov, V.A. Nasonova. GEOTAR-Media. М. 2008; 25. (In Russ.). Available at: http://kingmed.info/knigi/Revma- tologiya/book_2220/Revmatologiya_Natsionalnoe_ruko- vodstvo-Nasonov_EL_Nasonova_VA-2008-pdf. 8. Skakodub, A.A., Geppe, N.A., Admakin, O.I., Lyski- na, G.A. Chronic recurrent aphtous stomatitis in Behchet's disease in children. Clinical observation. "Doctor.RU" Pe- diatrics. 2017; 15(144); 26-30. (In Russ.). Available at: 8. Skakodub, A.A., Geppe, N.A., Admakin, O.I., Lyski- na, G.A. Chronic recurrent aphtous stomatitis in Behchet's disease in children. Clinical observation. "Doctor.RU" Pe- diatrics. 2017; 15(144); 26-30. (In Russ.). Available at: 5. A guide to pediatric rheumatology. Edited by N.A. Ge- ppe, N.S. Podchernyaeva, G.A. Lyskina. GEOTAR-Media. М. 5. A guide to pediatric rheumatology. Edited by N.A. Ge- ppe, N.S. Podchernyaeva, G.A. Lyskina. GEOTAR-Media. М. 4/2020 (76) Стоматология детского возраста и профилактика Оригинальная статья admakin1966@mail.ru ORCID: https://orcid.org/0000 -0002-5626-2961 Геппе Наталья Анатольевна, д.м.н., профессор, зав. кафедрой детских болезней лечебного факультета Первого МГМУ им. И.М. Сеченова Admakin Oleg I., MD, Head of the Department of Pre- vention and Public Dental Health – Institute of Dentistry FSAEI of HE I.M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federa- tion (Sechenov University), Moscow, Russian Federation Журналы издательской группы РПА РОССИЙСКАЯ ПАРОДОНТОЛОГИЧЕСКАЯ АССОЦИАЦИЯ Журналы издательской группы РПА ИЙСКАЯ ОДОНТОЛОГИЧЕСКАЯ ЦИАЦИЯ Журнал «Пародонтология» Стоимость подписки в печатном виде на 2021 год по России – 2700 рублей Подписной индекс в каталоге «Пресса России» – 18904 Стоимость подписки в электронном виде на 2020 год – 2500 рублей www.parodont.ru
https://openalex.org/W2942964300	https://revistaenfermagematual.com.br/index.php/revista/article/download/345/228	Portuguese	null	O paciente surdo e suas vivências no sistema de saúde: uma interface com a enfermagem	Revista Enfermagem Atual In Derme	2,019	cc-by	6,036	33 A RT I G O O R I G I N A L 33 A RT I G O O R I G I N A L 33 The deaf patient experiences in the health system: an interface with the nursing Vitor Machado Cavagna1 • Wesley Pereira de Jesus Silva2 •André Luiz de Souza Braga3 • Marilda Andrade4 Vitor Machado Cavagna1 • Wesley Pereira de Jesus Silva2 •André Luiz de Souza Braga3 • Marilda Andrade4 REsUmO Nos meios acadêmicos, a comunicação é um dos instrumentos básicos da enfermagem, porém, ao nos deparar com um paciente surdo, sem que tenhamos a capacitação necessária, o atendimento efetivo fi ca impossibilitado. Objetiva-se identifi car como se dá o atendimento ao paciente surdo e analisar as barreiras de comunicação do profi ssional de saúde nestes atendimentos. A metodologia utilizada foi de estudo descritivo, de caráter exploratório, com abordagem qualitativa, no qual se realizou um levantamento bibliográfi co sobre os aspectos gerais do processo de comunicação entre o profi ssional de saúde e o paciente surdo. Efetivou-se a coleta de dados através de um roteiro de entrevista semiestruturado, no Instituto Nacional de Educação de Surdos, localizado no bairro de Laranjeiras /RJ - Brasil. Os dados obtidos foram avaliados a partir do método de análise temática. Dos 15 participantes da pesquisa, 14 relataram situações de atendimento em saúde, nas quais, os profi ssionais não sabiam a Linguagem Brasileira de Sinais, retratando sentimento de exclusão neste paciente. Conclui-se a necessidade pela busca por capacitação dos profi ssionais de saúde no que tange à potencialização dos instrumentos utilizados nos processos de comunicação com esta demanda específi ca, oportunizando equidade e universalidade de acesso aos serviços de saúde. Palavras-chave: Surdez; Barreiras de Comunicação; Linguagem de Sinais; Sistema de Saúde; Enfermagem. Não houve fi nanciamento para a realização desta pesquisa. Texto aprovado no Comitê de Ética e Pesquisa do Hospital Universitário Antônio Pedro, da Universidade Federal Fluminense, no dia 03 de junho de 2011, e apresenta o nº 121/11. CAAE: 0126.0.258.258-11, não havendo confl itos de interesse. l 1 Enfermeiro Especialista em Saúde Pública. Mestrando em Ciências do Cuidado em Saúde. Escola de Enfermagem Aurora de Afonso Costa/ UFF. E-mail: vcavagna@hotmail. com Não houve fi nanciamento para a realização desta pesquisa. Texto aprovado no Comitê de Ética e Pesquisa do Hospital Universitário Antônio Pedro, da Universidade Federal Fluminense, no dia 03 de junho de 2011, e apresenta o nº 121/11. CAAE: 0126.0.258.258-11, não havendo confl itos de interesse. 1 Enfermeiro Especialista em Saúde Pública. Mestrando em Ciências do Cuidado em Saúde. Escola de Enfermagem Aurora de Afonso Costa/ UFF. E-mail: vcavagna@hotmail. com 2 Mestre em Ciências do Cuidado em Saúde – Enfermeiro do Centro Municipal de Saúde Dr.Albert Sabin. Secretaria Municipal de Saúde do Rio de Janeiro. E-mail: wesley. jesus@uol.com.br 3 Doutorando em Ciências do Cuidado em Saúde. Professor do Departamento de Fundamentos de Enfermagem e Administração. Escola de Enfermagem Aurora de Afonso Costa/UFF. E-mail: andre.braga@globo.com 4 Doutora em Enfermagem. Vice-diretora e Professora da Escola de Enfermagem Aurora de Afonso Costa/ UFF. E-mail: marildaandrade@uol.com.br Manuscrito baseado em trabalho de conclusão de curso de graduação em Enfermagem e Licenciatura, da Escola de Enfermagem Aurora de Afonso Costa. Niterói / RJ. Autores: Vitor Machado Cavagna, Marilda Andrade (orientadora), Wesley Pereira de Jesus Silva (coorientador). Título: O paciente surdo e suas vivências no sistema de saúde: uma interface com a enfermagem. Trabalho apresentado em dezembro de 2011. Método De acordo com pesquisas realizadas no ano de 20101, cerca de 46 milhões de brasileiros possuíam algum tipo de deficiência (23,9% da população), o que representa um percentual acima dos anos anteriores, quando se observou um contingente menor que 2%. Visto que passou-se a atribuir o título de deficiente não só àqueles que se consideram incapazes, mas também àqueles que reportaram uma grande dificuldade permanente como caminhar, ouvir e enxergar2. Trata-se de um estudo exploratório com abordagem qualitativa. O cenário da pesquisa foi o Instituto Nacional de Educação de Surdos (INES), localizado a Rua das Laranjeiras, nº232, no bairro das Laranjeiras, cidade do Rio de Janeiro - RJ - Brasil. Os participantes da pesquisa foram 15 estudantes surdos do ensino médio e de ambos os sexos. Os critérios de inclusão foram: alunos maiores de 18 anos e que estiveram presentes no cenário de pesquisa durante a coleta de dados. Já os critérios de exclusão foram: aqueles que nunca passaram por situações de atendimento hospitalar e sem nenhuma patologia associada. Nesse sentido, discorrer sobre deficiências depende de distintas percepções culturais e atitudes em relação às mesmas, como também na disponibilidade de serviços e legislação3, pois elas não são apenas resultado das condições saúde/doença, são também determinadas pelo contexto do meio ambiente físico e social. Desenvolveu-se um trabalho de campo, no qual houve a possibilidade de ter acesso ao local onde foi desenvolvida a pesquisa. A coleta de dados ocorreu por meio de observação participante e entrevista semiestruturada. A etapa de observação participante ocorreu durante as atividades desenvolvidas no local e, para a aplicação das entrevistas semiestruturadas, contou-se com a ajuda do intérprete no momento das entrevistas, sendo interlocutor entre o pesquisador e os participantes da pesquisa. A lei orgânica de saúde do Sistema Único de Saúde - SUS (Lei 8080/90) apresenta caráter de acessibilidade universal e gestão descentralizada, porém não engloba qualquer tipo de deficiência e irá tratar da ação governamental e das responsabilidades de cada setor a estes pacientes4. Determinando pleno acesso à saúde, à educação, a habilitação e reabilitação profissionais, ao trabalho, à cultura, ao desporto, ao turismo e ao lazer5. A importância e a legitimidade do trabalho do intérprete da Linguagem Brasileira de Sinais (LIBRAS) ainda encontra- se em processo de construção, pois tal ofício ainda é muito recente9. AbsTRAcT Contudo, não se preocupam em ressaltar a satisfação das necessidades básicas do ser humano6 como fatores indispensáveis para a promoção e preservação da saúde. Através de comunicação estabelecida com o paciente, o profissional pode compreendê-lo como ser integral e também perceber sua visão de mundo. Para desenvolver um bom processo de trabalho, faz-se necessário que os enfermeiros usem a comunicação de maneira efetiva e, assim, evitem falhas na assistência. Não se pode pensar sua comunicação a pessoas com habitualmente e, quando são hospita em ambiente estranho, com pessoas Assim, o estudo objetivou iden surdo vivencia o sistema de s prestado no contexto do Sistema e analisar as barreiras da comuni saúde com o surdo. Método Trata-se de um estudo explo qualitativa. O cenário da pesquisa de Educação de Surdos (INES), Laranjeiras, nº232, no bairro das L de Janeiro - RJ - Brasil. Os participantes da pesquisa surdos do ensino médio e de ambo inclusão foram: alunos maiores de presentes no cenário de pesqui dados. Já os critérios de exclusão fo passaram por situações de atendi nenhuma patologia associada. Desenvolveu-se um trabalho de possibilidade de ter acesso ao loc a pesquisa. A coleta de dados observação participante e entr A etapa de observação participa atividades desenvolvidas no loca entrevistas semiestruturadas, con intérprete no momento das entrev entre o pesquisador e os participa A importância e a legitimidade da Linguagem Brasileira de Sinais se em processo de construção, poi recente9. O intérprete tem o papel a comunicação entre os surdos e comunicação verbal e não verbal10 Para a interpretação dos dado a análise por categorias temática do sentido que compõem um proc presença ou frequência tenha al objeto analítico visado. Este trabalho obteve aprovaç e Pesquisa do Hospital Universi Universidade Federal Fluminense 2011, e apresenta o nº 121/11. 11. Foram atribuídos nomes fictíc pesquisa. Método O intérprete tem o papel de orientar e interpretar a comunicação entre os surdos e ouvintes e de fazer a comunicação verbal e não verbal10. Diante da crise observada no setor público de saúde, não só a hospitalização, como também qualquer outro ambiente que exponha o binômio saúde/doença, passa por uma questão política e social, de modo que muitos profissionais de saúde reúnem-se para discutir a origem da doença e buscar soluções para tal problema. Contudo, não se preocupam em ressaltar a satisfação das necessidades básicas do ser humano6 como fatores indispensáveis para a promoção e preservação da saúde. Para a interpretação dos dados coletados, realizou-se a análise por categorias temáticas8, na busca dos núcleos do sentido que compõem um processo comunicativo, cuja presença ou frequência tenha algum significado para o objeto analítico visado. Este trabalho obteve aprovação do Comitê de Ética e Pesquisa do Hospital Universitário Antônio Pedro, da Universidade Federal Fluminense, no dia 03 de junho de 2011, e apresenta o nº 121/11. CAAE: 0126.0.258.258- 11. Foram atribuídos nomes fictícios aos participantes da pesquisa. Através de comunicação estabelecida com o paciente, o profissional pode compreendê-lo como ser integral e também perceber sua visão de mundo. Para desenvolver um bom processo de trabalho, faz-se necessário que os enfermeiros usem a comunicação de maneira efetiva e, assim, evitem falhas na assistência. Não se pode pensar em atuação profissional sem levar em conta a importância do processo comunicativo e a união deste à prática assistencial7. Com isso, quando não há uma comunicação eficaz, não há como desenvolver a assistência pertinente a este paciente. Introdução sua comunicação a pessoas com as quais se relacionam habitualmente e, quando são hospitalizados8, passam a conviver em ambiente estranho, com pessoas que não os entendem. O desenvolvimento deste estudo deveu-se à reflexão sobre o atendimento à pessoa surda e sua inserção no sistema de saúde. Neste contexto, a comunicação se apresenta como um dos instrumentos básicos de trabalho do enfermeiro. Com isso, a contribuição quanto ao aprendizado de novos métodos de expressão com o paciente surdo é de extrema importância, pois se o profissional não consegue se comunicar com este cliente, inviabiliza-se o planejamento de cuidados para este usuário. Assim, o estudo objetivou identificar como o paciente surdo vivencia o sistema de saúde e o atendimento prestado no contexto do Sistema Único de Saúde (SUS), e analisar as barreiras da comunicação do profissional de saúde com o surdo. AbsTRAcT The communication is demonstrated in the academic times, as one of the basic instruments of the nursing, however, if we face a deaf patient and we do not have the necessary qualifi cation the effective attendance stays disabled. Objective to identify how the treatment to a deaf patient is given in the health system and analyze the professional’s communication barriers to the deaf patient. The used methodology was of descriptive study, with qualitative approach, in which a bibliographic survery happened on the general aspects of the communication process between the health professional and the deaf patient. It was accomplished, collects it of data through a semistructured script of interview, realized in the Instituto Nacional de Educação de Surdos (INES), nestled in Laranjeiras, RJ/Brazil. Of the 15 participants of the research, 14 had told situations of attendance in health, in which, the professionals did not know the Brazilian Language of Signals, depicting feeling of exclusion in this patient. It concludes that necessity for the search for health professionals’ qualifi cation in what it refers to the enhancement’s instruments used in the processes of communication with this specifi c demand, providing equity and universality of health services access. Keywords: Deafness; Communication Barriers; Signs Language; Health System; Nursing. NOTA 1 Enfermeiro Especialista em Saúde Pública. Mestrando em Ciências do Cuidado em Saúde. Escola de Enfermagem Aurora de Afonso Costa/ UFF. E-mail: vcavagna@hotmail. com do em Saúde – Enfermeiro do Centro Municipal de Saúde Dr.Albert Sabin. Secretaria Municipal de Saúde do Rio de Janeiro. E-mail: wesley. do Cuidado em Saúde. Professor do Departamento de Fundamentos de Enfermagem e Administração. Escola de Enfermagem Aurora de Afon raga@globo.com 4 Doutora em Enfermagem. Vice-diretora e Professora da Escola de Enfermagem Aurora de Afonso Costa/ UFF. E-mail: marildaandrade@uol.com.br 4 Doutora em Enfermagem. Vice-diretora e Professora da Escola de Enfermagem Aurora de Afonso Costa/ UFF. AbsTRAcT E-mail: marildaandrade@uol.com.br M it b d t b lh d l ã d d d ã E f Li i t d E l d E f A d Af C t Nit ói REVISTA ENFERMAGEM ATUAL \| 2017; 80 34 C a v a g n a V M , S i l v a W P J , B r a g a A L S , A n d r a d e M Introdução O desenvolvimento deste estudo deveu-se à reflexão sobre o atendimento à pessoa surda e sua inserção no sistema de saúde. Neste contexto, a comunicação se apresenta como um dos instrumentos básicos de trabalho do enfermeiro. Com isso, a contribuição quanto ao aprendizado de novos métodos de expressão com o paciente surdo é de extrema importância, pois se o profissional não consegue se comunicar com este cliente, inviabiliza-se o planejamento de cuidados para este usuário. De acordo com pesquisas realizadas no ano de 20101, cerca de 46 milhões de brasileiros possuíam algum tipo de deficiência (23,9% da população), o que representa um percentual acima dos anos anteriores, quando se observou um contingente menor que 2%. Visto que passou-se a atribuir o título de deficiente não só àqueles que se consideram incapazes, mas também àqueles que reportaram uma grande dificuldade permanente como caminhar, ouvir e enxergar2. Nesse sentido, discorrer sobre deficiências depende de distintas percepções culturais e atitudes em relação às mesmas, como também na disponibilidade de serviços e legislação3, pois elas não são apenas resultado das condições saúde/doença, são também determinadas pelo contexto do meio ambiente físico e social. A lei orgânica de saúde do Sistema Único de Saúde - SUS (Lei 8080/90) apresenta caráter de acessibilidade universal e gestão descentralizada, porém não engloba qualquer tipo de deficiência e irá tratar da ação governamental e das responsabilidades de cada setor a estes pacientes4. Determinando pleno acesso à saúde, à educação, a habilitação e reabilitação profissionais, ao trabalho, à cultura, ao desporto, ao turismo e ao lazer5. Diante da crise observada no setor público de saúde, não só a hospitalização, como também qualquer outro ambiente que exponha o binômio saúde/doença, passa por uma questão política e social, de modo que muitos profissionais de saúde reúnem-se para discutir a origem da doença e buscar soluções para tal problema. Aspectos gerais da população estudada Cada turma da instituição possui aproximadamente 15 alunos. Das turmas presentes no local no dia da pesquisa, 2 foram entrevistadas: 1º e 3º anos do ensino médio. O processo comunicativo torna-se mais sério quando se apresenta com pacientes surdos, pois estes restringem REVISTA ENFERMAGEM ATUAL \| 2017; 80 35 A RT I G O O R I G I N A L A RT I G O O R I G I N A L P a c i e n t e s u r d o e s i s t e m a d e s a ú d e abela 1. Turmas entrevistadas Entrevistados Ensino Médio 1º ano 2º ano 3º ano Curso Noturno 9 alunos - 4 alunos Curso Diurno - - 2 alunos Fonte: dados provenientes da pesquisa estratégia adotada por diversas empresas consiste na tentativa de justificar o descumprimento da lei de cotas4, sob o argumento de que suas atividades são caracterizadas por elevado grau de risco e periculosidade, o que tornaria não recomendável a contratação de pessoas com deficiência11. A maioria dos entrevistados (80%) se declarou solteira, possivelmente pela média da faixa etária que se encontravam e também por pesquisas que apontam que o matrimônio tem ocorrido no Brasil em idades mais avançadas. O padrão de idade de homens e mulheres disponíveis para o mercado matrimonial reflete as chances relativas de casamento e recasamento de cada um dos gêneros12. O relacionamento conjugal está associado à saúde e à qualidade de vida, principalmente nos anos de maturidade e velhice, porém o fato de um casamento durar não necessariamente significa que a união traga satisfação para os cônjuges13. A coleta de dados ocorreu após as 18 horas, devido à orientação da chefia de pesquisas da instituição e, neste horário, havia alunos de outros turnos sendo abordados sobre a participação no estudo, mostrando-se dispostos a contribuir com a pesquisa. Perfil dos Participantes Foram realizados 2 dias de entrevistas com os participantes do estudo, totalizando 15 participantes, os quais são caracterizados na tabela abaixo. Discussão A média da faixa etária encontrada foi 24,2 anos, sendo que 40% representam o sexo feminino e 60% masculino; a maioria dos entrevistados exerce atividades profissionais, o que demonstra que a limitação auditiva não tem se tornado entrave para inserção e permanência no mercado de trabalho, pois estão amparados por lei2 que estabelece a obrigatoriedade de empresas públicas e privadas de reservarem um quantitativo de vagas para esta parcela populacional. Como descrito anteriormente, a análise dos dados aconteceu a partir da confluência de sentido das respostas dadas após aplicação do roteiro de entrevistas, resultando nas seguintes categorias temáticas: (I) Comunicação em si – situação de atendimento em instituições de saúde; (II) Estratégias utilizadas para a comunicação com o profissional de saúde, (III) Conhecimento/desconhecimento por parte do profissional de saúde sobre a LIBRAS e (IV) Identificando a percepção do paciente em relação ao profissional. A seguir, apresenta-se a discussão dessas categorias. O trabalho não constitui mera forma de sobrevivência e a sua importância ultrapassa as questões de ordem prática, contribuindo para a dignidade humana11. A Tabela 2. Características gerais dos participantes Nome Idade Sexo Profissão Estado Civil Participante 1 20 F Rotulagem Solteira Participante 2 27 M Desempregado Solteiro Participante 3 22 F Desempregada Solteira Participante 4 20 M Estudante Solteiro Participante 5 25 M Estudante Solteiro Participante 6 23 M Aux. Administrativo Casado Participante 7 24 M Estoquista Solteiro Participante 8 35 F Aux. Operacional Solteira Participante 9 28 M Aux. de serv. gerais Solteiro Participante 10 22 M Estoquista Solteiro Participante 11 44 F Atendente Casada Participante 12 22 M Aux. Administrativo Solteiro Participante 13 25 F Operadora de fábrica Solteira Participante 14 22 M Desempregado Solteiro Participante 15 53 F Do lar Casada Fonte: dados provenientes da pesquisa Tabela 2. Características gerais dos participantes Nome Idade Sexo Profissão Estado Civil Participante 1 20 F Rotulagem Solteira Participante 2 27 M Desempregado Solteiro Participante 3 22 F Desempregada Solteira Participante 4 20 M Estudante Solteiro Participante 5 25 M Estudante Solteiro Participante 6 23 M Aux. Administrativo Casado Participante 7 24 M Estoquista Solteiro Participante 8 35 F Aux. Operacional Solteira Participante 9 28 M Aux. de serv. gerais Solteiro Participante 10 22 M Estoquista Solteiro Participante 11 44 F Atendente Casada Participante 12 22 M Aux. Discussão Administrativo Solteiro Participante 13 25 F Operadora de fábrica Solteira Participante 14 22 M Desempregado Solteiro Participante 15 53 F Do lar Casada Fonte: dados provenientes da pesquisa Tabela 2. Características gerais dos participantes REVISTA ENFERMAGEM ATUAL \| 2017; 80 36 C a v a g n a V M , S i l v a W P J , B r a g a A L S , A n d r a d e M I - Comunicação em si – situação de atendimento em instituições de saúde. A utilização da escrita também se apresentou como um recurso facilitador para a interação profissional-paciente, mas é válido salientar que, em muitos dos casos, os participantes do estudo relataram não entender a grafia dos profissionais. Entretanto, o que foi mais observado é a dificuldade dos próprios participantes em relação a língua portuguesa, visto que a LIBRAS é sua língua materna, e a língua portuguesa é um aprendizado acessório. Quando os participantes foram questionados acerca da vivência nas instituições de saúde, emergiram as seguintes falas: “Eu consegui me comunicar, porque domino a escrita [...] Geralmente quando tenho mais dificuldade, eu uso um intermediário, que é a minha mãe ou então uso intérprete.” (Participante 4) O intérprete da LIBRAS também aparece como intermediário, porém este é requisitado quando não se tem a possibilidade de ter uma pessoa da família presente na situação de atendimento em saúde. É imprescindível lembrar que os participantes se sentem excluídos nos atendimentos, pois os profissionais não têm nenhum tipo de interação com eles, apenas se comunicam com o intermediário e acabam esquecendo que o paciente surdo é seu verdadeiro foco de atendimento. “Eu tinha que chamar intérprete, porque nesse caso, eu não tinha ninguém da família e se eu fosse sozinho, a doença não era realmente tratada, porque eles não entendiam o que eu sentia de fato, às vezes eu entendi o que ele tava me falando, mas ele não chegava ao meu objetivo.” (Participante 5) “Eu já cheguei num hospital, ninguém teve nenhum tipo de relacionamento comigo. A minha irmã foi junto comigo, aí ela sabe sinalizar e eu sou um pouco oralizada.” (Participante 8) Quando acontecem situações em que não se fazem presentes intérpretes ou qualquer tipo de intermediário, o atendimento ao paciente se dá de maneira inespecífica, visto que as dificuldades apresentadas pelo paciente são muitas, como a falta de compreensão da grafia e dificuldades com a língua portuguesa. “Eu passo, mostro o que tá acontecendo comigo, eu passo pro papel, aí o doutor consegue entender o que está acontecendo comigo.” (Participante 9) “Quando tem algum problema sério comigo, eu chamo intérprete; quando é uma coisa mais básica, eu não vou pro hospital.” (Participante 10) II - Estratégias utilizadas para a comunicação com o profissional de saúde Dentre os recursos utilizados para a interação com o profissional de saúde, surgiram os seguintes depoimentos: “Eu cheguei na unidade médica com meu marido [...] eu tinha que ter alguém pra me comunicar com o doutor, só que este não deixou que meu marido entrasse comigo, então eu peguei e fui embora.”(Participante 11) “A minha mãe sempre está comigo, com isso, ela serve de estratégia. Eu sempre utilizo minha mãe para me comunicar.” (Participante 1) As dificuldades apresentadas nas respostas dos participantes quanto à vivência de utilização dos serviços de saúde nos fazem refletir acerca da necessidade de capacitação dos profissionais, visto que a grande maioria dos casos fez menção da presença de um acompanhante nas situações de atendimento. As barreiras de comunicação que se instauram entre o usuário dos serviços e o profissional justifica a necessidade de um acompanhante. “Todas as vezes que eu vou ao médico, sempre vou com o meu filho e o médico conversa com ele e o meu filho passa pra mim.” (Participante 15) “Gestos e mímicas são as estratégias utilizadas.” (Participante 2) “Escrita e leitura labial. Sempre gera um pouco de confusão, a comunicação não é perfeita”. (Participante 3) A saúde é um direito fundamental do ser humano, assegurado na lei. Sendo assim, cabe ao profissional de saúde se capacitar para promover a saúde da população, não excluindo este grupo específico (surdos), pois é dever do Estado garantir a saúde de toda a população14. Porém, a garantia de um atendimento inclusivo para este grupo deve resultar da capacitação profissional para um melhor atendimento, através do aprendizado da LIBRAS. “Ele começou a escrever na folha, eu com o português tenho um pouco de dificuldade [...] Ele tava escrevendo uma coisa que eu não tava entendendo. A comunicação foi muito pouca comigo.” (Participante 6) “É difícil, porque eles falam, falam e escrevem no papel, só que a gente não conhece português direito.” (Participante 10) “Não conseguem se comunicar comigo, tentam falar, escrevem e eu não entendo nada. Ele é profissional, eu sei, mas a letra dele é muito difícil de ser entendida.” (Participante 13) Há muitas barreiras no atendimento ao paciente surdo, entre as quais destacam-se as dificuldades linguísticas e a falta de confiança do paciente no profissional ouvinte, pois, muitas vezes, o surdo é comparado a deficientes mentais. “Ele escrevia, escrevia e eu não entendia nada. Eu pedia: Explica doutor, explica. III - Conhecimento /desconhecimento por parte do profissional de saúde sobre a LIBRAS Eu fico chateada, pois o surdo sofre muito .” (Participante 11) Em relação ao conhecimento e/ou desconhecimento da LIBRAS, obtiveram-se os seguintes resultados: dos 15 participantes, 14 relataram que os profissionais não sabiam/ desconheciam a linguagem de sinais. Apenas 1 relatou que o profissional sabia o alfabeto, porém não sinalizava. “Pra mim é normal, só que eu sei que existem pessoas que não sabem se comunicar comigo e é difícil, né? É muito importante que se tenha essa comunicação com a gente.” (Participante 12) “ É um ar de discriminação. Com o ouvinte é fácil, se fala o que pode e o que não pode, como está a saúde, mas pra gente é difícil”. (Participante 13) A falta de capacitação profissional é demonstrada na maioria dos casos apresentados no estudo, e mesmo no único caso que o profissional sabia “soletrar” o alfabeto na linguagem de sinais, o atendimento não se deu de forma efetiva, pois não conseguiu atender as expectativas do participante que relatou o caso, como se observa na fala seguinte: “Fico chateado devido a falta de intérprete e de comunicação com os profissionais.” (Participante 14) É notória a opinião dos participantes no que diz respeito à falta de comunicação dos profissionais; muitos se sentem destratados, indignados, chateados e discriminados, já que o atendimento não supre suas necessidades. Com isso, muitos deles procuravam as instituições de saúde, apenas nos momentos de extrema necessidade e, na maioria dos casos, não tinham atendimento satisfatório. “... uma vez eu fui a um ginecologista e este sabia soletrar, não sabia sinalizar; só sabia o alfabeto e então eu desisti...” (Participante 11). “... uma vez eu fui a um ginecologista e este sabia soletrar, não sabia sinalizar; só sabia o alfabeto e então eu desisti...” (Participante 11). Existem várias formas de se comunicar, porém existe a dificuldade dos enfermeiros em comunicar-se com pacientes através de meios não verbais. Os profissionais precisam ser eficientes no desempenho do seu papel, proporcionando melhoria na relação com este tipo de paciente18,7. Referindo-se à percepção do paciente acerca do atendimento nos serviços de saúde, há relatos que este expressa seus sentimentos como uma descrição particular de um momento vivenciado por ele19, considerando que sentimento é o símbolo utilizado para descrever a consciência de estar a “mercê” dos cuidados dos profissionais, porém, nem sempre, estes cuidados suprem suas necessidades. “Eu me sinto destratada.” (Participante 1) (Participante 10) “Eu me sinto destratada.” (Participante 1) um intermediário como estratégia facilitadora neste processo. Contudo, não descartam a escrita, estratégia em que encontram dificuldade de entendimento, devido a dois fatores: a grafia dos profissionais e ainda, a própria língua portuguesa. A utilização de gestos, mímicas, leitura labial e a fala lenta também foram mencionados em alguns casos, porém estes são apresentados como meios que, em algumas vezes, podem não demonstrar o entendimento esperado. “Parece que não tem nada de concreto, que não é um atendimento, não tem nada que me atenda de verdade.” (Participante 15) “O atendimento não é digno para a pessoa que é surda.” (Participante 2) “O atendimento não é digno para a pessoa que é surda.” (Participante 2) “Me sinto chateada, pois parece que o atendimento não é direcionado pra mim.” (Participante 3) A falta de iniciativa profissional para a interação com o paciente surdo é bem evidente, pois, na maioria dos casos apresentados, houve sempre uma estratégia por parte do paciente, o que nos faz refletir acerca da necessidade de maior envolvimento quanto à prestação do serviço de saúde e resolutividade dos problemas de saúde apresentados. Qualquer pessoa surda tem direito ao atendimento16, mas cabe aos profissionais ter as ferramentas mínimas para cuidar da pessoa portadora de um atendimento diferenciado e uma destas é se capacitar na linguagem de sinais. “Eu sinto como se toda sociedade excluísse.” (Participante 4) “Eu me sinto discriminado, sinto que o atendimento que deve ser prestado a mim, não é prestado.” (Participante 5) “É muito difícil esse atendimento, é um assunto pesado, é muito difícil esse relacionamento.” (Participante 6) “Tem gente que não tem experiência com a gente, é uma forma diferenciada, né? É muito difícil pra gente que é surdo.” (Participante 7) O fato de a sociedade ainda conceber a existência de barreiras na comunicação pode ser explicado pelas dificuldades que a população tem em enxergar as pessoas com deficiência como pessoas produtivas e por acreditarem que elas são inferiores17. Por isso, atribuírem uma predisposição negativa e fazem um julgamento depreciativo a esta parcela da população. “Os profissionais não dão muito valor, pois toda vez eu tenho que chamar minha irmã, e com isso eu fico muito incomodada.” (Participante 8) “Fico meio triste com isso, meio chateado com eles, por não ter essa comunicação [...] as pessoas têm que aprender a conviver com os surdos.” (Participante 9) “Eu fico com raiva disso, triste, chateado, com raiva. II - Estratégias utilizadas para a comunicação com o profissional de saúde Eu não entendi e fui pra casa, não adiantou eu ter ido lá.” (Participante 14) O encontro com o paciente surdo pode ser esporádico, porém o desafio dos profissionais de saúde pode ir além dos serviços especializados e as barreiras acima apresentadas podem colocar em risco a assistência proporcionada15. Com a finalidade de reduzir as dificuldades de comunicação, os participantes, em sua maioria, mencionam REVISTA ENFERMAGEM ATUAL \| 2017; 80 37 A RT I G O O R I G I N A L A RT I G O O R I G I N A L P a c i e n t e s u r d o e s i s t e m a d e s a ú d e Conclusão A percepção do paciente surdo acerca do profissional de saúde ainda é feita pelo distanciamento. Não se tem a total confiança para um atendimento, pois a falta de comunicação existente neste binômio se apresenta de maneira complexa. 6. Almeida IS, Rodrigues BMRD, Simões SMF. Desvelando o cotidiano do adolescente hospitalizado. Rev Bras de Enfermag. [Internet] Rio de Janeiro. 2005. [acesso atualizado em 02 novembro 2016]; mar-abr; 58(2):147-51. Disponível em: http://www.scielo.br/pdf/reben/v58n2/a03.pdf A humanização do cuidado deve ser idealizada dentro de perspectivas que incluam os que necessitam de atendimento especializado, conforme demonstrado neste estudo. As ações voltadas ainda se encontram deficitárias para esta parcela da população, evidenciando um despreparo profissional. 7. Pagliuca LMF, Fiúza NLG, Rebouças CBA. Aspectos da comunicação da enfermeira com o deficiente auditivo. Rev da Esc de Enfermag da USP. [Internet] São Paulo. 2007. [acesso atualizado em 02 novembro 2016]; 41(3):411-8 . Disponível em http://www.scielo.br/pdf/reeusp/v41n3/10.pdf A oferta de ferramentas mínimas na aprendizagem da LIBRAS oportunizariam aos profissionais uma forma inclusiva de atenção aos pacientes surdos. Flexibilizar o horário de trabalho destes profissionais para que possam se qualificar em relação aos aspectos da comunicação a torna efetiva, atendendo as expectativas do paciente em relação aos profissionais. Cabe a estes refletir acerca das necessidades dos pacientes, evitando constrangimentos e oportunizando uma melhor interação. 8. Garcia CL, Pereira HC, Marinho MNASB. Percepções da mulheres acerca do exame de prevenção câncer cérvico- uterino. Rev Bras de Enfermag. [Internet] São Paulo. 2010. [acesso atualizado em 05 novembro 2016]; v2, n.10. Disponível em http://www.sumarios.org/sites/default/files/ pdfs/60759_6438.PDF 9. Souza SRG, Figueiredo AM. Como elaborar projetos, monografias, dissertações e teses. 1ªEd. São Paulo. Lumen Juris 2010. p.30-5. A inserção da disciplina de LIBRAS no currículo acadêmico desvelou a relevância na construção de um profissional, através do processo comunicativo, com competências para reconhecer as necessidades desta parcela populacional em sua integralidade, e, assim, superar falhas nos serviços de saúde e credibilizar a relação profissional/paciente. 10. Araújo EABS, Ferraz FB. O conceito da pessoa com deficiência e seu impacto nas ações afirmativas brasileiras e no mercado de trabalho. In Anais do XIX Encontro Nacional do Compedi . Jun 09-12 [Internet] Fortaleza. 2010. [acesso atualizado em 05 novembro 2016]. Disponível em http://www.repositorio. ufc.br:8080/ri/bitstream/123456789/541/1/2010_eve_ fbferraz.pdf IV - Identificando a percepção do paciente em relação ao profissional A insatisfação presente na fala de todos os participantes nos faz refletir acerca da humanização no atendimento em saúde, visto que o despreparo profissional para a prestação de ações para este segmento populacional No que diz respeito à percepção do paciente sobre o atendimento profissional, apresentamos as seguintes falas: REVISTA ENFERMAGEM ATUAL \| 2017; 80 38 C a v a g n a V M , S i l v a W P J , B r a g a A L S , A n d r a d e M resulta no descrédito dos pacientes surdos em relação aos serviços de saúde, pois, como foi explicitado pelos próprios participantes deste estudo, a comunicação ainda não acontece de maneira efetiva, sempre existindo algum entrave neste processo. 4. Casa Civil. Subchefia de Assuntos Jurídicos – Decreto nº3298. Política Nacional para Integração da Pessoa com Deficiência. [Internet] Brasília. 1999. [acesso atualizado em 02 novembro 2016]; Disponível em http://www.planalto.gov.br/ccivil_03/ decreto/d3298.htm 4. Casa Civil. Subchefia de Assuntos Jurídicos – Decreto nº3298. Política Nacional para Integração da Pessoa com Deficiência. [Internet] Brasília. 1999. [acesso atualizado em 02 novembro 2016]; Disponível em http://www.planalto.gov.br/ccivil_03/ decreto/d3298.htm 5. Bernardes LCG, Maior IML, Spezia CH, Araújo TCCF. Pessoas com deficiência e políticas de saúde no Brasil: reflexões bioéticas. Rev Ciência e Saúde Colet. [Internet] Brasília. 2009. [acesso atualizado em 02 novembro 2016]; v.14(1). Disponível em http://biblioteca.unipac.br/cgi-bin/ wxis.exe?IsisScript=phl8/003.xis&cipar=valadares/phl. cip&bool=exp&opc=decorado&exp=PESSOAS%20COM%20 DEFICIENCIA&code=&lang= Referências 11. Ribeiro MCMA, Carneiro R. A inclusão indesejada: as empresas brasileiras face a lei de cotas para pessoas com deficiência no mercado de trabalho. Rev OES. 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W1998429145.txt	https://bmcinfectdis.biomedcentral.com/counter/pdf/10.1186/1471-2334-8-34	en		Surgical site infections in Italian Hospitals: a prospective multicenter study	BMC infectious diseases	2,008	cc-by	5,937	BMC Infectious Diseases BioMed Central Open Access Research article Surgical site infections in Italian Hospitals: a prospective multicenter study Nicola Petrosillo1, Cecilia MJ Drapeau1, Emanuele Nicastri2, Lorena Martini3, Giuseppe Ippolito3, Maria Luisa Moro4 and ANIPIO5 Address: 12nd Infectious Diseases Division, National Institute for Infectious Diseases "L. Spallanzani", Via Portuense, 292-00149 Rome, Italy, 24th Infectious Diseases Division, National Institute for Infectious Diseases "L. Spallanzani", Via Portuense, 292-00149 Rome, Italy, 3Epidemiology Department, National Institute for Infectious Diseases "L. Spallanzani", Via Portuense, 292-00149 Rome, Italy, 4Agenzia Sanitaria Regione EmiliaRomagna, Viale Aldo Moro, 21-40127 Bologna, Italy and 5ANIPIO: Italian Association of Infection Control Professionals: Full names and institutional addresses of the ANIPIO members are cited in the acknowledgment section Email: Nicola Petrosillo - petrosillo@inmi.it; Cecilia MJ Drapeau - c.drapeau@yahoo.it; Emanuele Nicastri - nicastri@inmi.it; Lorena Martini - lmartini@inmi.it; Giuseppe Ippolito - ippolito@inmi.it; Maria Luisa Moro - mlmoro@regione.emilia-romagna.it; ANIPIO - petrosillo@inmi.it * Corresponding author Published: 7 March 2008 BMC Infectious Diseases 2008, 8:34 doi:10.1186/1471-2334-8-34 Received: 19 July 2007 Accepted: 7 March 2008 This article is available from: http://www.biomedcentral.com/1471-2334/8/34 © 2008 Petrosillo 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: Surgical site infections (SSI) remain a major clinical problem in terms of morbidity, mortality, and hospital costs. Nearly 60% of SSI diagnosis occur in the postdischarge period. However, literature provides little information on risk factors associated to in-hospital and postdischarge SSI occurrence. A national prospective multicenter study was conducted with the aim of assessing the incidence of both in-hospital and postdisharge SSI, and the associated risk factors. Methods: In 2002, a one-month, prospective national multicenter surveillance study was conducted in General and Gynecological units of 48 Italian hospitals. Case ascertainment of SSI was carried out using standardized surveillance methodology. To assess potential risk factors for SSI we used a conditional logistic regression model. We also reported the odds ratios of in-hospital and postdischarge SSI. Results: SSI occurred in 241 (5.2%) of 4,665 patients, of which 148 (61.4%) during in-hospital, and 93 (38.6%) during postdischarge period. Of 93 postdischarge SSI, sixty-two (66.7%) and 31 (33.3%) were detected through telephone interview and questionnaire survey, respectively. Higher SSI incidence rates were observed in colon surgery (18.9%), gastric surgery (13.6%), and appendectomy (8.6%). If considering risk factors for SSI, at multivariate analysis we found that emergency interventions, NNIS risk score, preoperative hospital stay, and use of drains were significantly associated with SSI occurrence. Moreover, risk factors for total SSI were also associated to in-hospital SSI. Additionally, only NNIS, pre-operative hospital stay, use of drains, and antibiotic prophylaxis were associated with postdischarge SSI. Conclusion: Our study provided information on risk factors for SSI in a large population in general surgery setting in Italy. Standardized postdischarge surveillance detected 38.6% of all SSI. We also compared risk factors for in-hospital and postdischarge SSI, thus providing additional information to that of the current available literature. Finally, a large amount of postdischarge SSI were detected through telephone interview. The evaluation of the cost-effectiveness of the telephone interview as a postdischarge surveillance method could be an issue for further research. Page 1 of 9 (page number not for citation purposes) BMC Infectious Diseases 2008, 8:34 Background Surgical site infections (SSI) remain a major clinical problem in terms of morbidity, mortality [1], length of stay and hospital costs [2-4]. Nearly 60% of SSI diagnosis, ranging from 21 to 100%, [5-9] occur after hospital discharge and the trend increases as the length of postoperative hospital stay is getting shorter and the number of one day surgery procedures enlarges over time. According to current literature, active SSI surveillance is useful in reducing SSI incidence by surveillance-induced infection control efforts [10-12]. However, although representing a methodological tool of increasing importance for its large impact on SSI rates [5,13-16], it also represents a methodological challenge for clinicians and epidemiologists, because the patient is not constantly under medical observation, and microbiological investigation becomes difficult to perform after discharge. Indeed, a matter of concern is the choice of the most effective postdischarge surveillance (PDS) methodology to estimate SSI rates; actually, the incidence of postdischarge SSI could depend on which of the different PDS method is being performed. The authors of a recent systematic review of PDS methods reported that no valid and reliable method of SSI case ascertainment is available till now [17]. Most of the studies on postdischarge SSI aimed also to evaluate the associated risk factors, and interestingly, they suggested that most of the predictors of in-hospital SSI were not predictors of postdischarge SSI [7]. Particularly, the composite National Nosocomial Infection Surveillance (NNIS) risk score was found to be positively correlated to the risk of inhospital SSI [18], whereas other reports have documented a discrepancy between the predicted risk of infection by NNIS index score and the incidence of postdischarge SSI [7,8]. A national prospective multicenter study was conducted with the aim of assessing the incidence of both in-hospital and postdisharge SSI, and the associated risk factors. We aimed also to evaluate the performance of the NNIS risk index in predicting SSI occurrence in the Italian Surgical setting, and its validity in stratifying risk according to specific surgical procedures. http://www.biomedcentral.com/1471-2334/8/34 appendectomy (n = 238; 5.1%), vascular surgery (n = 16; 0.3%), and other minor interventions on genitourinary tract (n = 10; 0.21%). The following 6 surgical procedures included also the laparoscopic technique: cholecistectomy (n = 438; 56%), appendectomy (n = 24; 10%), colon surgery (n = 18; 4.9%), abdominal hysterectomy (n = 15; 4.2%), hernia repair (n = 39; 3.6%), and gastric surgery (n = 6; 3.6%). Table 1 summarizes the main characteristics of the study population. All the above listed procedures were included in the surveillance program. One month before starting the study, for each participating center, referent infection control personnel, including infection control practitioners, physicians, and nursing staff attended a training meeting on how to collect information and on patients' follow-up standardization. In-hospital SSI For each patient, infection control personnel used a questionnaire to collect information on demographic and operation specific characteristics, and on SSI occurrence. The following information was ascertained: demographic characteristics, dates of admission and discharge, operation characteristics (i.e. type, date, and duration of the surgical procedure, emergency or elective surgical procedures, wound contamination class according to the four-point wound infection score of the National Research Council [19], American Society of Anesthesiologists (ASA) physical status score [20], any use of endoscopic surgical approach, any prosthesis implant), antimicrobial perioperative prophylaxis, presence of drains, occurrence, date of onset and type of in-hospital SSI. SSI diagnosis was performed using the Centers for Diseases Control and Prevention (CDC) NNIS standardized criteria [21]. According to these criteria, SSI are classified as being either incisional or organ/space. Incisional SSI are divided into those involving skin and subcutaneous tissue (superficial incisional) and those involving deeper Table 1: Population and operation specific characteristics Total number of patients Males (%) 4,665 1630 (34.9%) Methods In 2002, a one-month, prospective national multicenter surveillance study was conducted in General and Gynecological units of 48 Italian hospitals on 4,665 consecutive patients undergoing the following surgical procedures: hernia repair (n = 1,079; 23.1%), caesarean section (n = 1,050; 22.5%), cholecystectomy (n = 781;16.7%), breast surgery (n = 436; 9.3%), colon surgery (n = 364; 7.8%), gastric surgery (n = 165; 3.5%), abdominal hysterectomy (n = 355; 7.6%), vaginal hysterectomy (n = 171;3.6%), Age, years ASA score Length of preoperative stay, days Length of postoperative stay, days Duration of intervention, hours NNIS index score Mean 53 2 2 6 1.3 0 Median 54 2 1 4 1 0 Range (18; 99) (1;5) (0; 69) (0; 377) (0.2; 8.5) (-1; 3) -1 refers to interventions without risk factors for the NNIS index score, performed laparoscopically. ASA = American Society of Anesthesiologists. NNIS = National Nosocomial Infection Surveillance. Page 2 of 9 (page number not for citation purposes) BMC Infectious Diseases 2008, 8:34 soft tissue of surgical incision (deep incisional SSI). Organ/space SSI involve any part of the anatomy other than incised body wall layers, that was opened or manipulated during an operation [21]. Moreover, the NNIS risk index score [22] was calculated by assigning one point each for a contaminated wound according to CDC definition [19], an ASA score ≥ 3, and surgical procedures lasting longer than the NNIS-derived 75th percentile for the duration of the procedure; whenever the procedure was done laparoscopically, the NNIS score was modified by subtracting 1 point, as more recently suggested by Gaynes et al. [23]. The laparoscopic surgical procedures without risk factors for the NNIS index score, where included in a new risk category defined as "M". Postdischarge SSI The last section of the questionnaire was dedicated to the PDS, which reported information collected within 30 days after the operative procedure. Case ascertainment of postdischarge SSI was carried out using the following active surveillance methods: 1) a follow-up questionnaire, that was given to the patient to be filled during follow-up visits by the hospital infection control personnel; if the patient was visited in another health care facility the physician was asked to fill out the questionnaire and to send it back to the reference center; 2) patients who missed follow-up visits were called by telephone by the infection control nurses to be interviewed for any wound signs and symptoms suggestive of SSI. In the first case, SSI were identified by using the CDC criteria [21]. For patients surveilled by telephone calling, SSI diagnosis was based on criteria that are included in CDC definition of SSI, at least for superficial infections. These criteria included one or more of the following self-reported conditions: 1) purulent wound secretion; 2) pain or tenderness, localized swelling, redness, or heat at the surgical site. To assess potential risk factors for SSI we performed a univariate analysis which included the following variables: gender, age, emergency interventions, NNIS score, any prosthesis placement, preoperative hospital stay, use of drains, any perioperative antimicrobial prophylaxis. All the covariates with a p-value less than 0.1 at univariate analysis were included in a multivariate conditional logistic regression model. Statistical analysis Multivariate statistical analysis was performed using SPSS (Version 11.0 Chigago Ilinois USA) statistical software; the continuous variables were analyzed by calculating the variance while the dichotomous variables were analyzed by using the Χ2 test. Results were expressed in terms of http://www.biomedcentral.com/1471-2334/8/34 Odds Ratio (OR) with their respective 95% Confidence Interval (CI). Each participating institution (see in the acknowledgment section) sought ethical clearance through their own Ethical Committee according to local regulations. Verbal informed consent was obtained. Results Surgical site infections occurred in 241 (5.2%) of 4,665 patients. One-hundred-forty-eight SSI (61.4%) occurred during hospital stay, and 93 (38.6%) within 30 days after discharge. Of the 148 in-hospital SSI, 87, 37, and 24 were classified as superficial, deep, and organ/space, respectively. Post-discharge SSI observed at medical follow-up were all superficial. Those SSI ascertained by telephone calling were not classified according to the CDC classification, due to lack of direct medical observation. However, the telephone interviewers referred that in about 90% of cases SSI could be classified as superficial. Table 2 reports the frequencies of SSI according to surgical procedure and NNIS risk index category. If excluding the procedures with unknown PDS status, the highest SSI incidence rates were observed in colon surgery (18.9%), gastric surgery (13.6%), and appendectomy (8.6%); the remaining surgical procedures had lower and similar SSI rates (range 3.2–4%). When calculating the OR of SSI for each NNIS risk index category, significant differences were observed between NNIS index category 0 and 1 for appendectomy: OR 24.38, 95% CI 3.21–510.30; p < 0.001, and for abdominal hysterectomy: OR 3.74, 95% CI 1.34–10.62; p = 0.003, and between NNIS index category M and >M for cholecistectomy: NNIS score 0: OR 3.46; 95% CI 1.00–13.06; p = 0.026; NNIS score 1: OR 4.68; 95% CI 1.29–18.41; p = 0.005; NNIS score 2 + 3: OR 5.15; 95% CI 1.04–25.39; p = 0.012. There was no significant difference between SSI rates in laparoscopic and open cholecistectomy (15/438; 3.4% vs. 15/339; 4.4%; OR 0.77; 95% CI 0.35–1.68; p = 0.473). For the other surgical procedures which also used the laparoscopic technique (i.e. gastric surgery, appendectomy, colon surgery, hernia repair, and abdominal hysterectomy), differences among the SSI rates between NNIS index category M and >M were not statistically significant. We found that 4,486 out of 4,665 enrolled patients, excluding those who developed an in-hospital SSI (n = 148), and those with a postoperative in-hospital stay longer than 30 days (n = 31), were eligible for PDS. A total of 4,228/4,486 (94.2%) patients underwent PDS, whereas 258 (5.8%) patients were lost to follow-up. One hundred Page 3 of 9 (page number not for citation purposes) BMC Infectious Diseases 2008, 8:34 http://www.biomedcentral.com/1471-2334/8/34 Table 2: Frequencies of SSI§ according to type of intervention and NNIS§§ index category. Surgical procedure Breast surgery Cesarean section Cholecistectomy Gastric surgery Appendectomy Colon surgery Hernia repair Abdominal hysterectomy Vaginal hysterectomy Total** number of procedures (n = 4,386) 406 1012 740 147 221 338 1014 346 162 N SSI (%) 13 (3.2%) 27 (2.6%) 30 (4%) 20 (13.6%) 19 (8.6%) 64 (18.9%) 41 (4%) 21 (6.1%) 6 (3.7%) N SSI (%) according to NNIS index category M 0 1 2+3 4/287 (1.4%) 1/3 (33.3%) 0/16 (0%) 2/4 (50%) 1/19 (5.3%) 1/12 (8.3%) - 10/281 (3.5%) 20/775 (2.6%) 11/236 (4.6%) 3/50 (6%) 1/108 (0.9%) 14/70 (20%) 20/641 (3.1%) 7/217 (3.2%) 3/117 (2.5%) 2/111 (1.8%) 6/218 (2.7%) 9/145 (6.2%) 1/63 (1.6%) 13/76 (17.1%) 24/151 (15.9%) 15/283 (5.3%) 11/91 (12.1%) 3/41 (7.3%) 1/10 (10%) 0/8 (0%) 4/59 (6.7%) 4/23 (17.4%) 3/18 (16.6%) 22/109 (20.2%) 4/46 (8.7%) 1/20 (5%) 0/2 (0%) § Surgical site infections. Nosocomial Infection Surveillance index category. Cardiovascular and other minor interventions are not reported because the denominator was too little (n = 21) to be included in statistical analysis. * The procedures with no postdischarge surveillance (258) were not included. M: no risk factor for NNIS index score plus laparoscopical procedure (NNIS = -1). §§National and eighty-four questionnaires were filled and returned back to the reference center; telephone interviews were carried out for the remaining 4,041 patients. Sixty-two (66.7%) and 31 (33.3%) of 93 postdischarge SSI were detected through telephone interview and questionnaire survey, respectively. At univariate analysis, age ≥ 55 years, emergency interventions, NNIS score > 0, use of prosthesis, pre-operative hospital stay > 1 day, and use of drains resulted significantly associated with SSI occurrence. At multivariate analysis, emergency interventions, NNIS score > 0, pre-operative hospital stay > 1 day, and use of drains remained significantly associated with SSI occurrence (Table 3). When stratifying risk factors for in-hospital and postdischarge SSI, we found that those factors that were significantly associated for total SSI, were also associated for inhospital SSI. However, if considering postdischarge SSI, only NNIS >1, pre-operative hospital stay >1, use of drains, and antibiotic prophylaxis were associated with postdischarge SSI (Table 4). Moreover, clean or clean/contaminated operations such as breast surgery, caesarean section, cholecistectomy, and hernia repair had postdischarge SSI rates from 1.2 to 3.3 times higher than those observed during the hospital stay. On the other hand, we found that contaminated operations had higher in-hospital SSI rates (Table 5). actually unknown in Europe and is likely to have been underestimated as a consequence of the variability of data collection, surveillance methods, and type of surgical procedures that are investigated. In the present study, the overall SSI incidence rate was 5.2% which is lower than that reported in other Italian (range 5.4%–12.8%) [24-26,9], and European [7,15] studies including the European Surveillance of Surgical site Infections HELICS – Improving patient Safety in Europe (IPSE) network [27], which used the NNIS definitions and surveillance methodology. In particular, if considering only general, gynecological (including caesarean section), and vascular surgery interventions, European SSI rates are higher and globally range from 6.34% to 14.8% [7,15]. However, for some types of interventions our SSI rates are higher than those reported in the literature. In our study, we found that the highest SSI incidence were observed in colon surgery (18.9%), gastric surgery (13.6%) and appendectomy (8.6%). These rates are about two fold higher than those reported by other national and European studies, and by the U.S. NNIS reports [10,9,24,28]. Moreover, the rates of SSI after cholecystectomies was very high, as compared to those mentioned in the HELICS network [27] These differences could depend on type, accuracy, and distribution of PDS methods [10,15,24,10], on losses in follow-up, and on the impossibility to assess the postdischarge response validity. Discussion In our study we used the NNIS methodology in order to standardize and compare our data with those published in the current literature. Indeed, according to a retrospective review on European studies [4], the true rate of SSI is In our study, we found that emergency interventions, NNIS index score, and pre-operative hospital stay were independently associated to overall SSI occurrence. These findings are not discordant with previous reports of the Page 4 of 9 (page number not for citation purposes) BMC Infectious Diseases 2008, 8:34 http://www.biomedcentral.com/1471-2334/8/34 Table 3: Risk factors for surgical site infection (SSI). Univariate analysis Variable Gender Male Female Age, years 18–36 37–54 55–70 >70 Emergency Interventions Yes No NNIS score M 0 1 2+3 Prosthesis Yes No Pre-operative hospital stay, days 0–1 >1 Drains No 1–3 days >3 days Perioperative prophylaxis Yes No Multivariate analysis Total § No. SSI/total§ Odds Ratio (95% CI^^) p* Odds Ratio (95% CI^^) p* 4,406 97/1521 144/2,885 1.30 (0.99–1.70) 0.054 1.13 (0.83–1.55) 0.429 4,172 35/1,101 45/1,004 67/1,107 88/960 1 1.43 (0.89–2.30) 1.96 (1.27–3.04) 3.07 (2.02–4.69) 0.118 0.001 <0.001 1.11 (0.72–1.69) 0.91 (0.63–1.31) 1.51 (0.91–2.51) 0.635 0.622 0.113 4,396 69/944 172/3,452 1.50 (1.11–2.03) 0.005 1.73 (1.22–2.44) 0.002 4,330 8/341 90/2,507 90/1,182 41/300 1 1.55 (0.72–3.48) 3.43 (1.59–7.71) 6.59 (2.91–15.50) 0.237 <0.001 <0.001 1.18 (0.77–1.82) 1.82 (1.14–2.90) 3.34 (1.41–7.93) 0.441 0.012 0.006 4,393 23/762 218/3,631 0.60 (0.39–0.91) 0.011 0.62 (0.38–1.04) 0.07 4,337 130/3,136 102/1,201 2.15 (1.63–2.83) <0.001 1.45 (1.06–1.98) 0.02 4,406 97/2,894 31/702 111/810 1 3.04 (2.31–3.99) 4.58 (3.41–6.15) <0.001 <0.001 2.39 (0.65–1.65) 2.17 (1.38–3.43) <0.001 <0.001 213/3,857 28/549 0.92 (0.60–1.40) 0.684 - - 4,406 § total number of procedures considered not equal to the study cohort (n = 4,665) for missing data; also patients with missing data for SSI occurrence during postdischarge (n = 258) were excluded from the analysis. ^^ 95% confidence intervals (CI). * p-value. M no risk factor for NNIS index score + laparoscopical procedure (NNIS = -1). NNIS = National Nosocomial Infection Surveillance. literature [18,22,24]; however, we provided additional details that deserve particular attention. First, when stratifying by NNIS index category according to operative procedure, we found no significant differences between NNIS index category, for most of the procedures except for appendectomy, abdominal hysterectomy (Table 2). Regarding laparoscopic procedures, it should be noticed that the small number of these procedures in some interventions was a limiting factor in the statistical analysis. Actually, conflicting data are reported in the literature about the predictive power of the NNIS index score, at least for specific interventions such as cesarean sections and breast interventions [10,8,29-31]. Furthermore, in agreement with our findings, in other reports [32,22] NNIS index score did not perform well for single surgical procedures, but it was found to perform better when taking into account a group of different interventions. On the contrary, according to the HELICS-IPSE most recent reports, the SSI increases with the NNIS index score, even for caesarean sections [27]. A possible explanation of our finding may be that the NNIS index score underestimates SSI incidence as it represents an inpatient risk score which does not include postdischarge surveillance variables. Therefore, NNIS index score may not represent a suitable tool for the SSI risk evaluation in particularly for clean interventions where SSI are likely to be detected during postdischarge surveillance, probably due to anticipated hospital discharge. Page 5 of 9 (page number not for citation purposes) BMC Infectious Diseases 2008, 8:34 http://www.biomedcentral.com/1471-2334/8/34 Table 4: Risk factors for in-hospital and postdischarge surgical site infection (SSI). In-hospital period Variable Gender Male Female Age (years) 18–36 37–54 55–70 >70 Emergency Intervention Post-discharge period Total § No. SSI/total§ Odds Ratio (95% CI^^) Total§S No. SSI/total §§ Odds Ratio (95% CI^^) 4,665 4,420 67/1,625 81/3,040 1.57 (1.12–2.21)* 15/1,163 26/1,057 44/1,162 60/1,038 1 1.93 (0.98–3.84)# 3.01 (1.62–5.69)* 4.70 (2.58–8.68)* 30/1,454 63/2,804 0.92 (0.58–1.45)# 20/1,086 19/978 23/1,063 28/900 1 1.06 (0.54–2.08)# 1.18 (0.62–2.25)# 1.71 (0.93–3.18)# 51/1,002 97/3,653 1.97 (1.37–2.82)* 18/903 75/3,355 0.89 (0.51–1.53)# 4/358 38/2,621 1 1.30 (0.44–4.32)# 4/336 52/2,459 1 1.79 (0.62–5.87)# 65/1,260 30/340 4.81 (1.67– 15.63)* 11.42 (3.86–38)* 25/1,108 11/263 1.92 (0.63–6.54)# 3.62 (1.05– 13.65)* 6/801 142/3,851 0.20 (0.08–0.46)* 17/756 76/3,489 1.03 (0.58–1.80)# 4,655 Yes No NNIS score § M 0–1 >1 Drains No 1–3 days >3 days Perioperativ e antibiotic prophylaxis Yes No 4,652 4,166 4,589 4,665 4,027 4,258 4,579 0 1 2+3 Prosthesis Yes No Pre-operative hospital stay (days) 4,258 4,245 4,196 72/3,300 69/1,289 2.54 (1.79–3.60)* 41/3,045 19/764 88/856 1 1.87 (1.04–3.33)* 8.40 (5.66–12.48)* 4,665 4,261 58/3,064 33/1,132 1.56 (0.99–2.45)* 56/2,853 12/686 23/722 1 0.89 (0.45–1.72)# 1.64 (0.97–2.75)* 74/3,718 19/540 1.80 (1.04–3.07)* 4,258 139/4,071 9/594 0.44 (0.21–0.89)* § total number of procedures surveilled during hospital stay not equal to the study cohort (n = 4,665) for missing data. NNIS = National Nosocomial Infection Surveillance. §§ total number of interventions surveilled during post-discharge, does not include missing data for SSI occurrence during postdischarge (n = 258), and patients with in-hospital SSI (n = 148) or with postoperative hospital stay ≥ 30 days (n = 25). ^^ 95% confidence intervals. * p-value < 0.05. # p value not significant. M: no risk factor + endoscopical procedure. Second, we observed that the use of drains was significantly associated with SSI occurrence independently of the length of drainage (i.e., 1–3 or < 3 days); additionally, we found that length of preoperative hospital stay > 1 day was associated with a significant risk of SSI. Of note, preoperative hospital may be independent from one day surgery procedures. This finding underlines the importance of performing day surgery procedures, whenever possible. Third, we found the SSI rate among laparoscopic cholecistectomy interventions was lower than the open technique but did not reached a statistical significance, contrarily to what reported in other reports [33]. In the present study, we also aimed to provide information on postdischarge SSI. Interestingly, we found that 38.6% of SSI were diagnosed after discharge; this rate is comparable to the 34.8% reported by Fiorio et al. [24] in Italian general surgery inpatients, although other similar surveillance studies [7-9] found considerably higher postdischarge SSI rates, ranging from 34.8 to 60%. We also observed that the higher ratio of postdischarge/in-hospi- Page 6 of 9 (page number not for citation purposes) BMC Infectious Diseases 2008, 8:34 http://www.biomedcentral.com/1471-2334/8/34 Table 5: Rates of in-hospital and postdischarge surgical site infections (SSIs) according to type of intervention. Surgical procedure N°Postdischarge SSI/total* (n = 4,407) Vascular surgery Breast surgery Cesarean section Cholecistectomy Gastric surgery Appendectomy Colon surgery Hernia repair Abdominal hysterectomy Vaginal hysterectomy Other genitourinary surgery 0/16 (0%) 10/406 (2.4%) 19/1012 (1.8%) 16/740 (2.1%) 5/147 (3.4%) 3/221 (1.4%) 6/338 (1.8%) 23/1014 (2.3%) 8/346 (2.3%) 3/162 (1.8%) 1/5 (20%) In-hospital SSI/total (n = 4,665) Posdischarge/In-hospital ratio 0/16 (0%) 3/437 (0.7%) 8/1050 (0.7%) 14/781 (1.8%) 15/165 (9.1%) 16/238 (6.7%) 58/364 (15.9%) 18/1079 (1.6%) 13/355 (3.7%) 3/171 (1.7%) 0/5 (0%) 3.4 2.5 1.2 0.4 0.2 0.1 1.4 0.6 1 - * The procedures with unknown PDS status (n = 258) were not included. tal SSI was found in clean interventions, as evidenced in other recent reports [7,9,15]. In particular, breast interventions, and cesarean sections had higher SSI incidence rates during the postdischarge period, ie 2.4% and 1.8%, respectively (Table 5). A possible explanation to this finding could be the shorter postoperative stay which characterizes the above mentioned interventions. Our finding should advice infection control practitioners of the need for improving PDS surveillance methods for targeted surgical procedures. Moreover, we were able to provide a better insight on the risk factors for postdischarge SSI, for which information from the literature is scant. Indeed, in our study we found that most predictors of in-hospital SSI were not predictors of postdischarge SSI, as already suggested by DelgadoRodriguez et al. [7] Only NNIS >1, preoperative hospital stay, and antibiotic prophylaxis were significantly associated with postdischarge diagnosis. The presence of drains was marginally predictive of postdischarge SSI, and only if drains remained for more than 3 days. These data are discordant with those of other similar studies [7,5] that failed to identify risk factors for postdischarge SSI, particularly on what concerns the validity of the NNIS index score. Another important finding of our study is the importance of telephone interview for the detection of postdischarge SSI. Indeed, almost 60% of postdischarge SSI were diagnosed by telephone interview, which underlines the importance of this cost-effective surveillance methodology for SSI detection. On the other hand, we are aware of the little supporting evidence regarding the validity and reliability of self-reported diagnosis, as suggested by other reports [17]. diabetes, cancer, immunosuppressive drugs. Moreover, another limitation of our study depends on the lack of standardized post-discharge surveillance methodologies, which could affect the validity of the post-discharge SSI rate. In fact, accurate, and standardized methods for defining and monitoring post-discharge SSI are needed to correct assess infection rates. Additionally, our study did not provide any information regarding the cost-effectiveness of telephone post-discharge surveillance, and on the economic impact of postdischarge SSI on public health. We suggest to evaluate the cost-effectiveness of such postdischarge method for further research. Finally, although most of the postdischarge SSI were superficial, we think that looking at them could make sense in terms of economic impact on public health as evidenced by recent studies [34,35]. Conclusion Our study provided information on risk factors for SSI occurrence in a large population in general surgery setting in Italy. Moreover, standardized postdischarge surveillance methodology was carried out, which detected 38.6% of all SSI. We were also able to compare risk factors for in-hospital and postdischarge SSI, thus providing additional information to that of the current available literature. Particularly, we found that NNIS >1, preoperative hospital stay >1 day, use of drains, and antibiotic prophylaxis were significantly associated to postdischarge SSI diagnosis. Moreover, a large amount of postdischarge SSI were detected through telephone interview, which underlines the importance of this PDS methodology for its costeffectiveness. Our study has some limitations including the lack of other possible host-related risk factors to be included in multivariate analysis such as body mass index, malnutrition, Page 7 of 9 (page number not for citation purposes) BMC Infectious Diseases 2008, 8:34 Competing interests Financial competing interests: NP is on the speakers' bureau for several companies including Merck Sharpe & Dohme, Aventis, Ethicon, GSK, Pfizer, AstraZeneca, Roche, Gilead. None of the other authors has financial competing interests. None of the authors has non financial competing interests to disclose. Authors' contributions NP, LM, and MLM conceived the study, contributed to its design, and to the manuscript draft and final version. CMJD contributed to the analysis of the data and to manuscript draft and final version. EN performed statistical analysis, and contributed to interpretation of data. ANIPIO group contributed to data collection. All authors read and approved the final manuscript. Acknowledgements http://www.biomedcentral.com/1471-2334/8/34 7. 8. 9. 10. 11. 12. 13. Work supported by Research project of Ministry of Health. Members of the ANIPIO group are: Roccamaria M. (Matera); Bonifacio M. (Matera), Bartocci L. (Roma); Alchieri S.(Crema); Briuglia R.(Messina); Palermo R.A (Taranto); Adriatico G., (Bari); Ballarino N, (Foggia); Pietraforte A. (Rieti); Batticiocca D. (Roma); Musolino M. (Roma); Tassoni V. (Tarquinia); Allochis M.C. (Pinerolo); Biagetti M. (Torino); Bacchetta B. (Borgomanero); Liggera L. (Savigliano); Lochi A. (Rivoli); Boux M. (Ivrea); Cappoia S. (Milano); Cerri F. (Vizzolo Predabissi); Burato E. (Mantova); Tentori C. (Lecco); Ruffato M. (Mirano); Bissolo E. (Verona); Marchetto R. (Rovigo); Germanis Cunja L. (Trieste); Stalio P. (Trieste); Mastaglia M. (Aosta); Olori M. (Ascoli Piceno); Colasanti P. (Pesaro); Giarrusso P. (S. Benedetto del Tronto); Palego M. (Terni); Metelli R.(Foligno); Zanni A. (Bologna); Bernardini V. (Pontedera); Sermenghi A. (Pescia); Rosadoni B. (Firenze); Caracolli C. (Valdichiana Cortona); Visani F.(Grosseto); Fabbri P.(Genova); Assensi M. (Imperia); Cattaneo M. (Genova); Greco F. (Pistoia); Cabutti S. (Alba e Brà Cuneo); Occelli P. (Cuneo); Boschetto M. (Padova); Daniela Venir, Guarneri S. (San Vito al Tagliamento); Amadori A., Bergamini R. (Forlì); Accorgi D., Ballotti M. (Prato); Bronzini B. (Macerata); Rosano A. (Poliporo); Maestri U. (Palermo); Dilani V. 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https://openalex.org/W3006394817	https://www.biorxiv.org/content/biorxiv/early/2019/05/02/626291.full.pdf	English	null	Differential Gene Expression between Fungal Mating Types Is Associated with Sequence Degeneration	Genome biology and evolution	2,020	cc-by	33,473	. CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 1 Differential gene expression is associated with 2 degeneration of mating-type chromosomes in 3 the absence of sexual antagonism 4 5 6 Wen-Juan Ma1, Fantin Carpentier2, Tatiana Giraud2, Michael Hood1 7 8 9 1. Department of Biology, Amherst College, Amherst, 01002 Massachusetts, USA 10 11 2. Ecologie Systématique Evolution, Bâtiment 360, Univ. Paris-Sud, AgroParisTech, CNRS, 12 Université Paris-Saclay, 91400, Orsay, France 13 14 15 16 17 18 19 20 21 22 * Corresponding authors: wenjuanma84@gmail.com, mhood@amherst.edu 23 24 25 26 27 28 29 30 31 32 33 Running title: Differential gene expression correlates with sequence degeneration 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 1 Differential gene expression is associated with 2 degeneration of mating-type chromosomes in 3 the absence of sexual antagonism 4 5 6 Wen-Juan Ma1, Fantin Carpentier2, Tatiana Giraud2, Michael Hood1 7 8 9 1. Department of Biology, Amherst College, Amherst, 01002 Massachusetts, USA 10 11 2. Ecologie Systématique Evolution, Bâtiment 360, Univ. Paris-Sud, AgroParisTech, CNRS, 12 Université Paris-Saclay, 91400, Orsay, France 13 14 15 16 17 18 19 20 21 22 * Corresponding authors: wenjuanma84@gmail.com, mhood@amherst.edu 23 24 25 26 27 28 29 30 31 32 33 Running title: Differential gene expression correlates with sequence degeneration 34 35 36 37 38 39 40 41 42 43 44 1 Differential gene expression is associated with 2 degeneration of mating-type chromosomes in 3 the absence of sexual antagonism Wen-Juan Ma1, Fantin Carpentier2, Tatiana Giraud2, Michael Hood1 1 1 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 49 Abstract 50 In animals and plants, differential expression of genes on sex chromosomes is widespread 51 and it is usually considered to result from sexually antagonistic selection; however differential 52 expression can also be caused by asymmetrical sequence degeneration in non-recombining sex 53 chromosomes, which has been very little studied. The anther-smut fungus Microbotryum 54 lychnidis-dioicae is ideal to investigate the extent to which differential gene expression is 55 associated with sequence degeneration because: 1) separate haploid cultures of opposite mating 56 types help identify differential expression, 2) its mating-type chromosomes display multiple 57 evolutionary strata reflecting successive events of gene linkage to the mating-type loci, and 3) 58 antagonistic selection is unlikely between isogamous haploid mating types. We therefore tested 59 the hypothesis that differential gene expression between mating types resulted from sequence 60 degeneration. We found that genes showing differential expression between haploid mating types 61 were enriched only on the oldest evolutionary strata of the mating-type chromosomes and were 62 associated with multiple signatures of sequence degeneration. We found that differential 63 expression between mating types was associated with elevated differences between alleles in 64 non-synonymous substitution rates, indels and premature stop codons, transposable element 65 insertions, and altered intron and GC content. Our findings strongly suggest that degenerative 66 mutations are important in the evolution of differential expression in non-recombining regions. 67 Our results are relevant for a broad range of taxa where mating compatibility or sex is determined 68 by genes located in large regions of recombination suppression, showing that differential 69 expression should not be taken as necessarily arising from antagonistic selection. 70 71 Key words: sex chromosomes, differential gene expression, sequence degeneration, haploid 72 culture, transposable elements, dN, premature stop codon, GC content, intron, Microbotryum 73 fungus 50 In animals and plants, differential expression of genes on sex chromosomes is widespread 51 and it is usually considered to result from sexually antagonistic selection; however differential 52 expression can also be caused by asymmetrical sequence degeneration in non-recombining sex 53 chromosomes, which has been very little studied. 49 Abstract The anther-smut fungus Microbotryum 54 lychnidis-dioicae is ideal to investigate the extent to which differential gene expression is 55 associated with sequence degeneration because: 1) separate haploid cultures of opposite mating 56 types help identify differential expression, 2) its mating-type chromosomes display multiple 57 evolutionary strata reflecting successive events of gene linkage to the mating-type loci, and 3) 58 antagonistic selection is unlikely between isogamous haploid mating types. We therefore tested 59 the hypothesis that differential gene expression between mating types resulted from sequence 60 degeneration. We found that genes showing differential expression between haploid mating types 61 were enriched only on the oldest evolutionary strata of the mating-type chromosomes and were 62 associated with multiple signatures of sequence degeneration. We found that differential 63 expression between mating types was associated with elevated differences between alleles in 64 non-synonymous substitution rates, indels and premature stop codons, transposable element 65 insertions, and altered intron and GC content. Our findings strongly suggest that degenerative 66 mutations are important in the evolution of differential expression in non-recombining regions. 67 Our results are relevant for a broad range of taxa where mating compatibility or sex is determined 68 by genes located in large regions of recombination suppression, showing that differential 69 expression should not be taken as necessarily arising from antagonistic selection. 70 71 Key words: sex chromosomes, differential gene expression, sequence degeneration, haploid 72 lt t bl l t dN t t d GC t t i t Mi b t 74 2 2 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 75 Author Summary 76 Differences between males and females, from morphology to behavior and physiology, are 77 considered to largely reflect differential expression of genes that maximize fitness benefits 78 relative to costs that are specific to one sex. However, there is an unexplored alternative to such 79 ‘sexually antagonistic selection’ to explain differential expression. Reproductive compatibility is 80 often determined by genes located in large non-recombining chromosomal regions, where 81 degenerative mutations are expected to accumulate and may separately affect the expression of 82 alternate alleles of genes. We tested the role of genetic degeneration in determining differential 83 expression between the isogamous haploid mating types of the anther-smut fungus, 84 Microbotryum lychnidis-dioicae, where sexually antagonistic selection is not a confounding 85 factor. We show that differentially expressed genes are highly enriched in the non-recombining 86 mating-type chromosomes, and that they are associated with various forms of degenerative 87 mutations, some of which indicate that the less expressed allele suffers greater mutational effects. 88 Our finding of the role for degenerative mutations in the evolution of differential expression is 89 relevant for a broad range of organisms where reproductive compatibility or sex is determined by 90 genes in regions of suppressed recombination, and shows that differential expression should not 91 be taken as necessarily arising from antagonistic selection. 76 Differences between males and females, from morphology to behavior and physiology, are 77 considered to largely reflect differential expression of genes that maximize fitness benefits 78 relative to costs that are specific to one sex. However, there is an unexplored alternative to such 79 ‘sexually antagonistic selection’ to explain differential expression. Reproductive compatibility is 80 often determined by genes located in large non-recombining chromosomal regions, where 81 degenerative mutations are expected to accumulate and may separately affect the expression of 82 alternate alleles of genes. We tested the role of genetic degeneration in determining differential 83 expression between the isogamous haploid mating types of the anther-smut fungus, 84 Microbotryum lychnidis-dioicae, where sexually antagonistic selection is not a confounding 85 factor. We show that differentially expressed genes are highly enriched in the non-recombining 86 mating-type chromosomes, and that they are associated with various forms of degenerative 87 mutations, some of which indicate that the less expressed allele suffers greater mutational effects. 75 Author Summary 88 Our finding of the role for degenerative mutations in the evolution of differential expression is 89 relevant for a broad range of organisms where reproductive compatibility or sex is determined by 90 genes in regions of suppressed recombination, and shows that differential expression should not 91 be taken as necessarily arising from antagonistic selection. 98 3 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 99 Introduction 99 Introduction 100 Sexual antagonism occurs when trait values that increase gene transmission through the male 101 function decrease gene transmission through the female function, or conversely [1–4]. 102 Differential gene expression between sexes is widely thought to be the primary means of 103 resolving the conflict of sexual antagonism, resulting in commonly called “sex-biased genes” 104 [5,6]. This adaptive explanation for the existence of sex-biased genes predominates the literature 105 [5–10], although empirical tests are rare and alternative hypotheses may explain the existence of 106 sex-biased genes in sex chromosomes. Non-recombining regions on sex chromosomes often 107 undergo degenerative changes that may also cause differential gene expression. Recombination 108 suppression indeed renders selection less effective due to reduced effective population size [11], 109 genetic hitchhiking of deleterious mutations with beneficial ones [2], and deleterious mutation 110 sheltering [12–14]. Over time, deleterious mutations accumulate in non-recombining regions, 111 e.g., in Y and W chromosomes [15–17], potentially including mutations that result in non-optimal 112 expression [16]. In contrast, X and Z chromosomes can recombine in the homogametic sex and 113 are thus less prone to degeneration. Sex-biased expression could thus result from degeneration, 114 without involving sexually antagonistic selection, although this hypothesis has received little 115 consideration [18,19]. For example, genes with differential expression on neo-sex chromosomes 116 of the passerine bird Sylvia communis [20], and in the mating-type chromosomes in the 117 hermaphroditic fungus Neurospora tetrasperma [21] were found to have elevated sequence 118 divergence between alleles. While those studies pointed to sexual antagonism as the likely cause 119 of differential expression between sexes and the relationship to sequence divergence between 120 alleles, accumulated mutations with degenerative effects remain an alternative possibility. 121 Several different types of mutations can cause sequence degeneration and alter gene 122 expression. Base pair substitutions and indels (insertion or deletion mutations) can change amino 123 acid sequences which can affect gene expression through modulation of the mRNA translation 124 [22], or disrupt promoter regions that impact transcriptional regulation [23]. Induction of early 100 Sexual antagonism occurs when trait values that increase gene transmission through the male 101 function decrease gene transmission through the female function, or conversely [1–4]. 102 Differential gene expression between sexes is widely thought to be the primary means of 103 resolving the conflict of sexual antagonism, resulting in commonly called “sex-biased genes” 104 [5,6]. 99 Introduction ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 125 stop codons that truncate protein length can lead to post-transcriptional regulatory negative 126 feedbacks upon expression (e.g. nonsense mediated decay; [24]). Transposable element insertion 127 in upstream promoter regions, or internal to genes, has long been recognized to have effects on 128 expression [25–29]. Epigenetic modifications, particularly cytosine methylation, contribute both 129 to heterochromatin formation and elevating mutation rates that reduce GC content [30–32]; thus 130 reduced GC content could represent a signature of methylation-induced gene silencing. Shorter 131 introns are more efficient for correcting transcription [33], such that changes in introns can 132 influence transcription rates, nuclear export, and transcript stability [34]. These forms of 133 degenerative changes are expected to accumulate under the reduced selection efficacy in non- 134 recombining regions. Yet, very few studies have addressed the relationship between sequence 135 degeneration and differential gene expression on sex chromosomes [5,35,36]. 136 The rarity of studies relating sequence degeneration to levels of differential gene expression 137 is likely due to several major challenges they face. Studying degenerative processes requires 138 assessing allele specific expression and comparing expression at various ages of sex chromosome 139 divergence, including young sex chromosomes where recombination suppression events are 140 recent. In commonly studied diploid organisms, however, sex chromosomes are old and already 141 highly degenerated, where sex-linked gene expression can be impacted by gene presence/absence 142 asymmetry between sex chromosomes and the resulting dosage compensation [37,38]. On the 143 other hand, the investigation of the early stages of sex chromosome differentiation is rendered 144 challenging by the lack of nucleotide differences which makes it difficult to confidently assigning 145 transcripts to alleles in diploid organisms [39]. In addition, sex chromosomes are challenging to 146 assemble in plants and animals, and the coverage-based method commonly used to identify sex- 147 linked genes [19,40] is not applicable in young sex chromosome systems without much gene loss. 148 For diploid systems with young sex chromosomes, sex-specific linkage maps are thus often 149 needed to assign alleles to X/Z or Y/W chromosomes. 125 stop codons that truncate protein length can lead to post-transcriptional regulatory negative 126 feedbacks upon expression (e.g. nonsense mediated decay; [24]). Transposable element insertion 127 in upstream promoter regions, or internal to genes, has long been recognized to have effects on 128 expression [25–29]. 99 Introduction This adaptive explanation for the existence of sex-biased genes predominates the literature 105 [5–10], although empirical tests are rare and alternative hypotheses may explain the existence of 106 sex-biased genes in sex chromosomes. Non-recombining regions on sex chromosomes often 107 undergo degenerative changes that may also cause differential gene expression. Recombination 108 suppression indeed renders selection less effective due to reduced effective population size [11], 109 genetic hitchhiking of deleterious mutations with beneficial ones [2], and deleterious mutation 110 sheltering [12–14]. Over time, deleterious mutations accumulate in non-recombining regions, 111 e.g., in Y and W chromosomes [15–17], potentially including mutations that result in non-optimal 112 expression [16]. In contrast, X and Z chromosomes can recombine in the homogametic sex and 113 are thus less prone to degeneration. Sex-biased expression could thus result from degeneration, 114 without involving sexually antagonistic selection, although this hypothesis has received little 115 consideration [18,19]. For example, genes with differential expression on neo-sex chromosomes 116 of the passerine bird Sylvia communis [20], and in the mating-type chromosomes in the 117 hermaphroditic fungus Neurospora tetrasperma [21] were found to have elevated sequence 118 divergence between alleles. While those studies pointed to sexual antagonism as the likely cause 119 of differential expression between sexes and the relationship to sequence divergence between 120 alleles, accumulated mutations with degenerative effects remain an alternative possibility. 121 Several different types of mutations can cause sequence degeneration and alter gene 122 expression. Base pair substitutions and indels (insertion or deletion mutations) can change amino 123 acid sequences which can affect gene expression through modulation of the mRNA translation 100 Sexual antagonism occurs when trait values that increase gene transmission through the male 101 function decrease gene transmission through the female function, or conversely [1–4]. 4 4 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. 99 Introduction Epigenetic modifications, particularly cytosine methylation, contribute both 129 to heterochromatin formation and elevating mutation rates that reduce GC content [30–32]; thus 130 reduced GC content could represent a signature of methylation-induced gene silencing. Shorter 131 introns are more efficient for correcting transcription [33], such that changes in introns can 132 influence transcription rates, nuclear export, and transcript stability [34]. These forms of 133 degenerative changes are expected to accumulate under the reduced selection efficacy in non- 134 recombining regions. Yet, very few studies have addressed the relationship between sequence 135 degeneration and differential gene expression on sex chromosomes [5,35,36]. 125 stop codons that truncate protein length can lead to post-transcriptional regulatory negative 126 feedbacks upon expression (e.g. nonsense mediated decay; [24]). Transposable element insertion 127 in upstream promoter regions, or internal to genes, has long been recognized to have effects on 128 expression [25–29]. Epigenetic modifications, particularly cytosine methylation, contribute both 129 to heterochromatin formation and elevating mutation rates that reduce GC content [30–32]; thus 130 reduced GC content could represent a signature of methylation-induced gene silencing. Shorter 131 introns are more efficient for correcting transcription [33], such that changes in introns can 132 influence transcription rates, nuclear export, and transcript stability [34]. These forms of 133 degenerative changes are expected to accumulate under the reduced selection efficacy in non- 134 recombining regions. Yet, very few studies have addressed the relationship between sequence 135 degeneration and differential gene expression on sex chromosomes [5,35,36]. 136 The rarity of studies relating sequence degeneration to levels of differential gene expression 137 is likely due to several major challenges they face. Studying degenerative processes requires 138 assessing allele specific expression and comparing expression at various ages of sex chromosome 139 divergence, including young sex chromosomes where recombination suppression events are 140 recent. In commonly studied diploid organisms, however, sex chromosomes are old and already 141 highly degenerated, where sex-linked gene expression can be impacted by gene presence/absence 142 asymmetry between sex chromosomes and the resulting dosage compensation [37,38]. On the 143 other hand, the investigation of the early stages of sex chromosome differentiation is rendered 144 challenging by the lack of nucleotide differences which makes it difficult to confidently assigning 145 transcripts to alleles in diploid organisms [39]. 99 Introduction In addition, sex chromosomes are challenging to 146 assemble in plants and animals, and the coverage-based method commonly used to identify sex- 147 linked genes [19,40] is not applicable in young sex chromosome systems without much gene loss. 148 For diploid systems with young sex chromosomes, sex-specific linkage maps are thus often 149 needed to assign alleles to X/Z or Y/W chromosomes. 136 The rarity of studies relating sequence degeneration to levels of differential gene expression 137 is likely due to several major challenges they face. Studying degenerative processes requires 138 assessing allele specific expression and comparing expression at various ages of sex chromosome 139 divergence, including young sex chromosomes where recombination suppression events are 140 recent. In commonly studied diploid organisms, however, sex chromosomes are old and already 141 highly degenerated, where sex-linked gene expression can be impacted by gene presence/absence 142 asymmetry between sex chromosomes and the resulting dosage compensation [37,38]. On the 143 other hand, the investigation of the early stages of sex chromosome differentiation is rendered 144 challenging by the lack of nucleotide differences which makes it difficult to confidently assigning 145 transcripts to alleles in diploid organisms [39]. In addition, sex chromosomes are challenging to 146 assemble in plants and animals, and the coverage-based method commonly used to identify sex- 147 linked genes [19,40] is not applicable in young sex chromosome systems without much gene loss. 148 For diploid systems with young sex chromosomes, sex-specific linkage maps are thus often 149 needed to assign alleles to X/Z or Y/W chromosomes. 5 5 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. 99 Introduction ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 150 Fungi can provide valuable insights into the relationship between sequence degeneration and 151 differential gene expression in sex or mating-type chromosomes, due to easy access to the 152 haploid phase where alternate mating types are expressed, the existence of young events of 153 recombination suppression in successive evolutionary strata, and the low potential for sexually 154 antagonistic traits [41–44]. The anther-smut fungi, in the genus Microbotryum, undergo mating in 155 the haploid phase via isogamous yeast-like cells of opposite mating types (a1 and a2), which can 156 be cultured separately to analyze expression levels of alleles [45]. The species Microbotryum 157 lychnidis-dioicae, causing anther-smut disease on the plant Silene latifolia, carry dimorphic 158 mating-type chromosomes that have been assembled at the chromosome-level scale 159 [42,43,46,47]. These mating-type chromosomes (a1, ~3.3Mb, and a2, ~4.0Mb, respectively) lack 160 recombination across 90% of their length [46,47]. Importantly, evolutionary strata of different 161 ages have been identified, i.e., regions with different levels of differentiation between mating 162 types as a result of an expanding process of recombination suppression over the past ca. 1.5 163 million years [43,44]. The non-recombining regions of the mating-type chromosomes in M. 164 lychnidis-dioicae are flanked by small recombining pseudo-autosomal regions (PARs). 150 Fungi can provide valuable insights into the relationship between sequence degeneration and 151 differential gene expression in sex or mating-type chromosomes, due to easy access to the 152 haploid phase where alternate mating types are expressed, the existence of young events of 153 recombination suppression in successive evolutionary strata, and the low potential for sexually 154 antagonistic traits [41–44]. The anther-smut fungi, in the genus Microbotryum, undergo mating i 155 the haploid phase via isogamous yeast-like cells of opposite mating types (a1 and a2), which can 156 be cultured separately to analyze expression levels of alleles [45]. The species Microbotryum 157 lychnidis-dioicae, causing anther-smut disease on the plant Silene latifolia, carry dimorphic 158 mating-type chromosomes that have been assembled at the chromosome-level scale 159 [42,43,46,47]. These mating-type chromosomes (a1, ~3.3Mb, and a2, ~4.0Mb, respectively) lack 160 recombination across 90% of their length [46,47]. Importantly, evolutionary strata of different 161 ages have been identified, i.e., regions with different levels of differentiation between mating 162 types as a result of an expanding process of recombination suppression over the past ca. 1.5 163 million years [43,44]. 99 Introduction The non-recombining regions of the mating-type chromosomes in M. 164 lychnidis-dioicae are flanked by small recombining pseudo-autosomal regions (PARs). 165 In M. lychnidis-dioicae, the two possible causes leading to differential gene expression 166 between the alternative haploid mating types are not equally probable, i.e. ‘mating-type 167 antagonistic selection’ (sensu [48]) and differential sequence degeneration between alleles. The 168 existence of genes under mating-type antagonistic selection in fungi would require fitness 169 differences associated with mating-type dimorphic traits. However, previous studies on M. 170 lychnidis-dioicae have shown that differences between the mating types, either developmental o 171 ecological, are lacking outside of the immediate process of gamete fusion [41,49–52]. Moreover 172 a recent study on gene expression and positive selection detected no evidence for mating-type 173 antagonistic selection [53]. This model system is therefore ideal to investigate the impact of 174 degeneration on differential gene expression between chromosomes determining reproductive 175 compatibility, notably without the confounding effect of sexual antagonism. 6 6 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 7 176 In this study, we therefore investigated whether mating-type specific differential gene 177 expression was related to differences between alleles for various signatures of degeneration in the 178 genome of M. lychnidis-dioicae. We then assessed the hypothesis that, for genes with differential 179 expression between mating types, the alleles showing lower expression levels would have higher 180 levels of degeneration footprints that are thought to be associated with disruption of gene 181 expression. The investigated degeneration signatures included differences between alleles in the 182 levels of non-synonymous sequence divergence, transposable element (TE) insertions, alteration 183 of predicted protein length, intron content, and GC content (as a predicted consequence of 184 epigenetic gene silencing) [27,28,33,54,55]. 99 Introduction As prior work indicated that non-recombining 185 regions of the mating-type chromosomes are enriched for signatures of sequence degeneration 186 compared to autosomes [42], we also investigated whether differential gene expression varied 187 among genomic compartments defined as autosomes, PARs, young evolutionary strata of the 188 mating-type chromosomes (including previously identified red and green strata, [43]), and old 189 evolutionary strata (blue, purple, orange and black strata, [43]). 190 191 Results 192 Allele identification and differential gene expression between a1 and a2 haploid genomes 193 Alleles of single-copy genes in M. lychnidis-dioicae were identified using the criterion of 1:1 194 reciprocal best BLASTp between a1 and a2 haploid genomes, based on the previously published 195 genome assembly and gene annotation [43,44]. Protein sequence identity of >70% was used 196 following evaluation of various identity thresholds (see details in Methods section). After 197 filtering out TE-related gene sequences, we identified 371 single-copy allelic pairs in mating-type 198 chromosomes and 9,025 in autosomes (S1 Table). 199 Using whole-genome RNA-seq data from separate a1 and a2 haploid mating-type cultures 200 under low nutrient conditions that resemble the natural haploid growth environment [45,56], 201 filtering for genes with significantly detectable expression recovered 8,549 single-copy genes for 176 In this study, we therefore investigated whether mating-type specific differential gene 177 expression was related to differences between alleles for various signatures of degeneration in the 178 genome of M. lychnidis-dioicae. We then assessed the hypothesis that, for genes with differential 179 expression between mating types, the alleles showing lower expression levels would have higher 180 levels of degeneration footprints that are thought to be associated with disruption of gene 181 expression. The investigated degeneration signatures included differences between alleles in the 182 levels of non-synonymous sequence divergence, transposable element (TE) insertions, alteration 183 of predicted protein length, intron content, and GC content (as a predicted consequence of 184 epigenetic gene silencing) [27,28,33,54,55]. As prior work indicated that non-recombining 185 regions of the mating-type chromosomes are enriched for signatures of sequence degeneration 186 compared to autosomes [42], we also investigated whether differential gene expression varied 187 among genomic compartments defined as autosomes, PARs, young evolutionary strata of the 188 mating-type chromosomes (including previously identified red and green strata, [43]), and old 189 evolutionary strata (blue, purple, orange and black strata, [43]). 99 Introduction Genes with differentia 221 expression between mating types showed significant enrichment in the old evolutionary strata 222 compared to autosomes, but there was no enrichment in the young evolutionary strata or the 223 PARs (Table 2). Similar patterns were observed for the comparisons in each of the a1 or a2 224 haploid genomes separately (S3 Table). Further post hoc assessments of degenerative traits are 225 presented in the following sections, including whether difference between alleles is oriented such 226 that the more affected allele is less expressed. 202 further analysis (342 on mating-type chromosomes and 8,207 on autosomes). The differential 203 gene expression profile (i.e. (\|Log2(a1/a2\|) significantly greater than zero with false discovery rate 204 (FDR) < 0.050, S1 Fig; S2 Table) revealed 392 genes (4.59% out of the 8,549 genes analyzed for 205 expression) that were significantly more highly expressed in the a1 haploid culture, and 203 206 (2.37%) that were significantly more highly expressed in a2 haploid culture. 99 Introduction 7 199 Using whole-genome RNA-seq data from separate a1 and a2 haploid mating-type cultures 200 under low nutrient conditions that resemble the natural haploid growth environment [45,56], 201 filtering for genes with significantly detectable expression recovered 8,549 single-copy genes for 201 filtering for genes with significantly detectable expression recovered 8,549 single-copy genes for 7 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 202 further analysis (342 on mating-type chromosomes and 8,207 on autosomes). The differential 203 gene expression profile (i.e. (\|Log2(a1/a2\|) significantly greater than zero with false discovery rat 204 (FDR) < 0.050, S1 Fig; S2 Table) revealed 392 genes (4.59% out of the 8,549 genes analyzed fo 205 expression) that were significantly more highly expressed in the a1 haploid culture, and 203 206 (2.37%) that were significantly more highly expressed in a2 haploid culture. 207 208 Differential gene expression and multiple signatures of sequence degeneration 209 Regression analysis (generalized linear model, GLM) revealed that the degree of differentia 210 expression (DE) between allele pairs of the two haploid mating types significantly increased with 211 increasing differences between alleles (using absolute values) in the various degeneration traits 212 examined (Table 1). The significant main-effect predictors of differential expression included 213 genomic compartment and differences between alleles in non-synonymous divergence (dN), 214 transposable element (TE) insertion number within 20kb (up and downstream), intron content 215 (proportional to coding sequence length), and overall GC content (GC0). Differences between 216 alleles in predicted protein length was not a significant main-effect predictor but was strongly 217 significant as an interaction term with genomic compartment and all other traits except intron 218 content (Table 1). Differential expression indeed increased with differences between alleles in 219 predicted protein length, but only in old evolutionary strata and when associated with higher 220 differences between alleles in dN, TE content, and GC0 (Table 1; S2 Fig). 228 Relationship between differential expression and elevated substitution rates 228 Relationship between differential expression and elevated substitution rates 229 Differentially expressed genes had greater sequence divergence between alleles than non- 230 differentially expressed (non-DE) genes within genomic compartments, specifically within the 231 old evolutionary strata of the mating-type chromosomes. DE genes had significantly higher non- 232 synonymous mutation rate (dN) and synonymous mutation rate (dS) between alleles than non-DE 233 genes within old evolutionary strata (Wilcoxon rank sum test for independent samples, dN: W = 234 1433, P < 0.001, dS: W = 1422, P < 0.001) (Fig 1A, S3 Fig, S4 Table). There was almost no 235 sequence divergence (dN or dS) between alleles on either autosomes or PARs for DE or non-DE 236 genes. The young evolutionary strata had only one DE gene, precluding comparison to non-DE 237 genes within this compartment. The old strata pattern held for genes in a1 or a2 cells considered 238 separately (S4A and S4B Fig). A tendency of higher dN/dS for DE genes compared to non-DE 239 genes was not significant within old strata (W = 1946, P = 0.611) (S5 Fig). 240 To test the hypothesis that the allele with lower expression would show a larger accumulation 241 of non-synonymous changes than the allele with higher expression, each allele in M. lychnidis- 242 dioicae was compared for sequence divergence with their ortholog in M. lagerheimii, which has 243 retained largely collinear and homozygous mating-type chromosomes, as the inferred ancestral 244 state in the Microbotryum genus [43,44]. Alleles in a1 haploid genome or in a2 haploid genome of 245 M. lychnidis-dioicae were compared for dN divergence accordingly with alleles from the M. 246 lagerheimii genome of the same mating type. Alleles having lower expression levels in M. 247 lychnidis-dioicae did not have significantly greater dN divergence from their ortholog in M. 248 lagerheimii than alleles with higher expression levels (W = 1,267, smallest P = 0.909) (S6 Fig, 249 S5 Table). 250 251 Relationship between differential expression and TE insertions 252 Differentially expressed genes were associated with greater differences between alleles for 253 TE insertions (within 20kb up and downstream) than alleles of non-DE genes across genomic 229 Differentially expressed genes had greater sequence divergence between alleles than non- 230 differentially expressed (non-DE) genes within genomic compartments, specifically within the 231 old evolutionary strata of the mating-type chromosomes. 208 Differential gene expression and multiple signatures of sequence degeneration 209 Regression analysis (generalized linear model, GLM) revealed that the degree of differential 210 expression (DE) between allele pairs of the two haploid mating types significantly increased with 211 increasing differences between alleles (using absolute values) in the various degeneration traits 212 examined (Table 1). The significant main-effect predictors of differential expression included 213 genomic compartment and differences between alleles in non-synonymous divergence (dN), 227 8 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 228 Relationship between differential expression and elevated substitution rates lychnidis-dioicae were compared for dN divergence accordingly with alleles from the M. 246 lagerheimii genome of the same mating type. Alleles having lower expression levels in M. 228 Relationship between differential expression and elevated substitution rates DE genes had significantly higher non- 232 synonymous mutation rate (dN) and synonymous mutation rate (dS) between alleles than non-DE 233 genes within old evolutionary strata (Wilcoxon rank sum test for independent samples, dN: W = 234 1433, P < 0.001, dS: W = 1422, P < 0.001) (Fig 1A, S3 Fig, S4 Table). There was almost no 235 sequence divergence (dN or dS) between alleles on either autosomes or PARs for DE or non-DE 236 genes. The young evolutionary strata had only one DE gene, precluding comparison to non-DE 237 genes within this compartment. The old strata pattern held for genes in a1 or a2 cells considered 238 separately (S4A and S4B Fig). A tendency of higher dN/dS for DE genes compared to non-DE 239 genes was not significant within old strata (W = 1946, P = 0.611) (S5 Fig). 240 To test the hypothesis that the allele with lower expression would show a larger accumulation 241 of non-synonymous changes than the allele with higher expression, each allele in M. lychnidis- 242 dioicae was compared for sequence divergence with their ortholog in M. lagerheimii, which has 243 retained largely collinear and homozygous mating-type chromosomes, as the inferred ancestral 244 state in the Microbotryum genus [43,44]. Alleles in a1 haploid genome or in a2 haploid genome of 245 M. lychnidis-dioicae were compared for dN divergence accordingly with alleles from the M. 246 lagerheimii genome of the same mating type. Alleles having lower expression levels in M. 247 lychnidis-dioicae did not have significantly greater dN divergence from their ortholog in M. 248 lagerheimii than alleles with higher expression levels (W = 1,267, smallest P = 0.909) (S6 Fig, 249 S5 Table). 250 251 Relationship between differential expression and TE insertions 252 Differentially expressed genes were associated with greater differences between alleles for 253 TE insertions (within 20kb up and downstream) than alleles of non-DE genes across genomic 240 To test the hypothesis that the allele with lower expression would show a larger accumulation 241 of non-synonymous changes than the allele with higher expression, each allele in M. lychnidis- 242 dioicae was compared for sequence divergence with their ortholog in M. lagerheimii, which has 243 retained largely collinear and homozygous mating-type chromosomes, as the inferred ancestral 244 state in the Microbotryum genus [43,44]. Alleles in a1 haploid genome or in a2 haploid genome of 245 M. 251 Relationship between differential expression and TE insertions Among DE genes, oriented TE insertion difference was a significant 264 predictor of the express ratio only in the window covering from 10kb upstream to the gene (Fig 265 2A, S7 Fig); alleles with more TE insertions having reduced expression (for this window, Wald 266 X2 = 6.674, P = 0.010, statistics of remaining windows in S6 Table). Among non-DE genes, none 267 of the windows was a significant predictor of variation in the expression ratio (S6 Table). 268 269 Relationship between differential expression and differences in predicted protein length 270 Differential gene expression was associated with the mutational changes that affect the 258 than the allele with higher expression, differences in TE insertions between alleles were 259 calculated as the TE number for the allele with lower expression minus the TE number for the 260 allele with higher expression; a positive value thus represented an excess of TEs in the less 261 expressed allele. This oriented TE number difference between alleles was tested as a predictor of 262 the expression ratio \|Log2(a1/a2)\| using a sliding window approach with a 15kb window size 263 overlapping by 5kb. Among DE genes, oriented TE insertion difference was a significant 264 predictor of the express ratio only in the window covering from 10kb upstream to the gene (Fig 265 2A, S7 Fig); alleles with more TE insertions having reduced expression (for this window, Wald 266 X2 = 6.674, P = 0.010, statistics of remaining windows in S6 Table). Among non-DE genes, none 267 of the windows was a significant predictor of variation in the expression ratio (S6 Table). 268 251 Relationship between differential expression and TE insertions 252 Differentially expressed genes were associated with greater differences between alleles for 253 TE insertions (within 20kb up and downstream) than alleles of non-DE genes across genomic 9 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 254 compartments. However, the difference was significant only in the autosomes (W = 313879, P < 255 0.001, Fig 1B), not in the PARs (W = 546, P < 0.192) or the old evolutionary strata (W = 4062, P 256 = 0.173); the comparison was not possible in young evolutionary strata. 254 compartments. However, the difference was significant only in the autosomes (W = 313879, P < 255 0.001, Fig 1B), not in the PARs (W = 546, P < 0.192) or the old evolutionary strata (W = 4062, P 256 = 0.173); the comparison was not possible in young evolutionary strata. 254 compartments. However, the difference was significant only in the autosomes (W = 313879, P < 255 0.001, Fig 1B), not in the PARs (W = 546, P < 0.192) or the old evolutionary strata (W = 4062, P 256 = 0.173); the comparison was not possible in young evolutionary strata. 257 To test the hypothesis that the allele with less expression would show more TE insertions 258 than the allele with higher expression, differences in TE insertions between alleles were 259 calculated as the TE number for the allele with lower expression minus the TE number for the 260 allele with higher expression; a positive value thus represented an excess of TEs in the less 261 expressed allele. This oriented TE number difference between alleles was tested as a predictor of 262 the expression ratio \|Log2(a1/a2)\| using a sliding window approach with a 15kb window size 263 overlapping by 5kb. 269 Relationship between differential expression and differences in predicted protein length Among genes with different protein lengths between 287 alleles in the old evolutionary strata, 44.6% (N = 56) of DE genes had different stop codon 288 positions between alleles, which was significantly higher than the 24.0% (N = 75) of non-DE 289 genes (two-proportion z-test, P = 0.018, S7B Fig). Similarly, DE genes in the autosomes were 290 marginally significantly more likely to have different stop codon positions between alleles than 291 non-DE genes, with 33.3% (N = 21) vs 10% (N = 40), respectively (P = 0.057, S8B Fig). Only 292 three frameshift mutations were observed among the 258 of genes examined with different 293 protein/coding sequence lengths, and thus frameshifts were not distinguishing features of DE 294 versus non-DE genes. 295 To test the hypothesis that the allele with less expression would show a truncation of protein 296 length compared to the allele with greater expression (i.e. by early stop codons or deletions), 297 differences in protein length between alleles were calculated as the ratio for the allele with higher 298 expression divided by the allele with lower expression; a larger ratio thus represented a shorter 299 length for the allele with lower expression. Among DE genes, this oriented metric of protein 300 length differences was a significant predictor of the differential expression degree as the ratio 301 \|Log2(a1/a2)\|, with alleles producing shorter proteins being less expressed (Wald X2 = 19.326, 302 two-tailed P < 0.001, Fig 2B). No significant relationship to expression level ratio was found for 303 the length ratios of non-DE genes (Wald X2 = 0.222, P = 0.638, Fig 2B). 304 305 Relationship between differential expression and intron content 285 Similarly, differences in the positions of stop codons contributed to protein length variation 286 more for DE genes than non-DE genes. Among genes with different protein lengths between 287 alleles in the old evolutionary strata, 44.6% (N = 56) of DE genes had different stop codon 288 positions between alleles, which was significantly higher than the 24.0% (N = 75) of non-DE 289 genes (two-proportion z-test, P = 0.018, S7B Fig). Similarly, DE genes in the autosomes were 290 marginally significantly more likely to have different stop codon positions between alleles than 291 non-DE genes, with 33.3% (N = 21) vs 10% (N = 40), respectively (P = 0.057, S8B Fig). 269 Relationship between differential expression and differences in predicted protein length 270 Differential gene expression was associated with the mutational changes that affect the 271 predicted protein length, including altered stop codon positions, indels, and indels causing 272 frameshifts. Within genomic compartment, alternate alleles of DE genes were significantly more 273 likely to produce proteins of different lengths than alleles of non-DE genes, particularly within 274 the old evolutionary strata (Two proportion Z test, z = 2.186, P = 0.029) and autosomes (z = 4.64, 275 P = 8.78e-06, Fig 1C, S7 Table); there were too few DE genes on PARs and young evolutionary 276 strata for statistical comparisons. 277 The various types of mutational changes that caused protein length variation between alleles 278 differed between DE and non-DE genes, as well as among genomic compartments. Among the 279 258 genes with different protein sequence lengths between alleles, all had indels. However, DE 10 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 280 genes in the old evolutionary strata and autosomes had significantly more indels than non-DE 281 genes; old strata mean indel number differed between alleles by 2.64 for DE genes and by 1.85 282 for non-DE genes (W = 2453.5, P = 0.013), and in autosomes alleles differed by a mean of 1.19 283 indels for DE genes and 1.03 for non-DE genes (W = 490.5, P = 0.025, S8A Fig); PARs and 284 young evolutionary strata could not be analyzed. 282 for non-DE genes (W = 2453.5, P = 0.013), and in autosomes alleles differed by a mean of 1.19 283 indels for DE genes and 1.03 for non-DE genes (W = 490.5, P = 0.025, S8A Fig); PARs and 284 young evolutionary strata could not be analyzed. 285 Similarly, differences in the positions of stop codons contributed to protein length variation 286 more for DE genes than non-DE genes. 269 Relationship between differential expression and differences in predicted protein length 330 To test the hypothesis that the allele with lower expression would show lower GC content 331 than the allele with higher expression, GC0 or GC3 differences between alleles were calculated 315 To test the hypothesis that the allele with lower expression would show a greater intron 316 content as a signature of degeneration, differences between alleles were calculated as the value 317 for the less expressed allele minus the value for the more expressed allele; a positive value thus 318 represented greater intron content for the less expressed allele. This oriented metric of intron 319 content differences between alleles was not a significant predictor of differential expression level 320 among DE genes (Wald X2 = 0.350, P = 0.554), or among non-DE genes (Wald X2 = 0.216, P = 321 0.642). 322 269 Relationship between differential expression and differences in predicted protein length Only 292 three frameshift mutations were observed among the 258 of genes examined with different 293 protein/coding sequence lengths, and thus frameshifts were not distinguishing features of DE 294 versus non-DE genes. 295 To test the hypothesis that the allele with less expression would show a truncation of protein 296 length compared to the allele with greater expression (i.e. by early stop codons or deletions), 297 differences in protein length between alleles were calculated as the ratio for the allele with higher 298 expression divided by the allele with lower expression; a larger ratio thus represented a shorter 299 length for the allele with lower expression. Among DE genes, this oriented metric of protein 300 length differences was a significant predictor of the differential expression degree as the ratio 301 \|Log2(a1/a2)\|, with alleles producing shorter proteins being less expressed (Wald X2 = 19.326, 302 two-tailed P < 0.001, Fig 2B). No significant relationship to expression level ratio was found for 303 the length ratios of non-DE genes (Wald X2 = 0.222, P = 0.638, Fig 2B). 304 305 Relationship between differential expression and intron content 295 To test the hypothesis that the allele with less expression would show a truncation of protein 296 length compared to the allele with greater expression (i.e. by early stop codons or deletions), 297 differences in protein length between alleles were calculated as the ratio for the allele with higher 298 expression divided by the allele with lower expression; a larger ratio thus represented a shorter 299 length for the allele with lower expression. Among DE genes, this oriented metric of protein 300 length differences was a significant predictor of the differential expression degree as the ratio 301 \|Log2(a1/a2)\|, with alleles producing shorter proteins being less expressed (Wald X2 = 19.326, 302 two-tailed P < 0.001, Fig 2B). No significant relationship to expression level ratio was found for 303 the length ratios of non-DE genes (Wald X2 = 0.222, P = 0.638, Fig 2B). 305 Relationship between differential expression and intron content 11 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . 269 Relationship between differential expression and differences in predicted protein length CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 306 Differential gene expression was associated with differences between alleles in intron 307 content, considering lower intron content to be favored by selection [33]. There were 308 significantly greater intron content differences between alleles for DE genes than for non-DE 309 genes; considering the ratio of intron to coding sequence lengths, alleles of DE gene overall 310 differed on average by 0.008 and alleles of non-DE genes differed by 0.002 (W = 2102758, P < 311 0.001). Alleles differed in intron content more for DE than non-DE genes within the autosomes 312 (W = 1920124, P = 0.033) and old evolutionary strata (W = 3205, P = 0.001) (Fig 1D, S8 Table), 313 but not within the PARs (W = 605, P = 0.888); the comparison in young evolutionary strata was 314 not possible. 315 To test the hypothesis that the allele with lower expression would show a greater intron 316 content as a signature of degeneration, differences between alleles were calculated as the value 317 for the less expressed allele minus the value for the more expressed allele; a positive value thus 318 represented greater intron content for the less expressed allele. This oriented metric of intron 319 content differences between alleles was not a significant predictor of differential expression level 320 among DE genes (Wald X2 = 0.350, P = 0.554), or among non-DE genes (Wald X2 = 0.216, P = 321 0.642). 322 323 Relationship between differential expression and GC content 324 Consistent with gene silencing by cytosine methylation contributing to decreased GC content 325 [30,32,57], DE genes had significantly greater overall GC0 differences between their alleles than 326 non-DE genes within the autosomes (W = 1907831, P < 0.001) and old evolutionary strata (W = 327 3010, P < 0.001) (Fig 1E). The comparison within the PARs was not significant (W = 578, P = 328 0.318); the comparison for young evolutionary strata was not possible. Analysis of third codon 329 position GC3 provided similar patterns and levels of significance (S9 Fig, S9 Table). 323 Relationship between differential expression and GC content 324 Consistent with gene silencing by cytosine methylation contributing to decreased GC content 325 [30,32,57], DE genes had significantly greater overall GC0 differences between their alleles than 326 non-DE genes within the autosomes (W = 1907831, P < 0.001) and old evolutionary strata (W = 327 3010, P < 0.001) (Fig 1E). The comparison within the PARs was not significant (W = 578, P = 328 0.318); the comparison for young evolutionary strata was not possible. Analysis of third codon 329 position GC3 provided similar patterns and levels of significance (S9 Fig, S9 Table). 324 Consistent with gene silencing by cytosine methylation contributing to decreased GC content 325 [30,32,57], DE genes had significantly greater overall GC0 differences between their alleles than 326 non-DE genes within the autosomes (W = 1907831, P < 0.001) and old evolutionary strata (W = 327 3010, P < 0.001) (Fig 1E). The comparison within the PARs was not significant (W = 578, P = 328 0.318); the comparison for young evolutionary strata was not possible. Analysis of third codon 329 position GC3 provided similar patterns and levels of significance (S9 Fig, S9 Table). 330 To test the hypothesis that the allele with lower expression would show lower GC content 331 than the allele with higher expression, GC0 or GC3 differences between alleles were calculated 330 To test the hypothesis that the allele with lower expression would show lower GC content 331 than the allele with higher expression, GC0 or GC3 differences between alleles were calculated 12 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 332 as the value for allele with higher expression minus the value for allele with lower expression; a 333 positive value thus represented reduced GC content for the allele with lower expression. 323 Relationship between differential expression and GC content To our knowledge, our study is the first to reveal an 345 association between a variety of degeneration measures and differential gene expression, doing so 346 in the anther-smut fungus M. lychnidis-dioicae where antagonistic selection is unlikely 347 [41,43,44,52,53], and thus does not constitute a confounding factor. Genes differentially 348 expressed between the haploid mating types were enriched on the oldest strata of the mating-type 349 chromosomes and displayed various forms of sequence (dN, dS, or GC content) or structural (TE 350 insertions, introns content, or protein length) heterozygosity at levels higher than non- 351 differentially expressed genes. These results across various forms of mutational change and 352 genomic compartments show importantly that differential gene expression is strongly associated 353 with sequence degeneration, and differential expression should therefore not be systematically 354 interpreted as a sign of antagonistic selection. 355 356 Differential gene expression between haploid mating types 339 Genomic regions controlling mating compatibility, whether non-recombining sex or mating- 340 type chromosomes, have been subject of intense research. This is because these regions 341 determine traits essential for fitness and because of their rapid evolutionary dynamics. Sequence 342 degeneration of non-recombining regions has been linked to major genomic features such as 343 chromosomal heteromorphism, dosage compensation or gene trafficking between non- 344 recombining regions and autosomes [14,37]. To our knowledge, our study is the first to reveal an 345 association between a variety of degeneration measures and differential gene expression, doing so 346 in the anther-smut fungus M. lychnidis-dioicae where antagonistic selection is unlikely 347 [41,43,44,52,53], and thus does not constitute a confounding factor. Genes differentially 348 expressed between the haploid mating types were enriched on the oldest strata of the mating-type 349 chromosomes and displayed various forms of sequence (dN, dS, or GC content) or structural (TE 350 insertions, introns content, or protein length) heterozygosity at levels higher than non- 351 differentially expressed genes. These results across various forms of mutational change and 352 genomic compartments show importantly that differential gene expression is strongly associated 353 with sequence degeneration, and differential expression should therefore not be systematically 354 interpreted as a sign of antagonistic selection. 323 Relationship between differential expression and GC content Among 334 DE genes, neither the oriented GC0 or GC3 differences between alleles were significant 335 predictors of the level of differential expression (GC0: Wald X2 = 1.039, P = 0.308, and GC3: 336 Wald X2 = 2.226, P = 0.136). 337 338 Discussion 339 Genomic regions controlling mating compatibility, whether non-recombining sex or mating- 340 type chromosomes, have been subject of intense research. This is because these regions 341 determine traits essential for fitness and because of their rapid evolutionary dynamics. Sequence 342 degeneration of non-recombining regions has been linked to major genomic features such as 343 chromosomal heteromorphism, dosage compensation or gene trafficking between non- 344 recombining regions and autosomes [14,37]. To our knowledge, our study is the first to reveal an 345 association between a variety of degeneration measures and differential gene expression, doing so 346 in the anther-smut fungus M. lychnidis-dioicae where antagonistic selection is unlikely 347 [41,43,44,52,53], and thus does not constitute a confounding factor. Genes differentially 348 expressed between the haploid mating types were enriched on the oldest strata of the mating-type 349 chromosomes and displayed various forms of sequence (dN, dS, or GC content) or structural (TE 350 insertions, introns content, or protein length) heterozygosity at levels higher than non- 351 differentially expressed genes. These results across various forms of mutational change and 352 genomic compartments show importantly that differential gene expression is strongly associated 353 with sequence degeneration, and differential expression should therefore not be systematically 354 interpreted as a sign of antagonistic selection. 332 as the value for allele with higher expression minus the value for allele with lower expression; a 333 positive value thus represented reduced GC content for the allele with lower expression. Among 334 DE genes, neither the oriented GC0 or GC3 differences between alleles were significant 335 predictors of the level of differential expression (GC0: Wald X2 = 1.039, P = 0.308, and GC3: 336 Wald X2 = 2.226, P = 0.136). 338 Discussion 339 Genomic regions controlling mating compatibility, whether non-recombining sex or mating- 340 type chromosomes, have been subject of intense research. This is because these regions 341 determine traits essential for fitness and because of their rapid evolutionary dynamics. Sequence 342 degeneration of non-recombining regions has been linked to major genomic features such as 343 chromosomal heteromorphism, dosage compensation or gene trafficking between non- 344 recombining regions and autosomes [14,37]. 356 Differential gene expression between haploid mating types 13 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 357 The proportion of genes with differential expression between haploid mating types of M. 358 lychnidis-dioicae was low, which is consistent with expectations based upon the lack of 359 dimorphism between mating types. The number of DE genes was slightly higher than in a 360 previous study based on the same dataset [42], likely due to an improved genome assembly and 361 non-recombining region identification. The 2.4~4.6% of genes with differential expression 362 between mating types of M. lychnidis-dioicae was similar to plant and animal non-reproductive 363 tissues, e.g. liver, spleen, leaves, roots [9,58–64]. However, this is much lower than in 364 reproductive tissues (e.g. ovaries or testes) of most animals and plants [5,6]. There seems to be an 365 overall positive relationship between proportions of sex-biased genes and levels of sexual 366 dimorphism across taxa [65], suggesting a possible correlation between phenotypic difference 367 between sexes and underlying transcriptional architecture. The proportion of sex-biased genes 368 was lower than the 12% found in the haploid sexes in the brown algae Ectocarpus which has a 369 low level of sexual dimorphism [66]. Among fungi, 3~4% of genes had differential expression 370 between mating types in the fungus Neurospora tetrasperma [21]. Therefore, the low percentage 371 of genes having differential expression between mating types in M. lychnidis-dioicae is consistent 372 with its life history having no ‘female’ or ‘male’ functions (isogamous gametes) and mating type 373 being controlled at the haploid stage without morphological or ecological differences that might 374 provide different trait optima between mating types [41,53,67]. 375 Differentially expressed (DE) genes were enriched in the mating-type chromosomes of M. 356 Differential gene expression between haploid mating types ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 383 expression [2,68–70]. Sex-biased gene expression is often considered the product of sexually 384 antagonistic selection across a broad range of taxa [5,6,20,21,71]. However, decades of research 385 have uncovered little genetic evidence directly supporting sexually antagonistic selection as being 386 the driving force for evolutionary strata of recombination suppression or for sex-biased 387 expression outside of the reproductive tissues themselves [7,19,58,72]. 383 expression [2,68–70]. Sex-biased gene expression is often considered the product of sexually 384 antagonistic selection across a broad range of taxa [5,6,20,21,71]. However, decades of research 385 have uncovered little genetic evidence directly supporting sexually antagonistic selection as being 386 the driving force for evolutionary strata of recombination suppression or for sex-biased 387 expression outside of the reproductive tissues themselves [7,19,58,72]. 387 expression outside of the reproductive tissues themselves [7,19,58,72]. 388 Our results, however, suggest a broader view of evolutionary forces, aside from sexual 389 antagonism, that can explain the occurrence of DE genes and their enrichment on chromosomes 390 determining reproductive compatibility. The role of sequence degeneration in differential 391 expression has so far been largely understudied but is likely the primary and powerful factor in 392 M. lychnidis-dioicae. The lack of female and male functions in Microbotryum fungi and a haploid 393 phase with very limited differences between mating types [41], likely explains the absence of 394 genes experiencing mating-type antagonistic selection [53]. In contrast, gene degeneration on 395 non-recombining sex or mating-type chromosomes occurs commonly among eukaryotic taxa due 396 to the combined influences of reduction in effective population size (Ne), Hill-Robertson 397 interference and sheltering effects in diploid organisms [2,5,42,68–70]. Sequence degeneration is 398 therefore a generally expected phenomenon in non-recombining regions, and the resulting 399 mutation accumulation may generate contrasting expression levels between differently affected 400 alleles. Certainly, sexually antagonistic selection and degeneration are not mutually exclusives 401 processes, and a general relationship between the amounts of sexual dimorphism and of DE genes 402 [5,6] supports the role for sexual antagonisms. However, several types of degeneration are found 403 in M. lychnidis-dioicae, in the absence of sexual antagonism, suggesting the potential for similar 404 in patterns associated with DE genes across diverse types of organisms. 356 Differential gene expression between haploid mating types 376 lychnidis-dioicae, which is also consistent with studies in animals and plants having 377 differentiated sex chromosomes (reviewed by [5]) Similarly in the fungus N tetrasperma DE 357 The proportion of genes with differential expression between haploid mating types of M. 358 lychnidis-dioicae was low, which is consistent with expectations based upon the lack of 359 dimorphism between mating types. The number of DE genes was slightly higher than in a 360 previous study based on the same dataset [42], likely due to an improved genome assembly and 361 non-recombining region identification. The 2.4~4.6% of genes with differential expression 362 between mating types of M. lychnidis-dioicae was similar to plant and animal non-reproductive 363 tissues, e.g. liver, spleen, leaves, roots [9,58–64]. However, this is much lower than in 364 reproductive tissues (e.g. ovaries or testes) of most animals and plants [5,6]. There seems to be an 365 overall positive relationship between proportions of sex-biased genes and levels of sexual 357 374 provide different trait optima between mating types [41,53,67]. 375 Differentially expressed (DE) genes were enriched in the mating-type chromosomes of M. 376 lychnidis-dioicae, which is also consistent with studies in animals and plants having 377 differentiated sex chromosomes (reviewed by [5]). Similarly, in the fungus N. tetrasperma, DE 378 genes were more frequently detected on mating-type chromosomes [21]. In animals and plants, 379 sexually antagonistic selection is accepted as an important force that can explain the stepwise 380 recombination suppression in sex chromosomes and formation of evolutionary strata. Indeed, 381 linkage of sexually-antagonistic genes to reproductive compatibility loci is considered 382 fundamental to the resolution of sexual conflict by allowing for sex-specific or sex-biased gene 375 Differentially expressed (DE) genes were enriched in the mating-type chromosomes of M. 376 lychnidis-dioicae, which is also consistent with studies in animals and plants having 14 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. 356 Differential gene expression between haploid mating types It is made available The copyright holder for this preprint (which was not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 409 effects, several of which were significant predictors in the overall regression model of differential 410 expression between mating types of M. lychnidis-dioicae. Some signatures of degeneration can 411 be directly connected to mechanisms known to reduce transcription levels. In particular, 412 transposable element (TE) insertion into genes or upstream have long been recognized to alter 413 gene expression [29,73]. TEs can disrupt promoter regions or other regulatory sequences internal 414 to genes [27,28]. In addition, epigenetic silencing, as a defense against TE proliferation, can 415 tighten local chromatin structure and inhibit access of transcriptional machinery [74,75]. In this 416 study, the differences between alleles in DE genes in the number of TE insertions were a 417 significant predictor of differential expression. Consistent with a direct effect upon differential 418 expression, the relative excess of TE insertions between alleles, specifically upstream of genes, 419 was associated with a lower expression level between alleles of DE genes. Similarly, the 420 introduction of early stop or non-sense codons is expected to reduce expression. Transcripts from 421 alleles with premature stop codons are affected by nonsense mediated decay, involving 422 degradation of mRNA and further components of the RNAi pathway than down-regulate 423 expression [54]. We found that alternative alleles were more likely to have differing stop codon 424 positions in DE genes than in non-DE genes, and, importantly, that the differential expression 425 was explained by the shorter allele having lower expression. This suggests that gene truncation, 426 by the gain of premature stop codons rather than the possibility of stop codon losses and read- 427 through transcripts, leads to differential expression. Both TEs and premature stop codons appear 428 to be important mutations changes that affect differential expression between alleles. 429 Oth h t i ti f DE i M l h idi di i l h i ti ll ti d t 409 effects, several of which were significant predictors in the overall regression model of differential 410 expression between mating types of M. lychnidis-dioicae. Some signatures of degeneration can 411 be directly connected to mechanisms known to reduce transcription levels. In particular, 412 transposable element (TE) insertion into genes or upstream have long been recognized to alter 413 gene expression [29,73]. 356 Differential gene expression between haploid mating types TEs can disrupt promoter regions or other regulatory sequences internal 414 to genes [27,28]. In addition, epigenetic silencing, as a defense against TE proliferation, can 415 tighten local chromatin structure and inhibit access of transcriptional machinery [74,75]. In this 416 study, the differences between alleles in DE genes in the number of TE insertions were a 417 significant predictor of differential expression. Consistent with a direct effect upon differential 418 expression, the relative excess of TE insertions between alleles, specifically upstream of genes, 419 was associated with a lower expression level between alleles of DE genes. Similarly, the 420 introduction of early stop or non-sense codons is expected to reduce expression. Transcripts from 421 alleles with premature stop codons are affected by nonsense mediated decay, involving 422 degradation of mRNA and further components of the RNAi pathway than down-regulate 423 expression [54]. We found that alternative alleles were more likely to have differing stop codon 424 positions in DE genes than in non-DE genes, and, importantly, that the differential expression 425 was explained by the shorter allele having lower expression. This suggests that gene truncation, 426 by the gain of premature stop codons rather than the possibility of stop codon losses and read- 427 through transcripts, leads to differential expression. Both TEs and premature stop codons appear 428 to be important mutations changes that affect differential expression between alleles. 429 Other characteristics of DE genes in M. lychnidis-dioicae are less mechanistically tied to 430 changes in expression levels but are nevertheless associated with sequence degeneration, directly 431 or indirectly. Most important among these characteristics was the degree of sequence divergence 432 between alleles. Alleles of DE genes were distinguished by markedly more non-synonymous and 429 Other characteristics of DE genes in M. lychnidis-dioicae are less mechanistically tied to 430 changes in expression levels but are nevertheless associated with sequence degeneration, directly 431 or indirectly. Most important among these characteristics was the degree of sequence divergence 432 between alleles. Alleles of DE genes were distinguished by markedly more non-synonymous and 433 synonymous base pair differences than alleles of non-DE genes, sequence divergence being a 434 positive predictor of the degree of differential gene expression. Similar results were demonstrated 429 Other characteristics of DE genes in M. lychnidis-dioicae are less mechanistically tied to 430 changes in expression levels but are nevertheless associated with sequence degeneration, directly 431 or indirectly. 356 Differential gene expression between haploid mating types 405 406 Various forms of degeneration 407 The properties of non-recombining regions that reduce the efficiency of selection (reduced 408 Ne, hitchhiking and sheltering) can lead to the fixation of various mutations having degenerative 388 Our results, however, suggest a broader view of evolutionary forces, aside from sexual 389 antagonism, that can explain the occurrence of DE genes and their enrichment on chromosomes 390 determining reproductive compatibility. The role of sequence degeneration in differential 391 expression has so far been largely understudied but is likely the primary and powerful factor in 392 M. lychnidis-dioicae. The lack of female and male functions in Microbotryum fungi and a haploid 393 phase with very limited differences between mating types [41], likely explains the absence of 394 genes experiencing mating-type antagonistic selection [53]. In contrast, gene degeneration on 395 non-recombining sex or mating-type chromosomes occurs commonly among eukaryotic taxa due 396 to the combined influences of reduction in effective population size (Ne), Hill-Robertson 397 interference and sheltering effects in diploid organisms [2,5,42,68–70]. Sequence degeneration is 398 therefore a generally expected phenomenon in non-recombining regions, and the resulting 399 mutation accumulation may generate contrasting expression levels between differently affected 400 alleles. Certainly, sexually antagonistic selection and degeneration are not mutually exclusives 401 processes, and a general relationship between the amounts of sexual dimorphism and of DE genes 402 [5,6] supports the role for sexual antagonisms. However, several types of degeneration are found 403 in M. lychnidis-dioicae, in the absence of sexual antagonism, suggesting the potential for similar 404 in patterns associated with DE genes across diverse types of organisms. 405 407 The properties of non-recombining regions that reduce the efficiency of selection (reduced 408 Ne, hitchhiking and sheltering) can lead to the fixation of various mutations having degenerative 407 The properties of non-recombining regions that reduce the efficiency of selection (reduced 408 Ne, hitchhiking and sheltering) can lead to the fixation of various mutations having degenerative 15 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 356 Differential gene expression between haploid mating types Most important among these characteristics was the degree of sequence divergence 432 between alleles. Alleles of DE genes were distinguished by markedly more non-synonymous and 433 synonymous base pair differences than alleles of non-DE genes, sequence divergence being a 434 positive predictor of the degree of differential gene expression. Similar results were demonstrated 16 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 435 in the anisogamous, hermaphroditic ascomycete N. tetrasperma, showing that differential gene 436 expression was positively correlated with sequence divergence between alleles of genes on 437 mating-type chromosomes [21]. Gene profiles in N. tetrasperma differed in their mating-type- 438 specific expression depending upon whether female or male reproduction was being induced by 439 the culture conditions, drawing analogy to sexual dimorphism commonly found in animals and 440 plants [21]. However, Microbotryum fungi do not have such male or female functions or 441 ecological differences between mating types, and therefore such an adaptive explanation for DE 442 genes is improbable. 435 in the anisogamous, hermaphroditic ascomycete N. tetrasperma, showing that differential gene 436 expression was positively correlated with sequence divergence between alleles of genes on 437 mating-type chromosomes [21]. Gene profiles in N. tetrasperma differed in their mating-type- 438 specific expression depending upon whether female or male reproduction was being induced by 439 the culture conditions, drawing analogy to sexual dimorphism commonly found in animals and 440 plants [21]. 356 Differential gene expression between haploid mating types Consistent with mating-type linkage 481 preserving heterozygosity, nearly the full range of mutational changes or footprints of 482 degeneration showed lowest levels in the autosomes and PARs and increasing through the young 461 degeneration types following recombination suppression on sex chromosome of Drosophila 462 albomicans [16,78]. Those studies indicate that reduced chromosome-wide gene expression 463 occurred as a first step in degeneration between non-recombining regions of a young sex 464 chromosome system [16,78]. While our findings are in support of the hypothesis for methylation- 465 mediated effects on differential expression, methylome analyses across the M. lychnidis-dioicae 466 genome should be investigated in more details. 461 degeneration types following recombination suppression on sex chromosome of Drosophila 462 albomicans [16,78]. Those studies indicate that reduced chromosome-wide gene expression 463 occurred as a first step in degeneration between non-recombining regions of a young sex 464 chromosome system [16,78]. While our findings are in support of the hypothesis for methylation- 465 mediated effects on differential expression, methylome analyses across the M. lychnidis-dioicae 466 genome should be investigated in more details. 356 Differential gene expression between haploid mating types However, Microbotryum fungi do not have such male or female functions or 441 ecological differences between mating types, and therefore such an adaptive explanation for DE 442 genes is improbable. 443 The remaining signatures of degeneration were most informative of how genes might evolve 444 as a consequence of differential expression. In general, gene expression levels are expected to 445 positively correlate with the strength of selection [55]; stronger selection and higher expression 446 levels have been correlated with reduced intron content [33]. It is also known for many 447 eukaryotes that introns can affect gene expression without functioning as a binding site for 448 transcription factors [76], by for example influencing the rate of transcription, nuclear export and 449 transcript stability [34]. Our data show that differences between alleles in intron content 450 positively predict differential expression level, which is consistent with these intron effects upon 451 gene expression, although the directional hypothesis testing was not significant. Additionally, our 452 data showed changes in GC content that are consistent with the consequences of suppression of 453 gene expression, specifically as predicted to result from epigenetic silencing through cytosine 454 methylation. Two important drivers of GC content changes are biased gene conversion and 455 methylcytosine-driven C-to-T mutation rates [31,32]; however, gene conversion that relies on 456 meiotic pairing is unlikely to cause the different mutation rates between alleles of single copy 457 genes [77]. While differences between alleles in GC content was a modestly significant 458 predictors of differential expression level compared to some other traits in the overall regression 459 model, we also found alleles of DE genes differed more in GC content than non-DE genes in the 460 old evolutionary strata. Methylation-mediated gene silencing has been presented as one of the 443 The remaining signatures of degeneration were most informative of how genes might evolve 444 as a consequence of differential expression. In general, gene expression levels are expected to 445 positively correlate with the strength of selection [55]; stronger selection and higher expression 446 levels have been correlated with reduced intron content [33]. It is also known for many 447 eukaryotes that introns can affect gene expression without functioning as a binding site for 448 transcription factors [76], by for example influencing the rate of transcription, nuclear export and 449 transcript stability [34]. Our data show that differences between alleles in intron content 17 . 356 Differential gene expression between haploid mating types CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 461 degeneration types following recombination suppression on sex chromosome of Drosophila 462 albomicans [16,78]. Those studies indicate that reduced chromosome-wide gene expression 463 occurred as a first step in degeneration between non-recombining regions of a young sex 464 chromosome system [16,78]. While our findings are in support of the hypothesis for methylation- 465 mediated effects on differential expression, methylome analyses across the M. lychnidis-dioicae 466 genome should be investigated in more details. 467 468 Degeneration across genomic compartments 469 The different forms of genetic degeneration in M. lychnidis-dioicae were not equally 470 represented among genomic compartments, perhaps reflecting the history of recombination 471 suppression. In this system, enrichment of DE genes on the mating-type chromosomes is unlikely 472 to be due to antagonistic selection, but rather to this region preserving heterozygosity in general. 473 As a matter of fact, enrichment of DE genes was significant only in the old evolutionary strata 474 and not in the younger strata, indicating it is a consequence and not a driver of recombination 475 suppression. 476 Mating between different haploid sexes or mating types ensures that all diploids are 477 heterogametic [79], and it has long been recognized that regions linked to mating type can 478 preserve heterozygosity [80]. In M. lychnidis-dioicae, the large non-recombining regions are in 479 fact highly heterozygous [43]. In contrast, the autosomes and PARs are largely homozygous, due 480 to the selfing mating system of M. lychnidis-dioicae [41,47]. 468 Degeneration across genomic compartments lychnidis-dioicae 493 showing the existence of deleterious recessive alleles that are linked to mating type and 494 preventing haploid growth [81,82], which may reflect outcomes of the mutational accumulation 495 described here. The recent discovery of multiple independent mating-type linkage events across 496 the Microbotryum genus [44] should allow further assessment of mutation accumulation and its 497 consequences for gene functions. Finally, it should be noted that the acquisition of one type of 498 degenerative mutation, particularly where expression is reduced, may relax selection and favor 499 the accumulation of additional sequence changes in that allele. Whether such feedback or cascade 500 dynamics further drive allele denervation should be addressed in further studies. 501 468 Degeneration across genomic compartments 469 The different forms of genetic degeneration in M. lychnidis-dioicae were not equally 470 represented among genomic compartments, perhaps reflecting the history of recombination 471 suppression. In this system, enrichment of DE genes on the mating-type chromosomes is unlikely 472 to be due to antagonistic selection, but rather to this region preserving heterozygosity in general. 473 As a matter of fact, enrichment of DE genes was significant only in the old evolutionary strata 474 and not in the younger strata, indicating it is a consequence and not a driver of recombination 475 suppression. 469 The different forms of genetic degeneration in M. lychnidis-dioicae were not equally 470 represented among genomic compartments, perhaps reflecting the history of recombination 471 suppression. In this system, enrichment of DE genes on the mating-type chromosomes is unlikely 472 to be due to antagonistic selection, but rather to this region preserving heterozygosity in general. 473 As a matter of fact, enrichment of DE genes was significant only in the old evolutionary strata 474 and not in the younger strata, indicating it is a consequence and not a driver of recombination 475 suppression. 476 Mating between different haploid sexes or mating types ensures that all diploids are 477 heterogametic [79], and it has long been recognized that regions linked to mating type can 478 preserve heterozygosity [80]. In M. lychnidis-dioicae, the large non-recombining regions are in 479 fact highly heterozygous [43]. In contrast, the autosomes and PARs are largely homozygous, due 480 to the selfing mating system of M. lychnidis-dioicae [41,47]. Consistent with mating-type linkage 481 preserving heterozygosity, nearly the full range of mutational changes or footprints of 482 degeneration showed lowest levels in the autosomes and PARs and increasing through the young 483 evolutionary strata to highest levels in the old evolutionary strata. Traits following this pattern 484 included differences between alleles in TE accumulation, base pair substitutions, protein length, 485 intron content and GC percentage. In most cases, these effects increased also for non-DE genes in 486 the non-recombining region of the mating-type chromosomes but to lower degrees than DE 18 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . 468 Degeneration across genomic compartments lychnidis-dioicae 493 showing the existence of deleterious recessive alleles that are linked to mating type and 494 preventing haploid growth [81,82], which may reflect outcomes of the mutational accumulation 495 described here. The recent discovery of multiple independent mating-type linkage events across 496 the Microbotryum genus [44] should allow further assessment of mutation accumulation and its 497 consequences for gene functions. Finally, it should be noted that the acquisition of one type of 498 degenerative mutation, particularly where expression is reduced, may relax selection and favor 499 the accumulation of additional sequence changes in that allele. Whether such feedback or cascade 500 dynamics further drive allele denervation should be addressed in further studies. 501 502 Insights across sexual eukaryotes 503 Microbotryum, and fungi in general, provide powerful models to investigate the genomics of 504 sequence degeneration and differential gene expression between sex-related chromosomes. The 505 ability to culture haploid genotypes helps to overcome issues of allele-specific expression 506 encountered in diploid systems. The genomes are small and readily assembled at the near- 507 chromosome level. Also, some pathogenic fungi like Microbotryum exhibit an obligately sexual 508 life cycle, where meiosis and haploid mating are required upon each instance of disease 509 transmission [41]. Furthermore, fungi often have a restricted range of evolutionary forces related 510 to a sexual life cycle (i.e. without sexually antagonistic selection and without asymmetrical 511 sheltering resulting from the haploid mating; [79]). Therefore, finding traits in common between 512 Microbotryum and familiar plant and animal systems help to identify fundamental evolutionarily 492 These degeneration patterns are consistent with prior studies on M. lychnidis-dioicae 493 showing the existence of deleterious recessive alleles that are linked to mating type and 494 preventing haploid growth [81,82], which may reflect outcomes of the mutational accumulation 495 described here. The recent discovery of multiple independent mating-type linkage events across 496 the Microbotryum genus [44] should allow further assessment of mutation accumulation and its 497 consequences for gene functions. Finally, it should be noted that the acquisition of one type of 498 degenerative mutation, particularly where expression is reduced, may relax selection and favor 499 the accumulation of additional sequence changes in that allele. Whether such feedback or cascade 500 dynamics further drive allele denervation should be addressed in further studies. 501 492 These degeneration patterns are consistent with prior studies on M. 468 Degeneration across genomic compartments CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 487 genes. Importantly, however, comparisons within genomic compartments repeatedly showed that 488 allele distinguishing mutations occurred more in association with DE genes than non-DE genes or 489 in the manner positively associated with levels of differential expression. Therefore, strong 490 evidence is shown for these degenerative changes being directly related to changes in expression 491 levels between alleles. 492 These degeneration patterns are consistent with prior studies on M. lychnidis-dioicae 493 showing the existence of deleterious recessive alleles that are linked to mating type and 494 preventing haploid growth [81,82], which may reflect outcomes of the mutational accumulation 495 described here. The recent discovery of multiple independent mating-type linkage events across 496 the Microbotryum genus [44] should allow further assessment of mutation accumulation and its 497 consequences for gene functions. Finally, it should be noted that the acquisition of one type of 498 degenerative mutation, particularly where expression is reduced, may relax selection and favor 499 the accumulation of additional sequence changes in that allele. Whether such feedback or cascade 500 dynamics further drive allele denervation should be addressed in further studies. 501 502 Insights across sexual eukaryotes 503 Microbotryum, and fungi in general, provide powerful models to investigate the genomics of 504 sequence degeneration and differential gene expression between sex related chromosomes The 487 genes. Importantly, however, comparisons within genomic compartments repeatedly showed that 488 allele distinguishing mutations occurred more in association with DE genes than non-DE genes or 489 in the manner positively associated with levels of differential expression. Therefore, strong 490 evidence is shown for these degenerative changes being directly related to changes in expression 491 levels between alleles. 488 allele distinguishing mutations occurred more in association with DE genes than non-DE genes or 489 in the manner positively associated with levels of differential expression. Therefore, strong 490 evidence is shown for these degenerative changes being directly related to changes in expression 491 levels between alleles. 492 These degeneration patterns are consistent with prior studies on M. 502 Insights across sexual eukaryotes 503 Microbotryum, and fungi in general, provide powerful models to investigate the genomics of 504 sequence degeneration and differential gene expression between sex-related chromosomes. The 505 ability to culture haploid genotypes helps to overcome issues of allele-specific expression 506 encountered in diploid systems. The genomes are small and readily assembled at the near- 507 chromosome level. Also, some pathogenic fungi like Microbotryum exhibit an obligately sexual 508 life cycle, where meiosis and haploid mating are required upon each instance of disease 509 transmission [41]. Furthermore, fungi often have a restricted range of evolutionary forces related 510 to a sexual life cycle (i.e. without sexually antagonistic selection and without asymmetrical 511 sheltering resulting from the haploid mating; [79]). Therefore, finding traits in common between 512 Microbotryum and familiar plant and animal systems help to identify fundamental evolutionarily 503 Microbotryum, and fungi in general, provide powerful models to investigate the genomics of 504 sequence degeneration and differential gene expression between sex-related chromosomes. The 505 ability to culture haploid genotypes helps to overcome issues of allele-specific expression 506 encountered in diploid systems. The genomes are small and readily assembled at the near- 507 chromosome level. Also, some pathogenic fungi like Microbotryum exhibit an obligately sexual 508 life cycle, where meiosis and haploid mating are required upon each instance of disease 509 transmission [41]. Furthermore, fungi often have a restricted range of evolutionary forces related 510 to a sexual life cycle (i.e. without sexually antagonistic selection and without asymmetrical 511 sheltering resulting from the haploid mating; [79]). Therefore, finding traits in common between 512 Microbotryum and familiar plant and animal systems help to identify fundamental evolutionarily 19 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 513 mechanisms for the genomics of reproductive compatibility [83]. In particular, the recent 514 demonstration of multiple evolutionary strata on the mating-type chromosomes of M. 502 Insights across sexual eukaryotes lychnidis- 515 dioicae and other Microbotryum species [43,44] reinforces the idea that mechanisms aside from 516 sexual antagonism are sufficient to drive such patterns of recombination cessation [84]. The 517 evolutionary strata in Microbotryum provide remarkable opportunities to study the accumulation 518 of sequence divergence traits over time. The current study further reveals differential expression 519 of genes as a common feature of non-recombining mating-type chromosomes and sex 520 chromosomes, where, in the absence of sexual antagonism, a major role for degenerative 521 mutations is indicated. 522 523 Conclusions 524 Our findings on differential gene expression being associated with various types of sequence 525 degeneration, and likely being a consequence of those, shed new lights on how differential gene 526 expression can evolve. In animals and plants, it is widely accepted that differential gene 527 expression on sex chromosomes is associated with sexually antagonistic selection [37,85]. Our 528 study shows that, in systems where sexual antagonistic selection is unlikely to occur, sequence 529 degeneration might readily lead to differential gene expression. Furthermore, the genes with 530 differential expression were highly enriched on mating-type chromosomes, similar to diverse 531 organisms where in contrast the separate sex functions have been cited as the cause. We further 532 found strong and consistent evidence of differential gene expression and its association with 533 various types of mutational changes, in particular TE insertions and premature stop codons, as 534 well as high levels of base pair substitutions, indels, intron and GC content that distinguish alleles 535 of DE genes. We show that sequence degeneration in fact largely characterizes DE genes 536 identified between fungal mating types, in the absence of sexually antagonistic selection, and 537 only on the old evolutionary strata. Our results help to uncover important patterns of gene 513 mechanisms for the genomics of reproductive compatibility [83]. In particular, the recent 514 demonstration of multiple evolutionary strata on the mating-type chromosomes of M. lychnidis- 515 dioicae and other Microbotryum species [43,44] reinforces the idea that mechanisms aside from 516 sexual antagonism are sufficient to drive such patterns of recombination cessation [84]. The 517 evolutionary strata in Microbotryum provide remarkable opportunities to study the accumulation 518 of sequence divergence traits over time. 502 Insights across sexual eukaryotes The current study further reveals differential expression 519 of genes as a common feature of non-recombining mating-type chromosomes and sex 520 chromosomes, where, in the absence of sexual antagonism, a major role for degenerative 521 mutations is indicated. 20 . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made availab The copyright holder for this preprint (which was this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 538 evolution relevant to a broad range of taxa where reproductive compatibility is determined in 539 extensive regions of recombination suppression. 540 541 Materials and Methods 542 Allele identification between a1 and a2 haploid genomes 543 In order to quantify differentially expressed genes between the two haploid genomes, the 544 alleles between a1 and a2 haploid genomes need to be identified for those genes. The genome 545 assembly and annotation of the same strain of M. lychnidis-dioicae have been published [43]. To 546 identify 1:1 single copy homologs in each haploid genome, the Reciprocal Best BLAST(p) Hits 547 (RBBH) python script (github.com/peterjc/galaxy_blast/tree/master/tools/blast_rbh) was applied 548 [86], with 50 percentage of length coverage. RBBH scripts also identified paralogs within each 549 haploid genome. A number of protein sequence alignment identity thresholds were tested, in 550 order to identify for the best strategy of maximizing the number of allele pair identification on the 551 non-recombining regions and while avoiding spurious BLAST results with low identity percent. 542 Allele identification between a1 and a2 haploid genomes 542 Allele identification between a1 and a2 haploid genomes 543 In order to quantify differentially expressed genes between the two haploid genomes, the 544 alleles between a1 and a2 haploid genomes need to be identified for those genes. The genome 545 assembly and annotation of the same strain of M. lychnidis-dioicae have been published [43]. To 546 identify 1:1 single copy homologs in each haploid genome, the Reciprocal Best BLAST(p) Hits 547 (RBBH) python script (github.com/peterjc/galaxy_blast/tree/master/tools/blast_rbh) was applied 548 [86], with 50 percentage of length coverage. RBBH scripts also identified paralogs within each 549 haploid genome. A number of protein sequence alignment identity thresholds were tested, in 550 order to identify for the best strategy of maximizing the number of allele pair identification on the 551 non-recombining regions and while avoiding spurious BLAST results with low identity percent. 552 Increasing the percent of protein sequence identity threshold from >70% to >85% resulted in a 553 decrease from 12.2% to 9.9% of single-copy genes on the mating-type chromosomes being 554 identified as differentially expressed genes (detailed below), while decreasing the threshold from 555 >70% to >30% resulted in only a marginal increase from 12.2% to 12.7%. The change in the 556 percentages of identified alleles that were differentially expressed on autosomes was negligible, 557 being 1.0%, 1.1% and 1.1% respectively for 80%, 70% and 30% thresholds (S10 Fig). Therefore, 558 the threshold of >70% protein sequence identity was used. To avoid potential bias due to paralogs 559 for identifying differential gene expression and other downstream analysis, genes with paralogs 560 within each haploid genome were filtered out and only single-copy allele pairs were retained for 561 downstream analysis. Genes were located to genomic compartments, including autosomes, 562 pseudo-autosomal regions (PARs), young evolutionary strata of the mating type chromosomes 543 In order to quantify differentially expressed genes between the two haploid genomes, the 544 alleles between a1 and a2 haploid genomes need to be identified for those genes. The genome 545 assembly and annotation of the same strain of M. lychnidis-dioicae have been published [43]. To 546 identify 1:1 single copy homologs in each haploid genome, the Reciprocal Best BLAST(p) Hits 547 (RBBH) python script (github.com/peterjc/galaxy_blast/tree/master/tools/blast_rbh) was applied 548 [86], with 50 percentage of length coverage. RBBH scripts also identified paralogs within each 549 haploid genome. 502 Insights across sexual eukaryotes 552 Increasing the percent of protein sequence identity threshold from >70% to >85% resulted in a 553 decrease from 12.2% to 9.9% of single-copy genes on the mating-type chromosomes being 554 identified as differentially expressed genes (detailed below), while decreasing the threshold from 555 >70% to >30% resulted in only a marginal increase from 12.2% to 12.7%. The change in the 556 percentages of identified alleles that were differentially expressed on autosomes was negligible, 557 being 1.0%, 1.1% and 1.1% respectively for 80%, 70% and 30% thresholds (S10 Fig). Therefore, 558 the threshold of >70% protein sequence identity was used. To avoid potential bias due to paralogs 559 for identifying differential gene expression and other downstream analysis, genes with paralogs 542 Allele identification between a1 and a2 haploid genomes A number of protein sequence alignment identity thresholds were tested, in 550 order to identify for the best strategy of maximizing the number of allele pair identification on the 551 non-recombining regions and while avoiding spurious BLAST results with low identity percent. 552 Increasing the percent of protein sequence identity threshold from >70% to >85% resulted in a 553 decrease from 12.2% to 9.9% of single-copy genes on the mating-type chromosomes being 554 identified as differentially expressed genes (detailed below), while decreasing the threshold from 555 >70% to >30% resulted in only a marginal increase from 12.2% to 12.7%. The change in the 556 percentages of identified alleles that were differentially expressed on autosomes was negligible, 557 being 1.0%, 1.1% and 1.1% respectively for 80%, 70% and 30% thresholds (S10 Fig). Therefore, 558 the threshold of >70% protein sequence identity was used. To avoid potential bias due to paralogs 559 for identifying differential gene expression and other downstream analysis, genes with paralogs 560 within each haploid genome were filtered out and only single-copy allele pairs were retained for 561 downstream analysis. Genes were located to genomic compartments, including autosomes, 562 pseudo-autosomal regions (PARs), young evolutionary strata of the mating type chromosomes 562 pseudo-autosomal regions (PARs), young evolutionary strata of the mating type chromosomes 21 . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made availa The copyright holder for this preprint (which wa this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 563 (including previously identified red and green strata; [43]) and old evolutionary strata (blue, 564 purple, orange and black strata; [43]). 565 566 Transposable element filtering 567 Transposable element (TE) annotation of both haploid genomes of M. 542 Allele identification between a1 and a2 haploid genomes lychnidis-dioicae was 568 published previously [87], and was used for analysis in this study. The coding sequence of each 569 gene from both a1 and a2 haploid genomes was search by BLAST(n) against the published 570 annotated TE consensus sequences of the same species, and alignment >80 percent of query 571 coverage (coding sequences) was used for identifications of TEs. The BLASTn output was 572 parsed using BASH scripts, and the coding sequences identified as TEs were removed from the 573 gene list for all further downstream analysis. 574 575 Identification of differentially expressed genes 576 RNAseq data and summary statistics of the datasets were described previously [45], and the 577 raw data of haploid culture growing separately in water agar conditions were downloaded from 578 the deposited NCBI database 579 (https://trace.ncbi.nlm.nih.gov/Traces/study/?acc=+PRJNA246470&go=go). First, the RNAseq 580 raw reads were quality assessed using FastQC v0.11.2 581 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and quality trimmed using 582 Trimmomatic v0.33 with default parameters for paired-end reads [88]. We filtered reads 583 containing adaptor sequences and trimmed reads if the sliding window average Phred score over 584 four bases was < 15 or if the leading/trailing bases had a Phred score < 3. Reads were then 585 removed post filtering if either read pair was < 36 bases. 586 To avoid possible bias for calling differential gene expression due to differences in homolog 587 length between a1 and a2, gaps differing between alleles by greater than 3bp were trimmed to 563 (including previously identified red and green strata; [43]) and old evolutionary strata (blue, 564 purple, orange and black strata; [43]). 567 Transposable element (TE) annotation of both haploid genomes of M. lychnidis-dioicae was 568 published previously [87], and was used for analysis in this study. The coding sequence of each 569 gene from both a1 and a2 haploid genomes was search by BLAST(n) against the published 570 annotated TE consensus sequences of the same species, and alignment >80 percent of query 571 coverage (coding sequences) was used for identifications of TEs. The BLASTn output was 572 parsed using BASH scripts, and the coding sequences identified as TEs were removed from the 573 gene list for all further downstream analysis. 575 Identification of differentially expressed genes ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 589 gaps from the ends of the alignment and inside the alignment, with inside gaps starting with the 590 closest to the end of the alignment (greater than the minimum gap size) until there are no gaps 591 larger than minimum gap size [89]. The trimmed allele pairs with equal length were used for read 592 mapping and calling differential gene expression. 589 gaps from the ends of the alignment and inside the alignment, with inside gaps starting with the 590 closest to the end of the alignment (greater than the minimum gap size) until there are no gaps 591 larger than minimum gap size [89]. The trimmed allele pairs with equal length were used for read 592 mapping and calling differential gene expression. 593 To quantify gene expression, we mapped the trimmed reads of haploid samples to the 594 trimmed homolog sequences of each haploid genome respectively with Kallisto v.0.43.0 [90]. 595 Read counts of the output from Kallisto mapping (e.g. using pseudo-alignment) were imported 596 for gene expression analysis in EdgeR v3.4 [91,92]. We filtered low counts and kept genes with 597 average Log(CPM) > 0 per sample, and CPM > 1 in half of the samples per haploid culture. We 598 then normalized the expression by trimmed mean of M values (TMM). We explored the libraries 599 of both haploid cultures in two dimensions using multi-dimensional scaling (MDS) plots (S11 600 Fig). Normalized expression counts for each sample were used to calculate differential expression 601 between mating types using standard measures. We first identified genes with differential 602 expression between mating types based on overall expression of the comparison group, and using 603 Benjamini-Hochberg correction for multiple-testing with false discovery rate (FDR) of 5%. 602 expression between mating types based on overall expression of the comparison group, and using 603 Benjamini-Hochberg correction for multiple-testing with false discovery rate (FDR) of 5%. 604 Differential expression between mating types was classified into four categories of fold changes, 605 namely 2 (low), 2-4 (mild), 4-8 (high), and > 8 (very high), and expressed as log2 ratio of a1-to-a2 606 expression (which has negative values for genes with higher a2 expression and positive values for 607 higher a1 expression). 575 Identification of differentially expressed genes 576 RNAseq data and summary statistics of the datasets were described previously [45], and the 577 raw data of haploid culture growing separately in water agar conditions were downloaded from 578 the deposited NCBI database 579 (https://trace.ncbi.nlm.nih.gov/Traces/study/?acc=+PRJNA246470&go=go). First, the RNAseq 580 raw reads were quality assessed using FastQC v0.11.2 581 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and quality trimmed using 582 Trimmomatic v0.33 with default parameters for paired-end reads [88]. We filtered reads 583 containing adaptor sequences and trimmed reads if the sliding window average Phred score over 584 four bases was < 15 or if the leading/trailing bases had a Phred score < 3. Reads were then 585 removed post filtering if either read pair was < 36 bases. 581 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and quality trimmed using 582 Trimmomatic v0.33 with default parameters for paired-end reads [88]. We filtered reads 583 containing adaptor sequences and trimmed reads if the sliding window average Phred score over 584 four bases was < 15 or if the leading/trailing bases had a Phred score < 3. Reads were then 585 removed post filtering if either read pair was < 36 bases. 581 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and quality trimmed using 582 Trimmomatic v0.33 with default parameters for paired-end reads [88]. We filtered reads 583 containing adaptor sequences and trimmed reads if the sliding window average Phred score over 584 four bases was < 15 or if the leading/trailing bases had a Phred score < 3. Reads were then 585 removed post filtering if either read pair was < 36 bases. 586 To avoid possible bias for calling differential gene expression due to differences in homolog 587 length between a1 and a2, gaps differing between alleles by greater than 3bp were trimmed to 588 keep the same length, using published custom Python script [89]. This trimming includes the 22 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. 575 Identification of differentially expressed genes 623 Other post hoc tests evaluating individual degeneration trait are described below. All statistical 624 analyses were conducted in SPSS v23 [94] and R v3.4.3 [95]. 615 two-way interactions terms for genomic compartments and the absolute value of differences 616 between alleles for sequence divergence (dN), transposable element insertions number within 617 20kb (up and downstream), predicted protein length, intron content and GC content. The absolute 618 value of the differences between alleles was calculated for each trait as detailed below. Model 619 family comparison was based upon minimizing Akaike’s Information Criterion and over/under- 620 dispersion using ratio of deviance/df; Tweedie, power 1.7 (approaching gamma distribution) 621 provided the best available fit for the expression ratio response variable. A best fit model was 622 selected using stepwise model selection, following removal of non-significant interaction terms. 623 Other post hoc tests evaluating individual degeneration trait are described below. All statistical 624 analyses were conducted in SPSS v23 [94] and R v3.4.3 [95]. 575 Identification of differentially expressed genes As suggested by [93], fold changes > 0 will be interpreted throughout, 604 Differential expression between mating types was classified into four categories of fold changes, 605 namely 2 (low), 2-4 (mild), 4-8 (high), and > 8 (very high), and expressed as log2 ratio of a1-to-a2 606 expression (which has negative values for genes with higher a2 expression and positive values for 607 higher a1 expression). As suggested by [93], fold changes > 0 will be interpreted throughout, 608 because we are working on haploid cell cultures and there are no possible scaling nor allometry 609 issues due to whole-body sampling. Thus, unless stated otherwise, both conditions FDR < 0.05 610 and \|log2FC\| > 0 will be met when calling mating-type bias. 611 The classification of genes as having differential expression between mating types or the 612 absolute values of gene expression ratio \|Log2(a1/a2)\| was used to assess relationships to various 613 forms of mutational changes. Generalized linear model (GLM) analysis was used to assess the 614 predictors of absolute values of expression ratio \|Log2(a1/a2)\|, with main effect variables and all 23 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 615 two-way interactions terms for genomic compartments and the absolute value of differences 616 between alleles for sequence divergence (dN), transposable element insertions number within 617 20kb (up and downstream), predicted protein length, intron content and GC content. The absolute 618 value of the differences between alleles was calculated for each trait as detailed below. Model 619 family comparison was based upon minimizing Akaike’s Information Criterion and over/under- 620 dispersion using ratio of deviance/df; Tweedie, power 1.7 (approaching gamma distribution) 621 provided the best available fit for the expression ratio response variable. A best fit model was 622 selected using stepwise model selection, following removal of non-significant interaction terms. 626 Relationship between differential expression and elevated substitution rates 627 Pairs of alleles between a1 and a2 mating types were aligned with PRANK (v170427) using 628 the codon model [96]. Each pair of allele alignment was then analyzed with codeml in PAML 629 [97] (runmode -2) to calculate the number of nonsynonymous substitutions per nonsynonymous 630 site (dN), the number of synonymous substitutions per synonymous site (dS), and the ratio of the 631 two (dN/dS), the latter excluding genes with dS value of zero. We then compared sequence 632 divergence between alleles using non-parametric Wilcoxon rank sum tests for DE versus non-DE 633 genes within genomic compartments. 627 Pairs of alleles between a1 and a2 mating types were aligned with PRANK (v170427) using 628 the codon model [96]. Each pair of allele alignment was then analyzed with codeml in PAML 629 [97] (runmode -2) to calculate the number of nonsynonymous substitutions per nonsynonymous 630 site (dN), the number of synonymous substitutions per synonymous site (dS), and the ratio of the 631 two (dN/dS), the latter excluding genes with dS value of zero. We then compared sequence 632 divergence between alleles using non-parametric Wilcoxon rank sum tests for DE versus non-DE 633 genes within genomic compartments. 632 divergence between alleles using non-parametric Wilcoxon rank sum tests for DE versus non-DE 633 genes within genomic compartments. 634 Also, the allele sequences were compared between M. lychnidis-dioicae and their orthologs in 635 M. lagerheimii, which has retained largely collinear and recombining mating-type chromosomes, 636 as the inferred ancestral state in the Microbotryum genus [43,44]. The single-copy orthologs for 637 a1 or a2 genomes between M. lychnidis-dioicea and M. lagerheimii were identified using RBBH 638 with 70 percent protein sequence coverage identity 634 Also, the allele sequences were compared between M. lychnidis-dioicae and their orthologs in 635 M. lagerheimii, which has retained largely collinear and recombining mating-type chromosomes, 636 as the inferred ancestral state in the Microbotryum genus [43,44]. The single-copy orthologs for 637 a1 or a2 genomes between M. lychnidis-dioicea and M. lagerheimii were identified using RBBH 638 with 70 percent protein sequence coverage identity 634 Also, the allele sequences were compared between M. lychnidis-dioicae and their orthologs in 635 M. lagerheimii, which has retained largely collinear and recombining mating-type chromosomes, 636 as the inferred ancestral state in the Microbotryum genus [43,44]. The single-copy orthologs for 637 a1 or a2 genomes between M. lychnidis-dioicea and M. 626 Relationship between differential expression and elevated substitution rates Oriented TE differences between alleles were calculated as the TE number for the allele 656 with lower expression minus the TE number for the higher expressed allele; a positive value thus 657 represented an excess of TEs in the lower expressed allele. A sliding window approach was used 658 with a window size of three adjacent intervals, progressing from upstream to downstream of the 659 genes. 660 661 Relationship between differential expression and differences in predicted protein length 662 We first verified whether there was bias in the gene prediction model across genomic 663 compartments, using the ratio of predicted coding sequence length divided by three times protein 664 sequence length, assessed using linear regression model. Coding sequencing length divided by . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 643 Relationship between differential expression and TE insertions 644 The TE annotation of the M. lychnidis-dioicae genome published previously [87] was used 645 for the analysis in this study. First, the TE insertion sites were assessed for each given focal gene, 646 upstream 0-5k, 5-10kb, 10-15kb, 15-20kb distance intervals, and downstream 0-5kb, 5-10kb, 10- 647 15kb and 15-20kb distance intervals using Bedtools window function for each indicated distance 648 window (https://bedtools.readthedocs.io/en/latest/content/tools/window.html). Both annotation 649 GFF3 files of gene models and TE annotations of M. lychnidis-dioicae were provided as input 650 files. The output files were parsed using Bash scripts. Wilcoxon rank sum tests were used to 651 compare TE insertions for DE and non-DE genes within genomic compartments. 644 The TE annotation of the M. lychnidis-dioicae genome published previously [87] was used 645 for the analysis in this study. First, the TE insertion sites were assessed for each given focal gene, 646 upstream 0-5k, 5-10kb, 10-15kb, 15-20kb distance intervals, and downstream 0-5kb, 5-10kb, 10- 647 15kb and 15-20kb distance intervals using Bedtools window function for each indicated distance 648 window (https://bedtools.readthedocs.io/en/latest/content/tools/window.html). Both annotation 649 GFF3 files of gene models and TE annotations of M. lychnidis-dioicae were provided as input 650 files. The output files were parsed using Bash scripts. Wilcoxon rank sum tests were used to 651 compare TE insertions for DE and non-DE genes within genomic compartments. 652 Also, a limited GLM model was used to assess the hypothesized directional association of TE 653 insertions and reducing allele expression (\|Log2(a1/a2)\|); this model contained genomic 654 compartment and oriented TE differences between alleles as main effects and their interaction 655 term. Oriented TE differences between alleles were calculated as the TE number for the allele 656 with lower expression minus the TE number for the higher expressed allele; a positive value thus 657 represented an excess of TEs in the lower expressed allele. A sliding window approach was used 658 with a window size of three adjacent intervals, progressing from upstream to downstream of the 659 genes. 660 661 R l ti hi b t diff ti l i d diff i di t d t i l th 626 Relationship between differential expression and elevated substitution rates lagerheimii were identified using RBBH 638 with 70 percent protein sequence coverage identity 639 (github.com/peterjc/galaxy_blast/tree/master/tools/blast_rbh, [86]). Wilcoxon rank sum test was 24 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 640 used to assess dN between orthlogs in M. lychnidis-dioicea and M. lagerheimii to evaluate the 641 hypothesis that the alleles with lower expression levels would have greater sequence divergence. 642 643 Relationship between differential expression and TE insertions 644 The TE annotation of the M. lychnidis-dioicae genome published previously [87] was used 645 for the analysis in this study. First, the TE insertion sites were assessed for each given focal gene, 646 upstream 0-5k, 5-10kb, 10-15kb, 15-20kb distance intervals, and downstream 0-5kb, 5-10kb, 10- 647 15kb and 15-20kb distance intervals using Bedtools window function for each indicated distance 648 window (https://bedtools.readthedocs.io/en/latest/content/tools/window.html). Both annotation 649 GFF3 files of gene models and TE annotations of M. lychnidis-dioicae were provided as input 650 files. The output files were parsed using Bash scripts. Wilcoxon rank sum tests were used to 651 compare TE insertions for DE and non-DE genes within genomic compartments. 652 Also, a limited GLM model was used to assess the hypothesized directional association of TE 653 insertions and reducing allele expression (\|Log2(a1/a2)\|); this model contained genomic 654 compartment and oriented TE differences between alleles as main effects and their interaction 655 term. 661 Relationship between differential expression and differences in predicted protein length The mutational causes of unequal protein lengths was assessed by 671 manually quantifying premature stop codons or indels using Geneious v8.1.7 [98]. A limited 672 GLM model was used to assess the hypothesized directional association of protein truncation and 673 reducing allele expression (\|Log2(a1/a2)\|); this model contained genomic compartment and 674 oriented predicted protein length differences between alleles as main effects and their interaction 675 term. Oriented predicted protein length differences between alleles were calculated as the ratio 676 for the allele with higher expression divided by the allele with lower expression; a larger ratio 677 thus represented a shorter length for the allele with lower expression. 661 Relationship between differential expression and differences in predicted protein length 662 We first verified whether there was bias in the gene prediction model across genomic 663 compartments, using the ratio of predicted coding sequence length divided by three times protein 664 sequence length, assessed using linear regression model. Coding sequencing length divided by 665 the length of predicted protein multiplied by 3 was consistently close to 1 and did not differ 25 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 666 among genomic compartments (autosome, PAR, young strata and old evolutionary strata; Linear 667 model, R2 = -5.50e-05, F-statistic = 0.869, P= 0.530, S12 Fig). We therefore calculated the ratio 668 of predicted protein length between allele pairs, and compared the proportions of genes in DE 669 and non-DE categories that had unequal lengths using two-proportion Z test for genes within 670 genomic compartments. The mutational causes of unequal protein lengths was assessed by 671 manually quantifying premature stop codons or indels using Geneious v8.1.7 [98]. A limited 672 GLM model was used to assess the hypothesized directional association of protein truncation and 673 reducing allele expression (\|Log2(a1/a2)\|); this model contained genomic compartment and 674 oriented predicted protein length differences between alleles as main effects and their interaction 675 term. Oriented predicted protein length differences between alleles were calculated as the ratio 676 for the allele with higher expression divided by the allele with lower expression; a larger ratio 677 thus represented a shorter length for the allele with lower expression. 678 666 among genomic compartments (autosome, PAR, young strata and old evolutionary strata; Linear 667 model, R2 = -5.50e-05, F-statistic = 0.869, P= 0.530, S12 Fig). We therefore calculated the ratio 668 of predicted protein length between allele pairs, and compared the proportions of genes in DE 669 and non-DE categories that had unequal lengths using two-proportion Z test for genes within 670 genomic compartments. 679 Relationship between differential expression and intron content 680 Using the published annotation gene models and coding sequences, we extracted the intron 681 number and mean intron length information from the annotation gff3 file, using Perl script 682 (https://bioops.info/2012/11/intron-size-gff3-perl/). We investigated the proportional differences 683 of the intron content for both DE and non-DE genes within genomic compartments using 684 Wilcoxon rank sum test. We also used a limited GLM model to test the hypothesized directional 685 association of greater intron content and reducing allele expression (\|Log2(a1/a2)\|); this model 686 contained genomic compartments and oriented intron content differences between alleles as main 687 effects and their interaction term. Oriented intron content differences between alleles were 688 calculated as the value for the lower expressed allele minus the value for the higher expressed 689 allele; a positive value thus represented greater intron content for the lower expressed allele. 690 691 Relationship between differential expression and GC content 680 Using the published annotation gene models and coding sequences, we extracted the intron 681 number and mean intron length information from the annotation gff3 file, using Perl script 682 (https://bioops.info/2012/11/intron-size-gff3-perl/). We investigated the proportional differences 683 of the intron content for both DE and non-DE genes within genomic compartments using 684 Wilcoxon rank sum test. We also used a limited GLM model to test the hypothesized directional 685 association of greater intron content and reducing allele expression (\|Log2(a1/a2)\|); this model 686 contained genomic compartments and oriented intron content differences between alleles as main 687 effects and their interaction term. Oriented intron content differences between alleles were 688 calculated as the value for the lower expressed allele minus the value for the higher expressed 689 allele; a positive value thus represented greater intron content for the lower expressed allele. 690 691 Relationship between differential expression and GC content 691 Relationship between differential expression and GC content CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 707 Fig 1. Comparisons of differentially expressed (DE) versus non-differentially expressed 708 (non-DE) genes between mating types of Microbotryum lychnidis-dioicae for various 709 degeneration-associated traits within genomic compartments. 707 Fig 1. Comparisons of differentially expressed (DE) versus non-differentially expressed 708 (non-DE) genes between mating types of Microbotryum lychnidis-dioicae for various 709 degeneration-associated traits within genomic compartments. 710 (A) Non-synonymous sequence divergence, dN, between alleles of DE and non-DE genes. (B) 711 Transposable element (TE) insertion number differences between alleles within 20kb (up and 712 downstream) of DE and non-DE genes. (C) Proportions of differentially expressed (DE) and non- 713 differentially expressed (non-DE) genes with different protein lengths between alleles. (D) Intron 714 content proportional differences between alleles of DE and non-DE genes. (E) Total GC content 715 (GC0) proportional differences between alleles of DE and non-DE genes. Analyzed allele 716 differences represent absolute values comparisons (i.e. unoriented with regard to allele expression 717 levels). Comparisons in panels A, C-E reflect Wilcoxon rank sum tests; panel B reflects a two- 718 proportion z-test. Significance levels shown as, **: P < 0.001, : P < 0.05; non-significant test 719 results shown in Supplementary Tables S4, S6-S9. Genomic compartments include autosomes, 720 pseudo-autosomal regions (PARs), young evolutionary strata (previously identified red and green 721 strata; [43]) and old evolutionary strata (blue, purple, orange and black strata; [43]). The notation 722 “a” indicates that the young evolutionary strata contained only one DE gene, precluding 723 comparisons to non-DE genes within this compartment. 724 691 Relationship between differential expression and GC content 26 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 692 We calculated the total GC percentage (GC0) and the GC percentage at the third position of 693 aminol acid (GC3) for alleles of each gene coding sequence using homemade awk scripts. We 694 investigated the differences of GC0 and GC3 for both DE and non-DE genes within genomic 695 compartments using Wilcoxon rank sum test. We also used a limited GLM model to test the 696 hypothesized directional association of reduced GC content and reducing allele expression 697 (\|Log2(a1/a2)\|); this model contained genomic compartments and oriented GC content differences 698 between alleles as main effects and their interaction term. Oriented GC content differences 699 between alleles were calculated as the value for the allele with higher expression minus the value 700 for the allele with lower expression; a positive value thus representing reduced GC content for the 701 allele with lower expression. 702 703 Ethics Statement 704 705 N/A 692 We calculated the total GC percentage (GC0) and the GC percentage at the third position of 693 aminol acid (GC3) for alleles of each gene coding sequence using homemade awk scripts. We 694 investigated the differences of GC0 and GC3 for both DE and non-DE genes within genomic 695 compartments using Wilcoxon rank sum test. We also used a limited GLM model to test the 696 hypothesized directional association of reduced GC content and reducing allele expression 697 (\|Log2(a1/a2)\|); this model contained genomic compartments and oriented GC content differences 698 between alleles as main effects and their interaction term. Oriented GC content differences 699 between alleles were calculated as the value for the allele with higher expression minus the value 700 for the allele with lower expression; a positive value thus representing reduced GC content for the 701 allele with lower expression. 692 27 . 725 Fig 2. Significant predictors of the degree of differential expression between mating types of 726 Microbotryum lychnidis-dioicae testing directional effects of degeneration-associated traits. 28 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 742 Supporting Information Legends 743 744 S1 Table. Number of single-copy genes with alleles in both a1 and a2 haploid genomes, with 745 70% protein sequence identity detection using reciprocal best BLASTp hits, before and 746 after filtering out genes with transposable element (TE)-related functions. 747 Filtering removed 192 paralogous genes within each haploid genome and genes with TE-related 748 functions, including 1750 and 1819 from a1 and a2 haploid genome, respectively. 749 750 S2 Table. Identification of differentially expressed (DE) genes with either a1-biased or a2- 751 biased expression (i.e., higher expression in a1 or a2, respectively) under various Log2(a1/a2) 752 criteria. 753 754 S3 Table. Numbers and percentages of genes with differential expression (DE) within 755 genomic compartments, including autosomes pseudo-autosomal regions (PARs), young 756 evolutionary strata (previously identified red and green strata; [43]) and old evolutionary 757 strata (blue, purple, orange and black strata; [43]). 758 Chi-squared test was used to assess whether DE genes were non-randomly distributed between 759 autosomes versus the genomic compartments on mating type chromosomes (MAT). P values 760 <0.05 are in bold. NA: not applicable. The young strata only had one DE gene, so statistic 761 comparisons could not be performed for these strata. 762 763 S4 Table. Wilcoxon rank sum test statistics for comparisons of mean non-synonymous 764 mutation rate (dN, A) and synonymous mutation rate (dS, B) of differentially expressed 765 genes (DE) versus non-differentially expressed genes (non-DE) within genomic 766 compartments, including autosomes pseudo-autosomal regions (PARs), young evolutionary 767 strata (previously identified red and green strata; [43]) and old evolutionary strata (blue, 768 purple, orange and black strata; [43]). 769 P values <0.05 are in bold. NA: not applicable. 725 Fig 2. Significant predictors of the degree of differential expression between mating types of 726 Microbotryum lychnidis-dioicae testing directional effects of degeneration-associated traits. NA: not applicable, as the young strata only had 770 one DE gene, statistical comparisons could not be performed for this compartment. 771 772 S5 Table. Wilcoxon rank sum test statistics for comparisons of divergence of alleles in 773 Microbotryum lychnidis-dioicae from orthologs in M. lagerheimii. 774 This test assessed whether the allele with lower expression in M. lychnidis-dioicae was more 775 divergent from orthologs in M. lagerheimii than the alleles with higher expression in M. 776 lychnidis-dioicae, considering non-synonymous mutation rate (dN), synonymous mutation rate . CC-BY 4.0 International license under a 772 S5 Table. Wilcoxon rank sum test statistics for comparisons of divergence of alleles in 773 Microbotryum lychnidis-dioicae from orthologs in M. lagerheimii. 29 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 777 (dS), and the ratio (dN/dS) within genomic compartments, including autosomes pseudo- 778 autosomal regions (PARs), young evolutionary strata (previously identified red and green strata; 779 [43]) and old evolutionary strata (blue, purple, orange and black strata; [43]). We calculated these 780 substitution rates for a1 and a2 alleles between these two species separately. NA: not applicable, 781 as the young strata only had one DE gene, statistical comparisons could not be performed for this 782 compartment. 783 784 S6 Table. Wilcoxon rank sum test statistics for comparisons of unoriented transposable 785 elements (TEs) insertion differences between alleles (within 20kb up and downstream) of 786 differentially expressed (DE) genes versus non-differentially expressed (non-DE) genes 787 within genomic compartments, including autosomes pseudo-autosomal regions (PARs), 788 young evolutionary strata (previously identified red and green strata; [43]) and old 789 evolutionary strata (blue, purple, orange and black strata; [43]). 790 P-values <0.05 are in bold. NA: not applicable, as the young strata only had one DE gene, 791 statistical comparisons could not be performed for this compartment. 792 793 S7 Table. Two proportion Z test for comparisons of unoriented protein length difference 794 between alleles of differentially expressed (DE) genes versus non-differentially expressed 795 (non-DE) genes within genomic compartments, including autosomes pseudo-autosomal 796 regions (PARs), young evolutionary strata (previously identified red and green strata; [43]) 797 and old evolutionary strata (blue, purple, orange and black strata; [43]). 798 P values <0.05 are in bold. NA: not applicable, as proportions * sample size was less than 5 for 799 the PARs and young strata, statistical comparisons could not be performed for these 800 compartments. 801 802 S8 Table. 772 S5 Table. Wilcoxon rank sum test statistics for comparisons of divergence of alleles in 773 Microbotryum lychnidis-dioicae from orthologs in M. lagerheimii. Wilcoxon rank sum test statistics for comparisons of unoriented differences in 803 intron content between alleles of differentially expressed (DE) versus non-differentially 804 expressed (non-DE) genes within genomic compartments, including autosomes pseudo- 805 autosomal regions (PARs), young evolutionary strata (previously identified red and green 806 strata; [43]) and old evolutionary strata (blue, purple, orange and black strata; [43]). 807 P values <0.05 are in bold. NA: not applicable, as the young strata only had one DE gene, 808 statistical comparisons could not be performed for this compartment. 809 777 (dS), and the ratio (dN/dS) within genomic compartments, including autosomes pseudo- 778 autosomal regions (PARs), young evolutionary strata (previously identified red and green strata; 779 [43]) and old evolutionary strata (blue, purple, orange and black strata; [43]). We calculated these 780 substitution rates for a1 and a2 alleles between these two species separately. NA: not applicable, 781 as the young strata only had one DE gene, statistical comparisons could not be performed for this 782 compartment. 790 P-values <0.05 are in bold. NA: not applicable, as the young strata only had one DE gene, 791 statistical comparisons could not be performed for this compartment. 30 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 810 S9 Table. Wilcoxon rank-sum test statistics for comparisons of unoriented differences in 811 overall GC content (GC0), and third codon position (GC3) between alleles of differentially 812 expressed (DE) versus non-differentially expressed (non-DE) genes within genomic 813 compartments, including autosomes pseudo-autosomal regions (PARs), young evolutionary 814 strata (previously identified red and green strata; [43]) and old evolutionary strata (blue, 815 purple, orange and black strata; [43]). 816 (A) Overall GC content. (B) Third codon position GC content. P values <0.05 are in bold. 826 S2 Fig. Interaction plots for pairs of predictor variables in overall GLM of differential gene 827 expression between mating types of Microbotryum lychnidis-dioicae. p g yp y y 828 Y-axes are GLM-predicted response values of differential expression ratio between allele pairs in 829 a1 and a2 haploid genomes, and x-axes are allele differences between allele pairs in a1 and a2 830 haploid genomes in predicted protein length as the predictor variable binned into levels of 831 interacting categorical predictor variable (i.e. panel A, genomic compartment) or other interacting 832 continuous predictor variables (i.e. panels B-D; the lowest bin being no differences between 833 alleles, and low and high bins being split at the median value among genes with non-zero 834 differences between alleles). (A) Interaction plot between protein length differences and genomic 835 compartment. Genomic compartments include autosomes, pseudo-autosomal regions (PARs), 836 young evolutionary strata (previously identified red and green strata; [43]) and old evolutionary 837 strata (blue, purple, orange and black strata; [43]). (B) Interaction plots between protein length 838 differences and differences in transposable elements (TEs) insertions. (C) Interaction plots 839 between protein length differences and non-synonymous mutation (dN) rate differences. (D) 840 Interaction plots between protein length differences and GC content differences. 841 772 S5 Table. Wilcoxon rank sum test statistics for comparisons of divergence of alleles in 773 Microbotryum lychnidis-dioicae from orthologs in M. lagerheimii. NA: 817 not applicable, as the young strata only had one DE gene, statistical comparisons could not be 818 performed for this compartment. 819 820 821 S1 Fig. Heatmap showing differentially expressed genes between haploid a1 culture and 822 haploid a2 of Microbotryum lychnidis-dioicae cultures under low nutrient condition. 823 Each column shows a replicated sample for each haploid cell culture. Z-score denotes the relative 824 gene expression level, and cluster shows the similar gene expression profiles. 825 826 S2 Fig. Interaction plots for pairs of predictor variables in overall GLM of differential gene 827 expression between mating types of Microbotryum lychnidis-dioicae. 828 Y-axes are GLM-predicted response values of differential expression ratio between allele pairs in 829 a1 and a2 haploid genomes, and x-axes are allele differences between allele pairs in a1 and a2 830 haploid genomes in predicted protein length as the predictor variable binned into levels of 831 interacting categorical predictor variable (i.e. panel A, genomic compartment) or other interacting 832 continuous predictor variables (i.e. panels B-D; the lowest bin being no differences between 833 alleles, and low and high bins being split at the median value among genes with non-zero 834 differences between alleles). (A) Interaction plot between protein length differences and genomic 835 compartment. Genomic compartments include autosomes, pseudo-autosomal regions (PARs), 836 young evolutionary strata (previously identified red and green strata; [43]) and old evolutionary 837 strata (blue, purple, orange and black strata; [43]). (B) Interaction plots between protein length 838 differences and differences in transposable elements (TEs) insertions. (C) Interaction plots 839 between protein length differences and non-synonymous mutation (dN) rate differences. (D) 840 Interaction plots between protein length differences and GC content differences. 841 842 S3 Fig. Boxplot of synonymous mutation rate (dS) for differentially expressed (DE) and 843 non-differentially expressed genes (Non-DE) of Microbotryum lychnidis-dioicae. 842 S3 Fig. Boxplot of synonymous mutation rate (dS) for differentially expressed (DE) and 843 non-differentially expressed genes (Non-DE) of Microbotryum lychnidis-dioicae. 31 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 844 Wilcoxon rank sum tests for comparisons of mean non-synonymous mutation rate (dN) of 845 differentially expressed genes (DE) versus non-differentially expressed genes (non-DE) within 846 genomic compartments: ‘*’: P < 0.001, other comparisons were not significant. Genomic 847 compartments include autosomes, pseudo-autosomal regions (PARs), young evolutionary strata 848 (previously identified red and green strata; [43]) and old evolutionary strata (blue, purple, orange 849 and black strata; [43]). 850 851 S4 Fig. Boxplot of differentially (a1-biased, a2-biased) and non-differentially expressed 852 genes (not-biased) and the sequence divergence between alleles of Microbotryum lychnidis- 853 dioicae. 854 Wilcoxon rank sum tests for comparisons of genes with higher allele expression in the a1 and a2 855 haploid mating type genomes separately to non-differentially expressed genes for the mean non- 856 synonymous mutation rate (dN) (A), synonymous mutation rate (dS) (B); NS: not significant, 857 ‘’: P < 0.001, ‘’: P < 0.01, ‘’: P < 0.5, ‘.’: P < 0.1, NS: not significant. As dN and dS of 858 almost all genes in autosome and PAR are zero, and there is only one DE gene on the young 859 strata, so no statistic test can be performed in these regions. Sample size for each genomic 860 compartment is listed either above or inside boxplot accordingly. Genomic compartments 861 include autosomes, pseudo-autosomal regions (PARs), young evolutionary strata (previously 862 identified red and green strata; [43]) and old evolutionary strata (blue, purple, orange and black 863 strata; [43]). 864 865 S5 Fig. Boxplot of differentially expressed (DE) and non-differentially expressed genes 866 (non-DE) and gene evolutionary rate dN/dS of Microbotryum lychnidis-dioicae. 867 Wilcoxon rank sum tests for comparisons of evolutionary rate dN/dS of differentially expressed 868 genes (DE) versus non-differentially expressed genes (non-DE) within genomic compartments; 869 NS: not significant. 842 S3 Fig. Boxplot of synonymous mutation rate (dS) for differentially expressed (DE) and 843 non-differentially expressed genes (Non-DE) of Microbotryum lychnidis-dioicae. As dN/dS of almost all genes in autosome and PAR are zero, and there is 870 only one DE gene on the young strata, so no statistic test can be performed in these regions. 871 Genomic compartments include autosomes, pseudo-autosomal regions (PARs), young 872 evolutionary strata (previously identified red and green strata; [43]) and old evolutionary strata 873 (blue, purple, orange and black strata; [43]). 874 875 S6 Fig. Boxplot of sequence divergence, non-synonymous mutation rate dN (A), 844 Wilcoxon rank sum tests for comparisons of mean non-synonymous mutation rate (dN) of 845 differentially expressed genes (DE) versus non-differentially expressed genes (non-DE) within 846 genomic compartments: ‘*’: P < 0.001, other comparisons were not significant. Genomic 847 compartments include autosomes, pseudo-autosomal regions (PARs), young evolutionary strata 848 (previously identified red and green strata; [43]) and old evolutionary strata (blue, purple, orange 849 and black strata; [43]). 65 S5 Fig. Boxplot of differentially expressed (DE) and non-differentially expressed genes 66 (non-DE) and gene evolutionary rate dN/dS of Microbotryum lychnidis-dioicae. 32 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 878 Alleles of differentially expressed genes with hypothesized higher (red - lower expressed allele) 879 and lower (blue - higher expressed allele) substitution rates and hypothesized equal (grey; non- 880 differentially expressed genes) mutation rates were pooled from a1 and a2 genomes and assessed 881 for divergences from orthologs in M. lagerheimii. Genomic compartments include autosomes, 882 pseudo-autosomal regions (PARs), young evolutionary strata (previously identified red and green 883 strata; [43]) and old evolutionary strata (blue, purple, orange and black strata; [43]). 884 885 S7 Fig. Dotplot of oriented differences of transposable element (TE) insertions and 886 differential gene expression between alleles of Microbotryum lychnidis-dioicae. 887 TE insertions are shown for sliding-window intervals from upstream to downstream of genes, 888 were differences between alleles were calculated as the TE number for the allele with lower 889 expression minus the TE number for the higher expressed allele; a positive value thus represented 890 an excess of TEs in the lower expressed allele. Sliding window intervals are shown as A: 891 upstream 20kb to 10kb, B: upstream 15kb to 5kb, C: upstream 10kb to gene, D: upstream 5kb to 892 downstream 5kb, E: gene to downstream 10kb, F: downstream 5kb to 15kb, and G: downstream 893 10kb to 20kb. 894 895 S8 Fig. Average indel numbers and proportions of genes with different stop codon positions 896 between alleles of differentially expressed genes of Microbotryum lychnidis-dioicae. 897 Among genes having alleles with different predicted protein lengths, boxplot of average indel 898 numbers for both differentially expressed (DE) and non-DE genes across various genomic 899 compartments (A), and barplot for proportions of genes with different stop codon positions for 900 both DE and non-DE genes across genomic compartments (B). 65 S5 Fig. Boxplot of differentially expressed (DE) and non-differentially expressed genes 66 (non-DE) and gene evolutionary rate dN/dS of Microbotryum lychnidis-dioicae. : P < 0.01, : P < 0.05, ‘.’: P < 901 0.1, NS: not significant. Genomic compartments include autosomes, pseudo-autosomal regions 902 (PARs), young evolutionary strata (previously identified red and green strata; [43]) and old 903 evolutionary strata (blue, purple, orange and black strata; [43]). 904 905 S9 Fig. Comparisons of differentially expressed (DE) and non-differentially expressed (non- 906 DE) genes between mating types of Microbotryum lychnidis-dioicae for differences between 907 alleles in third codon position GC content (GC3) within genomic compartments. 908 Wilcoxon rank sum test statistics for comparisons of unoriented differences in GC content of 909 third codon position (GC3) between alleles of differentially expressed (DE) versus non- 910 differentially expressed (non-DE) genes within genomic compartments; : P < 0.001, NS: non 911 significant. Genomic compartments include autosomes, pseudo-autosomal regions (PARs), young 878 Alleles of differentially expressed genes with hypothesized higher (red - lower expressed allele) 879 and lower (blue - higher expressed allele) substitution rates and hypothesized equal (grey; non- 880 differentially expressed genes) mutation rates were pooled from a1 and a2 genomes and assessed 881 for divergences from orthologs in M. lagerheimii. Genomic compartments include autosomes, 882 pseudo-autosomal regions (PARs), young evolutionary strata (previously identified red and green 883 strata; [43]) and old evolutionary strata (blue, purple, orange and black strata; [43]). 884 885 S7 Fig. Dotplot of oriented differences of transposable element (TE) insertions and 886 differential gene expression between alleles of Microbotryum lychnidis-dioicae. 905 S9 Fig. Comparisons of differentially expressed (DE) and non-differentially expressed (non- 906 DE) genes between mating types of Microbotryum lychnidis-dioicae for differences between 907 alleles in third codon position GC content (GC3) within genomic compartments. 908 Wilcoxon rank sum test statistics for comparisons of unoriented differences in GC content of 909 third codon position (GC3) between alleles of differentially expressed (DE) versus non- 910 differentially expressed (non-DE) genes within genomic compartments; : P < 0.001, NS: non 911 significant. Genomic compartments include autosomes, pseudo-autosomal regions (PARs), young 33 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 912 evolutionary strata (previously identified red and green strata; [43]) and old evolutionary strata 913 (blue, purple, orange and black strata; [43]). The notation “a” indicates that the young 914 evolutionary strata contained only one DE gene, precluding comparisons to non-DE genes within 915 this compartment. y y g p g p g 915 this compartment. 916 917 S10 Fig. Comparison of proportion (A) and number (B) of differentially expressed (DE) 918 genes detected between a1 and a2 haploid mating type genomes of Microbotryum lychnidis 919 dioicae. 920 Differentially expressed (DE) genes on mating-type chromosome (MAT) chromosomes and 921 autosomes (auto), at various percentage protein sequence identities used as threshold for 922 identification of alleles for genes in a1 and a2 haploid genomes. : P < 0.01 using Chi-square 923 test. All other comparisons of DE genes on mating-type chromosome and autosomes are not 924 significant. 925 926 S11 Fig. Multidimensional scaling (MDS) plot of RNAseq libraries of Microbotryum 927 lychnidis-dioicae. 928 Water denotes water agar (i.e. low nutrients) culture condition. 929 930 S12 Fig. The ratio index of coding sequence and protein sequence of Microbotryum 931 lychnidis-dioicae. 885 S7 Fig. Dotplot of oriented differences of transposable element (TE) insertions and 886 differential gene expression between alleles of Microbotryum lychnidis-dioicae. 932 The ratio of predicted coding sequence divided by the predicted protein sequence multiplied b 933 three for a1 and a2 alleles, among four genomic compartments. 934 935 936 Data access 937 We used published gene expression data to investigate the association of sequence degeneratio 938 and differential gene expression in Microbotryum lychnidis-dioicae ([42,45], 939 https://trace.ncbi.nlm.nih.gov/Traces/study/?acc=+PRJNA246470&go=go). We used publishe 940 genome assembly, gene predictions and assignations to genomic compartments [43,44]. We al 941 used published transposable elements identification in M. lychnidis-dioicae [87]. All relevant 942 scripts and data files to perform these analyses are deposited in Zenodo and Github 943 (https://github.com/Wen- 944 Juan/Differential_expression_associateswith_degeneration_Microbotryum_fungus), which wil 945 be released immediately upon manuscript acceptance. 9 https://trace.ncbi.nlm.nih.gov/Traces/study/?acc=+PRJNA246470&go=go). We used published 0 genome assembly, gene predictions and assignations to genomic compartments [43,44]. We also 1 used published transposable elements identification in M. lychnidis-dioicae [87]. All relevant 2 scripts and data files to perform these analyses are deposited in Zenodo and Github 3 (https://github.com/Wen- 944 Juan/Differential_expression_associateswith_degeneration_Microbotryum_fungus), which will 945 be released immediately upon manuscript acceptance. 34 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 946 Acknowledgements 947 948 We thank Darren J. Parker for assistance on python scripting and valuable discussions of the 949 results, and Ricardo C. Rodríguez de la Vega for insightful comments. The computations were 950 performed at the Vital-IT (http://www.vital-it.ch) Center for high-performance computing of the 951 SIB Swiss Institute of Bioinformatics. This work was supported by the NIH grant R15GM119092 952 to M. E. H., and the Louis D. Foundation award to T. G. 885 S7 Fig. Dotplot of oriented differences of transposable element (TE) insertions and 886 differential gene expression between alleles of Microbotryum lychnidis-dioicae. 953 954 References 955 1. 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It is made available The copyright holder for this preprint (which was not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 1228 Table 1. Output of a reduced best-fit generalized linear model (GLM) with differential gene 1229 expression (\|Log2(a1/a2)\|) as the response variable and as predictable variables genomic 1230 compartment and various degeneration traits, i.e., non-synonymous mutation rate (dN), 1231 transposable element (TE) insertions, protein length, intron content and GC content. P 1232 values <0.05 are in bold. NA: not applicable. Mol Biol Evol. 2015;32: 1581–1597. doi:10.1093/molbev/msv049 1233 GLM model output parameter Predictable variables and interaction terms Wald Chi- Square Degree of freedom (df) P value Regression coefficient (Intercept) 496.78 1 <0.001 NA Compartment 20.151 3 <0.001 NA dN 13.21 1 <0.001 5.081 TE insertions 8.405 1 0.004 0.044 Protein length 0.41 1 0.522 10.612 Intron content 10.209 1 0.001 0.768 GC content 4.233 1 0.040 0.499 Compartment Protein length 24.662 3 <0.001 NA dN * Protein length 13.36 1 <0.001 -50.726 TE insertions * Protein Length 8.398 1 0.004 -0.37 GC content * Protein length 10.801 1 0.001 -3.962 1234 GLM model output parameter Predictable variables and interaction terms Wald Chi- Square Degree of freedom (df) P value Regression coefficient (Intercept) 496.78 1 <0.001 NA Compartment 20.151 3 <0.001 NA dN 13.21 1 <0.001 5.081 TE insertions 8.405 1 0.004 0.044 Protein length 0.41 1 0.522 10.612 Intron content 10.209 1 0.001 0.768 GC content 4.233 1 0.040 0.499 Compartment * Protein length 24.662 3 <0.001 NA dN * Protein length 13.36 1 <0.001 -50.726 TE insertions * Protein Length 8.398 1 0.004 -0.37 GC content * Protein length 10.801 1 0.001 -3.962 43 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint 1237 Table 2. Numbers and percentages of genes with differential expression (DE) on the 1238 different genomic compartments on mating-type chromosomes and autosomes, and Fisher’s 1239 exact test for even distribution between DE genes on autosomes versus other genomic 1240 compartments, including pseudo-autosomal regions (PARs), young evolutionary strata 1241 (previously identified red and green strata; [43]) and old evolutionary strata (blue, purple, 1242 orange and black strata; [43]). P values <0.05 are in bold. NA: not applicable. Mol Biol Evol. 2015;32: 1581–1597. doi:10.1093/molbev/msv049 1243 Autosomes PAR Young strata Old Strata DE gene number 507 12 1 74 Total number 8207 114 29 198 Percentage 6.18% 10.53% 3.45% 37.37% Fisher's exact test (P value) NA 0.085 1 2.20E-16 1237 Table 2. Numbers and percentages of genes with differential expression (DE) on the 1238 different genomic compartments on mating-type chromosomes and autosomes, and Fisher’s 1239 exact test for even distribution between DE genes on autosomes versus other genomic 1240 compartments, including pseudo-autosomal regions (PARs), young evolutionary strata 1241 (previously identified red and green strata; [43]) and old evolutionary strata (blue, purple, 1242 orange and black strata; [43]). P values <0.05 are in bold. NA: not applicable. 1243 1237 Table 2. Numbers and percentages of genes with differential expression (DE) on the 1238 different genomic compartments on mating-type chromosomes and autosomes, and Fisher’s 1239 exact test for even distribution between DE genes on autosomes versus other genomic 1240 compartments, including pseudo-autosomal regions (PARs), young evolutionary strata 1241 (previously identified red and green strata; [43]) and old evolutionary strata (blue, purple, 1242 orange and black strata; [43]). P values <0.05 are in bold. NA: not applicable. 1243 Autosomes PAR Young strata Old Strata DE gene number 507 12 1 74 Total number 8207 114 29 198 Percentage 6.18% 10.53% 3.45% 37.37% Fisher's exact test (P value) NA 0.085 1 2.20E-16 44 . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under a 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 not this version posted May 2, 2019. ; doi.org/10.1101/626291 . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Mol Biol Evol. 2015;32: 1581–1597. doi:10.1093/molbev/msv049 It is made available The copyright holder for this preprint (which was not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint . CC-BY 4.0 International license under 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 not this version posted May 2, 2019. ; https://doi.org/10.1101/626291 doi: bioRxiv preprint
W4242410222.txt	https://www.qeios.com/read/F7BBQM/pdf	en		C-Circle	Definitions	2,020	cc-by	74	Qeios · Definition, February 2, 2020 Ope n Pe e r Re v ie w on Qe ios C-Circle National Cancer Institute Source National Cancer Institute. C-Circle. NCI T hesaurus. Code C105650. A stretch of telomeric DNA where the normally guanine-rich (T T AGGG)n overhanging sequence has gaps while the cytosine-rich (AAT CCC)n opposite strand remains intact. T he resulting partially single-stranded telomere can adopt a circular confirmation. Qeios ID: F7BBQM · https://doi.org/10.32388/F7BBQM 1/1
https://openalex.org/W4229365391	https://www.frontiersin.org/articles/10.3389/fvets.2022.870009/pdf	English	null	Expression and Immunogenicity of Recombinant African Swine Fever Virus Proteins Using the Semliki Forest Virus	Frontiers in veterinary science	2,022	cc-by	7,541	Expression and Immunogenicity of Recombinant African Swine Fever Virus Proteins Using the Semliki Forest Virus Niran Fang 1,2, Bin Yang 1, Ting Xu 2, Yanpeng Li 3, Huimin Li 4, Hanghui Zheng 1, Aiguo Zhang 5 and Ruiai Chen 1,2,3* 1 Zhaoqing Institute of Biotechnology Co. Ltd., Zhaoqing, China, 2 College of Veterinary Medicine, South China Agricultural University, Guangzhou, China, 3 Zhaoqing DaHuaNong Biology Medicine Co. Ltd., Zhaoqing, China, 4 Jinggangshan University, Jinggangshan, China, 5 Henan University of Animal Husbandry and Economy, Zhengzhou, China African swine fever virus (ASFV) is a large DNA virus belonging to the Asfarviridae family that damages the immune system of pigs, resulting in the death or slaughter of millions of animals worldwide. Recent modern techniques in ASFV vaccination have highlighted the potential of viral replicon particles (RPs), which can efﬁciently express foreign proteins and induce robust cellular and humoral immune responses compared with the existing vaccines. In this study, we established a Semliki Forest virus (SFV) vector by producing replication-defective viral particles. This vector was used to deliver RPs expressing ASFV antigens. SFV-RPs expressing ASFV p32 (SFV-p32) and p54 (SFV-p54) were tested in baby hamster kidney (BHK-21) cells. Proteins expression was evaluated via western blotting and indirect immunoﬂuorescence, while immunogenicity was evaluated in BALB/c mice. The resulting RPs exhibited high levels of protein expression and elicited robust humoral and cellular immune responses. INTRODUCTION Specialty section: This article was submitted to Veterinary Infectious Diseases, a section of the journal Frontiers in Veterinary Science African swine fever virus (ASFV) infection aﬀects domestic pigs and causes an acute, febrile, and highly infectious disease. No eﬀective vaccine against ASFV exists to date, and once the infection is detected, the animals must be slaughtered, imposing a large economic burden on the swine industry. ASFV was ﬁrst observed in Europe in 1957 and spread in Portugal in 1960 wherefrom the virus became established in the Iberian Peninsula and caused outbreaks across Western Europe, the Caribbean, and Brazil. Although ASFV was almost eradicated outside of Africa by the mid-1990s, in 2007 it was detected in Georgia wherefrom it spread throughout Eastern Asia and Eastern Europe, causing large-scale global economic losses. Before 2018, there were no reported cases of ASFV in China, but the government continued to monitor for the disease. In 2013, China joined the ASFV global alliance to strengthen the prevention and control of the disease. This initiated fundamental research eﬀorts with the ultimate goal of developing eﬀective vaccines against ASFV. Received: 05 February 2022 Accepted: 04 April 2022 Published: 09 May 2022 ORIGINAL RESEARCH published: 09 May 2022 doi: 10.3389/fvets.2022.870009 ORIGINAL RESEARCH published: 09 May 2022 doi: 10.3389/fvets.2022.870009 Keywords: African swine fever virus, Semliki Forest virus, replication-defective viral particles, immunogenicity, cellular immune response Edited by: Heather Lynne Wilson, University of Saskatchewan, Canada Reviewed by: Qin Zhao, Northwest A&F University, China Kenneth Lundstrom, PanTherapeutics, Switzerland Correspondence: Ruiai Chen chensa727@vip.126.com Edited by: Heather Lynne Wilson, University of Saskatchewan, Canada y Reviewed by: Qin Zhao, Northwest A&F University, China Kenneth Lundstrom, PanTherapeutics, Switzerland Reviewed by: Qin Zhao, Qin Zhao, Northwest A&F University, China Kenneth Lundstrom, PanTherapeutics, Switzerland Keywords: African swine fever virus, Semliki Forest virus, replication-defective viral particles, immunogenicity, cellular immune response Correspondence: Ruiai Chen chensa727@vip.126.com Citation: Fang N, Yang B, Xu T, Li Y, Li H, Zheng H, Zhang A and Chen R (2022) Expression and Immunogenicity of Recombinant African Swine Fever Virus Proteins Using the Semliki Forest Virus. Front. Vet. Sci. 9:870009. doi: 10.3389/fvets.2022.870009 In the earliest ASFV studies, attenuated viruses were found to protect from the disease, but their eﬃcacy in the ﬁeld was not adequate. In recent years, there has been a rapid drive to develop platforms for the expression of a variety of heterologous proteins in animal cells, and there are May 2022 \| Volume 9 \| Article 870009 Frontiers in Veterinary Science \| www.frontiersin.org Expressing ASFV Proteins Using SFV Fang et al. now many methods and vector expression platforms available for the expression of diﬀerent antigens. Vector expression platforms based on nucleic acids represent important new approaches for vaccine development, and DNA vaccines have the added advantage of expressing heterologous DNA sequences (1, 2). Recent improvements in vector systems have facilitated the development of virus-based mRNA vaccines (2, 3). RNA replicon vaccines are based on a vector platform derived from the Semliki Forest virus (SFV), which can replicate autonomously and induce systemic, cellular, and mucosal immunity (4). Murgia et al. (5) has described an alphavirus vector carrying replicon particle (RP)-30 and RP-54, using Venezuelan equine encephalitis virus (VEEV) rather than SFV, and with Vero cells rather than BHK-21 cells, which helped establish humoral immunity in swine. alphaviruses may infect many diﬀerent cell lines, indicating that cells, such as Chinese hamster ovary, primary chicken embryo ﬁbroblast, duck embryo ﬁbroblast, or human embryonic kidney (HEK) 293 (or 293T) cells, which can yield diﬀerent amounts of RPs, may be used in commercial vaccine production (4). Here we aimed to establish an SFV vector by producing replication-defective viral particles and utilized it to deliver RPs expressing ASFV antigens. SFV-RPs expressing ASFV p32 (SFV-p32) and p54 (SFV-p54) were tested in baby hamster kidney (BHK-21) cells. Proteins expression was evaluated via western blotting and indirect immunoﬂuorescence, while immunogenicity was evaluated in BALB/c mice. Construction of SFV Vector Plasmids Construction of SFV Vector Plasmids On an SFV vector platform, using the pSFVCs-LacZ containing the non-structural genome of SFV (Figure 1). The LacZ- encoding gene was removed using a BamHI restriction endonuclease to insert a heterologous gene into pSFVCs- LacZ. Enhanced green ﬂuorescent protein (EGFP)- and ASFV p32- or p54-encoding sequences were inserted into the vector plasmid to establish pSFVCs-EGFP, pSFV-EGFP, pSFV-p32, and pSFV-p54 (Figure 1) based on GenBank sequences for p32 and p54 (accession no. NP_042786 and NP_042818). The above heterologous proteins were inserted downstream of the 26S promoter. In a previous study, an SFV vector coding for foreign proteins, as well as a pSFV-helper1, was used to encode the SFV structural proteins and transcribe them into mRNA in vitro, these recombinant RNAs were electroporated into baby hamster kidney (BHK)-21 cells to express high levels of heterologous proteins (10). In the SFV replicon vector, SFV structural genes can be replaced with heterologous genes of interest, generating a self replicating RNA that can eﬃciently express the heterologous genes. Replicon RNA supplies the non-structural genes, which contain the packaging signal. The viral structural genes are provided by the helper RNA, which lacks part of the non- structural genes, including the packaging signal. Therefore, only the replicon RNA retains the packaging signal, and it is packaged into RPs through the expression of the structural genes supplied by the helper RNA. The resulting RPs can infect mammalian cells but only undergo one round of replication (10, 11). The alphavirus replicon system has been used to develop eﬀective inﬂuenza RP vaccines, which have been evaluated in chickens, pigs, and humans and elicited complete protection against lethal avian inﬂuenza (4, 12). Compared to conventional DNA vaccines, the replicon RNA system is self-amplifying, boosting cellular and humoral responses (4). The co-expression of genes encoding Ebola and Lassa virus-speciﬁc glycoproteins was used to produce an RP vaccine that protects guinea pigs against Ebola and Lassa viral challenges, indicating that RP vaccines may protect against multiple diseases (13). Another advantage of RP vaccines is that METHODS AND MATERIALS cDNA coding for a variety of viral proteins involved in ASFV infection, such as p32 and p54, can be cloned into the SFV vector. The p32 protein, which has a molecular weight of ∼30 kDa and is expressed in the early stages of viral infection, has a good level of antigenicity and can induce a robust immune response. A p32-speciﬁc blocking antibody inhibits the internalization of the virus, indicating that this viral component is also involved in the viral entry into cells (6). p54 is a type I transmembrane protein that crosses the inner envelope of the virus particle and forms a homodimer via a cysteine bridge (7). p54 helps the virus to bind to target cells (8) and stimulates the host to produce p54-speciﬁc antibodies. In ASFV-infected cells, p54 binds to the endoplasmic reticulum and uses its membrane to form the viral particle envelope. A special cross-reaction between p54 and the dynein light chain (DLC8) mediates intracellular transport of the virus, suggesting that p54 plays an important role in viral uptake and processing (9). Plasmids, Cells, and Animals pSFVCs-LacZ and pSFV-helper1 (plasmid numbers #92076 and #92073, respectively) were purchased from Addgene (Watertown, Massachusetts, USA). Fifteen 5-week-old SPF BALB/c mice were purchased from the Animal Experiment Center of Southern Medical University (Guangzhou, People’s Republic of China), and raised in individually ventilated cages within the SPF animal house of Zhaoqing DaHuaNong Biology Medicine Co. Ltd. (Zhaoqing, People’s Republic of China). The animal study was reviewed and approved by Animal Welfare and Ethical Censor Committee. Western Blotting g BHK-21 cells infected with SFV-p32 and SFV-p54 (RPs) for 36 h were collected and lysed on ice for 5 min. The lysate was mixed with loading buﬀer (5×) and boiled for 10 min. The mixture was loaded onto 12% polyacrylamide gels, electrophoresed for 120 min at 80 V, then transferred to a polyvinylidene diﬂuoride membrane via electroblotting for 90 min. The membranes were blocked with 5% BSA for 1 h at room temperature, then washed three times with PBST for 10 min each. They were incubated overnight at 4◦C with an anti-ASFV serum antibody diluted at 1:1,000, then washed and incubated for 1 h at 37◦C with a secondary goat anti-swine IgG (H+L) conjugated with horseradish peroxidase (ABclonal) diluted at 1:5,000. Finally, the membranes were washed three times and visualized using a FUSION FX EDGE SPECTRA imaging system (VILBER, Collégien, France). IFA SFV-p32 and SFV-p54 viral particles were collected every 6 h starting from 24 h after co-electroporation. Cells were cultured in plates with particles for 60 h and washed with phosphate- buﬀered saline containing Tween (PBST) three times, ﬁxed with formaldehyde for 10 min at 4◦C, then washed with PBST and permeabilized with 0.1% Triton X-100. They were then incubated for 2 h at 37◦C with 5% bovine serum albumin (BSA), followed by washing and 1 h incubation at 37◦C with an anti-ASFV serum antibody (Zhaoqing DaHuaNong Biology Medicine Co. Ltd., Zhaoqing, People’s Republic of China) diluted at 1:2500. The samples were then washed again and incubated for 1 h at 37◦C with IFTC-conjugated AﬃniPure Goat Anti-Swine IgG (H + L) (Jackson ImmunoResearch) diluted at 1:600 as the secondary antibody. They were then washed three times and observed under a ﬂuorescence microscope. To avoid interference from the fusion capsid protein during the speciﬁc expression of the target protein in the follow-up animal experiments, we removed the viral capsid protein and constructed the replicon plasmid pSFV-EGFP via homologous recombination using the pEASY R⃝-Basic Seamless Cloning and Assembly Kit (TransGen Biotech, Beijing, China). SFV-EGFP RP was then obtained using the above-described method for SFVCs- RP. Fluorescence signal intensity was compared with SFVCs-RPs 24 h after infection. SFVCs-RP Generation To obtain SFVCs-RP, the SFV expression vector plasmids described above were linearized using SpeI, then transcribed in vitro using the mMESSAGE mMACHINETM SP6 (Thermo Fisher Scientiﬁc, Waltham, MA, USA). pSFVCs-LacZ RNA and pSFVCs-EGFP RNA were electroporated into BHK-21 cells with pSFV-helper1 RNA at 100 V for 25 ms, respectively. BHK-21 cells were cultured in 1% fetal bovine serum (FBS)-containing Dulbecco’s Modiﬁed Eagle Medium (Gibco) at 37◦C until the cells adhered, then the medium was changed to serum-free medium. The viral particles were collected from the supernatant by centrifugation 36 h after co-electroporation, then ﬁltered through a 0.22 µm Millex-GP ﬁlter to generate SFVCs-LacZ and SFVCs-EGFP RPs. β-galactosidase was detected using in situ β-galactosidase Staining Kit (Beyotime, Shanghai, China), and EGFP was detected using a ﬂuorescence microscope. Frontiers in Veterinary Science \| www.frontiersin.org May 2022 \| Volume 9 \| Article 870009 2 Fang et al. Expressing ASFV Proteins Using SFV FIGURE 1 \| SFV expression vector system. We used the plasmid pSFVCs-LacZ, which contains the non-structural genome of SFV. To insert heterologous genes into the pSFVCs-LacZ plasmid, LacZ was removed via BamHI restriction endonuclease, EGFP- and p32-, p54-coding ASFV genes were inserted into the vector plasmid. We obtained pSFVCs-EGFP, pSFV-EGFP, pSFV-p32, and pSFV-p54. FIGURE 1 \| SFV expression vector system. We used the plasmid pSFVCs-LacZ, which contains the non-structural genome of SFV. To insert heterologous genes into the pSFVCs-LacZ plasmid, LacZ was removed via BamHI restriction endonuclease, EGFP- and p32-, p54-coding ASFV genes were inserted into the vector plasmid. We obtained pSFVCs-EGFP, pSFV-EGFP, pSFV-p32, and pSFV-p54. ASFV SFV-RP Generation To develop the plasmids expressing ASFV p32 and p54, we constructed the replicon plasmids pSFV-p32 and pSFV-p54 via homologous recombination using the pEASY R⃝-Basic Seamless Cloning and Assembly Kit and pSFV-EGFP as described above. pSFV-p32 and pSFV-p54 were linearized using SpeI and SpaI, respectively, and transcribed in vitro using the mMESSAGE mMACHINETM SP6 (Thermo Fisher Scientiﬁc). They were then electroporated into BHK-21 cells with the helper RNA at 100 V for 25 ms. SFV-p32 and SFV-p54 RPs were collected using the same method described above. To develop the plasmids expressing ASFV p32 and p54, we constructed the replicon plasmids pSFV-p32 and pSFV-p54 via homologous recombination using the pEASY R⃝-Basic Seamless Cloning and Assembly Kit and pSFV-EGFP as described above. pSFV-p32 and pSFV-p54 were linearized using SpeI and SpaI, respectively, and transcribed in vitro using the mMESSAGE mMACHINETM SP6 (Thermo Fisher Scientiﬁc). They were then electroporated into BHK-21 cells with the helper RNA at 100 V for 25 ms. SFV-p32 and SFV-p54 RPs were collected using the same method described above. ASFV SFV-RP Viral Titers and Detection of Infected BHK-21 Cells The viral supernatant was collected by centrifugation, every 6 h starting from 24 h after the replicon RNA electroporation into BHK-21 cells with helper RNA. Then, 100 µL dilutions of viral samples were added to 96-well plates containing BHK-21 cell monolayers. Following 60 h incubation at 37◦C under 5% CO2, viruses were detected based on the cytopathogenic eﬀect (CPE) and immunoﬂuorescence (IFA). ASFV SFV-RP titration was calculated using the Spearman-Karber method. May 2022 \| Volume 9 \| Article 870009 Frontiers in Veterinary Science \| www.frontiersin.org 3 Expressing ASFV Proteins Using SFV Fang et al. ASFV SFV-RP Immunization ASFV SFV-RP Immunization same dose (0.2 mL) [The SFV-p32 RP titer in BHK- 21 cells was 103.0 median tissue culture infectious dose (TCID50)/0.1 mL, and the SFV-p54 RPs titer was 103.5 TCID50/0.1 mL]. Blood samples were collected from the canthus before the ﬁrst immunization and 1 week after each Five-week-old BALB/c mice were immunized three times via intramuscular injections of SFV-32 RPs, SFV- 54 RPs, or PBS in the lateral thigh (n = 5/group). Each mouse was immunized at 2-week intervals at the pression of heterologous proteins in BHK-21 cells with SFVCs-RP infected. (A) β-galactosidase expression (×200). (B) EGFP expression (×200). (C) ted with SFVCs-EGFP RPs (×400) for 24 h. CPE was evaluated. nary Science \| www frontiersin org 4 May 2022 \| Volume 9 \| Article 870009 \| E i f h t l t i i BHK 21 ll ith SFVC RP i f t d (A) β l t id i ( 200) (B) EGFP i ( 200) (C) FIGURE 2 \| Expression of heterologous proteins in BHK-21 cells with SFVCs-RP infected. (A) β-galactosidase expression (×200). (B) EGFP expression (×200). (C) Cells were infected with SFVCs-EGFP RPs (×400) for 24 h. CPE was evaluated. FIGURE 2 \| Expression of heterologous proteins in BHK-21 cells with SFVCs-RP infected. (A) β-galactosidase expression (×200). (B) EGFP expression (×200). (C) Cells were infected with SFVCs-EGFP RPs (×400) for 24 h. CPE was evaluated. May 2022 \| Volume 9 \| Article 870009 Frontiers in Veterinary Science \| www.frontiersin.org 4 Expressing ASFV Proteins Using SFV Fang et al. immunization and stored at −80◦C. Mice were monitored and weighed daily. (Dakewe Biotech Co., Ltd., Shenzhen, China). The synthesized polypeptides were p32-1, DFNKV IRAHN FIQTI; p32-2, IRAHN FIQTI HGTPL; p54, ENLRQ RNTYT HKDLE (Sangon Bioengineering Co., Ltd., Shanghai, China). ASFV SFV-RP Viral Titers and Detection of Infected BHK-21 Cells Each group was assigned a positive control (10 µL PHA with resuspended cells), a negative control (without stimulant but with resuspended cells), and a background negative control (only the culture medium). Mice immunized with SFV-p32 RP (with stimulant polypeptides p32-1 and p32-2, the ﬁnal concentration is 60 µg/mL), and SFV- p54 RP (with stimulant polypeptide p54, the ﬁnal concentration is 60 µg/mL) (ﬁve mice each) were used. An immunospot image analyzer from the Dakewe Biotech Co., Ltd., Shenzhen, China was used. (Dakewe Biotech Co., Ltd., Shenzhen, China). The synthesized polypeptides were p32-1, DFNKV IRAHN FIQTI; p32-2, IRAHN FIQTI HGTPL; p54, ENLRQ RNTYT HKDLE (Sangon Bioengineering Co., Ltd., Shanghai, China). Each group was assigned a positive control (10 µL PHA with resuspended cells), a negative control (without stimulant but with resuspended cells), and a background negative control (only the culture medium). Mice immunized with SFV-p32 RP (with stimulant polypeptides p32-1 and p32-2, the ﬁnal concentration is 60 µg/mL), and SFV- p54 RP (with stimulant polypeptide p54, the ﬁnal concentration is 60 µg/mL) (ﬁve mice each) were used. An immunospot image analyzer from the Dakewe Biotech Co., Ltd., Shenzhen, China was used. Statistical Analysis y g ELISA for detecting anti-p54 antibody IgG was performed using AsurDxTM ASFV p54 Antibody Test Kit (BioStone Animal Health, Dallas, TX, USA). The microplates were coated with the ASFV p54 antigen. The serum samples were added and the microplates were washed ﬁve times. An anti-ASF monoclonal antibody (mAb)-biotin corjugate and HRP-tagged streptavidin were added. After incubation for 30 min at 37◦C, the plate was washed and the substrate was added. The color change was observed and the reaction was terminated, which caused another color change (blue to yellow). The speciﬁc absorption peak at 450 nm was measured using a Spectrophotometer Multiskan Skyhigh (Thermo Scientiﬁc). Three parallel experiments were performed for each sample. SPSS software (IBM SPSS Statistics for Windows, Version 20.0, IBM Corporation, Armonk, NY, USA) was used for statistical analysis. GraphPad Prism (version 9.2, GraphPad Software Inc., CA, USA) was used to analyze data. Signiﬁcance levels were represented by P-values (ns, non-signiﬁcant; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001). Enzyme-Linked Immune Absorbent Spot Assay y Mice were euthanized 2 weeks after the third immunization. Lymphocytes were aseptically isolated from the spleens for cellular immunity experiments. Interferon (IFN)-γ and interleukin (IL)-4 expressions were measured using anti- mouse IFN-γ- and anti-mouse IL-4-precoated ELISPOT kits Enzyme-Linked Immunosorbent Assay Enzyme Linked Immunosorbent Assay ELISA for detecting anti-p32 antibody IgG was performed with the serum samples described above using the ASFV p32 Antibodies Test Kit (sandwich ELISA) according to the manufacturer’s instructions (Zhaoqing DaHuaNong Biology Medicine Co. Ltd., Zhaoqing, China). Microplates were coated with the ASFV p32 antigen and the serum samples were added, followed by HRP-labeled ASFV antigen. After washing the plate, the substrate was added, and the blue color was observed. The reaction was then terminated, giving a yellow color. The speciﬁc absorption peak at 450 nm was observed using a Spectrophotometer Multiskan Skyhigh (Thermo Scientiﬁc). SFVCs-RP Production The SFVCs-LacZ and SFVCs-EGFP RPs were successfully packaged. The blue color due to β-galactosidase was observed in 80–90% of cells, while few control cells were stained (Figure 2A). EGFP signal was detected using a ﬂuorescence microscope, and more than half of the cells emitted green ﬂuorescence (Figure 2B). Cells infected for 24 h with SFVCs-LacZ and SFVCs- EGFP RPs showed evident CPE (Figure 2C). SFV-RP Production SFV-EGFP RPs were successfully packaged. BHK-21 cells were infected with SFV-EGFP RPs and SFVCs-EGFP RPs for 24 h, FIGURE 3 \| Fluorescence signal intensity was compared after removing the viral capsid protein. BHK-21 cells were infected with SFVCs-EGFP and SFV-EGFP RPs for 24 h, and ﬂuorescence signal intensities were compared (×200). FIGURE 3 \| Fluorescence signal intensity was compared after removing the viral capsid protein. BHK-21 cells were infected with SFVCs-EGFP and SFV-EGFP RPs for 24 h, and ﬂuorescence signal intensities were compared (×200). Frontiers in Veterinary Science \| www.frontiersin.org May 2022 \| Volume 9 \| Article 870009 5 Fang et al. Expressing ASFV Proteins Using SFV FIGURE 4 \| IFA and viral growth of ASFV SFV-RPs. (A) IFA of ASFV SFV-RPs infected BHK-21 cells after 60 h (×400). (B) Viral growth in ASFV SFV-RPs-infected viral growth of ASFV SFV-RPs. (A) IFA of ASFV SFV-RPs infected BHK-21 cells after 60 h (×400). (B) Viral growth in ASFV SFV-RPs-infec Science \| www.frontiersin.org 6 May 2022 \| Volume 9 \| Artic FIGURE 4 \| IFA and viral growth of ASFV SFV-RPs. (A) IFA of ASFV SFV-RPs infected BHK-21 cells after 60 h (×400). (B) Viral growth in ASFV SFV-RPs-infected BHK-21 cells. FIGURE 4 \| IFA and viral growth of ASFV SFV-RPs. (A) IFA of ASFV SFV-RPs infected BHK-21 cells after 60 h (×400). (B) Viral growth in ASFV SFV-RPs-infected BHK-21 cells. Frontiers in Veterinary Science \| www.frontiersin.org May 2022 \| Volume 9 \| Article 870009 6 Expressing ASFV Proteins Using SFV Fang et al. and ﬂuorescence signal intensities showed that the expression of EGFP in the former was not decreased compared with the latter (Figure 3), conﬁrming that the SFV vector could eﬃciently express foreign genes. FIGURE 5 \| Western blotting of ASFV SFV-RPs. ASFV p32 expression in SFV-p32 RP-infected BHK-21 cells (Lane 1), ASFV p54 expression in SFV-p54 RP-infected BHK-21 cells (Lane 2). ELISA Results Mice were immunized three times with SFV-p32 and SFV- p54 RPs. Serum collected 1 week after the second and third immunizations were tested using ELISA. As shown in Figure 7, SFV-p32 RP-infected mice had higher antibody levels after the third immunization compared to the second immunization. Similarly, SFV-p54 RP-infected mice produced IgG antibodies after the second and third immunizations (Figure 8). The results indicated that three immunizations induced robust antibody production. FIGURE 6 \| Comparative weight changes of immunized and non-immunized (PBS group) mice monitored for 35 days after the ﬁrst immunization. ns, non-signiﬁcant (P > 0.05). ASFV SFV-RP Western Blotting FIGURE 5 \| Western blotting of ASFV SFV-RPs. ASFV p32 expression in SFV-p32 RP-infected BHK-21 cells (Lane 1), ASFV p54 expression in SFV-p54 RP-infected BHK-21 cells (Lane 2). To test heterologous p32 and p54 protein expression, cells were lysed and analyzed via western blotting. The bands at 30 kDa conﬁrmed successful packaging of the SFV-p32 and SFV-p54 RPs and eﬃcient infection of BHK-21 cells (Figure 5). FIGURE 5 \| Western blotting of ASFV SFV-RPs. ASFV p32 expression in SFV-p32 RP-infected BHK-21 cells (Lane 1), ASFV p54 expression in SFV-p54 RP-infected BHK-21 cells (Lane 2). FIGURE 6 \| Comparative weight changes of immunized and non-immunized (PBS group) mice monitored for 35 days after the ﬁrst immunization. ns, non-signiﬁcant (P > 0.05). Mouse Weight Changes After Immunization As shown in Figure 6, the weight of the immunized group decreased slightly and began to increase gradually again 1 week later, while ﬁnally there was no diﬀerence in the weight and state of the mice compared to the PBS group after completing the third immunization. The result indicated there had no signiﬁcant eﬀect on the growth of mice after immunization with ASFV SFV-RPs. ASFV SFV-RP Production EGFP in the pSFV-EGFP was replaced with ASFV antigens p32 and p54 and electroporated into BHK-21 cells. SFV-p32 and SFV-p54 RPs were successfully packaged. IFA of Infected BHK-21 Cells and Viral Growth of ASFV SFV-RPs BHK-21 cells infected with SFV-p32 and SFV-p54 RPs for 60 h were detected using IFA. The results showed that infected cells exhibited green ﬂuorescence and CPE at a suitable viral titer (Figure 4A). The titers of SFV-p32 and SFV-p54 RPs in BHK-21 cells continuously increased from 24 to 36 h post-infection and peaked (103.0 TCID50/0.1 mL,103.5 TCID50/0.1 mL, respectively) 36 h post-infection (Figure 4B). The subsequent decrease was related to cell death. ELISPOT Results Our results show that replication-defective viral particles can be produced by packaging SFV replicon RNA and ﬁrst- generation helper RNA. The replication-defective viral particles were generated through ampliﬁcation and recombination within ∼24 h, with viral production peaking after 36 h. Due to the deletion of key sections of the SFV genome, the Two weeks after the third immunization, IFN-γ and IL- 4 expression were detected in lymphocytes using ELISPOT (Figure 9). The results showed that SFV-p32 and -p54 RP- injected mice had higher expression of both cytokines compared to the negative and background controls. May 2022 \| Volume 9 \| Article 870009 Frontiers in Veterinary Science \| www.frontiersin.org 7 Fang et al. Expressing ASFV Proteins Using SFV FIGURE 7 \| ELISA was used to detect anti-p32 IgG in each group. Cut off calculation: cut off = average value of negative control + 0.15. When the negative control value was ≤0.05, it was calculated as 0.05. Positive judgment: samples with OD value ≥cut off were judged as positive. Negative determination: samples with OD value < cut off were judged as negative. SFV-p32 RP-immunized mice are numbered 1–5, which are consistent with the numbers assigned to mice detected by pre-immune serum, the second immunization and the third immunization in the FIGURE. FIGURE 7 \| ELISA was used to detect anti-p32 IgG in each group. Cut off calculation: cut off = average value of negative control + 0.15. When the negative control value was ≤0.05, it was calculated as 0.05. Positive judgment: samples with OD value ≥cut off were judged as positive. Negative determination: samples with OD value < cut off were judged as negative. SFV-p32 RP-immunized mice are numbered 1–5, which are consistent with the numbers assigned to mice detected by pre-immune serum, the second immunization and the third immunization in the FIGURE. resulting replication-defective viral particles were only capable of performing one round of replication in infected cells (10). We initially produced SFVCs-EGFP RPs and found that over 50% of the cells expressed the EGFP. animal experiments. There is a need for safe and reproducible methods for vaccine development. In this study, we ﬁrst used 1% FBS in the medium after co-electroporation. Once the majority of the BHK-21 had adhered, they were cultured in a serum- free medium (growth kinetic parameters including cell density, viability, maximum growth density, and viral titer are not shown in this paper). ELISPOT Results We found that this approach was helpful for the RP production, and even subsequent animal experiments. Other researchers have reported the merits of using serum-free media in the production of vaccines and other biological products (2, 17). In the future, we will optimize our culture media to support viral proliferation better. p Sjöberg et al. (14) indicated that the ﬁrst 102 bases of the viral capsid gene play a role as a translational enhancer, and that SFV vectors that incorporate this RNA have improved heterologous protein synthesis. However, in the present study, the expression of the target protein was not reduced after the capsid-coding section was removed, which is diﬀerent from previous results (14). We believe that the diﬀerence in results may be explained in several ways. Viral structural protein synthesis might be inhibited after SFV-infection so that the translation enhancer does not play a corresponding role. It was reported that the synthesis of viral structural proteins in extensive amounts upon Sindbis- infection engages <25% of the synthesizing capacity used by the cell for steady state protein production and host protein synthesis stops almost completely 5 h after infection (14, 15). Furthermore, it was reported that a mildly acidic pH may lead to the hydrolysis of the SFV capsid (16). In addition, it is unclear which C’- gene fragment enhances translation initiation and whether the eﬀect results from an adaptation to the speciﬁc environment of SFV-infected cells (14). Further research is required to answer this question. In any case, to prevent the interference of the capsid protein on the speciﬁc expression of the target protein in subsequent animal experiments, the fused capsid protein should be removed. Therefore, we produced SFV- EGFP RPs. Finally, we produced ASFV SFV-RPs containing p32- and p54-encoding segments, which showed high levels of protein expression, implying that we were successful in establishing the SFV vector platform. In the present study, mice were immunized with SFV RPs containing ASFV antigens, which resulted in the robust induction of humoral and cell-mediated immune responses. Almost all mice inoculated with SFV-p32 and SFV-p54 (RPs) produced IgGs against the expressed antigen after the third immunization. Moreover, the levels of ASFV p32-speciﬁc IgG and ASFV p54-speciﬁc IgG remained high 5 months post immunization. The ELISPOT results showed that all immunized mice produced IFN-γ and IL-4, which indicates that the SFV- based vector vaccine induced a robust cell-mediated response. Frontiers in Veterinary Science \| www.frontiersin.org ELISPOT Results Although one mouse did not produce IgG antibodies after the third round of immunization with SFV-p54 RPs, it still exhibited a cellular immune response [Figure 9 (c-3, d-3)]. This might be because ASFV SFV-RPs are more eﬀective at inducing cellular rather than humoral immune responses. ILs mediate T and B lymphocyte activation, proliferation, and diﬀerentiation during the activation and regulation of the cellular immune response. Our data demonstrate that SFV-p32 and SFV-p54 RPs also elicited high IL-4 expression in mice, an important mediator of the Th1-type response. There is evidence that cytotoxic CD8+ T lymphocytes play an important role in protection against ASFV, The serum contains numerous heterologous proteins, making it diﬃcult to package RPs produced in cells for subsequent May 2022 \| Volume 9 \| Article 870009 8 Fang et al. Expressing ASFV Proteins Using SFV SA was used to detect anti-p54 IgG and calculate the inhibition percentage (IP) in each group. (A) ELISA results for each group. The mean OD of the was ≥0.5. (B) Calculation of the IP of all samples in each group. IP of all samples was calculated as follows: IP (%) = (1–OD450 test sample/Mean control)100. The IP (%) of the positive control was ≥55%. Serum/plasma samples were assessed as follows: IP ≤40% (negative), ASF antibodies he test samples; IP ≥50% ASF antibodies were present in the test samples; 50% > IP ≥40% doubtable, rechecked in 1–2 weeks. SFV-p54 mice are numbered 1–5, which are consistent with the numbers assigned to mice detected by pre-immune serum, the second immunization and the on in the FIGURE. FIGURE 8 \| ELISA was used to detect anti-p54 IgG and calculate the inhibition percentage (IP) in each group. (A) ELISA results for each group. The mean OD of the negative control was ≥0.5. (B) Calculation of the IP of all samples in each group. IP of all samples was calculated as follows: IP (%) = (1–OD450 test sample/Mean OD450 negative control)100. The IP (%) of the positive control was ≥55%. Serum/plasma samples were assessed as follows: IP ≤40% (negative), ASF antibodies were absent in the test samples; IP ≥50% ASF antibodies were present in the test samples; 50% > IP ≥40% doubtable, rechecked in 1–2 weeks. SFV-p54 RP-immunized mice are numbered 1–5, which are consistent with the numbers assigned to mice detected by pre-immune serum, the second immunization and the third immunization in the FIGURE. ELISPOT Results (B) s of SFV-p32 RP-immunized mice. (C) IFN-γ spots of SFV-p54 RP-immunized mice. (D) IL-4 spots of SFV-p54 RP-immunized mice. For all four panels, 1–5 xperimental groups and 6, 7, and 8 are the background control, negative control, and positive control, respectively. The numbers indicate the number of spots. FIGURE 9 \| ELISPOT was used to detect IFN-γ and IL-4 expression in SFV-p32 and -p54 RP-immunized mice. (A) IFN-γ spots of SFV-p32 RP-immunized mice. (B) IL-4 spots of SFV-p32 RP-immunized mice. (C) IFN-γ spots of SFV-p54 RP-immunized mice. (D) IL-4 spots of SFV-p54 RP-immunized mice. For all four panels, 1–5 are the experimental groups and 6, 7, and 8 are the background control, negative control, and positive control, respectively. The numbers indicate the number of spots. FIGURE 9 \| ELISPOT was used to detect IFN-γ and IL-4 expression in SFV-p32 and -p54 RP-immunized mice. (A) IFN-γ spots of SFV-p32 RP-immunized mice. (B) IL-4 spots of SFV-p32 RP-immunized mice. (C) IFN-γ spots of SFV-p54 RP-immunized mice. (D) IL-4 spots of SFV-p54 RP-immunized mice. For all four panels, 1–5 are the experimental groups and 6, 7, and 8 are the background control, negative control, and positive control, respectively. The numbers indicate the number of spots. of replicon particles and the study of cellular immune response, which we believe will be useful for further immunogenicity and protection experiments in pig models. (27). In this study, there was no recovery of infectious virus by blind passaging, IFA, or plaque assay; furthermore, no clinical symptoms of infection were observed in all inoculated animals, which indicate that use of this SFV vector system is valuable to express high levels of recombinant ASFV proteins and study the immunogenicity in pigs. It was reported that SFV-helper1 system is prone to generating the wild-type virus by recombination, however, using the pSFV-helper2 and the split helper can increase the safety of the system by decreasing recombination events (28– 31). Therefore, in the next stage, we will use two other systems and compare them with the SFV-helper1 system to improve applications in clinical trials. In addition, there is still room for further alphaviral vector optimization, such as replacing the SP6 Recent strategies have involved the modiﬁcation of the alphaviral vector 5′ terminal region to speciﬁcally identify cancer cells and induce their death (24). Although such research is still in its early stages, it holds promise for cancer treatment. ELISPOT Results FIGURE 8 \| ELISA was used to detect anti-p54 IgG and calculate the inhibition percentage (IP) in each group. (A) ELISA results for each group. The mean OD of the negative control was ≥0.5. (B) Calculation of the IP of all samples in each group. IP of all samples was calculated as follows: IP (%) = (1–OD450 test sample/Mean OD450 negative control)*100. The IP (%) of the positive control was ≥55%. Serum/plasma samples were assessed as follows: IP ≤40% (negative), ASF antibodies were absent in the test samples; IP ≥50% ASF antibodies were present in the test samples; 50% > IP ≥40% doubtable, rechecked in 1–2 weeks. SFV-p54 RP-immunized mice are numbered 1–5, which are consistent with the numbers assigned to mice detected by pre-immune serum, the second immunization and the third immunization in the FIGURE. and cell-mediated response is critical for clearing the infection (5, 18). chickens, pigs, and humans, and provided complete protection against lethal challenges (4, 12). It was reported that young pigs exhibited a protective humoral immune response following inﬂuenza hemagglutinin (HA) mRNA RPs vaccination (4, 22). In addition, robust humoral and cellular responses were elicited in mice, rabbits, and rhesus macaques which were given HA and neuraminidase (NA) mRNA RPs vaccines (4, 23). Though Murgia et al. (5) already described ASFV RP-30 and RP-54 using VEEV, which were tested for expression levels in Vero cells and immunogenicity in swine, there is a lack of relevant research on the cellular immune response. The present paper is diﬀerent from Murgia et al. (5) as it focused on the design and optimization The SFV-based vector vaccine is a defective single-cycle virus generated by substituting the structural protein-encoding genes from the SFV genome with the ASFV antigens and then electroporation this vector RNA into BHK-21 cells together with helper RNA. Numerous studies have documented the development of DNA and DNA replicon vaccines (19, 20). Although it was reported that DNA vaccines elicit humoral and cellular immune responses, they failed to protect animals from a virulent ASFV challenge (21). Moreover, RNA replicon vaccines based on the alphavirus replicon system have been evaluated in May 2022 \| Volume 9 \| Article 870009 Frontiers in Veterinary Science \| www.frontiersin.org 9 Fang et al. Expressing ASFV Proteins Using SFV 9 \| ELISPOT was used to detect IFN-γ and IL-4 expression in SFV-p32 and -p54 RP-immunized mice. (A) IFN-γ spots of SFV-p32 RP-immunized mice. Frontiers in Veterinary Science \| www.frontiersin.org REFERENCES 11. Berglund P, Sjöberg M, Garoﬀ H, Atkins GJ, Sheahan BJ, Liljeström P, et al. Semliki Forest expression system: production of conditionally infectious recombinant particles. Bio Technol. (1993) 11:916–20. doi: 10.1038/nbt0893-916 1. WolﬀJA, Malone RW, Williams P. Direct gene transfer into mouse muscle in vivo. Science. (1990) 247:1465–8. doi: 10.1126/science.1690918 1. 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Sindbis virus-induced inhibition of protein synthesis is partially reversed by medium containing an elevated potassium concentration. J Gen Virol. (1994) 75 (Pt 2):411–5. doi: 10.1099/0022-1317-75-2-411 6. Gomez-Puertas P, Rodriguez F, Oviedo JM, Brun A, Alonso C, Escribano JM, et al. The African swine fever virus proteins p54 and p30 are involved in two distinct steps of virus attachment and both contribute to the antibody-mediated protective immune response. Virology. (1998) 243:461– 71. doi: 10.1006/viro.1998.9068 16. Schlegel A, Schaller J, Jentsch P, Kempf C. Semliki forest virus core protein fragmentation: its possible role in nucleocapsid disassembly. Biosci Rep. (1993) 13:333–47. FUNDING This study was supported by the Technology Planning Project of Guangdong Province (grant number 2019B020211004). AUTHOR CONTRIBUTIONS We established an SFV vector platform to produce replication- defective viral particles containing ASFV antigens, which induced eﬀective antigen-speciﬁc humoral and cellular immune responses. The resulting RPs exhibited excellent biosafety and elicited a robust immune response due to the replication- defective nature of the viral particles and the use of a self- replicating RNA. However, in this study, we have not included any pig models. Nevertheless, we still believe that this paper will be useful for future immunogenicity research in pig models, providing a reference for the production of RNA replicon vaccines to elicit robust humoral and cellular immune responses. RC conceived the idea. NF did most of the experimental work and wrote the ﬁrst draft of the manuscript. BY, HZ, and TX made the statistical analyses and did parts of the experimental work. YL assembled the data. HL and AZ advised in the process of manuscript writing. All authors contributed to the article and approved the submitted version. ELISPOT Results Modiﬁed viruses can also be used in the research and development of cancer vaccines given the vast number of target proteins (25). Therapeutic alphaviral vector-derived cancer vaccines activate humoral and cellular immunity in the treatment of melanoma, as well as breast, mast cell, and prostate cancers (26). Despite the promise of viral vector vaccines, certain challenges remain May 2022 \| Volume 9 \| Article 870009 10 Expressing ASFV Proteins Using SFV Fang et al. ACKNOWLEDGMENTS The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material. We thank YL for providing the anti-ASFV serum. We thank Guangming Ma and Weifeng Zhong for providing the ASFV antibody test kit. ETHICS STATEMENT promoter with a cytomegalovirus (CMV) promoter, eliminating the in vitro transcription step, and minimizing RNA degradation, all of which could reduce costs and save eﬀort. The animal study was reviewed and approved by Animal Welfare and Ethical Censor Committee. REFERENCES Properties of Sindbis virus vectors produced with a chimeric split helper system. Int J Mol Med. (2005) 15:999– 1003. doi: 10.3892/ijmm.15.6.999 23. Hubby B, Talarico T, Maughan M, Reap EA, Berglund P, Kamrud KI, et al. Development and preclinical evaluation of an alphavirus replicon vaccine for inﬂuenza. Vaccine. (2007) 25:8180–9. doi: 10.1016/j.vaccine.2007.09.038 Conﬂict of Interest: NF, BY, HZ, and RC were employed by Zhaoqing Institute of Biotechnology Co. Ltd. YL and RC were employed by Zhaoqing DaHuaNong Biology Medicine Co. Ltd. 24. Ramachandran M, Yu D, Dyczynski M, Baskaran S, Zhang L, Lulla A, et al. Safe and eﬀective treatment of experimental neuroblastoma and glioblastoma using systemically delivered triple micro RNA- detargeted oncolytic semliki forest virus. Clin Cancer Res. (2017) 23:1519–30. doi: 10.1158/1078-0432.CCR-16-0925 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. 25. Schirmacher V, Forg P, Dalemans W, Chlichlia K, Zeng Y, Fournier P, et al. Intra-pinna anti-tumor vaccination with self-replicating infectious RNA or with DNA encoding a model tumor antigen and a cytokine. Gene Ther. (2000) 7:1137–47. doi: 10.1038/sj.gt.3301220 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 26. Ketola A, Hinkkanen A, Yongabi F, Furu P, Määttä AM, Liimatainen T, et al. Oncolytic Semliki forest virus vector as a novel candidate against unresectable osteosarcoma. Cancer Res. (2008) 68:8342–50. doi: 10.1158/0008-5472.CAN-08-0251 27. Smyth JW, Fleeton MN, Sheahan BJ, Atkins GJ. Treatment of rapidly growing K-BALB and CT26 mouse tumours using Semliki Forest virus and its derived vector. Gene Ther. (2005) 12:147–59. doi: 10.1038/sj.gt.3302390 Copyright © 2022 Fang, Yang, Xu, Li, Li, Zheng, Zhang and Chen. 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. 28. Tubulekas I, Liljeström P. REFERENCES doi: 10.1007/BF01150478 17. Fontana D, Kratje R, Etcheverrigaray M, Prieto C. Immunogenic virus-like particles continuously expressed in mammalian cells as a veterinary rabies vaccine candidate. Vaccine. (2015) 33:4238– 46. doi: 10.1016/j.vaccine.2015.03.088 7. Rodriguez F, Ley V, Gomez-Puertas P. The structural protein p54 is essential for African swine fever virus viability. Virus Res. (1996) 40:161– 7. doi: 10.1016/0168-1702(95)01268-0 18. Takamatsu HH, Denyer MS, Lacasta A, Stirling CM, Argilaguet JM, Netherton CL, et al. Cellular immunity in ASFV responses. Virus Res. 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A new generation of animal cell expression vectors based on the Semliki Forest Virus replicon. Biotechnology. (1991) 9:1356– 61. doi: 10.1038/nbt1291-1356 May 2022 \| Volume 9 \| Article 870009 Frontiers in Veterinary Science \| www.frontiersin.org 11 Fang et al. Expressing ASFV Proteins Using SFV 29. Smerdou C, Liljeström P. Two-helper RNA system for production of recombinant Semliki forest virus particles. J Virol. (1999) 73:1092– 8. doi: 10.1128/JVI.73.2.1092-1098.1999 21. Argilaguet JM, Pérez-Martín E, Nofrarías M, Gallardo C, Accensi F, Lacasta A, et al. DNA vaccination partially protects against African swine fever virus lethal challenge in the absence of antibodies. PLoS ONE. (2012) 7:942. doi: 10.1371/journal.pone.0040942 30. Rayner JO, Dryga SA, Kamrud KI. Alphavirus vectors and vaccination. Rev Med Virol. (2002) 12:279–96. doi: 10.1002/rmv.360 30. Rayner JO, Dryga SA, Kamrud KI. Alphavirus vectors and vaccination. Rev Med Virol. (2002) 12:279–96. doi: 10.1002/rmv.360 22. Erdman MM, Kamrud KI, Harris DL, Smith J. Alphavirus replicon particle vaccines developed for use in humans induce high levels of antibodies to inﬂuenza virus hemagglutinin in swine: proof of concept. Vaccine. (2010) 28:594–6. doi: 10.1016/j.vaccine.2009.10.015 31. Ketola A, Schlesinger S, Wahlfors J. Frontiers in Veterinary Science \| www.frontiersin.org REFERENCES Suppressors of cleavage-site mutations in the p62 envelope protein of Semliki Forest virus reveal dynamics in spike structure function. J Virol. (1998) 72:2825–31. doi: 10.1128/JVI.72.4.2825-28 31.1998 May 2022 \| Volume 9 \| Article 870009 Frontiers in Veterinary Science \| www.frontiersin.org 12
https://openalex.org/W4393132653	https://journal.unhas.ac.id/index.php/akrual/article/download/28280/11224	Indonesian	null	Telaah Kritis Konsep Keagenan dalam Tinjauan Perspektif Islam Emansipatoris	Deleted Journal	2,024	cc-by	7,808	Keywords: Agency Theory; Islamic Agency Theory; Agency Conflict; Emancipatory Abstrak: Penelitian ini bertujuan untuk mengkaji dan mengkritisi teori keagenan berdasarkan perspektif Islam. Jenis penelitian ini berkisar pada tinjauan literatur di mana sumber-sumber penting telah dikutip. Penelitian ini berupaya untuk meletakkan dasar konseptual dalam memahami konflik keagenan agar relevan dengan fenomena yang terjadi dalam entitas Islam. Makalah ini menyimpulkan bahwa prinsip Islam yang berlandaskan Tauhid bertolak belakang dengan konsep keagenan konvensional yang sarat akan asumsi perilaku manusia yang individualis. Asumsi yang muncul pada kedua konsep tersebut menghadirkan model hubungan dan persepsi yang berbeda antar pihak dalam menjalankan sebuah organisasi/perusahaan. Corak epistemologis yang berbeda satu sama lain menjadikan konsep keagenan konvensional kurang kompatibel jika digunakan dalam menganalisis fenomena yang berkaitan dengan entitas-entitas Islam. Dengan menggunakan perspektif Islam emansipatoris, konsep keagenan Islam dihadirkan dengan tetap berbasis etika Islam namun tidak luput memperhatikan sistem sosial yang hadir. Penelitian ini berkontribusi pada wacana yang sedang berlangsung tentang teori keagenan dengan menawarkan perspektif alternatif yang berakar pada prinsip dan nilai Islam. Penelitian lebih lanjut didorong untuk mengeksplorasi implikasi praktis dan aplikasi potensial dari keagenan Islam dalam konteks organisasi. Kata kunci: Teori Agensi; Teori Agensi Islam; Konflik Keagenan: Emansipatoris 10.26487/akrual.v17i01 Volume 17 Nomor 01, Januari 2024 P-ISSN: 1979-1712, E-ISSN: 2829-0615 Volume 17 Nomor 01, Januari 2024 P-ISSN: 1979-1712, E-ISSN: 2829-0615 Volume 17 Nomor 01, Januari 2024 P-ISSN: 1979-1712, E-ISSN: 2829-0615 This work is licensed under a Creative Commons Attribution 4.0 International License This work is licensed under a Creative Commons Attribution 4.0 International License This work is licensed under a Creative Commons Attribution 4.0 International License 1Nurul Jihan Hasril, 2 Alimuddin, 3Rahmawati HS 1 Departemen Akuntansi, Universitas Hasanuddin, Indonesia. E-mail: nuruljihanhasrill@gmail.com 2 Departemen Akuntansi, Universitas Hasanuddin, Indonesia. E-mail: alimuddin.febuh@gmail.com n Departemen Akuntansi, Universitas Hasanuddin, Indonesia. E-mail: rahma_hs@ymail.com Abstract: This research critically examines the agency theory through an Islamic lens. It adopts literature review approach, citing relevant and significant sources. The objective is to establish a conceptual framework for understanding agency conflicts. The findings indicate a fundamental divergence between Islamic principles and conventional agency concepts. Drawing on an emancipatory Islamic perspective, the research introduces the concept of Islamic agency, which upholds ethical considerations while acknowledging the prevailing social system. This study contributes to the ongoing discourse on agency theory by offering an alternative perspective rooted in Islamic principles and values. Further research is encouraged to explore the practical implications and potential applications of Islamic agency within organizational contexts. First Author’s Name, Second Author’s Name and Last Author’s Name. (Year). Telaah Kritis Konsep Keagenan dalam Tinjauan Perspektif Islam. Akrual: Jurnal Bisnis dan Akuntansi Kontemporer, 7(3), 1-18. DOI: 10.26487/akrual.v17i01.28280 P-ISSN: 1979-1712, E-ISSN: 2829-0615 P-ISSN: 1979-1712, E-ISSN: 2829-0615 menjelaskan serta memprediksi fenomena tersebut. Melihat definisi tersebut, posisi teori menjadi tumpuan yang diperlukan dalam menjelaskan bagaimana suatu fenomena dapat terjadi. Sebagai salah satu cabang ilmu pengetahuan, akuntansi juga menggunakan teori sebagai fondasi untuk kepentingan perkembangan ilmu akuntansi baik secara praktis maupun teoritis. Penggunaan teori sebagai landasan memungkinkan akuntansi bertransformasi dari waktu ke waktu guna menjawab permasalahan yang hadir. Wajah teori akuntansi hari ini masih erat pengaruhnya dengan pandangan positifis yang dominan berkembang di tahun 1970, di mana sejumlah artikel yang terbit di beberapa jurnal akuntansi didominasi oleh pendekatan yang berciri matematis dan pengujian hipotesis. Pendekatan positifis memberikan kontribusi yang signifikan terhadap perkembangan akuntansi. Adapun salah satu model pendekatan yang berkembang bersamaan dengan paradigma positifis ini ialah pendekatan teori agensi (agency theory). Di konteks ilmu akuntansi, teori agensi atau keagenan digunakan dalam menjelaskan konflik kepentingan yang terjadi antara principal dan agen di suatu organisasi maupun perusahaan. Setidaknya, teori ini mulai dipublikasikan oleh Jensen and Meckling di tahun 1976. Sejak itu, teori keagenan mulai dikembangkan dan diterapkan untuk membahas kebanyakan fenomena organisasi atau perusahaan seperti kompensasi, strategi akuisisi dan diversifikasi, hubungan dewan, maupun struktur kepemilikan dan pembiayaan (Eisenhardt, 1989). Walaupun penggunaannya yang populer dalam berbagai penelitian namun konsep keagenan berkembang menjadi konsep yang kontroversial khususnya pada model keagenan positifis. Heath (2009) menemukan bahwa terdapat beberapa potensi masalah dari hadirnya pemberlakuan motivasi berbasis kepentingan diri sendiri (self-interest) dan penekanan terhadap keunggulan pemegang saham atau principal yang hadir dalam hubungan antara principal dan agen. Panda dan Leepsa (2017) turut menjelaskan bahwa kepentingan principal yang hanya berkutat pada pemaksimalan pengembalian membuat peran mereka sangat terbatas sehingga muncul anggapan bahwa manajer sebagai seorang yang oportunistis. Htay dan Salman (2013) yang mendapati bahwa dalam situasi tertentu, rekomendasi yang diberikan oleh teori agensi tidak sesuai dengan temuan yang ada di lapangan. Chua (1986:609) menambahkan bahwa teori keagenan juga memilki ketidakmampuan dalam menjangkau persoalan struktural yang berakibat pada tidak terungkapnya potensi eksploitasi pada pekerja yang diakibatkan oleh distribusi akses sumber daya sosial dan ekonomi yang tidak merata. Teori keagenan sama sekali tidak obyektif dan secara samar memiliki kemampuan untuk merasionalkan, menormalisasi, dan melegitimasi berbagai macam instrumen yang digunakan untuk mengendalikan buruh seakan-akan kaum buruh memperoleh banyak manfaat dari sistem yang sesungguhnya sangat eksploitatif (Chwastiak, 1999). Degradasi dan eksploitasi tenaga kerja dalam masyarakat kapitalis bukanlah hasil dari proses evolusioner alami dalam organisasi sosial kerja semata. 1. Pendahuluan Kerlinger seperti dikutip (Belkaoui-Riahi, 2000:72) mendefinisikan teori sebagai sekumpulan gagasan (konsep), definisi, dan dalil yang menyajikan suatu pandangan sistematis tentang fenomena, dengan menjelaskan hubungan antar variabel yang ada dan bertujuan untuk 1 1 P-ISSN: 1979-1712, E-ISSN: 2829-0615 Zogning (2017) melihat bahwa teori keagenan mengadopsi nilai dari sistem ekonomi kapitalisme karena menghilangkan nilai-nilai kemanusiaan seperti rasa, intuisi, spiritual, saling menghormati, saling percaya dan kejujuran. Senada dengan hal tersebut, Shamsuddin dan Ismail (2013) memaparkan bahwa teori keagenan konvensional didominasi oleh kepentingan pribadi serta individualisme yang mencoba memaksimalkan utilitas milik principal yang berakibat pada kemunculan masalah keagenan yang tidak dapat diselesaikan. Menurut Zogning (2017), versi klasik dari teori keagenan menyatakan bahwa agen harus selalu bertindak demi kepentingan principal di mana terdapat anggapan bahwa kepentingan principal selalu dapat diterima atau bahwa agen harus sering bertindak bertentangan dengan etika demi memenuhi "kontrak" dalam hubungan keagenan. Dengan begitu teori keagenan dibangun dengan memandang manusia sebagai makhluk yang selalu bertindak atas naluri yang egois dan serakah (Ruslan, 2013). Di sisi lain, Islam memberikan perhatian terhadap persoalan terkait hubungan. Al-Quran sendiri memberikan pemahaman bahwa hubungan apa pun di dunia ini, terutama bagi umat Islam, harus dimulai dengan hubungan dengan Allah swt. Hubungan tersebut terjalin dalam bentuk penghambaan yang memiliki hakikat berupa ketaatan, ketundukan dan kepatuhan hanya kepada Allah swt. Khalifah merupakan makhluk yang melaksanakan tugas-tugas kehidupan di muka bumi secara optimal sesuai dengan kapasitasnya, dalam koridor ketentuan atau aturan yang telah ditetapkan Allah swt. baik dalam motivasi, bentuk tindakan maupun efek yang ditimbulkan (Budiyanti et al., 2020). Pada dasarnya, manusia diciptakan dengan mengemban tiga tugas utama yaitu menyembah kepada Allah swt. memakmurkan bumi, dan sebagai khalifah (Alimuddin, 2013). Pengangkatan predikat manusia sebagai khalifah berkaitan erat dengan potensi rasionalitas yang dimiliki oleh manusia. Kemampuan berpikir serta pengolahan ilmu pengetahuan memiliki pengaruh yang mendalam dibandingkan dengan makhluk ciptaan lainnya. Bersamaan dengan konsep khalifah, manusia merupakan pelaku ekonomi yang diperbolehkan untuk menggunakan sumber daya ekonomi atau harta benda dengan tanggung jawab dan tidak merugikan orang lain. Dalam pengertian kepemilikan Islam, kepemilikan mutlak dimiliki oleh Allah swt. yang memiliki konsekuensi terhadap peran seorang agen (wakil) sebagai wali tidak hanya bertanggung jawab kepada principal (muwakkil) tetapi juga kepada pencipta dan pengendali bumi yaitu Allah swt. (Shamsuddin dan Ismail, 2013) Sebagai imbas dari perkembangan akuntansi positifis yang berkelindan dengan dominasi ideologi kapitalisme, posisi konsep keagenan konvensional yang digunakan kebanyakan penelitian akuntansi didasari oleh nilai-nilai yang sarat akan individualistik, materialisitik, dan egoistik. Hal tersebut justru sangat kontras dengan ajaran Islam yang menawarkan cara pandang holistik yang berbasis epistemologi tauhid dalam bangunan ilmu pengetahuannya. P-ISSN: 1979-1712, E-ISSN: 2829-0615 Ini terbukti dari fakta bahwa sistem eksploitatif, seperti kapitalisme, harus dipertahankan dengan aturan ataupun propaganda, di mana ideologi dominan menanamkan keyakinan bahwa ketidakadilan dalam masyarakat tidak dapat dihindari. Model principal-agen sendiri telah menjadi paradigma populer dalam penelitian akuntansi yang melayani semua fungsi ideologi dominan tersebut (Chwastiak, 1999). 2 2 P-ISSN: 1979-1712, E-ISSN: 2829-0615 Realitas material ialah sebuah pemikiran yang mempertanyakan ideologi hegemonik yang bertolak pada kehidupan riil dan material atau mempertanyakan hegemoni yang bertolak pada realitas empiris sedangkan visi struktur ialah hal yang berkenaan dengan relasi, baik relasi kekuasaan dalam dunia produktif (majikan-buruh), maupun relasi hegemonik, dalam hubungan pemberi dan penerima narasi (ulama-umat), maupun relasi politik (penguasa-rakyat) (Verdiansyah, 2004) Perkembangan nalar Islam emansipatoris ini sangat dipengaruhi oleh pemikiran Hassan Hanafi – seorang pemikir dan filsuf muslim kontemporer- yang berpendapat bahwa pemikiran akidah Islam haruslah dibangun atas pengalaman di mana manusia hidup dan perlu dimulai dengan kajian atas problem yang menimpa manusia. Konsekuensinya, penguraian masalah tidak lagi terhenti pada persoalan ketuhanan yang jamak digunakan oleh para tradisionalis namun mulai berfokus pada penyelesaian masalah yang dihadapi manusia yang bersifat riil dan materiil. Keterbatasan yang hadir dalam konsep keagenan konvensional akhirnya mendorong untuk dilakukannya sejumlah penelitian dengan menggunakan perspektif yang berbeda. Perspektif Islam dipilih oleh beberapa peneliti untuk dijadikan sebagai cara pandang untuk memeriksa konsep yang menjelaskan hubungan antara principal dan agen. Beberapa penelitian tersebut antara lain Anggreini (2011) dan Agustin et al. (2020) yang menggunakan konsep amanah sebagai variabel dalam menjelaskan hubungan principal dan agen, Shamsuddin dan Ismail (2013) mengintegrasikan konsep tauhid sebagai basis kontrak yang tercipta dalam hubungan keagenan dan Htay dan Salman (2013) yang melakukan evaluasi terhadap implementasi dari konsep keagenan. Dalam kebanyakan penelitian tersebut, intervensi yang digunakan peneliti cenderung bercorak teosentris yang berakibat pada terbentuknya hubungan keagenan yang terkesan altruistik dan seakan terpisah dengan realitas keseharian manusia. Keterpisahan itu berujung pada pengabaian atas kritik sentral antara hubungan principal dan agen yang tersembunyi pada karakternya yang eksploitatif. Keterbatasan yang hadir dalam konsep keagenan konvensional akhirnya mendorong untuk dilakukannya sejumlah penelitian dengan menggunakan perspektif yang berbeda. Perspektif Islam dipilih oleh beberapa peneliti untuk dijadikan sebagai cara pandang untuk memeriksa konsep yang menjelaskan hubungan antara principal dan agen. Beberapa penelitian tersebut antara lain Anggreini (2011) dan Agustin et al. (2020) yang menggunakan konsep amanah sebagai variabel dalam menjelaskan hubungan principal dan agen, Shamsuddin dan Ismail (2013) mengintegrasikan konsep tauhid sebagai basis kontrak yang tercipta dalam hubungan keagenan dan Htay dan Salman (2013) yang melakukan evaluasi terhadap implementasi dari konsep keagenan. Dalam kebanyakan penelitian tersebut, intervensi yang digunakan peneliti cenderung bercorak teosentris yang berakibat pada terbentuknya hubungan keagenan yang terkesan altruistik dan seakan terpisah dengan realitas keseharian manusia. P-ISSN: 1979-1712, E-ISSN: 2829-0615 Gagasan mengenai tauhid ini selanjutnya dikenal juga dengan ilmu kalam yang perumusannya dibagi menjadi dua metode yaitu nalar tradisional dan nalar emansipatoris. Jenis pertama menerapkan kerangka berpikir yang bersifat melangit dengan berpijak pada teks di mana 3 3 P-ISSN: 1979-1712, E-ISSN: 2829-0615 P-ISSN: 1979-1712, E-ISSN: 2829-0615 realitas faktual harus sesuai dan tunduk pada teks yang berakibat pada model perumusan yang terpisah dengan realitas. Berbeda dengan tradisional, nalar ilmu kalam emansipatoris secara komprehensif tidak berhenti pada pembongkaran teks, tetapi teks dijadikan sebagai sarana pembebasan. Konsep emansipatoris ini hadir untuk mengatasi keterbatasan nalar tradisional dengan mengintegrasikan teori kritis ke dalam pembacaan teksnya sehingga perumusannya akan bersifat kontekstual dan sesuai dengan realitas material yang hadir (Dahlan, 2012) Perkembangan nalar Islam emansipatoris ini sangat dipengaruhi oleh pemikiran Hassan Hanafi – seorang pemikir dan filsuf muslim kontemporer- yang berpendapat bahwa pemikiran akidah Islam haruslah dibangun atas pengalaman di mana manusia hidup dan perlu dimulai dengan kajian atas problem yang menimpa manusia. Konsekuensinya, penguraian masalah tidak lagi terhenti pada persoalan ketuhanan yang jamak digunakan oleh para tradisionalis namun mulai berfokus pada penyelesaian masalah yang dihadapi manusia yang bersifat riil dan materiil. Penggunaan istilah emansipatoris tidak dapat dilepaskan dari sejarah teori kritis (Dahlan, 2012). Kritisisme digunakan sebagai kekuatan dalam mengungkap penindasan dengan memusatkan perhatiannya terhadap dua hal yaitu realitas material dan visi struktur. Realitas material ialah sebuah pemikiran yang mempertanyakan ideologi hegemonik yang bertolak pada kehidupan riil dan material atau mempertanyakan hegemoni yang bertolak pada realitas empiris sedangkan visi struktur ialah hal yang berkenaan dengan relasi, baik relasi kekuasaan dalam dunia produktif (majikan-buruh), maupun relasi hegemonik, dalam hubungan pemberi dan penerima narasi (ulama-umat), maupun relasi politik (penguasa-rakyat) (Verdiansyah, 2004) Perkembangan nalar Islam emansipatoris ini sangat dipengaruhi oleh pemikiran Hassan Hanafi – seorang pemikir dan filsuf muslim kontemporer- yang berpendapat bahwa pemikiran akidah Islam haruslah dibangun atas pengalaman di mana manusia hidup dan perlu dimulai dengan kajian atas problem yang menimpa manusia. Konsekuensinya, penguraian masalah tidak lagi terhenti pada persoalan ketuhanan yang jamak digunakan oleh para tradisionalis namun mulai berfokus pada penyelesaian masalah yang dihadapi manusia yang bersifat riil dan materiil. Penggunaan istilah emansipatoris tidak dapat dilepaskan dari sejarah teori kritis (Dahlan, 2012). Kritisisme digunakan sebagai kekuatan dalam mengungkap penindasan dengan memusatkan perhatiannya terhadap dua hal yaitu realitas material dan visi struktur. Kerangka Konseptual Kerangka Konseptual Gambar 1. Kerangka Konseptual Gambar 1. Kerangka Konseptual P-ISSN: 1979-1712, E-ISSN: 2829-0615 P-ISSN: 1979-1712, E-ISSN: 2829-0615 semata dan agen sebagai seorang yang oportunis dalam melakukan pekerjaannya berakibat pada hadirnya masalah keagenan. Jika ditelusuri, kehadiran asumsi tersebut tidak terlepas dari pandangan positifis yang bertalian dengan ideologi kapitalisme sebagai corak yang dominan pada saat teori agensi tersebut berkembang. semata dan agen sebagai seorang yang oportunis dalam melakukan pekerjaannya berakibat pada hadirnya masalah keagenan. Jika ditelusuri, kehadiran asumsi tersebut tidak terlepas dari pandangan positifis yang bertalian dengan ideologi kapitalisme sebagai corak yang dominan pada saat teori agensi tersebut berkembang. Berdasarkan kondisi yang dipaparkan sebelumnya, maka tujuan dari penelitian ini ialah untuk mengetahui konsep keagenan yang dapat digunakan dalam menganalisa fenomena yang terjadi pada entitas Islam. Hal ini diperlukan karena mengingat adanya perbedaan yang dianut oleh konsep keagenan konvensional. Selain itu, penelitian ini juga bertujuan untuk melakukan peninjauan kritis terhadap konsep keagenan secara umum dengan menggunakan perspektif Islam emansipatoris P-ISSN: 1979-1712, E-ISSN: 2829-0615 Keterpisahan itu berujung pada pengabaian atas kritik sentral antara hubungan principal dan agen yang tersembunyi pada karakternya yang eksploitatif. Penggunaan konsep keagenan yang jamak dalam mengurai permasalahan, membuatnya menjadi salah satu konsep penting khususnya ketika membahas persoalan yang berkaitan dengan tata kelola sebuah organisasi ataupun perusahaan. Namun di balik hal tersebut, penggunaan teori agensi ternyata menemui keterbatasan. Kehadiran asumsi yang menganggap principal sebagai sosok individualis karena hanya mementingkan keuntungannya 4 P-ISSN: 1979-1712, E-ISSN: 2829-0615 P-ISSN: 1979-1712, E-ISSN: 2829-0615 penelitian. Setelah mengoleksi literatur, koleksi yang sudah ada dibaca dan dirangkum dengan mencatat argumen yang penting dan relevan sebagai ringkasan hasil studi. Hasil rangkuman tersebut selanjutnya disusun dan dianalis agar selaras dengan fokus dari penelitian ini. Di tahap terakhir, hasil analisis dipaparkan dalam bentuk narasi maupun tabel. Pada gambar 2 memperlihatkan tahapan yang ditempuh dalam penelitian ini. Gambar 2. Tahapan Penelitian Gambar 2. Tahapan Penelitian 3.1. Meninjau Prinsip Ekonomi Islam dalam Konsep Keagenan 3.1.1. Perilaku Manusia dalam Hubungan Keagenan Islam 2. Metode Penelitian Penelitian ini bersifat kualitatif-eksploratif dengan pendekatan metode studi kepustakaan. Penelitian kualitatif merupakan upaya memahami secara mendalam terhadap suatu gejala kemudian menginterpretasikan dan menyimpulkan gejala-gejala tersebut sesuai dengan konteksnya sehingga dicapai suatu simpulan yang obyektif dan alamiah sesuai dengan gejala- gejala pada konteks tersebut (Suyitno, 2018:6). Studi eksplorasi diupayakan dikarenakan bahan yang berkaitan dengan topik penelitian masih kurang memadai dan relatif sedikit diketahui. Pengetahuan yang minim tersebut dapat disebabkan oleh hal yang sifatnya kontroversial ataupun kebaruannya sebagai suatu topik bahasan dalam suatu penelitian (Kumalasari dan Sudarma, 2013). Penelitian ini diawali dengan memformulasikan pertanyaan penelitian yang dilanjutkan dengan mencari sekaligus melakukan identifikasi terhadap literatur yang sesuai dengan topik 5 P-ISSN: 1979-1712, E-ISSN: 2829-0615 P-ISSN: 1979-1712, E-ISSN: 2829-0615 P-ISSN: 1979-1712, E-ISSN: 2829-0615 memastikan bahwa manajemen melaksanakan tugas terhadap pemegang saham secara efektif. Tujuan akhir dari setiap umat manusia di dunia ini adalah untuk mencari keridhaan Allah swt. dan setiap tindakan yang dilakukan oleh pihak mana pun akan diarahkan untuk mendapatkan Ridha Allah swt. dan dengan itu, tidak ada perilaku oportunis dalam memaksimalkan kepentingan diri sendiri dengan mengorbankan pihak lain yang diperbolehkan (Zainuldin etal., 2018:10) Shamsuddin dan Ismail (2013:540) menjelaskan bahwa terdapat beberapa nilai dalam Islam yang dapat memberikan dorongan yang kuat bagi para aktor dalam hubungan keagenan. Beberapa nilai tersebut ialah Pertama, Itqan (kesadaran dan pengetahuan) yaitu bahwa principal (muwakkil) dan agen (wakil) harus berkomitmen, berdedikasi untuk melakukan tugas. Kedua, Ikhlas (ketulusan) berarti para pihak dalam hubungan lembaga Islam harus ikhlas dalam menjalankan tugas yang diberikan yang tetap mengacu pada kemampuan para pihak untuk memberikan kontribusi keberhasilan bagi organisasi dan Ketiga, Amanah (kejujuran) yaitu posisi di mana semua aktor baik principal maupun agen jujur dalam tindakan, niat, ucapan, informasi dan penyelesaian masalah dalam perdagangan. Ini adalah nilai-nilai yang paling dibutuhkan dalam kontrak keagenan Islam untuk mengurangi, atau mungkin untuk menghilangkan masalah moral hazard dalam hubungan keagenan. Selain mengatur mengenai perilaku manusia, di dalam Islam dikenal juga sebuah prinsip yang disebut Umma yaitu sebuah prinsip yang menegaskan bahwa seorang Muslim tidak dapat bebas dari kepedulian terhadap orang lain. Prinsip tersebut mampu memperlihatkan posisi manusia sebagai individu sekaligus bagian dari sebuah komunitas di mana perilaku keegoisan dan individualisme mampu diredam. Maka dari itu aktivitas berbagi dan pendistribusian lebih diutamakan dibanding kepentingan individu (Kamla, 2006:252-253) Dengan menggunakan pendekatan politik Islam, Ceyhan dan Barca (2021:8) menjelaskan bahwa hubungan keagenan diperankan oleh tiga aktor di mana principal dan agen diposisikan sebagai orang yang dianggap bertanggung jawab terhadap Tuhan. Pertanyaan mendasar selanjutnya ialah bagaimana tanggung jawab tersebut dapat diterapkan dalam bidang manajerial. Hal tersebut dapat diterapkan dengan melakukan penekanan yang lebih pada tanggung jawab sosial. Terlepas dari tanggung jawab keagamaan individu (misalnya Shalat dan puasa), pada akhirnya setiap aktor perlu menempatkan kepentingan masyarakat yang membuat principal dan agen harus bekerja untuk masyarakat. Masalah krusial lainnya dalam hubungan keagenan ialah ketika terjadi perbedaan tujuan antara agen dan principal. Kedua pihak berusaha untuk memaksimalkan kepentingannya masing- masing. Kondisi ini dapat terlihat ketika agen tidak melakukan pekerjaan seperti yang diinginkan oleh principal dan ketika principal tidak membayar sesuai keinginan agen. Perbedaan kemaslahatan antara para pihak dapat diselesaikan melalui prinsip syura yang berarti musyawarah mufakat. 3.1.1. Perilaku Manusia dalam Hubungan Keagenan Islam Konsep tauhid dan Taqwa akan membentuk kehidupan seseorang dan memupuk perilakunya menjadi lebih bertanggung jawab dan beretika, karena ia sadar bahwa ia akan mempertanggungjawabkan apa yang ia lakukan di dunia ini dan pada gilirannya menentukan nasibnya di akhirat kelak. Dengan memiliki ketakwaan kepada Allah, memungkinkan seseorang untuk menyadari kehadiran dan sifat Allah yang akan menuntunnya untuk mengikuti perintah- perintah Allah dengan memenuhi semua kewajiban dan menghindari apa pun yang dilarang oleh-Nya. Setiap pikiran dan tindakannya akan diarahkan untuk meraih Ridha Allah. Seseorang yang memiliki ketakwaan dalam pikirannya memiliki pemahaman yang kuat tentang perannya untuk mengelola dunia sesuai dengan Syariah di mana ia akan mengintegrasikan kesejahteraan fisik dan nilai-nilai moral. Dalam kaitannya dengan mekanisme tata kelola perusahaan, Tauhid memainkan peran penting dalam memperbaiki masalah keagenan yang ada di dalam perusahaan perbankan Syariah. Sebagai contoh, seorang anggota dewan direksi yang memiliki keyakinan tauhid akan menyadari bahwa ia akan diawasi oleh Allah setiap saat, di mana setiap perbuatan baik akan mendapat pahala dan perbuatan buruk akan mendapat hukuman. Oleh karena itu, ia akan lebih berhati-hati dalam menjalankan tanggung jawabnya terhadap pihak-pihak yang berkepentingan. Karena ia takut melakukan kesalahan yang akan dipertanggungjawabkan kepada Allah di Hari Kiamat, ia akan berusaha dengan cara apa pun untuk menghindari kesempatan atau jalan menuju perbuatan buruk termasuk mencegah dirinya terlibat dalam kegiatan yang mengandung benturan kepentingan. Dengan demikian, biaya yang ditimbulkan dari masalah keagenan akan berkurang. Dalam hal peran pengawasan yang dilakukan oleh dewan direksi, mereka bertanggung jawab untuk memberikan arahan yang konstruktif dan 6 P-ISSN: 1979-1712, E-ISSN: 2829-0615 Principal dan agen harus menjalankan tugas dan tanggung jawabnya masing-masing sesuai kesepakatan bersama, baik mengenai tugas maupun kompensasinya. Kedua belah pihak harus menerapkan keterbukaan pikiran, rasa hormat, kepercayaan, penerimaan pandangan yang berbeda serta kebebasan untuk memberikan ide dan ekspresi. Konsep ini penting untuk menjamin keadilan, melindungi kepentingan kedua belah pihak dan menghindari nilai-nilai negatif arogansi dan ego. 7 P-ISSN: 1979-1712, E-ISSN: 2829-0615 P-ISSN: 1979-1712, E-ISSN: 2829-0615 Konsep keagenan konvensional mengadopsi gagasan bahwa agen dapat menggunakan informasi yang ia miliki untuk melawan kepentingan principal. Namun, dalam perspektif Islam, asimetris informasi antara aktor organisasi dapat dipersepsikan berbeda di mana principal dan agen yang kompeten secara moral akan mendorong agen untuk menggunakan informasi atau pengetahuannya dengan mempertimbangkan kepentingan semua orang, bukan melawan mereka. Dengan kata lain agen akan menggunakan informasinya untuk mewujudkan potensinya demi kepentingan orang lain. Logika yang serupa dapat juga berlaku bagi principal di mana mereka juga ingin bekerja secara maksimal dan turut memberikan kontribusi kepada masyarakat (Ceyhan dan Barca, 2021:12). 3.1.2. Komparasi Konsep Keagenan Konvensional dan Islam Teori keagenan didasarkan pada asumsi manusia rasional yang berusaha memaksimalkan utilitas individualnya. Berdasarkan asumsi ini, individu diharapkan menjadi oportunis dan termotivasi semata-mata oleh utilitas ekonomi. Namun, asumsi ini telah dikritik karena terlalu sederhana dan tidak mencerminkan lingkungan bisnis di dunia nyata. Lebih jauh lagi, model manusia yang diasumsikan dalam prinsip keagenan ini hanya sebuah penyederhanaan untuk permodelan matematis dan tidak sesuai untuk mencerminkan tuntutan eksistensi sosial. Selain itu, asumsi maksimisasi utilitas diri gagal menjelaskan kompleksitas tindakan manusia dan mengabaikan utilitas nilai-nilai yang lebih abstrak, seperti kejujuran, kewajiban untuk rajin, dan keadilan. Sejak pengembangan model keagenan, kemungkinan nilai etika dan sosial dalam mengurangi perilaku mementingkan diri sendiri mulai dibahas. Penelitian akuntansi sebelumnya, misalnya, Noreen (1988) berpendapat bahwa memasukkan etika dalam pengaturan principal-agen dapat meningkatkan kegunaan teori keagenan. Noreen (1988) berpendapat bahwa teori keagenan dapat menjadi "sarana yang tepat" dalam menjelaskan perilaku manajerial pada kenyataannya dengan memasukkan etika dalam diskusi akuntansi. Secara khusus, ia berpendapat bahwa kontrak keagenan saja tidak akan dapat membatasi perilaku oportunistik manajer, tetapi pertimbangan etika diperlukan untuk memastikan kelancaran fungsi pasar dan organisasi. Selain itu, Noreen (1988) menegaskan bahwa agama memainkan peran penting dalam menegakkan perilaku etis dengan memohon kepada Tuhan yang memiliki kekuatan untuk memberi penghargaan dan menghukum perilaku yang menyimpang. Hal ini menunjukkan bahwa agama adalah salah satu unsur penting yang mempengaruhi perilaku individu dan lebih jauh lagi mampu menahan tindakan yang mementingkan diri sendiri (Zainuldin et. al., 2018:12). Berikut pada Tabel 1 penulis menyajikan skema perbandingan antara perspektif yang digunakan pada konsep keagenan konvensional dengan perspektif etika Islam. Tabel 1. Skema Perbandingan Konsep Keagenan Konvensional dan Islam Elemen Konsep Perspektif Keagenan Konvensional Perspektif Etika Islam Ide Utama Hubungan principal-agen harus mencerminkan organisasi yang efisien dalam biaya informasi dan penanggulangan risiko Hubungan principal-agen mencerminkan pengelolaan sumber daya yang dipercayakan pada principal-agen (amanah) yang berorientasi terhadap kebaikan ekonomi dan moral masyarakat Kepentingan atas diri (self-interest) Kepentingan Umma Tabel 1. Skema Perbandingan Konsep Keagenan Konvensional dan Islam 8 P-ISSN: 1979-1712, E-ISSN: 2829-0615 Asumsi atas perilaku manusia Berperilaku rasional Berperilaku Taqwa, Itqad, Ikhlas, dan Amanah Penghindaran atas risiko Penghindaran risiko berbasis perilaku amanah Asumsi atas organisasi Konflik tujuan antar anggota Syura (Mutual Consultation) sebagai metode penyelesaian konflik Efisiensi sebagai kriteria efektivitas Efisiensi yang mengandalkan kemampuan principal-agen sebagai kriteria efektivitas Asimetris informasi antara principal dan agen Informasi yang tersedia digunakan untuk kepentingan semua aktor 3.2. Eksplorasi Konsep Keagenan dalam Perspektif Islam Emansipatoris Oleh karena itu, seorang Muslim bertanggung jawab untuk melaksanakan peran dan tanggung jawabnya sebagai pengemban amanah serta mematuhi ajaran Islam dalam semua aspek kehidupan mereka. dipertanggungjawabkan oleh seorang direktur Muslim sebagai pemilik (principal). Skema ini sangat berbeda pada perilaku direktur non-Muslim karena direktur non-Muslim percaya pada pandangan dunia sekuler yang memisahkan antara kegiatan keagamaan dan kegiatan sehari- hari. Oleh karena itu, seorang Muslim bertanggung jawab untuk melaksanakan peran dan tanggung jawabnya sebagai pengemban amanah serta mematuhi ajaran Islam dalam semua aspek kehidupan mereka. Kriteria ketiga dari Islam adalah kebajikan atau keunggulan. Kebajikan (Ihsan) atau kebaikan kepada orang lain didefinisikan sebagai suatu tindakan yang memberikan manfaat kepada orang lain selain orang yang menerima manfaat dari tindakan tersebut tanpa adanya kewajiban. Konsep ini merupakan inti dari akhlak Islam karena berfokus pada perilaku untuk kecintaan kepada Tuhan. Seperti yang telah dijelaskan sebelumnya, konsep ibadah dalam Islam mencakup segala upaya atau pekerjaan yang konstruktif. Hal ini menyiratkan bahwa karyawan muslim yang berkomitmen di tingkat organisasi mana pun harus melakukan pekerjaannya demi kecintaan kepada Allah swt. dan dengan kesadaran bahwa Allah swt. mengawasi perilakunya bahkan ketika atasannya tidak ada 3.2. Eksplorasi Konsep Keagenan dalam Perspektif Islam Emansipatoris 3.2.1. Sistem Akhlak Islam sebagai Basis Perilaku Keagenan Sebagai pengemban amanah Allah swt. di muka bumi, maka tindakan seorang muslim harus sesuai dengan ketentuan-ketentuan amanah tersebut. Penekanan pada peran manusia sebagai wali Allah swt. dan pada pekerjaan sebagai ibadah berikutnya mengondisikan perhatian pada pemangku kepentingan Islam terhadap bisnis dan mengarahkan pada kerangka akhlak yang berlaku di Islam. Menurut Beekun dan Badawi (2005:134-135) terdapat beberapa kriteria yang relevan ketika mengkaji aspek akhlak dari sudut pandang pemangku kepentingan yaitu keadilan (adl’) dan keseimbangan, kepercayaan, dan kebajikan. Pertama, kriteria keadilan yang digambarkan dengan dua kata dalam Al-Quran yaitu 'adl dan qist. Dalam Islam normatif, umat Islam didorong untuk berperilaku adil terhadap semua orang dan hal ini berkaitan erat dengan keimanan seseorang sebagai seorang Muslim. Pada saat yang sama, istilah 'adl juga berlaku untuk konsep keseimbangan dan kesetimbangan. Hal ini mengartikan bahwa melakukan segala sesuatu secara proporsional. Keadilan juga dijelaskan dalam Al Quran dengan kata penting lainnya yaitu Qist. Kata ini berarti bagian, porsi, ukuran, jatah, jumlah. Keadilan seperti yang digambarkan oleh kata qist berarti memberikan setiap orang dan segala sesuatu sesuai dengan haknya. Dengan demikian, Islam normatif mengajarkan bahwa seseorang harus bersikap adil dalam setiap aspek kehidupannya, terhadap semua orang dan segala sesuatu dan setiap saat. Secara keseluruhan, keadilan seperti yang dijelaskan oleh 'adl dan qist berarti menjaga keseimbangan antara kebutuhan tubuh, pikiran dan jiwa sambil memberikan semua orang dan segala sesuatu yang menjadi haknya. Kriteria kedua dari akhlak Islam berkaitan dengan konsep amanah atau kepercayaan. Hal ini mengartikan bahwa kekayaan dan sumber daya lain yang dimiliki manusia bukanlah miliknya tetapi telah dipinjamkan kepadanya oleh Allah swt. sebagai alat untuk memenuhi tanggung jawab perwalian. Implementasi model ini dapat diterjemahkan sebagai pelibatan Allah Swt. sebagai salah satu aktor yang memiliki kaitan erat dengan konsep akuntabilitas dalam Islam. Hameed (2000) mendefinisikan akuntabilitas Islam sebagai akuntabilitas ganda yaitu adanya akuntabilitas utama yang diakibatkan oleh peran manusia sebagai wali dari sumber daya Allah swt. dan akuntabilitas sekunder yang hadir antara pemilik atau investor dan manajer. Akuntabilitas primer sendiri merupakan akuntabilitas tambahan yang harus 9 9 P-ISSN: 1979-1712, E-ISSN: 2829-0615 P-ISSN: 1979-1712, E-ISSN: 2829-0615 dipertanggungjawabkan oleh seorang direktur Muslim sebagai pemilik (principal). Skema ini sangat berbeda pada perilaku direktur non-Muslim karena direktur non-Muslim percaya pada pandangan dunia sekuler yang memisahkan antara kegiatan keagamaan dan kegiatan sehari- hari. P-ISSN: 1979-1712, E-ISSN: 2829-0615 account/UIA) yang merupakan jenis kategori pembeda dalam skema penyediaan produk keuangan antara bank konvensional dan bank syariah. Kategori ini sering kali dijadikan topik utama dalam isu keagenan Islam dikarenakan terdapat hubungan keagenan di mana dana para deposan dikelola oleh pihak manajemen dan memberlakukan mekanisme bagi hasil sesuai dengan nisbah yang telah ditentukan sebelumnya (Tiby dan Grais, 2015:125-127) Dengan demikian, pemegang rekening investasi tidak terbatas atau UIA memiliki karakteristik yang mirip dengan pemegang saham. Mereka merupakan principal yang mempercayakan sumber daya mereka kepada agen, dalam hal ini manajemen lembaga keuangan syariah. Namun, perbedaan yang signifikan adalah bahwa agen ditunjuk oleh principal lain yaitu pemegang saham. Sementara pemegang saham dapat mempengaruhi perilaku bisnis melalui struktur dan proses tata kelola yang ada, pemegang UIA tidak memiliki akses yang sama untuk mengekspresikan pandangan mereka. Singkatnya, pemegang UIA merupakan kategori di mana mereka tidak memiliki suara institusional dalam pelaksanaan bisnis dan mereka mendelegasikan penunjukan agen mereka kepada principal lain yang kepentingannya mungkin tidak selalu sesuai dengan kepentingan mereka. Menurut Sakti et al. (2021:371) keunikan tata kelola perusahaan Islam terletak pada nilai-nilai dan norma-norma Islam yang spesifik dibandingkan dengan konsepsi tata kelola perusahaan konvensional. Jika dilihat secara kritis dari literatur-literatur yang ada mengenai tata kelola perusahaan Islam, setidaknya terdapat tiga model utama tata kelola perusahaan Islam, yaitu: (1) model berbasis tauhid yang mengacu pada mekanisme tata kelola perusahaan yang bersumber dari prinsip musyawarah (syura) di mana seluruh pemangku kepentingan digerakkan oleh tujuan yang sama, yaitu Tauhid atau keesaan Allah (2) model berbasis maqasid al-shari'ah yaitu praktik-praktik tata kelola perusahaan yang bertujuan untuk mewujudkan maqasid al-shari'ah dan (3) model tata kelola perusahaan yang berbasis pemangku kepentingan yang berkaitan dengan penerapan sistem nilai pemangku kepentingan dengan beberapa penyesuaian dengan norma-norma Islam untuk melindungi hak-hak pemangku kepentingan. Zainuldin et al. (2018:6) menjelaskan bahwa dalam struktur tata kelolanya, institusi layanan keuangan Islam memiliki lapisan tata kelola lain yaitu Dewan Pengawas Syariah (DPS). Pada dasarnya, DPS berfungsi sebagai mekanisme pengawasan dan pengendalian yang mencegah bank-bank syariah untuk terlibat dalam investasi yang tidak sesuai dengan syariah. Dalam memastikan bahwa keuntungan yang dihasilkan sesuai dengan parameter etika, DPS memiliki hak untuk memberi saran mengenai distribusi pendapatan atau pengeluaran di antara pemegang saham bank dan pemegang akun investasi (deposan). Sebagai bagian dari struktur tata kelola internal bank syariah, DPS meningkatkan kredibilitas lembaga di mata nasabah, deposan dan pemegang saham dengan cara memperkuat kredensial syariahnya. P-ISSN: 1979-1712, E-ISSN: 2829-0615 Selain itu, DPS bertanggung jawab untuk memantau aspek akhlak dan moral dari perilaku manajemen perusahaan yang akan mencegah individu untuk berperilaku tidak sesuai dengan prinsip moral yang berlaku dalam Islam. 3.2.2. Struktur Keagenan dalam Institusi Keuangan Islam Dasar pengorganisasian hubungan kontraktual antara para mitra sehubungan dengan pembagian tanggung jawab telah dipraktikkan sejak lama di mana Islam menetapkan konsep penulisan semua bentuk transaksi dan kontrak harus bebas dari eksploitasi. Pendasaran ini selanjutnya diterjemahkan ke dalam bentuk struktur tata Kelola yang merupakan topik yang bersinggungan dengan konsep keagenan. Islamic Financial Services Board (IFSB) mendefinisikan tata kelola perusahaan sebagai seperangkat hubungan antara manajemen perusahaan, dewan direksi, pemegang saham, dan pemangku kepentingan lainnya yang menyediakan struktur melalui bagaimana tujuan perusahaan ditetapkan serta cara-cara untuk mencapai tujuan tersebut ditentukan sesuai dengan prinsip-prinsip Islam. Menurut Tiby dan Grais (2005:124) lembaga yang menawarkan layanan keuangan Islam umumnya menerapkan struktur dan sistem tata kelola yang serupa dengan bisnis yang menggunakan standar keuangan konvensional dalam menangani masalah keagenan antara pemegang saham dan manajemen. Masalah tersebut menguak akibat konsentrasi kontrol atas keputusan eksekutif dan dewan direksi monolitik yang mungkin bias demi kepentingan tertentu kecuali jika diperiksa secara memadai. Sebagai contoh, manajemen dapat menggunakan keleluasaan dalam dana yang mereka kumpulkan untuk membiayai investasi tertentu dan memberikan imbal hasil yang lebih baik kepada pemegang saham dominan. Di samping pemegang saham, deposan merupakan kategori yang memiliki peran dalam struktur keagenan dalam lembaga penyedia layanan keuangan Islam. Umumnya, institusi keuangan Islam menawarkan tiga kategori besar rekening deposito yaitu pertama, investasi saat ini (current account) yang merupakan mekanisme penggunaan akun deposito untuk tujuan apa pun yang diizinkan oleh Syariah di mana pihak bank tidak berkewajiban untuk membayar bunga tetap atau bagi hasil kepada deposan dengan pengecualian hadiah (hibah) yang didistribusikan berdasarkan kebijaksanaan bank. Kedua, investasi terbatas (restricted investment account/RIA) yaitu pengoperasian rekening berdasarkan prinsip mudharabah dan penyesuaian mode investasi serta distribusi keuntungan dengan selera risiko dan kebutuhan masing-masing klien. Terakhir, ialah investasi tidak terbatas (unrestricted investment 10 P-ISSN: 1979-1712, E-ISSN: 2829-0615 P-ISSN: 1979-1712, E-ISSN: 2829-0615 P-ISSN: 1979-1712, E-ISSN: 2829-0615 P-ISSN: 1979-1712, E-ISSN: 2829-0615 mengenai topik tata kelola perusahaan yang berhubungan dengan hubungan hierarkis antara pemegang saham dan manajer. Keduanya mendapati bahwa hubungan bertingkat tersebut dilandasi oleh dua hal yaitu orientasi yang berpihak kepada para pemegang saham dan perihal hilangnya kontrol oleh pemegang saham akibat adanya pendelegasian wewenang kepada manajer. Selain berkutat dengan topik seputar tata kelola perusahaan, konsep keagenan juga turut memberikan kerangka kerja untuk lebih memahami dan mengevaluasi kegunaan konsep- konsep fundamental yang mendasari standar akuntansi keuangan. Penerapan konsep keagenan pada tema yang diperdebatkan dalam pengembangan kerangka konseptual oleh IASB ialah salah satunya yaitu pengukuran nilai wajar (fair value measurement) (Wagenhofer, 2015:353). mengenai topik tata kelola perusahaan yang berhubungan dengan hubungan hierarkis antara pemegang saham dan manajer. Keduanya mendapati bahwa hubungan bertingkat tersebut dilandasi oleh dua hal yaitu orientasi yang berpihak kepada para pemegang saham dan perihal hilangnya kontrol oleh pemegang saham akibat adanya pendelegasian wewenang kepada manajer. Selain berkutat dengan topik seputar tata kelola perusahaan, konsep keagenan juga turut memberikan kerangka kerja untuk lebih memahami dan mengevaluasi kegunaan konsep- konsep fundamental yang mendasari standar akuntansi keuangan. Penerapan konsep keagenan pada tema yang diperdebatkan dalam pengembangan kerangka konseptual oleh IASB ialah salah satunya yaitu pengukuran nilai wajar (fair value measurement) (Wagenhofer, 2015:353). Dalam diskursus ekonomi Islam sendiri, Khalid dan Sarea (2020) mengatakan bahwa masalah keagenan yang terjadi di institusi keuangan Islam cenderung lebih menantang daripada institusi yang berbasis konvensional. Hal ini disebabkan oleh ketidakpastian yang timbul dalam mekanisme pengembalian yang dimanifestasikan dalam tingkat suku bunga tetap atau mengambang. Di bank-bank Islam, pengembalian tidak dapat ditentukan sebelumnya sesuai dengan tingkat suku bunga dasar karena keuntungan maupun kerugian yang melekat pada risiko yang ditanggung bersama antara lembaga penyedia jasa keuangan dan nasabah mereka. Salah satu masalah keagenan yang banyak dibahas oleh para peneliti ialah pengaturan kontrak dari jenis rekening investasi mudharabah dan musharakah yang sering kali menimbulkan masalah keagenan antara pemegang rekening investasi yang memiliki hak arus kas dan pemegang saham yang memiliki hak kontrol. Larangan transaksi apa pun yang berhubungan dengan riba termasuk deposito berbunga mengharuskan bank-bank Islam untuk menawarkan rekening deposito yang didasarkan pada prinsip-prinsip mudarabah dan musharakah. Dalam kontrak mudarabah, rabb-ul-mal (deposan) menginvestasikan modal uang dan berhak menerima bagian keuntungan atau tidak mendapatkan keuntungan jika terjadi kerugian dari kumpulan aset di mana dana diinvestasikan, sedangkan dalam kontrak musharakah, keuntungan dan kerugian dibagi antara deposan dan bank. 3.2.3. Aplikasi Konsep Keagenan Islam terhadap Isu Akuntansi Menurut Aglietta dan Reberioux (2005:29) konsep keagenan memiliki pengaruh besar dalam perdebatan tata kelola perusahaan yang menjadi topik dalam isu akuntansi manajemen. Konsep ini telah membentuk kerangka interpretasi yang sekarang mendominasi terutama 11 P-ISSN: 1979-1712, E-ISSN: 2829-0615 P-ISSN: 1979-1712, E-ISSN: 2829-0615 P-ISSN: 1979-1712, E-ISSN: 2829-0615 memaksimalkan efektivitas kinerjanya ketika diberikan independensi penuh di berbagai dimensi. Dengan kata lain, auditor internal syariah yang menunjukkan independensi serta kemampuan mereka dalam mempertahankan sikap yang tepat dalam perencanaan program audit lebih mungkin untuk mencapai hasil organisasi yang positif bagi lembaga keuangan Islam daripada auditor internal syariah yang independensinya terlalu dibatasi. Penelitian tersebut menunjukkan bahwa konsep keagenan Islam dapat digunakan secara produktif sebagai dasar untuk membangun kerangka kerja analitis yang mampu mengukur dampak dari atribut multi- dimensi independensi terhadap efektivitas audit syariah internal. P-ISSN: 1979-1712, E-ISSN: 2829-0615 Tidak seperti bank konvensional, deposan tidak berbagi risiko dengan bank karena simpanan mereka telah dijamin pada tingkat tertentu, deposan musharaka dan mudharabah memiliki risiko kehilangan total pokok yang diinvestasikan kecuali jika kerugian tersebut disebabkan oleh kelalaian atau kesalahan yang terbukti dari pihak bank. Seperti halnya manajer bank konvensional, manajer bank syariah bertindak sebagai agen bagi pemegang saham dan pada saat yang sama, dalam kasus rekening investasi mudharabah dan musharaka, manajer bank syariah juga bertindak sebagai agen bagi pemegang rekening investasi. Hal ini akan mendorong aplikasi konsep keagenan Islam untuk mendiagnosis kemungkinan terjadinya konflik kepentingan yang dihadapi oleh manajer dalam menangani kepentingan pemegang saham dan pemegang rekening investasi. Selain mengatasi masalah keagenan yang terjadi di institusi keuangan Islam, konsep keagenan Islam dapat pula diaplikasikan dalam menganalisis perihal efektivitas pelaksanaan audit internal syariah dalam sebuah entitas Islam. Melalui prisma teori keagenan Islam, penelitian yang dilakukan oleh Khalid dan Sarea (2020) berupaya untuk mengeksplorasi hubungan konseptual antara independensi dan efektivitas audit syariah internal dengan mengajukan hipotesis bahwa masalah keagenan di Lembaga keuangan syariah melebihi masalah keagenan yang terjadi di lembaga keuangan konvensional. Keduanya menemukan bahwa konsep keagenan Islam mampu menginformasikan bahwa auditor internal Syariah mampu 12 P-ISSN: 1979-1712, E-ISSN: 2829-0615 Perbandingan Prinsip Konsep Keagenan dan Implikasinya Perspektif Prinsip Implikasi Konvensional Orientasi diri (self interest) Hubungan principal-agen hanya berbasis kontrak Analisa berbasis kontrak Analisa yang terfokus pada hubungan principal-agen semata Etika Islam Orientasi kepentingan Umma Hubungan principal-agen berbasis amanah Analisa yang berbasis akhlak dan moral Islam Hubungan yang hadir mengabaikan dan cenderung mempertahankan status quo Islam Emansipatoris Orientasi kepentingan Umma diatur dalam seperangkat nilai yang dipengaruhi oleh sistem sosial. Berpandangan bahwa hubungan keagenan dipengaruhi pula oleh sistem sosial Memungkinkan kehadiran analisa kritis terhadap konsep keagenan Islam melalui pemahaman mengenai sistem sosial Memungkinkan perluasan perspektif keagenan 3.2.4. Tinjauan Kritis atas Konsep Keagenan Islam Salah satu hipotesis yang dibangun oleh konsep keagenan Islam ialah bahwa pemberlakuan sistem akhlak dapat menurunkan perilaku oportunistik bagi principal maupun agen. Namun, temuan yang sebaliknya didapati oleh Alam et al. (2020) yang memperlihatkan bahwa lembaga keuangan Syariah tidak menunjukkan kecenderungan untuk mengurangi perilaku oportunistik manajerial. Sebaliknya, terdapat indikasi bahwa bank-bank konvensional yang besar dan memiliki leverage yang tinggi menunjukkan kepedulian yang lebih besar dibandingkan dengan ekspektasi religiusitas yang terdapat pada lembaga keuangan berbasis Islam. Temuan tersebut menyimpulkan bahwa biaya keagenan, baik syariah maupun konvensional, tidak dapat dihindari karena tata kelola yang ada saat ini tidak cukup memberikan solusi bagi perilaku oportunistik manajer, budaya atau nilai-nilai agama. Maka dari itu, Alam et al. (2020) menyarankan bahwa memiliki dewan pengawas syariah saja tidak cukup. Peran dewan pengawas syariah perlu ditingkatkan dengan tidak hanya berfokus pada aspek teknis dan legalitas transaksi bank syariah tetapi juga dengan menekankan kewajiban untuk terus mendorong penerapan akhlak Islam. Kerangka hukum yang tepat ditambah dengan pengawasan dan penegakan peraturan sangat penting untuk kerangka kerja tata kelola perusahaan syariah yang baik. Kritik yang diajukan terhadap konsep keagenan Islam lainnya terletak pada keperluan untuk melakukan perluasan perspektif keagenan. Hubungan keagenan disarankan untuk didekati tidak hanya dengan fokus individu tetapi juga harus mencakup dimensi sosial dan moral. Masalah keagenan bukan tidak bisa ditafsirkan ulang pada tingkat individu, kelompok, organisasi dan masyarakat. Dalam artian bahwa penerapan konsep keagenan Islam tidak dapat terbatas dalam konflik yang sifatnya horizontal namun dapat diperluas dengan menjelaskan konflik yang berbentuk vertikal (Ceyhan dan Barca, 2021:13) Salah satu usaha untuk memperluas perspektif tersebut ialah dengan memahami tatanan sosial masyarakat hari ini. Salah satu penelitian berkaitan dengan hal tersebut dilakukan oleh Ul-Haq et al. (2020:3) di mana mereka berupaya untuk menunjukkan proses-proses di mana kapitalisme mampu untuk merestrukturisasi dan mengooptasi sistem perbankan Islam. Melalui teori kooptasi, mereka menunjukkan proses-proses di mana kapitalisme neoliberal mampu merampas sistem alternatif yang berupaya dibangun oleh sistem keuangan Islam. Hal ini sangat kontras dengan Islam dengan epistemologi teistik dan kepercayaan kepada Allah swt. di mana pembebasan manusia berarti tunduk pada kehendak Allah swt. Dalam sistem kapitalis, individu dimodelkan sebagai pemaksimalan rasional yang mementingkan diri sendiri dengan mentalitas kompetitif dan hedonis sementara Islam mengasumsikan bahwa individu difokuskan pada kerja sama, empati, cinta, dan komunitas dengan mentalitas altruistik. 13 P-ISSN: 1979-1712, E-ISSN: 2829-0615 P-ISSN: 1979-1712, E-ISSN: 2829-0615 Keterikatan dengan modal pada akhirnya menantang batas-batas dan solidaritas yang ditawarkan oleh perbankan Islam sebagai suatu alternatif bagi kapitalisme. Perkembangan industri perbankan syariah mengindikasikan ketergantungannya pada wacana dan teknik kapitalis yang ada di mana terdapat kecenderungan jika industri perbankan Islam ini berkembang semakin besar dalam ukuran dan signifikansi ekonominya maka semakin mirip dengan industri perbankan konvensional yang berfokus pada efisiensi ekonomi, bukan pada misi awal yaitu keadilan sosial dan redistribusi (Ul-Haq et al., 2020:19). Meskipun sulit bagi industri perbankan syariah untuk mengubah arahnya dikarenakan skala dan cakupan aktivitasnya yang sangat besar dan tersebar di seluruh dunia, Ul-Haq et al. (2020:20) tetap menunjukkan bagaimana industri ini dapat tetap mematuhi prinsip dan semangat pedoman Islam. Pertama, perusahaan perlu menolak rasionalitas yang bersifat instrumental dari tujuan kinerja dan maksimalisasi kekayaan pemegang saham dan membangun tujuan yang bersifat non-kinerja. Dengan kata lain, diperlukan pelibatan yang aktif dengan tradisi Islam yang kaya untuk menghasilkan imajinasi radikal yang berfokus pada peningkatan keadilan sosial, mengurangi ketidaksetaraan, dan menegaskan nilai-nilai non-pasar. Selanjutnya, industri syariah mungkin akan memutuskan untuk melampaui mekanisme utang dan kembali ke model pembiayaan berbasis ekuitas dan model kemitraan dengan struktur tata kelola yang dimiliki bersama. Langkah ini membutuhkan banyak eksperimen dan merupakan jalan yang baik untuk penelitian di masa depan. Melalui Tabel 2 penulis mengurai perbedaan antara konsep keagenan konvensional, Islam dan Islam emansipatoris. Pada tabel tersebut penulis menyajikan prinsip serta implikasi teoretis dari ketiga konsep tersebut. Tabel 1. Perbandingan Prinsip Konsep Keagenan dan Implikasinya Perspektif Prinsip Implikasi Konvensional Orientasi diri (self interest) Hubungan principal-agen hanya berbasis kontrak Analisa berbasis kontrak Analisa yang terfokus pada hubungan principal-agen semata Etika Islam Orientasi kepentingan Umma Hubungan principal-agen berbasis amanah Analisa yang berbasis akhlak dan moral Islam Hubungan yang hadir mengabaikan dan cenderung mempertahankan status quo Islam Emansipatoris Orientasi kepentingan Umma diatur dalam seperangkat nilai yang dipengaruhi oleh sistem sosial. Berpandangan bahwa hubungan keagenan dipengaruhi pula oleh sistem sosial Memungkinkan kehadiran analisa kritis terhadap konsep keagenan Islam melalui pemahaman mengenai sistem sosial Memungkinkan perluasan perspektif keagenan Tabel 1. 4. Kesimpulan Meskipun konsep keagenan sangat realistis dan digunakan secara luas dalam banyak konteks, konsep ini masih menuai beragam kritik. Implikasi praktis yang dihadirkan oleh konsep keagenan banyak dikritik karena membawa organisasi pada perilaku yang tidak etis. Konsep keagenan dituduh menciptakan perilaku atau rasionalitas manusia yang rendah yang hanya memprioritaskan kepentingan diri sendiri dan menggunakan segala cara untuk mencapainya. 14 P-ISSN: 1979-1712, E-ISSN: 2829-0615 P-ISSN: 1979-1712, E-ISSN: 2829-0615 Perspektif manusianya dibatasi oleh asumsi individualisme, utilitarianisme, hedonisme, dan kontraktualisme. Perspektif manusianya dibatasi oleh asumsi individualisme, utilitarianisme, hedonisme, dan kontraktualisme. Dalam menanggapi isu tersebut, penerapan wacana Islam mengenai epistemologi tauhid sebagai prinsip dalam kontrak keagenan memiliki potensi besar untuk menciptakan hubungan yang berbeda antara pihak-pihak dalam kontrak keagenan. Teori keagenan konvensional lebih didominasi oleh kepentingan pribadi dan individualisme yang berusaha memaksimalkan utilitasnya sendiri sehingga menimbulkan masalah keagenan yang tidak dapat diselesaikan. Di sisi lain, hubungan keagenan Islam merupakan hubungan yang berpijak pada konsep khusus Tauhid sekaligus Tauhid sebagai fitur sosial yang berbasis moral seperti perilaku Ikhlas (ketulusan), Itqan (berpengetahuan), Amanah (kejujuran), Syura (saling berkonsultasi), dan keadilan. Dengan cara ini, seorang individu maupun sebuah institusi dapat mengintegrasikan keyakinan spiritual ke dalam organisasi untuk memastikan bisnis yang dijalankan sesuai dengan syariah Islam dengan tujuan pencapaian terbaik di dunia maupun di akhirat. Namun dalam perjalanannya, sistem ekonomi berbasis akhlak mengalami keterbatasan dalam mengungkap kemungkinan eksploitasi yang hadir dalam hubungan keagenan. Keterbatasan itu disebabkan oleh absennya analisa struktural yang menjadi persoalan utama dari melencengnya nilai-nilai Islam yang dianut oleh suatu institusi. Maka dari itu menjadi penting untuk mengintervensi diskusi ekonomi Islam dengan perspektif emansipatoris yang dapat membantu mengurai persoalan struktural yang hadir di tengah sistem sosial hari ini. Gagasan Islam emansipatoris hadir dengan keinginan untuk mendudukkan persoalan yang bersifat vertikal. Berbekal peninjauan secara kritis, didapati bahwa konsep keagenan Islam juga memiliki setidaknya dua keterbatasan yaitu pertama ketidaksesuaian hipotesis dengan data riil di lapangan dan yang kedua ialah ketidakluasan perspektif keagenan yang hanya berkutat pada konflik yang bersifat individual. Seturut berlakunya prinsip-prinsip Islam, maka menempatkan Allah swt. sebagai aktor dalam hubungan principal dan agen menjadi tidak terelakkan. Memosisikan Allah swt. sebagai aktor berkonsekuensi pada perhatian yang lebih utama pada dimensi sosial serta kerangka akhlak sebagai dasar dari setiap perilaku principal maupun agen. Perhatian yang lebih pada akhlak inilah yang menjadi salah satu pembeda utama antara konsep keagenan Islam dan konvensional. P-ISSN: 1979-1712, E-ISSN: 2829-0615 P-ISSN: 1979-1712, E-ISSN: 2829-0615 4. Kesimpulan Intervensi perspektif Islam emansipatoris berupaya untuk menggagas konsep keagenan yang tidak abai terhadap dimensi moral serta sosial yang turut memberi pengaruh terhadap masalah keagenan. Melalui pendekatan kritisnya yang turut menjadi karakteristik yang menonjol dari Islam emansipatoris, memungkinkan perluasan perspektif terhadap konsep keagenan Islam yang mampu melampaui persoalan yang bersifat horizontal. 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https://openalex.org/W4287889565	https://aclanthology.org/2022.semeval-1.32.pdf	English	null	CSECU-DSG at SemEval-2022 Task 3: Investigating the Taxonomic Relationship Between Two Arguments using Fusion of Multilingual Transformer Models	Proceedings of the 16th International Workshop on Semantic Evaluation (SemEval-2022)	2,022	cc-by	3,368	**The first two authors have equal contributions. CSECU-DSG at SemEval-2022 Task 3: Investigating the Taxonomic Relationship Between Two Arguments using Fusion of Multilingual Transformer Models Abdul Aziz, Md. Akram Hossain, and Abu Nowshed Chy Department of Computer Science and Engineering University of Chittagong, Chattogram-4331, Bangladesh {aziz.abdul.cu, akram.hossain.cse.cu}@gmail.com, and nowshed@cu.ac.bd Abdul Aziz, Md. Akram Hossain, and Abu Nowshed Chy Department of Computer Science and Engineering University of Chittagong, Chattogram-4331, Bangladesh {aziz.abdul.cu, akram.hossain.cse.cu}@gmail.com, and nowshed@cu.ac.bd Sentence Language Label / Score Sub-task 1 Apprezzo il vino , ma non il Chianti. It 1 J’ aime les chats, sauf les beagles. Fr 0 I like movies, but not comedies. En 1 I like earrings, except socks. En 0 Sub-task 2 Amo i merli pi ˜A¹ degli uccelli. It 1.9 J’aime les chats, et aussi les canards. Fr 6.17 I like cats, but not sparrows. En 4.77 Table 1: Examples of sub-task 1 and sub-task 2. Abstract Recognizing lexical relationships between words is one of the formidable tasks in compu- tational linguistics. It plays a vital role in the improvement of various NLP tasks. However, the diversity of word semantics, sentence struc- ture as well as word order information make it challenging to distill the relationship effec- tively. To address these challenges, SemEval- 2022 Task 3 introduced a shared task PreTENS focusing on semantic competence to determine the taxonomic relations between two nominal arguments. This paper presents our participa- tion in this task where we proposed an approach through exploiting an ensemble of multilingual transformer models. We employed two fine- tuned multilingual transformer models includ- ing XLM-RoBERTa and mBERT to train our model. To enhance the performance of individ- ual models, we fuse the predicted probability score of these two models using weighted arith- metic mean to generate a unified probability score. The experimental results showed that our proposed method achieved competitive per- formance among the participants’ methods. Table 1: Examples of sub-task 1 and sub-task 2. tual relation (Maksimov et al., 2018) to infer the lexical relation in between words. Considering this PreTENS shared task at Se- mEval 2022 (Zamparelli et al., 2022) introduce a new task that focuses mainly on semantic com- petence with specific attention on the estimating taxonomic relations between two nominal argu- ments. The taxonomic relation here means one argument is a supercategory of the other, or in ex- tensional terms, one argument is a superset of the other. The task is divided into two subtasks. The first one is a binary classification subtask, where a system needs to predict the acceptability label for given text considering the taxonomic relation. The second one is a regression subtask, where a system needs to predict the percentage of acceptability la- bel on a seven-point Likert-scale for a given text considering the same scenario. Besides, the task addresses the challenges of multilingual expression and comprises a dataset of three different languages including Italian (It), French (Fr), and English (En). To illustrate a clear view of the task definition and research goal, we articulate a few examples from different languages and corresponding labels for each subtask in Table 1. Proceedings of the 16th International Workshop on Semantic Evaluation (SemEval-2022), pages 255 - 259 July 14-15, 2022 ©2022 Association for Computational Linguistics 2.2 Multilingual BERT Multilingual-BERT (mBERT) (Devlin et al., 2019) is a version of BERT that is gaining popularity for effective contextual representation of textual con- tents in various multilingual tasks including natural language inference in cross-lingual characteristics, named entity recognition in multilingual corpora, and dependency parsing (Chi et al., 2020). It is pre- trained on 104 different languages in Wikipedia. It provides an effective path to zero-shot cross-lingual model transfer. 1 Introduction Lexical relations are regarded as the most important semantic relations to infer the meanings of words effectively (Khoo and Na, 2006). Therefore, iden- tifying such relations is beneficial to understand the semantic competence and distill the underlying context of the textual expression. If we consider the NLP applications, it has a significant impact on several tasks including semantic search, automatic question/answering, story generation, relation ex- traction, and machine translation (Barkan et al., 2020). However, most of the prior works focused mainly on syntactic (Luu et al., 2014) and contex- We articulate the rest of the contents as fol- lows: Section 2 describes our proposed approach 255 Multilingual BERT (mBERT) Feed-forward Linear Layer Probability Scores Figure 2: Multilingual BERT (mBERT) model. whereas, in Section 3, we present our experimental setup and conduct performance analysis against the various settings and participants’ methods. Finally, we conclude our work with some future directions in Section 4. https://huggingface.co/xlm-roberta-base 2 Proposed Framework In this section, we describe our proposed approach for the PreTENS shared task. Our goal is to deter- mine the semantic competence between two argu- ments focusing on the taxonomic relations. The task is articulated into both the binary classification subtask and the regression subtask. We depict the overview of our proposed framework in Figure 1. Multilingual BERT (mBERT) Text XLM-RoBERTa Multilingual BERT Fusion of Probability Scores Acceptability Label Probability Scores Probability Scores Figure 1: Overview of our proposed framework. Figure 2: Multilingual BERT (mBERT) model. Figure 2: Multilingual BERT (mBERT) model. possibility for a one-model-for-many-languages ap- proach rather than a single model per language. However, XLM-R maps the same sentence in dif- ferent languages to similar representations which is crucial for this PreTENS task to learn semantic competence in cross-lingual form. Here, we use the HuggingFace’s implementation of the XLM- RoBERTa-base model (Conneau et al., 2020). It is composed of 12-layers (i.e. transformer block), the dimension of hidden size is 768, the number of the self-attention head is 12, the size of vocabulary is 250K, and containing 270M parameters. Figure 1: Overview of our proposed framework. Given an input sentence containing two nominal arguments, we employ two transformer models in- cluding XLM-RoBERTa and multilingual BERT (mBERT) to extract the diverse contextual features. Later, a feed-forward linear layer is employed in each model to estimate the probability score of each class. In Figure 2, we illustrate an overview of the setup of mBERT transformer model. Finally, we fuse these models’ predictions by taking the weighted arithmetic mean of these scores to deter- mine the final label. 3.2 Experimental Settings Wi = xi ∗M + yi ∗R M + R We now describe the details of our experiments and set of parameters that we have used to design our proposed CSECU-DSG system for each subtask. xi and yi correspond to the mBERT and XLM- RoBERTa probability score, where M and R repre- sents their weight respectively. Wi (i.e. i = {0, 1}) is the unified probability score for each class. Parameter Optimal Value Subtask 1: Parameters used in both models Learning rate 3e-5 Max-len 100 Epoch 3 Batch size 16 Manual seed 4 Subtask 2: Parameters of XLM-RoBERTa Learning rate 3e-5 Max-len 100 Epoch 5 Batch size 8 Manual seed 4 Table 3: Model settings for each subtask. 2.1 XLM-RoBERTa The Facebook AI launched the XLM-RoBERTa as an upgrade to their initial XLM-100 model (Con- neau et al., 2020). It is a scaled cross-lingual sen- tence encoder. Using self-supervised training ap- proaches, it offers state-of-the-art performances in cross-lingual understanding where a model is taught in one language and then applied to multiple languages with no additional training data. This model showed increased performance on numer- ous NLP applications. XLM-RoBERTa creates the Multilingual-BERT representations are influ- enced by high-level grammatical features that are not manifested in any one input sentence which is critical to learn taxonomic relations in PreTENS task. In our approach, we employ the huggingface 256 implementation of the bert-base-multilingual-cased model and perform finetuning the model with the task-specific data. suppositions on the taxonomic status of their argu- ments A and B, e.g. comparatives (I like A more than B ), exemplifications (I like A, and in particu- lar B), generalizations (I like A, and B in general), and others. In Subtask 1, all samples are provided with an acceptability label either 1 or 0 where 1 stands for acceptable (i.e. the taxonomical rela- tions is compatible with the construction at issue) and 0 stands for not acceptable (not compatible). Besides, a subset of 1533 samples of the whole dataset i.e. 5% of the total and representative of the patterns considered, was used for the subtask 2. It was annotated via a crowdsourcing campaign on a seven point Likert-scale, ranging from 1 (not at all acceptable) to 7 (completely acceptable). The average judgment is considered as the final label for each sample. 2.3 Fusion of Transformer Models To enhance the performance of individual models, we fuse the predicted probability score of two mod- els to generate a unified probability score for each class. We use the weighted arithmetic mean to av- erage both model’s probability scores to determine the each class confidence of the fused model. After calculate the both class probability score of fused model, our system predict the best confidence class, which is our final label. The estimation is computed as follows: f(xi, yi) = ( 0, if W0 > W1 1, otherwise f(xi, yi) = ( 0, if W0 > W1 1, otherwise Wi = xi ∗M + yi ∗R M + R f(xi, yi) = ( 0, if W0 > W1 1, otherwise Wi = xi ∗M + yi ∗R M + R https://huggingface.co/bert-base-multilingual-cased 3.3 Evaluation Measures To assess the performance of the participants’ sys- tems, PreTENS task organizers (Zamparelli et al., 2022) used different strategies and metrics for sub- task 1 and subtask 2. Since the evaluation file contains instances from all three languages, the average of the F1-macro score from all the three languages is used as the global ranking score (GRS) to rank the participants’ systems. We can write this as follows: Table 4: Results of our proposed CSECU-DSG system on individual monolingual tracks (subtask 1). Language Spearman Cor. (Rho) It 0.207 Fr 0.081 En 0.191 All (CSECU-DSG) 0.160 Table 5: Results of our proposed CSECU-DSG system on individual monolingual tracks (subtask 2). Language Spearman Cor. (Rho) Global Rank Score, GRS = AS 3 where AS=(F1-macro (English) + F1-macro (French) + F1-macro (Italian)). Table 5: Results of our proposed CSECU-DSG system on individual monolingual tracks (subtask 2). In subtask 2, the average of the Spearman corre- lation (Rho) scores from all the three languages is used as the global ranking score (GRS) to rank the participants’ systems. We can write this as follows: Next, the obtained results of our proposed CSECU-DSG system in the PreTENS shared task along with other top performing and competitive participants systems in subtask 1 and subtask 2 are articulated in Table 6 and Table 7, respectively. Following the benchmark of the PreTENS shared task, participants’ systems are ranked based on the primary evaluation measures of each subtask. Global Rank Score, GRS = AS 3 where AS=(Rho (English) + Rho (French) + Rho (Italian)). 3.1 Dataset Description The organizers of the SemEval-2022 PreTENS shared task 3 (Zamparelli et al., 2022) provided a benchmark dataset to evaluate the performance of the participants’ systems. The dataset comprises in 3 languages including English, Italian, and French. The French and Italian are slightly adapted transla- tions of the English dataset. The dataset statistics is summarized in Table 2. Subtask 2: Parameters of XLM-RoBERTa Language Sub-task 1 Sub-task 2 Train Test Train Test It 5837 14560 524 1009 Fr 5837 14560 524 1009 En 5837 14560 524 1009 Total 17511 43680 1572 3027 Table 2: The statistics of the datasets used in PreTens shared task. Table 3: Model settings for each subtask. Table 3: Model settings for each subtask. In Subtask 1, we utilize the Huggingface (Wolf et al., 2019) implementation of the two state-of- the-art multilingual transformer models with fine- tuning, including XLM-RoBERTa and mBERT. We finetune these models with the provided training data. To generate the unified prediction, we fuse the probability score of each model as described in Section 2.3. Since XLM-R typically perform better than the mBERT, so we don’t count both model confidence weight as equal. However, from some sets of experimental result we choose the Table 2: The statistics of the datasets used in PreTens shared task. Dataset of each languages contained about 20K artificially generated text samples that enforces pre- 257 use only single transformer models instead of the fusion approach and failed to address and analyze the challenges of subtask 2 properly. weight R = 0.6 for XLM-RoBERTa and M = 0.4 for mBERT in equation 2.3. However, in Subtask 2, we only employed the XLM-RoBERTa model with finetuning strategy. The optimal parameter settings used in both subtasks are articulated in Table 3 and we used the default settings for the other parameters. Language F1-macro F1 It 91.113 90.620 Fr 90.732 90.334 En 91.506 91.189 All (CSECU-DSG) 91.117 - References Oren Barkan, Avi Caciularu, and Ido Dagan. 2020. Within-between lexical relation classification. In Proceedings of the 2020 Conference on Empirical Methods in Natural Language Processing (EMNLP), pages 3521–3527. Ethan A Chi, John Hewitt, and Christopher D Man- ning. 2020. Finding universal grammatical relations in multilingual bert. In Proceedings of the 58th An- nual Meeting of the Association for Computational Linguistics, pages 5564–5577. Table 7: Comparative results with other selected partici- pants (Sub-task 2). Alexis Conneau, Kartikay Khandelwal, Naman Goyal, Vishrav Chaudhary, Guillaume Wenzek, Francisco Guzm´an, ´Edouard Grave, Myle Ott, Luke Zettle- moyer, and Veselin Stoyanov. 2020. Unsupervised cross-lingual representation learning at scale. In Pro- ceedings of the 58th Annual Meeting of the Asso- ciation for Computational Linguistics, pages 8440– 8451. the challenges of the PreTENS shared task. How- ever, in subtask 2, our system obtained a poor score though we ranked 8th in this task. To further analyze the effectiveness of the com- ponents used in our approach, we estimate the performance of each model used in the fusion ap- proach in subtask 1. The results are reported in Ta- ble 8. It shows that our fusion strategy improves the overall performance of the model compared to the performance of the mBERT and XLM-RoBERTa. However, considering the individual model’s per- formances XLM-RoBERTa performed better com- pared to the mBERT. Jacob Devlin, Ming-Wei Chang, Kenton Lee, and Kristina Toutanova. 2019. Bert: Pre-training of deep bidirectional transformers for language understand- ing. In Proceedings of the 2019 Conference of the North American Chapter of the Association for Com- putational Linguistics: Human Language Technolo- gies, Volume 1, pages 4171–4186. Christopher SG Khoo and Jin-Cheon Na. 2006. Seman- tic relations in information science. Annual review of information science and technology, 40(1):157–228. Method F1-macro Global It Fr En CSECU-DSG 91.117 91.113 90.732 91.506 Performance of individual model XLM-RoBERTa 90.217 90.322 89.853 90.475 mBERT 88.076 86.477 88.288 89.463 Table 8: Performance analysis of individual model using subtask 1 test dataset. Anh Tuan Luu, Jung-jae Kim, and See Kiong Ng. 2014. Taxonomy construction using syntactic contextual evidence. In Proceedings of the 2014 Conference on Empirical Methods in Natural Language Processing (EMNLP), pages 810–819. NV Maksimov, AS Gavrilkina, VV Andronova, and IA Tazieva. 2018. Systematization and identification of semantic relations in ontologies for scientific and technical subject areas. Automatic Documentation and Mathematical Linguistics, 52(6):306–317. Table 8: Performance analysis of individual model using subtask 1 test dataset. References Thomas Wolf, Lysandre Debut, Victor Sanh, Julien Chaumond, Clement Delangue, Anthony Moi, Pier- ric Cistac, Tim Rault, R´emi Louf, Morgan Funtowicz, et al. 2019. HuggingFace’s transformers: State-of- the-art natural language processing. arXiv preprint arXiv:1910.03771. 3.4 Results and Analysis H ever, in subtask 2, our system obtained a poor s though we ranked 8th in this task. To further analyze the effectiveness of the c ponents used in our approach, we estimate performance of each model used in the fusion proach in subtask 1. The results are reported in ble 8. It shows that our fusion strategy improve overall performance of the model compared to performance of the mBERT and XLM-RoBE However, considering the individual model’s formances XLM-RoBERTa performed better c pared to the mBERT. Method F1-macro Global It Fr En CSECU-DSG 91.117 91.113 90.732 91.50 Performance of individual model XLM-RoBERTa 90.217 90.322 89.853 90.47 mBERT 88.076 86.477 88.288 89.46 Table 8: Performance analysis of individual model u subtask 1 test dataset. 4 Conclusion and Future Directions In this paper, we presented our proposed syste address the challenges of the PreTENS shared We employed the weighted fusion of two stat the-art multilingual transformer models predict Experimental results demonstrate the compete of our approach in Subtask 1. In the future, we have a plan to incorporate task specific features and technologies to add the challenges properly. We also have a pla explore the causal inference techniques to distill the taxonomic relation. Team (Rank) Spearman Correlation (Rho) Global It Fr En CSECU-DSG (8th) 0.160 0.207 0.081 0.191 Comparative performance of team based on Rho score LingJing (1st) 0.802 0.807 0.841 0.758 huawei zhangmin (3rd) 0.669 0.631 0.740 0.636 daydayemo (6th) 0.206 0.121 0.284 0.212 breaklikeafish (11th) 0.078 0.186 -0.010 0.059 thanet.markchom (13th) 0.056 0.089 -0.043 0.122 Baseline (4th) 0.309 0.344 0.317 0.265 Table 7: Comparative results with other selected partici- pants (Sub-task 2). 3.4 Results and Analysis Team (Rank) F1-macro Global It Fr En CSECU-DSG (4th) 91.117 91.113 90.732 91.506 Performance of team based on F1-macro score LingJing (1st) 94.485 93.047 93.236 97.172 injySarhanUU (3rd) 91.574 92.118 89.529 93.076 holdon (6th) 89.579 86.903 87.281 94.551 cnxupupup (10th) 86.676 86.755 86.390 86.883 breaklikeafish (15th) 77.985 74.398 82.345 77.213 Baseline (18th) 67.394 59.588 72.126 70.468 Table 6: Comparative results with other selected partici- pants (Sub-task 1). Team (Rank) F1-macro Global It Fr En CSECU-DSG (4th) 91.117 91.113 90.732 91.506 Performance of team based on F1-macro score LingJing (1st) 94.485 93.047 93.236 97.172 injySarhanUU (3rd) 91.574 92.118 89.529 93.076 holdon (6th) 89.579 86.903 87.281 94.551 cnxupupup (10th) 86.676 86.755 86.390 86.883 breaklikeafish (15th) 77.985 74.398 82.345 77.213 Baseline (18th) 67.394 59.588 72.126 70.468 Table 6: Comparative results with other selected partici- pants (Sub-task 1). In this section, we analyze the performance of our proposed approaches in the PreTENS shared sub- tasks. The dataset comprises of 3 different lan- guages including English, Italian, and French and the overall performance of the system is estimated considering the average score obtains in each lan- guages dataset. Considering this, we analyze the performance of our CSECU-DSG system, based on each language. The corresponding results for subtask 1 and subtask 2 are reported in Table 4 and Table 5, respectively. Results showed that in Subtask 1 our CSECU- DSG system achieved a pretty good score in all sets of datasets considering three languages. It demon- strates the generalizability of our approach in di- verse types of languages. However, our method limited to obtain a good score in Subtask 2 for all the datasets. One plausible reason behind this is to Table 6: Comparative results with other selected partici- pants (Sub-task 1). Results showed that our system ranked 4th among the participants’ systems in subtask 1. This deduces the efficacy of our approach in addressing 258 Team (Rank) Spearman Correlation (Rh Global It Fr E CSECU-DSG (8th) 0.160 0.207 0.081 0. Comparative performance of team based on Rho score LingJing (1st) 0.802 0.807 0.841 0. huawei zhangmin (3rd) 0.669 0.631 0.740 0. daydayemo (6th) 0.206 0.121 0.284 0. breaklikeafish (11th) 0.078 0.186 -0.010 0. thanet.markchom (13th) 0.056 0.089 -0.043 0. Baseline (4th) 0.309 0.344 0.317 0. Table 7: Comparative results with other selected pa pants (Sub-task 2). the challenges of the PreTENS shared task. 4 Conclusion and Future Directions In this paper, we presented our proposed system to address the challenges of the PreTENS shared task. We employed the weighted fusion of two state-of- the-art multilingual transformer models predictions. Experimental results demonstrate the competency of our approach in Subtask 1. Robertoa Zamparelli, Shammur Absar Chowdhury, Dominique Brunato, Cristiano Chesi, Felice Dell’Orletta, Arid Hasan, and Giulia Venturi. 2022. SemEval-2022 Task3 (PreTENS): Evaluating neural networks on presuppositional semantic knowledge. In Proceedings of the 16th International Workshop on Semantic Evaluation (SemEval-2022). Association for Computational Linguistics. We employed the weighted fusion of two state-of- the-art multilingual transformer models predictions. Experimental results demonstrate the competency of our approach in Subtask 1. In the future, we have a plan to incorporate the task specific features and technologies to address the challenges properly. We also have a plan to 259
https://openalex.org/W2531085542	https://europepmc.org/articles/pmc5059664?pdf=render	English	null	Airy beam self-focusing in a photorefractive medium	Scientific reports	2,016	cc-by	5,049	Noémi Wiersma1, Nicolas Marsal1,2, Marc Sciamanna1,2 & Delphine Wolfersberger1,2 The unique bending and shape-preserving properties of optical Airy beams offer a large range of applications in for example beam routing, optical waveguiding, particle manipulation and plasmonics. In these applications and others, the Airy beam may experience nonlinear light-matter interactions which in turn modify the Airy beam properties and propagation. A well-known example is light self- focusing that leads to the formation of spatial soliton. Here, we unveil experimentally the self-focusing properties of a 1D-Airy beam in a photorefractive crystal under focusing conditions. The transient evolution involves both self-bending and acceleration of the initially launched Airy beam due to the onset of an off-shooting soliton and the resulting nonlocal refractive index perturbation. Both the transient and stationary self-focusing properties can be tuned by varying the bias electric field, the injected Airy beam power and the background illumination. received: 27 June 2016 accepted: 19 September 2016 Published: 12 October 2016 Although being a truncated solution of the ideal Airy waveform, the optical Airy beam keeps its accelerating, non-spreading and self-healing properties over a finite distance1. The propagation of Airy beams in nonlinear media has been first studied in an unbiased photorefractive medium, where an optical beam is mainly subject to the diffusion effect. It has been shown that the peculiar asymmetrical Airy field distribution enables the Airy beam to undergo self-trapping, i.e. the diffraction of an Airy beam can be annihilated via the carrier diffusion effect in a nonlinear medium over a longer distance2,3. This observation also holds for Airy pulses4.f fh Besides the shape-preserving propagation of an Airy beam in diffusive media, the propagation direction of the accelerating beam can also be altered via an externally applied electric field. By studying further the impact of the nonlinearity of the medium on the propagation of an Airy beam, various theoretical as well as experimental studies have demonstrated that the shape and trajectory and therefore the acceleration of the Airy beam can be engineered via a refractive index variation5–7. The control of the Airy beam’s ballistic properties using either the medium nonlinearity or photonic lattices8,9 offers new possibilities in all-optical waveguiding or routing. yf g g g All these previous studies considered the nonlinear propagation of an Airy beam where the beam preserves its multi-lobe distribution and its curved trajectory (i.e. self-trapping). 1LMOPS, CentraleSupélec, Université de Lorraine, 57070 METZ, France. 2LMOPS, CentraleSupélec, Université Paris-Saclay, 57070 METZ, France. Correspondence and requests for materials should be addressed to D.W. (email: delphine.wolfersberger@centralesupelec.fr) www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Airy beam self-focusing in a photorefractive medium OPEN Noémi Wiersma1, Nicolas Marsal1,2, Marc Sciamanna1,2 & Delphine Wolfersberger1,2 Scientific Reports \| 6:35078 \| DOI: 10.1038/srep35078 Results O Our experiment consists of propagating a one-dimensional Airy beam (λ = 532 nm) into a photorefractive SBN-crystal with dimensions 5 mm5 mm1 cm (nSBN = 2.3) as depicted on Fig. 1. The one-dimensional Airy beam is generated using a cubic phase modulation on a spatial light modulator and is defined by the initial conditions: ψ(x)z=0,t=0 = Ai((x + xA)/xA)exp(a(x + xA)/xA), where xA = 10 μm is the main lobe’s waist and a = 0.04 the trun- cation parameter of the Airy beam. Under linear conditions, the beam propagates along the z-axis of the crystal with a transverse parabolic acceleration along the c-axis (parallel to the x-axis) of the photorefractive crystal. In our experiment we set x = 0 as the transverse output position of the linear Airy beam. < = − − x z L ( ) peak L z k x ( ) 4 A 2 2 3 (where k = 2πn/λ is the wave vector) mathematically describes the transverse parabolic acceleration. As a conse- quence, the Airy main lobe has initially been launched at −xpeak(z = 0) = 34 μm.i q , y y peak( ) μ When an external electrical bias field Ee is applied along the x-axis at t = 0 s, the optical Airy beam pho- toinduces a refractive index variation in the crystal through the Pockels effect. The photorefractive effect in the SBN-crystal induces both a focusing and a shift of the optical energy along the transverse c-axis thanks to mainly two contributions: the drift effect induced by the electrical bias field and the thermal diffusion effect14. To optimize the nonlinear photorefractive and solitonic effect of our system, the external electrical bias field is set at Ee = 4 kV/cm. In this nonlinear regime, the propagating 1D Airy beam turns into a so-called “off-shooting soliton” along the tangential direction (z-axis) and an accelerating beam13,15. The theoretical output position of the off-shooting soliton then matches with the input position of the main lobe of the Airy beam, i.e. no trans- verse shift along the x-axis. To analyze the dynamics of this nonlinear interaction, we image the output face of the crystal through a CCD camera. Figure 1(b–f) show the evolution of the intensity profile of the output beam versus time. Starting at t = 0 s, the intensity shifts towards the position of the linear second lobe, further towards the higher lobe’s orders [Fig. Noémi Wiersma1, Nicolas Marsal1,2, Marc Sciamanna1,2 & Delphine Wolfersberger1,2 When applying a small bias electric field to the nonlinear medium, the Airy beam still undergoes self-trapping and the main lobe is narrower while still accel- erating3. However, recent works have suggested the possibility to induce spatial solitons through self-focusing (and not self-trapping) of optical Airy beams3,10–13. When applying a larger bias electric field to the nonlinear medium, the Airy beam does not entirely turn into a soliton, but decomposes itself into a so-called off-shooting soliton and an accelerating wave packet. However these self-focusing properties of an optical Airy beam have not been experimentally demonstrated. In this Letter we experimentally demonstrate the existence of solitonic beam structures induced by an Airy beam under strong nonlinear self-focusing conditions. When applying a bias electric field in the direction of the c-axis of a photorefractive medium, the Airy beam splits into a weak accelerating structure and an off-shooting soliton propagating along the crystal without transverse acceleration. These results match the theoretical pre- dictions in literature3,10–13. By studying experimentally for the first time the transient self-focusing of the Airy beam, we unveil a two-steps dynamics. When applying an external bias voltage on the medium, the Airy beam first self-focuses into a solitonic structure as theoretically predicted. This solitonic beam coexists with an acceler- ating wave and, for longer times, relaxes into an Airy-like accelerating beam. By analyzing the properties of this off-shooting soliton build-up, we show that the onset of an off-shooting soliton shed from the initial Airy beam involves both self-bending and acceleration of the initially launched Airy beam. In addition, we demonstrate that both the transient and stationary self-focusing properties can be tuned by varying the bias electric field, the injected Airy beam power and an external background illumination applied on top of the photo refractive crystal [Fig. 1]. Scientific Reports \| 6:35078 \| DOI: 10.1038/srep35078 1 1 www.nature.com/scientificreports/ Figure 1. (a) Experimental setup. (b–f) Transverse intensity profile of the output beam under nonlinear focusing conditions (Ee = 4 kV/cm, PA = 400 μW) for increasing times. (g) 1D intensity profile along time. (h) Top-view sketch of the accelerating beam interacting with the off-shooting soliton, superimposed with their intensity profiles along time. Figure 1. (a) Experimental setup. (b–f) Transverse intensity profile of the output beam under nonlinear focusing conditions (Ee = 4 kV/cm, PA = 400 μW) for increasing times. (g) 1D intensity profile along time. Noémi Wiersma1, Nicolas Marsal1,2, Marc Sciamanna1,2 & Delphine Wolfersberger1,2 (h) Top-view sketch of the accelerating beam interacting with the off-shooting soliton, superimposed with their intensity profiles along time. Results O 1(b,c)] and finally reaches a maximum transverse shift x = −34 μm at t = 640 ms [Fig. 1(d)]. We will further refer to this position as the off-shooting soliton’s position (red dashed line). Then, on a longer time-scale a relaxation-type dynamics is observed towards a redistributed Airy-like profile similar to the input beam at t = 0 s [Fig. 1(e,f)]. The spatiotemporal dynamics of the nonlinearly propagating Airy beam can therefore be summarized in three stages. (i) First the output beam focuses towards the red dashed line of Fig. 1(g,h). (ii) Then we observe two co-existing beam’s structures [Fig. 1(g,h)]: the so-called off-shooting soliton at x/xA = −3.7 and an accelerating structure at x/xA = 0.5 with similar intensities. (iii) Finally the two previous solutions merge and form a new Airy-like structure on a longer time scale. Similar to the relaxation dynamics of a spatial soliton formed by a self-focused Gaussian beam14,16, the accelerating beam therefore relaxes for longer times into a less focused multi-lobe beam with a peak intensity that shifts back towards the +x-axis. t As we will now detail, the nonlinear interactions that emerge from the transient behavior of a single self-focused Airy beam can be characterized as an attraction of the accelerating structure towards the soli- tonic beam. Simultaneously the accelerating beam presents a tightening effect of the lobes. We show that both attraction and tightening effects can be tuned via the optical power of the initial Airy beam and a background illumination.l In order to characterize the attraction and deflection, we plot in Fig. 2 the nonlinear transverse intensity pro- file of the output beam for increasing times compared to the linear case at t = 0 s [Fig. 2(a)]. xd corresponds to the shift of the accelerating wave packet induced by the attraction of the off-shooting soliton with respect to the initial launched Airy beam. The position of the initial Airy main lobe defines the zero attraction position. Thus xd < 0 illustrates the attraction of the Airy beam towards the off-shooting soliton’s position (x/xA = −3.7, see Fig. 2(c,d)). It is worth mentioning that on Fig. 2(c–e) the output profile of the accelerating structure does not match with an Airy distribution anymore, but the output beam still presents secondary lobes. This is due to the multi-channel Scientific Reports \| 6:35078 \| DOI: 10.1038/srep35078 2 www.nature.com/scientificreports/ Figure 2. Results O 2(h), the tightening of the interlobes’ distance induces an increase of the acceleration rate from three (PA = 200 μW) to five times (PA = 900 μW) the initial value. h lf b d f h l b d h ff h l d h ( ) 0 ized peak acceleration for increasing input powers PA is calculated as follows: (∂2xpeak/∂z2)NL/(∂2xpeak/ ∂z2)lin = (xA/x0,NL)3. The resulting evolution is also displayed on Fig. 2(h). As shown by the data on Fig. 2(h), the tightening of the interlobes’ distance induces an increase of the acceleration rate from three (PA = 200 μW) to five times (PA = 900 μW) the initial value. ( A μ ) In summary, the self-bending of the accelerating beam towards the off-shooting soliton and its tightening effect are increased with the nonlinearity of the system during the transient self-focusing regime of an Airy beam. In particular the optical power enables to tune the effects of the self-focusing strength on the accelerating struc- ture, hence offering an easy-to-use control parameter for interactions between the solitonic beam and the accel- erating beam.f g As already mentioned, the photorefractive nonlinearity of our system can be tuned by different physical parameters such as the external electric bias field (Ee), the intensity of the launched beam (with π = I P x /(2 ) A A 2 in the main lobe) but also the so-called dark intensity of the photorefractive crystal via an external background illumination [Fig. 1]. Such an illumination tends to artificially increase the dark conductivity of the photorefrac- tive crystal which is initially very weak (I/Id ≈ ∞). In photorefractive systems using Gaussian beams it has been shown that Id plays a significant role during the self-focusing and solitonic regime17. In our Airy beam system, we question how such a background illumination may influence the previous results. Figure 3(a,b) depict the influ- ence of I/Id on the transient and corresponding steady state (t > 8 s) peak values of xd and x0. On Fig. 3(a) for I/Id = 15, the self-bending of the Airy beam observed previously is reduced in the transient regime from = −. x x 3 7 d noI A , d to = −. = x x 1 5 d I I A , / 15 d . Results O Experimental transient self-focusing of an Airy beam. (a–f) Intensity profile of the output beam along time, PA = 400 μW. (a) Linear intensity profile, (b–d) build-up of the off-shooting soliton, (e,f) relaxation into a multi-lobe stationary solution. The dashed lines correspond to the linear profile. (g) Attraction effect: transverse position of the output intensity peak versus time for different input powers. (h) Tightening effect: normalized acceleration and main lobe’s waist x0 of the Airy beam nonlinearly attracted towards its off-shooting soliton for increasing input power PA. Figure 2. Experimental transient self-focusing of an Airy beam. (a–f) Intensity profile of the output beam Figure 2. Experimental transient self-focusing of an Airy beam. (a–f) Intensity profile of the output beam along time, PA = 400 μW. (a) Linear intensity profile, (b–d) build-up of the off-shooting soliton, (e,f) relaxation into a multi-lobe stationary solution. The dashed lines correspond to the linear profile. (g) Attraction effect: transverse position of the output intensity peak versus time for different input powers. (h) Tightening effect: normalized acceleration and main lobe’s waist x0 of the Airy beam nonlinearly attracted towards its off-shooting soliton for increasing input power PA. waveguiding structure photoinduced by the multi-lobe structure of the Airy beam at t = 0 s. After t = 1.9 s, the solitonic structure vanishes and the intensity redistributes into an Airy-like profile [Fig. 2(f)]. Figure 2(g) details the temporal evolution of the transverse intensity peak’s position at the output of the crystal during the transient build-up regime of the off-shooting soliton (t < 800 ms, stages (i)–(ii)). Initially the Airy-distributed energy is mainly concentrated in the first lobe at x = 0. After the electrical switch-on of the focusing nonlinearity of the system at t = 0 s, the position corresponding to the peak intensity shifts towards the position of the higher lobes’ orders along the −x-axis (bending effect). Then, around t = 500 ms, the position of the peak intensity reaches a quasi-steady position corresponding to the location of the off-shooting soliton until, as depicted on Fig. 1(g), for longer times beyond t = 1 s, the position of the peak intensity shifts back into the position of the main lobe of the accelerating beam. g In a second step we now analyze whether the interactions between the solitonic and accelerating waves can be tuned by varying the nonlinearity. Scientific Reports \| 6:35078 \| DOI: 10.1038/srep35078 Results O In what follows we vary the intensity of the input Airy beam to tune the refractive index modulation depth and analyze the corresponding attraction and tightening effects. Figure 2(g) shows the evolution of the deflection of the accelerating beam for increasing Airy beam powers. Similarly to the self-focusing properties of Gaussian beams17,18, the transient time towards self-focusing is smaller when the input light intensity increases. In addition the increase of input power modifies the transient self-focusing properties. As depicted on Fig. 2(g), when the power increases from PA = 250 μW to PA = 300 μW, the maximum shift does not increase linearly, but jumps from the former second lobe’s position (x/xA = −2.25) to the theoretical output position of the off-shooting soliton. When further increasing the power (PA > 700 μW), the maximal bending of the beam still deviates but saturates at the third lobe’s position (x/xA = −3.7). By varying the optical power it is therefore possible to balance between diffraction and nonlinearity and tune our nonlinear system from a weak interaction (P ≤ 250 μW) to a strong attraction (P > 250 μW) between the accelerating wave packet and the soli- tonic structure. In addition, during the transient self-focusing regime we also observe a tightening of the lobes of the acceler- ating beam. Figure 2(h) depicts the normalized peak tightening rate x0/xA of the lobes of the accelerating beam for increasing power, where x0 is computed by fitting the intensity profile with an Airy beam profile [Fig. 2(a,b)]. As ( ) 2 In addition, during the transient self focusing regime we also observe a tightening of the lobes of the acceler ating beam. Figure 2(h) depicts the normalized peak tightening rate x0/xA of the lobes of the accelerating beam for increasing power, where x0 is computed by fitting the intensity profile with an Airy beam profile [Fig. 2(a,b)]. As the peak acceleration of an Airy beam is linked to the resulting lobes’ size x0 = = − ( ) x z L ( ) acc L k x 4 2 2 0 3 , the normal- ized peak acceleration for increasing input powers PA is calculated as follows: (∂2xpeak/∂z2)NL/(∂2xpeak/ ∂z2)lin = (xA/x0,NL)3. The resulting evolution is also displayed on Fig. 2(h). As shown by the data on Fig. Results O 1 suggested by Belić et al.19, where an optical beam F(x, t) propagates following the nonlinear wave propagation equation in a photorefractive SBN-crystal: ∂ + ∂ = Γ i F F E F, (1) z x 2 0 ∂ + ∂ = Γ i F F E F, z x 2 0 (1) = =             F x z F Ai x x a x x ( , 0) exp , (2) A A 0 (2) Γ = knx r E ( ) , A eff e 2 Γ = knx r E ( ) , (3) A eff e 2 (3) where Γ = 10 is the nonlinear photorefractive coupling strength, reff = 235pm/V is the effective compo- nent of the electro-optic tensor, Ee the external electric field and E0 = Esc/Ee is the homogeneous part of the x-component of the photorefractive space charge field. As the optical intensity modulates the space charge field, the steady-state E0 is equal to E0 = −I0/(1 + I0), I0 = \|F\|2. The time-dependency of the space charge field E0 is calculated from: τ∂ + = −\| \| + \| \| E E F F /(1 ), (4) t 0 0 2 2 (4) where τ = τ0/(1 + I0) is the relaxation time of the crystal, with τ0 the characteristic response time of the crystal. The numerical system is completely dimension-free, in particular the propagation z-axis is normalized to the diffraction length = L kx 2 d A 2 and the transverse x-axis is normalized to the beam’s waist being the lobe’s waist xA = 10 μm in the case of the Airy beam. We fix the truncation parameter a = 0.1 and vary the optical field ampli- tude F0.f 0 Similarly to the experiment, the Airy beam undergoes self-focusing into an off-shooting soliton (at x/xA = −5.5) and an accelerating beam (at x/xA = 0) [Fig. 3(c–e)]. The self-bending and the acceleration effects induced by the off-shooting soliton are also observed and can be enhanced by increasing the optical power and therefore the refractive index change [Fig. 3(f,g)]. The numerical results are in good qualitative agreement with the exper- imental observations of the nonlinear interactions between the off-shooting soliton and the accelerating beam (stages (i) and (ii)). Results O Contrary to the case without background illumination, where the Airy-like structure of stage (iii) is superimposed with the initial Airy beam [Fig. 2(f)], adding Id enables the Scientific Reports \| 6:35078 \| DOI: 10.1038/srep35078 3 www.nature.com/scientificreports/ Figure 3. (a,b) Influence of background illumination on the transient and final self-focusing beam: (a) for the transverse beam’s shift xd (attraction parameter) (b) and the interlobes’ distance x0 (cubic proportional to the acceleration). (c–g) Numerical study: Self-focusing of an Airy beam. (c) Intensity profile of the output beam along time; transverse intensity profiles in the (d) transient and (e) steady-state regime. The dashed lines in (d,e) correspond to the linear profile (t < 0 s). (f) Transverse position of the output intensity peak versus time for increasing optical power (via F0). (g) Acceleration effect: normalized acceleration and main lobe’s waist x0 of the Airy beam for increasing input intensity. Figure 3. (a,b) Influence of background illumination on the transient and final self-focusing beam: (a) for the transverse beam’s shift xd (attraction parameter) (b) and the interlobes’ distance x0 (cubic proportional to the acceleration). (c–g) Numerical study: Self-focusing of an Airy beam. (c) Intensity profile of the output beam along time; transverse intensity profiles in the (d) transient and (e) steady-state regime. The dashed lines in (d,e) correspond to the linear profile (t < 0 s). (f) Transverse position of the output intensity peak versus time for increasing optical power (via F0). (g) Acceleration effect: normalized acceleration and main lobe’s waist x0 of the Airy beam for increasing input intensity. accelerating beam to remain shifted even in the steady-state regime (maximum shift of −xA for I/Id = 30). As depicted on Fig. 3(b), the background illumination also influences the self-focusing effect both in the transient and the steady-state regime. In particular we still observe self-focusing of the accelerating beam after t > 8 s for I/Id = 45. d Our experimental results can be reproduced qualitatively well by numerical simulations. See Fig. 3(c–g). The nonlinear propagation of the Airy beam in the photorefractive crystal can be simulated by the normalized non-linear Schrödinger Eq. References l l J. Opt. B Quantum Semiclassical Opt. 6, S190 (2004). ( ) 20. Bekenstein, R., Schley, R., Mutzafi, M., Rotschild, C. & Segev, M. Optical simulations of gravitational effects in newton–schrödinger system. Nat. Phys. 11, 872–878 (2015). Acknowledgements g The authors acknowledge the support of Conseil Régional de Lorraine, Fondation Supélec, the IAP P7/35 (BELSPO) with the ‘Photonics@be’ project (2012–2017), Fonds européen de développement régional (FEDER) with the projects ‘PHOTON’ (2014-2015) and ‘APOLLO’ (2015), and Préfecture de Lorraine and Fonds National d’Aménagement et de Développement du Territoire (FNADT) with the project ‘APOLLO’. g The authors acknowledge the support of Conseil Régional de Lorraine, Fondation Supélec, the IAP P7/35 (BELSPO) with the ‘Photonics@be’ project (2012–2017), Fonds européen de développement régional (FEDER) with the projects ‘PHOTON’ (2014-2015) and ‘APOLLO’ (2015), and Préfecture de Lorraine and Fonds National d’Aménagement et de Développement du Territoire (FNADT) with the project ‘APOLLO’. Conclusion and Discussioni In summary, this work is the first experimental analysis of the self-focusing properties of an Airy beam in a photorefractive nonlinear medium. The transient self-focusing behavior shows nonlinear interactions between a soliton and an accelerating beam. These interactions result in both (i) an attraction and deflection effect into the off-shooting soliton’s position and (ii) a tightening of the interlobes distance which induces an increase of the acceleration rate. Both attraction and tightening effects can be tuned via the Airy beam intensity or the back- ground illumination. g Very recently, the nonlinear interaction between an accelerating (Airy) beam and a spatial soliton - created by the self-focusing of a Gaussian beam through thermal nonlinearity20 - was analyzed in the context of optical grav- itational lensing. Relying on the analogy with the Newton-Schrödinger model for quantum gravity, the authors relate the long-range interactions between the spatial soliton and the accelerating beam to the effects caused by a mass on light propagating in a gravitational field. In our configuration, where a soliton and an accelerating beam co-exist during the transient self-focusing regime of an Airy beam, the off-shooting soliton shed from the initial Airy beam similarly plays the role of a mass that attracts and deflects the remaining accelerating light from its own curved trajectory. The properties of this analogous gravitational lensing, i.e. deflection and acceleration, can be both controlled all-optically through the engineering of the optical photorefractive nonlinearity. Our obser- vations being very similar to those of ref. 20 but in a different system, our conclusion is therefore that the analogy with gravitational lensing effects is not limited to the Newton-Schrödinger framework but applies more generally to a nonlinear Schrödinger equation that accounts for a non-local nonlinearity. Besides its interest for the analogy with gravitation, the two-stages build-up dynamics of the focused beam provides a deeper insight into the subject of accelerating beams in nonlinear focusing media, and can be used to photoinduce multiple waveguide structures, as suggested in ref. 15. Author Contributions N.W. performed the experimental and numerical simulations under the supervision of D.W., N.M. and M.S. All the authors contributed to the data analysis and to the writing of the manuscript. N.W. performed the experimental and numerical simulations under the supervision of D.W., N.M. and M.S. All the authors contributed to the data analysis and to the writing of the manuscript. Additional Informationihi Results O We note that the numerical simulations do not reproduce the relaxation-type dynamics of the beam (stage (iii)). Although the main focus here is the nonlinear interaction between the accelerating beam and the off-shooting soliton well described by the model Eqs 1–4, this suggests that a more complete description of the relaxation process in self-focusing requires to account for other transport mechanisms as for example carrier diffusion. Scientific Reports \| 6:35078 \| DOI: 10.1038/srep35078 4 www.nature.com/scientificreports/ References l l g gi gy y p 2. Jia, S., Lee, J., Fleischer, J. W., Siviloglou, G. A. & Christodoulides, D. N. Diffusion-Trapped Airy Beams in Photorefractive Media. Phys. Rev. Lett. 104, 253904 (2010). 3. Kaminer, I., Segev, M. & Christodoulides, D. N. Self-accelerating self-trapped optical beams. Phys. Rev. Lett. 106, 213903 (2011). 4. Kaminer, I., Lumer, Y., Segev, M. & Christodoulides, D. N. Causality effects on accelerating light pulses. Opt. Express 19, 23132 (2011). ( ) 5. Hu, Y. et al. Optimal control of the ballistic motion of airy beams. Opt. Lett. 35, 2260 (2010). p y p 6. Ye, Z. et al. Acceleration control of Airy beams with optically induced refractive-index gradient. Opt. Lett. 36, 3230–3232 (2011). 7. Efremidis, N. K. Airy trajectory engineering in dynamic linear index potentials. Opt. Lett. 36, 3006 (2011). 8. Xiao, F. et al. Optical Bloch oscillations of an Airy beam in a photonic lattice with a linear transverse index gradient. Opt. Express 22, 22763 (2014). 9. Diebel, F. et al. Control of Airy-beam self-acceleration by photonic lattices. Phys. Rev. A 90, 033802 (2014). 10. Bekenstein, R. & Segev, M. Self-accelerating optical beams in highly nonlocal nonlinear media. Opt. Express19 19, 23706–23715 (2011). . Lotti, A. et al. Stationary nonlinear airy beams. Phys. Rev. A - At. M 2. Panagiotopoulos, P. et al. Nonlinear propagation dynamics of finite-energy airy beams. Phys. Rev. A - At. Mol. Opt. Phys. 86, 1–15 (2012). eshaping the trajectory and spectrum of nonlinear airy beams. Opt 13. Hu, Y. et al. Reshaping the trajectory and spectrum of nonlinea 4. Maufoy, J., Fressengeas, N., Wolfersberger, D. & Kugel, G. Simulation of the temporal behavior of soliton propagation in photorefractive media. Phys. Rev. E 59, 6116–6121 (1999). p y 5. Wiersma, N., Marsal, N., Sciamanna, M. & Wolfersberger, D. All-optical interconnects using airy beams. Opt. Lett. 39, 5997–6000 (2014). ( ) 16. Wolfersberger, D., Khelfaoui, N., Dan, C., Fressengeas, N. & Leblond, H. Fast photorefractive self-focusing in inp: fe semiconductor at infrared wavelengths. Appl. Phys. Lett. 92, 021106 (2008). g pp y , ( ) 7. Chen, Z., Segev, M. & Christodoulides, D. N. Optical spatial solitons: historical overview and recent advances. Reports Prog. Phys 75, 086401 (2012). 18. Petter, J., Weilnau, C., Denz, C., Stepken, A. & Kaiser, F. Self-bending of photorefractive solitons. Opt. Commun. 170, 291 (1999). ( ) 19. Belić, M. et al. Counterpropagating self-trapped beams in photorefractive crystals. g gi gy y p 2. Jia, S., Lee, J., Fleischer, J. W., Siviloglou, G. A. & Christodoulides, D. N. Diffusion-Trapped Airy Beams in Photorefractive Media Phys. Rev. Lett. 104, 253904 (2010). 1. Siviloglou, G. A. & Christodoulides, D. N. Accelerating finite energy Airy beams. Opt. Lett. 32, 979–981 (2007).f Scientific Reports \| 6:35078 \| DOI: 10.1038/srep35078 Additional Informationi 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: Wiersma, N. et al. Airy beam self-focusing in a photorefractive medium. Sci. Rep. 6, 35078; doi: 10.1038/srep35078 (2016). How to cite this article: Wiersma, N. et al. Airy beam self-focusing in a photorefractive medium. Sci. Rep. 6, 35078; doi: 10.1038/srep35078 (2016). Scientific Reports \| 6:35078 \| DOI: 10.1038/srep35078 5 www.nature.com/scientificreports/ 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/ © The Author(s) 2016 com/scientificreports/ 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/ www.nature.com/scientificreports/ 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/ © The Author(s) 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/ © The Author(s) 2016 © The Author(s) 2016 Scientific Reports \| 6:35078 \| DOI: 10.1038/srep35078 6
https://openalex.org/W4366189032	https://www.nature.com/articles/s41598-023-31850-y.pdf	English	null	Circulating proteins to predict COVID-19 severity	Scientific reports	2,023	cc-by	14,583	Circulating proteins to predict COVID‑19 severity OPEN Chen‑Yang Su 1,2,3,24, Sirui Zhou 1,4,24, Edgar Gonzalez‑Kozlova 5,24, Guillaume Butler‑Laporte 1,4, Elsa Brunet‑Ratnasingham 6, Tomoko Nakanishi 1,7,8,9, Wonseok Jeon 2, David R. Morrison 1, Laetitia Laurent 1, Jonathan Afilalo 1,4, Marc Afilalo 10, Danielle Henry 1, Yiheng Chen 1,7, Julia Carrasco‑Zanini 11, Yossi Farjoun 1, Maik Pietzner 11,18, Nofar Kimchi 1, Zaman Afrasiabi 1, Nardin Rezk 1, Meriem Bouab 1, Louis Petitjean 1, Charlotte Guzman 1, Xiaoqing Xue 1, Chris Tselios 1, Branka Vulesevic 1, Olumide Adeleye 1, Tala Abdullah 1, Noor Almamlouk 1, Yara Moussa 1, Chantal DeLuca 1, Naomi Duggan 1, Erwin Schurr 12, Nathalie Brassard 6, Madeleine Durand 6, Diane Marie Del Valle 13, Ryan Thompson 14, Mario A. Cedillo 15, Eric Schadt 14, Kai Nie 16, Nicole W. Simons 14, Konstantinos Mouskas 14, Nicolas Zaki 16, Manishkumar Patel 13, Hui Xie 16, Jocelyn Harris 16, Robert Marvin 16, Esther Cheng 14, Kevin Tuballes 13, Kimberly Argueta 16, Ieisha Scott 16, The Mount Sinai COVID-19 Biobank Team , Celia M. T. Greenwood 1,4, Clare Paterson 17, Michael A. Hinterberg 17, Claudia Langenberg 11,18, Vincenzo Forgetta 1, Joelle Pineau 2, Vincent Mooser 7, Thomas Marron 19, Noam D. Beckmann 14, Seunghee Kim‑schulze 16, Alexander W. Charney 20, Sacha Gnjatic 5,13,16, Daniel E. Kaufmann 6,21,22, Miriam Merad 13 & J. Brent Richards 1,4,7,23 Predicting COVID-19 severity is difficult, and the biological pathways involved are not fully understood. To approach this problem, we measured 4701 circulating human protein abundances in two independent cohorts totaling 986 individuals. We then trained prediction models including protein abundances and clinical risk factors to predict COVID-19 severity in 417 subjects and tested these models in a separate cohort of 569 individuals. For severe COVID-19, a baseline model including age and sex provided an area under the receiver operator curve (AUC) of 65% in the test cohort. Selecting 92 proteins from the 4701 unique protein abundances improved the AUC to 88% in the training cohort, which remained relatively stable in the testing cohort at 86%, suggesting good generalizability. Proteins selected from different COVID-19 severity were enriched for cytokine and cytokine receptors, 1Lady Davis Institute for Medical Research, Jewish General Hospital, Pavilion H‑413, 3755 Côte‑Ste‑Catherine Montréal, Montreal, QC H3T 1E2, Canada. 2Department of Computer Science, McGill University, Montréal, QC, Canada. 3Quantitative Life Sciences Program, McGill University, Montreal, Quebec, Canada. 4Department of Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal, QC, Canada. www.nature.com/scientificreports www.nature.com/scientificreports Results C h t Cohorts. To establish a proteomic-based prediction model for COVID-19 severity, we used the BQC19 cohort, which consisted of samples from two hospitals in Montreal, with proteomic measurements for train- ing and cross-validation. The final model was tested in an independent cohort from Mount Sinai hospital in New York City. Using the same SomaScan® assay, 4984 SOMAmer reagents measured the levels of 4701 dif- ferent circulating proteins in both the BQC19 and Mount Sinai cohorts. To train our models, we selected 417 individuals which included 313 nasal swab SARS-CoV-2 PCR positive patients with baseline samples collected within 14 days of symptom onset (mean and median time since symptom onset in COVID-19 patients = 7.0 days (SD = 3.96 days)). The BQC19 cohort also included an additional 104 individuals who presented to the same hospital sites with symptoms consistent with COVID-19 but had a negative SARS-CoV-2 PCR nasal swab. The Mount Sinai cohort consisted of 569 individuals with their earliest samples also collected within 14 days of symptom onset. Among them were 472 SARS-CoV-2 positive patients again confirmed by PCR, one patient confirmed by chest CT, and 96 SARS-CoV-2 negative individuals (89 with PCR confirmation). If multiple blood samples were collected from the same person, we used the samples collected at the time point closest to symptom onset. We chose to use samples close to symptom onset to reflect the proteome of acute COVID-19, rather than its recovery phase.h y p The demographic and clinical characteristics of the participants in the training and testing datasets are shown in Table 1. In the BQC19 cohort, the mean age across all samples was 65.3 years (SD = 18.4 years), and 52% of the cohort were men. In the Mount Sinai cohort, the mean age was 59.6 years (SD: 19.4 years), and 58.2% of the cohort were men. For the definition of COVID-19 severity, we focused on two levels: (1) severe COVID-19 was defined as individuals who died or required any form of oxygen supplementation; and (2) critical COVID-19, defined as individuals who died or experienced severe respiratory failure (requiring non-invasive ventilation, high flow oxygen therapy, intubation, or extracorporeal membrane oxygenation). Detailed definitions of these adverse outcomes are described in Methods. The overall study design is shown in Fig. 1, which outlines the training and testing stages of the study. Circulating proteins to predict COVID‑19 severity OPEN 5Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 6Research Centre of the Centre Hospitalier de L’Université de Montréal, Montreal, QC, Canada. 7Department of Human Genetics, McGill University, Montreal, QC, Canada. 8Graduate School of Medicine, McGill International Collaborative School in Genomic Medicine, Kyoto University, Kyoto, Japan. 9Japan Society for the Promotion of Science, Tokyo, Japan. 10Department of Emergency Medicine, Jewish General Hospital, McGill University, Montreal, QC, Canada. 11MRC Epidemiology Unit, School of Clinical Medicine, University of Cambridge, Cambridge, UK. 12Infectious Diseases and Immunity in Global Health Program, Research Institute of the McGill University Health Centre, Montréal, QC, Canada. 13Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 14Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 15Department of Radiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 16Human Immune Monitoring Center, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 17SomaLogic Operating Co., Inc., Boulder, CO, USA. 18Computational Medicine, Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany. 19Immunotherapy and Phase 1 Trials, Mount Sinai Hospital, New York, NY, USA. 20Mount Sinai Clinical Intelligence Center, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 21Department of Medicine, Université de Montréal, Montreal, QC, Canada. 22Division of Infectious Diseases, Department of Medicine, University Hospital of Lausanne and University of Lausanne, Lausanne, Switzerland. 23Department of Twin Research, King’s College London, London, UK. 24These authors contributed equally: Chen-Yang Su, Sirui Zhou and Edgar Gonzalez-Kozlova. A list of authors and their affiliations appears at the end of the paper. email: brent.richards@mcgill.ca \| https://doi.org/10.1038/s41598-023-31850-y Scientific Reports \| (2023) 13:6236 www.nature.com/scientificreports/ but more than half of the enriched pathways were not immune-related. Taken together, these findings suggest that circulating proteins measured at early stages of disease progression are reasonably accurate predictors of COVID-19 severity. Further research is needed to understand how to incorporate protein measurement into clinical care. but more than half of the enriched pathways were not immune-related. Taken together, these findings suggest that circulating proteins measured at early stages of disease progression are reasonably accurate predictors of COVID-19 severity. Further research is needed to understand how to incorporate protein measurement into clinical care. A remarkable feature of COVID-19 disease is its highly variable clinical course, where some individuals mani- fest severe disease or death, and others remain asymptomatic. Circulating proteins to predict COVID‑19 severity OPEN Several clinical and genetic risk factors explain a proportion of these outcomes1–6, yet most of the host biological causes of these adverse COVID-19 outcomes remain unknown.il Recent reports have identified some of the biologic pathways influencing risk of adverse COVID-19, such as immune responses7–11, interferon pathways12–14, and T-cell dysfunction15,16. However, many such studies have focused on narrow sets of pre-selected cytokines. One way to rapidly assess thousands of potential biomark- ers associated with the severity of COVID-19 is through the measurement of blood circulating proteins. Such circulating proteins may be useful because they can help to identify pathways influencing severity of disease. They may also identify individuals at high risk of a severe COVID-19 clinical course. Similarly, circulating proteomic biomarkers have recently been shown to serve as predictors of other common diseases17–24 including cardiovascular disease. They are also relevant in drug discovery because they are generally more accessible to pharmacological manipulation than intracellular proteins25–29. Thus, understanding the circulating proteins asso- ciated with COVID-19 severity may be helpful to address major challenges raised by the current pandemic15,30–41. y y p j g y p We undertook a large-scale study to assess the relationship of thousands of circulating proteins with COVID- 19 severity. To do so, we used machine learning methods to develop a predictive model of COVID-19 severity using the circulating blood protein abundances as predictors. Proteins were measured using 4984 nucleic acid aptamers (SOMAmer® reagents)42 targeting 4701 unique circulating human proteins in two cohorts collected from two countries, which in total included 986 individuals. The training cohort was comprised of 417 individuals from two sites of the Biobanque Québécoise de la COVID-19 (BQC19 cohort). This cohort was used to train a model to predict COVID-19 severity. This model was then tested in a separate test cohort from the Mount Sinai Hospital in New York City, which was similarly characterized for the same protein measurements and COVID-19 severity.hi y y y This large-scale study across two countries and two geographically separated cohorts identified circulating proteins associated with COVID-19 severity measured at a large-scale in well-characterized cohorts. These findings provide insights into the biological pathways influencing these outcomes and the ability of proteomics to predict these outcomes. Results C h t Consistent with recent successful large-scale genetic studies, we defined controls as all participants not meeting case criteria1. p p g A visualization of the distribution of individuals across COVID-19 severity outcomes in the BQC19 and Mount Sinai cohorts is provided in Supplementary Fig. 1. In the BQC19 training cohort 175 individuals were classified as severe cases and 242 individuals were controls. The controls for severe COVID-19 were comprised of 138 SARS-CoV-2 positive individuals not meeting case definition and 104 SARS-CoV-2 negative individuals. In the case of critical disease, 93 individuals out of 313 COVID-19 positive patients were classified as critical cases and 324 individuals were controls. The controls for critical COVID-19 cases were 220 SARS-CoV-2 posi- tive individuals not meeting case definition and 104 participants who were SARS-CoV-2 negative. Results C h t Severe—Cases individuals who tested positive for COVID-19 and died or required any type of respiratory support (including oxygen delivered by nasal prongs). Controls: individuals with COVID-19 but did not meet severe case criteria or individuals who presented with symptoms of COVID-19 but were SARS-CoV-2 PCR negative. Critical—Cases individuals who tested positive for COVID-19 and died or required respiratory support (intubation, continuous positive airway pressure, bilevel positive airway pressure, continuous external negative pressure, or high flow positive end expiratory pressure oxygen). Controls: individuals with COVID-19 but did not meet critical case criteria or individuals who presented with symptoms of COVID-19 but were SARS-CoV-2 PCR negative. Sample Processing Time time in hours between sample collection and sample freezing. CHUM Samples recruited from the Centre Hospitalier de l’Université de Montréal. JGH Samples recruited from the Jewish General Hospital. Table 1. Demographic characteristics of the participating cohorts. Mean (SD). N (%). When the counts of each cell do not sum to the total sample size, this is due to missing data or the patient answering, “I do not know”. Severe—Cases individuals who tested positive for COVID-19 and died or required any type of respiratory support (including oxygen delivered by nasal prongs). Controls: individuals with COVID-19 but did not meet severe case criteria or individuals who presented with symptoms of COVID-19 but were SARS-CoV-2 PCR negative. Critical—Cases individuals who tested positive for COVID-19 and died or required respiratory support (intubation, continuous positive airway pressure, bilevel positive airway pressure, continuous external negative pressure, or high flow positive end expiratory pressure oxygen). Controls: individuals with COVID-19 but did not meet critical case criteria or individuals who presented with symptoms of COVID-19 but were SARS-CoV-2 PCR negative. Sample Processing Time time in hours between sample collection and sample freezing. CHUM Samples recruited from the Centre Hospitalier de l’Université de Montréal. JGH Samples recruited from the Jewish General Hospital. critical disease 233 individuals were cases and 336 were controls. Generally, severe, or critical COVID-19 cases were older than controls in both the training dataset and the testing dataset. Males were also more likely to have severe or critical COVID-19 as compared to females (Table 1). The age and sex distribution of the participants stratified by case/control status for the two COVID-19 severity groups are shown in Supplementary Fig. 2. The distributions suggest that males who develop severe or critical COVID-19 are generally younger than females. Results C h t In the Mount Sinai testing cohort, 392 individuals were classified as severe cases and 177 individuals were controls while for Scientific Reports \| (2023) 13:6236 \| https://doi.org/10.1038/s41598-023-31850-y www.nature.com/scientificreports/ Dataset Training cohort (BQC19) COVID-19 severity Severe Critical All samples (n = 417) Cases (n = 175) Controls (n = 242) Cases (n = 93) Controls (n = 324) Age in years 65.3 (18.4) 67.6 (17.6) 63.7 (18.8) 66.7 (16.5) 64.9 (18.9) Female sex** 200 (48.0) 82 (46.9) 118 (48.8) 36 (38.7) 164 (50.6) Sample processing time (hours)* 9.7 (13.4) 7.5 (6.5) 11.3 (16.5) 7.0 (6.3) 10.4 (14.8) Hospital site CHUM 105 (25.2) 42 (24.0) 63 (26.0) 32 (34.4) 73 (22.5) JGH 312 (74.8) 133 (76.0) 179 (74.0) 61 (65.6) 251 (77.5) Diabetes No 288 (69.1) 100 (57.1) 188 (77.7) 54 (58.1) 234 (72.2) Yes 127 (30.5) 75 (42.9) 52 (21.5) 39 (41.9) 88 (27.2) Chronic obstructive pulmonary disease No 358 (85.9) 145 (82.9) 213 (88.0) 77 (82.8) 281 (86.7) Yes 57 (13.7) 30 (17.1) 27 (11.2) 16 (17.2) 41 (12.7) Chronic kidney disease No 363 (87.1) 151 (86.3) 212 (87.6) 77 (82.8) 286 (88.3) Yes 52 (12.5) 24 (13.7) 28 (11.6) 16 (17.2) 36 (11.1) Congestive heart failure No 355 (85.1) 152 (86.9) 203 (83.9) 80 (86.0) 275 (84.9) Yes 60 (14.4) 23 (13.1) 37 (15.3) 13 (14.0) 47 (14.5) Hypertension No 179 (42.9) 67 (38.3) 112 (46.3) 31 (33.3) 148 (45.7) Yes 235 (56.4) 107 (61.1) 128 (52.9) 61 (65.6) 174 (53.7) Liver disease No 403 (96.6) 172 (98.3) 231 (95.5) 91 (97.8) 312 (96.3) Yes 12 (2.9) 3 (1.7) 9 (3.7) 2 (2.2) 10 (3.1) Smoking status Current smoker 13 (3.1) 6 (3.4) 7 (2.9) 5 (5.4) 8 (2.5) Ex-smoker 47 (11.3) 21 (12.0) 26 (10.7) 11 (11.8) 36 (11.1) Never smoker 233 (55.9) 98 (56.0) 135 (55.8) 40 (43.0) 193 (59.6) Dataset Testing cohort (Mount Sinai) COVID-19 severity Severe Critical All samples (n = 569) Cases (n = 392) Controls (n = 177) Cases (n = 233) Controls (n = 336) Age in years* 59.6 (19.4) 63.0 (17.1) 52.1 (22.1) 63.7 (16.7) 56.8 (20.6) Female sex** 238 (41.8) 154 (39.3) 84 (47.5) 87 (37.3) 151 (44.9) Table 1. Demographic characteristics of the participating cohorts. Mean (SD). *N (%). When the counts of each cell do not sum to the total sample size, this is due to missing data or the patient answering, “I do not know”. Results C h t critical disease 233 individuals were cases and 336 were controls. Generally, severe, or critical COVID-19 cases were older than controls in both the training dataset and the testing dataset. Males were also more likely to have severe or critical COVID-19 as compared to females (Table 1). The age and sex distribution of the participants stratified by case/control status for the two COVID-19 severity groups are shown in Supplementary Fig. 2. The distributions suggest that males who develop severe or critical COVID-19 are generally younger than females. Association of protein abundance with COVID‑19 severity. In order to directly assess if any of the measured proteins were associated with COVID-19 severity, we used multivariable logistic regression to test the association of each of the 4984 SOMAmer reagents with the two COVID-19 outcomes while adjusting for age, sex, sample processing time, and hospital site in the BQC19 cohort. These variables were chosen because they https://doi.org/10.1038/s41598-023-31850-y Scientific Reports \| (2023) 13:6236 \| www.nature.com/scientificreports/ Figure 1. Overall Study design. Schematic of training and testing stages of this study. Severe COVID-19 is defined as death or use of any form of oxygen supplementation. Critical COVID-19 is defined as death or severe respiratory failure (non-invasive ventilation, high flow oxygen therapy, intubation, or extracorporeal membrane oxygenation). Figure 1. Overall Study design. Schematic of training and testing stages of this study. Severe COVID-19 is defined as death or use of any form of oxygen supplementation. Critical COVID-19 is defined as death or severe respiratory failure (non-invasive ventilation, high flow oxygen therapy, intubation, or extracorporeal membrane oxygenation). are readily available in the course of clinical care, representing the minimum set of variables to predict severity. Logistic regression identified 1531 SOMAmer reagents to be associated with severe COVID-19 (Supplementary Table 1) and 1592 SOMAmer reagents (Supplementary Table 2) to be associated with critical COVID-19 when using a Benjamini–Hochberg corrected p value of 0.01 (Supplementary Fig. 3). Model selection and performance using LASSO. One reason why many circulating proteins were associated with COVID-19 severity is that most of the protein levels were highly correlated with each other. Therefore, we used L1 regularized multivariable logistic regression models (LASSO)43 to select uncorrelated proteins that best predicted COVID-19 severity in the BQC19 training cohort. Results C h t We did so for three reasons: (1) LASSO performs well when the number of features is greater than the number of samples (as was the case in our experiment); (2) LASSO forces many correlated features to have a zero coefficient by randomly selecting one of the features (sometimes more than one) from a group of correlated features thereby preventing collinearity; (3) LASSO mitigates the possibility of overfitting43.ii g p yi g We first defined a baseline model which included only the four covariates in the logistic regression model: age, sex, sample processing time, and hospital site to predict COVID-19 severity. We then evaluated whether the addition of proteins would aid in identifying which patients developed severe COVID-19 by adding 4984 SOMAmer reagents to the baseline model. This model, which included baseline covariates and protein levels, is termed the “protein model”. To train both the baseline and the protein models, we performed 10 repeats of stratified fivefold cross-validation using LASSO logistic regression in the BQC19 cohort on both the severe and critical groups. We tuned the penalty parameter “lambda” across each of the 50 cross-validations and selected the lambda value corresponding to the model with the highest area under the receiver operator characteristic curve (AUC), which was averaged over the 50 cross-validation results. Results from the lambda parameter search are shown in Supplementary Fig. 4A, B. pp y g For the best performing model predicting severe COVID-19, we selected a log10 lambda value of − 1.5 which generated an average training AUC of 59% for the baseline model. We next selected a log10 lambda value of 1.0, which generated an average AUC of 88% for the protein model. For the best performing model predicting critical COVID-19, we selected log10 lambda values of − 2.0 and 1.0 corresponding to average cross-validation training AUC scores of 59% and 89% for the baseline and protein model, respectively (Fig. 2a, b). We then used these chosen lambda hyperparameters to build baseline and protein models for severe and critical COVID-19 using the entire BQC19 cohort and evaluated their performance in the independent external test cohort from Mount Sinai. Elastic net regularized logistic regression models were also trained on the BQC19 cohort but not tested on the Mount Sinai cohort due to negligible differences in training performance (Supplementary Note). Results C h t g gf g p ( pp y ) When testing the prediction of severe COVID-19 in the independent Mount Sinai cohort, AUC performance of the baseline model improved from 59% in the BQC19 training cohort to 65% in the Mount Sinai testing cohort. The AUC of the protein model decreased slightly between training and testing (88% vs. 86%).h h p g y g g ( ) The AUC of the protein model for predicting critical COVID-19 also decreased from a training score of 89% to 80% in the test set. In contrast, prediction of critical COVID-19 using the baseline model was consistent https://doi.org/10.1038/s41598-023-31850-y Scientific Reports \| (2023) 13:6236 \| www.nature.com/scientificreports/ a b c Figure 2. AUC score results. (a) L1 regularized logistic regression training and testing results for severe COVID-19 and (b) critical COVID-19. Blue and red are used to represent the protein model and baseline model, respectively, while solid and dotted lines represent the testing and training performance, respectively. i a b c Figure 2. AUC score results. (a) L1 regularized logistic regression training and testing results for severe COVID-19 and (b) critical COVID-19. Blue and red are used to represent the protein model and baseline model, respectively, while solid and dotted lines represent the testing and training performance, respectively. Shaded areas denote the 95% confidence intervals for the training cohort. (c) Two-by-two contingency table results from the test set are shown for predicting severe (top left, bottom left) and critical COVID-19 (top right, bottom right) using the protein model (blue) and baseline model (red). The threshold for predicting cases was determined during training using Youden’s J statistic which selects a threshold that maximizes the sum of the sensitivity and specificity score. PPV positive predictive value, NPV negative predictive value. c Figure 2. AUC score results. (a) L1 regularized logistic regression training and testing results for severe COVID-19 and (b) critical COVID-19. Blue and red are used to represent the protein model and baseline model, respectively, while solid and dotted lines represent the testing and training performance, respectively. Shaded areas denote the 95% confidence intervals for the training cohort. (c) Two-by-two contingency table results from the test set are shown for predicting severe (top left, bottom left) and critical COVID-19 (top right, bottom right) using the protein model (blue) and baseline model (red). Results C h t The protein model is sensitive (73.2%) at identifying severe COVID-19 cases and similarly sensitive (74.3%) at identifying critical COVID-19 cases. Further, the positive predictive value for severe COVID-19 was high at 88.9%, while the negative predic- tive value was 57.3% (Fig. 2c). These results suggest that a protein model could predict severe COVID-19 with relatively high confidence. Proteins prioritized by LASSO to predict COVID‑19 severity. To predict severe COVID-19, the best performing protein model selected 92 proteins along with age and sample processing time (Fig. 3, Supplemen- tary Table 3). Assessing the correlation of all 92 proteins, we found that, as expected, most of the proteins did not correlate with each other (mean absolute Spearman’s ρ = 0.17) (Supplementary Fig. 5A). Of 8464 total correla- tions (92 × 92), 8372 correlations (98.9%) had Spearman’s absolute ρ < 0.8. ( ), ( ) p ρ Next, when predicting critical COVID-19, the best performing protein model retained age, sample process- ing time, and 67 proteins (Fig. 3, Supplementary Table 4). The absolute effect estimates of these proteins were generally larger than the severe COVID-19 model proteins (mean: 0.081 vs. 0.077). As expected, the 67 selected proteins also showed low levels of correlation (mean absolute Spearman’s ρ = 0.15) (Supplementary Fig. 5B). Of 4489 total correlations (67 × 67), 4422 correlations (98.5%) had Spearman’s absolute ρ < 0.8. The correlation between the 92 and 67 proteins selected to best predict severe and critical COVID-19 is shown in Fig. 3. Out of 6164 total correlations (92 × 67), 6150 correlations (99.8%) had Spearman’s absolute ρ < 0.8. In general, proteins selected for predicting severe versus critical COVID-19 were not highly correlated (mean absolute Spearman’s ρ = 0.15). A hierarchically clustered heatmap after removal of the 14 common proteins in severe and critical COVID-19 showed that the proteins selected in predicting both outcomes were also generally uncorrelated with one another where 99.2% of the correlations had Spearman’s absolute ρ < 0.8 (Supplementary Fig. 5C).h p ρ ( pp y g ) The percent of selected proteins for the prediction of severe and critical COVID-19 that were cytokines or chemokines was only 5.4% and 4.5%, respectively. Cytokine IFNA7, as well as chemokines CXCL13, CXCL10, CCL7, and CCL8 were present in the proteins selected for predicting severe COVID-19. Three chemokines, CXCL13, CXCL10, and CCL7 were selected for predicting critical COVID-19. Results C h t These results suggest that while there were 14 overlapping proteins (SFTPD, CXCL10, RAB3A, NAGPA, CDH5, IFNA7, ZNRF3, CBS, CCL7, SETMAR, TNXB, CDHR1, CXCL13, and CBLN1), in general, the protein levels were uncorrelated with one another. Out of 6164 total correlations (92 × 67), 6150 correlations (99.8%) had a Spearman’s absolute ρ < 0.8. ◂ between training and test performance (AUC: 59%). The stability of these AUC estimates in the test cohort sug- gested that both protein models were robust.hi between training and test performance (AUC: 59%). The stability of these AUC estimates in the test cohort sug- gested that both protein models were robust.hi The classification performance of the baseline and protein models in the Mount Sinai cohort is shown as two-by-two contingency tables in Fig. 2c. The baseline and protein models used thresholds of 0.417 and 0.486 to predict severe COVID-19, respectively. These thresholds were selected by computing Youden’s J statistic during training and determine the threshold that maximized the sum of the sensitivity and specificity scores during training. The thresholds selected were roughly consistent with the case to control ratio in the BQC19 cohort used for training (175 cases, 242 controls). The protein model achieved a sensitivity of 73.2% compared to 61.0% for the baseline model, and a specificity of 79.7% compared to 60.5% for the baseline model, when predicting severe COVID-19 (Fig. 2c). g When predicting critical COVID-19 using the baseline and protein models, thresholds of 0.202 and 0.255 were used to predict cases, respectively, using the same method. The low threshold for predicting critical COVID- 19 cases is consistent with the case to control ratio in training which was 93–324 samples. The baseline model achieved a sensitivity/specificity score of 50.2%/57.4% while the protein model achieved 74.3%/69.6%, suggest- ing that the protein model trained to predict critical COVID-19 had decent power to classify true positives and true negatives (Fig. 2c). Furthermore, both the baseline and protein models demonstrated higher positive predictive values than negative predictive values when predicting severe COVID-19, compared to critical COVID-19. In contrast, these models produced higher negative predictive values than positive predictive values when predicting criti- cal COVID-19. Overall, these results suggest that the protein models predicting severe and critical COVID-19 both per- form reasonably well in terms of the trade-off between sensitivity and specificity. Results C h t The threshold for predicting cases was determined during training using Youden’s J statistic which selects a threshold that maximizes the sum of the sensitivity and specificity score. PPV positive predictive value, NPV negative predictive value. c c Figure 2. AUC score results. (a) L1 regularized logistic regression training and testing results for severe COVID-19 and (b) critical COVID-19. Blue and red are used to represent the protein model and baseline model, respectively, while solid and dotted lines represent the testing and training performance, respectively. Shaded areas denote the 95% confidence intervals for the training cohort. (c) Two-by-two contingency table results from the test set are shown for predicting severe (top left, bottom left) and critical COVID-19 (top right, bottom right) using the protein model (blue) and baseline model (red). The threshold for predicting cases was determined during training using Youden’s J statistic which selects a threshold that maximizes the sum of the sensitivity and specificity score. PPV positive predictive value, NPV negative predictive value. https://doi.org/10.1038/s41598-023-31850-y Scientific Reports \| (2023) 13:6236 \| www.nature.com/scientificreports/ https://doi.org/10.1038/s41598-023-31850-y Scientific Reports \| (2023) 13:6236 \| www.nature.com/scientificreports/ Figure 3. Feature importance and correlation of SOMAmers selected in the protein model to predict severe and critical COVID-19. (Left) Coefficient values of the 92 nonzero SOMAmer reagents in the final trained L1 regularized logistic regression protein model fitted to predict severe COVID-19. The original data contained 4984 SOMAmer reagents and 4 other variables: age, sex, sample processing time, and hospital site. 92 SOMAmer reagents remained within the model along with age and sample processing time which are not shown. The model was trained on the entire BQC19 cohort using lambda = 10.0 (log10 lambda = 1.0) which was the best lambda value found from the hyperparameter search. (Bottom) Coefficient values of the 67 nonzero SOMAmer reagents of the final trained L1 regularized logistic regression protein model fitted to predict critical COVID-19. The original data contained 4984 SOMAmer reagents and 4 variables age, sex, sample processing time, and hospital site. 67 SOMAmer reagents remained within the model along with age and sample processing time which are not shown. The model was trained on the entire BQC19 cohort using lambda = 10.0 (log10 lambda = 1.0) which was the best lambda value found from the hyperparameter search. (Right) Spearman’s rank correlations between the 92 proteins associated with severe COVID-19 and the 67 proteins associated with critical COVID-19. www.nature.com/scientificreports/ www.nature.com/scientificreports/ In addition, we clustered the 4984 SOMAmer reagents using uniform manifold approximation and projection (UMAP) to reduce the feature space to a 2-dimensional space and highlighted the position of the model-selected proteins predictive of either severe or critical COVID-19. Our results suggested that LASSO selected proteins were sparsely distributed across the clusters (Supplementary Fig. 6). This provides further evidence that: (1) few of the selected proteins are closely clustered with one another in UMAP space and (2) the proteins selected from the severe protein model and critical protein model were also quite distant from each other in UMAP space. Finally, we performed pathway analysis on common proteins with non-zero effect estimates that were included in the best predictive models of severe and critical COVID-19. Since LASSO is designed to pick one protein from a group of correlated proteins regardless of their biological relevance, we also included proteins highly correlated (Spearman’s absolute ρ > 0.75) with the proteins selected by LASSO for the enrichment analysis. As a result, 171 proteins were included in the severe group (92 LASSO selected proteins and 79 correlated proteins, Supplementary Table 5); and 96 proteins were included in the critical group (67 LASSO selected proteins and 29 correlated proteins, Supplementary Table 6). Among which, 32 proteins were common between the severe and critical groups. g We found that these 32 proteins were enriched in 35 pathways (g:SCS adjusted P value < 0.05), among which 15 were directly related to immune responses (Supplementary Fig. 7, Supplementary Table 7). Prominent path- ways included viral protein interaction with cytokines, cytokine and cytokine receptors (IL22RA1, TNFRSF10B, CCL7, CXCL10, CXCL13, adjusted P = 0.0008) and cytokine-cytokine receptor interactions (CD4, IL22RA1, TNFRSF10B, CCL7, CXCL10, CXCL13, IFNA7, adjusted P = 0.002). j Interestingly, more than half of enriched pathways were not related to immune response (e.g., signaling receptor binding, cell activation). This suggests that other non-immune response pathways influence COVID- 19 severity. In addition, some of these pathways included protein phosphorylation (CTSG, PRKCZ, PECAM1, CD4, CLEC7A, NPPA, TNFRSF10B, PRDX4, EPHA4, TNXB, CXCL10, IFNA7, CDH5) and glycosaminoglycan binding (CTSG, CCL7, TNXB, CXCL10, CXCL13), suggesting potential avenues to explore for drug development. Using clinical risk factors to predict COVID‑19 severity. In order to contrast the prediction capa- bilities of protein levels with established clinical risk factors, we performed two sensitivity analyses with results shown in Table 2. www.nature.com/scientificreports/ In the first analysis, we added six clinical risk factors to the baseline and protein models described in the main analyses. These clinical risk factors were diabetes, chronic obstructive pulmonary disease (COPD), chronic kidney disease, congestive heart failure, hypertension, and liver disease. The prevalence of these risk factors is shown in Table 1. Addition of these six clinical features to the baseline model improved the training AUC to 64% (from 59%) when predicting severe COVID-19 and to 61% (from 59%) when predicting critical COVID-19. However, add- ing these clinical risk factors to the protein model resulted in no change in the training AUC performance when predicting severe COVID-19 (AUC = 88% vs. 88%) and critical COVID-19 (AUC = 89% vs. 89%) (Table 2, Sup- plementary Fig. 8). 95 and 69 features with non-zero beta coefficient effect estimates were selected for the protein models predicting severe and critical COVID-19, respectively, in this sensitivity analysis (Supplementary Fig. 8). Comparing proteins selected by the protein model in this sensitivity analysis, only one protein, KIT, was added to the 94 features selected in the main analysis. For critical COVID-19, the 69 features selected remained the same. For the second sensitivity analysis, we augmented the first sensitivity analysis with an extra covariate for smok- ing status. Due to missing smoking information from the CHUM hospital site in the BQC19 cohort, only 312 samples were used in model training for the second sensitivity analysis. The results suggested that the addition of smoking and 6 clinical risk factors into the original baseline model composed of age, sex, sample processing time, and hospital site also slightly improved the training performance when predicting severe COVID-19 (AUC = 66% vs. 59%) and critical COVID-19 (AUC = 61% vs. 59%) (Table 2, Supplementary Fig. 9). When adding smoking and these 6 clinical risk factors to the protein model, we found that training performance actually decreased for severe COVID-19 (AUC = 85% vs. 88%) and critical COVID-19 (AUC = 85% vs. 89%). The non-zero beta coefficients of the proteins for severe and critical COVID-19 are shown in Supplementary Fig. 9 with a total of 79 and 51 features being selected, respectively. Comparing the 79 features selected by the protein model in this sensitivity analysis to the original 94 features selected previously when predicting severe COVID-19, we observed that only 48 features overlapped. www.nature.com/scientificreports/ Similarly, the 51 features selected by the protein model in this sensitivity analysis only had 28 features overlapping with the 69 features selected previously. The observed decrease in AUC and Addition of these six clinical features to the baseline model improved the training AUC to 64% (from 59%) when predicting severe COVID-19 and to 61% (from 59%) when predicting critical COVID-19. However, add- ing these clinical risk factors to the protein model resulted in no change in the training AUC performance when predicting severe COVID-19 (AUC = 88% vs. 88%) and critical COVID-19 (AUC = 89% vs. 89%) (Table 2, Sup- plementary Fig. 8). 95 and 69 features with non-zero beta coefficient effect estimates were selected for the protein models predicting severe and critical COVID-19, respectively, in this sensitivity analysis (Supplementary Fig. 8). Comparing proteins selected by the protein model in this sensitivity analysis, only one protein, KIT, was added to the 94 features selected in the main analysis. For critical COVID-19, the 69 features selected remained the same. F h d i i i l i d h fi i i i l i i h i f k y For the second sensitivity analysis, we augmented the first sensitivity analysis with an extra covariate for smok- ing status. Due to missing smoking information from the CHUM hospital site in the BQC19 cohort, only 312 samples were used in model training for the second sensitivity analysis. The results suggested that the addition of smoking and 6 clinical risk factors into the original baseline model composed of age, sex, sample processing time, and hospital site also slightly improved the training performance when predicting severe COVID-19 (AUC = 66% vs. 59%) and critical COVID-19 (AUC = 61% vs. 59%) (Table 2, Supplementary Fig. 9). When adding smoking and these 6 clinical risk factors to the protein model, we found that training performance actually decreased for severe COVID-19 (AUC = 85% vs. 88%) and critical COVID-19 (AUC = 85% vs. 89%). The non-zero beta coefficients of the proteins for severe and critical COVID-19 are shown in Supplementary Fig. 9 with a total of 79 and 51 features being selected, respectively. Comparing the 79 features selected by the protein model in this sensitivity analysis to the original 94 features selected previously when predicting severe COVID-19, we observed that only 48 features overlapped. Results C h t Importantly, among the 14 over- lapping proteins, those three chemokines, CXCL13, CXCL10, and CCL7 were selected for predicting both severe and critical COVID-19. In addition, SFTPD, a surfactant protein highly specific to the lung tissue was also among the overlapped proteins in predicting both COVID-19 severity groups. https://doi.org/10.1038/s41598-023-31850-y Scientific Reports \| (2023) 13:6236 \| www.nature.com/scientificreports/ Similarly, the 51 features selected by the protein model in this sensitivity analysis only had 28 features overlapping with the 69 features selected previously. The observed decrease in AUC and Table 2. Training AUC comparison. Main Analysis: Baseline model age, sex, sample processing time, and hospital site, Protein model baseline model and 4984 SOMAmer reagents. Main Analysis plus 6 clinical risk factors (CRFs): Baseline model + 6 CRFs age, sex, sample processing time, hospital site, and 6 comorbidities, Protein model + 6 CRFs baseline model + 6 CRFs and 4984 SOMAmer reagents. Main Analysis plus 7 clinical risk factors: Baseline model + 7 CRFs age, sex, sample processing time, hospital site, 6 comorbidities and smoking status, Protein model + 7 CRFs baseline model + 7 CRFs and 4984 SOMAmer reagents. Main analysis (n = 417) Main analysis plus 6 clinical risk factors (n = 417) Main analysis plus 7 clinical risk factors (n = 312) Baseline model Protein model Baseline model + 6 CRFs Protein model + 6 CRFs Baseline model + 7 CRFs Protein model + 7 CRFs Severe COVID-19 0.590 0.880 0.636 0.880 0.657 0.848 Critical COVID-19 0.591 0.890 0.607 0.890 0.606 0.848 Main analysis (n = 417) Main analysis plus 6 clinical risk factors (n = 417) Main analysis plus 7 clinical risk factors (n = 312) Baseline model Protein model Baseline model + 6 CRFs Protein model + 6 CRFs Baseline model + 7 CRFs Protein model + 7 CRFs Severe COVID-19 0.590 0.880 0.636 0.880 0.657 0.848 Critical COVID-19 0.591 0.890 0.607 0.890 0.606 0.848 Table 2. Training AUC comparison. Main Analysis: Baseline model age, sex, sample processing time, and hospital site, Protein model baseline model and 4984 SOMAmer reagents. Main Analysis plus 6 clinical risk factors (CRFs): Baseline model + 6 CRFs age, sex, sample processing time, hospital site, and 6 comorbidities, Protein model + 6 CRFs baseline model + 6 CRFs and 4984 SOMAmer reagents. Main Analysis plus 7 clinical risk factors: Baseline model + 7 CRFs age, sex, sample processing time, hospital site, 6 comorbidities and smoking status, Protein model + 7 CRFs baseline model + 7 CRFs and 4984 SOMAmer reagents. Table 2. Training AUC comparison. Main Analysis: Baseline model age, sex, sample processing time, and hospital site, Protein model baseline model and 4984 SOMAmer reagents. Discussion h l In this large-scale study testing the association of 4701 circulating proteins with severe and critical COVID-19, we found that a subset of these proteins were strong predictors of COVID-19 severity. Specifically, developing a model in 417 individuals and testing its performance in 569 separate samples from an independent external cohort, we demonstrated that a proteomic model was able to predict severe COVID-19, defined as requiring the use of oxygen, with an AUC of 86% and a positive predictive value of 89%. The addition of several commonly used clinical risk factors for COVID-19 severity did not improve the performance of this model. The identified proteins were strongly enriched for cytokine signaling and immune pathways, but also highlighted non-immune pathways. Importantly, sampling was performed on presentation, but the severity outcome is the worst the patient has been at any point and not the clinical status on presentation. Hence, this is in fact a true representation of the patients’ final outcomes, and our study predicts their clinical trajectory based on their protein status on presentation. Taken together, these findings demonstrate that circulating protein abundances are able to predict COVID-19 severity with reasonable accuracy. y y By including an independent cohort in this study, we implemented best practices for model development and validation44. An important aspect of any prediction model is the testing of the model in a cohort separate from the training cohort. Therefore, a strength of this study was that our samples were recruited from three separate hospitals, across two separate health care systems in two different countries. In this study, we used the same clinical risk factors and the exact same proteomic measurement procedure to both train and test the models. This increases the probability that the results presented are generalizable and not overfitted to the training data45. Differences in age and sex distributions in the training and testing cohorts were handled using 10 repeats of stratified five-fold cross validation to mitigate their effects on the generalizability of the model. Indeed, for severe COVID-19, there was little change in the AUC when comparing the training and test cohorts (88% vs. 86%). g p g g Further, most studies that have tested the association between protein levels and COVID-19 severity have focused on circulating cytokines and chemokines15,46–52. Discussion h l However, this difference is not expected to affect the validity of the results, as even with this genetic diversity, our model replicated well.h There are differences in genetic ancestry between the training and testing cohorts. The training cohort consists of individuals from two hospital sites in Montreal and may have different allele frequencies at loci related to the identified proteins compared to individuals from the testing cohort in New York. While Ashkenazi Jews are present in both Montreal and New York, major differences include the larger French-Canadian population in Montreal and larger percentage of African genetic ancestry individuals in New York56. However, this difference is not expected to affect the validity of the results, as even with this genetic diversity, our model replicated well. This study has important limitations. While the model was tested in a separate cohort, and generalized well, it should be tested in additional cohorts, especially in cohorts of diverse ancestry. The control population included individuals who were SARS-CoV-2 positive and had mild disease, in addition to individuals who were suspected to have COVID-19 but were SARS-CoV-2 negative. This means that the developed models provide insight into prediction of individuals who develop severe COVID-19 compared to mild COVID-19 and other acute diseases having symptoms consistent with COVID-19. Such control definitions reduce the potential for collider bias, but do not allow direct prediction of COVID-19 severity amongst only COVID-19 patients57. While critical COVID-19 case criteria allows the identification of biomarkers involved in critical illness, the specificity of the criteria limits the available cases for study, which limits statistical power, and requires consideration of the sensitivity–specificity trade-off. Hence, we defined severe COVID-19 which encompasses critical COVID-19 individuals and increased the available cases for model training. Deaths were included in both severe and critical COVID-19 case criteria to account for the effects of competing risk. The cause of death for individuals in both f This study has important limitations. While the model was tested in a separate cohort, and generalized well, it should be tested in additional cohorts, especially in cohorts of diverse ancestry. The control population included individuals who were SARS-CoV-2 positive and had mild disease, in addition to individuals who were suspected to have COVID-19 but were SARS-CoV-2 negative. This means that the developed models provide insight into prediction of individuals who develop severe COVID-19 compared to mild COVID-19 and other acute diseases having symptoms consistent with COVID-19. www.nature.com/scientificreports/ Main Analysis plus 6 clinical risk factors (CRFs): Baseline model + 6 CRFs age, sex, sample processing time, hospital site, and 6 comorbidities, Protein model + 6 CRFs baseline model + 6 CRFs and 4984 SOMAmer reagents. Main Analysis plus 7 clinical risk factors: Baseline model + 7 CRFs age, sex, sample processing time, hospital site, 6 comorbidities and smoking status, Protein model + 7 CRFs baseline model + 7 CRFs and 4984 SOMAmer reagents. Table 2. Training AUC comparison. Main Analysis: Baseline model age, sex, sample processing time, and hospital site, Protein model baseline model and 4984 SOMAmer reagents. Main Analysis plus 6 clinical risk factors (CRFs): Baseline model + 6 CRFs age, sex, sample processing time, hospital site, and 6 comorbidities, Protein model + 6 CRFs baseline model + 6 CRFs and 4984 SOMAmer reagents. Main Analysis plus 7 clinical risk factors: Baseline model + 7 CRFs age, sex, sample processing time, hospital site, 6 comorbidities and smoking status, Protein model + 7 CRFs baseline model + 7 CRFs and 4984 SOMAmer reagents. https://doi.org/10.1038/s41598-023-31850-y Scientific Reports \| (2023) 13:6236 \| www.nature.com/scientificreports/ fewer number of overlapping proteins when comparing main analyses and sensitivity analyses may be due to the reduction in sample size used for training.h The results from these sensitivity analyses suggest that the protein measurements are likely able to act as partial proxies of the tested clinical risk factors. The addition of the clinical risk factors that we assessed may improve the predictive performance for both COVID-19 severity groups when only demographic and sample processing parameters are available. However, when protein measurements are available, adding these extra clinical risk factors may add little for improving predictions. Discussion h l While this is a reasonable approach given the nature of the disease, we are unaware of any other studies that have tested the association of 4701 circulating proteins with COVID-19 severity. A recent study assessing thousands of proteins and their associations with COVID-19 severity achieved an AUC of 85%, but this was not tested in an independent cohort41.i y p Interestingly only 5 of the 14 proteins selected in the final model of both severe and critical COVID-19 were cytokines or chemokines. There were also proteins selected that were not specific to immune pathway proteins, such as glycosaminoglycan binding—a favourable set of targets for drug development. The lung-specific protein SFTPD was also selected and could potentially indicate the correlation of severity with lung tissue destruction. This suggests that many of the biological pathways that influence severity of COVID-19 may act distinctly from known cytokine and chemokine proteins. A major clinical challenge within the pandemic has been the triaging of patients to identify those most likely to require admission for hospitalization53. A common reason for hospitalization is the need for oxygen support. Currently, treating physicians are required to assess the need for admission using models with poor predictive performance. A model generated in China early in the pandemic to predict COVID-19 severity requires a medical history, chest X-ray, and extensive blood testing54. Further, the 4C Mortality Score was able to predict in-hospital mortality, but achieved an AUC of only 77%55. Approximately half of the patients enrolled in our study have developed severe or critical COVID-19 after their baseline proteomic measurement which were used as predic- tors in this study. This suggests circulating protein measurements could be considered for predicting COVID-19 severity, but this requires further study, including more sampling at the onset of symptoms.hfh There are differences in genetic ancestry between the training and testing cohorts. The training cohort consists of individuals from two hospital sites in Montreal and may have different allele frequencies at loci related to the identified proteins compared to individuals from the testing cohort in New York. While Ashkenazi Jews are present in both Montreal and New York, major differences include the larger French-Canadian population in Montreal and larger percentage of African genetic ancestry individuals in New York56. Methods h Cohorts. The Biobanque Québécoise de la COVID-19 (BQC19) is a Québec-wide biobank which was launched to enable research into the causes and consequences of COVID-19 disease (see bqc19.ca)60. For this study, we used results from 417 patients (313 SARS-CoV-2 nasal swab PCR positive patients and 104 individuals who presented with symptoms consistent with COVID-19 but had negative SARS-CoV-2 PCR nasal swabs) with available proteomic data from the SomaScan SomaLogic assay. The subjects were recruited at the Jewish General Hospital (JGH) and Centre Hospitalier de l’Université de Montréal (CHUM) in Montréal, Québec, Canada, both of which are university affiliated hospitals. For each individual, blood samples drawn at the earliest time point were used for training when an individual had multiple blood draws. Selecting the blood sample at the earliest time point reflects the protein measurements during the acute phase of COVID-19 disease. The demo- graphic characteristics of the participants in the BQC19 cohort who underwent SomaScan assays is detailed in Table 1. The demographic characteristics were obtained by medical chart review or patient interview performed by trained clinicians or trained research coordinators.h y The Mount Sinai cohort used in this study was composed of results from 569 patients made up of 472 SARS- CoV-2 positive patients and 89 SARS-CoV-2 negative patients confirmed through PCR tests, one COVID-19 positive patient diagnosed by a chest CT while the remaining 7 individuals were COVID-19 negative and did not have COVID-19 symptoms during specimen collection but may have had a history of exposure. The samples donated by the patients in the Mount Sinai cohort underwent the same proteomic data collection and profiling performed as in the BQC19 cohort. The subjects were recruited at the Mount Sinai Hospital in New York City which is affiliated with the Icahn School of Medicine. Table 1 lists the demographic and sample processing param- eters of participants in the Mount Sinai cohort that underwent SomaScan assays. Demographic characteristics were obtained similarly to that of the BQC19 cohort. Demographic, sample processing, and clinical variable definitions. Age and sex from the BQC19 and Mount Sinai cohorts were collected. Sample processing time and hospital site were collected for BQC19 samples with the former being a continuous variable that quantifies the time in hours between sample collection and sample freezing.h g The clinical variables were collected for the BQC19 cohort only. www.nature.com/scientificreports/ severe and critical COVID-19 groups is unknown. However, cause of death is generally hard to ascertain, dif- ficult to assess, and is an inherent limitation of the data collection process. When including clinical risk factors, smoking status was categorized as current, ex-, and never smokers instead of grouping current and ex-smokers together due to studies showing differences in risk for these two subgroups58,59. It may, however, be valuable to group current and ex-smokers together for sensitivity analysis in the future. We used LASSO for biomarker selection and while there are other approaches available for finding the best protein in each of the LASSO sets of proteins, this would require a different study design. For the purpose of external validation, this is not neces- sary. However, we recognize that other researchers may be interested in which proteins are associated with the ones selected by our models. Thus, we provide a list of highly correlated proteins with those selected by severe and critical COVID-19 in Supplementary Table 5 and 6, respectively. Last, the clinical translation of this study is hindered by the cost involved in measuring 4701 circulating proteins but could be improved by developing a specific assay to the selected proteins. pi y p In summary, circulating protein levels are strongly associated with COVID-19 severity and able to predict the need for oxygen supplementation or death with reasonable accuracy. Measured protein levels were superior to predicting COVID-19 severity when compared to nearly all clinical risk factors tested. Further research is needed to assess whether this proteomic approach can be applied in a clinical setting to assist in triaging patients for admission to hospital. Discussion h l Such control definitions reduce the potential for collider bias, but do not allow direct prediction of COVID-19 severity amongst only COVID-19 patients57. While critical COVID-19 case criteria allows the identification of biomarkers involved in critical illness, the specificity of the criteria limits the available cases for study, which limits statistical power, and requires consideration of the sensitivity–specificity trade-off. Hence, we defined severe COVID-19 which encompasses critical COVID-19 individuals and increased the available cases for model training. Deaths were included in both severe and critical COVID-19 case criteria to account for the effects of competing risk. The cause of death for individuals in both Scientific Reports \| (2023) 13:6236 \| https://doi.org/10.1038/s41598-023-31850-y www.nature.com/scientificreports/ Methods h The first model type is a “baseline model” which was trained using age, sex, sample process- ing time, and hospital site. The second model type is a “protein model” which is trained using age, sex, sample processing time, hospital site, and 4984 SOMAmer reagents. We used the baseline model in our analyses as a performance benchmark to compare the results of the protein model which we expected to perform better.i To predict the two COVID-19 severity groups defined above, we used LASSO regression and elastic nets. Elastic net models were only trained in the BQC19 cohort and not tested in the Mount Sinai cohort (Supple- mentary Note). Specifically, for LASSO regression we used L1 Regularized Logistic Regression (Sparse Logistic Regression) as implemented in the “LogisticRegression” module from Sci-kit learn version 0.24.1, a machine learning library, with the penalty set to “L1”. The L1 norm penalty adds a constraint to the effect estimates of the regression model by setting many variables to have a null effect or a coefficient of 0. This in turn allows a form of feature selection to occur and also prevents overfitting to the training dataset by forcing the model to be less complex. In addition, when multiple variables are correlated with one another such as in the case of highly correlated proteins, the penalty term from LASSO may select a single variable from the group, thus allowing a subset of uncorrelated proteins to be selected. It is important to note that although LASSO tends to select a single variable from a group of highly correlated variables, this property is not a certainty. LASSO may occasionally select more than one variable depending on the size of the dataset and the value of the penalty term. To train this model, the hyperparameter “lambda”, which controls the amount of L1 regularization to add to the model, was first tuned through cross-validation (details are described in the next section). A larger value of lambda increases the amount of L1 regularization and forces more of the variables to have a null effect. On the other hand, training the model on a smaller lambda value will result in a model with more nonzero coefficients. Cross‑validation and hyperparameter tuning. Methods h Clinical variables included smoking status and six different comorbidities: diabetes, COPD, chronic kidney disease, congestive heart failure, hypertension, and liver disease. All seven variables were collected as categorical values with the six comorbidities having three options (0 No, 1 Yes, and − 1 Don’t know) while smoking status contained 4 categories (0 Current Smoker, 1 Ex-smoker, 2 Never smoked, and − 1 Don’t know). Proteomic measurement using the SomaScan platform. Blood samples from both the BQC19 and Mount Sinai cohorts were collected using acid citrate dextrose (ACD) tubes. Proteomic measurement was performed at SomaLogic using the SomaScan v4.0 platform. In the BQC19 cohort, a total 1038 samples col- lected at different time points from 503 individuals were sent to SomaLogic for proteomic profiling as previously described2, while the Mount Sinai cohort contained 1200 samples collected at different time points from 592 individuals that were sent to SomaLogic for proteomic profiling. g p pi g SomaLogic uses the SomaScan proteomic platform which provides measurements on 4701 unique human circulating proteins using 4987 Slow Off-Rate Modified Aptamers (SOMAmer reagents) and quantifies protein levels in the form of relative fluorescence units (RFUs). Normalization and calibration steps were performed by SomaLogic to remove any systematic biases stemming from raw assays or samples. The normalization procedure involved three steps performed in a non-consecutive fashion: hybridization control normalization, intraplate median signal normalization, as well as plate scaling and calibration. More details on SomaLogic normalization can be found in their Technical Note61. Data preprocessing. A per-sample normalization process involved using a scale factor for a set of SOMA- mer reagents to compute against a reference value generated from the median of all calibrated, unnormalized samples, and then aggregating the results within a dilution. This was done because using a normal population https://doi.org/10.1038/s41598-023-31850-y Scientific Reports \| (2023) 13:6236 \| www.nature.com/scientificreports/ reference generated from EDTA plasma tubes would have been inappropriate for normalization, since samples in this study were from ACD plasma tubes. Due to the nature of the samples that were collected from patients during acute infection, we did not apply the recommended scale [0.4–2.5] to remove samples. The raw data- set composed of 5284 SOMAmer reagents was first processed with SomaLogic package SomaDataIO v3.1.0. We removed any SOMAmer reagents that represented non-human proteins or controls (NoneX, NonHuman, Spuriomer, HybControlElution, NonBiotin, NonCleavable) and retained 4984 unique SOMAmer reagents for analysis. Curation of samples from the longitudinal dataset. Methods h To investigate our primary study question, we focused on samples collected during the acute infection stage. Samples from the acute infection stage were defined as samples collected from SARS-CoV-2 PCR positive patients within 14 days of symptom onset. When an individual provided multiple samples collected within 14 days of symptom onset, the sample collected at the earliest timepoint was retained for analyses. Both the BQC19 and Mount Sinai samples adhered to this rule. COVID‑19 severity definitions. We defined two sets of severity groups for COVID-19: severe COVID-19 and critical COVID-19. Positive SARS-CoV-2 results were confirmed by SARS-CoV-2 viral nucleic acid ampli- fication tests (NAAT) from relevant biologic fluids. Cases for severe COVID-19 were defined as individuals who tested positive for COVID-19 and died or required any type of respiratory support (including oxygen delivered by nasal prongs) at any timepoint. Controls for severe COVID-19 were defined as individuals who did not meet these severe case criteria; thus, controls were individuals with COVID-19 but did not meet severe case criteria or were individuals who presented with symptoms of COVID-19 but were SARS-CoV-2 PCR negative. Cases for critical COVID-19 were defined as individuals who tested positive for COVID-19 and died or required invasive respiratory support (intubation, continuous positive airway pressure, bilevel positive airway pressure, continu- ous external negative pressure, or high flow positive end expiratory pressure oxygen) at any timepoint. Controls for critical COVID-19 were individuals with COVID-19 but did not meet critical case criteria or were individu- als who presented with symptoms of COVID-19 but were SARS-CoV-2 PCR negative. Multivariable logistic regression. Multivariable logistic regression models were used to test the asso- ciations of either severe or critical COVID-19 on four covariates along with each SOMAmer reagent: age, sex, sample processing time, and hospital site. We used R package “glm” to perform 4984 logistic regression models in the BQC19 cohort. We first applied a false discovery rate of P < 0.01 (corrected P values were determined using the Benjamini–Hochberg procedure62, p.adjust with method set to “BH” in R) to select a subset of proteins asso- ciated with severe or critical COVID-19. Volcano plots measuring the uncorrected − log10 P values as a function of the effect size estimates of each SOMAmer reagent were generated using the bioinfokit version 2.0.4 package in Python 3.7. Regularized logistic regression models. We defined two model types differing in the covariates used to train the model. www.nature.com/scientificreports/ will receive more weight and thus bias the results. The stratified cross-validation step was performed using the RepeatedStratifiedKFold function in Sci-kit learn. Due to the relatively small sample size of our training set (n = 417), we performed 10 repeats of this cross-validation process to stabilize the results from training. Each repeat first shuffled the entire training set then split the data into five folds which created more variability in the data used for training. will receive more weight and thus bias the results. The stratified cross-validation step was performed using the RepeatedStratifiedKFold function in Sci-kit learn. Due to the relatively small sample size of our training set (n = 417), we performed 10 repeats of this cross-validation process to stabilize the results from training. Each repeat first shuffled the entire training set then split the data into five folds which created more variability in the data used for training. g During training on the four folds, we standardized only the protein levels using the population standard deviation (i.e., dividing by the number of samples n) and use this mean and standard deviation to standardize the protein levels in the validation fold prior to validating. This prevents information leakage which can occur if standardization of protein levels was performed on the entire dataset rather than just the training folds. Age and sample processing time were treated as continuous variables, whereas sex and hospital site were treated using dummy variables (sex [0: Female, 1: Male], and hospital site [0: CHUM, 1: JGH]). We used the AUC to determine the model performance during cross-validation. To select the best value for the hyperparameter lambda, we compared the average AUC score (computed from 50 validation fold results) for all lambda values and selected the lambda value corresponding to the highest average AUC. Youden’s J statistic was calculated for each receiver operator characteristic (ROC) curve during training. This performance metric can be calculated by subtracting the false positive rate from the true positive rate for each data point on a ROC curve and taking the maximum value. The threshold which corresponds to this maximum Youden’s J statistic is the threshold that maximizes the sum of the sensitivity and specificity for that particular ROC curve. We computed the threshold corresponding to the maximum Youden’s J statistic for each of the 50 ROC curves and averaged the 50 thresholds to get a single threshold value. www.nature.com/scientificreports/ This averaged threshold value was computed for each of the baseline and protein models predicting severe and critical COVID-19 and used to produce two-by-two contingency tables and therefore sensitivity and specificity values in the Mount Sinai cohort during model testing. Model testing. We checked the generalizability of the baseline and protein models by testing them in an external, independent dataset from Mount Sinai. Protein measurements in the test dataset were first natural log transformed then standardized using the mean and standard deviation of the corresponding protein in the train- ing set. Similarly, age was not standardized and kept in years. Since samples were only from a single hospital, the hospital site parameter was left as is and did not need to be dummy encoded. The variable sample processing time, however, was absent from the testing set. For this reason, we imputed the sample processing time variable in the test cohort using the mean value of the sample processing time variable in the BQC19 training cohort. Protein correlations. Spearman’s Rank Order Correlation was used to determine the correlations between individual proteins in the BQC19 cohort. Heatmaps show magnitudes of correlation coefficients between values of − 1 and 1. Correlation heatmaps showing collected clusters such as in Supplementary Fig. 5 were generated using the ggcorrplot function in R with the parameter hc.order set to TRUE to perform hierarchical clustering. Moreover, we reduced the dimension of the correlation matrix of 4984 SOMAmer reagents to a 2-dimensional space using uniform manifold approximation and projection (UMAP) from the umap-learn 0.5.1 package using default parameters. We annotated the SOMAmer reagents selected from the protein model that were associated with severe COVID-19 and critical COVID-19 as well as the proteins that overlapped between the two severity groups. Pathway enrichment analyses. We used the web-based tool g:Profiler (https://biit.cs.ut.ee/gprofiler/ gost) to investigate the possible pathways of the selected proteins as good predictors for both critical and severe COVID-19 identified by LASSO. The g:SCS algorithm was used to estimate the threshold for enrichment against all annotated genes. We selected pathways and interaction databases including Gene Ontology, KEGG63,64, Reac- tome; TRANSFAC, miRTarBase, Human Protein Atlas, and CORUM. Sensitivity analyses. We tested the effect of six established clinical risk factors which included: diabetes, COPD, chronic kidney disease, congestive heart failure, hypertension, and liver disease in the BQC19 cohort to determine whether addition of comorbidities could improve prediction of COVID-19 severity. Methods h Due to the relatively small size of our training data- set from BQC19, we used 10 repeats of five-fold cross-validation to tune the hyperparameter, lambda, over 17 different lambdas (log10 values of lambda from − 2 to 2, incremented by 0.25). Each repeat of the five-fold cross-validation process involved splitting the dataset into five folds: training on four folds and validating the trained model on the final fold and performing the process five times to cover each validation fold. We used a stratified cross-validation approach, meaning that the train and validation folds maintained the same percentage of samples of each class (case/control) as the original data. This is important because of the unbalanced case/ control samples for critical COVID-19 (93 cases/324 controls). A standard five-fold cross-validation split may result in train and validation folds with varying proportions of cases and controls. Since classification algorithms tend to weight each sample equally, the class that is overrepresented, such as the controls in critical COVID-19, https://doi.org/10.1038/s41598-023-31850-y Scientific Reports \| (2023) 13:6236 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ We added these six additional covariates, with characteristics shown in Table 1, to the baseline and protein models to perform LASSO regression analysis. A total of 417 samples from the BQC19 cohort were used for training. We performed a second sensitivity analysis by adding smoking status along with these six established clini- cal variables to the baseline and protein models for LASSO regression analyses. Therefore, the baseline model contained covariates age, sex, sample processing time, hospital site, and seven clinical variables while the protein model contained all the baseline variables along with 4984 SOMAmer reagents. Since smoking status was not available from the CHUM hospital site, this sensitivity analysis only involved 312 samples from the BQC19 cohort that were collected at the JGH site.i Due to missing data, we imputed the values of samples: we first converted all six comorbidity features to binary values. Any value other than a “Yes” was converted to a “No” which may include missing values being converted to a “No”. For smoking status, we grouped all values into three categories: 0—Current Smoker, 1—Ex-smoker, and anything else (including missing values and − 1) was set as 2—Never smoked. Smoking status was dummy encoded and had one of the encoded variables dropped to prevent collinearity. For both sensitivity analyses, training of the L1 regularized logistic regression models used 10 repeats of stratified five-fold cross-validation as in the primary analysis. Ethics declarations. All contributing cohorts to the present analyses received ethics approval from their respective research ethics review boards. The Biobanque Québécoise de la COVID-19 (BQC19) received ethi- Scientific Reports \| (2023) 13:6236 \| https://doi.org/10.1038/s41598-023-31850-y www.nature.com/scientificreports/ cal approval from the institutional review board (IRB) of the JGH and the CHUM. This research was reviewed and approved by the Icahn School of Medicine at Mount Sinai Program for the Protection of Human Subjects (PPHS) under study number 20-00341. The Mount Sinai PPHS is an accredited IRB. This research was consid- ered minimal risk Human Subjects Research. All research was performed in accordance with relevant guide- lines/regulations and informed consent was obtained from all subjects and/or their legal guardians from which blood samples were taken. All methods used in this study were performed in accordance with the Declaration of Helsinki. Data availability y Code used in this analysis is available at https://github.com/chenyangsu/somalogic with additional informa- tion available upon request. The BQC19 is an Open Science Biobank. Instructions on how to access data for individuals from the BQC19 at the Jewish General Hospital site is available at https://www.mcgill.ca/genepi/ mcg-covid-19-biobank. Instructions on how to access data from other sites of the BQC19 is available at https:// www.bqc19.ca/en/access-data-samples. The proteomics data analyzed during the current study is available for download at https://drive.google.com/drive/folders/1xTMzgKJhkArL6XmqgcUaU3LW4kyxrBjv?usp=sharing. 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Scientific Reports \| (2023) 13:6236 \| https://doi.org/10.1038/s41598-023-31850-y Additional informationh Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-023-31850-y. Correspondence and requests for materials should be addressed to J.B.R. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2023 Acknowledgementsh g The Richards research group is supported by the Canadian Institutes of Health Research (CIHR: 365825; 409511, 100558), the Lady Davis Institute of the Jewish General Hospital, the Jewish General Hospital Foundation, the Canadian Foundation for Innovation, the NIH Foundation, Cancer Research UK, Genome Québec, the Public Health Agency of Canada, McGill University, Cancer Research UK [grant number C18281/A29019], and the Fonds de Recherche Québec Santé (FRQS). The Kaufmann lab’s COVID-19 work is supported by the Cana- dian Institutes of Health Research /CITF (VR2-173203 and VS1-175561), the American Foundation for AIDS Research (AmFAR 110068-68-RGCV), the Canadian Foundation for Innovation, and FRQS. Support from Calcul Québec and Compute Canada is acknowledged. TwinsUK is funded by the Welcome Trust, Medical Research Council, European Union, the National Institute for Health Research (NIHR)-funded BioResource, Clinical Research Facility, and Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust in partnership with King’s College London. These funding agencies had no role in the design, implementation, or interpretation of this study. The measurement of proteomics using the SomaLogic panel was supported by the McGill Interdisciplinary Initiative in Infection and Immunity (MI4) and in whole or in part, by the Bill & Melinda Gates Foundation [INV-017895] for the Mount Sinai cohort. J.B.R. and D.E.K. are supported by FRQS Mérite Clinical Research Scholarships. C.-Y.S. is supported by a Lady Davis Institute / TD Bank Studentship Award. https://doi.org/10.1038/s41598-023-31850-y Scientific Reports \| (2023) 13:6236 \| www.nature.com/scientificreports/ S.Z. is supported by a CIHR fellowship and an FRQS postdoctoral scholarship. G.B.L. is supported by a CIHR scholarship and a joint FRQS and Québec Ministry of Health and Social Services scholarship. T.N. is supported by Research Fellowships of the Japan Society for the Promotion of Science (JSPS) for Young Scientists. M.D. is supported by a clinician-researcher salary award from the FRQS. V.M. is supported by a Canada Excellence Research Chair. S.G. was supported by grants CA224319, DK124165, and CA196521. Members of the Mount Sinai COVID-19 Biobank Team are listed in Supplementary Table 8. Other authors part of the Mount Sinai Biobank Team that did not directly contribute to the paper are listed in Supplementary Table 8. S.Z. is supported by a CIHR fellowship and an FRQS postdoctoral scholarship. G.B.L. is supported by a CIHR scholarship and a joint FRQS and Québec Ministry of Health and Social Services scholarship. T.N. Author contributions d d Conception and design: C.Y.S., S.Z., W.J., J.P., and J.B.R.; Data analyses: C.Y.S., S.Z., and E.G.K.; Data acquisition: T.N., G.B.L., D.M., D.E.K., J.A., M.A., L.L., E.B.R., D.H., N.K., Z.A., N.R., M.B., L.P., C.G., X.X., C.T., B.V., O.A., T.A., N.A., N.B., M.D., K.N., N.W.S., K.M., D.M.D.V., N.Z., M.P., H.X., J.H., R.M., E.C., K.T., K.A., I.S., N.B., E.K., V.F., T.M., S.G., S.K.S., A.C., M.M., D.E.K., and J.B.R.; Interpretation of data: C.Y.S., S.Z., E.G.K., G.B.L., E.B.R., T.N., D.E.K., and J.B.R.; Funding acquisition: V.M., T.M., S.G., S.K.S., A.C., M.M., D.E.K., and J.B.R.; Methodol- ogy: C.Y.S., S.Z., E.G.K., C.M.T.G., C.P., M.H., J.C.Z.S., C.L., and J.B.R.; Project administration: D.M., V.F., A.C., M.M., D.E.K., J.B.R.; Validation: C.Y.S., S.Z., G.B.L., E.G.K., E.B.R., Y.C., Y.F., D.E.K., and J.B.R.; Visualization: C.Y.S. and S.Z.; Writing-original draft: C.Y.S., S.Z., E.G.K., G.B.L., E.B.R., T.N., D.E.K., and J.B.R.; Writing-review and editing: C.Y.S., S.Z., E.G.K., G.B.L., E.B.R., T.N., D.E.K., E.S., M.D., C.P., V.M., D.E.K., and J.B.R.; All authors were involved in further drafts of the manuscript and revised it critically for content. All authors gave final approval of the version to be published. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. Acknowledgementsh is supported by Research Fellowships of the Japan Society for the Promotion of Science (JSPS) for Young Scientists. M.D. is supported by a clinician-researcher salary award from the FRQS. V.M. is supported by a Canada Excellence Research Chair. S.G. was supported by grants CA224319, DK124165, and CA196521. Members of the Mount Sinai COVID-19 Biobank Team are listed in Supplementary Table 8. Other authors part of the Mount Sinai Biobank Team that did not directly contribute to the paper are listed in Supplementary Table 8. Competing interests J.B.R. has served as an advisor to GlaxoSmithKline and Deerfield Capital and is the Founder of 5 Prime Sciences. The Lady Davis Institute has previously received funding from GlaxoSmithKline, Eli Lilly, and Biogen for research programs at Dr. Richards’ laboratory unrelated to this manuscript. C.P. and M.H. are employees of SomaLogic. All other authors do not have any conflict of interest. TN has received speaking fees from Boehringer Ingelheim for talks unrelated to this research. S.G. reports other research funding from Bristol-Myers Squibb, Boehringer- Ingelheim, Celgene, Genentech, Regeneron, and Takeda. S.G. reports other research funding from Bristol-Myers Squibb, Boehringer-Ingelheim, Celgene, Genentech, Regeneron, and Takeda. The Mount Sinai COVID-19 Biobank Team Edgar Gonzalez‑Kozlova5,24, Diane Marie Del Valle13, Ryan Thompson14, Mario A. Cedillo15, Eric Schadt14, Kai Nie16, Nicole W. Simons14, Konstantinos Mouskas14, Nicolas Zaki16, Manishkumar Patel13, Hui Xie16, Jocelyn Harris16, Robert Marvin16, Esther Cheng14, Kevin Tuballes13, Kimberly Argueta16, Ieisha Scott16, Thomas Marron19, Noam D. Beckmann14, Seunghee Kim‑schulze16, Alexander W. Charney20, Sacha Gnjatic5,13,16 & Miriam Merad13 https://doi.org/10.1038/s41598-023-31850-y Scientific Reports \| (2023) 13:6236 \|
https://openalex.org/W3165739760	https://www.cairn.info/load_pdf.php?ID_ARTICLE=LAUTR_064_0004&download=1	French	null	Ondes de choc après l’assassinat de Samuel Paty	L'Autre	2,021	cc-by	2,188	Marie Rose Moro est pédopsychiatre, profes- seure de psychiatrie de l’enfant et de l’adoles- cent, cheffe de service de la Maison de Solenn – Maison des Adolescents, CESP, Inserm U1178, Université de Paris, APHP, Hôpital Cochin, directrice scientifique de la revue L’autre. L’horrible assassinat de Samuel Paty nous a toutes et tous pétrifié·e·s. Il nous a touché·e·s à plusieurs niveaux, dont celui de l’intime: qui d’entre nous n’a pas de dette à l’égard d’un·e enseignant·e qui nous a ouvert·e·s à d’autres horizons, rendu·e·s curieux du monde, initié·e·s aux savoirs? Samuel Paty n’est- il pas aussi la figure du professeur fin, intelligent, volontaire et engagé que nous admirons? Un homme a été tué, décapité; tué pour ce qu’il était, pour ses idées, pour son engagement professionnel. Nous qui avons tellement be- soin de nos enseignant·e·s, notamment pour nous enseigner l’Histoire. De ces éclaireurs et éclaireuses qui nous invitent à comprendre le monde. Cette mort arrive dans un enchaînement-déchaînement d’événements qui nous laissent K. O, comme si l’on assistait à un emballement de violence dont on cherche le sens, nous laissant sans voix, inquiet·e·s face à un avenir cruel et incertain. La tentation du repli serait grande, raffermie par un confinement qui nécessite de puiser en nous, chaque jour, plus d’efforts pour accomplir nos tâches quo- tidiennes. Au-delà de la violence des mots, il y a dans les mots « menace » et « déca- pitation », utilisés par ce journaliste, un effet de miroir avec la « banalisation » de la menace utilisée par certains jeunes. Non pas qu’elle soit banale en elle- même, mais on la voit brandie de plus en plus dans certaines circonstances. « Tu vas mourir comme Samuel Paty », dit le jeune Ilheb, condamné immédia- tement à la prison après avoir menacé un professeur à Nice3. Les jeunes choi- sissent bien entendu leurs mots à dessein pour faire peur aux adultes. Mais n’assistons-nous pas également à la naissance d’un « modèle d’inconduite », expression d’un « négativisme social » tel que Devereux (1977, p. 39) le définit, à savoir que même pour dire sa souffrance, pour s’opposer ou transgresser, il existe des normes dans une société donnée. « Parfois la culture elle-même fournit des directives explicites pour le mésusage des matériaux culturels, et cela tout particulièrement dans les situations de stress fréquents mais aty- piques. ÉDITORIAL ÉDITORIAL 1 Artigue-Cazcarra, E. (2020, novembre 10). Gi- ronde : pris en charge dans un foyer pour mineurs, il menace de décapitation des éducateurs. Sud- Ouest. https://www.su- douest.fr/2020/11/10/giron- de-pris-en-charge-dans- un-foyer-pour-mineurs-il- menace-de-decapitation- des-educateurs-8062019- 2897 4 \| L’autre, cliniques, cultures et sociétés, 2021, volume 22, n°1, pp. 4-6 3 Perrin, C. (2020, novem- bre 28). “Tu vas mourir comme Samuel Paty”: un jeune homme incarcéré après avoir menacé un prof à Nice. Nice-matin. www.nicematin.com/justi- ce/tu-vas-mourir-comme- samuel-paty-un-jeune- homme-incarcere-apres- avoir-menace-un-prof-a- nice-609749 2 Lorsque cet éditorial a été écrit la procédure judi- ciaire était toujours en cours. Claire MESTRE, Sevan MINASSIAN, Marie Rose MORO « Claire Mestre est psy- chiatre, anthropologue, CHU de Bordeaux, co-rédactrice en chef de la revue L’autre. Claire Mestre est psy- chiatre, anthropologue, CHU de Bordeaux, co-rédactrice en chef de la revue L’autre. « Il menace de décapitation des éducateurs »: dans le journal Sud- Ouest1, le titre claque comme une sentence concernant un jeune Mi- neur Non Accompagné (MNA)… Derrière cette histoire qui finit si mal devant la Présidente de l’audience des comparutions immédiates du tribunal correctionnel de Bordeaux2, se trame un drame terrible. En filigrane, un en- chaînement d’évènements souvent rencontrés par les professionnels qui ac- compagnent les MNA: des propos menaçants et graves, une radio du poignet qui « prouve » la majorité du jeune, donc l’abandon de la protection de l’ASE. Il faut également comprendre que ce jeune se retrouve irrémédiablement seul, alors que le journaliste évoque surtout une logique qui rend possible l’amal- game entre religion islamique-immigration-terrorisme. Sevan Minassian est pé- dopsychiatre, Maison de Solenn, Hôpital Cochin, APHP, Paris, France, Université de Paris, PCPP, F-92100 Boulo- gne-Billancourt, France. Sevan Minassian est pé- dopsychiatre, Maison de Solenn, Hôpital Cochin, APHP, Paris, France, Université de Paris, PCPP, F-92100 Boulo- gne-Billancourt, France. Ondes de choc après l’assassinat de Samuel Paty Claire MESTRE, Sevan MINASSIAN, Marie Rose MORO 7 Sur ce sujet, il est possi- ble d’écouter l’émission dif- fusée sur France Inter : Cherel, L. (2020, novembre 27). Assassinat de Samuel Paty : la fabrique d’un ter- roriste. France Inter. https://www.franceinter.fr/a ssassinat-de-samuel-paty- la-fabrique-d-un-terroriste Marie Rose Moro est La directive qui nous intéresse ici est la suivante: “Garde-toi de devenir fou, mais si tu le deviens, conduis-toi de telle ou telle manière”. Chaque so- ciété a des idées sur “comment les fous se conduisent” ». 2 Lorsque cet éditorial a été écrit la procédure judi- ciaire était toujours en cours. 3 Perrin, C. (2020, novem- bre 28). “Tu vas mourir comme Samuel Paty”: un jeune homme incarcéré après avoir menacé un prof à Nice. Nice-matin. www.nicematin.com/justi- ce/tu-vas-mourir-comme- samuel-paty-un-jeune- homme-incarcere-apres- avoir-menace-un-prof-a- nice-609749 Les caricatures du prophète auraient agi comme autant de critiques des valeurs de certains jeunes Musulmans dont les actes auraient eu comme ob- jectifs de porter atteinte à autrui et à la société en général. Éditorial Porter atteinte à un enseignant, une église ou un journal, est une position agressive et parfois mortelle vis-à-vis de la société et de ses représentants. Cette menace de décapitation risque désormais d’être brandie de façon banale pour provoquer la peur ou exprimer une désapprobation4. 4 Nous n’abordons pas ici ces effets sur le monde musulman qui mettent en danger des Français dans le monde entier. Ces effets graves demandent une analyse spécifique, fine, historique et géopolitique. Pourtant, entre ceux qui pointent un outrage lié aux caricatures et le pas- sage à l’acte violent, on trouve une multitude de trajectoires individuelles qu’il nous faut comprendre. Fabien Truong, ancien professeur de lycée en Seine- Saint-Denis devenu universitaire5, a restitué, suite à son immersion auprès de jeunes des quartiers populaires, ces trajectoires de façon très intéressante (Truong, 2017). Dans l’entretien qu’il donne au journal Le Monde6, il les relate et nous permet, à travers ses propos mesurés et précis, de sortir de la sidéra- tion, de la peur et du sentiment d’absurdité qui nous hante. Ce qui caractérise le plus souvent les trajectoires des terroristes avérés sont, nous dit-il, « les ex- périences de violence, la coupure, la clandestinité, les sentiments d’impasse et de persécution. » Ces blessures trouvent des réponses salvatrices dans les messages glanés sur les réseaux sociaux, hameçonnés par des vendeurs de haine qui sont passés maîtres dans la diffusion d’images et de messages for- matés par une idéologie religieuse marquée par la violence. Ces réponses at- tirent et construisent une idée de revanche habillée de religion. L’autre, cliniques, cultures et sociétés, 2021, volume 22, n°1 \| 5 Éditorial nement suscité par l’attaque devant les anciens locaux de Charlie Hebdo le vendredi 25 septembre 2020 et, d’autre part, le meurtre d’un de ses amis tchétchènes en pleine rue. Dans quelle mesure les sentiments d’humiliation et d’impasse, s’ajoutant à d’autres facteurs qui ne sont pas évoqués ici (l’expé- rience de la violence, le contexte familial), ont-ils influé sur le jeune tchétchène déjà très impulsif? Pour revenir au jeune MNA de Bordeaux, exclu de la protection de l’enfance et voué à la prison, les dessous de cette trajectoire sont également tragiques: issu d’une région camerounaise en guerre et ayant perdu sa famille, ayant eu des difficultés à faire reconnaître la minorité qu’il revendique, il a développé un « délire » apparu à partir d’une rencontre difficile avec l’hôpital. À l’évidence, il avait besoin de soins (qui, d’ailleurs, lui ont été proposés et prodigués). Son ex- éducateur nous raconte qu’il n’a jamais eu de gestes dangereux pendant son suivi. Ce qui semble toutefois avoir modifié un chemin migratoire déjà compliqué est la solitude due à un changement d’éducateur. Il serait ainsi dommage de criminaliser d’emblée des menaces qui pour- raient être désamorcées, en les remettant à leur juste place, sans avoir la han- tise d’affronter des jeunes provocateurs ou en prise avec le malheur. Critiquer la religion est un acte devenu dangereux. Dans le même temps, au sein de notre société laïque et surtout sécularisée, la religion est une res- source importante pour certains jeunes Musulmans (comme elle peut égale- ment l’être pour d’autres jeunes d’obédiences différentes). Elle donne un sens possible aux pertes, aux deuils, au déclassement, et nourrit l’estime de soi et des autres. Montrer du doigt uniquement le « séparatisme » religieux sans prendre en compte ni agir sur un « séparatisme » social dénoncé de longue date, contre le- quel on fait si peu, nourrit une guerre de mots et d’idéologies qui n’aidera pas tous ceux qui sont sur le terrain pour colmater les brèches de la société. Ces deux séparatismes ne sont pas liés mais ils peuvent se potentialiser. Enfin, faire l’amalgame entre migration et criminalité est simplement hon- teux. De nombreuses recherches en sciences sociales défont ce raccourci mis en exergue par des politiques, même si ces faits récents concernent des jeunes migrants. Tenir un discours juste, réaliste et distancié sur ces sujets n’est pas évident. Marie Rose Moro est Nous avons retrouvé cela dans les trajectoires des jeunes gens radicalisés qui s’engagent dans des combats idéologiques à l’intérieur même de la société française (Gut- ton & Moro, 2017). Ces idéologues homemade (de l’intérieur) se sentent en marge et discriminés par la société française avec le sentiment qu’ils n’ont plus rien à perdre (ibid.). 5 Fabien Truong est so- ciologue au Centre de Re- cherches Sociologiques et Politiques de Paris au sein de l’équipe Cultures et So- ciétés Urbaines (Cresppa- CSU), professeur agrégé au département de Socio- logie et d’Anthropologie de l’Université de Paris 8. 6 Truong, N. (2020, no- vembre 23). Fabien Truong : « Le drame de Conflans-Sainte-Honorine nous rappelle qu’une salle de classe n’est pas une arène politique publique ». Le Monde. www.lemonde.fr/ idees/article/2020/11/23/fa- bien-truong-le-drame-de- conflans-sainte-honorine- nous-rappelle-qu-une-sal- le-de-classe-n-est-pas-une- arene-politique-publique _6060745_3232.html 6 Truong, N. (2020, no- vembre 23). Fabien Truong : « Le drame de Conflans-Sainte-Honorine nous rappelle qu’une salle de classe n’est pas une arène politique publique ». Le Monde. www.lemonde.fr/ idees/article/2020/11/23/fa- bien-truong-le-drame-de- conflans-sainte-honorine- nous-rappelle-qu-une-sal- le-de-classe-n-est-pas-une- arene-politique-publique _6060745_3232.html Ainsi, dans le parcours du jeune Tchétchène assassin de Samuel Paty7, on retrouve des éléments qui ont (peut-être) nourri sa trajectoire jusqu’à la fin tra- gique que nous connaissons: petite enfance dans une Tchétchénie en conflit, vie dans un quartier nommé « prison sans retour » par les jeunes, passion pour la lutte - sport souvent pratiqué par les jeunes Tchétchènes - pour laquelle il fréquente un club. De tempérament impulsif, une bagarre certainement très violente (si l’on en croit le journaliste) l’amène à être exclu de son lycée profes- sionnel. C’est un tournant dans sa biographie: il entre alors dans le monde du travail et, surtout, quitte ses relations sociales habituelles, puis se nourrit, pro- bablement, de messages religieux extrémistes trouvés sur les réseaux sociaux. L’enquête journalistique met aussi en avant des « coïncidences » qui ont certai- nement joué un rôle favorisant le projet meurtrier: d’une part l’effet d’entraî- 5 L’autre, cliniques, cultures et sociétés, 2021, volume 22, n°1 \| 5 6 \| L’autre, cliniques, cultures et sociétés, 2021, volume 22, n°1 Altounian, J. (1977). Une Arménienne à l’école. Les Temps Modernes, 373-374, 37. Devereux, G. (1977). Normal et anormal. Dans G. Devereux, Essais d’ethnopsychiatrie générale (pp.1-83). Gallimard. Gutton, P., & Moro, M. R. (2017). Quand l’adolescent s’engage. Radicalité et construc- tion de soi. InPress. Kristeva, J., & Moro, M. R. (2020). Grandir c’est croire. Bayard. Truong, F. (2017). Loyautés radicales. La Découverte. n Crédits photographiques : P. 5 © Aurélien Adoue, Liberté, 31 mai 2019. CC BY 2.0 Éditorial Il le faut pourtant, pour que nos professeurs continuent leur travail. Et, plus lar- gement, pour que les soignants et tous les citoyens qui accompagnent les jeu- nes, qu’ils soient d’ici ou d’ailleurs, puissent continuer à porter haut les valeurs de l’accueil. Il est plus que nécessaire que l’école reste le lieu de la traduction, de l’analyse et, si possible, de la compréhension des catastrophes, des géno- cides et des guerres, par l’enseignement de l’Histoire et des histoires, comme l’a si bien écrit Janine Altounian (1977), fille d’un père arménien rescapé du génocide. Qu’elle reste le lieu de l’ouverture aux langues et aux religions comme références culturelles, de l’accueil de chacun, quelle que soit sa religion, et sur- tout d’apprentissage de la critique. g Pour grandir et s’inscrire dans une société, tout jeune a besoin de croire. Croire qu’il n’est pas seul et qu’il est inscrit dans le monde (Kristeva & Moro, 2020). En ayant près de lui un professeur qui croira en lui. l Bordeaux, Paris, le 1er décembre 2020 n Bibliographie Altounian, J. (1977). Une Arménienne à l’école. Les Temps Modernes, 373-374, 37. Devereux, G. (1977). Normal et anormal. Dans G. Devereux, Essais d’ethnopsychiatrie générale (pp.1-83). Gallimard. Gutton, P., & Moro, M. R. (2017). Quand l’adolescent s’engage. Radicalité et construc- tion de soi. InPress. Kristeva, J., & Moro, M. R. (2020). Grandir c’est croire. Bayard. Truong, F. (2017). Loyautés radicales. La Découverte. 6 \| L’autre, cliniques, cultures et sociétés, 2021, volume 22, n°1
https://openalex.org/W2007483766	https://hrcak.srce.hr/file/164630	English	null	Analysis of Ozone Data by Photochemical Pollution Indicators in Colorado	Croatica chemica acta	2,013	cc-by	3,166	INTRODUCTION layer of the atmosphere is going to rise.5,6 Ozone moni- toring is therefore imperative at least in the foreseeable future and, since it can be easily measured, various ozone–concentration–based indicators are already pro- posed.7 Photochemical pollution indicators based on the ratio of maximum and minimum daily value of hourly ozone volume fractions have already shown its potential to become good measure for the photochemical pollu- tion.8,9 Those proposed indicators, that have shown good predictive power in the case of central European, Medi- terranean (Italy, Slovenia, Croatia) and at the UK sets of stations, will be questioned and discussed here. For good comparison, it was decided that the same year range will be used as in the case of California.10 Cali- fornia is well known as one of the most problematic regions in the USA concerning ozone pollution. How- ever, it has been shown that, following the rapid urbani- sation and increased carbon fuel consumption, ozone is becoming major problem in the eastern parts of Rocky Mountains area as well,11 although exceedance in ozone levels is still considered highly related to natural causes, e.g. wildfires.12 According to the most recent analyses, oil and natural gas operations in Colorado represent high new source of volatile organic compounds which, in the end, causes elevated ozone values in the area.13 Various photochemical oxidants in the lower tropo- sphere, which are considered to be part of photochemi- cal smog, are widely recognised as dangerous to both plants and animals (including humans).1,2 That is espe- cially true for the most polluted areas in the world such as, for example, Pearl River Delta in China.3,4 As one of the most dangerous oxidants in atmosphere, ozone is one of the major tropospheric pollutants and an important component of the photochemical smog. It should not, however, be omitted its important role as a protector of the life on Earth as part of the stratospher- ic ozone layer. Major sources of the elevated ozone concentration in the troposphere are of anthropogenic origin. Whether as a result of a natural background generation of precursors (e.g. terpenes), direct genera- tion by wildfires or lightnings or as a result of human activities which produce higher quantities of ozone precursors, production of ozone is connected with several cycles. In those cycles, major ozone precur- sors, such as NOx (NO, NO2), CO and various VOCs (volatile organic compounds), participate. Analysis of Ozone Data by Photochemical Pollution Indicators in Colorado Brunislav Matasović,a,* Leo Klasinc,a,b Sean P. McGlynnb aDivision of Physical Chemistry, Ruđer Bošković Institute, Bijenička c. 54, HR-10002, Zagreb, Croatia bDepartment of Chemistry, Louisiana State University, 232 Choppin Hall, Baton Rouge, LA 70803, USA RECEIVED AUGUST 16, 2013; REVISED NOVEMBER 6, 2013; ACCEPTED NOVEMBER 7, 2013 Abstract. In order to assess the situation with air pollution by ozone in Colorado region during sunny part of the year (April to September), recently introduced photochemical pollution (PP) indicators have been applied on acquired ozone data, i.e. hourly ozone volume averages. PP indicators show very low values at the majority of stations except for the two located in Denver and Arvada which are located in the centre of the highly populated area covered by this assessment. Some other stations (e.g. National Renewable Ener- gy Labs) have somewhat high number of excess times during the growth period; total daily turnover of ozone is, however, rather low indicating absence of other photochemical pollutants (e.g. NOx) which, therefore, justify low values of PP indicators. Generally, our assessment method shows very low PP in the assessed area which is central, most populated part of the state of Colorado. (doi: 10.5562/cca2357) Keywords: photochemical pollution, pollution indicators, ozone, ozone precursors, growth season * Author to whom correspondence should be addressed. (E-mail: bmatasov@irb.hr) CROATICA CHEMICA ACTA CCACAA, ISSN 0011-1643, e-ISSN 1334-417X Croat. Chem. Acta 86 (3) (2013) 325–329. http://dx.doi.org/10.5562/cca2357 Original Scientific Article INTRODUCTION Since over- all pollution is essentially not reduced enough, it can be concluded that the level of ozone in the boundary 326 B. Matasović et al., Analysis of Ozone Data in Colorado Figure 1. Map of Colorado showing the locations of the monitoring sites. The names of the stations, abbreviations and their geo- graphical coordinates are given in Table 1. Figure 1. Map of Colorado showing the locations of the monitoring sites. The names of the stations, abbreviations and their geo- graphical coordinates are given in Table 1. Table 1. Geographical coordinates of monitoring sites Monitoring station Station abbrev. N Latitude W Longitude Altitude/ m Denver D 39.751761° 105.030681° 1621 Boulder B 39.957212° 105.238458° 1669 Highland Reservoir H 39.567887° 104.957193° 1747 Arvada A 39.800333° 105.099973° 1640 Welch W 39.638781° 105.139480° 1742 Natl. Renew. Energy Labs N 39.743724° 105.177989° 1832 Fort Collins CSU F 40.577470° 105.078920° 1524 Table 1. Geographical coordinates of monitoring sites Croat. Chem. Acta 86 (2013) 325. METHODS All of the used data were obtained from the US Envi- ronmental Protection Agency (http://www.epa.gov/ttn/ airs/airsaqs/detaildata/downloadaqsdata.htm). In Table 1 are given the locations of monitoring sites used in this paper. Locations are, also, shown on Figure 1. US EPA gives volume fractions in ppm, which are, for the sake of this analysis, all converted to ppb. All of the used data were obtained from the US Envi- ronmental Protection Agency (http://www.epa.gov/ttn/ airs/airsaqs/detaildata/downloadaqsdata.htm). In Table 1 are given the locations of monitoring sites used in this paper. Locations are, also, shown on Figure 1. US EPA gives volume fractions in ppm, which are, for the sake of this analysis, all converted to ppb. The calculation used in this article for the data analysis has been already described.8–10 It was, original- ly developed by analysing ten years of ozone data from European EMEP stations, but has proven applicable for other stations, too. Indicators that we used can be de- fined as: 1P RM A  (1) 2 exc (1 168 ) P R t N   (2) 3 1 2 P P P   (3) 1P RM A  (1) 2 exc (1 168 ) P R t N   (2) 3 1 2 P P P   (3) (1) (2) the arbitrary 1 hour/week average excess time that, if present, would double the R value. 3 1 2 P P P   (3) All indicators are based on daily maximum-to- minimum ratios. The minimum value is set to 0.8 if recorded as zero in accordance with the detection limit of the instruments in order to avoid division with zero which would be mathematically impossible. It is very important to see that indicators and R tend to have very high values in cases of near-zero values of minimum, therefore PPI (photochemical pollution indicators) have to be applied with care. It is, however, correct for indi- where R is the average of daily maximum-to-minimum ratios, M is the seasonal average of daily maximum values, A is the average of all seasonal data, texc is the duration in hours the limit of 80 ppb was exceeded (“excess time”) and N is the total number of hourly averages of ozone volume fractions measured over some period of time. The 168 factor in P2 arises from 327 B. RESULTS AND DISCUSSION The results for all stations are shown in Table 2. Data in the table include (in the order of the columns) location, name of the station followed by corresponding average values of the ozone volume fraction (A/ppb), average of daily maximum-to-minimum ratios (R), average of daily maxima (M/ppb), average excess time during which the 80 ppb limit was exceeded (texc/h) and averages of indi- cators P1, P2 and P3. The last column gives the total number of recorded and validated hourly average ozone volume fractions. Calculations were made for the period from April to September. For every station 6-year peri- ods from 2000 to 2005 has been covered. Somewhat higher average number of exceedances can be attributed to the situation in the year 2003 when all stations have many exceedances. Other results show low number of excess times at all stations even if we compare this number with those for Louisiana stations not to mention California. It is, however, still noticeable that two stations with the highest value of texc still have low value of P3 (17.6 at Higland Reservoir and 12.3 at National Renewable Energy Labs). Those two stations have small difference between daily maximum and min- imum fraction. Possible reason for that is a transfer of ozone from the nearby heavily polluted areas (Denver, Arvada) and lack of destruction of ozone in this area. In the case of Colorado stations, it cannot be con- cluded whether basic indicators P1 or P2 contribute to the combined indicator of P3 differently, as in the case of California where the influence of P2 is higher,10 or the same as in the moderate climate region.8 In this case with mountain climate, for the majority of stations this difference is negligible, while in the case of two stations – Highland Reservoir and National Renewable Energy Labs – contribution of P2 to the P3 is much higher. However, those two stations also have the highest num- ber of exceedances over the 80 ppb threshold which is the main contributor to the P2 indicator. Generally, it should be noted that all exceedances at all sites in the covered period are observed during summer season (i.e. April–to–September). Monthly averages and averages of daily maxima of ozone con- centrations for all stations are all significantly higher (up to double) for April–to–September period than for the October–to–March. METHODS Matasović et al., Analysis of Ozone Data in Colorado As can be seen from the data for the P3 indicator, the majority of these stations are much less photochemi- cally polluted than the stations in California. For the 5 stations value of P3 is lower than 40. In fact many sta- tions have the P3 indicator value less than 20. Only centrally positioned (in the middle of the most densely populated area of the state of Colorado) stations of Denver and Arvada have high P3 values of slightly over 100. Those two stations may be certainly proclaimed as polluted and one may also conclude that ranges estab- lished for the moderate climate region are more appro- priate here than those applied in California. Stations in California, which is also known as the very photochem- ically polluted area, have much higher P3 values compa- rable to those in the Pearl River Delta (maximum P3 value there was 277), while these stations show in al- most every case very low pollution. For further compar- ison, P3 values for three Louisiana stations (8-year aver- ages) were from 57 to 71.4 cators to show high values in cases where all ozone has been spent since that occur in the presence of various pollutant species in the atmosphere (e.g. NOx). Connec- tion between high PPI values with low minima can be seen from frequency distribution which is also shown in this article. Although these indicators have been developed in order to adjust simple maximum-to-minimum ratio of daily hourly volume fractions of ozone as a measure for a photochemical pollution, they are still very de- pendent on the R-value. R-value indirectly reflects the daily ozone turnover. Our indicators may also be a good measure for different effects which may occur on either organisms or materials. Croat. Chem. Acta 86 (2013) 325. RESULTS AND DISCUSSION This is consistent also with the already observed10 significant shift in the period of year with high yield of ozone; after 100 % here, April–to– September period covers over 95 % of high ozone Table 2. Seasonal (April-to-September) values of various parameters and PP-indicators for 7 monitoring sites in Colorado Station A / ppb R M / ppb texc / h P1 P2 P3 N Denver 27.9 48.0 58 31 100.4 109.1 102.7 25242 Boulder 37.6 4.5 58 31 6.9 10.0 8.1 25792 Highland Reservoir 40.5 8.6 62 47 13.2 24.1 17.6 25311 Arvada 28.4 45.5 58 37 92.7 111.7 100.3 25883 Welch 32.2 10.0 51 11 15.9 14.1 14.8 25738 Natl. Renewable Energy Labs 42.4 5.2 63 75 7.6 20.3 12.3 25810 Fort Collins CSU 30.9 21.0 55 19 38.3 36.3 37.0 24602 Croat. Chem. Acta 86 (2013) 325. B. Matasović et al., Analysis of Ozone Data in Colorado 328 P3(D) = 102.7 P3(B) = 8.1 P3(H) = 17.6 P3(A) = 100.3 P3(W) = 14.8 P3(N) = 12.3 P3(F) = 37.0 Figure 2. Histogram representations of frequency distributions of ambient ozone fractions (in 5 ppb intervals). Station abbrevia- tions are shown in Table 1. P3(H) = 17.6 P3(D) = 102.7 P3(B) = 8.1 P3(A) = 100.3 P3(W) = 14.8 P3(N) = 12.3 P3(F) = 37.0 Figure 2. Histogram representations of frequency distributions of ambient ozone fractions (in 5 ppb intervals). Station abbrevia- tions are shown in Table 1. of ozone values at different sites in the world.4,8–10 Although applied on the very diverse sets of data (e.g. sets of data from the areas with different climate, from subtropic highly polluted areas of China, over Mediter- ranean area and lowlands of Great Britain to the moun- tains and high plateaus of Colorado), simple PPIs such as P1, P2 and, the most importantly, P3 prove to be a good measurement for the assessment of a photochem- ical pollution. Low values are connected to the low PP, while high values have a direct connection with higher PP. Profiles of the distribution of the hourly ozone volume averages are also directly connected with the PPIs. The major problem about PPIs may be a non- linear relation between their high and low values. Much higher values (for example, for a ten or a hun- dred times) do not mean that the pollution is higher for the same level. Croat. Chem. Acta 86 (2013) 325. CONCLUSION 4. L. Klasinc, T. Cvitaš, S. P. McGlynn, M. Hu, X. Tang, and Y. Zhang, Croat. Chem. Acta 84 (2011) 11–16. The assessment of photochemical pollution on the sta- tions in the state of Colorado during the growth season from April–to–September using photochemical pollu- tion indicators based on hourly ozone volume fraction averages during the years 2000 to 2005 shows that those sites are generally far less polluted than any other previ- ously assessed using this method. Except for two sta- tions positioned in the densely populated areas, all other stations can be pronounced fairly clean. During another half of the year there are absolutely no photochemical pollution problems in this area which also can be seen from the fact that not a single exceedance of the 80 ppb threshold was observed during that part of the year. In all the other assessed areas which we covered in our previous papers9,10 at least few percent of the exceed- ance occurred during non-growth period. It was shown that the indicators reflect the distribution of hourly aver- age ozone volume fractions very well. High indicator values are associated with a significantly higher fre- quency of low hourly ozone volume data values. 5. D. S. Stevenson, F. J. Dentener, M. G. Schultz, K. Ellingsen, T. P. C. van Noije, O. Wild, G. Zeng, M. Amann, C. S. Atherton, N. Bell, D. J. Bergmann, I. Bey, T. Butler, J. Cofala, W. J. Collins, R. G. Derwent, R. M. Doherty, J. Drevet, H. J. Eskes, A. M. Fiore, M. Gauss, D. A. Hauglustaine, L. W. Horowitz, I. S. A. Isaaksen, M. C. Krol, J.-F. Lamarque, M. G. Lawrence, V. Montanaro, J.-F. Müller, G. Pitari, M. J. Prather, J. A. Pyle, S. Rast, J. M. Rodriquez, M. G. Sanderson, N. H. Savage, D. T. Shindell, S. E. Strahan, K. Sudo, and S. Szopa, J. Geophys. Res. - Atmos. 111 (2006) D08301. Montanaro, J.-F. Müller, G. Pitari, M. J. Prather, J. A. Pyle, S. Rast, J. M. Rodriquez, M. G. Sanderson, N. H. Savage, D. T. 6. H. E. Scheel, H. Areskoug, H. Geiss, B. Gomiscek, K. Granby, L. Haszpra, L. Klasinc, D. Kley, T. Laurila, A. Lindskog, M. Roemer, R. Schmitt, P. Simmonds, S. Solberg, and G. Toupance, J. Atmos. Chem. 28 (1997) 11–128. 7. E. Paoletti, A. de Marco, and S. Racalbuto, Environ. Monit. Assess. 128 (2007) 19–30. 8. E. Kovač-Andrić, G. Šorgo, N. REFERENCES probably has no physical meaning. Another potentially important relation of PPIs with the slope of ozone turnovers will be investigated in the future for all sites. 1. L.-W. Lao, Int. J. Environ. Health Res. 22(4) (2012) 287–304 2. J. Giles, Nature 435 (2005) 7. 2. J. Giles, Nature 435 (2005) 7. 3. Y. Tao, W. Huang, X. Huang, L. Zhong, S. E. Lu, Y. Li, L. Dai, Y. Zhang, and T. Zhu, Environ. Health Perspect. 120(3) (2012) 393–398. Acknowledgements. This work has been financially supported by the Ministry of Science, Education and Sports of the Re- public of Croatia (Project code 098-0982915-2947). Croat. Chem. Acta 86 (2013) 325. RESULTS AND DISCUSSION That is making difficult, at least for now, to give a correct range of PPIs for a polluted or non-polluted sites at the global scale (please compare references 8 and 10). It is, however, something that can be expected since the indicators are based on the average of daily maximum-to-minimum ratios of ozone hourly concentrations (volume fractions) which events in Europe, 91 % in Louisiana and in the most abundant June-November period only 84 % of the events in Pearl River Delta.4 Since low values of aver- age ozone hourly data critically affect the R-value and finally PP assessment, comparison of the shape of the ozone data frequency distribution with the P3 for Colo- rado stations was also taken into account. As it can be seen form the Figure 2., sites with low values of a P3 indicator have quasi-normal distribution of hourly ozone volume fractions averages, while those with higher values of P3, especially Denver and Arvada, have higher frequency of the very low ozone volume fractions which, logically, highly influences their P3 values. On the example of the Fort Collins station trans- formation from one to another distribution can easily be observed. If cumulative probability distribution is com- pared with P3 values, it can be seen that its shape trans- fers from sigmoidal to non-sigmoidal with the growth of P3. It can, therefore, be concluded that there is a strong correlation between the value of P3 and the shape of ozone data distribution. Finally, a global applicability of the PPI can be discussed as they were already used for the assessment Croat. Chem. Acta 86 (2013) 325. 329 B. Matasović et al., Analysis of Ozone Data in Colorado CONCLUSION Kezele, T. Cvitaš, and L. Klasinc, Environ. Monit. Assess. 165 (2010) 577–583. 9. B. Matasović, L. Klasinc, and T. Cvitaš, Croat. Chem. Acta 86 (2013) 57–64. 10. B. Matasović, T. Cvitaš, and L. Klasinc, Croat. Chem. Acta 85 (2012) 71–76. 11. R. Kohut, C. Flanagan, J. Cheatham, and E. Porter, West. N. Amer. Naturalist 72(1) (2012) 32–42. 12. D. Jaffe, Environ. Sci. Technol. 45(2) (2011) 432–438. 13. J. B. Gilman, B. M. Lerner, W. C. Kuster, and J. A. de Gouw, Environ. Sci. Technol. 47(3) (2013) 1297–1305.
https://openalex.org/W4287383892	https://awsjournal.org/wp-content/uploads/articles_xml/2675-9462-aws-40-e020220092/2675-9462-aws-40-e020220092.pdf	English	null	Native range efficacy assessment of Calophya terebinthifolii , a candidate biological control agent of Schinus terebinthifolia in Florida, USA	Advances in Weed Science	2,022	cc-by	5,914	Native range efficacy assessment of Calophya terebinthifolii, a candidate biological control agent of Schinus terebinthifolia in Florida, USA Native range efficacy assessment of Calophya terebinthifolii, a candidate biological control agent of Schinus terebinthifolia in Florida, USA James P. Cudaa* , Marcelo D. Vitorinob , Larissa Binib, Marcus M. Boenob, Edzard van Sentenc a Entomology & Nematology Department, IFAS, University of Florida, Gainesvsille, FL, USA. b Departamento de Engenharia Florestal, Universidade Regional de Blumenau, Blumenau, SC, Brazil. c IFAS Statistical Consulting Unit and Agronomy Department, University of Florida, Gainesville, FL, USA. to confirm the biological control potential of C. terebinthifolii. Methods: We used a randomized complete block design with four treatments (chemical exclusion [control], natural infestation, low and high psyllid densities) replicated in space. Results: At low densities, C. terebinthifolii significantly reduced flower production by 80.6% and 93.9%, respectively, compared to the chemical exclusion treatment. At high densities, the psyllid completely inhibited fruit production. Furthermore, leaf and stem biomass were significantly reduced following sustained attack by the psyllid C. terebinthifolii. Conclusions: Overall, our results confirmed that feeding damage by this psyllid negatively impacts above ground vegetative growth and reproduction of Brazilian peppertree. Therefore, C. terebinthifolii should be considered for introduction into Florida for biological control of this invasive shrub. Abstract: Background: Aroeira or Brazilian peppertree, Schinus terebinthifolia Raddi (Anacardiaceae: Rhoeae), is one of the worst invasive plants in California, Florida, and Hawaii, USA because of its wide distribution, adaptation to various habitats, toxicity, and demonstrated negative impacts on biodiversity. Chemical and mechanical methods provide only temporary control, are expensive, may damage native plants, and often create disturbance conducive to reinvasion by Brazilian peppertree and other invasive weeds. The leaflet galling psyllid Calophya terebinthifolii Burckhardt & Basset (Calophyidae: Calophyinae) is native to South America and recent studies have shown this insect attacks only Brazilian peppertree. Nymphs complete their development in open pit galls that create nutrient sinks, which divert resources away from normal plant growth and reproduction. Objective: The objective of this study was Keywords: Aroeira; Brazilian Peppertree; Invasive Shrub; Biocontrol; Field Impact Study formerly Planta Daninha formerly Planta Daninha RESEARCH ARTICLE formerly Planta Daninha * Corresponding author: <jcuda@ufl.edu> The invasive characteristics of Brazilian peppertree in the USA and globally are due to a variety of factors including allelopathy (Nickerson, Flory, 2015), hybrid vigor (Geiger et al., 2011), and the lack of competitive plant taxa or natural enemies (Hoshovsky, Randall, 2000). The absence of natural enemies provided the incentive for targeting Brazilian peppertree for classical biological control in Hawaii during the 1950s (Krauss, 1963; Yoshioka, Markin 1991). 1. Introduction Aroeira or Brazilian peppertree, Schinus terebinthifolia Raddi (Sapindales: Anacardiaceae), is a global invader. In the USA, this non-native woody shrub threatens the biodiversity of disturbed and natural ecosystems in California, Hawaii, Texas, Alabama, Georgia, and Florida (Center for Invasive Species and Ecosystem Health, 2020). For example, Brazilian peppertree has displaced several populations of two federal or state listed coastal Florida plant species: the beach jacquemontia, Jacquemontia reclinata House ex Small (Solanales: Convolvulaceae) and the beach star, Remirea maritima Aubl. (Poales: Cyperaceae) (Langeland et al., 2008). Furthermore, the existence of several federal-listed endangered and threatened native plants in Hawaii has been compromised due to the invasive habits of Brazilian peppertree (U.S. Fish and Wildlife Service, 1999). Brazilian peppertree was introduced into Florida from South America as an ornamental shrub in the late 1800s (Morton, 1978). It eventually escaped cultivation and invaded entire ecosystems in central and south Florida (Morton, 1978). Furthermore, other invasive species have benefitted from Brazilian peppertree’s rapid growth, prolific seed production and tolerance to environmental extremes (Ewel et al., 1982). For instance, Brazilian peppertree is a preferred novel host for the naturalized Caribbean root weevil, Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae), and the red -banded thrips, Selenothrips rubrocinctus Giard. (Thysanoptera: Thripidae) that are major pests of fruit trees as well as some landscape and crop plants in Florida and California (Morton, 1978; Jetter, Godfrey, 2009). Conflict of Interest: The authors declare that there is no conflict of interest regarding the publication of this manuscript. 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 that the original author and source are credited. Building on the Hawaii experience, classical biological control research was initiated in Florida during the mid-1980s (Wheeler et al., 2016). The long-term goal 2.1 Field site description Between 15-23 September 2015, a field site (altitude 67 m) was established at the Universidade Regional de Blumenau (FURB) experimental unit located in Gaspar, Santa Catarina, Brazil (S 26o 54.676’ and W 48o 56.194’). The region’s climate, which is similar to that of peninsular Florida, is classified as cfa (Koeppen) - subtropical, with hot summers, temperatures above 22ºC, average annual relative humidity of 84%, and well-distributed rainfall with an annual average of 1.670 mm (Alvares et al., 2013). The site was completely enclosed with chain link fencing for security. Potted plants from seeds collected from a single mother plant susceptible to psyllid attack (Diaz et al., 2014) were propagated at the Laboratory de Monitoramento e Proteção Florestal (LAMPF) greenhouse located at the FURB, Santa Catarina, Brazil, for 4-6 months prior to the experiment. Potted plants were transferred to the Gaspar field site and transplanted into the soil with a posthole digger. Using a tape measure, holes were placed 1.5 m apart within each row and 2.0 m apart between rows. In total, 16 seedlings were planted in each of 4 rows (64 plants total). All the seedlings were watered twice daily if no rain events occurred that day. A pre-release efficacy assessment or PREA was proposed by McClay and Balciunas (2005) as one approach for avoiding the selection of agents that are host specific but not sufficiently damaging to have a population level impact on the target weed. Although this assessment often is performed in the laboratory as indicated above, McClay and Balciunas (2005) recommend conducting field tests in the country of origin to measure the effect of the natural enemy on individual plants or per-capita effect. In the native range, densities of the candidate biological control agent can be readily manipulated via insecticide treatment or cages. Fitness of the infested plants is then compared to control plants where the insects have been chemically excluded. Moreover, insects and plants are exposed to natural biotic and abiotic factors that are eliminated in laboratory tests. Fitness parameters to be examined may include non-destructive measurements (e.g., plant height, stem diameter, flower/ fruit production, chlorophyll content, and survival), as well as destructive The experimental design was a randomized complete block (8 blocks per replicate with 4 treatments per block) and two replications. Treatments in each block were randomly assigned in each of the two replicates. Cuda JP, Vitorino MD, Bini L, Boeno MM, van Senten E measurements (e.g., flower and fruit production, changes in biomass). of this project is to introduce a complex of specialist natural enemies into Florida that are capable of selectively attacking and reducing the invasiveness of Brazilian peppertree (Wheeler et al., 2016). According to McClay and Balciunas (2005), there are several key elements that should be considered when performing a PREA in the insect’s native range. For instance, biological control agent density should approach outbreak levels; testing should continue until full effects of the agent become apparent; the study should be replicated to facilitate valid statistical design and analysis, and climatic and environmental conditions should match those of the proposed release area. Leaflet galling psyllids of the genus Calophya Löw (Hemiptera: Calophyidae) feed primarily on plants in the Anacardiacae, and several species are important natural enemies of Brazilian peppertree because they are host specific (Burkhardt et al., 2018). Calophya terebinthifolii Burckhardt & Basset, one of four species that attacks only Brazilian peppertree (Prade et al., 2021), is a polyvoltine species that reproduces continuously (Christ et al., 2013). Females deposit on average 55 eggs on new leaf flushes along leaflet margins, midribs, petiolules, and leaf buds (Christ et al., 2013). There are five nymphal instars, and a new generation is produced in about 44 days. At high densities, the psyllid galls induce leaf abscission (Prade et al., 2016). Maximum adult longevity is ~50 days (Prade et al., 2021). The objective of this two-year field study was to evaluate the impact (per-capita damage) of C. terebinthifolii on its host plant in southeastern Brazil using insecticide exclusion and short-term field cage releases. Results from these studies will be used to predict the damage to Brazilian peppertree following the release of Calophya spp. into Florida and elsewhere. In 1999, a group of weed biological control practitioners attending the X International Symposium on Biological Control of Weeds in Bozeman, Montana, adopted a Code of Best Practices for the discipline (Balciunas, 2000). The Code, which consists of 12 guidelines, covers all aspects of weed biological control. Guideline 3 (“Select agents with potential to control the target weed”) is particularly relevant because it is important to select agents that not only are safe to release, i.e., host specific but also are effective in controlling the target weed. Host range testing, which is a critical aspect of the selection process (Sheppard et al., 2005), does not address the efficacy issue. Impact studies, when they are performed, tend to be somewhat artificial in nature because of their relatively short duration and laboratory setting that mandates the use of caged potted plants (Vitorino et al., 2011; Cuda et al., 2016; Prade et al., 2016) that are not subjected to natural abiotic and biotic factors. https://doi.org/10.51694/AdvWeedSci/2022;40:00011 Copyright: 2022 Copyright: 2022 Copyright: 2022 1 2.2 Insect collections and field cage releases Except for the natural infestation treatment, each seedling in the four-psyllid density (low) and 16 psyllid density (high) treatments were caged for 2 weeks with the appropriate number of adult psyllids beginning with the high density treatment on 5 October 2015. Adult psyllids were collected at the restinga area in Itajaí, Santa Catarina, Brazil (S 26o 54.676’ and W 48o 56.194’) with a mouth aspirator and 9-dram snap cap plastic vials (maximum 8 psyllids/ vial). Field cages with open bottoms were constructed with 1/2” PVC pipe and 3-way fittings (30 cm x 30 cm x 60 cm or 12” x 12” x 24”). PVC cage frames were placed over rebar steel rods (0.95 cm x 33 cm or 3/8” x 13”) hammered into the soil for ~ half the length to anchor them. Frames were then covered with white chiffon cage covers (30 cm x 30 cm x 76 cm, BioQuip® Products, Inc, Compton, CA) to confine the psyllids. The cage covers were secured at the base with 4 cement bricks (1 per side). PVC frames and chiffon covers were carefully removed and transferred to either the low density treatment plants (4 psyllids per plant) or high density treatment plants (16 psyllids per plant) every two weeks and the appropriate number of adult psyllids added to each cage during the psyllid augmentation interval (8 months). In January 2016, half the Brazilian peppertree plants were randomly removed from both blocks due to canopy expansion, and larger new PVC frames and cage covers (60 cm x 60 cm x 200 cm or 24” x 24” x 80”) were installed to accommodate plant growth. Psyllid and plant parameters were assessed from all the plants including those that were removed every 2 weeks until January 2017 and comprised the following: number of adult psyllids per plant, number of leaves with one or more galls, and number of flowers and fruits per plant. Flowers and fruits produced on each plant were removed and recorded in the Spring (September 2016), and Fall (April, May 2017), which coincides with the reproductive phenology of Brazilian peppertree (Milani et al., 2021). 2.3 Data analysis Response data were analyzed using generalized linear mixed model methodology as implemented in SAS PROC GLIMMIX (SAS/STAT 15.1, SAS Institute, Cary, NC) using an appropriate distribution function viz. normal for leaf and stem biomass, and negative binomial for flower and fruit number, using the canonical link function for each distribution. Treatment was the sole fixed effect and block the sole random effect. Least squares means were calculated and transformed to the data scale using the link option of the LSMEANS statement in the above-named procedure. Means were compared using simple pairwise t-tests without any adjustment for multiple comparisons based on the recommendations by made by Milliken and Johnson (2009) and Saville (2015). Except for stem biomass (α = 0.1), all statistical analyses used a Type I error rate of α = 0.05. 2.1 Field site description Treatment positions were indicated by wooden stakes (100 cm x 5 cm x 5 cm). Tops of the stakes were spray painted with different colors to indicate each treatment: the control group (psyllids chemically excluded), natural infestation, low psyllid density (4 adults per plant) and high psyllid density (16 adults per plant). On 28 September 2015, all control plants were treated with imidicloprid insecticide (Adonis® 2F, Envincio, 2 Adv Weed Sci. 2022;40:e020220092 https://doi.org/10.51694/AdvWeedSci/2022;40:00011 https://doi.org/10.51694/AdvWeedSci/2022;40:00011 Native range impact of C. terebinthifolii Native range impact of C. terebinthifolii Cary, NC) according to label directions to exclude psyllid development. In total, 6 ml of insecticide were mixed in 244 ml of water (250 ml total volume) and was applied as a drench to the base of each control plant monthly; the concentration was increased as the plants grew as per label recommendations. Time release fertilizer (Osmocote Plus®, 15N-9P-12K, The Scotts Company, Marysville, OH) also was applied to all the plants (19 g per plant) after they were transplanted at Gaspar. variation were obtained. If the coefficient of variation was above 4%, additional subsamples were weighed until a value below 4% was reached. The average value of the weights of the subsamples was then used to obtain the total amount of fruit produced per tree. Total fruit production per tree was calculated using the Rule of Three (Cocker, 1702); the average weight equal to 50 fruits and the total weight of each tree per treatment was used to estimate the total amount of fruit in relation to the total weight. In June 2017, all leaves were removed and stems carefully cut down at the soil line, bagged separately by treatment in tight mesh plastic bags, and transported to the LAMPF for processing. Plant samples were washed and then dried in plastic bags at 70oC in a 2 m3 pilot wood drier (Benecke Industries, Timbó, Santa Catarina) until a constant weight was obtained (rechecked every 2 days). Bags were then transferred to hard plastic boxes (51 cm L x 31 cm W x 30 cm H), stacked inside the drier to complete the process and weighed to the nearest gm on a scale (Marte balanças eletrônicas, São Paulo, São Paulo state). https://doi.org/10.51694/AdvWeedSci/2022;40:00011 3. Results and Discussion terebinthifolii feeding although the impact was more subtle compared to the insect’s effect on reproduction. Psyllid feeding damage significantly reduced leaf dry matter (Figure 4, F3,84 = 3.3, P = 0.025) and substantially decreased stem dry matter (Figure 4, F3,84= 2.2, P = 0.095) in the low-density treatment. In a growth chamber study, feeding damage by the leaflet galling psyllid C. terebinthifolii decreased biomass accumulation (vegetative growth) of Brazilian peppertree by ~40% (Vitorino et al., 2011). Although the research by Vitorino et al. (2011) provided valuable insight into the psyllid’s potential impact to the growth of Brazilian peppertree under field conditions, this was a short-term experiment (3 months) conducted with potted Brazilian Final above-ground vegetative growth (biomass) of Brazilian peppertree also was affected by C. terebinthifolii feeding although the impact was more subtle compared to the insect’s effect on reproduction. Psyllid feeding damage significantly reduced leaf dry matter (Figure 4, F3,84 = 3.3, P = 0.025) and substantially decreased stem dry matter (Figure 4, F3,84= 2.2, P = 0.095) in the low-density treatment. However, only a few galled leaves were observed in this treatment group, which indicated the systemic insecticide imidacloprid effectively inhibited nymphal development for the duration of the experiment. Furthermore, all plants in each treatment group except for the controls were successfully attacked by the psyllid; there was no indication of the hypersensitivity response that was observed in Calophya latiforceps Burckhardt, a congener of C. terebinthifolii (Diaz et al., 2014). , In a growth chamber study, feeding damage by the leaflet galling psyllid C. terebinthifolii decreased biomass accumulation (vegetative growth) of Brazilian peppertree by ~40% (Vitorino et al., 2011). Although the research by Vitorino et al. (2011) provided valuable insight into the psyllid’s potential impact to the growth of Brazilian peppertree under field conditions, this was a short-term experiment (3 months) conducted with potted Brazilian Natural Low Density Sampling date High Density Exclusion Adults Galled Leaves Total Number 400 200 0 800 600 400 200 0 Sep 2015 Jan 2016 Jan 2017 May Sep Sep 2015 Jan 2016 Jan 2017 May Sep Sep 2015 Jan 2016 Jan 2017 May Sep Sep 2015 Jan 2016 Jan 2017 May Sep Figure 1 - Total number of adult Calophya terebinthifolii and galled leaves observed on plants in each treatment group, Septembe 2015- January 2017. Curves created with a penalized beta spline algorithm. 3. Results and Discussion On 29 October 2015, psyllid cages (PVC frames and covers) at the Gaspar field site were moved from all the high psyllid density plants back to the low-density plants. After removing the cage covers, eggs/crawlers of C. terebinthifolii were observed on one of the high psyllid density plants. Normal development of C. terebinthifolii was confirmed on 30 November 2015 with the appearance of mature nymphs and adults on leaflets of two of the high psyllid density treatment plants. These observations indicated the cage system was effective for augmenting psyllid populations during the experiment. Total fruit numbers were estimated using the following procedure. After sorting, total amount of fruits produced per plant in each block and treatment were weighed. Fruit size was standardized by sieving to separate smaller fruits to minimize size variation. Fruits were then weighed in 10 subsamples containing 50 fruits each and the variation coefficient was calculated until values below 4% error Adult psyllid populations remained relatively low until August 2016 and then exhibited an exponential increase in population growth that continued until January 2017 https://doi.org/10.51694/AdvWeedSci/2022;40:00011 Adv Weed Sci. 2022;40:e020220092 3 Cuda JP, Vitorino MD, Bini L, Boeno MM, van Senten E (Figure 1). Number of leaves with one or more galls likewise increased in all treatment groups from September 2016 to January 2017 except for the controls (Figure 1). Surprisingly, the highest number of adult psyllids (530) was observed on plants in the chemical exclusion (control) treatment in December 2017. This finding was not anticipated. However, neonicotinoid insecticides like imidacloprid used in this study have been associated with alteration of physiological processes in plants leading to greater absorption of water and nutrients, resulting in higher plant growth rates (Matiello, Almeida, 2000; Venancio et al., 2003). This could account for greater shoot production in the imidacloprid treated plants, which made them more attractive to the adult psyllids. There was a significant reduction in the average number of flowers (Figure 2, F3,7 =7.76, P = 0.0382) and fruits (Figure 3; F3,7 = 17.8, P =0.0018) produced per plant that was correlated with the adult psyllid treatments. In the high-density treatment, subsequent higher nymph and gall development probably induced substantial leaf abscission that resulted in complete inhibition of fruit production in Brazilian peppertree. Final above-ground vegetative growth (biomass) of Brazilian peppertree also was affected by C. 4. Conclusions The decrease in flowering and complete inhibition of fruit production that was observed at high psyllid densities has important implications for the management of Brazilian peppertree in Florida, USA. The invasive habit of Brazilian peppertree is directly correlated with fruit consumption and seed dispersal by birds and small mammals (Ewel et al., 1982). The ability of C. terebinthifolii Figure 2 - Effect of Calophya terebinthifolii on number of flowers per plant produced in each treatment group. Bars represent mean flower numbers ± SEM. Different letters denote statistical differences (F3,7 =7.76, P = 0.0382) 90000 70000 50000 40000 20000 0 a b c d Treatment Natural Exclusion Low Density High Density No. of Fruits 80000 60000 30000 10000 Figure 3 - Effect of Calophya terebinthifolii on number of estimated fruits (drupes) per plant produced in each treatment group. Bars represent mean fruit numbers ± SEM. Different letters denote statistical differences (F3,7 = 17.8, P = 0.0018) 90000 70000 50000 40000 20000 0 a b c d Treatment Natural Exclusion Low Density High Density No. of Fruits 80000 60000 30000 10000 5 Adv Weed Sci. 2022;40:e020220092 https://doi.org/10.51694/AdvWeedSci/2022;40:00011 90000 70000 50000 40000 20000 0 a b c d Treatment Natural Exclusion Low Density High Density No. of Fruits 80000 60000 30000 10000 Figure 3 - Effect of Calophya terebinthifolii on number of estimated fruits (drupes) per plant produced in each treatment group. Bars represent mean fruit numbers ± SEM. Different letters denote statistical differences (F3,7 = 17.8, P = 0.0018) Dry matter [g] 0 500 1000 1500 2000 0 500 1000 1500 2000 2500 a a b ab ab b a a Leaf Dry Matter Stem Dry Matter Treatment Natural Exclusion Low Density High Density Figure 4 - Effect of Calophya terebinthifolii on leaf and stem biomass (dry matter) per plant in each treatment group. Bars represent mean biomass (g) ± SEM. Different letters denote statistical differences (leaf, F3,84 = 3.3, P = 0.025; stem, F3,84= 2.2, P = 0.095) Dry matter [g] 0 500 1000 1500 2000 0 500 1000 1500 2000 2500 a a b ab ab b a a Leaf Dry Matter Stem Dry Matter Treatment Natural Exclusion Low Density High Density Figure 4 - Effect of Calophya terebinthifolii on leaf and stem biomass (dry matter) per plant in each treatment group. Bars represent mean biomass (g) ± SEM. 3. Results and Discussion Insecticide applications effectively excluded ga development on the control plants (exclusion, galled leaves) for the duration of the experiment despite the presence of comparable numbers of psyllid adults observed on all treatment plants Figure 1 - Total number of adult Calophya terebinthifolii and galled leaves observed on plants in each treatment group, September 2015- January 2017. Curves created with a penalized beta spline algorithm. Insecticide applications effectively excluded gall development on the control plants (exclusion, galled leaves) for the duration of the experiment despite the presence of comparable numbers of psyllid adults observed on all treatment plants 4 Adv Weed Sci. 2022;40:e020220092 https://doi.org/10.51694/AdvWeedSci/2022;40:00011 Native range impact of C. terebinthifolii Native range impact of C. terebinthifolii 250 200 150 100 50 0 a b b c Treatment Exclusion Natural Low Density High Density No. of Flowers Figure 2 - Effect of Calophya terebinthifolii on number of flowers per plant produced in each treatment group. Bars represent mean flower numbers ± SEM. Different letters denote statistical differences (F3,7 =7.76, P = 0.0382) peppertree seedlings under artificial light and growing conditions. More importantly, seedlings used in the growth chamber study were not of reproductive age. In contrast, the results of our 2-year field impact study showed that in addition to the slight reduction in biomass that was observed, reproduction of Brazilian peppertree was severely impacted by C. terebinthifolii. h 250 200 150 100 50 0 a b b c Treatment Exclusion Natural Low Density High Density No. of Flowers This type of impact has been observed with leaf galling insects in other systems. For example, Tectococcus ovatus Hempel from Brazil was released in Hawaii for biological control of strawberry guava, Psidium cattleianum Sabine in 2012 (Johnson, Chaney, 2014). This leaf galling insect weakens strawberry guava trees through its feeding and reduces its ability to fruit and set seed, thereby limiting its spread (U.S. Forest Service, 2016). In fact, one strawberry guava tree observed growing in a common garden at a laboratory in Brazil that was heavily infested with T. ovatus galls for 5 years ceased fruit production entirely (J.P. Cuda, pers. observ.). Funding This research was funded by the University of Florida Agricultural Experiment Station, the Florida Industrial and Phosphate Research Institute under Grant No. FIPR 12-03- 161, and the Fulbright Scholar Brazilian Scientific Mobility Program under Grant No. 48150097. The article processing charge was funded by the Department of Entomology & Nematology, IFAS, University of Florida, Gainesville, Florida, USA. Cuda JP, Vitorino MD, Bini L, Boeno MM, van Senten E Cuda JP, Vitorino MD, Bini L, Boeno MM, van Senten E Author’s contributions to restrict fruit production will eventually limit further spread and establishment of Brazilian peppertree if it is approved for release in Florida. All authors read and agreed to the published version of the manuscript. JPC and MDV: conceptualization of the manuscript and development of the methodology, funding acquisition and resources, project administration, supervision. JPC, MDV, LB, and MMB: data collection and curation. MDV, LB, MMB, and EVS: data analysis. JPC and EVS: data interpretation. JPC, MDV, and EVS: writing the original draft of the manuscript, review and editing. The results of our field research in Brazil with C. terebinthifolii also provide an indication of the potential impact on Brazilian peppertree by the congeneric C. latiforceps (Diaz et al., 2015). Calophya latiforceps was recommended for release in Florida in April 2016 by the federal interagency Technical Advisory Group for Biological Control of Weeds (TAG no. 15-02). The US Fish & Wildlife Service Biological Assessment was completed in January 2018 (U.S. Department of Agriculture, Animal and Plant Health Inspection Service USDA APHIS TAG, 2020). A permit from USDA APHIS PPQ for field release of C. latiforceps was issued in June 2019 and release from quarantine is anticipated in 2022. Acknowledgements We thank Mr. Firmino M. Santos for his assistance with maintenance of the plants and insect field cages at the FURB experimental unit in Gaspar. Finally, a petition requesting field release of C. terebinthifolii in Florida is in preparation for submission to the TAG. Because C. terebinthifolii is native to southern and southeastern Brazil (Burckhardt et al., 2018), it may be better adapted to cooler geographic areas in Florida that have been invaded by Brazilian peppertree (Christ et al., 2013). Conflict of Interest The authors declare there are no conflicts of interest regarding the publication of this manuscript. 4. Conclusions Different letters denote statistical differences (leaf, F3,84 = 3.3, P = 0.025; stem, F3,84= 2.2, P = 0.095) Dry matter [g] 0 500 1000 1500 2000 0 500 1000 1500 2000 2500 a a b ab ab b a a Leaf Dry Matter Stem Dry Matter Treatment Natural Exclusion Low Density High Density Stem Dry Matter Figure 4 - Effect of Calophya terebinthifolii on leaf and stem biomass (dry matter) per plant in each treatment group. Bars represent mean biomass (g) ± SEM. Different letters denote statistical differences (leaf, F3,84 = 3.3, P = 0.025; stem, F3,84= 2.2, P = 0.095) Figure 3 - Effect of Calophya terebinthifolii on number of estimated fruits (drupes) per plant produced in each treatment group. Bars represent mean fruit numbers ± SEM. 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https://openalex.org/W1868182489	https://www.scielo.br/j/rbepop/a/DNmw3FvHjFHtsscDjQ38Ftm/?lang=pt&format=pdf	Portuguese	null	Mundos distintos e realidades semelhantes: empregabilidade dos jovens no Nordeste e Sudeste brasileiros	Revista brasileira de estudos de população	2,015	cc-by	11,291	DOI http://dx.doi.org/10.1590/S0102-30982015000000019 DOI http://dx.doi.org/10.1590/S0102-30982015000000019 * Universidade Federal de Viçosa – UFV, Viçosa-MG, Brasil (mateus.neves@ufv.br). Universidade Federal de Viçosa – UFV, Viçosa-MG, Brasil (marcos.falcao@ufv.br). * Universidade Federal de Viçosa – UFV, Viçosa-MG, Brasil (jelima@ufv.br). R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 Palavras-chave: Empregabilidade. Jovens. Função logística. Universidade Federal de Viçosa – UFV, Viçosa-MG, Brasil (marcos.falcao@ufv.br). *** Universidade Federal de Viçosa – UFV, Viçosa-MG, Brasil (jelima@ufv.br). 1 A taxa de participação dos jovens de 15 a 24 anos, no Brasil, em 2006, era de 63,9%. Isso significa que cerca de dois em cada três jovens estavam trabalhando ou buscando ativamente uma ocupação (COSTANZI, 2009). 2 A frequência ao ensino médio na idade adequada abrange, atualmente, menos da metade dos jovens brasileiros de 15 a 17 anos, tendo em vista que cerca de um terço deles ainda está no ensino fundamental e cerca de 18% estão fora da escola (CASTRO; AQUINO, 2008). 3 Enquanto, em 2006, a taxa geral de desemprego (para os trabalhadores de 15 anos ou mais) era de 8,4% e a dos adultos correspondia a 5,6%, para os jovens essa cifra se elevava a 17,8%, sendo, portanto, aproximadamente 3,2 vezes superior à dos adultos e 2,1 vezes maior do que a taxa geral de desemprego (COSTANZI, 2009). Mundos distintos e realidades semelhantes: empregabilidade dos jovens no Nordeste e Sudeste brasileiros Mateus de Carvalho Reis Neves* Marcos Falcão Gonçalves João Eustáquio de Lima* O artigo aborda como certos fatores influenciaram a probabilidade da participação no mercado de trabalho de jovens de 15 a 24 anos, nas Regiões Nordeste e Sudeste, marcadas pela heterogeneidade entre si, em diversas dimensões. Para tanto, analisaram-se as razões de risco relativo e os efeitos marginais resultantes de uma função logística aplicada aos microdados da Pesquisa Nacional por Amostra de Domicílios – PNAD de 2012. Constatou-se que, a despeito das disparidades socioeconômicas presentes nestas regiões brasileiras, ambas exibiram comportamentos similares quanto ao efeito dos determinantes sobre a empregabilidade dos jovens. Ficou evidenciado que a escolaridade e a experiência profissional influenciam positivamente a participação no mercado de trabalho, sobretudo no Sudeste, onde um ano a mais de estudo eleva as chances de emprego em 16%. Ser mulher possui relação negativa com a probabilidade de trabalhar, especialmente no Nordeste, tendo em vista que os jovens do sexo masculino têm o dobro de chances de estarem empregados. Ainda, os jovens brancos do Sudeste possuem 28% menos probabilidade de estarem trabalhando, em relação aos não brancos. Tais resultados sugerem o caminho que podem trilhar políticas públicas, específicas para cada região, que visem mitigar o desemprego entre os jovens. Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Mundos distintos e realidades semelhantes Mundos distintos e realidades semelhantes Mundos distintos e realidades semelhantes Introdução As transformações nos modelos de produção, em curso nos países ao redor do mundo, fazem com que o desemprego inscreva-se como uma marca estrutural de diversas socieda- des contemporâneas, levando à indagação sobre a genealogia e a dimensão individual e coletiva dos processos em curso (GENNARI; ALBUQUERQUE, 2012). Ademais, Missio, Vieira e Iahn (2008) preconizam que a busca por maior produtividade do trabalho tem induzido a diminuição da oferta de empregos e a ampliação da base de exigências qualitativas do trabalhador – maior escolarização e proatividade – para que este esteja apto a ocupar as novas funções que vão surgindo com a mudança da base tecnológica do sistema produtivo. Sabe-se que entre os jovens concentra-se a maior parte dos indivíduos que buscam se incorporar ao mercado de trabalho pela primeira vez.1 Presente no contexto elucidativo acerca do elevado desemprego juvenil está justamente a dificuldade do jovem em obter o primeiro emprego. Ainda o desemprego juvenil é associado a um sistema de educação inadequado perante as já citadas, e cada vez maiores, exigências do mercado de trabalho e a incapacidade dos jovens em permanecer na escola.2 Outros autores, como Silva (2001), destacam a opção, por parte dos empresários, por trabalhadores adultos, que somam experiência e hábitos de trabalho mais sedimentados, tornando-se, portanto, mais um obstáculo aos jovens. Entretanto, de acordo com Costanzi (2009), não existe apenas uma juventude no Brasil. A heterogeneidade e as desigualdades que permeiam a sociedade brasileira tornam-se manifestas na situação dos jovens, mormente quando se considera o acesso a direitos, bens e serviços, que ampliam ou restringem as possibilidades de acesso a um trabalho decente. Existem, de fato, juventudes diversas, imersas em distintos cenários. As mulheres jovens, os jovens negros de ambos os sexos, bem como os jovens das regiões de baixa renda, ou de zonas rurais, por vezes, são molestados de forma mais severa pela exclusão social, pela falta de oportunidades e pelo déficit de oportunidades de emprego de qualidade.3 Assim, investigar algumas características relativas à participação dos jovens no mer- cado de trabalho, considerando algumas das diversidades regionais brasileiras, torna-se relevante para a compreensão de peculiaridades que auxiliem na elaboração de ações no sentido de uma melhor adequação dos jovens aos postos de trabalho. Pretende-se, portanto, a partir de dados da Pesquisa Nacional por Amostra de Domicílios (PNAD) de 2012, analisar alguns fatores associados à participação de jovens, com idade entre 15 e R. bras. R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 A taxa de participação dos jovens de 15 a 24 anos, no Brasil, em 2006, era de 63,9%. Isso significa que cerca de dois e ada três jovens estavam trabalhando ou buscando ativamente uma ocupação (COSTANZI, 2009). 4 Uma ilustração desta realidade pode ser a taxa de analfabetismo que, embora seja residual para os jovens de 15 a 24 anos nas Regiões Sul, Sudeste e Centro-Oeste, tem sua erradicação ainda se constituindo um desafio para o Nordeste, onde a taxa é de 2,4%, sendo ainda maior (3,3%) no caso dos homens jovens, em 2012. Ademais, também em 2012, a taxa de informalidade do emprego, entre os jovens, era de 34% no Sudeste, enquanto no Nordeste chegava a quase 70% (IBGE, 2013a). Dados e variáveis Os dados utilizados neste estudo foram retirados da Pesquisa Nacional por Amostra de Domicílios (PNAD), baseada em amostra probabilística de domicílios, com abrangência nacional, realizada pelo Instituto Brasileiro de Geografia e Estatística (IBGE), em setembro de 2012 (IBGE, 2013a). Conforme exposto por Leite e Silva (2002), a PNAD tem como finalidade produzir informações básicas, que possam ser empregadas no estudo de soluções que levem ao desenvolvimento do país. O plano amostral da pesquisa permite a obtenção de resultados para o total do Brasil, para cada uma das regiões geográficas e para cada estado brasileiro. Devido à natureza de sua coleta de dados, a pesquisa relata informações pormenorizadas acerca das condições socioeconômicas de cada um dos indivíduos residentes nos domi- cílios amostrados. Tendo como escopo deste trabalho os jovens residentes nas Regiões Nordeste (NE) e Sudeste (SE) do país, de ambos os sexos, com idade entre 15 e 24 anos, obteve-se uma amostra composta por 33.750 indivíduos (18.113 no NE e 15.637 no SE). Introdução Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 336 Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Mundos distintos e realidades semelhantes 24 anos, no mercado de trabalho de duas regiões brasileiras balizadas por grandes hete- rogeneidades socioeconômicas e demográficas: Sudeste e Nordeste.4 24 anos, no mercado de trabalho de duas regiões brasileiras balizadas por grandes hete- rogeneidades socioeconômicas e demográficas: Sudeste e Nordeste.4 No Sudeste então circunscritos alguns dos maiores polos urbanos e industriais, responsáveis pela maior parte do Produto Interno Bruto nacional, sendo a região mais afetada pela reestruturação produtiva. Por outro lado, o Nordeste, segundo Lima (2008), caracteriza-se pela baixa elasticidade emprego-produto, maior vulnerabilidade às variações climáticas, menores níveis de escolarização, rendimento e qualificação e grandes áreas pouco integradas à economia nacional. Assim, objetiva-se, com este estudo, examinar os diferenciais, entre as Regiões Nor- deste e Sudeste, de determinados fatores demográficos e socioeconômicos relacionados à participação dos jovens de 15 a 24 anos no mercado de trabalho. R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 5 A semana de referência da PNAD de 2012 foi a 23 a 29 de setembro daquele ano. Fatores determinantes para participação no mercado de trabalho Para determinar a participação do jovem no mercado de trabalho, foram consideradas três categorias relacionadas à situação ocupacional na qual o jovem poderia situar-se, no ano analisado, respeitando os critérios descritos nas notas técnicas da PNAD (IBGE, 2013b): estar ativo e empregado, ativo e desempregado ou inativo. Na categoria ativos R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 337 Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Mundos distintos e realidades semelhantes e empregados, enquadram-se os jovens que estavam efetivamente trabalhando (em ati- vidade remunerada ou não remunerada) no período de referência da PNAD de 2012, ou em parte dele (semana de referência5 ou período de referência de 365 dias). A categoria ativos e desempregados engloba aqueles que estavam desempregados, mas que tomaram providência na busca por emprego, também no período de referência. Por fim, inativos são os jovens que não estavam trabalhando e não haviam procurado emprego no período de referência. Vale destacar que nesta última categoria foram incluídos aqueles que estavam somente estudando, assim como os que realizavam afazeres domésticos sem remuneração. A probabilidade de os jovens estarem em uma destas categorias varia de acordo com as mais diversas características. Alguns destes atributos observáveis foram selecionados para uso neste artigo e são descritos a seguir. • Sexo – variável dummy que assume valor 1, quando o jovem é do sexo masculino, e 0, caso seja do sexo feminino. • Sexo – variável dummy que assume valor 1, quando o jovem é do sexo masculino, e 0, caso seja do sexo feminino. • Raça/cor – variável dummy que tem valor 1, quando o jovem é branco, e 0, caso contrário. • Filho – variável dummy que adquire valor 1, quando o jovem está na posição de filho da família, e 0, caso contrário (a partir da informação da pessoa de referência na família). • Cônjuge – variável dummy com valor 1, quando o jovem está na condição de cônjuge na família, e 0, caso contrário (a partir da informação da pessoa de referência na família). • Outro – variável dummy que assume valor 1, quando o jovem é outro parente, agre- gado, pensionista, empregado doméstico ou parente do empregado doméstico da pessoa de referência na família, e 0, caso contrário. R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 6 A escolha deste intervalo baseia-se na faixa etária usualmente empregada pelas Nações Unidas e Organização Internacional do Trabalho (2007) em estudos desta natureza, para motivos de comparabilidade. Entretanto, é importante salientar que a Constituição brasileira determina os 16 anos como idade mínima para o trabalho, admitindo, a partir dos 14 anos, apenas o trabalho na condição de aprendiz. Fatores determinantes para participação no mercado de trabalho • Chefe (categoria base) – variável dummy que tem valor 1, quando o jovem está na condição de pessoa de referência da família, e 0, caso contrário. • Situação de domicílio – variável dummy que apresenta valor 1, quando o jovem reside no meio urbano, e 0, caso resida no meio rural. • Experiência – anos de experiência do jovem no mercado de trabalho, somados os períodos de experiência profissional no emprego anterior, caso haja, e no emprego atual, caso haja. • Escolaridade – anos completos de estudo do jovem. • Escolaridade – anos completos de estudo do jovem. • Escolaridade x experiência – interação das variáveis experiência e escolaridade. • Componentes da família – número de componentes da família. • Renda – logaritmo natural do rendimento total per capita da família (somado 1 para rendimento zero). R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 338 Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Mundos distintos e realidades semelhantes • Situação ocupacional – variável dependente, que tem valor 0, quando o jovem per- tence à categoria inativa, 1, caso o jovem seja ativo e empregado, e 2, se estiver na categoria ativo e desempregado. • Situação ocupacional – variável dependente, que tem valor 0, quando o jovem per- tence à categoria inativa, 1, caso o jovem seja ativo e empregado, e 2, se estiver na categoria ativo e desempregado. Método Almejou-se definir quais características, entre aquelas selecionadas, foram relevan- tes para a participação no mercado de trabalho dos jovens de ambos os sexos de 15 a 24 anos,6 ou seja, buscou-se observar quais atributos o jovem e seus familiares possuíam (sexo, raça/cor, condição na família, situação de domicílio, experiência, escolaridade, ta- manho da família e renda familiar), tomando-os como fatores explicadores de sua relação com o mercado de trabalho em 2012. Além disso, procurou-se ressaltar as diversidades e similitudes regionais derivadas da comparação entre as Regiões Nordeste e Sudeste. Aplicações similares de modelos na determinação da participação dos indivíduos na força de trabalho, para o Brasil, foram abordadas por Mendonça et al. (2012), Silva e Kassouf (2002), Kassouf (1999, 1998), Fernandes e Picchetti (1999), entre outros. O instrumental econométrico utilizado, que possibilitou investigar as probabilidades de inatividade, emprego e desemprego, foi o logit multinomial, adequado para análise de variáveis dependentes categóricas, com J possíveis categorias, sem que haja alguma or- denação entre estas categorias. Desse modo, no modelo logit multinomial empregado, as categorias ocupacionais em que os jovens poderiam enquadrar-se foram definidas como: inativos (j=0); ativos e empregados (j=1); e ativos e desempregados (j=2). A estrutura destes modelos baseia-se na análise de probabilidades e, segundo Greene (2003), pode ser ilustrada na seguinte forma: (1) p g ob(Y=j) = F(X’,β) (1) Pj = Prob(Y=j) = F(X’,β) Pj = Prob(Y=j) = F(X’,β) (1) Onde j corresponde às categorias em que o jovem pode se situar. O conjunto do parâmetro β reflete o impacto das mudanças em x (matriz de características observáveis dos jovens) na probabilidade de determinado jovem se encontrar em uma das três categorias possíveis. p j g p Especifica-se o modelo logit multinomial, utilizado neste trabalho, como segue: (2) Prob Pj = j ∑k=0 (Yi = j) = e b j’xi e b k’xi , j = 0,1,2, ...J ( 2 ) ( 2 ) Onde Yi é a variável aleatória que indica a categoria ocupacional à qual o jovem pertence; Pj corresponde à probabilidade de o evento j ocorrer, o que é equivalente à probabilidade do jovem pertencer à categoria j; x representa a matriz de atributos dos jovens; e β refere-se ao vetor de parâmetros a ser estimado. R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 Método Onde Yi é a variável aleatória que indica a categoria ocupacional à qual o jovem pertence; Pj corresponde à probabilidade de o evento j ocorrer, o que é equivalente à probabilidade do jovem pertencer à categoria j; x representa a matriz de atributos dos jovens; e β refere-se ao vetor de parâmetros a ser estimado. 6 A escolha deste intervalo baseia-se na faixa etária usualmente empregada pelas Nações Unidas e Organização Internacional do Trabalho (2007) em estudos desta natureza, para motivos de comparabilidade. Entretanto, é importante salientar que a Constituição brasileira determina os 16 anos como idade mínima para o trabalho, admitindo, a partir dos 14 anos, apenas o trabalho na condição de aprendiz. R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 339 Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Mundos distintos e realidades semelhantes Nesse sentido, conforme o modelo descrito, a probabilidade de que ocorra certa resposta, ou seja, que o indivíduo se enquadre em uma das três categorias de ocupação definidas, varia entre os jovens de acordo com seus atributos observados.i Deve-se salientar que especificar o modelo conforme a equação (2) geraria uma inde- terminação, fato que pode ser resolvido escolhendo-se uma das formulações alternativas. Greene (2003) indica que uma normalização usual é supor igual a zero. Desse modo, as probabilidades seriam: (3) j ∑k=0 j) = e b j’xi e b k’xi , j = 0,1,2, ...J 1 + (3) (3) Prob Pj = j ∑k=0 (Yi = j) = e b j’xi e b k’xi , j = 0,1,2, ...J 1 + (3) e (4) Prob Pj = j ∑k=0 (Yi = 0) = e b k’xi 1 + 1 (4) (3) Prob Pj = j ∑k=0 (Yi = j) = e b j’xi e b k’xi , j = 0,1,2, ...J 1 + (3) e 1 (3) (4) ob j ∑k=0 (Yi = 0) = e b k’xi 1 + 1 (4) (4) Prob Pj = j ∑k=0 (Yi = 0) = e b k’xi 1 + 1 (4) (4) Neste trabalho, foi utilizado o método de Máxima Pseudoverossimilhança (MPV), tendo em vista características do plano amostral complexo da PNAD,7 implicando, diferentemente do método de Mínimos Quadrados Ordinários, por exemplo, coeficientes estimados que não possuem respostas marginais que possam ser assim interpretadas. 8 Para mais informações, ver Greene (2003, p. 722). 7 Para mais informações, ver Silva, Pessoa e Lila (2002). R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 Método Já a interpretação para uma variável categórica é dada pela relação de uma categoria com a outra (MENDONÇA et al., 2012).i A razão de chances no modelo multinomial logit é definida por: A razão de chances no m Prob (Y = j / x + 1) Prob (Y = k / x + 1) Prob (Y = j / x) Prob (Y = k / x) RRR = i Prob (Y = j / x + 1) Prob (Y = k / x + 1) Prob (Y = j / x) Prob (Y = k / x) RRR = (7) ( 7 ) Prob (Y = j / x + 1) Prob (Y = j / x + 1) Prob (Y = k / x + 1) Prob (Y = j / x) Prob (Y = k / x) RRR = (7) ( 7 ) ( 7 ) Segundo Powers e Xie (2000), o conceito de risco relativo assemelha-se ao de odds ratio, sendo o risco definido como a probabilidade de ocorrência de determinado evento em um intervalo de tempo. Visando facilitar sua interpretação, a odds ratio pode ser convertida em incremento percentual, conforme a expressão (8), com o resultado representando a probabilidade de mudança da categoria base para a categoria em análise como fruto de variações das características dos indivíduos. (odds 1) 100 (8) (odds – 1).100 (8) (8) Constata-se que o plano amostral adotado na PNAD é complexo, sendo formado por um processo de seleção da amostra que é executado envolvendo peso amostral, estratificação, conglomeração e probabilidades díspares de seleção (IBGE, 2013b). Para análise dos dados provenientes de processos de seleção amostral desta natureza, foram utilizados o Stata 13 (StataCorp, 2013a) e o seu comando survey.9 Neste trabalho, no intuito de destacar a importância de serem consideradas as características do plano amostral, foram calculadas as medidas MEFF para cada uma das variáveis apreciadas. Método Visando contornar esta limitação, foram calculados os efeitos marginais dos coeficientes, de modo a facilitar a avaliação dos resultados. Tais efeitos são obtidos pela diferenciação das equações (3) e (4): (5) δj = J ∑ k=0 дPj дxi Pj = βj Pk βk = Pj [ βj β ] (5) (5) Assim, de acordo com Greene (2003), os efeitos marginais são definidos conforme descrito na equação (5). Tem-se que todos os subvetores de β captam cada efeito margi- nal, por meio tanto das probabilidades quanto da média ponderada definida em δj. Esses valores podem ser computados a partir das estimativas dos parâmetros. Deve-se salientar que, para qualquer xk particular, δPj/δj xk pode não ter o mesmo sinal de βjk, ou seja, o sinal dos efeitos marginais pode não ser o mesmo dos coeficientes estimados.8 É possível complementar a abordagem dos efeitos marginais por meio de uma forma alternativa de análise, conhecida como odds-ratio ou razão de risco relativo (RRR). Define-se esta razão como a probabilidade de dado evento ocorrer, em relação a outro. Para tanto, considerando Pj a probabilidade de ocorrência do evento, e 1-Pj a probabilidade de não ocorrência, a razão de chances a favor de o evento ocorrer é dada pelo quociente: Pj 1 Pj (6) ( 6 ) ( 6 ) 340 Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Mundos distintos e realidades semelhantes Com a transformação do logit, a expressão (6) passa a ser conhecida como antilog do logit. Esta interpretação é idêntica à do logit binário. A interpretação de variáveis quanti- tativas é feita pelo incremento de uma unidade e pela comparação da categoria analisada com a categoria base. 9 Mais informações sobre a operacionalização deste comando e a aplicação da metodologia proposta neste artigo podem ser obtidas em StataCorp (2013b). R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 Análise descritiva dos dados Análise descritiva dos dados Antes de expor os resultados obtidos com a aplicação do modelo logit multinomial, as estatísticas descritivas das variáveis utilizadas podem ser visualizadas nas Tabelas 1 e 2. Notam-se distinções relativas à ocupação dos jovens residentes nas duas regiões estuda- das. Enquanto no Nordeste quase 46% dos jovens ainda não haviam buscado emprego, no Sudeste este número reduzia-se para pouco menos de 39%. Tal diferença reflete-se na quantidade de jovens que buscaram e obtiveram emprego, uma vez que, no Nordeste, estes eram pouco mais de 45%, enquanto no Sudeste ultrapassavam os 51%. Porém, em termos de jovens ativos e desempregados, nota-se semelhança entre as duas regiões, com valores R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 341 Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Mundos distintos e realidades semelhantes orno de 9%, revelando semelhança, em termos numéricos, quanto à desocupação da ulação analisada. em torno de 9%, revelando semelhança, em termos numéricos, quanto à desocupação da população analisada. TABELA 1 Distribuição dos jovens de 15 a 24 anos, segundo variáveis selecionadas Regiões Nordeste e Sudeste do Brasil – 2012 Variáveis Nordeste Sudeste % Erro-padrão % Erro-padrão Ocupação Inativos 0,4582 0,0050 0,3886 0,0048 Ativos e empregados 0,4520 0,0048 0,5164 0,0047 Ativos e desempregados 0,0899 0,0028 0,0950 0,0029 Sexo Mulheres (1) 0,5016 0,0039 0,4941 0,0043 Homens 0,4984 0,5059 Raça/cor Branca (1) 0,2624 0,0051 0,4982 0,0061 Não-branca 0,7376 0,5018 Situação na família Chefe (1) 0,1247 0,0027 0,1035 0,0026 Filho 0,6225 0,0054 0,6974 0,0048 Cônjuge 0,1264 0,0029 0,0983 0,0025 Outro 0,1264 0,0032 0,1007 0,0032 Situação de domicílio Urbano (1) 0,7272 0,0092 0,9308 0,0061 Rural 0,2728 0,0692 Fonte: IBGE. Pesquisa Nacional por Amostra de Domicílios – PNAD 2012. (1) Categorias bases que serão utilizadas no ajuste estatístico. TABELA 1 Relativamente às características demográficas, nota-se uma ligeira predominância das mulheres na composição da população jovem nordestina, sendo o contrário observado para o Sudeste. No que tange à raça/cor, grande parte dos jovens do Nordeste (74%) se declarou não branca (pretos, pardos, amarelos, indígenas e sem declaração), em profundo contraste com a composição do contingente jovem do Sudeste, onde o número de brancos era similar ao de não brancos. A posição do jovem em relação à pessoa de referência na família à qual pertence expres- sa algumas diferenças quanto às regiões analisadas. Análise descritiva dos dados Enquanto no Nordeste a quantidade de jovens na condição de responsáveis pela família era de 12%, tal proporção reduzia-se para 10% no Sudeste que, por sua vez, possuía maior parcela de jovens assumindo o papel de filhos (70%), enquanto no Nordeste 62% estavam nesta condição. Ainda, era maior o percentual de jovens nordestinos que eram cônjuges (13%, contra 10% no Sudeste). Refletindo sobre estas variáveis, em conjunto, pode-se inferir por uma precocidade ligei- ramente maior dos jovens do Nordeste quanto à responsabilidade que assumem perante o grupo familiar que integram. R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 342 Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Mundos distintos e realidades semelhantes Nesse sentido, as heterogeneidades entre as regiões são reforçadas com os dados sobre fecundidade das jovens de 15 a 19 anos, apresentados no estudo de Cavenaghi (2013), com taxa de 81,9 nascimentos por mil mulheres nesta faixa etária no Nordeste e 54,5 nascimentos no Sudeste, em 2010. No Brasil, essa taxa estava em 69,9 nascimentos por mil mulheres neste grupo de idade. Em perspectiva, entre 2000 e 2010, a taxa de fe- cundidade decresceu em 28,4% no Sudeste, de forma mais acentuada do que no Nordeste, onde a queda foi de 23,7%.10 Com relação à última variável considerada no estudo, apresentada na Tabela 1, o local de moradia de 93% dos jovens no Sudeste era o meio urbano, diferentemente do Nordeste, onde este percentual chega a 73%.11 A situação de domicílio dos jovens pode influenciar a empregabilidade de diversas formas. Por exemplo, autores como Silva e Kassouf (2002) e Mendonça et al. (2012) acreditam haver maior precocidade no acesso ao mercado de trabalho nas áreas rurais. A endogenia do ingresso na atividade rural familiar, como descrito por Brumer (2007), pode ser uma das explicações acerca deste processo. A Tabela 2 contém as estatísticas descritivas das variáveis contínuas consideradas neste estudo. O baixo valor médio para a experiência de trabalho, em torno de um ano, reflete a faixa etária dos investigados. Entretanto, a diferença de tempo de experiência notada entre os jovens das regiões consideradas (um ano no Nordeste e dez meses no Sudeste) pode refletir a situação de domicílio dos mesmos, visto que os maiores valores encontra- dos para a variável experiência concentram-se entre os jovens que residiam na zona rural. 10 Deve-se salientar que, apesar da menor taxa de fecundidade do Sudeste em relação ao Nordeste, esta ainda se encontra em níveis muito superiores aos encontrados em países mais desenvolvidos, considerando-se a faixa etária de 15 a 19 anos: 34 nascimentos por mil mulheres nos Estados Unidos, 7 por mil na França e 2,3 por mil na República da Coreia (CAVENAGHI, 2013). 11 Considerando-se o total da população, em 2010, residiam em meio urbano, no Sudeste, 92,95% dos habitantes, enquanto este número reduzia-se para 73,13% no Nordeste (IBGE, 2014). R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 12 De acordo com o estabelecido pelo Decreto da Presidência da República nº 7.655, de 23 de dezembro de 2011, o valor do salário mínimo brasileiro, em 2012, era de R$ 622,00 (BRASIL, 2011). R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 13 Esta medida avalia a tendência de um estimador usual (consistente), calculado sob hipótese de serem independentes e identicamente distribuídas (IID), em subestimar ou superestimar a variância verdadeira do estimador pontual (LEITE; SILVA, 2002). Para mais detalhes, ver Skinner, Holt e Smith (1989). 12 De acordo com o estabelecido pelo Decreto da Presidência da República nº 7.655, de 23 de dezembro de 2011, o valor do salário mínimo brasileiro, em 2012, era de R$ 622,00 (BRASIL, 2011). 13 Esta medida avalia a tendência de um estimador usual (consistente), calculado sob hipótese de serem independentes e identicamente distribuídas (IID), em subestimar ou superestimar a variância verdadeira do estimador pontual (LEITE; SILVA 2002) Para mais detalhes ver Skinner Holt e Smith (1989) Análise descritiva dos dados TABELA 2 Médias e erros-padrão da duração da participação no mercado de trabalho (experiência), número de componentes na família, rendimento total familiar per capita e anos completos de escolaridade dos jovens de 15 a 24 anos Regiões Nordeste e Sudeste do Brasil – 2012 Variáveis Nordeste Sudeste Média Erro-padrão Média Erro-padrão Anos de experiência 1,007 2,1668 0,8146 1,5806 Número de componentes da família 4,4149 1,8505 4,1307 1,5954 Renda total per capita (R$) 449,30 629,39 796,57 909,91 Escolaridade (em anos) 9,4737 3,1149 10,4212 2,7213 Fonte: IBGE. Pesquisa Nacional por Amostra de Domicílios – PNAD 2012. TABELA 2 i i ã O número médio de componentes das famílias consideradas não possui grande va- riação entre o Nordeste e o Sudeste, girando em torno de quatro pessoas. Entretanto, a análise do rendimento médio total per capita (renda total familiar dividida pelo número de componentes da família) evidenciou o abismo existe entre as regiões. Enquanto no R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 343 Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Mundos distintos e realidades semelhantes Sudeste este valor chegava a quase R$ 800,00, em média, sendo superior ao salário mí- nimo vigente em 2012,12 a renda familiar per capita dos jovens nordestinos não alcançava R$ 450,00, em média. Atendo-se à análise do nível educacional, cabe salientar que, para o Brasil, os dados demonstram que a juventude atual tem feito maior investimento em educação formal, o que seria reflexo da demanda do mercado de trabalho por níveis de educação formal mais elevados. Em que pesem estes avanços, há desigualdades expressivas no acesso à edu- cação (por faixa de renda, raça/cor, residência no rural/urbano e por região do país), além de problemas relativos à qualidade do sistema educacional. Um espelho desta realidade reflete as desigualdades regionais: mormente no Nordeste, mais do que em outras regiões do país, persiste uma elevada distorção idade-série, o que compromete o acesso ao ensino médio na idade adequada (CASTRO; AQUINO, 2008). Análise das razões de risco relativo na participação no mercado de trabalho Como evidenciado anteriormente, ao contrário de modelos como o de Mínimos Qua- drados Ordinários, os coeficientes estimados pelo logit multinomial não representam diretamente os efeitos marginais na variável dependente, devido às variações nas co- variáveis. Por este motivo, a exposição de tais coeficientes não possui muita utilidade e não foi feita neste trabalho. Assim, a apresentação e análise dos resultados provenientes do modelo logit multinomial foram realizadas à luz da razão de risco relativo (RRR) e dos efeitos marginais obtidos para cada uma das variáveis utilizadas.i Pela observação da Tabela 3, depreende-se que a maioria dos coeficientes RRR es- timados exibiu significância estatística. É importante salientar que, conjuntamente, os coeficientes foram estatisticamente diferentes de zero, já que o valor apresentado pela estatística F, que mensura a significância global do modelo, correspondeu a 66,44, para o modelo referente ao Nordeste, e 70,50, para o Sudeste. As estimativas do efeito do plano amostral indicam que sua correta consideração é essencial para obtenção de estimativas robustas e não viciadas. Nos modelos, praticamen- te todas as estimativas MEFF (Misspecification Effect)13 apresentaram-se superiores a 1, indicando que, caso a amostra complexa fosse considerada aleatória simples, as variân- cias das estimativas dos coeficientes seriam subestimadas. Em síntese, a estimação das razões de risco relativo permite verificar, dadas as mudanças das covariáveis, as chances que os jovens incluídos no estudo têm de pertencer à categoria inativa (categoria base) ou às demais categorias: ativo e empregado; e ativo e desempregado. R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 344 Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Mundos distintos e realidades semelhantes TABELA 3 Estimativas da Razão de Risco Relativo (RRR) e efeito do plano amostral (MEFF) para situação de participação no mercado de trabalho (1) para os jovens de 15 a 24 anos Regiões Nordeste e Sudeste do Brasil – 2012 Participação no mercado de trabalho e covariáveis Nordeste Sudeste RRR Erro- padrão MEFF RRR Erro- padrão MEFF Ativo e empregado Sexo (homens) 2,5245* 0,1268 1,3858 1,7409* 0,0777 0,9904 Raça/cor (brancos) 0,8939 0,0415 1,0606 0,7213* 0,0328 1,0666 Filho 0,1909* 0,0138 0,9681 0,1568* 0,0161 1,2863 Cônjuge 0,3150* 0,0268 0,9315 0,2486* 0,0320 1,3958 Outro 0,2386* 0,0225 1,2065 0,2435* 0,0304 1,3877 Situação de domicílio (urbano) 1,0794 ns 0,0635 1,2592 0,8858 ns 0,0766 0,9653 Experiência 2,1522* 0,4467 5,2892 15,1133* 6,4745 3,6066 Escolaridade 1,0777* 0,0106 1,6809 1,1582* 0,0133 1,4709 Esco. x Expe. R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 Análise das razões de risco relativo na participação no mercado de trabalho 1,0645* 0,0254 5,9371 0,9451 ns 0,0338 3,1142 Com. família 1,0562* 0,0136 1,1497 1,1138* 0,0166 1,0582 Renda 1,4558* 0,0401 1,8586 1,4963* 0,0461 2,0501 Ativo e desempregado Sexo (homens) 1,6002* 0,1092 1,4232 1,1566 0,0763 1,1152 Raça/cor (brancos) 0,7800* 0,0578 1,4056 0,7649* 0,0506 1,1610 Filho 0,6962* 0,0758 1,1856 0,5541* 0,0699 1,0867 Cônjuge 0,7343 0,0980 1,2772 0,5147* 0,0860 1,2421 Outro 0,7145* 0,0915 1,2081 0,4798* 0,0722 1,0826 Situação de domicílio (urbano) 2,3374* 0,2462 1,5037 1,6994* 0,2662 1,1277 Experiência 1,4274 ns 0,3475 2,7559 6,8595* 3,3795 2,9366 Escolaridade 1,1505* 0,0156 1,6843 1,1429* 0,0179 1,4360 Esco. x Expe. 1,0078 ns 0,0279 3,4226 0,9173 0,0397 2,7831 Com. família 0,9446* 0,0196 1,6402 1,0321 ns 0,0231 1,3833 Renda 0,8580* 0,0166 1,1303 0,9026* 0,0181 1,0848 Fonte: IBGE. Pesquisa Nacional por Amostra de Domicílios – PNAD 2012. Resultados da pesquisa. (1) Categoria base: Inativo; * Significativo a 10%; Significativo a 5%; * Significativo a 1%; e ns não significativo. TABELA 3 TABELA 3 Estimativas da Razão de Risco Relativo (RRR) e efeito do plano amostral (MEFF) para situação de participação no mercado de trabalho (1) para os jovens de 15 a 24 anos Regiões Nordeste e Sudeste do Brasil – 2012 Desse modo, na Tabela 3, nota-se que, quanto ao sexo dos jovens, no Nordeste a chance de os homens estarem na categoria ativo e empregado é duas vezes e meia supe- rior à das mulheres, enquanto no Sudeste esta chance é de 74%. Ainda, os jovens do sexo masculino têm suas chances de pertencer à categoria ativo e desempregado majoradas em 60% e 16%, no Nordeste e Sudeste, respectivamente, quando comparados às mulheres da categoria de inativos. Portanto, embora em ambas as regiões as mulheres jovens tenham menores chances de pertencerem às categorias ativas, estando empregadas ou procurando emprego, percebe-se maior diferenciação entre os sexos no Nordeste. Já com relação à raça/cor dos jovens, observa-se que, no Nordeste, aqueles que se declararam brancos possuem menos 11% e 22% de chances de pertencerem, respectiva- mente, às categorias ativo e empregado e ativo e desempregado, comparativamente aos R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 345 Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Mundos distintos e realidades semelhantes não brancos. Já no Sudeste, estes números são mais contundentes, principalmente na chance de pertencer à categoria ativo e empregado (menos 28%). Análise das razões de risco relativo na participação no mercado de trabalho Tais resultados indicam que pode haver estímulo aos jovens brancos em permanecerem inativos e não buscarem emprego, possivelmente somente estudando, sobretudo no Sudeste. Observando-se as variáveis binárias referentes à posição do jovem na família, em que a pessoa de referência é a categoria base, verifica-se a importância desta14 na família, mesmo que ainda com menos idade, pois assumir a posição de filho, cônjuge e outro reduz as chances de estar ativo e empregado, em comparação com a categoria base, tanto no Nordeste quanto no Sudeste. Em menor magnitude, há redução das chances de pertencer à categoria ativo e desempregado para os jovens que são filhos, cônjuges e outros, em ambas as regiões. Com relação à situação de domicílio, notou-se, tanto no Nordeste quanto no Sudeste, que o fato de os jovens morarem no meio urbano não foi significativo para explicar as chances de estarem na categoria ativo e empregado, quando comparados àqueles que residem na área rural. Porém, chama atenção o aumento em mais de duas vezes nas chances de os jovens pertencerem à categoria ativo e desempregado, caso morem no meio urbano, comparativamente a residir no meio rural, no Nordeste. Tal fator também aumenta a possibilidade de os jovens se enquadrarem nesta categoria em quase 70% no Sudeste. A análise destes efeitos demonstra que, especialmente no Nordeste, residir no meio urbano não traz vantagens relativas à obtenção de emprego, apesar de nada se poder afirmar quanto à maior facilidade para obtenção de emprego no campo e, muito menos, sobre a qualidade desta ocupação. Corroborando o que preconizam Silva e Kassouf (2002) com relação às variáveis ex- periência e escolaridade, que representam o estoque de capital humano contido em um indivíduo, estando o seu aumento associado à ampliação da produtividade e consequente elevação da probabilidade de obtenção de emprego, notou-se que incrementos nas variáveis experiência e escolaridade agiram no sentido esperado. Um ano a mais de experiência, por exemplo, aumenta, no Sudeste, em mais de 15 vezes a probabilidade de os jovens pertencerem à categoria ativo e empregado e eleva em 6 vezes a possibilidade de serem ativos e desempregados, demonstrando grande importância desta variável, mormente entre aqueles jovens que já trabalharam. Tal variável, para o Nordeste, aumenta em 2 vezes a chance de os jovens estarem empregados. Pessoa de referência é aquela responsável pela família, ou assim considerada pelos demais membros (IBGE, 2013b). Análise das razões de risco relativo na participação no mercado de trabalho Ademais, um ano adicional de estudo amplia em 8% as chances de se enquadrar na categoria ativo e empregado e em 15% na de ativo e desempregado, no Nordeste. Já no Sudeste, este ano a mais de estudo representa acréscimo de 16% nas chances de empregabilidade e de 14% na possibilidade de estar ativo e desempregado. Tais decorrências ressaltam a relevância das variáveis relacionadas ao conhecimento, notadamente no concorrido mercado de trabalho do Sudeste do país.i O número de componentes da família foi significativo apenas para explicar as chances de o jovem estar na categoria ativo e empregado, em ambas as regiões, com aumento de R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 346 Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Mundos distintos e realidades semelhantes 5,62% no Nordeste e 11,38% no Sudeste. No Nordeste esta variável estava significativamente relacionada às probabilidades de desemprego, reduzindo as chances em 5,54%, o que evi- dencia uma possível tendência, em famílias maiores, de obtenção de trabalho entre os jovens. Ainda quanto à análise da razão de risco relativo, percebe-se a importância de uma maior renda familiar nas chances de emprego dos jovens pesquisados, majorando, em am- bas as regiões, em mais de 45% as possibilidades de se enquadrarem na categoria ativo e empregado e, adicionalmente, reduzindo as chances de desemprego. Assim, depreende-se o que pode caracterizar-se como um círculo vicioso no que tange à variável renda, uma vez que maiores rendimentos familiares estariam relacionados a maiores chances de obtenção de emprego, nas duas regiões analisadas. 15 Entre os jovens de 20 a 24 anos, as mulheres possuíam, em média, 9,4 anos de estudo, contra 8,8 anos dos homens (COSTANZI, 2009). R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 Análise dos efeitos marginais Adicionalmente à análise das razões de risco relativo (RRR), foram obtidos, para as Regiões Nordeste e Sudeste, os efeitos marginais de cada variável, em seus respectivos pontos médios, para as três equações, em cada região considerada: inativo, ativo e em- pregado e ativo e desempregado. Conforme pode ser notado pela análise da Tabela 4, a maioria dos coeficientes exibiu significância estatística. Os efeitos evidenciaram que, para o Nordeste, caso o jovem seja do sexo masculino, tudo mais considerado constante, isto atua no sentido de diminuir a probabilidade de pertencer à categoria inativo em 18,4 pontos percentuais (p.p.), aumentando a probabili- dade de estar ativo e empregado em 19,45 p.p. Comportamento semelhante desta variável foi notado para o Sudeste, apesar da menor magnitude do efeito marginal. Nesta região, ser homem reduzia a inatividade em 8,1 p.p., ao passo em que elevava as chances de empregabilidade em 10,62 p.p. Estas implicações demonstram desvantagem relativa ao sexo feminino no preenchimento das vagas existentes no mercado de trabalho, sobretudo no Nordeste, corroborando o estudo de Silva e Kassouf (2002), que evidenciaram maior acesso ao mercado de trabalho por parte dos jovens do sexo masculino. Esta realidade fica mais visível em análise realizada pela Organização Internacional do Trabalho (OIT, 2007b), constatando que, na América Latina, 72% dos 22 milhões de jovens que não estudavam nem trabalhavam eram mulheres. Essa dupla “inatividade” pode estar relacionada à ma- ternidade precoce, que ainda é um fenômeno bastante frequente entre as jovens de baixa renda, e aos estereótipos de sexo, que atribuem às mulheres a responsabilidade principal (quando não exclusiva) pelas tarefas domésticas (COSTANZI, 2009). Entretanto, deve-se salientar que, a despeito do resultado encontrado em OIT (2007a), as mulheres brasileiras possuem, em geral, número médio de anos de estudo maior do que os homens, podendo também ser uma razão para a maior inatividade feminina, que permaneceria por mais tempo apenas estudando.15 R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 347 Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Análise dos efeitos marginais Mundos distintos e realidades semelhantes TABELA 4 Efeitos marginais para as equações de inatividade, emprego e desemprego dos jovens de 15 a 24 anos Regiões Nordeste e Sudeste do Brasil – 2012 Variáveis Inativo Ativo e empregado Ativo e desempregado Efeito marginal Erro- padrão Efeito marginal Erro- padrão Efeito marginal Erro- padrão Nordeste Sexo -0,1838* 0,0117 0,1945* 0,0119 -0,0107* 0,0052 Raça/cor 0,0277* 0,0099 -0,0147 ns 0,0105 -0,0130 0,0050 Filho 0,2907* 0,0157 -0,3447* 0,0152 0,0540* 0,0080 Cônjuge 0,2386* 0,0192 -0,2670* 0,0191 0,0285 0,0128 Outro 0,2918* 0,0210 -0,3289* 0,0198 0,0370* 0,0126 Situação de domicílio -0,0341* 0,0125 -0,0195 ns 0,0140 0,0536* 0,0064 Experiência -0,1529* 0,0429 0,1646* 0,0423 -0,0117 ns 0,0148 Escolaridade -0,0177* 0,0021 0,0107* 0,0023 0,0070* 0,0011 Esco. x Expe. -0,0119 0,0049 0,0145* 0,0047 -0,0026* 0,0015 Com. família -0,0088* 0,0027 0,0160* 0,0029 -0,0072* 0,0016 Renda -0,0664* 0,0060 0,0974* 0,0067 -0,0310* 0,0025 Sudeste Sexo -0,0811* 0,0076 0,1062* 0,0098 -0,0252* 0,0060 Raça/cor 0,0514* 0,0074 -0,0501* 0,0091 -0,0013 ns 0,0056 Filho 0,2253* 0,0143 -0,2946* 0,0156 0,0693* 0,0083 Cônjuge 0,2610* 0,0290 -0,2851* 0,0282 0,0241 ns 0,0156 Outro 0,2680* 0,0269 -0,2863* 0,0287 0,0182 ns 0,0143 Situação de domicílio 0,0091 ns 0,0130 -0,0541* 0,0163 0,0449* 0,0091 Experiência -0,4211* 0,0639 0,4374* 0,0660 -0,0163 ns 0,0307 Escolaridade -0,0234* 0,0021 0,0216* 0,0026 0,0017 ns 0,0014 Esco. x Expe. 0,0097* 0,0056 -0,0059 ns 0,0059 -0,0038 ns 0,0028 Com. família -0,0158* 0,0024 0,0204* 0,0031 -0,0046 0,0020 Renda -0,0548* 0,0053 0,0902* 0,0065 -0,0369* 0,0029 Fonte: IBGE. Pesquisa Nacional por Amostra de Domicílios – PNAD 2012. Resultados da pesquisa. Nota: * Significativo a 10%; Significativo a 5%; * Significativo a 1%; e ns não significativo. TABELA 4 TABELA 4 tos marginais para as equações de inatividade, emprego e desemprego dos jovens de 15 a 24 anos Regiões Nordeste e Sudeste do Brasil – 2012 Fonte: IBGE. Pesquisa Nacional por Amostra de Domicílios – PNAD 2012. Resultados da pesquisa. Nota: * Significativo a 10%; Significativo a 5%; * Significativo a 1%; e ns não significativo. A raça/cor dos jovens influencia da seguinte forma suas chances concernentes ao mer- cado de trabalho: no Nordeste, região com grande concentração de não brancos (pretos, pardos, amarelos ou indígenas), cœteris paribus, o jovem tem sua chance de ser inativo diminuída em 2,77 p.p., ao passo que aumentam suas chances de estar ativo e empregado (1,47 p.p.) e ativo e desempregado (1,3 p.p.). Análise dos efeitos marginais Estes efeitos são um pouco mais acentuados para o Sudeste, onde é quase idêntico o número de brancos e não brancos: relativamente aos brancos, os jovens não brancos têm diminuição de 5,14 p.p. na perspectiva de inati- vidade e acréscimo de 5,01 p.p. na possibilidade de obter emprego. Com relação às variáveis binárias referentes à posição dos jovens na família, em relação à categoria tomada como base (chefe), os resultados sugeriram que, para o Nordeste, a probabilidade de pertencer à categoria inativo tornou-se maior para o jovem no papel de filho (29,07 p.p.), cônjuge (23,86 p.p.) ou outro (29,18 p.p.), também atuando no sentido de aumentar as chances de estarem ativos e desempregados. Comportamento similar foi R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 348 Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Mundos distintos e realidades semelhantes observado para a Região Sudeste, onde ser filho, cônjuge ou outro ampliaram a chance de inatividade em 22,53 p.p., 26,10 p.p. e 26,80 p.p., respectivamente, reduzindo a probabi- lidade de obterem emprego. Nesse sentido, os chefes das famílias parecem beneficiar-se, perante os demais membros da família, tendo em vista a maior empregabilidade. Todavia, tal circunstância não se evidencia, necessariamente, vantajosa, pois uma das explicações para tal resultado indica que jovens de famílias ou domicílios com baixa renda per capita, que ocupam a posição de pessoas de referência, tendem a ter maior necessidade de in- gressar precocemente no mercado de trabalho para contribuir com a renda familiar, tendo, portanto, maiores dificuldades para continuar os estudos. Ademais, quando na chefia da família figuram os homens, a cônjuge do sexo feminino contribui assumindo a responsa- bilidade pelo trabalho reprodutivo e de cuidado na esfera doméstica, o que reduz suas chances de emprego (COSTANZI, 2009). Considerando o local onde o jovem reside (meio urbano ou meio rural), em ambas as regiões, morar na zona urbana diminui as chances de os jovens estarem ativos e desem- pregados, aumentando, no Sudeste (que possui alta taxa de urbanização), em 5,41 p.p. a possibilidade de estarem ativos e empregados. Os efeitos marginais ainda demonstraram que os jovens do Nordeste que habitam o meio rural têm reduzida em 3,41 p.p. a proba- bilidade de serem inativos. Tal resultado corrobora aquele obtido por Mendonça et al. (2012), na análise de mulheres jovens do Nordeste brasileiro. 16 Adicionalmente, segundo a estrutura ocupacional evidenciada pelo relatório da OIT (COSTANZI, 2009), 18,5% dos jovens de 15 a 24 anos ocupados estavam no setor agrícola. Em termos de sexo, enquanto 23,4% dos jovens trabalhavam no campo, este número decrescia para 11,2%, no caso das mulheres. R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 Análise dos efeitos marginais Deve-se salientar que, no meio rural, dadas as condições de recrutamento para o trabalho, geralmente, os jovens tornam-se ativos mais cedo.16 A experiência, importante variável determinante para a participação no mercado de trabalho, teve efeitos marginais, indicando que seu incremento eleva a chance de os jovens estarem na categoria ativo e empregado, notadamente no concorrido mercado da Região Sudeste, onde um ano a mais de experiência do jovem foi capaz de majorar a chance de estar ativo e empregado em 43,74 pontos percentuais, ao passo que reduz as chances de inatividade em 42,11 p.p., resultados que, segundo também comprovam Mendonça et al. (2012), confirmam a importância desta variável como determinante para o acesso ao mercado de trabalho. No Nordeste, apesar da menor magnitude dos efeitos, a variável experiência também reduz a inatividade (em 15,29 p.p.), elevando a empregabilidade (16,46 p.p.). De acordo com Silva e Kassouf (2002), dado que é natural que jovens possuam pouca experiência profissional, aqueles que tenham alguma vivência no mercado estão em vantagem em relação aos demais. Porém, em consonância com relatório da OIT (COSTANZI, 2009), o fato de tal resultado indicar o aumento da demanda por experiência profissional no mercado de trabalho acabaria, em última instância, por prejudicar os mais jovens. Todavia, a maior oportunidade aos adultos (mais experientes) do que aos jovens poderia ser contraditória com outra característica que parece estar presente no período, que é o R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 349 Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Mundos distintos e realidades semelhantes incremento da demanda por um nível mais elevado de educação formal, favorecendo os jovens, que possuem, como já demonstrado, maior escolaridade do que os adultos. Mas é imprescindível ponderar que um maior nível de escolaridade não será necessariamente mais valorizado no mercado de trabalho se a qualidade e a pertinência da educação estive- rem em questão. Por outro lado, em geral, a maior escolaridade dos jovens é diretamente proporcional às suas exigências para a participação no mercado de trabalho. Em outras palavras, os jovens que concluem o ensino superior buscam ocupações que lhes garantam melhores condições de trabalho e renda. A escolaridade, outra variável relevante para determinar o acesso ao mercado de traba- lho, atuou de forma semelhante à experiência nas três categorias analisadas. No Nordeste, um ano a mais de escolaridade agiu no sentido de diminuir em 1,77 p.p. 17 Enquanto 8,6% e 33% dos jovens na zona urbana tinham, respectivamente, 0 a 4, e 5 a 8 anos de estudo, tais percentuais se elevavam para 28,7% e 43,7% para a juventude no campo. 18 No Brasil, apenas 7,2% dos jovens brancos tinham de 0 a 4 anos de estudo e 29,5% possuíam de 5 a 8 anos. No caso dos jovens negros, estas cifras aumentavam, respectivamente, para 16,2% e 39,7% (COSTANZI, 2009). Análise dos efeitos marginais a probabilidade de o jovem estar inativo, elevando em 1,07 p.p. a chance de que seja ativo e empregado e em 0,7 p.p. a possibilidade de ser ativo e desempregado. No Sudeste, os efeitos marginais para a escolaridade não são tão elevados quanto aqueles apresentados pela variável experiência. Um ano a mais de escolaridade minimizou em 2,34 p.p. a chance de inatividade e elevou em 2,16 p.p. a empregabilidade, indicando efeitos novamente superiores aos do Nordeste. Entretanto, deve-se ponderar que, entre os jovens inativos, que por vezes assim se encontram justamente por estarem em período escolar, os anos de estudos adicionais lhes proporcionarão, quando de fato forem procurar emprego, maiores chances de obtê-lo, como já demonstrado nos resultados. Em termos das características inerentes à variável educacional, há numerosas evi- dências da existência de desigualdades que a permeiam, entre elas, aquelas tipicamente regionais. As Regiões Sul, Sudeste e Centro-Oeste apresentam os melhores índices. En- quanto, no Nordeste, a média de anos de estudo correspondia a 6,4 para os jovens de 15 a 17 anos, 7,5 para os de 18 e 19 anos e 7,8 para aqueles de 20 a 24 anos, no Sudeste essas mesmas cifras elevavam-se para, respectivamente, 7,7, 9,4 e 9,9 anos (COSTANZI, 2009). Outrossim, no Brasil, há uma desigualdade educacional considerável entre os jovens que estão na área rural e aqueles da zona urbana,17 além de expressiva inferioridade, em termos de anos de estudo, da população não branca,18 fato que se busca, mais recentemente, reverter por meio de políticas sociais de afirmação das populações mais vulneráveis. Porém, quando se deseja inferir acerca das razões mais apropriadas para esclarecer a baixa magnitude da influência da educação na empregabilidade, talvez deva-se considerar aquelas pertinentes à qualidade e ao (des)incentivo ao estudo. Afinal, a frequência ao ensino médio na idade adequada abrangia menos da metade dos jovens brasileiros de 15 a 17 anos, tendo em vista que cerca de um terço ainda encontra-se no ensino fundamental, quando deveria estar no ensino médio, e cerca de 18% estão fora da escola (CASTRO; AQUINO, 2008). R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 350 Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Análise dos efeitos marginais Mundos distintos e realidades semelhantes Nota-se, com relação às variáveis experiência e escolaridade, para o ano em análise, e considerando os jovens brasileiros na faixa etária de 15 a 24 anos, que o mercado de trabalho valoriza mais a experiência dos jovens do que a escolaridade. Pode-se também compreender este resultado como uma tendência de redução na taxa de participação da juventude, que está relacionada, basicamente, ao declínio da participação, no mercado de trabalho, dos mais jovens (15 a 19 anos), o que é positivo e, possivelmente, está re- lacionado ao aumento da escolaridade e à redução do trabalho infantil. Por outro lado, esta participação aumenta entre os 20 e os 24 anos (COSTANZI, 2009). Entretanto, alguns estudos, como o de Silva (2001), destacam, ainda, atitudes preconceituosas, como a opção, por parte dos empresários, por trabalhadores adultos, que somam experiência e hábitos de trabalho mais sedimentados, o que seria mais um obstáculo aos jovens, principalmente para a obtenção do primeiro emprego. O aumento no número de componentes da família levou ao acréscimo na probabili- dade de emprego (1,6 p.p., no Nordeste, e 2,04 p.p., no Sudeste) e redução nas chances de inatividade e desemprego, manifestando o conceito de que os jovens, muitas vezes, precisam trabalhar para auxiliar no sustento de seus lares, sobretudo aqueles que abrigam famílias mais numerosas, conforme argumentam Lima (2008) e Mendonça et al. (2012). Em ambas as regiões analisadas, os efeitos marginais da variável renda atuaram no sentido de reduzir as probabilidades de inatividade e desemprego, elevando a possibi- lidade de empregabilidade dos jovens avaliados, tudo mais considerado constante, em magnitudes similares, apesar do diferencial de renda observado entre o Nordeste e o Sudeste. Tais resultados são contrários à crença mais comum de que famílias com uma renda per capita maior podem proporcionar melhores condições para que seus filhos se preparem para o mercado de trabalho, possibilitando que se dediquem aos estudos, ele- vando, num primeiro momento, a inatividade. Porém, Scorzafave e Menezes-Filho (2001) demonstraram que, em famílias mais abastadas, há possibilidade de contratação de babás e pagamento de creches, liberando jovens mulheres para o trabalho. Além disso, a rede de contatos proporcionada aos jovens de famílias mais ricas poderia facilitar sua participação no mercado de trabalho. Análise dos efeitos marginais É válido salientar que, apesar de constituírem análises distintas, as razões de risco relativo e os efeitos marginais indicaram uma direção semelhante, nas Regiões Nordeste e Sudeste, com relação ao comportamento das variáveis consideradas determinantes para a situação ocupacional dos jovens brasileiros, no período examinado. R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 Considerações finais Abordou-se, neste trabalho, como certas características socioeconômicas e demográfi- cas estão associadas à empregabilidade de jovens de 15 a 24 anos, nas Regiões Nordeste e Sudeste do Brasil. Por meio das apreciações feitas, considerando os resultados tanto das razões de risco relativo quanto dos efeitos marginais, constatou-se que, apesar da grande R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 351 Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Mundos distintos e realidades semelhantes heterogeneidade que abrange o Nordeste e o Sudeste, evidenciada pela análise descritiva das variáveis abordadas, ambas as regiões apresentaram comportamentos semelhantes com relação ao sentido das implicações destes fatores associados à inatividade, à empre- gabilidade e ao desemprego dos jovens. O incremento do estoque de capital humano, com consequências favoráveis à produ- tividade dos trabalhadores de ambas as regiões, configura-se como fator crucial para a empregabilidade dos jovens. Com efeito, ressaltaram-se a escolaridade e a experiência profissional como fatores que influenciam positivamente a participação no mercado de trabalho, com impactos de maior magnitude no Sudeste. No sentido oposto, com influência negativa na probabilidade de estar empregado, ser do sexo feminino exibiu maior proeminência no Nordeste. Desse modo, nota-se a impor- tância do capital humano na região economicamente mais dinâmica do país, cujo mercado de trabalho prioriza a formação dos jovens como requisito para sua contratação, ao passo em que se percebe, no Nordeste, um maior alijamento das mulheres das oportunidades de emprego, refletindo, muito provavelmente, a ausência mais acentuada nesta região de mecanismos efetivos para mitigação de desigualdades. Com base neste diagnóstico, manifesta-se a necessidade de investimentos em iniciativas que priorizem a educação de qualidade, desde o ensino fundamental, possibilitando aos jovens de todas as raças/cores maior acesso ao ensino superior. A chance de adquirirem experiência profissional, por meio de ações que incentivem os empregadores a oferecerem a oportunidade do primeiro emprego aos jovens, condizentes com o objetivo do Programa Nacional de Estímulo ao Primeiro Emprego (PNPE), instituído pela Lei n. 10.748, de 22 de outubro de 2003, também mostra-se uma alternativa valiosa na busca pela redução do de- semprego juvenil. Ainda nesse contexto, a educação profissional técnica tem papel relevante, oferecendo conhecimento prático, que vem sendo valorizado pelo mercado de trabalho. Considerações finais Adicionalmente, campanhas educativas acerca das consequências da gravidez precoce, inserção da educação sexual integral na grade curricular das escolas, bem como políticas de planejamento reprodutivo que permitam aos jovens o acesso adequado e de qualidade aos métodos contraceptivos são extremamente importantes. Sobre esse ponto, destaca-se que, segundo Cavenaghi (2013), a porcentagem de jovens sexualmente ativas que utilizavam algum método anticonceptivo era alta tanto no Nordeste (80,4%) quanto no Sudeste (74,1%), em 2006, em comparação aos níveis de uso em outros países. No entanto, a eficácia do uso destes métodos não aparentava ser adequada, visto que 65,7% dos casos de gravidez dessas jovens no Nordeste e 64,9% no Sudeste foram declarados como não planejados para aquele momento ou não desejados. Adicionalmente, a provisão de uma ampla rede de creches e pré-escolas pode minimizar as atribulações ocasionadas pela presença de filhos, na participação de jovens, prioritariamente mulheres, no mercado de trabalho. Fundamentando-se nestes intuitos, faz-se imprescindível apoiar e desenvolver ações como a Política Nacional da Juventude, instituída por meio da Medida Provisória n. 238, de 1º de fevereiro de 2005, e o Programa Nacional de Inclusão de Jovens (ProJovem), tendo R. bras. Est. 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Different worlds and similar realities: youth employability in the Brazilian Northeast and Southeast Different worlds and similar realities: youth employability in the Brazilian Northeast and Southeast The focus of this paper is how certain factors influence the probability of participation into the labor market for young people, ages 15 to 24, in the Brazilian Northeast and Southeast, regions markedly different from each other, in several dimensions. To this end, it is analyzed the relative risk factors and the marginal effects of a logistic function applied to the microdata from the 2012 National Household Sample Survey (PNAD). Despite the socioeconomic disparities between the two Brazilian regions, they showed similarities in the effect of determining factors on employability. It was shown that education and work experience positively influenced the incorporation of youth into the labor market, mainly in the Brazilian Southeast, where one more year of schooling increases the chance of employment in 16%. Being female has a negative effect on the likelihood of employment, particularly in the Brazilian Northeast, where male youth have double chance of being employed compared to females. Yet, white youth living in the Southeast have 28% less chance of being employed compared to non-whites. These results suggest the paths that public policies could take, specific for each Brazilian region, to mitigate unemployment among young people. Keywords: Employability. Youth. Logistic function. Keywords: Employability. Youth. Logistic function. Sobre os autores Mateus de Carvalho Reis Neves é doutorando em Economia Aplicada na Universidade Federal de Viçosa (UFV), mestre em Economia Aplicada (UFV) e graduado em Gestão de Cooperativas (UFV). Mateus de Carvalho Reis Neves é doutorando em Economia Aplicada na Universidade Federal de Viçosa (UFV), mestre em Economia Aplicada (UFV) e graduado em Gestão de Cooperativas (UFV). Marcos Falcão Gonçalves é doutorando em Economia Aplicada na UFV, mestre em Economia Rural pela Universidade Federal do Ceará (UFC), especialista em Gestão da Inovação e Difusão Tecnológica em Arranjos Produtivos Locais pela Universidade Federal da Bahia (UFBA), especialista em Economia Financeira e Análise de Investimentos e graduado em Economia, ambos pela Universidade Estadual de Feira de Santana (UEFS). Economista do Escritório Técnico de Estudos Econômicos do Banco do Nordeste do Brasil (BNB/ETENE) e professor da Faculdade Cearense. Marcos Falcão Gonçalves é doutorando em Economia Aplicada na UFV, mestre em Economia Rural pela Universidade Federal do Ceará (UFC), especialista em Gestão da Inovação e Difusão Tecnológica em Arranjos Produtivos Locais pela Universidade Federal da Bahia (UFBA), especialista em Economia Financeira e Análise de Investimentos e graduado em Economia, ambos pela Universidade Estadual de Feira de Santana (UEFS). Economista do Escritório Técnico de Estudos Econômicos do Banco do Nordeste do Brasil (BNB/ETENE) e professor da Faculdade Cearense. R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 354 Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Mundos distintos e realidades semelhantes João Eustáquio de Lima é pós-doutor em Métodos Quantitativos, pela University of Florida, Ph.D. em Economia Rural pela Michigan State University e mestre em Economia Rural pela Universidade Federal de Viçosa (UFV). Professor titular do Departamento de Economia Rural da UFV. R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 Endereço para correspondência Mateus de Carvalho Reis Neves Departamento de Economia Rural Avenida Purdue, s/nº, campus universitário – Edifício Edson Potsch Magalhães 36570-900 – Viçosa-MG, Brasil Marcos Falcão Gonçalves Escritório Técnico de Estudos Econômicos do Banco do Nordeste do Brasil Av. Doutor Silas Munguba, 5.700, Bloco A2 térreo, Bairro Passaré 60743-902 – Fortaleza-CE, Brasil João Eustáquio de Lima Departamento de Economia Rural Avenida Purdue, s/nº, campus universitário – Edifício Edson Potsch Magalhães 36570-900 – Viçosa-MG, Brasil Mateus de Carvalho Reis Neves Departamento de Economia Rural Avenida Purdue, s/nº, campus universitário – Edifício Edson Potsch Magalhães 36570-900 – Viçosa-MG, Brasil Marcos Falcão Gonçalves Escritório Técnico de Estudos Econômicos do Banco do Nordeste do Brasil Av. Doutor Silas Munguba, 5.700, Bloco A2 térreo, Bairro Passaré 60743-902 – Fortaleza-CE, Brasil João Eustáquio de Lima Departamento de Economia Rural Avenida Purdue, s/nº, campus universitário – Edifício Edson Potsch Magalhães 36570-900 – Viçosa-MG, Brasil R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 Mundos distintos y realidades semejantes: empleabilidad de los jóvenes en el Nordeste y el Sudeste de Brasil Mundos distintos y realidades semejantes: empleabilidad de los jóvenes en el Nordeste y el Sudeste de Brasil En este artículo se aborda la forma en que ciertos factores influyen en la probabilidad de participación en el mercado de trabajo de los jóvenes de 15 a 24 años en las regiones del R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015 355 Mundos distintos e realidades semelhantes Neves, M.C.R., Gonçalves, M.F. e Lima, J.E. Nordeste y el Sudeste de Brasil, heterogéneas entre sí en varias dimensiones. Con este fin, se analizaron las razones de riesgo relativo y los efectos marginales resultantes de una función logística aplicada a los microdatos de la Pesquisa Nacional por Amostra de Domicílios (PNAD) de 2012. Se constató que, a pesar de las disparidades socioeconómicas de estas regiones del país, en ambas se registraron comportamientos similares respecto del efecto de los determinantes sobre la empleabilidad de los jóvenes. Se evidenció que la escolaridad y la experiencia laboral influyen positivamente en la participación en el mercado de trabajo, sobre todo en el Sudeste, donde un año de estudio o más eleva las posibilidades de estar empleado en un 16%. Ser mujer tiene una relación negativa con la probabilidad de trabajar, especialmente en el Nordeste, considerando que los varones de este grupo etario tienen el doble de probabilidad de estar empleados. Además, los jóvenes blancos del Sudeste poseen un 28% menos de probabilidad de estar trabajando que los que no lo son. Estos resultados sugieren el trayecto que pueden recorrer las políticas públicas específicas para cada región que apunten a reducir el desempleo entre los jóvenes. Palabras clave: Empleabilidad. Jóvenes. Función logística. Recebido para publicação em 17/01/2015 Recomendado para publicação 13/07/2015 Aceito para publicação em 02/08/2015 356 R. bras. Est. Pop., Rio de Janeiro, v. 32, n.2, p. 335-356, maio/ago. 2015
https://openalex.org/W2953334586	https://centaur.reading.ac.uk/85740/1/jmse-07-00069.pdf	English	null	Tool Use by Four Species of Indo-Pacific Sea Urchins	Journal of marine science and engineering	2,019	cc-by	9,178	It is advisable to refer to the publisher’s version if you intend to cite from the work. See Guidance on citing . Published version at: http://dx.doi.org/10.3390/jmse7030069 To link to this article DOI: http://dx.doi.org/10.3390/jmse7030069 It is advisable to refer to the publisher’s version if you intend to cite from the work. See Guidance on citing . Published version at: http://dx.doi.org/10.3390/jmse7030069 To link to this article DOI: http://dx.doi.org/10.3390/jmse7030069 Publisher: MDPI All outputs in CentAUR are protected by Intellectual Property Rights law, including copyright law. Copyright and IPR is retained by the creators or other copyright holders. Terms and conditions for use of this material are defined in the End User Agreement . www.reading.ac.uk/centaur Article Published Version Creative Commons: Attribution 4.0 (CC-BY) Creative Commons: Attribution 4.0 (CC-BY) Barrett, G. A. ORCID: https://orcid.org/0000-0003-1509-0179, Revell, D., Harding, L., Mills, I., Jorcin, A. and Stiefel, K. (2019) Tool Use by Four Species of Indo-Pacific Sea Urchins. Journal of Marine Science and Engineering, 7 (3). 69. ISSN 2077-1312 doi: https://doi.org/10.3390/jmse7030069 Available at https://centaur.reading.ac.uk/85740/ It is advisable to refer to the publisher’s version if you intend to cite from the work. See Guidance on citing . Published version at: http://dx.doi.org/10.3390/jmse7030069 To link to this article DOI: http://dx.doi.org/10.3390/jmse7030069 CentAUR Central Archive at the University of Reading Received: 5 February 2019; Accepted: 14 March 2019; Published: 18 March 2019 Abstract: We compared the covering behavior of four sea urchin species, Tripneustes gratilla, Pseudoboletia maculata, Toxopneustes pileolus, and Salmacis sphaeroides found in the waters of Malapascua Island, Cebu Province and Bolinao, Panagsinan Province, Philippines. Speciﬁcally, we measured the amount and type of covering material on each sea urchin, and in several cases, the recovery of debris material after stripping the animal of its cover. We found that Tripneustes gratilla and Salmacis sphaeroides have a higher afﬁnity for plant material, especially seagrass, compared to Pseudoboletia maculata and Toxopneustes pileolus, which prefer to cover themselves with coral rubble and other calciﬁed material. Only in Toxopneustes pileolus did we ﬁnd a signiﬁcant corresponding depth-dependent decrease in total cover area, conﬁrming previous work that covering behavior serves as a protection mechanism against UV radiation. We found no dependence of particle size on either species or size of sea urchin, but we observed that larger sea urchins generally carried more and heavier debris. We observed a transport mechanism of debris onto the echinoid body surface utilizing a combination of tube feet and spines. We compare our results to previous studies, comment on the phylogeny of sea urchin covering behavior, and discuss the interpretation of this behavior as animal tool use. Keywords: sea urchins; echinoid; echinoderms; covering behavior; animal tool use Glyn A. Barrett 1,2,* , Dominic Revell 2, Lucy Harding 2, Ian Mills 2, Axelle Jorcin 2 and Klaus M. Stiefel 2,3,4 Glyn A. Barrett 1,2,* , Dominic Revell 2, Lucy Harding 2, Ian Mills 2, Axelle Jorcin 2 and Klaus M. Stiefel 2,3,4 1 School of Biological Sciences, University of Reading, Reading RG6 6UR, UK 1 School of Biological Sciences, University of Reading, Reading RG6 6UR, UK School of Biological Sciences, University of Reading, Reading RG6 6UR, UK 2 People and The Sea, Logon, Daanbantayan, Cebu 6000, Philippines; domrevell@gmail.com (D.R.); lucy@peopleandthesea (L.H.); Ian@peopleandthesea.org (I.M.); axelle@peopleandthesea.org (A.J.); klaus@neurolinx.org (K.M.S.) 3 N li R h I i L J ll CA 92039 USA g y g g 2 People and The Sea, Logon, Daanbantayan, Cebu 6000, Philippines; domrevell@gmail.com (D.R.); lucy@peopleandthesea (L.H.); Ian@peopleandthesea.org (I.M.); axelle@peopleandthesea.org (A.J.); klaus@neurolinx.org (K.M.S.) 3 Neurolinx Research Institute, La Jolla, CA 92039, USA 4 Marine Science Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines 4 Marine Science Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines * Correspondence: glyn.barrett@reading.ac.uk * Correspondence: glyn.barrett@reading.ac.uk Reading’s research outputs online Journal of Marine Science and Engineering Journal of Marine Science and Engineering Journal of Marine Science and Engineering www.mdpi.com/journal/jmse J. Mar. Sci. Eng. 2019, 7, 69; doi:10.3390/jmse7030069 1. Introduction Several species of sea urchins cover their exposed body surfaces, in a special form of crypsis, with debris collected from their environment [1–5]. The debris can be biotic or abiotic in nature, and include scleractinian coral rubble, mollusc shells, sea grass, terrestrial leaves, and human rubbish. The sea urchins actively manipulate these debris fragments with their tube feet and combine these with their spines to transport them onto their upward facing body surface. Suggested reasons for this behavior are UV protection (demonstrated for Tripneustes gratilla [5,6]), olfactory camouﬂage (suggested for covering species living in light-deﬁcient deep-sea environments [7,8], and use as a ballast for weighing down of the animal by increasing the relative density of the sea urchin in the face of currents or surge (shown for Strongylocentrotus droebachiensis [2] and for Paracentrotus lividus [9]) as well as a direct anti-predatory function [1,10]. It has been shown that sea urchins increase their coverage if faced with stressors such as UV radiation and wave action [2,6]. Urchins were even capable of distinguishing between two types of artiﬁcial covering material, and preferred the type that was more conductive to UV protection [5]. Furthermore, since the covering material often acts as an effective platform for algal growth and is often moved from and across the main body of the animal J. Mar. Sci. Eng. 2019, 7, 69; doi:10.3390/jmse7030069 www.mdpi.com/journal/jmse J. Mar. Sci. Eng. 2019, 7, 69 2 of 12 toward the mouth, situated on the aboral surface, covering has been suggested as a food storage behavior [11,12]. Different species of sea urchins have been shown to be highly selective for the covering material used and make distinct choices for this [13]. For example, Toxopneustes pileolus showed a preference for the largest pieces of debris available amongst a range of differently sized substratum [14]. In tank studies, and given a choice of items of different sizes, Lytechinus variegatus selected those of intermediate size [15] in what the authors suggest is a trade-off between physiological energy expenditure and levels of UV protection. Individual choice of covering material in Paracentrotus lividus was found to be dependent on the size of the animal together with the availability of substrate [16], suggesting that covering behavior may differ dependent on habitat. 1. Introduction Whilst there are many studies on choice in the covering behavior of sea urchins, both at a species and individual level, there are few reports in the literature focusing on this behavior in a wider, ecological context whilst simultaneously considering multiple species sharing a common habitat. The aim of this study was to compare the covering behavior of four species of sea urchin, Tripneustes gratilla, Pseudoboletia maculata, Salmacis sphaeroides, and Toxopneustes pileolus, which are species found near reefs at shallow depths in the tropical Indo-Paciﬁc, and speciﬁcally the Philippines. These species partially overlap in their habitat use, and were often seen alongside each other; hence, they have access to the same types of covering material and forage that is available in similar concentrations and ratios throughout the range. This makes differences in their covering behavior all together intriguing. We combine ﬁeld observations of debris amount and type with video recordings of coverage material handling both in the ﬁeld and in aquaria. Given the active choice of coverage material and the situation-dependent loading of material, as well as the coordinated lifting of coverage material, we propose in the discussion that this behavior constitutes a rudimentary form of animal tool use. 2. Materials and Methods The radial position of the fragments determined in this way is an underestimation of the actual distance traveled, since the sea urchin bodies are compressed spheroids. However, relative measured speeds and positions are useful for comparative purposes. J. Mar. Sci. Eng. 2019, 7, x FOR PEER REVIEW 3 of 13 underestimation of the actual distance traveled, since the sea urchin bodies are compressed spheroids. However, relative measured speeds and positions are useful for comparative purposes. Figure 1. Examples of sea urchins covered in debris, photographed in their natural environment: Toxopneustes pileolus, Tripneustes gratilla, Salmacis sphaeroides, and Pseudoboletia maculata. Figure 1. Examples of sea urchins covered in debris, photographed in their natural environment: Toxopneustes pileolus, Tripneustes gratilla, Salmacis sphaeroides, and Pseudoboletia maculata. Figure 1. Examples of sea urchins covered in debris, photographed in their natural environment: Toxopneustes pileolus, Tripneustes gratilla, Salmacis sphaeroides, and Pseudoboletia maculata. Figure 1. Examples of sea urchins covered in debris, photographed in their natural environment: Toxopneustes pileolus, Tripneustes gratilla, Salmacis sphaeroides, and Pseudoboletia maculata. Six sea urchins each of the species Tripneustes gratilla and Salmacis sphaeroides were collected in Bolinao and maintained in aquaria at the Marine Lab of the University of the Philippines. The sea urchins were fed with seagrass and provided field-collected seagrass and coral rubble as coverage material. Close-up views of the covering behavior were filmed in the tanks. Six sea urchins each of the species Tripneustes gratilla and Salmacis sphaeroides were collected in Bolinao and maintained in aquaria at the Marine Lab of the University of the Philippines. The sea urchins were fed with seagrass and provided ﬁeld-collected seagrass and coral rubble as coverage material. Close-up views of the covering behavior were ﬁlmed in the tanks. a e ia C ose up ie s o e co e i g be a io e e i ed i e a ks Some of the video footage analyzed for this study can be accessed on our dedicated YouTube.com site as defined in supplementary materials. Some of the video footage analyzed for this study can be accessed on our dedicated YouTube.com site as deﬁned in Supplementary Materials. 2. Materials and Methods Echinoids were sampled on self-contained underwater breathing apparatus (SCUBA) at depths of between 2–16 m around Malapascua Island, Cebu Province, and Bolinao, Pangasinan Province, Philippines. All four investigated species are moderately common at and around the sampling sites. Of the six sites we sampled, one site was populated by four, three, and one species of the collector sea urchins we investigated, respectively, and the three remainder sites (including the sampling site in Bolinao) was populated by two species. In the ﬁeld, each sea urchin was photographed, measured (radial diameter), and carefully stripped of its attached debris. The debris was placed in a marked, plastic, Ziploc® bag and transported back to land via the research vessel. The “naked” sea urchin was then observed, and in many cases, ﬁlmed for up to 10 minutes, recording its covering behavior. If a sea urchin failed to re-cover itself, we would place debris comparable to the amount of debris removed from the animal to avoid any undue stress to the sea urchin, before moving to the next specimen. Post-dive, collected debris fragments were laid onto white slates with a ruler for scale and photographed directly from above. Individual pieces were counted, categorized, and surface area was determined using ImageJ® [17] image analysis software (Figure 1). Fragments were subdivided into the following categories: coral rubble, mollusc shells, tunicates, marine plants and algae, land plants, and human refuse. Fragments of debris were sun-dried for a period of 24 h and subsequently weighed using an electronic scale. To determine the speed of re-covering, we traced the position of selected pieces of debris during the sea urchin’s efforts to cover its body surface with debris. Footage was either shot in the ﬁeld (by a diver with a hand-held camera) or in concrete aquarium tanks (with a camera positioned above the tank). Video analysis was conducted with Tracker 5.0 Video Analysis and Modeling Tool software (GNU General Public License, ©2017 Douglas Brown). At intervals of 10 s, the position of the sea urchin’s pole and the pole-most point of the fragment were manually indicated, and the resulting positions were used for the analysis of fragment movement speed. At each time step, the scale bar 3 of 12 J. Mar. Sci. Eng. 2019, 7, 69 was also adjusted if necessary (if the distance from the camera to the sea urchin slightly changed in the case of hand-held footage). Statistical Analysis Statistical Analysis Levene’s test was used to assess the homogeneity of variance between individual sea urchin species. We used ANOVA to compare debris type preference between species of collector sea urchins and the size of the sea urchins, as determined by radial diameter and individual and global fragment size and weight. Linear regression was used to predict the depth-dependent total cover area per sea urchin species and test for significance in the size and weight of fragments on the overall size of the sea urchin. Levene’s test was used to assess the homogeneity of variance between individual sea urchin species. We used ANOVA to compare debris type preference between species of collector sea urchins and the size of the sea urchins, as determined by radial diameter and individual and global fragment size and weight. Linear regression was used to predict the depth-dependent total cover area per sea urchin species and test for signiﬁcance in the size and weight of fragments on the overall size of the sea urchin. 3. Results 3. Results (A) Absolute numbers of debris type per species; (B) Relative contribution in % of each type of debris per species; (C) Absolute numbers of fragments per species sorted between calciﬁed CaCO3 skeletons and organic material; (D) Relative contribution in % of CaCO3 skeletons and organic materials per species. Linear regression was calculated to predict the total cover area (cm2) based on depth (m) (Figure 3). A significant regression equation was found for T. pileolus (F(1, 14) = 7.443, p = 0.016), with an R2 of 0.347. T. pileolus predicted that the total cover area is equal to 248.4 – 10.1 (x) cm2 where (x) is depth measured in m. Therefore, sea urchin cover area decreased by an average of 10.1 cm2 for each meter of depth. Interestingly, depth was not a significant factor in influencing the total cover area for T. Linear regression was calculated to predict the total cover area (cm2) based on depth (m) (Figure 3). A signiﬁcant regression equation was found for T. pileolus (F(1, 14) = 7.443, p = 0.016), with an R2 of 0.347. T. pileolus predicted that the total cover area is equal to 248.4 – 10.1 (x) cm2 where (x) is depth measured in m. Therefore, sea urchin cover area decreased by an average of 10.1 cm2 for each meter of depth. Interestingly, depth was not a signiﬁcant factor in inﬂuencing the total cover area for T. gratilla, P. maculata, or S. sphaeroides. gratilla, P. maculata, or S. sphaeroides. We found significant differences in size, as defined by equatorial diameter, of species of sea urchin (ANOVA, F(3, 45) = 211.722, p < 0.001) with S. sphaeroides, P. maculata, T. gratilla, and T. pileolus at 6.3 cm, 9.6 cm, 9.7 cm, and 14.8 cm, respectively (Figure 4). Interestingly, the global mean size of fragments (5.6 ± 0.68 cm2) was independent of sea urchin species and the size of the sea urchin (ANOVA, F(1, 48) = 0.7, p = 0.407) (Figure 5). Linear regression was calculated to predict both the total number (n) and total weight (g) of fragments based on the diameter of the sea urchin (cm). Significant regression equations were found for both the total fragment number (F(1 47) = 10 094 p = 0 003) with We found signiﬁcant differences in size, as deﬁned by equatorial diameter, of species of sea urchin (ANOVA, F(3, 45) = 211.722, p < 0.001) with S. 3. Results 3. Results We sampled a total of 49 sea urchins, T. gratilla (n = 11), P. maculata (n = 10), S. sphaeroides (n = 12), and T. pileolus (n = 16) across six sites. We compared debris type preference between species of We sampled a total of 49 sea urchins, T. gratilla (n = 11), P. maculata (n = 10), S. sphaeroides (n = 12), and T. pileolus (n = 16) across six sites. We compared debris type preference between species of 4 of 12 J. Mar. Sci. Eng. 2019, 7, 69 collector sea urchins, and found signiﬁcant differences in coverage material most importantly between total organic (e.g., seagrass, rhodoliths, tunicates) (ANOVA, F(3, 45) = 7.198, p < 0.001) and total inorganic (e.g., coral, shell, rock) (ANOVA, F(3, 45) = 8.654, p < 0.001) materials (Figure 2). Post-hoc Tukey’s Honest Signiﬁcant Difference (HSD) analysis revealed that S. sphaeroides preferred seagrass as a covering material, whilst T. pileolus preferred coral fragments. J. Mar. Sci. Eng. 2019, 7, x FOR PEER REVIEW 4 of 13 total inorganic (e.g., coral, shell, rock) (ANOVA, F(3, 45) = 8.654, p < 0.001) materials (Figure 2). Post- hoc Tukey’s Honest Significant Difference (HSD) analysis revealed that S. sphaeroides preferred seagrass as a covering material, whilst T. pileolus preferred coral fragments. Figure 2. Sea urchin species-dependent preference in cover material. (A) Absolute numbers of debris type per species; (B) Relative contribution in % of each type of debris per species; (C) Absolute numbers of fragments per species sorted between calcified CaCO3 skeletons and organic material; (D) Relative contribution in % of CaCO3 skeletons and organic materials per species Figure 2. Sea urchin species-dependent preference in cover material. (A) Absolute numbers of debris type per species; (B) Relative contribution in % of each type of debris per species; (C) Absolute numbers of fragments per species sorted between calciﬁed CaCO3 skeletons and organic material; (D) Relative contribution in % of CaCO3 skeletons and organic materials per species. Figure 2. Sea urchin species-dependent preference in cover material. (A) Absolute numbers of debris type per species; (B) Relative contribution in % of each type of debris per species; (C) Absolute numbers of fragments per species sorted between calcified CaCO3 skeletons and organic material; (D) Relative contribution in % of CaCO3 skeletons and organic materials per species Figure 2. Sea urchin species-dependent preference in cover material. 3. Results 3. Results sphaeroides, P. maculata, T. gratilla, and T. pileolus at 6.3 cm, 9.6 cm, 9.7 cm, and 14.8 cm, respectively (Figure 4). Interestingly, the global mean size of fragments (5.6 ± 0.68 cm2) was independent of sea urchin species and the size of the sea urchin (ANOVA, F(1, 48) = 0.7, p = 0.407) (Figure 5). Linear regression was calculated to predict both the total number (n) and total weight (g) of fragments based on the diameter of the sea urchin (cm). Signiﬁcant regression equations were found for both the total fragment number (F(1, 47) = 10.094, p = 0.003), 5 of 12 J. Mar. Sci. Eng. 2019, 7, 69 with an R2 of 0.177, and the total weight of fragments (F(1, 47) = 3.997, p = 0.05), with an R2 of 0.078. The predicted total fragment number is equal to 8.818 + 0.071 (diameter) n when the diameter is measured in cm. The predicted total fragment weight is equal to 9.676 + 0.011 (diameter) g when the diameter is measured in cm. The total fragment number and total fragment weight increased, respectively, by 0.071 g and 0.011 g for each increase of 1 cm in diameter. J. Mar. Sci. Eng. 2019, 7, x FOR PEER REVIEW 5 of 13 is measured in cm. The total fragment number and total fragment weight increased, respectively, by 0 071 d 0 011 f h i f 1 i di t Figure 3. Depth influences the amount of debris on collector sea urchins. Fragment area for Tripneustes gratilla (red crosses), Pseudoboletia maculata (black circles), Salmacis sphaeroides (green squares), and Toxopneustes pileolus (blue triangles). Figure 3. Depth inﬂuences the amount of debris on collector sea urchins. Fragment area for Tripneustes gratilla (red crosses), Pseudoboletia maculata (black circles), Salmacis sphaeroides (green squares), and Toxopneustes pileolus (blue triangles). Figure 3. Depth influences the amount of debris on collector sea urchins. Fragment area for Tripneustes gratilla (red crosses), Pseudoboletia maculata (black circles), Salmacis sphaeroides (green squares), and Toxopneustes pileolus (blue triangles). Figure 3. Depth inﬂuences the amount of debris on collector sea urchins. Fragment area for Tripneustes gratilla (red crosses), Pseudoboletia maculata (black circles), Salmacis sphaeroides (green squares), and Toxopneustes pileolus (blue triangles). Figure 3. Depth influences the amount of debris on collector sea urchins. Fragment area for Tripneustes gratilla (red crosses), Pseudoboletia maculata (black circles), Salmacis sphaeroides (green squares), and Toxopneustes pileolus (blue triangles) Figure 3. 3. Results 3. Results Depth inﬂuences the amount of debris on collector sea urchins. Fragment area for Tripneustes gratilla (red crosses), Pseudoboletia maculata (black circles), Salmacis sphaeroides (green squares), and Toxopneustes pileolus (blue triangles). 6 of 12 6 f 13 of 13 6 of 12 f 13 of 13 J. Mar. Sci. Eng. 2019, 7, 69 J M S i E 2019 7 FOR . Mar. Sci. Eng. 2019, 7, x FOR Figure 4. Diameter (mean ± SEM) of collector sea urchins. Means with different letters are significantly different (Tukey’s HSD, p < 0.05). Figure 5. (A) Diameter vs. total fragments; (B) Diameter vs. average size of fragment (cm2); (C) Diameter vs. total weight of fragments (g); x-axis is diameter (cm); y-axis is log10 scale. (D) Average fragment weight per species. Figure 4. Diameter (mean ± SEM) of collector sea urchins. Means with different letters are signiﬁcantly different (Tukey’s HSD, p < 0.05). Figure 4. Diameter (mean ± SEM) of collector sea urchins. Means with different letters are significantly different (Tukey’s HSD, p < 0.05). Figure 5. (A) Diameter vs. total fragments; (B) Diameter vs. average size of fragment (cm2); (C) Diameter vs. total weight of fragments (g); x-axis is diameter (cm); y-axis is log10 scale. (D) Average fragment weight per species. Figure 5. (A) Diameter vs. total fragments; (B) Diameter vs. average size of fragment (cm2); (C) Diameter vs. total weight of fragments (g); x-axis is diameter (cm); y-axis is log10 scale. (D) Average fragment weight per species. Figure 4. Diameter (mean ± SEM) of collector sea urchins. Means with different letters are significantly Figure 4. Diameter (mean ± SEM) of collector sea urchins. Means with different letters are signiﬁcantly different (Tukey’s HSD, p < 0.05). Figure 4. Diameter (mean ± SEM) of collector sea urchins. Means with different letters are significantly different (Tukey’s HSD, p < 0.05). Figure 4 Diameter (mean ± SEM) of collector sea urchins Means with different letters are significantly Figure 4. Diameter (mean ± SEM) of collector sea urchins. Means with different letters are signiﬁcantly different (Tukey’s HSD, p < 0.05). Figure 4. Diameter (mean ± SEM) of collector sea urchins. Means with different letters are significantly different (Tukey’s HSD, p < 0.05). igure 4. Diameter (mean ± SEM) of collector sea urchins. Means with different letters are significantly Figure 4. Diameter (mean ± SEM) of collector sea urchins. 3. Results 3. Results 2019, 7, x FOR PEER REVIEW 7 of 13 After we removed the debris coverage from the body of a sea urchin, it would quickly grab new debris with its tube feet and place it on top of its body. The speed of fragment movement onto the sea urchin was similar between different species (Figure 6A). In the footage we analyzed, P. maculata moved coral rubble the fastest, at a mean distance of 2.0 cm min−1, followed by T. pileolus at 1.91 cm min−1, and T. gratilla at 0.99 cm min−1. The transport speed was not constant during the movement of a fragment, often with periods of fast transport being preceded and followed by period of slower movement (Figure 6B). Hence, despite the divergent global speeds noted above, instantaneous transport speed (as seen in the slopes of the fragment positions in Figure 6B) was relatively similar. Movements in the efforts of orienting individual fragments also affected the speed of its movement toward the dorsal pole of the echinoid. After we removed the debris coverage from the body of a sea urchin, it would quickly grab new debris with its tube feet and place it on top of its body. The speed of fragment movement onto the sea urchin was similar between different species (Figure 6A). In the footage we analyzed, P. maculata moved coral rubble the fastest, at a mean distance of 2.0 cm min−1, followed by T. pileolus at 1.91 cm min−1, and T. gratilla at 0.99 cm min−1. The transport speed was not constant during the movement of a fragment, often with periods of fast transport being preceded and followed by period of slower movement (Figure 6B). Hence, despite the divergent global speeds noted above, instantaneous transport speed (as seen in the slopes of the fragment positions in Figure 6B) was relatively similar. Movements in the efforts of orienting individual fragments also affected the speed of its movement toward the dorsal pole of the echinoid. Figure 6. Speed of recovery of debris cover of sea urchins. (A) Points indicating movement trajectory of the pole-most point of a coral rubble fragment on Toxopneustes pileolus (in the field). Note the scale bar in blue. Screen-shot from Tracker software; (B) Position of fragment relative to the dorsal pole of the sea urchin. Species are color coded (P. maculata, red; T. pileolus, blue; T. 3. Results 3. Results The footage was recorded in tanks (T. gratilla solid line, both S. sphaeroides) and in the field (all other recordings). Figure 6. Speed of recovery of debris cover of sea urchins. (A) Points indicating movement trajectory of the pole-most point of a coral rubble fragment on Toxopneustes pileolus (in the ﬁeld). Note the scale bar in blue. Screen-shot from Tracker software; (B) Position of fragment relative to the dorsal pole of the sea urchin. Species are color coded (P. maculata, red; T. pileolus, blue; T. gratilla, black), continuous and broken lines indicate fragments on different animals. The footage was recorded in tanks (T. gratilla solid line, both S. sphaeroides) and in the ﬁeld (all other recordings). The covering behavior is jointly carried out by spines and tube feet. Initially, the tube feet make searching rotational movements, until one of them comes into contact with a piece of debris. The tube foot is soon joined by several other tube feet, which jointly pull the fragment toward the sea urchin’s body (Figure 7A,B). It is not obvious from the video observations whether this is due to a directed recruitment (i.e., concerted effort) of tube feet by the echinoid nervous system, or simply due to the proximity of neighboring tube feet to the detected fragment. Next, the tube feet pull the debris fragment closer to the echinoid. The fragment is most commonly transported up onto the body of the sea urchin by a combined pulling movement of the tube feet and a pushing movement of the spines. Upward-angled spines keep the fragment from slipping off, while the spines above the fragment flatten out to allow fragments to pass over freely. The tube feet above the fragment are often seen pulling it toward the pole (anus) of the echinoid (Figure 7C,D). The pedicellariae are not involved in the transport process. The fragments reaching the top of the sea urchin naturally block the transport of further lower fragments, which is a process that repeats until the complete dorsal surface of the sea urchin is covered. We often observed that the sea urchins moved in one direction when stripped The covering behavior is jointly carried out by spines and tube feet. Initially, the tube feet make searching rotational movements, until one of them comes into contact with a piece of debris. 3. Results 3. Results Means with different letters are signiﬁcantly different (Tukey’s HSD, p < 0.05). igure 4. Diameter (mean ± SEM) of collector sea urchins. Means with different letters are significantly ifferent (Tukey’s HSD, p < 0.05). different (Tukey’s HSD, p < 0.05). Figure 5. (A) Diameter vs. total fragments; (B) Diameter vs. average size of fragment (cm2); (C) Diameter vs. total weight of fragments (g); x-axis is diameter (cm); y-axis is log10 scale. (D) Average Figure 5. (A) Diameter vs. total fragments; (B) Diameter vs. average size of fragment (cm2); (C) Diameter vs. total weight of fragments (g); x-axis is diameter (cm); y-axis is log10 scale. (D) Average fragment weight per species. Figure 5. (A) Diameter vs. total fragments; (B) Diameter vs. average size of fragment (cm2); (C) Diameter vs. total weight of fragments (g); x-axis is diameter (cm); y-axis is log10 scale. (D) Average fragment weight per species. different (Tukey s HSD, p < 0.05). Figure 5. (A) Diameter vs. total fragments; (B) Diameter vs. average size of fragment (cm2); (C) Diameter vs total weight of fragments (g); x-axis is diameter (cm); y-axis is log10 scale (D) Average igure 5. (A) Diameter vs. total fragments; (B) Diameter vs. average size of fragment (cm2); (C) Diameter vs. total weight of fragments (g); x-axis is diameter (cm); y-axis is log10 scale. (D) Average ragment weight per species. Figure 5. (A) Diameter vs. total fragments; (B) Diameter vs. average size of fragment (cm2); (C) Diameter vs. total weight of fragments (g); x-axis is diameter (cm); y-axis is log10 scale. (D) Average fragment weight per species. J. Mar. Sci. Eng. 2019, 7, 69 7 of 12 Mean fragment weight (±SEM) in g per species was as follows: Pseudoboletia maculata: 3.62 ± 0.54, Toxopneustes pileolus: 2.67 ± 0.34, Tripneustes gratilla: 5.6 ± 1.78, and Salmacis sphaeroides: 0.98 ± 0.32. An analysis of variance (ANOVA) of the observed fragment weights yielded signiﬁcant variation among sea urchin groups, F(3, 45) = 4.615, p = 0.007. A post-hoc Tukey test, denoted by letters above error bars, showed signiﬁcance among groups at p < 0.05. The mean difference in the fragment weight of S. sphaeroides and T. gratilla was signiﬁcant at the value of p = 0.004. J. Mar. Sci. Eng. 3. Results 3. Results gratilla, black), continuous and broken lines indicate fragments on different animals. The footage was recorded in tanks (T. gratilla solid line, both S. sphaeroides) and in the field (all other recordings). Figure 6. Speed of recovery of debris cover of sea urchins. (A) Points indicating movement trajectory of the pole-most point of a coral rubble fragment on Toxopneustes pileolus (in the ﬁeld). Note the scale bar in blue. Screen-shot from Tracker software; (B) Position of fragment relative to the dorsal pole of the sea urchin. Species are color coded (P. maculata, red; T. pileolus, blue; T. gratilla, black), continuous and broken lines indicate fragments on different animals. The footage was recorded in tanks (T. gratilla solid line, both S. sphaeroides) and in the ﬁeld (all other recordings). Figure 6. Speed of recovery of debris cover of sea urchins. (A) Points indicating movement trajectory of the pole-most point of a coral rubble fragment on Toxopneustes pileolus (in the field). Note the scale bar in blue. Screen-shot from Tracker software; (B) Position of fragment relative to the dorsal pole of the sea urchin. Species are color coded (P. maculata, red; T. pileolus, blue; T. gratilla, black), continuous and broken lines indicate fragments on different animals. The footage was recorded in tanks (T. gratilla solid line, both S. sphaeroides) and in the field (all other recordings). Figure 6. Speed of recovery of debris cover of sea urchins. (A) Points indicating movement trajectory of the pole-most point of a coral rubble fragment on Toxopneustes pileolus (in the ﬁeld). Note the scale bar in blue. Screen-shot from Tracker software; (B) Position of fragment relative to the dorsal pole of the sea urchin. Species are color coded (P. maculata, red; T. pileolus, blue; T. gratilla, black), continuous and broken lines indicate fragments on different animals. The footage was recorded in tanks (T. gratilla solid line, both S. sphaeroides) and in the ﬁeld (all other recordings). Figure 6. Speed of recovery of debris cover of sea urchins. (A) Points indicating movement trajectory of the pole-most point of a coral rubble fragment on Toxopneustes pileolus (in the field). Note the scale bar in blue. Screen-shot from Tracker software; (B) Position of fragment relative to the dorsal pole of the sea urchin. Species are color coded (P. maculata, red; T. pileolus, blue; T. gratilla, black), continuous and broken lines indicate fragments on different animals. 3. Results 3. Results The tube foot is soon joined by several other tube feet, which jointly pull the fragment toward the sea urchin’s body (Figure 7A,B). It is not obvious from the video observations whether this is due to a directed recruitment (i.e., concerted effort) of tube feet by the echinoid nervous system, or simply due to the proximity of neighboring tube feet to the detected fragment. Next, the tube feet pull the debris fragment closer to the echinoid. The fragment is most commonly transported up onto the body of the sea urchin by a combined pulling movement of the tube feet and a pushing movement of the spines. Upward-angled spines keep the fragment from slipping off, while the spines above the fragment ﬂatten out to allow fragments to pass over freely. The tube feet above the fragment are often seen pulling it toward the pole (anus) of the echinoid (Figure 7C,D). The pedicellariae are not involved in the transport process. The fragments reaching the top of the sea urchin naturally block the transport of further lower fragments, which is a process that repeats until the complete dorsal surface of the sea urchin is covered. We often observed that the sea urchins moved in one direction when stripped of J. Mar. Sci. Eng. 2019, 7, 69 8 of 12 covering material, whilst re-covering. This combined covering-walking behavior was observed both in the ﬁeld and in tanks. Mar. Sci. Eng. 2019, 7, x FOR PEER REVIEW 8 of 13 Figure 7. Spines and tube-feet are involved in the covering behavior in Tripneustes gratilla. Screen- shots of videos. (A) Several tube feet (one indicated by an arrow) jointly grab a coral rubble fragment in the initial phase of transport; (B) Two fragments held by tube feet (arrows); (C) Spines pushing a fragment upwards; (D) Spines pushing (downward arrow) and tube feet pulling (leftward arrow) a fragment. (A,D) are from footage recorded in the field; (B,C) were recorded in tanks. Figure 7. Spines and tube-feet are involved in the covering behavior in Tripneustes gratilla. Screen-shots of videos. (A) Several tube feet (one indicated by an arrow) jointly grab a coral rubble fragment in the initial phase of transport; (B) Two fragments held by tube feet (arrows); (C) Spines pushing a fragment upwards; (D) Spines pushing (downward arrow) and tube feet pulling (leftward arrow) a fragment. 3. Results 3. Results (A,D) are from footage recorded in the ﬁeld; (B,C) were recorded in tanks. Figure 7. Spines and tube-feet are involved in the covering behavior in Tripneustes gratilla. Screen- shots of videos. (A) Several tube feet (one indicated by an arrow) jointly grab a coral rubble fragment n the initial phase of transport; (B) Two fragments held by tube feet (arrows); (C) Spines pushing a ragment upwards; (D) Spines pushing (downward arrow) and tube feet pulling (leftward arrow) a ragment. (A,D) are from footage recorded in the field; (B,C) were recorded in tanks. Figure 7. Spines and tube-feet are involved in the covering behavior in Tripneustes gratilla. Screen-shots of videos. (A) Several tube feet (one indicated by an arrow) jointly grab a coral rubble fragment in the initial phase of transport; (B) Two fragments held by tube feet (arrows); (C) Spines pushing a fragment upwards; (D) Spines pushing (downward arrow) and tube feet pulling (leftward arrow) a fragment. (A,D) are from footage recorded in the ﬁeld; (B,C) were recorded in tanks. Several anecdotal observations during our study are worth mentioning. Two of the sea urchins arried seemingly living tunicates on their backs when first encountered. We found many sea urchins n areas with sand or coral rubble that was interspersed by rocks not higher than 30 cm. Tunicates sit n the edges of many of these rocks, exactly in a position where a passing sea urchin would be in the osition to impale them. Another observation we repeatedly made was that sea urchins stripped of ebris would crawl underneath the protective spines of groups of nearby Diadema sp. sea urchins. Also, when the collector sea urchins replaced the debris covering their bodies after we removed it, hey caused significant perturbation of the sand and rubble they “walked” over as if they were ttempting to dig themselves into the substrate. Then, the sea urchins were often followed by several pecies of wrasse (mostly Choris batuensis, Oxychelinus sp., Diproctacanthus sp.) and sand perch Parapercis sp.), presumably aiming to acquire food items from amongst the disturbed substrate. Several anecdotal observations during our study are worth mentioning. Two of the sea urchins carried seemingly living tunicates on their backs when ﬁrst encountered. We found many sea urchins in areas with sand or coral rubble that was interspersed by rocks not higher than 30 cm. 3. Results 3. Results Tunicates sit on the edges of many of these rocks, exactly in a position where a passing sea urchin would be in the position to impale them. Another observation we repeatedly made was that sea urchins stripped of debris would crawl underneath the protective spines of groups of nearby Diadema sp. sea urchins. Also, when the collector sea urchins replaced the debris covering their bodies after we removed it, they caused signiﬁcant perturbation of the sand and rubble they “walked” over as if they were attempting to dig themselves into the substrate. Then, the sea urchins were often followed by several species of wrasse (mostly Choris batuensis, Oxychelinus sp., Diproctacanthus sp.) and sand perch (Parapercis sp.), presumably aiming to acquire food items from amongst the disturbed substrate. 4.1. Echinoid Covering Behavior Evolution Phylogenetically, covering behavior occurs widely in Echinoidea. The species compared here belong to the families of the Toxopneustidae (Pseudoboletia maculata and Toxopneustes pileolus), and Temnopleuroidae (Tripneustes gratilla and Salmacis sphaeroides). Other echinoid families with members showing covering behavior are listed in Table 1. These families span several groups of echinoids outside of the more primitive pencil sea urchins (cidaroids) and the long-spined Diadema species (Figure 7). Among the Regularia (radially symmetrical sea urchins), covering behavior is found in several groups of the Echinacea. A big cluster of collector sea urchins is found in the Camarodonta, including collectors in the families of the Parechinidae, Strongylocentrotidae, Temnopleuridae, and Toxopneustidae. We found two mentions of irregular echinoids (asymmetric sea urchins, sand dollars, and their relatives) with covering behavior. One covering sea urchin is the deep-sea urchin Conolampas sigsbei, belonging to family Echinolampadidae in the Neognathostomata branch of the irregularia [3,19]. Also, Cystochinus loveni, another deep-sea species, was observed to cover itself with a layer of protists. The authors noted that the covering behavior was most likely speciﬁcally loaded by the echinoid, and speculated that camouﬂage and a weighing-down effect are the reasons for this covering behavior [20]. We are aware that many Irregularia bury themselves in the sediment [21]. This behavior, which likely shares mechanistic features with the covering behavior discussed here, is nevertheless distinct in its ecological role, and we treat the two behaviors separately. The ancestors of these collector sea urchin families split around 200 Ma ago in the lower Jurassic [22]. Since collecting behavior has not been observed in the majority of echinoids, it presumably evolved convergently multiple times in the lineages mentioned here (for an extensive discussion of convergent evolution, see [23]). Table 1. A list of echinoids showing covering behavior, assembled from the literature and from personal observations. We make no claim that this list is exhaustive. See also Figure 7. 4. Discussion Our study shows that the covering material of four species of collector sea urchin, Pseudoboletia maculata, Toxopneustes pileolus, Salmacis sphaeroides, and Tripneustes gratilla, is distinctively species-dependent. A previous study of the covering behavior of Tripneustes ventricosus and Lytechinus variegatus in the Caribbean found that the former prefers to cover itself with seagrass, even though it 9 of 12 J. Mar. Sci. Eng. 2019, 7, 69 uses other material independently of seagrass availability [13]. Our study comes to a similar conclusion, with 15% of the cover of the closely related Tripneustes gratilla being seagrass, compared to 0.5% and no seagrass cover for Toxopneustes pileolus and Pseudoboletia maculata, respectively. Salmacis sphaeroides had a higher preference for organic material with a total of 52%; 87% of this material was seagrass alone. Hence, our study conﬁrms a species-speciﬁc choice in covering material. That the fragment size was similar for different sea urchin species of different sizes points to similar covering mechanisms in all these species. A video analysis of fragment transport agrees with this conclusion, and shows a covering mechanism involving both spines and tube-feet. We only found a depth-dependent decrease in debris coverage in Toxopneustes pileolus, which conﬁrms the purpose of the covering behavior, in this species, as UV protection and a weighting down to counter water movement, as seen in previous studies [2,5,6,18]. The limited depth range at which we sampled the other species might have contributed to the lack of depth-dependence evidence in coverage. 4.2. Echinoid Covering Behavior as Tool Use An intriguing question is if the covering behavior shown by sea urchins can be classiﬁed as animal tool use. Animal tool use is notoriously difﬁcult to deﬁne, especially in borderline cases [28]. Following previous work on the matter of tool use in ﬁnches [29] and primates [30], St Amant and Horton in 2008 [31] expanded the deﬁnition by Shumaker et al. [32] and deﬁned tool use as “ . . . the exertion of control over a freely manipulable external object (the tool) with the goal of: (1.) altering the physical properties of another object, substance, surface or medium (the target, which may be the tool user or another organism) via a dynamic mechanical interaction; or (2.) mediating the ﬂow of information between the tool user and the environment or other organisms in the environment”. Collector sea urchins manipulate and freely control marine debris fragments of their choice with the goal of altering the physical properties of themselves (the tool user), for the sake of UV, predation, and water surge protection. They also mediate the ﬂow of information between the tool user and the environment or other organisms, by using the debris as camouﬂage. Thus, the collecting behavior of sea urchins falls into two categories, as given in Bentley-Condit and Smith (2010), namely, physical maintenance (i.e., the use of a tool to affect one’s appearance or body) and predator defense (i.e., the use of a tool to defend oneself). Bentley-Condit and Smith (2010) cited the placement of anemones on their shells by hermit crabs as an example of the latter, which is quite similar to the echinoderm behavior discussed here. Lawrence [33] argued that the covering behavior of Lytechinus variegatus is reﬂexive, rather than functional. This, in our opinion, lumps proximate and ultimate causes [34,35]. Certainly the proximal cause, which is also the physiological and behavioral explanation, is a reﬂexive loading of debris by the sea urchin. The animals achieve this reﬂexive loading without any form of understanding of their actions (see the following paragraph). However, the ultimate cause of the debris covering, the adaptive value of the behavior formed in evolution, is the use of debris as a tool, as indicated by the selectivity of debris collected and the modiﬁcation of debris cover in response to different environmental situations. 4.2. Echinoid Covering Behavior as Tool Use A signiﬁcant amount of investigation and experimentation has been directed to the question of how much reasoning and insight is behind animal tool use. Tool use by humans, and presumably apes and crows, derives from a mental model of the physical effects of the tool (for a good review, see [36]). However, such an internal model is not a condition for tool use, and Seed and Byrne (2010) reasoned that insect tool use (for example, ant lions ﬂicking sand at prey) comes about without an internal representation of the tool as an extension of the animal’s body. Collector sea urchins most likely have no mental representation of the tools they are using or the intended consequences of the tool use, since they lack a centralized brain. Nevertheless, several observations indicate that echinoid covering behavior is tool use, as deﬁned in the literature cited above: - The use of selected pieces of debris as a cover by sea urchins (as shown in this and other studies [5,13]). By no means is the covering material simply debris that gets stuck on the sea urchin, but it is there through the active selection and choice of the animal, as a consequence of the coordinated action of tube-feet and spines in response to the environment. - The adjustment of debris cover according to need (increased coverage in response to increased UV radiation [2], and the increased coverage at shallower depth as demonstrated in this study) also point to goal-directed tool-use behavior. - The adjustment of debris cover according to need (increased coverage in response to increased UV radiation [2], and the increased coverage at shallower depth as demonstrated in this study) also point to goal-directed tool-use behavior. - The difference in debris type between echinoid species points toward a speciﬁc form of tool use tailored to the needs and capabilities of the different sea urchin species. We are by no means the ﬁrst investigators to observe and describe the covering behavior of sea urchins, but the conceptual connection between this behavior and tool use has, to our knowledge, not been made previously, besides a passing mention in one other study [37]. We do believe that this behavior can be classiﬁed as tool use, adding the Echinodermata as a fourth phylum with members capable of tool use next to the Chordata (mainly primate mammals and corvid Aves), Arthropoda, and Molluska (primarily octopod cephalopods) [28]. 4.1. Echinoid Covering Behavior Evolution Order Family Genus Species Distribution Reference Camarodonta Parechinidae Paracentrotus lividus Mediterranean Sea and eastern Atlantic Ocean [16] Camarodonta Temnopleuridae Salmacis sphaeroides Tropical Indo-Paciﬁc [24] Camarodonta Toxopneustidae Lytechinus anamesus Caribbean [25] Camarodonta Toxopneustidae Lytechinus euerces Deep sea, Caribbean [26] Camarodonta Toxopneustidae Lytechinus variegatus Caribbean [13] Camarodonta Toxopneustidae Toxopneustes roseus Gulf of California [27] Camarodonta Toxopneustidae Tripneustes gratilla Tropical Indo-Paciﬁc This study Camarodonta Toxopneustidae Tripneustes ventricosus Caribbean [13] Camarodonta Toxopneustidae Pseudoboletia maculata Tropical Indo-Paciﬁc This study Camarodonta Toxopneustidae Toxopneustes pileolus Tropical Indo-Paciﬁc This study Echinoida Strongylocentrotidae Strongylocentrotus droebachiensis Temperate Atlantic [18] Echinoida Strongylocentrotidae Strongylocentrotus intermedius Temperate and cold northern hemisphere [1] Cassiduloida Echinolampadidae Conolampas sigsbei Deep sea, Caribbean [26] Spatangoida - Paleobrissus hilgardi Deep sea, Caribbean [26] Stomopneustoida Glyptocidaridae Glyptocidaris Crenularis Temperate and cold northern hemisphere [3] Table 1. A list of echinoids showing covering behavior, assembled from the literature and from personal observations. We make no claim that this list is exhaustive. See also Figure 7. 10 of 12 J. Mar. Sci. Eng. 2019, 7, 69 10 of 12 References 1. Agatsuma, Y. Effect of the covering behavior of the juvenile sea urchin Strongylocentrotus intermedius on predation by the spider crab Pugettia quadridens. Fish Sci. 2001, 67, 1181–1183. [CrossRef] 1. Agatsuma, Y. Effect of the covering behavior of the juvenile sea urchin Strongylocentrotus intermedius on predation by the spider crab Pugettia quadridens. Fish Sci. 2001, 67, 1181–1183. [CrossRef] 2. Dumont, C.P.; Drolet, D.; Deschênes, I.; Himmelman, J.H. Multiple factors explain the covering behaviour in the green sea urchin, Strongylocentrotus droebachiensis. Anim. Behav. 2007, 73, 979–986. [CrossRef] 2. Dumont, C.P.; Drolet, D.; Deschênes, I.; Himmelman, J.H. 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On the functional signiﬁcance of masking behaviour in sea urchins-an experiment with Paracentrotus lividus. Mar. Ecol. Prog. Ser. 2000, 205, 307–308. [CrossRef] 10. 4.2. Echinoid Covering Behavior as Tool Use Alternatively, the commonly used deﬁnitions of 11 of 12 J. Mar. Sci. Eng. 2019, 7, 69 tool use will have to be narrowed to exclude tool use without an explicit mental representation of the tool and its intended effects. tool use will have to be narrowed to exclude tool use without an explicit mental representation of the tool and its intended effects. Supplementary Materials: A video showcasing sea urchin covering behaviour through the use of spines and tube feet is available on our Youtube site (https://www.youtube.com/watch?v=GZw0mKy_un8&t=4). Author Contributions: All of the authors collected data; G.A.B. conceived the idea; G.A.B. and K.M.S. analyzed the data and wrote the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Acknowledgments: We thank everyone at People and the Sea and at the UP Bolinao Marine Lab for their support, as well as Cecilia Conaco and Patrick Cabaitan for their helpful discussion of the manuscript. Acknowledgments: We thank everyone at People and the Sea and at the UP Bolinao Marine Lab for their support, as well as Cecilia Conaco and Patrick Cabaitan for their helpful discussion of the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Zhao, C.; Ji, N.; Zhang, B.; Sun, P.; Feng, W.; Wei, J.; Chang, Y. Effects of covering behavior and exposure to a predatory crab Charybdis japonica on survival and HSP70 expression of juvenile sea urchins Strongylocentrotus intermedius. PLoS ONE 2014, 9, e97840. [CrossRef] 11. Dix, T.G. Covering response of the Echinoid Evechinus chloroticus (Val.). Pac. Sci. 1970, 24, 187–194. 12. 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W2770192776.txt	https://www.frontiersin.org/articles/10.3389/fsoc.2017.00019/pdf	en		Effects of Workplace Parent Management Training on Marital and Job Satisfaction among Iranian Working Parents	Frontiers in sociology	2,017	cc-by	4,010	Original Research published: 23 November 2017 doi: 10.3389/fsoc.2017.00019 Effects of Workplace Parent Management Training on Marital and Job Satisfaction among Iranian Working Parents Masoumeh Mousavi 1, Ramin Alavinezhad 2* and Arezoo Moosavi 3 1 Department of Clinical and Health Psychology, Faculty of Psychology, Shahid Beheshti University, Tehran, Iran, 2 Department of Psychology, Islamic Azad University, Central Branch, Tehran, Iran, 3 Islamic Azad University System, Tehran, Iran Background: Shifting the focus of parent management training (PMT) to parents and discussing implications for maximizing the outcomes of PMT for the entire family is new and promising. Objective: We aimed to examine the efficacy of work place PMT on job and marital satisfaction among staff members of an academic center. Edited by: Meichun Mohler-Kuo, University of Zurich, Switzerland Reviewed by: Costin Roventa, Hospital Prof. Dr. Al. Obregia, Romania Jude Uzoma Ohaeri, University of Nigeria, Nigeria Correspondence: Ramin Alavinezhad raminalavinezhad@yahoo.com Specialty section: This article was submitted to Public Mental Health, a section of the journal Frontiers in Sociology Received: 28 July 2017 Accepted: 06 November 2017 Published: 23 November 2017 Citation: Mousavi M, Alavinezhad R and Moosavi A (2017) Effects of Workplace Parent Management Training on Marital and Job Satisfaction among Iranian Working Parents. Front. Sociol. 2:19. doi: 10.3389/fsoc.2017.00019 Frontiers in Sociology \| www.frontiersin.org Methods: We held 8 PMT sessions (1.5 h each) for 20 staff members who were parents to children in the age range of 2–12 years. Dyadic Adjustment Scale (DAS) and Occupational Descriptive Index [Health and Safety Executive (HSE)] were used for baseline and post-intervention data gathering. DAS higher scores indicate higher marital adjustment satisfaction and higher HSE scores indicate higher occupational stress. To analyze changes in HSE and DAS scores over time, paired t-test and Wilcoxon signed rank test were used, respectively. Results: All DAS subscales show significant increased from baseline to the final session except for affectional expression which was not significant. We found no significant changes in total or subscale HSE scores among participants. Conclusion: Findings of this study underscore the role of psycho-education usage in work environment and provide evidence about the importance of designing interventions concerning working parents. Implications of PMT are discussed in the text. Keywords: parent management training, marital satisfaction, job satisfaction, Iranian working parents, PMT INTRODUCTION Training parents is known to be an effective way to change the behavior of their children, one known approach being “parent management training” or PMT (Westrupp et al., 2016). The time children usually spend with their parents and the relationship they have with each other is not comparable to anyone else. Hence, targeting parents in training sessions is considered as a main part of treatment provided for children’s behavioral problems, in specific “externalizing” behavior (Darcy and McCarthy, 2007; Lim and Kim, 2014). This is essentially indirect which means the trainer changes the behavior of the parent which in turn affects that of the child (Mauno et al., 2012). The most common 1 November 2017 \| Volume 2 \| Article 19 Mousavi et al. Effects of Workplace PMT disorder in children that is widely treated with this approach is oppositional defiant disorder and evidence also supports the effectiveness of training on parents whose children suffer from ADHD (Jacobs and Winslow, 2004; Darcy and McCarthy, 2007). There are many studies, however, that show the second hand effects of this procedure on the mental health of the parents and their general well-being. In one study, for instance, researchers found it helpful in treating depression among mothers of referred children (Mauno et al., 2012). PMT is considered to be effective even when conducted remotely and it is approximately as useful as in person (Vieira et al., 2016). In developed countries, most parents handle two different roles at the same time; the role of looking after their children while also pursuing duties of their jobs. There are fluid boundaries between work and family spheres which is discussed in different schools. Work–family enrichment is positively associated with their involvement in parental role while negative experiences in parenting would indeed influence one’s work negatively. Relationship frustration in the parent–child interactions affect perceived quality of attachment in the parent–child relationship and parenting sense of competence (Vieira et al., 2016). Several studies have investigated the positive and negative impact of work and family on each other. One study suggests that work has negative effects on family since they often compete for limited energy, temporal, and psychological resources (Lim and Kim, 2014). Another study was done for teachers and it was effective in reducing both types of work–family conflict, occupational stress, as well as increasing parental and work-related efficacy and parenting satisfaction (Bögels et al., 2014). Family systems theory postulates that there is a reciprocal association between functioning in the parental and spousal subsystems. There are many studies supporting this association. In one study, parenting stress has been found to negatively predict marital satisfaction for both fathers and mothers, while marital satisfaction was found to affect individual mental health, family functioning, and child development. In one research, it has been affirmed that parenting stress is a significant negative predictor of marital satisfaction, while parenting self-efficacy and co-parenting alliance are significant positive predictors of marital satisfaction and this applies to both fathers and mothers (Kwan et al., 2015). It has also been affirmed that co-parenting alliance, satisfaction with father involvement, and parenting self-efficacy were significantly and positively correlated with mothers’ marital satisfaction (Kwok et al., 2015). In this paper, we try to show the effects of PMT on two aspects of parents’ lives: the satisfaction of parents as a couple and their contentment in workplace. This indirect intervention has not been investigated systematically in Persian population, so we hypothesized that by enhancing child behavioral issues based on PMT, marital satisfaction will be increased and job stress will be reduced. The intervention was conducted as an indispensable educational course and was provided by a child and adolescent psychiatrist affiliated with the department of psychology in the Shahid Beheshti university. To meet the inclusion criteria, 20 parents of normal children in the age range of 2–12 years who were living together with the other parent and interested to attend PMT sessions participated in the study. Parents were recruited during a 1-day call in October 2015. Men and women were in the same group and sessions were held from October to December 2015. We invited parents for a baseline session to explain the goals of the study and set dates for an intake interview (session 1), also participant’s rights to privacy considered and written informed consent obtained after careful explanation of the study aims in the baseline introduction session. Our study was in accordance with the ethical standards and Shahid Beheshti ethic committee reviewed and approved this study. Eight sessions (1.5 h each) were designed to meet the purposes of the study. More than three absences from the sessions consider as exclusion criteria in our study. At the end of intervention, just 14 participants found with less than 3 absences. The psychotherapist start most sessions by teaching the group then the topic and participants possible questions discuss and the sessions come to the end by conclusions. It was a closed group and all sessions were held in a conference room located in the department of psychology and details on the provided content in each session are indicated in Table 1. Measures We used a two-part questionnaire to measure marital satisfaction and occupational stress; surveys were provided to participants both at baseline (pre-test) and in the last session (post-test). To measure marital adjustment satisfaction, we utilized the Persian version of Dyadic Adjustment Scale (DAS) (Spanier, 1976; South et al., 2009); that is a widely used 32-item instrument measuring four areas of relationship quality and adjustment: (1) dyadic satisfaction that measures the degree to which respondent feels satisfied with partner, (2) dyadic cohesion; TABLE 1 \| Brief report of parent management training sessions. 1 Intake interview (to gather comprehensive information about child’s presenting behavioral concerns); baseline questionnaire; training on completion of monitoring forms to track a specific child behavior in the following week 2 Review of session #1; check-up of first week monitoring forms; training on the concept of “empathy” and role playing 3 Review of previous sessions; check-up of monitoring forms; introduction of the concept of “encouragement” and the use of “star tables” 4 Review of previous sessions; check-up of monitoring forms; training on “how to manage misbehavior?” 5 Review of previous sessions; check-up of monitoring forms; discussion of the concept of “ordering and how to correct it?” MATERIALS AND METHODS 6 Review of previous sessions; check-up of monitoring forms; discussion on “how to manage our children in specific situations?” Setting and Participants 7 Review of previous sessions; check-up of monitoring forms; discussion on “children and their school assignments” This study was conducted among 20 parents selected from the staff members of the Shahid Beheshti University (main office). Frontiers in Sociology \| www.frontiersin.org 8 Review of all sessions; check-up of monitoring forms; helping parents to stay on their new disciplines; completion of post-test questionnaires 2 November 2017 \| Volume 2 \| Article 19 Mousavi et al. Effects of Workplace PMT RESULTS the degree of closeness and shared activities experienced by the couple, (3) dyadic consensus; the degree to which couples agree on matters of importance to their relationship, and (4) affectional expression; the degree to which respondent agrees with partner regarding emotional affection. Previous surveys have shown alpha coefficients ranging from 0.70 (for the 4-item Affectional Expression subscale) to 0.95 (for the 32-item total score; Median = 0.87). Stability coefficients ranged from 0.75 (Affectional Expression) to 0.87 (Total; Median = 0.81). Traditionally, 102 is the cutoff point for DAS score to diagnose distressed couples (Spanier and Thompson, 1982). And higher scores indicate higher marital adjustment satisfaction. A previous study (Hassan shahi, 1999) indicated that this questionnaire has an appropriate internal consistency and an adequate validity for the assessment of marital adjustment satisfaction in Persian sample. To measure occupational stress, we utilized the Health and Safety Executive (HSE) stress questionnaire that is designed under the supervision of the UK Department for Work Safety (Cousins et al., 2004). This questionnaire is a valid and reliable questionnaire for studying job stress that has been already standardized among Iranian population with a Cronbach’s Alpha ranging from 65 to 75% (Marzabady and Fesharaki, 2011). This questionnaire consists of 35 items and include 7 subscale: “demands,” “control,” “relationships,” “role,” “change,” “managers’ support,” and “peer support.” Higher scores in HSE questionnaire indicate higher occupational stress. General Characteristics From 20 staff members who attended the course, 6 completed less than 3 sessions and were omitted from the list. At the end of last session in post-test, 14 participants filled the questionnaires. Participants consisted of 12 (12/14) women and 2 (2/14) men; with the mean age of 36.14 years (SD = 4.38) and mean work experience of 11.84 years (SD = 3.41). This male to female ratio was consistent with gender distribution among the office staff. All participants had university education: 1/14 with associate degree, 10/14 with bachelor’s and 3/14 with master’s degree. Correlations Bivariate correlation analysis indicated that the two main variables of research (DAS and HSE mean scores) had no significant correlations (r = 0.012, p > 0.45). Correlations between two main variable of research and their subscales are shown in Table 2. Tests of Normality Based on Shapiro–Wilk test HSE scores showed normal distribution (Sig. = 0.2) but DAS scores did not have a normal distribution (Sig. < 0.041). DAS Scores Mean baseline DAS score was reported to be 102.64 (SD = 13.94). At baseline, 35.7% of participants gained scores lower than the cutoff point but in post-test this percentage decreased to 21.4%. Changes in DAS total and subscale scores measured by Wilcoxon signed rank test are shown in Table 3. Unlike the rest of subscales, there was no significant increase from baseline to final session in affectional expression (Z = −0.583, p > 0.05) and dyadic Consensus scores (Z = −1.43, p > 0.05). Data Analysis We analyzed data with Statistical Package for Social Sciences version 22.0 (SPSS vs.22.0, IBM Inc.). Continuous variables were shown in terms of mean (SD) and frequency (number and percentage). To check for possible correlations, we utilized bivariate correlation analysis to compare DAS and HSE scores. To analyze changes in HSE and DAS scores over time, paired t-test and Wilcoxon signed rank test were, respectively, used. Shapiro–Wilk test was used to check for normality of data. Level of significance was considered to be at 0.05. HSE Scores Mean HSE score at baseline was reported to be 119.71 (SD = 16.1). Changes in total and subscale HSE scores are also shown in TABLE 2 \| Correlations between two main variable of research and their subscales. 1 1. Demands 2. Control 3. Peer support 4. Manager support 5. Relationships 6. Role 7. Change 8. HSE 9. Consensus 10. Satisfaction 11. Affection Ex. 12. Cohesion 13. DAS 1 0.264 0.343 0.622 0.646 0.414 0.512 0.816 −0.267 −0.002 −0.183 0.492 0.004 2 3 1 0.221 0.445 0.441 −0.143 0.524 0.555* −0.053 −0.034 −0.113 −0.121 −0.804 1 0.578* 0.461 −0.151 0.347 0.607* −0.119 −0.021 −0.066 0.263 0.006 4 1 0.787 0.205 0.685 0.902 0.055 0.008 −0.044 0.398 0.125 5 6 1 0.076 0.599 0.832* 0.116 0.323 0.058 0.513 0.340 1 0.499 0.327 −0.213 −0.135 −0.360 0.118 −0.164 7 1 0.796** −0.471 −0.164 −0.472 0.090 −0.297 8 1 −0.174 0.001 −0.204 0.396 0.012 9 1 0.538* 0.722 0.308 0.812 10 1 0.802 0.335 0.874 11 1 0.377 0.890 12 13 1 0.594 1 p < 0.05. *p < 0.01. DAS, Dyadic Adjustment Scale; HSE, Health and Safety Executive. Frontiers in Sociology \| www.frontiersin.org 3 November 2017 \| Volume 2 \| Article 19 Mousavi et al. Effects of Workplace PMT we should mention that two subscales scores decreased from baseline to final sessions; this change may be attributed to intervention. Despite the large number of controlled studies attesting the efficacy of PMT fundamental questions remain about the magnitude, scope, and durability of impact (Kazdin, 2006). Marital satisfaction as a main outcome measure to this study was compared to more investigate these unknown areas of effect. Baseline DAS scores were borderline hence participants (working parents) were considered as non-distressed couples; however, their marital situation was not appraised to be satisfactory, not only based on DAS scores but also the observation throughout the intervention. In line with our findings, it is noted that parenting is a huge stress in the modern society especially when the mom works (Kwon, 2011; Kim, 2015). Comparisons of baseline and post-intervention DAS scores showed significant improvement in the present study that is in line with previous findings (Bögels et al., 2014). All marital satisfaction variables including “consensus,” “satisfaction,” “cohesion,” and DAS total scores had improved; except for “affectional expression” where the increment was not statistically significant. Contrary to our findings, a previous study found significant improvement in affectional expression after a similar intervention for both distressed and non-distressed couples (Eisenberg et al., 2011). We propose cultural differences in expressing affection as a probable explanation. When compared to an emotionally focused couple therapy intervention, different content of the intervention is supposedly the main factor leading to a different outcome in affectional expression scores (Najafi et al., 2015). Although participants had been reassured about anonymity and confidentiality of their responses, we assume privacy may have been a concern to affect affectional expression scores. Our findings should be considered in light of few limitations. First, shifting the focus of PMT to parents and discussing implications for maximizing the outcomes of PMT for the entire family was almost new and our study conducted without significant previous literature in this specific area. Second, measuring occupational stress by self-report questionnaire in participants work environment may cause bias in responding (as found in this study). Third, randomize sampling was not feasible and the sample was very low not only because of small number of university staff but also cause of exclusion criteria. It was a primary step to target marital satisfaction by applying PMT. Further research may conduct by a larger sample and randomize sampling, so results can be generalized to a large group of parents in need for intervention. “This research is sponsored by Shahid Beheshti University, department of health and clinical psychology.” TABLE 3 \| Comparison of total and subscale DAS scores in baseline and final sessions. Mean (SD) Consensus Satisfaction Affection Ex. Cohesion DAS Pre Post Z p Value 45.2 (5.2) 36 (6.5) 8.7 (2.01) 12.7 (3.7) 102.6 (13.9) 48 (7.3) 41.2 (4.6) 9.2 (2.1) 16.07 (3.7) 114.5 (15.3) −1.43 −2.55 −0.58 −2.90 −2.44 0.151 0.011 0.560 0.004 0.014 Ex., expression; DAS, Dyadic Adjustment Scale. TABLE 4 \| Comparison of total and subscale HSE scores in baseline and final sessions. Mean (SD) Demands Control Peer support Manager support Relationships Role Change HSE Pre Post T p Value 27.35 (4.9) 17.1 (3.09) 12.78 (3.3) 15 (4.2) 15.71 (2.5) 21.85 (2.1) 9.28 (2.09) 119.71 (16.1) 26.57 (3.4) 19 (3.4) 13.07 (2.8) 16.71 (3.02) 16 (2.7) 21.35 (2.2) 10 (1.8) 122.71 (14.09) 0.698 −1.605 −0.404 −1.812 −0.404 0.721 −1.735 −0.790 0.497 0.133 0.693 0.093 0.693 0.484 0.106 0.443 HSE, Health and Safety Executive. Table 4. Although “Demands” and “Role” scores had decreased in the final session compared to baseline, none of the scores showed significant difference before and after intervention. DISCUSSION Rather than changes in child behavior following PMT, we focused on parents to investigate implications for maximizing treatment effect in the entire family in this study. PMT can be defined as an approach to treating child behavior problems using “procedures in which parents are trained to alter their child’s behavior in the home (McMahon and Wells, 1989).” Marital and job satisfaction were the main outcome variables used to study the intervention effects. The choice of the two variables was based on a study that indicated the interdependent nature of family and work; stating the fact that experiences in one area affect the quality of life in the other (Sarantakos, 1996). Of relevance, evidence suggests that PMT has been successful in improving marital satisfaction among non-distressed couples (Dadds et al., 1987). In our study, baseline DAS score was borderline; highlighting the appropriate choice of model of training. Previous studies in Iran had shown similar findings with regard to the non-significant correlations between marital satisfaction and job stress (Rahimi et al., 2004; Sori et al., 2006). However, a recent research among flight attendants found positive correlation between job satisfaction and dyadic adjustment (Zamanifar et al., 2016). We found no significant changes in total or subscale HSE scores in this study. We suggest that the bonus considered by the management may have affected baseline HSE scores. However, Frontiers in Sociology \| www.frontiersin.org AUTHOR CONTRIBUTIONS The main idea and intervention of this research carried on by MM, but RA did the statistical analyze and other works like article writing. FUNDING Shahid Beheshti University provided the publication cost. 4 November 2017 \| Volume 2 \| Article 19 Mousavi et al. Effects of Workplace PMT REFERENCES McMahon, R. J., and Wells, K. C. (1989). “Conduct disorders,” in Treatment of Childhood Disorders, eds E. J. 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Work-family culture and job satisfaction: does gender and parenting status alter the relationship? Community Work Fam. 15, 101–129. doi:10.1080/13668803.2011.598733 Frontiers in Sociology \| www.frontiersin.org 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. Copyright © 2017 Mousavi, Alavinezhad and Moosavi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 5 November 2017 \| Volume 2 \| Article 19
https://openalex.org/W2604183555	https://europepmc.org/articles/pmc5429689?pdf=render	English	null	Enhanced interstitial fluid drainage in the hippocampus of spontaneously hypertensive rats	Scientific reports	2,017	cc-by	7,256	Enhanced interstitial fluid drainage in the hippocampus of spontaneously hypertensive rats Received: 9 December 2016 Accepted: 15 March 2017 Published: xx xx xxxx Hypertension is associated with cognitive decline and various forms of dementia, including Alzheimer’s disease. In animal models of hypertension, many of Alzheimer’s disease characteristics are recapitulated, including brain atrophy, cognitive decline, amyloid β accumulation and blood brain barrier dysfunction. Removal of amyloid β and other waste products depends in part on clearance via the brain interstitial fluid (ISF). Here we studied the impact of hypertension on ISF drainage, using spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). At 8 months, high (500 kD) and low (3 kD) fluorescent molecular weight tracers released passively into the hippocampus showed a drastically enhanced spreading in SHR. Tracer spreading was inhomogeneous, with accumulation at ISF-CSF borders, around arteries, and towards the stratum lacunosum moleculare. These locations stained positively for the astrocyte marker GFAP, and aquaporin 4. Despite enhanced dispersion, clearance of tracers was not affected in SHR. In conclusion, these data indicate enhanced bulk flow of ISF in the hippocampus of hypertensive rats. ISF drains along astrocytes towards the cerebrospinal fluid compartment, which leads to sieving of high molecular weight solutes. Sieving may lead to a local increase in the concentration of waste products and potentially promotes the aggregation of amyloid β. Microvascular dysfunction, including impaired neurovascular coupling and blood brain barrier (BBB) disrup- tion, occurs in both vascular dementia and Alzheimer’s Disease1. These changes may interfere with the clear- ance of potentially toxic waste products such as amyloid β, which are released in the brain interstitial fluid (ISF) and accumulate in the parenchyma and vessel walls1. Hypertension may aggravate neurodegenerative diseases2. Indeed, mid-life hypertension is associated with an increased risk of dementia3, while antihypertensive drugs have been found to reduce the risk to develop AD4, 5. Induction of high blood pressure by transverse aortic coarctation promotes amyloid β accumulation in the brain of mice6. Similarly, work in mouse models of AD showed that hypertension exacerbates AD-like pathology7–11. Spontaneously hypertensive rats (SHR) develop white matter damage, cognitive decline (novel object recognition) and BBB disruption, among other patholog- ical changes12. Stroke-prone SHR show an age-dependent deposition of amyloid β13. Thus, there is a strong link between hypertension and dementia in both human and animal studies. However, the underlying mechanisms for this relation are not well understood. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 9 December 2016 Accepted: 15 March 2017 Published: xx xx xxxx Scientific Reports \| 7: 744 \| DOI:10.1038/s41598-017-00861-x Results Hypertension is associated with a different ionic composition of the brain. Spontaneously hyper- tensive rats were lighter than normotensive WKY rats (Table 1). Also brain weight was lower in SHR as compared to WKY. As expected, both systolic and diastolic blood pressure were higher in SHR, while also heart rate was significantly increased in SHR as compared to WKY. SHR brains showed a tendency for increased water content, as based on the wet and dry brain weights (Table 1). As edema and altered fluid homeostasis may be more sensi- tively detected by altered ion concentrations, we measured the concentration of both sodium and potassium. This revealed a significant decrease in potassium content in the SHR brain. As the sodium content showed a small, non-significant increase, a highly significant increase in Na+/K+ ratio was present in SHR brain. Infusion of tracers into the hippocampus. To study the impact of hypertension on the spreading and removal of solutes from the brain extracellular space, we infused a small quantity of fluorescently labelled dex- trans into the hippocampus. We infused 2 µl of a mixture of a high and low molecular weight dextran. The com- bination of a large and a small dextran was used as an indication for diffusion or bulk flow. A large difference in distribution would suggest diffusion, whereas a comparable distribution would suggest bulk flow. For neither tracer did we observe any difference between SHR and WKY (Fig. 1). After using this approach however, we speculated that tracer spreading may have been affected by the infusion itself. We therefore decided to investigate the fate of tracers in the absence of pumping any volume of fluid into the tissue. Passive release of tracer in the hippocampus. In the second set of experiments we changed our approach and studied tracer spreading after passive release from the needle tip. After stereotactic placement of the needle in the hippocampus, both tracers were allowed to enter the brain from the needle tip by diffusion only, followed by further spreading through the interstitium. Under these conditions, we observed a significant difference in the distribution area between SHR and WKY rats. Thus, both tracers distributed over a 5–6 fold larger area in SHR as compared to WKY (Fig. 2). Remarkably, the distribution area of the small and large tracer was comparable in size for both SHR and WKY. Enhanced interstitial fluid drainage in the hippocampus of spontaneously hypertensive rats Whole brain sodium concentration was not different, but the potassium concentration was significantly lower in SHR brain as compared to WKY. Data are mean ± SEM. WKY SHR t-test Weight Body (gr) (n = 20) 404 ± 5 (n = 19) 372 ± 5 p < 0.001 Brain (gr) (n = 20) 2.26 ± 0.01 (n = 19) 2.12 ± 0.03 p < 0.001 Blood Pressure Systolic (n = 20) 163 ± 4 (n = 19) 189 ± 4 p < 0.001 Diastolic (n = 20) 118 ± 5 (n = 19) 146 ± 4 p < 0.001 BPM (n = 20) 378 ± 8 (n = 19) 460 ± 5 p < 0.001 Water content % (n = 6) 77.155 ± 0.021 (n = 6) 77.612 ± 0.1 0.052 Ions Na+ (mmol/ml Water) (n = 6) 0.060 ± 0.001 (n = 6) 0.062 ± 0.001 0.117 K+ (mmol/ml Water) (n = 6) 0.111 ± 0.002 (n = 6) 0.106 ± 0.001 0.004 Na+ to K+ ratio (n = 6) 0.539 ± 0.012 (n = 6) 0.592 ± 0.011 0.008 Table 1. Animal characteristics and brain composition. SHR body weight and brain weight were lower as compared to WKY. Systolic and diastolic blood pressure, and heart rate were elevated in SHR. Water content in SHR brain tended to be higher as compared to WKY. Whole brain sodium concentration was not different, but the potassium concentration was significantly lower in SHR brain as compared to WKY. Data are mean ± SEM. Table 1. Animal characteristics and brain composition. SHR body weight and brain weight were lower as compared to WKY. Systolic and diastolic blood pressure, and heart rate were elevated in SHR. Water content in SHR brain tended to be higher as compared to WKY. Whole brain sodium concentration was not different, but the potassium concentration was significantly lower in SHR brain as compared to WKY. Data are mean ± SEM. Table 1. Animal characteristics and brain composition. SHR body weight and brain weight were lower as compared to WKY. Systolic and diastolic blood pressure, and heart rate were elevated in SHR. Water content in SHR brain tended to be higher as compared to WKY. Whole brain sodium concentration was not different, but the potassium concentration was significantly lower in SHR brain as compared to WKY. Data are mean ± SEM. Enhanced interstitial fluid drainage in the hippocampus of spontaneously hypertensive rats As the clearance of waste products, including amyloid β, from the brain parenchyma depends on transport via the interstitial fluid and subsequent removal via the BBB and paravascular pathways14–17, we hypothesized that hypertension may alter fluid dynamics in the brain interstitium. This, in turn, could interfere with the clearance of solutes from the brain. Thus, in the present study we set out to determine the effect of hypertension on distribution and clearance of solutes via the interstitial fluid. For this purpose, we stud- ied the fate of fluorescent tracers released into the ISF of the hippocampus of normotensive and hypertensive rats. 1Department of Biomedical Engineering and Physics, Academic Medical Center, Amsterdam, The Netherlands. 2Division of Nephrology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. 3Department of Anatomy and Neurosciences, Neuroscience Campus Amsterdam, VU medical center, Amsterdam, The Netherlands. 4Department of Neurology, Academic Medical Centre, Amsterdam, Netherlands. 5Department of Neurology, Radboud University Medical Centre, Nijmegen, Netherlands. Correspondence and requests for materials should be addressed to E.N.T.P.B. (email: n.t.bakker@amc.uva.nl) Scientific Reports \| 7: 744 \| DOI:10.1038/s41598-017-00861-x Scientific Reports \| 7: 744 \| DOI:10.1038/s41598-017-00861-x www.nature.com/scientificreports/ WKY SHR t-test Weight Body (gr) (n = 20) 404 ± 5 (n = 19) 372 ± 5 p < 0.001 Brain (gr) (n = 20) 2.26 ± 0.01 (n = 19) 2.12 ± 0.03 p < 0.001 Blood Pressure Systolic (n = 20) 163 ± 4 (n = 19) 189 ± 4 p < 0.001 Diastolic (n = 20) 118 ± 5 (n = 19) 146 ± 4 p < 0.001 BPM (n = 20) 378 ± 8 (n = 19) 460 ± 5 p < 0.001 Water content % (n = 6) 77.155 ± 0.021 (n = 6) 77.612 ± 0.1 0.052 Ions Na+ (mmol/ml Water) (n = 6) 0.060 ± 0.001 (n = 6) 0.062 ± 0.001 0.117 K+ (mmol/ml Water) (n = 6) 0.111 ± 0.002 (n = 6) 0.106 ± 0.001 0.004 Na+ to K+ ratio (n = 6) 0.539 ± 0.012 (n = 6) 0.592 ± 0.011 0.008 Table 1. Animal characteristics and brain composition. SHR body weight and brain weight were lower as compared to WKY. Systolic and diastolic blood pressure, and heart rate were elevated in SHR. Water content in SHR brain tended to be higher as compared to WKY. Discussion In this study we investigated the impact of hypertension on the distribution and clearance of tracers from the brains of rats. In the first set of experiments we infused tracers into the hippocampus, using a small volume and low infusion speed to reduce the impact of the infusion itself. This resulted in a non-homogeneous distribution of tracers, but no difference in the distribution area between WKY and SHR. We then followed a still more subtle approach, and allowed tracers to spread passively from the needle tip. To the best of our knowledge, this approach has not been reported before. Although the distribution area was much smaller in this case, this uncovered a large difference in distribution area in SHR as compared to WKY. We believe that the impact of infusion is an issue that has been underestimated in many studies. Infusion of 1 microliter into the brain introduces a volume of 1 mm3. With the extracellular space being roughly 20% of the total volume, this 1 microliter replaces the ISF of 5 mm3 of brain tissue (total volume of a mouse hippocampus ≈25 mm3, rat hippocampus ≈100 mm3). The endogenous production rate of ISF is unknown and difficult to determine18, but the results of the current study indicates that even for very limited infusion rates, endogenous transport mechanisms are easily overwhelmed.hl y g y The presence of bulk flow of ISF is debated19. In the current study, the low and high molecular weight tracers distributed over similar areas, which was the case in both rat strains after passive release. In SHR, there was also a strong accumulation of particularly the high molecular weight tracer in specific areas. These two features are a clear indication that solutes are dispersed via bulk flow of ISF rather than by diffusion in SHR. The larger distri- bution area in SHR as compared to WKY after passive release of tracers also suggests that ISF flow is markedly enhanced in the hippocampus of SHR. It should however be noted that other factors could also play a role in the difference in distribution area, such as differences in extracellular matrix composition, which could lead to stick- ing or retention of tracers, and differences in tortuosity of the extracellular space. A further limitation is that a direct quantitative comparison of distribution areas between the two tracers is not straightforward. First of all, the fluorophores have different optical properties. Results This finding suggests that after release from the needle tip, tracers spread by bulk flow of ISF rather than by diffusion. Clearance of tracers from the hippocampus. The large difference in distribution area between SHR and WKY after passive release of tracers may influence the clearance of these tracers from the brain. Therefore, in the next set of experiments we quantified the removal of tracers from the brain. We first infused equal amounts of tracers into the hippocampus of SHR and WKY, and then allowed a 30 min period of spreading and clearance. Brain homogenates were then analysed for remaining fluorescence by spectrophotometry. Clearance was much higher for the low versus high molecular weight tracer. However, clearance of either tracer was not significantly different between SHR and WKY rats (Fig. 3). Pattern of tracer distribution. Tracer spreading was highly inhomogeneous. Figure 4 shows an example of tracer spreading after passive release in the hippocampus. The high molecular weight tracer spread along the stratum lacunosum moleculare (SLM) and further in the caudal direction following blood vessels, suggesting a highly preferential route for fluid movement. Based on myosin staining, these vessels were identified as arteries. Whether the tracers followed the arteries upstream or downstream could not be determined. Scientific Reports \| 7: 744 \| DOI:10.1038/s41598-017-00861-x 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Distribution of fluorescent tracers after infusion into the hippocampus. Panel A and B show coronal slices of WKY (left) and SHR (right) hippocampus at the infusion level. We did not observe a difference in the distribution area of either the low or high molecular weight tracer between WKY (N = 6) and SHR (n = 7). Mean data ± SEM are shown in panel C. In panel D the expected infusion site () is shown, based on the stereotactic coordinates for the rat brain. Scale bar 1 mm. Figure 1. Distribution of fluorescent tracers after infusion into the hippocampus. Panel A and B show coronal slices of WKY (left) and SHR (right) hippocampus at the infusion level. We did not observe a difference in the distribution area of either the low or high molecular weight tracer between WKY (N = 6) and SHR (n = 7). Mean data ± SEM are shown in panel C. In panel D the expected infusion site () is shown, based on the stereotactic coordinates for the rat brain. Scale bar 1 mm. Results Colocalization of tracers with aquaporin 4 and GFAP. The spreading of tracers via the SLM region and around arteries was also seen after infusion of the tracers, suggesting a low resistance pathway of ISF flow. However, in the case of infusion, tracers spread further and also accumulated at ISF-CSF borders. As these areas are known to be rich in astrocytes and their endfeet, we investigated the possible colocalization of tracer with astrocytes and aquaporin 4 (Aqp4). Indeed, we observed clear staining for the astrocyte marker GFAP and Aqp4 in areas of tracer accumulation (Fig. 5). Scientific Reports \| 7: 744 \| DOI:10.1038/s41598-017-00861-x Discussion Second, the distribution area is determined not only by spreading but also by simultaneous clearance. 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Distribution of tracers after passive release into the hippocampus. Panel A and B show representative coronal sections of WKY (left) and SHR (right) hippocampus at the level of needle insertion. Inserts show the overview of the whole sections. Panel C shows the quantification of the distribution area. Data are mean ± SEM. Indicates P ≤ 0.001. WKY = 8 SHR = 6. Scale bar 1 mm. Figure 2. Distribution of tracers after passive release into the hippocampus. Panel A and B show representative coronal sections of WKY (left) and SHR (right) hippocampus at the level of needle insertion. Inserts show the overview of the whole sections. Panel C shows the quantification of the distribution area. Data are mean ± SEM. Indicates P ≤ 0.001. WKY = 8 SHR = 6. Scale bar 1 mm. Figure 3. Clearance of tracers from the hippocampus. Clearance was calculated as the amount of tracer removed from the brain as percentage of the initially injected quantity. There was no difference in clearance for both the low and high molecular weight tracer in WKY as compared to SHR. Clearance was larger for the small tracer (3 kD) as compared to the large tracer (500 kD; P < 0.00001). WKY = 6 SHR = 6. Data are mean ± SEM. Figure 3. Clearance of tracers from the hippocampus. Clearance was calculated as the amount of tracer removed from the brain as percentage of the initially injected quantity. There was no difference in clearance for both the low and high molecular weight tracer in WKY as compared to SHR. Clearance was larger for the small tracer (3 kD) as compared to the large tracer (500 kD; P < 0.00001). WKY = 6 SHR = 6. Data are mean ± SEM. While the distribution area was different in size between infusion and passive release, the pattern was similar. In both cases, tracers spread and accumulated at the stratum lacunosum moleculare (SLM), around arteries, into the corpus callosum, and at the edges of the hippocampus including the border with the third ventricle. This was the case for both WKY and SHR, albeit this became only apparent after infusion in the WKY, since there was limited tracer spreading after passive release in this group. Discussion Arteries are invested with at least one, and perhaps two peri- or paravascular transport pathways15–17, 21. Our data indicate that in the present study, part of the tracers exited the hippocampus along arteries. This finding is consistent with data from Carare et al.16, and at variance with the view proposed by Iliff et al.17. We previously showed that these pathways around arteries connect to the CSF compartment, either at the subarachnoid space or via one of the cisterns that penetrate the brain22. Also in this study a similar anatomy was found, with most of the feeding arteries entering the parenchyma from the cisterns around the hippocampus. Thus, ISF appears to drain from the hippocampus into the CSF either directly via the 3rd ventricle, or indirectly via cisterns and paravascular spaces around arteries.hll y y The apparent increase in bulk flow in the SHR hippocampus raises the question what the origin of this fluid is. An obvious source for enhanced ISF flux would be leakage or secretion from the capillaries. Indeed, others have reported BBB leakage and ventricular enlargement in SHR12, 23. In agreement with this, we found a tendency for increased water content. This was accompanied by a shift in whole brain Na+/K+ ratio. Similar changes are seen in stroke, where an increase in brain tissue sodium concentration and a concomitant decrease in brain potassium concentration are associated with vasogenic edema24. Due to large concentration differences, whole brain sodium is mainly reflecting the extracellular sodium pool, whereas potassium is dominated by the intracellular pool. As we anticipate that the SHR maintains normal intra- and extracellular ion concentrations, these data point towards an expansion of the extracellular space in the brains of SHR. Although speculative, such an expansion of the extracellular space could further facilitate ISF flow due to a reduction in resistance. Taken together, the picture emerges that in SHR, BBB leakage and an increase in extracellular space are associated with enhanced flux of ISF from the capillaries into the CSF compartment.h p p We found that the small tracer was cleared to a larger extend than the large tracer. This was the case for both WKY and SHR. Tracers may have been eliminated across the BBB, via the ISF into the CSF, and possibly via the choroid plexus as noted previously15, 25. Discussion Immunostaining showed that these areas are rich in astrocytes, and strongly express aquaporin 4. Thus, it appears that ISF, containing tracers, travels from the paren- chyma towards these border zones where ISF mixes with CSF. While in general the ISF-CSF border is considered less tight than the BBB, barrier properties are apparently still significant and lead to accumulation of the tracers. Scientific Reports \| 7: 744 \| DOI:10.1038/s41598-017-00861-x 4 www.nature.com/scientificreports/ Figure 4. Tracer spreading within the hippocampus. Sequential sections of a SHR from the injection site (panel A; arrow indicates needle track) moving towards the caudal side of the brain (panels B–D). Tracer (500 kD) is shown in green. Panel B: 0.75 mm caudal from infusion area. Tracer spread along the SLM (arrows) and embedded arteries. Panel C: 1 mm from the infusion area, tracer co-localized with an artery. Panel D: 1.25 mm from the infusion area, stronger co-localization with the same artery. Dentate gyrus (DG); Stratum Lacunosum Molecolare (SLM); Paravascular Space (PVS). Stars indicate the same artery in the different panels. Blue: nuclear stain, magenta: smooth muscle myosin heavy chain, identifying the arterial nature of these vessels. Scale bar in A = 200 μm, B = 100 μm, C–D = 50 μm. Figure 4. Tracer spreading within the hippocampus. Sequential sections of a SHR from the injection site (panel A; arrow indicates needle track) moving towards the caudal side of the brain (panels B–D). Tracer (500 kD) is shown in green. Panel B: 0.75 mm caudal from infusion area. Tracer spread along the SLM (arrows) and embedded arteries. Panel C: 1 mm from the infusion area, tracer co-localized with an artery. Panel D: 1.25 mm from the infusion area, stronger co-localization with the same artery. Dentate gyrus (DG); Stratum Lacunosum Molecolare (SLM); Paravascular Space (PVS). Stars indicate the same artery in the different panels. Blue: nuclear stain, magenta: smooth muscle myosin heavy chain, identifying the arterial nature of these vessels. Scale bar in A = 200 μm, B = 100 μm, C–D = 50 μm. The presence of aquaporin 4 also suggests that facilitated water transport is necessary in these regions. This capac- ity may be provided by astrocytes, which are known to mediate water transport, particularly via aquaporin 4 in their endfeet20. It is interesting to note that arteries, but not veins, accumulated tracers. Scientific Reports \| 7: 744 \| DOI:10.1038/s41598-017-00861-x Discussion However, in view of the enhanced spreading of tracers in SHR, the absence of a difference in clearance between WKY and SHR for both tracers appears to be a contradictory finding. One possibility is that infusion of a relatively large volume of tracers overwhelms endogenous clearance mech- anisms, which obscures subtle differences in clearance rate between SHR and WKY. This would be a situation similar to the tracer spreading results in this study. Alternatively, enhanced ISF flow may not necessarily lead to enhanced solute removal. In that case, we speculate that enhanced ISF flow relates to an increase in water and ion fluxes, but not necessarily leads to more washout of larger molecules. These molecules may be retained within the tissue at barrier sites. Indeed, we observed strong accumulation of the tracers, particularly of the high molecular weight tracer, at the borders of ISF and CSF exchange. We therefore interpret these findings as a sieving effect, resulting from relatively easy passage of water and ions, and accumulation of tracer. Whether such a mechanism is relevant for accumulation of endogenous waste products remains to be established. It is however very tempting to speculate on such a phenomenon, as tracer accumulation as observed in our experiments mirrors the pattern of amyloid β accumulation in the hippocampus of a mouse model of Alzheimer’s disease26.ll y β pp p Studies on human brain interstitial fluid flux and hypertension are scarce. Enlarged perivascular spaces are considered a marker for impaired ISF drainage27 and correlate with vascular amyloid β deposition28. White mat- ter hyperintensities and enlarged perivascular spaces are also associated with dementia and may be explained by impaired drainage of ISF29. Another study using MRI suggested that systolic hypertension is associated with an increase in extracellular fluid30. Thus, there is only circumstantial evidence that ISF flow is altered in dementia and potentially aggravated by hypertension. Currently, treatment options therefore seem premature and await future studies that shed more light on ISF production, pathways, and impact of hypertension on clearance of waste products from the brain. Scientific Reports \| 7: 744 \| DOI:10.1038/s41598-017-00861-x 5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ ntificreports/ Figure 5. Co-localization of tracer accumulation with GFAP and aquaporin 4 staining. Panel A: High molecular weight tracer (green) distribution after infusion in the hippocampus of a WKY. Tracer accumulates along the stratum lacunosum molecolare (SLM) and at the boundaries of the hippocampus. Materials and Methods Animals. For this study 39 animals were used. Male normotensive Wistar Kyoto rats (WKY/NCrl) (n = 20) and male spontaneously hypertensive rats (SHR/NCrl) (n = 19) were obtained from Charles River at the age of 11 weeks. Animals were kept until 8 months of age, housed in groups and fed ad libitum with standard laboratory food and free access to water. Experiments were done during daytime, which is the sleep phase for rats. One ani- mal died during anesthesia, before the start of the experiment. All experimental protocols were approved by the Committee for Animal Experiments of the Academic Medical Center Amsterdam, and in accordance with the European Communities’ Council Directive 2010/63/EU. Blood pressure measurements. Blood pressure (BP) was measured non-invasively with a tail-cuff system (Kent Scientific, Torrington, Connecticut, USA). Rats were accustomed to handling and to the restrainer during a training period of 4 days before the BP measurements. During the measurements, the animals were placed in the restrainer on a heating pad, for at least 10 minutes to warm up the tail. In each animal, 4 to 10 BP measurements were made and averaged. In a subset of animals we also measured BP under KDA (see below) anaesthesia. This revealed no difference in BP between anaesthetized and awake animals (data not shown). Anaesthesia. In initial experiments, where tracers were infused into the brain by pumping, rats were anaes- thetized by intraperitoneal injection of 2 ml/kg of KDA mix, consisting of a combination of ketamine (75 mg/ kg, Nimatek, Eurovet), dexdomitor (0.5 mg/kg, Orion Pharma), and atropine (0.05 mg/kg, atropine sulphate, Eurovet) dissolved in PBS (Phosphate Buffered Saline, Lonza). Additional oxygen (99%) was administered via a nose cap to prevent hypoxia. As we experienced a more stable and adjustable level of anaesthesia with isoflurane, the remainder of the study was carried out with animals under anaesthesia with isoflurane (3% in O2) applied via an inhalation mask. General surgical procedure. The animals were anaesthetized and the scalp was shaved. Subsequently, heads were immobilized in a stereotactic frame (Stoelting) and the core body temperature was maintained using a heating pad. Ocular lubricant ointment (Duratears®, Alcon) was applied to keep the eyes hydrated. The temper- ature was monitored with a rectal thermometer (Greisinger Electronics) during the procedure. A small longitu- dinal skin incision was made on the skull and 10% xylocaine (AstraZeneca B.V.) was provided as additional local anaesthesia. Materials and Methods The periosteum was scratched off using a scalpel. Subsequently, according to the Paxinos and Watson Rat Brain atlas (6th edition, 2007), we defined the stereotactic coordinates for the CA1 region of the hippocampus as −4.0 mm caudal, 2.0 mm lateral, and 4.0 mm deep, from the bregma point. The depth was calculated taking into account the thickness of the skull. A small burr hole was drilled with a dental drill (W&H). Subsequently, a 33-gauge needle (Hamilton) connected to a polyethylene catheter was inserted into the hippocampus using the stereotactic device. The catheter was connected to a syringe filled with a mixture of green (500 kDa) and red (3 kDa) fluorescent dextran. Both tracers were used at a final concentration of 10 mg/mL. Infusion of tracers into the hippocampus. In this set of experiments, rats were anaesthetized with KDA mix. We infused 2 μl of the dextran mixture at a controlled flow rate of 0.066 μl/min using a syringe pump (Harvard Apparatus, Holliston, MA, USA), over a 30 minute period. After infusion, the syringe pump was stopped and the needle was removed 1 minute after the end of the infusion period. Subsequently, the animals were euthanized with an overdose of the anaesthetic and rapidly decapitated, after which the brains were carefully dissected. The brains were weighed and cut into three coronal blocks using an adult rat brain slicer matrix (Zinc instruments). The blocks were separately embedded in Tissue-Tek® (Sakura), snap frozen in liquid nitrogen, and stored at −80 °C. The brains were then cut in coronal sections, 5 µm thick, using a cryostat (Microm HM 560) and collected on SuperFrost slides (Menzel-Gläzer). After sectioning, sections were stored at −80 °C until they were used for immunohistochemistry. Passive tracer release in the hippocampus. In the remainder of the study, animals were anesthetized with isoflurane. For this set of experiments we inserted the needle via the burr hole and we let the tracers diffuse from the tip of the needle into the rat hippocampus for 30 minutes. At the end of the diffusion period the animals were euthanized and the brain processed as described for the infusion set of experiments. Confocal imaging. Prior to imaging brain slices were fixated in 3.7% paraformaldehyde (PFA) for 30 min- utes. Then, cell nuclei were stained with bisbenzimide (Sigma). Selected slices were additionally stained to iden- tify specific cell types and proteins. Discussion Panel B: Expression of the astrocyte marker GFAP. Panel C: Expression of aquaporin 4. Tracer, GFAP and Aqp4 colocalize at the suprapyramidal and infrapyramidal blades of the dentate gyrus and the ependyma layer at the third ventricle. DG: dentate gyrus. 3 V: third ventricle. Scale bar in A–C 400 µm. Panels D and E show a close up of the vessels present at the SLM region. Both GFAP and Aqp4 are expressed around vessels. Scale bar in D = 15 μm, E = 50 μm. Figure 5. Co-localization of tracer accumulation with GFAP and aquaporin 4 staining. Panel A: High molecular weight tracer (green) distribution after infusion in the hippocampus of a WKY. Tracer accumulates along the stratum lacunosum molecolare (SLM) and at the boundaries of the hippocampus. Panel B: Expression of the astrocyte marker GFAP. Panel C: Expression of aquaporin 4. Tracer, GFAP and Aqp4 colocalize at the suprapyramidal and infrapyramidal blades of the dentate gyrus and the ependyma layer at the third ventricle. DG: dentate gyrus. 3 V: third ventricle. Scale bar in A–C 400 µm. Panels D and E show a close up of the vessels present at the SLM region. Both GFAP and Aqp4 are expressed around vessels. Scale bar in D = 15 μm, E = 50 μm. Figure 5. Co-localization of tracer accumulation with GFAP and aquaporin 4 staining. Panel A: High molecular weight tracer (green) distribution after infusion in the hippocampus of a WKY. Tracer accumulates along the stratum lacunosum molecolare (SLM) and at the boundaries of the hippocampus. Panel B: Expression of the astrocyte marker GFAP. Panel C: Expression of aquaporin 4. Tracer, GFAP and Aqp4 colocalize at the suprapyramidal and infrapyramidal blades of the dentate gyrus and the ependyma layer at the third ventricle. DG: dentate gyrus. 3 V: third ventricle. Scale bar in A–C 400 µm. Panels D and E show a close up of the vessels present at the SLM region. Both GFAP and Aqp4 are expressed around vessels. Scale bar in D = 15 μm, E = 50 μm. 6 Scientific Reports \| 7: 744 \| DOI:10.1038/s41598-017-00861-x www.nature.com/scientificreports/ To conclude, we showed that ISF flow is greatly enhanced in the hippocampus of SHR rats as compared to normotensive WKY. In these animals, tracer distribution is consistent with bulk flow, rather than diffusion. Discussion This is based on the notion that high and low molecular weight tracers spread similarly, and strongly accumulate at border zones of ISF-CSF exchange. The retention of tracers in these areas can be explained by a sieving effect on ISF, which could be relevant for the accumulation of endogenous solutes and waste products such as amyloid β. Materials and Methods To discriminate between arteries and veins we stained for myosin heavy chain. Antibodies against GFAP and aquaporin 4 (Aqp4) were used to stain astrocytes and water channels. Fluorescent images were acquired using a confocal laser-scanning microscope (Leica TCS SP8), with a 20x objective for details and 10x objective for overviews. ImageJ software was used to quantify the distribution area of the tracers. Care was taken to prevent pixel saturation and to apply the same confocal settings to sections within each group of experiments. Clearance of tracer from the hippocampus. To quantify the clearance we first infused a known vol- ume (2 μl) of the dextran mixture at 0.066 μl/min for 30 min. Then, we allowed a subsequent 30 min period for Scientific Reports \| 7: 744 \| DOI:10.1038/s41598-017-00861-x 7 www.nature.com/scientificreports/ spreading and clearance of the tracers. We used active infusion of tracer in this set of experiments, because meas- uring clearance requires a similar and known quantity of tracer to start with in WKY and SHR. Animals were then euthanized rapidly and brains were carefully dissected and weighed. Brains were subsequently homogenized in 7 ml of RIPA buffer, using a manual potter and an automated blender to obtain a homogenous suspension. Fluorescence spectrometry was then performed and the total amount of both dyes in the brain homogenate was determined from integration of the spectra over the appropriate wavelengths. In order to normalize for possible differences in turbidity in the different samples, the spectrometry was repeated after addition of a known quantity of the tracers to the samples. Brain ashing procedure. We used snap frozen brain samples to analyze water, potassium, and sodium con- tent. To determine brain water content, we compared brain sample weights before and after desiccation at 90 °C for 48 hours. Next, samples were dry ashed for 40 hours at 450 °C. After ashing, all samples were dissolved in 5% HNO3. Sodium and potassium concentrations were determined by flame photometry. Reagents. Dextran, Texas Red-labelled (3 kD, Ex. 595 nm/Em. 615 nm) and dextran, fluorescein-labelled (500 kD, Ex. 494 nm/Em. 521), both lysine-fixable, were purchased from Molecular Probes-Life Technologies (Eugene, OR, USA). These dyes were dissolved in artificial cerebrospinal fluid (aCSF). In the passive release experiments we increased the final concentration of FITC dextran to 50 mg/ml. For immunohistochemistry we used antibodies against: smooth muscle myosin heavy chain 11 antibody (Abcam), Aqp4 (Millipore) and GFAP (Dako Cytomation, Glostrup). Materials and Methods Cell nuclei were stained with bisbenzimide (Sigma). Slices were mounted in fluo- rescent mounting medium (Dako). RIPA buffer consisted of 150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, in 50 mM Tris, pH 8.0. Statistics. Data are expressed as mean ± SEM. 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Nat Rev Neurosci 14, 265–277, doi:10.1038 nrn3468 (2013).i 21. Bakker, E. N. et al. Lymphatic Clearance of the Brain: Perivascular, Paravascular and Significance for Neurodegenerative Diseases. Cell Mol Neurobiol 36, 181–194, doi:10.1007/s10571-015-0273-8 (2016). 22. Bedussi, B. et al. Paravascular channels, cisterns, and the subarachnoid space in the rat brain: A single compartment with preferential pathways. J Cereb Blood Flow Metab (2016). Scientific Reports \| 7: 744 \| DOI:10.1038/s41598-017-00861-x 8 www.nature.com/scientificreports/ 3. Ritter, S. & Dinh, T. T. Progressive postnatal dilation of brain ventricles in spontaneously hypertensive rats. Brain Res 370, 327–332 doi:10.1016/0006-8993(86)90488-9 (1986).t 23. Ritter, S. & Dinh, T. T. Additional Informationh Competing Interests: The authors declare that they have no competing interests. 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. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2017 Scientific Reports \| 7: 744 \| DOI:10.1038/s41598-017-00861-x 9
W2164257781.txt	https://www.mdpi.com/1420-3049/16/3/2626/pdf?version=1403113037	en		Synthesis and In Vitro Antibacterial Activity of 7-(3-Amino-6,7-dihydro-2-methyl-2H-pyrazolo[4,3-c] Pyridin-5(4H)-yl)fluoroquinolone Derivatives	Molecules/Molecules online/Molecules annual	2,011	cc-by	3,721	Molecules 2011, 16, 2626-2635; doi:10.3390/molecules16032626 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Synthesis and In Vitro Antibacterial Activity of 7-(3-Amino-6,7-dihydro-2-methyl-2H-pyrazolo[4,3-c] Pyridin-5(4H)-yl)fluoroquinolone Derivatives Xin Guo 1,2, Ming Liang Liu 1, Hui Yuan Guo 1, Yu Cheng Wang 1 and Ju Xian Wang 1,* 1 2 Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China; E-Mail:guoxincq@hotmail.com * Author to whom correspondence should be addressed; E-Mail: wyc9999@gmail.com; Tel.: +86-10-63165263; Fax: +86-10-63165263. Received: 31 December 2010; in revised form: 16 March 2011 / Accepted: 18 March 2011 / Published: 22 March 2011 Abstract: A series of novel 7-(3-amino-6,7-dihydro-2-methyl-2H-pyrazolo[4,3-c]pyridin5(4H)-yl)fluoroquinolone derivatives were designed, synthesized and characterized by 1 H-NMR, MS and HRMS. These fluoroquinolones were evaluated for their in vitro antibacterial activity against representative Gram-positive and Gram-negative strains. Results reveal that most of the target compounds exhibit good growth inhibitory potency against methicillin-resistant Staphylococcus epidermidis (MRSE) (MIC: 0.25–4 μg/mL) and Streptococcus pneumoniae (MIC: 0.25–1 μg/mL). In addition, compound 8f is 8–128 fold more potent than the reference drugs gemifloxacin (GM), moxifloxacin (MX), ciprofloxacin (CP) and levofloxacin (LV) against methicillin-resistant Staphylococcus aureus 10-05 and Streptococcus hemolyticus 1002 and 2–64 fold more active against methicillin-sensitive Staphylococcus aureus 10-03 and 10-04. Keywords: fluoroquinolone; antibacterial activity; synthesis 2627 Molecules 2011, 16 1. Introduction Since the discovery of nalidixic acid by Lesher et al. in 1962 [1], the quinolones have evolved into an important class of antibacterial agents used mainly for the treatment of respiratory tract infections (RTI), urinary tract infections (UTI), sexually transmitted diseases (STD), gastrointestinal and abdominal infections, skin and soft tissue infections, and infections of the bone and joints, among many other uses [2]. These compounds act by binding to the quinolone-resistance-determining region (QRDR) in the catalytic domain of the topoisomerase II (DNA gyrase) or IV complex with DNA. Cell death is induced by trapping the topoisomerase protein-DNA complex thus disrupting normal DNA replication, inducing oxidative damage, and triggering cell-death mechanisms. DNA gyrase appears to be the primary target for quinolones in Gram-negative bacteria such as Escherichia coli, while topoisomerase IV is the primary target in Gram-positive bacteria such as Staphylococcus aureus [3,4]. Despite the large number of fluoroquinolones approved for the treatment of bacterial infections, there have been unabated efforts for the discovery of new quinolones with specific improved properties and most importantly, to overcome the growing problem of bacterial resistance. Furthermore, some of the side effects of quinolone antibacterials are unacceptable, for example, grepafloxacin was withdrawn from market due to increased cases of heart problems in clinical findings [5]. Similarly, Trovafloxacin was removed from the market due to its liver toxicity [5]. In a search for potent fluoroquinolone derivatives, it has been found that although some were based on modifications on other positions, the most successful compounds developed were based on modifications at C-7, and it has been found that the spectrum and level of antibacterial activity is highly affected by the nature of the C-7 substituent group [6]. In general, 5- and 6-membered nitrogen heterocycles including piperazinyl, pyrrolidinyl and piperidinyl type side chains have been proven to be the optimal substituents [7]. As part of an ongoing program to find potent and broad-spectrum antibacterial agents that display strong Gram-positive activities, we also have focused on introducing new functional groups to the piperidine ring [8-12]. Interestingly, IMB (Figure 1), a new 8-methoxylfluoroquinolone incorporating a 3-amino-4-methoxyiminopiperidine at C-7 position, shows excellent in vitro and in vivo antibacterial activities [8]. Figure 1. New 8-methoxylfluoroquinolone IMB. O COOH F H2N N N O H3CON CH3 In this paper a series of novel fluoroquinolone compounds containing both the piperidine ring and the 3-aminopyrazole ring at the 7-position were designed and synthesized. The 3-aminopyrazole 2628 Molecules 2011, 16 function group at the 3-positions of some fourth generation cephalosporins, such as cefoselis, was fused with a piperidine to furnish the 4,5,6,7-tetrahydro-2-methyl-2H-pyrazolo[4,3-c] pyridin-3-amine scaffold which can be readily obtained from the corresponding 4-oxopiperidine-3-carbonitrile in a single operation. Our primary objective was to optimize the potency of these compounds against Gram-positive and Gram-negative organisms. 2. Results and Discussion 2.1. Synthesis of 7-(3-amino-6,7-dihydro-2-methyl-2H-pyrazolo[4,3-c]pyridin-5(4H)-yl)fluoroquinolone derivatives The synthesis of novel fluoroquinolone derivatives 8a-i is outlined in Scheme 1. Addition reaction of ethyl 3-aminopropanoate hydrochloride (1) with acrylonitrile in the presence of sodium hydroxide gave the secondary amine 2, which was subsequently treated with di-tert-butoxycarbonyl dicarbonate (Boc2O) to produce Boc-protected cyano ester 3. Scheme 1. Synthesis of novel fluoroquinolones 8a-i. 8a: R1=cyclopropyl, X=N, R2=H 8b: R1=2,4-difluorophenyl, X=N, R2=H 8c: R1=C2H5, X=CF, R2=H 8d: R1=CH2CH2F, X=CF, R2=H 8e: R1=cyclopropyl, X=CF, R2=H 8f: R1=cyclopropyl, X=COCHF2, R2=H 8g: R1=cyclopropyl, X=COCH3, R2=H 8h: R1=cyclopropyl, X=COCH3, R2= NH2 8i: R1=cyclopropyl, X= O R2=H Reagents and Conditions: (i) MeOH, NaOH, acrylonitrile, 65 °C, 3 h; (ii) Boc2O, 55–60 °C, 1 h; (iii)NaH, toluene, 110 °C, 1.5 h; (iv) 40% NH2NHCH3/H2O, C2H5OH, r.t., 12 h; (v) MeOH, dry hydrogen chloride gas, r.t., 0.5 h; (vi) a: CH3CN, Et3N, 25–60 °C, 1-48 h; b: 5% NaOH/H2O, 40 °C, 0.5–2 h; c: 2 mol/L HCl, r.t. 2629 Molecules 2011, 16 Compound 3 was cyclized to the cyanoketone 4 by sodium hydride in refluxing toluene (overall yield of 83% for the three steps). Compound 4 was treated with methylhydrazine in ethanol to give the Boc-protected bicyclic amines 5, which upon deprotection gave the key intermediates 6 after treatment with hydrogen chloride gas [13,14]. Finally, the target compounds 8a-i were obtained by coupling the intermediates 6 with various boric acid chelates 7 containing quinolone and naphthyridone cores, and then hydrolysis of the chelating groups according to well-established literature procedures [11,12,15]. 2.2. Antibacterial activity of 7-(3-amino-6,7-dihydro-2-methyl-2H-pyrazolo[4,3-c]pyridin-5(4H)-yl)fluoroquinolone derivatives The in vitro antibacterial activity of the novel fluoroquinolones 8a-i against representative Gram-negative and Gram-positive strains was evaluated using standard techniques [16]. Minimum inhibitory concentration (MIC) of the synthesized compounds (the concentration of the compound required to cause complete inhibition of bacterial growth), along with those of the standard drugs gemifloxacin (GM), moxifloxacin (MX), ciprofloxacin (CP) and levofloxacin (LV) are given in Table 1. Table 1. In-vitro antibacterial activity of compounds 8a-i. Strains S.a.1 S.a.2 S.a.3 S.a.4 S.a.5 S.a.6 S.a.7 S.a.8 S.a.9 S.a.10 S.e.1 S.e.2 S.e.3 S.e.4 S.e.5 S.e.6 S.e.7 S.e.8 S.e9 S.e.10 S.p.1 S.p.2 S.p.3 S.h.1 S.h.2 8a 8b 8c 8d 0.5 128 64 128 16 64 4 8 16 8 1 1 0.5 0.25 2 64 128 128 64 32 0.25 0.5 1 64 64 0.25 >128 4 128 2 0.25 128 64 128 64 4 0.25 2 0.5 1 16 >128 16 128 64 0.25 0.25 0.5 128 >128 0.25 >128 0.5 128 4 16 16 32 16 32 32 1 4 2 2 1 8 2 1 2 0.25 0.25 32 32 >128 0.5 >128 0.25 128 128 128 128 128 2 128 16 0.25 0.5 1 4 0.25 4 0.5 0.25 2 0.25 0.25 0.5 128 >128 Compd. MIC (μg/mL) a) 8e 8f 8g 8h 8i 0.25 128 128 >128 128 8 32 8 4 8 1 0.25 0.5 2 2 128 64 128 128 128 0.25 0.25 32 32 128 GM MX LV 0.25 1 2 0.25 0.008 0.03 16 64 128 128 32 8 0.5 1 64 32 0.5 1 16 32 64 16 2 0.125 0.5 2 128 128 4 8 0.5 32 64 16 16 0.125 4 32 64 32 8 4 0.5 8 16 8 1 4 0.25 4 8 2 2 8 4 8 64 2 0.5 4 0.25 1 32 0.25 0.03 0.06 0.5 0.25 0.25 2 0.008 0.06 0.5 0.5 0.5 0.5 0.06 0.125 2 2 1 2 0.125 0.25 4 4 2 1 8 4 16 64 32 128 1 4 4 128 128 128 0.25 8 4 32 16 128 0.5 4 16 64 64 128 0.25 0.5 2 16 16 128 0.125 0.25 0.25 0.25 0.25 0.25 0.008 0.008 0.25 1 0.25 0.25 0.008 0.03 0.5 0.5 0.5 0.5 0.03 0.03 64 32 128 16 0.03 0.125 0.5 32 128 128 8 4 0.125 32 8 0.125 32 0.125 4 8 8 8 0.06 0.125 0.5 1 8 4 8 4 2 4 0.008 0.008 0.015 0.125 32 CP 0.25 64 0.25 0.25 64 0.25 32 1 16 1 0.25 2 4 2 16 0.03 16 2 1 8 0.06 0.008 0.03 0.06 64 2630 Molecules 2011, 16 Table 1. Cont. E.c.1 E.c.2 E.c.3 E.c.4 K.p.1 K.p.2 K.p.3 P.a.1 P.a.2 P.a.3 1 1 128 128 >128 >128 >128 >128 >128 >128 4 8 128 128 >128 >128 >128 >128 >128 >128 1 1 32 128 >128 >128 >128 128 >128 >128 2 0.25 128 128 >128 >128 >128 128 >128 >128 0.25 0.25 2 64 0.25 0.008 0.008 0.008 0.008 0.25 0.5 2 64 0.25 0.06 0.5 0.5 0.25 128 64 128 128 128 2 16 32 32 32 64 128 128 128 8 4 2 8 64 128 >128 128 >128 32 16 16 32 128 128 >128 128 >128 4 4 8 16 128 128 >128 128 >128 4 4 4 8 128 128 >128 128 128 4 2 8 64 128 128 >128 128 >128 2 8 16 8 128 128 >128 128 >128 16 4 32 128 a) The values were reproduced in three experiments; b)Abbreviations: S.a.1, Staphylococcus aureus ATCC259223; S.a.2, methicillin-resistant Staphylococcus aureus 10-02; S.a.3, methicillin-resistant Staphylococcus aureus 10-03; S.a.4, methicillin-resistant Staphylococcus aureus 10-04; S.a.5, methicillin-resistant Staphylococcus aureus 10-05; S.a.6, methicillin-sensitive Staphylococcus aureus 10-01; S.a.7, methicillin-sensitive Staphylococcus aureus 10-02; S.a.8, methicillin-sensitive Staphylococcus aureus 10-03; S.a.9, methicillin-sensitive Staphylococcus aureus 10-04; S.a.10, methicillin-sensitive Staphylococcus aureus 10-05; S.e.1, methicillin-resistant Staphylococcus epidermidis 10-1; S.e.2, methicillin-resistant Staphylococcus epidermidis 10-2; S.e.3, methicillin-resistant Staphylococcus epidermidis 10-3; S.e.4, methicillin-resistant Staphylococcus epidermidis 10-4; S.e.5, methicillin-resistant Staphylococcus epidermidis 10-5; S.e.6, methicillin-sensitive Staphylococcus epidermidis 10-1; S.e.7, methicillin-sensitive Staphylococcus epidermidis 10-2; S.e.8, methicillin-sensitive Staphylococcus epidermidis 10-3; S.e.9, methicillin-sensitive Staphylococcus epidermidis 10-4; S.e.10, methicillin-sensitive Staphylococcus epidermidis 10-5; S.p.1, Streptococcus pneumoniae 1001; S.p.2, Streptococcus pneumoniae 1003; S.p.3, Streptococcus pneumoniae 1004; S.h.1, Streptococcus hemolyticus 1001; S.h.2, Streptococcus hemolyticus 1002; E.c.1, Escherichia coli ATCC 25922; E.c.2, Escherichia coli 10-02; E.c.3, Escherichia coli 10-03; E.c.4, Escherichia coli 10-12; K.p.1, Klebsiella pneumoniae 10-1; K.p.2, Klebsiella pneumoniae 10-2; K.p.3, Klebsiella pneumoniae 1025; P.a.1, Pseudomonas aeruginosa ATCC27853; P.a.2, Pseudomonas aeruginosa 10-2; P.a.3, Pseudomonas aeruginosa 10-3. The novel fluoroquinolones 8a-i display generally rather weak potency against the tested Gram-negative strains, but most of them exhibit good potency in inhibiting the growth of methicillin-resistant Staphylococcus epidermidis (MRSE) (MIC: 0.25–4 μg/mL) and Streptococcus pneumoniae (MIC: 0.25–1 μg/mL). In particular, the most active compound 8f is 8–128 fold more potent than the reference drugs MX, GM, CP and LV against methicillin-resistant Staphylococcus aureus 10-05 and Streptococcus hemolyticus 1002 and 2–64 fold more against methicillin-sensitive Staphylococcus aureus 10-03 and 10-04. 3. Experimental 3.1. General All chemical reagents and solvents used in this study were purchased from Beihua Fine Chemicals Company (Beijing, China). Melting points were determined by X-5 digital display binocular microscope for melting-point tests (Gongyi, China) and are uncorrected. 1H-NMR spectra were Molecules 2011, 16 2631 recorded on a Varian Mercury-400 spectrometer using tetramethylsilane as internal standard. Electron spray ionization (ESI) mass spectra and high resolution mass spectra (HRMS) were recorded on a MDSSCIEX Q-Tap mass spectrometer. Merck silica gel ART5554 60F254 plates were used for analytical TLC. Column chromatography was carried out on silica gel HG/T2354-92 made in Haiyang Chemical Company (Qingdao, China). tert-Butyl 3-cyano-4-oxopiperidine-1-carboxylate (4). A mixture of ethyl 3-aminopropanoate hydrochloride (1, 184.3 g, 1.2 mol), sodium hydroxide (48.0 g, 1.2 mol) and methanol (600 mL) was stirred for 0.5 h, and then acrylonitrile (79.5 g, 1.5 mol) was added dropwise over a period of 40 min at room temperature. The reaction mixture was heated to 65 °C and stirred for 3 h to give the secondary amine 2, which was pure enough to be used for the next step without further purification. To the reaction mixture containing the amine 2 was added (Boc)2O (218.0 g, 1.0 mol) at room temperature, and the mixture was stirred at 55–60 °C for 1 h and filtered. The filtrate was concentrated under reduced pressure. The residue was diluted with ethyl acetate (400 mL), washed with water and then saturated brine, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford the cyano ester 3 as a colorless oil. To a refluxing suspension of sodium hydride (70%, 41.1 g, 1.2 mol) in dry toluene (600 mL) a solution of the cyano ester 3 (270 g, 1.0 mol) dissolved in dry toluene (200 mL) was added dropwise over a period of 0.5 h. The reaction mixture was stirred for 1 h at the same temperature, cooled to room temperature and then water (400 mL) was added slowly. The aqueous layer was separated and adjusted to pH 7 with 10% acetic acid. The solid obtained was filtered, washed twice with water and dried in vacuo to give the title compound 4 (224.2 g, 83%) as a yellow solid, m.p.: 97 °C 99 °C. 1H-NMR (CDCl3) δ 1.47 (9H, s, Boc-9H), 2.34–2.36 (2H, m, C5-H), 3.56–3.59 (2H, m, C6-H), 4.16–4.18 (2H, m, C2-H), 6.82 (1H, s, C3-H); MS (ESI) m/z 223 (M-H)+. tert-Butyl 3-amino-4,5,6,7-dihydro-2-methyl-2H-pyrazolo[4,3-c]pyridine)-carboxylate (5). A solution of tert-butyl 3-cyano-4-oxopiperidine-1-carboxylate (4, 2.2 g, 10.0 mmol) and methylhydrazine (0.5 g, 11.0 mmol) dissolved in ethanol (50 mL) was stirred for 12 h at room temperature to give the Boc-protected bicyclic compound 5 (1.5 g, 60%) as a yellow solid, m.p.:163–165 °C. 1H-NMR (CDCl3) δ 1.47 (9H, s, Boc-9H), 2.67–2.70 (2H, m, C7-H), 3.65 (2H, br., C6-H), 3.71 (3H, s, N-CH3), 4.27 (2H, br., C4-H); MS (ESI) m/z 253 (M+H)+. 4,5,6,7-Tetrahydro-2-methyl-2H-pyrazolo[4,3-c]pyridin-3-amine hydrochloride (6). Dried hydrogen chloride gas was bubbled through a solution of compound 5 (1.5 g, 6.0 mmol) in methanol (20 mL) at room temperature for 0.5 h, and then stirred for another 0.5 h at the same temperature. The resulting solid was collected by suction, and dried in vacuo to give the title compound 6 (0.6 g, 53%) as a yellow solid, mp: 252–254 °C. 1H-NMR (DMSO-d6) δ 2.72–2.75 (m, 2H, C7-H), 3.30–3.32 (m, 2H, C6-H), 3.53 (s, 3H, N-CH3), 3.86–3.88 (m, 2H, C4-H)；MS (ESI) m/z 153 (M+H)+. General procedure for the synthesis of 7-(3-amino-6,7-dihydro-2-methyl-2H-pyrazolo-[4,3-c]pyridin-5(4H)-yl)fluoroquinolone derivatives 8a-i: A mixture of 7 (1.0 mmol), 6 (1.5 mmol), triethylamine (8.0 mmol) and dry acetonitrile (20 mL) was stirred at 25~60 °C under an atmosphere of Molecules 2011, 16 2632 nitrogen for 1~48 h. The resulting solid was collected by suction, and dried in vacuo to give the title compounds 8a-i. 7-(3-Amino-6,7-dihydro-2-methyl-2H-pyrazolo[4,3-c]pyridin-5(4H)-yl)-1-cyclopropyl-6-fluoro-4-oxo1,4-dihydro-1,8-naphthyridine-3-carboxylic acid (8a). White solid (0.29 g, 73%), m.p.: >300 °C. 1 H-NMR (DMSO-d6) δ 1.07–1.30 (m, 4H, 2CH2), 2.69–2.72 (m, 2H, C7’-H), 3.45 (s, 3H, N-CH3), 3.71–3.74 (m, 1H, CH), 4.02–4.05 (m, 2H, C6’-H), 4.68 (s, 2H, C4’-H), 8.06 (d, J = 13.6 Hz, 1H, C5-H), 8.82 (1H, s, C2-H); HRMS (ESI) m/z (M+H)+: calcd for C19H20FN6O3+: 399.15809; found: 399.15777. 7-(3-Amino-6,7-dihydro-2-methyl-2H-pyrazolo[4,3-c]pyridin-5(4H)-yl)-1-(2,4-difluorophenyl)-6-fluor o-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid (8b). Yellow solid (0.37 g, 78%), m.p.: >300 °C. 1H-NMR (DMSO-d6) δ 2.07–2.34 (m, 2H, C7’-H), 3.41 (s, 3H, N-CH3), 4.491–4.495 (m, 2H, C6’-H), 5.10 (s, 2H, C4’-H), 7.34–7.39 (m, 1H, Ph-H), 7.60–7.66 (m, 1H, Ph-H), 7.79–7.85 (m, 1H, Ph-H), 8.14 (d, J = 13.2 Hz, 1H, C5-H), 8.85 (1H, s, C2-H); HRMS (ESI) m/z (M+H)+: calcd for C22H18F3N6O3+: 471.13925; found: 471.13972. 7-(3-Amino-6,7-dihydro-2-methyl-2H-pyrazolo[4,3-c]pyridin-5(4H)-yl)-1-ethyl-6,8-difluoro-4-oxo-1,4 -dihydroquinoline-3-carboxylic acid (8c). White solid (0.21 g, 52%), m.p.: >300 °C. 1H-NMR (DMSO-d6) δ 1.45 (t, J = 6.8 Hz, 3H, CH2CH3), 2.63 (br., 2H, C7’-H), 3.47 (s, 3H, N-CH3), 4.21 (s, 2H, C6’-H), 4.59–4.60 (m, 2H, CH2CH3), 5.09 (s, 2H, C4’-H), 7.86 (d, J = 12.0 Hz, 1H, C5-H), 8.92 (s, 1H, C2-H); HRMS (ESI) m/z (M+H)+: calcd for C19H20F2N5O3+: 404.15342; found: 404.15339. 7-(3-Amino-6,7-dihydro-2-methyl-2H-pyrazolo[4,3-c]pyridin-5(4H)-yl)-1-(2-fluoroethyl-6,8-difluoro-4 -oxo-1,4-dihydroquinoline-3-carboxylic acid (8d). Yellow solid (0.27 g, 64%), m.p.: >300 °C. 1 H-NMR (DMSO-d6) δ 2.60–2.62 (m, 2H, C7’-H), 3.45 (s, 3H, N-CH3), 3.52 (br., 2H, C6’-H), 4.19 (s, 2H, C7’-H), 4.83–5.07 (m, 4H, CH2CH2F), 7.88 (d, J = 12.4 Hz, 1H, C5-H), 8.85 (1H, s, C2-H); HRMS (ESI) m/z (M+H)+: calcd for C19H19F3N5O3+: 422.14400; found: 422.14732. 7-(3-Amino-6,7-dihydro-2-methyl-2H-pyrazolo[4,3-c]pyridin-5(4H)-yl)-1-cyclopropyl-6,8-difluoro-4-o xo-1,4-dihydroquinoline-3-carboxylic acid (8e). The title compound was obtained as a yellow solid (0.23 g, 56%), m.p.: >300 °C. 1H-NMR (DMSO-d6) δ 1.18–1.33 (m, 4H, 2CH2), 2.88–2.91 (m, 2H, C7’-H), 3.62 (br., 2H, C6’-H), 3.68 (s, 3H, N-CH3), 4.00–4.01 (m, 1H, CH), 4.32 (s, 2H, C4’-H), 7.92 (d, J = 11.2 Hz, 1H, C5-H), 8.78 (s, 1H, C2-H); HRMS (ESI) m/z (M+H)+: calcd for C20H20F2N5O3+: 416.15342; found: 416.15416. 7-(3-Amino-6,7-dihydro-2-methyl-2H-pyrazolo[4,3-c]pyridin-5(4H)-yl)-1-cyclopropyl-6-fluoro-8-diflu oromethoxyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (8f). Yellow solid (0.31 g, 68%), m.p.: >300 °C. 1H-NMR (DMSO-d6) δ 1.02–1.19 (m, 4H, 2CH2), 2.61–2.64 (m, 2H, C7’-H), 3.46 (s, 3H, N-CH3), 3.50–3.51 (m, 1H, CH), 4.07–4.15 (m, 2H, C6’-H), 5.11 (s, 2H, C4’-H), 6.92 (t, J = 73.6 Hz, 1H, OCHF2), 7.91 (d, J = 12.4 Hz, 1H, C5-H), 8.76 (s, 1H, C2-H); HRMS (ESI) m/z (M+H)+: calcd for C21H21F3N5O4+: 464.15456; found: 464.15348. 7-(3-Amino-6,7-dihydro-2-methyl-2H-pyrazolo[4,3-c]pyridin-5(4H)-yl)-1-cyclopropyl-6-fluoro-8-meth oxyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (8g). Yellow solid (0.14 g, 32%), m.p.: >300 °C. Molecules 2011, 16 2633 1 H-NMR (DMSO-d6) δ 1.01–1.15 (m, 4H, 2CH2), 2.62–2.65 (m, 2H, C7’-H), 3.30 (s, 3H, N-CH3), 3.42–3.64 (m, 2H, C6’-H), 3.70 (s, 3H, OCH3), 4.16 (s, 2H, C4’-H), 4.17–4.18 (m, 1H, CH), 7.75 (d, J = 12.0 Hz, 1H, C5-H), 8.69 (s, 1H, C2-H); HRMS (ESI) m/z (M+H)+: calcd for C21H23FN5O4+: 428.17341; found: 428.17379. 7-(3-Amino-6,7-dihydro-2-methyl-2H-pyrazolo[4,3-c]pyridin-5(4H)-yl)-1-cyclopropyl-5-amino-6-fluor o-8-methoxyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (8h). Yellow solid (0.08 g, 17%), m.p.: >300 °C. 1H-NMR (DMSO-d6) δ 0.93–1.11 (m, 4H, 2CH2), 2.09 (br., 2H, C7’-H), 2.64 (br., 2H, C6’-H), 3.46 (s, 3H, N-CH3), 3.52 (br., 1H, CH), 3.62 (s, 3H, OCH3), 4.17 (s, 2H, C4’-H), 8.67 (s, 1H, C2-H); HRMS (ESI) m/z (M+H)+: calcd for C21H23FN6O4+: 443.18431; found: 443.18522. 9-fluoro-3(S)-methyl-10-(3-amino-6,7-dihydro-2-methyl-2H-pyrazolo[4,3-c]pyridine-5(4H)-yl)-7-oxo2,3-dihydro-7H-pyrrido[1,2,3-d,e][1,4]benzoxazine-6-carboxylic acid (8i). Yellow solid (0.17 g, 41%), m.p.: >300 °C. 1H-NMR (DMSO-d6) δ 1.45 (d, J = 6.8 Hz, 3H, CH3), 2.55–2.65 (m, 2H, C7’-H), 3.30 (s, 3H, N-CH3), 4.13–4.16 (m, 2H, C6’-H), 4.34–4.37 (m, 1H, OCH2CHN), 4.46–4.49 (m, 2H, OCH2CHN), 5.01 (s, 2H, C4’-H), 7.60 (d, J = 12.4 Hz, 1H, C5-H), 8.95 (s, 1H, C2-H); HRMS (ESI) m/z (M+H)+: calcd for C20H21FN5O4+: 414.15776; found: 414.15497. 3.2. Antibacterial activity Compounds 8a-i were evaluated for their in vitro antibacterial activity in comparison to several reference drugs using the conventional agar-dilution method. Drugs (10.0 mg) were dissolved in 0.1 N sodium hydroxide (10 mL). Further progressive twofold serial dilution with melted Mueller-Hinton agar was performed to obtain the required concentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, and 0.03 μg/mL. Petri dishes were incubated with 104 colony forming units (cfu) and incubated at 35 °C for 18 h. The MIC was the lowest concentration of the test compound, which resulted in no visible growth on the plate. 4. Conclusions We report herein the synthesis of a series of novel 7-(3-amino-6,7-dihydro-2-methyl-2Hpyrazolo[4,3-c]pyridin-5(4H)-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid derivatives. The antibacterial activities of the newly synthesized compounds were evaluated. The test results reveal that most of the target compounds have good growth inhibiting activity against methicillin-resistant Staphylococcus epidermidis (MRSE) (MIC: 0.25–4 μg/mL) and Streptococcus pneumoniae (MIC: 0.25–1 μg/mL). In addition, compound 8f is 8–128 fold more potent than the reference drugs MX, GM, CP and LV against methicillin-resistant Staphylococcus aureus 10-05 and Streptococcus hemolyticus 1002 and 2–64 fold more active against methicillin-sensitive Staphylococcus aureus 10-03 and 10-04. 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https://openalex.org/W4221020270	https://www.researchsquare.com/article/rs-1314021/latest.pdf	English	null	What is the impact of COVID-19 pandemic years on Deliveries and Home Based New Born Care in India? A cross-sectional comparative research study, Jan 2018-2021Dec	Research Square (Research Square)	2,022	cc-by	9,731	What is the impact of COVID-19 pandemic years on Deliveries and Home Based New Born Care in India? A cross-sectional comparative research study, Jan 2018- 2021Dec Dr Piyush Kumar (  drpiyush003@gmail.com ) Health Department, Government of Bihar, India https://orcid.org/0000-0001-9857-478X ( p y @g ) h Department, Government of Bihar, India https://orcid.org/0000-0001-9857-478X Abstract The COVID-19 pandemic caused by SARS-CoV-2 has led to disruption of health services globally. I assessed the effect of the ongoing pandemic on the Deliveries and Home Based New Born Care (HBNC) in India. The author is curious and suspects a rise in maternal and neonatal mortality due to possible negative effects of COVID-19 on maternal institutional delivery and HBNC. Hence author decided to find out answer to the research question mentioned as title above. I conducted a retrospective cross-sectional comparative study among total numbers of women and newborn who have received delivery and HBNC care at different health facilities in 36 states and union territories across India during the pandemic years with pre-pandemic years (comparing 2020 and 2021(forecasted up to December 2021 with available data from January2021 to May 2021) with previous two years of pre- pandemic era i.e. 2018-2019). The data for the research study is extracted from electronic records of HMIS (health management information system) of MoHFW (ministry of health and family welfare), Govt. of India and analysed with stata and Microsoft office. This research study revealed that during covid-19 era there is increase in prevalence of 1.Total No. of Deliveries-TD (HD+ ID) registered per 100000 ANC registered,2.Total Number of Institutional Deliveries (ID) conducted (Including C-Sections) per 100000 Total deliveries3.Total Number of Home Deliveries attended by SBA per 100000 HD4.Total Number of newborns received 6 HBNC visits after Institutional Delivery per 100000 ID5.Total Number of newborns received 7 HBNC in HD per 100000 HD5.Total Number of PW given Tablet Misoprostol during HD per 100000 HD. This research study revealed that pandemic years have less number of home deliveries attended by SBA [(Skill Birth Attendant) (Doctor/Nurse/ANM/ Midwife)] as well as there is also decrease in HD. This research study revealed that pandemic years have less number of home deliveries attended by SBA [(Skill Birth Attendant) (Doctor/Nurse/ANM/ Midwife)] may be due to less number of deliveries. There is a (projected) decrease of 37.28% in mean number of home deliveries attended by SBA in 2021 as compared to 2018. This novel research study revealed that during pandemic years 2020 and 2021 all the variables reduced quantitatively except Number of PW given Tablet Misoprostol during HD which increased in 2020 (first pandemic year).The ongoing COVID-19 pandemic has had negative effects on Deliveries and Home Based New Born Care (HBNC) in India. Abstract More research is required to investigate the direct and indirect consequence of the pandemic on birth and provision of HBNC as well as the health facility type which are not performing well in providing respectful maternity care and newborn services during pandemic period. The facility based research analysis is under process by the author which will be available in next version of this article. Research Article Keywords: Home delivery, Institutional delivery, newborn, care, pandemic year, covid-19 Posted Date: March 2nd, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1314021/v3 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 Li Page 1/23 Objective The key aim of this research study was to find out the impact of COVID-19 pandemic years on Deliveries and Home Based New Born Care in India. To fulfil the aim of study the objective was to collect, analyse, interpret the data for the research question. Background/Rationale During this ongoing pandemic years countries expected with highest numbers of births during the 9 months since the declaration of pandemic are 1.India (20.1 million) 2.China (13.5 million), 3.Nigeria (6.4 million), 4.Pakistan (5 million) and 5.Indonesia (4 million). As per available data most of these countries listed above had a high neonatal mortality rates (NMR) even before the covid-19 pandemic and these levels may increase with COVID-19 situations [1]. Every year 25 million children are born in India accounting for one fifth of the global child births and every minute one baby dies. It is a fact that 46 per cent of all maternal mortality and 40 per cent of neonatal mortality happen during labour or in the first 24 hours after birth [2]. This research study is significant as death can be reduced by provision of good delivery care at institution/health facilities by enabling access to SBA (skilled birth attendants) and EMOC (emergency obstetric care) services. Home based newborn care (HBNC) in the post-new-born period can improve survival rates with early and exclusive breastfeeding and immunizing newborn against vaccine preventable diseases. Study Design The researcher conducted a retrospective cross-sectional comparative study among total numbers of women and newborn who have received delivery and HBNC care in different ways at different health facilities in 36 states and union territories across India during the pandemic (comparing 2020 and 2021(forecasted up to December 2021 with available data from January2021 to May 2021) with previous two years of pre-pandemic era i.e. 2018-2019). Background/Rationale As per the previous estimates of UNICEF (United Nations International Children's Emergency Fund) about 116 million babies will be born within 40 weeks of ongoing COVID-19 pandemic, straining health systems as well as medical supply chains globally – was declared as a pandemic on March 11[1]. Pregnant mothers as well as babies born during the ongoing COVID-19 pandemic are continued to be threatened by strained health systems involved in fighting with this novel disease. I assessed the effect of the ongoing pandemic on the Deliveries and Home Based New Born Care (HBNC) in India. The author is curious and suspects a rise in maternal and neonatal mortality due to possible negative effects of COVID-19 on maternal institutional delivery and HBNC. Hence author decided to find out answer to the research question mentioned as title above. Global containment measures like lockdowns etc; health centres overwhelmed with covid-19 response efforts added with supply and equipment shortages and a lack of sufficient skilled birth attendants because health workers, including midwives, are redeployed to treat Page 2/23 Page 2/23 COVID-19 patients [1]. During this ongoing pandemic years countries expected with highest numbers of births during the 9 months since the declaration of pandemic are 1.India (20.1 million) 2.China (13.5 million), 3.Nigeria (6.4 million), 4.Pakistan (5 million) and 5.Indonesia (4 million). As per available data most of these countries listed above had a high neonatal mortality rates (NMR) even before the covid-19 pandemic and these levels may increase with COVID-19 situations [1]. Every year 25 million children are born in India accounting for one fifth of the global child births and every minute one baby dies. It is a fact that 46 per cent of all maternal mortality and 40 per cent of neonatal mortality happen during labour or in the first 24 hours after birth [2]. This research study is significant as death can be reduced by provision of good delivery care at institution/health facilities by enabling access to SBA (skilled birth attendants) and EMOC (emergency obstetric care) services. Home based newborn care (HBNC) in the post-new-born period can improve survival rates with early and exclusive breastfeeding and immunizing newborn against vaccine preventable diseases. COVID-19 patients [1]. Setting Location – All India across 36 states and union territories. Study period – 1st January 2018 to 31st December 2021(forecast) Exposure - To know the impact of COVID-19 pandemic years on delivery and HBNC the following indicators of mentioned period are included as operational definition to assess impact on Deliveries and Home Based New Born Care in this research analysis (see – table – 1): Table – 1 – Indicators inclusion for operational definition to assess impact of covid-19 on delivery and HBNC Page 3/23 Page 3/23 Page 3/23 Variable Period Number of Home Deliveries attended by Skill Birth Attendant(SBA) (Doctor/Nurse/ANM/ Midwife) 1st Jan2018-31st Dec2021 Number of Home Deliveries attended by Non SBA (Trained Birth Attendant (TBA) /Relatives/etc.) 1st Jan2018-31st Dec2021 Number of PW given Tablet Misoprostol during home delivery 1st Jan2018-31st Dec2021 Number of newborns received 7 Home Based Newborn Care (HBNC) visits in case of Home delivery 1st Jan2018-31st Dec2021 Number of Institutional Deliveries conducted (Including C-Sections) 1st Jan2018-31st Dec2021 Out of total institutional deliveries number of women discharged within 48 hours of delivery 1st Jan2018-31st Dec2021 Number of newborns received 6 HBNC visits after Institutional Delivery 1st Jan2018-31st Dec2021 Follow up – The data is continuously extracted, observed and investigated for specificity, measurability, accuracy, reproducibility and timeliness. The available data is forecasted with excel to reduce bias arising due to lack of data availability up to December 2021 from HMIS. Data collection - The data is extracted from electronic records of HMIS (health management information system), MoHFW (ministry of health and family welfare), and Government of India. For this retrospective cross-sectional study, available online data from HMIS is collected from January 2018 to May 2021 forecasted up to December 2021 with Microsoft excel utilizing available data of 2021.Newborn who utilized HBNC and deliveries reported during this period were included in this study. Data analyses including statistical analysis were done with stata and Microsoft office software. Participants Eligibility Criteria – The eligibility criteria is listed below in table – 2 – for participants in this research study. All the eligible participants are those who are reported by electronic records of HMIS (health management information system), MoHFW (ministry of health and family welfare), and Government of India for variables mentioned in table – 2 below. Page 4/23 Table – 2 – Eligibility criteria or criteria for inclusion Variable Period Home Deliveries attended by Skill Birth Attendant(SBA) (Doctor/Nurse/ANM/ Midwife) 1st Jan2018-31st Dec2021 Home Deliveries attended by Non SBA (Trained Birth Attendant (TBA) /Relatives/etc.) 1st Jan2018-31st Dec2021 PW given Tablet Misoprostol during home delivery 1st Jan2018-31st Dec2021 Newborns received 7 Home Based Newborn Care (HBNC) visits in case of Home delivery 1st Jan2018-31st Dec2021 Institutional Deliveries conducted (Including C-Sections) 1st Jan2018-31st Dec2021 Out of total institutional deliveries number of women discharged within 48 hours of delivery 1st Jan2018-31st Dec2021 Newborns received 6 HBNC visits after Institutional Delivery 1st Jan2018-31st Dec2021 Page 4/23 Table – 2 – Eligibility criteria or criteria for inclusion Variable Period Home Deliveries attended by Skill Birth Attendant(SBA) (Doctor/Nurse/ANM/ Midwife) 1st Jan2018-31st Dec2021 Home Deliveries attended by Non SBA (Trained Birth Attendant (TBA) /Relatives/etc.) 1st Jan2018-31st Dec2021 PW given Tablet Misoprostol during home delivery 1st Jan2018-31st Dec2021 Newborns received 7 Home Based Newborn Care (HBNC) visits in case of Home delivery 1st Jan2018-31st Dec2021 Institutional Deliveries conducted (Including C-Sections) 1st Jan2018-31st Dec2021 Out of total institutional deliveries number of women discharged within 48 hours of delivery 1st Jan2018-31st Dec2021 Newborns received 6 HBNC visits after Institutional Delivery 1st Jan2018-31st Dec2021 Table – 2 – Eligibility criteria or criteria for inclusion Variable Period Home Deliveries attended by Skill Birth Attendant(SBA) (Doctor/Nurse/ANM/ Midwife) 1st Jan2018-31st Dec2021 Home Deliveries attended by Non SBA (Trained Birth Attendant (TBA) /Relatives/etc.) 1st Jan2018-31st Dec2021 PW given Tablet Misoprostol during home delivery 1st Jan2018-31st Dec2021 Newborns received 7 Home Based Newborn Care (HBNC) visits in case of Home delivery 1st Jan2018-31st Dec2021 Institutional Deliveries conducted (Including C-Sections) 1st Jan2018-31st Dec2021 Out of total institutional deliveries number of women discharged within 48 hours of delivery 1st Jan2018-31st Dec2021 Newborns received 6 HBNC visits after Institutional Delivery 1st Jan2018-31st Dec2021 Sources – Secondary data available from the electronic records of HMIS of MoHFW (Ministry of Health and Family Welfare), Government of India. Participants Method of Selection – A purposive sampling is done by the author for the research question mentioned in title above. The sample is collected continuously from accredited national source of HMIS of MoHFW (Ministry of Health and Family Welfare), Government of India. Variables The variables for this research study are mentioned in table 1 and 2 above. Data sources / Measurements Data Availability - For this novel study, accredited data is obtained from electronic records of HMIS of MoHFW (Ministry of Health and Family Welfare), Government of India. The link for the source is available below:- https://hmis.nhp.gov.in/#!/standardReports Bias – To reduce the bias the data is projected up to December 2021 with the Microsoft excel software utilizing available data for 2021. For further reduction of Bias the total number of pregnancy / HD / ID is taken into account (see – table-3). This will also show the trends of delivery and HBNC in the study period. Table – 3 – Total and Mean of Pregnancy and Delivery Period Total No. of pregnant women registered for ANC- Taken as total number of pregnancy Mean- No. of pregnant women Total HD Total ID Total No. of Deliveries (HD+ ID) Mean- Total Number of Deliveries Jan to Dec- 2018 28715284 7178821 1453777 19388411 20842188 1736849 Jan to Dec 2019 29239176 7309794 1243190 20328254 21571444 1797620 Jan to Dec 2020 27331061 6832765 1077371 19298327 20375698 1697975 Jan to Dec 2021 21891305 1824275 807260 16057132 16864392 1405366 Table – 3 – Total and Mean of Pregnancy and Delivery Period Total No. of pregnant women registered for ANC- Taken as total number of pregnancy Mean- No. of pregnant women Total HD Total ID Total No. of Deliveries (HD+ ID) Mean- Total Number of Deliveries Jan to Dec- 2018 28715284 7178821 1453777 19388411 20842188 1736849 Jan to Dec 2019 29239176 7309794 1243190 20328254 21571444 1797620 Jan to Dec 2020 27331061 6832765 1077371 19298327 20375698 1697975 Jan to Dec 2021 21891305 1824275 807260 16057132 16864392 1405366 Study Size – This research study size for different years are listed in above table number 3. Quantitative Variables – All the quantitative variables are listed in tables 1, 2, 3 above. Statistical Methods - Linear regression is used to forecast 2021 to predict future values from available past values of 2021. Study Size – This research study size for different years are listed in above table number 3. Quantitative Variables – All the quantitative variables are listed in tables 1, 2, 3 above. St ti ti l M th d Li i i d t f t 2021 t di t f t l f il bl t Study Size – This research study size for different years are listed in above table number 3. Data sources / Measurements Study Size – This research study size for different years are listed in above table number 3. Quantitative Variables – All the quantitative variables are listed in tables 1, 2, 3 above. Statistical Methods - Linear regression is used to forecast 2021 to predict future values from available past val of 2021. Study Size – This research study size for different years are listed in above table number 3. Quantitative Variables – All the quantitative variables are listed in tables 1, 2, 3 above. Statistical Methods - Linear regression is used to forecast 2021 to predict future values from available past values of 2021. Statistical Methods - Linear regression is used to forecast 2021 to predict future values from available past values Page 5/23 Results of Deliveries (HD+ ID) 20842188 21571444 20375698 16864392 Total Number of Institutional Deliveries conducted (Including C- Sections) 19388411 19388411 19298327 16057132 Total HD 1453777 1243190 1077371 807260 Total Number of Home Deliveries attended by SBA 255814 228079 197151 160443.4 Total Home Deliveries attended by Non SBA 1197963 1015111 880220 646816.6 Total (Out of total ID) women discharged within 48 hours 6803202 6890913 6483022 5382451 Total Number of PW given Tablet Misoprostol during HD 80621 74416 78722 63822.13 Total Number of newborns received 7 HBNC in HD 938660 936941 891089 802483.6 Total Number of newborns received 6 HBNC visits after Institutional Delivery 6405013 7981529 8651475 8355130 There is a declining trend in No. of pregnant women registered for ANC- Taken as total number of pregnancy during pandemic years 2020 and 2021 as compared to pre-pandemic years. A similar trend was observed in all other variables in this research study except in 2020 1. Number of PW given Tablet Misoprostol during HD increased in number compared to previous year 2019 by 4306 and 5.78639 percent (%) 2. number of newborns received 6 HBNC visits after Institutional Delivery HD increased in number compared to previous year 2019 by 669946 numbers and 8.393705 percent (%). There is a declining trend in No. of pregnant women registered for ANC- Taken as total number of pregnancy during pandemic years 2020 and 2021 as compared to pre-pandemic years. A similar trend was observed in all other variables in this research study except in 2020 1. Number of PW given Tablet Misoprostol during HD increased in number compared to previous year 2019 by 4306 and 5.78639 percent (%) 2. number of newborns received 6 HBNC visits after Institutional Delivery HD increased in number compared to previous year 2019 by 669946 numbers and 8.393705 percent (%). There is a declining trend in No. of pregnant women registered for ANC- Taken as total number of pregnancy during pandemic years 2020 and 2021 as compared to pre-pandemic years. A similar trend was observed in all other variables in this research study except in 2020 1. Number of PW given Tablet Misoprostol during HD increased in number compared to previous year 2019 by 4306 and 5.78639 percent (%) 2. number of newborns received 6 HBNC visits after Institutional Delivery HD increased in number compared to previous year 2019 by 669946 numbers and 8.393705 percent (%). Results Numbers of individuals at each stage of study The Total No. of pregnant women registered for ANC- Taken as total number of pregnancy for Jan to Dec-2018, Jan to Dec 2019, Jan to Dec 2020, and Jan to Dec 2021 was 28715284,29239176,27331061,21891305; respectively see table-4. This research study revealed that before start of pandemic in 2020, in which first case of COVID-19 was reported in India [3], year 2019 reported an increase in total number of pregnancy. During pandemic years this research study revealed a decrease in this numbers of several variables under study (see table – 4 and 5 and figure 1). Table – 4 – Total Numbers of quantitative variables under study in different years Period Jan to Dec- 2018 Jan to Dec 2019 Jan to Dec 2020 Jan to Dec 2021 Total No. of pregnant women registered for ANC- Taken as total number of pregnancy 28715284 29239176 27331061 21891305 Total No. of Deliveries (HD+ ID) 20842188 21571444 20375698 16864392 Total Number of Institutional Deliveries conducted (Including C- Sections) 19388411 19388411 19298327 16057132 Total HD 1453777 1243190 1077371 807260 Total Number of Home Deliveries attended by SBA 255814 228079 197151 160443.4 Total Home Deliveries attended by Non SBA 1197963 1015111 880220 646816.6 Total (Out of total ID) women discharged within 48 hours 6803202 6890913 6483022 5382451 Total Number of PW given Tablet Misoprostol during HD 80621 74416 78722 63822.13 Total Number of newborns received 7 HBNC in HD 938660 936941 891089 802483.6 Total Number of newborns received 6 HBNC visits after Institutional Delivery 6405013 7981529 8651475 8355130 Table – 4 – Total Numbers of quantitative variables under study in different years Period Jan to Dec- 2018 Jan to Dec 2019 Jan to Dec 2020 Jan to Dec 2021 Total No. of pregnant women registered for ANC- Taken as total number of pregnancy 28715284 29239176 27331061 21891305 Total No. Results Table – 5 – Increase and decrease in numbers and percentage of different variables taking 2018 base year Page 6/23 Page 6/23 Period Increase / Decrease 2019 to previous year 2018 - Numbers Increase / Decrease 2020 to previous year 2019 - Numbers Increase / Decrease 2021 to previous year 2020 - Numbers Increase / Decrease 2019 to previous year 2018- percent Increase / Decrease 2020 to previous year 2019 - percent Increase / Decrease 2021 to previous year 2020 - percent Total No. of pregnant women registered for ANC- Taken as total number of pregnancy 523892 -1908115 -5439756 1.824436074 -6.52588 -19.9032 Mean- No. of pregnant women 130973 -477029 -5008490 1.824436074 -6.52589 -73.3011 Total HD -210587 -165819 -270111 -14.48550913 -13.3382 -25.0713 Total ID 939843 -1029927 -3241195 4.847447271 -5.06648 -16.7952 Total No. of Deliveries (HD+ ID) 729256 -1195746 -3511306 3.498941666 -5.54319 -17.2328 Mean- Total Number of Deliveries 60771 -99645 -292609 3.498922474 -5.54316 -17.2328 Number of Home Deliveries attended by SBA -27735 -30928 -36707.6 -10.84186167 -13.5602 -18.619 Home Deliveries attended by Non SBA -182852 -134891 -233403 -15.26357659 -13.2883 -26.5165 Number of PW given Tablet Misoprostol during HD -6205 4306 -14899.9 -7.696505873 5.78639 -18.9272 Number of newborns received 7 HBNC in HD -1719 -45852 -88605.4 -0.183133403 -4.8938 -9.9435 Number of Institutional Deliveries conducted (Including C-Sections) 900000 -1000000 -3200000 4.639175258 -4.92611 -16.5803 Out of total ID women discharged within 48 hours 87711 -407891 -1100571 1.289260557 -5.91926 -16.9762 Number of newborns received 6 HBNC visits after Institutional Delivery 1576516 669946 -296345 24.61378299 8.393705 -3.42537 All variables have shown an increase in 2019 except 1.Total HD 2. Number of newborns received 7 HBNC in HD 3.Home Deliveries attended by Non SBA 4.Number of PW given Tablet Misoprostol during HD 5.Number of Home Deliveries attended by SBA. This novel research study revealed that during pandemic years 2020 and 2021 all the variables reduced quantitatively except 1.Number of PW given Tablet Misoprostol during HD 2. Number of newborns received 6 HBNC visits after Institutional Delivery 3.Total HD 4. Home Deliveries attended by Non SBA which increased little in 2020 see- table 5 and figure 1 (first pandemic year). The statistical analysis of All variables have shown an increase in 2019 except 1.Total HD 2. Number of newborns received 7 HBNC in HD 3.Home Deliveries attended by Non SBA 4.Number of PW given Tablet Misoprostol during HD 5.Number of Home Deliveries attended by SBA. Results This novel research study revealed that during pandemic years 2020 and 2021 all the variables reduced quantitatively except 1.Number of PW given Tablet Misoprostol during HD 2. Number of newborns received 6 HBNC visits after Institutional Delivery 3.Total HD 4. Home Deliveries attended by Non SBA which increased little in 2020 see- table 5 and figure 1 (first pandemic year). The statistical analysis of observation for years 2021, 2020, 2019 and 2018 is given in table- 6, 7, 8, and 9 and data for each year is given in 10, 11, 12, and 13. Page 7/23 Table –6-Summary- statistics-2021 (Jan to Dec) e –6-Summary- statistics-2021 (Jan to Dec) Table –6-Summary- statistics-2021 (Jan to Dec) Variable Obs Mean Std. Dev. Min Max No. of HD by SBA 12 13370.28 2235.476 10275.74 17301 No. of HD by non SBA 12 53901.39 12868.63 36336.46 75810 No. given Misoprostol-HD 12 5318.511 811.0819 4141.267 6629 No. received7 HBNC-HD 12 66873.63 7590.975 60215.57 81396 No. of ID (including C-secn) 12 1338094 195455.9 1207656 1826745 ID discharged <48hrs 12 448537.5 65624.69 401151 589308 No. received 6 HBNC-ID 12 696260.9 69886.11 646157.3 829656 Total estimation - 2021 Total estimation Number of obs = 12 Variable Total Std. Err. [95% Conf. Interval] No. of HD by SBA 160443.4 7743.915 143399.1 177487.6 No. of HD by non SBA 646816.6 44578.24 548700.6 744932.7 No. given Misoprostol-HD 63822.13 2809.67 57638.09 70006.17 No. received7 HBNC-HD 802483.6 26295.91 744606.7 860360.5 No. of ID (including C-secn) 1.61e+07 677079 1.46e+07 1.75e+07 ID discharged <48hrs \| 5382451 227330.6 4882099 5882802 No. received 6 HBNC-ID 8355130 242092.6 7822288 8887973 C.I (confidence interval) means – 2021 Variable Obs Mean Std. Err. [95% Conf. Interval] No. of HD by SBA 12 13370.28 645.3262 11949.93 14790.63 No. of HD by non SBA 12 53901.39 3714.853 45725.05 62077.72 No. given Misoprostol-HD 12 5318.511 234.1392 4803.174 5833.848 No. received7 HBNC-HD 12 66873.63 2191.326 62050.56 71696.71 No. of ID (including C-secn) 12 1338094 56423.25 1213908 1462281 ID discharged <48hrs \| 12 448537.5 18944.22 406841.6 490233.5 No. received 6 HBNC-ID 12 696260.9 20174.38 651857.4 740664.4 The mean number of home deliveries attended by SBA [(Skill Birth Attendant) (Doctor/Nurse/ANM/ Midwife)] during the pandemic year 2021 with total number of observation of 5 months forecasted up to December 2021across India was 13370.28, for years 2020, 2019, 2018 it was 16429.25, 19006.58, 21317.83 respectively wit total number of observation of 12 months across India for these three years. Results The mean number of home deliveries attended by SBA [(Skill Birth Attendant) (Doctor/Nurse/ANM/ Midwife)] during the pandemic year 2021 with total number of observation of 5 months forecasted up to December 2021across India was 13370.28, for years 2020, 2019, 2018 it was 16429.25, 19006.58, 21317.83 respectively with total number of observation of 12 months across India for these three years. Page 8/23 Table – 7-Summary- statistics-2020 (Jan to Dec) ble 7 Summary statistics 2020 (Jan to Dec) Table – 7-Summary- statistics-2020 (Jan to Dec) Variable \| Obs Mean Std. Dev. Min Max No. of HD by SBA \| 12 16429.25 3373.569 8047 20183 No. of HD by non SBA \| 12 73351.67 14621.01 33185 86457 No. given Misoprostol-HD \| 12 6560.167 1241.477 4251 8580 No. received7 HBNC-HD \| 12 74257.42 17327.82 28639 87890 No. of ID (including C-secn)\| 12 1608194 197666.5 1219729 1849115 ID discharged <48hrs \| 12 540251.8 91445.49 364659 648151 No. received 6 HBNC-ID \| 12 720956.3 123371.7 461874 869027 Total estimation - 2020 - Number of obs = 12 Variable \| Total Std. Err. [95% Conf. Interval] No. of HD by SBA \| 197151 11686.38 171429.4 222872.6 No. of HD by non SBA \| 880220 50648.66 768743 991697 No. given Misoprostol-HD \| 78722 4300.604 69256.43 88187.57 No. received7 HBNC-HD \| 891089 60025.33 758974.1 1023204 No. of ID (including C-secn) \| 1.93e+07 684736.8 1.78e+07 2.08e+07 ID discharged <48hrs \| 6483022 316776.5 5785802 7180242 No. received 6 HBNC-ID \| 8651475 427372.2 7710835 9592115 C.I (confidence interval) means – 2020 Variable \| Obs Mean Std. Err. [95% Conf. Interval] No. of HD by SBA \| 12 16429.25 973.8654 14285.79 18572.71 No. of HD by non SBA \| 12 73351.67 4220.722 64061.92 82641.41 No. given Misoprostol-HD \| 12 6560.167 358.3837 5771.37 7348.964 No. received7 HBNC-HD \| 12 74257.42 5002.111 63247.84 85266.99 No. of ID (including C-secn) \| 12 1608194 57061.4 1482603 1733785 ID discharged <48hrs \| 12 540251.8 26398.04 482150.1 598353.5 No. received 6 HBNC-ID \| 12 720956.3 35614.35 642569.6 799342.9 Table – 7-Summary- statistics-2020 (Jan to Dec) This research study revealed that pandemic years have less number of home deliveries attended by SBA [(Skill Birth Attendant) (Doctor/Nurse/ANM/ Midwife)]. There is a decrease of 37.28% in mean number of home deliveries attended by SBA in 2021 as compared to 2018. Results The details of data and statistical analysis are mentioned in table- 6, 7, 8, 9, 10, 11, 12, 13, 14 and the comparisons of various indicators are displayed in figures 1. The mean number of Number of Home Deliveries attended by Non SBA (Trained Birth Attendant (TBA) /Relatives/etc. during the pandemic year 2021 with total number of observation of 5 months forecasted up to December 2021across India was 53901.39, for years 2020, 2019, 2018 it was 73351.66, 84592.58, 99830.25 respectively with total number of observation of 12 months across India for these three years. This research study revealed that pandemic years have less number of home deliveries attended by Non SBA (Trained Birth Attendant (TBA) /Relatives/etc. There is a decrease of 33.74 % in mean number of home deliveries attended by Non-SBA in 2021 as compared to 2018. This research study revealed that pandemic years have less number of home deliveries attended by SBA [(Skill Birth Attendant) (Doctor/Nurse/ANM/ Midwife)]. There is a decrease of 37.28% in mean number of home deliveries attended by SBA in 2021 as compared to 2018. The details of data and statistical analysis are mentioned in table- 6, 7, 8, 9, 10, 11, 12, 13, 14 and the comparisons of various indicators are displayed in figures 1. The mean number of Number of Home Deliveries attended by Non SBA (Trained Birth Attendant (TBA) /Relatives/etc. during the pandemic year 2021 with total number of observation of 5 months forecasted up to December 2021across India was 53901.39, for years 2020, 2019, 2018 it was 73351.66, 84592.58, 99830.25 respectively with total number of observation of 12 months across India for these three years. This research study revealed that pandemic years have less number of home deliveries attended by Non SBA (Trained Birth Attendant (TBA) /Relatives/etc. There is a decrease of 33.74 % in mean number of home deliveries attended by Non-SBA in 2021 as compared to 2018. This research study revealed that pandemic years have less number of home deliveries attended by SBA [(Skill Birth Attendant) (Doctor/Nurse/ANM/ Midwife)]. There is a decrease of 37.28% in mean number of home deliveries attended by SBA in 2021 as compared to 2018. The details of data and statistical analysis are mentioned in table- 6, 7, 8, 9, 10, 11, 12, 13, 14 and the comparisons of various indicators are displayed in figures 1. Results The mean number of Number of Home Deliveries attended by Non SBA (Trained Birth Attendant (TBA) /Relatives/etc. during the pandemic year 2021 with total number of observation of 5 months forecasted up to December 2021across India was 53901.39, for years 2020, 2019, 2018 it was 73351.66, 84592.58, 99830.25 respectively with total number of observation of 12 months across India for these three years. This research study revealed that pandemic years have less number of home deliveries attended by Non SBA (Trained Birth Attendant (TBA) /Relatives/etc. There is a decrease of 33.74 % in mean number of home deliveries attended by Non-SBA in 2021 as compared to 2018. The mean number of Number of PW given Tablet Misoprostol during home delivery during the pandemic year 2021 with total number of observation of 5 months forecasted up to December 2021across India was 5318.511, for years 2020, 2019, 2018 it was 6560.16, 6201.33, and 6718.42 respectively with total number of observation of 12 months across India for these three years. This research study revealed that pandemic years have less number of Page 9/23 Page 9/23 PW given Tablet Misoprostol during home delivery. There is a decrease of 9.72 % in mean Number of PW given Tablet Misoprostol during home delivery in 2021 as compared to 2018. PW given Tablet Misoprostol during home delivery. There is a decrease of 9.72 % in mean Number of PW given Tablet Misoprostol during home delivery in 2021 as compared to 2018. ble – 8-Summary- statistics-2019 (Jan to Dec Table – 8-Summary- statistics-2019 (Jan to Dec) Variable \| Obs Mean Std. Dev. Min Max No. of HD by SBA \| 12 19006.58 1652.682 16387 20793 No. of HD by non SBA \| 12 84592.58 6727.217 70630 91917 No. given Misoprostol-HD \| 12 6201.333 531.5118 5253 6959 No. received7 HBNC-HD \| 12 78078.42 7856.47 63616 87138 No. of ID (including C-secn) 12 1694021 168219.1 1409794 1895697 ID discharged <48hrs \| 12 574242.8 72415.95 443409 671254 No. received 6 HBNC-ID \| 12 665127.4 85338.78 542151 781203 Total estimation -2019 Number of obs = 12 Variable \| Total Std. Err. [95% Conf. Interval] No. of HD by SBA \| 228079 5725.058 215478.2 240679.8 No. of HD by non SBA \| 1015111 23303.76 963819.8 1066402 No. given Misoprostol-HD \| 74416 1841.211 70363.52 78468.48 No. received7 HBNC-HD \| 936941 27215.61 877039.8 996842.2 No. Results There is a decrease of 8.34 % in mean number of Institutional Deliveries conducted (Including C-Sections) in 2021 as compared to 2018. observation of 12 months across India for these three years. This research study revealed that pandemic years have less number of Institutional Deliveries conducted (Including C-Sections). There is a decrease of 8.34 % in mean number of Institutional Deliveries conducted (Including C-Sections) in 2021 as compared to 2018. observation of 12 months across India for these three years. This research study revealed that pandemic years have less number of Institutional Deliveries conducted (Including C-Sections). There is a decrease of 8.34 % in mean number of Institutional Deliveries conducted (Including C-Sections) in 2021 as compared to 2018. Table – 9 - Summary- statistics-2018 (Jan to Dec) Table – 9 - Summary- statistics-2018 (Jan to Dec) Variable \| Obs Mean Std. Dev. Min Max No. of HD by SBA \| 12 21317.83 1730.307 19091 24266 No. of HD by non SBA \| 12 99830.25 9646.946 84554 116307 No. given Misoprostol-HD \| 12 6718.417 657.5543 5411 7564 No. received7 HBNC-HD \| 12 78221.67 7188.477 68640 87132 No. of ID (including C-secn) 12 1615701 176173.3 1363935 1856881 ID discharged <48hrs \| 12 566933.5 73511.3 448374 660925 No. received 6 HBNC-ID \| 12 533751.1 63534.34 440685 624944 Total estimation - 2018 Number of obs = 12 Variable Total Std. Err. [95% Conf. Interval] No. of HD by SBA \| 255814 5993.958 242621.4 269006.6 No. of HD by non SBA \| 1197963 33418 1124410 1271516 No. given Misoprostol-HD \| 80621 2277.835 75607.52 85634.48 No. received7 HBNC-HD \| 938660 24901.61 883851.9 993468.1 No. of ID (including C-secn) 1.94e+07 610282.2 1.80e+07 2.07e+07 ID discharged <48hrs \| 6803202 254650.6 6242720 7363684 No. received 6 HBNC-ID \| 6405013 220089.4 5920600 6889426 C.I (confidence interval) means – 2018 Variable \| Obs Mean Std. Err. [95% Conf. Interval] No. of HD by SBA \| 12 21317.83 499.4965 20218.45 22417.22 No. of HD by non SBA \| 12 99830.25 2784.833 93700.87 105959.6 No. given Misoprostol-HD \| 12 6718.417 189.8196 6300.627 7136.207 No. received7 HBNC-HD \| 12 78221.67 2075.135 73654.33 82789.01 No. of ID (including C-secn) 12 1615701 50856.85 1503766 1727636 ID discharged <48hrs \| 12 566933.5 21220.89 520226.6 613640.4 No. Results of ID (including C-secn) \| 2.03e+07 582728 1.90e+07 2.16e+07 ID discharged <48hrs \| 6890913 250856.2 6338782 7443044 No. received 6 HBNC-ID \| 7981529 295622.2 7330869 8632189 C.I (confidence interval) means – 2019 Variable \| Obs Mean Std. Err. [95% Conf. Interval] No. of HD by SBA \| 12 19006.58 477.0882 17956.52 20056.65 No. of HD by non SBA \| 12 84592.58 1941.98 80318.31 88866.85 No. given Misoprostol-HD \| 12 6201.333 153.4342 5863.627 6539.04 No. received7 HBNC-HD \| 12 78078.42 2267.967 73086.65 83070.18 No. of ID (including C-secn) \| 12 1694021 48560.67 1587140 1800902 ID discharged <48hrs \| 12 574242.8 20904.68 528231.8 620253.7 No. received 6 HBNC-ID \| 12 665127.4 24635.18 610905.7 719349.1 Table – 8-Summary- statistics-2019 (Jan to Dec The mean number of newborns who received 7 Home Based Newborn Care (HBNC) visits in case of Home delivery during the pandemic year 2021 with total number of observation of 5 months forecasted up to December 2021across India was 66873.63, for years 2020, 2019, 2018 it was 74257.42, 78078.42, and 78221.67 respectively with total number of observation of 12 months across India for these three years. This research study revealed that pandemic years have decreased number of newborns received 7 Home Based Newborn Care (HBNC) visits in case of Home delivery. There is a decrease of 6.47 % in mean Number of newborns received 7 Home Based Newborn Care (HBNC) visits in case of Home delivery in 2021 as compared to 2018. The researcher is pointing out the fact that less number of different variables during pandemic years may be due less number of pregnancy and resultant deliveries during covid-19era. The prevalence rate discussed at end will clear the scenario. The researcher is pointing out the fact that less number of different variables during pandemic years may be due less number of pregnancy and resultant deliveries during covid-19era. The prevalence rate discussed at end will clear the scenario. The mean number of Institutional Deliveries (ID) conducted (Including C-Sections)during the pandemic year 2021 with total number of observation of 5 months forecasted up to December 2021across India was 1338094, for years 2020, 2019, 2018 it was 1608193.92, 1694021.12, and 1615700.92 respectively with total number of Page 10/23 Page 10/23 observation of 12 months across India for these three years. This research study revealed that pandemic years have less number of Institutional Deliveries conducted (Including C-Sections). Results received 6 HBNC-ID \| 12 533751.1 18340.78 493383.3 574118.9 Table – 9 - Summary- statistics-2018 (Jan to Dec) The mean number of women discharged within 48 hours of delivery out of total ID during the pandemic year 2021 with total number of observation of 5 months forecasted up to December 2021across India was 448537.5, for years 2020, 2019, 2018 it was 540251.83, 574242.75, and 566933.5 respectively with total number of observation of 12 months across India for these three years. This research study revealed that pandemic years have less number of women discharged within 48 hours of delivery out of total ID. There is a decrease of 12.32 % in mean number of women discharged within 48 hours of delivery out of total ID in 2021 as compared to 2018. The mean number of newborns received 6 HBNC visits after Institutional Delivery during the pandemic year 2021 with total number of observation of 5 months forecasted up to December 2021across India was 696260.9, for years 2020, 2019, 2018 it was 720956.25, 665127.42, and 533751.08 respectively with total number of observation of 12 months across India for these three years. This research study revealed that pandemic years Page 11/23 Page 11/23 Page 11/23 have less number of newborns received 6 HBNC visits after Institutional Delivery. There is an increase of 40.03 % in mean number of newborns received 6 HBNC visits after Institutional Delivery in 2021 as compared to 2018. Table- 10- Prevalence of different study variables per 100000 in different years Period Jan to Dec- 2018 Jan to Dec 2019 Jan to Dec 2020 Jan to Dec 2021 Total No. of pregnant women registered for ANC- Taken as total number of pregnancy 28715284 29239176 27331061 21891305 Total No. Results of Deliveries-TD (HD+ ID) registered per 100000 ANC registered 72582.21092 73775.82734 74551.43436 77036.94229 Total Number of Institutional Deliveries(ID) conducted (Including C-Sections) per 100000 Total delivery 93024.83501 89879.98671 94712.47071 95213.22797 Total HD per 100000 TD 6975.164987 5763.128328 5287.529291 4786.772034 Total Number of Home Deliveries attended by SBA per 100000 HD 17596.50896 18346.27048 18299.26738 19875.05884 Total Home Deliveries attended by Non SBA per 100000 HD 82403.49104 81653.72952 81700.73262 80124.94116 Total (Out of total ID) women discharged within 48 hours per 100000 ID 35089.01271 35541.40151 33593.6996 33520.62498 Total Number of PW given Tablet Misoprostol during HD per 100000 HD 5545.623572 5985.891135 7306.860868 7906.019126 Total Number of newborns received 7 HBNC in HD per 100000 HD 64566.98655 75365.87328 82709.5773 99408.3195 Total Number of newborns received 6 HBNC visits after Institutional Delivery per 100000 ID 33035.26524 41166.49374 44830.18139 52033.76294 The prevalence of total No. of Deliveries-TD (HD+ ID) registered per 100000 ANC registered was 72582.21092, 73775.82734, 74551.43436, 77036.94229, for 2018, 2019, 2020 and 2021 respectively whereas prevalence for total number of Institutional Deliveries (ID) conducted (Including C-Sections) per 100000 total delivery was 93024.83501, 89879.98671, 94712.47071, 95213.22797 for the years 2018, 2019, 2020 and 2021 respectively see table-10 figure-2 and 3. Regarding total HD per 100000 TD the prevalence was 6975.164987, 5763.128328, 5287.529291, 4786.772034, and total Number of Home Deliveries attended by SBA per 100000 HD the prevalence were 17596.50896, 18346.27048, 18299.26738, and 19875.05884 for 2018, 2019, 2020 and 2021 respectively. Total Home Deliveries attended by Non SBA per 100000 HD was 82403.49104, 81653.72952, 81700.73262, 80124.94116, and total (Out of total ID) women discharged within 48 hours per 100000 ID were 35089.01271, 35541.40151, 33593.6996, 33520.62498, for 2018, 2019, 2020 and 2021 respectively. Total Number of PW given Tablet Misoprostol during HD per 100000 HD for 2018, 2019, 2020 and 2021 respectively was 5545.623572, 5985.891135, 7306.860868, 7906.019126, and total Number of newborns received 7 HBNC in HD per 100000 HD was 64566.98655, 75365.87328, 82709.5773, 99408.3195, respectively see table-10 figure-2 and 3. The total Number of newborns received 6 HBNC visits after Institutional Delivery per 100000 ID was 33035.26524, 41166.49374, 44830.18139, 52033.76294, for 2018, 2019, 2020 and 2021 respectively. This research study revealed that during covid-19 era there is increase in prevalence of following: The prevalence of total No. Study Strength and Limitation This is a novel cross-sectional retrospective research study based on accredited secondary data which was one of the limitations of this research study. Another limitation is data availability is not for latest time and date, hence it is forecasted using standard statistical method using excel. Accredited and established source of data recognized worldwide is not available for the same study anywhere else. The main strength is that the whole study is based on real time based accredited government data sources and this kind of research study is unique and not available for the context of India or other countries as found by the researcher of this novel study. Discussion Urgent attention and measures should be taken by government and policy makers to continue to provide maternal and newborn health services during ongoing COVID-19 pandemic. Government, policy makers should develop special need based guidelines, interventions-for the pregnant women and newborn. Continuous monitoring and routinely checking -the existing facilities health service in different levels of hospitals i.e. from local to tertiary specialised and rural-urban-public- private clinics. Legal regulations and strict enforcements so that healthcare institutions cannot deny providing maternal and newborn services. Training and capacity building of the health workers including everyone and to sort out the barriers to access maternal-newborn health services during ongoing COVID-19. Raising awareness among PW and relatives about visiting health care facilities along with the positive behavioural change of the health workers to work in pandemic situations can help in mitigation of negative impacts of covid-19. Results of Deliveries-TD (HD+ ID) registered per 100000 ANC registered Total Number of Institutional Deliveries (ID) conducted (Including C-Sections) per 100000 Total deliveries Total Number of Institutional Deliveries (ID) conducted (Including C-Sections) per 100000 Total deliveries Total Number of Institutional Deliveries (ID) conducted (Including C-Sections) per 100000 Total deliveries Total Number of Institutional Deliveries (ID) conducted (Including C-Sections) p Page 12/23 Page 12/23 Page 12/23 Total Number of Home Deliveries attended by SBA per 100000 HD Total Number of newborns received 6 HBNC visits after Institutional Delivery per 100000 ID Total Number of newborns received 7 HBNC in HD per 100000 HD Total Number of PW given Tablet Misoprostol during HD per 100000 HD Th i d i l f f ll i d i id 19 Total Number of Home Deliveries attended by SBA per 100000 HD Total Number of newborns received 6 HBNC visits after Institutional Delivery Total Number of newborns received 7 HBNC in HD per 100000 HD Total Number of PW given Tablet Misoprostol during HD per 100000 HD There is decrease in prevalence of following during covid-19 era: There is decrease in prevalence of following during covid-19 era: Total Home Deliveries attended by Non SBA per 100000 HD Total HD per 100000 TD and Total (Out of total ID) women discharged within 48 hours per 100000 ID. Total HD per 100000 TD and Total (Out of total ID) women discharged within 48 hours per 100000 ID. Results of Deliveries-TD (HD+ ID) registered per 100000 ANC registered was 72582.21092, 73775.82734, 74551.43436, 77036.94229, for 2018, 2019, 2020 and 2021 respectively whereas prevalence for total number of Institutional Deliveries (ID) conducted (Including C-Sections) per 100000 total delivery was 93024.83501, 89879.98671, 94712.47071, 95213.22797 for the years 2018, 2019, 2020 and 2021 respectively see table-10 figure-2 and 3. Regarding total HD per 100000 TD the prevalence was 6975.164987, 5763.128328, 5287.529291, 4786.772034, and total Number of Home Deliveries attended by SBA per 100000 HD the prevalence were 17596.50896, 18346.27048, 18299.26738, and 19875.05884 for 2018, 2019, 2020 and 2021 respectively. Total Home Deliveries attended by Non SBA per 100000 HD was 82403.49104, 81653.72952, 81700.73262, 80124.94116, and total (Out of total ID) women discharged within 48 hours per 100000 ID were 35089.01271, 35541.40151, 33593.6996, 33520.62498, for 2018, 2019, 2020 and 2021 respectively. Total Number of PW given Tablet Misoprostol during HD per 100000 HD for 2018, 2019, 2020 and 2021 respectively was 5545.623572, 5985.891135, 7306.860868, 7906.019126, and total Number of newborns received 7 HBNC in HD per 100000 HD was 64566.98655, 75365.87328, 82709.5773, 99408.3195, respectively see table-10 figure-2 and 3. The total Number of newborns received 6 HBNC visits after Institutional Delivery per 100000 ID was 33035.26524, 41166.49374, 44830.18139, 52033.76294, for 2018, 2019, 2020 and 2021 respectively. This research study revealed that during covid-19 era there is increase in prevalence of following: The prevalence of total No. of Deliveries-TD (HD+ ID) registered per 100000 ANC registered was 72582.21092, 73775.82734, 74551.43436, 77036.94229, for 2018, 2019, 2020 and 2021 respectively whereas prevalence for Total No. of Deliveries-TD (HD+ ID) registered per 100000 ANC registered Total No. of Deliveries-TD (HD+ ID) registered per 100000 ANC registered Total No. of Deliveries-TD (HD+ ID) registered per 100000 ANC registered Total No. Conclusion And Recommendation The ongoing COVID-19 pandemic has had less number of Deliveries and hence less Home Based New Born Care (HBNC) in India. More research is required to investigate the direct and indirect consequence of the pandemic on birth and provision of HBNC as well as the health facility type which are not performing well in providing respectful maternity care and newborn services during pandemic period. The facility based research analysis is under process by the author which will be available in next version of this article. The author recommends following: Establishment of exclusive specialised birth centres as pregnant women feels usually worried about giving birth in multipurpose hospitals. Establishment of exclusive specialised birth centres as pregnant women feels usually worried about giving birth in multipurpose hospitals. Helping pregnant women to receive ANC checkups, delivery care, postnatal care Helping pregnant women to receive ANC checkups, delivery care, postnatal care, COVID-19 care as needed; Page 13/23 Ensuring availability of skilled health workers with PPE (personal protective equipment), priority testing and vaccination so that they can deliver care to all pregnant women and newborn babies without spreading Ensuring availability of skilled health workers with PPE (personal protective equipment), priority testing and vaccination so that they can deliver care to all pregnant women and newborn babies without spreading Page 13/23 Page 13/23 infection during the pandemic; Application of infection prevention and control measures in health facilities during childbirth and care immediately after; Raising awareness among pregnant women through home visits, Encouraging women and their relatives in remote areas to use maternal health Encouraging women and their relatives in remote areas to use maternal health facilities and HBNC Encouraging women and their relatives in remote areas to use maternal health facilities and HBNC Training, protecting and capacity building of health workers with clean birth kits for home births when health f l l bl l d Encouraging women and their relatives in remote areas to use maternal health facilities and HBNC Training, protecting and capacity building of health workers with clean birth kits for home births when health facilities are not available or closed; Training, protecting and capacity building of health workers with clean birth kits for home births when health facilities are not available or closed; Allocating more resources to maternity and newborn services and supplies. Allocating more resources to maternity and newborn services and supplies. Conclusion And Recommendation Educating PW to protect themselves from exposure to covid-19, and take advice from the designated health facility if experience symptoms; Seek medical care timely from accredited health facility and avoid jhola-chaap(fake medical practioners) as well as quacks if they live in remote areas and have fever, cough or difficulty breathing; Continue breastfeeding their baby as the virus has not been found in samples of breast milk [4]. Wear a mask while feeding baby; wash hands; and routinely clean and disinfect surfaces; if not Covid positive continue to hold the newborn as well as perform skin-to-skin care; Ask designated doctor and health staff the safest place to give birth; Have a birth plan in advance to reduce stress and to ensure place on time; Routine immunizations, after the baby is born. -Author contact information Department of Health, Government of Bihar, MOBILE - +919955301119/+917677833752, Email drpiyush003@gmail.com Department of Health, Government of Bihar, MOBILE - +919955301119/+917677833752, Email drpiyush003@gmail.com Funding - Authors' contributions: The whole work is done by the Author - Dr Piyush Kumar, M.B.B.S., E.M.O.C., P.G.D.P.H.M., - Senior General Medical Officer- Bihar Health Services- Health Department- Government of Bihar, India. - Acknowledgements- I am thankful to Advocate Anupama my wife and daughters Aathmika-Atheeva for cooperation. This article is available only as pre-print and not published by any peer-reviewed journals. The author has written previously on this research which is available as preprint. There are chances of text and data overlapping with author previous works available as preprints only mentioned above in the references [5, 6, 7, 8, and 9, 10] -Financial Support & sponsorship: Nil Funding The author declares that no funds are taken from any individual or agency-institution for this research study. The author declares that no funds are taken from any individual or agency-institution for this research study. Note: - This article is available only as pre-print and not published by any peer-reviewed journals. The author has written previously on this research which is available as preprint. There are chances of text and data overlapping with my previous works available as preprints mentioned below in the references [5, 6, 7, 8, and 9, 10] -This version of paper has not been previously published in any peer reviewed journal and is not currently under consideration by any journal. The document is Microsoft word with English (United States) language & 3500 words excluding reference and declaration etc. (7027 words Total including all). -This version of paper has not been previously published in any peer reviewed journal and is not currently under consideration by any journal. The document is Microsoft word with English (United States) language & 3500 words excluding reference and declaration etc. (7027 words Total including all). - Ethics approval and consent to participate: Not applicable. This study has not involved any human or animals in real or for experiments. The submitted work does not contain any identifiable patient/participant information. - Ethics approval and consent to participate: Not applicable. This study has not involved any human or animals in real or for experiments. The submitted work does not contain any identifiable patient/participant information. -Consent for publication: The author provides consent for publication. -Consent for publication: The author provides consent for publication. -Availability of data and materials: Electronic records from HMIS (health management information system) of MoHFW (ministry of health and family welfare), Government of India. -Availability of data and materials: Electronic records from HMIS (health management information system) of MoHFW (ministry of health and family welfare), Government of India. -Conflicts of Interest/ Competing Interest: There are no conflicts / competing of interest cts of Interest/ Competing Interest: There are no conflicts / competing of interest Page 14/23 Page 14/23 - Funding-Self sponsored. No aid taken from individual or agency etc. - Authors' contributions: The whole work is done by the Author - Dr Piyush Kumar, M.B.B.S., E.M.O.C., P.G.D.P.H.M., - Senior General Medical Officer- Bihar Health Services- Health Department- Government of Bihar, India. 8. Author own preprints Kumar, D. (2022, January 31). What is the impact of COVID-19 pandemic years on Deliveries and Home Based New Born Care in India? Retrieved from osf.io/zkavs https://osf.io/zkavs/ 8. Author own preprints Kumar, D. (2022, January 31). What is the impact of COVID-19 pandemic years on Deliveries and Home Based New Born Care in India? Retrieved from osf.io/zkavs https://osf.io/zkavs/ 8. Author own preprints Kumar, D. (2022, January 31). What is the impact of COVID-19 pandemic years on Deliveries and Home Based New Born Care in India? Retrieved from osf.io/zkavs https://osf.io/zkavs/ 9. Author own preprints Dr Piyush Kumar. What is the impact of COVID-19 pandemic years on Deliveries and Home Based New Born Care in India? A cross-sectional comparative research study, Jan 2018-2021Dec, 18 February 2022, PREPRINT (Version 2) available at Research Square https://doi.org/10.21203/rs.3.rs-1314021/v2 10. Kumar, D. (2022, February 18). What is the impact of COVID-19 pandemic years on Deliveries and Home Based New Born Care in India? A cross-sectional comparative research study, Jan 2018- 2021Dec. https://doi.org/10.31219/osf.io/vxe49 Abbreviations Home Based New Born Care (HBNC); COVID-19- corona virus disease 2019; SARS-CoV-2- severe acute respiratory syndrome coronavirus 2; SBA [(Skill Birth Attendant); HMIS (health management information system); MoHFW (ministry of health and family welfare); HD – home delivery; PW- pregnant women; ID- institutional delivery; PPE (personal protective equipment); ANC – antenatal care; EMOC - emergency obstetric care; References 1. Pregnant mothers and babies born during COVID-19 pandemic threatened by strained health systems and disruptions in services - https://www.unicef.org/press-releases/pregnant-mothers-and-babies-born-during-covid-19- pandemic-threatened-strained-health 2. Newborn and child health – UNICEF – available at https://www.unicef.org/india/what-we-do/newborn-and-child- health 3. Dr. Piyush Kumar, What is the impact of Covid-19 on the Antenatal Care Services Utilization in Public-Private- Rural-Urban Hospitals of India during the COVID-19 Pandemic Period of 2020-2021 compared to pre-pandemic era 2018-2019? Dr. Piyush Kumar, MODERN APPLIED MEDICAL RESEARCH, 2022 2(2), pp.01-10; DOI Number: 10.36099/mamr.220522 ; HTML- https://gulfpublishers.com/text-html/242 ; 5.Author own preprints Kumar, Piyush and Kumar, Piyush, What is the Impact of COVID-19 Pandemic Years on Deliveries and Home Based New Born Care in India?. Available at SSRN: https://ssrn.com/abstract=4023226 or http://dx.doi.org/10.2139/ssrn.4023226 Page 15/23 Page 15/23 Annexure Annexure----Table –11, 12, 13, 14 (Total Observations) Table- 11 – Total Observation with forecast (Jun to Dec) for the year - 2021 Annexure----Table –11, 12, 13, 14 (Total Observations) Page 16/23 Page 16/23 2021 Number of Home Deliveries attended by Skill Birth Attendant(SBA) (Doctor/Nurse/ANM/ Midwife) Home Deliveries in no. Table-13 - Observation for the year 2019 Table- 14 - Observation for the year 2018 Annexure attended by Non SBA (Trained Birth Attendant (TBA) /Relatives/etc.) Number of PW given Tablet Misoprostol during home delivery Number of newborns received 7 Home Based Newborn Care (HBNC) visits in case of Home delivery Number of Institutional Deliveries conducted (Including C- Sections) Out of total institutional deliveries number of women discharged within 48 hours of delivery Number of newborns received 6 HBNC visits after Institutional Delivery Jan- 21 17301 75810 6629 81396 1615041 551895 829656 Feb- 21 15975 70376 6434 78307 1452146 517895 781658 Mar- 21 15791 68104 5826 77450 1826745 589308 817272 Apr- 21 13263 53151 5251 62140 1247082 422764 656825 May- 21 15125 63284 6189 66498 1263285 403324 651878 Jun- 21 13336.22747 53727.38473 5353.22939 64972.17675 1290810.996 436629.3658 684528.3665 Jul- 21 12899.79742 50847.59968 5191.408807 63609.23421 1252172.47 423582.2049 669984.7213 Aug- 21 12356.961 47832.20161 5067.426715 63168.86238 1224416.143 409968.2942 655277.1404 Sep- 21 12169.6793 46936.77135 4893.840417 62427.89444 1241652.183 414920.7519 659632.6303 Oct- 21 11122.22832 41081.52452 4468.987951 61464.34617 1227940.099 408929.0559 655365.6287 Nov- 21 10827.71983 39329.68095 4376.97079 60834.50528 1208184.943 402083.8468 646895.6104 Dec- 21 10275.73918 36336.45906 4141.266552 60215.56907 1207656.2 401150.9866 646157.2941 Table- 12 - Observation for the year 2020 Page 17/23 Page 17/23 Page 17/23 Indicator Number of Home Deliveries attended by SBA Home Deliveries attended by Non SBA Number of PW given Tablet Misoprostol during HD Number of newborns received 7 HBNC in HD Number of Institutional Deliveries conducted (Including C- Sections) Out of total ID women discharged within 48 hours Number of newborns received 6 HBNC visits after Institutional Delivery Total Jan 2020 17364 81458 6452 86904 1686421 564526 765405 Total Feb 2020 17321 75444 5690 84736 1534676 518676 738549 Total Mar 2020 14060 67611 5134 76170 1527869 511141 695368 Total April 2020 8047 33185 4251 28639 1219729 364659 461874 Total May 2020 14364 66359 5763 60682 1395381 435889 584777 Total Jun 2020 14946 67389 6253 64834 1409429 442182 618175 Total Jul 2020 15200 71088 6724 66954 1576238 517349 665568 Total Aug 2020 19355 80333 7479 75412 1725793 603254 740827 Total Sep 2020 20183 86457 8580 85804 1811424 648151 798730 Total Oct 2020 19388 84215 7991 86738 1849115 643160 852982 Total Nov 2020 18412 85274 7567 87890 1813584 627710 860193 Total Dec 2020 18511 81407 6838 86326 1748668 606325 869027 Table-13 - Observation for the year 2019 Table-13 - Observation for the year 2019 Page 18/23 Indicator Number of Home Deliveries attended by SBA Home Deliveries attended by Non SBA Number of PW given Tablet Misoprostol during HD Number of newborns received 7 HBNC in HD Number of Institutional Deliveries conducted (Including C- Sections) Out of total ID women discharged within 48 hours Number of newborns received 6 HBNC visits after Institutional Delivery Total Jan 2019 20559 90459 6959 77446 1660316 586521 609397 Total Feb 2019 19620 89852 6239 77616 1472340 523286 578978 Total March 2019 20325 91917 6504 80964 1717500 566155 614187 Total April 2019 16395 70630 5253 63616 1409794 443409 542151 Total May 2019 16387 75644 5370 67538 1544936 494842 581991 Total Jun 2019 17670 78944 5556 70976 1548487 501656 614269 Total Jul 2019 17582 82948 6255 72506 1710352 567274 662672 Total Aug 2019 20793 90982 6288 82663 1895697 671254 727880 Total Sep 2019 20572 88290 6305 85882 1871061 660643 760083 Total Oct 2019 20213 88023 6681 85424 1879063 652288 781203 Total Nov 2019 19661 85232 6490 85172 1848889 624935 752457 Total Dec 2019 18302 82190 6516 87138 1769819 598650 756261 Table- 14 - Observation for the year 2018 Table- 14 - Observation for the year 2018 Page 19/23 Page 19/23 Indicator Number of Home Deliveries attended by SBA Home Deliveries attended by Non SBA Number of PW given Tablet Misoprostol during HD Number of newborns received 7 HBNC in HD Number of Institutional Deliveries conducted (Including C- Sections) Out of total ID women discharged within 48 hours Number of newborns received 6 HBNC visits after Institutional Delivery Total Jan 2018 24266 116307 7564 85372 1537515 580841 495132 Total Feb 2018 22174 105130 6770 81164 1414812 533569 480260 Total March 2018 22682 104756 6826 83942 1559206 556698 505544 Total April 2018 19091 84554 5411 68640 1363935 448374 440685 Total May 2018 19981 89514 5599 69383 1479816 485796 478596 Total Jun 2018 19993 91476 6225 70263 1455342 478528 492563 Total Jul 2018 20255 95981 7194 74076 1590046 543513 512433 Total Aug 2018 21100 100816 7054 76342 1795922 643962 551799 Total Sep 2018 23731 110616 7066 87132 1845585 660925 596423 Total Oct 2018 22394 105652 7189 85570 1856881 660268 622508 Total Nov 2018 21046 103762 7084 85039 1802810 641385 624944 Total Dec 2018 19101 89399 6639 71737 1686541 569343 604126 Page 20/23 Figure 1 Comparisons and trends of different variables during pandemic and pre-pandemic years Figure 1 Comparisons and trends of different variables during pandemic and pre-pandemic years Comparisons and trends of different variables during pandemic and pre-pandemic years Page 21/23 Figure 2 Figure 2 Comparison and trends of Prevalence of different study variables per 100000 in different years Comparison and trends of Prevalence of different study variables per 100000 in different years Comparison and trends of Prevalence of different study variables per 100000 in different years Page 22/23 Figure 3 Figure 3 Comparison and trends of Prevalence of different study variables per 100000 in different years Comparison and trends of Prevalence of different study variables per 100000 in different years Page 23/23
https://openalex.org/W2055145672	https://zenodo.org/records/1791753/files/article.pdf	English	null	XXIX.—<i>Note on a variation in the number of genital pouches in</i> Thalassema neptuni, <i>Gaertner</i>	Annals & magazine of natural history	1,900	public-domain	1,174	XXIX.—Note on a variation in the number of genital pouches in Thalassema neptuni, Gaertner F.H. Stewart M.A. a a Gatty Marine Laboratory , St. Andrews Published online: 28 Sep 2009. To cite this article: F.H. Stewart M.A. (1900) XXIX.—Note on a variation in the number of genital pouches in Thalassema neptuni, Gaertner , Annals and Magazine of Natural History: Series 7, 6:32, 218-219, DOI: 10.1080/00222930008678366 To link to this article: http://dx.doi.org/10.1080/00222930008678366 To cite this article: F.H. Stewart M.A. (1900) XXIX.—Note on a variation in the number of genital pouches in Thalassema neptuni, Gaertner , Annals and Magazine of Natural History: Series 7, 6:32, 218-219, DOI: 10.1080/00222930008678366 To link to this article: http://dx.doi.org/10.1080/00222930008678366 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions ded by [New York University] at 18:29 15 April 2015 k University] at 18:29 15 April 2015 ty] at 18:29 15 April 2015 218 On tire Genital Poudtes in Thalassema neptuni. XXIX.--.Noteon a Variation in the Number of Genital Pouches in Thalassema neptani, Gaertner. By F. H. STEWAr:T, M.A., Garry Marine Laboratory, St. Andrews. WHILE dissecting a speclmenof Thalassema neptttn[, Gaertner, I observed that it varied fl'om the normal in possessing five genital pouches instead of four, one being placed in the middle dorsal line, the remaining four lying, as usual, two on each side. On a new Species of Bupresti&e. This point is illustrated by the accompanying diagram. It would appear from this comparison that the unpaired genital pouch is interpolated between the two normal pairs; and if this be granted it would have to be assumed either that a segment which does not normally bear pouches intervenes between the two which normally do, or that a partial dupli- cation of tile nephridia of one segment has occurred--a pheno- menon which we find in its complete form in the family of the Capitellida~. p Mr. Punnett, Assistant Professor of Natural History in the University of St. Andrews, who kindly gave me the abnormal and the various normal specimens, inibrms me that they were all obtained near Plymouth in the same locality. There can thus be no doubt that this case is an actual variation~ not a local variety. oaded by [New York University] at 18:29 15 April 2015 Downloaded by [New York University] at 18:29 XXX.--Deseriptlon of a new Species of Buprestidm. By CnAs. O. WATERI~OUSE, V.P.E.S. SPECIMENS of the species described below have been in the Museum collection for many years, separated as distinct fi'om .PMloptera quadrioeulota. A fresh specimen just brought from Upper Egypt by Mr. D. A. MacAlister shows that the differences between this and P. quadrioeulata are constant, and 1 therefore venture to give it a name, and I propose to call it .P. MaeAlisteri after ~he donor. PLEASE SCROLL DOWN FOR ARTICLE But on examination by means of" sections the pouch which appeared to be median proved to belong to the right side and to correspond to the anterior pouch on the left. Behind these on the right side came an unpaired nephridium, followed by a second normal pair. Downloaded by [New York University] at 18:29 15 April 2015 y p The question then arose, In what relations did the five nephridia of this specimen stand to the normal four ? Was the unpah'ed pouch interpolated between the normal two pairs, or did it represent the second normal pair, the fourth and fifth pouches being an additional pair ? It was obviously impossible to obtain any direct proof' on this point; but in order to obtain some indication I measured the distances between the successive nephridiopores in the abnormal and in several normal specimens, at the same time taking the distance between the genital hooks and the first parr of nephridiopores as a standard of comparison. In the abnormal specimen the distances were as tbtlows :--(a) Genital hooks to first pair of pores "5 millim. (b) First pair to unpaired "25 millim. (c) :First pair to second pair "7 millim. The relation of distance (a) to distance (b) is thus 2 : 1~ while that of (a) to (c) is 5 : 7. ( ) ( ) In the normal specimens the relation of the averages of the distances were :--Distance of genital hooks from first pair of pores to distance between two pairs as 2 to 3, i. e. "5 to '75. "25 × o o o Abnormal. × 0 o • 5 "7 × 0 0 Normal (mean). X 0 0 • 5 "7.5 ( X =genital hooks ; 0 = net)hridiopores. ) Abnormal. ( X =genital hooks ; 0 = net)hridiopores. ) 219 On a new Species of Bupresti&e. JPsiloptera MacAl/steri~ sp. n. P. quadrioculatce valde afllnis ; vitta obliqua thoraeis, elytrorumque vitta laterali cupreo-rubris; corpore subtus tomento griseo-albo induto, utrinque maculis majoribus circularibus ornate. Long. 20-27 mili. Very similar to _P. ~uadrioeulata~ but perhaps a little less convex. The general blackish-~eneous eolour is the same, but the impressions on the thorax and lateral stripe of the elytra are eoppery red. The front of the head is clothed with pale yellow pile. Tile thorax is moderately closely and strongly punctured, with a slight raised median line, and four rotundate, smooth~ black spots as in P. Tladrioculata~ but tile rugose surface surrounding them is coppery red and forms a distinct oblique band from the anterior spot to the bas% leaving a triangular space at the posterior angle slight]~y
https://openalex.org/W2965911581	https://www.frontiersin.org/articles/10.3389/fmats.2019.00169/pdf	English	null	Compressive Response of Non-slender Octet Carbon Microlattices	Frontiers in materials	2,019	cc-by	8,956	Compressive Response of Non-slender Octet Carbon Microlattices Akira Kudo 1, Diego Misseroni 2, Yuchen Wei 1 and Federico Bosi 3 1 Division of Engineering and Applied Science, California Institute of Technology, Pasadena, CA, United States, 2 Department of Civil, Environmental and Mechanical Engineering, University of Trento, Trento, Italy, 3 Department of Mechanical Engineering, University College London, London, United Kingdom Lattices are periodic three-dimensional architected solids designed at the micro and nano-scale to achieve unique properties not attainable by their constituent materials. The design of lightweight and strong structured solids by additive manufacturing requires the use of high-strength constituent materials and non-slender geometries to prevent strut elastic instabilities. Low slenderness carbon octet microlattices are obtained through pyrolysis of polymeric architectures manufactured with stereolithography technique. Their compressive behavior is numerically and experimentally investigated when the relative density ¯ρ ranges between 10 and 50%, with speciﬁc stiffness and strength approaching the limit of existing micro and nanoarchitectures. It is shown that additive manufacturing can introduce imperfections such as increased nodal volume, non-cubic unit cell, and orientation-dependent beam slenderness, all of which deeply affect the mechanical response of the lattice material. An accurate numerical modeling of non-slender octet lattices with signiﬁcant nodal volumes is demonstrated to overcome the limitations of classical analytical methods based on beam theory for the prediction of the lattice stiffness, strength and scaling laws. The presented numerical results and experimental methods provide new insights for the design of structural carbon architected materials toward ultra-strong and lightweight solids. Reviewed by: Reviewed by: Dongchan Jang, Korea Advanced Institute of Science and Technology (KAIST), South Korea Anastasiia O. Krushynska, University of Groningen, Netherlands Correspondence: Diego Misseroni diego.misseroni@unitn.it *Correspondence: Diego Misseroni diego.misseroni@unitn.it Keywords: architected materials, additive manufacturing, structural metamaterials, pyrolyzed lattices, mechanics Specialty section: Specialty section: This article was submitted to Mechanics of Materials, a section of the journal Frontiers in Materials Additive manufacturing has become one of the most promising technique to fabricate advanced materials and microstructures that exhibit properties unattained by homogeneous solids or conventionally manufactured architectures. The available 3D printing techniques have recently grown and comprise fused deposition modeling (FDM), direct ink writing (DIW), selective laser sintering (SLS), stereolithography (SLA), etc. Similarly, the selection of materials compatible with these processes has expanded and include thermoelastic polymers (Carneiro et al., 2015), transparent glasses (Nguyen et al., 2017), oxide ceramics (Wilkes et al., 2013), metallic alloys (Schwab et al., 2016), and composites (Spierings et al., 2015; Ni et al., 2018; Quintanilla et al., 2018). The precise micro- and nano-scale topology control achievable through additive manufacturing has allowed the development of unique functionalities to catalysis (Essa et al., 2017), batteries (Xia et al., 2016; Li et al., 2017), scaﬀolds (Maggi et al., 2017), biomedical implants (Murr et al., 2010), and Received: 14 January 2019 Accepted: 01 July 2019 Published: 31 July 2019 Edited by: Seunghwa Ryu, Edited by: Seunghwa Ryu, Korea Advanced Institute of Science and Technology (KAIST), South Korea ORIGINAL RESEARCH published: 31 July 2019 doi: 10.3389/fmats.2019.00169 Keywords: architected materials, additive manufacturing, structural metamaterials, pyrolyzed lattices, mechanics Citation: Kudo A, Misseroni D, Wei Y and Bosi F (2019) Compressive Response of Non-slender Octet Carbon Microlattices. Front. Mater. 6:169. doi: 10.3389/fmats.2019.00169 July 2019 \| Volume 6 \| Article 169 Frontiers in Materials \| www.frontiersin.org 1 Mechanics of Octet Carbon Microlattices Kudo et al. metamaterials (Hengsbach and Lantada, 2014; Misseroni et al., 2016; Bertoldi et al., 2017; Bilal et al., 2017). In particular, the ﬁeld of architected material has beneﬁted from the advancement of small-scale manufacturing that enables the design of multistable solids for energy storage (Shan et al., 2015), the evolution of phononic bandgap behavior (Sugino et al., 2015; Amendola et al., 2018) and the exploration of previously inaccessible mechanical property combinations (Bauer et al., 2016). Examples include structural metamaterials designed to achieve extremely lightweight and strong solids through a hierarchical design (Meza et al., 2015) or novel highly deformable and recoverable nanolattices made up of brittle materials (Meza et al., 2014). preventing the production of micro- and nano-architectures at a large scale. Carbon microlattices produced by self- propagating photopolymer waveguides (Jacobsen et al., 2011) and stereolithography (SLA) (Chen et al., 2017) overcome the scalability diﬃculties toward faster manufacturing of larger scale lattices. However, their mechanical performances are still limited, and the development of enhanced architected solids demands further understanding of the inﬂuence of the manufacturing-induced imperfection on the mechanics of 3D- printed carbon lattices. p The aim of this work is to manufacture stiﬀand strong non-slender octet carbon microlattices through digital light processing stereolithography (DLP-SLA), and to analytically, computationally, and experimentally investigate their compressive mechanical properties and scaling laws. We show that DLP-SLA 3D printing and pyrolysis techniques can aﬀect the designed lattice architecture introducing undesired features as increased nodal volume, non-cubic unit cell and diﬀerent strut slenderness depending on the beam orientation with respect to the printing direction. We investigate the inﬂuence of these factors on the compressive stiﬀness and strength of non-slender lattices with relative density ¯ρ that ranges between 10 and 50%. We prove the inappropriateness of classical analytical tools based on beam theory and the derived expressions for non-slender architectures with negligible eﬀective Poisson’s ratio, due to the topological features that are not accounted for in these formulations. Citation: We develop computational models that faithfully predicts the experimental lattice response by reproducing the manufactured geometry and we demonstrate that an accurate numerical modeling of non-slender octet lattices with signiﬁcant nodal volumes allows to identify the deviation from classical scaling laws and enables a proper design of advanced structural DLP-SLA 3D printed carbon architectures. p Structured solids can be classiﬁed as rigid or non-rigid architectures depending on their nodal connectivity, states of self stress, and mechanisms (Pellegrino and Calladine, 1986). The former includes octet lattices and shows a stretching dominated behavior, while the latter mostly presents a bending dominated response as demonstrated by pyramidal lattices. The response of architected materials has been extensively analyzed through the investigation of their constituent unit cells using beam theory to obtain the lattice eﬀective stiﬀness and strength scaling laws (Gibson and Ashby, 1997; Deshpande et al., 2001). These analytical tools have been proven to well predict the mechanical response of several lattices when the relative density ¯ρ is lower than 0.1 and the strut slenderness ratio r/l does not exceed 0.06 (Meza et al., 2017). However, some computational and experimental studies (Schaedler et al., 2011; Meza et al., 2015; Bauer et al., 2016) have recently reported deviations from the classical scaling laws due to non-slender struts and the inﬂuence of the node geometry (Portela et al., 2018), thus proposing diﬀerent scaling laws. The diﬃcult micro- and nano-scale fabrication of slender structured solids that obey to classical scaling laws motivates the investigation of non-slender architectures with pronounced nodal volume caused by an imperfect 3D printing. Therefore, the study of their mechanical properties is fundamental for the design of stronger lattices that do not suﬀer from strut elastic instabilities. Frontiers in Materials \| www.frontiersin.org 2.1. Sample Fabrication 2.1. Sample Fabrication p Three sets (A, B, and C) of carbon octet microlattices were manufactured by pyrolyzing polymeric lattices fabricated with a DLP-SLA Autodesk Ember 3D printer that employs a PR-48 transparent photoresist resin. The periodic polymeric 3D printed specimens consisted of a 10 × 3 × 6 (length × width × height) tassellation of 900 µm octet unit cell with three diﬀerent strut radii of rA = 52.8 µm, rB = 71.4 µm, and rC = 90.0 µm (Figure 1). The microlattices presented a theoretical relative density ¯ρA = 0.16, ¯ρB = 0.27, ¯ρC = 0.40, and a beam slenderness ratio of r/l A = 0.08, r/l B = 0.11, r/l C = 0.14. The DLP-SLA layers were deposited along the height direction and the bottom-most anchoring layer was designed to be thicker in order to sustain the microlattice. Prior to pyrolysis, the anchoring layer of each microstructure was removed using a razor blade to prevent lattice distortion, thus resulting in a 10 × 3 × 5 tassellation. The polymeric lattices were inserted in a fused quartz tube set on a Lindberg tube furnace (model 54357) and pyrolyzed under vacuum. During pyrolysis, the furnace temperature was ﬁrst raised to 300◦C and held constant for 4 h, then increased to 400◦C and maintained for 1 h, and ﬁnally elevated to 1,000◦C One of the most promising materials to fabricate extremely lightweight and resistant architected solids is carbon, which has recently become compatible with additive manufacturing processes. Direct ink writing (DIW) with printable inks that contain graphene, carbon nanotube, and graphene oxide (Fu et al., 2017) has been employed for the realization of ﬂexible, conductive, and chemically stable prototypes (Sun et al., 2013; Zhu et al., 2015; Yao et al., 2016; Zhang et al., 2016), while 3D-printed carbon ﬁber reinforced composites have been manufactured by means of FDM (Lewicki et al., 2017; Anwer and Naguib, 2018). Carbon nano- and micro-lattices are another form of 3D printed carbon which have demonstrated elevated structural performances. Architected carbon materials are obtained by pyrolyzing 3D-printed precursor, especially polymer lattices prepared by photocuring techniques. Carbon nanolattices fabricated through two-photon lithography have shown a strength comparable with the theoretical strength of ﬂaw insensitive glassy carbon (Bauer et al., 2016). July 2019 \| Volume 6 \| Article 169 2.2. Microstructural Characterization The polymeric and carbon microlattices obtained after 3D printing and pyrolysis were investigated using a Thermo- Fisher Versa 3D DualBeam Scanning Electron Microscopy (SEM). Specimen sizes, unit cell radii and strut diameters were measured to assess any imperfection and anisotropy introduced during fabrication. The compressive tests on the carbon microlattices were performed using an Instron 5569 electromechanical machine. The load P was applied by imposing a constant displacement rate of 2.5 µm/s on the 5 × 10 unit cells sample surface and was measured with an Instron 2525- 804 load cell (R.C. 10 kN). The compressive displacement 1y was evaluated with an LE-01 (Electronic Instrument Research) laser extensometer interfaced with the electromechanical testing frame for data synchronization. Five samples for each set of octet density were characterized and tested along the y-direction. The nominal lattice stress σ was obtained dividing the applied load P by the specimen footprint area, whereas the nominal strain ǫ was calculated from the initial sample height H as ǫ = 1y/H. 3.1. Manufacturing The geometrical features of the three sets of non-slender 3D- printed polymeric microlattices were investigated by using the SEM. From the images in Figure 3, it was observed that the additive manufacturing technique introduced undesirable lattice imperfections not present in the original computer-aided design (CAD) input ﬁles, where the lattices were constituted of cubic octet unit cells with strut radius r and length l. In particular, the height h of the unit cell resulted smaller than the width w, leading to a non-cubic unit cell. Therefore, the length l1 of the struts deposited along the width direction (x–y plane) was greater than the length l2 of the inclined struts 3D-printed along the height direction (z-direction). Similarly, the in-plane strut radius r1 resulted bigger than the out-of-plane beam radius r2. Moreover, 2.1. Sample Fabrication This printing technique solidiﬁes the polymeric precursor solution point- by-point at a submicron scale in a prolonged process, thus July 2019 \| Volume 6 \| Article 169 July 2019 \| Volume 6 \| Article 169 2 Kudo et al. Mechanics of Octet Carbon Microlattices FIGURE 1 \| (A) CAD of the microlattice constructed through a three-dimensional tasselation of octet unit cells. (B) A cubic octet unit cell with its geometric parameters l (strut length) and r (strut radius). FIGURE 1 \| (A) CAD of the microlattice constructed through a three-dimensional tasselation of octet unit cells. (B) A cubic octet unit cell with its geometric parameters l (strut length) and r (strut radius). diﬀerent relative density were modeled to investigate the stiﬀness and strength scaling laws. The microlattices were discretized with linear elastic second-order tetrahedral elements (C3D10) with Young’s modulus E = 25.38 GPa (Kudo et.al., in preparation) and Poisson coeﬃcient ν = 0.21 (Price and Kaae, 1969). Compressive forces were applied at the top nodes of the unit cell, with F acting on the central node and F/4 on the lateral nodes shared with four adjacent unit cells. The eﬀective lattice Young’s modulus E∗ y = E∗ x in the y-direction was calculated as the ratio between the average compressive stress on the unit cell σ avg y = 2F/wh and the average compressive strain ǫavg y = δy/w, where δy represents the displacement in the y-direction, w and h are the width and height of the unit cell (Figure 2). Similarly, the eﬀective Young’s modulus E∗ z in the z-direction was obtained as the ratio between the average compressive stress σ avg z = 2F/w2 and the average compressive strain ǫavg z = δz/h, where δz represents the displacement in the z-direction. Furthermore, a linear perturbation buckling analysis was conducted on each unit cell to assess the microlattices critical buckling strength. and kept constant for 4 h. This procedure, with all heating rates carried out at 10◦C/min, led to fully dense microlattices without gasiﬁed components and preserved the 3D printed topology. Frontiers in Materials \| www.frontiersin.org 2.3. Finite Element Analysis Full three-dimensional ﬁnite element analyses were performed in ABAQUS Standard 2018 in order to simulate the compressive behavior of the microlattices. Numerical simulations were carried out on representative octet unit cells loaded in the y-direction and constrained with boundary conditions that reproduce the response of the unit cell within the lattice. The three-dimensional unit cells were parametrically designed in SolidWorks to reproduce the measured geometrical parameters such as node and beam radii, unit cell height and width, and ﬁllet junction radii between nodes and struts. In addition to the unit cells representative of the fabricated microlattices, other unit cells with July 2019 \| Volume 6 \| Article 169 Frontiers in Materials \| www.frontiersin.org 3 Mechanics of Octet Carbon Microlattices Kudo et al. FIGURE 2 \| CAD isometric (A) and lateral (B) views of a representative 3D printed and pyrolyzed non-slender octet unit cell as modeled for the ﬁnite element analysis. The x-y in-plane beams present radius r1 and length l1, while the out-of-plane struts have radius r2 and length l2. The struts junction is characterized by the node radius rN and junction radius rj. FIGURE 2 \| CAD isometric (A) and lateral (B) views of a representative 3D printed and pyrolyzed non-slender octet unit cell as modeled for the ﬁnite element analysis. The x-y in-plane beams present radius r1 and length l1, while the out-of-plane struts have radius r2 and length l2. The struts junction is characterized by the node radius rN and junction radius rj. FIGURE 3 \| SEM images of the DLP-SLA manufactured polymeric microlattices A (A), B (B), and C (C) observed from the x-z plane. The imperfections introduced by additive manufacturing, visible in all the samples, are orientation dependent strut slenderness, increased nodal volume, and corrugation on the beam surface. FIGURE 3 \| SEM images of the DLP-SLA manufactured polymeric microlattices A (A), B (B), and C (C) observed from the x-z plane. The imperfections introduced by additive manufacturing, visible in all the samples, are orientation dependent strut slenderness, increased nodal volume, and corrugation on the beam surface. unit cell CAD geometry employed in the numerical simulations (Table 1). Figure 4 shows progressive magniﬁcation images which testify that the features present in the original polymeric lattices were preserved throughout the heat treatment for all lattices. Frontiers in Materials \| www.frontiersin.org 2.3. Finite Element Analysis It was observed that the strut slenderness after pyrolysis slightly decreased with respect to the polymeric lattices values (maximum deviation was ≈10%), hence the heat treatment did not signiﬁcantly modify the lattice geometry. The average ratio between the unit cell height and width was measured as (h/w)A = 0.87 and (h/w)B,C = 0.97, thus showing a quasi- cubic architecture for lattices B and C. The ratio between the out-of-plane and in-plane strut radii was r2/r1 ≃0.8 and the quasi-spherical nodes presented a radius rN ≃2r1. The highest magniﬁcation images report the lateral view of the octet unit cell and are paired with the CAD geometry, showing an excellent agreement between the manufactured and simulated unit cells (Figure 4, right). The only feature not reproduced in the CAD, and therefore not accounted for in the ﬁnite element analysis, was the strut corrugation. the intersection of twelve struts in a single point produced quasi- spherical nodes with radius rN and the microlattices presented unsought corrugated surfaces (Figure 3). The deviations from the originally designed lattices were caused by the DLP-SLA 3D printer, whose resolution was limited to 50 µm in the x- y plane and the layer thickness was 25 µm. For these reasons, stereolithography appears inadequate to precisely manufacture features as small as a few tens of microns, which would require other 3D printing techniques as two-photon lithography (Meza et al., 2014; Bauer et al., 2016). The average strut slenderness for the polymeric lattices was r1/l1 p,xy A = 0.10, r1/l1 p,xy B = 0.12, and r1/l1 p,xy C = 0.15 for the struts deposited in x-y plane, and for the inclined struts printed along the z-direction r2/l2 p,z A = 0.07, r2/l2 p,z B = 0.09, and r2/l2 p,z C = 0.12. By comparing the designed struts (from the CAD input ﬁles) and the beams of the manufactured lattices, we observed a decrease in the in- plane struts slenderness and an increase in the out-of-plane beam slenderness. This discrepancy is caused by the stereolithography 3D printing. The polymeric lattices were subjected to pyrolysis that led to a ∼70% linear shrinkage and produced pyrolytic carbon microlattices (Chen et al., 2017) (Figure 4, left). The three sets of pyrolyzed lattices were examined with SEM and their averaged geometrical parameters were used to deﬁne the octet 3.2. Relative Density y The relative density of the polymeric lattices was measured before pyrolysis as ¯ρp A = 0.17, ¯ρp B = 0.27, and ¯ρp C = 0.39, thus proving that 3D printing did not alter the desired relative density. After July 2019 \| Volume 6 \| Article 169 4 Mechanics of Octet Carbon Microlattices Kudo et al. TABLE 1 \| SEM measured geometrical parameters of the octet unit cell for the manufactured carbon microlattices. Unit cell w h r1 r2 rN rj ¯ρ ¯ρCAD ¯ρt [µm] [µm] [µm] [µm] [µm] [µm] [-] [-] [-] A 230.35 200.78 15.02 11.33 32.00 26.27 0.16 0.17 0.16 B 241.68 233.72 18.38 15.15 40.53 30.79 0.23 0.23 0.23 C 251.02 244.61 24.60 19.88 46.19 33.14 0.34 0.32 0.32 The experimental relative density ¯ρ is reported together with the relative density ¯ρCAD obtained from the CAD and the relative density ¯ρt calculated through the analytical expression (1). FIGURE 4 \| Optical microscopy (left), SEM characterization (center), and CAD model (right) of the manufactured microlattices A (A), B (B), and C (C) before and after pyrolysis. Progressive magniﬁcation images show that pyrolysis preserved the features of the polymeric lattices. The comparison between the fabricated carbon unit cell and the CAD model employed for computational analysis highlights the accuracy of the reproduced architectures. TABLE 1 \| SEM measured geometrical parameters of the octet unit cell for the manufactured carbon microlattices. TABLE 1 \| SEM measured geometrical parameters of the octet unit cell for the manufactured carbon microlattices. Unit cell w h r1 r2 rN rj ¯ρ ¯ρCAD ¯ρt [µm] [µm] [µm] [µm] [µm] [µm] [-] [-] [-] A 230.35 200.78 15.02 11.33 32.00 26.27 0.16 0.17 0.16 B 241.68 233.72 18.38 15.15 40.53 30.79 0.23 0.23 0.23 C 251.02 244.61 24.60 19.88 46.19 33.14 0.34 0.32 0.32 The experimental relative density ¯ρ is reported together with the relative density ¯ρCAD obtained from the CAD and the relative density ¯ρt calculated through the analytical expression (1). FIGURE 4 \| Optical microscopy (left), SEM characterization (center), and CAD model (right) of the manufactured microlattices A (A), B (B), and C (C) before and after pyrolysis. Progressive magniﬁcation images show that pyrolysis preserved the features of the polymeric lattices. The comparison between the fabricated carbon unit cell and the CAD model employed for computational analysis highlights the accuracy of the reproduced architectures. Frontiers in Materials \| www.frontiersin.org 3.3. Stiffness The eﬀective Young’s modulus E∗of an ideal octet-truss lattice with beam radius r and length l was ﬁrst examined by Deshpande et al. (2001) as a stretching dominated solid with pin-jointed struts. Later, several authors investigated the octet lattice as a frame architecture and demonstrated that the assumption of negligible bending eﬀects is valid for a relative density ¯ρ lower than 0.1 and strut slenderness r/l lower than 0.06 (Dong et al., 2015; He et al., 2017). From the analysis of the octet unit cell free to deform laterally due to Poisson eﬀect, the lattice stiﬀness is E∗= √ 2πE r l 2 1 + 3 r l 2 . (4) (4) It should be noticed that the derived formulation (4) predicts the eﬀective stiﬀness for non-slender lattices with negligible eﬀective Poisson’s ratio more accurately than the classical expression (2). However, it can be further improved by considering the manufacturing-induced imperfections as non-cubic unit cells, orientation-dependent beam slenderness, and increased nodal volume, in order to readily provide insights on the inﬂuence of each one of these features on the lattice behavior. Therefore, the eﬀective stiﬀness E∗predicted through Equation (4) is expected to underestimate the experimental microlattice eﬀective Young’s modulus as obtained from uniaxial compression tests, testifying the inaccuracy of currently available analytical techniques and justifying the use of numerical tools for the mechanical characterization of the manufactured lattices. E∗= 2 √ 2π 3 E r l 2 Kb, (2) (2) where E is the constituent material Young’s modulus, and Kb is a coeﬃcient that accounts for bending eﬀects. In particular, for truss architectures with pin-joined struts Kb = 1, while frame lattices that show bending eﬀects have nodal rigidity Kb > 1, and Kb can be written as Figure 5 reports the stress-strain curves of the three sets of fabricated carbon microlattices, with ﬁve specimens compressed for each relative density considered. It is observed that all samples showed a brittle linear elastic response after an initial toe region. An increase of the relative density yields an higher maximum stress and strain and a lower variability of the results. The eﬀective Young’s modulus E∗ y resulted E∗ y,A = 877.90 ± 48.57 MPa for A, E∗ y,B = 1565.43 ± 66.99 MPa for B, and E∗ y,C = 2464.81 ± 129.53 MPa for C. 3.2. Relative Density length l2 converge into spherical nodes of radius rN can be written as pyrolysis, the density ρ of the carbon microlattices was measured as ρA = 0.29 ± 0.02 g/cm3, ρB = 0.43 ± 0.02 g/cm3, and ρC = 0.62 ± 0.03 g/cm3 for the three sets of manufactured microlattices. Considering the density of pyrolytic carbon ρc = 1.85 g/cm3 (Kudo et.al., in preparation), their relative densities were ¯ρA = 0.16 ± 0.01, ¯ρB = 0.23 ± 0.01, and ¯ρC = 0.34 ± 0.02. The relative density of the carbon microlattices slightly decreased with respect to the polymeric lattices, as an eﬀect of the pyrolysis. The theoretical expression for the relative density of a non-cubic octet unit cell in which in-plane cylindrical struts of radius r1 and length l1 and out-of-plane cylindrical beams of radius r2 and ¯ρt = 2 √ 2π r2 1(l1 −2rN) + 2r2 2(l2 −2rN) l2 1l2 + 5 √ 2 3 π r3 N l2 1l2 , (1) where the ﬁrst term accounts for the struts volume while the second term considers the nodal volume. Although the previous relation neglects the node-strut junctions and the beam corrugations, it well approximates the experimental relative density, with a maximum error of 4% over the three sets July 2019 \| Volume 6 \| Article 169 Frontiers in Materials \| www.frontiersin.org Mechanics of Octet Carbon Microlattices Kudo et al. FIGURE 5 \| Experimental stress-strain curves for the three sets of microlattices considered: A (A), B (B), and C (C). Five samples were tested for each relative density considered. The insets show the modeled unit cells and their stress distribution during compression. FIGURE 5 \| Experimental stress-strain curves for the three sets of microlattices considered: A (A), B (B), and C (C). Five samples were tested for each relative density considered. The insets show the modeled unit cells and their stress distribution during compression. FIGURE 5 \| Experimental stress-strain curves for the three sets of microlattices considered: A (A), B (B), and C (C). Five samples considered. The insets show the modeled unit cells and their stress distribution during compression. strain curves for the three sets of microlattices considered: A (A), B (B), and C (C). Five samples were tested for each relative dens modeled unit cells and their stress distribution during compression. 3.2. Relative Density features contribute to limit the lateral displacement of the microlattices, which experimentally showed a negligible eﬀective Poisson’s ratio ν∗. Therefore, the eﬀective Young’s modulus of non-slender cubic lattices can be obtained by considering the bending eﬀects of a laterally constrained frame unit cell. Through this approximation, equivalent to a null eﬀective Poisson’s ratio, the unit cell eﬀective Young’s modulus E∗can be written as of microlattices (Table 1). The same table reports the relative density ¯ρCAD extrapolated from the unit cell drawing developed with the measured geometrical parameters. The CAD relative density resulted in excellent agreement with the measured relative density even if it does not consider strut corrugation. July 2019 \| Volume 6 \| Article 169 3.3. Stiffness Although the achieved stiﬀnesses do not represent the highest speciﬁc stiﬀness (stiﬀness to density ratio) ever recorded, they exceed the majority of natural materials and approach the values obtained for nanolattices (Zhang et al., 2019). Figure 6A shows the measured compressive stiﬀness as a function of the lattice relative density ¯ρ. The same ﬁgure reports the microlattice stiﬀness obtained from ﬁnite element analyses performed on representative octet unit cells with the geometrical Kb = 1 + 15 r l 2 + 36 r l 4 1 + 7 r l 2 . (3) (3) These expressions obtained by means of the beam theory predict that the eﬀective Poisson’s ratio ν∗is independent of the relative density and equal to 0.33. However, numerical simulations have proven that both rigid (Tancogne-Dejean et al., 2016) and non- rigid (Thiyagasundaram et al., 2010) architectures experience a decrease of ν with an increase of ¯ρ. Lattices with high relative density behave as bending-dominated structures with irrotational nodes and show limited lateral expansion when compressed. Similarly, the microlattices studied in this work presented a relative density ¯ρj greater than 0.1 and non-slender struts (ri/li)j > 0.06 for i = 1, 2 and j = A, B, C. These July 2019 \| Volume 6 \| Article 169 Frontiers in Materials \| www.frontiersin.org 6 Mechanics of Octet Carbon Microlattices Kudo et al. FIGURE 6 \| Effective Young’s modulus E∗of non-slender carbon microlattices reported as a function of the octet unit cell relative density ¯ρ. (A) Comparison between the stiffness measured from experiments (green/dot markers) and values predicted from ﬁnite element simulations (orange/diamond markers). Effective Young’s modulus E∗y (B) and E∗z (C) scaling laws obtained from numerical simulations for relative density that ranges between 10 and 50%. FIGURE 6 \| Effective Young’s modulus E∗of non-slender carbon microlattices reported as a function of the octet unit cell relative density ¯ρ. (A) Comparison between the stiffness measured from experiments (green/dot markers) and values predicted from ﬁnite element simulations (orange/diamond markers). Effective Young’s modulus E∗y (B) and E∗z (C) scaling laws obtained from numerical simulations for relative density that ranges between 10 and 50%. TABLE 2 \| Geometrical parameters of the octet unit cell employed in the numerical simulations. 3.3. Stiffness Microlattice w h r1 r2 rN rj ¯ρCAD ¯ρt [µm] [µm] [µm] [µm] [µm] [µm] [-] [-] 01 240.42 240.42 12.50 10.00 25.00 17.00 0.10 0.10 02 240.42 240.42 14.00 11.00 28.00 18.70 0.12 0.13 03 240.42 240.42 16.50 13.00 33.00 22.10 0.17 0.17 04 240.42 240.42 20.00 16.00 40.00 27.20 0.24 0.24 05 240.42 240.42 22.50 18.00 45.00 30.60 0.30 0.30 06 240.42 240.42 27.50 22.00 55.00 37.40 0.43 0.44 07 240.42 240.42 30.00 24.00 60.00 40.80 0.50 0.51 The relative density ¯ρCAD obtained from the modeled geometry is reported together with the density ¯ρt calculated through the analytical expression (1). TABLE 2 \| Geometrical parameters of the octet unit cell employed in the numerical simulations. unit cells with relative density ¯ρ that varied between 0.10 and 0.50 (Table 2). Similarly to the manufactured microlattices, we modeled cubic unit cells with node radius rN = 2r1, junction radius rj = 1.7r2, and that contain struts with diﬀerent in- plane and out-of-plane diameters, with r2 = 0.8r1. The obtained stiﬀness in the y and z directions are reported as a function of the relative density in Figures 6B,C. The eﬀective Young’s modulus of the considered non-slender octet lattices scales as E∗ y ∝¯ρ1.43 and E∗ z ∝¯ρ1.48 for the y and z directions respectively, whereas classical scaling law for stretching dominated octet-truss lattices predicts a scaling exponent equal to one (Fleck et al., 2010). Deviations from classical theory were also observed for diﬀerent rigid architectures when the strut slenderness ratio r/l was larger than 0.07, as a result of nodal contribution on the eﬀective lattice stiﬀness (Portela et al., 2018). The microlattices manufactured and analyzed in this work present beam slenderness that ranges between 0.065 and 0.17, pronounced nodal volumes and edge eﬀects caused by a limited number of unit cells along the height and width (Christodoulou, 2017). Therefore a scaling exponent mE > 1 testiﬁes that the lattices experience signiﬁcant bending during compression. The relative density ¯ρCAD obtained from the modeled geometry is reported together with the density ¯ρt calculated through the analytical expression (1). parameters measured from SEM (Table 1). The computational eﬀective Young’s modulus for the three investigated geometry resulted E∗ y,A = 1334.74 MPa, E∗ y,B = 1876.18 MPa, and E∗ y,C = 2849.78 MPa. Frontiers in Materials \| www.frontiersin.org 3.3. Stiffness The predicted values are in fair agreement with experimental measurements, with 33.5, 16.6, and 13.5% relative errors for the three sets of microlattices A, B, and C. The discrepancy between numerical predictions and measurements decreases with an increase of the sample relative density ¯ρ. This is primarily attributed to the manufacturing induced corrugation that were not modeled in the computational analysis and were less pronounced as the beam diameter and relative density increase. In particular, the average corrugation amplitude was measured as 18.4% of the oﬀ-plane beam diameter for the microlattice A, while it was limited to 11.2 and 8.7% for lattices B and C, respectively. The inﬂuence of each one of the manufacturing-induced imperfections on the lattice eﬀective stiﬀness was not considered because these features are inherently related, however, it is believed that the non-cubic unit cell had the most pronounced eﬀect for the microlattice A, while the nodal geometry had a signiﬁcant role for microlattices B and C, where the deviation from cubic unit cell is small. July 2019 \| Volume 6 \| Article 169 3.4. Strength The compressive strength of an octet-truss lattice material was determined considering the two possible failure mechanisms of elastic buckling or plastic yielding (Deshpande et al., 2001). The analysis of an octet-truss unit cell constituted of cylindrical beams reveals that the strut axial compressive stress σa is related to the vertical stress applied to the unit cell σv through the relation σv = 2 √ 2π r l 2 σa. (5) (5) If the lattice presents a low relative density, elastic buckling will be responsible of the octet-truss structural collapse when the axial stress σa reaches the Euler critical value σb = n2π2E 4 r l 2 , (6) (6) where n depends on the strut boundary conditions, with n = 1 for a pin-joined strut and n = 2 for a doubly clamped beam. In order to investigate the eﬀective stiﬀness scaling law, we performed other numerical simulations on non-slender octet July 2019 \| Volume 6 \| Article 169 7 Mechanics of Octet Carbon Microlattices Kudo et al. FIGURE 7 \| Compressive strength σ ∗of non-slender carbon microlattices reported as a function of the octet unit cell relative density ¯ρ. (A) Comparison between the strength measured from experiments (green/dot markers) and predicted failure σ ∗,f y (orange/diamond markers) and buckling σ ∗,b y (gray/square markers) strengths from ﬁnite element simulations. Fracture and buckling strengths scaling laws along the y-direction (B) and z-direction (C) obtained from numerical simulations for a relative density that ranges between 10 and 50%. FIGURE 7 \| Compressive strength σ ∗of non-slender carbon microlattices reported as a function of the octet unit cell relative density ¯ρ. (A) Comparison between the strength measured from experiments (green/dot markers) and predicted failure σ ∗,f y (orange/diamond markers) and buckling σ ∗,b y (gray/square markers) strengths from ﬁnite element simulations. Fracture and buckling strengths scaling laws along the y-direction (B) and z-direction (C) obtained from numerical simulations for a relative density that ranges between 10 and 50%. When the strut slenderness increases, buckling is preceded by strut failure (yielding) as the stress reaches the material failure (yield) strength σf . Therefore, the compressive strength for an octet-truss material can be expressed as Deshpande et al. (2001) are not accounted for Portela et al. (2018). 3.4. Strength Even though Equation (10) provides an improved strength estimation of octet lattices with negligible eﬀective Poisson’s ratio compared to Equation (7), the eﬀects of the manufacturing-induced imperfections are not fully reﬂected, and they were considered through numerical analyses. Therefore, more comprehensive analytical formulations need to be developed in order to assess the mechanics of imperfect lattices. The experimental strength of the octet microlattices σ ∗ y increased with the relative density as shown in Figure 7A, where σ ∗ y,A = 24.80 ± 4.26 MPa, σ ∗ y,B = 65.68 ± 4.45 MPa, and σ ∗ y,C = 122.74 ± 12.28 MPa refer to the three sets of lattices. It should be noticed that the attained speciﬁc strengths (strength to density ratio) exceed most of natural materials and approach the values obtained by carbon nanolattices (Bauer et al., 2016; Zhang et al., 2019), which represent the strongest architected materials ever realized. The achievement of strengths comparable to those of nanolattices testiﬁes that through the current manufacturing technique is possible to realize large- scale ultra-strong materials, overcoming the current scalability disadvantages of two-photon lithography nanofabrication. σ ∗= min (√ 2π3n2E 2 r l 4 ; 2 √ 2π r l 2 σf ) . (7) (7) The extension of the expression (7) to non-slender cubic frame lattices was obtained through the analysis of an octet unit cell constrained to deform laterally. Considering bending eﬀects, the relationship (5) between the strut axial stress σa and the vertical stress σv applied to the unit cell becomes The extension of the expression (7) to non-slender cubic frame lattices was obtained through the analysis of an octet unit cell constrained to deform laterally. Considering bending eﬀects, the relationship (5) between the strut axial stress σa and the vertical stress σv applied to the unit cell becomes σv = 2 √ 2π r l 2 1 + 3 r l 2 σa, (8) (8) while the dependence of σv on the maximum normal stress σm at the edges of the strut was obtained considering a combination of the bending moment and axial force eﬀects Considering the limitations of the analytical formulations, the complex microlattice stress distribution was assessed through numerical simulations, from which the maximum equivalent stress was obtained and was compared with the strength of the constituent material. July 2019 \| Volume 6 \| Article 169 3.4. Strength The bulk strength of pyrolytic carbon has been shown to be dependent on the specimen length- scale (Bullock and Kaae, 1979), with an increase as specimens dimensions decrease. This behavior depends on the probability of ﬁnding large critical ﬂaws within the materials, which decreases when the sample dimensions reduce. In brittle materials, the failure strength σf is inversely proportional to the square root of the pre-existing ﬂaw size σz = 2 √ 2π r l 2 1 + 3 r l 2 1 + 6 r l 2 σm. (9) (9) The octet-frame buckling strength was calculated by inserting Equation (6) into Equation (8), while the lattice failure (yield) strength is achieved when σm = σf . Hence, the compressive strength for a cubic octet-frame lattice material writes as σ ∗= min    √ 2π3n2E 2 r l 4 1 + 3 r l 2 ; 2 √ 2π r l 2 1 + 3 r l 2 1 + 6 r l 2 σf    . (10) σf = YKIc √πac , (11) (11) As observed for the stiﬀness calculation, simpliﬁed analytical expressions fail to capture the complex mechanical response of non-slender architectures as the orientation-dependent slenderness, non cubic unit cell and the nodal contribution where KIc = 0.91 MPa√m is the fracture toughness (Zhao et al., 1985; Brezny and Green, 1990), Y = 1 is a non-dimensional geometrical parameter for a semi-elliptical surface ﬂaw loaded in tension or bending (Bauer et al., 2016), and ac represents the July 2019 \| Volume 6 \| Article 169 Frontiers in Materials \| www.frontiersin.org Mechanics of Octet Carbon Microlattices Kudo et al. critical ﬂaw size. The strength of glassy carbon ﬁbers of 5 µm diameter made by carbonization of organic polymer reached 2 GPa, corresponding to a ﬂaw size of 66 nm (Kawamura and Jenkins, 1970). Since the strut diameters of the manufactured microlattices range between 20 and 50 µm, we assume that a material strength σf = 1.5 GPa can be reached, equivalent to a ﬂaw size of 0.12 µm. From the results of numerical simulations, it is possible to obtain the octet compressive stress along the y-direction that produces a critical equivalent stress within the unit cell. 3.4. Strength The numerical lattice failure strengths were found σ ∗,f y,A = 39.31 MPa, σ ∗,f y,B = 73.17 MPa, and σ ∗,f y,C = 102.83 MPa for the three sets of carbon microlattices and they are reported in Figure 7A together with the experimental data. Considering the uncertainties in the determination of the material failure strength σf and the lack of beam corrugation modeling in the ﬁnite element analysis, these results are in fair agreement with the measured values, with a maximum relative error of 36.9%. Furthermore, it should be noted that the numerical simulations were performed on a unit cell level, thus neglecting the edge eﬀects that a ﬁnite lattice experience. Numerical buckling analyses on the three octet unit cells conﬁrmed that the fracture strength was achieved before elastic buckling of the out-of-plane beams. The compressive stresses σ ∗,b y that trigger elastic instabilities were numerically found, with σ ∗,b y,A = 100.88 MPa, σ ∗,b y,B = 196.07 MPa and σ ∗,b y,C = 389.57 MPa. It can be seen that the numerical buckling strength σ ∗,b y,j is always greater than the fracture strength σ ∗,f y,j for the three microlattices j = A, B, C, thus excluding strut instability mechanisms. The computational analyses on non-slender octet unit cells performed to investigate the eﬀective stiﬀness scaling laws were employed to calculate the fracture and buckling strengths and their scaling laws. Figures 7B,C shows that for the y loading direction the fracture strength scales as σ ∗,f y ∝¯ρ0.97, and the buckling strength scales as σ ∗,b y ∝¯ρ2.49, while in the z-direction σ ∗,f z ∝ ¯ρ1.08 and σ ∗,b z ∝ ¯ρ2.59. As already noticed for the eﬀective stiﬀness behavior, the fracture and buckling strengths scaling laws deviate from the classical analysis of octet-truss architectures, where the scaling exponents are mf = 1 for fracture and mb = 2 for buckling (Deshpande et al., 2001). The diﬀerent scaling exponents obtained from the numerical Since the struts of the studied microlattices are non-slender, buckling is unlikely to cause failure before the onset of fracture. FIGURE 8 \| SEM fractography of the three sets of manufactures microlattices A (A), B (B), and C (C) after catastrophic mechanical compression tests. Progressive magniﬁcation images highlight the brittle fracture surfaces. Anwer, A., and Naguib, H. (2018). Multi-functional ﬂexible carbon ﬁber composites with controlled ﬁber alignment using additive manufacturing. Addit. Manuf. 22, 360–367. doi: 10.1016/j.addma.2018.05.013 Bauer, J., Schroer, A., Schwaiger, R., and Kraft, O. (2016). Approaching theoretical strength in glassy carbon nanolattices. Nat. Mater. 15, 438–443. doi: 10.1038/nmat4561 3.4. Strength FIGURE 8 \| SEM fractography of the three sets of manufactures microlattices A (A), B (B), and C (C) after catastrophic mechanical compression tests. Progressive magniﬁcation images highlight the brittle fracture surfaces. July 2019 \| Volume 6 \| Article 169 Frontiers in Materials \| www.frontiersin.org 9 Mechanics of Octet Carbon Microlattices Kudo et al. nonlinearities and viscoelastic eﬀects, independently of the manufacturing process. Although more accurate than classical octet-truss formulations when bending eﬀects are pronounced, the developed analytical construction remains inadequate for the prediction of the eﬀective stiﬀness and strength of the manufactured non-slender lattices, thus identifying the needs for future analytical investigations of structured solids with orientation dependent geometrical features and pronounced nodal connections, in order to develop optimized materials and assess the inﬂuence of these parameters on the lattice mechanical response. To overcome the identiﬁed limitations of the analytical tools, we have developed computational models that reproduce the unit cell geometry and capture the mechanical properties of the tested architectures. The numerical investigation has revealed the mechanics of low slenderness microlattices through the prediction of compressive stiﬀness, failure and buckling strengths, and their scaling laws, which provide a reliable method to estimate the mechanical properties of imperfectly manufactured octet lattices featuring the investigated topology. The presented manufacturing process and numerical methods represent tools to enhance the design of carbon architected materials toward strong and lightweight solids. simulations depend on the non-slender architecture and the pronounced nodal volume, and will help the evaluation of the mechanical properties prediction of any lattice with the same ratios between geometrical parameters. Lastly, it should be noted that the results of computational analysis on non-slender cubic octet unit cells with r2 = 0.8r1, rN = 2r2, and rj = 1.7r2 predict that failure due to elastic instability occurs when the relative density ¯ρ is lower than 0.12, corresponding to an out-of-plane strut slenderness r2/l2 = 0.065. Similarly to the results reported by Portela et al. (2018), from ﬁnite element analyses it was also observed that strain energy concentrates in the nodes and the stress concentration migrates toward the nodes as the relative density increases and beam slenderness decreases. Figure 8, left shows the SEM images of the three sets of manufactured microlattices after catastrophic compression tests, highlighting the failed components and the brittle fracture surfaces. 3.4. Strength As numerically predicted, the experimental fractographic examination proved that in the lighter microlattice A the nodes remained undamaged and only struts fractured (Figure 8A), while as density increases in lattices B and C node failures were observed (Figures 8B,C). Furthermore, it was commonly noticed among the three sets of samples that fracture within the struts did not follow the stereolithography induced corrugations. ACKNOWLEDGMENTS AK gratefully acknowledges the ﬁnancial support from the Resnick Sustainability Institute at the California Institute of Technology and from Prof. Julia R. Greer (Caltech) through the Vannevar-Bush Faculty Fellowship of the US Department of Defense. The authors thank Prof. Julia R. Greer’s research group (Caltech) and Prof. Sergio Pellegrino (Caltech) for their support in conducting experiments in their laboratories. FUNDING Vannevar-Bush Faculty Fellowship of the US Department of Defense. The Resnick Sustainability Institute Postdoctoral Fellowship of Caltech. Financial support from National Group of Mathematical Physics (GNFM-INdAM). 4. CONCLUSION AK, DM, and FB designed the research, discussed the results, and wrote the paper. DM and FB carried out the analytical and numerical analyses. AK manufactured the specimens. AK and YW performed the experiments. All authors reviewed the paper and gave ﬁnal approval for publication. In the present work, we have investigated the compressive behavior of stiﬀ and strong non-slender octet carbon microlattices obtained by pyrolyzing 3D-printed polymer architectures fabricated through stereolithography. We have shown that additive manufacturing can lead to imperfect lattices with signiﬁcant nodal volumes and strut slenderness dependent on the beam inclination with respect to the 3D- printing direction. The eﬀects of these features on the lattice structural response have been numerically and experimentally studied considering carbon microlattices with a relative density higher than 10%. The manufactured microarchitectures have achieved superior relative stiﬀness and strength that approach those of carbon nanolattices. Therefore, we have demonstrated that the employed manufacturing technique can lead a fast realization of large scale strong materials, currently not achievable through nanofabrication. We have extended the classical analytical tools based on beam theory to include non-slender cubic architectures that show a negligible eﬀective Poisson’s ratio. These formulations can be adopted for octet lattices constituted of any materials with negligible REFERENCES Portela, C., Greer, J., and Kochmann, D. (2018). Impact of node geometry on the eﬀective stiﬀness of non-slender three-dimensional truss lattice architectures. Extreme Mech. 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https://openalex.org/W2017372537	https://bmccancer.biomedcentral.com/counter/pdf/10.1186/1471-2407-13-321	English	null	Overexpression of peptide deformylase in breast, colon, and lung cancers	BMC cancer	2,013	cc-by	6,139	© 2013 Randhawa 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. * Correspondence: Katie.Reindl@ndsu.edu †Equal contributors 1Department of Biological Sciences, North Dakota State University, Fargo, ND, USA Full list of author information is available at the end of the article Overexpression of peptide deformylase in breast, colon, and lung cancers Harsharan Randhawa1†, Shireen Chikara1†, Drew Gehring1, Tuba Yildirim2, Jyotsana Menon3 an wa1†, Shireen Chikara1†, Drew Gehring1, Tuba Yildirim2, Jyotsana Menon3 and Katie M Reindl1* Abstract Background: Human mitochondrial peptide deformylase (PDF) has been proposed as a novel cancer therapeutic target. However, very little is known about its expression and regulation in human tissues. The purpose of this study was to characterize the expression pattern of PDF in cancerous tissues and to identify mechanisms that regulate its expression. Methods: The mRNA expression levels of PDF and methionine aminopeptidase 1D (MAP1D), an enzyme involved in a related pathway with PDF, were determined using tissue panels containing cDNA from patients with various types of cancer (breast, colon, kidney, liver, lung, ovarian, prostate, or thyroid) and human cell lines. Protein levels of PDF were also determined in 2 colon cancer patients via western blotting. Colon cancer cells were treated with inhibitors of ERK, Akt, and mTOR signaling pathways and the resulting effects on PDF and MAP1D mRNA levels were determined by qPCR for colon and lung cancer cell lines. Finally, the effects of a PDF inhibitor, actinonin, on the proliferation of breast, colon, and prostate cell lines were determined using the CyQUANT assay. Results: PDF and MAP1D mRNA levels were elevated in cancer cell lines compared to non-cancer lines. PDF mRNA levels were significantly increased in breast, colon, and lung cancer samples while MAP1D mRNA levels were increased in just colon cancers. The expression of PDF and MAP1D varied with stage in these cancers. Further, PDF protein expression was elevated in colon cancer tissue samples. Inhibition of the MEK/ERK, but not PI3K or mTOR, pathway reduced the expression of PDF and MAP1D in both colon and lung cancer cell lines. Further, inhibition of PDF with actinonin resulted in greater reduction of breast, colon, and prostate cancer cell proliferation than non-cancer cell lines. Conclusions: This is the first report showing that PDF is over-expressed in breast, colon, and lung cancers, and the first evidence that the MEK/ERK pathway plays a role in regulating the expression of PDF and MAP1D. The over-expression of PDF in several cancers and the inhibition of cancer cell growth by a PDF inhibitor suggest this enzyme may act as an oncogene to promote cancer cell proliferation. RESEARCH ARTICLE Open Access Open Access Randhawa et al. BMC Cancer 2013, 13:321 http://www.biomedcentral.com/1471-2407/13/321 Randhawa et al. BMC Cancer 2013, 13:321 Background the target of antimicrobial agents [2-5]. However, the recent discovery of PDF and a MAP isoform in the mitochondria of eukaryotes raises questions regarding their role in human cells [6-8]. In prokaryotic organisms, the N-terminal methionine ex- cision (NME) pathway is indispensible for proper protein functioning. This pathway involves two enzymes; peptide deformylase (PDF) which removes the formyl group from the initial methionine in nascent peptides, and methionine aminopeptidase (MAP) which subsequently removes the initial methionine [1]. Until recently, PDF was thought to exist only in prokaryotic organisms and hence has been Studies show that human PDF (HsPDF) can cleave the formyl group from an initiator methionine, but with reduced kinetics compared to the prokaryotic versions of the enzyme [2,8,9]. However, many of the respiratory Complex I peptides generated from mtDNA, putative substrates for PDF and MAP1D, retain their formylated initiator methionine [10]. In contrast, a recent report suggests that inhibition of PDF with actinonin results in reduced aerobic respiratory capacity by influencing the expression of proteins derived from the mtDNA [11]. * Correspondence: Katie.Reindl@ndsu.edu †Equal contributors 1Department of Biological Sciences, North Dakota State University, Fargo, ND, USA Full list of author information is available at the end of the article Page 2 of 7 Randhawa et al. BMC Cancer 2013, 13:321 http://www.biomedcentral.com/1471-2407/13/321 (Cambrex). PC-3 cells were grown in Ham’s F-12 K medium supplemented with 10% FBS. While there are conflicting views for their role in NME in humans, it is likely PDF and MAP1D have alternative functions. Indeed, RNA interference of MAP1D altered anchorage-dependent growth of colon cancer cells [12] and inhibition of PDF with actinonin and numerous analogs decreased proliferation of many cancer cells while having minimal effects on non-cancer cell lines [13]. Fur- ther, PDF inhibitors resulted in a reduced tumor volume in a mouse xenograft model using HL-60 [14]. These results have lead to recent studies focused on the design of inhibi- tors to target PDF in cancer [14-16]. Human tissue samples and cDNA TissueScan Cancer qPCR Arrays containing cDNA from normal and cancer tissue samples were purchased from Origene (Rockville, MA). The cDNA panels (cancer survey panel CSRT101, breast cancer panel BCRT101, matched colon cancer panel HCRT103, and matched lung cancer panel HLRT104), each had 48–96 samples per microplate. Equal loading of cDNA was verified by the manufacturer. Additionally, matched normal and colon cancer samples were obtained from two patients at the Veteran’s Affairs (VA) Hospital in Fargo, ND. This research was approved by the University of South Dakota and the North Dakota State University Institutional Review Board and performed according to the ethical guidelines imposed by these boards. Informed consent was obtained from each participant. Total RNA was isolated from human cell lines using the Fisher SurePrep Kit (Waltham, MA) and from human tissue samples using TRI Reagent (Molecular Research Center; Cincinnati, OH) as per the manufacturer’s sug- gestions. 100 ng of total RNA were reverse transcribed into cDNA using the qScript cDNA synthesis kit (Quanta Biosciences; Gaithersburg, MD). Despite these advances, little is known about the ex- pression and regulation of the NME enzymes in cancers. MAP1D is over-expressed in colon cancer [12], but no study has reported the expression of PDF in cancerous compared to normal tissues. Further, no study has described a mechanism that regulates human PDF or MAP1D expression. Therefore, the purpose of this study was to identify the expression profiles of PDF and MAP1D in human cancers compared to normal tissues and to identify a signaling pathway involved in regulating their expression. Given the role of human PDF and MAP1D in cancer cell growth and adhesion, we hypothesized that these proteins would be up-regulated in cancer cells and tissues compared to normal and their expression would be modulated by growth-regulatory pathways. In this paper, we report that PDF is elevated in breast, colon, and lung cancer tissues and MAP1D is elevated in colon cancer tissue samples compared to non-cancer controls. We also show that PDF and MAP1D mRNA expression is down- regulated when MEK/ERK signaling is disrupted. Signal transduction pathway inhibitors HT-29 colon cancer cells were seeded into a 6 well plate at 1.5 million cells per well and incubated overnight. The next day, the cells were treated for 5 hours with 10 μM U0126, 10 μM LY294002, or 10 μM rapamycin (all from Cell Signaling). Total RNA or total protein was collected from the cells for further analysis. QPCR All cell lines, unless otherwise noted, were obtained from ATCC (Manassas, VA) and cultured at 37°C with 5% carbon dioxide. Hs578Bst normal breast cells were maintained in Hybri-Care Medium (ATCC) supplemented with 1.5 g/L sodium bicarbonate (Sigma; St. Louis, MO), 30 ng/ml mouse EGF (BD Biosciences; San Jose, CA), and 10% fetal bovine serum (FBS; Atlanta Biologicals; Lawrenceville, GA). Hs578T breast cancer cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Thermo Scientific; Waltham, MA) supplemented with 0.01 mg/ml bovine insulin (Sigma) and 10% FBS. CCD- 18Co normal colon cells were maintained in Eagle's Minimum Essential Medium (EMEM; ATCC) supplemented with 10% FBS. HT-29 colon cancer cells were cultured in McCoy’s 5a (Thermo Scientific) medium supplemented with 10% FBS. Hs888Lu normal lung fibroblasts and A549 lung cancer cells were cultured in DMEM plus 10% FBS. PrEC normal prostate epithelial cells were obtained from Cambrex Corporation (East Rutherford, NJ) and propa- gated in PrEGM media with Bulletkit growth supplements Primers against human PDF and MAP1D were designed using Primer Express software (Applied Biosystems; Carlsbad, CA) and synthesized by Integrated DNA Technologies (Coralville, IA). Primer sequences were as follows; PDF forward AGGCGCTGTGTCGGGAGTGC, PDF reverse TCTCGCAGCCCTCGGGAAAG, MAP1D forward TATAGTTTTGCCGGCTGCAGT, MAP1D re- verse ATGTGCTTAGGAACCGGATGA, β-actin for- ward CAGCCATGTACGTTGCTATCCAGG, β-actin reverse AGGTCCAGACGCAGGATGGCATG. Steady- state mRNA levels of PDF or MAP1D were determined for all cDNAs by real-time PCR using PerfeCTa SYBR Green FastMix (Quanta Biosciences). The cycling parame- ters were 95°C for 10 min followed by 40 cycles of 95°C for 30 sec and 60°C for 1 min and a dissociation program that included 95°C for 1 min, 55°C for 30 sec, and 95°C for 30 sec ramping up at 0.2°C/sec. One distinct peak was observed for the primer sets. For the cell lines, qPCR standards were prepared using human PDF and Page 3 of 7 Randhawa et al. BMC Cancer 2013, 13:321 http://www.biomedcentral.com/1471-2407/13/321 cut-off of < 0.05 was used to determine statistical signifi- cance. For the cell lines, the PDF and MAP1D values were related to the standard curves for the respective targets to yield the approximate mRNA copy number/cell. These values were then normalized to β-actin values. The data are expressed as the average copy number ± SD for 3 rep- licates. A t-test comparing the PDF or MAP1D mRNA copy number in the cancer cell lines to the copy number in their respective normal cell lines. Toxicity assay Hs578Bst, Hs578T, CCD-18Co, HT-29, PrEC, and PC-3 cells were plated in 96-well microplates in growth medium at a density of 5,000 cells/well and incubated for 24 hours. The cells were then treated for 4 days with 0–250 μM actinonin. The CyQUANT (Life Technologies; Grand Island, NY) cell proliferation assay was performed according to the manufacturer’s instructions. Fluorescent readings were taken on day 4 to determine the percentage of viable cells. Each condition was performed with eight replicates, and the experiments were repeated three times. SDS-PAGE and western blotting Cell pellets or human tissue samples from the VA Hospital were lysed using an SDS lysis buffer (Cell Signaling Technologies, Danvers, MA) containing protease and phosphatases inhibitors (Roche; Indianapolis, IN). Samples were briefly sonicated to dissociate cell membranes. Fifty μg of total protein isolated from the human cell lines or tissues were separated on 10% SDS-polyacrylamide gels at 100 V for 1 hr. Proteins were transferred to nitrocellulose membranes at 100 V for 75 min at 4°C. Blots were then probed overnight at 4°C with primary antibodies. The PDF antibody was a kind gift from Carmela Giglione and Thierry Meinnel (Centre National de la Recherche Scientifique, Gif-sur-Yvette, France). The MAP1D antibody was obtained from R&D Systems (Minneapolis, MN). The total and phosphor-ERK antibodies were purchased from Cell Signaling. The next day, blots were rinsed with 1X TBS-tween (0.1%) and probed with anti-rabbit secondary antibody (Jackson Immuno Research; West Grove, PA) for 1 hr at room temperature. The western blots were analyzed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific; Rockford, IL) and images captured using the MultiImage™Light Cabinet (Alpha Innotech; San Leandro, CA). PDF levels were normalized to β-actin (Cell Signaling) expression. Immunoblots were performed in triplicate. Actinonin inhibits the growth of both cancer and non-cancer cell lines The effect of the PDF inhibitor actinonin on the prolifera- tion capacity of colon, breast, and prostate cancer and non-cancer cell lines was measured. Actinonin inhibited the proliferation of both cancer and non-cancer cell lines in a concentration-dependent manner, but had greater inhibition of cell proliferation in cancer cells compared QPCR For the cancer tissue cDNA plates, the average Ct value for all of the non- cancer tissue samples was set to 1. The data are expressed as the relative fold change in each individual sample compared to the average of these controls. A t-test was run for PDF mRNA expression in the cancer survey samples compared to their non-cancer controls. One-way ANOVA on ranks was done using Dunn’s method for multiple comparisons in the cancer stage I-III breast, colon, and lung samples compared to their normal tissues. A paired t-test was done to compare the effect of actinonin on the proliferation of the cancer cell line to the normal cell line. The data represent the percentage of viable cells ± SD for 8 replicates. Finally, a t-test was used to determine the effect of U0126 on the expression of PDF and MAP1D mRNA in 3 independent replicates. MAP1D full-length cDNA clones from Open Biosystems (Catalog numbers IHS1380-97652083 and MHS4426- 99238965, Huntsville, AL). The 1010 molecules/μL standard was serially diluted to 102 molecules/μL. The standards were run alongside the cDNA from the human cell lines in order to approximate the copy number of PDF or MAP1D in these cells. For the cDNA panels, fold-change in mRNA expression was calculated by comparing normalized threshold cycle numbers (CT) in the cancerous tissue compared to the normal tissues. The cell experiments were performed in triplicate. MAP1D full-length cDNA clones from Open Biosystems (Catalog numbers IHS1380-97652083 and MHS4426- 99238965, Huntsville, AL). The 1010 molecules/μL standard was serially diluted to 102 molecules/μL. The standards were run alongside the cDNA from the human cell lines in order to approximate the copy number of PDF or MAP1D in these cells. For the cDNA panels, fold-change in mRNA expression was calculated by comparing normalized threshold cycle numbers (CT) in the cancerous tissue compared to the normal tissues. The cell experiments were performed in triplicate. PDF and MAP1D expression is elevated in human cancer cell lines We compared the expression of PDF and MAP1D in four different types (breast, colon, lung, and prostate) of cancer cell lines to non-cancer cell lines. PDF mRNA expression was significantly higher in the HT-29 colon, A549 lung, and PC-3 prostate cancer cell lines compared to the CCD-18Co colon, Hs888Lu lung, and PrEC prostate non-cancer cell lines (Figure 1). MAP1D was significantly elevated in the PC-3 compared to PrEC cell line, but was not significantly different in the other pairs of cell lines (Figure 1). The Hs578Bst and Hs578T cell lines are a normal breast and breast cancer cell line isolated from the same patient. These cell lines did not significantly differ in their PDF or MAP1D expression, although PDF was slightly elevated and MAP1D was reduced. The data suggest that PDF and MAP1D expression varies across cell type and that they show altered expression in cancer compared to non-cancer cells. Statistical analysis PDF was significantly (denoted by “a”) elevated in the colon, lung, and prostate cancer cell lines compared to their respective normal cell lines while MAP1D was significantly (denoted by “b”) elevated only in the prostate cancer cell line. The data represent the average mRNA copy number for 3 replicate experiments ± SD. Additional tissue panels for breast, colon, and lung cancer patients were used to validate the previous results and to assess MAP1D levels in these cancer types. Colon and lung tissue panels contained 48 matched normal and cancer tissue samples from 24 cancer patients while the breast tissue panels contained 48 unmatched tissue samples that included 12 normal breast tissue controls and 36 breast cancer samples at various disease stages. Similar to the first results, PDF was elevated in breast, colon, and lung cancer samples and showed stage- dependent expression with the highest expression in late stage breast cancer, but early stage colon and lung cancers (Figure 4A). MAP1D mRNA expression was to non-cancer cells (Figure 2A-C). The IC50’s were 19.3, 17.3, and 113.5 μM for the Hs578T, HT-29, and PC-3 cancer cell lines, respectively while the IC50’s were 208, 31.9, and 207.4 μM for the Hs578Bst, CCD-18Co, and PrEC cells, respectively. While the IC50 was higher in the normal colon compared to the colon cancer cell line, the difference in the percentage of viable cells was not statistically significant. In contrast, actinonin significantly affected the growth of breast and prostate cancer cells to non-cancer cells (Figure 2A-C). The IC50’s were 19.3, 17.3, and 113.5 μM for the Hs578T, HT-29, and PC-3 cancer cell lines, respectively while the IC50’s were 208, 31.9, and 207.4 μM for the Hs578Bst, CCD-18Co, and PrEC cells, respectively. While the IC50 was higher in the normal colon compared to the colon cancer cell line, the difference in the percentage of viable cells was not statistically significant. In contrast, actinonin significantly affected the growth of breast and prostate cancer cells Figure 2 Actinonin inhibits the growth of cancer cell lines to a greater degree than non-cancer cell lines. (A) Normal breast (Hs578Bst) and breast cancer (Hs578T), (B) normal colon (CCD18Co) and colon cancer (HT29), and (C) normal prostate (PrEC) and prostate cancer (PC3) cell lines were treated with 0–250 μM actinonin for 96 hrs before the percentage of viable cells was determined. Statistical analysis SigmaPlot v12 software (Systat Software Inc.; Chicago, IL) was used for all statistical analyses. For all tests, a p-value Randhawa et al. BMC Cancer 2013, 13:321 http://www.biomedcentral.com/1471-2407/13/321 Page 4 of 7 Figure 1 PDF and MAP1D mRNA expression varies across human cell lines. The expression of PDF and MAP1D mRNA in normal breast (Hs578Bst), colon (CCD-18Co), lung (Hs888Lu), and prostate (PrEC) cell lines was compared to the expression in breast cancer (Hs578T), colon cancer (HT29), lung cancer (A549), and prostate cancer (PC3) cell lines, respectively. The mRNA copy numbers for PDF and MAP1D were related to cDNA standards for these two genes and then normalized to β-actin levels. PDF was significantly (denoted by “a”) elevated in the colon, lung, and prostate cancer cell lines compared to their respective normal cell lines while MAP1D was significantly (denoted by “b”) elevated only in the prostate cancer cell line. The data represent the average mRNA copy number for 3 replicate experiments ± SD. compared to their non-cancer cell controls. In general, the data suggest that inhibition of PDF by actinonin has a greater effect on proliferation of cancer cells compared to normal cells. PDF mRNA is elevated in many cancer tissues TissueScanTM Cancer qPCR Arrays containing cDNA from 96 tissue samples representing eight different cancers (breast, colon, kidney, liver, lung, ovary, prostate, thyroid) were used to determine PDF expression in cancer com- pared to non-cancer tissues. For each tissue type, the array contained 3 normal control tissues and 9 cancer tissues. With the exception of liver cancer that showed no change compared to control liver samples, PDF was at least slightly elevated in all cancer tissues compared to control, and PDF mRNA levels were significantly elevated in the breast, colon, and lung cancer tissue samples compared to their non-cancer samples (Figure 3). Breast cancer showed a 5.8-fold increase in expression of PDF while colon and lung showed a 3.5 and 3.4-fold increase in PDF expression, respectively. Figure 1 PDF and MAP1D mRNA expression varies across human cell lines. The expression of PDF and MAP1D mRNA in normal breast (Hs578Bst), colon (CCD-18Co), lung (Hs888Lu), and prostate (PrEC) cell lines was compared to the expression in breast cancer (Hs578T), colon cancer (HT29), lung cancer (A549), and prostate cancer (PC3) cell lines, respectively. The mRNA copy numbers for PDF and MAP1D were related to cDNA standards for these two genes and then normalized to β-actin levels. Statistical analysis The growth-inhibitory effect of actinonin was significantly greater in the breast cancer and prostate cancer cell lines than in their non-cancer control cell lines. The data represent the percentage of viable cells ± SD for 3 experiments with 8 replicates each. Figure 2 Actinonin inhibits the growth of cancer cell lines to a greater degree than non-cancer cell lines. (A) Normal breast (Hs578Bst) and breast cancer (Hs578T), (B) normal colon (CCD18Co) and colon cancer (HT29), and (C) normal prostate (PrEC) and prostate cancer (PC3) cell lines were treated with 0–250 μM actinonin for 96 hrs before the percentage of viable cells was determined. The growth-inhibitory effect of actinonin was significantly greater in the breast cancer and prostate cancer cell lines than in their non-cancer control cell lines. The data represent the percentage of viable cells ± SD for 3 experiments with 8 replicates each. Randhawa et al. BMC Cancer 2013, 13:321 http://www.biomedcentral.com/1471-2407/13/321 Randhawa et al. BMC Cancer 2013, 13:321 Page 5 of 7 http://www.biomedcentral.com/1471-2407/13/321 Figure 3 PDF mRNA is significantly elevated in breast, colon, and lung cancer. With the exception of liver, that showed equal expression, PDF was elevated in all cancers (●) compared to non-cancer/normal (○) tissues. All stages of disease were pooled for the cancer groups. Statistically significant differences () were observed in breast, colon, and lung cancers with expression values 5.8, 3.5, and 3.4-fold higher in cancer compared to normal tissues, respectively. Figure 3 PDF mRNA is significantly elevated in breast, colon, and lung cancer. With the exception of liver, that showed equal expression, PDF was elevated in all cancers (●) compared to non-cancer/normal (○) tissues. All stages of disease were pooled for the cancer groups. Statistically significant differences () were observed in breast, colon, and lung cancers with expression values 5.8, 3.5, and 3.4-fold higher in cancer compared to normal tissues, respectively. PDF protein levels are elevated in colon cancer tissues To verify that the increased PDF mRNA levels translated to increased PDF protein levels, we screened two sets of colon cancer tissues for PDF expression. Matched colon cancer and normal colon tissue sets were obtained from two pa- tients at the VA Hospital in Fargo, ND in accordance with IRB policies. Discussion PDF and MAP are essential enzymes in prokaryotic peptide synthesis, but their role in eukaryotic cells is less appreciated. Previous studies have suggested PDF and MAP1D as therapeutic targets for cancer treatment given their roles in modulating cell proliferation, adhesion, and aerobic respiration [11-13]. As a result, the goal of this research was to characterize the expression pattern of PDF and MAP1D in human cancer tissues in order to better understand their potential roles in these cancers. Over-expression of MAP1D has been previously observed in colon cancer tissues; 7 out of 8 colon cancer patients showed increased MAP1D mRNA expression and 9 out of 12 patients showed increased MAP1D protein expression [12]. Similarly, we also found that MAP1D was elevated in colon cancers, but not lung cancers. Interestingly we found that MAP1D mRNA expression was significantly reduced in breast cancer samples compared to normal breast tissue. This is the first report to suggest PDF is over-expressed in cancer, particularly breast, colon, and lung. Stage- dependent expression of PDF was observed in the tissue samples where higher expression was found in early stages of colon and lung cancer, but later stages of breast cancer. Early expression of PDF indicates it plays a role in the pro- liferation of tumor cells. The over-expression of PDF and MAP1D, particularly in early-stage colon cancer, suggests that these enzymes are important for cancer cell growth. these patients relative to their matched normal colon tissue (Figure 5). Statistical analysis Elevated PDF levels were found in the colon tumor samples for each patient. A β-actin antibody was used to confirm equal protein loading of the tissue samples for each patient. Two replicate experiments were performed and this image shows one representative experiment. Statistical analysis Western blotting for PDF revealed a striking elevation of PDF expression in the tumor sample of both of PDF protein levels are elevated in colon cancer tissues To verify that the increased PDF mRNA levels translated to increased PDF protein levels, we screened two sets of colon cancer tissues for PDF expression. Matched colon cancer and normal colon tissue sets were obtained from two pa- tients at the VA Hospital in Fargo, ND in accordance with IRB policies. Western blotting for PDF revealed a striking elevation of PDF expression in the tumor sample of both of elevated in early-stage colon cancer samples, and was surprisingly reduced in breast cancer samples compared to control samples (Figure 4B). There was no significant change in MAP1D mRNA levels in lung cancer samples at any stage compared to control. These results suggest PDF and MAP1D expression is altered in certain cancer tissues and that expression of these enzymes is correlated with the stage of disease. Figure 4 PDF and MAP1D mRNA expression varies with stage in breast, colon, and lung cancer samples. (A) PDF and (B) MAP1D mRNA expression is shown for normal (●) tissues relative to stage I (○), stage II (▼), and stage III (Δ) tissues for breast, colon, and lung cancer patients. PDF levels are significantly () elevated in late-stage breast, and early-stage colon and lung cancers while MAP1D levels are significantly increased in early-stage colon cancer, but decreased in breast cancer. Figure 4 PDF and MAP1D mRNA expression varies with stage in breast, colon, and lung cancer samples. (A) PDF and (B) MAP1D mRNA expression is shown for normal (●) tissues relative to stage I (○), stage II (▼), and stage III (Δ) tissues for breast, colon, and lung cancer patients. PDF levels are significantly () elevated in late-stage breast, and early-stage colon and lung cancers while MAP1D levels are significantly increased in early-stage colon cancer, but decreased in breast cancer. Page 6 of 7 Randhawa et al. BMC Cancer 2013, 13:321 http://www.biomedcentral.com/1471-2407/13/321 Randhawa et al. BMC Cancer 2013, 13:321 http://www.biomedcentral.com/1471-2407/13/321 rapamycin did not have an effect on PDF expression in HT-29 cells (Figure 6C). Figure 5 PDF protein expression is elevated in colon tumor tissues. Western blotting was done to determine the expression of PDF in colon cancer tissue samples (T) relative to normal colon tissue (N) from two patients. Inhibition of MEK/ERK results in reduced expression of PDF and MAP1D in colon cancer cells The regulation of PDF or MAP1D expression in human cells has not been previously studied. To understand potential mechanisms that regulate PDF and MAP1D gene expression, we used pharmacological inhibitors to target the MEK/ERK, PI3K, and mTOR signaling path- ways and determined their effects on PDF or MAP1D expression. Treatment of HT-29 colon cancer cells with the MEK inhibitor U0126 resulted in a 51% reduction in expression of PDF mRNA and a 47% reduction in MAP1D (Figure 6A). Western blotting confirmed that U0126 inhibited ERK signalling these cells (Figure 6B). Unlike U0126, the PI3K inhibitor LY294002 and mTOR inhibitor PDF and MAP1D are encoded in the nuclear genome (chromosome 16 and 2, respectively) and translocate to Figure 6 Inhibition of MEK down-regulates PDF and MAP1D mRNA expression in colon cancer cells. (A) Treatment of HT-29 colon cancer cells with 10 μM U for 5 hr resulted in about a 50% reduction in both PDF and MAP1D mRNA expression. (B) Western blot analysis confirmed that 5 hr treatment of HT-29 colon cancer cells with 10 μM U reduced phosphorylated ERK (pERK) levels. Total ERK (tERK) expression was determined in order to show equal protein loading. (C) Treatment of HT-29 cells with other inhibitors Ly294002 (LY) and rapamycin (Rap) does not affect PDF mRNA expression. These experiments were repeated 3 times and the data represent the average relative gene expression in the inhibitor-treated cells relative to the vehicle-treated controls. Statistically significant (p < 0.05) differences are denoted by . Figure 6 Inhibition of MEK down-regulates PDF and MAP1D mRNA expression in colon cancer cells. (A) Treatment of HT-29 colon cancer cells with 10 μM U for 5 hr resulted in about a 50% reduction in both PDF and MAP1D mRNA expression. (B) Western blot analysis confirmed that 5 hr treatment of HT-29 colon cancer cells with 10 μM U reduced phosphorylated ERK (pERK) levels. Total ERK (tERK) expression was determined in order to show equal protein loading. (C) Treatment of HT-29 cells with other inhibitors Ly294002 (LY) and rapamycin (Rap) does not affect PDF mRNA expression. These experiments were repeated 3 times and the data represent the average relative gene expression in the inhibitor-treated cells relative to the vehicle-treated controls. Statistically significant (p < 0.05) differences are denoted by . Randhawa et al. BMC Cancer 2013, 13:321 http://www.biomedcentral.com/1471-2407/13/321 Page 7 of 7 mitochondria [14]. Author details 1 1Department of Biological Sciences, North Dakota State University, Fargo, ND, USA. 2Department of Biology, Faculty of Art and Science, Amasya University, Amasya, Turkey. 3Ben May Department for Cancer Research, University of Chicago, Chicago, IL, USA. The authors have no competing interests in relation to this paper. 15. Antczak C, Shum D, Escobar S, Bassit B, Kim E, Seshan VE, Wu N, Yang G, Ouerfelli O, Li YM, et al: High-throughput identification of inhibitors of human mitochondrial peptide deformylase. J Biomol Screen 2007, 12(4):521–535. 15. Antczak C, Shum D, Escobar S, Bassit B, Kim E, Seshan VE, Wu N, Yang G, Ouerfelli O, Li YM, et al: High-throughput identification of inhibitors of human mitochondrial peptide deformylase. J Biomol Screen 2007, 12(4):521–535. Received: 16 July 2012 Accepted: 21 June 2013 Published: 1 July 2013 Received: 16 July 2012 Accepted: 21 June 2013 Published: 1 July 2013 Inhibition of MEK/ERK results in reduced expression of PDF and MAP1D in colon cancer cells It was interesting to find that the expression of both HsPDF and MAP1D was regulated by a similar pathway. Use of the MEK inhibitor U0126 resulted in about a 50% reduction in PDF and MAP1D expression in a human colon cell line. Conversely, rapamycin and LY294002 had little effect on PDF expression suggesting the MEK/ERK pathway specifically contributes to the expression of NME enzymes. A genetic and functional linkage of PDF and MAP1D has been shown in other animal genomes suggesting the tight regulation of NME ac- tivity in eukaryotic mitochondria [8]. The involvement of a growth-regulatory pathway in modulating PDF expression, provides further support that PDF promotes the growth of tumors and lends support to the pursuit of PDF in- hibitors as cancer therapies. Jessica Clairmont, William Becker, Mark Jensen, and Edward Sauter for their help collecting the human colon cancer tissues for this study. This study was supported by 2P20 RR015566 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH) to KMR and the NDSU Department of Biological Sciences. Authors’ contributions KR conceived, designed, and performed mRNA expression and actinonin experiments, analyzed the data, and wrote the manuscript. HR and SC performed mRNA and protein expression experiments, analyzed the data, and wrote the manuscript. DG and TY conducted gene regulation experiments using the signaling molecule inhibitors and analyzed the data. JM assisted with the design of the expression and regulation experiments and data analysis. All authors read and approved the final manuscript. 16. Escobar-Alvarez S, Goldgur Y, Yang G, Ouerfelli O, Li Y, Scheinberg DA: Structure and activity of human mitochondrial peptide deformylase, a novel cancer target. J Mol Biol 2009, 387(5):1211–1228. 16. Escobar-Alvarez S, Goldgur Y, Yang G, Ouerfelli O, Li Y, Scheinberg DA: Structure and activity of human mitochondrial peptide deformylase, a novel cancer target. J Mol Biol 2009, 387(5):1211–1228. doi:10.1186/1471-2407-13-321 Cite this article as: Randhawa et al.: Overexpression of peptide deformylase in breast, colon, and lung cancers. BMC Cancer 2013 13:321. doi:10.1186/1471-2407-13-321 Cite this article as: Randhawa et al.: Overexpression of peptide deformylase in breast, colon, and lung cancers. BMC Cancer 2013 1 References 1 S lbi i Curr Opin Pharmacol 2006, 6(5):445–452. 6. Giglione C, Serero A, Pierre M, Boisson B, Meinnel T: Identification of eukaryotic peptide deformylases reveals universality of N-terminal protein processing mechanisms. EMBO J 2000, 19(21):5916–5929. 7. Serero A, Giglione C, Meinnel T: Distinctive features of the two classes of eukaryotic peptide deformylases. J Mol Biol 2001, 314(4):695–708. 8. Serero A, Giglione C, Sardini A, Martinez-Sanz J, Meinnel T: An unusual peptide deformylase features in the human mitochondrial N-terminal methionine excision pathway. J Biol Chem 2003, 278(52):52953–52963. Abbreviations (ERK) E ll l (ERK): Extracellular-signal-regulated kinase; (MAP): Methionine aminopeptidase; (MEK): Mitogen-activated protein kinase kinase; (mtDNA): Mitochondrial DNA; (mTOR): Mammalian target of rapamycin; (NME): N-terminal methionine excision; (PDF): Peptide deformylase; (PI3K): Phosphatidylinositol 3-kinase. p g 13. Lee MD, She Y, Soskis MJ, Borella CP, Gardner JR, Hayes PA, Dy BM, Heaney ML, Philips MR, Bornmann WG, et al: Human mitochondrial peptide deformylase, a new anticancer target of actinonin-based antibiotics. J Clin Invest 2004, 114(8):1107–1116. 14. Antczak C, Shum D, Bassit B, Frattini MG, Li Y, Stanchina E, Scheinberg DA, Djaballah H: Identification of benzofuran-4,5-diones as novel and selective non-hydroxamic acid, non-peptidomimetic based inhibitors of human peptide deformylase. Bioorg Med Chem Lett 2011, 21(15):4528–4532. 14. Antczak C, Shum D, Bassit B, Frattini MG, Li Y, Stanchina E, Scheinberg DA, Djaballah H: Identification of benzofuran-4,5-diones as novel and selective non-hydroxamic acid, non-peptidomimetic based inhibitors of human peptide deformylase. Bioorg Med Chem Lett 2011, 21(15):4528–4532. Conclusions In conclusion, we found that PDF is up-regulated in several cancer types including breast, colon, and lung. Our data suggest that the MEK/ERK pathway contributes to the ex- pression of PDF and MAP1D colon cancer cells. Finally, we demonstrated that the PDF inhibitor actinonin inhibits the growth of cancer cell lines to a greater degree than non- cancer cell lines. These data suggest that PDF and MAP1D may function as oncogenes to promote tumor development and are potential selective targets for colon cancer therapy. y 9. Lee MD, Antczak C, Li Y, Sirotnak FM, Bornmann WG, Scheinberg DA: A new human peptide deformylase inhibitable by actinonin. Biochem Biophys Res Commun 2003, 312(2):309–315. 10. Carroll J, Fearnley IM, Walker JE: Definition of the mitochondrial proteome by measurement of molecular masses of membrane proteins. Proc Natl Acad Sci USA 2006, 103(44):16170–16175. 10. Carroll J, Fearnley IM, Walker JE: Definition of the mitochondrial proteome by measurement of molecular masses of membrane proteins. Proc Natl Acad Sci USA 2006, 103(44):16170–16175. 11. Escobar-Alvarez S, Gardner J, Sheth A, Manfredi G, Yang G, Ouerfelli O, Heaney ML, Scheinberg DA: Inhibition of human peptide deformylase disrupts mitochondrial function. Mol Cell Biol 2010, 30(21):5099–5109. 12. Leszczyniecka M, Bhatia U, Cueto M, Nirmala NR, Towbin H, Vattay A, Wang B, Zabludoff S, Phillips PE: MAP1D, a novel methionine aminopeptidase family member is overexpressed in colon cancer. Oncogene 2006, 25(24):3471–3478. References 1 S lbi i 1. Solbiati J, Chapman-Smith A, Miller JL, Miller CG, Cronan JE Jr: Processing of the N termini of nascent polypeptide chains requires deformylation prior to methionine removal. J Mol Biol 1999, 290(3):607–614. 1. Solbiati J, Chapman-Smith A, Miller JL, Miller CG, Cronan JE Jr: Processing of the N termini of nascent polypeptide chains requires deformylation prior to methionine removal. J Mol Biol 1999, 290(3):607–614. 2. Nguyen KT, Hu X, Colton C, Chakrabarti R, Zhu MX, Pei D: Characterization of a human peptide deformylase: implications for antibacterial drug design. Biochemistry 2003, 42(33):9952–9958. 2. Nguyen KT, Hu X, Colton C, Chakrabarti R, Zhu MX, Pei D: Characterization of a human peptide deformylase: implications for antibacterial drug design. Biochemistry 2003, 42(33):9952–9958. Lee et al. showed that the PDF inhibitor actinonin se- lectively inhibited the proliferation of numerous cancer cell lines while having a minimal effect on the growth of non-cancer cell lines [13]. Similarly, our data show that actinonin had significantly greater growth-inhibitory effects on breast and prostate cancer cells than non-cancer cell lines. These results suggest that PDF does play a role in the growth of cancer cells and may offer a selective target for cancer treatment. g y 3. Giglione C, Meinnel T: Peptide deformylase as an emerging target for antiparasitic agents. Expert Opin Ther Targets 2001, 5(1):41–57. 4. Giglione C, Pierre M, Meinnel T: Peptide deformylase as a target for new generation, broad spectrum antimicrobial agents. Mol Microbiol 2000, 36(6):1197–1205. g y 3. Giglione C, Meinnel T: Peptide deformylase as an emerging target for antiparasitic agents. Expert Opin Ther Targets 2001, 5(1):41–57. 4. Giglione C, Pierre M, Meinnel T: Peptide deformylase as a target for new generation, broad spectrum antimicrobial agents. Mol Microbiol 2000, 36(6):1197–1205. 5. Leeds JA, Dean CR: Peptide deformylase as an antibacterial target: a critical assessment. Curr Opin Pharmacol 2006, 6(5):445–452. 6. Giglione C, Serero A, Pierre M, Boisson B, Meinnel T: Identification of eukaryotic peptide deformylases reveals universality of N-terminal protein processing mechanisms. EMBO J 2000, 19(21):5916–5929. 7. Serero A, Giglione C, Meinnel T: Distinctive features of the two classes of eukaryotic peptide deformylases. J Mol Biol 2001, 314(4):695–708. 8. Serero A, Giglione C, Sardini A, Martinez-Sanz J, Meinnel T: An unusual peptide deformylase features in the human mitochondrial N-terminal methionine excision pathway. J Biol Chem 2003, 278(52):52953–52963. 5. Leeds JA, Dean CR: Peptide deformylase as an antibacterial target: a critical assessment. Competing interests Competing interests The authors have no competing interests in relation to this paper. The authors have no competing interests in relation to this paper Acknowledgements We are very grateful to Carmela Giglione and Thierry Meinnel of the Centre National de la Recherche Scientifique, Gif-sur-Yvette, France for kindly providing the PDF antibody. We would like to thank the Veteran’s Affairs Hospital in Fargo, ND and their research staff Jodie Haring, Candice Nelson,
https://openalex.org/W2150838974	https://europepmc.org/articles/pmc4109312?pdf=render	English	null	Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants	eLife	2,014	cc-by	10,988	Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants Satoshi Tsunoda1,2,4, Edward Avezov1,2,4, Alisa Zyryanova1,2,4, Tasuku Konno1,2,4, Leonardo Mendes-Silva3, Eduardo Pinho Melo3, Heather P Harding1,2,4, David Ron1,2,4 1Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom; 2Wellcome Trust MRC Institute of Metabolic Science, Cambridge, United Kingdom; 3Centre for Molecular and Structural Biomedicine, Universidade do Algarve, Faro, Portugal; 4NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom Abstract Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1CtoS purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1- roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER. DOI: 10.7554/eLife.03421.001 For correspondence: hph23@ cam.ac.uk (HPH); dr360@ medschl.cam.ac.uk (DR) Competing interests: See page 18 Funding: See page 18 Received: 20 May 2014 Accepted: 03 July 2014 Published: 29 July 2014 Reviewing editor: Matthew Freeman, University of Oxford, United Kingdom Copyright Tsunoda et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. For correspondence: hph23@ cam.ac.uk (HPH); dr360@ medschl.cam.ac.uk (DR) Competing interests: See page 18 Funding: See page 18 Received: 20 May 2014 Accepted: 03 July 2014 Published: 29 July 2014 Funding: See page 18 Received: 20 May 2014 Accepted: 03 July 2014 Published: 29 July 2014 elifesciences.org elifesciences.org RESEARCH ARTICLE elifesciences.org elifesciences.org Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants Introductioni Disulfide bonds have a critical role in stabilizing correctly folded secreted and membrane proteins (Fass, 2012). Dedicated enzymatic machinery, consisting of disulfide exchange catalysts of the protein disulfide isomerase (PDI) class, rapidly introduces disulfide bonds into nascent polypeptide chains. The recycling of PDIs back to their oxidized form is mediated by upstream oxidases, exemplified by ERO1 and back-up enzymes, such as PRDX4 and VKOR that exploit O2, H2O2, and vitamin K epoxide, respec tively, as downstream electron acceptors. Defects in components of this electron transfer chain mark edly affect protein folding homeostasis in the ER (Sevier and Kaiser, 2008; Rutkevich and Williams, 2012; Zito, 2013). Reviewing editor: Matthew Freeman, University of Oxford, United Kingdom The destruction of a poorly folded protein, via a process that also needs this protein's disulfide bonds to be broken down, was also not affected by a lack of reduced glutathione in the endoplasmic reticulum. p Furthermore, decreasing these levels of glutathione did not affect the unfolded protein response: a stress response in cells that are experiencing a build-up of unfolded or poorly folded proteins within the endoplasmic reticulum. As such, the findings of Tsunoda et al. challenge the importance of reduced glutathione in the endoplasmic reticulum and suggest that other chemical processes might be involved in editing disulfide bonds. Further work is now needed to investigate the other known processes that might complete this task instead to see which, if any, are involved. mixed disulfide by the C-terminal active site cysteine, in which PDI ‘escapes’ in its oxidized form, hav ing reduced its client protein (Walker et al., 1996). Both mechanisms require a pool of reduced PDI, the maintenance of which is not trivial, given the oxidative environment in the ER. This challenge is especially great in case of the second mechanism, which is a set-up for iterative cycles of net oxidation- reduction-oxidation (Schwaller et al., 2003). mixed disulfide by the C-terminal active site cysteine, in which PDI ‘escapes’ in its oxidized form, hav ing reduced its client protein (Walker et al., 1996). Both mechanisms require a pool of reduced PDI, the maintenance of which is not trivial, given the oxidative environment in the ER. This challenge is especially great in case of the second mechanism, which is a set-up for iterative cycles of net oxidation- reduction-oxidation (Schwaller et al., 2003). The crucial role of reduced PDI to oxidative protein folding is supported by in vitro experiments in which PDI's ability to accelerate folding is not monotonically increased by its oxidized fraction, but rather is optimal in a redox buffer that also contains a high concentration of reduced glutathione (Lyles and Gilbert, 1991). Yeast genetics reveal that disulfide shuffling (which requires reduced PDI) is PDI1's essential function, as an active-site mutant that has lost the ability to produce disulfides (but has selec tively retained activity as a disulfide reductase) is nonetheless able to rescue the lethal phenotype of pdi1 nullizygosity (Laboissiere et al., 1995). Reviewing editor: Matthew Freeman, University of Oxford, United Kingdom Reviewing editor: Matthew Freeman, University of Oxford, United Kingdom Copyright Tsunoda et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Many disulfides introduced by the oxidative machinery are not native and must be broken down in an editing process that is catalyzed by reduced forms of PDIs (Hatahet and Ruddock, 2009). This editing entails attack by the reduced N-terminal cysteine of PDI's active site on the misplaced disulfide following resolution of the mixed disulfide. The latter proceeds either by way of a zero-sum re-shuffling process entailing an attacking second reduced cysteine in the client polypeptide, which releases reduced PDI and establishes a new disulfide, or by a net reductive process involving an attack on the Tsunoda et al. eLife 2014;3:e03421. DOI: 10.7554/eLife.03421 1 of 20 Research article Research article Research article Cell biology eLife digest Proteins are basically strings of amino acids that have folded into a specific three-dimensional shape, and this shape is often important for the protein's function. Some proteins have bonds between pairs of cysteines—an amino acid that contains sulfur—in different parts of the protein to maintain its correct shape. p p In eukaryotes, such as plants and animals, these so-called ‘disulfide bonds’ are formed inside a structure within each cell called the endoplasmic reticulum, which is where many proteins are folded. Occasionally, disulfide bonds form in the wrong place in a protein, so they need to be broken and re-positioned—a process sometimes called editing—for the protein to fold correctly. It was widely assumed that a chemical called ‘reduced glutathione’ fuels the breaking of disulfide bonds in the endoplasmic reticulum, but to date few researchers have tried to test this assumption. p p Tsunoda et al. have now taken an enzyme that degrades glutathione elsewhere in the cell and modified it in a way that allows it to work inside the endoplasmic reticulum. When this modified enzyme was produced in human cells grown in the laboratory, it purged the endoplasmic reticulum of glutathione. However, the lack of glutathione had no effect on the folding of a large protein with 30 disulfide bonds, many of which need to be edited at one time or another for the protein to fold correctly. Tsunoda et al. eLife 2014;3:e03421. DOI: 10.7554/eLife.03421 A glutathione-degrading enzyme suited to the oxidizing conditions of the endoplasmic reticulum Kumar et al. recently reported that the mammalian pro-apoptotic gene ChaC1 encodes a glutathione- specific γ-glutamyl cyclotransferase that efficiently degrades glutathione (Kumar et al., 2012). We confirmed their observations by measuring the ability of purified murine ChaC1 (expressed in E. coli) to degrade glutathione in vitro: At submicromolar enzyme concentrations, recombinant ChaC1 was able to degrade a 10 mM solution of reduced glutathione within 1 hr (Figure 1A). The enzymatic activity was selective towards reduced glutathione (GSH) (Figure 1B). The inability of oxidized gluta thione (GSSG) to serve as a substrate for degradation correlated with its inability to bind a Chac1- based optical probe whose fluorescent resonance energy transfer (FRET) signal reflects substrate binding (Figure 1C–D and Figure 1—figure supplement 1). An E116Q mutation abolished all enzy matic activity (Figure 1E), as observed previously (Kumar et al., 2012). To exploit ChaC1 as a tool to purge the ER of glutathione, we targeted expression of this cyto solic enzyme to the ER, by fusing the coding sequence to an N-terminal cleavable signal peptide and a C-terminal KDEL ER retention signal. An N-terminal FLAG-M1 peptide tag was included, to facili tate detection of the enzyme. Cells transfected with a plasmid encoding ER-FLAG-ChaC1 expressed a protein of the expected mobility on reducing SDS-PAGE that reacted with the anti-FLAG anti body (Figure 2A) and resulted in a granular staining pattern that overlapped with that of the ER marker calreticulin (Figure 2B). However, unlike cytosolic ChaC1, which migrates at a position expected of the reduced monomer on non-reducing SDS-PAGE (Figure 2—figure supplement 1), ER-localized ChaC1 migrated as a heterogenous collection of species, consistent with inappro priate disulfide bond formation (compare the reducing and non-reducing SDS-PAGE in Figure 2A and Figure 2—figure supplement 1). A model of mouse ChaC1, based on the crystal structure of the homologous γ-glutamyl cyclotrans ferase (PDB 2RBH) (Kumar et al., 2012), suggested that none of its four cysteines is likely to play an important role in substrate recognition or catalysis. As expected, conversion of all four cysteines to serines resulted in a protein that no longer formed disulfides when introduced into the ER (Figure 2A, compare lanes 6 and 7 and Figure 2—figure supplement 1). Importantly, the cysteine-free enzyme (ChaC1CtoS) purified from E. coli retained its enzymatic activity (Figure 2C) and specificity for reduced glutathione (Figure 1D and Figure 1—figure supplement 1C). A glutathione-degrading enzyme suited to the oxidizing conditions of the endoplasmic reticulum There are no predicted N-linked gly cosylation sites in ChaC1CtoS to further corrupt protein structure when targeted to the ER, therefore, it seemed possible that ER-localized ChaC1CtoS might retain its enzymatic activity and breakdown gluta thione in the ER. Reviewing editor: Matthew Freeman, University of Oxford, United Kingdom The reductive facet of oxidative protein folding in the ER is especially important to the maturation of large proteins such as the low density lipoprotein receptor (LDL-R), in which it has been estimated that most disulfides that form early during biogenesis are non-native and must be rearranged before the protein clears ER quality control and traffics to the Golgi (Jansens et al., 2002). This editing pro cess appears to involve a specific PDI family member, ERdj5 (Oka et al., 2013). ERdj5 may have spe cialized in disulfide reduction, as its redox function also accelerates the clearance of misfolded ER proteins such as the null Hong Kong mutant α1-antitrypsin (NHK-A1AT) (Ushioda et al., 2008; Hagiwara et al., 2011), but the identity of ERdj5's reductase remains unknown. Reductive editing of disulfides is also observed in the E. coli periplasm. Where the transfer of elec trons from reduced thioredoxin in the cytosol maintains a reduced pool of the periplasmic isomerases, DsbC and DsbG. Electrons are conveyed across the inner-membrane space by a specialized transmem brane protein DsbD. This protein relay-based mechanism enables DsbC/DsbG-dependent disulfide shuffling despite the absence of a soluble small molecule redox buffer in the periplasmic space of gram negative bacteria (reviewed in Cho and Collet, 2013). By contrast, the mammalian ER contains Tsunoda et al. eLife 2014;3:e03421. DOI: 10.7554/eLife.03421 2 of 20 Research article Research article Research article Cell biology up to 15 mM glutathione (Montero et al., 2013) whose reduced form is widely believed to fuel the reductive aspects of secreted protein metabolism in eukaryotes, by serving as a terminal electron donor to reduce PDI family members (reviewed in Kojer and Riemer, 2014). To critically examine the role of ER glutathione in the reductive re-shuffling of non-native disulfides and in the reductive steps believed to be associated with degradation of misfolded ER proteins, we devised a method to selectively deplete the ER of glutathione and examined the consequences on the organelles' capacity to handle well-characterized sentinel proteins. Research article Cell biology Figure 1. Continued Figure 1. Continued Figure 1. Continued of the ability of ChaC1 to eliminate reduced (GSH) and oxidized glutathione (GSSG). Note that ChaC1 effectively eliminated reduced glutathione, but had no effect on oxidized glutathione. (C) Cartoon of the fluorescent resonance energy transfer (FRET) probe, OG-ChaC1-Cherry, used to detect substrate binding to ChaC1. Shown is a model of murine ChaC1 (UniProt Q8R3J5) residues 31–204, created by Phyre2 (Kelley and Sternberg, 2009) based on the crystal structure of γ-glutamyl cyclotransferase (PDB 2RBH). The side chain of Cys 92, which has been modified with the Oregon Green (OG) donor, is highlighted, as is the C-terminus of the protein, site of the fused mCherry fluorescent acceptor. (D) Time-resolved FRET signal (expressed as the ratio of the emission signal at 532 nm and 670 nm upon excitation at 480 nm) of the OG-ChaC1-Cherry probe [2.5 µM] following exposure to 10 mM reduced (GSH) or oxidized glutathione(GSSG). Where indicated, the sample was injected with dithiotreitol (DTT) to reduce the GSSG and convert it to a substrate for ChaC1. The biphasic change in FRET signal upon exposure to GSH is consistent with binding followed by breakdown of GSH by the probe, which retains its enzymatic activity. (E) Comparison of glutathione elimination by purified bacterially expressed wild-type and E116Q mutant ChaC1 in vitro. DOI: 10.7554/eLife.03421.003 The following figure supplements are available for figure 1: Figure supplement 1. Analysis of the substrate binding properties of ChaC1. DOI: 10.7554/eLife.03421.004 Oxidized glutathione levels recover rapidly following a reductive pulse in cultured mammalian cells (Appenzeller-Herzog et al., 2010). Therefore, the aforementioned in vitro observations indicating tight coupling of Grx1-roGFP2 to glutathione (and relative indifference to PDI) suggested that the rate of re-oxidation of an ER-localized Grx1-roGFP2 probe following a reductive pulse might be affected by the presence of glutathione in the ER lumen. To examine this further, we first compared the rate of re-oxidation of ER-localized roGFP2 with that of ER-localized Grx1-roGFP2 following a reductive pulse of dithiothreitol (DTT) and its washout. At steady-state both probes were highly oxidized and both were similarly reduced by the DTT pulse. However the recovery of ER-Grx1-roGFP2 was accelerated compared with ER-roGFP2 alone, with a half-time to recovery of 75.3 ± 7.7 s in case of the former and 167.3 ± 16.8 s in case of the latter (Figure 3E,F). Research article This observation is consistent with a direct contribu tion of lumenal glutathione to the kinetics of ER-Grx1-roGPF2 re-oxidation in vivo. Next, we compared the effect of ER-localized enzymatically-active ER-ChaC1CtoS and its catalytically dead counterpart, ER-ChaC1CtoS−E116Q on the rate of oxidation of ER-roGFP2 and ER-Grx1-roGFP2 fol lowing a reductive pulse and washout. To focus the measurements on cells co-expressing the roGFP probe and the ChaC1 enzyme, the latter was tagged at its C-terminus with mCherry. Oxidation of ER-roGFP2 was unaffected by the presence of active ChaC1, but the kinetic advantage of ER-Grx1- roGFP2 over the ER-roGFP2 alone was abolished by expression of an active, glutathione-degrading enzyme in the ER lumen (Figure 3G,H). We were unable to reproducibly measure glutathione concentrations in microsomal fractions of cultured cells; however, the effect of ChaC1 over-expression on total cellular glutathione was quantifi able. At similar levels of over-expression, cytosolic ChaC1 led to a marked depletion of total cellular glutathione levels, whereas ER-localized ChaC1CtoS had a more modest effect. However, ER-localized ChaC1CtoS markedly enhanced glutathione depletion by low concentrations of buthionine-sulfoxide (BSO, an inhibitor of the rate limiting step of glutathione biosynthesis, Figure 3I). To avoid the corrupt ing effect of an untransfected pool of cells, these ensemble measurements were conducted in stable clones homogenously and conditionally-expressing ChaC1 from a doxycycline-inducible transgene. They point to relatively slow equilibration of cytosolic and ER pools of glutathione in mammalian cells. Given that Grx1-roGFP2 reacts with glutathione with a Kmax0.5 in the 10−5 M range (Figure 3D) the effacement of its kinetic advantage over roGFP2 in the DTT washout experiment, indicated a profound and selective depletion of lumenal glutathione by the ER-targeted expression of active ChaC1, with modest effects on other cellular pools of glutathione. Purging the ER of glutathione Measuring the impact of ER-ChaC1CtoS on glutathione levels required an assay that would be selec tively sensitive to the ER pool of glutathione. Glutaredoxin (Grx1) has been shown to dramatically accelerate the interaction of a linked redox-sensitive green fluorescent protein (roGFP) with gluta thione, both in vivo and in vitro (Gutscher et al., 2008; Birk et al., 2013) (cartooned Figure 3A). We confirmed the reported ability of a linked Grx1 to accelerate the equilibration of roGFP with a gluta thione buffer: alone, reduced roGFP2 was only slowly oxidized by glutathione (Figure 3B), but the linked Grx1 markedly accelerated the oxidation of Grx1-roGFP2 (Figure 3, compare the red traces in panels B and C). The rate of probe oxidation by glutathione was concentration-dependent, with half- saturation (Kmax0.5) attained at ∼18 µM GSSG (Figure 3D). Importantly, the presence of oxidized PDI had a minor role in further accelerating the oxidation of Grx1-roGFP2, but dominated the oxidation kinetics of roGFP2 alone (Figure 3B,C). Tsunoda et al. eLife 2014;3:e03421. DOI: 10.7554/eLife.03421 3 of 20 Research article Research article Cell biology igure 1. ChaC1 efficiently and selectively degrades reduced glutathione. (A) A bar-graph representation of residual glutathione le ncubation of 10 mM glutathione with the indicated concentrations of bacterially expressed mouse ChaC1. Varying concentrations ssayed at a single time point (left panel) and varying initial concentrations of glutathione were assayed at different time points (right p igure 1. Continued on next page Figure 1. ChaC1 efficiently and selectively degrades reduced glutathione. (A) A bar-graph representation of residual glutathione levels following incubation of 10 mM glutathione with the indicated concentrations of bacterially expressed mouse ChaC1. Varying concentrations of enzymes were assayed at a single time point (left panel) and varying initial concentrations of glutathione were assayed at different time points (right panel). (B) Comparison Figure 1. Continued on next page Tsunoda et al. eLife 2014;3:e03421. DOI: 10.7554/eLife.03421 4 of 20 4 of 20 Research article Lumenal glutathione is dispensable to the reductive facets of ER protein folding f DOI: 10.7554/eLife.03421.006 Conversion of the LDL-R from its ER to post-ER form was unaffected by the presence of either active or inactive form of ER-ChaC1 and proceeded with a half-life of ∼1 hr in presence or absence of an active glutathione-depleting enzyme in the ER (Figure 4A). The non-reducing gel revealed identical accelerated mobility of LDL-R at the earliest time point, regardless of expression of ChaC1, indicating that glutathione depletion also had no drastic effect on the earlier oxidative phase of LDL-R matura tion (Figure 4A,B). LDL-R maturation was also unaffected by further global depletion of cellular glutathione, effected by the combined expression of ER-ChaC1CtoS and exposure to buthionine sulfoxide (BSO), which depleted glutathione globally (Figure 4C–E). These experiments, conducted in HeLa cells, where reductive editing of LDL-R disulfides was first discovered (Jansens et al., 2002), reinforces the dispen sability of glutathione to that process. To further explore the potential role of ER glutathione in the early oxidative steps of protein folding, we used highly sensitized mouse cells genetically deficient in the upstream thiol oxidases ERO1 and PRDX4 (Zito et al., 2010b, 2012) Expression of active ChaC1 in their ER had no effect on the kinetics of disulfide re-formation on ER-localized roGFP2 following a reductive DTT pulse (Figure 4F–G), fur ther attesting to the dispensability of glutathione to thiol oxidation in the mammalian ER. The degradation of misfolded NHK-A1AT requires the action of the reduced form of a specialized PDI, ERdj5 (Ushioda et al., 2008; Hagiwara et al., 2011). To determine if an ER pool of glutathione contributes to this process, we compared the half-life of C-terminally FLAG-tagged NHK-A1AT in cells expressing active ER-ChaC1CtoS and the catalytically inactive ER-ChaC1CtoS;E116Q. The stability of NHK- A1AT was unaffected by ChaC1 both in HeLa cells (Figure 5A,D) and in 293T cells (Figure 5B,E). Furthermore, depletion of total cellular pools of glutathione by coincidental exposure to BSO had no effect on NHK-A1AT half-life (Figure 5C,F), complementing the evidence for the dispensability of glutathione for ER-associated degradation of this redox-dependent substrate. Thiol redox reactions contribute to protein folding homeostasis in the endoplasmic reticulum. This is reflected in enhanced signaling in the endoplasmic reticulum unfolded protein response (UPR) in cells impaired in ER thiol redox (Frand and Kaiser, 1998; Pollard et al., 1998). Lumenal glutathione is dispensable to the reductive facets of ER protein folding g Maturation of the LDL-R in the ER entails significant rearrangement of its 30 disulfide bonds (Jansens et al., 2002) and involves the reduced form of the PDI family member ERdj5 (Oka et al., 2013). Maturation of the LDL-R can be tracked by the conversion of the relatively high mobility, glycosylated, ER form, to the lower-mobility post-ER form (reflective of Golgi sugar modifications) in pulse-chase labeling followed by immunoprecipitation. We thus compared the effects of ER expression of active ChaC1CtoS and the catalytically inactive ChaC1CtoS;E116Q mutant on the rate of maturation of co-expressed LDL-R tagged on its C-terminal, cytosolic facing domain, with a triple FLAG-tag. Tsunoda et al. eLife 2014;3:e03421. DOI: 10.7554/eLife.03421 5 of 20 Research article Research article Cell biology / f Figure 2. Cysteine-free ChaC1 is an active enzyme that can be targeted to the endoplasmic-r (A) Immunoblot of FLAG M1-tagged ER-localized wild type (WT), cysteine-free (CtoS), and compo enzymatically dead ChaC1 in N-ethyl maleimide-blocked lysates of transfected HeLa cells. Lanes Figure 2. Continued on next page Figure 2. Cysteine-free ChaC1 is an active enzyme that can be targeted to the endoplasmic-reticulum. (A) Immunoblot of FLAG M1-tagged ER-localized wild type (WT), cysteine-free (CtoS), and compound CtoS; E116Q enzymatically dead ChaC1 in N-ethyl maleimide-blocked lysates of transfected HeLa cells. Lanes 1–4 are from a Figure 2. Continued on next page Tsunoda et al. eLife 2014;3:e03421. DOI: 10.7554/eLife.03421 6 of 20 Research article Research article Cell biology Figure 2. Continued Figure 2. Continued Figure 2. Continued reducing and lanes 5–8 are from a non-reducing SDS-PAGE. Note the presence of high-molecular weight disulfide linked FLAG-tagged ChaC1 in cells transfected with the WT ER-targeted protein that is absent from those transfected with the cysteine-free, CtoS mutants. (B) Fluorescent photomicrographs of HeLa cells transfected with the indicated expression plasmids and immunostained for the FLAG tag (marking ChaC1) and calreticulin (CALR) as an ER marker. The merge panels show an overlap of the FLAG, CALR, and Hoechst 33,258 signal (to reveal the nuclei) at 630X with a close-up view in the right-most panel. (C) A bar-graph representation of residual glutathione levels following 60-min incubation of 10 mM glutathione with the indicated concentrations of bacterially expressed wild-type ChaC1, its cysteine free derivative, ChaC1CtoS, and its inactive mutant ChaC1CtoS;E116Q. DOI: 10.7554/eLife.03421.005 e following figure supplements are available for figure 2: Figure supplement 1. Replacement of cysteines with serines circumvents aberrant disulfide bond formation in ER-localized ChaC1. Lumenal glutathione is dispensable to the reductive facets of ER protein folding Therefore, to gain a more global view on the impact of ER glutathione depletion on ER protein folding homeostasis, we compared the effect on UPR activity of ER-targeted active and inactive ChaC1. These experiments made use of mCherry-tagged ChaC1CtoS, which retains its enzymatic activity, purges the ER of gluta thione (Figures 1D and 3G) and marks the ChaC1-expressing cells. Dual-channel FACS analysis revealed that neither active nor inactive ER-ChaC1CtoS-mCherry-KDEL measurably affected the basal activity of a stably integrated mammalian UPR reporter, CHOP::GFP (Novoa et al., 2001) (reflected in the absence of a shift to the right in the mCherry positive population of cells, Figure 6A, left column). Furthermore, the activity of the CHOP::GFP reporter, which was increased by tunicamycin, a toxin that perturbs protein folding homeostasis in the ER, was unaffected by ChaC1 (Figure 6A). The indifference of the UPR to ER-ChaC1CtoS is observed over a broad range of unfolded protein stress and over a broad range of ER-ChaC1CtoS-mCherry-KDEL expression, and sug gests that the lesson learned from the sentinel proteins, LDL-R and NHK-A1AT likely extend to the bulk of proteins that fold oxidatively in the ER under normal cell culture conditions. Tsunoda et al. eLife 2014;3:e03421. DOI: 10.7554/eLife.03421 7 of 20 Research article Research article Research article Cell biology Research article Figure 3. ER-targeted ChaC1 purges the organelle of its glutathione content. (A) Cartoon contrasting the slow coupling redox buffer (dashed lines) and the rapid coupling of Grx1-roGFP2 with the glutathione redox buffer (after Gutscher et a dependent changes in the ratio of reduced to oxidized roGFP2 detected optically as the ratio between the emission sign Figure 3 Continued on next page Figure 3. ER-targeted ChaC1 purges the organelle of its glutathione content. (A) Cartoon contrasting the slow coupling of roGFP2 with the glutathione redox buffer (dashed lines) and the rapid coupling of Grx1-roGFP2 with the glutathione redox buffer (after Gutscher et al., 2008). (B) Trace of time- dependent changes in the ratio of reduced to oxidized roGFP2 detected optically as the ratio between the emission signal (at 535 nm) upon excitation Figure 3. Continued on next page Figure 3. ER-targeted ChaC1 purges the organelle of its glutathione content. (A) Cartoon contrasting the slow coupling of roGFP2 with the glutathione redox buffer (dashed lines) and the rapid coupling of Grx1-roGFP2 with the glutathione redox buffer (after Gutscher et al., 2008). Research article Cell biology Figure 3. Continued Figure 3. Continued at 405 nm vs 488 nm (ex 405/488) following introduction of the fully reduced probe into the indicated solutions of oxidized glutathione (GSSG) or PDI and GSSG. (C) Similar trace of reduced Grx1-roGFP2. Note the indifference of roGFP2 and the marked responsiveness of Grx1-roGFP to oxidized glutathione. (D) Graph of the initial velocity of Grx1-roGFP2 oxidation as a function of GSSG concentration, fitted to Linweaver–Burk plot. Half-maximal velocity is observed at 18 µM GSSG. (E) Trace of time-dependent changes in the ratio of oxidized and reduced roGFP2 and Grx1-roGFP2 probes expressed in the ER of HeLa cells following a brief (1 min) reductive pulse with dithiothreitol (DTT, 2 mM) followed by a washout. (F) Bar diagram of the half-time to recovery of oxidized roGFP2 and Grx1-roGFP2 following the reductive DTT pulse. Shown are means ±SD (N = 4, p<0.01). (G) Trace of time-dependent changes in the ratio of oxidized to reduced probes expressed in the ER of HeLa cells alongside active or inactive ChaC1 (tagged at its C-terminus with mCherry to allow visualization of cells co-expressing the redox probes and the glutathione-depleting enzyme) following a brief reductive pulse with dithiothreitol and a washout. Note that the expression of active ChaC1 in the ER eliminates the kinetic advantage of Grx1-roGFP2 over roGFP2 in re-oxidation during the recovery from a DTT reductive pulse. (H) Bar diagram of the half-time to recovery of oxidized roGFP2 and Grx1-roGFP2 following the reductive DTT pulse in cells co-expressing active or inactive CHaC1. Shown are means ±SEM (N = 20, p<0.01). (I) Bar diagram of cellular glutathione levels following 36 hr of doxycycline (DOX) induction of cytosolic and ER localized active and inactive ChaC1 in the absence and presence of concomitant exposure to buthionine-sulfoxide (BSO, 50 µM). Also shown is a time course of total cellular glutathione following induction of active and inactive mCherry-KDEL-tagged ChaC1. The role of ER glutathione was further explored under conditions in which ER redox balance was perturbed by co-expression of a deregulated allele of the ER oxidase ERO1 (Sevier et al., 2007), which has been shown to hyper-oxidize the mammalian ER (Baker et al., 2008) and modestly activate the UPR (Hansen et al., 2012). Research article Introduction of the C104A; C133A human ERO1L (ERO1), expressed from a plasmid tagged with the CD2 surface marker indeed modestly activated the UPR, whether measured in cells stably expressing the CHOP::GFP transcriptional reporter (Figure 6B, reflected in the higher GFP levels of cells co-expressing the CD2 marker which tags the ERO1-expressing cells, panels 2, 4, and 7) or an XBP1::Venus splicing reporter (Iwawaki et al., 2004) (Figure 6—figure sup plement 1). However, co-expression of active ChaC1CtoS had no evident synergistic effect with ERO1* on UPR activity beyond that observed with the enzymatically inactive E116Q mutant enzyme. This is evident by first noting that in this co-transfection experiment most ERO1* expressing cells (CD2 posi tive) are also co-expressing the Cherry-tagged ChaC1CtoS (Figure 6B, panels 3 and 6) and then noting that the relationship between ERO1* expression (for which CD2 is a surrogate) and the CHOP::GFP signal is indistinguishable in cells co-expressing wild-type and enzymatically inactive ChaC1CtoS (Figure 6B panels 4 and 7). Similarly, the co-expression of ERO1* does not impart sensitivity to active ChaC1, as reflected by the observation that CHOP::GFP levels are unaffected by ChaC1CtoS in these double positive cells (Figure 6B panels 5 and 8). These findings indicate that glutathione is dispensa ble for the function of the ER even under hyperoxidizing conditions. Lumenal glutathione is dispensable to the reductive facets of ER protein folding (B) Trace of time- dependent changes in the ratio of reduced to oxidized roGFP2 detected optically as the ratio between the emission signal (at 535 nm) upon excitation Figure 3. Continued on next page Tsunoda et al. eLife 2014;3:e03421. DOI: 10.7554/eLife.03421 8 of 20 Research article Discussion DOI: 10.7554/eLife.03421 10 of 20 Research article Research article Cell biology Figure 4. Continued reducing SDS-PAGE. Cells were lysed at the end of a 30-min labeling pulse (lanes 1 and 6) or after an additional chase period (indicated). The mobility of the ER and Golgi forms of the LDL-R on reducing (left) and non-reducing (right) SDS-PAGE is indicated, as is the labeled ChaC1, which is also recovered in this anti-FLAG immunoprecipita tion. (B) Graphic presentation of the conversion of the ER to Golgi form of the LDL-R from ‘A’ above. Shown is a representative experiment reproduce three times with similar outcome. (C) Autoradiograph of an experiment similar in layout to that depicted in ‘A’ above. Where indicated, the cells were exposed to the glutathione synthesis inhibitor buthionine-sulfoxide (BSO, 100 µM, 20 hr) before the pulse-chase labeling. (D) Graphic presentation of the conversion of the ER to Golgi form of the LDL-R from ‘C’ above. Shown is a representative experiment reproduce three times with similar outcome. (E) Bar graph of total cellular glutathione in cells manipulated as in the experi ment described in ‘C’ (shown is the mean ±SD, n = 3, p<0.05). (F) Trace of time-dependent changes in the ratio of oxidized and reduced roGFP2 expressed in the ER of mouse embryonic fibroblasts with genetic lesions compromis ing disulfide bond formation (Ero1am/m; Ero1bm/m; Prdx4m/y) following a brief reductive pulse with dithiothreitol followed by a washout. The cells co-expressed ER-localized enzymatically active (ER-ChaC1CtoS) or inactive ChaC1 (ER-ChaC1CtoS;E116Q) marked at its C-terminus with an mCherry fluorescent probe. (G) Bar diagram of the half-time to recovery of oxidized roGFP2 following the reductive DTT pulse in ‘F’ above. Note that expression of active ChaC1 in the ER did not affect the rate of recovery of the sentinel disulfide in ER-localized roGFP2. DOI: 10 7554/eLife 03421 008 DOI: 10.7554/eLife.03421.008 How complete was depletion of ER glutathione by ChaC1? In vitro the re-oxidation of Grx1-roGFP2 is half-maximal at 18 µM glutathione (Figure 3D). Thus, the abolition of the kinetic advantage of Grx1- roGFP2 over roGFP2 in cells expressing enzymatically active ChaC1 argues that, despite the enzyme's relative low affinity for its substrate (in the millimolar range, Kumar et al., 2012; and Figure 1—figure supplement 1C,D), glutathione levels were purged to micromolar levels by the high concentration of the enzyme. Discussion This conclusion is also supported by the effects of ER ChaC1 on total cellular glutathione levels: At similar levels of expression, cytosolic ChaC1 led to near complete depletion of total cellular glutathione, whereas ER ChaC1 had a more modest effect on total cellular pools. Given the 10-fold greater volume of the cytosol over the ER (Stefan et al., 1987), the aforementioned observations indicate substantially higher local concentration of ER vs cytosolic ChaC1. Thus, the preservation of total cellular glutathione levels in cells expressing high levels of ChaC1 in their ER confirms that trans port of glutathione into the ER, from its site of synthesis in the cytosol, is slow (as suggested by Kumar et al., 2011), and therefore, that depletion of ER stores by ChaC1 is profound. The redundancy of glutathione could be explained by other small molecule thiols fueling reduction in the ER. These may even include cysteinyl-glycine, one of the glutathione breakdown product of ChaC1 action. Alternatively, eukaryotes may have a protein-driven apparatus akin to bacterial DsbD for ferrying reducing equivalents from the cytosol to reduce PDI family members. Despite its apparent dispensability, ER glutathione does equilibrate with protein thiols (Appenzeller- Herzog et al., 2010), indicating that it is a redox buffer in the organelle. However, it is possible that despite seemingly rapid equilibration of glutathione to protein thiol redox, the kinetics are insufficient to render glutathione essential and that other, even faster processes, maintain an adequate pool of reduced and oxidized PDIs in the mammalian ER, with glutathione following passively. It is noteworthy, in this vein, that ERO1-deficient cells, with compromised ER thiol oxidative power, have an elevated ratio of oxidized to reduced glutathione (Appenzeller-Herzog et al., 2010; Rutkevich and Williams, 2012). It has been speculated, reasonably, that this reflects the action of an alternative oxidative path way that kicks-in when the major oxidases are compromised and exploits oxidized glutathione to couple to protein thiol oxidation. However, our finding that ER glutathione depletion does not further compromise protein thiol oxidation kinetics in oxidase-deficient cells suggests that here too gluta thione follows passively the rearrangements in redox pathways and does not participate actively in their implementation. Across distant phyla, ER redox is not indifferent to cellular glutathione levels. Discussion Adapting a cytosolic enzyme that breaks down glutathione to function in the ER has afforded a means to selectively purge the organelle of glutathione and thereby assess the role of this otherwise abun dant tri-peptide in ER protein redox. Nearly complete depletion of ER glutathione had no measurable effect on the rate of disulfide bond formation in the highly sensitized experimental setting of cells deficient in ER thiol oxidases. More surprising was the lack of any measurable effect of glutathione depletion on processes that require a pool of reduced PDI enzymes: the disulfide shuffling-dependent maturation of nascent LDL-R and the degradation of a folding incompetent mutant NHK-A1AT. Furthermore, depletion of ER glutathione was without effect on two sensitive, broad-spectrum UPR reporters, arguing that glutathione is indeed dispensable to a broad range of processes required for protein folding homeostasis in the ER of cultured mammalian cells. The dispensability of glutathione to reductive processes in the ER has been hinted at previously: in yeast deletion of GSH1 (encoding gamma glutamylcysteine synthetase that performs the rate-limiting step in glutathione biosynthesis) does not adversely affect the maturation of the disulfide-bonded lysosomal hydrolyze CPY (Frand and Kaiser, 1998). However, the extent of ER glutathione depletion attained by this genetic manipulation is unclear and, given the essential role of cellular glutathione to yeast growth, may have been limited by toxicity. Similar reservations apply to the use of enzyme inhibi tors, such as BSO, which subject the cell to the consequences of glutathione depletion in other com partments where it has an essential role (Kumar et al., 2011), but whose effect on the ER pools of glutathione may be partial. Tsunoda et al. eLife 2014;3:e03421. DOI: 10.7554/eLife.03421 9 of 20 Research article Research article Research article Cell biology Figure 4. Maturation of the LDL-R receptor is unaffected by depletion of ER glutathione. (A) Autoradiograp metabolically labeled LDL receptor (LDL-R) immunopurified from HeLa cells co-expressing the FLAG-tagge and ER-localized enzymatically active (ER-ChaC1CtoS) or inactive ChaC1 (ER-ChaC1CtoS;E116Q) and resolved on a Figure 4. Continued on next page Figure 4. Maturation of the LDL-R receptor is unaffected by depletion of ER glutathione. (A) Autoradiograph of metabolically labeled LDL receptor (LDL-R) immunopurified from HeLa cells co-expressing the FLAG-tagged LDL-R and ER-localized enzymatically active (ER-ChaC1CtoS) or inactive ChaC1 (ER-ChaC1CtoS;E116Q) and resolved on a Figure 4. Continued on next page Tsunoda et al. eLife 2014;3:e03421. Discussion Depletion of glutathione, by GSH1 deletion, rescues the oxidative defect in ERO1-deficient yeast (Cuozzo and Kaiser, 1999), and cytosolic glutathione influences disulfide-bonding of glutenin in the ER of wheat endosperm (Lombardi et al., 2012). These observations, which fall short of directly indicting glutathione in reducing ER disulfides, nonetheless argue for participation of glutathione in a shared economy of reducing equivalents across the ER membrane. Prolonged (48 hr) depletion of glutathione by high concentra tions of BSO (1 mM) synergize with ER hyperoxidation to degrade cell viability (Hansen et al., 2012). Tsunoda et al. eLife 2014;3:e03421. DOI: 10.7554/eLife.03421 11 of 20 Research article Research article Research article Cell biology Figure 5. Clearance of misfolded null Hong Kong mutant alpha 1 anti-trypsin (NHK-A1AT) is unaffected by depletion of ER glutathione. (A) Autoradiograph of metabolically labeled NHK-A1AT immunopurified from HeLa cells co-expressing C-terminally FLAG-tagged NHK-A1AT and ER-localized enzymatically active (ER-ChaC1CtoS) or Figure 5. Continued on next page Figure 5. Clearance of misfolded null Hong Kong mutant alpha 1 anti-trypsin (NHK-A1AT) is unaffected by depletion of ER glutathione. (A) Autoradiograph of metabolically labeled NHK-A1AT immunopurified from HeLa cells co-expressing C-terminally FLAG-tagged NHK-A1AT and ER-localized enzymatically active (ER-ChaC1CtoS) o Figure 5. Continued on next page Figure 5. Clearance of misfolded null Hong Kong mutant alpha 1 anti-trypsin (NHK-A1AT) is unaffected by depletion of ER glutathione. (A) Autoradiograph of metabolically labeled NHK-A1AT immunopurified from HeLa cells co-expressing C-terminally FLAG-tagged NHK-A1AT and ER-localized enzymatically active (ER-ChaC1CtoS) or Figure 5. Continued on next page Tsunoda et al. eLife 2014;3:e03421. DOI: 10.7554/eLife.03421 12 of 20 Research article Research article Cell biology inactive ChaC1 (ER-ChaC1CtoS;E116Q) and resolved on a reducing or non-reducing SDS-PAGE. Cells were lysed at the end of a 30-min labeling pulse (lanes 1 and 5) or after an additional chase period (indicated). The mobility of the labeled NHK-A1AT, and the ER-chaperone BiP that co-purifies with it, are indicated, as is the labeled ChaC1, which is also recovered in this anti-FLAG immunoprecipitate. (B) Autoradiograph of samples recovered from 293T cells in an experimental design as in ‘A’. (C) Autoradiograph of samples recovered from HeLa cells in an experimental design as in ‘A’. Where indicated, cells were exposed to the glutathione synthesis inhibitor buthionine-sulfoxide (BSO, 100 µM, 20 hr) before the pulse-chase labeling. (D–F) Plot of time-dependent change in NHK-A1AT (monomer) signal from the reducing gels A–C above. Shown are representative experiments reproduced twice with similar outcome. Discussion DOI: 10.7554/eLife.03421.009 Further support for the idea of a shared economy of glutathione is provided by the phenotype of yeast over-expressing a plasma membrane glutathione transporter HGT1. Glutathione over-load in these cells compromises protein folding in the ER and triggers an unfolded protein response, the basis of which appears to be unregulated re-cycling of PDI from its oxidized to its reduced state imposed by the excessive glutathione (Kumar et al., 2011). While our state of ignorance in regard to the genes responsible for glutathione transport into the yeast ER preclude assigning a role to luminal glutathione in this consequences of cellular glutathione overload, the observations of Kumar et al. clearly argue that glutathione may participate in ER redox. In mammalian cells too, glutathione stands to impact the maturation of some secreted or mem brane proteins: In a study agnostic of its enzymatic activity, over-expression of wild-type cytosolic ChaC1 in mouse ganglionic eminence cells profoundly inhibited the maturation of the Notch precursor to its furin-cleaved form (Chi et al., 2012). Whereas anti-oxidants that restore impaired glutathione metabolism to normality have been shown to improve the capacity of liver cells to secrete factor VIII, a heavily disulfide bonded serum protein (Malhotra et al., 2008). Thus, conservatively interpreted, our observations lead to the conclusion that ER glutathione is not generally required to maintain protein thiol redox nor folding homeostasis in the ER of cultured mammalian cells. It may now be informative to examine the impact of an ER glutathione purge elicited by ER-localized ChaC1CtoS on the fate of specialized secretory cells and their specialized secretory cargo proteins. Plasmid construction Table 1 lists the plasmids used, their lab names, description, published reference, and a notation of their appearance in the figures. The mouse ChaC1 cDNA (IMAGE clone 4483043) was purchased from Source Bioscience. Bacterial expression vectors encoding C-terminally His 6X tagged wild type and E116Q mutant ChaC1 were constructed by PCR amplification. A mutant ChaC1 in which all four cysteines were converted to ser ines, CtoS (C92S, C169S, C190S, C212S), was synthesized as an artificial gene and shuttled into the mammalian and bacterial expression plasmids described in Table 1. Bacterial expression plasmids for roGFP2 (Hanson et al., 2004) and the glutaredoxin 1 fusion to roGFP2 (Gutscher et al., 2008) were gifts of James Remington (U Oregon) and Tobias Dick (DKFZ, Heidelebrg). ER localized mammalian expression plasmids of their counterparts were generated by deleting the E147b insertion and introducing an S65T mutation into ER_HA_Grx1_roGFP1iE_KDEL_ pcDNA3.1 (Birk et al., 2013) (a gift of Christian Appenzeller-Herzog, University of Basel). ER_roGFP2 was produced by deleting E147b from FLAGM1_roGFP2_iE_pCDNA3.1 (Avezov et al., 2013). Mammalian expression plasmids encoding ER localized FLAG M1-tagged ChaC1 with a KDEL ER retention signal at the C-terminus and mutant derivatives thereof were constructed by shuttling the ChaC1 coding sequence into the relevant plasmid backbone. Mammalian expression plasmids for ER-localized ChaC1 fused to the mCherry fluorescent protein were prepared using similar techniques. The hyperactive C104A: C133A allele of human ERO1L (ERO1A) was expressed from a modified pCDNA3 plasmid in which the coding sequence of the Neor marker had been replaced by human CD2. The hyperactive C104A: C133A allele of human ERO1L (ERO1A) was expressed from a modified pCDNA3 plasmid in which the coding sequence of the Neor marker had been replaced by human CD2. Research article Cell biology Figure 6. Continued Figure 6. Continued Figure 6. Continued reporting on the activity of the UPR (X-axis), was detected at 530 ± 30 nm following excitation at 488 nm, whereas mCherry fluorescent intensity, reporting on the level of ChaC1-mCherry-KDEL in the ER (Y axis), was detected at 610 ± 20 nm following excitation at 561 nm. Where indicated the cells were exposed to the ER stress-inducing agent tunicamycin for the indicated period of time. (B) Three-color FACScans as in ‘A’ of CHOP::GFP cells co-transfected with expression plasmids for a hyperactive mutant of ERO1 (C104A, C133A; ERO1) tagged by the cell-surface marker CD2 (decorated with an AF647-tagged antibody and detected by excitation at 640 nm and emission at 670 nm) with a wildtype or inactive mutants of ChaC1 fused to mCherry, as noted above. The axis of the scans are labeled with the cognate signals and panels numbered for ease of reference in the text. DOI: 10.7554/eLife.03421.010 The following figure supplements are available for figure 6: Figure supplement 1. Activity of the unfolded protein response (UPR) as measured by an XBP1-splicing reporter is also unaffected by depletion of ER glutathione. DOI: 10.7554/eLife.03421.011 DOI: 10.7554/eLife.03421.011 DOI: 10.7554/eLife.03421.011 by size exclusion chromatography on Superdex 200 (GE Healthcare, Chalfont St Giles, United Kingdom) in 20 mM Tris pH7.4, 100 mM NaCl, 10% Glycerol, 1 mM Tris(2-carboxyethyl)phosphine hydro chloride (TCEP). by size exclusion chromatography on Superdex 200 (GE Healthcare, Chalfont St Giles, United Kingdom) in 20 mM Tris pH7.4, 100 mM NaCl, 10% Glycerol, 1 mM Tris(2-carboxyethyl)phosphine hydro chloride (TCEP). Glutathione degradation by purified wild-type ChaC1 and its mutant derivatives was assayed using the 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) recycling assay of Tietze (1969) as modified by Griffith (1980). Mutant ChaC1 (C169S, C190S, C212S) fused at its C-terminus to an mCherry 6X His fluorescent protein was purified from E. coli and labeled at its single remaining cysteine (C92) with thiol reactive Oregon Green 488 iodoacetamide (Life Technologies), according to the manufacturer's instructions. Fluorescent resonance energy transfer (FRET) between the Oregon Green donor and the mCherry acceptor was measured at room temperature on TECAN 500 plate reader as the ratio between their fluorescence emission at 535 nm and 670 nm respectively when they were excited at 485 nm and 590 nm. The binding reaction was initiated by addition of reduced glutathione (G4251, Sigma) or oxi dized glutathione (G4626, Sigma). Research article Human PDI (PDIA1 18–508) and roGFP variants were expressed in the E. coli BL21 (DE3) strain, purified with Ni-NTA affinity chromatography, dialyzed into the reaction buffer, reduced by incubation with 20 mM of DTT, and then buffer exchanged on a PD-10 gel filtration column (GE Healthcare), as described previously (Avezov et al., 2013). Reduced PDI (5 μM) was equilibrated in 100 mM Tris–HCl, 150 mM NaCl, pH 7.5 degassed buffer before being added to samples containing roGFP variants in the reduced state followed by oxidized glutathione (100 μM; Sigma). The ratio of fluorescence emission at 535 nm of samples sequentially excited at 405 nm and 488 nm was measured using a Synergy 4 microplate reader (BioTek Instruments). Kinetic parameters were extracted by fitting the data to a Linweaver–Burk plot. Protein purification and in vitro enzymatic assays pi y y C-terminally 6X His-tagged mouse ChaC1 was expressed in BL21 pLysS E. coli at 30°C with 4 hr of induction with 1 mM IPTG and purified from the lysate by nickel affinity chromatography followed C-terminally 6X His-tagged mouse ChaC1 was expressed in BL21 pLysS E. coli at 30°C with 4 hr induction with 1 mM IPTG and purified from the lysate by nickel affinity chromatography follow Tsunoda et al. eLife 2014;3:e03421. DOI: 10.7554/eLife.03421 13 of 20 Research article Research article Research article Cell biology Figure 6. Activity of the unfolded protein response (UPR) is unaffected by depletion of ER glutathione. (A) Two-dimensional pl fluorescent intensity of individual CHO cells containing a stably-integrated UPR reporter, CHOP::GFP (Novoa et al., 2001), fo plasmids encoding ER-localized, active (ER-ChaC1CtoS-mCherry-KDEL) or inactive ChaC1 (ER-ChaC1CtoS;E116Q-mCherry-KDEL). G Figure 6. Continued on next page Figure 6. Activity of the unfolded protein response (UPR) is unaffected by depletion of ER glutathione. (A) Two-dimensional plots (FACScans) of fluorescent intensity of individual CHO cells containing a stably-integrated UPR reporter, CHOP::GFP (Novoa et al., 2001), following transfection with plasmids encoding ER-localized, active (ER-ChaC1CtoS-mCherry-KDEL) or inactive ChaC1 (ER-ChaC1CtoS;E116Q-mCherry-KDEL). GFP fluorescent intensity, Figure 6. Continued on next page Tsunoda et al. eLife 2014;3:e03421. DOI: 10.7554/eLife.03421 14 of 20 Research article Cell culture, transfection HeLa and HEK293T cells were cultured in DMEM supplemented with 10% FBS and maintained at 37°C with 5% CO2. Cells were electroporated with the indicated plasmids (5 μg of DNA/1 × 106 cells) utiliz ing the Neon transfection system or Lipofectamine LTX (both from Life Technologies) following the manufacturer's protocols. The effects of cytosolic and ER-localized ChaC1 on cellular glutathione pools were analyzed in stable clones of doxycycline-inducible Flp-In™ T-REx™ HEK 293T cells by the 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) recycling assay of Tietze (1969) as modified by Griffith (1980). The effects of ChaC1 on the activity of the unfolded protein response were studied by transient transfection of stable CHOP::GFP reporter Chinese Hamster Ovary cells (C30 clone of CHO K1 cells [Novoa et al., 2001]) or a stable clone of CHO K1 cells (XV8-1) expressing a Venus reporter of XBP1 splicing activity (Iwawaki et al., 2004) followed by multi-channel FACS analysis on an LSRFortessa (BD Bioscience) of the GFP or Venus signals, which reports on the intensity of the unfolded protein response and mCherry expression that reports on the level of expression of ER localized ChaC1. Where indicated, a CD2-marked expression plasmid encoding a hyper-active mutant of human ERO1L (C104A; C133A, Hansen et al., 2012) or CD2-marked empty vector was co-transfected and CD2 expression detected by surface staining with an AlexaFluor 647 conjugated mouse anti-human CD2 antibody, clone LT2 (MCA1194A647, AbD Serotec/Biorad). Tsunoda et al. eLife 2014;3:e03421. DOI: 10.7554/eLife.03421 15 of 20 Research article Research article Research article Cell biology Cell biology Table 1. List of the plasmids used in this study, their unique lab identifier, lab name, description, PMID of the relevant reference (if available), figure in which they first appear and cognate label in figure legend Table 1. Tsunoda et al. eLife 2014;3:e03421. DOI: 10.7554/eLife.03421 Cell culture, transfection List of the plasmids used in this study, their unique lab identifier, lab name, description, PMID of the relevant reference (if available), figure in which they first appear and cognate label in figure legend i g g ID Plasmid name Description Reference First appearance Label in figure 15 pFLAG-CMV1 Mammalian expression bovine trypsinogen signal peptide-FLAGM1 fusion PMID: 8024796 2A ER-FLAG_vector 242 roGFP2_pRSETB Bacterial expression of 6X His-tagged roGFP2 PMID: 14722062 3A roGFP2 836 mChac1_1-224- H6-pET30a Bacterial expression of mouse Chac1 C-terminal His-tagged This paper 1A ChaC1 888 pFLAG_mCherry_ KDEL_CMV1 ER localised FLAGM1- mCherry-KDEL in pFLAG-CMV1 This paper 6A ER-vector 915 mChaC1_1-224_ CtoS_pET30a Bacterial expression mouse Chac1 CtoS (C92S, C169S, C190S, C212S) C-terminal His-tagged This paper 2C ChaC1_CtoS 932 mChaC1_1-224_ E116Q_pET30a Bacterial expression of E116Q mutant mouse Chac1 C-terminal His-tagged This paper 1E ChaC1_E116Q 934 mChaC1_3XFLAG_ pCDNA5_FRT_TO Mammalian expression of C-term FLAG-tagged mouse ChaC1 This paper 3H Cyto-ChaC1 937 mChaC1_CtoS_ E116_pET30a Bacterial expression of E116Q cysteine to serine mutant mouse Chac1 C-terminal His-tagged This paper 2C ChaC1_CtoS_ E116Q 945 mChaC1_ E116Q_3XFLAG_ pCDNA5_FRT_TO Mammalian expression of C-term FLAG-tagged mouse ChaC1 E116Q mutant (Cyto-ChaC1_E116Q) This paper 3H Cyto-ChaC1_E116Q 950 FLAGM1_mChaC1_ CtoS_pCDNA5_ FRT_TO Mammalian expression ER-localised FLAG M1 tagged mouse CHAC1 CtoS (C92S, C169S, C190S, C212S) KDEL This paper 2A ER-FLAG_ChaC1_ CtoS 951 FLAGM1_mChaC1_ CtoS_E116Q_ pCDNA5_FRT_TO Mammalian expression ER-localised FLAG M1 tagged mouse CHAC1 CtoS (C92S, C169S, C190S, C212S) E116Q KDEL This paper 2A ER-FLAG_ChaC1_ CtoS_E116Q 974 FLAGM1_mChaC1_ WT_pCDNA5_ FRT_TO Mammalian expression ER-localised FLAG M1 tagged mouse CHAC1 KDEL This paper 2A ER-FLAG_ChaC1 988 FLAGM1_mChaC1_ CtoS_mCherry_ pCDNA5_FRT_TO Mammalian expression ER-localised FLAG M1 tagged mouse CHAC1 mCherry-KDEL, CtoS (C92S, C169S, C190S, C212S) This paper 3F ER-ChaC1_CtoS_ mCherry 993 mChaC1_CtoS_92C_ mCherry-pET30a Bacterial expression of mouse ChaC1-mCherry fusion, C-terminal His-tagged, (C169S, C190S, C212S) This paper 1D OG-ChaC1-Cherry probe 1028 FLAGM1_mChaC1_ CtoS_E116Q_ mCherry_pCDNA5 Mammalian expression ER-localised FLAG M1 tagged mouse CHAC1 mCherry-KDEL, CtoS, E116Q This paper 3F ER-ChaC1_CtoS_ E116Q_mCherry Table 1. Continued on next page Tsunoda et al. eLife 2014;3:e03421. DOI: 10.7554/eLife.03421 16 of 20 16 of 20 Research article Research article Cell biology Table 1. Continued Table 1. Immunofluorescence Transfected HeLa cells were grown on coverslips. 24 hr after transfection, the cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min at room temperature, permeabilized in PBS con taining 0.1% Triton X-100 for 30 min at room temperature, and blocked in 1% BSA in PBS for 30 min at room temperature. Anti-FLAG antibodies (1:1000 dilution in PBS) in combination with rabbit anti- calreticulin antibodies (a gift of Steven High, University of Manchester, 1:1000 dilution in PBS) fol lowed by goat anti-mouse and goat anti-rabbit secondary antibodies conjugated with DyLight 543 and DyLight 488 (1:1000 dilution in PBS, Jackson ImmunoResearch Laboratories), respectively. Nuclei were counter stained with Hoechst 33,342 (2 μg/ml in PBS) for 30 min at room temperature for counter-staining. Cell culture, transfection Continued ID Plasmid name Description Reference First appearance Label in figure 1037 mChaC1_CtoS_ S92C_E116Q_ mCherry-pET30a Bacterial expression of mouse ChaC1-mCherry fusion, C-terminal His-tagged, (C169S, C190S, C212S), E116Q This paper S1C OG-ChaC1_E116Q- Cherry 1052 ER_roGFP2_ pCDNA3.1 ER localized roGFP2 KDEL This paper 3D ER-roGFP2 1063 ER_Grx1_roGFP2_ KDEL_pCDNA3.1 ER localized Grx1 fused to roGFP2 KDEL PMID:23424194 3D ER-Grx1-roGFP2 1181 hLDLR_3XFLAG_ pCDNA5_FRT Mammalian expression plasmid of human LDL receptor, cytosolic tail tagged with a 3X FLAG tag PMID:12493918 4A FLAG-tagged LDL-R 1204 A1AT_NHK_ 3XFLAG_pCDNA5_ FRT_TO Mammalian expression plasmid of null Hong-Kong mutant a1-antitrypsin C-terminally tagged with 3XFLAG PMID:12736254 5A FLAG-tagged NHK-A1AT 1206 Grx1_roGFP2_pET30a Bacterial expression of Grx1-roGFP2 fusion protein PMID: 18469822 3B Grx1-roGFP2 1239 pCAX-F-XBP1∆DBD- venus XBP1 mini-cDNA with Venus fused the post-IRE1 spliced open reading frame PMID: 14702639 6C XBP1-Venus 1273 hERO1A_C104A_ C131A_pCDNA3-CD2 Mammalian expression plasmid encoding human hyperactive ERO1L (ERO1a) and a co-expressed human CD2 FACS marker PMID: 23027870 6B ERO1* Immunoblot analysis 24 hr after transfection, cells were washed twice with PBS and lysed for 30 min on ice in 1% Triton X-100, 20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1 mM PMSF, 3–7 TIU/L aprotinin, and 20 mM N-ethylmaleimide (NEM). The cellular lysate was centrifuged at 15,000 ×g for 15 min, and the supernatant was used for protein assay using a BCA protein assay reagent. Total proteins (30 μg) were separated on 12% SDS–polyacrylamide gels and electroblotted onto PVDF membrane. Primary anti bodies to the FLAG tag (Sigma Cat #F1804, 1:1000 dilution) or a rabbit antiserum to bacterially expressed H6-tagged mouse ChaC1 (residues 1–224, lab number UC8166, 1:1000 dilution) followed by IR800 conjugated secondary antibody and by scanning on a Licor Odyssey scanner. Confocal microscopy Cells co-transfected with the redox reporter (roGFP) and ChaC1-mCherry were analyzed by laser- scanning confocal microscopy system (510 Meta; Carl Zeiss) with a Plan-Apo- chromat 63× oil immer sion lens (NA 1.4), coupled to a microscope incubator, maintaining standard tissue culture conditions Tsunoda et al. eLife 2014;3:e03421. DOI: 10.7554/eLife.03421 17 of 20 Research article Research article Cell biology (Okolab). Fluorescence ratiometric intensity images (512 × 512 points, 16 bit) of live cells were acquired. A diode 405 nm and Argon 488 nm lasers (2 and 0.5% output respectively) were used for excitation of the ratiometric probes in the multitrack mode with an HFT 488/405 beam splitter, the signal was detected with 518–550 nm filters, the detector gain was arbitrary adjusted to yield an intensity ratio of the two channels approximating one. The recovery half-time was extracted from fitting the intensity ratio changes over time to an expo nential equation ( ) – = ( – ) 1 t I t A e τ , where I is intensity, t is time, τ is recovery half-time. Metabolic labeling and pulse-chase analysis Cells were co-transfected with ER_mChaC1 and the FLAG-tagged LDL receptor or mutant alpha1 antitrypsin NHK plasmids. 24 hr later, pulse-chase labeled, followed by anti-FLAG immunoprecipi tation (Sigma A2220), SDS-PAGE and autoradiography were conducted as previously described (Zito et al., 2010a). Where indicated, the glutathione synthesis inhibitor buthionine-sulfoxide (BSO, 100 µM, 20 hr) was added before the pulse-chase labeling. Purified protein intensities were quanti fied using ImageJ software. Images of gels were scaled to fit the page dimensions. Acknowledgements We are grateful to Tobias Dick and Christian Appenzeller-Herzog for the gift of ER_HA_Grx1_roGF P1iE_KDEL_pcDNA3.1 and Grx1-roGFP2_pQE60. Jim Remington for the gift of the roGFP2 cDNA, Ineke Braakman for the gift of the LDL receptor cDNA, Nobuko Hosokawa for the gift of mutant a1-anti trypsin cDNA and Steve High, University of Manchester for the gift of the calreticulin antiserum. Supported by a grant from the Wellcome Trust (Wellcome 084812/Z/08/Z) and the European Commission (EU FP7 Beta-Bat No: 277713) to DR, a Wellcome Trust Strategic Award for core facilities to the Cambridge Institute for Medical Research (Wellcome 100140) and the Fundação para a Ciência e Tecnologia, Portugal (project grant PTDC/QUI-BIQ/119677/2010) to EPM. DR is a Wellcome Trust Principal Research Fellow. We are grateful to Tobias Dick and Christian Appenzeller-Herzog for the gift of ER_HA_Grx1_roGF P1iE_KDEL_pcDNA3.1 and Grx1-roGFP2_pQE60. Jim Remington for the gift of the roGFP2 cDNA, Ineke Braakman for the gift of the LDL receptor cDNA, Nobuko Hosokawa for the gift of mutant a1-anti trypsin cDNA and Steve High, University of Manchester for the gift of the calreticulin antiserum. Supported by a grant from the Wellcome Trust (Wellcome 084812/Z/08/Z) and the European Commission (EU FP7 Beta-Bat No: 277713) to DR, a Wellcome Trust Strategic Award for core facilities to the Cambridge Institute for Medical Research (Wellcome 100140) and the Fundação para a Ciência e Tecnologia, Portugal (project grant PTDC/QUI-BIQ/119677/2010) to EPM. DR is a Wellcome Trust Principal Research Fellow. Competing interests p g DR: Reviewing editor, eLife. The other authors declare that no competing interests exist. Funding Funder Grant reference number Author Wellcome Trust Wellcome 084812/Z/08/Z David Ron European Commission EU FP7 Beta-Bat No: 277713 David Ron Wellcome Trust Wellcome 100140 David Ron Fundação para a Ciência e a Tecnologia PTDC/QUI-BIQ/119677/2010 Eduardo Pinho Melo The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. 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Author contributions ut o co t but o s ST, Designed, executed and interpreted the cell biological experiments shown in Figures 2, 4, 5 and 6 and the in vivo redox measurements of Figures 3 and 4; EA, Oversaw the design, execution and interpretation of the fluorescent based measurements in Figures 1, 3 and 4; AZ, Designed, executed, interpreted and led the biochemical and biophysical experiments characterizing ChaC1 in vitro, shown in Figure 1 and Figure 1—figure supplement 1; TK, Contributed to the design, execution, and interpretation of the in vivo redox measurements in Figures 3 & 4; LM-S, EPM, Contributed to the design, execution, and interpretation of the in vitro redox measurements of Figure 3; HPH, Oversaw and contributed to the design and interpretation of in vitro measurements of ChaC1's biochemical properties (Figure 1) and to execution of some of the cell biological experi ments (Figures 2 & 6) as well as to the writing and editing of the manuscript; DR, Conceived and oversaw the study as a whole, wrote the manuscript, designed and constructed expression plas mids for the study Tsunoda et al. eLife 2014;3:e03421. 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https://openalex.org/W2975980453	https://biblio.ugent.be/publication/8630307/file/8630311.pdf	English	null	Conceptualizing the Parliamentarization and Politicization of European Policies	Politics and governance	2,019	cc-by	8,770	Issue Issue This article is part of the issue “Out of the Shadows, Into the Limelight: Parliaments and Politicisation”, edited by Christine Neuhold (Maastricht University, The Netherlands) and Guri Rosén (University of Oslo, Norway). © 2019 by the author; licensee Cogitatio (Lisbon, Portugal). This article is licensed under a Creative Commons Attribu- tion 4.0 International License (CC BY). Politics and Governance (ISSN: 2183–2463) 2019, Volume 7, Issue 3, Pages 227–236 DOI: 10.17645/pag.v7i3.2144 Article Conceptualizing the Parliamentarization and Politicization of European Policies Niels Gheyle partment of Political Sciences, Ghent University, 9000 Ghent, Belgium; E-Mail: niels.gheyle@ugent.be Department of Political Sciences, Ghent University, 9000 Ghent, Belgium; E-Mail: niels.gheyle@ugent.be Submitted: 30 March 2019 \| Accepted: 14 June 2019 \| Published: 27 September 2019 Abstract In the past 20 years, two related literature strands have gradually moved centre stage of the attention of EU Studies scholars. The first is preoccupied with the ‘politicization of European integration’, a multi-faceted concept that aims to tie together a multitude of political and societal manifestations underlying an increasing controversiality of the EU. A second concerns the parliamentarization of the EU, referring to the changing (institutional) role and EU-related activities national parliaments engage in. The key point of this contribution is simple, but often overlooked: We can and should be seeing parliamentarization as a necessary, yet insufficient, component of a wider process of politicization. Doing so goes beyond the often ad hoc or pars pro toto theoretical assumptions in both literature strands, sheds new light on the normative consequences attached to these phenomena, and furthers a more complete understanding of how a ‘comprehensive’ politicization of European policies develops. Keywords Keywords communication; Europeanization; parliamentary; parliamentarization; politicization; national parliament communication; Europeanization; parliamentary; parliamentarization; politicization; national parliaments Politics and Governance, 2019, Volume 7, Issue 3, Pages 227–236 2. Parliamentarization of the EU tives and/or the European Parliament (Maurer & Wessels, 2001). The further strengthened formal role of national parliaments through the Lisbon Treaty (Neuhold & Smith, 2016) and the visible part some parliaments have played in recently salient episodes, such as the CETA-saga (Bollen, 2018), have only fuelled the attention. To make sense of the relationship between European in- tegration and domestic political systems, the concept of ‘Europeanization’ is commonly invoked, referring to “an incremental process re-orienting the direction and shape of politics to the degree that [EU] political and economic dynamics become part of the organisational logic of na- tional politics and policy-making” (Ladrech, 1994, p. 69). When the object of Europeanization is the national par- liamentary system, some authors speak of the degree of ‘parliamentarization’ of the EU. Herranz-Surrallés (2018), for example, defines parliamentarization as the “greater involvement of parliaments in scrutinizing and shaping regional and global governance” (p. 31). This is a broad interpretation, and as such, a ‘greater involvement’ can point both to the institutional ability to be involved in the shaping and scrutinizing of governance (e.g., Roederer- Rynning & Schimmelfennig, 2012), or to the actual prac- tice of shaping policy, in terms of scrutiny, coordination, or communication (e.g., Rozenberg & Hefftler, 2016). These ‘institutional’ responses, on the one hand, and the ‘attitudinal’ and ‘behavioural’ responses, on the other hand, are recognized categories of the Europeanization of national parliaments (Goetz & Meyer-Sahling, 2008). This contribution takes issue with the relationship between these two developments—(behavioural) parlia- mentarization and politicization—from a conceptual and analytical point of view. The key point is simple, but ar- guably often overlooked: We can and should be seeing (behavioural) parliamentarization as a necessary, yet in- sufficient, component of the politicization of European integration or specific policies, and not as separate, in- dependent, processes that react to each other. In other words, (behavioural) parliamentarization is not equal to politicization, but a ‘comprehensive’ understanding of politicization cannot exist without it. We ‘can’ make this link, first of all, because the con- ceptual and analytical contributions in both literature strands show very clear overlaps, which often go unrec- ognized when looking from one point of view. 2. Parliamentarization of the EU Crucially, we also ‘should’ re-conceptualize the relationship as fol- lows, as this: (i) goes beyond ad hoc or pars pro toto the- oretical assumptions that are sometimes used in these literature strands; (ii) sheds new light on the norma- tive consequences usually attached to them; and (iii) fur- thers a research agenda focused on a more complete understanding of how ‘comprehensive’ politicization of European policies develops. In sum, this discussion is more than a semantic exercise, as it benefits the schol- arly discussion of which role parliaments play in salient policies, while at the same time adding to our under- standing of the complex politicization concept—both key aims of this special issue. Early contributions came to the conclusion of a de- parliamentarization of the EU: (the appearance of) a decreasing and eroding role for national parliaments, increasingly unable to shape European governance (Raunio, 2009). The argument went that national parlia- ments were either uninterested in engaging with EU is- sues (Auel & Raunio, 2014b), or not able to do so effec- tively. Given that EU competences were simultaneously growing, this would result in them being the ‘losers of integration’ (Maurer & Wessels, 2001). Nevertheless, several scholars documented how na- tional parliaments actively started to ‘fight back’ by adapting institutionally to deal with this challenge of European integration (Raunio & Hix, 2000). They did so, for example, by establishing specific European Affairs Committees (EACs), increasing their informational rights with respect to EU or national executives, or establish- ing inter-parliamentary fora to be in a better position to scrutinize their governments (Raunio, 2009). A con- sensus emerged on the basic point that “national insti- tutions have made substantial efforts in order to cope with the requirements of the Union” (Wessels, Maurer, & Mittag, 2003, p. 414). Raunio (2009) backs this up by concluding in a review article that national parliaments are now generally in a much stronger position to scru- tinize their governments than they were in the 1990s. Several provisions in the Lisbon Treaty with respect to na- tional parliaments (such as the establishment of an ‘Early Warning System’) have further strengthened this devel- opment of ‘institutional parliamentarization’ (Neuhold & Smith, 2016). Beyond these specific conceptual considerations, this contribution has two more overarching goals. 1. Introduction set out the task of conceptualizing this complex phe- nomenon, constructing analytical frameworks to study its forms and appearances, explain its origins and dynam- ics, and evaluate the (normative) consequences (for con- ceptual overviews see De Wilde, Leupold, & Schmidtke, 2016; Gheyle, 2019). It is by now long overdue to state that the EU no longer flies under the political and societal radar. What was once characterized by a stance of indifference (or a ‘per- missive consensus’) by the wider public, has slowly but steadily turned into an expansion of the scope of conflict surrounding the EU (De Wilde, 2011a; Hooghe & Marks, 2009). This change has its origins somewhere around the mid-1990s and has become a focal point of scholarly at- tention since the failure of the Constitutional Treaty in 2004. ‘Politicization’ was argued to have kicked in, refer- ring to a state of political and societal debate character- ized by features such as an increasingly polarized pub- lic, frequent media visibility, electoral positioning, and Euro protests. All of this directed towards the EU itself or particularly salient policies, which together was seen as an “awakening of the sleeping giant” (Van der Eijk & Franklin, 2004). Since then, politicization scholars have During the same time period, a different but related literature strand focused on the role and EU-related activ- ities of national parliaments in the EU multilevel system. The emphasis here is on how national parliaments adapt, institutionally, to take pressures of European integration intoaccount,butalsoonthequestionofwhetherandhow they are actively engaging with EU policy-making. The lat- ter occurs by scrutinizing their governments, or commu- nicating European policies much more frequently—here labelled as ‘behavioural’ parliamentarization (Auel, 2015; Goetz & Meyer-Sahling, 2008). All of this despite or be- cause of the fact that they had initially been labelled the ‘losers of European integration’, to the benefit of execu- 227 Politics and Governance, 2019, Volume 7, Issue 3, Pages 227–236 Politics and Governance, 2019, Volume 7, Issue 3, Pages 227–236 2. Parliamentarization of the EU However, while it might be theoretically true that “na- tional parliaments provide a major space for public de- bate and are thus ideal arenas for the deliberation of im- portant European issues” (Auel, 2007, p. 498), there is not a great deal of empirical knowledge about whether they actually do so. National parliaments can play this role in different ways (Auel & Raunio, 2014b): informing the electorate, asking parliamentary questions, or mak- ing transcriptions of debates publicly available. A major role is however envisioned for plenary debates. The ple- nary is an ideal forum to articulate specific positions on a variety of (European) issues, commonly placing them next to other parties and politicians. The literature does, however, acknowledge that a translation of these de- bates to the mass media is often required to be able to make these debates actually visible to the public (Auel, Eisele, & Kinski, 2018). To evaluate actual parliamentary behaviour, sec- ondly, it makes sense to turn to the traditional parlia- mentary functions, to see how these have evolved in an EU context: electing governments, drafting legislation, scrutinizing the government, and communicating to the wider citizenry (Auel, Rozenberg, & Tacea, 2015). It is widely acknowledged that the first two of these func- tions do not readily apply to national parliaments, be- cause in an EU context the European Parliament has largely taken these over. Hence, the focus is on scrutiny of the government, and more recently on the commu- nicative function, to assess if and how ‘behavioural par- liamentarization’ takes place. Despite its theoretical importance, early results led to fairly depressing conclusions. In a special issue specifi- cally focused on the communicative function of national parliaments, Auel and Raunio (2014b) summarized that, generally speaking, national parliaments “seem not to live up to their task of bringing ‘Europe’ closer to the citi- zens or enabling them to make informed political choices and to exercise democratic control in EU affairs” (p. 10). Especially in plenary debates, the EU remains “a rare guest” (Auel & Raunio, p. 7). While some institutional determinants seem important (such as the formal rights granted to parliament, or the existence of a ‘talking’ in- stead of a ‘working’ parliament, see De Wilde, 2011b), it appears that incentive structures play a more decisive role in accounting for plenary debates and communica- tion in general (Auel & Raunio, 2014a; Auel et al., 2015). 2. Parliamentarization of the EU The first is to trigger more fine-grained conceptual and an- alytical reflection about two concepts that are—due to their popularity and importance—sometimes readily used without thorough conceptual consideration. Doing so runs the risk of applying the same broad labels to vary- ing empirical phenomena, hence undermining our collec- tive knowledge. Secondly, it aims to serve as a modest re- search agenda for studying behavioural parliamentariza- tion as connected to wider politicization dynamics. In do- ing so, it sets the scene for several empirically grounded contributions in this special issue. The article is structured as follows: Section 2 turns to the literature on the role and EU-related activities of national parliaments. Section 3 focuses on the ‘politiciza- tion’ phenomenon, unpacking its multi-faceted nature, manifestations, and settings. Section 4 then makes the ‘so what?’ of this contribution explicit: Why does it mat- ter that we see (behavioural) parliamentarization as a necessary, but insufficient, component of politicization? Section 5 ends with some concluding remarks and short- comings that can be taken up by further research. The more pressing question, then, is whether na- tional parliaments actually make use of these provi- Politics and Governance, 2019, Volume 7, Issue 3, Pages 227–236 228 sions to assert themselves in the EU multi-level system. Indeed, as Auel (2015) argues, institutional opportunities remain latent until they are utilized. Hence, to fully make sense of the parliamentarization of the EU, analyses of: (i) attitudes and role conceptions of members of parlia- ment (MPs); and (ii) their EU-related behaviour and activ- ities over time, are necessary. First, changes in attitudes and role conceptions of MPs can result in an increased willingness to actually be involved in EU policy or scrutiny of the government (Auel, 2015). In this respect, Wessels (2005) analysed contact patterns of German MPs, their involvement in EU policy-making and their perceptions of the control of power over time (1997–2003), and concluded that ‘attitudinal parliamentarization’ certainly takes place, but only at a very slow pace. of the EU polity as a whole. The extent to which they do this, “and are seen by citizens to carry out such actions” is hence arguably fundamental for the legitimacy of the political system (Auel & Raunio, 2014b, p. 3). 2. Parliamentarization of the EU When it comes to parliamentary control and over- sight, the main conclusion is one of differentiation across time and countries (Raunio, 2009; Winzen, 2012). Auel et al. (2015) tested six institutional and motivational hy- potheses on a unique dataset of 27 parliaments, and found that it was mainly the institutional strength in EU affairs and (more limitedly) the type of parliamen- tary system (majority or consensus) that explained the divergence in the time spent in EACs, and in drafting resolutions. However, Senninger (2017) also convinc- ingly shows that partisan competition has a defining influence on parliamentary EU oversight, as EU issue- based incentives across parties have been shown to explain the content and timing of oversight activities. National parliaments have over the years also estab- lished horizontal links with other parliaments, resulting in inter-parliamentary fora in order to be better able to follow European policies and share best practices (Raunio, 2009). In this respect, it is often the ‘public salience’ crite- rion that reappears in analyses of the communicative function, disguised in different topics (Auel, 2015). The topic of the euro crisis, for example, was shown to be a better predictor of parliamentary debates than some in- stitutional factors, simply because the public salience sur- rounding it was so high (Auel et al., 2015; Auel & Höing, 2014; Wendler, 2013). Miklin (2014) equally shows that in Germany and Austria, national parliaments discussed the EU Services Directive heavily, but only after the pub- lic salience of the issue had grown. In EU foreign policy as well, recent studies have found national parliaments in different Member States heavily debated EU free trade negotiations (with Canada and the US), but only after the public salience of these issues had grown significantly (Bollen, 2018; Gheyle, 2019). Furthermore, building on normative and democratic concerns, it is particularly the communicative function that has moved centre stage over the past five years (Auel & Raunio, 2014b). Democracy depends on a lively and viable public debate, where policy alternatives are presented to citizens to make informed choices. In this re- spect, national parliaments are argued to be in a unique position to translate EU policies, ‘bring the EU home’ and normalize its existence (Auel, 2007; Kröger & Bellamy, 2016). Politics and Governance, 2019, Volume 7, Issue 3, Pages 227–236 3. Politicization of EU Integration and Policies In the past 20 years, the so-called ‘permissive consensus’ towards European integration has abruptly come to an end (Hooghe & Marks, 2009). Different manifestations— listed in the introduction—are testimony of the fact that both support for and contestation against the EU have gradually become integral components of contemporary societal and political life (Zürn, Binder, & Ecker-Ehrhardt, 2012). This arguably has profound consequences for European integration and its democratic functioning. On the one hand, scholars argue that the fact that publics are now “looking over the shoulders” of EU elites, and that the EU has become an element of mass politics, constitutes a ‘constraining dissensus’ for further integra- tion (Hooghe & Marks, 2009). On the other hand, (pub- lic sphere) scholars see a large democratizing potential in frequently debating (and conflicting over) the EU, and in doing so providing citizens with alternatives, contes- tation, and options to fight for—the core elements of a vibrant democracy (Follesdal & Hix, 2006; Statham & Trenz, 2015). Whatever the stance, the politicization of European integration, in any case, seems to be under- stood as having a profound impact on further European (dis)integration, which makes it a key research topic for years to come. p g p In the first, widely-held approach, politicization is seen as an essentially discursive phenomenon that builds on political communication. As Hurrelmann, Gora and Wagner (2015) put it, in this approach, it is “not suffi- cient that actors become aware, or able to form opin- ions, but it must become salient in political communi- cation that seeks to influence decision-making” (p. 45). Applying the sub-processes here means seeing politiciza- tion as a visible, discursive, expansion of the scope of conflict, whereby different types of groups or political ac- tors publicly conflict over a certain topic, fuelled by dif- ferent opinions, ways of framing, or legitimations of the EU or its policies (Gheyle, 2019; Statham & Trenz, 2015). As such, the focus is on the “communicative processes that lead to an increasing intensity and controversy of debates” and on the settings or arenas where this can visibly play out (Schmidtke, 2014, p. 3). To speak of politicization in this view entails two things: (i) there are actors present who deliberately raise issues; and (ii) that these issues are raised in public, in front of an audience. 3. Politicization of EU Integration and Policies Politicization, therefore, does not happen automatically but builds on societal or political actors seeking publicity and resonance with a wider au- dience who are able to witness these actions (De Wilde, 2011a; Statham & Trenz, 2015). Where these discursive interventions can take place—and hence where we could eventually see evidence of politicization—are in those settings or arenas where there actually is a larger audi- ence able to follow an unfolding debate. Parliaments, the mass media, ‘on the streets’, town hall assemblies, or scientific conferences all fit that description (De Wilde, 2011a). Several authors have therefore come up with a classification of three types of settings in which to find and study politicization: institutional, intermediary, and citizen settings (Baglioni & Hurrelmann, 2016; De Wilde et al., 2016). From an academic point of view, the first tasks are therefore to conceptualize the politicization phe- nomenon and its relation to ‘politics’, as well as to analyt- ically capture its varying manifestations, in order to mea- sure its occurrence and extent. From a conceptual point of view, politicization implies a special case of ‘turning something political’, combining the visible and contested aspects of ‘the political’ (Palonen et al., 2019). The under- lying idea is hence that the existence of the EU, or its poli- cies and decisions, are debated and no longer escape the wider public’s attention (Rauh, 2016). This means that the commonly executive, elite-driven process of European in- tegration is no longer taken for granted, and frequently falls prey to heated and mass public debate (Rauh, 2016). Analytically, the starting point is often the contribution by De Wilde (2011a), who defined politicization as “an in- crease in polarization of opinions, interests or values and the extent to which they are publicly advanced towards the process of policy formulation within the EU” (p. 566). Given the broad scope of this operational definition, there is an on-going debate on how to measure and make f liti i ti ifi ll i t f th t f The institutional setting mainly comprises parliamen- tary arenas, where professional politicians engage in political debate about different (European) issues. The length of parliamentary debates or the polarization be- tween different parties about core EU policies is there- fore sometimes used as indicators of a politicized object (Green-Pedersen, 2012; Wendler, 2013; Wonka, 2016). 2. Parliamentarization of the EU As such, communication by national parliaments (and its members) can add to the democratic legitimacy All in all, the current literature documents that a pro- cess of institutional parliamentarization of the EU has definitely taken place while raising several questions re- garding the existence of a pattern of attitudinal and be- havioural parliamentarization. The former appears to be 229 Politics and Governance, 2019, Volume 7, Issue 3, Pages 227–236 ‘manifestations’ and ‘settings’ it comprises; the ways in which the phenomenon becomes visible for researchers to study its existence and extent (see Gheyle, 2019, for an exhaustive discussion). Various contributions have ini- tially led to a convergence around the idea that three sub-processes constitute politicization: salience, actor (or audience) expansion, and polarization (De Wilde et al., 2016). Nevertheless, the different interpretations that can be generated by these terms, have led to two overarching approaches to analysing politicization: as a purely (or predominantly) discursive phenomenon, or as a much more encompassing phenomenon. moving slowly, while the latter varies widely between countries, and according to specific (salient) episodes in time—especially when it comes to parliamentary com- munication and debates. In other words: The ability to deal with EU issues is, generally speaking, present, but incentive structures seem to inhibit their use. Following Raunio (2009), national parliaments may hence not be ‘late comers’ to the EU debate, but simply rationally not engaging having weighed up the pros and cons. Politics and Governance, 2019, Volume 7, Issue 3, Pages 227–236 3. Politicization of EU Integration and Policies The second intermediary setting serves as the link be- tween political decision-making and the broader citi- zenry and civil society. While there is some research on Given the broad scope of this operational definition, there is an on-going debate on how to measure and make sense of politicization, specifically in terms of the types of 230 Politics and Governance, 2019, Volume 7, Issue 3, Pages 227–236 party manifestos (Guinaudeau & Persico, 2013) and pub- lic protest (Hutter, Grande, & Kriesi, 2016), the main fo- cus is on the public sphere carried by the (mass) me- dia (De Wilde et al., 2016; Hoeglinger, 2016). With its wide audience reach, and its central place in contem- porary ‘audience democracies’ (Manin, 1997) the mass media arguably serves as the primus inter pares loca- tion to study politicization (Gheyle, 2019). Finally, the citizen setting has been least studied (but see Baglioni & Hurrelmann, 2016). It comprises arenas made up of laypeople engaging with politics, coming together, for ex- ample, in debating groups or on social media. The key insight here is that the three manifestations of politicization in the institutional setting largely overlap with the attitudinal and behavioural parliamentarization of the EU as introduced in the previous section. The par- liamentary communication function logically overlaps with political communication in the institutional setting. Increased parliamentary scrutiny of EU affairs is less visi- ble to the wider public but is still a political activity that implies a heightened importance of the EU or a certain policy. Even the increasing awareness of MPs about their role in multi-level policy-making, or about the impor- tance of a certain EU policy, can be seen as evidence of politicization (in the broader approach). In sum, different types of parliamentarization of the EU can be conceptu- alized as (as I will argue: necessary, yet insufficient) com- ponents of a broader politicization dynamic. The main focus in politicization research in all of these settings is on domestic arenas: national parlia- ments; domestic public (media) spheres; or domestic protest. This is because structural barriers, such as lan- guage differences or the nationally structured media sys- tems, still inhibit pan-European debates or conflict ex- pansion. For this reason, a multitude of institutional and agency-related variables are said to result in a “differen- tiated politicization of European governance, in which patterns vary substantially across settings and time” (De Wilde et al., 2016, p. 9). 3. Politicization of EU Integration and Policies This pattern has been con- firmed in several empirical studies (see Zürn, 2018, for an overview), and has fuelled the theoretical debate about which structural and agency related variables may ex- plain the divergence (Gheyle, 2019; Zürn, 2016). This means that the choice for the narrow or broad approach towards politicization is important to assess the relationship between parliamentarization and politi- cization. In the broad approach, attitudinal parliamen- tarization and the scrutiny aspect of behavioural par- liamentarization might be labelled particular manifesta- tions of the politicization of the EU. While this obviously has its merits to analyse longer-term dynamics in the EU multi-level polity, it can also lead to contradictory ten- dencies. Christiansen, Högenauer and Neuhold (2014), for example, theorized that institutional parliamentariza- tion, and the related scrutiny practices, may also result in increased bureaucratization and depoliticization, as tighter scrutiny practices may favour expert bureaucrats, to the detriment of more visible and polarized political discussions in plenary. Besides the discursive-centred approach, a second approach claims that politicization analytically implies much more than just public debate or political commu- nication (Hooghe & Marks, 2009; Rauh, 2016; Zürn et al., 2012). Driven by a different account of what ‘the politi- cal’ implies, it is argued that several other manifestations can also point to the fact that something is politicized (Zürn, 2016). Changing attitudes and beliefs about the EU (or core policies), various group activities, such as lob- bying, coalition formation, voting trends, or the (in cam- era) parliamentary scrutiny of European issues are in this sense also testimony of its politicization. While these ac- counts acknowledge that communication is important, and that politicization can play out in different settings, they argue we need to take these other types of mani- festation into account. Especially when we take a longer- term view of the politicization of the EU. In Table 1, three recurring manifestations are summarized, with examples of what could be seen as evidence of politicization. For this and other reasons (see Gheyle, 2019, Chapter 2), I argue it is most helpful to think of politi- cization in the narrow view set out above, as a primar- ily discursive phenomenon that builds on the other man- ifestations functioning as latent potentialities. This nar- row approach is also more suited to deal with short-term politicization phenomena, as is the case when focusing on European policies. 4.1. From ‘Partial’ to ‘Comprehensive’ Phenomena A first reason to study these phenomena together is that we no longer have to resort to ad hoc or pars pro toto theoretical assumptions related to the original concepts. The literature on parliamentary communication, for ex- ample, often has to include publicly salient topics, or the introduction of broad variables such as ‘Euroskepticism’ in order to explain the existence and extent of com- munication (e.g., Auel et al., 2015). Arguably, it cannot be the aim to identify—a priori—all possible salient is- sues in order to explain this parliamentary function, nor is ‘Euroskepticism’ a readily interpretable indication of why some issues pop up in parliamentary debates while others do not. Explaining parliamentary communication makes more sense from the point of view of the issue, rather than the institution or characteristics of the coun- try (even though these can play mediating roles). This could be attained by linking with the wider concept of ‘politicization’ of European policy. 3. Politicization of EU Integration and Policies Doing so also clarifies the relation- ship between (the communicative aspect of) behavioural parliamentarization and politicization. It is visible, polar- ized, parliamentary communication (most likely seen in plenary debates) that is considered the (necessary, yet insufficient) component of the broader (discursive) politi- cization of an EU issue. In the next section, I argue that Table 1. Examples of manifestations of politicization across different settings. Institutional setting Intermediary setting Citizen setting Beliefs/attitudes MP views on EU integration Expert views Public opinion Political activities Scrutiny or follow-up Associational activity, protest, Public protest, electoral of EU issues lobbying, coalition formation turnout, voting behaviour Political communication Parliamentary debates Mass media, party manifestos, Layperson’s discourse in plenary press releases Notes: Own elaboration, inspired by Zürn (2016) and Hurrelmann et al. (2015). ‘Narrow’ approach to politicization comprises the third row, while the broad approach comprises all three. Table 1. Examples of manifestations of politicization across different settings. Politics and Governance, 2019, Volume 7, Issue 3, Pages 227–236 231 politicization with political parties picking up an issue, ir- respective of whether the issue is visible to a wider pub- lic or not (e.g., Green-Pedersen, 2012; Hooghe & Marks, 2009). Herranz-Surrallés (2018) identifies an increasing polarization between political parties (in the European Parliament) and labels this as politicization. Miklin (2014) argues that we need to assess how ‘EU politicization’ can increase in order to affect the way national parlia- ments deal with these issues, implying that the former is something independent from parliamentary involve- ment. Zürn et al. (2012) label the increasing involvement of NGOs at the international level as politicization. Lastly, many contributions focus on the mass media setting as the most important location to study politicization but in doing so run the risk of narrowing the phenomenon to media visibility (see Zürn, 2016). we not only can but also should be seeing these phenom- ena as intertwined. 4. The Importance of an Integrated View In the previous part, I argued that it is analytically pos- sible to see (the communicative aspect of) behavioural parliamentarization as a component of a wider politiciza- tion phenomenon. In this penultimate section, I add that we also should be seeing the former as a necessary, yet insufficient, component of politicization, for three rea- sons: (i) it goes beyond often ad hoc or pars pro toto the- oretical assumptions in both literatures; (ii) it sheds new light on the normative consequences usually attached to both; and (iii) it furthers a more complete understanding of how ‘comprehensive’ politicization of European poli- cies unfolds. Recall that politicization is a complex, multi-faceted phenomenon, that aims to tie together different (types of) manifestations in different settings. There is there- fore arguably a risk in labelling something ‘politicization’ if it only manifests itself in one way or one particular setting. Just as a complex concept such as ‘democracy’ is not reducible to one constitutive element (such as free elections), so politicization is not present as soon as there is ‘protest’ or ‘media visibility’. It, therefore, makes sense to claim that political or societal debate in only one setting should be labelled a ‘partial’ form of politi- cization and if there is a spillover between different set- tings (parliaments and mass media) as ‘comprehensive’ politicization. Doing so would also (partly) address the thorny question of the ‘extent’ of politicization (What is high or low? Is there a threshold?), by arguing that we should at least be seeing parliamentary and medi- ated communication. Politics and Governance, 2019, Volume 7, Issue 3, Pages 227–236 4.2. Normative Consequences Besides theoretical and analytical reasons, there are also normative considerations that justify a closer connec- tion between behavioural parliamentarization and politi- cization. Recall that both politicization and parliamen- tarization imply a normative, democratic component: Widespread public debate, and the particular translation hereof in parliament, is argued to be a constitutive force of democratization. Here too, however, it seems incor- rect to assume that these far-reaching beneficial out- comes result from their partial manifestations only. Furthermore, national parliaments are not simply passive receivers of societal pressures that they must then translate into political action. They are an integral cog in a wider politicization dynamic, often able to decide whether to add visibility to certain EU issues, or whether to silence them. In his study of the EU Services Directive, Miklin (2014) for example, found that left parties aimed to “increase public pressure further and hence prevent the directive from being adopted” (p. 86). De Wilde (2014) states that “national parliaments can contribute to the politicization of Europe by exercising their com- municative role, but the main communicative arena that reaches citizens is constituted by mass media, rather than parliaments” (p. 46). It, therefore, seems almost in- evitable that the function of parliamentary communica- tion is studied in parallel with the study of mass media (Auel et al., 2018). For national parliamentary debates this is acknowl- edged, and intuitively clear: If parliamentary debates do not spill over beyond the assembly walls, it is quite dif- ficult to attach far-reaching normative consequences to the fact that they have taken place. Who saw them, what informing, aggregating, or polarizing function did they have? As Auel et al. (2018) succinctly put it: “Despite a remarkable increase in parliamentary involvement in EU affairs, the added value in terms of democratic legiti- macy will remain limited if citizens are not aware of their activities”. While social media may have a role to play In the politicization literature, secondly, it is often the case that evidence of one manifestation is taken to mean full-blown politicization. Some authors equate 232 Politics and Governance, 2019, Volume 7, Issue 3, Pages 227–236 velops. This firstly means looking for the conditions un- der which a spillover from wider societal debates to par- liamentary settings (or the other way around) occurs. 4.2. Normative Consequences If the existing power structures (such as governments or the European Commission) are disconnected from (or relatedly: irre- sponsive to) wider public debates, we should not hold public debates in such high (normative) regard simply be- cause they happen. This could just as well constitute a ‘dialogue of the deaf’ (Crespy, 2014), which in the end fu- els the (perceived) lack of legitimacy of the EU (Crespy, 2016). In sum, to genuinely speak of a politicized issue, outside debates need an inside translation, or they could evaporate in the already crowded public sphere. Political parties or specific MPs are therefore key play- ers. However, Kiiver (2007) argues that there is a range of trade-offs involved for MPs that impact their decisions to highlight EU issues, to take up contrasting positions, and to attempt to generate an impact on EU policy out- comes. Hence, further research should investigate not only if plenary debates, or political communication by MPs, about European issues takes place, but also what the underlying motivation is to do so, and when they ab- stain from it (see Senninger, 2017). This can be related to party variables, such as their ideology, or their place in government-opposition, but also to role division per- ceptions with the European Parliament, or issue-specific variables, such as distinctions between distributive, regu- latory or foreign policy issues, or the way parties are able to link (frame) EU policies to their domestic agenda. This point is backed up most explicitly by Kröger and Bellamy (2016), who argue that what is lacking, is not (primarily) democracy at the EU level, but a demo- cratic (re)connection between EU decision-making and the nationally-bound citizenry. In the current set-up, na- tional parliaments are crucial in order to domesticate and normalize the EU. Domestication, they argue, is already well underway, since in their view this over- laps with institutional parliamentarization (cf. Kröger & Bellamy, 2016). Normalization, however, means debat- ing “alternative EU policies by non-Euroskeptic parties ac- cording to their characteristic ideological commitments” (Kröger & Bellamy, 2016, p 14). Hence, the (beneficial) normative consequences scholars attach to politicization may only materialize depending on far-reaching domes- tication and normalization of the EU. This arguably has a better chance when the objects are specific EU policies, where citizens can readily observe the wider societal de- bates and how political parties deal with those in their domestic parliaments. 4.2. Normative Consequences Several of such ‘spillover’ variables have already been identified, but have not been placed into an overarch- ing framework due to the separate evolvement of these strands of literature. When it comes to the conditions generally considered necessary for political parties to pick up and openly communicate about EU issues, four variables are raised: (i) the issue must be salient; (ii) the party position must be in line with their voters; (iii) the party needs to be internally cohesive on the topic; and (iv) the competitors should take up alternative positions (Kröger & Bellamy, 2016; Miklin, 2014). here (even though there is doubt regarding the extent to which it can provide a balanced picture), the mass me- dia remains the primary transmission channel for carry- ing these debates to the wider public. The same reasoning applies for those contributions that study politicization as manifested in the primus in- ter pares setting, the mass media. While undoubtedly im- portant, the point is that the existence of polarized and mediated debates about EU issues may not be sufficient to attribute large normative consequences if, for exam- ple, these do not imply any domestic conflict (De Wilde & Lord, 2016). What deliberative, normative, consequences should we attach to those debates about and between foreign or European actors, making EU issues seem as re- mote, or as if played out between others, and not us? Interestingly, the spillover from parliamentary to wider societal settings (e.g., mass media) seems to be based on similar considerations. Auel et al. (2018) stud- ied different supply and (media) demand hypotheses about when parliamentary activity is covered in newspa- pers, and have found that there is a large role for parlia- mentary news supply. In other words: The media is cer- tainly interested in parliamentary debates about EU is- sues, but there have to be such events in the first place. p y Arguably, what matters from a normative point of view is a connection between the (deliberative) opinion-making process and the decision-making level (Papadopoulos, 2013, p. 77). MPs, and the parliament itself, play a major role in (selectively) increasing the resonance of this public debate, increasing (poten- tial) responsiveness by executive actors. 4.2. Normative Consequences Besides highlighting issues themselves, parties may, of course, react to publicly salient topics, which means we also need to take into account how societal debate expands from initial discursive interventions, to its en- trance in (semi-)public arenas and ultimately in parlia- ments (Palonen et al., 2019). Recent studies on the derail- ing of the Anti-Counterfeiting Trade Agreement (Crespy & Parks, 2017; Dür & Mateo, 2014) and the Transatlantic Trade and Investment Partnership (TTIP; Gheyle, 2019) have shown how interest groups and social movements succeeded in making complex foreign policy issues pub- licly salient, after which both national and European MPs picked it up. They point to normative framing strate- gies, pan-European coalition formation between groups, close ties with parliamentary actors, and outside lobby- ing efforts in terms of demonstrations and social me- dia campaigns. 4.3. Explanatory Potential Finally, we should be linking both phenomena together because it helps us advance a more complete under- standing of how this ‘comprehensive’ politicization de- While the previous points all suggest agency-related variables, we also need to take into account the struc- Politics and Governance, 2019, Volume 7, Issue 3, Pages 227–236 233 vance a more complete understanding and explanation of these phenomena. tural variables which inhibit or facilitate conflict expan- sion. In terms of parliamentary communication, for ex- ample, the either ex-ante or ex-post dealing with EU pol- icy issues matters significantly (De Wilde, 2011b). When the issue is initially left to executives and the adminis- tration, heated debates often follow at the end of the policy cycle. When close (ex-ante) scrutiny has been paid throughout, less conflict appears to emerge afterwards. Gheyle (2019), furthermore, shows that Irish advocacy groups faced enormous difficulties in getting media at- tention for their TTIP cause, in part because of a hos- tile media environment (in contrast with the very open and pluralist German media system). Hence, the intro- duction of political and/or discursive opportunity struc- tures should help explain the expansion or contraction of conflicts within and across settings. There are of course several limitations to this con- tribution. First of all, I have only focused on the role and activities of national parliaments, while their func- tioning and behaviour is also dependent on the place they have been granted (or carve out for themselves) in a multi-level parliamentary system. The relationship between these different levels is complex, even without questioning what multi-level politicization would look like (see Crum & Fossum, 2009). This contribution hope- fully serves as a conceptual and analytical stepping stone for further research dealing with these complexities. Secondly, as Table 1 shows, the citizen setting is also identified as a location for politicization to unfold, yet this is under-researched. An important consideration is if we should take this setting into account as well to make sure that any ‘comprehensive politicization’ we might witness, does not take place before a ‘blind audi- ence’. While it makes sense to assume that issues that are contested simultaneously in parliament and in the mass media are also on the public’s attention radar, we cannot rule out the possibility that citizens’ changing me- dia intake (e.g., through social media) fractures these links, and hence impacts the normative consequences at- tached to comprehensive politicization. 4.3. Explanatory Potential A modest research agenda should therefore primar- ily aim to disentangle (through theory-testing process- tracing or qualitative comparative analysis) which combi- nation of variables (or which mechanisms) explain ‘com- prehensive’ politicization. Such a case-based research de- sign is all the more important given that these ‘com- prehensive’ phenomena might take place much less fre- quently than we think. Kiiver (2007) even argues that we can at best expect ‘targeted’ politicization: some spe- cific EU policies, which attract deliberate attention by national parliaments, while the bulk of policies pass un- noticed. This is in line with the idea that politicization is essentially about how public and political attention— a scarce resource—flows (Palonen et al., 2019). A cer- tain politicized issue might detract attention, resources, and debate from another, pre-empting a comparative analysis of different issues. A qualitative research agenda unpacking the dynamics of successful ‘comprehensive’ politicized issues is, therefore, better suited than large N projects. Thirdly, the current contribution identifies overlap predominantly in terms of the location and manifesta- tions of politicization and parliamentarization but says little about the content of the debates that are being held. Any beneficial normative consequences attached to these phenomena obviously also depend on the way the EU is debated, and if position-taking and framing fol- low traditional political cleavages or if new ones are es- tablished (see e.g., Wendler, 2013). While it is here im- plied that contestation or ‘polarization’ is present as a necessary sub-process of politicization (and can arguably be seen as an empirical likelihood), empirical research should verify this in parallel, instead of only looking at where discursive interventions take place. Acknowledgments The author wishes to thank the three anonymous review- ers and the editorial team for their valuable feedback on the first version of this article. 5. Conclusions In this contribution, I have looked at the phenomena of (behavioural) parliamentarization and politicization from a conceptual and analytical angle in order to set the scene for the empirical contributions in this special issue. The key point I put forward is simple, but arguably often overlooked: We can and should be seeing (behavioural) parliamentarization as a necessary, yet insufficient, com- ponent of a wider politicization phenomenon, and not as separate processes that react to each other. By con- cisely reviewing both literature strands and deconstruct- ing these concepts, I have first of all shown that this is perfectly possible, analytically speaking. Conflict of Interests The author declares no conflict of interests. 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His research interests revolve around the (de)politicization of EU trade policy, European integration, and global governance. He finished his doctoral dissertation in 2019, focusing on the origins, dynamics, and consequences of the politicization of TTIP. 236 Politics and Governance, 2019, Volume 7, Issue 3, Pages 227–236
https://openalex.org/W4367460131	https://link.springer.com/content/pdf/10.1007/978-3-031-25820-6_11.pdf	English	null	Sparse Approximate Inverse Preconditioners	Nečas center series	2,023	cc-by	8,551	© The Author(s) 2023 J. Scott, M. T˚uma, Algorithms for Sparse Linear Systems, Neˇcas Center Series, https://doi.org/10.1007/978-3-031-25820-6_11 Chapter 11 Sparse Approximate Inverse Preconditioners While it is recognized that preconditioning the system often improves the convergence of a particular method, this is not always so. In particular, a successful preconditioner for one class of problems may prove ineffective on another class. – Gould & Scott (1998). There is, of course, no such concept as a best preconditioner ... However, every practitioner knows when they have a good preconditioner which enables feasible computation and solution of problems. In this sense, preconditioning will always be an art rather than a science. – Wathen (2015). Consider a preconditioner M based on an incomplete LU (or Cholesky) factorization of a matrix A. M−1, which represents an approximation of A−1, is applied by performing forward and back substitution steps; this can present a computational bottleneck. An alternative strategy is to directly approximate A−1 by explicitly com- puting M−1. Preconditioners of this kind are called sparse approximate inverse preconditioners. They constitute an important class of algebraic preconditioners that are complementary to the approaches discussed in the previous chapter. They can be attractive because when used with an iterative solver, they can require fewer iterations than standard incomplete factorization preconditioners that contain a similar number of entries while offering signiﬁcantly greater potential for parallel computations. From Theorem 7.3, the sparsity pattern of the inverse of an irreducible matrix A is dense, even when A is sparse. Therefore, if A is large, the exact computation of its inverse is not an option, and aggressive dropping is needed to obtain a sufﬁciently sparse approximation to A−1 that can be used as a preconditioner. Fortunately, for a wide class of problems of practical interest, many of the entries of A−1 are small in absolute value, so that approximating the inverse with a sparse M−1 may be feasible, although capturing the large (important) values of A−1 is a nontrivial task. Importantly, the computed M−1 can have nonzeros at positions that cannot be obtained by either a complete or an incomplete factorization, and this can be 205 11 Sparse Approximate Inverse Preconditioners 206 beneﬁcial. Furthermore, although A−1 is fully dense, the following result shows this is not the case for the factors of factorized inverses. beneﬁcial. Furthermore, although A−1 is fully dense, the following result show this is not the case for the factors of factorized inverses. Chapter 11 Sparse Approximate Inverse Preconditioners Theorem 11.1 (Bridson & Tang 1999; Benzi & T˚uma 2000) Assume the matrix A is SPD, and let L be its Cholesky factor. Then S{L−1} is the union of all entries (i, j) such that i is an ancestor of j in the elimination tree T (A). A consequence of this result is that L−1 need not be fully dense. Considering this implication algorithmically, if A is SPD, it may be advantageous to preorder A to limit the number of ancestors that the vertices in T (A) have. For example, nested dissection may be applied to S{A} (Section 8.4). If S{A} is nonsymmetric, then it may be possible to reduce ﬁll-in in the factors of A−1 by applying nested dissection to S{A + AT }. 11.1 Basic Approaches An obvious way to obtain an approximate inverse of A in factorized form is to compute an incomplete LU factorization of A and then perform an approximate inversion of the incomplete factors. For example, if incomplete factors L and U are available, approximate inverse factors can be found by solving the 2n triangular linear systems Lxi = ei, Uyi = ei, 1 ≤i ≤n, where ei is the i-th column of the identity matrix. These systems can all be solved independently, and hence, there is the potential for signiﬁcant parallelism. To reduce costs and to preserve sparsity in the approximate inverse factors, they may not need to be solved accurately. A disadvantage is that the computation of the preconditioner involves two levels of incompleteness, and because information from the incomplete factorization of A is passed into the second step, the loss of information can be excessive. Another straightforward approach is based on bordering. Let Aj denote the principal leading submatrix of A of order j (Aj = A1:j,1:j), and assume that its inverse factorization A−1 j = WjD−1 j ZT j is known. Here Wj and Zj are unit upper triangular matrices, and Dj is a diagonal matrix. Consider the following scheme: is known. Here Wj and Zj are unit upper triangular matrices, and Dj is a diagonal matrix. Consider the following scheme: ⎛ ⎝ ZT j 0 zT j+1 1 ⎞ ⎠ ⎛ ⎝ Aj A1:j,j+1 Aj+1,1:j aj+1,j+1 ⎞ ⎠ ⎛ ⎝ Wj wj+1 0 1 ⎞ ⎠= ⎛ ⎝ Dj 0 0 dj+1,j+1 ⎞ ⎠, 2 Approximate Inverses Based on Frobenius Norm Minimization 207 11.2 here for 1 ≤j < n wj+1 = −WjD−1 j ZT j A1:j,j+1, zj+1 = −ZjD−1 j W T j AT j+1,1:j, dj+1,j+1 = aj+1,j+1 + zT j+1Ajwj+1 + Aj+1,1:jwj+1 + zT j+1A1:j,j+1. where for 1 ≤j < n where for 1 ≤j < n wj+1 = −WjD−1 j ZT j A1:j,j+1, zj+1 = −ZjD−1 j W T j AT j+1,1:j, Starting from j = 1, this suggests a procedure for computing the inverse factors of A. Sparsity can be preserved by dropping some entries from the vectors wj+1 and zj+1 once they have been computed. Sparsity and the quality of the preconditioner can be inﬂuenced by preordering A. Starting from j = 1, this suggests a procedure for computing the inverse factors of A. 11.1 Basic Approaches Sparsity can be preserved by dropping some entries from the vectors wj+1 and zj+1 once they have been computed. Sparsity and the quality of the preconditioner can be inﬂuenced by preordering A. If A is symmetric, W = Z and the required work is halved. Furthermore, if A is SPD, then it can be shown that, in exact arithmetic, djj > 0 for all j and the process does not break down. In the general case, diagonal modiﬁcations may be required, which can limit the effectiveness of the resulting preconditioner. Observe that the computations of Z and W are tightly coupled, restricting the potential to exploit parallelism. At each step j, besides a matrix–vector product with Aj, four sparse matrix–vector products involving Wj, Zj and their transposes are needed; these account for most of the work. The implementation is simpliﬁed if access to the triangular factors is available by columns as well as by rows. 11.2 Approximate Inverses Based on Frobenius Norm Minimization It is clear from the above discussion that alternative techniques for constructing sparse approximate inverse preconditioners are needed. We start by looking at schemes based on Frobenius norm minimization. Historically, these were the ﬁrst to be proposed and offer the greatest potential for parallelism because both the construction of the preconditioner and its subsequent application can be performed in parallel. 11.2.1 SPAI Preconditioner To describe the sparse approximate inverse (SPAI) preconditioner, it is convenient to use the notation K = M−1. The basic idea is to compute K ≈A−1 with its columns denoted by kj as the solution of the problem of minimizing ∥I −AM−1∥2 F = ∥I −AK∥2 F = n j=1 ∥ej −Akj∥2 2, (11.1) (11.1) 208 11 Sparse Approximate Inverse Preconditioners over all K with pattern S. This produces a right approximate inverse. A left approximate inverse can be computed by solving a minimization problem for ∥I − KA∥F = ∥I −AT KT ∥F . This amounts to computing a right approximate inverse for AT and taking the transpose of the resulting matrix. For nonsymmetric matrices, the distinction between left and right approximate inverses can be important. Indeed, there are situations where it is difﬁcult to compute a good right approximate inverse but easy to ﬁnd a good left approximate inverse (or vice versa). In the following discussion, we assume that a right approximate inverse is being computed. The Frobenius norm is generally used because the minimization problem then reduces to least squares problems for the columns of K that can be computed independently and, if required, in parallel. Further, these least squares problems are all of small dimension when S is chosen to ensure K is sparse. Let J = {i \| kj(i) ̸= 0} be the set of indices of the nonzero entries in column kj. The set of indices of rows of A that can affect a product with column kj is I = {m \| Am,J ̸= 0}. Let \|I\| and \|J \| denote the number of entries in I and J , respectively, and letej = ej(I) be the vector of length \|I\| that is obtained by taking the entries of ej with row indices belonging to I. To solve (11.1) for kj, construct the \|I\| × \|J \| matrix A = AI,J and solve the small unconstrained least squares problem min kj ∥ej − Akj∥2 2. (11.2) (11.2) This can be done using a dense QR factorization of A. Extending kj to have length n by setting entries that are not in J to zero gives kj. A straightforward way to construct S that does not depend on a sophisticated initial choice (but could, for example, be the identity or be equal to S{A}) proceeds as follows. 11.2.1 SPAI Preconditioner Starting with a chosen column sparsity pattern J for kj, construct A, solve (11.2) for kj, set kj(J ) = kj, and deﬁne the residual vector rj = ej −A1:n,Jkj. rj = ej −A1:n,Jkj. If ∥rj∥2 ̸= 0, then kj is not equal to the j-th column of A−1, and a better approximation can be derived by augmenting J . To do this, let L = {l \| rj(l) ̸= 0} and deﬁne If ∥rj∥2 ̸= 0, then kj is not equal to the j-th column of A−1, and a better approximation can be derived by augmenting J . To do this, let L = {l \| rj(l) ̸= 0} and deﬁne J = {i \| AL,i ̸= 0} \ J . (11.3) (11.3) These are candidate indices that can be added to J , but as there may be many of them, they need to be chosen to most effectively reduce ∥rj∥2. A possible heuristic is to solve for each i ∈ J the minimization problem These are candidate indices that can be added to J , but as there may be many of them, they need to be chosen to most effectively reduce ∥rj∥2. A possible heuristic is to solve for each i ∈ J the minimization problem min μi \|\|rj −μiAei∥2 2. min μi \|\|rj −μiAei∥2 2. 11.2 Approximate Inverses Based on Frobenius Norm Minimization 209 This has the solution μi = rT j Aei/∥Aei∥2 2 with residual ∥rj∥2 −(rT j Aei)2/∥Aei∥2 2. Indices i ∈ J for which this is small are appended to J . The process can be repeated until either the required accuracy is attained or the maximum number of allowed entries in J is reached. Solving the unconstrained least squares problem (11.2) after extending A to AI∪I′, J ∪J ′ is typically performed using updating. Assume the QR factorization of A is A = AI,J = Q R 0 = Q1 Q2 R 0 , A = AI,J = Q R 0 = Q1 Q2 R 0 , where Q1 is \|I\| × \|J \|. Here Q is an orthogonal matrix and R is an upper triangular matrix. 11.2.1 SPAI Preconditioner 14: end for Determine new indices J ′ ⊂ J to add to J Determine new indices J ′ ⊂ J to add to J I′ = {m \| Am,J ′ ̸= 0} \ I I = I ∪I′ and J = J ∪J ′ I′ = {m \| Am,J ′ ̸= 0} \ I I = I ∪I′ and J = J ∪J ′ ▷Augment the sparsity pattern Construct new A = AI,J and new kj ▷Update the QR factorization rj = ej −A1:n,Jkj I′ = {m \| Am,J ′ ̸= 0} \ I I = I ∪I′ and J = J ∪J ′ ▷Augment the sparsity pattern Construct new A = AI,J and new kj ▷Update the QR factorization rj = ej −A1:n,Jkj 13: kj(J ) = kj ▷Extend kj to kj by setting entries not in J to zero. 14: end for A = ⎛ ⎜ ⎜ ⎜ ⎜ ⎝ 10 −2 −1 10 −2 −1 10 −2 −1 10 −2 −1 10 ⎞ ⎟ ⎟ ⎟ ⎟ ⎠ , A = ⎛ ⎝ 10 −2 −1 10 −1 ⎞ ⎠, k1 = 0.1020 0.0101 , r1 = ⎛ ⎜ ⎜ ⎜ ⎜ ⎝ 1.00 × 10−4 1.00 × 10−3 1.01 × 10−2 0 0 ⎞ ⎟ ⎟ ⎟ ⎟ ⎠ . A = ⎛ ⎜ ⎜ ⎝ 10 −2 −1 10 −2 −1 10 −1 ⎞ ⎟ ⎟ ⎠, k1 = ⎛ ⎝ 0.1021 0.0104 0.0010 ⎞ ⎠, r1 = ⎛ ⎜ ⎜ ⎜ ⎜ ⎝ 1.0 × 10−5 1.1 × 10−4 1.1 × 10−3 1.0 × 10−2 0 ⎞ ⎟ ⎟ ⎟ ⎟ ⎠ , k1 = ⎛ ⎜ ⎜ ⎜ ⎜ ⎝ 0.1021 0.0104 0.0010 0 0 ⎞ ⎟ ⎟ ⎟ ⎟ ⎠ . A = ⎛ ⎜ ⎜ ⎜ ⎜ ⎝ 10 −2 −1 10 −2 −1 10 −2 −1 10 −2 −1 10 ⎞ ⎟ ⎟ ⎟ ⎟ ⎠ , A = ⎛ ⎝ 10 −2 −1 10 −1 ⎞ ⎠, k1 = 0.1020 0.0101 , r1 = ⎛ ⎜ ⎜ ⎜ ⎜ ⎝ 1.00 × 10−4 1.00 × 10−3 1.01 × 10−2 0 0 ⎞ ⎟ ⎟ ⎟ ⎟ ⎠ . 11.2.1 SPAI Preconditioner The QR factorization of the extended matrix is AI∪I′, J ∪J ′ = A AI, J ′ AI′, J ′ = Q I ⎛ ⎝ R QT 1 AI, J ′ QT 2 AI, J ′ AI′, J ′ ⎞ ⎠ = Q I I Q′ ⎛ ⎝ R QT 1 AI, J ′ R′ 0 ⎞ ⎠, where Q′ and R′ are from the QR factorization of the (\|I′\| + \|I\| −\|J \|) × \|J ′\| matrix QT 2 AI, J ′ AI′, J ′ . Factorizing this matrix and updating the trailing QR factorization to get the new kj is much more efﬁcient than computing the QR factorization of the extended matrix from scratch. Factorizing this matrix and updating the trailing QR factorization to get the new kj is much more efﬁcient than computing the QR factorization of the extended matrix from scratch. Construction of the SPAI preconditioner is summarized in Algorithm 11.1. The maximum number of entries nzj that is permitted in kj must be at least as large as the number of entries in the initial sparsity pattern Jj. Updating can be used to compute a new kj for each pass through the while loop; the number of passes is typically small (for example, if a good initial sparsity pattern is available, a single pass may be sufﬁcient). The example in Figure 11.1 illustrates Algorithm 11.1. Starting with a tridiagonal matrix, it considers the computation of the ﬁrst column k1 of the inverse matrix K. The algorithm starts with J1 = {1, 2}. When A is symmetric, there is no guarantee that the computed K will be symmetric. One possibility is to use (K + KT )/2 to approximate A−1. The SPAI preconditioner is not sensitive to the ordering of A. This has the advantage that A can be partitioned and preordered in whatever way is convenient, for instance, 210 11 Sparse Approximate Inverse Preconditioners ALGORITHM 11.1 SPAI preconditioner (right-looking approach) Input: Nonsymmetric matrix A, a convergence tolerance η > 0, an initial sparsity pattern Jj and the maximum number nzj of permitted entries for column j of K (1 ≤j ≤n). 1 Output: K ≈A−1 with columns kj (1 ≤j ≤n). Output: K ≈A−1 with columns kj (1 ≤j ≤n). 11.2.1 SPAI Preconditioner 1: for j = 1 : n do ▷The columns may be computed in parallel 2: Set J = Jj and I = {m \| A(m, J ) ̸= 0}, ∥rj∥2 = ∞ 3: Construct A = AI,J and solve (11.2) for kj 4: rj = ej −A1:n,Jkj 5: while \|J \| < nzj and ∥rj∥2 > η do 6: Construct J given by (11.3) ▷ J is the candidate set 7: Determine new indices J ′ ⊂ J to add to J 1: for j = 1 : n do ▷The columns may be computed in parallel 2: Set J = Jj and I = {m \| A(m, J ) ̸= 0}, ∥rj∥2 = ∞ 3: Construct A = AI,J and solve (11.2) for kj 4: rj = ej −A1:n,Jkj 5: while \|J \| < nzj and ∥rj∥2 > η do ▷The columns may be computed in parallel 1: for j = 1 : n do ▷The columns may be co 2: Set J = Jj and I = {m \| A(m, J ) ̸= 0}, ∥rj∥2 = ∞ 3: Construct A = AI,J and solve (11.2) for kj 4: rj = ej −A1:n,Jkj 1: for j = 1 : n do ▷The columns may be computed in parallel 2: Set J = Jj and I = {m \| A(m, J ) ̸= 0}, ∥rj∥2 = ∞ 3: Construct A = AI,J and solve (11.2) for kj 4: rj = ej −A1:n,Jkj 5: while \|J \| < nzj and ∥rj∥2 > η do 6: Construct J given by (11.3) ▷ J is the candidate set 7: Determine new indices J ′ ⊂ J to add to J 8: I′ = {m \| Am,J ′ ̸= 0} \ I 9: I = I ∪I′ and J = J ∪J ′ ▷Augment the sparsity pattern 10: Construct new A = AI,J and new kj ▷Update the QR factorization 11: rj = ej −A1:n,Jkj 12: end while 13: kj(J ) = kj ▷Extend kj to kj by setting entries not in J to zero. 11.2.1 SPAI Preconditioner A = ⎛ ⎜ ⎜ ⎝ 10 −2 −1 10 −2 −1 10 −1 ⎞ ⎟ ⎟ ⎠, k1 = ⎛ ⎝ 0.1021 0.0104 0.0010 ⎞ ⎠, r1 = ⎛ ⎜ ⎜ ⎜ ⎜ ⎝ 1.0 × 10−5 1.1 × 10−4 1.1 × 10−3 1.0 × 10−2 0 ⎞ ⎟ ⎟ ⎟ ⎟ ⎠ , k1 = ⎛ ⎜ ⎜ ⎜ ⎜ ⎝ 0.1021 0.0104 0.0010 0 0 ⎞ ⎟ ⎟ ⎟ ⎟ ⎠ . Figure 11.1 An illustration of computing the ﬁrst column of a sparse approximate inverse using the SPAI algorithm with nz1 = 3. On the top line is the initial tridiagonal matrix A followed by the matrix A and the vectors k1 and r1 on the ﬁrst loop of Algorithm 11.1. The bottom line presents the updated matrix ˆA that is obtained on the second loop by adding the third row and column of A and the corresponding vectors k1 and r1 and, ﬁnally, k1. Here the numerical values have been appropriately rounded. Figure 11.1 An illustration of computing the ﬁrst column of a sparse approximate inverse using the SPAI algorithm with nz1 = 3. On the top line is the initial tridiagonal matrix A followed by the matrix A and the vectors k1 and r1 on the ﬁrst loop of Algorithm 11.1. The bottom line presents the updated matrix ˆA that is obtained on the second loop by adding the third row and column of A and the corresponding vectors k1 and r1 and, ﬁnally, k1. Here the numerical values have been appropriately rounded. to better suit the needs of a distributed implementation, without worrying about the impact on the subsequent convergence rate of the solver. The disadvantage is that orderings cannot be used to reduce ﬁll-in and/or improve the quality of this to better suit the needs of a distributed implementation, without worrying about the impact on the subsequent convergence rate of the solver. The disadvantage is that orderings cannot be used to reduce ﬁll-in and/or improve the quality of this Approximate Inverses Based on Frobenius Norm Minimization 211 11.2 approximate inverse. For instance, if A−1 has no small entries, SPAI will not ﬁnd a sparse K, and because the inverse of a permutation of A is just a permutation of A−1, no permutation of A will change this. 11.2.2 FSAI Preconditioner: SPD Case We next consider a class of preconditioners based on an incomplete inverse factor- ization of A−1. The factorized sparse approximate inverse (FSAI) preconditioner for an SPD matrix A is deﬁned as the product M−1 = GT G, M−1 = GT G, where the sparse lower triangular matrix G is an approximation of the inverse of the (complete) Cholesky factor L of A. Theoretically, a FSAI preconditioner is computed by choosing a lower triangular sparsity pattern SL and minimizing ∥I −GL∥2 F = tr (I −GL)T (I −GL) , (11.4) (11.4) over all G with sparsity pattern SL. Here tr denotes the matrix trace operator (that is, the sum of the entries on the diagonal). The computation of G can be performed without knowing L explicitly. Differentiating (11.4) with respect to the entries of G and setting to zero yields (GLLT )ij = (GA)ij = (LT )ij for all (i, j) ∈SL. (11.5) (11.5) Because LT is an upper triangular matrix while SL is a lower triangular pattern, the matrix equation (11.5) can be rewritten as (GA)ij = 0 i ̸= j, (i, j) ∈SL, lii i = j. (11.6) (11.6) G is not available from (11.6) because L is unknown. Instead, G is computed such that G is not available from (11.6) because L is unknown. Instead, G is computed such that (GA)ij = δi,j for all (i, j) ∈SL, (11.7) (11.7) where δi,j is the Kronecker delta function (δi,j = 1 if i = j and is equal to 0, otherwise). The FSAI factor G is then obtained by setting where δi,j is the Kronecker delta function (δi,j = 1 if i = j and is equal to 0, otherwise). The FSAI factor G is then obtained by setting G = DG, G = DG, 11 Sparse Approximate Inverse Preconditioners 212 where D is a diagonal scaling matrix. An appropriate choice for D is D = [diag(G)]−1/2, (11.8) (11.8) so that so that (GAGT )ii = 1, 1 ≤i ≤n. (GAGT )ii = 1, 1 ≤i ≤n. (GAGT )ii = 1, 1 ≤i ≤n. The following result shows that the FSAI preconditioner exists for any nonzero pattern SL that includes the main diagonal of A. Theorem 11.2 (Kolotilina & Yeremin 1993) Assume A is SPD. If the lower triangular sparsity pattern SL includes all diagonal positions, then G exists and is unique. ALGORITHM 11.2 FSAI preconditioner nput: SPD matrix A and a lower triangular sparsity pattern SL that includes all iagonal positions. g p Output: Lower triangular matrix G such that A−1 ≈GGT . Output: Lower triangular matrix G such that A−1 ≈GGT . 1: for i = 1 : n do 2: Construct Ii = {j \| (i, j) ∈SL}, AIi,Ii and set nzi = \|Ii\| 3: Solve AIi,Ii ¯gi = enzi 4: Scale gi = dii ¯gi with dii = ( ¯gi,nzi)−1/2 ▷¯gi,nzi is the last component of ¯gi 5: Extend gi to the row Gi,1:i by setting entries that are not in Ii to zero 6: end for 1: for i = 1 : n do 2: Construct Ii = {j \| (i, j) ∈SL}, AIi,Ii and set nzi = \|Ii\| 3: Solve AIi,Ii ¯gi = enzi i, i g zi 4: Scale gi = dii ¯gi with dii = ( ¯gi,nzi)−1/2 ▷¯gi,nzi is the last component of ¯g 5: Extend gi to the row Gi,1:i by setting entries that are not in Ii to zero 6: end for The performance of the FSAI preconditioner is highly dependent on the choice of SL. If entries are added to the pattern, then, as the following result shows, the preconditioner is more accurate, but it is also more expensive. Theorem 11.4 (Kolotilina et al. 2000) Let L be the Cholesky factor of the SPD matrix A. Given the lower triangular sparsity patterns SL1 and SL2 that include all diagonal positions, let the corresponding FSAI preconditioners computed using Algorithm 11.2 be G1 and G2, respectively. If SL1 ⊆SL2, then \|\|I −G2L\|\|F ≤\|\|I −G1L\|\|F . 11.2.2 FSAI Preconditioner: SPD Case Proof Set Ii = {j \| (i, j) ∈SL}, and let AIi, Ii denote the submatrix of order nzi = \|Ii\| of entries akl such that k, l ∈Ii. Let ¯gi and gi be dense vectors containing the nonzero coefﬁcients in row i of G and G, respectively. Using this notation, solving (11.7) decouples into solving n independent SPD linear systems AIi, Ii ¯gi = enzi, 1 ≤i ≤n, where the unit vectors are of length nzi. Moreover, (GAGT )ii = j∈Ii δi,jGij = Gii = (A−1 Ii,Ii)ii. This implies that the diagonal entries of D given by (11.8) are nonzero. Conse- quently, the computed rows of G exist and provide a unique solution. ⊓⊔ ⊓⊔ The procedure for computing a FSAI preconditioner is summarized in Algo- rithm 11.2. The computation of each row of G can be performed independently; thus, the algorithm is inherently parallel, but each application of the preconditioner requires the solution of triangular systems. The following theorem states that G computed using Algorithm 11.2 is in some sense optimal. Theorem 11.3 (Kolotilina et al. 2000) Let L be the Cholesky factor of the SPD matrix A. Given a lower triangular sparsity pattern SL that includes all diagonal positions, let G be the FSAI preconditioner computed using Algorithm 11.2. Then any lower triangular matrix G1 with its sparsity pattern contained in SL and (G1AGT 1 )ii = 1 (1 ≤i ≤n) satisﬁes Theorem 11.3 (Kolotilina et al. 2000) Let L be the Cholesky factor of the SPD matrix A. Given a lower triangular sparsity pattern SL that includes all diagonal positions, let G be the FSAI preconditioner computed using Algorithm 11.2. Then any lower triangular matrix G1 with its sparsity pattern contained in SL and (G1AGT 1 )ii = 1 (1 ≤i ≤n) satisﬁes \|\|I −GL\|\|F ≤\|\|I −G1L\|\|F . 11.2 Approximate Inverses Based on Frobenius Norm Minimization 213 11.2.4 Determining a Good Sparsity Pattern The role of the input pattern is to preserve sparsity by ﬁltering out entries of A−1 that contribute little to the quality of the preconditioner. For instance, it might be appropriate to ignore entries with a small absolute value, while retaining the largest ones. Unfortunately, the locations of large entries in A−1 are generally unknown, and this makes the a priori sparsity choice difﬁcult. A possible exception is when A is a banded SPD matrix. In this case, the entries of A−1 are bounded in an exponentially decaying manner along each row or column. Speciﬁcally, there exist 0 < ρ < 1 and a constant c such that for all i, j \|(A−1)ij\| ≤cρ\|i−j\|. The scalars ρ and c depend on the bandwidth and the condition number of A. For matrices with a large bandwidth and/or a high condition number, c can be very large and ρ close to one, indicating extremely slow decay. However, if the entries of A−1 can be shown to decay rapidly, then a banded M−1 should be a good approximation to A−1. In this case, SL can be chosen to correspond to a matrix with a prescribed bandwidth. A common choice for a general A is SL+SU = S{A}, motivated by the empirical observation that entries in A−1 that correspond to nonzero positions in A tend to be relatively large. However, this simple choice is not robust because entries of A−1 that lie outside S{A} can also be large. An alternative strategy based on the Neumann series expansion of A−1 is to use the pattern of a small power of A, i.e., S{A2} or S{A3}. By starting from the lower and upper triangular parts of A, this approach can be used to determine candidates SL and SU. While approximate inverses based on higher powers of A are often better than those corresponding to A, there is no guarantee they will result in good preconditioners. Furthermore, even small powers of A can be very dense, thus slowing down the construction and application of the preconditioner. A possible remedy is to use the power of a sparsiﬁed A. Alternatively, the pattern can be chosen dynamically by retaining the largest terms in each row of the preconditioner as it is computed, which is what the SPAI algorithm does. Another possibility is to implicitly determine SL + SU as follows. 11.2.3 FSAI Preconditioner: General Case The FSAI algorithm can be extended to a general matrix A. Two input sparsity patterns are required: a lower triangular sparsity pattern SL and an upper triangular sparsity pattern SU, both containing the diagonal positions. First, lower and upper triangular matrices GL and GU are computed such that (GLA)ij = δi,j for all (i, j) ∈SL, (AGU)ij = δi,j for all (i, j) ∈SU. Then D is obtained as the inverse of the diagonal of the matrix GLAGU, and the ﬁnal nonsymmetric FSAI factors are given by GL = GL and GU = GUD. The computation of the two approximate factors can be performed independently. Then D is obtained as the inverse of the diagonal of the matrix GLAGU, and the ﬁnal nonsymmetric FSAI factors are given by GL = GL and GU = GUD. The computation of the two approximate factors can be performed independently. This generalization is well deﬁned if, for example, A is nonsymmetric positive deﬁnite. There is also theory that extends existence to special classes of matrices, including M- and H-matrices. In more general cases, solutions to the reduced 214 11 Sparse Approximate Inverse Preconditioners systems may not exist, and modiﬁcations (such as perturbing the diagonal entries) are needed to circumvent breakdown. 11.2.4 Determining a Good Sparsity Pattern Starting with a simple sparsity pattern, compute the corresponding FSAI preconditioner G1. Then choose a pattern based on G1AGT 1 and apply the FSAI algorithm to G1AGT 1 to obtain G2. Finally, set the preconditioner to G2G1. Despite running the FSAI algorithm twice, this approach can be worthwhile. Unfortunately, the choice of the best technique for generating a FSAI preconditioner and its sparsity pattern is highly problem dependent. 215 11.3 Factorized Approximate Inverses Based on Incomplete Conjugation 11.3 Factorized Approximate Inverses Based on Incomplete Conjugation An alternative way to obtain a factorized approximate inverse is based on incom- plete conjugation (A-orthogonalization) in the SPD case and on incomplete A- biconjugation in the general case. For SPD matrices, the approach represents an approximate Gram–Schmidt orthogonalization that uses the A-inner product ⟨., .⟩A. An important attraction is that the sparsity patterns of the approximate inverse factors need not be speciﬁed in advance; instead, they are determined dynamically as the preconditioner is computed. 11.3.1 AINV Preconditioner: SPD Case Output: A−1 ≈ZD−1ZT with Z a unit upper triangular matrix and D a diagonal matrix with positive diagonal entries. 1: [z(0) 1 , . . . , z(0) n ] = [e1, . . . , en] ▷Initialise Z to hold the columns of the identity matrix 2: for j = 1 : n do 3: for k = 1 : j −1 do 4: α = Ak,1:n z(k−1) j /dkk 5: z(k) j = z(k−1) j −αz(k−1) k 6: Sparsify z(k) j ▷Drop entries from z(k) j 7: end for 8: djj = Aj,1:n z(j−1) j 9: end for 10: Z = [z(0) 1 , . . . , z(n−1) n ] ALGORITHM 11.3 AINV preconditioner (left-looking approach) Input: SPD matrix A and sparsifying rule. Output: A−1 ≈ZD−1ZT with Z a unit upper triangular matrix and D a diagonal matrix with positive diagonal entries. Output: A−1 ≈ZD−1ZT with Z a unit upper triangular matrix and D a diagonal matrix with positive diagonal entries. 1: [z(0) 1 , . . . , z(0) n ] = [e1, . . . , en] ▷Initialise Z to hold the columns of the identity matrix 11.3.1 AINV Preconditioner: SPD Case When A is an SPD matrix, the AINV preconditioner is deﬁned by an approximate inverse factorization of the form A−1 ≈M−1 = ZD−1ZT , where the matrix Z is unit upper triangular and D is a diagonal matrix with positive entries. The factor Z is a sparse approximation of the inverse of the LT factor in the square root-free factorization of A. Z and D are computed directly from A using an incomplete A-orthogonalization process applied to the columns of the identity matrix. If entries are not dropped, then a complete factorization of A−1 is computed and Z is signiﬁcantly denser than LT . To preserve sparsity, at each step of the computation, entries are discarded (for example, using a prescribed threshold, or according to the positions of the entries, or by retaining a chosen number of the largest entries in each column), resulting in an approximate factorization of A−1. largest entries in each column), resulting in an approximate factorization of A . There are several variants. Algorithms 11.3 and 11.4 outline left-looking and right-looking approaches, respectively. Practical implementations need to employ sparse matrix techniques. The left-looking scheme computes the j-th column zj of Z as a sparse linear combination of the previous columns z1, . . . , zj−1. The key is determining which multipliers (the α’s in Steps 4 and 5 of the two algorithms, respectively) are nonzero and need to be computed. This can be achieved very efﬁciently by having access to both the rows and columns of A (although the algorithm does not require that A is explicitly stored—only the capability of forming inner products involving the rows of A is required). For the right-looking approach, the crucial part for each j is the update of the sparse submatrix of Z composed of the columns j + 1 to n that are not yet fully computed. Here, only one row of A is used in the outer loop of the algorithm. Therefore, A can be generated on-the-ﬂy by rows. The DS format can be used to store the partially computed Z (Section 1.3.2). As with complete factorizations, the efﬁciency of the computation and application of AINV preconditioners can beneﬁt from incorporating blocking. 216 11 Sparse Approximate Inverse Preconditioners ALGORITHM 11.3 AINV preconditioner (left-looking approach) Input: SPD matrix A and sparsifying rule. 11.3.2 AINV Preconditioner: General Case In the general case, the AINV preconditioner is given by an approximate inverse factorization of the form A−1 ≈M−1 = WD−1ZT , where Z and W are unit upper triangular matrices and D is a diagonal matrix. Z and W are sparse approximations of the inverses of the LT and U factors in the LDU factorization of A, respectively. Starting from the columns of the identity matrix, A-biconjugation is used to compute the factors. Algorithm 11.5 outlines the right- looking approach. Note it offers two possibilities for computing the entries djj of D that are equivalent in exact arithmetic if the factorization is breakdown-free. The left-looking variant given in Algorithm 11.3 can be generalized in a similar way. where Z and W are unit upper triangular matrices and D is a diagonal matrix. Z and W are sparse approximations of the inverses of the LT and U factors in the LDU factorization of A, respectively. Starting from the columns of the identity matrix, A-biconjugation is used to compute the factors. Algorithm 11.5 outlines the right- looking approach. Note it offers two possibilities for computing the entries djj of D that are equivalent in exact arithmetic if the factorization is breakdown-free. The left-looking variant given in Algorithm 11.3 can be generalized in a similar way. Figure 11.2 illustrates the sparsity patterns of the AINV factors for a matrix arising in circuit simulation. S{A} is symmetric, but the values of the entries of A are nonsymmetric. The sparsity pattern S{W + ZT } is given, where W and Z are computed using Algorithm 11.5 with sparsiﬁcation based on a dropping tolerance of 0.5. Also given are the patterns S{L + U} and S{L−1 + U−1} for the incomplete factors L and U computed using Algorithm 10.2 (see Section 10.2) with a dropping tolerance of 0.1 and at most 10 entries in each row of L + U. Note that this dual dropping strategy is one of the most popular ways of employing 11.3 Factorized Approximate Inverses Based on Incomplete Conjugation 217 Figure 11.2 An example to illustrate the difference between the sparsity patterns of the AINV factors and those of the inverse of the ILU factors. 11.3.2 AINV Preconditioner: General Case The sparsity pattern S{A} of the matrix A is given (top left) together with the patterns of the factorized approximate inverse factors S{W +ZT } (top right), the ILU factors S{L + U} (bottom left), and their inverses S{L−1 + U−1} (bottom right). Figure 11.2 An example to illustrate the difference between the sparsity patterns of the AINV factors and those of the inverse of the ILU factors. The sparsity pattern S{A} of the matrix A is given (top left) together with the patterns of the factorized approximate inverse factors S{W +ZT } (top right), the ILU factors S{L + U} (bottom left), and their inverses S{L−1 + U−1} (bottom right). Algorithm 10.2; it is often denoted as ILUT(p, τ), where p is the maximum number of entries allowed in each row and τ is the dropping tolerance. In this example, the parameters were chosen so that the number of entries in both W + ZT and L + U is approximately equal, but the resulting sparsity patterns are clearly different. In particular, potentially important information is lost from S{L−1 + U−1}. Algorithm 10.2; it is often denoted as ILUT(p, τ), where p is the maximum number of entries allowed in each row and τ is the dropping tolerance. In this example, the parameters were chosen so that the number of entries in both W + ZT and L + U is approximately equal, but the resulting sparsity patterns are clearly different. In particular, potentially important information is lost from S{L−1 + U−1}. 11.3.3 SAINV: Stabilization of the AINV Method The following result is analogous to Theorem 9.4. The following result is analogous to Theorem 9.4. Theorem 11.5 (Benzi et al. 1996) If A is a nonsingular M- or H-matrix, then the AINV factorization of A does not break down. For more general matrices, breakdown can happen because of the occurrence of a zero djj or, in the SPD case, negative djj. In practice, exact zeros are unlikely but very small djj can occur (near breakdown), which may lead to uncontrolled growth in the size of entries in the incomplete factors and, because such entries are not dropped when using a threshold parameter, a large amount of ﬁll-in. The next theorem indicates how breakdown can be prevented when A is SPD through reformulating the A-orthogonalization. 218 11 Sparse Approximate Inverse Preconditioners ALGORITHM 11.4 AINV preconditioner (right-looking approach) Input: SPD matrix A and sparsifying rule. Output: A−1 ≈ZD−1ZT with Z a unit upper triangular matrix and D a diagonal matrix with positive diagonal entries. 1: [z(0) 1 , . . . , z(0) n ] = [e1, . . . , en] ▷Initialise Z to hold the columns of the identity matrix 2: for j = 1 : n do 3: djj = Aj,1:n z(j−1) j 4: for k = j + 1 : n do 5: α = Aj,1:n z(j−1) k /djj 6: z(j) k = z(j−1) k −αz(j−1) j 7: Sparsify z(j) k ▷Drop entries from z(j) k 8: end for 9: end for 10: Z = [z(0) 1 , . . . , z(n−1) n ] ALGORITHM 11.4 AINV preconditioner (right-looking approach) Input: SPD matrix A and sparsifying rule. Output: A−1 ≈ZD−1ZT with Z a unit upper triangular matrix and D a diagonal matrix with positive diagonal entries. Input: SPD matrix A and sparsifying rule. Output: A−1 ≈ZD−1ZT with Z a unit upper triangular matrix and D a diagonal matrix with positive diagonal entries. 1: [z(0) 1 , . . . , z(0) n ] = [e1, . . . , en] ▷Initialise Z to hold the columns of the identity matrix 2: for j = 1 : n do 3: djj = Aj,1:n z(j−1) j 4: for k = j + 1 : n do 5: α = Aj,1:n z(j−1) k /djj 6: z(j) k = z(j−1) k −αz(j−1) j 7: Sparsify z(j) k ▷Drop entries from z(j) k 8: end for 9: end for 10: Z = [z(0) 1 , . . . , z(n−1) n ] 1: [z(0) 1 , . The following result is analogous to Theorem 9.4. . . , z(0) n ] = [e1, . . . , en] ▷Initialise Z to hold the columns of the identity matrix ▷Drop entries from z(j) k ▷Drop entries from z(j) k Theorem 11.6 (Benzi et al. 2000; Kopal et al. 2012) Consider Algorithm 11.4 with no sparsiﬁcation (Step 7 is removed). The following identity holds Aj,1:n z(j−1) k ≡eT j Az(j−1) k = ⟨z(j−1) j , z(j−1) k ⟩A, 1 ≤j ≤k ≤n. Proof Because AZ = Z−T D and Z−T D is lower triangular, entries 1 to j −1 of the vector Az(j−1) k are equal to zero. Z is unit upper triangular so entries j + 1 to n of its j-th column z(j−1) j are also equal to zero. Thus, z(j−1) j can be written as the sum z + ej, where entries j to n of the vector z are zero. The result follows. ⊓⊔ This suggests using alternative computations within the AINV approach based on the whole of A instead of on its rows. The reformulation, which is called the stabilized AINV algorithm (SAINV), is outlined in Algorithm 11.6. It is breakdown-free for any SPD matrix A because the diagonal entries are djj = ⟨z(j−1) j , z(j−1) j ⟩A > 0. Practical experience shows that, while slightly more costly to compute, the SAINV algorithm gives higher quality preconditioners than the AINV algorithm. However, the computed diagonal entries can still be very small and may need to be modiﬁed. The factors Z and D obtained with no sparsiﬁcation can be used to compute the square root-free Cholesky factorization of A. The L factor of A and the inverse factor Z computed using Algorithm 11.6 without sparsiﬁcation satisfy 11.3 Factorized Approximate Inverses Based on Incomplete Conjugation 219 ALGORITHM 11.5 Nonsymmetric AINV preconditioner (right-looking approach) ALGORITHM 11.5 Nonsymmetric AINV preconditioner (right-looking approach) pp ) Input: Nonsymmetric matrix A and sparsifying rule. 1 1 T Input: Nonsymmetric matrix A and sparsifying rule. Output: A−1 ≈WD−1ZT with W and Z unit upper triangular matrices and D a diagonal matrix. Input: Nonsymmetric matrix A and sparsifying rule. Output: A−1 ≈WD−1ZT with W and Z unit upper triangular matrices and D a diagonal matrix. 1: [z(0) 1 , . . . , z(0) n ] = [e1, . . . , en] and [w(0) 1 , . . . , w(0) n ] = [e1, . . . , en] 2: for j = 1 : n do 3: djj = (A1:n,j)T z(j−1) j or djj = Aj,1:n w(j−1) j 4: for k = j + 1 : n do 5: α = (A1:n,j)T z(j−1) k /djj 6: z(j) k = z(j−1) k −αz(j−1) j 7: Sparsify z(j) k ▷Drop entries from z(j) k 8: β = Aj,1:n w(j−1) k /djj 9: w(j) k = w(j−1) k −βw(j−1) j 10: Sparsify w(j) k ▷Drop entries from w(j) k 11: end for 12: end for 13: Z = [z(0) 1 , . . . , z(n−1) n ] and W = [w(0) 1 , . . . , w(n−1) n ] 1: [z(0) 1 , . . . , z(0) n ] = [e1, . . . , en] and [w(0) 1 , . . . , w(0) n ] = [e1, . . . , en] 2: for j = 1 : n do 3: djj = (A1:n,j)T z(j−1) j or djj = Aj,1:n w(j−1) j 4: for k = j + 1 : n do 5: α = (A1:n,j)T z(j−1) k /djj 6: z(j) k = z(j−1) k −αz(j−1) j 7: Sparsify z(j) k ▷Drop entries from z(j) k 8: β = Aj,1:n w(j−1) k /djj 9: w(j) k = w(j−1) k −βw(j−1) j 10: Sparsify w(j) k ▷Drop entries from w(j) k 11: end for 12: end for 13: Z = [z(0) 1 , . . . , z(n−1) n ] and W = [w(0) 1 , . . . , w(n−1) n ] ▷Drop entries from z(j) k ▷Drop entries from z(j) k ▷Drop entries from w(j) k AZ = LD or L = AZD−1. ALGORITHM 11.6 SAINV preconditioner (right-looking approach) RITHM 11.6 SAINV preconditioner (right-looking approach) Input: SPD matrix A and sparsifying rule. Output: A−1 ≈ZD−1ZT with Z a unit upper triangular matrix and D a diagonal matrix with positive diagonal entries. 1: [z(0) 1 , . . . , z(0) n ] = [e1, . . . , en] 2: for j = 1 : n do 3: djj = ⟨z(j−1) j , z(j−1) j ⟩A 4: for k = j + 1 : n do 5: α = ⟨z(j−1) k , z(j−1) j ⟩A/djj 6: z(j) k = z(j−1) k −αz(j−1) j 7: Sparsify z(j) k ▷Drop entries from z(j) k 8: end for 9: end for 10: Z = [z(0) 1 , . . . , z(n−1) n ] ▷Drop entries from z(j) k Aj,1:n z(j−1) k = eT j Az(j−1) k = ⟨w(j−1) j , z(j−1) k ⟩A, (A1:n,j)T w(j−1) k = eT j AT w(j−1) k = ⟨z(j−1) j , w(j−1) k ⟩A, 1 ≤j ≤k ≤n. The nonsymmetric SAINV algorithm obtained using this reformulation can improve the preconditioner quality, but it is not guaranteed to be breakdown-free. ALGORITHM 11.5 Nonsymmetric AINV preconditioner (right-looking approach) Using djj = ⟨z(j−1) j , z(j−1) j ⟩A, and equating corresponding entries of AZD−1 and L, gives Using djj = ⟨z(j−1) j , z(j−1) j ⟩A, and equating corresponding entries of AZD−1 and L, gives Using djj = ⟨z(j−1) j , z(j−1) j ⟩A, and equating corresponding entries of AZD−1 and L, gives lij = ⟨z(j−1) j , z(j−1) i ⟩A ⟨z(j−1) j , z(j−1) j ⟩A , 1 ≤j ≤i ≤n. Thus, the SAINV algorithm generates the L factor of the square root-free Cholesky factorization of A as a by-product of orthogonalization in the inner product ⟨. , .⟩A at no extra cost and without breakdown. The stabilization strategy can be extended to the nonsymmetric AINV algorithm using the following result. Theorem 11.7 (Benzi & T˚uma 1998; Bollhöfer & Saad 2002) Consider Algo- rithm 11.5 with no sparsiﬁcation (Steps 7 and 10 removed). The following identities hold: 220 11 Sparse Approximate Inverse Preconditioners ALGORITHM 11.6 SAINV preconditioner (right-looking approach) Input: SPD matrix A and sparsifying rule. Output: A−1 ≈ZD−1ZT with Z a unit upper triangular matrix and D a diagonal matrix with positive diagonal entries. ALGORITHM 11.6 SAINV preconditioner (right-looking approach) 11.4 Notes and References Benzi & T˚uma (1999) present an early comparative study that puts preconditioning by approximate inverses into the context of alternative preconditioning techniques; see also Bollhöfer & Saad (2002, 2006), Benzi & T˚uma (2003), and Bru et al. (2008, 2010). The inverse by bordering method mentioned in Section 11.1 is from Saad (2003b). The ﬁrst use of approximate inverses based on Frobenius norm minimization is given by Benson (1973). A SPAI approach that can exploit a dynamically changing sparsity pattern S is introduced in Cosgrove et al. (1992); an independent and enhanced description is given in the inﬂuential paper by Grote & Huckle (1997). Later developments are presented in Holland et al. (2005), Jia & Zhang (2013), and Jia & Kang (2019). A comprehensive discussion on the choice of the sparsity pattern S can be found in Huckle (1999). Huckle & Kallischko (2007) consider modifying the SPAI method by probing or symmetrizing the approximate inverse 221 11.4 Notes and References and Bröker et al. (2001) look at using approximate inverses based on Frobenius norm minimization as smoothers for multigrid methods. Choosing sparsity patterns for a related approximate inverse with a particular emphasis on parallel computing is described by Chow (2000). For nonsymmetric matrices, MI12 within the HSL mathematical software library computes SPAI preconditioners (see Gould & Scott, 1998 for details and a discussion of the merits and limitations of the approach). An early parallel implementation is given by Barnard et al. (1999). Dehnavi et al. (2013) present an efﬁcient parallel implementation that uses GPUs and include comparisons with ParaSails (Chow, 2001). The latter handles SPD problems using a factored sparse approximate inverse and general problems with an unfactored sparse approximate inverse. A priori techniques determine S as a power of a sparsiﬁed matrix. Original work on the FSAI preconditioner is by Kolotilina & Yeremin (1986, 1993). Its use in solving systems on massively parallel computers is presented in Kolotilina et al. (1992), while an interesting iterative construction can be found in Kolotilina et al. (2000). A parallel variant called ISAI preconditioning that combines a Frobenius norm-based approach with traditional ILU preconditioning is proposed by Anzt et al. (2018). FSAI preconditioning has attracted signiﬁcant theoretical and practical attention. 11.4 Notes and References Recent contributions discuss not only its efﬁcacy but also parallel computation, the use of blocks, supernodes, and multilevel implementations (Ferronato et al., 2012, 2014; Janna & Ferronato, 2011; Janna et al., 2010, 2013, 2015; Ferronato & Pini, 2018; Magri et al., 2018). Many of these enhancements are exploited in the FSAIPACK software of Janna et al. (2015). The AINV preconditioner for SPD and nonsymmetric systems is introduced in Benzi et al. (1996) and Benzi & T˚uma (1998), respectively; see also Benzi et al. (1999) for a parallel implementation. However, the development of this type of preconditioner follows much earlier interest in factorized matrix inverses (for example, Morris, 1946 and Fox et al., 1948). For the SAINV algorithm, see Benzi et al. (2000) and Kharchenko et al. (2001). Theoretical and practical properties of the AINV and SAINV factorizations are studied in a series of papers by Kopal et al. (2012, 2016, 2020). Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this chapter are included in the chapter’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
https://openalex.org/W4306146065	https://zenodo.org/records/7193922/files/1Proceedings-13.07.2019-101-106.pdf	English	null	ERRORS IN ARITHMETIC COMPUTATION OF SIXTH STANDARD PUPILS OF BANTWAL TALUK	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	2,220	“Contemporary Issues and Challenges of Teacher Education” “Contemporary Issues and Challenges of Teacher Education” ERRORS IN ARITHMETIC COMPUTATION OF SIXTH STANDARD PUPILS OF BANTWAL TALUK Dr. Vijayalakshmi Naik Assistant Professor, College of Education, Srinivas University, Mangalore Email: vijaya2006123@rediffmail.com ABSTRACT The present study is titled as “Errors in arithmetic computation of sixth standard pupils of Bantwal taluk”. The central goal of education is not just leading to read and write but to develop the abilities to think and reason. Reasoning is the heart of education. This is more explicit in the education of Science and Mathematics than other fields. In the report of the Education Commission (1964-66) it is recommended that Sciences and Mathematics should be taught on the compulsory basis to all pupils as a part of general education during the first ten years of schooling. A difficulty in learning Mathematics is not generally a number based one but can be concept based or competency based. Some children learn to solve problems by mastery of steps in a procedure with a fixed order, and errors appear when the problem is presented in a different format or when the test is made up of a variety of problems. Survey method is used to select a sample of 600 students of sixth standard studying in 13 Kannada medium higher primary schools. A Diagnostic test is developed to assess the conceptual errors encountered in mathematical operations in selected areas of arithmetic. . The results of the statistical analyses reveal that a) Distribution of error scores in arithmetic computation for the total sample showed that pupils make maximum number of errors in division. b) Distribution of error scores based on gender showed that the total error scores of boys than that of girls. c) Distribution of error scores based on type of management of schools showed that the total error scores of government school pupils are higher than that of aided school pupils. This reveals that there are deficiencies in the teaching of arithmetic. To enhance teachers‟ use of student’s experiences, teacher education will need to focus on encouraging a variety of ways of teacher-student interaction during which students‟ mathematical ideas should be considered exhaustively. Keywords: Errors, arithmetic, computation, diagnostic test. Keywords: Errors, arithmetic, computation, diagnostic test. Institute of Education, Srinivas University Need for the study We have seen that mathematics has been as important part of the school curriculum ever because it gives the strong foundation to the education. Mathematics should be visualized as the vehicle to train a child to think, reason, analyse and articulate logically. Considering its role in the daily life of human beings arithmetic which is a branch of mathematics has special importance. Today, arithmetic is taught with special emphasis on its practical uses in life considering its importance gaining proficiency in arithmetic specially on process of operations like addition, subtraction, multiplication and division is a must. But it is fact that many go wrong or make mistakes even while doing simple calculations or computations. For this we have to go to the primary school level, where the children learn the basis of arithmetic computations. Pupils disability in arithmetic computation is a black mark to our educational system. A large number of studies are done on the concepts related to nature of arithmetic operations and along with effective and remedial teaching. But such studies are very few in our country. Hence there is a need to study what types of errors are committed by the pupils in arithmetic especially at the primary level. Objectives of the study 1. To find the distribution of errors of computation in selected areas of arithmetic of sixth standard pupils of Bantwal taluk based on a) Gender, b) Type of management of schools Methodology used 1. To find the distribution of errors of computation in selected areas of arithmetic of sixth standard pupils of Bantwal taluk based on a) Gender, b) Type of management of schools Introduction The present system of education geared to produce, citizens who can deal with the words, concepts and mathematical or scientific symbols which are necessary for success in a technological society. In classroom, where traditional teaching is followed, pupils are passive listeners and mathematics is learned with tears. Pupils fail to inquire into the subject and solve problems themselves. Institute of Education, Srinivas University 101 “Contemporary Issues and Challenges of Teacher Education” Sample of the study The sample of the study involved six hundred students studying in sixth standard of different schools of Kannada medium in bantwal taluk. Eleven schools which were selected randomly from a list of one hundred seventy schools. Five of these schools were government schools and six were aided schools. Tools used in the study To measure the only variable of the study ‘‘error in arithmetic computation’ in selected areas, the investigator constructed a test “a diagnostic test to detect the errors in arithmetic computation in mathematics”. The areas selected for arithmetic computations were fraction, decimal and percentages. Research method used Research method used Phase one a) Selection of the variables involved in the study b) Construction and validation of the diagnostic tool to measure the selected variable Phase two a) Selection of the sample involved in the study b) Collection of the data using the prepared tool Phase three a) Scoring the collected data using appropriate scoring procedure b) Analysis of the data using suitable statistical procedure Phase one a) Selection of the variables involved in the study b) Construction and validation of the diagnostic tool to measure the selected variable Phase two a) Selection of the sample involved in the study b) Collection of the data using the prepared tool Phase three a) Scoring the collected data using appropriate scoring procedure b) Analysis of the data using suitable statistical procedure Phase one a) Selection of the variables involved in the study b) Construction and validation of the diagnostic tool to measure the selected variable Phase two a) Selection of the sample involved in the study b) Collection of the data using the prepared tool Phase three a) Scoring the collected data using appropriate scoring procedure b) Analysis of the data using suitable statistical procedure Variables of the study In order to meet the objectives of the study the investigator selected the only variable ‘error in arithmetic computation’. Institute of Education, Srinivas University Methodology used The present study is a descriptive study whose purpose was to survey the ‘errors in arithmetic computation of sixth standard pupils of Bantwal taluk’. The errors in arithmetic computation involved the process of operations namely addition, subtraction, multiplication and division. Institute of Education, Srinivas University Institute of Education, Srinivas University 102 “Contemporary Issues and Challenges of Teacher Education” Analysis and interpretation of the study Distribution of error scores based on Gender Institute of Education, Srinivas University 104 “Contemporary Issues and Challenges of Teacher Education” From the table, it is evident that Boys error scores are higher than those of girls. Since the investigator was not able to find more related finding the present study could not be generalized. From the table, it is evident that Boys error scores are higher than those of girls. Since the investigator was not able to find more related finding the present study could not be generalized. C. Distribution of error scores based on types of management of schools GENDER SAMPLE PROCES S OF OPERATI ON MEAN ERRO R SCOR ES (%) SD GOVERNMENT 300 Addition 72.43 26.60 Subtractio n 75.53 25.82 Multiplicat ion 75.72 24.39 Division 94.32 18.96 Total 78.32 20.2 AIDEDD 300 Addition 60.94 26.61 Subtractio n 70.21 24.17 Multiplicat ion 68.85 24.82 Division 87.78 22.44 Total 71.64 21.00 Distribution of error scores based on types of management of schools From the table, it is evident that the government schools make more errors than that of aided schools in the process of operation addition, subtraction, multiplication and division. This may be due to the carelessness, due to carrying over confidence of the pupils in solving problems. Since the investigator was not able to find more related finding the present study could not be generalized. This may be due to the carelessness, due to carrying over confidence of the pupils in solving problems. Since the investigator was not able to find more related finding the present study could not be generalized. Analysis and interpretation of the study The objective of the study was to find the distribution of error scores in arithmetic computation of the sixth standard pupils of Bantwal Taluk in selected areas based on i) gender and ii) type of management of schools. This objective was analysed in three stages A. Distribution of error scores in arithmetic computation for the total sample B. Distribution of error scores based on Gender A. Distribution of error scores in arithmetic computation for the total sample A. Distribution of error scores in arithmetic computation for the total sample B Di t ib ti f b d G d B. Distribution of error scores based on Gender C. Distribution of error scores based on types of management of schools 103 “Contemporary Issues and Challenges of Teacher Education” A. Distribution of error scores in arithmetic computation for the total sample SAMPLE PROCESS OF OPERATION MEAN ERROR SCORES (%) 600 Addition 66.12 Subtraction 74.27 Multiplication 71.95 Division 90.73 Total 75.4 Distribution of error scores in arithmetic computation for the total sample From the table it is evident that error scores in the process of division are the highest. This may be due to lack of negligence towards division, not knowing the multiplication tables, cancellation of common numbers and so on. Teacher may not efficient enough to teach the process of division according to the difficulties of the pupils. Teachers may not give more drill work to the pupils in division problems because it takes more time to solve the problem. If more drill work is given then errors can be minimized. Total error score is very high. This indicates that the teachers need to concentrate on error scores along with achievement scores and remedial measures need to be taken for improving mathematics education. Since the studies were less to support the findings of the present study, it was not possible to arrive at any generalization. B. Distribution of error scores based on Gender GENDER SAMPLE PROCESS OF OPERATION MEAN ERROR SCORES (%) SD BOYS 300 Addition 70.86 26.60 Subtraction 76.78 25.82 Multiplication 75.10 24.39 Division 92.35 18.96 Total 78.12 20.2 GIRLS 300 Addition 62.02 28.59 Subtraction 71.78 27.09 Multiplication 68.84 27.20 Division 89.20 20.47 Total 72.71 21.50 B. Distribution of error scores based on Gender B. Major findings of the study 1. Distribution of error scores in arithmetic computation for the total sample showed that pupils make maximum number of errors in division 2. Distribution of error scores in based on gender showed that the total error scores of boys are higher than that of girls. 3. Distribution of error scores in based on types of schools showed that the total error scores of Government school pupils are higher than that of aided school pupils. Institute of Education, Srinivas University Institute of Education, Srinivas University 105 “Contemporary Issues and Challenges of Teacher Education” Educational implications of the study 1. It helps the school teachers to identify the common errors in arithmetic computation by the pupils in the process of operation 1. It helps the school teachers to identify the common errors in arithmetic computation by the pupils in the process of operation 2. The study provides suitable guidelines to the teachers as to how to analyze the answers of the student and thus help to providing remedial teaching. 3. Special attention could be given to the students of low achievement. Special programs and other supplementary materials should be provided to improve their achievement. 3. Special attention could be given to the students of low achievement. Special programs and other supplementary materials should be provided to improve their achievement. 4. Pupils should be given proper training to i) read problems carefully and comprehend the problems, ii) find out the appropriate steps in solving the problem. 4. Pupils should be given proper training to i) read problems carefully and comprehend the problems, ii) find out the appropriate steps in solving the problem. References: Lu Pien Cheng, ‘error analysis for arithmetic wordproblems-a case study of primary three students in Singapore school’ 2017 Rastogi S, ‘mathematical weakness causes and remeddy’, mittal publications, New Delhi 110059 (I di ) Lu Pien Cheng, ‘error analysis for arithmetic wordproblems-a case study of primary three students in Singapore school’ 2017 in Singapore school’ 2017 Rastogi S, ‘mathematical weakness causes and remeddy’, mittal publications, New Delhi 110059 (India). Journals: Anil kumar, ‘A study on the errors committed by the secondary school pupils in some basic concept of mathematics’, the progress of education, vol LXXII, September 1977. Stefanish, ‘An analysis of computational errors in the use of Division Algorithms by fourth grade students’ published in school-science and mathematics’, vol 92, D4 P201 205, april 1992. Anil kumar, ‘A study on the errors committed by the secondary school pupils in some basic concept of mathematics’, the progress of education, vol LXXII, September 1977. Stefanish, ‘An analysis of computational errors in the use of Division Algorithms by fourth grade students’ published in school-science and mathematics’, vol 92, D4 P201 205, april 1992. **************************** Institute of Education, Srinivas University 106
https://openalex.org/W4391964437	https://www.int-res.com/articles/aei2024/16/q016p145.pdf	English	null	Quantification of finfish assemblages associated with mussel and seaweed farms in southwest UK provides evidence of potential benefits to fisheries	Aquaculture environment interactions	2,024	cc-by	14,919	Corresponding author: sc718@exeter.ac.uk Quantification of finfish assemblages associated with mussel and seaweed farms in southwest UK provides evidence of potential benefits to fisheries Sophie Corrigan1,2,3,, Dan A. Smale3, Charles R. Tyler1,2, A. Ross Brown1,2 1Faculty of Health and Life Sciences, Geoffrey Pope Building, University of Exeter, Stocker Road, Exeter, Devon EX4 4QD, UK 2Sustainable Aquaculture Futures, University of Exeter, Exeter, Devon EX4 4QD, UK 3Marine Biological Association of the United Kingdom, The Laboratory, Citadel Hill, Plymouth PL1 2PB, UK ABSTRACT: Low trophic aquaculture, including shellfish and seaweed farming, offers a potentially sustainable food source and may provide additional environmental benefits, including the creation of new feeding, breeding and nursery areas for fish of commercial and ecological importance. How - ever, quantitative assessments of fish assemblages associated with aquaculture sites are lacking. We used pelagic baited remote underwater videos (BRUVs) and hook and line catches to survey summer fish assemblages at 2 integrated blue mussel Mytilus edulis and kelp (predominantly Sac- charina latissima) farms in southwest UK. We recorded at least 11 finfish species across the surveys, including several of commercial importance, with farmed mussels and/or kelps supporting signifi- cantly higher levels of abundance and richness than reference areas outside farm infrastructure. Farmed kelp provided temporary habitat due to seasonal harvesting schedules, whereas farmed mussels provided greater habitat stability due to overlapping interannual growth cycles. Stomach content analysis of fish caught at the farms revealed that some low trophic level species had high proportions of amphipods in their stomachs, which also dominated epibiont assemblages at the farms. Higher trophic level fish stomachs contained several lower trophic level fish species, suggest- ing that farms provide new foraging grounds and support secondary and tertiary production. Although not identified to species level, juvenile fish were abundant at both farms, suggesting potential provisioning of nursery or breeding grounds; however, this needs further verification. Overall, this study provides evidence that shellfish and seaweed aquaculture can support and enhance populations of commercially and ecologically important fish species through habitat pro- visioning. © The authors 2024. Open Access under Creative Commons by Attri- bution Licence. Use, distribution and reproduction are un restricted. Authors and original publication must be credited. Publisher: Inter-Research · www.int-res.com KEY WORDS: Restorative aquaculture · Shellfish farming · Kelp farming · BRUV · Biodiversity · Habitat · IMTA Vol. 16: 145–162, 2024 https://doi.org/10.3354/aei00478 Vol. 16: 145–162, 2024 https://doi.org/10.3354/aei00478 AQUACULTURE ENVIRONMENT INTERACTIONS Aquacult Environ Interact Published April 11 1. INTRODUCTION In areas devoid of structural features, such as natural reefs, shellfish farms may act as aggregating devices and form rel- atively stable artificial reef-like habitats for fish spe- cies, as they tend to have longer and more overlap- ping growth periods than temperate seaweed farms, which persist for only several months of the year (Cor- rigan et al. 2022). However, more evidence from both seaweed and shellfish farming is needed, including from integrated seaweed and shellfish co-cultivation sites (Wang et al. 2015, Sheehan et al. 2020, Under- wood & Jeffs 2023, United Nations Environment Pro- gramme 2023). secondary production of fish observed at oyster, mus- sel and seaweed aquaculture sites globally is esti- mated at 350–1000 kg ha−1 yr−1 relative to reference habitats, with additional value to commercial and rec- reational fishers of ~970–2850 USD ha−1 yr−1 (Barrett et al. 2022). Quantitative assessments of fish assem- blages associated with shellfish and seaweed farms, however, are largely lacking, and in particular very few studies have been conducted at seaweed farms in temperate regions (Callier et al. 2018, Kelly et al. 2020, Fariñas-Franco et al. 2021, Theuerkauf et al. 2021, Corrigan et al. 2022, Forbes et al. 2022). secondary production of fish observed at oyster, mus- sel and seaweed aquaculture sites globally is esti- mated at 350–1000 kg ha−1 yr−1 relative to reference habitats, with additional value to commercial and rec- reational fishers of ~970–2850 USD ha−1 yr−1 (Barrett et al. 2022). Quantitative assessments of fish assem- blages associated with shellfish and seaweed farms, however, are largely lacking, and in particular very few studies have been conducted at seaweed farms in temperate regions (Callier et al. 2018, Kelly et al. 2020, Fariñas-Franco et al. 2021, Theuerkauf et al. 2021, Corrigan et al. 2022, Forbes et al. 2022). g ) Shellfish and/or seaweed farms may provide novel structured habitat for fish species through both the provision of high densities of farmed organisms (i.e. bivalve shells and seaweed biomass) and their associ- ated epibiont communities, as well as farm infrastruc- ture (Callier et al. 2018, Barrett et al. 2019). This farmed biomass and infrastructure could provide valuable habitat in terms of spawning substrate, breeding grounds, shelter and/or food in the form of farmed biomass or associated epibionts, similar to wild populations of shellfish or seaweeds (Bergman et al. 2001, Callier et al. 2018, Theuerkauf et al. 2021). 1. INTRODUCTION tal benefits, such as improved water quality regula- tion and nutrient cycling (van der Schatte Olivier et al. 2020, United Nations Environment Programme 2023). Recent work has focused on the habitats pro- vided by shellfish and seaweed farms and their role in supporting coastal restoration efforts, particularly for fish species of commercial or ecological importance (Theuerkauf et al. 2021). The potential increase in Aquaculture, the farming of finfish, shellfish and algae in freshwater and saltwater environments, is one of the fastest growing food production systems globally (FAO 2022). Low-trophic or extractive aqua- culture, such as shellfish or seaweed farming, is increasingly recognised for its potential environmen- Publisher: Inter-Research · www.int-res.com Aquacult Environ Interact 16: 145–162, 2024 146 Recent reviews on the potential ecological benefits of seaweed farming have highlighted how farms will likely provide habitat distinct from natural seaweed beds, and so far the few reported effects on fish pop- ulations are highly variable between sites (Theuer- kauf et al. 2021, Corrigan et al. 2022, Forbes et al. 2022, United Nations Environment Programme 2023). For instance, increased abundances of fish have been reported in some shallow-bottom tropical seaweed farms compared to reference areas (e.g. Eklöf et al. 2006, Radulovich et al. 2015), whereas in other similar farms, either negative or no effects on fish popula- tions have been reported (as reviewed in Kelly et al. 2020). Little comparative evidence from suspended seaweed longline farms in temperate regions exists (Corrigan et al. 2022); however, a recent study in Maine, USA, found no difference in fish populations between kelp farms and reference sites, attributed to the seasonality of kelp farming in relation to fish migration patterns in the area (Schutt et al. 2023). Similarly, no effect on benthic fish assemblages was reported at a kelp farm in Sweden (Visch et al. 2020). Habitat provisioning for fish species at shellfish farms in temperate regions has been studied more widely, and although there is variation between sites, in gen- eral shellfish farms support comparable fish abun- dances and diversities to wild shellfish reefs (Callier et al. 2018, Ferriss et al. 2021, Martínez-Baena et al. 2022, Underwood & Jeffs 2023). 2.1. Study sites Surveys were conducted at and around 2 integrated blue mussel Mytilus edulis and kelp (predominantly sugar kelp Saccharina latissima) farms in south Corn- wall, UK (Fig. 1). Porthallow Bay farm (50°04’N, 5°04’W) is a small (~16 ha) site approximately 500 m from shore, while St Austell Bay farm (50°18’N, 4°43’W) is larger (~70 ha) and approximately 1– 1.5 km from shore, with a second blue mussel farm (~30 ha) neighbouring it to the west. Both farm areas are exposed to wind and wave action from the east and southeast, but are sheltered from the predominant southwesterly winds and North Atlantic swells. Both farms are suspended over seabed depths of 8 to 15 m (below chart datum), with the seabed below consisting of mixed rocky substrate, soft sediments and maerl Phymatolithon calcareum gravel. Reference areas were randomly selected and located 200 to 600 m away from farm sites, in areas which did not contain any structural pelagic habitat, but experienced similar environmental conditions and seabed depths to the respective farm (as per Sheehan et al. 2020). Neighbouring benthic habitats around both Porthallow Bay and St Austell Bay include natural kelp beds, seagrass meadows and P. calcareum beds, and both sites are located within the Falmouth Bay to St Austell Bay Special Protected Area (Crosby et al. 2023). Porthallow and St Austell Bay farms use long- line systems with ~200 m header lines anchored to the seabed and suspended 1–3 m below the surface, sup- porting either seaweed or mussel growth (Fig. 1). At both farms, mussel lines were seeded following the nat- ural spawning and settlement cycle of M. edulis, whereby as seawater temperatures rise (typically from March to May), mussels spawn and their larvae settle naturally onto the dropper ropes as spat, and continue to grow for ~18 mo before harvest, by which time new spat will have settled for the next harvesting season. Seaweed lines in Porthallow Bay were seeded in late October 2021 with predominantly S. latissima gameto- phytes, and several Alaria esculenta lines were also de- ployed. The seeded lines were then spaced at 2–3 m distances along the header lines. Seaweed lines in St Austell Bay were set in January–February 2022, whereby carrier lines were wrapped in twine with juve- nile S. latissima sporophytes attached. Several A. 1. INTRODUCTION Hence there is growing interest surrounding the potential for farms to act as ‘restorative’ or ‘regener- ative’ forms of aquaculture in areas where fish pop- ulations have been depleted due to degradation of natural habitats (Alleway et al. 2023). Shellfish and seaweed farming infrastructure may also restrict fish- ing activities in the immediate vicinity, which could benefit populations of fish and other organisms by ex - cluding extractive activities such as bottom trawling, thereby acting as de facto ‘no take zones’ with poten- tial spill-over benefits for fisheries in surrounding areas (Olsgard et al. 2008, Johnson et al. 2015, Wang et al. 2015, Gentry et al. 2020, Bridger et al. 2022). Farms may, however, induce changes in natural fish assemblages by altering food availability and quality (Oakes & Pondella 2009), or by replacing, modifying or damaging pre-existing habitats in the area, such as kelp forests, seagrass meadows or other important coastal habitats (McKindsey et al. 2011, Wood et al. 2017, Campbell et al. 2019, Kelly et al. 2020, Theuer- kauf et al. 2021, United Nations Environment Pro- gramme 2023). It is also uncertain how farming oper- ations and schedules may affect fish populations inhabiting farms. For example, in temperate seaweed farms, annual harvests remove cultivated biomass during summer months, which could disrupt impor- tant fish life stages such as spawning events and nurs- ery habitat provisioning for juvenile fish (Corrigan et al. 2022, Forbes et al. 2022). Most research on the ecological impacts of tem- perate shellfish and seaweed farms has focused on associated sessile and sedentary benthic organisms (Callier et al. 2018, Corrigan et al. 2022) rather than fish assemblages, which are highly mobile and diffi- cult to quantify due to their pronounced spatiotem- poral variability (Bicknell et al. 2019). Surveying coastal fishes in and around aquaculture sites pro- vides additional challenges, such as reduced visibil- ity and accessibility within farm infrastructure (Tonk et al. 2019, Sheehan et al. 2020, Bridger et al. 2022). Baited remote underwater video (BRUV) or camera Corrigan et al.: Finfish in mussel and seaweed farms 147 2. MATERIALS AND METHODS surveys are increasingly utilised for surveying fish assemblages in both natural and artificial habitats, as technological advances have made them rel- atively low cost and highly replicable (Langlois et al. 2010, 2018, Santana-Garcon et al. 2014, Tonk et al. 2019, Sheehan et al. 2020, Corrigan et al. 2022, Jackson-Bué et al. 2023). Furthermore, camera sur- veys have been de signed specifically for suspended aquaculture monitoring: for instance, low cost video systems have recently proved effective in monitor- ing pelagic fauna in a large offshore UK mussel farm (Sheehan et al. 2020) and seaweed farms in the Netherlands (Tonk et al. 2019), and Maine, USA (Schutt et al. 2023). Camera surveys can capture multiple species simultaneously and are effective at monitoring the abundance and diversity of fish as - semblages, as well as recording fish behaviour such as feeding. However, due to the relatively limited spatial and temporal coverage of static camera sys- tems, they inevitably capture only a subset of the entire fish assemblage and should therefore be com- plemented with other approaches where possible, such as fishing surveys that also enable dietary ana - lysis of caught individuals (Mallet & Pelletier 2014, Bicknell et al. 2016, 2019, Tonk et al. 2019, Corrigan et al. 2022). surveys are increasingly utilised for surveying fish assemblages in both natural and artificial habitats, as technological advances have made them rel- atively low cost and highly replicable (Langlois et al. 2010, 2018, Santana-Garcon et al. 2014, Tonk et al. 2019, Sheehan et al. 2020, Corrigan et al. 2022, Jackson-Bué et al. 2023). Furthermore, camera sur- veys have been de signed specifically for suspended aquaculture monitoring: for instance, low cost video systems have recently proved effective in monitor- ing pelagic fauna in a large offshore UK mussel farm (Sheehan et al. 2020) and seaweed farms in the Netherlands (Tonk et al. 2019), and Maine, USA (Schutt et al. 2023). Camera surveys can capture multiple species simultaneously and are effective at monitoring the abundance and diversity of fish as - semblages, as well as recording fish behaviour such as feeding. 2. MATERIALS AND METHODS However, due to the relatively limited spatial and temporal coverage of static camera sys- tems, they inevitably capture only a subset of the entire fish assemblage and should therefore be com- plemented with other approaches where possible, such as fishing surveys that also enable dietary ana - lysis of caught individuals (Mallet & Pelletier 2014, Bicknell et al. 2016, 2019, Tonk et al. 2019, Corrigan et al. 2022). 2.1. Study sites 2.1. Study sites escu- lenta lines were deployed using the same method, which were then spaced 2–3 m apart along the header lines. At both farms, the cultivated seaweed and mussel lines were frequently colonised with Saccorhiza poly- ) We quantified the structure of fish assemblages associated with farmed shellfish and seaweeds, and in comparable pelagic negative control sites, or refer- ence areas, over a summer season in southwest UK to determine whether aquaculture sites provide valu- able habitat for coastal fish species. We repeatedly surveyed 2 integrated mussel and kelp farms at 2 locations, using pelagic BRUVS and hook and line fishing to examine fish abundance and species rich- ness. We also analysed the stomach contents of cap- tured fish to assess whether they were using the farms as feeding grounds based on similarities to epibionts present on the farmed kelps and mussels. We com- pared fish assemblages before kelp harvest (May), during harvest (June) and after harvest (August) at one farm site, to determine whether kelp farm habitat was temporary as a result of the farming cycle. We expected that farms would support greater levels of finfish abundance and richness than reference areas, as they provide structurally complex habitat and elevated food supply through both cultivated bio- mass and associated epibionts. An increased under- standing of fish assemblages associated with mussel and seaweed farming will help determine whether low-trophic aquaculture sites can provide new or ‘restorative’ habitats for fish species of commercial or ecological importance. Aquacult Environ Interact 16: 145–162, 2024 148 Fig. 1. Maps of study area indicating (A) location of farm sites in Cornwall, southwest UK (red dots); (B) St Austell Bay farm (shaded blue rectangle on right, with a second smaller mussel farm to the west); (C) Porthallow Bay farm (shaded blue rectan- gle); (D) simplified diagram of the farm system with suspended seaweed and mussel lines (created with BioRender.com). Note seaweed and mussel lines were set on separate header lines, not together as shown in diagram Fig. 1. Maps of study area indicating (A) location of farm sites in Cornwall, southwest UK (red dots); (B) St Austell Bay farm (shaded blue rectangle on right, with a second smaller mussel farm to the west); (C) Porthallow Bay farm (shaded blue rectan- gle); (D) simplified diagram of the farm system with suspended seaweed and mussel lines (created with BioRender.com). 2.1. Study sites Note seaweed and mussel lines were set on separate header lines, not together as shown in diagram ported similar epibiont assemblages to the seeded S. latissima lines, along with smaller red and green algal species (Corrigan et al. 2023a,b). to give 4 independent replicates per treatment area per survey. After each deployment in a treatment area, BRUV units were moved to a new random location a minimum of 100 m away within the same treatment area, to ensure independence between the 4 replicates in each treatment (Ellis & Demartini 1995, Tonk et al. 2019). We also ensured that BRUVs in the seaweed and mussel treatments were >100 m apart to maintain independence between treatment areas. Reference areas were randomly selected 200 to 600 m away from farm sites to ensure they were not in fluenced by the farms (Ellis & Demartini 1995, Tonk et al. 2019). Deployments in all treatment areas occurred throughout daylight hours and across tidal cycles. Exact deployment locations of BRUV units in each survey treatment area were not consis- tent between survey dates; rather, for each survey, 4 new random locations were chosen within each treatment area. 2.2. BRUV design, deployment and processing At Porthallow Bay, surveys were conducted twice in May 2022, prior to kelp harvest, to compare assem- blages across the 3 treatment areas (seaweed lines, mussel lines and reference areas). In St Austell Bay, surveys were conducted 3 times throughout the summer of 2022 in each of the 3 treatment areas, to compare assemblages prior to kelp harvest (May), during kelp harvest (June) and after kelp harvest (August). The mussel line areas surveyed were not harvested at either site during the survey period. During each survey, BRUV units were deployed randomly throughout each treatment area 4 times, Corrigan et al.: Finfish in mussel and seaweed farms 149 survey. Fifty minutes of footage were analysed from the start of each video, once cameras had stabilised at their sampling depth following deployment. A 50 min period was chosen as this was the minimum length of footage recorded by any one camera, and most fish species are typically observed within the first 40 to 60 min of BRUV deployment (Unsworth et al. 2014, Jackson-Bué et al. 2023). Fish species were identified to the lowest taxonomic level possible, with species richness defined as number of different taxa observed per video (taxa richness). The maximum number of individuals from each species visible in a single frame (MaxN) was recorded as a conservative measure of relative abundance that reduces the possibility of double counting an individual (Cappo et al. 2003, Langlois et al. 2010). Total MaxN was calculated for each video as the sum of all MaxN values for each species observed. MaxN–min was also recorded as the maximum number of individuals of each species ob - served in the field of view at the same time per 1 min of video, and then averaged across the 50 min of video, to give the relative abundance (mean min–1) of every species and allow comparisons with similar studies (e.g. Bicknell et al. 2019, Sheehan et al. 2020). The total time fish of each species spent in view per video was also recorded in seconds (MaxT), along with the total time spent in view by all fish species per video (total MaxT). 2.2. BRUV design, deployment and processing BRUV units comprised 2 cameras (AKASO EK7000 Pro 4K Action Camera) placed back to back, which were each set to face a 35 cm bait arm holding a bait box with 100 g mixed bait (consisting of sardines or mackerel, mussels and prawns) to attract diverse fish species (Fig. 2). BRUV units were suspended at 3 m depths to correspond with seaweed and mussel growth while maintaining good visibility (Fig. 2), and bait was replenished for each deployment. BRUV units were set to record continuously for ~1 h, as re - stricted by battery life, and once deployed were avoided by the vessel for the hour to prevent disturb- ance. Visibility during each BRUV deployment was also measured using Secchi depth. Visibility in the water column remained sufficient to detect fish asso- ciated with the farm lines within a close proximity throughout surveys, ranging from 4 to 11 m (mean ± SE, 8 ± 0.34 m). In total, 118 of the 120 videos from the 60 BRUV unit deployments were successful, with footage from 2 videos being completely obscured by kelp biomass on the first survey in St Austell Bay farm (3 May 2022). In these 2 instances, only footage from the unob- scured cameras on the BRUV units were used. After this initial survey, the BRUV unit design used to sur- vey in the seaweed treatments was changed from the attached header line unit (Fig. 2B) to the independent unit (Fig. 2A) to distance the cameras slightly further from the kelp biomass and prevent further videos from being obscured. Shoals of lesser sand eels Ammodytes spp., which can comprise several species, were included in taxa richness estimates as a single species as a conserva- tive estimate. However, as shoals of Ammodytes spp. 2.3. Fishing and stomach content analysis To sample fish for stomach content analysis, hook and line fishing was conducted concurrently with BRUV surveys, using a range of lures and baits to attract different species. Fishing of the whole water column was conducted on an ad hoc basis around camera deployments, with approximately 30 min spent in each treatment area across the survey day (~1 tidal cycle). Up to 10 fish were caught per species per survey, and fish were terminated using schedule one procedure (destruction of the brain). Fish were mea- sured (maximum standard length, fork length, maxi- mum total length and body depth) and weighed. Fish stomachs were removed and their contents preserved in 70% ethanol for later identification. Each stomach sample was subsequently rinsed in fresh water and its contents identified to coarse taxonomic group, enu- merated (if applicable, e.g. whole organisms seen) and then weighed (blotted wet weight). Stomach contents were qualitatively compared with the struc- ture of epifaunal communities found on seaweed and mussels grown at Porthallow farm in 2020 (as per Cor- rigan et al. 2023b), to tentatively determine whether prey items were sourced from farm infrastructure. In June 2020, epibiont assemblages were quantified from kelp and mussel lines in Porthallow Bay; 50 cm of each line type was sampled from 2 to 2.5 m water depth, consistent with the growing depth of both spe- cies (Corrigan et al. 2023b). Three replicate sections from each line type were collected and epifaunal indi- viduals were identified down to the finest practicable taxonomic resolution (e.g. family) and blotted wet weight biomasses for each taxonomic group were recorded (Corrigan et al. 2023b). (p ) Variability in multivariate fish assemblage structure (MaxN, MaxN–min and MaxT) between factors was also examined using a 2-way PERMANOVA and visu- alised using metric multidimensional scaling (mMDS) ordination. Multivariate assemblages were examined using the models described above, but with permuta- tions based on Bray-Curtis resemblance matrices con- structed from each metric, with either a square root or fourth root transformation applied to down-weight the influence of highly abundant fish species and different dummy variables added to each metric (detailed in Table 1). SIMPER analysis was then performed to de- termine which taxa contributed most to the observed dissimilarity between treatments and survey dates in multivariate analysis. 2.2. BRUV design, deployment and processing observed were large, frequently consisting of >200 individuals, their abundance was not included in Footage from both cameras in each BRUV unit was analysed, as different fish species and abundances were observed between the 2 cameras; data were then averaged to yield 4 replicates per treatment per Buoy Bait arm with bait box on end with 100 g mixed bait Drop weight for mooring to seabed Stabilising vein 3 m Stabilising weight Hollow PVC pipe that rotates with prevailing current 0.35 m Rope 25 m Action cameras Not to scale Not to scale Bait arm with bait box on end with 100 g mixed bait Rope Header line Stabilising weight Action cameras 3 m 0.35 m A B Fig. 2. BRUV unit setup for (A) reference and seaweed areas and (B) farm deployments between mussel cultivation lines Buoy Bait arm with bait box on end with 100 g mixed bait Drop weight for mooring to seabed Stabilising vein 3 m Stabilising weight Hollow PVC pipe that rotates with prevailing current 0.35 m Rope 25 m Action cameras Not to scale A Not to scale Bait arm with bait box on end with 100 g mixed bait Rope Header line Stabilising weight Action cameras 3 m 0.35 m B B Fig. 2. BRUV unit setup for (A) reference and seaweed areas and (B) farm deployments between mussel cultivation lines Aquacult Environ Interact 16: 145–162, 2024 150 ware (Anderson et al. 2008, Clarke & Gorley 2015). Due to the different sampling schedules at Porthallow and St Austell Bay, data from each farm were analysed separately and qualitative comparisons were then made between sites. For comparisons of fish assem- blages between treatments (mussel lines, seaweed lines and reference areas), and over the sampling sea- son, univariate assemblage metrics (total MaxN, total MaxN–min, taxa richness and total MaxT) were exa - mined using 2-way permutational analyses of vari- ance (PERMANOVA) with treatment and survey date as fixed factors. For each univariate comparison, PER- MANOVAs with permutations (999 under an unre- stricted model) were based on Euclidean distances between untransformed data with different dummy variables added to each metric (detailed in Table 1). Pairwise tests in PERMANOVA were then conducted wherever main effects or interactions were significant (p < 0.05). MaxN estimates as this would heavily skew other fish species abundance estimates. The number of shoals of Ammodytes spp. 2.3. Fishing and stomach content analysis For both the univariate and mul- tivariate metrics, differences in within-treatment vari- ability between levels of factors were also examined using the permutational dispersion (PERMDISP) rou- tine. Where within-treatment dispersion differed be- tween groups, a more conservative p-value (p <0.01) was adopted for the main PERMANOVA test for that response variable (Anderson 2017). Values presented in the text and figures are means ± SE. 3.1. BRUV surveys 3.1. BRUV surveys Across all 118 BRUV recordings, a total of 1067 indi- viduals (excluding shoals of lesser sand eels Ammo - dytes spp.), representing at least 7 fish taxa were re - corded (including Ammodytes spp. as one taxo no mic group, and excluding unidentified juveniles) (Figs. 3 2.2. BRUV design, deployment and processing per video was recorded and com- pared across survey dates and treatments; however, no statistical differences were recorded, except for a higher number of Ammodytes spp. shoals in mussel lines than seaweed lines at St Austell Bay farm on 14 June 2022 (see Table S1 in the Supplement at www. int-res.com/articles/suppl/q016p145_supp.pdf). 2.4. Statistical analysis The statistical approaches described below involve univariate and multivariate permutational analyses using the PERMANOVA add on for PRIMER v7® soft- Corrigan et al.: Finfish in mussel and seaweed farms 151 Table 1. Results of univariate (Uv) and multivariate (Mv) PERMANOVAs to test for differences in fish assemblages between treatment areas — mussel (M), seaweed (S) and reference (R)— and survey dates at Porthallow and St Austell Bay farms. Aster- isks show significant differences; where significant differences between treatment and/or date were detected, the results of pairwise post hoc tests are shown. NA: not applicable Table 1. Results of univariate (Uv) and multivariate (Mv) PERMANOVAs to test for differences in fish assemblages between treatment areas — mussel (M), seaweed (S) and reference (R)— and survey dates at Porthallow and St Austell Bay farms. Aster- isks show significant differences; where significant differences between treatment and/or date were detected, the results of pairwise post hoc tests are shown. 2.4. Statistical analysis NA: not applicable Transformation Factor PERMANOVA PERMDISP Post-hoc significance df F p F p differences between treat- ments and survey dates Porthallow Bay (Uv) total MaxN Dummy variable Treatment 2 7.08 0.006* 5.64 0.04* M & S > R added = 1 Survey date 1 1.57 0.22 0.72 0.46 NA Treatment × 2 1.26 0.29 NA NA NA survey date (Uv) Taxa richness Dummy variable Treatment 2 13.17 0.001* 3.24 0.07 M & S > R added = 1 Survey date 1 1.27 0.27 0.12 0.71 NA Treatment × 2 0.97 0.41 NA NA NA survey date (Mv) MaxN Square root Treatment 2 10.06 0.001* 34.57 0.001* M & S - R Dummy variable Survey date 1 0.97 0.45 0.74 0.31 NA added = 1 Treatment × 2 1.06 0.41 NA NA NA survey date St Austell Bay (Uv) total MaxN Dummy variable Treatment 2 9.23 0.001* 7.27 0.001* M > S & R added = 1 Survey date 2 1.75 0.17 3.89 0.19 NA Treatment × 4 2.19 0.053 NA NA NA survey date (Uv) Taxa richness Dummy variable Treatment 2 13.96 0.001* 7.15 0.004* NA added = 1 Survey date 2 7.49 0.007* 2.19 0.26 NA Treatment × 4 10.13 0.001* NA NA 3 May: no difference survey date 14 June: M > S & R 16 August: M > S (Mv) MaxN Square root Treatment 2 18.22 0.001* 7.18 0.013* NA Dummy variable Survey date 2 8.35 0.001* 0.15 0.92 NA added = 1 Treatment × 4 5.31 0.001* NA NA 3 May: all different survey date 14 June: M – S & R 16 August: M – S & R May (Fig. 4). Total fish abundance (total MaxN) ranged from 0 to ~200 per video (0.0 to 7.4–min in terms of MaxN–min), with the highest average abundance at 55.0 ± 23.9 (5.7 ± 1.2–min in terms of MaxN–min) in the mussel lines in St Austell on 16 August (Figs. 4 & S1). This high abundance value was largely driven by T. trachurus, which were present in up to 68% (~34 min) of video footage captured from the mussel lines in August, giving the highest total MaxT value of the study (Table S1, Fig. S1). In general, throughout the season, total MaxN, MaxN–min, MaxT and taxa rich- ness were greater in the mussel and unharvested sea- weed lines than the reference areas (Figs. 2.4. Statistical analysis Taxa richness per video ranged from 0 to 5 taxa, with the highest average richness of 3.0 ± 0.5 re - corded in the mussel lines in Porthallow Bay on 31 Aquacult Environ Interact 16: 145–162, 2024 152 Fig. 3. Fish species observed using BRUV, ﬁshing or both in mussel, seaweed and reference areas across all surveys (May to August). Fish illustrations are adapted from Jackson-Bué et al. (2023) with permission. Mussel illustrations are created with BioRender.com; seaweed illustrations by S. Corrigan Fig. 3. Fish species observed using BRUV, ﬁshing or both in mussel, seaweed and reference areas across all surveys (May to August). Fish illustrations are adapted from Jackson-Bué et al. (2023) with permission. Mussel illustrations are created with BioRender.com; seaweed illustrations by S. Corrigan Fig. 3. Fish species observed using BRUV, ﬁshing or both in mussel, seaweed and reference areas across all surveys (May to August). Fish illustrations are adapted from Jackson-Bué et al. (2023) with permission. Mussel illustrations are created with BioRender.com; seaweed illustrations by S. Corrigan Fig. 3. Fish species observed using BRUV, ﬁshing or both in mussel, seaweed and reference areas across all surveys (May to August). Fish illustrations are adapted from Jackson-Bué et al. (2023) with permission. Mussel illustrations are created with BioRender.com; seaweed illustrations by S. Corrigan Fig. 3. Fish species observed using BRUV, ﬁshing or both in mussel, seaweed and reference areas across all surveys (May to August). Fish illustrations are adapted from Jackson-Bué et al. (2023) with permission. Mussel illustrations are created with BioRender.com; seaweed illustrations by S. Corrigan June and August, total MaxN–min was higher in mus- sel areas than both seaweed and reference areas, with few, or no, fish seen in the harvested seaweed areas of the farm (Table S1). Taxa richness and total MaxT were also lower in the seaweed areas after harvest than the mussel lines and were instead more compa- rable to the reference areas (Figs. 4 & S1, Tables 1 & S1). S1). In St Austell Bay, fish assemblages in all treat- ment areas were distinct from one another at the start of the survey season; however, as more seaweed was harvested over the season, seaweed areas became more similar to reference areas, and only fish assem- blages associated with mussel lines remained distinct in June and August (Figs. 5 & S2, Tables 1 & S1). 2.4. Statistical analysis 4 & S1, Tables 1 & S1). There was no significant interaction be tween survey date and treatment for total MaxN at either site (Table 1). However, in St Austell Bay, total MaxN–min was initially higher in both mussel and sea- weed lines than reference areas, whereas after the seaweed had been partially or totally harvested in May (Fig. 4). Total fish abundance (total MaxN) ranged from 0 to ~200 per video (0.0 to 7.4–min in terms of MaxN–min), with the highest average abundance at 55.0 ± 23.9 (5.7 ± 1.2–min in terms of MaxN–min) in the mussel lines in St Austell on 16 August (Figs. 4 & S1). This high abundance value was largely driven by T. trachurus, which were present in up to 68% (~34 min) of video footage captured from the mussel lines in August, giving the highest total MaxT value of the study (Table S1, Fig. S1). In general, throughout the season, total MaxN, MaxN–min, MaxT and taxa rich- ness were greater in the mussel and unharvested sea- weed lines than the reference areas (Figs. 4 & S1, Tables 1 & S1). There was no significant interaction be tween survey date and treatment for total MaxN at either site (Table 1). However, in St Austell Bay, total MaxN–min was initially higher in both mussel and sea- weed lines than reference areas, whereas after the seaweed had been partially or totally harvested in & 4). Horse mackerel Trachurus trachurus were the most abundant species recorded (~45%) in terms of MaxN; however, they were almost exclusively recorded in the mussel lines in St Austell Bay, particu- larly from June onwards (Fig. 4). Thick lipped grey mullet Chelon labrosus and greater sand eel or lance Hyperoplus lanceolatus were also abundant (~20 and ~15% of total fish abundance, respectively) in both farms, but were not recorded in reference areas, and became less abundant later in the season (Fig. 4). Juvenile fish, which could not be identified to species level as they were typically <5 cm in length (hereaf- ter ‘juveniles’), contributed ~12% of fish abundance and were present in all treatment areas; how ever, they were predominantly recorded inside the farms, par- ticularly in May (Fig. 4). 2.4. Statistical analysis SIMPER analysis showed that in Porthallow Bay, dif- ferences in MaxN and MaxN–min between farmed and reference areas were due to higher abundances of H. lanceolatus, C. labrosus and unidentified juveniles (Tables S3 & S5). These species also differed in abun- dances between the mussel and seaweed lines, with The multivariate analysis revealed that in Porthal- low Bay, both mussel and seaweed treatments hosted distinct fish assemblages (in terms of MaxN, MaxN–min and MaxT) compared to the reference areas on both survey dates (Figs. 5 & S2, Tables 1 & Corrigan et al.: Finfish in mussel and seaweed farms 153 0 10 20 30 40 50 60 70 80 Porthallow_SW_6thMay22 Porthallow_M_6thMay22 Porthallow_C_6thMay22 Porthallow_SW_31stMay22 Porthallow_M_31stMay22 Porthallow_C_31stMay22 ( e c n a d n u b A MaxN) 0 10 20 30 40 50 60 70 80 StAustell_SW_3rdMay22 StAustell_M_3rdMay22 StAustell_C_3rdMay22 StAustell_SW_14thJune22 StAustell_M_14thJune22 StAustell_C_14thJune22 StAustell_SW_16thAugust22 StAustell_M_16thAugust22 StAustell_C_16thAugust22 Abundance (MaxN) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Porthallow_SW_6thMay22 Porthallow_M_6thMay22 Porthallow_C_6thMay22 Porthallow_SW_31stMay22 Porthallow_M_31stMay22 Porthallow_C_31stMay22 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% StAustell_SW_3rdMay22 StAustell_M_3rdMay22 StAustell_C_3rdMay22 StAustell_SW_14thJune22 StAustell_M_14thJune22 StAustell_C_14thJune22 StAustell_SW_16thAugust22 StAustell_M_16thAugust22 StAustell_C_16thAugust22 Contribution of each taxon to total MaxN Contribution of each taxon to total MaxN 0 0.5 1 1.5 2 2.5 3 3.5 Porthallow_SW_6thMay22 Porthallow_M_6thMay22 Porthallow_C_6thMay22 Porthallow_SW_31stMay22 Porthallow_M_31stMay22 Porthallow_C_31stMay22 s s e n h cir a x a T 0 0.5 1 1.5 2 2.5 3 3.5 StAustell_SW_3rdMay22 StAustell_M_3rdMay22 StAustell_C_3rdMay22 StAustell_SW_14thJune22 StAustell_M_14thJune22 StAustell_C_14thJune22 StAustell_SW_16thAugust22 StAustell_M_16thAugust22 StAustell_C_16thAugust22 Taxa richness Sea bream Seabass Horse Mackerel Pollock Juveniles Lance Mullet Porthallow Bay St Austell Bay 6th May 2022 31st May 2022 3rd May 2022 14th June 2022 16th August 2022 Seaweed Mussel Reference Seaweed Mussel Reference Seaweed Mussel Reference Seaweed Mussel Reference Seaweed Mussel Reference A B C D E F Seaweed Mussel Reference H. lanceolatus T. trachurus S. cantharus P. pollachius D. labrax Unid. juveniles C. labrosus Treatment area: Fish species: a a a a b b b b a a a a a b a a b ab a a a a a a b b b Seaweed partially harvested Seaweed fully harvested Fig. 4. 2.4. Statistical analysis Differences in fish assemblages between seaweed, mussel and reference areas across survey dates at Porthallow Bay farm and St Austell Bay farm in terms of (A,B) taxa richness (including Ammodytes spp); (C,D) abundance based on MaxN (the maxi- mum number of individuals of each species visible in a single frame) of all species (excluding Ammodytes spp); (E,F) percentage contribution of each taxon to total abundance (excluding Ammodytes spp). Bars indicate mean values per area (unless otherwise stated) and error bars represent SE (n=4 per survey). Significant differences between treatments on a given survey date are denoted with letters. See Fig. 3 for full species names 0 0.5 1 1.5 2 2.5 3 3.5 Porthallow_SW_6thMay22 Porthallow_M_6thMay22 Porthallow_C_6thMay22 Porthallow_SW_31stMay22 Porthallow_M_31stMay22 Porthallow_C_31stMay22 s s e n h cir a x a T Porthallow Bay 6th May 2022 31st May 2022 A a a a a b b St Austell Bay A D C E 0 Porthallow_SW_6thMay22 Porthallow_M_6thMay22 Porthallow_C_6thMay22 Porthallow_SW_31stMay22 Porthallow_M_31stMay22 Porthallow_C_31stMay22 0 StAustell_SW_3rdMay22 StAustell_M_3rdMay22 StAustell_C_3rdMay22 StAustell_SW_14thJune22 StAustell_M_14thJune22 StAustell_C_14thJune22 StAustell_SW_16thAugust22 StAustell_M_16thAugust22 StAustell_C_16thAugust22 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Porthallow_SW_6thMay22 Porthallow_M_6thMay22 Porthallow_C_6thMay22 Porthallow_SW_31stMay22 Porthallow_M_31stMay22 Porthallow_C_31stMay22 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% StAustell_SW_3rdMay22 StAustell_M_3rdMay22 StAustell_C_3rdMay22 StAustell_SW_14thJune22 StAustell_M_14thJune22 StAustell_C_14thJune22 StAustell_SW_16thAugust22 StAustell_M_16thAugust22 StAustell_C_16thAugust22 Contribution of each taxon to total MaxN Contribution of each taxon to total MaxN Horse Mackerel Pollock Juveniles Lance Mullet Seaweed Mussel Reference Seaweed Mussel Reference Seaweed Mussel Reference Seaweed Mussel Reference Seaweed Mussel Reference E F H. lanceolatus T. trachurus P. pollachius Unid. juveniles C. labrosus F Fig. 4. Differences in fish assemblages between seaweed, mussel and reference areas across survey dates at Porthallow Bay farm and St Austell Bay farm in terms of (A,B) taxa richness (including Ammodytes spp); (C,D) abundance based on MaxN (the maxi- mum number of individuals of each species visible in a single frame) of all species (excluding Ammodytes spp); (E,F) percentage contribution of each taxon to total abundance (excluding Ammodytes spp). Bars indicate mean values per area (unless otherwise stated) and error bars represent SE (n=4 per survey). Significant differences between treatments on a given survey date are denoted with letters. See Fig. 3 for full species names Fig. 4. 2.4. Statistical analysis Differences in fish assemblages between seaweed, mussel and reference areas across survey dates at Porthallow Bay farm and St Austell Bay farm in terms of (A,B) taxa richness (including Ammodytes spp); (C,D) abundance based on MaxN (the maxi- mum number of individuals of each species visible in a single frame) of all species (excluding Ammodytes spp); (E,F) percentage contribution of each taxon to total abundance (excluding Ammodytes spp). Bars indicate mean values per area (unless otherwise stated) and error bars represent SE (n=4 per survey). Significant differences between treatments on a given survey date are denoted with letters. See Fig. 3 for full species names Aquacult Environ Interact 16: 145–162, 2024 154 i –100 –50 0 50 MDS1 MDS2 MDS2 –50 0 50 2D Stress: 0.19 –60 –40 –20 0 20 40 60 MDS1 –40 –20 0 20 40 2D Stress: 0.14 A B Treatment area & survey date: Seaweed Mussel 6/05/22 Reference Seaweed Mussel 31/05/22 Reference Seaweed Mussel 03/05/22 Reference Seaweed Mussel 14/06/22 Reference Seaweed Mussel 16/08/22 Reference Treatment area & survey date: T. trachurus Juveniles D. labrax C. labrosus S. cantharus H. lanceolatus Juveniles H. lanceolatus C. labrosus P. pollachius ig. 5. Metric multidimensional scaling (MDS) plots depicting multivariate analyses of fish assemblages between seaweed lines, mussel lines and reference areas across survey dates at (A) Porthallow Bay farm and (B) St Austell Bay farm. Assemblages repre- ent MaxN (the maximum number of individuals from each species visible in a single frame) of all fish species present, excluding mmodytes spp., with data square root transformed with a dummy variable of 1 added. Both plots are ordinated based on Bray- Curtis similarity matrices of species. n = 4 for each treatment in each survey. Vectors overlaying the plots are based on Pearson’s orrelation coefficients r > 0.2 for MaxN and depict species that drive separation among treatments in fish assemblages. Dates are given as d/mo/yr MDS2 –60 –40 –20 0 20 40 60 MDS1 –40 –20 0 20 40 2D Stress: 0.14 A Treatment area & survey date: Seaweed Mussel 6/05/22 Reference Seaweed Mussel 31/05/22 Reference Juveniles H. lanceolatus C. labrosus P. pollachius –100 –50 0 50 MDS1 MDS2 –50 0 50 2D Stress: 0.19 MDS1 B Seaweed Mussel 03/05/22 Reference Seaweed Mussel 14/06/22 Reference Seaweed Mussel 16/08/22 Reference Treatment area & survey date: T. trachurus Juveniles D. labrax C. labrosus S. cantharus H. lanceolatus B Fig. 5. 2.4. Statistical analysis Metric multidimensional scaling (MDS) plots depicting multivariate analyses of fish assemblages between seaweed lines, mussel lines and reference areas across survey dates at (A) Porthallow Bay farm and (B) St Austell Bay farm. Assemblages repre- sent MaxN (the maximum number of individuals from each species visible in a single frame) of all fish species present, excluding Ammodytes spp., with data square root transformed with a dummy variable of 1 added. Both plots are ordinated based on Bray- Curtis similarity matrices of species. n = 4 for each treatment in each survey. Vectors overlaying the plots are based on Pearson’s correlation coefficients r > 0.2 for MaxN and depict species that drive separation among treatments in fish assemblages. Dates are given as d/mo/yr the season (for both Max N and MaxN–min), although only a maximum of 3 ± 1.84 juveniles were reported in control areas (Tables S4 & S6). SIMPER analysis also revealed that across both farms, juvenile abun- dances were highest in early May, and in St Austell Bay, C. labrosus abundance declined over the survey season, while T. trachurus abundances increased (for both Max N and MaxN–min) (Figs. 4 & S1, Tables S3– S6). SIMPER analysis revealed that Ammodytes spp., H. lanceolatus and C. labrosus spent more time (MaxT) in the seaweed and mussel areas than the ref- higher abundances of C. labrosus observed in the sea- weed treatment (in terms of MaxN but not MaxN–min, which was the same for both treatments) and higher abundances of H. lanceolatus and juveniles observed in the mussel lines (for both Max N and MaxN–min) (Tables S3 & S5). In St Austell Bay, higher abundances of T. trachurus and H. lanceolatus distinguished fish assemblages in the mussel treatment from those in the seaweed and reference areas, while seaweed lines hosted higher abundances of C. labrosus but lower numbers of juveniles than reference areas throughout Corrigan et al.: Finfish in mussel and seaweed farms 155 consisting primarily of crabs, fish and worms, with 1 stomach containing a whole S. scombrus tail (Figs. 6 & S3). Differences between fish species in terms of prey composition were not assessed statistically (e.g. using prey accumulation curves), due to lack of replication for some species and within-species variation in stom- ach contents in relation to fish size and survey date (Table S2). 4. DISCUSSION Of the 8 wrasse indi- viduals sampled across the surveys, there was a high proportion (~34–95%) of mussel biomass in the stom- ach contents, particularly for L. bergylta, whereas L. mixtus stomach contents were dominated by crab bio- mass (Figs. 6 & S3). The 2 analysed samples of S. stel- laris, a predatory species, confirmed a mixed diet This study presents the first comparison of fish as - semblages between farmed mussels, kelp and non- farm reference areas in the UK, which, like other European countries, has a growing cultivation indus- try for both shellfish and seaweed species (Capuzzo & McKie 2016, Hughes & Black 2016). We found that farmed mussels and/or kelp lines hosted higher taxa richness and abundances of fish species than refer- ence areas away from farmed sites. However, as sea- weed biomass was harvested through the summer, valuable fish habitats were seemingly lost, whereas cultivated mussel lines offered more temporally stable habitat due to overlapping interannual growth cycles. Stomach content analysis revealed that sev- eral of the pelagic fish species inhabiting the farm had high proportions of amphipods in their diet (~40– 90%), consistent with the species recorded at the farms. Amphipods, primarily Jassa falcata, were the dominant epibiont found on both mussel and seaweed lines, occurring in huge numbers and contributing substantial biomass to secondary production at the farm sites (Corrigan et al. 2023a,b). Indeed, in several of the videos, pelagic fish were seen feeding on am - phi pods in the farm. Demersal wrasse species caught beneath farm infrastructure had high proportions of mussels in their stomachs, suggesting that they were also feeding directly from the farm or on mussels that had been detached from lines and accumulated on the seabed. However, further research (using stable iso- topes or biomarkers, for example) is needed to con- firm prey sources and trophic linkages. High abun- dances of juvenile fish were also recorded around both seaweed and mussel lines, suggesting farms could act as potential spawning and nursery grounds; however, these juveniles could not be identified to species from videos due to their small size (<5 cm) and further investigation is needed to confirm this. In total, 89 fish (94.7%) had stomach contents that could be analysed, with the remaining 5 being empty (Figs. 6 & S3, Table S2). Of the 4 pelagic species stud- ied (H. lanceolatus, T. trachurus, P. pollachius and S. 2.4. Statistical analysis Nevertheless, qualitative comparisons of stomach contents indicated notable differences in prey composition between even closely related spe- cies (e.g. L. mixtus versus L. bergylta) and between ju - veniles and sub-adults (e.g. for P. pollachius) (Fig. S3). erence areas at both sites, and T. trachurus spent more time in the mussel lines in St Austell Bay than the sea- weed and reference areas (Table S7 & S8). Juveniles also spent longer durations of time in the seaweed and mussel areas than reference areas in Porthallow; however, in St Austell Bay, they spent longer in refer- ence areas, predominantly due to a few individuals that were attracted to the bait box during 2 deploy- ments (Tables S7 & S8). With those exceptions, how - ever, fish did not generally seem to be attracted to or interact with the bait box. 4. DISCUSSION Across fishing surveys, 94 fish were caught, repre- senting 7 different species (Fig. 6, Table S2). These were primarily H. lanceolatus, with 10 caught per sur- vey; however, during the last survey in St Austell Bay in August, none were caught. A total of 7 fish species were caught around mussel lines, 6 around seaweed lines and only 1 in reference areas, despite compa- rable fishing effort across all 3 areas (Fig. 3). Compar- ing across sampling methods, fishing captured 4 ad ditional species not recorded on BRUVs, namely Atlan tic mackerel Scomber scombrus and 3 demersal species: Ballan wrasse Labrus bergylta, Cuckoo wrasse Labrus mixtus, and nursehound or bull huss Scyliorhinus stellaris. Five species recorded in BRUV videos were not caught using fishing methods (Fig. 3). Across fishing surveys, 94 fish were caught, repre- senting 7 different species (Fig. 6, Table S2). These were primarily H. lanceolatus, with 10 caught per sur- vey; however, during the last survey in St Austell Bay in August, none were caught. A total of 7 fish species were caught around mussel lines, 6 around seaweed lines and only 1 in reference areas, despite compa- rable fishing effort across all 3 areas (Fig. 3). Compar- ing across sampling methods, fishing captured 4 ad ditional species not recorded on BRUVs, namely Atlan tic mackerel Scomber scombrus and 3 demersal species: Ballan wrasse Labrus bergylta, Cuckoo wrasse Labrus mixtus, and nursehound or bull huss Scyliorhinus stellaris. Five species recorded in BRUV videos were not caught using fishing methods (Fig. 3). In total, 89 fish (94.7%) had stomach contents that could be analysed, with the remaining 5 being empty (Figs. 6 & S3, Table S2). Of the 4 pelagic species stud- ied (H. lanceolatus, T. trachurus, P. pollachius and S. scombrus), ~40–90% of stomach content biomass averaged across the surveys constituted amphipods, primarily Jassa falcata, with the remaining majority comprising fish species (Figs. 6 & S3). J. falcata amphi pods were also the primary colonising epibiont in terms of abundance and biomass on both seaweed and mussel lines at the Porthallow farm in 2020 (Cor- rigan et al. 2023a,b) (Fig. 6), which was consistent with epibionts observed on both farms in 2022 (S. Corrigan pers. obs.). On several occasions in the BRUV footage, H. lanceolatus, T. trachurus, P. polla- chius and Ammo dytes spp. could be seen feeding on amphipods present in the farm. 4. DISCUSSION scombrus), ~40–90% of stomach content biomass averaged across the surveys constituted amphipods, primarily Jassa falcata, with the remaining majority comprising fish species (Figs. 6 & S3). J. falcata amphi pods were also the primary colonising epibiont in terms of abundance and biomass on both seaweed and mussel lines at the Porthallow farm in 2020 (Cor- rigan et al. 2023a,b) (Fig. 6), which was consistent with epibionts observed on both farms in 2022 (S. Corrigan pers. obs.). On several occasions in the BRUV footage, H. lanceolatus, T. trachurus, P. polla- chius and Ammo dytes spp. could be seen feeding on amphipods present in the farm. Of the 8 wrasse indi- viduals sampled across the surveys, there was a high proportion (~34–95%) of mussel biomass in the stom- ach contents, particularly for L. bergylta, whereas L. mixtus stomach contents were dominated by crab bio- mass (Figs. 6 & S3). The 2 analysed samples of S. stel- laris, a predatory species, confirmed a mixed diet Aquacult Environ Interact 16: 145–162, 2024 156 q , O ll hi d d h h llfi h d Th fi h i f d b i hi d 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Fish species Other Gastropod Worm Bivalve other Mussel Crab Fish Amphipod 38 11 11 19 5 3 2 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Mussel Seaweed Line type A B Stomach content biomass Epifauna biomass H. lanceolatus T. trachurus S. scombrus P. pollachius L. bergylta L. mixtus S. stellaris Fig. 6. (A) Stomach contents of fish species caught over all surveys throughout the study, presented as percentage contrib- utions of total stomach content biomass (wet weight). Number of fish sampled per species is shown at the top of each bar. See Fig. 3 for full species names. (B) Percentage contributions of total epifauna biomass (wet weight) from mussel and seaweed lines sampled at Porthallow Bay farm in 2020 (adapted from Corrigan et al. 2023b) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Fish species Other Gastropod Worm Bivalve other Mussel 38 11 11 19 5 3 2 A Stomach content biomass H. lanceolatus T. trachurus S. scombrus P. pollachius L. bergylta L. mixtus S. 4. DISCUSSION stellaris A Fish species Crab Fish Amphipod 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Mussel Seaweed Line type B Epifauna biomass H B Fig. 6. (A) Stomach contents of fish species caught over all surveys throughout the study, presented as percentage contrib- utions of total stomach content biomass (wet weight). Number of fish sampled per species is shown at the top of each bar. See Fig. 3 for full species names. (B) Percentage contributions of total epifauna biomass (wet weight) from mussel and seaweed lines sampled at Porthallow Bay farm in 2020 (adapted from Corrigan et al. 2023b) Overall, this study demonstrates that shellfish and seaweed aquaculture may provide new, valuable hab- itat for fish species in coastal environments, particu- larly in areas that lack structural habitats that attract or maintain fish populations. The fish species found to be present in this study were consistent with those expected around the southwest UK for the time of year (Naylor 2021), and with those previously recorded at seaweed and mus- sel farm sites in Europe more widely (e.g. Sheehan et Corrigan et al.: Finfish in mussel and seaweed farms 157 north east Atlantic (Svetovidov 1986, Naylor 2021) (Table S9). Juvenile P. pollachius could be identified by their characteristic 3 dorsal fins, and several mature P. pollachius juveniles were caught around the mussel lines in St Austell Bay in August (Table S2), which might suggest that the farm could be providing a nursery or spawning ground for this species. Pre- viously, there has been uncertainty as to whether shellfish and seaweed farms enhance juvenile fish re- cruitment or simply aggregate existing adult fish pop- ulations from other local areas (Gentry et al. 2020). A recent study in New Zealand successfully used stand- ard monitoring units for the recruitment of fishes (SMURFs) as temporary settlement substrates to de- termine that juvenile recruitment and diversity was equivalent to natural habitats in both mussel mono- culture and mussel and seaweed co- cultivation sites (Underwood & Jeffs 2023). In the same study, for at least the most abundant species of fish present, the aquaculture sites had sufficient epibiont food re- sources to support growth from settlement to juvenile size classes. 4. DISCUSSION This, in turn, offers a promising new in- sight into how farms may create valuable habitat for fish across different life stages, particularly in relation to soft-bottom habitats that have limited structural complexity (Underwood & Jeffs 2023). al. 2020, Fariñas-Franco et al. 2021, Bridger et al. 2022, Clarke et al. 2023). The most abundant taxa found across our surveys was Trachurus trachurus, a commercial bentho-pelagic species which has also been reported as the dominant fish species in other European mussel and seaweed farms (Tonk et al. 2019, Sheehan et al. 2020, Fariñas-Franco et al. 2021, Bridger et al. 2022). In this study, T. trachurus were mainly observed around the mussel lines, particularly later in the season once the seaweed had been har- vested, suggesting a seasonal visiting pattern that has been observed in other fish species in temperate sea- weed farms in Maine, USA (Schutt et al. 2023). On several occasions T. trachurus were observed to be feeding on amphipods associated with the mussel lines, and the stomach contents analysed from the fish caught at the farms confirmed this as their main prey source, with the most predominant amphipod, J. fal- cata, consistent with that most commonly recorded at the farms (Corrigan et al. 2023a,b). Hyperoplus lance - o latus were also abundant during the surveys; how - ever, they became less common towards the end of the survey season, with none seen or caught in the last survey in August. H. lanceolatus are commer- cially and ecologically important due to their high fat content, which makes them desirable for marine predators including mammals, seabirds and pred- atory fish, and targets for fisheries that use H. lanceo- latus for fish meal, oil production, human consump- tion and fishing bait (Frimodt & Dore 1995, Winter et al. 2023). H. lanceolatus stomach contents sampled in this study were similarly dominated by J. falcata amphi pods and small fish, often juvenile H. lanceo - latus or Ammodytes spp., also frequently reported across the surveys. Chelon labrosus was also com- monly observed in the farm BRUV footage, particu- larly in May and June; however, none were caught using hook and line methods, and therefore their stomach contents could not be analysed. 4. DISCUSSION This demon- strates how different survey techniques are biased towards recording certain species and why a combi- nation of methods should be used to more holistically survey fish assemblages at farm sites in the future (Corrigan et al. 2022). Using a wider range of survey techniques, such as fi h t DNA th d l i ( S h tt t l al. 2020, Fariñas-Franco et al. 2021, Bridger et al. 2022, Clarke et al. 2023). The most abundant taxa found across our surveys was Trachurus trachurus, a commercial bentho-pelagic species which has also been reported as the dominant fish species in other European mussel and seaweed farms (Tonk et al. 2019, Sheehan et al. 2020, Fariñas-Franco et al. 2021, Bridger et al. 2022). In this study, T. trachurus were mainly observed around the mussel lines, particularly later in the season once the seaweed had been har- vested, suggesting a seasonal visiting pattern that has been observed in other fish species in temperate sea- weed farms in Maine, USA (Schutt et al. 2023). On several occasions T. trachurus were observed to be feeding on amphipods associated with the mussel lines, and the stomach contents analysed from the fish caught at the farms confirmed this as their main prey source, with the most predominant amphipod, J. fal- cata, consistent with that most commonly recorded at the farms (Corrigan et al. 2023a,b). Hyperoplus lance - o latus were also abundant during the surveys; how - ever, they became less common towards the end of the survey season, with none seen or caught in the last survey in August. H. lanceolatus are commer- cially and ecologically important due to their high fat content, which makes them desirable for marine predators including mammals, seabirds and pred- atory fish, and targets for fisheries that use H. lanceo- latus for fish meal, oil production, human consump- tion and fishing bait (Frimodt & Dore 1995, Winter et al. 2023). H. lanceolatus stomach contents sampled in this study were similarly dominated by J. falcata amphi pods and small fish, often juvenile H. lanceo - latus or Ammodytes spp., also frequently reported across the surveys. Chelon labrosus was also com- monly observed in the farm BRUV footage, particu- larly in May and June; however, none were caught using hook and line methods, and therefore their stomach contents could not be analysed. 4. DISCUSSION Evidence as to whether seaweed and/or mussel farms aggregate fish from neighbouring habitats or vice versa could be confirmed through medium to long-term telemetry studies, following the tagging of individual fish caught in the farm and surrounding areas (Bjordal & Johnstone 1993, Otterå & Skilbrei 2014, Arechavala- Lopez et al. 2015, McPeek et al. 2015). p ) In this study, we focused mainly on pelagic fish as - semblages, as these directly interact with farmed bio- mass suspended in the water column. However, in order to more comprehensively understand farm ef - fects on fish assemblages, demersal and benthic fish also need to be quantified (e.g. via use of BRUVS de- ployed on the seabed), as the abundance and richness of fish assemblages typically varies throughout the water column and with seabed depth and habitat (Schutt et al. 2023, Underwood & Jeffs 2023). Benthic surveys will also allow for comparisons of fish assem- blages between farms and naturally occurring shell fish reefs and seaweed beds. In the present study, no de- mersal species were caught in reference areas, com- pared to several wrasse (Labrus mixtus and L. bergylta) and 2 Scyliorhinus stellaris caught inside the farms, which may suggest that demersal fish abundances were lower outside the farm; however, this needs to be quantitatively as sessed in further surveys. A benthic BRUV survey conducted previously at St Austell Bay farm found 9 fish taxa in the farm area, including lesser sand eels, gadoids, wrasse species, and grey mullet Chelon labrosus (Clarke et al. 2023), which were con- sistent with our pelagic fish findings. This previous study, how ever, found no difference in species richness or MaxN between benthic farm or reference areas, which was attributed to high variability in the data and only 2 reference areas surveyed (Clarke et al. 2023). y It is necessary to understand how shellfish and sea- weed farms affect pelagic fish species, as they form an ecologically important prey source for larger marine predators, such as seabirds, mammals and sharks, which may also be attracted to farms if they support high abundances of pelagic fish (Callier et al. 2018). 4. DISCUSSION This demon- strates how different survey techniques are biased towards recording certain species and why a combi- nation of methods should be used to more holistically survey fish assemblages at farm sites in the future (Corrigan et al. 2022). p y ( ) In China, the abundance of juvenile fish at mussel farms was also not found to differ from natural rocky reefs, although assemblages at mussel farms were structurally more similar to those found in soft- bottom habitats than those associated with artificial and natural reefs (Wang et al. 2015). This contradicts the findings from New Zealand that showed no differ- ence in juvenile assemblages between a mussel farm and surrounding natural habitats, including soft- bottom areas and shellfish reefs (Underwood & Jeffs 2023), suggesting that aquaculture–environment inter actions may vary between sites. In future, col- lecting more data on size classes and ages of fish pre- sent at aquaculture sites will help determine the importance of farm habitat for fish at different life stages. Additionally long-term surveys with appropri- ate reference areas are needed to inform more ecosys- tem-based approaches to farm management. For instance, harvesting of cultivated biomass could be planned to avoid interrupting key life stages of fish species, such as juvenile recruitment windows, which would help maximise the habitat value of cultivation sites, rather than leading to increased mortality through the untimely removal of food and shelter, and increased predation (Barrett et al. 2022, Corrigan et al. 2022, Forbes et al. 2022, Schutt et al. 2023). However, given that the farms in Porthallow Bay and Using a wider range of survey techniques, such as fish traps or eDNA methodologies (e.g. Schutt et al. 2023, Underwood & Jeffs 2023), could also help to identify the many juvenile fish that were detected in the videos, which likely comprised several different species. Several fish species identified in this study spawn during the kelp farming period (typically No- vember through June), including Pollachius polla- chius, which spawns from January to May in the Aquacult Environ Interact 16: 145–162, 2024 158 St Austell Bay are situated in close proximity to other structured habitats, including seagrass meadows and kelp beds (Crosby et al. 2023), the fish associated with these farms would have the opportunity to migrate to these surrounding natural habitats after biomass has been harvested (Fariñas-Franco et al. 2021), although this was not tested in our study. 4. DISCUSSION Evidence as to whether seaweed and/or mussel farms aggregate fish from neighbouring habitats or vice versa could be confirmed through medium to long-term telemetry studies, following the tagging of individual fish caught in the farm and surrounding areas (Bjordal & Johnstone 1993, Otterå & Skilbrei 2014, Arechavala- Lopez et al. 2015, McPeek et al. 2015). Shellfish and seaweed farms can alter the benthic environment through shading, organic enrichment and bio deposition, increases in structural complexity, changes to hydrodynamic flow and increased sed- imentation (Mascorda Cabre et al. 2021, Corrigan et al. 2022). For mussel farms in particular, if they are placed over degraded or soft-bottom habitats, mussel fall-off or shell debris from the lines and farm infra - structure on the seabed can increase structural com- plexity, making the seabed more closely resemble natural reefs (Callier et al. 2018, Mascorda Cabre et al. 2021, Bridger et al. 2022). Indeed, in the UK, resto- ration success was seen in an area of seabed pre- viously degraded due to bottom trawling fisheries after only 4 yr of an offshore long-line mussel farm being implemented, with increased abundances of macroinvertebrate and fish species compared to ref- erence areas (Bridger et al. 2022). In areas where shellfish and seaweed farms have been placed over pre-existing structurally complex habitats such as sea grass meadows, however, there is concern that farms will have a negative effect on demersal fish as - semblages, particularly in tropical off-bottom sea- weed farms (United Nations Environment Pro- gramme 2023). Nevertheless, in temperate suspended seaweed farms, negligible impacts on seagrass beds have been reported (Walls et al. 2017, Fariñas-Franco et al. 2021), so effects are site and culturing method dependent. Given the degradation of many coastal habitats and the cost of their restoration, it is impor- tant to understand how aquaculture could have low cost restorative or regenerative benefits for fish spe- cies (Theuerkauf et al. 2019, Alleway et al. 2023). St Austell Bay are situated in close proximity to other structured habitats, including seagrass meadows and kelp beds (Crosby et al. 2023), the fish associated with these farms would have the opportunity to migrate to these surrounding natural habitats after biomass has been harvested (Fariñas-Franco et al. 2021), although this was not tested in our study. 4. DISCUSSION For example, seals and other pinnipeds may be attracted to mussel farms as they occasionally consume mussels as well as crabs and fish which are associated with them (Roycroft et al. 2004). Studies from Chile and Spain have reported dol- phins feeding on fish in mussel farms (Ribeiro et al. 2007, Díaz López & Methion 2017, Methion & López 2019), and in Maine, USA, predatory harbour seals and porpoises were also recorded on pelagic and benthic cameras around seaweed farms, suggesting that they are not excluded from farm areas (Schutt et al. 2023). In New Zealand, Australia and in another study in Chile, however, dolphins have been re ported to avoid shell- fish farms due to the presence of farm infrastructure (Markowitz et al. 2004, Watson-Capps & Mann 2005, Heinrich 2006, Pearson et al. 2012). It is therefore im- portant to understand how aquaculture may change natural fish assemblages and distributions to ensure farms are not displacing larger predators that depend on them (Callier et al. 2018). Porthallow and St Austell Bay farms supported sev- eral commercially important fish species, such as P. pollachius and S. scombrus, which are commonly targeted in both recreational and commercial fish- eries. Surrounding reference areas were less pop- ulated with commercial species, and additional spa- tial sampling is required in future to better resolve and quantify spill-over. Previous studies in Indonesia, Malaysia and the Philippines found a positive correla- tion between landings of herbivorous reef fishes and seaweed production at a national scale (Hehre & Meeuwig 2016), highlighting how large-scale low- trophic farming could support fisheries. It is unlikely that the small-scale shellfish and seaweed farms cur- rently in operation in Europe affect fisheries landings; however, as the footprint of cultivation in Europe in - creases in the future (Capuzzo & McKie 2016, Hughes & Black 2016), they may come to do so and studies are needed to investigate how fish from farms may con- tribute to local and regional fish stocks. servation and commercial im portance, and are listed as Near Threatened in Europe and Vulnerable in the Mediterranean on the IUCN Red List (Finucci et al. 2021). The stomach contents of both S. stellaris were typical of their normal predatory feeding patterns (Table S9), containing high proportions of crabs, fish and worms, with one sample containing a whole Scomber scombrus tail. S. 4. DISCUSSION scombrus are also an impor- tant commercial species and were themselves frequently caught around the farms. Their stomach contents consisted primarily of amphipods and smaller fish (including lesser sand eels also seen at the farms). This small insight into one aspect of the many potential trophic interactions at the farm highlights why further studies are needed to confirm wider trophic dynamics surrounding aquaculture systems and how they might have knock-on implications for broader ecosystem functioning. Previous research has assessed how other high trophic level species may be affected by farms, particularly in terms of food provisioning (Callier et al. 2018, Corrigan et al. 2022). For example, seals and other pinnipeds may be attracted to mussel farms as they occasionally consume mussels as well as crabs and fish which are associated with them (Roycroft et al. 2004). Studies from Chile and Spain have reported dol- phins feeding on fish in mussel farms (Ribeiro et al. 2007, Díaz López & Methion 2017, Methion & López 2019), and in Maine, USA, predatory harbour seals and porpoises were also recorded on pelagic and benthic cameras around seaweed farms, suggesting that they are not excluded from farm areas (Schutt et al. 2023). In New Zealand, Australia and in another study in Chile, however, dolphins have been re ported to avoid shell- fish farms due to the presence of farm infrastructure (Markowitz et al. 2004, Watson-Capps & Mann 2005, Heinrich 2006, Pearson et al. 2012). It is therefore im- portant to understand how aquaculture may change natural fish assemblages and distributions to ensure farms are not displacing larger predators that depend on them (Callier et al. 2018). 4. DISCUSSION At both farms in this study, fish from varying trophic levels with different feeding patterns were recorded (Table S9), indicating that shellfish and seaweed farms may interact with or attract a variety of fish species and functional groups, rather than just the herbi vorous species that have been the focus of previous studies conducted at tropical seaweed farms (Eklöf et al. 2006, Hehre & Meeuwig 2015, 2016, Mirera et al. 2020). In a mussel farm in Croatia, it was re ported that some pred- atory fish were present be cause they prey upon other smaller fish present at the farm (Šegvić-Bubić et al. 2011). In our study, 2 S. stellaris were caught below the seaweed and mussel lines in St Austell Bay. These are high-trophic level demersal predatory sharks of con- In 4 seaweed farms in Maine, USA, there were also no observable differences in benthic fish and inverte- brate assemblages between farms and reference areas, which were dominated by crab and lobster spe- cies (Schutt et al. 2023). These authors did however report differences in species assemblages between ben thic and pelagic areas within farms. This suggests that farms offer different habitat spatially between the benthos and the water column, with benthic habitats below farms potentially offering greater stability and similarity to reference sites than pelagic areas. Corrigan et al.: Finfish in mussel and seaweed farms 159 servation and commercial im portance, and are listed as Near Threatened in Europe and Vulnerable in the Mediterranean on the IUCN Red List (Finucci et al. 2021). The stomach contents of both S. stellaris were typical of their normal predatory feeding patterns (Table S9), containing high proportions of crabs, fish and worms, with one sample containing a whole Scomber scombrus tail. S. scombrus are also an impor- tant commercial species and were themselves frequently caught around the farms. Their stomach contents consisted primarily of amphipods and smaller fish (including lesser sand eels also seen at the farms). This small insight into one aspect of the many potential trophic interactions at the farm highlights why further studies are needed to confirm wider trophic dynamics surrounding aquaculture systems and how they might have knock-on implications for broader ecosystem functioning. Previous research has assessed how other high trophic level species may be affected by farms, particularly in terms of food provisioning (Callier et al. 2018, Corrigan et al. 2022). 5. CONCLUSION Front Ecol Environ 14: 424– 432 and ecosystem services these low-trophic aquacul- ture systems provide will help facilitate expansion of the industry and farmers should be encouraged to maintain habitat provisioning and increase environ- mental stewardship at their sites. Bicknell AWJ, Sheehan EV, Godley BJ, Doherty PD, Witt MJ (2019) Assessing the impact of introduced infrastructure at sea with cameras: a case study for spatial scale, time and statistical power. Mar Environ Res 147: 126– 137 Å and statistical power. Mar Environ Res 147: 126– 137 Å Acknowledgements. We thank Westcountry Mussels of Fowey, the Cornish Seaweed Company and BIOME Algae for their collaboration and access to their farm sites. We also thank Lily Burnet, Faith Gibbs, Margot Aubin, Shauna Corr, Catherine Wilding, Maxine Canvin and Emma Stuart from the Marine Biological Association, the Universities of Ply mouth and Exeter, and Plymouth Marine Laboratory for their help in data collection and sample processing. This research was funded by the Worshipful Company of Fish- mongers, Centre for Environment, Fisheries and Aquacul- ture Science (Cefas); the Marine Biological Association, Research Council UK (RCUK) (through the Aquaculture Research Collaborative Hub —UK), South West Partner- ship for Environmental and Economic Prosperity (SWEEP) (NE/P011217/1) and the University of Exeter. D.A.S. was supported by a UK Re search and Innovation (UKRI) Future Leaders Fellowship (MR/S032827/1). The APC was funded by UKRI. For the purpose of open access, the authors have ap plied a Creative Commons attribution (CC BY) licence to any author ac cepted manuscript version arising. The authors declare no competing interests. All animal work was carried out humanely and in accordance with the Uni- versity of Exeter Ethical Approvals Committee. The data- sets generated during and analysed during the present study are available from the corresponding author on rea- sonable request. Bjordal Å, Johnstone AD (1993) Local movements of saithe (Pollachius virens L.) in the vicinity of fish farm cages. ICES Mar Sci Symp 196: 143– 146 Bridger D, Attrill MJ, Davies BFR, Holmes LA and others (2022) The restoration potential of offshore mussel farming on degraded seabed habitat. Aquac Fish Fish 2: 437– 449 g q Callier MD, Byron CJ, Bengtson DA, Cranford PJ and others (2018) Attraction and repulsion of mobile wild organisms to finfish and shellfish aquaculture: a review. 5. CONCLUSION Rev Aqua- cult 10: 924– 949 Campbell I, Macleod A, Sahlmann C, Neves L and others (2019) The environmental risks associated with the devel- opment of seaweed farming in Europe — prioritizing key knowledge gaps. Front Mar Sci 6: 107 Cappo M, Harvey BE, Malcolm H, Speare P (2003) Potential of video techniques to monitor diversity, abundance and size of fish in studies of marine protected areas. In: Beumer J, Grant A, Smith D (eds) Aquatic protected areas. What works best and how do we know? University of Queensland, Cairns, p 455– 464 Capuzzo E, McKie T (2016) Seaweed in the UK and abroad — status, products, limitations, gaps and Cefas role. Cefas contract report FC0021. Centre for Environment, Fisheries & Aquaculture Science, Suffolk Clarke KR, Gorley RN (2015) Getting started with PRIMER v7. PRIMER-E, Plymouth Clarke LJ, Griffin RA, Domoney E, Smith HCM and others (2023) Low-impact rearing of a commercially valuable shellfish: sea-based container culture of European lobster Homarus gammarus in the United Kingdom. Aquacult Environ Interact 15: 215– 230 5. CONCLUSION Our results demonstrate that shellfish and seaweed farms located in southwest UK provide valuable hab- itats that support a high abundance and richness of coastal fish species, many of which are of commercial and/or ecological importance. It is likely that these farming structures provide shelter, feeding, breeding and/or nursery grounds; however, further investiga- tion is needed to confirm which of these are provi- sioned for different fish species and how this com- pares to natural kelp and shellfish beds. For nearshore pelagic environments, which are often degraded and lack any structural complexity in the water column, low-trophic farms may enhance fish numbers and help to regenerate fish populations, especially where they have been depleted. The habitat provisioning for seaweed cultivation lines, however, is markedly af - fected by harvesting, with dramatic declines in fish abundance and richness subsequent to crop removal highlighting that farms are not a replacement for nat- ural kelp habitats. In contrast, mussels that were grown in continuous overlapping cycles at the study farms provided temporally stable habitat for fish spe- cies for the duration of this study. Looking ahead, greater recognition by legislative and governmental bodies on the potential environmental benefits of low- trophic aquaculture sites is needed, together with the provision of financial subsidies to support their de - velopment as sustainable food production systems. Greater understanding of the environmental benefits Shellfish and seaweed farms may also displace fish- ing activity from an area, acting as de facto ‘no take zones’ (Wang et al. 2015), as the structure of longline mussel and seaweed farms ensures that fishermen cannot fish safely within the farm (Bridger et al. 2022). For instance, in China, the age structure of fish pop- ulations associated with caged fish farms was more di - verse than those sampled in reference areas, likely due to limited fishing in the area (Wang et al. 2015). Instead of fishing directly in the farm, fisheries may benefit from ‘spill-over’ effects, where an increase in fish abundance at farm sites spreads into surrounding fishing grounds (Bridger et al. 2022). We found that Aquacult Environ Interact 16: 145–162, 2024 160 Bicknell AWJ, Godley BJ, Sheehan EV, Votier SC, Witt MJ (2016) Camera technology for monitoring marine biodi- versity and human impact. 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https://openalex.org/W4365512349	https://journals.gen.tr/index.php/jsp/article/download/1886/1358	English	null	The effects of monocyte/high-density lipoprotein ratio on hospital stay in patients with mild acute pancreatitis	Health sciences quarterly	2,023	cc-by	3,156	Health Sciences Quarterly, Volume: 3 / Issue: 2 / Year: 2023 Volume: 3 Issue: 2 2023 E-ISSN: 2791-6022 https://journals.gen.tr/jsp Received: 2022-09-28 Accepted: 2023-01-17 Health Sciences Quarterly, Volume: 3 / Issue: 2 / Year: 2023 Volume: 3 Issue: 2 2023 E-ISSN: 2791-6022 https://journals.gen.tr/jsp Received: 2022-09-28 Accepted: 2023-01-17 ORIGINAL ARTICLE ORIGINAL ARTICLE The effects of monocyte/high-density lipoprotein ratio on hospital stay in patients with mild acute pancreatitis Alper Sarı1 Haydar Güngören1 Elif Dizen Kazan1 Semiha Orhan2 Mustafa Duran3 Sevnur Aysal Sarı4 Erhan Bozkurt1 Elif Dizen Kazan1 Sevnur Aysal Sarı4 1 Department of Internal Medical Sciences, Faculty of Medicine, Afyonkarahisar Health Sciences University. Afyonkarahisar / Türkiye rtment of Internal Medical Sciences, Faculty of Medicine, Afyonkarahisar Health Sciences University. Afyonkarahisar / 2 Intensive Care Unit, Faculty of Medicine, Afyonkarahisar Health Sciences University. Afyonkarahisar / Türkiye 3 Department of Hematology, Faculty of Medicine, Afyonkarahisar Health Sciences University. Afyonkarahisar / Türkiye 4 Department of Anesthesiology and Reanimation, Afyonkarahisar State Hospital. Afyonkarahisar / Türkiye Citation: Sarı A, Güngören H, Dizen Kazan E, Orhan S, Duran M, Aysal Sarı S, et al. The effects of monocyte/high-density lipoprotein ratio on hospital stay in patients with mild acute pancreatitis. Health Sci Q. 2023;3(2):69-74. https://doi.org/10.26900/hsq.1886 This work is licensed under a Creative Commons Attribution 4.0 International License. Corresponding Author: Alper Sarı Email: alpersari_@hotmail.com Introduction Acute pancreatitis progresses with cellular damage and inflammation of the pancreas; It is a condition that can lead to local and systemic complications. It can be in a self-limiting mild edematous form; It can show a wide clinical course from severe form with multi-organ involvement that can lead to local and systemic complications [1]. Predicting the severity of acute pancreatitis and the prognosis of the disease contributes to the reduction of disease- related morbidity and mortality. Many scoring systems such as Ranson, APACHE II, Balthazar, Glasgow are used to determine the severity of acute pancreatitis. Ranson scoring is one of the important scoring methods that has been used for many years and gives information about the mortality rate [2]. Abstract In this study, we aimed to evaluate the effect of monocyte/high-density lipoprotein (MHR), which is associated with systemic inflammation: on prolonged hospitalization in patients with mild acute pancreatitis. Patients hospitalized for acute edematous pancreatitis between 01.01.2021 and 31.12.2021 were retrospectively screened. Arrival Ranson scores of the patients were calculated. Those with a Ranson score <3 were considered as mild acute pancreatitis and were included in the study. Patients with mild acute pancreatitis were divided into 2 groups with a hospital stay of <8 days and ≥8 days. Monocyte/HDL, biochemical and metabolic parameters were compared between the groups. The study was conducted with a total of 39 patients, 23 male (59%) and 16 female (41%). While 28 (71.8%) of the patients were discharged within <8 days, 11 (28.2%) were hospitalized for ≥8 days. Group ≥8 days was considered as prolonged hospitalization. It was determined that the monocyte/HDL ratio was significantly higher in the group with prolonged hospitalization between the groups. In univariate analyzes, it was determined that the presence of Diabetes Mellitus and MHR increased the risk of prolonged hospitalization by 5.25 times and 1.085 times, respectively. In the multivariate analysis of these two parameters, MHR was found to be an independent risk factor for prolonged hospitalization. It was concluded that monocyte/HDL can be used as a simple and reliable parameter to predict the duration of hospitalization in patients with mild acute pancreatitis. Keywords: Acute pancreatitis, monocyte/high-density lipoprotein ratio, prolonged hospital stay This work is licensed under a Creative Commons Attribution 4.0 International License. Corresponding Author: Alper Sarı Email: alpersari_@hotmail.com 69 Sarı et al. literature, on the duration of hospitalization in acute pancreatitis. The aim of this study is to evaluate the effect of monocyte/HDL on prolonged hospitalization in acute pancreatitis. Materials and Methods Patients hospitalized with acute edematous pancreatitis between 01.01.2021-31.12.2021 in Afyonkarahisar Health Sciences University Faculty of Medicine Hospital Internal Diseases Clinic were retrospectively scanned from the hospital electronic file system. Hospitalization Ranson score of these patients; age >70 years, white blood cell (WBC) >18000 mm3, glucose >220 mg/dL, lactate dehydrogenase (LDH) >400 U/L, aspartate aminotransferase (AST) >250 U/L parameters. Patients with a Ranson score below 3 were considered as mild pancreatitis and were included in the study. The patients included in the study were divided into two groups with a hospital stay of <8 days and ≥8 days. Monocyte/ HDL and other parameters were compared between these two groups. Monocytes are cells that have an important role in cytokine synthesis. High-density lipoprotein (HDL) has an anti-inflammatory and antioxidant effect and has the effect of protecting the endothelium. The monocyte/HDL ratio has been widely used in recent years and has been shown as a new marker of inflammation(3). dated 04.02.2022 and numbered 2022/85. In the univariate analyzes, it was found that the presence of DM and MHR increased the risk of prolonged hospitalization by 5.25 times and 1.085 times, respectively. In the multivariate analysis of these two parameters, MHR was found to be an independent risk factor for prolonged hospitalization. Table 3 shows the regression analysis to identify risk factors for prolonged hospitalization. Results The study was conducted with a total of 39 patients, 23 male (59%) and 16 female (41%). The mean age of the study group was 53.1±20.2 years. While 28 (71.8%) of the patients were discharged within <8 days, 11 (28.2%) were hospitalized for ≥8 days. When the groups were compared in terms of demographic characteristics, comorbidities and the drugs they used, it was found that diabetes was significantly more common in patients with prolonged hospitalization. The general characteristics of the patients, their comorbidities and the comparison of the drugs they use are presented in Table 1. Statistical Analysis SPSS 26.0 package program was used for statistical analysis. Categorical variables were presented as percentages and frequencies. Continuous variables were expressed as mean±standard deviation. Conformity of continuous variables to normal distribution was checked with the Kolmogorov Smirnov test. Chi-square test was used to compare categorical variables between groups. Continuous variables were compared with Mann Whitney U test or independent sample t test. Logistic regression analysis was used to identify risk factors for prolonged hospitalization. Parameters with significant differences between groups and parameters with p<0.2 in comparison between groups were included in the logistic regression. Parameters found to be risk factors for prolonged hospitalization in univariate logistic regression analysis were included in the multivariate analysis. All the p values presented were bidirectional and the values with p<0.05 were expressed as statistically significant. Average length of stay in hospital in acute pancreatitis; It was determined as 4 days in mild severe forms and 9 days in severe pancreatitis [4]. Hospitalizations of 8 days or more are considered as prolonged hospitalization. In previous studies, the effects of diuretic, Angiotensin converting enzyme inhibitor (ACE) / Angiotensin receptor blocker (ARB) use, congestive heart failure, creatinine elevation, and fluid replacement given in the emergency department on the duration of hospitalization in patients with acute pancreatitis were investigated [5,6335 were included in the analysis. Baseline characteristics, determined by vital signs and laboratory parameters, were similar between the short and long hospitalization groups. However, the long hospitalization group received more intravenous crystalloid in the ED, and this group used more diuretics and more angiotensin- converting enzyme inhibitor and angiotensin- receptor blocker (ACEI/ARB]. There is no study evaluating the effect of monocyte/HDL, which is shown as a new inflammation marker in the This study was carried out according to the decision of Afyonkarahisar Health Sciences University Clinical Research Ethics Committee 70 Health Sciences Quarterly, Volume: 3 / Issue: 2 / Year: 2023 Fisher’s exact test Discussion Acute pancreatitis; it is one of the major reasons for hospitalization. With the various scoring systems used, the disease is classified according to its severity (mild, moderate, severe) and information about the prognosis can be obtained. In a community-based study in England, the average hospital stay for mild pancreatitis was found to be 4 days [4]. In some mild pancreatitis, this length of stay is much longer (≥8 days). The factors causing prolonged hospitalization in patients with mild pancreatitis have been tried to be revealed by previous studies. In this study, we aimed to investigate the effect of monocyte/HDL, which is used as a new generation inflammation istics comorbidities and the drugs they use When the groups were compared in terms of laboratory parameters, it was found that leukocytosis was more prominent in the group with prolonged hospitalization and the monocyte/HDL cholesterol ratio was found to be significantly higher. Comparisons of the patients in terms of laboratory parameters are presented in Table 2. Table 1. Comparison of the patients’ characteristics, comorbidities and the drugs they use. Feature Total (n= 39) <8 days (n= 28) ≥8 days (n= 11) p Age (mean±SD) 53,1±20,2 50,1±20,6 60,7±17,7 0,143 Male gender (%-n) 59-23 57,1-16 63,6-7 1 Ranson (mean±SD) 1±1,05 0,86±1,04 1,36±1,03 0,137 Diabetes mellitus (%-n) 35,9-14 25-7 63,6-7 0,033* Hypertension (%-n) 38,5-15 39,3-11 36,4-4 1* Coronary artery disease (%-n) 10,3-4 7,1-2 18,2-2 0,562* Heart failure (%-n) 7,7-3 3,6-1 18,2-2 0,187* Chronic renal failure (%-n) 5,1-2 0 18,2-2 0,074* ACE inhibitor (%-n) 20,5-8 14,3-4 36,4-4 0,188* ARB (%-n) 7,7-3 10,7-3 0 0,545* Diuretic (%-n) 23,1-9 17,9-5 36,4-4 0,238* Calcium channel blocker (%-n) 20,5-8 21,4-6 18,2-2 1* Metformin 17,9-7 14,3-4 27,3-3 0,379* DPP-4 inhibitor (%-n) 10,3-4 10,7-3 9,1-1 1* Insulin (%-n) 17,9-7 10,7-3 36,4-4 0,083* Acetyl salicylic acid (%-n) 12,8-5 7,1-2 27,3-3 0,125 *Fisher’s exact test 71 Sarı et al. Sarı et al. marker, on prolonged hospitalization in patients with mild pancreatitis. patients on prolonged hospitalization could not be demonstrated. The most important risk factor for prolonged hospitalization has been shown to be persistence of pancreatitis-related symptoms and oral intolerance [6a subset of mild acute pancreatitis (AP]. In 11 (28%) of 39 patients in our study, hospitalization was prolonged and, similar to this study, the comorbidities of the patients were found to be similar between both f l b Harkirat et al. Discussion In an article in which they evaluated the factors affecting prolonged hospitalization in patients with mild pancreatitis; prolonged hospitalization was observed in 20% of the patient group. Again, in this study, the effect of age, gender, and comorbidities of the Table 2. Comparison of the patients in terms of laboratory parameters. Parameter Total (n= 39) <8 days (n= 28) ≥8 days (n= 11) p Leukocytes (x109/uL) 11520±4260 11170±4013 12241±4929 0,031 Hemoglobin (gr/dL) 14,08±1,94 14,31±1,72 13,6±2,36 0,432 Platelets (x109/uL) 225,83±69,6 222,1±57,1 233,4±93,8 0,747 Amylase (U/L) 629,3±639,5 660,3±687,8 567,3±568,8 0,612 Lipase (U/L) 1455,8±2126,6 1610,7±2520,4 1146±1040,4 0,433 BUN (mg/dL) 18,27±26,3 18,86±32,1 17,1±7,6 0,567 AST (U/L) 79,9±222,3 105,6±271,2 28,6±16,2 0,842 Lactate dehydrogenase (U/L) 347,5±245,8 377,7±286,6 287,1±112,2 0,701 Calcium (mg/dL) 8,84±0,46 8,84±0,5 8,85±0,3 0,346 Glucose (mg/dL) 133,2±56,1 120,5±45,3 158,3±69,8 0,221 CRP (mg/L) 10,6±19,77 12,7±23,8 8,41±5,8 0,483 Absolute monocytes (x109/uL) 832,5±34,6 702,5±26,6 1092,5±355,2 <0,001 HDL cholesterol (mg/dL) 35,52±13,7 36,1±12,3 34,56±16,9 0,958 Monocyte/HDL ratio 26,73±15,2 21,28±10,9 37,63±17,1 0,001 In the univariate analyzes, it was found that the presence of DM and MHR increased the risk of Table 3. The results of the regression analysis performed to determine the risk factors for prolonged hospitalization. Table 3. The results of the regression analysis performed to determine the risk factors for prolonged hospitalization. Table 2. Comparison of the patients in terms of laboratory parameters. In the univariate analyzes, it was found that the presence of DM and MHR increased the risk of Table 3. The results of the regression analysis performed to determine the risk factors for prolonged hospitalization. prolonged hospitalization. prolonged hospitalization by 5.25 times and 1.085 times, respectively. In the multivariate analysis of these two parameters, MHR was found to be an independent risk factor for prolonged hospitalization. Table 3 shows the regression analysis to identify risk factors for prolonged hospitalization. Univariate analysis Multivariate analysis Parameter RR (%95 GA) p RR (%95 GA) p Age 1,028 (0,990-1,067) 0,146 Ranson 1,588 (0,809-3,119) 0,179 DM 5,250 (1,175-23,457) 0,03 3,896 (0,639-23,766) 0,141 Heart failure 6,000 (0,485-74,289) 0,163 KRF 50,2 (0-502,592) 0,999 ACE inh. Discussion 3,429 (0,677-17,353) 0,136 Insulin 4,762 (0,856-26,476) 0,075 ASA 4,875 (0,689-34,499) 0,113 Monocyte/HDL 1,085 (1,019-1,157) 0,011 1,082 (1,012-1,158) 0,021 DM: diyabetes mellitus, KRF: chronic renal failure ACE inh.: anjiyotensin converting enzym inhibitörs, DM: diyabetes mellitus, KRF: chronic renal failure ACE inh.: anjiyotensin converting enzym inhibitörs, ASA:acethylsalicylic acid DM: diyabetes mellitus, KRF: chronic renal failure ACE inh.: anjiyotensin converting enzym inhibitörs, DM: diyabetes mellitus, KRF: chronic renal failure ACE inh.: anjiyotensin converting enzym inhibitörs, ASA:acethylsalicylic acid 72 Health Sciences Quarterly, Volume: 3 / Issue: 2 / Year: 2023 shown to be closely related to the presence and prognosis of some cardiovascular diseases [10,11]. In our study, in the patient group with prolonged hospitalization; monocyte/HDL was found to be statistically higher than the other group. In the regression analysis, it was seen that monocyte/HDL is an independent risk factor over the length of hospital stay in acute pancreatitis. This can be explained by the fact that monocytes play an active role in inflammation, an increase in the number of monocytes is expected, and HDL is an antioxidant type of cholesterol. Peng et al. in a study in which they measured HDL and apolipoprotein A-1 levels in patients with severe acute pancreatitis, they found that HDL level showed an inverse correlation with the severity of pancreatitis. In the same study, HDL was also shown to be low in patients with organ damage in acute pancreatitis [12]. In our study, HDL levels were similar between groups. This situation can be explained by mild pancreatitis in both groups. patient groups. Although it differed between diabetes patient groups, it was not found to be an independent risk factor in multivariate analysis. In a study by Koçkan et al. investigating the determinants of prolonged hospitalization in patients with mild to moderate pancreatitis who applied to the emergency department, the use of diuretics was found to be higher in the group hospitalized long. Liver function tests and electrolytes were similar in both groups [5335 were included in the analysis. Baseline characteristics, determined by vital signs and laboratory parameters, were similar between the short and long hospitalization groups. However, the long hospitalization group received more intravenous crystalloid in the ED, and this group used more diuretics and more angiotensin- converting enzyme inhibitor and angiotensin- receptor blocker (ACEI/ARB]. Unlike in our study, no effect of diuretic use on hospital stay was observed. Funding No support of grants was used for the purpose of this research. Conflict of Interest No potential conflict of interest was reported by the authors. No potential conflict of interest was reported by the authors. Conclusion In conclusion, in mild acute pancreatitis, Monocyte/HDL may be helpful in predicting the length of stay in the hospital. There is a need for comprehensive prospective studies with more patient participation on this subject. Discussion Similar to this study, liver function tests and electrolytes were similar between the two groups. The retrospective planning of our study, the small number of patients, and the evaluation of pancreatitis severity only by Ranson score are among the limitations of our study. In recent years, many articles aiming to show the relationship between hematological inflammation parameters and the severity of acute pancreatitis have been published [7,8]. Kaya et al. in a study conducted by 418 acute pancreatitis patients admitted to the emergency department were evaluated. In this study, the Ranson score and the platelet lymphocyte ratio (PLR) ratio at the time of admission to the emergency department were compared and it was shown that PLR is an independent factor in predicting the severity of the disease [7]. Han et al. in a study conducted by; A positive correlation was found between the neutrophil lymphocyte ratio (NLR), another inflammation parameter, and Ranson criteria [9and theirs utility for predicting severity of AP was evaluated by receiver operator characteristic (ROC]. In a study by Önmez et al., NLR was shown to be an independent risk factor in showing the severity of acute pancreatitis [8]. 1. Ignatavicius P, Gulla A, Cernauskis K, Barauskas G, Dambrauskas Z. How severe is moderately severe acute pancreatitis? Clinical validation of revised 2012 Atlanta Classification. World J Gastroenterol. 2017;23(43):7785–90. doi: 10.3748/ References 1. Ignatavicius P, Gulla A, Cernauskis K, Barauskas G, Dambrauskas Z. How severe is moderately severe acute pancreatitis? Clinical validation of revised 2012 Atlanta Classification. World J Gastroenterol. 2017;23(43):7785–90. doi: 10.3748/ Monocyte/HDL is one of the new inflammation parameters frequently used in recent years [3]. In many previous studies, it can be a marker of inflammation and oxidative stress; it has been 73 Sarı et al. Fang JT, et al. Serum levels of apolipoprotein A-I and high-density lipoprotein can predict organ failure in acute pancreatitis. Crit Care. 2015;19(1):1-9. doi: 10.1186/s13054-015-0832-x. Fang JT, et al. Serum levels of apolipoprotein A-I and high-density lipoprotein can predict organ failure in acute pancreatitis. Crit Care. 2015;19(1):1-9. doi: 10.1186/s13054-015-0832-x. 12. Peng YS, Chen YC, Tian YC, Yang CW, Lien JM, Fang JT, et al. Serum levels of apolipoprotein A-I and high-density lipoprotein can predict organ failure in acute pancreatitis. Crit Care. 2015;19(1):1-9. doi: 10.1186/s13054-015-0832-x. wjg.v23.i43.7785. 2. Pitchumoni CS, Patel NM, Shah P. Factors influencing mortality in acute pancreatitis: Can we alter them? J Clin Gastroenterol. 2005;39(9):798- 814. doi: 10.1097/01.mcg.0000177257.87939.00. 3. Sari A, Ulu Ms, Kazan S, Tunca O, Kazan Ed. Comparison of monocyte / HDL ratio in routine hemodialysis and peritoneal dialysis patients. Dicle Tıp Derg. 2020;47:139-7. doi: 10.5798/ dicletip.706097. 4. PanWessex Study Group; Wessex Surgical Trainee Research Collaborative, Mirnezami A, Knight B, Moran B, Noble F, Branagan G, Primrose J, Pearson K, West M, Curtis N, Pucher P, Cuttress R, Pugh S, Underwood T. Population-based observational study of acute pancreatitis in southern England. Ann R Coll Surg Engl. 2019;101(7):487-94. doi: 10.1308/rcsann.2019.0055. 5. Koçkan E, Doğan NÖ, Pekdemir M, Yılmaz S, Yaka E. Predictors of prolonged hospitalization in patients with mild and moderate pancreatitis presenting to the emergency department. Medizinische Klin - Intensivmed und Notfallmedizin. 2022;117(6):459-65. doi: 10.1007/ s00063-021-00855-8. 6. Singh H, Gougol A, Mounzer R, Yadav D, Koutroumpakis E, Slivka A, et al. Which Patients with mild acute pancreatitis require prolonged hospitalization? Clin Transl Gastroenterol. 2017;8(12):E129. doi: 10.1038/ctg.2017.55. 7. Kaya Y, Düğeroğlu H, Çınar H. Akut pankreatitin şiddeti ile platelet lenfosit oranı arasındaki ilişki. FÜ Sağ Bil Tıp Derg. 2017;31(3):131-41. 8. Önmez A, Bilir E, Torun S. Akut pankreatit şiddeti ile trombosit lenfosit oranı, nötrofil lenfosit oranı, eritrosit dağılım genişliği ve ortalama platelet volümü arasındaki ilişki. Konuralp Tıp Derg. 2019;11(1):24-9. doi:10.18521/ktd.492274. 9. Han C, Zeng J, Lin R, Liu J, Qian W, Ding Z, et al. The utility of neutrophil to lymphocyte ratio and fluid sequestration as an early predictor of severe acute pancreatitis. Sci Rep. 2017;7(1):1-8. doi: 10.1038/s41598-017-10516-6. 10. Kanbay M, Solak Y, Unal HU, Kurt YG, Gok M, Cetinkaya H, et al. Monocyte count/HDL cholesterol ratio and cardiovascular events in patients with chronic kidney disease. Int Urol Nephrol. 2014;46(8):1619-25. doi: 10.1007/s11255- 014-0730-1. 11. Ucar FM. A potential marker of bare metal stent restenosis: Monocyte count - to- HDL cholesterol ratio. BMC Cardiovasc Disord. 2016;16(1):1-7. doi: 10.1186/s12872-016-0367-3. 12. Peng YS, Chen YC, Tian YC, Yang CW, Lien JM, 74
https://openalex.org/W2544131842	https://www.scielo.br/j/jaos/a/BSsFqS5GcW5w3DspKtVqdzv/?lang=en&format=pdf	English	null	Evaluation of the force generated by gradual deflection of orthodontic wires in conventional metallic, esthetic, and self-ligating brackets	Journal of Applied Oral Science	2,016	cc-by	4,066	www.scielo.br/jaos www.scielo.br/jaos http://dx.doi.org/10.1590/1678-775720150405 Evaluation of the force generated by gradual GHÀHFWLRQ RI RUWKRGRQWLF ZLUHV LQ FRQYHQWLRQDO metallic, esthetic, and self-ligating brackets ABSTRACT O EMHFWLYH7KHSXUSRVHRIWKLVVWXG\ZDVWRHYDOXDWHWKHGHÀHFWLRQIRUFHVRI1LWLQRO orthodontic wires placed in different types of brackets: metallic, reinforced polycarbonate with metallic slots, sapphire, passive and active self-ligating, by assessing strength values YDULDWLRQDFFRUGLQJWRJUDGXDOLQFUHDVHLQZLUHGLDPHWHUDQGGHÀHFWLRQDQGFRPSDULQJ GLIIHUHQWFRPELQDWLRQVLQWKHGLIIHUHQWGHÀHFWLRQV0DWHULDODQG0HWKRGV6SHFLPHQVZHUH set in a clinical simulation model and evaluated in a Universal Testing Machine (INSTRON 3342), using the ISO 15841 protocol. Data were subjected to One-way ANOVA, followed by Tukey tests (p<0.05). Results: Self-ligating brackets presented the most similar behavior to each other. For conventional brackets there was no consistent behavior for any of the GHÀHFWLRQVVWXGLHG&RQFOXVLRQV6HOIOLJDWLQJEUDFNHWVSUHVHQWHGWKHPRVWFRQVLVWHQWDQG predictable results while conventional brackets, as esthetic brackets, showed very different SDWWHUQVRIIRUFHV6HOIOLJDWLQJEUDFNHWVVKRZHGKLJKHUVWUHQJWKLQDOOGHÀHFWLRQVZKHQ compared with the others, in 0.020-inch wires. O Keywords: Orthodontic wires. Mechanical phenomena. Elasticity. Comparative study. Evaluation of the force generated by gradual GHÀHFWLRQ RI RUWKRGRQWLF ZLUHV LQ FRQYHQWLRQDO metallic, esthetic, and self-ligating brackets Manoela Fávaro FRANCISCONI, Guilherme JANSON, José Fernando Castanha HENRIQUES, Karina Maria Salvatore de FREITAS Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Odontopediatria, Ortodontia e Saúde Coletiva, Bauru, SP, Brasil. Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Odontopediatria, Ortodontia e Saúde Cole Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Odontopediatria, Ortodontia e Saúde Coletiva, Bauru, SP, Brasil. Corresponding address: Manoela Fávaro Francisconi - Departamento de Odontopediatria, Ortodontia e Saúde Coletiva Faculdade de Odontologia de Bauru - Universidade de São Paulo - Alameda Octávio Pinheiro Brisolla, 9-75 - Bauru - SP - 17012-901 - Brazil - Phone/Fax: 3235-8000/3235-8217 - e-mail: manuff@usp.br 6XEPLWWHG6HSWHPEHU0RGL¿FDWLRQ0DUFK$FFHSWHG$SULO J Appl Oral Sci. 2016;24(5):496-502 Corresponding address: Manoela Fávaro Francisconi - Departamento de Odontopediatria, Ortodontia e Saúde Coletiva Faculdade de Odontologia de Bauru - Universidade de São Paulo - Alameda Octávio Pinheiro Brisolla, 9-75 - Bauru - SP - 17012-901 - Brazil - Phone/Fax: 3235-8000/3235-8217 - e-mail: manuff@usp.br INTRODUCTION LQFUHDVLQJWKHHI¿FLHQF\RIWKHDOLJQPHQWUHVXOWLQJ in a shorter treatment time15,19. However, some studies showed that this reduced friction depends on the type and caliper of the wire and the degree of crowding30. Further studies and clinical evaluations DUHVWLOOQHFHVVDU\WRFRQ¿UPWKHVHEHQH¿WV24. Nowadays, having a natural and pleasant smile even during orthodontic treatment is one of patients’ main concerns. Devices combining acceptable esthetic and adequate technical performance, satisfying both the patient and the clinician expectations, have been developed2. Nevertheless, esthetic brackets show higher friction FRHI¿FLHQWVWKDQPHWDOOLFEUDFNHWVZKLFKFDQLPSDLU the desired movement35. Therefore, effectiveness of orthodontic movement results not only from different bracket systems, but also from a series of other factors, related to both the patient (teeth and supporting structures) and the type of mechanics applied. Teeth movement also depends on the action of orthodontic wires, which varies according to their structural and mechanical properties3. Self-ligating brackets, introduced as Russel’s accessories in the mid-1930s, are systems that present a mechanical device designed to close the edgewise slot33. Their use has become common in recent years. Manufactures claim several advantages in using these accessories, and the low friction seems to be the most studied among them11,15,18. Some studies confirm that there LV VLJQL¿FDQWO\ ORZHU IULFWLRQ LQ WKHVH EUDFNHWV Consequently, it is necessary to assess not only the friction related to different bracket systems, but also the behavior of different currently available orthodontic materials regarding the forces applied during orthodontic mechanics. Furthermore, the development of esthetic brackets with metal 496 J Appl Oral Sci. 2016;24(5):496-502 (YDOXDWLRQRIWKHIRUFHJHQHUDWHGE\JUDGXDOGHÀHFWLRQRIRUWKRGRQWLFZLUHVLQFRQYHQWLRQDOPHWDOOLFHVWKHWLFDQGVHOIOLJDWLQJEUDFNHW MATERIAL AND METHODS Records of the force released by the wire GHÀHFWLRQ ZHUH PDGH LQ DQG PP 'HÀHFWLRQ RI WKH ZLUH DWWDFKHG WR WKH EUDFNHW clinically corresponds to the beginning of treatment, when the teeth are poorly positioned and the wire is forced into the accessories slots. Depending on the degree of crowding, teeth experience more or less force to align the teeth. Methods - clinical simulation device Methods - clinical simulation device In order to internationally standardize the tests as adequately as possible, the methods used in this study followed the ISO 15841 Standard: Dentistry – Wires for use in orthodontics16 'HÀHFWLRQRIWKHRUWKRGRQWLFZLUHZDVSHUIRUPHG in a clinical simulation device representing all 10 teeth of the maxillary arch11. Figure 2 shows the clinical simulation device that was used in this study. This device consists of a parabola shaped acrylic UHVLQSODWHZLWK¿[HGDFU\OLFVWUXFWXUHVUHSUHVHQWLQJ the maxillary teeth (Figure 2). The parabola shape was determined by the wires, reducing the risk of GLYHUVHIRUFHVDULVLQJIURPWKHGHÀHFWLRQDSSOLHG in an unexpected way. Brackets were bonded with cyanoacrylate ester gel (Super Bonder, Loctite, São Paulo, SP, Brazil), positioned in the long axis of the acrylic devices parallel to the ground and at the same height. The wires had the same length12. The DFU\OLFVWUXFWXUHVZHUH¿[HGE\PHDQVRIWKUHDGHG screw in the bottom of the acrylic resin plate. 7KHGHÀHFWLRQWHVWVZHUHSHUIRUPHGXVLQJWKH Universal Testing Machine (Instron 3342), with a load cell of 10 N10 (Figure 3). This load cell has an accuracy of 0.5% of the reading value with the temperature of 25°C. In this study, the load cell was maintained at this temperature. Also according to the ISO standard, the tests were always performed at the same testing temperature of 36±1°C for all test groups26. To obtain this, an acrylic container with water at a temperature of 36±1°C, maintained with the aid of submersible heater with integral thermostat (Electronic Atman Heater, China) and checked by a decimal precision thermometer, with a limit of error of ± 0.2°C (Incoterm, reference 5097, São Paulo, SP, Brazil), was adapted to the testing machine24 (Figure 3). The tests were performed on the structure corresponding to the right maxillary central incisor (Figure 3). Unlike the others, this structure was not screwed, enabling its labio-lingual movement. It had a perforation, in which a metal cylinder was placed to activate it. MATERIAL AND METHODS FRPSRQHQWVFRPSULVHVDQHZ¿HOGIRUUHVHDUFK8. To make the best choice among the various brackets and orthodontic wires available, it is essential to know the magnitude of forces released by these wires and their behavior regarding the gradual LQFUHDVHLQZLUHGHÀHFWLRQ7. Material - experimental groups Material - experimental groups The sample used in this study consisted of 400 round-section Nitinol wires (Standard or Medium, GAC®, Bohemia, NY, USA) with 0.014, DQGLQFKGLDPHWHUVDQG¿YH different bracket types: conventional metallic, reinforced polycarbonate with metallic slots, sapphire, passive and active self-ligating brackets (Figure 1). Wires that showed very different behavior from the others were excluded21: they statistically represent outliers that would negatively interfere with the results. In conventional brackets, the wires were tied with “O” shaped elastomeric ligatures (GAC®; Bohemia, NY, USA). In the self- This in vitro VWXG\ DVVHVVHG GHÀHFWLRQ IRUFHV of round Nitinol orthodontic wires, placed in conventional metallic, reinforced polycarbonate, sapphire (Ormco, Glenda, CA, USA), and self- ligating brackets by using a clinical simulation model and following ISO 15841 as protocol. J Appl Oral Sci. 497 interfere with the results. In conventional brackets, the wires were tied with “O” shaped elastomeric ligatures (GAC®; Bohemia, NY, USA). In the self- Wires (GAC) Section Batch Bracket types Manufacturer Nitinol 0.014-inch 088637 Conventional metallic (Mini Diamond) Ormco GAC 0.016-inch 088649/ 091986 Conventional reinforced polycarbonate with metallic slots (Spirit) 0.018-inch 089071 Conventional sapphire (Inspire ICE) 0.020-inch 088679 Passive self-ligating (Damon Q) Active self-ligating (In-Ovation R) * All groups were tested at a controlled temperature of 36±1°C Figure 1- Sample used and test conditions Figure 2- Clinical simulation device 2016;24(5):496-502 Wires (GAC) Section Batch Bracket types Manufacturer Nitinol 0.014-inch 088637 Conventional metallic (Mini Diamond) Ormco GAC 0.016-inch 088649/ 091986 Conventional reinforced polycarbonate with metallic slots (Spirit) 0.018-inch 089071 Conventional sapphire (Inspire ICE) 0.020-inch 088679 Passive self-ligating (Damon Q) Active self-ligating (In-Ovation R) * All groups were tested at a controlled temperature of 36±1°C Figure 1- Sample used and test conditions J Appl Oral Sci. 497 Figure 2- Clinical simulation device 2016;24(5):496-502 Figure 2- Clinical simulation device J Appl Oral Sci. 497 2016;24(5):496-502 FRANCISCONI MF, JANSON G, HENRIQUES JFC, FREITAS KMS VSHHGRIWKHGHÀHFWLRQZDVPPPLQ ligating brackets the wires were tied by closing the passive (Damon Q) or active (In-Ovation R) systems. MATERIAL AND METHODS The tip of the activation head, attached to the testing machine, had a rounded FXWWR¿WWKHPHWDOF\OLQGHU'HÀHFWLRQRIWKHZLUH was performed without changing the inter bracket GLVWDQFHPPVLQFHWKHUHODWLRQGHÀHFWLRQIRUFH depends, among other things, on this distance. The Statistical analyses ANOVA and Tukey tests were used. nickel-titanium wires, except with the activation of 2 mm, in which the opposite occurred (Tables 1 and 2). Overall, Inspire ICE showed the highest deactivation forces while In-Ovation R showed the lowest. All statistical analyses were performed with Statistica software (Statistica for Windows – Release 7.0 - Copyright Statsoft Inc., Tulsa, OK, USA). Results were considered significant at p<0.05. All statistical analyses were performed with Statistica software (Statistica for Windows – Release 7.0 - Copyright Statsoft Inc., Tulsa, OK, USA). Results were considered significant at p<0.05. The deactivation forces were generally VLJQL¿FDQWO\KLJKHUZLWKFRQYHQWLRQDOWKDQZLWKVHOI ligating brackets, with 0.018-inch nickel-titanium wires (Table 3). Overall, Inspire ICE showed the highest deactivation forces while In-Ovation R showed the lowest. Statistical analyses The sample size, according to the ISO 15841 standards, is of six specimens in each group16. To minimize the chances of any technical error and increase reliability of the results, 20 specimens were chosen for each group. Outliers were excluded through a statistical program that provides the values to be deleted22. Normal distribution was evaluated with Kolmogorov-Smirnov tests. Because all variables showed a normal distribution, one-way Figure 3- Instron universal testing machine used in this study, with a load cell of 10 N. Acrylic container adapted to the Instron device n universal testing machine used in this study, with a load cell of 10 N. Acrylic container adapted to the Figure 3- Instron universal testing machine used in this study, with a load cell of 10 N. Acrylic container adapted to the Instron device J Appl Oral Sci. 498 2016;24(5):496-502 498 2016;24(5):496-50 J Appl Oral Sci. 498 'LIIHUHQWOHWWHUVUHSUHVHQWVWDWLVWLFDOO\VLJQL¿FDQWGLIIHUHQFHV RESULTS The deactivation forces were generally VLJQL¿FDQWO\ KLJKHU ZLWK FRQYHQWLRQDO WKDQ ZLWK self-ligating brackets, with 0.014 and 0.016-inch There was an inversion of the previous Table 1- Deactivation forces (cN) comparisons of the bracket types with 0.014-inch nickel-titanium wire GHÀHFWLRQV2QHZD\$QRYDIROORZHGE\7XNH\WHVWV Table 1- Deactivation forces (cN) comparisons of the bracket types with 0.014-inch nickel-titanium wire, in progressive GHÀHFWLRQV2QHZD\$QRYDIROORZHGE\7XNH\WHVWV es (cN) comparisons of the bracket types with 0.014-inch nickel-titanium wire, in progressive DIROORZHGE\7XNH\WHVWV 'HÀHFWLRQ (mm) Mini Diamond (N=15) Spirit (N=20) Inspire ICE (N=17) Damon Q (N=16) In-Ovation R (N=16) P Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) 0.5 123.78 (22.70)A 60.13 (32.77)B 97.23 (41.87)A 61.08 (5.61)B 39.15 (9.46)B <0.001 1.0 143.27 (10.56)AD 118.32 (8.74)B 149.73 (16.70)A 135.10 (5.96)D 100.27 (10.21)C <0.001 2.0 76.57 (10.36)A 92.63 (15.15)B 91.74 (20.94)B 156.59 (9.45)C 153.56 (10.02)C <0.001 3.0 391.28 (18.28)A 341.65 (15.03)B 498.63 (32.73)C 204.24 (9.52)D 190.19 (9.38)D <0.001 'LIIHUHQWOHWWHUVUHSUHVHQWVWDWLVWLFDOO\VLJQL¿FDQWGLIIHUHQFHV 'LIIHUHQWOHWWHUVUHSUHVHQWVWDWLVWLFDOO\VLJQL¿FDQWGLIIHUHQFHV 'LIIHUHQWOHWWHUVUHSUHVHQWVWDWLVWLFDOO\VLJQL¿FDQWGLIIHUHQFHV Table 2- Deactivation forces (cN) comparisons of the bracket types with 0.016-inch nickel-titanium wire, in progressive GHÀHFWLRQV2QHZD\$QRYDIROORZHGE\7XNH\WHVWV 'HÀHFWLRQ (mm) Mini Diamond (N=19) Spirit (N=17) Inspire ICE (N=17) Damon Q (N=18) In-Ovation R (N=20) P Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) 0.5 112.97 (65.22)A 59.69 (43.36)B 130.14 (53.65)A 86.58 (4.80)B 70.72 (16.22)B <0.001 1.0 231.49 (26.93)AD 184.01 (20.76)B 248.30 (24.89)A 226.47 (12.22)D 162.65 (19.95)C <0.001 2.0 218.93 (32.20)A 224.54 (25.33)A 232.22 (21.29)A 281.85 (15.04)B 273.24 (15.80)B <0.001 3.0 479.55 (31.76)A 459.29 (18.29)A 569.15 (38.27)B 372.87 (12.22)C 353.17 (21.67)C <0.001 'LIIHUHQWOHWWHUVUHSUHVHQWVWDWLVWLFDOO\VLJQL¿FDQWGLIIHUHQFHV Table 3- Deactivation forces (cN) comparisons of the bracket types with 0.018-inch nickel-titanium wire, in progressive GHÀHFWLRQV2QHZD\$QRYDIROORZHGE\7XNH\WHVWV 'HÀHFWLRQ (mm) Mini Diamond (N=16) Spirit (N=19) Inspire ICE (N=14) Damon Q (N=18) In-Ovation R (N=19) P Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) 0.5 158.85 (21.14)A 101.08 (53.22)B 141.52 (43.53)AC 108.70 (9.86)BC 96.85 (16.57)B <0.001 1.0 289.29 (17.81)AD 236.63 (26.26)B 269.06 (17.09)A 304.09 (12.66)D 209.14 (21.53)C <0.001 2.0 400.40 (31.08)A 351.94 (18.07)B 397.88 (37.32)A 392.24 (15.60)C 354.15 (22.05)B <0.001 3.0 626.94 (27.36)A 588.17 (32.19)B 674.28 (31.55)C 538.04 (17.69)E 486.04 (38.02)D <0.001 'LIIHUHQWOHWWHUVUHSUHVHQWVWDWLVWLFDOO\VLJQL¿FDQWGLIIHUHQFHV 'LIIHUHQWOHWWHUVUHSUHVHQWVWDWLVWLFDOO\VLJQL¿FDQWGLIIHUHQFHV J Appl Oral Sci. 499 2016;24(5):496-502 J Appl Oral Sci. RESULTS 499 2016;24(5):496-502 499 FRANCISCONI MF, JANSON G, HENRIQUES JFC, FREITAS KMS 'HÀHFWLRQ (mm) Mini Diamond (N=16) Spirit (N=18) Inspire ICE (N=19) Damon Q (N=18) In-Ovation R (N=20) P Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) 0.5 130.12 (34.33)A 58.56 (46.10)B 127.83 (66.48)A 159.03 (17.74)A 145.61 (26.12)A <0.001 1.0 249.64 (34.96)A 210.17 (26.03)B 259.66 (30.11)A 423.59 (36.33)D 344.09 (25.04)C <0.001 2.0 560.60 (20.07)A 410.95 (41.75)B 550.04 (39.91)A 774.27 (77.22)D 620.34 (52.04)C <0.001 3.0 804.86 (29.18)A 753.09 (24.42)B 815.67 (21.90)A ------ 858.98 (84.34)A <0.001 'LIIHUHQWOHWWHUVUHSUHVHQWVWDWLVWLFDOO\VLJQL¿FDQWGLIIHUHQFHV 5 b th th i d d th f f 1000 Table 4- Deactivation forces (cN) comparisons of the bracket types with 0.020-inch nickel-titanium wire, in progressive GHÀHFWLRQV2QHZD\$QRYDIROORZHGE\7XNH\WHVWV Table 4- Deactivation forces (cN) comparisons of the bracket types with 0.020-inch nickel-titanium wire, in progressive GHÀHFWLRQV2QHZD\$QRYDIROORZHGE\7XNH\WHVWV Table 4- Deactivation forces (cN) comparisons of the bracket types with 0.020-inch nickel-titanium wire, in progressive GHÀHFWLRQV2QHZD\$QRYDIROORZHGE\7XNH\WHVWV 'LIIHUHQWOHWWHUVUHSUHVHQWVWDWLVWLFDOO\VLJQL¿FDQWGLIIHUHQFHV ** n=5 because the other wires exceeded the force of 1000g to sliding, generated in tests with conventional bracket systems during unloading6,347KHVH¿QGLQJV DUHJURXQGHGLQSUHYLRXVVWXGLHVDQGFRQ¿UPWKDW NiTi wires, along with self-ligating bracket systems, JHQHUDWHVLJQL¿FDQWO\KLJKHUIRUFHVZKHQFRPSDUHG with conventional brackets1,9. tendency with 0.020-inch nickel-titanium wires. 7KHGHDFWLYDWLRQIRUFHVZHUHJHQHUDOO\VLJQL¿FDQWO\ higher with self-ligating than with conventional brackets (Table 4). Overall, Damon Q showed the highest deactivation forces while Spirit showed the lowest. The forces with the Damon Q with 3 mm of activation exceeded 1000g. However, in deflection of 3 mm, the self- ligating brackets presented the smallest forces, contradicting previous investigation27 (Table 1). This occurs because smaller diameter wires release smaller forces, since they are not completely pressed against the slots. In conventional brackets, even small diameter wires are pressed into the slots by the elastic tie. DISCUSSION Sample and methodology A clinical simulation device was used to approximate the laboratory results to clinical situations, providing more practical applications32. (YHQZLWKWKLVLQPLQGWKHVSHFL¿F,62VWDQGDUG was used for orthodontic wires laboratory tests16. &RQ¿UPLQJ SUHYLRXV UHVXOWV31, there were no differences in the discharge forces between Damon and In-Ovation self-ligating brackets with 0.014- LQFK1L7LZLUHVLQPRVWGHÀHFWLRQV7DEOH7KLV may be a consequence of the more uniform wire mechanical locking system than the wire tying process of conventional brackets, with elastomeric ligatures36. There may be more variation in the process of tying the elastomeric ligatures than when closing the self-ligating brackets. 7KHHODVWLFGHÀHFWLRQWHVWZDVFKRVHQEHFDXVH it is clinically closest to the orthodontists’ interests, since that is what they do when adapting a wire to the patient’s teeth. Although engineers work with parameters such as elastic modulus and yield value, the orthodontist is more concerned about knowing the force released regarding the amount RIGHÀHFWLRQ The results were not standardized between different bracket combinations with 0.016-inch Nitinol wires, showing that the design of the experimental testing qualitatively and quantitatively affects the discharge forces during leveling and alignment with superelastic NiTi wires28,36 (Table 2). Similarly, when these forces were evaluated in an experimental model with embedded bracket systems, the wires tended to lose their superelastic properties, showing variations of force27. Other VWXGLHVFRQ¿UPHGWKHVH¿QGLQJV24,25. J Appl Oral Sci. 2016;24(5):496-502 Results 1995;65:63-72. 8- De Franco DJ, Spiller RE Jr, von Fraunhofer JA. Frictional UHVLVWDQFHV XVLQJ WHÀRQFRDWHG OLJDWXUHV ZLWK YDULRXV EUDFNHW archwire combinations. Angle Orthod. 1995;65:63-72. Clinically, this explanation makes sense because friction increases the released force during loading, but decreases it during unloading23. Therefore, the device with higher friction generated less force, because friction hinders the return of the wire to its initial position during the discharge36. The presence of the brackets, the distance between them, the bands, and the crowding itself are factors that increase friction in the clinical setting. Thus, this large friction would be able to decrease the released force by the wire. Other studies also found higher frictional resistance of conventional brackets when compared with self-ligating ones5,13. 9- Franchi L, Baccetti T, Camporesi M, Giuntini V. Forces released by nonconventional bracket or ligature systems during alignment of buccally displaced teeth. Am J Orthod Dentofacial Orthop. 2009;136:316.e1-6. 10- Galvão MB, Camporesi M, Tortamano A, Dominguez GC, Defraia E. Frictional resistance in monocrystalline ceramic brackets with conventional and nonconventional elastomeric ligatures. Prog Orthod. 2013;14:9. XUJHO-$.HUU63RZHUV-0/H&URQH9)RUFHGHÀHFWLRQ properties of superelastic nickel-titanium archwires. Am J Orthod Dentofacial Orthop. 2001;120:378-82. +HPLQJZD\5:LOOLDPV5/+XQW-$5XGJH6-7KHLQÀXHQFH of bracket type on the force delivery of ni-ti archwires. Eur J Orthod. 2001;23:233-41. 13- Hiroce M, Fernandes DJ, Elias CN, Miguel JA. Sliding resistance of polycarbonate self-ligating brackets and stainless steel esthetic archwires. Prog Orthod. 2012;13:148-53. Conventional and esthetic brackets showed very different patterns of forces due to bracket composition. Thus, this result should be considered when choosing the type of brackets to be used according to the type of mechanics necessary in orthodontic treatment of each case. 14- Huang TH, Luk HS, Hsu YC, Kao CT. An in vitro comparison of the frictional forces between archwires and self-ligating brackets of passive and active types. Eur J Orthod. 2012;34:625-32. 15- Imai T, Watari F, Yamagata S, Kobayashi M, Nagayama K, Toyoizumi Y, et al. Mechanical properties and aesthetics of FRP orthodontic wire fabricated by hot drawing. Biomaterials. 1998;19:2195-200. An important aspect to be considered is the difficulty in extrapolating laboratorial findings regarding frictional forces to the clinical environment. Many different factors, such as occlusal forces and tightening or loosening the archwire in the brackets during treatment, may produce different force YDOXHVZLWKVLJQL¿FDQWFOLQLFDOUHOHYDQFH20. 16- International Organization for Standardization - ISO. ISO 15841: Dentistry - Wires for use in orthodontics. 1st ed. Geneva: ISO; 2006. 17- Krishnan M, Kalathil S, Abraham KM. Results Comparative evaluation of frictional forces in active and passive self-ligating brackets with various archwire alloys. Am J Orthod Dentofacial Orthop. 2009;136:675-82. CONCLUSIONS 18- Kumar S, Singh S, Hamsa PR, Ahmed S, Prasanthma, Bhatnagar A, et al. Evaluation of friction in orthodontics using various brackets and archwire combinations - an in vitro study. J Clin Diagn Res. 2014;8:ZC33-6. Conventional and esthetic brackets showed very different patterns of forces due to bracket composition; 19- Kusy RP. Orthodontic biomaterials: from the past to the present. Angle Orthod. 2002;72:501-12. .XV\ 53 :KLWOH\ -4 ,QÀXHQFH RI DUFKZLUH DQG EUDFNHW dimensions on sliding mechanics: derivations and determinations of the critical contact angles for binding. Eur J Orthod. 1999;21:199-208. Self-ligating brackets presented similar behavior to each other and showed higher strength in all GHÀHFWLRQV ZKHQ FRPSDUHG ZLWK WKH RWKHUV LQ 0.020-inch wires. 21- Lee SM, Hwang CJ. A comparative study of frictional force in self-ligating brackets according to the bracket-archwire angulation, bracket material, and wire type. Korean J Orthod. 2015;45:13-9. 22- Lee YS, Lee SJ, An H, Donatelli RE, Kim SH. Do Class III patients have a different growth spurt than the general population? Am J Orthod Dentofacial Orthop. 2012;142:679-89. 3- Burstone CJ. Variable-modulus orthodontics. Am J Orthod. 1981;80:1-16. Results In general, it was observed that the deactivation force increased with the increase in amount of deflection (Tables 1 to 4). These findings are consistent with other studies29. The results found in different combinations of the brackets with 0.014-inch nickel-titanium wires are in agreement with other studies in the literature that KDYHIRXQGHUUDWLFUHVXOWVLQGLIIHUHQWGHÀHFWLRQV large variation in the difference of force values, and VLJQL¿FDQWGLIIHUHQFHVEHWZHHQGLIIHUHQWW\SHVRI devices36 (Table 1). The self-ligating brackets continued delivering OHVVIRUFHLQWKHPPGHÀHFWLRQEHFDXVHRIWKH reasons previously provided (Table 2). 7KH VHOIOLJDWLQJ EUDFNHWV LQ GHÀHFWLRQ RI mm, presented the highest forces when compared with conventional brackets (Table 1). This result can be explained by assuming that part of the force is used to overcome the greater resistance The results of combinations of different bracket types with 0.018-inch Nitinol wires demonstrated WKDW'DPRQ4JHQHUDWHGVLJQL¿FDQWO\JUHDWHUIRUFHV when compared with In-Ovation R (Table 3). Other 500 2016;24(5):496-502 J Appl Oral Sci. (YDOXDWLRQRIWKHIRUFHJHQHUDWHGE\JUDGXDOGHÀHFWLRQRIRUWKRGRQWLFZLUHVLQFRQYHQWLRQDOPHWDOOLFHVWKHWLFDQGVHOIOLJDWLQJEUDFNHW studies4,21 also noticed higher forces when passive (Smart Clip) were compared with active self-ligating brackets (Time3). 4- Buzzoni R, Elias CN, Fernandes DJ, Miguel JA. Assessment of surface friction of self-ligating brackets under conditions of angulated traction. Dental Press J Orthod. 2012;17:51-6. 5- Cacciafesta V, Sfondrini MF, Ricciardi A, Scribante A, Klersy C, Auricchio F. Evaluation of friction of stainless steel and esthetic self-ligating brackets in various bracket-archwire combinations. Am J Orthod Dentofacial Orthop. 2003;124:395-402. Finally, different bracket combinations with 0.020-inch Nitinol wires showed again that Damon Q generated greater forces than In-Ovation R 7DEOH)URPPPRIGHÀHFWLRQWKHVHOIOLJDWLQJ brackets showed greater forces than the others. This result is consistent with another study in which friction was responsible for reducing the amount of released force36.This partially explains the fact that the highest average was generated by the simulation device with self-ligating brackets14,17. 6- Cordasco G, Lo Giudice A, Militi A, Nucera R, Triolo G, Matarese G. In vitro evaluation of resistance to sliding in self-ligating and conventional bracket systems during dental alignment. Korean J Orthod. 2012;42:218-24. 7- D'Antò V, Rongo R, Ametrano G, Spagnuolo G, Manzo P, Martina R, et al. Evaluation of surface roughness of orthodontic wires by means of atomic force microscopy. Angle Orthod. 2012;82:922-8. 7- D'Antò V, Rongo R, Ametrano G, Spagnuolo G, Manzo P, Martina R, et al. Evaluation of surface roughness of orthodontic wires by means of atomic force microscopy. Angle Orthod. 2012;82:922-8. 8- De Franco DJ, Spiller RE Jr, von Fraunhofer JA. Frictional UHVLVWDQFHV XVLQJ WHÀRQFRDWHG OLJDWXUHV ZLWK YDULRXV EUDFNHW archwire combinations. Angle Orthod. J Appl Oral Sci. 29- Pesce RE, Uribe F, Janakiraman N, Neace WP, Peterson DR, Nanda R. Evaluation of rotational control and forces generated GXULQJ ¿UVWRUGHU DUFKZLUH GHÀHFWLRQV D FRPSDULVRQ RI VHOI ligating and conventional brackets. Eur J Orthod. 2014;36:245-54. 30- Reznikov N, Har-Zion G, Barkana I, Abed Y, Redlich M. ,QÀXHQFH RI IULFWLRQ UHVLVWDQFH RQ H[SUHVVLRQ RI VXSHUHODVWLF properties of initial NiTi wires in “reduced friction” and conventional bracket systems. J Dent Biomech. 2010;2010:613142. 25- Montasser MA, El-Bialy T, Keilig L, Reimann S, Jager A, Bourauel C. Force loss in archwire-guided tooth movement of conventional and self-ligating brackets. Eur J Orthod. 2014;36:31-8. 26- Montasser MA, Keilig L, Bourauel C. Archwire diameter effect on tooth alignment with different bracket-archwire combinations. Am J Orthod Dentofacial Orthop. 2016;149:76-83. 27- Nucera R, Gatto E, Borsellino C, Aceto P, Fabiano F, Matarese HWDO,QÀXHQFHRIEUDFNHWVORWGHVLJQRQWKHIRUFHVUHOHDVHGE\ VXSHUHODVWLFQLFNHOWLWDQLXPDOLJQPHQWZLUHVLQGLIIHUHQWGHÀHFWLRQ FRQ¿JXUDWLRQV$QJOH2UWKRG 28- Parvizi F, Rock WP. The load/deflection characteristics of thermally activated orthodontic archwires. Eur J Orthod. 2003;25:417-21. 29- Pesce RE, Uribe F, Janakiraman N, Neace WP, Peterson DR, Nanda R. Evaluation of rotational control and forces generated GXULQJ ¿UVWRUGHU DUFKZLUH GHÀHFWLRQV D FRPSDULVRQ RI VHOI ligating and conventional brackets. Eur J Orthod. 2014;36:245-54. 30- Reznikov N, Har-Zion G, Barkana I, Abed Y, Redlich M. ,QÀXHQFH RI IULFWLRQ UHVLVWDQFH RQ H[SUHVVLRQ RI VXSHUHODVWLF properties of initial NiTi wires in “reduced friction” and conventional bracket systems. J Dent Biomech. 2010;2010:613142. 28- Parvizi F, Rock WP. The load/deflection characteristics of thermally activated orthodontic archwires. Eur J Orthod. 2003;25:417-21. REFERENCES 1- Badawi HM, Toogood RW, Carey JP, Heo G, Major PW. Three- dimensional orthodontic force measurements. Am J Orthod Dentofacial Orthop. 2009;136:518-28. 23- Liaw YC, Su YY, Lai YL, Lee SY. Stiffness and frictional resistance of a superelastic nickel-titanium orthodontic wire with low-stress hysteresis. Am J Orthod Dentofacial Orthop. 2007;131:578.e12-8. /RPEDUGR/0DUD¿RWL06WHIDQRQL)0ROOLFD)6LFLOLDQL* /RDGGHÀHFWLRQFKDUDFWHULVWLFVDQGIRUFHOHYHORIQLFNHOWLWDQLXP initial archwires. Angle Orthod. 2012;82:507-21. 23- Liaw YC, Su YY, Lai YL, Lee SY. Stiffness and frictional resistance of a superelastic nickel-titanium orthodontic wire with low-stress hysteresis. Am J Orthod Dentofacial Orthop. 2007;131:578.e12-8. /RPEDUGR/0DUD¿RWL06WHIDQRQL)0ROOLFD)6LFLOLDQL* /RDGGHÀHFWLRQFKDUDFWHULVWLFVDQGIRUFHOHYHORIQLFNHOWLWDQLXP initial archwires. Angle Orthod. 2012;82:507-21. 2- Birnie D. Ceramic brackets. Br J Orthod. 1990;17:71-4. 2- Birnie D. Ceramic brackets. Br J Orthod. 1990;17:71-4. 3- Burstone CJ. Variable-modulus orthodontics. Am J Orthod. 1981;80:1-16. 501 J Appl Oral Sci. 2016;24(5):496-502 FRANCISCONI MF, JANSON G, HENRIQUES JFC, FREITAS KMS 27- Nucera R, Gatto E, Borsellino C, Aceto P, Fabiano F, Matarese HWDO,QÀXHQFHRIEUDFNHWVORWGHVLJQRQWKHIRUFHVUHOHDVHGE\ VXSHUHODVWLFQLFNHOWLWDQLXPDOLJQPHQWZLUHVLQGLIIHUHQWGHÀHFWLRQ FRQ¿JXUDWLRQV$QJOH2UWKRG FRANCISCONI MF, JANSON G, HENRIQUES JFC, FREITAS KMS 31- Rino Neto J, Queiroz GV, Paiva JB, Ballester RY. Does self- OLJDWLQJ EUDFNHWV W\SH LQÀXHQFH WKH K\VWHUHVLV DFWLYDWLRQ DQG deactivation forces of superelastic NiTi archwires? Dental Press J Orthod. 2013;18:81-5. 25- Montasser MA, El-Bialy T, Keilig L, Reimann S, Jager A, Bourauel C. Force loss in archwire-guided tooth movement of conventional and self-ligating brackets. Eur J Orthod. 2014;36:31-8. 26- Montasser MA, Keilig L, Bourauel C. Archwire diameter effect on tooth alignment with different bracket-archwire combinations. Am J Orthod Dentofacial Orthop. 2016;149:76-83. 32- Sakima MT, Dalstra M, Melsen B. How does temperature LQÀXHQFHWKHSURSHUWLHVRIUHFWDQJXODUQLFNHOWLWDQLXPZLUHV"(XU J Orthod. 2006;28:282-91. 33- Stolzenberg J. The Russell attachment and its improved advantages. Int J Orthod Dent Child. 1935;21:837-40. 34- Thorstenson GA, Kusy RP. Effect of archwire size and material on the resistance to sliding of self-ligating brackets with second- order angulation in the dry state. Am J Orthod Dentofacial Orthop. 2002;122:295-305. 35- Tselepis M, Brockhurst P, West VC. The dynamic frictional resistance between orthodontic brackets and arch wires. Am J Orthod Dentofacial Orthop. 1994;106:131-8. :LONLQVRQ3''\VDUW36+RRG-$+HUELVRQ3/RDGGHÀHFWLRQ characteristics of superelastic nickel-titanium orthodontic wires. Am J Orthod Dentofacial Orthop. 2002;121:483-95. J Appl Oral Sci. 502 2016;24(5):496-502 502 2016;24 J Appl Oral Sci. 502
https://openalex.org/W2072256929	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0115869&type=printable	English	null	Influence of Genetic Variants in TPMT and COMT Associated with Cisplatin Induced Hearing Loss in Patients with Cancer: Two New Cohorts and a Meta-Analysis Reveal Significant Heterogeneity between Cohorts	PloS one	2,014	cc-by	5,856	RESEARCH ARTICLE Influence of Genetic Variants in TPMT and COMT Associated with Cisplatin Induced Hearing Loss in Patients with Cancer: Two New Cohorts and a Meta-Analysis Reveal Significant Heterogeneity between Cohorts Melanie M. Hagleitner1, Marieke J. H. Coenen2, Ana Patino-Garcia3, Eveline S. J. M. de Bont4, Anna Gonzalez-Neira5, Hanneke I. Vos1, Frank N. van Leeuwen1, Hans Gelderblom6, Peter M. Hoogerbrugge1, Henk-Jan Guchelaar7, Maroeska W. M. te Loo1 Melanie M. Hagleitner1, Marieke J. H. Coenen2, Ana Patino-Garcia3, Eveline S. J. M. de Bont4, Anna Gonzalez-Neira5, Hanneke I. Vos1, Frank N. van Leeuwen1, Hans Gelderblom6, Peter M. Hoogerbrugge1, Henk-Jan Guchelaar7, Maroeska W. M. te Loo1 1. Department of Pediatric Hematology and Oncology, Radboud University Medical Center, Nijmegen, The Netherlands, 2. Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands, 3. Department of Pediatrics, University of Navarra and University Clinic, Pamplona, Spain, 4. Department of Pediatric Hematology and Oncology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands, 5. Human Genotyping Unit-CeGen, Spanish National Cancer Research Center, Madrid, Spain, 6. Department of Clinical Oncology, Leiden University Medical Center, Leiden, The Netherlands, 7. Department of Clinical Pharmacy & Toxicology, Leiden University Medical Center, Leiden, The Netherlands OPEN ACCESS Citation: Hagleitner MM, Coenen MJH, Patino- Garcia A, de Bont ESJM, Gonzalez-Neira A, et al. (2014) Influence of Genetic Variants in TPMT and COMT Associated with Cisplatin Induced Hearing Loss in Patients with Cancer: Two New Cohorts and a Meta-Analysis Reveal Significant Heterogeneity between Cohorts. PLoS ONE 9(12): e115869. doi:10.1371/journal.pone.0115869 Melanie.Hagleitner@radboudumc.nl Role of Pharmacogenetics in Cancer Patients with Ototoxicity Introduction Cisplatin is an effective chemotherapeutic drug for several types of cancer such as ovarian cancer, lung cancer, osteosarcoma and neuroblastoma. Ototoxicity, characterized as a permanent, bilateral sensorineural hearing loss, is one of the most common side effects of cisplatin and occurs in 40–60% of patients [1]. Ototoxicity is one of the main reasons for dose reduction or termination of treatment with cisplatin treatment. Clinical risk factors for the development of cisplatin-induced ototoxicity have been described such as age younger than 5 years at diagnosis [2, 3], concurrent medication like carboplatin [4] as well as higher cumulative doses [2, 4], cranial radiation [5] and pre-existing renal dysfunction. Nevertheless, these clinical factors are not sufficient to reliably predict ototoxicity before the start of treatment. A candidate gene study in 166 pediatric patients suggested that genetic variants in the genes encoding thiopurine S-methyltransferase (TPMT) and catechol O-methyltransferase (COMT) can predict the development of cisplatin-induced ototoxicity and may explain the interindividual variability [6]. Two recently published studies, developed to validate these findings, showed inconsistent results. Pussegoda and colleagues confirmed the findings of Ross et al. for variants in the TPMT gene in a cohort of 155 patients however, with smaller effect sizes compared to the original findings [7]. In contrast, no association of TPMT or COMT and ototoxicity was found in a cohort of 213 patients with medulloblastoma [8]. Based on these three studies no recommendation for clinical implementation regarding genetic variants in TPMT and COMT and ototoxicity can be made. The aim of the current study is to provide a clearer picture of the genetic impact of TPMT and COMT variants on developing cisplatin-induced ototoxicity. Therefore we investigated two inde- pendent cohorts of 110 Dutch and 38 Spanish patients with osteosarcoma and performed a meta-analysis including previously published studies [6–8] resulting in a total population of 664 patients with cancer. PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 Abstract Treatment with cisplatin-containing chemotherapy regimens causes hearing loss in 40–60% of cancer patients. It has been suggested that genetic variants in the genes encoding thiopurine S-methyltransferase (TPMT) and catechol O- methyltransferase (COMT) can predict the development of cisplatin-induced ototoxicity and may explain interindividual variability in sensitivity to cisplatin- induced hearing loss. Two recently published studies however, sought to validate these findings and showed inconsistent results. The aim of this study was to evaluate the role of polymorphisms in the TPMT and COMT genes in cisplatin- induced ototoxicity. Therefore we investigated two independent cohorts of 110 Dutch and 38 Spanish patients with osteosarcoma and performed a meta-analysis including all previously published studies resulting in a total population of 664 patients with cancer. With this largest meta-analysis performed to date, we show that the influence of TPMT and COMT on the development of cisplatin-induced hearing loss may be less important than previously suggested. Editor: Pantelis G. Bagos, University of Central Greece, Greece Received: February 26, 2014 Accepted: December 2, 2014 Published: December 31, 2014 Copyright: 2014 Hagleitner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and repro- duction in any medium, provided the original author and source are credited. Funding: This project was supported by a grant from the Foundation KiKa, Amstelveen, The Netherlands and the Asociacio´n Espan˜ola Contra el Ca´ncer, Spain. 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. 1 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 Spanish cohort To enlarge sample size for meta-analyses, we additionally included an independent Spanish cohort of osteosarcoma patients who were treated with cisplatin-containing regimens. To prevent population bias, this cohort was analyzed separately. In total, 38 patients with osteosarcoma were included on the basis of availability of germline DNA and audiologic assessment at least one month after initial treatment. Data regarding ototoxic medication were available. The study was approved by the local ethics committee from the University of Navarra and University Clinic in Pamplona. Written informed consent was obtained from parents and/or patients. Role of Pharmacogenetics in Cancer Patients with Ototoxicity follow up time 2.3 years with range 1.1–5.5 years), DNA of paraffin-embedded samples was extracted as recently described [9]. The study was approved by the ethics committee from the Radboud University Medical Centre as the central committee for this study for the Netherlands and written informed consent was obtained from parents and/or patients Ototoxicity In both cohorts audiologic evaluations were prospectively performed at diagnosis, during therapy and after completion of therapy. First audiological follow-up was performed 1–3 months after completion of therapy and thereafter annually. All audiometric assessments were age appropriate performed by conventional or play audiometry under standardized conditions as part of routine clinical monitoring for cisplatin-related hearing loss. Hearing loss was retrospectively classified according to two grading systems: the National Cancer Institute CTCAE version 3.0 (http://ctep.cancer.gov/forms/CTCAEv3.pdf) and the new SIOP Boston ototoxicity scale which classifies hearing loss on the basis of absolute hearing levels in 4 grades [10]. The most recent audiologic assessment during follow-up period after the last cisplatin course was used for analysis. PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 Dutch cohort A cohort of 110 Dutch patients with high-grade osteosarcoma treated with cisplatin was recruited from the Radboud University Medical Centre, the University Medical Centre of Groningen and Leiden University Medical Center. All patients were treated with cisplatin with a median cumulative dose of 500 mg/ m2 (range: 100 to 600 mg/m2). Of patients included audiometric analysis were available at diagnose, during therapy and after completion of therapy. Data concerning administration of other potentially ototoxic medications such as furosemide, vancomycin, gentamicin, tobramycin, amphotericin B, carboplatin and vincristine, were recorded. In patients alive, DNA was extracted from blood using the QIAamp DNA Blood Midi kit (Qiagen Venlo, The Netherlands) or saliva (2 ml) using the Oragene DNA purification protocol (DNA Genotek, Kanata, Ontario, Canada). Of patients that passed away, (N539, 26.5%, median PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 2 / 13 Role of Pharmacogenetics in Cancer Patients with Ototoxicity Meta-analyses We searched PubMed for papers using the keywords: ‘ototoxicity’, ‘TPMT’ and ‘COMT’. Since the publication of Ross and colleagues [6] in 2009 only two other studies [7, 8] evaluated the association of the COMT and TPMT variants in cancer patients. Meta-analysis was performed on these three previously published studies and the two cohorts presented in this study were included. Of each of the five studies the CTCAE hearing loss criteria were used, excluding patients with grade 1 ototoxicity to better differentiate between cisplatin-induced ototoxicity and normal hearing. The studies were weighed using the inverse variance method, where larger studies with smaller standard errors have more weight than smaller studies with larger errors (Review manager 5.0, The Cochrane Collaboration, Oxford, UK). Heterogeneity among studies was examined with I2 statistics that can be interpreted as the proportion of total variation contributed by between- study variation. Allelic odds ratio (OR) were recalculated dependent on the number of cases and controls in each cohort and 95% confidence interval (CI) were estimated using a fixed effects model or random effects models in case large heterogeneity I2.50% was observed. SNP analysis In both cohorts, statistical differences regarding demographic data between patients who developed hearing loss (cases;.20 dB hearing loss above 4 kHz) and patients without hearing loss (controls; #20 dB hearing loss at all frequencies) were assessed by the Fischer exact test. Reported p-values are two-sided and are considered statistically significant if ,0.05. Associations between ototoxicity and potential confounders, such as age, gender, cumulative dosage of cisplatin and concomitant drugs were evaluated using a 262 table (in case of dichotomized variables) or linear regression (in case of linear variables) in SPSS version 20 (SPSS Inc., Chicago, Il). To assess the effect of the genetic variants on ototoxicity data were dichotomized in grade 0 vs. grade 2–4. Multivariate logistic regression analyses included vincristine exposure as confounder in PLINK [11] using the command –logistic. PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 Genotyping Previously associated variants in TPMT (rs12201199: C__31923406_10, rs1800460: C__30634116_20, rs1142345: C_____19567_20) and COMT (rs4646316: C__29193982_10, rs9332377: C__29614343_10) were genotyped using Taqman allelic discrimination assays according to the protocol of the manufacturer (Invitrogen, Bleiswijk, The Netherlands). After amplification, the fluorescent signal for allelic discrimination was determined using the 7500 Fast Real-time System (Invitrogen). Automated allele calling was performed by allelic discrimination plots using SDS 1.4 software (Invitrogen). 3 / 13 Dutch cohort A total of 110 patients with Dutch ancestry and newly diagnosed osteosarcoma were included of whom 42 (38.2%) developed.20 dB hearing loss above 4 kHz. Characteristics of patients divided in patients with hearing loss (cases;.20 dB hearing loss above 4 kHz) and patients without hearing loss (controls; #20 dB hearing loss at all frequencies) are depicted in Table 1. Baseline characteristics did not show statistically significant differences between the two groups. None of the patients included was treated with cranial irradiation or with concomitant ototoxic antibiotics. Only five patients were additionally treated with vincristine. PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 4 / 13 Dutch cohort Spanish cohort Cases Controls Cases Controls 5 Ototoxicity 5 No ototoxicity 5 Ototoxicity 5 No ototoxicity N542 N568 p-value N516 N522 p- value Age at diagnosis (y) 15 15 0.82 11.5 14 0.23 median, range (5–40) (7–39.3) (4–29) (7–28) Gender (male) 22 33 0.6 15 6 0.1 N, % (40%) (60%) (71.4%) (28.6%) Cumulative dose cisplatin (mg/m2) 500 480 1 504 515 0.32 median, range (100–600) (200–600) (120–870) (140–720) Concomitant drugs Vincristine (N, %) 3 (2.7%) 2 (1.8%) 0.3 2 (5.2%) 4 (10.5%) 0.13 Aminoglyocide antibiotics (N, %) 0 0 15 (68.2%) 10 (62.5%) 0.74 Otoprotectants (N, %) 0 0 0 0 doi:10.1371/journal.pone.0115869.t001 Role of Pharmacogenetics in Cancer Patients with Ototoxicity Table 1. Demographic data of the Dutch and Spanish cohort. Table 1. Demographic data of the Dutch and Spanish cohort. Table 1. Demographic data of the Dutch and Spanish cohort. Dutch cohort Spanish cohort Cases Controls Cases Controls 5 Ototoxicity 5 No ototoxicity 5 Ototoxicity 5 No ototoxicity N542 N568 p-value N516 N522 p- value Age at diagnosis (y) 15 15 0.82 11.5 14 0.23 median, range (5–40) (7–39.3) (4–29) (7–28) Gender (male) 22 33 0.6 15 6 0.1 N, % (40%) (60%) (71.4%) (28.6%) Cumulative dose cisplatin (mg/m2) 500 480 1 504 515 0.32 median, range (100–600) (200–600) (120–870) (140–720) Concomitant drugs Vincristine (N, %) 3 (2.7%) 2 (1.8%) 0.3 2 (5.2%) 4 (10.5%) 0.13 Aminoglyocide antibiotics (N, %) 0 0 15 (68.2%) 10 (62.5%) 0.74 Otoprotectants (N, %) 0 0 0 0 Other ototoxic chemotherapeutics were not administered. In 23 (20.9%) of all patients cumulative dose of cisplatin was reduced due to ototoxicity (N59, .20 dB hearing loss above 4 kHz) or other cisplatin-induced toxicity (N514). Baseline audiograms were available in 87 patients (79%) showing normal baseline hearing thresholds (#20 dB) in all patients. Median follow up period was 5.2 years (range 23–7763 days). Different ototoxicity grading systems were used to exclude biased dichotomisation. For the CTCAE criteria, 80 patients with baseline audiogram were included. Patients with grade 1 ototoxicity (N57) were excluded in order to use same inclusion/exclusion criteria as previous studies in literature [6–8]. Classification according to the CTCAE criteria showed in all but seven patients identical toxicity grades when compared to the SIOP grading system. Four patients with grade 1 and three patients with grade 2 ototoxicity according to the SIOP Boston ototoxicity scale were upgraded to grade 2 and 3 according the CTCAE criteria. Hearing loss grade 2–4 was seen in 23 patients graded according the CTCAE criteria (0 vs..25 dB at 4–8 kHz) and 22 according to the SIOP criteria (0 vs..20 dB at 4–8 kHz). Univariate analysis showed no association with non-genetic factors previously reported to be associated with ototoxicity: age (p50.73) and cumulative dose of cisplatin (p50.99). Role of Pharmacogenetics in Cancer Patients with Ototoxicity Other ototoxic chemotherapeutics were not administered. In 23 (20.9%) of all patients cumulative dose of cisplatin was reduced due to ototoxicity (N59, .20 dB hearing loss above 4 kHz) or other cisplatin-induced toxicity (N514). Baseline audiograms were available in 87 patients (79%) showing normal baseline hearing thresholds (#20 dB) in all patients. Median follow up period was 5.2 years (range 23–7763 days). Different ototoxicity grading systems were used to exclude biased dichotomisation. For the CTCAE criteria, 80 patients with baseline audiogram were included. Patients with grade 1 ototoxicity (N57) were excluded in order to use same inclusion/exclusion criteria as previous studies in literature [6–8]. Classification according to the CTCAE criteria showed in all but seven patients identical toxicity grades when compared to the SIOP grading system. Four patients with grade 1 and three patients with grade 2 ototoxicity according to the SIOP Boston ototoxicity scale were upgraded to grade 2 and 3 according the CTCAE criteria. Hearing loss grade 2–4 was seen in 23 patients graded according the CTCAE criteria (0 vs..25 dB at 4–8 kHz) and 22 according to the SIOP criteria (0 vs..20 dB at 4–8 kHz). Univariate analysis showed no association with non-genetic factors previously reported to be associated with ototoxicity: age (p50.73) and cumulative dose of cisplatin (p50.99). Spanish cohort To enlarge sample size for the meta-analyses we included another cohort of 38 Spanish patients of European ancestry with osteosarcoma (Table 1). No statistically significant differences concerning age, gender or cumulative dose of cisplatin was seen between patients with hearing loss (cases; .20 dB hearing loss above 4 kHz) and patients with normal hearing (controls; #20 dB hearing loss at Table 1. Demographic data of the Dutch and Spanish cohort. Meta-analyses So far, three studies with four independent cohorts studying the influence of TPMT and COMT on the development of ototoxicity have been published [6–8]. With this study we add two additional independent cohorts. In all studies the CTCAE criteria were reported. To better differentiate between cases and controls all studies excluded patients with grade 1 ototoxicity. In total, 664 patients were included in the meta-analyses. Table 2 shows the demographic data of the initial studies, as well as the number of patients per study that were included in the meta-analysis. In Figs. 1 and 2 odds ratios for each individual study are presented. The genetic variant rs4646316 in the COMT gene was the only significant association with ototoxicity (OR A versus T allele: 1.52, 95% CI: 1.16–1.99, P50.003; Fig. 2). The heterogeneity test showed a non-statistically significant heterogeneity with an I2 of 31%. Notably, there are substantial differences in the range of the ‘‘total events’’ captured between the meta-analyses which is related to incomplete genotyping in all studies, except for the study of Pussegoda and colleagues [7]. Genotyping yp g We genotyped three variants in the TPMT gene (rs12201199, rs1800460, and rs1142345) and two in the COMT gene (rs4646316, rs9332377). No deviation from Hardy–Weinberg equilibrium was observed. Irrespective of the grading system used (CTCAE or SIOP), association analysis using an additive genetic model correcting for usage of vincristine, showed no association between genetic variants in TPMT or COMT with an increased risk of ototoxicity in the Dutch cohort (S1 Table, S2Table and S3 Table). Role of Pharmacogenetics in Cancer Patients with Ototoxicity cranial irradiation. Regarding concomitant medication, all but six patients were additionally treated with vincristine and 23 patients received ototoxic antibiotics, such as vancomycin, gentamicin or tobramycin. In univariate analysis the use of these antibiotics was not a confounding factor to develop ototoxicity in this cohort (p50.74). As to the small sample size and to prevent population bias due to ethnicity, genotyping results of this cohort were analyzed as an independent cohort in the meta-analyses. Spanish cohort To enlarge sample size for the meta-analyses we included another cohort of 38 Spanish patients of European ancestry with osteosarcoma (Table 1). No statistically significant differences concerning age, gender or cumulative dose of cisplatin was seen between patients with hearing loss (cases; .20 dB hearing loss above 4 kHz) and patients with normal hearing (controls; #20 dB hearing loss at all frequencies). None of the included patients was additionally treated with 5 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 Discussion Although, the results of Ross and colleagues investigating the influence of variants in TPMT and COMT were very promising [6], the outcome of more recent studies argues against the premise that these variants might help to identify patients at risk for ototoxicity. In the present study, therefore, we aimed to gain a clearer picture on the role of these variants by adding additional data on the subjects, and by performing a meta-analysis including two new cohorts. PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 6 / 13 Role of Pharmacogenetics in Cancer Patients with Ototoxicity Table 2. Demographic data of patients with ototoxicity included for meta-analysis. Cases/Total Ethnicity Age (y) Types of cancer Gender of cases Cumulative dose Cranial irradiation Otoprotectant Vincristine Aminoglycocide N Median Male % Median (mg/m2) % used antibiotics Ross et al.6 106/162 Mixed population 6 Different cancers 67.0% 400 mg/m2 18.50% No information Yes Yes Pussegoda et al.7 87/155 Mixed population 6 Different cancers 49.4% 400 mg/m2 18% No information Yes Yes Yang et al., radiation cohort8 131/195 Mixed population 7 Medulloblastoma 68.8% 300 mg/m2* 100%* Yes Yes No information Yang et al., no radiation8 35/38 Mixed population 8 Medulloblastoma 65.0% 500 mg/m2 0 Yes Yes No information Hagleitner, this study 22/80 Dutch ancestry 15 Osteosarcoma 40.0% 500 mg/m2 0 No Yes No Patino-Garcia, this study 18/34 European ances- try 12 Osteosarcoma 55.3% 500 mg/m2 0 No Yes Yes data of total group as published by Yang et al. doi:10.1371/journal.pone.0115869.t002 Role of Pharmacogenetics in Cancer Patients with Oto Table 2. Demographic data of patients with ototoxicity included for meta-analysis. Cases/Total Ethnicity Age (y) Types of cancer Gender of cases Cumulative dose Cranial irradiation Otoprotectant Vincristine Aminoglycocide N Median Male % Median (mg/m2) % used antibiotics Ross et al.6 106/162 Mixed population 6 Different cancers 67.0% 400 mg/m2 18.50% No information Yes Yes Pussegoda et al.7 87/155 Mixed population 6 Different cancers 49.4% 400 mg/m2 18% No information Yes Yes Yang et al., radiation cohort8 131/195 Mixed population 7 Medulloblastoma 68.8% 300 mg/m2* 100%* Yes Yes No information Yang et al., no radiation8 35/38 Mixed population 8 Medulloblastoma 65.0% 500 mg/m2 0 Yes Yes No information Hagleitner, this study 22/80 Dutch ancestry 15 Osteosarcoma 40.0% 500 mg/m2 0 No Yes No Patino-Garcia, this study 18/34 European ances- try 12 Osteosarcoma 55.3% 500 mg/m2 0 No Yes Yes data of total group as Ro Table 2. Discussion Demographic data of patients with ototoxicity included for meta-analysis. Cases/Total Ethnicity Age (y) Types of cancer Gender of cases Cumulative dose Cra irra N Median Male % Median (mg/m2) % Ross et al.6 106/162 Mixed population 6 Different cancers 67.0% 400 mg/m2 18.5 Pussegoda et al.7 87/155 Mixed population 6 Different cancers 49.4% 400 mg/m2 18% Yang et al., radiation cohort8 131/195 Mixed population 7 Medulloblastoma 68.8% 300 mg/m2* 100 Yang et al., no radiation8 35/38 Mixed population 8 Medulloblastoma 65.0% 500 mg/m2 0 Hagleitner, this study 22/80 Dutch ancestry 15 Osteosarcoma 40.0% 500 mg/m2 0 Patino-Garcia, this study 18/34 European ances- try 12 Osteosarcoma 55.3% 500 mg/m2 0 *data of total group as published by Yang et al. doi:10.1371/journal.pone.0115869.t002 PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 7 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 7 / 13 Role of Pharmacogenetics in Cancer Patients with Ototoxicity Fig. 1. Forest plots genetic variants in TPMT gene. Fig. 1. Forest plots genetic variants in TPMT gene. doi:10.1371/journal.pone.0115869.g001 Our Dutch dataset of 110 uniformly treated patients with osteosarcoma, is a homogenous group without potentially confounding factors such as cranial radiation and concomitant medication. For classification of cisplatin-induced hearing loss, we preferred to use the SIOP Boston scale as it combines the two main types of measuring ototoxicity, namely changes of hearing from baseline as Our Dutch dataset of 110 uniformly treated patients with osteosarcoma, is a homogenous group without potentially confounding factors such as cranial radiation and concomitant medication. For classification of cisplatin-induced hearing loss, we preferred to use the SIOP Boston scale as it combines the two main types of measuring ototoxicity, namely changes of hearing from baseline as PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 8 / 13 Role of Pharmacogenetics in Cancer Patients with Ototoxicity Role of Pharmacogenetics in Cancer Patients with Ototoxicity Fig. 2. Forest plots genetic variants in COMT gene. doi:10.1371/journal.pone.0115869.g002 PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 Role of Pharmacogenetics in Cancer Patients with Ototoxicity significant. However, the effect was much smaller than reported in previously published studies. In the initial discovery cohort of Ross and colleagues the OR of this variant to develop ototoxicity was 2.5 (95% CI: 1.5–4.3) [6]. Whereas the combined effect of all studies included in the meta-analysis showed an OR of 1.52 (95% CI: 1.16–1.99). Three of the cohorts (excluding the initial study by Ross et al.) showed the same direction of effect for rs4646316, however, this was not statistically significant. The COMT gene encodes for the COMT enzyme which is involved in the inactivation of catecholamine neurotransmitters. Although, it has been shown that it is also highly expressed in sensory hair cells of the inner ear, its role regarding auditory function remains unclear [12]. In addition, the influence of the variant rs4646361 on the COMT enzyme has not been studied yet. Generally, the association between TPMT variants and cisplatin ototoxicity was unexpected [9], as it was not previously linked to cisplatin metabolism. Recent published data support the hypothesis that TPMT might have an influence on cisplatin metabolism by increasing levels of S-adenosylmethionine (SAM) [13]. Furthermore, a recent study demonstrated that functional TPMT variants were associated with progression-free survival in cisplatin-based chemotherapy outcomes in ovarian cancer [14] However in vivo studies in mice with different outcomes in ovarian cancer [14]. However, in vivo studies in mice with different TPMT genotypes showed no differences in hearing damage between TPMT wild- type and knockout mice [8]. y With the largest meta-analysis performed to date we show that the influence of TPMT and COMT on the development of cisplatin-induced hearing loss may be less pronounced than previously suggested. This lack of association observed might be attributable to heterogeneity between the different studies as shown in Table 2. First, different patient populations with different ethnicities were used in the studies which could account for the differences in the genetic associations in the meta-analysis. Second, in the study cohorts with clear positive association, a heterogeneous group of patients with respectively 9 and 12 different types of cancers was included [6, 7]. As a consequence, patients were treated with different treatment regimens sometimes including cranial irradiation and different co- medication. In these studies an analysis for specific patient subgroups was not performed probably due to the limited number of patients included for each subgroup. PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 Fig. 2. Forest plots genetic variants in COMT gene. Fig. 2. Forest plots genetic variants in COMT gene. Fig. 2. Forest plots genetic variants in COMT gene. doi:10.1371/journal.pone.0115869.g002 doi:10.1371/journal.pone.0115869.g002 well as absolute hearing levels [10]. As previously published studies used the CTCAE criteria for the classification of ototoxicity, we reclassified our data according to the CTCAE criteria, and excluded grade 1 ototoxicity to better distinguish between cases and controls. In the Dutch cohort, and also the other newly analyzed Spanish cohort, we did not observe any statistically significant association between the genetic variants in TPMT, COMT and ototoxicity. We did observe differences between the two cohorts such as lower ototoxicity rate in the Dutch cohort which might be explained by the inclusion of adults until the age of 40 years. It is known that young children are more susceptible to ototoxicity from cisplatin [3]. Furthermore, the Dutch cohort included patients who have already died. It might be that controls that died have become cases if they have survived longer. However, the median follow up time of these patients who have died was 2.3 years with a range between 1.1 and 5.5 years. Studies included in this meta- analysis showed universally that the majority of ototoxicity occurred between 0.5 and 6 months from start of cisplatin chemotherapy [6–8]. After meta-analyses of the genetic variants in TPMT and COMT including in total 664 patients only rs4646316 variant in the COMT gene remained statistically PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 9 / 13 Role of Pharmacogenetics in Cancer Patients with Ototoxicity important factor to develop cisplatin-induced hearing loss [3], which may be the reason for the lower incidence of ototoxicity in the osteosarcoma cohort as the median age at diagnosis is older compared to the other cohorts. At last, the use of otoprotective agents, such as amifostine, may be a potentially confounding factor. A Cochrane study on this topic was due to methodological limitations of individual studies unable to show evidence of effect to use amifostine as an otoprotective intervention [15]. However, there was also no evidence that it had no effect as otoprotectant. In the study of Yang et al the use of amifostine was linked to less ototoxicity [8]. y It is clear that all studies included in this meta-analysis have confounding variables which could potentially influence the development of ototoxicity. This is important given the inverse method of weighing studies which has been used in this meta-analysis. The studies of Pussegoda et al and Yang et al were consistently attributed the highest weighting. Pussegoda et al included a diverse population of different cancers with different treatment protocols, the youngest population in this meta-analysis with different follow-up time between cases (5 years follow-up) and controls (2 years follow-up). The radiation cohort of Yang et al patients received highly ototoxic cranial irradiation, but 90% also received an otoprotectant, and overall these patients received a significantly lower cumulative dose of cisplatin. Thus, the weight attributed to a study may affect the results of the meta-analysis, however this is something that cannot be circumvented in this type of analysis. Nevertheless, younger age and higher cumulative doses of cisplatin appear to be the most consistent risk factors for ototoxicity. However, an upfront risk stratification based on these two factors is unreliable as these factors seem to be protocol specific and dose adjustments may jeopardize treatment efficacy of cisplatin. The addition of otoprotective agents in combination with cisplatin might be an alternative treatment strategy to prevent hearing loss. Although, the mechanism of cisplatin-induced hearing loss is not fully understood, several preclinical studies have shown that in the cochlear cells cisplatin induces the production of toxic levels of reactive oxygen species (ROS) which can initiate cochlear cell death leading to hearing loss [16, 17]. Numerous studies have investigated a variety of antioxidant agents to protect cochlea cells from cisplatin damage. PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 Also the cohorts with clear negative association included patients with different cancer types, one study included patients with osteosarcoma (this study) as in the study of Yang et al. patients with medulloblastoma have been included which necessitates highly different treatment protocols regarding cumulative dose of cisplatin, cranial radiation and concomitant medication. Third, the different types of cancer were not equally distributed among patients with and without ototoxicity, for example in the cohort of solid tumors investigated by Yang and colleagues 35 of the 38 patients [8] were reported to have hearing loss which may lead to selection bias. Fourth, the median cumulative doses of cisplatin ranged from 300 to 500 mg/m2 in the different studies. However, neither in the study cohort of medulloblastomas with a relatively low cumulative dose of cisplatin [8] nor in osteosarcoma patients treated with higher doses of cisplatin (this study), an association was found. In addition, young age has recently been pointed out as an PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 10 / 13 doi:10.1371/journal.pone.0115869.s004 (DOC) doi:10.1371/journal.pone.0115869.s004 (DOC) Role of Pharmacogenetics in Cancer Patients with Ototoxicity cisplatin-induced ototoxicity and should therefore not guide clinical decision making for cisplatin dosing. Author Contributions Conceived and designed the experiments: MH MC H-JG MtL. Performed the experiments: MH MC HV. Analyzed the data: MC MH. Contributed reagents/ materials/analysis tools: AP-G EdB AG-N FL HG PH. Wrote the paper: MH MC AP-G EdB AG-N HV FL HG PH H-JG MtL. Supporting Information S1 Table. Minor allele frequency of Dutch cohort. doi:10.1371/journal.pone.0115869.s001 (DOCX) S2 Table. Association analyses of Dutch cohort. doi:10.1371/journal.pone.0115869.s002 (DOCX) S3 Table. Call rates of the Dutch and Spanish cohort for each variant according to the CTCAE ototoxicity criteria. d i 10 1371/j l 0115869 003 (DOCX) S3 Table. Call rates of the Dutch and Spanish cohort for each variant according to the CTCAE ototoxicity criteria. doi:10.1371/journal.pone.0115869.s003 (DOCX) PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 Among them, sodium thiosulfate, D or L-methionine, glutathione ester and amifostine were successfully tested in animal models [18, 19]. The efficacy of otoprotective agents in patients is however disappointing. For instance, sodium thiosulfate showed an unwanted compromised antitumor effect of cisplatin [20] and no otoprotection was observed in children with germ cell tumors treated with amifostine in combination with cisplatin [21]. It remains challenging to find the right balance to maintain antitumor effect of cisplatin and on the other hand to prevent unwanted adverse effects. Innovative studies translating basic science into clinical practice are needed to unravel both, the mechanism of cisplatin-induced hearing loss and identification of patients at risk to develop ototoxicity. Pharmacogenetics may help to find new target genes, unfortunately it seems that the genes TPMT and COMT play only a minor role in PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 11 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0115869 December 31, 2014 References 1. Knight KR, Kraemer DF, Neuwelt EA (2005) Ototoxicity in children receiving platinum chemotherapy: underestimating a commonly occurring toxicity that may influence academic and social development. J Clin Oncol 23: 8588–8596. 2. Li Y, Womer RB, Silber JH (2004) Predicting cisplaitn ototoxicity in children: the influence of age and the cumulative dose. 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https://openalex.org/W4360980767	https://ejb.springeropen.com/counter/pdf/10.1186/s43168-023-00189-3	English	null	Short-term evaluation of motor and sensory nerve conduction parameters in COVID-19-associated peripheral neuropathy patients	The Egyptian Journal of Bronchology	2,023	cc-by	5,504	© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS‐COV‐2) is mostly associated with upper and lower respiratory tract manifestations. However, coronavirus disease 19 (COVID-19) can result in a wide range of other systemic symptomatology, including neuropsychiatric, psychological, and psychosocial impairments. Literature regarding neurological compromise, including neuropathy and sensory and motor affection associated with COVID- 19, is still limited. This study aims to evaluate the sensory, motor neuropathy, and secondary neurological impairment among patients with mild to moderate coronavirus disease associated with peripheral neuropathy within 1 month. Methods Forty participants, including 20 mild to moderate COVID-19 patients with peripheral neuropathy and 20 age and gender-matched healthy volunteers, were recruited in this case/control study. Laboratory evaluation focused on C-reactive protein (CRP) and D-dimer levels. Oxygen saturation for all participants was recorded. The neurophysi- ological study included motor nerve study, sensory nerve study, and F wave study for upper and lower limbs were done. Results The two groups were similar regarding baseline data. Neurological symptoms’ onset in the COVID-19 group ranged from 4 to 24 days. Levels of CRP and D-dimer levels were significantly higher in patients versus the control group. Motor nerve conduction (MNC) amplitude and latency for the median nerve were significantly compromised among the COVID-19 group. The MNC latency and F wave latency for the posterior tibial nerve were significantly higher in the COVID-19 group. The CRP and D-dimer levels were associated with a significant positive correlation with a latency of median nerve MNC, sensory nerve conduction (SNC), and f-wave; latency of MNC and F wave of the posterior tibial nerve; and SNC latency for sural nerve. Conclusion neurological involvement can occur in mild to moderate cases of SARS-COV-2 infection and add to the burden of the disease. Neurological symptoms in the course of COVID-19 disease should be interpreted cautiously, and appropriate diagnosis, including nerve conduction studies and management, should be considered. Trial registration ClinicalTrials.gov. NCT05721040. Trial registration ClinicalTrials.gov. NCT05721040. Trial registration ClinicalTrials.gov. NCT05721040. Correspondence: Ahmad M. Shaddad Shaddad_ahmad@aun.edu.eg Full list of author information is available at the end of the article The Egyptian Journal of Bronchology The Egyptian Journal of Bronchology Shaddad et al. The Egyptian Journal of Bronchology (2023) 17:15 https://doi.org/10.1186/s43168-023-00189-3 Shaddad et al. Open Access Open Access Short‑term evaluation of motor and sensory nerve conduction parameters in COVID‑19‑associated peripheral neuropathy patients Ahmad M. Shaddad1 , Aliaë A. R. Mohammed Hussein1, Amal Mohamed Aly Tohamy2 and Waleed Gamal Elddine Khalil1 Keywords COVID-19, Nerve conduction studies, Peripheral neuropathy, Complication, Motor and sensory function, Nerve conduction velocity, Nerve conduction amplitude, Nerve conduction latency, F wave latency November 2020) with neurological symptoms sugges- tive of peripheral neuropathy were eligible for participa- tion in the current study. Exclusion criteria were patients presenting with neurological manifestations other than peripheral neuropathy, such as stroke, cerebral hemor- rhage, encephalitis, or meningitis. Patients with any med- ical condition or comorbidities that may affect the result of the study as diabetes, arthritis, and carpel tunnel syn- drome were excluded. Severe COVID-19 patients with a critical condition that needs hospitalization and ventila- tory support were also excluded from the study. Study design and participantsh Study design and participantsh This prospective case–control study was conducted in the Chest Department, Faculty of Medicine, Assiut University, and the Neurology Department, Faculty of Medicine, Assiut University, between October 2021 and April 2022. Twenty RT-PCR-confirmed mild to mod- erate COVID-19 patients with peripheral neuropathy presented within 1 month of acute disease at outpatient clinics of the Chest and Neurology Departments were recruited and twenty healthy age and gender-matched volunteers. The sensory nerve study was done for the median and sural nerves to estimate the peripheral neurological func- tion of both upper and lower limbs. The analysis was per- formed using stimulating ring electrodes placed over the middle and proximal phalanxes of the second and third fingers. In contrast, the recording electrode is placed over the palm in a position 1–2 cm proximal to the proxi- mal crease of the palm. The sensory nerve conduction study of the sural nerve was performed by placing sur- face recording electrodes over the posterior aspect of the calf at a point between the middle and lower thirds of the leg, just lateral of the midline. Stimulating electrodes are placed behind and below the lateral malleolus over the sural nerve. Sensory motor nerve conduction amplitude, latency, and velocity were recorded. Introduction Severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2), which first emerged in Wuhan, China, can result in a wide variety of symptomatology ranging from asymptomatic infection up to pneumonia, respiratory failure, or even death [1]. Respiratory involvement is the prevalent form of COVID-19 disease; however, growing evidence suggests neurological manifestations to be a direct consequence and even the presenting manifestation in a large propor- tion of patients [2]. Cerebrovascular diseases, including stroke, encephalitis, neuropathy, visual pathway abnor- mality, and Guillain-Barré syndrome, were recorded. Other common features encountered early in the course of infection were loss of smell (anosmia) and taste (ageu- sia) [3–6]. Neurophysiological study All studies were conducted using A Nihon Kohden Machine model 9400 (Tokyo, Japan).h This study aims to evaluate the pattern of motor and sensory, and neurological affections in mild to moder- ate COVID-19 with associated peripheral neuropathy patients and explore the relationship between inflamma- tory status and neurological deficits. The motor nerve study was performed in the median and posterior tibial nerve to assess the motor function of the upper and lower limbs. A standard procedure with concentric needle electrodes is used to assess motor nerve conduction velocity. Supramaximal intensity stim- ulation with a duration of 0.2 ms with a rate of one per second was performed. Compound motor action poten- tial (CMAP), motor nerve conduction velocity (MNCV), and nerve conduction latency were recorded.h Clinical and laboratory assessment All patients were subjected to intense history taking, including the onset of the COVID-19 infection and the start of neurological symptoms, careful clinical examina- tion, and neurological assessment. All patients and con- trol groups had oxygen saturation, CPR, and D-dimer measurement at the time of the study. Several theories emerged to explain neurological manifestations, including direct neuroinvasion by the coronavirus, [7, 8] explained through the activation of the angiotensin-converting enzyme 2 (ACE-2) receptor expressed in both capillary and neuronal endothelium [9]. Dysregulated immune response, [10, 11] hypoxemia, multiorgan involvement, and increased prothrombin time and coagulopathy are other explanations [12, 13].h Page 2 of 8 Shaddad et al. The Egyptian Journal of Bronchology (2023) 17:15 Keywords COVID-19, Nerve conduction studies, Peripheral neuropathy, Complication, Motor and sensory function, Nerve conduction velocity, Nerve conduction amplitude, Nerve conduction latency, F wave latency Inclusion and exclusion criteriai P value was significant if < 0.05 Data expressed as frequency (percentage) and mean (SD). P value was significant if < 0.05 Ethical considerations saturation in the COVID-19 group with the mean of 96.95 ± 1.35 and the control group of 98.5 ± 0.61 with a p value of 0.009 (Table 2). All participants subjected to the study were asked to apply informed written consent; the research’s nature, procedures, and possible side effects were clearly explained. The Faculty of Medicine, Assiut University’s ethical committee approved the study protocol under the Declaration of Helsinki. Regarding the nerve conduction study in both groups, there was a significant difference between the two groups in median nerve motor nerve conduction amplitude (MNCA) with a mean of 8.88 ± 2.6 and 9.04 ± 2.12 in the control group with a P value of 0.039. There was a sig- nificant statistical difference between the two groups in median nerve motor nerve conduction latency (MNCL) with a mean of 5.1 ± 1.34 and 3.29 ± 0.64 in the control Inclusion and exclusion criteriai All patients with confirmed RT-PCR COVID-19 infec- tion with mild to moderate severity based on the Egyp- tian Ministry of Health (MOH) protocol (version 1.4, Shaddad et al. The Egyptian Journal of Bronchology (2023) 17:15 Page 3 of 8 Shaddad et al. The Egyptian Journal of Bronchology (2023) 17:15 Table 1 Demographic data of the COVID-19 patients with peripheral neuropathy and control group (n = 40) Data expressed as frequency (percentage) and mean (SD). P value was significant if < 0.05 COVID-19 group N = 20 Control group N = 20 P value Gender Male 17 (85%) 16 (80%) 0.50 Female 3 (15%) 4 (20%) Age (years) Mean ± SD 45.05 ± 11.05 42.35 ± 11.05 0.745 Range (21–68) (20–59) Smoking Smoker 8 (40%) 9 (45%) 0.465 Non-smoker 11 (55%) 8 (40%) X smoker 1 (5%) 3 (15%) Education Literate 15 (75%) 17 (85%) 0.347 Illiterate 5 (25%) 3 (15%) Residence Urban 13 (65%) 16 (80%) 0.480 Rural 7 (35%) 4 (20%) The onset of neuro- logical symptoms (Range) 11.95 ± 5.90 (4–24) –- F wave study was performed for the upper limb in the median nerve by placing the recording electrode at the abductor pollices brevis muscle. In contrast, the record- ing electrodes were placed two cm distal near the ten- don insertion. For testing the F wave in the posterior tibial nerve for the lower limb, recording electrodes were placed over the abductor hullucis muscle, and the stimu- lating electrodes were placed at the malleolus. For F wave recording, a supramaximal stimulus was performed, ten stimuli were given, and their average was recorded. For all parameters of neurophysiological studies, the values below the 95th percentile or ± 2SD of control are considered abnormal. Diffuse axonal neuropathy was diagnosed by the reduction of CMAP amplitude with standard shape and duration and with normal or mini- mal disturbance of nerve conduction velocity. In con- trast, diffuse demyelinating neuropathy was diagnosed by increased nerve conduction latency, preserved ampli- tude, and average nerve conduction velocity. Statistical analysis and sample size were performed using the SPSS program (version 20, IBM and Armonk, New York). The Mann–Whitney test was used for contin- uous data, while chi2 test compared nominal data. Differ- ent correlations of continuous variables in the study were assessed with Spearman’s correlation. The sample size was estimated by Open Epi V.3.01 computer program. Data expressed as frequency (percentage) and mean (SD). Results There was a significant sta- tistical difference between the two groups in Posterior tibial nerve F wave latency with a mean of 52.28 ± 4.95 and 47.25 ± 3.17 in the control group with a P value of 0.002 (Table 3). nerve (MNCL) with r = 0.611, p value < 0.001; sural nerve (SNCL) with r = 0.624, p value < 0.001; and posterior tib- ial nerve F wave latency with r = 0.544, p value < 0.001. There was a positive correlation between D-dimer level and median nerve (MNCL) with r = 0.702, p value < 0.001; median nerve (SNCL) with r = 0.590, p value < 0.001; median nerve F wave latency with r = 0.418, p value < 0.007; posterior tibial nerve (MNCL) with r = 0.493, p value < 0.001; sural nerve (SNCL) with r = 0.657, p value < 0.001; and posterior tibial nerve F wave latency with r = 0.557, p value < 0.001. We correlated the laboratory data and age with the neurophysiological parameters. There was a positive cor- relation between CRP level and median nerve (MNCL) with r = 0.787, p value < 0.001; median nerve (SNCL) with r = 0.668, p value < 0.001; median nerve F wave latency with r = 0.386, p value < 0.014; posterior tibial There was a negative correlation between oxygen saturation and median nerve (SNCL) with r = − 0.542, p value < 0.001; median nerve F wave latency with r = − 0.364, p value < 0.021; posterior tibial nerve (MNCL) with r = − 0.565, p value < 0.001; sural nerve (SNCL) with r = − 0.640, p value < 0.001; posterior tibial nerve F wave latency with r = − 0.546, p value < 0.004; and positive correlation between oxygen saturation and median nerve (MNCL) with r = 0.619, p value < 0.001; median nerve (SNCV) with r = 0.328, p value 0.039; and posterior tibial nerve (CMAP) with r = 0.312, p value 0.049) (Table 4).i Table 3 Electrophysiological measurements of the COVID-19 group with the peripheral neuropathy and control group (n = 40) Data expressed as frequency (percentage) and mean (SD). Results In the current study, we recruited twenty COVID-19 patients with confirmed diagnoses by RT-PCR with neu- rological symptoms and twenty genders and age-matched healthy volunteers.h The demographic characteristics of the patients and the control group are shown in (Table 1). There was no sig- nificant difference between the two groups regarding age, gender, education, smoking, and residence. The mean onset of neurological symptoms is 11.95 ± 5.9 days and ranges from 4 to 24 days. Table 2 Laboratory data and oxygen saturation level of the COVID-19 patients with peripheral neuropathy and control group (n = 40) Table 2 Laboratory data and oxygen saturation level of the COVID-19 patients with peripheral neuropathy and control group (n = 40) Data expressed as frequency (percentage) and mean (SD). P value was significant if < 0.05 CRP C-reactive protein, CRP C-reactive protein, SpO2 Saturation of peripheral oxygen COVID-19 group N = 20 Control group N = 20 P value CRP 39.7 ± 7.46 6.30 ± 2.15 < 0.001* D-dimer 983.6 ± 324.88 315.30 ± 63.03 < 0.001* SpO2 96.95 ± 1.35 98.5 ± 0.61 0.009* Data expressed as frequency (percentage) and mean (SD). P value was significant if < 0.05 COVID-19 group N = 20 Control group N = 20 P value CRP 39.7 ± 7.46 6.30 ± 2.15 < 0.001* D-dimer 983.6 ± 324.88 315.30 ± 63.03 < 0.001* SpO2 96.95 ± 1.35 98.5 ± 0.61 0.009* There were significant differences between the patient group and the control group regarding; CRP level, where the mean value in the COVID-19 group was 39.7 ± 7.46 and 6.3 ± 2.15 in the control group with a p value of < 0.001, D-dimer with a mean value in the patients’ group of 983.6 ± 324.88 and the control group of 315.3 ± 63.03 with a p value of < 0.001 and oxygen Shaddad et al. The Egyptian Journal of Bronchology (2023) 17:15 Shaddad et al. The Egyptian Journal of Bronchology Page 4 of 8 group with a P value of < 0.001. There was a significant statistical difference between the two groups in posterior tibial nerve motor nerve conduction latency (MNCL) with a mean of 3.99 ± 0.99 and 3.06 ± 0.61 in the control group with a P value of 0.007. Results P value was significant if < 0.05 MNC Motor nerve conduction, SNC Sensory nerve conduction COVID-19 group N = 20 Control group N = 20 P value MNC amplitude (mV) 8.88 ± 2.60 9.04 ± 2.12 Median nerve 5.12–13.5 6.43–17.10 0.039* MNC latency (ms) 5.1 ± 1.34 3.29 ± 0.64 Median nerve 2.59–7.45 2.16–4.78 < 0.001* MNC velocity (m/s) 58.06 ± 6.4 58.18 ± 7.30 Median nerve 42.40–69.30 45.92–70.9 0.220 SNC amplitude (mV) 21.18 ± 8.76 21.19 ± 5.94 Median nerve 11.5–45.4 12.45–38 0.091 SNC latency (ms) 4.33 ± 0.91 3.47 ± 0.72 Median nerve 3.08–6.20 2.76–5.23 0.305 SNC velocity (m/s) 51.7 ± 5.62 51.99 ± 6.84 Median nerve 42.9 – 59.24 41.29–69.2 0.753 F wave latency (ms) 33.03 ± 4.59 30.14 ± 6.16 Median nerve 26.6–39.3 24.56–44.56 0.389 MNC amplitude (mV) 12.30 ± 2.15 12.84 ± 2.70 Post Tibial nerve 7.39–17.53 8.24–19.1 0.366 MNC latency (ms) 3.99 ± 0.99 3.06 ± 0.61 Post Tibial nerve 2.25–5.6 2.29–4.22 0.007* MNC velocity (m/s) 56.6 ± 5.44 57.05 ± 5.24 Post Tibial nerve 49.2–69.1 51.2–68.3 0.703 SNC amplitude (mV) 28.1 ± 4.44 28.25 ± 4.44 Sural nerve 21.2–35.3 21.3–36.2 0.956 SNC latency (m/s) 3.81 ± 0.6 3.01 ± 0.59 Sural nerve 2.68–4.70 2.11–3.98 0.760 SNC velocity (m/s) 56.7 ± 8.25 57.15 ± 6.05 Sural nerve 43.1–76.2 47.1–68.4 0.281 F wave latency (ms) 52.28 ± 4.95 47.25 ± 3.17 Post Tibial nerve 43.2–61.6 42.3–52.1 0.002* Table 3 Electrophysiological measurements of the COVID-19 group with the peripheral neuropathy and control group (n = 40) Stratification of the motor nerve conduction study of the COVID-19 group revealed that 10% of patients had motor axonal neuropathy, 15% mixed motor neuropa- thy, and 55% demyelinating motor neuropathy, while 20% of patients with a normal study (Fig. 1). Assessment of the sensory nerve conduction study showed that 5% of patients had sensory axonal neuropathy, 15% mixed sen- sory neuropathy, and 45% sensory demyelinating neurop- athy, while 35% of patients with a normal study (Fig. 2). Discussion In the current study, we enrolled twenty mild to moder- ate COVID-19 patients with peripheral neuropathy and compared them with 20 age and sex-matched healthy controls. Neurophysiological studies revealed a signifi- cant difference between the two groups in compound motor action potential and motor nerve conduction latency of the median nerve and motor nerve conduction latency of the posterior tibial nerve and F wave latency of the posterior tibial nerve. There was a positive cor- relation between oxygen saturation and SNC velocity of the median nerve, the MNC amplitude of the post-tibial nerve. There was a strong negative correlation between SpO2 and MNC latency, SNC latency, and F wave latency of the median nerve. Also, strong negative correlation between SpO2 and MNC latency of the posterior tibial nerve, SNC latency of the sural nerve, and F wave latency of the posterior tibial nerve.h There was a strong positive correlation between D-dimer level and CRP level with MNC, SNC, and F wave Shaddad et al. The Egyptian Journal of Bronchology (2023) 17:15 Page 5 of 8 Table 4 Correlation of age, CRP value, D-dimer value, and SpO2 with nerve conduction study results in COVID-19 cases with peripheral neuropathy (n = 20) Correlation for variables in the study was determined with spearman’s correlation. r correlation coefficient rho. Discussion P value: was significant if < 0.05 CRP C-reactive protein, SpO2 Saturation of peripheral oxygen, MNC Motor nerve conduction, SNC Sensory nerve conduction Age CRP D dimer SpO2 r P R P R P r P MNC amplitude Median nerve 0.055 0.735 − 0.079 0.629 − 0.143 0.379 0.149 0.358 MNC latency Median nerve − 0.089 0.585 0.787 < 0.001* 0.702 < 0.001* 0.619 < 0.001* MNC velocity Median nerve − 0.128 0.431 − 0.049 0.766 − 0.048 0.769 0.008 0.959 SNC amplitude Median nerve − 0.007 0.966 − 0.212 0.189 0.113 0.489 0.112 0.492 SNC latency Median nerve − 0.003 0.988 0.668 < 0.001* 0.590 < 0.001* − 0.542 < 0.001* SNC velocity Median nerve 0.061 0.710 − 0.082 0.616 − 0.121 0.457 0.328 0.039* F wave latency Median nerve 0.137 0.399 0.386 0.014* 0.418 0.007* − 0.364 0.021* MNC amplitude Post-tibial nerve − 0.300 0.060 − 0.051 0.757 − 0.076 0.642 0.312 0.049* MNC latency Post = tibial nerve − 0.002 0.992 0.611 < 0.001* 0.493 0.001* − 0.565 < 0.001* MNC velocity Post-tibial nerve − 0.015 0.929 − 0.068 0.676 − 0.212 0.188 0.197 0.223 SNC amplitude Sural nerve − 0.149 0.358 − 0.111 0.494 − 0.065 0.690 0.132 0.417 SNC latency Sural nerve 0.018 0.914 0.624 < 0.001* 0.657 < 0.001* − 0.640 < 0.001* SNC velocity Sural nerve 0.202 0.211 0.004 0.980 0.005 0.978 0.002 0.98 F wave latency Post-tibial nerve 0.046 0.777 0.544 < 0.001* 0.557 < 0.001* − 0.446 0.004* Table 4 Correlation of age, CRP value, D-dimer value, and SpO2 with nerve conduction study results in COVID-19 cases with peripheral neuropathy (n = 20) association between the presence of neurological symp- toms and SARS-COV-2 infection [15]. latencies of the median nerve, MNC and F wave latencies of the posterior tibial nerve, and SNC latency of the sural nerve. Motor axonal neuropathy was observed in 10% of patients; motor demyelinating neuropathy was observed in 55%, and mixed motor neuropathy in 15%. Sensory axonal neuropathy was observed in 5% of patients; demy- elinating sensory neuropathy was observed in 45%, and mixed sensory neuropathy in 15%. latencies of the median nerve, MNC and F wave latencies of the posterior tibial nerve, and SNC latency of the sural nerve. Motor axonal neuropathy was observed in 10% of patients; motor demyelinating neuropathy was observed in 55%, and mixed motor neuropathy in 15%. Discussion Sensory axonal neuropathy was observed in 5% of patients; demy- elinating sensory neuropathy was observed in 45%, and mixed sensory neuropathy in 15%. In an attempt to explain the pathophysiology of neu- rological affection in SARS-COV-2 infection; the inter- national human cell atlas community has reported increased expression of two key co-receptors during SARS-COV-2 infection, namely ACE2 and TMPRSS2 co- receptors [16]. Another theory is the para-infectious neu- rological syndrome due to SARS-COV-2 infection [17]. Neurological affection is observed throughout the course of the COVID-19 infection. A study by Mao and his colleagues on 214 COVID-19 patients observed the presence of neurological manifestations in 36% of the study group [14]. Although SAR-COV-2 is not docu- mented to be present in CSF of affected patients dur- ing the course of COVID-19 infection; there is a strong A possible etiological explanation is the host immune response to SARS-COV-2 and the develop- ment of cytokine storms [18]. Peripheral nervous system and muscle affection is common through the course of SARS-COV-2 infection with a median of 7 days (range 7–24) [19]. Shaddad et al. The Egyptian Journal of Bronchology (2023) 17:15 Page 6 of 8 Shaddad et al. The Egyptian Journal of Bronchology Fig. 1 Frequency and types of motor neuropathies among COVID-19 groups (n = 20) Fig. 1 Frequency and types of motor neuropathies among COVID-19 groups (n = 20) Fig. 2 Frequency and types of sensory neuropathies among COVID-19 groups (n = 20) Fig. 2 Frequency and types of sensory neuropathies among COVID-19 groups (n = 20) 2nd wave patients and the demyelinating with second- ary axonal affection in 13% of the 1st wave patients and 10.5% of 2nd wave patients [21]. In agreement with our study, EL-Leithy et al. enrolled 60 COVID-19 patients in 3 groups (20 mild, 20 moderate, and 20 severe cases) with a significant difference in upper and lower nerve conduction study between the patient groups and the control group, which were significantly correlated to the CRP level [20]. Ellul and colleagues using data available up to May 19, 2020. COVID-19 cases based on Johns Hopkins COVID- 19 Dashboard demonstrated that peripheral neuropathy is present in 0.05% of COVID-19 patients [16]. Elshebawy et al. Limitations Long-term follow-up can be done in the future to iden- tify the long-term neurological effects of SARS-COV-2. 3. Desforges M, Le Coupanec A, Dubeau P, Bourgouin A, Lajoie L, Dubé M et al (2019) Human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system. Viruses 12(1):14 Abbreviations ACE-2 Angiotensin-converting enzyme 2 COVID-19 Coronavirus disease 2019 CRP C-reactive protein CMAP Compound motor action potential EMG Electromyography MOH Ministry of Health MNC Motor nerve conduction MNCA Motor nerve conduction amplitude MNCL Motor nerve conduction latency MNCV Motor nerve conduction velocity RT-PCR Reverse transcription-polymerase chain reaction SARS‐COV‐2 Severe acute respiratory syndrome coronavirus 2 SNC Sensory nerve conduction SNCA Sensory nerve conduction amplitude SNCL Sensory nerve conduction latency SNCV Sensory nerve conduction velocity SPO2 Saturation of peripheral oxygen SPSS Statistical Package for Social Sciences Acknowledgements None. Authors’ contributions AMS, AARMH, AMAT, and WGEK: conception and design. AMS, AMAT, and WGEK: data collection. AMS and WGEK: statistical analysis. AMS, AARMH, AMAT, and WGEK: medical writing. The authors revised the manuscript. The authors read and approved the final manuscript. Funding None. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Abbreviations ACE-2 Angiotensin-converting enzyme 2 COVID-19 Coronavirus disease 2019 CRP C-reactive protein CMAP Compound motor action potential EMG Electromyography MOH Ministry of Health MNC Motor nerve conduction MNCA Motor nerve conduction amplitude MNCL Motor nerve conduction latency MNCV Motor nerve conduction velocity RT-PCR Reverse transcription-polymerase chain reaction SARS‐COV‐2 Severe acute respiratory syndrome coronavirus 2 SNC Sensory nerve conduction SNCA Sensory nerve conduction amplitude SNCL Sensory nerve conduction latency SNCV Sensory nerve conduction velocity SPO2 Saturation of peripheral oxygen SPSS Statistical Package for Social Sciences 4. Andalib S, Biller J, Di Napoli M, Moghimi N, McCullough LD, Rubinos CA, O’HanaNobleza C, Azarpazhooh MR, Catanese L, Elicer I, Jafari M (2021) Peripheral nervous system manifestations associated with COVID-19. Curr Neurol Neurosci Rep 21:1–4 5. Koskderelioglu A, Eskut N, Ortan P, Ozdemir HO, Tosun S (2022) Visual evoked potential and nerve conduction study findings in patients recov- ered from COVID-19. Neurol Sci 1:1–9 6. Lüers JC, Klußmann JP, Guntinas-Lichius O (2020) COVID-19-pandemie und das HNO-Fachgebiet: Worauf kommt es aktuell an? (The COVID-19 pandemic and otolaryngology: what it comes down to?). Laryngorhi- nootologie 99(5):287–91 7. Funding None. 13. Wang HY, Li XL, Yan ZR, Sun XP, Han J, Zhang BW (2020) Poten- tial neurological symptoms of COVID-19. Ther Adv Neurol Disord 13:1756286420917830 Author details 1 1 Chest Department, Faculty of Medicine, Assiut University, Assiut 71515, Egypt. 2 Neuropsychiatry Department, Faculty of Medicine, Assiut University, Assiut 71515, Egypt. Declarations the 15th day of affection. Muscle injury was present in 1.37% of patients, and symmetrical lower limb distal sensory neuropathy in 1.55% of the patients [22]. the 15th day of affection. Muscle injury was present in 1.37% of patients, and symmetrical lower limb distal sensory neuropathy in 1.55% of the patients [22]. Received: 13 February 2023 Accepted: 4 March 2023 Received: 13 February 2023 Accepted: 4 March 2023 Received: 13 February 2023 Accepted: 4 March 2023 References 1. Di Gennaro F, Pizzol D, Marotta C, Antunes M, Racalbuto V, Veronese N, Smith L (2020) Coronavirus diseases (COVID-19) current status and future perspectives: a narrative review. Int J Environ Res Public Health 17(8):2690 2. Chen N, Zhou M, Dong X, Qu J, Gong F, Han Y, Qiu Y, Wang J, Liu Y, Wei Y, Xia J, Yu T, Zhang X, Zhang L (2020) Epidemiological and clinical char- acteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. Lancet. 395(10223):507–13 Competing interests h h d l h Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Discussion enrolled 42 patients; 23 of them were in the first wave of COVID-19, and 19 were in the sec- ond wave; the characteristics of the study groups showed the presence of acute inflammatory demyelinating neu- ropathy in 47.8% of the 1st wave patients, and 73.7 in the In a Cohort of Egyptian Patients with COVID-19, Mekkawy and colleagues described peripheral nerv- ous manifestations in 29.04% of patients early from the 1st day to 7 days of infection and late from the 8th to Shaddad et al. The Egyptian Journal of Bronchology (2023) 17:15 Page 7 of 8 Consent for publication Not applicable. Consent for publication Not applicable. Authors’ contributions 11. Qin C, Zhou L, Hu Z, Zhang S, Yang S, Tao Y (2020) Desregulación de la respuesta inmune en pacientes con coronavirus 2019 (COVID-19) en Wuhan, China. Enferm Infecc Infecc Clín 71(15):762–768 AMS, AARMH, AMAT, and WGEK: conception and design. AMS, AMAT, and WGEK: data collection. AMS and WGEK: statistical analysis. AMS, AARMH, AMAT, and WGEK: medical writing. The authors revised the manuscript. The authors read and approved the final manuscript. 12. Kotfis K, Williams Roberson S, Wilson JE, Dabrowski W, Pun BT, Ely E (2020) COVID-19: ICU delirium management during SARS-CoV-2 pandemic. Crit Care 24(1):1–9 Limitations Desforges M, Le Coupanec A, Stodola JK, Meessen-Pinard M, Talbot PJ (2014) Human coronaviruses: viral and cellular factors involved in neu- roinvasiveness and neuropathogenesis. Virus Res 19(194):145–158 7. Desforges M, Le Coupanec A, Stodola JK, Meessen-Pinard M, Talbot PJ (2014) Human coronaviruses: viral and cellular factors involved in neu- roinvasiveness and neuropathogenesis. Virus Res 19(194):145–158 8. Natoli S, Oliveira V, Calabresi P, Maia LF, Pisani A (2020) Does SARS-Cov-2 invade the brain? Translational lessons from animal models. Eur J Neurol 27(9):1764–1773 8. Natoli S, Oliveira V, Calabresi P, Maia LF, Pisani A (2020) Does SARS-Cov-2 invade the brain? Translational lessons from animal models. Eur J Neurol 27(9):1764–1773 9. Wu Y, Xu X, Chen Z, Duan J, Hashimoto K, Yang L et al (2020) Nervous sys- tem involvement after infection with COVID-19 and other coronaviruses. Brain Behav Immun 87:18–22 9. Wu Y, Xu X, Chen Z, Duan J, Hashimoto K, Yang L et al (2020) Nervous sys- tem involvement after infection with COVID-19 and other coronaviruses. Brain Behav Immun 87:18–22 10. Toljan K (2020) Letter to the editor regarding the viewpoint “evidence of the COVID-19 virus targeting the CNS: tissue distribution, host–virus interaction, and proposed neurotropic mechanism.” ACS Chem Neurosci. 11(8):1192–4 Ethics approval and consent to participate: The study was approved by the institutional review board and ethical com- mittee of the Faculty of Medicine—Assiut University in compliance with the Helsinki declaration (IRB: 04–2023-300077). Bagnato and colleagues evaluated 21 patients after COVID-19 infection; neuromuscular affection was demonstrated in 17 patients. Affection varied between critical illness myopathy (CIM), critical illness polyneu- ropathy (CIP), Guillain-Barré syndrome, and peroneal nerve injury [23]. Conclusion Neurological affection including peripheral neuropa- thy associated with SARS-COV-2 should not be over- looked. Affected personnel may be limited in number but during a massive pandemic, this can have a tremen- dous effect. Careful short-term neurological assess- ment is needed for symptomatic patients and can result in early detection and appropriate management. Availability of data and materials Bagnato S, Ferraro M, Boccagni C, Battaglia G, D’Agostino T, Prestandrea C, Bellavia MA, Rubino F (2021) COVID-19 neuromuscular involvement in post-acute rehabilitation. Brain Sci 11(12):1611 23. Bagnato S, Ferraro M, Boccagni C, Battaglia G, D’Agostino T, Prestandrea C, Bellavia MA, Rubino F (2021) COVID-19 neuromuscular involvement in post-acute rehabilitation. Brain Sci 11(12):1611 Availability of data and materials 14. Mao L, Wang M, Chen S, He Q, Chang J, Hong C, Zhou Y, Wang D, Li Y, Jin H, Hu B (2020) Neurological manifestations of hospitalized patients with COVID- 19 in Wuhan, China: a retrospective case series study. MedRxiv 25:2020–2102 The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Page 8 of 8 Shaddad et al. 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J Formos Med Assoc 119:1119–1120 20 El-Leithy SA, Mohamed HA, El-Bably MM (2021) Nerve conduction studies in post-COVID-19 patients among Egyptian patients. Int J Curr Res Rev. https://doi.org/10.31782/IJCRR.2021.131245 g 21. Elshebawy H, Ezzeldin MY, Elzamarany EH (2021) Characteristics of COVID and post-COVID polyneuropathies in adults and pediatrics: an Egyptian sample. Egypt J Neurol Psychiatry Neurosurg 57:1–7 21. Elshebawy H, Ezzeldin MY, Elzamarany EH (2021) Characteristics of COVID and post-COVID polyneuropathies in adults and pediatrics: an Egyptian sample. Egypt J Neurol Psychiatry Neurosurg 57:1–7 22. Mekkawy DA, Hamdy S, Abdel-Naseer M, Shehata HS, Halfawy AA, Shalaby NM, Shehata GA, Ali AM, Elmazny A, Ahmed SM, Ismail JH (2022) Neurological manifestations in a cohort of Egyptian patients with COVID- 19: a prospective, multicenter, observational study. Brain Sci 12(1):74 22. 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https://openalex.org/W2756415577	https://inria.hal.science/hal-01679783/document	English	null	UX Professionals’ Definitions of Usability and UX – A Comparison Between Turkey, Finland, Denmark, France and Malaysia	Lecture notes in computer science	2,017	cc-by	10,829	To cite this version: Dorina Rajanen, Torkil Clemmensen, Netta Iivari, Yavuz Inal, Kerem Rizvanoğlu, et al.. UX Profes- sionals’ Definitions of Usability and UX – A Comparison Between Turkey, Finland, Denmark, France and Malaysia. 16th IFIP Conference on Human-Computer Interaction (INTERACT), Sep 2017, Bom- bay, India. pp.218-239, 10.1007/978-3-319-68059-0_14. hal-01679783 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01679783 https://inria.hal.science/hal-01679783v1 Submitted on 10 Jan 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 UX Professionals’ Definitions of Usability and UX – A Comparison between Turkey, Finland, Denmark, France and Malaysia Dorina Rajanen1, Torkil Clemmensen2, Netta Iivari1, Yavuz Inal3, Kerem Rızvanoğlu4, Ashok Sivaji5, and Amélie Roche6 Dorina Rajanen1, Torkil Clemmensen2, Netta Iivari1, Yavuz Inal3, Kerem Rızvanoğlu4, Ashok Sivaji5, and Amélie Roche6 1 University of Oulu, Oulu, Finland {dorina.rajanen, netta.iivari}@oulu.fi 2 Copenhagen Business School, Copenhagen, Denmark tc.itm@cbs.dk 3Atilim University, Ankara, Turkey yavuz.inal@atilim.edu.tr 4Galatasaray University, Istanbul, Turkey krizvanoglu@gmail.com 5MIMOS Technology Solutions, Kuala Lumpur, Federal Territory / WP, Malaysia ashok.sivaji@mimos.my 6ENSC, Bordeaux, France amelie.roche@ensc.fr Abstract. This paper examines the views of user experience (UX) professionals on the definitions of usability and UX, and compares the findings between countries and within different socio-cultural groups. A mixed-method analysis was employed on data gathered on 422 professionals through a survey in Tur- key, Finland, Denmark, France, and Malaysia. Usability appears to be an estab- lished concept, respondents across all countries agreeing on the importance of the ISO 9241-11 definition. There is also a tendency that UX professionals at- tach organizational perspective to usability. UX professionals diverge when de- fining UX, and there are systematic differences related to socio-cultural condi- tions. UX professionals in Finland and France incline more towards the defini- tion highlighting the experiential qualities, when compared to Turkey and Ma- laysia that incline towards the definition reflecting the ease of use, utility, at- tractiveness, and degree of usage. Further research should address the implica- tions of the diverse meanings and contexts of usability and UX. Keywords: User experience ∙ Usability ∙ UX professional ∙ Cross-cultural HCI Keywords: User experience ∙ Usability ∙ UX professional ∙ Cross-cultural HCI adfa, p. 1, 2011. © Springer-Verlag Berlin Heidelberg 2011 1 Introduction 1 As Human Computer Interaction (HCI) communities emerge all over the world, user experience (UX) professionals may find themselves as leaders in an emerging field, who have the opportunity to spread the word and to establish its meaning and value for many stakeholders. Currently, the UX field is not clearly defined and profession- als’ roles and competences are positioned along a continuum between the pure user- research for understanding and the applied design of objects, systems, or interactions1. Despite having established standards that define usability (ISO 9241-11; [1]) and UX (ISO 9241-210; [2]), HCI has so far failed to establish solid consensus about a scientific definition of usability and UX. The discipline of HCI appears to have ac- cepted various loosely defined notions of usability, see e.g., [3]. For UX, the contro- versy about the scientific use of the concept is even more obvious. Sustained efforts over the years have aimed at defining UX (see e.g., [4,5,6,7]), connecting UX to exist- ing HCI theory (e.g., [8,9,10]), or connecting UX to system development and design literature (e.g., [11,12,13]). However, it is fair to say that UX as a research area still can neither define the concept of UX, agree on how to capture the experiential quali- ties, or provide unified guidelines for experience design [7]. Our aim is to contribute to the clarification of the use of key concepts in the UX community. To this end, we focus on how UX professionals define usability and UX and on the socio-cultural factors that may influence the UX professionals’ perspec- tives. By socio-cultural factors, we refer to certain demographic and professional background variables that have been shown to influence the way usability and UX professionals understand and apply usability and UX concepts in their work, such as gender, educational background, country of work, job titles, hierarchical positions in the organization. Based on a survey conducted in five countries, this paper shows that UX professionals agree on the ISO definition of usability, but diverge when defining UX, and that there are systematic differences related to socio-cultural conditions. 2 UXPA (The User Experience Professionals Association). 1 http://interactions.acm.org/blog/view/ux-research-vs.-ux-design 2 1 http://interactions.acm.org/blog/view/ux-research-vs.-ux-design 2 UXPA (The User Experience Professionals Association). 3 ACM SIGCHI (ACM Special Interest Group on Human-Computer Interaction). 4 In this paper, quality in use has the same meaning as the broad view of usability expressed in ISO 9241-11 [1], in conformance also with [15,16,17]. 2 UX communities in Turkey, Malaysia, France, Finland and Denmark In this paper, we compare views of UX professionals from UX communities in Tur- key, Malaysia, France, Finland, and Denmark as these together represent geographic and cultural diversity. We relied on convenience sampling, executing the study in countries of the researchers showing initial interest in this study. However, we also intentionally included diversity into the sample. The selection includes countries with an extensive background in HCI (Finland, Denmark) and in ergonomics (France), as well as countries with a relatively recently established UX community (Turkey, Ma- laysia). Moreover, we intentionally wished to include cultural diversity into the sam- ple and tried to locate countries representing variety in terms of geographical position such as North-European, Central-European, South-East-European, and Asian. In Turkey, the dominant UX community is UXPA Turkey Chapter, which was launched in 2014 in Istanbul as a non-profit local chapter of the global UXPA2 to serve interaction designers, usability/UX professionals, HCI specialists, etc. In the email list of UXPA Turkey, there are more than 500 recipients, which present a varie- ty in terms of professional practice. In Malaysia, there is a recently established Human Computer Interaction Special Interest Group (SIGHCI) under the Human Factors and Ergonomics Society of Ma- laysia. The SIG plans to work with other technical committees and institutions in the development of usable products and services. In addition, UX Malaysia is an active and the largest UX-related social media group in Malaysia, comprising of UX practi- tioners in Malaysia and around the world. Founded in 2012, the group consists of 1897 members on Facebook. Another Facebook group known as SIGHCI Malaysia comprising of 75 members promotes HCI activities among Malaysian universities. In France, FLUPA (France-Luxembourg User Experience Professionals’ Associa- tion) was founded in 2008 as the France-Luxembourg branch of UXPA. In the email list, there are more than 500 recipients. In addition, Ergo IHM is a mailing list availa- ble in French community that reaches more than 800 HCI professionals and students. 3 In Finland, there is an ACM SIGCHI3 Chapter, namely SIGCHI Finland, founded in 2001. SIGCHI Finland is a scientific association that aims at gathering together researchers and practitioners in HCI, usability, and user experience in Finland. The email list includes around 450 recipients. In addition to SIGCHI Finland, there are several practitioner-oriented communities operating in Finland: IxDA Helsinki, IxDA Tampere, and KäytettävyysOSY, all having dedicated Facebook and LinkedIn groups that include several hundred members. 2 UX communities in Turkey, Malaysia, France, Finland and Denmark In Denmark, the dominant UX community is Sigchi.dk, which in 2015 changed its name to UX Denmark. Sigchi.dk (uxdanmark.dk) is associated with ACM SIGCHI and UXPA, but not a formal chapter of any of those. Sigchi.dk was launched in 1999 as a web site for interaction designers, usability professionals, HCI specialists, and so forth. The website uxdanmark.dk has about 1348 registered members from industry, government, and academia. The UX Denmark social media groups (LinkedIn UX Denmark and Facebook SIGCHI.dk page) have each about 491 members. 3.2 HCI definitions of UX The ISO 9241-210 [2] defines UX as “a person's perceptions and responses that result from the use or anticipated use of a product, system or service”. UX focuses on the individual experience in relation to the use of a product, rather than on the effective- ness and efficiency of achieving a goal in a context of use of a product [14], [20,21]. Bevan et al. [14], however, points out that satisfaction as an aspect of usability in- cludes aspects of UX and this clarification is to be added to a future revision of ISO 9241-11. Early efforts in HCI to formulate a shared UX definition for academic re- search (see e.g., [4]) ended up formulating a gap between those UX professionals who view UX as related to design issues and those who view UX as something to measure or capture [6,7]. Moreover, while some definitions of UX (e.g., the ISO standard for UX [2], [19]) explicitly mention the use of an interactive system, product, or service, other HCI researchers (and Don Norman5) focus on human experience with technolo- gy (see e.g., [23]). Moreover, while the original meaning of UX refers to momentary evaluation (see [20], [22]), Kujala et al. [22] explicitly aim at the evaluation of long- term experience with an interactive system, product, or service. Accordingly, there can be a difference in conceptualizing UX due to the scope; some definitions focus on (momentary) experiences of interaction with technology (e.g., [23]), while other focus on experience with long-term use and/or interaction with an interactive system, prod- uct, or service (see e.g., [22]). When referring to the long-term use of interactive sys- tems, product, and services, Kujala et al. point out the following UX attributes: attrac- tiveness of the system, ease of use, utility, and degree of usage [22]. This definition is referred to as system-oriented perspective of UX [24]. On the other hand, when refer- ring to interaction with technology, McCarthy and Wright define UX by four threads of experience: compositional, sensual, emotional, and spatio-temporal [23]; this view is referred to as human-oriented definition of UX [24]. 3.1 HCI definitions of usability Usability is a concept that stems from the research in ergonomics done in 80’s on the interactive systems, and gradually evolved into a definition of quality in use [14]. The current standard definition of usability adopted by the HCI community (ISO 9241- Part 11; [1]) reflects quality in use4 and stresses out the outcome the users gain by interacting with a system [14]. This definition states that usability is “the extent to which a product can be used by specified users to achieve specified goals with effec- tiveness, efficiency and satisfaction in a specified context of use” [1]. This definition and the definitions of each of its three aspects are supposed to be a common reference for HCI researchers and UX professionals alike. However, the meaning of the usabil- ity construct and its implications for how to measure usability appear to be undecided in HCI discipline (see e.g., [18]). Accordingly, studies of correlations among usability aspects have been a standard way to try to define usability, see e.g., [19], though not with much success. A meta-analysis of usability studies indicated diversity in conclu- sions on if and how different aspects of usability were correlated [17]. Hertzum [3] describes six different perspectives on usability: universal usability, situational usabil- ity, perceived usability, hedonic usability, organizational usability, and cultural usa- bility. While these six perspectives on usability have a shared essence, they differ in focus, scope, mindset, and the methods most appropriate for working with usability. 5 https://www.nngroup.com/articles/definition-user-experience/ 3.3 How usability and UX are construed by UX professionals Previous studies on how usability and UX are construed by UX professionals have examined system developers, users, and UX professionals’ operational understanding of usability and UX [24,25,26]. The focus in these studies was on these stakeholders’ understanding in use, which is different from giving definitions and explaining a con- cept such as usability and UX. In these studies [24,25,26], seventy-two participants across Europe (Denmark), India, and China elicited their personal constructs of quali- ty in use in the context of using their computers in everyday life; e.g., how they thought about the use of their own email system. The studies employed the repertory- grid method (see [27]). The findings [24,25,26] showed differences in how UX pro- fessionals think about their own user experiences, compared to how developers and users think about theirs. The differences included that UX professionals in general focused more on describing the human user, and in particular more on the human subjective user experience, than the two other stakeholder groups who focused more on the systems and the context of use. Interestingly, UX professionals were not as concerned with the context of use as the users. Clemmensen et al. [24] also found that all four UX conceptual classifications that were used to do content analysis of the participants’ answers (i.e., ISO 9241-210 user experience, objective vs. subjective UX, system-oriented UX, and human-experience of technology) could capture most of what participants said about their system use. In contrast, various views on usabil- ity definitions (i.e., ISO 9241-11 definition, utilitarian vs. experiential view, organiza- tional usability, and user experience) turned out to be hard to fit to half of what the participants said about their use of their own systems (see [25]). Thus, it was found that the concept of usability as described in the literature appeared to be much nar- rower than UX when trying to fit it to the words that system developers, end users, and UX professionals use to construe quality in use in the context of their own use of computers in everyday life. 3.4 Socio-cultural factors shaping UX professionals’ understanding and work practices 3.4 Socio-cultural factors shaping UX professionals’ understanding and work practices HCI research has revealed that various socio-cultural factors affect perception and practice of usability and UX. Previous studies conducted on Danish, Chinese, and Indian UX professionals showed that nationality has an influence on the way UX professionals think about and perceive usability and UX [24,25,26]. A study on usa- bility practices in game development in North-European countries (mostly Sweden, Finland, and UK) showed that more than 80% of Finnish game companies employ usability testing as compared to about 50% in the other surveyed countries [28]. A survey conducted in 2011 showed that while the UX practice has gained more atten- tion in Malaysia, UX professionals are new to the terminologies of usability and UX [29]. Research with users also found that nationality affects the way usability is un- derstood by users (e.g., Danish users emphasizing effectiveness and efficiency, while Chinese users, visual aspects [30]), and perceived (e.g., US users perceiving lower levels of user satisfaction, effectiveness and efficiency than Taiwanese users [31]). Cross-cultural usability studies also indicate that cultural issues shape UX work practices. Cultural factors influence usability evaluations [32,33,34] and participatory design sessions [35,36], as shown in studies carried out in different countries. Also organizational culture differences have been argued to shape UX work practices: usa- bility is understood and practiced in different ways in organizations with cultural differences [37,38]. Studies also show that very surprising and negative views may be attached to usability in organizations [39,40]. Factors defined by the professional profile such as educational background and ex- perience level have also been shown to influence the work practice. For example, the experience level of UX professionals may shape the outcomes of their work, such as in usability evaluation [41]. There is also a lot of diversity in the education of UX professionals – a multitude of disciplines contribute to and are relevant in UX work and this goes for the field of HCI overall (see e.g. [42,43,44]). Clemmensen [45] found that the UX community in Denmark mainly consisted of young people with less than ﬁve years of experience with usability work, and had an education in the social sciences or the humanities rather than a technical ﬁeld. Most respondents in the sur- vey had a keen interest in communication or participatory design. 3.4 Socio-cultural factors shaping UX professionals’ understanding and work practices Furthermore, the UX profession includes a variety of job titles emphasizing one or another aspect of work, and a variety of roles in the system development cycle. The label UX professional may refer to usability/UX designers, researchers, managers, engineers, and others (see e.g., [43], [46,47]). Such a variety in job titles indicates that these professionals may be engaged with very different concerns in their work. A review [48] of ISO standards that address usability evaluation pointed out that ISO 9241-11 [1] targeting especially usability and UX professionals, provides guidelines on usability evaluation in various stages of system development such as the require- ments, design, development, and use, but not in the post-implementation (mainte- nance) stage. On the other hand, ISO 13407 [49] (revised under ISO 9241-210 [2]) targeting designers of interactive systems, does not guide evaluation during the im- plementation stage. Standards targeting IT professionals, including software engi- neers, then again, refer also to evaluation at post-implementation stages such as sup- port and maintenance [48]. Sivaji et al. [50] found that also gender has an effect on the effectiveness of a method used in usability evaluation, in particular when gender interacts with the so- cial status of the users performing the evaluation. 3.5 This study Given the diversity of perspectives on usability and UX pointed out in our review, it is expected that different UX professionals may prefer different definitions of these concepts. Moreover, the diversity of socio-cultural conditions, which characterize and influence UX professionals’ mindset and work practices, is expected to be also re- flected in the diversity of ways UX professionals conceptualize usability and UX. In this study, we refer to socio-cultural conditions as being defined by the country of work, gender, educational background, experience level, hierarchical position in or- ganization, job title, role in system development, and similar other variables that form the demographic and professional background of UX professionals. These variables represent social and cultural factors that influence people’s mindset, attitude, and practices related to their profession. Based on the related work, we maintain that if there are common understandings of usability and UX among professionals, it is not clear which are the shared understand- ings or how UX professionals define these concepts. There seems to be many aspects that may be shaping these understandings: there may be a difference in understanding related to the history of UX within a country, and to the profile (gender, educational background, job title, and design process participation, etc.) UX professionals have. In this study, we inquire these understandings and definitions and their relations to the socio-cultural factors. We are especially interested in examining whether there are differences that can be accounted by the local communities’ different history in HCI and different cultural background as defined by the country of work. The overall aim is to clarify the use of key concepts in the UX community. Our research questions are:  RQ1: How usability is defined by UX professionals?  RQ2: How UX is defined by UX professionals?  RQ3: Does country of work have an impact on the way UX professionals define usability and UX?  RQ4: Do other socio-cultural factors than country of work (e.g., demographic and professional background) impact the way UX professionals define usability/UX? 4.1 Research design An online survey was administered over a period of eight weeks from January to March, 2016. Data were collected from the UX professionals working in Turkey, Denmark, France, Finland, and Malaysia. The survey was distributed in local lan- guages through the UX communities’ mailing lists and social media of each country. 4.2 Variables Dependent variables. To measure and capture UX professionals’ definitions and understanding of usability and UX, we asked the respondents to choose their position on a scale between two polar versions of usability and UX, respectively (see Table 1 and Table 2). For usability we chose the ISO 9241-11 definition [1] versus the Elliott and Kling’s organizational perspective on usability [51] (see also [3], [52]). The idea was that though the ISO definition is widely known, UX professionals working in companies and large organizations may prefer the organizational usability definition. Moreover, Bevan et al. [14] pointed out that organizational perspective should be included in the next revision of ISO 9241-11. For UX, we chose two definitions that have a different focus; the first is based on Kujala et al. [22], which is in line with ISO 9241-210 [2] and reflects a system- oriented definition of UX [21]. The second represents the McCarthy and Wright’s view on UX and focuses on the experience of interaction with technology [23], and represents a human-oriented view of UX [24]. As McCarthy and Wright’s view [23] is more on the human experience of using technology, we expected that as practition- ers, UX professionals would clearly prefer the system-oriented definition of UX. Regarding the capture of usability and UX understandings, respondents were also invited to provide their own definitions, which resulted in a relatively large amount of qualitative data to be coded and analyzed. Table 1. Definitions of usability rated in the survey Definitions of usability Based on 1: Usability describes how a product can support its users to be effective, efficient and satisfied in its use. ISO 9241-11, 1998 [1] 2: Usability describes the match between the product and the organization adopting it. Elliot & Kling, 1996 [51] Table 2. Definitions of UX rated in the survey Definitions of UX Based on 1: UX is the perceived attractiveness, ease of use, utility, and degree of usage of the product. Kujala et al., 2011 [22] 2: UX is the combined experience of the composition of the ele- ments, sensory qualities, related emotions, and the context. McCarthy & Wright, 2004 [23] Table 1. Definitions of usability rated in the survey Independent variables. 4.2 Variables To answer the research questions, the socio-cultural factors acting as independent variables were captured in terms of demographics (e.g., age, gender, education, occupation status, graduation field), and professional profile (HCI education, work experience, UX knowledge, and job characteristics such as job title, job position, stage in system development when involved). 4.3 Sample The target participants were practitioners who would self-identify as usability/UX professionals; they had to be knowledgeable about usability and UX in order to be able to answer the questions about their background. We aimed to include both in- house UX professionals and external consultants, and we had a question where partic- ipants had to identify as one of these groups. At the same time, our participants should have a local association, a country of work, so people from e.g., Norman Nielsen and other similar groups should participate in the survey only if they had a presence in the countries we aimed to include. To ensure the best sampling, we used local UX groups’ email lists, social media groups, and – to a wide extent – our own and our colleagues’ personal networks, so we utilized theory-based convenience sampling. 4.5 Data analysis For the data analysis, we employed a mixed-method approach. The quantitative data were coded to allow statistical data analysis in SPSS. Variables were not normally distributed, thus we used the Mann-Whitney test to compare the importance ratings given by the respondents in different countries. Wilcoxon signed-rank test was em- ployed to test whether the two definitions elicit significantly different importance scores within the same socio-cultural group (e.g., same hierarchical position). The qualitative data – obtained from open-ended questions – were coded in Excel and Nvivo using the content analysis method. In both categories of definitions, usabil- ity and UX, we labeled different aspects (attributes, descriptions, and perspectives) that were pointed out in previous studies (e.g., [3], [14], [24,25,26]). Moreover, other aspects that emerged in a data driven manner during the qualitative analysis of the definitions were also extracted such as subjectivity and objectivity of the constructs, and the customer perspective of usability and UX. 4.4 Questionnaire The questionnaire contained 62 questions that aimed to gather information related to the following seven categories: organization and work environment, usability/UX understanding, usability/UX activities and tools utilized in work, integration of usabil- ity/UX work, usability/UX communities, usability/UX in the country of work, and background including demographics and professional information. In this paper, in order to answer the research questions, we report data from 19 questions that focused on usability/UX understanding and on the background information. 5.1 Demographic and professional profile of UX professionals A total of 422 valid participants were retained for analysis after cleaning the data. The professionals are relatively mature regarding age (median = 34; M = 35.2; SD = 8.3), and have in average 5 years of experience in UX field (median = 5; M = 6.7; SD = 5.6). The average experience in the current job title position is 3 years (median = 3; M = 4.3; SD = 4.6). The age ranges from 19 to 66 years, and the total work experience from 0 to 43 years (median = 10; M = 11.2; SD = 7.9), thus the sample represents a wide and heterogeneous population of UX professionals in terms of age and work experience. The sample distribution by country was as follows: 21.3% of participants were from Turkey, 11.6% from Denmark, 15.4% from France, 20.4% from Finland, and 29.1% from Malaysia (see Table 3). Ten (2.4%) were classified as “Other” be- cause the respondents belonged to different countries than where the survey was con- ducted (e.g., Germany, Mexico). Among participants, 4.7% were of foreign nationali- ty relative to the survey country and 90.3% were locals; the rest did not disclose their nationality. Table 3 presents the demographics of the UX professionals participating in our survey; the first data column presents the figures for the entire sample, the second and third summarize the characteristics of the UX professionals who provided free-form definitions of usability and UX in the open-ended questions. The respondents who provided free-form definitions have similar profiles as the whole sample; however, regarding the country distribution, France is slightly more represented in the free- form definitions. Table 3. 5.1 Demographic and professional profile of UX professionals Demographic profile of UX professionals (N = 422) Entire sample (N = 422) Provided Usabil- ity definition (N =120) Provided UX definition (N = 104) n % n % n % Country of work Denmark 49 11.6 9 7.5 8 7.7 Finland 86 20.4 20 16.7 22 21.2 France 64 15.2 39 32.5 32 30.8 Malaysia 123 29.1 30 25.0 23 22.1 Turkey 90 21.3 18 15.0 16 15.4 Other 10 2.4 4 3.3 3 2.9 Gender Female 188 44.5 59 49.2 50 48.1 Male 213 50.5 61 50.8 54 51.9 Missing 21 5.0 0 0 0 0 Occupation status Employed 352 83.4 97 80.8 83 79.8 Freelance 16 3.8 7 5.8 5 4.8 Entrepreneur 30 7.1 6 5.0 7 6.7 Other 24 5.7 10 8.3 9 8.7 Education level Basic or diploma 21 5.0 1 .8 2 1.9 Bachelor degree 118 28.0 32 26.7 29 27.9 Master degree 213 50.5 72 60.0 63 60.6 PhD degree 49 11.6 15 12.5 10 9.6 Missing 21 5.0 0 0 0 0 Graduation field Computer/informationa 136 32.2 34 28.3 28 26.9 Media/communicationb 56 13.3 19 15.8 20 19.2 Psychology 29 6.9 15 12.5 8 7.7 Arts 23 5.5 6 5.0 4 3.8 Business/managementc 22 5.2 3 2.5 2 1.9 Electronic/automationd 18 4.3 2 1.7 2 1.9 Other 115 27.3 40 33.3 40 38.5 Missing 23 5.5 1 .8 0 0 Notes: a Computer and information sciences; b Media and Communication; c Business and management; d Electronic, automation and communication engineering, Electronics. Notes: a Computer and information sciences; b Media and Communication; c Business and management; d Electronic, automation and communication engineering, Electronics. Table 4 presents the professional profile of the survey respondents. Most UX pro- fessionals are involved in early stages or all stages of system development. Early stages included kick-off or initialization, requirements, and design. Late stages in- cluded development, testing, and implementation phases. Post-implementation was coded as not really involved in the development. According to their self-evaluation, the participants have medium or higher level of knowledge on UX, and most of them keep up with the evolution of the field by using different information media such as conferences, courses, books, blogs, magazines, and scientific articles. Most UX pro- fessionals have one or two types of formal HCI education such as HCI courses, the- ses, and/or project experience. The most common hierarchical position are lower or middle management, and top management. 5.1 Demographic and professional profile of UX professionals Regarding the job titles, there is a balance between titles specifying usability and UX (such as usability or UX specialist) and those that do not (such as product manager or service designer). Similar distributions are found in the groups providing own usability and UX definitions; however, a larger proportion of usability/UX jobs are found among the providers of UX definitions. Table 4. Professional profile of UX professionals (N = 422) Table 4. Professional profile of UX professionals (N = 422) Entire sample (N = 422) Provided Usa- bility definition (N =120) Provided UX definition (N = 104) n % n % n % HCI formal education No formal HCI 16 3.8 5 4.2 4 3.8 1 type 179 42.4 47 39.2 40 38.5 2 types 57 13.5 22 18.3 18 17.3 3 or more types 57 13.5 17 14.2 19 18.3 Missing 113 26.8 29 24.2 23 22.1 UX vocational education No vocational UX training 318 75.4 90 75.0 78 75.0 Vocational UX training 82 19.4 30 25.0 26 25.0 Missing 22 5.2 0 0 0 0 Job title UX or usability in job title 192 45.5 58 48.3 60 57.7 No UX or usability in title 199 47.2 61 50.8 43 41.3 Missing 31 7.3 1 0.8 1 1.0 Job hierarchy Entry level 34 8.1 8 6.7 6 5.8 Specialist 16 3.8 7 5.8 6 5.8 Lower/middle management 106 25.1 33 27.5 26 25.0 Top management 66 15.6 16 13.3 14 13.5 Outside hierarchy or other 26 6.2 13 10.8 8 7.7 Missing 174 41.2 43 35.8 44 42.3 Keeping up with UX field Keep up 355 84.1 112 93.3 98 94.2 Do not keep up 46 10.9 8 6.7 6 5.8 Missing 21 5.0 0 0 0 0 UX expertise level Novice 36 8.5 8 6.7 7 6.7 Little expertise 35 8.3 11 9.2 6 5.8 Medium expertise 119 28.2 45 37.5 30 28.8 Considerable expertise 149 35.3 38 31.7 38 36.5 Expert 62 14.7 18 15.0 23 22.1 Missing 21 5.0 0 0 0 0 Stage in system develop- ment (SD) Not involved in SD 20 4.7 5 4.2 3 2.9 Late stage 19 4.5 1 0.8 1 1.0 Early stage 174 41.2 52 43.3 44 42.3 All stages 206 48.8 61 50.8 55 52.9 Missing 3 0.7 1 0.8 1 1.0 5.2 Usability understanding Answering RQ1, “How usability is understood/defined by UX professionals?”, we found that most professionals (77.8% of 414 respondents who rated the definitions) preferred the ISO 9241-11 definition [1] rather than the organizational usability defi- nition by Elliott and Kling [51] (see Table 5). However, a relatively large number of respondents (76; 18.4%) found both definitions important. A substantial number of participants (120; 28%) have commented the existing def- initions or entered their own definitions on usability. Content analysis of the free-form usability definitions showed that 108 (90%) of the answers described usability by different attributes of usability or of usable systems/products/services (e.g., “effi- ciency”, “easy to use”), or requirements of usability or of usable systems/ products/ services (e.g., “you can use it without instructions”). These descriptions were ex- pressed as standalone definitions, explanations, or additions to the definitions 1 and 2 provided in the questionnaire. The rest of free-form answers (10%) were comments on the survey definitions, reference to standards, or some other personal insights about usability that were not interesting from the research point of view. Among the words used to describe usability, the most predominant were efficien- cy, effectiveness (also utility, usefulness, and helpfulness), functionality, ease of use (learnability, accessibility, cognitive load), accomplishing (user, business) goal, meet- ing needs, requirements and expectations of the user or business/organization. There were also references to attributes related to emotions and feelings (satisfaction, pleas- antness, stress-free, emotional load, enjoyable). In the free-form answers, 13 respond- ents referred to the concept of experience (use experience, user experience, and ser- vice experience) when discussing the concept of usability. Respondents also stressed that usability is a subjective and/or objective quality. Moreover, respondents pointed out that usability should also take into account the business/organization needs and goals and that usability is not only about users, but also about customers highlighting that users’ and customers’ requirements “are not always the same thing”. Table 5. Which definition is the most important? (N = 414) Table 5. Which definition is the most important? (N = 414) Ratings for Usability n (%) Ratings for UX n (%) Definition 1 is the most important 231 (55.8) 70 (16.9) Definition 1 is somewhat more important 91 (22.0) 44 (10.6) Both definitions are equally important 76 (18.4) 131 (31.6) Definition 2 is somewhat more important 12 (2.9) 92 (22.2) Definition 2 is the most important 4 (1.0) 77 (18.6) 5.3 UX understanding Answering RQ2, “How UX is understood/defined by UX professionals?”, we found that when contrasting the Kujala et al. [22] and McCarthy & Wright’s [23] defini- tions, participants were not in a consensus on the importance of these two definitions (Table 5). Many participants found both definitions important (31.6%). A higher pro- portion of respondents (40.8%) inclined towards the second definition highlighting the sensorial and emotion-related qualities. Overall, according to Sign test, the second definition was rated as statistically significantly more important across all data (Z = - 3.14; p = 0.002); however, according to Wilcoxon signed-rank test which takes into account also the magnitude of the differences between two paired scores, the prefer- ence towards the second definition failed to reach statistical significance at 0.05 (Z = - 1.69; p = 0.09). p ) Participants provided 104 definitions and/or clarifications as free-form answers, representing about a quarter of the total respondents (see Tables 3 and 4). Content analysis of the free-form UX definitions showed that 83 (80%) of the answers were descriptions of UX that referred to or reflected attributes or requirements of UX and/or of systems/products/services. We grouped the attributes and/or requirements in the following categories: 1) formal and aesthetic; 2) performance/operation-related; 3) information related, 4) emotion, feelings and cognitive; 5) experience related; and 6) other. The most predominant characterizations were those invoking emotions, feel- ings, experiences, performance and usability attributes. It was observed that, when describing UX with own words, UX professionals still addressed the performance and operation qualities of the product such as ease of use and effectiveness. We found also that professionals considered usability as a quality of UX and as being part of UX. Moreover, it was pointed out that the product has to match the goals, needs, and ex- pectations of the users. As anticipated, emotion- and experience-related qualities were frequently mentioned among the descriptions of UX. Interestingly, but not entirely unexpectedly, UX professionals pointed out descriptors such as fashion and branding, that we grouped in the category of formal and aesthetic qualities, and descriptors such as memory trace, sense making and meaning creation that we grouped with the emo- tional and cognitive attributes. Not the least, the references to business value, custom- ers, and company’s marketing strategy indicate the broad view on UX that transcends the boundaries of users’ satisfaction and reaches out to the company’s returns. 5.3 UX understanding When describing what UX is, respondents utilized various terms and conceptuali- zations such as: UX is (about) emotions/ feelings/ perception/ understanding of the user, UX is (about) (use/user) (overall/entire) experience, UX is a (user centered de- sign) method/methodology. Other participants referred to UX as being attribute(s) (related to the systems) such as satisfaction, ease of use, suitability, etc. (see above), a process, all aspects/dimensions of use/interaction, and results/effects/reactions. Fur- ther, other characterizations were found in terms of business branch, memory trace, aesthetic elements, and adaptation of tool to user. Regarding the organizational and business perspective, five respondents pointed out concepts such as business needs, company marketing’s strategy, business out- comes, and three respondents brought up the customer’s perspective by stating that the UX is defined by the customer, UX has to be designed in accordance with the customer requirements, and UX impacts customer’s system use. 5.4 Country specific usability and UX Finland 2915.500 0.004 Turkey Finland France vs. Malaysia 2503.000 0.000 Malaysia France Finland vs. Malaysia 4157.500 0.007 Malaysia Finland Denmark vs. France 1036.500 0.061 Denmark France France vs. Finland 2328.500 0.097 Finland France 5.5 Impact of demographic and professional profile on usability and UX understanding In answering RQ4, the Wilcoxon signed-rank test showed that, with regard to usabil- ity, there was a clear and significant consensus towards Definition 1 across all socio- cultural groups. However, with respect to UX, there was no clear consensus towards one definition across the social-cultural groups, thus different social-cultural profiles had different preferences towards the UX definitions as shown in Table 9. The upper part of Table 9 shows the profiles that rated Definition 2 as being more important. The lower part of table shows profiles that inclined towards Definition 1. UX profes- sionals involved early in system development (SD) or not really involved in SD showed a preference towards Definition 2, while people involved in late stages had a Table 6. Rating of Usability definition (% by country) Table 6. Rating of Usability definition (% by country) Denmark Finland France Malaysia Turkey Definition 1 is the most important 63.4% 59.3% 46.9% 57.7% 54.4% Definition 1 is somewhat more important 12.2% 31.4% 21.9% 13.0% 27.8% Both definitions are equally important 22.0% 9.3% 26.6% 24.4% 11.1% Definition 2 is somewhat more important 2.4% 0% 3.1% 4.9% 3.3% Definition 2 is the most important 0% 0% 1.6% 0% 3.3% Total % 100.0% 100.0% 100.0% 100.0% 100.0% Table 7. Rating of UX definition (% by country) Denmark Finland France Malaysia Turkey Definition 1 is the most important 17.1% 8.1% 6.3% 22.8% 25.6% Definition 1 is somewhat more important 9.8% 17.4% 4.7% 6.5% 13.3% Both definitions are equally important 29.3% 24.4% 31.3% 39.0% 31.1% Definition 2 is somewhat more important 22.0% 26.7% 25.0% 22.0% 15.6% Definition 2 is the most important 22.0% 23.3% 32.8% 9.8% 14.4% Total % 100.0% 100.0% 100.0% 100.0% 100.0% Table 8. Significant and near significant differences among countries Rating of UX Definitions Mann-Whitney U p Definition 1 more preferred by Definition 2 more preferred by Turkey vs. France 1773.500 0.000 Turkey France Turkey vs. Finland 2915.500 0.004 Turkey Finland France vs. Malaysia 2503.000 0.000 Malaysia France Finland vs. Malaysia 4157.500 0.007 Malaysia Finland Denmark vs. France 1036.500 0.061 Denmark France France vs. 5.4 Country specific usability and UX For answering RQ3, “Does country of work have an impact on the way UX profes- sionals understand/define usability and UX?”, we tested whether there were any sig- nificant differences in rating the importance of usability and UX definitions between countries. Table 6 shows the ratings of the usability definitions by country. There was a clear agreement among countries that the ISO definition of usability (Definition 1) is more important than the organizational definition; however, there were variations in the degree of importance and at those agreeing with both definitions. According to the Mann-Whitney test, there was a significant difference in the ratings between Finnish and French UX professionals, the former had stronger preferences towards the ISO definition compared to French respondents (U = 2184; p = 0.017). Table 7 shows the ratings of UX definitions by country and the medians of each definition’s ratings. There were statistically significant differences between France and Finland, on one hand, and Malaysia and Turkey, on the other hand (see Table 8). Turkey and Malaysia significantly preferred Definition 1 highlighting system-oriented UX, as compared to Finland and France who preferred the experiential definition. Slight differences, but not reaching statistical significance at 0.05 were observed be- tween Denmark and France, and between Finland and France (Table 8). Table 6. Rating of Usability definition (% by country) Denmark Finland France Malaysia Turkey Definition 1 is the most important 63.4% 59.3% 46.9% 57.7% 54.4% Definition 1 is somewhat more important 12.2% 31.4% 21.9% 13.0% 27.8% Both definitions are equally important 22.0% 9.3% 26.6% 24.4% 11.1% Definition 2 is somewhat more important 2.4% 0% 3.1% 4.9% 3.3% Definition 2 is the most important 0% 0% 1.6% 0% 3.3% Total % 100.0% 100.0% 100.0% 100.0% 100.0% Table 7. Rating of UX definition (% by country) Denmark Finland France Malaysia Turkey Definition 1 is the most important 17.1% 8.1% 6.3% 22.8% 25.6% Definition 1 is somewhat more important 9.8% 17.4% 4.7% 6.5% 13.3% Both definitions are equally important 29.3% 24.4% 31.3% 39.0% 31.1% Definition 2 is somewhat more important 22.0% 26.7% 25.0% 22.0% 15.6% Definition 2 is the most important 22.0% 23.3% 32.8% 9.8% 14.4% Total % 100.0% 100.0% 100.0% 100.0% 100.0% Table 8. Significant and near significant differences among countries Rating of UX Definitions Mann-Whitney U p Definition 1 more preferred by Definition 2 more preferred by Turkey vs. France 1773.500 0.000 Turkey France Turkey vs. 5.4 Country specific usability and UX Finland 2328.500 0.097 Finland France 5.5 Impact of demographic and professional profile on usability and UX understanding Table 7. Rating of UX definition (% by country) Denmark Finland France Malaysia Turkey Definition 1 is the most important 17.1% 8.1% 6.3% 22.8% 25.6% Definition 1 is somewhat more important 9.8% 17.4% 4.7% 6.5% 13.3% Both definitions are equally important 29.3% 24.4% 31.3% 39.0% 31.1% Definition 2 is somewhat more important 22.0% 26.7% 25.0% 22.0% 15.6% Definition 2 is the most important 22.0% 23.3% 32.8% 9.8% 14.4% Total % 100.0% 100.0% 100.0% 100.0% 100.0% Table 7. Rating of UX definition (% by country) Table 8. Significant and near significant differences among countries Rating of UX Definitions Mann-Whitney U p Definition 1 more preferred by Definition 2 more preferred by Turkey vs. France 1773.500 0.000 Turkey France Turkey vs. Finland 2915.500 0.004 Turkey Finland France vs. Malaysia 2503.000 0.000 Malaysia France Finland vs. Malaysia 4157.500 0.007 Malaysia Finland Denmark vs. France 1036.500 0.061 Denmark France France vs. Finland 2328.500 0.097 Finland France 5.5 Impact of demographic and professional profile on usability and UX understanding Table 8. Significant and near significant differences among countries 5.5 Impact of demographic and professional profile on usability and UX understanding 5.5 Impact of demographic and professional profile on usability and UX understanding In answering RQ4, the Wilcoxon signed-rank test showed that, with regard to usabil- ity, there was a clear and significant consensus towards Definition 1 across all socio- cultural groups. However, with respect to UX, there was no clear consensus towards one definition across the social-cultural groups, thus different social-cultural profiles had different preferences towards the UX definitions as shown in Table 9. The upper part of Table 9 shows the profiles that rated Definition 2 as being more important. The lower part of table shows profiles that inclined towards Definition 1. UX profes- sionals involved early in system development (SD) or not really involved in SD showed a preference towards Definition 2, while people involved in late stages had a preference towards Definition 1. Professionals in France and Finland had a signifi- cantly stronger preference for Definition 2 when compared to Definition 1. People self-evaluating themselves as having expert knowledge on UX had a stronger prefer- ence towards Definition 2, however the difference only approaching significance. Top management UX professionals tended to prefer Definition 2, while entry-level profes- sionals inclined towards Definition 1. Similar pattern was observed between people keeping up with the evolution of the UX field and people not keeping up, and be- tween professionals with Master degree and professionals with only Bachelor degree. There were stronger preferences for Definition 2 among those graduated in psycholo- gy, business and management, and fields classified as “others”. Males, professionals with usability or UX in the job title, and with work experience in the UX positions between 7 and 12 years strongly preferred Definition 2 to Definition 1. Table 9. 5.5 Impact of demographic and professional profile on usability and UX understanding Profiles that have a significant or near significant impact on UX definitions preference Type of profile Definition 2 preferred to Definition 1 Definition 1 preferred to Definition 2 n Z p Z p Early SD stages involvement 166 -1.96 0.050 Not involved in SD 20 -2.43 0.015 Finland 86 -2.81 0.005 France 64 -4.06 0.000 Expert level of UX knowledge 62 -1.80 0.072 Does keep up with UX field 355 -2.50 0.013 Usability/UX in job title 192 -2.99 0.003 Top management position 66 -1.81 0.070 Psychology as graduation field 29 -2.22 0.026 Business and management as graduation field 22 -1.72 0.085 Other grad fields 101 -2.19 0.029 Master degree 213 -3.19 0.001 Male 213 -2.23 0.026 UX work experience between 7 and 12 years 62 -2.86 0.004 Bachelor degree 118 -1.88 0.061 Entry level position 34 -1.66 0.097 Does not keep up with UX field 46 -1.80 0.071 Late SD stages involvement 19 -2.02 0.043 Table 9. Profiles that have a significant or near significant impact on UX definitions preference Type of profile Definition 2 preferred to Definition 1 Definition 1 preferred to Definition 2 n Z p Z p Early SD stages involvement 166 -1.96 0.050 Not involved in SD 20 -2.43 0.015 Finland 86 -2.81 0.005 France 64 -4.06 0.000 Expert level of UX knowledge 62 -1.80 0.072 Does keep up with UX field 355 -2.50 0.013 Usability/UX in job title 192 -2.99 0.003 Top management position 66 -1.81 0.070 Psychology as graduation field 29 -2.22 0.026 Business and management as graduation field 22 -1.72 0.085 Other grad fields 101 -2.19 0.029 Master degree 213 -3.19 0.001 Male 213 -2.23 0.026 UX work experience between 7 and 12 years 62 -2.86 0.004 Bachelor degree 118 -1.88 0.061 Entry level position 34 -1.66 0.097 Does not keep up with UX field 46 -1.80 0.071 Late SD stages involvement 19 -2.02 0.043 6 Discussion 6.1 Consensus about usability definition across countries and social-cultural groups There was a clear consensus towards the importance of ISO 9241-11 definition [1] of usability among the UX professionals across all countries and socio-cultural profiles analyzed in this paper. This indicates that the ISO definition of usability, reflecting individual empowerment of end users, is widely accepted and adopted in the UX community as pointed out also in [14]. Table 9. Profiles that have a significant or near significant impact on UX definitions preference 6.2 Organizational usability and other perspectives on usability The definition addressing organizational usability [51] was rated clearly less im- portant, however a relatively large number of UX professionals acknowledged the equal importance of both definitions. Moreover, the analysis of open answers showed that the UX professionals wished to extend the ISO usability definition with experi- ence, business, and organization related aspects. This shows that, though the ISO 9241-11 usability [1] is an established concept among professionals, the concept is still evolving. The announced forthcoming changes to ISO 9241-11 by Bevan and colleagues [14] to include organizational perspective are in line with our findings. The references to business, organizational, and customer perspective in the open answers as well as the acknowledged importance of both definitions among some UX profes- sionals show that defining usability by addressing the business benefits starts to be- come important. Thus, usability starts to be recognized as a success and strategic fac- tor for companies, in line with research on usability cost-benefit analysis models [53,54,55]. Some practitioners have also already adopted the customer perspective, which is in line with the recent emphasis on service design as opposed to physical product design [56]. Moreover, our results indicate that besides organizational usability, situational usa- bility, perceived usability, and hedonic usability [3] featured in the open answers. The variety of attributes in the free-form definitions shows that the diversity in HCI re- search [17] exists also among HCI practitioners. This has implications on how UX professionals actually operationalize the ISO 9241-11 definition [1] and measure the usability attributes in practice. 6.1 Consensus about usability definition across countries and social-cultural groups 6.1 Consensus about usability definition across countries and social-cultural groups 6.1 Consensus about usability definition across countries and social-cultural groups There was a clear consensus towards the importance of ISO 9241-11 definition [1] of usability among the UX professionals across all countries and socio-cultural profiles analyzed in this paper. This indicates that the ISO definition of usability, reflecting individual empowerment of end users, is widely accepted and adopted in the UX community as pointed out also in [14]. 6.3 Diversity in UX definitions There was no clear consensus as regards the UX definition among the UX profession- als; however, the preference towards the definition highlighting the experiential quali- ties during the use of a product [23] was approaching statistical significance when compared to the definition emphasizing system qualities in use [22]. Therefore, UX professionals generally preferred a human-oriented, experiential definition of UX, reflecting more consumer psychology than the work context. This result aligns with the original meaning of UX pointed out in [14], [20,21] and with the new emphasis on service design (see e.g., [56]). We anticipated a stronger preference for the definition reflecting system qualities in use given the fact that these are easier to capture and measure in practice; the re- sults showed that, indeed, certain socio-cultural groups of UX professionals preferred this definition. The comparison between countries showed that Turkey and Malaysia, which represent relatively young UX communities have a stronger preference towards the system-oriented UX definition versus the human-oriented one when compared to Finland and France. However, the preference for the former definition was not statis- tically significant within the countries, showing quite heterogeneous ratings. On the other hand, both Finnish and French communities showed stronger preferences to- wards the latter definition. Further analysis within each socio-cultural group showed that the system-oriented UX definition was preferred by profiles who reported late involvement in system development, and who might not yet have a firmly established foundation of UX knowledge and practice (they were graduates of Bachelor degree, worked in entry- level positions, and did not keep up with the UX field). On the other hand, the defini- tion stressing the experiential qualities was strongly preferred by socio-cultural pro- files that reported involvement in early stages of system development or were not really involved, and that had a stronger background in usability and UX studies and work. These findings may also relate to the organizational culture and background in us- ability work. Research has reported that organizations tend to start usability and UX work with usability testing in the end (e.g., [57]), while organizations should move towards starting usability and UX work early and continuing it thorough the phases of system development (e.g., [38], [47]). Thus, the maturity of organizations in terms of UX work, combined with the professional profile of the UX practitioners, would be interesting to examine in relation to the UX understandings of the practitioners. 6.3 Diversity in UX definitions The free-form definitions of UX revealed an extensive list of attributes. Unlike it was recommended by Bevan et al. [14], UX is viewed by some UX professionals as sharing characteristics with usability by addressing effectiveness, efficiency, and goals. The diversity of attributes assigned to UX by professionals parallels with the divergent discourses on defining UX in HCI (e.g., [4], [6,7]). This shows that research efforts of this kind are indeed necessary in order to clarify the true meaning of UX and ways to operationalize it and to measure it. 6.4 Implications for practice and research This study has implications for the design and evaluation of interactive systems, products, and services, as it points out that practitioners should be aware that usability and UX concepts are diverse, while pivotal in achieving the objective of excellence in user interface, quality in use, and service design (see e.g., [15,16], [56]). Diversity in the understandings of the UX professionals indicates that there likely is diversity in how UX professionals operationalize usability and UX in practice and in how they strive for high quality usability and UX in their design and evaluation practices. Con- cepts are still evolving as new dimensions and perspectives emerge; thus, practition- ers should keep up with the evolution of the field, and with the practices and concep- tualizations adopted by competitors. Our findings showed that our approach of including diversity in the sample in terms of UX community maturity and geographic location was beneficial for under- standing the perspectives and perceptions of both definitions of usability and UX. It is important for practitioners to observe and respond to the global trends across UX communities and countries. We reported also the near-significant results as they may indicate tendencies in the respective community, but also transitions from one per- spective to another. Longitudinal studies observing the evolution of views on UX within different socio-cultural groups would confirm or disconfirm the trends. Thus, our research points out that one could trace the development and adoption of usability and UX definitions based on the UX community maturity and geographical location. Further research including other countries would then complete the picture of per- spectives and perceptions of usability and UX. Thus, more countries with varying levels of usability and UX history should be included in further analyses to confirm the patterns observed in this study and to provide a mapping of how the field evolves. Further research should also investigate the implications that adopting one defini- tion or another has on the usability and UX work practices. We plan to extend the analysis to the activities, methods, and tools employed by UX professionals who pro- vided different views on usability and UX. Further research should also examine whether there are differences in usability and UX understanding due to organizational characteristics such as size, type, and culture. 1. ISO 9241-11: Ergonomic Requirements for Office Work with Visual Display Terminals (VDTs) - Part 11: Guidance on Usability. Geneva, CH: International Standard Organiza- tion (1998) 7 Conclusion This paper examined the views of UX professionals on the definitions of usability and UX, and compared the findings between five countries and within different socio- cultural groups. The paper contributes by showing that usability is now an established concept among UX professionals; the respondents across the five surveyed countries recognized the importance of the ISO 9241-11 definition. Moreover, the paper showed that UX professionals increasingly recognize usability as a construct im- portant for the organization, business, and customers, not just for users. The views on UX diverge among different socio-cultural groups when contrasting the experiential qualities with the system qualities in use. Especially UX professionals with a stronger socio-cultural background in usability and UX work preferred the experiential defini- tion. The paper contributes also by pointing out that UX professionals refer to a variety of characteristics and attributes associated with usability and UX that parallels the struggles in HCI research on finding the best ways to capture the essence of these concepts, as they evolve in time. These findings show that research is still needed on capturing and clarifying the meanings of usability and UX, as well as the implications of this diversity on the UX professionals’ design and evaluation practices. Practition- ers should be aware of the diversity of usability and UX definitions and adapt their practices to the global trends. We suggest also that further revisions of ISO 9241-11 should make the distinction between usability and UX clearer and firmer, and provide guidelines on using the two concepts in design and evaluation within organizations. 6.4 Implications for practice and research This research has not addressed the comparison of views regarding usability and UX, and we plan further analyses to assess the extent to which these views overlap. We plan also to address the time di- mension of usability and UX, and the views of UX professionals on this aspect. 8 References 2. ISO 9241-210: Ergonomics of Human-System Interaction - Part 210: Human-Centred De- sign for Interactive Systems. Geneva, CH: International Standard Organization (2010) 3. Hertzum, M.: Images of Usability. Int’l J. of Human-Computer Interaction 26(6), 567-600 (2010) 4. Law, E., Roto, V., Vermeeren, A. P., Kort, J., Hassenzahl, M.: Towards a Shared Defini- tion of User Experience. 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https://openalex.org/W2781765606	https://europepmc.org/articles/pmc5755024?pdf=render	English	null	Effect of GRM7 polymorphisms on the development of noise-induced hearing loss in Chinese Han workers: a nested case-control study	BMC medical genetics	2,018	cc-by	7,843	* Correspondence: chinastress@sina.com 2Henan Provincial Institute for Occupational Health, No.3, Kangfu Mid. St, Zhengzhou 450052, People’s Republic of China Full list of author information is available at the end of the article Yu et al. BMC Medical Genetics (2018) 19:4 DOI 10.1186/s12881-017-0515-3 Yu et al. BMC Medical Genetics (2018) 19:4 DOI 10.1186/s12881-017-0515-3 © 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. Effect of GRM7 polymorphisms on the development of noise-induced hearing loss in Chinese Han workers: a nested case-control study Peipei Yu1,2, Jie Jiao2, Guoshun Chen3, Wenhui Zhou2, Huanling Zhang3, Hui Wu2, Yanhong Li2, Guizhen Gu2, Yuxin Zheng4, Yue Yu5 and Shanfa Yu2* * Correspondence: chinastress@sina.com 2Henan Provincial Institute for Occupational Health, No.3, Kangfu Mid. St, Zhengzhou 450052, People’s Republic of China Full list of author information is available at the end of the article © 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. Methods Subjects p y It has been well known that NIHL is a complicated disease caused by the interaction of genetic and environ- mental factors. The environmental factors, such as noise exposure, organic solvents, ototoxic drugs, heat, vibra- tions, smoking and health relative factors (hypertension, high cholesterol, pigmentation) and so on, all have es- sential effect on the progress of NIHL and a lot of work on preventing the harmful effect induced by these fac- tors has been done. However, studies on human genetic factors which may be associated with the susceptibility to NIHL are relatively rare [7]. Researches on the knockout mice, for example Pjvk−/−[8], PMCA2+/−[9], P2RX2−/− [10], CDH23+/ -[11], SOD−/−[12], GPX1−/−[13], have indi- cated that the gene deficiency mice are more susceptible to NIHL. Current studies have shown that the polymor- phisms of potassium ion channel genes (like KCNQ4 and KCNE1) [14, 15], catalase (CAT), protocadherin 15 (PCDH15), myosin 14 (MYH14) [16] and heart shock protein (HSP70) [17] detected in different pop- ulations are significantly related to the development of NIHL. In addition, studies conducted by our research group has also found that heart shock protein (HSP70) [18], eye absent homolog 4 (EYA4) [19], suggestive POU- domain transcription POU4F3 and Grainyhead-like2 (GRHL2) [20] may be associated with the susceptibility to NIHL. A dynamic cohort was established in a big steel factory among lasting noise exposure workers On January 1, 2006. Occupational health examinations, hearing tests and questionnaire surveys were carried out among the 6886 works selected into the cohort in the first year. And then noise, heat, toxic and hazardous substances in the work environment were monitored and measured every year. Health examination and hearing test were conducted every two years for the studying population. Up to December 31, 2015, there were 6297 subjects completed more than twice health examinations and hearing tests and 817 subjects finished only once. In the process of follow-up, 559 subjects joined in the research and 331 subjects were loss to follow-up because of resig- nation or being transferred to other positions. Background type I spiral ganglion neurons [24, 25]. High concen- tration of glutamate is neurotoxic, which has been known to be associated with NIHL [21, 26]. Metabotropic glutamate receptor 7 (mGluR7) encoded by GRM7 can re- duce excessive glutamate release in the synaptic compart- ments to maintain their normal physiological concentration [21]. High-level noise exposure leads to the excessive re- lease of glutamate from the hair cells to the synaptic cleft and the high concentration of glutamate overstimulates the postsynaptic cells or dendrites, causing them excitatory poi- soning with the feature of swelling [27–29]. g With the widely spread of industrialization in the world, noise exposure is becoming more common in the indus- trial settings. This phenomenon is more noteworthy in developing countries. Nowadays, hearing loss due to oc- cupational noise exposure is still an intractable problem for both developing and developed countries, and a lot of studies on noise are mainly focused on occupational NIHL [1]. World Health Organization studies show that occupational NIHL, as the second occupational health hazard except unintentional injuries, accounts for 16% of occupational chronic diseases and 19% of the loss of health life induced by noise exposure in work place [2, 3]. About 22 million U.S. workers are exposed to harmful noise level in the working environment every year and NIHL is one of the most common occupa- tional diseases in the United States [4, 5]. In China, occupational NIHL is the third most harmful occupa- tional disease, accounting for one-sixth in all of the annual increased occupational diseases recently [6]. Hence, we hypothesize that GRM7 polymorphisms may have an effect on the development of NIHL. Then, we screen single nucleotide polymorphisms (SNPs) of GRM7 that may be related to the susceptibility of NIHL and carry out a nested case-control study in the occupa- tional populations to analyze the relationship between GRM7 polymorphisms and NIHL. Abstract Background: Noise-induced hearing loss (NIHL) is a complex, irreversible disease caused by the interaction of genetic and environmental factors. In recent years, a great many studies have been done to explore the NIHL susceptibility genes among humans. So far, high powerful detections have been founded that genes of potassium ion channel genes (KCNQ4 and KCNE1), catalase (CAT), protocadherin 15 (PCDH15), myosin 14 (MYH14) and heart shock protein (HSP70) which have been identified in more than one population may be associated with the susceptibility to NIHL. As for metabolic glutamate receptor7 gene (GRM7), a lot of researches mainly focus on age-related hearing loss (ARHL) and the results have shown that the polymorphisms of GRM7 are linked to the development of ARHL. However, little is known about the association of GRM7 and the susceptibility to NIHL. Therefore, the aim of this study was to explore the effect of GRM7 polymorphisms on the susceptibility to NIHL. Methods: A nested case-control study based on the cohort in a Chinese steel factory was implemented in 292 cases and 584 controls matched with the same sex, the age difference ≤5 years old, the same type of work, duration of occupational noise exposure ≤2 years. Five single nucleotide polymorphisms (SNPs) of GRM7 were gained through selecting and genotyping SNPs. Conditional logistic regression analysis was used to assess the main effect of GRM7 polymorphisms on the susceptibility to NIHL and the gene-by-environment interaction. Furthermore, the gene-by- gene interactions were analyzed by generalized multiple dimensionality reduction (GMDR). Results: This research discovered for the first time that the mutant allele C in rs1485175 of the GMR7 may decrease individuals’ susceptibility to NIHL. The interaction between rs1485175 and cumulative noise exposure (CNE) at high level was found after the stratification according to CNE (p/pbon = 0.014/0.007, OR = 0.550, 95% CI: 0.340–0.891). Permutation test of GMDR suggested that rs1920109, rs1485175 and rs9826579 in GRM7 might interact with each other in the process of developing NIHL (p = 0.037). Conclusions: The results suggest that the mutant allele C of rs1485175 in GRM7 may reduce the susceptibility to NIHL in Chinese Han population. Keywords: GRM7, Noise-induced hearing loss, Polymorphism Yu et al. BMC Medical Genetics (2018) 19:4 Page 2 of 10 Page 2 of 10 Methods Subjects In the cohort, there were 9 individuals with a history of being an airman, 76 with a history of being an artiller- ist, 53 with a history of head trauma, 3 with a history of blast exposure hearing damage, 10 with a history of ear- drum perforation, 1 with a history of taking ototoxic drugs, 32 with a familial history of deafness, 15 with a history of rubella, 4 with a history of Meniere’s syndrome, all of which were excluded in the case and control selecting. This research based on the cohort study with 1 case to 2 controls matched. The case group (hearing loss group) and the control group were selected with the level of occupational noise exposure ≥80 dB(A) and the time of accumulated occupational noise exposure ≥3 years. The inclusion of cases was that binaural average hearing thresh- old levels (HTLs) in high frequencies (3 kHz,4 kHz,6 kHz) ≥ 40 dB(A). The control group matched with the same sex, the age difference ≤5 years old and the same type of work, Up to now, when it comes to GRM7, previous stud- ies have shown that GRM7 polymorphisms are associ- ated with the susceptibility to hearing loss in the elderly [21–23], but the relationship between GRM7 polymorphisms and NIHL susceptibility remains to be further validated. At present, many researches have shown that glutamate is the main excitatory neuro- transmitter in the transmission of inner hair cells and Yu et al. BMC Medical Genetics (2018) 19:4 Page 3 of 10 0.5, 1, 2, 3, 4, 6 kHz. The surrounding should be quiet and the noise background value <25 dB(A). The results of the tests were collected by age and gender. duration of occupational noise exposure ≤2 years and was selected according to the HTL of any one ear in linguis- tic frequencies (0.5 kHz, 1 kHz, 2 kHz) < 25 dB(A) and average binaural HTL in high frequencies <35 dB(A). Finally, there were 292 cases and 584 controls entering into this study. Epidemiological survey A combinative method of investigators interviewing and respondents actively reporting were used to collect in- formation. Investigators had been professionally trained in advance. The questionnaire of this study mainly in- cluded the following aspects: (1) demographic character- istics: such as age, gender, date of birth, educational level, etc. (2) professional history: such as type of work, noise exposure duration in noise setting, environmental noise exposure level, etc. (3) living habits: such as, whether smoking and daily smoking levels, whether drinking and daily alcohol consumption, high-fat food intake, etc. (4) previous history of diseases affecting hearing: such as, ear trauma, tinnitus, sudden deafness, hypertension, etc. (5) history of ototoxic drug use, such as: aminoglycosides or vancomycin antibiotics, contain- ing cisplatin and other anti-tumor drugs, containing arsenic and other heavy metal drug use history and so on. The detailed questionnaire was offered in the supple- mentary file [see Additional file 1]. Calculation of CNE The equivalent continuous sound level (A) (LAeq, 8h) was measured using Noisepro multi-functional individ- ual noise dosimeters (NoisePro series, Quest Technolo- gies, USA) which were adjuested by type of QC-10 Sound calibrators before measurement. The noise do- simeters were set as weight of A, S (slow), the value of LAeq, 8h and then the CNE for every subjects was calcu- lated based on the Fig. 1 (the Footnotes of Fig. 1 at the end of the article) [6]. The study was approved by the Ethics Committee of Henan Provincial Institute for Occupational Health (Ethical approval no.: 2,013,003) and informed consent was signed by all study participants or their agents. SNP selection and genotyping SNPs were selected according to the 1000 Genomes Pro- ject resources (http://www.internationalgenome.org/) and relative literatures and the inclusion criteria were as follows: Smokers were those smoking at least one cigarette every day and more than 6 months, otherwise, they were regared as non-smokers. The criterion for judging drinkers was that subjects drank at least once per week more than 1 year, if not, they were thought as non- drinkers. (1)sites of SNPs laid in the area of GRM7 with the minor allele frequency (MAF) > 0.10 in the Chinese Han population; (2)the linkage disequilibrium (LD) method of SNPs with the pairwise r2 > 0.80. (2)the linkage disequilibrium (LD) method of SNPs with the pairwise r2 > 0.80. DNA extraction Peripheral blood (≥2 ml) was collected in the EDTA anti- coagulant tube and stored at minus 80 °C. The DNA of the peripheral blood was extracted using a 2 ml blood genomic DNA extraction kit (Shanghai Laifeng Biotech, Shanghai, Chnia). The concentration and purity of DNA were mea- sured using the NanoPhotometer P360 ultramicro spectro- photometer (Shanghai Boyibio Biotech, Shanghai, China). The prerequisites for subsequent DNA genotyping were that the A260/A280 value was between 1.8 and 2.0 and the concentration was >50 ng/μL. Hearing test and ear examination Five SNPs in GRM7 were selected in this study. Before the examination, all the subjects were required to leave the occupational noise environment for at least 12 h. 216 audiometers (Interacoustics AS Company, Denmark) calibrated previously were used to test bin- aural air and bone conduction threshold audiometry at y In this study, the SNPs were genetyped using the SNPscan multiplex SNP genotyping kits (Genesky Biopharm Technology, Shanghai, China). AB13730XL DNA analyser was used to detect the sequence and Fig. 1 The interpretation of CNE equation. Where Tref is equal to 1; n is the total number of different positions for the workers exposed to noise; i is the number of different posts; T is the time at different positions; LAeq, 8h is the equivalent continuous sound level of 8 h for different jobs Fig. 1 The interpretation of CNE equation. Where Tref is equal to 1; n is the total number of different positions for the workers exposed to noise; i is the number of different posts; T is the time at different positions; LAeq, 8h is the equivalent continuous sound level of 8 h for different jobs Page 4 of 10 Yu et al. BMC Medical Genetics (2018) 19:4 Page 4 of 10 GeneMapper 4.1 software (Applied Biosystems, USA) was utilized to analyze the genotype of SNP loci. (additive model, dominant model, recessive, codominant model) were established and a conditional logistic re- gression was implemented to analyse the relationship between the polymorphisms of GRM7 and the suscep- tibility to NIHL. The interactions of genetic and en- vironmental factors were also considered in logistic regression analysis by the option of multiplying inter- action effect, and if the interactions were significant, then the stratification would be carried out to analyze the main effects. The generalised multiple dimension- ality reduction software V.0.9 (GMDR V.0.9) was ap- plied to find the interaction among SNPs. The possible confounders, such as CNE, smoking, drink- ing, hypertension, were adjusted during the statistical analysis. The test level was α = 0.05 therefore it was Evaluation of the matching effects in the case and control groups by comparing the basic information distribution between them CNE: cumulative noise exposure Statistical analysis HTL is grouped by 5 dB(A) in both case and control groups. The control group ranges from 0 to 35 dB(A) and the case group is in the range of 40 to 85 dB(A) analysed by pearson χ2 test. The binaural average hear- ing threshold level (HTL) (3 kHz,4 kHz,6 kHz) in the case group was higher than that of the control group, which conformed to the design needs of our research (p < 0.001). What’s more, the proportion of tinnitus in the case group was higher than that of the control group with p < 0.001. Other comparisons in the case and con- trol groups, including the general demographic charac- teristics (age, sex, height, noise exposure duration), individual factors (smoking, drinking), disease history (hypertension), and the observing indicators (CNE, level of environmental noise exposure) were of no significant difference (p > 0.05). The proportional distribution of HTL in case and control groups was showed in Fig. 2 (the Footnotes of Fig. 2 at the end of the article). statistically significant if p < 0.05. All the testing hy- potheses were two-tailed. Bonferroni correction was used in the multiple comparing by pairs. Statistical analysis Data was analyzed using SPSS21.0 software. The con- tinuous variables were implemented tests of normality and all of them were not in accordance with normal dis- tribution, hence they were expressed by the median (range) and the differences between groups were ana- lysed by Wilcoxon rank sum test. All of the classification variables were expressed by frequencies (percentile) and the comparisons between groups were analysed by pear- son chi-square test.The control groups of SNPs were tested whether they were in line with Hardy-Weinberg equilibrium using Pearson’s χ2 test. Four genetic models Table 1 Basic Information Distribution in Case and Control Groups Variables Case (n = 292) Control (n = 584) Statistics p Age, year 20~30 45 (15.4%) 104 (17.8%) 30~40 79 (27.1%) 153 (26.2%) 40~50 134 (45.9%) 270 (46.2%) 50~60 34 (11.6%) 57(9.8%) 1.381‡ 0.710 Noise exposure duration, year 18.860 (8.500, 27.750) 18.509 (8.167, 26.917) −0.692§ 0.489 CNE, dB(A) 97.844 (94.686, 101.522) 97.767 (94.854, 101.195) −0.153§ 0.878 HTL, dB(A) † 50.980 (44.042, 55.833) 18.293 (12.500, 24.000) −24.153§ <0.001 Height, cm 170.366 (167.000, 174.750) 169.993 (166.000, 174.000) −1.004§ 0.315 Gender Male 281 (96.2%) 560 (95.9%) Female 11 (3.8%) 24 (4.1%) 0.060‡ 0.807 level of environmental noise exposure, dB(A) ≤85 121 (41.4%) 254 (43.5%) > 85 171 (58.6%) 330 (56.5%) 0.336‡ 0.562 Tinnitus Yes 196 (67.4%) 316 (54.2%) No 95 (32.6%) 267 (45.8%) 12.837‡ <0.001 Smoking Yes 181 (62.0%) 341 (58.4%) No 109 (38.0%) 243 (41.6%) 1.045‡ 0.307 Drinking Yes 203 (69.5%) 399 (68.3%) No 89 (30.5%) 185 (31.7%) 0.130‡ 0.718 Hypertension Yes 112 (38.4%) 242 (41.4%) No 180 (61.6%) 342 (58.6%) 0.768‡ 0.381 Evaluation of the matching effects in the case and control groups by comparing the basic information distribution between them CNE: cumulative noise exposure †HTL: the binaural average hearing threshold level in high frequencies ‡P hi t t Yu et al. BMC Medical Genetics (2018) 19:4 Page 5 of 10 Fig. 2 The proportional distribution of the binaural average hearing threshold levels (HTLs) at 3 kHz,4 kHz,6 kHz in case and control groups. HTL is grouped by 5 dB(A) in both case and control groups. The control group ranges from 0 to 35 dB(A) and the case group is in the range of 40 to 85 dB(A) Fig. 2 The proportional distribution of the binaural average hearing threshold levels (HTLs) at 3 kHz,4 kHz,6 kHz in case and control groups. Results Evaluation of matching effect in case and control groups In this study, a total of 876 people were involved, in which there were 292 cases and 584 controls, aging from 20.75 to 59.25 years old. As showed in Table 1 (after ref- erences), the matching effect was evaluated in the case and control groups through comparing the basic infor- mation distribution between them. By test of normality, the whole continuous variables did not conform to the normal distribution, so nonpara- metric test was used. The classification variables were Table 2 Basic Information of the Selected SNPs SNP Chromosomal position MAF Allele* χ2b p† Ancestral allele Mutant allele rs11920109 Chr3:7,212,686 0.4016 T = 0.4272 C = 0.5728 2.4271 0.2178 rs1485175 Chr3:7,620,789 0.4744 T = 0.5437 C = 0.4563 1.3677 0.2589 rs9819783 Chr3:7,208,213 0.3848 T = 0.4272 C = 0.5728 1.5317 0.3961 rs9826579 Chr3:7,782,371 0.3317 C = 0.1408 T = 0.8592 0.0185 0.9603 rs9877154 Chr3:7,159,406 0.4187 T = 0.3932 C = 0.6068 0.1975 0.9045 Hardy-Weinberg equilibrium test of all selected SNPs in the control group bPearson chi-square test is used to test whether the SNPs in the control group is in line with Hardy-Weinberg equilibrium The data comes from NCBI dbSNP and 1000 Genomes Browser (CHB) †Hardy-Weinberg Equilibrium Test of the control group by Pearson’s χ2 Hardy-Weinberg equilibrium test of all selected SNPs in the control group bPearson chi-square test is used to test whether the SNPs in the control group is in line with Hardy-Weinberg equilibrium The data comes from NCBI dbSNP and 1000 Genomes Browser (CHB) †Hardy-Weinberg Equilibrium Test of the control group by Pearson’s χ2 Yu et al. Effects of genetic models evaluated by conditional logistic regression , smoking, drinking, and hypertension are adjusted; CI: Confidence interval correction is used to adjust p values by means of 0.05 / 5 (5 selected SNPs) to get pbon values of 0.01, which means that it is significan Effects of genetic models evaluated by conditional logistic regression , g , g, g, yp j ; nferroni correction is used to adjust p values by means of 0.05 / 5 (5 selected SNPs) to get pbon values of 0.01, which means that it is s bon < 0.01 Effects of genetic models evaluated by conditional logistic regression CNE, height, smoking, drinking, and hypertension are adjusted; CI: Confidence interval , g , g, g, yp j ; †Bonferroni correction is used to adjust p values by means of 0.05 / 5 (5 selected SNPs) to get pbon values of 0.01, which means that it is significant in statistics if pbon < 0.01 y g g king, drinking, and hypertension are adjusted; CI: Confidence interv Results BMC Medical Genetics (2018) 19:4 Page 6 of 10 Table 3 Correlation of Genetic Models with Risk of Developing NIHL SNP Genotype Case Control OR (95%CI) p/pbon † n % n % rs11920109 TT 40 13.7 92 15.8 1 0.738/0.148 TC 153 52.4 297 50.9 1.182 (0.774, 1.806) 0.439/0.088 CC 99 33.9 194 33.3 1.141 (0.724, 1.800) 0.531/0.106 CC + TC 252 86.3 491 84.2 1.155 (0.736, 1.814) 0.443/0.089 TT + TC 193 66.1 389 66.7 1 CC 99 33.9 194 33.3 1.016 (0.745, 1.386) 0.920/0.184 TT/TC/CC 1.055 (0.850,1.309) 0.627/0.125 Allele C/T 1.039 (0.866, 1.246) 0.682/0.136 rs1485175 TT 103 35.5 169 29.1 1 0.029/0.006 TC 139 47.9 276 47.5 0.820 (0.593, 1.132) 0.227/0.045 CC 48 16.6 136 23.4 0.564(0.370, 0.860) 0.008/0.002 CC + TC 187 64.5 412 70.9 0.737 (0.544, 1.000) 0.050/0.010 TT + TC 242 83.4 445 76.6 1 CC 48 16.6 136 23.4 0.636 (0.437, 0.925) 0.018/0.004 TT/TC/CC 0.761 (0.620, 0.934) 0.009/0.002 Allele C/T 0.800 (0.666, 0.962) 0.017/0.003 rs9819783 TT 38 13.1 86 14.8 1 0.785/0.157 TC 148 50.9 288 49.5 1.163 (0.753, 1.797) 0.496/0.099 CC 105 36.1 208 35.7 1.150 (0.728, 1.816) 0.550/0.110 CC + TC 253 86.9 496 85.2 1.158 (0.765, 1.752) 0.489/0.098 TT + TC 186 63.9 374 64.3 1 CC 105 36.1 208 35.7 1.024 (0.751, 1.396) 0.881/0.176 TT/TC/CC 1.054 (0.848, 1.309) 0.637/0.127 Allele C/T 1.038 (0.864, 1.248) 0.688/0.138 rs9826579 CC 8 2.7 16 2.7 1 0.718/0.144 CT 73 25.1 162 27.8 0.938 (0.369, 2.386) 0.893/0.179 TT 210 72.2 404 69.4 1.072 (0.438, 2.622) 0.879/0.176 TT + CT 283 97.3 566 97.3 1.049 (0430, 2.559) 0.917/0.183 CC + CT 81 27.8 178 30.6 1 TT 210 72.2 404 69.4 1.136 (0.833, 1.549) 0.422/0.084 CC/CT/TT 1.106 (0.844, 1.451) 0.465/0.093 Allele T/C 1.086 (0.849, 1.390) 0.510/0.102 rs9877154 TT 41 14.0 86 14.7 1 0.712/0.142 TC 147 50.3 278 47.6 1.125 (0.737, 1.719) 0.585/0.117 CC 104 35.6 220 37.7 0.995 (0.636, 1.556) 0.983/0.197 CC + TC 251 86.0 498 85.3 1.071 (0.715, 1.604) 0.740/0.148 TT + TC 188 64.4 364 62.3 1 CC 104 35.6 220 37.7 0.909 (0.670, 1.233) 0.539/0.108 TT/TC/CC 0.973 (0.787, 1.202) 0.799/0.160 Allele C/T 0.980 (0.815, 1.177) 0.825/0.165 Effects of genetic models evaluated by conditional logistic regression CNE, height, smoking, drinking, and hypertension are adjusted; CI: Confidence interval †Bonferroni correction is used to adjust p values by means of 0.05 / 5 (5 selected SNPs) to get pbon values of 0.01, which means that it is significant in statistics f Table 3 Correlation of Genetic Models with Risk of Developing NIHL Yu et al. Results BMC Medical Genetics (2018) 19:4 Page 7 of 10 Table 4 Relationship between rs1485175 and NIHL layered by CNE Environmental factor Genotype Case Control OR (95% CI) p/pbon † n % n % CNE, dB (A) < 97 TT 44 30.8 81 29.5 1 0.846/0.423 TC 74 51.7 138 50.2 1.023 (0.700, 1.496) 0.906/0.453 CC 25 17.5 56 20.4 0.895 (0.546, 1.469) 0.662/0.331 > 97 TT 61 40.9 91 29.4 1 0.047/0.024 TC 65 43.6 138 44.7 0.784 (0.550, 1.118) 0.179/0.090 CC 23 15.4 80 25.9 0.550 (0.340, 0.891) 0.014/0.007 The interaction between genes and the environment (CNE) analyzed by layering Height, smoking, drinking, and hypertension are adjusted †The statistically significant p values are adjusted by Bonferroni correction through 0.05/2 (2 groups layered by CNE) and it is statistically significant if pbon < 0.025 Table 4 Relationship between rs1485175 and NIHL layered by CNE The interaction between genes and the environment (CNE) analyzed by layering Height, smoking, drinking, and hypertension are adjusted †The statistically significant p values are adjusted by Bonferroni correction through 0.05/2 (2 groups layered by CNE) and it is statistically significant if pbon < 0.025 Height, smoking, drinking, and hypertension are adjusted †The statistically significant p values are adjusted by Bonferroni correction through 0.05/2 (2 groups layered by CNE) and it is statistically significant if pbon < 0.025 Interaction Analysis between Genes and Environment The interaction between genes and the environment (CNE) was analyzed by the option of multiplying inter- action effect in logistic regression analysis. The analysis found that rs1485175 and CNE had a significant inter- action in statistics with p = 0.007 and OR = 0.997 (95% CI: 0.995–0.999). Then, the main genotype effect in each layer (< 97 dB (A) and > 97 dB(A)) was figured out. Sig- nificant differences were found in TT and CC with the CNE > 97 dB (A). The specific results of the analysis could be found in Table 4. Construction of genetic models and effect analysis Four genetic models were constructed for every SNP in this study: the additive model (wtwt, wtmt, mtmt), dom- inant model [(mtmt + wtmt) vs wtwt], recessive model [mtmt vs (wtwt + wtmt)], codominant model (wtwt vs wtmt vs mtmt) (wt: wild type, mt: mutant type) and then conditional logistic regression analysis was conducted to evaluate the effect of the genetic models. Results in Table 3 (after references) indicated that in additive model CC genotype of rs1485175 had a protective effect to the risk of developing NIHL with an adjusted OR of 0.564 (p/pbon = 0.008/0.002, 95% CI: 0.370–0.860). But statistical association with NIHL was not found in TC genotype. In dominant model of rs1485175, statistical significant difference with a decreased risk was found (p/ pbon= 0.050/0.010, OR = 0.737, 95% CI: 0.554–1.000) and the associations in same direction were also detected in the recessive/codominant model and the allele C/T with Assessment of genotype effects on NIHL the p/pbon value of 0.018/0.004 (OR = 0.636, 95%CI: 0.437– 0.925), 0.009/0.002 (OR = 0.761, 95%CI: 0.620–0.934) and 0.017/0.003 (OR = 0.800, 95%CI: 0.666–0.962), respectively. No significant associations were discovered in any other four SNPs between the case and control groups. Hardy-Weinberg equilibrium for control group Hardy-Weinberg equilibrium for control group Table 2 (after references) showed that p values in the control group of the selected SNPs were all above 0.05, which demonstrated that all SNPs in control group were in Hardy-Weinberg equilibrium (HWE) and the selected population of the control group was representative. Analysis of interaction effects among SNPs The generalized multifactor dimensionality reduction (GMDR) v0.9 was applied in this research to detect the interaction of the 5 selected SNPs in GRM7. Covariates, CNE, height, smoking, drinking, hypertension, were adjusted in the analysis by loading phenotype data. Table 5 (after references) provided the best model, testing balanced accuracy, cross-validation (CV) consistency, and Table 5 Results of the best model identified by GMDR Best model* Testing balanced accuracy (%) CV consistency † p‡ p§ rs1485175 52.62 10/10 0.6230 0.135 rs1485175, rs9877154 50.89 4/10 0.3770 0.141 rs11920109, rs1485175, rs9826579 53.55 9/10 0.0547 0.037 rs11920109, rs1485175, rs9819783, rs9826579 51.09 7/10 0.6230 0.126 rs11920109, rs1485175, rs9819783, rs9826579, rs9877154 50.59 10/10 0.6230 0.403 The analysis of the interaction of the 5 selected SNPs in GRM7 *CNE, height, smoking, drinking, hypertension are adjusted †CV consistency means cross-validation consistency ‡Based on sign test §Based on permutation test Page 8 of 10 Yu et al. BMC Medical Genetics (2018) 19:4 Page 8 of 10 p values by sign test. In all of the models the combination of rs1920109, rs1485175, rs9826579 formed the best model with a statistically significant p value of 0.0547, the maximum testing balanced accuracy of 53.55% and the biggest CV consistency (9/10). Permutation test appeared that the best model made of rs1920109, rs1485175, rs9826579 statistically significant (p = 0.037). Figure 3 (the Footnotes of Fig. 3 at the end of the article) showed the best model constituted by rs11920109, rs1485175, rs9826579. p values by sign test. In all of the models the combination of rs1920109, rs1485175, rs9826579 formed the best model with a statistically significant p value of 0.0547, the maximum testing balanced accuracy of 53.55% and the biggest CV consistency (9/10). Permutation test appeared that the best model made of rs1920109, rs1485175, rs9826579 statistically significant (p = 0.037). Figure 3 (the Footnotes of Fig. 3 at the end of the article) showed the best model constituted by rs11920109, rs1485175, rs9826579. who have CC genotype. But in our study we have not found the statistical correlation between the rs11928865 of GRM7 and the susceptibility to NIHL. p y Many researches have demonstrated that NIHL is a dis- ease resulted from the interaction of gene and environment. Therefore the interaction between rs1485175 and environ- mental factors and the interaction of rs1485175 with the other selected SNPs of GRM7 were analyzed. Discussion The whole genome association study has found that the T-allele of rs11928865 in GRM7 is associated with age- related hearing loss (ARHI) in European group, which is also confirmed in an elderly male Han Chinese popula- tion and rs779706 and rs779701, the SNPs within GRM7, are significant in the Finnish group [21, 30]. Al- though the polymorphisms of GRM7 are an important factor affecting ARHI, genetic polymorphisms are also common in general population and they should also been involved in the study of NIHL. Our results discovered for the first time that the mu- tant allele C in rs1485175 of the GMR7 had significant associations with NIHL among the additive, dominant, recessive, codominant models and the allele C/T. The result suggests that CC genotype might act as a protect- ive factor for the glutamate toxicity caused by high in- tensity noise exposure. That’s to say in the same harmful noisy environment, individuals with non-CC genotype in rs1485175 of GRM7 are more susceptible than those High glutamate concentration can lead to pro- grammed death of the spiral ganglion neurites and cells, which is also verified by incubating spiral ganglion explants with a caspase-3 inhibitor (an inhibitor of apoptosis) [27]. Therefore, the negative feedback regu- lation of glutamate is important in maintaining the normal transmission of the sound signal in the ear. The mGluR7, Group III mGlu receptors, is principally situated in the presynaptic membrane and negatively Fig. 3 Best model gained by the analysis of GMDR. The implications of bars and background color in each multifactor cell are as follows. The left bars represent the sum of scores in case and the right represent the control. High risk cells are expressed by black shadow if the ratio of the number of cases to the number of controls exceeds the preset value T, as low risk cells by light shadow if not more than the threshold and empty cells by no shadow which means no cases and controls. The multifactor cells labeled as “high risk” or “low risk” are then used to assess the classification and predication accuracy, thus identifying the best model in the subsequent steps Fig. 3 Best model gained by the analysis of GMDR. The implications of bars and background color in each multifactor cell are as follows. The left bars represent the sum of scores in case and the right represent the control. Analysis of interaction effects among SNPs The results of the gene-by-environment interaction analysis suggest that CNE at higher level (> 97 dB(A)) might interact with rs1485175 with the p value of 0.014 and OR of 0.550 (95% CI: 0.340–0.891). The risk of developing NIHL reduces by 0.447 after stratification according to CNE, which suggests that CNE may be a risk factor for NIHL and workers are more susceptible to noise at the higher level of CNE [6]. The gene-by-gene interaction data obtained through the software of GMDR showed that the best model made up of rs11920109, rs1485175, rs9826579, suggesting that the main effect of the GRM7 gene on susceptibility to NIHL we have found is the combination of these three genes; that is, the influence of rs1485175 on NIHL is dependent on the genotypes of the other two SNPs in GRM7. Competing interests Th h d l h The authors declare that they have no competing interests. Funding Thi This research was funded by the National Natural Science Foundation of China and National Science and Technology Support Program (81,372,940 and 2014BAI12B03). Conclusions Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. In summary, we find for the first time that the mutant al- lele C of rs1485175 in GRM7 may reduce the susceptibility of individuals to NIHL in Chinese Han population. At the same time, we also note that the gene-by-environmental and gene-by-gene interactions may affect the protective effect of mutant allele C in rs1485175 of GRM7. This finding, once verified by large studies, will have important implications in the prevention of NIHL in susceptible occupational population. Author details 1 1Department of Occupational and Environmental Health Hygiene, College of Public Health, Zhengzhou University, Zhengzhou, Henan, People’s Republic of China. 2Henan Provincial Institute for Occupational Health, No.3, Kangfu Mid. St, Zhengzhou 450052, People’s Republic of China. 3Wugang Institute for Occupational Health, Wugang, Henan, People’s Republic of China. 4College of Public Health, Qingdao University, Qingdao, Shandong, People’s Republic of China. 5National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing, People’s Republic of China. 1Department of Occupational and Environmental Health Hygiene, College of Public Health, Zhengzhou University, Zhengzhou, Henan, People’s Republic of China. 2Henan Provincial Institute for Occupational Health, No.3, Kangfu Mid. St, Zhengzhou 450052, People’s Republic of China. 3Wugang Institute for Occupational Health, Wugang, Henan, People’s Republic of China. 4 Control, Chinese Center for Disease Control and Prevention, Beijing, People’s Republic of China. References 1. Basner M, Babisch W, Davis A, Brink M, Clark C, Janssen S, Stansfeld S. Auditory and non-auditory effects of noise on health. Lancet. 2014; 383(9925):1325–32. 1. Basner M, Babisch W, Davis A, Brink M, Clark C, Janssen S, Stansfeld S. Auditory and non-auditory effects of noise on health. Lancet. 2014; 383(9925):1325–32. 1. Basner M, Babisch W, Davis A, Brink M, Clark C, Janssen S, Stansfeld S. Auditory and non-auditory effects of noise on health. Lancet. 2014; 383(9925):1325–32. 2. Corvalan. aPUJLSTC: Chapter 21 Selected occupational risk factors. comparative quantification of Health risks. global and regional Burden of disease attributable to selected major risk factors. 2004, Vol 2(1651–1802. 2. Corvalan. aPUJLSTC: Chapter 21 Selected occupational risk factors. comparative quantification of Health risks. global and regional Burden of disease attributable to selected major risk factors. 2004, Vol 2(1651–1802. Acknowledgements Not applicable It is possible that the T-allele mutation to C-allele in rs1485175 may take part in this process. In the normal inner hair cells and neurons, the mutant allele C in rs1485175 may increase the number of mGluR7 or enhance its sensitivity to glutamate in the presynaptic membrane, and then to accelerate the glutamate uptake or reduce the release of glutamate when exposed to high intense noise, thus avoiding the glutamate excitotoxicity. That may be the reason why individuals with CC genotype of rs1485175 in GRM7 are less susceptible to noise than non-CC genotype. CNE [6], height [32], smoking [33], drinking [34, 35], and hypertension [35], which are all hazardous factors for NIHL, were adjusted in the process of analyzing. Availability of data and materials The data that support the findings of this study are not publicly available due to the issue of intellectual property but are available from the corresponding author on reasonable request. Consent for publication Not applicable Consent for publication Not applicable Discussion High risk cells are expressed by black shadow if the ratio of the number of cases to the number of controls exceeds the preset value T, as low risk cells by light shadow if not more than the threshold and empty cells by no shadow which means no cases and controls. The multifactor cells labeled as “high risk” or “low risk” are then used to assess the classification and predication accuracy, thus identifying the best model in the subsequent steps Yu et al. BMC Medical Genetics (2018) 19:4 Page 9 of 10 Page 9 of 10 Weinberg equilibrium; LD: Linkage disequilibrium; MAF: Minor allele frequency; mGluR7: Metabotropic glutamate receptor 7; mt: Mutant type; NIHL: Noise-induced hearing loss; SNPs: Single nucleotide polymorphisms; wt: Wild type regulates the level of glutamate. The activation of mGluR7 can hinder glutamate releasing when exposed in high intensity noise level, which lowers the excitabil- ity of target cells and neurons, thus playing a protective role in glutamate excitotoxicity [31]. Received: 6 September 2017 Accepted: 19 December 2017 Received: 6 September 2017 Accepted: 19 December 2017 Received: 6 September 2017 Accepted: 19 December 2017 Additional file 1: The supplementary material was designed for this study. The data in the questionnaire titled “the Questionnaire of Occupational Health” and they contained the basic demographic information of the workers, the information of smoking and drinking, occupational history, the history of past disease and drug use, family history of deafness, work-related injuries and other information related to the occupational health. (DOCX 45 kb) Authors’ contributions SY made substantial contributions to the design of the study, the process of implementation and the revision of the final manuscript. YZ had offered many intellectual conceptions in the beginning of the subject and revised the final manuscript. GC, HZ and GG provided human and material support in the data collection and revised the final manuscript. JJ, WZ, HW and YL participated in the collection of data both in the epidemiological survey and the laboratory testing and revised the final manuscript. YY contributed to the interpretation of important information and the modification of this paper. PY has analyzed the data and written this paper. All authors have read and approved the final manuscript. Advantages for this study are as follows. Firstly, a nested case-control study used in our study can better overcome the selection bias and recall bias, as well as reducing costs. Secondly, more accurate measurements than previous studies are used, including the intensity of noise exposure, the diagnosis of NIHL and the principle of double blind and so on. Some limitations of this study should also be known. It has been known that exposing to noise coming from living environment and individual activities for a long time can also cause hearing impairment [36], but the effect is smaller and less hazardous compared with NIHL due to noise exposure in the working place [37]. Namely, the results of our study are authentic. Ethic approval and consent to participate The study was approved by the Ethics Committee of Henan Provincial Institute for Occupational Health (Ethical approval no.: 2,013,003) and informed consent was signed by all participants or their agents. The study was approved by the Ethics Committee of Henan Provincial Institute for Occupational Health (Ethical approval no.: 2,013,003) and informed consent was signed by all participants or their agents. Abbreviations ARHI A l 3. WHO: Protecting workers' health. 2014. [http://www.who.int/mediacentre/ factsheets/fs389/en/]. Accessed 25 June 2017. 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Int J Environ Res Public Health. 2016;13(561):14. 21. Friedman RA, Laer LV, Huentelman MJ, Sheth SS, Eyken EV, Corneveaux JJ, Tembe WD, Halperin RF, Thorburn AQ, Thys S, et al. GRM7 variants confer susceptibility to age-related hearing impairment. Hum Mol Genet. 2009; 18(4):785–96. 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 and we will help you at every step: 22. Newman DL, Fisher LM, Ohmen J, Parody R, Fong CT, Frisina ST, Mapes F, Eddins DA, Robert Frisina D, Frisina RD, Friedman RA. Abbreviations ARHI A l GRM7 variants associated with age-related hearing loss based on auditory perception. 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https://openalex.org/W3044475451	https://europepmc.org/articles/pmc7380644?pdf=render	English	null	Noninvasive imaging of tumor hypoxia after nanoparticle-mediated tumor vascular disruption	PloS one	2,020	cc-by	9,519	Abstract Citation: Virani NA, Kelada OJ, Kunjachan S, Detappe A, Kwon J, Hayashi J, et al. (2020) Noninvasive imaging of tumor hypoxia after nanoparticle-mediated tumor vascular disruption. PLoS ONE 15(7): e0236245. https://doi.org/ 10.1371/journal.pone.0236245 We have previously demonstrated that endothelial targeting of gold nanoparticles followed by external beam irradiation can cause specific tumor vascular disruption in mouse models of cancer. The induced vascular damage may lead to changes in tumor physiology, includ- ing tumor hypoxia, thereby compromising future therapeutic interventions. In this study, we investigate the dynamic changes in tumor hypoxia mediated by targeted gold nanoparticles and clinical radiation therapy (RT). By using noninvasive whole-body fluorescence imaging, tumor hypoxia was measured at baseline, on day 2 and day 13, post-tumor vascular disrup- tion. A 2.5-fold increase (P<0.05) in tumor hypoxia was measured two days after combined therapy, resolving by day 13. In addition, the combination of vascular-targeted gold nano- particles and radiation therapy resulted in a significant (P<0.05) suppression of tumor growth. This is the first study to demonstrate the tumor hypoxic physiological response and recovery after delivery of vascular-targeted gold nanoparticles followed by clinical radiation therapy in a human non-small cell lung cancer athymic Foxn1nu mouse model. Editor: Gayle E. Woloschak, Northwestern University Feinberg School of Medicine, UNITED STATES Editor: Gayle E. Woloschak, Northwestern University Feinberg School of Medicine, UNITED STATES Received: March 16, 2020 Accepted: July 1, 2020 Published: July 24, 2020 Copyright: © 2020 Virani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2020 Virani 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. Funding: This project was supported, in part, by award number R21CA188833 from the National Cancer Institute (NCI). The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the NCI or NIH. J.K. would like to acknowledge support from a Research Fellowship of the Japanese Society for the Promotion of Noninvasive imaging of tumor hypoxia after nanoparticle-mediated tumor vascular Needa A. ViraniID1☯, Olivia J. KeladaID1☯, Sijumon Kunjachan1, Alexandre Detappe1,2, Jihun KwonID1,3, Jennifer Hayashi1,4, Ana Vazquez-Pagan1,4, Douglas E. Biancur2, Thomas Ireland5, Rajiv Kumar1,4, Srinivas Sridhar1,4, G. Mike Makrigiorgos1, Ross I. Berbeco1 1 Department of Radiation Oncology, Brigham and Women’s Hospital, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts, United States of America, 2 Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston Massachusetts, United States of America, 3 Department of Radiation Oncology, Hokkaido University, Sapporo, Japan, 4 Nanomedicine Innovation Center and Department of Physics, Northeastern University, Boston, Massachusetts, United States of America, 5 LA-ICP-MS and ICP-ES Laboratories, Boston University, Boston, Massachusetts, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. navirani@bwh.harvard.edu PLOS ONE PLOS ONE RESEARCH ARTICLE Introduction A key component propagating tumor growth is the presence of high vascular infiltration and oxygen diffusion [1, 2]. As tumors progress beyond a few millimeters in size, rate of cell prolif- eration exceeds the rate of vasculature neoangiogenesis leading to areas of limited oxygen and nutrient supply. Due to the rapid neovascular growth rate, vessels are usually chaotic and abnormal with large pores allowing for passive accumulation of small molecules and PLOS ONE \| https://doi.org/10.1371/journal.pone.0236245 July 24, 2020 1 / 16 PLOS ONE Hypoxia imaging after vascular disruption therapy nanoparticles [3–5]. The importance of the vascular supply for continued tumor growth makes it a potential therapeutic target for both radiation and chemotherapy [6, 7]. Science (JSPS), and the JSPS Overseas Challenge Program for Young Researchers. This work was supported by JSPS KAKENHI Grant Number JP17J03616. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Science (JSPS), and the JSPS Overseas Challenge Program for Young Researchers. This work was supported by JSPS KAKENHI Grant Number JP17J03616. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Vascular targeting agents can either a) inhibit angiogenesis to control the development of blood vessels [6, 8], or b) restrict the function of existing blood vessels using vascular disrupt- ing agents (VDAs) [5, 9]. Multiple small molecule VDAs have been tested in clinical trials with limited success in translation [9–12]. A key downfall of VDA treatment alone is the remaining viable rim of tumor cells surrounding the necrotic, ischemic treated region leading to contin- ued tumor growth[5, 9]. Although some of these vascular targeting agents have shown to be effective when used in conjunction with conventional therapies, such as external beam radia- tion therapy, they have caused severe off-target toxicity [7, 8, 10–16]. In this work, we investi- gate vascular-targeted gold nanoparticles, which act as VDAs when irradiated after delivery. Combining a targeted VDA concept with the precision of modern image-guided radiation therapy enables double targeting and limits side effects by avoiding the activation of any gold nanoparticles that may accumulate in surrounding healthy tissue. This dual targeting strategy can minimize normal tissue toxicity and consequently improve the therapeutic benefit [17– 20]. Competing interests: The authors have declared that no competing interests exist. Introduction Metallic nanoparticles have been developed for use in combination with radiation therapy to intensify the damage to tumor cells via radiation dose amplification. This is due to the phys- ical interaction of low energy x-rays with high-Z elements resulting in the emission of both Auger electrons and short-range photoelectrons leading to local cell damage [21–24]. Gold nanoparticles (AuNP) are of particular interest as radiosensitizers due to their biocompatibil- ity. Gold nanoparticles have a high K-edge (81 keV), which enables local, controlled dose enhancement when coupled with conventional radiation therapy [25–27]. The ease of surface modification of nanoparticles and targeting capabilities to neovascular regions offers new pos- sibilities for anti-vascular therapies. Theoretical and experimental studies have shown that gold nanoparticles can impart con- siderable dose amplification to endothelial cells even without specific cellular uptake [18, 22, 28, 29]. Experimentally, we have previously shown that Arginylglycylaspartic acid (RGD) pep- tide surface modified gold nanoparticles boost local radiation doses due to the generation of short-range electrons resulting in tumor blood vessel disruption [30] and subsequent changes in tumor physiology indicative of a propensity for improved drug delivery [31]. RGD peptide has a strong affinity for αvβ3 and can be used as a tumor vasculature specific targeting agent. RGD is involved in protein and cell attachment, making it a prime candidate for the delivery of vasculature specific nanoparticles [30, 32]. While efficacy has been previously demonstrated, there is a lingering concern that vascular disruption therapy could lead to the development of hypoxic regions within the tumor where the high rate of oxygen demanded and consumed by the tumor and endothelial cells exceeds supply [33, 34]. The hypoxic tumor microenvironment hinders response to chemotherapy and radiation treatment resulting in an overall reduction in survival due to the reduction in cellular oxygenation [35]. Hypoxia further stimulates inducible factors leading to increased tumor pro- liferation (tumor invasion and metastases via tumor neovasculature) and a more aggressive phenotype [35–37]. The challenge of tumor hypoxia in cancer therapy has been known for over 60 years and is still a significant clinical problem today [38]. In the study presented, we investigated the impact of tumor vascular disruption on tumor hypoxia as the latter can lead to radiation resistance and is associated with a worse prognosis. To this end, we used vascular-targeted AuNP combined with clinical radiation (10Gy, 6MV) and performed longitudinal in vivo imaging of tumor hypoxia. Animal tumor model and in vivo studies Animal studies were approved by the Dana Farber Cancer Institute Institutional Animal Care and Use Committee (DFCI IACUC, Protocol Number 14–032) and conducted in full compli- ance with the Association for the Assessment and Accreditation of Laboratory Animal Care, governmental and institutional regulations and principles outlined in the United States Public Health Service Guide. 4–6 week old female athymic Foxn1nu mice (Charles River Laboratories, Wilmington, USA) were used. Mice weighing ~25 g were fed ad libitum with standard food pellets and water. Human lung adenocarcinoma cells, A549 (ATCC CCL-185, Manassas, VA), were grown in vitro in Roswell Park Memorial Institute medium (RPMI 1640; Gibco, Invitro- gen, USA), supplemented with 10% fetal bovine serum (FBS) (Invitrogen, USA) and 1% pen/ strep (10,000 U/mL penicillin; 10,000 μg/mL streptomycin, Invitrogen, USA). The culture was maintained at 37˚C, 5% CO2 and optimal humidity. Mice were inoculated with 3x106 cells/ 100 μL by subcutaneous injection into the dorsolateral left flank. Tumor volume was deter- mined with calipers using the formula: volume = (length × width2)/2 and a tumor size of 8 mm2 was obtained in ~2 weeks. Mice were euthanized when tumors were more than 10% of animal body weight or if any other signs of distress were identified (ulcerations, lethargy, etc). Chemical synthesis and characterization of functionalized targeted gold nanoparticles for tumor vascular targeting Standard procedures were used to prepare targeted gold nanoparticles and are described in greater detail elsewhere [39]. Colloidal gold was prepared by the reduction of chloroauric acid in the presence of a stabilizing/reducing agent, tetrakis(hydroxymethyl) phosphonium chlo- ride (THPC). The PEGylation of THPC stabilized gold nanoparticles was carried out by ligand exchange process using optimized ratios of three heterobifunctional PEGs (Thiol-PEG-Amine, Thiol-PEG-Carboxyl and Thiol-PEG-methoxy). The functionalized gold nanoparticles were further conjugated with Arginylglycylaspartic acid (RGD) peptide to the terminal carboxylic (−COOH) groups via EDC chemistry for vascular targeting and a near infrared dye (AF647) was attached for imaging. Subsequently, the resultant product, PEG-RGD-AuNP-AF647 (for the remainder of this study will be referred to as AuNP), was further purified and character- ized. Nanoparticles were purified using a membrane-filtration technique. AuNP was subjected to membrane dialysis by using 14 kDa cellulose membrane against purified double-distilled water to remove any traces of un-reacted EDC or other hydrolyzed products along with the un-reacted RGD molecules resulting in a final solution comprised of purified AuNP. Introduction Tumor regression studies were also performed on pre-clinical mouse models bearing human non-small cell lung cancer (NSCLC, A549) to investigate the potential therapeutic benefit of this dual-targeting approach. PLOS ONE \| https://doi.org/10.1371/journal.pone.0236245 July 24, 2020 2 / 16 PLOS ONE Hypoxia imaging after vascular disruption therapy Whole body hypoxia imaging Fluorescence imaging was performed on animals using the IVIS system (PerkinElmer, Boston, MA) prior to irradiation (0 h), as well as post-irradiation with a 6MV clinical beam (48 h and 13 days) after injection of a hypoxia-specific fluorophore marker (HypoxiSense680). Groups such as control (no AuNP / no IR), AuNP only, IR only and AuNP+IR were used for the stud- ies and imaged for longitudinal changes in tumor hypoxia. The Hypoxisense680 (HS680) imaging probe was administered as per the manufacturer’s protocol (PerkinElmer, Boston, MA). Each mouse was intravenously injected with 2 nmol (100 μL) of the reconstituted imaging probe 48 h prior to fluorescence imaging (FLI). For each imaging session, mice were anesthetized with an isoflurane/air mixture gas and positioned prone in the imaging cassette which was then placed into the imaging chamber (Em = 720 nm, Ex = 675 nm). The selection of excitation/emission wavelengths was chosen to effectively mini- mize the AF647 signal as seen in a representative excitation/emission spectra in S1 Fig. Auto- fluorescence background was subtracted by determining the mean tumor fluorescence signal in normalized counts in the image prior to injection. The collected fluorescence data were ana- lyzed using Living Image Software (version 4.2). Tumor regions of interest (ROI) were drawn, and a threshold was applied to all animals (equal to 30% of the mean tumor fluorescence of positive control mice) to calculate the hypoxic fraction of the tumors. AuNP biodistribution Mice were sacrificed at 1 h, 4 h, 12 h, 24 h, and 30 day time points post-intravenous (i.v.) injec- tion of ~1 mg/g of AuNP (as per previous dosing studies [30]) and the tumor and other vital organs were excised. Biodistribution of AuNP was measured by inductively coupled plasma mass spectrometry (IC-PMS) for determination of the optimal therapeutic time point for radi- ation delivery based on the accumulation of AuNP in the tumor and other organs. All samples were frozen with liquid nitrogen and then ground in an agate mortar and pestle. ~0.03 g of sample was weighed out and digested in 3 mL HCl and 1 mL HNO3 on a hotplate at 110˚C overnight. Once removed from the hot plate, 1 mL H2O2 was added slowly and completed in two 0.5 mL steps. Samples were dried on a hot plate overnight before adding 0.25 HCl. Then 0.3 mL HCl was added and allowed to sit overnight followed by 0.25 mL H2O2. Samples were then diluted with MQ water to reach a 3% HCl concentration solution. Just prior to ICP-MS, PLOS ONE \| https://doi.org/10.1371/journal.pone.0236245 July 24, 2020 3 / 16 PLOS ONE Hypoxia imaging after vascular disruption therapy samples were diluted further 100X. Standards were prepared using a pure Au standard and diluting with 3% HCl. Standards were made at concentrations of 0.1, 0.25, 0.5, 0.75, 1, 2.5, 5, 10 and 20 ppb. Transmission electron microscopy imaging High-resolution transmission electron microscopy (TEMJEOL 2010F, Philips Netherlands) was performed on cells and tumor samples. A549 cells were imaged 2 hours after incubation with 0.1 mg/mL AuNP. Afterward, samples were placed at 4˚C in dark conditions. High-reso- lution transmission electron microscopy was carried at magnifications of 30000x to visualize uptake of the AuNP. For in vivo samples, TEM imaging was performed on A549 lung tumor sections. Small tumor tissue fragments/ pieces (1–2 mm3) were obtained from the dissected whole tumor and fixed using a mixture of 2.5% glutaraldehyde in 0.2 M Sorensen buffer at pH 7. Using ultracryotome, thin sections were sliced and washed/stained in aqueous uranyl acetate (2%) for 2 h. The tissue was then placed at 4˚C in dark conditions and further dehydrated by 100% ethanol and propylene oxide to be embedded in liquid epoxy resin (Epon™). High-reso- lution electron microscopy was carried out at different magnifications ranging from 2500x - 10000x to visualize the accumulation of AuNP in tumor blood vessels 24 h post-AuNP injec- tion of 1mg/g AuNP. Clinical external beam radiation therapy Irradiations were performed 24 h after baseline tumor hypoxia imaging and injection of AuNP with a 6MV linear accelerator (TrueBeam, Varian Medical Systems, Palo Alto, CA). For clini- cal beam radiations, A549 tumor bearing mice in the IR and AuNP+IR groups were anesthe- tized with a mix of ketamine/xylazine (10:1) before irradiation. Eclipse (Varian Medical Systems, Palo Alto, CA) clinical treatment planning system was used to calculate the tumor dose distribution using the analytical anisotropic algorithm (AAA) for a 5 x 5 cm2 field size at a gantry angle of 180 degrees, SSD of 90 cm, dose rate of 600 MU/min and a prescription of 10 Gy to the center of the tumor. A 10-cm depth for the tumor was created with solid water PLOS ONE \| https://doi.org/10.1371/journal.pone.0236245 July 24, 2020 4 / 16 PLOS ONE Hypoxia imaging after vascular disruption therapy (CIRS, Norfolk, VA). Normal tissue was shielded by the positioning of the primary collimator as previously described [23, 40]. Tumor regression and time-to-tumor-doubling study Four groups (Control (-AuNP/-IR); AuNP only, IR only, and AuNP+IR) of 9–10 mice each were monitored for changes in tumor size for up to 50 days following tumor inoculation. Tumor response was measured by calipers using the formula: volume = (length × width2)/2. Mouse body weight was recorded, and behavior was monitored throughout the experiment to assess systemic toxicity. Mice were euthanized when tumors were more than 10% of animal body weight or if any other signs of distress were identified (ulcerations, lethargy, etc). Time- to-Tumor-Doubling (TTD) was defined as time for tumor volume to double relative to day of treatment. Statistical analysis Biodistribution and Hypoxisense statistical significance was evaluated using a standard stu- dent’s unpaired two-tailed t-test (P<0.05). Relative tumor volume statistical analysis was per- formed using Mann-Whitney nonparametric U test (P < 0.05) in GraphPad Prism (version 8). TTD statistical analysis was performed using a Log-Rank (Mantel-Cox) test (P < 0.05) in GraphPad Prism (version 8). Tumor hypoxia histological immunohistochemistry Histological examinations were carried out to investigate the effect of nanoparticles and 6MV clinical beam treatment (+/-AuNP; +/-IR) on tumor hypoxia. For tumor hypoxia staining, pimonidazole (Hypoxyprobe, Burlington, MA) was dissolved in neutral buffered saline and administrated i.v.at a concentration of 60 mg/kg body weight with mice sacrificed 90 min later. The excised tumor tissues were fixed in 10% formalin (neutral buffered) and embedded in paraffin. Thin tissue slices (5μm) were cut, and the sections were mounted for antigen retrieval. Standard IHC steps of de-paraffinizing and rehydrating was followed by staining with the mouse mAb FITC-labeled anti-pimonidazole antibody (Hypoxyprobe, Burlington, MA) and incubating for 1 h at 37 ˚C in the dark as per the manufacturer’s protocol. Following blocking and DAB steps, images were visualized using a Leica DMi8 (Leica Microsystems, Ger- many) widefield microscope with a high-resolution AxiocamMrm Rev.3 camera at 20x magnification. Toxicity histology For the assessment of treatment toxicity (+/-AuNP; +/-IR), mice bearing A549 subcutaneous tumors were injected with either saline or ~1 mg/g AuNP followed by 10 Gy radiation with a 220 kVp Small Animal Radiation Research Platform (SARRP, Xstrahl, Suwanee, GA) after 24 h. SARRP radiations were performed similar to previous studies [30]. Mice were then eutha- nized and vital organs (liver, lungs, spleen, kidney, and heart) were collected for histology 24 h post-treatment. H&E histological staining was carried out on all excised tissues. Results The conceptual design and the experimental set-up is depicted in Fig 1. Targeted gold nano- particles were co-functionalized with polyethylene glycol (PEG) and RGD. PEGylation pro- vides extended systemic circulation to allow AuNP to reach the tumor and RGD confers tumor vascular targeting in vivo [39]. High-resolution transmission electron microscopy (TEM) imaging showed AuNP to have spherical surface morphology with an average AuNP core diameter of~3–4 nm and DLS determined the hydrodynamic size to be ~10–12 nm, an appropriate size for renal clearance [41]. The absorption and fluorescence spectra of AuNP was λmax of 650/668 nm post-labeling respectively and the average zeta-potential (surface charge) was +7.93 mV in PBS (7.4 pH) [39]. The deposition and early uptake of AuNP clusters were observed in the A549 tumor cell vesicles in vitro after 2 h of incubation with 0.1 mg/mL AuNP (Fig 2A). Uptake in the tumor and tumor vasculature at 24 h post-AuNP intravenous injection were also visualized in TEM imaging (Fig 2B) to verify nanoparticles have accumulated within the tumor and tumor vascu- lature at time of radiation delivery. In Fig 2C, biodistribution studies confirmed maximum tumor accumulation of AuNP at 12 and 24 h, with no statistically significant difference between the time points. However, at 24 h significant (P<0.05) clearance of AuNP from the kidney was observed compared to 12 h, while accumulation in all other organs (liver, spleen, lungs, and heart) was not substantially different. Previous literature indicates that AuNPs are rapidly eliminated from the body via renal clearance [42, 43]. Other studies have also shown that the size, composition, shape, and surface charge of AuNP nanoparticles may also direct Fig 1. Experimental timeline and design. A schematic depiction of the experimental design to measure tumor hypoxia after radiation- induced tumor vascular damage is shown. In a human NSCLC xenograft model (A549) gold nanoparticles (AuNP, blue arrow) were i.v. administered to target tumor neovessels, and 10 Gy radiation was delivered (yellow arrow) 24 h post-AuNP injection. HypoxiSense680 fluorescence imaging (red arrow) was used to assess changes in tumor hypoxia pre (0 h) and post-treatment (48 h and 13 days) for all four groups (Control, AuNP, IR, and AuNP+IR). After imaging, mice were a) monitored for tumor progression or b) euthanized for immunohistochemistry studies to confirm tumor hypoxia. https://doi.org/10.1371/journal.pone.0236245.g001 Fig 1. Experimental timeline and design. Data availability All relevant data are within the manuscript. 5 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0236245 July 24, 2020 PLOS ONE Hypoxia imaging after vascular disruption therapy Results A schematic depiction of the experimental design to measure tumor hypoxia after radiation- induced tumor vascular damage is shown. In a human NSCLC xenograft model (A549) gold nanoparticles (AuNP, blue arrow) were i.v. administered to target tumor neovessels, and 10 Gy radiation was delivered (yellow arrow) 24 h post-AuNP injection. HypoxiSense680 fluorescence imaging (red arrow) was used to assess changes in tumor hypoxia pre (0 h) and post-treatment (48 h and 13 days) for all four groups (Control, AuNP, IR, and AuNP+IR). After imaging, mice were a) monitored for tumor progression or b) euthanized for immunohistochemistry studies to confirm tumor hypoxia. https://doi.org/10.1371/journal.pone.0236245.g001 6 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0236245 July 24, 2020 PLOS ONE Hypoxia imaging after vascular disruption therapy Fig 2. Tumor uptake and biodistribution studies. (A) Tumor uptake of AuNP at 2 h post-incubation in A549 cel was measured using high-resolution TEM. Localized uptake in several sub-cellular compartments was observed following 2 h incubation. (B) High-resolution transmission electron microscopy was carried out on ex vivo A549 lu tumor sections 24 h after 1mg/g AuNP injection. White arrows indicate tumor cell/vasculature uptake of AuNP (a magnification of 2500 - 10000x) and labels indicate the location of red blood cells and endothelial cells. (C) Longitudinal accumulation of AuNP in the tumor, kidney, liver, spleen, lungs, and heart was measured at 1, 4, 12, and 30 days after i.v. administration of ~1mg/g of AuNP (n = 2–5). The amount of Au was quantified using ICP-M and normalized with respective organ weights. Values represent mean ± SD, (P<0.05). https://doi.org/10.1371/journal.pone.0236245.g002 Fig 2. Tumor uptake and biodistribution studies. (A) Tumor uptake of AuNP at 2 h post-incubation in A549 cell was measured using high-resolution TEM. Localized uptake in several sub-cellular compartments was observed following 2 h incubation. (B) High-resolution transmission electron microscopy was carried out on ex vivo A549 lu tumor sections 24 h after 1mg/g AuNP injection. White arrows indicate tumor cell/vasculature uptake of AuNP (at magnification of 2500 - 10000x) and labels indicate the location of red blood cells and endothelial cells. (C) Longitudinal accumulation of AuNP in the tumor, kidney, liver, spleen, lungs, and heart was measured at 1, 4, 12, 2 and 30 days after i.v. administration of ~1mg/g of AuNP (n = 2–5). The amount of Au was quantified using ICP-M and normalized with respective organ weights. Values represent mean ± SD, (P<0.05). https://doi.org/10.1371/journal.pone.0236245.g002 Fig 2. Results Tumor uptake and biodistribution studies. (A) Tumor uptake of AuNP at 2 h post-incubation in A549 cel was measured using high-resolution TEM. Localized uptake in several sub-cellular compartments was observed following 2 h incubation. (B) High-resolution transmission electron microscopy was carried out on ex vivo A549 lu Fig 2. Tumor uptake and biodistribution studies. (A) Tumor uptake of AuNP at 2 h post-incubation in A549 cells was measured using high-resolution TEM. Localized uptake in several sub-cellular compartments was observed following 2 h incubation. (B) High-resolution transmission electron microscopy was carried out on ex vivo A549 lung tumor sections 24 h after 1mg/g AuNP injection. White arrows indicate tumor cell/vasculature uptake of AuNP (at a magnification of 2500 - 10000x) and labels indicate the location of red blood cells and endothelial cells. (C) Longitudinal accumulation of AuNP in the tumor, kidney, liver, spleen, lungs, and heart was measured at 1, 4, 12, 24 h, and 30 days after i.v. administration of ~1mg/g of AuNP (n = 2–5). The amount of Au was quantified using ICP-MS and normalized with respective organ weights. Values represent mean ± SD, (P<0.05). https://doi.org/10.1371/journal.pone.0236245.g002 clearance via the hepatobiliary pathway[44, 45]. Evaluation of treatment toxicity was assessed using H&E histological staining (Fig 3). At 24 h post treatment, H&E staining indicated no detectable toxicity or morphological changes to the surrounding organs (liver, lungs, spleen, 7 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0236245 July 24, 2020 PLOS ONE Hypoxia imaging after vascular disruption therapy Fig 3. Evaluation of treatment toxicity. H&E staining indicated no detectable toxicity in surrounding visceral organs such as spleen, kidney, lungs, heart, and liver due to either AuNP and/or radiation treatment. Scale bar: 50 μm. https://doi.org/10.1371/journal.pone.0236245.g003 Fig 3. Evaluation of treatment toxicity. H&E staining indicated no detectable toxicity in surrounding visceral organs such as spleen, kidney, lungs, heart, and liver due to either AuNP and/or radiation treatment. Scale bar: 50 μm. https://doi.org/10.1371/journal.pone.0236245.g003 https://doi.org/10.1371/journal.pone.0236245.g003 kidney, and heart) due to either AuNP and/or radiation, consistent with our previously reported results [30]. Evaluation of the therapeutic benefit demonstrated significant (P<0.05) tumor growth sup- pression as a result of the combined anti-vascular approach. There was no statistical difference between tumor growth in the control (no therapy) and AuNP or radiation only treatment groups (Fig 4A). The relative tumor growth 50 days post-inoculation was substantially reduced in the AuNP+IR treated group compared to all control groups. PLOS ONE \| https://doi.org/10.1371/journal.pone.0236245 July 24, 2020 Results (A) Tumor growth relative to tumor size on day of irradiation is shown. A significant (P<0.05) delay in tumor growth was observed for AuNP+IR treated mice compared to the controls up to 50 days (n = 9–10). Values represent mean growth ± SD. (B) Time-to-Tumor-Doubling (TTD) is shown with endpoint failure defined as tumor volume doubling relative to day of treatment. A significant (P<0.05) delay in doubling, which is an indicator of a progressive disease, is seen for mice treated with AuNP+IR compared to all control groups (n = 9–10). Fig 4. Therapeutic evaluation of tumor vascular disruption. (A) Tumor growth relative to tumor size on day of irradiation is shown. A significant (P<0.05) delay in tumor growth was observed for AuNP+IR treated mice compared to the controls up to 50 days (n = 9–10). Values represent mean growth ± SD. (B) Time-to-Tumor-Doubling (TTD) is shown with endpoint failure defined as tumor volume doubling relative to day of treatment. A significant (P<0.05) delay in doubling, which is an indicator of a progressive disease, is seen for mice treated with AuNP+IR compared to all control groups (n = 9–10). https://doi.org/10.1371/journal.pone.0236245.g004 control groups are likely due to the continued tumor growth. These findings were confirmed with representative pimonidazole staining that showed an increase in tumor hypoxia 48 h after AuNP+IR treatment (Fig 5D). Results Due to tumor ulceration occur- ring in all cohorts (including no treatment) necessitating pre-emptive euthanasia, Time-to- Tumor-Doubling (TTD) was accessed relative to day of treatment as a determinant of a pro- gressive disease. As seen in Fig 4B, mice treated with AuNP+IR (56.5 d) had significantly (P<0.05) delayed TTD compared to all control groups (33, 36.5, and 40 d for Control, AuNP, and IR, respectively). Given the trend in the tumor growth curves and TTD, one would reason- ably expect the AuNP+IR treated group to exhibit long-term survival. Mice with no tumor ulcerations in the AuNP+IR group survived over 110 days post-inoculation, with minimum change in tumor volume indicative of long-term tumor growth delay. This was further observed in a previous study using the same nanoparticle formulation and radiation regiment in a mouse model of pancreatic cancer [31]. We measured tumor hypoxia pre- and post-tumor vascular disruption at early and late time points. HypoxiSense680, a fluorescent probe that binds to carbonic anhydrase (CAIX) that is upregulated in hypoxic regions especially in non-small cell lung cancer [46], was utilized fol- lowing the manufacturer’s protocols. Mice were imaged 48 h post-injection to minimize noise from any unbound or non-specifically accumulated probe and maximize tumor hypoxic region uptake and retention. Whole body FLI showed a nearly 2.5-fold increase in Hypoxi- Sense680 signal at 48 h post-tumor vascular disruption for the AuNP+IR group (Fig 5A and 5B). However, in the IR-only group, this increase was substantially smaller compared to the non-treated controls. A comparison of the control and AuNP-only groups indicated that the AuNP contributed no additional fluorescence signal (Fig 5C). Measured increases in these PLOS ONE \| https://doi.org/10.1371/journal.pone.0236245 July 24, 2020 8 / 16 PLOS ONE Hypoxia imaging after vascular disruption therapy Fig 4. Therapeutic evaluation of tumor vascular disruption. (A) Tumor growth relative to tumor size on day of irradiation is shown. A significant (P<0.05) delay in tumor growth was observed for AuNP+IR treated mice compared to the controls up to 50 days (n = 9–10). Values represent mean growth ± SD. (B) Time-to-Tumor-Doubl (TTD) is shown with endpoint failure defined as tumor volume doubling relative to day of treatment. A significant (P<0.05) delay in doubling, which is an indicator of a progressive disease, is seen for mice treated with AuNP+IR compared to all control groups (n = 9–10). https://doi.org/10.1371/journal.pone.0236245.g004 Fig 4. Therapeutic evaluation of tumor vascular disruption. Discussion (B) Plots show the mean quantification of each tumor ROI (Radiation Efficiency ([photon/sec]/μWatt/cm2)) on each day of imaging (Day -1, 2 and 13). In the IR-only group, the images show a 1.5-fold increase in signal 48 h post-IR compared to baseline while 11 days later the tumor hypoxia remained stable. The AuNP+IR group showed a 2.5-fold increase (P<0.05) at 48 h post-IR and then a decrease to the level of IR-only signal 11 days later. Data presented as mean ± SD (n = 3). (C) The relative radiation efficiency intensity of HypoxiSense680 is similar in the control and GNP- only groups, indicating no interference from the attached fluorophore. (D) Qualitative histological assessment of tumor hypoxia by pimonidazole staining further confirmed the increase in the hypoxia following tumor vascular disruption at 48 h. Scale bar = 100 μm. htt //d i /10 1371/j l 0236245 005 y-induced tumor hypoxia in A549 non-small cell lung cancer xenografts. (A) Fluorescence imaging of tumor hypoxia in h l d h f d h h b f Fig 5. Measuring therapy-induced tumor hypoxia in A549 non-small cell lung cancer xenografts. (A) Fluorescence imaging of tumor hypoxia in representative mice from the IR-only and AuNP+IR groups with A549 xenografts. Mice were injected with HypoxiSense680 48 h before imaging. Tumor hypoxia is visualized pre- and post-10 Gy irradiation treatment. Representative images show the whole mouse and a magnified image of each tumor ROI. The color bar scale shows average radiant efficiency. (B) Plots show the mean quantification of each tumor ROI (Radiation Efficiency ([photon/sec]/μWatt/cm2)) on each day of imaging (Day -1, 2 and 13). In the IR-only group, the images show a 1.5-fold increase in signal 48 h post-IR compared to baseline while 11 days later the tumor hypoxia remained stable. The AuNP+IR group showed a 2.5-fold increase (P<0.05) at 48 h post-IR and then a decrease to the level of IR-only signal 11 days later. Data presented as mean ± SD (n = 3). (C) The relative radiation efficiency intensity of HypoxiSense680 is similar in the control and GNP- only groups, indicating no interference from the attached fluorophore. (D) Qualitative histological assessment of tumor hypoxia by pimonidazole staining further confirmed the increase in the hypoxia following tumor vascular disruption at 48 h. Scale bar = 100 μm. immunohistochemistry. Discussion Multiple studies have evaluated the role of radiation dose in either tumor cell or endothelial cell death as related to overall growth inhibition [47–49]. Studies have confirmed that low doses of radiation which are conventionally used in fractionated radiotherapy (2–5 Gy) only cause temporary changes to tumor vasculature, however doses > 8 Gy lead to endothelial cell death and vascular disruption [47–50]. Previous work in our lab has confirmed that a single dose of 10 Gy radiation increases vascular damage and permeation, which is further amplified with the addition of AuNPs [31, 51]. A study to evaluate the change in tumor perfusion in a human laryngeal squamous cell tumor indirectly correlated with the results presented of a decrease in perfusion post irradiation with a single dose of 10 Gy fol- lowed by some recovery in 7–11 days [52]. Previous work by Fuks and Kolesnick reported a potential mechanism for endothelial cell damage via the ceramide mediated apoptosis pathway that was increased at radiation doses of 8–10 Gy but no identifiable pathway changes < 3 Gy [53]. Our results indicating changes in hypoxia due to vascular disruption with a single dose of 10 Gy radiation and AuNP aligns with previous works confirming high doses of irradiation immunohistochemistry. Multiple studies have evaluated the role of radiation dose in either tumor cell or endothelial cell death as related to overall growth inhibition [47–49]. Studies have confirmed that low doses of radiation which are conventionally used in fractionated radiotherapy (2–5 Gy) only cause temporary changes to tumor vasculature, however doses > 8 Gy lead to endothelial cell death and vascular disruption [47–50]. Previous work in our lab has confirmed that a single dose of 10 Gy radiation increases vascular damage and permeation, which is further amplified with the addition of AuNPs [31, 51]. A study to evaluate the change in tumor perfusion in a human laryngeal squamous cell tumor indirectly correlated with the results presented of a decrease in perfusion post irradiation with a single dose of 10 Gy fol- lowed by some recovery in 7–11 days [52]. Previous work by Fuks and Kolesnick reported a potential mechanism for endothelial cell damage via the ceramide mediated apoptosis pathway that was increased at radiation doses of 8–10 Gy but no identifiable pathway changes < 3 Gy [53]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0236245 July 24, 2020 Discussion This is the first study to provide evidence that vascular targeted AuNPs combined with radia- tion causes a transient increase in tumor hypoxia but still results in better tumor control com- pared to radiation-alone treatment. Our experimental findings suggest that vascular targeted AuNPs combined with 10 Gy clinical 6 MV irradiation temporarily increased tumor hypoxia compared to control groups as confirmed by in vivo fluorescence imaging and PLOS ONE \| https://doi.org/10.1371/journal.pone.0236245 July 24, 2020 9 / 16 PLOS ONE Hypoxia imaging after vascular disruption therapy Fig 5. Measuring therapy-induced tumor hypoxia in A549 non-small cell lung cancer xenografts. (A) Fluorescence imaging of tumor hypoxia in representative mice from the IR-only and AuNP+IR groups with A549 xenografts. Mice were injected with HypoxiSense680 48 h before imaging. Tumor hypoxia is visualized pre- and post-10 Gy irradiation treatment. Representative images show the whole mouse and a magnified image of each tumor ROI. The color bar scale shows average radiant efficiency. (B) Plots show the mean quantification of each tumor ROI (Radiation Efficiency ([photon/sec]/μWatt/cm2)) on each day of imaging (Day -1, 2 and 13). In the IR-only group, the images show a 1.5-fold increase in signal 48 h post-IR compared to baseline while 11 days later the tumor hypoxia remained stable. The AuNP+IR group showed a 2.5-fold increase (P<0.05) at 48 h post-IR and then a decrease to the level of IR-only signal 11 days later. Data presented as mean ± SD (n = 3). (C) The relative radiation efficiency intensity of HypoxiSense680 is similar in the control and GNP- only groups, indicating no interference from the attached fluorophore. (D) Qualitative histological assessment of tumor hypoxia by pimonidazole staining further confirmed the increase in the hypoxia following tumor vascular disruption at 48 h. Scale bar = 100 μm. htt //d i /10 1371/j l 0236245 005 Fig 5. Measuring therapy-induced tumor hypoxia in A549 non-small cell lung cancer xenografts. (A) Fluorescence imaging Fig 5. Measuring therapy-induced tumor hypoxia in A549 non-small cell lung cancer xenografts. (A) Fluorescence imaging of tumor hypoxia in representative mice from the IR-only and AuNP+IR groups with A549 xenografts. Mice were injected with HypoxiSense680 48 h before imaging. Tumor hypoxia is visualized pre- and post-10 Gy irradiation treatment. Representative images show the whole mouse and a magnified image of each tumor ROI. The color bar scale shows average radiant efficiency. Discussion Our results indicating changes in hypoxia due to vascular disruption with a single dose of 10 Gy radiation and AuNP aligns with previous works confirming high doses of irradiation PLOS ONE \| https://doi.org/10.1371/journal.pone.0236245 July 24, 2020 10 / 16 PLOS ONE Hypoxia imaging after vascular disruption therapy leads to transient changes in tumor endothelial cells which are not seen in conventionally used < 2Gy fractionated therapy [47, 50]. Radiation therapy is dependent on the oxygen enhancement ratio (OER) which refers to the enhancement of ionizing radiation in the presence of oxygen [54]. Currently studied VDAs can hinder the efficacy of radiation therapy by reducing tumor oxygenation over a pro- longed period and migrating tumors towards a more aggressive phenotype. Here we have shown that although the underlying mechanism of treatment is still vascular disruption, over- all tumor growth was still hindered compared to control treatment and long term hypoxia was prevented. This transient vascular disruption was further confirmed by Kwon et al. demon- strating an increase in microbubble perfusion 24 hours post therapy followed by normalization to baseline levels, indicative of normalization of tumor vasculature flow [55]. The negative impact of hypoxia on radiation therapy has been known for decades [56, 57]. Fractionated radiation therapy takes advantage of time between doses to balance reoxygena- tion of cells versus repopulation of surviving cells to improve the efficacy of therapy [57, 58]. Tissue that is either hypoxic or anoxic during radiation are up to three times more likely to be radioresistant compared to oxygenated tissue [59]. A recent study compared 10–30 Gy radia- tion doses to vascular damage and microenvironment reoxygenation [57]. Similar to the work presented, they showed an increase in hypoxia markers, including HIF-1α, pimonidazole, and carbonic anhydrase 9, post-radiation followed by a decrease around 10 days post 20 Gy radia- tion [57]. They further showed that tumor reoxygenation was dose dependent and higher doses of radiation led to a rapid increase in hypoxic cells followed by reoxygenation of surviv- ing fractions [57]. This transient change in reoxygenation is crucial for conventionally use fractionation approaches and treatment planning. In the work presented here, we observed tumor growth suppression by combining targeted nanoparticles with radiation therapy, despite the transient increase in induced hypoxia, indicating that this could be a potential clini- cal strategy. PLOS ONE \| https://doi.org/10.1371/journal.pone.0236245 July 24, 2020 Discussion A recent study compared the impact of incorporating tumor hypoxia measure- ments in NSCLC patient prescribed dose calculations and identified the increased importance of hypoxic fractions within a tumor relative to overall tumor volume [56]. In summary, we have found that tumor vascular disruption due to the vascular targeting and radiation dose amplification properties of AuNPs resulted in a short-term transient increase in tumor hypoxia as well as long-term tumor growth suppression. Histology demon- strated no damage to normal tissues as a result of the therapy. As with many other anti-angio- genic or anti-vascular treatments, there are issues that still need to be addressed related to the optimal timing of subsequent therapies (radiation and/or chemotherapy) in order to minimize the impact of the induced hypoxia. Non-invasive molecular imaging techniques such as func- tional MRI, specifically blood oxygen level dependent (BOLD) MRI to continuously monitor hypoxia or dynamic contrast enhanced (DCE) MRI to monitor vascular permeability [60–63], photoacoustic imaging to monitor changes in hemodynamic characteristics [64], or more con- ventionally used positron emission tomography (PET) [65]could be used to further investigate earlier time points to resolve the kinetics of tumor hypoxia after vascular targeted AuNP and radiation therapy as well as determine an optimal dose schedule to maximize the OER during radiation therapy. In addition, future work incorporating an orthotopic NSCLC model can be studied to understand the microenvironment changes expected post-vascular disruption in a more clinically representative model. Recent studies comparing subcutaneous and orthotopic lung cancer models confirm the improved vascular functionality and in turn reduced hypoxia within orthotopic models [66, 67]. In this study we have shown that regardless of a transient hypoxic tumor microenvironment, tumor burden significantly decreased following vascular disruption therapy with targeted AuNP and IR. The increased vascularity of orthotopic tumors may improve AuNP distribution, subsequently further increasing the impact of IR. In PLOS ONE \| https://doi.org/10.1371/journal.pone.0236245 July 24, 2020 11 / 16 PLOS ONE Hypoxia imaging after vascular disruption therapy addition, the expected lower hypoxia in orthotopic tumors would increase the OER effect. Therefore, this therapy is expected to have a stronger impact in orthotopic versus subcutane- ous NSCLC models. The current study is the first step towards identifying a unique transient hypoxic tumor environment post-vascular disruption therapy, however further work with con- tinuous monitoring techniques could reveal opportunities to further improve radiation ther- apy outcomes. Supporting information S1 Fig. Representative AF647 and HS680 spectra. Representative spectra showing excitation and emission spectra of AF647 and HS680. The excitation and emission were chosen specifi- cally to minimize signal from AF647 when evaluating HS680 hypoxia florescence. (TIF) Software: Jihun Kwon. Supervision: Srinivas Sridhar, G. Mike Makrigiorgos, Ross I. Berbeco. Validation: Needa A. Virani, Olivia J. Kelada. Visualization: Needa A. Virani, Olivia J. Kelada, Jihun Kwon. Visualization: Needa A. Virani, Olivia J. Kelada, Jihun Kwon. Writing – original draft: Needa A. Virani, Olivia J. Kelada, Ross I. Berbeco. Writing – review & editing: Needa A. Virani, Olivia J. Kelada, Sijumon Kunjachan, Alexandre Detappe, Jihun Kwon, Ross I. Berbeco. Author Contributions Conceptualization: Needa A. Virani, Olivia J. Kelada, Ross I. Berbeco. Data curation: Needa A. Virani, Olivia J. Kelada, Alexandre Detappe, Jihun Kwon. Data curation: Needa A. Virani, Olivia J. Kelada, Alexandre Detappe, Jihun Kwon. Methodology: Needa A. Virani, Olivia J. Kelada, Sijumon Kunjachan, Alexandre Detappe, Jennifer Hayashi, Ana Vazquez-Pagan, Douglas E. Biancur, Thomas Ireland, Rajiv Kumar. Project administration: Needa A Virani Methodology: Needa A. Virani, Olivia J. Kelada, Sijumon Kunjachan, Alexandre Detappe, Jennifer Hayashi, Ana Vazquez-Pagan, Douglas E. Biancur, Thomas Ireland, Rajiv Kumar. P j t d i i t ti N d A Vi i Project administration: Needa A. Virani. Project administration: Needa A. Virani. Software: Jihun Kwon. 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https://openalex.org/W2769883989	https://bmcmededuc.biomedcentral.com/track/pdf/10.1186/s12909-017-1055-4	English	null	Medical student attitudes and educational interventions to prevent neurophobia: a longitudinal study	BMC medical education	2,017	cc-by	5,820	© 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. Shiels et al. BMC Medical Education (2017) 17:225 DOI 10.1186/s12909-017-1055-4 Shiels et al. BMC Medical Education (2017) 17:225 DOI 10.1186/s12909-017-1055-4 Open Access Abstract Background: With an aging American population, the burden of neurologic disease is intensifying and the decline in neurology residents and practicing neurologists is leaving these patients helpless and unable to find care. ‘Neurophobia’, a chronic illness that begins early in medical school, has been identified as a cause for the low number of neurology residents. Methods: A longitudinal study surveyed medical students at the beginning of their first year (M1) and then again at the beginning of their second year (M2). Three neuroscience educational interventions were studied: team based learning (TBL), case based teaching (CBT), and problem based learning (PBL). Participants provided self-reported neurophobia levels, attitudes about neuroscience, and the effectiveness of educational interventions. Results: A total of 446 students during M1 and 206 students during M2 participated in the survey. A significant change in self-reported neurophobia (p = 0.035) was observed from 19% in M1 to 26% in M2. Neuroscience knowledge and confidence managing a neurologic condition also significantly increased (p < 0.001 and p = 0.038 respectively). Perceived interest, difficulty, and desire to pursue a career in neuroscience did not a change significantly. Majority of students perceived CBT (76%), TBL (56%), and PBL (66%) beneficial. Only CBT demonstrated a statistical difference (p = 0.026) when stratified by self-reported change in neurophobia. Conclusion: An increase in neurophobia after completing a neuroscience was observed but the prevalence rate of 26% was lower than previous studies. Knowledge about neuroscience increased significantly and educational interventions were considered beneficial by students. Thus, interventions that increase knowledge and decrease neurophobia can lead to an increase in students pursuing neurology residencies. rds: Neuroscience, Caribbean, Team based learning, Case based teaching, Problem based learning Medical student attitudes and educational interventions to prevent neurophobia: a longitudinal study Lisa Shiels1, Pratish Majmundar1, Aleksander Zywot1,2* , John Sobotka1, Christine S. M. Lau1 and Tuula O. Jalonen1 a Shiels1, Pratish Majmundar1, Aleksander Zywot1,2* , John Sobotka1, Christine S. M. Lau1 and * Correspondence: aleksander.zywot@atlantichealth.org 1Department of Physiology and Neuroscience, St. George’s University School of Medicine, St. George’s, Grenada 2Department of Surgery, Morristown Medical Center, Morristown, NJ, USA Background It is a chronic illness that begins early in medical school [4]. Physicians and medical students alike often state that neurology is the most difficult subject in the med- ical school curriculum, and that their knowledge about the subject matter is limited, leading to a lack of confi- dence in managing neurology patients [5–7]. With an aging American population, the burden of neurologic disease is intensifying through an increase in both incidence and prevalence [1]. The decline in medical students pursuing residencies in neurosci- ence ultimately leads to less practicing neurologists, leaving these patients helpless and unable to find care [2–4]. The number of new neurologists is growing at a much slower pace compared to other specialties, in both the United States (US) and Europe [8]. From 2010 to 2015, the growth in both the number of students applying for and the number of programs offering residencies in the US in neurology (+1.3%) has paled in comparison to sur- gery (+4.6%), psychiatry (+4.9%), family medicine (+11.6%), and internal medicine (+23.4%) [9]. ‘Neurophobia’, a term first coined by Jozefowicz in 1994, describes medical students’ fear of neurology [2]. * Correspondence: aleksander.zywot@atlantichealth.org 1Department of Physiology and Neuroscience, St. George’s University School of Medicine, St. George’s, Grenada 2Department of Surgery, Morristown Medical Center, Morristown, NJ, USA Page 2 of 7 Shiels et al. BMC Medical Education (2017) 17:225 Since Jozefowicz’s original observation, researchers have been studying neurophobia to identify the cause and pro- vide a cure. Schon et al. [5] designed a clinical specialty atti- tudes survey with the aim of identifying attitudes and factors associated with neurophobia . Medical students and residents who participated in the survey reported that their knowledge for neurology was less than for other specialties and they perceived neurology to be the most difficult spe- cialty compared to cardiology, endocrinology, gastroenter- ology, geriatrics, respiratory medicine, and rheumatology [5]. Neurophobia was therefore postulated to, at least in part, be caused by the perceived difficulty of neurology and students’ poor knowledge of the subject [5]. Building on the findings of Schon et al., several studies integrated case based teaching (CBT) with the basic science portion of the neuroscience curriculum resulting in increased self- reported neuroscience knowledge and in turn decreased self-reported neurophobia levels [1, 6, 10]. Ridsdale et al. [4] employed team based learning (TBL) and demonstrated an improvement in student confidence in dealing with neurologic cases. TBL The objective of TBL is to have participants solve prob- lems by applying the knowledge they recently gained. Through self and team learning, students reinforce course concepts. TBL consists of four core steps [17, 18]. Educational interventions Previous studies have implemented educational inter- ventions such as CBT, TBL, and PBL, and have shown an increase in self-reported neuroscience knowledge. The aim of this study is therefore to assess the neuro- phobia level of incoming students, its evolution during the first year of medical school, and the perceived bene- fit of CBT, TBL, and PBL. Three educational interventions that previous studies identified to be beneficial in improving neuroscience knowledge and decreasing neurophobia were studied: TBL, CBT, and PBL [4–6, 8, 10–13, 15, 16]. Background Giles [11], Wiles [12] and others then highlighted the benefits of interactive learning and using problem based learning (PBL) to improve students’ under- standing of neuroscience [4, 11–13]. modules: neuroanatomy, cellular neuroscience, neurodeve- lopment, sensory systems, motor systems, regulatory sys- tems, and complex brain functions. The course was taught in a lecture hall sufficiently large to accommodate the entire class. Attendance was mandatory for all students. Two exams were administered, a midterm exam after the first three modules and a final exam at the end of the course. Approval to conduct the study was obtained from the SGU Institutional Review Board (IRB #14037) and in- formed consent forms were obtained from all partici- pants. Student participation was voluntary and had no effect on student grades, performance, or evaluation. All first semester medical students were invited to partici- pate in the survey (M1). Students were again invited to participate in the survey at the start of their second year following completion of the neuroscience class (M2). Students were grouped as either pre-neuroscience year one students (M1) or post-neuroscience year two stu- dents (M2). Cook et al. [14] established standards for medical education submissions which were used to guide data analysis, interpretation of results, and the reporting of findings. Participants and design A longitudinal study was conducted following the 2014 incoming class of medical students, from the start of medical school to the completion of the neuroscience portion of the curriculum, at St. George’s University (SGU) School of Medicine in Grenada, West Indies. SGU’s medical school curriculum is a 4-year program with 2 years of basic science and 2 years of clinical clerk- ships. SGU utilizes a subject based approach during the basic science portion comprising the first 2 years. Year one is divided into two terms with first term courses in Anatomy, Histology and Biochemistry, and second term courses in Neuroscience, Physiology, Genetics and Im- munology. Year two consists of three terms with a course in Behavioral Sciences in term three, Pathology, Microbiology, and Clinical Skills in term four, and Pathophysiology, Pharmacology, and an Advanced Clin- ical Skills in term five. TBL begins with the formation of permanent teams. Ideally teams should be created by instructors with a di- verse group of 5–7 students per team [17]. Next student knowledge is assessed with the Readiness Assurance Test (RAT) [17]. Several days prior to the start of the TBL session students are provided preparatory material and then at the start of the session students’ comprehension and knowledge are evaluated using the RAT. The in- structor then reviews the results of the RAT with the class, clarifying any misconceptions that exist. The third step of TBL is student participation in team based learn- ing activities [17]. These activities are designed to pro- mote group discussion, critical thinking, and knowledge application. The fourth and final step is the completion of an in-class assessment [17]. This process serves to both reinforce the material learned and to create ac- countability for each team member. Upon completion feedback is provided to once again aid in clarifying misconceptions. The neuroscience class at SGU was taught three times a week by three professors who rotated teaching the material based on their specialty. The course consisted of seven Page 3 of 7 Shiels et al. BMC Medical Education (2017) 17:225 Page 3 of 7 At St. George’s University, a total of 10 two-hour neuro- science TBL sessions were offered evenly throughout the semester. At the start of the semester each student was assigned to a team by the course professor in order to cre- ate a fair distribution of knowledge and diversity. CBT CBT CBT aims to make teaching student focused by having stu- dents actively process information in an attempt to solve a problem [19–21]. This form of learning forces students to process information, modify methods of thinking, and com- bine new knowledge with previous knowledge. In CBT, basic sciences and clinical sciences are integrated, making learning more effective and interesting for students. The first part of the questionnaire (Q1–8) collected minimal demographic information including sex, age, and current grade point average. Several questions then asked about educational background, previous neurosci- ence exposure, and learning preferences. A clinical case is first presented to students, providing basic patient history [21]. Next either questions follow, test- ing basic science and clinical knowledge, or a clinical change is presented and students are asked to reason through the processes of change [21]. Throughout the case a professor or clinical expert is present to guide the discus- sion, student reasoning, and clarify any misunderstandings. In part two of the questionnaire (Q9–14), the level of per- ceived neurophobia was assessed. Neurophobia was defined as medical students’ fear of neuroscience and directly mea- sured by having respondents rank their agreement with the following statement “I have an aversion to neuroscience”. g y y g Sixteen one-hour CBT sessions were held throughout the neuroscience semester at SGU. Four sessions were held during the first half of the semester and 12 were held in the second half. This uneven distribution was due to more clinical based lectures were held during the second half of the semester. CBT sessions were intended to facilitate the application of clinically relevant know- ledge gained during the preceding lectures and to help students develop their skills in critical clinical thinking and reasoning. Learning objectives were provided to stu- dents in advance and students were encouraged to re- view relevant lecture material. Case discussions were held in the lecture hall were student discussions were guided by faculty. For each case, students discussed the case history, reviewed examination findings and tech- niques, and rationalized through the diagnosis. Six questions from Schon et al.’s questionnaire were used to assess self-reported attitudes about neuroscience [5]. These questions evaluated: interest in neuroscience, know- ledge in neuroscience, difficulty of neuroscience, confidence in managing a clinical case pertaining to neuroscience, and the desire to pursue a career in neuroscience. A 5-Point Likert scale was used to grade responses. Participants and design Each team was comprised of about six students and lead by a student facilitators. Clinical experts and course professors were available and served as expert consultant if requested. Learning objects and resources for each session were pro- vided to students the week prior to the session. Students completed a self-assessment at the start of the session. At the end of every session each student, in collaboration with his or her team members, completed a short session exam- ination. Feedback was provided and explanations were offered to students. Students received a grade for participa- tion in each session and for completion of the end-of- session examination. Students who missed more than 20% of these sessions automatically failed the course. that contains some degree of foreign concepts [20, 22]. In PBL, similar to TBL, students are divided into groups and attempt to solve problems as a team [17]. Unlike TBL however, in PBL students solve problems to identify knowledge gaps [17]. Students then undertake self- directed studying to remedy areas of weakness. At SGU, 59 one-hour neuroscience lectures were of- fered to students with the last 10–15 min of every lec- ture reserved for a PBL session. Questions presented to students were designed to expand on the material pre- sented in the previous lecture. Faculty were present to guide discussion, explain concepts if necessary, and aid students in designing focused learning objectives. CBT Part three (Q15-17) was only administered to M2 stu- dents and comprised of three questions focused on the educational interventions. A 5-Point Likert scale was used to grade responses. Survey instrument h The survey instrument was based upon Schon et al.’s original questionnaire, which has since been imple- mented in multiple studies [5–7, 13, 23, 24]. A consor- tium of individuals at SGU from the biostatistics, psychology, and neuroscience departments met to design the questionnaire. The questionnaire contained three parts. Parts one and two were based on Schon et al.’s questionnaire and a third part was added to survey atti- tudes about educational intervention attitudes. Data collection and outcomes The survey questionnaire was administered twice to the same cohort of students, initially at the beginning of their first year of medical school (M1) and again when the stu- dents started their second year of medical school (M2). Parts one and two of the questionnaire were administered twice, during M1 and M2. Part three was only administered during M2. PBL PBL is a simpler learning strategy in which students are given a partial case and are guided in solving a problem Page 4 of 7 Shiels et al. BMC Medical Education (2017) 17:225 Table 1 Demographic characteristics participating medical students Unmatched Matched M1 frequency (%) M2 frequency (%) Frequency (%) N = 446 N = 206 N = 150 Gender Male 196 (49%) 61 (50%) 73 (51%) Female 204 (51%) 61 (50%) 71 (49%) Age < 23 24 (6%) 8 (5%) 9 (6%) 23–28 326 (78%) 140 (81%) 115 (80%) > 28 66 (16%) 25 (14%) 20 (14%) Educational training High school 11 (3%) 3 (2%) 3 (2%) Undergraduate degree 280 (67%) 116 (67%) 96 (68%) Graduate degree 127 (30%) 51 (31%) 42 (30%) Neuroscience familiarity No exposure 117 (27%) 36 (18%) 45 (30%) School/Work exposure 213 (48%) 129 (65%) 71 (48%) Neuroscience coursework 111 (25%) 35 (18%) 32 (22%) Abbreviations: M1 year one (pre-neuroscience) medical students, M2 year two (post-neuroscience) medical students, N number of participants Table 1 Demographic characteristics participating medical students Surveys were administered to the entire class with no exclusion based on age, sex, race, or prior educational experience. Student responses were submitted electron- ically and all responses were treated as confidential. Upon collection a unique electronic ID was assigned to participants which allowed for tracking students during the study. Consent to participate was provided on both occasions and participation was not dependent on a stu- dent answering all survey questions. Students who failed to answer to the question about neurophobia were ex- cluded from the study. The primary outcome was change in the level of self- reported neurophobia between M1 and M2. Neurophobia was defined as medical students’ fear of neuroscience and considered present when students answered positively to the statement “I have an aversion to neuroscience”. Secondary outcomes were effectiveness of the educational interventions (TBL, CBT, and PBL) and changes in self- reported attitudes about neurology between M1 and M2. Statistical analysis The data collected were coded and analyzed using Statis- tical Package for Social Sciences v. 20 (SPSS Inc. Chicago, IL, USA). Individual student responses were compared between groups using frequencies and Chi- square analysis. Matched analysis was conducted using a Wilcox Signed Rank test. A two-tailed p-value of <0.05 was considered statistically significant. Abbreviations: M1 year one (pre-neuroscience) medical students, M2 year two (post-neuroscience) medical students, N number of participants response for neurophobia in M1 and M2 was a 3/5 on the Likert scale (neither agree nor disagree) but M2 had an increased frequency of “strongly agree” (11%) and “agree” (15%) responses compared to M1 (7 and 12% respectively). A Wilcoxon signed-rank test demonstrated a significant difference in self-reported neurophobia (p = 0.035). Results A total of 446 students participated in the pre- neuroscience survey (M1) and 206 in the post- neuroscience survey (M2) (Table 1). A matched cohort, comprised of only students that answered both surveys, had a total of 150 respondents. Matched and unmatched cohorts had similar distributions in demographics and self-reported attitudes (Table 1). Perceived interest in neuroscience did not a change between M1 and M2. The median response in M1 and M2 cohorts was 4/5 on the Likert scale (moder- ate) and the Wilcoxon signed-rank test in the matched cohort did not find a significant difference (p = 0.327). Demographic characteristics In the matched cohort, the majority of students (80%) were 23–28 years of age with 51% males and 49% fe- males. Most students had previous exposure to neurosci- ence with 48% reporting school or work experience and 22% taking neuroscience courses. The unmatched cohort had a similar distribution. In the unmatched cohort, there was no significant difference between the M1 and M2 groups in baseline demographic factors including age (p = .901), gender (p = .888), or educational back- ground (p = . 963). During M2 the median response for neuroscience knowledge was a 3/5 on the Likert scale (good), which was an increase from the median response of 2/5 on the Likert scale (fair) in M1. The Wilcoxon signed-rank test showing a significant change (p < 0.001). Confidence in treating a neurologic con- dition also demonstrated a significant change (p = 0.038). Neuroscience was perceived equally diffi- cult during M1 and M2 with a median response of 2/ 5 (difficult) by both groups with no significant change in responses. Students’ desire to pursue a career in neuroscience was also did no demonstrate a signifi- cant change between M1 and M2. Discussion Neurophobia, defined as a medical student’s fear of neuroscience, is an affliction they will carry as a prac- ticing physician and can therefore have long-term impli- cations on patient care [2, 4]. Although the cause of neurohobia is multifactorial, studies have identified stu- dents’ lack of neuroscience knowledge as an associated factor [6]. Improving the medical educational curriculum is therefore considered the frontline intervention for preventing and treating neurophobia [6]. In this longitudinal survey, student’ self-reported atti- tudes towards neurology were measured and linked to self-reported neurophobia. Results showed a small but significant increase in neurophobia after exposure to the neuroscience course, providing further evidence that a medical school’s neuroscience education alters students’ perception of neurology. Jozefowic’s initial paper (1994) stated that 50% of stu- dents experienced neurophobia [2]. Follow-up studies identified neurophobia rates ranging from 18–47% [10, 25, 26]. Fantaneanu et al. [27] examined the subject fur- ther and found 32% of students neurophobic of academic neuroscience and 24% neurophobic of clinical neurosci- ence. The prevalence rate of 26% (agree or strongly agree) at SGU falls in the lower end of this spectrum. p Factors associated with neurophobia were compared to those in previously published values. Interest in neuroscience 51% during M2 (moderate or extreme) was similar to previous studies such as Sanya et al. (2010) who found that 49% of students were interested in neur- ology [28]. At SGU, knowledge about neuroscience in- creased after students completed the course from 11% in M1 (very good or excellent) to 31% in M2. Although this was a significant increase, studies such as Schon et al. have demonstrated that students consistently report hav- ing the lowest amount of knowledge about neuroscience compared to all other specialties examined [5]. Neuro- science was perceived to be less difficult at SGU com- pared to previous studies, with only 58% of students during M2 reporting the subject as a difficult or very dif- ficult. Abulaban et al. for instance reported that 86% of students considered the subject matter difficult [29]. Abbreviations: M1 year one (pre-neuroscience) medical students, M2 year two (post-neuroscience) medical students Effect of a neuroscience course on student attitudes Effect of a neuroscience course on student attitudes Self-reported neurophobia levels and neuroscience at- titudes were compared between M1 and M2 re- sponses in the matched cohort (Table 2). The median Shiels et al. BMC Medical Education (2017) 17:225 Page 5 of 7 O t f d ti l i t ti Table 2 Wilcox signed-rank analysis of self-reported neurophobia and neuroscience attitudes in matched M1 and M2 responses M1 frequency (%) M2 frequency (%) p-value Interest 0.327 None 21 (15%) 17 (17%) Slight 26 (18%) 11 (11%) Somewhat 40 (28%) 22 (22%) Moderate 36 (25%) 33 (33%) Extreme 19 (13%) 18 (18%) Knowledge 0.000 None 37 (27%) 8 (9%) Fair 53 (39%) 15 (16%) Good 32 (23%) 39 (43%) Very good 12 (9%) 24 (26%) Excellent 3 (2%) 5 (5%) Difficulty 0.057 Very difficult 35 (24%) 25 (27%) Difficult 71 (49%) 29 (31%) Neutral 34 (23%) 30 (32%) Easy 1 (1%) 6 (6%) Very easy 4 (3%) 3 (3%) Confidence 0.038 None 65 (46%) 29 (26%) Slight 41 (29%) 51 (46%) Somewhat 26 (18%) 29 (26%) Moderate 5 (4%) 2 (2%) Extreme 5 (4%) 1 (1%) Pursue career 0.063 Extremely unlikely 31 (22%) 38 (28%) Unlikely 35 (25%) 40 (30%) Neutral 59 (42%) 43 (32%) Likely 10 (7%) 8 (6%) Extremely likely 7 (5%) 5 (4%) Neurophobia 0.035 Strongly disagree 35 (23%) 20 (13%) Disagree 32 (21%) 44 (29%) Neither 61 (41%) 46 (31%) Agree 14 (9%) 23 (15%) Strongly agree 8 (5%) 17 (11%) Abbreviations: M1 year one (pre-neuroscience) medical students, M2 year two (post-neuroscience) medical students Table 2 Wilcox signed-rank analysis of self-reported neurophobia and neuroscience attitudes in matched M1 and M2 responses M1 frequency (%) M2 frequency (%) p-value Interest 0.327 Table 2 Wilcox signed-rank analysis of self-reported neurophobia and neuroscience attitudes in matched M1 and M2 responses analysis compared the perceived benefit of each educa- tional intervention stratified by the change in self- reported neurophobia levels from M1 to M2 (Table 3). CBT demonstrated a statistical difference (p = 0.026), while TBL (p = 0.623) and PBL (p = 0.425) did not. Outcomes of educational interventions Though the neurophobia prevalence rate of 26% at SGU is lower than most published studies, it does continue the trend of increasing neurophobia levels with increased years of medical education and exposure to the medical school neuroscience course [27]. Previous studies on neu- rophobia have shown that educational interventions can Most students in the matched cohort reported that each educational intervention implemented was beneficial in assisting with neuroscience studies (Table 3). The me- dian response was 4/5 (agree, 45%) for CBT, 4/5 (agree, 40%) for TBL, and 4/5 (agree, 40%) for PBL. Chi-square Shiels et al. BMC Medical Education (2017) 17:225 Page 6 of 7 Table 3 Chi-square analysis of perceived effectiveness of educational interventions stratified by change in self-reported neurophobia from M1 to M2 in a matched cohort of medical students Decreased neurophobia No change in neurophobia Increased neurophobia Total p-value CBT 0.026 Strongly disagree 2 (5%) 1 (2%) 0 (0%) 3 (3%) Disagree 6 (15%) 2 (4%) 3 (10%) 11 (9%) Neither 3 (8%) 3 (6%) 8 (26%) 14 (12%) Agree 19 (49%) 20 (42%) 14 (45%) 53 (45%) Strongly agree 9 (23%) 22 (46%) 6 (19%) 37 (31%) TBL 0.623 Strongly disagree 3 (9%) 5 (11%) 2 (6%) 10 (9%) Disagree 5 (15%) 3 (7%) 3 (10%) 11 (10%) Neither 11 (32%) 9 (20%) 7 (23%) 27 (25%) Agree 10 (29%) 18 (41%) 16 (52%) 44 (40%) Strongly agree 5 (15%) 9 (20%) 3 (10%) 17 (16%) PBL 0.425 Strongly disagree 2 (5%) 4 (7%) 1 (3%) 7 (5%) Disagree 6 (15%) 5 (9%) 7 (19%) 18 (14%) Neither 10 (24%) 6 (11%) 4 (11%) 20 (15%) Agree 15 (37%) 22 (40%) 16 (44%) 53 (40%) Strongly agree 8 (20%) 18 (33%) 8 (22%) 34 (26%) Abbreviations: CBT case based teaching, TBL team based learning, PBL problem based learning Table 3 Chi-square analysis of perceived effectiveness of educational interventions stratified by change in self-reported neurophobia from M1 to M2 in a matched cohort of medical students SGU students’ neurophobia significantly increased following the neuroscience courses. Thus, methods that decrease neu- rophobia and increase knowledge can lead to an increase in students pursuing neurology residencies. Future studies examining the effect of various educational models on expe- rienced neurophobia may provide further insight into ways to alter these sentiments by changes in curriculum. Availability of data and materials The datasets used and/or analyses during the current study are available from the corresponding author on reasonable request. Abbreviations Abbreviations CBT: Case based teaching; IRB: Insitutional Review Board; M1: First year medical student; M2: Second year medical student; PBL: Problem based learning; RAT: Readiness Assurance Test; SGU: St. George’s University; SPSS: Statistical Package for Social Sciences; TBL: Team based learning; US: United States Funding No funding was provided. Outcomes of educational interventions improve students’ knowledge of neurology and serve as a protective factor against neurophobia [1, 4, 6, 8, 10, 15]. The lower than expected level of neurophobia could therefore be in part due to the educational interventions implemented. Although all educational interventions were shown to be beneficial to the majority of the class, several limita- tions inhibit this study’s ability to generalize results. First, this study did not have a separate control group, which limits the ability to examine the effectiveness of each educational intervention independently. It therefore becomes difficult to separate the impact of the neurology course and the impact of educational interventions on the level of neurophobia. Furthermore, the neurology course at SGU is different from neurology courses in other universities and the effect of SGU’s neuroscience course on neurophobia and students’ attitudes differs. This study focused on first and second year students in- stead of third or fourth year clinical clerkship students, who are more focused on selecting a specialty for resi- dency. Lastly a precise instrument to measure neuropho- bia has yet to be designed and validated, thus making it difficult to compare between reported study findings. Abbreviations CBT: Case based teaching; IRB: Insitutional Review Board; M1: First year medical student; M2: Second year medical student; PBL: Problem based learning; RAT: Readiness Assurance Test; SGU: St. George’s University; SPSS: Statistical Package for Social Sciences; TBL: Team based learning; US: United States g Authors thank Dr. Ralph Jozefowicz, MD for his contributory input and suggestions, Dr. Omur Elci, MD, PhD for his guidance, knowledge, and expertise, and Professors Dirk Burkhardt, MD, MSc, PhD, Mark Clunes, PhD, and Sharmila Upadhya, MD for allowing the survey to be conducted in connection to their lecture time. Acknowledgements Authors thank Dr. Ralph Jozefowicz, MD for his contributory input and suggestions, Dr. Omur Elci, MD, PhD for his guidance, knowledge, and expertise, and Professors Dirk Burkhardt, MD, MSc, PhD, Mark Clunes, PhD, and Sharmila Upadhya, MD for allowing the survey to be conducted in connection to their lecture time. Competing interests The authors declare that they have no competing interests. 23. Youssef FF. Neurophobia and its implications: evidence from a Caribbean medical school. BMC Med Educ. 2009;9:39. doi:10.1186/1472-6920-9-39. Publisher’s Note 24. McCarron MO, Stevenson M, Loftus AM, McKeown P. Neurophobia among general practice trainees: the evidence, perceived causes and solutions. Clin Neurol Neurosurg. 2014;122:124–8. doi:10.1016/j.clineuro.2014.03.021. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 25. Kam K, Tan GSE, Tan K, Lim ECH, Koh NY, Tan NCK. Neurophobia in medical students and junior doctors–blame the GIK. Ann Acad Med Singap. 2013;42: 559–66. Received: 16 December 2016 Accepted: 2 November 2017 26. Gupta NB, Khadilkar SV, Bangar SS, Patil TR, Chaudhari CR. Neurology as career option among postgraduate medical students. Ann Indian Acad Neurol. 2013;16:478–82. doi:10.4103/0972-2327.120427. Consent for publication Not applicable. 22. Parmelee D, Michaelsen LK, Cook S, Hudes PD. Team-based learning: a practical guide: AMEE guide no. 65. Med Teach. 2012;34:e275–87. doi:10. 3109/0142159X.2012.651179. Ethics approval and consent to participate IRB approval from SGU IRB Board #14037. pp p p IRB approval from SGU IRB Board #14037. Written consent was obtained prior to administration of surveys. 21. Peiman S, Mirzazadeh A, Alizadeh M, Mortaz Hejri S, Najafi M-T, Tafakhori A, et al. A case based-shared teaching approach in undergraduate medical curriculum: a way for integration in basic and clinical sciences. Acta Med Iran. 2017;55:259–64. Conclusions LS - Drafting the article, analysis and interpretation of data, critical revision of the article. PM - Conception and design, Acquisition of data, drafting the article. AZ - Conception and design, data analysis, critical revision of the article. JS - Conception and design, acquisition of data, drafting the article. CL - Analysis and interpretation of data, critical revision of the article. TJ – Since the year 1994, researchers have examined Jozefowicz’s claims on the relationship between neurophobia and the medical school curriculum [2–6, 8, 10–12, 15, 16, 30]. At Page 7 of 7 Page 7 of 7 Shiels et al. BMC Medical Education (2017) 17:225 Project coordinator, conception and design, drafting the article, critical revision of the article. All authors have read and approved the final version of this manuscript. 19. Nair SP, Shah T, Seth S, Pandit N, Shah GV. Case based learning: a method for better understanding of biochemistry in medical students. J Clin Diagn Res. 2013;7:1576–8. doi:10.7860/JCDR/2013/5795.3212. 20. Aljarallah B, Hassan MS. Problem based learning (PBL) vs. case based curriculum in clinical clerkship, internal medicine innovated curriculum, student prospective. Int J Health Sci. 2015;9:147–52. References 1. Lim ECH, Seet RCS. Demystifying neurology: preventing “neurophobia” among medical students. Nat Clin Pract Neurol. 2008;4:462–3. doi:10.1038/ ncpneuro0849. 1. Lim ECH, Seet RCS. Demystifying neurology: preventing “neurophobia” among medical students. Nat Clin Pract Neurol. 2008;4:462–3. doi:10.1038/ ncpneuro0849. 27. Fantaneanu TA, Moreau K, Eady K, Clarkin C, DeMeulemeester C, Maclean H, et al. Neurophobia inception: a study of trainees’ perceptions of neurology education. Can J Neurol Sci. 2014;41:421–9. doi:10.1017/S0317167100018436. 2. Jozefowicz RF. Neurophobia: the fear of neurology among medical students. 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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. Tan NCK, Kandiah N, Chan YH, Umapathi T, Lee SH, Tan K. A controlled study of team-based learning for undergraduate clinical neurology education. BMC Med Educ. 2011;11:91. doi:10.1186/1472-6920-11-91. • 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. Anwar K, Shaikh AA, Sajid MR, Cahusac P, Alarifi NA, Al SA. Tackling student neurophobia in neurosciences block with team-based learning. Med Educ Online. 2015;20:28461. doi:10.3402/meo.v20.28461. 17. Michaelsen LK, Davidson N, Major CH. 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https://openalex.org/W4386515836	https://revistaabalf.com.br/index.html/index.php/rabalf/article/download/765/509	Portuguese	null	PRÁTICAS DE NUMERAMENTO COMO PRÁTICAS DISCURSIVAS	Revista Brasileira de Alfabetização	2,023	cc-by	9,693	RESUMO Este texto tem como objetivo discutir como os estudos que operam com o conceito de numeramento no Brasil se assumem como desdobramentos da perspectiva analítica e pedagógica que Magda Soares confere ao conceito de letramento. Esses estudos focalizam pessoas pouco ou não- escolarizadas ou grupos sociais de inserção recente no projeto educacional brasileiro apropriando- se de práticas discursivas da matemática hegemônica e, nessa apropriação, demarcando seu estranhamento com conhecimentos e ritos escolares. Destacam-se na base teórica dos estudos, nas opções metodológicas, nos caminhos analíticos e nas indicações para as práticas pedagógicas, as contribuições das elaborações conceituais e das propostas didáticas de Magda e a marca de seu compromisso político com a democratização e a qualidade da Educação. Refletindo também as contribuições metodológicas da pesquisadora Magda Soares, a discussão aqui proposta é confrontada com três eventos de numeramento, em que mulheres idosas alfabetizandas na EJA, uma criança num contexto não escolar e professores indígenas em formação, protagonizando práticas matemáticas, apropriam-se de práticas de letramento. Palavras-chave: Conceito de Letramento. Apropriação de práticas de numeramento. Práticas matemáticas como práticas discursivas. Constituição de sujeitos sociais. Magda Soares. Flávia Cristina Duarte Pôssas Grossi Instituto Federal de Minas Gerais fcdpossas@gmail.com Flávia Cristina Duarte Pôssas Grossi Instituto Federal de Minas Gerais fcdpossas@gmail.com Flávia Cristina Duarte Pôssas Grossi Instituto Federal de Minas Gerais fcdpossas@gmail.com Ruana Priscila da Silva Brito Universidade Federal de Minas Gerais ruanabrito@gmail.com Ruana Priscila da Silva Brito Universidade Federal de Minas Gerais ruanabrito@gmail.com NUMERACY PRACTICES AS DISCURSIVE PRACTICES: DEVELOPMENTS OF LITERACY STUDIES IN MATHEMATICS EDUCATION Maria da Conceição Ferreira Reis Fonseca Universidade Federal de Minas Gerais mcfrfon@gmail.com Maria da Conceição Ferreira Reis Fonseca Universidade Federal de Minas Gerais mcfrfon@gmail.com Introdução Assim como ocorreu ao uso do conceito de Letramento nos anos 1990 (SOARES, 1998), o uso do termo numeramento ou das expressões eventos de numeramento e práticas de numeramento em estu dos e em propostas de intervenção pedagógica no Brasil ainda hoje precisa ser precedido de algum esclarecimento que o estabeleça não como uma concessão a um modismo, mas como resposta a ne cessidades da reflexão sobre diversos aspectos da apropriação de práticas matemáticas que, como sujeitos sociais, as pessoas (crianças, adolescentes, jovens, pessoas adultas e idosas) protagonizam, em diferentes ambientes, que incluem os, mas não se restringem aos, contextos escolares. Esse uso indica uma disposição de reiterar o caráter sociocultural das práticas matemáticas e destacar sua inserção privilegiada nos modos de dizer o mundo (FREIRE, 1982) produzidos por sociedades que se tecem tendo que lidar – por adesão, em resistência, subjugadas, por inércia ou por sobrevivência – com os paradigmas do que se convencionou chamar de modernidade. De uma maneira geral, é apropriando-se de modos de dizer o mundo, que os sujeitos se confrontam com (e também produzem) diferentes condições de participação em práticas sociais. O conhecimento, o usufruto ou o enfrentamento dessas condições precisam ser considerados quando da elaboração, da proposição, do desenvolvimento, do acompanhamento e da avaliação de alternativas pedagógicas que visam promover a apropriação dessas práticas em contextos educativos, escolares ou não. Por isso, demandam ferramentas analíticas que ajudem a contemplar além daquelas condições e seus desdo bramentos, os próprios sujeitos constituindo-se enquanto protagonizam práticas sociais. É em resposta a essa demanda que compreendemos a meticulosa elaboração do conceito de letramento, seu didático compartilhamento e sua comprometida transformação em ação pedagógica, promovidos pela pesquisadora, pela formadora de docentes (e) pesquisadoras, pela autora de livros didáticos e textos científicos, pela professora e orientadora Magda Soares. Interpelando esse conceito, desenvolvendo-o, esclarecendo e explorando suas possibilidades analíticas e pedagógicas, divulgando-o e o traduzindo em compreensão dos sujeitos e dos processos que vivenciam, seu trabalho nos alerta para fatores e desdobramentos culturais e políticos relacionados ao destaque que a cultura escrita conquistou nas sociedades modernas, e que a faz pautar de tal forma as práticas socialmente valorizadas, a ponto de se caracterizarem tais sociedades como grafocêntricas (SOARES, 1998). conceptual elaborations and didactic proposals and the marks of her political commitment with the democratization and the quality of Education. Also reflecting the methodological contributions of the researcher Magda Soares, the discussion proposed here is confronted with three numeracy events, in which elderly literacy students, a child in a non-school context and indigenous teachers in training, leading mathematical practices, appropriate literacy practices. Key-words: Literacy Concept; Appropriation of Numeracy Practices; Math practices as discursive practices; Constitution of subjects; Magda Soares. conceptual elaborations and didactic proposals and the marks of her political commitment with the democratization and the quality of Education. Also reflecting the methodological contributions of the researcher Magda Soares, the discussion proposed here is confronted with three numeracy events, in which elderly literacy students, a child in a non-school context and indigenous teachers in training, leading mathematical practices, appropriate literacy practices. Key-words: Literacy Concept; Appropriation of Numeracy Practices; Math practices as discursive practices; Constitution of subjects; Magda Soares. ABSTRACT The purpose of this text is to discuss how Brazilian studies operating with the concept of numeracy assume themselves as developments of the analytical and pedagogical perspective that Magda Soares confers on the concept of literacy. These studies focus on people with little or no schooling or social groups that have recently entered the Brazilian educational project appropriating discursive practices of hegemonic mathematics and, in this appropriation, showing their estrangement from school knowledge and rites. The studies’ theoretical basis, their methodological options, their analytical paths and indications for the pedagogical practices highlight the contributions of Magda’s Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 1 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 Introdução Esse destaque da escrita como mediadora de uma grande diversidade de práticas sociais, pon dera Magda (SOARES, 1998), nos leva a procurar aportes teóricos para encarar a complexidade dos processos de apropriação das práticas de ler e de escrever, tanto no que se refere a procedimentos técnicos e comportamentos cognitivos relacionados ao funcionamento do sistema alfabético – con templados no termo alfabetização –, quanto no que decorre dos desafios, das potencialidades e das contradições advindas das dimensões social, cultural, tecnológica e política dessas práticas e de sua apropriação – que são especialmente focalizados quando se discute o letramento. Cabe esclarecer, entretanto, que a distinção desses termos só se justifica pela necessidade de se compreenderem Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 2 tanto os delicados processos que envolvem o aprendizado de elementos, padrões, regras e proce dimentos que configuram o sistema de escrita, quanto as habilidades complexas que se demandam de quem lê e escreve textos adequados a seu uso em situações diversas, e também a dimensão e a relevância social das práticas de leitura e de escrita numa sociedade grafocêntrica. Essa relevância permanece mesmo com os avanços tecnológicos que foram disponibilizando mídias variadas para registro e comunicação em áudio e vídeo, em interações remotas síncronas e assíncronas. Ainda que o texto escrito não seja a mídia adotada em muitas situações, ainda são os valores das culturas do escrito (GALVÃO, s.d.) que parametrizam as práticas socialmente prestigia das, incitando os que delas participam a se posicionarem em relação a classificações e controles, à acumulação e à padronização, a referências estéticas e a modos de significar, organizar, narrar, deduzir, avaliar, justificar e decidir, próprios dessas culturas. Entretanto, descrições, argumentos, narrativas, instruções ou previsões que constituem os múlti plos discursos, inclusive os textos escritos, que circulam nas diversas instâncias da vida social refle tem os modos como indivíduos e grupos que os produzem (e aqueles a quem presumivelmente se di rigem) se relacionam com o mundo e com a sociedade. É possível perceber como as representações, as referências, os argumentos, bem como os princípios de associação, classificação, padronização, ponderação e admissibilidade que estruturam ou, simplesmente, compõem esses discursos “estão frequentemente associados a ideias, símbolos e critérios que se relacionam com aquilo que aprende mos a chamar de ‘matemática’ no contexto escolar” (FONSECA, 2017a, p. 110). Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 Assim, ao tomarem o conceito de numeramento como uma dimensão do letramento, esses estudos reiteram que as práticas de letramento, especialmente nas sociedades ditas modernas, quase inevitavelmente, mobilizam conceitos, procedimentos ou princípios relacionados a conhe cimentos matemáticos (FONSECA, 2009, 2015; KNIJNIK; FONSECA, 2015), uma vez que, sendo as práticas de leitura e de escrita social e culturalmente determinadas pelo contexto em que são engendradas (SOARES, 2001; STREET, 2014), não estarão imunes ao poder dos critérios e da lin guagem matemáticos nos modos de relação entre pessoas e instituições nessa sociedade. Isso os leva a reconhecer que, além de grafocêntricas, as sociedades modernas são também quanticratas (FONSECA, 2017b). Assim, as situações sociais de uso da leitura e da escrita demandam cada vez mais conheci mentos que envolvem quantificação, medição, orientação e classificação (D’AMBRÓSIO, 1997; FON SECA, 2004), que compõem modos de usar a língua escrita e são por elas constituídas, não apenas porque as representações matemáticas estão presentes nos textos escritos, mas porque a própria cultura escrita, que constitui essas práticas “é também permeada por princípios calcados numa mes ma racionalidade que forja ou parametriza as práticas ditas numeradas e que é por elas reforçada” (FONSECA, 2009, p. 55). A opção por nomear tais práticas como práticas de numeramento, e não práticas matemáticas, tem, porém, a intenção de evitar que o peso da dimensão disciplinar da palavra matemática associe tais práticas àquelas que estabelecem uma relação de submissão aos princípios, regras e procedi mentos da matemática escolar, restringindo, assim, o olhar apenas para essas práticas valorizadas socialmente como hegemônicas e universais (FONSECA, 2015), o que não atende às intenções dos estudos ou das propostas de intervenção que os adotam. Essa perspectiva teórica tem orientado, de modo especial e explícito, estudos que destacam o caráter sociocultural da produção, dos usos e da circulação de conhecimentos matemáticos, não só daqueles mobilizados no contexto escolar, mas também daqueles que são forjados em outros es paços sociais e que mantêm com a(s) cultura(s) escrita(s) algum tipo de relação: Lima, 2007; Faria, 2007; Cabral, 2007, 2015; Souza, 2008; Ferreira, 2009; Adelino, 2009, 2018; Schneider, 2010; Simões, 2010, 2019; Vasconcelos, 2011; Brito, 2012, 2019; Lima, 2012; Silva, 2013; Carvalho, 2014; Mendon ça, 2014; Miranda, 2015; Sá, 2016; Lima, 2020; Novaes, 2020; Grossi, 2021; Felício De Jesus, 2021; Faria, 2023; Vanegas-Garcia, 2023; Pinheiro, 2023. Introdução Assim, a compreensão da constituição social, cultural e pragmática dos discursos em geral, e dos textos escritos em especial, promovida pelas elaborações e os desdobramentos do conceito de letramento na obra de Soares (1986; 1998; 2004; 2020), nos leva a conceber e nos possibilita explorar analítica e pedagogicamente a natureza discursiva das práticas matemáticas. Isso insere a preocu pação em refletir sobre relação das pessoas e dos grupos sociais com ideias, símbolos e critérios matemáticos num conjunto de esforços para se compreender a relação das pessoas com diferentes práticas sociais de uma sociedade grafocêntrica, explicitando sua impregnação por “modos cartesia nos de pensar o mundo” (FONSECA, 2017a, p. 112), investigando as intenções e os reflexos dessa impregnação, e promovendo uma apropriação crítica e democrática dessas práticas. É nesse sentido que nos propomos a discutir aqui como os estudos que operam com o conceito de numeramento no Brasil se assumem como desdobramentos da perspectiva analítica e pedagógica que Magda Soares confere ao conceito de letramento. Com efeito, os estudos do numeramento, especial mente como se desenvolveram neste país (CAMPETTI e DORNELLES, 2022), não se estabelecem como uma analogia no ensino de Matemática ao que seriam os estudos do letramento para o ensino de Portu guês, mas como uma dimensão dos estudos do Letramento, entendidos como modo de compreender e promover a participação das pessoas nas variadas práticas discursivas que compõem sua vida social. Esses estudos, de certa forma, distinguem-se de grande parte daqueles que encontramos na li teratura de língua inglesa, nos quais o termo numeracy costuma ser mobilizado para identificar e/ou descrever o domínio de habilidades matemáticas demandadas nos processos de quantificar, mensu rar, ordenar, localizar e classificar, considerando-as como uma “competência individual em Numeracy” (FONSECA, 2015, p. 6, destaques da autora), que precisa ser garantida e avaliada. A maioria dos usos do termo numeramento nos textos brasileiros, todavia, toma esse conceito em sua dimensão social, como um fenômeno cultural, pois os estudos consideram “a natureza relacional desse conceito e sua fertilidade na análise de práticas que se configuram nas relações entre pessoas e entre grupos e nas relações dessas e desses com o conhecimento que associamos à matemática” (FONSECA, 2015, p. 9). Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 3 “Eu tenho. Mas, vou falar a verdade, eu não sei olhar não” O primeiro dos eventos que aqui trazemos encontra-se em Grossi (2021) e foi oportunizado por uma atividade escolar, planejada pela professora de uma turma de mulheres idosas, alfabetizandas da Educação de Pessoas Jovens Adultas e Idosas (EJA). A professora propôs como exercício uma análise metalinguística do gênero discursivo rótulo com o objetivo de explicitar o conjunto de infor mações que esse tipo de texto veicula (porque interessa a quem o produz ou a quem o consome, e/ou para atender às exigências legais). Dentre as informações estudadas por aquelas estudantes, destacamos a interação discursiva que envolve a localização, a leitura e o uso da informação prazo de validade durante o estudo das informações veiculadas no rótulo de um bolo de banana industrializado, impresso em folha sulfite, e entregue para cada estudante da turma. Após a escolha do prazo de validade como primeiro elemento a ser estudado, a professora inicia uma conversa com as estudantes sobre a necessidade e a universalidade do registro da validade dos produtos em seu rótulo, destacando, principalmente, aqueles que são do ramo alimentício. Em segui da, a professora pergunta: “Vocês têm o hábito de ver a validade dos produtos que vocês compram?” A adesão a uma prática social hegemônica (conferir importância à leitura do prazo de validade antes da efetivação ou não da compra de algum produto) ecoa no enunciado da pergunta da professora sobre o costume das estudantes, quando as coloca como sujeitos (da prática e da oração gramatical) do verbo “ver” (a validade do produto estampada na embalagem) que, a rigor, envolve localizar, ler e considerar essa informação no rótulo. –“Eu tenho. Mas, vou falar a verdade, eu não sei olhar não” responde Dona Terezinha1, uma estudante de 64 anos de idade, que, nessa resposta, dissocia as três atividades que a professora parece ter condensado no verbo “ver”. Com efeito, a alfabetizanda afirma considerar a validade, mas confessa não saber ler essa informação (e, talvez, nem sequer localizá-la no rótulo). Essa dissociação denuncia interdições sofridas pela estudante ao longo da vida, as quais se estabelecem por sua con dição de mulher analfabeta nesse sistema de registro de datas, como consequência de sua exclusão do sistema escolar quando criança ou adolescente. Pessoas que estranham a escola apropriando-se de práticas matemáticas e a compreensão das práticas de numeramento como práticas de letramento Também como reflexo das contribuições metodológicas da pesquisadora e orientadora Magda Soa res, que sempre nos incitou a confrontar conceitos e teorias com a pragmática das empirias, convocamos três eventos de numeramento, que selecionamos dos estudos que consultamos, para emprestar sentidos e vitalidades à nossa discussão. As análises desses eventos permitem focalizar diferentes sujeitos, in seridos em também diferentes projetos e níveis de escolarização e em diferentes relações com a escola (mulheres idosas alfabetizando-se na escola, criança num contexto não escolar e professores indígenas em formação) que, protagonizando práticas matemáticas, apropriam-se de práticas de letramento. Tais trabalhos, ao tomarem as práticas de numeramento em sua dimensão sociocultural (YA SUKAWA et al, 2018), buscam compreender as próprias relações matemáticas como práticas cul turais, “estabelecidas na dinâmica das lutas sociais, com certas intenções, e em atendimento aos interesses de grupos em disputa” (FONSECA, 2017a, p. 112). Por isso, essa perspectiva se apresenta fértil para compreender a vivência escolar em suas relações com as práticas matemáticas, escolari zadas ou não, protagonizada por diferentes sujeitos que estranham uma escola que não foi projetada para o atendimento de suas demandas e para a potencialização e o confronto respeitoso de seus modos de dizer o mundo. Além disso, quando optam por mobilizar o conceito de práticas de numeramento, esses estudos procuram usufruir da fertilidade de tal conceito para compreender a própria constituição social dessas e desses estudantes da Educação Básica, que, embora marcados pela exclusão escolar, apresen tam-se como “protagonistas de práticas que questionam os critérios e os propósitos da escola que temos” (FONSECA, 2015, p. 10), quando se assumem como sujeitos de aprendizagem e de conheci mento, de memória e de esquecimento, de cultura e de projetos (GROSSI, 2021). Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 4 Dona Terezinha explicita sua tática (CERTEAU, 1998) para acesso e uso pragmático dessa informação e, desse modo, a estudante (e consumidora) reitera a dissociação entre o domínio dos códigos e a compreensão dos sistemas de escrita da validade, de um lado, e a prática de leitura, de outro. O posicionamento discursivo que Dona Terezinha assume desafia a lógica escolar que supõe a impossibilidade de participação na prática de leitura àqueles que não dominam o sistema de escrita utilizado. A esse raciocínio estão associados não só uma concepção de prática de leitura, mas também modos de ver o mundo e de valorar habilidades que sustentam essa concepção e, consequentemen te, as práticas escolares de ensino da leitura (SOARES, 2001). Ao aventar a possibilidade de pedir a uma pessoa (talvez, mais jovem do que ela, ou que ela suponha que domine as habilidades requeri das; talvez funcionária/o do estabelecimento ou outro/a consumidor/a) e manifestar sua disposição de fazê-lo, Dona Terezinha, a despeito de sua incapacidade de “olhar” a validade do produto que com pra, reitera o valor que atribui ao acesso àquela informação, seu conhecimento de que tal informação é veiculada naquele suporte, sua compreensão do que aquele texto comunica, sua capacidade de avaliá-lo e de usá-lo, bem como seu reconhecimento da posição que assume naquela interação, ao anunciar sua inserção e seu modo de participação naquela prática social. É nessa perspectiva que entendemos que o estudo realizado por Grossi (2021), confronta os desafios do reconhecimento e da garantia do direito das pessoas em processo de envelhecimento à educação escolar, considera a escola como agência privilegiada de letramento e reconhece as práticas matemáticas (tomadas como práticas discursivas e, como tal, socioculturais) como um componente decisivo na configuração das relações nas sociedades grafocêntricas. Por isso, ao investigar os modos pelos quais mulheres em processo de envelhecimento apropriam-se de tais práticas num contexto de alfabetização escolar, o estudo focaliza essas estudantes como mulheres de aprendizagem e de conhecimento, de vivências e de cultura, de memória e de esquecimento, de direitos e de expectativas. “Eu tenho. Mas, vou falar a verdade, eu não sei olhar não” A réplica da professora – “Não sabe não, né?” – sugere que ela não estaria tão surpresa com o fato de Dona Terezinha (e, possivelmente, outras estudantes da turma que silenciam diante de sua pergunta) não saber “olhar” a validade; mas a tréplica de Dona Terezinha confronta a possível conclusão de que ela estaria completamente distanciada daquela prática de leitura: – “Eu peço pra olhar pra mim”. 1 Grossi (2021) informa que a participante do estudo autorizou a utilização de seu verdadeiro nome no trabalho. 2021) informa que a participante do estudo autorizou a utilização de seu verdadeiro nome no trabalho. Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 5 2 Fernanda, que hoje é maior de idade, foi consultada e autorizou o uso de seu nome verdadeiro. 3 Letramento e Numeramento: Educação Matemática e Práticas de Leitura. Belo Horizonte: Ceale, 2014 https://www.ceale.fae.ufmg.br/ pages/view/palestras-do-pacto-2014.html 1a. Live Genial: Afinal o que é Numeramento? Belo Horizonte: Grupo de Estudos sobre Numeramento (GEN), 2023. https://www.youtube. com/watch?v=PrbG98xK-Q8 Fernanda, que hoje é maior de idade, foi consultada e autorizou o uso de seu nome verdadeiro. meramento: Educação Matemática e Práticas de Leitura. Belo Horizonte: Ceale, 2014 https://www.ceale.fae.ufmg.br s-do-pacto-2014.htmli al o que é Numeramento? Belo Horizonte: Grupo de Estudos sobre Numeramento (GEN), 2023. https://www.youtube 98xK-Q8 2 Fernanda, que hoje é maior de idade, foi consultada e autorizou o uso de seu nome verdadeiro. 3 Letramento e Numeramento: Educação Matemática e Práticas de Leitura. Belo Horizonte: Ceale, 2014 https://www.ceale.fae.ufmg.br/ 2 Fernanda, que hoje é maior de idade, foi consultada e autorizou o uso de seu nome verdadeiro. 3 Letramento e Numeramento: Educação Matemática e Práticas de Leitura. Belo Horizonte: Ceale, 2014 https://www.ceale.fae.ufmg.br/ pages/view/palestras-do-pacto-2014.html 1 Li G i l Afi l é N t ? B l H i t G d E t d b N t (GEN) 2023 htt // t b A incompreensão de criança de que a data que figura no registro do prazo de validade se refere ao último dia em que se pode, em tese, consumir o produto com segurança, e não à data a partir da qual se poderia fazê-lo, fez que com que a cena se tornasse pitoresca e fosse recontada, em tom jocoso, pelas pessoas adultas. Analisada, entretanto, como modos de apropriação de práticas de numeramento, o evento nos diz muito mais sobre o que Fernanda sabe da prática de escrita e de leitura de prazos de validade do que sobre o que ela não sabe. Fernanda sabe muitas coisas que poderíamos associar às habilidades do campo da alfabeti zação num sentido mais estrito. É o seu domínio do funcionamento do sistema alfabético, inclusive naquela ortografia abreviada, lhe permite ler VAL como “validade”... Fernanda conhece também o funcionamento de outros sistemas de registro, como o sistema numérico (que lhe permite ler corre tamente até mesmo 2011, que carrega a dificuldade do zero numa ordem intermediária) e o sistema de registro de datas (reconhecendo o primeiro valor como o que informa o dia; o segundo valor como relativo à identificação do mês conforme a sequência de meses do ano; e o terceiro como relativo ao ano). Nesse sentido, suas habilidades se diferem das de Dona Terezinha que confessa não saber “olhar” o prazo de validade. Em relação à interpretação do prazo de validade, Fernanda sabe ainda algo que Dona Terezinha talvez também soubesse àquela altura: inserir a data lida, no ciclo do calendário; isso permitiria a Dona Terezinha avaliar a informação lida por outra pessoa para tomar sua decisão de comprar ou não o produto, e, a Fernanda, permitiu concluir que, estando em fevereiro de 2011, “dez de abril de dois mil e onze” … “ainda não chegou”. Fernanda e Terezinha sabem também muitas coisas sobre o gênero “rótulo”, inclusive sobre as informações que devem ali conter e Fernanda sabe, inclusive, sobre onde localizar aquela que lhe interessa... Mas Fernanda e Dona Terezinha compartilham ainda um modo de apropriação da prática de escrever e ler prazos de validade (que, sendo mediada por representação e critérios matemáticos, chamamos de prática de numeramento). Não vai dar! Ainda não chegou O modo como Dona Terezinha protagoniza a prática de leitura da data de validade no rótulo dos produtos pode ser confrontado com a inserção de Fernanda2, uma criança de 8 anos, em prática semelhante, num evento que, extraído da crônica familiar, tem sido usado em iniciativas de formação docente3, justamente para discutir os conceitos de alfabetização matemática, alfabetismo matemático e numeramento. Passando o feriado de Carnaval de 2011 na casa da tia, Fernanda, inspirada pela tarde chuvosa, lhe propõe que façam pipoca. A tia pede, então, a Fernanda que busque um pacote de milho no armá rio. A sobrinha atende prontamente à solicitação da tia, mas a adverte:_ “Ih, tia, tem que olhar o prazo de validade”. A tia lhe dá razão e solicita que ela o faça: _ “Ah, é mesmo. Olha aí prá mim!” A menina, que já tinha o pacote nas mãos, vira-o para ver a face contrária à que estampava a marca do milho e acha o carimbo em que estava registrado: “VAL: 10/04/2011”. Ela lê: _ “Validade: dez de abril de dois mil e onze”. Exclama, então, desolada: _ “Ih tia, não vai dar… Ainda não chegou!” Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 6 Esse modo de apropriação considera o papel social da in formação impressa e valoriza seu poder de regulação, a ponto de nos fazer, por exemplo, abdicar de nossa própria avaliação visual, olfativa, tátil ou gustativa da qualidade do milho, para submeter nossa decisão de consumi-lo ou não à informação numérica registrada no pacote… A relação das comunidades indígenas com essas funções, entretanto, não é regida pelos mes mos valores, e isso desencadeia um salutar questionamento dos papéis que a Educação Estatística desempenhará no contexto da Educação Escolar Indígena. Nesse questionamento, colocam-se sob suspeita economia e objetividade; modos de articulação e regras gramaticais; explicações e causali dades; monitoramentos e limitações; comparações e identidades; relações com o passado e o futuro. Por outro lado, não se pode negligenciar a mobilização de ferramentas estatísticas em importantes frentes de luta dos povos indígenas, como é o caso do recurso a estudos demográficos e a diversos tipos de cadastros e registros das notificações de eventos diversos nas aldeias. Nesse sentido, povos tradicionais têm procurado não só aprender a lidar com conhecimentos, procedimentos e argumentos do campo da estatística, mas também produzir conhecimentos nesse campo, ressignificar, reorganizar ou elaborar novos procedimentos e usá-los pragmaticamente no confronto de argumentos e nas tomadas de decisão (TAHU KUKUTAI, 2012). i O movimento de apropriação dessas práticas de escrita e de leitura que se configuram como práticas estatísticas, por isso, não se restringe ao aprendizado de alguns conceitos ou dos procedimentos de cál culo de medidas de tendência central (média, moda, mediana, desvio padrão), mas envolve a produção e a negociação de diversos sentidos conferidos à estatística, ora relacionados a seus recursos para descri ção, ora a seu potencial para explicação, ora a sua utilização para controle, confronto ou projeção. Esses sentidos se podem identificar nos discursos de avaliação, adesão ou resistência à cultura estatística, pro duzidos por quem dela se apropria, e que fazem deles instâncias de incorporação e de tensionamento, de uso e de produção, de valorização e descrédito, de divulgação e silenciamento daquelas práticas. Por isso, nos parece coerente tomar as práticas estatísticas como práticas de letramento na abor dagem que lhes conferem Batista et al. (2015), segundo os quais, tais práticas envolvem um “conjunto de padrões culturais e os significados, bem como os distintos modos de atribuir significado” (BATISTA et al., 2015, p. 243). Essa compreensão das práticas de letramento como práticas culturais entende a cul tura como dinâmica e conformada pelas condições sociais. A apropriação dessas práticas supõe ação dos sujeitos na relação com os recursos culturais disponíveis. “Ah vou fazer dessa forma que eu acho mais viável, mais prático’ O terceiro e último evento que trazemos para subsidiar nossa reflexão, encontrado no estudo de Brito (2019), focaliza licenciandos e licenciandas Pataxó, Pataxó Hãhãhãe, Xakriabá, Guarani e Maxakali de um curso de Formação Intercultural para Educadores Indígenas apropriando-se de práticas estatísticas. Como naquele estudo, aqui nos interessa compreender essas práticas de nu meramento como práticas de letramento e considerar os conflitos e as potencialidades do confronto cultural que o curso oportuniza e, de certa forma, incentiva, e os modos como os sujeitos envolvidos o vivenciam. Descrever, explicar, controlar, contrastar e projetar são, sem dúvida, funções muito valorizadas na perspectiva de modernidade que rege as sociedades capitalistas, sendo possível compreender as habilidades que os currículos escolares elegem desenvolver como uma definição da contribuição da educação escolar para o atendimento das demandas das sociedades em que se inserem. Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 7 7 focar numa coisa sólida ali!’ Porque surgia... como é que posso dizer? ... Dali podiam filtrar mais coisa de um só tema. Pela opinião geral de todos os alunos, ficamos de pes quisar a questão da língua materna, o Patxohã, a língua materna, tema oficial. Aí, junto com eles, eu fui elaborando as perguntas, os questionários... Por exemplo: ‘quantas pessoas falam a língua na sua casa?’ E tudo...” (BRITO, 2019, p.203) focar numa coisa sólida ali!’ Porque surgia... como é que posso dizer? ... Dali podiam filtrar mais coisa de um só tema. Pela opinião geral de todos os alunos, ficamos de pes quisar a questão da língua materna, o Patxohã, a língua materna, tema oficial. Aí, junto com eles, eu fui elaborando as perguntas, os questionários... Por exemplo: ‘quantas pessoas falam a língua na sua casa?’ E tudo...” (BRITO, 2019, p.203) Antes de prosseguir reproduzindo o relato de Ronald, ocorre-nos destacar aqui o modo de toma da de decisão em relação ao tema que seria pesquisado pelos e pelas estudantes da escola indígena sob a condução do licenciando Pataxó. Quando confessa que não preparou papel para as crianças escolherem o tema, Ronald se refere à prática que foi realizada durante a Oficina na Universidade – votação em cédulas –, justificando sua não adoção por serem as crianças ainda pequenas. Todavia, a tomada de decisão por votação, em que a minoria se submete à vontade da maioria, não é mesmo a prática comum de tomada de decisão das comunidades indígenas. Nelas, as decisões coletivas são tomadas por consenso. Assim, a forma como Ronald narra que aconteceu a escolha do tema reflete muito mais o jeito próprio das comunidades indígenas de tomar decisões coletivas: abre-se o debate (“Aí fomos discutindo, foram surgindo bastante opiniões”), as crianças se engajam (“eles foram entusiasmando, queriam pesquisar coisas até mais além”), apontam-se alguns parâmetros (“Eu falei: ‘Vamos limitar nos demais trabalhos que foram apresentados por vocês, vamos focar numa coisa sólida ali!”), até que se chegue à decisão consensual (“Pela opinião geral de todos os alunos, ficamos de pesquisar a questão da língua materna, o Patxohã, a língua materna tema oficial”). i Esse primeiro relato já nos sugere que os procedimentos utilizados na Oficina realizada no mó dulo na Universidade, mesmo que tomados como referência, não seriam, porém, tão somente repro duzidos na aldeia, tampouco apenas adaptados às condições operacionais locais. Entretanto, se a análise dos processos de apropriação deve destacar a natureza ativa e transformadora do sujeito, não pode, porém, deixar de considerar o caráter coercitivo, mas também instrumental, das heranças culturais (ROCKWELL, 2005). i A cena que trazemos se configurou numa sessão ocorrida na Universidade em que os/as es tudantes indígenas deveriam narrar como eles/as haviam desenvolvido, na escola de sua aldeia, a proposta de orientar as crianças na realização de uma pesquisa de opinião, sobre um tema escolhido pelas próprias crianças. Nessa plenária, ocorrida na manhã de 16 de setembro de 2016, o licenciando Pataxó Ronald4, da Aldeia Velha, por iniciativa própria, foi o primeiro a apresentar seu relato: _ “Estou aqui apresentando... a comunidade indígena que estou residindo. A pesquisa foi feita na escola indígena Pataxó Trevo do Parque. É uma escola que só tem apenas três turma’. Na verdade, a educação infantil, segundo e terceiro seriado, e quarto e quin to seriado. Só tem duas salas a escola. Ah, sim... Eu peguei eh.... Como são crianças pequenas, Ensino Fundamental Um, eu não preparei papel pra eles escolherem diver sos temas. Eles ‘tão com aproximadamente dez anos, até de dez anos. Inicia a partir de quatro anos a Educação Infantil. Eu pedi um horário para a professora com as turmas e fiz um conjunto da escola. Como é escola pequena é mais fácil. E assim perguntei pra eles o que eles queriam pesquisar da comunidade. Aí fomos discutindo, foram surgindo bastante opiniões, eles foram entusiasmando, queriam pesquisar coisas até mais além. Eu falei: ‘Vamos limitar nos demais trabalhos que foram apresentados por vocês, vamos 19) informa que os/as participantes da pesquisa autorizaram a divulgação de seus nomes verdadeiros. Ronald também atendia Eu falei: Vamos limitar nos demais trabalhos que foram apresentados por vocês, vamos 4 Brito (2019) informa que os/as participantes da pesquisa autorizaram a divulgação de seus nomes verdadeiros. Ronald também atendia por seu apelido Rone. Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 8 Ao assumir, como sujeito social, o protagonismo da condução da pesquisa de opinião realizada por crianças da escola da aldeia, aquele licenciando indígena e o coletivo constituído na escola para fazer a pesquisa de opinião assumem padrões da cultura estatística, produzindo, entretanto, outros modos de lhes atribuir significado, o que configura um movimento de apropriação daquela prática de numeramento. Como, nessa introdução, Ronald já começa a descrever o processo de elaboração coletiva do instrumento da pesquisa (um texto do gênero questionário, com intenções e parâmetros técnicos bem definidos), a formadora o interrompe para demarcar o início da explicação dos procedimentos metodológicos: “Vocês elaboraram o questionário, então?” Ronald pega a deixa e prossegue o relato, contando como se elaborou o questionário e como e por quem ele foi respondido: É. ‘Quantas pessoas falam a língua na sua casa?’ ... Eh... ‘Você gosta mais da língua materna ou do português?’ ‘Você usa mais o português, no dia a dia, ou Patxohã, que é a nossa língua?’ Elaborou questionário com mais perguntas. Não está aqui o relató rio, mas pelo slide, se eu for fazer o relatório, eu consigo descrever tudo que a gente pesquisou. Tinha muito esboço no meu outro caderno... Então eles foram fazendo as la cunas, e tudo tal. Preencheu, fez o questionário, preencheu, aí mandei todos copiarem e responder. Cada um deu opinião, respondeu. Final todo mundo entregou a opinião, o que eles acharam daquele tema, das perguntas que eles mesmo criaram. ‘Ah, essa. Falam na minha casa, é essa’. ‘Se eu falo mais o português ou a língua materna no dia a dia?’ Aí daquelas perguntas que eles escolheram, cada um respondeu a sua própria, entregou essa folha pra mim, daí eu finalizei. Batia algum confronto com os cadernos, mochilas. E dali eu peguei e montei os slides, eh, com base, com gráfico do que foi ex traído das perguntas. Aí, dali deu umas porcentagens de algumas coisas, por exemplo, tantos por cento de família gostam de falar mais Patxohã do que o português no dia a dia, tantos por cento isso, tantos por cento aquilo. Os slides, botei as fotos, botei tam bém... apresentei a Escola, que escola indígena é multisseriada, as turmas que tem na escola. E finalizei com isso aí englobando tudo que eles queriam mostrar, com relação ao sistema da língua materna. A formadora intervém mais uma vez, como que para reiterar o compromisso do questionário com a in tenção da pesquisa de opinião: “O objetivo da pesquisa era o quê? Se as pessoas falam...”. Ronald acode: É assim, na verdade, como o tema era língua materna, era saber o nível de aprendizado deles, se a comunidade tem bastante falante... O objetivo era esse que eles queriam, até porque as crianças gostam bastante da língua materna. Aí, se deixar eles querem estudar mais a língua materna do que o português, no dia a dia. Eles têm mais entusiasmo do que os jovens, adolescentes, e adultos, entendeu? (BRITO, 2019, p.203) A escolha do tema e o tipo de questões elaboradas sugerem a apropriação do intuito de uma pesquisa de opinião – que é captar uma impressão do coletivo –, mas também de seu potencial de explicitar uma posição política. Foram elaboradas perguntas que produzem respostas quantificáveis, o que sugere que os elaboradores (as crianças orientadas por Rone) compreendem a dinâmica da produção de informações por meio dessas perguntas. Essa compreensão, porém, não é apenas do gênero textual questionário, e mais especificamente de como se formularem perguntas de resposta única e objetiva. Como pesquisadores, Rone e as crianças têm uma intencionalidade (“O objetivo era esse que eles queriam, até porque as crianças gostam bastante da língua materna”) e colocam a in vestigação estatística a serviço dessa intenção. Na sequência da interação, a questão da amostra pesquisada aparecerá na pergunta da pes quisadora (“Ô Rone, então eles não entrevistaram outros colegas da escola e nem da comunidade? Tudo na mesma...”), a que o licenciando responde: “Questões elaboradas por eles, cada um deu sua opinião. Então dali eu extraí o resultado da pesquisa.”; e no comentário da formadora: “Eles podem usar as mesmas perguntas e expandir.”, ao qual Rone adere, ainda que justificando o modo como efetivamente conduziu a pesquisa de opinião na escola da aldeia: É porque assim também eu pensei nessa hipótese. Mas como eu fiz uma junção das turmas, porque os professores às vezes, tinha um horário, eu tô fora da sala de aula, eu consegui esse espaço de horário, eu quis limitar ali, porque tem alguns que moram mais longe, vai de pé, é roça, e às vezes, não ia poder, sabe. Eu vou limitar aqui pra não ficar uma coisa expandida e não tenha um resultado. (BRITO, 2019, p.203) Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 9 Nesse sentido, na negociação que rege o processo de escolha do tema e sua delimitação, no esforço coletivo de elaboração do questionário, na decisão de restringir a amostra aos próprios es tudantes, considerando as limitações logísticas e o propósito de concluir o processo, na iniciativa de tabular ele mesmo as respostas, na produção dos resultados e na sua organização para publiciza-los, Rone coloca em funcionamento um modo culturalmente marcado de “compreensão e de utilização do contexto de um problema para propor investigações e esboçar conclusões, e reconhecer e entender o processo como um todo” (BEN-ZVI; GARFIELD, 2004, p. 7, tradução nossa)5, que caracterizam o tudantes, considerando as limitações logísticas e o propósito de concluir o processo, na iniciativa de tabular ele mesmo as respostas, na produção dos resultados e na sua organização para publiciza-los, Rone coloca em funcionamento um modo culturalmente marcado de “compreensão e de utilização do contexto de um problema para propor investigações e esboçar conclusões, e reconhecer e entender o processo como um todo” (BEN-ZVI; GARFIELD, 2004, p. 7, tradução nossa)5, que caracterizam o pensamento estatístico. Rone coloca em funcionamento um modo culturalmente marcado de “compreensão e de utilização do contexto de um problema para propor investigações e esboçar conclusões, e reconhecer e entender o processo como um todo” (BEN-ZVI; GARFIELD, 2004, p. 7, tradução nossa)5, que caracterizam o pensamento estatístico. Essa compreensão, entretanto, vai se constituindo ao longo do processo e leva em consideração a experiência vivida na Oficina no Curso de Formação e os modos como ele elabora a demanda da tarefa que deveria realizar na Aldeia e cujos resultados deveria depois apresentar aos colegas e à formadora. Mas também constituem essa compreensão a reflexão que ele faz (“Deu uma clareada na minha mente”) ponderando as intenções da pesquisa (“é o que eles estão querendo pesquisar, estão querendo mostrar”) e as condições materiais disponíveis (“aí falei: ‘ah vou fazer dessa forma que eu acho mais viável, mais prático”). Não é, entretanto, apenas o resultado dessa elaboração para a apresentação que o faz avaliar que o trabalho ficou “muito bom”: a avaliação positiva está relacionada a colocar toda aquela elabora ção para comunicar o processo da pesquisa e seus resultados a serviço daquilo que eles – o coletivo dos estudantes da escola e ele mesmo como seu porta-voz – queriam comunicar (“é o que eles estão querendo pesquisar, estão querendo mostrar. (BRITO, 2019, p.203) A indagação da pesquisadora, então se volta para os procedimentos de tratamento das respos tas aos questionários: “E quando você falou que fez um apanhando das respostas, a turma participou da contagem, da elaboração?” A indagação da pesquisadora, então se volta para os procedimentos de tratamento das respos tas aos questionários: “E quando você falou que fez um apanhando das respostas, a turma participou da contagem, da elaboração?” A resposta de Ronald nos diz muito sobre o seu processo de apropriação das práticas de letra mento ali envolvidas – às quais chamamos práticas de numeramento, por serem mediadas por ideias, procedimentos, conceitos e critérios do campo da Estatística: Isso. Eu fui anotando, foi elaborando no quadro lá. Aí depois eu montei os slides. Não ficou lá... Porque também, não vou mentir pra vocês. Até no dia de fazer, eu tinha muita dúvida sobre muitas coisas. Só conseguia lembrar dos slides que tinha que mostrar. Deu uma clareada na minha mente, aí falei: ‘ah vou fazer dessa forma que eu acho mais viável, mais prático; é o que eles estão querendo pesquisar, estão querendo mostrar’. E pra mim ficou excelente dessa forma. Não sei o ponto de vista de vocês. Rone menciona que assumiu o trabalho de tabulação, produção dos resultados e elaboração de gráficos e, assim, apropriando-se dos recursos que a estatística como ferramenta cultural oferece, usufrui deles na realização da intenção que motivou a pesquisa: “E finalizei com isso aí englobando tudo que eles queriam mostrar, com relação ao sistema da língua materna”. 1 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 10 E pra mim ficou excelente dessa forma”). Nesse sentido é que interpretamos esse relato de Ronald Pataxó como uma instância de apro priação não apenas de habilidades no manejo de conceitos e procedimentos estatísticos ou certa perícia em colocá-los em uso. Trouxemos aqui este evento para destacar o modo como esse licen ciando Pataxó, identificado com as dinâmicas da aldeia em que reside (a revitalização da língua materna e os diferentes engajamentos nessa revitalização, os modos de tomada de decisão por consenso, e não por votação, a escola como espaço de divulgação e sua organização em clas ses multisseriadas, o atendimento ao desejo das crianças de falarem sobre o Patxohã, o respeito à autoridade e à opinião do cacique etc.) apropria-se de “distintos modos de atribuir significado” (BATISTA et al., 2015, p. 243) às práticas estatísticas, o que nos faz considerar, aqui, esse proces so como apropriação de práticas de letramento, na perspectiva que Magda Soares confere a esse construto analítico e pedagógico. 5 “understanding and using the context of a problem to propose investigations and draw conclusions, and to recognize and understand the process as a whole”. Todavia, é importante destacar que os estudos que focalizam pessoas apropriando-se de práti cas de numeramento não limitam sua atenção investigativa aos processos de escolarização vivencia dos por crianças, por jovens ou por pessoas adultas e idosas, mas se voltam também para a busca de compreensões sobre o porquê de muitos desses sujeitos não reconhecerem nesses processos o atendimento de suas demandas genuínas de expressão verbal e não se sentirem convocados a assu mir o protagonismo dos processos de aprendizagem. Por isso, muitos estudos numeramento (assim como ocorre nos estudos do letramento em geral) têm se voltado para públicos que tiveram acesso mais recente à educação pública: crianças e jovens em situação de vulnerabilidade social6, pessoas jovens, adultas ou idosas a quem foi negado o direito à educação escolar quando crianças ou ado lescentes7, indígenas8, camponeses9, pessoas com deficiência e/ou com necessidades educacionais específicas10, entre outros. Muitos desses estudos, no modo como lidam com as práticas matemáticas como práticas sociais, enquadram-se no que Street (1984, 1993) caracterizou como o modelo de letramento ideológico, ou que Knijnik et al (2012) identificam como perspectiva Etnomatemática, assumindo que existem dife renças nas práticas de numeramento de grupos socioeconômica e culturalmente distintos devido às diferentes formas com que eles integram (ou não) a escrita e artefatos matemáticos no seu cotidiano, e que essas formas, por sua vez, mudam de acordo com as relações de poder estabelecidas e com os usos da leitura da escrita e da matemática nos diferentes contextos. A compreensão da dimensão cultural, social e política dos discursos que, assim, permeia as práticas de letramento, nelas incluídas as práticas de numeramento, desdobra-se na obra de Magda Soares e em sua atuação no cenário educacional na preocupação com a democratização da Edu cação. Essa preocupação, que seu legado insere ou potencializa nos estudos do letramento, refle te-se nos esforços que se promovem com vistas à efetividade das oportunidades de apropriação do sistema alfabético e dos sistemas de representação matemáticos (numéricos, métricos, estatísticos, algébricos, geométricos), e também de uma apropriação crítica da cultura escrita (nela incluída a cul tura matemática) socialmente valorizada. A promoção dessa apropriação supõe reconhecê-la como oportunidade de interação discursiva, espaço de elaboração, tensão, disputa, diálogo, cooperação, e mudança social. Considerações finais A utilização, nos estudos sobre apropriação de práticas de numeramento, do legado dos estu dos do campo do letramento e, de modo especial, da orientação que Magda Soares lhes conferiu considera que as práticas discursivas nas sociedades modernas em geral, mas, principalmente, naquelas que se forjam nos contextos escolares, são tão marcadas pela cultura escrita como o são pela quantificação. Mais do que isso, práticas de quantificação e suas correlatas (medição, ordenação, classificação, organização do espaço e das formas) estão referenciadas em um mesmo marco cultural que referencia as práticas de escrita e de leitura, e que estabelece os modos como indivíduos, grupos e instituições narram o mundo e as relações que nele se estabelecem e que o constituem. 5 “understanding and using the context of a problem to propose investigations and draw conclusions, and to recognize and understand the process as a whole”. Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 11 6 Cf. CABRAL, 2015; LIMA, 2020; FARIA, 2023. 7 Cf. LIMA, 2007; FARIA, 2007; CABRAL, 2007; SOUZA, 2008; FERREIRA, 2009; ADELINO, 2009; SCHNEIDER, 2010; SIMÕES, 2010, 2019; LIMA, 2012; SILVA, 2013; MIRANDA, 2015; GROSSI, 2021. 8 Cf. MENDES, 2001; BRITO, 2012; 2019; MENDONÇA, 2014 9 Cf. VASCONCELOS, 2011; SILVA, 2013; SÁ, 2016. 6 Cf. CABRAL, 2015; LIMA, 2020; FARIA, 2023. 7 Cf. LIMA, 2007; FARIA, 2007; CABRAL, 2007; SOUZA, 2008; FERREIRA, 2009; ADELINO, 2009; SCHNEIDER, 2010; SIMÕES, 2010, 2019; LIMA, 2012; SILVA, 2013; MIRANDA, 2015; GROSSI, 2021. Cf MENDES BRITO MENDONÇA BEN-ZVI, D.; GARFIELD, J. The challenge of developing statistical literacy, reasoning, and thinking. Dordrecht: Kluwer Academic Publishers, 2004. BEN-ZVI, D.; GARFIELD, J. The challenge of developing statistical literacy, reasoning, and thinking. Dordrecht: Kluwer Academic Publishers, 2004. BRITO, R. P. S. 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https://openalex.org/W2158685106	https://europepmc.org/articles/pmc3359232?pdf=render	English	null	Impact of genital warts on health related quality of life in men and women in mainland China: a multicenter hospital-based cross-sectional study	BMC public health	2,012	cc-by	7,715	© 2012 Shi 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: Information on the health-related quality of life (HRQoL) of patients with genital warts (GW) in populations in mainland China is still limited. The aim of the study was to use a generic instrument to measure the impact of genital warts on HRQoL in men and women in this setting. Methods: A multi-centre hospital-based cross-sectional study across 18 centers in China was conducted to interview patients using the European quality of life-5 dimension (EQ-5D) instrument; respondents’ demographic and clinical data were also collected. Results: A total of 1,358 GW patients (612 men, 746 women) were included in the analysis, with a mean age of 32.0 ± 10.6 years. 56.4% of the patients reported some problems in the dimension of Anxiety/Depression (highest), followed by Pain/Discomfort (24.7%) and Mobility (3.5%). The overall visual analogue scale (VAS) score of the study population was found to be 65.2 ± 22.0, and the EQ-5D index score was found to be 0.843 ± 0.129 using Japanese preference weights (the Chinese preference was unavailable yet). Patients with lower VAS means and EQ-5D index scores were more often female, living in urban area, and suffering multiple GW (all p values < 0.05), but the values did not differ notably by age (p values > 0.05). Conclusions: The HRQoL of patients with GW was substantially lower, compared to a national representative general population in China (VAS = ~80); the findings of different subgroups are informative for future GW prevention and control efforts. Impact of genital warts on health related quality of life in men and women in mainland China: a multicenter hospital-based cross-sectional study Ju-Fang Shi1,2, Dian-Ju Kang1,3, Shu-Zhen Qi4, Hai-Yan Wu5, Yan-Chun Liu6, Li-Jun Sun7, Li Li8, Ying Yang9, Qing Li10, Xiang-Xian Feng11, Li-Qin Zhang12, Jie Li13, Xiao-Li Li14, Yun Yang15, Mayinuer Niyazi16, Ai-Di Xu17, Jia-Hua Liu18, Qing Xiao19, Lian-Kun Li20, Xin-Zheng Wang21 and You-Lin Qiao1* * Correspondence: qiaoy@cicams.ac.cn 1Department of Cancer Epidemiology, Cancer Institute/Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, 17 South Panjiayuan LN, PO Box 2258, Beijing 100021, China Full list of author information is available at the end of the article Shi et al. BMC Public Health 2012, 12:153 Shi et al. BMC Public Health 2012, 12:153 Shi et al. BMC Public Health 2012, 12:153 http://www.biomedcentral.com/1471-2458/12/153 Open Access Background Data related to the less- developed centre in Southwest China were not available. Most of the study hospitals have had well established collaboration with CICAMS on a number of prior studies associated with HPV and cervical cancer. Figure 1 The distribution of study sites in China. The 11 higher level centers included Beijing (2 centers), Tianjin, Shenyang (Liaoning Province), Xi’an (Shaaxi Province), Changsha (Hunan Province), Chongqing, Shenzhen (Guangdong Province), Shanghai, Nanjing (Jiangsu Province) and Fuzhou (Fujian Province). The seven lower level centers included Dashiqiao County (Liaoning Province), Hetian County (Xinjiang Autonomous Region), Xinmi Prefecture (county-level, Henan Province), Panyu/Conghua County (Guangdong Province), Tonggu County (Jiangxi Province), Yangcheng County and Changzhi Prefecture (Shanxi Province). Data related to the less- developed centre in Southwest China were not available. Most of the study hospitals have had well established collaboration with CICAMS on a number of prior studies associated with HPV and cervical cancer. Figure 1 The distribution of study sites in China. The 11 higher level centers included Beijing (2 centers), Tianjin, Shenyang (Liaoning Province), Xi’an (Shaaxi Province), Changsha (Hunan Province), Chongqing, Shenzhen (Guangdong Province), Shanghai, Nanjing (Jiangsu Province) and Fuzhou (Fujian Province). The seven lower level centers included Dashiqiao County (Liaoning Province), Hetian County (Xinjiang Autonomous Region), Xinmi Prefecture (county-level, Henan Province), Panyu/Conghua County (Guangdong Province), Tonggu County (Jiangxi Province), Yangcheng County and Changzhi Prefecture (Shanxi Province). Data related to the less- developed centre in Southwest China were not available. Most of the study hospitals have had well established collaboration with CICAMS on a number of prior studies associated with HPV and cervical cancer. The Cancer Foundation of China (CFC) and the Can- cer Institute of Chinese Academy of Medical Sciences (CICAMS) jointly conducted a multi-centre hospital- based epidemiological and economic study on genital warts across China. As part of this broad project, the current study’s aim was to use the generic instrument EQ-5D to measure the impact of genital warts on health related quality of life in men and women in mainland China. Background and Hong Kong) [1]. However, the variability in the GW prevalence among different data sources from other Asia Pacific countries might suggest that under-reporting potentially existed in the population surveillance systems [1]. In addition, a high post-treatment recurrence rate of GW has been reported (40%) [4] and this characteristic of GW imposes a considerable sociopsychological bur- den (such as worries and concerns, negative emotional and sexual impact, shame or stigma, and worse interac- tions with partner and/or doctors [5,6]) and economic burden [7] on the patients. Genital warts (GW) is one of the most common sexually transmitted diseases and about 90% of GW are caused by infection with human papillomavirus (HPV)-6 and HPV-11 [1]. The incidence of GW is increasing globally although some developed countries, such as US and Australia, have reported relatively low rates ( < 0.3%) [2,3]. Information on the disease burden of GW in mainland China is still limited, but a relatively higher GW prevalence (0.5%) was reported in general popula- tions of other Asian regions (including India, Taiwan It has been estimated that multivalent prophylactic HPV vaccine has the potential to protect up to 83% of genital warts or condylomata acuminate in mainland China [8]. In an era of HPV vaccination, a number of * Correspondence: qiaoy@cicams.ac.cn 1Department of Cancer Epidemiology, Cancer Institute/Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, 17 South Panjiayuan LN, PO Box 2258, Beijing 100021, China Full list of author information is available at the end of the article Shi et al. BMC Public Health 2012, 12:153 http://www.biomedcentral.com/1471-2458/12/153 Page 2 of 9 Northwest Southwest Northeast North South Centre East Study site Figure 1 The distribution of study sites in China. The 11 higher level centers included Beijing (2 centers), Tianjin, Shenyang (Liaoning Province), Xi’an (Shaaxi Province), Changsha (Hunan Province), Chongqing, Shenzhen (Guangdong Province), Shanghai, Nanjing (Jiangsu Province) and Fuzhou (Fujian Province). The seven lower level centers included Dashiqiao County (Liaoning Province), Hetian County (Xinjiang Autonomous Region), Xinmi Prefecture (county-level, Henan Province), Panyu/Conghua County (Guangdong Province), Tonggu County (Jiangxi Province), Yangcheng County and Changzhi Prefecture (Shanxi Province). Data related to the less- developed centre in Southwest China were not available. Most of the study hospitals have had well established collaboration with CICAMS on a number of prior studies associated with HPV and cervical cancer. cost-effectiveness analyses (CEA) on multivalent HPV vaccines have been performed globally. Background Health related quality of life (HRQoL) contains self-reported measures of physical and psychological aspects, and an accurate impact analysis of genital warts on HRQoL was expected to contribute to the health economic evaluation on (HPV) vaccines protects against related HPV types. The extent of HRQoL of patients with GW has previously been quantified in different settings using generic instru- ments [6,9-11], including the European quality of life-5 dimension (EQ-5D) measure. EQ-5D is a standardized and commonly used instrument on HRQoL [12], and is also one of only a few measures recommended for use in cost-effectiveness analysis [13,14]. However, most of the quality of life studies using EQ-5D in China’s popu- lations were more focused on the general populations or patients with various chronic diseases [15-18], and assessment related to GW in this setting has not been performed. cost-effectiveness analyses (CEA) on multivalent HPV vaccines have been performed globally. Health related quality of life (HRQoL) contains self-reported measures of physical and psychological aspects, and an accurate impact analysis of genital warts on HRQoL was expected to contribute to the health economic evaluation on (HPV) vaccines protects against related HPV types. The extent of HRQoL of patients with GW has previously been quantified in different settings using generic instru- ments [6,9-11], including the European quality of life-5 dimension (EQ-5D) measure. EQ-5D is a standardized and commonly used instrument on HRQoL [12], and is also one of only a few measures recommended for use in cost-effectiveness analysis [13,14]. However, most of the quality of life studies using EQ-5D in China’s popu- lations were more focused on the general populations or patients with various chronic diseases [15-18], and assessment related to GW in this setting has not been performed. Northwest Southwest Northeast North South Centre East Study site Study site Figure 1 The distribution of study sites in China. The 11 higher level centers included Beijing (2 centers), Tianjin, Shenyang ( ) ’ ( h ) h h ( Figure 1 The distribution of study sites in China. The 11 higher level centers included Beijing (2 centers), Tianjin, Shenyang (Liaoning Province), Xi’an (Shaaxi Province), Changsha (Hunan Province), Chongqing, Shenzhen (Guangdong Province), Shanghai, Nanjing (Jiangsu Province) and Fuzhou (Fujian Province). The seven lower level centers included Dashiqiao County (Liaoning Province), Hetian County (Xinjiang Autonomous Region), Xinmi Prefecture (county-level, Henan Province), Panyu/Conghua County (Guangdong Province), Tonggu County (Jiangxi Province), Yangcheng County and Changzhi Prefecture (Shanxi Province). Methods The overall VAS mean among all cases was 65.2 (SD: 22.0), and men had a higher mean score than women (69.0 vs. 62.1, P < 0.001). To make com- parisons for all of the reported groups, relevant findings from a national population-based survey in China [15] were also added to Table 1 as references. After applying the preference weights of the Japanese, UK and US populations, the overall EQ-5D index scores of the cur- rent analysis were 0.843, 0.826 and 0.859, respectively; and similarly, men scored higher than women under every one of the three preference weight sets (all P values < 0.001) (Table 2). p The characteristics of the study population were described in general terms. Percentages of patients reporting any problems, age-specific means of EQ-5D VAS scores and their 95% confidence intervals were analyzed (overall and by sex), using Chi-square test or one-way ANOVA approach. Because of the absence of a EQ-5D preference weight set for the Chinese popula- tion, we applied the preference weight sets from other populations (the UK, US and Japan) [20-22] to estimate the mean EQ-5D index score of this GW population in China. Detailed VAS means and EQ-5D index scores by population characteristics were calculated and a one- way ANOVA approach was applied to detect the statis- tical differences between subgroups. In addition, Pearson correlation among VAS means and EQ-5D index scores using the three preference weight sets were character- ized using a univariate linear regression model. Since a very high association between weighted and non- weighted EQ scores has been reported [23], we then conducted a multivariate linear regression analysis to check the robustness of important variables, by input- ting variables with statistical significance confirmed in the non-weighted analysis. For the ordinary unordered categorical variables with more than two subgroups, dummy variables were applied and the subgroup with lowest mean/score (which was southwest in the region category and recurrent GW in the clinical status cate- gory) was chosen as the reference subgroup. All analysis used two tailed tests. A range of strengths of correlation between results from different analyses were characterized. On one hand, relatively low Pearson correlations R between EQ VAS mean and EQ-5D index scores were observed (0.371, 0.423, 0.412 using Japanese, UK and US prefer- ence weights respectively, all p values < 0.001). Methods BMC Public Health 2012, 12:153 http://www.biomedcentral.com/1471-2458/12/153 Shi et al. BMC Public Health 2012, 12:153 http://www.biomedcentral.com/1471-2458/12/153 Prior to the larger-scale survey, a pilot study was con- ducted in three sites (urban: Beijing; rural: Shanxi and Jiangxi); the initial study flowchart and questionnaire contents (excluding the EQ-5D questionnaire) were then modified and optimized based on the feedback from the pilot study. An investigators’ meeting was then organized by CICAMS and CFC to provide data collection training and to introduce the study protocol and an operations manual to all the co-principle investigators and the main project members from 18 sites. The meeting participants were then required to provide training to other project team members (including nurses and physicians at the clinics) in local individual centers, based on the study operation manual. The operations manual contained questionnaire instructions, coding, quality control pro- cess, collaborators’ responsibility, logistic instructions and other details in an operational level. CICAMS was responsible for all database building, logistical data checks (by interacting with local staff) and data analysis. Data collection and double entry and basic data checks were conducted in the 18 collaborative centers/hospitals. with a mean age of 32.0 years (SD: 10.6, range: 18-86). Of all study patients, 75.1% of them were enrolled from urban-based hospitals, 33.2% received college or higher education, 30.0% held health insurance, 22.2% were cur- rent frequent smokers, and their median monthly income was 1,400 Chinese Yuan (~200 US$). In addi- tion, 69.3% of the study subjects were married or living with their partners, and 54.3% have had two or more lifetime sexual partners. During the survey, the patients were basically categorized into three clinical statuses, including the initial clinical visit for GW, the follow-up visit within an episode, and visits for recurrent GW, with the proportions in each status being 76.0%, 10.8% and 13.3%, respectively. In all, 26.2% of the subjects were observed to have a single genital wart on their bodies, while the rest suffered multiple genital warts. Table 1 illustrates the results of the five study dimen- sions. For the whole study population, the dimension of Anxiety/Depression had the highest rate of self-reported problems (56.4%), followed by Pain/Discomfort (24.7%), Mobility (3.5%), Usual activities (2.8%) and Self-care (0.5%). Compared to the male patients, females tended to report more problems in three dimensions, including Anxiety/Depression (50.1% vs. 61.5%), Pain/Discomfort (20.6% vs. 28.0%), and Mobility (1.3% vs. 5.2%) (all P values < 0.005). Methods On the other hand, unsurprisingly, a slightly higher agreement (0.984, p < 0.001) was observed between EQ-5D index scores of UK and US preference weights compared to those of the other two pairs (Japan & UK: 0.817, Japan & US: 0.860, all p values < 0.001); this situation is potentially due to the cultural dissimilarity between Western and Asian populations. In addition to this, compared to the EQ-5D results for populations in the UK and US, the reported percentages of subjects’ pro- blems by each of the EQ-5D’s five dimensions among the Japanese general population were closer to those of Methods From July 2007 to July 2008, patients with genital warts were enrolled in 18 selected centers across seven geo- graphic regions of China (Northeast, North, Northwest, Central, Southwest, South and East China). Generally, within each region, one province-level centre in a more developed area, and one county-level centre in a less developed area were selected. In addition, four addi- tional study sites were selected from the North and East China regions in order to provide more information on more developed areas or metropolitan cities. Thus, a total of 11 higher level centers and 7 lower level centers eventually participated in the current survey (Figure 1). and diseases [12], and promising evidence for the mea- surement equivalence of EQ-5D’s English and Chinese versions has been reported [19]. EQ-5D contains five dimensions measurement, including Mobility, Self-care, Activities, Pain/Discomfort and Anxiety/Depression [12]. Three options are listed for each dimension: whether the subject has no problem, a moderate problem or an extreme problem. Separately, EQ-5D instrument also contains a visual analogue scale (VAS) which allows the study subjects themselves to assess and self-record their health status from 0 (the worst) to 100 (the best) [12]. Using the EQ-5D instrument (Chinese version), a face- to-face interview was administrated by a project staff member, or alternatively a capable patient completed the questionnaire by him or herself in the presence of a trained interviewer who answered any questions the par- ticipants had about the questionnaire. In addition, further patient information including place of residence, education, income, marriage status, whether or not the patient is a smoker, sexual behavior and clinical charac- teristics were also collected. Information from patients who did not accept the invitation was not recorded in the study. A total of 100 GW patients were initially required for each centre at a higher level and 50 patients at a lower level, and a convenience sampling method was applied within each centre. Patients who were currently diag- nosed with genital warts, aged 18 years or older and provided informed consent (no participation fee applied) were eligible for this study regardless of their gender or whether they were an incident or recurrent case. Central institutional review board approval was obtained from the Cancer Foundation of China. The EQ-5D instrument has several language versions and has been applied to a wide variety of populations Page 3 of 9 Page 3 of 9 Shi et al. Results A total of 1,358 outpatients with genital warts were included in the analysis (612 men and 746 women), Shi et al. BMC Public Health 2012, 12:153 http://www.biomedcentral.com/1471-2458/12/153 Page 4 of 9 Table 1 Percentages of subjects self-reported problems and means of visual analogue scale (VAS), total and by sex EQ-5D dimension The current study Findings from a national population-based survey in China, 2008 [15] Male Female Total Male Female Total* No. % No. % No. % % (N = 58,163) % (N = 62,540) % (N = 120,703) Mobility No 604 98.7 707 94.8 1,311 96.5 95.7 94.6 95.1 Moderate 8 1.3 38 5.1 46 3.4 4.0 5.0 4.5 Extreme 0 0.0 1 0.1 1 0.1 0.3 0.4 0.4 Self-care No 610 99.7 741 99.3 1,351 99.5 97.3 96.6 96.9 Moderate 2 0.3 5 0.7 7 0.5 2.3 3.0 2.7 Extreme 0 0.0 0 0.0 0 0.0 0.4 0.4 0.4 Usual activities No 596 97.4 723 96.9 1,319 97.1 96.0 94.9 95.4 Moderate 16 2.6 21 2.8 37 2.7 3.3 4.3 3.8 Extreme 0 0.0 2 0.3 2 0.1 0.7 0.8 0.8 Pain/Discomfort No 486 79.4 537 72.0 1,023 75.3 92.8 89.5 91.1 Moderate 122 19.9 205 27.5 327 24.1 6.9 10.0 8.5 Extreme 4 0.7 4 0.5 8 0.6 0.3 0.5 0.4 Anxiety/Depression No 305 49.8 287 38.5 592 43.6 94.8 92.9 93.8 Moderate 259 42.3 362 48.5 621 45.7 4.9 6.7 5.8 Extreme 48 7.8 97 13.0 145 10.7 0.3 0.4 0.4 VAS score 612 69.0 (mean) 746 62.1 (mean) 1,358 65.2 (mean) 80.9 (mean) 79.4 (mean) 80.1 (mean) Chi-square tests were performed, female patients tended to report more problems in three dimensions, including Anxiety/Depression, Pain/Discomfort and Mobility, when comparing to male patients (all P values < 0.005). * The percentages of “Total” group were estimated based on the reported findings for males and females with considering each group’s sample size. es of subjects self-reported problems and means of visual analogue scale (VAS), total and by sex f-reported problems and means of visual analogue scale (VAS), total and by sex lower VAS and EQ-5D index scores (one-way ANOVA results, all p values < 0.05); and a lower monthly income and the situation of recurrent GW probably contributed to a worse HRQoL of GW patients (P < 0.05 in the EQ-5D index score calculation, but P > 0.05 in the VAS analysis). Results Significant differences were not detected among subgroups by age (Figure 2), marital status, education, insurance or the number of lifetime sexual partners (all P values > 0.05). the general population in China [15,17]. For this reason, only the preference weight set of the Japanese popula- tion was applied when we calculated further characteris- tic-specific EQ-5D index scores in this analysis. Table 3 presents further detailed mean VAS and EQ-5D index scores for different subgroups. Briefly, female patients, living in urban areas, residing in the Southwest region, smoking less frequently and with multiple clini- cally-confirmed GW were statistically associated with In the multivariate linear regression analysis, similar results were observed (Table 4); when using the VAS mean as the dependent variable, the variables of gender, rural or urban, residence in the North or Central regions of China, and single/multiple GW were retained in the final model (all P values < 0.01, adjusted R square = 0.065). When placing EQ-5D index score as the dependent variable, more factors entered the model, including initi- ally occurred GW, monthly income, region of East, and region of Northeast (all P values < 0.001, adjusted R square = 0.117) (Table 4). The factors of smoking and monthly income were not statistically associated with the HRQoL of GW patients in the multivariate analysis. Table 2 The EQ-5D index score using different preference weights, total and by sex Table 2 The EQ-5D index score using different preference weights, total and by sex Preference weight set applied Male Female Total Mean SD Mean SD Mean SD Japan 0.864 0.13 0.827 0.126 0.843 0.129 UK 0.856 0.185 0.802 0.21 0.826 0.201 US 0.882 0.137 0.84 0.15 0.859 0.145 One-way ANOVA approach was applied to compare scores between men and women (all P values < 0.001) Shi et al. BMC Public Health 2012, 12:153 http://www.biomedcentral.com/1471-2458/12/153 Page 5 of 9 Page 5 of 9 Di i i t d d i d j bl f GW Table 3 Mean visual analogue scale (VAS) and EQ-5D index score, by population characteristics* Variable No. Results % VAS EQ-5D index score** Mean SD Sig.# Mean SD Sig.# Sex Male 612 45.1 69.0 21.3 < 0.001 0.864 0.130 < 0.001 Female 746 54.9 62.1 22.1 0.827 0.126 Region North 372 27.4 66.6 25.0 < 0.001 0.866 0.135 < 0.001 Northeast 128 9.4 64.0 24.7 0.897 0.125 Northwest 151 11.1 62.2 20.6 0.790 0.121 Central 133 9.8 72.3 12.8 0.871 0.120 Southwest 100 7.4 57.3 22.2 0.790 0.111 South 135 9.9 66.1 18.6 0.823 0.134 East 339 25.0 64.5 21.2 0.835 0.120 Setting Urban 1,020 75.1 63.2 22.4 < 0.001 0.837 0.126 < 0.005 Rural 338 24.9 71.0 19.6 0.862 0.137 Be married / living together Yes 941 69.3 65.0 22.2 > 0.05 0.844 0.130 > 0.05 No 417 30.7 65.5 21.6 0.843 0.128 Education Middle school or lower 408 30.0 66.3 20.7 > 0.05 0.842 0.132 > 0.05 High school 499 36.7 66.1 22.0 0.843 0.130 College or above 451 33.2 63.2 23.1 0.845 0.127 Monthly income (Chinese Yuan) * < 1000 364 26.8 63.5 22.4 > 0.05 0.827 0.133 < 0.005 1000~ 422 31.1 66.8 21.3 0.838 0.124 2000~ 223 16.4 65.8 22.8 0.852 0.131 3000~ 347 25.6 64.5 22.0 0.860 0.128 Any insurance coverage* Yes 432 31.8 65.8 21.8 > 0.05 0.842 0.129 > 0.05 No 925 68.1 65.0 22.0 0.844 0.131 Frequently smoking Yes 301 22.2 68.2 21.2 < 0.01 0.860 0.131 < 0.05 No 1,057 77.8 64.3 22.2 0.839 0.129 Number of lifetime sexual partners* 1 612 45.1 63.8 22.7 > 0.05 0.852 0.132 > 0.05 2 318 23.4 65.3 21.9 0.839 0.128 3 or more 421 31.0 67.0 21.0 0.834 0.126 Clinical status First clinical visit for the initially occurred GW 1,032 76.0 65.7 21.8 > 0.05 0.853 0.129 < 0.001 Follow-up visit for the initially occurred GW 146 10.8 64.1 24.4 0.822 0.126 Recurrent GW 180 13.3 63.9 21.0 0.804 0.124 Single or multiple genital warts Single 356 26.2 68.0 21.2 < 0.01 0.869 0.127 < 0.001 Multiple 1,002 73.8 64.2 22.2 0.835 0.129 * Some data missing; ** Japanese preference weights applied; # One-way ANOVA analysis. Table 3 Mean visual analogue scale (VAS) and EQ-5D index score, by population characteristics* anxiety and depression posed a major problem for GW patients, and the measurement of VAS suggested that the HRQoL of GW patients was substantially lowered. The current analysis using different preference weights provided a range of utility estimates (overall and Discussion The current work is a unique large-scale multi-centre study focusing on the HRQoL of male and female patients with GW using a generic and internationally comparable instrument, the EQ-5D. We found that Shi et al. BMC Public Health 2012, 12:153 http://www.biomedcentral.com/1471-2458/12/153 Page 6 of 9 The research group has published a parallel work con- ducted as another part of the whole GW project [5]. Both the studies explored the quality of life issue but from different perspectives and each study had its own emphasis. For example, firstly, the current study focused on both males and females, while the previous work mainly assessed female populations. Secondly, the cur- rent study was very focused on GW patients only, but the study by Wang et al included women who have a spectrum of HPV-related health statuses or laboratory diagnosis, including normal/abnormal pap smear, cervi- cal precancer, and HPV+/- after abnormal pap, and patients with GW were only one sub-group in the broad analysis [5]. Thirdly, the previous work used a newly- developed HPV-specific instrument, the HPV impact profile (HIP); it is more sensitive and can recognize some slight differences among the targeted HPV-related subgroup women, but it is more difficult to directly compare the findings of the HIP study and other quality of life studies using different instruments. Differently, the EQ-5D, used by the current analysis, is an easy-to- compare HRQoL instrument and commonly-used world widely [12]. Finally, compared to the HIP score out- comes, the EQ-5D index score outcomes reported by the current study were relatively more convertible when 50 55 60 65 70 75 80 85 90 18-19 20-24 25-29 30-34 35-39 40-44 45-49 50-84 VAS mean and 95% confidence interval Age groups (years) EQ-5D index score and 95% confidence interval 0.90 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 EQ-5D index score VAS mean Figure 2 Age-specific means of visual analogue scale (VAS) and EQ-5D index score. One-way ANOVA analyses were performed (P > 0.05); Japanese preference weights applied. Figure 2 Age-specific means of visual analogue scale (VAS) and EQ-5D index score. One-way ANOVA analyses were performed (P > 0.05); Japanese preference weights applied. characteristic-specific) for future detailed QALY- related cost-effectiveness evaluations. * Japanese preference weights applied. Discussion In contrast to the dimension of Anxiety/Depression, the dimensions of Mobility, Self- care and Usual Activities in this GW population are generally less impacted when compared to local and international general populations [15,17]; this situation is due partly to the reality that most of the studied GW patients (~93%) were younger than 50 years old, and were able to move, to take care of themselves, and to complete their usual activities with no difficulty. Also, the situation of lower rates of any problems in the three dimensions mentioned above is consistent with the find- ings of a UK GW study [9]. Due to the absence of an EQ-5D preference weight set in the Chinese population, three other populations’ pre- ference sets were applied to estimate the EQ-5D index scores in the current analysis. Our findings suggest that scores based on the Japanese preference weights could be regarded as the baseline utility values for future cost- effectiveness evaluations, whilst the results based on the UK and US populations could provide a plausible range of utilities estimates for sensitivity analysis. When com- paring our EQ-5D index scores to other international GW studies, a wide difference was noted but all our EQ-5D index scores using varied populations preference weights (0.826, 0.843 and 0.859) are within the range (0.76 - 0.91) of the available data from other populations [9-11]; the lowest utility value (0.76) of GW patients was from a Canadian study, and the highest value (0.91) was reported by Myers et al (conference abstract, details unavailable) which has been cited by a number of more recent cost-effectiveness evaluations of quadrivalent HPV vaccine [24-27]. Our EQ-5D index scores analysis also further supports the hypothesis that female patients suffered a larger decrease in quality of life than males and the scores did not differ notably among age groups, as we previously discussed in the VAS scores analysis. A large-scale survey included EQ-5D instrument was previously conducted based on a national representative sample in China in 2008, where the average VAS were found to be 80.9 for male and 79.4 for female [15]. Since HRQoL scores are very age-dependent but the subjects of current study mainly aged 15-49 years, we restricted the comparison to narrower age groups (15- 49 years, by 5-year age group); it was noted that the national survey reported relatively higher values of VAS score (81.4-89.8 for male, 79.2-89.6 for female). Discussion The observed differences between these GW cohorts in these settings might be explained by the differences in the VAS scores of the general populations behind them, which are 82.5, 78.7 and 80.1 for the UK, Canada and China, respectively. Based on results from other pub- lished works [9,15,17], it is not surprising to see that female GW patients had lower VAS scores than male patients. Although a VAS score declining with age was observed in a general population-based survey [15], our age curve of VAS scores shows a flat pattern, which is consistent with a prior UK GW study [9]. A potential reason for this inconsistency is that the majority of the GW patients were sexually active, and they might have a lower probability of susceptibility to a range of ageing diseases or situations. taking qualifying quality-adjusted life year (QALY) saved as the study outcome. The current analysis found that more than half of the GW patients in this survey were suffering anxiety and depression. This is a dramatically high proportion, when compared to the value of < 7% of the sampled general population (aged 15-49 years, by 5-year age group) across mainland China, as a part of the Chinese National Health Services Survey in 2008 (N = 120,703) [15]; and the proportion is also higher than that of some other countries (~ < 30%, pooled in a previous study by Wang et al) [17] and a group of GW patients in the UK (24%) [9]. This situation is due mainly to the relatively conservative culture and attitude to sex in China; being diagnosed with sexually transmitted dis- eases such as GW could be regarded as a big humilia- tion for patients and they usually would not let other people know and would not receive support even from their families. As expected, we also found that a high proportion of study cases were feeling pain and discom- fort (24.7%) when compared to the Chinese general population ( < 10%, in patients aged 15-49 years) [15]. Providing questionnaire interview to part of the patients after their treatment could increase the feeling of pain, but the current study did not distinguish the ordering of treatment and interview. However, discomfort is com- monly feeling in GW patients, which could explain some of this detriment. Discussion This study also determined the characteristics of patents with relatively lower quality of life, including being female, living in urban areas and suffering multiple GWs, which will be informative for future GW prevention and control efforts. Table 4 Mean VAS and EQ-5D index score, by population characteristics, using a multivariate linear regression approach Variable Variable definition Unstandardized Coefficients Standardized Coefficients t Sig. B SD Beta VAS mean Constant 63.721 1.455 43.795 0.000 Sex Woman = 0; Man = 1 7.451 1.176 0.168 6.335 0.000 Setting Rural = 0; Urban = 1 -6.459 1.361 -0.127 -4.747 0.000 Single/Multiple GW Multiple GW = 0; Single GW = 1 4.138 1.334 0.082 3.101 0.002 Region-Central Southwest = 0; Central = 1 8.653 2.042 0.117 4.237 0.000 Region-North Southwest = 0; North = 1 3.655 1.341 0.074 2.725 0.007 Region-East Southwest = 0; East = 1 0.032 0.010 0.108 3.145 0.002 EQ-5D index score* Constant 0.748 0.012 61.166 0.000 Sex Woman = 0; Man = 1 0.035 0.007 0.134 5.140 0.000 Setting Rural = 0; Urban = 1 -0.032 0.008 -0.106 -3.933 0.000 Single/Multiple GW Multiple GW = 0; Single GW = 1 0.029 0.008 0.098 3.749 0.000 Region-Central Southwest = 0; Central = 1 0.067 0.013 0.153 5.332 0.000 Region-North Southwest = 0; North = 1 0.069 0.009 0.238 7.813 0.000 Region-East Southwest = 0; East = 1 0.038 0.009 0.127 4.188 0.000 Region-Northeast Southwest = 0; Northeast = 1 0.087 0.013 0.196 6.905 0.000 Clinical status- Recurrent GW Recurrent GW = 0; Initially occurred GW = 1 0.031 0.008 0.103 3.968 0.000 Monthly income (Chinese Yuan) < 1000 = 1; 1000 ~ = 2; 2000 ~ = 3; 3000 ~ = 4 0.012 0.003 0.107 4.040 0.000 * Japanese preference weights applied. D index score, by population characteristics, using a multivariate linear regression Table 4 Mean VAS and EQ-5D index score, by population characteristics, using a multivariate line approach Shi et al. BMC Public Health 2012, 12:153 http://www.biomedcentral.com/1471-2458/12/153 Page 7 of 9 Page 7 of 9 using EQ-5D in other countries, the mean VAS scores of the current Chinese GW cohort are lower than the estimate of an Australian study (68.9, N = 40) and of a UK study (72, N = 81) [6,9], but somewhat closer to the estimate of a Canadian study (65.1, N = 39) [11]. Acknowledgements We wish to thank all members of the Chinese genital warts study group from Cancer Institute of Chinese Academy of Medical Sciences, Cancer Foundation of China and the 18 collaborative hospitals/universities. We also thank all the subjects who participated in this study. Dr. Karen Canfell and Mr. Robert Walker (Cancer Council NSW, Australia) are gratefully acknowledged for comments and reviewing this manuscript. The study was unconditionally funded by MSD China. We wish to thank all members of the Chinese genital warts study group from Cancer Institute of Chinese Academy of Medical Sciences, Cancer Foundation of China and the 18 collaborative hospitals/universities. We also thank all the subjects who participated in this study. Dr. Karen Canfell and Mr. Robert Walker (Cancer Council NSW, Australia) are gratefully acknowledged for comments and reviewing this manuscript. The study was unconditionally funded by MSD China. This analysis has some limitations. Firstly, selection biases could occur in this study, due to the convenient sampling approach we used; we also failed to collect information of non-attended patients and thus could not assess the differences in characteristics between the study participants and those who declined to participate. Secondly, use of the generic EQ-5D instrument, which is not sensitive to HPV-related diseases, potentially underestimates the negative impact from GW and a ceiling effect could occur. Thirdly, the current study is a questionnaire interview-based survey which is usually sensitive to the capacities of the interviewers and quality control, and some of the clinical physicians who admini- strated the interview had not directly received training provided by CICAMS, and quality-control could poten- tially vary among study centers to some extent. Another limitation of the study is that we did not have Chinese population specific preference weights and thus could not calculate the EQ-5D index scores accurately. Furthermore, combined with further data related to average duration of a clinical episode and the frequency of recurrence of GW, the detailed utilities findings from the current study would be informative for future cost- effectiveness evaluations related to quality-adjusted life years saved by new interventions against GW. Author details 1 1Department of Cancer Epidemiology, Cancer Institute/Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, 17 South Panjiayuan LN, PO Box 2258, Beijing 100021, China. 2Cancer Epidemiology Research Unit, Cancer Council NSW, New South Wales, Australia. 3Sichuan Center for Disease Control and Prevention, Chengdu, China. 4Institute of Dermatology, Chinese Academy of Medical Sciences, Peking Union Medical College, Nanjing, China. 5202 Hospital of Chinese PLA, Shenyang, China. 6Beijing Ditan Hospital, Beijing, China. 7Beijing You’an Hospital, Beijing, China. 8Daping Hospital, Third Military Medical University of Chinese PLA, Chongqing, China. 9Tangdu Hospital, Fourth Military Medical University of Chinese PLA, Xi’an, China. 10Shenzhen Women and Children Hospital, Shenzhen, China. 11Changzhi Medical College, Changzhi, China. 12General Hospital of Tianjin Medical University, Tianjin, China. 13Hunan Provincial People’s Hospital, Changsha, China. 14Xinmi Women and Children Hospital, Xinmi, China. 15Medical School of Yichun University, Yichun, China. 16People’s Hospital of Xinjiang Uygur Autonomous Region, Urumchi, China. 17Hongkou District Bureau of Health, Shanghai, China. 18Fujian Provincial Hospital, Fuzhou, China. 19Women and Children Hospital of Guangzhou City, Guangzhou, China. 20Liaoning Cancer Hospital, Shenyang, China. 21The 2nd People’s Hospital of Yangcheng County Shanxi China , p g y, Guangzhou, China. 20Liaoning Cancer Hospital, Shenyang, China. 21The 2nd People’s Hospital of Yangcheng County, Shanxi, China. Discussion The finding that the mean VAS scores in the current study are lower than those of Chinese general population (65.2 versus ~80 [15]) suggests that the HRQoL of patients with GW is notably lower than that of the gen- eral population. When compared to prior GW studies In addition to the variable of gender, other character- istics could also potentially affect the HRQoL of GW patients. Our finding that the urban patients suffered a heavier physiological burden than rural patients could be explained by the urban residents’ higher stress from job and mortgage payments and living a faster-paced life. This finding is identical with the results of another HRQoL analysis of HPV-related lesions (including GW) using a HPV-sensitive instrument [5]. It is not surpris- ing to observe that subjects in Southwest China had the Page 8 of 9 Shi et al. BMC Public Health 2012, 12:153 http://www.biomedcentral.com/1471-2458/12/153 lowest score, because a prior survey (N = 2,830) has reported that the general population in Guizhou Pro- vince in the Southwest region of China had a relatively low HRQoL (VAS: ~68 - ~ 80 in residents aged 15-49 years) [18]. It is also understandable that more patients with multiple genital warts reported lower scores than patients with only single GW. Although most of the dif- ferences detected between subgroups were marginally less than the mean of clinically important differences in EQ-5D (0.074) [28], they were still within the range (0.011-0.140) [28], and could potentially generate a rela- tively significant impact on mass public health interven- tion programs. As for other variables marginally associated with the HRQoL of GW patients, including initial or recurrent GW, smoking, patient’s monthly income and education level, they are beyond the scope of the current discussion, and more research needs to be done. intervention on GW patients, effectively reducing patients’ anxiety and depression can have a remarkably positive impact on the overall quality of life of GW patients. In addition, particular attention should prob- ably be given to female patients and patients from urban areas and who are suffering worse clinical situations in clinical practice. Abbreviations CFC C F CFC: Cancer Foundation of China; CICAMS: Cancer Institute of Chinese Academy of Medical Sciences; EQ-5D: European quality of life-5 dimension; HRQoL: Health-related quality of life; GW: Genital warts; HPV: Human Papillomavirus; VAS: Visual analogue scale. Conclusions Received: 20 October 2011 Accepted: 2 March 2012 Published: 2 March 2012 Received: 20 October 2011 Accepted: 2 March 2012 Published: 2 March 2012 Received: 20 October 2011 Accepted: 2 March 2012 Published: 2 March 2012 The HRQoL of patients with GW was substantially low- ered when compared to the general population in main- land China, and the quality of life was even worse among some sub-groups. It is vital to strengthen the public genital warts prevention program. In the era of HPV vaccination, although prophylactic HPV vaccines are not yet licensed in China, introducing a prophylactic vaccine protecting against HPV-6 and -11 would be fea- sible. Regarding the psychological effect of such an Authors’ contributions YLQ was the Project Investigator and supervised the conduct of the study. YLQ and JFS contributed to the study design. DJK, SZQ, HYW, YCL, LJS, LL, YY, QL, XXF, LQZ, JL, XLL, YY, MN, ADX, JHL, XQ, LKL and XZW collected the field data. JFS performed all the data analyses with input and comments from YLQ. JFS wrote the first draft of the paper with input and comments from all other authors. All authors read and approved the final manuscript. Competing interests 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. Pre-publication history privately insured patients in the United States. Sex Transm Dis 2004, 31:748-752. privately insured patients in the United States. Sex Transm Dis 2004, 31:748-752. The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2458/12/153/prepub The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2458/12/153/prepub 4. O’Mahony C: Genital warts: current and future management options. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit 24. Brisson M, Van d V, De WP, Boily MC: The potential cost-effectiveness of prophylactic human papillomavirus vaccines in Canada. Vaccine 2007, 25:5399-5408. Pre-publication history 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: 25. Kim JJ, Goldie SJ: Health and economic implications of HPV vaccination in the United States. N Engl J Med 2008, 359:821-832. 26. Kulasingam SL, Benard S, Barnabas RV, Largeron N, Myers ER: Adding a quadrivalent human papillomavirus vaccine to the UK cervical cancer screening programme: A cost-effectiveness analysis. Cost Eff Resour Alloc 2008, 6:4. 27. Elbasha EH, Dasbach EJ, Insinga RP: Model for assessing human papillomavirus vaccination strategies. Emerg Infect Dis 2007, 13:28-41. 28. Sinnott PL, Joyce VR, Barnett PG: Preference Measurement in Economic Analysis VA Palo Alto, Health Economics Resource Center: Guidebook. Menlo Park CA; 2007.
https://openalex.org/W1973777988	https://www.scielo.br/j/pab/a/gWJcRSdQNLfm4ytkBPgrqTJ/?lang=pt&format=pdf	Portuguese	null	Desenvolvimento ponderal de bezerras desmamadas em pastejo de Brachiaria decumbens com suplementação protéica e energética	Pesquisa Agropecuária Brasileira	2,005	cc-by	4,914	Desenvolvimento ponderal de bezerras desmamadas em pastejo de Brachiaria decumbens com suplementação protéica e energética José Luiz Viana Coutinho Filho(1), Célio Luiz Justo(1) e Roberto Molinari Peres(1) (1)Agência Paulista de Tecnologia dos Agronegócios (APTA), Unidade de Pesquisa e Desenvolvimento, Rodovia Washington Luiz, Km 445 Caixa Postal 1013, CEP 14025-990 São José do Rio Preto, SP. E-mail: coutinho@aptaregional.sp.gov.br, celiojusto@aptaregional.sp.gov.br molinari@aptaregional.sp.gov.br Resumo – Foi avaliado o efeito da suplementação a pasto, com concentrados fornecidos durante o ano (perío- dos de seca e águas), sobre o desenvolvimento ponderal de bezerras desmamadas. Foram utilizadas 36 bezerras da raça Santa Gertrudis, com média inicial de idade de 290 dias, média de peso vivo de 194 kg, mantidas em pasto de Brachiaria decumbens, distribuídas em um delineamento inteiramente casualizado, com dois tratamentos e 18 repetições. Foi oferecida mistura mineral o ano todo, ou suplemento com maior nível de proteína degradável na seca, e não-degradável nas águas. Os resultados obtidos para a média diária de ganho de peso vivo indicaram uma diferença significativa entre os tratamentos. O ganho de peso vivo, durante o ano, para o lote testemunha, foi de 0,504 kg animal-1 dia-1 e, para o lote suplementado foi de 0,561 kg. No período de seca, o ganho de peso vivo foi significativamente maior para os animais suplementados e, no período das águas não ocorreu diferença. A suplementação a pasto não apresentou vantagens bioeconômicas para se recomendar o seu uso. Nos dois tratamentos as bezerras alcançaram média de peso limite, para primeira cobertura, com idade próxima a 14 meses. Termos para indexação: bezerras de corte, ganho de peso, pasto, proteína degradável, suplemento. Ponderal performances of calves grazing Brachiaria decumbens and supplemented with protein and energetic diets Abstract – The concentrate supplementation effect upon ponderal performance of calves during the year (dry and wet seasons) was evaluated. Thirty-six Santa Gertrudis calves averaging 194 kg of live weight, with 290 days of age, grazing Brachiaria decumbens pasture, were used in a complete randomized design. The following supplements have been added ad libitum, fed during the experimental period: mineral mix, supplement based on degradable protein, during dry season, and supplement based on non-degradable protein (escape protein), during wet season. Results on average daily gain demonstrated a significant difference between treatments. The average daily gains were 0.504 and 0.561 kg animal-1 day-1 for the non-supplemented group (control group, fed mineral mix only), and supplemented group, respectively. During dry season, supplemented calves showed significantly higher average daily gain, and during wet season there was no difference. Pasture supplementation did not present bioeconomic advantage, then its use could not be recommended. In both treatments, calves reached the average weight limit, for first mating, at nearly 14 months of age. Index terms: beef calves, weight gain, pasture, degradable protein, supplement. Pesq. agropec. bras., Brasília, v.40, n.8, p.817-823, ago. 2005 Material e Métodos degradável, no desempenho de ruminantes, Hoover & Miller (1992) afirmaram que a eficiência dos microrga- nismos do rúmen é máxima, quando a matéria seca ingerida contém entre 10% e 13% de proteína degradável. Poppi & Mc Lennan (1995) acreditam, tam- bém, que apesar de não ocorrer falta de proteína na época das chuvas, pode haver uma resposta positiva à suplementação protéica (proteína não degradável no rúmen). O experimento foi conduzido de 1998 a 1999, na Uni- dade de Pesquisa e Desenvolvimento de Mirassol, per- tencente ao Pólo Regional de Desenvolvimento Tecnológico dos Agronegócios do Centro Norte (APTA – Secretaria da Agricultura e Abastecimento do Estado de São Paulo), localizada na região Noroeste do Estado, a 20°48'S e 49°23'W, e a 468 m de altitude. Se- gundo Köppen, essa região apresenta clima do tipo Aw, com estação chuvosa no verão, seguida de tempo ame- no e seco no inverno. A precipitação média anual, nos últimos 11 anos, 1993 a 2003, foi de 1.468 mm. O solo é identificado como Podzólico Vermelho-Amarelo distrófico, com relevo levemente ondulado. Milton et al. (1997) relataram que o uso de proteína não degradável no rúmen para a terminação de novilhos alimentados com milho, tem pouco valor, provavelmen- te em função da deficiência no atendimento dos requisi- tos em proteína metabolizável, por intermédio da prote- ína do milho, que escapa da degradação e da proteína microbiana produzida. Foram avaliados dois tipos de suplementação: um só de mistura mineral (MM), e outra protéica e energética (SPE), tendo sido priorizado, na época seca, o nível de proteína degradável no rúmen (PDR) e, nas águas, o de proteína não-degradável (PNDR), num delineamento inteiramente casualizado, com 2 tratamentos e 18 repe- tições. Na seca, a fonte específica de PDR foi a uréia e, nas águas, a fonte específica de PNDR foi a farinha de peixe. A utilização de proteína não degradável no rúmen (PNDR), na alimentação de bovinos, no sentido de me- lhorar o desempenho tanto em condições de pasto na época das águas, como de confinamento, já vem sendo estudada há mais de uma década. Poppi & McLennan (1995) afirmaram que suplementos energéticos e su- plementos com altos níveis de PNDR seriam benéfi- cos, para incrementar o ganho de peso em condições tropicais. Introdução cam que essa gramínea não tem potencial para atender às exigências nutricionais, para uma produção máxima no decorrer do ano (Euclides et al., 1997), a qual ficaria dependente de uma suplementação alimentar. As condições climáticas, nos trópicos, promovem uma ampla variação anual da quantidade e qualidade da forrageira das pastagens, que é a principal causa das idades avançadas de abate e das elevadas idades da primeira cobertura de bovinos de corte. Minson (1990) afirmou que forragem de baixa quali- dade (PB<7%), que é usual na seca, limitaria a ativida- de dos microrganismos no rúmen e, conseqüentemente, afetaria a digestibilidade e consumo da mesma, o que implicaria pior desempenho animal. Entre os pesquisa- dores que estudaram a influência do nível de proteína No Brasil, as pastagens de Brachiaria decumbens são as principais responsáveis pela produção de bovi- nos de corte, entretanto, resultados de pesquisas indi- Pesq. agropec. bras., Brasília, v.40, n.8, p.817-823, ago. 2005 J.L.V. Coutinho Filho et al. 818 Pesq. agropec. bras., Brasília, v.40, n.8, p.817-823, ago. 2005 Desenvolvimento ponderal de bezerras desmamadas Consumo diário médio dos suplementos e dos nu- trientes, nas épocas de suplementação. Tabela 2. Consumo diário médio dos suplementos e dos nu- trientes, nas épocas de suplementação. Ingestões Seca Águas SPE(1) MM(2) SPE MM Suplemento (kg animal ) -1 0,969 0,071 0,731 0,074 % PV 0,435 - 0,265 - PB (g animal ) -1 375 - 255 - PDR (g animal -1) 310 - 154 - PNDR (g animal -1) 65 - 101 - NDT (g animal -1) 594 - 425 - Eficiência de ganho (2) 0,69 - 0,621 - (1)Suplemento protéico energético. (2)Mistura mineral. (3)g GPV g-1 su- plemento consumido. Os dados foram submetidos à análise de variância e, para as comparações entre as médias, utilizou-se o tes- te de Tukey a 5% de probabilidade. Desenvolvimento ponderal de bezerras desmamadas Desenvolvimento ponderal de bezerras desmamadas os meses de agosto (107 mm) e outubro (219 mm) fo- ram atípicos, propiciando, assim, uma condição de pasto satisfatória para a época. Foram utilizadas 36 bezerras desmamadas, da raça Santa Gertrudis, com aproximadamente 290 dias de idade e 194 kg de peso vivo, todas oriundas do mesmo reba- nho e do mesmo manejo, que permaneceram sob pastejo durante todo o período experimental. O consumo médio diário dos suplementos, de nutri- entes e da mistura mineral estão apresentados na Tabe- la 2, e os valores referentes ao peso vivo e ganho de peso, na Tabela 3. O consumo da mistura mineral co- mercial, pelos animais do lote que não recebeu suplementação, foi de 71 e 74 g animal-1 dia-1, para seca e águas, respectivamente, as quais podem ser conside- radas acima do esperado, em razão da categoria animal utilizada. Um pasto de Brachiaria decumbens foi dividido em dois piquetes de 8,5 ha cada, com a lotação inicial de aproximadamente uma UA ha-1. Cada piquete possuía um cocho de concreto coberto, com comprimento que disponibilizava espaço de 50 cm animal-1. Foi realizado um rodízio dos animais nos piquetes a cada 14 dias, para minimizar o efeito das pastagens. Os pastos foram ma- nejados, a fim de não permitir que a altura fosse reduzi- da a menos de 20 cm. No final do período das águas, anterior ao período experimental, foi realizado um ma- nejo para aumentar a disponibilidade de MS para a épo- ca seca, de 3.500 a 4.000 kg ha-1. Não foi realizada análise estatística do consumo, pois, os animais foram alimentados em grupo. Considerando-se o ganho de peso médio diário, nos dois períodos avaliados, a diferença de 57 g a favor dos suplementados foi significativa; mas a diferença entre os pesos finais, 302 kg e 290 kg, não qualifica a suplementação em termos de resultados biológicos e econômicos no sistema de produção, com vistas à melhoria no desempenho ponderal e reprodutivo. Os animais foram pesados no início do experimen- to, no mês de julho, e a cada 28 dias, após jejum total de 18 horas. Nos dias de pesagens, eram retiradas as sobras do suplemento e da mistura mineral para o cál- culo do consumo médio nos períodos. Foram realizadas observações parciais a respeito do hábito de consumo do suplemento durante alguns períodos de pastejo. Tabela 2. Material e Métodos A mistura mineral oferecida foi comercial, recomen- dada para a categoria animal utilizada no experimento, com a seguinte composição por kg do produto: 126 g de Na, 194 g de Cl, 88 g de P, 120 g de Ca, 10,5 g de Mg, 1,8 g de Fe, 4 g de Zn, 1,5 g de Cu, 1,3 g de Mn, 0,055 g de Co, 0,075 g de I, 0,015 g de Se. Os suplementos ofe- recidos, nas duas épocas do ano, estão descritos na Ta- bela 1. Eles foram formulados de modo que sua compo- sição parcial, na época da seca, fosse 43% de proteína bruta (PB) (83,4% de PDR) e 68% de nutrientes digestíveis totais (NDT) e, nas águas, 39,5% de PB (43% de PNDR) e 65,7% de NDT. Os suplementos foram oferecidos ad libitum; por intermédio de ingredientes reguladores, previa-se um consumo de suplemento protéico e energético de aproximadamente 800 g animal-1 dia-1. De maneira semelhante à proteína, o fornecimento de um suplemento energético para ruminantes em pastejo, segundo Caton & Dhuyvetter (1997), geralmente melhora o ganho de peso vivo e a condição corporal (score), mas, na maioria das vezes, diminui a ingestão de forragem e a digestibilidade; baixos níveis de suplementação energética podem aumentar os valores desses parâmetros. Paulino et al. (1982) afirmaram que, de um modo geral, uma suplementação protéica teria condições de satisfazer as deficiências de proteína e de energia dos bovinos mantidos em pastagens, mas que a suplementação energética não supriria os requisitos protéicos. Tabela 1. Composição (%) dos suplementos. Tabela 1. Composição (%) dos suplementos. A suplementação de bovinos a pasto tem apresen- tado resultados variáveis, e requer, portanto, mais dados. O objetivo deste trabalho foi avaliar o manejo da suplementação protéica e energética para animais em condições de pasto, durante os períodos de seca e águas, em relação ao desenvolvimento de bezerras de corte. Pesq. agropec. bras., Brasília, v.40, n.8, p.817-823, ago. 2005 Desenvolvimento ponderal de bezerras desmamadas 819 Pesq. agropec. bras., Brasília, v.40, n.8, p.817-823, ago. 2005 Resultados e Discussão A precipitação pluviométrica no decorrer do experi- mento foi de 431 mm no período de seca, julho a outu- bro, e 1.178 mm no período de águas, novembro a fe- vereiro. Pode-se considerar que a seca foi amena, pois (1)Suplemento protéico energético. (2)Mistura mineral. (3)g GPV g-1 su- plemento consumido. Tabela 3. Peso vivo médio e ganho em peso diário, de bezerras desmamadas da raça Santa Gertrudis, suplementadas e não- suplementadas, nas épocas de seca e de águas. Tabela 3. Peso vivo médio e ganho em peso diário, de bezerras desmamadas da raça Santa Gertrudis, suplementadas e não- suplementadas, nas épocas de seca e de águas. ho em peso diário, de bezerras desmamadas da raça Santa Gertrudis, suplementadas e não- d á Tabela 3. Peso vivo médio e ganho em peso diário, de bezerras desmamadas da raça Santa Gertrudis, suplementadas e não- suplementadas, nas épocas de seca e de águas. (¹)CS: com suplemento. (²)SS: sem suplemento.(3)Médias seguidas de * e ** na mesma linha, diferem pelo teste de Tukey, respectivamente a 5% e 1% de probabilidade. (¹)CS: com suplemento. (²)SS: sem suplemento.(3)Médias seguidas de * e ** na mesma linha, diferem pelo teste de Tukey, respectivamente a 5% e 1% de probabilidade. Pesq. agropec. bras., Brasília, v.40, n.8, p.817-823, ago. 2005 820 J.L.V. Coutinho Filho et al. São poucos os trabalhos que abrangem os períodos de seca e águas, conjuntamente, com um mesmo grupo de animais. Na pesquisa de Thiago & Silva (2002) fo- ram avaliados 4 níveis de suplementação, 0, 500, 1000 e 2000 g dia-1, nos dois períodos do ano, mas com reagrupamento dos animais no início das águas. Os autores concluíram que o potencial das pastagens tropicais, para ganho de peso, pode ser melhorado por intermédio de manejo e suplementação alimentar, com melhor resultado na época de seca; o ganho compensa- tório seria uma possível explicação. No presente traba- lho não foi identificada essa característica, pois ao final da seca, a diferença média de peso a favor dos animais suplementados, era de aproximadamente 7 kg e, após o período das águas era de 12 kg. Resultados e Discussão Pela Tabela 2, pode-se observar que o nível de consumo do concentrado, em relação ao peso vivo (PV) dos animais, foi abaixo de 0,5%, o que, segundo Lalman & Gill citados por Grandini (2001), não implicaria efeito substitutivo; assim, a ingestão de grãos, na proporção de 0,25% a 0,5% do PV, não afetaria ou provocaria um pequeno aumento na ingestão de matéria seca e na digestibilidade da forra- gem, e poderia proporcionar um pequeno aumento no desempenho animal. o nível de precipitação registrado nessa época, e o outro seria, segundo Corsi (1993), a possibilidade de ter ocor- rido maior disponibilidade de forragem de melhor quali- dade no período seco, em relação ao chuvoso, aliada ao maior consumo de suplemento por animal, em relação ao PV observado no presente trabalho, 0,435% na seca, e 0,265% nas águas. Resultado semelhante foi obtido por Lourenço & Leme (1999): na seca, o GPV dos animais suplementados o ano todo foi de 719 g animal-1 dia-1 e, nas águas, foi de 592 g; os autores justificam o fato pe- las condições excepcionais de precipitação na época de seca e, também, pela adubação nitrogenada, realizada no final das águas. O ganho do peso médio, durante o ano, para os animais suplementados, foi de 635 g animal-1 dia-1, em função do consumo individual de suplemento de 756 g dia-1 (seca) e de 944 g dia-1 (águas). Esse ganho é superior ao obtido no presente trabalho, que foi de 561 g animal-1 dia-1. Apesar dos consumos médios diários de suplementos, durante o ano, terem sido iguais, 1.700 g, os consumos nas épocas foram invertidos, isto é, na seca foi de 969 g dia-1 e nas águas 731 g dia-1. Comparando-se as médias anuais de ganho de peso diário, entre os tratamentos, esses autores verificaram que os animais suplementados ganharam 239 g dia-1 a mais, do que os que consumiram apenas mistura mine- ral, resultado superior ao observado no presente traba- lho, onde a diferença foi de apenas 57 g dia-1, apesar dos níveis de PB e NDT dos suplementos serem seme- lhantes, aproximadamente 45% e 67,8% na seca, e 38% e 67% nas águas. No trabalho de Thiago & Silva (2002), realizado em uma pastagem de Brachiaria brizantha cv. Pesq. agropec. bras., Brasília, v.40, n.8, p.817-823, ago. 2005 Desenvolvimento ponderal de bezerras desmamadas Desenvolvimento ponderal de bezerras desmamadas 821 ma espécie forrageira, com disponibilidade de 6.800 kg ha-1, mas com ingestão de suplemento maior (0,3% e 0,6% do PV). aproximadamente 295 g a mais. Tal quantidade seria suficiente, segundo o NRC, 1984, para satisfazer as exigências de proteína, para elevar o GPV de uma be- zerra com 200 kg, de 200 g para 1 kg dia-1, desde que fosse satisfeita, também, a exigência de energia. Os autores concluem que o fornecimento de sal proteinado, NNP + proteína verdadeira, promove um significativo aumento no GPV, quando comparado com o fornecimento de mistura mineral exclusivo ou com uréia. O trabalho de Milton et al. (1997) conclui que, em alguns casos, a suplementação com PNDR não im- plica melhores desempenhos, e a justificativa seria a exigência de proteína metabolizável ser atendida pela somatória da proteína microbiana e da proteína de um alimento, por exemplo o milho, que escapa da degrada- ção ruminal. Tal afirmativa está relacionada com o pior desempenho obtido pelos animais, quando alimentados com altos níveis de concentrado e PNDR oriunda de farelo de glúten de milho e farinha de sangue. Essas ponderações estão de acordo com outros pesquisado- res como Sindt et al. (1993). Detmann et al. (2001) con- cluíram que a suplementação de animais, em condições de pasto, com alta disponibilidade de forragem e, por- tanto, com possibilidade de realizar um pastejo altamen- te seletivo, causaria diminuição da ingestão de matéria seca total (IMS) (21,2%), da ingestão de forragem (33,6%) e, também, da digestibilidade de alguns nutri- entes. No presente trabalho, estimou-se que o suplemento utilizado, na época das águas, tinha 39% de PB (16,50% de PNDR e 22,50% de PDR), o que proporcionou um GPV de 454 g animal-1 dia-1 e correspondeu a um ganho adicional, em relação ao grupo testemunha, de 48 g diárias; o animal ingeriu, via suplemento, 257 g de PB e 109 g de PNDR. Nessa linha de trabalho, Manella et al. (2002) obtiveram 782 g animal-1 dia-1 de GPV, com um diferencial positivo de 137 g, em relação ao grupo-tes- temunha, consumo médio de PB igual a 336 g dia-1, e de PNDR de 124 g dia-1; os bezerros utilizados nesse ex- perimento tinham peso inicial de 152 kg, tendo perma- necido em pasto de braquiarão, com pastejo rotacionado e adubação nitrogenada. Uma avaliação da eficiência da utilização de suple- mentos, realizada por Manella et al. (¹)Ganho de peso vivo no período. (2)Ingestão dos suplementos no perí- odo. (3)Custos dos suplementos e da mistura mineral ingeridas. (4)Valor da arroba = R$ 58,00; rendimento de carcaça = 52%. Resultados e Discussão Marandu, com um ano de formação, o nível de ingestão de su- plemento de 1000 g dia-1, aproximadamente 0,5% do PV – que corresponde ao nível observado no presente tra- balho na época seca (0,435%) –, imprimiu um diferen- cial maior de GPV (215 g animal-1 dia-1), em relação aos 67 g animal-1 dia-1. Cavaguti et al. (2000) trabalharam somente no perío- do da seca e realizaram um experimento com estrutura semelhante, isto é, suplementação em pasto de Braquiaria decumbens, bezerras mestiças (europeu x zebu) recém desmamadas, lotação semelhante e rodízio de piquetes para reduzir efeito de pasto. Comparando-se o nível de ingestão de nutrientes (PB e NDT), observou-se, no presente trabalho, que os ani- mais consumiram, via suplemento, 168 g a mais de PB e 81 g a menos de NDT. Porém, a qualidade do marandu, com mais de 8% de PB e, aproximadamente 55% de NDT, na matéria seca foliar (folhas e talos finos ver- des), possivelmente contribuiu, de modo significativo, para o diferencial de GPV observado entre os dois tra- balhos. No tratamento controle, onde foi oferecida apenas mis- tura mineral completa, o GPV de 148 g animal-1 dia-1 foi bem abaixo do obtido no presente trabalho, ou seja, 454 g animal-1 dia-1 a menos, provavelmente explicado pela baixa precipitação ocorrida no período. Nas épocas avaliadas, ocorreram resultados diferenci- ados no ganho de peso vivo dos animais, na época seca a suplementação imprimiu uma diferença significativa entre os ganhos médios, 0,669 x 0,602 kg animal-1 dia-1, e esses ganhos foram maiores que os obtidos no período de águas, inclusive com diferença significativa entre épocas, 0,635 x 0,430 kg animal-1 dia-1. Dois fatores poderiam explicar esse maior ganho no período de seca, um seria No tratamento do presente trabalho, com 8% de uréia, mais farelo de soja e mistura mineral, com 45% de PB, o qual corresponde ao tratamento de suplementação na época de seca, a diferença no GPV diário continuou a ser alta, 427 g , e poderia estar relacionada com a dife- rença na quantidade de PB ingerida diariamente, isto é, Desenvolvimento ponderal de bezerras desmamadas Desenvolvimento ponderal de bezerras desmamadas (2002), apresentou um cálculo de eficiência alimentar (kg de GPV kg-1 de concentrado consumido) igual a 1, para suplementação no ano todo, a qual é superior ao obtido no presente trabalho, que foi 0,66. Paulino et al. (1996) consideraram a possibilidade dos bovinos, de responderem ao aumento do suprimento de proteína intestinal, via proteção com tanino. Mbongo et al. (1994) citados por Poppi & McLennan (1995) afirma- ram que são necessárias aproximadamente 150 g de proteína extra, que sobrepassam para o intestino (PNDR), no sentido de se obter um acréscimo no ganho de peso 300 g dia-1 animal-1, para bezerros de 200 kg, acrésci- mo este bem superior aos citados, anteriormente, em nosso trabalho, e por Manella et al. (2002), de 48 g e 137 g, apesar de atenderem 72,7% e 82,7%, respecti- vamente, da exigência extra de proteína. Na Tabela 4, é apresentada uma avaliação econômi- ca do presente experimento, que avalia o valor da ven- da dos animais para abate, e os períodos das secas e águas, distintamente. Observa-se que não houve vanta- gem econômica com a utilização dos suplementos, quan- do comparamos esse tratamento com a utilização de apenas uma mistura mineral comercial, de boa qualida- de e própria para a categoria de animais em questão. Deve-se considerar, nessa discussão, que o período das Quanto à suplementação energética (milho) ou energético/protéica (farelo de algodão), na época das águas, com animais em pastejo de Brachiaria decumbens, Paulino et al. (2000) não observaram qual- quer efeito, quando avaliaram GPV e rendimento de carcaça; porém, a ingestão de matéria seca, via suple- mento, foi de aproximadamente 0,095% do PV, consi- derada de baixo nível, provavelmente em conseqüência de pastejo seletivo, permitido pela alta disponibilidade de forragem (9.950 kg ha-1), que foi determinante para o excelente desempenho dos animais (GPV = 1,2 kg animal-1 dia-1). Conclusão semelhante foi feita por Zervoudakis et al. (2001) que utilizaram a mes- Tabela 4. Avaliação econômica da utilização dos suplemen- tos, nos períodos seco e de águas, por animal. Pesq. agropec. bras., Brasília, v.40, n.8, p.817-823, ago. 2005 822 J.L.V. Coutinho Filho et al. secas não foi rigoroso, tendo chovido mais de 400 mm, e que a disponibilidade de matéria seca nos piquetes permitiu pastejo seletivo. Conclusão MILTON, C.T.; BRANDT JUNIOR, R.T.; TITGEMEYER, E.C.; KUHL, G.L. Effect of degradable and escape protein and roughage type on performance and carcass characteristics of finishing yearling steers. Journal of Animal Science, v.75, p.2834-2840, 1997. A suplementação a pasto, de bezerras desmamadas, na época das secas, com proteína degradável no rúmen, e nas águas, com proteína não-degradável no rúmen, não apresentou vantagens bioeconômicas que pudes- sem recomendar o seu uso. MINSON, D.J. Forage in ruminant nutrition. New York: Academic Press, 1990. 483p. NATIONAL RESEARCH COUNCIL (Washington, DC). Nutrient requirements of beef cattle. 6.ed. Washington: National Academy Press, 1984. 90p. Desenvolvimento ponderal de bezerras desmamadas Recria de bovinos Nelore em pastos de Brachiaria brizantha com suplementação protéica ou com acesso a banco de proteína de Leucaena lecocephala: bovinos Nelore em pastos de Brachiaria brizantha com suplementação protéica ou com acesso a banco de proteína de Leucaena lecocephala: desempenho animal. Revista Brasileira de Zootecnia, v.31, p.2274- 2282, 2002. desempenho animal. Revista Brasileira de Zootecnia, v.31, p.2274- 2282, 2002. Desenvolvimento ponderal de bezerras desmamadas O retorno econômico, em vir- tude da composição dos suplementos, principalmente pelo custo do farelo de soja, pode ser considerado baixo e bastante variável, de acordo com a disponibilidade e custo dos alimentos, e estratégia para aquisição dos mesmos. DETMANN, E.; PAULINO, M.F.; ZERVOUDAKIS, J.T.; VALADARES FILHO, S.C.; LANA, R.P.; QUEIROZ, D.S. Suplementação de novilhos mestiços durante a época das águas: parâmetros ingestivos e digestivos. Revista Brasileira de Zootecnia, v.30, p.1340-1349, 2001. DETMANN, E.; PAULINO, M.F.; ZERVOUDAKIS, J.T.; VALADARES FILHO, S.C.; LANA, R.P.; QUEIROZ, D.S. EUCLIDES, V.P.B.; EUCLIDES FILHO, K.; ARRUDA, Z.J.; FIGUEIREDO, G.R. Alternativas de suplementação para redução da idade de abate de bovinos em pastagem de Brachiaria decumbens. Campo Grande: Embrapa-CNPGC, 1997. 25p. (Circular Técnica, 25). Pode-se considerar numa avaliação econômica, a antecipação da estação de monta, por causa do melhor desempenho ponderal das fêmeas, em função da suplementação. Nesse sentido, o ANUALPEC (1997) relata que, considerando-se a vida inteira reprodutiva de uma fêmea, o resultado da antecipação da primeira parição, para 24 meses de idade, implica um ganho de 0,7 bezerro, além da diminuição na idade média do re- banho, a diminuição do tempo improdutivo das fêmeas e o maior descarte, em um rebanho estabilizado. GRANDINI, D.V. Produção de bovinos a pasto com suplementos protéicos e/ou energéticos. In: MATTOS, W.R.S.; PIRES, A.V.; HADDAD, C.M.; NUSSIO, L.G. A produção animal na visão dos brasileiros. Piracicaba: Fealq; Sociedade Brasileira de Zootecnia, 2001. p.235-245. HOOVER, W.H.; MILLER, T.K. Rumen digestive physiology and microbial biology. Virginia: West Virginia University, 1992. 36p. (Agric. Exp. Sta. Bull, 708T). LOURENÇO, A.J.; LEME, P.R. Desempenho animal em pastagens de Brachiaria brizantha associado a banco de proteína ou LOURENÇO, A.J.; LEME, P.R. Desempenho animal em pastagens de Brachiaria brizantha associado a banco de proteína ou suplementação alimentar. In: REUNIÃO DA SOCIEDADE BRASILEIRA DE ZOOTECNIA, 36., 1999, Porto Alegre. Anais. Porto Alegre: Sociedade Brasileira de Zootecnia; Gmosis, [1999]. 1 CD-ROM. No presente trabalho, tanto o lote de fêmeas suplementadas como o de não-suplementadas, alcança- ram os pesos limites para primeira cobertura no mês de dezembro, com idade aproximada de 14 meses, o que possibilitaria que parissem com a idade próxima de 24 meses. MANELLA, M.Q.; LOURENÇO, A.J.; LEME, P.R. Recria de bovinos Nelore em pastos de Brachiaria brizantha com suplementação protéica ou com acesso a banco de proteína de Leucaena lecocephala: desempenho animal. Revista Brasileira de Zootecnia, v.31, p.2274- 2282, 2002. MANELLA, M.Q.; LOURENÇO, A.J.; LEME, P.R. Pesq. agropec. bras., Brasília, v.40, n.8, p.817-823, ago. 2005 Referências PAULINO, M.F.; BORGES, L.E.; CARVALHO, P.P.; FREITAS, R.T.F. Fontes de proteína em suplementos múltiplos sobre o desenvolvimento de novilhos e novilhas mestiços em pastoreio durante a época das águas. In: REUNIÃO ANUAL DA SOCIEDADE BRASILEIRA DE ZOOTECNIA, 33., 1996, Fortaleza. Anais. Fortaleza: SBZ, 1996. v.1. p.12-14. PAULINO, M.F.; BORGES, L.E.; CARVALHO, P.P.; FREITAS, R.T.F. Fontes de proteína em suplementos múltiplos sobre o desenvolvimento de novilhos e novilhas mestiços em pastoreio Ã ANUALPEC 97. Anuário da pecuária brasileira. São Paulo: FNP Consultoria & Comércio, 1997. 329p. CATON, J.S.; DHUYVETTER, D.V. Influence of energy supplementation on grazing ruminants: requeriments and responses. Journal of Animal Science, v.75, p.533-542, 1997. BRASILEIRA DE ZOOTECNIA, 33., 1996, Fortaleza. Anais. Fortaleza: SBZ, 1996. v.1. p.12-14. CAVAGUTI, E.; ZANETI, M.A.; MORGULIS, S.C.F. Suplementação nitrogenada para bezerras de corte mantidas a pasto no período de estiagem. In: REUNIÃO ANUAL DA SOCIEDADE BRASILEIRA DE ZOOTECNIA, 37., 2000, Viçosa. Anais. Viçosa: Sociedade Brasileira de Zootecnia, 2000. 1 CD-ROM. CAVAGUTI, E.; ZANETI, M.A.; MORGULIS, S.C.F. Suplementação nitrogenada para bezerras de corte mantidas a pasto Ã PAULINO, M.F.; KABEYA, K.S.; VALADARES FILHO, S.C.; PEREIRA, O.G. 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(Ed.). Bovinocultura leiteira: fundamentos da exploração racional. 2.ed. Piracicaba: Fealq, 1993. p.399-411. PAULINO, M.F.; RECHFELD, O.A.M.; RUAS, J.R.M. Alguns aspectos da suplementação de bovinos de corte em regime de Pesq. agropec. bras., Brasília, v.40, n.8, p.817-823, ago. 2005 Desenvolvimento ponderal de bezerras desmamadas Desenvolvimento ponderal de bezerras desmamadas 823 pastagem durante a época seca. Informe Agropecuário, v.8, p.28- 31, 1982. pastagem durante a época seca. Informe Agropecuário, v.8, p.28- 31, 1982. Recebido em 5 de fevereiro de 2004 e aprovado em 22 de fevereiro de 2005 pastagem durante a época seca. Informe Agropecuário, v.8, p.28- 31, 1982. THIAGO, L.R.L.S.; SILVA, J.M. Suplementação a pasto: uma estratégia necessária para a produção do novilho precoce. In: DIA DE CAMPO SOBRE PARDO-SUÍÇO CORTE, 2002, Campo Grande. Anais eletrônicos. Campo Grande: Embrapa-CNPGC, 2002. Disponível em: <http://www.cnpgc.embrapa.br/eventos/2002/ dcpardo/index.html>. 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https://openalex.org/W2806562158	https://www.mdpi.com/2079-4991/8/6/398/pdf?version=1528343536	English	null	Pulse-Modulated Radio-Frequency Alternating-Current-Driven Atmospheric-Pressure Glow Discharge for Continuous-Flow Synthesis of Silver Nanoparticles and Evaluation of Their Cytotoxicity toward Human Melanoma Cells	Nanomaterials	2,018	cc-by	16,110	Received: 14 May 2018; Accepted: 30 May 2018; Published: 2 June 2018 Abstract: An innovative and environmentally friendly method for the synthesis of size-controlled silver nanoparticles (AgNPs) is presented. Pectin-stabilized AgNPs were synthesized in a plasma-reaction system in which pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) was operated in contact with a flowing liquid electrode. The use of pm-rf-APGD allows for better control of the size of AgNPs and their stability and monodispersity. AgNPs synthesized under defined operating conditions exhibited average sizes of 41.62 ± 12.08 nm and 10.38 ± 4.56 nm, as determined by dynamic light scattering and transmission electron microscopy (TEM), respectively. Energy-dispersive X-ray spectroscopy (EDS) confirmed that the nanoparticles were composed of metallic Ag. Furthermore, the ξ-potential of the AgNPs was shown to be −43.11 ± 0.96 mV, which will facilitate their application in biological systems. Between 70% and 90% of the cancerous cells of the human melanoma Hs 294T cell line underwent necrosis following treatment with the synthesized AgNPs. Furthermore, optical emission spectrometry (OES) identiﬁed reactive species, such as NO, NH, N2, O, and H, as pm-rf-APGD produced compounds that may be involved in the reduction of the Ag(I) ions. Keywords: cold atmospheric-pressure plasma; nanostructures; necrosis Anna Dzimitrowicz 1 ID , Aleksandra Bielawska-Pohl 2, George C. diCenzo 3 ID , Piotr Jamroz 1 ID , Jan Macioszczyk 4, Aleksandra Klimczak 2 and Pawel Pohl 1,* ID 1 Department of Analytical Chemistry and Chemical Metallurgy, Faculty of Chemistry, Wroclaw University of Science and Technology, Wybrzeze St. Wyspianskiego 27, 50-370 Wroclaw, Poland; anna.dzimitrowicz@pwr.edu.pl (A.D.); piotr.jamroz@pwr.edu.pl (P.J.) 2 Laboratory of Biology of Stem and Neoplastic Cells, Hirszfeld Institute of Immunology and Experimental Therapy Polish Academy of Science, R. Weigla 12, 53-114 Wroclaw, Poland; aleksandra.bielawska@iitd.pan.wroc.pl (A.B.-P.); klimczak@iitd.pan.wroc.pl (A.K.) 3 Department of Biology, University of Florence, via Madonna del Piano 6, 50017 Sesto Fiorentino, Italy; georgecolin.dicenzo@uniﬁ.it 4 Faculty of Microsystem Electronics and Photonics, Wroclaw University of Science and Technology, Wybrzeze St. Wyspianskiego 27, 50-370 Wroclaw, Poland; jan.macioszczyk@pwr.edu.pl 4 Faculty of Microsystem Electronics and Photonics, Wroclaw University of Science and Techn Wybrzeze St. Wyspianskiego 27, 50-370 Wroclaw, Poland; jan.macioszczyk@pwr.edu.pl y y y Wybrzeze St. Wyspianskiego 27, 50-370 Wroclaw, Poland; jan.macioszczyk@pwr.edu.pl * Correspondence: pawel.pohl@pwr.edu.pl; Tel.: +48-71-320-24-94 * Correspondence: pawel.pohl@pwr.edu.pl; Tel.: +48-71-320-24-94 nanomaterials nanomaterials nanomaterials Pulse-Modulated Radio-Frequency Alternating-Current-Driven Atmospheric-Pressure Glow Discharge for Continuous-Flow Synthesis of Silver Nanoparticles and Evaluation of Their Cytotoxicity toward Human Melanoma Cells Anna Dzimitrowicz 1 ID , Aleksandra Bielawska-Pohl 2, George C. diCenzo 3 ID , Piotr Jamroz 1 ID , Jan Macioszczyk 4, Aleksandra Klimczak 2 and Pawel Pohl 1,* ID www.mdpi.com/journal/nanomaterials 1. Introduction Since ancient times, the many special properties of metallic silver have been well known and widely utilized because of its antibacterial [1], conductive [2], and optical [3] applications. Special attention has been given to silver nanoparticles (AgNPs), as their high surface-area-to-volume ratio [4] increases their number of possible applications. Most commonly, AgNPs are utilized in surface-enhanced Raman spectroscopy [5] and metal-enhanced ﬂuorescence [6]. Furthermore, because of the excellent antibacterial activities of AgNPs, they are included in antibacterial clothing [7]. Nanomaterials 2018, 8, 398; doi:10.3390/nano8060398 www.mdpi.com/journal/nanomaterials www.mdpi.com/journal/nanomaterials 2 of 20 Nanomaterials 2018, 8, 398 One promising utilization of AgNPs, and nanoparticles in general, is their application in medicine, for example, serving as drug delivery vectors [8,9]. The antiproliferating and antitumor activities of AgNPs have been the focus of much scientific effort, having been reviewed by Banti et al. and Zhang et al. among others [10,11]. In particular, AgNPs have gained interest in the ﬁeld of nanomedicine because of their unique properties and their therapeutic potential in the treatment of at least some human cancers, with a particular focus on breast cancer [12,13]. Notably, a signiﬁcant difference in the effects of AgNPs on tumor and non-tumor cell lines has been observed [14], supporting their potential as a cancer therapy agent. The effects of AgNPs on cancer cells are dependent on the concentration [13] and size [15] of the AgNPs. For example, smaller AgNPs at a lower concentration induced apoptosis and necrosis in a human pancreatic ductal adenocarcinoma cell line, while larger AgNPs at higher concentrations induced autophagy in this cell line [15]. The cytotoxic and genotoxic effects of AgNPs against mammalian cells [16,17], as well as their antimicrobial properties [18], has led to increased demand for methods for the reproducible production of AgNPs with defined characteristics, such as size and shape. An intriguing technique for AgNP synthesis is the use of cold atmospheric-pressure plasmas (CAPPs) in direct interaction with AgNO3 solutions. The synthesis of AgNPs using CAPPs is a promising alternative to conventional “wet” chemical methods. It allows for the production of biocompatible AgNPs with higher monodispersity [19], without the use of potentially toxic reducing agents [20,21]. It additionally allows for the control of the optical and granulometric properties of the resulting nanomaterial, while minimizing the number of required steps and manipulations. 1. Introduction Among the different CAAPs applied for the production of Ag nanostructures directly in solutions, high-voltage direct-current-driven atmospheric-pressure glow discharge (dc-APGD) is the most widely used; however, the number of studies devoted to this topic is limited [22–33]. Considering experimental setups, stable dc-APGDs generated in contact with non-ﬂowing solutions have been sustained with the aid of Ar [23,29,31–33] or He [24,26–28,30] jets. Gases were introduced through Cu [29] or stainless steel [23–28,30–33] tubes or capillaries attached to the negative polarity outputs of high voltage direct current (HV-dc) power supplies. Solutions were positively biased by immersing graphite rods [24], Pt rods [29], or Pt foils [23,28,30–33] into them, to which the ground earth outputs of the supplies were attached, or through the use of another gaseous jet [27]. Generally, these systems use a stationary AgNO3 solution [20,23–27,30–34], and the reactors usually run for up to 30 min [31,32], resulting in the production of limited amounts of nanomaterials. However, a few continuous-ﬂow systems have also been developed that signiﬁcantly improve the production rate [19,22]. In the systems described above, the plasma generated in the gaps between gas-supporting capillaries and AgNO3 solutions is a rich source of high-energy electrons (eg−) that bombard the solution surface. After thermalization (energy loss through multibody interactions with water molecules) of the eg−from the gas phase, solvated electrons (eaq−) are formed in the liquid phase. Redox reactions mediated by the eaq−and other reactive species, such as hydrogen radicals (H), hydrogen peroxide (H2O2), singlet oxygen (O), nitrogen oxide (NO), and hydroxyls (OH), result in particle nucleation and the growth of AgNPs in the solution [22–30]. This process can lead to electrostatically stabilized AgNPs without the need for additional capping agents [22,30,33]. However, various stabilizers, such as pectin [19], dextran [29], fructose [30–32], sucrose [24], polyvinyl alcohol [23], polyacrylic acid [25], and sodium dodecyl sulfate [19,26,27], are often included to prevent undesirable agglomeration and precipitation of the Ag nanostructures. We are unaware of any studies making use of pulse-modulated radio-frequency alternating- current-driven atmospheric-pressure glow discharge (pm-rf-APGD) for the synthesis of nanomaterials. However, pm-rf-APGD presents many characteristics that make it an appealing alternative to dc-APGD for the synthesis of AgNPs. In pm-rf-APGD, there is a constant switching in the polarity of the electrodes, resulting in alternating injections of electrons (positive charge of the ﬂowing liquid electrode) and positive ions (negative charge of the ﬂowing liquid electrode). 2.1. Reagents and Solutions To obtain a 1000 mg·L−1 stock solution of Ag(I) ions, 0.3937 g of solid silver nitrate (AgNO3; Avantor Performance Materials, Gliwice, Poland) was dissolved in 250 mL of de-ionized water. The prepared stock solution was diluted 5, 2.86, or 2 times with de-ionized water, yielding ﬁnal Ag(I) ion concentrations of 200, 350, and 500 mg·L−1, respectively. Next, pectin (Agdia-Biofords, Evry Cedex, France), which is a biocompatible stabilizer, was added to aliquots of the working solutions to ﬁnal concentrations of 0%, 0.25%, or 0.50% (m/v). This non-toxic biopolymer was included with the aim to stabilize the synthesized Ag nanostructures as well as to prevent their aggregation and sedimentation. All of the reagents were of analytical grade or higher purity. De-ionized water was used in all experiments. 1. Introduction Both the electrons and positive ions contribute to the production of reactive oxygen and nitrogen species (RONS) responsible for 3 of 20 Nanomaterials 2018, 8, 398 the reduction of Ag(I) ions in the liquid phase. This characteristic may allow for the generation of AgNPs with improved monodispersity and long-term stability. By alternating between conditions convenient for reduction of the precursor of AgNPs (when the ﬂowing liquid electrode of pm-rf-APGD has positive polarity) and conditions permissive for AgNPs etching due to acidiﬁcation of the liquid (when the ﬂowing liquid electrode of pm-rf-APGD has negative polarity), pm-rf-APGD may allow for better control of the shape and size of the AgNPs [34]. Moreover, the use of pm-rf-APGD is expected to reduce the power consumption of the AgNPs synthesis procedure relative to the use of dc-APGD; when 30% or 70% duty cycles are used, the plasma is off for 70% or 30% of the time, respectively [34]. Here, the properties of pm-rf-APGD-mediated AgNPs are explored. The effects of ﬁve parameters of this novel plasma-reaction system on the wavelength of the maximum (λmax) of the localized surface plasmon resonance (LSPR) absorption band of the resultant AgNPs were examined using a response surface design. On the basis of the full quadratic response surface regression model, optimal conditions for the production of AgNPs of the largest and smallest sizes were selected, and the model was validated. Next, AgNPs produced under the conditions for the largest size were further characterized with respect to their optical and morphological properties and their cytotoxic activity toward human melanoma Hs 294T cells. Furthermore, the processes in the gas phase of the pm-rf-APGD were examined by optimal emission spectrometry (OES) in order to identify the reactive species possibly responsible for the synthesis of the Ag nanostructures. 2.3. Response Surface Model and Optimization of AgNP Synthesis UV/Vis absorption spectrophotometry to acquire the λmax va characteristically in the range of 400–750 nm for spherical AgNP To examine the effects of the operating parameters of the pm-rf-APGD plasma-reaction system on the location of λmax of the LSPR absorption band of the produced AgNPs, as well as to optimize this system so as to fabricate Ag nanostructures of a given size, the response surface methodology (RSM) was used to plan the experimental treatments and analyze the response surface. In this case, the Box–Behnken experimental design (BBD) was used, and the following ﬁve operating parameters were considered: the ﬂow rate of the ﬂowing liquid electrode solution (A) (mL·min−1), the precursor concentration in the ﬂowing liquid electrode solution (B) (mg·L−1), the stabilizer concentration (C) (% (m/v)), the frequency of pulse modulation of the rf current (D) (Hz), and the duty cycle (E) (%). The response surface design included 43 randomized experimental treatments at three different levels (−1, 0, and +1) of the operating parameters, counting three center points. The levels of parameters were arbitrarily selected and limited to the range in which stable operation of the pm-rf-APGD plasma-reaction system was obtained, that is, 3.0–6.0 mL·min−1 (A), 200–500 mg·L−1 of Ag(I) ions as AgNO3 (B), 0.00–0.50% (m/v) of pectin (C), 500–1500 Hz (D), and 30–70% (E). All experimental treatments were executed in one block within the response surface design matrix (with actual and coded values of the operating parameters) that is given in Table 1. 2.3. Response Surface Model and Optimization of AgNP Synthesis To examine the effects of the operating parameters of the pm-rf-APGD plasma-reaction system on the location of λmax of the LSPR absorption band of the produced AgNPs, as well as to optimize this system so as to fabricate Ag nanostructures of a given size, the response surface methodology (RSM) was used to plan the experimental treatments and analyze the response surface. In this case, the Box–Behnken experimental design (BBD) was used, and the following five operating parameters were considered: the flow rate of the flowing liquid electrode solution (A) (mL min−1), the precursor concentration in the flowing liquid electrode solution (B) (mg L−1), the stabilizer concentration (C) (% (m/v)), the frequency of pulse modulation of the rf current (D) (Hz), and the duty cycle (E) (%). The response surface design included 43 randomized experimental treatments at three different levels (−1, 0, and +1) of the operating parameters, counting three center points. 2.2. Plasma-Reaction System for the Continuous Synthesis of AgNPs Ag nanostructures were continuously produced in an innovative plasma-reaction system (Figure 1), in which APGD was generated between the surface of the ﬂowing liquid electrode and a pin-type tungsten electrode (outer diameter of 4.00 mm). The APGD was generated using the rf (50 kHz) voltage wave modulated at frequencies within 500–1500 Hz with duty cycles of 30–70%. The distance between the surface of the ﬂowing liquid electrode and the pin-type tungsten electrode was 4.00 mm. The rf voltage waveform was generated using a rf generator (Dora Electronics Equipment, Wroclaw, Poland). To charge the working solutions, a platinum wire was connected to the quartz-graphite capillary. Current and voltage measurements were made using a Rogowski coil and a voltage probe. The measured root-mean-square current was ~15 mA, while the root-mean-square voltage was 5 kV. The working solutions of the ﬂowing liquid electrode were introduced to the developed pm-rf-APGD plasma-reaction system by a four-channel peristaltic pump (Masterﬂex L\S, Cole-Parme, Vernon Hill, IL, USA), at ﬂow rates of 3.0–6.0 mL·min−1 via a quartz-graphite capillary (outer diameter of 6.00 mm). In the ﬂowing liquid electrode working solutions, the concentration of Ag(I) ions was between 200 and 500 mg·L−1, and the pectin concentration was within 0.00–0.50% (m/v). Flowing liquid electrode solutions containing Ag(I) ions of deﬁned concentrations (with or without pectin) were continuously introduced to the plasma-reaction system and treated by pm-rf-APGD. Plasma-treated solutions containing AgNPs were collected and subjected to analysis by UV/Vis absorption spectrophotometry 4 of 20 Nanomaterials 2018, 8, 398 to acquire the λmax value of the LSPR absorption band, characteristically in the range of 400–750 nm for spherical AgNPs [19,35]. Nanomaterials 2018, 8, x FOR PEER REVIEW 4 of 19 UV/Vi b i h i h λ l f h LSPR b i b d 2.3. Response Surface Model and Optimization of AgNP Synthesis UV/Vis absorption spectrophotometry to acquire the λmax va characteristically in the range of 400–750 nm for spherical AgNP The Box–Behnken response surface design with actual and coded values of operating parameters of the pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) plasma-reaction system for synthesis of silver nanoparticles (AgNPs), along with the randomized run order and the response, i.e., λmax of the localized surface plasmon resonance (LSPR) absorption band of AgNPs: (A) the ﬂow rate of the ﬂowing liquid electrode solution, (B) the precursor concentration, (C) the pectin concentration, (D) the frequency of pulse modulation of rf current, and (E) the duty cycle. 2.3. Response Surface Model and Optimization of AgNP Synthesis UV/Vis absorption spectrophotometry to acquire the λmax va characteristically in the range of 400–750 nm for spherical AgNP The levels of parameters were arbitrarily selected and limited to the range in which stable operation of the pm-rf-APGD plasma-reaction system was obtained, that is, 3.0–6.0 mL min−1 (A), 200–500 mg L−1 of Ag(I) ions as AgNO3 (B), 0.00–0.50% (m/v) of pectin (C), 500–1500 Hz (D), and 30–70% (E). All experimental treatments were executed in one block within the response surface design matrix (with actual and coded values of the operating parameters) that is given in Table 1. Figure 1. The scheme of the pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD)-based plasma-reaction system for the continuous production of silver nanoparticles (AgNPs). (A) Solution containing the AgNP precursor with or without pectin; (B) peristaltic pump; (C) quartz capillary; (D) graphite tube; (E) platinum wire; (F) pm-rf-APGD; (G) pin-type electrode; (H) quartz chamber; (I) radio-frequency (rf) voltage; (J) solution after pm-rf-APGD treatment containing AgNPs. Figure 1. The scheme of the pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD)-based plasma-reaction system for the continuous production of silver nanoparticles (AgNPs). (A) Solution containing the AgNP precursor with or without pectin; (B) peristaltic pump; (C) quartz capillary; (D) graphite tube; (E) platinum wire; (F) pm-rf-APGD; (G) pin-type electrode; (H) quartz chamber; (I) radio-frequency (rf) voltage; (J) solution after pm-rf-APGD treatment containing AgNPs. Figure 1. The scheme of the pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD)-based plasma-reaction system for the continuous production of silver nanoparticles (AgNPs). (A) Solution containing the AgNP precursor with or without pectin; (B) peristaltic pump; (C) quartz capillary; (D) graphite tube; (E) platinum wire; (F) pm-rf-APGD; (G) pin-type electrode; (H) quartz chamber; (I) radio-frequency (rf) voltage; (J) solution after pm-rf-APGD treatment containing AgNPs. Figure 1. The scheme of the pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD)-based plasma-reaction system for the continuous production of silver nanoparticles (AgNPs). (A) Solution containing the AgNP precursor with or without pectin; (B) peristaltic pump; (C) quartz capillary; (D) graphite tube; (E) platinum wire; (F) pm-rf-APGD; (G) pin-type electrode; (H) quartz chamber; (I) radio-frequency (rf) voltage; (J) solution after pm-rf-APGD treatment containing AgNPs. 5 of 20 Nanomaterials 2018, 8, 398 Table 1. 2.3. Response Surface Model and Optimization of AgNP Synthesis UV/Vis absorption spectrophotometry to acquire the λmax va characteristically in the range of 400–750 nm for spherical AgNP Run Order Actual (Coded) Levels of Operating Parameters Response λmax (nm) A (mL·min−1) B (mg·L−1) C (%(m/v)) D (Hz) E (%) 1 6.0 (+1) 350 (0) 0.25 (0) 1500 (+1) 50 (0) 401.9 2 4.5 (0) 350 (0) 0.25 (0) 500 (−1) 70 (+1) 404.0 3 3.0 (−1) 350 (0) 0.25 (0) 1500 (+1) 50 (0) 404.0 4 3.0 (−1) 350 (0) 0.25 (0) 500 (−1) 50 (0) 404.0 5 6.0 (+1) 350 (0) 0.00 (−1) 1000 (0) 50 (0) 406.1 6 a 4.5 (0) 350 (0) 0.25 (0) 1000 (0) 50 (0) 405.1 7 4.5 (0) 500 (+1) 0.00 (−1) 1000 (0) 50 (0) 402.5 8 4.5 (0) 200 (−1) 0.25 (0) 1500 (+1) 50 (0) 400.3 9 4.5 (0) 500 (+1) 0.25 (0) 500 (−1) 50 (0) 415.4 10 4.5 (0) 500 (+1) 0.50 (+1) 1000 (0) 50 (0) 411.8 11 4.5 (0) 350 (0) 0.50 (+1) 1500 (+1) 50 (0) 399.7 12 4.5 (0) 200 (−1) 0.25 (0) 500 (−1) 50 (0) 397.2 13 4.5 (0) 350 (0) 0.50 (+1) 1000 (0) 30 (−1) 394.7 14 4.5 (0) 350 (0) 0.00 (−1) 1000 (0) 30 (−1) 399.3 15 3.0 (−1) 350 (0) 0.50 (+1) 1000 (0) 50 (0) 409.0 16 6.0 (+1) 350 (0) 0.50 (+1) 1000 (0) 50 (0) 394.2 17 4.5 (0) 350 (0) 0.00 (−1) 1500 (+1) 50 (0) 400.8 18 3.0 (−1) 500 (+1) 0.25 (0) 1000 (0) 50 (0) 412.4 19 3.0 (−1) 350 (0) 0.25 (0) 1000 (0) 30 (−1) 404.0 20 4.5 (0) 350 (0) 0.50 (+1) 500 (−1) 50 (0) 401.0 21 4.5 (0) 350 (0) 0.00 (−1) 1000 (0) 70 (+1) 406.3 22 4.5 (0) 500 (+1) 0.25 (0) 1000 (0) 70 (+1) 415.9 23 6.0 (+1) 350 (0) 0.25 (0) 1000 (0) 70 (+1) 397.0 24 3.0 (−1) 350 (0) 0.25 (0) 1000 (0) 70 (+1) 403.1 25 4.5 (0) 200 (−1) 0.50 (+1) 1000 (0) 50 (0) 421.5 26 6.0 (+1) 350 (0) 0.25 (0) 500 (−1) 50 (0) 394.6 27 4.5 (0) 350 (0) 0.00 (−1) 500 (−1) 50 (0) 415.1 28 6.0 (+1) 500 (+1) 0.25 (0) 1000 (0) 50 (0) 411.4 29 4.5 (0) 350 (0) 0.25 (0) 1500 (+1) 30 (−1) 426.2 30 4.5 (0) 500 (+1) 0.25 (0) 1000 (0) 30 (−1) 407.7 31 6.0 (+1) 350 (0) 0.25 (0) 1000 (0) 30 (−1) 405.9 32 4.5 (0) 200 (−1) 0.25 (0) 1000 (0) 30 (−1) 384.3 33 3.0 (−1) 350 (0) 0.00 (−1) 1000 (0) 50 (0) 419.8 34 a 4.5 (0) 350 (0) 0.25 (0) 1000 (0) 50 (0) 397.5 35 4.5 (0) 350 (0) 0.25 (0) 500 (−1) 30 (−1) 392.5 36 4.5 (0) 200 (−1) 0.25 (0) 1000 (0) 70 (+1) 390.1 37 6.0 (+1) 200 (−1) 0.25 (0) 1000 (0) 50 (−1) 399.7 38 a 4.5 (0) 350 (0) 0.25 (0) 1000 (0) 50 (0) 395.3 39 4.5 (0) 350 (0) 0.50 (+1) 1000 (0) 70 (+1) 403.1 40 4.5 (0) 200 (−1) 0.00 (−1) 1000 (0) 50 (0) 403.5 41 4.5 (0) 500 (+1) 0.25 (0) 1500 (+1) 50 (0) 411.0 42 4.5 (0) 350 (0) 0.25 (0) 1500 (+1) 70 (+1) 406.2 43 3.0 (−1) 200 (−1) 0.25 (0) 1000 (0) 50 (0) 410.6 a Center point. 2.4. Characterization of the AgNPs Synthesized under Deﬁned Operating Conditions The optical properties of the Ag nanostructures were assessed using UV/Vis absorption spectrophotometry. The UV/Vis absorption spectra were acquired in the range from 260 to 1100 nm with a step of 0.1 nm, using a Specord 210 Plus (Analityk Jena, Jena, Germany) double-beam spectrophotometer. The UV/Vis absorption spectra were registered 1440 min after the pm-rf-APGD treatment. De-ionized water was used to zero the instrument. As the recorded UV/Vis absorption spectra were composed of more than a single band, the spectra were deconvoluted to resolve λmax of the LSPR absorption band. The acquired UV/Vis absorption spectra were resolved using OriginPro 8 software (OriginLab Corp., Northampton, MA, USA) and were ﬁtted by Gaussian functions, as was done by Calabrese et al. [36]. The size distribution by number and the polydispersity index of the synthesized Ag nanostructures (in reference to the mean hydrodynamic diameter) were estimated using a ZetaSizer Nano-ZS instrument (Malvern Instrument, Malvern, UK) with a detection angle of 173◦. The measurements were performed at 25 ◦C in optically homogenous polystyrene cuvettes. To reveal the surface charge of the AgNPs, the ξ-potential was measured with this instrument as well. To assess the results for the particle size distribution, polydispersity index, and ξ-potential, which represent the average of three measurements, Malvern Dispersion Technology Software (version 7.11) for ZetaSizer was applied. Next, the size and shape distributions, as well as the elemental composition and crystallographic structure, of the AgNPs produced under the optimal operating conditions were estimated via Tecnai G2 20 X-TWIN transmission electron microscopy (TEM) (FEI Co., Hillsboro, OR, USA) supported by energy-dispersive X-ray spectroscopy (EDS)(FEI Co., Hillsboro, OR, USA) and selected-area electron diffraction (SAED; AztecEnergy, Oxford Instrument, Abingdon, UK). One drop of proper solution was put onto a Cu grid (CF 400-Cu-UL, Electron Microscopy Sciences, Hatﬁeld, PA, USA) and evaporated to dry under infrared (IR) irradiation (95E, Philips Lighting, Pila, Poland). The size and shape distributions were assessed on the basis of the diameter of 60 single nanoparticles using FEI software (version 3.2, SP6 build 421, FEI Co., Hillsboro, OR, USA). 2.5. Optical Emission Spectrometry To identify reactive species generated in the gas phase of the pm-rf-APGD, OES measurements were performed. A Shamrock SR-500i (Andor, Belfast, UK) spectrometer with a Newton DU-920P-OE CCD camera (Andor, Belfast, UK) was used to resolve the radiation emitted by the pm-rf-APGD collimated onto the entrance slit (10 µm). 2.3. Response Surface Model and Optimization of AgNP Synthesis UV/Vis absorption spectrophotometry to acquire the λmax va characteristically in the range of 400–750 nm for spherical AgNP Actual (Coded) Levels of Operating Parameters To provide a good assessment of the curvature in the system, the acquired λmax values were modeled using a complete quadratic function, including the main and quadratic effects, and the two-way interactions. To lower the dimensionality of the model for easier interpretation, insigniﬁcant terms were eliminated using a forward-selection-of-terms algorithm. The suitability of the model was evaluated using an analysis of variance (ANOVA) test, on the basis of the p-values for each of the terms in the model as well as the values of the residual standard deviation (S) and the coefﬁcient of determination (R2). The model was also tested for lack-of-ﬁt, which provided evidence of the adequacy and efﬁcacy of the full quadratic regression model for approximation of the response surface. In such 6 of 20 Nanomaterials 2018, 8, 398 a case, the respective p-value for the lack-of-ﬁt test should ideally be high. Finally, the quality of the model and the reliability of its assumptions were checked by examining the residuals. To do this, scatter plots of the frequency distribution of the standardized residuals, as well as the standardized residuals versus ﬁtted values, were used for identifying outliers and/or non-contact variance. This provided a good way to look for severe non-normality or heteroscedasticity (unequal variation) in residuals in the model to ensure informed decisions about acceptance or rejection of the model. The response surface design was planned and analyzed using the Minitab 17 statistical software package (Minitab Ltd., Coventry, UK) for Windows 7 (32 bit). 2.9. Experimental Groups and Exposure Conditions In order to evaluate the effect of AgNPs on necrosis of the Hs 294T cell line, cells were treated with different concentrations of the reaction mixture compounds (for groups 2 and 3) or with the puriﬁed and non-puriﬁed Ag nanostructures (1, 5, 10, 50, and 100 µg·mL−1) for 24 h. As a control, culture medium was used for 24 h (group 1). The details of the experimental protocol are given in Table 2. Table 2. Experimental groups used in the in vitro tests. Table 2. Experimental groups used in the in vitro tests. Table 2. Experimental groups used in the in vitro tests. Serial No. Treatment/Compound Group 1 (Gr1) Cells treated with medium alone Group 2 (Gr2) Cells incubated with pectin at a concentration of 2500 mg·L−1 Group 3 (Gr3) Cells incubated with pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) activated water Group 4 (Gr4) Puriﬁed silver nanoparticles (AgNPs) solution Group 5 (Gr5) Solutions of Ag(I) ions and pectin before pm-rf-APGD treatment Group 6 (Gr6) Solutions of Ag(I) ions before pm-rf-APGD treatment Group 7 (Gr7) Non-puriﬁed AgNPs solution, containing unreacted Ag(I) ions N ti A 2.6. Puriﬁcation of AgNPs Dialysis was applied in order to purify the AgNPs from the pm-rf-APGD-treated reaction mixture, which also contained unreacted Ag(I) ions. The plasma-treated solution was transferred into a dialysis tube (molecular weight cut-off = 14,000 Da; Sigma-Aldrich, Poznan, Poland) and placed into 500 mL of de-ionized water. The dialysis was conducted for 24 h with magnetic stirring (WIGO, Pruszkow, Poland), as was done previously [19]. 7 of 20 Nanomaterials 2018, 8, 398 7 of 20 2.10. Necrotic Assay Necrosis was measured by ﬂow cytometry to assess the number of human cells incorporating propidium iodide compared to control cells treated only with culture medium alone. Brieﬂy, cells were cultured in medium containing fetal calf serum in 48-well plates and used in the experiments after reaching 80–90% conﬂuence. A deﬁned concentration of AgNPs was added to each well, and the cells were incubated for a further 24 h. A propidium iodide solution (1 µg·mL−1) was then added to each sample, and the dead cells were detected using ﬂow cytometry in a FL3 mode, i.e., red channel, λem = 620 nm. Data were analyzed using a FACSCalibur ﬂow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The percentage of necrotic target cells was calculated using the Flowing Software 2 program. All values are presented as the mean ± standard error of the mean of three independent experiments, each consisting of technical triplicates. The results were analyzed through Student’s t-tests using GraphPad Prism 5 software. The p-values for all investigated groups were calculated compared to the control group 1 (cells treated with medium alone). 2.7. Determining the Efﬁciency of AgNP Synthesis The yield of AgNPs following the pm-rf-APGD treatment was measured before and after dialysis. The yield was estimated by ﬂame atomic absorption spectrometry (FAAS) using a PerkinElmer 1100 B (Waltham, MA, USA), which is a single-beam ﬂame atomic absorption spectrometer. The FAAS measurements were carried out after the digestion of the AgNPs in a 65% (m/m) HNO3 solution (Avantor Performance Materials, Gliwice, Poland) at 100 ◦C for 30 min. 2.8. Cell Culture Conditions Hs 294T melanoma cells are of a continuous cell line established from metastatic melanoma isolated from a human lymph node. These cells were obtained from the American Type Culture Collection (ATCC HTB-140). The cell line was cultured as monolayers in high-glucose Dulbecco’s modified eagle medium (DMEM) culture medium supplemented with GlutaMAX-I, 10% heat-inactivated fetal bovine serum (BioMin Biotechnologia, Getzersdorf, Austria), 100 µg·mL−1 penicillin, and 100 µg·mL−1 streptomycin and was maintained in a cell culture incubator at 37 ◦C in 5% CO2. 2.9. Experimental Groups and Exposure Conditions 3.1. Response Surface Regression Model Outputs of analysis of variance (ANOVA) and the lack-of-ﬁt test for the response surface regression model established using the forward-selection-of-terms algorithm (α to enter = 0.25) for the pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) plasma-reaction system used for synthesis of silver nanoparticles (AgNPs) a. Table 3. Outputs of analysis of variance (ANOVA) and the lack-of-ﬁt test for the response surface regression model established using the forward-selection-of-terms algorithm (α to enter = 0.25) for the pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) plasma-reaction system used for synthesis of silver nanoparticles (AgNPs) a. Source of Data DF Adjusted SS Adjusted MS F-Value b p-Value Model 6 988.09 164.68 2.84 0.023 Linear 4 657.69 164.42 2.84 0.038 A 1 196.70 196.70 3.39 0.074 B 1 410.06 410.06 7.08 0.012 D 1 43.23 43.23 0.75 0.393 E 1 7.70 7.70 0.13 0.718 Square 1 82.33 82.33 1.42 0.241 E2 1 82.33 82.33 1.42 0.241 Two-way interactions 1 248.06 248.06 4.28 0.046 DE 1 248.06 248.06 4.28 0.046 Error 36 2086.15 57.95 - - Lack-of-ﬁt 34 2033.27 59.80 2.26 0.354 Pure error 2 52.88 26.44 - - Total 42 3074.24 - - - a DF: Degrees of freedom. SS: Sum of squares. MS: Mean of squares. A: The ﬂow rate of the ﬂowing liquid electrode solution. B: The precursor concentration. D: The frequency of pulse modulation of radio-frequency (rf) current. E: The duty cycle. b The value of the F-test for comparing model variance with residual (error) variance. a DF: Degrees of freedom. SS: Sum of squares. MS: Mean of squares. A: The ﬂow rate of the ﬂowing liquid electrode solution. B: The precursor concentration. D: The frequency of pulse modulation of radio-frequency (rf) current. E: The duty cycle. b The value of the F-test for comparing model variance with residual (error) variance. By using an α value of 0.25, it was possible to learn more about the effects of each of the entered factors on the response and on the terms already in the model [39,40]. The terms A (p = 0.074), B (p = 0.012), E2 (p = 0.241) and DE (p = 0.046) were statistically signiﬁcant in the model. Considering the hierarchy of the terms, the statistically insigniﬁcant terms D and E (p > 0.25) were also included in the model. 3.1. Response Surface Regression Model Nanostructures of different metals and sizes are able to absorb and reﬂect light of unique wavelengths, resulting in a LSPR absorption band centered around distinct wavelengths known as 8 of 20 Nanomaterials 2018, 8, 398 λmax [19]. In the case of spherical AgNPs, the LSPR absorption band typically occurs within the 400 to 750 nm range [14,19], with larger-sized AgNPs resulting in a red shift in the position of the λmax values. Therefore, to evaluate the effects of the operating parameters on the size of the synthesized AgNPs, 43 independent runs of AgNPs synthesis were performed with varying experimental conditions, and the position of λmax of the LSPR absorption band for each sample was recorded (Table 1). To evaluate the quality of the data, two scatter plots were prepared: (i) the range of the values of λmax measured under a given condition versus the mean of the λmax values, and (ii) the mean value of the λmax values versus the randomized run order. It was visually noted that the variability in the mean response between experimental treatments of the BBD was higher than the variability in the response within each treatment (for repeated measurements). Neither correlations nor trends in the scatter plots were observed. Thus, it was concluded that the variability in the results was associated with changes in the experimental conditions and that there was no need to stabilize the variance in the response through mathematical transformation [37,38]. To obtain the response surface, the values of λmax for the LSPR absorption band of the 43 AgNP solutions were approximated with a full quadratic polynomial model. The response surface regression model, developed with the aid of the forward-selection-of-terms algorithm, for the λmax values over the studied range of operating parameters was as follows (given in uncoded units): λmax = 341.8 −2.34A + 3.37 × 10−2B + 4.27 × 10−2D + 1.54E −7.16 × 10−3E2 −7.87 × 10−4DE. The accuracy of this regression model was tested by ANOVA and the lack-of-ﬁt test. The results of these analyses at α = 0.25 are given in Table 3. Table 3. Outputs of analysis of variance (ANOVA) and the lack-of-ﬁt test for the response surface regression model established using the forward-selection-of-terms algorithm (α to enter = 0.25) for the pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) plasma-reaction system used for synthesis of silver nanoparticles (AgNPs) a. Table 3. 3.1. Response Surface Regression Model The concentration of pectin in the ﬂowing liquid electrode solution, that is, term C, appeared to have no statistically signiﬁcant effect on the position of the λmax value of the LSPR absorption band of the synthesized AgNPs. The R2 value was 32.1%, which was relatively low but acceptable, as it is a measure of how close the data ﬁt the model and not a measure of the adequacy of the regression model. The p-value for the lack-of-ﬁt test was higher than 0.25 (p = 0.354) and was therefore statistically insigniﬁcant. The S-value was also relatively low, at 7.61. Therefore, on the basis of the mentioned statistics summarizing the regression model, it was concluded that there was no 9 of 20 e p- Nanomaterials 2018, 8, 398 synthesized AgNP of how close the d reason to reject the model nor evidence that it did not ﬁt the data. To ﬁnally check the goodness-of-ﬁt of the regression model, the residuals were examined by plotting the following: (i) the frequency distribution of the standardized residuals (Figure 2A), and (ii) the standardized residuals versus the ﬁtted values (Figure 2B). The distribution of the standardized residuals largely resembled a normal distribution with a mean of 0.015 and a standard deviation of 1.032 (n = 43). A random pattern of the residuals on both sides of zero was observed in the scatter plot of the standardized residuals versus the ﬁtted values, indicating that a correct polynomial function was used to model the response surface of the system. Except for two outliers (points 25 and 29), no unusual structures or patterns were observed in this scatter plot. These observations conﬁrmed the correctness of the model and the goodness-of-ﬁt of the empirical data with those established by the regression. The S-value was also relatively low, at 7.61. Therefore, on the basis of the mentioned statistics summarizing the regression model, it was concluded that there was no reason to reject the model nor evidence that it did not fit the data. To finally check the goodness-of-fit of the regression model, the residuals were examined by plotting the following: (i) the frequency distribution of the standardized residuals (Figure 2A), and (ii) the standardized residuals versus the fitted values (Figure 2B). The distribution of the standardized residuals largely resembled a normal distribution with a mean of 0.015 and a standard deviation of 1.032 (n = 43). 3.1. Response Surface Regression Model A random pattern of the residuals on both sides of zero was observed in the scatter plot of the standardized residuals versus the fitted values, indicating that a correct polynomial function was used to model the response surface of the system. Except for two outliers (points 25 and 29), no unusual structures or patterns were observed in this scatter plot. These observations confirmed the correctness of the model and the goodness-of-fit of the empirical data with those established by the regression. Figure 2. Frequency distribution graph of the standardized residuals (A) and a scatter plot of the standardized residuals versus the fitted values (B). h Eff f O Figure 2. Frequency distribution graph of the standardized residuals (A) and a scatter plot of the standardized residuals versus the ﬁtted values (B). Figure 2. Frequency distribution graph of the standardized residuals (A) and a scatter plot of the standardized residuals versus the fitted values (B). Figure 2. Frequency distribution graph of the standardized residuals (A) and a scatter plot of the standardized residuals versus the ﬁtted values (B). 3.2. The Effects of Operating Parameters The effects of the operating para 3.2. The Effects of Operating Parameters In addition, at higher Ag(I) concentrations, the ionic strength of the solution could 10 of 20 th o- Nanomaterials 2018, 8, 398 concentration of A the lo est λ alu destabilize the synthesized AgNPs [41]. Finally, increasing either the frequency of pulse modulation of the rf current or its duty cycle led to a gradual increase in the position of the λmax value of the LSPR absorption band. Hence, AgNPs of a greater particle size would be produced under these conditions. Both of these parameters directly affected the width of the pulses modulating the rf current in the on-and-off cycles and the duration of the interchanges of polarity set to the ﬂowing liquid electrode solution. At the lowest settings of these parameters (i.e., D: 500 Hz; E: 30%), the pulse width at a given modulation frequency will be the shortest, while the time spent in the off state of the cycle will be the longest. Considering that the solution was continuously replenished in the ﬂow-through system used, the above-mentioned conditions would have been responsible for the lowest production yield and the smallest size of the AgNPs. Particularly, the negative polarity of the ﬂowing liquid electrode solution and the bombardments of the solution surface with positive ions, for example, H2O+(g), could provide convenient conditions for the etching of AgNPs. This is because H2O molecules are ionized under these conditions (H2O+(g) + H2O = 2H2O+(aq) + eaq−), while the resultant H2O+(aq) ions recombine with water molecules, leading to the formation of H3O+(aq) ions and the acidiﬁcation of the solution (H2O+(aq) + H2O = H3O+(aq) + OH•(aq)) [37,38]. This would be a process competitive to the formation of AgNPs through the reduction of the Ag(I) ions; however, it appeared that the longest off time in the cycle was convenient for rearrangement of synthesized nanostructures, leading to improved control over their size. The effect of the stabilizer concentration (C) was established to be statistically insigniﬁcant. This could be due to electrostatic stabilization of AgNPs, that is, negative charging of the surface of the AgNPs by electrons “injected” from the discharge through the plasma–liquid interface. This explanation was also suggested by Patel et al. [42], who reported surfactant-free synthesis of AuNPs facilitated by dc-APGD operated between a He jet (acting as a cathode) and a non-ﬂowing solution of AuCl4−ions (acting as an anode). 3.2. The Effects of Operating Parameters The effects of the operating para 3.2. The Effects of Operating Parameters p g g [ ] g y, g Ag(I) ions, faster growth and further aggregation of AgNPs can take place as a result of the reduction of these ions on the surface of the AgNPs. This would make existing AgNPs larger rather then produce new particles in the solution. In addition, at higher Ag(I) concentrations, the ionic strength of the solution could destabilize the synthesized AgNPs [41]. Finally, increasing either the frequency of pulse modulation of the rf current or its duty cycle led to a gradual increase in the position of the λmax value of the LSPR absorption band. Hence, AgNPs of a greater particle size would be produced under these conditions. Both of these parameters directly affected the width of the pulses modulating the rf current in the on-and-off cycles and the duration of the interchanges of polarity set to the flowing liquid electrode solution. At the lowest settings of these parameters (i.e., D: 500 Hz; E: 30%), the pulse width at a given modulation frequency will be the shortest, while the time spent in the off state of the cycle will be the longest. Considering that the solution was continuously replenished in the flow-through system used, the above-mentioned conditions would have been responsible for the lowest production yield and the smallest size of the AgNPs. Particularly, the negative polarity of the flowing liquid electrode solution and the bombardments of the solution surface with positive ions, for example, H2O+(g), could provide convenient conditions for the etching of AgNPs. This is because H2O molecules are ionized under these conditions (H2O+(g) + H2O = 2H2O+(aq) + eaq−), while the resultant H2O+(aq) ions recombine with water molecules, leading to the formation of H3O+(aq) ions and the acidification of the solution (H2O+(aq) + H2O = H3O+(aq) + OH•(aq)) [37,38]. This would be a process competitive to the formation of AgNPs through the reduction of the Ag(I) ions; however, it appeared that the longest off time in the cycle was convenient for rearrangement of synthesized nanostructures, leading to improved control over their size. The effect of the stabilizer concentration (C) was established to be statistically insignificant. This could be due to electrostatic stabilization of AgNPs, that is, negative charging of the surface of the AgNPs by electrons “injected” from the discharge through the plasma–liquid interface. This explanation was also suggested by Patel et al. 3.2. The Effects of Operating Parameters The effects of the operating para 3.2. The Effects of Operating Parameters p g p p g ( A: the flow rate of the flowing liquid electrode solution; B: the precursor concentration in the flowing liquid electrode solution; D: the frequency of pulse modulation of the rf current; and E: the duty cycle) on the position of λmax for the LSPR absorption band of the synthesized AgNPs is shown in Figure 3. There was an inversely linear relationship between the flow rate of the flowing liquid electrode solution and the position of the λmax values; that is, a higher flow rate was associated with smaller AgNPs. As the flow rate increased, each milliliter of solution was exposed to the pm-rf-APGD for less time. Consequently, a smaller number of solvated electrons (eaq−) was likely available for The effects of the operating parameters included in the response surface regression model (i.e., A: the ﬂow rate of the ﬂowing liquid electrode solution; B: the precursor concentration in the ﬂowing liquid electrode solution; D: the frequency of pulse modulation of the rf current; and E: the duty cycle) on the position of λmax for the LSPR absorption band of the synthesized AgNPs is shown in Figure 3. There was an inversely linear relationship between the ﬂow rate of the ﬂowing liquid electrode solution and the position of the λmax values; that is, a higher ﬂow rate was associated with smaller AgNPs. As the ﬂow rate increased, each milliliter of solution was exposed to the pm-rf-APGD for less time. Consequently, a smaller number of solvated electrons (eaq−) was likely available for reduction of the Ag(I) ions in the plasma-treated solution; that is, Ag+ + eaq−= Ag0 [22–26,34]. Considering the effect of the precursor concentration in the ﬂowing liquid electrode solution, the lowest concentration of Ag(I) ions seemed to be preferable for obtaining small-sized AgNPs, that is, those with the lowest λmax value of their LSPR absorption band. This could be explained by the Finke–Watzky two-step mechanism of nucleation and growth of AgNPs [41]. Accordingly, at higher concentrations of Ag(I) ions, faster growth and further aggregation of AgNPs can take place as a result of the reduction of these ions on the surface of the AgNPs. This would make existing AgNPs larger rather then produce new particles in the solution. 3.3. Validation of the Response Surface Regression Model 3.3. Validation of the Response Surface Regression Model Graphical illustrations of the response surface regr Counter plots of the wavelength at maximum (λmax) of the localized surface plasmon resonance (LSPR) band of silver nanoparticles (AgNPs) synthesized using the pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) system for pairs of statistically signiﬁcant parameters in the developed response surface regression model (A–B, A–D, A–E, B–D, B–E, and D–E) at deﬁned hold values. A: the ﬂow rate of the ﬂowing liquid electrode (FLE) solution (mL·min−1); B: the precursor concentration in the FLE solution (mg·L−1); D: the frequency of pulse modulation of rf current (Hz); E: the duty cycle (%). Figure 4. Counter plots of the wavelength at maximum (λmax) of the localized surface plasmon resonance (LSPR) band of silver nanoparticles (AgNPs) synthesized using the pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) system for pairs of statistically significant parameters in the developed response surface regression model (A–B, A–D, A–E, B–D, B– E, and D–E) at defined hold values. A: the flow rate of the flowing liquid electrode (FLE) solution (mL min−1); B: the precursor concentration in the FLE solution (mg L−1); D: the frequency of pulse modulation of rf current (Hz); E: the duty cycle (%). Figure 4. Counter plots of the wavelength at maximum (λmax) of the localized surface plasmon resonance (LSPR) band of silver nanoparticles (AgNPs) synthesized using the pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) system for pairs of statistically signiﬁcant parameters in the developed response surface regression model (A–B, A–D, A–E, B–D, B–E, and D–E) at deﬁned hold values. A: the ﬂow rate of the ﬂowing liquid electrode (FLE) solution (mL·min−1); B: the precursor concentration in the FLE solution (mg·L−1); D: the frequency of pulse modulation of rf current (Hz); E: the duty cycle (%). According to the established regression model, the predicted value of λmax of the LSPR absorption band for condition 1 was 383.7 nm, with a standard error of fit of 5.7 nm and a 95% confidence interval of 372.2–395.3 nm. The pm-rf-APGD plasma-reaction system was run with the parameter settings of condition 1, the plasma-treated solutions were collected, and the positions of the λmax value of the LSPR absorption bands were determined (Figure 5). The mean value (n = 4) of the λmax values of the LSPR band was 374.6 ± 3.4 nm with a 95% confidence interval of 371.1–378.0 nm. 3.3. Validation of the Response Surface Regression Model 3.3. Validation of the Response Surface Regression Model Graphical illustrations of the response surface regr Graphical illustrations of the response surface regression model for the position of the λmax value for the LSPR absorption band of the synthesized AgNPs are given in Figure 4. These are given as contour plots of the λmax value for the following pairs of parameters at speciﬁc hold values: A–B, A–D, A–E, B–D, B–E, and D–E. To validate the developed regression model, two opposite sets of operating parameters were selected: condition 1, which led to the production of the smallest-size AgNPs (A: 6.0 mL·min−1; B: 200 mg·L−1; D: 500 Hz; E: 30%), and condition 2, which led to the production of the largest-size AgNPs (A: 3.0 mL·min−1; B: 500 mg·L−1; D: 1500 Hz; E: 30%). In both cases, pectin was included as a stabilizer in the ﬂowing liquid electrode solutions at a concentration of 0.25%. Graphical illustrations of the response surface regression model for the position of the λmax value for the LSPR absorption band of the synthesized AgNPs are given in Figure 4. These are given as contour plots of the λmax value for the following pairs of parameters at specific hold values: A–B, A– D, A–E, B–D, B–E, and D–E. To validate the developed regression model, two opposite sets of operating parameters were selected: condition 1, which led to the production of the smallest-size AgNPs (A: 6.0 mL·min−1; B: 200 mg·L−1; D: 500 Hz; E: 30%), and condition 2, which led to the production of the largest-size AgNPs (A: 3.0 mL·min−1; B: 500 mg·L−1; D: 1500 Hz; E: 30%). In both cases, pectin was included as a stabilizer in the flowing liquid electrode solutions at a concentration of 0.25%. Figure 4. Counter plots of the wavelength at maximum (λmax) of the localized surface plasmon resonance (LSPR) band of silver nanoparticles (AgNPs) synthesized using the pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) system for pairs of statistically significant parameters in the developed response surface regression model (A–B, A–D, A–E, B–D, B– E, and D–E) at defined hold values. A: the flow rate of the flowing liquid electrode (FLE) solution (mL min−1); B: the precursor concentration in the FLE solution (mg L−1); D: the frequency of pulse modulation of rf current (Hz); E: the duty cycle (%). Figure 4. 3.2. The Effects of Operating Parameters The effects of the operating para 3.2. The Effects of Operating Parameters [42], who reported surfactant-free synthesis of AuNPs facilitated by dc-APGD operated between a He jet (acting as a cathode) and a non-flowing solution of AuCl4−ions (acting as an anode) Figure 3. Graphical representation of the effects of the statistically significant parameters included in the response surface regression model on the wavelength at maximum (λmax) of the localized surface Figure 3. Graphical representation of the effects of the statistically signiﬁcant parameters included in the response surface regression model on the wavelength at maximum (λmax) of the localized surface plasmon resonance (LSPR) absorption band of the synthesized silver nanoparticles (AgNPs). Graphs demonstrate the effects of modifying variables when the operating conditions were otherwise optimal for the production of spherical AgNPs with the lowest (A) and highest (B) λmax. A: the ﬂow rate of the ﬂowing liquid electrode (FLE) solution (mL·min−1); B: the precursor concentration in the FLE solution (mg·L−1); D: the frequency of pulse modulation of radio-frequency (rf) current (Hz); E: the duty cycle (%). Figure 3. Graphical representation of the effects of the statistically significant parameters included in the response surface regression model on the wavelength at maximum (λmax) of the localized surface Figure 3. Graphical representation of the effects of the statistically signiﬁcant parameters included in the response surface regression model on the wavelength at maximum (λmax) of the localized surface plasmon resonance (LSPR) absorption band of the synthesized silver nanoparticles (AgNPs). Graphs demonstrate the effects of modifying variables when the operating conditions were otherwise optimal for the production of spherical AgNPs with the lowest (A) and highest (B) λmax. A: the ﬂow rate of the ﬂowing liquid electrode (FLE) solution (mL·min−1); B: the precursor concentration in the FLE solution (mg·L−1); D: the frequency of pulse modulation of radio-frequency (rf) current (Hz); E: the duty cycle (%). 11 of 20 11 of 20 Nanomaterials 2018, 8, 398 the duty cycle (%). 3.3. Validation of the Response Surface Regression Model 3.3. Validation of the Response Surface Regression Model Graphical illustrations of the response surface reg 3.3. Validation of the Response Surface Regression Model 3.3. Validation of the Response Surface Regression Model Graphical illustrations of the response surface regr UV/Vis absorption spectra of silver nanoparticles (AgNPs) synthesized with the aid of the pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) plasma- reaction under experimental conditions providing the smallest (condition 1) and the largest (condition 2) AgNPs according to the developed response surface regression model. Figure 5. UV/Vis absorption spectra of silver nanoparticles (AgNPs) synthesized with the aid of the pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) plasma-reaction under experimental conditions providing the smallest (condition 1) and the largest (condition 2) AgNPs according to the developed response surface regression model. Figure 5. UV/Vis absorption spectra of silver nanoparticles (AgNPs) synthesized with the aid of the pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) plasma- reaction under experimental conditions providing the smallest (condition 1) and the largest (condition 2) AgNPs according to the developed response surface regression model. Figure 5. UV/Vis absorption spectra of silver nanoparticles (AgNPs) synthesized with the aid of the pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) plasma-reaction under experimental conditions providing the smallest (condition 1) and the largest (condition 2) AgNPs according to the developed response surface regression model. As was done in previous work [42–44], the absorbance at λmax of the LSPR bands and their full width at half maximum (FWHM) values were recorded as measures of production yield and particle size distribution, respectively, of the AgNPs synthesized using conditions 1 and 2. Under condition 1, which was optimal for obtaining the smallest nanostructures, the absorbance was relatively low (0.21 ± 0.02), which was suggestive of a rather low production yield. The FWHM of the LSPR absorption band of these AgNPs was 106.8 ± 8.0 nm. Additionally, these AgNPs were stable for only 1 week, after which the color intensity of the solutions gradually decreased until they were colorless after 2 weeks. In the case of condition 2, which was optimal for the largest nanostructures, the measured absorbance was fairly high (1.16 ± 0.18), which was indicative of a much higher production yield than that using condition 1. Interestingly, the FWHM of the LSPR absorption bands for AgNPs produced under condition 2 (103.4 ± 6.2 nm) was very similar to that observed for AgNPs produced with condition 1. This suggested that a similar particle size distribution was achieved under both conditions, likely as a result of the electrostatic stabilization provided by the discharge as well as the presence of pectin. 3.3. Validation of the Response Surface Regression Model 3.3. Validation of the Response Surface Regression Model Graphical illustrations of the response surface regr The solutions of AgNPs produced using condition 2 remained stable even 6 weeks following plasma treatment, as the shape and absorbance of the LSPR absorption band did not change within this time period. Considering the production yields and the stabilities of the AgNPs synthesized under conditions 1 and 2, only the AgNPs produced under condition 2, optimal for production of the largest nanostructures, were further characterized. According to Mie’s scattering theory [45], a single intense LSPR absorption band, as was observed under condition 2, is expected in the UV/Vis absorption spectrum of monodisperse, spherical AgNPs. To finally prove that different reactive species are formed in the gas phase of the pm-rf-APGD As was done in previous work [42–44], the absorbance at λmax of the LSPR bands and their full width at half maximum (FWHM) values were recorded as measures of production yield and particle size distribution, respectively, of the AgNPs synthesized using conditions 1 and 2. Under condition 1, which was optimal for obtaining the smallest nanostructures, the absorbance was relatively low (0.21 ± 0.02), which was suggestive of a rather low production yield. The FWHM of the LSPR absorption band of these AgNPs was 106.8 ± 8.0 nm. Additionally, these AgNPs were stable for only 1 week, after which the color intensity of the solutions gradually decreased until they were colorless after 2 weeks. In the case of condition 2, which was optimal for the largest nanostructures, the measured absorbance was fairly high (1.16 ± 0.18), which was indicative of a much higher production yield than that using condition 1. Interestingly, the FWHM of the LSPR absorption bands for AgNPs produced under condition 2 (103.4 ± 6.2 nm) was very similar to that observed for AgNPs produced with condition 1. This suggested that a similar particle size distribution was achieved under both conditions, likely as a result of the electrostatic stabilization provided by the discharge as well as the presence of pectin. The solutions of AgNPs produced using condition 2 remained stable even 6 weeks following plasma treatment, as the shape and absorbance of the LSPR absorption band did not change within this time period. Considering the production yields and the stabilities of the AgNPs synthesized under conditions 1 and 2, only the AgNPs produced under condition 2, optimal for production of the largest nanostructures, were further characterized. 3.3. Validation of the Response Surface Regression Model 3.3. Validation of the Response Surface Regression Model Graphical illustrations of the response surface regr This corresponded well with the value predicted from the response surface regression model; the absolute error was 9.1 nm, which was lower than two standard errors of fit. Considering condition 2, the fitted value of λmax of the LSPR absorption band was 419.9 nm, with a standard error of fit of 5.7 nm and a 95% confidence interval of 408.3–431.5 nm. The plasma-reaction system was run with the parameters of condition 2, and the positions of the λmax values for the LSPR absorption band According to the established regression model, the predicted value of λmax of the LSPR absorption band for condition 1 was 383.7 nm, with a standard error of ﬁt of 5.7 nm and a 95% conﬁdence interval of 372.2–395.3 nm. The pm-rf-APGD plasma-reaction system was run with the parameter settings of condition 1, the plasma-treated solutions were collected, and the positions of the λmax value of the LSPR absorption bands were determined (Figure 5). The mean value (n = 4) of the λmax values of the LSPR band was 374.6 ± 3.4 nm with a 95% conﬁdence interval of 371.1–378.0 nm. This corresponded well with the value predicted from the response surface regression model; the absolute error was 9.1 nm, which was lower than two standard errors of ﬁt. Considering condition 2, the ﬁtted value of λmax of the LSPR absorption band was 419.9 nm, with a standard error of ﬁt of 5.7 nm and a 95% conﬁdence interval of 408.3–431.5 nm. The plasma-reaction system was run with the parameters of condition 2, and the positions of the λmax values for the LSPR absorption band of the synthesized AgNPs were determined (Figure 5). The mean (n = 4) λmax of the LSPR band was 415.8 ± 5.4 nm with a 95% conﬁdence interval of 410.4–421.2 nm. The absolute error between the predicted and empirical values of λmax was just 4.1 nm, which was lower than a single standard error of ﬁt. These tests thus conﬁrmed the usefulness of the developed response surface regression model for the control of the continuous-ﬂow, one-step production process of spherical AgNPs of a given size. 12 of 20 n model a given Nanomaterials 2018, 8, 398 for the control of the Figure 5. 3.4. Characterization of Synthesized AgNPs under Optimal Operating Conditions 3.4. Characterization of Synthesized AgNPs under Optimal Operating Conditions An aliquot of the AgNPs synthesized under condition 2 were purified by dialysis, and the purified and non-purified AgNPs were characterized by dynamic light scattering (DLS) to determine their average size by number, polydispersity index, and ξ-potential. The average sizes by number of the AgNPs were 32.78 ± 17.55 and 41.62 ± 12.08 nm, for the purified and non-purified AgNPs, respectively (Figure 6A,B). The similarity in sizes suggested that the purification process did not significantly influence the AgNPs population. Th l di iti f th ifi d d ifi d A NP d t i d t b 0 393 0 093 An aliquot of the AgNPs synthesized under condition 2 were puriﬁed by dialysis, and the puriﬁed and non-puriﬁed AgNPs were characterized by dynamic light scattering (DLS) to determine their average size by number, polydispersity index, and ξ-potential. The average sizes by number of the AgNPs were 32.78 ± 17.55 and 41.62 ± 12.08 nm, for the puriﬁed and non-puriﬁed AgNPs, respectively (Figure 6A,B). The similarity in sizes suggested that the puriﬁcation process did not signiﬁcantly inﬂuence the AgNPs population. The polydispersities of the purified and non-purified AgNPs were determined to be 0.393 ± 0.093 and 0.490 ± 0.050, respectively. The polydispersity index [48] is a measure of the width of the size distribution. When the polydispersity index value of a colloidal suspension is higher than 0.1, the dispersion exhibits polydispersity [49]. A polydispersity index value below 0.3 is favored for pharmaceutical AgNPs. Thus, the AgNPs synthesized by pm-rf-APGD exhibited moderate polydispersity. The higher polydispersity value of the non-purified AgNPs was associated with the presence of pectin, which can impact the Brownian motions and result in a higher polydispersity value being detected. The ξ-potential of the colloidal solutions was estimated in order to predict their stability, as it has been suggested that nanoparticles with negative or positive surface charges can overcome the van der Waals forces and avoid aggregation. The ξ-potentials of the purified and non- puriﬁed AgNPs were −37.8 ± 1.85 and −43.11 ± 0.96 mV, respectively. These results are supportive of these AgNPs being highly stable, consistent with the optical results reported above. Additionally, it The polydispersities of the puriﬁed and non-puriﬁed AgNPs were determined to be 0.393 ± 0.093 and 0.490 ± 0.050, respectively. The polydispersity index [48] is a measure of the width of the size distribution. 3.3. Validation of the Response Surface Regression Model 3.3. Validation of the Response Surface Regression Model Graphical illustrations of the response surface regr NH molecules, belonging to the A3Π-X3Σ system, were also present in the spectrum, as determined by the presence of the band heads at 336.0 (the transition 0–0) and at 337.0 13 of 20 system, rum as 13 of 20 system, rum as Nanomaterials 2018, 8, 398 nm (the transition 1– it d NH (the transition 1–1). Moreover, strong atomic Ag lines at 328.07 and 338.29 nm were identiﬁed, likely as a result of the formation of AgNPs in the liquid–plasma interfacial zone [46]. The NO and NH molecules were presumably produced as a result of the reaction of active nitrogen (N2 and N) with O and H radicals, respectively [47]. On the other hand, the main source of the OH radicals observed in the emission spectrum were likely dissociative processes of water (H2O) and its ions (e.g., H2O+ and H3O+), as was ﬁgured out on the basis of [37,38]. All reactive species, that is, NO, N2, OH, O, NH, N2+, and H, identiﬁed in the gas phase of pm-rf-APGD may certainly give rise to the formation of important active species in the liquid phase of the discharge that can be directly involved in the reduction of the Ag(I) ions to AgNPs. determined by the presence of the band heads at 336.0 (the transition 0 0) and at 337.0 (the transition 1–1). Moreover, strong atomic Ag lines at 328.07 and 338.29 nm were identified, likely as a result of the formation of AgNPs in the liquid–plasma interfacial zone [46]. The NO and NH molecules were presumably produced as a result of the reaction of active nitrogen (N2 and N) with O and H radicals, respectively [47]. On the other hand, the main source of the OH radicals observed in the emission spectrum were likely dissociative processes of water (H2O) and its ions (e.g., H2O+ and H3O+), as was figured out on the basis of [37,38]. All reactive species, that is, NO, N2, OH, O, NH, N2+, and H, identified in the gas phase of pm-rf-APGD may certainly give rise to the formation of important active species in the liquid phase of the discharge that can be directly involved in the reduction of the Ag(I) ions to AgNPs. Figure 6. The optical emission spectrometry (OES) spectrum of pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) generated under optimal operating conditions in the spectral range from 200 to 400 nm. Figure 6. 3.3. Validation of the Response Surface Regression Model 3.3. Validation of the Response Surface Regression Model Graphical illustrations of the response surface regr The optical emission spectrometry (OES) spectrum of pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) generated under optimal operating conditions in the spectral range from 200 to 400 nm. Figure 6. The optical emission spectrometry (OES) spectrum of pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) generated under optimal operating conditions in the spectral range from 200 to 400 nm. Figure 6. The optical emission spectrometry (OES) spectrum of pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) generated under optimal operating conditions in the spectral range from 200 to 400 nm. 3.3. Validation of the Response Surface Regression Model 3.3. Validation of the Response Surface Regression Model Graphical illustrations of the response surface regr According to Mie’s scattering theory [45], a single intense LSPR absorption band, as was observed under condition 2, is expected in the UV/Vis absorption spectrum of monodisperse, spherical AgNPs. To finally prove that different reactive species are formed in the gas phase of the pm rf APGD (as a result of alternating bombardments of the surface of the flowing liquid electrode with electrons and positive ions) and may contribute to the reduction of Ag(I) to AgNPs in the liquid phase as well as the formation of other species according to the reactions presented in the section 3.2., OES was used to acquire the emission spectra of this unique discharge system operating under condition 2. This spectrum is provided in Figure 6 and was recorded in the spectral range from 200 to 400 nm, in the near-liquid electrode zone. It was found that the UV region of the plasma-reaction system was dominated by the emission bands of N2 molecules (290–380 nm), which belong to the numerous transitions of the second positive system (C3Πu-B3Πg). Furthermore, bands of the NO γ-system (A2Σ+- To ﬁnally prove that different reactive species are formed in the gas phase of the pm-rf-APGD (as a result of alternating bombardments of the surface of the ﬂowing liquid electrode with electrons and positive ions) and may contribute to the reduction of Ag(I) to AgNPs in the liquid phase as well as the formation of other species according to the reactions presented in the Section 3.2., OES was used to acquire the emission spectra of this unique discharge system operating under condition 2. This spectrum is provided in Figure 6 and was recorded in the spectral range from 200 to 400 nm, in the near-liquid electrode zone. It was found that the UV region of the plasma-reaction system was dominated by the emission bands of N2 molecules (290–380 nm), which belong to the numerous transitions of the second positive system (C3Πu-B3Πg). Furthermore, bands of the NO γ-system (A2Σ+-X2Π) were observed in the 200–280 nm UV region. In addition, strong bands of OH molecules at 282.9 nm (the transition 1–1) and at 308.9 nm (the transition 0–0), which belong to the A2Σ+-X2Π system, were excited. 3.4. Characterization of Synthesized AgNPs under Optimal Operating Conditions 3.4. Characterization of Synthesized AgNPs under Optimal Operating Conditions When the polydispersity index value of a colloidal suspension is higher than 0.1, the dispersion exhibits polydispersity [49]. A polydispersity index value below 0.3 is favored for pharmaceutical AgNPs. Thus, the AgNPs synthesized by pm-rf-APGD exhibited moderate polydispersity. The higher polydispersity value of the non-puriﬁed AgNPs was associated with the presence of pectin, which can impact the Brownian motions and result in a higher polydispersity value being detected. The ξ-potential of the colloidal solutions was estimated in order to predict their stability, as it has been suggested that nanoparticles with negative or positive surface charges can overcome the van der Waals forces and avoid aggregation. The ξ-potentials of the puriﬁed and non-puriﬁed AgNPs were −37.8 ± 1.85 and −43.11 ± 0.96 mV, respectively. These results are supportive of these AgNPs being highly stable, consistent with the optical results reported above. Additionally, it is signiﬁcant that the AgNPs were negatively charged, as this can increase their ability to access tumor cells after injection into the circulatory system [50]. 14 of 20 19 14 of 20 f 19 Nanomaterials 2018, 8, 398 Nanomaterials 2018 8 The size and morphology of the non-puriﬁed AgNPs were further characterized using TEM. It was observed that the AgNPs produced using condition 2 were approximately spherical in shape and non-aggregated (Figure 7C,D). On the basis of the TEM measurements (Figure 7C,D), the average size of the AgNPs was 10.38 ± 4.56 nm. It was noted that on the basis of the TEM measurements, the AgNPs appeared to be uniform in size. The differences in the sizes of the AgNPs as measured by DLS and TEM were expected and could be explained by the principals of these techniques [51]. In the case of DLS, the average size is based on the metallic core of the nanoparticles as well as on any compounds connected to the core, such as a stabilizer [51], which in this case was pectin. In contrast, size estimation as performed by TEM only considers the metallic structures [51]. is significant that the AgNPs were negatively charged, as this can increase their ability to access tumor cells after injection into the circulatory system [50]. The size and morphology of the non-purified AgNPs were further characterized using TEM. It was observed that the AgNPs produced using condition 2 were approximately spherical in shape and non-aggregated (Figure 7C,D). 3.4. Characterization of Synthesized AgNPs under Optimal Operating Conditions 3.4. Characterization of Synthesized AgNPs under Optimal Operating Conditions On the basis of the TEM measurements (Figure 7C,D), the average size of the AgNPs was 10.38 ± 4.56 nm. It was noted that on the basis of the TEM measurements, the AgNPs appeared to be uniform in size. The differences in the sizes of the AgNPs as measured by DLS and TEM were expected and could be explained by the principals of these techniques [51]. In the case of DLS, the average size is based on the metallic core of the nanoparticles as well as on any compounds connected to the core, such as a stabilizer [51], which in this case was pectin. In contrast, i e e ti atio a e fo ed by TEM o ly o ide the etalli t u tu e [51] SAED analysis was performed in order to determine whether the structure of the synthesized AgNPs was crystalline or amorphous. On the basis of the SAED pattern (Figure 7E), the following d-spacings were calculated: 2.334, 2.083, 1.452, and 1.350 Å. These d-spacing values correspond to (110), (200), (220), and (311) Miller indices [52]. Thus, it was determined that the synthesized AgNPs had a face-centered-cubic (fcc) crystalline structure. size estimation as performed by TEM only considers the metallic structures [51]. SAED analysis was performed in order to determine whether the structure of the synthesized AgNPs was crystalline or amorphous. On the basis of the SAED pattern (Figure 7E), the following d- spacings were calculated: 2.334, 2.083, 1.452, and 1.350 Å. These d-spacing values correspond to (110), (200), (220), and (311) Miller indices [52]. Thus, it was determined that the synthesized AgNPs had a face-centered-cubic (fcc) crystalline structure. EDS analysis was conducted to reveal the elemental composition of the synthesized Ag nanostructures (Figure 7F). Peaks corresponding to metallic Ag were detected in the EDS spectrum, confirming the formation of AgNPs. The presence of O and C was also detected, presumably because of the presence of pectin, an organic compound that was included in the reaction mixture. The peaks located at 8000 and 8900 keV were associated with the Cu sample grid onto which the sample was coated. y EDS analysis was conducted to reveal the elemental composition of the synthesized Ag nanostructures (Figure 7F). Peaks corresponding to metallic Ag were detected in the EDS spectrum, confirming the formation of AgNPs. 3.5. Inﬂuence of the Synthesized AgNPs on Necrosis of Cancer Cells 3.5. Inﬂuence of the Synthesized AgNPs on Necrosis of Cancer Cells According to the American Cancer Society, cancer of the skin is the most common type of all cancers [53]. Melanoma skin cancer is responsible for the large majority of deaths related to skin cancer, with approximately 10,000 people expected to die from melanoma every year in the United States [53]. One of the most common treatments of melanoma cancer involves cell transfer therapies, based on antitumor lymphocytes and cytotoxic agents [54,55]. The cytotoxic agents might cause, for example, anemia and generation of cellular resistance [55]. A remedy to these drawbacks may be the use of AgNPs in the treatment of cancer. Previous studies have shown that nanoparticles in the range of 10–100 nm in size can have anticancer properties [56]. It is true that in vivo there are different types of cells within a tumor mass; however, all of them are activated and proliferated under a low O2 level as well as acidic pH conditions (hypoxic conditions and tumor microenvironment) [14,15]. Certainly, it is critical to incorporate the microenvironmental characteristics into the development of physiologically relevant in vitro models, which could provide reliable, quick, and low-cost methods for toxicity studies of the synthesized nanoparticles. The use of cell lines in the testing of the cytotoxicity of AgNPs is a common practice in the ﬁeld. Therefore, the activity of the AgNPs synthesized in this study using condition 2 against the human melanoma tumor cell line Hs 294T was tested (Table 2). As shown in Figure 8, necrosis of 70–100% of the tumor cell populations was detected following treatment with 5, 10, 50, and 100 µg·mL−1 puriﬁed or non-puriﬁed AgNPs compared to the control cell population treated only with culture medium (p < 0.0001). The majority of the tumor cells underwent necrosis within 24 h, with the ﬁrst symptoms of tumor cell death were observable as early as 6 h after the AgNPs’ application (data not shown). No necrosis was detected when a concentration of 1 µg·mL−1 of puriﬁed or non-puriﬁed AgNPs was used (Figure 8). Similar results were observed when the tumor cells were treated with free Ag(I) ions as when treated with AgNPs (Figure 8). 3.4. Characterization of Synthesized AgNPs under Optimal Operating Conditions 3.4. Characterization of Synthesized AgNPs under Optimal Operating Conditions The presence of O and C was also detected, presumably because of the presence of pectin, an organic compound that was included in the reaction mixture. The peaks located at 8000 and 8900 keV were associated with the Cu sample grid onto which the sample was coated. Figure 7. The morphology of silver nanoparticles (AgNPs) produced under optimal conditions. The histograms of size distribution by number of (A) purified AgNPs, and (B) non-purified AgNPs. (C,D) Representative transmission electron microscopy (TEM) micrographs of non-purified AgNPs, (E) the Figure 7. The morphology of silver nanoparticles (AgNPs) produced under optimal conditions. The histograms of size distribution by number of (A) puriﬁed AgNPs, and (B) non-puriﬁed AgNPs. (C,D) Representative transmission electron microscopy (TEM) micrographs of non-puriﬁed AgNPs, (E) the selected-area electron diffraction (SAED) pattern, and (F) the energy-dispersive X-ray spectroscopy (EDS) spectrum with prominent peaks labeled. Figure 7. The morphology of silver nanoparticles (AgNPs) produced under optimal conditions. The histograms of size distribution by number of (A) purified AgNPs, and (B) non-purified AgNPs. (C,D) Representative transmission electron microscopy (TEM) micrographs of non-purified AgNPs, (E) the Figure 7. The morphology of silver nanoparticles (AgNPs) produced under optimal conditions. The histograms of size distribution by number of (A) puriﬁed AgNPs, and (B) non-puriﬁed AgNPs. (C,D) Representative transmission electron microscopy (TEM) micrographs of non-puriﬁed AgNPs, (E) the selected-area electron diffraction (SAED) pattern, and (F) the energy-dispersive X-ray spectroscopy (EDS) spectrum with prominent peaks labeled. 15 of 20 Nanomaterials 2018, 8, 398 4. Conclusions I 4. Conclusions In summary, we have developed a simple, rapid, and versatile pm-rf-APGD-based method for the size-defined synthesis of stable-in-time AgNPs. As a result of the continuous characteristic of the investigated plasma-reaction system, as well as the low-cost of its utilization, this method provides an appealing alternative to those that are presently applied for the production of high amounts of long-term stable Ag nanostructures. We believe further development of this plasma-reaction system could lead to a high-volume AgNPs-synthesis method. The possibility of controlling the properties of AgNPs should allow for further applications, not only in the necrosis of human melanoma cancer cell lines, but also, for example, in the inactivation of pathogenic bacteria. h b A A A In summary, we have developed a simple, rapid, and versatile pm-rf-APGD-based method for the size-deﬁned synthesis of stable-in-time AgNPs. As a result of the continuous characteristic of the investigated plasma-reaction system, as well as the low-cost of its utilization, this method provides an appealing alternative to those that are presently applied for the production of high amounts of long-term stable Ag nanostructures. We believe further development of this plasma-reaction system could lead to a high-volume AgNPs-synthesis method. The possibility of controlling the properties of AgNPs should allow for further applications, not only in the necrosis of human melanoma cancer cell lines, but also, for example, in the inactivation of pathogenic bacteria. p p p p synthesis of AgNPs. P.P. carried out the statistical analyses. A.D. performed the detailed characterization of the obtained Ag nanostructures by UV/Vis, DLS, TEM, SAED, and EDS. A.B.-P. carried out all the biological experiments. A.D., A.B.-P., G.C.d., P.J., and P.P. summarized all acquired results. G.C.d. provided the graphical support. P.P., A.D., A.B.-P., and G.C.d. wrote the presented manuscript. P.J. and A.K. supervised all work and took part in discussions. Author Contributions: All authors planned the experiments. A.D. and J.M. performed the pm-rf-APGD-based synthesis of AgNPs. P.P. carried out the statistical analyses. A.D. performed the detailed characterization of the obtained Ag nanostructures by UV/Vis, DLS, TEM, SAED, and EDS. A.B.-P. carried out all the biological experiments. A.D., A.B.-P., G.C.d., P.J., and P.P. summarized all acquired results. G.C.d. provided the graphical support. P.P., A.D., A.B.-P., and G.C.d. wrote the presented manuscript. P.J. and A.K. supervised all work and took part in discussions. Funding: A.D., P.J., and P.P. 3.5. Inﬂuence of the Synthesized AgNPs on Necrosis of Cancer Cells In contrast, treatment of the tumor cells with pectin alone or with pm-rf-APGD-treated water alone had no effect on the rate of necrosis compared to the control sample, which had a rate of necrosis of ~10% of the cell population (Figure 8). It was reported that AgNPs had a higher inhibition efﬁcacy in tumor lines than in normal lines, which is due to the higher endocytic activity of tumor cells as compared to normal cells [14]. Therefore, additional experiments were performed, in which the antitumor activity of AgNPs against human ﬁbroblast isolated from skin (MSU-1.1) as well as human endothelial cells isolated from skin (HSkMEC.2) was tested in three different concentrations of AgNPs, that is, 0.01, 1, and 5 µg·mL−1. No cytotoxic effect of AgNPs even for the highest concentration of 5 µg·mL−1 toward any normal cells of skin origin was observed. The IC50 (inhibitory concentration, the concentration at which half of cells are necrotic) values were 7.8, 6.8, 4.3, and 6.6 µg·mL−1 for Gr4–Gr7, respectivly. This suggests that the melanoma cells were more sensitive to the solutions of Ag(I) ions before pm-rf-APGD treatment (Gr6) than to the solutions containing AgNPs. Overall, these data were consistent with the AgNPs synthesized by pm-rf-APGD inducing necrosis in human melanoma cancer cells, suggesting they may serve as an effective therapeutic agent for cancer treatment. The loss of cell viability following the AgNP treatment is believed to have been mediated through the release of Ag(I) ions within the tumor cells [57]. It is therefore not particularly surprising that treatment of the tumor cells with either AgNPs or free Ag(I) ions led to a strong reduction in cell viability, a result observed by both us and others [58]. The advantage of AgNP application lies in their selectivity toward tumor cells [59]. Whereas Ag(I) ions can display high toxicity to both cancerous and healthy cells, AgNPs can show greater speciﬁcity toward tumor cells, as the more acidic nature of their cytoplasm results in an elevated rate of Ag(I) ion release from the AgNPs relative to healthy cells [58,59]. It is important to consider that to fully determine the biological activity of nanoparticles, the correlation between material surface properties and cell functions must also be taken into account to eliminate undesired toxic effects of the nanoparticles [60–63]. 3.5. Inﬂuence of the Synthesized AgNPs on Necrosis of Cancer Cells 16 of 20 16 of 20 Nanomaterials 2018, 8, 398 , , nomaterials 2018, 8, x FOR PEER REVIEW 16 of Figure 8. Percentage of necrotic human melanoma Hs 294T cells. Human melanoma (Hs 294T) cancer cells were treated with 1, 5, 10, 50, and 100 μg mL−1 silver nanoparticles (AgNPs) or Ag(I) ions for 24 h or were treated with culture medium as a control (see Table 2 for a description of the groups). Percentages of necrotic cells were calculated as means ± the standard error of the mean of three independent experiments, each performed with triplicate wells for each treatment group. * p < 0.05; *** p < 0.0001. C l i Figure 8. Percentage of necrotic human melanoma Hs 294T cells. Human melanoma (Hs 294T) cancer cells were treated with 1, 5, 10, 50, and 100 µg·mL−1 silver nanoparticles (AgNPs) or Ag(I) ions for 24 h or were treated with culture medium as a control (see Table 2 for a description of the groups). Percentages of necrotic cells were calculated as means ± the standard error of the mean of three independent experiments, each performed with triplicate wells for each treatment group. * p < 0.05; *** p < 0.0001. Figure 8. Percentage of necrotic human melanoma Hs 294T cells. Human melanoma (Hs 294T) cancer cells were treated with 1, 5, 10, 50, and 100 μg mL−1 silver nanoparticles (AgNPs) or Ag(I) ions for 24 h or were treated with culture medium as a control (see Table 2 for a description of the groups). Percentages of necrotic cells were calculated as means ± the standard error of the mean of three independent experiments, each performed with triplicate wells for each treatment group. * p < 0.05; *** p < 0.0001. Figure 8. Percentage of necrotic human melanoma Hs 294T cells. Human melanoma (Hs 294T) cancer cells were treated with 1, 5, 10, 50, and 100 µg·mL−1 silver nanoparticles (AgNPs) or Ag(I) ions for 24 h or were treated with culture medium as a control (see Table 2 for a description of the groups). 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Furthermore, A.D. is supported by the Foundation for Polish Science (FNP), program START 022.2018. In addition, this work was ﬁnanced by a statutory activity subsidy from the Polish Ministry of Science and Higher Education for the Faculty of Chemistry of Wroclaw University of Technology. G.C.d. is supported by a Natural Sciences and Engineering Research Council of Canada Post-Doctoral Fellowship. Council of Canada Post-Doctoral Fellowship. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the Conﬂicts of Interest: The authors declare no conﬂict of interest. 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https://openalex.org/W4283028812	https://zenodo.org/records/6651112/files/ZDPP17093.pdf	Russian	null	ИШЛАБ ЧИҚАРИШ КОРХОНАЛАРИДА ИННОВАЦИОН ФАОЛИЯТИНИ МОЛИЯЛАШТИРИШ МЕХАНИЗМИНИНГ САМАРАДОРЛИГИНИ РИВОЖЛАНТИРИШ	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	1,352	ЯТИНИ ЯТИНИ ИШЛАБ ЧИҚАРИШ КОРХОНАЛАРИДА ИННОВАЦИОН ФАОЛИЯТИНИ МОЛИЯЛАШТИРИШ МЕХАНИЗМИНИНГ САМАРАДОРЛИГИНИ РИВОЖЛАНТИРИШ Суфиев Рустамжон1, Боймирзаев Одилжон Хасанбой ўғли2 1Фарғона давлат университети ўқитувчиси 2 Фарғона давлат университети магистри https://doi.org/10.5281/zenodo.6651112 Аннотация: Мамлакатимизда ишлаб чиқаришни модернизациялаш бўйича олиб борилаётган ислоҳотлар нафақат корхоналарда ишлатиладиган техника ва технологик жараёнларни янгилашни, балки бошқарув ва ҳисобини ташкил этишда инновацион ёндашувни талаб этади. Шу нуқтаи назардан корхоналарда ишлаб чиқарилган маҳсулотларни баҳолашнинг амалиётда қўлланиладиган айрим жиҳатларини тадқиқ этиш катта аҳамиятга эга. Калит сўзлар: Инновация, самарадорлик, ишлаб чиқариш, механизм Ишлаб чиқаришни инновация-техникавий ривожлантиришни бошқариш компания раҳбарияти юқори погонасида амалга оширилади. Унинг мақсади хўжалик субъектининг янги маҳсулот ишлаб чиқариш ва жорий этиш; ишлаб чиқариладиган маҳсулотни замонавийлаштириш ва такомиллаштириш; маҳсулот анаънавий турларини ишлаб чиқаришни янада ривожлантиришдан иборат. Хўжалик субъектлари инновация фаолиятини бошқаришда инновациялар стратегияси ва уни амалга ошириш чора-тадбирларини ишлаб чиқишга эътибор каратилмокда. Маҳсулот янги турларини яратиш ва ишлаб чиқариш фирма стратегиясининг устувор йўналиши бўлиб колмокда. Тадқиқот ва ишлаб чиқаришни бита жараёнга бирлаштирадиган ягона фан- техника мажмуини ташкил этиш, ишлаб чиқаришни инновация – техникавий ривожлантиришни бошқариш ҳозирги босқичдаги энг муҳим вазифадир. Бу эса «Фан - ишлаб чиқариш» цикли барча босқичларининг мустаҳкам богланишини тақозо килади. Фан - техника сотув яхлит тизимларини яратиш фан техника тараққиётни ва фирманинг бозор йўналишидаги эҳтиёжлари билан боғлиқ. Чунончи, ушбу яратувчилик ишлаб чиқариш булинмаси фаолиятини талабга ва истеъмолчилар эҳтиёжларига компаниянинг иқтисодий салоҳиятини мустаҳкамлашга жавоб берадиган маҳсулотлар яратиш йўли билан ишлаб чиқариш самарадорлигини оширишни таъминлайдиган соҳаларга йўналтирмокда. Шу муносабат билан ишлаб чиқаришни ривожлантириш қуйидаги асосий вазифаларни бажариш орқали, яъни: 378 «Zamonaviy dunyoda pedagogika va psixologiya» nomli ilmiy, masofaviy, onlayn konferensiya «Zamonaviy dunyoda pedagogika va psixologiya» nomli ilmiy, masofaviy, onlayn konferensiya ilmiy, masofaviy, onlayn konferensiya - инновация фаолияти режалари ва дастурларини ишлаб чиқиш; - янги маҳсулот ишлаб чиқиш жараёни ва унинг жорий этишини кузатиш; - янги маҳсулот яратиш лойихаларини куриб чиқиш; - ягона инновация сиёсатини юритиш; -ишлаб чиқариш булинмаларида шу борадиги фаолиятни мувофиқлаштириш; - инновация фаолятини дастурларини молиявий ва моддий ресурслар билан таъминлаш; -ишлаб чиқаришни инновация-техникавий ривожлантиришни бошқаришни малакали ходимлар билан таъминлаш; - ғоядан токи янги ишлаб чиқаришгача инновация муаммоларини ҳал этиш муайян гуруҳлар ташкил қилиш орқали амалга оширилади. Ушбу вазифаларни ҳал этишда янги инвестициялар манбаларини жалб этиш жуда муҳим. Республикамиздаги қишлоқ хўжалиги трамоги мисоллида курсак, бундай инвестициялар «Узқишлоқхўжаликмашхолдинг» корхоналарини инновация-техникавий ривожлантиришнинг қуйидаги босқичларида ишга солинди. Охирги ун йиллик давомида фан ва таълим сифат даражаси янги иқтисодий, ижтимоий ва сиёсий шароитлар ривожланмокда. Инновацион фаолиятининг ривожланишида асосий уринни иктисодиёт тармоқларига кириб бораётган фан эгаллайди. Ривожланган мамлакатларда, яъни ЕИ мамлакатларида 65% Японияда 71% АҚШда. 75 % изланишлар компаниялар томонидан амалга оширилади. Ўзбекистонда изланишлар олиб борувчи олий ўқув юртларининг улуши оз колмокда. Бундан фанга харажатлар ҳам жуда оз миқдорда (5% га якин ЯИМга нисбатан ЕИ да 21% Япония ва АҚШ да 15%) бўлмоқда - инновация фаолияти режалари ва дастурларини ишлаб чиқиш; - инновация фаолияти режалари ва дастурларини ишлаб чиқиш; - ягона инновация сиёсатини юритиш; Охирги ун йиллик давомида фан ва таълим сифат даражаси янги иқтисодий, ижтимоий ва сиёсий шароитлар ривожланмокда. Инновацион фаолиятининг ривожланишида асосий уринни иктисодиёт тармоқларига кириб бораётган фан эгаллайди. Ривожланган мамлакатларда, яъни ЕИ мамлакатларида 65% Японияда 71% АҚШда. 75 % изланишлар компаниялар томонидан амалга оширилади. Ўзбекистонда изланишлар олиб борувчи олий ўқув юртларининг улуши оз колмокда. Бундан фанга харажатлар ҳам жуда оз миқдорда (5% га якин ЯИМга нисбатан, ЕИ да 21%, Япония ва АҚШ да 15%) бўлмоқда. Фан инновацияларга оз даражада йўналитирилган. Миллий инновацион тизимнинг асосий элементларига илмий-техник соҳа, корхоналар, инновацион инфратузилма кириб, бир-бири билан якин алоқада эмас. Корхоналарда инновацион маданият етарли эмас, инвестицион ресурслар кам. Фандан фаол мутахассислар кетмокдалар ва бир вақтнинг узида ёшларининг келиш даражаси паст. Илмий - кадрлар ёш структураси кундан-кунга ёмонланмокда. Олимларнинг 48% и 50 дан ошган, Фан номзодларининг уртача ёши 53 ёш, Фан докторлариники эса 61, уртача ҳисобда ҳар бешинчи олим нафака ёшида. Утган асрнинг 70 йилларида интеллектуал харажатлар маҳсулот умумий қийматига нисбатан 3%ни ташкил килган. Бугунги кунда бу кўрсаткич 379 кич 70% 50% таҳлилчилар баҳолашига кура 2010 йилда Ушбу кўрсаткич 70% булади. XXI асрда айнан ахборот ва билим янги бойликлар ва юқори даромадлар келтиради ва жамиятнинг келажакдаги ривожланишга асос яратади. «Zamonaviy dunyoda pedagogika va psixologiya» nomli ilmiy, masofaviy, onlayn konferensiya Инновацияларнинг жамиятдаги ролини ҳисобга олиш зарур бўлиб, корхона фаолиятидаги асосий вазифа (маркетинг билан тенгма-тенг): - тадбиркорлик инструменти; - тадбиркорлик инструменти; - мустақил бизнес - мустақил бизнес. Инновациялар илмий ғоядан тайёр бозор маҳсулотига айланиши учун бир неча босқичларни босиб ўтади (1-расм). Товарнинг силжишни бир канча иштирокчиларининг узаро мураккаб таъсирлашуви натижасида юзага келади. Товарнинг силжишни бир канча иштирокчиларининг узаро мураккаб таъсирлашуви натижасида юзага келади. Ғоянинг интеллектуал инновацион маҳсулотга айланиши учун куйидиги шартлар бажрилиши лозим. Ғоянинг интеллектуал инновацион маҳсулотга айланиши учун куйидиги шартлар бажрилиши лозим. 1. Инновация бозорининг ривожланиш; 1. Инновация бозорининг ривожланиш; 2. Инновацион фаолиятни ҳимоялаш ва рағбатлантириш бўйича давлат дастурини ишлаб чиқиш. Омадли инновацион фаолиятнинг энг муҳим шарти давлат инновацион дастурини яратишдир. Бу дастур қуйидаги йўналишларни камраб олиши лозим: 380 1-расм Инновациянинг ҳаракатланиш йўли - қонунчилик: аниқ патент-лицензия қонунчилигини яртаиш, венчур капитал ва венчурли молиялаштириш бўйича ҳуқуқйи асос яратиш, солиқ қонунчилигига узгартиришлар киритиш; Инновациянинг ҳаракатланиш йўли Инновациянинг ҳаракатланиш йўли - қонунчилик: аниқ патент-лицензия қонунчилигини яртаиш, венчур капитал ва венчурли молиялаштириш бўйича ҳуқуқйи асос яратиш, солиқ қонунчилигига узгартиришлар киритиш; 380 лабалар, «Zamonaviy dunyoda pedagogika va psixologiya» nomli ilmiy, masofaviy, onlayn konferensiya - таълим: инновацион ишланмалар билан шуғулланувчи талабалар, аспирантлар ва олимларнинг фаолиятини қўллаб-қувватлаш, инновациялар соҳасида мутахассислар тайёрлаш; - таълим: инновацион ишланмалар билан шуғулланувчи талабалар, аспирантлар ва олимларнинг фаолиятини қўллаб-қувватлаш, инновациялар соҳасида мутахассислар тайёрлаш; - кичик фан билан боғлиқ тадбиркорликни қўллаб - қувватлаш. - кичик фан билан боғлиқ тадбиркорликни қўллаб - қувватлаш. Табиий ресурсларнинг чекланганлиги ва камабий бораётганлиги бозорда инновацион, яъни интеллектуал маҳсулотлар билан савдо килувчи ишлаб чиқариш корхоналар учун фойда келтиради. Яъни уларнинг фаолияти кенгайиб боради. Табиий ресурсларнинг чекланганлиги ва камабий бораётганлиги бозорда инновацион, яъни интеллектуал маҳсулотлар билан савдо килувчи ишлаб чиқариш корхоналар учун фойда келтиради. Яъни уларнинг фаолияти кенгайиб боради. Анъанавий муҳитда рақобатланишининг истиқболи йук. Шунинг учун жаҳон иқтисодиёти ривожланишидаги узгаришлар ва Ўзбекистон миллий хусусиятларидан келиб чиккан ҳолда фаолият юритиш лозим. Банклар ва кредит уюшмалари юқори рискли битимларни хоҳлашмайди ва инновацион лойиҳаларга кредит беришда кафиллик учун, яъни гаровга бирор нарса талаб қилишади. Лекин кичик корхоналар узининг дастбалки ривожланиш босқичида гаров учун мулк такдим этишга имконияти бўлмайди ва бу уларнинг ривожланишини оркага суради. Ушбу муаммони ҳал этишда бозор иқтисодиётига эга мамлакатларда фаолият юритадиган фондларнинг велчурли молиялаштиришдан фойдаланиш лозим. Лекин Ўзбекистонда бу молия соҳасидаги янги йўналиш ҳисобланади. Венчурли молиялаштиришнинг асосий вазифаси инновацион бизнесга маълум миқдордаги маблағни устав капиталидаги улуш ёки акция пакетининг бир қисми эвазига бериш ҳисобланади. Шубҳасиз, инвестор юқори рискка учрайди ва бу биринчи навбатда ўз маблағини қайтара ололмаслигига ҳам олиб келиши мумкин. Лекин риск учун мукофот бўлиб, инвестор дивиденлардан оладиган фойдаси ёки нархи кутарилган акцияни сотишдан келадиган фойда ҳисобланади. Инновацион фаолятни молиявий таъминлашни яхшилаш солиқ сиёсатини такомиллаштириш орқали эришилади. Бу тизимнинг моҳияти шундаки, солиқ миқдори билан маҳсулотнинг янги яратилган қиймати, олинган фойда ва мавжуд мол - мулк утрасида, бевосита боғлиқлик урнатиш ҳисобланади. Иқтисодиёти ривожланган мамлакатлар тажрибасига кўра, инновацион корхоналарга ҳам ишлаб чиқариш корхоналарига бериладиган барча имтиёзларни бериш лозим. Бундан ташқари, инновацион корхоналарга изланиш асбоб-усукунасини биринчи йилдаёк 50% ли амортизациялаш ва бошқа асосий фондларини тезлаштирилган амортизациялаш ҳуқуқий берилиши лозим. Саноатга ўз янгилигини жорий этувчи инновацион корхоналарга лицензия ва ноу - хаулар сотишда солиқ имтиёзлари 381 эса янги белгилаш ва ишлаб чиқаришни кенгайтирувчи корхоналарга эса янги ускунаси қийматини солиқ базасига киритмаслик ҳуқуқи берилиши лозим. Фойдаланилган адабиётлар: 6. Нехорошева Л.Н. Инновационные системы современной экономики. Л.Н. Нехорошева, Н.И. Богдан. - Минск: БГЭУ, 2003. Фойдаланилган адабиётлар: 1. Ўзбекистон Республикаси Президентининг 2018 йил 7 майдаги ПК-3698- сонли “Иқтисодиёт тармоқлари ва соҳаларига инновацияларни жорий этиш механизмларини такомиллаштириш бўйича қўшимча чора- тадбирлар тўғрисида”ги Қарори. 2. Мирзиёев Ш.М. “Танқидий таҳлил, катъий тартиб-интизом ва шахсий жавобгарлик - ҳар бир раҳбар фаолиятининг кундалик коидаси бўлиши керак”. -Т.: Ўзбекистон, 2017 йил. 3. Абулкосимов Х.Р., Хамраев О.Я. Иқтисодиётни давлат томонидан тартибга солиш. Укув қўлланма. - Т.: Иқтисод-молия, 2014 - 304-б. 4. Tolipov, A., & Teshabaeva, O. (2021). Innovative entrepreneurship is a factor in the development of the economy of modern production in Uzbekistan. Студенческий, (2-4), 96-99. 5. Тешабаева, О. Н., & Назарова, Л. Т. (2020). Классификация развития факторов инновационного потенциала предприятия в Узбекистане. In минтақа иқтисодиётини инвестициялашнинг молиявий-ҳуқуқий ва инновацион жиҳатлари (pp. 471-474). 6. Нехорошева Л.Н. Инновационные системы современной экономики. Л.Н. Нехорошева, Н.И. Богдан. - Минск: БГЭУ, 2003. 6. Нехорошева Л.Н. Инновационные системы современной экономики. Л.Н. Нехорошева, Н.И. Богдан. - Минск: БГЭУ, 2003. 382
https://openalex.org/W2962407470	https://biologicalproceduresonline.biomedcentral.com/track/pdf/10.1186/s12575-019-0103-3	English	null	Exploiting Differential Gene Expression to Discover Ionic and Osmotic-Associated Transcripts in the Halophyte Grass Aeluropus littoralis	Biological procedures online	2,019	cc-by	10,907	Exploiting Differential Gene Expression to Discover Ionic and Osmotic-Associated Transcripts in the Halophyte Grass Aeluropus littoralis Farzaneh Fatemi1,3, Seyyed Hamidreza Hashemi-petroudi1* , Ghorbanali Nematzadeh1, Hossein Askari2 and Mohammad Reza Abdollahi3 Correspondence: shr.hashemi@sanru.ac.ir; irahamidreza@yahoo.com Correspondence: shr.hashemi@sanru.ac.ir; irahamidreza@yahoo.com 1Department of Genetic Engineering and Molecular Biology, Genetic and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and Natural Resources University (SANRU), P.O. Box 578, Sari, Iran Full list of author information is available at the end of the article (2019) 21:14 (2019) 21:14 Fatemi et al. Biological Procedures Online https://doi.org/10.1186/s12575-019-0103-3 Abstract Background: Salinity as a most significant environmental challenges affects the growth and productivity of plants worldwide. In this study, the ionic and iso-osmotic effects of salt stress were investigated in Aeluropus littoralis L., a halophyte grass species from Poaceae family, by cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. To dissect the two different effects (ionic and osmotic) exerted by salt stress, various ionic agents including 200 and 400 mM sodium chloride (NaCl), 200 and 400 mM potassium chloride (KCl) as well as 280 and 406 gl−1 (−0.9 and −1.4 MPa) polyethylene glycol 6000 (PEG) as their iso-osmotic concentrations were applied. Results: Application of KCl and PEG significantly reduced the fresh weight (FW) of A. littoralis seedlings compared to control while NaCl treatment markedly enhanced the FW. At the transcriptome level, different observations of changes in gene expression have been made in response of A. littoralis to ionic and osmotic stresses. Out of 69 transcript derived fragments (TDFs), 42 TDFs belong to 9 different groups of genes involved in metabolism (11.6%), transcription (10.2%), ribosomal protein (8.7%), protein binding (8.7%) transporter (5.8%), translation (5.8%), signal transduction (4.3%), nucleosome assembly protein (2.9%) and catabolism (2.9%). The 44 and 28 percent of transcripts were expressed under ionic stress (NaCl-specific and KCl-specific) and osmotic stress (common with NaCl, KCl and PEG), respectively which indicating a greater response of plants to ionic stress than osmotic stress. Expression pattern of eight candidate TDFs including; SYP81, CAND1, KATN, ISB1, SAMDC, GLY1, HAK18 and ZF30 was evaluated by RT-qPCR at high salinity levels and recovery condition. Conclusion: Differential regulation of these TDFs was observed in root and shoot which confirm their role in salt stress tolerance and provide initial insights into the transcriptome of A. littoralis. Expression pattern of ionic and osmotic-related TDFs at A. littoralis can be taken as an indication of their functional relevance at different salt and drought stresses. Keywords: Salinity, Ionic effects, Osmotic effects, Aeluropus littoralis, cDNA-AFLP, RT-qPCR Background Various genes respond to salinity and drought stress in some species and their functions have been predicted by alignment to known orthologous genes [5]. g Crop production is adversely affected by various en- vironmental stresses. Many biochemical, physiological and molecular changes occur when plants frequently exposure to different stress conditions. These changes are resultant of massive regulations in the profile of gene expression [1]. Drought and salt stress physi- ology overlaps and cross-talks with each other. Salt stress generates lower water potential in the zone, making it difficult for the plant to absorb water lead- ing to dehydration of the cell and ultimately disrup- tion of osmotic equilibrium. Therefore, the form of a physiological drought is taken in the plant under the salt stress [2]. Na+, K+, H+ and Ca2+ are the major ions involved in signal transduction. Restoring the os- motic balance of the cell, damage repair and control by the maintenance of cellular homeostasis, detoxifi- cation and signaling to coordinate cell function are mechanisms that plants use in response to salinity and drought stresses [3, 4]. g g g Serial analysis of gene expression (SAGE), representa- tional difference analysis (RDA), differential display re- verse transcription-polymerase chain reaction (DD-RT- PCR), suppression subtractive hybridization (SSH), c- DNA microarray and cDNA-amplified fragment length polymorphism (cDNA-AFLP) are techniques that cur- rently are available for transcriptome analysis. Among these techniques, cDNA-AFLP as a transcriptome-wide screening tool [16] is an extremely efficient and less labor-intensive mRNA fingerprinting method for gene discovery [17] without any prerequisite knowledge about sequences [18]. This technique gives the possibil- ity to identify rarely expressed sequence tags (ESTs) [19]. So, it provides rapid and multiple comparisons of the plant response to different stress durations and in- tensities [20]. In this research, the strategy is based on the discrimin- ation of the whole salt stress effects into the ionic effect and osmotic effect. For this purpose, PEG was used as the non-ionic or iso-osmotic solution. We report the candidate genes that were differentially expressed in the roots under the NaCl, potassium chloride (KCl) and PEG treatments in A. littoralis using cDNA-AFLP method. The darkness condition was considered to omit the photosynthesis related genes due to its complexity and increasing the chance of identifying allocated genes to ionic and osmotic stresses. We also established a col- lection of stress-responsive ESTs in A. © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fatemi et al. Biological Procedures Online (2019) 21:14 Page 2 of 16 Fatemi et al. Biological Procedures Online (2019) 21:14 Background littoralis in NCBI GenBank and their possible functions and presumed biological implications were discussed based on the homology searches. This can help to identify salt and drought-inducible candidate genes in this halophyte plant for subsequent studies especially in the field of novel gene transfer. Salt stress consists of two main components including osmotic effect and ionic effect [5, 6]. Osmotic effect de- creased water absorption in the rhizosphere, while ionic effect results to imbalance or intercellular toxicity due to excess ions [7]. Many researchers have proved the exist- ence of the ionic and osmotic components of salt stress. Singh et al. [8], showed that sodium chloride (NaCl) was more harmful for germination of pea when used as an iso-osmotic solution of polyethylene glycol 6000 (PEG). A study on K+ fluxes in the mesophyll of bean leaf under the mannitol and iso-osmotic NaCl treatments showed that different mechanisms are involved in the concep- tion of ionic and osmotic components [9]. Halophytes are salt-tolerant plants growing exclusively in habitats with high salinity [10]. They can survive under the high salinities of NaCl. Aeluropus littoralis is a halophytic plant of Poaceae family. It is a monocotyle- donous halophyte which usually grows in the regions with intermediate to high salinity [11, 12]. A. littoralis can tolerate up to 600 mM NaCl [13] or 800 mM NaCl [11]. Aeluropus species are potentially known as pre- cious genetic resources due to accumulation of sodium and chloride ions in their over ground tissues and can improve our understanding about molecular mecha- nisms of salt and drought stress responses especially in cereals [14, 15]. Salinity stress increases the number and size of vacuole and also organelle density due to accu- mulating of Na+ and Cl−fractions. Therefore, A. littora- lis as a halophyte plant uses the same mechanism to overcome salt and drought stresses [11]. Many genes and biochemical–molecular mechanisms are involved in plant response to abiotic stress. Changes in gene expres- sion profile are induced by a complex of signal transduc- tion pathways that have not been determined clearly. Effect of Ionic and Osmotic Stresses on Plant Fresh Weight The FW of treated A.littoralis seedlings was evaluated in response to ionic and osmotic stresses. Both ionic and osmotic stresses induced by various levels of KCl and PEG significantly reduced the plant FW weight com- pared to control (Fig. 1 and Fig. 2) while use of 200 and 400 mM NaCl in liquid MS culture medium resulted in significant enhancement of FW compared to other treat- ments (Fig. 2). The reduction in plant FW was greater under PEG induced osmotic stress than stress induced by KCl treatments at their iso-osmotic concentrations (Fig. 2) . Also, use of 406 gl−1 PEG (400 mM or −1.4 MPa) in cul- ture medium resulted in more reduction in FW compared to 280 gl−1 PEG (200 Mm or −0.9 MPa) (Fig. 2). Page 3 of 16 Fatemi et al. Biological Procedures Online (2019) 21:1 (2019) 21:14 Fatemi et al. Biological Procedures Online Fig. 1 The effect of ionic and osmotic stresses on FW of A.littoralis in liquid MS culture medium. (a) Control culture medium, (b and c) culture media containing 200 and 400 mM NaCl, respectively, (d and e) culture media containing 200 and 400 mM KCl, respectively, (f and g), culture media supplemented with PEG 6000 preparing −0.9 and −1.4 MPa osmotic pressures, respectively Fig. 1 The effect of ionic and osmotic stresses on FW of A.littoralis in liquid MS culture medium. (a) Control culture medium, (b and c) culture media containing 200 and 400 mM NaCl, respectively, (d and e) culture media containing 200 and 400 mM KCl, respectively, (f and g), culture media supplemented with PEG 6000 preparing −0.9 and −1.4 MPa osmotic pressures, respectively Identification of Ionic and Osmotic Stress-Induced Transcripts Identification of Ionic and Osmotic Stress-Induced Transcripts root development. Finally, 69 readable sequences were determined as the ionic and osmotic responsive genes which shared homology with genes encoding known, un- known, hypothetical proteins. Approximately 60.9% of the TDFs were shared identity to reported sequences in database. Some of the sequences showed homology to The cDNA-AFLP technique was used to isolate ionic and osmotic-responsive genes from A. littoralis. Notice- able differences were observed in gene expression profile between ionic and osmotic effects of salt stress during Fig. 2 Effect of different ionic and osmotic treatments on FW of Aeluropus littoralis seedlings. Effect of Ionic and Osmotic Stresses on Plant Fresh Weight 3, No 22: JZ191103), are expressed in response to osmotic stress (common with NaCl, KCl and PEG) (Table 1 and Fig. 3). Among 69 deposited TDFs to the dbEST database, 42 TDFs are presented in Table 1 and Fig. 3 with the Gen- Bank accession numbers. The other 27 sequences were re- lated to hypothetical/unknown protein and no similarity match in GenBank. We found that ionic and osmotic re- lated TDFs was numerous in A. littoralis, especially the transcripts involved in metabolites and energy which should be considered to find out the mechanism of salt tolerance. Several TDFs like ribosomal proteins (Table 1 and Fig. 3, No 5, 12 and 32: JZ191088, JZ191056 and JZ191070), auxin response factor (Table 1 and Fig. 3, No 3: JZ191047), potassium transporter (Table 1 and Fig. 3, No 16 and 19: JZ191096 and JZ191100), NADH dehydro- genase (Table 1 and Fig. 3, No 41: JZ191107), S- adenosylmethionine decarboxylase (Table 1 and Fig. 3, No 25: JZ191058) and syntaxin (Table 1 and Fig. 3, No 4: JZ191048) are strong candidates that are specific to the ionic effects (NaCl and KCl). Some other TDFs such as transcription factors (Table 1 and Fig. 3, No 23: JZ191104), RNA binding protein (Table 1 and Fig. 3, No 30: JZ191081), cyclin (Table 1 and Fig. 3, No 33: JZ191083), translation initiation factor (Table 1 and Fig. 3, No 36: JZ191075), zinc finger CCCH domain (Table 1 and Fig. 3, No 20, 21 and 27: JZ191101, JZ191102 and JZ191061), ubi- quitin (Table 1 and Fig. 3, No 22: JZ191103), are expressed in response to osmotic stress (common with NaCl, KCl and PEG) (Table 1 and Fig. 3). TDFs or genomic sequences of Oryza sativa, Zea mays and Arabidopsis thaliana, but 8.7% of the TDFs did not show any significant similarity to nucleotide or amino acid sequences in the GenBank and classified as no sig- nificant matches. TDFs with known functions are listed in Table 1 and Fig. 3. TDFs or genomic sequences of Oryza sativa, Zea mays and Arabidopsis thaliana, but 8.7% of the TDFs did not show any significant similarity to nucleotide or amino acid sequences in the GenBank and classified as no sig- nificant matches. TDFs with known functions are listed in Table 1 and Fig. 3. Expression Patterns of the cDNA Fragments Expression Patterns of the cDNA Fragments The gene expression patterns of eight TDFs including SYP81, Syntaxin of plants 81; CAND1, Cullin- associated and neddylation-dissociated; KATN, Katanin p80 WD40; ISB1, Importin subunit beta-1; SAMDC, S- adenosylmethionine decarboxylase; GLY1, Glyoxalase I; HAK18, High-affinity potassium transporter; ZF30, Zinc finger CCCH domain-containing protein 30 were individually assessed in 600 mM of NaCl stress and re- covery condition by using RT-qPCR due to their im- portant role in ionic and osmotic stresses. It has been reported that at high salinity levels, the ionic effect domi- nates or equals to the osmotic effect [23]. Values were de- termined to statistically significant fold changes with 95% confidence (P = 0.05). Genes with fold changes are indi- cated in Fig. 6. In general, all the genes had significant dif- ference relative to control. At time-point 6 hps, the expression level of SYP81, KATN, SAMDC and ISB1 were higher while CAND1, GLY1, HAK18 and ZF30 were down-regulated in root (α < 0.05). All 8 genes followed the same pattern in 48 hps and 168 hps time-points except CAND1 and GLY (Fig. 6). Under recovery conditions at time-point 48 hpr, the expression level of CAND1, KATN, GLY1 and SAMDC increased relative to control while, the genes of SYP81, ISB1, HAK18 and ZF30 showed down- regulation in mRNA level. Similar to root analysis, Functional Determination of TDFs The sequence comparison of 69 readable TDFs against the database revealed that most of them had homology to genes with known functions (Fig. 5), whereas, 30.4% (21 TDFs) of TDFs belonged to either hypothetical or unknown function proteins, and 8.7% (6 TDFs) of TDFs showed no significant matches. To obtain a better insight into the identity and possible functional of these stress-induced TDFs, the functional categories of TDFs were assigned based on gene ontology. The transcripts were grouped into 9 functional categories related to bio- logical processes. The vast majority of annotations was involved in metabolism (11.6% - 8 TDFs), transcription (10.2% - 7 TDFs), ribosomal protein (8.7% - 6 TDFs) and protein binding (8.7% - 6 TDFs) (Fig. 5). Genes encoding proteins involved in transporter (5.8% - 4 TDFs), transla- tion (5.8% - 4 TDFs), signal transduction (4.3% - 3 TDFs), nucleosome assembly protein (2.9% - 2 TDFs) and catabolism (2.9% - 2 TDF) formed the second lar- gest groups. Differentially Expressed TDFs in Response to Ionic and Osmotic Treatments Effect of Ionic and Osmotic Stresses on Plant Fresh Weight The osmotic pressures, −0.9 and −1.4 MPa, prepared by 280 gl−1 and 407 gl−1 PEG 6000 solutions are iso-osmotic concentrations of 200 and 400 mM NaCl and KCl, respectively. The letters showed significantly difference at the 5% level according to Duncan’s multiple test. Significant differences between two bars marked with different letters Fig. 2 Effect of different ionic and osmotic treatments on FW of Aeluropus littoralis seedlings. The osmotic pressures, −0.9 and −1.4 MPa, prepared by 280 gl−1 and 407 gl−1 PEG 6000 solutions are iso-osmotic concentrations of 200 and 400 mM NaCl and KCl, respectively. The letters showed significantly difference at the 5% level according to Duncan’s multiple test. Significant differences between two bars marked with different letters Page 4 of 16 Fatemi et al. Biological Procedures Online (2019) 21:14 Page 4 of 16 Fatemi et al. Biological Procedures Online (2019) 21:14 Fatemi et al. Biological Procedures Online Among 69 deposited TDFs to the dbEST database, 42 TDFs are presented in Table 1 and Fig. 3 with the Gen- Bank accession numbers. The other 27 sequences were re- lated to hypothetical/unknown protein and no similarity match in GenBank. We found that ionic and osmotic re- lated TDFs was numerous in A. littoralis, especially the transcripts involved in metabolites and energy which should be considered to find out the mechanism of salt tolerance. Several TDFs like ribosomal proteins (Table 1 and Fig. 3, No 5, 12 and 32: JZ191088, JZ191056 and JZ191070), auxin response factor (Table 1 and Fig. 3, No 3: JZ191047), potassium transporter (Table 1 and Fig. 3, No 16 and 19: JZ191096 and JZ191100), NADH dehydro- genase (Table 1 and Fig. 3, No 41: JZ191107), S- adenosylmethionine decarboxylase (Table 1 and Fig. 3, No 25: JZ191058) and syntaxin (Table 1 and Fig. 3, No 4: JZ191048) are strong candidates that are specific to the ionic effects (NaCl and KCl). Some other TDFs such as transcription factors (Table 1 and Fig. 3, No 23: JZ191104), RNA binding protein (Table 1 and Fig. 3, No 30: JZ191081), cyclin (Table 1 and Fig. 3, No 33: JZ191083), translation initiation factor (Table 1 and Fig. 3, No 36: JZ191075), zinc finger CCCH domain (Table 1 and Fig. 3, No 20, 21 and 27: JZ191101, JZ191102 and JZ191061), ubi- quitin (Table 1 and Fig. Classification of Expression Patterns Different gene expression patterns were observed in the re- sponse of A. littoralis to ionic and osmotic stresses based on their presence/absence (qualitative variants). The TDFs classified into 6 categories. NaCl (specific to NaCl treat- ment), KCl (specific to KCl treatment), PEG (specific to PEG treatment), KCl/NaCl (common with ionic NaCl and KCl treatments) and KCl, NaCl/PEG (common with all treatments). TDFs in the NaCl and KCl categories, display the genes that are directly related to the ionic effect, which were not expressed in PEG treatment. Similarly, the KCl, NaCl/PEG category reflects the osmotic effect, and the PEG category should represent specific effects of PEG chemicals rather than the osmotic effect. Such extra ef- fects of PEG have been reported previously [21]. In the following classification (Fig. 4), a majority of the TDFs (44%) fell into the group that represented ionic response (NaCl 16%, KCL 16% and NaCl/KCl response 12%) whereas 28% of TDFs showed osmotic response. The rest of the TDFs belonged to PEG and control response (21 and 7% respectively) (Fig. 4). Differentially Expressed TDFs in Response to Ionic and Osmotic Treatments The cDNA-AFLP analysis showed that the root-specific TDFs were highly expressed in ionic and osmotic stresses. Fatemi et al. Biological Procedures Online (2019) 21:14 Page 5 of 16 Fatemi et al. Biological Procedures Online (2019) 21:14 Page 5 of 16 Table 1 List of TDFs induced in cDNA-AFL 200 mM NaCl (N1), 400 mM NaCl (N2), 200 m in root. Sequences were compared to sequ homology between the aligned sequences TDF Accession no. Treatments Length 1 JZ191042 P2 365 2 JZ191043 P2, P1, K2 350 3 JZ191047 N1, K1 312 4 JZ191048 C, K2 284 5 JZ191088 P2, N1, K1 307 6 JZ191049 P2 265 7 JZ191050 P1 261 8 JZ191051 P1, C, N1 260 9 JZ191052 P2, K1 260 10 JZ191054 P2, C 259 11 JZ191055 P2, N1 247 12 JZ191056 C, K2 259 13 JZ191092 P2 260 14 JZ191093 C 236 15 JZ191094 P2 231 16 JZ191096 C, N2 384 17 JZ191098 N2 212 18 JZ191099 N2 207 19 JZ191100 P2, C, N2 237 20 JZ191101 P1 180 21 JZ191102 P1 180 22 JZ191103 P2, P1, N2, K2 170 23 JZ191104 P2, N2, K1, K2 170 24 JZ191057 P1, C, N1, K1 159 25 JZ191058 C, K1 136 26 JZ191060 C 142 27 JZ191061 P2 133 28 JZ191062 C, N1, K1 126 29 JZ191063 C, N1, K1 142 30 JZ191081 P1, C, N1,K1, K2 523 32 JZ191070 P2, P1, C, N2, K2 391 33 JZ191083 P1, K1 362 34 JZ191072 N1, K1, K2 361 35 JZ191073 C, N1, K1, K2 357 36 JZ191075 P2, N1, K1, K2 329 37 JZ191064 C 566 38 JZ191065 N1, K1 234 39 JZ191066 K1 133 40 JZ191068 P2 403 41 JZ191107 C, N1 179 42 JZ191108 N2 152 Table 1 List of TDFs induced in cDNA-AFLP by KCl, NaCl and PEG treatments in roots of Aeluropus littoralis. TDFs were induced by 200 mM NaCl (N1), 400 mM NaCl (N2), 200 mM KCl (K1), 400 mM KCl (K2), −0.9 MPa/280 gl-1 PEG (P1) and −1.4 MPa/406 gl-1PEG (P2) in root. Sequences were compared to sequences in the GenBank database using the BLAST program. The E-value show the homology between the aligned sequences TDF Accession no. Differentially Expressed TDFs in Response to Ionic and Osmotic Treatments Treatments Length (bp) Homology to gene Name; accession number E-value 1 JZ191042 P2 365 DUF21 domain-containing protein (Brachypodium distachyon); XM_003568464 9e-33 2 JZ191043 P2, P1, K2 350 Genomic DNA, chromosome 4, BAC clone: OSIGBa0158F13 (Oryza sativa); CR855151.1 4.0 3 JZ191047 N1, K1 312 Auxin response factor 3-like (Glycine max); XM_003529306.1 0.88 4 JZ191048 C, K2 284 Syntaxin 81 (Zea mays); EU963152 6e-46 5 JZ191088 P2, N1, K1 307 40S ribosomal protein S3 (Glycine max); XM_003548212 8e-55 6 JZ191049 P2 265 Aspartic proteinase (Oryza sativa); BAA02242.1 1.4 7 JZ191050 P1 261 Chloroplast envelope membrane protein (Staurastrum punctulatum); YP_636444 3.5 8 JZ191051 P1, C, N1 260 Voltage-dependent anion-selective channel protein 4 (Oryza sativa); Q0JJV1.3 3e-29 9 JZ191052 P2, K1 260 Myb-like DNA-binding domain (Zea mays); NM_001157897.1 0.59 10 JZ191054 P2, C 259 Myb-like DNA-binding domain (Zea mays); NM_001157897.1 2.8 11 JZ191055 P2, N1 247 G-box binding protein (Oryza sativa); EU847024.1 0.15 12 JZ191056 C, K2 259 40S ribosomal protein S12 (Zea mays); EU957837.1 2e-42 13 JZ191092 P2 260 Importin subunit beta-1-like (Oryza sativa); XP_015619891.1 8e-44 14 JZ191093 C 236 Nucleolin 2 (Zea mays); ONM05985.1 3e-08 15 JZ191094 P2 231 Putative glyoxalase I (Oryza sativa); BAD28547.1 0.008 16 JZ191096 C, N2 384 Serine/threonine-protein kinase SIS8 (Brachypodium distachyon); XM_010235744.3 4e-13 17 JZ191098 N2 212 Golgin candidate 4-like (Brachypodium distachyon); XM_003574367.1 4e-13 18 JZ191099 N2 207 Calcineurin B-like-interacting protein kinase (Hordeum brevisubulatum); JX679077.1 9e-17 19 JZ191100 P2, C, N2 237 Potassium transporter (HAK18) (Brachypodium distachyon); XM_010241173.1 7e-13 20 JZ191101 P1 180 Zinc finger CCCH domain-containing protein (Oryza sativa); XP_015632054.1 8e-20 21 JZ191102 P1 180 Zinc finger CCCH domain-containing protein 24 (Oryza sativa); Q10EL1.1 6e-19 22 JZ191103 P2, P1, N2, K2 170 Ubiquitin-related modifier (Zea mays); NM_001149704.1 3e-37 23 JZ191104 P2, N2, K1, K2 170 AP2/EREBP transcription factor ERF-1 (Gossypium hirsutum); AY779339.1 0.034 24 JZ191057 P1, C, N1, K1 159 Cullin-associated nedd8-dissociated protein1 (Oryza sativa); EF575856.1 9e-16 25 JZ191058 C, K1 136 S-adenosylmethionine decarboxylase (Oryza sativa); JN944362.1 5e-20 26 JZ191060 C 142 Metal-nicotianamine transporter (Glycine max); XM_003548246.1 2.6 27 JZ191061 P2 133 Zinc finger CCCH domain-containing protein (Oryza sativa); XM_003518517.1 6.7 28 JZ191062 C, N1, K1 126 Nucleotide binding site leucine-rich repeat (Pyrus sinkiangensis); ACJ05259 6.8 29 JZ191063 C, N1, K1 142 DNA glycosylase/lyase 701 (Oryza sativa); FJ536320 7.8 30 JZ191081 P1, C, N1,K1, K2 523 RNA binding protein (Oryza sativa); AAP85377.1 1e-37 32 JZ191070 P2, P1, C, N2, K2 391 60S ribosomal protein L38 (Zea mays); NP_001152328.1 1e-41 33 JZ191083 P1, K1 362 Cyclint 2-like protein (Oryza sativa); XP_015627068.1 2e-15 34 JZ191072 N1, K1, K2 361 Nucleosome assembly protein (Brachypodium distachyon); XP_003568000.1 0.44 35 JZ191073 C, N1, K1, K2 357 Beta-galactosidase (Bathycoccus prasinos); CCO19627.1 5e-05 36 JZ191075 P2, N1, K1, K2 329 Eukaryotic translation initiation factor p28 (Zea mays); NP_001104917.1 2e-65 37 JZ191064 C 566 Katanin p80 WD40 (Brachypodium distachyon); XP_003579480 1e-51 38 JZ191065 N1, K1 234 Dehydrin Xero 2-like (Brassica rapa); XM_013864438.2 7.9 39 JZ191066 K1 133 Glutamate decarboxylase-like (Vitis vinifera); XM_002263045.2 6.7 40 JZ191068 P2 403 ADP-glucose pyrophosphorylase (Zea mays); HM749416.1 0.005 41 JZ191107 C, N1 179 NADH dehydrogenase subunit J (Passiflora incarnate); KT721860.1 2.5 g p g Homology to gene Name; accession number E-value DUF21 domain-containing protein (Brachypodium distachyon); XM_003568464 9e-33 Genomic DNA, chromosome 4, BAC clone: OSIGBa0158F13 (Oryza sativa); CR855151.1 4.0 Auxin response factor 3-like (Glycine max); XM_003529306.1 0.88 Syntaxin 81 (Zea mays); EU963152 6e-46 40S ribosomal protein S3 (Glycine max); XM_003548212 8e-55 Aspartic proteinase (Oryza sativa); BAA02242.1 1.4 Chloroplast envelope membrane protein (Staurastrum punctulatum); YP_636444 3.5 Voltage-dependent anion-selective channel protein 4 (Oryza sativa); Q0JJV1.3 3e-29 Myb-like DNA-binding domain (Zea mays); NM_001157897.1 0.59 Myb-like DNA-binding domain (Zea mays); NM_001157897.1 2.8 G-box binding protein (Oryza sativa); EU847024.1 0.15 40S ribosomal protein S12 (Zea mays); EU957837.1 2e-42 Importin subunit beta-1-like (Oryza sativa); XP_015619891.1 8e-44 Nucleolin 2 (Zea mays); ONM05985.1 3e-08 Putative glyoxalase I (Oryza sativa); BAD28547.1 0.008 Serine/threonine-protein kinase SIS8 (Brachypodium distachyon); XM_010235744.3 4e-13 Golgin candidate 4-like (Brachypodium distachyon); XM_003574367.1 4e-13 Calcineurin B-like-interacting protein kinase (Hordeum brevisubulatum); JX679077.1 9e-17 Potassium transporter (HAK18) (Brachypodium distachyon); XM_010241173.1 7e-13 Zinc finger CCCH domain-containing protein (Oryza sativa); XP_015632054.1 8e-20 Zinc finger CCCH domain-containing protein 24 (Oryza sativa); Q10EL1.1 6e-19 Ubiquitin-related modifier (Zea mays); NM_001149704.1 3e-37 AP2/EREBP transcription factor ERF-1 (Gossypium hirsutum); AY779339.1 0.034 Cullin-associated nedd8-dissociated protein1 (Oryza sativa); EF575856.1 9e-16 S-adenosylmethionine decarboxylase (Oryza sativa); JN944362.1 5e-20 Metal-nicotianamine transporter (Glycine max); XM_003548246.1 2.6 Zinc finger CCCH domain-containing protein (Oryza sativa); XM_003518517.1 6.7 Nucleotide binding site leucine-rich repeat (Pyrus sinkiangensis); ACJ05259 6.8 DNA glycosylase/lyase 701 (Oryza sativa); FJ536320 7.8 RNA binding protein (Oryza sativa); AAP85377.1 1e-37 60S ribosomal protein L38 (Zea mays); NP_001152328.1 1e-41 Cyclint 2-like protein (Oryza sativa); XP_015627068.1 2e-15 Nucleosome assembly protein (Brachypodium distachyon); XP_003568000.1 0.44 Beta-galactosidase (Bathycoccus prasinos); CCO19627.1 5e-05 Eukaryotic translation initiation factor p28 (Zea mays); NP_001104917.1 2e-65 Katanin p80 WD40 (Brachypodium distachyon); XP_003579480 1e-51 Dehydrin Xero 2-like (Brassica rapa); XM_013864438.2 7.9 Glutamate decarboxylase-like (Vitis vinifera); XM_002263045.2 6.7 ADP-glucose pyrophosphorylase (Zea mays); HM749416.1 0.005 NADH dehydrogenase subunit J (Passiflora incarnate); KT721860.1 2.5 Phytochrome C (Cenchrus americanus); JQ270557.1 0.027 Page 6 of 16 Fatemi et al. Differentially Expressed TDFs in Response to Ionic and Osmotic Treatments Biological Procedures Online (2019) 21:14 Fatemi et al. Biological Procedures Online Fig. 3 A representative picture of a silver-stained cDNA-AFLP gel showing the differential expression of the genes under different components of salt stress in Aeluropus littoralis. (a), (b), (c) and (d) are different sections of the main gel. Small black arrows show 42 TDFs described in Table 1 Fig. 3 A representative picture of a silver-stained cDNA-AFLP gel showing the differential expression of the genes under different components of salt stress in Aeluropus littoralis. (a), (b), (c) and (d) are different sections of the main gel. Small black arrows show 42 TDFs described in Table 1 Fig. 3 A representative picture of a silver-stained cDNA-AFLP gel showing the differential expression of the genes under different components of salt stress in Aeluropus littoralis. (a), (b), (c) and (d) are different sections of the main gel. Small black arrows show 42 TDFs described in Table 1 Page 7 of 16 (2019) 21:14 Page 7 of 16 Page 7 of 16 Fatemi et al. Biological Procedures Online (2019) 21:14 Fatemi et al. Biological Procedures Online Fig. 4 Expression patterns categories of Aeluropus littoralis roots in response to ionic and osmotic agents. The TDFs were classified into 6 groups based on presence/absence of bands KATN, HAK18 and ZF30 were observed among all ana- lyzed genes. The heat-map generated from RT-qPCR ex- pression data represented the differential transcript abundance of the eight candidate TDFs in salt stress and recovery condition in both leaf and root tissues (Fig. 7). The largest gene expression values are displayed in light green color while the dark blue showed smallest values. Furthermore, it has shown that the TDFs under investiga- tions cluster together based on their induction at different time-points (Fig. 7). Assigning subcellular localization to a protein is also an important step towards elucidating mo- lecular function and its interaction partners. For predict- ing protein subcellular localization of each TDF, the protein sequence of each TDF homologues in S. italica was analyzed by Plant-PLoc program (Table 2). Discussion In the present study, the molecular response of A. littoralis seedlings to different ionic agents (KCl and NaCl) and osmotic agent PEG, as their iso-osmotic con- centrations, were investigated to separate the ionic and osmotic effects of salinity. The plant FW was used in term of physiological growth index to evaluate toxicity in tolerant samples exposed to ionic and osmotic agents. Differentially expressed cDNA fragments from whole plant were assessed in etiolated samples to increase chance of isolating non photosynthesis-related genes. Also, expression pattern of eight candidate TDFs was evaluated at high salinity level to validate and confirm their functional relevance for regulating ion homeostasis and osmotic tolerance in A.littoralis. Putative gene Fig. 4 Expression patterns categories of Aeluropus littoralis roots in response to ionic and osmotic agents. The TDFs were classified into 6 groups based on presence/absence of bands expression values of eight genes across three time-points of leaf samples were also examined. The genes SYP81, KATN, SAMDC, ISB1, CAND1, HAK18 and ZF30 followed the same pattern in 6 hps and 48 hps time-points except GLY1. In 168 hps time-point, KATN, SAMDC, HAK18 and ZF30 were up-regulated whereas SYP81, ISB1, GLY1 and CAND1 were significantly down- regulated. In 48 hpr time-point, up-regulation of SYP81, Fig. 5 Functional percentage distribution of TDFs in Aeluropus littoralis based on gene functions obtained from Gene Ontology [22] Fig. 5 Functional percentage distribution of TDFs in Aeluropus littoralis based on gene functions obtained from Gene On (2019) 21:14 Fatemi et al. Biological Procedures Online (2019) 21:14 Page 8 of 16 Fatemi et al. Biological Procedures Online functions of ESTs were also classified as described by reduced the plant FW of A. littoralis. Several previous Fig. 6 Trend of regulated genes during different time-point of salt stress and recovery condition. SYP81, Syntaxin of plants 81; CAND1, Cullin- associated and neddylation-dissociated; KATN, Katanin p80 WD40; ISB1, Importin subunit beta-1; SAMDC, S-adenosylmethionine decarboxylase; GLY1, Glyoxalase I; HAK18, High-affinity potassium transporter; ZF30, Zinc finger CCCH domain-containing protein 30. hps and hpr are the abbreviated of hours post stress and hours post recovery, respectively. A single asterisk () and double asterisks () represent significant difference from the control (0 hps) (P < 0.05, n = 3) and very significant difference from the control (0 hps) (P < 0.01, n = 3), respectively. Discussion littoralis biological systems and especially for the large-scale ana- lysis of gene expression in plants [17]. Unlike DNA- microarray technology, cDNA-AFLP does not address transcript abundance exactly but it is helpful for identi- fying the change of transcript abundance without com- prehensive genomic information or prior sequence knowledge [27]. Here we used mRNA Capture Kit with one-tube method and the advantage of using streptavi- din-coated PCR tubes in which, mRNA reverse tran- scription and cDNA restriction can be performed in a one-tube format efficiently and eliminate several time- consuming steps. Salinity consists of multiple compo- nents including ionic and osmotic effects and this is the important reasons for the complexity of salt tolerance. cDNA-AFLP indicates the differences and similarities in gene expression profile between the ionic effect and osmotic effect successfully. In this study, cDNA-AFLP was used to detect the differences in gene expression profile between ionic or osmotic effects of salt stress. Transcript-derived fragments with potentially relevant function in ionic and osmotic tolerance were identified based on the differential expression pattern. However, limited information was obtained on the sequences and gene expression in A. littoralis. So, it was not surprising that 27 of the 69 TDFs identified as hypothetical or un- known proteins (30.4%) and 6 of them showed no simi- larity match (8.7%) to the presented data on NCBI. The results detected by cDNA-AFLP confirmed that all the selected TDFs from JZ191042 to JZ191110 showed spe- cific expression pattern. Similar results have been ob- tained from cDNA-AFLP analysis of salt-stressed soybean [7], such as, CCCH-type zinc finger protein, treatments at iso-osmotic concentrations indicated spe- cific ionic and non-ionic effects [24]. Osmotic stress in- duced by PEG exhibited more reduction in FW in comparison to ionic stress (NaCl and KCl treatments) at iso-osmotic levels indicating the inhibitory effect of os- motic stress than ionic stress [24]. On the other hand, different growth responses to dif- ferent concentrations of iso-osmotic salt solutions indi- cated specific ionic effects. Increasing the biomass production of the seedlings at 200 mM of NaCl treat- ment in compered to all treatment showed the halophyte nature of A. littoralis while reduction in FW at 400 mM of NaCl treatment indicated threshold of tolerance in this plant. Unexpectedly, KCl caused a greater inhibition of plant FW than control especially at 400 mM (−1.4 MPa) (Figs. Discussion The relative expression based on 2^-ΔΔCT is represented in the y axis and the time-point is represented in the χ axis Fig. 6 Trend of regulated genes during different time-point of salt stress and recovery condition. SYP81, Syntaxin of plants 81; CAND1, Cullin- associated and neddylation-dissociated; KATN, Katanin p80 WD40; ISB1, Importin subunit beta-1; SAMDC, S-adenosylmethionine decarboxylase; GLY1, Glyoxalase I; HAK18, High-affinity potassium transporter; ZF30, Zinc finger CCCH domain-containing protein 30. hps and hpr are the abbreviated of hours post stress and hours post recovery, respectively. A single asterisk () and double asterisks (*) represent significant difference from the control (0 hps) (P < 0.05, n = 3) and very significant difference from the control (0 hps) (P < 0.01, n = 3), respectively. The relative expression based on 2^-ΔΔCT is represented in the y axis and the time-point is represented in the χ axis Fig. 6 Trend of regulated genes during different time-point of salt stress and recovery condition. SYP81, Syntaxin of plants 81; CAND1, Cullin- associated and neddylation-dissociated; KATN, Katanin p80 WD40; ISB1, Importin subunit beta-1; SAMDC, S-adenosylmethionine decarboxylase; GLY1, Glyoxalase I; HAK18, High-affinity potassium transporter; ZF30, Zinc finger CCCH domain-containing protein 30. hps and hpr are the abbreviated of hours post stress and hours post recovery, respectively. A single asterisk () and double asterisks (**) represent significant difference from the control (0 hps) (P < 0.05, n = 3) and very significant difference from the control (0 hps) (P < 0.01, n = 3), respectively. The relative expression based on 2^-ΔΔCT is represented in the y axis and the time-point is represented in the χ axis functions of ESTs were also classified as described by Zouari et al. [15]. reduced the plant FW of A. littoralis. Several previous studies compared the effects of different salts and os- motic stresses in different plant species. Different re- sponses of plant FW to different ionic and non-ionic The present results showed that iso-osmotic stresses developed by NaCl, KCl and PEG agents significantly Fatemi et al. Biological Procedures Online (2019) 21:14 (2019) 21:14 Page 9 of 16 Fatemi et al. Biological Procedures Online Fig. 7 Heat-map showing RT–qPCR expression of eight TDFs against salt stress and recovery condition in leaf and root tissues of A. littoralis RT–qPCR expression of eight TDFs against salt stress and recovery condition in leaf and root tissues of A. Discussion 1 and 2) due to toxic effects of ionic ele- ments and a noticeable inhibition imposed by osmotic effect especially at −1.4 MPa PEG [25]. Decreasing of plant growth in response to KCl stress at both 200 and 400 mM, suggesting that 200 mM KCl could be con- sidered as threshold of tolerance. The result showed that potassium chloride is more toxic than sodium chloride and K+ ion would affect plant growth more than Na+ ion supporting the results of the research on Atriplex prostate [26]. So the inhibitory effect of salt stress and osmotic stress on plant growth and survival follow the pattern; PEG >KCl > NaCl. These findings are consistent with the results obtained from other re- searchers [24]. cDNA-AFLP is a whole transcriptome-wide technique for isolating tissue-specific genes in a wide range of (2019) 21:14 Page 10 of 16 Fatemi et al. Biological Procedures Online (2019) 21:14 Fatemi et al. Biological Procedures Online Table 2 The list of candidate TDFs and their gene specific primer sequences that were used for RT-qPCR. Prediction of subcellular localization was done base on their gene homologues in Setaria italica Gene symbol Name Function Gene homologues in Setaria italica Predicted subcellular location Primer sequence Amp. size SYP81 Syntaxin of plants 81 Vesicle trafficking protein that functions in the secretory pathway. XP_004976323 Nucleus CAGC ATGGCGTGGCTCTTAT AGCA TCTTGAAAGCGCATGG 90 CAND1 Cullin-associated and neddylation- dissociated Key assembly factor of SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complexes that promotes the exchange of the substrate-recognition F-box subunit in SCF complexes AT2G02560 cell-to-cell mo- bile RNA XP_004951789.1 Chloroplast TGGC AGTGACTACAGCATACGG ACTG CGCACAGAGCGGTACT 91 SAMDC S-adenosylmethionine decarboxylase Essential for polyamine homeostasis, and normal plant embryogenesis, growth and development. Discussion XP_004953064.1 Cytoplasm CCAT CCATGGTCCTGCTTTC GGGT TGAAGCCCATGACCTC 81 Katanin Katanin p80 WD40 Microtubule severing XP_012700331.1 Chloroplast TGAT CCCTCCCTTCCCAGTT CCTG AGCGAATGCGTAAACC 98 ISB1 Importin subunit beta-1 Protein transporter activity XP_004962709.1 Cytoplasm GCTC CAGCCAAATGTCAAGC GGTC TTGGTCAACAGCTTCAGG 86 GlyI Glyoxalase I Carbohydrate metabolic process XP_004952236.1 Chloroplast GTGG CATGGACTTGCTACGG CCGT GGCATCACAGAGGATT 92 HAK18 High-affinity potassium transporter Potassium ion transmembrane transporter activity XP_004956156.1 Plasma membrane GGCC AGACATTTCAGACCACA AGCC CTGATGACCGTGTTTC 99 ZF30 Zinc finger CCCH domain-containing protein 24 Regulation of transcription XP_004982091.1 Nucleus GCTC TTGTTGGCTCCCCTCT TCAC CATTTACGCCCCAATC 83 RPS3 40S ribosomal protein S3-like Structural constituent of ribosome involved in RNA methylation, photorespiration, translation XP_004972758.1 Chloroplast ATTC ACTGGCTGACCGGATG GTGC CAAGGGTTGTGAGGTC 107 UBQ Ubiquitin-like protein Biologically significant role in protein delivery to proteasomes and recruitment of proteasomes to transcription sites XP_004957594.1 Chloroplast CTTG GTCTGCTGTTGTCTTG CACG GTTCACTTATCCATCAC 200 EF1A Elongation factor-1 alpha Translation elongation factor activity XP_004984833.1 Cytoplasm TGCTGTCGGTGTCATCAA CTTC CATCAAACGCCTCATT 97 Page 11 of 16 (2019) 21:14 Fatemi et al. Biological Procedures Online (2019) 21:14 Fatemi et al. Biological Procedures Online Table 2 The list of candidate TDFs and their gene specific primer sequences that were used for RT-qPCR. Prediction of subcellular localization was done base on their gene homologues in Setaria italica (Continued) Gene symbol Name Function Gene homologues in Setaria italica Predicted subcellular location Primer sequence Amp. size U2SURP U2snRNP-associated SURP motif- containing protein-like RNA binding, required for spliceosome assembly to participate in splicing XP_004951689.1 Nucleus CGTG GATGAGATTGAGAGGAA TGGA GGACTACGGCTTCTA 199 GTF General transcription factor 3C polypeptide Involved in RNA polymerase III-mediated transcription XP_004975210.1 Nucleus TTCC AAGTGGCCATCAGGTT AAAG GGCTTCCTGCCTCTTG 108 Table 2 The list of candidate TDFs and their gene specific primer sequences that were used for RT-qPCR. Prediction of subcellular localization was done base on their gene homologues in Setaria italica (Continued) Gene symbol Name Function Gene homologues in Setaria italica Predicted subcellular location Primer sequence Amp. size U2SURP U2snRNP-associated SURP motif- containing protein-like RNA binding, required for spliceosome assembly to participate in splicing XP_004951689.1 Nucleus CGTG GATGAGATTGAGAGGAA TGGA GGACTACGGCTTCTA 199 GTF General transcription factor 3C polypeptide Involved in RNA polymerase III-mediated transcription XP_004975210.1 Nucleus TTCC AAGTGGCCATCAGGTT AAAG GGCTTCCTGCCTCTTG 108 Table 2 The list of candidate TDFs and their gene specific primer sequences that were used for RT-qPCR. Prediction of subcellular localization was done base on their gene homologues in Setaria italica (Continued) Gene symbol Name Function Gene homologues in Setaria italica Predicted subcellular location Primer sequence Amp. Discussion size U2SURP U2snRNP-associated SURP motif- containing protein-like RNA binding, required for spliceosome assembly to participate in splicing XP_004951689.1 Nucleus CGTG GATGAGATTGAGAGGAA TGGA GGACTACGGCTTCTA 199 GTF General transcription factor 3C polypeptide Involved in RNA polymerase III-mediated transcription XP_004975210.1 Nucleus TTCC AAGTGGCCATCAGGTT AAAG GGCTTCCTGCCTCTTG 108 Page 12 of 16 Page 12 of 16 Page 12 of 16 Fatemi et al. Biological Procedures Online (2019) 21:14 Fatemi et al. Biological Procedures Online ubiquitin, glutamate, envelope membrane protein, anion-selective channel protein, putative protein kinase, etc. aquaporins and plasma membrane K+ channels, which involved in the regulation of water homeostasis in the cell as a result playing an important role in osmotic adjustment during cell expansion and environmental stresses [34]. In cDNA-AFLP analysis, TDFs specific to ionic effect were remarkably more abundant in the root (44%) than those specific to the osmotic effect (28%) (Fig. 3). This indicates the importance of roots in stress conception. Previous studies have also noted that roots play an im- portant role for limiting ion accumulation in shoot [7]. In this research, TDFs of JZ191061, JZ191100, JZ191083 and JZ191048 were homologous to zinc finger CCCH domain, potassium transporter, cyclin, and syntaxin, re- spectively. These TDFs are candidates for regulating ion homeostasis and osmotic tolerance in A. littoralis. The RT-qPCR was done for these genes; SYP81, CAND1, KATN, ISB1, SAMDC, GLY1, HAK18, ZF30. The quanti- fication of given transcripts was performed to determine the changes in the transcript levels under exogenous treatments. In the present study, we have focused on gaining insight on the differential expression of some ESTs in response to salt stress and recovery condition which were estimated by RT-qPCR both in root and leaf tissues. Different responses were found to ionic and os- motic treatments. Interestingly, expression of some genes was induced by salt stress while also significantly repressed by recovery condition. The TDF JZ191083 exhibited similar pattern to cyclin proteins which regulate cell cycle and cell division but their exact function in abiotic stress are largely unknown. It is obvious that undesirable conditions prevent root growth due to regulation of cell division and cell cycle [35]. Cellular studies showed that growth is as a result of produced cells in the meristem and the final length of the cells at the end of the growth zone [36]. Discussion So, cell division partially specifies organ elongation rate by controlling the dividing cells number and also the average cell cycle dur- ation. From this viewpoint, salt stress decreases the rate of root elongation by reducing the final cells size and num- ber of dividing cells resulted in shortening the size of mer- istems [37]. Salt stress stimulates root cells to elongate closer to the root tip rapidly, resulting in reduced meri- stem size and stopped cells division at a smaller size [38]. Compartmentalization of sodium and chloride ions in vacuole is one of the major adaptive responses of plants to decrease the toxic effect in the cytoplasm [39]. y p Moreover, the significant number of genes related to photosynthetic metabolism is down- or up-regulated in response to drought and salt [40]. It has been proved that the high need for osmotic adjustment and the need for reducing photosynthetic activity caused the changes in ATP amounts which is a response to the stress in shoots of A. littoralis [41]. Therefore, it can be con- cluded that the high capacity of ATP is used to provide energy for tolerance-related strategies in A. littoralis, which is a highly energy requiring process [40]. Based on these results, the seedlings were grown in a darkness condition to omit the impact of photosynthesis-related genes due to its complexity and increasing the chance of identifying allocated genes to salinity and drought stresses. Our results emphasize the importance of mul- tiple effects of salt stress taking into consideration dis- tinguishing between the ionic and osmotic effects by cDNA-AFLP method. Since A. littoralis is a member of Poaceae family, understanding the mechanism of salt tolerance and detecting the key genes involved in salt tolerance would be very helpful for breeding and genetic engineering of salt tolerant varieties in other members of the grass family including maize, wheat, barley, and rice. Our classification of TDFs based on their specific re- sponse to ionic and osmotic stresses should facilitate the functional analysis of stress-responsive genes. The zinc finger CCCH domain plays a role during the osmotic stress. The TDF of JZ191061 was homologous to zinc finger CCCH domain-containing proteins from Glycine max, and found to be involved in different bio- logical processes including regulatory, signal transduc- tion, DNA and RNA binding, zinc ion binding, mRNA processing and various biotic and abiotic stress re- sponses [28, 29]. Discussion Investigation of the cis-elements in the promoter regions of the maize CCCH genes which response to stress showed similarity to two types of cis- elements, such as the ABA-responsive element and dehydration-responsive element. So, it can be concluded that the CCCH genes contain ABRE or DRE as the drought stress-responsive genes in their promoter se- quences [30]. The zinc finger protein encoding tran- scription factors was previously reported as stress- inducible genes by Zouari et al. [12]. The cDNA fragment JZ191048 was homologous to syntaxin 81 in Brachypodium distachyon. Physiological information showed that plant response to salinity and drought stresses is controlled by expression of a set of particular genes. The proteins derived from these genes are involved in plant protection against the adverse effects of stress and minimize the damage caused by them [31, 32]. The studies confirmed the key roles of syntaxin in mediating the vesicle traffick- ing between the plasma membrane and the Golgi [33]. They regulate transport and activity of both Plant Material Preparation p Seeds of A. littoralis were uncoated and sterilized for 1 min in 75% ethanol followed by 15 min in 2.5% commer- cial bleach, then rinsed three times with sterile distilled water. The decontaminated seeds were then placed in a flask containing 50 ml of full strength liquid MS medium [15] supplemented with 3% sucrose and vitamins. The pH of culture medium was adjusted to 5.8. Plantlets were grown in a growth chamber at 25 °C in the dark- ness condition and constant shaking (120 rpm) for seven days. These seven-day-old plantlets were subcultured and transferred to flasks containing 50 ml of liquid MS culture medium as described above. In order to study the ionic and osmotic effects, different concentrations of NaCl, KCl (200, 400 mM) and 280 and 406 gl−1 PEG 6000, as their iso-osmotic concentrations (produced − 0.9, −1.4 MPa), were added to subculture media accord- ing to Van’t Hoff equation [42]: (atm) π = icRT where, i is Van’t Hoff factor, which describes the number of parti- cles per molecule dissolved; c refers to molarity of solu- tion; R and T are universal gas constant (8.314472 LkPa/ molK) and absolute temperature in Kelvin, respectively. This equation gives the osmotic pressure in Bar which should be converted to MPa. RNA Extraction and cDNA-AFLP Procedure Total RNA from each stress treatment was isolated from about 100 mg of the frozen samples with the Trizol ex- traction kit (Invitrogen, USA) in three replications. Quality and quantity of isolated RNA were checked by agarose gel electrophoresis and spectrophotometer, re- spectively. The genomic DNA contamination was re- moved by DNase treatment (DNase I RNase-free, Thermo Scientific, USA). Root cells were lysed and mRNAs were isolated by capturing of poly(A+) RNA in streptavidin-coated tubes using a mRNA Capture kit (Roche, Switzerland). The first- and double-strand cDNA were synthesized by 5 μg of total RNA using a bi- otinylated oligo-dT primer in streptavidin-coated PCR tubes according to the manufacturer’s procedure. The restriction enzymes used for digestion of doubled strand cDNA were Taq I (Fermentase) and Mse I (Thermo Sci- entific, USA). The cDNA fragments released were puri- fied and subsequently ligated to Taq I and Mse I. The pre-amplification reaction on ligation products was car- ried out using primers corresponding to Taq І (5′- GACGATGAGTCCTGACCGA-3′) and Mse I (5′- GACGATGAGTCCTGAG-3′) adapters. Twelve primer combinations were used for selective amplification; the primers were Mse І + 2: CC/CA/CT and Taq І + 2: GG/ GC/AC/AT [18]. Selective amplification products (4 μl) were heat denatured and separated by electrophoresis in polyacrylamide gel (6%) containing 0.5X TBE. The gels were visualized by silver staining protocol and then were scanned by using imaging densitometer (GS-800, BioRad, USA). Conclusion In this study, we identified 69 stress-induced genes in A. littoralis. The detailed study of 42 genes expression Page 13 of 16 Fatemi et al. Biological Procedures Online (2019) 21:14 Fatemi et al. Biological Procedures Online (2019) 21:14 Fatemi et al. Biological Procedures Online under several treatments with eliminating the impact of photosynthesis indicated that most of the dedicated transcripts were expressed under ionic stress than osmotic stress which in turn showing a greater re- sponse of Aeluropus roots to ionic stress. Several novel stress-responsive genes expressed in A. littoralis indicated special mechanisms of stress adaptability in this halophyte species. Determination the function, expression and translation of stress-inducible genes is necessary to understand the molecular mechanisms and to improve stress tolerance of crops by genetic engineering. The differential gene expression patterns observed in the physiological side suggest a high plant specificity in order to increase the chance of ESTs in- volved in stress tolerance. Our classification of TDFs based on the specific response to ionic and osmotic stresses could facilitate the functional analysis of salt- responsive genes in future studies of A. littoralis. The role of hypothetical and unknown TDFs in salt and drought response will need to be characterized fur- ther, and the information gleaned from such studies is expected to improve stress tolerance of crops by genetic engineering. This work will be continued by ectopic expression of candidate TDFs in either eukaryotic or prokaryotic expression system. in liquid nitrogen, and stored at −80 °C for cDNA-AFLP analysis. The experiment was adjusted as a completely randomized design with five replications. The FW data were analyzed by analysis of variance (ANOVA). Signifi- cant differences between means were assessed by Dun- can’s multiple range test (DMRT) at p < 0.05. SPSS 16 software was used to test the significant differences among levels of treatments. Isolation, Cloning and Sequencing of Transcript Derived Fragments (TDFs) Discrimination of gene expression patterns to ionic and osmotic effects was assayed based on the presence/ab- sence (qualitative variants) of the visualized bands on gel electrophoresis. Gel profiles were quantified using Quan- tity One gel image analysis software (version 4.4.1, Bio- RAD, USA) resulted in measurement of band intensities per lane for each time interval. The interested bands were marked and cut from the dried gels. The excised bands were eluted in 50 μl double distilled sterile water at 95 °C for 15 min then hydrated overnight at 4 °C. The eluted TDFs were reamplified by using the same set of After 14 days, the grown plants under the stress condi- tions and control were harvested. The whole plant fresh weight (FW) was measured, and then was quickly frozen Page 14 of 16 Fatemi et al. Biological Procedures Online (2019) 21:14 Fatemi et al. Biological Procedures Online (2019) 21:14 Fatemi et al. Biological Procedures Online (2019) 21:14 Fatemi et al. Biological Procedures Online (2019) 21:14 Total RNA was extracted using TRIzol reagent (Invi- trogen Life Technologies, Karlsruhe, Germany) accord- ing to the manufacturer’s instructions. Equal quantity of RNA was used for cDNA synthesis. The cDNA was syn- thesized using the QuantiTect reverse transcription kit (Qiagen) according to the manufacturer’s instructions. The final cDNA reactions were diluted 1:5, and stored at −20 °C. Gene-specific primers (Table 2) were designed according to the obtained sequences of the candidate TDFs (Table 1) using the Primer3.0 web resource (http://frodo.wi.mit.edu/cgi-bin/primer3/pri-mer3_www. cgi). The primer specificity was evaluated by melt curve analysis, and size of the amplicons was tested by end- point PCR on 3% agarose gels. For normalization of ex- pression levels in Aeluropus littoralis, different sets of reference genes were selected for root and leaf samples according to the previous studies [13]. In this view, the three housekeeping genes (HKGs), namely RPS3, EF1A and UBQ were used as normalizer in root samples while two HKGs namely U2SURP and GTF were chosen for leaf samples. RT-qPCR was done by the Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific) with two-step cycling in CFX96 real-time PCR instrument (Bio-Rad, USA) according to the company’s suggestions. Data acquisition was performed during the annealing/ extension step. After amplification, all PCR reactions were subjected to a thermal melt with continuous fluor- escence measurement from 55 °C to 95 °C for dissoci- ation curve analysis. Acknowledgements We are grateful to all members of Genetic engineering and biology department, Genetic and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and Natural Resources University (SANRU) for their helpful discussion and technical assistance. Abbreviations AFLP Amplified AFLP: Amplified fragment length polymorphism; CAND1: Cullin-associated and neddylation-dissociated; FW: Fresh weight; GLY1: Glyoxalase I; HAK18: High-affinity potassium transporter; ISB1: Importin subunit beta-1; KATN: Katanin p80 WD40; KCl: Potassium chloride; NaCl: Sodium chloride; PEG: Polyethylene glycol; RT-qPCR: Reverse transcription–qPCR; SAMDC: S- adenosylmethionine decarboxylase; SYP81: Syntaxin of plants 81; TDF: Transcript derived fragment; ZF30: Zinc finger CCCH domain-containing protein 30 Isolation, Cloning and Sequencing of Transcript Derived Fragments (TDFs) At least one non-template control (NTC) was used for each primer pair master mix. The PCR efficiency were approximated by the shape of the PCR amplification plot, and based on similar amplifica- tion plots of target and reference genes, the 2^-ΔΔCT method were used for calculation of relative gene ex- pression ratio [44]. RT2 Profiler PCR Array Data Ana- lysis software (SABiosystems, (QIAGEN, Germany) was used to construct Heat-map. selective primers under the PCR conditions as men- tioned for AFLP. The PCR products were then checked on 0.5 x TBE 1.5% agarose gel. After confirming the size of the bands, the PCR products were ligated into the pTZ57R/T T/A cloning vector (InsTAclone PCR Cloning Kit, Thermo Scientific, USA), and were directly electro- transformed into DH5 competent E.coli cells. Colony PCR was done with M13 F/R primers and after confirm- ing the size of the band, they were sent for sequencing (GATC, Germany). Bioinformatics Analysis y For TDFs in silico analysis, sequences of vectors and adaptors were trimmed off by using the VecScreen pro- gram on the NCBI website (www.ncbi.nlm.nih.gov/ VecScreen). Translated sequences were analyzed for homology to publicly available GenBank non-redundant sequences databases (http.//www.ncbi.nlm.nih.gov/) using the BLASTX, BLASTN and TBLASX programs. Also, the Gene Ontology (http:// amigo1.geneontology. org/cgi-bin/amigo/go.cgi) and UniProt (http://www.uni- prot.org/) database (https://www.arabidopsis.org) were used to investigate the molecular function of each TDF and its role in biological processes as well as their loca- tion in the cell. Finally, 69 TDFs were deposited in the GenBank dbEST database under BioSample number SAMN01924517; library number LIBEST_028119 (69 ESTs with accession numbers JZ191042– JZ191110). For predicting each candidate TDFs protein subcellular localization, the gene homologues of each candidate TDF was found in Setaria italica by BLASTX program. By using the obtained full sequence of protein, the sub- cellular localization was predicted by Plant-PLoc pro- gram [43]. Reverse transcription–qPCR (RT-qPCR) p q ( q ) For RT-qPCR analysis, two-week-old seedlings were transferred to hydroponic culture containing Hoagland’s solution. The growth chamber conditions were 25 ± 2 C with 16 h light/8 h dark photoperiod at 600 μmol m−2s-l photon flux density using cool-white fluorescent light. The two-month-old seedlings were stressed by 600 mM of sodium chloride (received 100 mM sodium chloride per two days). After reaching to 600 mM, leaf and root samples were collected at three time-points including 6 h post stress (hps), 48 hps and 168 hps. In order to plant recovery, the remained plants were transferred to a so- dium chloride-free Hoagland’s solution, and then sam- ples were collected 48 h post recovery (hpr). Control samples were also taken from unstressed plants at the initial of time-points. All samples were immediately fro- zen in liquid nitrogen and stored at −70 °C for RT-qPCR analysis. Funding Thi 15. Zouari N, Ben Saad R, Legavre T, Azaza J, Sabau X, Jaoua M, Masmoudi K, Hassairi A. Identification and sequencing of ESTs from the halophyte grass Aeluropus littoralis. Gene. 2007;404:61–9. 15. Zouari N, Ben Saad R, Legavre T, Azaza J, Sabau X, Jaoua M, Masmoudi K, Hassairi A. Identification and sequencing of ESTs from the halophyte grass Aeluropus littoralis. Gene. 2007;404:61–9. u d g This research was supported by the Genetic and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and Natural Resources University (SANRU). 16. Ertani A, Schiavon M, Nardi S. Transcriptome-wide identification of differentially expressed genes in Solanum lycopersicon L. in response to an alfalfa-protein hydrolysate using microarrays. Front Plant Sci. 2017;8:1159. Author details 1 f Author details 1Department of Genetic Engineering and Molecular Biology, Genetic and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and Natural Resources University (SANRU), P.O. Box 578, Sari, Iran. 2Department of Biotechnology, Faculty of New Technologies and Energy Engineering, Shahid Beheshti University, Tehran, Iran. 3Department of Agronomy and Plant Breeding, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, Iran. 21. Gururani MA, Venkatesh J, Ghosh R, Strasser RJ, Ponpandian LN, Bae H. Chlorophyll-a fluorescence evaluation of PEG-induced osmotic stress on PSII activity in Arabidopsis plants expressing SIP1. Plant Biosyst. 2018;152:945–52. 1Department of Genetic Engineering and Molecular Biology, Genetic and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and Natural Resources University (SANRU), P.O. Box 578, Sari, Iran. 2Department of Biotechnology, Faculty of New Technologies and Energy h h d h h h 3 f 22. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene ontology: tool for the unification of biology. The gene ontology consortium. 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Ethics Approval and Consent to Participate This article does not contain any studies with human participants. Ethics Approval and Consent to Participate This article does not contain any studies with human participants. 18. Zhou C-P, Qi Y-P, You X, Yang L-T, Guo P, Ye X, Zhou X-X, Ke F-J, Chen L-S. Leaf cDNA-AFLP analysis of two citrus species differing in manganese tolerance in response to long-term manganese-toxicity. BMC Genet. 2013; 14:621. Consent for Publication Not applicable. Consent for Publication Not applicable. 19. Grover A, Sharma P. Development and use of molecular markers: past and present. Crit Rev Biotechnol. 2016;36:290–302. 20. Huang F, Peng M, Chen X, Li G, Di J, Zhao Y, Yang L, Chang R, Chen Y. cDNA-AFLP analysis of transcript derived fragments during seed development in castor bean (Ricinus communis L. Biotechnol Biotechnol Equip. 2018:1–7. Competing Interests h h d l h The authors declare that they have no competing interests. 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https://openalex.org/W4313305808	https://zenodo.org/records/7558889/files/med2022-11-01.pdf	English	null	Changes in the structural organization of spleen during short-term exposure of monosodium glutamate	European Scientific e-Journal	2,022	cc-by	4,829	DOI: 10.47451/med2022-11-01 The paper will be published in Crossref, ICI Copernicus, BASE, Academic Resource Index ResearchBib, J- Gate, ISI International Scientific Indexing, Zenodo, OpenAIRE, BASE, LORY, EBSCO, ADL, Mendeley, eLibrary, and WebArchive databases. Tetyana V. Garapko, Doctor of Medical Sciences, Docent, Department of Human Anatomy and Histology, Faculty of Medicine, Uzhhorod National University. Uzhhorod, Ukraine. ORCID 0000-0003-0596-9622. Lesia R. Mateshuk-Vatseba, Doctor of Medical Sciences, Professor, Department of Normal Anatomy, Danylo Halytskyi Lviv National Medical University. Lviv, Ukraine. ORCID 0000-0002-3466-5276. Andrii S. Holovatskyi, Doctor of Medical Sciences, Professor, Department of Human Anatomy and Histology, Faculty of Medicine, Uzhhorod National University. Uzhhorod, Ukraine. Anatolii I. Foros, Doctor of Philosophy, Department of Fundamental Medical Disciplines and Orthopedic Dentistry, Faculty of Dentistry, Uzhhorod National University. Uzhhorod, Ukraine. ORCID 0000-0003-0824-6702. Andrii S. Holovatskyi, Doctor of Medical Sciences, Professor, Department of Human Anatomy and Histology, Faculty of Medicine, Uzhhorod National University. Uzhhorod, Ukraine. Anatolii I. Foros, Doctor of Philosophy, Department of Fundamental Medical Disciplines and Orthopedic Dentistry, Faculty of Dentistry, Uzhhorod National University. Uzhhorod, Ukraine. ORCID 0000-0003-0824-6702. Changes in the structural organization of spleen during short-term exposure of monosodium glutamate Abstract: Monosodium glutamate is one of the most common food additives in the world. Its effect on the organs of the immune system is not sufficiently studied. The article presents and analyzes the data of an experimental study conducted on 40 white male and female rats of reproductive age (2.5-3.5 months old) weighing 125-195 g. The purpose of the study was to study the histological, morphometric and ultrastructural changes of the spleen under conditions of exposure to monosodium glutamate for four weeks. An experimental group of animals (10 male rats, 10 female rats), which was on a standard vivarium diet, was given monosodium glutamate at a dose of 0.07 g/kg of rat body weight every day for four weeks. In animals of the intact group, the structure of the spleen corresponded to the species norm. In the experimental group of animals, after four weeks of exposure to monosodium glutamate, an immunoinducing effect was observed with increased proliferation of activated lymphocytes and their further differentiation into plasma cells. There is a significant increase in the relative area of the germinal centers of splenic lymphoid nodules and the outer diameter of the central artery of the spleen. Arteries with a thickened wall, their lumen is full of blood. Degeneratively changed erythrocytes are located around the vessels. The veins have a deformed shape, are dilated, the lumen is also full of blood. The share of active macrophages, apoptically changed cells increases. The red pulp is full-blooded, filled with hemosiderin. Therefore, even a short-term daily exposure to monosodium glutamate, namely four weeks, causes changes in the structural organization of the spleen. Abstract: Monosodium glutamate is one of the most common food additives in the world. Its effect on the organs of the immune system is not sufficiently studied. The article presents and analyzes the data of an experimental study conducted on 40 white male and female rats of reproductive age (2.5-3.5 months old) weighing 125-195 g. The purpose of the study was to study the histological, morphometric and ultrastructural changes of the spleen under conditions of exposure to monosodium glutamate for four weeks. An experimental group of animals (10 male rats, 10 female rats), which was on a standard vivarium diet, was given monosodium glutamate at a dose of 0.07 g/kg of rat body weight every day for four weeks. Garapko, T. V., Mateshuk-Vatseba, L. R., Holovatskyi, A. S., & Foros, A. I. (2022). Changes in the structural organization of spleen during short-term exposure of monosodium glutamate. Actual Issues of Modern Science. European Scientific e-Journal, 23(8), 73-82. Ostrava: Tuculart Edition, European Institute for Innovation Development. Garapko, T. V., Mateshuk-Vatseba, L. R., Holovatskyi, A. S., & Foros, A. I. (2022). Changes in the structural organization of spleen during short-term exposure of monosodium glutamate. Actual Issues of Modern Science. European Scientific e-Journal, 23(8), 73-82. Ostrava: Tuculart Edition, European Institute for Innovation Development. p Гарапко, Т. В., Матешук-Вацеба, Л. Р., Головацький, А. С., Форос, А. I. (2022). Зміни в структурній організації селезінки при короткочасному впливі глутамату натрію. Actual Issues of Modern Science. European Scientific e-Journal, 23(8), 73-82. Ostrava: Tuculart Edition, European Institute for Innovation Development. Changes in the structural organization of spleen during short-term exposure of monosodium glutamate I i l f th i t t th t t f th l d d t th i I th Keywords: experiment, monosodium glutamate, spleen, white pulp, red pulp, lymphocytes. 1 1 Тетяна Василівна Гарапко, доктор медичних наук, доцент, кафедра анатомії людини та гістології, медичний факультет, Ужгородський національний університет. Ужгород, Україна. ORCID 0000-0003-0596-9622. Леся Ростиславівна Матешук-Вацеба, доктор медичних наук, професор, кафедра нормальної анатомії, Львівський національний медичний університет імені Данила Галицького. Львів, Україна. ORCID 0000-0002-3466-5276. Андрій Степанович Головацький, доктор медичних наук, професор, кафедра анатомії людини та гістології, медичний факультет, Ужгородський національний університет. Ужгород, Україна. натолій Ілліч Форос, доктор філософії, кафедра фундаментальних медичних дисциплін та ортопедичної стоматології, стоматологічний факультет, Ужгородський національний університет. Ужгород, Україна. ORCID 0000-0003-0824-6702. Анатолій Ілліч Форос, доктор філософії, кафедра фундаментальних медичних дисциплін та ортопедичної стоматології, стоматологічний факультет, Ужгородський національний університет. Ужгород, Україна. ORCID 0000-0003-0824-6702. Зміни в структурній організації селезінки при короткочасному впливі глутамату натрію Анотація: Глутамат натрію є однією з найбільш поширених харчових добавок у світі. Його вплив на органи імунної системи є не достатньо вивченим. В статті наведено та проаналізовано дані експериментального дослідження, проведеного на 40 білих щурах-самцях і самках репродуктивного віку (2,5-3,5-місячних) масою 125-195 г. Мета дослідження – вивчити гістологічні, морфометричні та ультраструктурні зміни селезінки в умовах впливу глутамату натрію впродовж чотирьох тижнів. Експериментальній групі тварин (10 щурів-самців, 10 щурів- самок), яка перебувала на стандартному харчовому раціоні віварію, впродовж чотирьох тижнів щодня додавали глутамат натрію в дозі 0,07 г/кг маси тіла щура. У тварин інтактної групи будова селезінки відповідала видовій нормі. В експериментальній групі тварин через чотири тижні дії глутамату натрію спостерігається iмуноіндукуючий ефект з посиленою проліферацією активованих лімфоцитів та їх подальшим диференціюванням у плазматичні клітини. Відбувається достовірне збільшення відносної площі зародкових центрів селезінкових лімфоїдних вузликів та зовнішнього діаметру центральної артерії селезінки. Артерії з потовщеною стінкою, їх просвіт повнокровний. Навколо судин розташовані дегенеративно змінені еритроцити. Вени мають деформовану форму, розширені, просвіт також повнокровний. Зростає частка активних макрофагів, апоптично змінених клітин. Червона пульпа повнокровна, заповнена гемосидерином. Отже, навіть короткотривалий щоденний вплив на організм глутамату натрію, а саме чотири тижні, викликає зміни структурної організації селезінки. Ключові слова: експеримент, глутамат натрію, селезінка, біла пульпа, червона пульпа, лімфоцит Introduction Monosodium glutamate is one of the most common food additives in the world. It is the monosodium salt of glutamic acid (Bautista et al., 2019). Belongs to taste enhancers, due to which it increases appetite. This leads to increased food intake, causing a high-calorie diet (Bhandari, 2018). The result of a high-calorie diet is excess body weight and obesity. Obesity contributes to the occurrence of numerous organ diseases, but the mechanisms of these processes remain unclear (Camacho & Ruppel, 2017). In the professional literature, obesity is considered a state of chronic inflammation, which is often associated with complications such as type two diabetes, cardiovascular disease, 2 hypertension, stroke, gallbladder disease, osteoarthritis, and psychosocial problems (Bibik, 2018; Escobedo & Oliver, 2017; Coppey, 2018). Numerous studies describe that a high-calorie diet leads to metabolic syndrome, insulin resistance, diabetes, splenomegaly, arterial hypertension, heart attacks, etc. (Finlayson, 2017; Buchan et al., 2018). In obesity, the functions of both T cells and B cells are impaired. Obesity-induced reduction of IL-10 synthesis in the spleen leads to inflammatory reactions in the kidneys and metabolic disturbances (Gotoh et al., 2017). Scientists conclude that monosodium glutamate causes metabolic disorders and contributes to the development of obesity. A number of separate studies on glutamate-induced obesity have been described in foreign publications (Bautista et al., 2019). The results of the study, which was conducted on eight-week-old rats that were on a high-calorie diet, showed an increase in blood levels of triglycerides, total cholesterol, low-density lipoproteins, and an increase in body weight (Farias et al., 2019). These are all signs of the development of obesity, which also negatively affects the organs of the immune system. The effect of monosodium glutamate on the organs of the immune system is not sufficiently studied. Therefore, this study is relevant, because it is these organs that react to the penetration of foreign antigens into the body (Escobedo & Oliver, 2017; Begay et al., 2022; Habashy et al., 2021). The purpose of the study was to study the histological, morphometric and electron microscopic changes in the spleen of monosodium glutamate rats for four weeks. The study was conducted on 40 white male and female rats of reproductive age (2.5-3.5 months old) weighing 125-195 g. The normal structure of the spleen was studied in 10 intact animals, of which there were 5 male rats and 5 female rats. The results of the study As the results of our study showed, the structure of the spleen in animals of the intact and control groups corresponded to the species norm. The organ is surrounded by a capsule. The parenchyma is made of white and red pulp (Figure 1). The white pulp contains splenic lymphoid nodules and lymphoid periarterial sheaths. The red pulp is a place of blood deposition, contains venous sinuses and splenic cords. This is where the destruction of spent erythrocytes and other blood cells takes place. Morphometric indicators are presented in Tables 1, 2 (Table 1; Table 2). Splenic lymphoid nodules consist of a periarterial, mantle, marginal zone and a germinal center. In the splenic cords, B-lymphocytes are transformed into plasma cells, and monocytes into macrophages. The lymphoid component of the spleen includes small, middle, and large T- and B-lymphocytes, macrophages, and plasma cells. All cells have a typical structure (Figure 2). After four weeks of the experiment, both male and female rats showed signs of antigenic stimulation on histological preparations of the spleen, as evidenced by hyperplasia of the lymphoid component of the spleen, in particular. the intensity of the formation of germinal centers increased, the processes of proliferation and differentiation of lymphoid cells increased (Figure 3). A large number of active macrophages and apoptically changed cells are observed on histological sections of spleen preparations. The number of cells with signs of mitosis decreased. The venous sinuses of the spleen are enlarged and full of blood, accumulating hemosiderin. The cytoplasm of active macrophages is filled with drops of hemosiderin (Figure 4). The vessels of the hemomicrocirculatory channel also undergo changes. Arteries with a thickened wall, probably due to edema, their lumen is full of blood. Degeneratively changed erythrocytes are located around the vessels. The veins have a deformed shape, are dilated, the lumen is also full of blood. The venous sinuses in the red pulp are enlarged and contain accumulations of hemosiderin. It was established by the morphometric method that the relative area of the white pulp of the spleen after four weeks of the experiment increases in comparison with the intact group of animals by only 1.4% in male rats and by 0.91% in female rats. The relative area of lymphoid nodules of the spleen decreases by 1.95% and 1.25%, respectively. The relative area of lymphoid periarterial sheaths decreased by 4.65% and 2.6%. Introduction The experimental group of animals included 10 male rats and 10 female rats. They received a standard vivarium diet, to which monosodium glutamate was added daily for four weeks at a dose of 0.07 g/kg of rat body weight. The animals had free access to water and food. The control group of animals consisted of 5 male rats and 5 female rats, which received a physiological solution (0.9% NaCl solution) with a standard vivarium diet. All experimental animals were kept in the vivarium of Lviv National Medical University named Danylo Halytsky. The research was conducted in accordance with the provisions of the European Convention on the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, 1986), Council of Europe Directives 86/609/EEC (1986), Law of Ukraine No. 3447-IV On the Protection of Animals from Cruelty Handling, general ethical principles of experiments on animals, adopted by the First National Congress of Ukraine on Bioethics (2001). Morphometric studies were performed during the required period of the experiment on histological preparations stained with hematoxylin and eosin. VideoTest-5.0, KAARA Image Base, Stepanizer and Microsoft Excel programs were used. Statistical processing of digital data was performed with the help of the “Excel” software using the parametric method. Preparation of spleen preparations for microscopic and ultrastructural examination was carried out according to well-known methods. Spleen pieces were fixed with a 1.5% solution of osmium tetroxide in a 0.2 M solution of sodium cacodylate at pH 7.2 for 2-2.5 hours in the cold. Sections were made on an ultramicrotome UMTP-6M with a diamond knife (DIATOM), double contrast was performed according to Reynolds and uranyl acetate. The submicroscopic study 3 was carried out using a TEM-100 transmission electron microscope. Photo documentation using a SONY-H9 digital camera. Semi-thin sections with a thickness of 1-2 μm were made on an ultramicrotome LKB-3 (Sweden). They were stained with methylene blue. The results of the study The relative area of the red pulp of the spleen is only 0.49% and 0.33% less than the parameters of the intact group of animals (Table 1; Table 2). The relative area of the mantle and marginal zones on the histological preparation of the spleen section is 0.98% in male rats and 1.36% in female rats less than the indicator of the intact group of animals, the germinal center increases compared to the intact group of animals by 15.21% and 14.91% (p<0.001), the periarterial zone is only 0.88% more in male rats and 2.93% less in female rats than the indicator of the intact group of animals. The outer diameter of the central arteries after four weeks of the experiment increases and is 44.94% and 40.25%, respectively, significantly (p<0.001) more than the parameters of the intact group of animals. 4 The inner diameter of the central arteries decreases and is 3.11% and 2.58% less than the indicator in animals of the intact group (Tables 1; Table 2). After four weeks of the experiment, during the electron microscopic study of the spleen of white male and female rats of reproductive age, many active macrophages were found, their cytoplasm is loaded with fragments of the nucleus of other cells, parts of “undigested” formed blood elements, and contains numerous phagosomes. The proportion of reticular cells and connective tissue fibers both in the splenic trabeculae and in the splenic cords has increased, the walls of the splenic sinuses are thickened (Figure 5). The reticular cells’ nucleus is elongated, the contour of the nuclear envelope is uneven, tortuous, the processes of the cell envelope are thinned. Blood capillaries are filled with erythrocytes, arranged in a “coin column”, presumably due to the narrowing of the lumen, which is associated with edema of the wall. Endotheliocyte nucleus in the wall of hemocapillaries are also enlarged, swollen, the basement membrane is thickened, signs of perivascular edema are revealed (Figure 6). Discussion After four weeks of daily exposure to monosodium glutamate, an immunoinducing effect is observed with increased proliferation of activated lymphocytes and their subsequent differentiation into plasma cells. This is the morphological prerequisite for increased synthesis of immunoglobulins. We think that all the changes mentioned by us are the primary reaction of the immune organs to the daily administration of monosodium glutamate. Signs of edema of the parenchyma of lymph nodes are that the intercellular space is expanded and contains vacuole- like structures. It has been described in the literature that under conditions of exposure to a high-calorie diet, the main changes in visceral adipose tissue occurred due to a change in the relative population of immune cells, which resulted in a higher percentage of macrophages, dendritic cells, and CD8+ T cells. A high-calorie diet had a greater effect on visceral adipose tissue than on subcutaneous In the visceral lymph node, as a result of the development of obesity, the populations of cells that suppress immune function are reduced and the populations of those that regulate/activate the immune response are increased (Magnuson et al., 2017). The authors in their research showed that a high-calorie diet, compared to a normal standard diet, reduced the expression of CD20, a surface molecule present on B cells that plays an important role in the immune response and produces IL-10 mainly in the spleen. Moreover, splenocyte proliferation stimulated by T-cell and B-cell mitogens was significantly lower in obese subjects (Gotoh et al., 2017). The authors found that acute irradiation of the spleen using the inverse intensity modulated radiotherapy source axis distance irradiation technique had a protective effect on rats with traumatic brain injury. The spleen of the rats was precisely irradiated by a 6 MV X-ray with a total dose of 8 Gy. Initiation of splenic irradiation four hours after traumatic brain injury reduced splenic inflammatory response, alleviated brain edema, and improved behavioral scores (Huang et al., 2021). When studying the distribution of connective tissue fibers in the spleen of diabetic rats and rats treated with vitamin C, the accumulation of collagen fibers was found in the splenic 5 trabeculae, in the capsule and around the central artery and venous sinuses of the spleen. There was thickening of the splenic trabeculae due to fibrosis, not edema as in our case. Reticular fibers accumulated in both the white and red pulp of the spleen. Discussion A partial rupture of elastic fibers in the arterial wall was observed. A slight thickening of the reticular fibers was found in the group of animals corrected with vitamin C, and the elastic fibers maintained their integrity and were better organised than in the group of animals with diabetes (Ozerkan, 2021). Similar changes were found by the authors when studying the effect of Nano-Cu, an additive to animal feed. It is a potential antibacterial and growth-promoting material that can be used as an additional additive to animal feed. However, with the widespread use of nano-Cu, the risk of developing their unknown toxic side effects is becoming increasingly likely. The study revealed nano-Cu induced obvious spleen damage and oxidative-inflammatory and immune changes associated with activation of several pro-inflammatory responses, oxy/antioxidants and modulation of CD3+CD4+/CD3+CD8+T cell subtypes in rat spleen. Nano-Cu is more immunotoxic than conventional Cu sources, so it is not suitable as a long-term animal feed additive (Xerong et al., 2019). The literature mentions that the spleen decreases in size after a stroke in rodents. Splenectomy two weeks before ischemic and hemorrhagic stroke in mice and rats shows a reduction in infarct volumes. Proinflammatory mediators are also increased in the spleen and subsequently in the brain after stroke (Seifert & Offner, 2018). Prospects for further development are related to the study of histological, morphometric and ultrastructural changes in the spleen of rats under conditions of long-term exposure to monosodium glutamate and correction. Conclusions As a result of a study conducted on male and female rats of reproductive age, it was found that even short-term daily exposure to monosodium glutamate, namely four weeks, causes changes in the structural organization of the spleen. An immunoinducing effect is observed with increased proliferation of activated lymphocytes and their subsequent differentiation into plasma cells. There is a significant increase in the relative area of the germinal centers of splenic lymphoid nodules and the outer diameter of the central artery of the spleen. The share of active macrophages, apoptically changed cells increases. The red pulp is full-blooded, filled with hemosiderin. Conflict of interest The authors declare no conflict of interest. References: Bautista, R. J. H., Mahmoud, A. M., Kоnigsberg, M., Guerrero N., & Guerrero, L. D. (2019). Obesity: Pathophysiology, monosodium glutamate-induced model and anti-obesity medicinal plants. Biomedicine & Pharmacotherapy, 111, 503-516. https://doi.org/10.1016/j.biopha.2018.12.108 Conflict of interest The authors declare no conflict of interest. References: References: Bautista, R. J. H., Mahmoud, A. M., Kоnigsberg, M., Guerrero N., & Guerrero, L. D. (2019). Obesity: Pathophysiology, monosodium glutamate-induced model and anti-obesity medicinal plants. Biomedicine & Pharmacotherapy, 111, 503-516. https://doi.org/10.1016/j.biopha.2018.12.108 6 6 Begay, V., Cirovic, B., Barker, A. J., Klopfleisch, R., Hart, D. W., Bennett, N. C., & Lewin, G. R. (2022). Immune competence and spleen size scale with colony status in the naked mole- rat. Open Biology, 12, 210292. https://doi.org/10.1098/rsob.210292 Bhandari, U. (2018). Effect of embelin in monosodium glutamate induced obesity in male neonatal Wistar rats. Atherosclerosis Supplements, 32, 138. https://doi.org/10.1016/j.atherosclerosissup.2018.04.423 Bibik, E. Y., Shipilova, N. V., & Demenko, A. V. (2018). Melatonin as an effective pharmacocorrector of alimentary obesity resulting from a long-therm excessive of intake of palm oil. Research Result: Pharmacology and Clinical Pharmacology, 4(1), 51-58. Buchan, L., Aubin Ch., Fisher, A. L., Hellings, A., Castro, M., Al-Nakkash, L., Broderick, T. L., & Plochocki, J. H. (2018). High-fat, high-sugar diet induces splenomegaly that is ameliorated with exercise and genistein treatment. BMC Research Notes, 11, 752-758. https://doi.org/10.1186/s13104-018-3862-z Camacho, S, & Ruppel, A. (2017). Is the calorie concept a real solution to the obesity epidemic? Glob Health Action, 10(1), 1289650. https://doi.org/10.1080/16549716.2017.1289650 Coppey, L., Shevalye, H., Obrosov, A., Davidson, E., & Yorek, M. (2018). Determination of peripheral neuropathy in high-fat diet fed low-dose streptozotocin-treated female C57Bl/6J mice and Sprague-Dawley rats. Diabetes Investigation, 9(5), 1033-1040. https://doi.org/10.1111/jdi.12814 Escobedo, N., & Oliver, G. (2017). The lymphatic vasculature: Its role in adipose metabolism and obesity. Conclusions Cell Metabolism, 26(4), 598-609. https://doi.org/10.1016/j.cmet.2017.07.020 Farias, T. S. M., Cruz, M. M., Sa, R. C. C., Severi, I., Perugini, J., & Senzacqua, M. (2019). Melatonin supplementation decreases hypertrophic obesity and inflammation induced by high-fat diet in mice. Front Endocrinology, 10, 750. https://doi.org/10.3389/fendo.2019.00750 Finlayson, G. (2017). Food addiction and obesity: unnecessary medicalization of hedonic overeating. Nature Reviews. Endocrinology, 13(8), 493-498. https://doi.org/10.1038/nrendo.2017.61 Gotoh, K., Fujiwara, K., Anai, M., Okamoto, M., Masaki, T., Kakuma, T., & Shibata, H. (2017). Role of spleen-derived IL-10 in prevention of systemic low-grade inflammation by obesity. Endocrine, 64, 375-378. https://doi.org/10.1507/endocrj.EJ17-0060 Habashy, N. H., Kodous, A. S., & Abu-Serie, M. M. (2021). Targeting ROS/NF-κB signaling pathway by the seedless black Vitis vinifera polyphenols in CCl4-intoxicated kidney, lung, brain, and spleen in rats. Scientific Reports, 11, 16575. https://doi.org/10.1038/s41598-021- 96008-0 Huang, X., Lu, Y., Li, L., Sun, T., Jiang, X., Li, M., & Zhang, T. (2021). Protective effect of acute splenic irradiation in rats with traumatic brain injury. Neuroreport, 32(8), 711-720. https://doi.org/10.1097/WNR.0000000000001650 Magnuson, A. M., Regan, D. P., Fouts, J. K., Booth, A. D., Dow, S. W., & Foster, M. T. (2017). Diet-induced obesity causes visceral, but not subcutaneous, lymph node hyperplasia via increases in specific immune cell populations. Cell Proliferation, 50(5), 12365. https://doi.org/10.1111/cpr.12365 7 7 Ozerkan, D., Ozsoy, N., Cebesoy, S., & Ozer, C. (2021). Distribution of spleen connective tissue fibers in diabetic and vitamin C treated diabetic rats. Biotechnic & Histochemistry, 96(5), 347- 353. https://doi.org/10.1080/10520295.2020.1795718 Seifert, H. A., & Offner, H. (2018). The splenic response to stroke: from rodents to stroke subjects. Neuroinflammation, 15, 195. https://doi.org/10.1186/s12974-018-1239-9 Xerong, Z., Luo, J., Tang, H., Zhao, L., Xu, M., Wang, Y., Yang, X., Chen, H., Li, Y., Ye, G., Shi, F., Lv, Ch., & Jing, B. (2019). The toxic effects and mechanisms of nano-Cu on the spleen of rats. International Journal of Molecular Sciences, 20(6), 1469. https://doi.org/10.3390/ijms20061469 8 8 Appendix Appendix Figure 2. Electron-microscopic organization of a fragment of the red pulp of the spleen of a white male rat of the intact group of animals. Electron micrograph. Approx. ×8000. Designation: 1 – erythrocytes; 2 – heterochromatin and euchromatin (3) in the nucleus of a small lymphocyte; 4 – cytoplasm of a small lymphocyte; 5 – heterochromatin and euchromatin (6) in the nucleus of a middle lymphocyte; 7 – cytoplasm of a middle lymphocyte Figure 1. A fragment of the spleen of a white male rat from the intact group of animals. Conclusions Figure 2. Electron-microscopic organization of a fragment of the red pulp of the spleen of a white male rat of the intact group of animals. Electron micrograph. Approx. ×8000. Designation: 1 – erythrocytes; 2 – heterochromatin and euchromatin (3) in the nucleus of a small lymphocyte; 4 – cytoplasm of a small lymphocyte; 5 – heterochromatin and euchromatin (6) in the nucleus of a middle lymphocyte; 7 – cytoplasm of a middle lymphocyte Figure 2. Electron-microscopic organization of a fragment of the red pulp of the spleen of a white male rat of the intact group of animals. Electron micrograph. Approx. ×8000. Designation: 1 – erythrocytes; 2 – heterochromatin and euchromatin (3) in the nucleus of a small lymphocyte; 4 – cytoplasm of a small lymphocyte; 5 – heterochromatin and euchromatin (6) in the nucleus of a middle lymphocyte; 7 – cytoplasm of a middle lymphocyte Staining with hematoxylin and eosin. Approx. ×100. Designation: 1 – germinal center of the lymphoid nodule; 2 – mantle and marginal zones of the lymphoid nodule; 3 – the central artery of the spleen; 4 – lymphoid periarterial sheath; 5 – red pulp; 6 – capsule of the spleen Figure 3. A fragment of the spleen of a white male rat after four weeks of monosodium glutamate exposure. Staining with hematoxylin and eosin. Approx. ×50. Designation: 1 – capsule of the spleen; 2 – germinal center of the lymphoid nodule; 3 – mantle and marginal zones of the lymphoid nodule; 4 – splenic trabeculae; 5 – lymphoid periarterial sheath; 6 – pulpal artery; 7 – full blood red pulp Figure 3. A fragment of the spleen of a white male rat after four weeks of monosodium glutamate exposure. Staining with hematoxylin and eosin. Approx. ×50. Designation: 1 – capsule of the spleen; 2 – germinal center of the lymphoid nodule; 3 – mantle and marginal zones of the lymphoid nodule; 4 – splenic trabeculae; 5 – lymphoid periarterial sheath; 6 – pulpal artery; 7 – full blood red pulp Figure 3. A fragment of the spleen of a white male rat after four weeks of monosodium glutamate exposure. Staining with hematoxylin and eosin. Approx. ×50. Conclusions Designation: 1 – capsule of the spleen; 2 – germinal center of the lymphoid nodule; 3 – mantle and marginal zones of the lymphoid nodule; 4 – splenic trabeculae; 5 – lymphoid periarterial sheath; 6 – pulpal artery; 7 – full blood red pulp Figure 4. A fragment of the red pulp of the spleen of a white male rat after four weeks of monosodium glutamate exposure. Semi-thin cut. Staining with methylene blue. Approx. ×1000. Designation: 1 – polysegmened neutrophil; 2 – accumulation of erythrocytes in the splenic sinus; 3 – reticular cell; 4 – perivascular edema; 5 – "active" macrophage; 6 – apoptically changed macrophage, overloaded with hemosiderin residues; 7 – accumulation of lymphocytes; 8 – splenic trabeculae; 9 – erythrocytes in the thickness of the red pulp 9 Figure 5. Electron-microscopic organization of a red pulp fragment of the spleen of a white female rat after four weeks of monosodium glutamate exposure. Electron micrograph. Approx. ×4000. Designation: 1 – nucleus and cytoplasm (2) of a small lymphocyte in the lumen of the splenic sinus; 2 – erythrocytes in the lumen of the splenic sinus; 4 – the nucleus of the interdigitating cell in the wall of the splenic sinus; 5 – thickened wall of the splenic sinus; 6 – osmiophilic inclusions; 7 – nucleus and cytoplasm (8) of a reticular cell; 9 – erythrocytes outside the sinus; 10 – perivascular edema Figure 6. Electron-microscopic organization of a fragment of the white pulp of the spleen of a white male rat after four weeks of monosodium glutamate exposure. Electron micrograph. Approx. ×6000. Designation: 1 – lymphocyte nucleus and cytoplasm; 3 – perivascular edema; 4 – thickened basal membrane of the hemocapillary; 5 – swollen endotheliocyte nucleus in the blood capillary wall; 6 – erythrocytes in the lumen of a blood capillary Figure 5. Electron-microscopic organization of a red pulp fragment of the spleen of a white female rat after four weeks of monosodium glutamate exposure. Electron micrograph. Approx. ×4000. Conclusions Designation: 1 – nucleus and cytoplasm (2) of a small lymphocyte in the lumen of the splenic sinus; 2 – erythrocytes in the lumen of the splenic sinus; 4 – the nucleus of the interdigitating cell in the wall of the splenic sinus; 5 – thickened wall of the splenic sinus; 6 – osmiophilic inclusions; 7 – nucleus and cytoplasm (8) of a reticular cell; 9 – erythrocytes outside the sinus; 10 – perivascular edema Figure 6. Electron-microscopic organization of a fragment of the white pulp of the spleen of a white male rat after four weeks of monosodium glutamate exposure. Electron micrograph. Approx. ×6000. Designation: 1 – lymphocyte nucleus and cytoplasm; 3 – perivascular edema; 4 – thickened basal membrane of the hemocapillary; 5 – swollen endotheliocyte nucleus in the blood capillary wall; 6 – erythrocytes in the lumen of a blood capillary Table 1. Morphometric parameters of the structural components of the spleen of the studied male rats (M ± m) 10 10 Table 2. Morphometric parameters of the structural components of the spleen of the studied female rats (M ± m) Table 2. Morphometric parameters of the structural components of the spleen of the studied female rats (M ± m) 11
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Digital Object Identifier (DOI): 10.1038/ncomms9901 Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: Nature Communications Auditory sequence processing reveals evolutionarily conserved regions of frontal cortex in macaques and humans Citation for published version: Smith, K 2015, 'Auditory sequence processing reveals evolutionarily conserved regions of frontal cortex in macaques and humans', Nature Communications, vol. 6, 8901. https://doi.org/10.1038/ncomms9901 ARTICLE Received 23 Apr 2015 \| Accepted 14 Oct 2015 \| Published 17 Nov 2015 Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Results g In a previous behavioural study, rhesus macaques (three of which participated in the current fMRI experiment) were exposed to representative, consistent sequences (Fig. 1a). In the following testing phase the animals showed stronger orienting responses to sequences containing violations of the ordering relationships, relative to novel, consistent sequences (Fig. 1b, (ref. 8)). Here we conducted a comparable behavioural experiment in humans and found that, after exposure, human participants showed similar behavioural sensitivity to the violation sequences (Fig. 1c). Additional analyses of the human and macaque behavioural data show that these results cannot be explained by simple learning strategies in either species, such as responding to highly salient violations of the ordering relationships early in the sequences (Supplementary Fig. 2). Instead, both species seemed to be sensitive to illegal transitions between elements throughout the sequences, and the effects generalized to novel consistent sequences not heard prior to the testing phase (compare effects for familiar versus novel consistent sequences in Supplementary Fig. 2). Furthermore, in both species we observed stronger responses to sequences containing more unexpected, unpre- dictable transitions (Supplementary Fig. 3). Altogether, the behavioural results demonstrate that the pattern of responses and strategies employed appear to be comparable across species (Fig. 1, Supplementary Figs 2 and 3), suggesting that macaques and humans respond similarly, based on the statistical probability with which the transitions between elements occur during the exposure phase. Human functional magnetic resonance imaging (fMRI) studies have provided further evidence for the link between sequence learning paradigms and language processing, demonstrating that certain sequencing operations can engage comparable brain areas to those seen in natural language tasks, including ‘perisylvian’ frontal, temporal and parietal areas in and around the Sylvian ﬁssure or lateral sulcus2–4,27–35. Sequences containing violations of learned local ordering relationships, for example the transitions between adjacent elements in a sequence, engage ventral and opercular regions of frontal cortex2,28, which form a part of the ventral temporal to frontal lobe processing pathway4,36–38. These regions are also involved in processing similar short-distance grammatical relationships within or between adjacent phrases in natural languages4,32–34. Therefore, the ventral frontal and opercular cortex (vFOC) has been proposed to play a critical role in initial stages of human syntactic processing4. Results Artiﬁcial grammars generate rule-based sequences of stimuli, which can be designed to investigate different types of sequencing computations10–12. Representative sequences are presented to the participant so that the statistical properties of the ordering relationships between the sequence elements can be learned, often referred to as statistical learning or sequence processing13,14. It is broadly accepted that these processes are linked to and important for understanding language learning13,15,16, as well as being useful for understanding the neurobiology of language and the frontal cortex2,17,18. This has led to a view of language acquisition involving a critical parallel, possibly implicit, process of statistical extraction14,15. Moreover, statistical learning has been shown to correlate with performance on language tasks and is impaired in agrammatic aphasic patients, suggesting that such sequence processing shares neural mechanisms with language-related processes in the human brain16,19. There are also a large number of studies showing that sound sequence analysis is a critical determinant of language disorder—so that ‘low-level’ auditory analysis is relevant to ‘high-level’ disease20,21. In addition, shared sequence processing capabilities have been identiﬁed in humans and non-human animals8,11,22–24, which are noted as candidate language precursor abilities in non-human primates25,26. Therefore, although language-speciﬁc processes are unique and can only be studied in humans, comparative sequence processing and statistical learning studies remain important for identifying language-related processes that are evolutionarily conserved (Supplementary Fig. 1 and Supplementary Note 1). Macaque and human behaviour. In artiﬁcial grammar learning or sequence learning paradigms, participants are exposed to representative rule-based sequences that are ‘consistent’ with the artiﬁcial grammar, following which they are tested with sequences that are either ‘consistent’ or that contain illegal ‘violation’ transitions10. Stronger responses to sequences that violate the legal ordering of the elements relative to consistent sequences can provide evidence that implicit learning of the relationships between the stimuli in the sequences has occurred4,12,14. The current experiments used stimuli based on an artiﬁcial grammar paradigm previously used to test human infants11 and non- human primates8,11,24. The paradigm consists of branching relationships between several obligatory and optional elements (Fig. 1a). This produces considerable variability in the transitional probabilities between elements and emulates some of the properties of language and bird song11 (Supplementary Note 1). The stimuli consist of ﬁve auditory nonsense words that are arranged into sequences that are either ‘consistent’ with the ordering relationships or which contain illegal, ‘violation’ transitions between elements (Fig. 1a). ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 ARTICLE C entral to debates about the neurobiological origins of language is the question of how far the neural systems supporting human language functions depend upon evolutionarily conserved processes that are also present in our primate relatives1–3. Human neuroimaging experiments have led to the development of a number of neurobiological models of language processing4–6 and hypotheses about the evolution of the brain areas that support these functions2,4,7–9. Testing such evolutionary hypotheses requires evidence from cross-species functional imaging studies, using paradigms that can both evaluate abilities present in human and non-human animals and which are known to engage language-related processes in the human brain. relationships would engage vFOC comparably in humans and monkeys2,4,7–9. We conducted comparative fMRI sequence processing experiments in rhesus macaques and humans to test this hypothesis by assessing similarities and differences in how key ventral frontal brain regions, particularly vFOC and areas 44/ 45, might be recruited across species. We identiﬁed striking cross- species functional correspondences in the sequence processing functions of the vFOC. These results raise the possibility that language-critical processes in modern humans are functionally integrated with an ancestral, domain-general system that is involved in sequence processing. NATURE COMMUNICATIONS \| 6:8901 \| DOI: 10.1038/ncomms9901 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:8901 \| DOI: 10.1038/ncomms9901 \| www.nature.com/naturecommunications Auditory sequence processing reveals evolutionarily conserved regions of frontal cortex in macaques and humans Benjamin Wilson1,2, Yukiko Kikuchi1,2, Li Sun1, David Hunter1, Frederic Dick3, Kenny Smith4, Alexander Thiele1, Timothy D. Grifﬁths1, William D. Marslen-Wilson5 & Christopher I. Petkov1,2 An evolutionary account of human language as a neurobiological system must distinguish between human-unique neurocognitive processes supporting language and evolutionarily conserved, domain-general processes that can be traced back to our primate ancestors. Neuroimaging studies across species may determine whether candidate neural processes are supported by homologous, functionally conserved brain areas or by different neurobiological substrates. Here we use functional magnetic resonance imaging in Rhesus macaques and humans to examine the brain regions involved in processing the ordering relationships between auditory nonsense words in rule-based sequences. We ﬁnd that key regions in the human ventral frontal and opercular cortex have functional counterparts in the monkey brain. These regions are also known to be associated with initial stages of human syntactic processing. This study raises the possibility that certain ventral frontal neural systems, which play a signiﬁcant role in language function in modern humans, originally evolved to support domain-general abilities involved in sequence processing. 1 Institute of Neuroscience, Henry Wellcome Building, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. 2 Centre for Behaviour and Evolution, Henry Wellcome Building, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. 3 Department of Psychological Sciences, Birkbeck University of London, London, WC1E 7HX, UK. 4 School of Philosophy, Psychology and Language Sciences, University of Edinburgh, Edinburgh, EH8 9AD, UK. 5 Department of Psychology, University of Cambridge, Cambridge, CB2 3EB, UK. Correspondence and requests for materials should be addressed to C.I.P. (email: chris.petkov@ncl.ac.uk). 1 NATURE COMMUNICATIONS \| 6:8901 \| DOI: 10.1038/ncomms9901 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| 6:8901 \| DOI: 10.1038/ncomms9901 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. & 2015 Macmillan Publishers Limited. All rights reserved. & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 (c) Mean proportion of trials (±s.e.m.) in which 12 humans reported the testing sequences as ‘different’ relative to those heard during exposure (t11 ¼ 15.437, Po0.001). For additional behavioural results, see Supplementary Figs 2 and 3. Figure 1 \| The artiﬁcial grammar and monkey and human behaviour. (a) The artiﬁcial grammar is represented by a feed-forward transition graph with non-deterministic branching points. Sequences are constructed from ﬁve auditory elements (nonsense words). Consistent sequences (strings of nonsense words) are generated by following any path of arrows from START to END. Violation sequences do not follow the arrows, for details see ref. 8. Experiments began with the participants being exposed to representative exposure sequences, followed by a testing phase evaluating responses to consistent and violation sequences. (b) Mean proportion of trials (±s.e.m.) on which the three Rhesus macaques made unambiguous looking responses towards a concealed audio speaker, which presented either consistent or violation testing sequences. Monkey 1 was tested with a video coding approach, as part of an experiment conducted with a group of 13 animals, and Monkeys 2 and 3 were also tested individually with eye-tracking, for further details see refs 8,24. The macaques responded more frequently and for longer durations to violation sequences than the consistent ones. Monkey 1’s responses were representative of the signiﬁcant effects seen in the group analyses (t12 ¼ 7.898, Po0.001). Monkeys 2 and 3 each showed signiﬁcantly more responses to the violation sequences (t24 ¼ 3.137, P ¼ 0.004; t24 ¼ 3.129, P ¼ 0.005). (c) Mean proportion of trials (±s.e.m.) in which 12 humans reported the testing sequences as ‘different’ relative to those heard during exposure (t11 ¼ 15.437, Po0.001). For additional behavioural results, see Supplementary Figs 2 and 3. them to implicitly learn the statistical properties of those sequences14,16 (Supplementary Figs 2 and 3). After the exposure phase, the macaques were then scanned with fMRI as they listened to testing sequences that were consistent with or that violated the rule-based ordering relationships (Fig. 1a; Methods). Figure 2 shows the brain regions sensitive to the violation sequences for each animal (contrast: violation versus consistent, see Methods and Supplementary Note 2) mapped onto a surface- rendered standard macaque template brain. Several signiﬁcant clusters (Po0.05, cluster corrected) occurred in corresponding anatomical regions across the animals. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 START END A C D F C G G Nonsense words Artificial grammar A C D F G ‘klor’ ‘biff’ ‘jux’ ‘cav’ ‘dupp’ Testing sequences Consistent Violation ACGFC ADCFCG ACFCG ADCGFC AFGCD AFCDGC FADGC DCAFGC Exposure sequences ACF ACFC ACGF ACGFC ADCF ADCFC ADCFCG ADCGF ACGFCG a a Artificial grammar D F G ‘jux’ ‘cav’ ‘dupp’ ADCFCG ACFCG ADCGFC AFCDGC FADGC DCAFGC ACGF ACGFC ADCFCG ADCGF ACGFCG 0 0.5 1 1.5 2 2.5 0 5 10 15 20 25 30 35 0 5 10 15 20 25 Consistent Violation 0 5 10 15 20 25 30 35 Consistent Violation Monkey 3 - Eye tracking Monkey 2 - Eye tracking Mean time looking at speaker (%) Mean time looking at speaker (%) Consistent Violation Mean time looking at speaker (s) Mean proportion of responsive trials (%) Monkey 1 - Response frequency Monkey 1 - Response duration Macaque behaviour Human group results Consistent Violation 0 20 40 60 %‘‘Different” 80 100 Human behaviour b c Figure 1 \| The artiﬁcial grammar and monkey and human behaviour. (a) The artiﬁcial grammar is represented by a feed-forward transition graph with non-deterministic branching points. Sequences are constructed from ﬁve auditory elements (nonsense words). Consistent sequences (strings of nonsense words) are generated by following any path of arrows from START to END. Violation sequences do not follow the arrows, for details see ref. 8. Experiments began with the participants being exposed to representative exposure sequences, followed by a testing phase evaluating responses to consistent and violation sequences. (b) Mean proportion of trials (±s.e.m.) on which the three Rhesus macaques made unambiguous looking responses towards a concealed audio speaker, which presented either consistent or violation testing sequences. Monkey 1 was tested with a video coding approach, as part of an experiment conducted with a group of 13 animals, and Monkeys 2 and 3 were also tested individually with eye-tracking, for further details see refs 8,24. The macaques responded more frequently and for longer durations to violation sequences than the consistent ones. Monkey 1’s responses were representative of the signiﬁcant effects seen in the group analyses (t12 ¼ 7.898, Po0.001). Monkeys 2 and 3 each showed signiﬁcantly more responses to the violation sequences (t24 ¼ 3.137, P ¼ 0.004; t24 ¼ 3.129, P ¼ 0.005). Results By comparison, sequences with more complex relationships between elements engage not only the vFOC but also the adjacent Brodmann Areas 44 and 45, which belong to a dorsal processing pathway4,36–39 and are involved in hierarchical processes in natural language2,6,27–31, such as the nesting of a phrase within another phrase in a sentence. Macaque fMRI. Functional MRI was used to reveal the brain regions associated with detecting sequence ordering violations in macaques and humans. Three rhesus macaques were ﬁrst exposed to a representative set of consistent sequences (Fig. 1a), allowing The combination of human neuroimaging and animal behavioural work on sequence learning has led to the hypothesis that the processing of sequences requiring the tracking of local NATURE COMMUNICATIONS \| 6:8901 \| DOI: 10.1038/ncomms9901 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 2 \| \| \| & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 (c) Mean proportion of trials (±s.e.m.) in which 12 humans reported the testing sequences as ‘different’ relative to those heard during exposure (t11 ¼ 15.437, Po0.001). For additional behavioural results, see Supplementary Figs 2 and 3. 0 0.5 1 1.5 2 2.5 0 5 10 15 20 25 30 35 0 5 10 15 20 25 Consistent Violation 0 5 10 15 20 25 30 35 Consistent Violation Monkey 3 - Eye tracking Monkey 2 - Eye tracking Mean time looking at speaker (%) Mean time looking at speaker (%) Consistent Violation Mean time looking at speaker (s) Mean proportion of responsive trials (%) Monkey 1 - Response frequency Monkey 1 - Response duration Macaque behaviour Human group results Consistent Violation 0 20 40 60 %‘‘Different” 80 100 Human behaviour b c 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Consistent Violation Monkey 2 - Eye tracking Mean time looking at speaker (%) Mean proportion of responsive trials (%) Monkey 1 - Response frequency Macaque b b Consistent Violation Figure 1 \| The artiﬁcial grammar and monkey and human behaviour. (a) The artiﬁcial grammar is represented by a feed-forward transition graph with non-deterministic branching points. Sequences are constructed from ﬁve auditory elements (nonsense words). Consistent sequences (strings of nonsense words) are generated by following any path of arrows from START to END. Violation sequences do not follow the arrows, for details see ref. 8. Experiments began with the participants being exposed to representative exposure sequences, followed by a testing phase evaluating responses to consistent and violation sequences. (b) Mean proportion of trials (±s.e.m.) on which the three Rhesus macaques made unambiguous looking responses towards a concealed audio speaker, which presented either consistent or violation testing sequences. Monkey 1 was tested with a video coding approach, as part of an experiment conducted with a group of 13 animals, and Monkeys 2 and 3 were also tested individually with eye-tracking, for further details see refs 8,24. The macaques responded more frequently and for longer durations to violation sequences than the consistent ones. Monkey 1’s responses were representative of the signiﬁcant effects seen in the group analyses (t12 ¼ 7.898, Po0.001). Monkeys 2 and 3 each showed signiﬁcantly more responses to the violation sequences (t24 ¼ 3.137, P ¼ 0.004; t24 ¼ 3.129, P ¼ 0.005). NATURE COMMUNICATIONS \| 6:8901 \| DOI: 10.1038/ncomms9901 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 A, anterior; D, dorsal; P, posterior; V, ventral, see also Supplementary Figs 5 Left lateral Right medial Left medial Right lateral BA46 Anterior intraparietal area Macaque fMRI contrast: violation - consistent Monkey 1: Monkey 2: Monkey 3: 2.3 4.9 Cluster corrected Z-score 10–2 10–5 p-value Area 45 Area 44 Insula Area 6v Area 7 TS2 Insula Area 6v Area 7 TS2 Nucleus accumbens Putamen Caudate nucleus Cingulate cortex Caudate nucleus Area 46 Putamen Area 45 Area 44 b a c b c M d d Figure 2 \| Macaque brain regions sensitive to sequence ordering violations. Statistical parametric maps of sensitivity to sequence violations (contrast: violation versus consistent) displayed in each of the three macaques (a–c) and in a majority consensus voxel-overlap map (d), all Po0.05 cluster corrected (see Methods). Results are displayed on rendered lateral and medial surface representations transformed to a standard monkey brain in register with a macaque stereotactic atlas (Methods); light grey: gyri; dark grey: sulci. A, anterior; D, dorsal; P, posterior; V, ventral, see also Supplementary Figs 5 and 6. (vFOC, areas 44/45); Hemisphere (left, right); and Monkey (three levels). A signiﬁcant main effect of condition (F1,4886 ¼ 108.1, Po0.001) showed increased activation to violation relative to consistent sequences in these ROIs. There was no signiﬁcant interaction between condition and ROI (F1,4886 ¼ 1.149, P ¼ 0.284), suggesting that although macaque vFOC is strongly sensitive to sequence violations, and consistently so across the three animals, areas 44/45 are also involved to some extent. Regarding the lateralization of results, there was no signiﬁcant interaction between Condition and Hemisphere (F1,4886 ¼ 3.37, P ¼ 0.07), suggesting that the effects in these areas are not signiﬁcantly lateralized to either hemisphere (see Supplementary Note 3 for a summary of lateralisation results). These analyses were complemented with analyses of the results in each of the animals individually (Fig. 3b–g). The individual animal results recapitulated the overall ﬁndings, showing signiﬁcant activation to violation sequences relative to consistent sequences in the vFOC in all of the animals, and that statistically signiﬁcant activation was also observed in areas 44/45 in two of the three macaques. exposed to randomly generated sequences of nonsense words in which every transition between nonsense word elements occurred equally frequently. Following this randomized exposure, the animal was tested with testing sequences and scanning methods identical to those used in the original experiment (Figs 2 and 3). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 Regions sensitive to sequence violations in all three animals included right ventral frontal cortex, involving opercular and dysgranular insular cortex, ventral to Areas 44/45 (Fig. 2). Even in monkey 3 (M3), who shows the more restricted pattern of activation of the three monkeys (Fig. 2c), there is clear engagement of the dysgranular insula in the frontal operculum (Supplementary Fig. 4). In a them to implicitly learn the statistical properties of those sequences14,16 (Supplementary Figs 2 and 3). After the exposure phase, the macaques were then scanned with fMRI as they listened to testing sequences that were consistent with or that violated the rule-based ordering relationships (Fig. 1a; Methods). Figure 2 shows the brain regions sensitive to the violation sequences for each animal (contrast: violation versus consistent, see Methods and Supplementary Note 2) mapped onto a surface- rendered standard macaque template brain. Several signiﬁcant clusters (Po0.05, cluster corrected) occurred in corresponding anatomical regions across the animals. Regions sensitive to sequence violations in all three animals included right ventral frontal cortex, involving opercular and dysgranular insular cortex, ventral to Areas 44/45 (Fig. 2). Even in monkey 3 (M3), who shows the more restricted pattern of activation of the three monkeys (Fig. 2c), there is clear engagement of the dysgranular insula in the frontal operculum (Supplementary Fig. 4). In a majority of the animals signiﬁcant clusters of activation were also observed in additional ventral frontal regions (surrounding and including ventral area 6v), the anterior temporal lobe (for example, area TS2), and posterior parietal cortex area 7 (see Table 1 for a list of all the signiﬁcantly activated brain areas). These effects were further evaluated using planned region of interest (ROI) analyses. First, separate ROIs for the vFOC (vFOC, green ROI in Fig. 3a) and the adjacent areas 44/45 (blue ROI in Fig. 3a) were deﬁned using accepted stereotactic coordinates for these regions in a macaque anatomical atlase (Methods). The vFOC ROI included ventral frontal cortical areas adjacent and inferior to areas 44/45, including the frontal operculum, ventral BA6v and dysgranular insular cortex, but excluding areas 44/45, much of area 47/12c (ref. 40) and all of area 49. NATURE COMMUNICATIONS \| 6:8901 \| DOI: 10.1038/ncomms9901 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 A voxel-based repeated measures (RM) analysis of variance (ANOVA) was used to evaluate effects in the ROIs with the factors: Condition (consistent and violation sequences); ROI 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 D V P A D V A P Left lateral Right medial Left medial Right lateral BA46 Anterior intraparietal area Macaque fMRI contrast: violation - consistent Monkey 1: Monkey 2: Monkey 3: Majority consensus voxel overlap map 2.3 4.9 Cluster corrected Z-score 10–2 10–5 p-value Area 45 Area 44 Insula Area 6v Area 7 TS2 Insula Area 6v Area 7 TS2 Insula Area 6v Area 7 TS2 Insula Area 6v Area 7 TS2 TS1 Nucleus accumbens Putamen Caudate nucleus Cingulate cortex Caudate nucleus Area 46 Putamen Area 45 Area 44 Area 45 Area 44 Area 45 Area 44 b a c d e 2 \| Macaque brain regions sensitive to sequence ordering violations. Statistical parametric maps of sensitivity to sequence violations (contrast: on versus consistent) displayed in each of the three macaques (a–c) and in a majority consensus voxel-overlap map (d), all Po0.05 cluster corrected Methods). Results are displayed on rendered lateral and medial surface representations transformed to a standard monkey brain in register with a que stereotactic atlas (Methods); light grey: gyri; dark grey: sulci. A, anterior; D, dorsal; P, posterior; V, ventral, see also Supplementary Figs 5 and 6. D V P A D V A P Left lateral Right medial Left medial Right lateral BA46 Anterior intraparietal area Macaque fMRI contrast: violation - consistent Monkey 1: Monkey 2: Monkey 3: Majority consensus voxel overlap map 2.3 4.9 Cluster corrected Z-score 10–2 10–5 p-value Area 45 Area 44 Insula Area 6v Area 7 TS2 Insula Area 6v Area 7 TS2 Insula Area 6v Area 7 TS2 Insula Area 6v Area 7 TS2 TS1 Nucleus accumbens Putamen Caudate nucleus Cingulate cortex Caudate nucleus Area 46 Putamen Area 45 Area 44 Area 45 Area 44 Area 45 Area 44 b a c d Figure 2 \| Macaque brain regions sensitive to sequence ordering violations. Statistical parametric maps of sensitivity to sequence violations (co violation versus consistent) displayed in each of the three macaques (a–c) and in a majority consensus voxel-overlap map (d), all Po0.05 cluster cor (see Methods). Results are displayed on rendered lateral and medial surface representations transformed to a standard monkey brain in register macaque stereotactic atlas (Methods); light grey: gyri; dark grey: sulci. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 z score Cluster P value Hemisphere x y z Ventral frontal cortex 46 22 # 12 3.55 1.90 " 10 # 4 Right Frontal opercular cortex, frontal orbital cortex, frontal pole Ventral frontal cortex # 40 34 # 10 3.26 5.61 " 10 # 3 Left Frontal opercular cortex, frontal orbital cortex, frontal pole Middle temporal gyrus, angular gyrus and lateral occipital cortex # 58 # 44 0 3.80 7.29 " 10 # 5 Left Middle temporal gyrus and angular gyrus 52 # 64 24 3.87 5.34 " 10 # 5 Right Lateral occipital cortex 24 # 82 # 26 3.38 3.62 " 10 # 4 Right Frontal pole # 10 60 20 3.81 6.92 " 10 # 5 Bilateral Cingulate gyrus 0 # 48 26 3.64 1.38 " 10 # 4 Bilateral NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 ARTICLE Human fMRI. Twelve human participants were exposed to the representative consistent sequences and then were scanned using fMRI while listening to the same consistent or violation stimulus sequences used to test the macaques (Fig. 1a; Methods). Large signiﬁcant clusters (Po0.05, cluster corrected) that were sensitive to violation relative to consistent sequences (contrast: violation versus consistent; Fig. 4 and Table 1) were observed in both hemispheres in the ventral frontal cortex, including the frontal operculum, but was not evident in areas 44/45. Our results are consistent with previous ﬁndings showing that human vFOC is engaged in processing sequences with local adjacent transitions between elements2. Strong bilateral activations were also observed in the posterior parietal cortex BA39 and middle temporal gyrus, regions which have also been implicated in language (Supplementary Fig. 6) and compared these results to those with sequences containing multiple violations (Figs 1a,2b and 3c,f). In comparison to the main experiment (Figs 2 and 3), here we observed weaker but similar activation of the vFOC, suggesting that the response of this region scales with the number of violations in the testing sequences and is evident at least to some extent even with single violations in sequences that are otherwise ‘consistent’ (Supplementary Fig. 6). Together these control experiments demonstrate a graded increase in the fMRI response in the vFOC. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 We conﬁrmed that the auditory fMRI responses were matched in power between the original experiment and this control experiment, with both datasets showing a comparable sound versus silence response in auditory cortex (Supplementary Fig. 5a,b). In this control experiment, the previously noted vFOC response to the violation versus consistent testing sequences (Figs 2a and 3b,e) disappeared (Supplementary Fig. 5c,e). This observation demonstrates that the ventral frontal cortex response in the main experiments depends on exposing the animals to sequences containing predictable statistical relationships that can subsequently be used to identify the violation sequences. Therefore, the responses do not result from general deviance detection responses elicited by perceptual properties of the testing sequences (Supplementary Note 4). Rather, the violation sequences, by design, violate the ordering relationships between the sequence elements, which are estab- lished by the statistical regularities in the sequences heard during the exposure phase. q Two additional control experiments with two of the macaques further demonstrate that the vFOC response, (1) depends on prior exposure to representative, consistent sequences and (2) scales with the number of violations that occur in the testing sequences. Speciﬁcally, in the ﬁrst control experiment, M1 was In the second control experiment, we scanned M2 with violation testing sequences each containing only a single violation 4 4 ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 (Supplementary Fig. 6) and compared these results to those with sequences containing multiple violations (Figs 1a,2b and 3c,f). In comparison to the main experiment (Figs 2 and 3), here we observed weaker but similar activation of the vFOC, suggesting that the response of this region scales with the number of violations in the testing sequences and is evident at least to some extent even with single violations in sequences that are otherwise ‘consistent’ (Supplementary Fig. 6). Together these control experiments demonstrate a graded increase in the fMRI response in the vFOC. These results cannot be explained by a binary Human fMRI. Twelve human participants were exposed to the representative consistent sequences and then were scanned using fMRI while listening to the same consistent or violation stimulus sequences used to test the macaques (Fig. 1a; Methods). Large signiﬁcant clusters (Po0.05, cluster corrected) that were sensitive to violation relative to consistent sequences (contrast: violation versus consistent; Fig. 4 and Table 1) were observed in both hemispheres in the ventral frontal cortex, including the frontal operculum, but was not evident in areas 44/45. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 Our results are consistent with previous ﬁndings showing that human vFOC is Table 1 \| Anatomical locations of signiﬁcantly activated clusters in R. macaques and human participants. R. Macaques Anatomical location Stereotactic coordinates Cluster corrected max. z score Cluster P value Hemisphere x y z Monkey 1 Ventral frontal cortex 25 26 20.5 3.55 6.04 " 10 # 6 Right Area 45, area 44, dysgranular insula, area 6, putamen Temporo-parietal regions 14.5 8 17.5 4.16 1.03 " 10 # 3 Right Area 7, caudate nucleus (posterior), posterior auditory cortex (Pro, CM) Cingulate cortex 8 14.5 25 3.08 3.46 " 10 # 3 Right Anterior/posterior cingulate cortex, caudate nucleus Caudate nucleus 1.5 24 18 3.08 3.41 " 10 # 2 Bilateral Monkey 2 Ventral frontal cortex and anterior temporal lobe 27 21.5 11.5 4.89 1.3 " 10 # 3 Right Area 6v, dysgranular insula, anterior auditory cortex (TS2), somatosensory regions 1 and 2 Frontal pole # 4 24.5 20 3.93 3.86 " 10 # 4 Left Anterior intrapartietal area # 19.5 12.5 29.5 3.88 1.83 " 10 # 2 Left Anterior and ventral intraparietal areas Caudate nucleus and putamen 5 32.5 20.5 3.87 1.45 " 10 # 2 Right Temporo-parietal regions 16 1.5 25 3.52 1.21 " 10 # 4 Right Area 7, posterior auditory cortex (TPT) Nucleus accumbens 2.5 20.5 7 3.49 4.23 " 10 # 2 Bilateral Auditory cortex and insula 22 16 10.5 3.10 2.61 " 10 # 4 Right Auditory cortex (R, RM), dysgranular insula, putamen Monkey 3 Frontal opercular insular and temporal cortex 26 19 7.5 3.60 3.17 " 10 # 2 Right Anterior auditory cortex (TS1, TS2), dysgranular insula, frontal operculum Majority consensus (overlapping signiﬁcant voxels in 2 þ monkeys) Dysgranular insula 20.5 17 17 Right Area 6v 24.5 27 19.5 Right Anterior auditory cortex (TS2) 26.5 20.5 11.5 Right Area 7 12 0.5 23 Right Caudate nucleus and putamen 14.5 24 17 Right Humans Anatomical location MNI coordinates Cluster corrected max. ncomms9901 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:8901 \| DOI: 10.1038/ncomms9901 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 These results cannot be explained by a binary categorical or ‘deviant’ response, elicited by any sequence that contains a violation, and they provide further evidence that the fMRI response reﬂects the processing of the statistical properties of the sequences, learned during the prior exposure phase. NATURE COMMUNICATIONS \| 6:8901 \| DOI: 10.1038/ncomms9901 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 5 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 Mean difference in normalised z score (violation - consistent) Mean difference in normalised z score (violation - consistent) c Monkey 2 d vFOC Monkey 3 b vFOC Monkey 1 0 0.1 * * * 0.02 0.04 0.06 0.08 Left Right Hemisphere * n.s. n.s. 0 0.02 0.04 0.06 0.08 0.1 Left Right Hemisphere * *** n.s. 0 0.02 0.04 0.06 0.08 0.1 Left Right Hemisphere f Areas 44/45 Monkey 2 g Areas 44/45 Monkey 3 e Monkey 1 * n.s. 0 0.05 0.1 0.15 n.s. Left Right Hemisphere n.s. n.s. n.s. 0 0.05 0.1 0.15 Left Right Hemisphere *** * n.s. –0.05 –0.05 –0.05 0 0.05 0.1 0.15 Left Right Hemisphere PrCO Insula Area 12 Area 6v Insula Somatosensory regions Area 12 Area 6v Insula Somatosensory regions Area 12 Gustatory Cortex Area 6v Insula Area 12 Somatosensory regions Gustatory cortex Area 6v Insula Area 44 Area 45 Area 12 Somatosensory regions Area 46 z = 18 z = 16 z = 13 z = 11 z = 10 Area 6v Somatosensory regions Area 46 Area 45 R L A P z = 20 Details of ROIs a Area 44 Area 45 Areas 44/45 vFOC gure 3 \| Macaque ROI results. (a) Bilateral anatomically deﬁned ROIs used for analyses: blue comprises anatomical areas 44 and 45; green comprises jacent vFOC areas, including anatomical areas PrCO, dysgranular insula and area 6v (Methods). Somatosensory and gustatory regions, and area 12 rbital frontal cortex) were excluded. (b–g) Normalized mean fMRI response differences (violation versus consistent) in the vFOC and Areas 44/45 gions-of-interest shown by hemisphere in each of the macaques. Details of statistical analyses: One-sample t-tests with Bonferroni correction, in Monkey eft vFOC: t366 ¼ 3.827, Po0.001; right vFOC: t404 ¼ 4.155, Po0.001; Monkey 2, left vFOC: t658 ¼ 2.401, P ¼ 0.034; right vFOC: t702 ¼ 12.052, Po0.001 onkey 3, left vFOC: t632 ¼ 1.523, P ¼ 0.256; right vFOC: t658 ¼ 3.818, Po0.001. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 –0.05 0 0.05 0.1 0.15 Left Right Hemisphere Areas 44/45 g Areas 44/45 Monkey 3 n.s. n.s. n.s. 0 0.05 0.1 0.15 Left Right Hemisphere –0.05 Mean difference in normalised z score (violation - consistent) f Areas 44/45 Monkey 2 g Areas 44/45 Monkey 3 e Monkey 1 * n.s. 0 0.05 0.1 0.15 n.s. Left Right Hemisphere n.s. n.s. n.s. 0 0.05 0.1 0.15 Left Right Hemisphere *** * n.s. –0.05 –0.05 –0.05 0 0.05 0.1 0.15 Left Right Hemisphere Areas 44/45 g f A e Figure 3 \| Macaque ROI results. (a) Bilateral anatomically deﬁned ROIs used for analyses: blue comprises anatomical areas 44 and 45; green comprises adjacent vFOC areas, including anatomical areas PrCO, dysgranular insula and area 6v (Methods). Somatosensory and gustatory regions, and area 12 (orbital frontal cortex) were excluded. (b–g) Normalized mean fMRI response differences (violation versus consistent) in the vFOC and Areas 44/45 regions-of-interest shown by hemisphere in each of the macaques. Details of statistical analyses: One-sample t-tests with Bonferroni correction, in Monkey 1, left vFOC: t366 ¼ 3.827, Po0.001; right vFOC: t404 ¼ 4.155, Po0.001; Monkey 2, left vFOC: t658 ¼ 2.401, P ¼ 0.034; right vFOC: t702 ¼ 12.052, Po0.001; Monkey 3, left vFOC: t632 ¼ 1.523, P ¼ 0.256; right vFOC: t658 ¼ 3.818, Po0.001. Two-sample t-tests, between hemispheres in vFOC, in Monkey 1: t770 ¼ 1.279, P ¼ 0.201; Monkey 2: t1360 ¼ 5.771, Po0.001; Monkey 3: t1290 ¼ 1.839, P ¼ 0.066. One-sample t-tests with Bonferroni correction, in Monkey 1, left areas 44/45: t192 ¼ 8.000, Po0.001; right areas 44/45: t181 ¼ 2.438, P ¼ 0.032; Monkey 2, left areas 44/45: t276 ¼ 0.05, P ¼ 1.0; right areas 44/45: t282 ¼ 2.372, P ¼ 0.036; Monkey 3, left areas 44/45: t265 ¼ 0.583, P ¼ 0.561; right areas 44/45: t270 ¼ 0.331, P ¼ 0.741. Two-sample t-tests, between hemispheres in areas 44/45, in Monkey 1: t373 ¼ 0.977, P ¼ 0.329; Monkey 2: t558 ¼ 1.556, Po0.120; Monkey 3: t535 ¼ 0.653, P ¼ 0.514, see also Supplementary Figs 5 and 6. Symbols: n.s.,not signiﬁcant; Po0.05; Po0.01; *Po0.001. processing35,41 (see Table 1 for the full list of signiﬁcantly activated brain areas). condition (consistent and violation sequences), ROI (vFOC, areas 44/45) and hemisphere (left, right). A signiﬁcant main effect of condition showed strong fMRI activity for violation sequences in these regions (F1,3149 ¼ 150.478, Po0.001). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 Two-sample t-tests, between hemispheres in vFOC, in Monkey 1: 70 ¼ 1.279, P ¼ 0.201; Monkey 2: t1360 ¼ 5.771, Po0.001; Monkey 3: t1290 ¼ 1.839, P ¼ 0.066. One-sample t-tests with Bonferroni correction, in Monkey 1 ft areas 44/45: t192 ¼ 8.000, Po0.001; right areas 44/45: t181 ¼ 2.438, P ¼ 0.032; Monkey 2, left areas 44/45: t276 ¼ 0.05, P ¼ 1.0; right areas 44/45 82 ¼ 2.372, P ¼ 0.036; Monkey 3, left areas 44/45: t265 ¼ 0.583, P ¼ 0.561; right areas 44/45: t270 ¼ 0.331, P ¼ 0.741. Two-sample t-tests, between mispheres in areas 44/45, in Monkey 1: t373 ¼ 0.977, P ¼ 0.329; Monkey 2: t558 ¼ 1.556, Po0.120; Monkey 3: t535 ¼ 0.653, P ¼ 0.514, see also pplementary Figs 5 and 6. Symbols: n.s.,not signiﬁcant; Po0.05; Po0.01; Po0.001. PrCO Insula Area 12 Area 6v Insula Somatosensory regions Area 12 Area 6v Insula Somatosensory regions Area 12 Gustatory Cortex Area 6v Insula Area 12 Somatosensory regions Gustatory cortex Area 6v Insula Area 44 Area 45 Area 12 Somatosensory regions Area 46 z = 18 z = 16 z = 13 z = 11 z = 10 Area 6v Somatosensory regions Area 46 Area 45 R L A P z = 20 Details of ROIs a Area 44 Area 45 PrCO Insula Area 12 Area 6v Insula Somatosensory regions Area 12 Area 6v Insula Somatosensory regions Area 12 Gustatory Cortex Area 6v Insula Area 12 Somatosensory regions Gustatory cortex Area 6v Area 44 rea 45 a 12 atosensory egions 8 z = 16 z = 13 z = 11 z = 10 Area 44 Area 45 c Monkey 2 d vFOC Monkey 3 0 0.1 * * 0.02 0.04 0.06 0.08 Left Right Hemisphere * n.s. n.s. 0 0.02 0.04 0.06 0.08 0.1 Left Right Hemisphere * * n.s. Left Right Hemisphere vFOC c Monkey 2 0 0.1 * *** * 0.02 0.04 0.06 0.08 Left Right Hemisphere vFOC Mean difference in normalised z score (violation - consistent) b vFOC Monkey 1 * * n.s. 0 0.02 0.04 0.06 0.08 0.1 Left Right Hemisphere b d c f Areas 44/45 Monkey 2 * n.s. 0 0.05 0.1 0.15 n.s. Left Right Hemisphere –0.05 Mean difference in normalised z score (violation - consistent) e Monkey 1 *** * n.s. NATURE COMMUNICATIONS \| 6:8901 \| DOI: 10.1038/ncomms9901 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 (b) vFOC (green) and areas 44/45 (blue) ROIs. (c) Normalized mean ROI voxel response differences (violation versus consistent) in the fMRI signal for vFOC and areas 44/45 in both left and right hemispheres of the human brain. Additional analyses to investigate activation in these two ROIs independently showed that the vFOC region was strongly sensitive to sequence violations bilaterally, while the areas 44/45 region showed no signiﬁcant sensitivity to the violation sequences. One-sample t-tests with Bonferroni correction, in left vFOC: t916 ¼ 12.930, Po0.001; right vFOC: t971 ¼ 11.705, Po0.001; two-sample t-test between hemispheres in vFOC: t1887 ¼ 1.244, P ¼ 0.214. One-sample t-tests with Bonferroni correction, in left areas 44/45: t613 ¼ # 0.359, P ¼ 1.0; right areas 44/45: t650 ¼ 1.064, P ¼ 0.576; two-sample t-test between hemispheres in areas 44/45: t1262 ¼ 1.087, P ¼ 0.277, see also Supplementary Fig. 8. Symbols: n.s.,not signiﬁcant; *Po0.001. Details of ROIs b De b b D V P A D V A P 2.3 4.2 Cluster corrected z score 10–2 10–5 P value Angular gyrus Angular gyrus Cingulate cortex Cingulate cortex Middle temporal gyrus Middle temporal gyrus Ventral frontal opercular cortex Ventral frontal opercular cortex Frontal pole Frontal pole Frontal pole Frontal pole Left lateral a Right lateral Left medial Right medial Human fMRI contrast: violation - consistent Dorsal frontal Dorsal frontal a c 0 0.05 0.1 0.15 –0.05 0 0.05 0.1 Mean difference in normalised z score (violation - consistent) Mean difference in normalised z score (violation - consistent) Areas 44/45 Left Right Hemisphere Left Right * * n.s. n.s. n.s. n.s. ROI Analyses vFOC c RO c Right Hemisphere Hemisphere nsitive to sequence ordering violations. (a) Human group (n ¼ 12) statistical parametric map of sensitivity to sequence s consistent), all Po0.05 cluster corrected. Results are displayed on rendered medial and lateral surface representations Figure 4 \| Human brain regions sensitive to sequence ordering violations. (a) Human group (n ¼ 12) statistical parametric map of sensitivity to sequence violations (contrast: violation versus consistent), all Po0.05 cluster corrected. Results are displayed on rendered medial and lateral surface representations (light grey: gyri; dark grey: sulci) transformed to the Montreal Neurological Institute standard brain and using human anatomical atlases (Methods). (b) vFOC (green) and areas 44/45 (blue) ROIs. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 The sensitivity to violation sequences was not signiﬁcantly lateralized, as shown by no interaction between condition and hemisphere (F1,3149 ¼ 2.469, P ¼ 0.116). Greater activation to violation sequences occurred in the vFOC relative to the areas 44/45 region, shown by an interaction between condition and ROI (F1,3149 ¼ 127.353, Po0.001). These results were supported by separate analyses of the effects in these two ROIs (Fig. 4c). As in the macaques, these analyses conﬁrm the central role of vFOC in both hemispheres in detecting AG sequence violations. These To evaluate the effects in vFOC in relation to the adjacent areas 44/45 and to allow direct cross-species comparisons, we conducted planned ROI analyses identical to those performed in the macaques. An ROI including areas 44 and 45 (blue in Fig. 4b) was deﬁned using accepted atlases of human anatomical regions in stereotactic coordinates (see Methods). A vFOC ROI (green in Fig. 4b), adjacent and inferior to areas 44/45, was also deﬁned, including the frontal operculum, insular cortex and additional ventral frontal regions, but excluding areas 44/45 and much of BA47. As in the macaques, a voxel-based RM-ANOVA was used to evaluate effects in the ROIs, with the factors: NATURE COMMUNICATIONS \| 6:8901 \| DOI: 10.1038/ncomms9901 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 6 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 D V P A D V A P 2.3 4.2 Cluster corrected z score 10–2 10–5 P value Angular gyrus Angular gyrus Cingulate cortex Cingulate cortex Middle temporal gyrus Middle temporal gyrus Ventral frontal opercular cortex Ventral frontal opercular cortex Frontal pole Frontal pole Frontal pole Frontal pole Left lateral a Right lateral Left medial Right medial Human fMRI contrast: violation - consistent Dorsal frontal Dorsal frontal Details of ROIs b c 0 0.05 0.1 0.15 –0.05 0 0.05 0.1 Mean difference in normalised z score (violation - consistent) Mean difference in normalised z score (violation - consistent) Areas 44/45 Left Right Hemisphere Left Right * * n.s. n.s. n.s. n.s. ROI Analyses vFOC Figure 4 \| Human brain regions sensitive to sequence ordering violations. (a) Human group (n ¼ 12) statistical parametric map of sensitivity to sequence violations (contrast: violation versus consistent), all Po0.05 cluster corrected. Results are displayed on rendered medial and lateral surface representations (light grey: gyri; dark grey: sulci) transformed to the Montreal Neurological Institute standard brain and using human anatomical atlases (Methods). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 (c) Normalized mean ROI voxel response differences (violation versus consistent) in the fMRI signal for vFOC and areas 44/45 in both left and right hemispheres of the human brain. Additional analyses to investigate activation in these two ROIs independently showed that the vFOC region was strongly sensitive to sequence violations bilaterally, while the areas 44/45 region showed no signiﬁcant sensitivity to the violation sequences. One-sample t-tests with Bonferroni correction, in left vFOC: t916 ¼ 12.930, Po0.001; right vFOC: t971 ¼ 11.705, Po0.001; two-sample t-test between hemispheres in vFOC: t1887 ¼ 1.244, P ¼ 0.214. One-sample t-tests with Bonferroni correction, in left areas 44/45: t613 ¼ # 0.359, P ¼ 1.0; right areas 44/45: t650 ¼ 1.064, P ¼ 0.576; two-sample t-test between hemispheres in areas 44/45: t1262 ¼ 1.087, P ¼ 0.277, see also Supplementary Fig. 8. Symbols: n.s.,not signiﬁcant; *Po0.001. Note, however, that the individual monkey (Fig. 3) and human (Fig. 5) ROI analyses show areas 44/45 to be more variable in effect across participants. The vFOC effects we observe are robust and stable in response to the violation sequences across humans and monkeys. These observations suggest substantial correspon- dence between the macaque and the human results associated with sequence processing, especially in vFOC. data also suggest that human areas 44/45 are not signiﬁcantly engaged by violation sequences at the group level. Conducting individual human ROI analyses, as was done with the macaques, shows that the vFOC sensitivity to violations of the sequence ordering relationships is robust in most (9/12) of the human participants but that the effect in areas 44/45 is more variable across participants, being evident in six of the twelve human participants (Fig. 5). NATURE COMMUNICATIONS \| 6:8901 \| DOI: 10.1038/ncomms9901 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 Results of individual human ROIs from areas (a) vFOC (green) and (b) areas 44/45 (blue), shown as the difference in activation to the ‘violation’ minus ‘consistent’ sequences in each of the 12 human participants individually, ranked by mean activation across both ROIs (±s.e.m.). The vFOC shows a more consistent sensitivity to violations of the sequencing relationships than the areas 44/45 ROI, see text. Possibly due to the high levels of task performance by all of the human participants (mean performance ¼ 91%, s.d. ¼ 2.7%), no signiﬁcant correlation was observed between behavioural performance and activation in either the vFOC (r ¼ # 0.19, P ¼ 0.56) or areas 44/45 (r ¼ 0.11, P ¼ 0.73) ROIs. vFOC is argued to represent an initial stage in neurobiological processes related to human syntax4. This region is involved in processing relationships within and between adjacent phrases in natural language4,32–34. Sequences that violate an artiﬁcial grammar designed to model similar adjacent syntactic relationships in natural language22 also engage these ventral frontal and opercular human brain areas2,28. Thus, prior human neuroimaging studies have suggested that the vFOC , which lies adjacent to but does not include areas 44/45, is involved in ‘local structure building’, thought to represent a key initial stage of human language processing2,4,7. By contrast, sequences that violate more complex sequence relationships engage this region and also areas 44/45 (refs 2,27–31). Areas 44/45 are particularly engaged by syntactically and semantically complex natural language processes4–6,42,43 as well as other complex domain- general sequencing processes3,44. In parallel, non-human primates have been shown to be able to detect illegal, violation transitions between at least adjacent sequence elements8,11,22,24. There is also some evidence that New World monkeys and apes are sensitive to non-adjacent relationships45–47, although there is a paucity of evidence that any non-human animals are able to recognize sequences which model more of the complexity of human language. These studies led to the novel hypothesis, tested here, that local sequence processing abilities, and the ventral frontal opercular regions that support them in humans, might be functionally conserved in certain extant non-human primates2,4,7, reﬂecting a common evolutionary origin. signiﬁcantly greater areas 44/45 ROI engagement in the macaques relative to the humans in response to violations of the sequencing relationships. It remains possible that if more human participants were tested signiﬁcant activation might have been observed in human areas 44/45 at the group level. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 However, we are careful not to over interpret the variable activation pattern in areas 44/45 in the current experiments. Therefore, the primary observation in this study is the statistically robust activation in the vFOC in both the humans and monkeys. These results provide evidence supporting the hypothesis that vFOC is comparably involved in sequence processing in macaques and humans. This region appears to be speciﬁcally involved in evaluating the transitions between sequence elements, in the context of the regularities learned during the prior exposure. To test the frontal operculum evolutionary hypothesis4 it was necessary to select a sequence processing task with an appropriate level of complexity, which both humans and macaques learn48. In this study, we used a moderately complex sequencing paradigm that can generate a range of legal sequences of different lengths, containing a wide range of transitions between elements (Fig. 1a, ref. 8). Our human fMRI results show that these sequencing operations are complex enough to activate ventral frontal opercular regions, consistent with earlier human fMRI studies of adjacent sequencing2,28. Furthermore, our behavioural results demonstrate a striking correspondence between the responses of the macaques and the humans (Fig. 1b,c, Supplementary Figs 2 and 3), suggesting that (unlike the behavioural pattern we previously observed in another species of monkey, common marmosets8) both macaques and humans produce a similar pattern of responses. Neither the responses of the humans or the macaques can plausibly be attributed to reliance on simple cues (such as the novelty of the testing sequences, Supplementary Fig. 2), or by trivial acoustical differences between the consistent and violation sequences (Supplementary Fig. 5). Furthermore, we demonstrated that both the humans and macaques respond more strongly to sequences containing less predictable transitions, suggesting that both species track the statistical properties of the transitions throughout the sequences (Supplementary Fig. 3). Our fMRI results reveal that ventral and opercular regions of frontal cortex are comparably involved in auditory sequence processing operations in humans and macaques. This vFOC activation was statistically robust, and was consistently observed across all three macaques and in the human participants at the whole brain and ROI levels, including with individual monkey and human ROIs (Figs 2–5, Supplementary Figs 4 and 5). These results suggest that the sequence processing function of the vFOC is shared by humans and macaques. The role of areas 44/45 in this task was variable both within and between species. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 areas 44/45 vFOC Mean difference in normalised z score (violation - consistent) Paticipant areas 44/45 Paticipant vFOC –0.15 –0.1 –0.05 0 0.05 0.1 0.15 0.2 0.25 9 5 6 3 2 8 11 10 1 4 12 7 3 2 9 12 6 1 8 5 10 4 11 7 Figure 5 \| Individual human ROI results. Results of individual human ROIs from areas (a) vFOC (green) and (b) areas 44/45 (blue), shown as the difference in activation to the ‘violation’ minus ‘consistent’ sequences in each of the 12 human participants individually, ranked by mean activation across both ROIs (±s.e.m.). The vFOC shows a more consistent sensitivity to violations of the sequencing relationships than the areas 44/45 ROI, see text. Possibly due to the high levels of task performance by all of the human participants (mean performance ¼ 91%, s.d. ¼ 2.7%), no signiﬁcant correlation was observed between behavioural performance and activation in either the vFOC (r ¼ # 0.19, P ¼ 0.56) or areas 44/45 (r ¼ 0.11, P ¼ 0.73) ROIs. areas 44/45 vFOC Mean difference in normalised z score (violation - consistent) Paticipant areas 44/45 Paticipant vFOC –0.15 –0.1 –0.05 0 0.05 0.1 0.15 0.2 0.25 9 5 6 3 2 8 11 10 1 4 12 7 3 2 9 12 6 1 8 5 10 4 11 7 areas 44/45 Paticipant areas 44/45 Figure 5 \| Individual human ROI results. Results of individual human ROIs from areas (a) vFOC (green) and (b) areas 44/45 (blue), shown as the difference in activation to the ‘violation’ minus ‘consistent’ sequences in each of the 12 human participants individually, ranked by mean activation across both ROIs (±s.e.m.). The vFOC shows a more consistent sensitivity to violations of the sequencing relationships than the areas 44/45 ROI, see text. Possibly due to the high levels of task performance by all of the human participants (mean performance ¼ 91%, s.d. ¼ 2.7%), no signiﬁcant correlation was observed between behavioural performance and activation in either the vFOC (r ¼ # 0.19, P ¼ 0.56) or areas 44/45 (r ¼ 0.11, P ¼ 0.73) ROIs. Figure 5 \| Individual human ROI results. NATURE COMMUNICATIONS \| 6:8901 \| DOI: 10.1038/ncomms9901 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. Discussion Macaque and human fMRI comparisons. The human and monkey ROI results were directly compared using an RM- ANOVA with the factors: condition (consistent, violation), spe- cies (human, monkey), ROI (areas 44/45, vFOC) and hemisphere (left, right). This showed a signiﬁcant overall sensitivity in the ROIs to violation relative to consistent sequences (main effect of condition: F1,8043 ¼ 234.6, Po0.001). A signiﬁcant condition by ROI interaction was observed (F1,8043 ¼ 90.452, Po0.001) con- ﬁrming that the vFOC is more strongly activated than areas 44/45 across both species. A two-way interaction was observed between condition and species (F1,8043 ¼ 8.277, P ¼ 0.004), indicating that the human brain is relatively more sensitive to the sequence ordering violations than the macaque brain. Furthermore, a sig- niﬁcant interaction between condition, ROI and species was observed (F1,8043 ¼ 52.2, Po0.001), showing that areas 44/45 were statistically more involved in the monkeys than in the humans. These cross-species RM-ANOVA results for the vFOC and areas 44/45 ROIs are supported by two separate RM-ANO- VAs including each ROI separately (Supplementary Note 5). This study provides evidence that regions of vFOC are comparably functionally engaged in monkeys and humans by sequences that violate the ordering relationships of an auditory artiﬁcial grammar8,9. We used a comparative sequence processing paradigm and fMRI to provide a ﬁrst test of a prominent neurobiological hypothesis about the evolution of human brain regions involved in initial syntax-related processes between words in a sentence or evaluating adjacent sequence elements2,4,7,9. Since non-human animals lack human language abilities, the results of this comparative human and monkey fMRI experiment inform us on the domain-general processes, not speciﬁc to language, which macaques and humans both possess and whose neural substrates are identiﬁed. The results of these comparative analyses provide evidence that regions of vFOC support evolutionarily conserved sequence processing functions. Our human fMRI results also link to the larger body of evidence that the frontal opercular cortex is associated with certain forms of human language processing and domain-general processes, as we consider. 7 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 NATURE COMMUNICATIONS \| 6:8901 \| DOI: 10.1038/ncomms9901 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited All rights reserved NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 8), and also to the macaque experiment (Figs 2 and 3). While it is possible that these or other testing differences (for example, the macaques and humans, respectively, received either juice or money as reward for participating) could obscure potential similarities between the species, the highly consistent results we observe in vFOC, in both species and in all of the monkeys, are remarkable given the inevitable methodolo- gical differences in how the species were tested (Supplementary Note 7). Our data suggest that the engagement of vFOC in both species is a key property of a common neural response to the violation of the sequencing relationships, and that this is present independent of the responses required by different tasks. Although many language-related processes are left-lateralized in humans (especially those involving complex syntax), these experiments revealed no signiﬁcantly left-lateralized effects in either species. While some of the results are suggestive of potential right lateralization, statistical testing failed to provide consistent evidence that sequence processing in the key frontal areas is signiﬁcantly lateralized in either macaques or humans. For a summary of the observations on lateralisation effects see Supplementary Note 3. Previous hypotheses about the evolu- tionary origins of the functions supported by the ventral frontal cortex have primarily focussed on the left hemisphere2,4,7. However, the human and macaque results reported here suggest that the right hemisphere also plays an important role in sequence processing. More generally, these observations of a lack of strong lateralization provide the ﬁrst direct comparative evidence in support of a ‘dual neurobiological language systems’ hypothesis6,57,58, but extend it in important ways. This hypothesis proposes that in modern humans, specializations for core syntactic language functions depend on a left-lateralized ventral fronto-temporal system, and that this left-lateralized system is functionally integrated with a more bilaterally distributed network, that has more general language-related functions, such as those that combine semantic and syntactic operations in natural language6. Given that our results are based on an artiﬁcial grammar or sequence learning paradigm8,9, the current ﬁndings suggest that the hypothesized bi-hemispheric system also supports sequence processing in both humans and monkeys, forming an important part of the ancestral systems that underpin aspects of language function in the modern human. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 Activation in this region was observed in two out of the three macaques (Fig. 3e,f). No signiﬁcant activation was seen at the group level in humans (Fig. 4c), however, individual analyses revealed that areas 44/45 were engaged in 6 of the 12 human participants (Fig. 5). Furthermore, in the human RM-ANOVA group results, the vFOC ROI was signiﬁcantly more responsive to the violation sequences than the areas 44/45 ROI, and the across species RM-ANOVA analyses showed an interaction indicative of The form of sequence processing required by this paradigm is markedly different to ‘deviance detection’ or ‘oddball’ responses elicited by the appearance of an unexpected sound or pattern of sounds in a stream of auditory stimuli49. In these tasks detecting the deviant stimuli involves noticing a perceptual difference in the stream. By comparison, our paradigm contains no ‘oddball’ NATURE COMMUNICATIONS \| 6:8901 \| DOI: 10.1038/ncomms9901 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 8 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 monkeys (Fig. 3). See Supplementary Fig. 7 for ROI results from this region in humans and monkeys. The focus of the parietal clusters that we observed appears to involve BA7 in macaques and BA39 in humans, both of which lie below the intraparietal sulcus. There has been considerable uncertainty as to which dorsal parietal regions are functionally homologous59, given that human BA7 lies above the intraparietal sulcus. Our results suggest a level of functional correspondence between BA7 in macaques and BA39. Relatedly, MRI-based estimates of connections in the inferior parietal lobule in humans and macaques have shown similarities between different aspects of the inferior parietal lobule in the two species60. It is interesting to note that in the human brain this parietal region is engaged by a wide range of language tasks4,32,61,62. However, neuropsychological and neuroimaging evidence (for example, ref. 41) suggests that the human inferior parietal lobule, although providing a substantial role in sentence comprehension, is not as critical to the core syntactic language functions as the perisylvian inferior/ventral frontal regions. Therefore, the precise role of the parietal region in our task might well differ in relation to the role of the vFOC in both species, and would need to be further studied in the future. sounds in any of our sequences: the same ﬁve nonsense word elements appear in both the violation and consistent sequences. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 Therefore none of the individual elements represent ‘deviants’ in the same way as in oddball tasks; only the ordering relationships between the same set of elements is regulated or violated. Furthermore, the two types of paradigms appear to engage different brain networks49 (Supplementary Note 6). pp y A number of studies highlight the correspondences that can be drawn between the prefrontal cortex in humans and non-human primates, as well as current areas of uncertainty. The earlier comparative neuroanatomical studies by Brodmann and more recent efforts have identiﬁed anatomical correspondences between many prefrontal regions, such as between areas 44 and 45 in humans and non-human primates50,51. Other active ﬁelds of research involve understanding the comparative connectivity of frontal and other areas that in humans are known to be involved in language-related processes39,52. Some authors have predicted that during human language evolution the interconnectivity between frontal and other areas increased53, which also predicts functional differences in otherwise cytoarchitectonically corresponding areas, such as area 44 (ref. 54). Our comparative behavioural and fMRI results in humans and macaques contribute to these efforts by providing functional insights on ventral frontal cortex regions, highlighting the role of the frontal operculum, which is a less well studied region of the frontal cortex. We also present results using both anatomically deﬁned ROI analyses (Figs 3 and 4) and whole- brain analyses, unconstrained by any particular parcellation scheme (Figs 2 and 4, Table 1), highlighting the correspondences in activation patterns at particular locations in the brains of the monkeys and humans across the species. This information could now be used as seed regions to determine the cross-species correspondences in anatomical pathways that are currently under question37,55,56. p It is also important to consider the impact of differences in the way the species were tested on the results, since it is not possible to conduct all measurement in the same way with both species. The behavioural results show that despite different testing methods after the exposure period, both species responded similarly to the statistical properties of the test sequences (Fig. 1, Supplementary Figs 2 and 3). Furthermore, fMRI data obtained from human participants scanned using the same ﬁxation task as the macaques produced activation in the ventral frontal cortex that is very similar to the effects observed using the button press task in the main human experiment (compare Fig. 4 with Supplementary Fig. & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:8901 \| DOI: 10.1038/ncomms9901 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:8901 \| DOI: 10.1038/ncomms9901 \| www.nature.com/naturecommunications Methods ll l All animal work and procedures performed were approved by the Animal Welfare and Ethical Review Body at Newcastle University and by the U.K. Home Ofﬁce. The work complies with the Animal Scientiﬁc Procedures Act (1986) on the care and use of animals in research, and with the European Directive on the protection of animals used in research (2010/63/EU). We support the principles on reporting animal research stated in the consortium on Animal Research Reporting of in vivo Experiments (ARRIVE). All persons involved in this project were Home Ofﬁce certiﬁed and the work was strictly regulated by the U.K. Home Ofﬁce. Human participants provided informed consent to participate in this study, which was approved by the human studies Ethical Review Body at Newcastle University and which conformed with the 2013 WMA Declaration of Helsinki. Three macaques were scanned for this experiment. All three of the macaques had participated in behavioural Experiment 1 (M1, M2 and M3, Fig. 1), and two in behavioural Experiment 2 using eye-tracking (M2 and M3). All the animals were male, aged 9, 14 and 6 years, respectively, weighing 6–12 kg, and lived in a colony of pair housed macaques. The animals were fMRI trained to complete trials of an established sparse-imaging/stimulation sequence with visual ﬁxation and without requirement of an explicit response during the auditory stimulation66,67. Each scanning session began with a 30 min exposure phase where the exposure sequences were presented binaurally over headphones (NordicNeuroLab) in a randomized order (rate of 9 sequences/min; inter-sequence interval ¼ 4 s; Fig. 1). The fMRI scanning was conducted using a sparse-imaging fMRI paradigm66, whereby the stimuli could be presented in relative silence and the stimulus-related volume followed each stimulus sequence by 3.5 s to account for the hemodynamic response lag68. In the scanner, the animals were presented with a randomized sampling of consistent and violation test sequences (40% each type of trial, Fig. 1) or silent trials (20% of trials). Each scanning run consisted of 30 trials, before which the monkeys were re-familiarized with the exposure sequences for 3 min. The number of trials obtained in each animal depended on their motivation to participate in the ﬁxation trials for a juice reward. Unexpected animal-related issues resulted in ﬁnal datasets with different numbers of completed trials (M1: 160 trials; M2: 1410 trials; M3: 780 trials). Methods ll l Nonetheless, all three animals showed signiﬁcant cluster corrected activity responses, including several clusters for M1 who had completed the fewest trials. We also noted that that there was no obvious relationship between the number of scanning runs and the sound versus silence response in auditory cortex (r ¼ 0.11, P40.9), or the violation versus consistent contrast in the vFOC (r ¼ # 0.30; P40.7). Stimuli. Each of the stimulus sequences (see Fig. 1a) was constructed by digitally combining recordings of naturally spoken nonsense words (that is, the elements in the artiﬁcial grammar sequences) produced by a female speaker and recorded with an Edirol R-09HR (Roland Corp.) sound recorder. The amplitude of the recorded sounds was r.m.s. balanced and the nonsense word stimuli were combined into sequences using customized Matlab scripts (100-ms inter-stimulus intervals). The experiments were coded in Matlab (Psychophysics Toolbox: http://psychtoolbox.- org/) and Cortex software (Salk Institute). All sounds were presented at B75 dB SPL (calibrated with an XL2 sound level meter, NTI Audio). For details on the sound delivery for the behavioural experiments, see: ref. 8. During scanning of both humans and monkeys, sound delivery was achieved through MRI-compatible headphones (NordicNeuroLab). The consistent and violation sequence were balanced for duration and element composition, for details see ref. 8. Brieﬂy, the duration of the naturally spoken nonsense word stimuli within the sequences was as follows: Klor ¼ 0.64 s; Jux ¼ 0.62 s; Cav ¼ 0.56 s; Biff ¼ 0.40 s; Dupp ¼ 0.39 s. The consistent and violation sequence sets were balanced in the number of elements in the sequences (Fig. 1a) and the mean lengths (±s.d.) of the sequences were comparable: consistent sequences, 3.14 (0.42) sec; violation sequences, 3.25 (0.28) sec. We conﬁrmed that there was no signiﬁcant difference in sequence sound duration between consistent and violation sequences (independent samples t-test, t6 ¼ 0.44, P ¼ 0.68), or in the duration of the individual elements present in con- sistent versus violation sequences (t42 ¼ 0.61, P ¼ 0.55; ref. 8). Macaque behavioural experiment 1: video coding. Thirteen rhesus macaques (age range: 4–16 year-old) in two group housed colonies were exposed to the nine exposure sequences presented in random order for 2 h the evening before the experiment (Fig. 1a). The following morning they were refamiliarized with these sequences for 10 min. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 any of our results (Supplementary Note 6), such processes may well engage the human and monkey vFOC once transitional regularities are encoded. Future studies might tease apart the contributions of different cognitive processes involved in various forms of sequence processing. Statistical or sequence learning paradigms remain appealing because they can create speciﬁc prediction errors (violations) in sequencing relationships (Supplementary Fig. 6; also see ref. 65) that can be studied at the neuronal level in animal models and, as we show, linked to processes in humans by way of comparative behaviour and neuroimaging (fMRI or electroencephalography65). Macaque behavioural experiment 2: eye-tracking. Three rhesus macaques (aged 6, 6 and 14-year-old) that had previously participated in the video-coding beha- vioural experiment were individually tested in an eye-tracking experiment using a head post for head immobilization and infra-red eye-tracking system (Arrington Research Inc.), for additional details see: ref. 8. Following 30 min of exposure to representative consistent sequences (Fig. 1), the animals participated in a simple ﬁxation task. After ﬁxating on a central ﬁxation spot for 2 s to obtain a juice reward, the ﬁxation spot would disappear and on 25% of trials the animals were presented with a randomly selected test sequence presented from either the right or left audio speaker. Looking responses toward the presenting speaker were recorded and the durations of these responses were analysed8. No feedback of any sort was given to the macaques for their behavioural responses to the testing sequences. g g g y In summary, the results of these experiments provide the ﬁrst evidence supporting the hypothesis that functions of key regions of vFOC are evolutionarily conserved in macaques and humans. These regions play an important role in sequence processing, and, as shown in a number of prior human neuroimaging studies, are also involved in analysing local grammatical relationships in the human brain. We conclude that vFOC in both species supports neuro-computational functions which include evaluating sensory sequences in relation to the probabilities with which the transitions between elements previously occurred. This raises the possibility that language-related processes in modern humans are functionally integrated with highly conserved, originally non-linguistic pro- cesses shared with our extant primate relatives. Human behavioural experiment. ARTICLE Twelve human participants (mean age 23; 6 female, 6 male, all right handed) were exposed for 5 min to the same exposure sequences as the monkeys (stimuli and rate of presentation were identical between experiments; Fig. 1). The participants then listened to the testing sequences (Fig. 1a). Following each testing stimulus sequence presentation the human par- ticipants responded by using button presses to classify the test sequence heard as either following the ‘same’ pattern as the exposure sequences (consistent) or a ‘different’ pattern (violation). No feedback of any sort was given to the participants for their behavioural responses to the testing sequences. Macaque MRI procedures. Measurements of the fMRI blood oxygen level dependant (BOLD) signal were made on a non-human primate dedicated, vertical 4.7 T research MRI scanner (Bruker BioSpin, Etlingen, Germany). The monkeys sat in a primate chair in the scanner. To minimize the impact of head movements on the MRI results, the subjects were acclimated to periods of head immobilization using a head post that had previously been surgically implanted under anaesthesia and in aseptic conditions (for additional details see refs 66,67). Signals were acquired using a birdcage RF coil. Functional data were acquired using a gradient- recalled echo planar imaging sequence with the following typical parameters: echo time, (TE): 21 ms; sparse imaging, volume acquisition time: 2 s, volume repetition time: 10 s; ﬂip angle: 90!; 16 slices, 2 mm thickness; in-plane ﬁeld of view: 12.8 " 9.6 cm, on a grid of 128 " 96 voxels, with a voxel resolution of 1 " 1 " 2 mm. Anatomical images were acquired in register with each functional scanning experiment using a 3D T1-weighted MDEFT sequence with parameters TE: 6 ms; repetition time: 750 ms; inversion delay: 700 ms; 22 slices; in-plane ﬁeld of view: 12 8 " 9 6 cm2 on a grid of 256 " 192 voxels with voxel resolution of NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 p p q y The extent to which language-speciﬁc and cognitive-domain- general processes overlap and share common processes in the human brain is an active area of research that informs us on the function of the frontal cortex. For example, Fedorenko et al.3, have suggested that domain-general subregions of area 44 lie next to regions that are more involved in language-speciﬁc functions3. Other studies have shown that the human inferior frontal gyrus and operculum are involved in cognitive processes such as attention and task control44,63,64. The type of comparative study that we conducted can help to link these disparate strands of research (Supplementary Fig. 1) and provide important inputs into future developments. For example, human neuroimaging studies aiming to distinguish domain-general from language- speciﬁc processes often use hierarchically organized natural language material. Our study points to the greater need for such studies to compare sequence processing tasks of different levels of complexity, some of which, as we show, can now be linked to a primate model system. Sequence processing paradigms can also readily incorporate visual or auditory stimuli and thus inform us on sequencing processes that are sensory modality independent. Moreover, although we found little evidence for non-speciﬁc deviance-detection responses in The neurobiological hypotheses that we tested here focussed on vFOC and areas 44/45. However, we also observed activation in broadly comparable perisylvian regions, including parietal regions, in the humans (Fig. 4) and two out of three of the 9 ARTICLE NATURE COMMUNICATIONS \| 6:8901 \| DOI: 10.1038/ncomms9901 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 In the scanner the participants were presented with the same consistent and violation test sequences as the macaques (40% each of type of trial, Fig. 3) and silent trials (20% of trials). As in the behavioural experiment, participants were asked to respond ‘same’ or ‘different’ using an MRI-compatible response box during the fMRI scanning. Each scanning run included 50 stimulus trials (200 trials total for each participant). Following each of the scanning runs (50 trials) the participants were re-exposed to the exposure sequences for 2–3 min before continuing with the next scanning run. g 19. Christiansen, M. H., Louise Kelly, M., Shillcock, R. C. & Greenﬁeld, K. Impaired artiﬁcial grammar learning in agrammatism. Cognition 116, 382–393 (2010). 20. Grube, M., Kumar, S., Cooper, F. E., Turton, S. & Grifﬁths, T. D. Auditory sequence analysis and phonological skill. Proc. Biol. Sci. 279, 4496–4504 (2012). y g 21. Goll, J. C., Crutch, S. J. & Warren, J. D. Central auditory disorders: toward a neuropsychology of auditory objects. Curr. Opin. Neurol. 23, 617 (2010). 22 Fit h W T & H M D C t ti l t i t t ti 22. Fitch, W. T. & Hauser, M. D. Computational constraints on syntacti processing in a nonhuman primate. Science 303, 377–380 (2004). 22. Fitch, W. T. & Hauser, M. D. Computational constraints on syntactic processing in a nonhuman primate. Science 303, 377–380 (2004). 23. Gentner, T. Q., Fenn, K. M., Margoliash, D. & Nusbaum, H. C. Recursive syntactic pattern learning by songbirds. Nature 440, 1204–1207 (2006). 24. Wilson, B., Smith, K. & Petkov, C. I. Mixed-complexity artiﬁcial grammar learning in humans and macaque monkeys: evaluating learning strategies. Eur. J. Neurosci. 41, 568–578 (2015). Human fMRI data analyses. We performed ﬁrst-level, ﬁxed-effects General Linear Analysis (FEAT analysis implemented in FSL) contrasting BOLD responses to violation versus consistent sequences at the individual level and registered to a standard template human brain (Montreal Neurological Institute, standard in FSL). The individual data were then analysed using a higher level mixed-effects group analysis using a cluster corrected (Po0.05) threshold level (5 mm smoothing, FWHM). Results were then registered and projected to a Free Surfer (http://sur- fer.nmr.mgh.harvard.edu/) surface-based representation of a template human brain. The results were highly comparable when the data were analysed using a ﬁxed-effects model. Table 1 also shows that the clusters (see peak z scores and P values) survive higher signiﬁcance thresholds. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 Grammatical pattern learning by human infants and cotton-top tamarin monkeys. Cognition 107, 479–500 (2008). 12. Pothos, E. M. Theories of artiﬁcial grammar learning. Psychol. Bull. 133, 227 (2007). 13. Frost, R., Armstrong, B. C., Siegelman, N. & Christiansen, M. H. Domain generality versus modality speciﬁcity: the paradox of statistical learning. Trends Cogn. Sci. 19, 117–125 (2015). 14. Saffran, J. R., Aslin, R. N. & Newport, E. L. Statistical learning by 8-month-old infants. Science 274, 1926–1928 (1996). Human MRI procedures. Measurements of the fMRI BOLD signal were made on a human dedicated clinical/research 3 T scanner (Phillips). Functional data were acquired using a gradient-recalled echo planar imaging sequence with the following typical parameters: echo time, TE: 30 ms; sparse imaging, volume acquisition time: 2 s, volume repetition time: 10 s; ﬂip angle: 90!; 28 slices, 4.5 mm thickness; in- plane ﬁeld of view: 19.2 " 19.2 cm2, on a grid of 64 " 64 voxels, with voxel reso- lution of 3 " 3 " 4.5 mm. T1-weighted anatomical images were acquired in register with each functional scanning experiment with parameters TE: 4.6 ms; repetition time: 9.6 ms; 150 slices; in plane ﬁeld of view 25.1 " 25.1 cm2, on a grid of 288 " 288 voxels; with voxel resolution of 0.87 " 0.87 " 1.2 mm. 15. Go´mez, R. L. & Gerken, L. Infant artiﬁcial language learning and language acquisition. Trends. Cogn. Sci. 4, 178–186 (2000). acquisition. Trends. Cogn. Sci. 4, 178–186 (2000) 16. Conway, C. M. & Pisoni, D. B. Neurocognitive basis of implicit learning of sequential structure and its relation to language processing. Ann. N. Y. Acad. Sci. 1145, 113–131 (2008). 17. Petersson, K. M., Folia, V. & Hagoort, P. What artiﬁcial grammar learning reveals about the neurobiology of syntax. Brain Lang. 120, 83–95 (2012). 18. Folia, V., Forkstam, C., Ingvar, M., Hagoort, P. & Petersson, K. M. Implicit artiﬁcial syntax processing: genes, preference, and bounded recursion. Biolinguistics 5, 105–132 (2011). The twelve participants (six female, six male) who took part in the behavioural experiment were scanned in the fMRI experiment between 1 and 2 weeks following behavioural testing. Before scanning the participants were presented with the exposure sequences in a randomized order for 10 min (rate of nine sequences permin; inter-sequence interval ¼ 4 s; Fig. 3a). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 harvard.edu/) surface-based representation of a standard template monkey brain69. For display purposes, a majority consensus map was used to summarize the macaque whole-brain results (Fig. 2d). This was created by identifying common voxels which were signiﬁcantly activated (Po0.05 cluster corrected) in at least two of the three animals and plotting the average z-score values on the rendered surfaces. 6. Marslen-Wilson, W. D., Bozic, M. & Tyler, L. K. in Cognitive neuroscience 5th Ed (edsGazzaniga, M. S., Davies, G., Ivry, R. B. & Mangun, G. R.) 639–648 (MIT Press, 2014). 7. Friederici, A. D. Processing local transitions versus long-distance syntactic hierarchies. Trends Cogn. Sci. 8, 245–247 (2004). g 8. Wilson, B. et al. Auditory artiﬁcial grammar learning in macaque and marmoset monkeys. J. Neurosci. 33, 18825–18835 (2013). ROIs were anatomically deﬁned in reference to a R. macaque atlas of anatomical regions in stereotactic coordinates40. One ROI consisted of areas 44 and 45. The other ROI consisted of the vFOC, including areas PrCO, area 6v and the dysgranular insula in the frontal operculum (see Fig. 3a). Excluded from this ROI were somatosensory areas 1, 2 and 3, the gustatory cortex, areas 44/45 and orbital frontal areas. These ROIs were then registered back to the animals’ own functional imaging data. This helped to avoid overinﬂating the number of voxels used for analysis (than if the higher resolution standard anatomical brain had been used). However, in this case, given the functional imaging scan resolution of 1 " 1 " 2 mm3 where the slice thickness is greater than the in-plane resolution, the animals’ results presented with some differences in the numbers of voxels in each ROI available for analysis. The average BOLD signal in response to violation versus consistent sequences was calculated for each voxel in the ROIs. These values were normalized based on the maximum activation observed in either ROI in each animal. These data were analysed with RM- ANOVAs and t-tests using Bonferroni corrections for multiple comparisons. marmoset monkeys. J. Neurosci. 33, 18825–18835 (2013). 9. Petkov, C. I. & Wilson, B. On the pursuit of the brain network for proto- syntactic learning in non-human primates: conceptual issues and neurobiological hypotheses. Philos. Trans. R. Soc. Lond., B, Biol. Sci. 367, 2077–2088 (2012). 10. Reber, A. S. Implicit learning of artiﬁcial grammars. J. Verbal Learning Verbal Behav. 6, 855–863 (1967). 11. Saffran, J. et al. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9901 No gender differences were observed in the fMRI results (Supplementary Note 8). 25. Fitch, W. T., Friederici, A. D. & Hagoort, P. Pattern perception and computational complexity: introduction to the special issue. Philos. Trans. R. Soc. Lond., B, Biol. Sci. 367, 1925–1932 (2012). 26. Hurford, J. R. The Origins of Grammar: Language in the Light of Evolution II (Oxford University Press, 2012). 27. Makuuchi, M., Bahlmann, J., Anwander, A. & Friederici, A. D. Segregating the core computational faculty of human language from working memory. Proc. Natl Acad. Sci. USA 106, 8362–8367 (2009). 28. Bahlmann, J., Schubotz, R. I. & Friederici, A. D. Hierarchical artiﬁcial grammar processing engages Broca’s area. Neuroimage 42, 525–534 (2008). 29. Petersson, K.-M., Folia, V. & Hagoort, P. What artiﬁcial grammar learning reveals about the neurobiology of syntax. Brain Lang. 120, 83–95 (2012). ROI analyses were conducted as follows. ROIs for Areas 44/45 and vFOC in both hemispheres were deﬁned on the standard brain. The human anatomical regions were deﬁned using human atlases of anatomical regions in stereotactic coordinates (Harvard-Oxford Cortical Structural Atlas and the Juelich Histological Atlas72) (ref. 70). The vFOC ROI was deﬁned encompassing parts of the inferior frontal cortex ventral to areas 44/45, including the frontal operculum and the insula, but excluding areas 44 and 45 (Fig. 4). To ensure the results were comparable across species, the average BOLD activation in each ROI was normalized and analysed in the same way as the macaque data. 30. Forkstam, C., Hagoort, P., Fernandez, G., Ingvar, M. & Petersson, K. M. Neural correlates of artiﬁcial syntactic structure classiﬁcation. Neuroimage 32, 956–967 (2006). g g correlates of artiﬁcial syntactic structure classiﬁcation. Neuroimage 32, 956–967 (2006). ( ) 31. Petersson, K. M., Forkstam, C. & Ingvar, M. Artiﬁcial syntactic violations activate Broca’s region. Cogn. Sci. 28, 383–407 (2004). g g 32. Friederici, A. D. & Kotz, S. A. The brain basis of syntactic processes: functional imaging and lesion studies. Neuroimage 20, S8–17 (2003). 33. Ni, W. et al. An event-related neuroimaging study distinguishing form and content in sentence processing. J. Cogn. Neurosci. 12, 120–133 (2000). ARTICLE ARTICLE Methods ll l They were then presented with 32 test sequences (4 ran- domized repetitions each of the 4 consistent and 4 violation sequences, Fig. 1a) from a concealed audio speaker. The macaques’ looking responses towards this speaker were video recorded. These responses were all independently blind-coded ofﬂine by three raters using a Likert scale. Orienting responses were calculated based on the proportion of trials on which at least two out of three of the raters agreed that an unambiguous response, caused by the stimulus presentation, had occurred8. Macaque fMRI data analysis. For each animal we performed a ﬁrst-level, ﬁxed- effects General Linear Model analysis (FEAT, FSL) contrasting BOLD responses to violation versus consistent sequences. This involved motion correction and regis- tration of the functional images to the macaque’s own high resolution anatomical scan, via intermediate scans including a lower resolution anatomical scan that was in register with the functional scans. The results of the ﬁrst-level GLM analyses were then registered to a standard macaque template brain69, which is in register with an accepted macaque atlas in stereotactic coordinates, to determine the anatomical ﬁelds within which the signiﬁcant activity clusters occurred40. The results were then combined into higher level analyses, evaluated at the cluster corrected (Po0.05) level (2 mm smoothing full-width half maximum, FWHM). 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https://openalex.org/W1976866991	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0004157&type=printable	English	null	A Novel Form of Memory for Auditory Fear Conditioning at a Low-Intensity Unconditioned Stimulus	PloS one	2,009	cc-by	9,224	Abstract Fear is one of the most potent emotional experiences and is an adaptive component of response to potentially threatening stimuli. On the other hand, too much or inappropriate fear accounts for many common psychiatric problems. Cumulative evidence suggests that the amygdala plays a central role in the acquisition, storage and expression of fear memory. Here, we developed an inducible striatal neuron ablation system in transgenic mice. The ablation of striatal neurons in the adult brain hardly affected the auditory fear learning under the standard condition in agreement with previous studies. When conditioned with a low-intensity unconditioned stimulus, however, the formation of long-term fear memory but not short- tem memory was impaired in striatal neuron-ablated mice. Consistently, the ablation of striatal neurons 24 h after conditioning with the low-intensity unconditioned stimulus, when the long-term fear memory was formed, diminished the retention of the long-term memory. Our results reveal a novel form of the auditory fear memory depending on striatal neurons at the low-intensity unconditioned stimulus. ation: Kishioka A, Fukushima F, Ito T, Kataoka H, Mori H, et al. (2009) A Novel Form of Memory for Auditory Fear Conditioni conditioned Stimulus. PLoS ONE 4(1): e4157. doi:10.1371/journal.pone.0004157 Editor: David S. Vicario, Rutgers University, United States of America Editor: David S. Vicario, Rutgers University, United States of America Received September 9, 2008; Accepted November 23, 2008; Published January 9, 2009 Copyright: 2009 Kishioka et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by Grant-in-Aid for Scientific Research on Priority Areas (Molecular Brain Science) and Global COE Program (Integrative Life Science Based on the Study of Biosignaling Mechanisms) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. F.F. was supported by Japan Society for the Promotion of Science. 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: mishina@m.u-tokyo.ac.jp * E-mail: mishina@m.u-tokyo.ac.jp ¤a Current address: Department of Molecular Neuroscience, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan ¤b Current address: Laboratory of Experimental Animal Model Research, National Center for Geriatrics and Gerontology, Obu, Japan cortico-amygdala pathway [13]. Abstract Evidence from many studies suggests that the amygdala—in particular, the lateral/basolateral nuclei—plays an essential role in the acquisition, storage and expression of fear memory [1,7,14–18]. Ayumi Kishioka1, Fumiaki Fukushima1, Tamae Ito1, Hirotaka Kataoka1, Hisashi Mori1¤a, Toshio Ikeda2¤b, Shigeyoshi Itohara2, Kenji Sakimura3, Masayoshi Mishina1* 1 Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan, 2 Laboratory of Behavioral Genetics, Brain Science Institute, RIKEN, Saitama, Japan, 3 Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata, Japan Introduction Fear is one of the most potent emotional experiences of our lifetime and is an adaptive component of response to potentially threatening stimuli, serving a function that is critical to the survival of higher vertebrates [1,2]. Too much or inappropriate fear, however, accounts for many common psychiatric problems [3–5]. A fearful experience can establish an emotional memory that results in permanent behavioral changes and emotional memories have been observed in many animal groups [6]. The brain mechanisms underlying fear are similar in different species and the fear system will respond similarly in a person or a rodent, using a limited set of defense response strategies [7]. The memory of learned fear can be assessed quantitatively using a Pavlovian fear- conditioning paradigm [1,2]. During fear conditioning, an initially neutral conditioned stimulus (CS, e.g. an auditory tone) acquires biological significance by becoming associated with an aversive unconditioned stimulus (US, e.g. a footshock). After learning this association, an animal responds to the previously neutral CS with a set of defensive behavioral responses, such as freezing. Anatomical tracing and lesion studies demonstrated the impor- tance of the amygdala for fear conditioning [8–10]. Subsequent physiological experiments showed that learning produces pro- longed synaptic modification in both of the inputs to the amygdala: the thalamo-amygdala pathway [11,12] and the Here, we developed an inducible striatal neuron ablation system in transgenic mice and examined the effect of striatal neuron ablation on auditory fear conditioning with different intensities of US. Under the standard condition, the ablation of striatal neurons in the adult brain hardly affected the auditory fear conditioning in agreement with previous studies [18–22]. We found, however, that under a weak condition, the formation of long-term auditory fear memory but not short-term memory was impaired by the ablation of striatal neurons. Our results suggest the presence of two forms of auditory fear memories distinguished by the US intensity and by the requirement of striatal neurons. Our finding that striatal neuron ablation diminished the auditory fear conditioning only when the US was weak is intriguing since the striatum is supposed to play a role in incorporating the positive or negative value of information into the determination of behavioral responses. A Novel Form of Memory for Auditory Fear Conditioning at a Low-Intensity Unconditioned Stimulus Ayumi Kishioka1, Fumiaki Fukushima1, Tamae Ito1, Hirotaka Kataoka1, Hisashi Mori1¤a, Toshio Ikeda2¤b, Shigeyoshi Itohara2, Kenji Sakimura3, Masayoshi Mishina1* PLoS ONE \| www.plosone.org Inducible ablation of striatal neurons We then crossed the Gc7-mCrePR mouse with a knock-in mouse (Eno2-STOP-DTA) in which the Cre-inducible diphtheria toxin A gene (DTA) was introduced into the neuron-specific enolase gene (Eno2) locus [26]. In Gng7+/mCrePR mice, one allele retains the intact Gng7 gene, and the other is inactivated by insertion of the CrePR gene. We injected 1 mg per g body weight of RU-486 into the peritoneum of Gc7-mCrePR6Eno2-STOP-DTA mice at postnatal day 42 (P42) to induce the recombinase activity of CrePR [24,25,27] (Fig. 2A). Mock-injected mice served as controls. Ten days after RU- 486 injection, TUNEL staining showed strong signals throughout the striatum, including the CP, NAc and olfactory tubercle (Fig. 2B). On the other hand, no TUNEL-signals were detectable in the striatum of the mock-injected mice. Both RU-486- and mock-treated mice showed faint TUNEL-signals in the olfactory bulb probably Medium-spiny projection neurons, the main output neurons, account for up to 90% of neurons in the striatum [29,30]. There were no detectable immunoreactivities for calbindin, a marker for medium-sized spiny neurons [31], in the mutant striatum (Fig. 4A). Medium-spiny projection neurons in the striatum can be largely subdivided into two groups: some that project to directly to the substantia nigra pars reticulata (SNr) (the direct pathway) express substance P; others that project to the same nucleus via the globus pallidus (GP) (the indirect pathway) express enkephalin [29]. These two neuropeptides are anterogradely transported to the axon terminals in the afferent regions [32]. There were no detectable immunoreactivities for substance P and enkephalin in Figure 1. Generation of Gc7-Cre and Gc7-mCrePR mice. A, Schema of the exon 4 region containing the translational initiation site of the Gng7 gene, targeting vector, and targeted allele. The targeting vector carries the cre or mCrePR gene and the neo gene flanked by two frt sequences. A, ApaI; EV, EcoRV; K, KpnI; S, SpeI. B, LacZ expression following Cre recombination. X-gal-staining of sagittal and coronal sections from Gng7+/cre; +/CAG- CAT-Z mice at postnatal day 14. Sections were counterstained with nuclear fast red. Abbreviations: Ce, cerebellum; Cx, cortex; Hi, hippocampus; Po, pons; St, striatum; Th, thalamus. Scale bars, 1 mm. doi:10.1371/journal.pone.0004157.g001 Figure 1. Generation of Gc7-Cre and Gc7-mCrePR mice. A, Schema of the exon 4 region containing the translational initiation site of the Gng7 gene, targeting vector, and targeted allele. The targeting vector carries the cre or mCrePR gene and the neo gene flanked by two frt sequences. Generation of striatum-specific Cre mouse lines The G-protein c7 subunit mRNA is expressed predominantly in medium spiny neurons of the caudate-putamen (CP) and nucleus PLoS ONE January 2009 \| Volume 4 \| Issue 1 \| e4157 PLoS ONE \| www.plosone.org 1 Novel Auditory Fear Memory due to the turnover of adult-generated olfactory granule cells [28]. In addition, Gc7-mCrePR mice exhibited no detectable TUNEL signals in the striatum upon RU-486 injection (data not shown). These results suggest that RU-486 treatment successfully induced recombination by CrePR, leading to cell ablation in the adult brain in the striatum-specific manner. Gc7-CrePR-mediated recombina- tion appeared to be critically dependent on target mice since b- galactosidase staining was hardly detectable in Gc7-mCrePR6 CAG-CAT-Z11 mice upon induction. accumbens (NAc) and neurons of the olfactory tubercle [23]. To develop a striatal neuron-specific gene manipulation system, we produced Gc7-Cre and Gc7-mCrePR mouse lines by inserting the gene encoding Cre recombinase or Cre recombinase-progesterone receptor fusing protein (CrePR) into the translational initiation site of the G-protein c7 subunit gene (Gng7) through homologous recombination in embryonic stem cells derived from the C57BL/6 strain [24] (Fig. 1A). We then crossed the Gc7-Cre and Gc7- mCrePR mice with the CAG-CAT-Z11 reporter mouse [25]. Brain slices prepared from Gc7-Cre6CAG-CAT-Z11 mice were stained for b-galactosidase activity to monitor the Cre recombi- nase activity. Strong b-galactosidase staining was found predom- inantly in the CP, NAc and olfactory tubercle. Faint signals were detected in the layer 5 of the neocortex and subiculum (Fig. 1B). On the other hand, no b-galactosidase staining was detectable in brain slices from Gc7-mCrePR6CAG-CAT-Z11 mice upon induction of Cre recombinase activity by RU-486 administration. Thirteen days after RU-486 treatment, TUNEL signals in the striatum became undetectable in Gc7-mCrePR6Eno2-STOP- DTA mice. We then quantitatively examined the ablation of striatal neurons by immunohistochemical staining for NeuN, a marker protein for neurons. The density of NeuN-positive neurons in the CP drastically decreased by 13 days after RU-486 injection (F6,54 = 99.5, P,0.001, one-way ANOVA) and remained at a very low level thereafter (Fig. 3A–C). The number of NeuN-positive cells in the NAc core and shell also decreased with a similar time course (Fig. 3B,D). However, NeuN immunostaining signals in other brain regions including the amygdala were comparable between mock- and RU-486-treated mice (Fig. 3B,E). January 2009 \| Volume 4 \| Issue 1 \| e4157 Inducible ablation of striatal neurons A, ApaI; EV, EcoRV; K, KpnI; S, SpeI. B, LacZ expression following Cre recombination. X-gal-staining of sagittal and coronal sections from Gng7+/cre; +/CAG- CAT-Z mice at postnatal day 14. Sections were counterstained with nuclear fast red. Abbreviations: Ce, cerebellum; Cx, cortex; Hi, hippocampus; Po, pons; St, striatum; Th, thalamus. Scale bars, 1 mm. doi:10.1371/journal.pone.0004157.g001 PLoS ONE \| www.plosone.org January 2009 \| Volume 4 \| Issue 1 \| e4157 2 Novel Auditory Fear Memory SNr and GP, respectively, of RU-486-treated mice (Fig. 4B,C), Motor activity Figure 2. Inducible ablation of striatal neurons. A, Schema for striatal neuron ablation induced by RU-486 administration. B, TUNEL staining (green) counterstained with DAPI (blue) in brain sections of control (left) and mutant (right) mice 10 days after mock and RU-486 administration, respectively. Scale bars, 1 mm. Abbreviations: Ce, cerebellum; Cx, cortex; Hi, hippocampus; Po, pons; St, striatum; Th, thalamus. doi:10.1371/journal.pone.0004157.g002 Figure 2. Inducible ablation of striatal neurons. A, Schema for striatal neuron ablation induced by RU-486 administration. B, TUNEL staining (green) counterstained with DAPI (blue) in brain sections of control (left) and mutant (right) mice 10 days after mock and RU-486 administration, respectively. Scale bars, 1 mm. Abbreviations: Ce, cerebellum; Cx, cortex; Hi, hippocampus; Po, pons; St, striatum; Th, thalamus. doi:10.1371/journal.pone.0004157.g002 Figure 2. Inducible ablation of striatal neurons. A, Schema for striatal neuron ablation induced by RU-486 administration. B, TUNEL staining (green) counterstained with DAPI (blue) in brain sections of control (left) and mutant (right) mice 10 days after mock and RU-486 administration, respectively. Scale bars, 1 mm. Abbreviations: Ce, cerebellum; Cx, cortex; Hi, hippocampus; Po, pons; St, striatum; Th, thalamus. doi:10.1371/journal.pone.0004157.g002 Impairment of auditory fear conditioning with a low- intensity footshock Mutant mice were subjected to auditory fear conditioning to examine the possible involvement of striatal neurons in the formation of the emotional memory. Fourteen days after RU-486 treatment, mutant mice were trained for auditory fear conditioning (Fig. 6A). Mice were given a single pairing of tone (CS) and footshock (US; 0.5 mA) on the conditioning day (Fig. 6B). Twenty-four hours after the conditioning, the mice were placed in a novel chamber. Six min after placement, the tone was delivered for 3 min. Mice exhibited a range of conditioned fear responses including freezing. Levels of freezing during the pre-tone period were comparable between mutant and control mice (F1,15 = 2.28, P = 0.15). Freezing responses to the tone were also similar between mutant and control mice (control, 31.665.1%; mutant, 28.065.1%; F1,15= 0.27, P = 0.61) (Fig. 6B). Thus, mutant mice successfully acquired fear memory under the standard condition despite of almost complete ablation of striatal medium spiny neurons. We further investigated the ability of mutant mice to acquire fear memory under a less intensive condition. Mice were trained with a single paring of the tone and a low-intensity footshock at 0.3 mA, and tested for the freezing response 24 h after training. Negligible levels of freezing were observed during the pre-tone period in control and mutant mice as well as RU-486-treated Gc7- mCrePR mice (RU-486 control). However, there were significant differences in the freezing responses across the CS presentation among 3 groups of mice (control, 29.764.9%; RU-486 control, 31.564.9%; mutant, 13.662.7%; F2,23 = 6.57, P = 0.006) (Fig. 6C). The freezing levels of mutant mice were much lower than those of control mice (P,0.05, mutant vs. control; P,0.01, mutant vs. RU-486 control; Post-hoc analysis). Comparable levels of freezing between control and RU-486-control mice indicated that treatment of RU-486 itself exerted little effect on the fear conditioning. There were no significant differences among control, RU-486 control, and mutant mice in pain thresholds for flinch and jump reactions (flinch, F2,16 = 0.094, P = 0.91, one-way ANOVA; jump, F2,16 = 0.021, P = 0.98) (Fig. 6D). The post-shock activity bursts [40] of mutant and control mice were also similar (at Figure 3. NeuN-immunohistochemstry. A, Immunohistochemical analysis for neuronal marker NeuN in control (left) and mutant (right) mice 13 days after mock and RU-486 administration, respectively. Scale bar, 1 mm. B, Higher magnification of NeuN-immunohistochemistry in various brain regions. Scale bars, 0.1 mm. Motor activity SNr and GP, respectively, of RU-486-treated mice (Fig. 4B,C), suggesting that any striatal output scarcely remains in the basal ganglia of the mutant mice. Along with the NeuN-immunohisto- chemistry, our results suggest that induction of CrePR-mediated DTA expression by RU-486 injection successfully ablated almost completely the medium spiny neurons that comprise approxi- mately 90% of the NeuN-positive striatal neurons within 13 days. In subsequent analyses, we used Gc7-mCrePR6Eno2-STOP- DTA mice from 13 to 22 days after RU-486 administration as striatal neuron-ablated mutant mice and corresponding mock- injected littermates served as controls. The striatum is intimately involved in motor control. The striatal neuron-ablated mutant mice showed no ataxic gait or tremor and could walk along a straight line as control did (control, n = 4; mutant, n = 4) (Fig. 5A). There was no significant difference in the performance in the stationary thin rod test [33] between mutant and control mice (F1,15 = 1.38, P = 0.26, repeated measures ANOVA) (Fig. 5C). Thus, the ablation of striatal neurons appeared to exert little effect on motor coordination under standard conditions at least for a week after loss of ,90% striatal neurons. In the accelerating rotarod test [34], both mutant and PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org January 2009 \| Volume 4 \| Issue 1 \| e4157 3 Novel Auditory Fear Memory control mice performed equally well in the first training session (F1,14 = 3.57, P = 0.08, one-way ANOVA) (Fig. 5D). Despite that approximately 90% of striatal neurons were ablated, the motor performance of the mutant mice appeared to be comparable to that of control mice in stationary thin rod and rotating rod tests. In subsequent sessions of the accelerating rotarod test, however, there was a significant difference in the retention time between two groups (F1,14 = 37.2, P,0.001, repeated measures ANOVA). Control mice showed a steady and rapid improvement in their performance over the training. In contrast, mutant mice failed to exhibit any improvements over trials, suggesting that the striatal neurons are indispensable for motor learning. Our results are consistent with the observation that striatum-specific NMDA receptor mutant mice showed impaired motor learning in an accelerating rotarod test [35]. In the open field test, the locomotor activity of mutant mice tended to be higher than that of control mice (F1,15 = 4.6, P = 0.05) (Fig. 5E). Figure 3. NeuN-immunohistochemstry. Motor activity A, Immunohistochemical analysis for neuronal marker NeuN in control (left) and mutant (right) mice 13 days after mock and RU-486 administration, respectively. Scale bar, 1 mm. B, Higher magnification of NeuN-immunohistochemistry in various brain regions. Scale bars, 0.1 mm. C, NeuN immunoreactive (NeuN+)-cell density in the CP after drug administration. n = 8–9 each. D, Densities of NeuN-positive cells in the NAc core (NAcC, open circles) and the NAc shell (NAcS, filled circles) after RU-486 treatment of Gng7+/mCrePR; +/Eno2-STOP- DTA mice (n = 8–9 each). E, Densities of NeuN-positive cells in the lateral amygdala (LA) of control and mutant mice 22 days after mock and RU-486 treatment, respectively (n = 15 each, F1,28 = 0.23, P = 0.64, one-way ANOVA). Abbreviations: Au, auditory cortex; CA1, hippocampal CA1 region; CP, caudate putamen; Cx, cortex; GP, globus pallidus; MGN, medial geniculate nucleus of thalamus; NAc, nucleus accumbens; OT, olfactory tubercle; PAG, periaqueductal gray; Sp, septum. The degeneration of striatal neurons is associated with Huntington’s disease [36,37] and dystonia [38,39]. Mutant mice, however, showed no abnormal clasping behavior induced by a tail suspension in a dystonic fashion (n = 6) (Fig. 5B); the clasping behavior was observed in the mutant mice 6 weeks after RU-486 injection. In addition, there were no easily recognizable movement disorders in mutant mice at least for a week after the drug-induced ablation of striatal neurons had been completed. Impairment of auditory fear conditioning with a low- intensity footshock C, NeuN immunoreactive (NeuN+)-cell density in the CP after drug administration. n = 8–9 each. D, Densities of NeuN-positive cells in the NAc core (NAcC, open circles) and the NAc shell (NAcS, filled circles) after RU-486 treatment of Gng7+/mCrePR; +/Eno2-STOP- DTA mice (n = 8–9 each). E, Densities of NeuN-positive cells in the lateral amygdala (LA) of control and mutant mice 22 days after mock and RU-486 treatment, respectively (n = 15 each, F1,28 = 0.23, P = 0.64, one-way ANOVA). Abbreviations: Au, auditory cortex; CA1, hippocampal CA1 region; CP, caudate putamen; Cx, cortex; GP, globus pallidus; MGN, medial geniculate nucleus of thalamus; NAc, nucleus accumbens; OT, olfactory tubercle; PAG, periaqueductal gray; Sp, septum. doi:10.1371/journal.pone.0004157.g003 PLoS ONE \| www.plosone.org January 2009 \| Volume 4 \| Issue 1 \| e4157 4 Novel Auditory Fear Memory Figure 4. Ablation of medium-spiny projection neurons in the striatum of mutant mice. A, Immunoreactivity for calbindin in the dorsal striatum of control (upper) and mutant (lower) mice. B, Immunoreactivity for tyrosine hydroxylase and substance P in substantia nigra of control and mutant mice. C, Immunoreactivity for GAD and enkephalin in GP of control and mutant mice. Abbreviations: CP, caudate putamen; GP, globus pallidus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; VTA, ventral tegmental area. Scale bars, 1 mm. doi:10.1371/journal.pone.0004157.g004 Figure 4. Ablation of medium-spiny projection neurons in the striatum of mutant mice. A, Immunoreactivity for calbindin in the dorsal striatum of control (upper) and mutant (lower) mice. B, Immunoreactivity for tyrosine hydroxylase and substance P in substantia nigra of control and mutant mice. C, Immunoreactivity for GAD and enkephalin in GP of control and mutant mice. Abbreviations: CP, caudate putamen; GP, globus pallidus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; VTA, ventral tegmental area. Scale bars, 1 mm. doi:10.1371/journal.pone.0004157.g004 0.3 mA, F2,31 = 3.30, P = 0.98; at 0.5 mA, F1,13 = 4.67, P = 0.23). These results suggest that striatal neurons are indispensable for efficient auditory fear conditioning with the low-intensity US. presentation than mock-injected mice (mock-injected mice, 37.663.9%; RU-486-injected mice, 11.562.6%; F1,13 = 41.9, P,0.001) (Fig. 7E). On the other hand, the ability of RU-486- injected mice to retain the acquired fear memory under the standard condition (0.5 mA) was comparable to that of mock- injected mice (mock-injected mice, 50.166.8%; RU-486-injected mice, 40.868.0%; F1,11 = 0.32, P = 0.58) (Fig. Impairment of auditory fear conditioning with a low- intensity footshock 7F), consistent with the observation that pre-conditioning ablation of striatal neurons hardly affected the auditory fear conditioning (Fig. 6B). These results suggest that the striatal neurons are required for the retention of fear memory previously acquired by the conditioning with the low-intensity US. Impairment of long-term fear memory To further examine the role of striatal neurons in fear conditioning, we trained mice under the weak condition (a single paring of tone and footshock at 0.3 mA), tested for short-term memory (STM) 1 or 3 h after training and then retested for long- term memory (LTM) 24 h after training [41] (Fig. 7A). The freezing responses of mutant mice were comparable to those of control mice 1 h after conditioning (control, 19.763.0%; mutant, 34.567.2%; F1,11= 3.20, P = 0.10, repeated measures ANOVA) (Fig. 7B left panel) as well as 3 h after conditioning (control, 20.663.6%; mutant, 24.267.1%; F1,8 = 0.51, P = 0.50) (Fig. 7C left panel). Twenty-four hours after training, however, mutant mice showed significantly smaller freezing responses than control mice (Fig. 7B right panel, control, 28.064.6%; mutant, 3.461.8%; F1,11 = 8.06, P = 0.02: Fig. 7C right panel, control, 17.764.0%; mutant, 2.961.2%; F1,8 = 46.7, P,0.001). These results suggest that the striatal neurons are involved selectively in the acquisition of LTM under the weak conditioning, but not in that of STM. The intact STM formation in mutant mice is consistent with no detectable alterations in the sensitivity to the electric footshock as above. Discussion Here, we show that striatal neurons can be selectively ablated upon induction in mice carrying Gng7-promoter-driven CrePR and Cre-dependent DTA genes. Despite that approximately 90% of striatal neurons were ablated, the motor performance of the mutant mice appeared to be comparable to that of control mice in stationary thin rod and rotating rod tests. However, the improvement of the mutant mice in the performance over trials was impaired in the accelerating rotarod test, suggesting the requirement of striatal neurons for motor learning. In addition, the mutant mice showed no abnormal behavior in the tail suspension test and there were no easily recognizable movement disorders in the mutant mice at least for a week after the drug-induced ablation of striatal neurons had been completed. Interestingly, however, the clasping behavior was observed 6 weeks after RU-486 injection. The motor phenotypes of mutant mice appeared later might be caused by secondary changes of the brain. It is known that dystonic symptoms occur a long time after brain injury, suggesting secondary changes [42,43]. PLoS ONE \| www.plosone.org Impairment of fear memory retention We further examined whether the ablation of striatal neurons affects the retention of previously acquired fear memory (Fig. 7D). Mice were first trained with a single paring of tone and footshock at 0.3 mA and placed back in the home cage. Twenty-four hours after conditioning when LTM was formed, the animals were treated with RU-486 for induction of striatal neuron ablation. When tested 14 days after the drug treatment, RU-486-injected mice showed significantly smaller freezing responses during tone One to several pairings of tones with footshocks at 0.5–2 mA are generally used for fear conditioning in rodents [18–22]. The January 2009 \| Volume 4 \| Issue 1 \| e4157 January 2009 \| Volume 4 \| Issue 1 \| e4157 5 Novel Auditory Fear Memory Figure 5. Performance of mutant mice in motor tests. A, Foot print of control (left) and mutant (right) mice. Scale bar, 2 cm. B, Tail suspension test of control (left) and mutant (right) mice. C, Performance of control (open circles, n = 9) and mutant (filled circles, n = 8) mice in the stationary thin rod test. D, Performance of control (open circles) and mutant (filled circles) mice in the accelerating rotarod (n = 8 each). E, Locomotor activity of control (open circles, n = 10) and mutant (filled circles, n = 7) mice in the openfield test. doi:10.1371/journal.pone.0004157.g005 Figure 5. Performance of mutant mice in motor tests. A, Foot print of control (left) and mutant (right) mice. Scale bar, 2 cm. B, Tail suspension test of control (left) and mutant (right) mice. C, Performance of control (open circles, n = 9) and mutant (filled circles, n = 8) mice in the stationary thin rod test. D, Performance of control (open circles) and mutant (filled circles) mice in the accelerating rotarod (n = 8 each). E, Locomotor activity of control (open circles, n = 10) and mutant (filled circles, n = 7) mice in the openfield test. doi:10.1371/journal.pone.0004157.g005 striatal neuron ablation hardly affected the auditory fear conditioning with a single pairing of tone with the footshock at 0.5 mA. Our results are consistent with previous ones that electrolytic or excitotoxic lesion of the striatum exerted little effect on the auditory fear conditioning [19–21]. On the other hand, a slight impairment of fear conditioning with 5 tone-footshock (0.5– 1 mA) pairings was reported for dorsal striatum- or NAc shell- lesioned rats [44,45]. PLoS ONE \| www.plosone.org Impairment of fear memory retention It will be difficult to ascertain whether the discrepant behavioral effects of classical lesion studies were caused by ablation of striatal neurons or other impairments. Our genetic ablation system specific for striatal neurons provides evidence supporting the view that the amygdala but not the striatum is essential for the auditory fear conditioning under the standard condition. neuron ablation simply disrupted the animal’s ability to make the freezing responses. Furthermore, the mutant mice showed the ability to acquire, retain and express the cued fear memory at least for 3 h after conditioning with the low-intensity US. It is to be noted with this respect that the induction of cell ablation was selective for striatal neurons, leaving the amygdala intact, which plays an essential role in the acquisition, storage and expression of fear memory [2,18,20]. Thus, the striatal neuron ablation appeared to impair the formation and/or retention of long-term fear memory rather than performance or acquisition and expression of fear memory. Consistently with this possibility, the ablation of striatal neurons after long-term fear memory formation, that is, 24 h after conditioning with the low-intensity US, diminished the retention of the LTM. In the present investigation, we found that when the tone was paired with the low-intensity footshock at 0.3 mA, the freezing responses 24 h after conditioning were significantly reduced in the striatal neuron-ablated mice. The impairment of tone-dependent fear conditioning with the low-intensity US itself does not reveal a specific role of these striatal neurons in either the learning or the performance of conditioned fear. However, the observation that the freezing responses of the mutant mice measured 1 h or 3 h after conditioning with the low-intensity US were comparable with those of control mice excluded the possibility that the striatal These results obtained by the use of an inducible striatal neuron-ablation system suggest the presence of at least two forms of the auditory fear memories distinguished by the US intensity and by the requirement of striatal neurons. Under the standard condition, auditory fear memory formation is hardly affected by the striatal neuron ablation, in agreement with previous studies showing that the amygdala but not the striatum plays a central role in the auditory fear conditioning [2,18–21]. Generation of striatum-specific Cre mice A full-length cDNA (210 bp) encoding the mouse G-protein c7 subunit was amplified with primers 59-GATGTCAGGTACTAA- CAACGTCGCCC-39 and 59-CTAGAGAATTATGCAAGGC- TTTTTGTCTTT-39 from a brain cDNA library from ICR mice. Using the cDNA fragment as a probe, we isolated a BAC clone containing the exon 4 and 5 of the Gng7 by screening a genomic DNA library of C57BL/6 mouse (Genome systems, St. Louis, MO). The 8.9 kb-SpeI-KpnI fragment from the BAC clone was inserted into the SpeI-KpnI sites of pBluescript II SK(+) (Stratagene, La Jolla, CA) to yield pGng7MET. The 523 bp EcoRI-AgeI fragment generated by 2-step-PCR using pGng7MET and pNCre [25] as templates and the 989 bp AgeI-EcoRI fragment from pNCre were cloned into the EcoRI site of pBluescript II SK(+) to yield pGng7Cre. The 1.4 kb EcoRI-SacI fragment from pGK1NeopA [27] was blunted and inserted into the EcoRV site of pGng7Cre to yield pCreNeo. Synthetic oligonucleotides (59- AGCTTTCAGGTACTAACAACGTCGCCCAGGCCCGGA- AGCTGGTGGAGCAGC-39 and 59-GCAGCTGCTCCACCA- GCTTCCGGGCCTGGGCGACGTTCTTAGTACCTGAA-39) and the 7.5 kb FspI-KpnI fragment from pGngMET were ligated with the HindIII (blunted)- and KpnI-digested pBluescript II SK(+) to yield pFSKP. The 7.5 kb EcoRV-KpnI fragment from pFSKP was ligated with the HindIII (blunted)- and KpnI-digested pCreNeo to yield pBTV. KpnI-digested pMC1DTApA was blunted and ligated, and the 4.3 kb NotI-HindIII fragment from the resulting plasmid was ligated with synthetic oligonucleotides, 59-GGCCGCGGTACCCGGGTCGACTTA-39 and 59-AGCT- TAAGTCGACCCGGGTACCGC-39, to yield pMC1DTApA2. The 11.1 kb NotI-KpnI fragment of pBTV was inserted into the NotI-KpnI sites of pMC1DTpA2 to yield targeting vector pGng7CreTV. The Cre coding sequence of the CrePR gene [25,27] was replaced by that of mammalian Cre with the optimal codon usage in mammals by 2-step PCR using pNCrePR [25] and pCXN-Cre [51]. The 1.9-kb fragment encoding mammalian CrePR (mCrePR) was cloned into the XbaI site of pEF-BOS [52] to yield pmNCrePR. The cre gene in pGngCreTV was replaced by the mCrePR gene to yield targeting vector pGngmCrePRTV. Figure 6. Impaired freezing responses of mutant mice after auditory fear conditioning with a low-intensity footshock. A, Experimental design. Mice were injected with RU-486 or vehicle. Fourteen days after treatment, the animals were subjected to auditory fear conditioning. B, Freezing responses of control (open circles, n = 9) and mutant (filled circles, n = 8) mice on the conditioning (left) and test (right) days. Auditory fear conditioning was carried out with the standard intensity of footshock (0.5 mA, an arrow). Solid lines represent tone. Impairment of fear memory retention When auditory fear conditioning was carried out with the low-intensity US, the formation of LTM but not STM became sensitive to striatal January 2009 \| Volume 4 \| Issue 1 \| e4157 January 2009 \| Volume 4 \| Issue 1 \| e4157 6 Novel Auditory Fear Memory Figure 6. Impaired freezing responses of mutant mice after auditory fear conditioning with a low-intensity footshock. A, Experimental design. Mice were injected with RU-486 or vehicle. Fourteen days after treatment, the animals were subjected to auditory fear conditioning. B, Freezing responses of control (open circles, n = 9) and mutant (filled circles, n = 8) mice on the conditioning (left) and test (right) days. Auditory fear conditioning was carried out with the standard intensity of footshock (0.5 mA, an arrow). Solid lines represent tone. C, Freezing responses of control (open circles, n = 8) and mutant (filled circles, n = 11) mice and RU-486-treated Gc7-mCrePR mice (RU- 486 control) (shaded triangles, n = 7) on the conditioning (left) and test (right) days. Auditory fear conditioning was carried out with a low intensity of footshock (0.3 mA, an arrow). Solid lines represent tone. D, Current thresholds of control (open bar), RU-486-control (shaded bar) and mutant (filled bar) mice for flinch and jump reactions (n = 6 each). doi:10.1371/journal.pone.0004157.g006 indirectly involved in the consolidation or retrieval of the long- term fear memory. While the contextual fear conditioning requires the hippocampus and amygdala, our results suggest further integration of brain systems for the emotional memory by showing the involvement of the striatum in the auditory fear conditioning at the weak US. Fear is an adaptive component of response to potentially threatening stimuli, but too much or inappropriate fear accounts for many common psychiatric problems, such as anxiety disorders [3–5]. Advances in basic and clinical neuroscience studies of fear are important for the development of strategies to treat and cure anxiety disorders [49,50]. Thus, the finding of a novel form of the auditory fear memory might have therapeutic implications. Generation of striatum-specific Cre mice C, Freezing responses of control (open circles, n = 8) and mutant (filled circles, n = 11) mice and RU-486-treated Gc7-mCrePR mice (RU- 486 control) (shaded triangles, n = 7) on the conditioning (left) and test (right) days. Auditory fear conditioning was carried out with a low intensity of footshock (0.3 mA, an arrow). Solid lines represent tone. D, Current thresholds of control (open bar), RU-486-control (shaded bar) and mutant (filled bar) mice for flinch and jump reactions (n = 6 each). doi:10.1371/journal.pone.0004157.g006 neuron ablation. Thus, our results reveal a novel form of the auditory fear memory depending on striatal neurons at the low- intensity US. When the US becomes weaker, it will be less threatening and more difficult to judge whether it is dangerous enough to be memorized for animals. Our finding that striatal neuron ablation diminished the auditory fear conditioning only when a footshock was weak is of interest in view that the striatum is supposed to play a role in incorporating the positive or negative value of information into the determination of behavioral responses [46–48]. It is possible, though not proven, that striatal neurons may be activated by the weak US and directly or neuron ablation. Thus, our results reveal a novel form of the auditory fear memory depending on striatal neurons at the low- intensity US. When the US becomes weaker, it will be less threatening and more difficult to judge whether it is dangerous enough to be memorized for animals. Our finding that striatal neuron ablation diminished the auditory fear conditioning only when a footshock was weak is of interest in view that the striatum is supposed to play a role in incorporating the positive or negative value of information into the determination of behavioral responses [46–48]. It is possible, though not proven, that striatal neurons may be activated by the weak US and directly or The targeting vectors were linearized by KpnI and electropo- rated into ES cells derived from the C57BL/6 strain [24,27]. Recombinant clones were identified by Southern blot analysis of genomic DNA using 0.25 kb AgeI-SpeI fragment from the BAC clone, 0.6 kb-PstI-PstI fragment from pPGK1-NeopA and 0.3 kb NdeI-SacI fragment from the BAC clone as 59 outer, neo, and 39 outer probes, respectively. Chimeric mice production was carried out essentially as described [24,27]. The Gng7Cre allele was identified by PCR using primers CreP1 and CreP2 [25]. Generation of striatum-specific Cre mice The PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org 7 January 2009 \| Volume 4 \| Issue 1 \| e4157 Novel Auditory Fear Memory Figure 7. Impairment of long-term fear memory. A, Experimental design. Mice were injected with RU-486 or vehicle. Fourteen days after treatment, the animals were subjected to auditory fear conditioning with a weak footshock at 0.3 mA. Freezing responses to tone were measured 1 or 3 h and 24 h after conditioning. B, Freezing responses of control (open circles, n = 8) and mutant (filled circles, n = 5) mice 1 h (left) and 24 h (right) after conditioning. C, Freezing responses of control (open circles, n = 6) and mutant (filled circles, n = 4) 3 h (left) and 24 h (right) after conditioning. D, Experimental design. Mice were subjected to auditory fear conditioning with a footshock at 0.3 mA or 0.5 mA. One day after conditioning, the conditioned mice were injected with RU-486 or vehicle. Their freezing responses were measured 14 days after drug treatment. E, Mice were subjected to auditory fear conditioning with a weak footshock at 0.3 mA. Freezing responses of mock-injected (open circles, n = 7) and RU-486-injected (filled circles, n = 8) mice on the conditioning (left) and test (right) days. F, Mice were subjected to auditory fear conditioning with the standard footshock at 0.5 mA. Freezing responses of mock- injected (open circles, n = 6) and RU-486-injected (filled circles, n = 7) mice on the conditioning (left) and test (right) days. doi:10.1371/journal.pone.0004157.g007 Figure 7. Impairment of long-term fear memory. A, Experimental design. Mice were injected with RU-486 or vehicle. Fourteen days after treatment, the animals were subjected to auditory fear conditioning with a weak footshock at 0.3 mA. Freezing responses to tone were measured 1 or 3 h and 24 h after conditioning. B, Freezing responses of control (open circles, n = 8) and mutant (filled circles, n = 5) mice 1 h (left) and 24 h (right) after conditioning. C, Freezing responses of control (open circles, n = 6) and mutant (filled circles, n = 4) 3 h (left) and 24 h (right) after conditioning. D, Experimental design. Mice were subjected to auditory fear conditioning with a footshock at 0.3 mA or 0.5 mA. One day after conditioning, the conditioned mice were injected with RU-486 or vehicle. Their freezing responses were measured 14 days after drug treatment. Induction of CrePR recombinase activity RU-486 (Sigma, St. Louis, MO) was suspended at a concentration of 50 mg/ml in water containing 0.25% carbox- ymethyl cellulose (Sigma) and 0.5% Tween 80 (Sigma). We injected 1 mg per g body weight of RU-486 into the peritoneum of mice at P42. Histochemistry Under deep pentobarbital anesthesia (100 mg/kg), animals were perfused transcardially with 4% paraformaldehyde in 0.1 M phos- phate buffered salts (PBS). b-Galactosidase staining was conducted as described previously [25]. Immunohistochemistry was performed as Generation of striatum-specific Cre mice E, Mice were subjected to auditory fear conditioning with a weak footshock at 0.3 mA. Freezing responses of mock-injected (open circles, n = 7) and RU-486-injected (filled circles, n = 8) mice on the conditioning (left) and test (right) days. F, Mice were subjected to auditory fear conditioning with the standard footshock at 0.5 mA. Freezing responses of mock- injected (open circles, n = 6) and RU-486-injected (filled circles, n = 7) mice on the conditioning (left) and test (right) days. doi:10.1371/journal.pone.0004157.g007 Gng7mCrePR allele was identified by PCR using primers 59-TATA- GGTACCCAGAAGTGAATTCGGTTCGC-39, 59-GGCGAC- GTTGTTAGTACCTGAC-39 and 59-GTGCAGCATGTTCA- GCTGGC-39. Gng7mCrePR allele was identified by PCR using primers 59-TATA- GGTACCCAGAAGTGAATTCGGTTCGC-39, 59-GGCGAC- GTTGTTAGTACCTGAC-39 and 59-GTGCAGCATGTTCA- GCTGGC-39. described previously [27] using antibodies against neuronal nuclei (NeuN), enkephalin, glutamic acid decarboxylase (GAD), substance P (Chemicon International, Temecula, CA), tyrosine hydroxyrase (Santacruz Biotechnology, Santa Cruz, CA), and calbindin. The numbers of NeuN-positive cells per 8.761022 mm2 were counted at the dorsolateral part of CP, dorsomedial part of NAc core, the medial part of the NAc shell (AP =1.2 mm from bregma), and the LA (AP = 21.7 mm) in the coronal brain sections. Only unequivocally stained cells were counted using the ImageJ software by two observers blind to the origin of the sections. Eno2-STOP-DTA mouse [26] was backcrossed 7 times to the C57BL/6 strain. The Eno2-STOP-DTA allele was identified by PCR using primers 59-AATTCTTAATTAAGGCGCGCGGG-39, 59- GTCAGAATTGAGGAAGAGCTGGGG-39 and 59-CACTGAG- GATTCTTCTGTGG-39. Breeding and maintenance of mice were carried out under institutional guidelines. Mice were fed ad libitum with standard laboratory chow and water in standard animal cages under a 12 h light/dark cycle. All animal procedures were approved by the Animal Care and the Use Committee of Graduate School of Medicine, the University of Tokyo (Approval #1721T062). Terminal deoxynucleotidyl transferase-mediated dUTP nick- end labeling (TUNEL) histochemistry was performed using ApopTag Fluorescein Direct In Situ Apoptosis Detection Kit (Chemicon International) according to the instructions of the manufacturer. In brief, sections were incubated in PBS containing 20 mg/ml proteinase K (Ambion, Austin, TX) at room temper- ature for 15 min, washed and stained using FITC-labeled dUTP and terminal deoxynucleotide transferase (TdT) (Chemicon International) at 37uC for 60 min. After TUNEL reaction was terminated, slides were mounted using Vectashield H-1500 mounting solution that contains DAPI (Vector, Burlingame, CA). Confocal images were obtained using confocal microscopes (TCS-SP5, Leica, Wetzlar, Germany). Fear conditioning A A clear conditioning chamber (10610610 cm) with polyvinyl chloride boards and a stainless steel rod floor that was composed of 14 stainless steel rods (2 mm in diameter spaced 7 mm apart) was surrounded by a sound-attenuating white chest (74 lux). Masking noise of 52 dB was provided by a ventilation fan. Mice were housed individually for 1 week before behavioral testing and were handled for 30 s everyday. On the conditioning day, mice were placed in the conditioning chamber for 2 min and then presented with a loud tone (65 dB, 10 kHz) for 1 min through a speaker on the ceiling of the conditioning chest. At the end of the tone presentation, the mice were given a scrambled electrical footshock (0.3 mA or 0.5 mA for 1 s). One minute after footshock, the mice were returned to their home cages. The conditioning chamber was cleaned with 70% ethanol between sessions. On the test day, mice were placed in a novel translucent acryl chamber with paper chips surrounded by a sound-attenuating black chest for 6 min and subsequently for 3 min in the presence of the tone. The test chamber was cleaned with benzalkonium (Ecolab, St. Paul, MN) between tests. All behaviors were monitored by a CCD camera (WAT-902B; Watec, Yamagata, Japan) attached to the ceiling of the chest. Eight bit grayscale images (90690 pixels) were captured at a rate of two frames per second and freezing behavior was automatically analyzed as an index of fear using IMAGE FZC software (O’Hara). Freezing behavior was defined as the absence of any visible movement of the body and vibrissae except for movement necessitated by respiration. Freezing time was sum- mated and the percentage of freezing was calculated per minute. To examine pain sensitivity, we measured current thresholds for reactions of mice to nociceptive shock, namely, flinch and jump [53]. Mice were given footshocks of increasing strength ranging from 0.05 to 0.5 mA in a stepwise manner by 0.05 mA with an interval of 30 s. recorded as 60 s. Six consecutive trials were performed with an intertrial interval of 1 h. The accelerating rotarod test was performed with an apparatus consisted of a 3.2 cm-diameter rod (RRAC-3002; O’Hara & Co., Tokyo, Japan) essentially as described [34]. References transmission in the cortical pathway to the lateral amygdala. Neuron 34: 289–300. transmission in the cortical pathway to the lateral amygdala. Neuron 34: 289–300. 1. Davis M (1992) The role of the amygdala in fear and anxiety. Annu Rev Neurosci 15: 353–375. 14. Fanselow MS, LeDoux JE (1999) Why we think plasticity underlying Pavlovian fear conditioning occurs in the basolateral amygdala. Neuron 23: 229–232. 2. LeDoux JE (2000) Emotion circuits in the brain. Annu Rev Neurosci 23: 155–184. 3. Kent JM, Rauch SL (2003) Neurocircuitry of anxiety disorders. Curr Psychiatry Rep 5: 266–273. 15. Fendt M, Fanselow MS (1999) The neuroanatomical and neurochemical basis of conditioned fear. Neurosci Biobehav Rev 23: 743–760. conditioned fear. Neurosci Biobehav Rev 23: 743–760 4. Millan MJ (2003) The neurobiology and control of anxious states. Prog Neurobiol 70: 83–244. 16. Maren S (2001) Neurobiology of Pavlovian fear conditioning. Annu Rev Neurosci 24: 897–931. 5. Uys JD, Stein DJ, Daniels WM, Harvey BH (2003) Animal models of anxiety disorders. Curr Psychiatry Rep 5: 274–281. 17. Maren S, Fanselow MS (1996) The amygdala and fear conditioning: has the nut been cracked? Neuron 16: 237–240. 6. Blanchard RJ, Yudko EB, Rodgers RJ, Blanchard DC (1993) Defense system psychopharmacology: an ethological approach to the pharmacology of fear and anxiety. Behav Brain Res 58: 155–165. 18. Maren S, Quirk GJ (2004) Neuronal signalling of fear memory. Nat Rev Neurosci 5: 844–852. 7. LeDoux JE (1996) The emotional brain: the mysterious underpinnings of emotional life. New York: Simon & Schuster. 384 p. 19. Haralambous T, Westbrook RF (1999) An infusion of bupivacaine into the nucleus accumbens disrupts the acquisition but not the expression of contextual fear conditioning. Behav Neurosci 113: 925–940. 8. Armony JL, Quirk GJ, LeDoux JE (1998) Differential effects of amygdala lesions on early and late plastic components of auditory cortex spike trains during fear conditioning. J Neurosci 18: 2592–2601. 20. LeDoux JE, Cicchetti P, Xagoraris A, Romanski LM (1990) The lateral amygdaloid nucleus: sensory interface of the amygdala in fear conditioning. J Neurosci 10: 1062–1069. 9. Kapp BS, Frysinger RC, Gallagher M, Haselton JR (1979) Amygdala central nucleus lesions: effect on heart rate conditioning in the rabbit. Physiol Behav 23: 1109–1117. 21. Riedel G, Harrington NR, Hall G, Macphail EM (1997) Nucleus accumbens lesions impair context, but not cue, conditioning in rats. Neuroreport 8: 2477–2481. 10. Statistics Data are expressed as mean6SEM. The statistics significance was evaluated using one-way or repeated measures ANOVA. When the interaction was significant, Fisher’s PLSD post hoc test was employed. The criterion for statistical significance was P,0.05. Fear conditioning A During the training period, mice were placed on the rotating rod starting at 5 rpm and gradually accelerated to 50 rpm at a rate 0.15 rpm/s. The latency to fall (retention time) was measured with cutoff time of 5 min. Mice were trained for 3 consecutive days, receiving 4 trials per day with an intertrial interval of 1 h. In the tail suspension test [54], mice were observed for 15 s. Abnormal movement was defined as any dystonic movement of the hindlimbs, forelimbs, or trunk with full clasping where limbs were pulled into the central body axis. In the open field test, locomotor activity was measured for 9 min in a square chamber (50650640 cm) with a CCD camera on the ceiling (OF4, O’Hara). Images were captured at a rate of one frame per second and walking distance was automatically measured by IMAGE OF4 software (O’Hara). Acknowledgments We thank Ms. R. Natsume for chimeric mouse preparation, Dr. S. Miyagawa for providing pCXN-Cre and Dr. M. Tsujita for the construction of pEF-mNCrePR. Fear conditioning A A computer-controlled fear conditioning system (CL-M2; O’Hara, Tokyo, Japan) was used in the fear conditioning PLoS ONE \| www.plosone.org January 2009 \| Volume 4 \| Issue 1 \| e4157 8 Novel Auditory Fear Memory experiments. A clear conditioning chamber (10610610 cm) with polyvinyl chloride boards and a stainless steel rod floor that was composed of 14 stainless steel rods (2 mm in diameter spaced 7 mm apart) was surrounded by a sound-attenuating white chest (74 lux). Masking noise of 52 dB was provided by a ventilation fan. Mice were housed individually for 1 week before behavioral testing and were handled for 30 s everyday. On the conditioning day, mice were placed in the conditioning chamber for 2 min and then presented with a loud tone (65 dB, 10 kHz) for 1 min through a speaker on the ceiling of the conditioning chest. At the end of the tone presentation, the mice were given a scrambled electrical footshock (0.3 mA or 0.5 mA for 1 s). One minute after footshock, the mice were returned to their home cages. The conditioning chamber was cleaned with 70% ethanol between sessions. On the test day, mice were placed in a novel translucent acryl chamber with paper chips surrounded by a sound-attenuating black chest for 6 min and subsequently for 3 min in the presence of the tone. The test chamber was cleaned with benzalkonium (Ecolab, St. Paul, MN) between tests. All behaviors were monitored by a CCD camera (WAT-902B; Watec, Yamagata, Japan) attached to the ceiling of the chest. Eight bit grayscale images (90690 pixels) were captured at a rate of two frames per second and freezing behavior was automatically analyzed as an index of fear using IMAGE FZC software (O’Hara). Freezing behavior was defined as the absence of any visible movement of the body and vibrissae except for movement necessitated by respiration. Freezing time was sum- mated and the percentage of freezing was calculated per minute. To examine pain sensitivity, we measured current thresholds for reactions of mice to nociceptive shock, namely, flinch and jump [53]. Mice were given footshocks of increasing strength ranging from 0.05 to 0.5 mA in a stepwise manner by 0.05 mA with an interval of 30 s. experiments. Author Contributions The stationary horizontal thin rod test was done as described [33]. 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https://openalex.org/W4391508154	https://egusphere.copernicus.org/preprints/2024/egusphere-2023-2841/egusphere-2023-2841.pdf	English	null	Comment on egusphere-2023-2841	null	2,024	cc-by	16,739	Role of the water balance constraint in the long short-term memory network: large-sample tests of rainfall-runoff prediction Qiang Li1, Tongtiegang Zhao1 1Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Key Laboratory for Water Security in the Guangdong-Hongkong-Macao Greater Bay Area, School of Civil Engineering, Sun Yat-Sen University, Guangzhou, China, Correspondence to: Tongtiegang Zhao (zhaottg@mail.sysu.edu.cn) Abstract. While deep learning (DL) models are effective in rainfall-runoff modelling, their dependence on data and lack of physical mechanisms can limit their use in hydrology. As there is yet no consensus on the consideration of the fundamental water balance for DL models, this paper presents an in-depth investigation of the effects of water balance constraint on the long-short term memory (LSTM) network. Specifically, based on the Catchment Attributes and Meteorology for Large-sample 10 Studies (CAMELS) dataset, the LSTM and its architecturally mass-conserving variant (MC-LSTM) are trained basin-wise to provide rainfall-runoff prediction and then the robustness of the LSTM and MC-LSTM against data sparsity, random parameters initialization and contrasting climate conditions are assessed across the contiguous United States. Through large- sample tests, the results show that the water balance constraint evidently improves the robustness of the basin-wise trained long-short term memory (LSTM) network. Specifically, based on the Catchment Attributes and Meteorology for Large-sample 10 Studies (CAMELS) dataset, the LSTM and its architecturally mass-conserving variant (MC-LSTM) are trained basin-wise to provide rainfall-runoff prediction and then the robustness of the LSTM and MC-LSTM against data sparsity, random parameters initialization and contrasting climate conditions are assessed across the contiguous United States. Through large- sample tests, the results show that the water balance constraint evidently improves the robustness of the basin-wise trained LSTM. On the one hand, as the amount of training data increases from 1 year to 15 years, the incorporation of the water 15 balance constraint into the LSTM network decreases the sensitivity from 95.0% to 32.7%. On the other hand, the water balance constraint contributes to the stability of the LSTM for 450 (85%) basins when there are 3 years’ training data. In the meantime, the water balance constraint improves the transferability of the LSTM from the driest years to the wettest years for 318 (67%) basins. Overall, the in-depth investigations of this paper facilitate insights into the use of DL models for rainfall-runoff modelling. 20 LSTM. On the one hand, as the amount of training data increases from 1 year to 15 years, the incorporation of the water 15 balance constraint into the LSTM network decreases the sensitivity from 95.0% to 32.7%. On the other hand, the water balance constraint contributes to the stability of the LSTM for 450 (85%) basins when there are 3 years’ training data. https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. Role of the water balance constraint in the long short-term memory network: large-sample tests of rainfall-runoff prediction Qiang Li1, Tongtiegang Zhao1 1Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Key Laboratory for Water Security in the Guangdong-Hongkong-Macao Greater Bay Area, School of Civil Engineering, Sun Yat-Sen University, Guangzhou, China, 5 Correspondence to: Tongtiegang Zhao (zhaottg@mail.sysu.edu.cn) Role of the water balance constraint in the long short-term memory network: large-sample tests of rainfall-runoff prediction Qiang Li1, Tongtiegang Zhao1 1Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Key Laboratory for Water Security in the Guangdong-Hongkong-Macao Greater Bay Area, School of Civil Engineering, Sun Yat-Sen University, Guangzhou, China, Correspondence to: Tongtiegang Zhao (zhaottg@mail.sysu.edu.cn) In the meantime, the water balance constraint improves the transferability of the LSTM from the driest years to the wettest years for 318 (67%) basins. Overall, the in-depth investigations of this paper facilitate insights into the use of DL models for rainfall-runoff modelling. 20 1 https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. 1 Introduction Deep learning (DL) has been increasingly used for rainfall-runoff modelling (Kratzert et al., 2018; Lees et al., 2021; Nearing et al., 2021; Shen, 2018; Tsai et al., 2021). Without explicit descriptions of the underlying physical processes and 30 related assumptions, DL models are set up to directly capture response patterns hidden in large datasets (Feng et al., 2020; LeCun et al., 2015). DL models have been shown to exhibit superiority in effectively simulating complex nonlinear systems across different fields owing to the rapid growth of available data and advances in computational capability (LeCun et al., 2015; Reichstein et al., 2019; Wang et al., 2023). Effective in dealing with the complexity and nonlinearity of rainfall-runoﬀ processes, DL models have become popular in hydrological applications (Frame et al., 2022; Gauch et al., 2021a; Nearing et 35 al., 2021; Kratzert et al., 2018). There are extensive uses of the long short-term memory (LSTM) network (Kratzert et al., 2018), the recurrent neural network (Nagesh Kumar et al., 2004), the gate recurrent unit (Zhang et al., 2021), the sequence-to- sequence model (Xiang et al., 2020) and the encoder-decoder model (Kao et al., 2020). processes, DL models have become popular in hydrological applications (Frame et al., 2022; Gauch et al., 2021a; Nearing et 35 al., 2021; Kratzert et al., 2018). There are extensive uses of the long short-term memory (LSTM) network (Kratzert et al., 2018), the recurrent neural network (Nagesh Kumar et al., 2004), the gate recurrent unit (Zhang et al., 2021), the sequence-to- sequence model (Xiang et al., 2020) and the encoder-decoder model (Kao et al., 2020). processes, DL models have become popular in hydrological applications (Frame et al., 2022; Gauch et al., 2021a; Nearing et 35 al., 2021; Kratzert et al., 2018). There are extensive uses of the long short-term memory (LSTM) network (Kratzert et al., 2018), the recurrent neural network (Nagesh Kumar et al., 2004), the gate recurrent unit (Zhang et al., 2021), the sequence-to- sequence model (Xiang et al., 2020) and the encoder-decoder model (Kao et al., 2020). The LSTM network is one of the most important DL models (Feng et al., 2021; Jiang et al., 2022; Kao et al., 2020; Lees et al., 2022; Razavi, 2021). https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. Short summary. The lack of physical mechanism is a critical issue for the use of popular deep learning models. This paper presents an in-depth investigation of the fundamental mass balance constraint for deep learning-based rainfall-runoff prediction. The robustness against data sparsity, random parameters initialization and contrasting climate conditions are detailed. The results highlight that the water balance constraint evidently improves the robustness in particular when there is limited training 25 data. 2 2 1 Introduction Due to the recurrent structure and unique gating mechanism (Hochreiter and Schmidhuber, 1997), 40 the LSTM network can account for not only nonlinear relationships but also temporal dependencies among variables (Jiang et al., 2022; Read et al., 2019). These inherent capabilities make the LSTM network well suited for modelling hydrologic dynamics, especially multi-scale memory effects such as the persistence and release of water from soil moisture and snowpack (Pokharel et al., 2023; Wi and Steinschneider, 2022). To date, substantial efforts have been made to exploit the predictive et al., 2022; Razavi, 2021). Due to the recurrent structure and unique gating mechanism (Hochreiter and Schmidhuber, 1997), 40 the LSTM network can account for not only nonlinear relationships but also temporal dependencies among variables (Jiang et al., 2022; Read et al., 2019). These inherent capabilities make the LSTM network well suited for modelling hydrologic dynamics, especially multi-scale memory effects such as the persistence and release of water from soil moisture and snowpack (Pokharel et al., 2023; Wi and Steinschneider, 2022). To date, substantial efforts have been made to exploit the predictive capability of the LSTM network (Jiang et al., 2022). Compared to process-based hydrologic models, the LSTM network has 45 been shown to be similarly effective or even better in rainfall-runoff prediction (Gauch et al., 2021a; Lees et al., 2021; Kratzert et al., 2018). There were thorough tests of Predictions in Ungauged Basins (PUB) (Kratzert et al., 2019a; Yin et al., 2021b), multistep predictions (Kao et al., 2020; Yin et al., 2021a; Xiang et al., 2020), predictions at multiple timescales (Gauch et al., 2021a) and regional modelling (Kratzert et al., 2019b; Feng et al., 2020). capability of the LSTM network (Jiang et al., 2022). Compared to process-based hydrologic models, the LSTM network has 45 been shown to be similarly effective or even better in rainfall-runoff prediction (Gauch et al., 2021a; Lees et al., 2021; Kratzert et al., 2018). There were thorough tests of Predictions in Ungauged Basins (PUB) (Kratzert et al., 2019a; Yin et al., 2021b), multistep predictions (Kao et al., 2020; Yin et al., 2021a; Xiang et al., 2020), predictions at multiple timescales (Gauch et al., 2021a) and regional modelling (Kratzert et al., 2019b; Feng et al., 2020). capability of the LSTM network (Jiang et al., 2022). 1 Introduction Compared to process-based hydrologic models, the LSTM network has 45 been shown to be similarly effective or even better in rainfall-runoff prediction (Gauch et al., 2021a; Lees et al., 2021; Kratzert et al., 2018). There were thorough tests of Predictions in Ungauged Basins (PUB) (Kratzert et al., 2019a; Yin et al., 2021b), multistep predictions (Kao et al., 2020; Yin et al., 2021a; Xiang et al., 2020), predictions at multiple timescales (Gauch et al., 2021a) and regional modelling (Kratzert et al., 2019b; Feng et al., 2020). 50 The lack of physical mechanism is a critical issue in the use of the LSTM network as it is a black box model (Read et al., 50 2019; Reichstein et al., 2019; Xie et al., 2021; Zhao et al., 2019). One the one hand, without explicit physical mechanism such as the conservation of mass and energy, the LSTM network cannot guarantee causal relationships as physical models can (Wang et al., 2023; Xie et al., 2021), which may lead to spurious and inaccurate prediction that is potential to violate water balance, particularly when extrapolating beyond the range of training data (Bhasme et al., 2022; Reichstein et al., 2019). This The lack of physical mechanism is a critical issue in the use of the LSTM network as it is a black box model (Read et al., 50 2019; Reichstein et al., 2019; Xie et al., 2021; Zhao et al., 2019). One the one hand, without explicit physical mechanism such as the conservation of mass and energy, the LSTM network cannot guarantee causal relationships as physical models can (Wang et al., 2023; Xie et al., 2021), which may lead to spurious and inaccurate prediction that is potential to violate water balance, particularly when extrapolating beyond the range of training data (Bhasme et al., 2022; Reichstein et al., 2019). This property reduces the credibility of the outputs of the LSTM network and limits its application (Cai et al., 2022; Read et al., 55 2019; Wang et al., 2023). On the other hand, the lack of physical mechanism leads to the heavy reliance of the LSTM network on available observations (Read et al., 2019; Xie et al., 2021). Usually, the LSTM network requires a large amount of training data to learn the dynamics of complex systems so as to achieve robust performance (Gauch et al., 2021b; Kratzert et al., 2019b; Tsai et al., 2021; Yang et al., 2020). 1 Introduction property reduces the credibility of the outputs of the LSTM network and limits its application (Cai et al., 2022; Read et al., 55 2019; Wang et al., 2023). On the other hand, the lack of physical mechanism leads to the heavy reliance of the LSTM network on available observations (Read et al., 2019; Xie et al., 2021). Usually, the LSTM network requires a large amount of training data to learn the dynamics of complex systems so as to achieve robust performance (Gauch et al., 2021b; Kratzert et al., 2019b; Tsai et al., 2021; Yang et al., 2020). property reduces the credibility of the outputs of the LSTM network and limits its application (Cai et al., 2022; Read et al., 55 2019; Wang et al., 2023). On the other hand, the lack of physical mechanism leads to the heavy reliance of the LSTM network on available observations (Read et al., 2019; Xie et al., 2021). Usually, the LSTM network requires a large amount of training data to learn the dynamics of complex systems so as to achieve robust performance (Gauch et al., 2021b; Kratzert et al., 2019b; Tsai et al., 2021; Yang et al., 2020). 55 3 3 To this end, large-sample tests for rainfall-runoff prediction are devised based on the Catchment Attributes and Meteorology for Large-sample Studies (CAMELS) dataset across the 70 contiguous United States. are devised based on the Catchment Attributes and Meteorology for Large-sample Studies (CAMELS) dataset across the 70 contiguous United States. https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. There is recently a growing attention to the water balance constraint for the LSTM network (Frame et al., 2023; Hoedt et 60 al., 2021; Nearing et al., 2020; Pokharel et al., 2023; Wi and Steinschneider, 2022). It has been found that the water balance constraint can enhance the accuracy and extrapolation ability of the LSTM network (Cai et al., 2022; Wang et al., 2023). Meanwhile, using an architecturally mass-conserving variant of the LSTM (MC-LSTM) (Hoedt et al., 2021), it has recently been observed that the water balance constraint can impair the predictive performance under extreme events (Frame et al., There is recently a growing attention to the water balance constraint for the LSTM network (Frame et al., 2023; Hoedt et 60 al., 2021; Nearing et al., 2020; Pokharel et al., 2023; Wi and Steinschneider, 2022). It has been found that the water balance constraint can enhance the accuracy and extrapolation ability of the LSTM network (Cai et al., 2022; Wang et al., 2023). Meanwhile, using an architecturally mass-conserving variant of the LSTM (MC-LSTM) (Hoedt et al., 2021), it has recently been observed that the water balance constraint can impair the predictive performance under extreme events (Frame et al., 60 2023, 2022). Therefore, there is yet no consensus on the effects of the water balance constraint on the use of the LSTM network. 65 Aiming to bridge the gap, this paper focuses on how the water balance constraint in model architecture affects the robustness of the basin-wise trained LSTM network for rainfall-runoff prediction. The objectives are (1) to investigate the robustness of the LSTM and MC-LSTM against data sparsity, (2) to assess their stability across random parameters initialization and (3) to verify their transferability under contrasting climate conditions. To this end, large-sample tests for rainfall-runoff prediction 2023, 2022). Therefore, there is yet no consensus on the effects of the water balance constraint on the use of the LSTM network. 65 Aiming to bridge the gap, this paper focuses on how the water balance constraint in model architecture affects the robustness of the basin-wise trained LSTM network for rainfall-runoff prediction. The objectives are (1) to investigate the robustness of the LSTM and MC-LSTM against data sparsity, (2) to assess their stability across random parameters initialization and (3) to verify their transferability under contrasting climate conditions. 2.2 Water balance constraint 85 The Theory-Guided Data Science (TGDS) (Faghmous et al., 2014; Faghmous and Kumar, 2014; Karpatne et al., 2017) has presented a new paradigm to incorporate physical constraints into DL models so that their predictions tend to be physically consistent (Jiang et al., 2020; Karniadakis et al., 2021; Read et al., 2019; Wang et al., 2023; Wi and Steinschneider, 2022). As one of the TGDS strategies, the mass-conserving LSTM (MC-LSTM) is an architecturally mass-conserving variant of the LSTM network (Hoedt et al., 2021). Specifically, the mass conservation constraint is incorporated into the architecture of the 90 LSTM network in order to enforce water balance in rainfall-runoff prediction (Frame et al., 2023, 2022; Hoedt et al., 2021; Nearing et al., 2021). LSTM network (Hoedt et al., 2021). Specifically, the mass conservation constraint is incorporated into the architecture of the 90 LSTM network in order to enforce water balance in rainfall-runoff prediction (Frame et al., 2023, 2022; Hoedt et al., 2021; Nearing et al., 2021). The MC-LSTM employs normalized activation functions and subtracts the output mass from the storage mass to enforce conservation laws in the architecture of the LSTM network. According to whether directly related to the mass, input variables The MC-LSTM employs normalized activation functions and subtracts the output mass from the storage mass to enforce conservation laws in the architecture of the LSTM network. According to whether directly related to the mass, input variables are distinguished between mass inputs (𝑥𝑡) and auxiliary inputs (𝑎𝑡). The normalized activation functions are used in the input 95 conservation laws in the architecture of the LSTM network. According to whether directly related to the mass, input variables are distinguished between mass inputs (𝑥𝑡) and auxiliary inputs (𝑎𝑡). The normalized activation functions are used in the input 95 gate (𝑖𝑡) and the forget gate (𝑅𝑡) to guarantee that mass is conserved from the mass inputs (𝑥𝑡) and the previous cell states (𝑐𝑡−1). Furthermore, the output mass (ℎ𝑡) is subtracted from the total mass (𝑚𝑡) through the output gate (𝑜𝑡) to keep mass conserved between the cell states (𝑐𝑡) and the output mass. https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. 2.1 LSTM The LSTM network takes a recurrent architecture, allowing information to be stored and passed over time steps through 75 the cell state vector (𝑐𝑡) and the hidden state vector (ℎ𝑡) (Hochreiter and Schmidhuber, 1997; Jiang et al., 2022). At each time step 𝑡, the recurrent unit utilizes the current input (𝑋𝑡) and previous hidden state (ℎ𝑡−1) to calculate three gates, the input gate (𝑖𝑡), forget gate (𝑓𝑡) and output gate (𝑜𝑡), which control what new information to add in, what previous information to forget and what current information to output, respectively. Finally, the hidden state (ℎ𝑡) is passes through a head layer to derive the final prediction. The above process can be formulated as follows: 80 The LSTM network takes a recurrent architecture, allowing information to be stored and passed over time steps through 75 the cell state vector (𝑐𝑡) and the hidden state vector (ℎ𝑡) (Hochreiter and Schmidhuber, 1997; Jiang et al., 2022). At each time step 𝑡, the recurrent unit utilizes the current input (𝑋𝑡) and previous hidden state (ℎ𝑡−1) to calculate three gates, the input gate (𝑖𝑡), forget gate (𝑓𝑡) and output gate (𝑜𝑡), which control what new information to add in, what previous information to forget and what current information to output, respectively. Finally, the hidden state (ℎ𝑡) is passes through a head layer to derive the final prediction. The above process can be formulated as follows: 80 { 𝑓𝑡= 𝜎(𝑊𝑥𝑓𝑋𝑡+ 𝑊ℎ𝑓ℎ𝑡−1 + 𝑏𝑓) 𝑐𝑡̃ = 𝑡𝑎𝑛ℎ(𝑊𝑥𝑐𝑋𝑡+ 𝑊ℎ𝑐ℎ𝑡−1 + 𝑏𝑐) 𝑖𝑡 = 𝜎(𝑊𝑥𝑖𝑋𝑡+ 𝑊ℎ𝑖ℎ𝑡−1 + 𝑏𝑖) 𝑐𝑡= 𝑓𝑡⊙𝑐𝑡−1 + 𝑖𝑡⊙𝑐𝑡̃ 𝑜𝑡= 𝜎(𝑊𝑥𝑜𝑋𝑡+ 𝑊ℎ𝑜ℎ𝑡−1 + 𝑏𝑜) ℎ𝑡= 𝑜𝑡⊙tanh(𝑐𝑡) (1) { 𝑓𝑡= 𝜎(𝑊𝑥𝑓𝑋𝑡+ 𝑊ℎ𝑓ℎ𝑡−1 + 𝑏𝑓) 𝑐𝑡̃ = 𝑡𝑎𝑛ℎ(𝑊𝑥𝑐𝑋𝑡+ 𝑊ℎ𝑐ℎ𝑡−1 + 𝑏𝑐) 𝑖𝑡 = 𝜎(𝑊𝑥𝑖𝑋𝑡+ 𝑊ℎ𝑖ℎ𝑡−1 + 𝑏𝑖) 𝑐𝑡= 𝑓𝑡⊙𝑐𝑡−1 + 𝑖𝑡⊙𝑐𝑡̃ 𝑜𝑡= 𝜎(𝑊𝑥𝑜𝑋𝑡+ 𝑊ℎ𝑜ℎ𝑡−1 + 𝑏𝑜) ℎ𝑡= 𝑜𝑡⊙tanh(𝑐𝑡) (1) (1) (1) where 𝑊 and 𝑏 respectively indicate learnable weights and bias to be calibrated during training period. Additionally, 𝜎, 𝑡𝑎𝑛ℎ and ⊙ represent the sigmoid function, the tanh function and the element-wise multiplication, respectively. where 𝑊 and 𝑏 respectively indicate learnable weights and bias to be calibrated during training period. Additionally, 𝜎, 𝑡𝑎𝑛ℎ and ⊙ represent the sigmoid function, the tanh function and the element-wise multiplication, respectively. 2.3 EXP-HYDRO The EXP-HYDRO model is employed to benchmark the performances of the LSTM and MC-LSTM. The EXP-HYDRO model is a daily conceptual hydrological model that strictly adheres to mass conservation (Patil and Stieglitz, 2014). It has 2 state variables referred to as a snow accumulation bucket (𝑆0) and a catchment bucket (𝑆1) with the water balance equation expressed as: 120 120 { 𝑑𝑆0 𝑑𝑡= 𝑃𝑠−𝑀 𝑑𝑆1 𝑑𝑡= 𝑃𝑟+ 𝑀−𝐸𝑇−𝑄 (11) (11) where 𝑀, 𝐸𝑇, 𝑄, 𝑃𝑠 and 𝑃𝑟 are 5 flux variables, representing the snowmelt (mm/day), evapotranspiration (mm/day), streamflow (mm/day), daily snowfall (mm/day) and rainfall (mm/day), respectively, calculated by 3 input variables (the daily precipitation (𝑃, mm/day), temperature (𝑇, ℃) and day length (𝐿𝑑𝑎𝑦, hour)). where 𝑀, 𝐸𝑇, 𝑄, 𝑃𝑠 and 𝑃𝑟 are 5 flux variables, representing the snowmelt (mm/day), evapotranspiration (mm/day), streamflow (mm/day), daily snowfall (mm/day) and rainfall (mm/day), respectively, calculated by 3 input variables (the daily precipitation (𝑃, mm/day), temperature (𝑇, ℃) and day length (𝐿𝑑𝑎𝑦, hour)). In this paper, the EXP-HYDRO model is wrapped with the DL architecture. That is, the non-analytically solvable ordinary 125 differential equations (ODEs) of the EXP-HYDRO model are incorporated into the DL model (Jiang et al., 2020). Therefore, the model parameters are learnable during training period like a DL model while the internal calculations follow the ODEs of the EXP-HYDRO model. Comparing to the MC-LSTM, the EXP-HYDRO model has more specific physical processes to distribute water. 125 130 https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. For unobserved mass sinks, e.g., evapotranspiration, the MC-LSTM takes a subset of the output mass vector to accumulate 110 the output water that does not convert to runoff. Given that, the runoff (𝑦𝑡) is the sum of the output mass vector, excluding that subset representing the unobserved mass sinks, shown in equation (8). Accordingly, the internal calculations of the MC-LSTM ensure strictly mass-conservation (here water balance) at any timesteps, between inputs (here precipitation), outputs (here runoff and other sinks) and cell states (here water storage) (Frame et al., 2023). For unobserved mass sinks, e.g., evapotranspiration, the MC-LSTM takes a subset of the output mass vector to accumulate 110 the output water that does not convert to runoff. Given that, the runoff (𝑦𝑡) is the sum of the output mass vector, excluding that subset representing the unobserved mass sinks, shown in equation (8). Accordingly, the internal calculations of the MC-LSTM ensure strictly mass-conservation (here water balance) at any timesteps, between inputs (here precipitation), outputs (here runoff and other sinks) and cell states (here water storage) (Frame et al., 2023). 115 2.2 Water balance constraint 85 Mathematically, the MC-LSTM is described as follows: 𝑖𝑡= 𝜎̃ (𝑊𝑖𝑎𝑡+ 𝑈𝑖 𝑐𝑡−1 ‖𝑐𝑡−1‖1 + 𝑉𝑖𝑥𝑡+ 𝑏𝑖) (2) 𝑜𝑡= 𝜎(𝑊𝑜𝑎𝑡+ 𝑈𝑜 𝑐𝑡−1 ‖𝑐𝑡−1‖1 + 𝑉𝑜𝑥𝑡+ 𝑏o) (3) 100 𝑅𝑡= 𝑅𝑒𝐿𝑈 ̃ (𝑊𝑟𝑎𝑡+ 𝑈𝑟 𝑐𝑡−1 ‖𝑐𝑡−1‖1 + 𝑉𝑟𝑥𝑡+ 𝑏𝑟) (4) 𝑚𝑡= 𝑅𝑡𝑐𝑡−1 + 𝑖𝑡𝑥𝑡 (5) 𝑐𝑡= (1 −𝑜𝑡) ⨀ 𝑚𝑡 (6) ℎ𝑡= 𝑜𝑡 ⨀ 𝑚𝑡 (7) 𝑦𝑡= ∑ℎ𝑖 𝑡 𝑛 𝑖=2 (8) 105 = 𝜎̃ (𝑊𝑖𝑎𝑡+ 𝑈𝑖 𝑐𝑡−1 ‖𝑐𝑡−1‖1 + 𝑉𝑖𝑥𝑡+ 𝑏𝑖) (2) = 𝜎(𝑊𝑜𝑎𝑡+ 𝑈𝑜 𝑐𝑡−1 ‖𝑐𝑡−1‖1 + 𝑉𝑜𝑥𝑡+ 𝑏o) (3) = 𝑅𝑒𝐿𝑈 ̃ (𝑊𝑟𝑎𝑡+ 𝑈𝑟 𝑐𝑡−1 ‖𝑐𝑡−1‖1 + 𝑉𝑟𝑥𝑡+ 𝑏𝑟) (4) 𝑚𝑡= 𝑅𝑡𝑐𝑡−1 + 𝑖𝑡𝑥𝑡 (5) 𝑐𝑡= (1 −𝑜𝑡) ⨀ 𝑚𝑡 (6) ℎ𝑡= 𝑜𝑡 ⨀ 𝑚𝑡 (7) 𝑦𝑡= ∑ℎ𝑖 𝑡 𝑛 𝑖=2 (8) (2) 100 (3) 105 (8) where 𝑊, 𝑈 and 𝑉 represent learnable weights; 𝑏 denotes the learnable bias; 𝜎̃ and 𝑅𝑒𝐿𝑈 ̃ indicate the normalized sigmoid function and the normalized ReLU function as Eq. (9) and Eq. (10), respectively. where 𝑊, 𝑈 and 𝑉 represent learnable weights; 𝑏 denotes the learnable bias; 𝜎̃ and 𝑅𝑒𝐿𝑈 ̃ indicate the normalized sigmoid function and the normalized ReLU function as Eq. (9) and Eq. (10), respectively. 𝜎̃(𝑖𝑘) = 𝜎(𝑖𝑘) ∑𝜎(𝑖𝑘) 𝑘 (9) 𝑅𝑒𝐿𝑈 ̃ (𝑠𝑘) = 𝑚𝑎𝑥(𝑠𝑘, 0) ∑𝑚𝑎𝑥(𝑠𝑘, 0) 𝑘 (10) 𝜎̃(𝑖𝑘) = 𝜎(𝑖𝑘) ∑𝜎(𝑖𝑘) 𝑘 (9) 𝜎̃(𝑖𝑘) = 𝜎(𝑖𝑘) ∑𝜎(𝑖𝑘) 𝑘 (9) 𝑅𝑒𝐿𝑈 ̃ (𝑠𝑘) = 𝑚𝑎𝑥(𝑠𝑘, 0) ∑𝑚𝑎𝑥(𝑠𝑘, 0) 𝑘 𝑅𝑒𝐿𝑈 ̃ (𝑠𝑘) = 𝑚𝑎𝑥(𝑠𝑘, 0) ∑𝑚𝑎𝑥(𝑠𝑘, 0) 𝑘 (10) (10) 5 https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. The three meteorological forcing data in the CAMELS dataset are derived from three different gridded data products, i.e., Daymet, Maurer and North American Land Data Assimilation System (NLDAS). The Daymet (Thornton et al., 1997) is chosen as the forcing inputs here because of its high spatial resolution (1 km × 1km) and better forcing quality (Feng et al., 2022; 140 Newman et al., 2015). For a direct comparison with the previous studies mentioned above, 531 basins are used, while other basins with an area greater than 2000 km2 or showing large discrepancies in their areas when calculated using different strategies are removed. In the LSTM, MC-LSTM and EXP-HYDRO modelling, the runoff is taken as the target variable and the precipitation, temperature, vapor pressure, solar radiation and day length as forcing variables. 145 3.1 The CAMELS dataset Large-sample tests are devised based on the CAMELS dataset that comprises daily streamflow observations, catchment attributes and three basin-averaged daily meteorological forcing inputs for 671 basins across the contiguous United States over the period from1980 to 2010 (Addor et al., 2017; Newman et al., 2015). The CAMELS dataset has been used to support 35 benchmark studies, generalization and application to other scenarios for its sufficiently long hydrometeorological time series and a large number of diverse basins (Feng et al., 2020; Frame et al., 2022; Kratzert et al., 2021, 2019b; Yin et al., 2021a). Large-sample tests are devised based on the CAMELS dataset that comprises daily streamflow observations, catchment attributes and three basin-averaged daily meteorological forcing inputs for 671 basins across the contiguous United States over the period from1980 to 2010 (Addor et al., 2017; Newman et al., 2015). The CAMELS dataset has been used to support 135 benchmark studies, generalization and application to other scenarios for its sufficiently long hydrometeorological time series and a large number of diverse basins (Feng et al., 2020; Frame et al., 2022; Kratzert et al., 2021, 2019b; Yin et al., 2021a). 135 6 3.2 Experimental design Four experiments are set up to assess the effects of the water balance constraint on the robustness of the basin-wise trained LSTM for rainfall-runoff prediction in different aspects (Fig.1). For each experiment, three types of models with different degrees of physical constraints, a standard LSTM, a MC-LSTM and a DL wrapped EXP-HYDRO, are trained independently Four experiments are set up to assess the effects of the water balance constraint on the robustness of the basin-wise trained LSTM for rainfall-runoff prediction in different aspects (Fig.1). For each experiment, three types of models with different degrees of physical constraints, a standard LSTM, a MC-LSTM and a DL wrapped EXP-HYDRO, are trained independently for each catchment. Experiment 1 tests the basic performances of the LSTM, MC-LSTM and EXP-HYDRO models in rainfall- 150 runoﬀ prediction for 531 basins. Experiment 2 aims to determine how the water balance constraint affects the sensitivity of the LSTM network to data sparsity. Based on the predictions from Experiment 1 and Experiment 2, Experiment 3 quantifies Four experiments are set up to assess the effects of the water balance constraint on the robustness of the basin-wise trained LSTM for rainfall-runoff prediction in different aspects (Fig.1). For each experiment, three types of models with different degrees of physical constraints, a standard LSTM, a MC-LSTM and a DL wrapped EXP-HYDRO, are trained independently for each catchment. Experiment 1 tests the basic performances of the LSTM, MC-LSTM and EXP-HYDRO models in rainfall- 150 runoﬀ prediction for 531 basins. Experiment 2 aims to determine how the water balance constraint affects the sensitivity of the LSTM network to data sparsity. Based on the predictions from Experiment 1 and Experiment 2, Experiment 3 quantifies the impact of the water balance constraint on the stability of the LSTM network against random parameters initialization. Experiment 4 is designed to assess the transferability of the three models under contrasting climate conditions and verify h th th t b l t i t h th t f bilit f th LSTM t k A di t th lt f E i t 155 for each catchment. Experiment 1 tests the basic performances of the LSTM, MC-LSTM and EXP-HYDRO models in rainfall- 150 runoﬀ prediction for 531 basins. Experiment 2 aims to determine how the water balance constraint affects the sensitivity of the LSTM network to data sparsity. https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. Figure 1. Schematic of (a) the split-sample for different sparse training datasets at each catchment and (b) (c) the modified DSST at 160 catchment 01022500 (USGS code) based on annual precipitation for hydrological year (1 October to 30 September). Figure 1. Schematic of (a) the split-sample for different sparse training datasets at each catchment and (b) (c) the modified DSST at 160 catchment 01022500 (USGS code) based on annual precipitation for hydrological year (1 October to 30 September). 3.2 Experimental design Based on the predictions from Experiment 1 and Experiment 2, Experiment 3 quantifies the impact of the water balance constraint on the stability of the LSTM network against random parameters initialization. Experiment 4 is designed to assess the transferability of the three models under contrasting climate conditions and verify for each catchment. Experiment 1 tests the basic performances of the LSTM, MC-LSTM and EXP-HYDRO models in rainfall- 150 runoﬀ prediction for 531 basins. Experiment 2 aims to determine how the water balance constraint affects the sensitivity of the LSTM network to data sparsity. Based on the predictions from Experiment 1 and Experiment 2, Experiment 3 quantifies the impact of the water balance constraint on the stability of the LSTM network against random parameters initialization. Experiment 4 is designed to assess the transferability of the three models under contrasting climate conditions and verify whether the water balance constraint enhances the transferability of the LSTM network. According to the results of Experiment 155 2, the training period length of Experiment 4 is set to be 6 years, which is considered to be sufficient to ensure transferability yet provide robust training. whether the water balance constraint enhances the transferability of the LSTM network. According to the results of Experiment 155 2, the training period length of Experiment 4 is set to be 6 years, which is considered to be sufficient to ensure transferability yet provide robust training. 7 Experiment 2: Sensitivity to data sparsity Trained and tested repeatedly 10 times with different 180 random seeds over the 531 basins, a total of 79,650 (10 × 3 × 5 × 531) models are obtained. Experiment 3: Stability against random parameters initialization Owing to the stochastic nature of the training process, the LSTM network can be disturbed by the initialization of model parameters (Kratzert et al., 2018). Accordingly, it is helpful to independently repeat the training and testing procedures for several times with different random seeds in order to eliminate the uncertainty caused by random initialization of model 185 parameters (Feng et al., 2020; Kratzert et al., 2019a). While the average performance of the ensemble models is then considered as the stable performance, the differences among the performances of models with different random seeds reflect the stability of the specific model against random parameters initialization. Experiment 3 calculates the standard deviation of the performances from the 10-member ensemble models in Experiment 1 and Experiment 2 as the stability of the model. period lengths ranging from 1 year to 12 years, as shown in Fig. 1a. Trained and tested repeatedly 10 times with different 180 random seeds over the 531 basins, a total of 79,650 (10 × 3 × 5 × 531) models are obtained. period lengths ranging from 1 year to 12 years, as shown in Fig. 1a. Trained and tested repeatedly 10 times with different 180 random seeds over the 531 basins, a total of 79,650 (10 × 3 × 5 × 531) models are obtained. Experiment 3: Stability against random parameters initialization Owing to the stochastic nature of the training process, the LSTM network can be disturbed by the initialization of model parameters (Kratzert et al., 2018). Accordingly, it is helpful to independently repeat the training and testing procedures for several times with different random seeds in order to eliminate the uncertainty caused by random initialization of model 185 parameters (Feng et al., 2020; Kratzert et al., 2019a). While the average performance of the ensemble models is then considered as the stable performance, the differences among the performances of models with different random seeds reflect the stability of the specific model against random parameters initialization. Experiment 3 calculates the standard deviation of the performances from the 10-member ensemble models in Experiment 1 and Experiment 2 as the stability of the model. Experiment 1: Performances of LSTM, MC-LSTM and EXP-HYDRO In order to test the predictive performances of the LSTM, MC-LSTM and EXP-HYDRO in modelling rainfall-runoﬀ processes, these models are trained separately for each of the 531 basins. The performances of the LSTM and MC-LSTM are 165 compared to quantify the general impact of the water balance constraint in model architecture to the LSTM network, while the EXP-HYDRO model serves as a benchmark. For each catchment, the training period covers 15 water years (from 1 October 1980 to 30 September 1995) and the testing period also covers 15 water years (from 1 October 1995 to 30 September 2010). Given the uncertainty caused by the random initialization of model parameters, the training and testing of each model are 8 8 Experiment 2: Sensitivity to data sparsity Owing to the stochastic nature of the training process, the LSTM network can be disturbed by the initialization of model parameters (Kratzert et al., 2018). Accordingly, it is helpful to independently repeat the training and testing procedures for several times with different random seeds in order to eliminate the uncertainty caused by random initialization of model 185 parameters (Feng et al., 2020; Kratzert et al., 2019a). While the average performance of the ensemble models is then considered as the stable performance, the differences among the performances of models with different random seeds reflect the stability of the specific model against random parameters initialization. Experiment 3 calculates the standard deviation of the performances from the 10-member ensemble models in Experiment 1 and Experiment 2 as the stability of the model. Experiment 4: Transferability under contrasting climate conditions 190 The transferability is examined through a modified version (Broderick et al., 2016) of differential split sample testing (DSST) (KLEMEŠ, 1986). There are three datasets, the training, the control (independent but similar to the training data) and the testing data (independent and opposite to the training and the control data). Models are trained based on the training data, and the differences in performances between the control (in-bound test) and the testing data (out-of-band test) are indicative of transferability (Broderick et al., 2016). For each catchment, two scenarios are conducted to examine the transferability 195 between the wettest and the driest hydrological years that identified from the total precipitation of hydrological years, while W/D (D/W) represents the scenario of training on the wettest (driest) years and then testing on the driest (wettest) years. In the W/D scenario, each model is trained using the first, third, fifth, sixth, eighth and tenth ranked wettest years, while model performance on the second, fourth, seventh and ninth ranked wettest years provides a benchmark to assess the transferability f h d l d h i d f h h d i h k d d i ( l f h b i 200 Experiment 4: Transferability under contrasting climate conditions 190 The transferability is examined through a modified version (Broderick et al., 2016) of differential split sample testing (DSST) (KLEMEŠ, 1986). There are three datasets, the training, the control (independent but similar to the training data) and the testing data (independent and opposite to the training and the control data). https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. repeated for 10 times with different random seeds. In total, Experiment 1 yields 15,930 models, 30 models for each of the 531 170 catchments. repeated for 10 times with different random seeds. In total, Experiment 1 yields 15,930 models, 30 models for each of the 531 170 catchments. Experiment 2: Sensitivity to data sparsity To determine how the water balance constraint affects the sensitivity of the LSTM network to data sparsity, the LSTM, MC-LSTM and EXP-HYDRO models are trained on various sparse training datasets and then evaluated on the same testing datasets. For each catchment, the training period and testing period in Experiment 1 are considered as the complete training 175 period and the testing period, respectively. Sparse training datasets are constructed for each basin by continually removing the data of entire water years from the earlier years of the whole training period instead of randomly removing, which can avoid excessive destruction of time dependency between data and simulate real scenarios that lack historical data (Read et al., 2019). Therefore, the sparse training datasets are set to 7%, 20%, 40%, 60% and 80% of the whole training data with the training period lengths ranging from 1 year to 12 years, as shown in Fig. 1a. Trained and tested repeatedly 10 times with different 180 random seeds over the 531 basins a total of 79 650 (10 × 3 × 5 × 531) models are obtained datasets. For each catchment, the training period and testing period in Experiment 1 are considered as the complete training 175 period and the testing period, respectively. Sparse training datasets are constructed for each basin by continually removing the data of entire water years from the earlier years of the whole training period instead of randomly removing, which can avoid excessive destruction of time dependency between data and simulate real scenarios that lack historical data (Read et al., 2019). Therefore, the sparse training datasets are set to 7%, 20%, 40%, 60% and 80% of the whole training data with the training datasets. For each catchment, the training period and testing period in Experiment 1 are considered as the complete training 175 period and the testing period, respectively. Sparse training datasets are constructed for each basin by continually removing the data of entire water years from the earlier years of the whole training period instead of randomly removing, which can avoid excessive destruction of time dependency between data and simulate real scenarios that lack historical data (Read et al., 2019). Therefore, the sparse training datasets are set to 7%, 20%, 40%, 60% and 80% of the whole training data with the training period lengths ranging from 1 year to 12 years, as shown in Fig. 1a. 3.3 Model training and evaluation 205 All input variables of the LSTM are normalized by removing the mean and scaling by the standard deviation. For the MC-LSTM, the auxiliary inputs (input variables excluding precipitation) are normalized while the mass input (precipitation) not. The MC-LSTM is architecturally constrained to the water balance so that they do not utilize dropout strategy. In order to compromise between maximumly reducing the uncertainty caused by different numbers of model parameters and achieving potentially more powerful predictions, the hidden sizes of the LSTM and MC-LSTM networks are set to 50 and 20, respectively, 210 so that their numbers of parameters differ by less than 0.1%. As the EXP-HYDRO model is a process-based model, there is no need for the DL wrapped EXP-HYDRO model to normalize their input variables and to set the hidden size or dropout rate. Excluding these settings above, the LSTM, MC-LSTM and EXP-HYDRO models in the four experiments have the same hyperparameters (shown in Table 1) and the same loss function: All input variables of the LSTM are normalized by removing the mean and scaling by the standard deviation. For the MC-LSTM, the auxiliary inputs (input variables excluding precipitation) are normalized while the mass input (precipitation) not. The MC-LSTM is architecturally constrained to the water balance so that they do not utilize dropout strategy. In order to compromise between maximumly reducing the uncertainty caused by different numbers of model parameters and achieving potentially more powerful predictions, the hidden sizes of the LSTM and MC-LSTM networks are set to 50 and 20, respectively, 210 potentially more powerful predictions, the hidden sizes of the LSTM and MC-LSTM networks are set to 50 and 20, respectively, 210 so that their numbers of parameters differ by less than 0.1%. As the EXP-HYDRO model is a process-based model, there is no need for the DL wrapped EXP-HYDRO model to normalize their input variables and to set the hidden size or dropout rate. Excluding these settings above, the LSTM, MC-LSTM and EXP-HYDRO models in the four experiments have the same hyperparameters (shown in Table 1) and the same loss function: 𝐹𝑉𝑈= ∑ (𝑦𝑛−𝑦̂𝑛)2 𝑁 𝑛=1 ∑ (𝑦𝑛−𝑦̅)2 𝑁 𝑛=1 𝐹𝑉𝑈= ∑ (𝑦𝑛−𝑦̂𝑛)2 𝑁 𝑛=1 ∑ (𝑦𝑛−𝑦̅)2 𝑁 𝑛=1 (12) 215 (12) where N is the number of samples; 𝑦̂ and 𝑦𝑛 represent the simulated runoff and its corresponding observation, respectively; 𝑦̅ is the averaged value of observed runoff. Table 1. https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. wettest years (as an example shown in Fig. 1c). Removing catchments without complete data from 1981 to 2010, 475 basins remain here, thus a total of 28,500 (10 × 3 × 2 × 475) models are trained and tested. wettest years (as an example shown in Fig. 1c). Removing catchments without complete data from 1981 to 2010, 475 basins remain here, thus a total of 28,500 (10 × 3 × 2 × 475) models are trained and tested. Experiment 2: Sensitivity to data sparsity Models are trained based on the training data, and the differences in performances between the control (in-bound test) and the testing data (out-of-band test) are indicative of transferability (Broderick et al., 2016). For each catchment, two scenarios are conducted to examine the transferability 195 between the wettest and the driest hydrological years that identified from the total precipitation of hydrological years, while W/D (D/W) represents the scenario of training on the wettest (driest) years and then testing on the driest (wettest) years. In the W/D scenario, each model is trained using the first, third, fifth, sixth, eighth and tenth ranked wettest years, while model performance on the second, fourth, seventh and ninth ranked wettest years provides a benchmark to assess the transferability f h d l d h i d f h h d i h k d d i ( l f h b i 200 of transferability (Broderick et al., 2016). For each catchment, two scenarios are conducted to examine the transferability 195 between the wettest and the driest hydrological years that identified from the total precipitation of hydrological years, while W/D (D/W) represents the scenario of training on the wettest (driest) years and then testing on the driest (wettest) years. In the W/D scenario, each model is trained using the first, third, fifth, sixth, eighth and tenth ranked wettest years, while model performance on the second, fourth, seventh and ninth ranked wettest years provides a benchmark to assess the transferability of the model tested on the contrasting second, fourth, seventh and ninth ranked driest years (as an example of the basin 200 01022500 shown in Fig. 1b). In the D/W scenario, the transferability assessment is conducted using the opposite driest and 9 The transferability of a model is better with a lower KGE difference under contrasting climate conditions. https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. The Kling-Gupta Efficiency (KGE) (Gupta et al., 2009) is used to quantify the performances of the rainfall-runoff predictions here. As shown below, KGE summarizes model performance in three key aspects: correlation, bias and variance. 𝐾𝐺𝐸= 1 −√(𝑟−1)2 + (𝛽−1)2 + (𝛾−1)2 (13) 𝑟−1)2 + (𝛽−1)2 + (𝛾−1)2 (13) 𝛽= 𝜇𝑠𝑖𝑚 𝜇𝑜𝑏𝑠 (14) 𝛾= 𝜎𝑠𝑖𝑚 𝜎𝑜𝑏𝑠 (15) (13) (15) 225 where 𝑟 is the correlation coefficient between simulations and observations; 𝛽 represents the ratio between mean simulations and mean observations; 𝛾 measures the relative variability in the simulations and observations; 𝜇 and 𝜎 represent the mean and standard deviation of the runoff series, respectively (Gupta et al., 2009).The value of KGE varies from negative inﬁnity to 1, with a value closer to 1 suggesting superior model performance. The model performance comprises two essential aspects: accuracy and robustness. The accuracy is calculated as the mean 230 KGE of the 10-member ensembles. The robustness is composed of three aspects: (a) the sensitivity to data sparsity, (b) the stability against random parameters initialization and (c) the transferability under contrasting climate conditions. In experiment 2, the sensitivity of a model at a catchment is estimated from the variation range in the KGE under varying sparse training data sets. A larger variation range suggests the high sensitivity of a model to data sparsity. In experiment 3, the stability of a model The model performance comprises two essential aspects: accuracy and robustness. The accuracy is calculated as the mean 230 KGE of the 10-member ensembles. The robustness is composed of three aspects: (a) the sensitivity to data sparsity, (b) the stability against random parameters initialization and (c) the transferability under contrasting climate conditions. In experiment 2, the sensitivity of a model at a catchment is estimated from the variation range in the KGE under varying sparse training data sets. A larger variation range suggests the high sensitivity of a model to data sparsity. In experiment 3, the stability of a model is calculated as the standard deviation of KGE values from 10-member ensembles, while a higher standard deviation of KGE 235 values indicates the worse stability of a model across random parameters initialization. In experiment 4, the transferability is estimated as the KGE difference between the control data and the corresponding testing data. 3.3 Model training and evaluation 205 Hyperparameters of the LSTM, MC-LSTM and EXP-HYDRO models Hyperparameter LSTM MC-LSTM EXP-HYDRO Batch size 256 256 256 Initial learning rate 0.01 0.01 0.01 Learning rate decay 0.3 0.3 0.3 Input time step (day) 365 365 365 Lead time (day) 1 1 1 Hidden size 50 20 - Dropout rate 0.4 - - Epoch Early stop Early stop Early stop Optimizer Adam Adam Adam Table 1. Hyperparameters of the LSTM, MC-LSTM and EXP-HYDRO models 220 10 4.1 Performances of LSTM, MC-LSTM and EXP-HYDRO The predictive performances of the LSTM, MC-LSTM and EXP-HYDRO models across the 531 catchments are examined by the KGE in Fig. 2. Specifically, the left panel illustrates the boxplot of the KGE values and the right panel displays the empirical cumulative distributions. It can be observed that there is marginal difference in the performances of the LSTM and MC-LSTM as the median KGE is respectively 0.676 and 0.679 (shown in the penultimate column in Table 2). In particular, 245 the distributions of mean KGE for the LSTM and MC-LSTM are similar, which is illustrated by their cumulative distribution th t l t l i i Fi 2 Th lt i di t th t th t b l t i t h littl i t th The predictive performances of the LSTM, MC-LSTM and EXP-HYDRO models across the 531 catchments are examined by the KGE in Fig. 2. Specifically, the left panel illustrates the boxplot of the KGE values and the right panel displays the empirical cumulative distributions. It can be observed that there is marginal difference in the performances of the LSTM The predictive performances of the LSTM, MC-LSTM and EXP-HYDRO models across the 531 catchments are examined by the KGE in Fig. 2. Specifically, the left panel illustrates the boxplot of the KGE values and the right panel displays the empirical cumulative distributions. It can be observed that there is marginal difference in the performances of the LSTM and MC-LSTM as the median KGE is respectively 0.676 and 0.679 (shown in the penultimate column in Table 2). In particular, 245 the distributions of mean KGE for the LSTM and MC-LSTM are similar, which is illustrated by their cumulative distribution curves that are close to overlapping in Fig. 2. These results indicate that the water balance constraint has little impact on the general accuracy of the LSTM network when it is trained with data of 15 years for a single basin, which is consistent with p g p and MC-LSTM as the median KGE is respectively 0.676 and 0.679 (shown in the penultimate column in Table 2). In particular, 245 the distributions of mean KGE for the LSTM and MC-LSTM are similar, which is illustrated by their cumulative distribution curves that are close to overlapping in Fig. 2. https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. previous studies (Hoedt et al., 2021; Nearing et al., 2020). In the meantime, the LSTM and MC-LSTM generally outperform the EXP-HYDRO model, which is visually shown by the rightward shifts of the empirical cumulative distributions for the 250 LSTM and MC-LSTM compared to the EXP-HYDRO models in Fig. 2. Figure 2. Empirical cumulative distributions and boxplot of the evaluation performances for the LSTM, MC-LSTM and EXP-HYDRO models across the 531 catchments. The performance of each model for each basin is summarized by the mean value of KGE across different 5 random seeds. Figure 2. Empirical cumulative distributions and boxplot of the evaluation performances for the LSTM, MC-LSTM and EXP-HYDRO models across the 531 catchments. The performance of each model for each basin is summarized by the mean value of KGE across different 5 random seeds. Figure 2. Empirical cumulative distributions and boxplot of the evaluation performances for the LSTM, MC-LSTM and EXP-HYDRO models across the 531 catchments. The performance of each model for each basin is summarized by the mean value of KGE across different 5 random seeds. Figure 2. Empirical cumulative distributions and boxplot of the evaluation performances for the LSTM, MC-LSTM and EXP-HYDRO models across the 531 catchments. The performance of each model for each basin is summarized by the mean value of KGE across different 55 random seeds. 4.1 Performances of LSTM, MC-LSTM and EXP-HYDRO These results indicate that the water balance constraint has little impact on the general accuracy of the LSTM network when it is trained with data of 15 years for a single basin, which is consistent with and MC-LSTM as the median KGE is respectively 0.676 and 0.679 (shown in the penultimate column in Table 2). In particular, 245 the distributions of mean KGE for the LSTM and MC-LSTM are similar, which is illustrated by their cumulative distribution curves that are close to overlapping in Fig. 2. These results indicate that the water balance constraint has little impact on the general accuracy of the LSTM network when it is trained with data of 15 years for a single basin, which is consistent with 11 4.2 Sensitivity to data sparsity In the testing period, the hydrographs of two basins where different models are trained wi In the testing period, the hydrographs of two basins where different models are trained with different amounts of training data are shown in Fig. 3. Not surprisingly, the LSTM network exhibits the largest variability of KGE values when the training 260 period length is 3 years. As the length of training data is increased from 3 years to 15 years, the variability of ensemble KGE values for the LSTM decreases, while there are less changes of the variability of the ensemble KGE values for the MC-LSTM and EXP-HYDRO models. In the basin 01439500, the MC-LSTM and EXP-HYDRO models outperform the LSTM with better goodness of fit and more narrow ranges of predictions. With the length of training data increased from 3 years to 15 data are shown in Fig. 3. Not surprisingly, the LSTM network exhibits the largest variability of KGE values when the training 260 period length is 3 years. As the length of training data is increased from 3 years to 15 years, the variability of ensemble KGE values for the LSTM decreases, while there are less changes of the variability of the ensemble KGE values for the MC-LSTM and EXP-HYDRO models. In the basin 01439500, the MC-LSTM and EXP-HYDRO models outperform the LSTM with better goodness of fit and more narrow ranges of predictions. With the length of training data increased from 3 years to 15 years, the LSTM becomes more accurate, and its prediction ranges narrow. In the basin 11532500, the LSTM, MC-LSTM and 265 EXP-HYDRO models fit well with both 3- and 15-year training data. The LSTM network becomes more accurate with more training data. years, the LSTM becomes more accurate, and its prediction ranges narrow. In the basin 11532500, the LSTM, MC-LSTM and 265 EXP-HYDRO models fit well with both 3- and 15-year training data. The LSTM network becomes more accurate with more training data. 12 https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. Figure 3. Hydrograph comparisons between different models with two different training period lengths. The bands of runoff represent the 270 prediction ranges of 10-member ensembles with different random seeds. https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. which is consistent with conclusions of the previous study that physical constraints reduce the data volume dependency of the 280 LSTM (Read et al., 2019; Wang et al., 2023). which is consistent with conclusions of the previous study that physical constraints reduce the data volume dependency of the 280 LSTM (Read et al., 2019; Wang et al., 2023). which is consistent with conclusions of the previous study that physical constraints reduce the data volume dependency of the 280 LSTM (Read et al., 2019; Wang et al., 2023). Figure 4. Ranges of the mean values of KGE for the LSTM, MC-LSTM and EXP-HYDRO models with different data sparsity across the 531 catchments. The markers, deep-colour bars and light-colour bars respectively represent the median, [25%, 75%] and [10%, 90%] inter- 285 quantile ranges over the 531 catchments. Figure 4. Ranges of the mean values of KGE for the LSTM, MC-LSTM and EXP-HYDRO models Figure 4. Ranges of the mean values of KGE for the LSTM, MC-LSTM and EXP-HYDRO models with different data sparsity across the 531 catchments. The markers, deep-colour bars and light-colour bars respectively represent the median, [25%, 75%] and [10%, 90%] inter- 285 quantile ranges over the 531 catchments. Figure 4. Ranges of the mean values of KGE for the LSTM, MC-LSTM and EXP-HYDRO models with different data sparsity across the 531 catchments. The markers, deep-colour bars and light-colour bars respectively represent the median, [25%, 75%] and [10%, 90%] inter- 285 quantile ranges over the 531 catchments. Table 2. Median values of mean KGE for the LSTM, MC-LSTM and EXP-HYDRO models with different data sparsity across the 531 catchments. Table 2. Median values of mean KGE for the LSTM, MC-LSTM and EXP-HYDRO models with different data sparsity across the 531 catchments. Table 2. Median values of mean KGE for the LSTM, MC-LSTM and EXP-HYDRO models with different data sparsity across the 531 catchments. 4.2 Sensitivity to data sparsity Figure 3 Hydrograph comparisons between different models with two different training period lengths The bands of runoff represent th 270 Figure 3. Hydrograph comparisons between different models with two different training period lengths. The bands of runoff represent the 270 prediction ranges of 10-member ensembles with different random seeds. Figure 4 presents an illustration of the KGE values across the 531 catchments under different data sparsity. It can be observed that the accuracy of the three models is affected by data sparsity and that the extent of the impacts varies. Specifically, as the amount of training data increases from 1 year to 15 years, the LSTM network benefits the most with median KGE 275 increased by 95.0% (shown in Table 2). By contrast, the MC-LSTM network is less affected and the median KGE is increased by 32.7%. Additionally, the accuracy of the LSTM rapidly increases by 0.459 (from 0.034 to 0.493) with the training data length increased from 1 to 3 years, while less than 0.123 (median KGE from 0.457 to 0.580) for the MC-LSTM. These results suggest that the incorporation of the water balance constraint reduces the sensitivity of the LSTM network to data sparsity, 13 https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. Training period (year) 1 3 6 9 12 15 Max ∆ (%) LSTM 0.034 0.493 0.606 0.649 0.663 0.676 95.0% MC-LSTM 0.457 0.580 0.633 0.660 0.669 0.679 32.7% EXP-HYDRO 0.530 0.605 0.626 0.624 0.629 0.631 16.0% Note: Max ∆ denotes the percentage change of median performance with training period length increased from 1 year to 15 years when the Training period (year) 1 3 6 9 12 15 Max ∆ (%) LSTM 0.034 0.493 0.606 0.649 0.663 0.676 95.0% MC-LSTM 0.457 0.580 0.633 0.660 0.669 0.679 32.7% EXP-HYDRO 0.530 0.605 0.626 0.624 0.629 0.631 16.0% Note: Max ∆ denotes the percentage change of median performance with training period length increased from 1 year to 15 years when the 290 performances of the models trained with data of 15 years is considered as the complete performance. 14 Table 3. Median values of standard deviation of KGE for the LSTM, MC-LSTM and EXP-HYDRO models with different data sparsity 310 across the 531 catchments. Training period (year) 1 3 6 9 12 15 Max ∆ (%) LSTM 0.146 0.073 0.044 0.038 0.038 0.037 -74.5% MC-LSTM 0.044 0.036 0.030 0.029 0.028 0.028 -36.6% EXP-HYDRO 0.044 0.026 0.020 0.020 0.019 0.020 -55.3% MC ∆ (%) -69.5% -51.0% -31.8% -25.4% -25.1% -24.0% - Note: MC ∆denotes the percentage difference in the median of the standard deviation of KGE between the MC-LSTM and LSTM. Max ∆denotes the percentage change of the standard deviation of KGE with training period length increased from 1 year to 15 years. Note: MC ∆denotes the percentage difference in the median of the standard deviation of KGE between the MC-LSTM and LSTM. Max ∆denotes the percentage change of the standard deviation of KGE with training period length increased from 1 year to 15 years. p g ff f f ∆denotes the percentage change of the standard deviation of KGE with training period length increased from 1 year to 15 years. 315 Figure 6 shows the change of the stability at individual basins for the LSTM and MC-LSTM. The MC-LSTM exhibits remarkably smaller standard deviation of KGE than the LSTM. Specifically, the MC-LSTM tends to be more stable at a total of 450 (85%), 386 (73%) and 366 (69%) basins when models are trained with data of 3 years, 9 years and 15 years, respectively. These results illustrate that the water balance constraint improves the stability of the LSTM network. In addition, the number of basins where the MC-LSTM is more stable than the LSTM has reduced with the training period length increased from 1 320 year to 15 years, and the differences (as shown in Table 3) in the median of the KGE standard deviation values between the MC-LSTM and LSTM decrease from -69.5% to -24.0%. The implication is that increasing the training data can narrow the stability differences between the LSTM and MC-LSTM, compensating for the instability of the LSTM network caused by the lack of the water balance constraint. Figure 6 shows the change of the stability at individual basins for the LSTM and MC-LSTM. The MC-LSTM exhibits remarkably smaller standard deviation of KGE than the LSTM. Specifically, the MC-LSTM tends to be more stable at a total of 450 (85%), 386 (73%) and 366 (69%) basins when models are trained with data of 3 years, 9 years and 15 years, respectively. 4.3 Stability against random parameters initialization 15 Training period (year) 1 3 6 9 12 15 Max ∆ (%) LSTM 0.146 0.073 0.044 0.038 0.038 0.037 -74.5% MC-LSTM 0.044 0.036 0.030 0.029 0.028 0.028 -36.6% EXP-HYDRO 0.044 0.026 0.020 0.020 0.019 0.020 -55.3% MC ∆ (%) -69.5% -51.0% -31.8% -25.4% -25.1% -24.0% - Note: MC ∆denotes the percentage difference in the median of the standard deviation of KGE between the MC-LSTM and LSTM. Max ∆denotes the percentage change of the standard deviation of KGE with training period length increased from 1 year to 15 years. https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. 4.3 Stability against random parameters initialization Across the 531 basins, the stability of the LSTM, MC-LSTM and EXP-HYDRO models with different extents of data sparsity is summarized in Fig. 5 and Table 3. Overall, the MC-LSTM has lower standard deviation values of KGE than the 295 LSTM for all sparsity levels of training data. This result suggests that adding the water balance constraint enhances the stability of the LSTM network against random parameters initialization. Specifically, the MC-LSTM, compared to the LSTM, experiences a reduction ranging from 69.5% to 24.0% in the median of the standard deviation of KGE, as shown in Table 3. In addition, the standard deviation values of KGE for the LSTM and MC-LSTM reduce with the training data increased, while the degree of reduction varies for each of the three models. The LSTM exhibits the largest magnitude of reduction in the 300 standard deviation of KGE with the length of training data increased from 1 year to 15 years, while the MC-LSTM and EXP- HYDRO models exhibit slighter reductions. These results also indicate that adding the water balance constraint reduces the sensitivity of the LSTM network to data sparsity. the degree of reduction varies for each of the three models. The LSTM exhibits the largest magnitude of reduction in the 300 standard deviation of KGE with the length of training data increased from 1 year to 15 years, while the MC-LSTM and EXP- HYDRO models exhibit slighter reductions. These results also indicate that adding the water balance constraint reduces the sensitivity of the LSTM network to data sparsity. 305 Figure 5. Ranges of the standard deviation values of KGE for the LSTM, MC-LSTM and EXP-HYDRO models across the 531 catchments under different data sparsity. The markers, deep-colour bars and light-colour bars respectively represent the median, [25%, 75%] and [10%, 90%] inter-quantile ranges over the 531 catchments. 05 305 Figure 5. Ranges of the standard deviation values of KGE for the LSTM, MC-LSTM and EXP-HYDRO models across the 531 catchments under different data sparsity. The markers, deep-colour bars and light-colour bars respectively represent the median, [25%, 75%] and [10%, 90%] inter-quantile ranges over the 531 catchments. Per-basin change of stability between the MC-LSTM and LSTM in the 531 basins. Red dots indicate basins where adding the water balance constraint improves the stability of the LSTM (darker indicates larger relative improvement), and blue dots indicate basins where there is a decrease in stability (darker indicates worse relative detriment). Note that a higher standard deviation (Std.) of KGE indicates the worse stability of a model across random parameters initialization. 330 Figure 6. Per-basin change of stability between the MC-LSTM and LSTM in the 531 basins. Red dots indicate basins where adding the water balance constraint improves the stability of the LSTM (darker indicates larger relative improvement), and blue dots indicate basins where there is a decrease in stability (darker indicates worse relative detriment). Note that a higher standard deviation (Std.) of KGE indicates the worse stability of a model across random parameters initialization. 30 These results illustrate that the water balance constraint improves the stability of the LSTM network. In addition, the number of basins where the MC-LSTM is more stable than the LSTM has reduced with the training period length increased from 1 320 year to 15 years, and the differences (as shown in Table 3) in the median of the KGE standard deviation values between the MC-LSTM and LSTM decrease from -69.5% to -24.0%. The implication is that increasing the training data can narrow the stability differences between the LSTM and MC-LSTM, compensating for the instability of the LSTM network caused by the lack of the water balance constraint. of basins where the MC-LSTM is more stable than the LSTM has reduced with the training period length increased from 1 320 year to 15 years, and the differences (as shown in Table 3) in the median of the KGE standard deviation values between the MC-LSTM and LSTM decrease from -69.5% to -24.0%. The implication is that increasing the training data can narrow the stability differences between the LSTM and MC-LSTM, compensating for the instability of the LSTM network caused by the lack of the water balance constraint. 325 16 https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. Figure 6. Per-basin change of stability between the MC-LSTM and LSTM in the 531 basins. Red dots indicate basins where adding the water balance constraint improves the stability of the LSTM (darker indicates larger relative improvement), and blue dots indicate basins where there is a decrease in stability (darker indicates worse relative detriment). Note that a higher standard deviation (Std.) of KGE indicates the worse stability of a model across random parameters initialization. Figure 6. Per-basin change of stability between the MC-LSTM and LSTM in the 531 basins. Red dots indicate basins where adding the Figure 6. Per-basin change of stability between the MC-LSTM and LSTM in the 531 basins. Red dots indicate basins where adding the water balance constraint improves the stability of the LSTM (darker indicates larger relative improvement), and blue dots indicate basins where there is a decrease in stability (darker indicates worse relative detriment). Note that a higher standard deviation (Std.) of KGE indicates the worse stability of a model across random parameters initialization. 330 Figure 6. 4.4 Transferability under contrasting climate conditions The KGE values for all models under the control and testing conditions across the 475 catchments are shown in Fig. 7 to examine the transferability between the wettest/driest years. In general, the MC-LSTM exhibits higher transferability than the LSTM. This result suggests that adding the water balance constraint advances the transferability of the LSTM under contrasting 335 climate conditions. In both W/D and D/W scenarios, the LSTM, MC-LSTM and EXP-HYDRO models illustrate close accuracy on the control data with KGE range of [0.634, 0.664] and [0.598, 0.602], as demonstrated in Table 4. When The KGE values for all models under the control and testing conditions across the 475 catchments are shown in Fig. 7 to examine the transferability between the wettest/driest years. In general, the MC-LSTM exhibits higher transferability than the LSTM. This result suggests that adding the water balance constraint advances the transferability of the LSTM under contrasting 335 climate conditions. In both W/D and D/W scenarios, the LSTM, MC-LSTM and EXP-HYDRO models illustrate close accuracy on the control data with KGE range of [0.634, 0.664] and [0.598, 0.602], as demonstrated in Table 4. When 17 https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. transferring to the testing data, the MC-LSTM outperforms the LSTM with median KGE of 0.576 in the W/D scenario and 0.515 in the D/W scenario, compared to the median KGE of 0.538 and 0.446 for the LSTM, respectively. In terms of median KGE differences between the control and testing data, the MC-LSTM exhibits less degradation in performance than the LSTM 340 with median KGE decreased by 13.3% in the W/D scenario and 15.4% in the D/W scenario, compared to the median KGE decreased by 17.4% and 25.4% for the LSTM, respectively. Figure 7. Ranges of KGE of the LSTM, MC-LSTM and EXP-HYDRO models across the 475 catchments for different DSST scenarios. 345 Delta denotes the KGE difference between the testing data (Testing) and control data (Control). Figure 7. Ranges of KGE of the LSTM, MC-LSTM and EXP-HYDRO models across the 475 catchments for different DSST scenarios. 345 Delta denotes the KGE difference between the testing data (Testing) and control data (Control). Table 4. Median KGE and median KGE differences (percent) between the control data and the testing data for different models in differ DSST scenarios across the 475 basins. Model W/D D/W Control Testing ∆ (%) Control Testing ∆ (%) LSTM 0.651 0.538 -17.4 0.598 0.446 -25.4 MC-LSTM 0.664 0.576 -13.3 0.609 0.515 -15.4 EXP-HYDRO 0.634 0.489 -23.0 0.602 0.590 -2.2 Note: ∆ denotes the median of the KGE differences between the control data and testing data. 350 able 4. Median KGE and median KGE differences (percent) between the control data and the testing data for d SST scenarios across the 475 basins. ian KGE differences (percent) between the control data and the testing data for different models in different basins. Note: ∆ denotes the median of the KGE differences between the control data and testing data. 350 The per-basin accuracy comparisons between the LSTM and MC-LSTM in the testing conditions are plotted in Fig. 8. In the D/W scenario, the MC-LSTM exhibits higher KGE values compared to the LSTM across 318 basins (67%). But for the 18 https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. W/D scenario, the number of basins with higher KGE for the MC-LSTM than the LSTM decreases to 262 (55%). The catchments where the MC-LSTM shows improved accuracy than the LSTM are mainly located in the central arid regions of 355 the United States. In these areas, the runoff generation is dominated by the inﬁltration-excess overland ﬂow, which is largely controlled by short-duration, high-intensity precipitation events (Berghuijs et al., 2016). These results suggest that the consideration of the water balance constraint improves the prediction of the LSTM transferred from the driest to the wettest years. For the prediction transferred from the wettest to the driest years, the LSTM itself demonstrates good accuracy and the W/D scenario, the number of basins with higher KGE for the MC-LSTM than the LSTM decreases to 262 (55%). The catchments where the MC-LSTM shows improved accuracy than the LSTM are mainly located in the central arid regions of 355 the United States. In these areas, the runoff generation is dominated by the inﬁltration-excess overland ﬂow, which is largely controlled by short-duration, high-intensity precipitation events (Berghuijs et al., 2016). These results suggest that the consideration of the water balance constraint improves the prediction of the LSTM transferred from the driest to the wettest years. For the prediction transferred from the wettest to the driest years, the LSTM itself demonstrates good accuracy and the improvement from enforcing the water balance constraint is not substantial. It is possibly due to the skewness and censoring 360 characteristics of hydroclimatic variables (Huang et al., 2023), with lower runoff values occurring much more frequently than higher values. This property makes the training data of the wettest years more similar to the testing data of the driest years, improvement from enforcing the water balance constraint is not substantial. It is possibly due to the skewness and censoring 360 characteristics of hydroclimatic variables (Huang et al., 2023), with lower runoff values occurring much more frequently than higher values. This property makes the training data of the wettest years more similar to the testing data of the driest years, thereby making it easier for the LSTM to transfer in the W/D scenario. improvement from enforcing the water balance constraint is not substantial. https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. It is possibly due to the skewness and censoring 360 characteristics of hydroclimatic variables (Huang et al., 2023), with lower runoff values occurring much more frequently than higher values. This property makes the training data of the wettest years more similar to the testing data of the driest years, thereby making it easier for the LSTM to transfer in the W/D scenario. 365 Figure 8. Per-basin change of accuracy between the MC-LSTM and LSTM at the 475 basins. Note that higher accuracy indicates higher transferability in testing conditions. Red dots indicate basins where adding the water balance constraint improved the transferability over the LSTM (darker indicates larger relative improvement), and blue dots indicate basins where the transferability decreases (darker indicates worse relative detriment). 365 Figure 8. Per-basin change of accuracy between the MC-LSTM and LSTM at the 475 basins. Note that higher accuracy indicates higher transferability in testing conditions. Red dots indicate basins where adding the water balance constraint improved the transferability over the LSTM (darker indicates larger relative improvement), and blue dots indicate basins where the transferability decreases (darker indicates worse relative detriment). 370 19 5 Discussion Though experiments here do not evaluate the impact of data 380 quality or uncertainty in model inputs on the quality of predictions, such efforts could provide additional insights beyond the scope of this paper (Read et al., 2019). Given that data is not always sufficient, the sensitivity of DL models when given scarce training data is essentially important (Feng et al., 2021; Gauch et al., 2021b). The TGDS provides effective tools for reducing data requirements of DL Given that data is not always sufficient, the sensitivity of DL models when given scarce training data is essentially important (Feng et al., 2021; Gauch et al., 2021b). The TGDS provides effective tools for reducing data requirements of DL models (Karniadakis et al., 2021; Karpatne et al., 2017; Read et al., 2019; Xie et al., 2021). In this paper, the incorporation of 385 the water balance constraint into the LSTM network provides direct guidance based on physical knowledge to the internal calculation processes, thus reducing the need to learn this specific physical mechanism form large amounts of data (Frame et al., 2023; Hoedt et al., 2021). Another TGDS strategy, which reconfigures the loss functions with physical penalties, allows DL models to obtain additional guidance to their training process from the physical mechanisms, also leading to less training data requirement (Wang et al 2023; Yang et al 2020; Zhao et al 2019) Moreover recent studies showed that using synthetic 390 models (Karniadakis et al., 2021; Karpatne et al., 2017; Read et al., 2019; Xie et al., 2021). In this paper, the incorporation of 385 the water balance constraint into the LSTM network provides direct guidance based on physical knowledge to the internal calculation processes, thus reducing the need to learn this specific physical mechanism form large amounts of data (Frame et al., 2023; Hoedt et al., 2021). Another TGDS strategy, which reconfigures the loss functions with physical penalties, allows DL models to obtain additional guidance to their training process from the physical mechanisms, also leading to less training data requirement (Wang et al., 2023; Yang et al., 2020; Zhao et al., 2019). Moreover, recent studies showed that using synthetic 390 data generated by physical models to pretrain DL models could also contribute to overcome the conditions of sparse observation data (Read et al., 2019; Xie et al., 2021; Zhang et al., 2022). 5 Discussion Furthermore, this paper also demonstrates the potential of enforcing physical constraints such as the water balance constraint to strengthen the robustness of DL models under random parameters initialization and climate change. data requirement (Wang et al., 2023; Yang et al., 2020; Zhao et al., 2019). Moreover, recent studies showed that using synthetic 390 data generated by physical models to pretrain DL models could also contribute to overcome the conditions of sparse observation data (Read et al., 2019; Xie et al., 2021; Zhang et al., 2022). Furthermore, this paper also demonstrates the potential of enforcing physical constraints such as the water balance constraint to strengthen the robustness of DL models under random parameters initialization and climate change. Although TGDS models can provide more accurate and robust predictions than pure DL models in basin-wise scale or 395 data scarce conditions, it deserves additional scrutiny when trained with data from a large number of diverse basins (Frame et al., 2022; Nearing et al., 2021; Wi and Steinschneider, 2022). Recent studies have illustrated that for DL models, physical constraints are effective in local models but offer little improvement in the regional models (Frame et al., 2023; Xie et al., 2021), even reduce predictive performance under extreme events (Frame et al., 2022). This outcome can be attributed to that Although TGDS models can provide more accurate and robust predictions than pure DL models in basin-wise scale or 395 data scarce conditions, it deserves additional scrutiny when trained with data from a large number of diverse basins (Frame et al., 2022; Nearing et al., 2021; Wi and Steinschneider, 2022). Recent studies have illustrated that for DL models, physical constraints are effective in local models but offer little improvement in the regional models (Frame et al., 2023; Xie et al., 2021), even reduce predictive performance under extreme events (Frame et al., 2022). This outcome can be attributed to that catchments with similar flood generating processes have some similar outliers (Bertola et al., 2023) and that extreme events 400 that did not occur frequently in one basin may occur in other basins (Xie et al., 2021). Therefore, there seems to be a compensating effect between data and knowledge on DL models, where the process knowledge is crucial for models trained with sparse data but less important with sufficient data. 5 Discussion It has been highlighted that more training data contributes to the performance of the LSTM network (Gauch et al., 2021b; Read et al., 2019; Wang et al., 2023; Xie et al., 2021). In this paper, the role of the training data in the performance of the MC- LSTM network is investigated through large-sample tests. The findings generally conform to previous findings that the LSTM trained to hundreds of basins allows for better streamflow predictions in a given basin compared to the LSTM only trained to 375 that specific basin or a smaller subset of basins (Jiang et al., 2020; Kratzert et al., 2019b), even for out-of-sample predictions (Gauch et al., 2021b; Xie et al., 2021). These results suggest that DL models can gain extrapolation ability from big data compensating the lack of physical mechanisms (Feng et al., 2021; Kratzert et al., 2019b; Nearing et al., 2019). Through extensive training with large amounts of data, DL models can advance hydrologic predictions, even without explicit physical trained to hundreds of basins allows for better streamflow predictions in a given basin compared to the LSTM only trained to 375 that specific basin or a smaller subset of basins (Jiang et al., 2020; Kratzert et al., 2019b), even for out-of-sample predictions (Gauch et al., 2021b; Xie et al., 2021). These results suggest that DL models can gain extrapolation ability from big data compensating the lack of physical mechanisms (Feng et al., 2021; Kratzert et al., 2019b; Nearing et al., 2019). Through extensive training with large amounts of data, DL models can advance hydrologic predictions, even without explicit physical mechanisms (Nearing et al., 2021; Wi and Steinschneider, 2022). Though experiments here do not evaluate the impact of data 380 quality or uncertainty in model inputs on the quality of predictions, such efforts could provide additional insights beyond the scope of this paper (Read et al., 2019). mechanisms (Nearing et al., 2021; Wi and Steinschneider, 2022). Though experiments here do not evaluate the impact of data 380 quality or uncertainty in model inputs on the quality of predictions, such efforts could provide additional insights beyond the scope of this paper (Read et al., 2019). mechanisms (Nearing et al., 2021; Wi and Steinschneider, 2022). https://doi.org/10.5194/egusphere-2023-2841 Preprint. Discussion started: 5 January 2024 c⃝Author(s) 2024. CC BY 4.0 License. systems respond to previously unobserved extreme events and climate conditions, large-sample hydrology is expected to enhance the performances of DL models for extreme events predictions and climate change projections (Bertola et al., 2023; 405 Wi and Steinschneider, 2022). 5 Discussion With much more available data to learn the patterns that hydrologic catchments with similar flood generating processes have some similar outliers (Bertola et al., 2023) and that extreme events 400 that did not occur frequently in one basin may occur in other basins (Xie et al., 2021). Therefore, there seems to be a compensating effect between data and knowledge on DL models, where the process knowledge is crucial for models trained with sparse data but less important with sufficient data. With much more available data to learn the patterns that hydrologic 20 Acknowledgements. This research is supported by the National Natural Science Foundation of China (U1911204, 51725905, 425 52130907, 51979295, 51861125203 and 52109046), the National Key Research and Development Program of China (2021YFC3001000) and the Guangdong Provincial Department of Science and Technology (2019ZT08G090). 6 Conclusions This paper is concentrated on the effects of the water balance constraint in model architecture on the robustness of the basin-wise trained LSTM for rainfall-runoff prediction. That is, large-sample tests based on CAMELS dataset are conducted 410 to assess the robustness of the LSTM and its architecturally mass-conserving variant (MC-LSTM) from three perspectives, i.e., the sensitivity to data sparsity, the stability across random parameters initialization and the transferability under contrasting climate conditions. The results show that the water balance constraint contributes to the robustness of the basin-wise trained LSTM. One finding is that for varying data sparsity of training data ranging from 1 year to 15 years, the addition of the water balance constraint decreases the sensitivity of the LSTM to data sparsity from 95.0% to 32.7%. Another finding is that the 415 water balance constraint is effective in improving the stability of the LSTM at 450 (85%) when available data are 3 years. The third finding is that the water balance constraint enhances the transferability of the LSTM from the driest years to the wettest years at 318 (67%) basins. The in-depth investigations of this paper facilitate insights into the use of the LSTM network and other DL models for rainfall-runoff modelling. balance constraint decreases the sensitivity of the LSTM to data sparsity from 95.0% to 32.7%. Another finding is that the 415 water balance constraint is effective in improving the stability of the LSTM at 450 (85%) when available data are 3 years. The third finding is that the water balance constraint enhances the transferability of the LSTM from the driest years to the wettest years at 318 (67%) basins. The in-depth investigations of this paper facilitate insights into the use of the LSTM network and other DL models for rainfall-runoff modelling. 420 ata availability. The CAMELS dataset is download from https://gdex.ucar.edu/dataset/camels.html References Cai, H., Liu, S., Shi, H., Zhou, Z., Jiang, S., and Babovic, V.: Toward improved lumped groundwater level predictions at catchment scale: Mutual integration of water balance mechanism and deep learning method, Journal of Hydrology, 613, 128495, https://doi.org/10.1016/j.jhydrol.2022.128495, 2022. 450 p g j j y Faghmous, J. H. and Kumar, V.: A big data guide to understanding climate change: the case for theory-guided data science, Big Data, 2, 155–163, https://doi.org/10.1089/big.2014.0026, 2014. p g j j y Faghmous, J. 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TZ and QL prepared the manuscript with contributions from the co-authors. Author contribution. TZ and QL designed the experiments. QL carried them out. QL and TZ developed the model code and performed the experiments. TZ and QL prepared the manuscript with contributions from the co-authors. Competing interests. The contact author has declared that none of the authors has any competing interests. Competing interests. The contact author has declared that none of the authors has any competing interests. Acknowledgements. This research is supported by the National Natural Science Foundation of China (U1911204, 51725905, 425 52130907, 51979295, 51861125203 and 52109046), the National Key Research and Development Program of China (2021YFC3001000) and the Guangdong Provincial Department of Science and Technology (2019ZT08G090). Acknowledgements. 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https://openalex.org/W4327530950	https://zenodo.org/records/7742002/files/MANUAL%20BOAS%20PRATICAS%202%20-%2014_03.pdf	Portuguese	null	Manual de boas práticas 2: PDDE e Ações Integradas: Gestão e Execução	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	6,974	MANUAL DE BOAS PRÁTICAS DE GESTÃO MANUAL DE BOAS PRÁTICAS DE GESTÃO MANUAL DE BOAS PRÁTICAS DE GESTÃO Anielson Barbosa da Silva Adriana Valéria Santos Diniz Maria Aparecida Nunes Pereira Wagner Junqueira de Araújo PDDE e Ações Integradas: Gestão e Execução João Pessoa-PB 2022 Lopes da Ponte Anielson Barbosa da Silva Adriana Valéria Santos Diniz Maria Aparecida Nunes Pereira Wagner Junqueira de Araújo João Pessoa-PB 2022 Lopes da Ponte ©2022 Universidade Federal da Paraíba Este Relatório foi elaborado pela equipe de pesquisadores (as) do Centro Colaborador de Apoio ao Monitoramento e à Gestão de Programas Educacionais – CECAMPE - da Região Nordeste, em parceria com o Fundo Nacional de Desenvolvimento da Educação – FNDE/MEC, por meio da Diretoria de Ações Educacionais/Coordenação Geral de Apoio à Manutenção Escolar. TED 9840/2020. Registro na PROPESQ/UFPB PID13079-2020. UNIVERSIDADE FEDERAL DA PARAÍBA - UFPB 2 UNIVERSIDADE FEDERAL DA PARAÍBA - UFPB Reitor Valdiney Veloso Gouveia Vice-Reitora Liana Filgueira Albuquerque Pró-Reitor de Pesquisa Valdir de Andrade Braga Pró-Reitoria de Extensão Berla Moreira de Moraes Coordenação Geral do CECAMPE-NE Adriana Valéria Santos Diniz Coordenação dos Eixos do CECAMPE-NE Maria Aparecida Nunes Pereira – Eixo Assistência Técnica Wagner Junqueira de Araújo – Eixo Monitoramento Ítalo Fittipaldi – Eixo Avaliação Autores Anielson Barbosa da Silva Adriana Valéria Santos Diniz Maria Aparecida Nunes Pereira Wagner Junqueira de Araújo Revisão e Diagramação Trindade Monografias e Edições Reitor 2 Como citar: SILVA, Anielson Barbosa da; DINIZ, Adriana Valéria Santos; PEREIRA, Maria Aparecida Nunes. Manual de boas práticas de gestão: PDDE e Ações Integradas: Estrutura e Planejamento. João Pessoa-PB, 2022. Como citar: SILVA, Anielson Barbosa da; DINIZ, Adriana Valéria Santos; PEREIRA, Maria Aparecida Nunes. Manual de boas práticas de gestão: PDDE e Ações Integradas: Estrutura e Planejamento. João Pessoa-PB, 2022. 3 SUMÁRIO 1. INTRODUÇÃO......................................................................................................4 2. CONTEXTO: BOAS PRÁTICAS DE EXECUÇÃO DO PDDE – CONHECENDO OS ENTRAVES E OS DESAFIOS ...................................................................................................... 6 3. A EXECUÇÃO COMO DETERMINANTE PARA A DISSEMINAÇÃO DE BOAS PRÁTICAS DE GESTÃO ............................................................................................................... 10 3.1. Vamos começar pelo plano anual de atividades! ............................................... 11 3.2. Agora, vamos conhecer o plano de aplicação de recursos! ................................. 12 3.3. Procedimentos técnico-operacionais para a execução dos recursos .................... 14 4. A CAPACITAÇÃO TÉCNICA: UM DOS FATORES DETERMINANTES PARA A GESTÃO DO PDDE ............................................................................................................. 18 5. CONSIDERAÇÕES ....................................................................................... 20 6. REFERÊNCIAS ............................................................................................. 21 SUMÁRIO 1. INTRODUÇÃO......................................................................................................4 2. CONTEXTO: BOAS PRÁTICAS DE EXECUÇÃO DO PDDE – CONHECENDO OS ENTRAVES E OS DESAFIOS ...................................................................................................... 6 3. A EXECUÇÃO COMO DETERMINANTE PARA A DISSEMINAÇÃO DE BOAS PRÁTICAS DE GESTÃO ............................................................................................................... 10 3.1. Vamos começar pelo plano anual de atividades! ............................................... 11 3.2. Agora, vamos conhecer o plano de aplicação de recursos! ................................. 12 3.3. Procedimentos técnico-operacionais para a execução dos recursos .................... 14 4. A CAPACITAÇÃO TÉCNICA: UM DOS FATORES DETERMINANTES PARA A GESTÃO DO PDDE ............................................................................................................. 18 5. CONSIDERAÇÕES ....................................................................................... 20 6. REFERÊNCIAS ............................................................................................. 21 .4 4 4 INTRODUÇÃO INTRODUÇÃO Este Manual é uma produção do eixo Assistência Técnica em conjunto com o Eixo Monitoramento do CECAMPE NORDESTE como parte integrante do produto voltado para a análise da evolução dos problemas de execução do PDDE (monitoramento) associado ao produto voltado para a sistematização e divulgação de boas práticas (assistência técnica). No âmbito do PDDE, caracteriza-se como boa prática toda e qualquer ação voltada à melhoria da gestão dos recursos financeiros do Programa e que esteja relacionada aos processos de adesão, execução, prestação de contas e participação social, resultando na elevação do Índice de Desempenho da Gestão Descentralizada do PDDE - IdeGES-PDDE. O IdeGES mensura o desempenho da gestão descentralizada do Programa em todo o país, com o objetivo de viabilizar iniciativas de monitoramento e avaliação, orientar a ação governamental para melhoria do desempenho do Programa, contribuir com o exercício do controle social e identificar experiências exitosas de gestão. Reitor O CECAMPE-NE, no período de 2021 e 2022, desenvolveu ações voltadas à elevação do IdeGES nos estados/municípios/escolas da região nordeste, por meio da assistência técnica nas modalidades presencial e a distância aos agentes envolvidos com a gestão dos recursos financeiros do PDDE e das Ações Integradas. No âmbito dessas ações de formação, mais especificamente o Curso a distância sem tutoria, foram aplicados questionários com o objetivo de identificar a percepção dos cursistas acerca do PDDE e das Ações Integradas. Foi possível constatar que apesar das especificidades de cada estado/município/escola, no geral, todos(as) relataram dificuldades muito semelhantes, dentre elas, o baixo conhecimento sobre a gestão do PDDE e Ações Integradas, envolvendo a adesão/atualização cadastral, a execução e a prestação de contas. 5 Este material visa, portanto, apresentar orientações voltadas ao desenvolvimento de ações que resultem em boas práticas de gestão, considerando os processos de adesão, execução e monitoramento do PDDE e das Ações Integradas, na região nordeste, a partir dos relatos dos participantes da pesquisa. Essas orientações estão sistematizadas em 3 (três) manuais: • PDDE e Ações Integradas: estrutura e planejamento; • PDDE e Ações Integradas: Gestão e Execução; • Sistema de Monitoramento da Gestão do PDDE. Neste volume, apresentaremos orientações relacionadas aos processos de gestão e Execução do PDDE e Ações Integradas, considerando as dificuldades que a comunidade escolar tem com essa etapa da gestão dos Programas. A proposta inicial era desenvolver toda uma análise da evolução dos problemas de execução, no âmbito do eixo monitoramento, para organizar um guia de perguntas e respostas, mas isso requer informações e, no contexto do projeto, enfrentamos uma limitação de acesso a algumas bases de dados, o que comprometeu em parte essa proposta. Optou-se, dessa forma, pelo trabalho conjunto com o eixo assistência técnica, ampliando o Manual de Boas Práticas que trata da qualidade da gestão e da execução do PDDE, demonstrando, a partir dos estudos e pesquisas desenvolvidas nos dois eixos, os entraves existentes e as suas formas de superação a fim de que se possa desenvolver boas e eficientes práticas de gestão do PDDE na Região Nordeste. Reitor Neste sentido, acreditamos que este material tem potencial para promover uma aprendizagem coletiva no âmbito dos programas de gestão descentralizada, uma vez que a disseminação de experiências envolvendo processos e práticas de gestão servirão de base para a reflexão de gestores, como também de referência para implementarem melhorias no âmbito das escolas, o que pode contribuir para otimização dos processos, melhoria do IdeGES, como também de outros indicadores de qualidade a exemplo do Índice de Desenvolvimento da Educação Básica – IDEB. 6 CONTEXTO: BOAS PRÁTICAS DE EXECUÇÃO DO PDDE – CONHECENDO OS ENTRAVES E OS DESAFIOS CONTEXTO: BOAS PRÁTICAS DE EXECUÇÃO DO PDDE – CONHECENDO OS ENTRAVES E OS DESAFIOS O O Programa Dinheiro Direto na Escola – PDDE Básico é considerado um dos programas mais exitosos de transferência direta de recursos às escolas, em quase 30 (trinta) anos de vigência, em todo o país. O Programa Dinheiro Direto na Escola – PDDE Básico é considerado um dos programas mais exitosos de transferência direta de recursos às escolas, em quase 30 (trinta) anos de vigência, em todo o país. Em caráter suplementar, transfere anualmente, às entidades participantes, recursos financeiros para serem executados nas seguintes finalidades: • Provimento das necessidades prioritárias dos estabelecimentos educacionais beneficiários que concorram para a garantia do funcionamento; • Promoção de melhorias na infraestrutura física e pedagógica; e • O incentivo da autogestão escolar e do exercício da cidadania, com a participação da comunidade no controle social. Além do PDDE Básico, existem as Ações Integradas, que consistem em Programas gerenciados pelas Secretarias do Ministério da Educação – MEC (Secretária de Alfabetização – Sealf, Secretaria de Educação Básica – SEB e Secretaria de Modalidades Especializadas – Semesp), com finalidades, objetos e públicos-alvo específicos, mas que seguem os mesmos moldes operacionais do PDDE Básico nos quesitos: forma de transferência dos repasses, modo de gestão dos recursos e modo de prestação de contas. Para receber os recursos do PDDE Básico e das Ações Integradas, as entidades participantes devem fazer a adesão/atualização cadastral, ter Unidade Executora Própria -UEX e não possuir pendências de Prestação de Contas. 7 região nordeste um montante de recursos de aproximadamente R$. 460.120.000,00. Desse total, apenas 51% foram executados neste ano, fato que demonstra, dentre outros, dificuldades no processo de execução desses recursos. (CECAMPE, 2022) No conjunto dos estados da região nordeste, verificamos, conforme dados do gráfico abaixo, que o nível de execução dos recursos encontra-se na média dos 40% a 50%. Observa-se uma queda no percentual de execução dos recursos no período de 2020 a 2021, em todos os estados, sendo a exceção o estado do Rio Grande do Norte, que ampliou seu percentual de 48% para 53%. O estado do Maranhão foi o que apresentou maior queda, oscilando de 52% em 2020 para 41% em 2021. CONTEXTO: BOAS PRÁTICAS DE EXECUÇÃO DO PDDE – CONHECENDO OS ENTRAVES E OS DESAFIOS el; 9 Para aprofundar um pouco mais a discussão, a Tabela 1, a seguir, apresenta informações sobre as principais dificuldades encontradas para o desenvolvimento das atividades de execução do PDDE, de acordo com os participantes dos cursos de capacitação in loco, realizadas pelo CECAMPE-NE nos nove estados da região Nordeste, no período de julho a setembro de 2022. Tabela 1 - Principais Dificuldades no Processo de Execução Tabela 1 - Principais Dificuldades no Processo de Execução Tabela 1 - Principais Dificuldades no Processo de Execução Dificuldades vivenciadas no processo de Execução do PDDE e/ou Ações Integradas 1º. Curso de Capacitação Presencial 2º. Curso de Capacitação Presencial 3º. Curso de Capacitação Presencial** N % n % n % Contratação de Serviços 1092 40,5 575 39,4 854 38,6 Pesquisa de Preços e Contratação 754 28,0 428 29,4 631 28,3 Elaboração do plano de aplicação dos recursos. 721 26,7 372 25,5 560 25,1 Levantamento da documentação das empresas para compra de materiais. 701 26,0 367 25,2 533 23,9 Planejamento participativo (participação da comunidade escolar no processo de execução de recursos). 819 30,4 323 22,2 490 22,0 Acompanhamento efetivo da comunidade escolar na execução dos recursos. 705 26,1 313 21,5 417 18,7 Diferenciação entre recursos de Capital e Custeio. 413 16,7 260 17,8 323 14,5 Preenchimento do formulário de consolidação de pesquisa de preços. 392 15,9 240 16,5 307 13,8 Compra de material permanente 393 14,6 204 14,0 273 12,2 Guarda de documentação 175 6,5 79 5,4 95 4,3 Não tenho dificuldades 353 13,71 269 18,4 473 21,2 * 2.697 respondentes 1.458 respondentes * 2.229 respondentes Fonte: CECAMPE-NE, 2022. Uma análise do quadro revela a percepção de 6.384 participantes das três primeiras edições do curso de capacitação, in loco, com gestores e técnicos que atuam na gestão de Unidades Executoras. Percebe-se que as principais dificuldades na execução são similares entre os participantes dos três cursos. A contratação de serviços, a pesquisa de preços, a elaboração do plano de aplicação de recursos são as três principais dificuldades. Esses dados demonstram a necessidade do CECAMPE/NE investir na elaboração de orientações que auxiliem os gestores a minimizarem essas dificuldades, pois só assim será possível pensar em boas práticas de gestão. CONTEXTO: BOAS PRÁTICAS DE EXECUÇÃO DO PDDE – CONHECENDO OS ENTRAVES E OS DESAFIOS (CECAMPE, 2022) A baixa execução no período de 2020 a 2021 pode estar associada ao fechamento das escolas em decorrência da Pandemia da Covid 19, contexto em que as escolas tiveram que reprogramar recursos não utilizados no exercício financeiro, incidindo na baixa execução e, consequentemente, na queda do IdeGES na região nordeste, de 6,8 em 2020 para 6,53 em 2021. (CECAMPE, 2022) 8 Fonte: CECAMPE, 2022. 8 Fonte: CECAMPE, 2022. Fonte: CECAMPE, 2022. Fonte: CECAMPE, 2022. 8 A queda no índice de execução pode estar associada também a dificuldades enfrentadas pelos agentes envolvidos com a gestão do PDDE no que se refere aos conhecimentos necessários à gestão e operacionalização do Programa. A queda no índice de execução pode estar associada também a dificuldades enfrentadas pelos agentes envolvidos com a gestão do PDDE no que se refere aos conhecimentos necessários à gestão e operacionalização do Programa. Pesquisa realizada com os cursistas ingressantes no Curso a distância sem tutoria, ofertado pelo CECAMPE-NE (2022) demonstrou que, num universo de 331 participantes, 49,3% indicaram ter um nível bom ou ótimo de conhecimento sobre o planejamento para aplicação dos recursos do PDDE, ou seja, em torno de 50%. Isso significa que aproximadamente 50% dos entrevistados possuem baixo ou nenhum conhecimento sobre o Programa, percentual bastante elevado. Os problemas identificados corroboram os questionamentos indicados pelos participantes dos Webnários, produzidos em 2021 e 2022, cujo formulário com questões sobre os problemas e questionamentos foi aplicado ao final de cada evento, os dados coletados foram tabulados e apresentados em formato de painéis de dados. A pesquisa evidenciou que os participantes apresentavam dificuldades no planejamento e na execução dos recursos, com destaque para a elaboração do Plano Anual de Atividades (59,3%) e do Plano de Aplicação de recursos (62,3%). (CECAMPE, 2022). Outras dificuldades relatadas foram: • aquisição de equipamentos, materiais e contratação de prestadores de serviços; • realização de pesquisa de preço; • diferenciação entre os recursos de capital e de custeio, além da alocação e utilização de forma correta e eficiente dos recursos; • elaboração das planilhas com orçamentos; • encontrar empresas prestadoras de serviços com produtos e preço acessível; • recebimento do recurso em várias contas; • irregularidades no comércio local e a dificuldade com a pesquisas de preço; • dificuldade em conseguir as informações com o Banco do Brasil. (Reclamação recorrente); • excesso de burocracia de alguns processos. CONTEXTO: BOAS PRÁTICAS DE EXECUÇÃO DO PDDE – CONHECENDO OS ENTRAVES E OS DESAFIOS Esses entraves, associado ao baixo ou nenhum conhecimento sobre o processo de execução de recursos por parte das entidades participantes, resultam em baixos índices de execução, acarretando na reprogramação dos saldos em contas, fato que compromete o alcance dos objetivos do Programa e na elevação do IdeGES. 10 A EXECUÇÃO COMO DETERMINANTE PARA A DISSEMINAÇÃO DE BOAS PRÁTICAS DE GESTÃO A EXECUÇÃO COMO DETERMINANTE PARA A DISSEMINAÇÃO DE BOAS PRÁTICAS DE GESTÃO A EXECUÇÃO COMO DETERMINANTE PARA A DISSEMINAÇÃO DE BOAS PRÁTICAS DE GESTÃO Então, agora que conhecemos os entraves para o desenvolvimento de boas práticas de execução, vamos orientá-los no desenvolvimento de ações voltadas à boa execução dos recursos do PDDE e das Ações Integradas! É importante lembrar que todas as orientações para que as entidades participantes possam fazer uma boa gestão dos recursos do PDDE e das Ações Integradas, encontram-se na Resolução 15, de 16 de setembro de 2021 e, no caso das Ações Integradas, contas Estrutura e Qualidade, em resoluções próprias. Para acessar esse conjunto de documentos, basta acessar o site do FNDE: https://www.gov.br/fnde/pt-br 11 1. Vamos começar pelo Plano Anual de Atividades! O Plano Anual de Atividades consiste num documento elaborado pela UEx, em conjunto com a comunidade escolar, com o objetivo de definir as prioridades e ações nas áreas administrativa, financeira, pedagógica e social, considerando os objetivos do PDDE e das Ações Integradas, quais sejam, melhoria da infraestrutura física e pedagógica da escola, autogestão administrativa, financeira e pedagógica, melhoria da qualidade do ensino e elevação dos índices de desempenho -IdeGES. Nesse processo, a participação da comunidade escolar é de fundamental importância pois é ela quem deve decidir, a partir do diagnóstico, como e onde devem ser empregados os recursos desses Programas. É importante lembrar que, antes de tudo, é necessário verificar se a escola está apta a receber os recursos do PDDE e das Ações Integradas, ou seja, fez a atualização cadastral (quando necessário), e se não possui pendências com prestação de contas. Confira o passo a passo para elaboração de um bom Plano Anual de Atividades: • Refletir sobre os objetivos e finalidades do Programa e a responsabilidade da comunidade no alcance desses objetivos. • Motivar a comunidade para que participem das discussões e sistematização das ações a serem desenvolvidas ao longo do ano/período. • Realizar um bom diagnóstico, buscando-se identificar os problemas, as dificuldades, os avanços e elencando as prioridades. • Fazer uma previsão do montante a ser recebido, considerando a soma do valor fixo (estabelecimento de ensino) com o valor variável, correspondente ao número de alunos conforme censo escolar do ano anterior. • Fazer uma previsão das despesas a partir da receita e das prioridades elencadas, considerando as categorias de custeio e capital. • Acompanhar, monitorar e avaliar o cumprimento das ações definidas no âmbito no Plano Anual de Atividades, visando o seu (re)planejamento. ATENÇÃO! ATENÇÃO! Para o atendimento das prioridades definidas, é importante que as entidades participantes (UEx e EM) informem até o dia 31 de dezembro, no PDDEWeb, os percentuais que desejarão receber em custeio e/ou capital no ano subsequente. Caso isso não aconteça, os percentuais a serem recebidos por cada entidade, nas categorias custeio e capital são: • UEx: 80% custeio e 20% Capital; • EM: 50% custeio e 50% capital. É importante destacar que o Plano Anual de Atividades deverá estar em consonância com o Projeto Político Pedagógico, concorrendo para a melhoria da qualidade do ensino, resultando em boas práticas no âmbito da execução do PDDE e das Ações Integradas. A EXECUÇÃO COMO DETERMINANTE PARA A DISSEMINAÇÃO DE BOAS PRÁTICAS DE GESTÃO • Registar, em ata, todas as decisões tomadas acerca das prioridades, ações e bens a serem adquiridos e serviços previstos de contratação. • Tornar público as decisões tomadas acerca das prioridades a serem atendidas pelas entidades participantes: bens e materiais a serem adquiridos e serviços a serem contratados. 12 12 2. Agora, vamos conhecer o Plano de Aplicação de Recursos! a Divulgação do Plano de Aplicação de recursos e qualquer informação referente à aplicação dos recursos do PDDE e das Ações Integradas: murais da escola, sites, redes sociais, dentre outros. 2. Agora, vamos conhecer o Plano de Aplicação de Recursos! • o Plano Anual de Atividades, como ponto de partida para a elaboração do Plano de Aplicação Financeira; • o Plano Anual de Atividades, como ponto de partida para a elaboração do Plano de Aplicação Financeira; • a previ são do montante a ser recebido à conta dos Programas, considerando o somatório do valor a ser repassado no ano, com eventuais saldos reprogramados de anos anteriores e rendimentos de aplicações no mercado financeiro; • a previ são do montante a ser recebido à conta dos Programas, considerando o somatório do valor a ser repassado no ano, com eventuais saldos reprogramados de anos anteriores e rendimentos de aplicações no mercado financeiro; saldos remanescentes nas contas bancárias das Ações Integradas ao PDDE, observando as categorias econômicas de custeio e de capital; • os percentuais definidos/recebidos nas categorias custeio e capital; os percentuais definidos/recebidos nas categorias custeio e capital; 13 • a Previsão de reprogramação de saldos de recursos financeiros, no caso de problemas no repasse dos recursos, tais como atrasos, problemas bancários ou necessidade de acúmulo para aquisição de bem com valor superior ao repassado ou contratação de serviços; o detalhamento das despesas a serem efetivadas no âmbito de cada categoria econômica; a previsão de recolhimento do Imposto de Renda quando da contratação de serviços por pessoas físicas, a serem executados nas finalidades do PDDE e das Ações Integradas; o prazo para a execução dos recursos recebidos à conta do PDDE e das Ações Integradas, ou seja, 31 de dezembro do ano em que tenha sido efetivado o crédito nas contas das UEx, EEx e EM; o prazo para a execução dos recursos recebidos à conta do PDDE e das Ações Integradas, ou seja, 31 de dezembro do ano em que tenha sido efetivado o crédito nas contas das UEx, EEx e EM; os dispêndios com fretes, seguros, entre outros que não sejam assegurados gratuitamente pelo fornecedor ou prestador de serviço; os dispêndios com fretes, seguros, entre outros que não sejam assegurados gratuitamente pelo fornecedor ou prestador de serviço; a previsão de despesas cartorárias decorrentes de alterações nos estatutos das UEx, bem como às relativas a recomposições de seus quatro membros; a Divulgação do Plano de Aplicação de recursos e qualquer informação referente à aplicação dos recursos do PDDE e das Ações Integradas: murais da escola, sites, redes sociais, dentre outros. 2. Agora, vamos conhecer o Plano de Aplicação de Recursos! O Plano de Aplicação de Recursos consiste num documento a ser elaborado a partir do Plano Anual de Atividades, no qual deverá constar as ações a serem realizadas, com a indicação das respectivas estimativas de custos, ou seja, é o planejamento da utilização dos recursos financeiros repassados à conta dos Programas. Compete às UEx, por meio da Assembleia Geral, Conselho Deliberativo e Diretoria, a elaboração, aprovação e execução do Plano de Aplicação de Recursos e, ao Conselho Fiscal, acompanhar, supervisionar e fiscalizar a aplicação dos recursos da Unidade Executora Própria. Mas, o que contemplar na elaboração do Plano de Aplicação de Recursos? Vamos conhecer alguns elementos, desse importante documento, apontados pelos participantes da pesquisa, como um dos entraves para a execução dos recursos do PDDE e das Ações Integradas! • • É importante lembrar: • • • do registro em ata de todas as decisões tomadas acerca do planejamento da execução dos recursos dos PDDE e das Ações Integradas; • do registro em ata de todas as decisões tomadas acerca do planejamento da execução dos recursos dos PDDE e das Ações Integradas; da execução dos recursos dos PDDE e das Ações Integradas; • do arquivamento em pastas físicas e /ou virtuais (Google Drive, pendrive, HD externo), de toda a documentação decorrente desse processo, bem como o próprio Plano de Aplicação Financeira. Elaborado e aprovado o Plano de Aplicação de Recursos, é hora de conhecer os procedimentos técnico-operacionais para a execução dos recursos! 14 3. Procedimentos técnico-operacionais para a execução dos recursos O FNDE transfere, sem a necessidade de convênio, acordo, contrato, ajuste ou instrumento congênere, recursos financeiros, em caráter suplementar, às entidades participantes para o atendimento dos objetivos e finalidades do PDDE e das Ações Integradas. Os recursos são repassados em 02 (duas) parcelas anuais, devendo o pagamento da primeira ser efetivado até 30 de abril e o da segunda até 30 de setembro. Devem ser utilizados de acordo com a categoria econômica a qual são destinados, ou seja, custeio e capital: Devem ser utilizados de acordo com a categoria econômica a qual são destinados, ou seja, custeio e capital: a) Custeio - são despesas efetivadas com a aquisição de materiais de consumo e à contratação de serviços para funcionamento e manutenção das escolas. De acordo com a Portaria 448/2002 material de consumo é aquele que, em razão do seu uso corrente, perde a sua identidade física e/ou tem sua utilização limitada a 02 (dois) anos. a) Custeio - são despesas efetivadas com a aquisição de materiais de consumo e à contratação de serviços para funcionamento e manutenção das escolas. De acordo com a Portaria 448/2002 material de consumo é aquele que, em razão do seu uso corrente, perde a sua identidade física e/ou tem sua utilização limitada a 02 (dois) anos. - Material de consumo: materiais de expediente, limpeza, construção etc.; Contratação de serviços: manutenção hidráulica elétrica jardinagem etc - Material de consumo: materiais de expediente, limpeza, construção etc.; - Contratação de serviços: manutenção hidráulica, elétrica, jardinagem etc. - Material de consumo: materiais de expediente, limpeza, construção etc.; - Contratação de serviços: manutenção hidráulica, elétrica, jardinagem etc. Material de consumo: materiais de expediente, limpeza, construção etc.; - Contratação de serviços: manutenção hidráulica, elétrica, jardinagem etc. b) Capital: referem-se às despesas realizadas com a aquisição de equipamentos e material permanente para as entidades participantes. É classificado como permanente o material que, em razão do seu uso corrente, não perde a sua identidade física, e ou tem uma durabilidade superior a 02 (dois) anos. (BRASIL, 2002) - Material permanente: eletrodomésticos, computadores, mobiliário, etc. - Material permanente: eletrodomésticos, computadores, mobiliário, etc. Mas, como e onde empregar os recursos recebidos? A Resolução 15/2021 traz o detalhamento da execução dos recursos! Mas, como e onde empregar os recursos recebidos? A Resolução 15/2021 traz o detalhamento da execução dos recursos! Vamos conhecer o detalhamento da execução dos recursos! Os recursos do PDDE e das Ações Integradas têm como objetivo a cobertura de despesas de custeio, manutenção e pequenos investimentos que concorram para a garantia do funcionamento e melhoria da infraestrutura física e pedagógica dos estabelecimentos de ensino beneficiários, devendo ser empregados no(a): I – aquisição de material permanente; II – realização de pequenos reparos, adequações e serviços necessários à manutenção, conservação e melhoria da estrutura física da unidade escolar; II – realização de pequenos reparos, adequações e serviços necessários à manutenção, conservação e melhoria da estrutura física da unidade escolar; III – aquisição de material de consumo; IV – avaliação de aprendizagem; V – implementação de projeto pedagógico; e VI – desenvolvimento de atividades educacionais. ATENÇÃO: ATENÇÃO: Os bens permanentes, adquiridos ou produzidos com os recursos transferidos às custas do PDDE e das Ações Integradas, deverão ser tombados e incorporados ao patrimônio das EEx e destinados ao uso dos respectivos estabelecimentos de ensino beneficiados para seu uso, guarda e conservação. É importante destacar que os recursos liberados na categoria custeio não poderão ser utilizados na categoria de capital e vice-versa. Caso isso ocorra, a entidade deverá submeter uma justificativa à avaliação do órgão responsável pela análise de prestação de contas. Se a justificativa não for aceita, os recursos devem ser devolvidos por meio da Guia de Recolhimento da União – GRU. 15 É PROIBIDO! I – implementação de outras ações que estejam sendo objeto de financiamento por outros programas executados pelo FNDE, exceto aquelas executadas sob a égide das normas do PDDE e Ações Integradas; II – gastos com pessoal; III – pagamento, a qualquer título, IV – cobertura de despesas com tarifas bancárias; e V – dispêndios com tributos federais, distritais, estaduais e municipais quando não incidentes sobre os bens adquiridos ou produzidos ou sobre os serviços contratados para a consecução dos objetivos do PDDE e Ações Integradas. Tendo clareza das diretrizes operacionais, é hora de atendermos as prioridades definidas no Plano de Aplicação de Recursos, ou seja, aquisição de materiais e bens e contratação de serviços. 16 Vamos às etapas! ❖ Levantamento e seleção das necessidades prioritárias, pela comunidade escolar, considerando as orientações legais de cada programa. Nessa etapa, faz-se necessário consultar o Plano de Atividades Anuais e o Plano de Aplicação de Recursos e registo em ata ❖ Levantamento e seleção das necessidades prioritárias, pela comunidade escolar, considerando as orientações legais de cada programa. Nessa etapa, faz-se necessário consultar o Plano de Atividades Anuais e o Plano de Aplicação de Recursos e registo em ata ❖ Levantamento e seleção das necessidades prioritárias, pela comunidade escolar, considerando as orientações legais de cada programa. Nessa etapa, faz-se necessário consultar o Plano de Atividades Anuais e o Plano de Aplicação de Recursos e registo em ata ❖ Realização de pesquisa de preços, preferencialmente no mercado local,registro no formulário "Rol de materiais, Bens e Serviços Prioritários". Seleção dos 03 (três) melhores orçamentos, no mínimo, a serem indicadas no formulário "Rol de Consolidação de Pesquisa de Preços" para apuração dos menores preços e definição de pessoa física ou jurídica à prestação de serviços. O registro em Ata é necessário nessa etapa. ❖ Realização de pesquisa de preços, preferencialmente no mercado local,registro no formulário "Rol de materiais, Bens e Serviços Prioritários". Seleção dos 03 (três) melhores orçamentos, no mínimo, a serem indicadas no formulário "Rol de Consolidação de Pesquisa de Preços" para apuração dos menores preços e definição de pessoa física ou jurídica à prestação de serviços. O registro em Ata é necessário nessa etapa. ❖ Escolha da melhor proposta - escolha mais vantajosa para o erário público. ❖ Escolha da melhor proposta - escolha mais vantajosa para o erário público ❖ Aquisição e/ou contratação - consultar, antes, o cadastro da empresa no site da Receita Federal. Atenção: Atenção: documentos comprobatórios de despesas deverão serem emitidos em nome da EEx, UEx ou da EM e conter, pelo menos, as seguintes informações: É PROIBIDO! Efetuar a compra ou contratação de serviços. No caso de pessoa jurídica, exigir documento fiscal original (nota fiscal, cupom fiscal, fatura etc.) e, para pessoa física, recibo, devidamente assinado, com os seguintes dados: nome, CPF, RG, endereço e contato telefônico. ❖ Aquisição e/ou contratação - consultar, antes, o cadastro da empresa no site da Receita Federal. Efetuar a compra ou contratação de serviços. No caso de pessoa jurídica, exigir documento fiscal original (nota fiscal, cupom fiscal, fatura etc.) e, para pessoa física, recibo, devidamente assinado, com os seguintes dados: nome, CPF, RG, endereço e contato telefônico. ❖ Aquisição e/ou contratação - consultar, antes, o cadastro da empresa no site da Receita Federal. Efetuar a compra ou contratação de serviços. No caso de pessoa jurídica, exigir documento fiscal original (nota fiscal, cupom fiscal, fatura etc.) e, para pessoa física, recibo, devidamente assinado, com os seguintes dados: nome, CPF, RG, endereço e contato telefônico. ❖ Guarda da documentação - 05 (cinco) anos, por meio físico ou digital, nas respectivas sedes, em boa ordem e organização, à disposição dos órgãos de controle externo e interno. ❖ Guarda da documentação - 05 (cinco) anos, por meio físico ou digital, nas respectivas sedes, em boa ordem e organização, à disposição dos órgãos de controle externo e interno. Vamos conferir! Vamos conferir! a) ROL DE MATERIAIS, BENS E/OU SERVIÇOS PRIORITÁRIOS PROGRAMA DINHEIRO DIRETO NA ESCOLA (PDDE - INSTRUÇÕES DE PREENCHIMENTO (Formulário a ser preenchido pela escola beneficiária para informar os materiais e bens e/ou serviços prioritários para atendimento de necessidades básicas). b) COMPROVANTE DE BENEFÍCIOS PROGRAMA DINHEIRO DIRETO NA ESCOLA (PDDE) INSTRUÇÕES DE PREENCHIMENTO (Formulário a ser preenchido pela Prefeitura Municipal (PM), Secretaria Distrital e Estadual de Educação (SEDUC) e Unidade Executora Própria (UEx) para registrar os materiais e bens fornecidos e/ou serviços prestados às escolas que mantém ou representam). b) COMPROVANTE DE BENEFÍCIOS PROGRAMA DINHEIRO DIRETO NA ESCOLA (PDDE) INSTRUÇÕES DE PREENCHIMENTO (Formulário a ser preenchido pela Prefeitura Municipal (PM), Secretaria Distrital e Estadual de Educação (SEDUC) e Unidade Executora Própria (UEx) para registrar os materiais e bens fornecidos e/ou serviços prestados às escolas que mantém ou representam). I –siglas FNDE e do PDDE e Ações Integradas; I –siglas FNDE e do PDDE e Ações Integradas; I –siglas FNDE e do PDDE e Ações Integradas; II –atesto do recebimento do material, do bem fornecido e/ou do serviço prestado à escola, com a data, a assinatura e a identificação do membro da UEx ou representante da EM que firmou o atesto; e III –registro de quitação da despesa efetivada, com a data, a assinatura e a identificação do representante legal do fornecedor do material ou bem ou do prestador do serviço. Atenção: É vedada a realização de pagamentos antes da efetiva entreg de materiais e bens e/ou prestação de serviços. Atenção: É vedada a realização de pagamentos antes da efetiva entrega de materiais e bens e/ou prestação de serviços. 17 Com a ampliação do comércio virtual, a aquisição de materiais e bens e/ou contratação de serviços, em empresas de comércio eletrônico pela internet, também é recomendada, desde que: Com a ampliação do comércio virtual, a aquisição de materiais e bens e/ou contratação de serviços, em empresas de comércio eletrônico pela internet, também é recomendada, desde que: Com a ampliação do comércio virtual, a aquisição de materiais e bens e/ou contratação de serviços, em empresas de comércio eletrônico pela internet, também é recomendada, desde que: • seja a única opção para se obter o bem/materiais e/ou contratação de serviços; • seja a única opção para se obter o bem/materiais e/ou contratação de serviços; • os preços praticados pelo comércio físico sejam superiores aos realizados no mercado on-line. • os preços praticados pelo comércio físico sejam superiores aos realizados no mercado on-line. As cotações de preços para aquisição de materiais e bens e/ou contratação de serviços on-line obedecem aos mesmos padrões para lojas físicas, no entanto, é necessária a demonstração de prints de tela, com os seguintes dados: a) especificidade do produto; b) valor do frete e; b) valor do frete e; c) tudo que possa influenciar a comparação com os outros locais nos quais serão realizadas cotação de preço. Em casos de compras on-line, é necessário verificar se há segurança na compra, para evitar a perda de recursos ou compras de produtos com qualidade diferente da adquirida. No site do FNDE (https://www.gov.br/fnde/pt-br/acesso-a-informacao/acoes-e- programas/programas/pdde/manuais-e-orientacoes-pdde) e nos anexos da Resolução 15/2021, é possível encontrar um conjunto de Formulários necessários à etapa da execução dos recursos do PDDE e das Ações Integradas, com as respectivas orientações para o correto preenchimento. c) CONSOLIDAÇÃO DE PESQUISAS DE PREÇOS; PROGRAMA DINHEIRO DIRETO NA ESCOLA (PDDE); INSTRUÇÕES DE PREENCHIMENTO. A elaboração do Plano Anual de Atividades e o Plano de Aplicação de Recursos pela comunidade escolar, a partir das demandas e prioridades identificadas, contribui com a boa execução financeira dos recursos do PDDE e das Ações Integradas. 18 Infelizmente, quando isso não acontece, as entidades correm o risco de reprogramar os recursos não utilizados e, ao se tratar da má utilização, devolvê-los ao FNDE. Nesse caso, os(as) gestores(as) à frente da gestão dos programas, serão responsabilizados. Infelizmente, quando isso não acontece, as entidades correm o risco de reprogramar os recursos não utilizados e, ao se tratar da má utilização, devolvê-los ao FNDE. Nesse caso, os(as) gestores(as) à frente da gestão dos programas, serão responsabilizados. Infelizmente, quando isso não acontece, as entidades correm o risco de reprogramar os recursos não utilizados e, ao se tratar da má utilização, devolvê-los ao FNDE. Nesse caso, os(as) gestores(as) à frente da gestão dos programas, serão responsabilizados. ● REPROGRAMAR QUANDO: Os recursos recebidos não forem utilizados até o dia 31 de dezembro. É necessário, para tanto, obedecer as classificações de custeio e capital nas quais foram repassados, para aplicação no exercício seguinte, com estrita observância de seu emprego nos objetivos da ação programática; ATENÇÃO: i l id d id ATENÇÃO: Qualquer pessoa, física ou jurídica poderá apresentar denúncia de irregularidades identificadas na aplicação dos recursos do PDDE e Ações Integradas ao FNDE, ao TCU, ao Sistema de Controle Interno do Poder Executivo Federal e ao Ministério Público, sendo assegurado o direito de sigilo dos dados pessoais. Vimos a importância de um bom planejamento para a correta execução dos recursos do PDDE e das Ações Integradas. É um processo fácil, mas que demanda da comunidade escolar conhecimento sobre os aspectos conceituais e operacionais desses programas, participação, engajamento da comunidade nas atividades a serem desenvolvidas, e tempo para se dedicar às atividades, uma vez que as atividades não são remuneradas, dentre outros. ● DEVOLVER AO FNDE QUANDO HOUVER: a) constatação de irregularidades na aplicação dos recursos do PDDE e das Ações Integradas. a) constatação de irregularidades na aplicação dos recursos do PDDE e das Ações Integradas. g b) depósitos indevidos feitos pelo FNDE; b) depósitos indevidos feitos pelo FNDE; ) p p c) paralisação das atividades ou extinção de EEx, UEx e EM; e, d) determinação do Poder Judiciário ou requisição do Ministério Público. A CAPACITAÇÃO TÉCNICA: UM DOS FATORES DETERMINANTES PARA A GESTÃO DO PDDE Considerando que as maiores dificuldades enfrentadas pelos que estão à frente desses programas se relacionam ao baixo ou nenhum nível de conhecimento sobre a gestão, faz-se necessária a participação dos representantes de entidades participantes em cursos de capacitação, como ponto de partida para a gestão do PDDE e das Ações Integradas. Vamos conhecer os impactos da capacitação na boa gestão desses programas! 19 Conforme Silva e Guerra (2022), existem 05 (cinco) fatores determinantes para o sucesso da gestão desses programas: a) cursos de capacitação; b) autonomia pedagógica, administrativa e financeira; c) controle social; d) gestão de recursos; e e) gestão participativa. A gestão do PDDE está intimamente vinculada ao nível de conhecimento sobre os processos de gestão da UEx. Nesse sentido, destacamos a necessidade e a importância da participação dos agentes envolvidos com a gestão do PDDE e das Ações Integradas, em cursos de capacitação que contribuam com o aumento do conhecimento sobre Programas, nos aspectos conceituais e operacionais. Pesquisa realizada com os 143 participantes que concluíram o curso do Curso a distância sem tutoria, ofertado pelo CECAMPE-NE (2022), dentre eles: diretores (40, %), vice-diretores (8,4%), coordenadores pedagógicos (4,9%), professores (11,2%), técnicos educacionais (5,6%), secretários escolares (2,8%), dentre outros segmentos, numa análise comparativa entre o nível de conhecimento no início do curso e no final, constatou aumento nos níveis de conhecimento que variaram entre 24,77% a 35,19%, sendo: ❖ 35,19% envolvendo o processo de adesão/habilitação; ❖ 31,03% o repasse de recursos; ❖ 27,96% o processo de planejamento da aplicação de recursos; ❖ 25,67% a execução dos recursos; ❖ 24,77% o processo de prestação de contas; e ❖ 31,23% o acompanhamento e o controle social dos recursos do PDDE. ❖ 35,19% envolvendo o processo de adesão/habilitação; Esses resultados demonstram que houve um gap (lacuna) positivo de incremento no nível de conhecimento sobre o processo de gestão do PDDE àqueles(as) que participaram do curso de capacitação a distância sem tutoria. (CECAMPE, 2022) Na avaliação de 98,7% dos respondentes do questionário, o curso foi avaliado como bom ou ótimo, além do destaque para as contribuições da capacitação à melhoria dos níveis de conhecimentos para os profissionais que dão suporte às escolas, prefeituras, Secretarias de Estado de Educação, dentre outras entidades, no tocante à gestão do PDDE e outros programas, a exemplo das Ações Integradas. Dessa forma, a capacitação permanente sobre o PDDE e as Ações Integradas deve ser uma política da unidade escolar para assegurar o bom e adequado uso dos recursos financeiros, com participação social, e voltado para a melhoria da educação pública. O CECAMPE NORDESTE oferece capacitação presencial e a distância. 20 o e e e apa ação p ese a e a d s â a Participe! Referências BRASIL. FNDE. Apresentação o que é o PDDE: Programa Dinheiro direto na Escola. 2014. Disponível em: https://www.fnde.gov.br/index.php/programas/pdde/sobre-o-plano-ou-programa/sobre- o-pdde. Acesso em: 10 mai. 2022. BRASIL. FNDE. Conselho Deliberativo. Resolução nº. 12, de 10 de maio de 1995. Brasília: FNDE, 1995. BRASIL. FNDE. Guia de Atuação do Centro Colaborador de Apoio ao Monitoramento e à Gestão de Programas Educacionais CECAMPE. Brasília/DF: 2020. BRASIL. FNDE. Resolução nº 15, de 16 de setembro de 2021. Dispõe sobre as orientações para o apoio técnico e financeiro, fiscalização e monitoramento na execução do Programa Dinheiro Direto na Escola - PDDE, em cumprimento ao disposto na Lei nº 11.947, de 16 de junho de 2009. Diário Oficial da União, Brasília, DF, 17 set. 2021. BRASIL. FNDE. Resolução nº 10, de 07 de outubro de 2020. Dispõe sobre a prorrogação dos prazos para o envio das prestações de contas de programas e ações educacionais executados ao FNDE, em virtude da situação de calamidade pública para enfrentamento da pandemia do novo coronavírus–Covid-19, e dá outras providências. Diário Oficial da União, Brasília, DF, 07 out. 2020. BRASIL. Lei nº 11.947, de 16 de junho de 2009. Dispõe sobre o atendimento da alimentação escolar e do Programa Dinheiro Direto na Escola aos alunos da educação básica. Diário Oficial da União, Brasília, DF, 16 jun. 2009. BRASIL. Manual de Orientação para constituição de Unidade Executora Própria. Ministério da Educação, Brasília, 2014. BRASIL. Medida Provisória nº. 1.784, de 14 de dezembro de 1998. Dispõe sobre o repasse de recursos financeiros do Programa Nacional de Alimentação Escolar, institui o PDDE, e dá outras providências. Diário Oficial da União, Brasília, DF, 14 dez.1998. BRASIL. Medida Provisória nº. 2.100-32, de 24 de maio de 2001. Dispõe sobre o repasse de recursos financeiros do PNAE, institui o PDDE, altera a Lei nº 9.533, de 10 de dezembro de 1997, que dispõe sobre programa de garantia de renda mínima, institui programas de apoio da União às ações dos Estados e Municípios, voltadas para o atendimento educacional, e dá outras providências. Diário Oficial da União, Brasília, DF, 25 maio 2001. BRASIL. Portaria 448, de 13 de setembro de 2002. Divulga o detalhamento das naturezas de despesas 339030, 339036, 339039 e 449052. Disponível em: https://sisweb.tesouro.gov.br/apex/f?p=2501:9::::9:P9_ID_PUBLICACAO:8754 . Acesso em: 11 dez. 2022. BRASIL. Ministério da Educação. Curso PDDE - Fundo Nacional de Desenvolvimento da Educação. Secretaria de Educação a Distância. 5 ed. Brasília: MEC, FNDE, 2013. Disponível em: http://cursos.fnde.gov.br/mdl07/pdf/ParteinicialdoCurso.pdf. Conclusão Neste manual, conhecemos algumas das dificuldades mais frequentes enfrentadas pelos gestores(as) escolares que participaram dos Webinários e do Curso a distância sem tutoria, ofertados pelo CECAMPE-NE e que têm sido responsáveis pela baixa execução dos recursos do PDDE e das Ações Integradas na região nordeste. Elaboração e execução do Plano Anual de Atividades e do Plano de Aplicação de Recursos, além de dificuldades na parte operacional, tais como: levantamento das prioridades da escola, pesquisa de preços, aquisição de bens e materiais e contratação de serviços, foram relatados pelos participantes como alguns dos entraves que prejudicam a gestão e a execução dos recursos recebidos pelas entidades participantes do PDDE e das Ações Integradas. A partir dessas dificuldades, buscamos sistematizar e apresentar neste Manual, orientações importantes aos gestores(as) de escolas públicas da região nordeste para que desenvolvam ações que contribuam com a boa gestão e execução dos recursos, evitando, dessa forma, a reprogramação e/ou devolução de recursos, fatores que comprometem o alcance dos objetivos dos referidos programas e com a queda do IdeGES. Agora, ficou fácil fazer o planejamento para a boa execução dos recursos. Vamos colocar a sua escola na lista das que melhor executam os recursos do PDDE e das Ações Integradas de modo a alcançar os objetivos, ou seja, a melhoria da infraestrutura física e pedagógica das escolas, a autogestão pedagógica, administrativa e financeira, com consequente elevação dos IdeGES e da melhoria da qualidade da educação pública. 21 Contem conosco! Contem conosco! CECAMPE-NE/UFPB/FNDE Referências Acesso em: 11 dez. 2022. CECAMPE NORDESTE. Painel IdeGES. Disponível em: https://www.cecampe.ufpb.br/paineisdeinformacoes. Acesso em: 28 out. 2022. 22 FNDE – FUNDO NACIONAL DE DESENVOLVIMENTO DA EDUCAÇÃO. O fortalecimento do Programa Dinheiro Direto na Escola na Região Nordeste como estratégia para a gestão democrática e para a qualidade da educação (Projeto Técnico). Brasília/DF, 2020. FNDE – FUNDO NACIONAL DE DESENVOLVIMENTO DA EDUCAÇÃO. O fortalecimento do Programa Dinheiro Direto na Escola na Região Nordeste como estratégia para a gestão democrática e para a qualidade da educação (Projeto Técnico). Brasília/DF, 2020. 23 PARCEIROS 23
https://openalex.org/W2120481021	https://europepmc.org/articles/pmc3843223?pdf=render	English	null	A penny for your thoughts: dimensions of self-generated thought content and relationships with individual differences in emotional wellbeing	Frontiers in psychology	2,013	cc-by	11,659	ORIGINAL RESEARCH ARTICLE published: 29 November 2013 doi: 10.3389/fpsyg.2013.00900 ORIGINAL RESEARCH ARTICLE bli h d 29 N b 2013 published: 29 November 2013 doi: 10.3389/fpsyg.2013.00900 Keywords: mind-wandering, autobiographical, rumination, depression, mindfulness Jessica R. Andrews-Hanna 1, Roselinde H. Kaiser 2, Amy E. J.Turner 1, Andrew E. Reineberg 2, Detre Godinez 1, Sona Dimidjian 2 and MarieT. Banich 1,2 1 Institute of Cognitive Science, University of Colorado Boulder, Boulder, CO, USA 2 Department of Psychology and Neuroscience, University of Colorado Boulder, Boulder, CO, USA Jessica R. Andrews-Hanna 1, Roselinde H. Kaiser 2, Amy E. J.Turner 1, Andrew E. Reineberg 2, Detre Godinez 1, Sona Dimidjian 2 and MarieT. Banich 1,2 1 Institute of Cognitive Science, University of Colorado Boulder, Boulder, CO, USA 2 Department of Psychology and Neuroscience, University of Colorado Boulder, Boulder, CO, USA Jessica R. Andrews-Hanna 1, Roselinde H. Kaiser 2, Amy E. J.Turner 1, Andrew E. Reineberg 2, Detre Godinez 1, Sona Dimidjian 2 and MarieT. Banich 1,2 1 Institute of Cognitive Science, University of Colorado Boulder, Boulder, CO, USA 2 Department of Psychology and Neuroscience, University of Colorado Boulder, Boulder, CO, USA A core aspect of human cognition involves overcoming the constraints of the present environment by mentally simulating another time, place, or perspective. Although these self-generated processes confer many beneﬁts, they can come at an important cost, and this cost is greater for some individuals than for others. Here we explore the possibility that the costs and beneﬁts of self-generated thought depend, in part, upon its phenomenological content. To test these hypotheses, we ﬁrst developed a novel thought sampling paradigm in which a large sample of young adults recalled several recurring thoughts and rated each thought on multiple content variables (i.e., valence, speciﬁcity, self-relevance, etc.). Next, we examined multi-level relationships among these content variables and used a hierarchical clustering approach to partition self- generated thought into distinct dimensions. Finally, we investigated whether these content dimensions predicted individual differences in the costs and beneﬁts of the experience, assessed with questionnaires measuring emotional health and wellbeing. Individuals who characterized their thoughts as more negative and more personally signiﬁcant scored higher on constructs associated with Depression and Trait Negative Affect, whereas those who characterized their thoughts as less speciﬁc scored higher on constructs linked to Rumination. In contrast, individuals who characterized their thoughts as more positive, less personally signiﬁcant, and more speciﬁc scored higher on constructs linked to improved wellbeing (Mindfulness). Collectively, these ﬁndings suggest that the content of people’s inner thoughts can (1) be productively examined, (2) be distilled into several major dimensions, and (3) account for a large portion of variability in their functional outcomes. Edited by: Edited by: Arnaud Delorme, Centre de Recherche Cerveau et Cognition, France November 2013 \| Volume 4 \| Article 900 \| 1 Reviewed by: Reviewed by: Arnaud Delorme, Centre de Recherche Cerveau et Cognition, France Julia W. Y. Kam, University of British Columbia, Canada Jonathan Jackson, Brandeis University, USA Correspondence: Jessica R. Andrews-Hanna, Institute of Cognitive Science, University of Colorado Boulder, 594 UCB, Boulder, CO 80309 USA e-mail: jandrewshanna@gmail.com Reviewed by: Arnaud Delorme, Centre de Recherche Cerveau et Cognition, France Julia W. Y. Kam, University of British Columbia, Canada Jonathan Jackson, Brandeis University, USA A penny for your thoughts: dimensions of self-generated thought content and relationships with individual differences in emotional wellbeing A penny for your thoughts: dimensions of self-generated thought content and relationships with individual differences in emotional wellbeing Jessica R. Andrews-Hanna 1, Roselinde H. Kaiser 2, Amy E. J.Turner 1, Andrew E. Reineberg 2, Detre Godinez 1, Sona Dimidjian 2 and MarieT. Banich 1,2 1 Institute of Cognitive Science, University of Colorado Boulder, Boulder, CO, USA 2 Department of Psychology and Neuroscience, University of Colorado Boulder, Boulder, CO, USA Correspondence: p Jessica R. Andrews-Hanna, Institute of Cognitive Science, University of Colorado Boulder, 594 UCB, Boulder, CO 80309 USA e-mail: jandrewshanna@gmail.com INTRODUCTION INTRODUCTION Moberly and Watkins, 2008; Watkins, 2008; Killingsworth and Gilbert, 2010; McVay and Kane, 2010; Mooneyham and Schooler, 2013; Smallwood and Andrews-Hanna, 2013). In fact, a variety of mental health disorders are deﬁned by impairments arising from self-generated thought (Harvey et al., 2004), and such ﬁndings suggest that the adaptive and maladaptive manifestation of the experience varies widely across individuals. Understanding the factors that give rise to this variability could help individuals har- ness the beneﬁcial aspects of self-generated thought and, in doing so, improve their psychological wellbeing. “You are today where your thoughts have brought you; you will be tomorrow where your thoughts take you.” – James Allen (1864–1912), author. A wealth of recent cognitive and neuroscientiﬁc research sug- gests that humans spend a remarkable amount of time engaged in self-generated thought (Singer, 1966; Klinger, 1971; Klinger and Cox, 1987; Kane et al., 2007; Killingsworth and Gilbert, 2010; Song and Wang, 2012), an internal mode of cognition characterized by its capacity to overcome the constraints of the external envi- ronment (Smallwood, 2013). Supporting its frequent occurrence, self-generated thought is associated with a wide variety of bene- ﬁts, enabling us to confront future challenges, solve problems, and navigate our social world (Smallwood and Schooler, 2006; Baars, 2010; Christoff et al.,2011; McMillan et al.,2013; Mooneyham and Schooler, 2013; Smallwood and Andrews-Hanna, 2013). At the same time, numerous studies suggest the experience can be asso- ciated with signiﬁcant costs, including disrupted task performance and poor psychological wellbeing (Smallwood and Schooler,2006; Here we tackle this research objective by testing recent hypothe- ses that the content of self-generated thought represents an important factor underlying its costs and beneﬁts (Watkins, 2008; Mar et al., 2012; Smallwood and Andrews-Hanna, 2013). The con- tent regulation hypothesis proposes that self-generated thoughts are particularly beneﬁcial for individuals who are able to regu- late the content of the experience to positive or productive topics (Smallwood and Andrews-Hanna, 2013). In line with this hypoth- esis, we predicted that individuals who primarily engaged in November 2013 \| Volume 4 \| Article 900 \| 1 www.frontiersin.org Self-generated thought and emotional wellbeing Andrews-Hanna et al. positive or productive thoughts would exhibit improved emo- tional health and wellbeing, whereas negative or unproductive thought content would be indicative of poor emotional health and wellbeing. Whereas existing studies primarily focus on the frequency of self-generated thought or a single aspect of thought content (i.e. MATERIALS AND PROCEDURE Thought sampling paradigm i l i ll i p p In a single session, all participants completed an autobiographi- cal thought sampling paradigm, developed for the present study to quantitatively assess the phenomenological content of a broad range of self-generated thoughts and enable computation of reli- able within-subject correlations. Participants recalled 36 different thoughts that had recently been on their mind in daily life, gen- erated a three-word description for each thought, and rated each thought on a variety of different content variables, including self- relevance, frequency of occurrence, importance/value, centrality to one’s sense of self-identity, valence, emotional intensity, goal orientation, social orientation, speciﬁcity, imagery, perspective taking, duration of the topic or event surrounding the thought, temporal orientation, and certainty that the event for which the thought pertains occurred or will occur (see Table 1 for precise questions). Both the nature of the instructions and the descrip- tions provided by participants (i.e., “My College GPA,” “Climb Pike’sPeak,”“MissMyParents”)suggestthatparticipantsprimarily generated self-generated thoughts that were independent of exter- nal stimuli (i.e., stimulus-independent) and similar in content to that observed during episodes of mind-wandering (Stawarczyk et al., 2011). To explore which aspects of thought content predict adap- tive functional outcomes, we examined individual differences in mindfulness – a purposeful, present-minded, and non-judgmental mode of awareness linked to improved health and emotional well- being (Brown and Ryan, 2003; Bishop, 2004). When measured as a trait, mindfulness seems to buffer against symptoms of depres- sion and rumination (Brown and Ryan, 2003), and elements of mindfulness have been incorporated into effective treatment therapiesforavarietyof mentalhealthdisorders,includingdepres- sion and anxiety (Kabat-Zinn, 1990; Segal et al., 2002; Baer, 2003). Mindful individuals report lower frequencies of negative automatic thoughts and a heightened ability to let go of such thoughts (Frewen et al., 2008; see also Evans and Segerstrom, INTRODUCTION valence) – typically assessed in the context of cued autobiographical memory retrieval paradigms, mood induction paradigms, or ongoing experimental tasks – we examined several aspects of self-generated thoughts that occupy participants’minds throughout their daily lives. By developing a novel thought sam- pling paradigm in which participants generate several recurring thoughts and rate each thought on numerous content-speciﬁc variables, we were able to (1) quantify normative estimates of thought content in a large group of adults, (2) examine multi-level relationships among content variables, and (3) explore which con- tent variables predicted unique variance in the costs and beneﬁts of the experience. 2011), and mindfulness-based interventions have been shown to alter the nature of self-generated thoughts in similar ways (Frewen et al., 2008). When applied to chronic depression, these thera- pies have also shown promise in increasing the speciﬁcity and perceived likelihood of achieving individuals’ life goals (Crane et al., 2012). Based on these ﬁndings, we predicted that dispo- sitional mindfulness would negatively correlate with depression and rumination, and would be associated with thought con- tent characterized as more positive, less self-focused, and more speciﬁc. MATERIAL AND METHODS PARTICIPANTS Seventy-six young adults (mean age: 21.6 years; 18–35 years; 38 male) participated in the study for paid compensation. To facilitate interdisciplinary analysis for future studies, participants were recruited from a larger database of individuals from the University of Colorado Boulder and the greater Boulder/Denver community who had previously completed a battery of self-report questionnaires, behavioral tasks, and functional neuroimaging measures in our laboratory. Though participants within this database were previously screened for an absence of a cur- rent or previous self-reported psychiatric illness, nearly 30% of participants exhibited scores on the Beck Depression Inventory indicative of mild to moderate depression (see below), con- sistent with prior ﬁndings that depressive symptoms often go undiagnosed and untreated (Eisenberg et al., 2007). All pro- cedures were carried out in accordance with the University of Colorado Boulder’s Institutional Review Board. p We focused on individual differences in depressive symptoms as an important marker of poor emotional wellbeing because (1) major depressive disorder (MDD) is the leading cause of disability worldwide (World Health Organization, 2008), (2) depressive symptoms predict signiﬁcant distress and impair- ment at subclinical levels of severity (Judd et al., 1998), and (3) individuals with a current or past history of depression often exhibit intrusive self-generated thoughts focused on negative self-schema (Beck, 1967; Watkins, 2008). Depressed individu- als also “overgeneralize” based on single instances (Beck, 1967) and exhibit overgeneral autobiographical memories that tend to be associated with a broad range of unconstructive conse- quences (Williams et al., 2007; Watkins, 2008). However, it is neither clear whether overgeneral memory applies to the types of self-generated thoughts that occur in daily life, nor whether these characteristics are driven by the affective symptoms of depression or the repetitive styles of thinking associated with the disorder. To disentangle the effects of negative affect and repetitive thought on the content of self-generated thought, we also examined individual differences in rumination, a repetitive style of thinking focused on “one’s symptoms of distress and the circumstances surrounding these symptoms” (Nolen-Hoeksema et al., 1997, 2008; Nolen-Hoeksema, 2000). We hypothesized that individuals with higher levels of depressive symptoms would exhibit thought content characterized as more negative, more self-focused and – to the degree that they also ruminate – less speciﬁc. Frontiers in Psychology \| Perception Science Questionnaires To assess individual differences in constructs relevant to men- tal health, a subset of participants (N = 70) completed November 2013 \| Volume 4 \| Article 900 \| 2 Frontiers in Psychology \| Perception Science \| 2 Self-generated thought and emotional wellbeing Andrews-Hanna et al. Table 1 \| Content variables. Content rating Question Scale Self-relevance I consider this thought to be highly self-relevant. 0 to 10, from strongly disagree to strongly agree Frequency Lately, it seems that this thought has been on my mind a great deal. 0 to 10, from strongly disagree to strongly agree Importance/value The topic of this thought is of great value or importance to me. 0 to 10, from strongly disagree to strongly agree Centrality This thought contributes to my sense of self-identity. 0 to 10, from strongly disagree to strongly agree Valence My own emotions pertaining to this thought are . . . 0 to 10, from very negative to very positive Intensity The intensity of my emotions pertaining to this thought are . . . 0 to 10, from not intense at all to very intense Goal-orientation This thought involves/involved reaching a particular goal of mine. 0 to 10, from strongly disagree to strongly agree Social-orientation I consider this thought to involve/concern other people (i.e., an upcoming date, vacation with friends). 0 to 10, from strongly disagree to strongly agree Detail/speciﬁcity I would characterize this thought as being tied to a highly detailed and speciﬁc event. 0 to 10, from strongly disagree to strongly agree Imagery When I experience this thought, my mental imagery is . . . 1 = Perfectly clear and as vivid as normal vision, 2 = Clea reasonably vivid, 3 = Moderately clear and vivid, 4 = Vagu dim, 5 = No imagery at all, you only “know” that you wer thinking of something Perspective When I experience this thought, I develop a mental image in which . . . 1 = I “see” things from my own perspective (like I would real world), 2 = I “see” myself from another perspective, 3 = neither Duration of topic This thought concerns an event or a topic that lasted or will likely last . . . 1 = many years, 2 = many months, 3 = a month, 4 = mu days, 5 = an entire day, 6 = minutes to hours Temporal orientation This event took/will take place. . Questionnaires I am certain that the event has taken or will take place for me 0 to 10, from strongly disagree to strongly agree Certainty The question assessing temporal orientation and certainty were only answered for thoughts that pertained to a speciﬁc event. Questionnaires assessing depressive symptoms and trait neg- ative affect included the Beck Depression Inventory, 2nd Edi- tion (BDI-2; Beck et al., 1996), the “Low Positive Affect” and “Loss of Interest” subscales of the Mood and Anxiety Symptom Questionnaire (MASQ; Watson and Clark, 1991; Watson et al., 1995a,b), and the “Negative” subscale of the Trait version of the Positive and Negative Affect Schedule (PANAS; Watson et al., 1988). All questionnaires loaded signiﬁcantly (p < 0.05) on the Depression/Negative Affect latent construct (Table 2) and provided a good ﬁt to the data (χ2 = 3.45, df = 2, CFI = 0.96). Note that there was considerable variability in scores across participants. Using previously recommended cutoff scores for the Beck Depres- sion Inventory (Beck et al., 1996), 71% of participants qualiﬁed as minimally depressed (scores 0–11), 22% as mildly depressed (scores 12–18), and 7% as moderately depressed (scores 19–29). a variety of self-report questionnaires related to emotional health and wellbeing. Whereas the thought sampling paradigm requires that participants retrieve speciﬁc examples of self- generated thoughts and rate those thoughts on a variety of different content variables, the mental health questionnaires assess more general aspects of a participant’s wellbeing includ- ing his/her behaviors, feelings, actions, and general styles of thinking. We used conﬁrmatory factor analyses (using maximum likelihood estimation) to isolate three latent con- structs deﬁned from a large body of clinical literature: Depres- sion/Negative Affect, trait Rumination, and trait Mindfulness (Table 2). The overall ﬁt of these models was estimated using a non-signiﬁcant χ2 and Bentler’s comparative ﬁt index (CFI) greater than 0.95 suggestive of an adequate ﬁt of the data. a variety of self-report questionnaires related to emotional health and wellbeing. Whereas the thought sampling paradigm requires that participants retrieve speciﬁc examples of self- generated thoughts and rate those thoughts on a variety of different content variables, the mental health questionnaires assess more general aspects of a participant’s wellbeing includ- ing his/her behaviors, feelings, actions, and general styles of thinking. Questionnaires . 1 = more than 3 years ago, 2 = within the past 3 years, 3 = within the past 365 days (a year’s time), 4 = within th 30 days (a month’s time), 5 = within the past 7 days (a we time), 6 = yesterday, 7 = earlier today, 8 = later today, 9 = tomorrow, 10 = within the next 7 days (a week’s time 11 = within the next 30 days (a month’s time) 12 = within Table 1 \| Content variables. Scale 0 to 10, from strongly disagree to strongly agree 0 to 10, from strongly disagree to strongly agree 0 to 10, from strongly disagree to strongly agree 0 to 10, from strongly disagree to strongly agree 0 to 10, from very negative to very positive 0 to 10, from not intense at all to very intense 0 to 10, from strongly disagree to strongly agree 0 to 10, from strongly disagree to strongly agree 1 = Perfectly clear and as vivid as normal vision, 2 = Clear and reasonably vivid, 3 = Moderately clear and vivid, 4 = Vague and dim, 5 = No imagery at all, you only “know” that you were thinking of something 1 = I “see” things from my own perspective (like I would in the real world), 2 = I “see” myself from another perspective, 3 = neither 1 = many years, 2 = many months, 3 = a month, 4 = multiple days, 5 = an entire day, 6 = minutes to hours 1 = more than 3 years ago, 2 = within the past 3 years, 3 = within the past 365 days (a year’s time), 4 = within the past 30 days (a month’s time), 5 = within the past 7 days (a week’s time), 6 = yesterday, 7 = earlier today, 8 = later today, 9 = tomorrow, 10 = within the next 7 days (a week’s time), 11 = within the next 30 days (a month’s time), 12 = within the next 365 days (a year’s time), 13 = within the next 3 years, 14 = more than 3 years from now. Questionnaires We used conﬁrmatory factor analyses (using maximum likelihood estimation) to isolate three latent con- structs deﬁned from a large body of clinical literature: Depres- sion/Negative Affect, trait Rumination, and trait Mindfulness (Table 2). The overall ﬁt of these models was estimated using a non-signiﬁcant χ2 and Bentler’s comparative ﬁt index (CFI) greater than 0.95 suggestive of an adequate ﬁt of the data. November 2013 \| Volume 4 \| Article 900 \| 3 www.frontiersin.org \| 3 Self-generated thought and emotional wellbeing Andrews-Hanna et al. Table 2 \| Constructs related to emotional health and wellbeing. Questionnaire Subscale Mean SD CFA latent construct Factor loading MASQ Loss of Interest 16.13 4.53 Depression/negative affect 0.34* MASQ Low Positive Affect 36.20 8.96 Depression/negative affect 0.37* BDI Total Score 8.85 6.08 Depression/negative affect 0.73* Trait PANAS Negative 17.92 5.66 Depression/negative affect 0.75* RRS Brooding 10.59 3.36 Rumination 0.80* RRS Depression-related 24.59 6.46 Rumination 0.97* RRS Reﬂection 10.50 3.68 Rumination 0.57* RRQ Rumination 40.69 8.18 Rumination 0.30* FFMQ Acting with awareness 24.62 5.51 Mindfulness 0.72* FFMQ Describing 26.90 5.99 Mindfulness 0.66* FFMQ Non-judging of experience 27.49 5.46 Mindfulness 0.69* FFMQ Non-reactivity to inner experience 22.28 3.98 Mindfulness 0.45* FFMQ Observing sensations, perceptions, thoughts, feelings 25.73 4.60 Mindfulness −0.15 CFA, conﬁrmatory factor analysis; MASQ, Mood and Anxiety Symptom Questionnaire; BDI, Beck Depression Inventory, 2nd Edition; PANAS, positive and negative affect schedule; RRS, Ruminative Responses Scale; RRQ, Rumination-Reﬂection Questionnaire; PSWQ, Penn State Worry Questionnaire; FFMQ, Five Facet Mindfulness Questionnaire; p < 0.05. Table 2 \| Constructs related to emotional health and wellbeing. Mood and Anxiety Symptom Questionnaire; BDI, Beck Depression Inventory, 2nd Edition; PANAS, positive and negative affect cale; RRQ, Rumination-Reﬂection Questionnaire; PSWQ, Penn State Worry Questionnaire; FFMQ, Five Facet Mindfulness CFA, conﬁrmatory factor analysis; MASQ, Mood and Anxiety Symptom Questionnaire; BDI, Beck Depression Inventory, 2nd Edition; PANAS, positive and negative affect schedule; RRS, Ruminative Responses Scale; RRQ, Rumination-Reﬂection Questionnaire; PSWQ, Penn State Worry Questionnaire; FFMQ, Five Facet Mindfulness Questionnaire; p < 0.05. As both the MASQ and the PSWQ have high test-retest reliabil- ity indicative of stable constructs (Stöber, 1998; Keogh and Reidy, 2000), we decided to include these questionnaires in our analyses. Questionnaires assessing trait rumination included “Brood- ing,” “Depression-Related,” and “Reﬂection-Related” subscales of the Ruminative Responses Scale (Roberts et al., 1998; Treynor et al., 2003), and the “Rumination” subscale of the Rumination-Reﬂection Questionnaire (RRQ; Trapnell and Camp- bell, 1999). Part 1: Normative estimates of thought content Part 1: Normative estimates of thought content The ﬁrst set of analyses sought to quantify normative estimates of thought content across the full group of 76 participants. To extract mean estimates of each content variable, we ﬁrst averaged partici- pants’ ratings across the 36 different thoughts for each participant separately. For each of the 12 content variables, we then averaged mean ratings across participants. Participants whose mean rating was more than 2.5 standard deviations above or below the group mean were included in ﬁgures for visual purposes only and were not included in calculations of the group mean. Questionnairesassessingdispositionalmindfulnessincludedall ﬁve subscales of the Five Facet Mindfulness Questionnaire (Baer et al., 2006). While the “Acting with Awareness,” “Describing,” “Non-judging of Experience,” and “Non-reactivity to Inner Expe- rience” subscales loaded highly onto the Mindfulness construct (p < 0.05), the “Observe” subscale loaded weakly onto the con- struct (p = 0.27; Table 2), consistent with prior observations (Baer et al., 2006). This model also provided an excellent ﬁt to the data (χ2 = 3.49, df = 5, CFI = 1). Part 2: Identifying major dimensions of thought content The second set of analyses sought to examine relationships among content variables within participants, and in doing so, identify the major dimensions of thought content. For each par- ticipant, we computed the relationship between each pair of content variables across his/her 36 different thoughts (i.e., speci- ﬁcity × self-relevancy,speciﬁcity × valence,etc.) using Spearman’s rho. Unlike Pearson’s R, Spearman’s rho does not assume the within-participant data are normally-distributed. Next, we aver- aged the 12 × 12 correlation matrices across participants to generate a group mean correlation matrix. Correlations were assessedforstatisticalsigniﬁcancebytransformingthemtoFisher’s z using the formula 0.5 × ln[(1 + r)/(1 −r)] and conduct- ing one-sample t-tests across the group to determine if each correlation was signiﬁcantly different from zero, using an alpha of p < 0.05. Given the large number of statistical tests, we also corrected for multiple comparisons using the Bonferroni Participants’ factor scores for the Depression/Negative Affect, Rumination, and Mindfulness latent constructs were designated as the dependent (outcome) variable in subsequent multiple regression analyses to explore relationships with the content of individuals’ typical mind-wandering episodes. Questionnaires All questionnaires loaded signiﬁcantly (p < 0.05) on the Rumination latent construct (Table 2) and provided an excellent ﬁt (χ2 = 0.63, df = 2, CFI = 1). Frontiers in Psychology \| Perception Science Part 3: Relationships with constructs relevant to mental health Part 3: Relationships with constructs relevant to mental health Our third objective was to examine relationships between indi- vidual differences in thought content and dimensional constructs linked to poor and satisfactory emotional health and wellbeing. In the subset of 70 participants who completed all mental health questionnaires, we ran three multiple linear regression analyses in which the participants’ thought content scores extracted from the hierarchical clustering analysis were entered together as inde- pendent predictors to predict participants’ factor scores from each of the Depression/Negative Affect, Rumination, and Mindfulness factors, respectively. Participants also characterized their thoughts as exhibiting a moderate degree of speciﬁcity and imagery. A ﬁrst person (“self”) visual perspective was adopted for 64.3% of thoughts (SD = 24.8%), while participants adopted a different perspec- tive for 18.7% of thoughts (SD = 16.0%), and did not adopt a particular perspective for 17.0% of thoughts (SD = 18.8%). On average, participants indicated that their thoughts pertained to topics or events that lasted (or will last) between multiple days and one month in duration. Because rumination and depression are often related, and becausebothdepressionandruminationoftenco-occurwithanxi- ety, we also explored the speciﬁcity of each construct’s relationship to self-generated thought by conducting part correlations. This procedure involved (1) removing from the dependent variable (i.e., Depression/Negative Affect) variance shared with the ques- tionnaires for which we wished to control (i.e., Anxious Arousal, Anxious Apprehension, and Rumination), and (2) using these standardized residuals as new dependent variables in subsequent multiple regressions with thought content scores as predictor variables. www.frontiersin.org Part 1: Normative estimates of thought content To examine the speciﬁcity of the relationships between Depression, Rumination, and thought content, we included questionnaires assessing indi- vidual differences in anxious arousal (the “Anxious Arousal” subscale of the MASQ) and anxious apprehension (the Penn State Worry Questionnaire, PSWQ; Meyer et al., 1990). We also assessed state affect immediately prior to the thought sampling paradigm using the “Positive” and “Negative” subscales of the State version of the PANAS questionnaire. Note that while the majority of other questionnaires were acquired in the same session subsequent to the thought sampling paradigm, the MASQ and the PSWQ were acquired as part of the larger study an average of 205 days earlier. November 2013 \| Volume 4 \| Article 900 \| 4 Frontiers in Psychology \| Perception Science Self-generated thought and emotional wellbeing Andrews-Hanna et al. method and assessed signiﬁcance against a corrected alpha of p < 0.0008. subscales of the State PANAS questionnaire were entered as covariates of non-interest. p To identify major content dimensions characterizing partici- pants’ self-generated thoughts, we applied hierarchical clustering analysis on the 12 × 12 group mean correlation matrix by ﬁrst generating a distance matrix using Euclidean distance. Content dimension scores for each participant were extracted by converting his/her mean rating for each content variable to a z-score using the group mean, and averaging the standardized content variable rat- ings pertaining to the same cluster. These scores were then used to explore relationships with constructs relevant to emotional health and wellbeing (see below). Note that questions assessing per- spective taking and certainty were excluded from the hierarchical clustering analyses. Perspective taking contained no intrinsic order in its response options, while certainty often yielded bimodal dis- tributions at the within-subject level, a strongly positively skewed distribution at the between-subject level (skewness = −0.97), and was only answered for a subset of questions that pertained to a particular event. These non-normal distributions arose because thoughts pertaining to past events were almost always answered with 100% certainty. All other content variables were normally distributed at the between-subject level, with a skewness between –0.5 and +0.5. All tests were conducted using two-tailed criteria (p < 0.05) and analyses were conducted using a combination of R statistical software (R Development Core Team,2010), SPSS (v21, IBM),and MPlus (Muthén and Muthén, 1998–2011). PART 1: NORMATIVE ESTIMATES OF THOUGHT CONTENT Although the phenomenological characteristics of self-generated thoughts varied widely across participants, several common themes emerged (Figure 1). On average, participants char- acterized their thoughts as highly self-relevant, of moderate importance or value, and moderately central to their sense of self-identity, yet they also indicated their thoughts were moder- ately social in nature. Participants characterized their thoughts as moderately recurrent, somewhat goal-oriented, of moderate emotional intensity, and positive in valence. Females rated their thoughts as more negative than males; t(74) = 2.94, p < 0.01. A majority of participants’ thoughts pertained to a particular event (mean = 77.4%, SD = 20.0%), although approximately one-quarter did not (mean = 22.6%, SD = 20.0%). Of those thoughts that pertained to a particular event, 59.8% were oriented toward the future (SD = 25.4%) and 40.2% were oriented toward the past (SD = 25.4%). On average, the events for which the thoughts pertained were expected to take place in the near future (today or tomorrow). PART 2: IDENTIFYING MAJOR DIMENSIONS OF THOUGHT CONTENT PART 2: IDENTIFYING MAJOR DIMENSIONS OF THOUGHT CONTENT As shown in Figure 2, several signiﬁcant relationships emerged between content variables at the within-subject level, and these relationships are reﬂected in the pattern of content dimensions identiﬁed from a hierarchical clustering analysis (Figure 3). One dimension, termed Level of Construal, refers to the style of abstract versus concrete processing adopted during self-generated thought, and includes questions pertaining to speciﬁcity, imagery and duration of the topic or event surrounding the thought. Social ori- entation also clustered with this dimension. Ratings for questions encompassed in the Level of Construal dimension were signiﬁ- cantly correlated with one another, with the exception of social orientation (which was only related to speciﬁcity and imagery; Figure 2). Thoughts rated as more detailed and speciﬁc were more likely to pertain to topics characterized by a shorter duration (i.e., a speciﬁc episode as opposed to a general topic), to be experienced with more vividness/imagery, and to involve other people. For each multiple regression, we computed Cook’s Distance, a multivariate measure of the statistical inﬂuence of an individual on the regression function (Cook, 1977). Outliers with a Cook’s D of > 4/N were removed from each analysis (Bollen and Jackman, 1987). In our ﬁnal set of analyses, we isolated those aspects of thought content that exhibited signiﬁcant relationships with emotional health and examined whether these relationships persisted after additionally controlling for state affect. Partial correlations were conducted between trait factor scores and each signiﬁcant content dimension separately, while participants’“Positive”and“Negative” A second dimension, termed Personal Signiﬁcance, included the following content ratings: self-relevance, centrality, frequency of occurrence, emotional intensity, value/importance, and goal November 2013 \| Volume 4 \| Article 900 \| 5 5 Self-generated thought and emotional wellbeing Andrews-Hanna et al. FIGURE 1 \| Phenomenological characteristics of self-generated thought. Individual data points represent each participant’s mean across 36 self-generated thoughts. Open circles represent outliers > 2.5 SD above or below the group mean (vertical bar) which are not included in calculations. PART 2: IDENTIFYING MAJOR DIMENSIONS OF THOUGHT CONTENT The scale for temporal orientation corresponds to: 1 = more than 3 years ago, 2 = within the past 3 years, 3 = within the past 365 days (a year’s time), 4 = within the past 30 days (a month’s time), 5 = within the past 7 days (a week’s time), 6 = yesterday, 7 = earlier today, 8 = later today, 9 = tomorrow, 10 = within the next 7 days (a week’s time), 11 = within the next 30 days (a month’s time), 12 = wit the next 365 days (a year’s time), 13 = within the next 3 years, 14 = more than 3 years from now. days (a year’s time), 4 = within the past 30 days (a month’s time), 5 = within the past 7 days (a week’s time), 6 = yesterday, 7 = earlier today, 8 = later today, 9 = tomorrow, 10 = within the next 7 days (a week’s time), 11 = within the next 30 days (a month’s time), 12 = within the next 365 days (a year’s time), 13 = within the next 3 years, 14 = more than 3 years from now. omenological characteristics of self-generated FIGURE 1 \| Phenomenological characteristics of self-generated thought. Individual data points represent each participant’s mean across 36 self-generated thoughts. Open circles represent outliers > 2.5 SD above or below the group mean (vertical bar) which are not included in calculations. The scale for temporal orientation corresponds to: 1 = more than 3 years ago, 2 = within the past 3 years, 3 = within the past 365 FIGURE 1 \| Phenomenological characteristics of self-generated thought. Individual data points represent each participant’s mean across 36 self-generated thoughts. Open circles represent outliers > 2.5 SD above or below the group mean (vertical bar) which are not included in calculations. The scale for temporal orientation corresponds to: 1 = more than 3 years ago, 2 = within the past 3 years, 3 = within the past 365 November 2013 \| Volume 4 \| Article 900 \| 6 Frontiers in Psychology \| Perception Science Self-generated thought and emotional wellbeing Andrews-Hanna et al. FIGURE 2 \| Relationships between content variables. Relationships between content variables were averaged across 76 participants. For scales corresponding to each content variable, see Figure 1 andTable 1. PART 2: IDENTIFYING MAJOR DIMENSIONS OF THOUGHT CONTENT Note that higher scores for Duration of Topic correspond to thoughts concerning topics or events lasting shorter durations. p < 0.05, p < 0.0008 (alpha corrected for multiple comparisons using the Bonferroni method). FIGURE 2 \| Relationships between content variables. Relationships between content variables were averaged across 76 participants. For scales corresponding to each content variable, see Figure 1 andTable 1. Note that higher scores for Duration of Topic correspond to thoughts concerning topics or events lasting shorter durations. p < 0.05, *p < 0.0008 (alpha corrected for multiple comparisons using the Bonferroni method). www.frontiersin.org PART 3: RELATIONSHIPS BETWEEN THOUGHT CONTENT AND EMOTIONAL WELLBEING Higher scores for the Level of Construal cluster correspond to thoughts that are more temporally and perceptually speciﬁc, as well as more socially oriented. Higher scores for the Personal Signiﬁcance cluster correspond to thoughts rated as more personally signiﬁcant. Higher scores for the Outlook cluster correspond to thoughts rated as more positive and future-oriented. On the y-axis, height is a measure of similarity or distance between content variables such that two variables forming a single cluster at a small height are more similar than two variables linked at a greater height. for the Outlook cluster correspond to thoughts rated as more positive and future-oriented. On the y-axis, height is a measure of similarity or distance between content variables such that two variables forming a single cluster at a small height are more similar than two variables linked at a greater height. FIGURE 3 \| Hierarchical clustering reveals major dimensions of thought content. Higher scores for the Level of Construal cluster correspond to thoughts that are more temporally and perceptually speciﬁc, as well as more socially oriented. Higher scores for the Personal Signiﬁcance cluster correspond to thoughts rated as more personally signiﬁcant. Higher scores F(4,63) = 3.24, p < 0.05, R2 = 0.17; Level of Construal: β = −0.35, t(60) = −2.77, p < 0.01. Depression/Negative Affect (Figure 4A). To examine the speciﬁcity of these ﬁndings for Depression/Negative Affect, we next controlled for statistical effects of anxious arousal, anxious apprehension, and Rumination on Depression/Negative Affect using a part cor- relation, and the effects were numerically stronger, R = 0.57, F(4,56) = 6.20, p < 0.001; Personal Signiﬁcance, β = 0.27, t(55) = 2.41, p < 0.05; Valence, β = −0.57, t(55) = −4.45, p < 0.001. The four types of thought content explained 21.2% of the variance in mindfulness, F(4,59) = 3.97, p < 0.01, with Level of Construal, Personal Signiﬁcance and Valence contributing independent variance (Level of Construal, β = 0.25, t(58) = 2.00, p = 0.05; Personal Signiﬁcance, β = −0.24, t(58) = −2.1, p < 0.05; Valence, β = 0.27, t(58) = 2.09, p < 0.05). Individu- als who rated their thoughts as more concrete and speciﬁc, less personally signiﬁcant, and more positive scored higher on dis- positional mindfulness questionnaires (Figure 4C). Both Depres- sion/Negative Affect and Rumination were negatively correlated with Mindfulness, r(64) = −0.68, p < 0.001 and r(64) = −0.28, p < 0.05. PART 3: RELATIONSHIPS BETWEEN THOUGHT CONTENT AND EMOTIONAL WELLBEING orientation. While all of these questions were signiﬁcantly posi- tively correlated (all r’s > 0.2, p’s < 0.001), self-relevance exhibited the strongest relationships with centrality and subjective value (Figure 2). Additionally, thoughts that were attributed high value were rated as more central to one’s sense of self-identity, higher in emotional intensity, and more recurrent. Goal-oriented thoughts were also rated as more self-relevant, more central to one’s sense of self-identity, more recurrent, more intense, and more important/valuable. The combined contribution of the 12 content variables explained a large amount of variance in individual differences in the three psychological constructs of interest. Speciﬁcally, thought content explained 45.8% of the variance in Depression/Negative Affect, 30.5% of the variance in Rumination, and 36.3% of the variance in Mindfulness. We next repeated the analyses after combining the content variables into the composite dimensions identiﬁed in Part 2, but splitting the dimension corresponding to Outlook into temporal orientation and valence because of the observed weak correlations between those content ratings and our a priori hypotheses linking valence to depression. Thus, four indepen- dent variables were entered together into each multiple regression (Level of Construal, Personal Signiﬁcance, Valence, and Temporal Orientation). A third dimension, termed Outlook, reﬂected signiﬁcant pos- itive relationships between temporal orientation and valence (Figures 2 and 3). Future-oriented thoughts were rated as more positive and past-oriented thoughts were rated as more negative. In addition to relationships between content variables within each content dimension (i.e., self-relevancy, value, etc.), relation- ships also emerged across variables belonging to different content dimensions (Figure 2). More future-oriented thoughts were gen- erally experienced with less speciﬁcity, while thoughts that were rated as more personally signiﬁcant pertained to topics of longer durations, yet were not necessarily less speciﬁc or vivid. Further- more, socially oriented thoughts were experienced with greater emotional intensity, and positive associations were observed between emotional intensity and both imagery and speciﬁcity. Collectively, these four types of thought content explained 33.0% of the variance in Depression/Negative Affect, R = 0.58, F(4,58) = 7.15, p < 0.001, with Personal Signiﬁcance and Valence contributing independent variance (Personal Signiﬁcance, β=0.22,t(57)=1.99,p=0.05;Valence, β=−0.57,t(57)=−4.65, p < 0.001). Individuals who rated their thoughts as more per- sonally signiﬁcant and more negative reported higher levels of November 2013 \| Volume 4 \| Article 900 \| 7 Self-generated thought and emotional wellbeing Andrews-Hanna et al. FIGURE 3 \| Hierarchical clustering reveals major dimensions of thought content. PART 3: RELATIONSHIPS BETWEEN THOUGHT CONTENT AND EMOTIONAL WELLBEING Depression/Negative Affect was marginally correlated with Rumination, r(63) = 0.22, p = 0.08, allowing us to examine relationships between self-generated thought content and each mental health construct speciﬁcally. Although a smaller percent- age of variance in Rumination was explained by the four classes of thought content, R = 0.38, F(4,62) = 2.68, p = 0.04, R2 = 14.7, Level of Construal was the only content dimension that contributed signiﬁcant independent variance to Rumination, β = −0.32, t(61) = −2.56, p < 0.05 (Figure 4B). As predicted, individu- als who generated more abstract and less speciﬁc thoughts were characterized by higher levels of Rumination. Importantly, the relationship between Level of Construal and Rumination became numerically stronger after controlling for comorbid effects of anx- iety and Depression/Negative Affect on Rumination, R = 0.41, Relationships with state affect Individual differences in Depression/Negative Affect were strongly predictive of state affect as assessed with the “positive” and “neg- ative” subscales of the state PANAS, R = 0.59, F(2,60) = 15.60, p < 0.001. However, state affect was not related to indi- vidual differences in Rumination, R = 0.13, F(2,66) = 0.58, p = 0.56. State affect was strongly related to Valence, R = 0.49, Frontiers in Psychology \| Perception Science November 2013 \| Volume 4 \| Article 900 \| 8 \| 8 Self-generated thought and emotional wellbeing Andrews-Hanna et al. FIGURE 4 \| Independent content predictors of constructs relevant for mental health. Positive βs are characterized by thoughts that are more concrete, more personally signiﬁcant, more future oriented and more positive (see scales in Figure 1). (A) Personal Signiﬁcance and Valence contributed a signiﬁcant amount of variance toward explaining Depression/Negative Affect. (B) Level of Construal was the only dimension that contributed a signiﬁcant amount of variance toward predicting individual differences in Rumination. (C) Level of Construal and Personal Signiﬁcance contributed a signiﬁcant amount of variance toward predicting Mindfulness, with a considerable additional contribution of Valence (β = 0.25, p = 0.054). p ≤0.05. (B) Level of Construal was the only dimension that contributed a signiﬁcant amount of variance toward predicting individual differences in Rumination. (C) Level of Construal and Personal Signiﬁcance contributed a signiﬁcant amount of variance toward predicting Mindfulness, with a considerable additional contribution of Valence (β = 0.25, p = 0.054). *p ≤0.05. FIGURE 4 \| Independent content predictors of constructs relevant for mental health. Positive βs are characterized by thoughts that are more concrete, more personally signiﬁcant, more future oriented and more positive (see scales in Figure 1). (A) Personal Signiﬁcance and Valence contributed a signiﬁcant amount of variance toward explaining Depression/Negative Affect. F(2,69) = 10.73, p < 0.001, but not to Personal Signiﬁcance, R = 0.12, F(2,68) = 0.49, p = 0.62. When controlling for state affect, the relationship between Depression/Negative Affect and Valence remained signiﬁcant (r(62) = −0.51, p < 0.001; partial r(58) = −0.44, p < 0.01). directions for research on self-generated thought and mental health. POSITIVE AND PRODUCTIVE MODES OF THOUGHT CONTENT REFLECT THE NORM IN A SAMPLE OF YOUNG ADULTS Measuring the content of self-generated thought across numerous thought samples, content variables, and participants allowed us to extract at least three dimensions of content: Level of Construal, Per- sonal Signiﬁcance, and Outlook. On average, participants’thoughts were moderately perceptually and temporally speciﬁc, consistent with prior studies (Klinger and Cox, 1987; Delamillieure et al., 2010; Stawarczyk et al., 2011, 2013). These concrete “low-level” construals have been linked to beneﬁcial outcomes encompassing effective problem solving, successful attainment of future goals, and enhanced self-regulatory behavior (Pham and Taylor, 1999; Watkins and Baracaia, 2002; Watkins and Moulds, 2005; Leary et al., 2006). Interestingly, socially oriented thoughts were also experienced with greater perceptual detail. Although not the main topicof thepresentmanuscript,theﬁndingthatparticipantsspend a notable amount of time thinking about other people is in line with prior studies (Mar et al., 2012; see also Ruby et al., under review) and provides strong support for theories highlighting the importance of social information for human cognition (Dunbar, 1998; Baumeister and Masicampo, 2010; Corballis, 2013). www.frontiersin.org DISCUSSION The current study indicates that self-generated thoughts can be well-characterized along three major content dimensions regard- ing their level of construal, their personal signiﬁcance, and the outlook they embody. Moreover, these dimensions explain a notable amount of variance in individual differences in emo- tional health and wellbeing. As such, our study speaks to a long-standing debate in psychology and neuroscience (e.g., Smallwood and Schooler, 2006; Watkins, 2008; McVay and Kane, 2010) by suggesting that the ability to regulate the con- tent of self-generated thought is a key factor underlying its costs and beneﬁts (Smallwood and Andrews-Hanna, 2013). Indi- viduals who characterized their thoughts as more personally signiﬁcant, more negative, and/or less detailed scored higher on constructs linked to poor psychological wellbeing (Depres- sion and Rumination), whereas those who characterized their thoughts as less personally signiﬁcant, more positive, and more speciﬁc scored higher on constructs linked to improved well- being (Mindfulness). Below, we synthesize our ﬁndings in the context of prior literature and conclude by discussing future Furthermore, participants’thoughts were highly personally sig- niﬁcant, somewhat goal-oriented, and attributed strong value, November 2013 \| Volume 4 \| Article 900 \| 9 www.frontiersin.org \| 9 Self-generated thought and emotional wellbeing Andrews-Hanna et al. resonating with theories that personal concerns form the basis of human thought content (Klinger, 1971, 2009; Fox et al., 2013; McMillan et al., 2013; Stawarczyk et al., 2013). resonating with theories that personal concerns form the basis of human thought content (Klinger, 1971, 2009; Fox et al., 2013; McMillan et al., 2013; Stawarczyk et al., 2013). rumination questionnaires pertain to negative content, it remains yet to be determined whether overgeneral patterns of thought also characterize other types of repetitive thought, independent of affective content. Rumination is strongly related to, yet distinct from, depression, and our observation that overgeneral thinking is particularly related to Rumination agrees well with studies report- ing elevated overgeneral memory following rumination induction (Watkins and Teasdale, 2001; Sutherland and Bryant, 2007). The relationship between overgeneral memory, rumination, and other types of repetitive thought marks an interesting area for future research. DISCUSSION Consistent with prior studies, participants rated their thoughts as somewhat positive (Killingsworth and Gilbert, 2010; Stawar- czyk et al., 2011, 2013; Fox et al., 2013) and future-oriented (Smallwood et al.,2009b;Andrews-Hanna et al.,2010a; Baird et al., 2011; Stawarczyk et al., 2011, 2013; Song and Wang, 2012; Fox et al., 2013; Ruby et al., 2013), supporting a growing body of work regarding the adaptive signiﬁcance of prospective thoughts (Gilbert and Wilson, 2007; Schacter et al., 2007; Suddendorf and Corballis,2007). Future-oriented thoughts were perceived as more positive than past-oriented thoughts, supporting prior ﬁndings linking retrospective focus to negative mood (Smallwood and O’Connor, 2011). MINDFUL ASPECTS OF THOUGHT CONTENT ARE RECIPROCALLY RELATED TO DEPRESSION AND RUMINATION In contrast to depression and rumination, dispositional mindful- ness and mindfulness meditation have been linked to improved health, cognitive ability, and psychological wellbeing (Brown and Ryan, 2003; Sedlmeier et al., 2012). Paralleling these ﬁndings, Mindfulness exhibited negative relationships with Depression and Rumination, and was partly explained by an opposing pattern of thought content, being characterized as more positive, less per- sonally signiﬁcant, and more speciﬁc. These results are consistent with prior research demonstrating that mindful individuals report higher levels of positive affect (Brown and Ryan, 2003), posi- tive or less negative thought content (Frewen et al., 2008; Evans and Segerstrom, 2011), and better emotion regulation ability (Farb et al., 2012). THOUGHT CONTENT RELATES TO EMOTIONAL HEALTH AND WELLBEING Providing strong support for the content regulation hypothesis (Smallwood and Andrews-Hanna, 2013), our ﬁndings indicate thatthoughtcontent isanimportantfactorunderlyingtheadaptive and maladaptive consequences of self-generated thought. Accord- ing to content regulation hypothesis, self-generated thoughts are primarily beneﬁcial when individuals are able to regulate the con- tent of the experience to positive or productive topics (Smallwood and Andrews-Hanna, 2013). Conversely, individuals who lack this capacity may experience maladaptive psychological consequences of self-generated thought. Of relevance, brief mindfulness inductions have been shown to dampen negative affect (Brown and Ryan, 2003; Huffziger et al., 2013), and many of the content variables that predicted increases in dispositional mindfulness in our study are central to mindfulness-based therapies for a variety of mental health disor- ders. For example, a key element of mindfulness-based cognitive therapy involves teaching people to become aware of the nature of their internal thoughts, view their thoughts from a more distant or “de-centered”perspective,andacceptthemforwhattheyarerather than the literal truth (Segal et al., 2002; Mennin and Fresco, 2013). When applied to recurrent depression,MBCT reduces relapse rates by nearly 50% (Teasdale et al., 2000; Watkins, 2008) and increases the speciﬁcity of individuals’ autobiographical memory (Williams et al., 2000, 2007). g g In line with these predictions, individuals who rated their thoughts as more negative in valence as well as more personally signiﬁcant scored higher on questionnaires assessing Depres- sion/Negative Affect. These ﬁndings are consistent with long-held theories that depressed individuals hold negative schemas about themselves, their future, and the environment (Beck, 1967), and that a dispositional style of self-focused thinking exacer- bates the duration and symptoms of depression (Smith and Greenberg, 1981; Larsen and Cowan, 1988). Depressed indi- viduals also exhibit attentional and mnemonic biases toward negative and self-referential information, difﬁculties clearing this information from working memory, and impaired performance particularly when the material is task-irrelevant (Gotlib and Joor- mann, 2010; Joormann, 2010). Further support for associations between self-focus and negative affect comes from studies linking self-focused attention to state and trait negative affect, particularly when the content of the self-focused thought is negative in nature (Smith and Greenberg, 1981; Mor and Winquist, 2002). Conse- quently, experimental inductions that amplify external focus and dampen internal focus help depressed individuals adopt a less pessimistic view of their future (Pyszczynski et al., 1987). Frontiers in Psychology \| Perception Science CONCLUSIONS AND FUTURE DIRECTIONS In summary, these ﬁndings provide strong evidence that the con- tent of people’s inner thoughts in daily life can be productively examined and distilled into several major dimensions. More- over, individual differences in thought content explain a notable amount of variance in constructs relevant to emotional health and wellbeing. As such, the present manuscript extends previous stud- ies that focus solely on the frequency with which self-generated thoughts occur, particularly because increases in frequency have been linked to both adaptive and maladaptive outcomes (i.e., McVay and Kane, 2009; Levinson et al., 2012). In contrast, the content characterizing self-generated thought may more precisely constrain the functional consequences of this human experience. Quantifying an individual’s full proﬁle of thought content – per- haps in combination with the frequency with which self-generated Although depressed individuals commonly exhibit overgeneral autobiographical memories (Williams et al., 2007; Watkins, 2008), the present results demonstrate that overgeneral styles of thinking were more strongly related to individual differences in Rumination than to Depression/Negative Affect. These ﬁndings suggest that the repetitive nature of self-generated thought rather than negative affect per se, may be most strongly linked to abstract levels of con- strual. However, since many of the questions contributing to the November 2013 \| Volume 4 \| Article 900 \| 10 Frontiers in Psychology \| Perception Science Self-generated thought and emotional wellbeing Andrews-Hanna et al. thoughtsoccur,and/orthedifﬁcultyof thetaskduringwhichunre- lated thoughts occurs (Smallwood and Andrews-Hanna, 2013) – might ultimately help researchers or clinicians distinguish between different trait dimensions and/or mental health disorders. Yarkoni for helpful discussion. This work was supported by the following grants from the National Institutes of Mental Health: F32 MH093985 (Jessica R. Andrews-Hanna) and P50 MH079485 (Marie T. Banich). Unfortunately, we were unable to fully address this last question using our sample of participants because none of our participants were diagnosed with a mental health disorder. 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Psychol. 4:900. doi: 10.3389/fpsyg.2013.00900 This article was submitted to Perception Science, a section of the journal Frontiers in Psychology. Watkins, E. R. (2008). Constructive and unconstructive repetitive thought. Psychol. Bull. 134, 163–206. doi: 10.1037/0033-2909.134.2.163 Copyright © 2013 Andrews-Hanna, Kaiser, Turner, Reineberg, Godinez, Dimidjian and Banich. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Watkins, E., and Baracaia, S. (2002). Rumination and social problem solving in depression. Behav. Res. Ther. 40, 1179–1189. doi: 10.1016/S0005-7967(01) 00098-5 Watkins, E., and Moulds, M. 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https://openalex.org/W4310064732	https://munin.uit.no/bitstream/10037/28236/2/article.pdf	English	null	Epidemiology and natural history in 101 subjects with FKRP-related limb-girdle muscular dystrophy R9. The Norwegian LGMDR9 cohort study (2020)	Neuromuscular disorders	2,023	cc-by	15,243	∗Corresponding author at: National Neuromuscular Centre Norway and Department of Neurology, University Hospital of North Norway HF (UNN), PO Box 100, N-9038, Tromsø, Norway. E-mail address: synnove.magnhild.jensen@unn.no (S.M. Jensen) . Neuromuscular Disorders journal homepage: www.elsevier.com/locate/nmd Epidemiology and natural history in 101 subjects with FKRP-related limb-girdle muscular dystrophy R9. The Norwegian LGMDR9 cohort study (2020) Bindoff c , d , e , Magnhild Rasmussen f , g , Kristin Ørstavik g , Christoffer Jonsrud h , Kristian Tveten i , Øivind Nilssen b , h , Marijke Van Ghelue b , h , Kjell Arne Arntzen a , b a National Neuromuscular Centre Norway and Department of Neurology, University Hospital of North Norway HF, Tromsø, PO Box 100, N-9038, Tromsø, Norway b Department of Clinical Medicine, University of Tromsø – The Artic University of Norway, PO Box 605 c Department of Clinical Medicine (K1), University of Bergen, N-5021, Bergen, Norway p f ( ), y f g , , g , y d Department of Neurology, Haukeland University Hospital, PO Box 1400, N-5021, Bergen, Norway p f gy y p g y National Unit of Newborn Screening and Advanced Laboratory Diagnostics, Oslo University Hospital, PO Box 4950 Nydalen, N-0424, Osl Department of Clinical Neurosciences for Children, Oslo University Hospital, PO Box 4950 Nydalen, N-0424, Oslo, Norway e National Unit of Newborn Screening and Advanced Laboratory Diagnostics, Oslo University Hospital, PO Box 4950 Nydalen, N-0424, Oslo, Norway f Department of Clinical Neurosciences for Children, Oslo University Hospital, PO Box 4950 Nydalen, N-0424, Oslo, Norway g U it f C it l d H dit N l C diti (EMAN) D t t f N l O l U i it H it l PO B 4950 N d l N 0424 e National Unit of Newborn Screening and Advanced Laboratory Diagnostics, Oslo University Hospital, PO Box 4950 Nydalen, N-0424, Oslo, Norwa f Department of Clinical Neurosciences for Children, Oslo University Hospital, PO Box 4950 Nydalen, N-0424, Oslo, Norway g Unit for Congenital and Hereditary Neuromuscular Conditions (EMAN), Department of Neurology, Oslo University Hospital, PO Box 4950 Nydalen Oslo Norway p f f , y p , y , , , y g Unit for Congenital and Hereditary Neuromuscular Conditions (EMAN), Department of Neurology, Oslo University Hospital, PO Box 4950 N Oslo, Norway , y h Department of Medical Genetics, Division of Child and Adolescent Health, University Hospital of North Norway HF, PO Box 55, N-9038, Tromsø, Norway i Department of Medical Genetics, Telemark Hospital Trust, PO Box 2900 Kjørbekk, N-3710, Skien, Norway j Department of Neurology, Hospital of Southern Norway, PO box 416 Lundsiden, 4604, Kristiansand S, Norway h Department of Medical Genetics, Division of Child and Adolescent Health, University Hospital of North Norway HF, PO Box 55, N-9038, Tr Department of Medical Genetics, Telemark Hospital Trust, PO Box 2900 Kjørbekk, N-3710, Skien, Norway D t t f N l H it l f S th N PO b 416 L d id 4604 K i ti d S N a b s t r a c t Article history: Received 22 February 2022 Revised 8 September 2022 Accepted 21 November 2022 Available online xxx Keywords: FKRP Fukutin-related protein Limb-girdle muscular dystrophies Cardiomyopathy Prevalence Gender-related LGMDR9 differences Article history: Received 22 February 2022 Revised 8 September 2022 Accepted 21 November 2022 Available online xxx Keywords: FKRP Fukutin-related protein Limb-girdle muscular dystrophies Cardiomyopathy Prevalence Gender-related LGMDR9 differences We aimed to investigate the epidemiology and natural history of FKRP-related limb-girdle muscular dystrophy R9 (LGMDR9) in Norway. We identiﬁed 153 genetically conﬁrmed subjects making the overall prevalence 2.84/10 0,0 0 0, the highest reported ﬁgure worldwide. Of the 153 subjects, 134 (88 %) were homozygous for FKRP c.826C > A giving a carrier frequency for this variant of 1/101 in Norway. Clinical questionnaires and patient notes from 101 subjects, including 88 c.826C > A homozygotes, were reviewed, and 43/101 subjects examined clinically. Age of onset in c.826C > A homozygotes demonstrated a bimodal distribution. Female subjects showed an increased cumulative probability of wheelchair dependency and need for ventilatory support. Across the cohort, the need for ventilatory support preceded wheelchair dependency in one third of the cases, usually due to sleep apnea. In c.826C > A homozygotes, occurrence of cardiomyopathy correlated positively with male gender but not with age or disease stage. This study highlights novel gender differences in both loss of ambulation, need for ventilatory support and the development of cardiomyopathy. Our results conﬁrm the need for vigilance in order to detect respiratory insuﬃciency and cardiac involvement, but indicate that these events affect males and females differently. © 2022 The Author(s). Published by Elsevier B.V. his is an open access article under the CC BY license ( http:/ ( ) y This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) Epidemiology and natural history in 101 subjects with FKRP-related limb-girdle muscular dystrophy R9. The Norwegian LGMDR9 cohort study (2020) Synnøve M. Jensen a , b , ∗, Kai Ivar Müller a , b , j , Svein Ivar Mellgren a , b , Laurence A. ARTICLE IN PRESS ARTICLE IN PRESS ARTICLE IN PRESS [m5G; December 13, 2022;4:40 ] JID: NMD https://doi.org/10.1016/j.nmd.2022.11.005 0960-8966/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) https://doi.org/10.1016/j.nmd.2022.11.005 0960-8966/© 2022 The Author(s). Publish 1 REDCap (Research Electronic Data Capture) is a secure, web-based software platform designed to support data capture for research studies, providing 1) an intuitive interface for validated data capture; 2) audit trails for tracking data manipulation and export procedures; 3) automated export procedures for seamless data downloads to common statistical packages; and 4) procedures for data integration and interoperability with external sources. 1. Introduction in the FKRP gene identiﬁed in 2001 [3] . In addition to limb girdle muscular dystrophy, FKRP - related diseases also include the very rare congenital muscular dystrophy type 1C (MDC1C), muscle-eye- brain disease (MEB) and Walker-Warburg syndrome (WWS) [ 4 , 5 ]. Fukutin Related Protein (FKRP) is a glycosyl transferase involved in the post-translational O -glycosylation of the sarcolemma protein α-dystroglycan and the N -glycosylation of the extracellular matrix (ECM) protein ﬁbronectin [ 4 , 6-8 ] in the Golgi. The limb-girdle muscular dystrophies (LGMDs) comprise a group of inherited muscle diseases characterized by progressive proximal weakness and muscle wasting [1] . The recessive LGMDR9, formerly known as LGMD2I [2] , is caused by pathogenic variants LGMDR9 is most frequent in North-European populations due to the distribution of the c.826C > A (p. Leu276Ile) variant. Analyses of neighbouring single nucleotide polymorphisms, performed on E-mail address: synnove.magnhild.jensen@unn.no (S.M. Jensen) . 22.11.005 s). Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) Please cite this article as: S.M. Jensen, K.I. Müller, S.I. Mellgren et al., Epidemiology and natural history in 101 subjects with FKRP-related limb-girdle muscular dystrophy R9 The Norwegian LGMDR9 cohort study (2020-), Neuromuscular Disorders, https://doi.org/10.1016/j. nmd 2022 11005 Please cite this article as: S.M. Jensen, K.I. Müller, S.I. Mellgren et al., Epidemiology and natural history in 101 subjects with FKRP-related limb-girdle muscular dystrophy R9 The Norwegian LGMDR9 cohort study (2020-), Neuromuscular Disorders, https://doi.org/10.1016/j. nmd 2022 11005 S.M. Jensen, K.I. Müller, S.I. Mellgren et al. Neuromuscular Disorders xxx (xxxx) xxx ARTICLE IN PRESS JID: NMD [m5G; December 13, 2022;4:40 ] ARTICLE IN PRESS [m5G; December 13, 2022;4:40 ] JID: NMD S.M. Jensen, K.I. Müller, S.I. Mellgren et al. Neuromuscular Disorders xxx (xxxx) xxx S.M. Jensen, K.I. Müller, S.I. Mellgren et al. families with FKRP c.826C > A, indicate that this variant has a common ancestral origin (founder) rather than being a mutational hot spot with recurrent events [ 9 , 10 ]. The allele frequencies of the c.826C > A variant differ in various populations: in the Swedish population it is 0.0046 (carrier frequency 1/109), in North-Western Europeans 0.0023 (carrier frequency 1/218), in Finns 0.0011 (carrier frequency 1/469) and in African/African-Americans < 0.0 0 034 (carrier frequency 1/1490) [11] . 2.3. Clinical data Data from patient notes and questionnaires as well as clinical data from those participating in The Norwegian LGMDR9 cohort study were registered. Participants were divided by genotype into c.826C > A homozygotes and non -c.826C > A homozygous groups. When analysing the natural history, gender was accounted for in both groups. Symptom onset was deﬁned as the ﬁrst symptoms or clinical signs of muscle disease including myoglobinuria, but not an incidental ﬁnding of elevated serum creatine kinase (CK) or transaminases (ALAT, ASAT). Achieved independent walking was used as the criterion to differentiate LGMDR9 from congenital muscular dystrophy type 1C. Ambulation was participant reported and recorded as wheelchair dependent or not and graded according to the maximum walking distance on ﬂat ground, the ability to climb stairs one ﬂoor, and the ability to run (Sect. 2.4). Ventilation was evaluated based on the need for ventilatory support while cardiomyopathy was assessed based on the results of routine echocardiography. The lower normal limit of left ventricular ejection fraction (LVEF) was set to 50 % in accordance with the reports. Natural history studies of LGMDR9 have mostly been based on limited sample-size although studies with 32-69 subjects have been reported in: Norway [15] , USA [ 17 , 18 ], UK [19] and Denmark [ 20 , 21 ]. A global patient registry containing > 300 registered subjects has also been established [22] . LGMDR9 manifests with a wide clinical variability and evidence suggests that this in part reﬂects the genotype: subjects homozygous for c.826C > A express a milder phenotype compared to compound heterozygous subjects [ 15 , 19 , 20 , 22 ]. Data from the global FKRP registry also showed a later onset (19 ± 12 versus 7 ± 7 years), a lower proportion of wheelchair dependency (18 % versus 40 %), and a later need for ventilatory support (47 ± 14 versus 33 ± 13 years) [22] . Cardiac studies predicted that c.826C > A homozygotes have a 50 % probability of cardiomyopathy by age 50 versus 20 years in c.826C > A compound heterozygotes [ 17 , 19 ]. Other studies found considerable variation in clinical severity within c.826C > A homozygotes [ 10 , 23 , 24 ] with age of onset ranging from 0-50 years [15] and variable age at loss of ambulation [22] . 2.4. Statistical analyses In the present study, we investigated the prevalence, the spectrum of variants and the clinical patterns of LGMDR9 in Norway. We searched for possible predictors for developing wheelchair dependency, need for ventilatory support and cardiomyopathy. Our goal was to provide data that may be useful for improving clinical practice, for clinical trials, and translational research. Study data were collected and managed using REDCap 1 electronic data capture tools hosted at UNN [ 27 , 28 ]. Statistical analyses were performed using IBM SPSS Statistics for Windows (Version 27.0. Armonk, NY: IBM Corp.). Wilson score was employed to estimate the conﬁdence intervals (CIs) of the lowest and highest county prevalence for comparison. The carrier frequency of the common c.826C > A variant in the population was calculated with the Hardy-Weinberg equation. Normally distributed continuous variables were described using the means and standard deviations, and non-parametric using medians and inter-quartile ranges (IQRs). Categorical variables were described as percentages. Pearson Chi-square test and Fisher ´s exact test with mid-p correction, as appropriate, were used to assess associations between categorical variables. Mann-Whitney U-test was used to assess the differences in continuous non-parametric distributed variables between two groups. Two-step cluster analysis was applied to assess the distribution of age at onset of the disease. For clinical endpoints with censored observations, Kaplan-Meier survival curves were estimated in speciﬁc subgroups using age as time scale. The events deﬁned were wheelchair dependency, commence of ventilatory support, and ﬁrst abnormal echocardiography, respectively. Negative subjects concerning wheelchair and ventilatory support were censored at the end of 2020, while for cardiomyopathy at last echocardiography, or at ischemic incidence (one participant). Log-rank test was used to compare survival curves between genders of similar genotype, while Cox regression between genotype groups to control 1. Introduction The allele has not been detected in the Asian population [11] who have a different spectrum of FKRP disease-causing variants [12–14] . The highest prevalence of LGMDR9 has been recorded in Norway: in 2009 the national prevalence was estimated to be 1.85/10 0,0 0 0 [15] . More recently the prevalence was found to be 5.88/10 0,0 0 0 in Northern Norway [16] . of the single nucleotide polymorphism, FKRP c.135C > T, previously found to be in linkage disequilibrium with c.826C > A, was also assessed in the Norwegian population of FKRP c.826C > A homozygotes [ 9 , 10 ]. No other FKRP-related phenotypes than LGMD were identiﬁed at the four relevant departments: The Medical Genetic Departments of UNN, HUS, THT and Oslo University Hospital. 2.1. Inclusion criteria All living subjects with genetically conﬁrmed LGMDR9 residing in Norway were included in the epidemiological part of the study. The subjects were identiﬁed through diagnostic patient registries at: The Medical Genetics Departments of the University Hospital of North Norway HF (UNN) and Telemark Hospital Trust (THT), the Department of Neurology at Haukeland University Hospital (HUS), the Norwegian Registry of Hereditary and Congenital Neuromuscular Disorders, and by the Global FKRP Registry. All were invited to participate in the observational study. Subjects ≥ 16 years of age were also invited to participate in the baseline examination of The Norwegian LGMDR9 cohort study at the National Neuromuscular Centre Norway, UNN. 2.5. Approval and patient consent We also found a subject who was compound heterozygous for FKRP c.166T > A and c.826C > A ( Table A 1). We concluded that the c.166T > A variant was likely pathogenic based on the recommendations of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (criteria PM2, PM3, PP2 og PP4) [29] . The patient manifests an LGMD phenotype with limb-girdle weakness, calf pseudohypertrophy, scapular winging, hyperCKemia and dilated cardiomyopathy. Symptoms began with post exercise myalgia at age 13 years. Histological features support a diagnosis of primary structural muscle disease but there was insuﬃcient material for additional staining including dystroglycan. EMG supports chronic myopathy. Muscle imaging has not been performed. The PP4-criterion was considered fulﬁlled when tests of genes with overlapping phenotypes disclosed no other ﬁndings. This patient was tested for variants using a neuromuscular NGS panel (328 genes) and MLPA of dystrophin and alpha-, beta-, delta and gamma-sarcoglycan. The FKRP c.135C > T/c.135C > T genotype was detected in all c.826C > A homozygotes who were genetically conﬁrmed at UNN (n = 127). For comparison, the frequency of the c.135C > T allele among Europeans (non-Finnish) is reported to be 0.14 and the homozygote frequency observed in the same population is 0.01 [11] . 2.2. Prevalence and genetic data The signiﬁcance level was set to 0.05. The Holm-Bonferroni method was applied due to multiple subgroup comparisons of the natural history (12 tests): age of onset and cumulative probability comparisons between both genotype and gender subgroups, and gender as an independent variable in the multivariate analyses. Due to the exploratory nature of the study, empirical p-values were reported while the multiple comparison test (MCT) was used to conﬁrm or not conﬁrm signiﬁcant p-values. Results with and without correction for multiple comparisons are discussed. for gender. Cox proportional Hazard regression models were applied to estimate hazard ratios (HRs). Fifty percent accumulated probabilities were estimated when applicable. Ambulation was both evaluated by wheelchair dependency as a binary variable, and by a 10-point (0-9) motor composite score (MCS). MCS was composed from the questionnaire consisting of three items: walking distance on ﬂat ground (0: unrestricted, 1: > 10 0 0 meters, 2: > 500 meters, 3: > 50 meters, 4: > a few steps, 5: zero or a few steps); ability to climb stairs one ﬂoor independently (0 = yes, 1 = manage with stair railing, 2 = not able); ability to run (0 = no problems, 1 = yes, but not fast, 2 = not able). (Cronbach ´s α = 0.73, corrected item-total correlation: 1. Item: 0.73, 2. Item: 0.80, 3. Item: 0.64). It was complemented with regression analyses of cross-sectional data in order to assess potential predictors and confounding effect. MCS as dependent continuous variable was assessed by linear regression. Multivariate binomial logistic regression with maximum-likelihood method was employed to estimate odds ratios (ORs) for cardiomyopathy, need for ventilatory support and wheelchair dependency as binary dependent variables. The independent variables assessed were age, gender, age at symptom onset, symptom duration, MCS for cardiomyopathy and ventilatory support, and ventilatory support for cardiomyopathy. Following simple regression analyses, the most relevant independent variables were selected for the multivariate analyses. The signiﬁcance level was set to 0.05. The Holm-Bonferroni method was applied due to multiple subgroup comparisons of the natural history (12 tests): age of onset and cumulative probability comparisons between both genotype and gender subgroups, and gender as an independent variable in the multivariate analyses. Due to the exploratory nature of the study, empirical p-values were reported while the multiple comparison test (MCT) was used to conﬁrm or not conﬁrm signiﬁcant p-values. Results with and without correction for multiple comparisons are discussed. Fig. 1. 2.2. Prevalence and genetic data Map of Norway showing minimum LGMDR9 prevalences and population sizes on national and county level as of 1 st of January 2021. Oslo was merged with Viken county due to the small area and similar prevalence (number 9). Colour intensity correlates with the whole number of cases with LGMDR9 per 10 0,0 0 0 population. Sources: Map from Wikimedia common (with adaptation), numbers from Statistics Norway. 3. Results 3.1. Prevalence and molecular data 2.5. Approval and patient consent 2.5. Approval and patient consent gnomAD population database [11] . The FKRP c.141_151del11 variant results in an out of frame deletion early in the coding exon 4, with a predicted consequence on the protein p.(Arg48Profs ∗9). This is a null allele causing FKRP truncation. Accordingly, the patient who is compound heterozygous for both this variant and c.826C > A most probably exclusively expresses the latter variant causing the LGMDR9 phenotype ( Table A 1). We also found a subject who was compound heterozygous for FKRP c.166T > A and c.826C > A ( Table A 1). We concluded that the c.166T > A variant was likely pathogenic based on the recommendations of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (criteria PM2, PM3, PP2 og PP4) [29] . The patient manifests an LGMD phenotype with limb-girdle weakness, calf pseudohypertrophy, scapular winging, hyperCKemia and dilated cardiomyopathy. Symptoms began with post exercise myalgia at age 13 years. Histological features support a diagnosis of primary structural muscle disease but there was insuﬃcient material for additional staining including dystroglycan. EMG supports chronic myopathy. Muscle imaging has not been performed. The PP4-criterion was considered fulﬁlled when tests of genes with overlapping phenotypes disclosed no other ﬁndings. This patient was tested for variants using a neuromuscular NGS panel (328 genes) and MLPA of dystrophin and alpha-, beta-, delta and gamma-sarcoglycan. The FKRP c.135C > T/c.135C > T genotype was detected in all c.826C > A homozygotes who were genetically conﬁrmed at UNN (n = 127). For comparison, the frequency of the c.135C > T allele among Europeans (non-Finnish) is reported to be 0.14 and the homozygote frequency observed in the same population is 0.01 [11] . All participants provided informed written consent for the collection and use of clinical data. The study was approved by the Ethical Review Board of North Norway (2018/1968/REK nord), and by the Data Protection Oﬃcer at UNN. gnomAD population database [11] . The FKRP c.141_151del11 variant results in an out of frame deletion early in the coding exon 4, with a predicted consequence on the protein p.(Arg48Profs ∗9). This is a null allele causing FKRP truncation. Accordingly, the patient who is compound heterozygous for both this variant and c.826C > A most probably exclusively expresses the latter variant causing the LGMDR9 phenotype ( Table A 1). 2.2. Prevalence and genetic data The minimum overall prevalences of LGMDR9 in Norway on January 1 st 2021 was calculated nationally and for each county, and separately for the paediatric and adult population. Minimum FKRP c.826C > A (NM_024301.5) allele- and carrier frequencies were also calculated. The population number was obtained from Statistics Norway [25] . Genotypes were recorded, and novel variants were registered in the ClinVar database [26] . The presence 2 ARTICLE IN PRESS [m5G; December 13, 2022;4:40 ] JID: NMD Neuromuscular Disorders xxx (xxxx) xxx S.M. Jensen, K.I. Müller, S.I. Mellgren et al. Neuromuscular Disorders xxx (xxxx) xxx Fig. 1. Map of Norway showing minimum LGMDR9 prevalences and population sizes on national and county level as of 1 st of January 2021. Oslo was merged with Viken county due to the small area and similar prevalence (number 9). Colour intensity correlates with the whole number of cases with LGMDR9 per 10 0,0 0 0 population. Sources: Map from Wikimedia common (with adaptation), numbers from Statistics Norway. for gender. Cox proportional Hazard regression models were applied to estimate hazard ratios (HRs). Fifty percent accumulated probabilities were estimated when applicable. Ambulation was both evaluated by wheelchair dependency as a binary variable, and by a 10-point (0-9) motor composite score (MCS). MCS was composed from the questionnaire consisting of three items: walking distance on ﬂat ground (0: unrestricted, 1: > 10 0 0 meters, 2: > 500 meters, 3: > 50 meters, 4: > a few steps, 5: zero or a few steps); ability to climb stairs one ﬂoor independently (0 = yes, 1 = manage with stair railing, 2 = not able); ability to run (0 = no problems, 1 = yes, but not fast, 2 = not able). (Cronbach ´s α = 0.73, corrected item-total correlation: 1. Item: 0.73, 2. Item: 0.80, 3. Item: 0.64). It was complemented with regression analyses of cross-sectional data in order to assess potential predictors and confounding effect. MCS as dependent continuous variable was assessed by linear regression. Multivariate binomial logistic regression with maximum-likelihood method was employed to estimate odds ratios (ORs) for cardiomyopathy, need for ventilatory support and wheelchair dependency as binary dependent variables. The independent variables assessed were age, gender, age at symptom onset, symptom duration, MCS for cardiomyopathy and ventilatory support, and ventilatory support for cardiomyopathy. Following simple regression analyses, the most relevant independent variables were selected for the multivariate analyses. 3.2.2. Clinical characteristics The most frequently reported clinical features in both genotype groups were proximal weakness, calf pseudohypertrophy, mild scapular winging, and tendo-achilles contractures. Additionally, scoliosis and multiple contractures were relatively frequent in the non -c.826C > A homozygous group ( Table 3 ). In this group there was also one subject with a thoracic kyphosis and another with a rigid spine. Plain X-rays of the spine were usually not performed. The scoliosis was in several cases speciﬁed in the patient notes as mild and only in two cases as pronounced. The frequency of self-reported scoliosis was 11/101. Of these 11 subjects, ﬁve had received no treatment, ﬁve physiotherapy and one both physiotherapy and corset. None had undergone scoliosis surgery. The clinical characteristics are summarized in Table 2 (demographics and natural history) and Table 3 (clinical features and symptoms). All participants achieved independent walking. In three subjects presenting after the age of one year, onset could be traced back to abnormalities in gross motor development in the ﬁrst year of life. One was a c.826C > A homozygote examined aged 10 years for abdominal pain and elevated CK and transaminases. It was found that she had diﬃculty sitting up. She had been late in sitting and did not roll or crawl independently. Independent walking was achieved at normal age, 15 months. The other two patients had other genotypes ( Table A 1, Subjects 1 and 3): despite their diagnostic work-up being at 3 and 6 years, signs were observed at 9 months (speciﬁcs missing) and 7 months (hypotonia) respectively, and the latter had delayed independent walking. Oropharyngeal symptoms were reported by 20 participants ( Table 3 ) of whom 11 had initiated ventilatory support. Dysarthria usually coexisted with dysphagia and both occurred at a median age of 40 years. Four subjects speciﬁed their diﬃculties as swallowing wrongly, and two as food getting stuck in the throat. Dysarthria was described as slurred speech by two subjects. One subject with diﬃculties chewing also required ventilation both night and day as well as percutaneous endoscopic gastrostomy (PEG). One c.826C > A homozygote and three non - c.826C > A homozygous subjects had PEG, all related to breathing diﬃculties including two subjects who needed it only temporarily. Two c.826C > A homozygous females reported cramps in the tongue and/ or throat. One subject was 24 years old with exertional myalgia from the age of 6 years. 3.1. Prevalence and molecular data On January 1 st , 2021, Norway ´s population was 5,391,369. We identiﬁed 153 living subjects with genetically conﬁrmed LGMDR9 giving a minimum point prevalence of 2.84/10 0,0 0 0 for the whole population. This could be further subdivided into 1.98/10 0,0 0 0 in the paediatric ( < 18 years), and 3.06/10 0,0 0 0 in the adult population. Prevalence was highest in the northern and central parts of Norway (counties 1-4, Fig. 1 ). The individual county prevalences ranged from 0.63/10 0,0 0 0 (CI: 0.24, 1.61) in the southwest to 8.32/10 0,0 0 0 (CI: 5.39, 12.85) in Nordland county in the north (county 5 and 2, Fig. 1 ). There was also regional clustering of LGMDR9 subjects within counties (not shown). Of the total 153 LGMDR9 subjects, 134 (87.6 %) were FKRP c.826C > A homozygotes (52 % females), 16 (10.4 %) carried the c.826C > A on one allele, while three subjects (2.0 %) did not have the c.826C > A variant. The total carrier frequency was calculated to 1/94 individuals, and the c.826C > A carrier frequency to 1/101. Two novel FKRP variants were identiﬁed: c.141_151del11 p.(Arg48Profs ∗9) and c.166T > A p.(Phe56Ile) ( Table 1 ). These have not been reported in the literature and are not present in the 3 ARTICLE IN PRESS [m5G; December 13, 2022;4:40 ] JID: NMD S.M. Jensen, K.I. Müller, S.I. Mellgren et al. 3.2.1. Participants CK values at diagnostic work-up were reported in 76/88 c.826C > A homozygotes and 12/13 subjects in the non -c.826C > A homozygous group. The median peak CK value was 6,0 0 0 U/L and 9,0 0 0 U/L, respectively, and the range between 206 and 42,0 0 0 U/L. One c.826C > A homozygote participant in her early 40s with ﬁfteen years of slowly progressive lower limb weakness did not have elevated CK values. In 18/100 participants, investigation for a muscle disorder was prompted by the ﬁnding of an elevated CK or transaminases detected during work-up for non-muscular complaints, most commonly abdominal pain (10/18). In 15 of these, signs or symptoms of muscle disease were present at initial examination. In four males (three c.826C > A homozygotes), diagnostic investigations occurred secondary to cardiac failure at the age of 20-35 years. Three of them had concomitant hyperCKemia (CK 3,0 0 0-10,0 0 0 U/L) (missing data on the fourth) and three had preceding muscle symptoms. p Of the 153 LGMDR9 individuals identiﬁed, 101 (66.0 %) participated in the observational study. Patient notes and questionnaires were obtained from all participants, and 43/101 (42.6 %) were also examined clinically. Of 101 participants, 88 were c.826C > A homozygotes. Thirteen participants harboured other genotypes and included 12 c.826C > A compound heterozygotes and one who was homozygous for c.160C > T ( Table A 1). The average age of c.826C > A homozygous participants was 43 ± 18 years compared to 31 ± 17 years (p = 0.037) for those with other genotypes. Symptom duration was 30 ± 19 and 23 ± 14 years, respectively (p = 0.22). It is likely that there is an overlap of participants between the current study and the two previous Norwegian studies [ 15 , 30 ], and the global registry [22] . 3.2. Clinical data 3.2. Clinical data 3.1. Prevalence and molecular data Neuromuscular Disorders xxx (xxxx) xxx Table 1 FKRP Variants (NM_024301.5) identiﬁed in the Norwegian population of LGMDR9 Table 1 FKRP Variants (NM_024301.5) identiﬁed in the Norwegian population of LGMDR9 N subjects (N fam) Variant on FKRP allele one Effect on FKRP protein FKRP variant in allele two Effect on FKRP protein 2 (1) c.160C > T p.(Arg54Trp) c.160C > T p.(Arg54Trp) 134 (107) c.826C > A p.(Leu276Ile) c.826C > A p.(Leu276Ile) 1 (1) c.826C > A p.(Leu276Ile) c.141_151del11 † p.(Arg48Profs ∗9) 1 (1) c.826C > A p.(Leu276Ile) c.166T > A † p.(Phe56Ile) 1 (1) c.826C > A p.(Leu276Ile) c.328C > T p.(Arg110Trp) 1 (1) c.826C > A p.(Leu276Ile) c.469G > C p.(Ala157Pro) 3 (3) c.826C > A p.(Leu276Ile) c.899T > C p.(Val300Ala) 1 (1) c.170_189del20 ‡ p.(Glu57Alafs ∗68) c.899T > C p.(Val300Ala) 7 (5) c.826C > A p.(Leu276Ile) c.962C > A p.(Ala321Glu) 2 (2) c.826C > A p.(Leu276Ile) c.1323T > G p.(Phe441Leu) † Novel variants ‡ Not registered in genetic database, but reported in a previous Norwegian study (Stensland et al, 2011) [15] . †Novel variants ‡ Not registered in genetic database, but reported in a previous Norwegian study (Stensland et al, 2011) [15] . infection. It was not reported whether there was full recovery or not. 3.2.2. Clinical characteristics She described cramps in the tongue and throat from the age of 10 years as well as a tendency for food to get stuck in her throat and having a slurred speech. Respiratory diﬃculties were, however, not reported. The second subject was a 51-year-old female with obstructive sleep apnea (OSA) who reported painful cramps in tongue or throat when The most common initial symptoms were lower limb weakness and exertional intolerance including myalgia, stiffness or cramps ( Table 3 ). Three subjects had a relatively sudden onset of symptoms. One c.826C > A homozygote who developed hip pain and lower limb weakness (CK unknown) at the age of 10 was initially considered to have a viral arthritis although no infection was conﬁrmed. Moreover, the gait diﬃculties persisted. Seventeen years later, she developed ankle arthritis and was diagnosed with sarcoidosis. The diagnosis of LGMDR9 was made some years later. Muscle biopsy was compatible with LGMDR9 including lack of α- dystroglycan expression. Another c.826C > A homozygote developed head drop and lower limb weakness aged 20 months following a fever. CK was 2,300 U/L. No infection was found and the lower limb weakness persisted. Similarly, a c.826C > A compound heterozygote ( Table A 1, Subject 10) had at the age of 3 years fever- related myoglobinuria and 4 days later hypotonia involving neck, truncal and lower limb muscles, a CK 18,0 0 0 U/L and no signs of 4 ARTICLE IN PRESS [m5G; December 13, 2022;4:40 ] Neuromuscular Disorders xxx (xxxx) xxx S.M. Jensen, K.I. Müller, S.I. Mellgren et al. S.M. Jensen, K.I. Müller, S.I. Mellgren et al. Table 2 Table 2 Demographic and natural history data of the LGMDR9 cohort (N = 101) 2 graphic and natural history data of the LGMDR9 cohort (N = 101) IQR = interquartile range, N.R = not reported, N.A.: not assessed, W/C = wheelchair dependency, NIV = non-invasive ventilatory support, CM = assumed LGMDR9-related cardiomyopathy, LVEF = left ventricular ejection fraction, ICD = Implantable Cardiac Deﬁbrillator, CRT-D = Cardiac Resynchronization Therapy-Device a Delayed relaxation (no comorbidity), mixed hypertrophy and dilatation (concomitant hypertension) b Electrical disturbances: Sinus bradycardia (2 males), atrial ﬂimmer/ﬂutter (1 female, 2 males), frequent ventricular extrasystoles (1 female, 1 male), ventricular tachycardia (1 male), ventricular dyssynchrony/ left bundle branch block (3 males), right bundle branch block (1 male), supraventricular extrasystoles/palpitations (3 females) c Cardiac involvement with cardiac medication: Angiotensin-Converting Enzyme Inhibitor (ACEI), α2-blocker, β-blocker, amiodarone, spironolactone, warfarin yawning. The cramps caused diﬃculties with swallowing and could last for hours or days. She also had troublesome cramps in the extremities triggered by minor physical exertion, sudden movement or trauma. It is unknown whether the cramps have been investigated further. c.826C > A homozygotes with cardiomyopathy, and palpitations or supraventricular extrasystoli in three c.826C > A homozygous females without cardiomyopathy ( Table 2 ). The earliest age at which cardiomyopathy was detected was in a 13-year-old c.826C > A homozygous boy with fractional shortening. At 17 years of age he developed a dilated cardiomyopathy with LVEF 50-55 % and MRI showing patchy ﬁbrosis. Cardioactive medication was then initiated. One c.826C > A homozygous male, with assumed LGMDR9-related dilated cardiomyopathy and cardiac implant (CRT-D), had concomitant pulmonary sarcoidosis, which could potentially have been the underlying cause. MRI showed ﬁbrosis subepicardial in the lateral wall and septum that was considered atypical for sarcoidosis. Another c.826C > A homozygous male with exertional stiffness in the thighs from the age of 12 years had, at the age of 14, increasing exertional stiffness, dyspnea and nausea and was diagnosed with rhabdomyolysis (CK > 20,0 0 0 U/L) and dilated cardiomyopathy. There was intramural contrast enhancement on MRI. Coronary CT scan and virus tests were initially negative. MRI one year later showed similar ﬁndings and virus testing was positive for respiratory syncytial virus, which c.826C > A homozygotes with cardiomyopathy, and palpitations or supraventricular extrasystoli in three c.826C > A homozygous females without cardiomyopathy ( Table 2 ). 3.2.2. Clinical characteristics Neuromuscular Disorders xxx (xxxx) xxx Table 2 Demographic and natural history data of the LGMDR9 cohort (N = 101) FKRP genotype c.826C > A / c.826C > A Other Gender Total Females Males Total Females Males N (%) 88/88 (100) 43/88 (48.9) 45/88 (51.1) 13/13 (100) 8/13 (61.5) 5/13 (38.5) Age (years) Mean ± SD [range] 43 ± 18 [8–78] 41 ± 19 44 ± 18 31 ± 17 [9–66] 23 ± 11 44 ± 19 Onset age (years) Median (IQR) [range] 8.5 (5-19) [0–45] 8 (6-20) 9 (5-20) 3 (1-12) [0–43] 2 (0-10) 7 (1.5-28) W/C n (%) 25/88 (28.4) 18/43 (41.9) 7/45 (15.6) 10/13 (76.9) 7/8 (87.5) 3/5 (60.0) Age at W/C (years) Median (IQR) [range] 36.5 (30-50) [15–60] 35.5 (31-49) 39 (22-50) 13 (9-24) [3–60] 11 (9-12) 11 (-) Years onset to W/C Median (IQR) [range] 29 (15-36) [5–49] 27.5 (17-36) 31 (13-42) 11 (8-13) [2–27] 14 (9-23) 12 (-) NIV initiated n (%) 27/88 (30.7) 19/43 (44.2) 8/45 (17.8) 5/13 (38.5) 2/8 (25.0) 3/5 (60.0) Discontinued 4/88 (4.5) 0/13 (0) Nocturnal 20/88 (22.7) 1/13 (7.7) Intermittently 3/88 (3.4) 2/13 (15.4) Continuously 0/88 (0) 2/13 (15.4) Age at NIV (years) Median (IQR) [range] 45.5 (39-56) [19–71] (N.R.: 1) 46 (43-56) (N.R.: 1) 38.5 (31-62) 27 (16-47) [8–63] 17.5 31 (-) Years W/C to NIV Median (IQR) [range] 10.0 (3-16) [-15-38] (n = 18) (N.R.: 1) 12 (6-17) (n = 13) (N.R.: 1) 0 (-9-12) (n = 5) 13.0 (4-18) [3-19] (n = 5) 9 (-) (n = 2) 16 (-) (n = 3) CM n (%) LVEF < 50 % Dilatation + borderline Other 26/84 (31.0) (N.A.: 4) 20/26 (76.9) (13 + 7)/26 (76.9) 2 a /26 (7.7) 7/41 (17.1) (N.A.: 2) 19/43 (44.2) (N.A.: 2) 5/13 (38.5) 5/5 (100) (1 + 0)/5 (20.0) 0/5 (0) 3/8 (37.5) 2/5 (40.0) Age at CM (years) Median (IQR) [range] 36.5 (26-47) [13–70] 39 (28-47) 35 (26-47) 24 (19-30) [18–31] 28 (-) 19 (-) Arrhythmia n (%) 12 b /88 (13.6) 5/43 (11.6) 7/45 (15.6) 0/13 (0) 0/8 (0) 0/5 (0) Cardiac medication n (%) ACEI/ α2-blocker + 0 ACEI/ α2-blocker + 1 ACEI/ α2-blocker + ≥2 19/87 (21.8) N.R.: 1 13/19 (68.4) 3/19 (15.8) 3 /19 (15.8) 4/43 (9.3) 15/44 (34.1) N.R.: 1 4/13 (30.8) 3/4 (75.0) 1/4 (25.0) 0/4 (0) 2/8 (25.0) 2/5 (40.0) Cardiac electrical implant n; at age (years) 3 0 ICD: 1; 38 CRT-D: 2; 43, 50 0 0 0 Cardiac transplant n; at age (years) 1 0 1; 57 0 0 0 IQR = interquartile range, N.R = not reported, N.A.: not assessed, W/C = wheelchair dependency, NIV = non-invasive ventilatory support, CM = assumed LGMDR9-related cardiomyopathy, LVEF = left ventricular ejection fraction, ICD = Implantable Cardiac Deﬁbrillator, CRT-D = Cardiac Resynchronization Therapy-Device a Delayed relaxation (no comorbidity), mixed hypertrophy and dilatation (concomitant hypertension) b Electrical disturbances: Sinus bradycardia (2 males), atrial ﬂimmer/ﬂutter (1 female, 2 males), frequent ventricular extrasystoles (1 female, 1 male), ventricular tachycardia (1 male), ventricular dyssynchrony/ left bundle branch block (3 males), right bundle branch block (1 male), supraventricular extrasystoles/palpitations (3 females) c Cardiac involvement with cardiac medication: Angiotensin-Converting Enzyme Inhibitor (ACEI), α2-blocker, β-blocker, amiodarone, spironolactone, warfarin Table 2 = not described a Pseudohypertrophy, other: thighs (quadriceps n = 10, unspeciﬁed n = 3), forearms (n = 3), trapezius (n = 2), gluteus (n = 1) b Contractures, other: elbows (n = 4), knees (n = 3), hips (n = 3), ﬁngers (n = 1) described a Pseudohypertrophy, other: thighs (quadriceps n = 10, unspeciﬁed n = 3), forearms (n = 3), trapezius (n = 2), gluteus (n = 1) b Contractures, other: elbows (n = 4), knees (n = 3), hips (n = 3), ﬁngers (n = 1) described a Pseudohypertrophy, other: thighs (quadriceps n = 10, unspeciﬁed n = 3), forearms (n = 3), trapezius (n = 2), gluteus (n = 1) b Contractures, other: elbows (n = 4), knees (n = 3), hips (n = 3), ﬁngers (n = 1) Survival analysis demonstrated lower cumulative probability of wheelchair dependency in males (HR 0.22 (CI: 0.09, 0.54), p < 0.001). Females reached a 50 % probability of wheelchair dependency by 49 years (CI: 37, 61). The male survival curve did not cross the 50 % level ( Fig. 3 A). Regression analysis showed that MCS correlated with female gender and disease duration ( Table B1 ) while age of onset showed no signiﬁcant correlation (p = 0.23). Wheelchair dependency was equally correlated with female gender, and disease duration was borderline signiﬁcant ( Table B2 ). Age of onset showed no signiﬁcant correlation (p = 0.25). MCT conﬁrmed the gender differences. is usually benign in this age group. He remained asymptomatic. Two years later, echocardiography showed normal ﬁndings despite taking no cardioactive medication. One c.826C > A homozygous male had dilated cardiomyopathy which was assumed to be post ischemic and was not included. Several participants had cardiovascular disease or risk factors. Myocardial infarction was reported in four males (8.0 %) and detected subclinically on scintigraphy in one female (2.0 %). One additional male and female also had coronary artery disease. Obesity was reported in ﬁve males (10.0 %) and 11 females (21.6 %). Hypertension was reported in 12 males (24.0 %) and four females (7.8 %), elevated cholesterol in four males (8.0 %) and two females (3.9 %) and diabetes mellitus in three males (6.0 %) and one female (2.0 %). Three males (6.0 %) and three females (5.9 %) were habitual smokers, and 12 males (24.0 %) and eight females (15.7 %) were former smokers. Table 2 Table 3 Clinical features of 101 LGMDR9 participants FKRP genotype c.826C > A / c.826C > A (n = 88) Other (n = 13) Onset symptom(s) n (%) Lower limb weakness 51/88 (58.0) 4/13 (30.8) Exertional pain, stiffness or cramps 33/88 (37.5) 2/13 (15.4) Exertional fatigue 9/88 (10.2) 2/13 (15.4) Exertion-induced myoglobinuria 8/88 (9.1) 0/13 (0) Upper limb weakness 5/88 (5.7) 1/13 (7.7) Delayed motor development 4/88 (4.5) 2/13 (15.4) Toe walking 2/88 (2.3) 1/13 (7.7) General fatigue 2/88 (2.3) 1/13 (7.7) Symptomatic cardiac failure 1/88 (1.1) 0/13 (0) Myoglobinuria n (%) 28/88 (31.8) N.D.: 10 3/13 (23.1) N.D.: 3 Hypertrophy Calves 53/88 (60.2) N.D.: 18 10/13 (76.9) N.D.: 2 Other a 16/88 (18.2) N.D.: 40 1/13 (7.7) N.D.: 8 Scapular winging n (%) 41/88 (46.6) N.D.: 8 9/13 (69.2) N.D.: 4 Contractures n (%) Tendo-achilles 32/88 (36.4) N.D.: 22 Tenotomy: n = 0 8/13 (61.5) N.D.: 1 Tenotomy: n = 1 Other b 4/88 (4.5) N.D.: 37 4/13 (30.8) N.D.: 3 Multiple lower limb 3/88 (3.4) 1/13 (7.7) Multiple lower + upper limbs 1/88 (1.1) 3/13 (23.1) Scoliosis n (%) 16/88 (18.2) 7/13 (53.8) Oropharyngeal symptoms n (%) 15/88 (17.0) Dysphagia 9/88 (10.2) Dysarthria 9/88 (10.2) Tongue/ throat cramps 2/88 (2.2) 5/13 (38.5) Dysphagia 5/13 (38.5) Dysarthria 1/13 (7.7) Jaw fatigue 1/13 (7.7) Chewing diﬃculties 1/13 (7.7) Macroglossia n (%) 9/88 (10.2) N.D.: 48 2/13 (15.4) N.D.: 9 Facial weakness n (%) 6/88 (6.8) N.D.: 19 2 /13 (15.4) N.D.: 1 The numbers in brackets indicate the percentage of participants with positive ﬁndings within each genotype subgroup. N.D. Table 2 The earliest age at which cardiomyopathy was detected was in a 13-year-old c.826C > A homozygous boy with fractional shortening. At 17 years of age he developed a dilated cardiomyopathy with LVEF 50-55 % and MRI showing patchy ﬁbrosis. Cardioactive medication was then initiated. One c.826C > A homozygous male, with assumed LGMDR9-related dilated cardiomyopathy and cardiac implant (CRT-D), had concomitant pulmonary sarcoidosis, which could potentially have been the underlying cause. MRI showed ﬁbrosis subepicardial in the lateral wall and septum that was considered atypical for sarcoidosis. Another c.826C > A homozygous male with exertional stiffness in the thighs from the age of 12 years had, at the age of 14, increasing exertional stiffness, dyspnea and nausea and was diagnosed with rhabdomyolysis (CK > 20,0 0 0 U/L) and dilated cardiomyopathy. There was intramural contrast enhancement on MRI. Coronary CT scan and virus tests were initially negative. MRI one year later showed similar ﬁndings and virus testing was positive for respiratory syncytial virus, which Wheelchair dependency was present in 35/101 participants, and non-invasive ventilatory support initiated in 32/101. The indication for ventilatory support was extrapulmonary restrictiveness in 15/32, OSA in 7/32, and both combined in 10/32. In two c.826C > A homozygotes age 52 and 68 years, ventilatory support was instituted after acute respiratory failure with hypercapnia. Both were wheelchair dependent. In 18/101 participants, pulmonary investigations were not performed, and it was uncertain in another three. None of these 21 subjects reported dyspnea. Cardiomyopathy with no other identiﬁed cause was recognized in 31/97 (32.0 %) participants. In 10 subjects, abnormalities were noticed on the ﬁrst echocardiography. Four c.826C > A homozygous males had a severe cardiomyopathy: three with an implanted electronic device, and one who required cardiac transplantation. Conduction abnormalities or arrhythmias were reported in nine 5 ARTICLE IN PRESS [m5G; December 13, 2022;4:40 ] JID: NMD Neuromuscular Disorders xxx (xxxx) xxx S.M. Jensen, K.I. Müller, S.I. Mellgren et al. Table 2 Accordingly, we observed that the four most severely affected cardiac patients were males 6 ARTICLE IN PRESS S.M. Jensen, K.I. Müller, S.I. Mellgren et al. Neuromuscular Disorders xxx (xxxx) xxx ARTICLE IN PRESS JID: NMD [m5G; December 13, 2022;4:40 ] Fig. 2. Histogram demonstrating the distribution of age at onset in the FKRP c.826C > A homozygous participants (n = 88). Subjects are divided in an early and late onset group according to a two-step cluster analysis. [m5G; December 13, 2022;4:40 ] S.M. Jensen, K.I. Müller, S.I. Mellgren et al. Neuromuscular Disorders xxx (xxxx) xxx Fig. 2. Histogram demonstrating the distribution of age at onset in the FKRP c.826C > A homozygous participants (n = 88). Subjects are divided in an early and late onset di l l i Neuromuscular Disorders xxx (xxxx) xxx Fig. 2. Histogram demonstrating the distribution of age at onset in the FKRP c.826C > A homozygous participants (n = 88). Sub group according to a two-step cluster analysis. Fig. 3. Kaplan-Meier curves showing cumulative probability with age of (A) being ambulant, (B) not having initiated ventilatory support, and (C) not having cardiomyopathy, respectively, in FKRP c.826C > A homozygous males (n = 45) versus females (n = 43). Fig. 3. Kaplan-Meier curves showing cumulative probability with age of (A) being ambulant, (B) not having initiated ventilatory support, and (C) not having cardiomyopathy, respectively, in FKRP c.826C > A homozygous males (n = 45) versus females (n = 43). (one cardiac transplanted and three with an implanted electronic device) and that three of them had relatively preserved ambulatory function with MCS 2 (cardiac transplanted), 2 and 1 (comorbidity: pulmonary sarcoidosis), respectively. Nevertheless, MCT did not conﬁrm the gender differences. to work-up was 6 years (IQR 2-13) excluding two subjects with incidental ﬁnding of elevated CK or transaminases and two who had a previously diagnosed sibling. Further, they demonstrated an increased cumulative probability of wheelchair dependency (HR 9.67 (CI: 4.19, 22.39), p < 0.001) and of requiring ventilatory support (HR 5.39 (CI: 1.84, 15.77), p = 0.002), but no signiﬁcant increase in respect to developing cardiomyopathy (HR 2.13 (CI: 0.81, 5.64), p = 0.13). The 50 % probability of wheelchair dependency was reached by the age of 20 (CI: 7, 33) and of cardiomyopathy development by 31 years (CI: 24, 38). Table 2 Median age of initiation of ventilatory support (46 years) was nearly a decade later than the median age of wheelchair dependency ( Fig. 4 ). Males showed lower accumulated probability of requiring ventilatory support (HR 0.26 (CI: 0.11, 0.65), p = 0.002). MCT conﬁrmed the gender difference. Females reached a 50 % probability of needing ventilatory support by the age of 52 years (CI: 44, 60), whereas males, in the age range of our cohort, did not reach the 50 % level ( Fig. 3 B). Ventilatory support was initiated in 10/26 cases in an ambulatory stage and 7/10 due to OSA. Multivariate regression analysis showed a positive correlation with age (p = 0.0011) and MCS (p = 0.0 0 03) but not with gender ( Table B3 ). Four more subjects had cardiac MRI. The MRI ﬁndings were consistent with the echocardiography ﬁndings showing dilatation and/ or reduced contractility. Additionally, one subject showed ﬁbrosis in the lateral wall, whereas in another subject apical ﬁbrosis was reported. 3.2.3. Natural history of the FKRP c.826C > A homozygous cohort The natural history data are summarized in Table 2 . In c.826C > A homozygotes, median age of symptom onset was 8.5 years. Interestingly, a two-step cluster analysis suggested a bimodal distribution with a predominant (75 %) early-onset subgroup (mean 7 years, range 0-17) and a late-onset subgroup (mean 29 years, range 20-45) ( Fig. 2 ) irrespective of gender (p = 0.52) (Fig. B.1). Diagnostic work-up started at a median age of 21 years (IQR 13-32). Median time from symptom onset to work-up was 10.5 years (IQR 4-20) excluding 16 subjects who had an incidental ﬁnding of elevated CK or transaminases and 14 with a previously diagnosed sibling. Age of cardiomyopathy detection was similar to that for becoming wheelchair dependent ( Fig. 4 ). In contrast to wheelchair dependency and ventilatory support, males had a higher cumulative probability of cardiomyopathy development (HR 2.71 (CI: 1.14, 6.46), p = 0.019). The predicted 50 % probability of cardiomyopathy development occurred at age 55 (CI: 44, 66) in males while the female survival curve did not cross the 50 % level ( Fig. 3 C). In the regression analysis, gender was the only signiﬁcant predictor variable for cardiomyopathy. Both age and MCS were non-signiﬁcant ( Table B4 ). Table 2 We could not predict a 50 % cumulative probability of ventilatory support by survival analysis ( Fig. A2 ), but ventilatory support was preceded by 3.2.4. Natural history of the non-c.826C > A homozygous cohort The natural history data are summarized in Table 2 . The non -c.826C > A homozygous group showed earlier symptom onset (median 3 years, p = 0.034) and faster progression than the c.826C > A homozygotes ( Fig. 4 ). Median age at diagnostic work- up was 7 years (IQR 3-20) (p = 0.005). Median time from onset 7 ARTICLE IN PRESS S.M. Jensen, K.I. Müller, S.I. Mellgren et al. Neuromuscular Disorders xxx (xxxx) xxx ARTICLE IN PRESS JID: NMD [m5G; December 13, 2022;4:40 ] Fig. 4. Box plot showing age at symptom onset, wheelchair dependency, cardiomyopathy and initiation of non-invasive ventilatory support (NIV) in subjects with FKRP c.826C > A homozygous genotype (n = 88) versus other FKRP genotypes (n = 13). [m5G; December 13, 2022;4:40 ] JID: NMD Fig. 4. Box plot showing age at symptom onset, wheelchair dependency, cardiomyopathy and initiation of non-invasive ventilatory support (NIV) in subjects with FKRP c.826C > A homozygous genotype (n = 88) versus other FKRP genotypes (n = 13). wheelchair dependency in ﬁve of ﬁve cases with a median interval of 13 years. MCT did not conﬁrm gender difference in age of onset but did so in cumulative probability of wheelchair dependency and ventilatory needs. north (county 2, Fig. 1 ). Interestingly, a recent study on the genetic distances between Norwegian counties, performed by hierarchical clustering of pair-wise F ST distances, demonstrates that counties 2, 3 and 4 ( Fig. 1 ) group together [32] . As the age and origin of the c.826C > A variant remains unclear, it is diﬃcult to explain the distribution of subjects. A north-south gradient in Europe may be explained by previous waves of European migration northward and eventually into the Scandinavian Peninsula. Small, isolated settlements with little intermixing could then explain why the variant shows a skewed accumulation in Norway. Genetic studies have demonstrated major genetic inﬂow to Norway from Central and Western European populations, especially the Germanic population [33] , but also geographic sub-structuring believed to be partially caused by geographic isolation by mountains and the sea [ 32 , 34 ]. The geographical distribution of subjects within counties appears as scattered clusters (not shown). 4.1. Prevalence and molecular data With a minimum prevalence of 2.84/10 0,0 0 0 (adult: 3.06/10 0,0 0 0, paediatric: 1.98/10 0,0 0 0), Norway has the highest reported prevalence of LGMDR9 worldwide. Moreover, we assume that the paediatric prevalence is underestimated due to the delay from symptom onset to diagnostic work-up caused by mild and nonspeciﬁc symptoms at onset. The high LGMDR9 prevalence in Norway reﬂects the high carrier frequency of the FKRP c.826C > A allele in the population, which we calculate to 1/101 (total carrier frequency 1/94). This is only slightly higher than the carrier frequency in the Swedish population (1/109) [11] , but more than twice the frequency in the non-Finnish, north-western European population (1/218) and 4-5 times the frequency in the Finnish population (1/469) [11] . There are also variations within Norway: the prevalence is relatively high in Northern-Norway and low in the southwest in accordance with previous regional epidemiological studies [ 16 , 31 ]. Additionally, we ﬁnd a relatively high prevalence in the central region (counties 3 and 4, Fig. 1 ), and the peak prevalence of 8.32/10 0,0 0 0 in Nordland county in the 3.2.5. Siblings In order to investigate to which extent clinical heterogeneity was present within families, siblings were compared. Among 12 c.826C > A homozygous sib-ships, median inter-sibling difference in age of onset was 5 years (range 0-10). MCS was similar in most siblings, but highly divergent in two sib-ships (male 36/female 40, male 69/female 71 years old, family number 6 and 7, Table C 1). The occurrence of cardiomyopathy appeared randomly since only one sibling was affected in seven of eight cases ( Table C 1). In the non -c.826C > A homozygous cohort, one sib-ship comprising a male and two females with the c.826C > A p.(Leu276Ile) / c.962C > A p.(Ala321Glu) genotype, the male exhibited a more severe course regarding both motor, respiratory and cardiac involvement ( Table A , Subject 7, 8 and 9). Table 2 These clusters likely emerged from migration, followed by historical isolation and subsequent genetic drift. The fact that also the Norwegian c.826C > A homozygotes are homozygous for the FKRP c.135C > A allele, previously shown to be in complete linkage disequilibrium with c.826C > A in other populations [ 9 , 10 ], supports the theory that c.826C > A is a founder variant. ARTICLE IN PRESS A register study did report earlier loss of running ability in females, and the tabulated data show a tendency towards higher wheelchair use and need for ventilatory support in females than males [22] . Interestingly, this is opposite to the tendency reported for other LGMDs: in Anoctamin 5-related LGMD, females appear less frequently affected [ 44 , 46 ] and in both Calpain- and Telethonin-related LGMD, males have more severe muscular impairment [ 41 , 54 ]. Likewise, a morphometric study showed more muscle ﬁber atrophy in males with Calpain- or Dysferlin-related LGMD, and suggested explanations were endocrinological and differing initial muscle mass [55] . Muscle MRI in LGMDR9 patients has shown some gender-speciﬁc patterns of involvement [56] and an MRI study of dysferlinopathy, showed that females had more severe involvement of several muscles of the lower limb [57] . These studies offer potential support for our ﬁndings. Gender was also analysed in two previous studies looking at cardiac involvement in LGMDR9, albeit in genotypically heterogenous cohorts. In one study an increased tendency for cardiomyopathy was found in males [19] , while in another study no association with gender was identiﬁed [17] . In the patients requiring ventilatory support, OSA was frequently diagnosed (53.1 %), although most often associated with a restrictive ventilatory defect. Whether OSA in these subjects is due to LGMDR9-related upper airway involvment, remains to be elucidated. In muscular dystrophies, the mechanisms of OSA are various, and OSA can be confounded by diaphragmatic events [50] . Furthermore, OSA is multifactorial, commonly related to high weight and increasing age, and prevalent in the general population [51] . As reduced mobility predisposes to weight gain, and several participants did have obesity, weight could be a signiﬁcant factor. In a cohort of patients with dysferlinopathy, OSA was reported in some patients and considered likely related to age and BMI [43] , which highlights the relevance of cofactors in the natural history. Gender-speciﬁc differences may also have explanations unrelated to the underlying muscle disease. Different self- reporting behavior, recognition and comorbidity can play a role. For example, levels of ambulation were mostly based on self-reported data. Recognition of ventilatory needs depends on the symptoms the patients report and their motivation to undergo a sleep study. Additionally, BMI could be a signiﬁcant factor in the need for ventilatory support, and our data did indicate that obesity was more frequent in female participants. ARTICLE IN PRESS ARTICLE IN PRESS [m5G; December 13, 2022;4:40 ] JID: NMD Neuromuscular Disorders xxx (xxxx) xxx S.M. Jensen, K.I. Müller, S.I. Mellgren et al. Exertional intolerance mimicking metabolic muscle disease was common in our LGMDR9 cohort, and similar to that described in a German LGMDR9 cohort [9] . This is diagnostically relevant, as is the high proportion (18.0 %) in whom the muscle disease was discovered by ﬁnding elevated CK or transaminases. That most of these patients also had unrecognized symptoms or signs of muscle disease highlights the need for CK analysis and appropriate clinical examination in patients with elevated transaminases to avoid misinterpretation as liver pathology. This was also addressed in a previous Norwegian study [30] . Pseudometabolic presentation and asymptomatic hyperCKemia are also frequent in anoctaminopathy [ 46 , 48 ]. The signiﬁcant latency from symptom onset to diagnostic work-up for a muscle disorder, especially in c.826C > A homozygotes (median 10.5 years), may reﬂect a slow disease progression. Delayed diagnoses was also reported recently in an American LGMDR9 cohort [18] . Similar to previous natural history studies, oropharyngeal symptoms were reported in our cohort: these included two cases with painful tongue cramps [35] , one case with dysphagia that resolved after the initiation of ventilatory support [23] , one with diﬃculties masticating food [49] , and four with reduced tongue strength [20] . We found that dysphagia and dysarthria tended to develop late in the disease course. between muscular involvement and respiratory function have been reported , [ 19 , 24 ]. As in previous studies, our data showed no correlation between cardiomyopathy development and loss of ambulation [ 19 , 24 , 35 , 36 ], nor with disease duration or age. We did, however, ﬁnd a positive correlation with male gender, although signiﬁcance was not conﬁrmed when we corrected for multiple subgroup analyses. Moreover, the most serious cases of cardiomyopathy in our study occurred in c.826C > A homozygous males. By contrast, in sarcoglycanopathy, the presence of cardiac involvement was found to be related to symptom duration [39] . Clinical gender dissimilarities in LGMDR9 were an unexpected ﬁnding. These differences cannot be explained by unequal gender representation, since the numbers of males and females diagnosed with LGMDR9 and their participation rates in this study were similar. ARTICLE IN PRESS The increased risk of cardiomyopathy in males may also be related to comorbidity and excluding other etiologies such as cardiovascular disease (CVD), alcohol overconsumption or viral myocarditis, is a challenge. Males, in general, have higher incidence of CVD, partly explained by the sex hormones [58] , but CVD may be under-recognized in females [59] . Accordingly, our comorbidity data showed a tendency of more CVD in males. Lastly, true LGMDR9-related gender differences can still be inﬂuenced by sex hormones and life style. Although it has been suggested that estrogen has a protective role of the skeletal muscle membrane [60] , there may be other relevant effects of sex hormones in LGMDR9. The fact that the skeletal and cardiac muscle involvement showed opposite relation to gender may indicate that these tissues are inﬂuenced by different factors. One could also hypothesize a cause-effect link where preserved ambulation leads to more physical exposure, which in turn puts strain on the cardiac muscle. Nutrition including deﬁciencies, metabolic health and exposure to toxins may also play a role in the disease progression. We analysed the disease course in c.826C > A homozygotes separately and found a median age of onset of 8.5 years, which is similar to a US cohort [18] . Further, we found a bimodal distribution with a second peak in the third decade of life raising the possibility of protective genetic or epigenetic factor(s) in the late-onset subpopulation. It should be remembered, however, that age of onset is subjective and deﬁned retrospectively. Deﬁning the age of onset can be diﬃcult in cases of gradual or non-speciﬁc symptoms such as poorer physical performance than peers. Also, time of recognition is likely inﬂuenced by the level of physical activity. We found that age of onset was not an independent predictor of wheelchair dependency, the need for ventilatory support or the risk of cardiomyopathy. Both age and symptom duration at onset of wheelchair dependency varied widely. Wheelchair dependency and need for ventilatory support correlated with female gender, and there was an inverse relationship between the ambulatory status and the need for ventilatory support. The gender difference in ventilatory needs was linked to the difference in ambulatory status. The need for ventilatory support usually occurred in the non-ambulatory stage analogous to other muscular dystrophies [ 52 , 53 ]. In sarcoglycanopathy, the need for ventilatory support is related to long disease duration and scoliosis [39] . 4.2. Clinical data Similar to previous studies, we found that a high proportion of our cohort had calf pseudohypertrophy [ 9 , 20 , 23 ], tendo- achilles contractures [ 20 , 30 , 35 ], cardiomyopathy [ 17 , 19 , 36 ] and need for ventilatory support [ 19 , 20 , 22 ]. Scapular winging was more frequent than previously reported [ 20 , 24 , 37 ], although often mild. Overlapping clinical features can be seen with the most common recessive LGMDs including calpainopathy (LGMDR1), dysferlinopathy (LGMDR2), sarcoglycanopathies (LGMDR3-R6) and anoctaminopathy (LGMDR12) [38] . Perhaps the clearest overlap is with the sarcoglycanopathies [ 39 , 40 ], although these tend to show earlier loss of ambulation [39] . In contrast, dilated cardiomyopathy and ventilatory needs are uncommon in subtype R1 [ 41 , 42 ], R2 [43] and R12 [44–46] . Furthermore, anoctaminopathy and dysferlinopathy are associated with a later onset; median 35 years (common type) [44] and 19 years [47] , respectively. 8 Declaration of Competing Interest None None The validity of data from patient notes is limited by the non- standardized follow-up and reporting, and consequently possible inaccuracies and underestimation of outcomes. Furthermore, when concerning cardiomyopathy, normal limit of LVEF was set to 50 % to agree with the echocardiography reports. Both 50 and 55 % are used in other studies. Considering the trend towards increased use of more sensitive technology, such as MRI, and a more liberal LVEF-threshold for cardioprotective treatment initiation, our results are conservative. Conversely, some cases with cardiomyopathy considered to be LGMDR9-related may have another etiology and thereby overestimate the risk. The lack of speciﬁc markers of LGMDR9-related cardiomyopathy is thus a limitation. Data concerning ambulation relied mainly on self-reports, which opens to misinterpretation and bias. In addition, we acknowledge that implementation of a validated scale for self-reported ambulation would be preferable, and objective measures optimal. The gender differences need validation through clinical testing, and the cardiorespiratory involvement needs to be assessed with a standardized protocol to obtain more accurate and comparable data. This is a work in progress, however only on the subset of participants who also consented to clinical participation. Analyses of the non -c.826C > A homozygous group were limited by power due to small sample size and genotype heterogeneity, and included one subject with a likely pathogenic variant. Elimination of this participant did not change the conclusions of the subgroup comparisons. Appendices Fig. A1. Histogram demonstrating distribution of age at onset in the FKRP c.826C > A homozygous participants divided into genders. 4.3. Strengths and limitations 4.3. Strengths and limitations With 101 participants in the observational study, our sample size is the largest outside the global register. A participation rate of 66 % is high but may still limit the generalization of the results. Our cohort had equal gender representation, paediadric and adult subjects, and data from all the Norwegian counties. The substantial number of c.826C > A homozygotes enabled us to perform a genotype-speciﬁc natural history analysis. Patient notes provided long-term data and, combined with the questionnaire, strengthened the data completeness and quality. Neuromuscular examination of 42.6 % of the participants compensated in part for incomplete data in patient notes. Funding This work was supported by the Northern Norway Regional Health Authority, Norwegian National Advisory Unit on Rare Disorders, Neuro Muscular Disorders Association of Norway, and Mr. Roar Orekåsa (private donor). Acknowledgment We thank the participants, the cooperating hospitals, Jorun Willumsen and Cecile Aronsen for their contribution to the collection of patient notes, Geir Ringvold for help with the ﬁgure formatting, the Department of Clinical Research at UNN for technical and statistical support, and the Global FKRP Registry for support with recruitment. Several of the authors are members of ERN Euro-NMD through Oslo University Hospital. ARTICLE IN PRESS The link to disease duration was similar in the current study, but the relationship with scoliosis was not assessed. Clinically, scoliosis was infrequent and often mild and since X-ray was usually not performed, it may be underdiagnosed or underestimated. There are few studies of the impact on respiration in patients with LGMDR9, although correlation between disease severity and need for ventilatory support [35] , and lack of correlation In the genotype-phenotype analyses our data were skewed with the non- c.826C > A homozygous subjects being relatively underrepresented and genetically heterogeneous. As expected from previous studies, non -c.826C > A homozygous subjects demonstrated a more severe disease course, although mild 9 ARTICLE IN PRESS [m5G; December 13, 2022;4:40 ] JID: NMD S.M. Jensen, K.I. Müller, S.I. Mellgren et al. Neuromuscular Disorders xxx (xxxx) xxx phenotypes or late onset of disease did also occur. While the numbers in our study were small, the risk of developing cardiomyopathy was not signiﬁcantly increased in the non- c.826C > A homozygous cohort. Previous studies have suggested both a higher risk [ 17 , 19 ], and no difference in risks for developing cardiomyopathy [36] . the phenotypes associated with c.826C > A homozygosity. Our data showed that initial symptoms commonly occur in the ﬁrst decade of life, and there is a signiﬁcant latency from onset to diagnostic work-up. This may indicate the need for increased awareness of muscle disorders in children. Both lower limb weakness and exertional myalgia were common early symptoms. Further, our results indicate that respiratory follow-up is increasingly important particularly as patients become wheelchair dependent, but that sleep-disordered breathing occurs to an uncertain extent also in ambulatory patients. Our data also support the importance of regularly cardiac assessment. The study provides insight that should be relevant to future and ongoing LGMDR9 clinical trials. The bimodal distribution of onset, the lack of correlation between skeletal and cardiac muscle involvement, and the gender correlations, are relevant ﬁndings in the search for disease modifying factors. We compared the 13 sib-ships among participants to address the question of environmental inﬂuence and found variability in age of onset (up to ten years), progression and cardiac involvement. Inter-sibling variability was also documented previously [ 10 , 23 , 24 ] and suggest that other genetic factors and/ or environmental factors may play a role in disease development. Table A1 [4] Walter MC, Petersen JA, Stucka R, Fischer D, Schröder R, Vorgerd M, et al. FKRP (826C > A) frequently causes limb-girdle muscular dystrophy in German patients. J Med Genet. 2004:41:e50. https://doi.org/10.1136/jmg.2003.013953 . [5] Stensland E, Lindal S, Jonsrud C, Torbergsen T, Bindoff LA, Rasmussen M, et al. Prevalence, mutation spectrum and phenotypic variability in Norwegian patients with Limb Girdle Muscular Dystrophy 2I. Neuromuscul Disord. 2011;21:41-6. https://doi.org/10.1016/j.nmd.2010.08.008 . [6] Rasmussen M SD, Breivik N, Mork M, Lindal S. Clinical and muscle biopsy ﬁndings in Norwegian paediatric patients with limb girdle muscular dystrophy 2I. Acta Paediatr. 2014;103(5):553-8. https://doi.org/10.1111/apa.12561 . [7] Henriques SF, Gicquel E, Marsolier J, Richard I. Functional and cellular localization diversity associated with Fukutin-related protein patient genetic variants. Hum Mutat. 2019;40:1874-1885. https://doi.org/10.1002/humu.23827 . [8] von der Hagen M, Kaindl AM, Koehler K, Mitzscherling P, Häusler HJ, Stoltenburg-Didinger G, et al. Limb girdle muscular dystrophy type 2I caused by a novel missense p // g/ /j g [5] Stensland E, Lindal S, Jonsrud C, Torbergsen T, Bindoff LA, Rasmussen M, et al. Prevalence, mutation spectrum and phenotypic variability in Norwegian patients with Limb Girdle Muscular Dystrophy 2I. Neuromuscul Disord. 2011;21:41-6. https://doi.org/10.1016/j.nmd.2010.08.008 . [6] Rasmussen M SD Breivik N Mork M Lindal S Clinical and muscle biopsy ﬁndings in Norwegian paediatric patients with limb girdle muscular dystrophy 2I Acta Paediatr ( ) p // g/ / p [7] Henriques SF, Gicquel E, Marsolier J, Richard I. Functional and cellular localization diversity associated with Fukutin-related protein patient genetic variants. Hum Mutat. 2019;40:1874-1885. https://doi.org/10.1002/humu.23827 . 8] von der Hagen M, Kaindl AM, Koehler K, Mitzscherling P, Häusler HJ, Stoltenburg-Didinger G, et al. Limb girdle muscular dystrophy type 2I caused by a novel missense mutation in the FKRP gene presenting as acute virus-associated myositis in infancy. Eur J Pediatr. 2006;165:62-3. https://doi.org/10.1007/s00431- 005- 1752- 6 . † Siblings, ‡ Lacking cardiac follow-up, § Indication: fatigue with slight sleep apnea [8] von der Hagen M, Kaindl AM, Koehler K, Mitzscherling P, Häusler HJ, Stoltenburg-Didinger G, et al. Limb girdle muscular dystrophy type 2I caused by a novel missense mutation in the FKRP gene presenting as acute virus-associated myositis in infancy. Eur J Pediatr. 2006;165:62-3. https://doi.org/10.1007/s00431- 005- 1752- 6 . Table A1 † Siblings, ‡ Lacking cardiac follow-up, § Indication: fatigue with slight sleep apnea Table B1 Motor composite score (MCS) (n = 88) Unadjusted β (CI) Unadjusted p-value Adjusted β (CI) β relative change (%) Adjusted p-value AGE (years) 0.09 (0.06, 0.12) < 0.001 ∗ 0.03 (-0.02, 0.08) - 67 0.21 YEARS SYMPTOMATIC 0.10 (0.08, 0.13) < 0.001 ∗ 0.08 (0.03, 0.08) - 23 0.001 ∗ GENDER (M) -1.52 (-2.82, - 0.23) 0.022 ∗ -1.69 (-2.70, -0.69) - 11 0.001 ∗ R 2 = 0.67, Z-residuals < ± 3, Cook ´s distances << 1.0. MCS: ambulation score from 0 (normal) to 9 ( = lost ambulation) (Details: Sect. 3.4), ∗p < 0.05, β = regression coeﬃcient, CI = 95 % conﬁdence interval, M = male gender. Unadjusted effects and p-values are calculated by simple regression analyses. Adjusted values mean that it is controlled for the other variables in the table. R 0.67, Z residuals < ± 3, Cook s distances << 1.0. MCS: ambulation score from 0 (normal) to 9 ( = lost ambulation) (Details: Sect. 3.4), ∗p < 0.05, β = regression coeﬃcient, CI = 95 % conﬁdence interval, M = male gender. Unadjusted effects and p-values are calculated by simple regression analyses. Adjusted values mean that it is controlled for the other variables in the table. Table A1 Table A1 Genotype-phenotype table of non- FKRP c.826C > A homozygous participants No. FKRP genotype [references of earlier identiﬁed variants] Gender Age MCS Age at onset (years) Spine Contr Age at W/C (years) Age at CM (years) Age at NIV (years) PEG Non c.826C > A: 1. c.160C > T p.(Arg54Trp) homozygous [1] F 11 9 0 + - 9 - - - c.826C > A compound with: 2. c.166T > A p.(Phe56Ile) M 24 4 13 - - - 20 - - 3. c.141_151 del 11 p.(Arg48Profs ∗9) F 11 8 0 + - 10 - - - 4. c.328C > T p.(Arg110Trp) [2] M 29 0 7 - - - - - - 5. c.899T > C p.(Val300Ala) [3–7] M 65 9 43 + - 60 - 63 - 6. F 24 0 11 - - - ‡ - - 7. † c.962C > A p.(Ala321Glu) [ 7 , 8 ] F 29 9 12 + - 23 24 - - 8. † F 34 9 8 + - 20 - - - 9. † M 36 9 2 + + 7 18 23 + 10. F 34 9 3 + + 14 31 27 + 11. M 62 9 1 + + 12 - 31 + 12. c.1323T > G p.(Phe441Leu) [5] F 9 6 1 + - 3 - 8 § - 13. F 30 8 1 - - 28 28 - - Table A1 Genotype-phenotype table of non- FKRP c.826C > A homozygous participants [1] Harel T, Goldberg Y, Shalev SA, Chervinski I, Oﬁr R, Birk OS. Limb-girdle muscular dystrophy 2I: phenotypic variability within a large consanguineous Bedouin family associated with a novel FKRP mutation. Eur J Hum Genet. 2004;12:38-43. https://doi.org/10.1038/sj.ejhg.5201087 . [1] Harel T, Goldberg Y, Shalev SA, Chervinski I, Oﬁr R, Birk OS. Limb-girdle muscular dystrophy 2I: phenotypic variability within a large consanguineous Bedouin family associated with a novel FKRP mutation. Eur J Hum Genet. 2004;12:38-43. https://doi.org/10.1038/sj.ejhg.5201087 . [2] Kang PB, Feener CA, Estrella E, Thorne M, White AJ, Darras AJ, et al. LGMD2I in a North American population. BMC Musculoskelet Disord. 2007;8,115. https://doi.org/10. 1186/1471-2474-8-115 . [3] de Paula F, Vieira N, Starling A, Yamamoto LU, Lima B, de Cássia Pavanello R, et al. Asymptomatic carriers for homozygous novel mutations in the FKRP gene: the other end of the spectrum. Eur J Hum Genet. 2003;11:923-30. https://doi.org/10.1038/sj.ejhg.5201066 . 5. Conclusions The Norwegian LGMDR9 prevalence remains the highest reported worldwide and is strongly linked to the high prevalence of the c.826C > A founder variant. Our study extends understanding of the clinical features and natural history of LGMDR9, particularly Fig. A1. Histogram demonstrating distribution of age at onset in the FKRP c.826C > A homozygous participants divided into genders. 10 10 S.M. Jensen, K.I. Müller, S.I. Mellgren et al. Neuromuscular Disorders xxx (xxxx) xxx ARTICLE IN PRESS JID: NMD [m5G; December 13, 2022;4:40 ] ARTICLE IN PRESS [m5G; December 13, 2022;4:40 ] S.M. Jensen, K.I. Müller, S.I. Mellgren et al. Fig. A2. Kaplan-Meier curves showing cumulative probability with age of not being wheelchair dependent, initiated non-invasive ventilatory support or developed cardiomyopathy, respectively, in FKRP c.826C > A homozygotes versus the cohort of other FKRP genotypes Fig. A2. Kaplan-Meier curves showing cumulative probability with age of not being wheelchair dependent, initiated non-invasive ventilatory support or developed cardiomyopathy, respectively, in FKRP c.826C > A homozygotes versus the cohort of other FKRP genotypes Table B3 Table B3 Initiated ventilatory support (n = 27/88 = 31 %) Unadjusted OR (CI) Unadjusted p-value Adjusted OR (CI) OR relative change (%) Adjusted p-value AGE (years) 1.08 (1.04, 1.12) < 0.001 ∗ 1.11 (1.04, 1,20) + 3.2 0.0011 ∗ YEARS SYMPTOMATIC 1.06 (1.02, 1.09) < 0.001 ∗ (0.90, 1.01) - 9.6 0.11 GENDER (M/F) 0.31 (0.12, 0.82) 0.015 ∗ 0.41 (0.10, 1.64) + 33 0.21 MCS 1.68 (1.36, 2.08) < 0.001 ∗ 1.63 (1.21, 2.19) - 3.3 0.0003 ∗ Nagelkerke R 2 = 0.58. Hosmer & Lemeshow Test: p = 0.52. Z-residuals < ± 3. Cook ´s distances << 1.0. ∗p < 0.05, OR = Odds ratio, CI = 95 % conﬁdence interval, M = males, F = females, MCS: ambulation score from 0 (normal) to 9 ( = lost ambulation) (Details: Sect. 3.4). Unadjusted effects and p-values are calculated by simple regression analyses. Adjusted values mean that it is controlled for the other variables in the table. Nagelkerke R = 0.58. Hosmer & Lemeshow Test: p = 0.52. Z residuals < ± 3. Cook s distances << 1.0. ∗p < 0.05, OR = Odds ratio, CI = 95 % conﬁdence interval, M = males, F = females, MCS: ambulation score from 0 (normal) to 9 ( = lost ambulation) (Details: Sect. 3.4). Unadjusted effects and p-values are calculated by simple regression analyses. Adjusted values mean that it is controlled for the other variables in the table. Table B4 Cardiomyopathy (n = 26/84 (31 %)) Unadjusted OR (CI) Unadjusted p-value Adjusted OR (CI) OR relative change (%) Adjusted p-value AGE (years) 1.01 (0.98, 1.03) 0.58 0.97 (0.92, 1.02) -4.0 0.21 YEARS SYMPTOMATIC 1.02 (0.99, 1.04) 0.24 1.05 (0.99, 1.11) + 3.2 0.092 GENDER (M/F) 3.85 (1.40, 10.58) 0.006 ∗ 3.71 (1.25, 11.00) -3.5 0.014 ∗ MCS Q 0.69 0.75 Q2 0.96 (0.24, 3.93) 0.96 0.65 (0.14, 3.10) -32 0.59 Q3 1.66 (0.43, 6.38) 0.46 1.25 (0.26, 6.00) -25 0.78 Q4 0.80 (0.20, 3.22) 0.75 0.64 (0.10, 3.99) -20 0.64 Nagelkerke R 2 = 0.17. Hosmer & Lemeshow Test: p = 0.99. One Z-residual > ± 3 (3.1). Cook ´s distances << 1.0. ∗p < 0.05, OR = Odds ratio, CI = 95 % conﬁdence interval, M = males, F = females, MCS: ambulation score from 0 (normal) to 9 ( = lost ambulation) (Details: Sect. Table B3 3.4), MCS Q = MCS quartile, Q2 = MCS 2-3, Q3 = MCS 4-7, Q4 = MCS 8-9. Unadjusted effects and p-values are calculated by simple regression analyses. Adjusted values mean that it is controlled for the other variables in the table. Table B4 C di Nagelkerke R 0.17. Hosmer & Lemeshow Test: p 0.99. One Z residual > ± 3 (3.1). Cook s distances << 1.0. ∗p < 0.05, OR = Odds ratio, CI = 95 % conﬁdence interval, M = males, F = females, MCS: ambulation score from 0 (normal) to 9 ( = lost ambulation) (Details: Sect. 3.4), MCS Q = MCS quartile, Q2 = MCS 2-3, Q3 = MCS 4-7, Q4 = MCS 8-9. Unadjusted effects and p-values are calculated by simple regression analyses. Adjusted values mean that it is controlled for the other variables in the table. Table C1 Natural history of sib-ships of patients homozygous for FKRP c. 826C > A Family No. Gender Age (years) MCS Age of onset (years) Age at W/C (years) Age at CM (years) Age at NIV (years) 1. F 46 4 10 - 44 - 1. F 50 3 20 - - - 2. F 33 1 28 - - - 2. M 29 0 24 - 27 - 3. M 35 3 7 - - - 3. M 31 1 13 - - - 4. F 25 3 6 - - - 4. M 23 4 13 - - - 5. M 46 3 7 - 46 - 5. M 49 4 13 - - - 6. M 69 2 15 - 49 (Transplant at 57) 65 6. F 71 9 7 50 - 62 7. M 36 1 32 - - - 7. F 40 9 25 38 - - 8. M 16 2 10 - - - 8. F 20 2 11 - - - 9. M 55 2 7 - - - 9. F 51 5 4 - - 44 10. M 35 6 1 - 34 - 10. F 30 7 1 - 28 - 11. M 18 1 12 - 14 (or myocarditis?) - 11. F 12 0 9 - - - 11. F 11 1 9 - - - 12. M 56 9 3 45 - 30 12. M 53 8 3 35 38 (ICD at 38) 38 MCS = ambulation score from 0 (normal) to 9 ( = lost ambulation) (Details; Sect. 3.4), W/C = wheelchair dependency, CM = cardiomyopathy, NIV = initiation of non-invasive ventilatory support, F = females, M = males, ICD = implantable cardioverter-deﬁbrillator - Signiﬁes that the outcome was not reached Table B2 Unadjusted OR (CI) Unadjusted p value Adjusted OR (CI) OR relative change (%) Adjusted p-value Table B1 MCS: ambulation score from 0 (normal) to 9 ( = lost ambulation) (Details: Sect. 3.4), ∗p < 0.05, β = regression coeﬃcient, CI = 95 % conﬁdence interval, M = male gender. Unadjusted effects and p-values are calculated by simple regression analyses. Adjusted values mean that it is controlled for the other variables in the table. 11 nsen, K.I. Müller, S.I. Mellgren et al. Neuromuscular Disorders xxx (xxxx) xxx ARTICLE IN PRESS MD [m5G; December 13, 2022;4:40 ] ARTICLE IN PRESS [m5G; December 13, 2022;4:40 ] JID: NMD S.M. Jensen, K.I. Müller, S.I. Mellgren et al. Neuromuscular Disorders xxx (xxxx) xxx Table B2 Wheelchair dependency (n = 25/88 (28 %)) Unadjusted OR (CI) Unadjusted p value Adjusted OR (CI) OR relative change (%) Adjusted p-value AGE (years) 1.05 (1.02, 1.09) < 0.001 ∗ 1.02 (0.96, 1.08) - 3.1 0.55 YEARS SYMPTOMATIC 1.06 (1.03, 1.09) < 0.001 ∗ 1.05 (0.99, 1.11) - 0.66 0.060 GENDER (M/F) 0.26 (0.09, 0.70) 0.006 ∗ 0.17 (0.05, 0.55) - 35 0.0015 ∗ Nagelkerke R 2 = 0.38. Hosmer and Lemeshow Test: p = 0.34. Cook ´s distances << 1.0. 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https://openalex.org/W2795171026	http://diposit.ub.edu/dspace/bitstream/2445/122302/1/679358.pdf	English	null	Memantine loaded PLGA PEGylated nanoparticles for Alzheimer’s disease: in vitro and in vivo characterization	Journal of nanobiotechnology	2,018	cc-by	11,443	© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Memantine, drug approved for moderate to severe Alzheimer’s disease, has not shown to be fully effective. In order to solve this issue, polylactic-co-glycolic (PLGA) nanoparticles could be a suitable solution to increase drug’s action on the target site as well as decrease adverse effects. For these reason, Memantine was loaded in biodegradable PLGA nanoparticles, produced by double emulsion method and surface-coated with polyethylene glycol. MEM–PEG–PLGA nanoparticles (NPs) were aimed to target the blood–brain barrier (BBB) upon oral administra‑ tion for the treatment of Alzheimer’s disease. Results: The production parameters were optimized by design of experiments. MEM–PEG–PLGA NPs showed a mean particle size below 200 nm (152.6 ± 0.5 nm), monomodal size distribution (polydispersity index, PI < 0.1) and negative surface charge (− 22.4 mV). Physicochemical characterization of NPs confirmed that the crystalline drug was dispersed inside the PLGA matrix. MEM–PEG–PLGA NPs were found to be non-cytotoxic on brain cell lines (bEnd.3 and astrocytes). Memantine followed a slower release profile from the NPs against the free drug solution, allowing to reduce drug administration frequency in vivo. Nanoparticles were able to cross BBB both in vitro and in vivo. Behavio‑ ral tests carried out on transgenic APPswe/PS1dE9 mice demonstrated to enhance the benefit of decreasing memory impairment when using MEM–PEG–PLGA NPs in comparison to the free drug solution. Histological studies confirmed that MEM–PEG–PLGA NPs reduced β-amyloid plaques and the associated inflammation characteristic of Alzheimer’s disease. Conclusions: Memantine NPs were suitable for Alzheimer’s disease and more effective than the free drug. Keywords: Memantine, Nanoparticles, Alzheimer’s disease, Brain targeting, APPswe/PS1dE9 mice, β-Amyloid plaques, bEnd.3, Astrocytes Memantine loaded PLGA PEGylated nanoparticles for Alzheimer’s disease: in vitro and in vivo characterization Elena Sánchez‑López1,2,3, Miren Ettcheto3,4, Maria Antonia Egea1,2, Marta Espina1,2, Amanda Cano1,2,3, Ana Cristina Calpena1,2, Antoni Camins3,4, Nuria Carmona1, Amélia M. Silva5,6, Eliana B. Souto7,8 and Maria Luisa García1,2 Elena Sánchez‑López1,2,3, Miren Ettcheto3,4, Maria Antonia Egea1,2, Marta Espina1,2, Amanda Cano1,2,3, Ana Cristina Calpena1,2, Antoni Camins3,4, Nuria Carmona1, Amélia M. Silva5,6, Eliana B. Souto7,8 and Maria Luisa García1,2 Correspondence: esanchezlopez@ub.edu 1 Department of Pharmacy, Pharmaceutical Technology and Physical Chemistry, Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain Full list of author information is available at the end of the article Journal of Nanobiotechnology Journal of Nanobiotechnology Sánchez‑López et al. J Nanobiotechnol (2018) 16:32 https://doi.org/10.1186/s12951-018-0356-z RESEARCH Memantine loaded PLGA PEGylated nanoparticles for Alzheimer’s disease: in vitro and in vivo characterization Elena Sánchez‑López1,2,3, Miren Ettcheto3,4, Maria Antonia Egea1,2, Marta Espina1,2, Amanda Cano1,2,3, Ana Cristina Calpena1,2, Antoni Camins3,4, Nuria Carmona1, Amélia M. Silva5,6, Eliana B. Souto7,8 and Maria Luisa García1,2 Open Access Background the only drug approved both in Europe and in the United States for moderate and severe degrees of the disease.h Alzheimer’s disease (AD) is the most prevalent neuro- degenerative disorder amongst patients over 65 years old [1]. Memantine hydrochloride (MEM), a low-affinity voltage-dependent uncompetitive antagonist to gluta- matergic N-methyl-d-aspartate (NMDA) receptors, is The clinical applications of nanoparticles (NPs) have proven great advantages for targeting and drug deliv- ery, in particular, for the management of AD since cur- rent therapeutic strategies are compromised by the tight junctions and endothelial cells of the blood–brain-barrier (BBB) [2]. Nanoparticles, with an average size below 200 nm, may represent an alternative for prolonged drug delivery across the BBB, given their capacity for endo- cytic transport [3, 4]. While a number of polymers have Full list of author information is available at the end of the article Sánchez‑López et al. J Nanobiotechnol (2018) 16:32 Page 2 of 16 already been used in the production of NPs, polyesters such as poly d,l-(lactic-co-glycolic) acid (PLGA), have been extensively applied for controlled drug delivery, including brain targeting [5, 6]. PLGA, which has been approved by the Food and Drug Administration, is one of the most successful biodegradable polymers because it undergoes hydrolysis to produce lactic and glycolic acid, easily cleared from the body [7]. In addition, advanced drug delivery systems based on PLGA NPs have recently demonstrated to be potential alternatives for the treat- ment neurodegenerative diseases [8]. A limitation on the use of PLGA NPs in drug delivery is, however, their fast uptake and clearance from the reticuloendothelial system (RES). To overcome the RES clearance, surface coating of NPs with poly (ethylene glycol) (PEG) has been recom- mended, an approach that has demonstrated to reduce NPs’ clearance significantly in vivo [9]. In addition, it has also been proven that such surface coating may increase NPs targeting and uptake through the BBB [10]. Load- ing drugs in PEG-PLGA NPs with a matrix structure is expected to prolong their circulation half-life compared to non-coated PLGA NPs, due to the presence of mobile and flexible PEG chains on their surface. their oral bioavailability [13, 14]. PEG surfacing PLGA NPs have enhanced mucus permeating properties, there- fore contributing to increase the drug’s bioavailability after oral administration [15]. In the present work, we report the development of a physicochemical stable, sustained-release MEM– PEG–PLGA NPs formulation, for the treatment of AD. Materials PLGA-PEG Resomer® RGP d 5055 was obtained from Boehringer Ingelheim, Germany and memantine (MEM) was purchased from Capotchem (Hangzhou, China). Water filtered through Millipore MilliQ system was used for all the experiments and all the other reagents were of analytical grade. Background Developed MEM–PEG–PLGA NPs are reported to be a non-invasive approach for brain targeting of MEM, with minimal adverse-side effects. The physicochemical stabil- ity of MEM after loading in PLGA NPs has been char- acterized by drug-polymer interaction studies, and by in vitro release profile. Cell viability was studied and the in vitro transport across the BBB was mapped. Trans- genic and non-transgenic mice were orally treated with MEM–PEG–PLGA NPs and compared with the results obtained after treatment with free drug solution. Brain and plasma drug concentrations were measured, whilst behavioural test and histological studies were undertaken to elucidate the therapeutic efficacy of MEM–PEG– PLGA NPs against free drug, for brain delivery. Methods l Memantine (MEM) is a good candidate for drug load- ing. MEM is an uncompetitive (open-channel) NMDA receptor antagonist with low-to-moderate affinity, which binds preferentially to the NMDA receptor-operated cation channels, overlapping the site of Magnesium [11]. Therefore, MEM decreases the excessive glutamate which causes neuronal death on AD patients [11]. Although this drug was found to improve patients’ cognition, global functioning behaviour and stage of dementia in com- parison to placebo groups, results obtained from meta- analysis of AD monotherapy translate its limited clinical benefits (i.e. the assessment scores were not statistically significant between treated and non-treated groups) [12]. In this sense, drug delivery systems would increase drug concentration on the target site probably enhancing it’s effects against AD. In addition, despite being well-toler- ated, MEM requires daily administration by the patients which, combined with the poor drug compliance, may also reduce the rates of successful treatment. EE(%) = Total amount of MEM −Free MEM Total amount of MEM (1) The quantification of MEM was performed in multi- ple reaction monitoring (MRM) mode using an ion trap mass spectrometer equipped with an atmospheric pres- sure electrospray ionization ion source, positive mode. The HPLC system was an Agilent 1100 series (Agilent Technologies, USA) coupled with a Brucker Ion Trap SL (Brucker Daltonics GmbH, Germany). MEM was sepa- rated on a reversed phase column (Kinetex de 2.6 μm 50 × 2.1 (Phenomenex) using methanol 0.1% formic acid in water 55:45 (v/v) as mobile phase. The flow rate was 1 ml/min at 45 °C [22]. For the study of the sonication parameters (Table 1) and concentration compounds (Table 2) two independ- ent full factorial designs were performed. The mean size (Z-AVE), PI and ZP of the NPs were studied and the effects and interactions between factors were calculated. According to the composite design matrix generated by Statgraphics Plus 5.1 software, a total of 16 experiments (8 factorial points, 6 axial points and two replicated center points) were required for each design. Production and physicochemical characterization of nanoparticlesi MEM loaded NPs were produced by a modified double emulsion method described elsewhere [16, 17]. Briefly, a predetermined amount of PLGA–PEG was dissolved in ethyl acetate (EA) forming the organic phase. Aqueous phase (w1) was obtained by dissolving MEM in deionized water. Sonication energy was applied to form the primary emulsion (w1/o). The w1/o emulsion was then dispersed in 2 ml of deionized water containing PVA. Secondary emulsion (w1/o/w2) was formed with ultrasound energy [18]. A volume of 2 ml of PVA (0.3%) were then added, under magnetic stirring, to stabilize the colloidal sys- tem. Solvent was evaporated and NPs were washed by centrifugation at 15,000 r.p.m. during 20 min. The load- ing of NPs with rhodamine followed the same procedure. Empty NPs were prepared using the same approach but without addition of drug into the inner water phase [19]. A sustained release formulation, based on PLGA NPs for oral administration, has been proposed to assure that the drug remains on the target site until the next patients’ intake of the medicine. MEM–PEG–PLGA NPs are expected to contribute to a time-stable dose on the brain, prolonging drug release, reducing administration fre- quency and decreasing the adverse-side effects. Compar- ing to other routes, and for chronic treatment schedules, oral administration offers comfort and improves patient’s compliance. Recent studies have demonstrated the added-value of loading drugs in PLGA NPs to enhance Mean average size (Z-AVE) and polydispersity index (PI) of NPs were determined by photon correlation spectroscopy (PCS) using a ZetaSizer Nano ZS (Mal- vern Instruments). Measurements were carried out by Sánchez‑López et al. J Nanobiotechnol (2018) 16:32 Page 3 of 16 Page 3 of 16 Italic values correspond to the optimized formulation of MEM loaded NPs Design of experiments triplicate at 180° in 10 mm diameter cells at a tempera- ture of 25 °C. Zeta potential (ZP) was calculated from electrophoretic mobility as described elsewhere [20, 21]. Drug concentration was determined indirectly. Pre- viously to the analysis, the non-loaded drug was sepa- rated from NPs by filtration/centrifugation at 14,000 r.p.m. (Mikro 22 Hettich Zentrifugen, Germany) using an Amicon® Ultra 0.5 centrifugal filter device (Amicon Mil- lipore Corporation, Ireland). The encapsulation efficiency (EE) was calculated by the difference between the total amount of drug and the free drug, present in the filtered fraction, using Eq. (1): Design of experiment (DoE) was used to optimize the developed formulation. Series of independent param- eters and their influence on NPs properties were studied, determining the effects and interactions between factors. The effect of a factor × (Ex), was calculated using Eq. (2): (2) Ex = X(+) − X(−) n/2 (2) n/2 where ∑X(+) stands for the sum of the factors at their highest level (+ 1), ∑X(−) is the sum of the factors at their lowest level (−1), and n/2 is the half of the number of measurements. Interactions between factors (factor 1: factor 2) were also calculated. To estimate an interaction between two factors, the effect of the first factor at the lowest level of the second factor has to be calculated and subtracted it from the effect of the first factor at the high- est level of the second factor. EE(%) = Total amount of MEM −Free MEM Total amount of MEM Italic values correspond to the optimized formulation of MEM loaded NPs Italic values correspond to the optimized formulation of MEM loaded NPs EE(%) = Total amount of MEM −Free MEM Total amount of MEM J Nanobiotechnol (2018) 16:32 Page 4 of 16 Table 2 Values of the matrix of a factorial design of concentration parameters and measured responses Italic values correspond to the optimized formulation of MEM loaded NPs c PLGA− PEG c MEM c PVA Zav (nm) PI ZP (mV) EE (%) Coded level (mg/ml) Coded level (mg/ml) Coded level (mg/ml) Factorial points F1 − 1 10 − 1 3 − 1 2.5 270.0 ± 2.4 0.081 ± 0.002 − 13.4 ± 0.93 50.45 F2 1 30 − 1 3 − 1 2.5 450.1 ± 6.6 0.306 ± 0.022 − 8.27 ± 0.16 60.84 F3 − 1 10 1 9 − 1 2.5 230.1 ± 3.2 0.034 ± 0.026 − 2.95 ± 0.23 98.98 F4 1 30 1 9 − 1 2.5 369.3 ± 3.4 0.287 ± 0.028 − 3.33 ± 0.13 98.76 F5 − 1 10 − 1 3 1 7.5 324.4 ± 3.8 0.188 ± 0.009 − 9.79 ± 0.27 57.74 F6 1 30 − 1 3 1 7.5 287.8 ± 4.6 0.139 ± 0.029 − 7.84 ± 0.25 72.07 F7 − 1 10 1 9 1 7.5 177.9 ± 2.9 0.034 ± 0.030 − 3.81 ± 0.44 81.23 F8 1 30 1 9 1 7.5 223.4 ± 0.6 0.063 ± 0.007 − 3.88 ± 0.17 84.87 Axial points F9 1.68 37 0 5 0 5 260.4 ± 1.6 0.102 ± 0.018 − 8.85 ± 0.53 66.08 F10 − 1.68 3 0 5 0 5 147.1 ± 0.7 0.032 ± 0.007 − 4.96 ± 0.17 69.52 F11 0 20 1.68 11 0 5 204.3 ± 2.5 0.081 ± 0.018 − 3.86 ± 0.27 52.84 F12 0 20 − 1.68 1 0 5 238.8 ± 0.7 0.077 ± 0.013 − 14.5 ± 0.45 55.33 F13 0 20 0 5 1.68 9.2 199.0 ± 2.1 0.071 ± 0.027 − 5.59 ± 0.19 57.27 F14 0 20 0 5 − 1.68 0.8 272.2 ± 2.1 0.103 ± 0.033 − 3.26 ± 0.19 65.65 Center points F15 0 20 0 5 0 5 213.1 ± 0.4 0.023 ± 0.024 − 6.03 ± 0.27 72.61 F16 0 20 0 5 0 5 211.7 ± 0.3 0.034 ± 0.023 − 5.96 ± 0.16 40.92 Table 2 Values of the matrix of a factorial design of concentration parameters and measured responses 2 Values of the matrix of a factorial design of concentration parameters and measured responses Table 2 Values of the matrix of a factorial design of concentration parameters and measured responses Italic values correspond to the optimized formulation of MEM loaded NPs exposed to CuK” radiation (45 kV, 40 mA, λ = 1.5418 Å) in the range (2θ) from 2° to 60° with a step size of 0.026° and a measuring time of 200 s per step. EE(%) = Total amount of MEM −Free MEM Total amount of MEM The studied Table 1 Values of the matrix of a factorial design of sonication parameters and measured responses Italic values correspond to the optimized formulation of MEM loaded NPs Amplitude 1st sonication time 2nd sonication time Zav (nm) PI ZP (mV) EE (%) Coded level (%) Coded level (s) Coded level (s) Factorial points F1 − 1 25.0 − 1 20.0 − 1 120.0 390.4 ± 2.2 0.213 ± 0.039 − 6.73 ± 0.04 3.46 F2 1 35.0 − 1 20.0 − 1 120.0 249.7 ± 4.7 0.069 ± 0.022 − 6.33 ± 0.49 9.59 F3 − 1 25.0 1 40.0 − 1 120.0 184.6 ± 0.7 0.125 ± 0.023 − 6.43 ± 0.45 42.57 F4 1 35.0 1 40.0 − 1 120.0 227.0 ± 2.6 0.057 ± 0.019 − 6.72 ± 0.33 36.89 F5 − 1 25.0 − 1 20.0 1 240.0 243.0 ± 0.9 0.194 ± 0.012 − 6.72 ± 0.24 7.43 F6 1 35.0 − 1 20.0 1 240.0 248.1 ± 1.9 0.053 ± 0.037 − 6.48 ± 0.15 14.69 F7 − 1 25.0 1 40.0 1 240.0 258.7 ± 4.5 0.198 ± 0.011 − 6.35 ± 0.33 22.63 F8 1 35.0 1 40.0 1 240.0 206.4 ± 1.2 0.061 ± 0.045 − 6.67 ± 0.30 2.88 Axial points F9 1.68 38.4 0 30.0 0 180.0 222.4 ± 2.4 0.033 ± 0.011 − 5.63 ± 0.37 39.12 F10 − 1.68 21.6 0 30.0 0 180.0 162.6 ± 0.4 0.262 ± 0.012 − 6.83 ± 0.37 39.36 F11 0 30.0 1.68 47.0 0 180.0 226.7 ± 4.4 0.236 ± 0.011 − 6.49 ± 0.25 19.94 F12 0 30.0 − 1.68 13.0 0 180.0 196.8 ± 2.5 0.103 ± 0.056 − 6.47 ± 0.55 43.10 F13 0 30.0 0 30.0 1.68 281.0 239.8 ± 0.7 0.056 ± 0.020 − 5.77 ± 0.47 23.39 F14 0 30.0 0 30.0 − 1.68 79.0 382.6 ± 5.2 0.221 ± 0.011 − 5.93 ± 0.21 33.95 Center points F15 0 30.0 0 30.0 0 180.0 220.1 ± 5.6 0.059 ± 0.019 − 5.36 ± 0.03 24.01 F16 0 30.0 0 30.0 0 180.0 222.1 ± 3.6 0.062 ± 0.021 − 5.36 ± 0.11 23.23 atrix of a factorial design of sonication parameters and measured responses Table 1 Values of the matrix of a factorial design of sonication parameters and Table 1 Values of the matrix of a factorial design of sonication parameters and measured responses Sánchez‑López et al. Nanospheres characterization and interaction studies Nanospheres characterization and interaction studies Morphology of the optimized formulation of MEM loaded NPs was determined by transmission elec- tron microscopy (TEM), performed on a JEOL 1010 microscope (Akishima, Japan). The physical state and chemical interactions between drug and polymers were studied by thermal and X-ray diffraction analyses. For the interaction studies, NPs were washed by centrifuga- tion and dried to constant weight previous to carry out the analysis. MEM thermal properties were studied by thermogravimetric (TG) analysis and differential thermal analysis (DTA) on a TASC 414/3 (Netsch, Thermal Anal- ysis). Temperature ranged from 25 to 600 °C at 10 °C/min and AlO3 pan was used as a reference. All experiments were carried out under nitrogen flow. Thermograms were obtained by differential scanning calorimetry (DSC) on a Mettler TA 4000 system (Greifensee, Switzerland) equipped with a DSC 25 cell. Samples were weighed (Mettler M3 Microbalance) in perforated aluminium pans and heated under a flow nitrogen at a rate of 10 °C/ min. X-Ray diffraction (XRD) was used to analyse the amorphous versus crystalline status of the samples. Com- pounds were sandwiched between polyester films and Storage stabilityh The stability of MEM–PEG–PLGA NPs stored at three different temperatures (4, 25 and 38 °C) was studied by light backscattering using Turbiscan®Lab operated at constant temperature. For this purpose, a glass measure- ment cell was filled with 20 ml of sample. The light source is a pulsed near infrared light and was received by a back- scattering detector at an angle of 45° from the incident beam. Backscattering data were acquired once a month for 24 h at intervals of 1 h. In addition to this technique, morphometric parameters (Z-AVE, PI and ZP) were also measured. EE(%) = Total amount of MEM −Free MEM Total amount of MEM Fourier-trans- formed infra-red (FTIR) spectra of different compounds were obtained using a Thermo Scientific Nicolet iZ10 with an ATR diamond and DTGS detector. experimental responses were the result of the individual influence and the interaction of the three independent variables. Trans‑epithelial electrical resistance studyh The brain vasculature is characterized by endothelial cells with strong tight junctions that limit paracellular diffu- sion of hydrophilic molecules selectively, according to their charge and size. When the movement of ions across the monolayer is restricted due to the proper function- ing of the barrier, an electric potential gradient on both sides is generated. Transepithelial electrical resistance (TEER) is an indicator of cell confluence, monolayer integrity and the formation of tight junctions between cells. Thus, TEER manual measurements were taken daily until a steady state was reached, employing epithe- lial EVOM2 voltmeter connected to a pair of electrodes STX2. The system operates with two electrodes, which can be applied directly to the inserts. To calculate TEER of each insert, Eq. (3) was used and values are expressed in Ω cm2. Cell culture Cells were thawed, grown, maintained and regularly observed under a microscope. Two cell lines were used, namely, mouse microvascular endothelial cells (bEnd.3) and astrocytes from brain rat cortex. Primary cultures of astrocytes were obtained from bank GAIKER-IK4 cul- ture. The bEnd.3 cells were maintained in their specific culture medium, DMEM + 10% FBS [24]. Cells and cor- responding culture medium were tempered at 37 °C, 1 ml of cells was diluted in 9 ml of medium and the cell sus- pension was centrifuged at 4 °C for 5 min at a speed of 130g. The supernatant was removed and the cells were re-suspended in culture medium. Cells were seeded in 75 cm2 flasks and kept in an incubator at 37 °C, an atmos- phere of 5% CO2 and a relative humidity of 95%. Once removed and washed, the cells were seeded with fresh medium. (3) TEER = Ωcell monolayer −ΩFilter (without cells) × [Filter Surface] (3) Co-culture experiments were carried out in 24-well plates. Inserts were removed and placed in new media plates with Hanks and 0.5% bovine serum albumin (BSA). Apical media was removed, washed with Hanks and MEM–PEG–PLGA NPs were added (dissolved in 0.5% BSA Hanks) in the apical part of the inserts and left for 1 h. Furthermore, to verify that MEM–PEG–PLGA NPs did not compromise membrane integrity, a compound with low paracellular permeability, Lucifer yellow (LY), was added at the end of the study. Membrane integrity (with LY) was determined calculating the permeability coefficient by using the clearance principle which allows a permeability value independent of concentration. In vitro drug release In vitro drug release of MEM–PEG–PLGA NPs was stud- ied against free MEM in PBS, in a bulk equilibrium direct dialysis bag technique under sink conditions for 36 h (n = 6). Briefly, a volume of 5 ml of each formulation was placed directly into a dialysis bag (cellulose membrane, 12–14 kDa, size 3,20/32’’ diameter, Iberlabo) and each Sánchez‑López et al. J Nanobiotechnol (2018) 16:32 Page 5 of 16 used with a semipermeable membrane of 0.4 μm pore. For co-culture experiments, endothelial cells were seeded in the apical part of the inserts. A semipermeable filter was placed, and in the basolateral compartment cells from primary cultures of rat astrocytes were added. bag was placed on 150 ml of isotonic phosphate-buffered saline (PBS) 0.1 M, pH 7.4 at 37 °C. At predetermined intervals, 1 ml of sample was withdrawn from the stirred release medium and simultaneously replaced with 1 ml of fresh buffer at the same temperature. Akaike’s infor- mation criteria was determined as an indicator of the suitability of the model for a given dataset. The model associated to the smallest Akaike’s information criteria value is considered as giving the best fit of the set of data [23]. In vivo studies M l APP Male APPswe/PS1dE9 (APP/PS1) and C57BL/6 mice were used for the in vivo studies. APP/PS1 animals co- express a Swedish (K594M/N595L) mutation of a chi- meric mouse/human APP (Mo/HuAPP695swe), together with the human exon-9-deleted variant of PS1 (PS1- dE9), allowing these mice to secrete elevated amounts of human Aβ peptide [24]. The animals were kept under controlled temperature, humidity and light con- ditions with food and water provided ad libitum. Mice were treated in accordance with the European Commu- nity Council Directive 86/609/EEC and the procedures Cytotoxicity studies Alamar blue reduction was used as a parameter for cell viability. This assay is based on that viable and metaboli- cally active cells reduce resazurin to resorufin, which is released into the culture medium. This conversion is intracellular, facilitated by oxidoreductases of mitochon- drial, microsomal and cytosolic origin. In a toxic event, where a loss of cell viability and proliferation occurs, the cells that comprise the epithelial tissue lose the ability to reduce resazurin. Therefore, resazurin reduction ratio is directly proportional to the number of viable cells. Absorbance was determined at λ of 570 and 620 nm, reduced and oxidized form, respectively [24]. Data were analysed by calculating the percentage of Alamar blue reduction and expressed as percentage of control (untreated), as reported before [25]. In vitro transport across the BBB In vitro BBB models have become a standard tool for esti- mating the ability of drugs to bypass the BBB at the early stage of drug development. In the present work, endothe- lial cell-based models were optimized by co-culturing the endothelial cells with astrocytes in Transwell systems, as shown in Fig. 1. Polycarbonate transwell inserts were Sánchez‑López et al. J Nanobiotechnol (2018) 16:32 Page 6 of 16 Fig. 1 DoE of sonication parameters a surface plot of MEM-PLGA-PEG NPs PI and b Pareto’s chart of the effect of sonication parameters on ZP Fig 1 DoE of sonication parameters a s rface plot of MEM PLGA PEG NPs PI and b Pareto’s chart of the effect of sonication parameters on ZP Fig. 1 DoE of sonication parameters a surface plot of MEM-PLGA-PEG NPs PI and b Pareto’s chart of the effect of sonication parameters on ZP linking PLGA to Rhodamine. NPs were orally adminis- tered for 1 week on alternate days and after 24 h of clear- ance mice were anesthetized with sodium pentobarbital and perfused with 4% paraformaldehyde. Brains were stored at 4 °C overnight dehydrated in 30% phosphate- buffered sucrose solution. Samples were preserved at − 80 °C and coronal sections of 20 μm were obtained by a cryostat (Leica Microsystems, Wetzlar, Germany). Samples were visualized using a fluorescence microscope using Rhodamine filter (BX41 Laboratory Microscope, Melville, NY-Olympus America Inc). established by the Departament d’Agricultura, Ramade- ria i Pesca of the Generalitat de Catalunya. Every effort was made to minimize animal suffering and to reduce the number of animals used. 6 month-old animals, divided into six groups, were used for the present study, with at least 10 WT and 10 APP/PS1 transgenic mice, per group. In each genotype group, the mice were treated either with untreated water, with free MEM or with MEM NPs (Additional file 1: Figure S1). Mice were treated for 2 months with MEM at therapeutic dose (30 mg/kg/ day). MEM–PEG–PLGA NPs were orally administered in alternate days calculating the volume for each animal previously weighted followed by a regime of untreated water. After the in vivo testing, the animals were sacri- ficed. Prior to sacrifice, mice were left fasting for at least 8 h and in the case of treated mice. At least 6 mice in each group were used for histological studies [26]. In vitro transport across the BBB Previously to the study of the therapeutic effects, drug at steady state levels was quantified. Blood samples were extracted from the facial vein and samples were centri- fuged during 20 min at 2000 r.p.m. adding EDTA (10 μl K2EDTA 18 mg/ml) to avoid blood coagulation. Mice were sacrificed by cervical dislocation. Amantadine was added as internal standard and MEM extraction was carried out using organic solvents (t-butyl methyl ether and diethyl ether–chloroform for brain and blood sam- ples, respectively). Solvents were evaporated under Nanoparticles brain distribution To tackle the NPs uptake and visualize them in the target tissue in vivo, MEM NPs were fluorescently labelled by Sánchez‑López et al. J Nanobiotechnol (2018) 16:32 Page 7 of 16 β-amyloid plaques was quantified. For each mouse, four fields per section with the highest density of plaques were chosen as representative and were averaged [31]. nitrogen flow and samples were reconstituted with methanol [27, 28]. Samples were analysed as described previously (section NPs production). The analyses were carried out using the parent to daughter combinations of m/z 180 > 163 (MEM) and m/z 152 > 135 (amantadine) [22]. Statistical analysis All of the data are presented as the mean ± S.D. Two-way ANOVA followed by Tukey post hoc test was performed for multi-group comparison. Student’s t test was used for two-group comparisons. Statistical significance was set at p < 0.05 by using GraphPad Prism version 5.00 for Win- dows, GraphPad Software, San Diego California USA. Optimization of NPs’ parameters Optimization of NPs’ parameters Double emulsion evaporation method was chosen for the production of PLGA NPs due to its suitability for the loading of hydrophilic drugs, such as MEM. Since the mean particle size is a critical parameter to assure that NPs are absorbed in the gastrointestinal tract and achieve the BBB, the aim of the factorial design was to produce MEM–PEG–PLGA NPs with a mean size 100 and 200 nm. Since MEM is insoluble in ethyl acetate, this organic solvent has been used for the preparation of w1/o/w2 emulsions, allowing the retention of the drug in the inner aqueous phase. From preliminary studies, the addition of small amount of polyvinyl alcohol (PVA, 0.3%) after the second emulsification process was shown to contribute to the decrease of the mean size of NPs from 264.6 nm (with a bimodal distribution) down to 220.1 nm (with a monomodal distribution, PI < 0.1). These results were attributed to the delay of the solvent diffusion to the outer aqueous phase upon addition of the aqueous PVA solution, limiting the risk of droplets agglutination and polymer precipitation [19]. The results obtained from the full factorial designed performed for the selection of the appropriate sonication parameters (i.e. wave amplitude and sonication time) are shown in Table 1. Morris water mazeh The Morris water maze (MWM) test was conducted in a circular tank filled with water at 21 ± 2 °C and divided into four equal quadrants. A white platform was sub- merged below the water surface in the middle of the northeast quadrant. The behavioural data were acquired and analysed using a computerized video tracking sys- tem. The procedure of the behaviour assessment con- sisted of a six-day navigation testing session and a probe trail. Mice received five trials per day for 6 successive days continuing with the same drug regime. Animals were placed into the maze facing the tank wall at water-level. They were allowed to swim freely for 60 s to seek the invisible platform and allowed to remain there for 10 s. If a mouse failed to find the platform, it was guided to it and left there for 30 s. The probe trial was performed the day after the last training test. In the probe test, the hid- den platform was removed, and the mice were released from the southwest quadrant and allowed to swim for 60 s. Results were calculated individually for each animal [29]. Immunohistochemistry studies y Mice were anesthetized with sodium pentobarbital and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (PBS) after the probe trial. Brains were stored in 4% paraformaldehyde at 4 °C overnight then dehy- drated in 30% phosphate-buffered sucrose solution for cryoprotection. Samples were preserved at − 80 °C and coronal sections of 20 µm were obtained by a cryostat (Leica Microsystems, Wetzlar, Germany). Sections were incubated overnight at 4 °C with the rabbit anti-GFAP (1:2000; Dako, Glostrup, Denmark) primary antibody, and sequentially incubated for 2 h with Alexa Fluor 594 goat anti-rabbit antibody at room temperature (1:500; Invitrogen, Eugene, OR, USA). Staining of β-Amyloid plaques was performed using Thioflavin S (ThS 0.002%, Sigma-Aldrich) to compare β-amyloid plaque den- sity among different treatment groups. Sections were counterstained with 0.1 μg/ml Hoechst 33,258 (Sigma- Aldrich, St Louis, MO, USA) and rinsed afterwards with PBS 0.1 M [30]. ThS-stained β-amyloid plaques were visualized using a fluorescence microscope with a fluo- rescence filter (BX41 Laboratory Microscope, Melville, NY-Olympus America Inc). For each image, the propor- tion of total image area covered by fluorescently stained Factorial design demonstrates that the interaction between 1st and 2nd sonication times increased the mean size of NPs. As a consequence, a lower 1st sonica- tion time was chosen to obtain NPs smaller than 200 nm. High amplitudes, around 38%, are mandatory to obtain small and monodispersed NPs (Fig. 2a) [19]. As shown in Fig. 1b, amplitude was a significant parameter regard- ing particles surface charge, establishing a trend where increasing the amplitude causes a slight increase of the negative charge and, subsequently, the creation of more stable particles. Therefore, the maximum amplitude (F9, Table 1) would be applied.h The optimized concentration parameters of the for- mulation compounds have also been studied (Table 2). The increase of the polymer concentration caused the increase of both z-AVE and PI (Fig. 3a). Indeed, the Page 8 of 16 Sánchez‑López et al. J Nanobiotechnol (2018) 16:32 Fig. 2 DoE of concentration parameters; a surface plot of MEM –PLGA-PEG NPs z-AVE and b effect of compounds concentration on ZP Fig. 2 DoE of concentration parameters; a surface plot of MEM –PLGA-PEG NPs z-AVE and b effect of compounds concentration on ZP Fig. 2 DoE of concentration parameters; a surface plot of MEM –PLGA-PEG NPs z-AVE and b effect of compounds concen a statistically significant relationship between EE and MEM concentration was established. Characterization of NPs and interaction studiesi reduction of the attractive forces among the polymer chains. MEM displayed a melting transition followed by decomposition between 190 and 322 °C, exhibiting a thermal event comprising both phenomena. DSC analy- sis of MEM–PEG–PLGA NPs displayed an endothermic event corresponding to the Tg of the polymer occurring at 50.56 °C. The increasing of the Tg of the polymer has been attributed to the incorporation of an alkaline drug, which causes interactions between the carboxylic groups of the polymer. In vitro and in vivo drug release profiles are highly dependent on the physical state of the drug inside the NPs. TG and DTA were therefore used to study the inter- action between MEM and polymers. MEM shown to be stable at low temperatures probably due to the pres- ence of the anhydrous [33]. TG profile of MEM exhibited a weight loss starting at 290 °C, and finishing at 354 °C, which correspond to the complete degradation of drug. (Additional file 1: Figure S2). DTA showed an onset of endothermic event at 280 °C followed by a maximum at 352 °C being these results similar to those obtained by DSC. A thermal decomposition of MEM was shown to occur in two steps, corresponding the latter to a final oxi- dative degradation. Results from XRD studies are shown in Fig. 3b. Drug powder diffraction pattern showed sharp crystalline peaks, whereas PEG–PLGA showed an amorphous pro- file. MEM–PEG–PLGA NPs displayed a profile similar as PEG–PLGA, but a slight attenuated peak corresponding to the drug was also observed. The surfactant displayed a semi-crystalline pattern, not present in the formulation. This fact demonstrates the effectiveness of the centrifu- gation process, confirmed by FTIR analysis (Fig. 3c). This suggests that the surfactant acts only as adjuvant in the NPs production, stabilizing the freshly prepared parti- cles while it is not entrapped in the polymer because it was effectively removed by centrifugation. This property is relevant since a high surfactant concentration may DSC curves of MEM, PVA, PEG–PLGA, MEM–PEG– PLGA NPs, and physical mixture of MEM are depicted in Fig. 3a. PVA exhibits two endothermic peaks, corre- sponding to the melting (192.86 °C) and decomposition (318.61 °C) events, respectively. PEG-PLGA onset transi- tion temperature (Tg) takes place around 44.50 °C. PLGA without PEG chains exhibited a Tg around 54.18 °C. Immunohistochemistry studies According to the factorial design data, a suitable formulation was achieved with a minimum concentration of polymer (10 mg/ml), an upper-intermediate drug concentration (9 mg/ml) and a maximum amount of PVA (7.5 mg/ml) (F7, Table 2). After ultracentrifugation at 15,000 r.p.m. for 20 min of the optimized formulation, NPs kept their size properties (z-AVE of 152.6 ± 0.5 nm and PI 0.043 ± 0.009), although ZP was more negative (− 22.4 ± 0.5 mV), which was attributed to the removal of surfactant molecules from the surface of the particles. Detailed structure of MEM– PEG–PLGA NPs was further characterized by TEM, which confirmed the spherical shape and smooth surface of NPs (Additional file 1: Figure S1). higher the viscosity of the inner aqueous phase of the pri- mary emulsion (w1/o), the less efficient is the reduction of the emulsion droplet size during the second emulsifi- cation step (w1/o/w2) [16, 17]. The higher the PVA con- centration, the smaller the NPs obtained (Fig. 2a). These results suggest that the optimized PVA concentration should be able to ensure enough surfactant molecules to cover the interface between the organic phase and the external aqueous phase, improving the protection of the droplets from coalescence [17]. Pareto’s chart (Fig. 2b) shows that MEM concentra- tion influenced the surface electrical charge of NPs sig- nificantly. MEM has an amine group which can easily be protonated and decrease negative surface charge caused by the polymer [32, 33]. However, while high MEM concentrations negatively affect the particles stability, Sánchez‑López et al. J Nanobiotechnol (2018) 16:32 Page 9 of 16 Fig. 3 MEM-PLGA-PEG NPs interaction studies a differential scanning calorimetry analysis, b X-ray diffraction and c Fourier transformed infra-red analysis Fig. 3 MEM-PLGA-PEG NPs interaction studies a differential scanning calorimetry analysis, b X-ray diffraction and c Fourier transformed infra-red analysis Fig. 3 MEM-PLGA-PEG NPs interaction studies a differential scanning calorimetry analysis, b X-ray diffraction and c Fourier transformed infra-red analysis Fig. 3 MEM-PLGA-PEG NPs interaction studies a differential scanning calorimetry analysis, b X-ray diffraction and c Fourier analysis Characterization of NPs and interaction studiesi Characterization of NPs and interaction studiesi The presence of PEG chains produced a decrease of the Tg values, attributed to the plasticizing effect based on the Sánchez‑López et al. J Nanobiotechnol (2018) 16:32 Page 10 of 16 induce toxicity by establishing an interconnected net- work with the polymer [34]. amine corresponding peak associated with N–H stretch- ing bond. As reported by other authors, the peak at 1648 cm−1 indicates presence of C–O group attached to –NH [36]. FTIR analysis (Fig. 3c) does not show any evidence of chemical interaction or strong bond formation between MEM and PEG–PLGA or between NPs and surfactant. The stretching band of the polymer carbonyl groups (C=O) was observed at 1740 cm−1, whereas the first pol- ymer bands are due to C–O PLGA–PEG bonds [35]. The bond at 2950 cm−1 clearly indicates the presence of C–H (ethylene glycol). PVA exhibits a number of absorption peaks at 2900, 1324, 843 and 1084 and 3237 cm−1 due to C–H stretching, C–H bending and C–O stretching, which are not depicted in the profile obtained for MEM– PEG–PLGA NPs. Around 3000 cm−1 MEM showed the In vitro and in vivo transport across the BBB In vitro and in vivo transport across the BBB Results show that 40% of the initial MEM–PEG–PLGA NPs were retained by the cell membrane of the in vitro model within 1 h of incubation, whereas only 30% of the initial MEM was found inside the barrier. Drug per- meability coefficient (Pe) in this model was 0.933. This fact indicates that NPs retained in this tissue would be able, either to achieve a slow drug release from there, or to partially cross through it and release the drug into the basolateral media. TEM images demonstrate that the part of MEM–PEG–PLGA NPs achieving the BBB remained spherical and non-aggregated with an average size below 200 nm (Fig. 7a). In addition, Lucifer Yellow (LY) was used as control at the end of the study, show- ing that these systems do not cause disruption of the BBB as no increase of paracellular passage of LY was observed (Pe < 1 in all the experiments). In addition, other authors that develop similar drug delivery systems for CNS diseases also demonstrate that the NPs were able to effectively transport the drugs. Year ago, Alyaut- dinvand colleagues developed Polibutylcianoacrilate NPs encapsulating Loperamide (which is unable to pass through the BBB), showing that the drug delivery systems (around 290 nm and PI below 0.1) were effective [38]. More recently, Liu and co-workers developed PEGylated Storage stability Stability of the developed NPs at different temperatures (4, 25 and 38 °C) was also monitored. Samples stored at 4 and 25 °C remained visually unchanged during the first 6 months of storage. Samples stored at 38 °C were completely transparent and unstable by the end of the 1st month because of the degradation of the polymer induced by higher temperatures (Fig. 4a). 4 Backscattering profile of MEM-PLGA-PEG NPs stored for 6 months; a 38 °C, b 25 °C and c 4 °C Sánchez‑López et al. J Nanobiotechnol (2018) 16:32 Page 11 of 16 Fig. 6 Cell viability assessment using Alamar blue on brain cell lines Samples stored at 4 and 25 °C kept their ZP and size parameters (z-AVE and PI) for 6 months. No statistically significant differences were found between formulations stored at 4 and at 25 °C. Backscattering profiles at both temperatures were similar to those obtained by the end of the 1st month, but NPs stored at 25 °C showed a slight decrease of the light scattered percentage corresponding to the bottom of the sample, which was not observed at 4 °C (Fig. 4b, c). This result was attributed to a slight NPs sedimentation process being preferential the particles’ storage at 4 °C. In vitro drug release Fig. 6 Cell viability assessment using Alamar blue on brain cell lines In vitro drug release was analysed against a drug solu- tion in PBS (free MEM). Free MEM release was faster than the observed for MEM–PEG–PLGA NPs (Fig. 5). The optimized formulation showed an immediate release (burst release) attributed to the non-loaded MEM frac- tion which is weakly bound to the NPs’ surface, because of the PEG coating [37]. After this initial phase, the drug displayed a sustained release diffusing slowly from the polymeric matrix into the release medium. Akaike’s information criterion for hyperbola adjustment was 64.97 for MEM-loaded NPs and 86.8 for free drug. Parameters corresponding to hyperbola adjustment were analysed. Kd, equilibrium dissociation constant, expressed in con- centration, corresponding to MEM–PEG–PLGA NPs was almost twice (0.74 for drug loaded NPs and 0.38 for the free drug) than Kd obtained for the free drug, con- firming the slower release of the drug from the colloidal system. reason together they are considered as a suitable model to test nanoparticles cytotoxicity. Following the incuba- tion for 24 h, MEM–PEG–PLGA NPs did not show any measurable toxic effect (Fig. 6). These results confirm that the developed particles are biocompatible with both endothelial glial brain cells. In addition, the slight amount of PVA that could remain after centrifugation process did not induce any toxicity nor influenced the normal growth of both epithelial cell lines within the assessed doses. Cytotoxicity studies Cell viability of MEM–PEG–PLGA NPs was measured in bEnd.3 (brain endothelial cells) and rat astrocyte pri- mary cultures. These cells form the BBB and, for this Fig. 5 In vitro release profile of MEM from PBS solution or MEM- PLGA-PEG NPs. Mean parameters were obtained adjusting data to hyperbola equation Fig. 5 In vitro release profile of MEM from PBS solution or MEM- PLGA-PEG NPs. Mean parameters were obtained adjusting data to hyperbola equation Page 12 of 16 Sánchez‑López et al. J Nanobiotechnol (2018) 16:32 Page 12 of 16 of these mechanisms [42]. The advantage that offer the entrapment on polymeric NPs has been demonstrated by several authors. For instance, Mittal et al. developed similar drug delivery systems to be administered orally and demonstrated their ability to be absorbed by the GI and provide therapeutic blood levels [43]. It has also been demonstrated that, in addition of NPs size, the sol- vent and the stabilized used has an effect on drug release. Other authors, such as Sahana and co-workers develop similar drug delivery systems with a mean size around 270 nm using ethyl acetate and PVA obtaining a pro- longed drug release for 3 days after their administration and Kalaria and collegues also achieve superior effectivity of oral PLGA NPs compared with the free drug in vivo [44, 45]. Moreover, NP surface characteristics can be tai- lored to optimize mucoadhesion [42]. As demonstrated by Tobío and co-workers, PEGylation of polymeric NPs of a mena size around 160 nm, imparts additional pro- tection against enzyme induced aggregation and deg- radation in simulated GI fluids in vitro [46]. Previous publications of our group demonstrated that PLGA PEGylated NPs were suitable for GI absortion [47] show- ing the NPs groups and increased behavioral and molec- ular effectivity. Fig. 7 a TEM pictures of MEM–PEG–PLGA NPs on the basolateral compartment of the BBB transport model after one hour of incuba‑ tion, b Hipocampus of WT mice with PLGA-Rho NPs f In addition, NPs demonstrated to be able to reach the hippocampus confirming that this drug delivery systems could be an effective strategy to achieve an increased drug release. Furthermore, plasma levels after a single NPs dose were recorded achieving MEM concentrations in plasma around 883.02 ng/ml after 40 h of clearance. Fig. Cytotoxicity studies 7 a TEM pictures of MEM–PEG–PLGA NPs on the basolateral compartment of the BBB transport model after one hour of incuba‑ tion, b Hipocampus of WT mice with PLGA-Rho NPs Morris water maze test Th ff f NPs to cross the BBB with an average size smaller than 200 nm, able to cross the BBB [39]. The author’s labelled the NPs fluorescently obtaining qualitative results and no disruption of the BBB was reported. Hereby, we provide behavioral and molecular data that gives solid evidence of the enhancement of the drug activity provided by the NPs [39]. In addition, spherical shape demonstrated to increase their transport across the BBB [40]. Moreover, grafting NPs with PEG decreases protein adsorption and slows down the nanomaterials clearance improving blood circulation time; as a consequence, PEGylated NPs accu- mulate more efficiently in the brain [41]. The effects of MEM treatment on the animal’s behaviour were assessed with the MWM test (Fig. 8). The overall ANOVA for the training days revealed both a genotype (APP/PS1 against WT showed significant differences, p < 0.01) and a drug effect (treated vs untreated APP/PS1 mice) on mice spatial learning capacities. Escape latency on the test day results are shown in Fig. 8a. Untreated APP mice showed a significant increase on scape latency compared to MEM loaded NPs group (p < 0.01). In addi- tion, mice treated with NPs revealed an improvement on spatial learning memory compared with free MEM (no statistically significant differences). This indicates that the developed nanosystems constitute a suitable strategy for the delivery of drugs for AD. fi As can be observed in Fig. 7b, Rhodamine labelled NPs were able to be absorbed by the GI, cross the BBB and reach the brain upon oral administration. Oral delivery is the most common route of administration and NP drug carriers that can shield drugs from degradation and deliver them enabling more efficient and sustained drug delivery [42]. Particles synthesized from commonly used polymers, such as PLGA, may achieve mucoadhe- sion via hydrogen bonding, polymer entanglements with mucins, hydrophobic interactions, or a combination As shown in Fig. 8b, MEM-loaded NPs groups fol- lowed a more direct path until platform, than the rest of transgenic groups. As expected, significant differences were obtained with APP/PS1 untreated group and MEM- loaded NPs (p < 0.01). Regarding time percentage in the platform quadrant (data not shown), APP/PS1 mice treated with MEM-loaded NPs presented an average Sánchez‑López et al. J Nanobiotechnol (2018) 16:32 Page 13 of 16 Fig. 8 Morris water maze results on the probe trail of the APP/PS1 mice. Morris water maze test Th ff f a Escape latency and b representative swimming path of transgenic mice. Data represent mean ± SD; p < 0.05, p < 0.01, p < 0.001, *p < 0.0001 Fig. 8 Morris water maze results on the probe trail of the APP/PS1 mice. a Escape latency and b representative swimming path of transgenic mice. Data represent mean ± SD; p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 Fig. 8 development. Moreover, plaques were surrounded by a high inflammatory state characteristic of AD. MEM– PEG–PLGA NPs groups showed fewer plaques and also inflammation degree was lower than the rest of trans- genic groups [48]. These results are in agreement with behavioural assays, indicating that MEM restored cog- nition by decreasing insoluble amyloid plaques and the inflammatory response associated with AD. of 37.22% of the time, whereas transgenic mice treated with MEM spend a 24.72% of the time revealing that oral MEM-loaded NPs restore cognition more effectively than the free drug. Immunohistochemistryh The formation of Aβ plaques, which is a pathologic hall- mark of AD, could be observed by Thioflavin˗S staining. Several studies confirmed that MEM decrease the num- ber of amyloid plaques, therefore, histological studies to observe plaque development would be of great relevance. Figure 9a shows the results corresponding to amyloid plaques counting of APP/PS1 mice. WT groups did not develop β-amyloid plaques. APP/PS1 mice treated with NPs developed some plaques, but the number was significantly lower than those obtained for the rest of transgenic groups (p < 0.001 against untreated mice and p < 0.01 against MEM mice). Conclusions In this study, factorial design allowed to obtain NPs with an average size lower than 200 nm and PI < 0.1, character- istic of monodispersed systems, suitable to be absorbed by the gastrointestinal tract and release the drug across the BBB. The optimized formulation was obtained by adding 7.5 mg/ml of surfactant, a low polymer concen- tration and a high drug amount. NPs were washed by ultracentrifugation process and effective surfactant elimi- nation was demonstrated both by XRD and FTIR since no PVA bands were observed in the NPs profile. This sug- gests that the surfactant only acts as an adjuvant in the Figure 9b depicts the microscopic images after immu- nohistochemically staining of insoluble β-plaque devel- opment. APP untreated mice showed a greater plaque Sánchez‑López et al. J Nanobiotechnol (2018) 16:32 Page 14 of 16 Fig. 9 a Immunohistochemically (cortex) staining of amyloid plaques (green) and GFAP (red) of WT and APP/PS1 mice (untreated, MEM free and MEM loaded NPs). Bar reference equivalent to 100 µm. and b Amyloid plaques counting of APP/PS1 mice. Data represent mean ± SD; p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 Fig. 9 a Immunohistochemically (cortex) staining of amyloid plaques (green) and GFAP (red) of WT and APP/PS1 mice (untreated, MEM free and MEM loaded NPs). Bar reference equivalent to 100 µm. and b Amyloid plaques counting of APP/PS1 mice. Data represent mean ± SD; p < 0.05, p < 0.01, p < 0.001, ***p < 0.0001 NPs production, stabilizing the colloidal suspension and it is not entrapped in the polymer since it was effectively removed by centrifugation. This is an increase outcome since a high surfactant amount may induce toxicity by establishing an interconnected network with the polymer. MEM–PEG–PLGA NPs raised the Tg of the polymer, thus confirming the drug loading within the particles. Moreover, no evidence of strong bond or chemical inter- action between drug and polymer was found. MEM– PEG–PLGA NPs did not show the drug melting and decomposition process observed in the physical mixture, confirming that the drug loaded into NPs was in the form of either a molecular dispersion or in a solid solution. properties. Moreover, reduced administration frequency (on alternate days) demonstrated to be adequate to achieve brain therapeutic concentrations of drug. Conclusions Behav- ioural and histological studies of APP/PS1 and WT mice treated with NPs in alternate days showed a better effect of NPs groups against free MEM treatment improving both learning capacities and β-amyloid brain plaques on APP/PS1 animals. This can be attributed to the sustained release obtained with MEM–PEG–PLGA NPs that pro- vide a stable drug amount into the target organ. In summary, MEM–PEG–PLGA NPs could be a promising alternative towards a better treatment of AD patients since NPs have demonstrated to be capable to provide a more effective treatment than free MEM. MEM–PEG–PLGA NPs showed to be physically sta- ble upon 6 months storage both at 25 and 4 °C, being preferable 4 °C storage due to a slight NPs sedimenta- tion process observed in the backscattering profile. The developed formulation presented a slow in vitro release profile at 37 °C against free drug both fitting to hyperbola equation. This could be due to a first fast drug release (burst effect) provided by the drug accumulated onto the NPs surface, followed by a released caused by the drug entrapped into the polymeric matrix.h AD: Alzheimer’s disease; Aβ: β-amyloid; MEM: memantine; PLGA: poly(lactic-co-glycolic acid); FDA: Food and Drug Administration; NPs: nanoparticles; PEG: poly(ethylene glycol); PVA: polyvinyl alcohol; Z-AVE: mean average size; PI: polydispersity index; ZP: zeta potential; EE: encapsulation efficiency; TEM: transmission electron microscopy; Pe: permeability across cell monolayer; APP/PS1: APPswe/PS1dE9 mice. Additional file Additional file 1: Figure S1. MEM-PLGA-PEG NPs transmission electron microscopy and size distribution obtained by dynamic light scattering. Figure S2. MEM thermogravimetric and differential thermal analysis. Fig- ure S3. A) Experimental groups involved on the study, B) In vivo Timeline. Figure S4. Escape latency results of the Morris water maze test on the probe trail of the WT mice. Additional file 1: Figure S1. MEM-PLGA-PEG NPs transmission electron microscopy and size distribution obtained by dynamic light scattering. Figure S2. MEM thermogravimetric and differential thermal analysis. Fig- ure S3. A) Experimental groups involved on the study, B) In vivo Timeline. Figure S4. Escape latency results of the Morris water maze test on the probe trail of the WT mice. The in vitro and in vivo results for brain drug levels showed clear evidence that the developed systems provide a sustained delivery of the drug into the target tissue. The developed colloidal systems increase drug amount into the target organ and confirm the suitability of the NPs for oral administration attributed to the bioadhesive polymer Additional file 1: Figure S1. MEM-PLGA-PEG NPs transmission electron microscopy and size distribution obtained by dynamic light scattering. Figure S2. MEM thermogravimetric and differential thermal analysis. Fig- ure S3. A) Experimental groups involved on the study, B) In vivo Timeline. Figure S4. Escape latency results of the Morris water maze test on the probe trail of the WT mice. Abbreviations l h Page 15 of 16 Sánchez‑López et al. J Nanobiotechnol (2018) 16:32 Consent for publication Not applicable. 14. Zhu S, Chen S, Gao Y, Guo F, Li F, Xie B, et al. Enhanced oral bioavail‑ ability of insulin using PLGA nanoparticles co-modified with cell- penetrating peptides and Engrailed secretion peptide. Drug Deliv. 2016;23(6):1980–91. References 1. Nakamura Y, Kitamura S, Homma A, Shiosakai K, Matsui D. 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Author details 1 1 Department of Pharmacy, Pharmaceutical Technology and Physical Chem‑ istry, Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain. 2 Institute of Nanoscience and Nanotechnology (IN2UB), Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain. 3 Networking Research Centre of Neurodegenerative Disease (CIBERNED), Instituto de Salud Juan Carlos III, Madrid, Spain. 4 Department of Pharmacology and Therapeutic Chemistry, Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain. 5 Department of Biology and Environment, School of Life and Envi‑ ronmental Sciences (ECVA, UTAD), University of Trás-os-Montes and Alto Douro, Quinta de Prados, 5001‑801 Vila Real, Portugal. 6 Centre for Research and Technology of Agro‑Environmental and Biological Sciences, University of Trás-os-Montes and Alto Douro, CITAB-UTAD, 5001‑801 Vila Real, Portugal. 7 Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra (FFUC), Polo das Ciencias da Saúde Azinhaga de Santa Comba, 3000‑548 Coimbra, Portugal. 8 REQUIMTE/LAQV, Group of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal. y 5. 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https://openalex.org/W2291593108	https://educationaltechnologyjournal.springeropen.com/track/pdf/10.1186/s41239-016-0010-5	English	null	Applying action research in CAD teaching to improve the learning experience and academic level	International journal of educational technology in higher education	2,016	cc-by	6,847	© 2016 Gracia-Ibáñez and Vergara. 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. Applying action research in CAD teaching to improve the learning experience and academic level Veronica Gracia-Ibáñez* and Margarita Vergara Veronica Gracia-Ibáñez* and Margarita Vergara * Correspondence: vgracia@uji.es Departamento de Ingeniería Mecánica y Construcción, Universitat Jaume I, Avda. Sos Baynat, s/n, E12071 Castellón, Spain Abstract A project involving innovation in university education is described in this paper. It was implemented in a Computer-Aided Design (CAD) course on the Bachelor’s Degree in Industrial Design and Product Development Engineering. Several studies have been published about CAD curricula, yet to the best of our knowledge nothing on applying Action Research (AR) to CAD teaching. The project was carried out over a two-year period, after detecting that academic results were not very good even when the course was not very demanding. In the first year, an experiment based on the AR methodology was planned and put into practice. We took the figure of the teacher-researcher as our own, owing to our conviction that teachers themselves must study their own work and reflect on it critically. Encouraged by the good results, throughout the following year, more changes were made in order to further improve the learning experience. The main actions were to develop self-learning material and to use rubrics for assessment, together with an increase in the level of difficulty in some parts of the evaluation. A detailed chronological description of the actions that were carried out, the implied motivations and both the expected results and those actually obtained are presented. Keywords: CAD, improving academic results, level of difficulty, Action Research methodology * Correspondence: vgracia@uji.es Departamento de Ingeniería Mecánica y Construcción, Universitat Jaume I, Avda. Sos Baynat, s/n, E12071 Castellón, Spain Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 DOI 10.1186/s41239-016-0010-5 Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 DOI 10.1186/s41239-016-0010-5 Open Access Open Access Introduction The teaching of Computer-Aided Design (CAD) is such a broad field of study (Asperl, 2005) that there is no consensus about what to include in the curricula, since these dif- fer depending on the degree and on the level and age of the CAD learners. Further- more, some studies (Field, 2004) emphasise the importance of continuing evolution in the training and educational needs of users of CAD systems. From the point of view that today’s student is tomorrow’s engineer, descriptions have also been put forward re- garding what CAD curricula should be like depending on the role CAD will play in the student’s future (as a user, an application developer, a software developer or a CAD manager) (Ye, Peng, Chen, & Cai, 2004). An analysis of the situation of CAD teaching in Spain (Rubio Garcıa R, Gallego Santos R, Suárez Quirós J, Álvarez Peñın PI, 2005) showed that, since the Organic Law of Universities1 came into force, each teacher uses different methodologies and tools to teach CAD. In that study, the authors presented Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 Page 2 of 13 Page 2 of 13 the results of a survey about different issues carried out on over 600 teachers. It re- vealed that, in Spanish universities, there was no agreement on the desirable number of students in the labs, the time to be dedicated to practical sessions, the CAD software to be used or its type (parametric or not) or on the system of evaluation. Hence, the au- thors classified the ways of assessing students into three groups: by means of practical exercises that are delivered and revised on a weekly base; by means of a final examin- ation in which the student has to solve a problem by using the computer to draw a so- lution within a given time; or by means of an end-of-year assignment. The tendency seemed to be to give more weight to students’ daily work than to the traditional final examinations. On the other hand, there are also several studies that deal with improving different CAD-related abilities (spatial abilities, visualisation skills or the relationship between 2D drawings and 3D models) using different technologies and methodologies (Contero M, Naya F, Company P, Saorín JL, 2006; Contero M, Naya F, Company P, Saorín JL, Conesa J, 2005; Plumed, Calleja, Varley, & Martin, 2013). Furthermore, Rossignac (2004) presented some particular examples of teaching CAD based on what he calls education-driven research (EDR). In these examples, new concepts are introduced by mean of analogies or metaphors; expressive notation and evocative names are used to facilitate memorisation, and the focus is placed only on essential aspects, thereby sim- plifying concepts until they are well understood. One interesting aspect of teaching CAD (Chester, 2007) is the difference between command knowledge (knowing the tools provided by the software and the procedure for using them) and strategic knowledge (knowing the methods that can be used to achieve a specific task and how to choose one of them), with strategic knowledge being more effective in the development of CAD expertise, especially when no previous CAD instruction has been provided. Another important aspect in teaching CAD (Asperl, 2005) is the choice of software. Often, CAD software applications have limitations (tools fail to work the way they should, the user interface is not appropriate, the software is not affordable for students, and so forth) that impose a specific way of working. Knowing exactly how knowledge is transferred and what way of teaching is best for each type of competence will almost certainly lead to an improvement in CAD educa- tion. Teaching and researching, understood as an integrated activity, gives rise to the figure of the teacher-researcher. This figure was first noted by Stenhouse (Rudduck & Hopkins, 1985), a British pedagogue who argued that action research refers to the re- search conducted by teachers on their own practice in order to find ways to improve students’ learning. Teachers’ research, he claimed, should also aim to make a contribu- tion to the theory of education that is accessible to other teachers and enters into the critical discourse of the profession (Groundwater-Smith & Mockler, 2011). Rudduck and Hopkins (1985) developed a paradigm of action research that aimed to “demystify and democratise research”, as a response to their perception that educa- tional research was not contributing effectively to classroom practitioners. It seems pretty clear to us that the best contributions that research in education can make are the contributions that can be transferred to actual educational practice. Although this methodology is well known in the field of Social Sciences, it is still little known in most technological areas such as Engineering (Marqués Andrés & Ferrández Berrueco, Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 Page 3 of 13 Page 3 of 13 2011). The Action Research (AR) methodology (Marqués Andrés & Ferrández Berrueco, 2011; Riel & Whitehead, 2011; Riel, 2010) implies following five phases throughout its implementation: identifying the focus, i.e., what we want to improve; analysing the problem or the issues than can be improved; developing an action plan with indicators to measure the impact of the action; carrying out the actions; and, fi- nally, observing and reflecting upon it by analysing the data collected. Successful indi- cators would point out that the planned action has obtained the expected outcome. This article presents a case study in which the AR methodology was applied to a CAD course in a particular context (available software, degree, size of lab groups, and education programme). The course is taught to students in the second year of the Bachelor’s Degree in Industrial Design and Product Development Engineering at Jaume I University (UJI) in Castellón, Spain. It has been taught for three years since the degree started in the 2011/2012 academic year. The project described here was carried out for the two years following the start of the degree, after detecting that the academic results in the first year were not very good even when the level of difficulty of assessment ac- tivities was not very high. This situation is not a desirable one for a committed teacher. In the first year of its implementation (2012/2013), an experiment based on the AR methodology was planned and developed. Encouraged by good results, throughout the second year of its implementation (2013/2014), more changes were made in order to improve the learning experience. A detailed chronological description of the actions carried out, the implied motivations and both the expected results and those actually obtained are presented. Our focus was on finding methodologies to improve academic results while increasing the level of difficulty. If academic results improve, despite a higher level of difficulty of the assessment activities, the learning experience will be bet- ter and the target competences will be achieved. Even though the applied AR method- ology aims to detect local needs, the changes in methodology and evaluation that have been made are based in the principles of the most ingrained pedagogical theory of knowledge: constructivism, in its different forms (Coll, 1996). We have taken from Pia- get the idea that humans have to create their own knowledge (Zaphiris & Ioannou, 2014), from Ausubel (1968) that we have to start from prior knowledge for learning to be significant, and from Vygotsky (1978) the idea of scaffolded learning. Consequently, our results can be applied to other subjects or situations to improve the learning ex- perience. The main idea is to provide resources to help students achieve significant learning by working on their own and by making their own efforts. The action plan was intended to be carried out over two years: in the first academic year, the focus was on the practical part, whereas in the second year, the focus was also on the theoretical part. The indicators employed to decide whether the action imple- mented worked as expected were the academic results (final marks) and the students’ perceptions of the planned actions and the teachers’ perceptions of the experiment. Material and methods The CAD course is taught in coordination with three other courses of the degree. The global aims of the four coordinated courses, related to Technical Drawing and Graphic Communication, are that students have to develop the following competences and achieve the following learning outcomes: Page 4 of 13 Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 Page 4 of 13 Ability to apply spatial representation systems (multiviews and pictorial projections), and graphic representation techniques. Knowledge of descriptive geometry and metric geometry. Ability to sketch, and ability to draw and model with CAD. Skills and capabilities for engineering technical drawings. The first two competences, the ability to sketch and some skills for engineering tech- nical drawings are worked on intensively in the first year of the degree, through two one-semester courses (Technical Drawing and Graphics I & II). In the second year of the degree, in the CAD I course, the one for which we created the experience described here, students have to develop the abilities needed to draw with CAD applications and to improve their skills and capabilities for engineering technical drawings, using mainly the 2D drawing module of a CAD commercial software package. In the third year, the aim of the CAD II course is to improve those same abilities and skills but instead using 3D modelling (virtual models of products in 3D or virtual prototypes). The educational programme, or syllabus, of the course must follow certain rules with regard to content, to time dedicated to specific activities (theory and lab classes, per- sonal work) and to evaluation, which were approved in the initial definition of the de- gree and could not be changed. The content can be summarised as knowledge of CAD tools for generating, editing and sharing engineering drawings, as well as the develop- ment of technical drawings with the 2D drafting module of a widely used commercial CAD system. The study load of the course is 6 ECTS credits and Table 1 shows the number of hours set per activity, including both class attendance and personal work. Finally, with regard to the assessment of the students, the degree establishes a max- imum weight of 20 % of the final mark for practical assignments carried out at home, which implies that at least 80 % of the final mark has to be achieved through exams. Material and methods Within this context, we have detailed the CAD I curricula in a more specific list that includes typical drawing aids of CAD systems, coordinate systems and geometric trans- formations of figures, primitives in CAD (groups, layers, advanced primitives such as patterns, text and tables), graphic formats, storage and exchange of information, di- mensioning and parametric drawing, curves, schematic drawings, project drawings and an introduction to 3D. Table 1 Activities scheduled for the CAD I course Activities Classroom hours Personal work hours Specific activity (proposed by teachers) Theory 12 0 Theory sessions (12 x 1 hours/week) Practical part (laboratory) and Tutoring 37 + 1 0 Practical sessions (≈13 x 3 hours/week) including project tutoring Evaluation 10 0 Practical evaluation (2 x 3 hours) + Final Exam (4 hours) Personal work 0 60 Weekly voluntary exercises (14 weeks x 2.5 hours/ week) + Graphic Project (25 hours) Exam preparation 0 30 Theoretical concepts study, solving previous year’s exams Total hours (number credits * 25) = 150 60 90 Table 1 Activities scheduled for the CAD I course Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 The product selected is a toy car powered by a friction motor Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 Page 6 of 13 Page 6 of 13 Ken Bain (Bain, 2007), we do not consider good teachers to be those who prepare their students well for an exam, but instead those who expect their students to achieve valu- able learning, and that is what we pursue. In the first year of the experiment, we fo- cused on the practical part and greater importance was given to the graphic project in order to reach the desired competences. Both the practical part and the graphic project bear a closer relationship to real problems that students are likely to find in their pro- fessional lives. Our five AR phases were as follows: Our aim was to find methodologies to improve academic results while increasing the level of difficulty of the assessment activities. If academic results improve, despite a higher level of difficulty, the learning experience will be better and the target compe- tences would probably be achieved (phase 1). On analysing the problem (phase 2), the action plan (phase 3) was intended to be de- veloped over two years. In the first year, the focus was on the practical part of the sub- ject, whereas the second academic year also focused on the theory part. Basically, two actions were carried out (phase 4): making changes in the methodology and increasing the level of difficulty of the assessment activities. From the results ob- tained, an in-depth reflection was made (phase 5) and the conclusions shown in this work were drawn. Changes in the methodology were taken into account by providing the students with more learning resources. Throughout the first year, some tutorials (Fig. 2) were devel- oped for the practical sessions as learning support material. More than 25 tutorials Fig. 2 Example of a tutorial provided for the practical sessions. Content is not relevant due to its specificity on the CAD I subject. It is shown in order to point out the methodology used in solving problems. The steps always follow the same pattern: statement of the problem, strategy to solve the problem, detailed procedure and conclusions to highlight important learned things to remember. Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 Page 5 of 13 Page 5 of 13 Table 1 also shows the methodology we followed for each time period and activity. Time spent on attending classes consists of theory sessions presenting general concepts of CAD according to the detailed curricula, and the related practical sessions where those concepts can be applied. The software used is AutoCAD©, which is available at the university with a campus licence and students can also have a legal copy through the university. In the practical sessions, certain problems are proposed, the commands of the program needed to solve them are presented, and the different ways of solving them are discussed in terms of efficacy to ensure that strategic knowledge can be achieved. Personal work at home includes weekly voluntary exercises (similar to the problems proposed in the lab session) and a mandatory graphic design project, in which technical drawings of an assigned existing product have to be produced (Fig. 1) as an end-of-year assignment. Given that the weight of the exams in the final mark is so high, there are two mid- term practical ones and a final theory and practice one. Hence, the final mark is com- posed of the mark obtained in the exams and the mark for the graphic project. During the course prior to carrying out the project, several issues were found: Listening to the students, it became quite clear that there was a shortage of learning material. From the results obtained, especially the graphic design projects handed in, it was easy to deduce that these results were unsatisfactory, even though the level of difficulty of the assessment activities was not very high. Hence, we felt compelled to do something. It is important to note that we did not feel compelled to do something because of the desire for students to have good grades. Like Fig. 1 Example of a technical drawing from a graphic project (reproduced with the student’s permission). The product selected is a toy car powered by a friction motor Fig. 1 Example of a technical drawing from a graphic project (reproduced with the student’s permission). Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 The tutorials were provided in pdf format and each rectangle in this figure corresponds to a page of the pdf Fig. 2 Example of a tutorial provided for the practical sessions. Content is not relevant due to its specificity on the CAD I subject. It is shown in order to point out the methodology used in solving problems. The steps always follow the same pattern: statement of the problem, strategy to solve the problem, detailed procedure and conclusions to highlight important learned things to remember. The tutorials were provided in pdf format and each rectangle in this figure corresponds to a page of the pdf Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 Page 7 of 13 Page 7 of 13 Page 7 of 13 were produced, ranging from 9 to 38 pages. Each tutorial formulates a problem and ex- plains a strategy to solve it and how to do it step by step, emphasising the use of the most appropriate tools in CAD software to achieve it in a more efficient way. Thus, the students could work on their own. They could also review those aspects that had been practised in class. Hence, the action developed was aimed at giving students more in- formation to allow them to undertake self-learning. The experiment focused on con- structivist learning, self-learning and meaningful learning (Novak, 2004) through solving problems/real cases (Coll, Mauri, & Onrubia Goñi, 2006) as methodologies to produce improvements. This should presumably lead to higher academic performance. Encouraged by the good results obtained in the practical skills in the first year (for a description, see below under Results), the second year of the study was devoted to im- proving the theoretical skills, also by creating more material or improving it. More than 15 detailed documents were produced, ranging from 15 to 59 pages. In this case, be- sides the written support for theoretical classes, weekly online self-correcting question- naires (Fig. 3) were developed as a support to the continuous learning of theoretical concepts. Furthermore, as supplementary material to help in focusing on the objectives, in the first year, the assessment criteria for the practical exams were clearly specified and ex- plained to students, and in the second year, a rubric for the assessment of the graphic project was also provided (Fig. Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 4), together with the formulation of the project in order to better guide its execution. It has already been demonstrated that it is of utmost im- portance to specify the assessment criteria clearly (Sanmartí, 2007). Regarding the second action carried out, we increased the level of difficulty in two di- rections, by means of insisting on higher quality in the graphic project and also by re- quiring a minimum mark. In the first year of its implementation, we increased the difficulty of the assessment activities, both in terms of the quality of the graphic design project and in the exams. The level of difficulty for the project was increased by requiring more quality and con- tent and, in the exams, by requiring a minimum mark of 4 out of 10 in each part. In the second year, the requirement of 4 out of 10 was also added in the project. Fig. 3 Example of some of the questions on the weekly online self-correcting questionnaires (12 questionnaires with 7 to 14 questions in each one) Page 8 of 13 Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 Fig. 4 Rubric for the Graphic Project To encourage the students, a higher weight was given to the project in the final mark by increasing the percentage up to the maximum allowed, that is to say, 20 %. All these conditions are listed in Table 2. Objectivity was taken into account by triangulating data when establishing the indica- tors to decide whether the actions implemented gave the expected results: academic outcomes (final marks), students’ perceptions of the planned actions and teachers’ per- ceptions of the experiment. Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 Page 9 of 13 Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 Page 9 of 13 Page 9 of 13 Table 2 Evolution of assessment over the years of implementation First call (January) Second call (June) Year PE (%) GP (%) TP (%) PP (%) Additional requirements PE (%) GP (%) TP (%) PP (%) Additional requirements Previous year 10 10 32 48 32 68 First year 10 20 25 45 TP ≥4 25 75 TP ≥4 PP ≥4 PP ≥4 Second year 10 20 25 45 TP ≥4 20 25 55 TP ≥4 PP ≥4 PP ≥4 GP ≥4 PE Practical exercises during course (2), GP Graphic Project TP Theory part of the final exam, PP Practical part of the final exam Table 2 Evolution of assessment over the years of implementation In the case of academic outcomes, an increase in the number of students who pass or an increase of marks would indicate that the actions implemented gave the expected results. First, Pearson’s Chi Square tests (Siegel, 1988) were applied to check significant differences in the percentage of students who did or did not pass in the different years. Second, the final marks neither follow normal distributions for the years of implemen- tation (before or after the experiment) nor present homogeneity of variances. There- fore, instead of an ANOVA, a Kruskal-Wallis test (Siegel, 1988) was performed to check whether the distribution of the final marks in the three years was significantly different (dependent variable: the final marks obtained [best mark of each call for all the students, those who passed or not]; independent variable: year of implementation). The students’ and the teachers’ perceptions were also taken into account. A survey was conducted in order to include the students’ perceptions. The questions included dealt mainly with the usefulness of the learning materials provided and their opinions about the methodology as a whole. In particular, three questions asked about the tuto- rials of the practical sessions, three about the graphic project and three about the on- line self-correcting questionnaires. A Likert scale from 1 to 5 was used. Furthermore, open-ended questions were included for all the three aspects and for the midterm exams. Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 All the teachers of the course (three or four depending on the year) had previ- ous and ongoing meetings to coordinate methodology and evaluation criteria and, above all, a final meeting to discuss their individual perceptions about the implementa- tion of the experiment. Results Data obtained from the three viewpoints are presented below. Figure 5 shows a graph with the percentage of students who passed (out of those who took the exams). An initial Pearson’s Chi Square test, with the three years and the number of students who passed or failed, produced a significant value of p = 0.049 indi- cating that a significant statistical difference exists. Therefore, to check which years were different (post-hoc), three subsequent Pearson Chi Square tests were performed including each pair of years (to have 2×2 contingency tables). The significant values were 0.017 for Previous/First Year, 0.724 for Previous/Second Year, and 0.042 for First/ Second Year, which means that statistical differences in the percentage of students that passed the exams exist between the first year of implementation and the other two. Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 Page 10 of 13 Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 Page 10 of 13 Page 10 of 13 Fig. 5 Graph of the percentage of students (out of those who took the exams) who have passed over the years Fig. 5 Graph of the percentage of students (out of those who took the exams) who have passed over the years Table 3 shows descriptive statistics of marks across the years of implementation. The results of the Kruskal-Wallis test showed significant differences in the statistical distri- bution of the marks between the previous year and the other two. The significant values were <0.001 for Previous/First Year, <0.001 for Previous/Second Year, and 0.821 for First/Second Year. The existence of significant differences between the previous year and the other two years of implementation can be inferred. Besides the academic results, especially the final marks, two more indicators were also considered: teachers’ observations and students’ perceptions. Figure 6 shows the results of the survey conducted on the students, aggregated by methodology (practical tutorials for practical sessions, graphic project and the ques- tionnaires for theory concepts): the confidence interval of mean opinions over the two years of the implementation of the plan. The open-ended questions also offered some good insights. The main complaint reflected in them was about the (according to them, still too small) weight of the pro- ject in the final mark. Discussion From the results obtained and in relation to the first indicator, it can be observed that the percentage of students who passed at all the exam calls increased significantly in the first year of implementation (Fig. 5 and statistical test). However, the percentage of Table 3 Descriptive statistics of marks: mean and standard deviation, along with quartiles for all students (those who passed and those who did not, considering their best mark in both calls) based on a total of 10 points Year Mean SD P25 P50 P75 Previous year 5.2 1.2 5.0 5.3 5.9 First year 5.9 1.2 5.1 6.1 6.7 Second year 5.9 1.5 5.0 6.0 7.0 Table 3 Descriptive statistics of marks: mean and standard deviation, along with quartiles for all students (those who passed and those who did not, considering their best mark in both calls) based on a total of 10 points p Year Mean SD P25 P50 P75 Previous year 5.2 1.2 5.0 5.3 5.9 First year 5.9 1.2 5.1 6.1 6.7 Second year 5.9 1.5 5.0 6.0 7.0 Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 Page 11 of 13 Fig. 6 95 % CI for the mean of the questionnaire. The scale was a Likert scale from 1 to 5 Fig. 6 95 % CI for the mean of the questionnaire. The scale was a Likert scale from 1 to 5 students who passed at the first call in that case was reduced, probably due to the new requirement of the minimum mark of 4 in the theoretical exam without any additional support and the higher level of difficulty in the exam. For the second year of the experi- ment, the level of difficulty in the theory part was increased, because we expected a better result on the weekly online questionnaires. The number of students who passed increased in comparison to the previous year, but decreased if compared with the first year. This result reinforces the idea that the changes made produced a benefit because of a significant increase of the students who passed among those who took the exam, especially in the first year of the implementation. Also worthy of note is that despite the more demanding requirements, the percentage of students who passed and the average mark both increased as a result of the imple- mentation of these methodologies. Discussion However, what attracts the most attention in this case is that the standard deviation increased in the second year. This may be due to a different reaction of the students when increasing the level of difficulty: those who make a greater effort get higher marks, but those who do not get lower ones; hence there is more variation in the marks. The results from the Kruskal-Wallis test, showing significant differences between marks of the previous year (before the implementation of the experiment) and marks of the first and second years (after the implementation of experiment) reinforces the fact that the experiment carried out produced significant improvements. The results are in accordance with the ones expected according to literature. The ac- tions carried out tried to support students by means of scaffolding their learning (Vygotsky, 1978) by facilitating ways to create their knowledge. Our purpose was far from simply giving the students knowledge to be accepted and memorised. Instead, it was to give them strategies and resources to make them create their own knowledge and to enable them to use it to solve problems. In Piaget’s words: “… education means Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 Page 12 of 13 making creators… You have to make inventors, innovators—not conformists” (Bringuier, 1980). Actions taken in this sense seem to have worked properly. If we take a look at the students’ responses to the survey, they indicate a clear accept- ance of the methodology. It is stimulating to see that the students’ perceptions more or less coincide with the teachers’ perception. The teachers observed an improvement in receptivity to the subject by the students and a clear improvement in the level, which was reflected above all in the project in which the level of quality increased, as more complicated products were successfully represented (as can be seen in Fig. 1). Obvi- ously, the students’ higher implication and effective work achieved with these actions have been accompanied by a great deal of effort by the teachers. We strongly believe that good levels of coordination among teachers, hard work in preparing valuable ma- terial or a thorough description of the evaluation criteria are some of the key points for success. However, all this hard work is highly rewarded when an improvement in the quality of learning and an increase in the students’ satisfaction are observed. Conclusions From the experiments carried out, it can be deduced that the methodology employed has produced the expected effects, which means an improvement in academic results despite a higher level of difficulty of the assessment activities. It is remarkable that the perception that students have of the subject has also been improved. Although the ex- perience has been developed in teaching CAD, the methodology may be applied to any specific subject or country. As a final conclusion we would say that by: Providing varied self-learning material Detailing evaluation criteria in each part (including the use of rubrics) Requiring a higher level (quality, complexity) Demanding minimum marks, but giving a mark for everything better academic results can be achieved, together with a good level of appreciation by students. Endnotes 1The Organic Law of Universities came into force in 2001 in Spain for defining, among other aspects, the rules for Spanish Universities to adapt their degree courses to the requirements of the European Higher Education Area (Bologna Process). Authors information Verónica Gracia-Ibáñez is a lecturer in Computer-Aided Design at Jaume I University (UJI) in Castellón, Spain. Her professional background has been in the field of industrial engineering projects. Since her engagement as a part- time lecturer in 2011, she has developed an interest in researching both in the field of biomechanics, in which she is currently working on her PhD thesis, as well as in the field of education, as shown by her participation in two educational innovation projects, two publications at international congresses on education and the co-authorship of a textbook. Margarita Vergara is Full Professor in Computer-Aided Design and Ergonomics at Jaume I University (UJI) in Castellón, Spain. Her professional research interests are biomechanics of the hand and the ergonomic evaluation of product design, as well as ways to improve her teaching. Thus, she has taught Computer-Aided Design and Graphics in different Engineering Degrees for more than 20 years and has authored 18 textbooks. As a result of her interest in improving teaching, she has published one paper in an international peer-reviewed journal and presented 18 communications at congresses and conferences on teaching. Authors’ information ó bá Page 13 of 13 Page 13 of 13 Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 Gracia-Ibáñez and Vergara International Journal of Educational Technology in Higher Education (2016) 13:9 References Asperl, A. (2005). How to teach CAD. Computer-Aided Design and Applications, 2(1–4), 459–468. doi:10.1080/16864360. 2005.10738395 Ausubel, D. P. (1968). Educational Psychology: A Cognitive View. 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Designing and Developing Novel Learning Experiences. doi:10.1007/978-3-319-07482-5 Submit your manuscript to a journal and beneﬁ t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the ﬁ eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com Submit your manuscript to a journal and beneﬁ t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the ﬁ eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com Submit your manuscript to a journal and beneﬁ t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the ﬁ eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com
https://openalex.org/W3217122706	https://ejournal.upi.edu/index.php/cakrawaladini/article/download/40304/16877	English	null	LOCAL WISDOM IN LARO NG LAHI AS FOUNDATION IN GAME-BASED PEDAGOGY	Cakrawala Dini	2,021	cc-by-sa	8,950	LOCAL WISDOM IN LARO NG LAHI AS FOUNDATION IN GAME- BASED PEDAGOGY Roel V. Avila1 1Professor, Philippine Normal University South Luzon Abstract: Filipino traditional games (Laro ng Lahi) are reflective of the lifestyle of a locality and form part of significant national heritage. Albeit modern technology seems to challenge the existence of Laro ng Lahi, these traditional games can actually be transformed to adapt the present demands of the cyberspace. Employing the hermeneutic phenomenology and educational research & development, the author paired the past investigations with his observations though the use of Hermeneutic Cycle as guide. Data obtained from the online survey accomplished by thirty-one (31) pre-service teachers (Generation Z, selected using the purposive sampling) provided connection between the online documents and the local practices and lived experiences of the respondents. Respondents are still familiar with piko (taplak gunung), agawan kuta (bentengan), and tubigan (gobak sodor) since they played such games before and they were aware of the the Laro ng Lahi benefits. Thus, they preferred to have Laro ng Lahi as activity tools in school subjects, and to convert academic knowledge and skills into learning course lessons. Upon finalizing the mechanics of each of the three Filipino traditional games, and the data provided by the respondents, the researcher conceptualized the game-based pedagogy and developed lessons using the local wisdom as the basis. p g Keyword: local wisdom, traditional games, laro ng lahi, foundational basis, game-based pedagogy Abstrak: Permainan tradisional Filipina (Laro ng Lahi) merefleksikan gaya hidup suatu daerah dan merupakan bagian dari warisan nasional yang signifikan. Meski teknologi modern seolah menantang keberadaan Laro ng Lahi, permainan tradisional ini sebenarnya dapat ditransformasikan untuk mengadaptasi tuntutan dunia maya saat ini. Tujuan penelitian ini adalah mengembangkan pelajaran dari kearifan lokal yang digali dari permainan tradisional lokal. Metode yang digunakan yaitu fenomenologi hermeneutik dan R&D, penulis memasangkan penyelidikan masa lalu dengan pengamatannya melalui penggunaan Siklus Hermeneutik sebagai panduan. Data yang diperoleh dari survei online yang dilakukan oleh tiga puluh satu (31) guru pra-jabatan menyediakan hubungan antara dokumen online dan praktik lokal dan pengalaman langsung responden. Temuan: Responden masih mengenal piko (taplak gunung), agawan kuta (bentengan), dan tubigan (gobak sodor) sejak mereka memainkan permainan tersebut sebelumnya dan mereka mengetahui manfaat Laro ng Lahi. Oleh karena itu, mereka lebih memilih Laro ng Lahi sebagai alat aktivitas dalam mata pelajaran sekolah, dan untuk mengubah pengetahuan dan keterampilan akademik menjadi pelajaran kursus dalam pembelajaran. Cakrawala Dini: Jurnal Pendidikan Anak Usia Dini \| p-ISSN 2087-1317 \| e-ISSN 2621-8321 Vol. 12. No.2 November 2021 \| Hal 107-124 Cakrawala Dini: Jurnal Pendidikan Anak Usia Dini \| p-ISSN 2087-1317 \| e-ISSN 2621-8321 Vol. 12. No.2 November 2021 \| Hal 107-124 Kata Kunci: Kearifan lokal, Permainan tradisional, laro ng lahi, basis dasar, Permainan berbasis pedagogik 1 Philippine Normal University South Luzon, Email: pnusl.avila.rv@gmail.com Roel V. Avila: Local Wisdom in Laro Ng Lahi As Foundation in Game-Based Pedagogy 107 1. Background of the study 1. Background of the study 1. Background of the study Stuck on their gadgets that cause the decline of their engagement in physical activities offered by traditional games or Laro ng Lahi as Filipinos call them (Aguado, 2012), the children rarely play traditional games due to the advancement of technology (Bakar, et al., 2008; Estole, 2018; Miller & Kuhaneck, 2008). Thus, UN encourages each country to preserve its cultural treasure (UNESCO, 2004). b. Which of the given Filipino traditional games have they experienced playing? c. Do the respondents’ agree on the benefits the Filipino traditional games provide? d. Do the pre-service teachers prefer to have Filipino traditional games as activity tools in school subjects? e. Do the pre-service teachers agree to convert academic knowledge and skills gained through Laro ng Lahi into learning course lessons? Traditional games can still be preserved (Khan, et al., 2018) by employing continual engagement in the respective houses and integrating such in the classrooms (Prestoza, et al., 2020) by using ICT so that the new generations can understand (unesco.org, 2015). f. Which among the given Filipino traditional games are the respondents’ favorite game? g. What are the reasons why the respondents picked their respective favorite Laro ng Lahi? The problem lies on the failure of the kids of Generation Z (Cobanoglul, Tagrikulu, & Gul, 2018) to get health benefits provided by the traditional games (WHO, 2018) which are beneficial to a child’s growth and development, cultural values and teaching practices (Kun, & Mat Nayan, 2018; Prestoza, et al., 2020). Laro ng Lahi is an escape for a stressful day, gives happiness to minds, and improves health (Prakash, 2012). All these show that engagement in traditional games is highly beneficial. There has to be some ways on how to preserve these indigenous games to suit the characteristics of the new generation. h. What local wisdom can serve as bases in the development of game-based pedagogy? 3. Scope and Limitation This investigation covers the Filipino Traditional Games or Laro ng Lahi and the latent local wisdom therein in the light of Filipino culture to serve as foundational basis of developing a game- based pedagogy aimed at enhancing academic knowledge and skills. Due to pandemic, the data were gathered through online survey instrument accomplished by select pre-service teachers. Only three Laro ng Lahi, namely piko, agawan kuta, and tubigan were selected for the sake of focus and brevity. The researcher decided to engage in extracting the local wisdom in Laro ng Lahi as basic foundation in developing game-based pedagogy LOCAL WISDOM IN LARO NG LAHI AS FOUNDATION IN GAME- BASED PEDAGOGY Kesimpulan: Setelah menyelesaikan mekanisme masing-masing dari tiga permainan tradisional Filipina, dan data yang diberikan oleh responden, peneliti membuat konsep pedagogi berbasis permainan dan mengembangkan pelajaran dengan menggunakan kearifan lokal sebagai dasar. Kata Kunci: Kearifan lokal, Permainan tradisional, laro ng lahi, basis dasar, Permainan berbasis pedagogik 1 Philippine Normal University South Luzon, Email: pnusl.avila.rv@gmail.com Roel V. Avila: Local Wisdom in Laro Ng Lahi As Foundation in Game-Based Pedagogy 107 INTRODUCTION gunung), agawan kuta (bentengan), and tubigan (gobak sodor) the pre- service teachers are familiar with? gunung), agawan kuta (bentengan), and tubigan (gobak sodor) the pre- service teachers are familiar with? RESULTS AND DISCUSSION Familiarity with the Select Laro ng Lahi. No respondents are not familiar with piko, agawan base, and tubigan. Figure 1. Pre-service Teachers’ Familiarity with piko (taplak gunung), agawan kuta (bentengan), and tubigan (gobak sodor) 1. Researach Design The author employed the hermeneutic phenomenology based on the existing documents which are paired with the author’s observations (Mays & Brown, 1972; Kneller, 1984), and the participants’ lived experiences. Employing the Hermeneutic Cycle, the author engaged in 1) Reading and viewing To present, discuss, and extract local wisdom from the select Philippine traditional games to come up with the formal statement on their significance as foundational basis in developing game- based pedagogy. a. Which among the given Philippine traditional games such as piko (taplak a. Which among the given Philippine traditional games such as piko (taplak 108 Cakrawala Dini: Vol. 12 No. 2, November 2021 of the Documents; 2) Reflective Writing; and 3) Interpreting (Kafle, 2011) or explicitation of data. Afterwards, the researcher used Borg and Gall’s (2003) educational research and development (R&D) in developing game-based pedagogy. It was tested during the Summer Program 2021 international conference hosted by Universitas Pendidikan Indonesia on September 1-11, 2021. 2. Participants Data obtained from the online survey accomplished by thirty-one (31) pre-service teachers provided connection between the online documents and the local practices and lived experiences of the respondents. Being members of the Generation Z (and students of Foreign Language), they were selected using the purposive sampling based on the geographical location of their home municipalities. Figure 1. Pre-service Teachers’ Familiarity with piko (taplak gunung), agawan kuta (bentengan), and tubigan (gobak sodor) Playing the games means familiarity with such games. Compared to the study of Asuncion (2019) which revealed that 56.66 percent of research participants still play traditional Filipino games, all respondents in the present study provided a much higher percentage (100%), proving the claim of Prestoza, et al. (2020) that Laro ng Lahi are still played by children despite their access to advanced technologies. Prestoza, et al., (2020) further expounded that the elementary school teachers usually observe these Laro ng Lahi that are still played by pupils: Tumbang Preso, Sipa, Patintero, Tagu-Taguan, Chinese Garter, Sungka, Luksong Tinik, Luksong Baka, Holen, 3. Research Instrument 3. Research Instrument Modifying Kun and Mat Nayan’s (2018) survey questions, the researcher contextualized the instrument and supported each item with related literature. The researcher used descriptive coding using the thematic analysis by identifying, analyzing, and interpreting patterns of meaning within the qualitative data as similarities and differences of responses are highlighted (Brenowitz & Tuttle, 2003; Hermans, et al., 2008; Hu, et al., 2003; King, 2004) Tug of War, Agawang Sisiw, Bum- Bum Lata, Agawan ng Panyo, Agawan ng Base, and Siyato. 4. Development of Course Lessons based on Laro ng Lahi Upon finalizing the mechanics of each of the three Filipino traditional games, and the data provided by the respondents, the researcher conceptualized the game-based pedagogy and developed lessons using the local wisdom as the basis. Upon finalizing the mechanics of each of the three Filipino traditional games, and the data provided by the respondents, the researcher Roel V. Avila: Local Wisdom in Laro Ng Lahi As Foundation in Game-Based Pedagogy 109 Roel V. Avila: Local Wisdom in Laro Ng Lahi As Foundation in Game-Based Pedagogy 109 Figure 3.1 Benefits on body and brain gained through Laro ng Lahi Experienced Playing Laro ng Lahi. Figure 2. Experiences in Playing the Select Laro ng Lahi The respondents showed 100% agreement to the specified benefits. Today’s kids fail to get health benefits provided by the traditional games. In order to attain the cardiorespiratory and muscular fitness, bone health, and cardiovascular and metabolic health biomarkers, child’s growth and development, cultural values and teaching practices as benefits of playing traditional games (WHO, 2018; Kun, & Mat Nayan, 2018) children must be guided to accumulate at least 60 minutes of physical activity daily. Playing the Laro ng Lahi develops motor skills, body coordination, exercise locomotor, apply skill-related activities, and enhance mental capacity (Prestoza, et al., 2020), besides, Laro ng Lahi is an escape for a stressful day, gives happiness to minds, and improves health (Prakash, 2012). Figure 2. Experiences in Playing the Select Laro ng Lahi All respondents experienced playing the select games. People value their experiences because they were able to connect the old generation to them (young generation), socialize with and create sense of togetherness, integrate friends and families in workplace, neighborhood, and generations, and open up the mind to diverse perspectives (Khan, et al., 2018). Playing the games of the old generation by the new generation is cultural preservation that needs scaffolding. Despite being members of Generation Z, the respondents have both the technological expertise and physical activeness by playing traditional games, contradicting the claim of Cobanoglul, et al., (2018). However, this gap between Generation X and Generation Z is continuously widening due to the gadgets the latter have. Benefits from Laro ng Lahi on Personal Discipline and Socialization Figure 3.2. Benefits on personal discipline and socialization gained through Laro ng Lahi Figure 3.2. Benefits on personal discipline and socialization gained through Laro ng Lahi Figure 3.2. Benefits on personal discipline and socialization gained through Laro ng Lahi Benefits on Body and Brain Gained through Laro ng Lahi Benefits on Body and Brain Gained through Laro ng Lahi 110 Cakrawala Dini: Vol. 12 No. 2, November 2021 Personal discipline and socialization are learned by the 96.8% of the respondents. Traditional games, as talking games, are capable of improving students’ communication and collaborative skills (Suhaebah, 2019; Kolb, 1984). As children are entertained in their engagement in games, cognitive (Piaget, 1962; Vygotsky, 1966) and psychosocial developments (Elkind, 2007; Erikson, 110 Cakrawala Dini: Vol. 12 No. 2, November 2021 110 Cakrawala Dini: Vol. 12 No. Experienced Playing Laro ng Lahi. 2, November 2021 1950) occur. Playing game is an indispensable part of children’s development (Foley, 2008) and is the springboard to the attainment of some universal values (Aypay, 2016). Figure 4. Preference to have Filipino traditional children’s games as activity tools in any school subject. Respondents wanted to have Filipino traditional games as activity tools in any school subject (90.3%). Not connecting the games played outside by the learners to the lessons inside the classroom contributes to the boredom which they experience. If the claim of Aguado (2012) is true that traditional Filipino street games are alive in the Philippines, the teachers have to utilize them for increased motivation. While members of Generation Z are still into traditional games (Asuncion, 2019; Khan, et al., 2018), the teachers can grasp the opportunity to use them (Prestoza, et al., 2020) even in teaching values (Aypay, 2016), for the sake of their happiness and health (Elkind, 2007), by helping to improve the children’s basic abilities (Saputra & Ekawati, 2017), by tapping the media (Kun & May Nayan, 2018), by preserving them (Estole, 2018; Magna Kultura, 2012), by knowing the kids’ play preferences (Miller & Kuhaneck, 2008), and to develop learners’ communication and collaborative skills (Suhaebah, 2019). Related literature and studies strongly support the idea of making traditional games as activity tools in teaching school subjects. 112 Cakrawala Dini: Vol. 12 No. 2, November 2021 Benefits from Laro ng Lahi on Building Team Spirit Benefits from Laro ng Lahi on Building Team Spirit Figure 3.3. Team spirit gained through Laro ng Lahi Figure 3.3. Team spirit gained through Laro ng Lahi Almost all (96%) of the respondents agreed that Laro ng Lahi enhance team spirit. Studies have shown that traditional games can play a significant role in children’s growth and development, which help to improve the basic skills of children, such as physical health, thinking skills, interpersonal and social skills (Saputra, 2017; Maryani, 2009). Laro ng Lahi establishes children’s self- confidence, positive attitude, sportsmanship, camaraderie, and friendship (Prestoza, et al., 2020). By playing, socialization skills are honed and the concept of “others” as different from their selves is discovered. Conversion of Academic Knowledge and Skills Gained through the Laro ng Lahi into Learning Course Lessons Figure 5. Preference on the conversion of knowledge and skills gained through Laro ng Lahi into learning course lessons. Laro ng Lahi as Activity Tools in School Subjects. Roel V. Avila: Local Wisdom in Laro Ng Lahi As Foundation in Game-Based Pedagogy 111 Figure 5. Preference on the conversion of knowledge and skills gained through Laro ng Lahi into learning course lessons. Figure 5. Preference on the conversion of knowledge and skills gained through Laro ng Lahi into learning course lessons. Figure 5. Preference on the conversion of knowledge and skills gained through Laro ng Lahi into learning course lessons. Roel V. Avila: Local Wisdom in Laro Ng Lahi As Foundation in Game-Based Pedagogy 111 Roel V. Avila: Local Wisdom in Laro Ng Lahi As Foundation in Game-Based Pedagogy 111 A big chunk of responses (83.9%) supports the idea of converting knowledge and skills gained through Laro ng Lahi into learning course lessons. The other chunk (16.1%) implies questioning how it can be done. Designing and validating Laro ng Lahi as game-based academic activity are the foci of some researchers (Del Carmen, et al., 2015). Morales (2017) explored indigenous games as basis in Physics activities, Suhaebah (2019) in teaching Social Studies, conforming to UNESCO (2018) in its desire to mobilize countries to incorporate traditional children’s games in classrooms, which Estole (2018) followed by recommending for Laro ng Lahi inclusion to DepEd programs. Respondents’ Favorite Games Respondents’ Favorite Games Figure 6. Respondents’ favorite Filipino traditional game Figure 6. Respondents’ favorite Filipino traditional game Respondents’ favorite games as to rank are Tubigan (54.8%), Piko (25.8%), and Agawan Base (19.4%). Owing to the nature of Tubigan - collaborative, exciting, challenging, gender-fair, and movement-based, Tubigan becomes the favorite game. Miller and Kuhaneck (2008) stressed the importance of considering the perceptions of the players as well as their play preferences. It is a sound action of a teacher to always ask for students’ game preferences. 112 Cakrawala Dini: Vol. 12 No. 2, November 2021 Table 1. Reasons for Picking Favorite Laro ng Lahi FAVORITE LARO NG LAHI REASON (IN THEMES) PIKO − memories with friends and cousins − does not consume much energy − simplicity of the game − players’ persistence − helps body to be active − mind-body coordination − enhances discipline − requires focus AGAWAN BASE (KUTA) − a form of body exercise − enhances leadership skills − promotes sportsmanship − helps think of tactics − promotes team spirit (camaraderie) − can involve more players to have more fun − fun and exciting − develops communicative skills TUBIGAN (PATINTERO) − develops personality − helps possess strategic thinking − fun and challenging − exciting − develops alertness − requires team coordination − improves socialization skills − allows creativity − often played − improves cognitive and psychomotor skills − Requires agility and flexibility − enhances communicative skills − player needs to think fast and be alert every time Table 1 contains the words of the respondents as answers to the question – What are your reasons for picking the f it L L hi? Th Tubigan); they are entertaining; they reflect local culture; they are in tune with the existing living styles of the locality (Kh t l 2018) It i tl − memories with friends and cousins − does not consume much energy − simplicity of the game − players’ persistence − helps body to be active − mind-body coordination − enhances discipline − requires focus TUBIGAN (PATINTERO) − Requires agility and flexibility − enhances communicative skills − player needs to think fast and be alert every time Tubigan); they are entertaining; they reflect local culture; they are in tune with the existing living styles of the locality (Khan, et al., 2018). It is consequently recommended that these three traditional games be the subjects of game-based pedagogy. Respondents’ Favorite Games Table 1 contains the words of the respondents as answers to the question – What are your reasons for picking the favorite Laro ng Lahi? The answers were reduced into themes. Piko, Agawan Base, and Tubigan are favorite games because they are not much physically demanding; they do not require more resources (especially Roel V. Avila: Local Wisdom in Laro Ng Lahi As Foundation in Game-Based Pedagogy 113 Roel V. Avila: Local Wisdom in Laro Ng Lahi As Foundation in Game-Based Pedagogy 113 Table 2. Local Wisdom in Piko as Foundational Basis in the Development of Game-based Pedagogy GLOBAL GAME LARO NG LAHI LOCAL WISDOM GAME-BASED PEDAGOGY Hopscotch PIKO Homestead Learning New Language Game’s Objective Owning each box Occupying field To win each category Players Individual (M or F) Ideal Number = 5 Individual Talent & Strategy Reviewing Lessons Materials Needed: Clearing tool Plain ground (tiled, cemented, soil) Line markers. Chalk, tape, string, Pamato (Pucks) stone, piece of jar, slipper. Unoccupied field Land markers Module Powerpoint Slides Access to Virtual Room Rules: 1.Join the manohan 2. Respect succession of turns 3. Pamato must be identical in size 4. No stepping on the line 5. Don’t touch the ground 6. Don’t step on other’s house. 7.Wait for your turn 1. Daig ng maagap ang masipag (Being early bests being hardworking) 2. Concept of “Nauna” (As in ordinal position among siblings) 3. Use the right tool 4. Respect ecosystem. 5. No unnecessary cutting of trees 6. Respect private property 7. There is learning in waiting by observing. 1. First to answer question 2. Type on the chat box No use of translator Don’t answer other’s Q How to play the game: 1. Join the manohan. 2.Wait for your turn 3. Throw your Pamato on Box 1; Hop to Box 2; jump to Boxes 3 & 4; Hop to Box 5; jump to Boxes 6 & 7; Hop to Box 8 and turn around. Retrace your path; reach for Pamato; skip Box 1; land on Starting Line; Turn back on Playing field; gently throw pamato; mark your house where pamato landed; rest and wait for Round 2. 114 Cakrawala Dini: Vol. 12 No. 2, November 2021 Respondents’ Favorite Games Concept of “Dayo” Clearing the field the right way Nasa Dios ang awa, nasa tao ang gawa Throwing of pamato (Gawa) Where pamato landed (Awa) Resting (Ginhawa) Waiting (Pagbibigay-daan) Balik-bayan = Bayani Field is owned by worthy owner Determining the best caretaker Owned by its tiller Recognized Answer each Q correctly Pass through all levels Reserve appeal Claim the selected Category Pedagogy. Piko has a resemblance to the global game called Hopschotch. Obviously, hopscotch is cutting a large Table 2 shows four columns with the headings Global Game, Laro ng Lahi, Local Wisdom, and Game-based 114 Cakrawala Dini: Vol. 12 No. 2, November 2021 114 Cakrawala Dini: Vol. 12 No. 2, November 2021 area into small pieces (Whiteman, 2005). To apply its basic concept locally, Piko could mean occupying a certain field out of a bigger one according to the specifications of the laws on agrarian reform and agriculture. happen to the whole family (Millington & Maxfield, 1906); the Tagalogs used to utter these lines: Puno, puno, paghinga ay pigilin/ Pansumandali laang, ikaw ay aputulin/ Mula sa ‘yong tuod, ikaw ay masupling/ (Tree, tree, hold your breath; I will momentarily cut you down; from your stump, you are expected to sprout.) (Avila, 2020). These are but two of the local wisdom which must be considered by a homesteader. There are bits of local wisdom involved in the process of occupying certain fields, such as “Daig ng maagap ang masipag!” (The early beat the hardworking!), ecocentrism (of animism by the pre-hispanic Philippines) over anthropocentrism (of Christianity by the Spanish conquistadores). The caretaker of a certain field becomes worthy only after considering local-wisdom-related principles, i.e. [1] Sa marunong magbungkal, ang lupa ay ibibigay (The land belongs to someone who tills it), [2] Asking permission before cutting down an old tree: Ilocanos utter “Bari Bari/Dika agunget pari/Ta pumukan kami/Iti pabakirda kami// (Do not feel bad my friend for we cut as we are ordered.); the Visayans believe that if they were to cut down the trees before they had induced the spirits to move away, something bad will Figure 7. Sample slide on game-based pedagogy using Piko. Figure 7. Sample slide on game-based pedagogy using Piko. Table 3. Roel V. Avila: Local Wisdom in Laro Ng Lahi As Foundation in Game-Based Pedagogy 115 Respondents’ Favorite Games Local Wisdom in Agawan Base as Foundational Basis in the Development of Game-based Pedagogy GLOBAL GAME LARO NG LAHI LOCAL WISDOM GAME-BASED PEDAGOGY Prisoner’s Base Agawan Base Agawan Kuta Pangangayaw Game’s Objective: Get the opponent’s flag 1. Pananamsam 2. Paghihiganti 3. Pamahiin 4. Karangalan Team Victory Individual Victory Players: 1. Runner 2. Strategist 3. Bait 4. Savior 5. Sentinel Agorang 1 Agorang 2 Agorang 3 1.Matulin (Quick) 2.Maparaan (Creative) 3.Maisog (Brave) 4.Mapagkalinga (Caring) 5.Mapagmanman (Observant) Agorang 1 = former raha (raja) Agorang 2 = former bagani (berani) 1. Fastest to translate; 2. Finding ways how to translate correctly; 3. Volunteers when nobody in the team wants to answer; 4. Provides help to the recognized team mate; 5. Looks for opponents who attempt to cheat; Agorang 1 = Supplies sentences to translate; cal Wisdom in Agawan Base as Foundational Basis in the Development of Game-based Roel V. Avila: Local Wisdom in Laro Ng Lahi As Foundation in Game-Based Pedagogy 115 Agorang 3 = babaylan/guro (guru) heads in giving decisions to issues; Agorang 2 = Proclaims each decision; helps in the analysis of each issue; Agorang 3 = Checks the correctness of each sentence. Materials: 1. Playing field at least as big as a basketball court 2. Two posts 3. Two flags 1. Banwa 2. Puod 3. Bandila Internet connection Virtual Room Chat box Rules: 1. Same number of players 2. Fresher player defeats older player 3. Touching the post = refreshed 4. Plant the flag beside the post 5. Tag1 (tagged by the enemy) = captured 6. Tag2 (tagged by teammate) = refreshed 7. Tag3 (tagged by teammate) = saved 8. Tag4 (tagged again by enemy) = recaptured as slave 9. Slave can’t be saved by teammates 10. Slave can serve as spy to steal the flag (opponent’s tag prevents the slave) 1. Patas na pagkakataon (equal chance) 2. Kabataan: Youth over old age in war 3. Pakikiugnay: Constant communication with the group 4. Bandila: Flag represents the entire tribe 5. Pagkakabihag: Captivity with purpose is not to be ashamed of. 6. Pang-agdong: Reinforcement renews the goal 7. Utang na Loob = Life for life 8. Muling Pagkabihag = Recapture, with purpose, is heroism. 9. Ang taong walang lululuning banig ay hindi takot mamatay 10. Alipin ma’y may silbi rin: A slave can still help his village. 1. Equal number of questions and equal level of difficulty 2. Respondents’ Favorite Games There is no “weak” member if the leader knows their varied talents; 10. Agorangs or Elders can give wise counsels if they do not have personal interests; 11. Formal declaration of war is important; 12. Although war is anchored on deception, word of honor, based on the agreement, must be observed; 2. 3. 4. 5. 6. 7. 8. 9. 10 1 12 2. The odd number of Agorangs is ideal since they rule by majority vote; three is better than five; 2. Odd number is good; even number can slide to deadlock; 3. Even number among the students to form two teams is good; 3. Even number is divisible equally by 2; 4. Agorangs must have noses for potential student leaders; 5. Every game has the G factor; 4. Wise people know who has the potential to lead; 6. Leader’s choice can spell the difference; p 5. These two players shall engage in bato-bato-pik (scissors-paper- stone); 5. Game of chance elevates the issue to Bathala to decide justly; 7. Grouping provides the sense of belongingness; 8. Virtual flag of the losing team must be attached to the flag pole of the winning team; 6. Leader is given the freedom to choose; 6. Winner in every attempt shall choose a member until members are complete; 7. Base or kuta gives occupants sense of ownership and responsibility; 9. Translating sentences is made easy by pooling ideas together; 7. Members of Team A shall proceed to their base; 8. Attaching the flag to the pole is symbolic of their common goal; 10. How can the Agorangs know the correct translation if they lack knowledge about it? 8. Members of Team B shall proceed to the opposite base; 9. There is no “weak” member if the leader knows their varied talents; 9. The leader shall determine the roles the members shall play; 11. Agreement to engage in competition is important; 10. Agorangs or Elders can give wise counsels if they do not have personal interests; 12. Losing with grace is more important than winning by deception;winner. 10. Agorangs shall decide issues democratically. 11. Formal declaration of war is important; 11. Respondents’ Favorite Games Leader is given the freedom to choose; 7. Base or kuta gives occupants sense of ownership and responsibility; 8. Attaching the flag to the pole is symbolic of their common goal; 9. There is no “weak” member if the leader knows their varied talents; 10. Agorangs or Elders can give wise counsels if they do not have personal interests; 11. Formal declaration of war is important; 12. Although war is anchored on deception, word of honor, based on the agreement, must be observed; 2. Odd number is good; even number can slide to deadlock; 3. Even number among the students to form two teams is good; 4. Agorangs must have noses for potential student leaders; 5. Every game has the G factor; 6. Leader’s choice can spell the difference; 7. Grouping provides the sense of belongingness; 8. Virtual flag of the losing team must be attached to the flag pole of the winning team; 9. Translating sentences is made easy by pooling ideas together; 10. How can the Agorangs know the correct translation if they lack knowledge about it? 11. Agreement to engage in competition is important; 12. Losing with grace is more important than winning by deception;winner. 2. Three of them must agree to serve as Agorang; 3. The rest must be even number. If odd number, one may volunteer to join the Agorangs; 4. Agorangs shall select two potential leaders; 5. These two players shall engage in bato-bato-pik (scissors-paper- stone); 6. Winner in every attempt shall choose a member until members are complete; 7. Members of Team A shall proceed to their base; 8. Members of Team B shall proceed to the opposite base; 9. The leader shall determine the roles the members shall play; 10. Agorangs shall decide issues democratically. 11. Group that got the other team’s flag is the winner; 12. Losing team members, as slaves, shall “pay homage” to their new rajah. 2. The odd number of Agorangs is ideal since they rule by majority vote; three is better than five; 3. Even number is divisible equally by 2; 4. Wise people know who has the potential to lead; 5. Game of chance elevates the issue to Bathala to decide justly; 6. Leader is given the freedom to choose; 7. Base or kuta gives occupants sense of ownership and responsibility; 8. Attaching the flag to the pole is symbolic of their common goal; 9. Roel V. Avila: Local Wisdom in Laro Ng Lahi As Foundation in Game-Based Pedagogy 117 Respondents’ Favorite Games Students present in the virtual room are prioritized 3. Get assistance from team members 4. Flag is kept with utmost care. Do not underestimate the importance of the game. 5. Providing wrong translation is chance to learn the right one. 6. Listening to team mate’s help is precious. 7. Team mate who helped you translate should be appreciated. 8. Committing two errors gives higher degree of learning the right answer. 9. A student who used to commit errors is not anymore afraid to commit another one. 10. If a student does not know how to translate, s/he can still extend assistance to the team. How to play the game: 1. One player shall get the total number of children present; 1. Importance of census in each barangay; 1. Finding out the number of students who are virtually present; 5. Providing wrong translation is chance to learn the right one. 6. Listening to team mate’s help is precious. 7. Team mate who helped you translate should be appreciated. 7. Utang na Loob = Life for life 8. Committing two errors gives higher degree of learning the right answer. 8. Muling Pagkabihag = Recapture, with purpose, is heroism. 9. A student who used to commit errors is not anymore afraid to commit another one. 116 Cakrawala Dini: Vol. 12 No. 2, November 2021 116 Cakrawala Dini: Vol. 12 No. 2, November 2021 2. Three of them must agree to serve as Agorang; 3. The rest must be even number. If odd number, one may volunteer to join the Agorangs; 4. Agorangs shall select two potential leaders; 5. These two players shall engage in bato-bato-pik (scissors-paper- stone); 6. Winner in every attempt shall choose a member until members are complete; 7. Members of Team A shall proceed to their base; 8. Members of Team B shall proceed to the opposite base; 9. The leader shall determine the roles the members shall play; 10. Agorangs shall decide issues democratically. 11. Group that got the other team’s flag is the winner; 12. Losing team members, as slaves, shall “pay homage” to their new rajah. 2. The odd number of Agorangs is ideal since they rule by majority vote; three is better than five; 3. Even number is divisible equally by 2; 4. Wise people know who has the potential to lead; 5. Game of chance elevates the issue to Bathala to decide justly; 6. Respondents’ Favorite Games Group that got the other team’s flag is the winner; Objective is to capture the base by getting the flag (called Pangangayaw in early Philippines where spoils of war, such as gold and animals, as well as some citizens Table 3 presents the mechanics of the game, Agawan Kuta, with the local wisdom serving as basis in developing a game-based pedagogy. The Game’s Roel V. Avila: Local Wisdom in Laro Ng Lahi As Foundation in Game-Based Pedagogy 117 to be enslaved). It can serve as basis of game-based pedagogy by limiting the game to negotiation where translation of sentences occur (in the case of Foreign Language course). skills, experiences, fame, etc. and coming back to the homeland. This is the concept of bayani or hero according to Salazar (2004). As applied to game-based pedagogy, Agawan Kuta is attempt to win the game and go home with the “spoils of war” as the old Tagalog saying goes: “Mga bihag at bulawan ay para sa mga nagtagumpay” (To the victor belongs the spoils of war), meaning, if the course is Foreign Language, finishing the course by learning the language, that is, considering the rules of war (Lu, 2018) specifically safeguarding religious sites (UNAOC, 2019), and not destroying cultural heritage (Stone, 2020). Sentences have to be reflective of religious tolerance and other sensitive principles. Players of Agawan Kuta, who, ideally, must have respective roles to play, ought to learn much from the war their game mimics particularly in terms of strategies. There is really truth in the old saying: “To the victor belongs the spoils of war” or in Filipino parlance: “Mga bihag at bulawan ay para sa mga nagtagumpay”. Each team consists of at least 5 members with the following characteristics: [1] Matulin (Quick), [2] Maparaan (Creative), [3] Maisog (Brave), [4] Mapagkalinga (Caring), and [5] Mapagmanman (Observant). In the Game-based Pedagogy, each Agorang has a clear role to fulfill: Agorang 1 = Supplies sentences to translate; heads in giving decisions to issues; Agorang 2 = Proclaims each decision; helps in the analysis of each issue; and Agorang 3 = Checks the correctness of each sentence. Figure 8. Graph on Agawan Kuta showing Q & A as judged by the Agorang The concept of Pangangayaw (Invasion) has evolved at present: Pangingibang-bayan at paghahanap ng ginhawa (Villan & Esquejo, 2021). Respondents’ Favorite Games In other words, Pangangayaw which is figuratively represented by Agawan Kuta (Prisoner’s Base) may positively mean going abroad to get wealth, knowledge, Figure 8. Graph on Agawan Kuta showing Q & A as judged by the Agorang Table 4. Local Wisdom in Tubigan as Foundational Basis in the Development of Game-based Pedagogy GLOBAL GAME LARO NG LAHI LOCAL WISDOM GAME-BASED PEDAGOGY Block & Tag Game Tubigan or Patintero Tago Nang Tago (TNT) Tubigan Game’s Objective: One Team: to pass through the guards back and forth Other Team: to tag any member of the Team trying to pass through 1.Staying illegally in a foreign land 2.Catching illegal immigrants Concept: Tagarito, Dayo Surviving Interrogation by using the language of another group Players: 4 players each team For One Team: Hiding, Feigning, Collaborating, 4 members each team have respective roles to fulfill Table 4. Local Wisdom in Tubigan as Foundational Basis in the Development of Game-based 118 Cakrawala Dini: Vol. 12 No. 2, November 2021 118 Cakrawala Dini: Vol. 12 No. 2, November 2021 Panguna: Player guarding the first line Pangalawa: Player guarding the second line Pangatlo: Player guarding the third line Panggitna: Player guarding the middle line intersecting the three lines For Another Team: Pang-uyo Panggulo Panglusot Panggoyo Fooling, Deceiving, Bribing, Escaping Although war is always anchored on deception, there are actions which must be avoided, such as destroying cultural heritage, including relics and images considered holy. War treaties Fear of the Umalagad (pantheon of gods) Materials: Playing field: tiled, cemented or not If tiled, masking tape is used to form lines; If cemented, chalk is needed to draw lines; If not, water is poured as lines Banwa Ibayong-dagat Ibang lupain Internet connection Virtual Room Chat box Rules: 1. Equal number of members 2. Guard always stands on the line. 3. The only allowed parts of the body to legally tag an immigrant are the hands; 4. Immigrants may use their hands to prevent the hands of the guards from tagging them; 5. No immigrant shall pass through using the space outside the boundary lines; 6. Any immigrant may negotiate with any guard if the guard is his/her friend. 7. A guard who says “pass” to an immigrant is not 1. Equal chance 2. A person outside his/her territory will render illegal use of authority; 3. A non- commissioned person cannot usurp authority; 4. The right to self- preservation 5. Respondents’ Favorite Games Winner is decided by scores according to the agreed number of rounds. 1. Two opposing groups always engage in conflicts; 2. Toss coin connotes subscribing to the concept of fate; 3. Each barangay (village), city, province, or nation has clear boundary lines which are guarded for security purposes; 4. Tagging is finding, confronting, and arresting the dayo; 5. Balik-bayan is a local hero (bayani); 6. Since that foreign land has something that gives kaginhawahan for the immigrants, they return to the place; 7. If any member of the group is arrested, it means the end of the attempt; Other members return to their homeland; 8. In their homeland, they take part in guarding their territory from illegal immigrants. 1. Two mem 2. Toss the IT 3. Each quest interr 4. Findi guard Team confr askin prove confr alien 5. If the to langu his/h given pass 6. Virtu of T one m (one- forth each confr immi 7. Any group wron the e journ score 8. As f retur use langu mem oppo 4. The IT Team shall prevent the opposing team by tagging each player of the opposing team to cross each line and come back successfully 5. If the one asked is able to answer in that language (it is not his/her native tongue) is given permission to pass through; 5. Any member of the opposing team that successfully returned to the starting line brings a score. 5. Balik-bayan is a local hero (bayani); 6. Virtually, one member of Team A confronts one member of Team B (one-on-one) in going forth. In going back, each guard shall confront a different immigrant. 6. Since that foreign land has something that gives 6. All members of Team B shall again position themselves in the starting line to try to repeat the journey. kaginhawahan for the immigrants, they return to the place; 7. Any member of the group which gives a wrong answer signals the end of the Team’s journey without a score; 7. If any member of Team B is tagged legally, Team B shall become the IT and Team A shall cross the lines this time. 7. If any member of the group is arrested, it means the end of the attempt; Other members return to their homeland; 8. As former immigrants return home, they will use their native language to interrogate members of the opposing team. Respondents’ Favorite Games Winner is decided by scores according to the agreed number of rounds. 1. Two opposing groups always engage in conflicts; 2. Toss coin connotes subscribing to the concept of fate; 3. Each barangay (village), city, province, or nation has clear boundary lines which are guarded for security purposes; 4. Tagging is finding, confronting, and arresting the dayo; 5. Balik-bayan is a local hero (bayani); 6. Since that foreign land has something that gives kaginhawahan for the immigrants, they return to the place; 7. If any member of the group is arrested, it means the end of the attempt; Other members return to their homeland; 8. In their homeland, they take part in guarding their territory from illegal immigrants. 1. Two teams with four members each 2. Toss coin to determine the IT Team; 3. Each line stores questions for interrogation; 4. Finding one player, the guard (using his Team’s language) confronts him/her by asking questions to prove the one confronted is an illegal alien; 5. If the one asked is able to answer in that language (it is not his/her native tongue) is given permission to pass through; 6. Virtually, one member of Team A confronts one member of Team B (one-on-one) in going forth. In going back, each guard shall confront a different immigrant. 7. Any member of the group which gives a wrong answer signals the end of the Team’s journey without a score; 8. As former immigrants return home, they will use their native language to interrogate members of the opposing team. allowed to tag his/her friend. How to play the game: 1. Two teams with four members each; 2. Toss coin will determine which team will serve as IT. 3. Team A (as IT), will position themselves sentinel guarding each line (First Liner, Second Liner, Third Liner, and Middle Liner) 4. The IT Team shall prevent the opposing team by tagging each player of the opposing team to cross each line and come back successfully 5. Any member of the opposing team that successfully returned to the starting line brings a score. 6. All members of Team B shall again position themselves in the starting line to try to repeat the journey. 7. If any member of Team B is tagged legally, Team B shall become the IT and Team A shall cross the lines this time. 8. Respondents’ Favorite Games The space outside the boundary line belongs to another country; 6. Negotiation may involve bribe or relationships; 7. “May isang salita” (Palabra de honor or word of honor) 1. Two teams with equal number of members; 2. No question or answer shall emanate from the audience; 3. A person who is not an authority is not allowed to interrogate a player; 4. An interrogated player has the right to clarify each question; 5. No player shall ask the audience or use translator when the game is played; 6. Each player has the right to negotiate or to have mutual individual agreement; 7. “Pass” is only uttered once every round by only one member of the concerned team. 4. An interrogated player has the right to clarify each question; 4. Immigrants may use their hands to prevent the hands of the guards from tagging them; 5. The space outside the boundary line belongs to another country; 5. No player shall ask the audience or use translator when the game is played; 5. No immigrant shall pass through using the space outside the boundary lines; 6. Negotiation may involve bribe or relationships; 6. Each player has the right to negotiate or to have mutual individual agreement; 6. Any immigrant may negotiate with any guard if the guard is his/her friend. 7. “May isang salita” (Palabra de honor or word of honor) 7. “Pass” is only uttered once every round by only one member of the concerned team. Roel V. Avila: Local Wisdom in Laro Ng Lahi As Foundation in Game-Based Pedagogy 119 allowed to tag his/her friend. How to play the game: 1. Two teams with four members each; 2. Toss coin will determine which team will serve as IT. 3. Team A (as IT), will position themselves sentinel guarding each line (First Liner, Second Liner, Third Liner, and Middle Liner) 4. The IT Team shall prevent the opposing team by tagging each player of the opposing team to cross each line and come back successfully 5. Any member of the opposing team that successfully returned to the starting line brings a score. 6. All members of Team B shall again position themselves in the starting line to try to repeat the journey. 7. If any member of Team B is tagged legally, Team B shall become the IT and Team A shall cross the lines this time. 8. Respondents’ Favorite Games 120 Cakrawala Dini: Vol. 12 No. 2, November 2021 lifestyle of a locality, and modern technology can be of great help in the development of a game-based pedagogy that is anchored on local wisdom. This study used hermeneutic phenomenology and R & D on the beginning and last parts, respectively. Generation Z are still familiar with the Laro ng Lahi by playing them at home and schools. They prefer Tubigan, Piko, and Agawan Kuta for several reasons, mostly, on being beneficial to brain and body, personal discipline and socialization, and building team spirit. They are amenable to the conversion of knowledge and skills gained through Laro ng Lahi into learning course lessons, thereby leading into a game-based pedagogy. Table 4 presents the mechanics of Tubigan and the metaphorized local wisdom as basis of developing game- based pedagogy. The mechanics enumerates the materials needed and the steps in playing Tubigan. As metaphorized, Tubigan taps the existing local wisdom, such as the concepts: tagarito (citizen), dayo (foreigner), balikbayan (return to country), kaginhawahan (comfort), Tago Nang Tago (hiding and escaping from authorities), and karapatang mabuhay (right to self-preservation). Developing a pedagogy based on Tubigan best fits interrogations using the atmosphere of “immigrant-immigration officer” interaction Figure 9.1. Graph showing the Filipino value Palabra de Honor in Tubigan Figure 9.2. Graph showing a TNT and a player trying to cross the last line. g p g gy Respondents are still familiar with piko (taplak gunung), agawan kuta (bentengan), and tubigan (gobak sodor) since they played such games before and they were aware of the the Laro ng Lahi benefits. Thus, they preferred to have Laro ng Lahi as activity tools in school subjects, and to convert academic knowledge and skills into learning course lessons. Upon finalizing the mechanics of each of the three Filipino traditional games, and the data provided by the respondents, the researcher conceptualized the game-based pedagogy and developed lessons using the local wisdom as the basis. Figure 9.1. Graph showing the Filipino value Palabra de Honor in Tubigan Figure 9.1. Graph showing the Filipino value Palabra de Honor in Tubigan Figure 9.1. Graph showing the Filipino value Palabra de Honor in Tubigan Figure 9.2. Graph showing a TNT and a player trying to cross the last line. Local Wisdom can be extracted from Laro ng Lahi to serve as foundational basis for game-based pedagogy. REFERENCES Figure 9.2. Graph showing a TNT and a player trying to cross the last line. Aguado, D. (2012). 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https://openalex.org/W4380576974	https://eprints.whiterose.ac.uk/200472/1/s41467-023-39251-5.pdf	English	null	Mean-shift exploration in shape assembly of robot swarms	Nature communications	2,023	cc-by	14,555	This is a repository copy of Mean-shift exploration in shape assembly of robot swarms. White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/200472/ Version: Published Version Article: Sun, G. orcid.org/0000-0002-8729-2856, Zhou, R., Ma, Z. et al. (4 more authors) (2023) Mean-shift exploration in shape assembly of robot swarms. Nature Communications, 14 (1). 3476. ISSN 2041-1723 https://doi.org/10.1038/s41467-023-39251-5 This is a repository copy of Mean-shift exploration in shape assembly of robot s Version: Published Version Version: Published Version Article: Sun, G. orcid.org/0000-0002-8729-2856, Zhou, R., Ma, Z. et al. (4 more authors) (2023) Mean-shift exploration in shape assembly of robot swarms. Nature Communications, 14 (1). 3476. ISSN 2041-1723 https://doi.org/10.1038/s41467-023-39251-5 https://doi.org/10.1038/s41467-023-39251-5 Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Article https://doi.org/10.1038/s41467-023-39251-5 Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ Mean-shift exploration in shape assembly of robot swarms Guibin Sun 1,2, Rui Zhou1, Zhao Ma2, Yongqi Li2, Roderich Groß 3, Zhang Chen 4 & Shiyu Zhao 2,5,6,7 Received: 20 October 2022 Accepted: 6 June 2023 Check for updates The fascinating collective behaviors of biological systems have inspired extensive studies on shape assembly of robot swarms. Here, we propose a strategy for shape assembly of robot swarms based on the idea of mean-shift exploration: when a robot is surrounded by neighboring robots and unoccu- pied locations, it would actively give up its current location by exploring the highest density of nearby unoccupied locations in the desired shape. This idea is realized by adapting the mean-shift algorithm, which is an optimization technique widely used in machine learning for locating the maxima of a den- sity function. The proposed strategy empowers robot swarms to assemble highly complex shapes with strong adaptability, as veriﬁed by experiments with swarms of 50 ground robots. The comparison between the proposed strategy and the state-of-the-art demonstrates its high efﬁciency especially for large-scale swarms. The proposed strategy can also be adapted to generate interesting behaviors including shape regeneration, cooperative cargo trans- portation, and complex environment exploration. In nature, groups of insects and animals can self-assemble various spatial shapes that are functional for the groups adapting to the environments1–3. As a remarkable example, army ants can assemble shapes to transport food cooperatively or construct bridges using their bodies to overcome spatial gaps4,5. These shape assembly beha- viors are generated spontaneously by local interactions among the individuals. They exhibit strong adaptability to individual faults and can be easily scaled up to groups of thousands or millions of individuals1,3. centralized goal assignment is inefﬁcient to support large-scale swarms since the computational complexity increases rapidly as the number of robots increases15,16. Moreover, when robots fail to function normally, additional algorithms for fault-tolerant detection and goal re-assignment are required to handle such situations17. As a compar- ison, distributed goal assignment can support large-scale swarms by decomposing the centralized assignment into multiple local ones11,12. It also exhibits better robustness to robot faults. However, since dis- tributed goal assignments are based on locally sensed information, conﬂicts among local assignments are inevitable and must be resolved by sophisticated algorithms such as local task swapping11,12. In nature, groups of insects and animals can self-assemble various spatial shapes that are functional for the groups adapting to the environments1–3. 1School of Automation Science and Electrical Engineering, Beihang University, Beijing, China. 2School of Engineering, Westlake University, Hangzhou, China. 3Department of Automatic Control and Systems Engineering, The University of Shefﬁeld, Shefﬁeld, UK. 4Department of Automation, Tsinghua University, Beijing, China. 5Research Center for Industries of the Future, Westlake University, Hangzhou, China. 6 Key Laboratory of Coastal Environment and Resources of Zhejiang Province, Westlake University, Hangzhou, China. 7Westlake Institute for Advanced Study, Hangzhou, China. e-mail: zhaoshiyu@westlake.edu.cn A human-swarm graphical interface Here, we propose a strategy for shape assembly of robot swarms based on the idea of mean-shift exploration: when a robot is sur- rounded by neighboring robots and unoccupied locations, it would actively give up its current location by exploring the highest density of nearby unoccupied locations in the desired shape. This idea does not rely on goal assignment. It is realized by adapting the mean-shift algorithm26–28, which is an optimization technique widely used in machine learning for locating the maxima of a density function. Moreover, a distributed negotiation mechanism is designed to allow robots to negotiate the ﬁnal desired shape with their neighbors in a distributed manner. This negotiation mechanism enables the swarm to The ﬁrst step of shape assembly is to specify a desired geometric shape. We designed a human-swarm interface that allows a human operator to specify the desired shape by drawing or loading a binary graphical image (Fig. 1a). The graphical image is converted to a black- white binary grid, where the black cells in the grid correspond to the locations that should be occupied by the robots. The binary grid is then converted to a grayscale grid by the distance transformation algorithm29 so that the inﬂuence of the desired shape is gradually expanded and the robots can move into the shape more smoothly (Fig. 1a). With this human-swarm interface, the operator can specify the desired shape in a graphical way without specifying the physical Distributed interpretation negotiation Desired shape Distributed interpretation negotiation Distributed control strategy Each robot interprets the desired shape locally and may have different initial interpretations. Specify as a black-white grid by the human-swarm interface. sense r Initial position All robots negotiate the desired shape in a distributed manner. Finally, all interpretations reach a consensus. Convert to a gray-level grid. Send the gray-level grid to all robots without centroid location or orientation parameters. Blue robot: select the nearest gray cell as its local target. Red robot: select the darkest neighboring cell as its local target. Blue robot: avoid collision with neighbors. Red robot: align its velocity with neighbors. Blue robot: move into the deep of the desired shape. Red robot: explore unoccupied cells in the desired shape. Shape-entering command Shape-exploring command Interaction command a c Different Initial interpretations b Fig. 1 \| An illustration of the proposed shape assembly strategy. a The human- swarm interface. A human-swarm graphical interface The desired shape is speciﬁed via the interface and sent to all the robots as a nonparametric grid. b The robots have different initial interpretations of the translation and orientation of the desired shape. They gradually reach a consensus via the proposed distributed negotiation algorithm. c Examples to illustrate the three control commands in the proposed control strategy. Details o the algorithms can be found in Methods and Sections 1 to 3 in the Supplementary Information. desired shape locally and may have different initial interpretations Each robot interprets t Different Initial interpretations b Finally, all interpretations reach a consensus. sense r Initial position All robots negotiate the desired shape in a distributed manner. Specify as a black-white grid by the human-swarm interface. Finally, all interpretations reach a consensus. All robots negotiate the desired shape in a distributed manner. Different Initial interpretations Distributed control strategy Blue robot: avoid collision with neighbors. Red robot: align its velocity with neighbors. Interaction command Convert to a gray-level grid. Send the gray-level grid to all robots without centroid location or orientation parameters. Blue robot: move into the deep of the desired shape. Red robot: explore unoccupied cells in the desired shape Shape-exploring command Blue robot: select the nearest gray cell as its local target. Red robot: select the darkest neighboring cell as its local target. Shape-entering command c Interaction command c Shape-entering command c Shape-exploring command Shape-exploring command Blue robot: select the nearest gray cell as its local target. Red robot: select the darkest neighboring cell as its local target. Blue robot: move into the deep o the desired shape. Red robot: explore unoccupied cells in the desired shape. Blue robot: avoid collision with neighbors. Red robot: align its velocity with neighbors. Blue robot: move into the deep o the desired shape. Red robot: explore unoccupied cells in the desired shape. Blue robot: move into the deep of the desired shape. Send the gray-level grid to all robots without centroid location or orientation parameters. Send the gray-level grid to all robots without centroid location or orientation parameters. g Red robot: align its velocity with neighbors. Fig. 1 \| An illustration of the proposed shape assembly strategy. a The human- swarm interface. The desired shape is speciﬁed via the interface and sent to all the robots as a nonparametric grid. b The robots have different initial interpretations of the translation and orientation of the desired shape. Mean-shift exploration in shape assembly of robot swarms Instead, edge detection is realized in a distributed manner by fusing the beliefs of a robot with its neighbors. However, since the robots cannot self-localize themselves relative to the desired shape, they make use of random walks to search for the edges, which would lead to random trajectories. Another class of methods that do not require goal assignment is based on artiﬁcial potential ﬁelds22–25. One limitation of this class of methods is that robots may easily get trapped in local minima, making it difﬁcult to assemble nonconvex complex shapes. on a small number of pre-localized seed robots. As a consequence of the local positioning system, the proposed edge-following control method requires that only the robots on the edge of a swarm can move while those inside must stay stationary. The method in ref. 19 can generate swarm shapes spontaneously from a reaction-diffusion net- work similar to embryogenesis in nature. However, this method is not able to generate user-speciﬁed shapes precisely. The method in ref. 21 can aggregate robots on the frontier of shapes based on saliency detection. The user-deﬁned shape is speciﬁed by a digital light pro- jector. An interesting feature of this method is that it does not require centralized edge detectors. Instead, edge detection is realized in a distributed manner by fusing the beliefs of a robot with its neighbors. However, since the robots cannot self-localize themselves relative to the desired shape, they make use of random walks to search for the edges, which would lead to random trajectories. Another class of methods that do not require goal assignment is based on artiﬁcial potential ﬁelds22–25. One limitation of this class of methods is that robots may easily get trapped in local minima, making it difﬁcult to assemble nonconvex complex shapes. maneuver while maintaining a desired shape based on a small number of informed robots. The proposed strategy empowers robot swarms to assemble nonconvex complex shapes with strong adaptability and high efﬁciency, as veriﬁed by numerical simulation results and real- world experiments with swarms of 50 ground robots. The strategy can be adapted to generate interesting behaviors including shape regen- eration, cooperative cargo transportation, and complex environment exploration. Mean-shift exploration in shape assembly of robot swarms As a remarkable example, army ants can assemble shapes to transport food cooperatively or construct bridges using their bodies to overcome spatial gaps4,5. These shape assembly beha- viors are generated spontaneously by local interactions among the individuals. They exhibit strong adaptability to individual faults and can be easily scaled up to groups of thousands or millions of individuals1,3. The fascinating collective behaviors of biological systems have inspired extensive studies on shape assembly of robot swarms6–9. One class of strategies widely studied in the literature are based on goal assignment in either centralized or distributed ways10–12. Once a swarm of robots are assigned unique goal locations in a desired shape, the consequent task is simply to plan collision-free trajectories for the robots to reach their goal locations10 or conduct distributed formation control based on locally sensed information6,13,14. It is notable that Another class of strategies for shape assembly that have also attracted extensive research attention are free of goal assignment18–21. For instance, the method proposed in ref. 18 can assemble complex shapes using thousands of homogeneous robots. An interesting fea- ture of this method is that it does not rely on external global posi- tioning systems.Instead, it establishes a local positioning system based 1School of Automation Science and Electrical Engineering, Beihang University, Beijing, China. 2School of Engineering, Westlake University, Hangzhou, China. 3Department of Automatic Control and Systems Engineering, The University of Shefﬁeld, Shefﬁeld, UK. 4Department of Automation, Tsinghua University, Beijing, China. 5Research Center for Industries of the Future, Westlake University, Hangzhou, China. 6 Key Laboratory of Coastal Environment and Resources of Zhejiang Province, Westlake University, Hangzhou, China. 7Westlake Institute for Advanced Study, Hangzhou, China. e-mail: zhaoshiyu@westlake.edu.cn Nature Communications\| (2023) 14:3476 1 Article https://doi.org/10.1038/s41467-023-39251-5 on a small number of pre-localized seed robots. As a consequence of the local positioning system, the proposed edge-following control method requires that only the robots on the edge of a swarm can move while those inside must stay stationary. The method in ref. 19 can generate swarm shapes spontaneously from a reaction-diffusion net- work similar to embryogenesis in nature. However, this method is not able to generate user-speciﬁed shapes precisely. The method in ref. 21 can aggregate robots on the frontier of shapes based on saliency detection. The user-deﬁned shape is speciﬁed by a digital light pro- jector. An interesting feature of this method is that it does not require centralized edge detectors. Results The proposed shape assembly strategy consists of three components. The ﬁrst is a human-swarm graphical interface that can specify user- deﬁned shapes. The second is a distributed negotiation process that can autonomously reach an agreement among the robots on the ﬁnal location and orientation of the swarm shape. The third, which is the core of the proposed strategy, is a distributed control algorithm. The three components are detailed as follows. A human-swarm graphical interface They gradually reach a consensus via the proposed distributed negotiation algorithm. c Examples to illustrate the three control commands in the proposed control strategy. Details of the algorithms can be found in Methods and Sections 1 to 3 in the Supplementary Information. consensus via the proposed distributed negotiation algorithm. c Examples to illustrate the three control commands in the proposed control strategy. Details of the algorithms can be found in Methods and Sections 1 to 3 in the Supplementary Information. Nature Communications\| (2023) 14:3476 2 Article https://doi.org/10.1038/s41467-023-39251-5 cases. In the ﬁrst case where robot i is near the edge of the desired shape so that the cells within its sensing radius are either black or gray, vexp i would drive the robot toward the highest density of black cells. In this way, the robot is attracted into the shape. In the second case where robot i is already inside the desired shape so that the cells within its sensing radius are all black, vexp i would drive the robot toward the direction with the highest density of the unoccupied black cells. A cell is deﬁned as unoccupied if the distance between its center and any robot is greater than half of the robot’s collision avoidance radius (F 3B). The shape-exploring command is the key to resolving inter-robot competition. When a robot is surrounded by neighboring robots and unoccupied locations, it would actively give up its current location by exploring the highest density of nearby unoccupied locations. parameters of the grid. The parameters such as the physical size of each cell can be autonomously generated. Moreover, the number of cells in the grid does not have to be the same as the number of robots because the proposed method can handle mismatches between the grid and robot numbers (Fig. 4 as shown later). Although the desired shape is represented as a discrete grid, the robots can move freely across the boundaries of the cells in the grid. More information can be found in Methods and Section 1 in the Supplementary Information. Platforms and experimental setup We implemented the proposed strategy on a swarm of 50 holonomic wheeled robots (Fig. 2a, b). Details of the robotic platforms are given in Section 5.1 in the Supplementary Information. Each robot can exhibit its velocity command by using a LED belt inside the robot’s dome. In particular, each LED can show four colors: red, green, blue, and white (Fig. 2a). The numbers of LEDs showing the four colors correspond to the magnitudes of vent i ,vexp i ,vint i , and vi, respectively (Section 5.1 in the Supplementary Information). The experiments were conducted in an indoor environment with the support of a motion capture system (Fig. 2c, d). To implement the proposed strategy, each robot needs to acquire certain information about its neighbors and sense its sur- roundings to identify unoccupied cells and obstacles. These functions are realized through a parallel multi-thread system in a distributed manner (Fig. 2e). More speciﬁcally, each robot is assigned an inde- pendent control thread running onthe workstation and communicates with its thread using wireless routers. Although all the information is available in the workstation, the control command for each robot merely uses the local information that each robot is supposed to have. Details of the experimental setup are given in Section 5.2 in the Sup- plementary Information. Distributed control of shape assembly The aim of each robot is to move into the desired shape. This process is highly dynamic because its goal location is time-varying due to the dynamical convergence process of its interpretation and, more importantly, the avoidance of inter-robot collision or goal location competition. In our method, each robot executes the same control strategy based on its locally sensed information so as to enter the desired shape while avoiding collision. In particular, the velocity command for the ith robot is vi = vent i + vexp i + vint i , where vent i , vexp i , and vint i represent the shape-entering, shape-explor- ing, and interaction velocity commands, respectively. The roles of the three velocity commands are explained as follows, whereas their mathematical expressions are given in Methods. Distributed negotiation on the desired shape Once the grayscale grid has been generated by the human-swarm interface, it is sent to all the robots and stored in each robot’s memory. The memory sizes of the grids used in our work are around tens of kilobytes (details are given in Section 1 in the Supplementary Infor- mation), which is affordable for mainstream embedded computers nowadays. The Euclidean parameters of the desired shape including the position or orientation are not assigned manually. Instead, the robots negotiate with each other to autonomously determine them in a distributed manner. In particular, each robot ﬁrst interprets the desired shape based on its local interest. For example, every robot may initially interpret itself as the center of the desired shape (Fig. 1b), which is locally optimal for each robot in the sense that they do not need to move because they are already inside the desired shape. While the initial interpretations of different robots may conﬂict, each robot negotiates its interpretation with its neighbors through local wireless communication by a distributed consensus algorithm. With the negotiation algorithm, all the robots would eventually reach a con- sensus on the translation and orientation of the ﬁnal desired shape (Fig. 1b). By introducing a small number of informed robots, the swarm can reach a consensus on a user-speciﬁed trajectory of the desired shape. More information about shape negotiation is given in Methods and Section 2 and Section 6.1 in the Supplementary Information. The interaction command vint i consists of two sub-terms (Fig. 1c and Methods). The ﬁrst is a collision-avoidance term that generates repulsive velocity commands when robot i is too close to its sur- roundings. The second is a velocity-alignment term that aligns the velocity of robot i with its neighbors. The velocity-alignment term plays two roles. First, it can reduce velocity mismatches among the robots to reduce the chances of inter-robot collision. Second, since the desired shape may maneuver across in the motion space, the velocity- alignment term is necessary for robot i to track the velocity of the moving desired shape. Complex shape assembly The proposed strategy is able to assemble nonconvex complex shapes. One representative complex shape is the snowﬂake as shown in Fig. 3a. The snowﬂake shape has 6 major branches and 18 minor branches, making the shape highly nonconvex and challenging to assemble. The proposed strategy can assemble the snowﬂake shape, which is quantitatively veriﬁed by the fact that the coverage rate converges to 100% (Fig. 3a). The coverage rate and other metrics to evaluate the assembly performance are deﬁned in Methods. The shape-exploring control term of the strategy plays a key role in this process. In particular, when this term is disabled, the swarm fails to assemble the snowﬂake shape since many robots get trapped in local minima and the coverage rate drops sharply to 75% (Fig. 3a). The reason for the failure is that a robot would stop moving and hover near the boundary once it has entered the desired shape in the absence of this term. As a result, it may block those robots behind from entering the shape (Fig. 3b). By contrast, with this term, a robot would continue moving toward the deep of the desired shape after passing the boundary so that those robots behind it The shape-entering command vent i aims to steer robot i to the desired shape by seeking the darkest grid cells around them (Fig. 1c and Methods). More speciﬁcally, when robot i is too far away from the desired shape, vent i would drive the robot toward the closest gray cell. When robot i has entered the area of the gray cells, vent i would push the robot toward one of the neighboring gray cells that has the lowest gray level. In this way, the robot can gradually reach a black cell in the desired shape. It is notable that vent i would vanish once a robot steps into the desired shape area. In this case, the robot would stop moving and hence block those robots behind. To resolve this problem, the following velocity command vexp i is designed so that the robots can continue moving into the deep of the desired shape. The shape-exploring command vexp i , which is realized by an adapted version of the mean-shift algorithm27, aims to push each robot into the desired shape and then explore unoccupied cells inside the desired shape (Fig. 1c and Methods). Nature Communications\| (2023) 14:3476 Adaptability to swarm scale variants The proposed strategy is adaptive to the variant of robot number in a swarm. When some robots leave the swarm, the remaining robots can autonomously assemble the desired shape without any goal re- assignment or fault-tolerant control. A representative example is the shape regeneration behavior as demonstrated in Fig. 5, where the desired geometric shape is a starﬁsh. After one arm of the starﬁsh shape has been removed, the rest of the swarm spontaneously grows a new arm and assembles the starﬁsh shape again. The adaptability is due to the shape-exploring term, by which the robots can actively search unoccupied cells and hence replace the roles of the removed robots. In the following, the efﬁciency of the proposed method is com- pared with that of two state-of-the-art methods. The ﬁrst is the assignment-based method in ref. 12 and the second is the assignment- free method in ref. 21. Both of the methods are decentralized. The method in ref. 12 and our proposed method require external global positioning, whereas the one in ref. 21 does not. The comparison results are presented in Fig. 4 and Supplementary Fig. 8. It is shown that, given the same initial conﬁgurations, the three methods have similar convergence times when the number of robots is as small as 20. However, as the number of robots increases, the convergence times of the state-of-the-art methods increase rapidly while that of the pro- posed one increases slightly. Speciﬁcally, when the number of robots is as large as 300, the convergence time of the proposed method is at least 20 times shorter than the others. The reason why the method in ref. 12 requires longer convergence time is that it executes local goal swaps constantly and the robots must move along the grid lines. By contrast, the proposed method does not rely on goal assignment due to the mean-shift exploration control. It also allows the robots to move continuously in the working space. The merit of the method in ref. 21 is that it does not rely on any global or local positioning system. How- ever, since the robots do not know their positions relative to the desired shape, they make use of random walks to search for the edges Notably, the newly generated starﬁsh has less number of robots than the original one, verifying that the proposed strategy can assemble the same shape with different numbers of robots. Complex shape assembly More speciﬁcally, there are two Nature Communications\| (2023) 14:3476 3 Article https://doi.org/10.1038/s41467-023-39251-5 Implementation architecture A swarm of 50 robots a Red: shape-entering command Green: shape-exploring command Blue: interaction command White Experimental setup Rainbow robot platform d b c Color LED belt WI-FI transceiver Marker Master controller Mecanum wheel e … … … Interpretation negotiation Own interpretation Others’ negotiations Others’ motion states Velocity observation Own position Own velocity Workstation Thread Thread Thread … … Assembly control … … … Motion capture system Motion Controller IMU DC Motors Commands Robot Robot Robot … Robot swarm Motion capture system Robot swarm Ultra-wide angle camera Workstation Wireless router x y z Human-swarm interface … Wireless router Fig. 2 \| Platforms and implementation setup. a The speciﬁcally designed Rainbow robots. Each robot can show different colors to reﬂect the ingredients of its control commands. b Hardware components of robot platform. See Section 5.1 in the Supplementary Information for more details. c A swarm of 50 Rainbow robots. d An illustration of the experimental setup. The detailed functions of each experiment element are given in Section 5.2 in the Supplementary Information. e The multi- thread implementation architecture. Each robot corresponds to a unique contro thread in the workstation, and the threads are executed in a parallel way. Rainbow robot platform b Color LED belt WI-FI transceiver Marker Master controller Mecanum wheel A swarm of 50 robots c a Red: shape-entering command Green: shape-exploring command Blue: interaction command White A swarm of 50 robots b Rainbow robot platform c Implementation architecture Mecanum wheel e … … … Interpretation negotiation Own interpretation Others’ negotiations Others’ motion states Velocity observation Own position Own velocity Workstation Thread Thread Thread … … Assembly control … … … Motion capture system Motion Controller IMU DC Motors Commands Robot Robot Robot … Robot swarm Human-swarm interface … Wireless router Experimental setup d Motion capture system Robot swarm Ultra-wide angle camera Workstation Wireless router x y z d Experimental setup e Motion capture system Ultra-wide angle camera Robot swarm Fig. 2 \| Platforms and implementation setup. a The speciﬁcally designed Rainbow robots. Each robot can show different colors to reﬂect the ingredients of its control commands. b Hardware components of robot platform. See Section 5.1 in the Supplementary Information for more details. c A swarm of 50 Rainbow robots. d An illustration of the experimental setup. Complex shape assembly The detailed functions of each experimental element are given in Section 5.2 in the Supplementary Information. e The multi- thread implementation architecture. Each robot corresponds to a unique control thread in the workstation, and the threads are executed in a parallel way. of the desired shape, which would lead to random and long trajectories. can enter the shape smoothly (Fig. 3b). In addition to the shape- exploring term, the shape-entering term in the proposed strategy also plays a necessary role to steer all the robots into the desired shape. As shown in Fig. 3b, not all the robots can enter the shape in the absence of this shape-entering term even though the shape can still be fulﬁlled by a subset of the swarm. The proposed strategy exhibits smooth swarming motion in shape forming and switching tasks. As shown in the experimental results in Fig. 3c, the swarm can assemble different nonconvex shapes and switch from one to another smoothly. can enter the shape smoothly (Fig. 3b). In addition to the shape- exploring term, the shape-entering term in the proposed strategy also plays a necessary role to steer all the robots into the desired shape. As shown in Fig. 3b, not all the robots can enter the shape in the absence of this shape-entering term even though the shape can still be fulﬁlled by a subset of the swarm. The proposed strategy exhibits smooth swarming motion in shape forming and switching tasks. As shown in the experimental results in Fig. 3c, the swarm can assemble different nonconvex shapes and switch from one to another smoothly. Nature Communications\| (2023) 14:3476 Adaptability to swarm scale variants See Supplementary Movies 1 and 2. 10 0 0 50 100 Time (s) Entering rate M2 (%) 2.5 5 7.5 87.50% 43.45% Successful shape assembly Assembly of a ring shape from a linear configuration b Failure without shape-exploring Failure without shape-entering y-axis (m) 0 20 x-axis (m) 20 Initial position Final position Lack exploring Full controlling Lack entering 10 0 0 50 100 Time (s) Coverage rate M1 (%) 2.5 5 7.5 80.72% 10 0 0 2.5 5 Time (s) Uniformity M3 (m2) 2.5 5 7.5 Assembly of a ring shape from a linear configuration b 1 Coverage rate M1 (%) 5 10 Entering rate M2 (%) t o h s p a n S y r o t c e j a r T r o rr e n o it a it o g e N Fig. 3 \| Complex shape assembly by the proposed strategy. a Experimental results of 50 real Rainbow robots assembling a complex snowﬂake shape. The shape-exploring control term is necessary since the shape assembly task would fail in the absence of the term as shown in the rightmost subﬁgure. b Numerical simulation of 16 robots assembling a ring shape starting from an initial linear conﬁguration. Both the shape-exploring and shape-entering control terms are necessary since shape assembly would fail in the absence of either of them. c Experimental results of 50 real Rainbow robots assembling different shapes in a sequence. The distributed negotiation process among the robots converges fast. The shape switches smoothly due to the ability of active exploration and inde- pendence of goal assignment of the proposed strategy. See Supplementary Movies 1 and 2. necessary since shape assembly would fail in the absence of either of them. c Experimental results of 50 real Rainbow robots assembling different shapes in a sequence. The distributed negotiation process among the robots converges fast. The shape switches smoothly due to the ability of active exploration and inde- pendence of goal assignment of the proposed strategy. See Supplementary Movies 1 and 2. trajectory. The interpretations of the rest uninformed robots would gradually converge to the informed ones so that all the robots reach a consensus on the desired trajectory of the shape (see the details of the algorithm in Section 2 in the Supplementary Information). While the robots attempt to assemble the desired moving shape, they automatically transport the cargo encircled in the center (Fig. 6b). Adaptability to swarm scale variants In fact, the strategy exhibits stable performance in the presence of mismatches between the number of robots, denoted as nrobot, and the number of black cells in the desired shape, denoted as ncell (Fig. 5d). For the starﬁsh example, it is shown by the statistical results in Fig. 5e that the proposed strategy can successfully assemble the desired shape with a wide range of ncell/nrobot. Speciﬁcally, the coverage rate of robots for the desired shape is greater than 93% and the entering rate remains 100% when the ratio ncell/nrobot varies from 0.45 to 128.06. As a con- sequence, the strategy avoids the requirement of the condition nrobot = ncell, a condition widely adopted in shape assembly methods11, and hence makes the strategy more adaptive. In addition, when the swarm scale increases from nrobot = 16 to 1024, the convergence time of the entire swarm increases mildly as shown in the rightmost sub- ﬁgure of Fig. 4E, verifying the high motion efﬁciency of the strategy. Adaptability to swarm scale variants Nature Communications\| (2023) 14:3476 4 Article https://doi.org/10.1038/s41467-023-39251-5 Minimum / Maximum Average error 88 0 -2 0 2 Time (s) y-position error (m) 22 44 66 110 Average error Minimum / Maximum 88 0 0 2 4 Time (s) Orientation error (rad) 22 44 66 110 88 0 -2 0 2 Time (s) x- ) m ( r o rr e n o iti s o p 22 44 66 110 Minimum / Maximum Average error 10 0 0 50 100 Time (s) Entering rate M2 (%) 2.5 5 7.5 87.50% 43.45% Successful shape assembly Assembly of a ring shape from a linear configuration t o h s p a n S a c Time = 20 s Time = 40 s Time = 60 s Time = 80 s Time = 110 s Time = 0 s b Initial configuration Successful shape assembly Failure without shape-exploring Assembly of a complex snowflake shape Failure without shape-exploring Failure without shape-entering y r o t c e j a r T y-axis (m) 0 20 x-axis (m) 20 Initial position Final position Lack exploring Full controlling Lack entering 10 0 0 50 100 Time (s) Coverage rate M1 (%) 2.5 5 7.5 80.72% 10 0 0 2.5 5 Time (s) Uniformity M3 (m2) 2.5 5 7.5 12 0 0 50 100 Time (s) M e t a r e g a r e v o C 1 (%) 3 6 9 15 12 0 0 50 100 Time (s) 3 6 9 15 0.4 0.2 Velocity (m/s) r o rr e n o it a it o g e N Fig. 3 \| Complex shape assembly by the proposed strategy. a Experimental results of 50 real Rainbow robots assembling a complex snowﬂake shape. The shape-exploring control term is necessary since the shape assembly task would fail n the absence of the term as shown in the rightmost subﬁgure. b Numerical simulation of 16 robots assembling a ring shape starting from an initial linear conﬁguration. Both the shape-exploring and shape-entering control terms are necessary since shape assembly would fail in the absence of either of them. c Experimental results of 50 real Rainbow robots assembling different shapes i sequence. The distributed negotiation process among the robots converges fa The shape switches smoothly due to the ability of active exploration and inde- pendence of goal assignment of the proposed strategy. Adaptability to swarm scale variants Since it is difﬁcult for all the robots evenly distribute along the edge, the convergence time is deﬁned as the time when the convergence rate reaches 70%. The parameters of the three algo- rithms are given in Supplementary Table 2. Article https://doi.org/10.1038/s41467-023-39251-5 https://doi.org/10.1038/s41467-023-39251-5 Article 5.5 -0.1 11.1 16.7 22.3 y-axis (m) x-axis (m) 5.5 11.1 16.7 22.3 -0.1 Initial position Final position a The same initial configuration t n e m n g i s s A - d e s a b Snapshot of the swarm at 2s Snapshot of the swarm at 9s Final swarm and trajectory x-axis (m) 5.5 11.1 16.7 22.3 -0.1 x-axis (m) 5.5 11.1 16.7 22.3 -0.1 5.5 -0.1 11.1 16.7 22.3 y-axis (m) t n e m n g i s s A -free 5.5 -0.1 11.1 16.7 22.3 y-axis (m) d e s o p o r p r u O x-axis (m) 5.5 11.1 16.7 22.3 -0.1 Final swarm and trajectory a s c irt e m e c n a m r o fr e P ( ) Assignment-based Assignment-free Our proposed ( ) ( ) ) % ( e t a r e c n e g r e v n o C Time (s) 0 120.5 100 241.5 361.5 482 0 50 b ( ) Convergence time (s) Number of robots nrobot 0 500 100 300 20 250 Assignment-based Minimum / maximum Assignment-free Our proposed Fig. 4 \| Comparison between the proposed method, the decentralized assignment-based method in ref. 12 and the decentralized assignment-free method in ref 21 a Snapshots and trajectories of the shape assembly processes by the time when the convergence rate is equal to 100%. For the assignment-free method in ref. Adaptability to swarm scale variants The velocity alignment component in the interaction control term of the proposed strategy is essential for all the robots tracking the maneuvering swarm shape. Cooperative transportation and shape maneuvering In nature, ants can surround and transport a piece of food that is much larger than their individual body size in a cooperative manner4. The proposed strategy can be applied to cooperative cargo trans- portation. To do that, we can specify a hollow shape centered at a cargo (Fig. 6a). When the robots assemble the hollow shape, they would encircle the cargo tightly. The consequent task is to steer the swarm to move collectively while maintaining the desired shape. In particular, we introduce a small number of informed robots that know the desired translational and rotational trajectory of the shape. The informed robots have counterparts in ant swarms4, where some leader ants can guide the others to transport food to a goal location. During the inter-robot negotiation process, the informed robots play a stubborn role by insisting on their knowledge of the desired The number of informed robots is required to be at least one. The more informed robots there are, the faster the negotiation process can converge. Adaptability to swarm scale variants As shown in Supplementary Figure 6, seven informed robots are randomly selected out of 128 ones, which can reach a consensus on the time-varying translation and orientation of Nature Communications\| (2023) 14:3476 5 s c irt e m e c n a m r o fr e P 5.5 -0.1 11.1 16.7 22.3 y-axis (m) x-axis (m) 5.5 11.1 16.7 22.3 -0.1 Initial position Final position a The same initial configuration t n e m n g i s s A - d e s a b Snapshot of the swarm at 2s Snapshot of the swarm at 9s Assignment-based Assignment-free Our proposed Final swarm and trajectory x-axis (m) 5.5 11.1 16.7 22.3 -0.1 x-axis (m) 5.5 11.1 16.7 22.3 -0.1 5.5 -0.1 11.1 16.7 22.3 y-axis (m) t n e m n g i s s A -free 5.5 -0.1 11.1 16.7 22.3 y-axis (m) d e s o p o r p r u O ) % ( e t a r e c n e g r e v n o C Time (s) 0 120.5 100 241.5 361.5 482 0 50 b x-axis (m) 5.5 11.1 16.7 22.3 -0.1 Convergence time (s) Number of robots nrobot 0 500 100 300 20 250 Assignment-based Minimum / maximum Assignment-free Our proposed Fig. 4 \| Comparison between the proposed method, the decentralized assignment-based method in ref. 12 and the decentralized assignment-free method in ref. 21. a Snapshots and trajectories of the shape assembly processes by he three methods. There are 300 robots assembling the shape of “N''. b Statistics of convergence rate and convergence time. Each average value is calculated based on 0 trials. For the proposed method and the assignment-based one in ref. 12, the convergence rate is deﬁned as the ratio between the number of robots that are nside the shape and the total number of robots. The convergence time is deﬁned as the time when the convergence rate is equal to 100%. For the assignment-free method in ref. 21, the convergence rate is deﬁned as the ratio between the number of robots that have reached the edge and the total number of robots. The width of the border, a parameter in the method in ref. 21, is set to zero so that the robots can aggregate evenly around the shape as much as possible. Adaptability to swarm scale variants 21, the convergence rate is deﬁned as the ratio between the number of robots that have reached the edge and the total number of robots The width of Number of robots nrobot 0 00 100 300 20 50 Assignment-based Minimum / maximum Assignment-free Our proposed b s c irt e m e c n a m r o fr e P Assignment-based Assignment-free Our proposed ) % ( e t a r e c n e g r e v n o C Time (s) 0 120.5 100 241.5 361.5 482 0 50 b Convergence time (s) 0 500 250 s c irt e m e c n a m r o fr e P Assignment-based Assignment-free Our proposed ) % ( e t a r e c n e g r e v n o C Time (s) 0 120.5 100 241.5 361.5 482 0 50 b Convergence time (s) Number of robots 0 500 100 20 250 Assignment-based Minimum / maximum Assignment-free Our proposed omparison between the proposed method, the decentralized Fig. 4 \| Comparison between the proposed method, the decentralized assignment-based method in ref. 12 and the decentralized assignment-free method in ref. 21. a Snapshots and trajectories of the shape assembly processes by the three methods. There are 300 robots assembling the shape of “N''. b Statistics of convergence rate and convergence time. Each average value is calculated based on 10 trials. For the proposed method and the assignment-based one in ref. 12, the convergence rate is deﬁned as the ratio between the number of robots that are inside the shape and the total number of robots. The convergence time is deﬁned as Fig. 4 \| Comparison between the proposed method, the decentralized assignment-based method in ref. 12 and the decentralized assignment-free the time when the convergence rate is equal to 100%. For the assignment-free method in ref. 21, the convergence rate is deﬁned as the ratio between the number of robots that have reached the edge and the total number of robots. The width of the border, a parameter in the method in ref. 21, is set to zero so that the robots can aggregate evenly around the shape as much as possible. Since it is difﬁcult for all the robots evenly distribute along the edge, the convergence time is deﬁned as the time when the convergence rate reaches 70%. Adaptability to swarm scale variants The parameters of the three algo- rithms are given in Supplementary Table 2. the time when the convergence rate is equal to 100%. For the assignment-free method in ref. 21, the convergence rate is deﬁned as the ratio between the number of robots that have reached the edge and the total number of robots. The width of the border, a parameter in the method in ref. 21, is set to zero so that the robots can aggregate evenly around the shape as much as possible. Since it is difﬁcult for all the robots evenly distribute along the edge, the convergence time is deﬁned as the time when the convergence rate reaches 70%. The parameters of the three algo- rithms are given in Supplementary Table 2. assignment based method in ref. 12 and the decentralized assignment free method in ref. 21. a Snapshots and trajectories of the shape assembly processes by the three methods. There are 300 robots assembling the shape of “N''. b Statistics of convergence rate and convergence time. Each average value is calculated based on 10 trials. For the proposed method and the assignment-based one in ref. 12, the convergence rate is deﬁned as the ratio between the number of robots that are inside the shape and the total number of robots. The convergence time is deﬁned as Complex environment exploration the desired shape efﬁciently. As a consequence, the swarm can maneuver while the overall shape varies. Being able to track moving shapes is an important feature of the proposed strategy. By contrast, the state-of-the-art methods for homogeneous swarms are only applicable to static shapes10,12,18,19,30. Although there is a rich body of control-theoretic methods that can achieve maneuvering formations14,31,32, these methods require goal assignments or unique robot identities. The proposed shape assembly strategy can be applied to environ- ment exploration tasks, in which a swarm must evenly fulﬁll an environment while avoiding obstacles. As a representative example, a swarm can ﬂood into a room through a narrow passage without getting trapped at the entrance (Fig. 7a). This example mimics the process of pedestrians entering a passenger elevator, which well demonstrates the idea of mean-shift exploration of the proposed Nature Communications\| (2023) 14:3476 6 Coverage rate M1 (%) ncell/nrobot 95 90 100 Case 1 Case 3 Case 2 Case 4 3.5 Time (s) 7.5 10.5 14 80 60 0 100 Coverage rate M1 (%) 3.5 Time (s) 7.5 10.5 14 70 40 0 100 Entering rate M2 (%) 3.5 Time (s) 7.5 10.5 14 80 60 0 100 Uniformity M3 (m2) Initial position Final position 27 x-axis (m) 30 33 36 27 24 24 30 33 36 y-axis (m) 18 x-axis (m) 30 42 54 18 6 6 30 42 54 14 x-axis (m) 30 46 62 14 -2 -2 30 46 62 10 x-axis (m) 30 50 70 10 -10 -10 30 50 70 Case 1: nrobot=16, ncell=2049 Case 2: nrobot=256, ncell=115 Case 3: nrobot=512, ncell=521 Case 4: nrobot=1024, ncell=2060 a b c 1 2 3 4 5 6 7 8 Starfish Initial shape Starfish formed One arm removed Starfish reformed Arms Exploring Regeneration Arm growing Arm growing d Time = 5.5 s Time = 7.0 s Time = 7.8 s Time = 14.0 s Average Minimum / Maximum Average Minimum / Maximum Average Minimum / Maximum Number of robots nrobot 8 0 16 16 Convergence time (s) 32 64 128 256 512 768 1024 Entering rate M2 (%) 95 90 100 ncell/nrobot e Fig. 5 \| Adaptability of the proposed strategy. a The desired shape is a starﬁsh33. b, c Experimental results of 50 real Rainbow robots assembling a starﬁsh shape. Complex environment exploration a The desired shape is a starﬁsh33. b, c Experimental results of 50 real Rainbow robots assembling a starﬁsh shape. If one arm of the starﬁsh shape is removed, the swarm spontaneously grows a new arm without any goal re-assignment or fault-tolerant control thanks to the shape- exploring control term. See Supplementary Movie 3. d It is shown by numerical simulation that the strategy shows stable performance in the presence of the mismatch between nrobot and ncell, quantitatively veriﬁed by the three metrics of coverage rate, entering rate, and uniformity. e Statistical results of 512 simulations of the starﬁsh assembly task given mismatched values of nrobot and ncell. In each simulation, nrobot and ncell take values respectively from {16, 32, 64, 128, 256, 512, 768, 1024} and {115, 521, 920, 1321, 1623, 2049, 2060, 2682}. Each pair of nrobot an ncell further corresponds to eight trials with random initializations (including robots' locations and their interpretations of the desired shape). The 8 × 8 × 8 = 512 statistical results show that the proposed strategy exhibits stable performance in terms of the evaluation metrics in the presence of strong mis- matches between nrobot and ncell. a Starfish Arms b c 1 2 3 4 5 6 7 8 Initial shape Starfish formed One arm removed Starfish reformed Exploring Regeneration Arm growing Arm growing b a c c 8 d Case 4: nrobot=1024, ncell=2060 Case 1 Case 3 Case 2 Case 4 3.5 Time (s) 7.5 10.5 14 80 60 0 100 Coverage rate M1 (%) Time 5.5 s Coverage rate M1 (%) ncell/nrobot 95 90 100 Average Minimum / Maximum e Average Minimum / Maximum Entering rate M2 (%) 95 90 100 ncell/nrobot of the starﬁsh assembly task given mismatched values of nrobot and ncell. In each simulation, nrobot and ncell take values respectively from {16, 32, 64, 128, 256, 512, 768, 1024} and {115, 521, 920, 1321, 1623, 2049, 2060, 2682}. Each pair of nrobot and ncell further corresponds to eight trials with random initializations (including robots' locations and their interpretations of the desired shape). The 8 × 8 × 8 = 512 statistical results show that the proposed strategy exhibits stable performance in terms of the evaluation metrics in the presence of strong mis- matches between nrobot and ncell. Fig. 5 \| Adaptability of the proposed strategy. a The desired shape is a starﬁsh33. Complex environment exploration b, c Experimental results of 50 real Rainbow robots assembling a starﬁsh shape. If one arm of the starﬁsh shape is removed, the swarm spontaneously grows a new arm without any goal re-assignment or fault-tolerant control thanks to the shape- exploring control term. See Supplementary Movie 3. d It is shown by numerical simulation that the strategy shows stable performance in the presence of the mismatch between nrobot and ncell, quantitatively veriﬁed by the three metrics of coverage rate, entering rate, and uniformity. e Statistical results of 512 simulations of the starﬁsh assembly task given mismatched values of nrobot and ncell. In each simulation, nrobot and ncell take values respectively from {16, 32, 64, 128, 256, 512, of the starﬁsh assembly task given mismatched values of nrobot and ncell. In each simulation, nrobot and ncell take values respectively from {16, 32, 64, 128, 256, 512, 768, 1024} and {115, 521, 920, 1321, 1623, 2049, 2060, 2682}. Each pair of nrobot and ncell further corresponds to eight trials with random initializations (including robots' locations and their interpretations of the desired shape). The 8 × 8 × 8 = 512 statistical results show that the proposed strategy exhibits stable performance in terms of the evaluation metrics in the presence of strong mis- matches between nrobot and ncell. strategy: The pedestrians that enter the elevator ﬁrst should move into the deep rather than staying near the entrance to block the people behind. That is, a robot inside the desired shape would stop exploring when it has no neighbors inside its sensing radius. As a result, the robots would not explore too fast to depart from the group. This feature is useful in practice because robots may have to explore an environ- ment while keeping sufﬁciently close to others to maintain wireless connections. In addition, the system is resilient to robot additions or removals. For example, if the connectivity of the swarm is broken due to the removal of some robots, the isolated ones that are already in the maze would stop moving. As more and more robots That is, a robot inside the desired shape would stop exploring when it has no neighbors inside its sensing radius. As a result, the robots would not explore too fast to depart from the group. Nature Communications\| (2023) 14:3476 Complex environment exploration If one arm of the starﬁsh shape is removed, the swarm spontaneously grows a new arm without any goal re-assignment or fault-tolerant control thanks to the shape- exploring control term. See Supplementary Movie 3. d It is shown by numerical simulation that the strategy shows stable performance in the presence of the mismatch between nrobot and ncell, quantitatively veriﬁed by the three metrics of coverage rate, entering rate, and uniformity. e Statistical results of 512 simulations of the starﬁsh assembly task given mismatched values of nrobot and ncell. In each simulation, nrobot and ncell take values respectively from {16, 32, 64, 128, 256, 512 768, 1024} and {115, 521, 920, 1321, 1623, 2049, 2060, 2682}. Each pair of nrobot an ncell further corresponds to eight trials with random initializations (including robots' locations and their interpretations of the desired shape). The 8 × 8 × 8 = 512 statistical results show that the proposed strategy exhibits stable performance in terms of the evaluation metrics in the presence of strong mis- matches between nrobot and ncell. Article https://doi.org/10.1038/s41467-023-39251 Article https://doi.org/10.1038/s41467-023-39251-5 Coverage rate M1 (%) ncell/nrobot 95 90 100 Case 1 Case 3 Case 2 Case 4 3.5 Time (s) 7.5 10.5 14 80 60 0 100 Coverage rate M1 (%) 3.5 Time (s) 7.5 10.5 14 70 40 0 100 Entering rate M2 (%) 3.5 Time (s) 7.5 10.5 14 80 60 0 100 Uniformity M3 (m2) Initial position Final position 27 x-axis (m) 30 33 36 27 24 24 30 33 36 y-axis (m) 18 x-axis (m) 30 42 54 18 6 6 30 42 54 14 x-axis (m) 30 46 62 14 -2 -2 30 46 62 10 x-axis (m) 30 50 70 10 -10 -10 30 50 70 Case 1: nrobot=16, ncell=2049 Case 2: nrobot=256, ncell=115 Case 3: nrobot=512, ncell=521 Case 4: nrobot=1024, ncell=2060 a b c 1 2 3 4 5 6 7 8 Starfish Initial shape Starfish formed One arm removed Starfish reformed Arms Exploring Regeneration Arm growing Arm growing d Time = 5.5 s Time = 7.0 s Time = 7.8 s Time = 14.0 s Average Minimum / Maximum Average Minimum / Maximum Average Minimum / Maximum Number of robots nrobot 8 0 16 16 Convergence time (s) 32 64 128 256 512 768 1024 Entering rate M2 (%) 95 90 100 ncell/nrobot e Fig. 5 \| Adaptability of the proposed strategy. Discussion The distributed negotiation and control strategy proposed in this work is free of both centralized and decentralized goal assignment proce- dures. This strategy introduces a mean-shift exploration mechanism so that each robot can actively explore unoccupied locations around them to avoid inter-robot competition for the same locations. The mean-shift exploration is achieved through an adapted version of the mean-shift algorithm so that each robot keeps seeking the highest density of unoccupied cells. It is noticed that the mean-shift algorithm is also adopted in ref. 25 to achieve shape assembly. However, the mean-shift algorithm therein is used as an alternative to generating attractive and repulsive forces and hence is not able to resolve the inter-robot competition problem. One limitation of the proposed strategy is that it can only assemble shapes with single connected components. That is because the mean-shift exploration of each robot is based on local sensing of unoccupied locations. Since the sensing range of each robot is limited, if two disconnected shape components are far away from each other, it is difﬁcult for a robot to explore unoccupied locations across different shape components. This problem may be potentially solved in several ways in practice. The ﬁrst is to globally assign the robots to different components in advance. This way requires a centralized assignment process. A simple distributed solution is to add a line segment to bridge two disconnected components so that the entire shape becomes connected. In this way, each robot can explore the bridge to move from one shape component to another. Experimental results veriﬁed that the proposed strategy can assemble highly nonconvex shapes and exhibits strong adaptability against mismatches between the number of robots and the number of cells in the desired shape. The strategy can also be applied to solve some challenging tasks such as cooperative cargo transportation and environment exploration with minimal modiﬁcations of the algo- rithms. It, therefore, provides a promising method for efﬁcient and adaptive shape assembly tasks of robot swarms. The desired shape in a shape assembly task is a global constraint for all the robots. The global constraint sets a fundamental require- ment that certain global information must be used during the process of shape assembly. However, the required global information may exhibit in different forms in different methods. Complex environment exploration This feature is useful in practice because robots may have to explore an environ- ment while keeping sufﬁciently close to others to maintain wireless connections. In addition, the system is resilient to robot additions or removals. For example, if the connectivity of the swarm is broken due to the removal of some robots, the isolated ones that are already in the maze would stop moving. As more and more robots The proposed strategy can also be used to explore more com- plex environments such as a maze as shown in Fig. 7b. Although there are many nonconvex corners in the maze, the swarm can successfully fulﬁll the maze without getting deadlocked at any corner, verifying the strong exploration ability of the proposed strategy. The strategy can well balance the exploration speed and inter-robot connectivity. Nature Communications\| (2023) 14:3476 7 7 Article https://doi.org/10.1038/s41467-023-39251-5 a Desired shape Rotation Translation b Desired shape Cargo Informed robots Greater entering action Less shape- entering action Stop moving Shape-entering action Initial shape Greater shape- exploring action Interacting with neighbor Fig. 6 \| Cooperative cargo transportation by a swarm of eight real Rainbow robots. a The desired shape is speciﬁed as a hollow rectangle centered at the cargo. b The process of cooperative transportation by the swarm. When the robots assemble the desired shape, they would encircle the cargo tightly. Since the desired shape is moving, the robots would transport the cargo while tracking the desired moving shape. The entire process is led by two informed robots. The color of each robot indicates its velocity command. See Supplementary Movies 4. a Desired shape b Desired shape Cargo Stop moving Initial shape Fig. 6 \| Cooperative cargo transportation by a swarm of eight real Rainbow robots. a The desired shape is speciﬁed as a hollow rectangle centered at the cargo. b The process of cooperative transportation by the swarm. When the robots assemble the desired shape, they would encircle the cargo tightly. Since the desired Rotation Translation b Informed robots Greater entering action Less shape- entering action Stop moving Shape-entering action Initial shape Greater shape- exploring action Interacting with neighbor ht real Rainbow ntered at the cargo. en the robots y. Since the desired shape is moving, the robots would transport the cargo while tracking the desired moving shape. The entire process is led by two informed robots. The color of each robot indicates its velocity command. Complex environment exploration See Supplementary Movies 4. Rotation Translation b Informed robots Greater entering action Less shape- entering action Stop moving Shape-entering action Initial shape Greater shape- exploring action Interacting with neighbor b Desired shape Cargo Cargo Desired shape Fig. 6 \| Cooperative cargo transportation by a swarm of eight real Rainbow robots. a The desired shape is speciﬁed as a hollow rectangle centered at the cargo. b The process of cooperative transportation by the swarm. When the robots assemble the desired shape, they would encircle the cargo tightly. Since the desired shape is moving, the robots would transport the cargo while tracking the desired moving shape. The entire process is led by two informed robots. The color of each robot indicates its velocity command. See Supplementary Movies 4. move into the maze, the isolated ones would get connected to the others again. The proposed negotiation and control algorithms are applied to assemble 2D shapes in this work. They can be extended to 3D scenarios since these algorithms are based on vector calculations where the position and velocity vectors are not restricted to 2D. However, a new human-swarm interface that can specify 3D shapes is required. In addition to user-speciﬁed geometric shapes, the proposed algorithms may be extended to assemble shapes speciﬁed in a natural way. For example, a swarm of robots may be used to clean an area of chemical pollutants in a water body. Here, the area of the densest pollutants can be treated as the desired shape and the density change in the nearby water can be regarded as the gradient. In this case, the shape is “spe- ciﬁed” by the environment naturally and the proposed algorithms can be potentially applied. Discussion For example, in the proposed strategy and the state-of-the-art ones10,12,30, the required global information is that every robot has a common sense on a global reference frame. With thisglobal information, every robot can evaluate its location relative to the desired shape. In practice, the global refer- ence frame can be obtained by GPS in outdoor open spaces. If the global reference frame is unavailable, there must be alternatives to acquire global information. For example, the method in ref. 18 replaces the global reference frame by using a relative positioning system based on four seed robots. Moreover, we achieved the environment exploration tasks by treating them as shape assembly tasks, where the map of the environ- ment is known and speciﬁed as the desired shape. While the map of the environment to be explored may not be available in practical search and rescue tasks, the proposed strategy can be applied if a minimal mod- iﬁcation is made so that any regions unoccupied by obstacles are treated as desired shape regions to fulﬁll. Finally, although the algo- rithms proposed in this work are fully distributed, the physical robots in our experiments were provided data from a centralized motion capture system. It will be a promising research direction to develop robotic hardware systems with onboard sensing units to realize fully distributed shape assembly tasks of large-scale robot swarms. Nature Communications\| (2023) 14:3476 8 8 Article https://doi.org/10.1038/s41467-023-39251-5 a b 1 2 3 4 5 6 Obstacles 27 robots Entrance Entering Exploring Interaction Collision points Balanced and stop Fig. 7 \| Environment exploration by the proposed strategy. a Experiments of 27 real Rainbow robots ﬂooding into a room through a narrow passage. This scenario mimics the process of pedestrians entering a passenger elevator: The pedestrians that enter the elevator ﬁrst should move into the deep rather than staying near the a b 1 2 3 4 5 6 Obstacles 36 robots Entrance 7 6 5 4 3 2 1 Obstacles 27 robots Entrance Entering Exploring Interaction Collision points Balanced and stop Fig. 7 \| Environment exploration by the proposed strategy. a Experiments of 27 real Rainbow robots ﬂooding into a room through a narrow passage. This scenario mimics the process of pedestrians entering a passenger elevator: The pedestrians that enter the elevator ﬁrst should move into the deep rather than staying near the entrance to block the people behind. Specify a desired shape as a black-white grid The proposed human-swarm interface allows the user to either load a predesigned image or manually draw one in the workspace (Supple- mentary Figure 1). Either way, we can obtain a binary grid with black and white cells. Each cell of the grid is described by two basic para- meters ρ and ξρ. Here, ρ = (ρx, ρy) is the column and row indexes of the cell and ξρ ∈{0, 1} represents the color of the cell: ξρ = 0 if the cell is black and ξρ = 1 if the cell is white. The desired shape corresponds to the set of black cells. More information is given in Section 1 in the Supplementary Information. Methods Parameterization of the size of each cell in the grid The desired shape represented by a grid obtained from the human- swarm interface is merely graphical. Some important parameters of the desired shape are to be determined automatically. The ﬁrst para- meter is the size length of each cell denoted as ℓcell. On the one hand, the total area of all the black cells is ‘2 cellncell. On the other hand, let ravoid be the collision avoidance distance between the centers of any two robots. It is expected that the distance among each pair of robots in the desired shape is equal to ravoid (Supplementary Figure 2c). As a result, the space occupied by each robot could be approximated by a circle with the center at the robot and the radius as ravoid/2. Thus, the area occupied by all the nrobot robots is πðravoid=2Þ2nrobot. The above two areas are expected to be equal so that the robots could cover the desired shape (Supplementary Figure 2c): Discussion b Experiments of 36 real Rainbow robots exploring a complex maze without getting deadlocked at any nonconvex corners See Supplementary Movies 5 and 6. a 1 2 3 4 5 6 Obstacles 27 robots Entrance Entering Exploring Interaction Collision points Balanced and stop Fig. 7 \| Environment exploration by the proposed strategy. a Experiments of 2 real Rainbow robots ﬂooding into a room through a narrow passage. This scenar mimics the process of pedestrians entering a passenger elevator: The pedestrian that enter the elevator ﬁrst should move into the deep rather than staying near th b Obstacles 36 robots Entrance 1 b a 7 6 5 4 3 2 3 5 5 6 entrance to block the people behind. b Experiments of 36 real Rainbow robots exploring a complex maze without getting deadlocked at any nonconvex corners. See Supplementary Movies 5 and 6. Fig. 7 \| Environment exploration by the proposed strategy. a Experiments of 27 real Rainbow robots ﬂooding into a room through a narrow passage. This scenario mimics the process of pedestrians entering a passenger elevator: The pedestrians that enter the elevator ﬁrst should move into the deep rather than staying near the Nature Communications\| (2023) 14:3476 https://doi.org/10.1038/s41467-023-39251-5 Supplementary Figure 3a. In particular, it is designed as Supplementary Figure 3a. In particular, it is designed as more general cases where ncell and nrobot may mismatch (see examples in Fig. 5 and Supplementary Figure 5). more general cases where ncell and nrobot may mismatch (see examples in Fig. 5 and Supplementary Figure 5). vent i = κ1ξρi pT,i pi k pT,i pi k + vρo,i ð5Þ ð5Þ Shape-exploration velocity command Here, sign( ⋅) and ∣⋅∣denote the sign and the absolute value of a real number. In addition, c1 > 0 and 0 < α < 1 are two constant gains. There are two terms in (3). The ﬁrst term in (3) is the average of the deﬁciency of the position interpretations between robot i and its neighbors. Here, sign( ⋅) and ∣⋅∣denote the sign and the absolute value of a real number. In addition, c1 > 0 and 0 < α < 1 are two constant gains. The role of the ﬁrst term is to drive pρo,i ! pρo,j where j 2 N i. The second term is the average of the velocities of the neighboring robots. Its role is to drive vρo,i ! vρo,j where j 2 N i. Since α < 1, consensus can be achieved in a ﬁnite time. This is important for speeding up the negotiation process. The convergence analysis is given in Theorem 1 in the Supplementary Information. The velocity command in (6) encourages robot i to explore unoccupied black cells. Here, Mneigh i is deﬁned as the set of all the unoccupied black cells that are within the sensing radius rsense of the robot (see Supplementary Figure 3b) and κ2 = σ2. In addition, when robot i is close to the boundary of the shape so that there are non-black cells within the sensing radius, on top of exploring unoccupied black cells, the velocity command also aims to push robot i into the desired shape. In this case, Mneigh i includes all the black cells no matter whe- ther they are occupied or not and κ2 = σ1. Regarding the orientation negotiation, let ϕi be the interpretation of ϕ by robot i. The initial values of ϕi could be randomly selected or based on task-oriented requirements. The distributed orientation negotiation algorithm is Interaction velocity command The aim of the interaction velocity command vint i in (7) is to achieve collision avoidance and velocity alignment. To that end, it is designed as _ϕi = c2 ∣N i∣ X j2N i signðϕi ϕjÞ∣ϕi ϕj∣α + 1 ∣N i∣ X j2N i _ϕj ð4Þ ð4Þ ð4Þ as vint i = κ3 X j2N i ∪Oi μð∣∣pi pj∣∣Þ pi pj \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} first term X j2N i 1 ∣N i∣vi vj \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} second term ð7Þ where c2 is a positive constant coefﬁcient. This algorithm has the same structure as (3) and can be analyzed analogously. ð7Þ If the desired shape is required to track a speciﬁed trajectory, we can introduce informed robots to achieve that. Details are given in Section 2 in Supplementary Information. where κ3 is a positive control gain. where κ3 is a positive control gain. The ﬁrst term in (7) is a weighted sum of pi −pj, where pi is the position of robot i and pj is the position of the neighboring robot j or a collision point j. Here, j 2 N i ∪Oi where N i is the set of neighboring robots and Oi is the set of collision points (see Supplementary Parameterization of the position and orientation of the desired shape Another two parameters of the desired shape are the center position and orientation. There is no centralized assignment of the position or orientation. The robots can negotiate the two parameters in a dis- tributed manner. where pi is the position of robot i and pT,i is a target location for robot i to move toward. Hence, pT,ipi kpT,ipik is a unit vector pointing from pi to pT,i. Therefore, the ﬁrst term in (5) drives the robot toward its target location. Here, κ1ξρi is a control gain where ρi is the index of the cell that is closest to pi, ξρi is the gray level of that cell, and κ1 > 0 is a constant gain. The second term vρo,i in (5) is the local interpretation of robot i on the moving velocity of the entire shape. This term is necessary when the desired shape is moving. The calculation of the cell index ρi and the target location pT,i is given in Section 3 in the Sup- plementary Information. Before proceeding further, we need to introduce some necessary notations that will be used frequently later. Suppose there are nrobot mobile robots in R2 and nrobot ≥2. Each robot is regarded as a circle with the radius as rbody. Let pi 2 R2 be the position of the center point of robot i in a global coordinate frame. The dynamic model of each robot is assumed to be _pi = vi where i = 1, …, nrobot. Here, vi is the velocity command to be designed. When the distance between two robots is less than a threshold rsense, the two robots could share information with each other. The information network deﬁnes an undirected graph G = ðV, EÞ, which consists of a vertex set V = f1, . . . , nrobotg and an edge set E V × V such that E = fði, jÞ :k pi pj k < rsense, j≠ig. Here, ∥⋅∥is the Euclidean norm. The set of neighbors of robot i is N i = fj 2 V : ði, jÞ 2 Eg. Convert the desired shape to a grayscale grid The purpose to convert the black-white grid to a grayscale grid is to expand the inﬂuence scope of the desired shape so that the robots can move into the desired shape more smoothly. The gray conversion is based on the distance transformation algorithm29. Speciﬁcally, we expand the set of black cells out by h cells to generate an h-level grayscale grid. The gray level of each cell denoted as ξρ is calculated based on a local parallel method described in ref. 29. In particular, for any cell ρ in the grid, its gray value is calculated iteratively by π 4 r2 avoidnrobot ≈‘2 cellncell from which ℓcell can be solved as from which ℓcell can be solved as ‘cell = ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ π 4 nrobot ncell r ravoid: ð2Þ ð2Þ ξk ρ = min ρ02Mρ ξk1 ρ0 + 1 h , k = 1, 2, . . . , h 1: ð1Þ Equation (2) indicates that, when nrobot and ravoid are given, ℓcell is inversely proportional to ﬃﬃﬃﬃﬃﬃﬃﬃﬃ ncell p . ð1Þ It is notable that ncell is speciﬁed by the user when drawing a shape in the human-swarm interface. If ncell = nrobot, equation (2) becomes ‘cell = ﬃﬃﬃπ 4 p ravoid ≈ravoid, which means the side length of each cell is equal to the avoidance distance. In this case, each robot can approximately occupy one cell. However, our method does not necessarily require ncell = nrobot, which is a strict condition. The proposed method allows Here, the superscript k denotes the k-th iteration. In every iteration, we conduct (1) for each cell. We need at most h −1 iterations to obtain the ﬁnal grayscale grid29. Here, Mρ is a 3 × 3 mask centered at the cell ρ. Illustrative examples and more information can be found in Section 1 in the Supplementary Information. Nature Communications\| (2023) 14:3476 9 9 Article https://doi.org/10.1038/s41467-023-39251-5 Nature Communications\| (2023) 14:3476 Shape-exploration velocity command The shape-exploration velocity command vexp i in (6) aims to push robot i into the desired shape and explore unoccupied black cells. Here, a cell is deﬁned as occupied if the distance between its center and any robot is less than ravoid/2; and unoccupied otherwise. In particular, we employ the mean-shift concept26 to design vexp i as i Suppose ρo is the cell located closest to the center of the desired shape. Let pρo and vρo be the position and velocity of the center point of cell ρo in a global reference frame. Let ϕ denote the orientation angle of the desired shape. Then, the position and orientation of the desired shape could be represented by pρo and ϕ, respectively. vexp i = P ρ2Mneigh i κ2ψðk pρ pi k =rsenseÞ pρ pi P ρ2Mneigh i ψðk pρ pi k =rsenseÞ ð6Þ ð6Þ ρo Every robot has its own interpretation of pρo and vρo, denoted as pρo,i and vρo,i. Initially, pρo,iðt0Þ = piðt0Þ and vρo,iðt0Þ = viðt0Þ, which means that each robot initially treats itself as the center of the desired group shape. The interpretations of different robots can gradually reach a consensus by the following distributed negotiation algorithm: which is a normalized weighted average of pρ −pi, where pi is robot i’s position and pρ is a position of a valid cell whose index is ρ. Here, ρ belongs to the set Mneigh i . The factor κ2 ∈{σ1, σ2} with σ1, σ2 > 0 is a positive constant. The weight for pρ −pi is a function ψ deﬁned as ψðzÞ = 1, z ≤0 1 2 ð1 + cos πzÞ, 0 < z < 1 0, z ≥1 8 > < > : : vρo,i = c1 ∣N i∣ X j2N i signðpρo,i pρo,jÞ∣pρo,i pρo,j∣α + 1 ∣N i∣ X j2N i vρo,j: ð3Þ ð3Þ This function is monotonically decreasing from 1 to 0 as z increases. As a result, the weight ψ(∥pρ −pi∥/rsense) is large when the distance between pρ and pi is small. Hence, more weights are given to the cells that are closer to robot i. There are two terms in (3). The ﬁrst term in (3) is the average of the deﬁciency of the position interpretations between robot i and its neighbors. References 1. Anderson, C., Theraulaz, G. & Deneubourg, J. L. Self-assemblages in insect societies. Insectes Soc. 49, 99–110 (2002). 2. Chen, Y. & Kolokolnikov, T. A minimal model of predator-swarm interactions. J. R. Soc. Interface 11, 20131208 (2014). 2. Chen, Y. & Kolokolnikov, T. A minimal model of predator-swarm interactions. J. R. Soc. Interface 11, 20131208 (2014). M1 = nocc ncell × 100% M1 = nocc ncell × 100% 3. Vicsek, T. & Zafeiris, A. Collective motion. Phys. Rep. 517, 71–140 (2012). 4. Gelblum, A. et al. Ant groups optimally amplify the effect of transiently informed individuals. Nat. Commun. 6, 7729 (2015). 4. Gelblum, A. et al. Ant groups optimally amplify the effect of transiently informed individuals. Nat. Commun. 6, 7729 (2015). where nocc is the number of black cells occupied by robots. If all black cells are occupied, that is nocc = ncell, then M1 = 100%. If no black cells are occupied, then M1 = 0. 5. Berman, S., Lindsey, Q., Sakar, M. S., Kumar, V. & Pratt, S. C. Experimental study and modeling of group retrieval in ants as an approach to collective transport in swarm robotic systems. Proc. IEEE 99, 1470–1481 (2011). 5. Berman, S., Lindsey, Q., Sakar, M. S., Kumar, V. & Pratt, S. C. Experimental study and modeling of group retrieval in ants as an approach to collective transport in swarm robotic systems. Proc. IEEE 99, 1470–1481 (2011). The second metric, entering rate, is the proportion of the robots that have entered the desired shape. Here, a robot is said to be inside the shape if it occupies a black cell. The metric is deﬁned as 6. Ren, W., Beard, R. W. & Atkins, E. M. Information consensus in multi- vehicle cooperative control. IEEE Control Syst. Mag. 27, 71–82 (2007). 6. Ren, W., Beard, R. W. & Atkins, E. M. Information consensus in multi- vehicle cooperative control. IEEE Control Syst. Mag. 27, 71–82 (2007). M2 = nin nrobot × 100% 7. Brambilla, M., Ferrante, E., Birattari, M. & Dorigo, M. Swarm robotics: a review from the swarm engineering perspective. Swarm Intell. 7, 1–41 (2013). 7. Brambilla, M., Ferrante, E., Birattari, M. & Dorigo, M. Swarm robotics: a review from the swarm engineering perspective. Swarm Intell. 7, 1–41 (2013). where nin is the number of robots inside the desired shape. Article Figure 3c). The weight μ(∥pi −pj∥) is deﬁned as Figure 3c). The weight μ(∥pi −pj∥) is deﬁned as charts, the lower and upper edges of the bar represent the minimum and maximum, and the shaded region represents the average. μðk pi pj kÞ = ravoid kpipjk 1, k pi pj k ≤ravoid 0, k pi pj k > ravoid ( : We implemented the simulation in Matlab. Details of the simula- tion setup and parameters used are given in Section 4 in the Supple- mentary Information. In our experiments, we use 50 holonomic wheeled robots. We implemented the experiments in Visual Studio using C++. Details of the experimental setup, robotic platform, and parameter settings are given in Section 5 in the Supplementary Information. By deﬁnition, when ∥pi −pj∥is close to zero, the weight μ(∥pi −pj∥) methods to inﬁnity. When ∥pi −pj∥is close to ravoid, the weight μ(∥pi −pj∥) monotonically decreases to zero. Since pi −pj is a vector pointing from pj to pi, the ﬁrst term is a repulsive velocity that pushes robot i away from its surroundings to avoid a collision. The second term in (7) aims to align robot i’s velocity with its neighbors. Velocity alignment can help reduce the chance of inter-robot collision. In the meantime, it is necessary when the entire shape needs to track a moving trajectory. By deﬁnition, when ∥pi −pj∥is close to zero, the weight μ(∥pi −pj∥) methods to inﬁnity. When ∥pi −pj∥is close to ravoid, the weight μ(∥pi −pj∥) monotonically decreases to zero. Since pi −pj is a vector pointing from pj to pi, the ﬁrst term is a repulsive velocity that pushes robot i away from its surroundings to avoid a collision. The second term in (7) aims to align robot i’s velocity with its neighbors. Velocity alignment can help reduce the chance of inter-robot collision. In the meantime, it is necessary when the entire shape needs to track a moving trajectory. References If all robots are inside the desired shape, then M2 = 100%, and otherwise M2 < 100%. 8. Oh, K. K., Park, M. C. & Ahn, H. S. A survey of multi-agent formation control. Automatica 53, 424–440 (2015). 8. Oh, K. K., Park, M. C. & Ahn, H. S. A survey of multi-agent formation control. Automatica 53, 424–440 (2015). The third metric, distribution uniformity, measures the distribu- tion uniformity of the robots in the desired shape. Denote rmin ,i = minj2N i k pi pj k as the minimum distance from robot i to its neighbors. Then, the metric is deﬁned as 9. Oh, H., Shirazi, A. R., Sun, C. & Jin, Y. Bio-inspired self-organising multi-robot pattern formation: a review. Rob. Autom. Syst. 91, 83–100 (2017). 10. Alonso-Mora, J., Breitenmoser, A., Ruﬂi, M., Siegwart, R. & Beards- ley, P. Image and animation display with multiple mobile robots. Int. J. Robot. Res. 31, 753–773 (2012). M3 = X n i = 1 ðrmin ,i rmin ,iÞ2 M3 = X n i = 1 ðrmin ,i rmin ,iÞ2 11. Sakurama, K. & Ahn, H. S. Multi-agent coordination over local indexes via clique-based distributed assignment. Automatica 112, 108670 (2020). where rmin ,i = 1 n Pn i = 1 rmin,i. If the distance between every pair of robots is the same, then M3 = 0; otherwise, M3 > 0. 12. Wang, H. & Rubenstein, M. Shape formation in homogeneous swarms using local task swapping. IEEE Trans. Robot. 36, 597–612 (2020). The fourth metric, velocity polarization, is to measure the polar- ization of the velocities of the robots. The metric is deﬁned as: 13. Anderson, B. D., Yu, C., Fidan, B. & Hendrickx, J. M. Rigid graph control architectures for autonomous formations. IEEE Control Syst. Mag. 28, 48–63 (2008). M4 = ∣Pn i = 1 vi∣ Pn i = 1∣vi∣: 14. Zhao, S. & Zelazo, D. Bearing rigidity theory and its applications for control and estimation of network systems: Life beyond distance rigidity. IEEE Control Syst. Mag. 39, 66–83 (2019). If all robots move with the same velocity, then M4 = 1. If the robots’ velocities are random, then M4 is close to zero. 15. Kuhn, H. W. The Hungarian method for the assignment problem. Naval Res. Logistics 2, 83–97 (1955). Data availability The data used in this study are available from the corresponding author upon request. Code availability The code of the human-swarm interface used in this study is publicly available at https://github.com/WestlakeAerialRobotics/Human- swarm-interface(DOI: 10.5281/zenodo.7960508). Performance metrics To evaluate the performance of the proposed strategy, we consider the following four metrics. The ﬁrst metric, coverage rate, is to evaluate the proportion of the black cells in the desired shape that are occupied by robots. Here, a cell is deﬁned as occupied if the distance between its center and any robot is less than ravoid/2. The metric is deﬁned as Shape-entering velocity command The shape-entering velocity command vent i in (5) aims to drive robot i into its interpretation of the desired shape, as depicted in Nature Communications\| (2023) 14:3476 10 Article https://doi.org/10.1038/s41467-023-39251-5 Reprints and permissions information is available at http://www.nature.com/reprints 29. Borgefors, G. Distance transformations in digital images. Comput. Vis. Graph. Image Process. 34, 344–371 (1986). Publisher’s note Springer Nature remains neutral with regard to jur- isdictional claims in published maps and institutional afﬁliations. 30. Morgan, D., Subramanian, G. P., Chung, S. J. & Hadaegh, F. Y. Swarm assignment and trajectory optimization using variable- swarm, distributed auction assignment and sequential convex programming. Int. J. Robot. Res. 35, 1261–1285 (2016). 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/. 31. Zhao, S. Afﬁne formation maneuver control of multiagent systems. IEEE Trans. Autom. Control 63, 4140–4155 (2018). 32. Dong, X., Yu, B., Shi, Z. & Zhong, Y. Time-varying formation control for unmanned aerial vehicles: Theories and applications. IEEE Trans. Control Syst. Technol. 23, 340–348 (2014). 33. Yang, T. et al. A damage-tolerant, dual-scale, single-crystalline microlattice in the knobby starﬁsh, protoreaster nodosus. Science 375, 647–652 (2022). Statistics and reproducibility 16. Bertsekas, D. P. The auction algorithm: a distributed relaxation method for the assignment problem. Ann. Oper. Res. 14, 105–123 (1988). Data analysis was done by using the native functions of Originlab. All the statistical results are displayed in the format of “average ± minimum/maximum”. During the statistical analysis, no statistical method was used to predetermine sample size, and no data were excluded. All statistical trials used random initialization of robots’ locations and their interpretations of the desired shape. In all bar 17. Kamel, M. A., Yu, X. & Zhang, Y. Formation control and coordination of multiple unmanned ground vehicles in normal and faulty situa- tions: a review. Annu. Rev. Control 49, 128–144 (2020). Nature Communications\| (2023) 14:3476 11 Article https://doi.org/10.1038/s41467-023-39251-5 18. Rubenstein, M., Cornejo, A. & Nagpal, R. Programmable self- assembly in a thousand-robot swarm. Science 345, 795–799 (2014). discussions. This work was supported by the STI 2030-Major Projects (Grant No. 2022ZD0208804), the Beihang Zhuoyue Postdoctoral Fel- low Program (G.S.), the Hangzhou Key Technology Research and Development Program (Grant No. 20200416A16), and Research Center for Industries of the Future at Westlake University (Grant No. WU2022C027). 19. Slavkov, I. et al. Morphogenesis in robot swarms. Sci. Robot. 3, eaau9178 (2018). 20. Gauci, M., Nagpal, R. & Rubenstein, M. Programmable self- disassembly for shape formation in large-scale robot collectives, pp. 573–586 (Springer, Cambridge, 2018). Competing interests 23. Sabattini, L., Secchi, C. & Fantuzzi, C. Arbitrarily shaped formations of mobile robots: artiﬁcial potential ﬁelds and coordinate transfor- mation. Auto. Robots 30, 385 (2011). Author contributions 21. Alhafnawi, M., Hauert, S. & O’Dowd, P. Self-organised saliency detection and representation in robot swarms. IEEE Robot. Autom. Lett. 6, 1487–1494 (2021). S.Z., G.S., and R.Z. designed research; G.S., Z.M., Y.L., and S.Z. per- formed research; G.S., S.Z., R.G., and Z.C. analyzed data; S.Z., G.S., R.G., and Z.C. wrote the paper. 22. Hsieh, M. A., Kumar, V. & Chaimowicz, L. Decentralized controllers for shape generation with robotic swarms. Robotica 26, 691–701 (2008). Competing interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-39251-5. 24. Hou, S. P. & Cheah, C. C. Dynamic compound shape control of robot swarm. IET Control Theory Appl. 6, 454–460 (2012). 25. Vickery, C. & Salehi, S.A. A mean shift-based pattern formation algorithm for robot swarms. in 7th Int. Conf. Autom., Robot. Appl., pp. 16–20 (Prague, Czech Republic, IEEE, 2021). Correspondence and requests for materials should be addressed to Shiyu Zhao. Correspondence and requests for materials should be addressed to Shiyu Zhao. 26. Fukunaga, K. & Hostetler, L. The estimation of the gradient of a density function, with applications in pattern recognition. IEEE Trans. Inf. Theory 21, 32–40 (1975). Peer review information Nature Communications thanks Nicola Bastia- nello, Sabine Hauert, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. 27. Cheng, Y. Mean shift, mode seeking, and clustering. IEEE Trans. Pattern Anal. Mach. Intell. 17, 790–799 (1995). 28. Comaniciu, D. & Meer, P. Mean shift: a robust approach toward feature space analysis. IEEE Trans. Pattern Anal. Mach. Intell. 24, 603–619 (2002). Acknowledgements We thank Fei Chen, Canlun Zheng, Weicong Zhang, Zian Ning, Ye Zheng, and Yin Zhang at Westlake University for their help in the experiments, and Chen Wei at Beihang University for helpful © The Author(s) 2023 Nature Communications\| (2023) 14:3476 Nature Communications\| (2023) 14:3476 12
W3081007965.txt	https://zenodo.org/record/4062210/files/rechercheseducations-10562.pdf	fr		Perception de la continuité pédagogique des participants à un webinaire sur l’apprentissage à distance dans un contexte de confinement	Recherches & éducations/Recherches & éducations/Éduquer	2,020	cc-by	6,293	Recherches & éducations HS \| Juillet 2020 Quelle éducation avec la Covid-19 Perception de la continuité pédagogique des participants à un webinaire sur l’apprentissage à distance dans un contexte de confinement Sarah Descamps, Sabrin Housni, Gwendydd Piret, Audrey Kumps, Karim Boumazguida, Marie Dumont, Laëtitia Dragone, Gaëtan Temperman et Bruno De Lièvre Édition électronique URL : http://journals.openedition.org/rechercheseducations/10562 ISSN : 1760-7760 Éditeur Société Binet Simon Ce document vous est offert par Université de Mons Référence électronique Sarah Descamps, Sabrin Housni, Gwendydd Piret, Audrey Kumps, Karim Boumazguida, Marie Dumont, Laëtitia Dragone, Gaëtan Temperman et Bruno De Lièvre, « Perception de la continuité pédagogique des participants à un webinaire sur l’apprentissage à distance dans un contexte de confinement », Recherches & éducations [En ligne], HS \| Juillet 2020, mis en ligne le , consulté le 01 octobre 2020. URL : http://journals.openedition.org/rechercheseducations/10562 Ce document a été généré automatiquement le 1 octobre 2020. Propriété intellectuelle Perception de la continuité pédagogique des participants à un webinaire sur l... Perception de la continuité pédagogique des participants à un webinaire sur l’apprentissage à distance dans un contexte de confinement Sarah Descamps, Sabrin Housni, Gwendydd Piret, Audrey Kumps, Karim Boumazguida, Marie Dumont, Laëtitia Dragone, Gaëtan Temperman et Bruno De Lièvre Introduction 1 Depuis janvier 2020, l’actualité nationale et internationale est dominée par la propagation de la maladie infectieuse appelée COVID-19. Dans ce contexte extraordinaire, des mesures fortes ont été prises par les autorités des États les plus touchés. De ce fait, la Belgique est confinée depuis le samedi 14 mars pour limiter considérablement les contacts entre les personnes, et ce, dans le but de ralentir la propagation du virus et de réduire le risque que les services médicaux concernés ne puissent faire face à l’afflux de patients. Ces mesures entraînent, entre autres, la fermeture des écoles et occasionnent de nombreux bouleversements en termes de méthodes d’enseignement. En effet, les cours étant suspendus, les activités à domicile ont dû être envisagées (Circulaire 7541, 2020). Bien que les activités en classe soient stoppées, les politiques éducatives montrent une volonté d’assurer l’éducation des élèves en confinement. Si, pour l’enseignement obligatoire, les enseignants ont pour mission de respecter la logique “remédiation-consolidation-dépassement” dans les apprentissages (Circulaire 7541, 2020), dans l’enseignement supérieur, le mot d’ordre est d’organiser les enseignements à distance. Ainsi, les attentes du milieu scolaire sont assez diversifiées, allant d’un simple appel hebdomadaire, à l’envoi de devoirs par courriel, en passant par Recherches & éducations, HS \| Juillet 2020 1 Perception de la continuité pédagogique des participants à un webinaire sur l... des cours en ligne aux horaires prévus. Pourtant, tous les enseignants ne sont pas égaux face aux tâches numériques proposées (UNESCO, 2020). Certains éprouvent des difficultés techniques, d’autres pédagogiques ou encore organisationnelles et communicationnelles avec cette pratique si particulière qu’est l’enseignement à distance. D’autres, par contre, y voient une opportunité d’explorer et de tester de nouvelles pratiques pédagogiques. Ces différents facteurs sont donc soit la source de frustrations soit l’origine d’une accélération de l’usage du numérique pédagogique, ce qui a amené différents acteurs à exprimer leur ressenti via les réseaux sociaux, les forums… pour partager leurs pratiques ou pour chercher des réponses à leurs besoins.. Dans ce contexte, cet article propose une analyse de la perception de 154 acteurs éducatifs qui ont participé à une conférence live1 organisée et suivie par plus de 400 personnes, le jeudi 26 mars, soit 12 jours après le début du confinement, par “MUMONS, Sciences, Arts & Curiosités !”, structure de l’Université de Mons dans le domaine de la culture et de la diffusion des sciences et des technologies. Lors de celle-ci, Bruno De Lièvre et Gaëtan Temperman proposent d’accompagner les enseignants dans leur réflexion sur l’enseignement à distance. Cette conférence est l’occasion d’aller plus loin que la recommandation d’une liste d’outils : elle présente des exemples de tâches sur lesquelles ils pourraient se baser pour alimenter leurs propres scénarios pédagogiques et rendre actifs les apprenants. L’objectif principal est de mettre en évidence d’une part, des principes pédagogiques (Dillenbourg, 2016) pour scénariser l’apprentissage (création et enchainement de tâches, identification d’outils numériques, etc.) ; d’autre part, de décrire les modalités de suivi indispensables pour l’encadrer de manière efficace (Quintin, 2008). Ancrages théoriques 2 De Lièvre, Depover et Dillenbourg (2006) ainsi que Quintin (2008) mettent en évidence l’apport positif d’interventions du tuteur pour faciliter la progression des apprenants. Pour eux, l’idée d’intervenir en cours d’apprentissage correspond à l’approche suggérée par Bruner (1998). Elle a pour but d’accompagner les apprenants (« guide on the side ») dans leurs démarches. Elles sont susceptibles d’avoir un impact important sur leurs performances en cours et en fin d’apprentissage. En termes de contenu, la régulation peut concerner différents aspects complémentaires. Elle peut traiter à la fois des aspects pédagogiques, organisationnels et socioaffectifs (relationnels), ainsi que des aspects techniques quand on mobilise les outils numériques. Le soutien pédagogique passe d’abord par l’explicitation des objectifs à atteindre dans le cadre de la formation. Les aspects pédagogiques portent sur des clarifications au niveau du contenu à maîtriser et sur des aides pour restructurer la matière. Sur le plan évaluatif, il correspond à la rédaction de feedbacks formatifs comme aides à l’apprentissage. Les interventions de type pédagogique permettent d’attirer l’attention sur les points importants et d’encourager les apprenants à construire leur savoir. Sur le plan organisationnel, les interventions concernent l’état d’avancement dans la réalisation de la tâche. Elles facilitent la gestion du temps qui constitue une difficulté importante à surmonter pour les apprenants à distance. Dans cette perspective, elles amènent également les apprenants à prendre du recul par rapport à la planification des tâches, au partage de celles-ci ainsi qu’à l’utilisation pertinente des outils mis à disposition. En ce qui concerne les aspects relationnels, les interventions prennent en compte d’une Recherches & éducations, HS \| Juillet 2020 2 Perception de la continuité pédagogique des participants à un webinaire sur l... part, la dimension socioaffective comme la facilitation de connaissance mutuelle qu’ont les apprenants les uns des autres et la mise en évidence des apports positifs de chacun ; d’autre part, la dynamique motivationnelle comme l’encouragement et la relance des étudiants de manière régulière à participer au processus. Quintin (2008) met en évidence que le tutorat ciblé sur la dimension socioaffective a un effet positif sur le processus en ce qui concerne la cohésion du groupe. Il ressort également des travaux de Quintin (2008) que ce meilleur climat relationnel influence positivement la qualité du produit de l’apprentissage réalisé en équipe et, corollairement, la progression individuelle des apprenants au terme de l’apprentissage. Enfin, les aspects techniques font référence aux unités d’interventions relevant de la résolution de problèmes de maîtrise des outils numériques (Quintin, 2008). Méthodologie Objectif de la recherche et prise de données 3 L’objectif de la recherche étant d’établir un état des lieux des perceptions des acteurs éducatifs, nous nous sommes posé la question principale suivante : comment se déroulent la continuité pédagogique et la mise en place des modalités d’enseignement à distance pour les différents acteurs de l’éducation ? Afin d’apporter des éléments de réponse à cette question et rendre la conférence interactive, l’outil Wooclap 2 a été utilisé pour récolter différentes données. Cinq questions ont été posées aux participants pendant la conférence. D’abord, le profil des participants a été collecté : parents, enseignants, responsables d’établissement, étudiants ou élèves ou encore grands-parents. Le format en ligne permettant une plus large diffusion de la conférence, la proposition "autre" a été ajoutée. La troisième question concernait leur ressenti quant à la continuité pédagogique : elle se déroule plutôt bien ; elle est viable dans cette situation d’urgence ; elle est très difficile à vivre ; sans avis. Ensuite, les répondants se sont positionnés sur une échelle de Likert de 1 (pas du tout d’accord) à 5 (tout à fait d’accord) pour identifier, en fonction de leur statut (parents, enseignants, etc..), leur niveau d’intégration des contraintes de la continuité pédagogique. Enfin, la dernière question a permis aux participants d’indiquer les aspects de la continuité pédagogique qu’ils estiment bien fonctionner ou qui nécessitent des améliorations. Échantillon 4 L’échantillon se compose majoritairement de participants issus de Belgique francophone. L’analyse des questions se base sur un nombre de réponses variable. En effet, cette variabilité est liée à l’instrument de récolte utilisé : Wooclap. Chaque participant n’a pas nécessairement répondu à toutes les questions. De ce fait, sur les 400 participants, seuls 154 ont répondu à l’ensemble des questions posées lors de la conférence (pas encore connectés au moment de la question, l’outil Wooclap pas encore pris en main, problème technique ou encore souhait de ne pas participer). Parmi ces 154 répondants, 57 sont des enseignants, 1 est parent, 13 sont des apprenants (étudiants ou élèves), 3 sont des responsables d’établissement et 13 sont repris dans la catégorie “autre” . Il est intéressant de noter que certains participants ont mentionné qu’ils occupent plusieurs rôles dans cette continuité pédagogique. Ainsi, 23 participants Recherches & éducations, HS \| Juillet 2020 3 Perception de la continuité pédagogique des participants à un webinaire sur l... ont précisé qu’ils remplissaient les rôles à la fois d’enseignants, de parents ou d’étudiants. Enfin, 44 participants n’ont pas répondu à cette question (Figure 1). Figure 1 - Répartition des sujets en fonction de leur rôle éducatif 5 Nous relevons que certains sujets choisissent un seul rôle, tandis que d’autres annoncent en assumer plusieurs durant la continuité pédagogique. Il convient, néanmoins, de faire preuve de prudence avec ces données. En effet, un participant pouvait choisir un seul rôle alors qu’il en remplit plusieurs (chef d’établissement et parent). Résultats 6 Lorsque l’on questionne l’échantillon, il apparaît clairement que la continuité pédagogique n’est facile à vivre pour personne. Sur la figure 2, nous observons que plus le taux est proche de 100 %, plus la situation semble facile à vivre, plus le taux est proche de 0 %, plus la situation semble difficile à vivre. Nous observons que toutes les moyennes sont inférieures à 50 % et par conséquent, elles nous indiquent que cette situation semble difficilement tenable, plus particulièrement pour les enseignants (37 %) qui présentent le taux de satisfaction le plus faible de notre échantillon. Recherches & éducations, HS \| Juillet 2020 4 Perception de la continuité pédagogique des participants à un webinaire sur l... Figure 2 - La perception des sujets quant à l’accommodement à la continuité pédagogique selon leur fonction 7 Lors de cette conférence, il nous a paru intéressant de questionner les participants sur leur ressenti : “Concernant la continuité pédagogique, vous diriez qu’elle...” (Figure 3). Alors que dans la question précédente, la situation est perçue comme assez difficile à vivre pour les enseignants, la majorité d’entre eux (N = 57 ; 52 %) estime que la situation est viable dans cette situation d’urgence (31 %) ou se déroule plutôt bien (10 %). Au contraire 6,36 % des enseignants pensent que cette situation est difficile à vivre. Enfin, quelques enseignants n’émettent pas d’avis (2,73 %). Ces constats corroborent avec ceux de Haag (2020, p. 3) selon lesquels les propos témoignent d’une capacité de résilience dans des circonstances inédites. Selon les apprenants de l’échantillon (N = 13 ; 12 %), la situation est assez positive. Parmi les 12 % de l’échantillon représenté par des apprenants, 8 % estiment que la situation est viable, et 1 % qu’elle se déroule bien. Au contraire, pour 2,73 %, elle semble difficile à vivre. Pour les responsables d’établissement (N = 3 ; 3 %), la situation est viable dans l’urgence (1,82 %). À l’inverse, pour le seul parent de l’échantillon (N = 1 ; 1 %) cette situation est très difficile à vivre. Pour les personnes qui possèdent plusieurs statuts (N = 22 ; 23 % - enseignant et parent, enseignant et étudiant ou encore enseignant, étudiant et parent), la situation semble moins positive. En effet, pour 5,46 % d’entre eux, la situation semble très difficile à vivre. Nous émettons l’hypothèse que cette difficulté provient de la surcharge de travail liée à la multiplicité des tâches inhérentes à leurs profils. Ainsi, quand un enseignant se retrouve à la maison avec sa famille, il doit, en plus de la charge liée à son emploi, assurer la gestion de ses enfants au niveau des besoins primaires, affectifs et scolaires, mais aussi les tâches liées à la gestion du quotidien. Au contraire, 8,19 % pensent que la situation est viable et 3,64 % qu’elle se déroule plutôt bien. Enfin, les participants ne se retrouvant dans aucun des profils précités (N = 13 ; 12 %) se montrent plus mitigés. Pour 2,73 %, la situation est très difficile à vivre, 4,55 % pensent que la continuité pédagogique est viable et 1,82 % pensent qu’elle se déroule bien. Finalement, la figure 3 met en avant une perception assez positive pour les enseignants, les apprenants et les responsables d’établissement ; assez mitigée pour les personnes présentant plusieurs profils simultanément, ainsi que pour le profil “autre” ; enfin une perception plutôt négative pour les parents. Recherches & éducations, HS \| Juillet 2020 5 Perception de la continuité pédagogique des participants à un webinaire sur l... Figure 3 - La perception du déroulement de la continuité pédagogique des sujets en fonction de leur rôle éducatif 8 Les enseignants et les directions d’écoles ont fait preuve de réactivité et de créativité afin de poursuivre les apprentissages à distance, dans un contexte inédit les amenant, voire les contraignant, à s’approprier de nouveaux outils (Capitanescu Bennetti & Connac, 2020). Nous imaginons que ce passage forcé et en urgence à de nouvelles pratiques d’enseignement peut être difficile à vivre pour certains enseignants. Par ailleurs, ces nouvelles conditions de travail peuvent créer de l’anxiété, un sentiment d’incompétence numérique, des remises en question et de la frustration (Goyette, 2020). Néanmoins comme le rappelle Saint-Fuscien (2017), le caractère exceptionnel de certains événements comme cette crise sanitaire est l’occasion de repenser les pratiques d’enseignement et peut être source d’innovations pédagogiques. Concernant les apprenants, l’étude de Haag (2020) apporte des éléments de réponse sur le vécu des élèves face à cette nouvelle situation d’enseignement à distance. Bien qu’ils ne souhaitent pas que l’école à distance devienne la norme, près de sept élèves sur dix mettent en évidence des éléments positifs comme un gain d’autonomie, une nouvelle forme d’apprentissage (travaux manuels, outils numériques…), une meilleure qualité de vie (rythme de sommeil, alimentation, temps de repos)… Le rôle des parents se retrouve lui modifié, passant d’une supervision des devoirs à un soutien plus important, étant celui « … qui traduit, reformule, organise les consignes et enseigne au bout du compte » (Capitanescu Benneti & D’Addona, 2020, p. 20) et ce, parfois avec une anxiété relative à cette nouvelle compétence que certains doivent prendre en charge. Cette nouvelle dynamique dans laquelle s’inscrivent les élèves et leurs parents peut conduire à des tensions, l’exigence des performances scolaires satisfaisantes pouvant être plus intensément vécue au sein de cette relation parents / enfants. Nous ajoutons à ceci les difficultés inhérentes à l’organisation familiale entre le télétravail, la gestion des tâches scolaires et les contraintes techniques, tant pour l’espace temps que les espaces de travail (Ibid.). Lors de la conférence, il a été demandé aux participants d’identifier ce qui fonctionnait adéquatement ou ce qui devait être amélioré dans la continuité pédagogique, 12 jours après son commencement (Figure 4). Afin de dissocier leurs réponses, chaque répondant a indiqué une colonne ad hoc un “+” si son avis était positif et “-” s’’il s’agissait d’une difficulté ou d’un manque. Les verbatims récoltés ont ensuite été Recherches & éducations, HS \| Juillet 2020 6 Perception de la continuité pédagogique des participants à un webinaire sur l... analysés en fonction des quatre catégories d’interventions tutorales de Quintin (2008) : technique, pédagogique, organisationnelle et socioaffective. L’examen de la figure 4 montre que le nombre d’unités de sens négatifs (N = 153) est plus élevé que le nombre d’unités de sens positifs (N =108). Ce résultat est cohérent avec la difficulté perçue concernant la mise en oeuvre actuelle de la continuité pédagogique (cf. Figure 2). Figure 4 - Répartition des verbatims en fonction des quatre catégories d’interventions tutorales de Quintin (2008) 9 Concernant l’aspect technique de la continuité pédagogique, de nombreux éléments devraient être améliorés (Technique négatif (T-) = 50 (en rouge) ; Technique positif (T+) = 21 (en bleu)), selon l’avis des répondants. En effet, ces derniers relèvent des manques liés à l’utilisation et à l’intégration du numérique à distance. Les personnes interrogées regrettent l’absence de formation aux TIC, ainsi que le manque d’outils adaptés à leur besoin. Les enseignants et les étudiants manquent de compétences numériques. Ce besoin avait déjà été souligné par Frau-Meigs (2020) qui démontre l’urgence de former aux compétences numériques et médiatiques. Cependant, cette crise sanitaire a mis en lumière un élément déjà connu : le manque de compétences techniques des enseignants (Papi, 2012 ; Loisy, 2017 ; Nogry et Sort, 2016). Nous avançons l’idée que cette crise oblige les formateurs à renforcer le développement des compétences numériques dans la formation initiale et continue des enseignants. Dans la gestion technique de la continuité pédagogique, plusieurs participants identifient la fracture numérique comme un frein. En effet, ils sont préoccupés de pouvoir donner à tous leurs élèves ou étudiants le même suivi alors que certaines familles n’ont pas l’accès à l’outil informatique ou à internet. Depuis, le début du confinement cette inégalité a été mise en évidence à de nombreuses reprises (FrauMeigs, 2020 ; The Education Foundation, 2020 ; Barras, 2020). L’UNESCO (2020) a d’ailleurs tiré la sonnette d’alarme en indiquant que d’un point de vue mondial la moitié des apprenants, n’ont pas accès à un ordinateur à domicile et 43 % n’ont pas internet à la maison. Recherches & éducations, HS \| Juillet 2020 7 Perception de la continuité pédagogique des participants à un webinaire sur l... Malgré ces avis négatifs, les répondants ont découvert le potentiel numérique d’outils faciles à prendre en main comme les Lives, Messenger ou encore les visioconférences. Ils apprécient l’opportunité de pouvoir monter en compétence en utilisant de nouveaux outils et en créant du contenu numérique plus élaboré, ainsi que la facilité de pouvoir accéder aux TICs pour mettre en place cet accompagnement à distance. La catégorie liée aux aspects pédagogiques est celle où les répondants sont les plus mitigés (pédagogique-positif (P+) = 41 ; pédagogique- négatif (P-) = 44). En effet, le passage de cours en présentiel à des cours entièrement à distance a dû s’opérer dans l’urgence et l’enseignant n’y était pas préparé (Cerisier, 2020). Nos participants citent d’ailleurs plusieurs difficultés à cette adaptation comme l’explication de certaines notions, les consignes peu claires, la gestion des travaux, le manque de moyens pour récupérer l’attention de l’étudiant ou encore la gestion de grands groupes. Pourtant, ces différents aspects de la gestion pédagogique sont depuis longtemps pris en compte dans la scénarisation des formations à distance ou hybride. Prenons, par exemple, la gestion de grands groupes. Des enseignants insistent sur le manque d’outils adaptés ou encore la difficulté d’interaction. Pourtant, la propagation dans le paysage éducatif des MOOCs, dont le M signifie Massive, démontre que le grand nombre d’apprenants est surtout une composante à prendre en compte lors de la conception de dispositifs, plutôt qu’un frein à l’apprentissage (Depover, Karsenti, Komis, 2017). Nos répondants identifient également l’absence de feedback comme étant une faiblesse de cette continuité pédagogique, qui impacte aussi bien les enseignants (l’absence de feedback continu rend très difficile l’adoption des tâches d’apprentissage) que les apprenants (l’absence de feedback tend à démotiver les enfants). Ce constat est logique comme le montre Hattie (2007), le feedback est l’un des moyens pour mettre en place l’étayage, et il provoque un effet positif sur le sentiment d’efficacité des étudiants. La qualité de continuité pédagogique est différente en fonction du type de cours : pour les cours théoriques, ça se passe bien. Pour les cours pratiques, c’est plus compliqué. Bobroff, Bouquet et Delabre (2020) ont cependant identifié différentes alternatives pour envisager ce changement : les simulations numériques, des tests grandeur nature ou encore le smartphone qui possède des capteurs très performants (par exemple, mesurer la vitesse de rotation). Les auteurs soulignent que les étudiants sont ravis de ce changement de routine et de cette possibilité de travailler en autonomie. Dans le cadre du cours de sciences, cette expérimentation à distance permet de sortir du laboratoire et de s’emparer du quotidien. Cette adaptation a demandé aux enseignants de repenser leur enseignement de fond en comble. Pour Frau-Meigs (2020), la crise sanitaire a permis de réaliser qu’une routine s’était installée dans les cours en présence alors que l’e-learning permet d’enseigner autrement, de varier les pratiques et de rendre l’élève acteur de son apprentissage. De plus, les étudiants peuvent progresser à leur rythme et pas à celui imposé à tous. Les résultats de Haag (2020) vont dans le même sens. Les élèves aussi avancent que cette période permet d’apprendre différemment, d’aller à leur rythme et de gagner en autonomie. Dans son étude, Quintin (2008) identifie l’organisation comme l’une des quatre composantes de la mise en place et de l’encadrement des formations à distance. Quand l’on interroge les participants sur la continuité pédagogique, un grand nombre (organisationnel-négatif (O-) = 42 ; organisationnel-positif (O+) = 10) met en avant des difficultés à s’organiser et à gérer le temps. La mise en place en urgence de dispositifs d’apprentissage à distance qui demande beaucoup de temps a surchargé les enseignants. De plus, pour ceux qui sont parents, il est difficile d’organiser son propre travail à distance Recherches & éducations, HS \| Juillet 2020 8 Perception de la continuité pédagogique des participants à un webinaire sur l... et celui de ses enfants. En bref, comme le cite Frau-Meigs (2020), la continuité pédagogique qui pourrait être active s’est transformée en télétravail subi. Majoritairement constitués d’enseignants, nos participants pointent du doigt cette difficulté de planification. Pourtant, du côté des apprenants, les résultats de Haag (2020) montrent que les élèves apprécient ce gain d’autonomie rendu possible par la crise sanitaire notamment en apprenant à s’organiser. Au final, ce qui fonctionne le mieux dans la continuité pédagogique, c’est le maintien du lien social, du contact entre les élèves et les professeurs (socioaffective positif (S+) = 36 ; socioaffective – négatif (S-) = 17). D’ailleurs, les élèves sont motivés à garder le contact et les enseignants à participer à la continuité pédagogique. L’école n’est pas un lieu uniquement d’apprentissage et la crise sanitaire a permis de réaffirmer ce rôle social de l’école (Frau-Meigs, 2020). En effet, en ce temps de crise, l’enseignant doit être un accompagnateur à la fois empathique et vigilant, et manifester une e-présence (Hadji, 2020). L’aspect socioaffectif est la seule catégorie qui obtient plus de résultats positifs que négatifs. En effet, les répondants soulignent la solidarité entre les enseignants et le partage de ressources rendu possible notamment grâce aux réseaux sociaux. La solidarité avait d’ailleurs déjà été retenue comme l’un des aspects positifs de cette crise par les élèves de l’étude de Haag (2020). La crise du COVID-19 a réveillé les démarches d’intelligences collectives dans des domaines variés (Santolini, 2020) : médecine, économie et également dans l’enseignement. Cependant, en raison de ce climat anxiogène et de la distanciation sociale, l’isolement rend difficile le travail à domicile et la motivation autorégulée. Dans la suite, nous mettons en lien les catégories établies par Quintin (2008) qui ont toutes été subdivisées en sous-catégorie “positive” ou “négative” selon l’orientation des réponses données par les participants. Pour y parvenir, nous nous appuyons sur une analyse de correspondances multiples. Cette démarche permet de synthétiser sous la forme d’un nuage de points les liaisons entre des variables qualitatives. Sur le graphe, des proximités entre celles-ci traduisent une association forte entre les réponses. À l’inverse, des écarts importants traduisent une opposition entre ces catégories. Tout en restant prudents par rapport à cette mise en correspondance, dans la mesure où notre échantillon n’est pas représentatif, plusieurs résultats instructifs peuvent être mis en évidence à partir de la figure 5 et des verbatims des participants. Recherches & éducations, HS \| Juillet 2020 9 Perception de la continuité pédagogique des participants à un webinaire sur l... Figure 5 - Analyse de correspondances multiples des catégories d’interventions tutorales (Quintin, 2008) 10 Lorsque les participants formulent un avis dans une des dimensions (pédagogique, technique, socioaffective ou organisationnelle), celui-ci est tranché. Il est soit positif, à l’instar de ce verbatim : “accompagnement [P+] ; accès aux TIC [T+]”, soit négatif, par exemple : “ Il est indispensable que l’on nous fournisse des outils de qualité et spécifiques [T-] ; Pas d’outils adaptés aux grands groupes (> 200) [T-]”. Il ressort aussi que quand un avis est négatif, il l’est généralement pour l’ensemble des dimensions. Toutefois, une relation plus forte est constatée entre les avis négatifs P- et T-. Cela signifie que quand un participant invoque un avis négatif sur le plan pédagogique, il a tendance à évoquer également un avis négatif sur le plan technique. Le verbatim suivant illustre bien ce cas de figure : “plus compliqué de rester impliqué [P-] ; peut être compliqué pour les parents [sic] d’accompagner les enfants dans leurs apprentissages [P-] ; certaines familles n’ont pas l’accès à l’outil informatique [T-] ”. Cela signifierait que, dans le cas de ces répondants, dont 35,80 % sont des enseignants, la non-maîtrise des aspects techniques aurait des répercussions directes au niveau pédagogique, et mettrait cet aspect en difficulté. On peut penser que les aspects techniques liés à l’usage du numérique constituent une condition à toute action pédagogique et cette situation est évidemment renforcée dans le contexte d’une mise à distance dans l’urgence. Autre tendance dans les avis négatifs, nous remarquons qu’une opinion sur le plan social est souvent liée à un avis sur le plan organisationnel, comme en témoigne le verbatim suivant : “surcharge ! [O-] ; difficultés au niveau de l’agenda [O-] ; parfois dur de travailler chez soi …[O-] ; stage en télétravail (isolement !) [S-]”. Ainsi, le fait de connaître des difficultés au niveau social (isolement, gestion familiale, etc.) renforcerait la difficulté à s’organiser et vice-versa. Pour les participants qui invoquent des aspects négatifs, mais aussi des aspects positifs, l’aspect positif qui est le plus souvent associé relève du plan social. Concrètement, une Recherches & éducations, HS \| Juillet 2020 10 Perception de la continuité pédagogique des participants à un webinaire sur l... personne invoque que “ (il est)...difficile d’exploiter les différents supports et ressources [T-] ; le parent n’est pas le prof [P-] ; difficile à mettre en place [O-] ; beaucoup de partage de ressources [S+] ; trop de stress, on se sent surchargé, perdu [O-] ; c’est difficile de travailler dans ce contexte [P-]”. On peut dès lors avancer que si une personne se trouve dans une situation qu’elle estime compliquée et négative, l’aspect le plus positif qui est invoqué relève du niveau social. À propos des recommandations que nous pourrions formuler, nous invoquerions que, dans la mise en place de dispositifs à distance, la dimension sociale constitue un facteur important pour que l’apprentissage ait lieu. Cette observation corrobore les conclusions de Quintin (2008) selon lequel les interventions tutorales de type socioaffectif impactent davantage les apprentissages dans un contexte d’apprentissage à distance. Par ailleurs, les aspects pédagogiques positifs semblent occuper une place centrale et sont liés aux aspects positifs des trois autres dimensions . Ainsi, les aspects pédagogiques positifs sont tantôt associés à des aspects organisationnels positifs : “ L’aide des professeurs [P+] ; la disponibilité des professeurs [O+]”, tantôt à des aspects techniques positifs : “l’obligation de tester des moyens numériques nouveaux [T+] ; la nécessité de repenser son enseignement de fond en comble [P+]”. L’analyse de correspondances multiples montre néanmoins que les avis positifs sur le plan pédagogique (P+) s’accompagnent le plus souvent d’avis positifs sur le plan social (S+) : “intérêt des élèves pour utiliser des outils numériques pour l’apprentissage [P+] / être en contact direct avec les élèves via le numérique [S+]” ou encore “Le lien social [S+] ; un suivi plus différencié [P+] ; mise en place de pratiques probantes en matière de partages [P+]”. Cela signifierait que pour les répondants, en majorité des enseignants, le pédagogique est intimement lié au social. Ils n’envisagent pas une transmission de la matière sans interactions avec leurs élèves. En effet, l’école a toujours eu un rôle allant bien au-delà de la simple transmission de connaissances (Blanchard & Cayonnette-Remblière, 2016) et relève également de la motivation à s’engager dans les apprentissages (Viau, 2005). Pour interpréter nos données encore plus finement, nous avons essayé d’établir des relations entre le profil de répondants (Figure 1), le ressenti exprimé sur la manière dont la continuité pédagogique est vécue (Figure 2 & Figure 3) et l’avis des participants à propos de ces quatre dimensions (Figure 4). Aucune relation significative n’a toutefois été observée. Ni la fonction ni la perception de la continuité pédagogique ne semblent moduler l’avis sur l’importance de l’encadrement en cours de processus d’apprentissage. Ce résultat nous parait logique étant donné le caractère opportuniste de notre échantillon. Conclusion 11 Tout en restant prudent dans la mesure où il s’agit d’un échantillon motivé par la question éducative, notre analyse des déclarations des participants du webinaire fait ressortir plusieurs résultats intéressants par rapport à la mise à distance de situations d’enseignement dans un contexte de pandémie. Le passage à un enseignement à distance nécessite une maîtrise de compétences numériques par les enseignants. Force de constater que cela ne va pas de soi et que cela a pu être un obstacle dans les pratiques d’enseignement dans ce contexte de crise sanitaire. Ce passage forcé a, néanmoins, permis aux enseignants de repenser leurs pratiques d’enseignement en profondeur et les amène à partager ces nouvelles pratiques, à collaborer et à s’entraider, notamment via les réseaux sociaux. Recherches & éducations, HS \| Juillet 2020 11 Perception de la continuité pédagogique des participants à un webinaire sur l... Ce partage de ressources, la solidarité née durant cette période de confinement et de manière plus générale les aspects socioaffectifs tiennent une place centrale dans les témoignages des participants. En effet, selon eux, il s’agit là de l’unique aspect qui présente dans la continuité pédagogique plus d’aspects positifs que d’aspects négatifs. D’ailleurs, pour certains participants, alors que la situation de confinement s’avère compliquée à plusieurs niveaux (pédagogiques, techniques et/ou organisationnels) les seuls aspects positifs invoqués sont de cet ordre. Ces éléments mettent en avant que l’école n’est pas uniquement un lieu d’instruction et d’apprentissage, mais que sa composante sociale demeure fondamentale pour assurer le suivi pédagogique (même (ou encore plus) à distance). En outre, l’aspect organisationnel semble primordial dans la mise en place de l’école à distance, tant pour les enseignants consacrant un investissement de temps important que pour les parents alternant télétravail et gestion des tâches scolaires de leurs enfants. Nos résultats montrent d’ailleurs que les lacunes organisationnelles sont liées aux lacunes sociales. Les enseignants sont aussi conscients que tous leurs élèves ne bénéficient pas du même suivi et du même accès aux outils informatiques. Leur crainte est que cette fracture numérique ne rende la continuité pédagogique inéquitable. Enfin, nous nous interrogeons sur les usages numériques que feront les enseignants dans leur classe à la sortie du confinement. Poursuivront-ils leurs initiatives d’adoption des outils numériques ? Exprimeront-ils toujours des besoins de formations aux compétences numériques ? Les enseignants redoutent les inégalités causées par la fracture numérique. Seront-elles amplifiées au retour de ceux-ci en classe ? De nouvelles inégalités vont-elles surgir à la suite de ce confinement ? Autant de questions qui devront être traitées de manière urgente après cette période de pandémie. BIBLIOGRAPHIE Barras, H. (2020). Évaluer dans l’urgence, en repensant sa planification à l’aide des principes issus de la gestion de crises. Évaluer. Journal international de recherche en éducation et formation, Numéro Hors-série, 1, 17-24. Blanchard, M., & Cayouette-Remblière, J. (2016). Sociologie de l’école. 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(2020, 17 mars), Covid-19 : heurs et malheurs de la continuité pédagogique à la française. The Conversation. Consulté à l’adresse https://theconversation.com/covid-19-heurs-etmalheurs-de-la-continuite-pedagogique-a-la-francaise-133820 Circulaire 7541 : Coronavirus Covid-19 : décision du Conseil National de sécurité du 15 avril 2020. Fédération Wallonie-Bruxelles, 16 avril 2020. Consulté à l’adresse http://enseignement.be/upload/ circulaires/000000000003/FWB%20-%20Circulaire%207541%20(7794_20200416_164436).pdf De Lièvre, B., Depover, C., & Dillenbourg, P. (2006). The relationship between tutoring mode and learners’ use of help tools in distance education. Instructional science, 34(2), 97-129. Depover, C., Karsenti, T., & Komis, V. (2017). Pour comprendre les MOOCs : Nature, enjeux et perspectives. Presse de l’Université du Québec. Dillenbourg, P. (2015). Orchestration graphs : Modeling scalable education. Lausanne : EPFL Press. Frau-Meigs, D. (2020, 2 mai). 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Lien vers la conférence : https://youtu.be/TztVsWMT3J4 2. Wooclap est un outil interactif de télévote, qui facilite la participation et l'interaction avec un public : les participants répondent en temps réel à des questions et peuvent également poser des questions par télévoteur pendant la présentation. RÉSUMÉS La crise sanitaire internationale a contraint le gouvernement belge à fermer les établissements d’enseignement et de formation pour limiter la propagation du COVID-19. Cette décision a amené les acteurs du système éducatif à redéfinir leurs pratiques. Différentes initiatives ont vu le jour pour permettre une continuité pédagogique. Dans le cadre d’une conférence en ligne organisée par le “MUMONS, nous avons recueilli les perceptions des participants (enseignants, parents, étudiants, etc.) au moyen du système de vote en ligne « Wooclap ». Les résultats de nos analyses montrent que la continuité pédagogique par la mise à distance des situations d’enseignement et d’apprentissage se révèle difficile à mettre en œuvre pour les participants. Les aspects techniques semblent constituer un obstacle important pour y parvenir dans l’urgence. Par ailleurs, notre analyse de contenu nous permet de mettre en évidence des proximités et des oppositions à la fois logiques et contre-intuitives entre les différentes dimensions (organisation, socio-affectif, technique, pédagogique) pour soutenir les apprentissages. INDEX Mots-clés : Covid-19, continuité pédagogique, enseignement à distance, perceptions, Wooclap AUTEURS SARAH DESCAMPS Université de Mons à la faculté des sciences psychologiques et de l’éducation dans le service d’ingénierie pédagogique et numérique éducatif (IPN) SABRIN HOUSNI Université de Mons à la faculté des sciences psychologiques et de l’éducation dans le service d’ingénierie pédagogique et numérique éducatif (IPN) Recherches & éducations, HS \| Juillet 2020 14 Perception de la continuité pédagogique des participants à un webinaire sur l... GWENDYDD PIRET Université de Mons à la faculté des sciences psychologiques et de l’éducation dans le service d’ingénierie pédagogique et numérique éducatif (IPN) AUDREY KUMPS Université de Mons à la faculté des sciences psychologiques et de l’éducation dans le service d’ingénierie pédagogique et numérique éducatif (IPN) KARIM BOUMAZGUIDA Université de Mons à la faculté des sciences psychologiques et de l’éducation dans le service d’ingénierie pédagogique et numérique éducatif (IPN) MARIE DUMONT Université de Mons à la faculté des sciences psychologiques et de l’éducation dans le service d’ingénierie pédagogique et numérique éducatif (IPN) LAËTITIA DRAGONE Université de Mons à la faculté des sciences psychologiques et de l’éducation dans le service d’ingénierie pédagogique et numérique éducatif (IPN) GAËTAN TEMPERMAN Université de Mons à la faculté des sciences psychologiques et de l’éducation dans le service d’ingénierie pédagogique et numérique éducatif (IPN) BRUNO DE LIÈVRE Université de Mons à la faculté des sciences psychologiques et de l’éducation dans le service d’ingénierie pédagogique et numérique éducatif (IPN) Recherches & éducations, HS \| Juillet 2020 15
https://openalex.org/W3214761100	https://www.mdpi.com/1420-3049/26/22/7022/pdf?version=1637843412	English	null	Therapeutic Deep Eutectic Systems towards the Treatment of Tuberculosis and Colorectal Cancer: Opportunities and Challenges	Molecules/Molecules online/Molecules annual	2,021	cc-by	7,744	Citation: Oliveira, F.; Santos, F.; Duarte, A.R.C. Therapeutic Deep Eutectic Systems towards the Treatment of Tuberculosis and Colorectal Cancer: Opportunities and Challenges. Molecules 2021, 26, 7022. https://doi.org/10.3390/ molecules26227022 Keywords: therapeutic deep eutectic systems; active pharmaceutical ingredient; tuberculosis; colorectal cancer; drug delivery Received: 12 October 2021 Accepted: 17 November 2021 Published: 20 November 2021 1. Introduction Green chemistry and sustainable processes are nowadays paramount principles which industry must have present in their production lines [1]. The constant search for new methods with lower environmental risks is a transversal concern to the industry, and the pharma- ceutical sector is no exception. For pharmaceutical and biomedical companies, particularly in the quest for new substances or carriers for drug delivery, to enhance the solubility or permeability, avoid polymorphism, decrease toxicity, and improve pharmacokinetics, green chemistry can provide powerful innovative solutions [2]. Nowadays, even though intense research and development are being undertaken, it is more and more difﬁcult to synthesize new molecules with enhanced therapeutic efﬁcacy to substitute the “old” ones. Consequently, pharmaceutical sciences and drug development research have been giving particular emphasis on the enhancement of existing drug properties. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Therapeutic Deep Eutectic Systems towards the Treatment of Tuberculosis and Colorectal Cancer: Opportunities and Challenges Filipe Oliveira , Filipa Santos and Ana Rita C. Duarte * Associated Laboratory for Green Chemistry (LAQV) of the Network of Chemistry and Technology (REQUIMTE), Chemistry Department, NOVA School of Science and Technology, 2829-516 Caparica, Portugal; fsn.oliveira@campus.fct.unl.pt (F.O.); mfca.santos@campus.fct.unl.pt (F.S.) * Correspondence: aduarte@fct.unl.pt; Tel.: +351-212-948-300 Associated Laboratory for Green Chemistry (LAQV) of the Network of Chemistry and Technology (REQUIMTE), Chemistry Department, NOVA School of Science and Technology, 2829-516 Caparica, Portugal; fsn.oliveira@campus.fct.unl.pt (F.O.); mfca.santos@campus.fct.unl.pt (F.S.) * Correspondence: aduarte@fct.unl.pt; Tel.: +351-212-948-300 Associated Laboratory for Green Chemistry (LAQV) of the Network of Chemistry and Technology (REQUIMTE), Chemistry Department, NOVA School of Science and Technology, 2829-516 Caparica, Portugal; fsn.oliveira@campus.fct.unl.pt (F.O.); mfca.santos@campus.fct.unl.pt (F.S.) * Correspondence: aduarte@fct.unl.pt; Tel.: +351-212-948-300 Abstract: What if a new technology based on therapeutic deep eutectic systems would disrupt the current treatment of major economic and socially burden diseases? The classical deﬁnition of eutectic systems is that they are the combination of two or more compounds that interact via hydrogen bonds, from which results a melting temperature depression in comparison with that of its individual components. Therapeutic deep eutectic systems are deﬁned as eutectic systems in which at least one of the individual components is an active pharmaceutical ingredient, or a eutectic system in which the active pharmaceutical ingredient is dissolved. Current literature reports on tuberculosis have been mostly based on the most common anti-tuberculosis drugs prescribed. Using eutectic systems based on naturally occurring molecules known for their anti-microbial activity may also present a promising therapeutic strategy able to cope with the prevalence of Mycobacterium tuberculosis and prevent the appearance of multidrug resistance strains. With regards to colorectal cancer, literature has been unravelling combinations of terpenes with anti-inﬂammatory drugs that are selectively cytotoxic towards colorectal cancer cells and do not compromise the viability of normal intestinal cells. This technology could contribute to preventing tumor growth and metastasis while providing a patient compliance therapeutics, which will be crucial to the success of overcoming the challenges presented by cancers. Citation: Oliveira, F.; Santos, F.; Duarte, A.R.C. Therapeutic Deep Eutectic Systems towards the Treatment of Tuberculosis and Colorectal Cancer: Opportunities and Challenges. Molecules 2021, 26, 7022. https://doi.org/10.3390/ molecules26227022 Academic Editor: Mara G. Freire Received: 12 October 2021 Accepted: 17 November 2021 Published: 20 November 2021 molecules molecules molecules 2. Green Chemistry and Deep Eutectic Systems In 1998, Warner and Anastas introduced the concept of green chemistry by listing its 12 principles. In organic synthesis and chemical engineering processes, the use of solvents should be minimized and solvents should be regarded only as auxiliary substances, hence https://www.mdpi.com/journal/molecules Molecules 2021, 26, 7022. https://doi.org/10.3390/molecules26227022 Molecules 2021, 26, 7022 2 of 11 being used in very low amounts. Whenever it is not possible to replace solvents completely and their use is required, they should be innocuous [3,4]. Consequently, in the past two decades, the research on safe and environmentally benign solvents has been increasingly growing. Several substances, such as water, ionic liquids (IL), supercritical ﬂuids (SCF), and more recently deep eutectic systems (DES), have been considered as alternative green solvents [5–9]. DES were ﬁrstly reported in 2003 by Abbott and co-workers, as a novel class of green solvents with similar performance to IL [7,10–12]. Eutectic systems have been categorized in different classes according to the nature of their constituents. DES were ﬁrst classiﬁed as type (I), a combination of a quaternarium salt with a non-hydrated metal halides; type (II), a combination of a quaternarium salt with a hydrated metal halides; type (III), a combination of a quaternarium salt (HBA) and a hydrogen bond donor (HBD); and type (IV), a combination of a metal chloride and a hydrogen bond donor [13]. Overall, all classes of DES encompass eutectic systems with a deep depression on the system’s melting point, when in comparison with its individual components, and a deviation from the ideality. After this ﬁrst classiﬁcation in these four types, other classiﬁcations and nomenclatures have been described. This is the case, for instance, of the term low transition temperature mixtures (LTTM), described by Francisco and co-workers, referring to mixtures of two or more compounds which include not only the systems with changes in their melting temperatures, but also associations of compounds which lead to any other changes on the thermal events in relation to the initial raw materials [14]. Under the deﬁnition of LTTM fall the well-known natural deep eutectic systems (NADES), which arose from the work developed in 2011 by Choi and co-workers. The preparation of more than 30 viscous liquids has been reported from combinations of sugars, natural carboxylic acids, choline chloride, and/or water [15,16]. 2. Green Chemistry and Deep Eutectic Systems In our daily life, we often handle NADES without even knowing it; honey and maple syrup are an example of that, in which mixtures of sugars form eutectic systems that are liquid at room temperature. Such examples may be used in food and nutritional supplements, but also in medical formulations, since they often present interesting bioactivities, are biodegradable, and present, most of the time, a low toxicity [13]. 3. Therapeutic Deep Eutectic Systems Therapeutic deep eutectic systems (THEDES) are described as eutectic systems in which one of the components is an API, or a system where active pharmaceutical ingre- dients (API) can be dispersed. A brief literature search in the ISI Web of Knowledge (03.08.2021) for “deep eutectic solvents” reveals 6040 references (590 reviews), with 59 ref- erences (18 of which reviews) on “deep eutectic systems pharmaceutics”, and in which Portugal is the leading country in the ﬁeld. g g y The ﬁrst studies reported in the literature refer to the possibility to dissolve API in DES. Stott and co-workers observed that DES could incorporate an API; therefore, DES acts as a delivery system while simultaneously improving the characteristics of the API itself [17,18]. Other molecules of interest to the pharmaceutical industry have, since then, been the object of study. Shen and co-workers focused on the use of a eutectic mixture of borneol:menthol for the intestinal absorption of daidzein [19]. Morrison et al. tested NADES as potential solubilization media for benzoic acid, griseofulvin, danazol, itraconazole, and AMG517, and the results showed a signiﬁcant increase between 5 to 22,000-fold in the solubility of the API in NADES compared to water [20]. Li et al. have investigated other drugs, such as itraconazole, piroxicam, lidocaine, and posaconazole, and the solubility enhanced up to 6700-fold, 430-fold, 28-fold, and 6400-fold, respectively, when compared to their solubility in water [21]. Shekaari et al., in another study, analyzed the solubility enhancement of acetaminophen in NADES [22]. Goud et al. explored the formation of eutectic mixtures with curcumin, which could potentially be used as therapeutics for Alzheimer’s [23]. p y p Several NADES and THEDES with different bioactivities have been investigated namely as antibacterial and antibioﬁlm, antifungal and anticancer agents, as well as wound Several NADES and THEDES with different bioactivities have been investigated namely as antibacterial and antibioﬁlm, antifungal and anticancer agents, as well as wound Molecules 2021, 26, 7022 3 of 11 healing enhancers. Table 1 summarizes some of the work carried out and the major aims of each study. healing enhancers. Table 1 summarizes some of the work carried out and the major aims of each study. Table 1. Summary of different eutectic systems reported in the literature and their aim. 3. Therapeutic Deep Eutectic Systems THEDES Aim Reference Choline Chloride:Acetylsalicylic Acid Choline Chloride:Phenylacetic Acid Menthol:Benzoic Acid Menthol:Acetylsalicylic Acid Menthol:Phenylacetic Acid Preparation of THEDES to study their bioavailability [24,25] Menthol:Ibuprofen Incorporation of THEDES with NSAID in a polymeric matrix [26] Choline Chloride:Mandelic Acid THEDES preparation and encapsulation in ﬁber membranes [27] Choline Chloride:Ascorbic Acid Preparation of NADES and enhancement of dexamethasone solubility [28] Citric Acid:Ethambutol:H2O Preparation of THEDES with API for TB [29] Citric Acid:L-Arginine:H2O Encapsulation of THEDES in a lipidic carrier [30] Menthol:Lauric Acid Menthol:Myristic Acid Menthol:Stearic Acid Development of THEDES with terpenes and fatty acids with antimicrobial and wound healing properties [31] Lauric Acid:Myristic Acid Preparation of eutectic blends and loading onto gauzes [32] Capric Acid:Lauric acid Capric Acid:Stearic Acid Capric Acid:Myristic Acid Preparation of different DES with fatty acids with antimicrobial activity and bioﬁlm detachment [33] Limonene:ibuprofen Perillyl alcohol:ibuprofen Menthol: ibuprofen Thymol:ibuprofen Preparation of THEDES with terpenes + NSAIDS and evaluation of their activity against CRC cells [34–36] The use of THEDES revealed, hence, to be a powerful tool in pharmaceutical sciences enhancing the properties of existing drugs and contributing to the development of new formulations that could be more effective and easily administered [5]. However, this scientiﬁc area is still largely unexplored. 4. THEDES for Tuberculosis Treatment p Mycobacterium tuberculosis (Mtb) is the cause of TB, and e and preventive disease, it is still reported by the W ) as one of the deadliest infections worldwide, having k eople in 2019 [37]. The WHO established policies to p er of cases of TB and end with deaths triggered by this gation of new drugs for TB treatment as well as more ef In 2014, the WHO established a strategy to control and end TB by 2035 called the “End TB Strategy”, which is based in the development of sustainable goals, such as: (i) creation of integrated and patient-centered care and prevention strategies; (ii) creation of strong policies and supportive systems with participation of governments, communities and private investors; and (iii) promotion of the research and innovation to help accelerate the decline rates of global TB incidence and completely eliminate the TB epidemic. The anti-TB drugs that started to be investigated in the 1940s and in 1960s gave rise to a treatment that combines four of the ﬁrst line anti-TB drugs and requires its administration for several months (a minimum of 6 months). This drug regimen is still the primary drug combination used as ﬁrst-line treatment of TB and it is considered the more effective treatment available today. igation of new drugs for TB treatment as well as more ef ates that, for R&D, at least an extra US $1.1 billion per y velopment of new tools for this problem [37]. Moreove pandemic caused by the coronavirus (SARS-CoV-2), c ess made towards the decrease of global TB incidence, i ated mortality [37]. Even though 14 vaccines, 22 drugs ments are being studied in clinical trials, the drugs that p ts are still the same that were designed 50 years ago. M THEDES have been used in research for pursuing improvements in TB therapy (Table 2). For instance, in our previous work we described an increase on the solubility of ethambutol and L-arginine when combined as a THEDES with citric acid [29]. Zakrewsky et al. reported a eutectic system based on choline bicarbonate and geranic acid (1:2 molar ratio) with antimicrobial activity towards Mtb drug-resistant strains [38]. 4. THEDES for Tuberculosis Treatment Tuberculosis (TB) is described as an ancient disease that has been associated with humans at least since the Neolithic revolution and is characterized by its epidemic cycles with emergent outbreaks that then recede (Figure 1). Throughout modern history, TB has been associated with high density population areas living with poor sanitation. In Europe, TB had become an epidemic in the 18th and 19th centuries where it often revealed a seasonal incidence pattern. Public health measures, vaccination, and antibiotics (such as streptomycin) represented effective measures towards the control of TB. Nevertheless, the rise of drug resistant bacterial strains in the last three decades have push backwards the control and complete elimination of TB. Mycobacterium tuberculosis (Mtb) is the cause of TB, and although it is considered a curable and preventive disease, it is still reported by the World Health Organization (WHO) as one of the deadliest infections worldwide, having killed an average of 1.4 million people in 2019 [37]. The WHO established policies to promote the decrease of the number of cases of TB and end with deaths triggered by this infection, encouraging the investigation of new drugs for TB treatment as well as more effective therapies. The WHO estimates that, for R&D, at least an extra US $1.1 billion per year is needed to accelerate the development of new tools for this problem [37]. Moreover, other factors, such as the recent pandemic caused by the coronavirus (SARS-CoV-2), can lead to a reversal of the progress made towards the decrease of global TB incidence, increasing its incidence and associated mortality [37]. 4 of 11 Molecules 2021, 26, 7022 Even though 14 vaccines, 22 drugs, and several combinatorial treatments are being studied in clinical trials, the drugs that present the best therapeutic outputs are still the same that were designed 50 years ago. Moreover, the indiscriminate use of antibiotics leads to the development of multi-resistant strains and consequent antibiotic resistance. The nonspeciﬁc antimicrobial activity of NADES is able to compromise the bacterial agents’ viability while avoiding the risk of inducing resistance. Figure 1 Schematic representation of the site of infection Figure 1. Schematic representation of the site of infection in TB. Created with BioRender.com. Figure 1 Schematic representation of the site of infection Figure 1. Schematic representation of the site of infection in TB. Created with BioRender.com. 4. THEDES for Tuberculosis Treatment Another study reported the use anti-TB drugs (pyrazinamide and isoniazid) to obtain binary and ternary solid eutectic compositions from a co-crystallization of these drugs with succinic acid and fumaric acid, showing that during co-crystallization synthesis the formation of eutectic mixtures could occur when cohesive interactions are predominant and the size/shape of the components are mismatched [39]. If the adhesive interactions dominating the re- sulting compound are characterized as a cocrystal, and yet if both interactions, adhesive and cohesive, are balanced and a match of size/shape between the components occurs, a Molecules 2021, 26, 7022 5 of 11 solid solution is formed [39]. Furthermore, Rajbongshi et al. also studied the formation of eutectics through crystallization processes with pyrazinamide and different aromatic carboxylic acids characterized by a “V” shape phase diagram [40]. solid solution is formed [39]. Furthermore, Rajbongshi et al. also studied the formation of eutectics through crystallization processes with pyrazinamide and different aromatic carboxylic acids characterized by a “V” shape phase diagram [40]. Table 2. Summary of the THEDES prepared for anti-tuberculosis treatment. THEDES Reference Citric Acid:Ethambutol:H2O [25] Choline bicarbonate: geranic acid (1:2) [38] Pyrazinamide:succinic acid Isoniazid:succinic acid [39] Pyrazinamide: Nicotinic acid (2:1) Pyrazinamide:isonicotinic acid (5:1) Pyrazinamide: saccharin (4:1) [37,40] Table 2. Summary of the THEDES prepared for anti-tuberculosis treatment. The study of eutectics for pharmaceutical applications have been increasing along the years and, particularly, in antimicrobial applications, such as for TB therapy, and has showed promising results with improvements in anti-TB drugs properties. How- ever, little advances have been reported in the mechanisms underlying these observa- tions. Monteiro and co-workers described a possible mechanism for the system Citric Acid:Ethambutol:H2O [41], unveiling a possible entry pathway for ethambutol promoted by citric acid through a recently published mycobacterial ABC (Rv1819c, PDB ID: 6TQE) transporter [42]. This transporter has been described to be involved in the uptake of hy- drophilic compounds through the inner mycobacterial membrane. The results of this study suggest that the citric acid molecules interact with R287 while, at the same time, the negatively charged carboxylate groups strengthen the interaction between ethambutol and Rv1819c. Furthermore, the development of alternative formulations that can lead to new and more effective administration routes is another challenge in this quest for more efﬁcient anti-TB therapies. 4. THEDES for Tuberculosis Treatment The major problem in TB therapeutics is the long time needed for the treatment, which often leads to patients ceasing the treatment and, therefore, leading to the appearance of multi-resistant strains. One of the major concerns in TB treatment and prevention is related to drug-resistance that could emerge by spontaneous mutations of bacteria and trigger resistance to one or more effective anti-TB drugs, like the ones that are administered as ﬁrst-line treatment such as isoniazid and rifampicin (multidrug-resistant tuberculosis, or rifampicin-resistant TB) [43,44] This problem could be even more serious when multidrug-resistant TB and drug-resistance to at least one of second-line anti-TB drugs occurs (extensively drug-resistant TB) [37,43,44] This may be even more severe when the bacteria do not respond to either the ﬁrst-line or second-line of anti-TB drugs and are termed totally drug-resistant TB. These cases leave patients without additional treatment options that could be safe and effective [37]. Moreover, drugs used in second and third-line of treatment are, in general, less effective, more toxic, and comprise higher costs, as they will increase the time of the treatment for approximately 18–20 months. The immense variety of natural molecules accessible, coupled with the advantages of designing tailor- made systems, has led to the development of several NADES applications in the past years, particularly working as promising antimicrobial agents and promoters of bioﬁlm detach- ment. Several groups have reported the antimicrobial activity of eutectic systems, however, most of them do not rely solely on natural occurring molecules. Silva and co-workers revealed the use of fatty acids, such as capric acid, myristic acid, stearic acid, and lauric acid in NADES formulation, rendering systems with antimicrobial properties. The most promising system towards Gram-positive bacteria and C. albicans was capric acid:lauric acid (2:1). This system seems to be highly interesting as well for the detachment of bioﬁlms of several types of microorganisms [32]. Giving the high incidence and mortality of TB, alongside with all the research that has been carried out to discover effective treatments, exploring combinatorial approaches might be the path to follow. It became evident that Molecules 2021, 26, 7022 and/or 6 of 11 gy TB t combinatorial therapies are able to achieve target-speciﬁcity, increased bioavailability, pro- vide lower toxicity towards normal tissues, promote drug controlled released, and are efﬁcient in formulation for a speciﬁc route of administration while in compliance with the green chemistry metrics. 5. THEDES for Colorectal Cancer Treatment , thy lifestyle choices making Cancer is also a high burden economic and social devastating disease, in which mortality has been comparatively rising and has already overcome cardiovascular diseases as the main cause of dead in certain countries [45]. According to the WHO, in 2019 cancer was responsible for almost 10 million deaths worldwide, indicating that one in every six deaths is cancer related [46]. This sharp increase is due to several key factors, such as infections, chronic malnutrition, lack of access to medical care, but also population aging and unhealthy lifestyle choices, making cancer a burden both for the rapidly developing countries but also developed ones. Colorectal cancer (CRC) is the third most commonly diagnosed cancer and, if detected early and treated accordingly, has a high cure rate [46]. Nonetheless, it occupies the second position in the deadliest cancers [46]. Therefore, the high mortality rate and poor prognosis of CRC reveals the ineffectiveness of current therapies in controlling the disease progression, particularly in the case of advanced or metastatic phases (Figure 2). Thus, the need for alternative approaches to eliminate premalignant lesions, control their progression to cancer, and to avoid cancer recurrence are of at most importance. thy lifestyle choices, making cancer a burden both for t ut also developed ones. Colorectal cancer (CRC) is the cancer and, if detected early and treated accordingly, ha s, it occupies the second position in the deadliest cance ity rate and poor prognosis of CRC reveals the ineffecti trolling the disease progression, particularly in the case s (Figure 2). Thus, the need for alternative approaches s, control their progression to cancer, and to avoid canc tance. Figure 2. Schematic representation of progression of colorectal cancer (CRC). Created with BioRender.com. Figure 2. Schematic representation of progression of colorectal cancer (CRC). Created with BioRender.com. ematic representation of progression of colorectal cancer (CR The cytotoxicity of different NADES based on ammonium- and choline chloride for cell lines, such as human colon adenocarcinoma cell line (HT29), human malignant melanoma cell line (A375), among others, has been reported [47]. Table 3 presents a summary of the THEDES systems together with their reported anti-cancer activity. hematic representation of progression of colorectal cancer (CRC . The cytotoxicity of different NADES based on ammonium- and choline chloride for cell lines, such as human colon adenocarcinoma cell line (HT29), human malignant melanoma cell line (A375), among others, has been reported [47]. 4. THEDES for Tuberculosis Treatment One example of combinatorial technologies associates the use of THEDES design for TB therapy and SCF technology. This work, presented by our group, describes the preparation of THEDES with L-Arginine (described as an adjuvant in TB therapy) and the design of inhalable particles through SCF technology (PGSS), achieving an encapsulation efﬁciency of approximately 75%, revealing promising results for encapsu- lation of THEDES [30]. This is just one example of what could be achieved by combining different strategies like nanotechnology with green solvents and/or SCF technology for the formulation of nanoparticles in TB therapy. for Colorectal Cancer Treatment r is also a high burden economic and social devastating d een comparatively rising and has already overcome card use of dead in certain countries [45]. According to the sible for almost 10 million deaths worldwide, indicatin ncer related [46]. This sharp increase is due to several k ronic malnutrition lack of access to medical care but 5. THEDES for Colorectal Cancer Treatment , thy lifestyle choices making Table 3 presents a summary of the THEDES systems together with their reported anti-cancer activity. 7 of 11 Molecules 2021, 26, 7022 Table 3. Summary of reported THEDES with anticancer activity. THEDES Reported anti-cancer activity Reference Choline Chloride:Glycerine Choline Chloride: Ethylene glycol Choline Chloride:Urea Choline Chloride:Triethylene glycol Human breast cancer cell line (MCF-7); Human prostate cancer cell line (PC3); Human malignant melanoma cell line (A375); Human liver hepatocellular cell line (HepG2); Human colon adenocarcinoma cell line (HT29); Carcinoma-derived human oral keratinocyte cells (H413) [48] Choline Chloride:fructose Choline Chloride:glucose N,N-diethylethanol ammonium chloride:Triethylene glycol MCF-7, PC3, A375, Human gastric adenocarcinoma (AGS), Human uterus adenocarcinoma (HelaS3), Human hepatic cell lines (WRL-68) [47] Piperidinyl:diethylstilbestrol Pyrrolidinyl:diethylstilbestrol Human Breast Carcinoma cells (MCF-7) [49] Limonene:ibuprofen Perillyl alcohol:ibuprofen Menthol: ibuprofen Thymol:ibuprofen HT29 [30–32] Table 3. Summary of reported THEDES with anticancer activity. The anticancer activity of THEDES has to be evaluated against the effect of the systems in so-called “normal” cell lines, i.e., cell lines which mimic a healthy environment. Figure 3 represents the EC50 values reported for the different systems, in which their cytotoxicity is concerned, i.e., effective concentration to reduce cell viability in 50%, towards healthy cells and their antiproliferative activity towards cancer cells. The mechanisms of action of the systems with anti-cancer reported activity are still unclear, despite some hints that have been disclosed. For example, Mbous and co-workers report that NADESs activity, namely choline chloride:fructose, choline chloride:glucose and N,N-diethyl ethanolammonium chloride:triethylene glycol, by increasing membrane porosity and redox stress [47]. When it comes to targeting CRC cells, terpenes such as limonene, perillyl alcohol, menthol, and thymol, amongst others, combined with non-steroidal anti-inﬂammatory drugs (NSAID) have been studied and have shown great promise. Interestingly, it has been demonstrated that, when compared to its individual components, a eutectic mixture results in a new entity with different and enhanced properties, thus causing a more se- lective and severe impact on cancer cells [47]. However, the speciﬁc cytotoxic action of THEDES towards both malignant and non-malignant cells is still unclear [34,47,50]. These combinations with NSAIDs arise from the ﬁnding that NSAIDs reduce CRC and tumor recurrence risk by 40–50% [51]. Terpenes are plant secondary metabolites which may have an important role in the pursuit of natural molecules with anticancer potential by relying on their displayed cytotoxic action. Among the cancer hallmarks, there is the sustaining of proliferative signals and resisting cell death. 5. THEDES for Colorectal Cancer Treatment , thy lifestyle choices making In CRC, limonene has been reported to induce apoptosis via mitochondrial pathways and affecting the PI3K/Akt signaling pathway for survival and apoptosis [52]. Our ﬁrst studies, as reported by Pereira and co-workers, have demonstrated that IBU:LIM (1:4) has a dose-dependent effect and that the mechanism of action is completely different from isolated IBU and LIM. Furthermore, the assessment of intracellular reactive oxygen species (ROS) and nitric oxide (NO) production indicate that the lowest tested concentration of IBU:LIM (1:4) was able to protect HT29 cells from oxidative stress, showing anti-inﬂammatory effects through the inhibition of ROS and NO production [34]. Perillyl alcohol, menthol, and thymol are other examples of terpenes with an associative cytotoxic action towards cancer cells. In CRC, the epithelial–mesenchymal transition is widely associated with angiogenesis and a chronic inﬂammatory state [53]. Cox-2 is upregulated in CRC and the production of Cox-2-derived prostaglandin is known to promote β-catenin activation [53]. Moreover, it has been reported in the literature that the aberrant activation of the (Wnt)/β-catenin signaling pathway is known to be responsible Molecules 2021, 26, 7022 8 of 11 for approximately 90% of CRC cases [54,55]. β-catenin plays an important role in cell-to-cell adhesion and gene transcription regulation [55]. Only through the understanding of the speciﬁc cytotoxic action of THEDES towards both malignant and non-malignant cells will be possible to design adequate delivery systems to successfully target CRC and provide a signiﬁcant contribution to decrease the associated number of deaths. 8 of 12 Figure 3. THEDES cytotoxicity and antiproliferative activity measured as the effective concentra- tion to inhibit 50% of cell growth (EC50). Figure 3. THEDES cytotoxicity and antiproliferative activity measured as the effective concentration to inhibit 50% of cell growth (EC50). Figure 3. THEDES cytotoxicity and antiproliferative activity measured as the effective concentra- tion to inhibit 50% of cell growth (EC50). Figure 3. THEDES cytotoxicity and antiproliferative activity measured as the effective concentration to inhibit 50% of cell growth (EC50). Figure 3. THEDES cytotoxicity and antiproliferative activity measured as the effective concentra- tion to inhibit 50% of cell growth (EC50). Figure 3. THEDES cytotoxicity and antiproliferative activity measured as the effective concentration to inhibit 50% of cell growth (EC50). The mechanisms of action of the system ar despite some hints that have been d 6. Conclusions and Outlook Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. Sample Availability: Samples of the oregano oil used in this study are available from the authors. Abbreviations API Active pharmaceutical ingredient CRC Colorectal cancer DES Deep eutectic system HBA Hydrogen bond acceptor HBD Hydrogen bond donor IL Ionic liquid LTTM Low transition temperature mixtures Mtb Mycobacterium tuberculosis NADES Natural deep eutectic system NO Nitric oxide NSAID Non-steroidal anti-inﬂammatory drug PGSS Particles from gas saturated solutions ROS Reactive oxygen species TB Tuberculosis THEDES Therapeutic deep eutectic system SCF Supercritical ﬂuids WHO World Health Organization Institutional Review Board Statement: Not applicable. API Active pharmaceutical ingredient CRC Colorectal cancer DES Deep eutectic system HBA Hydrogen bond acceptor HBD Hydrogen bond donor IL Ionic liquid LTTM Low transition temperature mixtures Mtb Mycobacterium tuberculosis NADES Natural deep eutectic system NO Nitric oxide NSAID Non-steroidal anti-inﬂammatory drug PGSS Particles from gas saturated solutions ROS Reactive oxygen species TB Tuberculosis THEDES Therapeutic deep eutectic system SCF Supercritical ﬂuids WHO World Health Organization References 1. Sheldon, R.A. The E factor 25 years on: The rise of green chemistry and sustainability. Green Chem. 2017, 19, 18–43. [CrossRef] 2. Tucker, G.; DeSilva, B.; Dressman, J.; Ito, M.; Kumamoto, T.; Mager, D.; Mahler, H.C.; Maitland-van der Zee, A.H.; Pauletti, G.M.; Sasaki, H.; et al. Current Challenges and Potential Opportunities for the Pharmaceutical Sciences to Make Global Impact: An FIP Perspective. J. Pharm. Sci. 2016, 105, 2489–2497. [CrossRef] Sheldon, R.A. 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Interestingly, it has demonstrated that, when compared to its individual components, a eutectic mixture In pharmaceutical sciences, therapeutic deep eutectic systems are still in their infancy and exciting developments may be foreseen in the coming years. The studies carried out so far pave the way towards new ﬁndings, particularly those concerning the enhancement of drug bioavailability, either by increasing the solubility of the API in aqueous solutions or by increasing its permeability through relevant biological barriers (skin, intestinal wall, or other biological tissues). Side by side, with these advances go along new ﬁndings on the molecular understanding of the interactions that take place. From the formation of hydrotopes to enhance the API solubility, to the interactions of the THEDES with biological membranes and tissues, major advances are to be expected when these interactions are better understood. With regards to the advances in particular therapeutic applications, such as TB and CRC, which are the ones described in this manuscript, together with the advances in the fundamental knowledge on deep eutectic systems, the understating of the mechanisms of action of these liquid formulations and their differences in comparison Molecules 2021, 26, 7022 9 of 11 9 of 11 to the solid APIs will help boost the adoption of these green technology platform by the pharmaceutical industry. Author Contributions: Conceptualization, investigation, writing—original draft preparation, writing— review and editing, F.O., F.S. and A.R.C.D.; supervision, A.R.C.D.; project administration, A.R.C.D.; funding acquisition; A.R.C.D. All authors have read and agreed to the published version of the manuscript. Funding: This project has received funding from the European Union’s Horizon 2020 (European Research Council) under grant agreement No. ERC-2016-CoG 725034 and was supported by the Asso- ciate Laboratory for Green Chemistry-LAQV which is ﬁnanced by national funds from FCT/MCTES (UIDB/50006/2020) and co-ﬁnanced by the ERDF under the PT2020 Partnership Agreement (POCI- 01-0145-FEDER 007265). Filipe Oliveira acknowledges his PhD grant awarded by FCT—Fundação para a Ciência e Tecnologia (REF: 2021.07780.BD). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. References Shen, Q.; Li, X.; Li, W.; Zhao, X. Enhanced Intestinal Absorption of Daidzein by Borneol / Menthol Eutectic Mixture and Microemulsion. Aaps Pharmscitech. 2011, 12, 1044–1049. [CrossRef] p 20. Morrison, H.G.; Sun, C.C.; Neervannan, S. Characterization of thermal behavior of deep eutectic solvents and their potential as drug solubilization vehicles. Int. J. 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https://openalex.org/W1985395991	https://europepmc.org/articles/pmc3382273?pdf=render	English	null	Application of Genomic Tools in Plant Breeding	Current genomics	2,012	cc-by	15,977	Address correspondence to this author at the Instituto de Conservación y Mejora de la Agrodiversidad Valenciana, Universitat Politècnica de València, Camino de Vera 14, 46022 Valencia, Spain; Tel: +34963879424; Fax: +34963879422; E-mail: jprohens@btc.upv.es Application of Genomic Tools in Plant Breeding A.M. Pérez-de-Castro, S. Vilanova, J. Cañizares, L. Pascual, J.M. Blanca, M.J. Díez, J. Prohens and B. Picó Instituto de Conservación y Mejora de la Agrodiversidad Valenciana, Universitat Politècnica de València, Camino de Vera 14, 46022 Valencia, Spain Instituto de Conservación y Mejora de la Agrodiversidad Valenciana, Universitat Politècnica de València, Camino de Vera 14, 46022 Valencia, Spain Abstract: Plant breeding has been very successful in developing improved varieties using conventional tools and methodologies. Nowadays, the availability of genomic tools and resources is leading to a new revolution of plant breeding, as they facilitate the study of the genotype and its relationship with the phenotype, in particular for complex traits. Next Generation Sequencing (NGS) technologies are allowing the mass sequencing of genomes and transcriptomes, which is producing a vast array of genomic information. The analysis of NGS data by means of bioinformatics developments allows discovering new genes and regulatory sequences and their positions, and makes available large collections of molecular markers. Genome-wide expression studies provide breeders with an understanding of the molecular basis of complex traits. Genomic approaches include TILLING and EcoTILLING, which make possible to screen mutant and germplasm collections for allelic variants in target genes. Re-sequencing of genomes is very useful for the genome-wide discovery of markers amenable for high-throughput genotyping platforms, like SSRs and SNPs, or the construction of high density genetic maps. All these tools and resources facilitate studying the genetic diversity, which is important for germplasm management, enhancement and use. Also, they allow the identification of markers linked to genes and QTLs, using a diversity of techniques like bulked segregant analysis (BSA), fine genetic mapping, or association mapping. These new markers are used for marker assisted selection, including marker assisted backcross selection, ‘breeding by design’, or new strategies, like genomic selection. In conclusion, advances in genomics are providing breeders with new tools and methodologies that allow a great leap forward in plant breeding, including the ‘superdomestication’ of crops and the genetic dissection and breeding for complex traits. Received on: June 17, 2011 - Revised on: September 16, 2011 - Accepted on: October 11, 2011 Received on: June 17, 2011 - Revised on: September 16, 2011 - Accepted on: October 11, 2011 Keywords: Bioinformatics, complex traits, genetic maps, marker assisted selection, molecular markers, next-generation- sequencing, quantitative trait loci. Keywords: Bioinformatics, complex traits, genetic maps, marker assisted selection, mole sequencing, quantitative trait loci. ©2012 Bentham Science Publishers 179 179 Current Genomics, 2012, 13, 179-195 INTRODUCTION improving and accelerating the breeding process in many ways (e.g., association mapping, marker assisted selection, ‘breeding by design’, gene pyramiding, genomic selection, etc.) [5-7]. The Sanger technology has been the predominant sequencing method for the past thirty years. It has been used to sequence several genomes as well as many transcriptomes. The first international collaborative project resulted in the whole genome sequence of the model plant Arabidopsis thaliana [11]. Since then, reference genomes of selected genotypes were completed in a limited number of crops such as rice [12], maize [13], sorghum [14], populous [15], grapevine [16], papaya [17], or soybean [18]. The transcriptomes of most major crops, to a greater or lesser extent, were also sequenced. A global view of the genomes and transcriptomes sequenced can be obtained from the Gene Index Project (http://compbio.dfci.harvard.edu/tgi/plant. html) or in the NCBI Unigene database (http://www.ncbi. nlm.nih.gov/unigene). Genomics approaches are particularly useful when dealing with complex traits, as these traits usually have a multi-genic nature and an important environmental influence. Thanks to these technological improvements it is now feasible for a small laboratory to generate in a short time span (e.g., several months) enough molecular data to obtain a set of mapped quantitative trait loci (QTLs), even in a species lacking any previous genomic information [8]. Genomic tools are thus facilitating the detection of QTLs and the identification of existing favorable alleles of small effect, which have frequently remained unnoticed and have not been included in the gene pool used for breeding [9, 10]. In this review, we present and discuss the most relevant advances in the development and application of genomic tools for plant breeding, in particular for complex traits. Firstly, we introduce the most relevant genomic tools and secondly we provide examples of the application of these tools to plant breeding. The objective is to provide modern breeders with an updated synthetic view of how genomics can effectively improve the efficiency of breeding programs. The field of genomics has changed with the arrival of NGS technologies [19]. These new technologies have reduced the cost of sequencing by more than one thousand times compared to Sanger technology, by avoiding time- consuming and tedious traditional cloning steps and making possible to perform millions of sequencing reactions in parallel (Table 1). Among the “second generation” technologies, the 454 (Roche, http://www.454.com) and Illumina (Illumina, http://www.illumina.com) platforms are already profusely used to sequence crop species. INTRODUCTION genome, and which represents a paradigm shift, by facilitating the direct study of the genotype and its relationship with the phenotype [2]. While classical genetics revolutionized plant breeding at the beginning of the 20th century, genomics is leading to a new revolution in plant breeding at the beginning of the 21th century. Ever since the beginnings of the domestication of plants, some 10,000 years ago, plant breeding has been extremely successful in developing crops and varieties that have contributed to the development of modern societies, and have successively beaten (neo-)Malthusian predictions [1]. Application of conventional pre-genomics scientific breeding methodologies has led to the development of modern cultivars, which have contributed to the dramatic improvement of yield of most major crops since the middle of the 20th century. The success of plant breeding in the last century has relied in the utilization of natural and mutant induced genetic variation and in the efficient selection, by using suitable breeding methods, of the favorable genetic combinations. In this respect, the evaluation and identification of genetic variants of interest as well as the selection methodologies used have largely been based in the phenotypic evaluation. The field of genomics and its application to plant breeding are developing very quickly. The combination of conventional breeding techniques with genomic tools and approaches is leading to a new genomics-based plant breeding. In this new plant breeding context, genomics will be essential to develop more efficient plant cultivars, which are necessary, according to FAO, for the new 'greener revolution' needed to feed the world’s growing population while preserving natural resources. One of the main pillars of genomic breeding is the development of high-throughput DNA sequencing technologies, collectively known as next generation sequencing (NGS) methods. These and other technical revolutions provide genome-wide molecular tools for breeders (large collections of markers, high-throughput genotyping strategies, high density genetic maps, new experimental populations, etc.) that can be incorporated into existing breeding methods [2-5]. Recent advances in genomics are producing new plant breeding methodologies, Nowadays, genomics provides breeders with a new set of tools and techniques that allow the study of the whole 1-/12 $58.00+.00 Current Genomics, 2012, Vol. 13, No. 3 180 Pérez-de-Castro et al. diverse libraries, it is difficult to identify all genes and transcripts variants. Despite these limitations, ESTs collections have been very useful for breeders. INTRODUCTION Others, like Solid (Applied Biosystems, http://www.appliedbiosystems. com/technologies), have been less exploited in plants. By using these NGS technologies, the sequencing capacity of laboratories is continuously increasing. For instance, one High-Seq 2000 Illumina Sequencer is able to generate 55 Gb per day, which is roughly eighteen times the size of the human genome. Moreover, new, “third generation” platforms are being developed and incorporated to sequencing projects, such as PacBio RS (Pacific Biosciences, http://www.pacificbiosciences.com), Helicos (Helicos, http://www.helicosbio.com), or Ion Torrent (Life Technologies, http://www.iontorrent.com). The sequence obtained by NGS are generally deposited in the NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/ unigene). Bioinformatics NGS technologies are facilitating sequencing projects, but have brought new challenges, as millions of short DNA reads have to be analysed and assembled [19]. Also, genetic maps, genotypes, or expression information at a genomic scale have to be processed in order to obtain the relevant biological information. Therefore, it is necessary to develop new bioinformatics tools (algorithms and software), which allow the analyses of huge amounts of genome-wide data, but it is also necessary to change the approaches used to understand complex biological traits [25, 26]. The algorithms and methods used to store and process raw genomic data generated by the different technological platforms will depend on the type of data being processed and on the result expected. In any case, once the relevant information is obtained by the bioinformaticians, results have to be made available to the breeders by using an interface as easy and friendly as possible [25]. To provide access to this information, the generation of an easily browseable web site is a common and usually successful approach. Several general purpose web databases exist to make the relevant biological information available to the researchers and breeders (Table 2), like GenBank (http://www.ncbi.nlm.nih.gov/genbank/), EBML (http:// www.ebi.ac.uk/embl/), DDBJ (http://www.ddbj.nig.ac.jp/) and Swiss-prot (http://expasy.org/sprot/). These latter databases are devoted to store information about any species, but several other more specific databases focused on species of interest to the breeders also exist, like the Sgn (http://solgenomics.net/), Phytozome (http://www.phyto- zome.net/), Gramene (http://www.gramene.org/) or CropNet (http://ukcrop.net/), which hold information that could have more specific use for breeding programs. The field of sequence analysis has a long tradition and has enabled the assembly of many genome sequences obtained by Sanger sequencing. Nowadays, the huge amount of sequence reads generated by NGS and the low quality of individual reads requires new software tools and algorithms that allow dealing with these data in an efficient way [27]. We consider this to be a limiting factor for this kind of analyses right now. Although in the last years great advancements in the sequence processing algorithms have been achieved, the software currently available requires improvements in many cases. Two of the most common analyses carried out on these NGS reads are genome assembly and annotation and mapping. Genome assembly is a complex task requiring powerful computers and skilled bioinformaticians [25]. Genome and Transcriptome Sequencing The availability of the whole genome sequence of a crop is of great utility for plant breeding, but until recently it has been an unachievable goal for most crops. This privilege was restricted to a reduced number of model species with small genomes and to projects with an important investment in time and resources, but now has extended to an increasing number of crops. However, it is also true that for important cultivated species with large and complex genomes such as wheat, sugarcane, or coffee, the sequencing of the whole genome is very challenging and may take several years before a draft is completed. Given the high cost of whole genome sequencing, transcriptome sequencing has been a cheaper alternative. The cDNA sequences (expressed sequence tags, ESTs) provide relevant information about the genes expressed in a certain tissue or organ, at a given stage of development and under particular environmental conditions. ESTs sequencing projects do not provide information about non-coding sequences and, even using Nowadays, it is feasible to sequence most crop genomes (excluding those with a very large and complex genome) with a relatively modest budget, by combining Sanger with NGS technologies. Sequencing projects for 135 plant Table 1. Comparison of the Main Characteristics of the Conventional Sanger and Some of the Most Currently Used Next Generation Sequencing (NGS) Technologies and Approximate Sequencing Cost (in US $ Per Mbp) Technology Read Length (bp) Mbp per run Cost ($/Mbp) Sanger 1000 0.001 3000.00 454 Roche 450 450 66.00 Illumina Hi-Seq2000 100 270000 0.07 Solid 5500xl 50 270000 0.07 e Main Characteristics of the Conventional Sanger and Some of the Most Currently Used Next cing (NGS) Technologies and Approximate Sequencing Cost (in US $ Per Mbp) Current Genomics, 2012, Vol. 13, No. 3 181 Genomic Tools in Plant Breeding 181 genomes, most of them corresponding to cultivated species or wild relatives, have been completely sequenced (3), are being assembled (27) or are in progress (105), as reported at the NCBI database (http://www.ncbi.nlm.nih.gov/genomes/ leuks.cgi). Other databases including plant genome sequences are Plant GDB (http://www.plantgdb.org) and Phytozome (http://www.phytozome.net). A fully sequenced and well annotated genome provides useful tools for the breeders, as it allows the discovery of genes, determining their position and function, as well as the development of large marker collections and high resolution maps. Genome and Transcriptome Sequencing In the cases where only a draft sequence is available, its usefulness depends on the quality of the assembly. For instance on many occasions thousands of scaffolds are obtained but they are not anchored to the genetic map, which difficults the use of the information. Many transcriptomes have also been sequenced, a number of them in species for which no previous sequence information was available. Sweet potato [20], squash [21], pigeonpea [22], or buckwheat [23] represent some examples published in the last months. These assays are showing the great complexity of plant transcriptomes, allowing the identification of rare transcript variants that are being used to improve gene annotation and our knowledge of gene function and regulation. computer requirements are usually less demanding and the limiting factor could be the storage capacity. Some commonly used mappers are Bowtie, BWA, and TopHat. Once the reads are aligned, single nucleotide polymorphism (SNPs) can be detected by using the SAMtools or the GigaBayes SNP callers [28]. The open source software mainly used by the bioinformaticians is cumbersome for users not well versed in the Unix command line operating system. Some commercial proprietary solutions easier to use have been developed (e.g., LaserGene or CLC Genomics Workbench), but they have not been widely embraced by the breeders. Galaxy is an open source project devoted to create an easy to use web interface to the open source CLI based applications used in this area. An important amount of work has been devoted in this field to the creation of standard and open file formats capable of storing information regarding sequence alignment and modelling (SAM) [24], SNP calls using variant call format (VCF; http://1000genomes.org/wiki/doku.php?id= 1000_genomes: analysis:vcf4.0), genomic regions with browser extensible data (BED, http://genome.ucsc.edu/FAQ/ FAQformat# format1) and genomic annotations using the general feature format (GFF; http://www.sequenceontology. org/resources/gff3.html]). These open and standard formats allow the interoperability of the different software tools that are being actively developed and used. In addition, the computer requirements might be strong as some analyses require a large amount of RAM memory or storage capability. Bioinformatics In particular, the RAM memory requirements of the computers used to assemble eukariotic genomes could hinder the application of this technique by small laboratories. Some of the most commonly used assemblers are Roche's 454 Gsassembler, Celera Assembler, and Mira. Once a reference genome is available in the species it is common to study its variation [19]. To do this, a mapper software is commonly used instead of an assembler software. A mapper tries to align every read against the reference genome. This process is much simpler and faster than the assembly. In this case the Expression Studies, from Microarrays to RNA-seq New genomic tools are also of interest to expand and accelerate gene expression studies. The analysis of gene expression has provided a rich source of biological information, which allows breeders to understand the molecular basis of complex plant processes, leading to the identification of new targets for manipulating these processes. 182 Current Genomics, 2012, Vol. 13, No. 3 Pérez-de-Castro et al. 182 Table 2. Some Important Databases and Repositories of Genomic Information of Interest for Breeders Database Description URL Genbank General public sequence repository http://www.ncbi.nlm.nih.gov/genbank/ EMBL General public sequence repository http://www.ebi.ac.uk/embl/ DDBJ General public sequence repository http://www.ddbj.nig.ac.jp UniProt Protein sequences and functional information http://www.uniprot.org/ NCBI Biomedical and genomical information http://www.ncbi.nlm.nih.gov/ Gene Index Project Transcriptome repository http://compbio.dfci.harvard.edu/tgi/ GOLD Repository of genomes databases http://genomesonline.org/cgi-bin/GOLD/bin/gold.cgi Phytozome Genomic plant database http://www.phytozome.net/ Plantgdb Genomic plant database http://www.plantgdb.org CropNet Genomic plant database http://ukcrop.net/ SGN Solanaceae information resource http://solgenomics.net/ Gramene Grass information resource http://www.gramene.org/ MaizeGDB Maize infornation resource http://www.maizegdb.org/ Tair Arabidopsis information resource http://www.arabidopsis.org/ CotthonDB Cotton information resource http://cottondb.org/ CPGR Phytopathogen genomic resource http://cpgr.plantbiology.msu.edu/ . Some Important Databases and Repositories of Genomic Information of Interest for Breeders Gene expression studies were at first based on the classical Northern blot method that only allowed the quantification of tens of genes simultaneously. The QRT- PCR is a more affordable and quantitative technique; but the number of genes analyzed by experiment is also limited [29]. Other approaches allowing the study of thousands of genes were differential display and cDNA amplified fragment length polymorphisms (cDNA-AFLPs) [30]. However, these methods are not really quantitative and are limited by the ability of the developed libraries to capture low-abundance transcripts. Other methods that overcome part of these problems are the serial analysis of gene expression (SAGE) [31] and massively parallel signature sequencing (MPSS) [32]. Nevertheless, the most employed methods at present to analyze transcript profiling are the hybridization-based platforms or microarrays [33]. Expression arrays have several advantages when compared with other methods. They can measure tens of thousands of different transcripts in the same reaction, they are semi-quantitative and sensitive to low-abundance transcripts if those are represented in a given array. The most extensive data are from the model species A. thaliana [37], but an increasing number of studies in crops like maize, wheat, rice, barley, or soybean are already available. All published expression data are public and disposables in databases as GEO (http://www.ncbi.nlm.nih. gov/geo/) [38], ArrayExpress (http://www.ebi.ac.uk/array- express/) [39] or species specific databases. Re-Sequencing for SNPs Discovery and Use in Genotyping Platforms One of the most interesting applications of NGS for plant breeders is the discovery of genetic variation. Now it is possible to sequence rapidly multiple individuals within a species with limited technical expertise and at minimal cost. The parallel development of computational pipeline tools is greatly accelerating the accurate mining of these sequences for genetic variants that can be converted into genetic markers, mainly microsatellites or simple sequence repeats (SSRs) and SNPs [70]. SSRs and SNPs are now the predominant markers in plant genetic analysis. SNPs are more abundant, stable, amenable to automation, and increasingly cost-effective, thus are fast becoming the marker system of choice in modern genomics research [71]. Another important source of variability corresponds to the artificial mutant collections developed in several crop species. These artificial collections are created by radiation, chemical mutagenesis, or transgenic and insertion methods. Artificial mutations, mostly obtained by radiation and chemical methods, were used in plant breeding in the pre- genomics era, but new technologies are allowing the development of other types of collections [49]. For instance, the transferred DNA tagged lines and transposon tagged lines have been used to develop mutant collections in several species such as the model plant Arabidopsis (The Arabidopsis Information Resource; http://www.arabidopsis. org) or rice (International Rice Functional Consortium; http://irfgc.irri.org). Gene silencing technologies, using RNA interference, have also been used to create gene specific mutant collections in Arabidopsis, like the AGRIKOLA project (http://www.agrikola.org). The artificial mutant collections frequently contain variability not present in the natural collections, and so are also very useful for the study and development of new traits. The genome-wide SNPs discovery by massive re- sequencing has been performed in model species with small genomes, such as Arabidopsis thaliana, where the 1001 Genomes project (http://www.1001genomes.org) [72] is attempting to unveil the whole-genome sequence variation in this reference plant. Similar re-sequencing efforts are becoming possible in rice, maize, grape, soybean, poplar etc. by sequencing sets of related genotypes, individually or pooled, within each species (elite cultivars, breeding lines, ecotypes, landraces, and/or weedy and wild relatives of a crop) [73-76]. The higher complexity in architecture and repeat content of these genomes has made necessary the use of approaches for genomic complexity reduction that also reduce sequencing cost [77]. Identification of SNPs is also very challenging in species with high levels of heterozygosity and/or with complex ploidy levels, as pseudo- SNPs are identified by misassembly of paralogs [78-80]. Re-Sequencing for SNPs Discovery and Use in Genotyping Platforms In order to facilitate the identification of the accessions of interest in these collections, a genetic reverse approach has been used. Targeting Induced Local Lesions in Genomes (TILLING) [50] is able to identify all allelic variants of a DNA region present in an artificial mutant collection. A similar procedure called ecotype TILLING (EcoTILLING) [51] can be used to identify allelic variants for targeting genes in natural collections. These two methods are based on the use of endonucleases, such as CEL I or Endo I, that recognize and cut mismatches in the double helix of DNA [52, 53]. Since the TILLING and EcoTILLING techniques identify all allelic variants for a certain genomic region, the phenotypic characterization effort can be concentrated in a reduced number of accessions with different variants. Obviously, the success of the identification of variation useful for breeding programmes will depend on the right selection of target genes. The availability of sequences coming from NGS sequencing projects and the information provided by gene expression studies is significantly increasing the number and quality of candidates for TILLING and EcoTILLING studies. Both Roche 454 and Illumina GA have been mostly used for genome re-sequencing. The aligment difficulties often associtated to the use of short Illumina GA reads (Table 1) are less problematic in species for which available reference genomes facilitates SNPs calling and genome positioning of genetic variation [81]. For most of these species, limited collections of SSRs and SNPs were available from early re- sequencing efforts, previous to the advent of NGS, but new genome-wide re-sequencing is enlarging the SNP pools and making them more representative of the range of natural variation. For an increasing number of species with high societal or economic value NGS genome re-sequencing is providing the first SSRs and SNPs resources. Examples are the grain amaranths (Amaranthus sp.), important pseudocereals, appreciated for their nutritional quality and tolerance to abiotic stress [82], for which no prior genome information existed. In these cases the combination of several sequencing techniques, and the use of paired-end sequencing facilitates SNP discovery. Roche 454 and Illumina GA were combined for high-throughput SNP discovery in common bean [83] and also Solid was used to sequence diploid wheat species, These procedures have been successfully used in many crops [54]. For example, TILLING has been applied to Arabidopsis [55], Lotus [56], barley [57], maize [58], pea [59], and melon [60]. Expression Studies, from Microarrays to RNA-seq These data can be really useful when analyzing gene expression in these species or other crops [40]. Microarrays make use of the existing EST collections and genome sequence data. The vast increase provided by NGS in the number of sequences opens the possibilities of expression studies in a large number of species lacking previous sequence information. Also, deep NGS sequencing of RNA transcripts (RNA-seq) is emerging as an alternative to microarray studies to quantify gene expression [41, 42]. RNA-seq does not depend on genome annotation or on the probes contained in the array platform. This technology is also very useful to improve genome annotation, improving the detection of rare transcripts and splicing variants and the mapping of exon/intron boundaries. Moreover, RNA-seq avoids bias introduced during hybridization of microarrays and saturation level problems, haa a greater sensibility, and shows high reproducibility [41, 43]. This approach has been already used in different crops with different breeding objectives, leading to the identification of genes involved in several metabolic pathways, disease response, fruit development, etc. [44-47]. All these studies show the potential of RNA-seq for complex traits breeding. However, RNA-seq is an emerging technology and the methods used to analyze this kind of data are still being developed. There are several web resources that facilitate microarray data analysis (e.g., http://babelomics.bioinfo.cipf.es/) [34] or even web pages where the breeder can download experiments already performed and analyzed. There are also software packages specializated in microarrays analysis as the Bioconductor (http://www.bioconductor.org/help/work- flows/oligo-arrays/) or MeV (ttp://www.tm4.org/mev/) [35]. Probably one of the most useful database is Genevestigator (https://www.genevestigator.com/gv/ doc/plant_biotech.jsp) [36], which contains microarray data from different species. Current Genomics, 2012, Vol. 13, No. 3 183 Genomic Tools in Plant Breeding 183 Mutant and Germplasm Collections in the Genomics Era: TILLING and EcoTILLING using both genebank collections and natural populations [65]. These assays used gene targets involved in different processes. Many studies have been focused on detecting allelic variants in genes most related to organoleptic quality [66, 67] or disease resistance [68, 69]. Plant breeding requires genetic variability to be selected in order to increase the frequencies of favourable alleles and genetic combinations. Sources of natural genetic variability can be found within the crop, mostly in the form of landraces, and also in the wild relatives. Although many landraces have been substituted by modern and uniform cultivars and genetic erosion has taken place in wild materials, gene banks preserve many of these materials, which constitute an important reservoir of genetic variation useful for breeding [48]. Re-Sequencing for SNPs Discovery and Use in Genotyping Platforms Re-Sequencing for SNPs Discovery and Use in Genotyping Platforms Rice was the first crop for which EcoTILLING was applied [61]. Subsequently, EcoTILLING has been used in other crops and wild relatives, like barley [62], wheat [63], or the wild peanut Arachis duranensis [64], 184 Current Genomics, 2012, Vol. 13, No. 3 Pérez-de-Castro et al. which are donors of the subgenomes of modern hexaploid bread wheat [84]. candidate genes involved in complex biological processes of interest to plant breeders. For example, the transcriptomes of two resistant and one susceptible lines of water yam, a major staple crop in Africa, under anthracnose infection, were successfully sequenced detecting SNPs in genes putatively involved in pathogen response [94]. Also, two alfalfa genotypes contrasting for cellulose and lignin content were sequenced, which allowed selecting SNPs useful to improve alfalfa as a forage crop and cellulosic feedstock [95]. Most of the effort in species lacking genomic resources is being made through transcriptome re-sequencing, as an alternative way of genome complexity reduction. Targeted amplicon re-sequencing is another strategy for discovering SNPs in specific genes [78]. One of the first examples of deep transcriptome sequencing was a study with two maize inbred lines [85]. This first study was followed by a large and rapidly increasing number of projects using non-model crops, some of them with large, complex, polyploid, and uncharacterized genomes, including forest trees, like Eucalyptus [86], oak [87], several polyploid crops, like oilseed rape [79], oats [80], coffee [88], and sweet potato [89], non-model grain legumes as chickpea and chickling pea [90], tomato [91], or several cucurbits, including Cucurbita spp. [21], cucumber [92], and melon [93]. The use of genome and transcriptome sequencing for SNP discovery has resulted in large SNPs collections in most of the crops. These large collections are being validated and applied for different purposes such as map construction, map saturation, genome-wide diversity studies, association mapping etc. (Table 3). Some of the most important achievements will be described in later sections. There are many SNPs genotyping techniques, which are more or less appropriate for different scales of individuals/SNPs to be genotyped [107]. The implementation of marker-assisted breeding strategies often requires the generation of thousands of genotypes per population. Thus, one practical way of optimizing the use of these large SNPs collections is using them with cost- effective platforms for medium to high density genotyping. Re-Sequencing for SNPs Discovery and Use in Genotyping Platforms Genotyping assays usually require a previous process of selection of a set of SNPs, among the hundreds/tens of thousands detected, that are appropriate for the assay objectives. semiautomated or fully automated SNP genotyping [108, 109]. Genotyping assays usually require a previous process of selection of a set of SNPs, among the hundreds/tens of thousands detected, that are appropriate for the assay objectives. The markers derived from NGS can also be useful to position sequence scaffolds onto physical maps. In this respect, a new method combining deep sequencing and the bin mapping strategy has been described [117]. The SNPs identified by re-sequencing genomic libraries from selected progeny individuals, derived from a cross between two closely related diploid strawberry species, were used to anchor 92.8% of the Fragaria genome to the genetic map. Results highlighted the potential of this methodology to obtain a robust framework for the anchoring of the genome sequence without the requirement of a high density physical mapping or a well-saturated genetic map. The Illumina GoldenGate assays have been the most widely used for mid-throughput applications. SNPs platforms with 384, 768, or 1536 SNPs are available for a number of species (Table 3). Popularity is also increasing for the Sequenom Mass array and the KASPar genotyping chemistry [82, 110]. Expanded arrays with tens of thousands SNPs for high-throughput applications have been also developed with the Infinium technology in maize, grape, tomato, pine, and poplar and are under development in soybean and several Rosaceae crops (apple, peach, and cherry) [74, 111]. Whole-genome re-sequencing at different coverage levels is being increasingly applied for map construction using different strategies. As an example, a genetic map for rice has been constructed using whole genome re-sequencing of 150 RILs [118]. These authors concluded that the sequencing-based method was approximately 35 times more precise in recombination breakpoint determination than PCR-based markers maps. Also, the whole genome of 128 chromosome segment substitution lines (CSSLs) of rice was re-sequenced and used it for the construction of an ultra-high quality physical map in this crop [119]. Based on low coverage re-sequencing, a new mapping strategy that allows inferring the parental genotypes of the assayed RILs population has been proposed [120]. An ultra-high density linkage map was obtained with this method and the quality of the map was evaluated by using it to identify a QTL controlling grain width. Construction of High Density Genetic Maps One of the main applications of genomic advances is the development of high density genetic maps. The high-density map construction involves the location of hundreds or even thousand markers in the different linkage groups. In these maps the coverage should be very high and no large gaps must be present. NGS technologies and high-throughput genotyping platforms have allowed the improvement of genetic maps by increasing markers density. Several works include the integration of new markers, basically SNPs derived from re-sequencing studies, into previously developed genetic maps, both in diploid and polyploidy species [80, 112]. Golden Gate has been the most widely used platform. It has been estimated that this genotyping platform is 100-fold faster than gel-based methods for increasing 2-3 times maize map density [101]. Also Sequenom-based SNP-typing assays are starting to be applied. In a recent study, a total of 1.016 SNPs, identified via comparative next-generation transcriptomic sequencing, were successfully mapped by genotyping 297 maize recombinant inbred lines (RILs) [113]. Other genotyping strategies based on arrays hybridization, such as the single- feature polymorphisms (SFP), variants detected by a single probe in oligonucleotide arrays, are speeding up genetic map construction. This technique has been used for the construction of a high-density linkage map in species poorly characterized, like Eucalyptus [114]. The newly developed maps, enriched in sequence-based markers are facilitating comparative mapping. Recent examples are high density SNPs maps of barley compared with wheat and rice [98, 115]. Re-Sequencing for SNPs Discovery and Use in Genotyping Platforms Further applications of new sequence-based denser genetic maps to QTL discovery and marker assisted selection (MAS) will be discussed later. Re-Sequencing for SNPs Discovery and Use in Genotyping Platforms A large number of commercial platforms are available for These studies employ normalized/non-normalized cDNA libraries generated from single or multi-tissues samples, and derived from single or pooled genotypes, combined or not with multiplex identifier barcodes that allow allele origin identification. Sequencing is often focused on selected genotypes subjected to specific conditions, to detect SNPs in Table 3. Some Examples of the Utility of Molecular Markers Developed by Means of High-throughput Genomics Techniques for the Breeding of Important Crops Crop Markers Plant Material Use for Breeding Reference Rice (Oryza sativa) ~3.6·106 SNPs 517 rice landraces Association studies for 14 agronomic traits [96] Barley (Hordeum vulgare) 1,536 SNPs 768 breeding lines Association studies for Fusarium head blight resistance [97] 3,072 SNPs 336 DH lines and 213 germplasm selections High-density genetic map construction and MAF estimation [98] Maize (Zea mays) 8,590 SNPs 553 elite maize inbred lines Association studies for oleic acid content [99] 1,106 SNPs 5,000 RILs Association studies for resistance to southern leaf blight [100] 1,536 SNPs 154 maize inbred lines Diversity studies [101] Grapevine (Vitis vinifera) 94 SNPs and 7 indels 148 grape varieties Association studies for muscat flavor candidate gene VvDXS [102] 9000 SNPs 10 cultivated Vitis vinifera and 7 wild Vitis spp. Diversity and population structure studies [74] Pea (Pisum sativum) 384 SNPs 91 RIL mapping population and 373 Pisum accessions Linkage map construction and diversity studies. [103] Wheat (Triticum aestivum) 874 DArT markers winter wheat core collection of 96 accessions Association studies for 20 agronomic traits [104] 1,536 SNPs 478 spring and winter wheat cultivars Diversity studies [105] White spruce (Picea glauca) 944 SNPs 492 individuals Association studies with 549candidate genes and 25 wood quality traits [106] Table 3. Some Examples of the Utility of Molecular Markers Developed by Means of High-throughput Genomics Techniques for the Breeding of Important Crops Utility of Molecular Markers Developed by Means of High-throughput Genomics Techniques for Table 3. Some Examples of the Utility of Molecular Markers Developed by Means of High-throughput Genomics Techniques for the Breeding of Important Crops Table 3. Some Examples of the Utility of Molecular Markers Developed by Means of High-through the Breeding of Important Crops Genomic Tools in Plant Breeding Current Genomics, 2012, Vol. 13, No. 3 Current Genomics, 2012, Vol. 13, No. 3 18 Current Genomics, 2012, Vol. 13, No. 3 1 185 semiautomated or fully automated SNP genotyping [108, 109]. Genome-Wide Genetic Diversity Studies [122] identified and genotyped several million sequence polymorphisms among 27 diverse maize inbred lines. This study allowed the discovery of regions with highly suppressed recombination that appear to have influenced the effectiveness of selection during maize inbred development and may be a major component of heterosis. Also, highly differentiated regions were found that probably contained loci that are key to geographic adaptation. Also in legumes, the Medicago HapMap Project, that consist in the sequencing the whole-genomes of 30 inbred lines, will explore the genetic basis of symbiosis, creating a robust platform for genome-scale association mapping [123]. NGS and high-resolution maps have led to a significant improvement in the identification of molecular markers linked to specific genes and to QTLs. The most important advantage comes from the dense genome coverage, which allows the identification of markers closely linked to any target genomic region, with the advantages that this tight linkage provides. Methods already used in the pre-genomics era to facilitate the identification of markers linked to single loci, such as bulked segregant analysis (BSA), are now optimized. For example, a GoldenGate assay has been combined with BSA to significantly accelerate mapping of the dominant resistance locus to soybean rust Rpp3 [127]. In this respect, there is an increasing number of reports on exploitation of NGS technologies to identify molecular markers tightly linked to major genes. For example, a fine genetic mapping of the single dominant scab resistance gene (Ccu) in RILs of cucumber (Cucumis sativus) has been conducted [128]. The resistant cucumber genome was sequenced with Solexa/Illumina NGS and compared with the susceptible cucumber genome. As a result, three insertion/deletion (indel) markers closely linked to the Ccu locus where obtained. A detailed study of the region delimited by markers revealed four resistance gene analogs as possible candidates for Ccu. The diversity panels can include representatives of close or more distantly related species to check if these sequence- based SNP assays also work in related species [74, 82]. Sometimes sets of SNPs specifically developed for detecting genetic diversity among closely related cultivars are used in genotyping platforms. For example, despite the large amounts of SNPs available in rice obtained from the comparison of the two reference genomic sequences (one japonica and one indica variety) [124], extremely low levels of DNA polymorphism were detected within japonica cultivars. Genome-Wide Genetic Diversity Studies One of the main challenges in agricultural genetics is to access and use the tremendous genetic variation present in germplasm collections and in the wild relatives. A significant part of this variation remains untapped because of the difficulties in effectively identifying genetic differences in large collections. Some traits, with high heritability and of simple characterization, are easy to select for. However, desirable allelic variants and genetic combinations for complex traits are difficult to identify. Recent advances in genotyping are enabling genome-wide diversity studies capable of better capturing the spectrum of variability in natural and breeding populations. Most of the mid- to high-throughput genotyping platforms described above are being used for studies on diversity and population structure in the corresponding crops (Table 3). By using representative diversity panels, polymorphism rates for individual SNP markers, minor allele frequencies (MAFs), etc. are estimated, facilitating the selection of those SNPs with biological interest and highly polymorphic in the different groups. For example, the Infinium arrays developed in some of these crops are being used to create haplotype maps for vast germplasm collections, such as the 18,000 accessions of the USDA soybean germplasm collection [121]. The decrease of sequencing costs is also allowing the detection of new types of genetic markers useful for increasing the density of genetic maps.In this respect, restriction-site associated DNA (RAD) is a kind of marker which detects genetic variation adjacent to restriction enzyme cleavage sites across a target genome. These markers are produced after NGS sequencing of genomic libraries obtained after digestion with different restrictases. As an example of the utility of this technique, a total of 445 RAD markers distributed across all seven barley chromosomes were located, which was very useful for linkage map construction in this crop [116]. 186 Current Genomics, 2012, Vol. 13, No. 3 Pérez-de-Castro et al. Identification of Molecular Markers Linked to Single Genes and QTLs Haplotype maps (hapmap) of entire collections are useful to identify rare, potentially valuable, alleles. Hapmap projects are undergoing in a number of species such as the “rice diversity project” (http://www.ricehapmap.org/ index.aspx) aimed to develop a 10,000 SNP chip for rice and create a haplotype map to document the differences in allelic variation within and between the different subpopulations of O. sativa and its progenitor O. rufipogon. Large-scale genetic diversity studies have also been accomplished in maize. Gore et al. Genome-Wide Genetic Diversity Studies A whole-genome sequencing of an elite Japanese rice cultivar, closely related to the reference japonica variety, has been conducted and the SNP information obtained by comparison of the two japonica sequences was applied to develop a high-throughput genotyping array used for genotyping a set of representative Japanese cultivars [125]. These experiments are useful for understanding the role of selection and breeding in the distribution of genetic variation across the crop genome. In fact, this assay led to the identification of several haplotype blocks which are inherited from traditional landraces to current improved varieties. Moreover, it was found that, as predicted, modern breeding practices have generally decreased genetic diversity. On the practical level, the distribution of genetic diversity in modern cultivars plays an important role in the choice of specific mapping and crop improvement strategies. QTL detection has traditionally been conducted by linkage mapping. NGS technologies are significantly contributing to increase accuracy in detection of QTLs. They allow increases in many orders of magnitude of the number of markers mapped, ensuring high mapping resolution, and also aid in the development of mapping populations, such as RILs, near isogenic lines (NILs), and CSSLs, more appropriated for QTLs detection. These populations have conventionally been constructed and genotyped using a limited number of markers. There are increasing reports describing accurate QTLs mapping with different NGS or high-throughput genotyping strategies. For example, a high density rice map constructed by whole-genome re-sequencing of a RILs population, was used to identify four QTLs controlling plant height [90]. On a different study [129] an ultra-high density genetic map based on SNPs, obtained with Illumina GA, was compared with a linkage map based on RFLPs/SSRs in rice. The positions of several cloned genes, two major QTLs for grain length and grain width, and a QTL for pigmentation were evaluated in a RIL population, arising the expected result that the SNPs map detected more QTLs and more accurately than a RFLPs/SSRs based linkage map. Genome-wide survey of genetic diversity is useful to elucidate the causative genetic differences that give rise to observed phenotypic variation providing a foundation for dissecting complex traits through genome-wide association studies. However, its utility is limited if phenotypic data are not available. Not just genomics and transcriptomics, but the other 'omics' disciplines, like proteomics and metabolomics, are useful to understand how the changes in the genotype lead to differences in the final phenotype. Genome-Wide Genetic Diversity Studies Phenomics, which uses high-throughput technologies to characterize germplasm, is being developed and will help to deal with this issue [126]. QTL detection based on the linkage analysis method has the disadvantage that the number of recombination events is limited to the generations needed to develop the mapping population. Association mapping or linkage disequilibrium (LD) mapping is a new powerful approach to map complex traits. This method identifies genetic loci associated with phenotypic trait variation in a collection of individuals. Association mapping uses the natural diversity, which represents many more recombination events occurred in the Current Genomics, 2012, Vol. 13, No. 3 18 Genomic Tools in Plant Breeding 187 history of the population, providing better resolution. Nowadays, two association mapping methodologies are in use: candidate gene association, where a good understanding of the biochemistry and genetics of the trait is needed, and whole genome scan, also called genome-wide association (GWA) studies. New genomic advances are providing the higher density of genetic markers required to ensure enough coverage to detect linkage between markers and a causal locus. Also the decrease of sequencing costs (Table 1) has allowed the use of whole genome sequencing in current studies [130]. recombination. This is today feasible in many crop species in which NGS cDNA sequencing is being conducted. Some of these studies perform expression profiling, identifying candidates and associated gene targeted markers. MAS is also frequently applied to perform background selection in the context of backcrossing programmes. Background selection consists in the identification of plants with lower contents in donor genome to continue the breeding scheme, in order to achieve the recovery of the recipient genome. The use of background markers facilitates the quick recovery of the recurrent parent genome [139]. Background selection is being used extensively in rice breeding. High-density genome maps are being effectively used in background analysis. For example, background selection integrated with foreground selection of bacterial blight resistance (xa13 and Xa21 genes), amylose content (waxy gene) and fertility restorer gene has been performed in order to identify superior lines with maximum recovery of Basmati rice genome along with the quality traits and minimum non-targeted genomic introgressions of the donor chromosomes [140]. Nevertheless, association mapping is just rising in model species and major crops. Maize is the most widely studied crop regarding association analysis. Many candidate genes have been successfully associated to morphological or quality traits. Genome-Wide Genetic Diversity Studies As an example, candidate genes Dwarf8, Vgt1 and ZmRap2.7 were successfully associated to flowering time [131]. Other candidate genes have been associated, among others, to forage quality, carotenoid content, oil content and kernel quality [132-135]. GWA studies have been more limited, probably due to the large genome of maize (2300 Mbp) and the great number of markers needed to cover it. The first study identified a fatty acid desaturase gene (fad2) associated with increased oleic acid levels [99]. More recently, other authors found 32 QTLs associated with southern leaf blight disease resistance [100]. In some cases, the problem of recovering the genetic background of the recurrent parent arises because of the linkage drag, that is, the introgression of chromosome regions with deleterious effects which are tightly linked to the gene or QTL of interest. The detection of QTLs responsible of the negative effects and the localization of molecular markers tightly associated to them can be an efficient way to break the genetic drag. A well known example concerns canola (rapeseed) breeding, which began with the discovery of germplasm with low erucic acid content in seeds of a spring forage cultivar in the 1950’s. The problem arose because a high association between low erucic acid content and low seed oil content exists. The recent availability of high-density molecular maps has allowed the detection of several QTLs associated to both traits. Moreover, the identification of molecular markers very tightly linked to the QTLs made possible to break the linkage drag between the low oil content and erucic acid concentration in seeds in the process for breeding new high seed oil content canola cultivars [141]. Examples of association mapping approaches in other crops are more limited. Studies based on the candidate gene approach have been reported in some crops, like grape, or conifers [102, 106]. However, GWA studies have only been developed either in the model species A. thaliana [136] or in major crops such as rice [96], barley [97], or wheat [104]. Some articles also describe successful mapping processes combining classical linkage mapping and association mapping [137]. Although genetic association mapping is in its early steps, it is a promising tool for the dissection of complex traits in crop plants. Marker Assisted Backcross Selection Marker assisted selection (MAS) is an indirect process where selection is carried out on the basis of a marker instead of the trait itself. The successful application of MAS relies on the tight association between the marker and the major gene or QTL responsible for the trait. As we have described before, the new genomic tools accelerate the identification of markers tightly linked to target genomic regions. On the other hand, the new dense genotyping platforms available today accelerate the genotyping of large amounts of samples during the MAS process in a rapid and economically feasible manner. MAS can take benefit from these technologies, speeding up the release of new varieties. Frequently, current breeding programmes involve the introgression of more than one gene or QTL controlling traits of interest into one genetic background, in a process that is called pyramiding. The most useful application of MAS in the process of pyramiding is related to the introgression of different genes or QTLs whose effect on the phenotype is undistinguishable. The accumulation of genes from different sources which confer resistance against the same disease is an example, and is indeed one of the most widespread applications of gene pyramiding [142]. The main advantages of recent advances in plant genomics incorporated into gene pyramiding will be related to two different aspects. On one hand, the number of plants to be analyzed in a gene pyramiding programme must be increased as the number of loci of interest is higher, to ensure with a reasonable likelihood that the genotype combining favorable alleles is present in the population [143]. In this sense, the availability of genotyping platforms will provide the possibility to screen larger generations. On the other hand, In spite of the close linkage between the marker and the gene, the possibility of recombination limits the use of MAS. The use of intragenic markers, also called functional markers, can help to overcome this limitation [138]. NGS sequencing projects produce large collections of functional markers. These markers enhance real gene assisted breeding, reducing the possibility of losing the desirable trait due to 188 Current Genomics, 2012, Vol. 13, No. 3 188 Pérez-de-Castro et al. Fig. (1). Genomic selection scheme. Information on phenotype and genotype for a reference population allows estimating parameters for the model. This model explains phenotype based on all markers analyzed. Marker Assisted Backcross Selection The model predicts the phenotype of plants in a breeding population on the basis of the genotyping results: this is the genomic estimated breeding value (GEBV), used to select the desired phenotypes. REFERENCE POPULATION phenotype genotype AACATCTAGGCTATATAAGCT CCATAAAAATAAGTTCACAATT CATATGCACTTACTTAAGGCA TGATGGATATATGTTCTCAAA CACATAGCTCCATTTTAGAAAT AACAGCTAGGCTATATAAGCT CCATAAAAATAAGTTCATAATT CATATGCACTTACTTAAGGCA TGATGGATATATGTTCTCAAA CACATAGCTCCATTTTAGAAAT AACAGCTAGTCTATATAAGCT CCATAAAAATAAGTTCACAATT CATATGCACTTA···AAGGCATG ATGGATATATGTTCTCAAACA CAAAGCTCCATTTTAGAAAT AACACCTAGGCTATATAAGCT CCATAAAAATAAGTTCACAATT CATATGCACTTACTTAAGGCA TGATGGATATATGTTCTCAAA CACATAGCTCCATTTTAGAAAT PARAMETERS OF THE MODEL BREEDING POPULATION genotyping GEBV SELECTION AACAGCTAGTCTATATAAGCT CCATAAAAATAAGTTCACAATT CATATGCACTTA···AAGGCATG ATGGATATATGTTCTCAAACA CAAAGCTCCATTTTAGAAAT Phenotype explained by all markers 1 2 3 4 Phenotype PARAMETERS OF THE MODEL Phenotype explained by all markers BREEDING POPULATION GEBV Fig. (1). Genomic selection scheme. Information on phenotype and genotype for a reference population allows estimating parameters for the model. This model explains phenotype based on all markers analyzed. The model predicts the phenotype of plants in a breeding population on the basis of the genotyping results: this is the genomic estimated breeding value (GEBV), used to select the desired phenotypes. the efficiency of the process strongly depends on the tightness of the linkage between markers used and the target genes or QTLs. Again, identification of functional markers will circumvent this limitation. discussed, other possibilities such as GWA studies allow a more efficient way to accomplish this first step, avoiding limitations of biparental populations. The second step of the process consists in the identification of allelic variation for the locus of interest and the assignation of the phenotypic value to each of them. This step cannot be based on biparental populations, given that only two alleles per locus are segregating in this case. The analysis should then include plant materials representing the variability of the species. Genotypic and phenotypic data for each plant are required. ‘Breeding by Design’ The possibility to predict the outcome of a set of crosses on the basis of molecular markers information is known as ‘breeding by design’ [6]. The process includes three steps: mapping loci involved in all agronomically relevant traits, assessment of the allelic variation at those loci, and, finally, breeding by design. In the method as initially described by Peleman and van der Voort [6], the first step was proposed to be completed by either using mapping populations segregating for the trait of interest or based on a candidate gene approach (mainly exploiting information from model plant species and increasing understanding of gene function). Also linkage disequilibrium (LD) mapping was suggested, focused on the region previously identified as related to the trait (‘targeted LD mapping’). Currently, as previously As previously stated, high level of saturation with markers is not the limiting factor in most cases, and so currently the restrictions mostly come from the phenotyping step. Strictly speaking, ‘breeding by design’ exploits information obtained in the previous steps: once the loci of interest have been mapped, and the contribution of each allelic variant has been determined, crosses can be established to generate superior genotypes which combine all favourable alleles. Application of this breeding strategy Current Genomics, 2012, Vol. 13, No. 3 189 Genomic Tools in Plant Breeding has been used for different crops and with different objectives, such as breeding for heading date in rice [144] or seed length in soybean [145]. This procedure has also been used in patent applications; as an example, ‘breeding by design’ has been reported as part of the development of higher quality maize varieties. However, the most effective application of the ‘breeding by design’ approach will come from the incorporation of the most advanced genomic tools into the process, which will allow the improvement of the predictions. the utilization of this approach allows detecting the chromosome regions involved in a given trait, as markers with greater effect on the phenotype will indicate the presence of a QTL for this trait [152]. Some studies go one step farther and propose the application of MAS prior to phenotyping. This approach involves the use of prior indices, i.e., marker selection indices which have been constructed from a given phenotyped and genotyped population and are applied to different populations which have not been phenotyped [129]. CONCLUSIONS For some major crops the pace experimented for genetic gains in yield and other complex traits in the 20th century will be difficult to be maintained if only existing pre- genomics technologies are used [153]. However, plant breeding is a dynamic science and, fortunately, genomics resources and tools are already available and are helping to give another quantitative leap in plant breeding. In this respect many advances are already taking place, and the superdomestication, i.e., “the processes that lead to a domesticate with dramatically increased yield that could not be selected in natural environments from naturally occurring variation without recourse to new technologies” [10], will require the combination of conventional breeding with crop genomics. Also, genomic tools and approaches will help conventional breeding in achieving important advances in the breeding of crops that from the point of view of genetic improvement have remained either orphaned or neglected [8]. Therefore, while conventional pre-genomics plant breeding has been, is, and will be successful at improving our crops, the application of genomic tools and resources to practical plant breeding will push forward the genetic gains obtained by breeding programmes. New genomic advances, many of which are already being developed, will make easier for breeders to obtain new cultivars with improved characteristics, either by facilitating selection or by improving the variation available for breeders by using precision breeding approaches. In particular, the present and new genomics tools are of great value for the genetic dissection and breeding of complex traits. Simulation studies carried out using maize proved the usefulness of genomic selection applied to an initial cross between an adapted line and exotic germplasm. With 512 markers and a reference population of 288 F2 plants evaluated in six different environments, it was possible to obtain good selection response after 7-8 generations. [149]. Also with maize, simulations showed that response to selection was 18 to 43% larger for genomic selection than for marker assisted recurrent selection [150]. Response obtained when using genomic selection can be lower than response by phenotypic selection. However, the reduction in cycle length due to early MAS results in an increase of gain per time unit. This reduction is even more accused for species with a long generation interval, such as tree species [148]. ‘Breeding by Design’ The decrease in the costs of genotyping provides the appropriate scenario for this strategy to become cost-effective. Genomic Selection MAS strategies described so far require the identification of markers associated to the traits of interest. This represents one of the weaknesses of traditional MAS approaches [146]. Nevertheless, MAS can also be applied eluding this step, using an approach known as genomic (or genome-wide) selection (Fig. 1). The method was first described in 2001 [147], as an attempt to exploit information generated from emerging genotyping technologies. Genomic selection is based on simultaneous estimation of effects on phenotype of all loci, haplotypes, and markers available. The difference with other MAS methods relies on the fact that no previous selection of markers with effects on phenotype is developed [148]. Genomic selection requires the availability of phenotypic and genotypic data for the reference population. This data set will allow estimating the parameters for the model, so that the differences at the phenotype level are explained by the markers analysed. Once the model is established, application to breeding populations makes possible to determine the genomic value of each individual, i.e., the expected phenotype based on the genotypic data. The requirement is the availability of enough molecular markers to provide good genome coverage [5, 146]. In any case, even the identification of the QTLs responsible for a certain trait does not imply the identification of the gene or genes controlling the trait itself or the understanding of the mode of action. Models applied in genomic selection are useful to predict breeding values and, in some cases, detect regions associated to a trait, but further work is necessary from this point to identify the gene or genes responsible for the phenotypic variability observed. From plant breeders’ perspective, the availability of molecular markers which allow MAS to be applied is generally sufficient. However, development of the new high throughput -omics technologies has provided breeders with new strategies to search for candidate genes, mainly based on microarray for differential gene expression, being the possibility to explore more genes the most important advantage. Future exploitation of these strategies could facilitate the identification of candidate genes underlying the traits of interest and make MAS more efficient. ABBREVIATIONS ABBREVIATIONS AFLP = Amplified Fragment Length Polymorphism BED = Browser Extensible Data BSA = Bulked Segregant Analysis CSSL = Chromosome Segment Substitution Line DArT = Diversity Arrays Technology EcoTILLING = Ecotype TILLING EST = Expressed Sequence Tag GFF = General Feature Format GWA = Genome Wide Association LD = Linkage Disequilibrium MAF = Minor Allele Frequency MAS = Marker Assisted Selection MPSS = Massively Parallel Signature Sequencing NGS = Next Generation Sequencing NIL = Near Isogenic Line QTL = Quantitative Trait Locus RAD = Restriction-Site Associated DNA RIL = Recombinant Inbred Line RNA-seq = Sequencing of RNA Transcripts SAGE = Serial Analysis of Gene Expression SAM = Sequence Alignment and Modelling SPF = Single-Feature Polymorphism SNP = Single Nucleotide Polymorphism SSR = Simple Sequence Repeat TILLING = Targeting Induced Local Lesions on Genomes VCF = Variant Call Format [9] Morgante, M.; Salamini, F. From plant genomics to breeding practice. Curr. Opin. Biotechnol., 2003, 14, 214-219. p p [10] Vaughan, D.A.; Balász, E.; Heslop-Harrison, J.S. From crop domestication to super-domestication. Ann. Bot., 2007, 100, 893- 901. [11] The Arabidopsis Genome Initiative. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature, 2010, 408, 796–815. [12] International Rice Genome Sequencing Project. The map-based sequence of the rice genome. Nature, 2005, 436, 793-800. 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Tuskan, G.A.; Difazio, S,; Jansson, S.; Bohlmann, J.; Grigoriev, I.; Hellsten, U.; Putnam, N.; Ralph, S.; Rombauts, S.; Salamov, A.; Schein, J.; Sterck, L.; Aerts, A.; Bhalerao, R.R.; Bhalerao, R.P.; Blaudez, D.; Boerjan, W.; Brun, A.; Brunner, A.; Busov, V.; Campbell, M.; Carlson, J.; Chalot, M.; Chapman, J.; Chen, G.L. CONCLUSIONS The availability of phenotypic databases for different crops has allowed the comparison of predictions about the genotypic value obtained using genomic selection with the true genotypic value as shown by the phenotypic manifestation of the trait. In a study developed with phenotypic and genotypic data from Arabidopsis, maize and barley, results obtained were more accurate when genome- wide selection was carried out, if compared with results derived of previous selection of markers with effects on the phenotype [151]. Although when applying genomic selection there is no need to previously identify QTLs controlling a certain trait, 190 Pérez-de-Castro et al. 190 Current Genomics, 2012, Vol. 13, No. 3 ABBREVIATIONS Cooper, D.; Coutinho, P.M.; Couturier, J.; Covert, S.; Cronk, Q.; Cunningham, R.; Davis, J.; Degroeve, S.; Déjardin, A.; Depamphilis, C.; Detter, J.; Dirks, B.; Dubchak, I.; Duplessis, S.; Ehlting, J.; Ellis, B.; Gendler, K.; Goodstein, D.; Gribskov, M.; Grimwood, J.; Groover, A.; Gunter, L.; Hamberger, B.; Heinze, B.; ACKNOWLEDGEMENTS [5] Lorenz, A.J.; Chao, S.; Asoro, F.G.; Heffner, E.L.; Hayashi, T.; Iwata, H.; Smith, K.P.; Sorrells, M.K.; Jannink, J.L. Genomic selection in plant breeding: knowledge and prospects. Adv. Agron., 2011, 110, 77-123. This contribution has been partially funded by the Ministerio de Ciencia y Tecnología (AGL2008-05114, AGL2009-07257, and PIM2010PKB-00691), and Instituto Nacional de Investigación y Tecnología Agraria y Agroalimentaria (RTA2008-00035-C02-02 and RTA2011- 00044-C02-2) and FEDER funds (EU). [6] Peleman, J.D.; van der Voort, J.R. Breeding by design. Trends Plant Sci., 2003, 8, 330-334. y g ( AGL2009-07257, and PIM2010PKB-00691), and Instituto Nacional de Investigación y Tecnología Agraria y Agroalimentaria (RTA2008-00035-C02-02 and RTA2011- 00044-C02-2) and FEDER funds (EU). [7] Collard, B.C.Y.; Mackill, D.J. Marker-assisted selection: an approach for precision plant breeding in the twenty-first century. Phil. Transact. Royal Soc. B, 2008, 363, 557-572. [8] Varshney, R.K.; Glaszmann, J.C.; Leung, H.; Ribaut, J.M. More genomic resources for less-studied crops. Trends Biotechnol., 2010, 28, 452-460. 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https://openalex.org/W3201516651	https://europepmc.org/articles/pmc8488483?pdf=render	English	null	Multiple brain metastases in a patient with ypT0N0 micropapillary urothelial carcinoma of the bladder	Urology case reports	2,021	cc-by	2,003	Multiple brain metastases in a patient with ypT0N0 micropapillary urothelial carcinoma of the bladder Srikar Kuppa a, Jeffrey Wei a, Marshall Strother b, Curtis Miyamoto c, Michael Weaver d, Alexander Kutikov b,* Srikar Kuppa a, Jeffrey Wei a, Marshall Strother b, Curtis Miyamoto c, Michael Weaver Alexander Kutikov b,* a Department of Urology, Temple University School of Medicine, Philadelphia, PA, USA b Division of Urologic Oncology, Fox Chase Cancer Center, Philadelphia, PA, USA c Department of Radiation Oncology, Temple University School of Medicine, Philadelphia, PA, USA d Department of Neurosurgery, Temple University School of Medicine, Philadelphia, PA, USA a Department of Urology, Temple University School of Medicine, Philadelphia, PA, USA b Division of Urologic Oncology, Fox Chase Cancer Center, Philadelphia, PA, USA c Department of Radiation Oncology, Temple University School of Medicine, Philadelphia, PA, USA d Department of Neurosurgery, Temple University School of Medicine, Philadelphia, PA, USA A R T I C L E I N F O Radical cystectomy (RC) after neoadjuvant chemotherapy (NAC) is the gold standard for management of muscle- invasive bladder cancer (MIBC). Patients without residual tumor at the time of extirpative surgery (ypT0) have excellent prognosis. Distant metastases in this population are rare. We present a unique case of a patient with ypT0N0 urothelial carcinoma (UC) with rapid development of metastasis to the brain. Keywords: Muscle invasive bladder cancer Neoadjuvant chemotherapy Brain metastass to thigh, florine-18 2-fluoro-2-deoxy-D-glucose-positron emission to mography/computed tomography (FDG-PET/CT) scan demonstrated uptake in the known bladder mass as well as two suspicious right pelvic lymph nodes. At an outside cancer center, the patient was started on a NAC regimen with six cycles of gemcitabine, cisplatin, and paclitaxel. Due to paclitaxel-induced neuropathy, the patient finished the last two cycles with gemcitabine and cisplatin alone. Two weeks after completing NAC, the patient developed a lower extremity deep venous thrombosis (DVT). He was started on apixaban and an inferior vena cava filter was placed. Seven months following his initial presentation, and 8 weeks after completing NAC, the patient underwent RC with a Studer- type ileal neobladder urinary diversion. The pelvic lymph node dissec tion was intentionally limited to calibrate perioperative risks in view of the patient’s recent DVT. Pathologic examination of the bladder revealed no residual tumor, negative margins, and 8 negative lymph nodes, including those that revealed increased FDG uptake on pre- operative PET CT. Final staging was ypT0N0. Patient recovered well from surgery with excellent neobladder function. A 5-month post- operative CT of the chest and CT urogram demonstrated no evidence of disease. 1. Introduction Neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC) is the standard of care for muscle-invasive bladder cancer (MIBC). In fact, some 30% of patients who undergo RC after NAC, do not harbor residual malignancy in the bladder (ypT0).1 These patients with ypT0 pathology exhibit superior survival compared to those with residual cancer.2 Additionally, lymph node recurrence and metachronous solid organ metastases in these patients are very uncommon.2 In this report, we present, to our knowledge, the first documented case of a patient with ypT0N0M0 UC who developed symptomatic brain metastases 8 months following RC. * Corresponding author. Fox Chase Cancer Center, Division of Urologic Oncology, 333 Cottman Avenue, Philadelphia, PA, 19111, USA. E-mail address: alexander.kutikov@fccc.edu (A. Kutikov). Urology Case Reports 39 (2021) 101838 Urology Case Reports 39 (2021) 101838 Available online 13 September 2021 2214-4420/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.eucr.2021.101838 Received 5 August 2021; Received in revised form 30 August 2021; Accepted 3 September 2021 3. Discussion While ypT0 stage is associated with excellent prognosis, with re ported 5-year disease-free survival of 84–88%,2 this case represents an unexpected scenario for a patient with ypT0N0 UC following NAC and RC.1 Though uncommon, recurrence in ypT0 disease is usually due to regional lymph node spread and is seen in 3–7% of patients.1 Typical distant metastatic sites of UC include bone, lung, liver, and peritoneum.1 Our patient had no evidence of disease on routine surveillance at 5-months CT chest/abdomen/pelvis. The pelvic lymph nodes apparent on the initial PET-CT imaging were resected and there were no con cerning findings of follow up scans. Patient’s brain metastases were discovered when the patient became symptomatic. Fig. 2. 8-Months Post-Operative CT Abdomen and Pelvis with Contrast Demonstrating Intact Neobladder with no Evidence of Lymphadenopathy or Metastatic Disease. A) Coronal. B) Axial. Fig. 2. 8-Months Post-Operative CT Abdomen and Pelvis with Contrast Demonstrating Intact Neobladder with no Evidence of Lymphadenopathy or Metastatic Disease. A) Coronal. B) Axial. Brain metastases in patients with bladder cancer are rare, and are both a diagnostic and therapeutic challenge. Description of brain me tastases from UC are sparse, being found in an estimated 1–3% of met astatic UC patients.3 Moreover, the negative whole body FDG-PET/CT scan prior to RC could be explained by either a rapidly growing mass that developed following cystectomy or reflect physiologically-high FDG uptake by the brain masking an underlying metabolically active small metastatic lesion.4 Given rapid kinetics of new brain lesions develop ment following resection, the former scenario is most likely. Fig. 2. 8-Months Post-Operative CT Abdomen and Pelvis with Contrast Demonstrating Intact Neobladder with no Evidence of Lymphadenopathy or Metastatic Disease. A) Coronal. B) Axial. Surgical intervention for bladder UC brain metastases is rarely re ported since most patients present with concomitant extracranial dis ease. Fokas et al. reported that in a study of 62 patients with brain metastasis, metastasectomy with radiotherapy was not associated with a survival advantage over radiotherapy alone with a median overall sur vival of 9.6 vs. 8.9 months (p < 0.70).3 Resection in this case was un dertaken for diagnostic purposes given that patient’s pre-test probability for harboring intracranial metastases was low due to ypT0 pathology and lack of other metastatic sites. of surveillance.5 Kassouf et al. 2. Case presentation A 58-year-old man with a 35 pack-year smoking history presented to his primary care physician with mild abdominal pain, dysuria, and urinary frequency. Urinalysis showed microhematuria and urine cytology and fluorescence in situ hybridization (FISH) testing suggested UC. Computed tomography urogram (CT) confirmed a bladder mass and metastatic workup was consolidated with negative chest imaging. Approximately 8 months after RC, the patient presented with nausea, vomiting, dizziness, weakness and new onset of urinary incontinence. Repeat CT chest/abdomen/pelvis again showed intact neobladder with no evidence of lymphadenopathy or metastatic disease (Fig. 2). How ever, MRI of the brain found a large tumor in the left cerebellar The patient underwent transurethral resection of bladder tumor (TURBT) at an outside facility, which documented high grade muscle- invasive UC with lymphovascular invasion (LVI) and 20% micro papillary features (Fig. 1). The tumor was incompletely resected. Skull S. Kuppa et al. Urology Case Reports 39 (2021) 101838 Fig. 2. 8-Months Post-Operative CT Abdomen and Pelvis with Contrast Demonstrating Intact Neobladder with no Evidence of Lymphadenopathy or Metastatic Disease. A) Coronal. B) Axial. hemisphere and a smaller lesion in the right posterior frontal para sagittal cortex (Fig. 3). The left cerebellar mass measured 4.9 × 3.4 × 2.6 cm with surrounding vasogenic edema resulting in mass effect com pressing the ventricle. Due to the size and symptomatic nature of the cerebellar lesion, the patient underwent a left craniotomy with resection of the left cerebellar tumor, which revealed metastatic urothelial car cinoma with micropapillary features. Three weeks post-craniotomy, MRI of the brain showed a size increase in the parasagittal cortex lesion two new punctate lesions in the parasagittal right frontal lobe and the right parietal lobe which were suspicious for plural metastases. The patient received stereotactic radiosurgery (SRS) for the remaining le sions via gamma-knife and linac-based SRS. He recovered well post- craniotomy and his current treatment plan includes radiation therapy with immunotherapy and close radiographic surveillance. The patient is now 16 months post-RC with latest urology follow up at 15 months post- RC. 3. Discussion performed multivariate analysis of pa tients with ypT0 UC and found LVI in TURBT specimens predicted a significantly worse prognosis than TURBT specimens without LVI.2 Moreover, this patient had micropapillary features on histology for both the TURBT and craniotomy specimens. Like LVI, micropapillary features represent a highly aggressive UC subtype.5 Declaration of competing interest None. Acknowledgments Aeen Asghar, Rami Almefty, Brandon Bachert, Yulan Gong, Aileen Arriola Support/financial disclosures None. 4. Conclusion Because clinical outcomes can vary in patients with ypT0 disease, this case demonstrates the importance of risk-stratifying such patients to inform clinicians’ suspicion of metastatic disease and the necessary level Patients who undergo NAC and have no residual tumor upon RC Patients who undergo NAC and have no residual tumor upon RC Fig. 1. TURBT Specimen Histology. A) Urothelial carcinoma with Micropapillary Features, 100x. B) Urothelial Carcinoma with Micropapillary Features and Solid- Pattern Urothelial Carcinoma, 100x. C) Urothelial Carcinoma with Micropapillary Features and Lymphovascular Invasion, 100x. Fig. 1. TURBT Specimen Histology. A) Urothelial carcinoma with Micropapillary Features, 100x. B) Urothelial Carcinoma with Micropapillary Features and Solid- Pattern Urothelial Carcinoma, 100x. C) Urothelial Carcinoma with Micropapillary Features and Lymphovascular Invasion, 100x. 2 Urology Case Reports 39 (2021) 101838 S. Kuppa et al. S. Kuppa et al. S. Kuppa et al. ( T0) i ll h i l i O dd Fig. 3. T1-weighted post-contrast brain MRI images. A) Lesion in right poste rior frontal parasagittal cortex (arrow). B) Left cerebellar lesion (arrow) with marked vasogenic edema and compression of the fourth ventricle. C) Post- craniotomy imaging showing new punctate lesion (arrow). D) Post- craniotomy imaging showing increased size of right posterior frontal para sagittal cortex lesion (arrow). E) Post-craniotomy and radiotherapy imaging of right posterior frontal parasagittal cortex lesion site (arrow). F) Post- craniotomy and radiotherapy imaging of left cerebellar lesion site (arrow). to the evolving literature on optimal management of such patients and reinforces the importance of future work in risk stratification and treatment of patients with variant histology. Support/financial disclosures (pT0) typically have superior oncologic outcomes. Our case report adds References 1. Magers MJ, Lopez-Beltran A, Montironi R, Williamson SR, Kaimakliotis HZ, Cheng L. Staging of bladder cancer. Histopathology. 2019;74(1):112–134. https://doi.org/ 10.1111/his.13734. 2. Kassouf W, Spiess PE, Brown GA, et al. P0 stage at radical cystectomy for bladder cancer is associated with improved outcome independent of traditional clinical risk factors. Eur Urol. 2007;52(3):769–774. https://doi.org/10.1016/j. eururo.2007.03.086. 3. Fokas E, Henzel M, Engenhart-Cabillic R. A comparison of radiotherapy with radiotherapy plus surgery for brain metastases from urinary bladder cancer: analysis of 62 patients. Strahlenther Onkol Organ Dtsch Rontgengesellschaft Al. 2010;186(10): 565–571. https://doi.org/10.1007/s00066-010-2159-5. 3. Fokas E, Henzel M, Engenhart-Cabillic R. A comparison of radiotherapy with radiotherapy plus surgery for brain metastases from urinary bladder cancer: analysis of 62 patients. Strahlenther Onkol Organ Dtsch Rontgengesellschaft Al. 2010;186(10): 565–571. https://doi.org/10.1007/s00066-010-2159-5. 4. Hasan O, Nguyen M, Son H, Puri S. Role of FDG PET/CT in detecting unsuspected brain metastases during routine staging work up in oncology practice and its impact on patient management. J Nucl Med. 2014;55(suppl 1), 1303-1303. 4. Hasan O, Nguyen M, Son H, Puri S. Role of FDG PET/CT in detecting unsuspected brain metastases during routine staging work up in oncology practice and its impact on patient management. J Nucl Med. 2014;55(suppl 1), 1303-1303. p g pp 5. Black PC, Brown GA, Dinney CPN. The impact of variant histology on the outcome of bladder cancer treated with curative intent. Urol Oncol Semin Orig Investig. 2009;27 (1):3–7. https://doi.org/10.1016/j.urolonc.2007.07.010. g 5. Black PC, Brown GA, Dinney CPN. The impact of variant histology on the outcome of bladder cancer treated with curative intent. Urol Oncol Semin Orig Investig. 2009;27 (1):3–7. https://doi.org/10.1016/j.urolonc.2007.07.010. Fig. 3. T1-weighted post-contrast brain MRI images. A) Lesion in right poste rior frontal parasagittal cortex (arrow). B) Left cerebellar lesion (arrow) with marked vasogenic edema and compression of the fourth ventricle. C) Post- craniotomy imaging showing new punctate lesion (arrow). D) Post- craniotomy imaging showing increased size of right posterior frontal para sagittal cortex lesion (arrow). E) Post-craniotomy and radiotherapy imaging of right posterior frontal parasagittal cortex lesion site (arrow). F) Post- craniotomy and radiotherapy imaging of left cerebellar lesion site (arrow).
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https://openalex.org/W3018449359	https://publikationen.bibliothek.kit.edu/1000118694/71217716	English	null	Structural, Electronic and Magnetic Properties of a Few Nanometer-Thick Superconducting NdBa2Cu3O7 Films	Nanomaterials	2,020	cc-by	5,734	Received: 30 March 2020; Accepted: 21 April 2020; Published: 24 April 2020 Abstract: Epitaxial ﬁlms of high critical temperature (Tc) cuprate superconductors preserve their transport properties even when their thickness is reduced to a few nanometers. However, when approaching the single crystalline unit cell (u.c.) of thickness, Tc decreases and eventually, superconductivity is lost. Strain originating from the mismatch with the substrate, electronic reconstruction at the interface and alteration of the chemical composition and of doping can be the cause of such changes. Here, we use resonant inelastic x-ray scattering at the Cu L3 edge to study the crystal ﬁeld and spin excitations of NdBa2Cu3O7−x ultrathin ﬁlms grown on SrTiO3, comparing 1, 2 and 80 u.c.-thick samples. We ﬁnd that even at extremely low thicknesses, the strength of the in-plane superexchange interaction is mostly preserved, with just a slight decrease in the 1 u.c. with respect to the 80 u.c.-thick sample. We also observe spectroscopic signatures for a decrease of the hole-doping at low thickness, consistent with the expansion of the c-axis lattice parameter and oxygen deﬁciency in the chains of the ﬁrst unit cell, determined by high-resolution transmission microscopy and x-ray diffraction. Keywords: unit cell-thick ﬁlms; superconductivity; resonant inelastic x-ray scattering Structural, Electronic and Magnetic Properties of a Few Nanometer-Thick Superconducting NdBa2Cu3O7 Films Marco Moretti Sala 1,,† , Marco Salluzzo 2,†, Matteo Minola 3,† , Gabriella Maria De Luca 2,4, Greta Dellea 1, Vesna Srot 3, Yi Wang 3, Peter A. van Aken 3 , Matthieu Le Tacon 5, Bernhard Keimer 3, Claudia Dallera 1, Lucio Braicovich 1 and Giacomo Ghiringhelli 1,6, 1 Dipartimento di Fisica, Politecnico di Milano, Piazza Leonardo da Vinci 32, I-20133 Milano, Italy; greta.dellea@gmail.com (G.D.); claudia.dallera@polimi.it (C.D.); lucio.braicovich@polimi.it (L.B.) 1 Dipartimento di Fisica, Politecnico di Milano, Piazza Leonardo da Vinci 32, I-20133 Milano, Italy; greta.dellea@gmail.com (G.D.); claudia.dallera@polimi.it (C.D.); lucio.braicovich@polimi.it (L.B.) 2 1 Dipartimento di Fisica, Politecnico di Milano, Piazza Leonardo da Vinci 32, I-20133 Milano, Italy; greta.dellea@gmail.com (G.D.); claudia.dallera@polimi.it (C.D.); lucio.braicovich@polimi.it (L.B.) 2 CNR-SPIN, Complesso Monte Sant’Angelo-Via Cinthia, I-80126 Napoli, Italy; marco.salluzzo@spin.cnr.it (M.S.); gabriellamaria.deluca@unina.it (G.M.D.L.) g g p p 2 CNR-SPIN, Complesso Monte Sant’Angelo-Via Cinthia, I-80126 Napoli, Italy; marco.salluzzo@spin.cnr.it (M.S.); gabriellamaria.deluca@unina.it (G.M.D.L.) p ( ) g ( ) 3 Max Planck Institut für Festkörperforschung, Heisenbergstrasse 1, D-70569 Stuttgart, Germany; m.minola@fkf.mpg.de (M.M.); V.Srot@fkf.mpg.de (V.S.); y.wang@fkf.mpg.de (Y.W.); p vanaken@fkf mpg de (PA vA ); B Keimer@fkf mpg de (B K ) 4 Dipartimento di Fisica “E. Pancini”, Università Degli Studi di Napoli “Federico II”, Complesso Monte Sant’Angelo Via Cinthia I 80126 Napoli Italy 5 Institute for Quantum Materials and Technologies, Karlsruhe Institute of Technology, Hermann-v.-Helmholtz Platz 1, 76344 Eggenstein-Leopoldshafen, Germany; matthieu.tacon@kit.edu 6 CNR SPIN Dipartimento di Fisica Politecnico di Milano Piazza Leonardo da Vinci 32 I 20133 Milano Italy p * Correspondence: marco.moretti@polimi.it (M.M.S.); giacomo.ghiringhelli@polimi.it (G.G.) † These authors contributed equally to this work. † These authors contributed equally to this work. nanomaterials nanomaterials nanomaterials nanomaterials 1. Introduction The quest for miniaturization of technological devices has pushed the development of nanotechnology, a ﬁeld of applied research that deals with materials whose properties are intimately related to, and strongly dependent on their lateral size and shape. Ultimately, when the process of miniaturization is taken to the extreme, the resulting material could be made by one or few of its building blocks, which in the case of crystalline solids is the compound unit cell, i.e., the smallest group Nanomaterials 2020, 10, 817; doi:10.3390/nano10040817 www.mdpi.com/journal/nanomaterials 2 of 9 Nanomaterials 2020, 10, 817 of atoms that possesses the overall symmetry of the crystal. Within this framework, we investigated the properties of unconventional high-temperature superconductors in the form of ﬁlms, with variable thicknesses down to the single unit cell, in order to monitor the evolution of their physical properties as a function of thickness and possibly isolate new phenomena. High-Tc superconductors are materials that can have critical superconducting temperatures exceeding that of the boiling point of liquid nitrogen, and mostly include copper- and iron-based materials. They are “unconventional” superconductors, because their properties cannot be accounted for by the conventional Bardeen-Cooper-Schrieffer (BCS) theory of superconductivity [1]. High-temperature superconductivity was discovered in 1986 by Bednorz and Müller in an oxide of Ba, La and Cu [2] and ever since physicists worldwide have been trying to explain its mechanism, which remains to date a subject of intense debate. Here, we focus our attention on ultrathin ﬁlms of NdBa2Cu3O7−x, the member of a wide class of superconducting copper oxides (Nd,Y)Ba2Cu3O7−x with critical temperatures above 90 K at ambient pressure. It is known that the transport properties of cuprate epitaxial ﬁlms tend to degrade at extremely low thicknesses, in particular Tc decreases and eventually superconductivity is lost [3–6]. Despite a quasi-perfect lattice matching with the substrates, that ensures high-quality crystalline growth above few nanometers, at very low coverage epitaxial strain might be imposing a non-negligible deformation to the cuprate structure [7]. Moreover, at the interface the electronic structure might feel the symmetry breaking and undergo some degree of reconstruction. Finally, and even more importantly, the actual chemical composition might be altered, leading to an effective doping of the CuO2 planes different from that of thicker ﬁlms. A good evidence of the latter effect for the NBCO and YBCO families has been given in the recent literature, based on transport and structural studies [4,6]. 1. Introduction What is missing is a spectroscopic analysis. Resonant inelastic x-ray scattering is ideally suited for this purpose, as it probes the crystal ﬁeld (dd) excitations that are related to local coordination of the Cu ions [8–10], and the spin excitations (paramagnons) [11–15], whose dispersion is linked to the in-plane superexchange interaction. The latter is particularly signiﬁcant for ultrathin ﬁlms: is the antiferromagnetic short and medium range order preserved also in the single unit cell limit, where only 2 CuO2 planes are present, or is the quasi-perfect two-dimensionality of the system an obstacle to such order? In order to correlate the changes in the crystallographic, electronic and magnetic properties of the ﬁlms as a function of thickness, we carried out systematic structural and spectroscopic investigations. Note that RIXS measurements on low dimensional materials were reported previously in a superlattice consisting of 25 repetitions of La2CuO4/LaAlO3 building block layers, corresponding to an effective probed thickness of 33 nm [16]. Here, on the contrary, we probe a single layer of cuprate material, thus opening the way to the study of 2D-atomic materials by RIXS down to the single elementary unit cell. 2. Materials and Methods Films of NdBa2Cu3O7−x were grown on TiO2-terminated and (001)-oriented SrTiO3 substrates by high pure oxygen pressure diode sputtering [4,6]. In particular, we focus here on the 1, 2 and 80 u.c.-thick ﬁlms: the growth rate was accurately calibrated by systematic x-ray reﬂectivity measurements on samples deposited with different deposition time, from 2 to 120 min, and resulted to be 0.5 unit cells per minute. The uncertainty in the ﬁlm thickness is only half unit cell, as conﬁrmed by high-resolution scanning transmission electron microscopy (STEM) [17–19]. The quality of the crystalline structure is shown in the STEM images of Figure 1. In order to preserve the ﬁlm quality, the epitaxial ﬁlms were covered with a 2.4 nm-thick amorphous layer of NdBa2Cu3O7−x with the same nominal stoichiometry of the ﬁlm, deposited at room temperature, which provides a barrier against external perturbation and prevents change in the oxygen stoichiometry. As shown in Figure 2 (left panel), the X-ray absorption spectra of an amorphous (2.4 nm) NBCO is characterized by a very small absorption due to copper, but does not give any signiﬁcant contribution to the RIXS measurements at the peak of the Cu L3 absorption of the crystalline samples. Transport data shown in Figure 2 (right panel) show that a 1 u.c.-thick ﬁlm is not superconducting, while superconductivity is observed 3 of 9 Nanomaterials 2020, 10, 817 already in the 2 u.c.-thick sample, with a reduced Tc of 30 K, while the maximum Tc of 93 K is recovered already in 9 u.c.-thick samples, even when capped with the amorphous layer. 2 nm 2 nm 2 nm 2 nm O Cu Nd Ba (a) (b) (c) (d) STO NBCO Amorphous (e) (f) Figure 1. Scanning Transmission Electron Microscopy (STEM) images of epitaxial NBCO ultrathin ﬁlms grown on STO with an overlay of the NBCO structural model. Images were obtained with the high-angle annular dark-ﬁeld (HAADF) (panels a,c) and annular bright-ﬁeld (ABF) (panels b,d) techniques. Panels (a,b) are for the 1 u.c. thick ﬁlm, panels (c,d) for the 2 u.c. thick ﬁlm. The details of atom position assignment is shown in panel (f). The scheme of the ﬁlm growth structure is shown in panel (e). 2 nm 2 nm 2 nm 2 nm O Cu Nd Ba (a) (b) (c) (d) STO NBCO Amorphous (e) (f) Figure 1. 3. Results and Discussion NdBa2Cu3O7 in bulk form has an orthorhombic crystal structure with lattice parameters a = 3.87 Å, b = 3.91 Å and c = 11.74 Å and with CuO chains running along the b-axis separating bilayer units of CuO2 planes. XRD measurements show that above a threshold ﬁlm thickness of about 60 u.c. the NdBa2Cu3O7−x ﬁlm is fully relaxed and its lattice parameters match those of bulk samples. Below 60 u.c., instead, NdBa2Cu3O7−x ﬁlms are subject to a small in-plane tensile strain and adopt a tetragonal crystal structure with in-plane lattice parameters perfectly matched with the STO substrate (a = b = 3.905 Å) and short CuO chains running along both a and b axis. This suggests a clean pseudomorphic growth during the epitaxial deposition. Nevertheless, despite the sign of the strain, the c-axis increases as the ﬁlm thickness decreases. For samples capped with the amorphous NBCO, the maximum value of the c-axis is 11.80 Å in 1 u.c.-thick ﬁlms. The combination of transport and x-ray diffraction results suggests a direct correlation between the c lattice parameter and the doping level of NdBa2Cu3O7−x: in particular, as for other high-Tc cuprates of the so-called 123-family, the c-axis expands when the oxygen stoichiometry in the chains decreases. Thus, the c-axis is one-to-one related to the oxygen stoichiometry and to the effective doping level of NdBa2Cu3O7−x. In our case, we estimate x = 0.6, 0.5 and about 0 for 1, 2 and 80 u.c.-thick ﬁlms, respectively, consistent with transport measurements and with the chemical analysis by high-resolution electron microscopy [17,19]. The Figure 3b shows representative RIXS spectra of 1, 2 and 80 u.c.-thick ﬁlms at momentum transfer q∥= 0.376 r.l.u.. The acquisition time was 8 minutes for the 80 u.c.-thick sample and 30 min for the thinner ones, but data were normalized to the area of the spectrum in the energy region between 1 and 3 eV energy loss because of the large absolute count-rate variation between the various samples. However, we ﬁrst of all would like to emphasise the strong light-matter interaction that characterises RIXS, as opposed to the weak neutron-matter interaction in inelastic neutron scattering (INS), which makes RIXS suitable to study electronic, including magnetic, excitations in layers with thicknesses down to the single unit cell, in a reasonable amount of time. 2. Materials and Methods Scanning Transmission Electron Microscopy (STEM) images of epitaxial NBCO ultrathin ﬁlms grown on STO with an overlay of the NBCO structural model. Images were obtained with the high-angle annular dark-ﬁeld (HAADF) (panels a,c) and annular bright-ﬁeld (ABF) (panels b,d) techniques. Panels (a,b) are for the 1 u.c. thick ﬁlm, panels (c,d) for the 2 u.c. thick ﬁlm. The details of atom position assignment is shown in panel (f). The scheme of the ﬁlm growth structure is shown in panel (e). Figure 2. Panel (a) XAS measurements in total electron yield (TEY) mode of 2 u.c.-thick NdBa2Cu3O7−x ﬁlm covered with a 2.4 nm-thick amorphous layer of the same material (red line) and of the 2.4 nm-thick amorphous layer alone (black line). Panel (b) Resistivity measurements of 1 u.c. (black symbols), 2 u.c. (red symbols), 9 u.c. (gray symbols) -thick NdBa2Cu3O7−x ﬁlms covered with a 2.4 nm-thick amorphous layer of the same material and of 80 u.c.-thick NdBa2Cu3O7−x ﬁlm (blue symbols) with no capping layer. Figure 2. Panel (a) XAS measurements in total electron yield (TEY) mode of 2 u.c.-thick NdBa2Cu3O7−x ﬁlm covered with a 2.4 nm-thick amorphous layer of the same material (red line) and of the 2.4 nm-thick amorphous layer alone (black line). Panel (b) Resistivity measurements of 1 u.c. (black symbols), 2 u.c. (red symbols), 9 u.c. (gray symbols) -thick NdBa2Cu3O7−x ﬁlms covered with a 2.4 nm-thick amorphous layer of the same material and of 80 u.c.-thick NdBa2Cu3O7−x ﬁlm (blue symbols) with no capping layer. X-ray diffraction (XRD) θ/2θ measurements were performed by a conventional four-circle diffractometer (modiﬁed RIKAGU D-Max) equipped with a Cu-x-ray anode source. Transmission electron microscopy (TEM) measurements were carried out at the Stuttgart Center for Electron Microscopy of the Max Planck Institute for Solid State Research in Stuttgart (D). Resonant inelastic x-ray scattering (RIXS) measurements were performed at the ADRESS beam line [20] of the Swiss Light Source at the Paul Scherrer Institute (SLS-PSI) in Villigen (CH) using the SAXES [21] spectrometer. In order to obtain a reasonable count rate also on ultrathin ﬁlms, the combined energy resolution was set to 190 meV full-width-at-half-maximum at 931 eV, corresponding to the main peak in the Cu L3 edge x-ray absorption (XAS) proﬁle of crystalline NdBa2Cu3O7−x. The polarization of the incoming photons 4 of 9 Nanomaterials 2020, 10, 817 was horizontal in the laboratory reference frame and parallel to the scattering plane. 2. Materials and Methods The scattering angle was set to 130◦in order to maximize the available momentum transfer. All measurements were performed at T = 10 K, so that possible radiation damage effects were minimized. 3. Results and Discussion u.) Energy loss (eV) 2 1 0 0 10 20 30 x10 Intensity (ph/min) x0.1 80 u.c. 2 u.c. 1 u.c. amorph 0.5 0.0 q// Intensity (arb.u.) Energy loss (eV) 0.407 0.376 0.342 0.305 0.266 0.225 0.182 0.138 1 u.c. (a) (b) Figure 3. Panel (a) RIXS measurements of magnetic excitations in 1 u.c.-thick NdBa2Cu3O7−x ﬁlm as a function of momentum transfer q∥. Panel (b) Comparison between the RIXS spectra at q∥= 0.376 r.l.u. of 1, 2 and 80 u.c.-thick NdBa2Cu3O7−x ﬁlms after normalization to the area of the dd excitations. Also shown is the RIXS spectrum of a 2.4 nm-thick amorphous layer of NdBa2Cu3O7−x, rescaled by a factor 10. The inset shows the same data normalized to the acquisition time. We now turn to the analysis of the magnetic excitations, which in superconducting cuprates are heavily damped spin-excitations in the absence of long-range magnetic ordering and are, therefore, named paramagnons. Figure 3a shows that magnetic excitations disperse as a function of the momentum transfer q∥. The low-energy region of the RIXS spectra was ﬁtted with two curves, an energy-resolution limited Gaussian for the elastic line and an asymmetric Voigt function for the paramagnon. Details of the ﬁt for q∥= 0.376 r.l.u. are reproduced in Figure 4a–c. We note that the elastic line intensity increases as the ﬁlm thickness is reduced, thus suggesting a growing non-resonant elastic scattering contribution from the substrate. 1.0 0.5 0.0 Energy loss (eV) data elastic paramagnon total background 1.0 0.5 0.0 Intensity, norm. to dd area (arb.u.) Energy loss (eV) -1.0 -0.5 0.0 Energy loss (eV) 80 u.c. 2 u.c. 1 u.c. (a) (b) (c) Figure 4. Panels (a–c): Fit to the RIXS spectra of 80, 2 and 1 u.c-thick NdBa2Cu3O7−x ﬁlms at q∥= 0.376 r.l.u. Figure 4. Panels (a–c): Fit to the RIXS spectra of 80, 2 and 1 u.c-thick NdBa2Cu3O7−x ﬁlms at q∥= 0.376 l Figure 4. Panels (a–c): Fit to the RIXS spectra of 80, 2 and 1 u.c-thick NdBa2Cu3O7−x ﬁlms at q∥= 0.376 r.l.u. A summary of the main ﬁtting parameters used to ﬁt the RIXS data summarized in Figure 3 is provided in Figure 5. 3. Results and Discussion The spectra in Figure 3 are characterized by a quasi-elastic line at zero energy loss, spin excitations around 0.3 eV [22], while the most intense excitations at around 1.5 eV are crystal-ﬁeld (or dd) transitions [10,21,22]. The inset in the same ﬁgure shows that the contribution of the amorphous NdBa2Cu3O7 protective layer to the RIXS signal is negligible. Indeed, the XAS spectra of crystalline and amorphous NdBa2Cu3O7 show absorption resonances at distinct energies: this difference provides chemical sensitivity to RIXS and can be used to distinguish the contribution of Cu ions in different chemical environments. First, we discuss the effect of the ﬁlm thickness on dd excitations, i.e., transitions between the crystal ﬁeld split states of Cu2+. For bulk, insulating NdBa2Cu3O6, past RIXS studies allowed to determine with precision the following sequence of transition energies: the 3z2 −r2, xy and zx/yz states of the 3d9 hole are found at 1.98, 1.52 and 1.75 eV, respectively [10]. In the presence of doping, however, the corresponding peaks broaden and merge in an asymmetric, triangular feature, making the assignment of each individual ﬁnal state impossible. The shape of the crystal-ﬁeld excitation peak, therefore, reﬂects the degree of doping in the system: in our case, we observe a gradual evolution of its shape from very broad and asymmetric to rather sharp and symmetric as the ﬁlm thickness is reduced, consistent with the variation of effective doping level invoked before. 5 of 9 Nanomaterials 2020, 10, 817 4 3 2 1 0 80 u.c. 2 u.c. 1 u.c. Normalized Intensity (arb. u.) Energy loss (eV) 2 1 0 0 10 20 30 x10 Intensity (ph/min) x0.1 80 u.c. 2 u.c. 1 u.c. amorph 0.5 0.0 q// Intensity (arb.u.) Energy loss (eV) 0.407 0.376 0.342 0.305 0.266 0.225 0.182 0.138 1 u.c. (a) (b) Figure 3. Panel (a) RIXS measurements of magnetic excitations in 1 u.c.-thick NdBa2Cu3O7−x ﬁlm as a function of momentum transfer q∥. Panel (b) Comparison between the RIXS spectra at q∥= 0.376 r.l.u. of 1, 2 and 80 u.c.-thick NdBa2Cu3O7−x ﬁlms after normalization to the area of the dd excitations. Also shown is the RIXS spectrum of a 2.4 nm-thick amorphous layer of NdBa2Cu3O7−x, rescaled by a factor 10. The inset shows the same data normalized to the acquisition time. 4 3 2 1 0 80 u.c. 2 u.c. 1 u.c. Normalized Intensity (arb. 3. Results and Discussion In particular, Figure 5a shows that the paramagnon energy slightly diminishes with the ﬁlm thickness: the experimental data points are well ﬁtted to the dispersion curve of optical magnons within the framework of a simple Heisenberg model, which provides the main in-plane (and out-of-plane) magnetic coupling(s) J∥(and J⊥, respectively). Note that, despite few authors have adopted a different interpretation of the spin excitations in doped cuprates [23], this approach is fully justiﬁed by a number of previous studies showing that, although damped, the spin excitations in doped cuprates remain well deﬁned, preserve their spectral weight up to very high doping levels beyond optimal and perfectly reproduce the magnon dispersion observed in the AF insulators[12,13,24]. We ﬁnd that J∥evolves from 98 meV for the 1 u.c.-thick ﬁlm, to 110 meV for the 2 u.c., to 114 meV 6 of 9 Nanomaterials 2020, 10, 817 for the 80 u.c.-thick ﬁlm (the out of plane coupling has been ﬁxed at 7 meV as in Ref. [25]). This little change can be consistent with the small variation of the in-plane lattice parameters vs. ﬁlm thickness. On the other hand, Figure 5b evidences a large variation of the paramagnon width at all transferred momenta: in particular, it is drastically reduced in the thinnest ﬁlms as compared to the 80 u.c.-thick one. Again, this observation is consistent with the thickness dependence of the effective doping level and, in particular, with the evidence coming from transport measurements that the 1 u.c.-thick ﬁlm is the least-doped sample. We note that in the data presented here, the mild tensile strain is playing only a secondary role in the determination of the thickness dependence of the crystal ﬁeld and spin excitation energy. In other cases, e.g., for CaCuO2 [26] and La2CuO4 grown on different substrates [27], the larger strain leads to sizable changes of the hopping integrals that govern the 3d orbital energy splitting and the superexchange parameter J∥. On the contrary, here the dominant effect is the oxygen deﬁciency and correspondingly lower doping level of the ultrathin ﬁlms, which leads to modiﬁcations of the RIXS spectra similar to those observed in bulk crystals, i.e., the broadening of the magnetic peak, with no or little variation of its energy position as a function of doping [28]. 0.1 0.2 0.3 0.4 0 100 200 300 400 FWHM (meV) q// (r.l.u.) 80 u.c. 2 u.c. 1 u.c. 3. Results and Discussion 0.1 0.2 0.3 0.4 0 50 100 150 200 250 Energy (meV) q// (r.l.u.) 80 u.c. 2 u.c. 1 u.c. (a) (b) Figure 5. (a) Dispersion and (b) FWHM of magnetic excitations in 1 (black circles), 2 (red diamonds) and 80 (blue squares) u.c.-thick NdBa2Cu3O7−x ﬁlms. Continuous (dotted) lines show the corresponding optical (acoustic) magnetic branch used to ﬁt the dispersion. 0.1 0.2 0.3 0.4 0 100 200 300 400 FWHM (meV) q// (r.l.u.) 80 u.c. 2 u.c. 1 u.c. (b) 0.1 0.2 0.3 0.4 0 50 100 150 200 250 Energy (meV) q// (r.l.u.) 80 u.c. 2 u.c. 1 u.c. (a) Figure 5. (a) Dispersion and (b) FWHM of magnetic excitations in 1 (black circles), 2 (red diamonds) and 80 (blue squares) u.c.-thick NdBa2Cu3O7−x ﬁlms. Continuous (dotted) lines show the corresponding optical (acoustic) magnetic branch used to ﬁt the dispersion. 4. Conclusions We were able to measure the RIXS response of NdBa2Cu3O7−x ﬁlms, with thicknesses down to the single unit cell, i.e., the smallest fundamental constituent of matter in crystalline form. This very fact proves the extreme sensitivity of RIXS in the study of nanometer-sized materials. Speciﬁcally, our measurements probe both magnetic and crystal-ﬁeld (or dd) excitations and provide evidence of stoichiometry variations vs. thickness in nominally chemically equivalent ﬁlms: in particular, we ﬁnd that the effective doping of the NdBa2Cu3O7−x ﬁlms decreases as the ﬁlm thickness is reduced. Our results are consistent with previous transport and structural investigations, thus providing a coherent picture of the electronic properties of ultrathin NdBa2Cu3O7−x ﬁlms. These results suggest that in cuprates, the degradation of superconducting properties when approaching the single unit cell thickness limit is related to the actual doping at the interface with the substrate rather than to the modiﬁcation of the Mott-Hubbard physics in the CuO2 planes. Namely, the realization of almost ideal 2D superconductivity is not impeded by the supposed disruption of the strong antiferromagnetic background in the planes. On the contrary, short and medium range magnetic correlation persists down to 1 unit cell, leaving space for the Cooper pairing mediated by spin-ﬂuctuation to be at play also in the extreme case of a single CuO2 plane [29], a scenario realized with half u.c.-thick Bi2Sr2CaCu2O8+x [30] and recently with Bi2Sr2CaCu2O8+δ in the form of 1 unit cell thick ﬂakes [31]. 7 of 9 Nanomaterials 2020, 10, 817 Author Contributions: G.G., L.B., M.L.T., B.K. and M.S. conceived the experiment; M.S. and G.M.D.L. grew and characterized the samples; M.M., M.S., G.D., G.G. performed the RIXS experiment; M.M., M.M.S. and G.G. analysed the RIXS spectra; V.S., Y.W. and P.A.v.A. performed the TEM measurements and analyzed them; M.M.S., C.D., M.S. and G.G. wrote the manuscript with inputs from all coauthors; M.L.T., B.K., G.G. supervised the project. All authors have read and agreed to the published version of the manuscript. Funding: RIXS data were collected at the beam line ADRESS of the Swiss Light Source at the Paul Scherrer Institute (Switzerland) using the SAXES spectrometer jointly designed and built by the PSI and the Politecnico di Milano. M.M.S., C.D. and G.G. acknowledge support by project PRIN2017 “Quantum-2D” ID 2017Z8TS5B of the Ministry for University and Research (MIUR) of Italy. References 1. Keimer, B.; Kivelson, S.A.; Norman, M.R.; Uchida, S.; Zaanen, J. From quantum matter to high-temperature superconductivity in copper oxides. Nature 2015, 518, 179–186. [CrossRef] 2. Bednorz, J.G.; Müller, K.A. Possible highTc superconductivity in the Ba-La-Cu-O system. Zeitschrift für Physik B Condensed Matter 1986, 64, 189–193. [CrossRef] 3. Triscone, J.M.; Karkut, M.G.; Antognazza, L.; Brunner, O.; Fischer, O. Y-Ba-Cu-O/Dy-Ba-Cu-O superlattices: A ﬁrst step towards the artiﬁcial construction of high-Tc superconductors. Phys. Rev. Lett. 1989, 63, 1016–1019. [CrossRef] 4. 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Braicovich, L.; van den Brink, J.; Bisogni, V.; Moretti Sala, M.; Ament, L.J.P.; Brookes, N.B.; De Luca, G.M.; Salluzzo, M.; Schmitt, T.; Strocov, V.N.; et al. Magnetic Excitations and Phase Separation in the Underdoped La2−xSrxCuO4 Superconductor Measured by Resonant Inelastic X-Ray Scattering. Phys. Rev. Lett. 4. Conclusions This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 823717-ESTEEM3. Acknowledgments: C.D., M.M.S. and G.G. wish to recall and acknowledge their friendship and sincere admiration for Claudio Ferrero, a unique friend at ESRF. Claudio was the one person we were sure we would ﬁnd in his ofﬁce or around the ring, any time of the day and of the night, and would always have time for us, to chat or to discuss physics, or sometimes even to go for dinner... before returning to work again. 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https://openalex.org/W2968937301	https://nottingham-repository.worktribe.com/preview/2434840/pzz107.pdf	English	null	Responsiveness and Minimal Important Change for Pain and Disability Outcome Measures in Pregnancy-Related Low Back and Pelvic Girdle Pain	Physical therapy	2,019	cc-by	8,393	TITLE: Responsiveness and Minimal Important Change for Pain and Disability Outcome Measures in Pregnancy-Related Low Back and Pelvic Girdle Pain TITLE: Responsiveness and Minimal Important Change for Pain and Disability Outcome Measures in Pregnancy-Related Low Back and Pelvic Girdle Pain SECTION/TOC CATEGORY: Women’s Health AUTHOR BYLINE: Reuben Ogollah, Annette Bishop, Martyn Lewis, Margreth Grotle, Nadine E. Foster AUTHOR BYLINE: Reuben Ogollah, Annette Bishop, Martyn Lewis, Margreth Grotle, Nadine E. Foster AUTHOR INFORMATION: AUTHOR INFORMATION: R. Ogollah, PhD, Nottingham Clinical Trials Unit, School of Medicine, University of Nottingham, D Floor, South Block, Queens Medical Centre, Nottingham NG7 2UH, United Kingdom. Address all correspondence to Dr Ogollah at: reuben.ogollah@nottingham.ac.uk. A. Bishop, PT, PhD, Research Institute for Primary Care and Health Sciences, Keele University, Keele, Staffordshire, United Kingdom. M. Lewis, PhD, Research Institute for Primary Care and Health Sciences, Keele University; and Keele Clinical Trials Unit, Keele University. M. Grotle, PT, PhD, Department of Physiotherapy, Oslo Metropolitan University, Oslo, Norway; and FORMI, Oslo University Hospital, Oslo, Norway. N.E. Foster, PT, BSc(Hons), FCSP, DPhil, Research Institute for Primary Care and Health Sciences, Keele University; and Keele Clinical Trials Unit, Keele University. KEYWORDS: Pregnancy-Related Low Back Pain and Pelvic Girdle Pain, Minimally Important Change, Responsiveness, Patient-Based Outcome Measures ACCEPTED: March 25, 2019 SUBMITTED: July 10, 2018 1 1 Background. Pregnancy-related low back pain and pelvic girdle pain (LBP/PGP) are common and negatively impact the lives of many pregnant women. Several patient-based outcome instruments measure treatment effect but there is no consensus about which measure to use with women who have these pain presentations. Objective. The objective was to compare the responsiveness of 3 outcome measures in LBP/PGP: Oswestry Disability Index-version 2.0 (ODI), Pelvic Girdle Questionnaire (PGQ), and 0-10 numerical rating scale for pain severity (NRS); and to estimate a minimal important change (MIC) for these measures in pregnancy-related LBP/PGP. Design. This was a methodology study using data from a pilot randomised trial (RCT). Methods. Women (n = 124) with pregnancy-related LBP/PGP were recruited to a pilot RCT evaluating the benefit of adding acupuncture to standard care and 90 completed 8-weeks follow-up. Responsiveness was evaluated by examining correlation between change score and the external anchor (6-point global perceived effect scale) and by using receiver operating characteristic (ROC) curve analysis. MIC was estimated using anchor-based methods. Results. All measures showed good responsiveness, with areas under ROC curve ranging from 0.77 to 0.90. The estimated MICs were 3.1, 11.0, 9.4, 13.3, and 1.3 for ODI, PGQ-total, PGQ-activity, PGQ- symptoms, and NRS, respectively. All the measures, apart from ODI, had MICs larger than the measurement error. Limitations. Lack of optimal “gold standard” or external criterion for assessing responsiveness and MIC was a limitation of this study. Limitations. Lack of optimal “gold standard” or external criterion for assessing responsiveness and MIC was a limitation of this study. Conclusion. All 3 outcome measures demonstrated good responsiveness. MICs were derived for each instrument. The PGQ at 8 weeks post-randomisation was identified as an appropriate outcome measure for pregnancy-related LBP/PGP since it is specific to these pain presentations and assesses both activity limitations and symptoms. The NRS is an efficient, shorter alternative. 1 This can be evaluated by determining the minimal important change (MIC), defined as the minimal amount of change that is important to the patient.11,12 Analysing the responsiveness and MICs of an outcome measure is a continuous process that is strongly recommended to strengthen its properties and expand its applicability.13 3 Since two main aims of treatment for pregnancy-related LBP and PGP are to relieve pain and to improve functional ability, we included PROMs that are frequently used to capture pain and impact of pain on everyday activities (measured using both the Oswestry Disability Index and the Pelvic Girdle Questionnaire (PGQ). The responsiveness for the ODI and NRS has been established for the non- specific LBP population, but not in pregnancy-related LBP or PGP. The Pelvic Girdle Questionnaire (PGQ) is a recent outcome measure developed for both pregnant and postpartum women and its responsiveness and MIC have been established for women in late pregnancy and transitioning to postpartum,14 however, no previous study has established the properties of this instrument entirely during the course of pregnancy within a trial context. The previous study14 recruited women in the last trimester of their pregnancy and within 3 months after delivery and therefore their assessment of the measurement properties are more relevant to the context of natural history or epidemiological cohort studies rather than clinical trials of treatment for LBP and PGP during pregnancy. In order to aid decision-making about outcome measures for use in clinical trials of treatment effectiveness for pregnancy-related LBP and PGP, it is important to determine the responsiveness and MICs of the PROMs used to capture pain and impact of pain in women seeking treatment. This can be best done by choosing a population of women seeking treatment for pregnancy-related LBP and PGP and a time-point during pregnancy in the evaluation of responsiveness and MICs. The purpose of this study was twofold: (1) to compare the responsiveness of three PROMs during pregnancy in patients with pregnancy-related LBP and PGP: the Oswestry Disability Index-version 2.0 (ODI), the Pelvic Girdle Questionnaire (PGQ), and a 0-10 numerical rating scale (NRS) for pain severity, and (2) to estimate the optimal cut-off values for a MIC for these measures with patients who have pregnancy-related LBP and PGP. 1 2 Pregnancy-related low back pain (LBP) and pelvic girdle pain (PGP) are common conditions1,2 and impact, both socially and economically, the daily lives of many pregnant women.3 The incidence and point prevalence of pregnancy-related LBP and PGP reported in literature vary, partly because there is a variation in the criteria employed by various studies for the diagnosis of these conditions and the designs of the studies, but estimates range from 24% to 90% of women experiencing LBP or PGP at some stage during their pregnancy.1,4-6 The two pain presentations may occur separately or together and typically increase with advancing pregnancy.2 Treatment of pregnancy-related LBP and PGP is often aimed at reducing the pain and improving function. Therefore, assessing whether pain or function has changed over time is a vital objective of measurements in clinical practice and health research. Outcome measures for LBP and for the disability caused by LBP have become important standards for evaluation of interventions due to their frequent use in the assessment of the effectiveness of treatments.7 There have been several consensus developing initiatives to agree on core outcome domains and core outcome measures for non-specific LBP,7,8 however, there is a lack of consensus on what measure to use for pregnancy-related LBP and PGP as outcome measures that have been validated for non-specific LBP are not necessarily the most appropriate for pregnancy-related LBP and PGP.9 There is therefore a need for a direct comparison between LBP outcome measures in order to inform the choice of the most appropriate outcome measure to use in pregnancy-related LBP and PGP. An important measurement property of a patient reported outcome measure (PROM) is its ability to detect clinical change over time. This can be evaluated by determining the responsiveness of the PROM, defined as the ability of an instrument to detect change over time in the construct to be measured.10 A related question of interest is whether the observed changes within patients are clinically relevant. [H2]Study participants This study used the data from a three-arm parallel pilot randomised controlled trial (RCT) that assessed the feasibility of a future large RCT testing the additional benefit of acupuncture to standard 4 4 4 care for pregnancy-related LBP and PGP– the EASE Back pilot trial15 (trial registration ISRCTN49955124). Full details of the EASE Back pilot trial are available elsewhere,15,16 including the full details of the definitions of LBP and PGP we used. Here we give brief details. The EASE Back trial was funded following a commissioned call by the UK’s National Institute for Health Research (NIHR) Health Technology Assessment programme, focused on pregnancy-related LBP with or without PGP. Women aged 18 years and over were included if they had pregnancy-related LBP defined as self-reported pain in the lumbar area (between the 12th rib and the gluteal fold) with or without PGP (defined by pain between the posterior iliac crest and the gluteal fold, particularly in the vicinity of the sacro-iliac joints), were under the care of participating NHS sites and GP practices, at 13 to 31 weeks gestation, were naïve to acupuncture treatment, able to read and communicate in English and were willing to participate. Women who had LBP episodes before this pregnancy were included as long as the current episode of LBP was either attributed to, or made worse as a result of, this pregnancy. Women were excluded if they had previously had any form of acupuncture, were at high risk of miscarriage or pre-term labour, had pre-eclampsia, previous history of surgery to the spine or pelvis, and had contra-indications to the treatments, pain in the anterior pelvic region only, or a current urinary tract infection. Eligible women who gave written informed consent to participate were randomised in a 1:1:1 ratio, to one of the three treatment arms: standard care alone (comprehensive self-management booklet and an option to access EASE Back physiotherapy care if needed); standard care plus a course of true acupuncture; and standard care plus a course of non-penetrating acupuncture, delivered by physiotherapists. Ethical approval for the EASE Back pilot trial was granted by NRES Committee West Midlands - Staffordshire (reference 13/WM/00). [H2]Sample size [H2]Outcome measures The EASE Back pilot trial did not have a primary clinical outcome. Instead, two outcome measures capturing data on physical function and one instrument of pain severity were included in the pilot trial, in order to help inform the decision about a primary outcome measure for a future main trial. Several factors were taken into consideration in examining the performance of these measures. These included the amount of missing data at the item and scale levels; precision of the outcome measures; any evidence of floor or ceiling effects; their responsiveness to change; and MICs. Evaluation of these factors is the focus of this paper. The PROMs used included: [H2]Sample size In line with pilot trial recommendations17 no formal sample size calculation was conducted for the EASE Back pilot trial for the key clinical outcomes. However, we aimed to recruit a large number of patients to obtain an acceptable number of patients for the responsiveness and MIC analysis based on the guidelines for sample sizes in studies on validity and responsiveness.13 A total of 124 pregnant 5 5 5 women were randomised and provided baseline data, while 90 also provided data on the three outcome measures at 8 weeks follow-up. [H2]Data collection At baseline, patients completed a questionnaire containing sociodemographic data and PROMs. At 8 weeks post-randomisation, patients completed the same outcome measures used at baseline assessment and a patient’s Global Perceived Effect (GPE) scale for change of the pregnancy-related LBP/PGP and disability measured using a 6-point ordinal scale: (1) completely recovered, (2) much improved, (3) somewhat improved, (4) same, (5) somewhat worse, and (6) much worse. The decision to collect outcome data at 8 weeks post-randomisation was timed to ensure that most women would not yet have given birth to their baby. [H3]Numerical rating scale (NRS) for pain The NRS asks patients to rate their pain severity on an 11-point scale where 0 indicates no pain and 10 indicates worst imaginable pain. Pain severity was measured using the mean of three 0 to 10 NRS for least, usual and current pain over the previous 2 weeks.23 [H3]Oswestry disability index (ODI), version 2.0 The ODI18,19 is a self-administered questionnaire containing 10 sections, each containing six statements that are scored from 0 to 5, with 0 representing no difficulty in the activity and 5 representing maximal difficulty. The scores from each section are totalled and divided by the total possible score to obtain a final percentage of disability, with a higher percent indicating greater disability. Decreasing scores for the ODI over time denote improvement. The ODI is a valid, reliable, and responsive clinical tool for analysing disability status in individuals with LBP.20 6 6 The PGQ is a validated tool that captures self-reported pain and disability specifically for pregnant and post-partum women.21,22 It has two subscales capturing activity limitations and symptoms. Items are scored on a 4-point descriptive scale, and item scores are summed and transformed to yield a score of 0 to 100, where 100 is the worst possible score. The PGQ has been shown to have acceptable responsiveness in women with PGP, LBP, or both.14 The PGQ is a validated tool that captures self-reported pain and disability specifically for pregnant and post-partum women.21,22 It has two subscales capturing activity limitations and symptoms. Items are scored on a 4-point descriptive scale, and item scores are summed and transformed to yield a score of 0 to 100, where 100 is the worst possible score. The PGQ has been shown to have acceptable responsiveness in women with PGP, LBP, or both.14 [H2]Responsiveness Due to variations in method of assessing responsiveness, we adhere to the COnsensus based Standards for the selection of health Measurement Instruments (COSMIN)10 recommendations in this paper. First, we determined the correlation between the change score and the rank of the self-rated GPE scale using Spearman’s coefficient, where we hypothesised that there would be moderate-to-high correlations between change score and the GPE scale if they measure the same construct. Second, we used a receiver operating characteristic (ROC) curve analysis to plot the sensitivity versus 1-specificity for multiple cut-off points of the outcome measures against the external criterion. In the absence of a gold standard, the GPE scale, dichotomised into “improved” (combining categories 1 and 2) and not improved (combining categories 3-6) was used as the external criterion since this categorisation has been recommended as the optimal cut-off.29 This measure, despite criticisms that it is a retrospective measure of change30 and is influenced by current status,31 has been shown to provide reliable assessments of health transition in people with musculoskeletal pain.31 An area under the ROC curve (AUC) was used to assess the ability of the instruments to discriminate between patients who are considered to be improved and patients who are not considered to be improved according to the external criterion.32 [H2]Analysis All analyses were performed using Stata version-14.24 For a better comparison of the measures with differing scoring intervals, all scales were transformed to cover an interval ranging from 0–100. Change in scores was obtained by subtracting the 8 week follow-up scores from the baseline scores. Descriptive statistics (mean and standard deviations (SDs) or frequencies and percentages, as appropriate) were used to summarise the characteristics of participants who returned questionnaires at baseline and those who returned both baseline and follow-up questionnaires and to summarise the distribution of the change scores for the different outcomes according to whether the patients were classified as improved. Box plots were used to visualise the distribution of change score by the categories of GPE scale. The proportion of missing data at the item and scale level was computed. Since the percentage of missing (or not applicable) data for the questionnaire items was very minimal, the scores were recalculated as a percentage of the remaining items whenever an item was missing as recommended by the questionnaire developers; hence all the participants followed up had a score on all the instruments. Floor and ceiling effects were considered present if more than 15% of respondents achieved the lowest or highest possible score, respectively.25 Since the percentage of missing (or not applicable) data for the questionnaire items was very minimal, the scores were recalculated as a percentage of the remaining items whenever an item was missing as recommended by the questionnaire developers; hence all the participants followed up had a score on all the instruments. Floor and ceiling effects were considered present if more than 15% of respondents achieved the lowest or highest possible score, respectively.25 7 The precision of the measures was evaluated using standard error of measurement (SEM),26,27 calculated as SDඥ(1 −𝑟𝑟)28, where SD is the standard deviation of the baseline scores and r is the test-retest reliability coefficient obtained from a previous similar study.22 The minimum detectable 7 change (MDC), defined as the lowest change, for an individual, that exceeds measurement error and noise at a 95% confidence level was calculated as 1.96√2SEM=2.77*SEM.28 [H2]MIC The MIC was assessed using the anchor based approach to give an indication of the importance of the observed change by participating women. We used the ROC curve analysis where each instrument was considered as the diagnostic test, and the anchor as the gold standard, to distinguish patients with important improvement from patients with no important change.33 We calculated sensitivity and specificity for each possible cut-off value of the change score and obtained an ROC curve. The MIC was defined as the optimal ROC cut-off point, that is, the change in score that is associated with the smallest amount of misclassification. 8 8 [H2]ROLE OF THE FUNDING SOURCE: The EASE Back study was funded by the National Institute for Health Research (NIHR) Health Technology Assessment Programme (HTA) (grant ref. no. 10/69/05) and supported by an NIHR Research Professorship to Professor Foster (NIHR-RP-011- 015), who is an NIHR Senior Investigator. The funders played no role in the conduct, analysis, or reporting of this study, and the views expressed are those of the authors and not necessarily those of the National Health Service, the NIHR, or the UK Department of Health. 9 [H2]Baseline characteristics A total of 124 pregnant women (mean (SD) age 28 (5.3) years) were randomised and provided baseline data and 90 provided follow-up data at 8 weeks for all 3 measures. Characteristics of these participants have previously been reported.15 As described in the report, a comparison of key baseline characteristics (age and index of multiple deprivations) captured from the anonymised data on women who were ineligible or declined participation with the EASE Back trial participants showed that there was reasonable overall comparability in these key baseline characteristics, indicating that our study sample was generally representative. Only 14% of participants had given birth by the time they returned the 8-weeks follow-up questionnaire. Table 1 shows the characteristics of the participants who completed questionnaires at baseline and those who provided data at both baseline and at 8-weeks follow-up. The subset of patients at 8-weeks follow-up was similar to the baseline sample in age, gestation week, pain location, pain duration, pain severity, and disability scores, but had a higher proportion of patients in full/part-time jobs and a slightly higher proportion of patients who were married. 1 The percentage is for the item missing data, for example, 35 items were missing out of a total of 1240 ODI scale items for the 124 participants. Similarly, there were 191 missing items (6.2%) out of possible 3100 items (25 x 124) for PGQ. [H2]Responsiveness The Spearman’s correlation coefficient between the change score and the GPE scale for each measure was moderate to high ranging from 0.59 for PGQ activity subscale to 0.77 for pain severity (Tab. 3). All the measures showed good responsiveness (Fig. 2), with AUCs ranging between 0.77 and 0.90, indicating that all these measures are responsive over time in this patient population. Pain severity and the PGQ-symptoms subscale showed higher AUC compared to other measures. Exploratory subgroup analyses of the correlations by trial arms (Supplementary Tab. 1) and gestational weeks (Supplementary Tab. 2) showed no major differences between the study arms (especially between the standard care and true acupuncture) or gestational weeks, showing that randomisation and gestational week had no major effect on the measurement properties. [H2]Change score from baseline [H2]Change score from baseline Table 2 shows the mean (SD) change score for improved and unimproved patients for all the three outcome measures. Table 2 shows the mean (SD) change score for improved and unimproved patients for all the three outcome measures. Figure 1 shows the box plots of change scores for categories of GPE. Patients who reported complete recovery or much improved showed the highest change in all the measures while those who reported worsening showed deterioration on all the measures. Hence, change scores on all the measures corresponded to the magnitude of change as rated by patients on the GPE. [H2]Missing data, floor and ceiling effect There were no missing data for pain severity, but minimal amounts of missing data for the ODI and PGQ. The proportion of the questionnaire items with missing data for all participants at baseline was 2.8% for ODI1 and 6.2% for the PGQ. At follow-up the proportion was 7.8% and 8.7% for the ODI and PGQ, respectively. No floor or ceiling effects were shown in any of the measures– the total score for ODI ranged from 10% (scored by n=1) to 66% (scored by n=1) while the total PGQ score ranged from 13.6 (scored by n=1) to 85.3 (scored by n=1). 10 be at least higher than the SEM), we observe that the obtained MICs were all larger than the SEM except for the ODI. [H2]SEM, MDC and MIC estimates Table 4 presents the SEM, MDC95 values, and the MIC identified by the optimal cut-off change in the ROC analysis together with the sensitivity and specificity at the cut-off for each outcome measure. The MIC ranged from 3.1 for ODI to 13.3 for pain severity (0–100 scale). When we relate the calculated MICs to the imprecision of the outcome measures assessed by the SEM (as we would want the MIC to 11 be at least higher than the SEM), we observe that the obtained MICs were all larger than the SEM except for the ODI. be at least higher than the SEM), we observe that the obtained MICs were all larger than the SEM except for the ODI. be at least higher than the SEM), we observe that the obtained MICs were all larger than the SEM except for the ODI. 12 [H2]Summary of findings This study provided a head-to-head comparison of the responsiveness of three PROMs, a 0-10 NRS for pain severity, the ODI and PGQ, and estimated the MIC for each. We found that all the three outcome measures had acceptable responsiveness based on a moderate-to-high correlation with the anchor and an AUC of more than 0.7, with NRS having the highest responsiveness, followed by the PGQ- symptoms subscale. Missing data in the three outcome measures was very minimal. No floor or ceiling effects were observed, indicating that the PROMs may be able to distinguish patients with the lowest or highest possible score. Our study also estimated the MICs for the ODI, PGQ-total score, PGQ-activity subscale, PGQ- symptom subscale and 0-10 NRS pain severity as 3, 11, 9, 13 and 1.3 points, respectively. This indicates that when used at individual level, a change score smaller than these MIC values should be regarded as insignificant and only a change above these values can be considered as ‘‘clinically relevant’’ change. However, it’s worth noting that the obtained MIC for the ODI was almost the same as the SEM value indicating that for this measure there might be a risk that an obtained MIC is an expression of the imprecision of the instrument, and not a true change. [H2]Comparison with previous studies [H2]Comparison with previous studies Previous studies have compared the responsiveness of PROMs measuring activity limitations in people with non-specific LBP,20,34 however, to the best of our knowledge, only one previous study14 has evaluated the responsiveness and MICs of these questionnaires in pregnant women who report PGP. Key differences between that previous study14 and our study include: (1) study design – the previous study was a prospective cohort of women recruited from maternity centres, compared to our randomised trial comparing effectiveness of treatments in women seeking treatment for their pregnancy-related LBP and PGP; (2) study population – our study recruited women seeking treatment for pregnancy-related LBP and PGP who were between 13 to 31 weeks pregnant whereas Stuge et al’s14 study recruited a consecutive sample of women from maternity centres in the last trimester of 13 pregnancy whose status was changing from pregnant to post-partum, irrespective of whether they had pain or were seeking treatment for it; and (3) timing of outcome assessment – our follow-up time- point was deliberately chosen to capture change in pain and function whilst women were still pregnant, whereas Stuge et al’s14 follow-up time-point was post-partum. Hence our study addresses a different question than the previous study,14 as we focused on the responsiveness and MIC of the three outcome measures in women seeking treatment for pregnancy-related LBP and PGP, we included a different population of women and used a time-point during pregnancy in our evaluation of responsiveness and MIC. The latter point is important given the relevance to future clinical trials of treatment effectiveness of interventions for pregnancy-related LBP and PGP. Estimates of MIC have previously been reported for the ODI in non-specific LBP patients29,35 and for the ODI and PGQ in pregnant women transitioning to the post-partum period.14 We found the MIC for the ODI to be 3.1 points. This value is lower than the MIC values previously reported in the non- specific LBP literature, where MICs of at least 10 points have been recommended.29 This suggests that the ODI might not be a sensitive measure of activity limitations among pregnant women. Our MIC values for the PGQ and its subscales were also lower than the ones found for women proceeding from being pregnant to post-partum14. [H2]Implications [H2]Implications Despite having many outcome measures with which to assess non-specific LBP, currently there is no measure specifically for pregnancy-related LBP and PGP so comparing three measures and recommending the most appropriate one provides guidance for the future assessment of these pain presentations. The MIC is not a fixed attribute and usually fluctuates based on what is interpreted as important to the patient,26 therefore, it is important to derive these values from the population of interest. Condition specific outcomes have been shown to have several advantages as they are intended to have very relevant content for the specific problem in that population and are therefore more likely to detect important changes that occur over time.39 Our findings provide evidence regarding which outcome measures to recommend for assessing pain and function in women with pregnancy-related LBP and PGP. All the three measures showed acceptable responsiveness in our study. However, the MIC for the ODI was of similar magnitude to the measurement error thus questioning its use. [H2]Comparison with previous studies These differences in the MIC values may be explained by the fact that MIC cannot be considered a fixed property of an outcome measure36 as it depends on the setting of the study in which the measure is used. Hence for our study population, an important change for pregnant women is not necessarily the same as an important change for the women as they transition from being pregnant to after giving birth. For pregnant women, activity limitation is often accepted as a ‘normal’ part of pregnancy, therefore, even a small improvement can be clinically meaningful to them, this may explain the lower MIC values obtained in our study. The MIC value for pain severity measured by the NRS in non-specific LBP has also been previously reported in the literature to vary widely from 0.7 to 4 points.29,37,38 However, there are no studies that have evaluated the MIC for the NRS for pain severity specifically in pregnant women, so we cannot make a direct comparison. Our MIC value for NRS pain severity of 1.3 points falls within the range for the general non-specific LBP population suggested by other authors.29,37 14 [H2]Future research Since the number of patients who reported being unchanged was small, we relied on the reliability parameters from a previous study to calculate the SEM. A future test-retest study with a similar population should be carried out in order to confirm our findings. A future study with larger sample size could use other anchor based methods such as the mean change method based on patients who identify themselves to be ‘‘somewhat improved’’ on the patient-reported GPE scale. [H2]Strengths and limitations A limitation of this study, which also applies to nearly all the responsiveness and MIC studies,40,41 is the lack of a universally accepted methodology to determine responsiveness and MIC, which can result in a wide range of reported MIC values for a single outcome measure depending on the methodology chosen. However, we have adhered to the COSMIN recommendations which attempt to unify the methods for evaluating responsiveness and MIC. A further limitation of our study is the use of the GPE scale as the sole external criterion as there is no universally acceptable definition of “minimally important” using this anchor; hence our MICs would differ if a different anchor was chosen. However, it has been argued that for the GPE scale, the MIC is most appropriately defined in terms of at least “much improved” instead of including “somewhat improved,”'29 and this is the approach we took. Reassuringly, our results showed that change scores on all three outcome measures corresponded to the magnitude of change as perceived by patients. Further limitations of our study involve the inclusion of women reporting pain in areas consistent with a presentation of LBP, with or without PGP (as this was the population stipulated by the research funder), therefore, it is possible that the findings for the PGQ may be different than if we had included a sample of women who had PGP only; 15 however, there is no reliable method to distinguish clearly between these patient groups and in clinical practice as in this study, women present with both. [H1]Conclusion Our analysis, comparing the ODI, PGQ and pain NRS, showed that all three outcome measures were responsive to change in women with pregnancy-related LBP and PGP and hence are suitable for measuring the effectiveness of treatments; of the three tested, the NRS for pain severity was the most responsive. The MIC of the ODI was comparable to its imprecision, hence its utility may be questionable. We identified the PGQ at 8 weeks post-randomisation as an appropriate outcome measure for pregnancy-related LBP and PGP. The NRS for pain severity is an efficient, shorter alternative. CLINICAL TRIAL REGISTRATION This study used data from a pilot randomized clinical trial registered in the ISRCTN registry (ISRCTN49955124). This study used data from a pilot randomized clinical trial registered in the ISRCTN registry (ISRCTN49955124). AUTHOR CONTRIBUTIONS AND ACKNOWLEDGMENTS AUTHOR CONTRIBUTIONS AND ACKNOWLEDGMENTS The authors contributed to the manuscript as follows: conception and design: RO, AB, ML, MG, NEF; analysis and interpretation of the data: RO, AB, ML, MG, NEF; first drafting of the article: RO; revisions and final approval of the article: All. Concept/idea/research design: R. Ogollah, A. Bishop, M. Lewis, M. Grotle, N.E Writing: R. Ogollah, A. Bishop, M. Lewis, M. Grotle, N.E. Foster Data collection: A. Bishop, N.E. Foster Data analysis: R. Ogollah, A. Bishop, M. Grotle, N.E. Foster Project management: A. Bishop, M. Lewis Fund procurement: A. Bishop, N.E. Foster Consultation (including review of manuscript before submitting): M. Lewis The authors acknowledge funding for this study from the National Institute for Health Research (NIHR) Health Technology Assessment Programme (HTA), support from managers and clinicians in the University Hospital of the North Midlands (UHNM), the Staffordshire and Stoke-on-Trent Partnership NHS Trust; the CaRE for Women’s Health Network in the West Midlands of England, the NIHR Clinical Research Network (CRN), and participating clinicians and pregnant women. The authors also thank the research midwifery team at UHNM and the research nurse team within the CRN 16 for their help in identifying, recruiting, and conducting follow-up with women; the physical therapists who delivered the interventions; and the physical therapy administrators who organized EASE Back study appointments. for their help in identifying, recruiting, and conducting follow-up with women; the physical therapists who delivered the interventions; and the physical therapy administrators who organized EASE Back study appointments. ETHICS APPROVAL Ethics approval for the EASE Back pilot trial was granted by the National Research Ethics Service (NRES) Committee, West Midlands, Staffordshire (ref. no. 13/WM/00). FUNDING The EASE Back study was funded by the National Institute for Health Research (NIHR) Health Technology Assessment Programme (HTA) (grant ref. no. 10/69/05) and supported by an NIHR Research Professorship to Professor Foster (NIHR-RP-011-015), who is an NIHR Senior Investigator. References 1. Vermani E, Mittal R, Weeks A. Pelvic girdle pain and low back pain in pregnancy: a review. Pain Practice. 2010;10:60-71. 2. Liddle SD, Pennick V. Interventions for preventing and treating low‐back and pelvic pain during pregnancy. Cochrane Database Syst Rev. 2015;(9):CD001139. 3. Mogren I. Perceived health, sick leave, psychosocial situation, and sexual life in women with low-back pain and pelvic pain during pregnancy. Acta Obstetricia et Gynecologica Scandinavica. 2006;85:647-656. 4. Vleeming A, Albert H, Östgaard H, Sturesson B, Stuge B. European guidelines for the diagnosis and treatment of pelvic girdle pain. Eur Spine J. 2008;17:794-819. 5. Wu WH, Meijer OG, Uegaki K, et al. Pregnancy-related pelvic girdle pain (PPP), I: terminology, clinical presentation, and prevalence. Eur Spine J. 2004;13:575-589. 6. Pierce H, Homer CS, Dahlen HG, King J. Pregnancy-related lumbopelvic pain: listening to Australian women. Nurs Res Pract. 2012;2012:387428. 7. Chiarotto A, Deyo RA, Terwee CB, et al. Core outcome domains for clinical trials in non-specific low back pain. Eur Spine J. 2015;24:1127-1142. 8. Chiarotto A, Boers M, Deyo RA, et al. Core outcome measurement instruments for clinical trials in nonspecific low back pain. Pain. 2018;159:481-495. 9. Boissonnault JS. A review of self-report functional outcome measures in selected obstetric physical therapy interventions. J Womens Health Phys Therap. 2009;33:7-12. 10. Mokkink LB, Terwee CB, Patrick DL, et al. The COSMIN study reached international consensus on taxonomy, terminology, and definitions of measurement properties for health-related patient-reported outcomes. J Clin Epidemiol. 2010;63:737-745. 11. Jaeschke R, Singer J, Guyatt GH. Measurement of health status. Ascertaining the minimal clinically important difference. Control Clin Trials. 1989;10:407-415. 12. de Vet HC, Terwee CB, Ostelo RW, Beckerman H, Knol DL, Bouter LM. Minimal changes in health status questionnaires: distinction between minimally detectable change and minimally important change. Health Qual Life Outcomes. 2006;4:54. 13. de Vet HCW, Terwee CB, Mokkink LB, Knol DL. Measurement in Medicine: A Practical Guide: Cambridge University Press; 2011. 14. Stuge B, Jenssen HK, Grotle M. The pelvic girdle questionnaire: responsiveness and minimal important change in women with pregnancy-related pelvic girdle pain, low back pain, or both. Phys Ther. 2017;97:1103-1113. 15. Foster NE, Bishop A, Bartlam B, et al. Evaluating Acupuncture and Standard carE for pregnant women with Back pain (EASE Back): a feasibility study and pilot randomised trial. Health Technol Assess. 2016;20:1-236. 16. Bishop A, Ogollah R, Bartlam B, et al. DISCLOSURES AND PRESENTATIONS The authors completed the ICJME Form for Disclosure of Potential Conflicts of Interest and reported no conflicts of interest. The only publications arising from this work are in form of abstracts presented as posters at the International Back and Neck Pain Forum, Buxton, Derbyshire, UK, in June 2016. The full details of the EASE Back feasibility and pilot study have been published previously in an NIHR HTA monograph: Foster NE, Bishop A, Bartlam B, et al. Evaluating acupuncture and standard carE for pregnant women with back pain (EASE Back): a feasibility study and pilot randomized trial. Health Technol Assess. 2016;20:1-236. The authors completed the ICJME Form for Disclosure of Potential Conflicts of Interest and reported no conflicts of interest. The only publications arising from this work are in form of abstracts presented as posters at the International Back and Neck Pain Forum, Buxton, Derbyshire, UK, in June 2016. The full details of the EASE Back feasibility and pilot study have been published previously in an NIHR HTA monograph: Foster NE, Bishop A, Bartlam B, et al. Evaluating acupuncture and standard carE for pregnant women with back pain (EASE Back): a feasibility study and pilot randomized trial. Health Technol Assess. 2016;20:1-236. 17 References Evaluating acupuncture and standard care for pregnant women with back pain: the EASE Back pilot randomised controlled trial (ISRCTN49955124). Pilot Feasibility Stud. 2016;2:72. 17. Arain M, Campbell MJ, Cooper CL, Lancaster GA. What is a pilot or feasibility study? A review of current practice and editorial policy. BMC Med Res Methodol. 2010;10:67. 18 18. Fairbank JC, Couper J, Davies JB, O'Brien JP. The Oswestry low back pain disability questionnaire. Physiotherapy. 1980;66:271-273. 19. Fairbank JC, Pynsent PB. The Oswestry Disability Index. Spine (Phila Pa 1976). 2000;25:2940-2952; discussion 2952. 20. Davidson M, Keating JL. A Comparison of five low back disability questionnaires: reliability and responsiveness. Phys Ther. 2002;82:8-24. 21. Stuge B, Garratt A, Krogstad Jenssen H, Grotle M. The pelvic girdle questionnaire: a condition-specific instrument for assessing activity limitations and symptoms in people with pelvic girdle pain. Phys Ther. 2011;91:1096-1108. 22. Grotle M, Garratt AM, Krogstad Jenssen H, Stuge B. Reliability and construct validity of self-report questionnaires for patients with pelvic girdle pain. Phys Ther. 2012;92:111-123. 23. Dunn KM, Jordan KP, Croft PR. Recall of medication use, self-care activities and pain intensity: a comparison of daily diaries and self-report questionnaires among low back pain patients. Prim Health Care Res Dev. 2010;11:93-102. 24. Stata Statistical Software: Release 14 [computer program]: College Station, TX: StataCorp LP; 2015. 25. Terwee CB, Bot SD, de Boer MR, et al. Quality criteria were proposed for measurement properties of health status questionnaires. J Clin Epidemiol. 2007;60:34-42. 26. Wyrwich KW, Tierney WM, Wolinsky FD. Further evidence supporting an SEM-based criterion for identifying meaningful intra-individual changes in health-related quality of life. J Clin Epidemiol. 1999;52:861-873. 27. Crosby RD, Kolotkin RL, Williams GR. Defining clinically meaningful change in health-related quality of life. J Clin Epidemiol. 2003;56:395-407. 28. de Vet HC, Terwee CB, Knol DL, Bouter LM. When to use agreement versus reliability measures. J Clin Epidemiol. 2006;59:1033-1039. 29. Ostelo RW, de Vet HC. Clinically important outcomes in low back pain. Best Pract Res Clin Rheumatol. 2005;19:593-607. 30. Norman GR, Stratford P, Regehr G. Methodological problems in the retrospective computation of responsiveness to change: the lesson of Cronbach. J Clin Epidemiol. 1997;50:869-879. 31. Kamper SJ, Ostelo RW, Knol DL, Maher CG, de Vet HC, Hancock MJ. Global perceived effect scales provided reliable assessments of health transition in people with musculoskeletal disorders, but ratings are strongly influenced by current status. J Clin Epidemiol. 2010;63:760-766 e761. 32. Deyo RA, Diehr P, Patrick DL. References Reproducibility and responsiveness of health status measures statistics and strategies for evaluation. Control Clin Trials. 1991;12:S142-S158. 33. Deyo RA, Centor RM. Assessing the responsiveness of functional scales to clinical change: an analogy to diagnostic test performance. J Chronic Dis. 1986;39:897-906. 34. van der Roer N, Ostelo RW, Bekkering GE, van Tulder MW, de Vet HC. Minimal clinically important change for pain intensity, functional status, and general health status in patients with nonspecific low back pain. Spine (Phila Pa 1976). 2006;31:578-582. 35. Ostelo RW, Deyo RA, Stratford P, et al. Interpreting change scores for pain and functional status in low back pain: towards international consensus regarding minimal important change. Spine (Phila Pa 1976). 2008;33:90-94. 19 36. Terwee CB, Dekker FW, Wiersinga WM, Prummel MF, Bossuyt PM. On assessing responsiveness of health-related quality of life instruments: guidelines for instrument evaluation. Qual Life Res. 2003;12:349-362. 37. Maughan EF, Lewis JS. Outcome measures in chronic low back pain. Eur Spine J. 2010;19:1484-1494. 38. Salaffi F, Stancati A, Silvestri CA, Ciapetti A, Grassi W. Minimal clinically important changes in chronic musculoskeletal pain intensity measured on a numerical rating scale. Eur J Pain. 2004;8:283-291. 39. Patrick DL, Deyo RA. Generic and disease-specific measures in assessing health status and quality of life. Med Care. 1989;27(3 Suppl):S217-S232. 40. de Vet HC, Ostelo RW, Terwee CB, et al. Minimally important change determined by a visual method integrating an anchor-based and a distribution-based approach. Qual Life Res. 2007;16:131-142. 41. Terwee CB, Roorda LD, Dekker J, et al. Mind the MIC: large variation among populations and methods. J Clin Epidemiol. 2010;63:524-534. References Table 1: Baseline Characteristics of Participants Who Returned Questionnaires at Baseline and Those Who Provided Data at Both Baseline and at 8-Weeks Follow-upa Baseline Characteristics Participants Randomized and Providing Baseline Data (n = 124) Participants Returning Complete 8-Weeks Follow-up Questionnaire on All 3 Outcome Measures (n = 90) Age (years), mean (SD) 28.3 (5.3) 29.2 (5.3) Highest qualification: degree/postgraduate, n (%) 45 (36.3) 40 (44.4) Gestation weeks at inclusion: 24+, n (%) 52 (41.9) 37 (41.1) Married, n (%) 54 (43.6) 46 (51.1) Working (full-time or part-time), n (%) 87 (70.2) 71 (78.9) Pre-pregnancy BMI, n (%) Normal/ underweight 47 (39.8) 37 (44.1) Overweight 36 (30.5) 27 (32.1) Obese/ morbidly obese 35 (29.7) 20 (23.8) Pain location (manikin)b, n (%) LBP only 23 (18.6) 17 (18.9) LBP with anterior PGP 18 (14.5) 13 (14.4) LBP with anterior PGP and pain elsewhere 36 (29.0) 22 (24.4) LBP and pain elsewhere 47 (37.9) 38 (42.2) Duration of episode: > 6 weeks, n (%) 68 (54.8) 46 (51.1) Pain severity (mean of 3 0 - 10 NRS), mean (SD) 4.6 (1.7) 4.3 (1.6) Oswestry Disability Index (0 - 100) c, mean (SD) 34.4 (12.9) 32.3 (11.7) Pelvic Girdle Questionnaire (0 - 100) d, mean (SD) Total 53.3 (17.0) 51.6 (17.1) /ptj/advance-article-abstract/doi/10.1093/ptj/pzz107/5544636 by University of Nottingham user on 15 August 2019 LBP only LBP and pain elsewhere Duration of episode: > 6 weeks, n (%) 20 Symptom subscale Hence, 44 (49%) were classified as improved and 46 (51%) as not improved. Key Outcomes Mean (SD) Change Score From Baseline Improved (n = 44)b Not Improved (n = 46) ODI score (0-100) 14.9 (12.2) -3.1 (15.5) PGQ (0-100) Total 21.8 (16.2) -0.8 (21.6) Activity subscale 20.1 (16.8) -1.6 (22.9) Symptom subscale 28.3 (19.2) 2.2 (21.0) Pain severity (mean of 3 NRS)b 29.9 (15.6) -2.8 (20.5) aODI = Oswestry Disability Index; PGQ = Pelvic Girdle Questionnaire; SD = standard deviation bAt 8 weeks follow-up, 6 participants (7%) reported complete recovery, 38 (42%) much improved, 17 (19%) somewhat improved, 7 (8%) the same, 11 (12%) somewhat worse and 11 (12%) much worse. Hence, 44 (49%) were classified as improved and 46 (51%) as not improved. Table 3: AUC (With 95% CI), The Spearman Correlation Coefficient Between the Change Score and the Rank of the Self-Rated GPE Scale for Each Measure (N=90) and the Median Change Score Amongst Patients Who Rated Themselves as “Somewhat Improved”a Key Outcomes Correlation with GPE Scaleb AUC (95% CI) Median (IQR) Change Score for the “Somewhat Improved” GPE Category (n=17) ODI score (0-100) -0.67 0.83 (0.74, 0.92) 8.0 (-6.2, 17.1) PGQ (0-100) Total -0.62 0.79 (0.70, 0.89) 11.0 (3.9, 29.2) Activity subscale -0.59 0.77 (0.67, 0.87) 8.8 (-1.5, 33.3) Symptom subscale -0.66 0.83 (0.74, 0.92) 13.3 (0.0, 20.0) Pain severity (mean of 3 NRS)b -0.77 0.90 (0.84, 0.97) 6.7 (0.0, 20.0) aODI = Oswestry Disability Index; PGQ = Pelvic Girdle Questionnaire; GPE = Global Perceived Effect; IQR = interquartile range; AUC = area under the curve; CI = confidence interval bThe negative sign for the correlation coefficient indicates that an increase in change score (improvement) for each of the measures is .com/ptj/advance-article-abstract/doi/10.1093/ptj/pzz107/5544636 by University of Nottingham user on 15 August 2019 Key Outcomes Mean (SD) Change Score From Baseline Improved (n = 44)b Not Improved (n = 46) ODI score (0-100) 14.9 (12.2) -3.1 (15.5) PGQ (0-100) Total 21.8 (16.2) -0.8 (21.6) Activity subscale 20.1 (16.8) -1.6 (22.9) Symptom subscale 28.3 (19.2) 2.2 (21.0) Pain severity (mean of 3 NRS)b 29.9 (15.6) -2.8 (20.5) aODI = Oswestry Disability Index; PGQ = Pelvic Girdle Questionnaire; SD = standard deviation bAt 8 weeks follow-up, 6 participants (7%) reported complete recovery, 38 (42%) much improved, 17 (19%) somewhat improved, 7 (8%) the same, 11 (12%) somewhat worse and 11 (12%) much worse. Symptom subscale aLBP = low back pain; BMI = body mass index; PGP = pelvic girdle pain; SD = standard deviation b th aLBP = low back pain; BMI = body mass index; PGP = pelvic girdle pain; SD = standard deviation bLBP was defined as self-reported pain in the lumbar spine area (between the 12th rib and the gluteal fold); PGP was defined by pain between the posterior iliac crest and the gluteal fold, particularly in the vicinity of the sacroiliac joints; Pain elsewhere was pain not in the areas described by the definitions of LBP or PGP e.g. pain in the thoracic spine, pain in the shoulder, etc. cOswestry Disability Index- has 10 sections with scores ranging from 0 to 5 in each section, item scores are summed and transformed to yield a score of 0 to 100 where 100 is the worst possible score. dPelvic Girdle Questionnaire- items are scored on a 4-point scale and item scores are summed and transformed to yield a score of 0 to 100 where 100 is the worst possible score. stionnaire- items are scored on a 4-point scale and item scores are summed and transformed to yield a score of 0 to he worst possible score. Table 2: Mean Change Scores (SD) for the 3 Outcome Measures According to Whether They Were Classified as Improved or Not Based on Their Answer on the GPE Scale.a Table 2: Mean Change Scores (SD) for the 3 Outcome Measures According to Whether They Were Classified as Improved or Not Based on Their Answer on the GPE Scale.a Table 2: Mean Change Scores (SD) for the 3 Outcome Measures According to Whether They Were Classified as Improved or Not Based on Their Answer on the GPE Scale.a Key Outcomes Mean (SD) Change Score From Baseline Improved (n = 44)b Not Improved (n = 46) ODI score (0-100) 14.9 (12.2) -3.1 (15.5) PGQ (0-100) Total 21.8 (16.2) -0.8 (21.6) Activity subscale 20.1 (16.8) -1.6 (22.9) Symptom subscale 28.3 (19.2) 2.2 (21.0) Pain severity (mean of 3 NRS)b 29.9 (15.6) -2.8 (20.5) aODI = Oswestry Disability Index; PGQ = Pelvic Girdle Questionnaire; SD = standard deviation bAt 8 weeks follow-up, 6 participants (7%) reported complete recovery, 38 (42%) much improved, 17 (19%) somewhat improved, 7 (8%) the same, 11 (12%) somewhat worse and 11 (12%) much worse. area under the curve; CI confidence interval bThe negative sign for the correlation coefficient indicates that an increase in change score (improvement) for each of the measures is associated with lower rating (better improvement) on the GPE scale. Symptom subscale Hence, 44 (49%) were classified as improved and 46 (51%) as not improved. Table 3: AUC (With 95% CI), The Spearman Correlation Coefficient Between the Change Score and the Rank of the Self-Rated GPE Scale for Each Measure (N=90) and the Median Change Score Amongst Patients Who Rated Themselves as “Somewhat Improved”a Key Outcomes Correlation with GPE Scaleb AUC (95% CI) Median (IQR) Change Score for the “Somewhat Improved” GPE Category (n=17) ODI score (0-100) -0.67 0.83 (0.74, 0.92) 8.0 (-6.2, 17.1) PGQ (0-100) Total -0.62 0.79 (0.70, 0.89) 11.0 (3.9, 29.2) Activity subscale -0.59 0.77 (0.67, 0.87) 8.8 (-1.5, 33.3) Symptom subscale -0.66 0.83 (0.74, 0.92) 13.3 (0.0, 20.0) Pain severity (mean of 3 NRS)b -0.77 0.90 (0.84, 0.97) 6.7 (0.0, 20.0) aODI = Oswestry Disability Index; PGQ = Pelvic Girdle Questionnaire; GPE = Global Perceived Effect; IQR = interquartile range; AUC = area under the curve; CI = confidence interval bThe negative sign for the correlation coefficient indicates that an increase in change score (improvement) for each of the measures is associated with lower rating (better improvement) on the GPE scale. 36 by University of Nottingham user on 15 August 2019 21 Table 4: Measurement Error, MDC, and MIC Values for Each of the Three Outcome Measures Key Outcome Measures MIC ROC SEM MDCb AUC (95% CI) Optimal Cut-Point Sensitivity and Specificity at Cut-Point ODI score (0-100) 0.83 (0.74, 0.92) 3.1 0.89; 0.74 3.1 8.68 PGQ (0-100) Total 0.80 (0.70, 0.89) 11.0 0.77; 0.74 4.5 12.46 Activity subscale 0.77 (0.67, 0.87) 9.4 0.77; 0.72 4.7 13.12 Symptom subscale 0.83 (0.74, 0.92) 13.3 0.84; 0.72 5.5 15.29 Pain severity (mean of 3 NRS) c 0.90 (0.84, 0.97) 13.3 0.87; 0.85 5.2 20.44 aAUC = area under the curve; CI = confidence interval; MIC = minimal important change; MDC = minimum detectable change ODI = Oswestry Disability Index; PGQ = Pelvic Girdle Questionnaire; ROC = receiver operating characteristic; SEM = standard error of measurement. bThe MDC95 represents the smallest change score that could be said to represent a real change beyond measurement error with 95% confidence in one individual. For example, the MDC95 value for ODI indicates that in 95% of the cases, patients will have experienced real change (beyond measurement error) if their score has changed by at least 8.68 points on the ODI. cThe NRS has been transformed to 0-100 for comparability with other outcome measures. Symptom subscale For SEM computation, a reliability of 0.94 was used for ODI, 0.9 for pain severity, 0.93 for PGQ total, 0.93 for PGQ-activity and 0.91 for PGQ-symptoms subscales obtained from a previous validation study. asurement Error, MDC, and MIC Values for Each of the Three Outcome Measures Downloaded from https://academic.oup.com/ptj/advance-article-abstract/doi/10.1093/ptj/pzz easu e e t. bThe MDC95 represents the smallest change score that could be said to represent a real change beyond measurement error with 95% confidence in one individual. For example, the MDC95 value for ODI indicates that in 95% of the cases, patients will have experienced real change (beyond measurement error) if their score has changed by at least 8.68 points on the ODI. cThe NRS has been transformed to 0-100 for comparability with other outcome measures. For SEM computation, a reliability of 0.94 was used for ODI, 0.9 for pain severity, 0.93 for PGQ total, 0.93 for PGQ-activity and 0.91 for PGQ-symptoms subscales obtained from a previous validation study. cThe NRS has been transformed to 0-100 for comparability with other outcome measures. For SEM computation, a reliability of 0.94 was used for ODI, 0.9 for pain severity, 0.93 for PGQ total, 0.93 for PGQ-activity and 0.91 for PGQ-symptoms subscales obtained from a previous validation study. 22 Figure Legend Figure 1: Box plots of the distribution of change scores (baseline minus follow-up) for the different outcome measures in relation to categories of GPE scale. ODI = Oswestry Disability Index; PGQ = Pelvic Girdle Questionnaire. Downloaded from https://academic.oup.com/ptj/advance-article-abstract/doi/10.1093/ptj/pzz1 cle-abstract/doi/10.1093/ptj/pzz107/5544636 by University of Nottingham user on 15 August 2019 23 Figure 2: The receiver operating characteristic (ROC) curves of key outcome measures against global perceived effect. AUC = area under the curve; ODI = Oswestry Disability Index; PGQ = Pelvic Girdle Questionnaire. Downloaded from https://academic.oup.com/ptj/advance-article-abstract/doi/10.1093/ptj/pz Downloaded from https://academic.oup.com/ptj/advance-article-abstract/doi/10.1093/ptj/pzz Downloaded from https://academic.oup.com/ptj/advance-article-abstract/doi/10.1093/ptj/pzz1 24
https://openalex.org/W2775167938	https://www.frontiersin.org/articles/10.3389/fimmu.2017.01745/pdf	English	null	Aging, Obesity, and Inflammatory Age-Related Diseases	Frontiers in immunology	2,017	cc-by	10,029	Aging, Obesity, and inflammatory Age-Related Diseases The increase in the prevalence of obesity represents a worldwide phenomenon in all age groups and is pathologically and genetically correlated with several metabolic and cardiovascular diseases, representing the most frequent age-related diseases. Obesity superimposed on aging drastically increases chronic low-grade inflammation (inflammag- ing), which is an important link between obesity, insulin resistance, and age-associated diseases. Immune cells of both the innate and the adaptive immune systems infiltrate the adipose tissue (AT) and during obesity induce inflammatory responses associated with metabolic switches and changes in phenotypes and function of immune cell subsets. Obesity poses new health problems especially when it occurs in the context of other diseases, many of them frequently affect elderly subjects. An emerging problem is the decreased proportion of patients with obesity achieving clinical response to therapy. In this review, we will discuss the reciprocal influences of immune cell and AT inflamma- tion in aging and age-associated diseases and the complex relationship of nutrient and energy-sensing homeostatic checkpoints, which contribute to shape the phenotype of the AT. We will specifically examine type-2 diabetes, rheumatoid arthritis, osteoarthritis, cognitive impairment, and dementia, where obesity plays a significant role, also in shap- ing some clinical aspects. Mini Review Mini Review published: 07 December 2017 doi: 10.3389/fimmu.2017.01745 Edited by: Valquiria Bueno, Federal University of São Paulo, Brazil Reviewed by: Oreste Gualillo, Servicio Gallego de Salud, Spain Pasquale Maffia, University of Glasgow, United Kingdom Keywords: aging, obesity, inflammation, type-2 diabetes, rheumatoid arthritis Keywords: aging, obesity, inflammation, type-2 diabetes, rheumatoid arthritis *Correspondence: Daniela Frasca dfrasca@med.miami.edu INTRODUCTION The increase in prevalence of overweight and obesity represents a worldwide phenomenon, which is associated with several chronic diseases such as type-2 diabetes (T2D), cancer, rheumatoid arthritis and osteoarthritis (OA), cognitive impairment and dementia, and those affecting the cardiovascular (CV) system.hf Specialty section: This article was submitted to Inflammation, a section of the journal Frontiers in Immunology Received: 24 October 2017 Accepted: 23 November 2017 Published: 07 December 2017 Citation: Frasca D, Blomberg BB and Paganelli R (2017) Aging, Obesity, and Inflammatory Age- Related Diseases. Front. Immunol. 8:1745. doi: 10.3389/fimmu.2017.01745 Specialty section: This article was submitted to Inflammation, a section of the journal Frontiers in Immunology The global obesity pandemic affects all age groups. Recent studies examining body mass index (BMI) data in 68 million people in 195 countries showed both increased prevalence and disease burden of high BMI subjects globally over the past 20 years (1). Although the prevalence of obesity among children is lower than in adults, its rate of increase exceeds that of adults (2). The global burden of disease related to high BMI is calculated in individuals without underlying conditions, and it increases at a slower pace in adults mainly because of the reduction of other risk factors for CV diseases and for effective clinical intervention. However, increased BMI has been shown to be pathogenetically related to several diseases. Among these, insulin resistance (IR) and T2D have a strong link to obesity, and the metabolic syndrome represents a cluster of risk factors for severe CV events (coronary artery disease, stroke). Obesity superimposed on aging represents an additional risk factor for older age groups in which the prevalence of chronic disease as well Received: 24 October 2017 Accepted: 23 November 2017 Published: 07 December 2017 Immune Cell Infiltration in the Obese AT Immune Cell Infiltration in the Obese AT Data from obese mice and humans have indicated that the hypertrophied AT becomes heavily infiltrated by a variety of immune cells displaying a pro-inflammatory phenotype, char- acterized by secretion of SASP markers (32), and their numbers inversely correlate with insulin sensitivity. Cells with an anti- inflammatory phenotype have also been reported in the obese AT, but these cells are present at low frequencies. These are B1 B cells producing IL-10 (15, 33) and innate lymphoid cells type 2, which produce large amounts of Th2 cytokines such as IL-4, IL-5, and IL-13 (34). Tregs have also been reported but only in the lean AT (35).i Production of Pro-inflammatory Mediators in the Obese AT Mediators in the Obese AT High levels of TNF-α in the AT are associated with chronically elevated basal lipolysis, the process of hydrolysis of tryglyc- erides to release FFAs and lipids (22). These provide chronic stimulation to macrophages leading to FFA-induced TNF-α production, causing IR. It has been proposed that adipocyte- derived TNF-α contributes to elevated levels of FFAs in the blood of obese individuals (22), and neutralization of TNF-α in vivo in obese mice decreases circulating levels of FFAs (23). TNF-α has also been shown to reduce the expression of proteins stabilizing lipid droplet (perilipins) (24), leading to ectopic lipid deposition in insulin-sensitive tissues. Lipids and lipid-derived molecules have direct effects on insulin-sensitive tissues and induce IR (25).l Inflammaging is an important link among obesity, IR, aging, and age-associated diseases such as cognitive impairment, athero- sclerosis, cancer, and autoimmunity. Elevated pro-inflammatory cytokines are associated with decreased insulin sensitivity. Chronic low-grade (sterile) inflammation causes IR, which leads to the transition from metabolically normal obesity to metabolic syndrome. This occurs through both systemic inflammation and metaflammation (12), a process whereby excess nutrients promote chronic low-grade inflammation, and whose metabolic hallmarks are high levels of lipids, free fatty acids (FFAs), glucose, and reactive oxygen species (ROS). Other major pro-inflammatory cytokines released by the obese AT are IFN-γ secreted by CD8+ T cells (26) and NK cells (27) and IL-17 secreted by CD4+ T cells (28). Citation: December 2017 \| Volume 8 \| Article 1745 December 2017 \| Volume 8 \| Article 1745 1 Frontiers in Immunology \| www.frontiersin.org Aging, Obesity, Inflammation, Diseases Frasca et al. as the occurrence of complications increases (3–5). The disease burden of high BMI in children (≤18 years of age) has not been addressed in the same detail.h It is not completely clear how obesity causes the develop- ment of IR. Although many molecular mechanisms have been proposed, including ER stress, oxidative stress, dysregulation of lipid homeostasis, mitochondrial dysfunction, hypoxia, and impairment of the insulin signaling pathway in insulin-respon- sive cells, there is evidence that obesity-induced inflammation may be a key factor for IR (21). Figure 1 summarizes the main pathways leading to inflammation in the obese AT. The aging process is characterized by a state of chronic inflammation, known as inflammaging. Several factors contrib- ute to inflammaging, including polymorphisms in the promoter regions of pro-inflammatory genes, chronic stimulation of immune cells with viruses such as cytomegalovirus, changes in the gut microbiome, and increased permeability from the intestine [reviewed in Ref. (6)]. It has been recently proposed that continuous engagement of innate receptors by endogeneous signals such as damage-associated molecular patterns drives a chronic state of background inflammation, which needs to be counterbalanced by anti-inflammatory mechanisms. Cellular senescence and the acquisition of the senescence-associated secretory phenotype (SASP) by fibroblasts (7) and endothelial (8) and immune cells (9–11) has also been pinpointed as a sig- nificant contributor to inflammaging. Cell senescence induces the accumulation of terminally differentiated B, T, and NK cells with dysregulated function through the activation of pathways integrating senescence and energy-sensing signals.l Hypoxia and Release of “Self” Antigens in the Obese AT During the development of obesity, the supply of oxygen to the expanding AT becomes inadequate, resulting in areas of hypoxia (29, 30). This phenomenon of poorly oxygenated AT not only activates the transcription factor hypoxia-inducible factor-1α (HIF-1α) and further release of pro-inflammatory cytokines (31) but also induces cell death and release of “self” antigens, which stimulate class switch and the production of IgG pathogenic antibodies. Hypoxia in the AT has been the only mechanism suggested so far for the release of “self” antigens in the obese AT. Immune cells of the innate and adaptive immune systems infiltrate insulin responsive tissues, such as the visceral adipose tissue (VAT) and with obesity incite inflammatory responses. Immune cells (macrophages, T, B, NK, NKT cells, and neutro- phils) have been implicated in adipose tissue (AT) inflamma- tion and IR (13–17). Inflammation leads to local and systemic increases in pro-inflammatory molecules, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, interferon (IFN)-γ, inflammatory adipokines, chemokines, and FFAs [reviewed in Ref. (16)]. LINKS OF OBESITY TO INSULIN RESISTANCE (IR) AND T2D One mechanisms for the release of “self”-antigens in the obese AT is the decreased supply of oxygen, resulting in areas of hypoxia, which leads to further release of pro-inflammatory cytokines, as well as to the release of “self”-antigens, such as intracellular proteins, cell-free DNA, and lipids. FIGURE 1 \| Model for regulation of inflammatory pathways in the obese adipose tissue (AT). Adipocytes (AD) in the obese AT are highly inflammatory and secrete several pro-inflammatory cytokines and chemokines, which recruit immune cells, thus contributing to the establishment and maintenance of local and systemic inflammation. Among these inflammatory mediators, tumor necrosis factor (TNF)-α released by both AD and immune cells induces lipolysis and release of free fatty acids (FFAs), which activate tissue-resident macrophages (MΦ) to release cytokines and chemokines. FFAs are also released in blood and cause both insulin resistance and inflammation in major insulin target tissues. Immune cells recruited to the obese AT differentiate into inflammatory subsets and secrete additional pro-inflammatory mediators. We hypothesize that these cells would generate suboptimal immune responses in obese individuals by circulating to peripheral lymphoid organs. Pathogenic antibodies may be secreted by B cells in the AT. These antibodies may form immune complexes with “self”-antigens, which in turn activate complement and Fc receptors on immune cells, leading to enhanced local inflammation, remodeling of the AT, impairment of adipocyte function and nutrient metabolism, and exacerbation of obesity-associated conditions. These antibodies can also exert additional detrimental effects both locally and systemically targeting distinct clusters of self proteins. One mechanisms for the release of “self”-antigens in the obese AT is the decreased supply of oxygen, resulting in areas of hypoxia, which leads to further release of pro-inflammatory cytokines, as well as to the release of “self”-antigens, such as intracellular proteins, cell-free DNA, and lipids. recruitment of immune cells to the AT. However, adipocytes also secrete pro-inflammatory mediators (cytokines, chemokines, and adipokines) and in larger amounts compared with immune cells (36). Therefore, with obesity, a crosstalk between adipocytes and the immune cells infiltrating the AT contributes to the establishment of chronic inflammation, a prerequisite for IR. Macrophages in the AT are almost exclusively M1, they depend on glycolysis for their inflammatory function, and their stimula- tion in the AT induces glucose transporter expression and glucose intake and utilization (37). Hypoxia (via HIF-1α) potentiates glycolysis and stabilizes the inflammatory phenotype (38). LINKS OF OBESITY TO INSULIN RESISTANCE (IR) AND T2D IR is the lack of appropriate response to circulating insulin in several tissues, including liver, muscle, and AT (18). It frequently associates with obesity, hypertension (integrating features of the metabolic syndrome), and CV disease and typically precedes the onset of T2D. In the pancreas, β-cells adapt to hyperglycemia with an expansion of the total β-cell mass and with increased secretion of insulin (hyperinsulinemia), which is able not only to control normal levels of glycemia but also can induce β-cell stress, causing β-cell failure, and then T2D (19). Poor glycemic control in individuals with T2D results in severe complications, such as renal failure, blindness, neuropathy, and CV disorders (20). Macrophage infiltration within the AT has been consid- ered a major driver of inflammation, due to the secretion of pro-inflammatory cytokines and chemokines involved in the Frontiers in Immunology \| www.frontiersin.org December 2017 \| Volume 8 \| Article 1745 2 Aging, Obesity, Inflammation, Diseases Frasca et al. FIGURE 1 \| Model for regulation of inflammatory pathways in the obese adipose tissue (AT). Adipocytes (AD) in the obese AT are highly inflammatory and secrete several pro-inflammatory cytokines and chemokines, which recruit immune cells, thus contributing to the establishment and maintenance of local and systemic inflammation. Among these inflammatory mediators, tumor necrosis factor (TNF)-α released by both AD and immune cells induces lipolysis and release of free fatty acids (FFAs), which activate tissue-resident macrophages (MΦ) to release cytokines and chemokines. FFAs are also released in blood and cause both insulin resistance and inflammation in major insulin target tissues. Immune cells recruited to the obese AT differentiate into inflammatory subsets and secrete additional pro-inflammatory mediators. We hypothesize that these cells would generate suboptimal immune responses in obese individuals by circulating to peripheral lymphoid organs. Pathogenic antibodies may be secreted by B cells in the AT. These antibodies may form immune complexes with “self”-antigens, which in turn activate complement and Fc receptors on immune cells, leading to enhanced local inflammation, remodeling of the AT, impairment of adipocyte function and nutrient metabolism, and exacerbation of obesity-associated conditions. These antibodies can also exert additional detrimental effects both locally and systemically targeting distinct clusters of self proteins. Frontiers in Immunology \| www.frontiersin.org LINKS OF OBESITY TO INSULIN RESISTANCE (IR) AND T2D In M1 macrophages, the inflammasome NLRP3 activates caspase 1 and the secretion of IL-1β (39), which is directly toxic to pancreatic β-cells and induces IR (40). Increased inflammasome activity has been reported in monocyte-derived macrophages from T2D patients (41). high levels of membrane glucose transporters and are highly glycolytic (44), a trait supporting inflammatory responses.h l Interferon-γ, the signature Th1 cytokine, induces macrophages and T cells to secrete chemokines, which recruit immune cells to the obese AT (45, 46). Moreover, IFN-γ facilitates the M2 to M1 polarization (47) and decreases insulin receptor signaling by reducing the expression of insulin receptors and glucose trans- porters (48). IFN-γ production is regulated by T-bet, a T-box family transcription factor first identified as a transcriptional inducer of IFN-γ in CD4+ T cells (49). T-bet plays a critical role in the development of IR in animal models of obesity, and T-bet- deficient mice fed a high-fat diet are refractory to the induction of IR (50). These mice show improved insulin sensitivity and glucose tolerance, reduced numbers of immune cells in the AT (CD4+/CD8+ T cells, NK cells, and macrophages), and reduced production of pro-inflammatory cytokines per gram of fat (IFN-γ, TNF-α, IL-1β, and IL-6). T cells in the AT are Th1 CD4+ and IFN-γ-producing CD8+ T cells (26). These promote secretion of pro-inflammatory cytokines from M1 macrophages leading to both local and systemic IR (42). Similar to macrophages, T cell subset skewing in the AT occurs through modulation of substrate metabolism regulated by hormones (leptin) and intracellular nutrient sensing kinases, such as AMPK/mTOR (43). Th1 CD4+ T cells express Obese and T2D patients have alterations in the composition of their microbiome, with reduced proportions of Bacteroidetes (beneficial bacteria) in obese versus lean individuals (51). Moreover, it has been reported that the gut microflora regulates the development of obesity in animal models (52). T-bet regu- lates mucosal T cell activation (53), and T-bet deficiency alters December 2017 \| Volume 8 \| Article 1745 Frontiers in Immunology \| www.frontiersin.org 3 Aging, Obesity, Inflammation, Diseases Frasca et al. some studies, increased BMI had the opposite effect of reduced mortality (70, 73), which has been described as the “obesity paradox” (74). Moreover, in overweight RA patients, progression of bone destruction was reduced (75, 76), the number of swollen joints is not increased, and better quality of life has been reported (77). LINKS OF OBESITY TO INSULIN RESISTANCE (IR) AND T2D Weight loss and cachexia represent major determinants for a greater risk of death (78) and worse quality of life (77), thus strengthening the paradoxical observation of lower mortality in obese patients. However, follow-up studies have demonstrated that in RA patients with a history of obesity reduced BMI is strongly associated with death. Therefore, the “obesity paradox” does not entail a biologically protective role of obesity (73), raising the question whether the use of BMI is a valid tool for assessing obesity in RA (65).f the composition of microflora (54). T-bet deficiency may also alter the microbiome in individuals with obesity leading to the inflammatory and metabolic processes that regulate T2D. B cells also accumulate in the obese AT (15, 55, 56). B cell recruitment can initiate T cell-induced M1 polarization and IR. Obesity and hyperglycemia have direct influence on antibody production, and IgG secretion from inflamed VAT modulate the function of resident macrophages. It has been reported that B cells in AT are induced to produce pathogenic IgG autoan- tibodies, due to the dysregulated expression of autoantigens by hypoxic adipocytes. B cells also support the activation of inflammatory T cells, which are the main pathogenic drivers in systemic inflammation and IR. Recently, a new lymphoid tissue called fat-associated lym- phoid clusters (FALCs) has been identified in the mesenteric AT of mice and humans. FALCs are rapidly induced after inflamma- tory stimuli and support B cell proliferation and differentiation regulating antibody production within the AT (57). g y High BMI contributes to disease activity in RA by affecting both biomechanics and the metabolic status, and obese RA patients show worse subjective assessment of symptoms (79). Hyperglycemia, as a part of the metabolic syndrome, is more common in early RA (80), whereas active RA shows decreased lipid levels (81) despite an increased risk of CV events, due to the lipid-lowering effect of systemic inflammation (82). An increase in VAT, e.g., the epicardial fat (83), and the more abundant mac- rophage infiltrate are associated with systemic inflammation, metabolic syndrome, and IR (84). Anti-TNF-α therapy improves insulin sensitivity in RA patients who are resistant, but despite controlling inflammation, it does not achieve the same extent of improvement in obese RA patients (85). In addition, even when therapy succeeds in the control of disease activity, it fails to restore the altered body composition and improve physical function (86). OBESITY AND RHEUMATOID ARTHRITIS (RA): EVIDENCES AND MECHANISTIC LINKS RA is a debilitating chronic autoimmune disease that causes synovial inflammation and destruction of joints including the cartilage and the adjacent bone. It generally occurs between the fourth and sixth decades of life and affects more women than men. It is characterized by joint stiffness, pain, and swelling and is accompanied by extraarticular manifestations and systemic inflammation. RA has been associated with muscle wasting and cachexia due to uncontrolled inflammation driven by TNF-α, which fuels hypercatabolism (58). However, BMI rarely falls below normal because loss of lean tissue is compensated by increased AT, and this characterizes rheumatoid cachexia, also called “cachectic obesity” (59, 60). It has been observed that in RA patients, despite adequate nutrient intake, but inflammatory cytokine dominance and reduced activity due to pain, joint deformity, and decreased muscle strength (61), cachexia appears to be similar to that occurring in aged subjects with disability. Abnormal body composition in RA can be defined as a sarco- penic obesity (62) with characteristic changes that in the elderly contribute to frailty (63, 64). Moreover, the percentage of obese RA patients has increased (65), and the impact of obesity on RA has become a relevant issue not much for the negligible risk of developing RA (66), but for its negative effects on disease activity, response to therapy, and CV risk. Obese RA patients are indeed less likely to achieve sustained remission in response to therapy with conventional chemical (4) or biologic (TNF-α inhibitors) agents (67). Despite opposite results with some treatments (68), obesity decreases the rate of remission in RA and negatively affects disease activity (69) and patient-reported outcomes dur- ing therapy (70). Since weight reduction may have possible contraindications (lower BMI being associated with accelerated mortality in RA), and the assessment of the inflammatory milieu of VAT in RA patients is still incomplete, much research has been devoted to uncovering the metabolic changes occurring in the develop- ment and chronicization of RA. This field has been recently reviewed (90) and can be summarized in the two distinct stages of early and chronic RA. In the first stage, there is a high metabolic demand in all cell types involved, due to proliferative signaling, angiogenesis, cellular de-differentiation, and unbal- anced bone turnover. LINKS OF OBESITY TO INSULIN RESISTANCE (IR) AND T2D Adipokines (leptin, adiponectin, visfatin, resistin, and chemerin) have been postulated to be the mediators linking AT and RA activity (87). Adipokine imbalance may underlie the higher degree of inflammation (88), the levels of autoantibodies (leptin and adiponectin differentially regulate the generation of Treg cells, which are abundant in normal VAT), and also the lower amount of bone resorption observed in obese patients (89). On another level, the association of RA with both the metabolic syndrome and atherosclerosis is probably also medi- ated by VAT through altered secretion of adipokines. Therefore, adipokines contribute decisively to the systemic inflammation underlying RA, which represents an independent risk factor for CV diseases. OBESITY AND RHEUMATOID ARTHRITIS (RA): EVIDENCES AND MECHANISTIC LINKS However, in RA T cells, at variance with other types of inflammatory metabolic changes, the glycolytic pathway is reduced in favor of the pentose phosphate shunt (91), reduced ROS generation, and decreased AMPK func- tion. In early stages, AMPK activation (e.g., by Metformin) The “Obesity Paradox” in RA Obesity represents an important link with comorbidities such as metabolic syndrome (71) and CV diseases (72); however, in Role of B Cells in RA Pathogenesish These cells increased even more significantly after B cell depletion therapy (BCDT) with rituximab. There is also agree- ment on the fact that response to BCDT relies on elimination of memory B cells, and their repopulation, along with transitional B lymphocytes, may predict relapse (103). The role of homeostatic lymphoproliferation of both memory B cells and the extent of BCDT in bone marrow and synovial tissue represent critical points still to be elucidated. Since it has been observed that CD4+ T cell activation decrease after BCDT (104), changes in not only B cell subsets but also T cell subsets may underlie the response of RA patients to therapy. The Treg compartment is less affected by RA treatments (105), but the presence of Breg lymphocytes (decreased in untreated RA) seem to play a role in balancing immune abnormalities and predict the treatment outcome (106). Cytokine production by B cell subsets is also relevant to RA pathogenesis and disease activity [reviewed in Ref. (107)], with inflammatory cytokines predominating in untreated severe RA, as activated memory B cells preferentially secrete TNF-α, whereas BCDT induced a shift to subpopulations producing IL-10. The In patients younger than 60 years of age with symptomatic OA, joint pain and disabilities are less recognized as inevitable consequences of growing old, compared to OA patients older than 70 years (117). Several factors contribute to the devel- opment of OA: acute injury (including fracture), excessive mechanical overloading (113, 118), diabetes, and chronic tobacco smoking, all playing a role in the amplification of senescence-inducing stresses (118–121). These factors develop before symptoms appearance and may cause early onset of OA; multimorbidity including OA and obesity can be seen at an adult age (122). The prevalence of arthritis is increasing, with 29.3% ever reported doctor-diagnosed arthritis in individuals aged 45–64 years versus 49.6% in individuals aged 65 or older in the United States (123). However, obesity prevalence did not change significantly over time among middle-aged and younger adults with doctor-diagnosed arthritis (124) despite increasing significantly over time among older adults with RA and remaining also higher when compared with adults without RA. Obesity impacts progression of OA and has a negative influence on outcomes (125). Exercise and loss of at least 10% of body weight can effectively lead to improvement in symptoms, pain relief, and physical function. Role of B Cells in RA Pathogenesish g The key role played by autoreactive B cells is highlighted by the presence of diagnostic autoantibodies, and rheumatoid factor (RF) (99) and anticyclic-citrullinated peptide antibodies (ACPAs) (100) are well-established indicators of disease and disease sever- ity and may precede the onset of disease. The role of B cells in RA pathogenesis in the context of overweight/obesity has not been addressed yet and deserves thorough attention. A primary defect in early B cell tolerance has been detected since the major- ity of naive B lymphocytes express polyreactive autoantibodies, including RF and ACPA. These B cells are resistant to Fas-induced apoptosis and therefore not suppressed by Treg (101). However, B cells are involved in RA by other mechanisms, in a bidirectional support of helper T lymphocytes, as self-antigen-presenting cells, with the release of inflammatory mediators, and with the promo- tion of lymphoid neogenesis (which is prominent in RA synovitis). RF+ B cells are able to take up IgG-containing immune complexes and present antigen to T cells, thus activating a reciprocally rein- forced response (102).h In the elderly, arthritis is frequently associated with other diseases with multiple aging or degenerative features (109). OA and RA share common features in elderly patients and significantly con- tribute to disability (110). OA is usually differentiated from RA by age at diagnosis, duration of morning stiffness, pattern of joint involvement, and radiographic findings. Distinguishing between the diseases can be challenging, but in the >60 years of age group, OA is by far more common. Despite the fact that OA directly correlates with age, the real cause of this association is not clear, and OA development can be separated into aging-dependent and aging-independent processes (111–114). Both increased produc- tion of matrix metalloproteinases and cytokines, reduced levels of collagen type II synthesis, and increased production of ROS induce age-related changes in chondrocytes (114). These changes alter cartilage function, and sarcopenia further leads to decreased joint stability (115). Cellular senescence, impaired regeneration, and repair are recognized factors contributing to cartilage dam- age with aging (115, 116). The phenotype of B lymphocytes in RA has been extensively studied in peripheral blood and in synovial tissue, with some discordant data owing to examination of different stages of the disease. The general consensus is the increased presence of memory (CD27+) B cells with an activated (CD95+, CD21low) phe- notype both in peripheral blood and in the synovial compartment (103). The “Obesity Paradox” in RA y Obesity represents an important link with comorbidities such as metabolic syndrome (71) and CV diseases (72); however, in December 2017 \| Volume 8 \| Article 1745 Frontiers in Immunology \| www.frontiersin.org 4 Aging, Obesity, Inflammation, Diseases Frasca et al. recent identification of a subset of B cells able to produce large amounts of RANKL (108) provides a mechanistic link between activated memory B cells and bone resorption through induction of osteoclastogenesis. It is relevant to mention that ACPAs are associated with more joint and bone damage and that therapy does not eliminate ACPA-producing autoreactive B cells in the synovial tissue. The central role of B lymphocytes in RA patho- genesis and in tissue damage makes these cells and their products attractive targets for treatment; however, there is still uncertainty about the beneficial or even protective effects of B cell subsets. may be an attractive target because its activity is decreased in several tissues of obese or IR patients. In the late (erosive) stage of RA, the inflamed joint is a hypermetabolic lesion (90), T cells undergo a metabolic switch to aerobic glycolysis due to hypoxic conditions, and mitochondrial dysfunction with increased lactate production causes acidification of the synovia. The reprogramming of T cells accounts for pro-inflammatory Th1/Th17 phenotypes and premature T cell aging (92). Several aspects of immunosenescence have been found to be relevant in RA pathogenesis (93–96), and rejuvenation of the immune system has been proposed as therapy, including mTOR inhibi- tors (97, 98). Frontiers in Immunology \| www.frontiersin.org COGNITIVE IMPAIRMENT, DEMENTIA, AND RELATIONSHIP TO AGE- ASSOCIATED DISEASES AND OBESITY in this case (135). A recent systematic review and meta-analysis suggests a positive association between obesity in mid-life and later dementia but the opposite in late life (136). A successive study confirmed the association of mid-life obesity and dementia, but that of being underweight and dementia remained contro- versial (137). It is difficult to draw a clear distinction between visceral adiposity and total body fat in most studies, and this is reflected on the paucity of mechanistic hypotheses supported by experimental data. The attention has been focused on the role of several adipokines and mainly the two major hormones produced by the AT, leptin and adiponectin, that interact directly with the brain (138). They have the capability to cross the blood–brain barrier and influence dementia processes within the brain (139), but evidence for a direct role is missing. Another postulated link is through altered gut microbial flora, which may participate in the development of obesity, T2D, and subsequent initiation of AD (140). Also lacking is the evidence that weight reduction in mid-life may produce beneficial effects on demen- tia development. However, in older adults, regular exercise pro- vides numerous health benefits that include improvements in blood pressure, coronary artery disease, diabetes, lipid profile, OA, osteoporosis, mood, neurocognitive function, and overall morbidity so that studies in this area should be encouraged. Our summary of conditions where inflammation, obesity, and aging converge in defining particular features and outcomes of disease must also briefly mention the dementias, whose preva- lence has been reported to be declining among older US adults between 2000 and 2012 (127). However, dementia rates are grow- ing at an alarming proportion in most regions of the world and are related to population aging (128). Prevalence varies in coun- tries with different mean population ages. However, differences persist after adjusting for age (129). The decline in the United States occurred in those older than 65 years and was related to increased number of years in education despite the age- and sex- adjusted increase in the prevalence of hypertension, diabetes, and obesity in the same years. There is a long unresolved debate on the prodromal phase of the neurodegenerative disorders with inflammatory features, such as Alzheimer’s dementia, but it is undisputed that prevalence of dementias of all types increase with old age, from about 2–3% among those aged 70–75 years to 20–25% among those aged 85 years or more (130). Role of B Cells in RA Pathogenesish Physical activity may reactivate a regenerative process by mobilizing stem cells and increase proteoglycan production, restoring the cartilage structure (113, 115, 126). December 2017 \| Volume 8 \| Article 1745 5 Aging, Obesity, Inflammation, Diseases Frasca et al. CONCLUDING REMARKS Immunity and metabolism are highly integrated factors in aging and age-related diseases. This is an expanding field of investiga- tion. Obesity and related complications are a major global epi- demic. Scientific research must be a crucial part of the solution to understand all implications of obesity, but this research is still in its initial phase. The investigation of the mechanisms whereby inflammation and immune activation disrupt a functional immune response adds novel insights to the understanding of the relationship between inflammation and long-term metabolic disease outcome and opens new ways for effective therapeutic interventions. Taken together for the two most frequent types of dementia (Alzheimer’s and Vascular) (129), vascular risk factors such as dietary fat intake, high cholesterol, obesity, T2D, and hyperten- sion have emerged as the most important determinants (132). Vascular risk is seldom isolated and is accompanied by alterations in hormonal metabolism. Overweight/obesity, due to excess AT, increase the CV risk and also for late-onset dementia. This is exemplified in the prodromal phases of dementia, as vascular and metabolic parameters decline in direct relation to cognitive impairment and in a way which seems to differ from that occur- ring in “normal” aging. With regard to obesity, its presence at midlife is associated with an increased risk of dementia and Alzheimer’s later in life (133), and in particular central obesity in midlife increases the risk of dementia independent of diabetes and CV comorbidities (134). The risk is reversed when late-life BMI is considered: underweight persons had an increased risk of dementia, whereas being overweight was not associated and being obese reduced the risk of dementia compared with normal BMI. This has been dubbed as an “obesity paradox” also COGNITIVE IMPAIRMENT, DEMENTIA, AND RELATIONSHIP TO AGE- ASSOCIATED DISEASES AND OBESITY The known risk factors include obesity, depression, diabetes, decreased physical activity, hypertension, smoking, hypercholesterolemia, coronary heart disease, and alcohol use; and assessment of these provide an estimate of the risk of developing dementia (131) despite the fact that in the oldest-old (80–97 years old), these factors did not increase the risk for dementia, so that age plays a major role. FUNDING This study was supported by NIH AG-32576 (BBB), AI096446, AG042826, and AG032576 (BBB and DF). This study was supported by NIH AG-32576 (BBB), AI096446, AG042826, and AG032576 (BBB and DF). This study was supported by NIH AG-32576 (BBB), AI096446, AG042826, and AG032576 (BBB and DF). AUTHOR CONTRIBUTIONS All authors were involved in writing the article and had final approval of the submitted version. 3. 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Expansion of activated peripheral blood memory B cells in rheumatoid arthritis, impact of B cell depletion therapy, and biomarkers of response. PLoS One (2015) 10(6):e0128269. doi:10.1371/journal.pone.0128269 126. Rahmati M, Nalesso G, Mobasheri A, Mozafari M. Aging and osteoarthritis: central role of the extracellular matrix. Ageing Res Rev (2017) 40:20–30. doi:10.1016/j.arr.2017.07.004 December 2017 \| Volume 8 \| Article 1745 Frontiers in Immunology \| www.frontiersin.org 9 Aging, Obesity, Inflammation, Diseases Frasca et al. 127. Langa KM, Larson EB, Crimmins EM, Faul JD, Levine DA, Kabeto MU, et al. A comparison of the prevalence of dementia in the United States in 2000 and 2012. JAMA Intern Med (2017) 177(1):51–8. doi:10.1001/ jamainternmed.2016.6807 136. Pedditizi E, Peters R, Beckett N. The risk of overweight/obesity in mid-life and late life for the development of dementia: a systematic review and meta-analysis of longitudinal studies. Age Ageing (2016) 45(1):14–21. doi:10.1093/ageing/afv151 137. Albanese E, Launer LJ, Egger M, Prince MJ, Giannakopoulos P, Wolters FJ, et al. Body mass index in midlife and dementia: systematic review and meta-regression analysis of 589,649 men and women followed in longitu- dinal studies. Alzheimers Dement (Amst) (2017) 8:165–78. doi:10.1016/j. dadm.2017.05.007 128. Kalaria RN, Maestre GE, Arizaga R, Friedland RP, Galasko D, Hall K, et al. Alzheimer’s disease and vascular dementia in developing countries: prev- alence, management, and risk factors. Lancet Neurol (2008) 7(9):812–26. doi:10.1016/S1474-4422(08)70169-8 129. Rizzi L, Rosset I, Roriz-Cruz M. Global epidemiology of dementia: Alzheimer’s and vascular types. Biomed Res Int (2014) 2014:908915. doi:10.1155/2014/908915 138. Kiliaan AJ, Arnoldussen IA, Gustafson DR. Adipokines: a link between obesity and dementia? Lancet Neurol (2014) 13(9):913–23. doi:10.1016/ S1474-4422(14)70085-7 130. Copyright © 2017 Frasca, Blomberg and Paganelli. This is an open-access arti cle distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic prac tice. No use, distribution or reproduction is permitted which does not comply with these terms. REFERENCES Ferri CP, Prince M, Brayne C, Brodaty H, Fratiglioni L, Ganguli M, et al. Global prevalence of dementia: a Delphi consensus study. Lancet (2005) 366(9503):2112–7. doi:10.1016/S0140-6736(05)67889-0 139. Arnoldussen IA, Kiliaan AJ, Gustafson DR. Obesity and dementia: adipokines interact with the brain. Eur Neuropsychopharmacol (2014) 24(12):1982–99. doi:10.1016/j.euroneuro.2014.03.002 131. Vos SJB, van Boxtel MPJ, Schiepers OJG, Deckers K, de Vugt M, Carriere I, et al. Modifiable risk factors for prevention of dementia in midlife, late life and the oldest-old: validation of the libra index. J Alzheimers Dis (2017) 58(2):537–47. doi:10.3233/JAD-161208 140. Naseer MI, Bibi F, Alqahtani MH, Chaudhary AG, Azhar EI, Kamal MA, et al. Role of gut microbiota in obesity, type 2 diabetes and Alzheimer’s disease. CNS Neurol Disord Drug Targets (2014) 13(2):305–11. doi:10.2174/ 18715273113126660147 132. Villringer A. The path from obesity and hypertension to dementia. Adv Exp Med Biol (2015) 821:5. doi:10.1007/978-3-319-08939-3_3 133. Kivipelto M, Ngandu T, Fratiglioni L, Viitanen M, Kareholt I, Winblad B, et al. Obesity and vascular risk factors at midlife and the risk of dementia and Alzheimer disease. Arch Neurol (2005) 62(10):1556–60. doi:10.1001/ archneur.62.10.1556 Conflict of Interest Statement: The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Conflict of Interest Statement: The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 134. Whitmer RA, Gustafson DR, Barrett-Connor E, Haan MN, Gunderson EP, Yaffe K. Central obesity and increased risk of dementia more than three decades later. Neurology (2008) 71(14):1057–64. doi:10.1212/01. wnl.0000306313.89165.ef Copyright © 2017 Frasca, Blomberg and Paganelli. This is an open-access arti cle distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic prac tice. No use, distribution or reproduction is permitted which does not comply with these terms. 135. Fitzpatrick AL, Kuller LH, Lopez OL, Diehr P, O’Meara ES, Longstreth WT Jr, et al. Midlife and late-life obesity and the risk of dementia: cardiovascular health study. Arch Neurol (2009) 66(3):336–42. doi:10.1001/archneurol. 2008.582 December 2017 \| Volume 8 \| Article 1745 Frontiers in Immunology \| www.frontiersin.org 10
https://openalex.org/W4210894517	http://romanica.upol.cz/doi/10.5507/ro.2021.017.pdf	Spanish; Castilian	null	Jorge Isaacs' María as a subversion of Romanticism in New Granada	Romanica Olomucensia	2,021	cc-by-sa	10,088	Facultad de Ciencias Económicas y Administrativas, Universidad de Medellín, Carrera 87, N° 30 – 65, Medellín – Colombia jcherrera@udemedellin.edu.co Facultad de Ciencias Económicas y Administrativas, Universidad de Medellín, Carrera 87, N° 30 – 65, Medellín – Colombia jcherrera@udemedellin.edu.co MARÍA DE JORGE ISAACS COMO SUBVERSIÓN DEL ROMANTICISMO EN LA NUEVA GRANADA Juan Carlos Herrera Ruiz omanica Olomucensia 33/2 (2021): 241-257, doi: 10.5507/ro.2021.0017 (CC BY-NC-ND 4.0) 1 «María, testimonio vigente del romanticismo americano» (1966). 2 Ello lo constata Rafael Gutiérrez Girardot en el Tomo I de sus Ensayos de literatura colombiana (2011: 152); adicionalmente, en un estudio sobre la novela colombiana, Pineda Botero documenta la muy positiva recepción que tuvo la obra desde su publicación y las numerosas ediciones que aparecieron en el resto del continente hacia finales del siglo XIX (1999: 199). 3 Y con esto alude a la frecuente postulación de María como una especie de meritorio, pero tardío, eco americano de Paul et Virginia (1787), de J.H. Bernardin de Saint-Pierre, de Jocelyn (1836), de Alphonse de Lamartine, pero sobre todo de Atala (1801), de Chateaubriand. 4 A guisa de ejemplos, citamos tres trabajos ya clásicos: Comunidades imaginadas. Reflexiones sobre el origen y la difusión del nacionalismo (1993) de Benedict Anderson, Nación y narración: entre la ilusión de una iden tidad y las diferencias culturales (2010) de Homi K. Bhabha, y la investigación de Doris Sommer Ficciones Jorge Isaacs’ María as a subversion of Romanticism in New Granada Abstract: The article analyses aspects of the slavery relations of production portrayed in Jorge Isaacs’ María (1867) as an expression of altered criticism towards the canon of Romanticism and as a form of cultural resistance to the hegemonic discourse of the politi cal elites in the founding literature of the 19th century. The work carried out by black char acters from the master’s point of view is traced through some keys derived from the Hege lian dialectic of the master and the slave, noting how their subjectivities are confronted and how, in the unfolding of the narrative voice, desire is restored to the slave. The acquisition of self-awareness on the part of the slave and the recovery of the ancestral memory of their African cultural roots through oral tradition imply the breaking of the historical cycle that legitimizes the power of the ruling class, while at the same time evidencing the crisis of slavery as a social and economic institution. It is concluded that the novel constitutes a particularly “subversive” attempt to signify the imaginary around slavery, as well as the notion of the “black African”, within the framework of an American literary tradition that refracts the social, political, and economic contradictions in the historical development of capitalism. Keywords: Jorge Isaacs’ María; slavery; dialectic of the master and the slave; Hispano- American Romanticism; alter criticism Resumen: El artículo analiza aspectos de las relaciones de producción esclavista represen tados en la novela María de Jorge Isaacs (1867) como expresión de alteridad crítica frente al canon del romanticismo y como forma de resistencia cultural ante el discurso hegemónico de las élites políticas en la literatura fundacional del siglo XIX. Se realiza un rastreo del trabajo que realizan personajes negros desde el punto de vista del amo a través de algu nas claves derivadas de la dialéctica hegeliana del amo y el esclavo, constatando cómo se confrontan sus subjetividades y cómo desde la voz narrativa se restituye el deseo al esclavo. La adquisición de una autoconciencia por parte del esclavo y la recuperación de Romanica Olomucensia 33/2 (2021): 241-257, doi: 10.5507/ro.2021.0017 (CC BY-NC-ND 4.0) Juan Carlos Herrera Ruiz la memoria ancestral de sus raíces culturales africanas por medio de la tradición oral im plica la ruptura del ciclo histórico que legitima el poder de la clase dominante, a la vez que evidencia la crisis del esclavismo como institución social y económica. Jorge Isaacs’ María as a subversion of Romanticism in New Granada Se concluye que la novela constituye una tentativa particularmente «subversiva» por resignificar el imagi nario en torno a la esclavitud, así como la noción de lo «negro-africano», en el marco de una tradición literaria americana que refracta las contradicciones de orden social, político y económico inherentes al desarrollo histórico del capitalismo. Palabras clave: María de Jorge Isaacs; esclavismo; dialéctica del amo y el esclavo, romanti cismo hispanoamericano, alteridad crítica 1. Introducción En un artículo escrito en ocasión del centenario de María de Jorge Isaacs,1 el narra dor y africanista colombiano Manuel Zapata Olivella aborda la dimensión política de una de las novelas más populares del siglo XIX hispanoamericano, y acaso tam bién la más leída en ese ámbito.2 Más allá de remarcar el acento americanista y de argumentar, sobre un copioso corpus de obras literarias con África como tema, la emancipación estética de Europa, en esta su tesis sobre María encontramos algunos temas y problemas en torno a la esclavitud que apenas quedan insinuados, y que entran en el interés de este artículo en tanto abren la perspectiva de una alteridad crítica, si cabe la expresión, o de una forma de resistencia cultural ante un discurso producido por las élites letradas: a contrapelo del canon de María que fijara el con notado americanista dominicano Pedro Henríquez Ureña en sus Corrientes literarias en la América hispánica, como la expresión más acabada del romanticismo español en América (1994: 152-153), Zapata Olivella sugiere que el mérito y la vigencia de la novela residen por fuera de su relación con «los valores eternos del romance»,3 y agrega que «hay otro substrato de igual o mayor hondura que el amor y la poesía en sus páginas: el patriotismo que enmarca a María sólidamente en el romanticismo americano» (1966: 15). ( ) Pero este «patriotismo» que postula Zapata Olivella de cara a la emergencia de un «romanticismo americano» no se congracia con la línea de análisis histórico-bi bliográfico que, más tarde en el siglo XX, atribuyó a la novela decimonónica ameri cana una función servil al proyecto de fundación de Estados nacionales, calcado del modelo europeo, ni con la correspondiente consolidación de una identidad nacio nal en las recién independizadas repúblicas hispanoamericanas;4 tampoco se alinea 242 María de Jorge Isaacs como subversión del romanticismo en la Nueva Granada maquinalmente con un encriptado sentimiento de lo nacional derivado de la exis tencia de un «alma étnica», sino más bien con el conocimiento de las relaciones de producción y de las condiciones económicas y sociales, de contenido distinto, que han enfrentado las comunidades afrodescendientes en su lucha contra la esclavitud y que se encuentran refractadas en María. 1. Introducción Ello a más de subrayar que tras el anti cipo antillano de Sab, de la cubana Gertrudis Gómez de Avellaneda, publicada en España en 1841, María sería la primera novela con temática negra escrita en tierra firme (Zapata Olivella 1966: 20). La recepción de Zapata Olivella y su intervención crítica sobre María configuran, de por sí, un principio de subversión ante un patrón de recepción que favorece los logros políticos y económicos de la clase dominante; de ahí que tampoco escapa a su atención el origen blanco del autor de la novela, ni su subjetividad, blanca también, producto de su época, que permanece siempre en la otra orilla de los intereses de clase. Dicho lo anterior y sobre la base de algunas claves que entrega Zapata Olivella, este ejercicio constituye una tentativa por resignificar la presencia de África en María como un pensamiento primigenio, un retorno al origen de la historia y de la cultura, que se pone en marcha en tanto se integran al relato personajes negros y mestizos, hombres y mujeres, a los que se atribuye corporeidad, nombre, ocasionalmente voz, y, aunque el resultado sea incipiente, se concede también una subjetividad y una forma de conciencia de clase. La representación de las prácticas sociales africanas en María, si vale decir así, están subordinadas a las relaciones de producción en un contexto en el que aún es moral y políticamente legítimo poseer esclavos, si bien en su elusiva historicidad se adivina que ese esclavismo, como institución social, está entrando en crisis;5 una señal de ello es que al esclavo, sin propiedad ni ideología, se le restituye el deseo, en el umbral de una lucha histórica que enfrenta, por un lado, a un amo atrapado en la servidumbre de sus pasiones tristes,6 esto es, carente ya de poder y sin control de sus emociones y, de otro lado, a un siervo que deviene dueño de su propia conciencia y subvierte así la condición de subordinado. p p y En esa dirección se rastrea un flujo de transformación y reproducción de la ma teria en el que participan esclavos y amos, de canales de comunicación de cara al encuentro de sus voluntades y subjetividades, siguiendo la dialéctica hegeliana del amo y el esclavo. fundacionales: las novelas nacionales de América Latina, con un apartado, este último libro, titulado «El mal de María», en que evidencia una instrumentalización del sentimiento del amor como insumo para la creación de escenarios históricos convergentes de lo político y lo erótico, en lo que llama una «intimi dad nacional» (Sommer 2004: 230). 5 María se publica en 1867, mientras que la abolición de la esclavitud en la Nueva Granada se decreta en 1852. 6 Esta es una evocación del principio que expuso Baruch Spinoza en el Libro III de su Ética con relación a los afectos fundamentales, que hacen diferir la esencia y la naturaleza de un individuo con relación a las de otro sobre la base del deseo de cada uno: la alegría y la tristeza son pasiones que, en tanto au mentan o disminuyen, favorecen o reprimen la potencia de cada individuo con relación a los demás, o sea, el esfuerzo por preservar su propio ser (Spinoza 2001: 257). 1. Introducción Parece claro que, junto a este encuentro de voluntades y deseos, gravita en María una advertencia velada en torno al abolicionismo, en el marco de la construcción de nación en la que negros y mulatos son incluidos, pero esta vez 243 Juan Carlos Herrera Ruiz como relación dialéctica de amor y de odio: el momento en que está en juego la con dición original de los agentes de clase y los límites de poder sobre la voluntad y el deseo del otro. Como complemento a este enfoque, se apunta también a generar una interpretación alternativa de la novela como mediación artística entre la historia del esclavismo, que está en la base de la fundación nacional neogranadina, y una recu peración de la memoria del mundo africano a través de la tradición oral. como relación dialéctica de amor y de odio: el momento en que está en juego la con dición original de los agentes de clase y los límites de poder sobre la voluntad y el deseo del otro. Como complemento a este enfoque, se apunta también a generar una interpretación alternativa de la novela como mediación artística entre la historia del esclavismo, que está en la base de la fundación nacional neogranadina, y una recu peración de la memoria del mundo africano a través de la tradición oral. p Necesario es considerar desde un principio que la elección de motivos africanos como insumo para la narración en María fuera producto, además del contacto fre cuente con afrodescendientes neogranadinos, del interés intelectual de Jorge Isaacs (1837-1895) por la antropología académica, estrechamente ligada en sus orígenes al colonialismo británico en África: esta intuición es compartida por Zapata Olivella, que, pese a reconocer al antropólogo en el poeta, en lo que respecta a María, rechaza la idea de que la ciencia del primero supeditara la lírica del segundo. Conviene a este respecto recordar que Isaacs también escribió obras del tipo que hoy llamaríamos «etnográficas», producto de su vida entre grupos indígenas y de su participación en misiones de exploración científica en la Nueva Granada; con este propósito realizó, por ejemplo, extensos recorridos por la Guajira y la Sierra Nevada de Santa Marta, cuyos informes se compilaron en un volumen titulado Hulleras de Aracataca, o el tra bajo etnolingüístico publicado en 1884 Estudio sobre las tribus indígenas del Magdalena (Rueda 2007: 350). 7 Apegados estrictamente a la narración, los únicos personajes nacidos en África son Feliciana, que antes de llegar a América se llamaba Nay, y su marido Sinar. Los demás personajes negros han nacido en América; en cuanto a estos referenciamos una cantidad que estimamos representativa para los fines del artículo, mas no la totalidad de los presentes en la novela. 2. El trabajo, el amor y el odio en las relaciones entre amos y esclavos en María Como ya dijimos, María es una de las novelas más populares y leídas en el ámbito hispanoamericano; ha sido además considerada la novela nacional de Colombia por su centralidad en la formación de una tradición literaria nacional.8 La historia tiene lugar en la segunda mitad del siglo XIX en el marco de una sociedad rural organi zada en torno a la gran hacienda productiva, institución social, política y económica heredada de la época de dominación española, y plenamente vigente en sus prácticas de señorío y servidumbre, no obstante la Nueva Granada fuese ya, al menos sobre el papel, una República emancipada. En el seno de una familia de ascendencia europea surge el amor entre dos jóvenes: Efraín y su prima María; tras fugaces años de solaz en medio del más idílico paisaje americano, Efraín marcha primero a la capital Bo gotá y luego a Inglaterra, a continuar sus estudios de medicina, interrumpiendo así el noviazgo con María, pero no sin antes haber formalizado con esta el compromiso matrimonial una vez regresara. Ya desde edad temprana, la familia había advertido que María padecía de una enfermedad que la hacía demasiado frágil y que finalmen te le causaría la muerte, encontrándose Efraín en el extranjero. El angustioso regreso de este a la patria marca el final dramático de la novela. Al margen de la parte de la historia que atañe al amor entre los amos, el tema de la esclavitud en el plano de la ficción dentro de la narración es advertido por Zapata Olivella como un trazo «ágil y seguro» de una situación social e histórica en la que los límites de la servidumbre y la esclavitud son imprecisos (1966: 20). En efecto, dicho trazo se desenvuelve a lo largo del relato, pero muy especialmente en «la Nouvelle de Nay y Sinar dentro de María», como acierta en llamarla Gustavo Mejía en el prólogo a la edición de Biblioteca Ayacucho,9 aludiendo a los episodios intercalados entre los capítulos XL-XLIII, segmento narrativo que se instrumenta liza como evocación implícita del amor frustrado de Efraín y María, pero esta vez representado por seres africanos: la fiel Nay y su esposo, separados por la fuerza de la tragedia y de la tiranía. 8 Valga anotar que la novela mantiene su peso y vigencia en el debate socioliterario colombiano y en el interés de sus académicos: prueba de ello es la aparición de tres ediciones críticas en el marco del joven siglo XXI: María Teresa Cristina (2007); Flor M. Rodríguez-Arenas (2008); Mario Carvajal (2017). 9 Edición que citaremos en adelante. 1. Introducción De ahí que en María converjan la antropología filosófica, enten dida como punto de vista de la conciencia sobre la fenomenología, y una especie de sensibilidad romántica del narrador por la exuberancia de la naturaleza africana que, en función de una pretensión universalista, imagina e incorpora a su creación lírica sobre la exuberancia de la naturaleza americana, esto por estimarlas ontológi camente análogas en un proceso de emergencia del espíritu en la naturaleza. Adicionalmente se orienta nuestra atención hacia los trazos característicos de per sonajes africanos o afrodescendientes7 –y de su carácter– en María, especialmente a partir de algunas enunciaciones o categorías, en las que la noción de lo africano viene nombrada como adjetivo de origen, como corporeidad o incluso como una forma de subjetividad; de otra parte, lo africano también se materializa en el trabajo de los esclavos y en el modo en que se relacionan con los amos en el marco de ese modelo económico esclavista: desde el trabajo de producir alimentos, como sembrar, cosechar, cuidar y sacrificar los animales, cazar, pescar, hasta otras tareas más del or den doméstico como limpiar o cocinar, pasando por el cuidado y la educación de los niños de su amos, a quienes han de entretener, acompañar, consolar y servir en todo. q p y Finalmente se postula que el trabajo y la subjetividad del esclavo en la visión del amo, junto al bosquejo de relaciones de producción que se ensaya en María, son una refracción de la autoconciencia que le es restituida al esclavo, en el marco dialéctico de las relaciones entre señorío y servidumbre, como las entendió Hegel; ello en tanto suponen el encuentro de dos individualidades en una nueva etapa en la formación 244 María de Jorge Isaacs como subversión del romanticismo en la Nueva Granada de la conciencia histórica del yo y en la búsqueda de la libertad de la autoconciencia. En María se levanta la posibilidad del surgimiento de una conciencia de clase en el esclavo cuando se trazan los contornos de una clase dominante decadente y parasi taria, los amos blancos, que se hunde en sus propias penas y en un vivir superfluo, fantasmagórico. 2. El trabajo, el amor y el odio en las relaciones entre amos y esclavos en María g y La primera referencia textual a la esclavitud se sitúa en el marco del Capítulo III, en el que se describe la cena que en familia tuvo Efraín tras su regreso de Bogotá, al final de la cual «los esclavos levantaron los manteles; uno de ellos rezó el Padre nues tro, y sus amos completamos la oración» (Isaacs 1978: 6). Los esclavos son la fuerza 245 Juan Carlos Herrera Ruiz motriz de la hacienda azucarera del padre de Efraín y parte de su patrimonio como hacendado; la existencia de esclavos en la forma en que se los describe en ese régi men señorial denota la cotidiana jerarquización de las relaciones de producción, así como la evangelización cristiana de la que son objeto. Entiéndase aquí por «relacio nes de producción» el dispositivo social que desencadena las fuerzas de producción y las relaciones sociales de propiedad que de ahí derivan, como lo esbozó Marx en la introducción a su Crítica de la economía política (2008: 284). p ( ) En el capítulo IV, el narrador evoca su niñez cuando se dormía mientras escu chaba «los cuentos maravillosos del esclavo Pedro» (Isaacs 1978: 7). Pedro, uno de los personajes más cercanos a Efraín, tan cercano que le narra cuentos, hace parte de aquellos «esclavos» que más adelante en el capítulo V describe como «bien ves tidos y contentos, hasta donde es posible estarlo en la servidumbre, eran sumisos y afectuosos para con sus amos» (8), en un cuadro casi infantil que pareciera ser, en principio, una apología o idealización del orden social esclavista. Justamente Efraín piensa en Pedro como aquel «buen amigo y fiel ayo» (9), que ya era viejo y a quien no encontró a su regreso; el mismo que le había dicho: «amito mío, ya no te veré más» (9) antes de la partida del amo para Bogotá, pues presentía la cercana muerte. ( ) p p g p p También en el capítulo V aparece Bruno, «un joven negro de notable postura» (9), quien se va a casar con Remigia, obteniendo para tal fin el consentimiento y el regalo del amo (el padre de Efraín); también se alude a la ceremonia del matrimonio, don de se introduce a «Julián, el esclavo capitán de la cuadrilla» (9), quien ostenta una jerarquía más alta entre los esclavos. 10 Esclavo al que se le ha concedido la libertad. 11 Tipo de falda. 2. El trabajo, el amor y el odio en las relaciones entre amos y esclavos en María Se aclara que entre los negros hay «esclavos» y «manumisos» (10).10 y ( ) En el baile del matrimonio emerge otro elemento mestizo: «hermosos bambucos [...] entonado por finas voces de negritos [...] había en sus cantos tan sentida combi nación de melancólicos, alegres y ligeros acordes; los versos que cantaban eran tan extremadamente sencillos, que el más culto diletante hubiera escuchado en éxtasis aquella música semisalvaje» (10). También en este cuadro de la celebración del ma trimonio se describe el traje de los novios, por ejemplo, el de Remigia, como un «fo llao11 de boleros azules, tumbadillo de flores rojas, camisa blanca bordada de negro y gargantilla y zarcillos de cristal color rubí» (10) buscando con ello caracterizar un tipo social a partir de las vestimentas festivas de los esclavos, pero de paso aspectos relacionados con la subjetividad y el deseo del esclavo, que palpita escondido tras el adorno y el atavío. y En el capítulo XV aparece «el negrito Juan Ángel» (23), que «aquietaba» el caballo en el que en medio de la noche Efraín debía ir a buscar al doctor Mayn, en vista de un «acceso» que sufre María por su enfermedad. El mismo personaje «Juan Ángel» reaparece en el capítulo XVI, despertando a Efraín al amanecer, en medio de las angustias de este por el progresivo empeoramiento de la salud de María: «[...] me arrepentí de haber enviado al negrito, quien silbando y tarareando bambucos iba a internarse en la primera mancha del bosque» (29). 246 María de Jorge Isaacs como subversión del romanticismo en la Nueva Granada Nótese en el «me arrepentí» un principio de negación de su conciencia de clase en Efraín, que se hará reiterativo; se trata, en suma, de la aparición de una fisura en el sistema histórico y estético que media las relaciones entre amo y esclavo. Y es justo ahí donde resulta atendible la fórmula que entrega Hegel en el capítulo IV de su Fenomenología de espíritu con respecto al «reconocimiento de la autoconciencia», que es para y depende del reconocimiento mutuo en la autoconciencia de otro ser; ello significa, en función del trazo de un carácter tipificado en Efraín y su medio, que la autoconciencia (de clase) se ha perdido a sí misma pues ahora ella se encuentra como un ser distinto. 2. El trabajo, el amor y el odio en las relaciones entre amos y esclavos en María Pero puede significar, inversamente o en función del carácter del siervo, que la autoconciencia ha suprimido y superado al otro al no reconocerlo como un ser (Wesen), sino que lo que ella ve en la otra autoconciencia es a sí misma (Hegel 2009: 286-287). 12 Crespos rubios. 13 Joven mestiza o mulata. 14 Mochila. 13 Joven mestiza o mulata. 12 Crespos rubios. 13 Joven mestiza o mulata. 14 Mochila. 3. El inicio del fin de la subordinación Aquí se pintan una vez más las tareas asignadas a los esclavos, aunque pareciera que el narrador busca insinuar un contraste entre el modo en que él y su padre tratan a los esclavos con el modo, más severo, en que los tratan otros esclavistas. con no volver!» (43). Aquí se pintan una vez más las tareas asignadas a los esclavos, aunque pareciera que el narrador busca insinuar un contraste entre el modo en que él y su padre tratan a los esclavos con el modo, más severo, en que los tratan otros esclavistas. Ya en el capítulo XXII, «el negrito Juan Ángel» es objeto de burlas por parte de Braulio, por haber tenido miedo de regresar al sitio en que estaba prevista la caza del tigre: «¿Con que no volviste por miedo? –le pregunto Braulio riendo [...] Este dio rienda suelta a una estrepitosa risotada, diciéndole al fin al negrito aterrado: –¡Y es tuviste todo el día metido entre estos matorrales como un conejo!» (50). Las burlas continuaron y Braulio hizo parecer a Juan Ángel como un cobarde (51), y justamente mientras contempla la escena Efraín reflexiona: Tenía yo un cariño especial al negrito: él contaba a la sazón doce años; era simpático y casi pudiera decirse que bello. Aunque inteligente, su índole tenía algo de huraño. La vida que hasta entonces había llevado no era la adecuada para dar rienda suelta a su carácter, pues mediaban motivos para mimarlo. Feliciana, su madre, criada que había desempeñado en la familia funciones de aya y disfrutado de todas las consideraciones de tal, procuró siempre hacer de su hijo un buen paje para mí. Más fuera del servicio de mesa y de cámara y de habilidad para preparar el café (51). La composición presenta al esclavo en su minoría de edad e inmadurez intelectual, aunque no solo por los años con que cuenta, sino también por su condición de ne gro a quien el amo toma cariño especial, casi compasión paternalista. 15 A propósito, G. Mejía comenta que Juan Ángel habría sido un personaje real que efectivamente estuvo al servicio de la familia del autor hasta 1850, cuando «se fugó de la casa de los Isaacs» (en Isaacs 1978: 51). 3. El inicio del fin de la subordinación Más personajes negros reaparecen en el capítulo XIX, en el marco de la visita de Efraín a la casa de Emigdio, un joven amigo de su clase: «Vinieron en mi auxilio dos negros, varón y mujer: él sin más vestido que unos calzones, mostraba la espalda atlética luciente con el sudor peculiar de la raza; ella con follao de fula azul y por camisa un pañuelo anudado hacia la nuca y cogido con la pretina, el cual le cubría el pecho» (Isaacs 1978: 34). Nótese una vez más el foco sobre el vestido característi co de los esclavos y sobre su fisionomía desde una mirada determinista: «el sudor particular de la raza». A la misma pareja se referirá a renglón seguido, para exaltar que «no solo el negro sino también su compañera iba armada con rejos de enlazar» (34) y que eran expertos en el manejo de potros, tareas que aparecen como propias de la servidumbre. En el saludo de Efraín con Emigdio, este último le responde que por ser día de matanza «estaba racionando a los negros, que es una friega» (36), aludiendo al tipo de faenas productivas de las que se ocupan los esclavos, caracterizando en particular a uno: «¡Chotó! –gritó; y a poco se presentó un negrito medio desnudo, pasas monas12 y un brazo seco y lleno de cicatrices» (36). La imagen de «Choto» evoca el sufrimiento físico y las marcas que dejan el tipo de tareas destinadas a los esclavos. En una con versación subsiguiente a la faena, Emigdio le confiesa secretamente a Efraín haberse «enamorado locamente de una ñapanguita13», romance que debe ocultar a su padre pues «no era una señora» (39), esto es, no completamente blanca, configurándose así, una vez más, la estratificación socio-racial característica de la época y de ese mundo esclavista. El negrito Juan Ángel reaparece en el capítulo XXI, nuevamente acompa ñando a Efraín en una visita a José y su sobrino Braulio, que tenía como propósito planear una jornada de caza a un tigre; el papel del «negrito» consistía en cargar una mochila con legumbres: «Y tú, –qué haces ahí que no te largas, negritico –dijo José a Juan Ángel. Carga con la guambía14 y vete, para que vuelvas pronto [...] ¡Cuidado 247 Juan Carlos Herrera Ruiz con no volver!» (43). 3. El inicio del fin de la subordinación No se debe soslayar, sin embargo, el temporal foco en la personalidad del esclavo, buscando de algún modo darle un matiz algo más elevado con relación a otros esclavos y aproxi mándolo, si cabe la expresión, a los rasgos de la personalidad que también tendría un blanco,15 pero de paso anuncia la pérdida de autoconciencia por parte del amo: empieza a dejar de ver al siervo como tal debido a la existencia de una autocon ciencia que ya le atribuyó. También en la cita anterior se introduce otro personaje, «Feliciana», quien, como se verá más adelante, nació en África. Á La imagen del muchachito temeroso, Juan Ángel, se refuerza poco después, cuan do le ordenan vaciar el contenido de una bolsa que le hicieron cargar, en la que estaba junto a otros restos la cabeza del tigre que había sido recientemente cazado: [...] mandó al negrito que los sacase; y trataba de hacerlo así cuando dio un grito de terror y un salto de venado sorprendido. Cada uno de los circunstantes quiso averiguar lo que había pasado. Juan Ángel, de espaldas contra la pared, los ojos tamaños y señalando con los brazos extendidos exclamó: –¡El tigre! (52). Una primera interpretación de la polisemia contenida en esta escena pondría de manifiesto que, entre amo y esclavo, el papel del «salvaje» corresponde decidida mente al primero, por lo cruel de su conducta; pero vista bajo el lente de Hegel, encontramos que el papel del siervo se verifica en aquel que por salvar la vida re nuncia a la libertad: «Es sólo en el exponer y en el arriesgar la vida mediante lo que se acredita la libertad» (Hegel 2009: 293). El miedo resaltado de Juan Ángel y su 248 María de Jorge Isaacs como subversión del romanticismo en la Nueva Granada reacción sensible hacia la muerte, incluso la de un animal, es un matiz del carácter que se ensaya del ser dominado: el jovencito antepone a su deseo el temor a morir. 3. El inicio del fin de la subordinación Inversamente, el lente dialéctico permite concebir que para el amo blanco, Braulio tomemos por caso, el deseo es más fuerte que el temor a morir y por eso es él quien somete al esclavo, cuya consciencia y deseo sustrae, pues no pueden existir por fuera de su propia autoconciencia, es decir, que el papel del señor se verifica, de nuevo ci tando a Hegel, en «la conciencia que es para sí» (294), o aquel que niega la existencia de la conciencia y del deseo del otro. No obstante, tras este cuadro de derrota moral del negro en la escena de caza, emerge un desasosiego en la conciencia del narrador, señal de la crisis moral de su clase, al sopesar el alcance de su relación (la de Efraín) con un esclavo ahora provisto de autoconciencia y de deseo: comprende con amar gura que, en el encuentro de sus voluntades, la más débil, es decir, la de Juan Ángel, había sido la que había amado más, influenciada por sus emociones. q p En El ser y la nada Sartre traza un esquema relacional análogo, recogiendo justa mente la idea hegeliana de la dialéctica del amo y el esclavo, para evidenciar que cada sujeto construye una relación con el otro en forma asimétrica. En este sentido las palabras, las actitudes, las acciones y las decisiones, hacen parte de un sistema complejo a través del cual se mantiene el equilibrio de las relaciones humanas y de las estructuras de poder a lo largo de la historia, relaciones expresadas sobre todo desde la corporeidad, pues «ese objeto que otro es para mí, y ese objeto que yo soy para otro, se manifiesta como cuerpos» (Sartre 1979: 354). En el caso de María son ellos mismos, los cuerpos, el método de confirmación de la existencia material de los esclavos al que apela reiterativamente la conciencia del amo. q p En plena consonancia con este principio de la asimetría relacional ligada a lo cor poral, nótese el comentario de Efraín en cita precedente sobre el cuerpo de Juan Ángel: «casi pudiera decirse que bello”, o su inquietud frente a la vida del «negrito», vida que, hasta entonces, «no era la adecuada para dar rienda suelta a su carácter» (Isaacs 1978: 51). ¿A cuál carácter, o más bien, a qué deseo no expresado en la novela se refiere la voz narrativa? 3. El inicio del fin de la subordinación En cualquier caso, allí se concretiza una ruptura liminar con el viejo régimen señorial, de jerarquías y de conciencias, siguiendo de nuevo a Sartre, «la esencia objetiva de nuestra existencia», que implica la aceptación del otro y su deseo, como fundamento de nuestra propia esencia (1979: 439); y más allá, se trata de una aceptación de la libertad de cara a los cambios que inevitablemente trae consigo el paso del tiempo, o Zeitgeist, dicho en clave hegeliana. g p p g g En los capítulos XXV y XXVI aparece nuevamente Juan Ángel, cumpliendo labo res de servidumbre y cuidado de animales: «apostado dentro de la quebrada de la Honda con dos de los cuatro perros que he mandado traer de Santa Elena [...] para evitar que se nos escape el venado a la novillera [...] Braulio, después de ensillarnos los caballos ayudado por Juan Ángel se puso en marcha con éste para desempeñar la parte que le tocaba de la batida» (Isaacs 1978: 67). En el capítulo XXVII se introduce otro personaje afrodescendiente, Estefana, quien puso flores en el estanque en que Efraín tomaba su baño: «Era Estefana una negra de doce años, hija de esclavos nuestros: su índole y belleza la hacían simpática para todos. 249 Juan Carlos Herrera Ruiz Tenía un afecto fanático por su señorita María» (72-73). No deja de llamar nuevamente la atención el énfasis en la «belleza» de la esclava, como reafirmación de la predomi nancia de lo corpóreo en las relaciones de producción. Piénsese al punto en la figura de la niña que perfuma el agua en la que Efraín toma el baño, transferencia del propio deseo al acto de servidumbre: el señor se satisface con el goce y el disfrute de las cosas objeto de deseo, y para ello interpone al siervo entre su deseo y las cosas (la materia) del mundo; el señor es «el poder [Macht] sobre ese ser, pues él en la lucha demostró que sólo él consideraba ese ser como lo negativo» (Hegel 2009: 295). En Efraín, ese patrón de relación ha entrado en crisis y ha perdido el poder al no negar el deseo y la conciencia de la joven sierva. 16 Visibilizar por esta vía las estructuras de poder y las jerarquías vigentes, aun entre los negros, puede interpretarse tentativamente como un matiz diferencial «subversivo» de la novela. Véase un eco con temporáneo de esta simbología en el cuento «Buen viaje, señor presidente» (1992) de Gabriel García Márquez, en la que utiliza una figura similar al representar el pasado lejano de un personaje negro, Lazara, esposa de Homero, como la descendiente de un rey Yoruba. 3. El inicio del fin de la subordinación Se corresponde con la figura del amo atrapado en el cono de sombras de una emergente «conciencia desgraciada», como la llama Hegel, pues se encuentra disociada de sí misma y constituye una contradicción de su esencia, de su ser [Wesen], «sin más remedio que tener en una conciencia siempre también la otra» (313). 4. La Nouvelle de Nay y Sinar dentro de María La orden que da Efraín para que aumentaran «el número de esclavas que le servían» a su sierva hace de preludio a la presentación de la vida pasada de Feliciana, quien en su nativa África había sido, como se verá más adelante, una noble en el equivalente de la nomenclatura de los blancos;16 250 María de Jorge Isaacs como subversión del romanticismo en la Nueva Granada de otra parte, se entiende que Feliciana había estado encargada del cuidado de los niños de la casa y que la relación con estos había sido muy cercana.17 y q y A renglón seguido, el narrador reproduce la historia de Feliciana que «con rústi co y patético lenguaje», entretuvo algunas veladas de su infancia (116). La historia cuenta el enfrentamiento entre dos naciones africanas: los Achanti y los Achimis. Antes de llegar a América como esclava, Feliciana se llamaba Nay, y era hija de Magmahú, jefe guerrero al servicio del rey Achanti Say Tuto Kuamina, hasta el mo mento en que a manos del propio Magmahú muere Orsué, caudillo este último de los Achimis y padre de Sinar (117). y p ( ) Magmahú fue objeto de envidia e intrigas por parte de otros guerreros que veían con desconfianza el favor que este obtuvo del rey Achanti, quien le había encargado los ejércitos para pelear contra los ingleses, a los que casi logra derrotar de no ser por la traición y la deserción de algunos. Por esta razón el rey Achanti hace la paz con los ingleses y degrada a Magmahú (117). Por su parte, el joven Sinar fue hecho esclavo por los Achanti y puesto al servicio de Nay, única en conocer el origen no ble de aquel. Entre ambos surge secretamente el amor y en una ocasión propicia Sinar invita a Nay a huir con él hacia su país y le promete que la hará su reina y su única mujer. Nay le pide que espere a recibir el consentimiento de su padre. Sinar marcha a la guerra contra los ingleses al servicio de Magmahú y es herido (118-119). Desilusionado Magmahú por no ganar la guerra decide abandonar aquel reino y ex patriarse en Gambia, pero antes quiere hacer un ritual en el río Tandó para conjurar su mal destino y sacrificar allí algunos guerreros, entre estos a Sinar. 4. La Nouvelle de Nay y Sinar dentro de María En el capítulo XXXVIII, mientras el narrador evoca junto a las hermanas y María los relatos que hacía su madre sobre su tierra natal, el Chocó, aparece de nuevo Juan Ángel: «interrumpió aquella conversación la llegada de Juan Ángel, quien venía del pueblo trayendo la correspondencia» (Isaacs 1978: 110). Una de las cartas contenía la noticia que anticipaba la partida de Efraín para Europa. En el capítulo XXXIX apare ce nuevamente Feliciana, la madre de Juan Ángel, quien se encontraba agonizante en una de las haciendas de la familia: «Llevarás a Juan Ángel, para que vea a su madre» (114), dice la madre de Efraín a este. Este capítulo cierra con el compromiso formal entre Efraín y María, lo que sirve de preludio al episodio intercalado en los capítulos siguientes (XL, XLI, XLII y XLIII), que se ocupan de la historia de Nay (Feliciana) y Sinar, su marido africano, con lo que se alude implícitamente al romance de Efraín y María, ello como parte de un relato exótico trasplantado de África a América, que condensa de paso la dialéctica del amo y el esclavo en su sentido más purista, esto es, como motor de la historia y de la cultura. y La referida historia se inicia cuando Efraín llega «a las haciendas» y se entera de parte del médico que Feliciana estaba por morir: «Di orden para que aumentaran el número de esclavas que le servían; hice colocarla en una pieza más cómoda» (115). Pero ¿quién es Feliciana?: «Aquella mujer iba a morir lejos de su patria; aquella mujer que tan dulce afecto me había tenido desde que fuera a nuestra casa; en cuyos brazos se durmió María tantas veces siendo niña...» (116). 17 En una aproximación a la diáspora africana narrada desde varios puntos de vista en la tradición no velística colombiana, Henao-Restrepo (2020: 74) recoge memorias de «la educación de niños que des pertaron al mundo de la mano de nanas negras», citando entre otros ejemplos María y las novelas y cuentos de Tomás Carrasquilla, pero alude también a Casa Grande & Senzala de Gilberto Freyre y a Go Down, Moses, antología de relatos que Willian Faulkner dedicó como muestra de gratitud a su nana negra Mammy Caroline Barr. 4. La Nouvelle de Nay y Sinar dentro de María paisaje metafísico que enmarca el drama de Atala y Chactas en Atala, entre otras ana logías fenomenológicas con la novela de Chateaubriand en María, que se explican en parte por la influencia que tuvo aquel entre las elites letradas latinoamericanas del siglo XIX18 (Altamirano & Myers 2008), pero en especial sobre Isaacs, como ad virtió agudamente Brown en el siguiente comentario a la edición inglesa de María: «The Frenchman idealizes the North American Indian, while his Colombian admirer crosses the Atlantic in the opposite direction to idealize some noble West Africans» (1947: 326). ( ) Pero más allá de las afinidades electivas de Isaacs con el «genio del cristianis mo», así se lo nombra en el capítulo XII, cabe resaltar tanto en Atala como en María la ligazón entre el sentimiento del paisaje metamorfoseado, en un ser mujer, y la pasión: «como influencia y como correspondencia, de intercambio entre la mujer y la naturaleza», como señala perspicazmente Vinciguerra (1968: 55), al sacar a la luz esta simbología antropológica en María y al significarla en clave hegeliana, esto es, como emergencia del espíritu, de mujer, en la naturaleza, pero también como relación de producción y asunción de autoconciencia: «Puede decirse que constituye uno de los elementos esenciales de la economía y del porvenir de la acción interior» (56). Sin embargo, y al margen de que ciertos simbolismos en María evoquen cua dros de carga emotiva pintados en Atala, esto es, idilios entre seres inocentes, amores que se enfrentan a la muerte, naturaleza exuberante, conviene recordar a este punto que mientras Chateaubriand se limita a reafirmar, desde la ficción literaria, el mito del buen salvaje que busca la felicidad lejos de los vicios de la civilización, Isaacs fija la acción de los jóvenes amantes en África, donde históricamente se inicia el proceso de la esclavitud y sus horrores (con la captura de Nay y Sinar) para ulteriormente trasladarse a América. Allí se concretiza la importancia conferida al tema en la nove la de cara a un debate social, político y económico que interesa a la fundación de la nación a lo largo del siglo XIX. 18 En 1850 se publica la traducción española del Ensayo sobre las revoluciones, obra en la que el Vizconde de Chateaubriand formula una serie de interrogantes sobre la naturaleza de las revoluciones y sus consecuencias en la vida de los pueblos a lo largo de la historia, incluyendo desde luego la francesa. 4. La Nouvelle de Nay y Sinar dentro de María Al enterarse Nay pierde transitoriamente el conocimiento y menciona el nombre de Sinar, Mag mahú descubre el secreto amor entre los jóvenes y los perdona; por su origen noble acepta a Sinar en la familia y lo convida a la travesía a él también para escapar de Cumasia, tierra de los Achanti, hasta llegar al país de los Kombu-Manez, en Gambia, donde los recibieron, y de paso pusieron a Magmahú y a Sinar a su servicio en la guerra contra los Cambez, enemigos que solían entregar a sus prisioneros de guerra, y a veces también a sus súbditos, como esclavos a los blancos, a cambio de «armas, pólvora, sal, fierro y aguardiente» (119-120). Estando en el país de los Kombu-Manez, a la sombra de un árbol baobab, Nay y Sinar conocen a unos misioneros franceses que venían del país de los Achimis, de donde era Sinar; los misioneros buscaban enterrar el cadáver de un sacerdote muerto y Sinar ayuda a que los jefes Kombu-Manez consintieran. Estos misioneros logran de paso convertir a Nay y a Sinar al cristianismo antes de la boda de estos (121-123). En esta figura de la cristianización de nativos, como en todo el relato que narra la odisea de los amantes africanos en su desventurado viaje hacia la muerte, se configura un nuevo trasunto metamorfoseado, esta vez del cuadro natural y del 251 Juan Carlos Herrera Ruiz paisaje metafísico que enmarca el drama de Atala y Chactas en Atala, entre otras ana logías fenomenológicas con la novela de Chateaubriand en María, que se explican en parte por la influencia que tuvo aquel entre las elites letradas latinoamericanas del siglo XIX18 (Altamirano & Myers 2008), pero en especial sobre Isaacs, como ad virtió agudamente Brown en el siguiente comentario a la edición inglesa de María: «The Frenchman idealizes the North American Indian, while his Colombian admirer crosses the Atlantic in the opposite direction to idealize some noble West Africans» (1947: 326). 19 El reino de este mundo (1949) del cubano Alejo Carpentier, Changó, el gran putas (1983) del colombiano Manuel Zapata Olivella, A gloriosa familia (1997) del angoleño Artur Carlos Pepeleta y Un defeito de cor (2006) de la brasileña Ana María Gonçalves. 20 Conocida también como «Novela histórica contemporánea», es una corriente a partir de la cual se renueva el interés por la reflexión histórica en la novelística latinoamericana. Para conocer en detalle aspectos teóricos y metodológicos a los que responde la categoría recomendamos el libro de Magda lena Perkowska: Historias híbridas. La nueva novela histórica (1985-2000) ante las teorías posmodernas de la historia (2008). 5. Evocación y memoria de África en el lado de Acá En el marco de los estudios culturales que abordan novelas históricas con África como tema, pertinente es apelar aquí al trabajo de Daiana Nascimento dos Santos, El océano de fronteras invisibles (2015), puntualmente en su tercer capítulo a cuyo tí tulo aludimos en este acápite. Allí se abordan cuatro novelas de siglo XX19 en su relación con las imágenes de África, a partir de una memoria colectica que se recons truye sobre la base del discurso oral. Al margen de que pertenezcan al siglo XX, la relectura de estas obras entrega una valiosa herramienta de cara a dilucidar un aspecto fundamental de la vida de Nay, narrada por ella misma: nos pone de frente a la validez de la tradición oral como forma de evocación de África, que se expresa con mayor o menor acento dentro de la tradición literaria poscolonial americana, ello no solo porque alimenta la multi plicidad de connotaciones de la esclavitud que surgen con la novela histórica desde el siglo XIX, tanto en lengua española como portuguesa, sino también porque se in corpora de manera crítica al fluido diálogo entre historia y literatura que caracteriza este género de narrativa. g Pero más allá de la referida reconstrucción del pasado a partir del discurso oral, el análisis de Nascimento dos Santos incorpora un elemento adicional en favor de una relectura de la ficción novelada de los autores que aborda –y por extensión también de Isaacs– en clave de resistencia cultural: la historia de Nay puede ser entendida como reminiscencia de las diásporas, y si se quiere también como invocación de una lucha social que inicia justamente en el «reunir los fragmentos dispersos de nuestra expe riencia en el mundo», como anota Sañudo Caicedo (2019: 98) en su tentativa por sacar a la luz un diálogo estético y político entre María y la novela de la también colombiana Adelaida Fernández Ochoa Afuera crece un mundo (2017), en la que se «rescata» a Nay, la esclava manumisa en la ficción de Isaacs, para convertirla en mujer autónoma de su cuerpo y de su destino, protagonista de una historia narrada en primera persona y de una lucha contra la esclavitud que contempla el viaje de regreso a Gambia. 4. La Nouvelle de Nay y Sinar dentro de María g g Terminada la fiesta por el matrimonio de Nay y Sinar, el pueblo es atacado por los enemigos Cambez, quienes dan muerte a Magmahú y toman prisioneros a Nay y a Sinar para llevarlos al mar y entregarlos a los europeos como esclavos; los aman tes son separados para siempre y Nay va a dar al Darién colombiano, allí es entrega da a un irlandés y a su mujer que le toma cariño y entiende que Nay se ha cristiani zado (Isaacs 1978: 125-126). Nay descubre que está en embarazo de Sinar, su hijo será Juan Ángel, quien na cerá allí mismo. Se entera también de que en aquel país, desde 1821, los hijos de los esclavos serían libres a partir de los 18 años. Esta situación coincide con el tránsito por aquel lugar de un inglés y una pequeña niña, ellos son el padre de Efraín y su sobrina, María, que entonces se llamaba aun Ester por su origen judío; regresaban de Jamaica e iban camino al Cauca, tras la desaparición de los padres de aquella. El padre de Efraín se compadece del destino que le espera a la joven mujer y decide 252 María de Jorge Isaacs como subversión del romanticismo en la Nueva Granada comprarla y llevarla consigo, hacer de ella su aya y cambiarle el nombre por el de Feliciana (128-130). 5. Evocación y memoria de África en el lado de Acá j g Claramente afín a los rasgos que definen la nueva novela histórica latinoamerica na (NNH)20, Afuera crece un mundo marca un punto de inflexión con respecto a no velas como María, en las que «lo subversivo» llega apenas hasta visibilizar las rela ciones de poder entre amos y esclavos, insinuándose en sus rupturas, pero sin explí citamente controvertir o contestar dicho sistema social. En la novela de Fernández 253 Juan Carlos Herrera Ruiz Ochoa, así como en las que aborda Nascimento dos Santos en su estudio, se relee y se reescribe la historia oficial siguiendo a África como un hilo que permite reconstruir la experiencia del esclavismo, conjurar la alteración de sus significados y resignificar su pasado traumático con lo que entrega la memoria: no solo a través del cuento que en la enunciación del blanco cruza el océano para enfocarse temporalmente en el personaje secundario de Nay, como en el procedimiento que emplea Isaacs, sino para «volver a imaginar el mundo africano desde las prácticas discursivas ances trales, con el propósito de resistir simbólicamente a la hegemonía del discurso del poder colonial, subvirtiéndolo mediante sus propias subjetividades» (Nascimento dos Santos 2015: 53). ) Terminado el relato de la vida de Nay, Efraín describe la muerte de Feliciana: «luego las esclavas la vistieron y la colocaron en un ataúd, cubierta desde la gargan ta hasta los pies de un lino blanco, fue puesta en una mesa enlutada [...] Juan Ángel a la cabecera de la mesa derramaba lágrimas sobre la frente de su madre» (Isaacs 1978: 132). No podemos soslayar aquí, a propósito de la obra de Fernández Ochoa, que en esta Juan Ángel es nombrado como «Sundiata», nombre africano, cuya resti tución de la libertad –y de la autoconciencia si lo decimos en clave hegeliana– pasa necesariamente por el retorno a «su única patria posible» (Sañudo Caicedo 2019: 111), junto a su madre, en busca de Sinar, padre y esposo, pero también de la lengua, de los rituales y de los saberes africanos, configurando así un «canto paralelo», que toma distancia frente a la novela de Isaacs, pero también y sobre todo frente al dis curso hegemónico de la historia oficial. 21 «Boga», el que rema, figura muy común en la literatura colombiana del siglo XIX. El canto que entonan los bogas «Cortico y Laureán» en este capítulo se corresponde intertextualmente con uno de los versos del poema «Canción del boga ausente», del poeta afrocolombiano Candelario Obeso; este y otros poe mas aparecerán publicados en 1877 como parte de una antología titulada Cantos populares de mi tierra. 5. Evocación y memoria de África en el lado de Acá Este trazo opuesto al del tiempo vacío y he terogéneo que se impuso desde la ficción fundacional decimonónica, agrega Sañudo Caicedo, supone la incorporación de una mirada heterogénea sobre el imaginario construido en torno a «lo negro africano» y a todo el eje articulador de los discursos narrativos en torno a la esclavitud y a las leyes abolicionistas en el siglo XIX. Final mente, y siempre en ocasión del funeral de Feliciana, Efraín agrega: «Mandé orden al capitán de la cuadrilla de esclavos para que aquella noche la trajese a rezar a casa [...] Terminado el rosario, una esclava entonó la primera estrofa de una esas salves llenas de dolorosa melancolía y los desgarradores lamentos de algún corazón escla vo que oró» (Isaacs 1978: 132). Nótese, además, cómo el amo ordena que los esclavos hagan ellos mismos el ritual funeral y oren, pero él mismo no manifiesta hacerlo, aunque da a entender que cumple con el deber de inducir a la pena como parte de una formación y de un aprendizaje cristianos por el que deben pasar los esclavos. En el carácter y en la figura de Feliciana, tanto la temprana liberación por parte del pa dre de Efraín, como el homenaje funerario de este son refracción de la fragmentación de una conciencia (de clase) que debería ser «indivisa», como puntualiza Hegel, pero que ahora aparece duplicada (o doble): «ella misma es el mirar de una autoconcien cia en otra, y ella misma es ambas» (2009: 314). y Pero al margen de esto, la muerte solitaria de Nay pareciera una anticipación de la muerte de María, y la muerte de María, como intuimos, no es otra que la muerte de aquel régimen señorial. Luego de cumplido el ritual «Los ocho esclavos que condu cían el cadáver, y yo, nos pusimos en marcha [...] El ataúd estaba ya en el fondo de 254 María de Jorge Isaacs como subversión del romanticismo en la Nueva Granada la fosa; uno de los esclavos le echó encima la primera palada de tierra» (Isaacs 1978: 133). Los esclavos finalizan el trabajo mortuorio y del enterramiento, y el número de «ocho», entre otros tantos esclavos que se mencionan, da cuenta de la cantidad de la servidumbre. 5. Evocación y memoria de África en el lado de Acá Finalmente, en el capítulo LVII se enmarca el regreso de Efraín a la patria tras la noticia de la enfermedad de María; en el viaje tiene que atravesar por paisajes y parajes que hacen lenta e interrumpida la marcha. En la etapa final del viaje por río hacia al Valle aparece la nostalgia por «los bogas21 que conducían a las canoas mi equipaje [...] ¡Cortico! ¡Laureán!... cuidármelo mucho como cosa mía. –Sí mi amo– contestaron a dúo los dos negros» (173). Las canciones de los bogas fijadas como intertextos en María tampoco escapan a la atención de Nascimento dos Santos, a los que lee como relatos emergentes de tradiciones culturales en función de una autoafirmación, de «resistencias, enunciaciones y miradas que provienen de ex periencias en común, vinculadas a través de la trata negrera, de la descolonización, del sometimiento y de los cuentos que cruzaron los océanos» (2015: 66-67). 6. Conclusión En el mismo plano dialéctico de la relación entre amo y siervo, aunque solo intuida por el amo, la certeza de «lo verdadero» reside en el trabajo que realiza el siervo, pues solo el trabajo pone en contacto con el mundo real y es «por ese medio como la conciencia viene a sí misma» y descubre la relación asimétrica que separa al amo del siervo (298). de la voz narrativa, la del amo, lo que no significa, empero, que el resultado de esta introspección no sea un anticipo, o si se quiere un preludio artísticamente elabora do, de la cercana e inevitable inversión de roles en la dialéctica del amo y el esclavo. En el mismo plano dialéctico de la relación entre amo y siervo, aunque solo intuida por el amo, la certeza de «lo verdadero» reside en el trabajo que realiza el siervo, pues solo el trabajo pone en contacto con el mundo real y es «por ese medio como la conciencia viene a sí misma» y descubre la relación asimétrica que separa al amo del siervo (298). ( ) En la novela subyace la posibilidad de una revisión del discurso histórico hege mónico en torno al esclavismo en el marco de la fundación de la nación, por medio de una reivindicación de la recuperación de la tradición oral implícita en la ficción literaria, en la que se reconstruye la memoria colectiva, como forma de resisten cia cultural, desde el punto de vista y a partir de la subjetividad de sus personajes «negroafricanos». 6. Conclusión Hasta aquí se ha focalizado la atención en una parte representativa de los persona jes afrodescendientes a los que la voz narrativa atribuye un nombre en relación con el trabajo y el deseo del amo, pero a los cuales también se atribuye un deseo y una autoconciencia. Cabe aclarar que a lo largo del relato se enuncian muchas veces de manera genérica a «los o las esclavos(as)», «los o las negros(as)» sin especificar nombres propios; en otras ocasiones la referencia al esclavo o esclava, al negro o la negra, aluden a personajes ya conocidos y nombrados, como por ejemplo «Juan Ángel», al que en repetidas oportunidades se le nombra simplemente como «el negrito». g En las relaciones de producción que se configuran entre amos y esclavos en la novela María estos últimos realizan prácticamente la totalidad de las funciones y del trabajo que determinan la riqueza y la apetencia de los amos. Los esclavos, al haber sacrificado ellos mismos su deseo y libertad al miedo a la muerte, y aun privados de una autoconciencia, asumen todavía la posición de quien transforma la materia en sus diversas manifestaciones, poniendo en marcha con ello los ciclos de producción y reproducción de los que se alimenta la cultura, y en última instancia, también la historia, entendida como devenir material. Recuérdese, al punto, que para Hegel el siervo es aquel que por salvar la vida renuncia a la libertad: «Es sólo en el exponer y en el arriesgar la vida mediante lo que se acredita la libertad» (Hegel 2009: 293). El desarrollo dialéctico que se da paralelo a la transformación de la materia pro voca una ruptura del tiempo histórico que incide en la subjetividad, entiéndase en clave hegeliana, en la autoconciencia, que media las relaciones entre amos y esclavos en la novela María. Dicha ruptura es percibida y racionalizada desde la conciencia 255 Juan Carlos Herrera Ruiz de la voz narrativa, la del amo, lo que no significa, empero, que el resultado de esta introspección no sea un anticipo, o si se quiere un preludio artísticamente elabora do, de la cercana e inevitable inversión de roles en la dialéctica del amo y el esclavo. Agradecimientos Este artículo deriva del proyecto de investigación «Relaciones de producción escla vista en la literatura colombiana del siglo XIX», desarrollado en el marco de una es tancia posdoctoral de su autor en la Professur für Romanische Literaturwissenschaft / Hispanistik (Institut für Romanistik) de la Facultad de Filosofía y Ciencias de la Cultura (Geistes- und Kulturwissenschaften, GuK) de la Universidad de Bamberg, Alemania, en 2021, bajo la dirección del Prof. Dr. Enrique Rodrigues-Moura y el auspicio del Servicio Alemán de Intercambio Académico (Deutsche Akademische Austauschdienst, DAAD). Referencias bibliográficas ltamirano, Carlos – Myers, Jorge (2008), Historia de los intelectuales en América Latina I. La ciudad letrada, de la conquista al modernismo, Buenos Aires: Katz. q Brown, Donald F. (1947), «Chateaubriand and the Story of Feliciana in Jorge Isaac q rown, Donald F. (1947), «Chateaubriand and the Story of Feliciana in Jorge Isaacs’ María», Modern Languages Notes 62/5, 326-329. g g / Chateaubriand, François-René (1980) [1801], Atala/René, Madrid: Magisterio. ( ) g Fernández Ochoa, Adelaida (2017) [2015], Afuera crece un mundo, Bogotá: Seix Barral García Márquez, Gabriel (1992), Doce cuentos peregrinos, Buenos Aires: Suramericana Gutiérrez Girardot, Rafael (2011), Ensayos de literatura colombiana I, Medellín: Edicio nes UNAULA. Hegel, Georg Wilhelm Friedrich (2009) [1807], Fenomenología del espíritu, Valencia: Pre- Textos. Henao-Restrepo, Darío (2020), «Improntas africanas: la negredumbre en la novela colombiana», Revista Ciencias Sociales 30, 73-95. Henríquez Ureña, Pedro (1994) [1949], Corrientes literarias en la América Hispánica, México: F.C.E. Isaacs, Jorge (1978) [1867], María, Caracas: Biblioteca Ayacucho. Marx, Karl (2008) [1859], Contribución a la crítica de la economía política, México: Siglo XXI Editores. 256 María de Jorge Isaacs como subversión del romanticismo en la Nueva Granada Perkowska, Magdalena (2008), Historias híbridas. La nueva novela histórica (1985-2000) ante las teorías posmodernas de la historia, Madrid: Iberoamericana/Vervuert. Á p Pineda Botero, Álvaro (1999), La fábula y el desastre. Estudios críticos sobre la nove colombiana 1850-1931, Medellín: EAFIT. Rueda Enciso, José Eduardo (2007), «Jorge Isaacs: de la literatura a la etnología», Bole tín de Antropología Universidad de Antioquia 21/38, 337-356. Santos, Daiana Nascimento dos (2015), El Océano de fronteras invisibles: relecturas sobre (¿el fin? de) la esclavitud en la novela contemporánea, Madrid: Editorial Verbum. Sañudo Caicedo, Juana M. (2019), «Fisuras Heterotópicas en la nación: María (Isaacs) y Afuera crece un mundo (Fernández)», Communitas 3/6, 98-118. Sartre, Jean-Paul (1979) [1943], El ser y la nada, Buenos Aires: Losada. Sommer, Doris (2004), Ficciones fundacionales. Las novelas nacionales de América latina, Bogotá: F.C.E. Spinoza, Baruch (2001) [1677], Ética: demostrada según el orden geométrico, Madrid: Alianza. Vinciguerra, Marie-Jean (1968), «La “Atala” de Chateaubriand y la “María” de Isaacs», Ideas y Valores Universidad Nacional 30, 53-61. Zapata Olivella, Manuel (1966), «María, testimonio vigente del romanticismo ameri cano», Letras nacionales 14, 14-44. 257
https://openalex.org/W4297052478	http://psiholoska-obzorja.si/arhiv_clanki/2022/educ22_Versteeg.pdf	English	null	Creating education that fits students: Present-Day Challenges for the EFPA Standing Committee on Psychology in Education	Psihološka obzorja	2,022	cc-by	3,904	Creating education that fits students: Present-Day Challenges for the EFPA Standing Committee on Psychology in Education Margreet Versteeg School psychologist in the Netherlands, member of the Dutch Association for Psychologists, NIP, Abstract: This article gives information about the Standing Committee on Psychology in Education (SC PiE), the history of the SC PiE and its organisation. It also describes the aims and the work of the SC PiE. The SC PiE focuses on the psychologist’s role in the educational system, the psychologist’s training in the educational system related to the Specialist EuroPsy Certificate on Educational Psychology, the importance of inclusion in education and evidence-based knowledge and processes. The SC PiE gives information and promotes these topics through publications, symposia and presentations. The committee undertakes some limited research. The activities are described in this article. The EFPA Standing Committee Psychology in Education has been invited by the Slovenian Psychologist’s Association to contribute to a volume of their Scientific and Professional Journal “Horizons of Psychology” (2022) with the theme “Psychology in Education: Challenges, choices and changes”. The topic comprises articles about psychology in education from European countries. The special topic is issued on the occasion of the European Congress of Psychology, ECP, 2022 in Ljubljana. The theme of the ECP is "Psychology as the Hub Science: Opportunities and Responsibility". Keywords: EFPA Standing Committee on Psychology in Education, school psychologist, educational psychology, inclusion, evidence- b d k l d Keywords: EFPA Standing Committee on Psychology in Education, school psychologist, educational psychology, inclusion, evidence- based knowledge Psihološka obzorja / Horizons of Psychology, 31, 448–452 (2022) © Društvo psihologov Slovenije, ISSN 2350-5141 Mnenje / Opinion Psihološka obzorja / Horizons of Psychology, 31, 448–452 (2022) © Društvo psihologov Slovenije, ISSN 2350-5141 Mnenje / Opinion Psihološka obzorja / Horizons of Psychology, 31, 448–452 (2022) © Društvo psihologov Slovenije, ISSN 2350-5141 Mnenje / Opinion DOI: 10.20419/2022.31.552 CC: 3500, 3410 UDK: 37.015.3 Naslov/Address: ga. Margreet Versteeg, EFPA Sc PiE, NIP the Netherlands, Arthur van Schendelstraat 650 , 3511 MJ Utrecht, The Netherlands, e-mail: mmbversteeg@hotmail.nl ga. Margreet Versteeg, EFPA Sc PiE, NIP the Netherlands, Arthur van Schendelstraat 650 , 3511 MJ Utrecht, The il b t @h t il l Naslov/Address: ga. Margreet Versteeg, EFPA Sc PiE, NIP the Netherlands, Arthur van Schendelstraat 650 , 3511 MJ Utrecht, The Netherlands, e-mail: mmbversteeg@hotmail.nl Članek je licenciran pod pogoji Creative Commons Attribution 4.0 International licence (CC-BY licenca). The article is licensed under a Creative Commons Attribution 4.0 International License (CC-BY license). anek je licenciran pod pogoji Creative Commons Attribution 4.0 International licence (CC-BY licenca). e article is licensed under a Creative Commons Attribution 4.0 International License (CC-BY license). Ustvarjanje učencem prilagojenega izobraževanja: Izzivi za Stalni komite za psihologijo v izobraževanju pri EFPA g g šolska psihologinja na Nizozemskem, članica Dutch Association for Psychologists, NIP, in v letih 2011–2021 predstavnica te zveze pri EFPA Standing Committee Psychology in Education Povzetek: Članek predstavlja Stalni komite za psihologijo v izobraževanju (SC PiE), njegovo zgodovino in organiziranost ter opisuje njegove cilje in delo. SC Pie se ukvarja z vlogo psihologov v izobraževalnem sistemu, njihovim usposabljanjem znotraj Sistema, kot se povezuje s specialitičnim certifikatom EuroPsy iz pedagoške psihologije, pomembnostjo vključevanja psihologov v izobraževanje ter z znanjem in proseci, ki temeljijo na empiričnih dokazih. SC PiE informira in promovira naštete tematike skozi publikacije, simpozije in predstavitve, poleg tega pa izvaja tudi nekatere omejene raziskave. Dejavnosti komiteja so predstavljene v članku. Stalni komite za psihologijo v izobraževanju pri EFPA je Društvo psihologov Slovenije povabilo, da prispeva k znanstveni reviji Psihološka obzorja v okviru posebne teme Psihologija v izobraževanju: izzivi, izbire in spremembe. Posebna tema vključuje članke o psihologiji v izobraževanju iz različnih evropskih držav in je bila izdana ob robu Evropskega kongresa za psihologijo (ECP 2022) ki je potekal v Ljubljani pod naslovom Psihologija kot vozliščna znanost: priložnosti in odgovornosti. Ključne besede: Stalni komite za psihologijo v izobraževanju pri EFPA, šolski psihologi, pedagoška psihologija, inklucija, na dokazih temelječe znanje Ključne besede: Stalni komite za psihologijo v izobraževanju pri EFPA, šolski psihologi, pedagoška psihologi lni komite za psihologijo v izobraževanju pri EFPA, šolski psihologi, pedagoška psihologija, inklucija, na dokaz Članek je licenciran pod pogoji Creative Commons Attribution 4.0 International licence (CC-BY licenca). The article is licensed under a Creative Commons Attribution 4.0 International License (CC-BY license). 449 Creating education that fits students This article discusses the work of the European Federation of Psychology Associations (EFPA) Standing Committee on Psychology in Education (SC PiE). The EFPA SC PiE is one of the working groups of the EFPA. The field of the SC PiE is education in the broadest sense of the word. The field covers day-care, preschool, primary education, secondary education, vocational education, higher education, and organisations that support these kinds of education. The field of education is interesting but complex. The field of education is interesting because it is concerned with all the peoples of Europe, as well as connections with people across the whole world. Life-long learning covers the youngest child to the most elderly. The complexity is seen in education at a European level but also at the national level in European countries. History From the beginning of the network the SC PiE had three crucial challenges. In short, these challenges are the psychologist’s role in the educational system, inclusion in education, and evidence-based knowledge and processes in education. In 2007 psychologists from 8 countries started a working group called the Network of European Psychologists in the Educational System (NEPES). Since the start of NEPES, it had connections with EFPA and ISPA, International School Psychologists Association. In the NEPES regulations of 2008 the working group formulated eleven aims (NEPES, 2008). Three of them are: to promote psychology as a science and profession in the educational system in cooperation with EFPA and ISPA, to promote communication and cooperation between psychologists and their organisations working in the educational system, and to promote children’s rights. In the beginning NEPES was also a discussion-forum where the differences in the work of the psychologists in the educational system between countries were highlighted and discussed. Aims of the SC PiE The role of the SC Psychology in Education (EFPA, SC PiE, 2022, para. 2) is to analyse and appraise the developments in research and professional practice of psychologists in educational contexts across Europe. Ustvarjanje učencem prilagojenega izobraževanja: Izzivi za Stalni komite za psihologijo v izobraževanju pri EFPA Different people and organisations are involved and contribute to the complexity of education, for example students, parents, teachers, but also the management of schools, municipalities and counties. Various kinds of educational systems, legislation, research about teaching, and the many visions on education also contribute to this complexity. The EFPA SC PiE carries out projects, writes position papers, and gives presentations and lectures at national and international conferences. The SC PiE has organised meetings with school psychologists of national departments of psychologist’s associations about this interesting, large and complex field. The focus of the network was concerned with the practical psychological work in the school system. In 2009 NEPES got the status of a task force of EFPA. The working group NEPES adapted to the new EFPA structures at that time. In 2011 the network evolved into a standing committee of EFPA. This change meant, for example, that the committee had to write an activity plan and a report every two years. Both are presented at the General Assembly of EFPA (EFPA, 2022). The convenor of the network NEPES was responsible for writing these documents based on the results of the discussions, for organizing the meetings, and keep in touch with the Head Office of EFPA and organisations outside EFPA. The organisation of the SC PIE has evolved during the time towards sharing responsibilities for one or more of the activities for all members. Every active representative has a task of writing documents, organising meetings and being active in a subgroup. The meetings of the SC PiE evolved from once a year a physical meeting to two physical meetings a year, to in the Corona period 2020 and 2021, fourteen online meetings: major, in-between and subgroup meetings. What is the EFPA SC PiE? To these ends, the SC PiE will :i EFPA is the European Federation of Psychology Associations. Thirty-eight national associations of psychologists work together and are united in EFPA. These member associations participate in the various EFPA working groups with more than 300 volunteers nominated by the member associations. The EFPA Standing Committee on Psychology in Education is one of the sixteen permanent working groups of EFPA. Active members in the SC PiE represent twenty-eight member associations. The SC PiE has 11 observer representatives from member associations who are informed about the work of the SC PiE. The representatives in the SC PiE are all psychologists who can offer expertise and have experience working in school and educational system as psychologists, researchers or counsellors. – Disseminate scientific knowledge about psychology in education – Exchange experiences of good practices of psychologists in education – Promote inclusiveness in education – Promote inclusiveness in education – Promote and support the best interests of vulnerable young people in educational systems y g p p y – Adopt both preventative and reactive approaches in educational contexts – Provide a platform for sharing best practices of psychologists in education across Europe and the wider world. Challenge 2. Promotion of inclusion in education Another important challenge of the Sc PiE is to promote inclusion in education. The psychologists have an important role in implementing inclusive education. From the beginning, the work of the SC PiE has been based on the needs of society, the UN Convention on the Rights of the Child (United Nations, 1989), the UN Convention on the Rights of People with Disabilities (United Nations, 2008) and The Salamanca Statement and Framework for Action on Special Needs Education (United Nations Educational, Scientific and Cultural Organisation [UNESCO] & Ministry of Education and Science Spain, 1994). All European countries ratified both conventions, but in many nations, the inclusion of all children in the educational system is not a reality. p ( , ) In 2009 the network NEPES participated in the Life Long Learning Programme of the EU. In this project, the network worked on ensuring the quality of psychologists’ training and work in education. The project report “European School Psychologists Improve Life Long Learning, ESPIL” (NEPES, 2010) gave the educational system recommendations about the psychologist’s role, European quality standards for the education training of psychologists and recommendations about European quality standards for the psychological service. These recommendations are a step toward the development of the Specialist EuroPsy Certificate for Educational Psychology. Further work from NEPES includes overviews of the agreements and differences in role, training and legislation in European countries. In 2009 NEPES published the “Preliminary Report School Psychology in Secondary Schools across Europe” (NEPES, 2009b). In this report, the network gave an overview of the psychologist’s role in guidance and career counselling in schools in Europe. Also, in 2009 NEPES published an overview of mental health projects carried out by psychologists in the educational system of the countries represented in the SC “Psychologists supporting Mental Health in Youth and Education” (NEPES, 2009a). This included descriptions of these mental health projects. The network also produced overviews and surveys that were not published. In 2012 NEPES produced an overview of training and courses of Universities with a special focus on a study of psychology in education -, school psychology, “EU reference guide of universities with offers in the area of psychology in education”. In 2013 a survey was made NEPES and the SC PiE attribute an important role to psychologists working in education on implementing inclusion. During the NEPES period, the network participated in many conferences. Challenge 1. The role of the psychologist in the educational system The first challenge relates to the training and role of the psychologist in the educational system, the systems to get work and the regulated working conditions in education. Every country in Europe has its own educational system. Therefore, the psychologist’s role in this educational system is different in all European countries. For example, the role can be focused on mental health service, student counselling, 450 M. Versteeg career guidance and counselling, training of teachers and staff, crisis management, teaching social emotional learning, etc. (Arnold & Horan, 2017). about the field and the role of the psychologist in education, a survey about the competencies needed in the work of the psychologist in education and in 2014, a survey about tasks in the work of the psychologist in education. ( ) In the Bologna Statement (1999, as cited in Europsy, 2016), twenty-nine countries agreed to reform the structures of their higher education systems towards convergence. Nevertheless, higher education courses at universities continue to fit in with the education system of the individual European country. The fit to the national educational system is one of the reasons why the training of the psychologist working in the educational system is different in all European countries. A psychologist, who works in education, needs a university master’s degree of psychology in most European countries. However, in some states of Germany, the psychologist can only work in education when s/he has qualified teacher training and experience as a teacher. In many countries, school- or educational psychology courses are organised at master’s, post-master’s or doctoral levels, for example, in Cyprus, Lithuania, the Netherlands, the UK and Russia. However, in some other countries, there are no courses at all. In 2017 the SC PiE published the book “Inclusive Educational Practice in Europe: psychological perspectives” (Arnold & Horan, 2017). On the first two pages of every chapter is an overview of the key facts underpinning the work of the psychologists in schools of each of 13 European countries. The differences in training, legislation, role and tasks of psychologists between the national educational systems have the consequence that there is no commonality in terminology, concern and structure as a basis for the work of the Sc PiE. The big challenge for the SC PiE is to come to more unified and quality assure the work. Challenge 1. The role of the psychologist in the educational system In general, the European Certificate in Psychology Basic, EuroPsy Basic (Lunt et al., 2015), can be used to get more consistency in the quality of the work. For example, the association of psychologists in Portugal and Spain use the Europsy basic. But the SC PiE has the ambition to develop and promote a standard of post- academic education and professional training at a higher level for the specialist psychologist working in education, the Specialist EuroPsy Certificate on Educational Psychology. Not only is the training and role of the psychologist in the educational system different in every country, but also the legislative structures for psychologists working in the educational system are not the same in European countries. For example, Austria, Czech Republic, Denmark, and the United Kingdom have legislation defining the psychologist’s role in education. However, in the Netherlands, in general, no such legislation underpins the work of the psychologist in education, with one exception the advice for admission to special education (Arnold & Horan, 2017). Challenge 2. Promotion of inclusion in education They contributed presentations about mental health for youth, the ESPIL project, the improvement of special needs education in European schools, mainstreaming of childrens’ rights and child health and rights education for health professionals, including psychologists. These subjects are affiliated with inclusion in education. As a result, the core business of the SC PiE evolved into two main subjects, the rights of the child and inclusion in education (Kant, 2013). The SC PiE gives a vision on inclusion in the report “Psychologists Supporting Inclusive Education” (SC PiE, 2012). Inclusive education is education in which all children can participate regardless of features that can lead to discrimination. Inclusive education aims to ensure equal development focussed opportunities for every child in education. Furthermore, inclusive education values the 451 Creating education that fits students richness and the benefits of diversity. In many organisations and states, the limited definition of inclusion in education is used: inclusion of students with disabilities, special needs. i representatives of NEPES and the SC PiE. They did research with surveys and questionnaires. The results of this research were presented in seminars during national and European meetings. In these lectures and the reports of NEPES highlighted the good practices of countries in the field of mental health in education, guidance and career counselling in schools, special needs projects, early childhood education and social-emotional learning. i , p The SC PiE handles the comprehensive definition of inclusion in education in the report “Psychologists Supporting Inclusive Education” (SC PiE, 2012) and the position paper of 2012. In the report, representatives describe the degree of inclusiveness in education in their country. They give examples of good practices of their country. Some countries have inclusive education based on the legislation of the country, for example, Italy and Denmark. Important in all publications, lectures, and presentations about inclusive education is the information about the psychologist’s role in implementing inclusion in education. The SC PiE promoted inclusion in education and the psychologist’s role by lectures on this theme at conferences in Porto, Bratislava, Cyprus and Graz. Challenge 2. Promotion of inclusion in education The SC PiE promoted inclusion in education also with organising symposia during the European Congress for Psychologists, ECP, in Amsterdam 2017, “Inclusive Education in Europe: prevention and early intervention”, in Moscow 2019 “, Psychology in Education: creating a future together” and in Ljubljana 2022, “The impact of COVID-19 on children, families, and schools: Implications for psychology in education professional’s roles during the recovery phase”. After the first decade, the composition of representatives in the SC PiE changed to a committee with much more teachers/researchers of higher education than practitioners. This change gave an opportunity to do more research. At the ECP Moscow 2019, the SC PiE organised two symposia, “Evidence in School Psychology: What Counts?” and “Psychology in Education: Creating the future together.” Representatives of different countries presented their research on the ECP in Moscow. They promoted in these symposia the use of evidence-based knowledge in education in the lectures. The research reports presented are described in articles in the Journal “Psychology in Russia: State of Art” (Arnold & Schad, 2019). The presentations of research were an impetus for joint research. For the next decade, the SC PiE developed several research plans. The SC PiE started in 2020 collaboration with the American Psychological Association [APA], Coalition for Psychology in Schools and Education. The APA Coalition has researched what principles are used during a teaching in primary education. The research results are the TOP 20 Principles from Psychology in Schools and Education (APA, Coalition for Psychology in Schools and Education, 2015). p g y p The SC PiE published three issues about inclusion in education in European countries 1. The book “Inclusive Educational Practice in Europe: psychological perspectives” (Arnold & Horan, 2017), 2. The Journal “Psychology in Russia: State of Art”, volume 2019 – 4 (Arnold & Schad, 2019), 3. The Journal “Horizons of Psychology”, volume 2022 (Juriševič et al., 2022). The book “Inclusive Educational Practice in Europe: psychological perspectives” (Arnold & Horan, 2017) describes the development of inclusion in education in 13 countries of Europe. Psychologists, representatives in the SC PiE, working in schools and other educational settings from these 13 different European states, provide a picture of the progress in the inclusion process in their country. The SC PiE uses a comprehensive definition of inclusion in education in this publication. Challenge 2. Promotion of inclusion in education The Journal “Psychology in Russia: State of the Art” (Arnold & Schad, 2019) and The Journal “Horizons of Psychology” (Juriševič et al., 2022) contain papers that are reports of research. The results of the research contribute to the implementation of inclusion in education. As a result of this collaboration, the SC PiE started to research the use of evidence-based knowledge and processes in education by teachers. Representatives of several European countries participate in this research. The results of this research are presented during the ECP Ljubljana 2022 in the symposium “ Use of Psychological Science in Schools from a Euro-North American Perspective.” The journal “Horizons of Psychology” (2022) includes the research reports and presentations from the SC PiE during the ECP. Through the activities, research and presentations by the SC PiE, the committee disseminates scientific knowledge about psychology in education. All the activities mentioned in the description of the challenges contribute to fulfilling the aims of the SC PiE (EFPA, SC PiE, 2022, para. 2): “The role of the SC on Psychology in Education is to analyse and appraise the developments in research and professional practice of psychologists in educational contexts across Europe”. Challenge 3. The use of scientific knowledge and good practices in education Challenge 3. includes the research described and the promotion of evidence-based knowledge in education in challenge 1. The psychologist’s role in the educational system and challenge 2. The promotion of inclusion in education. The kind of research and promotion of evidence-based knowledge depends not only on the subject, but also on the development and composition of the SC PiE. The composition of the SC PiE was in the first decade, a mixture of practitioners and teachers/researchers in higher education. The first challenge described the investigation of the psychologist’s task, training, role in the educational system, the requirements to get work, and the regulated working conditions in education by Prispelo/Received: 13. 5. 2022 Sprejeto/Accepted: 27. 5. 2022 References American Psychological Association, Coalition for Psychology in Schools and Education. (2015). Top 20 principles from psychology for preK–12 teaching and learning. https://www.apa.org/ed/schools/teaching- learning/top-twenty-principles.pdf Arnold, C., & Horan, J. (Eds.). (2017). Inclusive educational practice in Europe: Psychological perspectives. Trentham Books. 452 M. Versteeg Arnold, C., & Schad, E. (Eds.). (2019). Educational psychology from a contemporary perspective [Special issue]. Psychology in Russia: State of the Art, 12(4). https://psychologyinrussia.com/volumes/12_4_2019.php European Federation of Psychologists’ Associations. (2022). Recent Reports. http://psyineducation.efpa.eu/ recent-reports/ European Federation of Psychologists’ Associations, Standing Committee on Psychology in Education. (2022). Aims. http://psyineducation.efpa.eu/introduction/ Europsy. (2016, October 29). Europsy and Bologna. EFPA. https://www.europsy.eu/policies/europsy-and-bologna Juriševič, M., Versteeg, M., & Arnold, C. (Eds.). (2022). Psychology in education: Challenges, choices and changes [Special topic]. Horizons of Psychology, 31. http://psiholoska-obzorja.si/en/?l=2022 Kant, M. (2013). Report 2013 of the Standing Committee Psychology in Education. EFPA. Lunt, I., Peiro, J. M., Poortinga, Y. H., & Roe, R. A. (2015). Europsy – Standards and quality in education for psychologists. Gottingen. Network of European Psychologists in the Educational System. (2008). NEPES Regulations (1/2008). Network of European Psychologists in the Educational System. (2009a). Psychologists supporting mental health in youth and education. Brussels. Network of European Psychologists in the Educational System. (2009b). Preliminary report School psychology in secondary schools across Europe. Brussels. Network of European Psychologists in the Educational System. (2010). Education, training, professional, profile and service of psychologists in the European educational system. Report European school psychologists improve lifelong learning, ESPIL. Brussels. https://docplayer. net/15662662-Education-training-professional- profile-and-service-of-psychologists-in-the-european- educational-system.html Standing Committee Psychology in Education. (2012). Psychologists supporting inclusive education. Brussels. United Nations. (1989). Convention on the rights of the child. https://www.unicef.org/child-rights-convention United Nations. (2008). Convention on the Rights of People with Disabilities (CRPD). https://www.un.org/ development/desa/disabilities/convention-on-the-rights- of-persons-with-disabilities.html. (accessed, February 16, 2022). i United Nations Educational, Scientific and Cultural Organisation & Ministry of Education and Science Spain. (1994). The Salamanca statement and framework for action on special needs education. https://unesdoc. unesco.org/ark:/48223/pf0000098427 Prispelo/Received: 13. 5. 2022 Sprejeto/Accepted: 27. 5. 2022 Prispelo/Received: 13. 5. 2022 Sprejeto/Accepted: 27. 5. 2022
https://openalex.org/W3036790841	https://www.frontiersin.org/articles/10.3389/fmicb.2020.01385/pdf	English	null	Response Characteristics of Nitrifying Bacteria and Archaea Community Involved in Nitrogen Removal and Bioelectricity Generation in Integrated Tidal Flow Constructed Wetland-Microbial Fuel Cell	Frontiers in microbiology	2,020	cc-by	11,406	Edited by: Shubiao Wu, Aarhus Institute of Advanced Studies, Denmark Edited by: Shubiao Wu, Aarhus Institute of Advanced Studies, Denmark Reviewed by: Yifeng Zhang, Technical University of Denmark, Denmark Mikk Espenberg, University of Tartu, Estonia Haiming Wu, Northwest A&F University, China Reviewed by: Yifeng Zhang, Technical University of Denmark, Denmark Mikk Espenberg, University of Tartu, Estonia Haiming Wu, Northwest A&F University, China Reviewed by: Yifeng Zhang, Technical University of Denmark, Denmark Mikk Espenberg, University of Tartu, Estonia Haiming Wu, Northwest A&F University, China Correspondence: Jianjun Lian jjlian85@126.com Yibin Cui cyb@nies.org †These authors have contributed equally to this work Reviewed by: Yifeng Zhang, Technical University of Denmark, Denmark Mikk Espenberg, University of Tartu, Estonia Haiming Wu, Northwest A&F University, China Correspondence: Jianjun Lian jjlian85@126.com Yibin Cui cyb@nies.org †These authors have contributed equally to this work Longmian Wang1†, Qingqing Pang1†, Fuquan Peng1, Aiguo Zhang1, Ying Zhou2, Jianjun Lian3, Yimin Zhang1, Fei Yang1, Yueming Zhu1, Chengcheng Ding1, Xiang Zhu1, Yiping Li2 and Yibin Cui1 1 Nanjing Institute of Environmental Sciences, Ministry of Ecology and Environment, Nanjing, China, 2 College of Environment, Hohai University, Nanjing, China, 3 College of Energy and Environment, Anhui University of Technology, Ma’anshan, China This study explores nitrogen removal performance, bioelectricity generation, and the response of microbial community in two novel tidal ﬂow constructed wetland- microbial fuel cells (TFCW-MFCs) when treating synthetic wastewater under two different chemical oxygen demand/total nitrogen (COD/TN, or simpliﬁed as C/N) ratios (10:1 and 5:1). The results showed that they achieved high and stable COD, NH4+-N, and TN removal efﬁciencies. Besides, TN removal rate of TFCW-MFC was increased by 5–10% compared with that of traditional CW-MFC. Molecular biological analysis revealed that during the stabilization period, a low C/N ratio remarkably promoted diversities of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in the cathode layer, whereas a high one enhanced the richness of nitrite-oxidizing bacteria (NOB) in each medium; the dominant genera in AOA, AOB, and NOB were Candidatus Nitrosotenuis, Nitrosomonas, and Nitrobacter. Moreover, a high C/N ratio facilitated the growth of Nitrosomonas, while it inhibited the growth of Candidatus Nitrosotenuis. The distribution of microbial community structures in NOB was separated by space rather than time or C/N ratio, except for Nitrobacter. This is caused by the differences of pH, dissolved oxygen (DO), and nitrogen concentration. ORIGINAL RESEARCH published: 23 June 2020 doi: 10.3389/fmicb.2020.01385 The response of microbial community characteristics to nitrogen transformations and bioelectricity generation demonstrated that TN concentration is signiﬁcantly negatively correlated with AOA-shannon, AOA-chao, 16S rRNA V4−V5-shannon, and 16S rRNA V4−V5-chao, particularly due to the crucial functions of Nitrosopumilus, Planctomyces, and Aquicella. Additionally, voltage output was primarily inﬂuenced by microorganisms in the genera of Correspondence: Jianjun Lian jjlian85@126.com Yibin Cui cyb@nies.org †These authors have contributed equally to this work Correspondence: Jianjun Lian jjlian85@126.com Yibin Cui cyb@nies.org †These authors have contributed equally to this work Correspondence: Jianjun Lian jjlian85@126.com Yibin Cui cyb@nies.org †These authors have contributed equally to this work Specialty section: This article was submitted to Microbiotechnology, a section of the journal Frontiers in Microbiology Specialty section: This article was submitted to Microbiotechnology, a section of the journal Frontiers in Microbiology Received: 04 March 2020 Accepted: 29 May 2020 Published: 23 June 2020 Received: 04 March 2020 Accepted: 29 May 2020 Published: 23 June 2020 INTRODUCTION enhance the nitrogen removal (Zhi and Ji, 2014). However, the nitrogen removal pathway and the underlying mechanism that governs the nitrogen transformation process of TFCW-MFC are not clearly understood. Earth’s biogeochemical cycles have been largely aﬀected by human activities (Yu et al., 2019), especially by a large amount of nitrogen released from agriculture and animal husbandry, which is the root of many environmental issues such as eutrophication and drinking water pollution. To this end, various methods have been purposed to remove nitrogen. However, these traditional nitrogen removal approaches (i.e., activated sludge process, trickling ﬁlter, and oxidation pond) have some limitations, such as low removal rate or high operating cost (Tanwar et al., 2008). It is acknowledged that total nitrogen (TN) in wastewater mainly consists of ammonia (NH4+-N), nitrite (NO2−-N), and nitrate (NO3−-N). Generally, nitriﬁcation, denitriﬁcation, and anaerobic ammonia oxidation (anammox) processes are the typical biological pathways used for nitrogen elimination (Zhi et al., 2015). Also, removal of nitrogen in wastewater has been investigated by biological method, and/or its combination with natural and ecological approaches (Wu et al., 2017). With two prominent challenges nowadyas, i.e., energy scarcity and non-point sourced nitrogen, ﬁnding economical and sustainable nitrogen removal technologies is particularly diﬃcult. Generally, nitriﬁcation is a biological oxidation of ammonium to nitrite and nitrate, and is the ﬁrst and most crucial step in TN removal. It is conducted in two sequential steps via several microorganisms that are phylogenetically distinct, including ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and nitrite-oxidizing bacteria (NOB) (Zhang et al., 2011). Oxidation of NH4+-N is considered mainly driven by chemolithoautotrophic AOB (Guimaraes et al., 2017). However, a recent study has shown that AOA, which is ubiquitously distributed in aquatic environments, often has higher abundance than AOB, and its abundance is correlated with nitriﬁcation rates (Leininger et al., 2006). On the other hand, oxidation of NO2−-N (to NO3−-N) depends mainly on NOB (Zhang et al., 2019). The quantitative relationships between ammonia oxidation rate and nitrogen functional genes (AOA and AOB) in TFCW have been analyzed (Li et al., 2015). However, the accuracy of AOA, AOB, and NOB compositions in TFCW-MFC remains unconﬁrmed, and the interactions between the dominant genera and environmental factors have yet to be established; these have limited our ability to optimize the treatment processes. INTRODUCTION Inﬂuent chemical oxygen demand/total nitrogen (COD/TN; generally simpliﬁed as C/N) ratio has been considered as a key factor inﬂuencing the nitrogen removal in CWs (Meng et al., 2018). It is also an important parameter aﬀecting the activity of AOB and NOB (Li et al., 2017). The recommended optimal inﬂuent C/N ratio for the removal of nitrogen (from approximately 80% TN) by CW-MFC is 5.37 (Wang et al., 2019). Unfortunately, to date, related research conducted using TFCW-MFC has not been reported. p y Constructed wetlands (CWs) have been extensively designed and studied as a sustainable technology to treat water bodies contaminated with low-concentration nitrogen (Zhi et al., 2015). However, many CWs only limitedly reduce nitrogen levels with ﬂuctuating results; thus, an improved treatment method is urgently needed (Liu et al., 2015). Vertical ﬂow constructed wetlands (VFCWs) and horizontal ﬂow constructed wetlands (HFCWs) are the initial designs of modiﬁed constructed wetlands. Due to low TN removal eﬃciency of a single- stage CW, some multi-stage treatment systems, such as HF- VFCWs or VF-HFCWs, have also been developed (Vymazal, 2007, 2013). However, these systems are practically diﬃcult to use due to complex operation conditions and a huge demand for land. To ﬁll this knowledge gap, two laboratory-scale TFCW- MFCs were developed, and their nitrogen removal performance and bioelectricity generation from synthetic wastewater under two diﬀerent C/N ratios were examined. The spatiotemporal variations of the diversity and community structure of AOA, AOB, NOB, and 16S rRNA V4−V5 region at ﬁlters and electrodes were investigated using the high-throughput sequencing. The relationships between the genetic characteristics of the four types of microorganisms (i.e., diversity and relative abundance of dominant genera) and eﬄuent indexes [i.e., pH, dissolved oxygen (DO), temperature (T), NH4+-N, NO3−-N, NO2−-N, and TN] were also determined. This work provides comprehensive theoretical support for improving TN removal through the optimization of main functional microbial composition and populations in TFCW-MFC. Recently, an expanded system consisting of a microbial fuel cell (MFC) coupled to a CW (CW-MFC) is becoming increasingly popular because it allows simultaneous wastewater treatment and electricity generation (Yadav et al., 2012; Doherty et al., 2015). CW and MFC share the redox gradient between an anaerobic anode and an aerobic cathode, in addition to electricity produced via MFC; thus, they have enhanced removal eﬃciencies (Srivastava et al., 2015). Citation: Wang L, Pang Q, Peng F, Zhang A, Zhou Y, Lian J, Zhang Y, Yang F, Zhu Y, Ding C, Zhu X, Li Y and Cui Y (2020) Response Characteristics of Nitrifying Bacteria and Archaea Community Involved in Nitrogen Removal and Bioelectricity Generation in Integrated Tidal Flow Constructed Wetland-Microbial Fuel Cell. Front. Microbiol. 11:1385. doi: 10.3389/fmicb.2020.01385 Wang L, Pang Q, Peng F, Zhang A, Zhou Y, Lian J, Zhang Y, Yang F, Zhu Y, Ding C, Zhu X, Li Y and Cui Y (2020) Response Characteristics of Nitrifying Bacteria and Archaea Community Involved in Nitrogen Removal and Bioelectricity Generation in Integrated Tidal Flow Constructed Wetland-Microbial Fuel Cell. Front. Microbiol. 11:1385. doi: 10.3389/fmicb.2020.01385 June 2020 \| Volume 11 \| Article 1385 1 Frontiers in Microbiology \| www.frontiersin.org Wang et al. Nitrogen Removal and Bioelectricity Generation Nitrosopumilus, Nitrosospira, Altererythrobacter, Gemmata, and Aquicella. This study not only presents an applicable tool to treat high nitrogen-containing wastewater, but also provides a theoretical basis for the use of TFCW-MFC and the regulation of microbial community in nitrogen removal and electricity production. Nitrosopumilus, Nitrosospira, Altererythrobacter, Gemmata, and Aquicella. This study not only presents an applicable tool to treat high nitrogen-containing wastewater, but also provides a theoretical basis for the use of TFCW-MFC and the regulation of microbial community in nitrogen removal and electricity production. Keywords: nitriﬁcation, tidal ﬂow constructed wetland, microbial fuel cell, microbial community structure, electricity generation Keywords: nitriﬁcation, tidal ﬂow constructed wetland, microbial fuel cell, microbial community structure, electricity generation Frontiers in Microbiology \| www.frontiersin.org INTRODUCTION Tidal ﬂow constructed wetland (TFCW), operated through a rhythmic sequential cycle of a “feeding/ﬂooding” phase and a “draining/resting” phase, has been proposed as a compact and eﬃcient method that can June 2020 \| Volume 11 \| Article 1385 Frontiers in Microbiology \| www.frontiersin.org 2 Nitrogen Removal and Bioelectricity Generation Wang et al. Inoculation and Operation of TFCW-MFC ocu a o a d Ope a o o C C Prior to the operation, the TFCW-MFC device was inoculated (at the anode compartment) with conventional activated sludge (1.1 L) obtained from the Jiang Xinzhou wastewater treatment plant (Nanjing, China). Sodium acetate (1.0 g/L) was mixed with the supernatant at a ratio of 1:1, and the mixture was pumped continuously into the bottom of the system at a ﬂow rate of 0.02 m3/d. Fifty percent of the eﬄuent was pumped back to the device to enrich the electrogens in the anode layer. After 1 month of operation, the maximum voltages were found stable, indicating that TFCW-MFC has been successfully started. Ten strains of cattail planted for 1 month at an ambient temperature were transferred to the cathode layer during the start-up of TFCW-MFC. where J is the mean current density (mA/m2), P is the power density (mW/m2), S is the surface area of the anode electrode (m2), U is the voltage (V), and R is the external resistance (). MATERIALS AND METHODS After a 5-h ﬂooding period, the treated wastewater was drained from the bottom of the devise into the water storage tank for 12 min using a high pressure pump; afterward, the TFCW-MFC was rested for up to 4 h. Thereafter, the treated wastewater in water storage tank was pumped back into TFCW-MFC about 2 h in the bottom-up ﬂow mode. After submerging 5 h, the wastewater was discharged from the bottom of the device as a full cycle accomplishment. The complete hydraulic retention of this system including recirculation was 18.4 h. Water samples were collected from the inﬂuent tank and the total water outlet once every 2 days and then analyzed. Device Conﬁguration g Two identical integrated TFCW-MFCs, namely Device A (inﬂuent C/N = 10:1) and Device B (inﬂuent C/N = 5:1), were constructed in the Laboratory of Watershed Water Environment at the Nanjing Institute of Environmental Sciences, Nanjing, China. Each TFCW-MFC was covered with a black adhesive tape and was placed at room temperature (22◦C). The main structure of TFCW-MFC was made from a polyacrylic plastic cylinder (internal diameter = 50 cm, height = 80 cm). Five layers were constructed from the bottom to the top including: a support layer made of cobble (diameter = 15–30 mm) with a depth of 15 cm; a quartz sand II layer (diameter = 5– 10 mm) with a depth of 25 cm; an anode layer with a depth of 10 cm; a quartz sand I layer (diameter = 5–10 mm) with a depth of 25 cm; and a cathode layer with a depth of 5 cm. The anode and cathode layers were constructed using granular activated carbon (GAC; speciﬁc surface area = 450– 800 m2/g, diameter = 2–5 mm; Jiangsu Zhuxi Activated Carbon Co., Ltd., Jiangsu, China) and stainless-steel mesh (0.3-mm thickness, 1.7-mm diameter; Nanjing Zhongdong Chemical Glass Instrument Co., Ltd., Nanjing, China), respectively. The anode and cathode layers were connected by a 1-mm titanium wire connecting to a 1000- external resistance. Cattail (Typha latifolia L.), as the wetland plant, was planted in the cathode layer, which would be combined with plant roots to promote oxygen transport to create better aerobic conditions (Oodally et al., 2019). Four sampling ports (each with a diameter of 40 mm) arranged in the cathode, quartz sand I, anode, and quartz sand II layers, were used to collect ﬁlters or electrodes, so as to analyze the characteristics of the related bacteria. Sample Collection and Analysis Sample Collection and Analysis Samples of inﬂuents and eﬄuents were collected for wastewater treatment analysis every 2 days using a 250-mL beaker. COD, NH4+-N, NO2−-N, NO3−-N were detected by the closed reﬂux titrimetric method, Nessler’s reagent colorimetric method, colorimetric method and ultraviolet spectrophotometric screening method, respectively, according to the Standard Methods (American Public Health Association [APHA], 2005). TN was determined by spectrophotometry with alkaline potassium persulfate ultraviolet. DO, pH, and temperature were measured using a portable multiparameter water quality monitoring (HQ40d, HACH, United States). The voltage was monitored through a Digital Voltage acquisition system (Keithley 2700, Tektronix, United States) and was recorded once per minute. The internal resistance was determined and calculated from the polarization curves, and the power density curve of CW-MFC was calculated according to Eqs. (1, 2) (Logan, 2008). Samples of inﬂuents and eﬄuents were collected for wastewater treatment analysis every 2 days using a 250-mL beaker. COD, NH4+-N, NO2−-N, NO3−-N were detected by the closed reﬂux titrimetric method, Nessler’s reagent colorimetric method, colorimetric method and ultraviolet spectrophotometric screening method, respectively, according to the Standard Methods (American Public Health Association [APHA], 2005). J = U SR (1) P = U2 SR (2) J = U SR (1) (1) P = U2 SR (2) (2) Frontiers in Microbiology \| www.frontiersin.org High-Throughput Sequencing Analysis Microorganism samples were collected from the four substrate- sampling ports at days 30 and 90 in Table 1. DNA extraction was performed using 0.25 g soil by the Fast DNA spin kit for soil (MO BIO Laboratories, Carlsbad, CA, United States) following the manufacturer’s protocol. The extraction was taken from each obtained DNA for agarose gel electrophoresis (1.2%) testing. High-quality DNAs with clear bright bands (approximate 30 ng/µL DNA content) could be used as templates for polymerase chain reaction (PCR). AOA, AOB, NOB, and V4−V5 region of 16S rRNA were ampliﬁed by PCR using universal microbial primers (Supplementary Table S2). The PCR ampliﬁcation condition was as follows: initial denaturation at 90–95◦C for 2 min; 30–35 cycles of denaturation at 90–95◦C for 30 s, annealing at 50–60◦C for 30 s, and extension at 70– 75◦C for 30 s; and ﬁnal extension at 70–75◦C for 5 min. The After the system was stable, two TFCW-MFCs were operated with two diﬀerent C/N ratios, and synthetic wastewater was then introduced into the systems, which were processed for another 90 days (Supplementary Table S1). Figure 1 shows the schematic diagram of the TFCW-MFC operation. At the beginning, 40 L of synthetic wastewater was fed into the bottom of TFCW-MFC using a peristaltic pump (BT100-1L, Baoding Longer Precision Pump Co., Ltd., Baoding, China). June 2020 \| Volume 11 \| Article 1385 Frontiers in Microbiology \| www.frontiersin.org 3 Nitrogen Removal and Bioelectricity Generation Wang et al. FIGURE 1 \| Schematic diagram of TFCW-MFC during a typical cycle mode. FIGURE 1 \| Schematic diagram of TFCW-MFC during a typical cycle mode. TABLE 1 \| Locations of different sampling points in TFCW-MFC. TABLE 1 \| Locations of different sampling points in TFCW-MFC. Day C/N S1 S2 S3 S4 30 10:1 Ac1 Au1 Aa1 Ab1 5:1 Bc1 Bu1 Ba1 Bb1 90 10:1 Ac2 Au2 Aa2 Ab2 5:1 Bc2 Bu2 Ba2 Bb2 A −represents the devices with inﬂuent C/N = 10:1; B −represents the devices with inﬂuent C/N = 5:1; c, u, a, b mean cathode layer, upper layer, anode layer, bottom layer, respectively; (1) operation on the 30th day; (2) operation on the 90th day. TABLE 1 \| Locations of different sampling points in TFCW-MFC. between microbial communities was analyzed based on the phylogenetic information using the Weighted UniFrac (Wang et al., 2017). High-Throughput Sequencing Analysis The relationships between microbial communities were determined based on the distance matrix using the principal coordinates analysis (PCoA). Analysis of environmental factors that had the most eﬀects on the microbial community structure was conducted using the redundancy analysis (RDA) by Canoco software (version 4.5) (Zhang et al., 2019). The impact of each environmental factor on the microorganism was analyzed based on the RDA data using the variation partitioning analysis (VPA) by Canoco. A −represents the devices with inﬂuent C/N = 10:1; B −represents the devices with inﬂuent C/N = 5:1; c, u, a, b mean cathode layer, upper layer, anode layer, bottom layer, respectively; (1) operation on the 30th day; (2) operation on the 90th day. Frontiers in Microbiology \| www.frontiersin.org Statistical Analysis sequences with a similarity threshold of 97% were clustered into operational taxonomic units (OTUs). The sequencing was performed using an Illumina Miseq (2 × 300) platform by the Personal Biotechnology Co., Ltd. (Shanghai, China). Data analyses were conducted using Microsoft Excel 2010, and the ﬁgures were prepared using Origin 2016. Means among all treatment groups were compared by one-way analysis of variance (ANOVA) using SPSS 19.0 software package. Based on the assumption of normal distribution, the Pearson correlation, which was used to determine the relationships between alpha diversity at the genus level and eﬄuent nitrogen concentration, and between nitrogen removal and the relative abundance of the dominant genera of each of the four types of microorganisms in 16 sample sizes, was also calculated using SPSS 19.0. P < 0.05 and P < 0.01 indicate the signiﬁcant diﬀerences. In addition, Alpha diversity indexes (including Chao and Shannon indexes) were calculated based on the OTU numbers using quantitative insights into microbial ecology (QIIME) (Caporaso et al., 2010). The Chao indexes were mainly used to reﬂect the species richness between samples. The larger the index value, the more abundant the species; The Shannon indexes mainly reﬂected the diversity of the communities in the samples. The larger the Shannon index, the richer the diversity. The diﬀerence June 2020 \| Volume 11 \| Article 1385 Frontiers in Microbiology \| www.frontiersin.org 4 Nitrogen Removal and Bioelectricity Generation Wang et al. respectively, during the stabilization period (Figure 2A). The obtained COD removal eﬃciencies were higher than previous studies of the CW-MFC to treat livestock sewage, which have typical removal eﬃciencies ranging from 71 to 81% (Zhao et al., 2013; Doherty et al., 2015; Liu et al., 2020). The COD removal load of Device A was 40.26 g/m2·d, which was higher than that reported previously (9.8 g/m2·d by Albuquerque et al. (2009), 29.20 g/m2·d by Konnerup et al. (2009). This enhanced COD removal eﬃciency is likely attributed to the intensiﬁed oxygen supply generated by the tidal ﬂow operation (Oon et al., 2017). the P-values for multiple testing remain uncorrected, including the False Discovery Rate procedure, due to the small number of samples in this study. Electricity Generation The voltage curve of TFCW-MFC under diﬀerent inﬂuent C/N ratios is shown in Figure 3. According to the curve, Device A and Device B had the average voltages of 40.25 and 26.85 mV, respectively. Moreover, the maximum power densities of the system operated with C/N ratios of 10:1 and 5:1 were 25.78 and 16.97 mW/m2, respectively, while the current densities were 0.18 and 0.11 A/m2, respectively, and the internal resistances were 151 and 174 , respectively. This clearly indicated that low internal resistance causes the increase in power density. Similar and high power density values have been reported by previous studies in Table 2: Wang et al. (2017) have reported a value of 21.53 mW/m2 in CW-MFC planted with T. orientalis; Corbella et al. (2015) have reported a maximum value of 36 mW/m2, which was harvested by implementing MFC in HFCW during the treatment of eﬄuent from a hydrolytic up-ﬂow sludge blanket reactor. Low power density obtained by TFCW-MFC may be attributed to the following three reasons: (1) a small ratio of electrode surface to CW-MFC system volume may obstruct the accumulation of electrochemically active microorganisms on the electrode; (2) electrons may be largely depleted due to high concentrations of electron acceptors (NO2−-N, NO3−-N, oxygen, etc.) presented in the bioﬁlter devices (Wang et al., 2016); and (3) spacing between anode and cathode electrodes (25 cm) may be too large, as has been described by Oon et al. (2017), who observed that the voltage output in up-ﬂow constructed wetland-MFC tends to decrease when the spacing between anode and cathode electrodes are increased from 15 to 30 cm. Hiegemann et al. (2017) have also reported that the voltage output increases with the increase of organic concentration, as the voltage output is dependent on the electrons and protons transferred from carbon sources at the anode. However, excessive organic loading is not conducive to electricity generation because the multiplication of methanogens can inhibit the power generation (Martin et al., 2010), or organics g As shown in Figure 2, the removal of nitrogen other than NO3−-N in Device B was stable after 30 days of operation, which indicates that the microorganisms used in nitrogen removal are mature. The average removal rate of TN (82.39%) and NO3−- N (52.16%) in Device A were higher than those in Device B (79.25% of TN, 36.72% of NO3−-N). Wastewater Treatment Performance Wastewater Treatment Performance Wastewater Treatment Performance Device A and Device B were stable after 30 days of operation, and they achieved COD removal eﬃciencies of 97.66 and 98.36%, Device A and Device B were stable after 30 days of operation, and they achieved COD removal eﬃciencies of 97.66 and 98.36%, IGURE 2 \| Average removal efﬁciency and change in concentrations of COD (A), TN (B), NH4+-N (C), NO3–-N (D), NO2−−−N, (E) with various C/N ratios. ge in concentrations of COD (A), TN (B), NH4+-N (C), NO3–-N (D), NO2−−−N, (E) with various C/N ratios. FIGURE 2 \| Average removal efﬁciency and change in concentrations of COD (A), TN (B), NH4+-N (C), NO3–-N (D), NO2−−−N moval efﬁciency and change in concentrations of COD (A), TN (B), NH4+-N (C), NO3–-N (D), NO2−−−N, (E) with various C/N ratio June 2020 \| Volume 11 \| Article 1385 June 2020 \| Volume 11 \| Article 1385 Frontiers in Microbiology \| www.frontiersin.org er 2 m2 m3 3 2 m2 m2 m3 2 2 3 m3 3 CW-MFCs. ) C/N Hydraulic retention time (d) COD removal rate (%) TN removal rate (%) Max. powe 7.25 0.9−1.25 76.5 49.7 9.4 mW/m2 − 3.2 80−100 − 0.15 mW/m − 1 90.9 − 43.63 mW/m 9.25 1 64 58 0.28 W/m3 7.8 0.17 61 − 36 mW/m2 10 4 57.4 − 8.08 mW/m 3.77 2 80 74 21.53 mW/m − 1 98 − 184.75 mW/m 4.72 40 69 69 112 mW/m − 3 82.32 82.46 3.71 W/m2 6.4 − 97 − 229 mW/m 3 2 88.07 − 496.4 mW/m 22 0.63 99.5 90 25 mW/m3 5.4 2 64.04 88.78 6.09 mW/m 10 0.77 97.66 82.39 25.78 mW/m 5 0.77 98.36% 79.25 16.97 mW/m Nitrogen Removal and Bioelectricity Generation Wang et al. FIGURE 3 \| Voltage output of two TFCW-MFC devices operated with various C/N ratios. genes is attributed to their environmental adaptions to the balance condition of nitriﬁcation and denitriﬁcation via oxygen transfer, as well as their related distinct ecological niches (Zhi et al., 2015). Thus, the increase in gene abundance is more likely to be the reason accounting for the enhanced denitrifying activity. Furthermore, better TN removal rates (90% and 87%) in Table 2 were obtained using CW-MFC in latest reports (Srivastava et al., 2020; Tao et al., 2020). Wastewater Treatment Performance Compared with the results in the literature, the advantage of this study was that the eﬃcient TN removal could be achieved under relatively low C/N ratios and short retention time. However, the NO3−- N removal rate is low because high DO is not conducive to denitrifying microorganism growth. Although the concentration diﬀerences of NO2−-N in eﬄuents between Device A and B were found, the NO2−-N removal rates in both systems were more than 90%. Since NO2−-N is in the intermediate valence state of nitrogen transformation and it has an unstable characteristic, the impact of C/N ratio on NO2−-N attenuation could be negligible. Nonetheless, exorbitant carbon source concentration inhibits denitriﬁcation instead of boosting it (Liu et al., 2012). Thus, the C/N ratio should be further optimized to achieve higher NO3−-N removal rate. FIGURE 3 \| Voltage output of two TFCW-MFC devices operated with various C/N ratios. The eﬄuent DO in both Device A (5.31 mg/L) and Device B (4.83 mg/L) were higher than the inﬂuent DO, which may be due to the oxygen intake from the atmosphere via employing the tidal ﬂow mode. Meanwhile, the direct reduction by heterotrophic and electroactive microorganisms in the root area and plant uptake in the system also improved COD degradation rates (Yang et al., 2020). Additionally, wastewater treatment by the coupled MFC has been reported to be more eﬀective than that by CWs. Compared with traditional CWs, the biodegradation of organic compounds is enhanced by the transfer of electrons from the anode to the cathode. Study has also shown that CW- MFC is superior to conventional CW concerning the COD removal rate (Wang et al., 2016). Therefore, it is highly likely that high-eﬃcient removal of COD by TFCW is due to the integration of MFC. Electricity Generation By contrast, the removal rates of NH4+-N in both devices were similar with values of higher than 95% (95.94% in Device A, 97.13% in Device B). The removal rates of NH4+-N and TN were similar, inferring that the removal of TN may be in the form of NH4+-N and a certain amount of the eﬄuent NO3−-N may be accumulated. This indicates that the loss of carbon source may lead to incomplete denitriﬁcation, thus causing the accumulation of NO3−-N. The TN removal rate of TFCW-MFC was higher than that of CW-MFC previously reported (74% has been reported by Wang et al., 2017, 69% has been reported by Wu et al., 2017; Table 2). This observation further demonstrated that the developed device has higher TN removal eﬃciency, which may be caused by the use of tidal ﬂow. It could increase the abundance of nitrogen-transformation genes. The coenrichment of these June 2020 \| Volume 11 \| Article 1385 Frontiers in Microbiology \| www.frontiersin.org 7 Nitrogen Removal and Bioelectricity Generation Wang et al. FIGURE 4 \| Relative change of Chao indexes of panels (A) AOA, (B) AOB, (C) NOB, and (D) 16S rRNA V4–V5 region, at different sampling points of TFCW-MFCs. The horizontal line in the box represents the median. The size of the box represents the data dispersion (Interquartile range = IQR). FIGURE 4 \| Relative change of Chao indexes of panels (A) AOA, (B) AOB, (C) NOB, and (D) 16S rRNA V4–V5 region, at different sampling points of TFCW-MFCs. The horizontal line in the box represents the median. The size of the box represents the data dispersion (Interquartile range = IQR). FIGURE 4 \| Relative change of Chao indexes of panels (A) AOA, (B) AOB, (C) NOB, and (D) 16S rRNA V4–V5 region, at differen The horizontal line in the box represents the median. The size of the box represents the data dispersion (Interquartile range = IQR AOB had a higher abundance than AOA, indicating that the microenvironment of the ﬁlter is more suitable for AOB growth. While one study has reported that AOA in many marine and terrestrial ecosystems is more abundant by one to two orders of magnitude than AOB (Mincer et al., 2007); by contrast, another study has indicated that AOB is more dominant (Liu et al., 2014). Electricity Generation This inconsistent result may be primarily related to the high- strength NH4+-N at the inﬂuent, beneﬁcial to AOB rather than AOA reproduction. Furthermore, the spatial distribution of each type of microorganisms is diﬀerent and it changes over time, due to the collaborative functions of nitrogen concentration, pH, and temperature (Fierer et al., 2009; Hallin et al., 2009; Fan et al., 2011; Zhang et al., 2019). The microbial richness in the upper layer of V4−V5 region was higher than that in the lower layer, which may mainly be due to the aerobic microsites adjacent to plant roots and exudates from rhizomes, where the oxygen released from the Cattail’s root into the cathode could promote may not be completely oxidized at the anode, thereby generating an anaerobic environment that causes the CW-MFC to have decreased power output or even stop working (Villaseñor et al., 2013). It is worth noting that the voltage output of both devices increased after 30 days of operation, but thereafter gradually decreased until it became stable. This indicated that high electrogenic microbial activity or organic concentration can promote electricity generation in the system. After 40 days of operation, the denitrifying microorganism in the system may become more abundant than the electrogenic microorganism; consequently, the electricity-producing ability was inhibited. Diversity of Microbial Community y y The Chao index was used to evaluate the richness of microbial community structure. As shown in Figures 4A–D, the Chao indexes of the two systems decreased in the following order: 16S rRNA V4−V5 region > AOB > NOB > AOA. Interestingly, June 2020 \| Volume 11 \| Article 1385 Frontiers in Microbiology \| www.frontiersin.org 8 Nitrogen Removal and Bioelectricity Generation Wang et al. FIGURE 5 \| Difference in distribution of microbial community of panels (A) AOA, (B) AOB, (C) NOB, and (D) 16S rRNA V4–V5 region in different samples in the two devices. Data were obtained based on the phylogenetic lineages using the weighted Fast UniFrac PCoA. The red ellipse indicates that different samples with similar microbial community structure are clustered together. FIGURE 5 \| Difference in distribution of microbial community of panels (A) AOA, (B) AOB, (C) NOB, and (D) 16S rRNA V4–V5 region in different samples in the two devices. Data were obtained based on the phylogenetic lineages using the weighted Fast UniFrac PCoA. The red ellipse indicates that different samples with similar microbial community structure are clustered together. Bb1 (3.1) within AOB, Aa1 (2.53), Ba1 (1.75) within NOB, and Ab1 (3.56) and Bb1 (5.58) within the V4−V5 region. It is possible that at the beginning of the operation, carbon and nitrogen concentrations may have a direct or indirect eﬀect on the microorganism in the anode and the bottom layers of the two devices. In contrast, the microbial community structure was relatively stable after 90 days, and the lower inﬂuent C/N ratio (5:1) appeared to facilitate the growth of AOA and AOB in the cathode layer, as well as that of all types of microorganisms in the V4−V5 region. The microorganism in the cathode layer is more diverse, which is likely due to the higher DO (Wu et al., 2011; Supplementary Figure S1) caused by the reoxygenation ability of the tidal ﬂow, or there may be a large number of aerobic electrogenic microorganisms in AOA and AOB. The inﬂuent C/N ratio had little eﬀect on the diversity of microorganisms in the same layer. the growth of microorganisms. The richness of NOB in each substrate layer in Device A was higher than that in Device B (Figure 4C), indicating that a higher C/N ratio is beneﬁcial to the survival of NOB. Frontiers in Microbiology \| www.frontiersin.org Diversity of Microbial Community The richness of AOA and AOB were higher in the anode layer on the 90th day than that on the 30th day, due to that the exoelectrogenic microorganism and AOA/AOB gradually adapted to coexist on the anode in a facultative oxygen environment (DO = 0.9–1.5 mg/L at the anode in Supplementary Figure S1). This result is consistent with the observation by Xu et al. (2018), who have reported that the microbial richness in the anode is the highest in the CW-MFC system. As shown in Supplementary Figure S2 and Supplementary Tables S3–S6, the diversity of microorganisms in the system became unstable after 30 days, and the C/N ratio had a greater inﬂuence on the space-time distribution of all four types of microorganisms in the same layer, especially Ab1 (1.42) and June 2020 \| Volume 11 \| Article 1385 Frontiers in Microbiology \| www.frontiersin.org 9 Nitrogen Removal and Bioelectricity Generation Wang et al. FIGURE 6 \| Bar plot and cluster tree of microbial community of panels (A) AOA, (B) AOB, (C) NOB, and (D) 16S rRNA V4–V5 region. Comparative Analysis of Microbial Community Structure The diﬀerences in microbial community structure of the two in device B, which is likely to be caused by the instability of the system at the initial operation of the tidal ﬂow, and the oxygen concentration in the lower layer of the device may be insuﬃcient. Additionally, the microbial community structure of AOA, AOB, NOB, and V4−V5 region were changed by 82.96%, 84.1%, 92.27%, and 65.94%, respectively. These results indicated that the microbial community structure is largely separated by space, which is consistent with the bar plot and cluster tree shown in Figure 6. The C/N ratio also had inﬂuence on the structure of the four types of microorganisms. While that of Nitrobacter, which belongs to NOB, was not aﬀected by the C/N ratio, the relative abundances of Candidatus Nitrosotenuis (belongs to AOA) and Nitrosomonas (belongs to AOB) in the cathode layer were signiﬁcantly promoted by a low C/N ratio (5:1) during the stabilization period. This result is consistent with the inﬂuence of C/N ratio on the diversity of the microbial community, indicating that these predominant genera may cause the change of diversity. Further, as shown in Figure 6, a low C/N ratio (5:1) signiﬁcantly increased the relative abundance of Nitrosopumilus (belongs to AOA) in all layers. Diversity of Microbial Community Although the discovery of anammox genera, the role FIGURE 6 \| Bar plot and cluster tree of microbial community of panels (A) AOA, (B) AOB, (C) NOB, and (D) 16S rRNA V4–V5 region. Diversity of Microbial Community AOA was found to participate in ammonia removal processes; however, its functions and contributions in the processes under the inﬂuence of MFC remain unclear. These results show that at a low C/N ratio (5:1), the cathode layer is beneﬁcial to the survival of the dominant archaea AOA. On the other hand, a high C/N ratio (10:1) promoted the relative abundance of Nitrosomonas (belongs to AOB) in the anode and the upper layer during the stabilization period. This may be caused by the increased production of carbon dioxide in the anode layer, and AOB may consume a large amount of inorganic nitrogen while immobilizing carbon dioxide (He et al., 2016). These data indicate that the C/N ratio has a signiﬁcant eﬀect on the spatial distribution of the dominant bacteria AOB. The results further showed that a low C/N ratio is more suitable for the growth of Candidatus Nitrosotenuis (belongs to AOA), whereas a high C/N ratio is beneﬁcial to the growth of Nitrosomonas (belongs to AOB). It is likely that Nitrosomonas can better adapt to a highly polluted environment than Candidatus Nitrosotenuis, as has been described by a previous study (Fan et al., 2011). FIGURE 6 \| Bar plot and cluster tree of microbial community of panels (A) AOA, (B) AOB, (C) NOB, and (D) 16S rRNA V4–V5 region. As shown in Figure 6C, the relative abundance of Nitrobacter in each sample was the highest (from 86.95 to 99.99%). This is consistent with the previous study, which has described that Nitrobacter is the single abundant genus in NOB (Zhang et al., 2019). However, it is opposite to another study using laboratory-scale devices, which has reported that Nitrospira is the predominant genus in NOB (Sayess et al., 2013). The diﬀerence is likely due to that Nitrospira can better adapt to low nitrite and oxygen concentrations, and Nitrobacter can outcompete Nitrospira at high substrate concentration (Maixner et al., 2006). The genera in the V4−V5 region was more abundant than those in other microorganisms, and the dominant microorganisms included Phormidium, Planctomyces, Gemmata, and Aquicella (Figure 6D). Trichococcus and Rhodopseudomonas preferred a high C/N ratio, while Planctomyces and Gemmata, which are anammox bacteria (Xu et al., 2018), were less aﬀected by the C/N ratio. Comparative Analysis of Microbial Community Structure The diﬀerences in microbial community structure of the two systems were evaluated based on the phylogenetic lineages by the weighted Fast UniFrac PCoA. As shown in Figure 5, there was a certain loss of AOA in Device A and of AOB June 2020 \| Volume 11 \| Article 1385 Frontiers in Microbiology \| www.frontiersin.org 10 Nitrogen Removal and Bioelectricity Generation Wang et al. FIGURE 7 \| RDA analysis curves of panels AOA (A), AOB (B), NOB (C), and 16S rRNA V4-V5 region (D). Symbols indicate the samples and arrows represent environmental variables. Group A represents Device A operated with a C/N ratio = 10:1, and Group B represents Device B operated with a C/N ratio = 5:1. Environmental variables were chosen based on the signiﬁcance determined from individual RDA results. FIGURE 7 \| RDA analysis curves of panels AOA (A), AOB (B), NOB (C), and 16S rRNA V4-V5 region (D). Symbols indicate the samples and arrows represent environmental variables. Group A represents Device A operated with a C/N ratio = 10:1, and Group B represents Device B operated with a C/N ratio = 5:1. Environmental variables were chosen based on the signiﬁcance determined from individual RDA results. by the general chemical indexes (pH and DO, 29%), and temperature (21%); (2) AOB community structure was explained by the nitrogen concentration (12%), and the shared fraction between pH, DO, and nitrogen concentration accounted for 16%; (3) NOB community structure was explained by the shared fraction between pH, DO, and nitrogen concentration (37%), and the shared fraction between nitrogen concentration and temperature accounted for 33%; and (4) the community structure of V4−V5 region was explained by nitrogen concentration (14%), and the shared fraction between pH, DO, and nitrogen concentration accounted for 12%. These data were subjected to the RAD analysis to determine the correlation between speciﬁc environmental factors and the community structure of of NH4+-N removal via anaerobic ammonium oxidation in TFCW-MFC was negligible in the presence of relatively high DO throughout the tidal ﬂow. TABLE 3 \| Pearson correlation between alpha diversity at the genus level, voltage, and efﬂuent nitrogen concentration (n = 16). AOA−chao AOB−chao NOB-chao V4−V5-chao AOA-shannon AOB-shannon NOB-shannon V4−V5-shannon TN NH4+-N NO3−-N NO2−-N ABLE 3 \| Pearson correlation between alpha diversity at the genus level, voltage, and efﬂuent nitrogen concentration (n = 16). AOA−chao AOB−chao NOB-chao V4−V5-chao AOA-shannon AOB-shannon NOB-shannon V4−V5-shannon TN NH4+-N NO3−-N NO2−-N OB-chao −0.154a OB-chao −0.316 0.682* 4-V5-chao 0.839 −0.371 −0.379 OA-shannon 0.872 −0.297 −0.433 0.821 OB-shannon −0.150 0.812 0.522* −0.333 −0.274 OB-shannon −0.293 0.146 0.383 −0.173 −0.264 0.117 4-V5-shannon 0.721 −0.325 −0.341 0.927 0.747** −0.264 −0.185 N −0.608* 0.477 0.460 −0.646 −0.693 0.220 0.175 −0.596* H4+-N −0.688** −0.413 −0.543* −0.826 −0.746 0.296 0.096 −0.744 0.828 O3−-N 0.265 −0.232 0.233 0.392 0.234 −0.123 0.457 0.303 −0.169 −0.552* O2−-N −0.122 0.213 −0.188 −0.181 −0.005 0.542* −0.424 −0.212 −0.179 0.013 −0.550* oltage 0.740 −0.402 −0.639 0.825 0.477 −0.085 −0.244 0.758 −0.819 −0.775 0.951* 0.757 coefﬁcient of association (R); signiﬁcant correlation (P < 0.05); signiﬁcant correlation (P < 0.01). Bold values mean signiﬁcance correlation including p < 0.05 and p < 0.01. the four types of microorganisms under the inﬂuence of diﬀerent C/N ratios. The RDA results indicated that the residual concentration of NO2−-N is positively correlated with AOA, AOB, NOB, and microorganisms in the V4−V5 region in the lower layer of the two devices. Additionally, the residual concentration of TN and NH4+-N, and temperature were positively correlated with AOB, NOB, and the microorganisms in the V4−V5 region of Device A, but not with AOA. Moreover, the pH, DO, and residual concentration of NO3−-N were positively correlated with AOA, AOB, and NOB in the upper layer of the two devices at day 90. This is consistent with the results reported by Zhou et al. (2016), who have observed that multiple physicochemical parameters are involved in the relative abundance of nitrifying microorganisms grown in the same environment. Combined with the VPA results, we may conclude that the ecological niche of TFCW-MFC device is mainly inﬂuenced by pH, which serves as the crucial factor that changes the diversities and compositions of all four microbial communities. It appears that each microbial species has its own optimal survival environment; for example, the optimal conditions for Nitrosomonas (belong to AOB) are 35◦C and pH 8.1 and those for Nitrobacter (belongs to NOB) are 38◦C and pH 7.9 (Grunditz and Dalhammar, 2011). At day 90, the eﬄuent pH and temperature in Device A were 7.47 and 23.5◦C, respectively, while those in Device B were 7.34 and 23.1◦C, respectively. TABLE 3 \| Pearson correlation between alpha diversity at the genus level, voltage, and efﬂuent nitrogen concentration (n = 16). AOA−chao AOB−chao NOB-chao V4−V5-chao AOA-shannon AOB-shannon NOB-shannon V4−V5-shannon TN NH4+-N NO3−-N NO2−-N The second most inﬂuential factor is DO, which plays an important role as an electron acceptor for nitrifying microorganisms; thus, it can determine microbial growth and reproductive rate (Yuan and Blackall, 2002). DO is also a key factor aﬀecting the positive correlation between NO3−-N and microorganisms in the upper layer, and NO2−-N and microorganisms in the lower layer. AOB population size has been reported to increase with increasing soil NH4+-N concentration (Okano et al., 2004), and AOB and NOB growths rely on the energy provided by NH4+-N and NO2−-N (Winkler et al., 2012). However, the amount of AOA is not correlated with ammonia concentration (Di et al., 2009), therefore the eﬀects of NH4+-N concentration on AOA, AOB and NOB are diﬀerent. By considering the eﬀects of voltage on microbial community, voltage had a greater impact on the V4−V5 region microorganisms than AOA, AOB, and NOB; it was also positively correlated with the V4−V5 region microorganisms in Device A only at day 90 and in Device B only at day 30. The V4−V5 region comprises many exoelectrogenic microorganisms (Corbella et al., 2015); it is likely that electrons are largely depleted due to the relatively high concentrations of electron acceptors such as NO2−-N, NO3−-N, oxygen, etc., Wang et al. (2016). Correlation Between Single or Multiple Environmental Variables and Microbial Community Structure The eﬀects of environmental variables, including nitrogen concentration, pH, DO, temperature, and voltage, on microbial community structure were analyzed using VPA (Supplementary Figure S3) and RDA (Figure 7). According to the VPA diagram (Supplementary Figure S3), the following results were observed; (1) AOA community structure was explained June 2020 \| Volume 11 \| Article 1385 Frontiers in Microbiology \| www.frontiersin.org 11 Nitrogen Removal and Bioelectricity Generation Wang et al. Pearson Correlation Between Microbial Characteristic Parameters, Nitrogen Removal, and Voltage Output Planctomyces (V4−V5 region) belongs to the anammox bacteria that can use NH4+-N as an electron donor and NO3−-N/NO2−-N as an electron acceptor to convert nitrogen compounds in water into nitrogen. The correlation between the alpha diversity of each genus and NH4+-N is similar to that between the alpha diversity of genus and TN. Concentration of NH4+-N was signiﬁcantly negatively correlated with V4−V5-chao, V4−V5- shannon, AOA-chao, and AOA-shannon with R = −0.826, −0.744, −0.688, and −0.746, respectively (P < 0.01), as well as with NOB-chao with R = −0.543 (P < 0.05). By contrast, it was signiﬁcantly positively correlated with TN concentration with R = 0.828 (P < 0.01). The correlation between the genus and nitrogen removal (Table 4) further indicated that NH4+-N concentration was signiﬁcantly negatively correlated with Candidatus Nitrosotenuis (belongs AOA), Nitrobacter (belongs NOB), and Planctomyces (belongs to V4−V5 region microorganisms) with R-values of −0.617 (P < 0.05), −0.648 (P < 0.01), and −0.819 (P < 0.01), respectively. Candidatus Nitrosotenuis was the dominant genus of AOA, and a low C/N ratio could signiﬁcantly promote its relative abundance in the cathode layer during the stabilization period; thus, this genus is more conducive to the conversion of NH4+-N. Furthermore, as the most abundant genus of NOB, Nitrobacter is suitable for NH4+-N removal in wastewater, which has also been conﬁrmed by our previous study (Zhang et al., 2019). The eﬄuent concentration of NO2−-N was signiﬁcantly positively correlated with AOB-shannon with R = 0.542 with Candidatus Nitrosotenuis, Nitrososphaera, and Gemmata with R = 0.527, 0.566, and 0.614, respectively (P < 0.05). This demonstrated that the abundance of ammonia oxidizing bacteria increases with increasing eﬄuent NO2−-N and NO3−- N concentrations. Gemmata belongs to anammox bacteria, which can convert ammonia nitrogen using NO2−-N as an electron acceptor; thus a negative correlation between NO2−-N and Gemmata was observed. Furthermore, voltage was signiﬁcantly positively correlated with the eﬄuent NO3−-N concentration, Nitrosopumilus, Nitrosospira, Altererythrobacter and Aquicella with R = 0.951, 0.983, 0.963, 0.963, and 0.962 (P < 0.05), respectively, while was signiﬁcantly negatively correlated with Gemmata (R = −0.999; P < 0.01). These data indicate the above genera and the electrogenic bacteria in the system may work synergistically so that the eﬀect of the electrogenic bacteria is promoted, causing the voltage output to increase. Pearson Correlation Between Microbial Characteristic Parameters, Nitrogen Removal, and Voltage Output Concentration of NH4+-N was signiﬁcantly negatively correlated with V4−V5-chao, V4−V5- shannon, AOA-chao, and AOA-shannon with R = −0.826, −0.744, −0.688, and −0.746, respectively (P < 0.01), as well as with NOB-chao with R = −0.543 (P < 0.05). By contrast, it was signiﬁcantly positively correlated with TN concentration with R = 0.828 (P < 0.01). The correlation between the genus and nitrogen removal (Table 4) further indicated that NH4+-N concentration was signiﬁcantly negatively correlated with Candidatus Nitrosotenuis (belongs AOA), Nitrobacter (belongs NOB), and Planctomyces (belongs to V4−V5 region microorganisms) with R-values of −0.617 (P < 0.05), −0.648 (P < 0.01), and −0.819 (P < 0.01), respectively. Candidatus Nitrosotenuis was the dominant genus of AOA, and a low C/N ratio could signiﬁcantly promote its relative abundance in the cathode layer during the stabilization period; thus, this genus is more conducive to the conversion of NH4+-N. Furthermore, as the most abundant genus of NOB, Nitrobacter is suitable for NH4+-N removal in wastewater, which has also been conﬁrmed by our previous study (Zhang et al., 2019). Pearson Correlation Between Microbial Characteristic Parameters, Nitrogen Removal, and Voltage Output To further study the eﬀect of microorganisms on nitrogen removal and bioelectricity generation, the relationship between alpha diversity index, voltage output, and eﬄuent nitrogen concentration was analyzed by the Pearson correlation. As shown June 2020 \| Volume 11 \| Article 1385 Frontiers in Microbiology \| www.frontiersin.org 12 microorganisms, voltage, and efﬂuent nitrogen concentration (n = 16). a Alterery throbacter Nitro bacter Phor midium Planc tomyces Gem mata Aqui cella TN NH 1 0.219 1 0.212 −0.115 −0.174 0.219 1 0.218 0.399 −0.139 1 0.068 0.230 −0.316 0.629* −0.290 0.047 −0.176 0.737 1 0.653 0.313 0.152 0.289 −0.100 1 −0.422 −0.424 0.220 −0.666** −0.234 −0.572* 1 −0.287 −0.648 −0.109 −0.819 −0.443 −0.446 0.828** 1 −0.263 0.200 0.436 0.463 0.614* −0.040 −0.169 −0. 0.579* 0.131 0.028 −0.384 −0.698** 0.575* −0.179 0.0 0.963* 0.439 −0.064 0.854 −0.999** 0.962* −0.819 −0. ation (P < 0.01). The selected genus accounts for >10% of the total composition of each gene. Bold NH 8 1 9 −0. 9 0. 9 −0. Bold N 8 1 9 −0. 9 0. 9 −0. Bold Nitrogen Removal and Bioelectricity Generation Wang et al. in Table 3, TN concentration was signiﬁcantly negatively correlated with V4−V5-chao and AOA-shannon with R-values of −0.646 and −0.693, respectively (P < 0.01), as well as with AOA-chao and V4−V5-shannon with R-values of −0.608 and −0.596, respectively (P < 0.05). This is mainly due to the dominant genera of AOA and V4−V5 region. The Pearson correlation analysis was employed to further clarify the correlation between the dominant microorganisms and nitrogen removal. The analysis was performed using the high- abundance microorganism only the genus with >10% of the total genus composition), and the eﬄuent nitrogen concentrations from the devices using each of these microorganisms were then compared. As shown in Table 4, TN concentration was signiﬁcantly negatively correlated with Nitrosopumilus (belongs to AOA) and Aquicella (belongs to V4−V5 region) with R-values of −0.529 and −0.572, respectively (P < 0.05), as well as with Planctomyces (V4−V5 region) with R-value of −0.666 (P < 0.01). Nitrosopumilus is a dominant genus of AOA, and a low C/N ratio could signiﬁcantly promote its relative abundance in all layers during the stabilization period. Therefore, Nitrosopumilus, which was especially rich in anode and cathode layers, could accelerate the nitriﬁcation reaction and greatly promote the conversion of nitrogen. Pearson Correlation Between Microbial Characteristic Parameters, Nitrogen Removal, and Voltage Output On the other hand, organic matter can be oxidized by electrogens at the anode of MFC, and the produced electrons can ﬂow to the cathode by an external circuit (Puig et al., 2012). Consequently, NH4+-N loses its electrons and is converted to NO2−-N and NO3−-N by the action of microorganisms, as indicated by a higher abundance and diversity of the microorganism at the cathode and anode layers. Gemmata, which belongs to the phylum Planctomycetes, contains ﬁbrillar nucleoid surrounded by electron-dense granules (Fuerst and Richard, 1991) that may have an adverse eﬀect on the electrogenic microorganism in the system. In spite of these data, the distribution characteristics of the electrogenic microorganisms and their contribution to the electricity production have not been analyzed. Thus, studies on the topics should be further conducted. in Table 3, TN concentration was signiﬁcantly negatively correlated with V4−V5-chao and AOA-shannon with R-values of −0.646 and −0.693, respectively (P < 0.01), as well as with AOA-chao and V4−V5-shannon with R-values of −0.608 and −0.596, respectively (P < 0.05). This is mainly due to the dominant genera of AOA and V4−V5 region. The Pearson correlation analysis was employed to further clarify the correlation between the dominant microorganisms and nitrogen removal. The analysis was performed using the high- abundance microorganism only the genus with >10% of the total genus composition), and the eﬄuent nitrogen concentrations from the devices using each of these microorganisms were then compared. As shown in Table 4, TN concentration was signiﬁcantly negatively correlated with Nitrosopumilus (belongs to AOA) and Aquicella (belongs to V4−V5 region) with R-values of −0.529 and −0.572, respectively (P < 0.05), as well as with Planctomyces (V4−V5 region) with R-value of −0.666 (P < 0.01). Nitrosopumilus is a dominant genus of AOA, and a low C/N ratio could signiﬁcantly promote its relative abundance in all layers during the stabilization period. Therefore, Nitrosopumilus, which was especially rich in anode and cathode layers, could accelerate the nitriﬁcation reaction and greatly promote the conversion of nitrogen. Planctomyces (V4−V5 region) belongs to the anammox bacteria that can use NH4+-N as an electron donor and NO3−-N/NO2−-N as an electron acceptor to convert nitrogen compounds in water into nitrogen. The correlation between the alpha diversity of each genus and NH4+-N is similar to that between the alpha diversity of genus and TN. REFERENCES Fuerst, J. A., and Richard, I. W. (1991). Membrane-bounded nucleoid in the eubacterium Gemmata obscuriglobus. Proc. Natl. Acad. Sci. U.S.A. 88, 8184– 8188. doi: 10.1073/pnas.88.18.8184 Albuquerque, A., Arendacz, M., Gajewska, M., Obarska-Pempkowiak, H., Randerson, P., and Kowalik, P. (2009). Removal of organic matter and nitrogen in an horizontal subsurface ﬂow (HSSF) constructed wetland under transient loads. Water Sci. 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This study was supported by the Innovative Team Project of the Nanjing Institute of Environmental Sciences, MEE (GYZX200101), the National Natural Science Foundation of China (Nos. 51778265 and 51709001), the Major Science and Technology Program for Water Control and Treatment (2017ZX07204001), Key R&D Program of Ningxia Province (Special Project for S&T Cooperation with unit outside the province in 2019) (2019BFG02028), the Research Project for Environmental Science and Technology of Ningxia (2018-005), and the Nature Science Foundation of Anhui Province of China (1708085QD81). SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2020.01385/full#supplementary-material CONCLUSION In summary, we demonstrated that the removal of COD, NH4+- N, and TN from wastewater with diﬀerent C/N ratios (10:1 and 5:1) using TFCW-MFC were not signiﬁcantly diﬀerent. Compared to the conventional CW-MFC, our TFCW-MFC achieved a high and stable TN removal eﬃciency (77–89%) from wastewater containing various C/N ratios. Moreover, the high-throughput sequencing analysis conﬁrmed that a low C/N led to enhanced AOA and AOB diversities in the cathode layer during the stabilization period, causing the community structure of the dominant genera to change drastically. A low C/N ratio (5:1) signiﬁcantly promoted the relative abundance of Candidatus Nitrosotenuis, Nitrosopumilus (both belong to AOA) and Nitrosomonas (belongs to AOB) in the cathode layer during the stabilization period. On the other hand, a high C/N ratio (10:1) promoted the relative abundance of Nitrosomonas (belongs to AOB) in anode and upper layers. We further observed that multiple environmental variables collaboratively contributed to change of microbial compositions; for instance, pH, DO, and nitrogen concentration could potentially change the spatial distribution of the microbial community structure. The results further revealed that eﬄuent nitrogen concentration could be reduced through changes of certain alpha diversities indexes of microorganisms or relative abundances of the dominant genera. The eﬄuent concentration of NO2−-N was signiﬁcantly positively correlated with AOB-shannon with R = 0.542 (P < 0.05), while may be related with the dominant genus Altererythrobacter (R = 0.579; P < 0.05). Additionally, NO2−- N concentration was also signiﬁcantly positively correlated with Nitrosopumilus and Aquicella with R-values of 0.525 and 0.575, respectively (P < 0.05), while was negatively correlated with Gemmata with R-value of −0.698 (P < 0.01). 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The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Zhou, X. H., Li, Y. M., Zhang, J. P., Liu, B., Wang, M. Y., Zhou, Y. W., et al. (2016). Diversity, abundance and community structure of ammonia-oxidizing archaea and bacteria in riparian sediment of Zhenjiang ancient canal. Ecol. Eng. 90, 447–458. doi: 10.1016/j.ecoleng.2016.01.068 June 2020 \| Volume 11 \| Article 1385 Frontiers in Microbiology \| www.frontiersin.org 17
https://openalex.org/W2792652901	https://www.mdpi.com/2072-4292/10/3/467/pdf?version=1525337842	English	null	Potential of Combining Optical and Dual Polarimetric SAR Data for Improving Mangrove Species Discrimination Using Rotation Forest	Remote sensing	2,018	cc-by	11,551	Hongsheng Zhang 1,2, Ting Wang 1, Mingfeng Liu 1, Mingming Jia 3 ID , Hui Lin 1,2,4,, LM Chu 5 and Adam Thomas Devlin 1 Hongsheng Zhang 1,2, Ting Wang 1, Mingfeng Liu 1, Mingming Jia 3 ID , Hui Lin 1,2,4,, LM Chu 5 and Adam Thomas Devlin 1 1 Institute of Space and Earth Information Science, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong; zhangstream@gmail.com (H.Z.); wangtingwhu@126.com (T.W.); mingfengliu@link.cuhk.edu.hk (M.L.); adam_devlin@cuhk.edu.hk (A.T.D.) 1 Institute of Space and Earth Information Science, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong; zhangstream@gmail.com (H.Z.); wangtingwhu@126.com (T.W.); mingfengliu@link.cuhk.edu.hk (M.L.); adam_devlin@cuhk.edu.hk (A.T.D.) 2 Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen 518057, China 3 Key Laboratory of Wetland Ecology and Environment, Northeast Institute of Geography and Agro Chinese Academy of Sciences, No. 4888, Shengbei Street, Changchun 130102, China; jiamingming@i y g g j g g g 4 Key Laboratory of Poyang Lake Wetland and Watershed Research, Ministry of Education, Jiangxi Normal University, Nanchang 330022, China g y g 5 School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; leemanchu@cuhk.edu.hk 5 School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; leemanchu@cuhk.edu.hk * Correspondence: huilin@cuhk.edu.hk; Tel.: +852-39436010 Received: 18 November 2017; Accepted: 13 March 2018; Published: 16 March 2018 Abstract: Classification of mangrove species using satellite images is important for investigating the spatial distribution of mangroves at community and species levels on local, regional and global scales. Hence, studies of mangrove deforestation and reforestation are imperative to support the conservation of mangrove forests. However, accurate discrimination of mangrove species remains challenging due to many factors such as data resolution, species number and spectral confusion between species. In this study, three different combinations of datasets were designed from Worldview-3 and Radarsat-2 data to classify four mangrove species, Kandelia obovate (KO), Avicennia marina (AM), Acanthus ilicifolius (AI) and Aegiceras corniculatum (AC). Then, the Rotation Forest (RoF) method was employed to classify the four mangrove species. Results indicated the benefits of dual polarimetric SAR data with an improvement of accuracy by 2–3%, which can be useful for more accurate large-scale mapping of mangrove species. Moreover, the difficulty of classifying different mangrove species, in order of increasing difficulty, was identified as KO < AM < AI < AC. Dual polarimetric SAR data are recognized to improve the classification of AI and AC species. Although this improvement is not remarkable, it is consistent for all three methods. Hongsheng Zhang 1,2, Ting Wang 1, Mingfeng Liu 1, Mingming Jia 3 ID , Hui Lin 1,2,4,*, LM Chu 5 and Adam Thomas Devlin 1 The improvement can be particularly important for large-scale mapping of mangrove forest at the species level. These findings also provide useful guidance for future studies using multi-source satellite data for mangrove monitoring and conservation. Keywords: mangrove species; rotation forest; Worldview-3; Radarsat-2; Hong Kong wetlands www.mdpi.com/journal/remotesensing remote sensing remote sensing remote sensing 1. Introduction Mangrove forests comprise one of the most productive and important planetary ecosystems because of their ecological, biological and socioeconomic significance [1–3]. They provide important ecological services including: (1) shoreline stabilization to reduce the impacts of natural disasters; (2) habitat provision for invertebrates, fish and other coastal wildlife; and (3) carbon sequestration for the ocean [1]. However, mangrove forest cover has decreased dramatically during the past century due to various human activities such as urbanization, agriculture conversion and tourism [1,2]. Monitoring the spatial distribution of mangrove forests is imperative to understand their vegetation dynamics better, such as Remote Sens. 2018, 10, 467; doi:10.3390/rs10030467 www.mdpi.com/journal/remotesensing 2 of 15 Remote Sens. 2018, 10, 467 succession, deforestation, stand density and health conditions, as well as further understanding the ecological services they provide [1,2,4,5]. Conventionally, a number of approaches has been proposed to monitor and analyze mangrove forests using remote sensing data from various data sources at different spatial, spectral and temporal resolutions. Regarding the remote sensing data sources, most previous studies can be grouped into two main categories, those employing optical data (e.g., multispectral and hyperspectral data) and those using Synthetic Aperture Radar (SAR) data (single- and multi-polarimetric data). High resolution satellite data have been used for discrimination of mangrove species [6–11], since they contained rich textural information allowing identification of various species. Hyperspectral satellite data were also intensively employed for mangrove species discrimination [12–14]. Additionally, coarser resolution satellite data were applied to map the spatial dynamics of mangrove forests using mixed analysis and object-oriented approaches [1,4,5,15–17]. Additionally, SAR satellite data have been increasingly used to study mangroves since longer wavelength (e.g., C and L bands) active microwave signals are sensitive to the surface and vertical structures and, hence, biomass of mangrove forests. Different frequencies (C-/L-/P-band) of microwave radar have also been used to examine different mangrove species (Laguncularia, Avicennia and Rhizophora) using airborne multi-polarimetric SAR data [18–22]. The tree density, basal area, tree height, tree DBH and biomass were used to test the responses from different frequencies of SAR data. With a shorter wavelength and less penetration, the C-band was found to be more sensitive to crown characteristics, canopy structure and canopy heterogeneity [18–20]. The radiative nature of C-band backscatters was investigated in different vegetation, such as wheat and burned forests regarding their canopy [23,24]. 1. Introduction This radiative nature was also modelled based on the C-band backscattering with different vegetation, such as the Michigan Microwave Canopy Scattering (MIMICS) model towards the soybean canopy using Spaceborne Imaging Radar-C (SIR-C) data [25] and the Water Cloud Model (WCM) based on the boreal forests using ERS-1 data [26]. Polarimetric data were applied to study mangrove species by analyzing different polarizations in the coast of French Guiana [19]. Biophysical parameters such as Leaf Area Index (LAI) and biomass were also calculated from multi-polarimetric ENVISAT and ALOS data to study the health of mangroves [27,28]. In particular, optical and SAR remote sensing data provide complementary information to each other [29–31], and thus, their combined use can enhance better accuracy for mangrove monitoring. Both Landsat and Radarsat-1 data were applied to conduct a comprehensive classification over coastal forests in the Brazilian Amazon Region [32]. Landsat and ENVISAT were also combined together to analyze mangrove conditions [33]. More recently, hyperspectral data were combined with ENVISAT data to map mangrove species in Hong Kong [34,35]. However, the dual polarimetric SAR data are yet to be investigated to explore their potentials for mangrove species classification. This study aims to evaluate this potential regarding different mangrove species (Kandelia obovate, Avicennia marina, Acanthus ilicifolius and Aegiceras corniculatum) using the rotation forest method, and thus provide a technical reference for studies employing the C-band dual polarimetric Radarsat-2 data for mangrove monitoring. 2.1. Study Site The Mai Po Marshes Nature Reserve (MPMNR), 22◦29′23.75′′N, 114◦1′42.71′′E, was selected as the study site for mangrove species discrimination in this study. It is located in the northwestern part in the New Territories, Hong Kong, bordering on the inner Deep Bay between Shenzhen and Hong Kong (Figure 1). The Mai Po and inner Deep Bay are listed as a Wetland of International Importance under the Ramsar Convention, which was the ﬁrst Ramsar Site Strategy and Management Plan (RSMP) in Hong Kong [16,35,36]. There are around 350 ha of mangrove forests in the Core Zone of the MPMNR. The whole wetland is under the inﬂuence of a humid subtropical climate with rainy and cloudy weather throughout the year, which has hindered the applications of any form of optical satellite remote sensing [16,37]. Additionally, the area is located on the Pearl River Estuary (PRE), which has been affected by the dramatic urbanization of the Pearl River Delta (PRD) region, which is 3 of 15 (PRE), hich is Remote Sens. 2018, 10, 467 satellite remote sensi which has been affect affected by serious water and air pollution. Moreover, sea level change and temperature increase as a result of climate change threaten wetlands through sedimentation and erosion in the intertidal area [36]. To improve the management of the region and enhance the ecological values of the wetland, the MPMNR is structured into ﬁve different zones by the Agriculture, Fisheries and Conservation Department (AFCD), including the Core Zone, Biodiversity Management Zone, Wise Use Zone, Public Access Zone and Private Land Zone. y p g p a result of climate change threaten wetlands through sedimentation and erosion in the intertidal area [36]. To improve the management of the region and enhance the ecological values of the wetland, the MPMNR is structured into five different zones by the Agriculture, Fisheries and Conservation Department (AFCD), including the Core Zone, Biodiversity Management Zone, Wise Use Zone, Public Access Zone and Private Land Zone. (a) Figure 1. Location of the study site in Hong Kong: (a) map showing its geographical setting in the Pearl River Estuary (PRE), (b) a fine-scale map of the Mai Po Marshes Nature Reserve (MPMNR), modified from the government report published by the Agriculture, Fisheries and Conservation Department (AFCD, 2011), and (c) a true color image from Worldview-3 data. (a) (b) (c) Figure 1. 2 2 Field Data 2.2. Field Data The MPMNR is the biggest mangrove forest in Hong Kong and one of the few extensive mangroves in southern China [16,38,39]. Among these management zones, the Core Zone was selected as the major study site where a total of seven mangrove species were reported by the local government [35,36]. However, only four species distributed over a wide area can be identified from satellite images (Figure 2), including Kandelia obovate (KO), Avicennia marina (AM), Acanthus ilicifolius (AI) and Aegiceras corniculatum (AC). A set of data was collected by two rounds of field surveys over the MPMNR on 11 July 2013 and 10 November 2015. Since the Core Zone area is strictly protected by the Hong Kong government, a quadrat survey was not allowed to be used to investigate the biomass of mangroves. Only GPS receivers and cameras were used to record the field data by marking the species of mangroves during our two survey rounds. A total of 718 samples were collected for six classes, including Mudflat (MUD), Water body (WAT) and four mangrove species, namely KO, AM, AI and AC. In particular, the collection time of field data and satellite data, to be discussed in the next section, had a time difference of about two years. However, the Core Zone has been highly protected since 1995 [36,40], indicating minimal impacts from local human activities. Based on previous investigations of the mangrove biomass that demonstrated that the biomass of local mangrove forests The MPMNR is the biggest mangrove forest in Hong Kong and one of the few extensive mangroves in southern China [16,38,39]. Among these management zones, the Core Zone was selected as the major study site where a total of seven mangrove species were reported by the local government [35,36]. However, only four species distributed over a wide area can be identified from satellite images (Figure 2), including Kandelia obovate (KO), Avicennia marina (AM), Acanthus ilicifolius (AI) and Aegiceras corniculatum (AC). A set of data was collected by two rounds of field surveys over the MPMNR on 11 July 2013 and 10 November 2015. Since the Core Zone area is strictly protected by the Hong Kong government, a quadrat survey was not allowed to be used to investigate the biomass of mangroves. Only GPS receivers and cameras were used to record the field data by marking the species of mangroves during our two survey rounds. 2.1. Study Site Location of the study site in Hong Kong: (a) map showing its geographical setting in the Pearl River Estuary (PRE), (b) a ﬁne-scale map of the Mai Po Marshes Nature Reserve (MPMNR), modiﬁed from the government report published by the Agriculture, Fisheries and Conservation Department (AFCD, 2011), and (c) a true color image from Worldview-3 data. (b) (b) (c) (c) Figure 1. Location of the study site in Hong Kong: (a) map showing its geographical setting in the Pearl River Estuary (PRE), (b) a fine-scale map of the Mai Po Marshes Nature Reserve (MPMNR), modified from the government report published by the Agriculture, Fisheries and Conservation Department (AFCD, 2011), and (c) a true color image from Worldview-3 data Figure 1. Location of the study site in Hong Kong: (a) map showing its geographical setting in the Pearl River Estuary (PRE), (b) a ﬁne-scale map of the Mai Po Marshes Nature Reserve (MPMNR), modiﬁed from the government report published by the Agriculture, Fisheries and Conservation Department (AFCD, 2011), and (c) a true color image from Worldview-3 data. 2 3 Dataset and Preprocessing 2.3. Dataset and Preprocessing 2.3. Dataset and Preprocessing Worldview-3 data were employed as the optical data, while the dual polarimetric SAR data from Radarsat-2 were used as the SAR data to classify the four mangrove species. The Worldview-3 data, collected on 1 January 2015, have eight multispectral bands at 1.6-m resolution and one panchromatic band at 0.4-m resolution. To pre-process the eight-band product, a radiometric correction has been conducted by the data provider DigitalGlobe Corporate (U.S.). Additionally, a calibration was applied to convert the data into absolute radiance in the units of (uW)/(cm2 × nm × sr), using the metadata file provided by the data provider. The calibration was conducted by the software ENVI 5.0 with the metadata file. The Radarsat-2 data used in this study are dual-polarimetric products with HH and HV polarizations, collected on 8 November 2014. The resolution of geo-referenced Radarsat- 2 data is about 8 m. To preprocess the SAR images, basic radiometric correction and calibration processes were conducted, and thus, the data were transformed to units of dB. Considering the uncertainty of the speckle noises in SAR images, speckle noises were filtered by the enhanced Lee filter, with a window size of 3 × 3 pixels. Multi-look techniques were also applied with two looks in the azimuthal direction and one look in the range direction. There was approximately a two-month difference of the imaging dates between the Worldview-3 and Radarsat-2 data; however, as they occurred in the same season, we assumed that there was no significant change of the mangrove forests within this period. Finally, both the optical and SAR data were geocoded and co-registered using the Universal Transverse Mercator (UTM) projection (Zone 49N) and Datum World Geodetic System 1984 (WGS84). The detailed parameters of the data are provided in Table 1. Since the two datasets were of different spatial resolutions, according to our previous experiences in integrating ti l d SAR d t [29] th W ld i 3 d t l d t 8 th R d t 2 d t Worldview-3 data were employed as the optical data, while the dual polarimetric SAR data from Radarsat-2 were used as the SAR data to classify the four mangrove species. The Worldview-3 data, collected on 1 January 2015, have eight multispectral bands at 1.6-m resolution and one panchromatic band at 0.4-m resolution. 2 2 Field Data 2.2. Field Data A total of 718 samples were collected for six classes, including Mudflat (MUD), Water body (WAT) and four mangrove species, namely KO, AM, AI and AC. In particular, the collection time of field data and satellite data, to be discussed in the next section, had a time difference of about two years. However, the Core Zone has been highly protected since 1995 [36,40], indicating minimal impacts from local human activities. Based on previous investigations of the mangrove biomass that demonstrated that the biomass of local mangrove forests did not change significantly [41,42], we assume that the two-year difference between the field data and satellite data does not have a significant impact on our study. 4 of 15 d data Remote Sens. 2018, 10, 467 did not change signif d t llit d t d Figure 2. Photos of the four mangrove species given close consideration in the field survey, (a) is Kandelia obovate (KO), (b) is Avicennia marina (AM), (c) is Acanthus ilicifolius (AI) and (d) is Aegiceras corniculatum (AC). Figure 2. Photosof thefourmangrovespecies givencloseconsideration inthefield survey, (a)is Kandeliaobovate (KO), (b) is Avicennia marina (AM), (c) is Acanthus ilicifolius (AI) and (d) is Aegiceras corniculatum (AC). Figure 2. Photos of the four mangrove species given close consideration in the field survey, (a) is Kandelia obovate (KO), (b) is Avicennia marina (AM), (c) is Acanthus ilicifolius (AI) and (d) is Aegiceras corniculatum (AC) Figure 2. Photosof thefourmangrovespecies givencloseconsideration inthefield survey, (a)is Kandeliaobovate (KO), (b) is Avicennia marina (AM), (c) is Acanthus ilicifolius (AI) and (d) is Aegiceras corniculatum (AC). 2 3 Dataset and Preprocessing 2.3. Dataset and Preprocessing To pre-process the eight-band product, a radiometric correction has been conducted by the data provider DigitalGlobe Corporate (U.S.). Additionally, a calibration was applied to convert the data into absolute radiance in the units of (uW)/(cm2 × nm × sr), using the metadata file provided by the data provider. The calibration was conducted by the software ENVI 5.0 with the metadata file. The Radarsat-2 data used in this study are dual-polarimetric products with HH and HV polarizations, collected on 8 November 2014. The resolution of geo-referenced Radarsat-2 data is about 8 m. To preprocess the SAR images, basic radiometric correction and calibration processes were conducted, and thus, the data were transformed to units of dB. Considering the uncertainty of the speckle noises in SAR images, speckle noises were filtered by the enhanced Lee filter, with a window size of 3 × 3 pixels. Multi-look techniques were also applied with two looks in the azimuthal direction and one look in the range direction. There was approximately a two-month difference of the imaging dates between the Worldview-3 and Radarsat-2 data; however, as they occurred in the same season, we assumed that there was no significant change of the mangrove forests within this period. Finally, both the optical and SAR data were geocoded and co-registered using the Universal Transverse Mercator (UTM) projection (Zone 49N) and Datum World Geodetic System 1984 (WGS84). The detailed parameters of the data are provided in Table 1. Since the two datasets were of different spatial resolutions, according to our previous experiences in integrating optical and SAR data [29], the Worldview-3 data were resampled to 8 m as the Radarsat-2 data. 5 of 15 Remote Sens. 2018, 10, 467 Table 1. Details of the remote sensing data. REVIEW Table 1. Details of the remote sensing data. REVIEW Satellite Date Spectral/Polarizations Resolution Worldview-3 1 January 2015 Coastal: 400–450 nm 1.6 m Blue: 450–510 nm Green: 510–580 nm Yellow: 585–625 nm Red: 630–690 nm Red Edge: 705–745 nm Near Infrared 1: 770–895 nm Near Infrared 2: 860–1040 nm Radarsat-2 8 November 2014 HH, HV 8 m te Sens. 2018, 10, x FOR PEER REVIEW 5 o Table 1. Details of the remote sensing data. 2 3 Dataset and Preprocessing 2.3. Dataset and Preprocessing Satellite Date Spectral/Polarizations Resolution Worldview-3 1 January 2015 Coastal: 400–450 nm 1.6 m Blue: 450–510 nm Green: 510–580 nm Yellow: 585–625 nm Red: 630–690 nm Red Edge: 705–745 nm Near Infrared 1: 770–895 nm Near Infrared 2: 860–1040 nm Radarsat-2 8 November 2014 HH HV 8 m Spectral/Polarizations Resolution he remote sensing data. 3. Methods 3. Meth The methodological framework used in this study is illustrated in Figure 3. There are four main parts: the preprocessing of satellite data, the feature extraction for optical and SAR data, the mangrove species classiﬁcation and the accuracy assessment using the ﬁeld data. The preprocessing of both optical and SAR data was detailed in Section 2.3, and this section will describe the other three parts of the methods. 3. Methods The methodological framework used in this study is illustrated in Figure 3. There are four main parts: the preprocessing of satellite data, the feature extraction for optical and SAR data, the mangrove species classification and the accuracy assessment using the field data. The preprocessing of both optical and SAR data was detailed in Section 2.3, and this section will describe the other three parts of the methods. Optical Data SAR Data Preprocessing Preprocessing Alpha-entropy decomposition Optical-SAR co-registration Geocoding Feature extraction: NDVI, NDWI, GLCM Feature extraction: GLCM RoF, RF, SVM Field Data Mangrove Species Classification Accuracy Assessment Figure 3. Methodological framework of mangrove species discrimination. RoF, Rotation Forest. Figure 3. Methodological framework of mangrove species discrimination. RoF, Rotation Forest. Figure 3. Methodological framework of mangrove species discrimination. RoF, Rotation Forest. Figure 3. Methodological framework of mangrove species discrimination. RoF, Rotation Forest. 3.1. Feature Extraction from Optical and Polarimetric SAR Data Spectral features were extracted from optical data by analy 3.1. Feature Extraction from Optical and Polarimetric SAR Data 3.1. Feature Extraction from Optical and Polarimetric SAR Data Spectral features were extracted from optical data by analy 3.1. Feature Extraction from Optical and Polarimetric SAR Data Spectral features were extracted from optical data by analyzing the spectral reflectance or digital numbers for each band based on a single pixel. Spectral features are often based on some of the important bands that characterize the features of one land cover type, such as vegetation, water or soil. In this study, the Normalized Difference Vegetation Index (NDVI) and Normalized Difference Water Index (NDWI) were employed as the spectral features for optical data [43,44]. There are two different ways of calculating NDVI and NDWI, corresponding to the standard band NIR1 (772–890 nm) and the additional band NIR2 (866–954 nm) in Worldview-3 data. Spectral features were extracted from optical data by analyzing the spectral reﬂectance or digital numbers for each band based on a single pixel. Spectral features are often based on some of the important bands that characterize the features of one land cover type, such as vegetation, water or soil. In this study, the Normalized Difference Vegetation Index (NDVI) and Normalized Difference Water Index (NDWI) were employed as the spectral features for optical data [43,44]. There are two different ways of calculating NDVI and NDWI, corresponding to the standard band NIR1 (772–890 nm) and the additional band NIR2 (866–954 nm) in Worldview-3 data. 6 of 15 Remote Sens. 2018, 10, 467 Textural features are often beneﬁcial as land targets in high spatial resolution images, as they provide rich spatial details. In this study, as shown in Figure 3, texture features were extracted from both optical and SAR data. Generally, there are numerous textural measurement approaches performed in other studies, while the Grey Level Co-occurrence Matrix (GLCM) approach [45] has been widely employed and proven to be helpful in many study cases. One typical issue when applying GLCM analysis is the determination of the window size to calculate the co-occurrence matrix and the texture-related parameters of the intended target. Since the aim of this study is to evaluate the potential of polarimetric features from SAR data and the complementation between optical and SAR data for mangrove species mapping, we did not apply a comprehensive analysis of the optimization of these GLCM parameters. Empirically, we applied a 3-by-3 window to calculate the GLCM, and eight texture measures based on GLCM were employed, including mean value, variance, homogeneity, contrast, dissimilarity, entropy, angular second moment and correlation. 3.1. Feature Extraction from Optical and Polarimetric SAR Data Spectral features were extracted from optical data by analy 3.1. Feature Extraction from Optical and Polarimetric SAR Data In polarimetric SAR data, the size and shape of mangrove leaves differ from one species to another, while the height and density of mangrove stands also differ among species. These mangrove tree features may lead to different scattering mechanisms, which are reﬂected in the polarimetric features. Various scattering types can be characterized from different decomposition components, including the surface scattering, volume scattering and double bounce scattering. In general, dual polarimetric SAR data can be represented in the form of a scattering matrix (S), which can be further transformed into a coherency matrix (T). Polarimetric features can be extracted from the polarimetric SAR data based on the decomposition of the covariance matrix or coherency matrix by applying various decomposition methods. It should be noted that the polarimetric decomposition was applied before the SAR data were geocoded, while the texture features were extracted after the geocoding and the co-registration, as shown in Figure 3. " # T = " ⟨SHHS∗ HH⟩ SHHS∗ HV ⟨SHVS∗ HH⟩ SHVS∗ HV # (1) (1) In this study, the alpha-entropy decomposition based on the Cloude–Pottier decomposition approach has been widely applied to classify polarimetric SAR data [46,47]. The alpha, entropy (H) and anisotropy (A) are calculated based on the eigenvalues (λ1 and λ2) and eigenvectors of coherency matrix T (Equations (2) and (3)) and are able to reﬂect the scattering information and the complexity of land surfaces. π Alpha = α(P1 −P2) + P2 π 2 (2) H = P1 log2 P1 + P2 log2 P2 (3) A = λ1 −λ2 λ1 + λ2 (4) (2) (3) (4) 3.2. Mangrove Species Classiﬁcation Using Rotation Forest 3.2. Mangrove Species Classiﬁcation Using Rotation Forest 3.2. Mangrove Species Classiﬁcation Using Rotation Forest 3.2. Mangrove Species Classiﬁcation Using Rotation Forest 3.2. Mangrove Species Classiﬁcation Using Rotation Forest Rotation Forest (RoF) is an ensemble machine learning method based on a base decision tree classifier by randomly splitting the samples into K subsets, and Principal Component Analysis (PCA) is applied to each of the subsets for feature extraction [48]. RoF has been applied to the classification of remote sensing images [49]. The detailed technical steps to build up an RoF can be found in [48,49]. In this study, the Classification And Regression Tree (CART) method was selected as the base classifier. Typically, two key parameters need to be set to achieve good performance of RoF, the number of subsets, K, and the percentage of information used for building up the classifier after the transformation of PCA. In this study, K was set to be 30 by empirically testing a series of different values from 10–50. The percentage of preserved information after PCA was set to 75% according to the recommendation in [49], where it was reported that too small of a percentage value cannot maintain the useful information while too large of a percentage value cannot yield an obvious improvement of RoF. In order to evaluate the impacts of these new methods on the mangrove species classification, another two advanced machine learning methodologies, Support Vector Machine (SVM) [50] and Random Forest 7 of 15 Remote Sens. 2018, 10, 467 (RF) [51] were comparatively employed to carry out the classification. Both SVM and RF were set with optimized parameters based on previous studies. For instance, for the SVM method, the Radial Basis Function (RBF) was used as the kernel function. Additionally, there are two key parameters that can influence the performance of SVM, the gamma (G) in RBF and the penalty (C) during the identification of support vectors. In this study, the optimal G and C were chosen using an empirical grid-search method that was previously used in our research [52]. Moreover, a one-against-rest strategy was employed to implement the multi-class SVM [53]. For the RF method, the performance depends on two key parameters, the number of decision trees (T) and the number of variables/features (m), to determine the decision rules at each node. In our previous study, we analyzed the optimal setting of T and m by combining optical and SAR data. 3.3. Validation and Accuracy Assessment Result validation was applied to the four mangroves species using the reference samples described in Section 2.2. Since a supervised classiﬁcation approach was applied, the reference samples were separated into two parts; model calibration and results validation, with 80% for training and 20% for testing. The detailed distribution of all samples among different classes is shown in Table 2. Compared to mangrove species identiﬁcation, water and mudﬂat designations were much easier to classify. Therefore, the accuracy would be higher than that using six classes. To represent the accuracy of the classiﬁcation, the most commonly-used technique is a Confusion Matrix (CM), which is utilized to look into the class confusion between various mangrove species. Two CM-based accuracy indices, the overall accuracy and the kappa coefﬁcient, were calculated. Moreover, since the two decision tree-based methods, RF and RoF, have technical features, a 10-fold cross-validation was performed, and the Student’s t-test was used to evaluate the signiﬁcance of classiﬁcation accuracy from RF and RoF, to assess the contribution of using RoF in this study. Table 2. Details of the training and testing samples. MUD, mudﬂat. Class Training (Samples) Testing (Samples) Total (Samples) KO 80 20 100 AM 114 29 143 AI 97 25 122 AC 52 14 66 MUD 105 27 132 WATER 124 31 155 Total 572 146 718 4. Results Table 2. Details of the training and testing samples. MUD, mudﬂat. 3.2. Mangrove Species Classiﬁcation Using Rotation Forest This study followed the recommendation that T should be bigger than 20 and m should be determined by Equation (5), where M is the total number of features for each sample [37]. In particular, RoF is different from RF in its technical details, although both of them construct a set of decision trees to form a forest with a certain process of randomness. First, RoF applies PCA on each subset of samples for feature extraction, while RF applies random selection of features instead of feature extraction. Second, RoF adopts CART to construct the decision trees, while RF uses a different tree construction method from CART. m = √ M + 1 (5) (5) m = √ M + 1 4. Results 4.1. Mangrove Species Classiﬁcation 4.1. Mangrove Species Classiﬁcation The Worldview 3 and Radarsat-2 data were designed in three different groups: (1) original eight-band Worldview 3 data (WV3), (2) original Worldview 3 data and its related spectral and textural Features (WV3F) and (3) WV3F and the polarimetric features described in Section 2 from the Radarsat-2 data (WV3FRS2), using three different machine learning methods, SVM, RF and RoF. Figure 4 compares the mangrove species classification results from different datasets and methods. The Worldview 3 and Radarsat-2 data were designed in three different groups: (1) original eight-band Worldview 3 data (WV3), (2) original Worldview 3 data and its related spectral and textural Features (WV3F) and (3) WV3F and the polarimetric features described in Section 2 from the Radarsat-2 data (WV3FRS2), using three different machine learning methods, SVM, RF and RoF. Figure 4 compares the mangrove species classification results from different datasets and methods. 8 of 15 d RoF. hods Remote Sens. 2018, 10, 467 Radarsat-2 data (WV Fi 4 th Figure 4. Mangrove species classification using SVM (a–c), random forest (d–f) and rotation forest (g–i) with different datasets; (a,d,g) for original Worldview 3 data (WV3); (b,e,h) for original Worldview 3 data and all related Features (WV3F); (c,f,i) for all the Worldview 3 data, related features and the polarimetric features from Radarsat 2 data (WV3FRS2). WAT denotes Water body and BKG denotes background Figure 4. Mangrove species classification using SVM (a–c), random forest (d–f) and rotation forest (g–i) with different datasets; (a,d,g) for original Worldview 3 data (WV3); (b,e,h) for original Worldview 3 data and all related Features (WV3F); (c,f,i) for all the Worldview 3 data, related features and the polarimetric features from Radarsat 2 data (WV3FRS2). WAT denotes Water body and BKG denotes background. Figure 4. Mangrove species classification using SVM (a–c), random forest (d–f) and rotation forest (g–i) with different datasets; (a,d,g) for original Worldview 3 data (WV3); (b,e,h) for original Worldview 3 data and all related Features (WV3F); (c,f,i) for all the Worldview 3 data, related features and the polarimetric features from Radarsat 2 data (WV3FRS2). WAT denotes Water body d BKG d t b k d Figure 4. 4.1. Mangrove Species Classiﬁcation When using only the Worldview data (Figure 4a), the AC species cannot be separated from KO and AM. The RoF method obtained the best results for every dataset. Additionally, RF demonstrated moderate performance among the three methods, though some overestimation of AC can be observed from Figure 4e,f. Finally, when using only the WV3 data, the AC species was generally underestimated, and some of the sea water was incorrectly classified as MUD when using RF and RoF. With the additional use of features from WV3, i.e., WV3F, this incorrect MUD was removed when using RoF. However, the advantage from further use of dual polarimetric data cannot be easily recognized from Figure 4. Therefore a quantitative assessment of the classification was provided in the Section 4 2 A set of observations is summarized in Figure 4. SVM performed the worst among the three methods, with a notable underestimation of the AC species in all three datasets. When using only the Worldview data (Figure 4a), the AC species cannot be separated from KO and AM. The RoF method obtained the best results for every dataset. Additionally, RF demonstrated moderate performance among the three methods, though some overestimation of AC can be observed from Figure 4e,f. Finally, when using only the WV3 data, the AC species was generally underestimated, and some of the sea water was incorrectly classified as MUD when using RF and RoF. With the additional use of features from WV3, i.e., WV3F, this incorrect MUD was removed when using RoF. However, the advantage from further use of dual polarimetric data cannot be easily recognized from Figure 4. Therefore, a quantitative assessment of the classification was provided in the Section 4.2. 4.1. Mangrove Species Classiﬁcation Mangrove species classification using SVM (a–c), random forest (d–f) and rotation forest (g–i) with different datasets; (a,d,g) for original Worldview 3 data (WV3); (b,e,h) for original Worldview 3 data and all related Features (WV3F); (c,f,i) for all the Worldview 3 data, related features and the polarimetric features from Radarsat 2 data (WV3FRS2). WAT denotes Water body and BKG denotes background. A set of observations is summarized in Figure 4. SVM performed the worst among the three methods, with a notable underestimation of the AC species in all three datasets. When using only the Worldview data (Figure 4a), the AC species cannot be separated from KO and AM. The RoF method obtained the best results for every dataset. Additionally, RF demonstrated moderate performance among the three methods, though some overestimation of AC can be observed from Figure 4e,f. Finally, when using only the WV3 data, the AC species was generally underestimated, and some of the sea water was incorrectly classified as MUD when using RF and RoF. With the additional use of features from WV3, i.e., WV3F, this incorrect MUD was removed when using RoF. However, the advantage from further use of dual polarimetric data cannot be easily recognized from Figure 4. Therefore a quantitative assessment of the classification was provided in the Section 4 2 A set of observations is summarized in Figure 4. SVM performed the worst among the three methods, with a notable underestimation of the AC species in all three datasets. When using only the Worldview data (Figure 4a), the AC species cannot be separated from KO and AM. The RoF method obtained the best results for every dataset. Additionally, RF demonstrated moderate performance among the three methods, though some overestimation of AC can be observed from Figure 4e,f. Finally, when using only the WV3 data, the AC species was generally underestimated, and some of the sea water was incorrectly classified as MUD when using RF and RoF. With the additional use of features from WV3, i.e., WV3F, this incorrect MUD was removed when using RoF. However, the advantage from further use of dual polarimetric data cannot be easily recognized from Figure 4. Therefore, a quantitative assessment of the classification was provided in the Section 4.2. A set of observations is summarized in Figure 4. SVM performed the worst among the three methods, with a notable underestimation of the AC species in all three datasets. q 4 2 Accuracy Assessment of the Classification 4.2. Accuracy Assessment of the Classiﬁcation The highest accuracy was obtained from RoF using the WV3FRS2 dataset, with an overall accuracy of 85.23% and a kappa value of 0.7955. Table 3 Confusion matrix for mangrove species classifications (in pixels) Table 3. Confusion matrix for mangrove species classiﬁcations (in pixels). Table 3. Confusion matrix for mangrove species classifications (in pixels). Reference WV3 WV3F WV3FRS2 Classified KO AM AI AC KO AM AI AC KO AM AI AC SVM KO 17 3 0 0 18 0 0 0 17 0 0 0 AM 3 19 12 5 2 26 3 6 3 26 2 6 AI 0 7 13 9 0 3 19 3 0 3 20 1 AC 0 0 0 0 0 0 3 5 0 0 3 7 OA: 55.68%; kappa: 0.3746 OA: 77.27%; kappa: 0.6842 OA: 79.55%; kappa: 0.7167 RF KO 17 3 0 0 18 0 0 0 19 0 0 0 AM 3 20 9 1 2 27 4 3 1 27 4 3 AI 0 3 13 5 0 2 20 3 0 2 20 3 AC 0 3 3 8 0 0 1 8 0 0 1 8 OA: 65.91%; kappa: 0.5341 OA: 82.95%; kappa: 0.7638 OA: 84.09%; kappa: 0.7799 RoF KO 18 1 0 1 18 0 0 0 19 0 0 0 AM 2 20 8 3 2 27 3 3 1 28 4 3 AI 0 4 13 6 0 2 21 4 0 1 20 3 AC 0 4 3 4 0 0 1 7 0 0 1 8 OA: 63.22%; kappa: 0.4944 OA: 82.95%; kappa: 0.7635 OA: 85.23%; kappa: 0.7955 In order to further investigate the complementary information from dual polarimetric SAR data regarding different mangrove species, the producer’s and user’s accuracy of the classification [54,55] Table 3. Confusion matrix for mangrove species classiﬁcations (in pixels). q 4 2 Accuracy Assessment of the Classification 4.2. Accuracy Assessment of the Classiﬁcation 4.2. Accuracy Assessment of the Classification The overall accuracy and kappa coefficient based on the CM were calculated and are compared in Figure 5. Consistent with the results highlighted in Figure 4, the lowest accuracy came from SVM, while the highest accuracy was from RoF. This is also consistent with the datasets of WV3F and The overall accuracy and kappa coefﬁcient based on the CM were calculated and are compared in Figure 5. Consistent with the results highlighted in Figure 4, the lowest accuracy came from SVM, while the highest accuracy was from RoF. This is also consistent with the datasets of WV3F and WV3FRS2. For the dataset WV3, RF performed the best. Furthermore, both the overall accuracy and kappa coefﬁcient demonstrated the advantages of the use of features from WV3 and dual polarimetric SAR data, with accuracies of WV3 < WV3F < WV3FRS2. This observation indicated that dual polarimetric SAR data were able to compensate optical data to improve the classiﬁcation of mangrove species, using different machine learning methods. Remote Sens. 2018, 10, 467 dual polarimetric SA mangrove species, us 9 of 15 tion of 9 of 15 tion of Figure 5. Overall accuracy and kappa coefficient. Figure 5. Overall accuracy and kappa coefﬁcient. Figure 5. Overall accuracy and kappa coefficient. Figure 5. Overall accuracy and kappa coefﬁcient. For more details about the accuracy assessment, the confusion matrix for all classifications is provided in Table 3, corresponding to the results in Figures 4 and 5. It is consistent that misclassifications of the mangrove species AI and AC were obvious using only the WV3 data for all methods. This misclassification was improved substantially by adding the features from the WV3 data and was further improved after adding the Radarsat-2 images. The highest accuracy was obtained from RoF using the WV3FRS2 dataset, with an overall accuracy of 85.23% and a kappa value of 0 7955 For more details about the accuracy assessment, the confusion matrix for all classifications is provided in Table 3, corresponding to the results in Figures 4 and 5. It is consistent that misclassifications of the mangrove species AI and AC were obvious using only the WV3 data for all methods. This misclassification was improved substantially by adding the features from the WV3 data and was further improved after adding the Radarsat-2 images. q 4 2 Accuracy Assessment of the Classification 4.2. Accuracy Assessment of the Classiﬁcation Reference WV3 WV3F WV3FRS2 Classiﬁed KO AM AI AC KO AM AI AC KO AM AI AC SVM KO 17 3 0 0 18 0 0 0 17 0 0 0 AM 3 19 12 5 2 26 3 6 3 26 2 6 AI 0 7 13 9 0 3 19 3 0 3 20 1 AC 0 0 0 0 0 0 3 5 0 0 3 7 OA: 55.68%; kappa: 0.3746 OA: 77.27%; kappa: 0.6842 OA: 79.55%; kappa: 0.7167 RF KO 17 3 0 0 18 0 0 0 19 0 0 0 AM 3 20 9 1 2 27 4 3 1 27 4 3 AI 0 3 13 5 0 2 20 3 0 2 20 3 AC 0 3 3 8 0 0 1 8 0 0 1 8 OA: 65.91%; kappa: 0.5341 OA: 82.95%; kappa: 0.7638 OA: 84.09%; kappa: 0.7799 RoF KO 18 1 0 1 18 0 0 0 19 0 0 0 AM 2 20 8 3 2 27 3 3 1 28 4 3 AI 0 4 13 6 0 2 21 4 0 1 20 3 AC 0 4 3 4 0 0 1 7 0 0 1 8 OA: 63.22%; kappa: 0.4944 OA: 82.95%; kappa: 0.7635 OA: 85.23%; kappa: 0.7955 using different datasets and different methods are shown in Figure 6. The producer’s and user’s accuracy indicated that KO and AM species were easier to identify compared with other species. All three different datasets can obtain adequate accuracy for KO and AM species. KO and AM are often taller than AI and AC in the MPMNR mangrove forest, which can be reflected in the reflectance and texture in the WV3 data. However, AI and AC were not identified as easily, especially when using only the WV3 data, where the SVM method was not able to classify AC species. This can also be observed in Figure 4. With the addition of texture and spectral features from the WV3 data, the In order to further investigate the complementary information from dual polarimetric SAR data regarding different mangrove species, the producer’s and user’s accuracy of the classiﬁcation [54,55] using different datasets and different methods are shown in Figure 6. The producer’s and user’s accuracy indicated that KO and AM species were easier to identify compared with other species. All three different datasets can obtain adequate accuracy for KO and AM species. q 4 2 Accuracy Assessment of the Classification 4.2. Accuracy Assessment of the Classiﬁcation KO and AM are often taller than AI and AC in the MPMNR mangrove forest, which can be reﬂected in the reﬂectance and texture in the WV3 data. However, AI and AC were not identiﬁed as easily, especially when using only the WV3 data, where the SVM method was not able to classify AC species. This can also be observed in Figure 4. With the addition of texture and spectral features from the WV3 data, the producer’s accuracy and user’s accuracy for AI and AC were noticeably increased. Furthermore, by combining the dual polarimetric data, the producer’s accuracy of AC was increased. Generally, an order of species 10 of 15 ore, by 10 of 15 ore, by Remote Sens. 2018, 10, 467 producer’s accuracy a can be concluded from Figure 6, that is KO < AM < AI < AC, with increasing difﬁculty of identiﬁcation from satellite data in this study. order of species can be concluded from Figure 6, that is KO < AM < AI < AC, with increasing difficulty of identification from satellite data in this study. y Figure 6. Producer’s and user’s accuracies of the classification. Figure 6. Producer’s and user’s accuracies of the classiﬁcation. Figure 6. Producer’s and user’s accuracies of the classification. Figure 6. Producer’s and user’s accuracies of the classiﬁcation. Figure 6. Producer’s and user’s accuracies of the classification. Figure 6. Producer’s and user’s accuracies of the classiﬁcation. 5.1. Contributions from the Dual Polarimetric SAR Data 5.1. Contributions from the Dual Polarimetric SAR Data Mangrove forests comprise one of the most productive and important ecosystems because they provide important ecological services for shoreline stabilization, habitat provision for invertebrates, fish and other coastal wildlife and carbon sequestration for the ocean. Monitoring the spatial distribution of mangrove forests from remote sensing is imperative to better understand their ecological services, while accurate assessment of the mangroves’ distribution remains challenging, especially at the species level. This study assessed the complementary role of SAR data towards optical data for the improvement of mangrove classification. The overall accuracy was improved from 63.22% (WV3) to 82.95% (WV3F) and 85.23% (WV3FRS2), while the kappa coefficient improved from 0.4944 (WV3) to 0.7635 (WV3F) and 0.7955 (WV3FRS2). Although the improvement may be small (about 3%), it may be important for more efficient large-scale monitoring of mangrove forest. In terms of overall accuracy, for an area of 350 ha of mangroves (the case in this study), a 3% improvement can be 7–10.5 ha from incorrect mapping to correct mapping. If the mangrove forest Mangrove forests comprise one of the most productive and important ecosystems because they provide important ecological services for shoreline stabilization, habitat provision for invertebrates, fish and other coastal wildlife and carbon sequestration for the ocean. Monitoring the spatial distribution of mangrove forests from remote sensing is imperative to better understand their ecological services, while accurate assessment of the mangroves’ distribution remains challenging, especially at the species level. This study assessed the complementary role of SAR data towards optical data for the improvement of mangrove classification. The overall accuracy was improved from 63.22% (WV3) to 82.95% (WV3F) and 85.23% (WV3FRS2), while the kappa coefficient improved from 0.4944 (WV3) to 0.7635 (WV3F) and 0.7955 (WV3FRS2). Although the improvement may be small (about 3%), it may be important for more efficient large-scale monitoring of mangrove forest. In terms of overall accuracy, for an area of 350 ha of mangroves (the case in this study), a 3% improvement can be 7–10.5 ha from incorrect mapping to correct mapping. If the mangrove forest reaches 10,000 ha, this corrected area can be up to 300 ha. 11 of 15 Remote Sens. 2018, 10, 467 This improvement can be further explained from the producer’s and user’s accuracies over different species, showing that dual polarimetric SAR data mainly contributed to the identification of AI and AC species. 5.1. Contributions from the Dual Polarimetric SAR Data 5.1. Contributions from the Dual Polarimetric SAR Data Generally, different mangrove species have high spectral confusions in their optical reflectance, leading to the difficulty of accurate classification using optical data alone. However, SAR is sensitive to the geometric properties of different species, including tree height, shape and size of leaves and shape and size of the canopy. For instance, KO is often among the tallest trees in the mangrove community, while AI often locates at the bottom layer. AM and AC appears similar in their size and shape of the leaves. All these geometric properties can be easier reflected in SAR data and thus compensate the classification of different species. Regarding the backscattering mechanism, the dominant scattering of microwave (e.g., C-band in this study) depends greatly on the mangrove height and biomass, which have been reported to vary significantly among different mangrove species [20,22,41,42]. For instance, the biomass of KO is much higher than AM, while the biomass of AC is higher than KO in the study area [41,42], which can be reflected in the backscattering coefficient of the dual-polarization C-band SAR data. 5.2. Factors Affecting Accuracy Assessment Due to the mangrove forest conservation policy of the local government, reference data collection is often limited, and thus, large amounts of reference data are difficult to collect. Consequently, small sample sizes of reference samples are typical [16,35,56,57]. In this study, only 718 samples were collected over the MPMNR, among which 80% were used for training the classifiers and 20% for validation and accuracy assessment (see Table 2). The small size of mangrove species samples of field data in MPMNR using remote sensing was also reported in previous literature [16,35]. However, this small sample size may decrease the reliability of the accuracy assessment from a statistical point of view. In this study, the classification results derived from the combined SAR and optical data (WV3FRS2) using RF and RoF are noticeably better than the results from other data combinations with all three methods (Table 3). However, the accuracy from WV3FRS2 and RF (OA: 84.09%; kappa: 0.7799) and the accuracy from WV3FRS2 and RoF (OA: 85.23%; kappa: 0.7955) are close, indicating a possible small improvement from using RoF compared with RF. In order to assess the significance of this improvement, we further conducted a 10-fold cross-validation classification of WV3FRS2 data using RoF and RF (Table 4). The OA of RF varied from 76.74–88.37% with a mean of 82.36%, while the OA using RoF varied from 76.74–90.70% with a mean of 85.61%. The kappa coefficient from RF varied from 0.6747–0.8404 with a mean of 0.7567, while the kappa value from RoF from 0.6866–0.8720 with a mean of 0.8021. The standard deviation of OA from RoF is less than that from RF, indicating that RoF is more stable. Furthermore, a Student’s t-test was applied to the OA and kappa coefficient. The t-value is 0.0096 for the OA values and 0.0087 for the kappa values between RF and RoF, indicating that the accuracies from these two methods are significantly different, and thus, the improvement of RoF over RF is significant. Table 4. Statistics of accuracy assessment from a 10-fold cross-validation using RF and RoF. WV3FRS2 Minimum Maximum Mean Standard Deviation RF OA 76.74% 88.37% 82.36% 4.69 Kappa 0.6747 0.8404 0.7567 0.06 RoF OA 76.74% 90.70% 85.61% 4.38 Kappa 0.6866 0.8720 0.8021 0.06 5.3. Comments about Datasets and Methods Table 4. Statistics of accuracy assessment from a 10-fold cross-validation using RF and RoF. 5.3. Comments about Datasets and Methods 5.3. Comments about Datasets and Methods In this study, the potential benefits of combining SAR and optical data for improving mangrove species classification in this manuscript were investigated. There are two main practical reasons. First, regarding the SAR datasets, only dual polarimetric SAR data were used. Fully-polarimetric SAR data could have provided much more backscattering features of different species, but the majority of currently-available SAR data, such as the free-access Sentinel-1 data, are dual polarimetric. Investigating 12 of 15 Remote Sens. 2018, 10, 467 the dual polarimetric SAR data can also be useful for practical use, especially for those cases with only free-access SAR data. With the increasing availability of SAR data free to the public (e.g., Sentinel 1 and ALOS), a large amount of SAR data at different resolutions is ready to be used covering the whole Earth’s surface. Thus, it is practical to support operational mangrove forest monitoring efforts. Moreover, the processing and computation of such big SAR data are also being constantly improved with a rapid development of various machine learning techniques (e.g., deep learning). Therefore, the cost of adding SAR data as an additional dataset is not very high for mangrove species classification and is worth the effort even for the small improvements in accuracy found here. Considering the benefits derived from increasing accuracy, it should be useful to add polarimetric SAR data to compensate optical data for improving species classification of mangroves. Second, regarding the methodology, there are many promising methods that still have not been applied to mangrove species classiﬁcation from a combined dataset of multi-source remote sensing data. However, it is a gradual procedure to test and validate the new and advanced methods to improve the species classiﬁcation step by step. In this paper, the Rotation Forest (RoF) method is employed, as we hypothesized that it can be more useful than the frequently-used Random Forest (RF). Speciﬁcally, three typical methods were compared: SVM, RF and RoF. Although SVM has been one of the most popular classiﬁers with wide applications, it did not demonstrate good performance compared with RF and RoF. This may be caused by the high confusion among different mangrove species, leading to difﬁculty in learning the support vectors. However, RF and RoF were based on the ensemble of decision trees, which can be made more robust for non-linear and highly confused classes. Furthermore, different classiﬁers performed differently over the species. 5.3. Comments about Datasets and Methods For instance, as observed from the experiments, RoF is more useful for identifying KO and AM species, while RF is more helpful for classifying AC species. Therefore, it would be useful to further investigate the advantages and disadvantages of each classiﬁer over different mangrove species. Author Contributions: H.Z., T.W. and H.L. conceived of and designed the experiments. H.Z. and M.L. performed the experiments. M.J., H.L., L.M.C. and A.T.D. analyzed the data and results. H.Z. wrote the paper. A.T.D. revised and edited the paper. References [CrossRef] 6. Wang, L.; Sousa, W.P.; Gong, P. Integration of object-based and pixel-based classiﬁcation for mapping mangroves with ikonos imagery. Int. J. Remote Sens. 2004, 25, 5655–5668. [CrossRef] 6. Wang, L.; Sousa, W.P.; Gong, P. Integration of object-based and pixel-based classiﬁcation for mapping mangroves with ikonos imagery. Int. J. Remote Sens. 2004, 25, 5655–5668. [CrossRef] 7. Wang, L.; Sousa, W.P.; Gong, P.; Biging, G.S. Comparison of Ikonos and Quickbird images for mapping mangrove species on the caribbean coast of panama. Remote Sens. Environ. 2004, 91, 432–440. [CrossRef] 7. Wang, L.; Sousa, W.P.; Gong, P.; Biging, G.S. Comparison of Ikonos and Quickbird images for mapping mangrove species on the caribbean coast of panama. Remote Sens. Environ. 2004, 91, 432–440. [CrossRef] 8. Heenkenda, M.K.; Joyce, K.E.; Maier, S.W.; Bartolo, R. Mangrove species identiﬁcation: Comparing Worldview-2 with aerial photographs. Remote Sens. 2014, 6, 6064–6088. [CrossRef] 8. Heenkenda, M.K.; Joyce, K.E.; Maier, S.W.; Bartolo, R. Mangrove species identiﬁcation: Comparing Worldview-2 with aerial photographs. Remote Sens. 2014, 6, 6064–6088. [CrossRef] 9. Heumann, B.W. An object-based classiﬁcation of mangroves using a hybrid decision tree-support vector machine approach. Remote Sens. 2011, 3, 2440–2460. [CrossRef] 10. Wang, T.; Zhang, H.S.; Lin, H.; Fang, C.Y. Textural-spectral feature-based species classiﬁcation of mangroves in Mai Po Nature Reserve from Worldview-3 imagery. Remote Sens. 2016, 8, 24. [CrossRef] 11. Wan, L.; Zhang, H.; Wang, T.; Li, G.; Lin, H. Mangrove species discrimination from very high resolution imagery using gaussian markov random ﬁeld model. Wetlands 2017. accepted. 2. Wang, L.; Sousa, W.P. Distinguishing mangrove species with laboratory measurements of hyperspectral reﬂectance. Int. J. 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Satellite remote sensing of mangrove forests: Recent advances and future opportunities. P Ph G 35 1 [C R f] 2. Duke, N.C.; Meynecke, J.O.; Dittmann, S.; Ellison, A.M.; Anger, K.; Berger, U.; Cannicci, S.; Diele, K.; Ewel, K.C.; Field, C.D.; et al. A world without mangroves? Science 2007, 317, 41–42. [CrossRef] [PubMed] Ewel, K.C.; Field, C.D.; et al. A world without mangroves? Science 2007, 317, 41 42. [CrossRef] [PubMed] 3. Heumann, B.W. Satellite remote sensing of mangrove forests: Recent advances and future opportunities. Prog. Phys. Geogr. 2011, 35, 87–108. [CrossRef] 3. Heumann, B.W. Satellite remote sensing of mangrove forests: Recent advances and future opportunities. Prog. Phys. Geogr. 2011, 35, 87–108. [CrossRef] 4. Rogers, K.; Lymburner, L.; Salum, R.; Brooke, B.P.; Woodroffe, C.D. Mapping of mangrove extent and zonation using high and low tide composites of Landsat data. Hydrobiologia 2017, 803, 49–68. [CrossRef] 4. 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This study investigated the potential of the Worldview-3 with an eight-band product and Radarsat-2 with a dual-polarimetric SAR product for mangrove species classification. Three different combinations of datasets were designed, including WV3, WV3F and WV3FRS2. Then, RoF was applied to classify the four mangrove species. Experiments indicated the effectiveness of different datasets with an increasing accuracy as WV3 < WV3F < WV3FRS2, showing the benefits of dual polarimetric SAR data. Additionally, the difficulty of classifying different mangrove species was also identified as KO < AM < AI < AC, in order of increasing difficulty. Dual polarimetric SAR data were recognized to improve the classification of AI and AC species. Moreover, RoF turned out to be more suitable than SVM and RF. Although this improvement is not remarkable, the improvement is consistent for all three methods. Furthermore, a 10-fold cross-validation with a Student’s t-test was performed and indicated that the improvement of using RoF is statistically significant compared with RF. The improvement can be particularly important for large-scale mapping of mangrove forest at the species level. These findings also provide useful references for future studies using multi-source satellite data for mangrove monitoring and conservation. Acknowledgments: This study was jointly supported by the National Natural Science Foundation of China (41401370 and 41671378), the Collaborative Innovation Center for Major Security Issues of Jiangxi Province and Monitoring Implementation (No. JXS-EW-00), the Research Grants Council (RGC) General Research Fund (CUHK 14601515, CUHK 14635916 and CUHK 14605917) and the Key Laboratory of Spatial Data Mining & Information Sharing of Ministry of Education, Fuzhou University (No. 2016LSDMIS02). 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https://openalex.org/W4388535665	https://press.umsida.ac.id/index.php/umsidapress/article/download/978-623-464-053-3/1140	Indonesian	null	Buku Monograf Pembiayaan Yang Berkelanjutan Untuk Meningkatkan Kinerja UMKM yang Terdampak Pandemi Covid-19	Umsida Press eBooks	2,022	cc-by	9,820	Redaksi : Redaksi : Universitas Muhammadiyah Sidoarjo Jl. Mojopahit 666 B Sidoarjo, Jawa Timur Monograf Pembiayaan Yang Berkelanjutan Untuk Meningkatkan Kinerja UMKM yang Terdampak Pandemi Covid-19 Penulis: Sriyono ISBN: 978-623-464-053-3 ISBN: 978-623-464-053-3 Cetakan pertama, Desember 2022 © Hak cipta dilindungi undang-undang Dilarang memperbanyak karya tulis ini dengan suatu apapun tanpa ijin tertulis dari penerbit. KATA PEGANTAR Puji syukur penulis panjatkan kehadirat Alloh SWT, yang telah melimpahkan rahmat dan ridho-Nya sehingga BUKU MONOGRAF dengan judul “Pembiayaan Yang Berkelanjutan Untuk Meningkatkan Kinerja UMKM yang Terdampak Pandemi Covid-19 “dapat penulis selesaikan. Buku ini disusun berdasarkan hasil penelitian dan kajian literatur yang bersumber pada berbagai artikel jurnal Internasional relevan terkait. Nilai kebaruan penelitian ini adalah Buku ini disusun dengan memberikan solusi yang terbaik kapada UMKM tentang masalah pembiayaan. Biasanya masalah pembiayaan selalu berhubungan dengan pihak Lembaga Keuangan atau perbankan. Pada akhirnya pihak UMKM tidak mampu untuk memenuhi syarat yang diajukan oleh pihak perbankkan sehingga UMKM memenuhi kesulitan. Pada buku ini di bahas bagaimana UMKM dapat memperoleh pembiayaan dengan mudah dari pihak ke tiga tanpa melalui syarat yang diberikan. Akhirnya saya berharap semoga buku ini dapat memberikan manfaat kepada sesama manusia dan besar harapan penulis agar pembaca dapat menjadikan buku Pembiayaan Yang Berkelanjutan Untuk Meningkatkan Kinerja UMKM yang Terdampak Pandemi Covid-19 ini dapat digunakan sebagai buku referensi untuk pemecahan Permasalahan pada UMKM yang terdampak Covid 19 Terima kasih penulis ucapkan kepada semua pihak yang tidak dapat saya sampaikan satu persatu atas bantuannya dalam menyelesaiakn buku referensi ini, semoga segala amal yang diberikan mendapat pahala yang besar Penulis Penulis PENDAHULUAN 1.1.Latar Belakang DAFTAR ISI KATA PENGANTAR .................................................. iii DAFTAR ISI ................................................................. v I. PENDAHULUAN ...................................................... 1 1.1 Latar Belakang ...................................................... 1 II. TINJAUAN PUSTAKA .......................................... 14 2.1 Koperasi dan Usaha Mikro Kecil Menengah (KUMKM) ................................................................. 14 2.2 Permasalahan Pemberdayaan UMKM ................. 23 2.3 Teori Model Daya Tarik ...................................... 25 2.4 Teori stakeholder ................................................. 27 2.5 Pengembangan Usaha .......................................... 27 2.6 Usaha Mikro Kecil dan Menengah (UMKM) ...... 28 2.7 Bantuan Sosial ..................................................... 29 2.8 Insentif pajak (tax incentive) ............................... 31 2.9 Modal Kerja ......................................................... 32 III. PEMBAHASAN ..................................................... 33 IV. KESIMPULAN ....................................................... 46 DAFTAR PUSTAKA .................................................... 49 1.1.Latar Belakang Pandemi Coronavirus Disease 19 atau lebih dikenal dengan singkatan Covid-19 telah memberikan perubahan yang sangat besar terhadap tatanan sosial dan ekonomi di seluruh dunia. Perubahan tersebut telah menimbulkan beberapa gejolak di tengah-tengah masyarakat yang sebelumnya hidup secara normal dengan kebiasaan dan budaya masing-masing. Namun dengan munculnya Covid-19 yang pertama di Wuhan, Tiongkok yang mulai merebak pada tahun 2019 lalu telah menjadi permasalahan global yang membutuhkan penanganan bersama antar Negara tak terkecuali Indonesia.Menurut laporan Organisation for Economic Co-operation and Development (OECD), (2020), dampak ekonomi akibat pandemik Covid-19 bagi perekonomian Asia yang berkembang (Emerging Asia) mengalami pertumbuhan negatif rata-rata minus 2,9% pada tahun 2020. Sementara perekonomian di ASEAN tambah laporan tersebut mengatakan pertumbuhan ekonomi rata-rata minus 2,8% atau lebih rendah di Asia.Kementerian 1 1 Keuangan (Kemenkeu)Republik Indonesia sendiri menurunkan laporan yang mengatakan bahwa tekanan yang dihadapi ekonomi global akibat COVID-19 berada pada magnitude yang sangat tinggi dan terburuk sejak krisis keuangan global di 2008/2009. I nternational Monetary Fund (IMF) mengestimasi potensi kerugian dunia akibat pandemik COVID-19 secara kumulatif di tahun 2020 dan 2021 mencapai USD9 triliun atau lebih besar dari gabungan ukuran ekonomi Jepang dan Jerman. Perubahan perekonomian global tersebut tentu saja berdampak terhadap ekonomi domestik Indonesia hingga perekonomian ke level daerah seperti tingkat provinsi dan kabupaten/kota. Pada tataran perekonomian daerah, dampak yang berpengaruh signifikan yaitu terhadap Usaha Mikro Kecil dan Menengah (UMKM). Bahkan OECD menyebutkan pandemi ini berimplikasi terhadap ancaman krisis ekonomi besar yang ditandai dengan terhentinya aktivitas produksi di banyak negara, jatuhnya tingkat konsumsi masyarakat, hilangnya kepercayaan konsumen, jatuhnya bursa saham yang pada akhirnya mengarah kepada ketidakpastian. 2 2 Pandemi Covid-19 saat ini telah berubah menjadi tantangan yang sangat berat bagi bangsa Indonesia. Tantangan terberat bagi perkembangan sosial, ekonomi khususnya UMKM, dan kesejahteraan bahkan tak jarang menjadi tantangan politik. Sehingga pemerintah merespon situasi tersebut dengan menjalankan beberapa kebijakan yang dapat membantu roda ekonomi makro dan mikro seperti UMKM agar dapat bertahan dan terus bergerak di tengah krisis dimana kondisi UMKM Indonesia Usaha skala kecil di Indonesia merupakan subyek diskusi dan menjadi perhatian pemerintah karena perusahaan kecil tersebut menyebar dimana-mana, dan dapat memberi kesempatan kerja yang potensial. Para ahli ekonomi sudah lama menyadari bahwa sektor industri kecil sebagai salah satu karakteristik keberhasilan dan pertumbuhan ekonomi. Industri kecil menyumbang pembangunan dengan berbagai jalan, menciptakan kesempatan kerja, untuk perluasan angakatan kerja bagi urbanisasi, dan menyediakan fleksibilitas kebutuhan serta inovasi dalam perekonomian secara keseluruhan. 1.1.Latar Belakang Secara kuantitas, UMKM memang unggul, hal ini didasarkan pada fakta bahwa sebagian besar usaha di 3 Indonesia (lebih dari 99 %) berbentuk usaha skala kecil dan menengah (UMKM). Namun secara jumlah omset dan aset, apabila keseluruhan omset dan aset UMKM di Indonesia digabungkan, belum tentu jumlahnya dapat menyaingi satu perusahaan berskala nasional. Data-data tersebut menunjukkan bahwa UMKM berada di sebagian besar sektor usaha yang ada di Indonesia. Apabila mau dicermati lebih jauh, pengembangan sektor swasta, khususnya UMKM, perlu untuk dilakukan mengingat sektor ini memiliki potensi untuk menjaga kestabilan perekonomian, peningkatan tenaga kerja, meningkatkan PDB, mengembangkan dunia usaha, dan penambahan APBN dan APBD melalui perpajakan. Namun secara jumlah omset dan aset, apabila keseluruhan omset dan aset UMKM di Indonesia digabungkan, belum tentu jumlahnya dapat menyaingi satu perusahaan berskala nasional. Data-data tersebut menunjukkan bahwa UMKM berada di sebagian besar sektor usaha yang ada di Indonesia. Penyebaran kelompok usaha kecil ini berupa usaha/rumah tangga. Hal ini menunjukkan bahwa tingkat penyerapan tenaga kerja usaha kecil lebih besar dibandingkan dengan tingkat penyerapan tenaga kerja pada usaha besar dan menengah. Gambaran di atas nampaknya sudah cukup untuk menafikkan pikiran bahwa UMKM adalah usaha yang tidak penting, hanya untuk orang-orang tidak berpendidikan. Justru mungkin inilah saat bagi kita yang sudah menyadari begitu dahsyatnya ketangguhan UMKM, untuk mulai memberikan 4 perhatian yang lebih serius di dalam sektor ini. Bila kita melihat UMKM yang ada di Negara lain, salahsatunya adalah Korea Selatan yang berhasil mengembangkan UKM. Negara ini mendefinisikan UKM sebagai usaha yang jumlah tenaga kerjanya di bawah 300 orang dan jumlah assetnya kurang dari US $ 60 juta. Saat ini keadaan UMKM di Indonesia semakin menurun, ini dikarenakan kesalahan pengurusan dan kurangnya perhatian pemerintah. Alasannya, pelaksanaan program pemberdayaan UMKM berikut anggarannya yang sangat melimpah tiap tahun dinilai tidak efektif. Ini terbukti dari kenyataan bahwa sektor UMKM yang mampu menyediakan 99,46% lapangan pekerjaan baru, namun kontribusinya baru 43,42% dari seluruh nilai transaksi perekonomian Indonesia setiap tahunnya. Peran UMKM nampak belum begitu dirasakan, adapun kebijakan pemerintah yang salah satunya yaitu dengan menggelontorkan stimulus ekonomi yang sangat besar melalui instrumen fiskal dan moneter. Pemerintah memberikan berbagai bantuan baik secara langsung maupun tidak langsung terhadap pelaku UMKM yang jumlahnya mencapai 60 juta unit lebih yang 5 tersebar di seluruh Indonesia. Langkah-langkah kebijakan tersebut dilakukan dengan tujuan utama yakni akselerasi atau mempercepat penanganan COVID-19 hingga upaya mitigasi dampak ekonomi dan keuangan. 1.1.Latar Belakang Mengenai bantuan yang disediakan bagi UMKM sebagaimana dipaparkan di atas, pemerintah mengoptimalkan peran Kementerian Koperasi dan UKM (Kemenkop dan UKM), Kementerian Perindustrian (Kemenperin), dan Kementerian Keuangan (Kemenkeu) yang merupakan lembaga teknis yang ditugaskan oleh presiden hingga melibatkan lembaga keuangan perbankan. Diantara bantuan yang terkait langsung dengan permasalahan yang dihadapi UMKM adalah aspek permodalan dan pinjaman. Peran pemerintah sangatlah dibutuhkan untuk memberikan stimulasi kepada para pelaku usaha yang berdasarkan peraturan yang berlaku. Teori daya tarik industri dan teori stakeholder digunakan sebagai acuan dalam penelitian ini. Teori daya tarik industri menjelaskan bahwa pemerintah melakukan usaha untuk mengembalikan kondisi dengan memberikan insentif dan subsidi Lincoln (2015). Sedangkan teori stakeholder menekankan adanya hubungan searah antara pemangku kepentingan dan stakeholder, 6 6 dimana pelaku usaha sebagai pemangku kepentingan dan pemerintah, masyarakat, kreditor, serta pihak lainnya merupakan stakeholder (Ghozali 2020). Bank Indonesia memperkirakan pada tahun 2020 pertumbuhan ekonomi dan laju inflasi sebesar 4,2 – 4,6 persen. Namun nyatanya angka pertumbuhan ekonomi dan laju inflasi tahun 2020 mengalami penurunan. Salah satu sebabnya karena salah satu sektor strategis dalam perekonomian nasional yaitu UMKM yang mengalami permasalahan dalam kegiatan usahanya sebagai dampak pandemi dari sisi penawaran dan permintaan. Menurut OEDC (2020) dan Febrantara (2020) dalam sisi penawaran UMKM menghadapi permasalahan tenaga kerja akibat dari pemberlakuan Pembatasan Sosial Berskala Besar (PSBB). Sesangkan pada sisi permintaan, yaitu berkurangnya permintaan barang dan jasa yang menyebabkan permasalahan likuiditas pelaku usaha. Meningkatnya permasalahan yang dihadapi oleh pelaku UMKM menjadi topik pembahasan yang menarik untuk diteliti lebih dalam penyebabnya dan bagaimana penanganannya. Terdapat empat permasalahan ekonomi yang dihadapi pelaku UMKM pada masa pandemi yaitu penurunan penjualan sebagai akibat 7 aturan PSBB, kesulitan permodalan karena perputaran modal yang sulit, hambatan distribusi produk, dan kesulitan bahan baku. Melihat permasalahan yang dihadapi UMKM maka pemerintah mengambil kebijakan dalam pemberian bantuan sosial kepada pelaku usaha; memberikan insentif pajak, melakukan relaksasi dan restrukturasi kredit; perluasan pembiayaan modal kerja; menempatkan kementrian, BUMN, dan Pemerintah Daerah sebagai penyangga produk UMKM; dan pelatihan secara e-learning. Kebijakan yang dilakukan pemerintah ini sebagai bentuk implementasi dari teori model daya tarik industri. Dalam teori model daya tarik adanya insentif dan subsidi memiliki tujuan untuk mengembalikan posisi pasar. Pemberian bantuan sosial yang dilakukan pemerintah sebagai salah satu bentuk penyelamatan UMKM pada masa pandemi Covid-19. Program penyaluran bantuan sosial tersebut dalam bentuk kemitraan UMKM, produksi jamu dan masker kain, pelonggaran kredit, dukungan bahan baku, dan distribusi paket sembako. 1.1.Latar Belakang Adanya pemberian bantuan sosial tersebut diharapkan akan membantu para pelaku UMKM untuk tetap menjalankan usaha ditengah masa pandemi Covid-19. 8 8 Kepedulian pemerintah juga dalam bentuk pemberian insentif pajak bagi UMKM. Adanya insentif pajak diharapkan dapat menekan biaya operasional sehingga perekonomian dapat bertahan ditengah pandemi Covid-19. Masalah perluasan modal kerja juga dirancang oleh pemerintah bagi pelaku UMKM yang terdampak Covid-19 yang sudah terhubung dengan lembaga pembiayaan atau perbankan pada UMKM yang belum pernah mendapatkan pembiayaan dari lembaga keuangan. Bagi pelaku UMKM yang bersifat bankable penyalurannya melalui progran perluasan Kredit Usaha Rakyat (KUR), sedangkan bagi yang tidak bankable penyalurannya dengan jalur Kredit Ultramikro (UMi), Permodalan Nasional Madani Membina Keluarga Sejahtera (PNM Mekar), Lembaga Pengelola Dana Bergulir (LPDB), hingga penerima bantuan permodalan dari beberapa kementrian. Kepedulian pemerintah juga dalam bentuk pemberian insentif pajak bagi UMKM. Adanya insentif pajak diharapkan dapat menekan biaya operasional sehingga perekonomian dapat bertahan ditengah pandemi Covid-19. Masalah perluasan modal kerja juga dirancang oleh pemerintah bagi pelaku UMKM yang terdampak Covid-19 yang sudah terhubung dengan lembaga pembiayaan atau perbankan pada UMKM yang belum pernah mendapatkan pembiayaan dari lembaga keuangan. Bagi pelaku UMKM yang bersifat bankable penyalurannya melalui progran perluasan Kredit Usaha Rakyat (KUR), sedangkan bagi yang tidak bankable penyalurannya dengan jalur Kredit Ultramikro (UMi), Permodalan Nasional Madani Membina Keluarga Sejahtera (PNM Mekar), Lembaga Pengelola Dana Bergulir (LPDB), hingga penerima bantuan permodalan dari beberapa kementrian. Hal ini dilakukan agar dapat digunakan UMKM sebagai modal usaha di masa pandemi Covid-19. Hasil penelitian Sugiri (2020) menemukan bahwa pemberian bantuan sosial, insentif pajak, perluasan modal kerja berpengaruh positif terhadap perkembangan UMKM pada masa pandemi Covid-19. Penelitian Iping (2020) menemukan 9 9 bahwa secara ekonomi pemberian bantuan sosial memberikan dampak yang signifikan terhadap kelangsungan usaha UMKM. Berbeda hasil oleh Bhagas (2016) menemukan bantuan pemerintah tidak berpengaruh terhadap pendapatan 52 UMKM Sulampita Kota Semarang. Terkait dengan insentif pajak, penelitian Kumala et al. (2020) dan Selvi (2020) menemukan bahwa insentif pajak berpengaruh terhadap pelaku UMKM. Namun Tandilino et al (2016) menemukan penerapan pajak terasa memberatkan UMKM di kota Kendari sehingga menurunkan penghasilan dan perkembangan UMKM. Khairiyah Ra’ida, Yotasa dan Akhmadi Heru (2019) menemukan bahwa adanya insentif pajak ternyata menjadikan UMKM belum secara sukarela membayar pajak karena merasa belum mendapatkan manfaat secara langsung, sehingga berimbas pada rendahnya penerimaan pajak dari sektor UMKM. Penelitian perluasan modal kerja dilakukan Hutahaean (2020) menemukan bahwa modal kerja berpengaruh terhadap pelaku UMKM.. 1.1.Latar Belakang Sejalan dengan Polandos et al (2019) juga menemukan modal usaha berpengaruh positif terhadap pendapatan pengusaha UMKM di Kecamatan Langowan Timur. 10 Namun Gonibala et al (2019) menemukan modal pinjaman tidak mempunyai pengaruh terhadap pendapatan UMKM di Kota Kotamobagu Kab. Bolaang Mongondow Provinsi Sulawesi Utara. Demikian juga Saragih, I dan Nasution (2015) menemukan modal pinjaman pinjaman kredit usaha rakyat (KUR) terbukti menurunkan tingkat pendapatan pengusaha UMKM Kabupaten Toba Samosir. Keterbaruan dalam penelitian ini yaitu menggunakan pendekatan teori model daya tarik industri untuk menjelaskan pengembangan UMKM dilihat dari kebijakan pemerintah. Namun Gonibala et al (2019) menemukan modal pinjaman tidak mempunyai pengaruh terhadap pendapatan UMKM di Kota Kotamobagu Kab. Bolaang Mongondow Provinsi Sulawesi Utara. Demikian juga Saragih, I dan Nasution (2015) menemukan modal pinjaman pinjaman kredit usaha rakyat (KUR) terbukti menurunkan tingkat pendapatan pengusaha UMKM Kabupaten Toba Samosir. Keterbaruan dalam penelitian ini yaitu menggunakan pendekatan teori model daya tarik industri untuk menjelaskan pengembangan UMKM dilihat dari kebijakan pemerintah. Pendekatan teori ini lebih sesuai digunakan pada masa pandemi Covid 19, dimana sebelumnya belum pernah dipakai oleh peneliti lain dengan topik yang serupa. Hasil penelitian ini memberikan informasi tambahan pemerintah dalam menyiapkan skema besar untuk program perlindungan dan pemulihan ekonomi bagi UMKM yang terdampak Covid-19 serta mengevaluasi strategi bisnis UMKM menuju model bisnis UMKM yang modern dengan tetap menjaga protokol kesehatan. Pendekatan teori ini lebih sesuai digunakan pada masa pandemi Covid 19, dimana sebelumnya belum pernah dipakai oleh peneliti lain dengan topik yang serupa. Hasil penelitian ini memberikan informasi tambahan pemerintah dalam menyiapkan skema besar untuk program perlindungan dan pemulihan ekonomi bagi UMKM yang terdampak Covid-19 serta mengevaluasi strategi bisnis UMKM menuju model bisnis UMKM yang modern dengan tetap menjaga protokol kesehatan. Pemerintah pun telah berupaya untuk menyediakan insentif melalui program Pemulihan Ekonomi Nasional (PEN) yang telah dilakukan sejak tahun 2020 dan masih 11 berlanjut sampai saat ini dengan realisasi PEN untuk tahun 2020 mencapai Rp112,84 triliun yang telah dibagikan kepada lebih dari 30 juta UMKM di seluruh Indonesia7 . Sementara untuk tahun 2021, Pemerintah juga telah menganggarkan PEN untuk mendukung UMKM dengan dana sebesar Rp 121,90 triliun untuk menjaga kelanjutan momentum pemulihan ekonomi. Pemerintah berharap semoga Program PEN ini dapat mendorong UMKM untuk kembali pulih di masa pandemi. Program PEN untuk mendukung UMKM pada tahun 2020 tercatat telah berhasil menjadi bantalan dukungan bagi dunia usaha, khususnya bagi sektor informal dan UMKM untuk bertahan dalam menghadapi dampak pandemi. 1.1.Latar Belakang Selain itu, ini juga dapat membantu dalam menekan penurunan tenaga kerja. Dilansir dari data BPS per Agustus 2020, terdapat penciptaan kesempatan kerja baru dengan penambahan 0,76 juta orang yang membuka usaha dan kenaikan 4,55 juta buruh informal. Pemulihan ekonomi di Indonesia terus berlanjut dengan mendapat dukungan dari konsumsi Pemerintah yang mendorong perbaikan pada konsumsi rumah tangga dan investasi. Membaiknya permintaan domestik telah direspon dengan peningkatan 12 aktivitas produksi di berbagai sektor usaha. Pemerintah juga terus berupaya untuk melakukan perbaikan dan peningkatan efektivitas dalam penanganan Covid-19 agar dapat terus mendorong terjadinya perbaikan akitivitas ekonomi. Berdasarkan pada uraian tersebut di atas, Penelitiam ini bertujuan untuk mengkaji Upaya Pemerintah Dalam Membantu Pelaku Usaha UMKM yang Terdampak Pandemi Covid-19. 13 BAB II TINJAUAN PUSTAKA 2.1 Koperasi dan Usaha Mikro Kecil Menengah (KUMKM) KUMKM merupakan bagian dunia usaha yang melakukan kegiatan dan domisili usahanya di Indonesia. KUMKM memiliki peran dalam pembangunan ekonomi kalangan menengah ke bawah. KUMKM di Indonesia juga memiliki andil besar dalam penyerapan tenaga kerja dan pendapatan nasional. Terdapat berbagai definisi yang berbeda mengenai KUMKM berdasarkan kepentingan lembaga yang menjelaskan definisi, definisi tersebut diantaranya: a. Departemen Koperasi dan Usaha Kecil Menengah (UU No. 9 Tahun 1995) Usaha Kecil (UK), termasuk Usaha Mikro (UMI) adalah entitas usaha yang mempunyai memiliki kekayaan bersih paling banyak Rp. 200.000.000,- tidak termasuk tanah dan bangunan tempat usaha, dan memiliki penjualan tahunan paling banyak Rp 1.000.000.000,- sementara itu, Usaha Menengah (UM) merupakan entitas usaha milik warga negara Indonesia yang memiliki kekayaan bersih antara Rp 200.000.000 s.d. Rp 10.000.000.000,- tidak termasuk tanah dan bangunan. 14 b. Badan Pusat Statistik Nasional BPS memberikan definisi UMKM berdasarkan kuantitas tenaga kerja. Usaha kecil merupakan entitas usaha yang memiliki jumlah tenaga kerja 5 s.d. 19 orang. Sedangkan usaha menengah merupakan entitas usaha yang memiliki tenaga kerja 20 s.d. 99 orang. c. Bank Indonesia (BI) UMKM adalah perusahaan atau industri dengan karakteristik berupa: 1. Modalnya kurang dari Rp 20 juta. 2. Untuk satu putaran dari usahanya hanya membutuhkan dana Rp 5 juta. 2. Untuk satu putaran dari usahanya hanya membutuhkan dana Rp 5 juta. 3. Memiliki aset maksimum Rp 600 juta di luar tanah dan bangunan. 3. Memiliki aset maksimum Rp 600 juta di luar tanah dan bangunan. 4. Omzet tahunan kurang lebih Rp 1 miliar. d. Keppres No. 16/1994 UKM adalah perusahaan yang memiliki kekayaan bersih maksimal Rp. 400 juta 4. Omzet tahunan kurang lebih Rp 1 miliar. d. Keppres No. 16/1994 UKM adalah perusahaan yang memiliki kekayaan bersih maksimal Rp. 400 juta d. Departemen Perindustrian dan perdagangan UMKM harus memiliki kriteria sebagai berikut: 1. Perusahaan memiliki aset maksimal Rp 600 juta di luar tanah dan bangunan 2. Perusahaan memiliki modal kerja di bawah Rp 25 juta 15 e. Departemen Keuangan UMKM adalah perusahaan yang memiliki omzet maksimal Rp. 600 juta per tahun dan atau aset maksimum Rp 600 juta di luar tanah dan bangunan e. Departemen Keuangan UMKM adalah perusahaan yang memiliki omzet maksimal Rp. 600 juta per tahun dan atau aset maksimum Rp 600 juta di luar tanah dan bangunan f. BAB II TINJAUAN PUSTAKA Menurut Undang-Undang Nomor 20 tahun 2008 tentang Usaha Mikro, Kecil dan Menengah: Pengertian UMKM meliputi: Pengertian UMKM meliputi: Pengertian UMKM meliputi: 1. Usaha Mikro adalah usaha produktif milik orang perorangan atau badan usaha perorangan yang memenuhi kriteria Usaha Mikro sebagaimana diatur dalam Undang- Undang. 2. Usaha Kecil adalah usaha ekonomi produktif berdiri sendiri, yang dilakukan oleh orang perorangan atau badan usaha yang bukan merupakan anak perusahaan atau bukan cabang perusahaan yang dimiliki, dikuasai, atau menjadi bagian baik langsung maupun tidak langsung dari usaha menengah atau usaha besar yang memenuhi kriteria Usaha Kecil sebagaimana dimaksud dalam UndangUndang ini 2. Usaha Kecil adalah usaha ekonomi produktif berdiri sendiri, yang dilakukan oleh orang perorangan atau badan usaha yang bukan merupakan anak perusahaan atau bukan cabang perusahaan yang dimiliki, dikuasai, atau menjadi bagian baik langsung maupun tidak langsung dari usaha menengah atau usaha besar yang memenuhi kriteria Usaha Kecil sebagaimana dimaksud dalam UndangUndang ini 3. Usaha Menengah adalah usaha ekonomi produktif yang berdiri sendiri, yang dilakukan oleh orang perseorangan atau badan usaha yang bukan merupakan anak perusahaan atau cabang perusahaan yang dimiliki, dikuasai, atau menjadi bagian baik langsung maupun tidak langsung 3. Usaha Menengah adalah usaha ekonomi produktif yang berdiri sendiri, yang dilakukan oleh orang perseorangan atau badan usaha yang bukan merupakan anak perusahaan atau cabang perusahaan yang dimiliki, dikuasai, atau menjadi bagian baik langsung maupun tidak langsung 16 dengan usaha kecil atau usaha besar dengan jumlah kekayaaan bersih atau hasil penjualan tahunan sebagaimana di atur dalam Undang-Undang ini. Bentuk Pembinaan Bagi Usaha Kecil dan Menengah sebagai Upaya Untuk meningkatkan Pendapatan. Sebagai sarana bantuan serta bentuk nyata pembinaan usaha kecil yang tercatat selama ini diantaranya adalah: dengan usaha kecil atau usaha besar dengan jumlah kekayaaan bersih atau hasil penjualan tahunan sebagaimana di atur dalam Undang-Undang ini. Bentuk Pembinaan Bagi Usaha Kecil dan Menengah sebagai Upaya Untuk meningkatkan Pendapatan. Sebagai sarana bantuan serta bentuk nyata pembinaan usaha kecil yang tercatat selama ini diantaranya adalah: 1. Sistem kemitraan usaha 2. Dana pembinaan BUMN 1-5 persen dari keuntungan bersih. 3. Pembentukan lembaga penjamin kredit usaha kecil. 4. Fasilitas kredit perbankan khususnya untuk pengusaha kecil 5. Kredit tanpa agunan (kredit kelayakan usaha). 5. Kredit tanpa agunan (kredit kelayakan usaha). 6. Pembentukan proyek pengembangan usaha kecil. 6. Pembentukan proyek pengembangan usaha kecil. 7. Pembentukan proyek pengembangan hubungan bank dengan kelompok swadaya masyarakat. 7. Pembentukan proyek pengembangan hubungan bank dengan kelompok swadaya masyarakat. 8. BAB II TINJAUAN PUSTAKA Pembentukan forum komunikasi perbankan untuk pengembangan usaha kecil. 8. Pembentukan forum komunikasi perbankan untuk pengembangan usaha kecil. Usaha kecil dianggap sebagai kegiatan ekonomi yang tepat dalam pembangunan di negara yang sedang berkembang, karena : 17 - Usaha kecil mendorong munculnya kewirausahaan domestik dan sekaligus menghemat sumber daya negara. - Usaha kecil menggunakan teknologi padat karya, sehingga dapat menciptakan lebih banyak kesempatan kerja dibandingkan yang disediakan oleh perusahaan berskala besar. - Usaha kecil dapat didirikan, dioperasikan dan memberi hasil dengan cepat. - Pengembangan usaha kecil dapat mendorong proses desentralisasi interregional dan intra regional, karena usaha kecil dapat berlokasi di kota-kota kecil dan pedesaaan. - Usaha kecil memungkinkan tercapainya objektif ekonomi dan sosial politik. Dana Bergulir Dana bergulir merupakan dana yang dipinjamkan untuk dikelola dan digulirkan kepada masyarakat oleh pengguna anggaran atau Kuasa Pengguna Anggaran yang bertujuan meningkatkan ekonomi rakyat dan tujuan lainnya. Adapun karakteristik dari dana bergulir menurut Simanjutak dkk (2008) adalah sebagai berikut: 18 1. Dana tersebut merupakan bagiandari keuangan negara/daerah. Dana bergulir dapat bersumber dari Anggaran Pendapatan dan Belanja Negara/ Anggaran Pendapatan dan Belanja Daerah (APBN/APBD) dan luar APBN/ APBD misalnya dari masyarakat atau hibah dari luar negeri. Sesuai dengan Undang-Undang Nomor 17 tahun 2003 tentang Keuangan Negara, dana bergulir yang berasal dari luar APBN, diakui sebagai kekayaan negara/daerah jika dana itu diberikan dan atau diterima atas nama pemerintah/pemerintah daerah. 2. Dana tersebut dicantumkan dalam APBN/APBD dan atau laporan keuangan. Sesuai dengan UndangUndang Nomor 1 tahun 2004 tentang perbendaharaan Negara Menyatakan semua pengeluaran negar/daerah dimasukkan dalam APBN/APBD. Oleh sebab itu alokasi anggaran untuk dana bergulir harus dimasukkan ke dalam APBN/APBD. Pencantuman alokasi anggaran untuk dana bergulir harus dicantumkan dalam APBN/APBD awal atau revisi APBN/APBD (APBN-P atau APBD Perubahan). 3. Dana tersebut harus dikuasai, dimiliki dan /atau dikendalikan oleh Pengguna Anggaran/Kuasa Pengguna Anggaran (PA/KPA). Pengertian dikuasai dan atau 3. Dana tersebut harus dikuasai, dimiliki dan /atau dikendalikan oleh Pengguna Anggaran/Kuasa Pengguna Anggaran (PA/KPA). Pengertian dikuasai dan atau 19 dimiliki mempunyai makna yang luas yaitu PA/KPA mempunyai hak kepemilikan atau penguasaan atas dana bergulir, sementara dikendalikan maksutnya adalah PA/KPA mempunyai kewenangan dalam melakukan pembinaan, monitoring, pengawasan atau kegiatan lain dalam rangka pemberdayaan dana bergulir. 4. Dana tersebut merupakan dana yang disalurkan kepada masyarakat ditagih kembali dari masyarakat dengan atau tanpa nilai tambah, selanjutnya dana disalurkan kembali kepada masyarakat demikian seterusnya (bergulir). 4. Dana tersebut merupakan dana yang disalurkan kepada masyarakat ditagih kembali dari masyarakat dengan atau tanpa nilai tambah, selanjutnya dana disalurkan kembali kepada masyarakat demikian seterusnya (bergulir). 5. Pemerintah dapat menarik kembali dana bergulir. Dana yang digulirkan oleh pemerintah dapat ditagih oleh Kementrian Negara/Lembaga baik untuk dihentikan pergulirannya atau digulirkan kembali kepada masyarakat. Tujuan Penyaluran Dana Bergulir Dana bergulir digunakan untuk membantu penguatan modal kepada Koperasi usaha mikro, usaha kecil, dan usaha menengah sehingga terwujud pengembangan dan kemandirian pelaku usaha guna mempercepat pertumbuhan dan pemerataan perekonomian daerah. Peraturan Gubernur Jawa Timur No 21 Tahun 2012 pasal 2: 20 20 1. Fasilitasi dana bergulir yang bersumber dari anggaran Pendapatan dan Belanja Daerah Provinsi Jawa Timur. Pelaksanaan kegiatannya dikoordinasikan oleh kelompok Kerja Pengelolaan Dana Bergulir Propinsi Jawa Timur. 2. Dana Bergulir Pengelolaan dana bergulir yang diberikan dinas/Badan/Biro yang bertugas sebagai Sekretariat dengan tugas menerima permohonan menyeleksi kelengkapan administrasi kelayakan usaha dari Usaha Mikro Kecil, Menengah dan Koperasi (UMKMK) 3. Dalam pengelolaan dana bergulir sebagaimana yang dimaksud pada ayat 1, Pelaksanaan Survey kelayakan usaha dari Usaha Mikro Kecil, Menengah dan Koperasi (UMKMK) dilakukan oleh Bank pelaksana. Peraturan Gubernur Jawa Timur No 21 Tahun 2012 pasal 5: 1) Dana berguler yang disalurkan oleh Bank Pelaksana dilakukan dengan pengikatan dan pengadministrasian nasabah/Debitur. 2) Dana Bergulir sebagaimana yang dimaksud pada ayat 1 dapat dijamin oleh PT Jamkrida. 3) Usaha Mikro, Kecil, menengah dan Koperasi (UMKMK) yang dijamin oleh PT Jamkrida adalah 3) Usaha Mikro, Kecil, menengah dan Koperasi (UMKMK) yang dijamin oleh PT Jamkrida adalah 21 yang layak usaha tetapi tidak cukup memenuhi angunan/ jaminan sesuai dengan peraturan ini. Peraturan Gubernur Jawa Timur No 21 Tahun 2012 pasal 7 1) Pengadministrasian sebagaimana dimaksud dalam pasal 5 ayat 1 diberikan biaya administrasi dilakukan dalam bentuk program kegiatan pada Dinas/ Badan/Biro yang bersangkutan mekutanlalui Dkutanokumen Pelaksana Anggaran (DPA) SKPD masingmasing. 1) Pengadministrasian sebagaimana dimaksud dalam pasal 5 ayat 1 diberikan biaya administrasi dilakukan dalam bentuk program kegiatan pada Dinas/ Badan/Biro yang bersangkutan mekutanlalui Dkutanokumen Pelaksana Anggaran (DPA) SKPD masingmasing. 2) Terhadap penjaminan sebagaimana dimaksud pasal 5 ayat 2 diberikan imbal Jasa Penjaminan (IJP) sebesar 1 % (satu persen) per tahun dari plafon kredit yang dibebankan kepada UMKMK melalui Bank pelaksana. Peraturan Gubernur Jawa Timur No 21 Tahun 2012 pasal 10: Penyelesaian dana bergulir yang direalisasi sebelum berlakunya Peraturan ini tetap menggunakan ketentuan sesuai dengan Peraturan Gubernur nomer 66 Tahun 2009 Pedoman Umum Pengelolaan Dana Bergulir Propinsi Jawa Timur. Sasaran Dana Bergulir Adapun sasaran dari dana bergulir adalah sebagai berikut : 2) Terhadap penjaminan sebagaimana dimaksud pasal 5 ayat 2 diberikan imbal Jasa Penjaminan (IJP) sebesar 1 % (satu persen) per tahun dari plafon kredit yang dibebankan kepada UMKMK melalui Bank pelaksana. 2) Terhadap penjaminan sebagaimana dimaksud pasal 5 ayat 2 diberikan imbal Jasa Penjaminan (IJP) sebesar 1 % (satu persen) per tahun dari plafon kredit yang dibebankan kepada UMKMK melalui Bank pelaksana. Peraturan Gubernur Jawa Timur No 21 Tahun 2012 pasal 10: Penyelesaian dana bergulir yang direalisasi sebelum berlakunya Peraturan ini tetap menggunakan ketentuan sesuai dengan Peraturan Gubernur nomer 66 Tahun 2009 Pedoman Umum Pengelolaan Dana Bergulir Propinsi Jawa Timur. Dana Bergulir Sasaran Dana Bergulir Adapun sasaran dari dana bergulir adalah sebagai berikut : • Koperasi • Koperasi • Usaha mikro • Usaha kecil • Usaha kecil 22 • Usaha menengah • Usaha menengah 2.2 Permasalahan Pemberdayaan UMKM Beberapa permasalahan yang tengah di hadapi oleh sektor UMKM dewasa ini antara lain : 1) Perlunya perluasan jaringan kerjasama dan kemitraan dengan pihak-pihak lain terkait untuk mengoptimalkan serta mensinergikan berbagai upaya penanggulangan kemiskinan, baik di dalam maupun di luar negeri. 2) Perlunya peningkatan upaya pemberdayaan Usaha Mikro, Kecil, dan Menengah (UMKM) melalui intensifikasi dan perluasan penyaluran kredit dari lembaga perbankan/keuangan kepada UMKM secara sistemik dan berkelanjutan. Terkait dengan hal ini, diprioritaskan untuk membuka akses lembaga perbankan dan keuangan untuk pengembangan UMKM, menyederhanakan prosedur dan persyaratan dan penjaminan usaha/kredit khususnya yang diberikan oleh pemerintah daerah. 2) Perlunya peningkatan upaya pemberdayaan Usaha Mikro, Kecil, dan Menengah (UMKM) melalui intensifikasi dan perluasan penyaluran kredit dari lembaga perbankan/keuangan kepada UMKM secara sistemik dan berkelanjutan. Terkait dengan hal ini, diprioritaskan untuk membuka akses lembaga perbankan dan keuangan untuk pengembangan UMKM, menyederhanakan prosedur dan persyaratan dan penjaminan usaha/kredit khususnya yang diberikan oleh pemerintah daerah. 3) Peningkatan intensitas upaya pemberdayaan, pelatihan, dan pengembangan Konsultan Keuangan/Pendamping UMKM Mitra Bank (KKMB) guna meningkatkan ketersediaan tenaga pendamping usaha mikro. Terkait 23 dengan hal ini, diperlukan upaya penyusunan panduan dan kebijakan untuk menentukan kedudukan serta penghargaan terhadap jasa profesional KKMB oleh Bank Indonesia sebagai lembaga pembina. 4) Penguatan peran Lembaga Keuangan Mikro (LKM) dalam sistem perbankan nasional terutama untuk memperluas penyaluran kredit terutama untuk segmen usaha mikro dan kecil. Dalam hal ini perlu diprioritaskan untuk menyiapkan landasan regulasi berupa Undang-Undang tentang LKM. 4) Penguatan peran Lembaga Keuangan Mikro (LKM) dalam sistem perbankan nasional terutama untuk memperluas penyaluran kredit terutama untuk segmen usaha mikro dan kecil. Dalam hal ini perlu diprioritaskan untuk menyiapkan landasan regulasi berupa Undang-Undang tentang LKM. 5) Peranan Bank Indonesia dalam pengembangan UMKM mengalami perubahan paradigma, namun bukan berarti kebijakan dan strategi untuk mendukung UMKM menjadi berkurang tetapi disesuaikan dengan perundangan baru yang berlaku. Oleh karenanya, kebijakan Bank Indonesia dalam pengembangan dan pemberdayaan UMKM adalah dalam rangka untuk mendorong peningkatan fungsi intermediasi perbankan serta untuk mendukung sistem perbankan yang sehat. 6) Inovasi produk layanan kredit/keuangan mikro harus terus dikembangkan oleh perbankan sehingga mampu menjembatani kesenjangan antara aspek kehati-hatian 24 (prudential) dan aspek potensi UMKM (yang masih belum bankable). (prudential) dan aspek potensi UMKM (yang masih belum bankable). 7) Perlunya penyediaan sumberdaya manusia pengelola Satgas Daerah KKMB dalam bentuk staf Unit Bantuan Teknis (UBT) jumlah cukup, memiliki kompetensi, kapabilitas yang memadai, dan bekerja secara full time untuk menggerakkan dan mempercepat operasionalisasi Satgas Daerah KKMB. • Usaha menengah 8) Perlunya peningkatan intensitas forum-forum komunikasi diantara Bank, KKMB, Usahawan, dan stakeholder terkait lainnya sebagai media untuk menyamakan persepsi terkait dengan implementasi penyaluran kredit untuk UMKM, terutama kredit usaha mikro. 9) Perlunya peningkatan optimalitas pemanfaatan dana-dana BUMN (melalui Dana Program Kemitraan dan Bina Lingkungan) untuk membiayai pengembangan dan pemberdayaan UMKM terutama melalui pembiayaan usaha mikro di daerah. 9) Perlunya peningkatan optimalitas pemanfaatan dana-dana BUMN (melalui Dana Program Kemitraan dan Bina Lingkungan) untuk membiayai pengembangan dan pemberdayaan UMKM terutama melalui pembiayaan usaha mikro di daerah. 2.3 Teori Model Daya Tarik Menurut Lincoln (2015) pembangunan ekonomi daerah adalah suatu prosedur yang dilakukan oleh pemerintah dan 25 masyarakat dalam mengelola sumberdaya dengan membentuk mitra antara pemerintah daerah dengan sektor swasta yang dapat menciptakan lapangan kerja baru dan merangsang perkembangan ekonomi di wilayahnya. Peran pemerintah dalam pembangunan daerah sebagai stimulator dimana pemerintah daerah menciptakan stimulasi dan pengembangan usaha melalui tindakan khusus yang dapat mempengaruhi para pelaku usaha. Salah satu teori pembangunan ekonomi daerah yang sesuai dengan penelitian ini yaitu teori model daya tarik. Implementasi teori ini yaitu dengan dikeluarkannya PMK No.86 Tahun 2020 dan PMK No. 81/PMK.05/2012. Dalam teori ini pemerintah memberikan subsidi dan insentif bagi para pelaku usaha. Pemberian tersebut sebagai salah satu bentuk usaha untuk mempertahankan usaha para pelaku UMKM di masa pandemi Covid-19 yang telah memberikan dampak yang cukup signifikan penurunan pendapatan sehingga melemahkan pertumbuhan usaha pada suatu wilayah. Adanya pemberian subsidi dan insentif bertujuan untuk mengembalikan posisi pasar . Oleh karenanya teori ini sejalan dengan arah penelitian yang mengamati pemberian subsidi dan insentif untuk pelaku UMKM yang berupa pemberian 26 bantuansosial, insentif pajak, dan perluasan modal kerja yang bertujuan untuk mengembangkan usaha pada masa pandemi Covid-19 khususnya di wilayah Sidoarjo. 2.4 Teori stakeholder menekankan adanya hubungan searah antara pemangku kepentingan dan stakeholder, dimana pelaku usaha sebagai pemangku kepentingan dan pemerintah,masyarakat, kreditor, serta pihak lainnya merupakan stakeholder (Ghozali 2020) 2.5 Pengembangan Usaha Menurut Fadilah (2012) Pengembangan usaha merupakan suatu tugas dan proses persiapan analitis mengenai peluang, dukungan, dan pemantauan, tetapi tidak termasuk keputusan strategi dan implementasi dari peluang pertumbuhan usaha. Maka dapat disimpulkan bahwa pengembangan usaha merupakan suatu bentuk usaha yang dilakukan oleh pelaku usaha untuk meningkatkan usahanya. Suatu pengembangan dilakukan sebagai upaya pelaku bisnis untuk memperluas dan mempertahankan bisnis tersebut agar dapat berjalan dengan baik. Jika akan dilakukan pengembangan bisnis dibutuhkan dukungan dari berbagai aspek seperti bidang produksi dan pengolaan, pemasaran, 27 SDM, teknologi, dan lain – lain. Tahapan dalam pengembangan usaha yaitu: Identifikasi peluang, merumuskan alternatif usaha, selektif alternatif, pelaksanaan alternatif pilihan, dan evalusi. Tahapan dalam pengembangan usaha tersebut dilakukan untuk mengurangi risiko dalam kegiatan usaha. 2.6 Usaha Mikro Kecil dan Menengah (UMKM) Usaha mikro, kecil, dan menengah merupakan suatu bentuk usaha yang dilakukan oleh orang pribadi maupun badan usaha dan memiliki kriteria khusus dalam pengelompokan jenis usahanya. 2.3 Teori Model Daya Tarik Pengelompokan UMKM diatur oleh UU No.20 Tahun 2008 yaitu: 2.6 Usaha Mikro Kecil dan Menengah (UMKM) Usaha mikro, kecil, dan menengah merupakan suatu bentuk usaha yang dilakukan oleh orang pribadi maupun badan usaha dan memiliki kriteria khusus dalam pengelompokan jenis usahanya. Pengelompokan UMKM diatur oleh UU No.20 Tahun 2008 yaitu: 1. Usaha Mikro merupakan usaha produktif yang dimiliki oleh perorangan atau badan usaha perorangan yang memiliki kriteria kekayaan bersih paling sebanyak Rp 50.000.000 dan hasil penjualan tahunan paling banyak Rp 300.000.000. 2. Usaha Kecil merupakan usaha produktif yang berdiri sendiri, yang dikelola oleh perorangan atau badan usaha yang bukan bagian dari anak perusahaan maupun cabang. Memiliki kriteria yaitu kekayaan paling banyak Rp 28 500.000.000 dan hasil penjualan tahunan paling banyak Rp 2.500.000.000. 500.000.000 dan hasil penjualan tahunan paling banyak Rp 2.500.000.000. 3. Usaha Menengah merupakan usaha produktif yang berdiri sendiri, dikelola oleh perorangan atau badan usaha yang bukan anak perusahaan atau cabang perusahaan. Memiliki kriteria yaitu kekayaan paling banyak Rp 10.000.000.000 tidak termasuk tanah dan bangunan tempat usaha dan memiliki hasil penjualan tahunan paling banyak Rp 50.000.000.000. Dominasi UMKM di Indonesia perlu mendapat perhatian khusus karena kontribusi UMKM terhadap perekonomian nasional yang cukup besar (Pakpahan 2020). Terdapat tiga peran UMKM sangat penting dalam kehidupan masyarakat kecil yakni sarana mengentaskan kemiskinan, meratakan tingkat perekonomian rakyat kecil, kontribusi devisa bagi Negara (Prasetyo, Anton dan Huda 2019). 2.7 Bantuan Sosial Berdasarkan penelitian sebelumnya yang dilakukan oleh Rahmansyah et al (2020) menyatakan bahwa masalah yang dihadapi dalam penyaluran bantuan sosial biasanya meliputi alokasi anggaran ditiap kementrian berbeda,data yang penerima bantuan yang tidak 29 terintegrasi,dan sistem penyaluran bantuan sosial tersebut. Implikasi dari penelitian ini adalah dibutuhkannya kerjasama,koordinasi, dan harmonisasi antara pemerintah pusat,daerah, dan lembaga swasta agar tepat sasaran, efektif, dan efisien serta didukung oleh sistem yang baik,terintegrasi,transparan, dan akuntabilitas dalam penyaluran dana bantuan sosial tersebut. Kebijakan program bantuan sosial bagi UMKM yang terdampak Covid-19 di Kota Semarang memiliki beberapa bentuk bantuan yaitu, pemberian bahan baku produksi,bantuan langsung tunai, dan pembelian produk UMKM oleh pemerintah Kota Semarang (Apriyanti 2020). Menurut Permendagri No.39 Tahun 2012 Pasal 1 bahwa bantuan sosial merupakan pemberian bantuan berupa uang/barang dari pemerintah daerah kepada individu, keluarga, kelompok dan /atau masyarakat yang sifatnya tidak secara terus menurus dan selektif. Pemberian bantuan sosial tersebut bertujuan untuk mengurangi risiko sosial yang ditimbulkan dari kondisi tertentu. Dengan kata lain bantuan sosial merupakan suatu penyaluran dana yang dimiliki pemerintah yang diberikan kepada masyarakat dengan syarat tertentu. 2.3 Teori Model Daya Tarik Adapun tujuan dari pemberian bantuan sosial tertuang dalam Peraturan Menteri Keuangan (PMK) Nomor 30 81/PMK.05/2012 yaitu sebagai rehabilitasi sosial, perlindungan sosial, pemberdayaan sosial, jaminan sosial, penanggulangan kemiskinan, dan penanggulangan bencana. Insentif Pajak Insentif pajak merupakan suatu kebijakan yang diambil pemerintah dan ditujukan kepada individu atau organisasi guna memudahkan dan mendorong wajib pajak untuk meningkatkan kepatuhan dalam melaksanakan kewajiban perpajakannya. Menurut Winardi (2011) 81/PMK.05/2012 2.8 Insentif pajak (tax incentive) merupakan bentuk penawaran dari pemerintah , malalui pemanfaat pajak, dalam kegiatan tertentu, seperti kontribusi uang atau harta untuk kegiatan yang berkualitas. Pemberlakukan insentif pajak merupakan sesuatu hal yang memberikan keuntungan bagi pelaku bisnis, adanya kebijakan tersebut maka keuntungan pengusaha sebagai pelaku ekonomi menjadi semakin besar. Pemberian insentif pajak untuk pelaku UMKM yang mengalami dampak pandemi Covid-19 diatur oleh PMK No.86 Tahun 2020. Insentif pajak dengan menyampaikan atau permohonan secara online melalui www.pajak.go.id. 31 2.9 Modal Kerja Pengertian modal kerja merupakan modal yang dimiliki perusahaan atau organisasi untuk kegiatan operasinalnya (Kasmir 2016). Modal kerja juga dapat diartikan sebagai aset lancar yang dimilik oleh perusahaan yang digunakan sebagai dana yang digunakan dalam kegiatan operasi perusahaan. Modal kerja memiliki peranan yang sangat penting bagi operasional suatu perusahaan. Oleh karenanya setiap pelaku usaha berusaha memenuhi kebutuhan modal kerjanya untuk tercapainya pemaksimalan perolehan labanya. Menurut Kasmir (2016) terdapat beberapa faktor yang mempengaruhi modal kerja yaitu jenis perusahaan dan syarat kredit. Pada masa pandemi seperti ini modal kerja menjadi bagian yang sangat penting terhadap keberlangsungan para pelaku usaha, karena tidak sedikit pelaku usaha yang tidak memperoleh pinjaman modal kerja akibat tidak terpenuhinya syarat kredit yang ditetapkan oleh bank maupun bukan bank. Pemerintah dalam hal ini mengeluarkan peraturan mengenai kebijakan penyaluran modal kerja untuk pelaku UMKM yaitu PMK Nomor 104/PMK.05/2020 UMKM. 2.9 Modal Kerja Pengertian modal kerja merupakan modal yang dimiliki perusahaan atau organisasi untuk kegiatan operasinalnya (Kasmir 2016). Modal kerja juga dapat diartikan sebagai aset lancar yang dimilik oleh perusahaan yang digunakan sebagai dana yang digunakan dalam kegiatan operasi perusahaan. Modal kerja memiliki peranan yang sangat penting bagi operasional suatu perusahaan. Oleh karenanya setiap pelaku usaha berusaha memenuhi kebutuhan modal kerjanya untuk tercapainya pemaksimalan perolehan labanya. Menurut Kasmir (2016) terdapat beberapa faktor yang mempengaruhi modal kerja yaitu jenis perusahaan dan syarat kredit. 2.3 Teori Model Daya Tarik Pada masa pandemi seperti ini modal kerja menjadi bagian yang sangat penting terhadap keberlangsungan para pelaku usaha, karena tidak sedikit pelaku usaha yang tidak memperoleh pinjaman modal kerja akibat tidak terpenuhinya syarat kredit yang ditetapkan oleh bank maupun bukan bank. Pemerintah dalam hal ini mengeluarkan peraturan mengenai kebijakan penyaluran modal kerja untuk pelaku UMKM yaitu PMK Nomor 104/PMK.05/2020 UMKM. 32 PEMBAHASAN UMKM yang tersebar di Indonesia memiliki beragam jenis dan kegiatan, keseluruhannya memberikan kontribusi yang tidak sedikit terhadap wilayah dan daerah masing- masing dimana UMKM tersebut beroperasi. Disaat pandemi covid19 ini jumlah UMKM yang beroperasi mulai menurun seperti yang disampaikan oleh sekretaris Kementrian Koperasi dan UKM “Selama pandemi ini jujur saja banyak yang terhenti usahanya, sekitar 30 persen yang usahanya terganggu. Sedangkan yang memang terganggu tapi menciptakan inovasi-inovasi kreatif sekitar 50-70 persen, meskipun mereka terkena dampak,” kata Sekretaris Kementerian Koperasi dan UKM Rully Indrawan. Survei Badan Pusat Statistik (BPS) bertajuk Dampak Covid-19 terhadap Pelaku Usaha yang digelar 10-26 Juli 2020, menemukan 42 persen pelaku usaha hanya dapat bertahan selama tiga bulan, yaitu sejak Juli sampai Oktober 2020. Sedangkan 58 persen lainnya masih bisa bertahan di atas 3 bulan. Estimasi ini muncul bila tidak ada perubahan operasional dan intervensi dari pemerintah. Survei dengan responden 34.559 pelaku usaha terdiri dari 25.256 usaha 33 mikro dan kecil (UMK), 6.821 usaha menengah dan besar (UMB), dan 2.482 di sektor pertanian.3 Survei yang sama menyebut, ada pelbagai bantuan yang dibutuhkan pelaku usaha. Bagi UMK, yang paling dibutuhkan adalah modal usaha yaitu sekitar 69,02 persen. Mereka juga perlu bantuan berupa keringanan tagihan listrik (41,18 persen); relaksasi pembayaran pinjaman (29,98 persen); penundaan pembayaran pajak (15,07 persen); dan kemudahan administrasi pengajuan pinjaman (17,21 persen). Pemerintah sebelumnya telah menyediakan insentif bagi UMKM dalam program pemulihan ekonomi nasional (PEN). Jumlahnya pun cukup besar yaitu sekitar Rp123,46 triliun. Dari jumlah tersebut, bantuan diberikan melalui pelbagai program seperti: penempatan dana di perbankan untuk modal kerja, subsidi bunga kredit, bantuan presiden (banpres) produktif sebesar Rp2,4 juta, dan sebagainya. Bantuan-bantuan tersebut diharapkan dapat meringankan beban UMKM terutama dalam modal kerja agar mereka bisa kembali beroperasi dan memberikan kontribusi pada pertumbuhan ekonomi Indonesia. 1. Upaya Pemerintah dalam membantu pelaku usaha UMKM yang terdampak Pndemi Covid-19 34 Meningkat atau menurunnya pertumbuhan ekonomi di Indonesia dipengaruhi oleh berbagai faktor, salah satunya keterlibatan UMKM. Sesuai dengan UUD 1945 Pasal 33 ayat (4), UMKM merupakan bagian dari perekonomian nasional yang berwawasan kemandirian dan memiliki potensi besar untuk meningkatkan kesejahteraan masyarakat. UMKM memiliki peran yang signifikan terhadap pertumbuhan ekonomi negara. Data Kementerian Koperasi dan UKM, jumlah UMKM saat ini mencapai 64,19 juta dengan kontribusi terhadap PDB sebesar 61,97% atau senilai 8.573,89 triliun rupiah. Kontribusi UMKM terhadap perekonomian Indonesia meliputi kemampuan menyerap 97% dari total tenaga kerja yang ada serta dapat menghimpun sampai 60,4% dari total investasi. PEMBAHASAN Tingginya jumlah UMKM di Indonesia tidak terlepas dari berbagai tantangan serta kondisi pandemi Covid19 yang mendorong perubahan pada pola konsumsi barang dan jasa menjadi momentum untuk mengakselerasi transformasi digital. Pentingnya Peran UMKM Terhadap Perekonomian Indonesia yaitu sebagai salah satu pilar perekonomian Indonesia, UMKM memiliki peran yang signifikan yaitu UMKM memiliki kontribusi besar terhadap PDB yaitu 61,97% dari total PDB nasional 35 atau setara dengan Rp. 8.500 triliun pada tahun 2020, UMKM menyerap tenaga kerja dalam jumlah yang besar yaitu 97% dari daya serap dunia usaha pada tahun 2020. Jumlah UMKM yang banyak berbanding lurus dengan banyaknya lapangan pekerjaan di Indonesia sehingga UMKM memiliki andil besar dalam penyerapan tenaga kerja, UMKM menyerap kredit terbesar pada tahun 2018 sebesar kurang lebih Rp. 1 triliun Usaha Mikro, Kecil, dan Menengah merupakan kegiatan usaha yang mampu memperluas lapangan kerja dan memberikan pelayanan ekonomi secara luas kepada masyarakat, dan dapat berperan dalam proses pemerataan dan peningkatan pendapatan masyarakat, mendorong pertumbuhan ekonomi, dan berperan dalam mewujudkan stabilitas nasional. Usaha Mikro, Kecil, dan Menengah adalah salah satu pilar utama ekonomi nasional yang harus memperoleh kesempatan utama, dukungan, perlindungan dan pengembangan seluas-luasnya sebagai wujud keberpihakan yang tegas kepada kelompok usaha ekonomi rakyat, tanpa mengabaikan peranan Usaha Besar dan Badan Usaha Milik Negara. Meskipun Usaha Mikro, Kecil, dan Menengah telah menunjukkan peranannya dalam perekonomian nasional, namun masih menghadapi berbagai hambatan dan kendala, 36 baik yang bersifat internal maupun eksternal, dalam hal produksi dan pengolahan, pemasaran, sumber daya manusia, desain dan teknologi, permodalan, serta iklim usaha. Guna meningkatkan kesempatan, kemampuan, dan perlindungan Usaha Mikro, Kecil, dan Menengah, telah ditetapkan berbagai kebijakan tentang pencadangan usaha, pendanaan, dan pengembangannya namun belum optimal. Hal itu dikarenakan kebijakan tersebut belum dapat memberikan perlindungan, kepastian berusaha, dan fasilitas yang memadai untuk pemberdayaan Usaha Mikro, Kecil, dan Menengah. Sehubungan dengan itu Usaha Mikro, Kecil, dan Menengah perlu diberdayakan dengan cara penumbuhan iklim usaha yang mendukung pengembangan Usaha Mikro, Kecil, dan Menengah dan pengembangan dan pembinaan Usaha Mikro, Kecil, dan Menengah. Sebagai upaya untuk meningkatkan kemampuan dan peran serta kelembagaan Usaha Mikro, Kecil, dan Menengah dalam perekonomian nasional, maka pemberdayaan tersebut perlu dilaksanakan oleh Pemerintah, Pemerintah Daerah, Dunia Usaha, dan masyarakat secara menyeluruh, sinergis, dan berkesinambungan. Dalam memberdayakan Usaha Mikro, Kecil, dan Menengah, seluruh peraturan perundang- baik yang bersifat internal maupun eksternal, dalam hal produksi dan pengolahan, pemasaran, sumber daya manusia, desain dan teknologi, permodalan, serta iklim usaha. PEMBAHASAN p p g , p , y , desain dan teknologi, permodalan, serta iklim usaha. Guna meningkatkan kesempatan, kemampuan, dan perlindungan Usaha Mikro, Kecil, dan Menengah, telah ditetapkan berbagai kebijakan tentang pencadangan usaha, pendanaan, dan pengembangannya namun belum optimal. Hal itu dikarenakan kebijakan tersebut belum dapat memberikan perlindungan, kepastian berusaha, dan fasilitas yang memadai untuk pemberdayaan Usaha Mikro, Kecil, dan Menengah. Sehubungan dengan itu Usaha Mikro, Kecil, dan Menengah perlu diberdayakan dengan cara penumbuhan iklim usaha yang mendukung pengembangan Usaha Mikro, Kecil, dan Menengah dan pengembangan dan pembinaan Usaha Mikro, Kecil, dan Menengah. Sebagai upaya untuk meningkatkan kemampuan dan peran serta kelembagaan Usaha Mikro, Kecil, dan Menengah dalam perekonomian nasional, maka pemberdayaan tersebut perlu dilaksanakan oleh Pemerintah, Pemerintah Daerah, Dunia Usaha, dan masyarakat secara menyeluruh, sinergis, dan berkesinambungan. Dalam memberdayakan Usaha Mikro, Kecil, dan Menengah, seluruh peraturan perundang- 37 undangan yang berkaitan dengan Usaha Mikro, Kecil, dan Menengah merupakan suatu kesatuan yang saling melengkapi g p Menurut Tulus Tambunan dalam bukunya yang berjudul Usaha Mikro Kecil dan Menengah di Indonesia, ada beberapa alasan yang mengemukakan pentingnya UMKM bagi perekonomian nasional: Jumlah UMKM yang sangat banyak dan tersebar di perkotaan maupun pedesaan bahkan hingga di pelosok terpencil. UMKM tergolong sangat padat karya, mempunyai potensi pertumbuhan kesempatan kerja yang besar dan peningkatan pendapatan. UMKM banyak terdapat dalam sektor pertanian yang secara tidak langsung mendukung pembangunan. UMKM membantu dalam menampung banyak pekerja yang memiliki tingkat pendidikan yang rendah. Dalam kondisi krisis ekonomi, UMKM mampu untuk bertahan, seperti yang terjadi pada tahun 1997/1998. Menjadi titik awal mobilitas investasi di pedesaan sekaligus wadah bagi peningkatan kemampuan wiraswasta. Menjadi alat untuk mengalihkan pengeluaran konsumsi warga pedesaan menjadi tabungan. UMKM mampu menyediakan barang-barang kebutuhan relatif murah. Melalui beragam jenis investasi dan penanaman Menurut Tulus Tambunan dalam bukunya yang berjudul Usaha Mikro Kecil dan Menengah di Indonesia, ada beberapa alasan yang mengemukakan pentingnya UMKM bagi perekonomian nasional: Jumlah UMKM yang sangat banyak dan tersebar di perkotaan maupun pedesaan bahkan hingga di pelosok terpencil. UMKM tergolong sangat padat karya, mempunyai potensi pertumbuhan kesempatan kerja yang besar dan peningkatan pendapatan. UMKM banyak terdapat dalam sektor pertanian yang secara tidak langsung mendukung pembangunan. UMKM membantu dalam menampung banyak pekerja yang memiliki tingkat pendidikan yang rendah. Dalam kondisi krisis ekonomi, UMKM mampu untuk bertahan, seperti yang terjadi pada tahun 1997/1998. Menjadi titik awal mobilitas investasi di pedesaan sekaligus wadah bagi peningkatan kemampuan wiraswasta. PEMBAHASAN Menjadi alat untuk mengalihkan pengeluaran konsumsi warga pedesaan menjadi tabungan. UMKM mampu menyediakan barang-barang kebutuhan relatif murah. Melalui beragam jenis investasi dan penanaman 38 modal, UMKM mampu dan cepat beradaptasi dalam kemajuan zaman. Memiliki tingkat fleksibilitas yang tinggi. Pemerintah pun menyediakan insentif dukungan bagi UMKM melalui program Pemulihan Ekonomi Nasional (PEN) di 2020 dan dilanjutkan di 2021. Realisasi PEN untuk mendukung UMKM sebesar Rp 112,84 triliun telah dinikmati oleh lebih dari 30 juta UMKM pada 2020. Pemerintah juga telah menganggarkan PEN untuk mendukung UMKM dengan dana sebesar Rp 121,90 triliun untuk menjaga kelanjutan momentum pemulihan ekonomi. Pemerintah berharap semoga Program PEN ini dapat mendorong UMKM untuk kembali pulih di masa pandemi. Program PEN untuk mendukung UMKM pada tahun 2020 tercatat telah berhasil menjadi bantalan dukungan bagi dunia usaha, khususnya bagi sektor informal dan UMKM untuk bertahan dalam menghadapi dampak pandemi. Program ini diharapkan dapat membantu menekan penurunan tenaga kerja. Pasalnya, berdasarkan data BPS per Agustus 2020, terdapat penciptaan kesempatan kerja baru dengan penambahan 760 ribu orang yang membuka usaha dan kenaikan 4,55 juta buruh informal. Pemerintah juga terus berupaya mendorong para pelaku UMKM untuk on board ke 39 platform digital melalui Program Gerakan Nasional Bangga Buatan Indonesia (Gernas BBI), dimana hingga akhir 2020 sudah terdapat 11,7 juta UMKM on boarding. Pada 2030 targetnya jumlah UMKM yang go digital akan mencapai 30 juta. Perluasan ekspor produk Indonesia bagi UMKM juga dilakukan melalui ASEAN Online Sale Day (AOSD) di 2020. Dari 64,19 juta UMKM di Indonesia, sebanyak 64,13 juta masih merupakan UMK yang masih berada di sektor informal sehingga perlu didorong untuk bertransformasi ke sektor formal. Pemerintah sudah menyiapkan berbagai program dan kebijakan baik dalam konteks Pemulihan Ekonomi Nasional maupun beberapa program yang ke depannya kita harapkan betul-betul dapat memberikan kemudahan, perlindungan dan pemberdayaan bagi UMKM Program Pemulihan Ekonomi Nasional (PEN) merupakan salah satu program yang dicetuskan pemerintah untuk memulihkan ekonomi Indonesia akibat dampak Covid-19. Program ini juga merupakan respon pemerintah atas penurunan aktivitas masyarakat yang terdampak, khususnya sektor informal atau UMKM. Program ini dibuat berdasarkan PP Nomor 23 tahun 2020 yang kemudian diubah menjadi PP Nomor 43 tahun 2020. PEMBAHASAN Upaya pemerintah untuk memajukan 40 UMKM, diantaranya adalah merupakan rincian program PEN untuk UMKM, yaitu: Subsidi bunga/margin, Belanja Imbal Jasa Penjaminan (IJP), Penempatan Dana Pemerintah di perbankan, Penjaminan loss limit kredit UMKM, Pajang penghasilan final UMKM ditanggung pemerintah, Pembiayaan investasi kepada koperasi melalui Lembaga Pengelola Dana Bergulir (LPDB) koperasi UMKM dan Program Bantuan Presiden (Banpres) Produktif Usaha Mikro. Upaya lain dari pemerintah untuk memajukan UMKM yaitu program Kredit Usaha Rakyat (KUR) yang disalurkan melalui lembaga keuangan dengan pola penjaminan. Adapun biaya jasa (suku bunga) atas kredit/pembiayaan modal kerja disubsidi oleh pemerintah. Tujuan KUR adalah untuk meningkatkan meningkatkan akses pembiayaan dan memperkuat permodalan UMKM. Gerakan Nasional Bangga Buatan Indonesia (Gernas BBI) merupakan salah satu program pemerintah sebagai upaya pemerintah untuk memajukan UMKM yang diluncurkan pada tahun 2020. Tujuan Gernas BBI yaitu mendorong national branding produk lokal unggulan untuk menciptakan industri baru dan tentunya meningkatkan pertumbuhan ekonomi. Melalui 41 program ini, pemerintah mendorong pelaku UMKM untuk bergabung ke platform digital. 2. Hambatan-hambatan dan solusi dari Pemerintah dalam membantu pelaku usaha UMKM yang terdampak pandemic Covid-19 Walaupun UMKM siap melakukan ekspor tetapi masih mengalami berbagai kendala seperti minimnya pengetahuan pasar luar negeri, konsistensi kualitas dan kapasitas produk, sertifikasi, hingga kendala logistik. Pemerintah terus berupaya untuk membantu UMKM agar dapat mengatasi kendala-kendala tersebut. Kementerian, Lembaga dan para pihak terkait lainnya telah meluncurkan Program Penciptaan 500 ribu Eksportir Baru hingga tahun 2035. Permasalahan UMKM Akibat Pandemi diantaranya adalah Penurunan jumlah UMKM dan kontribusi UMKM terhadap PDB Indonesia disebabkan oleh pandemi sejak 2020 lalu. Beberapa permasalahan yang dialami diantaranya adalah perubahan pola konsumsi barang dan jasa masyarakat dimasa pandemi dari offline ke online, UMKM mengalami permasalahan tenaga kerja akibat pemberlakuan Pembatasan Sosial Berskala Besar (PSBB) hambatan distribusi produk program ini, pemerintah mendorong pelaku UMKM untuk bergabung ke platform digital. program ini, pemerintah mendorong pelaku UMKM untuk bergabung ke platform digital. 2. Hambatan-hambatan dan solusi dari Pemerintah dalam membantu pelaku usaha UMKM yang terdampak pandemic Covid-19 Walaupun UMKM siap melakukan ekspor tetapi masih mengalami berbagai kendala seperti minimnya pengetahuan pasar luar negeri, konsistensi kualitas dan kapasitas produk, sertifikasi, hingga kendala logistik. Pemerintah terus berupaya untuk membantu UMKM agar dapat mengatasi kendala-kendala tersebut. Kementerian, Lembaga dan para pihak terkait lainnya telah meluncurkan Program Penciptaan 500 ribu Eksportir Baru hingga tahun 2035. Permasalahan UMKM Akibat Pandemi diantaranya adalah Penurunan jumlah UMKM dan kontribusi UMKM terhadap PDB Indonesia disebabkan oleh pandemi sejak 2020 lalu. PEMBAHASAN Beberapa permasalahan yang dialami diantaranya adalah perubahan pola konsumsi barang dan jasa masyarakat dimasa pandemi dari offline ke online, UMKM mengalami permasalahan tenaga kerja akibat pemberlakuan Pembatasan Sosial Berskala Besar (PSBB), hambatan distribusi produk dan kesulitan bahan baku produksi. Dari total keseluruhan 42 jumlah UMKM di Indonesia, sebanyak 64,13 juta merupakan UMK yang masih berada di sektor informal sehingga perlu didorong untuk bertransformasi ke sektor formal. Indonesia masih memiliki kendala dalam perizinan yang rumit serta tumpang tindih antara regulasi di tingkat pusat dan daerah. Pemerintah berupaya mengakomodir permasalahan tersebut melalui penyusunan UU Cipta Kerja yang telah disahkan pada tahun 2020. Salah satu substansi yang diatur adalah mengenai kemudahan, perlindungan dan pemberdayaan UMKM. Pemerintah berharap melalui UU Cipta Kerja, UMKM dapat terus berkembang dan berdaya saing. Hasil survey dari beberapa lembaga (BPS, Bappenas, dan World Bank) menunjukkan bahwa pandemi ini menyebabkan banyak UMKM kesulitan melunasi pinjaman serta membayar tagihan listrik, gas, dan gaji karyawan. Beberapa diantaranya sampai harus melakukan PHK. Kendala lain yang dialami UMKM, antara lain sulitnya memperoleh bahan baku, permodalan, pelanggan menurun, distribusi dan produksi terhambat. Salah satu kendala perkembangan UMKM menurutnya adalah permasalahan perizinan yang rumit dengan banyaknya regulasi pusat dan daerah atau hiper- regulasi yang mengatur perizinan di berbagai sektor yang 43 menyebabkan disharmoni, tumpang tindih, tidak operasional, dan sektoral. Adanya UU Cipta Kerja diharapkan dapat memberikan kemudahan, pelindungan, dan pemberdayaan UMKM yang berdaya saing. Salah satu solusi penting pemulihan UMKM adalah insentif bagi UMKM melalui program Pemulihan Ekonomi Nasional (PEN) pemerintah pusat di 2020 dan dilanjutkan di 2021. Hasilnya adalah sebagian sektor informal dan UMKM dapat bertahan menghadapi dampak pandemi covid-19. Artinya tidak mengalami krisis yang sangat berat dibandingkan beberapa industri besar. Program ini diharapkan dapat membantu menekan penurunan Pemutusan Hak Kerja (PHK) pada UMKM. Dalam penyaluran dana PEN atau dukungan UMKM secara khususnya, pemerintah harus memastikan bahwa dana dukungan dapat disalurkan secara cepat dan tepat sasaran. Permasalahan yang dihadapai saat ini adalah kurang terintegrasinya data UMKM yang ada. Selain itu, skema dukungan UMKM melalui subsidi bunga Kredit Usaha Rakyat (KUR) juga perlu mendapat perhatian lebih mengingat masih banyaknya UMKM yang masih belum tersentuh layanan perbankan. Pandemi covid-19 berdampak besar pada penurunan perekonomian nasional termasuk 44 sektor UMKM. Namun, seiring menurunnya jumlah penularan covid-19 dan berjalannya program vaksinasi menumbuhkan optimisme sektor UMKM dapat pulih pada tahun 2021 ini. PEMBAHASAN Pemerintah sejak 2020 mengeluarkan berbagai kebijakan dalam mendukung pemulihan sektor UMKM antara lain penyaluran PEN bagi sektor UMKM, program Gernas BBI, program vaksinasi, restrukturisasi kredit dan rencana pembentukan holding BUMN ultra mikro. DPR RI melalui komisi terkait perlu memberikan dukungan dan perhatian pada pemerintah dalam melaksanakan kebijakan yang diambil guna memulihkan sektor UMKM agar momentum pertumbuhan ekonomi di tahun 2021 ini dapat terwujud. 45 BAB IV KESIMPULAN Upaya pemerintah dalam membantu pelaku usaha UMKM yang terdampak pandemi covid-19, yaitu Program Pemulihan Ekonomi Nasional (PEN) merupakan salah satu program yang dicetuskan pemerintah untuk memulihkan ekonomi Indonesia akibat dampak Covid-19. Program ini juga merupakan respon pemerintah atas penurunan aktivitas masyarakat yang terdampak, khususnya sektor informal atau UMKM. Program ini dibuat berdasarkan PP Nomor 23 tahun 2020 yang kemudian diubah menjadi PP Nomor 43 tahun 2020. Upaya pemerintah untuk memajukan UMKM di era pandemi covid-19, diantaranya adalah merupakan rincian program PEN untuk UMKM, yaitu: Subsidi bunga/margin, Belanja Imbal Jasa Penjaminan (IJP), Penempatan Dana Pemerintah di perbankan, Penjaminan loss limit kredit UMKM, Pajak penghasilan final UMKM ditanggung pemerintah, Pembiayaan investasi kepada koperasi melalui Lembaga Pengelola Dana Bergulir (LPDB) koperasi UMKM dan Program Bantuan Presiden (Banpres) Produktif Usaha Mikro.Upaya lain dari pemerintah untuk memajukan UMKM yaitu program Kredit Usaha Rakyat (KUR) yang disalurkan melalui lembaga keuangan dengan pola penjaminan. 46 Hambatan-hambatan dan solusi dari pemerintah dalam membantu pelaku usaha UMKM yang terdampak pandemi covid-19 yaitu hambatan seperti minimnya pengetahuan pasar luar negeri, konsistensi kualitas dan kapasitas produk, sertifikasi, hingga kendala logistik. Pemerintah terus berupaya untuk membantu UMKM agar dapat mengatasi hambatanhambatan tersebut. Pandemi ini menyebabkan banyak UMKM kesulitan melunasi pinjaman serta membayar tagihan listrik, gas, dan gaji karyawan. Beberapa diantaranya sampai harus melakukan PHK. Kendala lain yang dialami UMKM, antara lain sulitnya memperoleh bahan baku, permodalan, pelanggan menurun, distribusi dan produksi terhambat. Selain itu permasalahan UMKM Akibat Pandemi diantaranya adalah Penurunan jumlah UMKM dan kontribusi UMKM terhadap PDB Indonesia disebabkan oleh pandemi sejak 2020 lalu. Beberapa permasalahan yang dialami diantaranya adalah perubahan pola konsumsi barang dan jasa masyarakat dimasa pandemi dari offline ke online, UMKM mengalami permasalahan tenaga kerja akibat pemberlakuan Pembatasan Sosial Berskala Besar (PSBB), hambatan distribusi produk dan kesulitan bahan baku produksi. Salah satu solusi penting pemulihan UMKM adalah insentif bagi 47 UMKM melalui program Pemulihan Ekonomi Nasional (PEN) pemerintah pusat di 2020 dan dilanjutkan di 2021. Hasilnya adalah sebagian sektor informal dan UMKM dapat bertahan menghadapi dampak pandemi covid-19. Artinya tidak mengalami krisis yang sangat berat dibandingkan beberapa industri besar. Program ini diharapkan dapat membantu menekan penurunan Pemutusan Hubungan Kerja (PHK) pada UMKM. 48 DAFTAR PUSTAKA Abor, J., & Quartey, P. 2010. Issues in SME development in Ghana and South Africa. International Research Journal of Finance and Economics, (39), 218-228. Accra Noble, B. F. 2003.,Auditing strategic environmental assessment in Canada. Journal of Environmental Assessment Policyand Management 5:127–147 Azis, abdul dan A. herani rusland 2009. Peranan Bank Indonesiadi Dalam Mendukung Pengembangan Usaha Mikro, Kecil, dan Menengah, Seri Kebanksentralan, No 21, Pusat Pendidikan dan Studi Kebanksentralan (ppsk) Bank Indonesia. Arthur, P. 2003. The implications of state policy for micro-enterprise development. In: Tettey, Wisdom; Puplampu, Korbla; Berman, Bruce 2003. 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Pendidikan S1 diselesaikan di Fakultas Sains dan Teknologi Unair tahun 1986, S2 diselesaikan di Magister Manajemen Program Pascasarjana Univeritas Sebelas Maret Solo (2005), dan S3 diselesikan di Fakultas Eonomi dan Bisnis Universitas Program Studi ESP Universitas Airlangga Surabaya (2012). Pengalaman mengajar dimulai sejak selesai S2 di Solo dan menjadi Dosen Luar biasa dibeberapa Perguruan Tinggi PTN dan PTS di Surabaya mulai tahun 2007 hingga tahun 2011, dan sampai saat ini mengajar di Pascasarjana dan FEB Universitas Muhammadiyah Sidoarjo Penulis juga penah menjadi Konsultan Lingkungan (Sertifikasi Amdal UNS 2003), Konsultan Bisnis (Sertifikasi Auditor UNS, 2004), Konsultan Appraisel Properti ( Sertifikasi MAPPI 2008 ), serta Sertifikasi untuk UKM dan Koperasi. Selain itu juga menjadi Asesor BKD, serta 54 memiliki Sertifikasi Reviewer Artikel dan Penelitian. Disamping itu juga aktif di Ikatan Sarjana Ekonomi Indonesia (ISEI), Asosiasi Manajemen Perguruan Tinggi Muhammadiyah (APSMA) dan Asosiasi Dosen Indonesia (ADI) serta di KADIN Kabupaten Sidoarjo Buku Pengantar Manajemen Kontribusi Luther Gullick terhadap Manajeman Publik adalah merupakan Karya Pertama Penulis. kemudian disusul Buku ke Dua Pengantar Manajemen Resiko, Manajemen Strategi dan Resiko, dan Financial Technologi. Disamping sebagai Dosen, penulis juga aktif mengikuti Seminar Internasional dan Nasional serta melakukan Penelitian dan Pengabdian Masyarakat serta menjadi Nara Sumber untuk beberapa kegiatan dengan UMKM. Disamping itu juga aktif membimbing mahasiswa pada program MBKM maupun PKM memiliki Sertifikasi Reviewer Artikel dan Penelitian. Disamping itu juga aktif di Ikatan Sarjana Ekonomi Indonesia (ISEI), Asosiasi Manajemen Perguruan Tinggi Muhammadiyah (APSMA) dan Asosiasi Dosen Indonesia (ADI) serta di KADIN Kabupaten Sidoarjo Buku Pengantar Manajemen Kontribusi Luther Gullick terhadap Manajeman Publik adalah merupakan Karya Pertama Penulis. kemudian disusul Buku ke Dua Pengantar Manajemen Resiko, Manajemen Strategi dan Resiko, dan Financial Technologi. Disamping sebagai Dosen, penulis juga aktif mengikuti Seminar Internasional dan Nasional serta melakukan Penelitian dan Pengabdian Masyarakat serta menjadi Nara Sumber untuk beberapa kegiatan dengan UMKM. Disamping itu juga aktif membimbing mahasiswa pada program MBKM maupun PKM 55 55 56 UMSIDA PRESS Universitas Muhammadiyah Sidoarjo Jl. Mojopahit No. 666 B Sidoarjo Sidoarjo, JAWA TIMUR UMSIDA PRESS Universitas Muhammadiyah Sidoarjo Jl. Mojopahit No. 666 B Sidoarjo Sidoarjo, JAWA TIMUR 56
https://openalex.org/W2161183565	https://sajhrm.co.za/index.php/sajhrm/article/download/277/311	English	null	Conflict between work and nonwork roles of employees in the mining industry: Prevalence and differences between demographic groups	SA journal of human resource management	2,011	cc-by	13,297	How to cite this article: How to cite this article: Steyl, B., & Koekemoer, E. (2011). Conflict between work and nonwork roles of employees in the mining industry: Prevalence and differences between demographic groups. SA Journal of Human Resource Management/SA Tydskrif vir Menslikehulpbronbestuur, 9(1), Art. #277, 14 pages. doi:10.4102/sajhrm.v9i1.277 Contribution/value-add: Organisations are able to focus interventions and programmes that specifically address the problem of work–nonwork conflict in specific roles and for different demographic groups. Introduction There is growing evidence that workers have faced increased pressures at work and in their personal lives during the past few years (Brink & De la Rey, 2001; Geurts & Demerouti, 2003). According to Geurts, Rutte, and Peeters (1999), the interdependence between these domains is increasingly recognised. This shows that, apart from work demands, workers also face increasing demands in their family or private lives (also called ‘nonwork’ domains). Dates: Received: 28 Jan. 2010 Accepted: 19 Feb. 2011 Published: 20 June 2011 Republished: 22 Sept. 2011 Practical/managerial implications: Organisations need to recognise the negative interference or conflict between work and nonwork roles for different demographic groups and address the prevalent work–nonwork conflicts in their organisations. Conflict between work and nonwork roles of employees in the mining industry: Prevalence and differences between demographic groups Orientation: International researchers have increasingly recognised the interaction between work and nonwork roles as an interesting and important topic. Research purpose: The purpose of this study was to investigate the prevalence of different work–nonwork conflict subscales and differences between demographic groups in work– nonwork conflict. Affiliations: 1School of Human Resource Management, Faculty of Economic and Management Sciences, North-West University, South Africa Motivation for the study: Several studies have shown that demographic groups differ in their experiences of the interaction between work and family life. This may also be true of conflict between work and nonwork roles. The prevalence of work–nonwork conflict and nonwork– work conflict is also very important for organisations that may find the results very valuable for developing organisational and individual interventions and performance management in organisations. 2School of Human Resource Sciences, Faculty of Economic and Management Sciences, North-West University, Potchefstroom campus, South Africa Research design, approach and method: The researchers chose a random sample of mining employees (n = 245) from a platinum mine in Rustenburg. The researchers used self-developed items similar to items developed in the Work–nonwork Interference Scale of Koekemoer, Mostert and Rothmann (2010) to measure conflict between work and various nonwork roles. The researchers used descriptive statistics, paired-sample t-tests, multivariate analysis of variance and one-way analysis of variance to analyse the data. Correspondence to: Eileen Koekemoer Email: Eileen.Koekemoer@nwu.ac.za Postal address: Private Bag X6001, Internal Box 114, Potchefstroom 2531, South Africa Dates: Received: 28 Jan. 2010 Accepted: 19 Feb. 2011 Published: 20 June 2011 Republished: 22 Sept. 2011 Main findings: Work–nonwork conflict was more prevalent than nonwork–work conflict. Work–family conflict was more prevalent than work–domestic conflict and work–religion/ spirituality conflict. The researchers found significant differences for marital status and language groups about work–nonwork conflict. Results showed that participants who spoke African languages experienced higher levels of private–work conflict. Note: i In addition to the possible prevalence of work–nonwork conflict and nonwork–work conflict, several studies have shown that demographic groups may differ in their experiences of the interaction between work and family (De Klerk & Mostert, 2010; Donald & Linington, 2008; Geurts & Demerouti, 2003; Greenhaus & Parasuraman, 1999; Grzywacz & Marks, 2000; Matthews & Power, 2002; Oldfield & Mostert, 2005; Pieterse & Mostert, 2005; Rost & Mostert, 2007; Van Tonder, 2005). Workers from different demographic groups have different working environments. They are also involved in different nonwork domains that they may experience differently (Keene & Reynolds, 2005; Greenhaus & Powell, 2003; Nikandrou, Panayotopoulou & Apospori, 2008). Having several roles can make life even more complex (Tingey, Kiger & Riley, 1996). Workers have to address several demands that could conflict (Geurts & Demerouti, 2003; Nordemark, 2002; Premeaux et al., 2007; Small & Riley, 1990). Therefore, the involvement of workers in several roles can be very stressful. With few exceptions, the more one exhibits the behaviours particular roles expect, the more conflicting demands can arise from these roles. They could lead to work–nonwork interference or conflict (Carlson, Kacmar & Williams, 2000; Katz & Kahn, 1978; Olson- Buchanan & Boswell, 2005; Premaux et al., 2007; Tingey et al., 1996). There might be various reasons why workers from different demographic groups (gender, age, language, qualifications, marital and parental status) have different experiences of work–nonwork conflict (Baca Zinn, 1990; Desrochers, Andreassi & Thompson, 2002; Donald & Linington, 2008; Duxbury & Higgins, 2001; Greenhaus & Parasuraman, 1999; Grzywacz & Marks, 2000; Kreiner, 2006; Nasurdin & Hsia, 2008; Wallis & Prince, 2003). Most research and empirical studies on the interaction between work and personal life has shown that the interference or conflict that arises in the work domain is more prevalent than the interference or conflict that arises in the family or home domain (Bond, Galinksy & Swanberg, 1998; Eagles, Miles & Icenogle, 1997; Eby et al., 2005; Frone, 2003; Frone, Russel & Cooper, 1992; Geurts & Demerouti, 2003; Grzywacz & Marks, 2000; Gutek, Klepa & Searle, 1991; Mesmer-Magnus & Viswesvaran, 2005; Rost & Mostert, 2007). Some could be individual preferences, individual personal or traditional roles, personal goals, or personal home or household situations (Day & Chamberlain, 2006; Matthews & Power, 2002; Nikandrou et al., 2008; Schulteiss, 2006). Note: i This article is republished with an amended affiliation of Betsie Steyl. This article is republished with an amended affiliation of Betsie Steyl. This is mainly because of the demographic and structural changes in family and workforce structures, both nationally in transformation and technological developments as well as high unemployment rates (Cavaleros, Van Vuuren & Visser, 2002) and internationally (Geurts et al., 1999; Geurts & Demerouti, 2003). As a result, there is an increase in the number of women and dual-earner families entering the workforce (Greenhaus & Parasuraman, 1999; Stevens, Minnotte, Mannon & Kiger, 2007). As a result, increasing demands in their work and nonwork domains confront more and more employees and many of their daily work difficulties become incompatible with their private responsibilities (Jansen, Peeters, De Jonge, Houkes & Tummers, 2004). © 2011. The Authors. Licensee: OpenJournals Publishing. This work is licensed under the Creative Commons Attribution License. © 2011. The Authors. Licensee: OpenJournals Publishing. This work is licensed under the Creative Commons Attribution License. d http://www.sajhrm.ac.za d http://www.sajhrm.ac.za Page 2 of 14 Consequently, the potential for interference or conflict occurring between a worker’s work and personal life, also known as work–family conflict (WFC), is increasing (Byron, 2005; Eby, Casper, Lockwood, Bordeaux & Brinley, 2005; Greenhaus & Beutell, 1985; Greenhaus & Powell, 2003; Mesmer-Magnus & Viswesvaran, 2005). However, according to Westman and Piotrkowski (1999), as well as Premeaux, Adkins and Mossholder (2007), one can only reach an adequate understanding of the interaction between work and personal life when one views it in the context of multiple roles outside work. These are also called nonwork roles, like domestic or home roles and religious or spiritual roles. Alternatively, the prevalence of work–family or home conflict (or work–nonwork conflict) may suggest that the interference or conflict arising in the work domain is also more prevalent than the interference or conflict arising in the different nonwork roles (family, religion or spirituality and home or domestic). This might result in more work–nonwork conflict than nonwork–work conflict. Alternatively, the prevalence of work–family or home conflict (or work–nonwork conflict) may suggest that the interference or conflict arising in the work domain is also more prevalent than the interference or conflict arising in the different nonwork roles (family, religion or spirituality and home or domestic). This might result in more work–nonwork conflict than nonwork–work conflict. Note: i Because the importance and meaning (also called ‘saliency’) that workers attach to certain roles in their private lives differ from person to person, the interaction between work and nonwork roles might also differ between workers from different demographic groups (Frone, 2003; Geurts & Demerouti, 2003). These researchers suggest that workers are more likely to give preference to work-related matters. This results in less involvement in their home or family domains (Frone, 2003). This might mean that their work domains are less flexible than the home or family domains or that employees might perceive their work roles as more important (Carlson & Kacmar, 2000; Day & Chamberlain, 2006; Greenhaus & Powell, 2003). Consequently, employees might invest more emotion and time in their work. This could result in less investment in their private lives or nonwork domains and cause interference or conflict between their work and nonwork domains, known as work–nonwork conflict (Carlson et al., 2000; Premeaux et al., 2007). The general objectives of this study were to investigate: • the prevalence of different work–nonwork conflicts and nonwork–work conflicts and their subscales • demographic differences (gender, age, language, and qualifications, marital and parental status) in work– nonwork conflict in a sample of mining employees. Trends from the literature Work–nonwork conflict In addition, their experiences of the conflict between the different nonwork roles might differ. The work domain may interfere more with the more important nonwork roles (like those of parents or spouses). Work–family conflict may be more prevalent than work–domestic conflict because of the importance of the family (Frone, Russell & Cooper, 1992; Luchetta, 1995; McClellan & Uys, 2009). Greenhaus and Beutell (1985) initially defined WFC as: Greenhaus and Beutell (1985) initially defined WFC as: a form of inter-role conflict in which the role pressures from the work and family domains are mutually incompatible – in such way that participation in the work (or family) role is made more difficult by virtue of participation in the family (or work) role. (Greenhaus & Beutell, 1985, p. 77) (Greenhaus & Beutell, 1985, p. 77) http://www.sajhrm.ac.za doi:10.4102/sajhrm.v9i1.277 Page 3 of 14 that adequate performance in one role jeopardises adequate performance in the other. The restricted resources people have to fulfil these role demands are often in a state of imbalance. This leads to conflict between domains (Greenhaus & Beutell, 1985). This definition suggests a bidirectional dimension in which the work domain can interfere with the family domain and the family domain can interfere with the work domain. Although this definition is widely accepted, this broad definition does not differentiate between the interaction or conflict between work and nonwork roles. Kirchmeyer (1992) elaborated on this issue by stating that researchers need to accept people as parents, spouses or members of the community and not just as workers. Many workers, and especially employed parents, struggle to combine their obligations to the work domain with those to the nonwork or personal domains (Bailyn & Harrington, 2004; Byron, 2005; Geurts & Demerouti, 2003). This relates to identity theory. It suggests that a particular situation evokes a particular identity (that also relates to roles) and the commitment of the person to the different identities or roles that make up the self-concept determines that identity (Stryker, 1987). Therefore, people or workers can engage in a variety of roles or identities (parental, spousal, domestic and religious) outside their work. The general demands of each role comprise the responsibilities, requirements, duties, commitments and expectations related to performance in a given domain or role (Bulger, Matthews & Hoffman, 2007; Netemeyer, Boles & Mcmurrian, 1996; McClellan & Uys, 2009; Voydanoff, 2007). Trends from the literature Work–nonwork conflict According to Greenhaus and Beutell (1985), the type of WFC could result from the role characteristics that affect the time involvement, strain or behaviour in one domain, but which are incompatible with the role characteristics of the other (work vs. home). Thoits (1991) also stated that people who participate in different roles have various identities that are organised in a hierarchy of centrality. People derive more meaning and purpose from participating in a more salient role (Thoits, 1991). Therefore, they will invest more time or emotion in that identity or role (Stryker & Serpe, 1994). According to Wiley (1991), people could experience stress when performing the behaviours and confirm the salient identity as poor. Therefore, inter-role strain or conflict will arise when people perceive conflicting and competing expectancies from the two or more roles they perform (Holahan & Gilbert, 1979). It makes work–nonwork conflict possible. The three forms of work–family conflict or family–work conflict the literature identifies mainly are (Greenhaus & Beutell, 1985): • strain-based conflict l Prevalence of work–nonwork conflict and nonwork–work conflict • behaviour-based conflict l • time-based conflict. Various research and empirical studies have shown that the negative interference of work to home is more prevalent than the negative interference of home to work (Bond et al., 1998; Frone, 2003; Frone et al., 1992; Geurts & Demerouti, 2003; Grzywacz & Marks, 2000; Rost & Mostert, 2007). Because of the forced structure and obligatory nature of work, workers are more likely to emphasise work over private and family matters. This reduces the amount of effort they invest at home rather than at work (Frone et al., 1992; Gutek et al., 1991). Since the initial definition of Greenhaus and Beutell (1985), research on the interaction between work and family life has progressed substantially. It has introduced newer terms like work–life integration, work–family interaction, work–home interaction, work–life interference, work–nonwork interface and work–family integration (Bailyn & Harrington, 2004; Carlson & Frone, 2003; Curbow, McDonnell, Spratt, Griffen & Agnew, 2003; Desrochers, Hilton & Larwood, 2005; Eby et al., 2005; Geurts & Dikkers, 2002; Geurts et al., 2005; Lewis & Cooper, 2005; Olson-Buchanan & Boswell, 2006; Premeaux et al., 2007; Thompson & Bunderson, 2001). Geurts et al. (2005) support this; they suggest that the home domain may offer more opportunity to adjust one’s behaviour to one’s present needs than the work domain does. This might also be the cause of the conflict between the work and various nonwork roles. Although researchers have studied all these terms well, the main limitation of the terms is that they exclude nonwork roles other than family or home life that may interact or interfere with the work domain (Bellavia & Frone, 2005; Frone, 2003; Geurts & Demerouti, 2003; Tetrick & Buffardi, 2006). From the perspective of identity theory, it is possible that, because of the saliency of the work role over other nonwork roles, people will invest more time and effort in their work. This might interfere, or conflict with, some nonwork roles (Carlson & Kacmar, 2000; Day & Chamberlain, 2006; Greenhause & Powell, 2003; Perrone, Webb & Jackson, 2007; Wiley, 1991). Multiple roles and role identity theory as theoretical frameworks A very important aspect in work–nonwork conflict research is the different roles people have to participate in or perform in their personal lives (their nonwork roles). For example, people might attach high saliency to roles like parenting, religion or spirituality. However, because their work is so demanding and because there are inadequate opportunities to engage in these personal roles, specific According to Barnett and Baruch (1985), role conflict might occur when the demands from two or more roles are such http://www.sajhrm.ac.za doi:10.4102/sajhrm.v9i1.277 Page 4 of 14 nurturing and rearing their children as well as providing emotional support instead of only being breadwinners (Hand & Lewis, 2002; Kaufman & Uhlenberg, 2000; Root & Wooten, 2008). work–nonwork conflict may arise and be more apparent or prevalent than others (like work–parent conflict and work– spirituality conflict). For some people, some nonwork roles might be more prevalent or salient and lead to more specific work–nonwork conflict than other nonwork roles do. Once again, the saliency people attach to these roles becomes relevant. It is possible that the saliency of family roles has changed amongst fathers, possibly leading to more work– family conflict. In addition, fathers might value specific nonwork roles, like religion or spirituality, more than women do and, because of their demanding work, are unable to attend to these roles. This might lead to specific work– nonwork conflict. Therefore, it makes sense that there might be differences in the interaction between work and other specific nonwork roles for men and women. Based on the theoretical framework and literature review on the prevalence of work–nonwork conflict, the researchers formulated two hypotheses: • the direction of work–nonwork conflict is more prevalent than nonwork–work conflict (Hypothesis 1a) • within each direction of conflict (work–nonwork conflict and nonwork–work conflict), some specific subscales of conflict are more prevalent than others and depend on the saliency of the nonwork roles (Hypothesis 1b). Most studies found no relationship between WFC and different age groups (Frone et al., 1997; Kinnunen & Mauno, 1998; Pieterse & Mostert, 2005; Van Tonder, 2005). However, Oldfield and Mostert (2005) found that older people (those between the ages of 50 and 69) experienced statistically significant lower levels of WFC than their younger counterparts (those between the ages of 22 and 39) did. The younger group seems to experience the highest level of WFC. Multiple roles and role identity theory as theoretical frameworks Furthermore, Duxbury and Higgins (2001) reported that participants between 36 and 55 experienced more work– home conflict. Rost and Mostert (2007) found that younger employees (between the ages of 26 and 35 years of age) experienced statistically significant lower levels of positive work–home interference than older employees (between 46 and 65 years of age) did, whilst older employees experienced statistically significant lower levels of work–home conflict than younger employees did. Grzywacz and Marks (2000) also found more family–work conflict in younger men than in older men, whilst younger women reported less WFC than older women. Demographic differences and work–nonwork conflict There is considerable literature on WFC and the differences in work–nonwork conflict between demographic groups (gender, age, language, qualifications as well as marital and parental status [See the overview in Frone, 2003]). The evidence of gender differences in WFC is mixed (De Klerk & Mostert, 2010; Frone, 2003; Geurts & Demerouti, 2003; Nasurdin & Hsia, 2008; Nordenmark, 2002). Some research has shown that men and women generally report similar levels of WFC and family–work conflict, or FWC (Carnicer, Sánchez, Pérez & Jiménez, 2004; Demerouti et al., 2004; Frone, 2003). Other researchers have found that women experience more WFC than men do (Frone et al., 1992; Hammer et al., 1997). On the other hand, Oldfield and Mostert (2005), as well as Rost and Mostert (2007), observed that men experienced higher levels of WFC than women did. In addition, Pieterse and Mostert (2005) found a significant difference between men and women on WFC. This shows that men reported a higher level of WFC than was the case with women. This discrepancy in results between age groups might be attributed to the saliency of roles for specific age groups. Some older employees might feel that their family or personal roles are becoming more important to them because they already have achieved their career goals or positions. Alternatively, younger employees might still feel the need to prove themselves in their work and personal lives. Therefore, it is possible that people from different age groups also differ in their experiences of the influences of other nonwork and work roles. One might attribute these differences in the experience of WFC for men and women to the traditional roles of people. Previously, women had more of a parenting role and rarely participated in work roles (Greenhaus & Parasuraman, 1999). However, more women are now entering the workforce. This might cause clashes between their more traditional parenting roles and their work roles and lead to WFC (Day & Chamberlain, 2006; Doumas, Margolin & John, 2008; Plaisier et al., 2008). As far as differences between language and ethnic groups are concerned, Rost and Mostert (2007) found that English- speaking participants experienced higher levels of work– home conflict than speakers of Afrikaans and African languages did. Grzcywacz and Marks (2000) and Van Tonder (2005) also found that White and African groups have higher levels of work–home conflict than coloured and Indian participants do. Demographic differences and work–nonwork conflict In addition, more working fathers are now taking over some parenting responsibilities and helping to care for their children. This might also lead to conflicting role demands (Daly & Palkovitz, 2004; Heraty et al., 2008; Lingard & Francis, 2005; Root & Wooten, 2008). Furthermore, according to Baxter (2007), the roles associated with men and fathers became multifaceted. Men fulfil the role of breadwinners, must spend time with their families and must support and assist their spouses. Fathers are now more involved in In addition, more working fathers are now taking over some parenting responsibilities and helping to care for their children. This might also lead to conflicting role demands (Daly & Palkovitz, 2004; Heraty et al., 2008; Lingard & Francis, 2005; Root & Wooten, 2008). Furthermore, according to Baxter (2007), the roles associated with men and fathers became multifaceted. Men fulfil the role of breadwinners, must spend time with their families and must support and assist their spouses. Fathers are now more involved in These findings contradict the findings of Kinnunen and Mauno (1998); Frone et al. (1997) and Pieterse and Mostert (2005). They found no differences in work–home conflict and http://www.sajhrm.ac.za doi:10.4102/sajhrm.v9i1.277 doi:10.4102/sajhrm.v9i1.277 Page 5 of 14 language groups. However, because household and family situations differ so much in the different cultural or language groups, differences in the experiences of the interaction or interference between work and family or home are possible. Furthermore, one can view the responsibilities of parenting (caring for children) as added demands on people (Day & Chamberlain, 2006). Oldfield and Mostert (2005) reported that working parents appear to have higher levels of WFC than married people without children do. Grzywacz, Almeida and McDonald (2002) found that having a child (aged between 6 and 18) was associated with less positive spill over from family to work compared to being childless. Couples without children can act independently as they do not have children to look after (Duxbury & Higgins, 2001). Therefore, it seems that the parental status of people will influence the degree of work–nonwork conflict they experience. Furthermore, the saliency that people from different language groups attach to their different nonwork roles might differ. It is possible that the work role is more important than the family roles or vice versa for specific language groups. This will influence the interaction between these roles. The research of Frone et al. The potential value of the study Work–nonwork conflict is an important topic of research amongst various occupational and demographic groups. It can also play a significant role in a mining environment, which is widely acknowledged as very stressful and demanding (Singer, 2002). WFC research that considered marital status as a demographic variable found that single men and women report less WFC than married men and women. However, it also found that being unmarried was strongly associated with less positive spill over from home to work (Grzywacz & Marks, 2000). This supports the findings of Herman and Gyllstrom (1977) that married people experienced more work–family conflict than unmarried people did. Because of its important contribution to the economy of South Africa, various companies in the mining industry need to maintain competitive advantage whilst complying with the demands of change. They consequently impose various forms of stressors on their employees. Amongst the present stressors are the consequences of a demanding work environment that tend to spill over into people’s personal lives and could negatively influence their well-being (Brough, 2003). In a stressful mining environment, the different roles employees may have outside work (those of parents, spouses, religion or spirituality, and home or domestic) could interfere with their work and vice versa. Demerouti et al. (2004) found that people who lived alone have more work–family conflict than those who lived with a spouse. However, Oldfield and Mostert (2005) reported no significant differences between married and unmarried people. The reasons for the differences could be people’s perceptions of marriage and the value, or meaning, that they attach to their spouses or partners and their perceptions of their responsibilities in a marriage (Day & Chamberlain, 2006). These might differ from person to person and it is possible that married people view their marriages as relationships that entail additional demands. Alternatively, single people might feel that, because they do not have a partner or spouse to help them at home, they experience more strain and demands and, ultimately, more work–family conflict. When there is work–nonwork conflict, organisations need to focus more specifically on programmes and interventions to address the problem. More specifically, organisations need to know which forms of work–nonwork conflict cause problems to their employees and which specific nonwork roles are very demanding for them. The potential value of the study Šverko, Arambašić, and Galešić (2002) stated that more and more companies have adopted various family-responsive policies and other programmes to support their employees (like paid maternity leave) to address the issue. With regard to parental status, employees with families often miss career opportunities when they need to put their family responsibilities before their work (Rothbard & Edwards, 2003). This suggests parent–work conflict. There are also indications that women with children experience more conflict between work and family compared to childless women and men (Crouter, 1984; Grzywacz & Marks, 2000). The saliency that people attach to their parenting roles is important here. Organisations, that know which nonwork roles and nonwork–work conflict are more prevalent, will also be able to attend to the problems better. Demographic differences and work–nonwork conflict (1997) and Pieterse and Mostert (2005) found no significant relationships between qualifications and work–home conflict. However, Rost and Mostert (2007) found that employees with tertiary qualifications experienced significantly higher levels of positive work–nonwork interference than employees with postgraduate degrees did. Oldfield and Mostert (2005) also found that people with tertiary qualifications appear to experience lower levels of negative work–home conflict and home–work conflict than those with secondary education. It can be assumed that people with higher qualifications attach more value to their work role, seek higher qualifications and therefore do not accept interference with their work role. The researchers formulated one hypothesis based on the differences between demographic groups. It is that there are differences in work–nonwork conflict for demographic groups based on gender, age, language, qualifications, marital status and parental status (Hypothesis 2). Table 1 gives the characteristics of the participants. Table 1 gives the characteristics of the participants. Most of the participants were women (55.1%), Afrikaans- speaking or English-speaking (63.7%) and between the ages of 20 and 39 years (30.6%). Most were either White (54.3%) or African (38.8%). Of the participants, 55.5% had tertiary education qualifications. Most of the participants were married (61.2%) and had children (58.4%). The sample included employees from different Patterson grade levels (C1–D4). Of the participants, 18.4% were on a C Upper (C4) level whilst 18% were on a C Lower (C1) level. work and various nonwork roles (like spousal, parental, religious or spiritual and home or domestic roles) and 24 items to measure conflict between nonwork roles (spousal, parental, religious or spiritual and domestic) and work. More specifically, the researchers developed six items for each proposed subscale in both directions: work–nonwork conflict and nonwork–work conflict; work–spouse conflict (‘… your work interferes with your relationship with your spouse or partner’); work–parent conflict (‘… your job makes it hard for you to have a good relationship with your child[ren]’); work–religion or spirituality conflict (‘… your work environment does not encourage your religious or spiritual beliefs’); work–domestic conflict (‘… your job interferes with your domestic responsibilities at home’); spouse–work conflict (‘… your relationship with your spouse or partner interferes with your work’); parent–work conflict (‘… your work suffers because you need to take care of your child[ren]’); religion or spirituality–work conflict (‘… your religious or spiritual commitments interfere with your work schedule’); and domestic–work conflict (‘… you have to rearrange your work schedule because of your domestic responsibilities’). Research design Research approach This was a quantitative study in which the researchers used a cross-sectional survey design to achieve their research http://www.sajhrm.ac.za doi:10.4102/sajhrm.v9i1.277 doi:10.4102/sajhrm.v9i1.277 Page 6 of 14 TABLE 1: Characteristics of participants (n = 245). Item Category f % Gender Male 110 44.9 Female 135 55.1 Age 20–29 years 74 30.2 30–39 years 75 30.6 40–49 years 55 22.4 50 years and older 38 15.5 Missing values 3 1.2 Race or ethnicity White 133 54.3 African 95 38.8 Coloured 13 5.3 Indian 4 1.6 Language Afrikaans and English 156 63.7 African language 89 36.3 Qualifications Secondary education 109 44.5 Tertiary education 136 55.5 Level of position (Patterson grading scale) C1 — supervisory level 44 18 C2 – supervisory level 36 14.7 C3 – supervisory level 23 9.4 C4 – supervisory level 45 18.4 C5 – middle management 24 9.8 D1 – middle management 25 10.2 D2 – middle management 27 11 D3 – middle management 9 3.7 D4 – middle management 12 4.9 Marital status Single 92 37.6 Married 150 61.2 Missing values 3 1.2 Parental status With children 143 58.4 Without children 99 40.4 Missing values 3 1.2 C1–D4, patterson grade levels. f, frequency. TABLE 1: Characteristics of participants (n = 245). objectives. Cross-sectional research designs involve measuring all variables for all cases within a narrow time span so that the measurements are contemporaneous. The researchers collected data at only one point in time and compared different participants (Du Plooy, 2001). They collected the primary data through surveys. Research participants and sampling The researchers took random samples (n = 245) of employees working in a platinum mine in the Rustenburg area. The response rate was 49%. According to DeVellis (2003), samples should be large enough to eliminate subject variance as a significant concern. Researchers usually regard a sample of 300 as adequate. However, scales have been successfully developed and used with smaller samples. In order to identify and establish the sample population, the researchers obtained lists of all workers from middle managers in the human resource department of the mining company. The researchers asked workers in Patterson grade levels C1–D4 (middle management) to participate in the study and gave them questionnaires to complete. They chose workers from these Patterson levels only because workers from these grade levels in a mining environment are literate and could complete the questionnaires. preliminary analysis before analysing the data, the researchers used two exploratory factor analyses to determine the construct validity of the self-developed items that measure work– nonwork conflict. The researchers analysed the items that measure the two directions of conflict (work–nonwork conflict items and nonwork–work conflict items) separately with exploratory factor analyses where they applied an oblique rotation method. The researchers released no personal information that could lead to the identification of participants. Booklets were completed anonymously and only included numbers for record-keeping purposes. The researchers also informed the participants that, if they participated in the research and completed the questionnaire, they were giving consent to the researchers to use the data for research purposes only. Page 7 of 14 whether the means of two groups are statistically different from each other. A biographical questionnaire: The biographical characteristics the researchers measured were age, race, educational level, household situation (marital status and/ or having children or not) and type of position. With the biographical information the researchers obtained, they were able to compare the differences in work–nonwork conflict and nonwork–work conflict for the different biographical groups in the platinum mine. The researchers used multivariate analysis of variance (MANOVA) to determine the significance of the differences between the work-nonwork conflict scales of different demographic groups. MANOVA is the counterpart of analysis of variance (ANOVA). It covers cases where more than one dependent variable occurs and where one cannot simply combine the dependent variables. Exploratory factor analyses on the 24 items that measure work–nonwork conflict Firstly, the researchers performed exploratory factor analyses on the 24 items that measure work–nonwork conflict. For this initial factor analysis on the work–nonwork conflict items, the researchers used multiple criteria to determine the number of factors. The researchers gave participants three weeks to complete the questionnaires. Afterwards the researchers collected the questionnaires personally on an arranged date. The initial specific criteria the researchers used to determine the number of factors were factors with eigenvalues greater than 1 (Kaiser’s criterion), cumulative percentages of variance, communalities and a scree plot of the factor eigenvalues. Research procedure The researchers gave the HR manager a research protocol letter explaining the research procedure and requesting participation before the start of the study. After the researchers obtained approval for the project, the participants were voluntarily involved in the research process. The researchers used Wilk’s Lambda to test whether the population mean vectors for all groups were likely to be identical to those of the sample mean vectors for the different groups (Field, 2005; Tabachnick & Fidell, 2007). When an effect was significant (p ≤ 0.05) in MANOVA, the researchers used one-way analysis of variance to discover which dependent variables had been affected. ANOVA expresses the tests of interests as estimates of variance (Muller & Fetterman, 2002). The researchers distributed questionnaires. The questionnaires included letters that explained the goal and importance of the study as well as a list of contact persons for enquiries. The letters also informed participants about the confidentiality and anonymity of their participation. Measuring instruments The researchers used the following measuring instruments in the empirical study. Items to measure work–nonwork conflict: The researchers used self-developed items to measure the conflict between work and various nonwork roles. These items were similar to preliminary items used in developing the Work–nonwork Interference Scale of Koekemoer, Mostert, and Rothmann (2010). These items measure conflict in both directions, namely work- to-nonwork and nonwork-to-work. The researchers phrased all the items ‘How often does it happen that …’ and used a 4-point scale that ranged from 0 (‘never’) to 3 (‘always’). The researchers developed 24 items to measure conflict between The results section reports the construct validity of these items as preliminary analyses. doi:10.4102/sajhrm.v9i1.277 doi:10.4102/sajhrm.v9i1.277 http://www.sajhrm.ac.za Results The researchers kept all the data confidential. Only the researchers involved in the study could capture or analyse the data. The researchers protected the completed questionnaires at all times and kept them in a safe and secure location (locked cupboards in the office of the main researcher). Statistical analysis One should check them for redundancy in the item wording that could result in within-factor correlated measurement errors. Based on these criteria, the researchers excluded three items from the additional factor analyses that followed. These were work–spouse item 1, work–spouse item 2 and work– religion/spirituality item 6. Table 2 (factor analysis on 21 items) gives the final factor analysis for the items that measure work–nonwork conflict. It reports only the final items that the researchers retained. Table 2 does not include the three items that the researchers excluded. Nevertheless, three factors still emerged and measured the same three scales as before: work–family conflict, work–domestic conflict and work–religion/ spirituality conflict. Secondly, the researchers analysed the 24 items that measure nonwork–work conflict using an additional and separate exploratory factor analysis. Table 3 gives the factor loadings and percentage of variance for these 24 nonwork–work conflict items. Two factors emerged from the nonwork– work conflict items. The researchers labelled them ‘family– work conflict’ and ‘private–work conflict’ (see Table 3). The researchers obtained high factor loadings and communalities for all the items. Although the researchers used the same criteria as they did for the work–nonwork conflict items, they retained all 24 items. They developed the final two scales for nonwork–work conflict. TABLE 3: Factor loadings, communalities, percentage variance for the nonwork to work conflict scale items. TABLE 3: Factor loadings, communalities, percentage variance for the nonwork to work conflict scale items. Statistical analysis The researchers performed the statistical analysis with the SPSS program (SPSS Inc., 2005). In the preliminary analyses, the researchers performed exploratory factor analyses to determine the construct validity of the self-developed items that measured work–nonwork conflict. Here they used Cronbach alpha coefficients to assess the reliability of the instruments. Using these initial criteria, three factors emerged from these items. The researchers labelled these factors ‘work–family interference’, ‘work–domestic interference’ and ‘work– religion or spirituality interference’. The researchers also used descriptive statistics (means, standard deviations, skewness and kurtosis) and inferential statistics to analyse the data. In order to refine the results from this initial factor analysis, the researchers used additional criteria to determine the number of items to retain for the work–nonwork conflict scale. They retained items with loadings greater than 0.45 on at least one factor (Stephens & Sommer, 1996). In addition, according to Netemeyer, Boles, and Mcmurrian (1996) and In order to determine the prevalence of the work–nonwork conflict and nonwork–work conflict subscales, the researchers used paired-sample t-tests. Paired-sample t-tests assess doi:10.4102/sajhrm.v9i1.277 http://www.sajhrm.ac.za Page 8 of 14 TABLE 2: Factor loadings, communalities and percentage variance for the work to nonwork conflict scale items (final factor analysis on 21 items). Items Factor labels Communalities F1 F2 F3 Work–spouse item 3 0.52 0.33 0.08 0.74 Work–spouse item 4 0.87 0.09 -0.07 0.77 Work–spouse item 5 0.55 0.22 0.08 0.69 Work–spouse item 6 0.67 0.08 0.07 0.72 Work–parent item 1 0.45 0.21 0.21 0.72 Work–parent item 2 0.53 0.05 0.27 0.73 Work–parent item 3 0.82 -0.07 0.07 0.7 Work–parent item 4 0.74 0.19 -0.02 0.75 Work–parent item 5 0.92 -0.11 0.01 0.76 Work–parent item 6 0.58 0.03 0.16 0.58 Work–domestic item 1 -0.05 0.03 0.92 0.78 Work–domestic item 2 0.03 0.02 0.87 0.81 Work–domestic item 3 0 -0.01 0.86 0.75 Work–domestic item 4 0.24 0.19 0.42 0.66 Work–domestic item 5 0.05 0.14 0.71 0.75 Work–domestic item 6 0.28 -0.07 0.61 0.66 Work–religion or spirituality item 1 -0.06 0.74 0.12 0.61 Work–religion or spirituality item 2 0.18 0.68 0.04 0.76 Work–religion or spirituality item 3 -0.12 0.94 0.1 0.79 Work–religion or spirituality item 4 0.21 0.58 0.05 0.68 Work–religion or spirituality item 5 0.19 0.75 -0.11 0.69 Percentage variance 59.4 5.42 3.77 - F1, work–family conflict; F2, work–domestic conflict; F3, work–religion/spirituality conflict. Bagozzi and Yi (1988), it is not advisable to retain items with extremely high factor loadings (> 0.9). Statistical analysis Items Factor labels Communalities F1 F2 Spouse–work item 1 0.75 0.1 0.79 Spouse–work item 2 0.97 -0.11 0.81 Spouse–work item 3 0.67 0.13 0.75 Spouse–work item 4 0.71 0.14 0.73 Spouse–work item 5 0.77 0.17 0.88 Spouse–work item 6 0.83 0.03 0.83 Parent–work item 1 0.9 -0.08 0.72 Parent–work item 2 0.81 0.06 0.81 Parent–work item 3 0.83 -0.05 0.69 Parent–work item 4 0.69 0.22 0.84 Parent–work item 5 0.89 -0.03 0.88 Parent–work item 6 0.85 0.03 0.82 Domestic–work item 1 0.31 0.5 0.71 Domestic–work item 2 0.17 0.68 0.76 Domestic–work item 3 0.18 0.69 0.76 Domestic–work item 4 0.2 0.57 0.77 Domestic–work item 5 0.19 0.67 0.79 Domestic–work item 6 0.24 0.66 0.82 Religion or spirituality–work item 1 -0.01 0.89 0.86 Religion or spirituality–work item 2 -0.07 0.9 0.83 Religion or spirituality–work item 3 -0.11 0.89 0.74 Religion or spirituality–work item 4 0 0.88 0.84 Religion or spirituality–work item 5 -0.06 0.95 0.99 Religion or spirituality–work item 6 0.02 0.85 0.8 Percentage variance 66.42 5.05 - F1, family–work conflict; F2, private–work conflict. Descriptive statistics After the preliminary analyses, the researchers estimated the descriptive statistics and Cronbach alpha coefficients for the work–nonwork conflict and nonwork–work conflict subscales (see Table 4). Table 4 shows that the researchers obtained acceptable Cronbach alpha coefficients for all the subscales compared to the guideline of α > 0.7 (Nunnally & Bernstein, 1994). Prevalence In order to determine the prevalence of the work–nonwork conflict and nonwork–work conflict subscales, the researchers performed separate prevalence analyses. Firstly, it was important to establish which direction of conflict is more prevalent (work–nonwork conflict or nonwork–work conflict by testing Hypothesis 1a). TABLE 4: Descriptive statistics and Cronbach alpha coefficients of the work–nonwork conflict and nonwork–work conflict subscales (n = 245). TABLE 4: Descriptive statistics and Cronbach alpha coefficients of the work–nonwork conflict and nonwork–work conflict subscales (n = 245). Item Mean SD Cronbach alpha Work–nonwork conflict subscales Work–family conflict scale 1 0.72 0.95 Work–domestic conflict scale 1.99 0.68 0.93 Work–religion or spirituality conflict scale 0.82 0.76 0.91 Nonwork–work conflict subscales Family–work conflict scale 0.8 0.7 0.93 Private–work conflict scale 0.58 0.68 0.92 SD, standard deviation. Secondly, it was important to establish, within each direction of conflict, which specific work–nonwork roles and which specific nonwork–work roles were more prevalent by testing Hypothesis 1b. Table 5 gives the results of the different paired-sample t-tests to: to: http://www.sajhrm.ac.za • establish which direction of conflict is more prevalent TABLE 5: Paired-sample t-tests for the prevalence of the direction of conflict (work–nonwork conflict and nonwork–work conflict) and the prevalence of specific work–nonwork conflict and specific nonwork–work conflict. TABLE 5: Paired-sample t-tests for the prevalence of the direction of conflict (work–nonwork conflict and nonwork–work conflict) and the prevalence of specific work–nonwork conflict and specific nonwork–work conflict. TABLE 5: Paired-sample t-tests for the prevalence of the direction of conflict (work–nonwork conflict and nonwork–work conflict) and the prevalence of specific work–nonwork conflict and specific nonwork–work conflict. Item t df Significance Prevalence of the direction of conflict (work–nonwork conflict and nonwork–work conflict) Work–family (M = 1.01) vs. family–work (M = 0.8) 5.12 198 0* Work–domestic (M = 0.99) vs. private–work (M = 0.58) -9.79 244 0* Work–religion/spirituality (M = 0.82) vs. private–work (M = 0.58) -5.17 244 0* Prevalence of specific work–nonwork conflict and specific nonwork–work conflict subscales Work–family (M = 1) vs. work–domestic (M = 1) 0.04 200 0.97 Work–family (M = 1) vs. work–religion (M = 0.81) 5.44 200 0* Work–domestic (M = 0.99) vs. work–religion (M = 0.82) 4.75 244 0* Family–work (M = 0.8) vs. private–work (M = 0.59) 7.05 198 0* M, mean value; t, t-value; df, degree of freedom. , p ≤ 0.05 denotes statistically significant. Prevalence Table 7 gives the results of the ANOVA based on language. Differences between demographic groups Table 7 showed statistically significant differences between levels of private–work conflict. African-speaking participants experienced higher levels of private–work conflict compared to Afrikaans-speaking and English-speaking participants. Table 8 gives the results of the ANOVA based on marital status. After the analyses of prevalence, the researchers used MANOVA (multivariate analysis of variance) statistics to determine the differences between demographic groups with regard to work–nonwork conflict and nonwork–work conflict. The demographic groups that the researchers compared were gender, age, language, qualifications, marital and parental status. Table 8 showed statistically significant differences between levels of private–work conflict. Single participants’ experienced higher levels of private–work conflict compared to married ones. The researchers analysed the results for statistical significance using Wilk’s Lambda statistics (Field, 2005; Tabachnick & Fidell, 2007). Table 6 gives the results of the MANOVA analyses. Prevalence • obtain the results of the paired-sample t-tests to indicate specifically, within the direction of conflict, which work– nonwork roles and which nonwork–work roles are more prevalent. With regard to the prevalent direction of conflict (work– nonwork conflict and nonwork–work conflict), paired- sample t-tests revealed that employees reported more work– family conflict (M = 1) than family–work conflict (M = 0.8, t(500) = 5.12, p < 0.01). The results also showed that workers experienced significantly more work–domestic conflict (M = 0.99) than private–work conflict (M = 0.58, t(500) = -9.79, p < 0.01) and more work–religion or spirituality conflict (M = 0.82) than private–work conflict (M = 0.58, t(500) = -5.17, p < 0.01). These results support Hypothesis 1a. k–domestic (M = 0.99) vs. private–work 0.58) -9.79 244 0 k–religion/spirituality (M = 0.82) rivate–work (M = 0.58) -5.17 244 0* alence of specific work–nonwork conflict and specific nonwork–work conflict cales k–family (M = 1) vs. work–domestic 1) 0.04 200 0.97 k–family (M = 1) vs. work–religion 5.44 200 0* Prevalence of specific work–nonwork conflict and specific nonwork–work conflict subscales With regard to the prevalence of the different specific work–nonwork conflict subscales, workers experienced more work–family conflict (M = 1) than work–religion or spirituality conflict (M = 0.81, t(500) = 5.44, p <0.01) and more work–domestic (M = 0.99) than work–religion or spirituality (M = 0.82, t(500) = 4.75 p < 0.01). The researchers found no significant differences in the prevalence of work–family conflict (M = 1) and work–domestic conflict (M = 1, t(500) = 0.04, p > 0.01). With regard to the prevalence of the two nonwork–work conflict subscales, results indicated more family–work conflict (M = 0.8) than private–work conflict (M = 0.59, t(500) = 7.05, p < 0.01). Therefore, these results support Hypothesis 1b. nonwork conflict between the gender, age, qualifications or parental status of the employees. However, the researchers found statistically significant differences (p <0.05) with language groups and marital status. The researchers used ANOVA to analyse further the relationship between work– nonwork conflict and the demographic variable levels that showed a statistically significant difference. nonwork conflict between the gender, age, qualifications or parental status of the employees. However, the researchers found statistically significant differences (p <0.05) with language groups and marital status. The researchers used ANOVA to analyse further the relationship between work– nonwork conflict and the demographic variable levels that showed a statistically significant difference. p a, The term ‘work–nonwork conflict’ is used here as a global concept in the MANOVA analyses, and incorporates ‘nonwork–work conflict’. , p ≤ 0.05 denotes statistically significant. Discussion In addition, work–family conflict might be more prevalent than work–domestic conflict because of the family salience that employees have (Frone, Russell & Cooper, 1992; Luchetta, 1995; McClellan & Uys, 2009). An alternative explanation might be that, for employees in this study, the work domain might be more salient than the nonwork domains. As a result, employees might invest more time and energy in their work domain. This might affect their time and investment in nonwork domains. Although work–nonwork conflict was more prevalent amongst mining employees, the results also showed the prevalence of the specific subscales of nonwork–work conflict. Conflict that originates from family roles (including parental and spousal roles) was higher than the conflict that originates from the private ones (including religious and domestic roles). Therefore, family roles are more likely to interfere with employees’ work than their domestic responsibilities or their religious or spiritual roles do. Burke and Reitzes (1981) stated that the saliency people attach to their identities or roles influences how much effort they put into each role and how well they perform in each. According to Stryker (1968), the various identities that make up the self exist in a hierarchy of salience. People are most likely to call upon the identities they rank highest in situations that involve different aspects of the self. In this study, the workers probably rated their family identities highest. Therefore, work interferes most negatively on family life. More specifically, employees in this study experienced more work–family conflict than work–domestic conflict and work–religion or spirituality conflict. An explanation for this might be that, unlike with domestic roles, persons with family roles are not always able to plan according to a schedule because some unplanned situations may occur in these family roles. Parents, for example, may not always foresee their children becoming ill. This might force employees to turn up late for work or to apply for family responsibility leave. However, employees also experienced more work–domestic conflict than work–religion or spirituality conflict. This shows that the work environment conflicts most with the family domain and least with religion or spirituality. This might indicate that employees value their family lives and domestic roles more than their religious or spiritual ones. Therefore, because they are unable to participate in these domains because of their high workloads or the mandatory nature of the mining work environment, the conflict in these domains is more prevalent. Table 6 gives the results of the MANOVA analyses. These results provide partial support for Hypothesis 2. They show differences for work–nonwork conflict for some of the proposed demographic groups (marital status and language groups). The analysis of the Wilk’s Lambda values showed no statistically significant differences (p < 0.05) in work– TABLE 6: MANOVA – differences in work–nonwork conflict of demographic groupsa. Variable Wilk’s Lambda values F df p Partial Eta squared Gender 0.97 1.01 5 0.41 0.03 Age 0.92 1.06 15 0.39 0.03 Language 0.92 3.52 5 0.01 0.08 Qualification 0.96 1.58 5 0.17 0.04 Marital status 0.89 4.7 5 0* 0.11 Parental status 0.98 0.93 5 0.46 0.02 MANOVA, multivariate analysis of variance; F, F-value; df, degree of freedom; Eta, describes the ratio of variance explained in the dependent variable by a predictor whilst controlling for other predictors. a, The term ‘work–nonwork conflict’ is used here as a global concept in the MANOVA analyses, and incorporates ‘nonwork–work conflict’. , p ≤ 0.05 denotes statistically significant. TABLE 6: MANOVA – differences in work–nonwork conflict of demographic groupsa MANOVA, multivariate analysis of variance; F, F-value; df, degree of freedom; Eta, describes the ratio of variance explained in the dependent variable by a predictor whilst controlling for other predictors. a, The term ‘work–nonwork conflict’ is used here as a global concept in the MANOVA analyses, and incorporates ‘nonwork–work conflict’. , p ≤0.05 denotes statistically significant. p a, The term ‘work–nonwork conflict’ is used here as a global concept in the MANOVA analyses, and incorporates ‘nonwork–work conflict’. , p ≤ 0.05 denotes statistically significant. http://www.sajhrm.ac.za TABLE 7: ANOVA – differences in work–nonwork conflict based on language. TABLE 7: ANOVA – differences in work–nonwork conflict based on language. Item Mean values Afrikaans and English Mean values African languages p Partial Eta squared Work–family conflict 0.97 1.08 0.28 0.01 Work–domestic conflict 1.02 0.98 0.68 0 Work–religion conflict 0.74 0.95 0.06 0.02 Family–work conflict 0.75 0.89 0.16 0.01 Private–work conflict 0.5 0.73 0.02 0.03 ANOVA, analysis of variance; Eta squared, describes the ratio of variance explained in the dependent variable by a predictor whilst controlling for other predictors. , p ≤ 0.05 denotes statistically significant. Discussion TABLE 7: ANOVA – differences in work–nonwork conflict based o The general objective was to investigate the prevalence and demographic differences in work–nonwork conflict in a mining environment. The first specific objective related to the overall and specific prevalence of work–nonwork conflict and nonwork–work conflict and their subscales. The results showed that mining employees reported more work–nonwork conflict than nonwork–work conflict. ANOVA, analysis of variance; Eta squared, describes the ratio of variance explained in the dependent variable by a predictor whilst controlling for other predictors. , p ≤ 0.05 denotes statistically significant. TABLE 8: ANOVA – differences in work–nonwork conflict based on marital status. Item Mean values single Mean values married p Partial Eta squared Work–family conflict 1.09 0.98 0.34 0.01 Work–domestic conflict 0.97 1.02 0.62 0 Work–religion conflict 0.99 0.77 0.07 0.2 Family–work conflict 0.96 0.74 0.06 0.02 Private–work conflict 0.85 0.51 0* 0.05 ANOVA, analysis of variance; Eta squared, describes the ratio of variance explained in the dependent variable by a predictor whilst controlling for other predictors. , p ≤ 0.05 denotes statistically significant. One might attribute the prevalent direction of conflict (work– nonwork over nonwork–work) to the mandatory nature of work so that employees tend to prioritise work over family or nonwork matters (Frone et al., 1992; Gutek et al., 1991). Miners also work in severely stressful situations and are exposed to various demands like shift work, unplanned overtime, suffering and death, job pressures and emotional stressors (Singer, 2002). They might feel that they are unable to change any of these factors. Consequently, the interference or conflict from their work domain is more prevalent and supports Hypothesis 1a. ANOVA, analysis of variance; Eta squared, describes the ratio of variance explained in the dependent variable by a predictor whilst controlling for other predictors. , p ≤ 0.05 denotes statistically significant. 2003; Grzywacz & Marks, 2000; Rost & Mostert, 2007). In addition, work–family conflict might be more prevalent than work–domestic conflict because of the family salience that employees have (Frone, Russell & Cooper, 1992; Luchetta, 1995; McClellan & Uys, 2009). 2003; Grzywacz & Marks, 2000; Rost & Mostert, 2007). In addition, work–family conflict might be more prevalent than work–domestic conflict because of the family salience that employees have (Frone, Russell & Cooper, 1992; Luchetta, 1995; McClellan & Uys, 2009). 2003; Grzywacz & Marks, 2000; Rost & Mostert, 2007). Discussion In addition, according to the Employment Equity Act, Act 55 of 1998 (EEA), persons may not be unfairly discriminated against on the grounds of their religions (Venter, 2004). Therefore, employees might feel that they can practise their religions at work without prejudice and this might reduced their conflict. These results, on the prevalence of the specific subscales of work–nonwork conflict and nonwork–work conflict, support hypotheses 1b. These findings seem to be consistent with other empirical studies, which showed that interference from the work domain is more prevalent than interference from the home domain (Bond et al., 1998; Frone, 2003; Geurts & Demerouti, The second specific objective of this study related to the possible differences between work–nonwork conflict and http://www.sajhrm.ac.za doi:10.4102/sajhrm.v9i1.277 Page 11 of 14 languages (Brink & De la Rey, 2001; Schreuder & Theron, 2001). They are usually very traditional and do not always want to adapt to changes in their way of life, thus causing African men to struggle to accept this new lifestyle. For example, it might be difficult for some African men to accept that their wives want to follow careers when their own mothers and grandmothers never worked. This can also explain why there are high levels of private–work conflict. nonwork–work conflict for various demographic groups (gender, age, language, qualifications, marital and parental status) in the mining industry. Overall, the results showed significant differences in work–nonwork conflict based on employees’ language and marital status. Therefore, they support Hypothesis 2 partially. Although the researchers expected differences for gender, age, qualifications and parental status, based on previous studies (Oldfield & Mostert, 2005; Rost & Mostert, 2007; Van Tonder, 2005), they found no statistically significant differences. These differences contradict the findings of Frone et al. (1997), and Pieterse and Mostert (2005). They found no differences in work–home interaction between different language groups, even though they had small and economically homogeneous samples. One could suggest various reasons to explain the lack of differences in the different demographic groups. These could include the homogeneity of the sample (the distribution of demographic groups was not always equal) and the structure of the work environment (all participants were from the same Patterson levels and possibly age groups). Discussion In addition, the possible perceptions of women in the work domain (women are working in a ‘male environment’) and because more fathers are becoming more active in family matters (Daly & Palkovitz, 2004; Heraty et al., 2008; Lingard & Francis, 2005; Root & Wooten, 2008) might explain the lack of differences. Unmarried workers experienced higher levels of private– work conflict than married ones. Grzywacz and Marks (2000) also showed that unmarried people tend to experience less positive work–family interaction. According to Ross, Mirowsky and Goldsteen (1990), unmarried people have higher levels of depression, anxiety and other forms of psychological distress than married people do. A person who lives alone may be isolated from important social and economic ties (Mirowsky & Ross, 1989). These ties may help to create security, belonging and direction. Without them, a person may feel lonely and unprotected. A partner or spouse who helps with domestic tasks, family matters or gives emotional support might provide a buffer against private–work conflict. Although the researchers found no differences for some demographic groups, the differences based on language showed that participants who spoke African languages experienced higher levels of private–work conflict compared to Afrikaans-speaking and English-speaking participants. Unmarried people also have to handle their responsibilities and conflict without the support of spouses and can consequently experience higher levels of private–work conflict. Unmarried people will most probably have family identities that are lower in their salience hierarchy. This means that the saliency unmarried people attach to their family roles is not as high as the saliency married people attach to their family roles. Therefore, unmarried workers’ commitment to their family roles, and the effort they put into them, might be less. A possible reason for this may be that each cultural or language group has different characteristics that influence how it understands conditions and situations. Rost and Mostert (2007) also stated that the cultural differences and backgrounds might influence the way workers perceive the working environment. People who speak African languages are more orientated towards society and more often merge the boundaries between work and home. As a result, they experience more conflict between private and work life (Rost & Mostert, 2007). Alternatively, married employees may occasionally miss career opportunities when they need to put their marital responsibilities ahead of their work. Conclusion In conclusion, these results suggest that there are different work–nonwork conflicts. They also suggest that people experience interference between life roles (family, parental, spousal, religious or spiritual and home or domestic) differently. For example, organisations can offer flexible working hours, childcare facilities or parental leave. Another example of an intervention to address the problem of private–work conflict may be extra family responsibility leave specifically for family and/or community funerals. Workers will experience less stress and less work–nonwork conflict if they know there are programmes to address these issues. The results also showed that different demographic groups differ in their experiences of work–nonwork conflict and nonwork–work conflict. Work–nonwork conflict is more prevalent than nonwork-conflict and some specific work– nonwork role conflicts are more common. For example, work–family conflict is more prevalent than work–domestic conflict and work–domestic conflict is more common than work–religion conflict. These specific interventions will result in the long-term well- being of employees and make organisations more successful. Discussion For example, when employers offer promotions that will cause employees to be away from home from time to time, unmarried people will be more motivated to take the promotion. On the other hand, married employees might reject promotions in order to maintain favourable relationships with their families. For example, Oldfield and Mostert (2005) stated that it is considered disrespectful in the African culture not to attend family and/or community funerals. Therefore, they have more family demands to address. In addition, a higher percentage of African children live with their grandparents (Amoateng, Heaton & Kalule-Sabiti, 2007). They usually live in different provinces to the parents, compared to other race groups. This may mean that the parents miss birthdays, sporting events or parent-teacher meetings. This, in turn, may cause higher levels of conflict between private and work life. Furthermore, married employees share responsibilities and decide how to handle conflict together. Married people also have higher household incomes (Bianchi & Spain, 1986). This may mean that marital quality or spousal support is an important buffer to job-related stress (Barnett, 1996) and can have an effect on levels of private–work conflict. In addition, Grzywacz and Marks (2000) stated that family-related social support correlated positively with home–work interaction. Affirmative action policies have also led to more women and dual-earning families entering the workforce, thereby changing the traditional role of men who speak African http://www.sajhrm.ac.za doi:10.4102/sajhrm.v9i1.277 doi:10.4102/sajhrm.v9i1.277 occupational groups. It could also explain why there are few statistically significant differences between the different demographic groups in work–nonwork role conflict. Implications for managers This study yielded promising and valuable findings that organisations and their managers could use. Some recommendations that emerged from this study include that the mining industry should: Even though the instrument is valid and reliable, it can be a limitation because it is new and should be tested in different environments. • provide support in terms of resources • provide support in terms of resources • effectively manage work–nonwork conflicts that are conducive to helping employees align their work and nonwork domains. Limitations of the study Although this current study yielded promising results, it has limitations. The experiences of nonwork–work conflict differ for different language groups. Participants who speak African languages experience more private–work conflict than family–work conflict. The main limitation is the cross-sectional design of the study. Cross-sectional studies mean that one can make no concrete decisions about the cause-and-effect relationship between the variables (Field, 2005). Suggestions for future research The researchers recommend larger sample sizes and that questionnaires are administered to various occupational groups in South Africa. Training people to use the most effective ways of coping with demands in their work environments may reduce their work–family conflicts. Future studies could also investigate the possible positive interaction between work and other life roles of people. Studies of interference could also be combined with certain antecedents and consequences. The mining industry must be aware that the work domain is interfering with the different roles employees fulfil outside work (parental, spousal, religious or spiritual and home or domestic) and vice versa. Finally, the researchers recommend that longitudinal research designs are used in work–nonwork conflict research because, for many people, work–nonwork conflict undoubtedly fluctuates over time. More specifically, managers and supervisors must be aware that the negative interference between work and family roles are more prevalent than that between work and other life roles. Therefore, managers should focus on interventions and programmes that address this problem specifically. These interventions and programmes will help to prevent crises and to help with the everyday management of employees. 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https://openalex.org/W2755706921	https://www.ijmhr.org/ijpr.5.5/IJPR.2017.197.pdf	English	null	DOES MCCONNELL TAPING OR THE STABILITY THROUGH EXTERNAL ROTATION OF THE FEMUR (SERF) STRAP AFFECT REARFOOT PLANTAR LOADING PATTERNS DURING WALKING IN HEALTHY ADULTS?	International journal of physiotherapy and research/International journal of physiotherapy research	2,017	cc-by-sa	4,980	International Journal of Physiotherapy and Research, Int J Physiother Res 2017, Vol 5(5):2314-19. ISSN 2321-1822 DOI: https://dx.doi.org/10.16965/ijpr.2017.1927 International Journal of Physiotherapy and Research, Int J Physiother Res 2017, Vol 5(5):2314-19. ISSN 2321-1822 DOI: https://dx.doi.org/10.16965/ijpr.2017.1927 Original Research Article DOES MCCONNELL TAPING OR THE STABILITY THROUGH EXTER- NAL ROTATION OF THE FEMUR (SERF) STRAP AFFECT REARFOOT PLANTAR LOADING PATTERNS DURING WALKING IN HEALTHY ADULTS? Somruthai Poomsalood , Karen Hambly School of Sport and Exercise Sciences, University of Kent, Medway Building, Chatham Maritime, Chatham, Kent, United Kingdom, ME4 4AG. ABSTRACT ISSN 2321-1822 2314 INTRODUCTION Quick Response code Access this Article online International Journal of Physiotherapy and Research ISSN 2321- 1822 www.ijmhr.org/ijpr.html DOI: 10.16965/ijpr.2017.197 Received: 00-07-2017 Peer Review: 00-07-2017 Revised: None Accepted: 02-08-2017 Published (O): 11-09-2017 Published (P): 11-10-2017 proposed to immediately reduce pain following application in individuals with patellofemoral pain syndrome (PFPS) [1-3] . Patellar taping has become widely used since the introduction of the original approach by Jenny McConnell in Changes in patellofemoral joint biomechanics have the potential to influence function of the lower extremity. Medial patellar taping is an inexpensive treatment option that has been Quick Response code DOI: 10.16965/ijpr.2017.197 DOI: 10.16965/ijpr.2017.197 Address for correspondence: Somruthai Poomsalood, MSc, School of Sport and Exercise Sciences, University of Kent, Medway Building, Chatham Maritime, Chatham, Kent, United Kingdom, ME4 4AG. Telephone number: +44 (0)1634 88 (8903) E-Mail: sp620@kent.ac.uk Int J Physiother Res 2017;5(5):2314-19. ISSN 2321-1822 ABSTRACT Background: Changes in patellofemoral joint biomechanics have the potential to influence function of the lower extremity. McConnell taping has been proposed to reduce pain in individuals with patellofemoral pain syn- drome (PFPS). It is also believed to improve vastus medialis oblique (VMO) muscle, patellofemoral alignment, and stride length. The stability through external rotation of the femur (SERF) strap has been developed to pull the femur externally to stabilise the patellofemoral joint, in order to reduce patellofemoral pain and improve lower limb kinematics. A lack of literature has examined effects of these two treatment methods on plantar pressures. Therefore, the aim of this study was to investigate the effects of McConnell taping and the SERF strap on rearfoot plantar loading patterns during walking in healthy adults. Materials and Methods: Twenty-three participants (12 males and 11 females, age: 26.52±6.4 years) were ran- domly tested under 3 conditions: 1) no tape, 2) McConnell taping, and 3) SERF strap. Each participant was instructed to walk on a 2 m pressure plate at their own natural pace. Three valid stance phases of the right foot were recorded for each condition. Maximum pressures of medial heel and lateral heel, contact area of medial heel and lateral heel, initial heel contact, foot axis angle, and centre of pressure were collected. Results: There were significant differences of maximum pressures of lateral heel (p = 0.011) with McConnell taping condition and the SERF strap condition demonstrating higher pressures than the no-tape condition (p = 0.042, p = 0.010 respectively). However, significant differences of other variables were not found. Conclusion: The differences of maximum pressures of lateral heel between the conditions could be a clinical role for McConnell taping or SERF strap use in reducing rearfoot pronation in individuals with lower extremity problems especially PFPS. KEY WORDS: Patellofemoral pain syndrome, Plantar pressure, Foot pronation, Medial patellar taping, SERF strap Address for correspondence: Somruthai Poomsalood, MSc, School of Sport and Exercise Sciences, University of Kent, Medway Building, Chatham Maritime, Chatham, Kent, United Kingdom, ME4 4AG. Telephone number: +44 (0)1634 88 (8903) E-Mail: sp620@kent.ac.uk Int J Physiother Res 2017;5(5):2314-19. INTRODUCTION When the femur rotates medially, the compression between the lateral side of the patella and the femur increases, resulting in increased patellofemoral joint stress [13-18]. The forces on the knee during weight- bearing activities are transmitted from the foot to the knee. Hence, loading of the knee and patellofemoral joint can be influenced by force and loading patterns at the foot [17]. By cor- recting excessive rearfoot pronation, external tibial rotation is increased, eliciting a relative medial patellar glide [19]. Higher loading on the medial areas of the plantar surface of the foot during running and other weight-bearing activi- ties have been suggested to be important fac- tors for lower limb injuries [14, 17]. Willems et al. (2006) [20] and Willems et al. (2007) [21] found increased pronation, accompanied with more pressure on the medial side of the rearfoot in healthy people who developed exercise- related lower leg pain during barefoot running. These findings are interesting because a greater medial foot-loading pattern may increase lat- eral force on the patellofemoral joint. Participants and study design: The sample size was calculated using GPower 3.1.9.2 [25] with ANOVA repeated measures, within factors at the power of 0.85, medium effect size (0.3), and a 0.05 alpha level. A minimum sample size was 22 but 23 participants (12 males and 11 females) were recruited in this cross-sectional study. The average age of the participants was 26.5±6.4 years with an average weight of 69.7±14.8 kg and an average height of 171.7±8.9 cm. All participants were healthy in- dividuals aged between 18 and 35 years who had engaged in physical activities for 2 hours or more per week during the previous 12 months before the beginning of the study with no dis- crepancy of 1 cm or greater in lower leg length [26] and normal arch of foot measured by using the plantar arch index (A/B) [27]. Participants had no history of surgery or injury involving the lower leg, ankle or foot in the last 12 and 6 months respectively [20] and no knee pain with activity [28]. The Stability through External Rotation of the Femur ®(SERF) strap (Don Joy Orthopedics Inc, Vista, CA) has been developed to pull the femur into external rotation to stabilise the patellofemoral joint, in order to reduce patellofemoral pain and improve lower limb ki- nematics during dynamic activities [22-24]. INTRODUCTION proposed to immediately reduce pain following application in individuals with patellofemoral pain syndrome (PFPS) [1-3] . Patellar taping has become widely used since the introduction of the original approach by Jenny McConnell in Changes in patellofemoral joint biomechanics have the potential to influence function of the lower extremity. Medial patellar taping is an inexpensive treatment option that has been Int J Physiother Res 2017;5(5):2314-19. ISSN 2321-1822 2314 Somruthai Poomsalood, Karen Hambly. DOES MCCONNELL TAPING OR THE STABILITY THROUGH EXTERNAL ROTATION OF THE FEMUR (SERF) STRAP AFFECT REARFOOT PLANTAR LOADING PATTERNS DURING WALKING IN HEALTHY ADULTS 1984 [4]. It is believed that the reduction of pain following the medial tape application is associ- ated with alterations in patellofemoral joint reaction forces [1]. In addition to pain reduc- tion, medial patellar taping has also been proposed to improve the activity of vastus medialis oblique (VMO) and facilitating strength- ening exercises of quadriceps femoris muscle [5-8]. Patellofemoral alignment [9-11] and stride length [12] during ramp ascent have also been shown to be improved after medial taping ap- plication. However, there is a lack of litera- ture examining the effects of medial patellar taping on plantar pressures during walking. rotation of the femur and tibia from excessive rearfoot pronation, it is proposed that the appli- cation of the SERF strap should alter plantar load- ing patterns by pulling the femur laterally resulting in a reduction in medial tibial rotation and foot pronation. Although McConnell taping has become very popular for patellofemoral pain management, relatively little is known regard- ing its effect on rearfoot pressures [19]. There- fore, the aims of this study were to investigate the effects of both McConnell taping and the SERF strap on rearfoot plantar loading patterns during walking in healthy adults. On the basis of the review and the evidence that is presented, the McConnell taping has been associated with changing of patellofemoral joint reaction force and the SERF strap has been developed to pull the femur laterally so the hypotheses of this study were that both McConnell taping and the SERF strap would alter rearfoot plantar loading patterns during walking. Excessive and/or prolonged rearfoot pronation has been shown to lead to excessive medial rotation of the tibia and the femur in a closed kinetic chain [13]. Int J Physiother Res 2017;5(5):2314-19. ISSN 2321-1822 INTRODUCTION Since it has been suggested that abnormal patellar tracking may be the result of excessive internal Procedures: All participants were randomly tested in a walking activity under 3 conditions: 1) no tape, 2) McConnell taping, and 3) SERF strap. Starting with the participants lying on their back, a rolled-up towel was placed under the 2315 Int J Physiother Res 2017;5(5):2314-19. ISSN 2321-1822 Somruthai Poomsalood, Karen Hambly. DOES MCCONNELL TAPING OR THE STABILITY THROUGH EXTERNAL ROTATION OF THE FEMUR (SERF) STRAP AFFECT REARFOOT PLANTAR LOADING PATTERNS DURING WALKING IN HEALTHY ADULTS deviation (SD). Maximum foot pressures of me- dial heel (MH) and lateral heel (LH) were normalised by the participants’ body weight. The data were checked for normal distributions us- ing Shapiro-Wilk test. Maximum pressures of MH and LH and initial heel contact were not nor- mally distributed so they were analysed by Fried- man test and Wilcoxon Signed Rank test. Con- tact area of MH and LH, foot axis angle, and centre of pressure (COP) were analysed by re- peated measures ANOVA and Tukey’s Post-Hoc test as the data were normally distributed. The data were analysed using SPSS 16.0 (Norusis/ SPSS Inc., Chicago, IL, USA). right knee. Before the tape application, the participant’s knee were shaved using a razor. Dur- ing the tape application, an approximately 15 cm length of hypoallergenic tape (5 cm wide) was first applied on the knee with no tension to avoid allergic reactions [29]. A 10 cm length of zinc oxide tape (3.8 cm wide) as commonly used for McConnell taping was placed over the hypoallergenic tape on the lateral patellar bor- der and the other end of the tape was medially pulled over the patella and secured near the medial femoral condyle [19]. Wrinkles of the skin at the inner aspect of the knee (Figure 1) were used as an indication that the patella had been moved medially. In the SERF strap condition, the SERF strap was applied on the right leg while the participants sat on a chair. The strap was wrapped around the lower limb from the knee to the waist (Figure 2). The tensioning of the strap and the direction of pull was to facilitate lateral rotation of the femur. Fig. 1: Application of McConnell taping (medial patellar taping) on the right knee Fig. 2: Application of the stability through external rotation of the femur (SERF) strap on the right knee. Int J Physiother Res 2017;5(5):2314-19. ISSN 2321-1822 INTRODUCTION The participants gave written informed consent and the study was approved by School of Sport and Exercise Sciences Research Ethics and Ad- visory Groups (SSES REAG), University of Kent at Medway (Ethics reference: 143-2014_2015). Data were collected using a 2 m footscan pres- sure plate (RSscan International, Belgium). Be- fore the testing started, all participants were acquainted with the data collection procedures including walking on the pressure plate (5-6 tri- als with no tape) until they felt comfortable [30]. During the study, each participant was instructed to walk on the pressure plate at their own natu- ral pace while looking straight ahead and not towards the floor [15]. The participants were asked to start walking with their left foot first so that a completed right foot step was re- corded. The participants performed all tests barefoot. The condition order (no tape, McConnell taping, and SERF strap) was assigned randomly between participants to control poten- tial order effects. Three valid stance phases of the right foot were recorded for each condition. A trial was considered valid when the entire right foot was captured. Participants were given 3 minutes to recover between each of the condi- tions [22]. Fig. 2: Application of the stability through external rotation of the femur (SERF) strap on the right knee. Twenty-three participants’ foot pressures (12 men and 11 women) were collected in this study. There were statistically significant differences of maximum pressures of LH (p = 0.019) with McConnell taping condition demonstrating RESULTS RESULTS The demons strap changes plantar load participants supports the ies to evaluate the effect plantar pressure in people Significant differences of sures between the 3 cond in the study. However, th play higher MH maximu McConnell taping, althoug cant. A larger sample size a significant differenc pressures between the 3 Excessive rearfoot pron associated with PFPS d individuals with PFPS contact areas at the med From the results of the McConnell taping and S reduce contact areas of during walking (no tap McConnell taping: 16.57± 16.66±2.82 cm2, p = 0.4 differences were not stat Type II error may have occ sample size in this stud reduction in contact area during walking may be clin McConnell taping and the to reduce foot axis angle d M C ll t i t to evaluate the effects of nd SERF strap on rearfoot terns during walking. The study were that there were es of maximum pressures e no-tape condition and = 0.042) and between the nd the SERF strap condition ndings result in the accep- hesis that both McConnell RF strap have significant plantar loading patterns believed to unload abnor- oft tissue around the , to improve patellar align- ove lower limb mechanics 9]. In the present study, the Connell taping increased s of LH during walking. The th Nyland et al. (2002) [19] nt differences on anterior- ar force location of the fore- ntar force onset with the ng a more forefoot directed The SERF strap has been used to assist lower limb kinematics and support femoral abduction and external rotation. In the present study, maxi- mum pressure of LH while wearing the SERF strap was significantly higher than maximum pressure of LH with the no-tape condition dur- ing walking. A possible reason is that the SERF strap could be pulling the femur and the tibia laterally and, in so doing, reducing pronation of the foot and increasing the lateral loading of the rearfoot. The demonstration that the SERF strap changes plantar loading patterns in healthy participants supports the need for future stud- ies to evaluate the effect of the SERF strap on plantar pressure in people with PFPS. Table 1: Mean differences of maximum pressures of MH and LH, contact area of MH and LH, initial heel contact, foot axis angle, and COP during walking. Int J Physiother Res 2017;5(5):2314-19. ISSN 2321-1822 RESULTS Twenty-three participants’ foot pressures (12 men and 11 women) were collected in this study. There were statistically significant differences of maximum pressures of LH (p = 0.019) with McConnell taping condition demonstrating Twenty-three participants’ foot pressures (12 men and 11 women) were collected in this study. There were statistically significant differences of maximum pressures of LH (p = 0.019) with McConnell taping condition demonstrating Statistical analysis: All rear foot plantar load- ing patterns were expressed as mean±standard Int J Physiother Res 2017;5(5):2314-19. ISSN 2321-1822 2316 Somruthai Poomsalood, Karen Hambly. DOES MCCONNELL TAPING OR THE STABILITY THROUGH EXTERNAL ROTATION OF THE FEMUR (SERF) STRAP AFFECT REARFOOT PLANTAR LOADING PATTERNS DURING WALKING IN HEALTHY ADULTS higher maximum LH pressure than the no-tape condition (p = 0.042) and the SERF strap condition demonstrating higher maximum LH pressure than the no-tape condition (p = 0.010). However, significant differences of maximum pressures of MH, contact area of MH and LH, initial heel contact, foot axis angle, and COP were not found between the treatment condi- tions (Table 1). peak plantar force location and delaying peak plantar force onset following initial ground contact when applying McConnell taping on basketball players while running and dribbling a basketball before the lay-up. The results of this study suggest that McConnell taping has an effect on distal lower extremity function by shifting peak planter force anteriorly towards the forefoot and delaying peak plantar force onset. o tape condition (p 0.010). t differences of maximum ontact area of MH and LH, foot axis angle, and COP ween the treatment condi- es of maximum pressures of MH MH and LH, initial heel contact, P during walking. tape McConnell SERF p-value 3±0.03 0.114±0.04 0.121±0.06 0.068 5±0.03 0.112±0.05* 0.113±0.05* 0.019** 4±2.69 16.57±2.79 16.66±2.82 0.422 4±2.31 14.63±2.49 14.77±2.45 0.273 5±14.95 58.42±13.66 60.48±15.42 0.486 9±8.57 11.77±8.55 12.06±8.50 0.216 0±2.21 -0.95±2.60 -1.36±1.97 0.146 , * Wilcoxon signed rank test No tape) 05 basketball players while r a basketball before the l this study suggest that M an effect on distal lower shifting peak planter force forefoot and delaying pea The SERF strap has been limb kinematics and suppo and external rotation. In th mum pressure of LH wh strap was significantly h pressure of LH with the n ing walking. A possible re strap could be pulling the laterally and, in so doing, the foot and increasing t the rearfoot. Somruthai Poomsalood, Karen Hambly. DOES MCCONNELL TAPING OR THE STABILITY THROUGH EXTERNAL ROTATION OF THE FEMUR (SERF) STRAP AFFECT REARFOOT PLANTAR LOADING PATTERNS DURING WALKING IN HEALTHY ADULTS (2010) [32] evaluated the foot posture in 15 young healthy volunteers and 15 volunteers with PFPS and found that sig- nificantly greater pronated foot posture between subtalar joint neutral and relaxed stance were indicated in PFPS group compared to healthy group. Levinger and Gilleard (2007) [33] measured rearfoot, tibia motion, and ground reaction force during the stance phase of walk- ing in patients with PFPS and healthy individu- als. The results indicated prolonged rearfoot eversion during the stance phase of walking in PFPS participants. Since individuals with PFPS produce higher foot pronation than healthy individuals, McConnell taping and SERF strap could be choices for individuals with PFPS to reduce foot pronation. SERF strap: 12.06±8.50 degrees, p = 0.216) which means that foot abduction and pronation decreased. Barton et al. (2010) [32] evaluated the foot posture in 15 young healthy volunteers and 15 volunteers with PFPS and found that sig- nificantly greater pronated foot posture between subtalar joint neutral and relaxed stance were indicated in PFPS group compared to healthy group. Levinger and Gilleard (2007) [33] measured rearfoot, tibia motion, and ground reaction force during the stance phase of walk- ing in patients with PFPS and healthy individu- als. The results indicated prolonged rearfoot eversion during the stance phase of walking in PFPS participants. Since individuals with PFPS produce higher foot pronation than healthy individuals, McConnell taping and SERF strap could be choices for individuals with PFPS to reduce foot pronation. REFERENCES [1]. Mostamand J, Bader DL, Hudson Z. Reliability test- ing of the patellofemoral joint reaction force (PFJRF) measurement during double-legged squatting in healthy subjects: a pilot study. J Bodyw Mov Ther 2012;16(2):217-23. [1]. Mostamand J, Bader DL, Hudson Z. Reliability test- ing of the patellofemoral joint reaction force (PFJRF) measurement during double-legged squatting in healthy subjects: a pilot study. J Bodyw Mov Ther 2012;16(2):217-23. [2]. Callaghan MJ, Selfe J, McHenry A, Oldham JA. Effects of patellar taping on knee joint proprioception in patients with patellofemoral pain syndrome. Man Ther 2008;13(3):192-9. [3]. Hinman RS, Bennell KL, Crossley KM, McConnell J. Immediate effects of adhesive tape on pain and dis- ability in individuals with knee osteoarthritis. Rheu- matology (Oxford) 2003;42(7):865-9. [4]. Campolo M, Babu J, Dmochowska K, Scariah S, Varughese J. A comparison of two taping techniques (kinesio and mcconnell) and their effect on ante- rior knee pain during functional activities. Int J Sports Phys Ther 2013;8(2):105-10. [5]. Christou EA. Patellar taping increases vastus me- dialis oblique activity in the presence of patellofemoral pain. J Electromyogr Kinesiol 2004;14(4):495-504. ABBREVIATIONS [10]. Somes S, Worrell TW, Corey B, Lngersol CD. Effects of patellar taping on patellar position in the open and closed kinetic chain: a preliminary study. J Sport Rehabil 1997;6(4):299-308. PFPS – Patellofemoral pain syndrome SERF – The Stability through External Rotation of the Femur [11]. Worrell T, Ingersoll CD, Bockrath-Pugliese K, Minis P. Effect of patellar taping and bracing on patellar position as determined by MRI in patients with patellofemoral pain. J Athl Train 1998;33(1):16-20. [12]. Powers CM, Landel R, Sosnick T, Kirby J, Mengel K, Cheney A, et al. The effects of patellar taping on stride characteristics and joint motion in subjects with patellofemoral pain. J Orthop Sports Phys Ther 1997;26(6):286-91. COP – Centre of pressure CONCLUSION [6]. Herrington L, Payton C. Effects of Corrective Taping of the Patella on Patients with Patellofemoral Pain. Physiotherapy 1997;83(11):566-72. The applications of McConnell taping and the SERF strap resulted in significantly higher LH maximum pressures compared to the no-tape condition during walking. These results suggest that there could be a clinical role for McConnell taping or SERF strap use in reducing rearfoot pronation in people with lower extremity prob- lems especially PFPS. Further studies should fo- cus on the effect of the SERF strap and McConnell taping on plantar pressures in pa- tients with PFPS. [7]. Kowall MG, Kolk G, Nuber GW, Cassisi JE, Stern SH. Patellar taping in the treatment of patellofemoral pain. A prospective randomized study. Am J Sports Med 1996;24(1):61-6. [8]. Verma C, Krishnan V. Comparison between Mc Connell patellar taping and conventional physio-therapy treatment in the management of patellofemoral pain syndrome-A randomised controlled trial. JKIMSU 2012;1(2):95-104. [9]. Larsen B, Andreasen E, Urfer A, Mickelson MR, Newhouse KE. Patellar taping: a radiographic ex- amination of the medial glide technique. Am J Sports Med 1995;23(4):465-71. Somruthai Poomsalood, Karen Hambly. DOES MCCONNELL TAPING OR THE STABILITY THROUGH EXTERNAL ROTATION OF THE FEMUR (SERF) STRAP AFFECT REARFOOT PLANTAR LOADING PATTERNS DURING WALKING IN HEALTHY ADULTS Somruthai Poomsalood, Karen Hambly. DOES MCCONNELL TAPING OR THE STABILITY THROUGH EXTERNAL ROTATION OF THE FEMUR (SERF) STRAP AFFECT REARFOOT PLANTAR LOADING PATTERNS DURING WALKING IN HEALTHY ADULTS Somruthai Poomsalood, Karen Hambly. DOES MCCONNELL TAPING OR THE STABILITY THROUGH EXTERNAL ROTATION OF THE FEMUR (SERF) STRAP AFFECT REARFOOT PLANTAR LOADING PATTERNS DURING WALKING IN HEALTHY ADULTS Somruthai Poomsalood, Karen Hambly. DOES MCCONNELL TAPING OR THE STABILITY THROUGH EXTERNAL ROTATION OF THE FEMUR (SERF) STRAP AFFECT REARFOOT PLANTAR LOADING PATTERNS DURING WALKING IN HEALTHY ADULTS SERF strap: 12.06±8.50 degrees, p = 0.216) which means that foot abduction and pronation decreased. Barton et al. (2010) [32] evaluated the foot posture in 15 young healthy volunteers and 15 volunteers with PFPS and found that sig- nificantly greater pronated foot posture between subtalar joint neutral and relaxed stance were indicated in PFPS group compared to healthy group. Levinger and Gilleard (2007) [33] measured rearfoot, tibia motion, and ground reaction force during the stance phase of walk- ing in patients with PFPS and healthy individu- als. The results indicated prolonged rearfoot eversion during the stance phase of walking in PFPS participants. Since individuals with PFPS produce higher foot pronation than healthy individuals, McConnell taping and SERF strap could be choices for individuals with PFPS to reduce foot pronation. SERF strap: 12.06±8.50 degrees, p = 0.216) which means that foot abduction and pronation decreased. Barton et al. (2010) [32] evaluated the foot posture in 15 young healthy volunteers and 15 volunteers with PFPS and found that sig- nificantly greater pronated foot posture between subtalar joint neutral and relaxed stance were indicated in PFPS group compared to healthy group. Levinger and Gilleard (2007) [33] measured rearfoot, tibia motion, and ground reaction force during the stance phase of walk- ing in patients with PFPS and healthy individu- als. The results indicated prolonged rearfoot eversion during the stance phase of walking in PFPS participants. Since individuals with PFPS produce higher foot pronation than healthy individuals, McConnell taping and SERF strap could be choices for individuals with PFPS to reduce foot pronation. SERF strap: 12.06±8.50 degrees, p = 0.216) which means that foot abduction and pronation decreased. Barton et al. DISCUSSION Significant differences of MH maximum pres- sures between the 3 conditions were not found in the study. However, the SERF strap did dis- play higher MH maximum pressure than the McConnell taping, although this was not signifi- cant. A larger sample size may have resulted in a significant difference in MH maximum pressures between the 3 conditions. Excessive rearfoot pronation is frequently associated with PFPS development [31] so individuals with PFPS present with larger contact areas at the medial rearfoot [14, 28]. From the results of the present study, both McConnell taping and SERF strap tended to reduce contact areas of the medial rearfoot during walking (no tape: 16.74±2.69 cm2, McConnell taping: 16.57±2.79 cm2, SERF strap: 16.66±2.82 cm2, p = 0.422). Although these differences were not statistically significant, a Type II error may have occurred due to the small sample size in this study. Nevertheless, the reduction in contact area of the medial rearfoot during walking may be clinically significant. Both McConnell taping and the SERF strap also tended to reduce foot axis angle (no tape: 12.69±8.57 degrees, McConnell taping: 11.77±8.55 degrees, McConnell taping is believed to unload abnor- mally stressed soft tissue around the patellofemoral joint, to improve patellar align- ment, and to improve lower limb mechanics including the foot [29]. In the present study, the application of McConnell taping increased maximum pressures of LH during walking. The results are in line with Nyland et al. (2002) [19] who found significant differences on anterior- posterior peak plantar force location of the fore- foot and peak plantar force onset with the participants displaying a more forefoot directed Int J Physiother Res 2017;5(5):2314-19. ISSN 2321-1822 2317 ACKNOWLEDGEMENTS The authors would like to thank all healthy indi- viduals who participated in this study and staff at the School of Sport and Exercise Sciences, University of Kent for their support. [13]. Powers CM. The influence of abnormal hip me- chanics on knee injury: a biomechanical perspec- tive. J Orthop Sports Phys Ther 2010;40(2):42-51. [14]. Aliberti S, Costa Mde S, Passaro Ade C, Arnone AC, Hirata R, Sacco IC. Influence of patellofemoral pain syndrome on plantar pressure in the foot rollover process during gait. Clinics (Sao Paulo) 2011;66(3):367-72. Int J Physiother Res 2017;5(5):2314-19. ISSN 2321-1822 Funding: School of Sport and Exercise Sciences, University of Kent, United Kingdom Conflicts of interest: None 2318 Int J Physiother Res 2017;5(5):2314-19. ISSN 2321-1822 Somruthai Poomsalood, Karen Hambly. DOES MCCONNELL TAPING OR THE STABILITY THROUGH EXTERNAL ROTATION OF THE FEMUR (SERF) STRAP AFFECT REARFOOT PLANTAR LOADING PATTERNS DURING WALKING IN HEALTHY ADULTS Somruthai Poomsalood, Karen Hambly. DOES MCCONNELL TAPING OR THE STABILITY THROUGH EXTERNAL ROTATION OF THE FEMUR (SERF) STRAP AFFECT REARFOOT PLANTAR LOADING PATTERNS DURING WALKING IN HEALTHY ADULTS Somruthai Poomsalood, Karen Hambly. DOES MCCONNELL TAPING OR THE STABILITY THROUGH EXTERNAL ROTATION OF THE FEMUR (SERF) STRAP AFFECT REARFOOT PLANTAR LOADING PATTERNS DURING WALKING IN HEALTHY ADULTS [15]. Bek N, Kinikli GI, Callaghan MJ, Atay OA. Foot bio- mechanics and initial effects of infrapatellar strap on gait parameters in patients with unilateral patellofemoral pain syndrome. Foot (Edinb) 2011;21(3):114-8. [25]. Faul F, Erdfelder E, Lang AG, Buchner A. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods 2007;39(2):175-91. [26]. Arroyo-Morales M, Olea N, Martinez M, Moreno- Lorenzo C, Diaz-Rodriguez L, Hidalgo-Lozano A. Ef- fects of myofascial release after high-intensity ex- ercise: a randomized clinical trial. J Manipulative Physiol Ther 2008;31(3):217-23. [16]. Boling MC, Padua DA, Marshall SW, Guskiewicz K, Pyne S, Beutler A. A prospective investigation of bio- mechanical risk factors for patellofemoral pain syndrome: the Joint Undertaking to Monitor and Prevent ACL Injury (JUMP-ACL) cohort. Am J Sports Med 2009;37(11):2108-16. [27]. Huang YC, Wang LY, Wang HC, Chang KL, Leong CP. The relationship between the flexible flatfoot and plantar fasciitis: ultrasonographic evaluation. Chang Gung Med J 2004;27(6):443-8. [17]. Rathleff MS, Richter C, Brushoj C, Bencke J, Bandholm T, Holmich P, et al. Increased medial foot loading during drop jump in subjects with patellofemoral pain. Knee Surg Sports Traumatol Arthrosc 2014;22(10):2301-7. [28]. ACKNOWLEDGEMENTS Aliberti S, Costa MS, Passaro AC, Arnone AC, Sacco IC. Medial contact and smaller plantar loads char- acterize individuals with Patellofemoral Pain Syn- drome during stair descent. Phys Ther Sport 2010;11(1):30-4. [18]. Willson JD, Ellis ED, Kernozek TW. Plantar loading characteristics during walking in females with and without patellofemoral pain. J Am Podiatr Med Assoc 2015;105(1):1-7. [29]. Whittingham M, Palmer S, Macmillan F. Effects of taping on pain and function in patellofemoral pain syndrome: a randomized controlled trial. J Orthop Sports Phys Ther 2004;34(9):504-10. [19]. Nyland JA, Ullery LR, Caborn DN. Medial patellar taping changes the peak plantar force location and timing of female basketball players. Gait Posture 2002;15(2):146-52. [30]. Franklyn-Miller A, Bilzon J, Wilson C, McCrory P. Can RSScan footscan((R)) D3D software predict in- jury in a military population following plantar pres- sure assessment? A prospective cohort study. Foot (Edinb) 2014;24(1):6-10. [20]. Willems TM, De Clercq D, Delbaere K, Vanderstraeten G, De Cock A, Witvrouw E. A prospective study of gait related risk factors for exercise-related lower leg pain. Gait Posture 2006;23(1):91-8. [31]. Barton CJ, Levinger P, Webster KE, Menz HB. Walking kinematics in individuals with patellofemoral pain syndrome: a case-control study. Gait Posture 2011;33(2):286-91. [21]. Willems TM, Witvrouw E, De Cock A, De Clercq D. Gait-related risk factors for exercise-related lower- leg pain during shod running. Med Sci Sports Exerc 2007;39(2):330-9. [32]. Barton C, Bonanno D, Levinger P, Menz H. Determin- ing the reliability and sensitivity of foot posture measures in individuals with patellofemoral pain syndrome. J Orthop Sports Phys 2010;40(3):A30. [22].Herrington L. Effect of a SERF strap on pain and knee- valgus angle during unilateral squat and step land- ing in patellofemoral patients. J Sport Rehabil 2013;22(1):27-32. [33]. Levinger P, Gilleard W. Tibia and rearfoot motion and ground reaction forces in subjects with patellofemoral pain syndrome during walking. Gait Posture 2007;25(1):2-8. [23]. Wallace D, Barr C. The effect of hip bracing on gait in patients with medial knee osteoarthritis. Arthri- tis 2012;2012:240376. [24]. Austin AB, Souza RB, Meyer JL, Powers CM. Identifi- cation of abnormal hip motion associated with ac- etabular labral pathology. J Orthop Sports Phys Ther 2008;38(9):558-65. [24]. Austin AB, Souza RB, Meyer JL, Powers CM. Identifi- cation of abnormal hip motion associated with ac- etabular labral pathology. J Orthop Sports Phys Ther 2008;38(9):558-65. 2319
https://openalex.org/W4388234143	https://www.researchsquare.com/article/rs-3512528/latest.pdf	English	null	Causal Relationship Study Between Basal Metabolic Rate and the Risk of Dental Caries: Evidence from Genome-Wide Association Analysis	Research Square (Research Square)	2,023	cc-by	5,672	Conclusion Utilizing two-sample Mendelian Randomization (MR) analysis, our study establishes a genetic causal link between basal metabolic rate and dental caries, thereby offering invaluable insights for the comprehensive, lifelong management of dental caries. Methods This study employed two-sample Mendelian randomization (MR) to investigate the genetic causal relationship between BMR and dental caries. Summary data for genome-wide association studies (GWAS) on BMR were sourced from the UK Biobank, while GWAS summary data for dental caries were obtained from the FinnGen Consortium. Inverse variance weighted (IVW), MR Egger, weighted median, simple mode, and weighted mode were used for MR analysis. Additionally, Cochrane’s Q test was employed to detect heterogeneity in MR estimates.Horizontal pleiotropy was examined using MR-Egger intercept test and MR-PRESSO test, and leave-one-out analysis was conducted to assess the sensitivity of this association. Results: The IVW method indicated that basal metabolic rate is an independent risk factor for dental caries (P = 0.029). Horizontal pleiotropy is unlikely to bias the causal relationship (P > 0.05), and no evidence of heterogeneity was found between genetic variants (P > 0.05). Lastly, leave-one- out analysis revealed the stability and robustness of this association. Research Article Additional Declarations: No competing interests reported. Page 1/14 Page 1/14 Page 1/14 Background Basal metabolic rate (BMR) may be a potential risk factor for dental caries; however, existing observational studies have failed to provide direct evidence of a causal relationship between basal metabolic rate and dental caries. 1. Background Dental caries represents a pervasive, chronic oral condition with global implications. This pathology not only exacerbates existing systemic diseases but also potentially instigates new ones, thereby substantially diminishing the quality of life and imposing a significant economic burden[1]. Despite ongoing efforts, current epidemiological data indicate that the prevention and treatment of dental caries continue to confront formidable obstacles, as evidenced by its persistently high prevalence[2]. A 2017 study published in The Lancet disclosed that dental caries holds the distinction of being the most prevalent and second most incident condition among 328 diseases surveyed, affecting an estimated 2.44 billion individuals globally with persistent dental caries[3]. Furthermore, data from the fourth National Oral Health Survey in mainland China elucidates that the prevalence of primary tooth caries in children aged five has surged to 71.9%, marking a 5.9% escalation compared to a decade earlier[4]. Page 2/14 Concurrently, the prevalence of permanent tooth caries in 12-year-old children has reached 38.5%, registering a 9.6% increment from ten years prior[5]. Accordingly, optimizing disease management strategies constitutes a critical element in the holistic management of dental caries across all age groups and life stages[6]. Metabolism constitutes a cornerstone of biological function. In order to sustain physiological homeostasis, the human body metabolizes nutrients derived from dietary intake for energy production[7]. Fluctuations in body weight result from an incongruity between total caloric consumption and energy expenditure. Notably, basal metabolic rate (BMR) contributes to approximately 70% of daily energy outlay, the efficiency of which can be quantified via the BMR metric[8]. Owing to its pivotal role, basal metabolic rate (BMR) has attracted substantial scholarly scrutiny, in addition to eliciting public interest. A multitude of factors exert influence over BMR, inclusive of body weight, age, gender, and body composition. Lower BMR is correlated with a heightened susceptibility to obesity, insulin resistance, and Type 2 diabetes[9– 11], implicating it as an adverse determinant of health. Conversely, a higher BMR is implicated in an augmented risk of multimorbidity among the elderly population, as well as an increased mortality rate[12]. Furthermore, a higher BMR also escalates the likelihood of cardiovascular diseases[13], serving as a potential harbinger of declining health. Despite the dual implications of basal metabolic rate (BMR) for health outcomes—both advantageous and deleterious—its role in the etiopathogenesis of dental caries remains enigmatic. Observational studies grapple with mitigating the confounding variables and reverse causality that obfuscate these relationships. 1. Background According to prevailing etiological frameworks, dental caries arises from a intricate temporal interplay among host-specific factors, oral microbiota, and fermentable carbohydrates[14]. Fluctuations in basal metabolic rate (BMR) may have ramifications for systemic health, which could subsequently modulate the physiological mechanisms governing oral tissues[15]. This cascade may engender shifts in the oral microbiome, thereby influencing oral health and potentially contributing to the initiation and progression of dental caries. Nevertheless, conclusive evidence elucidating the causal linkage between BMR and dental caries remains scant. Mendelian Randomization (MR) serves as an investigative approach that employs genetic polymorphisms as instrumental variables to probe the causative associations between exposures and pathologies[16]. By harnessing the stochastic assignment of genetic variants during meiosis, MR mitigates the confounding effects that can obfuscate genuine relationships, thus circumventing the pitfalls of reverse causality. This methodology offers the potential to elucidate the causal nexus between basal metabolic rate (BMR) and dental caries. In the current investigation, a two-sample Mendelian Randomization analysis, predicated on extensive Genome-Wide Association Study (GWAS) data, was employed to examine the causal nexus between basal metabolic rate (BMR) and dental caries. This research paradigm aims to ascertain whether BMR constitutes a modifiable risk determinant for dental caries and to furnish empirical evidence to inform prospective prevention and intervention protocols. Addressing this query augments our comprehension of Page 3/14 Page 3/14 the etiological factors underpinning dental caries and yields salient insights for the development and enactment of public health directives. 2. Methods A two-sample Mendelian randomization analysis was employed to probe the causal nexus between basal metabolic rate (BMR) and dental caries within the conceptual framework delineated in Fig. 1. Distinct from single-sample Mendelian randomization, the two-sample methodology leverages data from disparate, independent Genome-Wide Association Studies (GWAS), thereby enhancing the efficiency and robustness of the analysis. A two-sample Mendelian randomization analysis was employed to probe the causal nexus between basal metabolic rate (BMR) and dental caries within the conceptual framework delineated in Fig. 1. Distinct from single-sample Mendelian randomization, the two-sample methodology leverages data from disparate, independent Genome-Wide Association Studies (GWAS), thereby enhancing the efficiency and robustness of the analysis. The GWAS summary statistics for basal metabolic rate are derived from the UK Biobank (2017) dataset, encompassing a total sample size of 331,307 individuals. For dental caries, the summary data are sourced from the FinnGen consortium's 2021 release, which comprises 4,170 cases alongside 195,395 controls. All study participants are of European descent. Figure 1 Directed acyclic graphs for Mendelian randomization. The directed acyclic graph presents the causal relationship among the instrumental variables (single nucleotide polymorphisms, or SNPs), the exposure variable (basal metabolic rate, BMR), the outcome variable (dental caries), and potential confounding factors (G). Mendelian randomization mandates that the selected genetic instrumental variants adhere to three critical assumptions. 2.2 Instrumental Variable Selection To carry out a Mendelian Randomization (MR) analysis, compliance with three fundamental assumptions is indispensable: relevance, independence, and exclusion restriction. Accordingly, all Instrumental Variables (IVs) slated for subsequent analyses are subjected to meticulous vetting. Initially, Single Nucleotide Polymorphisms (SNPs) with a strong correlation to the exposure variable (p-value < 5×10^-8) are selected, while those with an F-statistic below 10 are eliminated. This strategy serves to ensure statistical significance and minimize the potential for weak IV bias.The F-statistic equation used in this study is F = R^2 × (N-2) / (1-R^2), where R^2 is the proportion of variance in the exposure explained by each IV. R^2 = 2×EAF×(1-EAF)×Beta^2, where N represents the number of particiPants, Beta is the allelic effect size and EAF is the effect allele frequency.Secondly, to counter the possibility of bias induced by higher levels of linkage disequilibrium among Instrumental Variables (IVs), clumping algorithms are executed, adhering to criteria r^2 < 0.001 and a physical window of 10,000 kb, to ensure their statistical independence. Thirdly, Phenoscanner V2 is utilized to omit Single Nucleotide Polymorphisms (SNPs) that Page 4/14 Page 4/14 are confounded with other factors or outcomes, thereby confirming that the IVs exert influence on the outcome solely via the exposure. Lastly, harmonization of the exposure and outcome datasets is undertaken to affirm that the effect alleles are congruent, while palindromic SNPs with intermediate allele frequencies are excluded. 2.3 Statistical Analysis Five distinct analytical approaches were utilized to investigate the genetic correlation between basal metabolic rate and dental caries, namely: MR-Egger regression, weighted median, inverse-variance weighted (IVW), simple mode, and weighted mode methods. Among these, the IVW technique presupposes the validity of all single nucleotide polymorphisms (SNPs) included in the analysis, thus potentially providing the most accurate estimations. As a result, IVW serves as the primary analytical framework for this study. Subsequently, statistical power was ascertained using the online platform https://shiny.cnsgenomics.com/mRnd/, which is predicated on the research by Brion Marie-Jo and colleagues, and facilitates the calculation of statistical power in Mendelian Randomization (MR) studies through non-central parameter-based methods.The power calculation requires the total R^2 values for each IV (the variance in the exposure explained by each IV), with the formula for calculating R^2 as previously described. To ensure the robustness of the results, multiple tests were administered, including Cochrane's Q test for the identification of heterogeneity in the associations. Additionally, funnel plots served as a graphical tool to illustrate heterogeneity via symmetry assessment. Pleiotropic effects were scrutinized through both MR-Egger intercept tests and MR-PRESSO (Mendelian Randomization Pleiotropy RESidual Sum and Outlier) global tests. Notably, MR-PRESSO possesses the capacity to discern outliers within the associations, facilitating subsequent estimations post their exclusion. Leave-one-out sensitivity analyses were executed to examine the influence of individual single nucleotide polymorphisms (SNPs) on the observed associations. By systematically omitting one SNP at a time, this methodology ascertains the robustness of the associations by evaluating the effects conferred by the remaining SNPs. All statistical computations were conducted utilizing R software, version 4.3.1, in conjunction with the TwoSampleMR package. A significance level of p < 0.05 was adopted to demarcate statistical relevance. 3.1 Selection of Instrumental Variables Initially, single nucleotide polymorphisms (SNPs) demonstrating a high correlation with the exposure variable(P < 5×10^-8, F-value > 10) were isolated, and their independence was substantiated by enforcing a linkage disequilibrium threshold (r^2 < 0.001, physical window = 10,000 kb). Subsequent to this selection procedure, a cumulative total of 389 SNPs were ascertained as preliminary instrumental variables (IVs). Remarkably, no SNPs were excluded during the vetting process via Phenoscanner V2, thereby corroborating that the chosen IV set was devoid of SNPs correlated with either the outcome or potential confounding variables. Upon harmonization of exposure and outcome data—details of which are delineated in Supplementary Table S1—a total of 21 palindromic single nucleotide polymorphisms Page 5/14 Page 5/14 (SNPs) exhibiting intermediate allele frequencies were excluded. Consequently, 368 SNPs were retained for Mendelian randomization analyses to augment the reliability and validity of the instrumental variables (IVs). 3.2 Mendelian Randomization Analysis Table 1 elucidates the methodologies and outcomes of our Mendelian randomization (MR) investigation, wherein the random-effects Inverse Variance Weighted (IVW) technique serves as the primary analytical modality for probing the genetic linkage between basal metabolic rate and dental caries. The data gleaned from the forest plot substantiates basal metabolic rate as an autonomous risk determinant for dental caries, manifesting a statistically significant, positive causal association (P = 0.029, odds ratio [OR] [95% Confidence Interval (CI)] = 1.208 [1.019–1.4329]) (refer to Supplementary Table S2 for details). Although the ancillary quartet of analytical methodologies failed to uncover a significant association (P > 0.05), they uniformly indicated a positive directional tendency between basal metabolic rate and dental caries. Remarkably, the Mendelian statistical power calculated for the causal linkage between basal metabolic rate and dental caries stands at 1.0. Table 1 The MR results by five methods. Exposure Outcome Method SNP(n) OR OR95CI(Lower-Upper) P-value BMR Caries MR-Egger 368 1.202 0.787–1.835 0.394 BMR Caries Weighted median 368 1.291 0.992–1.679 0.058 BMR Caries IVW 368 1.208 1.019–1.432 0.029 BMR Caries Simple mode 368 1.452 0.688–3.064 0.328 BMR Caries Weighted mode 368 1.261 0.784–2.027 0.339 BMR: basal metabolic rate. IVM: Inverse variance weighted. Table 1: In this table, IVW was regarded as major method, and it showed BMR was an independent risk factor for dental caries. Table 1: In this table, IVW was regarded as major method, and it showed BMR was an independent risk factor for dental caries. To validate the robustness of our research outcomes, we executed a series of data sensitivity analyses. An inaugural application of Cochrane's Q test was undertaken to ascertain heterogeneity; it yielded no evidence to substantiate heterogeneity in the association between basal metabolic rate and dental caries (IVW Q = 359.220,P = 0.604). Subsequent results from both the MR-Egger intercept test (Egger intercept = 8.578773e-05, SE = 0.034, P = 0.98, Fig. 2A) and the MR-PRESSO global test (P = 0.637) corroborated the absence of horizontal pleiotropy in said association. Furthermore, the MR-PRESSO outcomes exhibited no outliers in the course of Mendelian randomization analyses. Visual inspection of the funnel plot confirmed the symmetry of the Single Nucleotide Polymorphisms (SNPs) (Fig. 2B). Additionally, the leave- one-out sensitivity test corroborated that no individual SNP unduly influenced our Mendelian Page 6/14 Page 6/14 randomization outcomes (Supplementary Table S3), thereby affirming the stability and reliability of our findings. 3.2 Mendelian Randomization Analysis The observed consistency and variances in the outcomes underscore the imperative of selecting judicious Mendelian randomization (MR) methodologies for research endeavors. While individual methods may exhibit nuanced differences, our investigation collectively accentuates the putative linkage between basal metabolic rate and dental caries. Consequently, this work serves as a valuable referential framework for ensuing research, advocating for the employment of more intricate analytical techniques to elucidate complex causative relationships. Figure 2 Causal Associations between Basal Metabolic Rate (BMR) and Dental Caries Outcomes: A) The scatter plot elucidates the causal relationship between BMR and dental caries, employing various Mendelian randomization (MR) techniques. Each plotted point symbolizes an instrumental variant. B) The funnel plot demonstrates the symmetry of Single Nucleotide Polymorphisms (SNPs). 4. Discussion Employing two-sample Mendelian Randomization (MR) analysis grounded on expansive genome-wide association study (GWAS) datasets, we probed the causative link between basal metabolic rate (BMR) and dental caries. Our findings indicate that a genetic predisposition for higher BMR is concomitant with an augmented risk of developing dental caries. This observed association between BMR and dental caries posits that targeted interventions to modulate BMR could serve as a viable strategy for attenuating both the prevalence and severity of dental caries, thereby bearing implications for sustained oral health management. The paucity of research exploring the nexus between basal metabolic rate (BMR) and dental caries highlights an exigent imperative for more comprehensive investigations in this domain. A pivotal study, utilizing a nationally representative U.S. sample, ascertained a significant association between severe periodontitis and metabolic syndrome (MetS), thereby catalyzing subsequent rigorous inquiries into the interplay between metabolic dysregulation and oral health[17]. Complementing this, a large cross- sectional study encompassing 13,998 Chinese health screening participants, aged 45 to 65, demonstrated a statistically meaningful correlation between MetS and dental caries—especially salient among those manifesting aberrations in BMR[18]. Several studies have demonstrated a correlation between low body mass index (BMI) and the prevalence of dental caries, whereas others have identified an inverse relationship between overweight conditions and dental caries[19,20]. Utilizing statistical modeling, Chala et al. elucidated a U-shaped correlation between BMI and dental caries, thereby suggesting that dental caries are associated with both underweight and overweight states[21]. Variations in basal metabolic rate (BMR) have been shown to induce systemic physiological changes, including fluctuations in body weight and waist circumference. Existing literature posits that higher BMR is correlated with specific facets of metabolic syndrome[22], thereby intimating a complex and nuanced interrelationship between BMR and dental caries. The present study aims to investigate this intricate The paucity of research exploring the nexus between basal metabolic rate (BMR) and dental caries highlights an exigent imperative for more comprehensive investigations in this domain. A pivotal study, utilizing a nationally representative U.S. sample, ascertained a significant association between severe periodontitis and metabolic syndrome (MetS), thereby catalyzing subsequent rigorous inquiries into the interplay between metabolic dysregulation and oral health[17]. Complementing this, a large cross- sectional study encompassing 13,998 Chinese health screening participants, aged 45 to 65, Page 7/14 Page 7/14 relationship between BMR and dental caries, offering novel insights into the multifaceted interplay between oral health and systemic metabolism. 4. Discussion Nonetheless, observational studies have yet to definitively establish a causal association between basal metabolic rate and dental caries. Basal metabolic rate frequently coexists with comorbid conditions like cardiovascular diseases and diabetes and is often concomitant with various lifestyle factors and genetic predispositions that could impact oral health[13,23]. These concurrent conditions could potentially confound the observed positive correlation between basal metabolic rate and dental caries. Genome-Wide Association Studies (GWAS) serve as a pivotal tool in investigating complex diseases, allowing for the discernment of individual genes or gene clusters that extend beyond the scope of single-gene association studies. These studies also corroborate earlier research while introducing novel avenues for exploration. Leveraging a comprehensive Mendelian randomization analysis within the framework of large-scale GWAS, our investigation furnishes compelling genetic evidence that establishes a causal link between basal metabolic rate and dental caries. This assertion is further buttressed by an Inverse Variance Weighted (IVW) value of 0.029, highlighting the potential causal nexus between the two variables under study. The present study elucidates a positive causal association between basal metabolic rate and the susceptibility to dental caries, as evidenced through Mendelian randomization experiments. Specifically, higher basal metabolic rates are implicated in heightened risks of developing dental caries. This relationship can be explicated by several underlying biological mechanisms. Primarily, variations in basal metabolic rate may modulate the composition of oral microbiota, thereby fostering a milieu conducive to the proliferation of pathogenic microorganisms[24]. Secondly, an augmented basal metabolic rate may correlate with higher levels of energy metabolism, subsequently engendering an increase in acidic metabolic byproducts. These acidic byproducts may translocate to the oral cavity via the bloodstream, thereby perturbing the oral pH equilibrium. Such acidification of the oral microenvironment is conducive to the proliferation of cariogenic bacteria, consequently elevating the risk of dental caries[25]. The present study elucidates a positive causal association between basal metabolic rate and the susceptibility to dental caries, as evidenced through Mendelian randomization experiments. Specifically, higher basal metabolic rates are implicated in heightened risks of developing dental caries. This relationship can be explicated by several underlying biological mechanisms. Primarily, variations in basal metabolic rate may modulate the composition of oral microbiota, thereby fostering a milieu conducive to the proliferation of pathogenic microorganisms[24]. Secondly, an augmented basal metabolic rate may correlate with higher levels of energy metabolism, subsequently engendering an increase in acidic metabolic byproducts. 4. Discussion These acidic byproducts may translocate to the oral cavity via the bloodstream, thereby perturbing the oral pH equilibrium. Such acidification of the oral microenvironment is conducive to the proliferation of cariogenic bacteria, consequently elevating the risk of dental caries[25]. Simultaneously, a heightened basal metabolic rate may induce compositional alterations in saliva, attenuating its antimicrobial efficacy and amplifying the susceptibility to dental caries. Saliva serves a pivotal role in maintaining oral health, being replete with antimicrobial agents, minerals, and proteins that preserve the integrity of oral tissues[26]. Furthermore, basal metabolic rate may be correlated with an individual's dietary predilections and nutritional status. Individuals possessing an higher basal metabolic rate (BMR) may exhibit augmented energy requirements, thereby necessitating a more frequent consumption of food, particularly fermentable carbohydrates[27]. Such dietary patterns could potentiate acidogenesis in the oral cavity, thereby amplifying the risk of dental caries. Additionally, a heightened BMR may be concomitant with escalated immune system activity, engendering excessive inflammatory responses within the oral microenvironment and thus elevating the likelihood of damage to oral tissues and dental caries[28]. Moreover, variations in BMR may be influenced by genetic predispositions, with specific genes concurrently affecting metabolic rates and oral health status. This phenomenon may be evidenced through genetic determinants affecting enamel microarchitecture or the biosynthesis of particular salivary constituents, which in turn influence the prevalence of dental caries[29]. These findings to the proliferation of cariogenic bacteria, consequently elevating the risk of dental caries[25]. Simultaneously, a heightened basal metabolic rate may induce compositional alterations in saliva, attenuating its antimicrobial efficacy and amplifying the susceptibility to dental caries. Saliva serves a pivotal role in maintaining oral health, being replete with antimicrobial agents, minerals, and proteins that preserve the integrity of oral tissues[26]. Furthermore, basal metabolic rate may be correlated with an individual's dietary predilections and nutritional status. Individuals possessing an higher basal metabolic rate (BMR) may exhibit augmented energy requirements, thereby necessitating a more frequent consumption of food, particularly fermentable carbohydrates[27]. Such dietary patterns could potentiate acidogenesis in the oral cavity, thereby amplifying the risk of dental caries. Additionally, a heightened BMR may be concomitant with escalated immune system activity, engendering excessive inflammatory responses within the oral microenvironment and thus elevating the likelihood of damage to oral tissues and dental caries[28]. Moreover, variations in BMR may be influenced by genetic predispositions, with specific genes concurrently affecting metabolic rates and oral health status. 4. Discussion This phenomenon may be evidenced through genetic determinants affecting enamel microarchitecture or the biosynthesis of particular salivary constituents, which in turn influence the prevalence of dental caries[29]. These findings Page 8/14 Page 8/14 elucidate the intricate relationship between basal metabolic rate and oral health, furnishing substantive data for ongoing investigations into the prevention and management of dental caries. The actual association between these variables is likely to be mediated by more intricate biological pathways. Future experimental studies, coupled with molecular-level analyses, will be instrumental in deepening our understanding of the causal linkage between basal metabolic rate and dental caries, as well as in delineating the specific biological mechanisms at play. This study presents multiple strengths. Foremost, to the best of our understanding, this investigation represents the inaugural application of large-scale Genome-Wide Association Studies (GWAS) to elucidate the causal nexus between basal metabolic rate and dental caries. Utilizing a two-sample Mendelian Randomization (MR) methodology allows us to circumvent certain limitations inherent to observational studies, such as reverse causality, confounding variables, and assorted biases. Secondly, all instrumental variables deployed for MR analyses underwent rigorous vetting, thereby guaranteeing the precision of our outcomes. Lastly, we employed a multifaceted array of techniques to assess sensitivity, examine multicollinearity across multiple dimensions, and scrutinize heterogeneity. Collectively, these assessments corroborate the stability and dependability of the association between basal metabolic rate and dental caries. Nonetheless, this investigation is encumbered by several constraints. Firstly, the cohort in the Genome- Wide Association Study (GWAS) is exclusively comprised of individuals of European descent, thus casting doubt on the generalizability of our results to diverse populations and geographic locales. Secondly, while employing MR-Egger intercept and MR-PRESSO global tests to mitigate the multicollinearity of genetic variations, residual confounding variables, including educational attainment, obesity status, and nutritional factors, could still infuse bias into our findings. Lastly, the reliance of our Mendelian Randomization (MR) analysis on foundational GWAS meta-analytical data precludes stratified analyses across different nations, ethnic groups, or age brackets. Consequently, the observed effects of basal metabolic rate in this MR investigation may not be extrapolatable to demographically distinct populations, particularly in terms of ethnicity and age. MR-Egger intercept and MR-PRESSO global tests to mitigate the 5. Conclusion We have substantiated a causal linkage between basal metabolic rate and the incidence of dental caries, implicating shared biological mechanisms in this association. These findings intimate that therapeutic approaches for dental caries ought to prioritize the identification and management of patient-specific risk determinants, alongside the treatment of extant lesions via bespoke, progressively phased interventions. In summation, this manuscript furnishes invaluable perspectives for the efficacious life-course management of dental caries and delineates novel avenues for forthcoming investigations into the nexus between metabolic rate and oral health. Acknowledgments This research has been conducted using the UK Biobank and FinnGen Consortium. The authors thank the participants and coordinators for this unique datase. Funding ：This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author Contributions ：Study conception and design, data acquisition and analysis, drafted the manuscript: Chengxi Li ; interpreted data and made critical revisions of the manuscript: Xue Wang . All authors read and approved the fnal manuscript. Declarations of interest：The authors declare no competing interests. Declarations of interest：The authors declare no competing interests. Declarations of interest：The authors declare no competing interests. Data Availability Statement:The original contributions presented in the study are included in the article/Supplementary Material, and further inquiries can be directed to the corresponding author. Declarations Page 9/14 References 1. Peres MA, Macpherson LMD, Weyant RJ, et al. Oral diseases: a global public health challenge [published correction appears in Lancet. 2019 Sep 21;394(10203):1010]. 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Lazzer S, Bedogni G, Lafortuna CL, et al. Relationship between basal metabolic rate, gender, age, and body composition in 8,780 white obese subjects. Obesity (Silver Spring). 2010;18(1): 71-78. doi:10.1038/oby.2009.162. 28. Mobley C, Marshall TA, Milgrom P, Coldwell SE. The contribution of dietary factors to dental caries and disparities in caries. Acad Pediatr. 2009;9(6):410-414. doi:10.1016/j.acap.2009.09.008 28. Mobley C, Marshall TA, Milgrom P, Coldwell SE. The contribution of dietary factors to dental caries and disparities in caries. Acad Pediatr. 2009;9(6):410-414. doi:10.1016/j.acap.2009.09.008 29. Opal S, Garg S, Jain J, Walia I. Genetic factors affecting dental caries risk. Aust Dent J. 2015; 60(1):2- 11. doi:10.1111/adj.12262. 29. Opal S, Garg S, Jain J, Walia I. Genetic factors affecting dental caries risk. Aust Dent J. 2015; 60(1):2- 11. doi:10.1111/adj.12262. Figures Page 12/14 Figure 1 Directed acyclic graphs for Mendelian randomization. The directed acyclic graph presents the causal relationship among the instrumental variables (single nucleotide polymorphisms, or SNPs), the exposure variable (basal metabolic rate, BMR), the outcome variable (dental caries), and potential confounding factors (G). Mendelian randomization mandates that the selected genetic instrumental variants adhere to three critical assumptions. Figure 2 Causal Associations between Basal Metabolic Rate (BMR) and Dental Caries Outcomes: A) The scatter plot elucidates the causal relationship between BMR and dental caries, employing various Mendelian randomization (MR) techniques. Each plotted point symbolizes an instrumental variant. B) The funnel plot demonstrates the symmetry of Single Nucleotide Polymorphisms (SNPs). Causal Associations between Basal Metabolic Rate (BMR) and Dental Caries Outcomes: A) The scatter plot elucidates the causal relationship between BMR and dental caries, employing various Mendelian randomization (MR) techniques. Each plotted point symbolizes an instrumental variant. B) The funnel plot demonstrates the symmetry of Single Nucleotide Polymorphisms (SNPs). Figure 1 Directed acyclic graphs for Mendelian randomization. The directed acyclic graph presents the causal relationship among the instrumental variables (single nucleotide polymorphisms, or SNPs), the exposure variable (basal metabolic rate, BMR), the outcome variable (dental caries), and potential confounding factors (G). Mendelian randomization mandates that the selected genetic instrumental variants adhere to three critical assumptions. Page 13/14 Page 13/14 Page 13/14 Figure 2 Causal Associations between Basal Metabolic Rate (BMR) and Dental Caries Outcomes: A) The scatter plot elucidates the causal relationship between BMR and dental caries, employing various Mendelian randomization (MR) techniques. Each plotted point symbolizes an instrumental variant. B) The funnel plot demonstrates the symmetry of Single Nucleotide Polymorphisms (SNPs). Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryTableS1.csv SupplementaryTableS2.png SupplementaryTableS3.png Page 14/14
https://openalex.org/W4365483810	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0284354&type=printable	English	null	The effects of short messages encouraging prevention behaviors early in the COVID-19 pandemic	PloS one	2,023	cc-by	8,102	The effects of short messages encouraging prevention behaviors early in the COVID-19 pandemic Sophia L. PinkID1, Michael N. Stagnaro2, James ChuID3, Joseph S. Mernyk1, Jan G. Voelkel1, Robb Willer1 1 Department of Sociology, Stanford University, Palo Alto, California, United States of America, 2 Sloan School of Management, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 3 Department of Sociology, Columbia University, New York, New York, United States of America sopink@wharton.upenn.edu * sopink@wharton.upenn.edu a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 PLOS ONE RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract Effectively addressing public health crises like the COVID-19 pandemic requires persuad- ing the mass public to change their behavior in significant ways. Many efforts to encour- age behavior change–such as public service announcements, social media posts, and billboards–involve short, persuasive appeals, yet the effectiveness of these messages is unclear. Early in the COVID-19 pandemic, we tested whether short messages could increase intentions to comply with public health guidelines. To identify promising mes- sages, we conducted two pretests (n = 1,596) in which participants rated the persuasive- ness of 56 unique messages: 31 based on the persuasion and social influence literatures and 25 drawn from a pool of crowdsourced messages generated by online respondents. The four top-rated messages emphasized: (1) civic responsibility to reciprocate the sacri- fices of health care workers, (2) caring for the elderly and vulnerable, (3) a specific, sym- pathetic victim, and (4) limited health care system capacity. We then conducted three well-powered, pre-registered experiments (total n = 3,719) testing whether these four top-rated messages, and a standard public health message based on language from the CDC, increased intentions to comply with public health guidelines, such as masking in public spaces. In Study 1, we found the four messages and the standard public health message significantly outperformed a null control. In Studies 2 and 3, we compared the effects of persuasive messages to the standard public health message, finding that none consistently out-performed the standard message. This is in line with other research showing minimal persuasive effects of short messages after the very early stages of the pandemic. Across our studies, we found that (1) short messages can increase intentions to comply with public health guidelines, but (2) short messages featuring persuasive tech- niques from the social science literature did not substantially outperform standard public health messages. PLOS ONE PLOS ONE Introduction The coronavirus pandemic has infected more than 500 million people globally, claimed the lives of more than 6 million people [1], severely affected economic growth and employment in nearly every country, and changed everyday life as we know it. To ensure engagement in behaviors that reduce the spread of COVID-19, organizations and governments must generate public health messages that are compelling and persuasive. This is of contemporary impor- tance, as novel variants of SARS-CoV-2 evade vaccines more effectively than previous strains, so behavioral compliance with public health guidelines–such as masking–has remained criti- cally important [2]. In addition, future pandemics and other public health crises will require public health messaging campaigns. A substantial body of work preceding the COVID-19 pandemic has tested whether short messages—such as those found on social media, public service announcements, or billboards —can impact people’s health behaviors [3–5]. Researchers across many disciplines have stud- ied how to create messages that persuade people to engage in behaviors to protect their own and others’ health [6, 7]. Beyond public health, there is a vast, multidisciplinary literature on effective persuasive techniques [8]. Nevertheless, it is unclear the extent to which short mes- sages were persuasive in the context of the early stages of the COVID-19 pandemic in the United States, and if they were, which persuasive strategies were most effective at promoting behavior change. The COVID-19 pandemic differs in many ways from recent public health crises—it has directly impacted every American’s life, required sudden and drastic behavior change, and occurred in a deeply polarized country with historically-low trust in media [9] and government [10]. People have been saturated with often conflicting messaging from a variety of news sources, the federal government, local and state governments, employers, schools, friends, and family. Consistent with this, views of the coronavirus pandemic have polarized along political lines [11], reported skepticism about the seriousness of COVID-19 is high, and conspiracy theories regarding its origins have proliferated [12]. Past research sug- gests that people would be most likely to be persuaded by messaging campaigns early in the pandemic, as messaging campaigns are less effective when issues have been salient for a longer time period [13–15]. OPEN ACCESS Citation: Pink SL, Stagnaro MN, Chu J, Mernyk JS, Voelkel JG, Willer R (2023) The effects of short messages encouraging prevention behaviors early in the COVID-19 pandemic. PLoS ONE 18(4): e0284354. https://doi.org/10.1371/journal. pone.0284354 Editor: Sergio A. Useche, University of Valencia: Universitat de Valencia, SPAIN Received: May 30, 2022 Accepted: March 29, 2023 Published: April 14, 2023 Editor: Sergio A. Useche, University of Valencia: Universitat de Valencia, SPAIN Received: May 30, 2022 Accepted: March 29, 2023 Published: April 14, 2023 Copyright: © 2023 Pink 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 data and analysis files are available in an Open Science Framework database at this link: https://osf.io/eqr4w/. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. 1 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0284354 April 14, 2023 PLOS ONE The effects of short messages encouraging prevention behaviors early in the COVID-19 pandemic Persuasive strategies Many studies have been conducted across the behavioral and social sciences investigating which persuasive strategies used in short messages are most effective in encouraging public health behaviors. This literature spans many disciplines, making it difficult to review compre- hensively. There are several messaging approaches and debates that are common to much of the literature about prevention behaviors. First, some public health research has found that prosocial messages are more likely to change people’s behavior than messaging that appeals to self-interest [23, 24]. However, other work has shown that self-interested messages are just as effective, if not more effective, in con- texts such as vaccination [25]. It is unclear which approach would be more effective in the con- text of the COVID-19 pandemic. Second, past research has found that stories of identifiable victims are more persuasive than statistics about larger groups, although there has been limited research on this approach in the context of public health behaviors [26]. Third, social proof— indicating that many other people are complying with guidelines—has been shown to be effec- tive for prompting behavior change, although much of the behavior change research has been done in the context of energy savings [27]. Fourth, reciprocity messaging—the desire to help people who have helped you—is another approach that has been shown to be effective for per- suasion [28]. Fifth, moral messaging—appealing to an individual’s moral foundations (e.g. harm/care, authority) can convince people to change their mind on polarized issues, such as climate change, universal healthcare, military spending, and other politically-charged topics [29, 30]. This may work in the context of the COVID-19 pandemic given the politicization of the virus, but past work has tested the effect of moral reframing on support for policies and candidates, not public health behaviors. Researchers have also begun to analyze which types of persuasive messaging have been more effective within the context of the COVID-19 pandemic [24, 31–33]. However, we do not know the relative impact of all of these messages, because the studies have been conducted using different outcomes, different contexts, and different sample populations. Short messages as a medium of persuasion Short messages are one of the most important forms of persuasion available for agencies seek- ing to increase public health behavior compliance. There are many media through which per- suasive appeals occur, but short messages, such as short radio scripts, video ads, text messages, or text on billboards, are ubiquitous. Research shows that short messages, which are scalable and consumable even to those with limited time to pay attention, are key for reaching the gen- eral public [16]. A meta-analysis of mass-media public health campaigns involving short mes- sages found that messaging campaigns lead to approximately a 5% increase in the number of people performing the intended behavior [3]. Other reviews have also found that mass media campaigns have small to moderate effects on health behaviors [17], and that short messages can impact behaviors related to physical activity, sexual health, treatment-seeking, and smok- ing cessation [4, 5]. The field of social marketing [18, 19] has studied how to use communication strategies to persuade people to change their public health behaviors. Research on social media campaigns, has found that these campaigns can spread messaging, but there has been limited work on the extent to which social media campaigns change behavior [20, 21]. The effect of social media campaigns may also depend on audience skepticism—past work has found that consumers PLOS ONE \| https://doi.org/10.1371/journal.pone.0284354 April 14, 2023 2 / 15 PLOS ONE The effects of short messages encouraging prevention behaviors early in the COVID-19 pandemic who are more skeptical about advertising like advertising less and are less responsive to adver- tising than those who are less skeptical [22]. However, the context of the COVID-19 pandemic has differed from the contexts of much of this past research in terms of scale, quantity of information people are receiving, and the politicization of the virus. Given this unique context, it is important to test whether short mes- sages impacted prevention behaviors during the COVID-19 pandemic. PLOS ONE \| https://doi.org/10.1371/journal.pone.0284354 April 14, 2023 The present research The studies that follow had three main objectives. Our first objective was to systematically identify messages with high potential for persuasiveness from a large set of possible messages in the early stages of the pandemic. In two pre-tests (total n = 1,596), we tested how persuasive participants found 31 messages based on past research and 25 messages drawn from 600 crowdsourced messages, for a total of 56 messages. Our second objective was to test whether reading a single persuasive message would impact intentions to engage in COVID-19 preventative behaviors, compared to a null control (i.e., seeing no message). In Study 1, which was pre-registered, we examined whether the four top- rated messages from the pre-tests and a standard public health message increased intentions to engage in preventive behaviors. We tested the messages in a between-subjects format, using the difference between people’s reported past behaviors before seeing a message and intended future behaviors after seeing a message as the dependent variable. 3 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0284354 April 14, 2023 PLOS ONE The effects of short messages encouraging prevention behaviors early in the COVID-19 pandemic Our third objective was to test whether specific persuasive strategies can outperform a stan- dard public health message with a description of the virus and recommended prevention behaviors. Using a standard message as an “active control”, we isolated the effects of a particu- lar messaging approach, independent of the effect of receiving a reminder of the virus and desirable responses. In Studies 2 and 3, which were pre-registered, we tested whether any of the top-performing persuasive messages from the pre-tests could out-perform a standard pub- lic health message. Pretests Our goal for the pretests was to identify promising messages to test further. All studies were approved by the Stanford University Institutional Review Board, and we obtained informed consent from all research participants. Method In the first pretest of 598 participants, fielded in March of 2020, we measured perceptions of the persuasiveness of 24 short messages aimed at convincing people to stay home to prevent the spread of COVID-19. In the second pre-test of 998 participants, we used this same method, but added 7 additional messages from literature and 25 messages crowdsourced from partici- pants in the first pre-test. We recruited participants from Mechanical Turk and Lucid (See S1 Appendix for more details about participants and recruitment). In the second pre-test, we also only included participants who indicated that they were not highly compliant with COVID behaviors, in order to find out which messages were most persuasive to those who most needed to change their behavior (see S1 Appendix). The messages were based on a wide range of both general and prior research on persuasion and messaging, and research specific to the current pandemic. All messages were two to four sentences long and advocated for staying home to prevent the spread of coronavirus. See S1 Appendix for message text. The control message stated, “Coronavirus is a respiratory illness that can spread from person to person. The virus is thought to spread mainly between people who are in close contact with one another. You can help prevent the spread of COVID-19. Stay home and avoid contact with others when you must go out.” Each participant rated 10 messages for persuasiveness. The rating was a composite of three items measuring perceived persuasiveness (e.g. “How convincing do you find this reason for social distancing (staying home during the coronavirus pandemic)?”) on a scale from 1 to 9. PLOS ONE \| https://doi.org/10.1371/journal.pone.0284354 April 14, 2023 PLOS ONE PLOS ONE Table 1. Messages that outperformed control in merged analysis of Studies 1 and 2. Message Number Text Effect size, Pre-test 1 Effect size, Pre-test 2 Effect size, Merged Analysis 6: Reciprocity Doctors, nurses, and other health care workers are working around the clock, risking their lives to care for patients with the coronavirus. Working long hours in highly infectious environments, many of them are falling ill. As our health care workers put their lives on the line, we can do our part simply by staying home and limiting physical contact with others 0.60* 0.39 0.54* (0.11) (0.094) (0.067) 15: Resource scarcity Public health officials tell us that we must slow the spread of the coronavirus so numbers of sick people don’t overwhelm our doctors, nurses, and hospitals. If we don’t slow the spread, cases will increase rapidly, suddenly spiking beyond what the health care system can handle. We all can do our part to slow the spread by staying inside and avoiding contact with others when we must go out. If we take action to slow the spread now, we will save lives. 0.59* 0.21 0.46*** (0.11) (0.094) (0.067) 7: Sympathetic victim A few weeks ago, Fiona was a healthy 26-year-old with no medical complications. Then she suddenly came down with a bad cough and a feeling like she could not breathe. She tested positive for COVID-19, and is now hospitalized, receiving oxygen from a ventilator, and fighting for her life. This could be any of us. Please stay home and protect yourself against this virus! 0.35* 0.19 0.32*** (0.11) (0.095) (0.067) 0: Protecting vulnerable The sick, elderly, and immuno-compromised need our help. We all have a choice: If we go out, we risk the lives of others. But by staying home we can protect those most likely to be harmed. Stay home to protect those who are vulnerable! 0.34* 0.17 0.29 * (0.11) (0.095) (0.066) 8: Speed of transmission On average, each person passes on the coronavirus to 2 to 3 people, who then pass it on to more people, and so on. If you break a chain of transmission, you can single-handedly save lives and prevent the suffering of potentially dozens of people. Stay home as much as you can, and break the transmission chain! 0.48* -0.02 0.29*** (0.11) (0.092) (0.067) Beta values are non-standardized. Study 1 In this study, we tested whether the four messages that were rated as most persuasive in the pre-tests would lead people who are not fully compliant to change their behaviors, compared to both a null control (no message) and a standard public health message. PLOS ONE All reported p-values are adjusted using the Holm correction for multiple hypothesis testing. * p < .05; p < .01; * p < .001 Results We used a mixed-effects model to calculate the main effects of message condition on persua- siveness, with a control message as the reference. The Level 1 effect was the message, and the Level 2 effect was the participant. We included controls for gender, age, race, education, and income. The outcome variable was perceived persuasiveness, which was the coefficient value associated with each message, non-standardized. (See S1 Appendix for full equation). We used the Holm method to adjust p-values to account for multiple comparisons. In a merged analysis of the two pre-tests, the five most persuasive messages emphasized responsibility to reciprocate sacrifices of healthcare workers (Message #6, β6 = 0.54, pholm < .001), risk of overwhelming healthcare resources (Message #15, β15 = 0.46, pholm < .001), the story of a sympathetic victim (Message #7, β7 = .32, pholm < .001), protecting the vulnerable (Message #0, β0 = .29, pholm < .001), and the speed of transmission (Message #8, β8 = 0.29, pholm < .001). (Table 1) See Fig 1 for effect sizes of all messages when we pooled the data and included a dummy to indicate the first or second pre-test. 4 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0284354 April 14, 2023 The effects of short messages encouraging prevention behaviors early in the COVID-19 pandemic PLOS ONE \| https://doi.org/10.1371/journal.pone.0284354 April 14, 2023 Method Design and participants. This was a pre-registered, 6-condition, between-subjects study, with 4 treatment messages compared to a null control (no message) and an “active control” message that has a reminder of the virus and recommended behaviors. The active control mes- sage was the same as the control message in the pre-tests. We used both a null control and an active control in order to distinguish the effect of a specific message from the effect of remind- ing people of recommended behaviors. The four treatment messages were the messages that were significantly more persuasive than the control in the merged analysis of both pre-tests, and more persuasive than the control in the second pre-test, which focused on participants which were lower in compliance with public health behaviors (Messages #6, #7, #15, and #0). We made small edits to the messages to bring them up to date with current public health guidelines. We used GPower to calculate that 250 participants per condition would allow us to detect an effect size of Cohen’s d = 0.3 (Power = 80%, alpha = 0.05). Therefore, we targeted a sample size of 1500. 5 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0284354 April 14, 2023 PLOS ONE The effects of short messages encouraging prevention behaviors early in the COVID-19 pandemic Fig 1. Effect size of messages relative to control in merged analysis of Studies 1 and 2 (95% confidence intervals). See S1 Appendix for message number key. Fig 1. Effect size of messages relative to control in merged analysis of Studies 1 and 2 (95% confidence intervals). See S1 Appendix for message number key. essages relative to control in merged analysis of Studies 1 and 2 (95% confidence intervals). See S1 Appendix for message Fig 1. Effect size of messages relative to control in merged analysis of Studies 1 and 2 (95% confidence intervals). See S1 Appendix for message number key. Fig 1. Effect size of messages relative to control in merged analysis of Studies 1 and 2 (95% confidence intervals). See S number key. https://doi.org/10.1371/journal.pone.0284354.g001 https://doi.org/10.1371/journal.pone.0284354.g001 The study was fielded between May 22–23, 2020 using Lucid Marketplace. 5,180 partici- pants began the survey. Based on our pre-registered exclusion criteria, we excluded partici- pants who failed either an attention check at the start of the survey or one towards the end of the survey. PLOS ONE \| https://doi.org/10.1371/journal.pone.0284354 April 14, 2023 Study 2 We tested the persuasive messages again in Study 2. As noted above, one concern from Study 1 was that the behaviors the message called for may have been outdated. Strict stay-at-home orders were being lifted, and most public health officials were advocating for new guidelines, such as wearing face masks. Thus, in Study 2, we kept the same messaging, but modified the recommended behaviors. Method After excluding participants who failed the first attention check, 4,149 remained (See S1 Appendix for attention check item). Only low and mid-level compliers with public health guidelines qualified to complete the survey. That is, we included people who reported engaging in at least two of the six behaviors: washed hands fewer than 10 times per day, cleaned surfaces fewer than 3 times a day, left the house for non-essential reasons at least once in past week, and marked less than 50 on a scale of 1 (Never) - 100 (Extremely often) for how frequently they kept six feet away from others, wore a face mask, and avoided touching their face when they left their home. We also excluded participants who failed the second simple attention check, which was at the end of the survey. There was no differential attrition by condition based on this exclusion. After excluding participants for low attention and high COVID compliance, this left 1,627 par- ticipants. (48% male, median age = 47, 42% conservative, 30% moderate, 28% liberal). For the analyses that did not include the null control message, we also excluded participants who spent less than two seconds reading the message to account for inattentive participants, again follow- ing our pre-registered exclusion criteria. 1,072 participants met the inclusion criterion across the five conditions that were not the null control. Procedure. Change in intended behaviors. After reading the message, participants indi- cated how often, in the next week, they intended to engage in the same six behaviors that they were asked about earlier (staying at home, wearing a mask, etc.). The items were on the same scale as the behavioral questions participants answered before reading the message, but were 6 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0284354 April 14, 2023 PLOS ONE The effects of short messages encouraging prevention behaviors early in the COVID-19 pandemic about intended future behaviors instead of past behaviors (see S1 Appendix). We took the dif- ference between participants’ intended behaviors and past behaviors on each of the six items and normalized each one to be from 0–1. The main dependent variable was the unweighted average of these six normalized difference scores. We chose this variable because we wanted to measure whether the message caused people to take more precautionary behaviors in the future than they did in the past. Results We ran two multiple linear regressions with the average normalized difference score across the six behaviors as the outcome variable. In the first model, the null control was the reference group. The additional covariates included in the model were 1) normalized measures of age, income, and 2) dummy variables for gender, education level and race/ethnicity. Compared to the null control, three messages and the active control message caused an increase in inten- tions to comply with public health guidelines, and the one message that was not significant showed trending effects (Table 2). Next, we conducted our pre-registered analysis. This was the same model, but excluding the null control and instead using the active control as the reference group. None of the four messages caused a significantly greater increase in preventative behaviors intentions than the active control (Table 2). Discussion Participants who read any message advocating for COVID-19 preventive behaviors, including the active control message, reported significantly greater increases in intentions to engage in preventive behaviors than those in the null control condition. This suggests that seeing a reminder of the pandemic and recommended behaviors had an effect. However, no messages were significantly more persuasive than the active control message. This implies that the per- suasive strategies had minimal effects. One possible reason for this is that we conducted the study at a time when many states were relaxing shelter-in-place orders for the first time since the start of the pandemic [34], and instead advocating for additional behaviors like wearing face masks. The messages may have seemed contradictory to “reopening” messaging prevalent at the time. In addition, Message #6 may not have seemed accurate—several participants expressed suspicion regarding whether healthcare systems were heavily impacted at that time. Method Design and participants. Study 2 was a pre-registered, 5-condition study, comparing four treatment messages to an active control message. We adapted the same four messages from Study 1 to call for three new behaviors instead of asking people to stay home: physically distancing from others, wearing a mask, and washing hands after returning home (see S1 Appendix for message texts). 7 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0284354 April 14, 2023 PLOS ONE The effects of short messages encouraging prevention behaviors early in the COVID-19 pandemic Table 2. Regression from Study 1, aggregate difference score as outcome. Variable Model 1 Model 2 Null control as reference group Active control as reference group Active control 0.106 -- (.046) Message 6: Reciprocity towards healthcare workers 0.081. 0.0098 (.046) (.052) Message 7: Sympathetic victim 0.165* 0.080 (.045) (.050) Message 15: Resource scarcity 0.159* 0.0695 (.046) (.053) Message 0: Protecting the vulnerable 0.159* 0.0337 (.047) (.053) Normalized Age -0.0159 -0.024 (.014) (.017) Male -0.142* -.125*** (.027) (.033) Education: HS or less 0.0783* 0.091. (0.038) (.048) Education: Some college 0.033 0.028 (.034) (.042) Education: Postgraduate -.004 -0.025 (.045) (.055) Race: Asian 0.077 0.079 (.060) (.076) Race: Black 0.212* 0.292* (.047) (.063) Race: Hispanic 0.125** 0.159* (.058) (.080) Race: Other 0.142 0.152 (.09) (.10) Income -4.0e-08 9.2e-08 (2.8e-07) (3.52e-07) Adjusted R-squared 0.042 0.041 Number of observations 1627 1072 https://doi.org/10.1371/journal.pone.0284354.t002 https://doi.org/10.1371/journal.pone.0284354.t002 We fielded the study on Mechanical Turk from June 29 –July 1, 2020. At this point, the number of new COVID-19 cases per day had increased beyond the prior peak in April. Using GPower, we found that a sample of 1,530 participants would allow us to detect a small effect size of Cohen’s d = 0.25 (Power = 80%, alpha = 0.05). We calibrated to a smaller effect size than in Study 1 given that there were no significant differences between messages in Study 1. We excluded participants who failed the same attention checks as in Study 1 –the two sim- ple attention checks as well as those who spent less than 2 seconds reading the message. We 8 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0284354 April 14, 2023 PLOS ONE The effects of short messages encouraging prevention behaviors early in the COVID-19 pandemic also excluded participants who were highly compliant with the three behaviors in the message. Results Consistent with our pre-registration, we ran the same regression model as in Study 1 to iden- tify which messages caused a greater increase in intentions to engage in preventive behaviors. The most persuasive message was the reciprocity message (Message #6) (β = 2.47, p = .02), which was significantly more persuasive than the active control. No other messages were more persuasive than the active control (Table 3). Most of the effect of the reciprocity message on intended compliance was driven by individ- uals low in skepticism about COVID-19. Among participants with skepticism rating below 50, those who viewed the reciprocity message (Message #6) (β = 3.49, p = .03) and the message about resource scarcity (Message #15) (β = 3.08, p = 0.05) reported a greater increase in inten- tions to comply compared to the active control message. There was no significant effect of any message among participants with skepticism scores greater than 50. Method Participants reported how often they engaged in the three behaviors when they last left their place of residence, on a scale from 0 (Never) to 100 (Extremely often). We allowed participants to enter the study whose average on these three items was less than or equal to 93 out of 100. We chose this criterion because based on pilot data suggesting 50–60% of respondents would qualify. We slackened these criteria for two reasons. First, we did not see large differences in which messages were persuasive to the full sample in the first pretest and the low-compilers in the second pretest. Second, it would be beneficial to have 100% compliance on these behaviors, since participants can comply with them under almost all circumstances. Anyone who is not highly compliant has room for improvement. 3,058 unique respondents began the survey. 1,666 passed the first simple attention check and the filter for low-compliers. Of those, 1,531 passed the end attention check, and 1,421 par- ticipants spent at least two seconds on the page with the messages (58% male, median age = 34, 43% conservative, 19% moderate, 37% liberal). Note that this average age is much lower than the average age in Study 2, which reflects recruitment from different survey platforms. Procedure. The procedure was similar to that of Study 1, but with new items before the messages to measure respondents’ skepticism toward COVID-19. To measure skepticism of COVID, participants answered two items on scales ranging from 0 (Strongly disagree) to 100 (Strongly agree) measuring how skeptical people were about the severity of COVID-19 (e.g. “Most people are overreacting to COVID-19.”) (α = 0.93). To measure change in intended behaviors, after reading the messages, participants indicated how often they intended to engage in the three behaviors listed in the message the next time they left their place of residence. We calculated the difference between the intended actions and reported past actions for each activ- ity, and the main dependent variable was the average of these three differences (Mean = 5.0, Median = 7.0). Discussion The reciprocity message, which was most persuasive in the pre-tests, was significantly more persuasive than the control in Study 2. However, the same message was not significantly more persuasive than the null control in Study 1. There are several reasons why results may have dif- fered in Studies 1 and 2. First, the messages encouraged behaviors that were more in line with public health recommendations, whereas Study 1 messages may have contradicted other infor- mation respondents were receiving. Second, cases were rising when Study 2 was fielded (26), so it may have seemed more realistic that hospitals could be overwhelmed and there was there- fore a need to reciprocate the actions of healthcare workers. 9 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0284354 April 14, 2023 PLOS ONE The effects of short messages encouraging prevention behaviors early in the COVID-19 pandemic Table 3. Regression from Study 2 and 3, difference score (scale from 0–100) as outcome. Variable Study 2 Study 3 Message 6 2.47 -0.765 (1.08) (0.89) Message 7 0.52 -- (1.07) Message 15 1.76 -- (1.08) Message 0 1.11 -- (1.08) Normalized Age -0.21 2.82 (0.328) (0.5) Gender: Male -1.44* -2.64** (0.69) (0.91) Education: HS or less 2.7* 1.45 (1.25) (1.64) Education: Some college 2.35** 1.00 (0.85) (1.15) Education: Post-graduate 0.66 1.27 (0.94) (1.27) Race: Asian 2.56* 1.41 (1.25) (1.57) Race: Black 0.584 1.12 (1.12) (1.41) Race: Hispanic 0.62 3.96 (1.77) (1.73) Race: Other -1.92 4.84 (3.07) (5.35) Income -6.24e-06 2.5e-6 (7.99e-06) (9.4e-6) Adjusted R-squared 0.01 0.003 Number of observations 1427 568 https://doi.org/10.1371/journal.pone.0284354.t003 Study 3 Given the different effects in Studies 1 and 2, we conducted a partial replication of Study 2, fea- turing only the reciprocity message and the active control. General discussion Across two within-subjects pre-tests where participants rated the persuasiveness of many short messages, several messages were significantly more persuasive than a control message. We then tested four messages in a between-subjects study, and all four led to an increase in inten- tions to comply with public health guidelines, compared to seeing no message. This suggests that seeing any of several different types of reminders of the virus and behaviors can increase intentions to engage in prevention behaviors. However, across three studies, none of the four messages consistently led to an increase in intentions to comply with public health guidelines compared to a standard public health message. We find that changes in the persuasive approach from short messages do not significantly affect participants’ intentions to increase compliance with public health guidelines. A message which emphasized civic responsibility to reciprocate sacrifices made by healthcare workers was rated most persuasive in the pre-tests out of 24 and 56 messages, respectively. It also showed a significant effect in Study 2. Yet it did not show an effect in a pre-registered replication. There are several possible reasons why no messages were consistently more persuasive than a standard public health message. Coronavirus was in the news constantly and impacted almost every American. Small differences between short messages may not be persuasive in such a saturated information environment. It could be that there was a “persuasion window” early on when short messages could change behaviors, but the messages were no longer effec- tive later on due to the overwhelming volume of messaging and the crystallization of views. These null effects are consistent with other research on COVID-19 messaging. One study tested seven different messages designed to reflect moral values and relate these values to COVID-19. None of these messages significantly affected attitudes, intentions, or behaviors compared to a control condition [32]. Other work found that within a month of the COVID- 19 shutdowns, prosocial messages were no longer effective at changing intended behaviors [24]. Beyond public health, research on the effectiveness of political ads, another salient issue, has also found that political ads for Presidential candidates and other salient elections have minimal effects [14, 15, 32, 35], and an experimental prime affected support for Trump’s can- didacy in early 2016 but then had null effects later in the year [13]. Discussion The null effect may be because the earlier evidence for the reciprocity message were false posi- tives. Alternatively, other changes in the world–e.g., crystallization of Americans’ views of the pandemic–may have led this message to no longer increase intentions to engage in preventive behaviors. Method Design and participants. This pre-registered study used a two-condition, between-sub- jects design. Otherwise, the procedure was the same as Study 2. We expected a small effect size, so using G*Power, we found that 620 participants would allow us to detect an effect size of Cohen’s d = 0.2 (80% power, alpha = 0.05). 10 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0284354 April 14, 2023 PLOS ONE The effects of short messages encouraging prevention behaviors early in the COVID-19 pandemic The study was fielded on Mechanical Turk from July 14–15, 2020, when the number of new COVID-19 cases reported per day was increasing rapidly (25). Of the 1,212 participants who began the survey, 648 passed the behavior filter and simple attention item, 597 responded cor- rectly to the end attention check, and 568 spent at least 2 second reading the message (52% male, median age = 34, 46% conservative, 18% moderate, 36% liberal). Results. Using the same multiple linear regression as Study 2, Message #6 was not signifi- cantly more persuasive than the control (Table 3) in a two-tailed test. Though we conducted the study under nearly identical conditions approximately two weeks later, we did not find a significant effect of Message #6 on increase in intentions to comply. PLOS ONE \| https://doi.org/10.1371/journal.pone.0284354 April 14, 2023 General discussion Our results affirm prior research that it is unlikely any single, short, message would persuade people to change their behavior during the COVID-19 pandemic. PLOS ONE \| https://doi.org/10.1371/journal.pone.0284354 April 14, 2023 11 / 15 PLOS ONE The effects of short messages encouraging prevention behaviors early in the COVID-19 pandemic Our exploratory analysis in Study 2 is are broadly consistent with other work on COVID- 19 messaging by showing that skepticism may be a moderator. In Study 2, we found that highly to moderately skeptical individuals were less likely to change their behavior in response to the short messages than individuals who were less skeptical. This is consistent with work [36] which found that people tend to think that individuals with slightly negative attitudes towards a behavior will be most persuadable, but actually, people with slightly positive attitudes towards a behavior are more persuadable. In addition, we found that many messages in the pretests were rated as significantly less per- suasive than a control message in our pre-tests. Messages evoking national identity, collective action, patriotism, analogies to war, religion, and purity were not perceived as highly persua- sive. Likewise, messages highlighting vivid costs of not following guidelines (e.g., killing others, citations by law enforcement) were generally perceived to be less persuasive than the control. It may be that these messages read as direct persuasive efforts for certain subgroups, and prior work on direct vs. peripheral appeals suggest that some people often resist direct appeals [37]. Or, it might be that these messages are perceived as less authoritative. This finding is consistent with research [38] which found that messaging that were similar to the types of messages par- ticipants in the study expected from a health provider (not surprising, casual, or interactive) tended to be more effective at encouraging vaccination than surprising ones. This finding is also consistent with research on the “formality effect” [39], which found that formal messag- ing from government bodies was more effective than informal messaging for influencing behavior. Limitations These experiments were conservative tests of messaging effects because their brevity may have led to minimal impact on participants. Participants read a very short message, without a clearly identified source, one time. The messages might be more persuasive if they were longer, deliv- ered multiple times, or were conveyed via a more compelling medium, such as video. Simi- larly, one-on-one conversations [40] or receiving the message from a trusted source [41, 42] might have been more persuasive than decontextualized messages. It is possible that some of the persuasive strategies from the messages are effective, but only if delivered differently. Another limitation is that we relied on self-reported past and intended future behaviors. Despite our care to include active controls, our dependent variable may still be susceptible to social desirability bias. In addition, the pandemic affected different regions of the country at different points in time, and health guidelines varied substantially by region. Some messages may have been effective in certain regions at certain times, but our studies were insufficiently powered to analyze regional differences. Data curation: Joseph S. Mernyk. Data curation: Joseph S. Mernyk. Data curation: Joseph S. Mernyk. Formal analysis: Sophia L. Pink, Jan G. Voelkel. Formal analysis: Sophia L. Pink, Jan G. Voelkel. Funding acquisition: Robb Willer. Investigation: Robb Willer. Methodology: Sophia L. Pink, Michael N. Stagnaro, James Chu, Joseph S. Mernyk, Jan G. Voelkel, Robb Willer. Supervision: James Chu. Supervision: James Chu. Writing – original draft: Sophia L. Pink. Writing – original draft: Sophia L. Pink. Writing – review & editing: Sophia L. Pink, Michael N. Stagnaro, James Chu, Joseph S. Mer- nyk, Jan G. Voelkel, Robb Willer. Future areas of research This work points towards several possible areas of future research. First, more work could study whether there is a “persuasion window” after a crisis. There may be a short period of time after the start of a crisis, and before opinions crystallize, where messaging is effective at changing behavior. Second, more work could study heterogeneous treatment effects to find out which groups are most impacted by short messages. We find evidence suggesting that skepticism could moderate the relationship between messages and intended behavior change, and further research could study how baseline skepticism affects receptiveness to messages. Third, further work could study the effect of receiving multiple messages, longer messages, or more vivid messages. These may be more impactful than one-time, short messages and 12 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0284354 April 14, 2023 PLOS ONE The effects of short messages encouraging prevention behaviors early in the COVID-19 pandemic therefore be better tests. Fourth, future work could use adaptive experimental designs to effi- ciently test a larger number of messages. Conclusion Across two pre-tests and three experiments conducted throughout five months of the corona- virus pandemic, results on the persuasiveness of different messaging strategies were inconsis- tent. We found that seeing a message with any reminder of the virus and prevention behaviors outperformed seeing no message at all. However, across three studies, none of the top-rated messages consistently outperformed a standard public health message. We generally found that the highest performing message highlighted reciprocity towards healthcare workers, but this effect did not replicate in the final study. Messages that are more frequent, more vivid, or from more credible sources may be more effective at increasing intentions to engage in pre- vention behaviors. These findings suggest that small changes in messaging are largely incapa- ble of affecting intended compliance, even during early stages of the pandemic. Experimental protocols All experimental protocols were approved by the Stanford University Internal Review Board and were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from all participants, and all participants were over the age of 18. Supporting information S1 Appendix. Additional methods and results. (PDF) S1 Appendix. Additional methods and results. (PDF) Author Contributions Conceptualization: Michael N. Stagnaro, James Chu, Joseph S. Mernyk, Jan G. Voelkel, Robb Willer. References 1. Covid map: Coronavirus cases, deaths, vaccinations by country. BBC News. 26 Apr 2022. https://www. bbc.com/news/world-51235105. 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https://openalex.org/W2076950612	https://orca.cardiff.ac.uk/id/eprint/28796/1/Legare%202008.pdf	English	null	Translating shared decision-making into health care clinical practices: Proof of concepts	Implementation science	2,008	cc-by	5,459	BioMed Central BioMed Central Op Study protocol Translating shared decision-making into health care clinical practices: Proof of concepts France Légaré1, Glyn Elwyn2, Martin Fishbein3, Pierre Frémont1, Dominick Frosch4, Marie-Pierre Gagnon1, David A Kenny5, Michel Labrecque1, Dawn Stacey6, Sylvie St-Jacques1 and Trudy van der Weijden7 Address: 1Centre hospitalier universitaire de Québec, Hôpital St-François D'Assise, Unité de recherche évaluative, 10 rue de l'Espinay, Québec, Québec, G1L 3L5, Canada, 2Department of Primary Care and Public Health, School of Medicine, Cardiff University, Neuadd Meirionnydd, Heath Park CF 14 4YS, UK, 3Annenberg School for Communication, University of Pennsylvania, 3620 Walnut Street, Philadelphia, PA 19104, USA, 4UCLA Med-GIM & HSR, BOX 951736, 911 Broxton, Los Angeles, CA 90095-1736, USA, 5Department of Psychology, University of Connecticut, 406 Babbidge Road Unit 1020 Storrs, CT 06269-1020, USA, 6School of Nursing, University of Ottawa, 451 Smyth, Room RGN 3247A Ottawa, ON K1H 8M5, Canada and 7Department of General Practice/School of Public Health and Primary Care Caphri, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands Email: France Légaré - france.legare@mfa.ulaval.ca; Glyn Elwyn - elwyng@cardiff.ac.uk; Martin Fishbein - mfishbein@asc.upenn.edu; Pierre Frémont - pierre.fremont@crchul.ulaval.ca; Dominick Frosch - dfrosch@mednet.ucla.edu; Marie- Pierre Gagnon - david.kenny@uconn.edu; David A Kenny - marie-pierre@fsi.ulaval.ca; Michel Labrecque - michel.labrecque@mfa.ulaval.ca; Dawn Stacey - dstacey@uottawa.ca; Sylvie St-Jacques - sylvie.st-jacques@crsfa.ulaval.ca; Trudy van der Weijden - trudy.vanderweijden@hag.unimass.nl * Corresponding author Received: 3 December 2007 Accepted: 14 January 2008 Received: 3 December 2007 Accepted: 14 January 2008 Published: 14 January 2008 Implementation Science 2008, 3:2 doi:10.1186/1748-5908-3-2 This article is available from: http://www.implementationscience.com/content/3/1/2 © 2008 Légaré et al; licensee BioMed Central Ltd. g ; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Implementation Science Open Access Background situation. These results suggest that health professionals might be selecting, a priori, certain patients for whom they believe that SDM is feasible or functional. This is of some concern because physicians may misjudge patients' desire for active involvement in decision-making [17]. These results highlight the importance of the patient's input for successful implementation of SDM and patient decision aids in clinical practice. Hence, the concomitant evalua- tion of patients' and providers' perception of the decision- making process (dyadic decision-making) remains una- voidable for those interested in a comprehensive under- standing of clinical decision-making [18]. g With the increased emphasis on engagement of patients as partners in their care, there is a need to determine effective ways to involve patients in the process by which health- related decisions are made in clinical settings. The health decision-making process is complex, as it brings together a health professional, considered a scientific content expert, and an individual, considered an expert in his own personal values [1]. It is in this context that there is con- siderable interest today in the process of shared decision- making (SDM) [2]. SDM is defined as a decision-making process jointly shared by patients and their health care provider [3], and is said to be the crux of patient-centered care [4]. It relies on the best evidence about risks and ben- efits associated with all available options (including doing nothing) and on the values and preferences of patients, without excluding those of health professionals [5]. Therefore, it includes the following components: establishing a context in which patients' views about treat- ment options are valued and deemed necessary; reviewing the patient's preferences for role in decision-making; transferring technical information; making sure patients understand this information; helping patients base their preference on the best evidence; eliciting patients' prefer- ences; sharing treatment recommendations; and making explicit the component of uncertainty in the clinical deci- sion-making process [6]. However, a recent systematic review identified 161 conceptual definitions of SDM, thus suggesting that SDM as a concept is still an object of ongo- ing research [7]. In recent years, social cognitive theoretical models have been used to improve our understanding of health care behaviors [19,20] and health care professionals' behav- iors [21-23]. Background At the time this research protocol was pro- posed, most of the studies that had been conducted to improve our understanding of the implementation of SDM in clinical practice had no clear theoretical basis. This is of some concern because it has been acknowledged that more attention needs to be given to the combination of different theories that could help us understand profes- sional behaviours [14,24] and design effective implemen- tation strategies [25]. Nonetheless, when social cognitive theoretical models have been used to study health care- related behaviors, such as communication during a con- sultation or the patient's adherence to medical advice, groups of patients and groups of health professionals have been studied separately as if living in separate worlds. This is a source of concern because 'the right thing to do' may only emerge in the course of the professional's contact with patients or clients [26]. Considering simultaneously both perspectives of the decision-making process is a log- ical approach for conceptualizing SDM and its implemen- tation in clinical practice, as well as for identifying which aspects should be jointly evaluated by patients and their providers [27]. Patient decision aids and decision coaching are effective interventions to support patients to engage in SDM. When compared to usual care, decision aids reduce patients' pas- sivity in the decision-making process, improve patients' knowledge about clinical options, increase realistic expec- tations, reduce decisional conflict and the number of indi- viduals who remain undecided, increase satisfaction with the decision-making process, and increase congruence between patient preferences and clinical options selected [8]. Moreover, notwithstanding the preferred role of patients, active participation of patients in the decision- making process correlates with improved quality of life measured three years after the decision [9]. However, the study of dyads poses specific conceptual as well as methodological issues [28], and thus several chal- lenges in advancing knowledge in this area remain, including the lack of consensus on which aspects should be jointly evaluated by patients and their providers; the absence of standardized measures with established psy- chometric properties; and the failure to take into account the clustering of patients under health providers [29]. In the majority of the studies pertaining to the relationship between a patient and a health care provider, very few have adequately addressed these methodological issues. http://www.implementationscience.com/content/3/1/2 http://www.implementationscience.com/content/3/1/2 http://www.implementationscience.com/content/3/1/2 Implementation Science 2008, 3:2 Page 2 of 6 (page number not for citation purposes) Abstract Background: There is considerable interest today in shared decision-making (SDM), defined as a decision-making process jointly shared by patients and their health care provider. However, the data show that SDM has not been broadly adopted yet. Consequently, the main goal of this proposal is to bring together the resources and the expertise needed to develop an interdisciplinary and international research team on the implementation of SDM in clinical practice using a theory-based dyadic perspective. Methods: Participants include researchers from Canada, US, UK, and Netherlands, representing medicine, nursing, psychology, community health and epidemiology. In order to develop a collaborative research network that takes advantage of the expertise of the team members, the following research activities are planned: 1) establish networking and on-going communication through internet-based forum, conference calls, and a bi-weekly e-bulletin; 2) hold a two- day workshop with two key experts (one in theoretical underpinnings of behavioral change, and a second in dyadic data analysis), and invite all investigators to present their views on the challenges related to the implementation of SDM in clinical practices; 3) conduct a secondary analyses of existing dyadic datasets to ensure that discussion among team members is grounded in empirical data; 4) build capacity with involvement of graduate students in the workshop and online forum; and 5) elaborate a position paper and an international multi-site study protocol. Discussion: This study protocol aims to inform researchers, educators, and clinicians interested in improving their understanding of effective strategies to implement shared decision-making in clinical practice using a theory-based dyadic perspective. Page 1 of 6 (page number not for citation purposes) (page number not for citation purposes) http://www.implementationscience.com/content/3/1/2 3) Perform secondary analyses of existing dyadic datasets 3) Perform secondary analyses of existing dyadic datasets One of the purposes of the workshop is to use existing dyadic datasets to explore the research questions pre- sented above. This will ensure that the team's discussions are grounded in data. A dyadic dataset is defined as a data- set that include data on both members of a dyad that is a pair of two individuals. When only one member of the dyad is measured, the design is termed one-sided. When both members are measured on the same variable, the design is termed two-sided or reciprocal. Three different types of dyadic designs can be identified: 1) standard dyadic design in which each individual is linked to one and only one other individual in the sample; 2) one-with- many design in which one individual is linked to many other individuals; and 3) Social Relation Model design in which each individual is paired with multiple others, and each of these others is also paired with multiple others Other collaborators from the US are the two key invited presenters at the two-day workshop. Together, they will bring extensive expertise on the theoretical underpinnings of implementing behavioral change [44-46], the study of interpersonal influences [28] and the analysis of dyadic data [47]. 1) Foster ongoing communication among members of this international research network Consequently, the main goal of this new international collaboration is to bring together the resources and the expertise needed to develop an interdisciplinary and inter- national research team dedicated to the study of imple- menting SDM in clinical practice using a theory-based dyadic perspective. Its objectives are: 1) to develop a col- laborative research network in this area; 2) to test new strategies to analyze dyadic data and explore the impact of such analysis on the theoretical underpinnings guiding the implementation of SDM in clinical practice; and 3) to define a research agenda and best practices regarding the implementation of SDM in clinical practice. At the outset of the project, using internet-based forum or conference calls hosted by the group at Université Laval, all participants discuss a similar definition of the prob- lems and challenges with implementing SDM, including methodological issues with analysis of dyadic data. Partic- ipants share relevant literature within the group and start to think about how this applies to the identified prob- lems/challenges. Relevant collated documents are used to create a knowledgebase that can be shared through a web- site. An e-journal club dedicated to the critical appraisal of relevant health-related dyadic studies is proposed. It is possible that other issues that are truly unique to SDM will be identified. Ongoing communication is encouraged through a bi-weekly e-bulletin that is sent to all partici- pants. Methods Participants i i p Participants include researchers from Canada, US, UK, and Netherlands representing medicine, nursing, psychol- ogy, community health, and epidemiology. Team mem- bers from Canada contribute to this project by: 1) coordinating the proposed international collaboration; 2) hosting the workshop; 3) providing the necessary moni- toring and on-going support that is required for an inter- national research group to evolve and develop; 4) hosting the internet-based forum and collating relevant material to be shared with the team members; 5) sharing their experience and expertise in the development of a dyadic approach to the implementation of SDM in clinical prac- tice and the data management of large existing datasets; 6) offering a unique perspective to implementing SDM in nursing clinical practice [33,34]; and 7) providing data- sets to be used during the workshop. 2) Provide a workshop A d k h A two-day workshop in Quebec City will be based on the previous work and expertise of participants. Each partici- pant will be asked to prepare a short presentation outlin- ing how they propose to address the following three research questions: 1) What are the most appropriate the- oretical frameworks to assess how health professionals and patients engage in SDM, and what are the most appropriate theoretical frameworks to guide implementa- tion of SDM in clinical practice? 2) What are the most appropriate measures to assess how health professionals and patients concomitantly engage in SDM, and what is the impact of SDM on both? 3) What are the most appro- priate strategies and frameworks to analyze dyadic data that are nested under health professionals? Team members from other countries contribute to this project by: 1) providing extensive expertise in SDM at both the conceptual and methodological levels [6,13,35- 37] and in implementation sciences [38-42]; 2) sharing their experience in producing and conducting clinical tri- als evaluating patient decision aids [43] and implementa- tion strategies [38-42]; and 3) providing datasets to be used during the workshop. http://www.implementationscience.com/content/3/1/2 http://www.implementationscience.com/content/3/1/2 Implementation Science 2008, 3:2 practice because many dyadic processes are at play: patient-health provider, patient-family member, and health provider-health provider, to name only a few. implementation expertise as well as on the extensive clin- ical research background in SDM of the investigators involved in the project, we propose to: 1) Foster ongoing communication among members of this international research network Page 3 of 6 (page number not for citation purposes) Background The expertise, analytical strategies, and theoretical frame- works for studying dyads that have emerged in relation- ship studies [28,30-32] have the potential to enhance the theoretical underpinnings and the research methods for studying the implementation process of SDM in clinical The data show that SDM has not been broadly adopted yet [10-13]. There are major barriers to overcome in the goal of diffusion or dissemination of new approaches in clini- cal practice [14,15]. In a systematic review of barriers and facilitators to implementing SDM and patient decision aids in clinical practice as perceived by health profession- als [16], among 28 unique studies that had collected data from 15 countries, the three most often reported barriers were: time constraints, lack of applicability due to patient characteristics, and lack of applicability due to the clinical Page 2 of 6 (page number not for citation purposes) Page 2 of 6 (page number not for citation purposes) http://www.implementationscience.com/content/3/1/2 http://www.implementationscience.com/content/3/1/2 http://www.implementationscience.com/content/3/1/2 Implementation Science 2008, 3:2 [47]. In this project, secondary analyses of existing dyadic datasets with a reciprocal one-with-many design will be favoured. published. The team will develop an international multi- site study protocol that is based on the work accom- plished during this project. The overarching goal of this study is to support both health professionals and individ- uals to engage in SDM. Based on the strong record of research excellence of all co-investigators and on existing dyadic data sets to be analyzed during the workshop, our research team is firmly convinced that it will attract fund- ing for future projects. Sources of data Previous trials and ongoing pilot trials of SDM in primary care were selected because they include the same measures at both the practitioner and patient levels. FL will provide a data set of 122 primary care providers and their 923 patients [48], and a data set of about 15 family practition- ers and 51 pregnant women facing a decision about pre- natal testing (on-going study). FL and ML will provide a data set of 36 to 60 family practitioners and 450 to 750 patients facing a decision about the use of antibiotics in acute respiratory infections [49]. DF will provide a dataset of about eight general practitioners and 164 adults facing a decision about prostate cancer and colorectal screening (ongoing study). Discussion 'G d h i 'Good theories determine what one can see and discover in nature. Cutting-edge research methods and statistical techniques can influence what scientists see and discover in their data but also inform and change the way in which scientists think theoretically'[47]. This study protocol aims to inform researchers, educators, policy makers, and clinicians interested in designing and/or conducting implementation studies of SDM in clinical practice using a theory-based dyadic perspective. Although some inter- national collaboration has been initiated between some of the team members, there are currently no coordinated efforts to enhance the research capacity at the interna- tional level to create a knowledgebase for implementing SDM in clinical practice using a theory-based dyadic per- spective. Also, to the best of our knowledge, the proposed project does not duplicate other current international research effort in the area of implementation of SDM in clinical practice using a theory-based as well as a dyadic perspective. Therefore, this international collaboration addresses the many challenges associated with the system- atic failure of implementing change in clinical practice by ensuring that future implementation research will take into account that the health professional's position is one that is ultimately 'relationship-centered' [54], and thus needs to be appraised within a dyadic perspective. Data collected and variables assessed Two datasets have data based on the Integrative Model of Behaviour [50] including the following variables: inten- tion, attitude, social norm, and self-efficacy regarding engaging in SDM from the perspective of both providers and patients. The two datasets will be pooled. Based on the Ottawa Decision Support Framework [51,52], three datasets have data from the Decisional Conflict Scale [53], which was administered to both providers and patients after a specific clinical encounter. Based on the existing lit- erature, all constructs that will be used in the planned analyses have excellent psychometrics in both languages (French and English) in both providers and patients. Data analysis Existing datasets will be combined. Proper handling of missing data will be ensured and simple descriptive statis- tics will be computed. Diverse dyadic indexes will then be tested between constructs assessed both in patients and providers [47]. The Actor-Partner-Interdependence Model (APIM) will be used to assess concomitantly in patients and providers the relationship between constructs [31]. The deliverables of this Canadian Institute of Health Research (CIHR) funded research initiative are many: International and interdisciplinary group of researchers dedicated to implementing SDM in clinical practice using a dyadic perspective; conceptual and analytical approaches that will be used in future implementation of SDM in clinical practice studies; secondary data analyses of existing dyadic datasets; capacity building; a position paper defining a research agenda and best practices regarding the implementation of SDM in clinical practice; and a protocol for an international multi-site study on the implementation of SDM clinical practice. 4) Build capacity When and where possible, graduate students of the co- investigators will be invited to join the think tank ses- sions, participate in the e-journal club using the internet- based forum, and attend the two-day workshop. If appro- priate, graduate students will be invited to participate in data synthesis and hypothesis testing activities. 5) Elaborate a position paper and an international multi-site study protocol Research activities In order to develop a collaborative research network that draws upon the extensive theoretical, methodological and Page 3 of 6 (page number not for citation purposes) Page 3 of 6 (page number not for citation purposes) http://www.implementationscience.com/content/3/1/2 5) Elaborate a position paper and an international multi-site study protocol In line with four of the eleven priority research themes of the Institute of Health Services and Policy Research of the Canadian Institute of Health Research, these deliverables are important as they will: Provide innovative insight on how to successfully implement change in clinical practices A position paper defining a research agenda and best prac- tices regarding the implementation of SDM in clinical practice using a theory-based dyadic perspective will be Page 4 of 6 (page number not for citation purposes) http://www.implementationscience.com/content/3/1/2 Implementation Science 2008, 3:2 using a theory-based dyadic perspective; be helpful for future research on new models of collaborative care within the workforce environment related to health care provider-patient dyads; serve as a strategy to increase qual- ity of care and patient safety; and reinforce a patient-cen- tered care approach, one that highly values relationships [55]. Lastly, this international research initiative is in line with research priorities on social interactions of the Cana- dian Institute for Advanced Research whose mission is to 'incubate ideas that go on to revolutionize the interna- tional research community, and change the lives of people all over the world.' In summary, the proposed initiative is of foremost importance since it fosters a critical mass of research activities within an international network on the implementation of SDM in clinical practice and high- lights a new paradigm in implementation science by putting forward a theory-based dyadic perspective. low-up of women with breast cancer. Psychooncology 2006, 15:9-19. low-up of women with breast cancer. Psychooncology 2006, 15:9-19. 10. 10. Makoul G, Arntson P, Schofield T: Health promotion in primary care: physician-patient communication and decision making about prescription medications. Soc Sci Med 1995, 41:1241-1254. 11. McKinstry B: Do patients wish to be involved in decision mak- ing in the consultation? A cross sectional survey with video vignettes. BMJ 2000, 321:867-871. 12. Guimond P, Bunn H, O'Connor AM, Jacobsen MJ, Tait VK, Drake ER, Graham ID, Stacey D, Elmslie T: Validation of a tool to assess health practitioners' decision support and communication skills. Patient Educ Couns 2003, 50:235-245. 13. Elwyn G, Edwards A, Wensing M, Hood K, Atwell C, Grol R: Shared decision making: developing the OPTION scale for measur- ing patient involvement. Qual Saf Health Care 2003, 12:93-99. g p Q f 14. Grol R, Grimshaw J: From best evidence to best practice: effec- tive implementation of change in patients' care. Lancet 2003, 362:1225-1230. 15. Acknowledgements This study is funded by the Canadian Institutes of Health Research (CIHR 2007–2008; DCO190GP grant # 165691-OPD-). It also receives financial support from the Improved Clinical Effectiveness through Behavioral Research Group (ICEBeRG). FL is Tier Two Canada Research Chair in Implementation of Shared Decision-making in Primary Care. MPG is CIHR new investigator. ML is Fonds de la Recherche en Santé du Québec senior clinical scientist. y 20. Légaré F, Godin G, Dodin S, Turcot-Lemay L, Laperrière L: Adher- ence to hormone replacement therapy: a longitudinal study using the theory of planned behaviour. Psychology and Health 2003, 18:351-371. 21. Légaré F, Godin G, Ringa V, Dodin S, Turcot L, Norton J: Variation in the psychosocial determinants of the intention to pre- scribe hormone therapy: a survey of GPs and gynaecologists in France and Quebec. BMC Med Inform Decis Mak 2005, 5:31. 22. Gagnon MP, Godin G, Gagne C, Fortin JP, Lamothe L, Reinharz D, Cloutier A: An adaptation of the theory of interpersonal behaviour to the study of telemedicine adoption by physi- cians. Int J Med Inf 2003, 71:103-115. Authors' contributions 18. 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References Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 6 of 6 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "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 48. Légaré F, O'Connor AM, Graham ID, Wells GA, Tremblay S: Impact of the Ottawa Decision Support Framework on the Agree- ment and the Difference between Patients' and Physicians' Decisional Conflict. Med Decis Making 2006, 26:373-390. Decisional Conflict. Med Decis Making 2006, 26:373- 49. Légaré F, Labrecque M, Leblanc A, Thivierge R, Godin G, Laurier C, Cote L, O'Connor AM, Allain-Boule N, Rousseau J, Tapp S: Does training family physicians in shared decision making pro- mote optimal use of antibiotics for acute respiratory infec- tions? 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https://openalex.org/W2897454901	https://www.shs-conferences.org/10.1051/shsconf/20185001177/pdf	Latin	null	Representation of “Other” in Russian and German Media Discourse	SHS web of conferences	2,018	cc-by	5,237	Alteration strategy The alteration strategy is intended to compare representatives of "our" group with those who opposes us, shares other values, has a different worldview and at the same time reveals similar features [7]. The alteration strategy results in the use of two tactics: contrasting and distancing. In the philosophical sense otherness is the result of a discursive process by which a dominant in-group constructs one or many dominated out-groups by stigmatizing a difference – real or imagined – presented as a negation of identity and thus a motive for potential discrimination. The creation of otherness consists of applying a principle that allows individuals to be classified into two hierarchical groups: them and us. Otherness and identity are two inseparable sides oft he same coin. The Other only exists relative to the Self, and vice versa [4]. The Contrasting tactic. The contrasting tactic involves the thematisation of the comparison and its explicit expression. To implement this tactic, in the media discourse the communicative turn of drawing a parallel and the communicative turn of hierarchy are used. The analysis of the empiric material has shown that as actual "other" in the media discourses of Russia and Germany are perceived representatives of different generations, non-traditional sexual orientation, disabled people, as well as a separate category of citizens who have a migration background. It is worth noting that perception of their otherness has different types of manifestation. otherness is the way of defining an identity in relation to others. The identity is mainly a product of social, cultural, political and other ways of construction through different approaches. The idea of otherness is representative to sociological analyses of how majority and minority identities are built. The asymmetry in power relationships is central to the construction of otherness [5, 6]. Let us look at the examples of developing "otherness" in media discourses of both countries. Turn of drawing a parallel allows to explicate the "otherness" of the described subject. To establish the idea of difference with the goal of self-identification, it is necessary to actualize in the minds of members of the dominant social group certain differentiating schemes, the list of which can vary in different societies. Representation of “Other” in Russian and German Media Discourse Marina Milovanova, and Marina Svinkina* Marina Milovanova, and Marina Svinkina* Volgograd State University, 400062, Universitetskiy Av., 100, Volgograd, Russia Abstract. The article is devoted to the opposition “we vs. they” in Russian and German linguistic cultures. Analysis of linguistic means representing this opposition allows identifying the national and cultural specificity of perception of the identified phenomena in non- relative linguistic cultures. It is shown that the most frequently used strategies in the Russian and German media discourse are alteration and empathization. The most popular tactics and communicative turns of the particular strategies of representation of the “other” were exposed in the both media discourses. The discourse and contrastive analysis of Russian and German newspaper texts is used as the research method. 1 Introduction The analysis of Russian and German press articles over the period 2016 to 2018 has shown that the otherness can be performed by means of alteration strategy, which refers to the human instincts. In modern society mass media texts are matched by a certain way, become one of the main sources of stereotypes [1]. Analysis of the representation of social reality in both Russia and Germany allows us to note the tendency of the media to manipulate micro-differences [2]. When trying to characterize the "other" in a media discourse, the following three aspects are of main importance: 1. "the other" is an outsider; 2. he or she differs from us, but we are not enemies; 3. Relationships with "others" can take different forms: from friendship and love, to polemics and confrontation [3]. https://doi.org/10.1051/shsconf/20185001177 https://doi.org/10.1051/shsconf/20185001177 SHS Web of Conferences 50, 01177 (2018) CILDIAH-2018 © 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/). g Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 ses/by/4.0/). 2 Alteration strategy: comparative analysis in the german and in the russian discourse The language means of implementing the turn of drawing a parallel are: – cross nominative lexical items, which call the phenomena in the framework of one concept (opposition * Corresponding author: svinkina29@gmail.com * Corresponding author: svinkina29@gmail.com The particular turn is characterized by the use of metaphors, symbolizing the top and bottom of society: Как не скатиться в черную пропасть инвалидности / How not to fall into the black abyss of disability; Как челябинка помогла колясочнику подняться с самого дна / How resident of Chelyabinsk helped the wheelchair to rise from the very bottom; die Jungen drängen langsam, aber sicher nach oben. – complex sentences, emphasizing the semantic parallelism, based on comparison: Молодые не могут, старики не хотят / Young can not, old people do not want; Alte wollen Brexit, Junge die EU; Die Welt ist anders – die Jugend auch; Wir Normalen – die Behinderten/ – complex sentences, emphasizing the semantic parallelism, based on comparison: Молодые не могут, старики не хотят / Young can not, old people do not want; Alte wollen Brexit, Junge die EU; Die Welt ist anders – die Jugend auch; Wir Normalen – die Behinderten/ – estimated adjectives with a given semantics of "otherness": Что делать, если в классе оказался «нестандартный» ученик?/ What should be done if there is a "non-standard" student in the classroom?; Besondere Kinder machen besondere Sorgen / Special children disturb special. – estimated adjectives with a given semantics of "otherness": Что делать, если в классе оказался «нестандартный» ученик?/ What should be done if there is a "non-standard" student in the classroom?; Besondere Kinder machen besondere Sorgen / Special children disturb special. – lexical items, indicating different temporal location of objects (antonymicity of time intervals within the opposition "we vs. they"): Разница между моим поколением и поколением моих родителей была намного меньше, чем между мной теперешней и нынешними тридцатилетними / The difference between my generation and the generation of my parents was much less than between me at the present age and current thirty years old people; Die damaligen Kinder verbrachten ihre Freizeit zusammen und spielten gemeinsam auf dem Schulhof. Die Jugend von heute geht auf die Straße, um zu demonstrieren; Damals «Popper» – heute «Hipster» / At that time «Popper» - today «Hipster»; The disparity between "normal" people, personifying the majority, and all others is verbalized with the help of an indirect comparison in favor of representetives of in- group: Здесь они, как и многие другие (но здоровые) дети, занимаются в театральной студии / Here they, like many other (but healthy) children, are engaged in a theater studio; Вход воспрещён. * Corresponding author: svinkina29@gmail.com https://doi.org/10.1051/shsconf/20185001177 SHS Web of Conferences 50, 01177 (2018) CILDIAH-2018 – precedent phenomena, known only within the insiders group: В детстве у нас были диафильмы, уличная игра «Казаки – разбойники», черные металлические двуствольные пистолеты и передача «АБВГДейка»/ In childhood we had filmstrips, street game "Cossacks - robbers", black metal double-barrel pistols and the television programme called "ABVGDeyka"; Was ist ein Trabi auf einem Berg? Ein Rätsel für die heutige Generation / What is a Trabi on a mountain? A puzzle for the present generation. – precedent phenomena, known only within the insiders group: В детстве у нас были диафильмы, уличная игра «Казаки – разбойники», черные металлические двуствольные пистолеты и передача «АБВГДейка»/ In childhood we had filmstrips, street game "Cossacks - robbers", black metal double-barrel pistols and the television programme called "ABVGDeyka"; Was ist ein Trabi auf einem Berg? Ein Rätsel für die heutige Generation / What is a Trabi on a mountain? A puzzle for the present generation. – precedent phenomena, known only within the insiders group: В детстве у нас были диафильмы, уличная игра «Казаки – разбойники», черные металлические двуствольные пистолеты и передача «АБВГДейка»/ In childhood we had filmstrips, street game "Cossacks - robbers", black metal double-barrel pistols and the television programme called "ABVGDeyka"; Was ist ein Trabi auf einem Berg? Ein Rätsel für die heutige Generation / What is a Trabi on a mountain? A puzzle for the present generation. "we vs. they"): молодежь vs. более старшее поколение, поколение наших родителей, cтарики; старшее поколение vs. миллениалы, поколение Z / youth vs. the older generation, the generation of our parents, the elderly; older generation vs. milleniales, generation Z; Die Jungen vs. ältere Generation, Senioren, Silberne Reserven, Rentner; люди с традиционной и нетрадиционной ориентацией, гетеро- и гомосексуалы, сексуальное большинство и меньшинство / people with traditional and non- traditional orientation, heterosexual and homosexual, sexual majority and minority; Hetero -, Bi- und Heterosexuelle, Angehörige sexueller Minderheiten oder Mehrheiten, Menschen mit traditionellen sexuellen Orientierung und LGBT / Hetero -, bi - and heterosexuals, members of sexual minorities or majorities, people with traditional sexual orientation and LGBT people ; здоровые люди vs. инвалиды, лица с ограниченными физическими возможностями, люди с отклонениями, калеки / healthy people vs. disabled people, people with disabilities, cripples; Gesunde oder normale Menschen vs. Behinderten / Healthy or normal people vs. disabled. The name of the comparison object is usually not specific, in the generalized form "we vs. they". * Corresponding author: svinkina29@gmail.com p f p g It should be noted that the use of such time markers is typical only for the representation of "otherness" in the opposition "young vs. old", which logically follows from the most differentiating criterion: age. The opposition on the basis of sexual orientation is the most ambivalent: in the German press, representatives of the LGBT community are viewed as other, while the tonality of the Russian media discourse, ranging from a sharply negative to a moderately critical assessment of "such people," does not allow us to define them as "others" or as "aliens". Let us provide some examples: И объединяются такие нетрадиционалы в ЛГБТ-движения, и начинают на каждом углу выпячивать свои особенности, и требуют к себе внимания/ And such non-traditionalists join in the LGBT movement, and begin to stick out their features at every corner, and demand attention to themselves. And decently nothing comes to mind?; Когда он произнёс эти слова – «Мама, я гей», сердце моё упало. Я даже в ужасе подумала, что лучше бы самое плохое из моих предположений оказалось правдой / When he said these words - "Mom, I'm gay," my heart sank. I even in horror thought that it would be better the worst of my assumptions turned out to be true; Москве не нужны гейские улицы, как в Манчестере / Moscow does not need gay streets, as in Manchester. – personal and possessive pronouns, the use of which is reduced to opposition us vs. they, ours vs. their: Против этой идеи выступает старшее поколение с их "советской ностальгией"; Наши деды выиграли войну, наши отцы строили БАМ, за считанные годы воздвигали города на крайнем севере, покоряли космос. А что оставят после себя ваши? / Against this idea is the older generation with their "Soviet nostalgia"; Our grandfathers won the war, our fathers built the Baikal-Amur Mainline (BAM), in a few years erected cities in the far north, conquered space. And what will leave your relatives behind? Hierarchy turn. When analysing tactics and techniques for constructing the image of the "other" in the national discourse, it is necessary to emphasize the tradition of monologue in the communicative process of otherness representation. The hierarchy turn allows us to identify a higher position of the representatives of the "own" group in relation to "others." All assessments are made only on behalf of in-group. Hybridization turn The hybridization turn is build on the idea of the ethnicity as a social parameter of correlation with others. For modern society, hybrid ethnoidentity is characteristic as a sociocultural construct created, in particular, by the media. Harvard University professor L. Abu-Lughod introduced the term "Halfies" into scientific usage, using it in relation to people whose national or cultural identity is mixed in view of the migration past, other origin or nurture, as a result of long-term studies in another country as well. Under migration, the researcher understands the fundamentally new availability transportation means and communication tools that leads to the formation of so- called "transnational migration" [8]. Citizens of developed countries, inhabitants of megacities, representatives of intercultural diversity in the process of self-identification tend to use a "hyphenated form" by the nomination of ethnicity. The hybridization turn is implemented in the German media discourse by means of the national ethnonyms: Deutsch-Amerikaner (persons having US citizenship, but German origin), Deutsch- Türken (persons of Turkish origin who hold German citizenship or live in the territory of Germany for a long time), Deutsch-Iraner (Iranian, living in Germany for a long time or having the German citizenship); confessional ethnonyms: Euro-Muslime (European Moslem); ethnonyms which refer to the particular institution: Unions-Bürger (EU citizen). The advantage of "hypheneted form" is the opportunity to combine two or more national and cultural identity into one syntagm without belittling the significance of each component. M. Sher points out that the whole essence of European culture is revealed in the productive rapprochement of antagonistic positions and the complementarity of In these headlines, the emphasis is placed on the place of residence of immigrants, which is implemented through the use of a hyphen or the addition to an ethnonym an adjective made up from geographical names. In the German media discourse, attention is drawn to ethnonymic units, which are based on the names of the federal states of Germany: Baden-Württembergerin ist die Zweitschönste «Miss Germany» / inhabitant of Baden- Württemberg is the second most beautiful “Miss Germany”, Ich bin kein Deutscher, ich bin Bayer / I'm not a German, I'm a Bavarian. O.M. Hauer-Tyukarkian explains these linguistic facts of territorial identity with the phenomenon of regionalization of the self-perception of German citizens [10]. After all the value attitude to the past is always contextually conditioned, it is the product of social relations; it differs in increasing importance in the modern society. * Corresponding author: svinkina29@gmail.com Почему детсады не берут в обычные группы детей с инвалидностью? / No entry. Why kindergartens do not admit children with disabilities to the normal groups?; Normale Arbeitsplätze für Menschen mit Behinderung; verbs with subordination semantics: Инвалиды сносили унижения и подчинялись 2 SHS Web of Conferences 50, 01177 (2018) CILDIAH-2018 https://doi.org/10.1051/shsconf/20185001177 differences. A researcher regards this manifestation of cultural identity as a special form of representation of ethnic dialogue in the language [9]. However, the lack of a "hyphenated form" of identity consists in the isolation of a smaller social group through its specific nomination from a large ethnic, national community, and in creating the ground for a discourse of hate in the society on the basis of "own vs. alien". In the Russian media discourse ethnonyms consisting of two or more lexical items are also present, as evidenced by the following headlines: На МКС полетят русский киргиз и американский китаец / The Russian Kirghiz and the American Chinese will fly to the International Space Station (ISS); Русский татарин /Russian Tatar; Российский гастарбайтер прислуживал английской королеве / Russian gastarbeiter served the English queen. Unlike the dominating in the German language, the hyphenated form of writing (bilexeme), the form of the binary substantive- attributive phrase in Russian possesses the transparency of the main and dependent word. In the role of the main constituent of the phrase is usually presented a noun. нелепому закону отделения / Persons with disabilities suffered humiliation and obeyed the absurd law of separation; геям остается смириться с неприязнью / gays have to reconcile disfavor. нелепому закону отделения / Persons with disabilities suffered humiliation and obeyed the absurd law of separation; геям остается смириться с неприязнью / gays have to reconcile disfavor. "Otherness" can be manifested in a carelessly patronizing attitude toward "the other". In the media discourse, predicative elements with an illusory positive evaluation (irony) are used for this purpose: Молодые, амбициозные такие, деятельные! // Чем опасны молодые начальники / Young, such ambitious, active//How dangerous are young chiefs? ; suffixes giving a diminutive-pejorative connotation; dysphemisms: нашлись два... таких своеобразных сапиенса – Владимир Климов из Свердловска и Николай Алексеев из Москвы, которые объявили о проведении гей-парадов в ряде столиц субъектов Северного Кавказа / there were two ... such peculiar sapiens – Vladimir Klimov from Sverdlovsk and Nikolay Alekseev from Moscow who announced gay parades in a number of towns in the North Caucasus. Distancing tactic. * Corresponding author: svinkina29@gmail.com This tactic represents "otherness", expressed in the form of a soft conflict discourse and is indicated by the password "I'm not like you". To verbalize the distancing tactic the hybridization turn and the negation of belongingness turn are used. The ambivalence of personal identity manifests itself not only at the national macro level, but also at the regional micro level of the nomination, i.e. the degree of particularization of ethnic specifics and differences is increasing. For instance: Türkisch-Neuköllnerin hilft den geflüchteten Frauen, Stuttgarter Grieche zahlt Schmiergeld bei Krankenhausbesuch, Berliner Russen gelten mit ihrem Fleiß und Bildungswillen als Migrations- Musterknaben; Московский таджик покорил СМИ своим благородством / The Moscow-based Tajik subjugated the media with his nobility; Философия дагестанской кухни от московский дагестанцев / Philosophy of Dagestanian cuisine from Moscow Dagestanis; Волгоградские таджики отпразднуют «Навруз» / Volgograd Tajiks will celebrate Navruz . Empathization strategy The argumentation tactic is used in media discourses in Russia and Germany; it appeals to the nature of otherness with the intention of explaining what are the specific features of the "other". To verbalize the desired meanings allows the communicative turn of "human story" and the turn of statistics. A similar trend is observed in media discourses of both countries: ̆̆ The "human story" turn is aimed at personalization of information, referring to specific stories from the life of "others" in order to minimise their difference in the eyes of society. – lexical items особый, особенный / besonderer (special) as regards the “others”: Обсуждается вопросы доставки особых людей до шествия, ведь добраться будет не просто, дороги будут перекрыты / The issue of pickup services for special people to the procession are discussed, because all roads will be blocked; В Тотальном диктанте поучаствуют особенные люди / Special people will take part in the national project “Total dictation”; Besondere Menschen–besondere Fahrräder; die Welt der Autisten als ein Kosmos der ganz besonderen Menschen // Geniale Störung: Wir alle sind so anders. – lexical items особый, особенный / besonderer (special) as regards the “others”: Обсуждается вопросы доставки особых людей до шествия, ведь добраться будет не просто, дороги будут перекрыты / The issue of pickup services for special people to the procession are discussed, because all roads will be blocked; В Тотальном диктанте поучаствуют особенные люди / Special people will take part in the national project “Total dictation”; Besondere Menschen–besondere Fahrräder; die Welt der Autisten als ein Kosmos der ganz besonderen Menschen // Geniale Störung: Wir alle sind so anders. Lexical items история (history), исповедь (confession), Bekenntnis (confession) as part of the headlines prepare addressee for the perception of a new information in a form of confession: Исповедь мини- леди, как живут женщины-карлики / Confession of a mini-lady, how do dwarf-women live; eine Geschichte der Behinderten / A story of a disabled person. The fatic intent of authors of such positive stories can also be expressed in identification statements, which – negation “no”: Нет! Мы не ошибка природы: монологи трансгендеров / No! We are not a mistake of nature: transgender monologues; Nein! ich bin nicht behindert. perform the function of an illustrative example: Nina Waskowski. Musikerin mit Behinderung stellt sich vor / Nina Waskowski. Musician with disability introduces herself; appeals in the form of verbs in the imperative mood: Behinderung: Wer darf leben? Hybridization turn "National" is a sensitive issue for Germany, "being a German" for a long time meant to be responsible for the outbreak of World War II. Identified ethnonyms in the Russian and German media discourses are characterized by a high content capacity and expand our perception of the image formation of a person of another ethno-culture, adapted to living conditions in a new homeland. Using bilexems in German allows minimizing 3 3 SHS Web of Conferences 50, 01177 (2018) CILDIAH-2018 https://doi.org/10.1051/shsconf/20185001177 the distance between the titular nation and a new citizen of Germany who has a different ethnic origin. Therefore, within the alteration strategy in the media discourses of both countries, a wide range of language means and stylistic devices is used. The negation of belongingness turn is based on the desire to dissociate from the "label". To verbalize this turn the following language means are used: 3 Empathization strategy: comparative analysis in the german and in the russian discourse – negative particles “не / nicht” and a negative pronoun kein(e): Мы не старики, старики – не мы / We are not old people, old people are not we; Мы не инвалиды, мы как все / We are not disabled, we are like everyone else; Ohne Arme, ohne Beine: ich bin nicht behindert, ich werde behindert! The otherness can be also expressed by means of the empathization strategy, which aims to share someone else's feelings or experiences by imagining what it would be like to be in that person's situation. Denial of stereotyped images witnesses to the unwillingness of "others" to be considered to be a minority: Лучше быть здоровым, чем больным. Лучше быть молодым, чем старым. Лучше быть белым, чем черным. И да: лучше быть гетеросексуальным // Ты гомосексуалист. Это ненормально. It is better to be healthy than sick. It is better to be young than old. Better to be white than black. And yes: it's better to be heterosexual // You're a homosexual. It is not normal. Empathization strategy Petersburg for sighted people. painting. She is keen on photography, mosaic, icon painting. Another consequence of the limited mobility of disabled people is the impossibility to play sports. In the German media discourse we find the following counterargument: Empathic statements are mostly positively marked - their illocutionary force is aimed at supporting the partner in conversation or improving his state of health in order to establish an emotional contact that would allow avoiding difficult communication. Sport ist mein Lieblingsfach. Mein liebstes Spiel ist Merkball, wo man die anderen mit einem Ball abschießen muss // Behinderung: Was man sich wünscht / Sport is my favorite subject. My favorite game is Merkball, where you have to shoot the others with a ball // Disability: What do you desire? In the Russian and German media discourse there are some metaphors that create the image of "others", in particular the disabled people. The first metaphorical model represents a disabled person as a hero. The texts emphasize the courage, striking persistence of "such" people and love of life; Дети с синдромом Дауна стали героями великих картин / Children with Down syndrome became heroes of great paintings; Einbeinige Asha Noppeney läuft Marathon; Tanzen trotz Handicup / Dancing despite handicap. The potential of turn of statistics is that the numbers and statistical data are considered as an objective information that can convince the addressee in one or another opinion. The particular turn is implemented by means of the linguistic units denoting "the other," as well as the numbers. For example: По статистике, различные ограничения по здоровью имеют более 120 тыс. приморцев / According to statistics, various restrictions on health have more than 120 thousand inhabitants of Primorye; По статистике, в России более 12 млн людей с инвалидностью и еще порядка 40 млн маломобильных граждан. Это означает, что практически в каждом московском дворе проживает хотя бы один человек с инвалидностью, просто мы об этом не задумываемся /According to statistics, there are more than 12 million people with disabilities in Russia and about 40 million mobility impaired people. This means that practically every Moscow yard has at least one person with a disability, we just do not think about it. Another metaphorical model delivers the image of a disabled person as a victim. Empathization strategy The pity allows detaching yourself mentally from the situation with the participation of disabled people and putting yourself above "them." Бедные люди: под Волгоградом ограбили семью инвалидов / Poor people: a family of disabled people was robbed near Volgograd; Тяжелая судьба ученого- инвалида / Hard luck of a scientist with a disability; Natalia Wodjanova: Topmodel verteidigt behinderte Schwester / Natalia Wodjanova: Top model defends handicapped sister. Often, disability is also associated with a heavy burden. Here are some examples of the metaphor: a disabled person is a burden to relatives: Ребенок- инвалид – крест для родителей? / Does a disabled child cast a long shadow on its parents?; Behinderung: viele Ehen zerbrechen an Sorgenkindern wie Lotta / Disability: many marriages break up with such troubled children like Lotta. Compassion tactic. This tactic is built on the objectivization of the emotional and evaluative attitude of communicants to each other, which can manifest itself in a person's ability to recognize the emotional state of the "other", in listening, feeling his / her emotional experiences and expressing willingness to provide support. By creating the image of disabled people, passive structures emphasizing the helplessness predominate. pp Expression of empathy turn: It must be noted that these metaphorical models are more characteristic for the Russian media discourse. In the German press there were only several cases, which is largely due to the traditions already established in the society in relation to people with disabilities. – lexical items with an emotionally evaluative content of "empathy", primarily evaluative adjectives: Несмотря на инвалидность он милый, симпатичный парень / Despite his disability, he is a nice, handsome guy; Gregor Demblin ist Manager von zwei Unternehmen. Seinen Rollstuhl hat er dabei zum Werbemittel gemacht / Gregor Demblin is the manager of two companies. He has turned his wheelchair into an advertising medium; – lexical items with an emotionally evaluative content of "empathy", primarily evaluative adjectives: Несмотря на инвалидность он милый, симпатичный парень / Despite his disability, he is a nice, handsome guy; Gregor Demblin ist Manager von zwei Unternehmen. Seinen Rollstuhl hat er dabei zum Werbemittel gemacht / Gregor Demblin is the manager of two companies. He has turned his wheelchair into an advertising medium; Empathization strategy Debattieren Sie mit uns! / Disability: who is allowed to live? Let us debate an issue! The peculiarity of this turn is that narrator's perspective is changing. "Others" deny their belonging to the marginal social group and "insiders" usually prevent the entry of the other in their social network. This concept is confirmed by metaphors with the meaning of duality: Zwischen den Stühlen / fall between two stools; Трансгендер: нахожусь на распутье / Transgender: I am man in the middle; verbs and participles with the intolerance semantics: мисгендерить / to misgender; ausgrenzen / to exclude, sich distanzieren / to distance oneself, abkoppeln / to separate; вынужденные уехать / enforced leaving. The examples below show that the generalizations offered by those who construct the problem can raise doubts, and the stereotypical notions in the society are not always true. One of the image-making elements of a "disabled person" in the Russian media discourse is its limited mobility, which leads to the inability to broaden the horizons. In the following example, as a counterargument, the active activity of a disabled person with a diagnosis of cerebral palsy is described: Галя З. объездила почти всю Европу, побывала во многих городах России, работает, занимается волонтерством и постоянно учится: сейчас заканчивает художественный колледж по специальности художник-прикладник, мастер росписи. Активно увлекается фотографией, мозаикой, иконописью. / Galya Z. traveled throughout Europe, visited many cities of Russia, worked, was a volunteer and is constantly studying. Now she graduates from an art college, specializing in applied art, master of A distinctive feature of the German society is a more tolerant attitude towards people of non-traditional orientation, as evidenced by the use in the media discourse of predominantly neutral or positively colored vocabulary (in the Russian language euphemisms and peyorativs predominate), as well as a large proportion of publications with statements of famous people about their other sexual orientation, where such "otherness" acquires a positive connotation: Ich bin stolz, schwul zu sein. Ich bin stolz, Republikaner zu sein / I am proud to be a gay. I am proud to be a Republican. 4 SHS Web of Conferences 50, 01177 (2018) CILDIAH-2018 https://doi.org/10.1051/shsconf/20185001177 Незрячий гид водит экскурсии по Петербургу для зрячих / Walks in the dark. A blind guide guides excursions around St. Petersburg for sighted people. Незрячий гид водит экскурсии по Петербургу для зрячих / Walks in the dark. A blind guide guides excursions around St. Conclusions – verbs expressing positive feelings and emotions in relation to the addressee: уважать, ценить, восхищаться, achten, respektieren, ehren / to respect, to admire; – verbs expressing positive feelings and emotions in relation to the addressee: уважать, ценить, восхищаться, achten, respektieren, ehren / to respect, to admire; Analysis of the representation of "otherness" in the media discourse of Russia and Germany allowed us to distinguish two main strategies: alteration and empathization. As "others" in the Russian and German media act as representatives of younger / older generation, people who have a non-traditional sexual orientation, disabled people. In the German media discourse, the group of "others" may also include German citizens who have a migration background. The turn of drawing a parallel is common for the Russian and German press, while the hierarchization turn is more presented in the Russian media discourse. Objectivization of "otherness" in the media discourses of both countries takes shape by – sensory verbs: Besondere Menschen erkennt man nicht, man fühlt sie / You can not recognize special people, you feel them; ̆ – understanding operators: Понимаю, что найти работу сейчас нелегко, тем более инвалиду / I understand that it is not easy for a disabled person to find a job, especially; Homosexualität: Keine Angst, wir verstehen Sie / Homosexuality: do not worry, we understand you; – association with "others": imagine yourself in the shoes of people in the wheelchair!; Прогулки в темноте. 5 SHS Web of Conferences 50, 01177 (2018) CILDIAH-2018 https://doi.org/10.1051/shsconf/20185001177 using lexical items within the opposition "we vs. they", comparative constructions, ethnonyms, euphemisms, constructions with a particle not (не /nicht). In the Russian media discourse we have to add to this list metaphors symbolizing the “top” and “bottom” of society and lexical items with suffixes giving a diminutive-pejorative connotation. Within a framework of the emphatization strategy in the media discourses of both countries, we should highlight the "human story" turn, used as a particular manifestation of the argumentation tactic. Such a way of presentation of "otherness" is relatively new for Russian discourse, but reveals a similar narrative rhetoric with German discourse. The empatization strategy can be articulated by means of positively colored lexical items, numerals, metaphors "invalid equals to hero", "disabled person equals to a victim". References 1. E. A. Repina, M. R. Zheltukhina, N. A. Kovaleva, T.G. Popova, C. G. Caselles, XLinguae 11 (2), 557- 585 (2018) 2. B. Dubin, Pro et Contra 1(44), 6-19 (2009) 3. G. Ghazaryan, Foreign Languages at Higher Education 1(16), 14-18 (2014). 4. J. Miller, The Journal of Religion 62 (3), 299-304 (1982) 5. R. Scollon, S.W. Scollon, Intercultural communication: A discourse approach (Wiley, Maiden, 2001). 6. M. V. Milovanova, M. Yu. Svinkina, Alienation as foreignization strategy in international and national media discourse in Russia and Germany, ASSEHR 97 (2017) ̈ 7. H. Turk, Jahrbuch für Internationale Germanistik 1(22), 8-31 (1990) 8. Abu-Lughod, L. Writing against Culture. Washington, Research Press, (1991) 9. M. Scheer, Bindestrich-Deutsche? Mehrfachzugehörigkeit und Beheimatungspraktiken im Alltag. TVV Verlag, Tübingen, (2014) 10. O.M. Hauer-Tjukarkina, Politia 4(71), 67-78, (2013) 6 6
https://openalex.org/W4283210473	https://journal.rniito.org/jour/article/download/1798/pdf	Russian	null	The Editor-in-Chief Appeal to the Issue Nо 2 for 2022 of the Journal “Traumatology and Orthopedics of Russia”	Travmatologiâ i ortopediâ Rossii	2,022	cc-by	742	ОТ РЕДАКтора / EDITORIAL Редакционная статья Редакционная статья https://doi.org/10.17816/2311-2905-1798 Тихилов Р.М. Обращение главного редактора к выходу второго выпуска журнала «Травматология и ортопедия России» за 2022 год. Травматология и ортопедия России. 2022;28(2):5-6. https://doi.org/10.17816/2311-2905-1798. Cite as: Tikhilov R.M. [The Editor-in-Chief Appeal to the Issue Nо 2 for 2022 of the Journal “Traumatology and Orthopedics of Russia”]. Travmatologiya i ortopediya Rossii [Traumatology and Orthopedics of Russia]. 2022;28(2):5-6. https://doi.org/10.17816/2311-2905-1798. (In Russian).  Уважаемые читатели! Представляем очередной выпуск журнала, в ко- тором нашли отражение самые разные проблемы нашей и смежных с ней специальностей. Традиционная для нашего журнала тема эн- допротезирования в этом выпуске представлена работой А.Н. Цеда с соавторами, в которой анали- зируются особенности выполнения этой операции у пациентов с первичной идиопатической тромбо- цитопенией, заключающиеся в более длительной предоперационной подготовке, увеличении интра- операционной кровопотери и объема переливания гемокомпонентов. Свой метод устранения посттравматической контрактуры коленного сустава с использова- нием ортопедического гексапода представили С.А. Рохоев с соавторами. Детская травматология и ортопедия представ- лена в этом выпуске работами наших коллег из Центра им. Г.И. Турнера. П.И. Бортулев с соавто- рами представили результаты сравнительного ис- следования эффективности двух видов остеото- мии при дисплазии тазобедренного сустава — по Salter и по Pemberton. С.Ю. Семенов с соавторами выявили изменения рентгенометрических па- раметров при нестабильности дистального лу- челоктевого сустава у детей, а также их влияние на планирование хирургического вмешательства и прогнозирование восстановления анатомии и функции предплечья. исследований. В этом выпуске эксперименталь- ные исследования представлены двумя статьями. А.В. Петряйкин с соавторами представили резуль- таты сравнения четырех денситометров разной геометрии и фирм-производителей при модели- ровании различного водно-жирового окружения. М.В. Стогов с соавторами провели исследование на животных, показавшее, что применение нагру- зок в зоне имплантации чрескожных имплантатов положительно сказывается на их приживаемости и снижает обсемененность этой области. исследований. В этом выпуске эксперименталь- ные исследования представлены двумя статьями. А.В. Петряйкин с соавторами представили резуль- таты сравнения четырех денситометров разной геометрии и фирм-производителей при модели- ровании различного водно-жирового окружения. М.В. Стогов с соавторами провели исследование на животных, показавшее, что применение нагру- зок в зоне имплантации чрескожных имплантатов положительно сказывается на их приживаемости и снижает обсемененность этой области. В разделе «Дискуссии» представлены различ- ные взгляды и подходы к выполнению экстренных и плановых операций остеосинтеза пациентам с переломами длинных костей в крупных городах Российской Федерации. Приглашаем наших коллег присоединиться к обсуждению этой актуальной проблемы. Как всегда, мы отобрали наиболее интерес- ные и редкие клинические наблюдения, а также представляем вашему вниманию интересный об- зор литературы по проблеме лечения пациентов с контрактурой Дюпюитрена. Уважаемые коллеги, несмотря на то, что Министерство образования и науки России в сло- жившихся условиях приостановило до декабря 2022 года учет индексации публикаций отече- ственных ученых в международных базах дан- Наше издание является научно-практическим, и мы стараемся уделять достаточно внимания экспериментальным и теоретическим работам, которые являются базой будущих клинических Cite as: Tikhilov R.M. Тихилов Рашид Муртузалиевич / Rashid M. Tikhilov; e-mail: rtikhilov@gmail.com  © Тихилов Р.М., 2022 © Tikhilov R.M., 2022 Cite as: Tikhilov R.M. [The Editor-in-Chief Appeal to the Issue Nо 2 for 2022 of the Journal “Traumatology and Orthopedics of Russia”]. Travmatologiya i ortopediya Rossii [Traumatology and Orthopedics of Russia]. 2022;28(2):5-6. https://doi.org/10.17816/2311-2905-1798. (In Russian). Уважаемые читатели! [The Editor-in-Chief Appeal to the Issue Nо 2 for 2022 of the Journal “Traumatology and Orthopedics of Russia”]. Travmatologiya i ortopediya Rossii [Traumatology and Orthopedics of Russia]. 2022;28(2):5-6. https://doi.org/10.17816/2311-2905-1798. (In Russian). © Тихилов Р.М., 2022 © Tikhilov R.M., 2022 5 травматология и ортопедия россии / Traumatology and orthopedics of Russia 2022;28(2) травматология и ортопедия россии / Traumatology and orthopedics of Russia 2022;28(2) ОТ РЕДАКтора / EDITORIAL ОТ РЕДАКтора / EDITORIAL ных, в том числе и в Web of Science, куда входит наш журнал, мы приняли решение сохранить принятые в журнале международные критерии оценки качества поступающих статей. Сейчас в Российской Федерации создается новая система оценки результативности научных исследований и разработок, в которой будут учитываться публи- кации в высокорейтинговых отечественных из- даниях, входящих в ядро РИНЦ и Russian Science Citation Index. Наш журнал входит в обе эти базы, и задача редакции и редколлегии — сохранить вы- сокий рейтинг журнала. А сделать это можно, толь- ко публикуя высококачественные востребованные читателями статьи. даниях, входящих в ядро РИНЦ и Russian Science Citation Index. Наш журнал входит в обе эти базы, и задача редакции и редколлегии — сохранить вы- сокий рейтинг журнала. А сделать это можно, толь- ко публикуя высококачественные востребованные читателями статьи. Будем рады увидеть ваши работы на страницах нашего журнала. С уважением, главный редактор журнала «Травматология и ортопедия России» член-корреспондент РАН профессор Р.М. Тихилов травматология и ортопедия россии / Traumatology and orthopedics of Russia 6 2022;28(2)
https://openalex.org/W2055506903	https://zenodo.org/records/1660317/files/article.pdf	English	null	On the Effect of External Conditions on the Reproduction of Daphnia	The American naturalist	1,910	public-domain	3,014	y 2 Wheil the daplhidls were crowded in dishes of the same pool water they soon began to die, owing to the accumulation of their excretions. When 'From the Zoological Laboratory of the University of Missouri and the Histological Laboratory of Cornell IUniversity Medical College, New York City. ON THE EFFECT OF EXTERINAL CONDITIONS ON THE REPIRODUCTION OF DAPHNIA1 DR. J. F. MeCLENDON DR. J. F. MeCLENDON CORNELL AMEDICAL COLLEGE SINCE in tile great maJority of organisms only the germ cells are capable of reproducing tile entire indi- vTidual, tile question as to -what differences exist between the germ and body cells, and how they arise, is of gen- eral interest. Therefore any\ chaange of conditions which affects the germ cells or their relation to the body cells deserves special study. In Daph2ia external conditions not only affect tile relation of tile germ cells to the body cells, but tiley affect the egg cells in such. a manner as to determine whlethler they do or do not need fertilization Tile purpose of tile preseilt paper is not merely to acid the results of my\T experiments to those of other investi- gators, but to tentatively arrange tile available data under a general working lypotlhesis in tile ilope tilat somne more direct method of investigating the relation of the germ alcid body cells be dev\ised. g Last spring (Ma1:rch 10) I began experiments on the effects of environment on Daphnia pulex, De Geer, with- out knowing that W\Toltereck was working on tile same line. Tile material came fronl a sliall Pool investigated bv Dr. W. C. Curtis and containllng a single strain of this anld 1o other species of Daphjtia. For the first few -weeks some ice remained on tile pool and the tempera- ture did not much exceed 40 C.; after this it rose steadily to ab out 20? by the endi of May. Specimens from the pool were exanlined at intervals as a control on tile ex- perimnent S.2 404 This content downloaded from 187.038.063.047 on February 18, 2018 20:54:15 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-a 405 REPRODUCTION OF DAPHANIA No. 523] No. 523] EFFECT OF ENVIRONMENT ON DIFFERENTIAL GROWTH By differential growth I mean the unequal growth of different parts, viz., the germ and body cells. Only parthenogenetic females were used, and each was kept separately in the same quantity of water. All measure- ments were made at sexual maturity, i. e., when the first eggs appeared in the brood pouch. Warren3 found that under uniform conditions there was a slight varia- bility, but Woltereck showed that these fluctuating varia- tions were very small, though he did find mutations as rare occurrences. Nutrition.-I had in the laboratory a pure culture of a unicellular green alga which the daphnids ate readily. This alga did not remain entirely suspended in the water, but as the daphnids fed on the bottom as well as while swimming, and stirred up the algae, it can not be said that most of the food was out of their reach. Those with a superabundance of food were larger at sexual maturity and had a shorter spine than those with insufficient food, and conversely. The smaller size and longer spine of the starved daphnids are characteristic of immature stages. g Temnperature.-Three sets of experiments were transferred suddenly to artesian tap water many died, though with a gradual change all lived. The composition of the tap water was as follows: Ca, .148 X 10-1 molecular; Mg, .1 X 10-8 molecular; CO3, .045 X 10-8 molec- ular; SO4, .146 X 10-8 molecular; Cl, .055 X 10-8 molecular. Besides these were very small quantities of silica, clay, iron, ammonia and nitrates, and traces of lithium and potassium. Beside the carbonates the water when drawn from the tap was super-saturated with carbon dioxide. In order to find the cause of death from change of water I added various amounts of molecular solutions of certain salts. The toxicity of cations increased as follows: NA<1/2 Ca<K, and of anions: Cl<1/2 SO4<HCO3<1/2 HPO4. But as K became toxic only on 1/100 and HPO4 on 1/500 molecular concen- tration there must be other toxic substances than salts in the water. The carbon dioxide in the water killed crayfish and was probably the most toxic constituent to the daphnids. 8"An Observation on Inheritance in Parthenogenesis," Proc. Boy. Soc. London, 1899, LXV, and "On the Reactions of Daphnia magna to certain changes in Environment,"I Quart. Jour. Mier. Sc., 1900. 8"An Observation on Inheritance in Parthenogenesis," Proc. Boy. Soc. I IUeber das Abhuingigkeitsverhuiltnis zwischen del Bau der Plankton- organismen mid den specifischeii Gewiclit des Siisswassers, I Biol. Centlb., 1900, XX, pp. 606-619, 644-656, and " Studier over de Danske soers Plankton,'' Copenhagen, 1904. '5' Experimientelle Untersuchlngen iiber den Saisonpolynmorphisnlius bei Daphniden,'' Archiv f. Enftwicklulagseiech. d. Organqisom-en, 1904, XVII, p. 415. EFFECT OF ENVIRONMENT ON DIFFERENTIAL GROWTH London, 1899, LXV, and "On the Reactions of Daphnia magna to certain changes in Environment,"I Quart. Jour. Mier. Sc., 1900. This content downloaded from 187.038.063.047 on February 18, 2018 20:54:15 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t- THE AMERICAN NATURALIST [VOL. XLIV 406 THE AMERICAN NATURALIST started: at 4-10, 19-200 and 29-310 C. The results were as follows: Average Body Length. Ratio of Average Spine Time from Hatching Length to Body Length. to Sexual Matnrity. 4-100 22 0.24 35 days 19-210 20 0. 5 14 ` 29-31? 17.7 0.27 6 " 1 unit =2, millimeters. All were given a surplus of food daily,. It may be observed that a higher temperature has the same effect as insufficient food. Salts.-Since salts have such a marked effect on the development of marine and some fresh-water animals, I placed claplmids in the strongest solutions of various salts that they would live in (without acclimatization). The effects in two months (four generations) were Un7 ioticeable. Liygt.-Cultures were kept in the dark and in diffuse and direct sunlight, but n1o effect was observed. A number of observers have recorded season-poly- morphism in daphnlmids. Wesenberg-Lundc4 pointed out that when the specific gravity and consequent buoyancy of the water decreased by heat in summer-the body of the daphnids became smaller or were provided with outgrowths, so as to offer a greater resistance to sinking. Wolfgang Ostwaldc5 produced, all at the same time, the forms that occurred in nature at different seasons, by varying the temperature. - He emphasizedl the fact that rise in temperature loweredl the internal viscosity of the water. He found that in the warm cultures the daphnids often became productive at an undeveloped stage and, as is true generally, reproduction retarded body growth. This content downloaded from 187.038.063.047 on February 18, 2018 20:54:15 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t- REPRODUCTION OF DAPHNIA 407 No. 523] No. 523] Woltereck6 maintains that Ostwald's results were due to the fact that at a higher temperature the daphnids need more food. Woltereck caused decrease in body length both by starving and by increasing the tempera- ture, but the latter was not effective with an optimum supply of food. lIe found that more food than the optimum produced effects similar to starving. Raising the internal viscosity of the water by adding quince gum produced no effect. 6 "'Ueber naturliche und kunstliehe Varietaitenbildung bei Daphniden,'" Veprh. Deetsch. Zool. Gesell., 1908, p. 234; adl "'Weitere experinmentel Uiitersuehlngen iiber Artverandenirng, speziell fiber das Wesen quantitativer Artuntersehiede bei Daphniden,'" ibid., 1909, p. 110. p 7 vvUeber experimenetelle Untersuelhungen z Fragen der Fortpflanzung, Variation und Vererbung bei Daphniden," VeFih. Deutsch. Zool. Gesell., 1909, p. 281. This content downloaded from 187.038.063.047 on February 18, 2018 20:54:15 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t- 6 "'Ueber naturliche und kunstliehe Varietaitenbildung bei Daphniden,'" Veprh. Deetsch. Zool. Gesell., 1908, p. 234; adl "'Weitere experinmentel Uiitersuehlngen iiber Artverandenirng, speziell fiber das Wesen quantitativer Artuntersehiede bei Daphniden,'" ibid., 1909, p. 110. 7 vvUeber experimenetelle Untersuelhungen z Fragen der Fortpflanzung, Variation und Vererbung bei Daphniden," VeFih. Deutsch. Zool. Gesell., 1909, p. 281. I " Doclekas neuer Cladoceren nebst einer kurzen iibersicht cler Clacloceren- fauna B6hmens," Sitz. Ber. math. natm'wv. Wien, 1875. - ''Zur Kenntnis des Einfulsses der ausseren Lebensbeclingungen auf die Organisation der Tiere," Zeit. wiss. Zool., 1877, XXIX. '1'"Beitrdge zur Naturgeschichte der Daphnoiden, VII,'" Zeits. wiss. Zool., XXXIII, p. 111. 11 " Geseclechtbeetinmi-encde Ursachen be! den Daphnilen,'" Biol. Cen- tralb., 1909, XXV, pp. 529_536. EFFECT OF ENVIRONMENT ON DIFFERENTIAL GROWTH He showed that though feeding in- fluenced the differential growth, there was a cyclical tendency for this to vary, viz., season-polymorphism However, the effects of prolonged abundant feeding were inherited to some degree. One might interpret these results in different ways. It is known that the temperature coefficients for various chemical reactions are slightly different. Possibly the mean of the temperature coefficients for the processes in the development of the reproductive organs is higher than the same for the body wall, and at a higher tem- perature the germ cells would develop faster. However, under adverse conditions the "affinity" of the repro- ductive organs for nutriment is greater than that of the rest of the body, so with deficient food the body wall is retarded more than the germ cells in development. The higher temperature may be considered an adverse condi- tion since the mortality is greater. In this way starv- ing has the same effect as a higher temperature. g g p Langerhan s7 found that accumulation of excretions caused shortening of the spine in daphnicls. I do not know what relation this bears to the above results. 408 THE AMERICAN NATURALIST [VOL. XLIV THE AMERICAN NATURALIST I " Doclekas neuer Cladoceren nebst einer kurzen iibersicht cler Clacloceren- fauna B6hmens," Sitz. Ber. math. natm'wv. Wien, 1875. - ''Zur Kenntnis des Einfulsses der ausseren Lebensbeclingungen auf die Organisation der Tiere," Zeit. wiss. Zool., 1877, XXIX. 11 " Geseclechtbeetinmi-encde Ursachen be! den Daphnilen,'" Biol. Cen- tralb., 1909, XXV, pp. 529_536. '1'"Beitrdge zur Naturgeschichte der Daphnoiden, VII,'" Zeits. wiss. Zool., XXXIII, p. 111. EFFECT OF ENVIRONMENT ON THE LIFE CYCLE In most species of daphniiids, generations of partheno- genetic females alternate with generations of males and females which produce eggs that must be fertilized, and either frozen, dried or kept a long time before they will develop (resting or " winter " eggs). In different species the number of successive parthenogenetic gen- erations varies. In some all are, and in some none are, parthenogenetic. I found that heat hastened the appearance of sexual forms, as did starving or the accumulation of excretory products. All of these factors might be combined in the dr-ying up of a pond, as heat would aid in drying, and driving would concentrate the daphnids and their ex- cretions, and concentration of the daphnids would cause them to eat up the algae faster than they could multiply. However, by keeping the culture cold, fresh or well-fed, or all combined, I could delay but not prevent the ap- pearance of sexual forms. p Kurzs said the drying up of the water caused the ap- pearance of sexual forms, and Schrnankewitz9 suggested that it was the increase in salts. Weismaannt0 tested both of these hypotheses and concluded that they were Wro1g01. He also tried the effect of food and tempera- ture, with varying results. He concluded that the life cycle was fixed for each species and variety. Issako- witz" concluded that cold favored the appearance of sexual forms and warmth favored the parthenogenetic Also, hunger favored the appearance of sexual forms and abundant food the parthenogenetic. It may be that cold retarded multiplication of the food plant or the This content downloaded from 187.038.063.047 on February 18, 2018 20:54:15 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-a REPRODUCTION OF DAPHNIA 409 No. 523] No. 523] movements of the clapimidcs so that they did not keep the algae stirred up in the water sufficiently to get at them. The parthenogenetic egg arises from follr cells, but a large number of cells enter into the composi- tion of the fertilizable egg. If the latter egg is not fertilized it is absorbed and, as Issakowitsch noted, fur- nishes food for the development of parthenogenetic eggs. Woltereckl 2 found that starving hastened the appear- ance of sexual reproduction, but a concentration of food above the optimum produced results similar to starving. 12Loc. cit. 13 oc cit. This content downloaded from 187.038.063.047 on February 18, 2018 20:54:15 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-an EFFECT OF ENVIRONMENT ON THE LIFE CYCLE He found, as Weismann maintained, a cyclical tendency toward the alternation of sexual and parthenogenet,> generations which, contrary to Weismann, was tempo- rarily influenced by nutrition, and the effects of constant nutrition over a long period was inherited to some ex- tent. Langerlhans1" found that the accumulation of excre- tions caused the decrease in numbers of parthenogenetic females in the autumn and thinks that the appearance of sexual forms is due to the same cause. The life cycle of a daphnid is, therefore, an hereditary tendency, but can be influenced by nutrition and probably by- temperature and the accumulation of excretions. Nutrition is the mnost important factor, and former ex- periments on the effect of temperature and the drying -ip of the water were complicated by secondary effects on concentration of food and excretory products. Discussion of Results.-Two views might be held as to the origin of the differences between the germ and body cells: the differences might be the result of differ- ence in position in the embryo, or of unequal mitoses. In the parasitic copepods I found the primary germ cell to arise by an unequal mitosis at the fifth cleavage of the egg. The germ cell when first formed is one thirty- second of the total number of cells, but owing to the more This content downloaded from 187.038.063.047 on February 18, 2018 20:54:15 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-a 410 THE AMERICAN NATURALIST [VOL. XLIV THE AMERICAN NATURALIST [VOL. XLIV [VOL. XLIV rapid division of the body cells this ratio decreases. In fact the chief difference between the yolk-free body cells and tlhe (yolk-free) germ cells is the slow rate of division of the latter. Finally, the eggs will not divide at al] un- less specially stimulated, by fertilization. The question now arises: what causes the cell to divide. Sacks found that plant cells divide when they have reached a certain size. This rule has been extended to animals, and the final size of the cell found to be determined by the ratio of nucleus to cytoplasm. This rule may apply to the germ cells, since it appears that after the egg cell, pri- mary oocyte, reaches a certain size any additional food absorbed does not cause growth of the protoplasm, but is precipitatedas yolk. This content downloaded from 187.038.063.047 on February 18, 2018 20:54:15 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-an EFFECT OF ENVIRONMENT ON THE LIFE CYCLE p p y If the egg is properly stimulated, rapid growth of protoplasm and cell divisions follow. Firomn the study of artificial parthenogenesis it appears probable that stimuli which lead to development of the egg increases the permeability of its plasma mienibrane. If this be true we may say that the gernm cells are distinguishec by the fact that their plasma membranes are poorly permeable and retard those reactions between the cell contents and envirolnelnt which lead to growth and cell division. In other words, the optimum intensity of stim- ulation toward growth and division is higher for the germ cells than for the body cells. g This difference is probably due to a difference in the colloids of the cell, which in animals could be explained as the result of an unequal mitosis. This explanation may 1e modified so as to apply to plants. Klebs has shown that those conditions which are adverse to vege- tative growth of plants (too strong stimuli?) call forth flowers. Perhaps there are slight differences in the sensitiveness of plant cells to stimuli, and as the stimuli increase, those initially least sensitive cells acquire further immunity to the stimulus, whereas those initially more sensitive cells are overstinmlated and weakened. Thus the difference between germ and body cells is grad- This content downloaded from 187.038.063.047 on February 18, 2018 20:54:15 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-an REPRODUCTION OF DAPHNIA 411 No. 523] No. 523] ally acquired. The Malpighian layer of the skin may be stimulated to proliferate more rapidly, but if the stimuli are too strong the growth will be retarded instead of increased. On gradually increasing the stimulus imp munity to it may be acquired. To apply this hypothesis to the daphnids: conditions which are adverse to the growth of the body cells, such as extremes of temperature (viz., high temperatures) or of concentration of excretory products, or disorded nu- trition, either fail to retard the development of the germ cells or stimulate their development, so that in either case the daphnid becomes sexually mature at a less de- veloped stage. Under the less extreme conditions the eggs develop on receiving the slight stimulus incident to their transfer to the brood pouch, but under the more extreme conditions those eggs which develop at all must be stimulated by fertilization before they develop. EFFECT OF ENVIRONMENT ON THE LIFE CYCLE These two types of eggs may perhaps develop from two kinds of cells, or the sexual egg may arise from the same kind of cell producing the parthenogenetic egg by ac- quiring an immunity to slight stimuli. Whereas more than one cell goes to make up a single egg; only one nucleus is retained, and it may be said that one cell is the eag cell and the remainder furnish its food. This content downloaded from 187.038.063.047 on February 18, 2018 20:54:15 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-an
https://openalex.org/W2299789761	https://europepmc.org/articles/pmc4811074?pdf=render	English	null	Hiatus Hernia: A Rare Cause of Acute Pancreatitis	Case reports in medicine	2,016	cc-by	2,477	1. Introduction abdominal pain and vomiting for 1 day. He was in his usual state of health prior to an acute onset of epigastric pain which was dull and crampy in nature, 8/10 in intensity and associated with nausea and 10–12 episodes of nonbloody vomiting. He denied any other complaints. In the ER, his blood pressure (BP) was 165/91 mm of Hg, temperature (𝑇) was 98 F, heart rate (HR) was 85/min, and the respiratory rate (RR) was 18. His abdomen was soft, with mild epigastric tenderness and normal bowel sounds. The rest of the physical exam was unremarkable. Hiatal hernia (HH) refers to herniation of elements of the abdominal cavity through the esophageal hiatus of the diaphragm.h The stomach is the most common organ to herniate. Other less common herniations include transverse colon, small intestine, and spleen. Herniation of the pancreas through the hiatus is an extremely rare occurrence. Pancreatic herniation leading to acute pancreatitis is an even more rare condition. In the world literature, from 1958 to 2011, 12 cases have been reported [1]. All reported patients were sympto- matic except for one case reported by Katz et al. [2]. His initial blood work revealed a lipase of 2950. Com- puted tomography (CT) of abdomen/pelvis with radio con- trast (Figures 1 and 2) was done which showed a large hiatal hernia containing the entire stomach, the first part of the duodenum, and most of the body and the tail of the pancreas. This was associated with peripancreatic inflammation highly suspicious for acute pancreatitis.h We describe a rare case of an overtly symptomatic patient diagnosed with acute pancreatitis who was found to have herniation of the stomach, the body, and the tail of the pancreas and the first part of the duodenum. The patient underwent an esophagogastroduodenoscopy (EGD) on day 4 of admission which revealed erythematous mucosa of the fundus and body of the stomach, Barrett’s esophagus, and a sliding HH. Biopsy revealed squamo- columnar mucosa with mild chronic inactive inflammation Correspondence should be addressed to Shruti Patel; spatel12@numc.edu Correspondence should be addressed to Shruti Patel; spatel12@numc.edu Received 23 December 2015; Accepted 7 February 2016 Academic Editor: Tobias Keck Copyright © 2016 Shruti Patel 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. Hiatal hernia (HH) is the herniation of elements of the abdominal cavity through the esophageal hiatus of the diaphragm. A giant HH with pancreatic prolapse is very rare and its causing pancreatitis is an even more extraordinary condition. We describe a case of a 65-year-old man diagnosed with acute pancreatitis secondary to pancreatic herniation. In these cases, acute pancreatitis may be caused by the diaphragmatic crura impinging upon the pancreas and leading to repetitive trauma as it crosses the hernia; intermittent folding of the main pancreatic duct; ischemia associated with stretching at its vascular pedicle; or total pancreatic incarceration. Asymptomatic hernia may not require any treatment, while multiple studies have supported the recommendation of early elective repair as a safer route in symptomatic patients. In summary, though rare, pancreatic herniation should be considered as a cause of acute pancreatitis. A high index of suspicion for complications is warranted in cases like these. Hindawi Publishing Corporation Case Reports in Medicine Volume 2016, Article ID 2531925, 4 pages http://dx.doi.org/10.1155/2016/2531925 Hindawi Publishing Corporation Case Reports in Medicine Volume 2016, Article ID 2531925, 4 pages http://dx.doi.org/10.1155/2016/2531925 Hindawi Publishing Corporation Case Reports in Medicine Volume 2016, Article ID 2531925, 4 pages http://dx.doi.org/10.1155/2016/2531925 2. Case Presentation A 65-year-old Caucasian male with no known past medical history came to the emergency room (ER) complaining of 2 Case Reports in Medicine Figure 1: Coronal plane; hiatal hernia; herniation of the stomach, the body and tail of the pancreas, and the first part of the duodenum. leads to increased laxity of the posterior adhering fascia thereby mobilizing the pancreas, hence causing herniation. Overall, herniation of the pancreas is extraordinary with only 12 cases reported until now. HH are more common in Western countries [5]. Burkitt and James suggest that the Western, fiber-depleted diet leads to a state of chronic constipation and straining during bowel movement, which could explain the higher incidence of this condition in Western countries. Hiatal hernias are more common in women than in men. The frequency of HH increases with age, from 10% in patients younger than 40 years to 70% in patients older than 70 years.i y p y HH is classified into two types depending on the position of the gastroesophageal (GE) junction and the extent of her- niated stomach: sliding hernias and paraesophageal hernias [6, 7]. Type I, also called sliding hernias, occurs when the GE junction migrates through the hiatus into the posterior mediastinum. It is the most common type, accounting for 85– 95% of all the cases [8, 9]. Types II, III, and IV, also called paraesophageal hernias, are less common and account for the remaining 5–15% of the cases [10, 11]. Type II occurs when the GE junction is in its normal position and the fundus herniates through the hiatus along its side. Type III is a combination of types I and II wherein there is a protrusion of the stomach through the hiatus along with a displaced GE junction. Type IV is defined by herniation of the stomach with other organs into the chest. Figure 1: Coronal plane; hiatal hernia; herniation of the stomach, the body and tail of the pancreas, and the first part of the duodenum. Figure 2: Sagittal plane; diaphragmatic crura impinging the pan- creas. HH may be asymptomatic or patients might complain of heartburn, belching, dysphagia, abdominal pain, nausea, chest pain, or cough. In most patients, the cause of the HH is unknown. Some people are born with a weakness or an especially large hiatus. 2. Case Presentation It is believed that increased pressure in the abdomen from chronic coughing, straining during bowel movements, pregnancy and delivery, obesity, and abdominal ascites may contribute to the development of the HH. Rarely, iatrogenic or traumatic diaphragmatic hernia (DH) may occur. They account for less than 1% of all the DH [12]. Iatrogenic hernias can occur due to alterations in the normal anatomy from surgical dissection of the hiatus. This may be due to disruption of a previous hiatal closure, postoperative gastric dilatation, disruption of the phrenoesophageal mem- brane by operative dissection, and failure to recognize eso- phageal shortening or an existing hiatal defect [13]. Etiologies of iatrogenic diaphragmatic defects include Ivor Lewis proce- dure, antireflux procedures, esophagomyotomy, partial gas- trectomy, gastric banding procedures, and misguided chest tubes or thoracoabdominal incisions in which the diaphragm is taken down [14]. Figure 2: Sagittal plane; diaphragmatic crura impinging the pan- creas. and no helicobacter pylori identified. The patient refused any surgical intervention. He clinically improved and was discharged with outpatient follow-up. References [1] T. J¨ager, D. Neureiter, C. Nawara, A. Dinnewitzer, D. ¨Ofner, and W. Lamad´e, “Intrathoracic major duodenal papilla with tran- shiatal herniation of the pancreas and duodenum: a case report and review of the literature,” World Journal of Gastrointestinal Surgery, vol. 5, no. 6, pp. 202–206, 2013. [2] M. Katz, E. Atar, and P. Herskovitz, “Asymptomatic diaphrag- matic hiatal herniation of the pancreas,” Journal of Computer Assisted Tomography, vol. 26, no. 4, pp. 524–525, 2002. [3] K. Suzuki, Y. Izumi, T. Ryotokuji, A. Miura, T. Kato, and M. Tokura, “Giant hiatal hernia with pancreatic prolapse: report of a case,” SAGES, http://www.sages.org/meetings/annual-meet- ing/abstracts-archive/giant-hiatal-hernia-with-pancreatic-pro- lapse-report-of-a-case/. [4] P. Chevailler, E. Peten, C. Pellgrino et al., “Hiatal hernia with pancreatic volvulus: a rare cause of acute pancreatitis,” American Journal of Roentgenology, vol. 177, pp. 373–374, 2001. [5] D. P. Burkitt and P. A. James, “Low-residue diets and hiatus hernia,” The Lancet, vol. 302, no. 7821, pp. 128–130, 1973. [6] M. O. Mitiek and R. S. Andrade, “Giant hiatal hernia,” Annals of Thoracic Surgery, vol. 89, no. 6, pp. S2168–S2173, 2010. The risk of these hernias becoming incarcerated, leading to strangulation or perforation, is approximately 5%. Elective repair often is advised in symptomatic patients. In a study by Sihvo et al. [20], of the 563 patients in the surgical group, the overall mortality was 2.7% whereas mortality of the 67 patients in the conservative treatment group was 16.4%. 13% of the deaths might have been avoided with elective surgical intervention. Of the 32 patients who died, over half had type III or type IV hiatal hernias; 4 patients had type II hiatal hernias, with the remaining 3 deceased having an unknown type. In a Swiss study [21], of the 354 laparoscopic paraesophageal hernia repairs, age at 70 years or older was significantly associated with postoperative morbidity (24.4%) and mortality (2.4%) relative to those younger than 70 years. These findings support early elective repair as a safer route in symptomatic patients. [7] O. Awais and J. D. Luketich, “Management of giant parae- sophageal hernia,” Minerva Chirurgica, vol. 64, no. 2, pp. 159– 168, 2009. [8] C. Schieman and S. C. Grondin, “Paraesophageal hernia: clin- ical presentation, evaluation, and management controversies,” Thoracic Surgery Clinics, vol. 19, no. 4, pp. 473–484, 2009.i [9] P. J. Kahrilas, H. C. Kim, and J. E. Case Reports in Medicine Case Reports in Medicine Many diagnostic modalities are available including plain chest X-ray film, barium upper gastrointestinal series, CT imaging, and EGD. Asymptomatic hernia might not require any treatment. HH with gastroesophageal reflux disease can be managed medically. Symptomatic HH (e.g., chest pain and dysphagia), HH with severe esophagitis, and types II, III, and IV HH might require surgical intervention which includes reduction of the hernia, closure of the hiatal defect, and antireflux procedure. Hiatal hernias can be repaired by a transabdominal or transthoracic approach [18]. Laparo- scopic hiatal hernia repair and open transabdominal repair are equally effective. Laparoscopic repair is the preferred approach for the majority of the hiatal hernias as it is associated with reduced rate of perioperative morbidity and mortality and shorter hospital stays [19]. The operative principles of a hernia repair are the same for laparoscopic and open approaches. They involve the dissection of the sac and reduction of the hernia followed by complete excision of the sac from the mediastinum. After the esophagus is mobilized, the hiatal defect is closed by interrupted nonabsorbable sutures (primary repair) if the defect is small or using a mesh for the larger defects. Gastric fixation, such as anterior gastropexy may safely be used in addition to hiatal repair to prevent recurrence. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. References Pandolfino, “Approaches to the diagnosis and grading of hiatal hernia,” Best Practice and Research: Clinical Gastroenterology, vol. 22, no. 4, pp. 601–616, 2008. [10] P. J. Kahrilas, “Hiatus hernia causes reflux: fact or fiction?” Gullet, vol. 3, supplement, pp. 21–30, 1993. [11] G. Peridikis and R. A. Hinder, “Paraesophageal hiatal hernia,” in Hernia, L. M. Nyhus and R. E. Condon, Eds., p. 544, JB Lippincott, Philadelphia, Pa, USA, 1995. [12] S. Eren and F. C¸iris¸, “Diaphragmatic hernia: diagnostic approaches with review of the literature,” European Journal of Radiology, vol. 54, no. 3, pp. 448–459, 2005. Pancreatitis caused in our patient was treated with fluids, analgesia, antiemetics, and gradual advancement of diet as tolerated by the patient. Our patient did not undergo surgery since the patient only wanted conservative management at that time. [13] J. M. Streitz Jr. and F. H. Ellis Jr., “Iatrogenic paraesophageal hiatus hernia,” The Annals of Thoracic Surgery, vol. 50, no. 3, pp. 446–449, 1990. [14] M. M. Hutter and D. W. Rattner, “Paraesophageal and other complex diaphragmatic hernias,” in Shackelford’s Surgery of the Alimentary Tract, C. J. Yeo, Ed., pp. 549–562, Saunders Elsevier, Philadelphia, Pa, USA, 2007. [15] M. J. Oliver, A. R. M. Wilson, and L. Kapila, “Acute pancreatitis and gastric volvulus occurring in a congenital diaphragmatic hernia,” Journal of Pediatric Surgery, vol. 25, no. 12, pp. 1240– 1241, 1990. 3. Discussion HH corresponds to the transient or permanent migration of the stomach through the diaphragm into the chest which may be accompanied by other abdominal organs as in this case. The most common organs to accompany the stomach are the colon (usually the splenic flexure), loops of small bowel, and omentum. A large HH is a hernia that includes at least 30% of the stomach in the chest. Many reports of giant HH have been published, but a giant HH with pancreatic prolapse is extremely rare [3]. The head of the pancreas and the duodenum are typically held down by the ligament of Treitz and hence herniation of the pancreas usually does not occur [4]. It is proposed that stretching of the transverse mesocolon HH can be complicated by intermittent bleeding episodes from associated esophagitis, erosions, and esophageal ulcers; iron deficiency anemia; incarceration; strangulation and perforation. Acute pancreatitis complicating a diaphragmatic hernia is rare and multiple theories have been proposed to explain it. Acute pancreatitis occurring in the case of a pan- creatic hernia may be caused by repetitive trauma as it crosses the hernia, ischemia associated with stretching at its vascular pedicle, [15, 16], or intermittent folding of the main pancreatic duct. Total incarceration of the pancreas may also contribute to pancreatitis [17]. 3 4. Conclusion Pancreatic herniation is a rare entity which may lead to even rarer complications of pancreatitis. Symptomatic herniation is best treated with surgery. Our patient’s large hernia was not surgically repaired, despite our recommendations and an explanation of the potentially fatal risk. Elective surgery is found to be safer than emergent surgery in a patient that has suffered a serious complication. [16] R. J. Cuschieri and W. A. Wilson, “Incarcerated Bochdalek hernia presenting as acute pancreatitis,” British Journal of Surgery, vol. 68, no. 9, article 669, 1981.k [17] N. J. Kafka, I. M. Leitman, and J. Tromba, “Acute pancreatitis secondary to incarcerated paraesophageal hernia,” Surgery, vol. 115, no. 5, pp. 653–655, 1994. 4 Case Reports in Medicine [18] J. B. Pearson and J. G. Gray, “Oesophageal hiatus hernia: long- term results of the conventional thoracic operation,” British Journal of Surgery, vol. 54, no. 6, pp. 530–533, 1967. [19] F. Yano, R. J. Stadlhuber, K. Tsuboi, J. Gerhardt, C. J. Filipi, and S. K. Mittal, “Outcomes of surgical treatment of intrathoracic stomach,” Diseases of the Esophagus, vol. 22, no. 3, pp. 284–288, 2009. [20] E. I. Sihvo, J. A. Salo, J. V. R¨as¨anen, and T. K. Rantanen, “Fatal complications of adult paraesophageal hernia: a population- based study,” Journal of Thoracic and Cardiovascular Surgery, vol. 137, no. 2, pp. 419–424, 2009. [21] H. J. Larusson, U. Zingg, D. Hahnloser, K. Delport, B. Seifert, and D. Oertli, “Predictive factors for morbidity and mortality in patients undergoing laparoscopic paraesophageal hernia repair: age, ASA score and operation type influence morbidity,” World Journal of Surgery, vol. 33, no. 5, pp. 980–985, 2009.
https://openalex.org/W2792229663	https://fermet.misis.ru/jour/article/download/1237/1065	Russian	null	MOVEMENT SPECIFICS OF A GERM WITH COMPLEX SHAPE ON THE PELLETIZING DISK IN IRON-ORE PELLETS PRODUCTION	Izvestiâ vysših učebnyh zavedenij. Černaâ metallurgiâ	2,018	cc-by	6,228	Павловец В.М., к.т.н., доцент кафедры теплоэнергетики и экологии ( pavlowets.victor@yandex.ru ) Герасимук А.В., аспирант кафедры электротехники, электропривода и промышленной электроники ( heavymetal7200@gmail.com ) Павловец В.М., к.т.н., доцент кафедры теплоэнергетики и экологии ( pavlowets.victor@yandex.ru ) Герасимук А.В., аспирант кафедры электротехники, электропривода и промышленной электроники ( heavymetal7200@gmail.com ) Сибирский государственный индустриальный университет (654007, Россия, Новокузнецк, Кемеровская обл., ул. Кирова, 42) ОСОБЕННОСТИ ДВИЖЕНИЯ ЗАРОДЫША СЛОЖНОЙ ФОРМЫ НА ТАРЕЛЬЧАТОМ ОКОМКОВАТЕЛЕ В ПРОИЗВОДСТВЕ ЖЕЛЕЗОРУДНЫХ ОКАТЫШЕЙ Павловец В.М., к.т.н., доцент кафедры теплоэнергетики и экологии ( pavlowets.victor@yandex.ru ) Герасимук А.В., аспирант кафедры электротехники, электропривода и промышленной электроники ( heavymetal7200@gmail.com ) Сибирский государственный индустриальный университет (654007, Россия, Новокузнецк, Кемеровская обл., ул. Кирова, 42) Аннотация. Рассмотрены особенности движения зародыша сложной формы на тарельчатом окомкователе в производстве железорудных окаты- шей. В результате проведенного анализ сил, действующих на кубический зародыш при его перекате на шихтовом гарнисаже (ШГ) в усло- виях пластической деформации, построена математическая модель, позволяющая определить оптимальные параметры работы окомкова- теля для производства окатышей по технологии принудительного зародышеобразования. Рассмотрены два частных случая устойчивости зародыша на ШГ окомкователя и условия преодоления устойчивости, связанные с формированием переката. Устойчивость зародыша или, напротив, его перекат определяются горизонтальным положением его центра массы относительно опорного ребра, расположенного по- перек вероятного направления переката. Если линия действующей силы (силы тяжести) переместится за опорное ребро и опорную плос кость, то сформируется момент силы, который выведет зародыш из состояния устойчивости и сформирует перекат зародыша. Первым частным случаем устойчивости зародыша на наклонной плоскости является отсутствие переката на ШГ при минимальной величине цент робежной силы, что соответствует расположению зародыша в центральных областях зоны окомкователя, или работе рабочего органа с низкой частотой вращения (n < 3 об/мин). Более общим случаем выхода зародыша из состояния устойчивости в режим переката на наклон- ной плоскости ШГ является работа окомкователя при сравнительно высокой величине центробежной силы (n >3 об/мин) и расположение зародыша в периферийных областях рабочей зоны окомкователя. Для указанных случаев сформулированы условия переката зародышей на ШГ в рабочей зоне окомкователя и корректирующие мероприятия в условиях пластической деформации при смятии его углов и ребер. Показаны особенности движения зародыша в рабочей зоне тарели окомкователя. Отмечены условия для корректировки режима принуди- тельного зародышеобразования и озвучены мероприятия по целенаправленному воздействию на коэффициент смятия зародыша. Установ- лено, что для организации режима переката зародыша сложной формы угол наклона тарели окомкователя к горизонту следует назначать в зависимости от частоты его вращения и коэффициента смятия зародышей. Ключевые слова: зародыш сложной формы, коэффициент смятия зародыша, перекат зародышей, тарельчатый окомкователь, шихтовый гарни- саж, принудительное зародышеобразование, напыление влажной шихты, железорудные окатыши. Металлургические технологии МЕТАЛЛУРГИЧЕСКИЕ ТЕХНОЛОГИИ Металлургические технологии МЕТАЛЛУРГИЧЕСКИЕ ТЕХНОЛОГИИ ISSN: 0368-0797. Известия высших учебных заведений. Черная металлургия. 2018. Том 61. № 2. С. 87 – 95. © 2018. Павловец В.М., Герасимук А.В. ISSN: 0368-0797. Известия высших учебных заведений. Черная металлургия. 2018. Том 61. № 2. С. 87 – 95. © 2018. Павловец В.М., Герасимук А.В. ISSN: 0368-0797. Известия высших учебных заведений. Черная металлургия. 2018. Том 61. № 2. С. 87 – 95. © 2018. Павловец В.М., Герасимук А.В. УДК 669.162.12:622 DOI: 10.17073/0368-0797-2018-2-87-95 Это также справедливо в от- ношении зародыша со смятыми (деформированными) углами и ребрами (рис. 1, б) и в отношении зародыша, на котором при перекате частично сформирована обо- лочка окатыша (рис. 1, в). Положение устойчивости характеризуется углом переката γк , составленным из перпендикуляра на опорную плоскость ШГ и линии действующей силы. Графически этот угол можно за- фиксировать при γ = γк . Выход из состояния устойчи- вости и формирование переката происходят при γ > γк . Для этих условий у зародыша правильной кубической формы угол переката превышает 45° (рис. 1, а). тать с более высокой удельной производительностью. Для организации режима переката зародышей сложной формы и формирования окатышей на промышленном окомкователе необходимо задать соответствующий угол наклона тарели к горизонту и ее частоту вращения в зависимости от степени деформации зародыша [2]. Для этого требуется проанализировать характер сило- вого воздействия на зародыш сложной формы в режиме переката со стороны сил, формирующихся вращаю- щейся тарелью окомкователя. ШГ при минимальной величине центробежной силы, что соответствует расположению зародыша в цент- ральных областях зоны окомкователя или работе рабо- чего органа с низкой частотой вращения (n < 3 об/мин) (рис. 1). В этом случае устойчивость зародыша или, на- против, его перекат определяются горизонтальным по- ложением его центра массы (Ц) относительно опорного ребра OО1 , расположенного поперек вероятного на- правления переката (рис. 1). Если линия действующей силы (в данном случае силы тяжести) переместится за опорное ребро OО1 , то сформируется момент силы, который выведет зародыш из состояния устойчивости и сформирует его перекат. Это также справедливо в от- ношении зародыша со смятыми (деформированными) углами и ребрами (рис. 1, б) и в отношении зародыша, на котором при перекате частично сформирована обо- лочка окатыша (рис. 1, в). Положение устойчивости характеризуется углом переката γк , составленным из перпендикуляра на опорную плоскость ШГ и линии действующей силы. Графически этот угол можно за- фиксировать при γ = γк . Выход из состояния устойчи- вости и формирование переката происходят при γ > γк . Для этих условий у зародыша правильной кубической формы угол переката превышает 45° (рис. 1, а). Целью настоящей работы является анализ сил, действующих на кубический зародыш при его перекате на шихтовом гарнисаже в условиях его пластической деформации, и определение рабочих параметров рабо- ты окомкователя. DOI: 10.17073/0368-0797-2018-2-87-95 DOI: 10.17073/0368-0797-2018-2-87-95 Производство железорудных окатышей в ряде стран с развитой металлургической промышленностью являет- ся основным направлением в технике подготовки сырья к доменной плавке и металлизации [1 – 4]. Причем зна- чительная доля тонкодисперсного концентрата и вторич- ного техногенного сырья в производстве окатышей ком- куется в тарельчатых окомкователях [1, 2]. В последнее время этот тип окомкователя предложено использовать в промышленности для окомкования аглошихты [5, 6]. Результаты исследований и выводы, приведенные в этих источниках, свидетельствуют о повышении качества окомкованного продукта в тарельчатых окомкователях и увеличении производительности агломашин. Резервные возможности окомковательного оборудования для полу- чения окатышей предложено использовать в технологии принудительного зародышеобразования [1, 2]. шей сложной формы, близкой к кубической, в холостой зоне тарели и создание оболочки окатышей путем их доокомкования в рабочей зоне окомкователя в режиме переката [7, 8]. Принудительное зародышеобразование основано на технике напыления влажной шихты сжатым воздухом на шихтовый гарнисаж (ШГ) с получением плотного слоя шихты, который механическим путем принуди- тельно делится на мерные зародышевые фрагменты. Эта технология экспериментально опробована в лабо- раторных условиях на окомкователях малого диамет ра (0,6 и 1,0 м) и, в отличие от известной технологии, позволяет получать окатыши с дифференцированным распределением пористости по их сечению с более высокой долей открытых пор [9]. В процессе принуди- тельного зародышеобразования происходит удаление влаги, способное снизить влажность окатышей после окомкования на 0,5 – 2,5 %, а окомкователь может рабо- Получение сырых окатышей по этой технологии включает две основные стадии: формирование зароды- 87 Известия высших учебных заведений. Черная металлургия. 2018. Том 61. № 2 тать с более высокой удельной производительностью. Для организации режима переката зародышей сложной формы и формирования окатышей на промышленном окомкователе необходимо задать соответствующий угол наклона тарели к горизонту и ее частоту вращения в зависимости от степени деформации зародыша [2]. Для этого требуется проанализировать характер сило- вого воздействия на зародыш сложной формы в режиме переката со стороны сил, формирующихся вращаю- щейся тарелью окомкователя. ШГ при минимальной величине центробежной силы, что соответствует расположению зародыша в цент- ральных областях зоны окомкователя или работе рабо- чего органа с низкой частотой вращения (n < 3 об/мин) (рис. 1). В этом случае устойчивость зародыша или, на- против, его перекат определяются горизонтальным по- ложением его центра массы (Ц) относительно опорного ребра OО1 , расположенного поперек вероятного на- правления переката (рис. 1). Если линия действующей силы (в данном случае силы тяжести) переместится за опорное ребро OО1 , то сформируется момент силы, который выведет зародыш из состояния устойчивости и сформирует его перекат. Fig. 1. Diagrams of the germ stability (a – в) and of the roll regime (г – е) in the CS; germs: a, г – with regular cubic form; б, д – with partially crushed corners and edges; в, e – with completely crushed corners and edges Fig. 1. Diagrams of the germ stability (a – в) and of the roll regime (г – е) in the CS; germs: l bi f б д i h i ll h d d d i h l l h d g g g y ( ) g ( ) ; g gular cubic form; б, д – with partially crushed corners and edges; в, e – with completely crushed corners and edges (1) (1) тy Момент сил, вызывающих перекат зародыша через опорное ребро, описывается уравнением где hзр – размер ребра несмятого зародыша правильной кубической формы, м; hзр1 – размер ребра зародыша с частично смятыми углами и гранями, м (рис. 1). MΣ = 0,5 hзр FΣ . (5) (5) Численное значение Kсм может варьироваться от единицы у несмятого зародыша кубической формы до нуля у зародыша с полностью смятыми углами и ребра- ми, на которые одновременно накатывается влажная шихта. В процессе переката его плоские стороны округ ляются и становятся выпуклыми. В конце технологии окомкования форма окатыша приближается к форме шара (рис. 1, в). где FΣ – суммарная сила, вызывающая перекат зароды- ша, Н. Для этого момента суммарной силы плечом являет- ся расстояние 0,5hзр. р Момент силы FтZ , препятствующей перекату заро- дыша, описывается уравнением Угол переката зародыша с частично смятыми углами и ребрами может быть определен в состоянии устойчи- вости зародыша на ШГ геометрическими построения ми из треугольника ЦЦ’О (рис. 1, б) как арктангенс от- ношения его сторон: MтZ = 0,5 Kсм hзр FтZ . (6) (6) Для этого момента силы плечом является расстоя- ние 0,5Kсм hзр . Для этого момента силы плечом является расстоя- ние 0,5Kсм hзр . см зр Силы, влияющие на устойчивость и перекат зароды- ша, определяются по выражениям (2) (2) FтY = mg sin γк; (7) (8) FтZ = mg cos γк , (9) (7) Для рассматриваемого частного случая величины углов переката γк для коэффициентов Kсм , равных 0; 0,25; 0,5; 0,75; 1,0, соответственно равны 0; 14°; 26,6°; 36,9°; 45°. (8) FтZ = mg cos γк , (9) (9) Более общим случаем выхода зародыша из состоя ния устойчивости в режим переката на наклонной плоскости ШГ является работа окомкователя при срав- нительно высокой центробежной силе (n > 3 об/мин) и расположении зародыша в периферийных областях рабочей зоны окомкователя (квадранты Ι и ΙΙ) (рис. 2). где m – масса зародыша, кг; g – ускорение свободно- го падения, м/c2; ω – угловая частота вращения, рад/c; n – частота вращения тарели окомкователя, об/мин. n – частота вращения тарели окомкователя, об/мин. Суммарная сила FΣ формируется как результирую- щая между проекцией силы тяжести FтY и центробеж- ной силой Fц . Она определяется по известному выра- жению [10, 15] Устойчивость зародыша на вращающемся ШГ опре- деляется балансом моментов сил, вызывающих пере- кат, и сил, препятствующих перекату. DOI: 10.17073/0368-0797-2018-2-87-95 Для анализа сил, действующих на кубический за- родыш при его перекате, условно приняли, что смятие углов и граней зародыша не сопровождается изменением его массы и центра тяжести, а действием силы трения на ШГ пренебрегли вследствие ее малой величины по сравнению силами, вызывающими перекат. В расчете не учитывали ударное взаимодействие зародышей между собой в слое, которое в общем случае может как пре- пятствовать, так и способствовать перекату, и которое достаточно трудно учесть в расчете. Предполагали, что плоскость ШГ является абсолютно ровной и не дефор- мируется в ходе переката от соударений с зародышами. Для зародыша с частично смятыми углами и ребра- ми угол переката зависит от степени его деформации, которую определяли по коэффициенту смятия (дефор- мации) зародыша Kсм , вычисляемого по выражению Частным случаем устойчивости зародыша на на- клонной плоскости является отсутствие переката на Рис. 1. Схемы, иллюстрирующие устойчивость (а – в) зародыша и режим переката (г – е) на шахтный гарнисаж. Зародыши: а, г – правильной кубической формы; б, д – с частично смятыми углами и ребрами; в, е – с полностью смятыми углами и ребрами Рис. 1. Схемы, иллюстрирующие устойчивость (а – в) зародыша и режим переката (г – е) на шахтный гарнисаж. Зародыши: а, г – правильной кубической формы; б, д – с частично смятыми углами и ребрами; в, е – с полностью смятыми углами и ребрами Fig. 1. Diagrams of the germ stability (a – в) and of the roll regime (г – е) in the CS; germs: a, г – with regular cubic form; б, д – with partially crushed corners and edges; в, e – with completely crushed corners and edges 88 Металлургические технологии (1) ранте I и препятствует перекату в квадранте II, так как ее проекция на ось Y(R) в этом квадранте противона- правлена силе Fтy. (1) Баланс моментов этих сил представляет собой равенство [10 – 12]: (10) (3) (10) (3) где MΣ и MΣ0 – моменты сил, вызывающих перекат заро- дыша и препятствующих его перекату, Н·м. где θ – угол между силами Fц и FтY . С помощью угла θ определяются положение заро- дыша на ШГ и область рабочей зоны окомкователя в квадрантах Ι и ΙΙ до того момента, когда начинается пе- рекат зародыша. При движении зародыша в этой зоне от точки 1 к точке 2, в которой происходит его перекат, угол θ увеличивается от нуля до 180° (рис. 2). Для того, чтобы произошел выход зародыша из со- стояния устойчивости в режим переката, необходимо выполнить условие [13, 14] MΣ > MΣ0 . (4) (4) Равенство (3) с учетом выражений (5) – (10) примет вид К силам, вызывающим перекат зародыша через опорное ребро ОО1 , относятся: сила Fтy , являющаяся проекцией силы тяжести на ось Y(R), и центробежная сила Fц . К силе, препятствующей перекату зародыша через опорное ребро ОО1 , относится сила Fтz , которая является проекцией силы тяжести на ось Z и ориенти- рована перпендикулярно ШГ и силам Fц и Fтy . Центро- бежная сила способствует перекату зародыша в квад вид (11) (11) Решая уравнение (11), получим выражение для на- хождения угла переката γк : 89 Известия высших учебных заведений. Черная металлургия. 2018. Том 61. № 2 Рис. 2. Схема сил и моментов сил, действующих на зародыш, расположенный на тарели окомкователя до начала переката Fi 2 S h f f d t f f ti th l t d th ll ti di k b f th b i i f th ll Рис. 2. Схема сил и моментов сил, действующих на зародыш, расположенный на тарели окомкователя до начала переката Fig. 2. Scheme of forces and moments of forces acting on the germ located on the pelletizer disk before the beginning of the roll (12) Анализируя формулу (10), можно отметить, что суммарная сила представляет собой периодическую функцию угла поворота θ и может иметь как знакопе- ременный (при FтY < Fц , рис. 3, а), так и пульсирующий характер (при FтY ≥ Fц , рис. 3, б). (12) ц Можно заключить, что при угле θ = 0 ÷ 90° (квад рант Ι) условия для переката зародыша по ШГ наиболее благоприятные, так как сила тяжести FтY и центробеж- ная сила Fц направлены под острым углом друг к другу, в результате чего уменьшается значение результирующей силы. (1) В квадранте ΙΙ при θ = 90 – 180° указанные силы располагаются под тупым углом, тем самым уменьша- ют результирующую силу. Поэтому для расчетов угла Задавая угол θ в этом уравнении от нуля до 180° и соответственно область ШГ рабочей зоны окомкова- теля в квадрантах Ι и ΙΙ, можно вычислить значение угла γк , превысив которое можно осуществить перекат зародыша на ШГ. Рис. 3. Зависимости суммарной силы FΣ от угла θ. Штриховая линия соответствует углам поворота тарели, при которых зародыш не может находиться в квадрантах I и II Рис. 3. Зависимости суммарной силы FΣ от угла θ. Штриховая линия соответствует углам поворота тарели, при которых зародыш не может находиться в квадрантах I и II Fig. 3. Dependences of the total force FΣ on the angle θ. The dashed line corresponds to the rotation angles of the plate at which the germ cannot be in the quadrants I and II 90 Металлургические технологии переката γк была выбрана точка 2 с углом θ = 180°, в которой реализуются наименее благоприятные условия для переката, так как в ней силы FтY и Fц направлены диаметрально противоположно друг другу и суммар- ная сила FΣ имеет минимальное значение. Дополни- тельным условием для выбора угла θ = 180° (точка 2) явились требования технологии [16 – 19], согласно ко- торым комкуемый материал должен полностью зани- мать площадь рабочей зоны тарели, вследствие чего перекат зародышей должен быть реализован в верхней точке вертикального диаметра тарели. Результаты рас- четов представлены в виде безразмерного комплекса N, составленного из отношения рассчитанного угла γк и угла γ, заданного в технологическом процессе: ем угла наклона тарели γ свыше 45° параметр N резко снижается, и область возможного переката зародышей расширяется. При N > 1 перекат зародыша по ШГ не- возможен, поскольку процесс переката несмятого за- родыша (Kсм = 1,0) протекает при N < 1, то угол γ для n = 3 об/мин должен быть не менее 45° (рис. 4, а). С увеличением параметра n значение угла γ должно быть скорректировано в сторону увеличения. В част- ности, для n = 12 об/мин угол γ должен быть более 55° (рис. 4, г). Результаты расчетов показывают, что при- нудительно изменяемый коэффициент смятия заро- дышей может быть фактором, влияющим на рабочие параметры окомкователя. Установлено (рис. 4), что с уменьшением коэффициента смятия зародыша и при- ближения его формы к сферической область переката по величине угла γк расширяется, а перекат возможен при меньших углах γ наклона тарели к горизонту. Fig. 4. Dependence of the parameter N on the germ crushing factor Kcm and the angle of disk inclination γ at the rotation frequencies n, rpm, equaling to 3 (a), 6 (б), 9 (в) and 12 (г) (1) Dependence of the parameter N on the rotation frequency n and the angle γ for the factors Kcm , equaling to 1.0 (a); 0.75 (б); 0.5 (в) and 0.25 (г) meter N on the rotation frequency n and the angle γ for the factors Kcm , equaling to 1.0 (a); 0.75 (б); 0.5 (в) and 0.25 (г) Fig. 5. Dependence of the parameter N on the rotation frequency n and the angle γ for the ются. Для того, чтобы скорректировать режим переката (N < 1), используя полученные данные, необходимо с ростом угла γ на каждые 5° (при условии γк < γ) частоту вращения тарели окомкователя увеличивать примерно на 3 об/мин. Следует заметить, что режим зародыше- образования в цикле: напыление шихты на ШГ с по- лучением плотного напыленного слоя – деление слоя на зародыши – деформация зародышей, также должен быть скорректирован, поскольку все процессы проте- кают на одном вращающемся днище тарели одновре- менно. Коррекция напыления может быть проведена достаточно оперативно с помощью таких параметров, как угол атаки ВШС, расстояние до напыленного слоя и др. [2]. В ряде источников [20 – 22] коррекцию напы- ления наряду с указанными параметрами предложено осуществлять с помощью кинематических параметров, включающих траекторию и скорость перемещения на- пыляющего устройства, регулирование расхода дис- персной фазы за счет изменения скорости вращения шнекового питателя и др. ремещается из квадранта ΙΙ в квадрант Ι, что реально происходит на практике [1, 2]. Следует подчеркнуть, что значения Kсм и γк могут измениться на стадии зародышеобразования в холостой зоне тарели оком- кователя, о чем свидетельствуют эксперименталь- ные данные [2]. В частности, на этой стадии можно организовать предварительное смятие углов и ре- бер зародышей механическим путем, что приведет к изменению коэффициента Kсм , значение которого можно вычислить, проведя физический эксперимент. Рассчитанное значение угла γк для стадии переката и роста массы зародышей в рабочей зоне окомкователя потребует корректировки угла γ и частоты вращения тарели окомкователя. ремещается из квадранта ΙΙ в квадрант Ι, что реально происходит на практике [1, 2]. Следует подчеркнуть, что значения Kсм и γк могут измениться на стадии зародышеобразования в холостой зоне тарели оком- кователя, о чем свидетельствуют эксперименталь- ные данные [2]. В частности, на этой стадии можно организовать предварительное смятие углов и ре- бер зародышей механическим путем, что приведет к изменению коэффициента Kсм , значение которого можно вычислить, проведя физический эксперимент. (1) Это характерно для зародышей даже с малым коэффици- ентом смятия (Kсм = 0,7), у которых угол γ может быть уменьшен с 45 до 35° (n = 3 об/мин). Результаты расчетов представлены на рис. 4 – 7. Безразмерный комплекс N позволяет в зависимости от коэффициента смятия и частоты вращения тарели окомкователя прогнозировать условия переката за- родыша на ШГ и назначить соответствующий угол γ наклона днища тарели к горизонту. Зависимость N = f (Kсм ) (рис. 4) показывает, что перекат зародыша по ШГ возможен при N < 1. Видно, что с увеличени- В процессе сфероидезации зародыша параметры Kсм и γк изменяются довольно существенно, а назна- чаемый угол γ естественно остается неизменным в течение всего технологического процесса. Это значит, что траектория движения зародыша меняется, и он пе- Рис. 4. Зависимость параметра N от коэффициента смятия зародыша Kсм и угла наклона тарели γ при частотах вращения n, об/мин, равных 3 (а), 6 (б), 9 (в) и 12 (г) Fig. 4. Dependence of the parameter N on the germ crushing factor Kcm and the angle of disk inclination γ at the rotation frequencies n, rpm, equaling to 3 (a), 6 (б), 9 (в) and 12 (г) Рис. 4. Зависимость параметра N от коэффициента смятия зародыша Kсм и угла наклона тарели γ при частотах вращения n, об/мин, равных 3 (а), 6 (б), 9 (в) и 12 (г) Fig. 4. Dependence of the parameter N on the germ crushing factor Kcm and the angle of disk inclination γ at the rotation frequencies n, rpm, equaling to 3 (a), 6 (б), 9 (в) and 12 (г) 91 91 Известия высших учебных заведений. Черная металлургия. 2018. Том 61. № 2 Рис. 5. Зависимость параметра N от частоты вращения n и угла γ при коэффициентах Kсм , равных 1,0 (а); 0,75 (б); 0,5 (в) и 0,25 (г) Fig. 5. Dependence of the parameter N on the rotation frequency n and the angle γ for the factors Kcm , equaling to 1.0 (a); 0.75 (б); 0.5 (в) and 0.25 (г) метра N от частоты вращения n и угла γ при коэффициентах Kсм , равных 1,0 (а); 0,75 (б); 0,5 (в) и 0,25 (г) Рис. 5. Зависимость параметра N от частоты вращения n и угла γ при коэффициентах Kсм , равных 1,0 (а); 0,75 (б); 0,5 (в) и 0,25 (г) Fig. 5. (1) Рассчитанное значение угла γк для стадии переката и роста массы зародышей в рабочей зоне окомкователя потребует корректировки угла γ и частоты вращения тарели окомкователя. С увеличением частоты вращения тарели окомкова- теля область стабильного переката зародышей (N < 1) существенно снижается (рис. 5). Вернуть процесс пе- реката в его стабильную зону возможно с увеличением угла наклона тарели свыше 45°. Заметим, что на произ- водстве это наиболее мобильный параметр воздействия на технологический процесс. При перемещении зародыша от точки 1 к точке 2 (на рис. 7 угол θ меняется от 30 до 180°) расчетный угол переката γк увеличивается с разной интенсивностью при работе тарели окомкователя на различных часто- тах вращения. Это значит, что для соблюдения условия N < 1 следует одновременно увеличивать и угол γ, что С ростом угла наклона тарели окомкователя γ пара- метр N уменьшается, и зона стабильного переката заро- дышей расширяется (рис. 6). Данные этого рисунка до- полняют ранее полученные выводы о том, что с ростом частоты вращения тарели параметры N и γк увеличива- 92 Металлургические технологии Рис. 6. Зависимость параметра N от угла γ и коэффициента Kсм при частотах вращения тарели n, об/мин, равных 3,0 (а), 6 (б), 9 (в) и 12 (г) Fig. 6. Dependence of the parameter N on the angle γ and the factor Kcm at the rotation frequencies of the plates n, rpm, equaling to 3 (a), 6 (б), 9 (в) and 12 (г) Рис. 6. Зависимость параметра N от угла γ и коэффициента Kсм при частотах вращения тарели n, о Зависимость параметра N от угла γ и коэффициента Kсм при частотах вращения тарели n, об/мин, равных 3,0 (а), 6 (б), 9 ( Fig. 6. Dependence of the parameter N on the angle γ and the factor Kcm at the rotation frequencies of the plates n, rpm, equaling to 3 (a), 6 (б), 9 (в) and 12 (г) Fig. 6. Dependence of the parameter N on the angle γ and the factor Kcm at the rotation frequencies of the plates n, rpm, equaling to 3 (a), 6 (б), 9 (в) and 12 (г) физически сделать невозможно. В этом случае следует подобрать такую частоту вращения тарели окомковате- ля, при которой угол переката γк изменялся бы в наи- более узких пределах. Результаты расчетов показывают, что это возможно реализовать на тарелях, работающих с низкой частотой вращения. (1) В частности, для часто- ты вращения n = 3 об/мин (Kсм = 1,0) угол γк меняется в наиболее узком интервале значений (γк = 44 – 46°). Это позволяет на практике сделать соответствующую кор- ректировку угла γ при условии N < 1. Установлено, что для организации режима переката зародыша сложной формы угол наклона тарели оком- кователя к горизонту следует назначать в зависимости от частоты его вращения и коэффициента смятия за- родышей. БИБЛИОГРАФИЧЕСКИЙ СПИСОК 1. Павловец В.М. Окатыши в технологии экстракции металлов из руд: Учебн. пособие. – Новокузнецк: Изд. центр СибГИУ, 2014. – 345 c. 1. Павловец В.М. Окатыши в технологии экстракции металлов из руд: Учебн. пособие. – Новокузнецк: Изд. центр СибГИУ, 2014. – 345 c. 2. Павловец В.М. Расширение функциональных возможностей аг- регатов для подготовки железорудного сырья к металлургичес кой плавке. – Новокузнецк: Изд. центр СибГИУ, 2016. – 373 c. Выводы. В результате проведенного анализ сил, действующих на кубический зародыш при его перека- те на ШГ в условиях пластической деформации, пост роена математическая модель, позволяющая опреде- лить оптимальные параметры работы окомкователя для производства окатышей по технологии принуди- тельного зародышеобразования. Сформулированы условия переката зародышей на ШГ в рабочей зоне окомкователя и корректирующие мероприятия в усло- виях пластической деформации при смятии его углов и ребер. Показаны особенности движения зародыша в рабочей зоне тарели окомкователя. Отмечены условия для корректировки режима принудительного зароды- шеобразования и мероприятия по целенаправленно- му воздействию на коэффициент смятия зародыша. Выводы. В результате проведенного анализ сил, действующих на кубический зародыш при его перека- те на ШГ в условиях пластической деформации, пост роена математическая модель, позволяющая опреде- лить оптимальные параметры работы окомкователя для производства окатышей по технологии принуди- тельного зародышеобразования. Сформулированы условия переката зародышей на ШГ в рабочей зоне окомкователя и корректирующие мероприятия в усло- виях пластической деформации при смятии его углов и ребер. Показаны особенности движения зародыша в рабочей зоне тарели окомкователя. Отмечены условия для корректировки режима принудительного зароды- шеобразования и мероприятия по целенаправленно- му воздействию на коэффициент смятия зародыша. 3. Frantes K. North American Iron Mines Running Flat Out to Meet Domestic and Worldwide Demand // Skillings’ Mining Review. 2005. Vol. 94. No. 7. Pр. 6 – 21. 3. Frantes K. North American Iron Mines Running Flat Out to Meet Domestic and Worldwide Demand // Skillings’ Mining Review. 2005. Vol. 94. No. 7. Pр. 6 – 21. 4. BASICS IN MINERAL PROCESSING: Metso Corporation, 2015. – 752 p. 5. Chikashi Kamijo, Masaki Hara, Takazo Kawaguchi et al. Sinter feed granulation improvement technique by pan pelletizer // Zairyo to Prosesu = CAMP ISIJ. 2010. Vol. 23. No. 1. P. 5. to Prosesu = CAMP ISIJ. 2010. Vol. 23. No. 1. P. 5. 6. Kei Watanabe, Masahide Yasukawa, Chikashi Kamisaka et al. Improvement of productivity by installing P-type separate granula tion process at Wakayama no. 5 sintering plank // Zairyo to Prosesu = CAMP ISIJ. 2010. Vol. 23. No. 1. P. 6. БИБЛИОГРАФИЧЕСКИЙ СПИСОК = CAMP ISIJ. 2010. Vol. 23. No. 1. P. 6. 7. Павловец В.М. Исследование процесса получения влажных окатышей с использованием принудительного зародышеобра- зования // Изв. вуз. Черная металлургия. 2010. № 6. С. 15 – 20. 93 Известия высших учебных заведений. Черная металлургия. 2018. Том 61. № 2 Рис. 7. Зависимость угла переката γк от угла θ и частоты вращения тарели окомкователя n при коэффициентах Kсм , равных 1,0 (а); 0,75 (б); 0,5 (в) и 0,25 (г) Fig. 7. Dependence of the roll angle γk on the angle θ and rotation frequency of the disc pelletizer at coefficients Kcm , equaling to 1.0 (a); 0.75 (б); 0.5 (в) and 0.25 (г) Рис. 7. Зависимость угла переката γк от угла θ и частоты вращения тарели окомкователя n при коэффициентах Kсм , равных 1,0 (а); 0,75 (б); 0,5 (в) и 0,25 (г) Fig. 7. Dependence of the roll angle γk on the angle θ and rotation frequency of the disc pelletizer at coefficients Kcm , equaling to 1.0 (a); 0.75 (б); 0.5 (в) and 0.25 (г) 8. Павловец. В.М. Развитие принципов принудительного зароды- шеобразования в процессе получения железорудных окатышей // Изв. вуз. Черная металлургия. 2014. № 10. С. 15 – 22. 16. Интенсификация производства и улучшение качества окаты- шей / Ю.С. Юсфин, Н.Ф. Пашков, П.К. Антоненко, Р.М. Жак. – М.: Металлургия, 1994. – 240 с. 17. Ручкин И.Е. Производство железорудных окатышей. – М.: Ме- таллургия, 1976. – 184 с. 9. Павловец В.М. Формирование структуры железорудных окаты- шей, полученных с использованием теплосилового напыления влажной шихты // Изв. вуз. Черная металлургия. 2016. № 12. С. 857 – 863. 18. Маерчак Ш. Производство окатышей. – М.: Металлургия, 1982. – 232 с. 10. Яблонский А.А., Никифорова В.М. Курс теоретической меха- ники. Ч. 1. – М.: Высшая школа, 1966. – 429 c. 19. Коротич В.И. Теоретические основы окомкования железоруд- ных материалов. – М.: Металлургия, 1966. – 152 с. 20. Goejen J.G., Miller R.A., Brindley W.J., Leissler G.W. A simulation technique for predicting defects of thermal sprayed coatings: NASA Technical Memorandum TM-106939, 1995. 11. Sunil Golwala. Lecture Notes on Classical Mechanics for Physics 106ab. Publisher: CreateSpace Independent Publishing Platform, 2014. – 396 p. 21. Hansbo A. and Nylén P. Models for the simulation of spray deposi tion and robot motion optimization in thermal spraying of rotating objects // Surface Coatings Technol. 1999 Vol. 122. Pp. 191 – 201.fi 12. John R. Taylor. Classical mechanics. Siberian State Industrial University, Russia, Novokuznetsk Siberian State Industrial University, Russia, Novokuznetsk Abstract. The movement specifics of a germ with complex shape on the pelletizing disk in production of iron-ore pellets are considered. As a result of the analysis of the forces acting on the cubic germ during its roll on the charge scull (CS) in the conditions of plastic deformation, a mathematical model was build that allows the optimal parameters of the pelletizer to be determined for the production of pellets based on the technology of a forced germ formation. Two particular cases of germ stability on the pelletizer CS and the conditions for the over- coming of the stability associated with the formation of a roll are considered. The germ stability, or conversely, its roll is determined by the horizontal position of its mass center relative to the support edge located across the probable direction of the roll. If the line of the acting force (gravity force) moves behind the support edge and the reference plane, a force moment will be generated which will lead the germ out of the stability state and form a germ roll. The first special case of germ stability on the inclined plane is the absence of a roll on the CS with a minimum value of the centrifugal force correspond- ing to the location of the germ in the central areas of the pelletizing zone or to operation of a working element with a low rotation speed (n < 3 rpm). A more general case of the transition of the germ from the state of stability into the roll regime on the inclined plane of CS is the work of the pelletizer when centrifugal force is relatively high (n > 3 rpm) and location of the germ in peripheral areas of the work- ing zone of the pelletizer. For these cases the conditions for germs roll on the CS in the pelletizer working zone and correcting measures in the conditions of plastic deformation during the corners and edges crushing are formulated. The features of the germ movement in the working zone of the pelletizing disk are shown. The conditions for correcting the regime of forced germ formation are provided, and the measures are indicated for the purposeful impact on the crushing factor of a germ. Siberian State Industrial University, Russia, Novokuznetsk It was established that for the organization of the regime of a germ roll with complex shape the angle of disk inclina- tion to the horizon should be set depending on the frequency of its rotation and the crushing factor of germs. 7. Pavlovets V.M. Moisture-laden pellets production with forced nucleation process. Izvestiya VUZov. Chernaya metallurgiya = Iz- vestiya. Ferrous Metallurgy. 2010, no. 6, pp. 15–20. (In Russ.). 8. Pavlovets V.M. Development of principles of forced nucleation in the production process of iron ore pellets. Izvestiya VUZov. Cher- naya metallurgiya = Izvestiya. Ferrous Metallurgy. 2014, no. 10, pp. 15–22. (In Russ.). 9. Pavlovets V.M. Formation of the structure of iron ore pellets pro- duced using heat-spraying of wet charge. Izvestiya VUZov. Cher- naya metallurgiya = Izvestiya. Ferrous Metallurgy. 2016, no. 12, pp. 857–863. (In Russ.). 10. Yablonskii A.A., Nikiforova V.M. Kurs teoreticheskoi mekhaniki. Ch. 1 [Course of theoretical mechanics. Part 1]. Moscow: Vysshaya shkola, 1966, 429 p. (In Russ.). 11. Sunil Golwala. Lecture notes on classical mechanics for physics 106ab. Publisher: CreateSpace Independent Publishing Platform, 2014, 396 p. 12. John R. Taylor. Classical mechanics. Publisher: University Science Books, 2005, 786 p. 13. James L., Meriam L., Kraige G. Engineering mechanics. Vol. 1. Statics. Wiley; 7 edition, 2011, 544 p. 14. James L., Meriam L., Kraige G. Engineering mechanics. Vol. 2. Dy- namics. Wiley; 7 edition, 2012, 752 p. 15. Demidovich B.P., Kudryavtsev V.A. Kratkii kurs vysshei matema- tiki. Uchebnoe posobie dlya vuzov [A short course in higher mathe matics. Textbook for universities]. Moscow: Astrel’; AST, 2001, 656 p. (In Russ.).ii 16. Yusfin Yu.S. etc. Intensifikatsiya proizvodstva i uluchshenie kachest- va okatyshei [Intensification of production and improvement of the quality of pellets]. Moscow: Metallurgiya, 1994, 240 p. (In Russ.). 17. Ruchkin I.E. Proizvodstvo zhelezorudnykh okatyshei [Production of iron-ore pellets]. Moscow: Metallurgiya, 1976, 184 p. (In Russ.). 18. Maerchak Sh. Proizvodstvo okatyshei [Production of pellets]. Mos- cow: Metallurgiya, 1982, 232 p. (In Russ.). 19. Korotich V.I. Teoreticheskie osnovy okomkovaniya zhelezorudnykh materialov [Theoretical foundations for the palletization of iron-ore materials]. Moscow: Metallurgiya, 1966, 152 p. (In Russ.). Keywords: germ with complex shape, germ crushing factor, germ roll, pel- letizer, charge skull, forced germ formation, spraying of wet charge, iron-ore pellets. 20. Goejen J.G., Miller R.A., Brindley W.J., Leissler G.W. A simulation technique for predicting defects of thermal sprayed coatings. NASA Technical Memorandum TM-106939, 1995. DOI: 10.17073/0368-0797-2018-2-87-95 REFERENCES 21. Hansbo A., Nylén P. Models for the simulation of spray deposition and robot motion optimization in thermal spraying of rotating ob- jects. Surface Coatings Technol. 1999, vol. 122, pp. 191–201. fi 1. Pavlovets V.M. Okatyshi v tekhnologii ekstraktsii metallov iz rud: uchebn. posobie [Pellets in the technology of metals extraction from ores: Manual]. Novokuznetsk: SibGIU, 2014, 345 p. (In Russ.). 1. Pavlovets V.M. Okatyshi v tekhnologii ekstraktsii metallov iz rud: uchebn. posobie [Pellets in the technology of metals extraction from ores: Manual]. Novokuznetsk: SibGIU, 2014, 345 p. (In Russ.). 22. Ensz М.Т., Griffith М.L., Reckaway D.Е. Critical issues for func- tionally graded material deposition by laser engineered net shaping. Available at URL: http://edge.cs.drexel.edu/GICL/people/schroe der/references/mpif02me.pdf 2. Pavlovets V.M. Rasshirenie funktsional’nykh vozmozhnostei agre- gatov dlya podgotovki zhelezorudnogo syr’ya k metallurgicheskoi plavke [Expansion of functional capabilities of aggregates for the preparation of iron-ore raw materials for metallurgical smelting]. Novokuznetsk: SibGIU, 2016, 373 p. (In Russ.).l 2. Pavlovets V.M. Rasshirenie funktsional’nykh vozmozhnostei agre- gatov dlya podgotovki zhelezorudnogo syr’ya k metallurgicheskoi plavke [Expansion of functional capabilities of aggregates for the preparation of iron-ore raw materials for metallurgical smelting]. Novokuznetsk: SibGIU, 2016, 373 p. (In Russ.).l БИБЛИОГРАФИЧЕСКИЙ СПИСОК – Publisher: University Scien ce Books, 2005. – 786 p. 13. James L., Meriam L., Kraige G. Engineering Mechanics. Vol. 1. Statics. Wiley; 7 edition, 2011. – 544 p. 22. Ensz М.Т., Griffith М.L., Reckaway D.Е. Critical issues for func tionally graded material deposition by laser engineered net shaping. http://edge.cs.drexel.edu/GICL/people/schroeder/references/ mpif02me.pdf 14. James L., Meriam L., Kraige G. Engineering Mechanics. Vol. 2. Dynamics. Wiley; 7 edition, 2012. – 752 p. 15. Демидович Б.П., Кудрявцев В.А. Краткий курс высшей мате- матики. Учебное пособие для вузов. – М.: Астрель; АСТ, 2001. – 656 c. Поступила 17 февраля 2017 г. 94 Металлургические технологии Izvestiya VUZov. Chernaya Metallurgiya = Izvestiya. Ferrous Metallurgy. 2018. Vol. ernaya Metallurgiya = Izvestiya. Ferrous Metallurgy. 2018. Vol. 61. No. 2, pp. 87–95. V.M. Pavlovets, A.V. Gerasimuk 6. Kei Watanabe, Masahide Yasukawa, Chikashi Kamisaka etc. Im- provement of productivity by installing P-type separate granulation process at Wakayama no. 5 sintering plank. Zairyo to Prosesu = CAMP ISIJ. 2010, vol. 23, no. 1, pp. 6. 6. Kei Watanabe, Masahide Yasukawa, Chikashi Kamisaka etc. Im- provement of productivity by installing P-type separate granulation process at Wakayama no. 5 sintering plank. Zairyo to Prosesu = CAMP ISIJ. 2010, vol. 23, no. 1, pp. 6. MOVEMENT SPECIFICS OF A GERM WITH COMPLEX SHAPE ON THE PELLETIZING DISK IN IRON-ORE PELLETS PRODUCTION V.M. Pavlovets, A.V. Gerasimuk Information about the authors: Novokuznetsk: SibGIU, 2016, 373 p. (In Russ.). V.M. Pavlovets, Cand. Sci., (Eng.), Assist. Professor of the Chair “Thermal Power and Ecology” ( pavlowets.victor@yandex.ru ) A.V. Gerasimuk, Postgraduate of the Chair of Electrical Engineering, Electric Drive and Industrial Electronics ( heavymetal7200@gmail.com ) 3. Frantes K. North American iron mines running flat out to meet domestic and worldwide demand. Skillings’ Mining Review. 2005, vol. 94, no. 7, pр. 6–21. asics in mineral processing: Metso Corporation. 2015, 752 p 4. Basics in mineral processing: Metso Corporation. 2015, 752 p. 5. Chikashi Kamijo, Masaki Hara, Takazo Kawaguchi etc. Sinter feed granulation improvement technique by pan pelletizer. Zairyo to Prosesu = CAMP ISIJ. 2010, vol. 23, no. 1, pp. 5. 5. Chikashi Kamijo, Masaki Hara, Takazo Kawaguchi etc. Sinter feed granulation improvement technique by pan pelletizer. Zairyo to Prosesu = CAMP ISIJ. 2010, vol. 23, no. 1, pp. 5. Received February 17, 2017 Received February 17, 2017 95
https://openalex.org/W4282931820	https://publicera.kb.se/tgv/article/download/3964/3589	Swedish	null	Marginaliserade manskroppar: Bilder av feta arbetarklassmän	Tidskrift för genusvetenskap	2,006	cc-by	12,380	Den traditionella bilden av arbetarklassmannens kropp är hård och muskulös, präglad av fysiskt arbete. Anna Johansson analyserar här en annan bilden av en mjuk och fet manskropp som blivit allt vanligare på TV och i filmer. Den traditionella bilden av arbetarklassmannens kropp är hård och muskulös, präglad av fysiskt arbete. Anna Johansson analyserar här en annan bilden av en mjuk och fet manskropp som blivit allt vanligare på TV och i filmer. Marginaliserade manskroppar: Bilder av feta arbetarklassmän Anna Johansson I vissa fall i samma utsatta situationer och poser som kvinnor och barn vanligtvis brukar avbildas i, eller i poser som skulle kunna vara maskulina men som motsägs och undermine- ras av nakenheten, den bulliga fetman, ansikts- uttrycken, klädseln och attributen. Han leker med dominerande och stereotypa föreställ- ningar om maskulinitet och män, särskilt vit, heterosexuell maskulinitet. maskulinitet som antas vara i "kris". Som illu- strerande exempel skall jag främst använda två brittiska filmer: The Full Monty (sv. Allt eller inget) från 1997 och All or Nothing från 2002.4 Dessa båda filmer sätter, som så många andra brittiska filmer på senare år, arbe- tarklassens (anti-)hjälte i fokus och uppmärk- sammar de konflikter som vita arbetar- klassmän ställs inför i en situation av ekono- misk och social marginalisering.5 I båda film- erna är huvudpersonerna antingen helt eller delvis arbetslösa och utestängda från möjlig- heten att konstruera sin maskulinitet runt de centrala, och traditionella, identiteterna av yrkesman och familjeförsörjare. De manliga huvudpersonerna är också överviktiga eller feta och är positionerade, eller positionerar sig själva, som marginaliserade genom sin kropps- liga avvikelse. På så sätt förkroppsligar de vad Robert Connell definierar som marginalisera- de maskuliniteter vilka är hierarkisk unde- rordnade en hegemonisk maskulinitet.6 Medan The Full Monty medvetet problemati- serar genusrelationer och genusidentiteter finns temat mer implicit närvarande i All or Nothing. Själv är jag intresserad av hur de överviktiga, feta manliga rollfigurerna får iscensätta olika former och relationer av mas- kulinitet men också av femininitet, och hur det på en och samma gång sker en reproduktion och underminering av rådande genusordningar. Historiskt sett har den manliga kroppens nakenhet varit osynlig både i bildkonst, foto- grafi, film och TV. Mäns kroppslighet - sär- skilt penis - har dolts och förnekats. Så har fö- reställningen om den ideala falliska kroppen, den hårda, disciplinerade och ogenomträngliga manskroppen, och dess maktposition som självklar norm, kunnat upprätthållas.2- Tynans fotografier av sin egen kropp är ett aktuellt exempel på kulturella representationer av manliga kroppar som utmanar denna makt- position. Bilderna synliggör inte bara den manliga kroppen som sårbar utan det är också ett synliggörande av en typ av manskropp som många män känner igen sig i, men som sällan eller aldrig avbildas eftersom den avviker från normen. För Steven Tynans kropp är inte bara naken, den är också överviktig. Marginaliserade manskroppar: Bilder av feta arbetarklassmän Anna Johansson En bild på en naken man. Jag betraktar en bild på en medelålders, vit man med små hängande bröst och en stor rund mage. Kulturhuset hösten 2.003. Tynan visar sig själv i en serie bilder där han är naken, eller delvis klädd, i olika miljöer ute och inne, i eller utan- för sitt hem i England: ihopklämd på en lek- sakstraktor med den tjocka, vita magen häng- ande över det lilla styret och med ett uttrycks- löst ansikte med nästan sorgsen blick, eller med ansträngt ansiktsuttryck med sonen på en gunghäst. På en rad bilder är han naken så när som på ett par stora rutiga kalsonger; på en bild med en öl i handen, dumt stirrande, på en annan bild med knutna nävar och argt ansiktsuttryck i gummistövlar. På ytterligare en annan har han kalsongerna högt uppdragna med penisen hängande utanför, slappt mot ena benet. Han poserar också som en kvinnlig nakenmodell på en bänk och sitter naken i gru- set med ryggen mot en mur och benen uträckta framför sig, liksom uppallad som en docka - eller placerad, som ett passivt, lydigt barn. En bild på en naken man. En bild på en naken man. Jag betraktar en bild på en medelålders, vit man med små hängande bröst och en stor rund mage. Han står lite framåtlutad, med ena handen kupad över könet, den andra handen lätt knuten. Han ser obekväm och krampaktig ut, ansiktet är sammandraget, ögonen näs- tan ihopknipna. Det känns ovant att se en man se så sårbar och tafatt ut. Att se en tjock, fet, överviktig manskropp så naken och exponerad. Jag tittar ytterligare. Nu ser jag att mannen står med fötterna i en vattenfylld barnbas- säng. En gul liten anka flyter vid kanten. Jag fnissar överraskat och förtjust. "Vad absurt, och modigt" vet jag att jag tänker. Och på något sätt så ytterst rörande. Detta var några av mina reaktioner jag när jag såg den brittiske fotografen Steven Tynans bilder av sig själv i utställningen Astronaut på "Jag beslöt mig för att göra mig själv till offret", skriver Tynan. 1 Han har medvetet Kropp I 9 Kropp I 9 framställt sig själv som tafatt, sårbar och löj- lig. Marginaliserade manskroppar: Bilder av feta arbetarklassmän Anna Johansson Den här artikeln undersöker kulturella betydelser av den överviktiga, feta manskrop- pen i ett senmodernt, västerländskt samhälle där manlig fetma, och särskilt den stora magen, i allt större utsträckning tycks ha för- lorat sina positiva betydelser som tecken på välstånd, pondus och makt och snarare ses som tecken på fysiskt och moraliskt förfall, förlust av kontroll och på underordning.3 Mer specifikt är syftet att undersöka hur den över- viktiga manskroppen i film och på TV kommit att tjäna som symbol för en underminerad vit, heterosexuell arbetarklassmaskulinitet - en I filmerna jag valt sätts den vita arbe- tarklassmaskulinitetens kroppslighet på kar- tan, i relation till förlusten av en kropp som traditionellt formats genom kroppsarbete. De skildrar en demaskuliniserad och feminiserad manskropp - en kropp som i sin avvikelse på en och samma gång kan ses som ett hot mot den hegemoniska manskroppen och som ett löfte om nya former av maskulinitet. I denna 781 I Kvinnovetenskaplig tidskrift 1.06 781 I Kvinnovetenskaplig tidskrift 1.06 781 I Kvinnovetenskaplig tidskrift 1.06 kvinnor kommer rusande och hjälper honom att lägga sig ned. Tröjan glider upp och blottar en fettvalk. ambivalens finns också vägen ur den dikoto- misering som Peter Lehman ser i bilderna av den manliga kroppen: som fångade mellan å ena sidan en uppvisning av en fallisk, beund- rad maskulin kropp och å andra sidan en kol- laps av en sårbar, ömkansvärd och komisk manskropp.7 Detta är en av de mest dramatiska scenerna från filmen All or Nothing, ett tungt och lång- samt berättat relationsdrama av Mike Leigh som utspelar sig i ett nedgånget och fattigt arbetarklassområde i Londons utkanter. Som ofta i Leighs filmer utforskas en familjs var- dagsliv, i denna film står familjen Basset i fokus med pappan Phil (Timothy Spall), mam- man Penny (Lesley Manville), dottern Rachel och sonen Rory, alla feta utom mamman. Sce- nen som skildrar Rory när han får en hjärt- infarkt framstår som en bild av en blottad och extremt utsatt manskropp. Den fungerar också i filmen som en slags vändpunkt inte bara för den unge mannen utan för hela famil- jen. Här sätts familjens existens och livsvillkor på sin spets. All or Nothing har blivit mycket populär bland medelklasspubliken i London och även utomlands, men är relativt okänd för svenska tittare.9 Manlig fetma i film och TV tade redan på nittiotalet har Doug (Kevin James) som huvudkaraktär, en man som arbe- tar som chaufför på en budfirma, lever till- sammans med sin (slanka och framgångsrika) fru Carrie (Leah Remini) och hennes far. Karaktären Doug framställs tydligt som en "soffpotatis" genom att han säger saker som "Vänner hindrar en bara från att se på T V " och är ohämmat förtjust i - och upptagen av - mat. Dougs övervikt och ätande är teman som ofta dyker upp som föremål för skämt och kommentarer, inte minst av honom själv: "Jag skulle lika gärna kunna heta Fatty McButter- pants". Att hans övervikt är en källa till iden- tifikation för fansen kan märkas på kommen- tarerna på olika hemsidor där han bland annat beskrivs som "en vanlig överviktig m a n " . I 4 Enligt medieforskaren Jerry Mosher har fetma under det senaste decenniet börjat synliggöras på film och än mer i T V . 1 1 Mellan 1 9 9 5 - 1 9 9 6 släpptes i USA inte mindre än fem filmer med feta män och pojkar i huvudrollerna och med fetma som tema: Heavy, Heavyweighters, Angus, The Nutty Professor och Thinner. Som Mosher poängterar har detta en stor betydel- se eftersom det markerar en medvetenhet om fetma och ger feta män karaktärer att identifi- era sig med. Om fetma på bioduken lätt framstår som grotesk, och i filmer ofta används för att repre- sentera det avvikande, har manlig fetma inom TV-mediet alltmer fått representera "the ordi- nary guy", den vanlige mannen. I sina studier av feta manliga rollfigurer inom olika TV-gen- rer som situationskomedier (Norm i Skål, Drew Carey, Dan Conner i Roseanne), men också i deckare (Frank Cannon och Nero Wolfe), konstaterar Mosher att dessa rollfigu- rer alltid antingen är arbetarklass; mekaniker, byggjobbare, chaufförer, eller lägre tjänste- män i utkanten av en korporativ kultur. Redan med karaktären Archie Bunker i serien All in the Family på 70-talet hade manlig fetma etablerats som TV-symbol för hur den vita, heterosexuella maskuliniteten hade förlorat sina tydliga konturer och blivit "mjuk och impotent". 1 2 Även den populära svenska situationsko- medin Svensson, Svensson från mitten av nit- tiotalet har en överviktig man i huvudrollen, brevbäraren Gustav (Allan Svensson). Gustav skildras som en vanlig svensk familjefar som bor i radhus i en mindre stad, av arbetarklass- ursprung, socialdemokrat och fotbollsfantast. Manlig fetma i film och TV Han framstår också som självgod, ego- centrisk, barnslig och löjlig och utan någon som helst auktoritet varken som far eller make. Hans fru (Suzanne Reuter) har i egen- skap av bankdirektör inte bara högre samhäl- lelig status utan är också den som har rollen som familjens överhuvud. Precis som i Kung- en av Queens skämtar man om Gustavs ätan- de och kropp. 15 Som en tydlig illustration av Moshers reso- nemang kan man se hur det i svensk T V (främst i T V 3, TV 4 och Kanal 5) idag visas anmärkningsvärt många amerikanska situa- tionskomedier med säte i hemmet där just överviktiga män spelar rollen som make/far. Jims värld (James Belushi), Listen up (Jason Alexander), Universums mittpunkt (John Goodman) och Kungen av Queens är bara några av dem. 13 Kungen av Queens som star- Scener En överviktig, vit man sitter ensam i mörkret i ett trädgårdsskjul. Han har bar överkropp och är insvept i plastfolie. Han mumsar sorgset på en bit choklad. Detta är en av de roligaste men också en av de mer sorgliga scenerna i filmen The Full Monty. Den före detta stålverksarbetaren Dave (Mark Addy) som inte bara är arbetslös utan också tjock och impotent, framställs här i en typiskt feminin position: som tröstätande i smyg under en desperat bantningskur. Filmens handling kretsar runt sex arbetslö- sa män i Sheffield, med Gaz (Robert Carlyle) som huvudperson. I sin desperata kamp för att tjäna pengar och återerövra någon form av kontroll och stolthet som män, beslutar de sig för att göra som den manliga stripgruppen Chippendales, som uppträder för fulla hus på Working Men's Club. Regissören Peter Catta- neos komedi blev hyllad och en stor publik- succé. Den är mer än en film, den är ett feno- men. Plötsligt kände vanliga män runtom i England att det inte bara var möjligt att ta av sig kläderna inför publik, det var något att sträva efter.8 I de beskrivna scenerna konstrueras den feta manskroppen som ett passivt (och femi- niserat) objekt att aktivt iaktta, granska och värdera. Dave är dels betraktad av sin fru Jean som står i fönstret, dels av kameran som zoomat in honom i närbild, halvnaken. Rory är filmad ovanifrån av kameran medan han stapplar runt och faller på gräsmattan. Han är även observerad ovanifrån av de kvinnli- ga grannarna. Detta är exempel på hur den "manliga blicken", som en betraktarposi- tion, inte nödvändigtvis är knuten till män som tittar på kvinnor, utan en position som även intas av kvinnor som tittar på män eller av män som betraktar sig själva eller varand- r a . 1 0 En ung fet man i träningsoverall stapplar runt på en gräsmatta, omgiven av fler- våningshus. Han håller sig för hjärtat och skakar. Hans ansikte är förvridet. Två Kropp I 111 Är fetma en mansfråga? I båda dessa serier får den fete mannen sym- bolisera en ifrågasatt och underminerad vit, heterosexuell arbetarklassmaskulinitet - en man som inte längre är herre över sitt hus och som ofta framställs som löjlig. Samtidigt kon- strueras den manliga övervikten också som en 781 I Kvinnovetenskaplig tidskrift 1.06 781 I Kvinnovetenskaplig tidskrift 1.06 781 I Kvinnovetenskaplig tidskrift 1.06 del av den "normale" arbetarklassmannens liv och identitet - en man som är hårt arbetande och som gärna ligger på soffan och tittar på TV när han är ledig, en man som är storkonsu- ment av mat och som inte reflekterar särskilt mycket över sitt utseende eller hälsa. Kropp, vikt och mat representeras inte som ett pro- blem - som för de kvinnliga rollfigurerna - och är inte kopplad vare sig till estetik, till sex- uell attraktivitet eller till hälsa. Denna fram- ställning av den överviktige arbetarklassman- nen stämmer också väl med resultaten av de få studier som hittills har gjorts om överviktiga män. Dessa visar att överviktiga män generellt inte har lika negativ kroppsuppfattning som överviktiga kvinnor. Överviktiga män uppfat- tar sig i högre grad än överviktiga kvinnor som "normalviktiga", är nöjda med sin vikt och är inte benägna att försöka förändra sitt beteende för att gå ned i vikt. Feta män ser sig själva som överviktiga snarare än feta. Vissa forskare har framfört hypotesen att männen snarare ser sig som "stora och starka" än som " f e t a " . 1 6 Frågor runt ätande och vikt har hittills, både i Sverige och i övriga västvärlden, i var- dagsförståelse, klinisk praktik, samhällsdebatt och forskning, uppfattats som kopplat till kvinnor och femininitet.18 Den estetiska och moraliska plikten att vakta sin vikt och forma sin kropp med den slanka, fettfria kroppen som norm, att underkasta sig "slankhetens tyranni", har främst definierats som kvinnors plikt. Och under Orbach paroll "Fat is a Femi- nist Issue" (Fetma är en kvinnofråga) har femi- nistisk forskning kopplat slankhetens tyranni och kvinnors problematiska förhållande till kropp, vikt och ätande till relationer av man- lig makt och kvinnlig underordning, till den kvinnliga kroppens objektifiering och dess position som den avvikande, bristfälliga och problematiska Andra som skall kontrolleras, tuktas och d i s c i p l i n e r a s . Är fetma en mansfråga? Man häv- dar här att många män idag lider av "Ado- niskomplexet" - en beteckning som hämtats från den grekiska mytologin där den muskulö- sa grekiske halvguden Adonis representerade den ideala maskulina kroppen.25 Att den muskulösa kroppen är ett domine- rande ideal för den manliga kroppen kan för- klaras med att muskler symboliserar hälsa, styrka, kraft, dominans, oberoende, aggressi- vitet, kompetens och sexuell virilitet, egenska- per eller karakteristika som traditionellt ses som maskulina.2-8 I en värld där mäns roller har förändrats och deras maktpositioner underminerats kan strävan efter en hård, mus- kulös kropp ses som ett försök att (re-)etablera eller förstärka den manliga dominansen; Som ett försök att bevara traditionella uppfatt- ningar om maskulinitet.29 Är fetma en mansfråga? K v i n n o r s uppta- genhet av sin vikt har analyserats som uttryck för en så kallad objektifierad kroppsmedve- tenhet vilken inkluderar kroppsövervakning, internalisering av kulturella kroppsideal och en idé om att kunna (och att man borde) forma sin kropp i enlighet med dessa ideal.20 De manliga huvudkaraktärernas kroppar får i hög grad sin mening i relation till de kvin- nor de är ihopparade med. Inte i någon av de situationskomedier där mannen är överviktig eller fet är kvinnan det också. r7 Tvärtom är de kvinnliga karaktärerna alla slanka och följer konventionella utseendenormer. Kanske kan man säga att för att de manliga karaktärerna inte skall framstå främst som patetiska icke- män är det av central betydelse att deras kvinnliga partners uppfattas som sexuellt attraktiva och slanka. Medan manlig övervikt på en och samma gång blir konstruerad som en symbol för avvikelse och normalitet tycks kvinnlig övervikt och fetma fortfarande ses främst som grotesk, hotfull, avvikande och inte möjlig (eller önskvärd?) att identifiera sig med för TV-publiken. Under senare år har mäns kroppar och maskuliniteters förkroppsligande synliggjorts och mäns förhållande till kropp, vikt och ätan- de uppmärksammats. Man har också börjat hävda att fetma även är en " m a n s f r å g a " . 2 1 Sander L Gilman skriver i Fat Boys, en av de få böcker om manlig fetma som hittills skrivits, att den feta manliga kroppen är förknippad med, och producerar andra betydelser, än den feta kvinnliga kroppen. Dessa betydelser är mångfasetterade, motsägelsefulla och hittills inte undersökta i någon större utsträckning.22 Nutida betydelser av den feta manliga krop- pen i det västerländska samhället behöver sät- tas i relation till den ökande objektifieringen och kommersialiseringen av den manliga krop- Kropp I 113 pen, ett fenomen som lett Susan Faludi till att mynta begreppet "ornamental maskulinitet" - en maskulinitet där ytan och utseendet betyder allt.z3 I takt med denna objektifiering tycks också mäns, i synnerhet vita medelklassmäns, upptagenhet av och missnöje med sina kroppar öka.24 I boken The Adonis Complex. The Sec- ret Crisis ofMale Body Obsesssion analyserar man bland annat utifrån kliniska exempel nor- damerikanska mäns ökande "besatthet" av kropp och utseende i termer av en "kris" som man menar hittills inte har uppmärksammats inom forskning och klinisk praktik. Fetmadiskursen 3 3 misk och social makt, ställs i skarp kontrast till kvinnorna som arbetar och försörjer familjer- na och till och med tar över Working Men's Club. De sex männen i filmen representerar alla på olika sätt marginaliserade kroppar: åldran- de, svarta, homosexuella och överviktiga kroppar som tidigare varit vana vid kroppsar- bete men nu är improduktiva: den fete Dave som har blivit impotent, den rödhårige och bleke före detta säkerhetsvakten Lomper som försöker ta livet av sig och som visar sig vara bög, den äldre före detta förmannen Gerald som inte vågar berätta för sin fru att han för- lorat arbetet och upprätthåller fasaden av lägre medelklass, Horse som är en grånande, svart man som även han är impotent och hant- verkaren Guy som har en muskulös, attraktiv manskropp och stor penis men är bög. Gaz, som är den ende som är vit, heterosexuell, ung och slank (och vars penis aldrig kommenteras) har suttit i fängelse, är frånskild och håller på att förlora rätten att ens träffa sin son. Det är utifrån denna marginaliserade position, och den desperation de alla upplever, som de bör- jar sin resa tillsammans. Bilderna av fetma som fenomen - och män- niskors sätt att förstå detta fenomen - skapas till viss del genom användningen av metaforer. Ett idag vanligt sätt att framställa fetma i media är i termer av en "epidemi". Trots att fetma inte är en epidemisk sjukdom (eller inte ens givet en "sjukdom") talar man om en "fet- maepidemi". 34 I sin klassiska bok Sjukdom som metafor från slutet av sjuttiotalet, skriver Susan Sontag om hur epidemiska sjukdomar används som en symbol för samhällelig oord- ning; för förfall, karaktärssvaghet och mora- lisk upplösning.3 5 Genom att framställa fetma som en epide- mi frammanas bilden av en hotfull och okon- trollerbar kraft som hotar världens befolk- ning, inte bara i termer av fysisk nedbrytning utan också moraliskt. Med denna association till natur, kaos och upplösning, en kroppslig- het som löper amok, blir fetma och den feta kroppen i många sammanhang konstruerad som feminin. Och det är också när den tjocka, feta manskroppen tydligt kodas som feminin som den kan fungera som en kraftfull symbol för en underminerad vit, heterosexuell arbe- tarklassmaskulinitet. Fetmadiskursen Ur detta perspektiv kan också den närmast paranoida skräcken för fetma som råder i den västerländska kulturen tolkas som en skräck för uppluckring av den hegemoniska maskuli- na kroppen, en skräck för att bli invaderad av "kvinnlig smitta". Om hårdhet i den kvinnli- ga kroppen blivit möjlig - till och med erotise- rad, ses mäns mjukhet fortfarande som avvi- kande och stigmatiseras som "feminin".3° Som Thomas Johansson skriver: "Den hårda kroppen skyr det som hotar dess världsbild. Allt mjukt, 'feminint', 'moderligt' och kontur- löst står i klar motsatsställning till denna kropp". 3 1 Och så blir "kriget mot fetman" en del av ett krig mot en feminisering av den man- liga kroppen. Enligt studier associeras den rektangulära och hårda manskroppen med egenskaper som mod, styrka, hälsa och intelligens medan män med rund och mjuk kropp ses som smutsiga, slarviga, beroende av andra, lata, ensamma, mindre intelligenta och mindre fysiskt attrak- tiva. Även den alltför smala manskroppen ses som avvikande och tunna män uppfattas som nervösa, sjukliga, räddhågsna, veka och omanliga. 2 6 Genom fokusgruppsamtal med pojkar och unga män har man undersökt hur de upplever sina kroppar, vilka kroppsideal de har och vilka försök de gör att förändra sin kroppsform och vikt genom träning och diet. Resultaten visade att ett gemensamt kropps- ideal var att vara vältränad och muskulös. Samtidigt ville man inte vara "för muskulös". Alla grupper uttryckte en negativ syn på över- vikt. Man skuldbelade de överviktiga och ansåg det självklart att det var naturligt att reta och förlöjliga dem. Fetma associerades med att förlora kontroll över kroppen och med brist på viljestyrka.27 Allt fler samhällsforskåre och debattörer uppmärksammar att det idag råder en starkt moraliserande "fat is bad"-diskurs som stig- matiserar feta människor som en avvikande och moraliskt suspekt grupp. Fetma definieras som äckligt, fult, ohälsosamt, obscent och överviktiga och feta människor ses som dumma, lata, infantila, oansvariga och impulsstyrda. Man kan säga att det råder en slags moralisk panik runt fetma.3 2 Enligt Paul Campos handlar den massiva fokuseringen på övervikt och fetma under senare år lika myck- et om politik och kultur som medicin. I boken The Obesity Myth jämför Campos den massi- va kampanj mot fetma och den stigmatisering av feta människor som försiggår i dagens USA med de rashygieniska rörelserna under trettio- talet. Fetmadiskursen De är alla anti-hjältar men som tidigare analyser av filmen påpekat finns det också en hierarkisering inom gruppen.37 I berättelsens mitt finns de tre vita, heterosexuella männen medan de homosexuella männen och den svar- te Horse är perifera och skildras på ett relativt stereotypt sätt (Horse är till exempel bra på att dansa). Om man däremot fokuserar på berät- telsen om just Dave, kan man se att hans över- viktiga kropp är den kropp som framställs som mest sårbar av de olika manskropparna. Hans marginalisering, mer tydligt än de andras, blir konstruerad genom hans fetma. Under filmens gång blir hans fetma stigmatiserad och han är föremål för kommentarer, inte bara genom att 781 I Kvinnovetenskaplig tidskrift 1.06 Bantning Under en sekvens i filmen befinner sig alla männen hemma hos Gerald. De står och trängs i ett av rummen då Gaz meddelar dem att det nu är dags att öva på att klä av sig och visa sina kroppar inför varandra. Alla protes- terar, mest Dave som är uppenbart illa till mods över att visa sin kropp. Men som Gaz säger "Mina damer, vi är strippor!". Daves kropp feminiseras bland annat genom hur karaktären är placerad i och rör sig i rummet. Medan de andra manliga karaktä- rerna först och främst skildras i offentliga rum, på arbetsförmedlingen, utomhus, i replo- kalerna eller i hemmiljö inneslutna i en homo- social gemenskap, är Dave i flera scener ensam när hans delvis nakna kropp zoomas in. Dave är den enda karaktär som visas i sovrummet, i sängen, i badrummet; i rum som symboliserar intimitet och det feminina. Han är också den karaktär som visas delvis avklädd i flest sce- ner, förutom scenerna i de gemensamma av- klädningarna. Dave (tar av sig sin tröja): Titta inte. Och skratta inte era djävlar. Vad skall jag göra med detta (tar sig på magen). Gaz: Det är inte alls så illa (paus) fram- ifrån. Gerald (med eftertryck): Fetma, David, är en kvinnofråga! Dave:Vad menar du med det? Gerald: Jag vet inte. Men det är det. Dave (i defensiv ton): Jag försöker verk- ligen. Jag försöker banta. De andra tittar alla på honom med miss- tro. Dave: Det känns som jag har bantat hela mitt liv. Ju mindre jag äter desto fetare blir jag. Dave (tar av sig sin tröja): Titta inte. Och skratta inte era djävlar. Vad skall jag göra med detta (tar sig på magen). Gaz: Det är inte alls så illa (paus) fram- ifrån. Gerald (med eftertryck): Fetma, David, är en kvinnofråga! Man kan säga att Dave representerar och artikulerar en objektifierad kroppsmedveten- het, ett förhållningssätt som traditionellt är kopplade till kvinnor och femininitet.3 8 Genom hela filmen kommenterar han sin kropp på ett negativt sätt, han skäms över och är upptagen främst av sin stora mage, som han klappar på, granskar och i en rad scener för- söker dölja. Maskulinitet i kris The Full Monty är en film som av en rad fors- kare kopplats till maskulinitetens kris och till en lek med genusidentiteter.3é Filmen präglas av en problematisering av den maskulina iden- titeten där de arbetslösa männen, utan ekono- Kropp I 115 han i rå, skämtsam ton blir kallad "tjockis", "fet idiot", "feta svin" av Gaz och de andra männen, till och med Gazs son uppmanar honom att banta, utan också för att Daves karaktär själv ett flertal gånger kallar sig "fet djävel". Med fokus på karaktären Dave blir det också uppenbart att hans (typiskt kvinnli- ga) skamfyllda upptagenhet av och missnöje med kroppen hotar att underminera det gemensamma manliga projektet. Han är den som ställer sig utanför, drar sig undan och iso- lerar sig. I relation till de andra männen, särskilt den handlingskraftige och riskbenägne Gaz, framställs Dave i många situationer som passiv och uppgiven. Horse i sin oro för att penisen skall vara slapp på scen - tycks Dave i högre grad vara fokuse- rad på sexuell attraktion och granskar sin kropp utifrån dess estetiska kvaliteter, utifrån att bli betraktad och eftertraktad. Män som erotiska och estetiska objekt (...) Dave: Jag är inte ett kycklingben, Gerald. Gaz: Du virar det runt, minskar fettet... Nu deltar alla och visar med olika gester hur man sveper in sig i plastfolie. Gerald: Hon lever för det. Män som erotiska och estetiska objekt Ytterligare en central episod utspelar sig hemma hos Gerald då gruppen skall prova ut scenkläder och de tillsammans kommer till insikt om att deras kroppar kommer att bli granskade på samma sätt som de själva grans- kar kvinnors kroppar. Den som för det på tal och formulerar denna insikt är Dave. I sin bok Skönhetsmyten definierar Naomi Wolf bantning som "kvintessensen av norma- tiv kvinnlighet". Att banta är ett sätt för kvin- nor att disciplinera sin kropp för att uppnå rådande utseende och kroppsideal och bli en "riktig" kvinna. 39 Männen befinner sig i Geralds frus solari- um; Guy ligger och solar, Lomper dansar runt i rummet, tittar på fruns anti-rynkkräm och Horse sitter på en motionscykel. Dave bläddrar i en herrtidning och männen kommenterar tjej- ernas kroppar. Här utför man ritualer som kan sägas vara förknippade med normativ masku- linitet: de motionerar tillsammans, tittar på kvinnors kroppar tillsammans men de utför också ritualer som syftar till den vård av krop- pen, estetisering som förknippas med normativ kvinnlighet. Både solandet och Lompers intres- se för hudkräm riktar fokus på kroppens yta, huden, en del av kroppen som män av tradition aldrig ägnat sig åt. Dessa senare aktiviteter symboliserar hur de, som kvinnor, håller på att omvandla sig till estetiska och erotiska objekt, att visas upp för kritiska blickar (i denna situa- tion är de karaktärer som är mest aktiva i att överskrida könsgränserna de två män som sedan visar sig vara homosexuella). Bantning framställs och uppfattas fort- farande som något kopplat till kvinnor och femininitet. Att bekymra sig om hur kroppen ser ut och över fettinnehållet i mat uppfattas gärna av män som något som kvinnor gör och som något "kärringaktigt". En "riktig man" bantar inte. I den mån män bantar betonar de att det är ett målinriktat och rationellt pro- jekt.4° Det är denna förståelse som The Full Monty anspelar på och som gör det så komiskt med Dave som inför sina manliga vänner talar om hur han bantat hela sitt liv. Bantning Medan Gerald och Horse mer uppenbart är upptagna av "manliga" bekym- mer relaterade till sexuell prestation - Gerald i sin oro för att han skall ha stånd på scen och Vad betyder Geralds kommentar "Fetma är en kvinnofråga?" Att Dave inte skall bry sig, för det är kvinnors fetma som är ett problem, inte mäns? Och om Dave talar om sin fetma är han "som en kvinna". Eller att det inte finns utrymme för mäns bekymmer över sina krop- par? Dave svarar genom att försäkra alla att 781 I Kvinnovetenskaplig tidskrift 1.06 781 I Kvinnovetenskaplig tidskrift 1.06 han verkligen försöker gå ned i vikt, han för- söker verkligen banta, men att han ändå inte blir smalare. tidigt är det också tydligt att Dave själv och de andra männen i denna situation betraktar Daves kropp med en ny typ av objektifierande och bedömande blick. De synar nu hans kropp utifrån ett antagandet att han medvetet skulle kunna - och borde - forma sin kropp för att uppnå de kulturella slankhetsidealen. Gerald (förklarar uppmuntrande): En kompis till Linda hade en massa plast omkring sig på en hälsoklubb. Hon gick ned massor. Det var som magi. Gerald (förklarar uppmuntrande): En kompis till Linda hade en massa plast omkring sig på en hälsoklubb. Hon gick ned massor. Det var som magi. (...) Dave: Jag är inte ett kycklingben, Gerald. Gaz: Du virar det runt, minskar fettet... Nu deltar alla och visar med olika gester hur man sveper in sig i plastfolie. Gerald: Hon lever för det. Den mjuka penisen Flera av männen i filmen framställs som ytterst sårbara och maktlösa i sin relation till de kvinnliga karaktärerna, vilka främst fyller funktionen av att vara kontrollerande och bedömande: Gaz är underkastad sin före detta frus kritiska granskning av honom som far och hon har makten att ge och ta ifrån honom sonen. Lomper är kontrollerad av sin gamla mamma, Gerald försöker tillfredställa sin frus behov av ekonomisk och social status genom att låtsas arbeta och handla på kredit. Detta är en spännande scen. Dave är återigen den som problematiserar den manliga krop- pen och formulerar nu något de andra aldrig överhuvudtaget tänkt på: att deras kroppar skulle kunna granskas och bedömas av kvin- nor på samma sätt som de själva granskar och bedömer kvinnors kroppar. Hur utsatta de är i denna position som erotiska objekt blir än tydligare i nästa replikskifte då Lomper, uppenbart skakad, säger: Dave är den enda som skildras i en sexuell och nära relation med en kvinna, sin fru Jean (Lesley Sharp). Redan i början av filmen säger Jean till en av sina väninnor: "Det är som han gett upp. Arbetet, mig, allting". I Daves egen fantasi är han misslyckad och oattraktiv och hans fru är därför intresserad av andra män, kanske till och med otrogen med en kollega. Medan hans hustru på olika sätt försöker närma sig honom och visa kärlek drar han sig bara undan i skam. Lomper: Jag sa inget om hennes person- lighet. Hon är säkert ganska trevlig. Dave (replikerar snabbt): Och de kommer inte att säga något om din personlighet hel- ler. Vilket är tur eftersom du är ett svin. (...) Dave: Det är det här de tittar på (Dave pekar på sin kropp). Och jag skall säga dig en sak, polarn. Det finns anti-rynkkräm, men det finns ingen anti-fettsvin kräm. Dave: Det är det här de tittar på (Dave pekar på sin kropp). Och jag skall säga dig en sak, polarn. Det finns anti-rynkkräm, men det finns ingen anti-fettsvin kräm. I en scen ser vi honom i badrummet i en klassiskt kvinnlig position, framför spegeln med bar överkropp. Han granskar sin kropp med en ängslig och kritisk blick, klämmer oro- lig på sin mage. Under tiden som han tafatt försöker tala med sin fru ligger hon i sängen i en typiskt manlig position, påklädd, lite otå- Det är inte deras egenskaper eller prestationer, utan deras utseende som skall bedömas. Män som erotiska och estetiska objekt Här positione- ras Dave som feminin i förhållande till de andra, mer maskulina männen som varken känner till eller har praktiserat denna typiskt "kvinnliga" form av kroppslig disciplinering. Deras okunnighet markeras tydligt av Geralds råd till Dave, råd som framstår som hysteriskt roliga för kvinnliga åskådare som vanligtvis är experter på olika bantningsmetoder. Sam- Mitt under diskussionen runt den kvinnli- ga modellens bröst säger Dave: Jag bara hoppas att de är mer förstående än vi. Kropp I 117 Horse slutar cykla. Guy lyfter upp locket till solariet. Horse: Vad? Dave: De kommer att titta på oss så där. Gerald stirrar. Dave: Tänk nästa fredag om 400 kvinnor säger: "Han är för fet, han är för gammal, han har en bröstkorg som en liten sparv". Lomper tittar på sitt bara bröst. Horse (tyst): Det skulle de inte. Dave: Varför inte? Han sa ju att hennes bröst är för stora. Lomper (tveksamt): Det är annorlunda... Vi är grabbar. Dave: Ja. Och? Horse slutar cykla. Guy lyfter upp locket till solariet. Horse slutar cykla. Guy lyfter upp locket till solariet. blicken. I den nya intimitet som skapas mellan männen, i det problematiserande talet om kroppen, det skygga betraktandet av varandras kroppar och i blandningen av ritualerna, är det den fete Dave, i positionen som den feminisera- de Andre, som anger tonen. Aktivt och med auktoritet problematiserar han den manliga kroppen som objekt. Om han i den tidigare sce- nen var osäker och skamfylld i sin avkläddhet, talar han här om sin fetma med rå humor.41 Vändpunkten - från skam till stolthet I nästa scen befinner sig Dave med Gaz och hans son Nathan på det shoppingcenter där Jean arbetar. Dave står bakom godishyllan och tittar på Jean som skämtar med en manlig arbetskamrat. Han är svartsjuk och uttrycker självförakt för att han inte har något jobb och att Jean hellre föredrar någon annan. Först frå- gar han Nathan om han har mintchoklad kvar men han svarar bara "Försvinn, du bantar", därefter tigger han karameller av Nathan, men får inga. På utvägen när de springer ut med fil- merna de stulit stoppar Dave karameller i munnen. Han lägger sig bredvid Jean i dubbel- sängen. Jean: (Lägger ifrån sig tidningen, rullar över och tittar honom i ögonen) Jag bryr mig inte om ifall de är svarta , vita eller regnbågsfärgade, jag är gift med dig. Dave: Visst (med hopplöshet i rösten). God natt (han vänder ryggen mot Jean) Jean: Varför skulle jag vilja ha någon annan (klappar honom på bröstet och pussar honom på axeln, han ser obekväm ut). Min store man. Jean: (Lägger ifrån sig tidningen, rullar över och tittar honom i ögonen) Jag bryr mig inte om ifall de är svarta , vita eller regnbågsfärgade, jag är gift med dig. Inte bara är Dave den enda karaktär som äter i filmen utan i flera scener ser vi honom tröstäta sötsaker. En stor aptit på "riktig" mat som kött, symboliserar maskulinitet medan ett småätande av sötsaker däremot ses som ett typiskt generande och impulsstyrt ätande - ett feminint ätande. 43 Dave: Visst (med hopplöshet i rösten). God natt (han vänder ryggen mot Jean) Jean: Varför skulle jag vilja ha någon annan (klappar honom på bröstet och pussar honom på axeln, han ser obekväm ut). Min store man. Skildringen av karaktären Dave är trots allt inte entydig. I flera scener lämnar Dave posi- tionen av passivitet, självförakt och skam och visar sig aktiv, kraftfull och stolt. När skuld- indrivare kommer till Gerald för att hämta TV:n är det Dave som bredbent står längst fram i gruppen av män, iförd endast kalsong- erna, för att skrämma bort indrivarna. När Gaz ännu en gång kallar Dave för fet idiot så jagar Dave honom, håller fast honom och säger med vrede: "Kalla mig inte fet idiot igen". Dave: Jeani (bedjande och tar bort hennes hand). Jag är helt slut. Den mjuka penisen De är nu beroende av bekräftelse från den kvinnliga 781 I Kvinnovetenskaplig tidskrift 1.0 lig, "vad vill han nu då". Daves rumsliga pla- cering är viktig eftersom badrummet är en plats som är förknippat med den öppna krop- pen, kroppsliga flöden och vätskor och där- med också förknippat med kvinnlighet.42 potens och en hård fallisk kropp. Daves egen penis är mjuk och förknippad med en "mjuk" kropp, passiv, improduktiv på alla sätt - vilket är motsatsen till maskulinitet. Dave represente- rar här den kastrerade mannen och hans över- viktiga kropp blir en metafor för hans brist på potens och makt. Dave: Du, Jean. Jean:Ja? Dave: Har du varit ute med en svart man? Jean: Det vet du att jag inte har. Dave: Men om du skulle leta efter en ny man då? Om du skulle det till exempel, skulle du fundera på det? Jean: Vad är det med dig? Dave: Skulle du det? Jean: Mår du bra? Ja, det kanske jag skulle. Dave: Så det är sant? Jean: Vad är sant? Dave: Att de har bra kroppar. Han lägger sig bredvid Jean i dubbel- sängen. Jean: (Lägger ifrån sig tidningen, rullar över och tittar honom i ögonen) Jag bryr mig inte om ifall de är svarta , vita eller regnbågsfärgade, jag är gift med dig. Dave: Visst (med hopplöshet i rösten). God natt (han vänder ryggen mot Jean) Jean: Varför skulle jag vilja ha någon annan (klappar honom på bröstet och pussar honom på axeln, han ser obekväm ut). Min store man. Dave: Jeani (bedjande och tar bort hennes hand). Jag är helt slut. Det är konstigt hur tröttsamt det är att inte göra något (släcker lampan, ligger och stirrar ut i mörkret, hon lägger sig tillbaka på sin sida av sängen). denna scen blir Daves osäkerhet runt sin se ella prestation formulerad. Den svarte man Dave: Du, Jean. Jean:Ja? Dave: Har du varit ute med en svart man? Jean: Det vet du att jag inte har. Dave: Men om du skulle leta efter en ny man då? Om du skulle det till exempel, skulle du fundera på det? Jean: Vad är det med dig? Dave: Skulle du det? Jean: Mår du bra? Ja, det kanske jag skulle. Dave: Så det är sant? Jean: Vad är sant? Dave: Att de har bra kroppar. Han lägger sig bredvid Jean i dubbel- sängen. Kropp I 119 Kropp I 119 Kropp I 119 Denna uppvisning av kraftfull maskulinitet där hans kropp konstrueras som "stor och stark" snarare än "fet och svag" är dock inget bestående. Han återfaller återigen i orörlighet och passivitet. Detta består till slutet av filmen, den dag då stripshowen skall gå av stapeln. Dave kommer hem efter att ha smitit från job- bet som säkerhetsvakt, men har ändå bestämt sig för att inte vara med och uppträda. När han kommer in i sovrummet sitter Jean på sängen och gråter. Hon har hittat babylotion och tangatrosor och dragit slutsatsen att Dave är otrogen. Hon är förtvivlad och arg och slår på honom medan han försöker förklara att det inte handlar om någon annan kvinna utan att han har varit med Gaz och övat på att dansa. Att de hade tänkt tjäna pengar som strippor. När han ser hennes misstroende ansiktsuttryck säger han i lite defensiv ton: han får kraft att inte bara gå ut på scenen med de andra utan att också ta ledarrollen och vara en slags konferencier. Filmen slutar precis i det ögonblick då männen visar the full monty, och kastar ut de hattar som dolt deras kön till den jublande kvinnliga publiken. Vi vet inte om deras peni- sar är erigerade, men det finns ett under- liggande antagande om potens. Som Jane Addelston visar i sin analys av bland annat The Full Monty är den främsta vägen för mar- ginaliserade män att återerövra status, att visa sin faktiska eller metaforiska penis, den ulti- mata symbolen för maskulinitet. Det räcker att ge sken av att ha en.44 Till skillnad från de andra männen är det inte denna uppvisning inför den kvinnliga publikens blickar som ger Dave stoltheten som man åter. Det är istället mötet med Jean. Vändpunkten - från skam till stolthet Det är konstigt hur tröttsamt det är att inte göra något (släcker lampan, ligger och stirrar ut i mörkret, hon lägger sig tillbaka på sin sida av sängen). I denna scen blir Daves osäkerhet runt sin sex- uella prestation formulerad. Den svarte man- nen symboliserar här en erigerad, hård, stor penis som markör för maskulinitet, sexualitet, Den kännande manskroppen Vi var inte så dåliga. Bara det att jag inte kunde. Redan i inledningen av filmen All or Nothing markeras att identiteten som arbetarklass är centralt för filmen. Första scenen visar huvud- karaktärerna på sina arbetsplatser: dottern Rachel som systematiskt tvättar korridoren på ålderdomshemmet och vänligt frågar en äldre kvinna om hon behöver hjälp, Penny, frun och mamman med trötthet och hopplöshet ristad in i det smala, lite snipiga ansiktet vid sin snabbköpskassa, familjefadern Phil som med en orolig min och halvöppen mun kör sin taxi- bil med olika passagerare i baksätet, fulla, grälande, skrikande. Den hopplöshet och stumhet som är kopplad till familjens sociala och ekonomiska marginalisering tycks för- kroppsligas i de tre familjemedlemmarnas fetma: fadern, dottern och sonen. På olika sätt representerar de passivitet och misslyckanden. kunde. Jean: Varför inte? Dave: Titta på mig! Jean: Och? Dave: Jeani, vem vill se det här dansa? (med tårar i ögonen). Jean: (tittar stint på honom med förgråtna ögon) Jag, Dave. Jag vill! Hon pussar honom och håller om honom. Han lutar sig mot henne. De omfamnar varandra. Här sker det en vändpunkt. Det är genom att han står emotionellt naken inför Jean med sin skam och möter hennes kärleksfulla och accepterande blick som han slutar skämmas för sin kropp. Han förvandlas här från den impotente, "fete djäveln" till "Bamse", Jeans stora och starka man som går rak i ryggen och är handlingskraftig. Det är genom detta möte 1 de första scenerna skapas en bild av Phil som motsatsen till det hegemoniska maskuli- 781 I Kvinnovetenskaplig tidskrift 1.06 nitetsidealet. Hans stora, tunga kropp med ölmagen hängande över byxkanten, det pluf- siga ansiktet och det feta och stripiga håret markerar tillhörigheten till en underordnad klass där man inte har råd med eller värderar disciplineringen av kroppen. Hans ansiktsut- tryck signalerar dumhet och osäkerhet och när han talar är det långsamt med en tyst och tvekande röst. Phils brist på maskulin kraft, aggressivitet och aktiv hållning ställs också i motsats till kollegan som när han krockar med sin bil blir högljutt arg, skyller ifrån sig, svär, och protesterar mot chefen när han ger honom order. I sin vänlighet och omsorg erbjuder sig Phil köra kollegan hem. Och medan denne fortsätter svära över den kvinnliga passagera- ren som enligt honom orsakat olyckan, och över kvinnor i allmänhet, sitter Phil tyst med ett godmodigt uttryck i ansiktet. Den kännande manskroppen Nedsjunken i pubens soffhörna och med en öl i handen filo- soferar han sedan med stilla röst, tittar ned som om han talade för sig själv: kopplat till kvinnliga kvaliteter. Med bland annat kriminalpsykologen Fitz i den engelska serien Cracker som exempel menar Gilman att den fete mannens tankeprocess framstår som känslomässig och intuitiv snarare än analytisk och objektiv. Han är empatisk med offren och även med förövarna, snarare än kyligt analy- tisk.4 5 Phil börjar också sin tankebana i fun- deringar kring vilken betydelse den kvinnliga passageraren hade för vännens öde. Även i relation till sin fru positioneras Phil som feminin. Medan frun skildras som distan- serad och sammanbiten i sina hushållsgöro- mål, försöker Phil skapa familjegemenskap och kontakt genom att berätta små anekdoter om sina passagerare och visa omsorg genom att med mjuk röst fråga sina barn hur de mår. Det är dock i försöken att leva upp till den tra- ditionella rollen som familjeförsörjare som hans maskulinitet verkligen sätts i gungning. Till skillnad från fru och dotter som båda har fast heltidslön är Phils lön liten och instabil. För att kunna betala veckohyran för sin bil måste han utsätta sig för förödmjukelsen att tigga småmynt av sin fru, be sin dotter om att få låna pengar och att muta sin son för att han skall flytta på sig där han somnat i soffan för att han själv skall kunna leta småpengar som ramlat ned mellan soffkuddarna. Han lufsar runt i huset i sin pinsamma jakt på pengar, ursäktar sig gång på gång. I scenen där han frå- gar frun sitter hon i sängen och löser korsord, med glasögon på näsan. Han står tafatt i dör- ren som en tiggare: Hon kanske gjorde dig en tjänst. Om det inte hade hänt kanske du hade kört på ett barn i nästa hörn. Vad är det man säger 'Ödet är oberäkneligt.' Visste man vad som skulle hända skulle man inte kliva ur sängen. Sånt är livet. Och han fortsätter suckande: Klockan tickar, jorden snurrar. Tidvattnet kommer in och ut. Man föds, man dör. Det är allt. Hon kanske gjorde dig en tjänst. Om det inte hade hänt kanske du hade kört på ett barn i nästa hörn. Vad är det man säger 'Ödet är oberäkneligt.' Visste man vad som skulle hända skulle man inte kliva ur sängen. Sånt är livet. Han framstår här som en filosof och grubbla- re. Den kännande manskroppen Men precis som Sander Gilman påpekar i sin analys av den fete detektiven, är det skill- nad mellan den feta, tänkande kroppen och den smale filosofen. Medan den fete tänkarens kropp framstår som feminin i kontrast till den smala, är också den fete mannens tänkande Han framstår här som en filosof och grubbla- re. Men precis som Sander Gilman påpekar i sin analys av den fete detektiven, är det skill- nad mellan den feta, tänkande kroppen och den smale filosofen. Medan den fete tänkarens kropp framstår som feminin i kontrast till den smala, är också den fete mannens tänkande Phil: Jag har inte haft en så bra vecka. Har du något över? (tittar oroligt på frun) Får jag låna lite? Hon är tyst. Ser sträng ut. Phil: Jag betalar tillbaka i helgen. Penny: Varför går du inte upp tidigare och Kropp I 21 kör folk till jobbet och flygplatsen? Phil: Ja, det förstås... Penny: De är nere i min väska. stapplar runt och faller på en gräsmatta omgi- ven av hus, underordnad genom att han är synlig för (kvinnornas) vakande blickar utan att själv se. Phil framstår som en förlorare, en man utan handlingskraft och det är uppenbart att både han själv och Penny har givit upp. De talar inte heller längre med varandra utan lever sida vid sida i stumhet, trötthet och isolering. I dominerande maskulinitetskonstruktio- ner hänger sjukdom ihop med svaghet, fysiska defekter, förlust av kontroll och passivitet - och därmed med omanlighet. Efter kollapsen på gräsmattan skildras Rory som halvsittande i en sjukhussäng med den fetlagda överkrop- pen bar och med tydliga bröst. Sedan ses han i ett antal scener liggande i sjukhussängen, flämtande och med magen i vädret, flankerad av kvinnor som betraktar (kontrollerar) honom med moderlig omsorg. Också läkarna tar kontrollen över hans kropp. En vänlig och skämtande, men också lite nedlåtande läkare säger att nu är det slut med rökning, hambur- gare och friterad mat och han måste ta medi- cin hela livet. Rory blir en slags symbol för en farlig och ohälsosam livsstil som män, och särskilt underklassens män är förknippade med: fet mat, kött, rökning och alkohol.46 Den kollapsade manskroppen "Ja", säger han, "Jag älskar henne". Han upprepar det, som för sig själv och ler lite. Han filosoferar vidare om kär- lek och ensamhet och konstaterar att: "Vi föds ensamma och dör ensamma". Och som om han då och där fått en uppenbarelse, stänger han av radion och mobilen och kör ut till havet. Där står han och bara tittar. När han kommer till- baka till sin fattiga del av London konfronteras han med nyheten om Rorys hjärtattack och får en utskällning av Penny för att han inte gick att få tag på. Då de trötta och tyngda kommer hem från sjukhuset tillsammans med dottern undrar Penny återigen varför han gjort sig oåtkomlig. Han berättar då att han hade "fått nog av allt". Hennes reaktion blir ilska; vad har han att få nog av, han gör ju inget? Själv både arbetar hon för försörjningen och gör allt hushållsarbetet. Han säger inte mycket först men frågar till slut med nedböjt huvud om hon älskar honom. Trots att hon säger att hon inte förstår vad det har med saken att göra fortsätter han: till Londons West End. Han samtalar med den kvinnliga passageraren som ställer frågan till Phil om han älskar sin fru. "Ja", säger han, "Jag älskar henne". Han upprepar det, som för sig själv och ler lite. Han filosoferar vidare om kär- lek och ensamhet och konstaterar att: "Vi föds ensamma och dör ensamma". Och som om han då och där fått en uppenbarelse, stänger han av radion och mobilen och kör ut till havet. Där står han och bara tittar. När han kommer till- baka till sin fattiga del av London konfronteras han med nyheten om Rorys hjärtattack och får en utskällning av Penny för att han inte gick att få tag på. Då de trötta och tyngda kommer hem från sjukhuset tillsammans med dottern undrar Penny återigen varför han gjort sig oåtkomlig. Han berättar då att han hade "fått nog av allt". Hennes reaktion blir ilska; vad har han att få nog av, han gör ju inget? Själv både arbetar hon för försörjningen och gör allt hushållsarbetet. Han säger inte mycket först men frågar till slut med nedböjt huvud om hon älskar honom. Trots att hon säger att hon inte förstår vad det har med saken att göra fortsätter han: Det är inte rättvist. Varför säger du detta till mig? till mig? Den kollapsade manskroppen Sonen Rory (James Corden) är den manliga karaktär vars kropp görs mest sårbar. Han har gått ur skolan och är arbetslös och skildras som en arg och isolerad ung man som främst ligger framför TV:n och äter eller sover. Han är enbart konsument, av mat, TV och cigaret- ter, utan att producera något. I flera scener zoomar kameran in enbart hans bulliga bak- del och lår där han vältrar sig i soffan. Han visas aldrig i någon kontakt med jämnåriga tjejer eller killar utan skildras endast i relation till sina familjemedlemmar eller ensam på går- den med en cigarett eller boll. Med Rorys hjärtinfarkt och hans nya posi- tion som "sjuk" och "patient" förändras han. Det blir en vändpunkt inte bara för familjen i stort utan också för karaktären Rory som per- son. Från att ha framställts som en ung man i ett konstant uppror och som en som glupar i sig mat blir han nu ett lydigt och snällt barn som äter broccoli och annan sund (omanlig) kost han har fått sig ordinerad. Han är inte längre aggressiv och gapig, utan bara lite but- ter och tystlåten. Nu är han sin mammas lille pojke igen, passiv och utan egen vilja. I egen- skap av fet pojke blir rollfiguren Rory både avsexualiserad och infantiliserad. I några scener får han omotiverade raseri- utbrott och skriker och svär antingen åt kil- larna på gården eller åt sin mamma. Hans aggressivitet är inte skildrad som maskulint kraftfull och farlig, som handlar om makt och dominans, utan mer som en aggressivitet av ett barnsligt eller hysteriskt slag: obehärskad och irrationell. Hans röst går upp i falsett när han skriker. Det är i en sådan här situation som Rory får sin hjärtinfarkt. Några killar tar hans boll och skriker retsamt åt honom medan han jagar dem "Hör du Fetto". När han börjar flämta och ta sig för hjärtat springer de sin väg och han är ensam kvar. Rorys främsta roll i filmen är att genom sin fetma inta positionen som en kollapsad och fysiskt defekt manskropp. Han Vändpunkten - den öppnade manskroppen Parallellt med att Rory visas liggande i smärtor på gräsmattan skildras Phil under en körning in 781 I Kvinnovetenskaplig tidskrift 1.06 781 I Kvinnovetenskaplig tidskrift 1.06 781 I Kvinnovetenskaplig tidskrift 1.06 till Londons West End. Han samtalar med den kvinnliga passageraren som ställer frågan till Phil om han älskar sin fru. Den kollapsade manskroppen Han sitter med nedböjd huvud igen: Phil: För det är outhärdligt (hans ord är nästan ohörbara av gråt). Penny: Vad är det som är outhärdligt. Phil: Du älskar mig inte längre, gör du det? Älskar du mig? Jag måste få veta. Hon gråter, ser stum ut. Han gråter allt- mer ljudligt hulkande. Phil (med nedböjt huvud): Om du inte äls- kar mig längre kan jag lika gärna ge mig av. Penny: Vart skall du ta vägen? Phil: Jag vet inte. Om jag gör dig olyck- lig... Jag är inte bra på något, jag tjänar inte tillräckligt. Jag vet att jag är en besvi- kelse för dig och går dig på nerverna. (Ropar) Det är som om någonting har dött (gråter så att axlarna skakar). Jag känner mig som ett gammalt träd som inte får något vatten. Han sitter med nedböjd huvud igen: Phil: För det är outhärdligt (hans ord är nästan ohörbara av gråt). Hon gråter. Han snorar, torkar ögonen. Phil: När vi först träffades kunde jag inte tro på det. En söt tjej som du med en tjock- is som jag. Folk tittade på oss. Det kändes toppen. Vi har inte mycket men vi har varandra... Och det räcker (gråter). Men om du inte vill ha mig har vi ingenting. (Ropar) Då är vi ingen familj så är det bara. Phil (med intensiv röst): Du har inte älskat mig på flera år. Phil (med intensiv röst): Du har inte älskat mig på flera år. Hon är tyst och lyssnar. Phil (med darrande röst): Du tycker inte om mig, du respekterar mig inte. Du talar till mig med förakt. Phil (med darrande röst): Du tycker inte om mig, du respekterar mig inte. Du talar till mig med förakt. Hennes ögon fylls av tårar, uppspärrade, men hon säger ingenting. I den här scenen är det Phil som gråtande och snörande bryter den stumhet som råder mellan dem, talar om hur han känner, att han känner sig som en förlorare som hon föraktar, om brist på respekt och kärlek och att höra ihop och att "ha varandra". Phils feta manskropp framställs som en läckande, öppen och genomsläpplig kropp där sekret som tårar och snor får fritt utlopp. Han är också "öppen" i bemärkelsen att han "läcker" tal om känslor. Penny (med eftertryck och desperation i rösten): Det gör jag inte alls. Phil (gråtande): Jo, det gör du! Den kollapsade manskroppen Penny: Jag talar inte alls till dig på det sättet. Phil: Du är inte medveten om det men du gör det. Phil: Du är inte medveten om det men du gör det. Penny (gråter högt och ropar): Dumheter. Penny (gråter högt och ropar): Dumheter. Kropp I 23 Det är som han själv säger till Penny när han efteråt ber om ursäkt och förklarar "Jag kunde inte hålla det inom mig". Denna öppna kropp står i motsats till en sluten, ogenom- tränglig manskropp som är disciplinerad till att upprätta gränser mot omvärlden, inte til- låts vara sårbar, och framför allt inte uttrycka sorg och gråt.47 Phils öppenhet står också i motsats till den kvinnliga karaktären, hustrun Penny. Medan Phil målande beskriver sitt känslotillstånd och dramatisk ger uttryck för sina känslor, framstår Penny som tillbaka- hållen, utan ord, stum och oförmögen att han- tera situationen. Hon gråter våldsamt, prote- sterar och ställer motfrågor men kan inte besvara Phils frågor eller beskriva sina egna känslor. sluten och fallisk med nödvändighet uppfattas som omanlig, och därmed ömkansvärd och lite patetisk. Finns det då inte andra sätt att betrakta den feta manskroppen på? Jo, jag fin- ner i de två filmer som har analyserats i den här texten en komplexitet och mångtydighet i representationen av den feta manliga arbe- tarklasskroppen som uppmanar till alternati- va seenden. För även om filmerna använder denna kropp främst som symbol för avvikelse från en hegemonisk maskulinitet, och som symbol för manlig förödmjukelse och margi- nalisering, visar berättelserna om Dave och Phil också på flera olika vägar ut ur den under- ordnade positionen. En av de vägar som stakas ut är en strävan att leva upp till de normer som är förknippad med det traditionellt maskulina inom vit arbe- tarklass. I anknytning till de tidigare resone- mangen om den manliga fetmans normaliser- ing inom amerikanska situationskomedier, blir det tydligt att rollen som god försörjare och sexuellt presterande make (i relation till både kvinnor och andra män) är helt avgöran- de för de feta, manliga karaktärerna i deras kamp att upprätthålla positionen som hetero- sexuella, vita arbetarklassmän. Det är genom att arbeta och försörja familjen och prestera ^exuellt som Daves och Phils överviktiga/feta kroppar kan definieras som positivt maskuli- na i bemärkelsen "stora och starka". Den kollapsade manskroppen Särskilt tydligt är det i filmernas respektive slutscener; när Dave med stor pondus som "The Big M a n " kliver upp på scenen i uniform och Jean, i takt med att han tar av sig kläderna, blir allt- mer flickaktigt förtjust och extatisk, eller när Penny tittar på Phil med ny respekt och beund- ran efter att han har visat handlingskraft och tagit körningar fem på morgonen för att börja spara till en familjesemester. På så sätt kan man säga att filmerna i mycket bekräftar och I näst sista scenen går Penny till slut fram till Phil och tar hans hand. Hon berättar att hon hela tiden känner sig isolerad, ensam. Han säger att han känner likadant. De ser varandra i ögonen och kysser varandra och skrattar. När de dagen efter sitter tillsammans med dottern vid Rorys sjukbädd ser de båda annorlunda ut och uppför sig annorlunda mot varandra. Phil har rent hår och har för första gången gått upp tidigt för en körning till flyg- platsen. Penny tittar på honom med glädje och ömhet. De skrattar. De är tillsammans. Avslutning När jag som kvinnlig betraktare första gången ser fotografierna på en naken Steve Tynan blir jag rörd och känner ömhet. Lite medlidande. Likaså när jag ser filmkaraktären Dave in- svept i plastfolie, Rory i sjuksängen och Phils tunga lufsande. Jag vill nästan vända bort blicken. Bespara dem skammen att bli betittade som ömkansvärda och patetiska. Som offer. Här tycks jag själv fastna i en betraktar- position där den manskropp som inte är hård, ningar för nya former att "göra genus" på. 48 Det vi får möjlighet att se är hur den fete man- nen genom en praktik av kommunikation och relaterande upphör att symbolisera den vita, heterosexuella arbetarklassmannens unde- rordning, i relation till dominerande maskuli- nitetskonstruktioner. Vi får se hur han istället, som ett aktivt subjekt, kommer att förkropps- liga löften om kärlek, närhet, stolthet, respekt och hopp. reproducerar dominerande maskulinitetskon- struktioner. Och medan The Full Monty hyllar den homosociala gemenskapen, hyllar All or Nothing familjegemenskapen. Samtidigt visar filmerna på en annan väg, ett alternativ, där de feta manliga karaktärer- na utför handlingar och iscensätter positioner som traditionellt är kopplad till kvinnor och femininitet (och även till medelklassmaskuli- nitet, den nye mannen), och där de därmed gör maskulinitet på nya sätt. Scenerna som före- går slutet, då Dave respektive Phil, utifrån sina positioner som feta män, öppet erkänner sin skam, visar sin sårbarhet, längtan och sitt beroende av kärlek och acceptans från sina partners framstår som de verkliga vändpunkt- erna - och den förvandling de genomgår då de blir sedda av de kvinnor de älskar. Då Phil grå- tande och snörande ropar till Penny "Jag kän- ner mig som ett gammalt dött träd som inte fått något vatten", då Dave naket frågar sin fru "Vem vill se det här dansa?". Här sker ett slags överskridande av stereotypa framställ- ningar av män och maskulinitet, där de feta männen i slutändan varken är representerade som passiva, feminiserade offer eller "riktiga män". 781 I Kvinnovetenskaplig tidskrift 1.0 Noter Jag använder inte de medicinska distinktio- nerna mellan överviktig och fet utifrån Body Mass Index utan använder omväxlande ter- merna fet och överviktig . Min avsikt är att undersöka de kulturella betydelserna av fetma/övervikt och i det sammanhanget ges inte termerna någon given eller fast defini- tion. mat og belse i möte med menn som opplever övervikt, Institutt for Sosiologi og Sam- funnsgeografi, Olso universitet 2004. Jag använder inte de medicinska distinktio- nerna mellan överviktig och fet utifrån Body Mass Index utan använder omväxlande ter- merna fet och överviktig . Min avsikt är att undersöka de kulturella betydelserna av fetma/övervikt och i det sammanhanget ges inte termerna någon given eller fast defini- tion. 13 Jag håller för närvarande på med en analys av betydelser av genus, fetma/slankhet, mat och ätande i Kungen av Queens, som är den första i raden av denna typ av sit-coms, karak- teriserade som "The Beauty and the Best". 14 Se http//boards.sonypictures.com/boards/ achive/index. 11/10/0 5. 5 Santiago Fouz Hernåndez: "Phallic Matters? Ewan McGregor and the representations of the male body in Peter Greenaway's 'The Pillow Book'", Men and Masculinities vol. 8, 2005:2. 5 Santiago Fouz Hernåndez: "Phallic Matters? Ewan McGregor and the representations of the male body in Peter Greenaway's 'The Pillow Book'", Men and Masculinities vol. 8, 2005:2. 15 Jag håller för närvarande på med en analys av genus, fetma/slankhet, mat och ätande i Svensson, Svensson. 6 Robert W Connell: Masculinities, Polity Press 1995. 16 Se Thomas F Cash & Karen L Hicks: "Being Fat versus Thinking Fat. Relationships with Body Image, Eating Behaviours and Well- being", Cognitive Therapy and Research, vol.14, 1990:3, Kerry E Mc Pherson & Jane D Turnbull: "Body Image Satisfaction in Scottish men and its Implications for Promoting Health Behaviours", Internatio- nal Journal of Men's Health, 2005 och Marlene B Schwartz & Kelly D Brownell: "Obesity and Body Image", International Journal of Obsesity vol. 26, 2004:1. Se också www.en.Wikipedia.org/wiki/body image 19/12/05. 6 Robert W Connell: Masculinities, Polity Press 1995. 7 Peter Lehman: "Crying over the Melodrama Penis: Melodrama and Male Nudity in Films of the 90", Masculinity. Bodies, movies, cul- ture, red. Peter Lehman, Routledge 2001. 7 Peter Lehman: "Crying over the Melodrama Penis: Melodrama and Male Nudity in Films of the 90", Masculinity. Bodies, movies, cul- ture, red. Peter Lehman, Routledge 2001. 8 Kelly Farrell: "Naked Nation. Noter 1 Utställningskort Astronaut. Fotografier av Steven Tynan. Kulturhuset, Stockholm 2003. 1 Utställningskort Astronaut. Fotografier av Steven Tynan. Kulturhuset, Stockholm 2003. Se till exempel Susan Bordo: The Male Body. A New Look at Men in Public and Private. Farrar, Straus and Giroux 1999, s.15-35. Denna text är en del i ett pågående bokpro- jekt som har arbetstiteln "Maskulinitet, kropp och hunger. Berättelser om manlig fetma." Här kommer jag bland annat att undersöka varierande och motsägelsefulla betydelser av manlig fetma, både historiska och samtida, främst inom den västerländska kulturen. Som ett exempel på negativa, sam- tida betydelser av manlig fetma tar jag medieframställningar av finansmannen Jan Stenbeck samt statsminister Göran Persson. Som ett exempel på positiva betydelser tar jag Bearkulturen, en subkultur bland bogar där normen är att vara stor och hårig. För litteratur runt manlig fetma se Sander L. Gil- man: Fat Boys. A Slim Book, University of Nebraska Press 2004, Jeffery Mosher: "Set- ting Free the Bears: Refiguring Fat Men on Television", Bodies Out ofBound. Fatness and Trangression, red. J E Braziel & K Le Besco, University of California Press 2001a samt Marianne Inez Lien: Menn og slanking. En kvalitativ studie av maskulinitet, kropp, 3 Detta innebär inte nödvändigtvis ett ifrå- gasättande av manlig dominans och kvinnlig underordning. I The Full Monty är de kvinnli- ga karaktärerna mycket uppenbart bara bifi- gurer med funktion att väcka känslor och reaktioner hos männen och driva dem till vissa handlingar. Jeans roll är till exempel främst att vara hinder eller stöd för Dave i hans kamp för återupprätta någon slags stolthet som man. Snarare än att ifrågasätta maktrelationerna mellan könen fungerar de överskridande framställningarna som ett sätt att vidga spekt- rat i skildringen av män och maskulinitet, något som i sin tur kanske kan skapa öpp- Kropp I 25 "Having Their Cake and Eating It Too: Fat Acceptance Films and the Production of Meaning", red. J Lewis, The End of Cinema as we know it. American Film in the Nineties. New York University Press 2001b. T 2 Mosher 200 ih. "Having Their Cake and Eating It Too: Fat Acceptance Films and the Production of Meaning", red. J Lewis, The End of Cinema as we know it. American Film in the Nineties. New York University Press 2001b. T 2 Mosher 200 ih. mat og belse i möte med menn som opplever övervikt, Institutt for Sosiologi og Sam- funnsgeografi, Olso universitet 2004. Noter The Full Monty, Working Class Masculinity, and the British Image", Men and Masculinities, vol. 6, 2003:2. 9 Gilman 2004. 9 Gilman 2004. 10 Teorin om "the male gaze" publicerades först i Laura Mulveys klassiska artikel "Visual Pleasure and Narrative Cinema", Screen, vol. 16, 1975:3. För svensk översätt- ning se bland annat "Spelfilmen och lusten att se", Modern filmteori, red. L G Anders- son &c E Hedling, Studentlitteratur t995. Se också vidare i Modern filmteori för artiklar runt feministiska filmteori och utvecklingen efter Mulveys teori till exempel Janet Bergström & Mary Ann Doane: "Den kvinnliga åskådaren: kontexter och tendenser". 10 Teorin om "the male gaze" publicerades först i Laura Mulveys klassiska artikel "Visual Pleasure and Narrative Cinema", Screen, vol. 16, 1975:3. För svensk översätt- ning se bland annat "Spelfilmen och lusten att se", Modern filmteori, red. L G Anders- son &c E Hedling, Studentlitteratur t995. Se också vidare i Modern filmteori för artiklar runt feministiska filmteori och utvecklingen efter Mulveys teori till exempel Janet Bergström & Mary Ann Doane: "Den kvinnliga åskådaren: kontexter och tendenser". 17 Här ser jag Roseanne som tillhörande en annan kategori än ovanstående situations- komedier eftersom det här är den feta kvin- nan, (Roseanne Barr) som är huvudperso- nen snarare än hennes man Dan (John Goodman). 18 För tidig feministisk nordamerikansk littera- tur i ämnet se till exempel. Susy Orbach: Fetma är en kvinnofråga, Prisma 1980. Kim Chernin: The Obsession. Reflections on the Tyranny of Slenderness, Harper &c Row 1 1 Mosher 2001a samt Jeffery Mosher: 781 I Kvinnovetenskaplig tidskrift 1.0 30 För diskussioner runt genus, kropp, hårdhet och mjukhet, se Bordo 1995 s.36-68, Tho- mas Johansson: Det första könet? Mans- forskning som reflexivt projekt, Studentlitte- ratur 2000 s.101-103. 1982, Susan Bordo: Unbearable Weight. Western Culture, and the Body. University of California Press 1993. För senare littera- tur i Sverige se Anna Johansson: Elefant i nylonstrumpor. Om kvinnlighet, kropp och hunger, Anamma 1999. För fokus på mäns fetma se Gilman 2004. 31 Johansson 2000, s.103. 3 2 Se Peter N. Stearns: Fat History. Bodies and Beauties in the Modern West, New York University Press 1997, Johansson 1999, Jana Evans Braziel &C Kathleen LeBesco: "Edi- tor's Introduction", Bodies Out of Bound. Fatness and Trangression, red. Jana Evans Braziel & Katleen LeBesco , University of California Press 2001. 19 Orbach 1980, Chernin 1982, Bordo 1993, Johansson 1999. Kropp I 27 Kropp I 27 Kropp I 27 41 För intressant analys av männens samtalssti- lar i filmen se Halvorsen 2000. 41 För intressant analys av männens samtalssti- lar i filmen se Halvorsen 2000. 41 För intressant analys av männens samtalssti- lar i filmen se Halvorsen 2000. 41 För intressant analys av männens samtalssti- lar i filmen se Halvorsen 2000. movies are used as illustrating examples, both dealing with the male working class anti-hero as facing social and economic marginalisation and loss of power: The Full Monty by Peter Cattaneo (1997) and All or Nothing (2002) by Mike Leigh. Both these movies have leading male characters who are fat/overweight. They are not only repre- sented as marginalised because of unemploy- ment and an inability to construct masculinity around work and the identity as provider, but they are also positioned as marginalised because of theirdeviant bodies. 42 Robyn Longhurst: Bodies. Exploring Fluid Boundaries. Routledge 2001, s.66-90. 42 Robyn Longhurst: Bodies. Exploring Fluid Boundaries. Routledge 2001, s.66-90. 43 Bordo 1993, Johansson 1999. 43 Bordo 1993, Johansson 1999. 43 Bordo 1993, Johansson 1999. 44 Addelston 1999. 44 Addelston 1999. 44 Addelston 1999. 45 Gilman 2004. 46 Om mäns hälsa och maskulinitet se till exempel Jonathan Watson: Male Bodies. Health, Culture and Identity. Open Univer- sity Press 2000. 46 Om mäns hälsa och maskulinitet se till exempel Jonathan Watson: Male Bodies. Health, Culture and Identity. Open Univer- sity Press 2000. 47 För temat genus, kropp, öppenhet/slutenhet se Longhurst 2001, s. 1-32, Jorun Solheim: Den öppna kroppen. Om könssymbolik i modern kultur, Daidalos 2001. The movies are analysed within the social and cultural context of an increasing visibility (and nomalization) of the fat man in movies and sit- coms (primarily in the USA), the increasing media objectification of the male body, weight, eating and appearance as becoming issues for men, and finally, the highly moralizing and stigmatizing "fat is bad" discourse which dominates Western cul- ture. The analysis focus on how both the character Dave in The Full Monty and the character Phil and hisson Rory in All or /Vof/7/'ngare feminized in rela- tion to the other male characters and in relation to women. They are represented as without tradi- tional male characteristics such as assertiveness, aggressivity and sexual potency. Kropp I 27 For example Dave is depicted as being ashamed of his body, deeply concerned of his weight and appearance, occupied with finding ways to reshape his body through dieting etc. He manifests an objectified body consciousness, usually associated with women and femininity. 48 Se Marie Nordberg för inspirerande resone- mang i artikeln "'Kvinnlig maskulinitet" och manlig femininitet'. En möjlighet att överskrida könsdikotomin?", Kvinnoveten- skaplig tidskrift 2004:1-2. gender studies, masculinity, body, overweight, film, working-class gender studies, masculinity, body, overweight, film, working-class Nyckelord genusvetenskap, maskulinitet, kropp, fetma, film, arbetarklass genusvetenskap, maskulinitet, kropp, fetma, film, arbetarklass Noter 20 Nita Mary McKinley: "Feminist Perspecti- ves and Objectified Body Consciousness", Body Image. A Handbook ofTheory, Research and Clinical Practice, red. Thomas F Pruzinsky & Thomas Cash, The Guildford Press 2002. 33 Paul Campos: The Obesity Myth. Why America's Obsession with Weight is Hazar- dous to your Health. Gotham Books 2004. 21 Bordo 1999, Gilman 2004. 21 Bordo 1999, Gilman 2004. 22 Gilman 2004. 22 Gilman 2004. 34 Se till exempel Sahlgrenska universitetssjuk- hus hemsida om fetma där man har "En glo- bal epidemi" som en rubrik, www.obesi- tas.med.gu.se/fetma. 13/12/05. 23 Susan Faludi: Ställd. Föräderiet mot mannen, Ordfront 2000. 24 Bordo 1999, Sarah Grogan: Body Image: Understanding Body Dissatisfaction in Men, Woman and Children, Routledge 1999. 3 5 Susan Sontag: Sjukdom som metafor, Brom- bergs 1981. 25 Roberto Oliviardia, Harrison G. Pope & Katherine A Phillips: The Adonis Complex. The Secret Crisis of Male Body Obsesssion, Free Press 2000. 36 Farrell 2003, Judi Addelston: "Doing the Full Monty with Dirk and Jane: Using the Phallus to Validate Marginalized Masculini- ties", The Journal of Men's Studies vol.7, T999:3> Kristin Halvorsen:"Maskuliniteter på blå resept. Maskuliniteter og menns sam- talepraksiser i filmen The Full Monty". Kvinneforskning. Empirisk forskning etter poststrukturalismen 2000:1. 26 Chris Wienke: "Negotiating the Male Body: Men, Masculinity, and Cultural Ideals", The journal of Men's Studies, vol. 6, 1998:3. 27 Sarah Grogan & Helen Richards: "Body Image: Focus Groups with Boys and Men", Men and Masculinities, vol. 4 , 2002:3. 37 Se till exempel Halvorsen 2000. 28 Kennneth R Dutton: The Perfectible Body. The Western Ideal of Physical Development, Cassell 1995, Wienke 1998. 37 Se till exempel Halvorsen 2000. 38 McKinley 2000. 38 McKinley 2000. 39 Naomi Wolf: Skönhetsmyten. Hur föreställ- ningar om skönhet används mot kvinnor. Natur & Kultur 1982. 29 Se till exempel Roberto 01iviardia:"Body Image and Muscularity", Body Image. A Handbook ofTheory, Research and Clinical Practice, red. Thomas F Cash & Thomas Pruzinsky, The Guildford Press 2002. 40 Lien 2004. Enligt siffror från England ban- tar 24,8 procent av de brittiska männen jäm- fört med 42,8 procent av kvinnorna. www.news.bbc.com 5/11/04. 40 Lien 2004. Enligt siffror från England ban- tar 24,8 procent av de brittiska männen jäm- fört med 42,8 procent av kvinnorna. www.news.bbc.com 5/11/04. Summary Marginalized male bodies: pictures of fat working- class men, by Anna Johansson, PhD in Sociology, gestalt therapist and teacher at the Gestat-Academy in Scandinavia. In the conclusion Johansson argues that both movies represent the fat male body in a ambiva- lent and contradicting way, outlining different alternatives for the fat characters to deal with mar- ginalization and loss of masculine power. One way is to try to fulfil the traditional norm of working class masculinity, to be hard working and sexually potent, and be able to earn the epithet of "big and This artide is analysing contemporary cultural menings of the fat male body. More specifically it is looking into how the fat, male body is used as a cultural symbol for the crisis of a white, hetero- sexual working-class masculinity, in relation to the hegemonic masculine (and fallic) body as hard, muscular and impermeable. Two British 781 I Kvinnovetenskaplig tidskrift 1.06 781 I Kvinnovetenskaplig tidskrift 1.06 strong" rather than "fat and weak". The other way is to "do" masculinity in new ways and to perform femininity. Johansson argues that the actual turn- ing points for the characters of Dave and Phil, the shift from shame and passivity to pride and activ- ity, are the scenes when they openly acknowledge their shame and vulnerability, and when they are met with acceptance and love. Although none of the movies challenge male dominance, the way the representations of the fat, white, heterosexual working dass man transgress stereotypes, could possibly open up for alternative representations of masculinity. Through a practice of communica- tion the fat male figure ceases to symbolize the subordination of the white, heterosexual working dass man in relation to a hegemonic ideal. Instead he becomes an adive subject, embodying hope, respect, closeness and pride. Anna Johansson Gestalt-Akademin i Skandinavien Västerlånggatan 53 111 29 Stockholm anna.johansson@gestaltakademin.se Anna Johansson Anna Johansson Gestalt-Akademin i Skandinavien Västerlånggatan 53 111 29 Stockholm anna.johansson@gestaltakademin.se
W4225522781.txt	https://www.nomos-elibrary.de/10.5771/9783748913177-111.pdf?download_full_pdf=1&page=1	de		Kapitel 2: Europäische und internationale Leitlinien	Nomos Verlagsgesellschaft mbH & Co. KG eBooks	2,022	cc-by	15,154	Kapitel 2: Europäische und internationale Leitlinien Nachdem die Grundlagen der Forschungsfrage erörtert sind und insbesondere die Regulierungsbedürftigkeit der Problematik geschlechterdiskriminierender Werbung hervorgehoben ist, widmet sich das folgende Kapitel einer staatenübergreifenden Perspektive. In einer globalisierten Welt gewinnen internationale und supranationale Vereinigungen und Institutionen zunehmend an Gewicht. Die nationalen Regulierungssysteme der einzelnen Staaten können daher nicht ohne den Blick auf mehrstaatliche Referenzrahmen analysiert werden. Die Vorgaben der Europäischen Union spielen unabhängig von den nationalen Rechtsstrukturen und den individuellen kulturellen Gegebenheiten eine wichtige Rolle für die Regelungslandschaft der Mitgliedstaaten. Viele Rechtserneuerungen im Bereich der Medienregulierung folgen dem Ziel der europäischen Integration.447 Die Regulierungsprozesse und die Regulierungspraxis werden von diesem Phänomen beeinflusst.448 Daneben sind internationale völkerrechtliche Gremien und Institutionen wie beispielsweise diejenigen der Vereinten Nationen prägend für Teilbereiche der nationalen Rechtsentwicklungen. Das Werberecht wird ebenfalls von verschiedenen Rechtsquellen geformt, die unter anderem von internationalen Organisationen oder europäischen Institutionen stammen.449 Es ist das Ziel des folgenden Abschnitts, europäische und internationale Leitlinien zu geschlechterdiskriminierender Wirtschaftswerbung herauszuarbeiten. Geschlechterdiskriminierende Werbeinhalte sowie verwandte Themenfelder werden in völkerrechtlichen Beschlüssen (A.), Vorschriften der EU und Zusammenkünften der EU-Organe (B.) ebenso wie im Rahmen der internationalen Werbeselbstkontrolle (C.) behandelt. Die nachfolgende Befassung mit diesen bildet sowohl die Grundlage für die Erfassung des Einflusses internationaler und europäischer Vorgaben auf die nationalen Lösungsansätze im dritten Kapitel als auch für die Erörterung der möglichen Regulierungs- und Harmonisierungsvarianten im vierten Kapitel. 447 Vgl. Seufert/Gundlach, Medienregulierung in Deutschland, S. 300 f. 448 So auch Siegert/Brecheis, Werbung in der Medien- und Informationsgesellschaft, S. 101. 449 Ebenda; Seufert/Gundlach, Medienregulierung in Deutschland, S. 300 f. 111 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien A. Völkerrecht Das Völkerrecht spielt auf dem Weg zur Geschlechtergleichstellung eine wichtige Rolle. Zwar sind die Bindung und der Prozess völkerrechtlicher Konventionen und Vorschriften nicht mit der innerstaatlichen Pflicht zur Umsetzung von EU-Richtlinien vergleichbar. Dennoch setzen einige Bestimmungen wichtige Impulse und einige der völkerrechtlichen Vorgaben können als richtungsweisend für die europäische Anti-Diskriminierungspolitik beschrieben werden. In diesem Zusammenhang sind die Übereinkommen der Vereinten Nationen, vor allem die Convention on the Elimination of All Forms of Discrimination against Women (Konvention zur Beseitigung jeder Form von Diskriminierung der Frau), die letztmalig 1995 abgehaltene Weltfrauenkonferenz der Vereinten Nationen sowie die Regelungen und die Entschließungen des Europarats zu nennen, die nachfolgend untersucht werden. I. Vereinte Nationen Gleichstellung und Antidiskriminierungsfragen werden in zahlreichen Entschließungen, Programmen und Ausschüssen der Vereinten Nationen behandelt. Diese wirken sich auf nationale Regulierungsansätze und -strategien aus. Erst im Jahr 2019 veröffentlichte das Bundesministerium für Familie, Senioren, Frauen und Jugend ein Handbuch zur Frauenrechtskonvention der Vereinten Nationen aus dem Jahr 1979.450 Die Convention on the Elimination of All Forms of Discrimination Against Women (CEDAW) vom 18. Dezember 1979 ist, um es mit den Worten der ehemaligen Bundesministerin Giffey auszudrücken, das „erste und bis heute umfassendste internationale Übereinkommen zu den Menschenrechten speziell von Frauen“.451 Die CEDAW wird in anderen deutschen sowie internationalen Berichten als das „wichtigste völkerrechtliche Menschenrechtsinstrument 450 Bundesministerium für Familie, Senioren, Frauen und Jugend, Mit RECHT zur Gleichstellung!. 451 Ebenda, siehe Vorwort, S. 5. 112 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb A. Völkerrecht für Frauen“452 oder als die „Magna Charta der UN-Frauenrechte“453 beschrieben.454 Das Übereinkommen geht maßgeblich auf die Forderungen nach einem internationalen Rechtsinstrument zur Bekämpfung der Diskriminierung der Frau zurück, die bei der ersten Weltfrauenkonferenz im Jahr 1975 in Mexiko aufgestellt wurden.455 Inzwischen haben 189 Staaten das Abkommen ratifiziert, unter anderem Deutschland als einer der ersten Staaten bereits im Jahr 1985.456 Es bleibt zu berücksichtigen, dass die völkerrechtlichen Bestimmungen der Vereinten Nationen ausschließlich die Frauendiskriminierung behandeln und die Diskriminierung von Männern auf Grund des Geschlechts vom Wortlaut der CEDAW nicht erfasst wird.457 452 https://www.institut-fuer-menschenrechte.de/menschenrechtsinstrumente/verei nte-nationen/menschenrechtsabkommen/frauenrechtskonvention-cedaw/. 453 Vgl. Dackweiler, Europäische Gleichstellungspolitik, KJ 2017, 297 (300). 454 In Art. 5 a) der Vereinbarung wird festgelegt, dass die Vertragsstaaten „alle geeigneten Maßnahmen, um einen Wandel in den sozialen und kulturellen Verhaltensmustern von Mann und Frau zu bewirken, um so zur Beseitigung von Vorurteilen sowie von herkömmlichen und allen sonstigen auf der Vorstellung von der Unterlegenheit oder Überlegenheit des einen oder anderen Geschlechts oder der stereotypen Rollenverteilung von Mann und Frau beruhenden Praktiken zu gelangen“, https://www.institut-fuer-menschenrechte.de/fileadmin/use r_upload/PDF-Dateien/Pakte_Konventionen/CEDAW/cedaw_de.pdf; hiermit wird sich deutlich gegen Frauendiskriminierung und Geschlechterstereotypen in den Medien und der Werbung gestellt. 455 Die Weltfrauenkonferenz sollte den Fortschritt im Bereich der Geschlechtergleichstellung fördern, der seit der Gründung der VN 1945 zu kurz gekommen war. Neben der Möglichkeit Themen zur Gleichberechtigung und zur Beendigung jeglicher Form der Diskriminierung zu diskutieren, sollte die Konferenz ebenfalls dazu dienen einen gemeinsamen Aktionsplan auszuarbeiten. Die abschließende Deklaration der Aktionsplattform von Mexiko – die CEDAW – wurde sodann 1979 von der VN-Generalversammlung angenommen. Nordmeyer, 20 Jahre Weltfrauenkonferenz von Beijing, UN 2015, 261 (261 f.). 456 https://www.bmfsfj.de/bmfsfj/aktuelles/alle-meldungen/dr--franziska-giffey---w ir-sind-noch-lange-nicht-am-ziel-/141854; die USA hat das Übereinkommen nie ratifiziert; Somalia, Iran und der Vatikan haben nicht unterschrieben (https://w ww1.wdr.de/stichtag/stichtag-un-weltfrauenkonferenz-100.html). 457 Dazu auch CEDAW General recommendation No. 25, on article 4, paragraph 1, Abs. 5. 113 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien 1. Entwicklung von Frauenrechten: Weltfrauenkonferenzen Nachdem die Vereinten Nationen das Jahr 1975 zum „Jahr der Frau“ ausgerufen hatten, wurden im Rahmen der ersten Weltfrauenkonferenz die drei Hauptthemen Gleichstellung, Entwicklung und Frieden diskutiert. Bloch beschreibt die Ereignisse im historischen Kontext der Frauenrechte der Vereinten Nationen als „Zäsur [...], deren Bedeutung nicht hoch genug bewertet werden kann“.458 Weitere Weltfrauenkonferenzen fanden 1980 in Kopenhagen, 1985 in Nairobi sowie zuletzt 1995 in Peking statt. Die Konferenzen werden vorrangig als Möglichkeit wahrgenommen, Fraueninteressen weltweit zu koordinieren. Die bisherigen Konferenzen haben die Erarbeitung von Themen, Definitionen und Zielsetzungen erlaubt, die nicht nur für die Organisation und normative Arbeit der Vereinten Nationen von Bedeutung sind, sondern ebenfalls die Grundlage für die Bekämpfung von Frauendiskriminierungen und insbesondere für Lobbyarbeit in dem Themenfeld bilden.459 Jede der vier Weltfrauenkonferenz brachte wichtige Fortschritte auf dem Weg zur Gleichberechtigung.460 Der Abschlussbericht von 1995 aus Peking enthält Ausführungen zur Darstellung von Frauen in den Medien (Abschnitt J: Frauen und Medien). Es wird unter anderem das strategische Ziel der „Förderung der ausgewogenen und nichtstereotypen Darstellung von Frauen in den Medien“ aufgeführt.461 Hierbei werden die „klischeefreie Darstellung von Frauen“462 sowie die „Einführung von berufsständischen Richtlinien und Verhaltensregeln, im Einklang mit der Meinungsfreiheit, die sich mit gewalttätigen, erniedrigenden oder pornographischen Darstellungen von Frauen in den Medien, insbesondere auch in der Werbung, befassen“,463 als notwendige Maßnahmen auf nationaler sowie internationaler Ebene gefordert. Svensson bemängelt die „moderate Ausgestaltung“ des Abschlussberichts, die darauf basiere, dass gesetzliche Verbote von Geschlechterstereotypen nach den Annahmen der „westlichen Welt“ eine Bedrohung für die Meinungsfreiheit darstellen würden. Als Folge werde in den meisten 458 Bloch, UN-Woman, S. 27. 459 Die Vereinten Nationen und Frauen, UN-BASIS-INFORMATIONEN 51, S. 3. 460 Ebenda, S. 3 f.; dazu ausführlich Bloch, UN-Woman, S. 29 ff. 461 https://www.un.org/Depts/german/conf/beijing/anh_2_10.html#iv-j. 462 Siehe 244. a) Hs. 2 (Abschnitt J). 463 Siehe 244. b) (Abschnitt J). 114 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb A. Völkerrecht Staaten Selbstregulierung auf der Grundlage privater Verhaltenskodizes einer gesetzlichen Regelung vorgezogen.464 Im Kern ist das Vorgehen in Bezug auf Werbeinhalte zwar in der Tat als eher zurückhaltend zu beschreiben. Dabei ist jedoch zu berücksichtigen, dass die Darstellung von Frauen in den Medien nur einen Teilbereich der in dem Bericht diskutierten Aufgaben und Ziele umfasst. Zudem ist die Festlegung von Bestimmungen in einem derart großen Staatenbündnis wie dem der Vereinten Nationen eine besondere Herausforderung, die nicht ohne Kompromisse auskommen kann. Es ist daher naheliegend, dass in einem solchen Rahmen nur begrenzt konkrete staatenübergreifende Lösungsansätze erarbeitet werden können. Statt weiterer Konferenzen wurde im Anschluss an die Konferenz in Peking eine regelmäßige Berichterstattungspflicht der Staaten465 und eine damit einhergehende Überprüfung der Einhaltung und Realisierung der Ziele durch die Vereinten Nationen statuiert.466 Nordmeyer erklärt die „wenig ambitionierte Veranstaltungsform“ der Konferenz im Jahr 2015467 einerseits mit der mangelnden Finanzierung, andererseits mit der allgemeinen Sorge, dass eine Neuverhandlung der Aktionsplattform aufgrund der „sich immer deutlicher abzeichnenden fundamentalistischen und religiösen frauenfeindlichen Strömungen vieler Regierungen“ nur mit einem Rückschritt einhergehen könnte.468 Dennoch verzeichneten viele der Staatenberichte eine, wenn auch häufig als zu langsam beschriebene, Verbes- 464 Svensson/Edstörm, Freedom of Expression vs. Gender Equality, TRF 2014, 479 (488). 465 Für Deutschland befinden sich die Berichte etwa in Erklärungen aus 2010 (Peking + 15, siehe Entschließung des Europäischen Parlaments vom 25.02.2010 zu Peking + 15-UN-Plattform für Maßnahmen zur Gleichstellung der Geschlechter, ABl. C 348 E, 21.12.2010, S. 11–14), 2015 (Peking + 20, siehe https://www .unwomen.org/en/csw/previous-sessions/csw59-2015/preparations#Regional) und 2020 (Peking + 25, Aktueller Bericht der UN siehe https://www.unwomen. org/-/media/headquarters/attachments/sections/library/publications/2020/gender -equality-womens-rights-in-review-en.pdf?la=en&vs=934). 466 Das Übereinkommen sieht in Art. 18 vor, dass die Vertragsstaaten in regelmäßigen Abständen einer Berichtspflicht nachkommen, die von dem CEDAW-Ausschuss kontrolliert wird. Seit 1999 gibt es zudem ein Fakultativprotokoll, das die Individualbeschwerde vorsieht. Nordmeyer, 20 Jahre Weltfrauenkonferenz von Beijing, UN 2015, 261 (261). 467 Zunächst hatte der UN-Generalsekretär Ban Ki-moon eine fünfte Weltfrauenkonferenz vorgeschlagen. Dies wurde jedoch nicht weiterverfolgt, sondern auf regionale Überprüfungskonferenzen von fünf UN-Regionalkommissionen reduziert. 468 Nordmeyer, 20 Jahre Weltfrauenkonferenz von Beijing, UN 2015, 261 (263). 115 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien serung der „politischen und gesellschaftlichen Rahmenbedingungen für Frauen“. Im Bereich der Medien seien die Stellung und die Einflussmöglichkeiten von Frauen jedoch noch sehr gering und auch die Darstellungen von Frauen in den Medien sei noch überwiegend von negativen und entwürdigenden Bildern geprägt.469 2. Convention on the Elimination of All Forms of Discrimination Against Women Das Kernelement für das internationale Bemühen um Geschlechtergleichstellung bildet die CEDAW. Für alle Staaten, die die Konvention ratifiziert haben, besteht eine völkerrechtliche Verpflichtung gegenüber den anderen Vertragsstaaten, Maßnahmen zu ergreifen, um die Vorgaben und Ziele zu erreichen.470 In Deutschland wird die völkerrechtliche Dimension durch eine innerstaatliche Geltung nach Art. 59 Abs. 2 S. 1 GG umgesetzt,471 so dass die Bestimmungen nach überwiegender Auffassung einem einfachen Bundesgesetz gleichzusetzen sind.472 Eine Besonderheit des Übereinkommens besteht darin, dass die Vertragsstaaten verpflichtet werden, gegen jegliche Form der Diskriminierung von Frauen vorzugehen. Mithin sind nicht nur solche Diskriminierungen erfasst, die durch den Staat erfolgen, sondern gleichermaßen Diskriminierungen, die durch andere Personen, Organisationen oder Unternehmen verursacht werden. Zudem bezieht sich die Bestimmung nicht allein auf rechtliche Diskriminierungen. Vielmehr ist jede tatsächliche Diskriminierung von Frauen in allen Lebensbereichen, also vom Arbeitsmarkt bis hin zu sozialen und privaten Sphären, umfasst.473 Nach Rodi sei die CEDAW „von der Einsicht geprägt, dass Diskriminierung von einem sozialen Um- 469 Ebenda (264) 470 Rodi, Bekämpfung von Geschlechterstereotypen durch die Frauenrechtskonvention der Vereinten Nationen, S. 61. 471 Deutschland hat die CEDAW 1985 ratifiziert (https://www.bmfsfj.de/bmfsfj/the men/gleichstellung/internationale-gleichstellungspolitk/vn-frauenrechtskonvent ion-cedaw-staatenberichtsverfahren-und-dokumente-80794). 472 Rodi, Bekämpfung von Geschlechterstereotypen durch die Frauenrechtskonvention der Vereinten Nationen, S. 62. 473 Dazu siehe auch Tertinegg, Sexismus in der Werbung, S. 53 f. 116 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb A. Völkerrecht feld mit kulturell beeinflussten Rollenbildern und Rollenerwartungen an Frauen und Männer bedingt wird“.474 Art. 1 der CEDAW definiert die Diskriminierung von Frauen als „jede Unterscheidung, jeder Ausschluss oder jede Beschränkung aufgrund des Geschlechts, die bewirkt oder bezweckt, dass die Anerkennung oder die Ausübung der Menschenrechte und Grundfreiheiten im politischen, wirtschaftlichen, sozialen, kulturellen, zivilen oder in jedem anderen Bereich durch Frauen, unabhängig von ihrem Familienstand, auf der Grundlage der Gleichheit von Männern und Frauen beeinträchtigt oder zunichte gemacht werden“. Dadurch wird die direkte und die indirekte Diskriminierung von Frauen sowohl auf der Grundlage ihres biologischen Geschlechts („Sex“) als auch auf der Grundlage ihres sozialen Geschlechtes („Gender“) untersagt. Gender wird als diejenigen Unterschiede verstanden, die auf Grundlage der sozial konstruierten Identitäten, Eigenschaften und Rollen für Frauen und Männer und deren sozialer und kultureller Bedeutung für die biologischen Unterschiede bestehen, und die zu hierarchischen Beziehungen zwischen Frauen und Männern und zur Verteilung von Macht und Rechten führen, die Männer begünstigen und Frauen benachteiligen. Dazu heißt es, dass „[d]iese gesellschaftliche Positionierung von Frauen und Männern [...] durch politische, wirtschaftliche, kulturelle, soziale, religiöse, ideologische und umweltbedingte Faktoren beeinflusst [wird] und [...] durch Kultur, Gesellschaft und Gemeinschaft verändert werden [kann]“.475 Direkte Diskriminierung wird als eine unterschiedliche Behandlung definiert, die ausdrücklich auf dem Geschlecht und den geschlechtsspezifischen Unterschieden beruht, wohingegen indirekte Diskriminierung als scheinbar geschlechterneutrale Maßnahme beschrieben wird, die in der Praxis aber eine diskriminierende Wirkung auf Frauen hat, da bereits bestehende Ungleichheiten durch die scheinbar neutrale Maßnahme nicht behoben werden.476 Darüber hinaus wird in Art. 5 a) der CEDAW festgelegt, dass die Vertragsstaaten „alle geeigneten Maßnahmen ergreifen sollen, um einen Wandel in den sozialen und kulturellen Verhaltensmustern von Mann und Frau zu bewirken und so Vorurteile sowie alle sonstigen Vorstellungen und dazugehörigen Praktiken, die auf der Idee der Unterlegenheit oder 474 Rodi, Bekämpfung von Geschlechterstereotypen durch die Frauenrechtskonvention der Vereinten Nationen, S. 53. 475 CEDAW General recommendation No. 28, CEDAW/C/GC/28, 16.12.10, Abs. 5. 476 Ebenda, Abs. 16. 117 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien Überlegenheit des einen oder anderen Geschlechts oder der stereotypen Rollenverteilung von Mann und Frau beruhen, zu beseitigen“. Interessant ist hierbei, dass nicht nur auf weibliche, sondern auch auf bestehende männliche Stereotypen hingewiesen wird. Möglicherweise wurde dadurch auf den Umstand aufmerksam gemacht, dass die weiblichen und männlichen Attribute in der Regel Gegensatzpaare bilden, die sinnvollerweise zusammen betrachtet werden sollten. Insgesamt bestimmt die Regelung die Vertragsstaaten dazu, traditionellen Geschlechterrollen und Geschlechterstereotypen aktiv entgegenzuwirken, wenn sie zu einer Diskriminierung führen. Damit enthält die CEDAW zwar keine expliziten Ausführungen zu geschlechterdiskriminierender Werbung oder zur Darstellung von Frauen und Männern in den Medien, jedoch weitreichende Empfehlungen, die nicht nur die Diskriminierung von Frauen im Allgemeinen, sondern auch ausdrücklich Geschlechterstereotype betreffen.477 Rodi erkennt in Art. 5 CEDAW die „zentrale Norm für die Bekämpfung von Geschlechterstereotypen“.478 Die Norm stelle fest, dass „geschlechtsspezifische Arbeitsteilung“ ebenso wie „stereotype Elternrollen“ das Ziel der Geschlechtergleichstellung nachteilig beeinflussen würden.479 Gleichwohl merkt Rodi ebenfalls an, dass die Norm häufig restriktiv ausgelegt würde und sich die Verpflichtung folglich auf „Informations- und Bildungskampagnen“ beschränken würde.480 Holtmaat erkennt in der Konvention das Potenzial, nicht nur ein Zeichen gegen Geschlechterdiskriminierung zu setzen, sondern die Vertragsstaaten auch dazu zu motivieren, strukturelle Diskriminierung aufgrund des Geschlechts aktiv zu bekämpfen.481 Dennoch hebt sie ebenfalls hervor, dass insbesondere die Verpflichtung, Geschlechterstereotype und feste elterliche Geschlechterrollen zu ändern, in vielen Staaten auf Widerstand stoßen könnte.482 So hänge die Art, wie die Geschlechterbeziehungen in einem Land strukturiert sind, eng mit dessen nationaler Identität zusam- 477 Dazu auch Svensson/Edstörm, Freedom of Expression vs. Gender Equality, TFR 2014, 479 (485). 478 Rodi, Bekämpfung von Geschlechterstereotypen durch die Frauenrechtskonvention der Vereinten Nationen, S. 55. 479 Ebenda, S. 56. 480 Ebenda. 481 Holtmaat, The CEDAW: A holistic approach to women’s equality and freedom, CUP 2013, 95 (96). 482 Insbesondere die islamischen Staaten wollen einer Berichtspflicht nicht zustimmen, da sie Konflikte mit der Scharia erkennen. Nordmeyer, 20 Jahre Weltfrauenkonferenz von Beijing, UN 2015, 261 (262). 118 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb A. Völkerrecht men. Die Bereitschaft souveräne Befugnisse aufzugeben und internationale Richtlinien umzusetzen, sei vor allem bei Regelungsinhalten, die Identitätsfaktoren berühren, problematisch.483 Zudem kritisiert sie, dass insbesondere die Rolle von Geschlechterstereotypen in Bezug auf Geschlechterdiskriminierung nicht hinreichend konkretisiert wäre. So werden Geschlechterstereotypen stellenweise als eine Ursache für Diskriminierung beschrieben, demgegenüber an anderen Stellen mit Geschlechterdiskriminierung gleichgesetzt.484 Im Ergebnis fasst Holtmaat zusammen, dass Art. 5 zwar ein „revolutionäres Instrument“ zur Bekämpfung der Ursachen der Diskriminierung von Frauen sei, zu seiner wirksamen Nutzung jedoch ein lokaler und transnationaler Dialog aller Akteur:innen notwendig wäre.485 Ähnlich argumentiert Rodi, dass die Vertragsstaaten nach Art. 5 CEDAW grundsätzlich dazu verpflichtet seien, Maßnahmen zur Beseitigung von stereotypen geschlechtlichen Rollenvorstellungen zu treffen. Gleichwohl könnten sie das Mittel dafür frei wählen. Des Weiteren würden sich die Forderungen des CEDAW-Ausschusses nach der aktuellen Situation auf Bildungs- und Informationskampagnen beschränken. Im Ergebnis bestehe zum einen ein zu großer Spielraum der Vertragsstaaten und es fehle zum anderen an konkreten Verpflichtungen mit einforderbaren subjektiven Rechten für Betroffene.486 Obgleich die Bedenken der Autorinnen zu würdigen sind, darf nicht unberücksichtigt bleiben, dass die CEDAW ein internationales Übereinkommen ist, das von Staaten aus der ganzen Welt von Ägypten über Nigeria bis nach China ratifiziert wurde. Zwar wären konkretere Vorgaben bezüglich bestimmter Geschlechterbilder oder Maßnahmen stellenweise wünschenswert. Gleichwohl müssen diese auch konsensfähig sein. Insbesondere streng religiöse Staaten sowie solche, in denen traditionelle Geschlechterrollen nach wie vor tief in der Kultur verankert sind, dürfen bei der Betrachtung und Maßstabsbildung nicht außen vor gelassen werden, wenn es das Ziel ist, möglichst viele Länder hinter der Konvention zu 483 Holtmaat, The CEDAW: A holistic approach to women’s equality and freedom, CUP 2013, 95 (97, 118 f.). 484 Ebenda (109); dazu auch Bloch, UN-Woman, S. 115 f. 485 Holtmaat, The CEDAW: A holistic approach to women’s equality and freedom, CUP 2013, 95 (123); ähnlich Rodi, Bekämpfung von Geschlechterstereotypen durch die Frauenrechtskonvention der Vereinten Nationen, S. 75 f. 486 Rodi, Bekämpfung von Geschlechterstereotypen durch die Frauenrechtskonvention der Vereinten Nationen, S. 67. 119 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien vereinen. Dass hierfür ein gewisses Abstraktionsniveau erreicht werden muss, ist folglich kaum zu vermeiden. 3. Zwischenfazit Die Vereinten Nationen spielen eine wichtige Rolle für die Geschlechtergleichstellung. Die CEDAW ist insbesondere aus einer historischen Perspektive ein Kernelement der weltweiten Gleichstellungspolitik. Die Definition der Diskriminierung der Frau in Art. 1 CEDAW legt einen wichtigen Baustein, ebenso wie die in diesem Zusammenhang bestehende Existenz hierarchischer Geschlechterverhältnisse oder stereotyper Rollenverteilungen, die im Rahmen von Art. 5 CEDAW geltend gemacht werden. Gleichwohl ist auch der weite Rahmen der Konvention erwähnenswert, der vorrangig einen Grundtenor ohne konkretere Aspekte setzt. In Bezug auf geschlechterdiskriminierende Werbung kann die Regelung zwar herangezogen werden. Sie entfaltet jedoch keinen prägenden Charakter, wenn es um explizite Darstellungen und Werbebilder oder Regulierungsstrategien geht. Nichtsdestoweniger werden die Grundsätze von einigen Nationen bei dem Kampf zur Gleichstellung der Geschlechter als Ankerpunkt genutzt und insbesondere die regelmäßige Berichterstattungspflicht bindet die Staaten, die das Abkommen ratifiziert haben, sich wiederholt mit den Zielen der CEDAW und den nationalen Fortschritten diesbezüglich auseinanderzusetzen. Hinzukommend erscheint es in Anbetracht der Vielzahl der unterschiedlichen geschichtlich und kulturell tief verankerten Rollenbilder, die innerhalb der Mitgliedsstaaten existieren, zweifelhaft, dass die bestehenden Differenzen durch die CEDAW oder jede andere vergleichbare internationale Konvention überbrückt werden könnten. Vielmehr ist es naheliegend, dass ein derartiges Übereinkommen notwendigerweise ausreichend Raum für die verschiedenen kulturellen Besonderheiten seiner Mitgliedsstaaten lassen muss, um übereinstimmend beschlossen werden zu können. II. Europarat Der Europarat beschreibt sich selbst als „Europas führende Organisation für Menschenrechte“.487 Der 1949 gegründeten internationalen Organisati487 https://www.coe.int/de/web/about-us/who-we-are. 120 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb A. Völkerrecht on gehören heute 47 Mitgliedstaaten an,488 darunter alle 27 Mitgliedstaaten der Europäischen Union. Nach Art. 1 a des Gründungsstatuts dient der Europarat dem Ziel, eine engere Verbindung zwischen seinen Mitgliedern herzustellen, um die Ideale und Grundsätze, die ihr gemeinsames Erbe sind, insbesondere den Schutz der Menschenrechte, der Demokratie und der Rechtsstaatlichkeit, zu wahren und zu verwirklichen und ihren wirtschaftlichen und sozialen Fortschritt zu fördern.489 Gleichermaßen gehört die Gleichstellung der Geschlechter heute zu den Zielen des Europarats.490 1. Europäische Menschenrechtskonvention Bei der Konvention zum Schutze der Menschenrechte und Grundfreiheiten (EMRK), die 1950 vom Europarat verabschiedet wurde, handelt es sich um einen völkerrechtlichen Vertrag zum Schutz der Menschenrechte und Grundfreiheiten zwischen den Mitgliedstaaten des Europarates. Die innerstaatliche Geltung der EMRK richtet sich nach nationalem Recht. In Deutschland hat sie als völkerrechtlicher Vertrag nach Art. 59 Abs. 2 S. 1 GG den Rang eines einfachen Bundesgesetzes.491 Ein Spannungsfeld ergibt sich für den Untersuchungsgegenstand dieser Abhandlung dadurch, dass die EMRK sowohl den Schutz von Wirtschaftswerbung als auch den Schutz vor Diskriminierung gewährleistet. Mithin können sich nicht nur diejenigen, die in konkreten Werbebildern eine Geschlechterdiskriminierung erkennen, sondern auch die Werbetreibenden auf die EMRK berufen. Wirtschaftswerbung fällt in den Schutzbereich der Meinungsäußerungsfreiheit nach Art. 10 Abs. 1 S. 1 EMRK, der als einheitliches Kommunikationsgrundrecht ausgelegt wird.492 Gemäß dem prägenden Fall Casado Coca vs. Spain kommt es für die Eröffnung des Schutzbereiches des Art. 10 nicht darauf an, ob eine Äußerung mit Gewinnerzielungsabsicht erfolgt oder nicht. Vielmehr könne nach Ansicht des Europäischen Ge- 488 https://www.coe.int/de/web/about-us/founding-fathers. 489 Statute of the Council of Europe, European Treaty Series – No. 1, London, 5. V. 1949. 490 https://www.coe.int/en/web/genderequality. 491 Karpenstein/Mayer/Mayer, EMRK Einl. Rn. 78; Jarass/Pieroth/Jarass, Art. 59 Rn. 9. 492 Vgl. Doukas, Werbefreiheit und Werbebeschränkungen, S. 168; dazu auch Arnold, Wirtschaftswerbung und die Meinungsfreiheit des Grundgesetzes, S. 114, 116. 121 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien richtshofs für Menschenrechte (EGMR) eine Unterscheidung in diesem Sinne als Diskriminierung nach Art. 14 EMRK ausgelegt werden.493 Nach Art. 10 Abs. 2 EMRK kann die Meinungsäußerungsfreiheit jedoch eingeschränkt werden, wobei rein kommerzielle Äußerungen stärkeren Einschränkungen zugänglich sind als politische Meinungsäußerungen.494 Gleichermaßen ist das generelle Diskriminierungsverbot nach Art. 14 EMRK in Bezug auf Geschlechtergleichstellung zu berücksichtigen. Danach sind alle durch die Konvention anerkannten Rechte und Freiheiten „ohne Diskriminierung insbesondere wegen des Geschlechts“ zu gewährleisten. Aufgrund seines akzessorischen Charakters kann eine Verletzung des Diskriminierungsverbots nur in Verbindung mit einem Freiheitsrecht gerügt werden.495 Sein Anwendungsbereich und seine Reichweite werden daher als beschränkt beschrieben.496 Art. 1 des zwölften Zusatzprotokolls497 statuiert demgegenüber ein nichtakzessorisches Diskriminierungsverbot, welches bislang allerdings nur von wenigen Staaten ratifiziert wurde. Deutschland beispielsweise hat das Protokoll zwar unterschrieben, es bis heute jedoch nicht ratifiziert.498 2. Empfehlungen und Entschließungen zu Geschlechterdarstellungen in der Werbung Neben den Vorschriften der EMRK lassen sich auch einzelne Ausführungen des Europarats zu der Thematik geschlechterdiskriminierender Werbung finden. Sie umfassen Empfehlungen (recommendations) und Ent- 493 EGMR v. 24.02.1994, Nr. 15450/89 – Casado Coca v. Spain, S. 13; dazu auch Arnold, Wirtschaftswerbung und die Meinungsfreiheit des Grundgesetzes, S. 117. 494 Schricker/Henning-Bodewig, Elemente einer Harmonisierung des Rechts des unlauteren Wettbewerbs in der Europäischen Union, WRP 2001, 1367 (1374). 495 Karpenstein/Mayer/Sauer, EMRK Art. 14 Rn. 14. 496 Lembke, Menschenrechtliche Diskriminierungsverbote, S. 140. 497 Protokoll Nr. 12 zur Konvention zum Schutz der Menschenrechte und Grundfreiheiten, Council of Europe, Sammlung Europäischer Verträge – Nr. 177, Rom, 4.XI.2000. 498 Unterschriften und Ratifikationsstand des Vertrags 177, Protokoll Nr. 12 zur Konvention zum Schutze der Menschenrechte und Grundfreiheiten, Council of Europe, 10.06.2021. 122 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb A. Völkerrecht schließungen (resolutions) der Parlamentarischen Versammlung499 des Europarats und des Ministerkomitees500. Während Entschließungen der Parlamentarischen Versammlung in der Regel einen Beschluss zu einer Sachfrage enthalten, zu deren Erörterung sie ermächtigt wurde, umfassen Empfehlungen regelmäßig einen Vorschlag der Versammlung an das Ministerkomitee. Weder Empfehlungen noch Entschließungen sind rechtlich verbindlich. Aufgrund der Zusammensetzung der Parlamentarischen Versammlung, deren Mitglieder in der Regel zugleich eine politische Kontrollfunktion in den Mitgliedsstaaten einnehmen, kommt den Beschlüssen jedoch eine politische Bedeutung zu.501 In Bezug auf die deutsche Gesetzgebung kann bei diesen Beschlüssen von einer „gewissen Initiativwirkung“ oder zumindest einem mittelbaren Einfluss gesprochen werden.502 a. Ministerkomitee In Bezug auf den Untersuchungsgegenstand existieren drei Empfehlungen des Ministerkomitees. In der Empfehlung von 1984 betonte das Entscheidungsorgan die wichtige soziale Rolle der Darstellung von Frauen und Männern in den Medien, insbesondere in der (Fernseh-)Werbung.503 Darin werden grundlegende Prinzipien für Fernsehwerbung statuiert. So heißt es etwa, dass Fernsehwerbung mit einem Verantwortungsbewusst499 Die Parlamentarische Versammlung ist nach Art. 10 der Satzung des Europarates in Verbindung mit Art. 22 S. 1 der Satzung das beratende Organ des Europarates; siehe Europarat, Satzung vom 05.05.1949, https://rm.coe.int/1680306051; vgl. Deutscher Bundestag, Sachstandsbericht 6 2016, WD 2-3000-087/16, Die Bedeutung von Entschließungen der Parlamentarischen Versammlung des Europarats und Konventionen des Europarates für die Gesetzgebung in Deutschland, S. 10. 500 Das Ministerkomitee ist das Entscheidungsorgan des Europarates, das die Politik des Europarates und das Tätigkeitsprogramm der Organisation bestimmt, siehe https://www.coe.int/de/web/about-us/structure. 501 Dazu siehe auch Sachstandsbericht, Deutscher Bundestag 6 2016, WD 2- 3000 – 087/16, Die Bedeutung von Entschließungen der Parlamentarischen Versammlung des Europarats und Konventionen des Europarates für die Gesetzgebung in Deutschland, S. 10. 502 Ebenda, S. 12. 503 Council of Europe, Committee of Ministers, Recommendation No. R (84) 3 of the Committee of Ministers to Member States on principles of television advertising, adopted by the Committee of Ministers on 23 February 1984 at the 367th meeting of the Ministers' Deputies. 123 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien sein gegenüber der Gesellschaft erstellt werden und dabei die moralischen Werte berücksichtigen werden sollen.504 Eine weitere Auseinandersetzung erfolgte 1995 mit der „Entschließung zur Darstellung der Frau und des Mannes in Werbung und Medien“.505 In dieser wird unter anderem auf das Potenzial von Werbung und Medien hingewiesen, eine Änderung von sozialen Verhaltensweisen herbeizuführen, indem sie vielfältige Frauen- und Männerrollen abbildet. Explizit heißt es beispielsweise, „dass geschlechtsspezifische Rollenklischees in Werbung und Medien Teil der Ungleichheiten sind, die die Haltungen gegenüber der Gleichstellung von Männern und Frauen beeinflussen, und dass es deshalb wichtig ist, die Gleichstellung von Männern und Frauen in allen Bereichen des gesellschaftlichen Lebens zu fördern“.506 Ergänzend werden die Mitgliedsstaaten aufgefordert, die Werbeagenturen zu ermutigen, neue Konzepte für die Darstellung der Vielfalt der Rollen von Frauen und Männern anzustrengen.507 In einer Empfehlung aus dem Jahr 2013 adressierte das Ministerkomitee des Europarats die Problematik erneut und forderte die Mitgliedsstaaten auf, die Regulierungs- und Selbstregulierungsstrategien weiter auszuarbeiten. Medienunternehmen sollen ein nicht stereotypes Bild von Frauen und Männern porträtieren, sexistische Werbung vermeiden und Inhalte unterlassen, die zu Diskriminierung aufgrund des Geschlechts, Aufstachelung zu Hass und geschlechtsspezifischer Gewalt führen könnten.508 b. Parlamentarische Versammlung Weitere Entschließungen stammen von der Parlamentarischen Versammlung des Europarates. Die darin gemachten Ausführungen beziehen sich ausschließlich auf das Bild der Frau. Im Jahr 2002 wurde beispielsweise das 504 Ebenda, I. General principle No. 1. 505 Entschließung des Rates und der im Rat vereinigten Vertreter der Regierungen der Mitgliedstaaten vom 5. Oktober 1995 zur Darstellung der Frau und des Mannes in Werbung und Medien, ABl. Nr. C 296/15, 10.11.1995. 506 Ebenda, I. Nr. 2. 507 Ebenda, II. Nr. 2.7. a). 508 Recommendation CM/Rec (2013) 1 of the Committee of Ministers to member States on gender equality and media, 1176th meeting of the Ministers’ Deputies, Guidelines B. 4. 124 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb A. Völkerrecht „Bild der Frau in den Medien“ in einer Empfehlung adressiert.509 In der Empfehlung wurde festgestellt, dass sich im Allgemeinen zwar Fortschritte zeigen, das Bild der Frau in den Medien jedoch in vielen europäischen Staaten häufig noch stereotyp und sexistisch sei. So würden Frauen häufig als Sexobjekte dargestellt oder nur mit dem Haushalt oder Familienleben in Verbindung gebracht.510 Ergänzend müssten die Staaten den Begriff „Sexismus“, der als Negierung der gleichen Würde von Menschen aufgrund ihres Geschlechts zu definieren sei, in ihren Gesetzen verankern, um das Phänomen effektiv bekämpfen zu können. Es sei sinnvoll, die Selbstregulierung im Bereich der Werbung durch ein System der Berufsethik zu stärken.511 Eine Entschließung von 2007 fokussiert sich schließlich explizit auf das „Bild der Frau in der Werbung“.512 Das Werbebild der Frau stehe nach wie vor im Widerspruch zu ihrer tatsächlichen Rolle in der heutigen Gesellschaft. Sie würde nicht nur in Situationen abgebildet, die erniedrigend, entwürdigend oder sogar gewalttätig sind und die die Menschenwürde verletzen, sondern auch auf die Rolle von bloßen Konsumgütern, Sexobjekten oder veralteten Stereotypen reduziert.513 Als Gegenmaßnahmen wird einerseits auf das Bedürfnis von weitergehenden Bildungs- und Sensibilisierungsmaßnahmen hingewiesen, andererseits die Notwendigkeit der Ausweitung der Befugnisse von Frauenrechtsorganisationen sowie der Stärkung der Werbeselbstregulierung betont.514 Die Forderungen wurden 2010 erneut aufgegriffen und mit der Entschließung „Kampf gegen sexistische Stereotypen in den Medien“ als notwendige Maßnahme bei der Bekämpfung des nach wie vor bestehenden Problems der Darstellung der Geschlechter in den Medien angeführt.515 509 Parliamentary Assembly, Image of women in the media, Assembly debate on 24 April 2002 (13th Sitting), Recommendation 1555 (2002). 510 Ebenda, Rn. 2. 511 Ebenda, Rn. 10. 512 Parliamentary Assembly, The image of women in advertising, Resolution 1557 (2007), Assembly debate on 26 June 2007 (21st Sitting) (see Doc. 11286). 513 Ebenda, Rn. 1 ff. 514 Ebenda, Rn. 12. 515 Parliamentary Assembly, Combating sexist stereotypes in the media, Resolution 1751 (2010). 125 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien 3. Zwischenfazit Die Würdigungen durch die Organe des Europarates sind gegenüber den Maßnahmen der Vereinten Nationen bereits als gestaltender zu bewerten. So wurde nicht nur die Gefahr um das Bestehen der stereotypen, teils sexistischen Darstellung von Frauen und Männern in den Medien, insbesondere auch in der Werbung, hervorgehoben, sondern konkrete Maßnahmen zur Bekämpfung dieser Diskriminierungen vorgeschlagen. Neben dem Ausbau und der Stärkung der Werbeselbstregulierung wurde vor allem auf die Notwendigkeit von Sensibilisierungs- und Lehrmaßnahmen hingewiesen. Die im Vergleich zu der CEDAW erfolgten konkreteren Ausführungen könnten als weiteres Indiz dafür gewertet werden, dass ein kleineres Staatenbündnis das Spektrum der möglichen Handlungsformen erweitert. Aufgrund der rechtlichen Unverbindlichkeit der Maßnahmen wird es im Folgenden jedoch zu hinterfragen sein, inwieweit die Staaten den Empfehlungen nachkommen oder sich bei der Regulierung geschlechterdiskriminierender Werbeinhalte auf sie beziehen. B. Europäische Union Für die Mitgliedstaaten der Europäischen Union bilden sowohl die Entschließungen und Richtlinien des europäischen Sekundärrechts als auch die Grundwerte der Staatengemeinschaft, die im europäischen Primärrecht zusammengefasst sind, die wichtigsten Einflussquellen in Bezug auf die Werberegulierungssysteme. Insbesondere die wettbewerbsrechtlichen Vorgaben und die Strukturen und Ziele der Gleichstellungspolitik der Europäischen Union sind diesbezüglich von Relevanz. Dabei taucht die Problematik geschlechterdiskriminierender Werbung im Zusammenhang mit „der Verpflichtung zu einer aktiven Gleichstellungspolitik“ der Mitgliedstaaten der Europäischen Union kontinuierlich auf.516 Die Staaten verpflichten sich, die Gleichstellung der Geschlechter zu fördern und Diskriminierung aufgrund des Geschlechts zu unterbinden.517 Hierfür formulieren das Europäische Parlament, der Rat der Europäischen Union sowie die Europäische Kommission Zwischenziele und konkrete Maßnahmen, 516 Dazu vgl. Dackweiler, Europäische Gleichstellungspolitik, KJ 2017, 297 (297). 517 So auch Konsunen/Asikainen/Gústafsdóttir u.a., Regulation of Gender: Discriminatory advertising in the Nordic countries, S. 16. 126 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb B. Europäische Union die es in Verbindung mit kompetenzrechtlichen Fragestellungen zu untersuchen gilt. I. Grundwerte und Ziele im europäischen Primärrecht 1. Geschlechtergleichheit und Diskriminierungsschutz Wie im Folgenden gezeigt wird, sieht das ideologische Fundament der Europäischen Union, welches sich insbesondere im Vertrag über die Europäische Union (EUV), im Vertrag über die Arbeitsweise der Europäischen Union (AEUV) und in der Europäischen Grundrechte-Charta (GRCh) niederschlägt, die Gleichstellung von Frauen und Männer vor und hat sich zum Ziel gesetzt, jeder Art von Geschlechterdiskriminierung entgegenzutreten. Das Ziel der Gleichstellung von Frauen und Männern war bereits im Vertrag zur Gründung der Europäischen Gemeinschaft (EG-Vertrag) verankert (Art. 2, Art. 3 Abs. 2 EG-Vertrag). Die Europäische Kommission definiert Geschlechtergleichheit als „Situation, in der alle Menschen ihre persönlichen Fähigkeiten frei entwickeln und freie Entscheidungen treffen können, ohne durch strikte geschlechtsspezifische Rollen eingeschränkt zu werden, und in der die unterschiedlichen Verhaltensweisen, die unterschiedlichen Ziele und die unterschiedlichen Bedürfnisse von Frauen und Männern in gleicher Weise berücksichtigt, anerkannt und gefördert werden“.518 Dabei sei die formale oder juristische Gleichheit jedoch nur der erste Schritt zur materiellen (de facto) Gleichheit der Geschlechter. Bestimmungen zur Gleichheit von Männern und Frauen finden sich mehrfach im europäischen Primärrecht.519 Das Gebot der Nichtdiskriminierung aus Art. 21 Abs. 1 GRCh statuiert, dass „Diskriminierungen insbesondere wegen des Geschlechts [...] verboten“ sind. Zudem ist nach Art. 23 GRCh die „Gleichheit von Männern und Frauen [...] in allen Bereichen, einschließlich der Beschäftigung, der Arbeit und des Arbeitsentgelts, sicherzustellen.“ Art. 2 S. 2 EUV legt fest, dass die Werte, auf denen der Zusammenschluss der Union basiert, „die Achtung der Menschenwürde, Freiheit, Demokratie, Gleichheit, Rechtsstaatlichkeit und die Wahrung der 518 Europäische Kommission, 100 Begriffe aus der Gleichstellungspolitik, S. 33. 519 Dazu auch Heselhaus/Nowak/von der Decken, Handbuch der Europäischen Grundrechte, S. 1050 Rn. 1. 127 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien Menschenrechte einschließlich der Rechte der Personen, die Minderheiten angehören“ sind. Weiterhin sind die „Werte [...] allen Mitgliedstaaten in einer Gesellschaft gemeinsam, die sich durch Pluralismus, Nichtdiskriminierung, Toleranz, Gerechtigkeit, Solidarität und die Gleichheit von Frauen und Männern auszeichnet“. Nach Art. 3 Abs. 3 EUV ist es das Ziel der Union, „soziale Ausgrenzung und Diskriminierungen“ zu bekämpfen und die „Gleichstellung von Frauen und Männern“ zu fördern. Weitere Bestimmungen zur Geschlechterdiskriminierung finden sich in Art. 10 und Art. 19 Abs. 1 AEUV. Demnach sind Diskriminierungen aus Gründen des Geschlechts zu bekämpfen, wobei nicht nur Rahmenbedingungen zu schaffen sind, sondern auch aktiv gegen Diskriminierungen vorgegangen werden muss. Daneben wirkt die Europäische Union gemäß Art. 8 AEUV bei all ihren Tätigkeiten darauf hin, Ungleichheiten zu beseitigen und die Gleichstellung von Männern und Frauen zu fördern. Die Gleichstellung der Geschlechter und der Grundsatz der Nichtdiskriminierung haben mithin einen hohen, wenn nicht sogar elementaren, Stellenwert in dem im europäischen Primärrecht verankerten Wertegefüge der Europäischen Union. 2. Werbung im europäischen Kommunikations- und Wirtschaftsgefüge Neben dem Ziel der Geschlechtergleichheit und des Diskriminierungsschutzes sind Werbeinhalte im Rechtsgefüge der Europäischen Union ebenfalls geschützt. Der Werbung kommt im Binnenmarkt eine wichtige Bedeutung zu und Werbeverbote sind von jeher umstritten.520 Wirtschaftswerbung wird durch europäisches Recht, insbesondere die EU-Grundrechtecharta, geschützt. Dabei sind nicht nur die Berufsfreiheit (Art. 15 GRCh) und die unternehmerische Freiheit (Art. 16 GRCh) relevant, sondern auch die Freiheit der Meinungsäußerung und die Informationsfreiheit (Art. 11 GRCh), in deren Schutzbereich auf europäischer Ebene jede Art der Werbung, also auch Wirtschaftswerbung, fällt.521 Der Europäische Gerichtshof (EuGH) trennt bei der Bewertung zwischen Werbeinhalten, die allein wirtschaftliche Informationen enthalten und daher einer höheren Kontrolldichte unterliegen, und solchen, die politische Inhalte implizieren 520 Heselhaus/Nowak/Kühling, Handbuch der Europäischen Grundrechte, S. 596, Rn. 25. 521 Verfassungsmäßigkeit eines umfassenden Verbots der Werbung für Tabakerzeugnisse, Deutscher Bundestag, WD 10 – 3000 – 023/16, 12.04.2016, S. 6. 128 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb B. Europäische Union und für die der Einschätzungs- und Bewertungsspielraum daher begrenzt ist.522 In den Bestimmungen der nationalen Verfassungen spiegelt sich der grundsätzliche Schutz der Werbung demgegenüber nur teilweise wider.523 Während beispielsweise Schweden in der Verfassung ausdrücklich auf die Garantie der Kommunikationsfreiheit Bezug nimmt und auch kommerzielle Kommunikation in großen Teilen schützt,524 ist die reine Wirtschaftswerbung in Frankreich nach überwiegender Auffassung nicht von dem verfassungsrechtlichen Schutz der Meinungsfreiheit umfasst.525 Auch in Italien haben sich Jurist:innen lange Zeit die Frage gestellt, ob kommerzielle Werbung von der Verfassung geschützt wird, wobei aufgrund der wirtschaftlichen Ausrichtung zu strengeren Grenzen als bei anderen Formen der Kommunikation tendiert wird.526 In Dänemark bietet die Meinungsäußerungsfreiheit der Werbung einen beträchtlichen Spielraum, der sich jedoch an Erwägungen des öffentlichen Interesses, wie etwa der Bekämpfung der Geschlechterdiskriminierung nach dem Gleichstellungsgesetz, messen lassen muss.527 In Deutschland ist Wirtschaftswerbung in der Regel nur teilweise von der Meinungsfreiheit geschützt.528 In den Verfassungen anderer Staaten ist der Umfang des Schutzes umstritten oder noch ungeklärt.529 Nach der Ansicht von Kühling wird der Schutz der Kommunikationsfreiheit im Bereich der Wirtschaftswerbung auf europäischer Ebene weiter gezogen als in den meisten Rechtsordnungen der EU-Mitgliedsstaaten, da das Unionsrecht in hohem Maße wirtschaftliche Bereiche betreffe.530 Kühling argumentiert, dass jede Art von Werbeverbot einer „staatlich möglichst unbeschränkten Meinungsbildung der Verbraucher im Hinblick auf 522 Streinz, Europäisches und deutsches Verfassungsrecht: Erfassung von geschäftlicher Werbung, S. 99. 523 Gleichwohl ist hervorzuheben, dass die Meinungsfreiheit in den Verfassungen aller Mitgliedsstaaten der EU eine zentrale, „überragende Stellung im Grundrechtsgefüge einnimmt“ (Dreier/Schulze-Fielitz, GG Art. 5 Abs. 1-2 Rn. 34). 524 Dazu vgl. Svensson/Edstörm, Freedom of Expression vs. Gender Equality, TFR 2014, 479 (494). 525 Dazu Doukas, Werbefreiheit und Werbebeschränkungen, S. 225 ff. 526 Vgl. D'Ippolito, Controllare la pubblicità, S. 16. 527 Konsunen/Asikainen/Gústafsdóttir u.a., Regulation of Gender: Discriminatory advertising in the Nordic countries, S. 33. 528 Ausführlich siehe Kapitel 3, B. I. 2. a. 529 Dazu Doukas, Werbefreiheit und Werbebeschränkungen, S. 225 ff. 530 Heselhaus/Nowak/Kühling, Handbuch der Europäischen Grundrechte, S. 596, Rn. 26 f. 129 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien Entscheidungen auf den Produktmärkten“ entgegenstehen würde.531 Damit sei eine Rückkopplung an das Demokratieprinzip gegeben, die nicht allein aus einer Reflexwirkung der „Wirtschaftswerbung als Finanzierungsgrundlage für offene Kommunikation in den für die Demokratie wesentlichen Medien hervorgeht“.532 Im Ergebnis schützt die unionsrechtliche Kommunikationsfreiheit auch Wirtschafswerbung.533 Daneben ist zu berücksichtigen, dass nationale Vorschriften, die Werbemaßnahmen inhaltlich begrenzen, ein praktisches Hindernis für den Binnenmarkt sein können.534 Regelungen, die Werbeinhalte beschränken, müssen sich deswegen an verschiedenen Bestimmungen des Unionsrechts messen lassen. Insbesondere sind hierbei die Vorgaben zum freien Warenverkehr aus Art. 34 und Art. 36 AEUV relevant. Dies gilt gleichermaßen für europäische Maßnahmen, wenn sie zu Beschränkungen des Handels zwischen den Mitgliedstaaten führen können.535 3. Kollision der Rechtsgüter Damit kollidieren die Ziele der Geschlechtergleichstellung und des Diskriminierungsschutzes auf dem Gebiet der Wirtschaftswerbung mit den Grundwerten der Kommunikations-, Werbe- und unternehmerischen Freiheit.536 Zwar dürften Bilder, die den Kern der Menschenwürde berühren, regelmäßig Vorrang vor der Freiheit der Meinungsäußerung oder der Freiheit der unternehmerischen Betätigung haben. Wenn nur ein Anstandsoder Sittlichkeitsverstoß vorliegt, fällt die Bewertung hingegen in der Regel anders aus.537 Beiden Rechtspositionen kommt ein außerordentlich hoher Rang im unionsrechtlichen Rechtsgefüge zu.538 Vorliegend kann 531 Ebenda. 532 Ebenda. 533 Ebenda, S. 596, Rn. 28 m. w. N. 534 Dazu Doukas, Werbefreiheit und Werbebeschränkungen, S. 42. 535 Streinz, Europäisches und deutsches Verfassungsrecht: Erfassung von geschäftlicher Werbung, S. 124; ergänzend zur Beeinträchtigung der Warenverkehrsfreiheit siehe EuGH v. 24.11.1993, C-267/91, C-268/91, NJW 1994, 121 (Keck und Mithouard); EuGH v. 20.02.1979, 120/78, NJW 1979, 1766 (Cassis de Dijon); EuGH, 11.07.1974, 8/74, NJW 1975, 515 (Dassonville). 536 Dazu auch Dackweiler, Europäische Gleichstellungspolitik, KJ 2017, 297 (301). 537 Zur verhältnismäßigen Beeinträchtigung von Wirtschaftswerbung siehe Heselhaus/Nowak/Kühling, Handbuch der Europäischen Grundrechte, S. 596, Rn. 84. 538 Heselhaus/Nowak/Kühling, Handbuch der Europäischen Grundrechte, S. 596, Rn. 91. 130 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb B. Europäische Union damit keine abschließende Antwort auf einen grundsätzlichen Vorrang eines der betroffenen europäischen Rechtsgüter gegeben werden. Festzuhalten bleibt allein, dass sowohl der Diskriminierungsschutz und das Ziel der Geschlechtergleichheit als auch die Freiheit der Wirtschaftswerbung in der europäischen Werteordnung eine bedeutende Rolle einnehmen. Entscheidungen des EuGHs zu verschiedenen Werbebestimmungen, insbesondere zu diversen Tabakwerberichtlinien, suggerieren jedoch, dass begrenzte Werbeverbote grundsätzlich möglich sind, soweit eine hinreichende Kompetenzgrundlage besteht und die Vorschrift aus Gründen des Allgemeinwohls angemessen erscheint.539 Auch im Fall geschlechterdiskriminierender Werbung wäre ein begrenztes Verbot konkreter Werbebilder mithin nicht ausgeschlossen. II. Europäisches Sekundärrecht Die Problematik geschlechterdiskriminierender Werbeinhalte wurde von den Organen der Europäischen Union in mehreren Berichten nicht nur anerkannt, sondern auch ihre Ambitionen, dieser entgegenzuwirken, aufgezeigt.540 Die Möglichkeit rechtsverbindliche Richtlinien und Verordnungen zu erlassen,541 stellt einen Vorteil zur fehlenden innerstaatlichen Umsetzungspflicht, die in der Regel bei internationalen Übereinkünften besteht, dar.542 Geschlechterdiskriminierende Werbung und die damit in Zusammenhang stehenden Konflikte und Herausforderungen wurden seit den Neunzigerjahren vermehrt in verschiedenen Rechtsakten der EU-Organe adressiert.543 Bei den Rechtsakten kann nach Art. 288 AEUV zwischen verbindlichen, also Verordnungen, Richtlinien und Beschlüssen, und nicht ver539 Siehe bspw. EuGH, Rs. C-376/98, Urteil v. 05.10.2000, Bundesrepublik Deutschland gegen Europäisches Parlament und Rat der Europäischen Union, Rn. 98, 104, 111. 540 Holtz-Bacha, Falsche (Vor)Bilder? Frauen und Männer in der Werbung, S. 11; zur Rolle der Europäischen Union bei den Vereinten Nationen, vgl. https://ww w.consilium.europa.eu/de/policies/unga/. 541 Holtz-Bacha, Falsche (Vor)Bilder? Frauen und Männer in der Werbung, S. 11; dazu auch Beckmann, Werbeselbstdisziplin in Deutschland und Europa, WRP 1991, 702 (703). 542 So auch Dackweiler, Europäische Gleichstellungspolitik, KJ 2017, 297 (297); zu den EU-Rechtsakten siehe bspw. https://europa.eu/european-union/law/legal-act s_de. 543 Dackweiler, Europäische Gleichstellungspolitik, KJ 2017, 297 (301). 131 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien bindlichen, also Empfehlungen und Stellungnahmen,544 unterschieden werden. Nach Klein sei seit Ende der Neunzigerjahre eine zunehmende Bedeutung unverbindlicher Rechtsakte auszumachen. Deren Erfolg hänge jedoch von „der Umsetzungsbereitschaft der Mitgliedsstaaten ab, die nicht in jedem Fall besonders hoch“ sei.545 Gleichwohl sei die Geschlechtergleichstellung eine Materie, die nicht durch Rechtsakte alleine herzustellen sei, sondern es bedürfe auch einer Bewusstseinsbildung, „die durch Maßnahmen und Aktionsprogramme gefördert“ werden könne.546 Damit sind sowohl die verbindlichen Vorschriften als auch die nicht verbindlichen Rechtsakte sowie die verschiedenen Kampagnen, Maßnahmen und Programme der Europäischen Union entscheidend. Dementsprechend wird im Folgenden zwischen diesen beiden Handlungsalternativen getrennt sowie im zweiten Abschnitt zwischen den Handlungsorganen, also dem Europäischen Parlament, dem Rat der Europäischen Union und der Europäischen Kommission. Entscheidungen der Europäischen Gerichte, die sich explizit mit geschlechterdiskriminierender Werbung auseinandersetzen, gibt es keine. 1. Verbindliche Rechtsakte Bevor auf konkrete Vorgaben, insbesondere relevante Richtlinien der Europäischen Union in Bezug auf geschlechterdiskriminierende Werbung eingegangen wird, sind die Kompetenzverteilung und die Frage zu erörtern, inwiefern europäische Lösungsansätze im Kontext geschlechterdiskriminierender Werbung zulässig sind. Eine vollumfassende Auseinandersetzung mit dem Kompetenzrecht der Organe der Europäischen Union in Bezug auf die Thematik, insbesondere zur Möglichkeit einer rechtsverbindlichen Bestimmung gegen geschlechterdiskriminierende Werbung auf europäischer Ebene würde jedoch den Rahmen der vorliegenden Arbeit sprengen, so dass Fragen zur Regelungskompetenz nur in den Grundzügen behandelt werden können. 544 Siehe https://europa.eu/european-union/law/legal-acts_de. 545 Klein, Geschlechterverhältnisse, Geschlechterpolitik und Gleichstellungspolitik in der Europäischen Union, S. 24. 546 Ebenda, S. 27, 63 f. 132 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb B. Europäische Union a. Regelungskompetenz Für die Abgrenzung der Zuständigkeiten der Union gilt nach Art. 5 EUV der Grundsatz der begrenzten Einzelermächtigung. Danach können Organe der Europäischen Union in rechtlich zulässiger Weise in der Regel nur normgebend tätig werden, wenn in konkreten Vertragsbestimmungen eine entsprechende Ermächtigung in dem zu regelnden Bereich vorgesehen ist.547 Das Werberecht ist kein eigener Regelungsbereich, sondern wird je nach Inhalt von unterschiedlichen Regelungsgegenständen und Sektoren erfasst.548 Insbesondere spielen das Wirtschaftsrecht und das Medienrecht, welches selbst wiederum einen Doppelcharakter als Kultur- und Wirtschaftsgut aufweist,549 eine wichtige Rolle und werden daher nachfolgend in Bezug auf eine Richtlinie zu geschlechterdiskriminierender Werbung näher beleuchtet. aa) Wirtschaftsrecht Die Regelungskompetenz für das Wirtschaftsrecht liegt bei den Mitgliedsstaaten.550 Jedoch können die EU-Beschlussorgane, also das Europäische Parlament551 und der Rat der Europäischen Union552, nach Art. 114 AEUV nach Anhörung des Wirtschafts- und Sozialausschusses Maßnahmen zur 547 Calliess/Ruffert/Calliess, EUV/AEUV, EU-Vertrag Art. 5 Rn. 6 f. 548 Teilweise wird auch von einem „europäischen Umstieg“ der Regulierung von einer „vertikalen Unterteilung der rechtlichen Rahmenbedingungen“, also durch die Trennung zwischen unterschiedlichen Sektoren (bspw. Rundfunkregulierung gegenüber Regulierung der Telekommunikation) hin zu einem „horizontalen Ansatz“, der im Rahmen der Regulierung nach Inhalt und der Übertragung trennt, gesprochen. Roßnagel/Kleist/Scheuer, Die Reform der Regulierung elektronischer Medien in Europa, S. 183. 549 Burggraf/Gerlach/Wiesner, Europäische Medienregulierung im Spannungsfeld zwischen EU- und mitgliedstaatlicher Kompetenz, MP 2018, 496 (496). 550 Vgl. Seufert/Gundlach, Medienregulierung in Deutschland, S. 301. 551 Das Europäische Parlament wird seit 1979 von den Bürger:innen der Mitgliedsstaaten gewählt. Als einziges direkt gewähltes Organ der EU vertritt es damit die Interessen des „Europäischen Volkes“ in besonderem Maße und nimmt eine wichtige Rolle in der EU-Gesetzgebung ein. Siehe dazu etwa Klein, Geschlechterverhältnisse, Geschlechterpolitik und Gleichstellungspolitik in der Europäischen Union, S. 38. 552 Neben dem Europäischen Parlament gehört die Gesetzgebung auch zu den Hauptaufgaben des Rates der Europäischen Union. Er vertritt die nationalen 133 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien Rechtsangleichung erlassen, die die Errichtung und das Funktionieren des Binnenmarkts zum Gegenstand haben. Art. 115 AEUV sieht ferner vor, dass der Rat gemäß einem besonderen Gesetzgebungsverfahren einstimmig und nach Anhörung des Europäischen Parlaments und des Wirtschafts- und Sozialausschusses unbeschadet des Art. 114 AEUV Richtlinien für die Angleichung derjenigen Rechts- und Verwaltungsvorschriften der Mitgliedstaaten erlässt, die sich unmittelbar auf die Errichtung oder das Funktionieren des Binnenmarkts auswirken. Daraus folgend könnten die EU-Beschlussorgane zur Schaffung eines einheitlichen EU-Binnenmarktes harmonisierende Richtlinien zur Angleichung der nationalen Rechtsvorschriften im Wirtschaftsrecht erlassen.553 Seufert und Gundlach stellen beispielsweise fest, dass es sich bei den meisten der Änderungen im deutschen Wirtschaftsrecht, die in den letzten Jahren vorgenommenen wurden, um werberelevante Bestimmungen handelt, die auf EU-Richtlinien zurückzuführen sind.554 Möglich wäre eine Richtlinie zu geschlechterdiskriminierender Wirtschaftswerbung dann, wenn sich aufgrund nationaler Bestimmungen Wettbewerbshindernisse für den Binnenmarkt ergeben würden. bb) Medienrecht und Kulturangelegenheiten Das Medienrecht, worunter auch das Presserecht, das Rundfunkrecht und das Telemedienrecht fällt, enthält ebenfalls Regeln für die Werbung.555 Wie das Wirtschaftsrecht fällt die Regulierungskompetenz jedoch in die Zuständigkeit der Mitgliedstaaten,556 so dass ein Vorgehen der EU-Be- Interessen der Mitgliedsstaaten. Siehe etwa Klein, Geschlechterverhältnisse, Geschlechterpolitik und Gleichstellungspolitik in der Europäischen Union, S. 37. 553 Vgl. Seufert/Gundlach, Medienregulierung in Deutschland, S. 301. 554 So bspw. Richtlinie des Europäischen Parlaments über unlautere Geschäftspraktiken von Unternehmen gegenüber Verbrauchern im Binnenmarkt, 05/2005 (Richtlinie 2005/29/EG); die Richtlinie des Europäischen Parlaments und des Rates über irreführende und vergleichende Werbung, 12/2006 (Richtlinie 2006/14/EG); die Richtlinie des Europäischen Parlaments und des Rates über die Verarbeitung personenbezogener Daten und den Schutz der Privatsphäre in der elektronischen Kommunikation, 12/2009 (Richtlinien 2002/S8/EG bzw. Richtlinie 2009/136/EG). Seufert/Gundlach, Medienregulierung in Deutschland, S. 301. 555 Vgl. Seufert/Gundlach, Medienregulierung in Deutschland, S. 302. 556 Burggraf/Gerlach/Wiesner, Europäische Medienregulierung im Spannungsfeld zwischen EU- und mitgliedstaatlicher Kompetenz, MP 2018, 496 (496). 134 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb B. Europäische Union schlussorgane wiederum nur zur Angleichung der Vorschriften im Binnenmarkt nach Art. 114 f. AEUV möglich wäre. Gleichwohl illustriert die Praxis auch hier, dass durchaus Werbebestimmungen des Medienrechts, insbesondere des Rundfunkrechts, auf EU-Richtlinien gründen. Beispielhaft ist die EU-Fernsehrichtlinie bzw. die Richtlinie des Europäischen Parlaments und des Rates zu audiovisuellen Mediendiensten (AVMD-Richtlinie)557 zu nennen.558 Diese sieht etwa vor, dass die Mitgliedstaaten nicht die Weiterverbreitung von audiovisuellen Mediendiensten aus anderen Mitgliedstaaten in ihrem Hoheitsgebiet behindern dürfen (Art. 3) oder, dass die Mitgliedsstaaten sicherzustellen haben, dass audiovisuelle Mediendienste nicht die körperliche, geistige oder sittliche Entwicklung von Minderjährigen beeinträchtigen (Art. 6 a). Zu berücksichtigen ist diesbezüglich jedoch, dass die Richtlinien nicht im Bereich der Kulturpolitik, sondern auf Basis des Wirtschaftsrechts erlassen wurden, da die EU ausdrücklich keine Regelungen zu Kulturfragen treffen darf.559 In Relation zu Kulturangelegenheiten sind mehrere europäische Vorschriften zu berücksichtigen. Art. 167 Abs. 1 AEUV legt fest, dass die Union die „Entfaltung der Kulturen der Mitgliedstaaten unter Wahrung ihrer nationalen und regionalen Vielfalt sowie gleichzeitiger Hervorhebung des gemeinsamen kulturellen Erbes“ unterstützt. Nach Art. 167 Abs. 4 AEUV trägt die Union „bei ihrer Tätigkeit aufgrund anderer Bestimmungen der Verträge den kulturellen Aspekten Rechnung, insbesondere zur Wahrung und Förderung der Vielfalt ihrer Kulturen“. Oberstes Maß ist damit stets das kulturelle Erbe und die Vielfalt Europas.560 Der Kulturschutz kann sich ebenfalls auf den allgemeinen europäischen Integrations- und Harmonisierungsprozess auswirken. So betonen Kirsch und Kleiner, dass die Mitgliedsstaaten bei der Umsetzung europäischer Vorgaben in nationales Recht bestimmte eigene Begebenheiten und Vorstellungen berücksichtigen können.561 Weiterhin beinhaltet Art. 3 EUV nicht nur den allgemeinen Grundsatz, dass die Geschlechtergleichstellung 557 Richtlinie 2018/1808/EU des Europäischen Parlaments und des Rates zur Änderung der Richtlinie 2010/13/EU zur Koordinierung bestimmter Rechts- und Verwaltungsvorschriften der Mitgliedstaaten über die Bereitstellung audiovisueller Mediendienste (Richtlinie über audiovisuelle Mediendienste) im Hinblick auf sich verändernde Marktgegebenheiten, ABl. L 3030/69, 28.11.2018. 558 Seufert/Gundlach, Medienregulierung in Deutschland, S. 302. 559 Ebenda. 560 Ausführlich zur Kulturklausel siehe Knodel, Medien und Europa, S. 123 f.; ebenfalls https://europa.eu/european-union/topics/culture_de. 561 Klein/Kirschner, Werberegulierung in Europa, S. 42. 135 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien von allen Mitgliedsstaaten gefördert werden soll, sondern auch eine Regelung, wonach die Mitgliedsstaaten „den Reichtum ihrer kulturellen und sprachlichen Vielfalt“ wahren und „für den Schutz und die Entwicklung des kulturellen Erbes Europas“ eintreten sollen.562 Neben dem grundsätzlichen Ziel, die Gleichstellung von Frauen und Männern in der gesamten Europäischen Union zu erreichen, sind damit kulturelle Besonderheiten der einzelnen Staaten zu berücksichtigen. Gleichwohl spielen einige europäische Regeln in die Materie hinein, so dass der Regelungsbereich als „dynamisches, nicht immer konfliktfreies Spannungsfeld mitgliedstaatlicher und gemeinschaftlicher Politik und Gesetzgebung“ beschrieben wird.563 Klein argumentiert, dass die Geschlechterverhältnisse innerhalb der Europäischen Union auf der einen Seite „von Entwicklungen, die über die EU-Entwicklung hinausgehen und sich fördernd oder hemmend auf Geschlechtergerechtigkeit auswirken können“ und auf der anderen Seite „von historisch-kulturell geprägten Geschlechterbildern in einzelnen Mitgliedsstaaten, die hartnäckig und veränderungsresistent sind, beeinflusst werden“.564 Illustrativ für das Konfliktpotenzial im Werbe- und Medienbereich war die Richtlinie „Fernsehen ohne Grenzen“, die nach langen Beratungen im Oktober 1989 beschlossen wurde. Die Beratungen waren von kontroversen Auseinandersetzungen zwischen der Kommission und den Mitgliedstaaten geprägt, die mehrfach die fehlende EU-Kompetenz gerügt hatten.565 cc) Zwischenfazit Welche konkreten Möglichkeiten die EU-Beschlussorgane zur Regelung einheitlicher Bestimmungen zu geschlechterdiskriminierender Werbung im Binnenmarkt durch eine Richtlinie hätten, ist unklar. Fest steht, dass keine originäre gemeinschaftliche Kompetenz existiert. Der Erlass einer Vorschrift auf Grundlage des Wirtschaftsrechts wäre zwar grundsätzlich möglich, im Ergebnis gleichwohl zweifelhaft. Zum einen ist es unwahrscheinlich, dass eine Wettbewerbsverzerrung droht, nur weil einige Natio- 562 Dazu auch Knodel, Medien und Europa, S. 39. 563 Burggraf/Gerlach/Wiesner, Europäische Medienregulierung im Spannungsfeld zwischen EU- und mitgliedstaatlicher Kompetenz, MP 2018, 496 (496). 564 Klein, Geschlechterverhältnisse, Geschlechterpolitik und Gleichstellungspolitik in der Europäischen Union, S. 25. 565 Holtz-Bacha, bpb, 09.12.2016. 136 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb B. Europäische Union nen andere Geschlechterbilder in der Werbung zulassen. Zum anderen könnte eine entsprechende Regelung unter Berücksichtigung von Kulturund Medienangelegenheiten unzulässig sein. Nichtsdestoweniger bleibt festzuhalten, dass der Erlass einer Vorschrift zu geschlechterdiskriminierender Wirtschaftswerbung trotz kontroverser Teilaspekte – ähnlich wie bei der Richtlinie „Fernsehen ohne Grenzen“ – auch nicht vollkommen ausgeschlossen ist. Insbesondere für den Fall, dass die grenzüberschreitende Werbung weiterhin zunehmen und die Problematik sich gleichermaßen verschärfen sollte, wäre eine einheitliche Regelung auf EU-Ebene angemessen. b. Relevante Richtlinien Dementsprechend gibt es auf Gemeinschaftsebene keine allgemeine Vorschrift zur Regulierung von Werbung. Die Bestimmungen sind über mehrere Richtlinien und Verordnungen verstreut und beziehen sich in der Regel auf einzelne Produkte oder konkrete Verbreitungsmedien.566 In Bezug auf den Schutz der:des Konsument:innen ist insbesondere die „Richtlinie über irreführende und vergleichende Werbung“ zu nennen, die jedoch keine Regelungen zu stereotypen oder diskriminierenden Inhalten enthält.567 Verbindliche Rechtsakte, die Geschlechterdarstellungen in den Medien und der Werbung aufgreifen, gibt es trotz der Auseinandersetzung der EU-Organe mit der Problematik kaum. Einzige rechtlich verbindliche Vorschrift ist die 1989568 erlassene „Richtlinie Fernsehen ohne Grenzen“569, die 2007 zur „Richtlinie über audiovisuelle Mediendienste“570 erweitert und zuletzt 2018571 geändert wurde. Sie regelt die Fernsehwerbung in 566 Henning-Bodewig, Ethics, Taste and Decency in Advertising, S. 68. 567 Richtlinie 2006/114/EG des Europäischen Parlaments und des Rates vom 12.12.2006 über irreführende und vergleichende Werbung. 568 Erste Versuche eine vergleichbare Regelung einzuführen gab es bereits 1978 und 1979 in den Vorschlägen zu einer Richtlinie für irreführende und unlautere Werbung. Ausführlich Schricker/Henning-Bodewig, Elemente einer Harmonisierung des Rechts des unlauteren Wettbewerbs in der Europäischen Union, WRP 2001, 1367 (1372, 1395 f., 1397). 569 Richtlinie 89/552/EWG des Europäischen Parlaments und des Rates vom 03.10.1989 zur Koordinierung bestimmter Rechts- und Verwaltungsvorschriften der Mitgliedstaaten über die Ausübung der Fernsehtätigkeit. 570 Richtlinie 2007/65/EG über audiovisuelle Mediendienste. 571 Richtlinie 2018/1808/EU des Europäischen Parlaments und des Rates vom 14.11.2018 zur Änderung der Richtlinie 2010/13/EU zur Koordinierung be- 137 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien Europa weitestgehend einheitlich.572 Dabei enthält sie den Grundsatz, dass audio-visuelle Kommunikation „die Menschenwürde nicht verletzen“ und nach Art. 9 Abs. 1 c) (ii) keine „Diskriminierung aufgrund von Geschlecht, Rasse oder ethnischer Herkunft, Staatsangehörigkeit, Religion oder Glauben, Behinderung, Alter oder sexueller Ausrichtung beinhalten oder fördern“ darf.573 Das Verbot ist ein „Sonderfall“ im europäischen Rechtsgefüge, der 1989 mit der „befürchteten Suggestivwirkung“ der Fernsehwerbung begründet wurde.574 Demgegenüber enthält beispielsweise die EU-Richtlinie zur „Verwirklichung des Grundsatzes der Gleichbehandlung von Männern und Frauen beim Zugang zu und bei der Versorgung mit Gütern und Dienstleistungen“ keine verbindliche Regelung zu geschlechterdiskriminierenden Werbeinhalten, sondern schließt die Anwendung der Richtlinie auf den Inhalt von Medien und Werbung gemäß Art. 3 Abs. 3 vielmehr explizit aus.575 Auch in den Erwägungsgründen zu der Richtlinie zu unlauteren Geschäftspraktiken wird festgestellt, dass sich die Richtlinie nicht auf „Fragen der guten Sitten und des Anstands“ bezieht, da sie „in den Mitgliedstaaten sehr unterschiedlich“ seien.576 Demnach wird die Regulierung derartiger Fragen den Mitgliedstaaten überlassen. Die Fokussierung auf die Werbeselbstkontrolle und Bildungskampagnen gehe nach Henning-Bodewig auf die schnelleren und flexibleren Beurteilungs- und Sanktionsmechanismen zurück.577 Dackweiler begründet die Beschränkung auf „weiche stimmter Rechts- und Verwaltungsvorschriften der Mitgliedstaaten über die Bereitstellung audiovisueller Mediendienste (Richtlinie über audiovisuelle Mediendienste) im Hinblick auf sich verändernde Marktgegebenheiten. 572 Dazu auch Klein/Kirschner, Werberegulierung in Europa, S. 41. 573 Richtlinie 2018/1808/EU des Europäischen Parlaments und des Rates vom 14.11.2018 zur Änderung der Richtlinie 2010/13/EU zur Koordinierung bestimmter Rechts- und Verwaltungsvorschriften der Mitgliedstaaten über die Bereitstellung audiovisueller Mediendienste (Richtlinie über audiovisuelle Mediendienste) im Hinblick auf sich verändernde Marktgegebenheiten, Art. 9 AVMD-RL (1) c) i) ii). 574 Henning-Bodewig, „Sexistische Werbung“ – ein Fall für den Gesetzgeber?, S. 13 f. 575 Richtlinie 2004/113/EG vom 13.12.2004 zur „Verwirklichung des Grundsatzes der Gleichbehandlung von Männern und Frauen beim Zugang zu und bei der Versorgung mit Gütern und Dienstleistungen“; dazu auch Dackweiler, Europäische Gleichstellungspolitik, KJ 2017, 297 (300). 576 Richtlinie 2005/29/EG des Europäischen Parlaments und des Rates vom 11.05.2005 über unlautere Geschäftspraktiken im binnenmarktinternen Geschäftsverkehr zwischen Unternehmen und Verbrauchern, ABl. Nr. L 149/22 vom 11.06.2005. 577 Henning-Bodewig, „Sexistische Werbung“ – ein Fall für den Gesetzgeber?, S. 13 f. 138 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb B. Europäische Union Maßnahmen“ hingegen mit der Meinungs- und Pressefreiheit, die „in keiner Weise angetastet werden“ dürfe.578 c. Zwischenfazit Im Ergebnis fehlt es auf Gemeinschaftsebene an einheitlichen Bestimmungen für die Begrenzung von Geschlechterbildern in der Werbung, ebenso wie an übergreifenden Kontroll- und Durchsetzungsvorgaben. Einzig die Richtlinie über audiovisuelle Mediendienste zieht eine Grenze, indem sie ein medienspezifisches Verbot geschlechterdiskriminierender Werbung statuiert, welches jedoch nicht näher spezifiziert wird. Die Zurückhaltung des europäischen Gesetzgebers kann auf die Vermutung gestützt werden, dass er kulturelle, verfassungsrechtliche sowie praktische Besonderheiten der Mitgliedsstaaten nicht beschränken will. 2. Unverbindliche Rechtsakte Im Gegensatz zu dem Mangel rechtsverbindlicher Vorschriften zu Geschlechterdarstellungen in der Werbung existiert eine Vielzahl an unverbindlichen Empfehlungen und Entschließungen der EU-Organe zu der Thematik. Dabei handelt es sich um Dokumente, die keine Rechtswirkung beabsichtigen, sondern das Ziel verfolgen, einen politischen Standpunkt in Zusammenhang mit den Tätigkeitsbereichen der Europäischen Union zum Ausdruck zu bringen. Daneben können die unverbindlichen Empfehlungen und Entschließungen jedoch auch dazu dienen, die Mitgliedstaaten oder andere EU-Institution dazu aufzufordern, Maßnahmen zu einer bestimmten Frage oder zu einem bestimmten Problem zu ergreifen. Während das Parlament zwischen Entschließungen und Empfehlungen wählen kann, kann der Rat auf Schlussfolgerungen und Entschließungen zurückgreifen. Die Kommission kann als ähnliche Mittel Grünbücher oder Fahrpläne zur Anregung von Diskussionen über bestimmte Themen auf EU-Ebene veröffentlichen.579 Die unverbindlichen Rechtsakte der EU-Organe zu geschlechterdiskriminierender Werbung schaffen einen wichtigen Grundkonsens, der Leitlinien für den Umgang mit der Problematik auf- 578 Dackweiler, Europäische Gleichstellungspolitik, KJ 2017, 297 (301). 579 Dazu siehe etwa https://www.consilium.europa.eu/de/council-eu/conclusions-re solutions/. 139 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien zeigt und möglicherweise bereits als ausreichende Grundlage für nationale Regelungen zu erachten sein könnte. a. Europäisches Parlament Bereits 1997 erkannte das Parlament den unzureichenden Diskriminierungsschutz von Frauen in der Werbung und den Medien an und verfasste einen Entschließungsbericht, in dem verschiedene Maßnahmen diesbezüglich vorgeschlagen wurden. In dem Bericht „Diskriminierung von Frauen in der Werbung“ von 1997 wurde zunächst auf die Rolle der Werbung bei der Verwirklichung von Geschlechtergleichberechtigung eingegangen. Dabei wurde darauf hingewiesen, dass insbesondere pornographische Inhalte und die ungerechtfertigte Ausbeutung des weiblichen Körpers zu kommerziellen Zwecken die Würde von Frauen besonders schwerwiegend verletzen können. Ebenfalls wurde die Auffassung vertreten, dass die Medien das soziale Verhalten beeinflussen, zu einem Mentalitätswandel führen und zur Verwirklichung der Gleichstellung beitragen können, wenn sie vielfältige und nicht-stereotype Geschlechterrollen beinhalten. Dafür schlug der Bericht eine Reihe von legislativen Maßnahmen und Selbstregulierungskodizes vor.580 So werden etwa „die nationalen werbedisziplinären Einrichtungen [aufgefordert], die Verbraucher verstärkt über Ziele, Funktionsweise und Grundsätze der Werbekontrolle und ihre Beschwerdemöglichkeiten aufzuklären“. Die beteiligten Industriebranchen werden ermutigt, „Mindeststandards für nichtdiskriminierende Werbung in den Selbstkontroll-Kodex aufzunehmen“.581 Ebenso wird „die Weiterentwicklung der Europäischen Allianz der Werbekontrolle (EASA) zu einem echten Selbstkontroll-Organ der europäischen Werbewirtschaft [empfohlen], das die Aufgabe hat, europäische Grundsätze der Werbedisziplin, u.a. gegen diskriminierende Werbung, aufzustellen, und als Selbstregulierungsorgan bei grenzüberschreitenden Klagen mit Kontroll- und Empfehlungsbefugnissen ausgestattet ist“.582 Ergänzend wird die Europäische Kommission ermutigt, „alle Beteiligten auf[zufordern], einen Selbstkontroll-Kodex gegen jede Form der Diskriminierung für die Werbewirtschaft auszuarbeiten“, eine „realitätsbezogenen Darstellung der heutigen Frau [...] in den 580 Entschließung vom 16.09.1997 zur Diskriminierung von Frauen in der Werbung, ABl. Nr. C 304, 06.10.1997, S. 60. 581 Ebenda, Rn. 16. 582 Ebenda, Rn. 17. 140 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb B. Europäische Union Medien und in der Werbung“ zu fördern und in ihren Studien zum neuen Geschlechterrollenverständnis die Werbung miteinzubeziehen.583 Gleichermaßen wird die Kommission angeregt, den existierenden europäischen Gesetzesrahmen im Bereich der Werbung zu überarbeiten, wobei die Notwendigkeit betont wird, eine größere Konvergenz der nationalen Vorschriften zu schaffen und dadurch ein angemessenes Schutzniveau der Interessen der Allgemeinheit zu garantieren.584 Im Mai 2008 veröffentlichte das Europäische Parlament einen Bericht „über die Auswirkungen von Marketing und Werbung auf die Gleichstellung von Frauen und Männern“.585 Auch in diesem Bericht betonte das Parlament, dass die Auswirkungen von Werbeinhalten auf Aspekte der geschlechtsspezifischen Sozialisation und der Verhaltens- und Meinungsbildung der Bürger:innen nicht zu unterschätzen seien. Dabei wurde verstärkt auf die Gefahr diskriminierender und erniedrigender Werbebotschaften hingewiesen. Die Darstellung von Geschlechterstereotypen sei als Hindernis für eine moderne und gleichberechtigte Gesellschaft anzuerkennen.586 Die federführende schwedische Europaabgeordnete Svensson hatte noch einige wesentlich strengere Maßnahmen gefordert, wie einen Verhaltenskodex für die Werbebranche, nationale Gremien zur Medienüberwachung oder das Verbot, anorektische Models in der Werbung abzubilden587 Diese Forderungen konnten sich letztlich jedoch nicht durchsetzen. Die Berichterstattung einer deutschen Zeitung formuliert dazu: „Geschlechterdiskriminierung – EU will Hausfrau aus Werbung verbannen“.588 Weiterhin heißt es: „Keine Hausfrau hinter dem Herd, kein starker Hausmann, der den Rasen trimmt. Sexistische Klischees in der Werbung sollen künftig tabu sein.“ Zuletzt wird hervorgehoben, dass der Beschluss des Europaparlaments von der Werbewirtschaft aufgrund der befürchteten, weitergehenden Maßnahmen „trotzdem mit einer gewissen Erleichterung aufgenommen wurde“.589 In einem weiteren Bericht zu einer Entschließung im Dezember 2010 zum Einfluss der Werbung auf das Verbraucherverhalten wird erneut da- 583 Ebenda, Rn. 21 f. 584 Ebenda, Rn. 24. 585 Bericht vom 29.05.2008 über die Auswirkungen von Marketing und Werbung auf die Gleichstellung von Frauen und Männern(2008/2038/INI), ABl. A6– 0199/2008. 586 Ebenda. 587 Kafsack, F.A.Z., 04.09.2008. 588 Ebenda. 589 Ebenda. 141 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien rauf hingewiesen, dass „Reklame ein wirksames Instrument zur Bekämpfung von Stereotypen und Vorurteilen aufgrund von Rassismus, Sexismus und Fremdenfeindlichkeit darstellen kann“590 und „Reklame häufig einseitige und/oder verfälschte Inhalte transportiert, die stereotypisierte Vorurteile in Bezug auf das Geschlecht verfestigen, wodurch Gleichstellungsstrategien, die diese Ungleichheiten beseitigen sollen, unterminiert werden“591. Zudem werden die Kommission und die Mitgliedstaaten aufgefordert, „durch geeignete Mittel sicherzustellen, dass die Medien- und die Werbefachleute die Achtung der Menschenwürde gewährleisten und sich Darstellungen, die unmittelbar oder mittelbar diskriminierend oder klischeehaft sind, sowie einer Aufstachelung zum Hass aufgrund des Geschlechts, der Rasse, der ethnischen Herkunft, des Alters, der Religion oder Weltanschauung, der sexuellen Veranlagung, einer Behinderung oder des sozialen Status [beinhaltet], widersetzen“592. Abschließend werden Erkenntnisse und Maßnahmen zur „Gewährleistung der Gleichstellung von Frauen und Männern und der menschlichen Würde in der Werbung“ spezifiziert.593 Dazu gehört beispielsweise, dass „Werbung ein wirksames Instrument für die Hinterfragung von Klischees und die Auseinandersetzung mit ihnen sowie ein Mittel zur Abwehr von Rassismus, Sexismus und Diskriminierung sein kann, dem in den heutigen multikulturellen Gesellschaften entscheidende Bedeutung zukommt“.594 Ferner wird festgestellt, dass „Werbung oft diskriminierende und/oder entwürdigende Botschaften vermittelt, die auf allen Arten von Geschlechterklischees beruhen und die den Strategien zur Förderung der Gleichstellung von Frauen und Männern abträglich sind“595 und dass „die Darstellung gesunder Rollenmodelle demnach einen positiven Einfluss auf die gesellschaftlichen Vorstellungen in Fragen wie Geschlechterrollen, Körperbild und Normalität haben kann“596. In einer Entschließung zum „Abbau von Geschlechterstereotypen in der EU“ aus dem Jahr 2013 wird gleichermaßen erwähnt, dass „geschlechts- 590 Entschließung des Europäischen Parlaments vom 15.12.2010 zum Einfluss der Werbung auf das Verbraucherverhalten (2010/2052(INI)), 2012/ABl. C 169 E/08, 15.06.2012, Erwägungsgrund N. 591 Ebenda, Erwägungsgrund O. 592 Ebenda, Bewertung 15. 593 Ebenda, Gewährleistung der Gleichstellung von Frauen und Männern und der menschlichen Würde in der Werbung (32.-37.). 594 Ebenda, 33. 595 Ebenda, 36. 596 Ebenda, 37. 142 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb B. Europäische Union spezifische Diskriminierung in [...] der Werbung immer noch häufig vorkommt und die Reproduktion von Geschlechterstereotypen erleichtert, insbesondere indem Frauen als Sexobjekte dargestellt werden, um Geschäfte zu fördern“597. Dabei werden die „möglichen Auswirkungen auf die geschlechtsspezifische Sozialisierung“598 als problematisch hervorgehoben. Es wird eine verstärkte Zusammenarbeit der Kommission, der Mitgliedstaaten, der Zivilgesellschaft und der Selbstregulierungsorgane der Werbebranche bei der „Bekämpfung solcher Praktiken“ und der Gewährleistung der „Achtung der menschlichen Würde und Integrität“ in der Werbung gefordert.599 Insgesamt wird die wichtige Rolle der Medien und des Werbesektors in Bezug auf Geschlechterstereotype anerkannt. b. Rat der Europäischen Union Ebenso hat sich der Rat der Europäischen Union bereits mehrfach mit geschlechterdiskriminierenden Werbeinhalten auseinandergesetzt. In einer der ersten Entschließungen zu der Problematik aus dem Jahr 1995 heißt es beispielsweise, dass „geschlechtsspezifische Rollenklischees in Werbung und Medien Teil der Ungleichheiten sind, die die Haltungen gegenüber der Gleichstellung von Männern und Frauen beeinflussen, und dass es deshalb wichtig ist, die Gleichstellung von Männern und Frauen in allen Bereichen des gesellschaftlichen Lebens zu fördern“. Es wird ausgeführt, dass „Werbung und Medien einen wichtigen Beitrag zur Änderung der Verhaltensweisen in der Gesellschaft leisten können, indem sie die Vielfalt der Rollen und des Potenzials von Frauen und Männern, ihre Beteiligung an allen Bereichen des gesellschaftlichen Lebens und eine ausgewogene Aufteilung der Verantwortung in Familie, Beruf und Gesellschaft zwischen Frauen und Männern widerspiegeln“ und dass „Werbung und Medien die Menschenwürde nicht verletzen und keine Diskriminierung aufgrund des Geschlechts enthalten dürfen“. Weiterhin soll „unter Beachtung ihrer Verfassungsvorschriften und/oder ihrer nationalen Sichtweisen und Gepflo- 597 Entschließung des Europäischen Parlaments vom 12.03.2013 zum Abbau von Geschlechterstereotypen in der EU (2012/2116(INI)), ABl. 2016/C 036/03, 29.01.2016, Erwägungsgrund J. Weiter heißt es darin, dass „Frauen zum Beispiel in der Werbung nur 27 Prozent der gezeigten Angestellten und Fachkräfte ausmachen, wohingegen Rollen in denen Hausarbeit und Kinderbetreuung vorkommen zu 60 Prozent mit Frauen besetzt sind“. 598 Ebenda, Erwägungsgrund J., EU-Maßnahme, Medien und Kultur, Nr. 7. 599 Ebenda, EU-Maßnahme, Medien und Kultur, Nr. 9. 143 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien genheiten“ ein realistisches und differenziertes Bild der Möglichkeiten und Fähigkeiten von Frauen und Männern in der Gesellschaft durch geeignete Maßnahmen gefördert werden. Dafür soll insbesondere auf die „Entwicklung und Umsetzung von Verhaltenskodizes zur freiwilligen Selbstkontrolle“ gesetzt werden.600 Zudem erließ der Europäischen Rat 2006 einen „Europäischen Pakt für die Gleichstellung der Geschlechter“, in welchem er die Unerlässlichkeit der Geschlechtergleichstellungspolitik für Wirtschaftswachstum, Wohlstand und Wettbewerbsfähigkeit betonte.601 Eine weitere Schlussfolgerung befasste sich mit der „stärkeren Förderung von Frauen als Entscheidungsträger in den Medien“. Laut dieser haben die Medien „ein enormes Potenzial“, beispielsweise durch eine „ausgewogene und nicht stereotype Darstellung von Frauen und Männern“ oder durch die „Darstellung von Frauen als Entscheidungsträger und in Führungspositionen“ in der Werbung auf „positive Weise zur Erreichung der Gleichstellung der Geschlechter auf allen Ebenen beizutragen“.602 Als aktuelleres Beispiel ist die Entschließung des Europäischen Parlaments vom 25. Februar 2010 zu „Peking + 15 – UN-Plattform für Maßnahmen zur Gleichstellung der Geschlechter“ zu nennen.603 Darin wird sich auf die CEDAW bezogen und unter anderem festgestellt, „dass die strategischen Ziele der Aktionsplattform von Peking trotz aller Bemühungen bislang nicht erreicht worden sind, dass Ungleichheit und Geschlechterstereotype fortbestehen und Frauen in den in der Plattform angesprochenen Bereichen weiterhin eine den Männern untergeordnete Position einneh- 600 Entschließung des Rates und der im Rat vereinigten Vertreter der Regierungen der Mitgliedsstaaten vom 05.10.1995 zur Darstellung der Frau und des Mannes in Werbung und Medien (95/C 296/06). Siehe ABl. C 296, 10.11.1995, S. 15–16; dazu auch Klein, Geschlechterverhältnisse, Geschlechterpolitik und Gleichstellungspolitik in der Europäischen Union, S. 54. 601 Rat der Europäischen Union, ABl. 7775/1/06 REV 1, Concl. 1, 2006, S. 12, Rn. 40 und Anlage II. 602 Stärkere Förderung von Frauen als Entscheidungsträger in den Medien, Schlussfolgerungen des Rates der Europäischen Union, ABl. 11470/13 SOC 451, 24.06.2013, Nr. 16. 603 Entschließung des Europäischen Parlaments vom 25.02.2010 zu Peking + 15 – UN-Plattform für Maßnahmen zur Gleichstellung der Geschlechter (2010/C 348 E/03), ABl. C 348 E/11, 21.02.10; siehe dazu auch Klein, Geschlechterverhältnisse, Geschlechterpolitik und Gleichstellungspolitik in der Europäischen Union, S. 54. 144 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb B. Europäische Union men“.604 Gleichermaßen werden die Kommission und die Mitgliedstaaten aufgefordert, „spezifische politische Maßnahmen zur Gleichstellung der Geschlechter, einschließlich aktiver Maßnahmen, anzunehmen und umzusetzen, um die Erreichung einer faktischen Gleichstellung der Geschlechter zu beschleunigen und die umfassende Wahrnehmung aller Menschenrechte durch Frauen und Mädchen zu fördern“.605 c. Europäische Kommission Schließlich ist das Thema auch bei der Europäischen Kommission nicht unbeachtet geblieben. So erließ sie 2006 einen „Fahrplan für die Gleichstellung von Frauen und Männern“, der als Rahmen für die Gleichstellungspolitik gelten sollte und spezifische Maßnahmen für den Zeitraum zwischen 2006 und 2010 vorsah.606 Unter anderem solle Geschlechterstereotypen in den Medien entgegengewirkt werden, um „ein realistisches Bild der Fähigkeiten und Potenziale von Frauen und Männern in der modernen Gesellschaft“ zu zeichnen. Dafür sei es jedoch unerlässlich, in den Dialog mit EU-Bürger:innen zu treten und das Bewusstsein für die Geschlechtergleichstellung zu stärken.607 In keinem der nachfolgenden Pläne608 wurde die Problematik adressiert. Mittlerweile ist die „Strategie für die Gleichstellung der Geschlechter 2020–2025“ in Kraft.609 Sie sieht unter anderem vor, eine EU-weite Kommunikationskampagne gegen Geschlechterstereotypen zu starten.610 604 Entschließung des Europäischen Parlaments vom 25.02.2010 zu Peking + 15 – UN-Plattform für Maßnahmen zur Gleichstellung der Geschlechter (2010/C 348 E/03), ABl. C 348 E/11, 21.02.2010, A. 605 Ebenda, 16. 606 Europäischen Kommission, Ein Fahrplan für die Gleichstellung von Frauen und Männern, KOM (2006) 92 und Folgenabschätzung SEK (2006) 0275. 607 Europäischen Kommission, Ein Fahrplan für die Gleichstellung von Frauen und Männern, KOM (2006) 92, S. 9. 608 Europäischen Kommission, Strategie für die Gleichstellung von Frauen und Männern 2010–2015, KOM (2010) 491, (SEK (2010) 1079; SEK (2010) 1080); Europäischen Kommission, Strategisches Engagement für die Gleichstellung der Geschlechter (2016–2019), SWD (2015) 278. 609 Europäischen Kommission, Eine Union der Gleichheit: Strategie für die Gleichstellung der Geschlechter 2020–2025, KOM (2020) 152. 610 Ebenda, S. 7. 145 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien Unlängst befasste sich 2018 eine von Frankreich ausgerichtete Konferenz mit dem Gegenstand „Frauen und die Medien“.611 Dabei wurden die „Wirksamkeit von Beschwerdemechanismen“ sowie ein mögliches „Vorgehen gegen Geschlechterstereotypen und Sexismus in Medieninhalten“ diskutiert.612 In dem Zusammenhang ging es um die unterschiedlichen nationalen Ansätze von staatlichen Überwachungsinstanzen über Ombudsstellen bis hin zu Werberäten. Es wurden die Schwierigkeiten der Bürger:innenbeteiligung und die teilweise mangelnden Sanktionsmöglichkeiten im Selbstregulierungsverfahren hervorgehoben. Die Bekämpfung von Geschlechterstereotypen sei insbesondere aufgrund der sich konstant verändernden kulturellen Denkweise und der daraus resultierenden neuen Bewertungsperspektiven eine herausfordernde Aufgabe.613 d. Zwischenfazit In den Entschließungen des Europäischen Parlaments, die sich von 1997 bis 2013 erstrecken, wird geschlechterdiskriminierende Werbung als „europäisches Problem“ gewürdigt und festgestellt, dass in keinem Staat ausreichende Schutzmaßnahmen existieren, und zwar weder auf gesetzlicher noch auf untergesetzlicher Ebene. Ergänzend werden die Gefahren diskriminierender Werbeinhalte in Bezug auf den Sozialisationsprozess betont und Geschlechterstereotype als Schwierigkeit hervorgehoben. Zwar liegt der Schwerpunkt der Debatte auf dem Bild der Frau. Während 1997 jedoch noch ausschließlich das Bild der Frau diskutiert wurde, werden seitdem zunehmend auch problematische Männerdarstellungen kritisiert. Das Europäische Parlament weist nicht nur auf die Notwendigkeit eines Tätigwerdens der Mitgliedsstaaten hin, sondern macht selbst konkrete Lösungsvorschläge. Insgesamt beschränken sich die Vorschläge allerdings auf unverbindliche Empfehlungen und überwiegend „weiche“ Maßnahmen wie die Erstellung von Werbeselbstregulierungskodizes. Parallel dazu handelt es sich bei den Maßnahmen des Rats der Europäischen Union und der Europäischen Kommission gleichermaßen um unverbindliche Rechtsakte, die zwar die Problematik von konkreten Werbebildern anerkennen und sich insbesondere mit Geschlechterstereotypen auseinandersetzen, jedoch kaum eigene Lösungsvorschläge unterbreiten. 611 Europäische Kommission, Frauen und die Medien. 612 Ebenda, S. 15. 613 Ebenda. 146 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb B. Europäische Union 3. Tendenzen und Bewertung Die Analyse hat verdeutlicht, dass sich ein Bewusstsein für die Problematik geschlechterdiskriminierender Werbung auf EU-Ebene erstmals Anfang der Neunzigerjahre erkennen ließ. Hand in Hand mit der verstärkten Aufmerksamkeit, die feministischen Theorien und Genderkonzepten in dieser Zeit erstmals in der breiteren Öffentlichkeit gewidmet wurde,614 erfolgten die ersten Auseinandersetzungen mit dem Thema. Zwischen 2000 und 2010 hat sich die Würdigung der Problematik weiter verdichtet. Fast alle EU-Organe befassen sich in dieser Zeitspanne mit der Materie geschlechterdiskriminierender Werbung. Die Bezugnahme auf sozialpsychologische und kommunikationswissenschaftliche Studien, die die negativen Konsequenzen sexistischer Werbebilder zum Gegenstand haben, werden als Argumentationsgrundlagen herangezogen und erste Vorschläge für Gegenmaßnahmen werden unterbreitet. Es scheint als würde das Problem ernst genommen und sich eines Entgegenwirkens bemüht. Gleichwohl scheint die Auseinandersetzung mit den Regulierungsfragen hier ihren Höhepunkt erreicht zu haben, denn seitdem ebbt die Anzahl an Entschließungen diesbezüglich ab. Dass alles Relevante gesagt ist, erscheint unwahrscheinlich. Doch warum stagnieren die Prozesse auf europäischer Ebene? Gerade in Anbetracht der gestiegenen geschlechtsbezogenen Sensibilität der Gesellschaft und den vielfältigen medienwirksamen Debatten zum Thema Geschlechtergleichheit und Geschlechterdiskriminierung ist die Entwicklung ungewöhnlich.615 Zwar könnte vorgebracht werden, dass die kulturelle Verankerung von Geschlechterbildern als Grund herangezogen wurde, der Problematik vorrangig auf nationaler und nicht mehr auf europäischer Ebene zu begegnen. Jedoch erscheint diese Überlegung auf Grundlage des langen Zeitraums, in dem sich auf EU-Ebene mit der Fragestellung auseinandergesetzt wurde, zweifelhaft. So wurde der Aufbau nationaler Werbeselbstregulierungsinstanzen zwar vielfach als effektvolle Maßnahme betont. Gleichermaßen wurde allerdings auch der Ausbau der europäischen Werbeselbstkontrolle und ein weiteres Tätigwerden der Europäischen Kommission gefordert, so dass ein Abstellen auf die Förderung nationaler Lösungsansätze als Grund für sinkende Entschließungszahlen weniger naheliegend ist als es auf den ersten Blick erscheinen mag. 614 Von Bargen, Von Welle zu Welle, abrufbar unter: https://www.boell.de/de/2018/ 07/03/von-welle-zu-welle. 615 Ebenda. 147 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien Demgegenüber könnte als weiterer möglicher Grund in Erwägung gezogen werden, dass die gestiegene Sensibilität für Geschlechterungleichheit dazu geführt hat, dass viele weitere Problematiken, die mit Geschlechterdiskriminierung in der Gesellschaft zusammenhängen, kenntlich gemacht und sie von den EU-Organen – im Vergleich zu der Problematik geschlechterdiskriminierender Wirtschaftswerbung – als existenzieller wahrgenommen wurden. Insbesondere die Förderung von Frauen auf dem Arbeitsmarkt, die Überbrückung von „Gender-Pay-Gaps“ und die Verhinderung von geschlechtsspezifischer Gewalt waren beispielsweise viel diskutierte Themen.616 Inwieweit diese die Geschlechterdarstellung in der Werbung als Diskussionsthema verdrängt haben, kann jedoch ebenfalls nur spekuliert werden, so dass an dieser Stelle offenbleiben muss, warum seit der zweiten Hälfte der Zehner-Jahre eine weniger starke Forcierung der Problematik auf EU-Ebene erfolgte. Als weitere Erkenntnis der Analyse hat sich gezeigt, dass auf europäischer Ebene bislang überwiegend unverbindliche Rechtsakte zu Geschlechterdarstellungen in der Werbung erlassen wurden. Dies könnte einerseits mit einer unzureichenden Regelungskompetenz der EU-Organe auf dem Gebiet geschlechterdiskriminierender Werbung in Verbindung stehen. Dackweiler beispielsweise erklärt, dass die „Verbindlichkeitslücke“ mit dem Grundsatz der „kulturellen Ausnahme“ zusammenhänge, wonach der Bereich kultureller Bildung und Medien nicht in den Zuständigkeitsbereich der EU-Gesetzgebung falle.617 Andererseits könnte jedoch die schwere Greifbarkeit der Problematik verantwortlich für die Zurückhaltung der EU-Organe sein. Diese ergibt sich nach dem bisherigen Erkenntnisstand aus dem komplexen Definitionsprozess und der nationalen Prägung der mit geschlechterdiskriminierender Werbung in Verbindung stehenden Inhalte. Unabhängig vom Fehlen rechtsverbindlicher Strukturen bilden die unverbindlichen Maßnahmen einen ersten Referenzrahmen für nationale Initiativen der Mitgliedsstaaten. Die Maßnahmen lenken den Blick auf konkrete Aspekte der Problematik und animieren die Mitgliedstaaten, gegen geschlechterdiskriminierende vorzugehen. Ergänzend ist den Ausführungen von Klein und Kirschner zu folgen, die erklären, dass verbind- 616 Siehe dazu etwa Europäische Kommission, Striving for a Union of Equality: The Gender Equality Strategy 2020–2025; ebenso Europäische Kommission, Strategic Engagement for Gender Equality 2016–2019. 617 Dackweiler, Europäische Gleichstellungspolitik, KJ 2017, 297 (300). 148 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb C. Internationale Werbeselbstregulierung liche Vorschriften ohnehin „lediglich die Basis für ethisches Verhalten bilden“ können und „auch in einer sich ausdehnenden Europäischen Union [nur] einen Grundkonsens schaffen [können], auf de[m] landes- und kulturspezifische Sonderregelungen aufbauen“.618 Daraus folgend sollten die „Regelungen der Europäischen Union [...] keinesfalls als Maximalforderung interpretiert werden“, da es auf EU-Ebene eine Herausforderung darstelle den „richtigen Grad der Regulierung zu finden, ohne dabei zu stark in die Autonomie und die Gesellschaft eines Landes einzugreifen“.619 Möglicherweise sind rechtsunverbindliche Vorschriften für den gewählten Regulierungsgegenstand demnach kein zweitrangiges Regulierungsinstrument, sondern schaffen eine Orientierungsgrundlage, auf der weitergehende Maßnahmen aufbauen können. Eine weiterführende Erörterung und Bewertung europäischer Möglichkeiten zur Lösung der Problematik erfolgt im vierten Kapitel. C. Internationale Werbeselbstregulierung Die Untersuchung der bestehenden völkerrechtlichen Vorgaben und der Rechtsakte der EU-Organe hat verdeutlicht, dass weitgehend auf rechtsverbindliche Maßnahmen verzichtet und das Potenzial der Werbeselbstregulierung als Mittel zur Bekämpfung geschlechterdiskriminierender Werbeinhalte hervorgehoben wurde. Der Nutzen der freiwilligen Selbstkontrolle wurde als Alternative zu verwaltungsaufwändigen rechtlichen Regelungen herausgestellt und der Ausbau und die Förderung nationaler Selbstregulierungsorganisationen betont.620 In fast allen Mitgliedstaaten der Europäischen Union existieren Werbeselbstregulierungsinstanzen. Allerdings ist die Bedeutung der Werbeselbstregulierung von Staat zu Staat sehr unterschiedlich. Die Differenziertheit wettbewerbs- oder werberechtlicher Gesetze spielt hierfür eine tragende Rolle.621 Neben den einzelnen nationalen Bestimmungen und Organisationen gibt es staatenübergreifenden 618 Klein/Kirschner, Werberegulierung in Europa, S. 44 f. 619 Ebenda. 620 Etwa in den Richtlinien 84/450/EWG zur irreführenden Werbung und der Richtlinie 2005/29/EG zu unlauteren Geschäftspraktiken wird die Rolle von Verhaltenskodizes der freiwilligen Werbeselbstkontrolle als Möglichkeit gerichtliche oder administrative Maßnahmen zu vermeiden ausdrücklich genannt. 621 Dazu vgl. Schricker/Henning-Bodewig, Elemente einer Harmonisierung des Rechts des unlauteren Wettbewerbs in der Europäischen Union, WRP 2001, 1367 (1369). 149 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien Institutionen, die die administrative Arbeit auf dem Gebiet der Werbeselbstkontrolle erleichtern. Zudem setzen sie gemeinsame internationale oder europäische Standards, an denen sich ein Großteil der nationalen Verhaltenskodizes orientiert. In Bezug auf internationale und europäische Leitlinien sind daher die Standards der Werbeselbstregulierung der International Chamber of Commerce (ICC; Internationale Handelskammer), der International Council for Advertising Self-Regulation (ICAS; Internationaler Rat für Werbeselbstregulierung) und der European Advertising Standards Alliance (EASA; Europäische Allianz der Werbeselbstkontrolle) zu untersuchen. I. ICC Advertising and Marketing Communications Code Einen ersten wichtigen Rahmen für die weltweite Werbeselbstregulierung bildet der ICC Advertising and Marketing Communications Code (ICC-Kodex; Werbe- und Marketingkodex der Internationalen Handelskammer).622 Er wurde erstmals 1937 veröffentlicht und setzt eigenen Angaben zufolge internationale branchenübergreifende Mindeststandards in Bezug auf verantwortungsvolle Werbung. Der Kodex wird stetig überarbeitet und dem aktuellen Werbegeschehen angepasst. Im Jahr 2018 wurde er in seiner aktuellen elften Fassung ausgearbeitet.623 Die Regelungen des ICC-Kodex präzisieren unter anderem, welche Verhaltensweisen von Werbetreibenden als unethisch zu erachten sind.624 Darüber hinaus dienen sie der Reduzierung detaillierter gesetzlicher Vorschriften.625 Das Regelwerk ist nicht nur selbst Grundlage für die Bewertung von Werbeinhalten, sondern fungiert gleichermaßen als Ankerpunkt bei der Entwicklung von nationalen Werbekodizes. Staaten können sich bei der Formulierung ihrer Verhaltensregeln inhaltlich am ICC-Kodex 622 Die Internationale Handelskammer ist ein weltweit tätiger, nichtstaatlicher Wirtschaftsverband, der aus einer Vielfalt von Organisationen, Firmen und Einzelpersonen des Wirtschaftslebens besteht. Heute umfasst der Verband mehr als 45 Millionen Unternehmen in über 100 Ländern und Bürositze in mehr als 90 (https://iccwbo.org/about-us/). 623 https://www.iccgermany.de/standards-incotermsr/verhaltensrichtlinien/werbe-u nd-marketingstandards/. 624 Siehe Vorwort ICC-Kodex 2018, S. i; dazu auch Felser, Werbe- und Konsumpsychologie, S. 19. 625 Dazu ebenfalls Svensson/Edstörm, Freedom of Expression vs. Gender Equality, TFR 2014, 479 (493). 150 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb C. Internationale Werbeselbstregulierung orientieren. Im europäischen Raum gehen fast alle Verhaltenskodizes der Werbeselbstregulierungsorganisationen auf den ICC-Kodex zurück.626 Hierzu führt die Internationale Handelskammer aus, dass ihre „Kodizes ausreichend flexibel sind, um unterschiedliche kulturelle und soziale Regeln und Normen in Einklang zu bringen“.627 Wichtigste Vorschrift für den Kontext dieser Abhandlung ist Art. 2 des ICC-Kodex, der die soziale Verantwortung von Werbemaßnahmen regelt. Demnach soll Werbung „die Würde des Menschen respektieren und keinerlei Form von Diskriminierung anregen oder stillschweigend dulden, einschließlich jedweder Diskriminierung, die auf Rasse, ethnischer Herkunft, Religion, Geschlecht, Alter, Behinderung oder sexueller Orientierung basiert“. Die Auslegung des Regelwerks soll „sinngemäß“ und „detailgenau“ erfolgen sowie in „Wissen, Erfahrung und charakteristischen Fähigkeiten des typischen Verbrauchers beurteilt werden“.628 Zudem sind „soziale, kulturelle und sprachliche Faktoren“ zu berücksichtigen.629 Die Formulierung aus Art. 2 ist zwar eindeutig dahingehend zu verstehen, dass menschenwürdeverletzende und diskriminierende Werbeinhalte nicht mit dem Prinzip der sozialen Verantwortung übereinstimmen. Jedoch geht nicht hervor, welche konkreten Inhalte als menschenwürdeverletzend oder (geschlechter-)diskriminierend anzuerkennen sind. Aufgrund der Angaben in der Auslegungshilfe der ICC liegt die Vermutung nahe, dass die Bewertung, welche Inhalte menschenwürdeverletzend oder geschlechterdiskriminierend sind, nach nationalen Maßstäben erfolgen soll. Der Internationalen Handelskammer kommt durch den Erlass des ICCKodex bei der Setzung internationaler Standards im Gebiet der Werbeselbstregulierung eine wichtige Rolle zu. Die Grundsätze spiegeln allgemeingültige Vorstellungen über eine verantwortungsvolle Kommunikation wider. Aufgrund der sehr weiten Formulierungen dienen sie jedoch 626 Vgl. Henning-Bodewig, Ethics, Taste and Decency in Advertising, S. 67; siehe auch https://www.iccgermany.de/standards-incotermsr/verhaltensrichtlinien/we rbe-und-marketingstandards/; ebenso https://zaw.de/selbstregulierung/internatio nale-werbeselbstkontrolle/. 627 ICC-Kodex 2018, Grenzüberschreitende Kommunikation – Ursprung und Rechtsprechung, S. 5. 628 Ebenda, Auslegung, S. 6. 629 Ebenda. 151 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien vorrangig als „erster Anker“ und nicht „zur Konkretisierung von Begriffen wie Diskriminierung oder Anstand“.630 II. International Council for Ad Self-Regulation Der International Council for Ad Self-Regulation (ICAS; Internationaler Rat für Werbeselbstregulierung) setzt sich aus Werbeselbstregulierungsorganisationen verschiedener Staaten zusammen. Zu den europäischen Mitgliedern zählen beispielsweise die Werbeselbstregulierungsorganisationen aus Großbritannien, Spanien und Frankreich.631 Die Organisation agiert als globale Plattform und setzt sich für die Förderung der Werbeselbstregulierung ein.632 Dabei richtet sie sich maßgeblich nach den Standards des ICC-Kodexes. Der ICAS bestimmt, dass nationale Vorschriften in ihrer Formulierung, Auslegung und Anwendung flexibel genug sein sollten, um sich an neue Formen von Werbetechniken und -technologien anzupassen.633 Darüber hinaus bewertet er Werbeselbstregulierung als am effektivsten, wenn sie auf nationaler Ebene durchgeführt wird. So würden die Merkmale eines gegebenen Selbstregulierungssystems regelmäßig von lokalen Faktoren wie bestehenden kulturellen Traditionen, dem geschäftlichen Umfeld und den gesetzlichen Anforderungen abhängen.634 Eigene Standards oder Richtlinien gibt der ICAS nicht heraus. Damit ist die Organisation in erster Linie als Bündnis zur Erleichterung administrativer Aspekte der Werbeselbstregulierung anzuerkennen. Die Betonung lokaler Faktoren lässt, ähnlich wie der ICC-Kodex, darauf schließen, dass die Organisation vorrangig Strukturen zur Förderung nationaler Selbstregulierungsorganisationen schafft. Einen Einfluss auf die Regulierung geschlechterdiskriminierender Werbung in Europa hat sie demnach nicht. 630 Henning-Bodewig, Ethics, Taste and Decency in Advertising, S. 67. 631 Der Deutsche Werberat als deutsche Werbeselbstregulierungsorganisation ist kein Mitglied. 632 https://icas.global/about/. 633 https://icas.global/advertising-self-regulation/national-standards/. 634 https://icas.global/advertising-self-regulation/how-does-self-regulatory-organizati ons-work/. 152 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb C. Internationale Werbeselbstregulierung III. European Advertising Standards Alliance Eine weitere Organisation, die auf dem Gebiet der Werberegulierung auf untergesetzlicher Ebene tätig ist, ist die 1992 gegründete European Advertising Standards Alliance (EASA; Europäische Allianz der Werbeselbstkontrolle).635 Die EASA ist ein Zusammenschluss der europäischen Werbeselbstregulierungsorganisationen636 sowie weiterer in der Branche tätiger Unternehmen. Ihre Entstehung geht maßgeblich auf die Bemühungen des EU-Wettbewerbskommissars Sir Brittan zurück, der durch die Einführung des Binnenmarktes die Notwendigkeit eines grenzüberschreitenden Systems erkannte.637 Da die Werbeindustrie eine detaillierte Gesetzgebung ablehnte, forderte er sie auf, Wege zu finden, um die durch die Schaffung des Binnenmarktes aufgeworfenen Probleme durch Selbstregulierung zu lösen und so gesetzliche Vorgaben zu vermeiden.638 Beckmann begründete das Bedürfnis nach einer europäischen Werbeselbstregulierungsorganisation ebenfalls mit der Zunahme grenzüberschreitender Werbung, welcher nur mit einem europaweiten System der Werbeselbstkontrolle begegnet werden könnte.639 Ein Jahr später wurde die EASA gegründet. Auf einer Konferenz im Jahr 1997 würdigten Vertreter der Werbeindustrie die Fortschritte und Errungenschaften der EASA, und 1998 erkannte Sir Brittan an, dass die Arbeit der EASA „den wahrgenommenen Bedarf an gesetzgeberischen Eingriffen verringert“ hatte.640 635 www.easa-alliance.org. 636 Mitglieder sind alle Selbstregulierungsgremien der EU-Nationen ebenso wie weitere aus dem europäischen Raum und einige wenige aus dem nicht-europäischen Raum (https://www.easa-alliance.org/members/europe). 637 Latzer/Just/Sauerwein u.a, Selbst- und Ko-Regulierung im Mediamatiksektor, S. 88; Gray, Das funktionierende Cross-Border Complaints System der EASA, S. 93. 638 https://www.eesc.europa.eu/en/policies/policy-areas/enterprise/database-self-and -co-regulation-initiatives/88. 639 Beckmann, Werbeselbstdisziplin in Deutschland und Europa, WRP 1991, 702 (703). 640 https://www.eesc.europa.eu/en/policies/policy-areas/enterprise/database-self-and -co-regulation-initiatives/88. 153 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien Abb. 7: Werbeselbstregulierung in Europa Heute bildet die EASA ein Netzwerk aus 40 Organisationen.641 Darunter befinden sich 27 europäische Werbeselbstkontrollvereinigungen (Abb. 7) wie beispielsweise der Zentralverband der deutschen Werbewirtschaft, die britische Advertising Standards Authority und die französische Autorité de Régulation Professionnelle de la Publicité sowie 13 weitere Zusammenschlüsse aus dem Mediensektor.642 Ihr Tätigkeitsspektrum umfasst in erster Linie die Bearbeitung von grenzüberschreitenden Beschwerden („cross border complaints“). Dabei führt sie das Beschwerdeverfahren jedoch nicht selbst durch, sondern leitet die Beschwerde nach dem Herkunftslandprinzip an das Land weiter, aus dem die Werbung stammt.643 Zwar ist die Zahl an grenzüberschreitenden Beschwerden mit 332 im Jahr 2019 auf den ersten Blick noch sehr gering. Im Vergleich zum Vorjahr ist sie jedoch um siebzig Prozent angewachsen, wodurch ein steigender Trend zu 641 https://www.easa-alliance.org/about-easa/what-we-do. 642 So etwa Werbende, Werbeagenturen und Medienunternehmen. 643 Diese gewähren die Möglichkeit durch ein Beschwerdesystem gegen grenzüberschreitende Werbung vorzugehen. Das jeweilige zuständige nationale Gremium bewertet die Werbung sodann nach nationalen Standards. Vgl. Klein/Kirschner, Werberegulierung in Europa, S. 25; dazu auch Suhr, Europäische Presse-Selbstkontrolle, S. 24. 154 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb C. Internationale Werbeselbstregulierung erkennen ist.644 Geschlechterdiskriminierende Werbung macht circa acht Prozent der Beschwerden aus.645 Daneben fördert die EASA den Austausch zwischen den Werbeselbstkontrollorganisationen sowie die Durchsetzung der ethischen Werbestandards durch Pressearbeit und informiert zu gesetzlichen Vorschriften und Regelungen der Selbstkontrolleinrichtungen. Das Ziel der Organisation ist es, das allgemeine Bewusstsein für die Werbeselbstregulierung zu stärken.646 Der Erlass eines europäischen Kodexes oder eines gemeinsamen Bewertungsprozesses wurde bislang nicht angestrebt. Vielmehr spricht sich die EASA dafür aus, die Selbstregulierung allein nationalen Bestimmungen zu unterwerfen. Begründet wird dies mit staatenspezifischen Besonderheiten in Kultur, Recht und Wirtschaft.647 So würden die einzelnen nationalen Regelwerke, insbesondere die Struktur und das Verfahren, maßgeblich von zwei Faktoren abhängen. Zum einen würden sie von den Traditionen des Landes beeinflusst, also welche kulturellen, kommerziellen und rechtlichen Gegebenheiten berücksichtigt werden müssen. Zum anderen sei das nationale Verhältnis zwischen gesetzlicher Regulierung und Selbstregulierung entscheidend. Demnach könne sich Selbstregulierung nur so weit entwickeln, wie es die Gesetzeslandschaft zulassen würde. Demgegenüber sei die Ausformulierung der Kodizes in allen Staaten vergleichbar, was auch mit deren Orientierung an dem ICC-Kodex zusammenhängen würde. Ergänzend würden alle Mitgliedsorganisationen einen hohen Standard des Verbraucherschutzes gewährleisten. Dennoch gäbe es kein „gängiges Mittel“ für die Erreichung dieses Ziels, so dass die Allianz insgesamt für das Prinzip „Einheit durch Vielfalt“ stehe. Deswegen beschränkt sich die EASA ihren Angaben zufolge darauf, bei dem Aufbau nationaler Selbstre- 644 EASA Statistics Complaints report 2019, European Trends in Advertising Complaints, Copy Advice and Pre-Clearance, S. 3, 19. 645 EASA Annual Review 2020, S. 16. 646 Klein/Kirschner, Werberegulierung in Europa, S. 24 f. 647 In einem Bericht von 2019 wird darauf hingewiesen, dass die Beschwerden zu „anstößigen Inhalten“ maßgeblich von „lokalen Empfindlichkeiten“ und „kultureller Narrativen“ geprägt sind, die zwischen den Staaten oft erheblich divergieren (siehe EASA Statistics Complaints report 2019, European Trends in Advertising Complaints, Copy Advice and Pre-Clearance, S. 13). 155 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien gulierungsbehörden zu helfen und für einen übergreifenden Qualitätsstandard zu sorgen.648 Um die allgemeinen Qualitätsstandards zu erreichen, hat die EASA 2004 den Advertising Self-Regulation Charter (Charta der Selbstverantwortung) herausgegeben, in der sich die Mitglieder anhand bestimmter allgemeiner Grundsätze zur wirksamen Selbstregulierung verpflichten und angeben, das Vertrauen in verantwortungsvolle Werbung für Verbraucher:innen, Wettbewerber:innen und die Gesellschaft maximieren zu wollen. Weiterhin wollen sie aktiv für die höchsten ethischen Standards der Werbebranche eintreten, wofür sie in der Charta zehn Grundsätze formuliert haben. Darunter fallen umfassende und effektive Kodizes auf der Grundlage der ICC-Regeln, welche durch die Konsultation mit interessierten Parteien weiterentwickelt werden, ebenso wie die Einbeziehung von unabhängigen, nicht-staatlichen Laien in den Beschwerdebeurteilungsprozess. Vorgesehen sind außerdem die Bereitstellung von Beratungen und Schulungen für Fachleute der Branche, die die Standards erhöhen sollen, sowie die wirksame Sensibilisierung von Industrie und Verbraucher:innen für das Selbstregulierungssystem.649 Der Deutsche Werberat beschreibt die Vorgaben der Charta als „Empfehlungen für ein optimales Verfahren in der praktischen Arbeit der Werbeselbstkontrolle“.650 Geschlechterdarstellungen in der Werbung und geschlechterdiskriminierende Werbeinhalte wurden von der EASA und ihren Mitgliedern nur in Grundzügen thematisiert. Die Organisation verweist auf den Grundsatz, dass „Frauen und Männer in der Werbung positiv und verantwortungsbewusst dargestellt“ und die Darstellung der Geschlechter in der Werbung „mit Sorgfalt behandelt werden“ sollte. Dabei bezieht sie sich in erster Linie auf die Regeln der ICC, insbesondere auf Art. 2 des ICC-Kodex zur sozialen Verantwortung. Nach Angabe der EASA beinhalten alle europäischen Kodizes für die Werbepraxis den Wortlaut des Art. 2 oder eine vergleichbare Regel. Viele der Kodizes würden inhaltlich sogar noch weiter gehen, „indem sie spezifische Regeln für die Darstellung des Geschlechts enthalten“. Zuletzt betont die EASA erneut, dass die unterschiedlichen 648 Siehe Präambel, Advertising Self-Regulation Charter, EASA Summit, 2004 (https://www.easa-alliance.org/sites/default/files/SR_CHARTER_ENG_0.pdf); dazu auch Klein/Kirschner, Werberegulierung in Europa, S. 24. 649 Advertising Self-Regulation Charter, EASA Summit, 25.06.2004, S. 2 (https://w ww.easa-alliance.org/sites/default/files/SR_CHARTER_ENG_0.pdf); die zehn Grundsätze befinden sich zudem im Original im Anhang. 650 Deutscher Werberat, Jahrbuch 2021, S. 54. 156 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb C. Internationale Werbeselbstregulierung Regeln in den einzelnen Staaten die verschiedenen kulturellen Werte und sozialen Gepflogenheiten, die innerhalb der EU und in Europa bestehen, widerspiegeln würden.651 Auch ein Blick auf die aktuelle Werbepraxis spiegelt diese Ansicht zu Teilen wider. So vertreiben Unternehmen ein und dasselbe Produkt in den verschiedenen Mitgliedsstaaten häufig unter einem anderen Namen oder durch unterschiedliche Marketingstrategien, die genau auf den kulturellen Kontext abgestimmt sind. Die deutsche Speiseeis-Marke „Langnese“ wird etwa in Österreich unter dem Namen „Eskimo“, in Dänemark unter dem Namen „Frisko“ und in den USA und Kanada unter dem Namen „Good Humor“ geführt, um landessprachliche Aspekte besser einzubinden.652 Weiterhin löste beispielsweise eine Werbung der Firma Volkswagen aus dem Jahr 1998, die Leonardo da Vincis Abendmahl neu interpretierte, lebhafte Kontroversen in Frankreich aus (Abb. 8). Darin wurde eine Parodie des Gemäldes mit dem Slogan versehen: „Mes amis, réjouissons-nous car une nouvelle Golf est née.“ Etwa: „Meine Freunde, lasst uns jubeln, denn ein neuer Golf ist geboren.“ In einem Beitrag dazu heißt es, dass Volkswagen auf dem „harten Weg“ hätte erfahren müssen, dass ihre Kampagne völlig ungeeignet für den lokalen französischen Markt gewesen sei. Das Unternehmen hätte die „kulturellen Einstellungen und Denkweisen der Menschen vor Ort“ nicht hinreichend beachtet, so dass es sich schließlich vor Gericht gegen die französische Bischofskonferenz (CEF) hätte verteidigen müssen, die die Kampagne als empörende und unrechtmäßige Störung ihres katholischen Glaubens empfunden hatte.653 651 https://www.easa-alliance.org/issues/gender. 652 https://www.slogans.de/magazin/andere-laender-andere-marken-47. 653 https://www.lebeninfrankreich.info/werbung-in-frankreich-marketing-kommun ikation-und-kulturelle-unterschiede/; dazu ebenfalls https://www.protestantisme etimages.com/Zur-Verteidigung-verbotener-Bilder.html. 157 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien Abb. 8: Volkswagen, Frankreich, 1998 Lingenfelder spricht diesbezüglich von einem „Vorhandensein [...] interkultureller Divergenzen“ und einem „positiven [sowie] negativen Herkunftslandeffekt“.654 Danach müssten Werbetreibende sich der unterschiedlichen Bedeutungen etwa von „Zeichen, Farben, Symbolen und Wörtern“ bewusst sein und ihre Werbebotschaften gegebenenfalls national anpassen. Insbesondere Werbung mit Humor und Aussagen, die an das traditionelle Rollenverständnis von Männern und Frauen anknüpfen würden, würden in den verschiedenen Ländern Europas unterschiedlich bewertet werden.655 Ergänzend könnte somit vorgebracht werden, dass die Werbebranche nur begrenzt von einheitlichen Regelungen profitieren würde, da sie zwar keine nur auf spezielle Staaten ausgerichtete Werbung mehr schalten müsste, diese Vorgehensweise jedoch regelmäßig zu ihrem eigenen Vorteil nutzt. Obgleich es auch zunehmend Beispiele für Werbeinhalte gibt, die in mehreren Staaten in gleicher oder zumindest sehr ähnlicher Weise 654 Lingenfelder, Die Internationalisierung im europäischen Einzelhandel, S. 417. 655 Ebenda. 158 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb D. Zusammenfassung laufen,656 illustriert der Fall von Volkswagen, dass ein solches Vorgehen missglücken kann, wenn nationale und kulturelle Besonderheiten wie etwa religiöse Ansichten nicht hinreichend gewürdigt werden.657 Möglicherweise liegt darin ein weiterer Grund für den fehlenden Impetus der Werbebranche einen gemeinsamen europäischen Kodex auf Ebene der Selbstregulierung zu erlassen. Abschließend kann festgehalten werden, dass die EASA zwar einen wichtigen strukturellen Rahmen für die Werbeselbstregulierung in Europa bildet. Da ihr Arbeitsschwerpunkt in administrativen Tätigkeiten liegt, kommt ihr bei der Begrenzung geschlechterdiskriminierender Werbung jedoch bislang keine nennenswerte Rolle zu. D. Zusammenfassung Zusammenfassend hat die Materie geschlechterdiskriminierender Werbung auf internationaler ebenso wie auf europäischer Ebene bereits mehrfach Beachtung gefunden. Im Rahmen der Auseinandersetzung der beteiligten Organe mit den damit in Zusammenhang stehenden Problemfeldern wird weitestgehend anerkannt, dass gegen geschlechterdiskriminierende Werbebilder vorgegangen werden muss. So wird die diskriminierende Darstellung von Frauen und Männern in der Werbung als ein Hindernis auf dem Weg zur Geschlechtergleichstellung angesehen und auch die konkreten Auswirkungen bestimmter Werbebilder sind nach den Ausführungen der Vereinten Nationen, des Europarats und der EU-Organe nicht zu unterschätzen. Es werden nicht nur Darstellungen mit Gewalt oder Erniedrigung aufgeführt, sondern auch solche, die von stereotypen oder klischeehaften Geschlechtervorstellungen geprägt sind. Zumeist liegt der Fokus bezüglich der kritisierten Werbeinhalte auf dem Bild der Frau. Der vielfachen Würdigung und den teilweise sehr ausführlichen Beiträgen der EU-Organe steht ein Defizit an konkreten Regelungen und Vorgaben gegenüber. Die europäischen Bestimmungen bilden eher vage Leitlinien als einen verbindlichen Rechtsrahmen. So wird geschlechterdiskriminie- 656 Illustrativ siehe Carglass-Werbung aus Holland, Frankreich, Italien und England: https://www.seitvertreib.de/2010/08/17/wie-die-gleiche-werbung-in-4-lan dern-aussieht/; dazu siehe auch Bendt/Fantapié Altobelli/Sander, Internationales Marketingmanagement, S. 268. 657 Althans, Die Übertragbarkeit von Werbekonzeptionen auf internationale Märkte, S. 94, 104 f. 159 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb Kapitel 2: Europäische und internationale Leitlinien rende Werbung zwar als Problem hervorgehoben. Anstelle konkreter Maßnahmen und rechtsverbindlicher Richtlinien wird sich jedoch auf Feststellungen, Empfehlungen und Aufforderungen beschränkt, die jeweils an keine weiterführenden Konsequenzen geknüpft sind. Das dadurch geschaffene Grundgerüst kann damit allenfalls als Basis für nationale Individuallösungen einzelner Staaten gewertet werden, das den Mitgliedsstaaten einen weiten Spielraum bei dem Umgang mit den aufgeworfenen Thesen und den Zielsetzungen lässt. Einzig ein verstärkter Hinweis auf die Werbeselbstregulierung zieht sich wie ein roter Faden durch die unverbindlichen Rechtsakte. Ähnliches lässt sich für die CEDAW der Vereinten Nationen und die Ausführungen des Europarats anführen, welche zwar richtungsweisende Leitlinien schaffen, im Kern jedoch bislang nicht über symbolische Ansätze hinausgehen. Die Verbände der Werbeselbstregulierung wie die EASA bieten demgegenüber wichtige strukturelle Rahmenbedingungen. Die Bestimmungen der internationalen Werbeselbstregulierung wie der ICC-Marketing-Kodex oder der Advertising Self-Regulation Charter dienen als solide Grundlage für den Aufbau nationaler Werbeselbstregulierungsorganisationen und nationaler Verhaltenskodizes. Welche Geschlechterbilder als diskriminierend erachten werden können und welche Gegenmaßnahmen unternommen werden sollen, bleibt jedoch ebenfalls offen. Die Grundsätze, wann eine Geschlechterdiskriminierung vorliegt, müssen nach dem Vorbringen der Institutionen national unter Berücksichtigung der kulturellen Besonderheiten ausgefüllt werden. Ob insoweit Harmonisierungseffekte bestehen, erscheint somit zweifelhaft. Daraus folgend kann in Bezug auf das angestrebte Ziel eines europäischen Lösungsansatzes, dem im vierten Kapitel nachgegangen wird, bereits festgestellt werden, dass die Entwicklung konkreterer Richtlinien zur Werbeselbstregulierung in Anbetracht des weiten Spielraums gewinnbringend wäre, der den einzelnen Staaten sowohl von Seiten internationaler und europäischer Leitlinien als auch von der internationalen Werbeselbstregulierung bei der Definition geschlechterdiskriminierender Werbung und beim Erlass rechtsverbindlicher Maßnahmen oder unverbindlicher Empfehlungen und Verhaltenskodizes eingeräumt wird. Es könnten staatenübergreifende Grundstandards etabliert werden, ohne unverhältnismäßig in nationale, insbesondere kulturelle Charakteristiken einzugreifen. Nichtsdestoweniger dürfen die kulturellen Charakteristiken auch im Rahmen der Selbstregulierung nicht völlig außer Acht gelassen werden. So liegt die Vermutung nahe, dass sich konkretisierende Standards bislang noch nicht durchgesetzt haben, weil Werbung und Geschlechterrollen 160 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb D. Zusammenfassung stark von lokalen Kulturen geprägt werden, die die Etablierung gemeinsamer Grundsätze erschweren. Je mehr Staaten involviert sind, desto schwieriger wird es einen Grundkonsens zu finden. Eine ähnliche Problematik zeigt sich auf EU-Ebene, wo kulturelle Aspekte als Argument gegen eingriffsintensivere, insbesondere verbindliche, Maßnahmen zur Regulierung geschlechterdiskriminierender Werbung genannt werden. Hinzukommend ist seit den Nullerjahren eine rückläufige Tendenz auch bei den weichen Maßnahmen und Empfehlungen zu dem Thema festzustellen, die im Hinblick auf die in den letzten Jahren erstarkte Geschlechterdebatte und die Einflüsse von Bewegungen wie „#Metoo“ noch viel Raum für weiterführende Studien lässt. Abschließend ist – trotz der zu beobachtenden Entwicklungen – festzuhalten, dass die Problematik geschlechterdiskriminierender Werbung bereits vielfach auf internationaler und europäischer Ebene adressiert wurde. Gleichwohl konnten keine klaren Vorgaben für die Regulierung geschlechterdiskriminierender Wirtschaftswerbung, insbesondere für konkrete Geschlechterbilder in der Werbung, vorgefunden werden. Während dies im Rahmen von internationalen Konventionen auf die Größe und die immense kulturelle Vielfalt der Mitgliedsstaaten zurückgeführt werden kann, spielen bei der EU mit hoher Wahrscheinlichkeit kompetenzrechtliche Herausforderungen eine wichtige Rolle. Im Rahmen der internationalen Werbeselbstregulierung könnten ebenfalls kulturelle Erwägungen verantwortlich für das Ausbleiben weiterführender Maßnahmen sein. Inwieweit sich die Anerkennung des Problems auf internationaler und europäischer Ebene in den einzelnen Staaten im Rahmen ihrer Regulierungspraxis niederschlägt, kann bei der sich anschließenden Erforschung der nationalen Lösungsansätze aufgrund des weiten Anwendungsspielraums und der Auslegungsoffenheit der Vorgaben nur begrenzt beantwortet werden. So ist es ohne eine ausdrückliche Bezugnahme auf eine bestehende internationale oder europäische Empfehlung nur schwer möglich eine konkrete nationale Maßnahme mit dieser in Verbindung zu bringen. Gleichwohl wird das folgende Kapitel zeigen, dass sich die verstärkte Förderung der Werbeselbstregulierung ebenso wie die durch die EASA und durch die ICC geschaffenen strukturellen Grundlagen in der nationalen Regulierungspraxis in Bezug auf geschlechterdiskriminierende Wirtschaftswerbung widerspiegeln. 161 https://doi.org/10.5771/9783748913177-111, am 29.06.2024, 13:50:25 Open Access – - https://www.nomos-elibrary.de/agb
https://openalex.org/W2922471166	https://escholarship.org/content/qt9054v8vm/qt9054v8vm.pdf?t=qankkw	English	null	Author Correction: Strong indirect coupling between graphene-based mechanical resonators via a phonon cavity	Nature communications	2,019	cc-by	972	UC Merced UC Merced Previously Published Works UC Merced UC Merced Previously Published Works Title Author Correction: Strong indirect coupling between graphene-based mechanical resonators via a phonon cavity Permalink https://escholarship.org/uc/item/9054v8vm Journal Nature Communications, 10(1) ISSN 2041-1723 Authors Luo, Gang Zhang, Zhuo-Zhi Deng, Guang-Wei et al. Publication Date 2019 DOI 10.1038/s41467-019-09240-8 Peer reviewed UC Merced UC Merced Previously Published Works Title Author Correction: Strong indirect coupling between graphene-based mechanical resonators via a phonon cavity Permalink https://escholarship.org/uc/item/9054v8vm Journal Nature Communications, 10(1) ISSN 2041-1723 Authors Luo, Gang Zhang, Zhuo-Zhi Deng, Guang-Wei et al. Publication Date 2019 DOI 10.1038/s41467-019-09240-8 Peer reviewed UC Merced UC Merced Previously Published Works Title Author Correction: Strong indirect coupling between grap resonators via a phonon cavity Permalink https://escholarship.org/uc/item/9054v8vm Journal Nature Communications, 10(1) ISSN 2041-1723 Authors Luo, Gang Zhang, Zhuo-Zhi Deng, Guang-Wei et al. Publication Date 2019 DOI 10.1038/s41467-019-09240-8 Peer reviewed UC Merced UC Merced Previously Published Title Author Correction: Strong indirect coupling be resonators via a phonon cavity Permalink https://escholarship.org/uc/item/9054v8vm Journal Nature Communications, 10(1) ISSN 2041-1723 Authors Luo, Gang Zhang, Zhuo-Zhi Deng, Guang-Wei et al. Publication Date 2019 DOI 10.1038/s41467-019-09240-8 Peer reviewed Title Title Author Correction: Strong indirect coupling between graphene-based mechanical resonators via a phonon cavity 1 CAS Key Laboratory of Quantum Information, University of Science and Technology of China, Hefei, 230026 Anhui, China. 2 Synergetic Innovation Center of Quantum Information and Quantum Physics, University of Science and Technology of China, Hefei, 230026 Anhui, China. 3 School of Nature Sciences, University of California, Merced, CA 95343, USA. These authors contributed equally: Gang Luo and Zhuo-Zhi Zhang. Correspondence and requests for materials should be addressed to G.-W.D. (email: gwdeng@ustc.edu.cn) or to L.T. (email: ltian@ucmerced.edu) or to G.-P.G. (email: gpguo@ustc.edu.cn) Correction to: Nature Communications https://doi.org/10.1038/s41467-018-02854-4; published onli The original version of this Article contained an error in the last sentence of the second paragraph of the ‘Raman-like coupling between well-separated resonators’ section of the Results, which incorrectly read ‘On the contrary, when the detuning Δ12 is lowered to ~180 kHz, a distinct avoided level crossing between modes R1 and R3 is observed, as shown inside the dashed circle in Fig. 2b.’ The correct version states ‘Δ12/2π’ in place of ‘Δ12.’ Additionally, the ﬁrst sentence of the ‘Theory of three-mode coupling’ section of the Methods originally incorrectly read ‘We describe this three-mode system with the Hamiltonian (ħ = 4).’ The correct version states ‘ħ = 1’ instead of ‘ħ = 4’. Equation 8 was originally missing a factor of ‘a2’ from the second term, and incorrectly read: aΔ ± ¼ ðΩ12a1 ± ωΔ0 Δ ð Þ þ Ω23a3Þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2ωΔ0ðωΔ0 ΔÞ p : aΔ ± ¼ ðΩ12a1 ± ωΔ0 Δ ð Þ þ Ω23a3Þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2ωΔ0ðωΔ0 ΔÞ p : The correct form of Equation 8 is: aΔ ± ¼ ðΩ12a1 ± ωΔ0 Δ ð Þa2 þ Ω23a3Þ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2ωΔ0ðωΔ0 ΔÞ p : The eleventh and twelfth sentences of the second paragraph of the ‘Theory of three-mode coupling’ section of the Methods originally incorrectly read ‘The nearly degenerate modes can be viewed as a hybridization of α1 and α3 with an effective coupling Ω13 αy 1α3 þ αy 3α1 =2. The magnitude of this effective coupling is equal to the frequency splitting between the nearly degenerate modes, with Ω3 ¼ ðΩ2 12 þ Ω2 23Þ=2Δ.’ These sentences have been replaced with ‘The nearly degenerate modes can be viewed as a hybridization of α1 and α3 with an effective splitting ðΩ2 12 þ Ω2 23Þ=2Δ.’ In Equation 10, the term on the left-hand side was originally incorrectly given as ‘M’. The corrected v he left-hand side was originally incorrectly given as ‘M’. The corrected version changes this to ‘Meff’. Finally, the original version of this Article contained an error in Fig. 3b, in which the units on the y-axis were incorrectly given as ‘MHz’, rather than the correct ‘kHz’. This has been corrected in both the PDF and HTML versions of the Article. 1 CAS Key Laboratory of Quantum Information, University of Science and Technology of China, Hefei, 230026 Anhui, China. Author Correction: Strong indirect coupling between graphene-based mechanical resonators via a phonon cavity Gang Luo1,2, Zhuo-Zhi Zhang 1,2, Guang-Wei Deng 1,2, Hai-Ou Li1,2, Gang Cao1,2, Ming Xiao1,2, Guang-Can Guo1,2, Lin Tian 3 & Guo-Ping Guo 1,2 Powered by the California Digital Library University of California Powered by the California Digital Library University of California eScholarship.org Correction to: Nature Communications https://doi.org/10.1038/s41467-018-02854-4; published onli 2 Synergetic Innovation Center of Quantum Information and Quantum Physics, University of Science and Technology of China, Hefei, 230026 Anhui, China. 3 School of Nature Sciences, University of California, Merced, CA 95343, USA. These authors contributed equally: Gang Luo and Zhuo-Zhi Zhang. Correspondence and requests for materials should be addressed to G.-W.D. (email: gwdeng@ustc.edu.cn) or to L.T. (email: ltian@ucmerced.edu) or to G.-P.G. (email: gpguo@ustc.edu.cn) NATURE COMMUNICATIONS \| (2019) 10:1343 \| https://doi.org/10.1038/s41467-019-09240-8 \| www.nature.com/naturecommunications 1 © The Author(s) 2019 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-09240-8 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/. © The Author(s) 2019 NATURE COMMUNICATIONS \| (2019) 10:1343 \| https://doi.org/10.1038/s41467-019-09240-8 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| (2019) 10:1343 \| https://doi.org/10.1038/s41467-019-09240-8 \| www.nature.com/naturecommunicatio 2
https://openalex.org/W4386412105	https://www.bio-conferences.org/articles/bioconf/pdf/2023/10/bioconf_ebwff2023_10009.pdf	English	null	Factors development of teachers’ competence in improving the quality of education	Bio web of conferences/BIO web of conferences	2,023	cc-by	3,734	* Corresponding author: Fayzieyva1936@mail.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). Shakhnoza Iskandarova1 , Mukhabbat Fayziyeva1, Gulnara Asadova1 , Xilola Mursaidova1, Akram Sadivakasov1 and Durdona Mustafoyeva2 1Tashkent Pediatric Medical Institute, Tashkent, Uzbekistan 2Tashkent Institute of Irrigation and Agricultural Mechanization Engineers” National Research University, Tashkent, Uzbekistan 1Tashkent Pediatric Medical Institute, Tashkent, Uzbekistan 2Tashkent Institute of Irrigation and Agricultural Mechanization Engineers” National Research University, Tashkent, Uzbekistan Abstract. Modern strategic changes in the socio-economic and political spheres of education, which have reduced the standard of living in our country, pose new challenges to the higher school teacher. They include concepts such as the need to master the structure of a new subject in education, ongoing changes and the acquisition of innovative technologies and methods of education that take into account the activity and independence of education recipients. BIO Web of Conferences 65, 10009 (2023) EBWFF 2023 BIO Web of Conferences 65, 10009 (2023) EBWFF 2023 https://doi.org/10.1051/bioconf/20236510009 1 Introduction Changing the sphere of knowledge implies the acquisition of knowledge in the field of modern industrial and social technologies, the development of independent solutions, as well as the ability to predict the consequences of their occurrence, the ability to cooperate and influence the composition and nature of pedagogical activity with professional qualities. In this regard, the relentless search for teachers on their own has become an urgent requirement. It is necessary to evaluate the work on the development of the teacher's potential, the continuous professional conflicts of the teacher's position, the knowledge of new ministers in professional denominations, students and other persons with knowledge and professional competencies, as well as the renewal of the training of teaching staff and the implementation of strategic directions for the development of modern education. g p It is necessary to evaluate the work on the development of the teacher's potential, the continuous professional conflicts of the teacher's position, the knowledge of new ministers in professional denominations, students and other persons with knowledge and professional competencies, as well as the renewal of the training of teaching staff and the implementation of strategic directions for the development of modern education. G.K.Britell, R.M.Jueger, W.E.Blahk, N.Muslimov[1], A.Abdugadirov, K.T.Ummatalieva, M.Urazova and in the scientific research of others, relevant conclusions were formed, which were studied and based on the scope of the study [2-7]. In particular, German scientists who conducted research on the professional competence of teaching staff of higher educational institutions G. Lehmann, W. Nieke, S Blomeke, A Kopka and Brindt assess competence as a necessary unit of professional knowledge, skills https://doi.org/10.1051/bioconf/20236510009 BIO Web of Conferences 65, 10009 (2023) EBWFF 2023 and professional ethics, recognizing social analysis, diagnosis of pedagogical situations, reflection and professional action as its main components. and professional ethics, recognizing social analysis, diagnosis of pedagogical situations, reflection and professional action as its main components. 1 Introduction Stages of competence development Socio- synergetic criterion Acmeological- value criterion Reflexive criterion (reflexive-motivational) - the activity of personal views; as a source of the mechanism of development of the educational process, it manifests itself as the principle of "liberation + visualization"; empathy, acceptance, creativity and the ability to reflect a system of measures reflecting the development of education; organizational and structural consistency and the teacher-teacher tradition for reforming the system; pedagogical fascia through system-synergetic interaction of the audience orientation towards achieving the highest level of professional and pedagogical activity; increased attention to universal values; reflection of the values of poverty and debt in educational activities, activation of the influence of acmeological views on the process of self- awareness Оrganizational and economic mechanism rating system for evaluating the activity of a teacher in the field of special sciences of higher education; proposals for additional expenses for monthly wages; implementation of author's professional development programs Development of innovative activity in the process of professional development : motivation and synergy of reflection in advanced training courses, that is, activation of the necessary motivations; facilitate the repetition of the reflex; increase activity in the content of pedagogical activity Socio- synergetic criterion Acmeological- value criterion orientation towards achieving the highest level of professional and pedagogical activity; increased attention to universal values; reflection of the values of poverty and debt in educational activities, activation of the influence of acmeological views on the process of self- awareness orientation towards achieving the highest level of professional and pedagogical activity; increased attention to universal values; reflection of the values of poverty and debt in educational activities, activation of the influence of acmeological views on the process of self- awareness (reflexive-motivational) - the activity of personal views; as a source of the mechanism of development of the educational process, it manifests itself as the principle of "liberation + visualization"; empathy, acceptance, creativity and the ability to reflect a system of measures reflecting the development of education; organizational and structural consistency and the teacher-teacher tradition for reforming the system; pedagogical fascia through system-synergetic interaction of the audience Оrganizational and economic mechanism Development of innovative activity in the process of professional development : motivation and synergy of reflection in advanced training courses, that is, activation of the necessary motivations; facilitate the repetition of the reflex; increase activity in the content of pedagogical activity Fig. 1. 1 Introduction In particular, according to researchers, the professional competence of a teacher consi in his: In particular, according to researchers, the professional competence of a teacher consists in his: - possess special scientific and theoretical knowledge in the field of science and educati - possess special scientific and theoretical knowledge in the field of science and education, as well as the ability to effectively solve tasks; - possess special scientific and theoretical knowledge in the field of science and educati as well as the ability to effectively solve tasks; as well as the ability to effectively solve tasks; - to find an effective solution to specific pedagogical situations arising from the interests of teachers on the basis of special professional ethics; - to find an effective solution to specific pedagogical situations arising from the interests of teachers on the basis of special professional ethics; - for each pedagogical act in the performance of the above professional duties is determined by a sense of responsibility and responsibility to each person [8]. - for each pedagogical act in the performance of the above professional duties is determined by a sense of responsibility and responsibility to each person [8]. It is known that if the development of basic knowledge, skills and abilities in future types of professional activity occupies an important place in the professional development of a specialist, then the improvement of activities, the quality and level of its implementation among teaching staff engaged in real practical activities are considered important factors. Because in these processes, deep knowledge is defined as complete knowledge of one's own work, the essence of the work performed, the dependencies, relationships existing in it, means and methods of achieving the goals set. The idea of having deep knowledge can include the skills of an individual to move freely in difficult professional conditions, manage their subject and object components, introduce new methods of activity, technologies. This, in turn, allows us to ensure the highest level of social and professional effectiveness of teaching staff. Here are the stages of competence development we have reflected in Figure 1. 2 2 BIO Web of Conferences 65, 10009 (2023) EBWFF 2023 https://doi.org/10.1051/bioconf/20236510009 Fig. 1. 1 Introduction Stages of competence development p p g The development of competencies is manifested and develops in such manifestations as pedagogical and scientific activity of the teacher, universal values in the team, personal reflection, self-awareness and self-esteem, taking into account reality, real circumstances and changes in the process. Social-synergetic criteria are developed on the basis of the systemic- synergetic significance of the audience through a purposeful pedagogical hobby. This criterion includes the goal of the level of educational activity, the gradual development of education and the formation of a competent specialist through the principle of adherence to the traditions of the teacher, the development of integrative and educational activities through the regular use of interactive methods and training. In addition, the specialty is an indicator not only of the professional competence of teachers of natural sciences, but also of the control and development of the quality of pedagogical knowledge The acmeological criterion provides active access to various educational activities that are socially motivated and increase the creative activity of the individual, this criterion includes such concepts as acceptance of existence, systematic and directed thinking, the influence of actual creative activity on intellectual reflex, orientation of education to personality and systematic increase in educational innovation [9]. The reflexive criterion is determined by the content of the activity with an approach to personality, which ensures an improvement in the quality of the educational process in the process of professional 3 BIO Web of Conferences 65, 10009 (2023) EBWFF 2023 https://doi.org/10.1051/bioconf/20236510009 development; provides convenience and variability of educational programs in accordance with the needs of the audience; skills of self-awareness and activation in the development of personality, traditional practical assistance in the development of personal and professional potential through these laws. development; provides convenience and variability of educational programs in accordance with the needs of the audience; skills of self-awareness and activation in the development of personality, traditional practical assistance in the development of personal and professional potential through these laws. The reflexive criterion is determined by the content of the activity with an approach to personality, which ensures an improvement in the quality of the educational process in the process of professional development; provides convenience and variability of educational programs in accordance with the needs of the audience; skills of self-awareness and activation in the development of personality, traditional practical assistance in the development of personal and professional potential through these laws [10]. 2 Methods As part of the development of qualifications and skills, the concept of "competence" entered the educational sphere as a result of psychological research. Therefore, competence means "having a plan of action in non-standard situations, the expert himself in unexpected situations, entering into communication, a new way of interacting with competitors, performing ambiguous tasks, using information full of contradictions, consistently developing and complex processes." Also, the competence is the continuous enrichment of special knowledge, the study of new information, the understanding of specific social requirements, the search for new information, their processing and analysis It is necessary to clarify beforehand the changes that have occurred in the qualifications system, which has a high professional level and is found and recognized ready to work at this level, for teaching staff, to the extent that the problems that have arisen should be eliminated. Of course, in order for a teacher to be mature, such concepts as professional, pedagogical and technological competence should be reflected in it, we will consider them in turn. Competence is a set of knowledge, skills, qualifications, views on the field, values and qualities of a person, the ability to show qualifications or influence it. Knowledge of the types of competence in pedagogy and psychology; self-improvement; ability to plan, evaluate and restore communication in the educational process; formation of motivation among students; knowledge of ICT; innovation in the educational environment; excellent knowledge of their own science. Personal competence includes such concepts as: communication; tolerance; leadership; activity and initiative; flexibility; commitment to a healthy lifestyle; responsiveness; skill; humanism [11]. 4 BIO Web of Conferences 65, 10009 (2023) EBWFF 2023 https://doi.org/10.1051/bioconf/20236510009 BIO Web of Conferences 65, 10009 (2023) EBWFF 2023 https://doi.org/10.1051/bioconf/20236510009 management of the learning environment and the learning process in the classroom, the planning of the coverage of the content of the subject, the assessment of the achievements and development of students, motivation and assistance to students, as well as the development of students' knowledge. management of the learning environment and the learning process in the classroom, the planning of the coverage of the content of the subject, the assessment of the achievements and development of students, motivation and assistance to students, as well as the development of students' knowledge. The teaching skills of another teacher are determined by the following requirements:: Professional dignity and erudition; * Psychological and pedagogical training; * Psychological and pedagogical training; * The ability to teach teachers independent thinking and the acquisition of new knowledg * The ability to teach teachers independent * Knowledge of forms and types of educational literature; * Mastering new pedagogical and information technologies, Thus, within the framework of personality-oriented methodological work aimed at the development of pedagogical activity and the personality of teachers, as well as at improving the professional competence of lyceum teachers, a proactive approach was developed in the organization of the use of pedagogical technologies had a dual purpose: g p g g g p p 1. Correctional: identification and elimination of difficulties in the pedagogical activity of specific teachers based on the development of pedagogical technologies. 1. Correctional: identification and elimination of difficulties in the pedagogical activity of specific teachers based on the development of pedagogical technologies. 2. Prognostic, carried out in two directions: the identification of new difficulties of teachers in the organization of theoretical training and educational and production activities and the selection of appropriate technologies to eliminate them; the choice of undeveloped skills, skills and competencies of students and the selection of technologies that form them [12-13]. 2. Prognostic, carried out in two directions: the identification of new difficulties of teachers in the organization of theoretical training and educational and production activities and the selection of appropriate technologies to eliminate them; the choice of undeveloped skills, skills and competencies of students and the selection of technologies that form them [12-13]. BIO Web of Conferences 65, 10009 (2023) EBWFF 2023 Such gradual development of pedagogical technologies, their systematization, from the point of view of conscious application, is the basis for the formation of the readiness of the teaching staff and each teacher to transition from the traditional organization of the educational process to competence-based learning. Strengthening, improving and expanding the sphere of education at the modern stage it is necessary to include the country in the list of priority areas of development of the educational system, the reason for which it is the most basic condition for overcoming persistent situations in society, taking into account the economy, culture and science Modeling of the professional development process can be considered optimal if the work is carried out in the following areas: • the target table highlights a number of skills, skills, and competencies that teachers can form with the same technology, and we plan to implement it in this cycle; • the target table highlights a number of skills, skills, and competencies that teachers can form with the same technology, and we plan to implement it in this cycle; • information support of the educational process begins with the author's programs, which are gradually developed on the basis of content modeling technology and modular technologies. The program determines the number of hours needed to study each module. The allocation of hours requires separate testing, because it can be adequate or inadequate to the goals set, and is determined by the complexity of competence and the effectiveness of the applied pedagogical technology. Teachers are engaged in the creation of an electronic textbook, a methodological guide on the subject; workbooks, develop training exercises and other forms that ensure the formation of students' developed competence; for the transition to competence-based education, correction and development of pedagogical activity and the personality of the teacher is necessary, which requires clear step-by-step planning, a personality-oriented approach and the organization of monitoring; • to determine the effectiveness of teachers' professional development, monitoring tools are being developed to determine at the stage of entrance, current, and final control, the dynamics of the development of professional competence, pedagogical activity, the personality of the teacher;. • for the organization of a personality-oriented approach to the professional development of teachers and the implementation of the activity principle in its organization, it is necessary to choose a set of methods and forms, training sessions for the development of pedagogical technologies. 3 Results and discussion As for special competence, such qualities mean knowledge of special methods of a teacher in their subject; stratification of education; knowledge of the needs of students; knowledge of the characteristics of different ages. The concept of didactic competence includes the 5 BIO Web of Conferences 65, 10009 (2023) EBWFF 2023 BIO Web of Conferences 65, 10009 (2023) EBWFF 2023 The activity of a teacher in the monitoring mode has a cyclical nature, is carried 6 BIO Web of Conferences 65, 10009 (2023) EBWFF 2023 https://doi.org/10.1051/bioconf/20236510009 out strictly in stages with regular monitoring of completed tasks and mastered types of pedagogical activity competencies are a set of tasks that a specialist must solve, and competence is a set of competencies that he must possess. Competence is a kind of integrative complex characteristic of a personality, its ability to solve specific practical problems arising in various spheres of life. The importance of the competence approach is that it involves the transition from the assessment of knowledge to the assessment of competencies. Thus, the theoretical substantiation of the features of the development of cultural competence of teachers in the system of advanced training is based on the following approaches: systemic, activity-based, personality-oriented [14]. out strictly in stages with regular monitoring of completed tasks and mastered types of pedagogical activity competencies are a set of tasks that a specialist must solve, and competence is a set of competencies that he must possess. Competence is a kind of integrative complex characteristic of a personality, its ability to solve specific practical problems arising in various spheres of life. The importance of the competence approach is that it involves the transition from the assessment of knowledge to the assessment of competencies. Thus, the theoretical substantiation of the features of the development of cultural competence of teachers in the system of advanced training is based on the following approaches: systemic, activity-based, personality-oriented [14]. In order to reveal the features of the development of cultural competence of teachers in the system of professional development, the most significant is the cultural approach, which consists in considering, along with the competence approach, that the basic factor of professional and personal development of teachers in the "lifelong education" model is the formation of their own professional and general personal competence. In this regard, in the model of development of professional competence of teachers of specialized sciences, we have divided the following components: purposeful, meaningful and productive components. 1. The purpose of the target component is to develop the professional competence of teachers of specialized sciences, which includes goals and objectives. 4 Conclusion The main goal is to develop the professional competence of teachers of specialized sciences in advanced training courses. And the main tasks were identified as follows: analysis of the development of professional competence and the definition of pedagogical conditions, analysis of the current state of advanced training courses and the current state of the theoretical and methodological foundations of the Bund and modern approaches, as well as clarification and justification of pedagogical conditions; development of an improved methodology for improving educational and methodological support. In conclusion, in order to achieve the necessary level of professionalism in the field of modern society, it is necessary to have a continuous professional base. This gives a continuous effect for the professional growth of a modern teacher, which includes such concepts as professional, personal competence. The implementation of the competence approach is a set of actions through which the formation of competencies is carried out during the learning process, their assessment: the formation of competencies by modules; the level differentiation of students' competencies and their presentation in the form of a competence schedule; the definition of all types of training sessions (both classroom and independent), on the basis of which competencies are supposed to be formed; the construction and organization of the educational process focused on the formation of competencies; building competency assessment systems and their components, including the development of assessment methods and procedures, the choice of means for documenting learning outcomes. 3. N. N. Azizkho'jaeva, Pedagogical technology and pedagogical skills, Tashkent, 432 (2003) References 1. Decree of the President of the Republic of Uzbekistan "On the Strategy of further development of the Republic of Uzbekistan", Collection of Legislation of the Republic of Uzbekistan, Tashkent, 39 (2017) 1. Decree of the President of the Republic of Uzbekistan "On the Strategy of further development of the Republic of Uzbekistan", Collection of Legislation of the Republic of Uzbekistan, Tashkent, 39 (2017) 2. I. A. Zimnyaya, Pedagogical psychology, Mocow, Logos, 382 (2000) 2. I. A. Zimnyaya, Pedagogical psychology, Mocow, Logos, 382 (2000) 3. N. N. Azizkho'jaeva, Pedagogical technology and pedagogical skills, Tashkent, 432 (2003) 7 BIO Web of Conferences 65, 10009 (2023) EBWFF 2023 https://doi.org/10.1051/bioconf/20236510009 4. B. L. Farberman, Advanced pedagogical technologies, Tashkent Own.R.F.A. Science, 128 (2000) 4. B. L. Farberman, Advanced pedagogical technologies, Tashkent Own.R.F.A. Science, 128 (2000) 5. J. G'. Yoldoshev, Training is a factor in education, Teacher, 136 (1993) 6. I. Z. Karabaevna, K. D. Omonovich, K. N. Murodillaevich, S. U. Normuminovna, A. O. Mahmatqulovich, Formation of a system of methods of technical thinking future engineers. J. of Critical Rev., 787-794 (2020) 7. O. V. Panferova, Features of the individuality of the child and adaptation to the school: avtoref. dis. cand. psychol. nauk, Moscow, 26 (1996) 8. S. A. Boltaboev, M. M. Ismailova, Course work on the methodology of vocational education. (methodical guide), Tashkent TDPU named after Nizami, (2001) 9. K. Davlatov, Practical training from the theory and methodology of Labor and vocational education, upbringing and choice of profession: revenge for the students of the Pedagogical Institute, Tashkent, "Teacher" (1995) 10. N. A. Muslimov, Professional formation of teachers of future vocational education. Monographs, Tashkent: Science (2004) 11. N. A. Muslimov, Some problems of vocational training teachers training J. Pedagogical education (2002) 12. Z. Karabaevna, A. Musurmanova, R. Xamroevich, Improving the competence of future vocational education teachers based on modular-rating education. International Journal of Engineering and Advanced Technology, 9(1), 6903–6906 (2019) 13. A. Isakov, B. Tukhtamishev, R. Choriev, IOP Conference Series: Earth and Environmental Science, 614(1), 012006 (2020) 14. O. Glovatskii, J. Rashidov, В. Kholbutaev, K. Tuychiev, Achieving reliability and energy savings in operate of pumping stations. In E3S Web of Conferences, EDP Sciences, 264 (2021) 8 8
https://openalex.org/W2897925958	https://inria.hal.science/hal-02274145/document	English	null	Learning Time Analysis - Case Study in the IT Sector in the Czech Republic	Lecture notes in computer science	2,018	cc-by	4,010	To cite this version: Vaclav Zubr, Hana Mohelska. Learning Time Analysis - Case Study in the IT Sector in the Czech Republic. 17th Conference on e-Business, e-Services and e-Society (I3E), Oct 2018, Kuwait City, Kuwait. pp.21-29, 10.1007/978-3-030-02131-3_3. hal-02274145 Learning Time Analysis - Case Study in the IT Sector in the Czech Republic Vaclav Zubr, Hana Mohelska To cite this version: Vaclav Zubr, Hana Mohelska. Learning Time Analysis - Case Study in the IT Sector in the Czech Republic. 17th Conference on e-Business, e-Services and e-Society (I3E), Oct 2018, Kuwait City, Kuwait. pp.21-29, 10.1007/978-3-030-02131-3_3. hal-02274145 Distributed under a Creative Commons Attribution 4.0 International License adfa, p. 1, 2011. © Springer-Verlag Berlin Heidelberg 2011 Learning Time Analysis - Case Study in the IT Sector in the Czech Republic Vaclav Zubr1[0000-0002-4049-9999] and Hana Mohelska1[0000-0003-0441-0712] 1 The University of Hradec Kralove, Faculty of Informatics and Management, Hradec Kralove, Czech Republic vaclav.zubr@uhk.cz Abstract. For learning organisations, acquiring knowledge is one of the key ac- tivities. Then for example, the acquired knowledge will allow organisations greater flexibility or a strategic advantage. As small and medium-sized organi- sations in the Czech Republic are of great importance from the employment of people viewpoint, this survey is focused on education in these organisations. The aim of this study is to evaluate learning time in small and medium-sized organizations in the Czech Republic and to compare the learning time of gen- eral staff and managers in these organizations. When comparing results with foreign studies, it can be argued that the results obtained correlate with each other and are satisfactory. In this study, there was a statistically significant dif- ference found between people who are learning at least 1-10 hours per month and those who are not. At the same time, the positive influence of learning on the evaluation of some dimensions was found. Keywords: IT sector, learning time, small and medium-sized organizations, general staff learning, managers´ learning Learning Time Analysis - Case Study in the IT Sector in the Czech Republic Vaclav Zubr1[0000-0002-4049-9999] and Hana Mohelska1[0000-0003-0441-0712] 1 The University of Hradec Kralove, Faculty of Informatics and Management, Hradec Kralove, Czech Republic vaclav.zubr@uhk.cz Learning Time Analysis - Case Study in the IT Sector in the Czech Republic Vaclav Zubr1[0000-0002-4049-9999] and Hana Mohelska1[0000-0003-0441-0712] 1 The University of Hradec Kralove, Faculty of Informatics and Management, Hradec Kralove, Czech Republic vaclav.zubr@uhk.cz HAL Id: hal-02274145 https://inria.hal.science/hal-02274145v1 Submitted on 29 Aug 2019 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1.1 Small and medium-sized organizations in the Czech Republic The importance of small and medium-sized organizations in the Czech Republic is relatively high given the high percentage of people they employ (more than 70% of employees in the private sector) [5]. Small and medium-sized organizations are defined as organizations that employ up to 250 people. In detail, small and medium-sized organizations can be divided into small enter-prises (also micro-companies) with 1 to 9 employees, small organizations with 10 to 49 employees and medium organizations employ between 50 and 250 peo- ple [4, 5]. 1 Introduction 1 The definition of a "learning organisation" has been described by several authors over the years. For example, a learning organisation is defined in the book of The Fifth Discipline by Peter Senge as: "... an organisation whereby people continually improve their abilities and achieve the results they truly desire where they find support, new and dynamic models of thinking where collective thinking and inspiration are very welcome, and where people still learn how to learn." [13] From other sources, a learning organisation can be characterised as an organisation that acquires knowledge and innovates fast enough to survive and prosper in a rapidly changing environment, supports continuous employee education, critical thinking as well as risk-taking in the application of new ideas, as well as the dissemination of new knowledge for an organisation in order to incorporate them into day-to-day activities [3]. Learning then becomes an integral part of the whole work process. Work and learning are interconnected in the process of continual improvement. A learning or- ganisation doesn’t rely on learning as a by-product of routine work but is actively supported, facilitated and rewarded. Interaction between individuals is then a key aspect of organisational learning [17]. According to several studies, more factors are involved in good functioning of the learning organisation concept (management, learning communities, inner compliance, empowering individuals, organising culture, self-development, teamwork, sharing information, creating knowledge, building reliable learning dimensions and innova- tion or facilitating leadership) [19]. Learning is one of the basic activities for the learning organisation concept and can be carried out at individual, group or organisa- tional levels [18]. Organisational learning is the result of an interactive and interdependent process. This type of learning is based on organisational memory (past knowledge and experi- ence) and is carried out through common knowledge and mental models of individual company members. Individuals and groups in the organisation are articles through which organisational learning takes place [8, 9]. 1.2 Measurement of learning organisation We can use a large number of tools to measure and diagnose learning organisations. The used tool depends on the different definitions of the learning organisation. The definition of learning organisation by Marsick and Watkins [15] is also one of these tools. Table 1. Seven learning organisation dimensions No of Dimension Name 1 Create continuous learning opportunities 2 Promote inquiry and dialogue 3 Encourage collaboration and team learning 4 Create systems to capture and share learning 5 Empower people toward a collective vision 6 Connect the organisation to its environment 7 Provide strategic leadership for learning Source: own processing by [7, 9] Table 1. Seven learning organisation dimensions According to Marsick and Watkins, there are seven dimensions that characterise the learning organisation culture. Individual dimensions then represent the efforts of organisations to create learning opportunities for all employees, the effort to create a platform supporting dialogues, reactions and experiments among members, team learning, vision sharing or strategic leadership [12]. All dimensions are interconnected, which can aggravate statistical evaluation of analyses [16]. When comparing organisations with dimensions, we can see a correla- tion between dimensions and knowledge and financial performance [9, 13]. In the Czech Republic, the topics of introducing a learning organisation and the level of learning in organisations haven’t been significantly addressed yet. The miss- ing data about learning situation in organizations can lead to worsen market position of the organization. Therefore the aim of this study is to evaluate learning time in small and medium-sized organizations in the Czech Republic and to compare the learning time of general staff and managers in these organizations. According to the published foreign studies [16], using the Dimension of a Learning Organisation ques- tionnaire seems to be satisfying tool to evaluate the level of learning in organizations. To maintain the validity of this study it was conducted the cross-section questionnaire survey with using the Dimensions of a Learning Organization questionnaire. 2 Methodology For each dimension, Cronbach confi- dence coefficient was calculated using IBM SPSS Statistics Version 24. The Alpha coefficient ranged from 0.683 to 0.860 for each dimension. Overall, the value of the coefficient was 0.933. The calculated values of the Cronbach coefficient appear to be satisfactory (the coefficient higher than 0.7 is "satisfactory") [6]. Individual dimen- sions were assessed by the respondents on the 6-point Likert scale. In order to verify the clarity of the questionnaire, a pilot study was initially carried out. This pilot study was attended by a total of 20 students from the combined form of follow-up Master's degree in Information Management. The final version of the ques- tionnaire was created using "docs.google.com". In total, 2,884 respondents were ad- dressed. Approximately 250 of the e-mail addresses no longer existed, 25 respondents are not currently in business. The obtained data was analysed using Microsoft Excel 2016 and IBM SPSS Statis- tics version 24 using descriptive statistics, parametric and non-parametric tests at confidence levels α = 0.01 and α = 0.05. 2 Methodology At the beginning of the research, an in-depth data analysis was carried out analysing articles from books and journals searched using web databases (Web of Science, Sco- pus, Sage Journals, Emerald Insight, Science Direct, Wiley Online Library, Taylor & Francis, etc.) with related issues. Based on the synthesis of the obtained data, the search keywords were chosen: learning organisation, learning organisation perform- ance, building a learning organisation, DLOQ, Dimensions of a learning organisation questionnaire study, etc. To comparison this study with published foreign studies [16] a cross-section ques- tionnaire survey was conducted between December 2017 and February 2018. This survey was focused on small and medium-sized organizations in the IT sector in the Czech Republic. The respondents were sent a questionnaire via e-mail addresses ob- tained from the Albertina Business and Marketing Database [2]. The size of the or- ganization and the sector of activity were selected as a business selection criterion. The business sectors were entered by the CZ-NACE code, the predominant activity, specifically: [10] J – Information and communication activities – 62.0 – Activities in the Informa- tion Technology field – 62.01 – Programming - 62.02 – Information Technology Consultancy - 62.03 – Computer Equipment Management – 62.09 – Other IT ac- tivities For this survey, a Dimension of a Learning Organisation questionnaire was se- lected in a 21-issue questionnaire version focusing on the 7 dimensions of a learning organisation [9]. Thanks to its expansion, this questionnaire is easily comparable to foreign studies. This questionnaire also provides adequate measurement results with its focus on the seven dimensions of a learning organisation. To maintain the validity of the questionnaire, the questionnaire was translated by two independent translators from English into Czech and then back to English. At the same time, retaining the meaning of the questionnaire was considered. For each dimension, Cronbach confi- dence coefficient was calculated using IBM SPSS Statistics Version 24. The Alpha coefficient ranged from 0.683 to 0.860 for each dimension. Overall, the value of the coefficient was 0.933. The calculated values of the Cronbach coefficient appear to be satisfactory (the coefficient higher than 0.7 is "satisfactory") [6]. Individual dimen- sions were assessed by the respondents on the 6-point Likert scale. from English into Czech and then back to English. At the same time, retaining the meaning of the questionnaire was considered. 3 Results In order to verify the questionnaire understands, a pilot study was carried out involv- ing 20 students from the combined form of Master's degree in Information Manage- ment at the Faculty of Informatics and Management in Hradec Králové. These re- spondents are employed in the following areas: software development, telecommuni- cations, IT, electronics production, internet sale, sales, law, health, advertising, gov- ernment, work with children, transport and logistics, sports, energy and heating. The data from the pilot study was evaluated using Microsoft Excel 2016 and the IBM SPSS Statistics version 24 statistical programme. After evaluating the pilot survey, information on the organisation’s size and the position in the organisation was added to the questionnaire. Table 2. Cronbach alpha for each dimension Table 2. Cronbach alpha for each dimension Table 2. Cronbach alpha for each dimension Dimension Cronbach α D1: Creating opportunities for systematic learning 0.721 D2: Support for polling and dialogue 0.860 D3: Encourage team learning and collaboration 0.761 D4: Creating systems for capturing and sharing learning 0.683 D5: Motivating people for a collective vision 0.796 D6: System interfaces 0.765 D7: Strategic guidance for learning 0.791 Total 0.933 Source: own processing Using the Cronbach alpha reliability indicator, the reliability of each dimension was determined. All dimensions except dimension 4 met the required reliability value, total reliability is relatively high (α = 0.933). Although dimension 4 (Creating systems for capturing and sharing learning) didn’t reach 0.7 value, it’s significantly close to this value (0.017 difference), so this value can also be considered satisfactory. A total of 2,884 respondents from small and medium-sized companies from the Czech Republic with a focus on information technology activities were addressed. The study was attended by a total of 201 respondents (return on questionnaires was 6.97%). Organisations employing up to 10 employees (32.3%), up to 50 employees (45.3%) and 250 employees (22.4%) were represented in the study. General staff accounted for 35.8% of the respondents and managers accounted for 64.2% of the respondents. The respondents were made up of 137 men and 64 women. The largest age group were respondents aged 39 (6.0%), then 38 years (5.5%) and 47 years (5.0%). Regarding the distribution of respondents to age groups, the 31-40 years age group (37.8%) were represented most, followed by 57 respondents in the 41-50 years age group (28.4%) and 32 respondents in the 51-60 years age group (15.9%). 3 Results In terms of education, respondents with a university education (72.1%) were represented most, followed by secondary school graduates (20.9%). The respondents reported employment time in the organisation up to five years (32.8%), followed by 11 - 15 years (21.4%). Only two respondents worked in organi- sations for a longer time - one respondent for 31 years and the other for 36 years. When comparing the evaluation of individual questions, one can say that none of the questions received less than half of the points. The respondents rated the worse questions 10 and 11: "My organisation creates systems to measure the difference be- tween actual and expected performance" with an average rating of 3.386 and "My organisation makes all evaluations available to all employees" with an average rating of 3.236. The greatest indecision through the evaluation by respondents was ex- pressed at question 20: "In my organisation, managers look for opportunities for fur- ther education.", when they identified two values on the Likert scale from which the average was calculated. The majority of respondents devote from 1 to 10 hours per-month to education that’s related to employment. On average, employees spend 13 hours on education. The relationship between the training time and job position is shown in Figure 1. Fig. 1. Training time (per month) by worker's position (Source: own processing) 10% 54% 21% 6% 10% 2% 54% 26% 9% 10% 0% 10% 20% 30% 40% 50% 60% 0 hours a month 1 – 10 h. a month 11 – 20 h. a month 21 – 35 h. a month 36 or more h. Month Staff member Executive member Fig. 1. Training time (per month) by worker's position (Source: own processing) The differences between managers and ordinary staff members are noticeable in the case where employees don’t learn. The poorer attitude of ordinary staff members in regard to education could be justified by insufficient motivation for further education or by the fact that the organisation doesn’t require further training. For dimensions 3, 4, and 5, the statement asserts that positive dimension ratings are growing up to 35 hours per-month while investing more time in education per-month is slightly declin- ing. 4 Discussion The extension of the learning organisation concept in the Czech Republic hasn’t been described yet. This paper is focused on evaluation of the learning organisation con- cept in Information Technology organisations registered in the Czech Republic. Overall, nearly the study consisted of 70% men and approximately 30% women. This result was expected with respect to the field chosen for the study and is in-line with the reported male/female statistics in the IT sector [11]. When comparing the learning time of the ordinary staff members and managers, it’s clear that managers learn more than regular staff. The managers’ higher education is probably directly related to their higher assessment of the individual learning or- ganisation dimensions (Figure 1). If we compare the learning time per-month with the average assessment of the in- dividual dimensions, we find that there is a statistically significant difference (p = 0.00074) between non-learning staff and the employees who learn 36 or more hours per-month at the materiality level of α = 0.01. At the same time, for dimensions 3, 4, and 5, positive dimensional ratings are growing up to 35 hours per-month, while in- vesting more time in education per-month slightly decreases (Table 3). If employees were learning more than 36 hours per-month, training could eventually take place at the expense of employment, which may have resulted in lower dimensions evaluation. If we compare the average results of each dimension, we see that all dimensions correlate very closely with one another, which is consistent with Watkins, O'Neil [16]. If we compare the average results of each dimension, we see that all dimensions correlate very closely with one another, which is consistent with Watkins, O'Neil [16]. Table 4 below shows an example of studies conducted abroad, along with an aver- age rating score for each dimension. The total number of respondents in the compara- ti t di N 2854 correlate very closely with one another, which is consistent with Watkins, O Neil [16]. Table 4 below shows an example of studies conducted abroad, along with an aver- age rating score for each dimension. The total number of respondents in the compara- tive studies was N = 2854. Table 4. 3 Results The T-test reveals a statistically significant difference between people devoting "0 hours" a month to education and the other groups of respondents who devote to education ("1-10 hours" p = 0.00028; "11-20 hours" p = 0.00025; " 21-35 hours "p = 0.000092;" more than 36 hours "(p = 0.00074, α = 0.01). Table 3. Comparison of respondents' responses with different intensity of education Aver- age of D1 Aver- age of D2 Aver- age of D3 Aver- age of D4 Aver- age of D5 Aver- age of D6 Aver- age of D7 0 hours a month 2.741 3.463 3.778 3.148 3.111 3.370 3.296 1 – 10 h. a month 4.482 4.346 4.125 3.557 4.335 3.888 4.280 11 – 20 h. a month 4.396 4.444 4.347 3.493 4.403 3.948 4.462 21 – 35 h. a month 4.600 4.578 4.444 3.622 4.622 4.078 4.378 36 or more h. Month 5.100 4.725 4.167 3.550 4.442 4.400 4.667 Source: own processing Table 3. Comparison of respondents' responses with different intensity of education Source: own processing by [16] 4 Discussion Comparing DLOQ results with other studies Study Author N D1 D2 D3 D4 D5 D6 D7 Watkins and Marsick 389 3.94 3.91 3.98 3.50 3.74 4.00 4.13 Selden 142 5.01 4.05 4.09 3.44 3.83 4.17 4.49 McHargue 264 4.16 4.15 4.33 3.78 4.20 4.35 4.73 Lien, Yang, Li 79 3.97 4.05 4.00 4.13 4.08 4.01 4.26 Hernandez 906 3.94 4.16 4.01 4.09 4.21 3.96 4.27 Maria 628 4.05 4.08 3.84 3.96 3.79 3.98 4.21 Ellinger 208 4.12 4.04 4.13 3.70 3.93 4.19 4.26 Milton. Watkins 37 4.26 4.35 4.32 3.13 4.15 3.99 4.42 Weighted average 4.06 4.09 4.01 3.86 3.99 4.04 4.29 Zubr 201 4.45 4.39 4.19 3.52 4.33 3.94 4.33 Source: own processing by [16] Table 4. Comparing DLOQ results with other studies 5 Conclusion The aim of this study was to evaluate learning time in small and medium-sized or- ganizations in the Czech Republic and to compare the learning time of general staff and managers in these organizations. To obtain the results we used the Dimensions of a Learning organization questionnaire according to other published studies. This ques- tionnaire was distributed via e-mail between the general staff and managers of small and medium-sized organizations in IT sector in the Czech Republic. If we compare this study’s results with studies already conducted, we can say that the results ob- tained from organisations from the IT sector in the Czech Republic are satisfactory. The results obtained in 5 of 7 dimensions are higher than the weighted average of foreign studies that have been conducted. Compared to the weighted average of re- sults, it can be concluded that organisations in the IT sector in the Czech Republic meet most of the learning organisation’s dimensions better than organisations in for- eign studies. Higher ranking of organisations should give organisations a greater stra- tegic advantage [16]. Organisations in the Czech Republic only have a lower average score in two dimensions. Specifically, Dimension 4 "Creating systems for learning and sharing learning" and Dimension 6 "System interfaces". There was no statistically significant difference between the individual dimensions of the learning organisation concept (p = 0.658, α = 0.05) when comparing the average value from the compara- tive studies with the completed study in the Czech Republic. The training period per- month has a positive impact on the evaluation of individual dimensions. When assessing individual dimensions of the learning organisation, the differences between managers and ordinary staff members were observed. In the follow-up re- search, it would be appropriate to analyse several small and medium-sized organisa- tions to look for a larger sample of management as well as ordinary staff members. At the same time, it would be useful to mutually compare DLOQ results across the vari- ous fields of action. Acknowledgement The paper was written with the support of the specific project 6/2018 grant "Determi- nants of Cognitive Processes Impacting the Work Performance" granted by the Uni- versity of Hradec Králové, Czech Republic and thanks to help of students Majid Ziaei Nafchi. 1. Adamec J (2010) Základní principy koncepce učící se organizace a jejich přijímání. http://emi.mvso.cz/EMI/2010-02/07%20Adamec/Adamec.pdf 2. 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https://openalex.org/W4301374193	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0274083&type=printable	English	null	A multi-dimensional measure of pro-environmental behavior for use across populations with varying levels of environmental involvement in the United States	PloS one	2,022	public-domain	13,027	PLOS ONE PLOS ONE Editor: Carlos Andres Trujillo, Universidad de Los Andes, COLOMBIA Received: August 23, 2021 Accepted: August 22, 2022 Published: October 4, 2022 Received: August 23, 2021 Accepted: August 22, 2022 Published: October 4, 2022 Received: August 23, 2021 Accepted: August 22, 2022 Published: October 4, 2022 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0274083 A multi-dimensional measure of pro- environmental behavior for use across populations with varying levels of environmental involvement in the United States Timothy J. MateerID1, Theresa N. Melton1, Zachary D. Miller2, Ben Lawhon3, Jennifer P. Agans1, B. Derrick Taff1 Timothy J. MateerID1, Theresa N. Melton1, Zachary D. Miller2, Ben Lawhon3, Jennifer P. Agans1, B. Derrick Taff1 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Recreation, Park, and Tourism Management, The Pennsylvania State University, University Park, Philadelphia, United States of America, 2 Intermountain Region-National Park Service, Philadelphia, Pennsylvania, United States of America, 3 Leave No Trace Center for Outdoor Ethics, Boulder, Colorado, United States of America * tjm715@psu.edu Abstract Citation: Mateer TJ, Melton TN, Miller ZD, Lawhon B, Agans JP, Taff BD (2022) A multi-dimensional measure of pro-environmental behavior for use across populations with varying levels of environmental involvement in the United States. PLoS ONE 17(10): e0274083. https://doi.org/ 10.1371/journal.pone.0274083 Researchers continue to explore ways to understand and promote pro-environmental behavior (PEB) amongst various populations. Despite this shared goal, much debate exists on the operationalization and the dimensionality of PEB and how it is measured. This piece- meal approach to measurement has limited the ability to draw conclusions across studies. We address limitations associated with previous measures of PEB by developing a multi- dimensional scale that is validated across both a general population of individuals residing in the United States as well as a group of individuals associated with a pro-environmental organization. Exploratory and confirmatory factor analyses and reliability estimation were conducted for the developed measure across these two populations. Measurement invari- ance testing was also utilized to assess the psychometric stability of the scale across the two groups. Results indicated an 11 item scale was best fitting with two sub-scales: private and public behaviors. Implications for research and practice are discussed. Editor: Carlos Andres Trujillo, Universidad de Los Andes, COLOMBIA 1. Introduction Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. As social-ecological systems continue to be plagued by multi-faceted, “wicked” environmental problems [1, 2], human behavior exists at the forefront of many of these environmental issues as well as their solutions [3, 4]. Given all human behavior impacts the natural world either directly or indirectly [5], environmental psychologists and environmental social psychologists are often at the forefront of efforts to understand and reduce the adverse ecological impacts associated with human behavior in various ways [6]. In linking human behavior to its environ- mental impacts, a variety of terms have been used in the academic literature including environmentally responsible behavior [7], environmentally significant behavior [5], general Data Availability Statement: All relevant data are within the paper and its Supporting information files. Data Availability Statement: All relevant data are within the paper and its Supporting information files. 1 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 PLOS ONE Measuring pro-environmental behavior in the United States ecological behavior [8], and pro-environmental behavior (PEB) [9, 10], the latter of which will be used throughout this study. As PEB becomes better understood and promoted, social-eco- logical systems can become more sustainable and resilient as well. Funding: The authors received support for this research from the Leave No Trace Center for Outdoor Ethics (https://lnt.org/). As one of the co- authors of this manuscript is employed by the supporting organization, the funder had a role in study design, data collection, and manuscript preparation. Measuring PEB effectively has significant implications for designing educational and psy- chological interventions aiming to encourage sustainable behavior within various populations [3, 11]. Be it an examination of barriers to acting in a pro-environmental manner [12] or for- mulating interventions to shift habitual behaviors towards more environmentally-friendly practices [13], environmental psychology has established itself as a major contributor to the conversation around behavioral change and environmentalism. While this body of research contributes important insight into how and why people behave in a pro-environmental man- ner, research on PEB has been broadly limited by inconsistencies in how it has been measured across studies. 1. Introduction While some studies require researchers to measure specific, isolated behaviors [14, 15], many studies aim to understand how other social or psychological factors are related to more generalized PEB [16, 17]. The latter category is specifically constrained by many stud- ies utilizing varied measures of general PEB. For example, [18] asserts that in a review of 49 studies measuring PEB through multi-item scales, 42 unique scales were utilized. As [19] fur- ther note, “many of these scales are ad hoc measures of unknown psychometric quality that have been developed for a particular research project” (p. 93). Such practices do not diminish the unique findings of these various studies, but the field of environmental psychology is lim- ited in its understanding of broader trends due to this piecemeal approach to measurement. Competing interests: The authors have declared that no competing interests exist. In looking to other established scales in the field of environmental psychology, standardized measurements allow for findings to be more easily generalized for other psychological and behavioral constructs. For example, the New Ecological Paradigm represents a “gold standard” for measuring environmental attitudes that is broadly used across studies [20]. Similarly, Clay- ton’s Environmental Identity scale [21] represents a general measure of environmental identity that is also utilized regularly (also see [22] for an updated environmental identity measure). Regarding measures of PEB specifically, [19] assert the General Ecological Behavior scale [8] “can probably be considered the best established of these domain-general propensity mea- sures” (p. 93). However, this scale, while having advanced PEB research considerably in the past two decades, contains some measures which may be considered outdated or only tangen- tially linked to PEB (e.g., “If possible, I do not insist on my right of way and make the traffic stop before entering a crosswalk”). Given social and cultural perceptions of PEB have evolved in the two decades since the General Ecological Behavior scale was developed, updated mea- sures of PEB have been created but display some limitations when being utilized in general populations. For example, some have been developed for specific sub-groups [23] or have dis- played tenuous reliability and validity by categorizing behaviors by their environmental impact rather than their psychological properties [18]. Many scales measuring general PEB in the field of environmental psychology have been uti- lized in populations with a range of environmental orientations, values, and attitudes [16, 17, 24–26]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 2.1 Understanding and measuring pro-environmental behavior Understanding and promoting PEB has been a central topic in the field of environmental psy- chology [5, 9, 10, 19, 29]. While prior research focused heavily on these environmentally- focused behaviors, some debate exists in the field about what is and is not considered PEB [30]. For example, Stern [5] adopts an intention-oriented approach to understanding PEB, stating it is “defined from the actor’s standpoint as behavior that is undertaken with the inten- tion to change (normally, to benefit) the environment” (p. 408). Alternatively, Steg and Vlek [11] assert that PEB consists of actions taken that benefit or minimize the harm done to the natural environment, with the intention behind the behavior not being as heavily emphasized in their definition. As exemplified by Truelove and Gillis [9], other motivations such as saving money or benefiting one’s health may also encourage individuals to shift their behaviors in a pro-environmental manner. Given these divergent definitions, there may be some behaviors that would fit into one of the provided definitions of PEB but not the other (e.g., riding a bike to work specifically to save money on gas would likely not fall under Stern’s [5] definition of PEB but would be encompassed by that provided by Steg and Vlek [11]). Many recent studies [12, 18, 19, 23] have endorsed a conceptualization of PEB similar to that provided by Steg and Vlek [11], recognizing that benefits to the natural environment exist regardless of the social or psychological pathway that encourages their enactment [3, 10, 31, 32]. Expanding our under- standing of PEB beyond an intention-oriented conceptualization allows for PEB to be encour- aged through multiple pathways [4, 13, 31]. In conjunction with the debate on how to define PEB within the academic literature, other studies have also explored the social and psychological pathways of enacting general PEB and how these pathways influence the frequency at which different categories of behaviors are enacted [5, 23, 33, 34]. While many studies have aimed to measure general PEB through improvised unidimensional scales [17, 25, 35], such approaches fail to recognize the various factors that may inhibit or facilitate individuals’ enactment of some forms of PEB in compari- son to others [9, 12, 34]. As Larson et al. [23] outline, behavioral difficulty, structural factors, and social influences all may influence whether certain clusters of behaviors more or less likely to be carried out by specific individuals. 1. Introduction If a general PEB scale is to be effectively employed across these disparate groups, it should be developed and tested amongst the various populations within which it will ulti- mately be utilized [27]. With that in mind, this study aims to develop an updated measure of general PEB for both a general population of individuals living in the United States and a pop- ulation associated with a pro-environmental organization (sampled from the Leave No Trace Center for Outdoor Ethics, to be discussed later). Furthermore, measurement invariance test- ing examines the psychometric stability of the developed scale across the two groups [28]. While we specifically develop this general PEB scale in the social, cultural, and infrastructural context of the United States, it may also potentially provide a basis for measuring general PEB While we specifically develop this general PEB scale in the social, cultural, and infrastructural context of the United States, it may also potentially provide a basis for measuring general PEB in other countries as well. 2 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 PLOS ONE Measuring pro-environmental behavior in the United States PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 2.1 Understanding and measuring pro-environmental behavior For example, individuals living in a city with a well- established recycling system may easily be able to enact that specific PEB but may have diffi- culty enacting another behavior such as biking to work due to a lack of accessible bike lanes in the area. As certain behaviors may be easier or more difficult to enact for various populations, it is imperative to develop tools for measuring PEB within the populations with which they will be ultimately utilized [36–39]. This is generally done by utilizing sub-scales to represent the real- world differences in how different clusters of behaviors are enacted as a result of various psy- chological, social, and infrastructural factors. Each sub-scale is utilized to capture a different dimension of general PEB. The heterogeneity in PEB scales developed within different popula- tions may provide insight into this divergent dimensionality of environmentally-oriented behaviors (see the Supplementary Materials of Lange and Dewitte [19] for an extensive list of previously developed PEB scales and their various dimensions). For example, Gkargkavouzi et al. [33] found PEB to exist as six unique factors within a population of Greek citizens: civic actions, policy support, transportation choices, household setting, and consumer behavior. Alternatively, Larson et al. [23] found four dimensions of PEB within residents living in rural communities of the United States: conservation lifestyle, land stewardship, social environmen- talism, and environmental citizenship. It is likely that the various social, infrastructural, and psychological contexts unique to these communities influence the dimensionality of how PEB PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 3 / 22 PLOS ONE Measuring pro-environmental behavior in the United States is perceived and enacted [9, 23, 40]. Extensive research has aimed to understand PEB across groups with varying psychological and social orientations to the natural world [10, 41, 42], but a scale measuring general PEB has not been developed across these disparate groups. 2.2 Scale development theory There is an overall need for researchers to psychometrically evaluate measures prior to their use, an approach that has frequently been lacking in research examining PEB. To confidently identify what behaviors contribute to an individual’s PEB, researchers must utilize scales that have demonstrated both their reliability and accuracy in measuring the construct of interest [27]. Psychometric testing, which examines a scale’s reliability and validity, establishes confi- dence in the ability of a scale to accurately capture information on the construct researchers hope to be measuring; it is, therefore, a critical first step to conducting research. This notion of validity, or accuracy of the measure, is not absolute [43]. Although researchers often discuss the fact that the validity of a scale can be evaluated for a specific purpose, such as measuring an individual’s tendency to engage in PEB, the validity of a measure may differ based on the pop- ulation included in the study as well [37, 43]. Not only does one’s context influence the behaviors with which they engage, constructs are understood and operationalized differently based on an individual’s setting and culture [36, 37]. For this reason, researchers [27] argue for the importance of evaluating the quality of a measure in the population within which findings are to be generalized, and caution against extending research to different groups without first testing that the instrument functions equally well within that specific population [38, 39]. However, previous PEB scales have mostly been developed within either a convenience sample of university students [18] or through online survey panels [44]. When adopted for other research, these PEB scales are generally uti- lized in populations that vary greatly from the populations used for scale development. PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 4. Methods All research procedures that involved human subjects were approved by the Pennsylvania State University Institutional Review Board (STUDY00015401). Oral or written consent was not obtained as all data were analyzed anonymously. 3. Study purpose RQ1: What self-reported behaviors form a reliable and valid measure of general PEB within the unique social, cultural, and infrastructural context of the United States? RQ2: What unique dimensions of general PEB are best represented as sub-scales within the gen- eral measure of PEB that is developed? RQ2: What unique dimensions of general PEB are best represented as sub-scales within the gen- eral measure of PEB that is developed? RQ3: What measures of general PEB form a psychometrically stable scale across a population of individuals sampled from a pro-environmental organization and a population of individuals sampled to represent the general population of the United States? RQ3: What measures of general PEB form a psychometrically stable scale across a population of individuals sampled from a pro-environmental organization and a population of individuals sampled to represent the general population of the United States? 3. Study purpose Given the various limitations associated with measuring PEB in the field of environmental psy- chology, the ability to extend findings to inform psychologically-grounded interventions to promote desired behaviors is also constrained [9]. This is especially important as research interest continues to grow around processes that support the adoption of PEB generally, such as promoting behavioral spillover [45, 46]. Lange and Dewitte [19] note that self-reported PEB measures are primarily used by personality psychologists aiming to connect these measures to other psychological constructs such as environmental values and identity, emphasizing the importance of using self-reported behavioral measures that are psychologically grounded as well. Taken collectively, there is a clear need to design a PEB scale rooted in the social and psy- chological processes of the communities within which they will be utilized, both to promote more effective research as well as to help practitioners design more effective psychological interventions. Given this need, we aimed to develop a scale that measures a breadth of mean- ingful PEB while also considering the psychological dimensions of these behaviors. Specifi- cally, we aim to establish a PEB scale that is psychometrically sound both within the general population of the United States and for a population of individuals sampled from a pro-envi- ronmental organization also based in the United States. In specifically sampling individuals from a pro-environmental organization, we surveyed individuals from the Leave No Trace Center for Outdoor Ethics, a prominent non-profit organization promoting responsible out- door recreation behaviors in the United States [47]. This organization was intentionally chosen as the source of one of the two samples as outdoor recreation involvement and pariticpation in environmental groups has been linked to pro-environmental attitudes and behaviors in PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 4 / 22 PLOS ONE Measuring pro-environmental behavior in the United States previous research [42, 48, 49]. Given many studies aim to understand and compare PEB within populations that have various social and psychological orientation towards the natural world [25, 26, 35, 48], this study aims to develop a scale that is psychometrically stable and useful across these different populations. This study was specifically informed by the following three research questions: RQ1: What self-reported behaviors form a reliable and valid measure of general PEB within the unique social, cultural, and infrastructural context of the United States? PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 4.1 Preliminary scale development Initial scale items were aggregated from a range of previous studies measuring various aspects of PEB [8, 17, 18, 23, 50, 51]. These studies were intentionally reviewed by experts in the field of environmental psychology, communication, and education [27] to determine the breadth of behaviors that fall within Steg and Vlek’s [11] impact-oriented definition of PEB. This defini- tion was considered simultaneously with the variety of ways previous research has attempted to characterize PEB such as activist-oriented behaviors [52], land stewardship [23, 53, 54], pri- vate-sphere behaviors [5], and socially-oriented behaviors [55]. After reviewing how PEB was measured in these previous studies, 27 initial scale items were extracted to represent a range of potential behaviors for preliminary analysis. These initial items are outlined in S1 Appendix. For these scale items, individuals were prompted to “Please rate how frequently you have par- ticipated in the following activities by selecting the appropriate point from the scale below.” Seven Likert-scale response options were provided ranging from “Never” to “As frequently as possible.” These initial items were piloted within a population of 305 individuals recruited from Qual- trics market research panels [56]. All research processes for this pilot study, as well as subse- quent data collection and analyses, were approved by the Pennsylvania State University Institutional Review Board. A quota sampling approach [57] was utilized to match recruited individuals to several broader demographic characteristics of the United States population as determined by the United States Census Bureau [58]. Specifically, demographic variables were matched for age, gender, and household income, each of which has been found to influence pro-environmental behavior in previous research [29]. Given that the goal of this preliminary test was to determine an initial scale for more rigor- ous analysis within two larger populations (one representing the general population of the United States and one sampled from a pro-environmental organization), this study phase uti- lized both exploratory and confirmatory techniques to eliminate redundant items as well as those that did not fit well within the psychometric properties relative to the broader scale. 5 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 PLOS ONE Measuring pro-environmental behavior in the United States Table 1. Preliminary PEB items retained following the pilot study, including results from reliability analyses and confirmatory factor analyses. 4.1 Preliminary scale development Latent Construct Item Label Please rate how frequently you have participated in the following activities Λ Mean SD Private Behaviors Priv-PEB-1 Bought environmentally friendly and/or energy efficient products 0.77 3.84 1.78 Priv-PEB-2 Walked or rode a bike when traveling short distances 0.55 3.38 2.17 Priv-PEB-3 Reused or mended items rather than throwing them away 0.57 4.19 1.69 Priv-PEB-4 Composted food or yard and garden refuse 0.66 2.23 2.24 Priv-PEB-5 Avoided buying products with excessive packaging 0.80 3.26 1.98 Priv-PEB-6 Bought organic vegetables 0.63 3.27 2.04 Priv-PEB-7 Used rechargeable batteries 0.54 3.40 2.07 Priv-PEB-8 Minimized use of heating or air conditioning to limit energy use 0.59 3.93 1.85 Priv-PEB-9 Car-pooled when traveling to a destination 0.61 2.77 2.15 Cronbach’s Alpha 0.86 Public Behaviors Pub-PEB-1 Talked to others in your community about environmental issues 0.83 2.46 2.12 Pub-PEB-2 Worked with others to address an environmental problem or issue 0.86 2.11 2.13 Pub-PEB-3 Participated as an active member in a local environmental group 0.82 1.49 1.90 Pub-PEB-4 Signed a petition about an environmental issue 0.77 2.43 2.18 Pub-PEB-5 Donated money to support local environmental protection 0.85 2.30 2.17 Cronbach’s Alpha 0.91 Global Fit Indices: S2 = 180.34, df = 76, p<0.001; RMSEA = 0.069; SRMR = 0.048; CFI = 0.950 https://doi org/10 1371/journal pone 0274083 t001 Specifically, descriptive statistics, exploratory factor analyses, confirmatory factor analyses, and reliability analyses were all used to examine the psychometric properties of these initial items. For more information on analysis processes and sample demographics, please see S2 Appendix. A final confirmatory factor analysis was primarily utilized at this stage to indicate preliminary discriminant validity for the scale [59]. Discriminant validity indicates the under- lying sub-scale structure of a survey, which can be displayed through item loadings and latent construct relationships of a confirmatory factor analysis [60]. Items retained following this pilot study, including sub-scale structure, reliability statistics, and results from the confirma- tory factor analysis are outlined in Table 1. PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 4.2 Sample populations Following the pilot study, two primary samples were utilized to further develop the prelimi- nary scale outlined in the previous section: a larger online sample representative of the United States population and a sample of individuals recruited from the Leave No Trace Center for Outdoor Ethics email list. For the representative sample of United States residents, 1043 indi- viduals residing in the United States were recruited from Qualtrics market research panels [56], referred to as QUAL from here onward. Age, gender, race/ethnicity, and household income were matched using a quota sampling procedure [57] to demographic information col- lected from the United States census [58]. Like the other demographic variables, race/ethnicity has been found to potentially influence environmental concern in previous research [61], therefore an additional quota for race/ethnicity was intentionally added after the large percent- age of White respondents was recruited from Qualtrics for the pilot study. Surveys were also distributed to an email list of 22,180 individuals provided by the Leave No Trace Center for Outdoor Ethics. This group will be referred to as LNT from here onward. Individuals were added to this list by registering as members of the organization, attending a training workshop, or taking a week-long course to become a master educator through the organization. The PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 6 / 22 PLOS ONE Measuring pro-environmental behavior in the United States online list is mostly composed of avid outdoor recreationists who participate in 8 to 12 hours of outdoor recreation per week [62]. online list is mostly composed of avid outdoor recreationists who participate in 8 to 12 hours of outdoor recreation per week [62]. 4.3 Measures Online surveys were distributed to the two populations outlined in the previous section (QUAL and LNT). These online surveys included several batteries of questions including items measuring demographic characteristics of the sample and PEB. Demographic informa- tion was collected from participants, including their gender, age, household income, level of education, and race/ethnicity. For QUAL, these demographic variables were utilized in the quota sampling procedure to ensure the representativeness of the population to the broader demographics of the United States. Additionally, the 14 items measuring different types of PEB, outlined in Table 1, were also included in the online survey. Individuals taking the survey were prompted: “Please rate how frequently you have participated in the following activities in the past six months by selecting the appropriate point from the scale below.” In contrast to the prompt utilized in the pilot study, this prompt specifically provides a time bound within which participants were asked to consider their behaviors to reduce the arbitrary nature of the measurement [63]. Six months was chosen specifically as it provided a broad enough time period for individuals to participate in PEB’s that may occur less frequently (e.g., donating money to an environmental organiza- tion) while also limiting the reflection period to a reasonable period for individuals to recall. Again, seven options were provided on a Likert-scale ranging from “Never” to “As frequently as possible.” These extreme end points were chosen intentionally to maximize scale variance [64]. Several additional measures were included within the online survey measuring environ- mental identity, outdoor recreation habits, and environmental values. These measures were utilized for an alternate study for which data were also being collected. This parallel study aimed to understand how involvement in environmentally-conscious outdoor recreation prac- tices influenced the adoption of PEB’s in other life domains. A similar population structure combining avid outdoor recreationists with a United States census-matched population was also utilized for this alternate study. PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 4.4 Data analysis Several analytical steps were taken to further develop a psychometrically valid and reliable scale from the items initially outlined in Table 1. All data analyses were carried out in IBM SPSS 26 or R Statistical Analysis Software. Analyses that utilized confirmatory factor analyses were carried out using the AMOS extension of IBM SPSS 26. First, an exploratory factor analysis was run for both QUAL and LNT to determine whether the two latent constructs indicated from the initial pilot sample held within the two broader samples, especially because they differed from the pilot sample regarding several demographic characteristics (outlined in the Sample Populations section). This analysis was carried out to specifically address research questions #1 and #2. For each of these samples, a principle com- ponent analysis (PCA) was utilized to determine the appropriate number of factors to extract from the larger scale. While not technically a form of exploratory factor analysis, this initial PCA determined the number of factors to extract by taking advantage of all available variance within the data, providing a more general approach to understanding the measures prior to determining factor loadings via further analysis. Identifying the number of factors to extract was done utilizing a combination of theoretical interpretation, examination of corresponding scree plots, and Kaiser’s Rule [65]. Once the number of factors were determined, principle axis PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 7 / 22 PLOS ONE Measuring pro-environmental behavior in the United States factors (PAF) was utilized to examine how individual items related to the underlying latent constructs represented by the scale. This sequence of steps (PCA followed by PAF) followed the exploratory factor analysis approach outlined by [66]. A cutoff value for factor loadings of 0.32 was utilized, as items below are less likely to have a statistically meaningful relationship with the associated latent construct [67]. Second, in order to further address research questions #1 and #2, two confirmatory factor analyses were run, one for QUAL one for LNT, to establish the factors structure of the PEB items. Like the confirmatory factor analysis conducted within the pilot study, the S2 statistic was used to assess model fit [68]. Additionally, other global fit indices utilized to assess model fit were: RMSEA 0.10 [68]; SRMR 0.08 [68]; and CFI 0.90 [69]. 4.4 Data analysis Also in parallel to the pilot study, factor loading were considered adequate if statistically significant and with load- ings over 0.30 [70]. Bias-corrected confidence intervals (95% confidence interval computed by 5000 bootstrap resamples) helped to minimize the likelihood of Type 1 Error for factor load- ings [71]. Third, to explore the psychometric stability of the final measure between a general popula- tion of individuals living in the United States and members of the Leave No Trace Center for Outdoor Ethics (addressing research question #3), invariance testing was utilized to compare the psychometric equivalence of the measure between the QUAL and LNT groups. On the importance of exploring measurement invariance between groups for which the measure will ultimately be utilized, [28] state, “meaning is essentially conventionalized, and so different groups can apply different meanings to the same cognition or behavior. Appropriate and proper comparison of a construct between groups or across times, therefore, depends first on ensuring equivalence of meaning of the construct” (p. 72). Therefore, configural, metric, and scalar invariance [28] were explored between the LNT and QUAL groups. This is done through an iterative process of sequentially imposing greater constraints on a multi-group CFA that incorporates both groups of interest [72]. Configural invariance explored whether the basic item-construct structure was the same between the two groups. Broadly, configural invariance suggests the same items load on to the same latent constructs across the two groups of interest [28, 73]. This was determined by assessing model fit using the same fit indices out- lined for the confirmatory factor analysis in the previous paragraph. Metric invariance then constrained item loadings to be equivalent between the two groups and determined whether this constraint significantly reduced model fit when compared to the configural model. Theo- retically, metric invariance suggests that not only the same items load on to the same latent constructs across the two groups (as explored in configural invariance), but the same items contribute to the same latent construct in a similar pattern across the two groups [28, 73]. Finally, scalar invariance constrained item intercepts between QUAL and LNT. Scalar invari- ance explores whether differences in latent constructs between the two groups adequately cap- tures mean differences in the shared variance across the measured items (i.e., higher levels of a measured item in one group result in a higher level of the corresponding latent construct) [28, 73]. PLOS ONE PLOS ONE Table 2. Demographic characteristics of the two collected samples. Demographic Variables n Percentage of Sample n Percentage of Sample LNT Mean age = 47.3 (SD = 16.2) QUAL Mean age = 45.6 (SD = 17.1) Gender Female 847 42.8 541 51.9 Male 1019 51.5 498 47.7 Non-binary 21 1.1 4 0.4 Missing 91 4.6 0 0.0 Ethnicity White 1725 87.2 643 61.6 Hispanic or Latino/Latina/Latinx 47 2.4 188 18.8 Black or African American 8 0.4 127 12.2 Native American, American Indian, or Alaska Native 16 0.8 10 1.0 Asian or Pacific Islander 38 1.9 58 5.6 Other 40 2.0 17 1.6 Missing 104 5.3 0 0.0 Household Income Less than $10,000 52 2.6 76 7.3 $10,000-$19,999 73 3.7 74 7.1 $20,000-$29,999 97 4.9 107 10.3 $30,000-$39,999 133 6.7 85 8.1 $40,000-$49,999 115 5.8 82 7.9 $50,000-$59,999 125 6.3 84 8.1 $60,000-$69,999 147 7.4 82 7.9 $70,000-$79,999 119 6.0 79 7.6 $80,000-$89,999 114 5.8 51 4.9 $90,000-$99,999 112 5.7 47 4.5 $100,000-$149,999 390 19.7 176 16.9 More than $150,000 312 15.8 100 9.6 Missing 189 9.6 0 0.0 Education Elementary 0 0 1 0.1 Some high school 1 0.1 33 3.2 GED or high school graduate 54 2.7 213 20.4 Some college or technical school 364 18.4 373 35.8 Four-year college graduate 785 39.7 262 25.1 Graduate degree 681 34.4 161 15.4 Missing 93 4.7 0 0.0 https://doi.org/10.1371/journal.pone.0274083.t002 4.4 Data analysis Reduction in model fit for the scalar invariance model was compared to the metric invari- ance model, with all outlined procedures matching those outlined in Putnick and Bornstein [28]. Since S2 is sensitive to large sample sizes, reduction in model fit was assessed utilizing alter- native fit indices, specifically the cutoff measures developed by Cheung and Rensvold [74] and Chen [75]. Changes in fit indices should not exceed -0.010 for CFI and 0.015 for RMSEA. Additionally, for SRMR, changes should be less than or equal to 0.030 for metric invariance and 0.015 for scalar invariance. If model fit failed to meet thresholds of advancing to the next strictest model, constraints were selectively released based off meaningful differences between the two groups. Model fit was then reassessed for the partially restricted model. PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 8 / 22 Measuring pro-environmental behavior in the United States PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 5.1 Sample characteristics and descriptive statistics Once the number of factors to extract were determined using PCA, PAF was utilized to fur- ther examine the nature of these latent constructs and how individual items loaded onto them for each population. An oblique rotation was utilized as the two latent factors were highly cor- related with each other (0.61 for QUAL and 0.55 for LNT). Pub PEB 4 2.51 2.14 0 3.31 2.12 10.8 Pub-PEB-5 2.43 1.99 0 3.58 1.88 10.6 https://doi.org/10.1371/journal.pone.0274083.t003 Table 4. Eigenvalues for each factor extracted from LNT sample and GEN sample. LNT 4.63 1.41 0.99 0.91 0.85 0.79 0.72 0.69 0.64 0.57 0.53 0.48 0.43 0.35 QUAL 6.42 1.43 0.79 0.69 0.67 0.63 0.62 0.53 0.49 0.42 0.40 0.35 0.30 0.26 https://doi.org/10.1371/journal.pone.0274083.t004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 10 / 22 higher in LNT, further supporting prior work showing that individuals invested in environmen- tal organizations and outdoor recreation self-report higher levels of PEB [25, 35]. While there was no missing data in QUAL, missing data was reported across the PEB items for LNT. Little’s MCAR Test [76] was utilized to explore whether data for the PEB items was missing randomly within LNT, an assumption that was confirmed by the analysis (S2 = 270.85, df = 269, p = 0.46). Once data were confirmed to be missing completely at random for LNT, the individuals with missing data on PEB measures were deleted listwise, leaving 1719 individuals for analysis. 5.1 Sample characteristics and descriptive statistics Of the distributed surveys, 1043 completed surveys were returned for QUAL while 1978 sur- veys were returned for LNT. QUAL was primarily white (61.6%), had a slight majority of females (51.9%), and had a mean age of 45.6 years old. Like QUAL, LNT was primarily white (though at a much higher percentage at 87.2%) and had a similar mean age of 47.3 years old. LNT deviated from QUAL in being primarily male (51.5%). Additionally, LNT generally had a higher household income and education level than those in QUAL. Detailed demographic information for both samples is provided in Table 2. Descriptive statistics for the 14 items measuring PEB are outlined in Table 3, and item means are outlined for both QUAL and LNT. As expected, scores on the PEB measures were generally PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 9 / 22 PLOS ONE Measuring pro-environmental behavior in the United States higher in LNT, further supporting prior work showing that individuals invested in environmen- tal organizations and outdoor recreation self-report higher levels of PEB [25, 35]. While there was no missing data in QUAL, missing data was reported across the PEB items for LNT. Little’s MCAR Test [76] was utilized to explore whether data for the PEB items was missing randomly within LNT, an assumption that was confirmed by the analysis (S2 = 270.85, df = 269, p = 0.46). Once data were confirmed to be missing completely at random for LNT, the individuals with d d l d l l d d l f l Table 3. Descriptive statistics for QUAL and LNT for PEB items. QUAL LNT Item Label Mean SD % Missing Mean SD % Missing Priv-PEB-1 3.85 1.64 0 4.94 1.21 10.5 Priv-PEB-2 3.54 2.00 0 4.46 1.63 10.4 Priv-PEB-3 4.10 1.68 0 4.99 1.20 10.4 Priv-PEB-4 2.72 2.18 0 3.72 2.31 10.7 Priv-PEB-5 3.46 1.86 0 4.36 1.55 10.4 Priv-PEB-6 3.42 1.92 0 4.06 1.78 10.4 Priv-PEB-7 3.42 1.97 0 3.56 1.92 10.6 Priv-PEB-8 3.85 1.74 0 4.62 1.44 10.5 Priv-PEB-9 2.90 2.06 0 3.82 1.84 10.8 Pub-PEB-1 2.56 1.99 0 4.04 1.67 10.7 Pub-PEB-2 2.37 1.94 0 3.54 1.85 10.8 Pub-PEB-3 1.94 2.00 0 2.98 2.14 10.5 Pub-PEB-4 2.51 2.14 0 3.31 2.12 10.8 Pub-PEB-5 2.43 1.99 0 3.58 1.88 10.6 https://doi.org/10.1371/journal.pone.0274083.t003 Table 3. Descriptive statistics for QUAL and LNT for PEB items. 5.1 Sample characteristics and descriptive statistics QUAL LNT Item Label Mean SD % Missing Mean SD % Missing Priv-PEB-1 3.85 1.64 0 4.94 1.21 10.5 Priv-PEB-2 3.54 2.00 0 4.46 1.63 10.4 Priv-PEB-3 4.10 1.68 0 4.99 1.20 10.4 Priv-PEB-4 2.72 2.18 0 3.72 2.31 10.7 Priv-PEB-5 3.46 1.86 0 4.36 1.55 10.4 Priv-PEB-6 3.42 1.92 0 4.06 1.78 10.4 Priv-PEB-7 3.42 1.97 0 3.56 1.92 10.6 Priv-PEB-8 3.85 1.74 0 4.62 1.44 10.5 Priv-PEB-9 2.90 2.06 0 3.82 1.84 10.8 Pub-PEB-1 2.56 1.99 0 4.04 1.67 10.7 Pub-PEB-2 2.37 1.94 0 3.54 1.85 10.8 Pub-PEB-3 1.94 2.00 0 2.98 2.14 10.5 Pub-PEB-4 2.51 2.14 0 3.31 2.12 10.8 Pub-PEB-5 2.43 1.99 0 3.58 1.88 10.6 https://doi.org/10.1371/journal.pone.0274083.t003 Table 3. Descriptive statistics for QUAL and LNT for PEB items. higher in LNT, further supporting prior work showing that individuals invested in environmen- tal organizations and outdoor recreation self-report higher levels of PEB [25, 35]. While there was no missing data in QUAL, missing data was reported across the PEB items for LNT. Little’s MCAR Test [76] was utilized to explore whether data for the PEB items was missing randomly within LNT, an assumption that was confirmed by the analysis (S2 = 270.85, df = 269, p = 0.46). Once data were confirmed to be missing completely at random for LNT, the individuals with missing data on PEB measures were deleted listwise, leaving 1719 individuals for analysis. 5.2 Exploratory factor analyses Exploratory factor analyses were utilized to further understand the underlying psychological dimensions of the 14 PEB items retained from the pilot sample in both the QUAL and LNT samples. Utilizing the Kaiser Rule [65] and examining the corresponding scree plot for each of the two PCAs, both samples suggested that two underlying latent constructs existed within the broader scale measuring PEB. For LNT, eigenvalues were 4.63 and 1.41 for the first two factors, respectively, with all other factors loading below the cutoff value of 1. For QUAL, the eigenval- ues were 6.42 and 1.43. Similarly, all other factors failed to meet the threshold of exceeding 1. This statistical evidence also aligned with previous empirical evidence [24, 77] and with our pilot study that indicated two latent constructs existed within the concept of PEB: Private Behaviors and Public Behaviors. Eigenvalues for all extracted factors for both PCAs are out- lined in Table 4. https://doi.org/10.1371/journal.pone.0274083.t003 5.2 Exploratory factor analyses Item loadings on two extracted factors for both QUAL and LNT samples. Item factor loadings for each latent construct are outlined in Table 5. After examining the item loadings on each latent factor, three problematic items were identified between the QUAL and LNT samples. For QUAL, Priv-PEB-4 and Priv-PEB-9 both cross-loaded consider- ably, with a slightly higher loading on the factor that seemed to align with Public Behaviors rather than Private Behaviors. This contrasted with what was indicated by the findings from the pilot study. Specifically, Priv-PEB-4 asked about composting behavior, a behavior that is constrained by infrastructural availability for some individuals (e.g., those living in urban envi- ronments), which could pose a significant limitation to performing a behavior even if behav- ioral intent was present [31]. Additionally, Priv-PEB-9 inquired about carpooling behaviors. While in some regards this behavior could take place in a private setting, it also required the participation of others. Given this practical justification and inconsistent statistical perfor- mance across the two groups, both items were dropped from further analysis. Additionally, Priv-PEB-7, which asked about the use of rechargeable batteries, did not meet the pre-deter- mined loading threshold of 0.32 [67] for the LNT sample. Given the goal of this study is to develop a scale measuring PEB that can be generalized between general and pro-environmen- tal groups within the United States, this item was also dropped. In broadly examining the remaining 11 item loadings, they aligned with the two latent fac- tors of Private Behaviors and Public Behaviors observed in the pilot study. With this, the six remaining items from the Private Behaviors sub-scale and the five items making up the Public Behaviors sub-scale established from the pilot study were maintained for further analysis. 5.2 Exploratory factor analyses Exploratory factor analyses were utilized to further understand the underlying psychological dimensions of the 14 PEB items retained from the pilot sample in both the QUAL and LNT samples. Utilizing the Kaiser Rule [65] and examining the corresponding scree plot for each of the two PCAs, both samples suggested that two underlying latent constructs existed within the broader scale measuring PEB. For LNT, eigenvalues were 4.63 and 1.41 for the first two factors, respectively, with all other factors loading below the cutoff value of 1. For QUAL, the eigenval- ues were 6.42 and 1.43. Similarly, all other factors failed to meet the threshold of exceeding 1. This statistical evidence also aligned with previous empirical evidence [24, 77] and with our pilot study that indicated two latent constructs existed within the concept of PEB: Private Behaviors and Public Behaviors. Eigenvalues for all extracted factors for both PCAs are out- lined in Table 4. Once the number of factors to extract were determined using PCA, PAF was utilized to fur- ther examine the nature of these latent constructs and how individual items loaded onto them for each population. An oblique rotation was utilized as the two latent factors were highly cor- related with each other (0.61 for QUAL and 0.55 for LNT). Table 4. Eigenvalues for each factor extracted from LNT sample and GEN sample. LNT 4.63 1.41 0.99 0.91 0.85 0.79 0.72 0.69 0.64 0.57 0.53 0.48 0.43 0.35 QUAL 6.42 1.43 0.79 0.69 0.67 0.63 0.62 0.53 0.49 0.42 0.40 0.35 0.30 0.26 https://doi.org/10.1371/journal.pone.0274083.t004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 10 / 22 Table 4. Eigenvalues for each factor extracted from LNT sample and GEN sample. PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 10 / 22 PLOS ONE Measuring pro-environmental behavior in the United States Table 5. Item loadings on two extracted factors for both QUAL and LNT samples. QUAL LNT Factor 1 Factor 2 Factor 1 Factor 2 Priv-PEB-1 0.74 0.04 0.67 0.01 Priv-PEB-2 0.41 0.22 0.45 0.05 Priv-PEB-3 0.72 -0.09 0.56 -0.04 Priv-PEB-4 0.29 0.36 0.32 0.07 Priv-PEB-5 0.68 0.09 0.70 0.00 Priv-PEB-6 0.42 0.25 0.51 0.02 Priv-PEB-7 0.43 0.17 0.28 0.15 Priv-PEB-8 0.67 -0.08 0.56 -0.06 Priv-PEB-9 0.29 0.37 0.37 0.10 Pub-PEB-1 0.08 0.77 0.16 0.59 Pub-PEB-2 0.01 0.84 0.02 0.77 Pub-PEB-3 -0.12 0.92 -0.10 0.83 Pub-PEB-4 0.10 0.67 0.31 0.37 Pub-PEB-5 0.06 0.75 0.17 0.48 https://doi.org/10.1371/journal.pone.0274083.t005 Table 5. 5.4 Measurement invariance Invariance testing was conducted between QUAL and LNT to explore the psychometric stabil- ity of the PEB measures and their associated latent constructs across the two populations. The configural invariance model was found to have appropriate model fit when examining all fit indices aside from the S2 measure (S2 = 626.786, df = 86, p<0.001; RMSEA = 0.048; SRMR = 0.0471; CFI = 0.950). Given these appropriate fit statistics, configural invariance for the PEB measures between the two groups was supported. In comparing the metric invariance model to the configural invariance model, corresponding changes in model fit fell within the pre-established thresholds for RMSEA and SRMR measures but exceeded the appropriate level of change for the CFI measures. The fit indices for the metric invariance model were S2 = 800.686, df = 97, p<0.001; RMSEA = 0.051; SRMR = 0.0687; CFI = 0.935. The -0.015 change in CFI exceeded the threshold of -0.010 established by both Cheung and Rensvold [74] and Chen [75]. [ ] Given the PEB measures and the associated latent constructs were found to be metric non- invariant, single item loadings were released in a stepwise manner to understand whether par- tial metric invariance could be established. These items were released specifically by examining potential theoretical differences between the QUAL and LNT populations. Ulti- mately, constraints on four item loadings were released: Priv-PEB-2, Pub-PEB-1, Pub-PEB-3, and Pub-PEB-5. Priv-PEB-2 specifically addresses walking and biking behaviors in commuting to nearby destinations. Since prior research has indicated that the LNT population regularly partakes in high levels of outdoor recreation on a weekly basis [62], the unique nature of the LNT population may have resulted in loading differences for this item. Additionally, Pub- PEB-1 and Pub-PEB-3 may be directly influenced by the nature of individuals’ involvement with the Leave No Trace Center for Outdoor Ethics. These items specifically address whether individuals talk to others about environmental issues and whether they participate as a regular member in an environmental organization. Given many individuals within the LNT sample are involved with the Leave No Trace Center for Outdoor Ethics as educators, this may have resulted in unique difference for factor loadings on these two items as well when compared to a general population. 5.3 Confirmatory factor analyses Confirmatory factor analyses were utilized to analyze the relationship between the 11 items retained after the exploratory factor analysis for both the QUAL and LNT populations. Model fit indices and factor loadings are outlined for each population in the following paragraphs. Labels for the two latent constructs, Private Behaviors and Public Behaviors, were retained from the pilot study for the confirmatory factor analysis. Each of these corresponding labels continued to capture the theoretical concept represented by their corresponding items effectively. 11 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 PLOS ONE Measuring pro-environmental behavior in the United States For QUAL, S2 indicated poor model fit (S2 = 211.082, df = 43, p<0.001), a result likely linked to the large sample size. Other global fit indices alternatively indicated good model fit though: RMSEA = 0.061; SRMR = 0.044; CFI = 0.969. All factor loadings also met appropriate thresholds (0.30) on the corresponding latent construct and were significant. For LNT, S2 val- ues also indicated poor model fit (S2 = 415.713, df = 43, p<0.001), though the large LNT sam- ple size also likely contributed to this result [68]. Alternative global fit indices also met necessary thresholds: RMSEA = 0.071; SRMR = 0.047; CFI = 0.929. Like QUAL, all items loaded on the related latent constructs at appropriate levels (0.30) while also being statistically significant. With this, both original confirmatory factor analysis models were retained without modification for QUAL and LNT. Models for both QUAL and LNT populations are outlined in Figs 1 and 2, respectively. 5.4 Measurement invariance Lastly, LNT had much higher household income levels than QUAL, potentially influencing difference in how individuals rated frequency of participation in Priv- PEB-5 which asks about donating money to support environmental protection. Given the PEB measures and the associated latent constructs were found to be metric non- invariant, single item loadings were released in a stepwise manner to understand whether par- tial metric invariance could be established. These items were released specifically by When item loadings were released between groups for these four items, partial metric invariance was supported as changes in fit indices fell within all appropriate thresholds (S2 = 742.186, df = 93, p<0.001; RMSEA = 0.050; SRMR = 0.0615; CFI = 0.940). Releasing factor loadings for four items was considered appropriate as previous research has indicated that constraints should be maintained on at least half of the items for partial metric invariance to PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 12 / 22 PLOS ONE Measuring pro-environmental behavior in the United States Fig 1. Confirmatory factor analysis diagram for QUAL with standardized estimates; global fit indices: S2 = 211.082, df = 43, p<0.001; RMSEA = 0.061; SRMR = 0.044; CFI = 0.969. Fig 1. Confirmatory factor analysis diagram for QUAL with standardized estimates; global fit indices: S2 = 211.082, df = 43, p<0.001; RMSEA = 0.061; SRMR = 0.044; CFI = 0.969. https://doi org/10 1371/journal pone 0274083 g001 https://doi.org/10.1371/journal.pone.0274083.g001 be confirmed [78, 79]. Partial metric invariance indicates that item loadings were generally comparable across the LNT and QUAL groups, excluding the four items that were allowed to vary between groups (theoretical justification in why these items may not load in a similar pat- tern across groups being outlined previously). Given poor model fit and change in fit indices greatly exceeding appropriate thresholds when constraining for item intercepts, it was con- firmed that the items were scalar noninvariant across the two groups. Model fit indices are fur- ther outlined in Table 6. Achieving partial metric invariance, but not scalar invariance, indicates that the proposed scale behaves similarly across the two groups, but some limitations exist in this similarity. The four indicated items (Priv-PEB-2, Pub-PEB-1, Pub-PEB-3, and be confirmed [78, 79]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 5.4 Measurement invariance Partial metric invariance indicates that item loadings were generally comparable across the LNT and QUAL groups, excluding the four items that were allowed to vary between groups (theoretical justification in why these items may not load in a similar pat- tern across groups being outlined previously). Given poor model fit and change in fit indices greatly exceeding appropriate thresholds when constraining for item intercepts, it was con- firmed that the items were scalar noninvariant across the two groups. Model fit indices are fur- ther outlined in Table 6. Achieving partial metric invariance, but not scalar invariance, indicates that the proposed scale behaves similarly across the two groups, but some limitations exist in this similarity. The four indicated items (Priv-PEB-2, Pub-PEB-1, Pub-PEB-3, and PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 13 / 22 PLOS ONE Measuring pro-environmental behavior in the United States Fig 2. Confirmatory factor analysis diagram for LNT with standardized estimates; global fit indices: S2 = 415.713, df = 43, p<0.001; RMSEA = 0.071; SRMR = 0.047; CFI = 0.929. Fig 2. Confirmatory factor analysis diagram for LNT with standardized estimates; global fit indices: S2 = 415.713, df = 43, p<0.001; RMSEA = 0.071; SRMR = 0.047; CFI = 0.929. Fig 2. Confirmatory factor analysis diagram for LNT with standardized estimates; global fit indices: S2 = 415.713, df = 43, p<0.001; RMSEA = 0.071; SRMR = 0.047; CFI = 0.929. htt //d i /10 1371/j l 0274083 002 https://doi.org/10.1371/journal.pone.0274083.g002 https://doi.org/10.1371/journal.pone.0274083.g002 Pub-PEB-5) load differently on the Private Behaviors and Public Behaviors latent constructs. Additionally, failing the test for scalar invariance indicates that an increase or decrease in an item’s intercept does not uniformly influence that latent construct between the LNT and QUAL groups. While partial metric invariance indicates that the developed scale behaves simi- larly across the two groups of interest, limitations to this similarity should be acknowledged. PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 6. Discussion This study provides researchers and practitioners with a reliable and valid scale to measure general PEB in the United States across groups with different levels of involvement in pro- environmental organizations or activities. Results mostly supported scale reliability and valid- ity for both the LNT and QUAL groups. Additionally, partial metric invariance also indicated a level of consistency in how PEB was perceived across the different populations as well. These findings may allow for scale comparability across these different groups, though it should be noted that not all factor loadings and intercepts related to latent constructs in the same manner across groups. This scale overcomes limitations generally attributed to other measures of PEB in prior research, primarily a lack of rigorous psychometric testing in populations of interest to encourage use across studies. In this generalized scale, the sub-scale structure is split between private and public behaviors. This represents a simplification in how behaviors have been categorized when compared to previous scales measuring PEB, which have included a larger number of behavioral sub-scales [18, 23, 33, 81]. Indicators of reliability and validity for the developed scale were appropriate across both a population of individuals involved in a pro-environmental organization and a general popula- tion of individuals in the United States. Establishing these metrics between the two groups rep- resents an important step forward in developing a scale measuring self-reported PEB. This is especially important as many studies measure behaviors within or between populations that have a range of involvement in environmental organizations, outdoor recreation activities, or psychosocial factors related to the behaviors themselves [25, 26, 44]. The establishment of par- tial metric invariance for the developed scale across these two populations further indicates that there is a level of psychometric stability across these divergent groups as well [28]. It is helpful to establish this consistency in the psychometric performance of the scale as differences in self-reported PEB across groups in future studies can more definitively be linked to changes in variables of interest rather than unaccounted for differences in how the scale is interpreted between groups [28]. Specifically, for this study, various practical differences between the QUAL and LNT groups may explain why full metric invariance was not achieved. PLOS ONE PLOS ONE Table 6. Models testing measurement invariance for QUAL and LNT samples. Model S2 df p RMSEA SRMR CFI Configural 626.786 86 <0.001 0.048 0.0471 0.950 Partial Metric 742.186 93 <0.001 0.050 0.0615 0.940 Metric 800.686 97 <0.001 0.051 0.0687 0.935 Scalar 1433.003 108 <0.001 0.067 0.1026 0.877 Partial metric invariance released constraints on four item loadings: Priv-PEB-2, Pub-PEB-1, Pub-PEB-3, and Pub-PEB-5 Table 6. Models testing measurement invariance for QUAL and LNT samples. Partial metric invariance released constraints on four item loadings: Priv-PEB-2, Pub-PEB-1, Pub-PEB-3, and Pub-PEB-5 Private Behaviors sub-scale in QUAL was 0.82, while the reliability measure for the Public Behaviors sub-scale in the same group was 0.90. For LNT, the Private Behaviors sub-scale had a reliability of 0.74. The Public Behaviors sub-scale had a reliability of 0.80 in LNT. Private Behaviors sub-scale in QUAL was 0.82, while the reliability measure for the Public Behaviors sub-scale in the same group was 0.90. For LNT, the Private Behaviors sub-scale had a reliability of 0.74. The Public Behaviors sub-scale had a reliability of 0.80 in LNT. Private Behaviors sub-scale in QUAL was 0.82, while the reliability measure for the Public Behaviors sub-scale in the same group was 0.90. For LNT, the Private Behaviors sub-scale had a reliability of 0.74. The Public Behaviors sub-scale had a reliability of 0.80 in LNT. Private Behaviors sub-scale in QUAL was 0.82, while the reliability measure for the Public Behaviors sub-scale in the same group was 0.90. For LNT, the Private Behaviors sub-scale had a reliability of 0.74. The Public Behaviors sub-scale had a reliability of 0.80 in LNT. 5.5 Reliability analysis Scale reliabilities were calculated using Cronbach’s Alpha [80] for each of the Private Behaviors and Public Behaviors sub-scales for both QUAL and LNT. The reliability measure for the PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 14 / 22 Measuring pro-environmental behavior in the United States 6. Discussion In developing this novel measure, a bi-dimensional sub-scale structure, in line with the public/private split highlighted by some other PEB research [24, 77], is supported as a psycho- metrically sound way to understand the multi-dimensional nature of PEB across these differ- ent groups. This is somewhat unsurprising given the significant role social norms have been found to play in influencing how PEB is enacted in various circumstances [31, 82, 83]. Public behaviors are generally social in nature, and this may be a contributing factor to the underlying PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 15 / 22 PLOS ONE Measuring pro-environmental behavior in the United States sub-scale structure. Despite the influence social norms have been found to have on PEB, they have not been the primary factor determining the structure of a widely used PEB scale previ- ously. Though this sub-scale structure has not been represented in previous developed scales (in contrast to more theoretical work on the nature of PEB; [31, 82, 83]), measuring general PEB through these two dimensions was determined to be psychometrically consistent across LNT and QUAL groups. g In looking to other PEB measures utilizing sub-scales that have received widespread use previously, the bi-dimensional structure of the scale developed in this study generally repre- sents a simplification in categorizing self-reported behaviors. Markle [18] asserts PEB should be measured via four sub-scales (conservation, environmental citizenship, food, and transpor- tation), while Kaiser [81] asserts there should be seven sub-scales (prosocial behavior, ecologi- cal garbage removal, water and power conservation, ecologically aware consumer behavior, garbage inhibition, volunteering in nature protection activities, and ecological automobile use). Evidence from this study suggests that these previously established measures may over specify the multi-dimensional nature of PEB when understanding it in the broad context of the general United States population. For example, in an alternative conceptualization of PEB by Larson et al. [23], “land stewardship” was an important aspect of PEB that is notably absent from the scale developed in this study. Given that Larson et al. [23] developed their scale within a rural population in the United States, accessibility or ownership of natural areas is likely more widespread throughout their population. When understanding PEB within the broader population of the United States, these behaviors may not be as commonplace due to restricted access to natural areas. 6. Discussion For example, composting behavior was eventually removed from our scale due to its cross-loading between sub-scales amongst the QUAL group in the exploratory factor analysis, indicating there may be some confounding factors influencing how this behavior is perceived and enacted. This aligns with findings by Huddart-Kennedy et al. [51], suggesting that land stewardship behaviors (a broader PEB category that can include composting) are enacted less frequently in populations outside of rural areas. While generaliz- ing to the United States population may lead to a reduced level of nuance within the sub-scale structure of the developed measure, two dimensions were found to be reliable and valid across both the LNT and QUAL populations. In comparison to previously developed scales, the scale developed here may oversimplify the nature of PEB to a degree. There is certainly less breadth of behaviors represented in the final scale in comparison to the initial 27 items utilized in the pilot study (outlined in S1 Appendix). This simplification is somewhat of a trade-off for other aspects of the developed scale that may be beneficial: the generally stable psychometric proper- ties across groups with varying environmental orientations and its brief nature. Additionally, findings from this study further support the assertion made by Larson et al. [23] that PEB is not a unidimensional construct and therefore should not be measured as such. This simplified practice diminishes the behavioral and psychological complexity of PEB, a layer of intricacy that should not be lost if future studies are to most effectively understand PEB. 7. Limitations Several limitations must be acknowledged when drawing conclusions and implications from the findings of this study. Firstly, the PEB scale was developed solely within populations resid- ing in the United States. The behaviors and associated sub-scale structure are therefore repre- sentative of how PEB is perceived and enacted in the United States. Application of this scale to other populations should be done with care and additional psychometric testing, acknowledg- ing that the associated social and psychological factors influencing PEB may differ across cul- tures and locations. This newly developed scale may provide direction in how to measure PEB PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 16 / 22 PLOS ONE Measuring pro-environmental behavior in the United States in other populations, but measurement invariance should be established first [28]. Establishing such consistency across different countries and regions represents a potential step forward for future research. Secondly, while partial metric invariance was established between the LNT and QUAL groups, these findings indicate that divergences remain in how the developed scale is perceived and interpreted between the two different groups. While this degree of psychometric stability between groups presents a considerable step forward in developing a scale that can be used across populations with differing levels of involvement in environmentally-oriented causes, there remain limitations to the scale’s performance as well. Item loadings on latent constructs across groups were not fully stable, and difference in intercepts did not correspond to uniform differences in latent constructs across the two groups. This indicates that future applications of this measure across groups with varying involvement in pro-environmental organizations may need to consider that some differences may be a result of how sub-scales are perceived rather than true differences in behavior. Thirdly, it should be noted that this study did not utilize a new sample to further confirm scale structure after the primary analyses done across the QUAL and LNT groups. Utilizing an additional sample to determine the scale structure would further support the psychometric sta- bility of the developed PEB scale. Despite the potential benefits of recruiting an additional pop- ulation of individuals to test scale properties, the three populations (pilot study group, QUAL, and LNT) utilized throughout the presented study suggest initial evidence for utilizing the items and sub-scale structure in future research. 7. Limitations While the presented scale could further be confirmed in a new group of individuals, this step may only add marginal evidence for scale stability and structure. Given limited time and resources allocated to the scale development process, this additional step may not be justified by the amount of additional information that it would provide. 8. Conclusion While a range of studies have been focused on how to measure self-reported PEB previously, this study represents a step forward in developing a psychometrically sound scale that can be more readily utilized in populations with a range of orientations towards the natural environ- ment. While some previous PEB studies have aimed to establish measurement consistency across different populations such as between individuals residing in different countries [8], this scale development process took the novel approach of developing a scale that is explicitly designed to be consistent across populations with different levels of involvement with a pro- environmental organization. Additionally, the range of behaviors measured relative to the scale’s short length (11 items) represents a tool for future researchers to readily incorporate into future studies without significantly increasing measurement burden. Considering the plethora of research focused on understanding and encouraging PEB within the field of environmental psychology, the use of a consistent scale across these studies can aid greatly in more holistically understanding trends across and between studies. This, in turn, allows for researchers and practitioners to better develop a body of collective knowledge on how to encourage PEB in various populations. Developing consistent, reliable, and valid measurement practices represents a necessary step towards achieving that goal. S1 Data. Anonymized data utilized in study procedures. (CSV) S1 Data. Anonymized data utilized in study procedures. (CSV) Acknowledgments The views expressed in this article are the responsibility of the authors and do not necessarily represent the opinions or policy of the National Park Service. The views expressed in this article are the responsibility of the authors and do not necessarily represent the opinions or policy of the National Park Service. Author Contributions Conceptualization: Timothy J. Mateer, Zachary D. Miller, Ben Lawhon, Jennifer P. Agans, B. Derrick Taff. Data curation: Timothy J. Mateer, Jennifer P. Agans, B. Derrick Taff. Formal analysis: Timothy J. Mateer, Theresa N. Melton, Zachary D. Miller, Jennifer P. Agans. Formal analysis: Timothy J. Mateer, Theresa N. Melton, Zachary D. Miller, Jennifer P. Agans. Funding acquisition: Ben Lawhon, B. Derrick Taff. Formal analysis: Timothy J. Mateer, Theresa N. Melton, Zachary D. Miller, Jennifer P. Agans. Funding acquisition: Ben Lawhon, B. Derrick Taff. Investigation: Zachary D. Miller, Ben Lawhon. Methodology: Timothy J. Mateer, Theresa N. Melton, Zachary D. Miller, Ben Lawhon, Jenni- fer P. Agans. Project administration: Timothy J. Mateer, Ben Lawhon, B. Derrick Taff. Supervision: Ben Lawhon, B. Derrick Taff. Writing – original draft: Timothy J. Mateer, Theresa N. Melton. Writing – review & editing: Timothy J. Mateer, Theresa N. Melton, Jennifer P. Agans, B. Der- rick Taff. Supporting information 17 / 22 17 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0274083 October 4, 2022 PLOS ONE Measuring pro-environmental behavior in the United States S1 Appendix. (DOCX) S2 Appendix. (DOCX) S3 Appendix. (DOCX) References 1. 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https://openalex.org/W3091516960	https://journal.unnes.ac.id/nju/index.php/jpii/article/download/25046/10782	English	null	Investigating the Purposes of Thought Experiments: Based on the Students’ Performance	Jurnal Pendidikan IPA Indonesia/JPPI : Jurnal Perndidikan IPA Indonesia	2,020	cc-by	7,068	http://journal.unnes.ac.id/index.php/jpii http://journal.unnes.ac.id/index.php/jpii Correspondence Address E-mail: hartono.b.b@unismuh.ac.id ABSTRACT The objective of this study is to examine the students’ purposes in conducting thought experiments while solv ing physics problems. There were 12 pre- and in-service physics teachers as the participants in this study and divided into three groups with four students in each. Physics problem-solving activities were used as a context for observing students’ processes in doing TEs. The results of the data analysis showed that there were three types of purposes of the students in doing the TEs during physics problem-solving activities: prediction, verification, and explanation. Therefore, it can be concluded that in the context of problem-solving, students design and run thought experiments as a creative ability tool to (1) predict solutions to the problem, (2) verify whether their hy pothesis is correct or incorrect, (3) provide a detailed explanation to their hypothesis. Based on this study, we dis cuss the differences and similarities in the purposes of scientists and students in doing thought experiments. The importance and implications of thought experiments for current and future physics teachers are also discussed in the last part of this study. © 2020 Science Education Study Program FMIPA UNNES Semarang Keywords: physics teacher; problem-solving; purpose; thought experiment JPII 9 (3) (2020) 351-360 JPII 9 (3) (2020) 351-360 © 2020 Science Education Study Program FMIPA UNNES Semarang H. Bancong1 and J. Song2 Physics Education Department, Universitas Muhammadiyah Makassar, Makassar, Indonesia 2Center for Educational Research, Seoul National University, Seoul, South Korea 1Physics Education Department, Universitas Muhammadiyah Makassar, Makassar, Indonesia 2Center for Educational Research, Seoul National University, Seoul, South Korea INTRODUCTION Therefore, the objective of this study is to examine the purposes of students in doing TEs. In our previous study (Bancong & Song, 2020b), we had explored the processes of TEs carried out by students when they were working colla boratively to solve physics problems provided. The findings revealed that given the context of collaborative problem-solving activities, students could design, discuss, share opinions, rethink, and review their TEs, which signifies that the TEs can be established and directed together even though they initially appear in personalized ver sions. Furthermore, the study also showed that in the process of evaluating the results of TEs, students employed four sources of evaluation, na mely conceptual understanding, past-daily expe rience, logical reasoning, and conceptual-logical inference (Bancong & Song, 2020b). This study, therefore, is a follow up to the previous one. In this study, we present physics problems for small groups of students. While solving the problem, we carefully observe the processes of students doing TEs and investigating their purposes. So, the research question in this study was, “what are the students’ purposes in conducting TEs while solving physics problems?” y Since TEs have a central role in the his tory of science and philosophy (Machery, 2011; Asikainen & Hirvonen, 2014; Arfini et al., 2019), some researchers are interested in studying the role of TEs in education, especially physics edu cation. For instance, Velentzas & Halkia (2011) implemented a TE of Heisenberg’s Microscope as an instructional tool for teaching uncertainty principles to upper secondary education students. They also implemented the TE of Einstein’s eleva tor and Einstein’s train for teaching the theory of relativity (Velentzas & Halkia, 2013). The results showed that the utilization of TEs could enable students to recognize the situations which refer to a world outside of their daily. The use of TEs can also reveal students’ hidden reasoning when working on complex physics problems (Kösem & Özdemir, 2014) and can deepen their understan ding of physics concepts (Klassen, 2006; Galili, 2009). Several physics researchers have carefully investigated the use of TEs in high school physics textbooks (e.g., Bancong & Song, 2018). Recent ly, Bancong & Song (2020a) has identified the fac tors that influence students’ in constructing TEs: students’ bodily knowledge, students’ imaginary visual knowledge, opposing ideas, similar ideas, and support from group mates. These five factors encourage students to do TEs when they are sol ving physics problems collaboratively. INTRODUCTION physics, especially in the fields of relativity and quantum mechanics. Throughout the history of physics, thought experiments (TEs) plays an essential role in the development of physics theories. There are seve ral examples where physicists used TEs to sup port their hypotheses or to criticize existing the ories. For example, Albert Einstein introduced a TE from a train to support his hypothesis about time dilation (Einstein, 1905) or Erwin Schrödin ger introduced a TE about a cat in a sealed box that may be simultaneously both alive and dead in order to refute Copenhagen’s interpre tation of quantum superposition (Schrödinger, 1935). Einstein’s train, Einstein’s twin paradox, Schrodinger’s cat, Heisenberg’s microscope, and EPR paradox are just a few examples of popu lar TEs used by physicists in developing modern According to the most quoted definition, TEs are experiments in the mind laboratory that involve mental activities (Brown, 1991). TEs are a continuum of real experiments (REs) that are car ried out in the mind’s laboratory and be observed using the mind’s eye (Galili, 2009; Buzzoni, 2013; 2019; Stuart, 2016) as opposed to the ideas that TEs is fiction (e.g., Elgin 2014; Meynell, 2014; Cameron, 2015). Marco Buzzoni, in a number of his effort, has been trying to convince us that TEs and REs are identical in principle and at least in physics; TEs are impossible without REs, and vice versa (e.g., Buzzoni, 2013; 2019). He argued that “(empirical) TEs without REs are empty; REs without TEs are blind” (Buzzoni, 2013, p. 100). TEs are thought and experimental activities (Galili, 2009; Bancong & Song, 2020b). Thought activities refer to modeling the imaginary worlds H. Bancong and J. Song / JPII 9 (3) (2020) 351-360 352 related to daily experiences of the experimenter and the scientific theories, while experiment ac tivities refer to experimental activities in the la boratory, such as manipulating objects, variables, and making observations. Reiner & Burko (2003) and Brown (2006) suggested several similar steps of TEs: visualize imaginary worlds, perform ex periments, and describe the results. These three activities as the steps of conducting TEs perfor med by individuals. a way that it can provide us with complete and more precise information of how students con duct TEs, as well as generate ideas about what needs to be done in helping students to develop and enhance their TEs experiences. INTRODUCTION In short, several studies have shown that TEs have an es sential role not only for developing physics theo ries but also for teaching and learning of physics. METHODS As mentioned earlier, this study’s objective was to examine the students’ aims in doing TEs while being involved in solving physics problems. Therefore, the qualitative approach was conside red the best methodology to achieve the objective. Merriam & Tisdell (2016) stated that qualitative research is all about creating an understanding of how people make sense out of their lives, depict the process (rather than the outcome or product) of meaning-making, and describe how people interpret what they have experienced. Likewise, Miles et al. (2014) stated that in qualitative rese arch, researchers took data about participants’ perceptions through a process of deep attention and empathic understanding. However, some questions related to the use of TEs for students in schools have not been clear ly answered. For example, if physicists generally use TEs as a creative ability device to support and refute the existing theories or to produce new the ories (Brown, 1991; Galili, 2009; Thagard, 2014; Fehige & Stuart, 2014), do students use TEs with the same goals as scientists? If not, then what is the function of this imagination tool for students as a representation of non-experts?. Previous studies have not provided a clear description of students’ goals in doing TEs. Meanwhile, under standing the process and purposes of students in conducting TEs is significantly beneficial in Participants Information about Participants Group Members Gender Status Furthermore, students’ notes and the researcher’s notes were used as additional data resources to ensure the validity of the data. The students’ notes were those written and drawn freely by the participants while doing the TEs or discussing with the other participants. Meanwhi le, observation notes collected by the researcher were outlines about the steps or procedures of TEs the students conducted during problem-sol ving activities. These two kinds of notes helped to justify TEs processes described in audio and video recordings transcripts. To sum up, there are three methods of collecting data used in this re search, namely audio and video recorded during the process of discussion, students’ notes, and the researcher’s observation notes. The following Figure 1 shows a diagram of the data collection processes. Participants Considering the purpose of the study, we selected 12 pre- and in-service physics teachers as participants. Pre-service physics teachers are prospective physics teachers who are pursuing teacher education. They are the 7th-semester stu H. Bancong and J. Song / JPII 9 (3) (2020) 351-360 353 dents at Universitas Muhammadiyah Makassar. Instead, in-service physics teachers are students who have obtained a bachelor’s degree in physics education and have taught physics at school, and for now, they are pursuing master education to improve and develop their knowledge and skills continuously. These participants were then divi ded into three groups, with four students in each group. Adhering to the principles of research et hics, we used only the initials of the participants’ names to protect their identity. Additional infor mation regarding the participants in each group can be seen in Table 1. physics, which was designed to activate students’ imaginary world so that it allows them to conduct TEs while solving these problems. We picked up some potential problems from that book for discussing. After that, we conducted a pilot stu dy with several students to evaluate whether the potential problems can make students carry out TEs while solving those problems. Based on the results, we selected the five most potential phy sics problems that stimulated and triggered the students to carry out TEs as instruments in this study. In order to collect data, first of all, we or ganized students into groups and presented the physics problems. We also provided each group with blank sheets of paper that they could use to write or to draw whatever comes up in their mind during the physics problem-solving process. In these activities, we carefully observed students’ processes in solving these problems and identified the TEs that occurred. All of these activities were recorded using two cameras, from the front and behind the participants. Because each group had to work on five problems, we conducted observa tion and recording five times for each group. Table 1. Information about Participants Group Members Gender Status 1 AN Female In-service teacher AF Female In-service teacher AE Female In-service teacher FA Female In-service teacher 2 MY Male In-service teacher AA Male In-service teacher SS Female Pre-service teacher DS Female Pre-service teacher 3 NN Female Pre-service teacher RR Female Pre-service teacher NI Female Pre-service teacher MH Male Pre-service teacher Instrument and Procedures Table 1. Instrument and Procedures Several studies have mentioned that both scientists and students used TEs as an imagina tion tool when they are solving problems (Cle ment, 2009; Kösem & Özdemir, 2014). Therefo re, we used physics problem-solving activities to see how students deal with the problems by using TEs. Those physics problems were adapted from a book entitled Thinking Physics Is Gedanken Physics, written by Epstein (1995). The term “Gedanken experimente” was used in the book as a synonym for TE. This book provides a set of problems in Figure 1. The Process of Data Collection Figure 1. The Process of Data Collection Figure 1. The Process of Data Collection H. Bancong and J. Song / JPII 9 (3) (2020) 351-360 354 Merriam & Tisdell (2016) suggest that re searchers act as participant-observers in collec ting qualitative data. By using this method, the researchers have sufficient space to access many people and detailed information. However, we realized that the involvement of the researcher might create effects on the process. process were then clustered under some specific categories. We read the transcripts repeatedly to ensure that we found all intentions conveyed by the students either implicitly or explicitly. Final ly, through the deductive method, the three main categories of students’ goal for doing TEs when solving physics problems were grouped into pre diction, verification, and explanation. Therefore, to reduce the possibility of inte ractive discourse occurred between the researcher and the participants in this study, we minimized the intervention. In this case, the researcher enga ged in dialogue only when needed, as the students were sometimes confused about what to do. The forms of interventions that the researcher carried out were asking participants if the problem given was clear or unclear, responding to participants’ questions related to the problem, asking them what they thought about something to clarify their thinking, and asking them about what they can conclude after solving the problem given. p Miles et al. (2014) suggested peer review as a technique of checking the reliability of the analysis. Therefore, we invited two physics edu cation researchers to identify the processes and purposes of students conducting TEs. The two researchers separately analyzed 75% of the total transcripts of the audio and video recordings that were obtained. We examined and organized the results of the analysis until about 92% agreement is reached. Data Analysis As the primary data sources were the au dio and video recordings of each group, transc ribing activity was the first step done in the data analysis process. In this step, not a single utteran ce in the conversations carried out by participants during the physics problem-solving activities was missed. For the next step, we reviewed the audio and video recordings in order to obtain a more in- depth insight of the TEs that appeared after iden tifying the TEs features in the transcripts of episo des of physics problem-solving activities. When we recognized a discourse that looked like a TE, we paid more attention to the episode in the vi deo. Third, the discourse that has been identified as TEs in the second step was then confirmed by using the researcher’s observational notes and students’ notes. These two kinds of notes helped to determine and highlight the TEs parts in the data transcripts. We then watched and listened to the video repeatedly to make sure that there are no TEs episodes that are missed from the second step. Instrument and Procedures We also discussed the results with the participants to check whether there was any mi sinterpretation of the data. Besides, we also re ceived positive responses from academics when presenting the findings of this study at an inter national conference. Table 2. Distribution of the Students’ Purposes in Conducting TEs 1Imagine if we are inside a ship that is straight-moving at a constant speed. But, because we cannot see outside, we do not know whether we are moving or not. 2If I drop something, a pen, or whatever in the ship. Let us say gravity exists there, 3then the thing I drop will fall straight down, not left behind. 2 NI: Yeah, we will not realize that we are moving. 3 NN: Yes, it is similar to this [the situation in the Problem] 4 MH: So, for me, the woman who feels that she is not moving is the one in the straight-moving box. How about you guys? 5 R= Researcher; 1 = visualize imaginary world; 2 = perform experiment; 3 = describe the results RESULTS AND DISCUSSION The results of the data analysis showed that predicting, verifying, and explaining are three main reasons for students doing TEs while working on physics problems. When the students utilized a TE to deal with the challenge they are working on, the motivation behind students’ TE goals is coded as “prediction.” Conversely, when students utilized a TE to check whether their as sumption or hypothesis is correct or incorrect, at that point, the reason for students’ TE is coded as “verification.” Lastly, when students utilized a TE to give further clarifications about their as sumption or hypothesis, at that point, the motiva tion behind students’ TE goals is coded as “exp lanation”. Table 2 below presents the distribution of the students’ purposes in doing TEs while solving physics problems. As we can see, 4 of the 8 TEs carried out by group 1 were used to provide furt her explanation for their hypothesis. The others are used to predict the answers to the given ques tions and to prove their hypotheses. When the re searcher finished reading the problem, group 1, whose members were all in-service physics teach ers, tended to propose hypotheses or assumptions first as answers to the problems they faced. Their hypothesis was based on their prior knowledge or experience. They then designed and conducted TEs to be used as a creative ability device to pro vide detailed explanations of their hypothesis. Reiner & Burko (2003) and Brown (2006), as referred earlier, suggested three procedures of TEs generally performed by individuals while solving physics problems, namely 1) illustrating imaginary worlds, 2) conducting experiments, and 3) explaining the results. After determining the TEs episodes in the transcript, we went furt her to analyze the students’ objectives in conduc ting TEs. To conduct the analysis, Miles et al. (2014) proposed coding activity as the first step to summarize parts of the data in the raw transcrip tions. The codes resulted in the first cycle coding H. Bancong and J. Song / JPII 9 (3) (2020) 351-360 355 Table 2. Distribution of the Students’ Purposes in Conducting TEs Purposes of TEs Participant/Problem (P) Group 1 Group 2 Group 3 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 Prediction ** * * * Verification * * * ** * Explanation * * * * * ** * * Table 2. Table 2. Distribution of the Students’ Purposes in Conducting TEs Furthermore, 4 of the 7 TEs generated by group 2 during physics problem-solving activities were also used to provide further explanation of their hypothesis. The rest of the TEs generated by group 2 were used to prove their hypothesis as the temporary answers to the problems they faced. Group 2 members were a combination of in-service and pre-service physics teachers. to the problem. The transcripts presented below is a piece of the transcript of problem-solving activities from group 3 while solving the second problem. The problem presented an illustration of two scientists sitting in different boxes. The students were required to think and determine which scientist in can recognize her motion in the space: the scientist who is in the straight-moving box or the one in the smoothly-turning box, as illustrated in Figure 2. Conversely, 7 out of 9 TEs generated by Group 3 are instead used to predict the solution to the problems they face. There was only 1 TE generated by group 3, which aims to verify their hypothesis and another one to provide a more detailed explanation of their hypothesis. The members of group 3 were all pre-service physics teachers. When the researcher has read out the problem, group 3 members generally did not have an assumption or a hypothesis as a temporary answer to the problem. Thus, group 3 tends to directly visualize the imaginary world and then design a TE when they solved the problem. The results of their TE was then used as a solution to the problem they faced. The further explanation of these three purposes of TEs is presented in the sections below. g Figure 2. Scientists in the Box as an Illustration of Problem 2. Adapted from Thinking physics is Gedanken Physics (p. 104), by L. C. Epstein, 1995, insight press Figure 2. Scientists in the Box as an Illustration of Problem 2. Adapted from Thinking physics is Gedanken Physics (p. 104), by L. C. Epstein, 1995, insight press R: We move to Problem 2 [the researcher then reads the question]. So, what do you think about this problem? Which scientist can detect her motion? 1 R: R: We move to Problem 2 [the researcher then reads the question]. So, what do you think about this problem? Which scientist can detect her motion? 1 [. . .] MH: Aha, I think the situation is like this. RESULTS AND DISCUSSION Distribution of the Students’ Purposes in Conducting TEs Prediction h The results of data analysis showed some evidence that students used TEs as a creative abi lity tool in guessing the answers to the questions given. In this case, when students were faced with physics problems, they did not immediate ly propose hypotheses or assumptions as tempo rary answers to the problems. Instead, students first used their imagination, designed a TE, and described the results. Hadzigeorgiou (2016) sta ted that although one can imagine without being creative, one cannot be creative without being imaginative. Students then used the results of the TE as solutions to the problem given. The words, such as “I think the situation is like this,” “that it is,” or “aha,” are typical words that were often said by students when they were designing and running TE to predict solutions to the problems they face. The following is an example in which the student utilized a TE to presume an answer MH: Aha, I think the situation is like this. 1Imagine if we are inside a ship that is straight-moving at a constant speed. But, because we cannot see outside, we do not know whether we are moving or not. 2If I drop something, a pen, or whatever in the ship. Let us say gravity exists there, 3then the thing I drop will fall straight down, not left behind. 2 H. Bancong and J. Song / JPII 9 (3) (2020) 351-360 356 Figure 3. Trolley in the Rain as an Illustration of Problem 1. Adapted from Thinking physics is Gedanken Physics (p. 87), by L. C. Epstein, 1995, insight press As we can see in line 2, MH was doing a TE to find out the answer to the given problem. MH immediately imagined an imaginary world by saying, “I think the situation was like this” when the researcher finished reading the prob lem (Line 1). At this moment, MH did not have a hypothesis or a prior assumption as a solution to the question given. MH suddenly visualized an imaginary world, and then designed and run experiments in his mind and achieves the results. The words “imagine if” were highlighted as a sign of visualization, which indicates that MH was starting to visualize an imaginary world. As expected, MH visualized an imaginary world by imagining himself inside a ship that moves straight ahead. Prediction h In my view, the speed of the trolley will get affected. 1Imagine if we played with a toy car. Imagine If the toy car is launched on an inclined plane. 2If I push this trolley, consider a toy car [pointing to the trolley’s image on the problem given], 3then it will slide, right? Now, 1imagine if 2we filled this toy car with stones, for example. 3Its speed is faster than those that are not filled, right? So, a toy car that is filled with stones will slide faster than those that are not filled. 6 Prediction h Because MH could not see out side, he did not know whether he was moving or not. In this situation, MH then experimented by dropping several pens on a ship that was moving at a constant speed. MH saw the pen that was dropped would still fall perpendicularly by using his mind’s eye, just like when the ship was still not moving. MH then used the result of this TE as a solution to the problem given. MH then con cluded that the scientist in the straight-moving box would not feel that she is moving. The case described above shows that the students make use of the TEs as a tool of imagination to figure out the solution to the problem as stated by Sorensen (2016), TEs as a tool of the imagination for inves tigating the nature of science. Figure 3. Trolley in the Rain as an Illustration of Problem 1. Adapted from Thinking physics is Gedanken Physics (p. 87), by L. C. Epstein, 1995, insight press Figure 3. Trolley in the Rain as an Illustration of Problem 1. Adapted from Thinking physics is Gedanken Physics (p. 87), by L. C. Epstein, 1995, insight press AA Well, in my opinion, there is the effect of rainwater on the trolley. 2 AA Well, in my opinion, there is the effect of rainwater on the trolley. 2 SS Really? For me, not. The speed of the trolley will remain constant. 3 SS Really? For me, not. The speed of the trolley will remain constant. 3 MY So, is there no effect of rainwater on the trol ley’s speed? 4 MY So, is there no effect of rainwater on the trol ley’s speed? 4 SS Yes, I think so. Right [while looking at AA]? 5 AA No. In my view, the speed of the trolley will get affected. 1Imagine if we played with a toy car. Imagine If the toy car is launched on an inclined plane. 2If I push this trolley, consider a toy car [pointing to the trolley’s image on the problem given], 3then it will slide, right? Now, 1imagine if 2we filled this toy car with stones, for example. 3Its speed is faster than those that are not filled, right? So, a toy car that is filled with stones will slide faster than those that are not filled. 6 AA No. Verification Like this 6 [This picture shows a circular path of the objects in the trajec tory written by MY while working on problem 3] Verification The results of data analysis also show that students used TEs as a creative ability device to verify their hypotheses that have been proposed earlier as a temporary answer to a given problem. The students constructed a hypothesis based on their daily experiences or scientific theories. As the researcher completed the reading of the prob lem, some students put forward assumptions or hypotheses first as a solution to the problem they are facing. They then conducted TEs to verify whether their assumptions or hypotheses were correct or incorrect. The words, such as “right?” “maybe?” “Won’t we?” or “ isn’t it “ are typical words that were often said by participants when they were designing and running TEs to verify their hypotheses or assumptions. The transcript presented below when group 2 was working on problem 1 is an example of this case. Problem 1 was asking whether there is the effect of rain on the trolley’s speed, as illustrated in Figure 3. MY As we can see in the transcript, AA did a TE (Line 6) to prove his hypothesis that the speed of the trolley will be affected (Lines 2 and 6). At the beginning of the discourse, all participants H. Bancong and J. Song / JPII 9 (3) (2020) 351-360 357 closest to the trajectory of the after coming out of point 2, as illustrated in Figure 4. closest to the trajectory of the after coming out of point 2, as illustrated in Figure 4. actively participated in understanding the prob lem. When MY asked AA, AA said that there is an effect of rainwater on the trolley’s speed (Line 2). SS suddenly responded and rejected AA’s assumption. According to SS, the trolley’s speed will always remain constant in the heavy rain (Line 3). It means that there is no effect of rainwater on the trolley’s speed. However, AA rejected SS’s opinion and hypothesized that the rainwater would affect to the trolley’s speed. To prove his hypothesis, AA then experimented with his mind. actively participated in understanding the prob lem. When MY asked AA, AA said that there is an effect of rainwater on the trolley’s speed (Line 2). SS suddenly responded and rejected AA’s assumption. According to SS, the trolley’s speed will always remain constant in the heavy rain (Line 3). It means that there is no effect of rainwater on the trolley’s speed. Verification However, AA rejected SS’s opinion and hypothesized that the rainwater would affect to the trolley’s speed. To prove his hypothesis, AA then experimented with his mind. Figure 4. Ball in the Semicircular Channel as an Illustration of Problem 3 Figure 4. Ball in the Semicircular Channel as an Illustration of Problem 3 The words “imagine if” uttered by AA (line 6) were indicators of visualization. It indi cated that AA was starting to visualize an imagi nary world. As we can see in the transcript above, AA experimented with his mind by launching the toy cars. First, AA visualizes an imaginary world by imagining himself launching toy cars that were filled with stones and without stones on an inclined plane. By using his mind’s eye, AA then observed the speed of these two toy cars. AA noted that a toy car filled with stones would slide faster than those without stones. This TE proved that its mass influences the velocity of an object. Similar to the trolley that moves in heavy rain. The speed of the trolley will slow down and con tinue to slow down until it may stop because its mass increases continuously. After conducting a TE, AA then invited the rest of the team to ret hink and review the opinion together to achieve mutual understanding. The example described above shows that students use TEs as an imaging tool to verify their hypothesis. R . . . So, which is the path of the ball after exiting at point 2? 1 MY I think it is B. 2 DS Yes, B. 3 R Why? 4 MY Because of the vector of velocity. 1if, try to imagine, for example, 2the ball is suspended and is rotating above [while demonstrating it with his hand] 3then is suddenly released. The ball is pointing out, like B. That is what is in my mind, what about you? 5 [. . .] MY Yes, the direction of the velocity is always like that. Its direction is always perpendicu lar to radial acceleration. Like this 6 [This picture shows a circular path of the objects in the trajec tory written by MY while working on problem 3] MY MY Yes, the direction of the velocity is always like that. Its direction is always perpendicu lar to radial acceleration. Explanation Lastly, the results of the data analysis sho wed that some students designed and conducted TEs to be used as a creative ability device in pro viding further explanations for their hypotheses. For this purpose, students did not directly desig ned and run TEs when they are facing a prob lem but instead proposed hypotheses first. They constructed hypotheses based on scientific theo ries or their daily experiences. After constructing hypotheses as to the temporary answer to the problem, they then run TEs to be used to support their hypothesis. The words, such as “to illustra te,” “for instance,” and “for example,” are typical words that were often said by students when they were designing and running TEs for this purpose. The transcript presented below when group 2 was working on problem 3 is an example in which the students used TE to provide more explanation for their hypothesis. Problem 3 asked about a hori zontal plane, which of the path representations is [This picture shows a circular path of the objects in the trajec tory written by MY while working on problem 3] After listening carefully to the problem read by the researcher (Line 1), MY suddenly hy pothesized that the solution to the problem given was B, as shown in the transcript above. DS also supported MY’s assumption by saying, “yes, B.” These two participants proposed answers to the issue provided without any attempt to do TE first. When the researcher asked, why did you choose B ?, MY answered that it is the direction of the velocity vector (Lines 2-5). This hypothesis pro posing by MY was based on his prior knowledge when studying physics. MY then did a TE by first H. Bancong and J. Song / JPII 9 (3) (2020) 351-360 358 saying the word “for example” to provide further explanation of his hypothesis. students carried out more than one TEs for one physics problem, as shown in Table 2. This may indicate that TE is a natural process that occurs suddenly as a reaction to the problem. As we can see, MY visualized the imagi nary world by using the words “if” and “try to imagine.” He then experimented with his mind and showed with his left-hand spinning above. In his experiment, MY tied a ball with a rope then rotated it on the top. REFERENCES The result of this study also shows that both scientists and students use TEs to support their hypotheses further. Students used TEs to ve rify whether their hypothesis is correct or incor rect or to provide further explanation about their hypothesis. Similarly, scientists also used TEs as cognitive tools in developing and supporting their hypotheses and theories (Galili, 2009; Thagard, 2014; Fehige & Stuart, 2014). Also, both scien tists and students used TEs as an argumentation tool. The contradictory opinions among the stu dents in the discussion may bring a positive im pact as it can stimulate them to explore different views. In the end, this situation is expected to be able to increase the students’ motivation to carry out a TE in order to find support for their ideas and, at the same time, reject their opponent’s ide as. Likewise, in some cases, physicists also used TEs to counter opposing ideas and simultaneous ly generate new ideas. TE of Galileo’s free-falling body and Einstein’s magnet and conductor are examples of this case. This kind of TE is termed as platonic TE (Brown, 1991). Arfini, S., Casadio, C., & Magnani, L. (2019). Igno rance-preserving mental models thought exper iments as abductive metaphors. Foundations of Science, 24, 391–409. Asikainen, M. A., & Hirvonen, P. E. (2014). Probing pre- and in-service physics teachers’ knowledge using the double-slit thought experiment. Sci ence & Education, 23, 1811–1833. Bancong, H., & Song, J. (2018). Do physics textbooks present the ideas of thought experiments?: A case in Indonesia. Jurnal Pendidikan IPA Indo nesia, 7(1), 25–33. Bancong, H., & Song, J. (2020a). Factors triggering thought experiments in small group physics problem-solving activities. New Physics: Sae Mulli, 70(5), 466–480. Bancong, H., & Song, J. (2020b). Exploring how stu dents construct collaborative thought experi ments during physics problem-solving activi ties. Science & Education, 29(3), 617-645. Brown, J. R. (1991). The laboratory of the mind: thought experiments in the natural sciences. New York, NY: Routledge. Brown, J. R. (2006). The promise and perils of thought experiments. Interchange, 37(1–2), 63–75. Buzzoni, M. (2013). On thought experiments and the Kantian a priori in the natural sciences: A re ply to Yiftach J.H. Fehige. Epistemologia, 36(2), 277–293. H. Bancong and J. Song / JPII 9 (3) (2020) 351-360 359 detailed explanation of their hypothesis as a tem porary answer to the problem at hand. Although both physicists and students uti lized TEs when solving complex issues, they sho wed different basis of performing TEs. Students simply design and run TEs intuitively without any plans or prior intention. The processes of the stu dents’ TEs was undoubtedly not in the same way as what the prominent scientists had been doing, such as Schrodinger’s cat, which is used by Erwin Schrodinger to disapprove the Copenhagen’s in terpretation of quantum mechanics (Schrödinger, 1935) or Einstein’s magnet and conductor to re fute Maxwell’s view of ether theory and simulta neously produced the relativity theory (Einstein, 1905). The TEs conducted by the students are less complex in which they are applied mainly to comprehend the situation in the proposed prob lem. Moreover, the TEs generated by students are mostly validated based on their experience. It is in contrast to the TEs produced by scientists that are based on robust scientific theories. Based on the results of this study, we would recommend introducing TEs to physics te achers and prospective physics teachers as it can be a useful creative ability tool for teaching phy sics at schools. Physics materials, such as thermo dynamics, quantum, and relativity theories, have great potential to introduce and integrate TEs in teaching physics at schools. We also thought for the authors of school physics textbooks would introduce and present TEs in detail. Physics tex tbooks play an important role in introducing TEs due to the tendency of today’s physics teachers to teach physics based on textbooks. Overall, TE is an essential part of the history and philosophy of physics and, therefore, an essential inclusion in the physics teaching process in schools. Explanation By using his mind’s eyes, MY saw that when the ball was released, then the ball would come out perpendicularly, like point B, on the given problem. To support the results of the TE, MY then evaluated it using the concept of circular motion (Line 6). MY believed that the ball would come out perpendicularly based on the velocity vector concept that he has learned before. In the physics concept, the acceleration vector always points to the circle center, while the velocity vector points to the direction of motion. Thus, this example shows that students perform TEs as an imaging tool to be used to provide more explanation for their hypothesis. y p The study has shown that while working on physics problems, the participants utilized TEs as a creative ability tool to guess the solution to the given problem, to verify their assumption or hypothesis, and to give detailed explanations to their assumption or hypothesis. In prediction, when participants were faced with physics prob lems, they first constructed an imaginary world and then designed and run experiments. The re sults of experiments conducted by participants in their minds were then used as solutions to the problems. In verification, when students were fa ced with physics problems, they first proposed a hypothesis or an assumption as a solution to the problem. Students’ hypothesis is based on their daily experiences or scientific theories. The stu dents then designed and run TEs to verify whet her their hypothesis or assumption was correct or incorrect. In explanation, students also proposed a hypothesis first, and then designed and run a TE to provide further explanation of their hy pothesis. Figure 5 below illustrated the students’ purposes in conducting TEs while working on physics problems. The findings of this study support the pre vious studies that both scientists and students used TEs as a cognitive tool when they are sol ving physics problems (e.g., Clement, 2009; Kö sem & Özdemir, 2014). All participants in this study—regardless of their status—were able to design and run experiments in their minds. The re are some moments in this study where the Figure 5. Illustration of the Students’ Purposes in Conducting TEs while Solving Physics Problem Figure 5. Illustration of the Students’ Purposes in Conducting TEs while Solving Physics Problem H. Bancong and J. Song / JPII 9 (3) (2020) 351-360 CONCLUSION This study concluded that in the context of problem-solving, students use TEs as a creative ability device for three purposes: prediction, veri fication, and explanation. This study has shown that students not only design and conduct TEs to predict the solutions to the problems they are fa cing but also to confirm and prove whether their hypothesis is correct or incorrect. Some students used TEs as a creative ability device to provide a Buzzoni, M. (2019). Thought experiments in philoso phy: A neo-Kantian and experimentalist point of view. Topoi, 38(4), 771–779. Cameron, R. 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On the origins of the philosophy of thought experiments: The Fore run. Perspectives on Science, 22(2), 179-220. Schrödinger, E. (1935). Die gegenwärtige situation in der Quantenmechanik (The present situation in quantum mechanics). Naturwissenschaften, 23(48), 807–812. Galili, I. (2009). Thought experiments: Determining their meaning. Science & Education, 18(1), 1–23. Hadzigeorgiou, Y. (2016). Imaginative science education: The central role of imagination in science education. Basel, Switzerland: Springer. Sorensen, R. (2016). Thought experiment and imagi nation. In A. Kind (Ed.), The routledge handbook of philosophy of imagination (pp. 420-436). Lon don, England: Routledge. Klassen, S. (2006). The science thought experiment: How might it be used profitably in the class room? Interchange, 37(1-2), 77–96. Stuart, M. T. (2016). Norton and the logic of thought experiments. Axiomathes, 26, 451–466. Kösem, Ş. D., & Özdemir, Ö. F. (2014). 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https://openalex.org/W2063710291	https://europepmc.org/articles/pmc3806743?pdf=render	English	null	Effects of 2′-O-Methyl Nucleotide Substitution on EcoRI Endonuclease Cleavage Activities	PloS one	2,013	cc-by	8,137	Introduction Nucleic acid chemistry has seen a remarkable progress in developing novel nucleotide derivatives [1]. With chemical modifications on bases, ribose or phosphate group, these nucleotide derivatives have demonstrated many unusual prop- erties. Particularly, the 29-modified nucleotides have attracted a great attention. Typical examples include 29-O-methyl nucleo- tide (29-OMeN) and locked nucleic acid (LNA). In the case of 29-OMeN, the –OH group on the C29 atom of the native nucleotide is replaced by a –OCH3 group, generating a larger steric hindrance [2]. LNA is characterized by a 29-O, 49-C- methylene-b-D-ribofuranosyl unit which creates a bicyclic ribosyl structure [3]. The most distinctive feature of 29-OMeN and LNA is that they have a constrained C39-endo pucker conformation that is dominant in A-form DNA and RNA (Fig. 1). When incorporated into oligonucleotides, LNA dem- onstrates an enhanced binding affinity toward their comple- mentary DNA targets, whereas 29-OMeN binds to complemen- tary RNA nucleotides more favorably [4,5,6]. Thus, LNA- modified oligonucleotides can improve the identification of mismatched base pairs [7] and resistance against the exonucle- ase digestion which benefits their lifetimes in vivo applications [8,9]. On the other hand, 29-OMeN exhibits a faster hybridization dynamics than usual nucleotides [10]. Further- more, both LNA and 29-OMeN demonstrate the capability of These experimental results promote our interest in knowing how these nucleotide derivatives affect the DNA-protein interac- tions, particularly, interactions with type II restriction endonucle- ases. Restriction endonuclease EcoRI presents a good model suitable for this study, since it has been characterized very well functionally and structurally [12,13,14,15]. EcoRI in a homodi- meric form recognizes the DNA palindrome sequence -GAATTC- and cleaves the -O39-P- bond between G1 and A2 positions, generating two DNA fragments with 4-nt tails at the 59-ends. This recognition is so specific that non-cognate sequences differing from the canonical recognition sequence by as little as one base-pair could be discriminated unambiguously. The crystal structure of EcoRI-d(TCGCGAATTCGCG) com- plex has furnished a great amount of information regarding the DNA-EcoRI interactions [16]. This global structure shows that, upon binding, the DNA conformation is distorted significantly from the classical B-form. The distortion, however, is not uniformly distributed over the recognition sequence; instead they concentrate in three locations known as neokinks (Fig. 2). Type I neokink lies in the center of the EcoRI recognition sequence GAA- TTC, and unwinds systematically the entire duplex by 25u. Abstract To investigate the effect of sugar pucker conformation on DNA-protein interactions, we used 29-O-methyl nucleotide (29- OMeN) to modify the EcoRI recognition sequence -TGAATTCT-, and monitored the enzymatic cleavage process using FRET method. The 29-O-methyl nucleotide has a C39-endo sugar pucker conformation different from the C29-endo sugar pucker conformation of native DNA nucleotides. The initial reaction velocities were measured and the kinetic parameters, Km and Vmax were derived using Michaelis-Menten equation. Experimental results showed that 29-OMeN substitutions for the EcoRI recognition sequence decreased the cleavage efficiency for A2, A3 and T4 substitutions significantly, and 29-OMeN substitution for T5 residue inhibited the enzymatic activity completely. In contrast, substitutions for G1 and C6 could maintain the original activity. 29-fluoro nucleic acid (29-FNA) and locked nucleic acid (LNA) having similar C39-endo sugar pucker conformation also demonstrated similar enzymatic results. This position-dependent enzymatic cleavage property might be attributed to the phosphate backbone distortion caused by the switch from C29-endo to C39-endo sugar pucker conformation, and was interpreted on the basis of the DNA-EcoRI structure. These 29-modified nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property will have potential applications in genetic engineering and biomedicine. Citation: Zhao G, Zhao B, Tong Z, Mu R, Guan Y (2013) Effects of 29-O-Methyl Nucleotide Substitution on EcoRI Endonuclease Cleavage Activities. PLoS ONE 8(10): e77111. doi:10.1371/journal.pone.0077111 Editor: Maxim Antopolsky, University of Helsinki, Finland Received June 5, 2013; Accepted August 30, 2013; Published October 23, 2013 i h h l h l d b d d h f h b h h , Tong Z, Mu R, Guan Y (2013) Effects of 29-O-Methyl Nucleotide Substitution on EcoRI Endonuclease Cleavage Activities. PLoS ONE 8(10) nal.pone.0077111 Received June 5, 2013; Accepted August 30, 2013; Published October 23, 2013 Copyright: 2013 Zhao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from the National Natural Science Foundation of China (No. 31070705) and Doctoral Startup Found of Liaoning Province (No. 20131139). 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 * E-mail: yfguan@mail.cmu.edu.cn modulating structure transitions of the G-quadruplexes between the parallel and the anti-parallel folding topologies [11]. Guojie Zhao1, Bin Zhao2, Zhaoxue Tong1, Runqing Mu3, Yifu Guan1* 1 Key Laboratory of Medical Cell Biology (Ministry of Education), Department of Biochemistry and Molecular Biology, China Medical University, Shenyang, China, 2 Department of Movement Science, Shenyang Sport University, Shenyang, China, 3 Department of Laboratory Medicine, First Affiliated Hospital, China Medical University, Shenyang, China Introduction Thus, type I neokink enlarges the major groove and facilitates the enzyme to access to the substrate. Consequently, type I neokink is October 2013 \| Volume 8 \| Issue 10 \| e77111 1 PLOS ONE \| www.plosone.org Effects of 29-OMe Nucleotide on EcoRI Cleavage Figure 1. Chemical structures of DNA, RNA, LNA, 29-OMeN and 29-FNA. doi:10.1371/journal.pone.0077111.g001 Figure 1. Chemical structures of DNA, RNA, LNA, 29-OMeN and 29-FNA. doi:10.1371/journal.pone.0077111.g001 Figure 1. Chemical structures of DNA, RNA, LNA, 29-OMeN and 29-FNA. doi:10.1371/journal.pone.0077111.g001 Preparation of Oligonucleotides In this experiment, three types of oligonucleotides have been used to form the substrate for EcoRI enzyme. A 45-mer containing the EcoRI recognition sequence -GAATTC- was referred to as template, a 26-mer with a fluorophore (FITC) at the 59-end was called as F-ON, and a 17-mer with a quencher (Dabcyl) at the 39- end was called as Q-ON. To evaluate the enzymatic cleavage efficiency, 29-OMeN, 29-FNA and LNA were used to modify the EcoRI recognition sequence (Table 1). These oligonucleotides were purchased from Sangon Biotech Co. Ltd. (Shanghai, China), and prepared at the concentration of 100 mM in ddH2O. These stock solutions were stored in 220uC. In designing the oligonucleotides, we have carefully examined the sequences by using sequence analysis software to eliminate the possible formation of mismatches and hairpins. Materials and Methods truly critical for the enzyme recognition since the a-helixes of EcoRI enzyme can only fit into the expanded major groove around type I neokink [16,17]. Two type II neokinks flank the recognition sequence -GAATTC-. Type II neokinks introduce a large bend of 23u between helical axes. This bend includes a rolling motion toward the minor groove and, importantly, it presents an A–B junction. Considering these structural features, we speculated that site-specific substitutions of 29-modified nucleotides for the EcoRI recognition sequence will change the contacts between the DNA substrates and enzyme EcoRI, and consequently, the enzyme- catalyzed cleavage might be enhanced or decreased without scarifying the recognition specificity. Furthermore, recombination DNA technology has become a routine approach to clone DNA fragments and to generate novel DNA constructs. Thus, regulation of enzymatic activity, either the cleavage efficiency or the linkage efficiency, at the designated nucleotide sequences could provide more flexible options for genetic engineering and pharmaceutical applications. truly critical for the enzyme recognition since the a-helixes of EcoRI enzyme can only fit into the expanded major groove around type I neokink [16,17]. Two type II neokinks flank the recognition sequence -GAATTC-. Type II neokinks introduce a large bend of 23u between helical axes. This bend includes a rolling motion toward the minor groove and, importantly, it presents an A–B junction. Considering these structural features, we speculated that site-specific substitutions of 29-modified nucleotides for the EcoRI recognition sequence will change the contacts between the DNA substrates and enzyme EcoRI, and consequently, the enzyme- catalyzed cleavage might be enhanced or decreased without scarifying the recognition specificity. Furthermore, recombination DNA technology has become a routine approach to clone DNA fragments and to generate novel DNA constructs. Thus, regulation of enzymatic activity, either the cleavage efficiency or the linkage efficiency, at the designated nucleotide sequences could provide more flexible options for genetic engineering and pharmaceutical applications. FRET Analysis of Enzymatic Cleavage In the present study, we have demonstrated that the enzymatic cleavage activity can be modulated by 29-OMeN substitutions for the EcoRI recognition sequence. Six nucleotides of the EcoRI recognition sequence and two nucleotides flanking the recognition sequence have been substituted by 29-OMeN once a time. The initial reaction velocities were measured and the steady state kinetic parameters, Km and Vmax, were determined. These 29-OMeN substitutions at different positions showed significantly different effects on the binding affinity and the enzymatic cleavage efficiency. The effects of 29-OMeN substitutions have been compared with that of LNA, 29-FNA and other nucleobase derivatives. Mechanisms have been proposed to interpret the unusual enzymatic behavior on the basis of the crystal structure of EcoRI-DNA complex. These results suggested that 29-OMeN substitution could be used as a regulatory element to regulate the EcoRI enzymatic activity in vitro. This novel property provides an application potential in bioengineering and biomedicine. The substrate of enzyme EcoRI is composed of the template, F- ON, and Q-ON at the same concentration of 50 nM in 100 ml buffer containing 50 mM Tris, 10 mM MgCl2, 1 mM DTT, and Table 1. Sequences of oligonucleotides used in this study. Table 1. Sequences of oligonucleotides used in this study. Table 1. Sequences of oligonucleotides used in this study. Name Sequences* Template 59-ATACGCATACCTGTGAATTCTGGCTAAAAGCACACGCACGGAGAC T-T(21) 59-ATACGCATACCTGtGAATTCTGGCTAAAAGCACACGCACGGAGAC T-G1 59-ATACGCATACCTGTgAATTCTGGCTAAAAGCACACGCACGGAGAC T-A2 59-ATACGCATACCTGTGaATTCTGGCTAAAAGCACACGCACGGAGAC T-A3 59-ATACGCATACCTGTGAaTTCTGGCTAAAAGCACACGCACGGAGAC T-T4 59-ATACGCATACCTGTGAAtTCTGGCTAAAAGCACACGCACGGAGAC T-T5 59-ATACGCATACCTGTGAATtCTGGCTAAAAGCACACGCACGGAGAC T-C6 59-ATACGCATACCTGTGAATTcTGGCTAAAAGCACACGCACGGAGAC T-T7 59-ATACGCATACCTGTGAATTCtGGCTAAAAGCACACGCACGGAGAC F-ON 39-TATGCGTATGGACACTTAAGACCGAT-FITC Q-ON 39-Dabcyl-TCGTGTGCGTGCCTCTG The nucleotide derivatives 29-OMeN, 29-FNA and LNA are in lower case, and DNA nucleotides are in upper case, respectively. F-ON is 59 labeled by FITC. Q- ON is 39 labeled by Dabcyl. For convenience, the sequences of F-ON and Q-ON are written from the 39-end to the 59-end. The EcoRI recognition sequence is underlined. doi:10.1371/journal.pone.0077111.t001 Figure 2. Illustration of three neokinks in EcoRI-bonded duplex. doi:10.1371/journal.pone.0077111.g002 The nucleotide derivatives 29-OMeN, 29-FNA and LNA are in lower case, and DNA nucleotides are in upper case, respectively. F-ON is 59 labeled by FITC. Q- ON is 39 labeled by Dabcyl. For convenience, the sequences of F-ON and Q-ON are written from the 39-end to the 59-end. The EcoRI recognition sequence is underlined. Figure 2. Illustration of three neokinks in EcoRI-bonded duplex. doi:10.1371/journal.pone.0077111.g002 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org October 2013 \| Volume 8 \| Issue 10 \| e77111 2 Effects of 29-OMe Nucleotide on EcoRI Cleavage 100 mM NaCl (pH 7.5). The template and F-ON were mixed first, and fluorescence was measured as F0. After adding Q-ON, stable EcoRI substrates were formed. In this complex, the quencher and the fluorophore were so close in space that there was very low fluorescence signal defined as F1. Once EcoRI was added into the substrate solution, it started to cleave the recognition sequence formed by F-ON and the template. The cleaved 59-portion of F-ON was too short to hybridize firmly with the template and then released, generating a fluorescence signal Ft (Fig. 3). The relative fluorescence intensity (RF) was calculated using formula RF = (Ft2F1)/(F02F1). 100 mM NaCl (pH 7.5). The template and F-ON were mixed first, and fluorescence was measured as F0. After adding Q-ON, stable EcoRI substrates were formed. In this complex, the quencher and the fluorophore were so close in space that there was very low fluorescence signal defined as F1. Once EcoRI was added into the substrate solution, it started to cleave the recognition sequence formed by F-ON and the template. The cleaved 59-portion of F-ON was too short to hybridize firmly with the template and then released, generating a fluorescence signal Ft (Fig. 3). The relative fluorescence intensity (RF) was calculated using formula RF = (Ft2F1)/(F02F1). the fluorescence intensities increased monotonically as the reaction was progressing, and at higher concentrations, the fluorescence intensities increased rapidly and then became saturated, exhibiting the typical rectangular hyperbolic behavior (Fig. 4A). The initial reaction velocities were then calculated, and showed a linear relationship with the enzyme concentrations (Fig. 4B). Further- more, by plotting the reaction velocities with respect to the substrate concentrations and fitting these data to the hyperbolic curves, kinetic parameters Km and Vmax were estimated to be 97.6 nM and 0.103 nM/s, respectively. These values were compatible with the data reported previously with small margins [19,22]. Determination of Kinetic Parameters The substrate was prepared at the concentration of 0.2,5.0 folds of Km, which was estimated from the initial rates of the different substrate concentrations. Initial velocities (V0) of each reaction were determined from the slope of the intensity curves of the fluorescence signal with respect to the time. The substrate concentration of 50,800 nM and the initial velocity were plotted by using non-linear curve fitting and linear (Lineweaver-Burk) curve fitting, and Km and Vmax were calculated from Michaelis- Menten equation. Data presented here are averaged results of three independent measurements. To understand the molecular interactions involved in this enzymatic cleavage, we have measured the initial reaction velocities of EcoRI at different concentrations of the 29-OMeN modified substrates, and determined their steady state kinetic parameters, Km and Vmax (Fig. 6 and Table 2). In general, the binding of protein-DNA was reduced in most modified positions, as shown the majority of modified positions presented increased Km, though some of increases were small. The Vmax values increased for G1 and C6 positions, while substitutions at other positions showed decreased activities by different percentages. The ability to discriminate substrate, also referred to as the specificity constant, can be evaluated by Vmax/Km. In comparison with the control (the unsubstituted substrate), the values of Vmax/Km varied from the lowest 0.3 of A3 position to the highest 1.2 of C6 position. Position-dependent Enzymatic Cleavage Fig. 5 shows the initial reaction velocities of EcoRI when the EcoRI recognition sequence nucleotides are modified with 29- OMeN. In comparison with the unmodified sequence, substitu- tions at G1 and C6 positions maintaind the initial reaction velocities, whereas substitutions at T(21), A2, A3, T4 and T7 positions reduced the cleavage efficiency significantly by 50,80%. The most astonished result was that the EcoRI cleavage activity has been completely diminished by 29-OMeN substitution at T5 position. It has been known that the lengths of the duplex substrates as well as the nucleotides flanking the recognition sequence at 59- or 39-end could exert direct or indirect effects on the kinetic values, e.g. by affecting the stability of the duplex [23,24] or the rate-limiting step of the product release [25]. In this experiment, we have performed a heating-annealing cycle to ensure the formation of three-oligonucleotide substrates before measurement. Fluorescence signals were recorded at 37uC on Microplate Reader (Infinite M200, Tecan, USA) with excitation wavelength at 480 nm and emission wavelength at 524 nm. The signals were collected every 15 seconds with an integration time of 20 microseconds, and measurements were performed by monitoring the fluorescence emission with time over ,20 min. Results Concentration Dependence of Enzymatic Cleavage Discussion negative charge dispersion and reactivity of different chemical groups have been considered to be responsible for these changes [30,31,32,33]. To identify the interactions involved in determining the sequence-specified recognition, different base derivatives have been utilized to make site-specific substitutions of d(-GAATTC-) sequence, including hypoxanthine [22], 5-Br-C [26], 5-Br-dC [22], 5-Br-U [22], 8-Br-dA [27], 2,6-diaminopurine [22], 6- methyl-dA [22], 5-methyl-dA [22] and 7-deaza-dA [27,28,29]. dU or 5-Br-dT substitution has concluded that T5 position is more important than T4 position, and it is possibly attributed to the steric hindrance of 5-CH3 group on thymine residue [22]. Modification at either A2 or A3 position by 6-methyl-dA has led to no enzymatic cleavage [22]. Furthermore, phosphate deriva- tives of methylphosphonate, O-ethyl-phosphotriester and phos- phorothioate have been used to modify the phosphate groups of the EcoRI recognition sequence. Each of these chiral derivatives creates two different spatial orientations of the substituents at the phosphorous atom and results in enzymatic activity variations from complete inactivation to significant enhancement. The In comparison with these investigations, the effects of 29- modified nucleotide derivatives on the recognition and hydrolytic cleavage of EcoRI have much less explored. 29-fluoro-29-deox- yguanosine (29-F-dG) and 29-chloro-29-deoxyguanosine (29-Cl-dG) have been the only two 29-modified nucleotide derivatives so far, and only the G1 residue of the EcoRI sequence has been modified. Modification of the residue flanking the recognition sequence at the 39-end by L-nucleotide (ribose in L-homochirality) has shown different enzymatic behavior from the normal one [19]. In structure biology, six torsion angles along the phosphate backbone -P-O59-C59-C49-C39-O39-P- of DNA strand, namely a, b, c, d, e and f, are used to characterize the backbone conformations of DNA and RNA, and each one has been restricted in a well defined range for different conformations of DNA duplexes. The C39 atom is directly involved in torsion angles d and e, thus, the C39-endo sugar pucker conformation of 29-modified nucleotides will Figure 5. Effects of 29-OMeN substitution on EcoRI cleavage. (A) Fluorescence intensity changes of the 29-OMeN-modified EcoRI sequences with respect to reaction time at the EcoRI concentration of 1.2 unit/ml. (B) Initial reaction velocities calculated based on the fluorescence curves in Fig. 5A. doi:10.1371/journal.pone.0077111.g005 Figure 5. Effects of 29-OMeN substitution on EcoRI cleavage. (A) Fluorescence intensity changes of the 29-OMeN-modified EcoRI sequences with respect to reaction time at the EcoRI concentration of 1.2 unit/ml. (B) Initial reaction velocities calculated based on the fluorescence curves in Fig. 5A. Concentration Dependence of Enzymatic Cleavage FRET approach has been established previously for monitoring the restriction endonucease cleavage activity [18,19,20,21]. To validate this method and to determine the enzyme concentration adequate for experiments in the current study, we tested the effects of the enzyme concentration on the cleavage efficiency for the native nucleotide sequence. EcoRI concentrations in the range of 19 U/ml to 1200 U/ml were examined. At lower concentrations, Figure 3. Illustrative diagram showing the principle of the FRET-based assay for endonuclease cleavage measurement. doi:10.1371/journal.pone.0077111.g003 Figure 3. Illustrative diagram showing the principle of the FRET-based assay for endonuclease cleavage measurement. doi:10 1371/journal pone 0077111 g003 m showing the principle of the FRET-based assay for endonuclease cleavage measurement. 1 003 Figure 3. Illustrative diagram showing the principle of the FRET-based assay for endonuclease cleavage measurement. doi:10.1371/journal.pone.0077111.g003 October 2013 \| Volume 8 \| Issue 10 \| e77111 PLOS ONE \| www.plosone.org Effects of 29-OMe Nucleotide on EcoRI Cleavage Figure 4. Effects of enzyme concentration on EcoRI cleavage. (A) Fluorescence intensity changes with respect to the reaction time for different EcoRI enzyme concentrations. (B) The effect of EcoRI concentration on the initial reaction velocity for the unsubstituted EcoRI recognition sequence. doi:10.1371/journal.pone.0077111.g004 Figure 4. Effects of enzyme concentration on EcoRI cleavage. (A) Fluorescence intensity changes with respect to the reaction time for different EcoRI enzyme concentrations. (B) The effect of EcoRI concentration on the initial reaction velocity for the unsubstituted EcoRI recognition sequence. doi:10.1371/journal.pone.0077111.g004 A2 Position A2 position plays an important role in sequence recognition and cleavage. In the catalytic center, N6 and N7 of A2 nucleotide interact with Glu144 and Arg145 through hydrogen bonds which contribute to the specific recognition of adenine. The phosphate of A2 is also fixed by Asp91 and Glu111 through Mg2+ for hydrolyzing phosphoester bond. 29-OMeN substitution for A2 residue has reduced both Km and Vmax. The specificity constant decreased slightly due mainly to the decreased Vmax. The phenomena of better binding alone with the reduced Vmax have also observed in other studies [37,38]. The alteration of A2 phosphate group in the catalytic center can exert direct inhibitory effect on the cleavage. Structural analysis has pinpointed another possibility. The phosphate group of -A2pA3- forms a hydrogen bond with His114. The His114 distrubance induced by backbone change could interfere with the adjacent residues Glu111 and Lys113 which are the elements at the catalytic center. Moreover, it has been proposed that Gln115, another key residue adjacent to His114, plays an important role in coupling the specific DNA binding to the catalytic cleavage [39]. Though Vmax of this position presented the lowest value of all six substituted positions except T5, the reduced extent is still below our expectation when taking into account many of the most important residues mentioned above. This gives us an impression that an elaborate and flexible interaction network must have the ability to compensate the reduced Vmax. In addition, we have proposed another interpretation. Based on the crystal structure, the most possible residue to interact with C29 moiety of A2 position is Arg145, since the distance of C29 atom to the nearby residue Arg145 of EcoRI was determined to be 4.69 A˚ (1CKQ.pdb). The electronegativity of 29-OMe can also increase its interaction with this basic amino acid. Since both the bases and backbones of A2 position interact with functional groups of EcoRI in a more complex format, the retardation by C29 derivatives might be a mixed effect. Further studies are under conduct currently. A previous study by Gohda et al. has selected 29-fluoro-29- deoxyguanosine (29-F-dG), 29-chloro-29-deoxyguanosine (29-Cl- dG) and guanosine (rG) to substitute G1 residue of the d(GGAATTCC) sequence, and examined their effects on EcoRI cleavage activities [36]. 29-F-dG modified sequence has shown a similar property to that of normal DNA substrate: 100% cleavage for both the unmodified and the 29-F-dG modified d(GGAATTCC) sequence in 60 min. G1 Position G1 is just on the edge of the type II neokink. Crystal structures of several EcoRI-DNA complexes have revealed that DNA duplex is surrounded by both direct and indirect recognitions, i.e. both phosphate backbone and bases are bonded by functional elements of enzyme. The O6 and N7 atoms of G1 nucleotide interact with Arg200 of EcoRI. Both of the two non-bridging oxygen atoms of G1 phosphate group are bonded with the conserved residues Lys89, Lys148 and Asn149. Previous study has reported that hypoxanthine substitution of G1 increased both Km and kcat [22]. Hypoxanthine substitution removed away the C2-NH2 group from minor groove and left functional groups in major groove untouched. It has been explained in terms of the backbone conformation alternation of type II neokink caused by hypoxan- thine substitution in minor groove. Our results showed that the 29- OMeN substitution for G1 nucleotide also increased Km as well as Vmax, suggesting a reduced binding affinity and an enhanced cleavage efficiency. Since the G1 position is just on the edge of type II neokink and must experience the A–B form transition. Thus, 29-OMeN substitution at G1 position might arrange the substrate backbone to an energetically favored conformation, facilitating the organization of the catalytic center in the catalytic step and benefiting the EcoRI cleavage. Accelerated product releasing may also attribute to the increased Vmax. Effects of 29-OMe Nucleotide on EcoRI Cleavage It has also been found that Lys130 and Arg203 form hydrogen bonds with phosphate groups [34]. Furthermore, each strand of the DNA duplex interacts with two subunits of homodimeric EcoRI with comparable energies [35]. Effects of 29-OMe Nucleotide on EcoRI Cleavage Table 2. Kinetic parameters for different 29-OMeN substituted EcoRI sequences. Substitution position Km (nM) Vmax (nM/s) Vmax/Km (1/s) GAATTC 97.6 0.103 0.0011 (1.0) gAATTC 161.8 0.185 0.0011 (1.1) GaATTC 60.9 0.049 0.0008 (0.8) GAaTTC 248.4 0.081 0.0003 (0.3) GAAtTC 104.6 0.093 0.0009 (0.8) GAATtC N/A N/A N/A GAATTc 246.2 0.293 0.0012 (1.1) Letters and capitals represent 29-OMeN and DNA nucleotide, respectively. In parentheses are the relative values of modified substrate compared to unmodified control. doi:10.1371/journal.pone.0077111.t002 changes (29-Cl-dG.rG.29-F-dG) coincided with the observed cleavage activity as well as the van der Waals volumes of the 29- substituents (Cl.OH.F). Thus, the steric hindrance of the 29- substituent has been considered to be the cause of the conforma- tional changes of the phosphate backbone and consequent changes of enzymatic activities. They have also used the 29-Cl-dG substituted and rG substituted octamers as inhibitors in this study, and concluded that the substituted octamers behaved like a competitive inhibitor [36]. Table 2. Kinetic parameters for different 29-OMeN substituted EcoRI sequences. Table 2. Kinetic parameters for different 29-OMeN substituted EcoRI sequences. In the currrent study, we also made substitutions at this G1 position using 29-FNA. This 29-FNA substitution reduced the initial reaction velocity by about ,30% (Fig. 7A). Considering the difference in size and charge of substituent group, the electroneg- ativity as well as the van der Waals radius might compromise the favorable conformation by 29-fluoro modification. We also used the base derivative O6-Me-dG to substitute for G1 nucleotide and observed a 50% reduction in the cleavage efficiency (Fig. 7B). The methylation of O6-dG can disrupt the hydrogen bond between O6 and Arg200. The steric hindrance of methyl group may also cause a damage of adjacent interactions. Therefore, O6-Me-dG substitution destabilized enzyme-DNA complex and decreased cleavage rate. Previous studies have revealed that O6 and N7 of G1 residue interacted with Arg200 of EcoRI [17] and removal of N7 by substitution of 7-deaza-dG decreased the cleavage significantly [28]. In comparison with these experimental data, the current data might suggest that sugar pucker conformation switch has less effect in this position on regulating the enzymatic cleavage than that of the nucleobase modifications. specific recognition. It has also been found that Lys130 and Arg203 form hydrogen bonds with phosphate groups [34]. Furthermore, each strand of the DNA duplex interacts with two subunits of homodimeric EcoRI with comparable energies [35]. specific recognition. Discussion doi:10.1371/journal.pone.0077111.g005 doi:10.1371/journal.pone.0077111.g005 October 2013 \| Volume 8 \| Issue 10 \| e77111 October 2013 \| Volume 8 \| Issue 10 \| e77111 PLOS ONE \| www.plosone.org 4 Effects of 29-OMe Nucleotide on EcoRI Cleavage Figure 6. Steady state kinetic analysis of EcoRI cleavage of unmodified and 29-OMeN modified DNA. (A) Unmodified DNA. (B) Modification at G1 position. (C) Modification at A2 position. (D) Modification at A3 position. (E) Modification at T4 position. (F) Modification at C6 position. The line is non-linear curve fitting of the initial velocities at different duplex substrate concentrations. Inset is Lineweaver-Burk plots of the data. Letters and capitals represent 29-OMeN and DNA nucleotide, respectively. doi:10.1371/journal.pone.0077111.g006 Figure 6. Steady state kinetic analysis of EcoRI cleavage of unmodified and 29-OMeN modified DNA. (A) Unmodified DNA. (B) Modification at G1 position. (C) Modification at A2 position. (D) Modification at A3 position. (E) Modification at T4 position. (F) Modification at C6 position. The line is non-linear curve fitting of the initial velocities at different duplex substrate concentrations. Inset is Lineweaver-Burk plots of the data. Letters and capitals represent 29-OMeN and DNA nucleotide, respectively. doi:10.1371/journal.pone.0077111.g006 inevitably alter the duplex phosphate backbone, and consequently, create different effects on either the binding affinity with the substrate or the cleavage efficiency of EcoRI. the molecular interactions involved in enzymatic cleavage. The EcoRI-d(TCGCGAATTCGCG) complex has shown a tight and complementary contact between enzyme and the major groove of the DNA duplex. There are twelve EcoRI-purine hydrogen bonds as well as a set of van der Waals contacts responsible for the Crystal structures of several EcoRI-DNA complexes have been reported. They have provided a structural basis for interpreting October 2013 \| Volume 8 \| Issue 10 \| e77111 PLOS ONE \| www.plosone.org 5 Effects of 29-OMe Nucleotide on EcoRI Cleavage A3 Position A3 position experiences the central type I neokink which unwinds duplex in -A3pT4- by 25u, resulting in a wider major groove and a narrower minor groove. Since EcoRI access DNA duplex through major groove, the enlarged major groove is necessary for enzyme’s entry. In addition, A3 is also the nucleotide whose base can be directly read out by protein, i.e. Arg145 forms hydrogen bond with N7 of A3 nucleotide. In previous study, 2,6- diaminopurine (2,6-AP) has been used to substitute for this position and introduced C2-NH2 into minor groove, and increased Km and decreased kcat have been observed [22]. It has been suggested that an additional C2-NH2 in minor groove as a bulkier obstacle interferes with type I neokink formation. In our study, a significant increase in Km and obvious decrease in Vmax were observed in the situation of 29-OMeN substitution at the A3 position, thus gave rise to the lowest specificity constant. C39-endo sugar pucker facilitate A-form duplexes which have even narrowed major groove in comparison with B-form. This is surely troublesome for the duplex binding by enzyme, and could diminish the binding affinity as well as the cleavage rate. Further crystal structural analysis showed that the distance of the C29 atom of A3 residue to adjacent residue Ala142 of EcoRI is 4.32 A˚ (1CKQ.pdb). This would also increase a possibility of steric hindrance. In the case of 29-FNA substitution, the modified substrates showed the reduced cleavage rates by about 40% which is similar to that of 29-OMeN modification (Fig. 7A). Thus, the retardation by 29-OMeN and 29-FNA substitutions might be due mainly to the backbone conformation change which disturb the type I neokink, and inhibit the binding and cleavage of EcoRI. T4 Position 29-OMeN substitution for T4 residue increased Km slightly and decreased Vmax in a small margin, and the specificity constant decreased slightly in comparison with the native substrate. Previous studies have showed that the C5-CH3 group of T4 residue interacted with Gly140, Ala142 and Gln115 through hydrophobic interaction [42], and dU substitution for T4 residue diminished the hydrophobic interaction and resulted in an increased Km and almost unchanged kcat which is similar to 29- OMeN substitution in our study [22]. Thus, 29-OMeN substitu- tion for T4 residue created a relatively minor impact on the enzymatic cleavage behavior. The interaction of phosphate backbone with Lys117 and Gly116 may be also disturbed by C39-endo conformation. Similarly, 29-FNA substitution for T4 residue declined the enzymatic cleavage at a similar level (Fig. 7A). Figure 7. Effects of other analog substitutions on EcoRI cleavage. (A) The effects of 29-FNA substitution at different positions on the reaction initial velocity. (B) Comparison of initial reaction velocities of different nucleotide derivatives at G1 position. (C) Comparison of initial reaction velocities of different nucleotide derivatives at A2 position. doi:10.1371/journal.pone.0077111.g007 No literature could be found regarding the substitution of the A2 residue by 29-modified nucleotide derivatives so far, thus, we used chiral methylphosphonates in previous studies for compar- ison [30]. Chiral methylphosphonates, namely Sp-Pm and Rp-Pm, have replaced the phosphate group of -A2pA3-. Experimental Effects of 29-OMe Nucleotide on EcoRI Cleavage N lit t ld b f d di th b tit ti f th Figure 7. Effects of other analog substitutions on EcoRI cleavage. (A) The effects of 29-FNA substitution at different positions on the reaction initial velocity. (B) Comparison of initial reaction velocities of different nucleotide derivatives at G1 position. (C) Comparison of initial reaction velocities of different nucleotide derivatives at A2 position. doi:10.1371/journal.pone.0077111.g007 results have shown that the binding affinities were impaired by both Sp-Pm and Rp-Pm substitutions, whereas the cleavage efficiencies were also reduced to quite different extent. It has been proposed that the Rp-oxygen had no binding partner while Sp- oxygen was hydrogen-bounded with His114 and a localized water molecule [30]. The C39-endo conformation of the 29-OMeN substitution at the A2 position in the present study could influence the local backbone of -A2pA3-, thus altered the position of His114. We also made 29-FNA and LNA substitutions for A2 residue, and observed the reduced cleavage ability by about 50,60% (Fig. 7C). Because they share the same C39-endo sugar pucker conformation as 29-OMeN [4,40,41], the backbone distortion may play a major role in this cleavage rate reduction. A2 Position In contrast, rG and 29- Cl-dG modifications have repressed the cleavage differently: only 5% for rG and no cleavage at all for 29-Cl-dG in 24 h. Since these four octamers exhibit similar binding affinity (1026–1027 M), the differences in cleavage activity may be attributed to the catalytic reaction instead of the binding process. They have also used 31P- NMR method to analyze the change of the phosphate backbone conformation, and observed that the order of the conformation October 2013 \| Volume 8 \| Issue 10 \| e77111 PLOS ONE \| www.plosone.org 6 Effects of 29-OMe Nucleotide on EcoRI Cleavage T5 Position To our surprise, 29-OMeN substitution for T5 residue diminished the EcoRI enzymatic activity completely (Fig. 5). No Km and Vmax could be measured. In previous studies, using dU or October 2013 \| Volume 8 \| Issue 10 \| e77111 PLOS ONE \| www.plosone.org 7 Effects of 29-OMe Nucleotide on EcoRI Cleavage 5-Br-dT substitution, T5 residue has been found to be more important than T4 residue which was also true for 29-OMeN substitution in our study. The dU substitution deleting C5-CH3 of T5 but not T4 position has abolished cleavage, which has been suggested to be attributed to the interaction of C5-CH3 group, or a conformation alteration of N-glycosidic bonds in dA?dU pairing, moreover, in which the paired dA of dU is just involved in the cleavage site. [22]. Both the C5-CH3 of T4 and T5 were surrounded by hydrophobic groups of protein functional elements in enzyme-DNA complex crystal structure, such as Gly140, Ala142, and Gln115. However, in our study, the inhibition of cleavage was so strong that hydrophobic interaction is not enough to interpret the observation. It is more likely that this substitution is followed by breakdown of cooperative mechanism, where one alteration causes other more losses of protein-DNA interactions which involved in the elaborated interaction networks of the complex. In this mechanism, the effects can even spread to the unmodified strand. (We will discuss this point later.) Though the T5 position does not fall into the three neokinks of protein-DNA complex, the backbone conformation change by C39-endo pucker might be an incentive for further breakdown of networks. This explanation has also been suggested by dU substitution at T5 position [22]. In addition, we also pay attention to the C29 substitution moiety of T5 position. Structural analysis showed that the nearest residue to the C29 moiety of T5 position is Met137 (3.9 A˚ (1CKQ.pdb)) which is located at the end of an extended chain, and the –OCH3 moiety at C29 might push extended chain away by steric hindrance of Met137 to disturb key interactions between extended chain and bases. In comparison with 29-OMeN modification, substitution of the T5 residue by 29-FNA only decreased but not abolished enzymatic cleavage (Fig. 7A). unnatural L-nucleotide (ribose in L-homochirality) residues at the 39-flanking site of the EcoRI recognition sequence has been evaluated, showing a larger Vmax value (2.5 folds) and a smaller Km value (,3 folds) compared with normal D-nucleotide (ribose in D-homochirality) one. Residues Flanking 59- and 39-termini Residues Flanking 5 and 3 termini 29-OMeN substitutions for T(21) and T7 residues decreased the initial reaction velocities significantly (Fig. 5). These results implied that, in addition to the hexameric sequence, residues flanking the recognition sequence are also important for the hydrolytic cleavage. Crystal structure has suggested that the phosphates of pNpG1 interact with residues in b-bridge, such as Ser86, Ser87 and Lys89. It is believed that the C39-endo conformation switch at the T(21) position distorted these local phosphate backbone contacts between EcoRI and DNA substrate and changed enzymatic properties. These results are consistent with a previous study that the phosphate contacts at pGAATTC play a crucial role in the recognition [29]. In addition, the effect of T5 Position It has been attributed to the L-nucleotide induced preferable conformational alternation for binding and catalysis by EcoRI [19]. Moreover, phosphothioate substitution of C6pN has shown that both Sp and Rp substituents give very poor substrates [30]. The interaction of Lys130 has been proposed for the reason. The 29-OMeN substitution of T7 also presented significantly reduced cleavage rate, and might share similar mechanism with that of phosphothioate substitution. Interstranded Interaction Phosphate derivatives have been extensively studied to manifest the enzymatic cleavage mechanism. R- and S-methylphosphonates (known as Rp-Pm and Sp-Pm) single substitutions for each phosphate of d(GACpGpApApTpTpCpGTC) have decreased the binding affinity as well as the enzymatic cleavage [30]. R- or S- phosphorothioates (known as R-Ps or S-Ps) have produced significant stereospecific effects on EcoRI binding and cleavage [33] [37] [45]. The cleavage inhibition has been considered as the consequence of structural alteration such as a minor displacement of Asp91 and Glu111 [33]. Substitutions of O-ethyl phospho- triester derivative have also demonstrated a position-dependent resistance to enzymatic cleavage [45], although ethylation of this phosphate weakly interfered with binding of the enzyme [46]. The absolute configuration assigned as Rp has shown that the ethoxy group was oriented into the major groove of the DNA molecule (‘‘inward’’ orientation), whereas the Sp configuration was oriented ‘‘outward’’ of the DNA molecule [45]. Structure analysis has shown that the phosphate group between two thymine residues was located almost directly across the scissile bond on the complementary strand, therefore, it was proposed the esterification of this phosphate group could influence the cleavage of the scissile bond on the opposite strand rather than on the modified strand. To prove it, Koziolkiewicz and Stec have used heteroduplexes of d(AAGAATTCCC)/d(GGGAATTCTT) and d(AAGAATp(Oet)- TCCC)/d(GGGAATTCTT). Results have shown that the pres- ence of an O-ethyl group between the two thymine residues in strand d(AAGAATp(Oet)TCCC) inhibited the cleavage of the opposite unmodified strand more effectively than cleavage of the modified strand [45]. C6 Position 29-OMeN substitution for C6 residue increased both Km and Vmax significantly, resulting in an almost unchanged specificity constant. Many base analog substitutions at C6 position have given poor substrates, such as 5-methylcytocine and 5-bromocy- tocine [22]. It has been suggested that 5-methylation decrease the flexibility and mobility of duplex backbone in C6 position and then reduced catalysis [43,44]. Since just like G1 residue, C6 residue is on the edge of type II neokink, the flexibility and distortion at this site are very important for the binding and cleavage of EcoRI. Thus, the reduced flexibility and mobility of C6 backbone will prevent cleavage. In the case of 29-OMeN substitution of this study, the C39-endo conformation of 29-OMeN could set the local structure to an energetically favored A-form for enzymatic cleavage. Consequently, an almost unchanged cleavage rate was observed in this position. In comparison, 29-FNA modifications at C6 position produced similar initial reaction velocities to that of 29-OMeN substitution (Fig. 7A). As shown in this study, the 29-OMeN substitution for T5 residue on the template strand has completely inhibited the cleavage. It was interesting because the inhibitoriest effect occurred at the T5 residue rather than the A2 residue (Fig. 5). The same phenomenon was also observed when the 29-OMeN substitution for T5 residue on the F-ON strand (data not shown). The 29-OMeN substitution for A2 residue on the F-ON strand reduced the catalytic activity significantly although not completely, and the double-substitution of 29-OMeN for T5 residues on both strands exhibited a similar 100% inhibition (data not shown). By analogy to the Koziolkiewicz and Stec’s conclusion, we speculated that the T5 residue might transfer the sugar pucker conformational changes to the unmod- ified strand through the interstrand T5-A2 basepair interaction, inhibiting the cleavage of the unmodified strand. Further investigations are needed to prove this speculation. References Campbell JM, Bacon TA, Wickstrom E (1990) Oligodeoxynucleoside phosphor- othioate stability in subcellular extracts, culture media, sera and cerebrospinal fluid. J Biochem Biophys Methods 20: 259–267. 29. Lesser DR, Kurpiewski MR, Waters T, Connolly BA, Jen-Jacobson L (1993) Facilitated distortion of the DNA site enhances EcoRI endonuclease-DNA recognition. Proc Natl Acad Sci U S A 90: 7548–7552. 9. Phillips MI, Zhang YC (2000) Basic principles of using antisense oligonucleotides in vivo. Methods Enzymol 313: 46–56. 30. 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(2001) Solution conformation of EcoRI restriction endonuclease changes upon binding of cognate DNA and Mg2+ cofactor. Biochemistry 40: 683–692. protein-protein interactions. Curr Opin Struct Biol 14: 104– 35. References 1. Kurreck J (2003) Antisense technologies. Improvement through novel chemical modifications. Eur J Biochem 270: 1628–1644. 1. Kurreck J (2003) Antisense technologies. Improvement through novel chemical modifications. Eur J Biochem 270: 1628–1644. 22. Brennan CA, Van Cleve MD, Gumport RI (1986) The effects of base analogue substitutions on the cleavage by the EcoRI restriction endonuclease of octadeoxyribonucleotides containing modified EcoRI recognition sequences. J Biol Chem 261: 7270–7278. 2. Prakash TP, Bhat B (2007) 29-Modified oligonucleotides for antisense therapeutics. Curr Top Med Chem 7: 641–649. 2. Prakash TP, Bhat B (2007) 29-Modified oligonucleotides for antisense therapeutics. Curr Top Med Chem 7: 641–649. J 23. 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Seela F, Driller H (1986) Palindromic oligonucleotides containing 7-deaza-29- deoxyguanosine: solid-phase synthesis of d[(p)GGAATTCC] octamers and recognition by the endodeoxyribonuclease EcoRI. Nucleic Acids Res 14: 2319– 2332. 8. Author Contributions Conceived and designed the experiments: GZ YG. Performed the experiments: GZ BZ ZT RM. Analyzed the data: GZ BZ ZT RM YG. Contributed reagents/materials/analysis tools: YG. Wrote the paper: GZ YG. Biological Significance Synthetic biology has become very attractive as an emerging field. Synthetic biology utilizes extensively tool enzymes, such as restriction endonucleases and ligases to construct new DNA sequences by cleaving and jointing nucleotide fragments. Therefore, October 2013 \| Volume 8 \| Issue 10 \| e77111 PLOS ONE \| www.plosone.org 8 Effects of 29-OMe Nucleotide on EcoRI Cleavage assembling a biological circuit using different biological elements and parts is a challenging test, where repetitive cleavage and jointing will be performed. Thus, enhancement of cleavage efficiency under one condition and repression of cleavage under another condition could offer operation flexibility to optimize the new DNA constructs. Our study shows that substitutions of 29-OMeN, 29- FNA and LNA for nucleotides of EcoRI recognition sequence could provide the feasibility to regulate the cleavage efficiency of EcoRI in varied extent in vitro. It is our hope to exploit this kind of characteristics of C29-modified nucleotide derivatives in synthetic biology. showed that 29-OMeN substitutions for the EcoRI recognition sequence could decrease the cleavage efficiency for A2, A3 and T4 substitutions, and preserve the original cleavage efficiency for G1 and C6 substitutions. 29-OMeN substitution for T5 residue, however, diminished the enzymatic activity completely. LNA and 29-FNA also demonstrated similar results. The distorted phosphate backbone by a switch from C29-endo to C39-endo sugar pucker conformation might be responsible for these observed cleavage activity changes. Conclusions We have conducted a study on the effect of 29-OMeN substituted substrates on the EcoRI enzymatic activity. Our results 41. Williams DM, Benseler F, Eckstein F (1991) Properties of 29-fluorothymidine- containing oligonucleotides: interaction with restriction endonuclease EcoRV. Biochemistry 30: 4001–4009. y 42. 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Koziolkiewicz M, Stec WJ (1992) Application of phosphate-backbone-modified oligonucleotides in the studies on EcoRI endonuclease mechanism of action. Biochemistry 31: 9460–9466. 46. Lesser DR, Kurpiewski MR, Jen-Jacobson L (1990) The energetic basis of specificity in the Eco RI endonuclease–DNA interaction. Science 250: 776–786. References Sen S, Nilsson L (1999) Structure, interaction, dynamics and solvent effects on the DNA-EcoRI complex in aqueous solution from molecular dynamics simulation. Biophys J 77: 1782–1800. 16. Frederick CA, Grable J, Melia M, Samudzi C, Jen-Jacobson L, et al. (1984) Kinked DNA in crystalline complex with EcoRI endonuclease. Nature 309: 327–331. 36. Gohda K, Matsuo N, Oda Y, Ikehara M, Uesugi S (1997) Effects of 29- substituents of the first deoxyguanosine residue in the recognition sequence of EcoRI restriction endonuclease activity. J Biochem 121: 219–224. 17. McClarin JA, Frederick CA, Wang BC, Greene P, Boyer HW, et al. (1986) Structure of the DNA-Eco RI endonuclease recognition complex at 3 A resolution. Science 234: 1526–1541. 37. Lesser DR, Grajkowski A, Kurpiewski MR, Koziolkiewicz M, Stec WJ, et al. (1992) Stereoselective interaction with chiral phosphorothioates at the central DNA kink of the EcoRI endonuclease-GAATTC complex. J Biol Chem 267: 24810–24818. 18. Li X, Song C, Zhao M, Li Y (2008) Continuous monitoring of restriction endonuclease cleavage activity by universal molecular beacon light quenching coupled with real-time polymerase chain reaction. Anal Biochem 381: 1–7. 38. Milligan JF, Uhlenbeck OC (1989) Determination of RNA-protein contacts using thiophosphate substitutions. Biochemistry 28: 2849–2855. 19. Urata H, Tamaki C, Matsuno M, Wada S, Akagi M (2009) FRET-based kinetic analysis of highly reactive heterochiral DNA toward EcoRI endonuclease. Biochem Biophys Res Commun 390: 192–195. 39. Jeltsch A, Alves J, Oelgeschlager T, Wolfes H, Maass G, et al. (1993) Mutational analysis of the function of Gln115 in the EcoRI restriction endonuclease, a critical amino acid for recognition of the inner thymidine residue in the sequence -GAATTC- and for coupling specific DNA binding to catalysis. J Mol Biol 229: 221–234. 20. Huang Y, Zhao S, Shi M, Chen J, Chen ZF, et al. (2011) Intermolecular and intramolecular quencher based quantum dot nanoprobes for multiplexed detection of endonuclease activity and inhibition. Anal Chem 83: 8913–8918. 21. Eisenschmidt K, Lanio T, Jeltsch A, Pingoud A (2002) A fluorimetric assay for on-line detection of DNA cleavage by restriction endonucleases. J Biotechnol 96: 185–191. 40. Pieken WA, Olsen DB, Benseler F, Aurup H, Eckstein F (1991) Kinetic characterization of ribonuclease-resistant 29-modified hammerhead ribozymes. Science 253: 314–317. 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https://openalex.org/W3119010174	https://www.frontiersin.org/articles/10.3389/fphar.2020.612071/pdf	English	null	A Review of Authenticity and Authentication of Commercial Ginseng Herbal Medicines and Food Supplements	Frontiers in pharmacology	2,021	cc-by	7,677	MINI REVIEW MINI REVIEW published: 11 January 2021 doi: 10.3389/fphar.2020.612071 A Review of Authenticity and Authentication of Commercial Ginseng Herbal Medicines and Food Supplements Mihael Cristin Ichim 1* and Hugo J. de Boer 2 1“Stejarul” Research Centre for Biological Sciences, National Institute of Research and Development for Biological Sciences, Piatra Neamt, Romania, 2Natural History Museum, University of Oslo, Oslo, Norway Ginseng traditional medicines and food supplements are the globally top selling herbal products. Panax ginseng, Panax quinquefolius and Panax notoginseng are the main commercial ginseng species in herbal medicine. Prices of ginseng products vary widely based on the species, quality, and purity of the used ginseng, and this provides a strong driver for intentional adulteration. Our systematic literature search has reviewed the authenticity results of 507 ginseng-containing commercial herbal products sold in 12 countries scattered across six continents. The analysis of the botanical and chemical identity of all these products shows that 76% are authentic while 24% were reported as adulterated. The number of commercial products as well as the percentage of adulteration varies signiﬁcantly between continents, being highest in South America (100%) and Australia (75%), and lower in Europe (35%), North America (23%), Asia (21%) and Africa (0%). At a national level, from the ﬁve countries for which more than 10 products have been successfully authenticated, the highest percentage of adulterated ginseng products were purchased from Taiwan (49%), followed by Italy (37%), China (21%), and USA (12%), while all products bought in South Korea were reported to be authentic. In most cases, labeled Panax species were substituted with other Panax species, but substitution of ginseng root, the medicinally recommended plant part, with leaves, stems or ﬂowers was also reported. Efﬁcient and practical authentication using biomarkers to distinguish the main ginseng varieties and secondary metabolite spectra for age determination are essential to combat adulteration in the global marketplace. Edited by: Sukvinder Kaur Bhamra, University of Kent, United Kingdom Reviewed by: Zemede Asfaw, Addis Ababa University, Ethiopia Anthony Booker, University of Westminster, United Kingdom Reviewed by: Zemede Asfaw, Addis Ababa University, Ethiopia Anthony Booker, University of Westminster, United Kingdom Correspondence: Mihael Cristin Ichim cichim@hotmail.com Specialty section: This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology Received: 30 September 2020 Accepted: 25 November 2020 Published: 11 January 2021 Specialty section: This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology Specialty section: This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology Received: 30 September 2020 Accepted: 25 November 2020 Published: 11 January 2021 Citation: Ichim MC and de Boer HJ (2020) A Review of Authenticity and Authentication of Commercial Ginseng Herbal Medicines and Food Supplements. Front. Pharmacol. 11:612071. doi: 10.3389/fphar.2020.612071 Received: 30 September 2020 Accepted: 25 November 2020 Published: 11 January 2021 Keywords: ginseng, Panax, identiﬁcation, adulteration, contamination, herbal product, herbal medicine, food supplement Selection Process and Criteria Selection Process and Criteria A total of 3,683 records were identiﬁed, with WoS 1,277, PubMed 692, Scopus 1,480, ScienceDirect 234 respectively, as well as another 49 records from other sources. Out of these a total 1,023 records were retained after removing duplicates. After screening, 869 records were excluded. The highly diverse reporting formats of the authenticity results made it necessary to deﬁne unifying criteria for selection and retain only the relevant articles in our review. The remaining 154 full-text articles were assessed for eligibility using the assessment criteria: 1) The reported samples had to be “herbal products” in the broad sense. The widest possible range of commercial names was used for the searches and accepted for inclusion in the review; 2) The analyzed products had to be “commercial.” The “cost-free,” “gifts” or the “donated” products were excluded from our analysis; 3) The products had to be allocated speciﬁcally to a “country” or “territory” (i.e., Hong Kong, European Union); 4) The “authentic” or “adulterated” conclusion was drawn by the authors of the reviewed articles; 5) All authentication methods were accepted. After applying these selection criteria, 120 articles were excluded because they did not report authentication results of commercial herbal products. This objective literature search identiﬁed 34 peer-reviewed publications reporting authentication of a total of 507 ginseng-containing commercial herbal products (Table 1). Authentication of botanical identity was reported for 468 products in 29 studies, and 13 of them used only DNA- based methods, mostly DNA barcoding. In another 13 studies only chemical methods were used, while the remainder used a combination of genetic and chemical authentication methods. Fifty-seven percent of studies (17 out of 30) reported authentication results for ginseng products purchased from China. The integrity of the chemical composition of 39 ginseng products was reported in ﬁve additional peer- reviewed studies. In the global medicinal plant trade, the economic value of ginseng is estimated to be more than US$ 2.1 billion (Manzanilla et al., 2018). Prices vary widely based on the quality and the quantity of the ginseng root. The ginseng cultivar, locality, growth condition (cultivated or harvested from the wild), and plant age are some of the most important factors which inﬂuence the quality and properties of P. ginseng (Kim et al., 2012). It was shown previously that P. Citation: These ginseng varieties are sold in different stages of processing from raw materials through highly processed products that have lost their botanical morphological characteristics to extracts differentiated mainly by ginsenoside content (Yang and Wu, 2016). In the global market, many different formulations of these herbs are available, including gelcaps, capsules, teas, tinctures, slices to eat in salads, powders, as well as entire roots. There are a wide variety of products that contain ginseng such as toothpaste, cigarettes, soaps, cosmetics, beverages (including beer), coffee, baby food, candies, and gum (Morgan and Cupp, 2010). credibility of the whole supply chain. The development of efﬁcient and practical authentication using biomarkers to distinguish the main ginseng varieties and secondary metabolite spectra for age determination are essential to combat adulteration. This review aims to provide an overview of the state of the art in ginseng authentication, while also highlighting current strengths and limitations. Citation: Ichim MC and de Boer HJ (2020) A Review of Authenticity and Authentication of Commercial Ginseng Herbal Medicines and Food Supplements. Front. Pharmacol. 11:612071. doi: 10.3389/fphar.2020.612071 Ginseng is used collectively to refer to several plant species, mainly in the Araliaceae genera Panax L. and Eleutherococcus Maxim. In China, the ginseng preparations have been used for thousands of years in traditional medicine (Robbins, 1998). Demand for ginseng roots in the 18th century intensiﬁed the wild harvest of the main species Panax ginseng C. A. Mey. (Araliaceae) (Korean ginseng), and nearly extirpated it from the wild (Millspaugh, 1974), but also fuelled a rapid expansion in wild-harvesting of Panax quinquefolius L. (Araliaceae) (American ginseng) that in turn destroyed Ginseng is used collectively to refer to several plant species, mainly in the Araliaceae genera Panax L. and Eleutherococcus Maxim. In China, the ginseng preparations have been used for thousands of years in traditional medicine (Robbins, 1998). Demand for ginseng roots in the 18th century intensiﬁed the wild harvest of the main species Panax ginseng C. A. Mey. (Araliaceae) (Korean ginseng), and nearly extirpated it from the wild (Millspaugh, 1974), but also fuelled a rapid expansion in wild-harvesting of Panax quinquefolius L. (Araliaceae) (American ginseng) that in turn destroyed January 2021 \| Volume 11 \| Article 612071 Frontiers in Pharmacology \| www.frontiersin.org Authenticity of Commercial Ginseng Products Ichim and de Boe wild populations in North America (Kimmens, 1975). There are currently 13 recognized ginseng species, but new taxa at species, subspecies and variety level are continuously published (Manzanilla et al., 2018). The most commonly used species in the genus are P. ginseng, P. quinquefolius, Panax notoginseng (Burkill) F. H. Chen (Araliaceae) (Chinese ginseng), Panax japonicus (T. Nees) C. A. Mey. (Araliaceae) (Japanese ginseng), Panax pseudoginseng Wall. (Araliaceae) (Himalayan ginseng) and Panax vietnamensis Ha and Grushv. (Araliaceae) (Vietnamese ginseng) (Yang and Wu, 2016). The majority of commercialized ginseng material is from cultivation and controlled sustainable wild harvest, whereas material from uncontrolled depletive wild harvesting plays a minor and decreasing role. P. ginseng, P. quinquefolius and P. notoginseng are the three most widely used species in herbal medicine, and are traded as either red ginseng (Ginseng Radix et Rhizoma Rubra), white ginseng (Ginseng Radix et Rhizoma), American ginseng (Panacis Quinquefolii Radix), or notoginseng (Notoginseng Radix et Rhizoma). Search Strategy gy Four databases (Web of Science, PubMed, Scopus, and ScienceDirect) were systematically searched for peer reviewed articles using keywords combined with Boolean operators: [(ginseng OR Panax) AND (identiﬁcation OR authentication OR authenticity OR authentic OR adulteration OR contamination OR substitution)] following the PRISMA guidelines (Moher et al., 2009). After the search was performed (on 17 February 2020), weekly updates were received and taken into consideration as the option “search alert” has been activated for all four literature databases. Frontiers in Pharmacology \| www.frontiersin.org Selection Process and Criteria ginseng possesses different active ingredients and differentiated curative effects depending on the age of the plants, that is why the older ginseng plants are sold at a much higher price than the younger ones (Kim et al., 2011; Yang et al., 2012; Pan et al., 2013). Tenfold and more price differences among the main Panax processed varieties is the main driver for intentional adulteration (Choi et al., 2007). Adulteration using low-price varieties, low grade material from above-ground parts or processing waste, alternative species, or nothing at all, could bring huge proﬁts through deceptive or illegal trade (Huang et al., 2017). The authentic herbal medicines and food supplements should be non-adulterated (Simmler et al., 2018) while the inadvertent or intentional adulteration (Simmler et al., 2018) includes contamination, product substitution and the use of ﬁllers (Shanmughanandhan et al., 2016). Efﬁcient discrimination methods for ginseng varieties is necessary to combat fraud. Product adulteration and substitution are severe and widespread problems in the ginseng market, given the signiﬁcant difference in medicinal value and economic beneﬁts of different ginseng preparations (Zhao et al., 2020). The adulteration interferes with the proper and correct use of ginseng products and compromises the January 2021 \| Volume 11 \| Article 612071 Frontiers in Pharmacology \| www.frontiersin.org 2 Authenticity of Commercial Ginseng Products Ichim and de Boe TABLE 1 \| The authenticity of ginseng commercial herbal products sold on the global market. Country/ territory Labeled and authenticated Panax sp./type of herbal product Identiﬁed adulterant Product composition Products Authentication methods Ref Total Authentic Adulterated no no no Botanical identity Brazil P. ginseng Pfafﬁa spp. Root products (0.2% ginsenoside Rg1 and 0.1% ginsenoside Rb1, HPLC) 5 0 5 DNA barcoding (Palhares et al., 2015) China P. ginseng, P. quinquefolius, P. notoginseng n/a Batches of TCM compound preparations (e.g. pill, bag, injection, capsule, tablet, powder, dripping pill) 40 38 2 LC–MS (Yang et al., 2016) China P. ginseng P. quinquefolius Batches of CPMs containing ginseng products 24 19 5 DNA barcoding (Liu et al., 2016) China P. ginseng P. quinquefolius Ginseng Radix et Rhizoma samples 15 12 3 DNA barcoding (Han et al., 2016) China P. notoginseng n/a Baches of CPM Shuxiong tablets prepared from Notoginseng Radix et Rhizoma), Carthami Flos, and Chuanxiong Rhizoma 12 12 0 UPLC/QTOF- Fast DDA (Yao et al., 2016) China P. ginseng, P. January 2021 \| Volume 11 \| Article 612071 Selection Process and Criteria quinquefolius n/a CPMs containing ginseng or American ginseng 11 8 3 MAS-PCR (Cheng et al., 2015) China P. ginseng, P. quinquefolius, P. notoginseng n/a TCHMs multi- ingredient ginseng preparations, Sheng Mai Yin (SMY) capsule and granules 11 6 5 HPTLC (Xie et al., 2006) China P. notoginseng ﬂower of P. notoginseng TCM preparations with P. notoginseng powder (dry roots and rootstock) 10 9 1 UPLC/Qtof MS (Liu et al., 2015) China P. ginseng n/a Ginseng containing samples 10 8 2 PCR (Zhou et al., 2016) China P. ginseng P. quinquefolius, Platycodon grandiﬂorum (Jacq.) A.DC. (Campanulaceae), Physochlaina infundibularisKuang (Solanaceae), Phytolacca acinosa Roxb. (Phytolaccaceae) Root samples 8 5 3 NIR barcode (Dong et al., 2020) China P. ginseng n/a Ginseng Radix et Rhizoma (dried roots and rhizomes of P. ginseng) 3 3 0 DNA barcoding (Zhang et al., 2019) China P. ginseng, P. notoginseng n/a steamed roots of P. ginseng; powdered roots of P. notoginseng 2 2 0 DNA barcoding (Dong et al., 2014) China P. ginseng n/a Shihu Yeguang Pills containing Ginseng Radix Et Rhizoma 1 1 0 DNA barcoding (Jiang et al., 2019) (Continued) TABLE 1 \| The authenticity of ginseng commercial herbal products sold on the global market. January 2021 \| Volume 11 \| Article 612071 Frontiers in Pharmacology \| www.frontiersin.org Authenticity of Commercial Ginseng Products Ichim and de Boe TABLE 1 \| (Continued) The authenticity of ginseng commercial herbal products sold on the global market. Country/ territory Labeled and authenticated Panax sp./type of herbal product Identiﬁed adulterant Product composition Products Authentication methods Ref Total Authentic Adulterated no no no China P. ginseng n/a Batches of Asian ginseng 11 11 0 UPLC/Q-TOF-MS (Li et al., 2010) China P. quinquefolius n/a Batches of American ginseng 7 7 0 Canada P. quinquefolius n/a Batches of American ginseng 5 5 0 United States P. quinquefolius n/a Batches of American ginseng 4 4 0 China P. quinquefolius P. ginseng Batches of American ginseng preparations 13 0 13 China P. ginseng leaf/stem Root extract 1 0 1 HPTLC, HPLC (Govindaraghavan, 2017) European Union P. ginseng P. ginseng leaf or other plant parts, P. quinquefolius roots Root extracts, stem/ leaf extract, berry extract 12 6 6 Australia P. ginseng P. ginseng leaf or other plant parts Capsules (extract or herb), tablet 4 1 3 South Korea P. ginseng n/a P. ginseng extract, tea, capsule 3 3 0 Multiplex PCR (Tian et al., 2020) China P. Frontiers in Pharmacology \| www.frontiersin.org Selection Process and Criteria ginseng, P. quinquefolius n/a P. ginseng extract, tea, capsule; P. quinquefolius extract, tea, capsule 6 6 0 United States P. ginseng, P. quinquefolius n/a P. quinquefolius extract, tea, capsule 3 3 0 Hong Kong P. ginseng n/a Ready-to-serve ginseng soup 1 1 0 Multiplex PCR, DNA sequencing (Lo et al., 2015) Hong Kong P. ginseng, P. quinquefolius n/a Dried roots, powders, tea granules 7 7 0 PCR, HAD (Jiang et al., 2014) Italy P. ginseng, P. quinquefolius n/a Raw materials (as body root, root tails and root prongs), capsules and tablets (containing dried extract) 19 12 7 RFLP, HPLC (Del Serrone et al., 2006) South Korea P. ginseng n/a decoctions, beverages, capsules, tablets 61 61 0 HPLC, UPLC–DAD–ESI- IT-TOF-MS (Choi et al., 2018) South Korea P. ginseng n/a Bak-Ho-Ga-Insam- Tang resources containing P. ginseng 14 14 0 Gradient PCR (Shim et al., 2005) Taiwan P. ginseng not composed of 6 years old ginseng radix only White ginseng radix sliced material, powder, capsules 7 6 1 1H-NMR (Lin et al., 2010) China P. ginseng n/a white ginseng radix sliced material 1 1 0 Taiwan P. ginseng Panacis quinquefolii Radix Chinese medical preparations containing Ginseng Radix 58 27 31 nested PCR, RFLP, DNA sequencing (Lu et al., 2010) United Kingdom P. ginseng, P. quinquefolius, P. notoginseng n/a American ginseng, white Asian ginseng, sanchi ginseng 8 8 0 LC-MS (Kite et al., 2003) United States P. ginseng Astragalus propinquus Schischkin (Leguminosae) Single ingredient HMP 1 0 1 DNA barcoding (Molina et al., 2018) (Continued) TABLE 1 \| (Continued) The authenticity of ginseng commercial herbal products sold on the global market. January 2021 \| Volume 11 \| Article 612071 Frontiers in Pharmacology \| www.frontiersin.org Authenticity of Commercial Ginseng Products Ichim and de Boe RESULTS The analysis of the botanical and chemical identity of 507 ginseng- containing commercial herbal products shows that 76% (n 386) are authentic while 24% (n 121) were reported as adulterated. These ginseng commercial herbal products were purchased from 12 countries scattered across six continents: Asia (n 375), North America (n 81), Europe (n 40), South America (n 5), Australia (n 4), and Africa (n 2). Selection Process and Criteria Among continents, the number of commercial products as well as the percentage of adulteration varies signiﬁcantly, being the highest in South America (100%, n 5) and Australia (75%, n 3), and lower in Europe (35%, n 14), North America (23%, n 19), Asia (21%, n 80) and Africa (0%) (Table 1). TABLE 1 \| (Continued) The authenticity of ginseng commercial herbal products sold on the global market. Country/ territory Labeled and authenticated Panax sp./type of herbal product Identiﬁed adulterant Product composition Products Authentication methods Ref Total Authentic Adulterated no no no United States P. ginseng, P. quinquefolius n/a Ground P. quinquefolius root (capsule), red P. ginseng root extract (liquid vial) 2 2 0 UPLC/QTOF-MS (Yuk et al., 2016) United States P. quinquefolius P. ginseng American ginseng products 6 4 2 HPLC (Yu et al., 2014) United States P. ginseng, P. quinquefolius soybean American and Korean ginseng fresh or dried roots, powders, capsules, tablets 24 22 2 PCR, RAPD, HPLC (Mihalov et al., 2000) China P. ginseng, P. quinquefolius n/a American and Korean ginseng dried root 2 2 0 United States and Canada P. ginseng, P. quinquefolius P. quinquefolius NHPs containing Red Korean, Korean, American, sand ginseng (capsules, tablets, roots, carved roots, extracts, teas, and dried and shredded products 36 22 14 DNA barcoding (Wallace et al., 2012) Total 468 358 110 Chemical composition China Functional food for relieving physical fatigue Testosterone, adalaﬁl, sildenaﬁl Fur seal ginseng pills with complex herbal composition, including P. ginseng 16 13 3 HPLC (Wang et al., 2019) China Antidiabetic functional food Tolbutamide, glimepiride, metformin Bitter melon and ginseng soft gels containing also containing American ginseng root 16 13 3 UPLC-Q-Orbitrap- MS/MS (Xie et al., 2019) China Herbal medicines for male sexual health Sildenaﬁl, hongdenaﬁl, vardenaﬁl, homosildenaﬁl Complex herbal composition, including Radix et rhizoma ginseng or Radix et rhizoma ginseng rubra (tablet, capsule, pills, soft gel capsule) 4 0 4 TLC, HPLC-MS (Cai et al., 2010) Saudi Arabia Dietary supplements for weight loss, slimming and as a stimulant or stamina enhancer Theobromine, theophylline, pseudoephedrine, caffeine, hydrochlorothiazide, yohimbine Ginseng extract (capsule), Korean Ginseng (capsule) 2 2 0 UHPLC–DAD (Ahmad et al., 2020) Sweden Dietary supplement Ephedrine Ginseng preparation 1 0 1 n/a (Cui et al., 1994) Total 39 28 11 TOTAL 507 386 121 TABLE 1 \| (Continued) The authenticity of ginseng commercial herbal products sold on the global market. RESULTS quinquefolius in China (Han et al., 2016; Liu et al., 2016; Dong et al., 2020) and Taiwan (Lu et al., 2010), and vice-versa in United States (Li et al., 2010; Yu et al., 2014), so that the species most highly valued in that country is substituted by other ginseng species. The adulteration of traditional ginseng medicinal products included the substitution of ginseng root, the medicinally recommended plant part, with leaves, stems (Govindaraghavan, 2017) or even ﬂowers (Liu et al., 2015), all replacing the high value herbal material with less costly ones. Liu et al. (2017) showed that ginsenoside ratios could be used to determine the age of cultivated Panax ginseng with 30-fold differences in some ginsenosides with age. Premium ginseng commercial products sold on the Taiwanese market have been reported to be adulterated with inferior plant material (Lin et al., 2010). Economically motivated adulteration of commercial ginseng products includes the use of soybean as the only plant ingredient identiﬁed in commercial products sold in the United States (Mihalov et al., 2000) thus conﬁrming the reported widespread adulteration of commercial herbal products on the North American market (Newmaster et al., 2013). Analysis of authentication results per country (n 459 commercial ginseng products were clearly allocated to a single country) show that the number of samples purchased and tested for each country varies. In China, 224 ginseng products were successfully authenticated, representing almost half (49%) of all reported samples worldwide, while South Korea (n 78), Taiwan (n 65) and United States (n 40) follow distantly. All of these countries, except Taiwan, are the three major ginseng cultivating countries in the world. This data also suggests the importance of these herbal products in Asian traditional medicine systems is mirrored by the interest of the scientiﬁc community to develop and test new methods on market products. Much smaller numbers of tested commercial ginseng products were reported for Italy (n 19), Hong Kong (n 8), United Kingdom (n 8), Brazil (n 5), Canada (n 5), Australia (n 4), Saudi Arabia (n 2), and Sweden (n 1), but they support and reconﬁrm the widespread interest for ginseng-containing products on the global market. RESULTS America (n 81), Europe (n 40), South America (n 5), Australia (n 4), and Africa (n 2). Among continents, the number of commercial products as well as the percentage of adulteration varies signiﬁcantly, being the highest in South America (100%, n 5) and Australia (75%, n 3), and lower in Europe (35%, n 14), North America (23%, n 19), Asia (21%, n 80) and Africa (0%) (Table 1). The analysis of the botanical and chemical identity of 507 ginseng- containing commercial herbal products shows that 76% (n 386) are authentic while 24% (n 121) were reported as adulterated. These ginseng commercial herbal products were purchased from 12 countries scattered across six continents: Asia (n 375), North January 2021 \| Volume 11 \| Article 612071 Frontiers in Pharmacology \| www.frontiersin.org 5 Authenticity of Commercial Ginseng Products Ichim and de Boe TABLE 2 \| National distribution of the ginseng commercial ginseng herbal products and their authenticity. Country Products (total) Authentic/adulterated no no % no %* China 224 176 79% 48 21% South Korea 78 78 100% 0 0% Taiwan 65 33 51% 32 49% United States 40 35 88% 5 12% Italy 19 12 63% 7 37% Hong Kong 8 8 100% 0 0% United Kingdom 8 8 100% 0 0% Brazil 5 0 0% 5 100% Canada 5 5 100% 0 0% Australia 4 1 25% 3 75% Saudi Arabia 2 2 100% 0 0% Sweden 1 0 0% 1 100% *The percentage values were rounded to the nearest whole number. TABLE 2 \| National distribution of the ginseng commercial ginseng herbal products and their authenticity. adulterated with respect to the labeled species or declared chemical composition. Adulterated ginseng products are present across many regions and countries, and this conﬁrms a recent global analysis of DNA-based authenticity testing, both in terms of adulteration percentage and geographic spread (Ichim, 2019). In addition, our analysis further conﬁrms the widespread presence of adulterated commercial herbal products in traditional medicine systems, including Traditional Chinese Medicine (TCM) (Han et al., 2016) and Ayurvedic medicine (Seethapathy et al., 2019). ( p y ) In most cases, labeled Panax species are substituted with other Panax species. P. ginseng is replaced by P. RESULTS The widespread use of the ginseng vernacular name across many countries and continents, including for species not from the same genus or even family (Osathanunkul and Madesis, 2019), is a catalyst conducive to adulteration. In Brazil for example, ginseng products have been found to be adulterated with a completely unrelated species from another family, Pfafﬁa spp., albeit locally known as Brazilian ginseng (Palhares et al., 2015). The inadvertent contamination through plant misidentiﬁcation during harvesting or cross-contamination during processing but also the intentional and fraudulent use of ﬁller species or cheaper substitutes, present on the herbal product market (Jordan et al., 2010; Sgamma et al., 2017; Ichim et al., 2020), also affects the highly valued ginseng-containing food supplements and traditional medicines across the globalized market. The presence of substitutes or ﬁller species in some cases reﬂects intentional, economically motivated and fraudulent practices by producers or vendors although the European Pharmacopoeia (Ph. Eur.) and the United States Pharmacopeia (USP) as well as some monographs for herbal raw materials, allow a certain amount (e.g., 2% in USP) of foreign organic matter (Parveen et al., 2016; Sgamma et al., 2017) as acceptable accidental contamination. Counterfeiting by adding synthetic prescription drugs as chemical adulterants is another form of falsiﬁcation, and Calahan et al. (2016) found that 28% of authenticated ginseng The ginseng authenticity reported for the 12 countries represented in our review range widely from country to country. The ﬁve countries for which more than 10 products have been successfully authenticated account together for 84% of all ginseng products analyzed worldwide. Out of these countries, the highest percentage of adulterated ginseng products were purchased from Taiwan (49%), followed by Italy (37%), China (21%), United States (12%), while all the products bought from the South Korean market were reported to be authentic. Notably, six out of the seven remaining countries, each with less than 10 authenticated commercial samples, have all their products reported either as authentic (i.e., Canada, Hong Kong, Saudi Arabia, United Kingdom), or adulterated (i.e., Brazil, Sweden) while the ginseng products purchased from Australia were reported to be 25% authentic and 75% adulterated (Table 2). Authenticity of Commercial Ginseng Herbal Products Sold on the Global Market Authenticity of Commercial Ginseng Herbal Products Sold on the Global Market Overall, the peer-reviewed publications show that one in each four ginseng containing herbal products sold on the market is January 2021 \| Volume 11 \| Article 612071 Frontiers in Pharmacology \| www.frontiersin.org 6 Authenticity of Commercial Ginseng Products Ichim and de Boe ginseng may also produce effects similar to those of estrogen (Anadón et al., 2016). Moreover, women may experience additional side effects, such as vaginal bleeding and breast tenderness. Most of these side effects are serious enough to warrant stopping taking ginseng in breast cancer patients (Sellami et al., 2018). A rare adverse drug reaction to herbal and dietary supplementation, the drug-induced liver injury (DILI), was reported as result from ingestion of ginseng for premenopausal symptoms (Lin et al., 2018). Recently, a website with a critically reviewed database presenting reported cases of ginseng-drug interactions was publicly launched (Wu et al., 2019). products in their study were adulterated. Apart from the many cases of adulteration and general low quality of commercial herbal products, P. ginseng was also reported, along with some other plant species, as adulterant of a herbal product sold in Australia, supposedly to contain only Eucalyptus radiata A. Cunn. ex DC. (Myrtaceae) and Melaleuca alternifolia (Maiden and Betche) Cheel (Myrtaceae). This latter case suggests either accidental contamination through poor manufacturing process or intentional adulteration for achieving an expected physiological or pharmacological effect (Hoban et al., 2020). Ginseng-Drug Interactions, Abuse and Negative Effects of Long-Term Use on Human Health The herbal medicines differ considerably from conventional medicines, and they pose a variety of challenges to their pharmacovigilance. For herbal pharmacovigilance, four main challenges: 1) Substitution and adulteration; 2) Nomenclature of herbals and ingredients of plant origin; 3) Lack of monitoring; and 4) Standardization, have been identiﬁed (de Boer et al., 2015). This review highlights that all four apply to ginseng commercial products, and support the challenge of detecting signiﬁcant adverse drug reactions in a timely manner to protect consumers. The presence of unlabeled species, plant extracts or synthetic chemical compounds might negatively interact with other medicinal plants, food supplements or prescription drugs and will pose signiﬁcant risks for human health (Jordan et al., 2010). Adverse drug reactions (ADRs) due to herb–drug interactions (HDI) can appear in patients taking herbs and prescribed medications concomitantly (Awortwe et al., 2018). Ginseng can interfere with various drugs, such as digoxin, insulin, anticoagulants, and monoamine oxidase inhibitors (Sellami et al., 2018). Pharmacovigilance relies heavily on ADR reporting, and despite initiatives to stimulate reporting of suspected ADRs associated with herbal medicines, numbers of herbal ADR reports are relatively low. ADRs Under- reporting is likely to be speciﬁc for herbal medicines, since their users usually do not look for medical advice about their use of such products, or report if they experience any adverse effects (Barnes, 2003). CONCLUSION Despite being the most valuable herbal product in terms of market value share, ginseng products are poorly regulated. Several authentication studies have shown that adulteration is not uncommon and not limited to any speciﬁc country of origin. The high market value of ginseng provides an incentive for fraudulent actors to generate proﬁts at the expense of gullible consumers and honest producers, wholesalers and retailers. Although authentication using traditional, pharmacopoeial, analytical methods such as TLC, HPLC and NMR can be used for advanced quality control, the standard authentication protocols are insufﬁcient for efﬁciently detecting species adulteration, adulteration with synthetic pharmaceuticals and spiking of low quality products with marker compounds. Cutting-edge approaches enable distinction of age-speciﬁc metabolite spectra, quantiﬁcation of active ingredients and accurate identiﬁcation of ginseng species, but these are not yet in widespread use. The speciﬁc active constituents in Panax herbs, the ginsenosides, have been shown to improve immune function, reduce mental stress, and stabilize blood pressure while ginseng products are used as an endurance performance enhancer (Sellami et al., 2018). Nevertheless, authentic ginseng products does not represent a doping concern for athletes, as there were no positive tests for any International Olympic Committee (IOC) banned or restricted substances in any of the subjects after the ingestion of commercially available, proprietary ginseng root extract product (Goel et al., 2004). Adverse drug reactions to ginseng are associated with high doses and long-term usage (Kiefer and Pantuso, 2003; Sellami et al., 2018). The ginseng abuse syndrome includes edema, decreased appetite, depression, and hypotension (∼10%), hypertension (17%), sleeplessness (20%), nervousness (25%), skin eruption (25%), and morning diarrhea (35%) (Paik and Lee, 2015). 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https://openalex.org/W3015494688	https://www.medrxiv.org/content/medrxiv/early/2020/04/15/2020.04.09.20059014.full.pdf	English	null	How much of SARS-CoV-2 Infections is India detecting? A model-based estimation	medRxiv (Cold Spring Harbor Laboratory)	2,020	cc-by	2,705	. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 15, 2020. ; https://doi.org/10.1101/2020.04.09.20059014 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 15, 2020. ; https://doi.org/10.1101/2020.04.09.20059014 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 15, 2020. ; https://doi.org/10.1101/2020.04.09.20059014 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 15, 2020. ; https://doi.org/10.1101/2020.04.09.20059014 doi: medRxiv preprint reprint reports new research that has not been certified by peer review and should not be used to guide clinical practice How much of SARS-CoV-2 Infections is India detecting? A model-based estimation First Author: Srinivas Goli, Ph.D. (Corresponding Author) Australia India Institute (AII) NGN Research Fellow UWA Public Policy Institute University of Western Australia (UWA) 35 Stirling Highway Perth WA 6009 Australia Email: srinivas.goli@uwa.edu.au T +61 8 6488 2914, M +61 41`6271232 & Assistant Professor, Population Studies Centre for the Study of Regional Development Room No. 102, School of Social Sciences (SSS-III) Jawaharlal Nehru University (JNU) New Delhi-110067 Phone No: 011 26738798 The University of Western Australia (M251), 35 Stirling Highway, 6009 Perth, Australia Email: srinivas.goli@uwa.edu.au; sirispeaks2u@gmail.com http://orcid.org/0000-0002-8481-484X Second Author: K.S. James, PhD Director & Senior Professor International Institute for Population Sciences (IIPS), Govandi Station Road, Deonar, Mumbai – 400088, India Phone No: 022-42372888 Email: ksjames@iips.net; ksjames@gmail.com NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 15, 2020. ; https://doi.org/10.1101/2020.04.09.20059014 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 15, 2020. ; https://doi.org/10.1101/2020.04.09.20059014 doi: medRxiv preprint Abstract: Background and Rationale: Amid SARS-CoV-2 outbreak, the low number of infections for a population size of 1.38 billion is widely discussed, but with no definite answers. Methods: We used the model proposed by Bommer and Vollmer to assess the quality of official case records. The infection fatality rates were taken from Verity et al (2020). Age distribution of the population for India and states are taken from the Census of India (2011). Reported number of deaths and SARS-CoV-2 confirmed cases from https://www.covid19india.org. The reported numbers of samples tests were collected from the reports of the Indian Council for Medical Research (ICMR). Results: The findings suggest that India is detecting just 3.6% of the total number of infections with a huge variation across its states. Among 13 states which have more than 100 COVID-19 cases, the detection rate varies from 81.9% (of 410 estimated infections) in Kerala to 0.8% (of 35487 estimated infections) in Madhya Pradesh and 2.4% (of 7431 estimated infections) in Gujarat. Conclusion: As the study reports a lower number of deaths and higher recovery rates in the states with a high detection rate, thus suggest that India must enhance its testing capacity and go for widespread testing. Late detection puts patients in greater need of mechanical ventilation and ICU care, which imposes greater costs on the health system. The country should also adopt population-level random testing to assess the prevalence of the infection. Introduction and rationale The low numbers of SARS-CoV-2 cases in India given its population size presents a conundrum that is widely discussed, but with no definite answers. The low testing rate is often cited as a possible explanation for the low observed positive cases. There is considerable interest and need to understand the true rate of infection, but at the same time, it is difficult to obtain unless random tests are carried out at the population level. Since that may involve a long waiting period, what can be the possible predicted rates from available models across countries? In this context, we used the model proposed by Bommer and Vollmer to assess the quality of official case records. Model Researchers from the University of Gottingen used age-specific infection fatality rates from a study published in The Lancet Infectious Diseases journal5,1 to estimate the total number of infections. The details of Bommer and Vollmer (2020) model are presented elsewhere5. We have adjusted the age-specific infection fatality rates presented in the Lancet with the age distribution of the population of India and 13 states which have more than 100 confirmed cases as on 8th April. The Lancet study reported perfect ascertainment between observed and estimated cases in the age group 50-59 years. We apply the infection fatality rate of this age group to all ages assuming infection attack rate is uniform for population across the age- groups in India. The number of infections is estimated as the number of deaths till April, 08, 2020 divided by the adjusted infection fatality rate. Based on the global evidence, we assumed that the mean duration between the onsets of symptoms to deaths is 14 days. Thus, it yields the total number of infections two weeks prior to April 8. The detection rate is estimated as the number of confirmed cases divided by the estimated number of infections. Further, by taking data from reports of the ICMR, we have also adjusted the detection rates with the sum of the difference in the rate of change in testing done on a daily basis to derive a more realistic figure of the total number of infections (confirmed + undetected infections) for India as of April 8. Results Our estimates suggest that the total number of COVID-19 infections in India could be around 1, 59,939 instead of the 5,480 confirmed cases as on 8th April. As a result, the detection rate of infections for the country as a whole remains merely at 3.6%, below the world average of 6%. Based on the evidence from other countries, we assume that a major share (85%) of these cases may be those with milder symptoms, who may not have ended up in hospitals6. However, we found a huge variation within India. Among 13 states which have more than 100 COVID-19 cases, the detection rate varies from 81.9% (of 410 estimated infections) in Kerala to 0.8% (of 35487 estimated infections) in Madhya Pradesh and 2.4% (of 7431 estimated infections) in Gujarat. The detection rate in Maharashtra (the biggest COVID-19 infected state) is also very low with 1.8% (of 61,268 estimated infections). However, Rajasthan is moderately doing well in terms of the detection rate of infections by confirming 15.8% of total estimated infections. Even, economically and health systems wise better-off states such as Andhra Pradesh, Telangana, Tamil Nadu and Delhi are performing poorly in terms of detection rate; thus, experiencing more deaths than Rajasthan and Uttar Pradesh where more individuals were tested than estimated infections (Figure1 & Table 1). Nearly 19.7% of all the COVID-19 infected cases are from Maharashtra alone, while its low detection rates pose a greater threat of COVID-19 related mortality in the state. Currently, the state contributes to 40% of the COVID-19-related deaths, and if the low detection rate continues, this number is likely to rise even further. Data We have used data from multiple sources. The infection fatality rates were taken from Verity et al. (2020)1. Age distribution of the population for India and states are taken from the Census of India (2011)2. Reported number of deaths and SARS-CoV-2 confirmed cases from https://www.covid19india.org3. The reported numbers of samples tests were collected from the reports of the Indian Council for Medical Research (ICMR)4. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 15, 2020. ; https://doi.org/10.1101/2020.04.09.20059014 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 15, 2020. ; https://doi.org/10.1101/2020.04.09.20059014 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuit (which was not certified by peer review) The copyright holder for this prep this version posted April 15, 2020. ; https://doi.org/10.1101/2020.04.09.20059014 doi: medRxiv preprint Conclusion Because states with a high detection rate (Kerala and Rajasthan) show lower death rates, we suggest that India must enhance its testing capacity and go for widespread testing. Late detection puts patients in greater need of mechanical ventilation and ICU care, which imposes greater costs on the health system. Early detection and avoidance of critical care for a majority of infected patients is an important way of avoiding overcrowding on the limited health care resources available in the country. The country should also adopt population-level random testing to assess the prevalence of the infection. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 15, 2020. ; https://doi.org/10.1101/2020.04.09.20059014 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 15, 2020. ; https://doi.org/10.1101/2020.04.09.20059014 doi: medRxiv preprint Also, there is a need to put out age-wise and co-morbidity characteristics of the patients who died from COVID-19 in the domain of epidemiologists, public health scientists and demographers to assess the relative risk of the infected population based on demographic characteristics. Such rapid assessments are critical to control the damage early and to ensure that resources are used efficiently. References 1. Verity et al. “Estimates of the severity of coronavirus disease 2019: a model-based analysis. The Lancet Infectious Diseases 2020. DOI: 10.1016/S1473-3099(20)30243-7. 1. Verity et al. “Estimates of the severity of coronavirus disease 2019: a model-based analysis. The Lancet Infectious Diseases 2020. DOI: 10.1016/S1473-3099(20)30243-7. 2. COVID19India https://www.covid19india.org, 2020 2. COVID19India https://www.covid19india.org, 2020 2. COVID19India https://www.covid19india.org, 2020 2. COVID19India https://www.covid19india.org, 2020 3. Office of RGI and Census Commissioner, Census of India, 2011. Office of Registrar General of India, Government of India, New Delhi. 4. ICMR. ICMR Website of Testing Update for various dates, https://icmr.nic.in/sites/default/files/whats_new/ICMR_testing_update_08April_9PM_IST.pdf 5. Bommer C & Vollmer S (2020) Average detection rate of SARS-CoV-2 infections is estimated around six percent. www.uni-goettingen.de/en/606540.html. 6. Schwalbe, N., We could be vastly overestimating the death rate for COVID-19. Here's why, 2020, https://www.weforum.org/agenda/2020/04/we-could-be-vastly-overestimating-the-death- rate-for-covid-19-heres-why/ . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 15, 2020. ; https://doi.org/10.1101/2020.04.09.20059014 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 15, 2020. ; https://doi.org/10.1101/2020.04.09.20059014 doi: medRxiv preprint Figure 1: State-wise estimated total number of COVID-19 infections, Total tested samples and Detection rate in India as on 8th April, 2020. 61268 15490 17722 11674 2299 410 3368 11149 35487 1821 7431 1825 3644 150939 17563 5305 8464 1319 12279 11232 5255 4704 2812 6580 3552 2318 1551 101068 0 20000 40000 60000 80000 100000 120000 140000 160000 M aharashtra Tam il Nadu Delhi Telangana Rajasthan Kerala Uttar Pradesh Andhra Pradesh M adhya Pradesh Karnataka Gujarat Haryana Jam m u & Kashm ir India Total Estim ated infections and Tested sam ples Estimated number of infections (as on 08/04/2020) Total tested samples (ICMR) 1: State-wise estimated total number of COVID-19 infections, Total tested samples tection rate in India as on 8th April, 2020. Figure 1: State-wise estimated total number of COVID-19 infections, Total tested samples and Detection rate in India as on 8th April, 2020. Figure 1: State-wise estimated total number of COVID-19 infections, Total tested samples and Detection rate in India as on 8th April, 2020. tal number of i ed of ns 020) Estimated number o infection on 26/03/20 3 4 130 1514 29 1334 36 2270 43 2546 43 558 138 345 43 894 10 694 23 5768 55 1134 43 3658 21 507 14 836 730 41204 total number of eaths, we modell ed cases as on 2 ty rate from ver mber of in Estimated number of infections on 26/03/202 4 15141 1334 2270 2546 558 345 894 694 5768 1134 3658 507 836 41204 umber of e modelle s as on 26 from veri ( 03/2020 4 15141 1334 2270 2546 558 345 894 694 5768 1134 3658 507 836 41204 ber of in modelled s on 26/ m verity 15141 1334 2270 2546 558 345 894 694 5768 1134 3658 507 836 41204 mber of modell as on 2 m ver 2. COVID19India https://www.covid19india.org, 2020 61268 15490 17722 11674 2299 410 3368 11149 35487 1821 7431 1825 3644 150939 17563 5305 8464 1319 12279 11232 5255 4704 2812 6580 3552 2318 1551 101068 0 20000 40000 60000 80000 100000 120000 140000 160000 M aharashtra Tam il Nadu Delhi Telangana Rajasthan Kerala Uttar Pradesh Andhra Pradesh M adhya Pradesh Karnataka Gujarat Haryana Jam m u & Kashm ir India Total Estim ated infections and Tested sam ples Estimated number of infections (as on 08/04/2020) Total tested samples (ICMR) 836 4120 ber of model as on 2 m ver total number of infections and detection rate in India as of 8th April, 2020. med r of ons 2020) Estimated number of infections (as on 26/03/2020)2 Estimated detection rate (%)3 Number of confirmed cases (as on 8/04/2020) Adjusted detection rate (%) ♣ Estimated number of infections (as on 08/04/2020)4 Total tested samples (ICMR) Da for sam 3 4 5 6 7 8 9 130 15141 0.86 1078 1.76 61268 17563 29 1334 2.17 690 4.45 15490 5305 36 2270 1.59 576 3.25 17722 8464 43 2546 1.69 404 3.46 11674 1319 43 558 7.70 363 15.79 2299 12279 138 345 39.96 336 81.88 410 11232 43 894 4.81 332 9.86 3368 5255 10 694 1.44 329 2.95 11149 4704 23 5768 0.40 290 0.82 35487 2812 55 1134 4.85 181 9.94 1821 6580 43 3658 1.18 179 2.41 7431 3552 21 507 4.14 155 8.49 1825 2318 14 836 1.67 125 3.43 3644 1551 730 41204 1.77 5480 3.63 150939 101068 ts total number of infections for the states with more than 25 deaths. However, considering th deaths, we modelled the estimates for all those states which greater than 100 cases on 8th April, ated cases as on 26/03/2020 has been adjusted for the growth in the number of tests performe ality rate from verity et al. (2020) after adjusting them to age-distribution of the population of ing t April orm on o D fo sa 9 3 5 4 9 9 2 5 4 2 0 2 8 8 ring t April form ion o

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